Synthesis and biological evaluation of isoxazolyl-sulfonamides: A non-cytotoxic scaffold active against Trypanosoma cruzi, Leishmania amazonensis and Herpes Simplex Virus

Article abstract of DOI:10.1016/j.bmcl.2018.08.040

In this study we report the synthesis, characterization, biological evaluation, and druglikeness assessment of a series of 20 novel isoxazolyl-sulfonamides, obtained by a four-step synthetic route. The compounds had their activity against Trypanosoma cruz

Full text of DOI:10.1016/j.bmcl.2018.08.040

Accepted Manuscript
Synthesis and biological evaluation of isoxazolyl-sulfonamides: a non-cytotoxic
scaffold active against Trypanosoma cruzi, Leishmania amazonensis and Her-
pes Simplex Virus
Rafael da Rosa, Lara Almida Zimmermann, Milene Höehr de Moraes, Naira
Fernanda Zanchett Schneider, Alice Schappo, Claudia Maria de Oliveira
Simões, Mario Steindel, Eloir Paulo Schenkel, Lílian Sibelle Campos Bernardes
PII:
S0960-894X(18)30717-0
https://doi.org/10.1016/j.bmcl.2018.08.040
BMCL 26020
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 July 2018
27 August 2018
30 August 2018
Please cite this article as: da Rosa, R., Zimmermann, L.A., de Moraes, M.H., Schneider, N.F.Z., Schappo, A., de
Oliveira Simões, C.M., Steindel, M., Schenkel, E.P., Bernardes, L.S.C., Synthesis and biological evaluation of
isoxazolyl-sulfonamides: a non-cytotoxic scaffold active against Trypanosoma cruzi, Leishmania amazonensis and
Herpes Simplex Virus, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.
2018.08.040
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1
Synthesis and biological evaluation of isoxazolyl-sulfonamides: a non-cytotoxic scaffold
active against Trypanosoma cruzi, Leishmania amazonensis and Herpes Simplex Virus.
Rafael da Rosaa,d , Lara Almida Zimmermanna,d, Milene Höehr de Moraesb, Naira Fernanda Zanchett
Schneiderc, Alice Schappoa, Claudia Maria de Oliveira Simõesc, Mario Steindelb, Eloir Paulo
Schenkela, Lílian Sibelle Campos Bernardesa*
aGraduate Program in Pharmacy, Pharmaceutical 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.
cApplied Virology Laboratory, Department of Microbiology, Immunology and Parasitology, Federal
University of Santa Catarina, Florianópolis, 88040-900 SC, Brazil.
dThese authors contributed equally to this work
*Corresponding author: l.bernardes@ufsc.br
Highlights
●
●
●
Synthesis and characterization of 20 isoxazolyl-sulfonamides are reported.
The compounds were active against T. cruzi, L. amazonensis, and HSV-1.
Druglikeness evaluation showed potential for further pre-clinical development.
Abstract
In this study we report the synthesis, characterization, biological evaluation, and druglikeness
assessment of a series of 20 novel isoxazolyl-sulfonamides, obtained by a four-step synthetic route.
The compounds had their activity against Trypanosoma cruzi, Leishmania amazonensis, Herpes
Simplex Virus type 1 and cytotoxicity evaluated in phenotypic assays. All compounds have drug-like
properties, showed low cytotoxicity and were promising regarding all other biological activities
reported herein, especially the inhibitory activity against T. cruzi. The compounds 8 and 16 showed
significant potency and selectivity against T. cruzi (GI50=14.3 µM, SI>34.8 and GI50=11.6 µM, SI=29.1,
respectively). These values, close to the values of the reference drug benznidazole (GI50=10.2 µM),
suggest that compounds 8 and 16 represent promising candidates for further pre-clinical development
targeting Chagas disease.
Keywords: isoxazole; sulfonamide; Trypanosoma cruzi; Leishmania amazonensis; Herpes Simplex
Virus

