Pre-clinical evidences of the antileishmanial effects of diselenides and selenocyanates

Article abstract of DOI:10.1016/j.bmcl.2020.127371

A series of thirty-one selenocompounds covering a wide chemical space was assessed for in vitro leishmanicidal activities against Leishmania infantum amastigotes. The cytotoxicity of those compounds was also evaluated on human THP-1 cells. Interestingly most tested derivatives were active in the low micromolar range and seven of them (A.I.3, A.I.7, B.I.1, B.I.2, C.I.7 C.I.8 and C.II.8) stood out for selectivity indexes higher than the ones exhibited by reference compounds mitelfosine and edelfosine. These leader compounds were evaluated against infected macrophages and their trypanothione reductase (TryR) inhibition potency was measured to further approach the mechanism by which they caused their action. Among them diselenide tested structures were pointed out for their ability to reduce infection rates. Three of the leader compounds inhibited TryR effectively, therefore this enzyme may be implicated in the mechanism of action by which these compounds cause their leishmanicidal effect.

Full text of DOI:10.1016/j.bmcl.2020.127371

Bioorganic&MedicinalChemistryLetters30(2020)127371
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pre-clinical evidences of the antileishmanial effects of diselenides and
selenocyanates
Pablo Garnicaa,d, Mikel Etxebeste-Mitxeltorenaa,b,d, Daniel Planoa,b, Esther Morenoa,b
,
⁎
Socorro Espuelasa,b, Juan Antonio Palopa, Antonio Jiménez-Ruizc, Carmen Sanmartína,b,
a University of Navarra, School of Pharmacy and Nutrition, Department of Pharmaceutical Technology and Chemistry, Irunlarrea 1, 31008 Pamplona, Spain
b Institute of Tropical Health, University of Navarra, Pamplona, Spain
c Department of Biochemistry and Molecular Biology, Universidad de Alcalá, Carretera Madrid-Barcelona km 33,600, E-28871 Alcalá de Henares, Madrid, Spain
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of thirty-one selenocompounds covering a wide chemical space was assessed for in vitro leishmanicidal
activities against Leishmania infantum amastigotes. The cytotoxicity of those compounds was also evaluated on
human THP-1 cells. Interestingly most tested derivatives were active in the low micromolar range and seven of
them (A.I.3, A.I.7, B.I.1, B.I.2, C.I.7 C.I.8 and C.II.8) stood out for selectivity indexes higher than the ones
exhibited by reference compounds mitelfosine and edelfosine. These leader compounds were evaluated against
infected macrophages and their trypanothione reductase (TryR) inhibition potency was measured to further
approach the mechanism by which they caused their action. Among them diselenide tested structures were
pointed out for their ability to reduce infection rates. Three of the leader compounds inhibited TryR effectively,
therefore this enzyme may be implicated in the mechanism of action by which these compounds cause their
leishmanicidal effect.
Diselenides
Leishmanicidal agents
Selenocompounds
Selenocyanates
Leishmaniasis is one tropical fatal disease caused by different spe-
cies of protozoa belonging to the genus Leishmania. It is transmitted to
humans by the bite of female phlebotomine and it is endemic in 98
countries. In spite of the existance of around 53 species of Leishmania,
only 20 are capable of infecting humans1.
privileged chemical element to design new leishmanicidal agents. In
this context, in the past years, our research group has reported a set of
organoselenium compounds with this activity 14–21. Different chemical
frameworks have been explored such as selenocyanate, diselenide, se-
lenourea and methylseleno moieties. Among these series new anti-
kinetoplastid derivatives bis(4-aminophenyl)diselenide and 4-amino-
phenylselenocyanate exhibited interesting properties as antiparasitic
agents18. With these observations in mind, we decided to modulate bis
(4-aminophenyl)diselenide and 4-aminophenylselenocyanate with dif-
ferent derivatizations (Fig. 1) with the aim to improve pharmacological
properties obtaining maximal 3D structural enrichment as well as
providing us with information on the effects of electrostatic modula-
tion. Acylselenoureas (Series A) have been included due to their re-
Leishmaniasis can present three main clinical forms: cutaneous,
which is the most common and causes skin sores; visceral or kala-azar,
which is the most serious form and affects several internal organs
(spleen, liver and bone marrow) and mucocutaneous, which destroys
the mucous membranes of nose, mouth and throat2–4. Drugs including
pentavalent antimonial derivatives such as Pentostam® and Glu-
cantime® or more recently amphotericin B, miltefosine, paramomycin,
sitamaquine and edelfosine have several complications such as toxicity,
side effects, low efficacy, drug resistances and high costs, among
others5–9. These reasons reinforce the urgent need of new treatments
cently described presence in structures with potent leishmanicidal ac-
20
tivity
.
and new targets that must be explored for treating this illness10,11
.
The presence of amide groups in Series C as a result of the reaction
of the parent compounds with anhydrides provided us with a carboxylic
residue that enabled us to explore the formation of imides (Series B).
The synthetic pathway followed to yield those derivatives is described
in Scheme 1.
In the search of new drugs growing evidence suggests a connection
between selenium (Se) and parasites, particularly trypanosomatids. In
fact, some parasites have been proven to express selenoproteins and
metabolize Se12,13. This indicates the potential importance of Se as a
⁎ Corresponding author at: University of Navarra, School of Pharmacy and Nutrition, Department of Pharmaceutical Technology and Chemistry, Irunlarrea 1,
31008 Pamplona, Spain.
E-mail address: sanmartin@unav.es (C. Sanmartín).
d Authors have contributed equally to this article.
https://doi.org/10.1016/j.bmcl.2020.127371
Received 15 May 2020; Received in revised form 25 June 2020; Accepted 26 June 2020
Availableonline01July2020
0960-894X/©2020PublishedbyElsevierLtd.

