In vitro antileishmanial activity of aza-scorpiand macrocycles. Inhibition of the antioxidant enzyme iron superoxide dismutase

Article abstract of DOI:10.1039/c5ra21262f

The in vitro leishmanicidal activity of a series of nine aza-scorpiand-like macrocycles, recently synthesized, was tested on Leishmania infantum, Leishmania braziliensis and Leishmania donovani parasites,? using promastigotes and intracellular amastigotes forms. The cytotoxicity of the tested compounds on J774.2 macrophage cells was also measured. Four of the tested compounds (1, 2, 8 and 9) showed selectivity indexes higher than those of the reference drug Glucantime for the three Leishmania species. Moreover, the data on infection rates and on amastigotes showed that compounds 1, 2, 8 and 9 are the most active against the three Leishmania species. The changes in the excretion product profile of parasites treated with the four compounds (1, 2, 8 and 9) were also consistent with substantial cytoplasmic alterations. On the other hand, the most active compounds were potent inhibitors of Fe-SOD in the three parasite species considered, whereas their impact on human CuZn-SOD was low. The high activity, low toxicity, stability, low cost of the starting materials and straightforward synthesis make these compounds appropriate molecules for the development of affordable anti-leishmanicidal agents.

Full text of DOI:10.1039/c5ra21262f

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In vitro antileishmanial activity of aza-scorpiand
macrocycles. Inhibition of the antioxidant enzyme
iron superoxide dismutase†
Cite this: RSC Adv., 2016, 6, 17446
Clotilde Marın,a Mario Inclan,b Inmaculada Ramırez-Macıas,a M. Teresa Albelda,c
´
´
´
´
Rocio Canas,a M Paz Clares,b Jorge Gonzalez-Garcıa,b Maria Jose Rosales,a
˜
´
´
a
b
a
*
*
Kristina Urbanova, Enrique Garcıa-Espana and Manuel Sanchez-Moreno
´
˜
´
The in vitro leishmanicidal activity of a series of nine aza-scorpiand-like macrocycles, recently synthesized,
was tested on Leishmania infantum, Leishmania braziliensis and Leishmania donovani parasites, using
promastigotes and intracellular amastigotes forms. The cytotoxicity of the tested compounds on J774.2
macrophage cells was also measured. Four of the tested compounds (1, 2, 8 and 9) showed selectivity
indexes higher than those of the reference drug Glucantime for the three Leishmania species. Moreover,
the data on infection rates and on amastigotes showed that compounds 1, 2, 8 and 9 are the most active
against the three Leishmania species. The changes in the excretion product profile of parasites treated
with the four compounds (1, 2, 8 and 9) were also consistent with substantial cytoplasmic alterations. On
the other hand, the most active compounds were potent inhibitors of Fe-SOD in the three parasite
species considered, whereas their impact on human CuZn-SOD was low. The high activity, low toxicity,
stability, low cost of the starting materials and straightforward synthesis make these compounds
appropriate molecules for the development of affordable anti-leishmanicidal agents.
Received 13th October 2015
Accepted 28th January 2016
DOI: 10.1039/c5ra21262f
www.rsc.org/advances
triggers localized self-healing skin lesions. In mucocutaneous
leishmaniasis (ML), also known as espundia, the infection
Introduction
spreads in the mucous membranes of the mouth and nose,
becoming fatal in some cases. However, the most serious
among them is undoubtedly visceral leishmaniasis (VL), also
known as kala azar, which is oen lethal if untreated, since the
parasites infect the liver and spleen and cause anaemia by
severely affecting the immune system.3 Those clinical
syndromes are caused by different Leishmania subspecies. The
most representative are: (i) L. major, which originates CL and is
found in North Africa, India, China or the Middle East; (ii)
L. braziliensis, one of the main species causing ML, most
common in South America (Bolivia, Brazil and Peru), (iii)
L. donovani complex, responsible for the fearsome VL, which is
mainly constituted by zoonotic L. infantum, found in Europe,
North Africa and Latin America and (iv) anthroponotic L.