2
Since the discovery of Prontosil (4-[(2,4-diaminophenyl)diazenyl]benzenesulfonamide) as an
antibacterial agent in 1932, sulfa drugs have a prominent place in therapeutics1. Soon after,
sulfonamide-containing antidiabetics2 and carbonic anhydrase inhibitor drugs3,4 reached the market
and remain relevant until current days. In recent decades, research on these classes continues
active5–9 although it also has moved to distinct applications. Sulfa-bearing compounds displaying anti-
inflammatory (COX-2 inhibitors)10, anti-proliferative11–14, anti-parasitary15–18, anti-A fibrillogenisis19,20
,
among other properties, have been reported and showcase the versatility of their biological potential.
Our research group has been working on the synthesis of heterocyclic compounds based on
natural scaffolds to obtain new trypanocidal and leishmanicidal agents. In a previous work, a series of
3,5-disubstituted isoxazole derivatives was reported active against T. cruzi, and in an effort to expand
the understanding of the structure-activity relationship, isoxazole-triazole bis-heterocyclic derivatives
were later synthesized and assayed against the parasite21,22. Considering that sulfonamide moieties
are frequent in numerous molecules of medicinal interest, we planned derivatives containing this
functional group between the aromatic rings present in the previously reported molecules. The
biological potential of these compounds was assessed by their cytotoxic, trypanocidal, leishmanicidal
and antiherpes activities together with a druglikeness evaluation assisted by in silico tools.
Synthetically, the derivatives were obtained in four steps from 5-hydroxymethyl-3-phenyl-
isoxazole derivatives (Compound a, Scheme 1)23.
Scheme 1. Synthesis of isoxazolyl-sulfonamide compounds. i) SOCl2, CH2Cl2, N2, r.t. ii) NaN3, DMF,
r.t. iii) TPP, THF, r.t. iv) sulfonyl chloride, pyridine, CH2Cl2, r.t.
In the first step, the hydroxyl group was converted into a chlorine (Compound b), which is a
suitable leaving group for the SN2 reaction with sodium azide in the second step (Compound c). The
azide derivative was reduced to an amine through the Staudinger reaction using triphenylphosphine in
THF (Compound d) and, finally, the amine derivatives were coupled with sulfonyl chlorides leading to
the isoxazolyl-sulfonamide derivatives 1-20 in satisfactory yields (from 17% to 62%). The final

3
1
compounds were characterized by H and 13C NMR spectroscopy and HRMS. The spectral details
are described in Supplementary Data.
The biological evaluation of derivatives 1-20 started by measuring the effects of compounds on
cell viability (VERO, THP-1, A549 and HCT-8 cell lines), using either MTT or sulforhodamine B
assays. The compounds were tested at the maximum concentration of 500 µM, since a low
cytotoxicity is desired because it would contribute to high selectivity indexes (SI) in the other activities.
Most of the compounds had no effects on cell viability even at the highest concentration tested.
Compounds 5, 11, 13, and 16 showed, in general, low cytotoxicity, which should not impact their
biological application. Their concentration values responsible for reducing cell viability by 50% (CC50
µM) can be found in Table 1.
Table 1. Cytotoxicity of compounds 5, 11, 13 and 16.
ID
5
A549a
HCT-8a
VEROa
THP-1b
>500
>500
>331.1
(130.2 – 842.2)
397.2
>500
11
13
16
327.3
(232.2 – 461.3)
357.8
(246.3 – 519.7)
283.5
373.8
(219.7 – 536.2)
436.9
(279.3 – 683.3)
>500
299.8
(283.0 – 317.0)
>500
(191.5 – 524.0)
>500
387.0
339.1
(192.7 – 417.2)
(188.6 – 793.9)
(321.0 – 357.0)
a,bData represent the mean (95% confidence interval) of two or three independent experiments, respectively, and
are expressed as CC50 values.
Next, we aimed to investigate compounds 1-20 as potential hits that could be developed to
treat Chagas disease - a life-threatening condition caused by the protozoan parasite Trypanosoma
cruzi. Although the disease is highly prevalent in Latin America countries, benznidazole (Rochagan®)
and nifurtimox (Lampit®) are the only drugs currently available for pharmacological treatment and both
show limited efficacy24–26. Having in mind the urgency of identifying new chemical entities which are
active against the parasite, we used intracellular amastigotes of T. cruzi (Tulahuen strain expressing
ß-galactosidase) cultivated in THP-1 cells to evaluate the trypanocidal activity of the compounds
using a colorimetric assay. Phenotypic approaches are highly recommended in drug discovery for
neglected tropical diseases once they allow identifying cell-permeating compounds that might affect
parasites by reaching different molecular targets27–29
.
Compounds 1-20 were screened at 50 µM and, then, if the growth inhibition (%GI) was higher
than 50%, they were serially diluted to determine the exact concentration responsible for inhibiting
50% of the parasite growth (GI50). Table 2 shows that 14 out of 20 compounds inhibited the parasite
growth to some extent and four of them presented GI50 values lower than 50 µM.
Table 2. Trypanocidal screening of isoxazolyl-sulfonamides 1-20.
ID
%GI (50 µM)
GI50 (µM)
SI