P. Garnica, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127371
Fig. 1. Structures for the Se-containing derivatives tested in this work.
Scheme 1. General procedure of synthesis.
Among the potential targets for the treatment of leishmaniasis we
of oxidative stress. In this sense, all Trypanosomatids, including
Leishmania, present diverse regulatory pathways to protect them from
the oxidative damage. Trypanothione Reductase (TryR) is one of the
focused on the redox regulatory systems. Parasites are highly sensitive
to reactive oxygen species (ROS), which are produced under conditions
2

P. Garnica, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127371
most relevant and studied enzymes that plays an important role in the
survival of the parasite, due to its implication in the redox system. This
enzyme catalyzes the reduction of trypanothione disulfide to trypa-
nothione. Due to its absence in the human host, TryR is considered a
promising target for the development of new drugs that improve the
efficacy, potency and safety of the reference drugs such as miltefosine
and edelfosine14,16,22. On the basis of the chemical analogy of sulfur
and selenium, we decided to explore diselenide and selenocyanate de-
rivatives to generate new TryR inhibitors.
Table 1
IC50 values for compounds on amastigotes and cytotoxic activity on THP-1 cell
line.
Code
IC50 (mean
Amastigotes
> 25
SEM), μM
SI
THP-1
A.I.1
> 25
> 25
> 25
> 25
> 25
> 25
7.44
n.d.
A.I.2
15.03
2.27
13.48
> 25
9.22
0.67
0.35
4.55
5.57
14.6
9.98
3.08
6.18
1.54
2.07
10.40
> 25
4.57
7.9
0.78
0.91
2.21
> 1.66
> 11.01
> 1.85
n.d
A.I.3
A.I.4
In this work, we assess the antileishmanial effect of a library of
thirty-one organoseleno compounds (Fig. 1) against Leishmania in-
fantum axenic amastigotes as well as the cytotoxicity against THP-1
cells in order to evaluate their selectivity. Furthermore, the most active
compounds were evaluated in L. infantum-infected macrophages and
have been tested to study the potential role played by trypanothione
reductase as putative target.
A.I.5
A.I.6
0.95
0.15
0.06
0.71
0.80
0.90
0.75
0.43
0.19
0.25
0.21
> 2.77
11.19
3.10
A.I.7
1.47
0.07
A.I.8
1.07
A.I.9
22.16
> 25
> 25
13.45
12.77
8.50
4.87
A.I.10
A.I.11
A.I.12
A.I.13
A.I.14
B.I.1
> 4.48
> 1.71
1.35
1.26
1.50
The synthesis of this second-generation derivatives followed two
different synthetic pathways. Briefly, the formation of acylselenoureas
(Series A) involved acylisoselenocyanates as intermediates that were
then reacted with bis(4-aminopheny)diselenide following a synthetic
pathway previously described by our research group23. On the other
hand, derivatives containing amide acids (Series C) or imides (Series
B) were formed by the reaction of symmetric anhydrides with either bis
(4-aminophenydiselenide) to yield compounds coded C.I. or 4-amino-
phenyselenocyanate for derivatives coded C.II 24. The structure of these
derivatives was determined by microanalysis, 1H and 13C NMR spec-
troscopy, infrared spectroscopy and mass spectrometry and met the
required purity to be tested in order to assess their biological activity.
The leishmanicidal activity of thirty-one synthesized Se-containing
compounds was analyzed against Leishmania Infantum axenic amasti-
gotes at 24 h treatment time. This form of the parasite was selected as
amastigotes are responsible for clinical manifestations in humans.
Miltefosine and edelfosine were used as reference drugs. Three in-
dependent experiments were performed according to a previously de-
scribed procedure14 and results are expressed as IC50 values in Table 1.
Selectivity is a compulsory property for any antileishmanial drug as
toxicity in human cells is a non-desired effect in drug development. In
order to asses this factor, cytotoxicity obtained after 24 h of exposure to
the compounds in THP-1 cells was tested according to previously de-
scribed procedure14. Selectivity index (SI) was calculated as the ratio of
IC50 value obtained for THP-1 cells and IC50 values obtained for L. in-
fantum amastigotes.
4.15
0.40
1.34
15.23
22.97
8.53
2.28
1.16
9.91
B.I.2
11.08
0.82
B.I.3
0.21
0.99
1.36
C.I.1
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
> 25
21.53
18.5
n.d.
C.I.2
> 5.47
> 3.16
> 2.06
> 12.31
> 19.13
n.d.
C.I.3
0.23
C.I.5
12.10
2.03
1.31
> 25
> 25
11.33
> 25
> 25
10.66
10.61
3.22
2.84
0.82
1.72
C.I.7
0.36
0.16
C.I.8
C.II.1
C.II.2
C.II.3
C.II.4
C.II.5
C.II.6
C.II.7
C.II.8
miltefosine
edelfosine
n.d.