donovani, present in East Africa and the Indian subcontinent.4,5
Because leishmaniasis is a severe health problem, mainly
(but not only) in developing countries, it is necessary to develop
drugs that adequately treat and prevent the disease. The current
list of available drugs is limited in number and clearly unsat-
isfactory due to toxicity, generation of resistance and/or the very
high costs involved. Pentavalent antimonials, mainly meglu-
mine antimoniate (Glucantime) and sodium stibogluconate,
have been the usual drugs of choice in the treatment of VL and
CL for more than 60 years. However, acquired resistance has
been reported, especially in India, with failure rates of 65% and
Leishmaniasis is a parasitic disease caused by protozoan para-
sites of the genus Leishmania and transmitted by sandies
commonly found in tropical and subtropical regions of the
world. It is estimated that 350 million people are at risk of
infection worldwide and the annual incidence of the disease
has been calculated to range between 1.5 and 2 million with
about 70 000 deaths every year.1 It is widely spread in almost 100
countries of Latin America, the Indian subcontinent, east Africa
and the Mediterranean Basin.2 However, the principal etiolog-
ical agents, vectors, animal reservoirs and clinical syndromes
may be different in each continent. However, it has to be
stressed that these data are probably very conservative because
of under reporting and misdiagnosis. There are different clin-
ical manifestations of this disease. The most widespread, but
also the least dangerous is cutaneous leishmaniasis (CL), which
aDepartamento de Parasitologıa, Instituto de Investigacion Biosanitaria
(ibs.GRANADA), Hospitales Universitarios De Granada/Universidad de Granada,
Granada, Spain. E-mail: msanchem@ugr.es; Fax: +34-958-243-862; Tel: +34-958-
´
´
242-369
b
´
´
Instituto de Ciencia Molecular, Departamento de Quımica Inorganica, Universidad de
Valencia, E-46980 Paterna (Valencia), Spain. E-mail: enrique.garcia-es@uv.es; Fax:
+34-96-354-3274; Tel: +34-963-544879
c
´
GIBI 230, Hospital Universitario y Politecnico La Fe, Valencia, Spain
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c5ra21262f
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side-effects including serious cardiotoxicity in about 10% of
patients.1,6 Second-line drugs, like pentamidine and ampho-
tericin B have not experienced widespread use due to toxicity
and cost. Pentamidine is associated with hypotension, hypo-
glycemia and diabetes, and also causes nephrotoxicity. Milte-
fosine is used in the treatment of VL infections and is less
harmful than pentamidine, but the ease with which resistant
mutants are obtained is worrying.7 The most recent line of
drugs are uconazole, paromomycin and sitamaquine, but they
also exhibit toxicity problems (i.e. hepatotoxicity with paromo-
mycin or uconazole, methemoglobinemia with sitamaquine)
and it remains to be seen how these agents stand up in face of
the pandemic of resistance.8,9 Due to the growing resistance to
chemotherapy, which is a drawback shown by most of the
compounds mentioned above, drug combinations are now
being explored as a path to delay or prevent the emergence of
resistance, increase efficacy and shorten the course of
treatment.9
In the search of new effective anti-leishmanial drugs able to
broaden the narrow spectrum of compounds currently avail-
able, potential targets that perform functions essential for
parasite survival are being investigated. Among them, antioxi-
dant enzymes preventing oxidative stress that would otherwise
lead to death of the parasite are in the spotlight. Within that
group, trypanothione reductase is currently receiving special
consideration.10,11 However, another basic defense mechanism
against oxidation is provided by the iron superoxide dismutase
enzyme (Fe-SOD), which is located in the cytosol, the mito-
chondria and glycosomes of the parasite and plays an important
role in the dismutation of harmful superoxide radicals into
oxygen and hydrogen peroxide.12 One further advantage of Fe-
SOD as a drug target is that it is not present in humans, who
use copper/zinc and manganese superoxide dismutase enzymes
(Cu, Zn and Mn-SOD) for the same purposes. Therefore, mole-
cules able to inhibit selectively Fe-SOD could be promising
candidates as leishmanicidal agents.13 Since their prosthetic
groups are essential in all processes regulated by enzymes,
alterations in the active centre induced by dissociation of the
iron atom or by modications in the coordination geometry
could effectively deactivate its antioxidant action and, therefore,
inuence both the growth and survival of the parasite cells.