4
1
2
5.8±1.1
16.6±4.0
NA
>50
>50
-
-
3
NA
-
4
34.0±1.2
NA
>50
-
5
NA
-
6
NA
NA
-
7
44.5±4.9
85.8±1.0
25.6±14.8
27.4±2.5
72.8±1.9
53.9±6.7
NA
>50
-
8
14.3±0.9
>50
>34.8
9
-
10
11
12
13
14
15
16
17
18
19
20
BZ
N
>50
-
42.4±0.9
>50
7.1
-
NA
-
14.2±0.7
NA
>50
-
NA
-
86.3±0.5
9.8±1.0
NA
11.6±1.0
>50
29.1
-
NA
-
-
7.8±1.0
76.2±1.2
>50
38.6±1.3
>12.9
96.6±0.2a
10.2±0.3
>49.1
a
Data represent the mean ± SD of three independent experiments. Growth inhibition at 20 µM; SI: selectivity
index = CC50 THP-1/GI50; NA: not active at the tested concentration; BZN: benznidazole.
The obtained results suggest that a nitro substituent at the sulfonamidic aromatic ring plays an
important role in the trypanocidal activity, once compounds 8, 12, 16, and 20 are among the most
potent. However, it is important to highlight that compound 4 does not show a relevant trypanocidal
activity in spite of having this substituent. The main difference between compound 4 and the other
compounds is the presence of substituents in the other phenyl ring, such as in compounds 8, 12, 16,
and 20, suggesting that this might be a requirement for a higher trypanocidal activity due to
pharmacodynamic factors. Considering the other unsubstituted compounds 1-3, this hypothesis
strengthens, once their activity is among the weakest in the entire series.
As an electron withdrawing group, the nitro substituent decreases the pKa of the sulfonamide
acidic hydrogen assisting the hydrosolubility of the molecules. The highest aqueous solubility of the
active compounds could indicate that pharmacokinetic factors also might be fundamental for this
activity (Figure 1).

5
Figure 1. Suggested SAR for the trypanocidal activity of compounds 1-20.
Interestingly, the fluoro-substituted compound 11 also showed a good trypanocidal activity,
which might be related to the high electronegativity of fluorine. On the other hand, the fluoro-
substituted molecules 3, 7, 15, and 19 showed different behaviors; implying that the presence of
methoxy groups in the other aromatic ring could have a determining role in their biological application.
Overall, compound 16 was the most active of the series showing a GI50 value comparable with
that of the reference drug benznidazole (11.6 µM and 10.2 µM, respectively), and displays a high
selectivity index (SI= 29.1). The potency in combination with selectivity demonstrates that compound
16 has the potential to be further investigated regarding its activity against T. cruzi.
Altogether with Chagas disease, leishmaniasis, which is caused by Leishmania parasites, is
also a neglected kinetoplastid disease that has a great impact on socioeconomic indicators in
developing countries30,31. Considering the promising results obtained from the trypanocidal screening,
the activity against intracellular amastigotes of L. amazonensis (LTB0016 strain expressing ß-
galactosidase) was also assessed.
Table 3. Leishmanicidal screening of isoxazolyl-sulfonamides 1-20.
ID
1
%GI (50 µM)
10.9±2.6
17.2±0.7
13.2±1.0
18.4±0.5
2.0±0.5
6.4±2.0
14.0±1.4
48.7±0.4
4.1±1.0
NA
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
6.6±2.4
10.2±4.0
11.4±3.8
30.7±8.6
12.8±2.2
43.4±2.0