0.84
> 2.20
n.d.
n.d.
0.51
1.00
> 2.34
> 2.35
7.76
0.46
0.63
0.60
0.16
0.10
0.13
6.51
4.96
6.01
Table 2
IC50 for the selected compounds against TryR inhibition.
Code
IC50 (mean
SEM), μM
A.I.7
9.22
1.50
0.01
B.I.1
0.23
B.I.2
21.81
18.51
25.00
0.54
2.55
C.I.7
0.16
0.69
Data obtained revealed potent bioactivity for seven of the analyzed
compounds (A.I.3, A.I.7, A.I.8, B.I.1, B.I.2, C.I.7 and C.I.8) which met
the threshold stated by the reference drug miltefosine (IC50 = 2.84 μM)
in terms of potency. Derivatives A.I.7 and A.I.8 even exceeded the
edelfosine standard for this assay (IC50 = 0.82 μM). Interestingly, only
diselenide derivatives matched or surpassed the potency obtained for
the reference drugs. The results found for compounds A.I.7 and A.I.8
were comparable to or lower than those obtained for bis(4-amino-
phenyl)diselenide (IC50 = 0.65 μM), common fragment for diselenide
derivatives18. If we make the corresponding comparison of seleno-
cyante derivatives with their parent drug 4-aminophenylselenocyanate
(IC50 = 9.29 μM)18, compound C.II.8 stood out for the significant an-
tikinetoplastid activity (see Table 2).
C.I.8
C.II.8
mepacrine
0.13
16.99
1.18
infection significantly at 3 μM concentration after 48 h treatment time
as shown in Fig. 2. Compounds A.I.7, B.I.2 and C.I.8 reduced infection
rates to values under the ones obtained for the treatment of the re-
ference drug edelfosine. Five of the tested compounds correspond to
diselenide derivatives, all of them exhibited infection reduction po-
tential in the range of the one found for edelfosine. On the other hand,
selenocyanate derivative C.II.8 did not meet this standard, again this
fact pointed to diselenide moiety over selenocyanate in general terms of
potential as leishmanicidal structures.
Low toxicitiy is required for a good antileishmanial drug, conse-
quently IC50 values obtained for THP-1 and SI have to carefully be
observed in order to satisfy this condition. Compounds A.I.3, A.I.7,
B.I.1, B.I.2, C.I.7, C.I.8 and C.II.8 outperformed miltefosine and
edelfosine (SI = 6.51 and 6.01 respectively) in terms of selectivity
(Table 1).
To further determine the potential mechanism of action of the six
leader compounds we assessed their ability to inhibit TryR. Six different
concentrations ranging from 75 to 0.1 μM were screened. Mepacrine
was used as a positive control due to its proven TryR inhibition prop-
erties. Three out of the six tested compounds (A.I.7, B.I.1 and C.II.8)
exceeded inhibition potency of mepacrine25, compounds B.I.1 and
C.II.8 were 30-fold more active than the positive control. The re-
maining tested compounds B.I.2, C.I.7 and C.I.8 showed IC50 values in
the same order of magnitude as the positive control mepacrine. When
compounds were ranked following their IC50 for this inhibition assay
aking into consideration both potency and selectivity, seven deri-
vatives (A.I.3, A.I.7, B.I.1, B.I.2, C.I.7, C.I.8 and C.II.8) were selected
to be assayed in amastigote-infected THP-1 cells. Compound A.I.3
presented reproducibility issues under the conditions used for this assay
and therefore had to be discarded. All tested compounds reduced
3

P. Garnica, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127371
Declaration of Competing Interest
70
60
50
40
30
20
10
0
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
*
Acknowledgments
*
*
*
*
The research leading to these results has received funding from “la
Caixa” Banking Foundation. P. Garnica wishes to express his gratitude
to the Asociación de Amigos de la Universidad de Navarra for the pre-
doctoral fellowship. Furthermore, the authors wish to express their
gratitude to the Plan de Investigación de la Universidad de Navarra,
PIUNA (Ref 2014–26) as well as Caixa Foundation-UNED, Roviralta and
Ubesol for financial support for the project.
*
*
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.
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A
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B
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Appendix A. Supplementary data
e
d
e
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmcl.2020.127371.
Fig. 2. Infection rates of THP-1 cells by amastigotes with 3 μM dose at 48 h
treatment,
(mean
autofluorescence
correction
applied.
Aggregate
results
SEM; n = 3) expressed as fold induction relative to control cells
treated with DMSO. *p < 0.05.
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