Fig. 1 Chemical structure of the tested aza-scorpiand-like macro-
cycles compound complexes.
against macrophages. We compared these new compounds with
Glucantime as a reference drug. The effect of the compounds on
the ultrastructure of Leishmania spp. was also studied by trans-
mission electronic microscopy (TEM) experiments to conrm the
1
type of damage caused to the parasite. H-NMR analysis of the
nature and percentage of excreted metabolites was performed to
obtain information about the inhibitory effect of our compounds
on the glycolytic pathway since this represents the prime energy
source of the parasite. Previously, we have demonstrated that
these derivatives are good inhibitors of Fe-SOD, not only in
T. cruzi,17 but also in Leishmania spp.16 An evaluation of their
effectiveness as putative inhibitors of Fe-SOD in relation to
human CuZn-SOD is presented.
Results and discussion
Synthesis of the compounds
The synthesis of 1–9 was accomplished as described previ-
Regarding this point, in the last few years, our research ously14,18,19 following a modication of the Richman–Atkins
group has designed a new family of polyamine compounds procedure.20 First, pertosylated polyamine tris(2-aminoethyl)
consisting of a macrocyclic pyridinophane core substituted with amine was reacted with 2,6-bis(bromomethyl)pyridine in
side chains containing additional donor atoms (Fig. 1).14 These a 1 : 1 molar ratio using potassium carbonate (K2CO3) as the
macrocycles are termed scorpiand ligands because the side base in reuxing acetonitrile (CH3CN). Detosylation was carried
chain can fold towards the macrocyclic core following the out with HBr/HAc/PhOH. Compounds 1, 2, 3, 4 and 9 were ob-
binding of target guest species such as protons, anions or metal tained by reacting this intermediate, in its free amine form, with
ions.14,15
the corresponding carboxaldehydes in dry ethanol followed by
Recently, we have studied the in vitro action of several of aza- in situ reaction with sodium borohydride (NaBH4). The
scorpiand-like macrocyclic compounds against Trypanosoma compounds were nally precipitated as hydrochloride salts
cruzi and two species of Leishmania (L. infantum and L. brazil- (Scheme S1, ESI†).
iensis). The results showed that these macrocycles are potentially
The reaction of the pertosylated intermediate with N-
promising agents for the treatment of trypanosomatid infec- (3-bromopropyl)phthalimide in a 1 : 1 molar ratio using K2CO3
tions.16,17 In the present work, we tested the in vitro leishmani- as the base in reuxing CH3CN followed by deprotection and
cidal (against L. infantum, L. braziliensis and L. donovani) activity detosylation, carried out with hydrazine monohydrate and HBr/
of another new 9 macrocycles of this family and their toxicity HAc/PhOH, respectively, yielded
a second intermediate
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(Scheme S1, ESI†). Compounds 5, 6, 7 and 8 were obtained by
reacting this intermediate, in its free amine form, with the
corresponding carboxaldehydes in dry ethanol followed by in
situ reaction with NaBH4. The compounds were nally precipi-
tated as the hydrochloride salts.
Interaction with iron
As discussed in the introduction, one of the potential targets of
new anti-leishmanial drugs is the iron superoxide dismutase
enzyme (Fe-SOD). For this reason it is interesting to evaluate the
affinity of these chelating ligands for iron. We have previously
reported on the affinity towards Fe(II) of some of the compounds
studied here, which showed remarkable stability. As an
example, here we report for the rst time on the interaction of
compound 6 with Fe(II), determined by potentiometric titra-
tions. This compound behaves as a good iron chelator, as
shown by its high stability constants (Table S1, ESI†). The
distribution diagram (Fig. 2), built upon these constants, shows
how at intermediate pH values all the Fe(II) is complexed by the
ligand, mostly as the [FeL]2+ complex. This means that if the
ligand approaches close enough to the active site of Fe-SOD,
some competition might be established with the primary
ligands of the active centre for the capture of the metal ions.
Fig. 3 Effect of aza-scorpiand macrocycles derivatives 1, 2, 8 and 9 on
the infection and growth rates of Leishmania infantum. (A) Rates of
infection; (B) mean numbers of amastigotes of L. infantum per infected
J774 A.2 macrophage cell (at IC25 conc.). Values are the means of three
separate experiments.