6
17
18
19
20
AB
9.3±0.9
2.0±0.5
15.7±1.7
25.2±3.0
92.1±0.9a
a
Data represent the mean ± SD of three independent experiments. Growth inhibition at 2 µM; NA: not active at
the tested concentration; AB: amphotericin B.
Except for compound 10, all other compounds slightly affected the parasite growth (%GI > 0)
even though their GI50 values are higher than 50 µM in all cases (Table 3). And, similarly to the results
of the trypanocidal screening, compounds 8 and 16 were also the most active against L.
amazonensis.
In order to guide us in exploring other biological activities of compounds 1-20, a publicly
available virtual prediction tool (SEA – Similarity Ensemble Approach, http://sea.bkslab.org) was used
to locate molecular targets in which these compounds could bind33. Among different proteins, SEA
suggested us to target the inhibition of the catalytic subunit of the DNA polymerase of Human
herpesvirus 6A (HHV-6A). The Herpesviridae family comprises eight viruses highly prevalent in the
population, including the causative agent of herpes labialis (Herpes Simplex Virus type 1, HSV-1). It is
known that the catalytic subunit of HHV-6A’s DNA polymerase displays around 62% of similarity and
37% of identity with the homologous enzyme of HSV-1. Therefore, we initially screened compounds
1-20 for their ability to inhibit HSV-1 replication (Table 4)34,35. The antiherpes screening was
performed using a plaque number reduction assay36. Vero cells were inoculated with HSV-1 (KOS
strain) and treated with compounds 1-20 at 100 µM to determine the percentages of replication
inhibition (%RI 100 µM). Then, the most active compounds were assayed at different concentrations
to determine their RI50 values (concentration that inhibits 50% of viral replication).
Table 4. Anti-HSV-1 screening of isoxazolyl-sulfonamides 1-20.
ID
CC50 (µM) Vero
cells
%RI (100 µM)
RI50 (µM)
SI
1
2
3
4
5
>500
25.3±12.4
17.8±2.3
40.2±8.7
10.4±3.4
>100
>100
-
>500
-
>500
>100
-
-
>500
>100
331.1
(130.2 – 842.2)
>500
95.9±6.3
>3.4
65.0±7.5
32.0±4.1
100±8.8
60.0±7.9
NA
6
7
>100
87.4±9.7
137.4±10.3
NA
-
>5.7
>3.6
-
>500
>500
>500
>500
8
9
10
2.0±0,9
>100
-