Toxicity values against J774.2 macrophage aer 72 h of culture
were also calculated. Results obtained for the reference drug
Glucantime were included in all cases for comparison.
It was shown that the leishmanicidal activities against the
In vitro antileishmanial evaluation
In a rst step we assayed the in vitro biological activity of
compounds 1–9 (Fig. 3) against three signicant species of more indicative intracellular forms of the parasites were higher
Leishmania: L. infantum, L. braziliensis and L. donovani on both than those found for Glucantime with the compounds tested
extra- and intracellular forms of the parasites (Fig. S1–S6, ESI†). (comp. 1, 2, 7, 8 and 9), whereas the effect on extracellular forms
Extracellular forms are more commonly used due to the ease of was more random. But more interesting were the toxicity data in
working with them, but are less indicative of leishmanicidal mammalian cells, in the case of compound 7 is quite toxic and
activity. The use of intracellular forms is more cumbersome but therefore not be considered for further studies, the other four
gives more accurate results, as promastigotes are converted to compounds tested were found to be much less toxic for
amastigotes in vertebrate host cells,21 Intracellular assays were macrophages than the reference drug. Thus, compound 1 and 9
performed by infecting macrophage cells with promastigotes, were 15-fold less cytotoxic than Glucantime, the compounds 2
which transformed into amastigotes within 1 day aer infec- and 8 were also about 10-fold less cytotoxic. In order to obtain
tion. Table 1 shows the IC50 values obtained aer 72 h of a more accurate picture of the features commented on above,
exposure when compounds 1–9 were tested on extra- and we show in Table 2 the selectivity index values calculated from
intracellular forms of L. infantum, L. braziliensis and L. donovani. the data in Table 1, since they are very illustrative of the in vitro
potential of the compounds tested with respect to the reference
drug. The number of times that the SI of each compound
exceeded the SI of Glucantime is also shown in parenthesis. In
this Table 2 the differences between Glucantime and the tested
compounds are clearly revealed. All four (1, 2, 8 and 9)
compounds exhibited substantially better SI values than the
reference drug in the three Leishmania spp. tested and, in the
most remarkable case, the SI of the compound 2 on the intra-
cellular form of L. donovani exceeded that of Glucantime by 303-
fold, a relevant data point which is by far the best SI value ob-
tained with the different series of related heterocyclic systems
tested by us against parasites in previous research.22–26 The SI
displayed in Table 2 are also very illustrative about the differ-
ence in behaviour of different compounds, which recurs in the
three species and in both extra and intracellular forms, since 1,
Fig. 2 Distribution diagram of the Fe(II)–6 system ([Fe2+] ¼ [6] ¼ 1 ꢀ
2, 8 and 9 always show substantially better results than the rest
10ꢁ3 M).
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Table 1 In vitro activity and toxicity for the aza-scorpiand like macrocycles derivatives on extra- and intracellular forms of Leishmania spp.a
IC50b mM
Leishmania infantum
Leishmania braziliensis
Leishmania donovani
Promastigote
forms
Amastigote
forms
Promastigote
forms
Amastigote
forms
Promastigote
forms
Amastigote
forms
Toxicity IC50
Compounds
macrophagec (mM)
Glucantime®
18.0 ꢂ 3.1
2.3 ꢂ 0.3
3.7 ꢂ 0.2
57.3 ꢂ 3.2
12.1 ꢂ 1.0
25.4 ꢂ 3.1
15.6 ꢂ 0.7
9.5 ꢂ 0.7
4.8 ꢂ 0.3
11.7 ꢂ 1.4
24.2 ꢂ 2.6
3.3 ꢂ 0.6
2.9 ꢂ 1.0
23.0 ꢂ 3.7
8.6 ꢂ 0.9
23.8 ꢂ 2.4
13.7 ꢂ 1.3
9.0 ꢂ 0.8
3.0 ꢂ 0.1
6.2 ꢂ 0.8
25.6 ꢂ 1.6
2.1 ꢂ 0.2
2.3 ꢂ 0.5
32.9 ꢂ 4.3
15.6 ꢂ 0.8
25.2 ꢂ 3.5
23.2 ꢂ 3.4
17.5 ꢂ 2.3
1.7 ꢂ 0.2
1.4 ꢂ 0.2
30.4 ꢂ 6.1
2.7 ꢂ 0.6
4.2 ꢂ 1.1
15.0 ꢂ 2.3
14.1 ꢂ 1.3
20.6 ꢂ 1.7
15.3 ꢂ 1.6
14.6 ꢂ 1.1
2.8 ꢂ 0.4
3.6 ꢂ 0.4
27.4 ꢂ 3.8
14.1 ꢂ 1.2
18.5 ꢂ 2.5
49.5 ꢂ 5.1
13.5 ꢂ 1.5
23.4 ꢂ 3.0
14.0 ꢂ 1.3
7.4 ꢂ 0.5
31.4 ꢂ 4.1
4.83 ꢂ 0.6
1.1 ꢂ 0.0
19.5 ꢂ 2.3
10.7 ꢂ 0.8
16.9 ꢂ 0.9
12.5 ꢂ 1.0
6.9 ꢂ 0.2
4.6 ꢂ 0.7
3.2 ꢂ 0.3
15.2 ꢂ 1.3
231.8 ꢂ 12.6
166.9 ꢂ 11.2
181.3 ꢂ 10.7
92.4 ꢂ 6.8
1
2
3
4
5
6
7
8
9
144.9 ꢂ 12.4
117.3 ꢂ 9.8
97.4 ꢂ 8.1
8.4 ꢂ 0.3
140.0 ꢂ 11.3
221.3 ꢂ 16.9
12.5 ꢂ 0.9
a
b
Results are averages of three separate determinations. IC50 ¼ the concentration required to give 50% inhibition, calculated by non-linear
c
regression analysis from the Kc values at concentrations employed (1, 10, 25, 50 and 100 mM). Towards macrophage cell aer 72 h of culture of
Leishmania spp.
of the tested compounds. In tests performed on the L. infantum
In order to gain better insight into the activities of these
specie, the SI of 1 exceeded that of the reference drug by 126- compounds (1, 2, 8 and 9), their effect on the infectivity and
and 117-fold for the extra- and intracellular forms, respectively, intracellular replication of amastigotes was subsequently
by 184- and 172-fold with L. braziliensis, and by 33 and 97-fold determined. Macrophage cells were grown and infected with
when 1 was tested on L. donovani. The compound 2 has similar promastigotes in the stationary phase. The parasites invaded
SI to 1; the respective values obtained were 56- and 96-fold on the cells and underwent morphological conversion to amasti-
the L. infantum for the extra- and intracellular forms, respec- gotes within 1 day aer infection. On day 10, the rate of host cell
tively, 121–79-fold on extra- and intracellular forms the L. bra- infection reached its maximum (control experiment). We used
ziliensis; while on L. donovani, values of 18- and 303-fold were the IC25 of each product as the test dosage. Different authors
found respectively. Compounds 8 and 9 were equally effective have claimed that compounds with SI values less than 20-fold
against the three species of Leishmania and against both extra- those of the reference drug should be discarded as candidates
and intracellular forms. Comp. 8 has 36 year 78-fold SI increases for more advanced leishmanicidal tests due to their poor
on L. infantum, 137 and 100-fold on L. braziliensis and 33- and selectivity against mammalian cells.27
61-fold against L. donovani. Comp. 9 also presented results
As shown in Fig. 3A, when compounds 1, 2, 8 and 9 were
similar or slightly higher than 8 against the two forms of the added to macrophages infected with L. infantum promastigotes,
parasite and off the three species tested.