7
11
397.2
>100
-
(191.5 – 524.0)
22.8±5.8
NA
12
13
14
15
16
>500
>500
>500
>500
NA
>100
-
19.1±7.1
NA
-
NA
-
>2.9
-
70.1±11.4
173.2±12.5
387.0
(188.6 – 793.9)
25.3±5.7
>100
17
18
>500
>500
>500
>500
>2000
NA
NA
NA
NA
-
NA
NA
-
-
19
NA
-
-
20
NA
ACV
1.29±0.38
>1500
Data represent the mean ± SD or mean (95% confidence interval) of two independent experiments. SI: selectivity
index = CC50 Vero/RI50; NA: not active at the tested concentration; ACV: Acyclovir.
Within the group of 13 active molecules, compounds 5, 7, 8, and 15 showed a significant anti-
herpes activity inhibiting ≥ 60% of the HSV-1 replication at the tested concentration. Furthermore, the
compounds 5 and 7 presented the lowest RI50 values among the evaluated compounds (RI50= 95.9
and 87.4, respectively), and their SI values demonstrated that they are acting selectively against
HSV-1 replication. Although the inhibition of DNA polymerase was not directly assessed in this assay,
we could identify promising compounds to upcoming investigations regarding their mode of action.
In addition to potency, different factors are crucial in the decision of moving new chemical
entities through the preclinical drug development pipeline. Physicochemical and structural descriptors
are often associated to ADME properties, which are a frequent cause of failure in drug development
process37. Toxicological concerns also impact how the pace of development is dictated and might
restrict the application of a bioactive hit38.
Aided by the software DataWarrior 4.7.2 (http://www.openmolecules.org), we calculated
molecular weight (MW), ClogP, number of hydrogen bond acceptors (HBA), number of hydrogen
bond donors (HBD), number of rotatable bonds (nROTB) and topological polar surface area (tPSA) of
compounds 1-20 (Supplementary Data, p. S32). These properties are associated to Lipinski’s Rule of
Five and Veber rules, used as predictors of oral bioavailability, a desired aspect for bioactive
molecules aiming to proceed in development39,40. The results have shown that most compounds fit all
criteria of these rules, with the exception of compounds 12, 16, and 20 that have more than 140 Ų of
polar surface area due to a combination of polar aromatic substituents.
Regarding toxicity, Hughes (2008) demonstrated that physicochemical properties, specifically
ClogP and tPSA, could be related to in vivo adverse outcomes. In a set of preclinical molecules, those
with ClogP> 3 and tPSA< 75 Ų showed 2.5 more likelihood of being toxic, possibly because of the
increased probability of an off-target binding41. Moreover, DataWarrior 4.7.2 also provides a toxicity
risk assessment by means of a fragment-based approach, being possible to predict whether

8
tumorigenic, mutagenic, and irritative functions are present in chemical structures. The data obtained
for compounds 1-20 suggest that they are not prone to present toxic effects, thereby, positively
influencing their druglikeness.
In conclusion, twenty non-cytotoxic isoxazolyl-sulfonamides were obtained through a four-step
synthetic route from 5-hydroxymethyl-3-phenyl-isoxazole derivatives, previously described by our
research group. To the best of our knowledge, all final compounds are not yet described in literature42
and were phenotypically assayed against Herpes Simplex Virus type 1, and intracellular amastigotes
of T. cruzi and L. amazonensis.
Our results have shown that the isoxazolyl-sulfonamide scaffold is promising regarding all
biological activities studied herein, especially the inhibitory activity against T. cruzi. Compounds 8 and
16 were the most trypanocidal compounds, presenting high selectivity and potency near to those of
the reference drug benznidazole. Furthermore, compound 7 was the most active in the anti-HSV-1
screening giving us an opportunity to continue improving this scaffold to obtain more potent
antiherpetic derivatives. Also, all compounds could be ranked as highly drug-like, once they possess
features that are related to adequate permeation, solubility, and low toxicity.
Acknowledgments
This work was supported by the Brazilian funding agencies FAPESC/CNPq (PRONEX, grant number
2671/2012-9), CNPq (grant number 407626/2016-6). The authors would like to thank CNPq and
CAPES for their research fellowships as well as Luis Otávio Zamoner (FCFRP, USP, Brazil), Vinícius
Palaretti (FFLCH, USP, Brazil) and Louis Pergaud Sandjo (CIF, UFSC, Brazil) for the spectral
analyses.
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Highlights (3 to 5 of 85 characters each)
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Synthesis and characterization of 20 isoxazolyl-sulfonamides are reported.
The compounds were active against T. cruzi, L. amazonensis, and HSV-1.
Druglikeness evaluation showed potential for further pre-clinical development.

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