the infection rate decreased signicantly with respect to the
control and, furthermore, the four compounds were also
Table 2 Selectivity index found for the aza-scorpiand like macrocycles derivatives on extracellular and intracellular forms of Leishmania spp.a
SIb
Leishmania infantum
Leishmania braziliensis
Leishmania donovani
Compounds
Glucantime® 0.8
Promastigote forms Amastigote forms Promastigote forms Amastigote forms Promastigote forms Amastigote forms
0.6
0.6
0.5
0.5
0.5
1
2
3
4
5
6
7
8
9
100.8 (126)
45.1 (56)
3.2 (4)
7.6 (10)
5.7 (7)
7.5 (9)
10.3 (13)
29.2 (36)
18.9 (24)
70.2 (117)
57.6 (96)
7.9 (13)
10.7 (18)
6.1 (10)
8.6 (14)
10.8 (18)
46.7 (78)
35.7 (59)
110.4 (184)
72.6 (121)
5.5 (9)
5.9 (10)
5.8 (10)
5.1 (8)
5.6 (9)
82.4 (137)
158.1 (263)
85.9 (172)
39.7 (79)
12.1 (24)
6.6 (13)
7.0 (14)
7.7 (15)
6.7 (13)
50.0 (100)
61.5 (123)
16.4 (33)
9.0 (18)
3.7 (7)
6.8 (14)
6.2 (12)
8.4 (17)
13.2 (26)
16.7 (33)
17.7 (35)
48.3 (97)
151.7 (303)
9.3 (19)
8.6 (17)
8.6 (17)
9.4 (19)
14.1 (28)
30.4 (61)
69.2 (138)
a
b
Results are averages of three separate determinations. Selectivity index ¼ IC50 macrophage cell/IC50 extracellular and intracellular form of
parasite. In brackets: number of times that compound exceeds the reference drug SI (on extracellular and intracellular forms of Leishmania spp.).
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Fig. 4 Effect of aza-scorpiand macrocycles derivatives 1, 2, 8 and 9 on
the infection and growth rates of Leishmania braziliensis. (A) Rates of
infection; (B), mean numbers of amastigotes of L. braziliensis, per
infected J774 A.2 macrophage cell (at IC25 conc.). Values are the
means of three separate experiments.
Fig. 5 Effect of aza-scorpiand macrocycles derivatives 1, 2, 8 and 9 on
the infection and growth rates of Leishmania donovani. (A) Rates of
infection; (B), mean numbers of amastigotes of L. donovani, per
infected J774 A.2 macrophage cell (at IC25 conc.). Values are the
means of three separate experiments.
remarkably more effective in decreasing infectivity than Glu-
cantime (86%, 83%, 88% and 76% for 1, 2, 8 and 9, respectively,
versus only 26% for the reference drug). A measure of the
average number of amastigotes per infected macrophage
(Fig. 3B) led to similar conclusions: all four compounds were
more effective than Glucantime (with only a 30% decrease), and
amastigote numbers of 51%, 68%, 77% and 72% were found for
1, 2, 8 and 9 respectively.
Infection rates (Fig. 4A) and amastigote numbers (Fig. 4B)
obtained on L. braziliensis also showed that, in both cases, the
four compounds were clearly more effective than Glucantime,
and also in both cases the order of effectiveness was 8 > 9 > 2 >
1 > Glucantime, since the infectivity rates calculated were: 77%,
72%, 68%, 51% and 47%, respectively. The decreases in amas-
tigote numbers were: 65%, 60%, 63%, 76% and 39% for 1, 2, 8, 9
and Glucantime respectively. Finally, similar results were found
on L. donovani, with infectivity rates of 70%, 66%, 59%, 53%
and 30% (Fig. 5A); and decreases in the number of amastigotes
of: 50%, 63%, 42%, 79% and 32% (Fig. 5B) for 1, 2, 8, 9 and
Glucantime, respectively. From all these data it can be
concluded that: (i) all four compounds are substantially more
active than Glucantime against the three Leishmania species
tested; (ii) the results are in full accordance with related data
described for T. cruzi parasites.21
most relevant structural features observed in control and
treated cells of promastigotes of the three species. As a general
observation, the four tested compounds were responsible for
the death of a high percentage of individuals and many other
abnormalities such as vacuolization, disintegration of the cell
cytoplasm and rupture of the parasites.
Fig. 6 corresponds to L. infantum, and it can be seen that
when the parasites were treated with 1, 2 and 9 the disturbances
were more notable than when other species where treated
(Fig. 6, plates B–F) compared to the control (Fig. 6, plate A). The
most effective compound was 1 (plates B and C). Aer treatment
with 1 many parasites were found with reduced size. The
examination evidenced that these parasites had very altered
organelles. Others were broken or had strange shapes, and a few
had swollen kinetoplast and many vacuoles. With comp. 8 (D)
many parasites had their cytoplasmic membrane broken, the
agellar pocket and mitochondria very swollen and large
vacuoles. The treatment with 2 (E) showed that many parasites
were very small and were heavily vacuolated. Finally, comp. 9 (F)
caused a reduction in the number of ribosomes, some appeared
with intense vacuolization and some lled of glycosomes in
higher number than usual.
L. braziliensis was greatly affected by the four compounds as
shown in (Fig. 7 plates B, C, D, E and F) compared to control
(Fig. 7 plate A). Thus, compound 1 (Fig. 7, plate B) caused
intense vacuolization, disorganization, and the parasites were
lled of glycosomes. In some of these promastigotes there was
only cytoplasmic membrane. 8 was the compound most effec-
tive (Fig. 7 plates C, D) for the damages that caused on the
promastigotes, as with all the compounds the cytoplasm full of
vacuoles but also could be observed changes in the morphology
of the parasite. They had strange shapes, looking crashed or
amoeboid and structures like mitochondria and kinetoplastos
Ultrastructural alterations
Since all these compounds showed remarkable leishmanicidal
activity, major damage to parasite cells could be inferred. In
order to conrm this, a transmission electron microscopy
(TEM) study on the promastigote forms of the three Leishmania
species under consideration was performed. As expected,
signicant morphological alterations were observed compared
with untreated control cells. Fig. 6, 7 and 8 display some of the
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Fig. 6 Ultrastructural alterations observed by TEM in promastigotes of
the Leishmania infantum after treatment with compounds 1 (plates B
and C), 2 (plate E), 8 (plate D) or 9 (plate F). Abbreviations: dead
parasites (D), flagellum (F), glycosomes (G), kinetoplasts (K), vacuoles
(V), mitochondria (M), nucleus (N), ribosomes (R), flagellar pocket,
distorted membranes (single arrow), cellular rest (RE). Scale bars are 1.0
mm in all cases.
Fig. 7 Ultrastructural alterations observed by TEM in promastigotes of
the Leishmania braziliensis after treatment with compounds 1 (plate B),
2 (plate E), 8 (plates C and D) or 9 (plate F). Abbreviations: dead
parasites (D), flagellum (F), glycosomes (G), kinetoplasts (K), vacuoles
(V), mitochondria (M), nucleus (N), ribosomes (R), flagellar pocket,
distorted membranes (single arrow), cellular rest (RE). Scale bars are 1.0
mm in all cases. Scale bars are 1.0 mm in all cases.
were extremely swollen. Furthermore, there were many broken
parasites (Fig. 7 plate D), and large and extended glycosomes.
Comp. 2 also caused similar changes but the highlight were just
the completely altered morphology and the strange appearance
that had many promastigotes (Fig. 7, plate E). These parasites
had also abnormal organelles, lled vacuoles, some of these
strange organelles that could correspond to degenerate mito-
chondria. Most normal structures could not be assessed.
Compound 9 was also effective on promastigotes (Fig. 7, plate F),
in this case highly vacuolated cytoplasm, few glycosomes
together with swollen mitochondria were the main disturbances.
According to the results obtained by TEM (Fig. 8) and
compared to a control culture (Fig. 8, plate A), the compounds
tested turned out to be the most effective on L. donovani. The
compound 8 (Fig. 8, plate B) caused a drastic decrease in the
viability of promastigotes. Actually their cytoplasmic contents
were very little electron-appeared and it was observed only
ribosomes and glycosomes. Their appearance was practically
dead bodies. Compound 1 was the most effective (Fig. 8, plates
C, D), with few ribosomes and they all were full of empty
vacuoles, mitochondria were empty and very swollen. L. dono-
vani was also similarly affected by compound 2. Finally
compound 9 showed identical effect to the ones above as shown
in the image (Fig. 8, plate F). The parasites were completely
empty of content or very few ribosomes without cytoplasmic
organelles or some very altered beyond recognition.
Metabolite excretion effect
Since trypanosomatids are unable to completely degrade
glucose to CO2 under aerobic conditions, they excrete much of
the hexose skeleton into the medium as partially oxidized
fragments in the form of fermented metabolites. The nature
and percentages of those excretion products depend on the
pathway used for glucose metabolism by each species.28,29 The
nal products of glucose catabolism in Leishmania are usually
CO2, succinate, acetate, D-lactate, L-alanine and, in some cases,
ethanol.30 Among them, succinate is the most relevant, because
its main role is to maintain the glycosomal redox balance,
allowing the reoxidation of NADH produced in the glycolytic
pathway. Succinic fermentation requires only half of the phos-
phoenolpyruvate produced to maintain the NAD+/NADH
balance, and the remaining pyruvate is converted inside the
mitochondrion and the cytosol into acetate, D-lactate, L-alanine
or ethanol, according to the degradation pathway followed by
a specic species.31
We reported above that the most active compounds 1, 2, 8
and 9 caused great damage to the mitochondria of the parasites
in the three Leishmania species, so that their highly disruptive
action presumably affected their glucose metabolism and,
consequently, modied the percentages of the nal excretion
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differences were found in parasites treated with the derivatives,
whereas the presence of benznidazole did not lead to signicant
alterations in fuel metabolism (data not shown).
The most relevant nding was that the production of succi-
nate, the key catabolite in glucose metabolism, was substan-
tially inhibited by the four compounds in L. donovani (Table 3).
In L. braziliensis, there is no reduction in the succinate peak
observed, instead of it, with some of the compounds there is
a slight increase of its production. A similar behavior, of lesser
magnitude, was found in the L. infantum species. In fact, the
relevant succinate inhibition values obtained agreed with the
TEM evidence, indicating that these derivatives severely damage
the mitochondrial structures of the parasites and, therefore,
succinate production is substantially affected in L. donovani.
With regard to the less signicant excretion products shown
in Table 3, the formation of L-alanine and D-lactate were also
remarkably inhibited in L. donovani by both 2, 8 and 9, which
produced inhibitory values ranging from 13% to 45%. Although
variations in the secondary excretion products were in general
more erratic in L. infantum and L. braziliensis, increases of the
formation of L-alanine and D-lactate was always seen with
compounds 8 and 9, to a greater or lesser extent. The only
exceptions found were increases of 9% and 20%, respectively, in
the production of acetate when parasites of those two series
Fig. 8 Ultrastructural alterations observed by TEM in promastigotes of
the Leishmania donovani after treatment with compounds 1 (plate C
and D), 2 (plate E), 8 (plates B) or 9 (plate F). Abbreviations: dead
parasites (D), flagellum (F), glycosomes (G), kinetoplasts (K), vacuoles
(V), mitochondria (M), nucleus (N), ribosomes (R), flagellar pocket,
distorted membranes (single arrow), cellular rest (RE). Scale bars are 1.0
mm in all cases. Scale bars are 1.0 mm in all cases.
1
were treated with compound 4. In summary, the H NMR data
on catabolites are indicative of substantial alterations in
glucose metabolism in the three species. It is feasible that the
small size of these compounds allows them to easily penetrate
the cristae (tubular invaginations of the inner mitochondrial
membrane),32 leading to subsequent changes in the metabolic
pathway. Concerning the variations in acetate production, it is
well known that this end-product originates in the mitochon-
dria of Leishmania species through a collateral pathway via
pyruvate which is regulated by the pyruvate dehydrogenase
complex,30 so that 2 and 4 could be interfering in a different way
with that enzyme complex in both the L. infantum and L. bra-
ziliensis species.
products formed. Therefore, we registered the 1H NMR spectra
of promastigotes from the L. infantum, L. braziliensis and
L. donovani species aer treatment with the four compounds
with signicant leishmanicidal activity, and the nal excretion
products were identied qualitatively and quantitatively. The
spectra obtained were compared with those from promastigotes
maintained in cell-free medium (control) for four days aer
inoculation with the parasites. Variations in the height of the
signals corresponding to the most signicant catabolites are
displayed in Fig. S7–S9 of the ESI.† The expected presence of
acetate, D-lactate, L-alanine and succinate was conrmed in the
control experiments performed on all three species, and the
major metabolite was succinate in all cases, in agreement with
data reported in the literature.32 However, noteworthy
SOD enzymatic inhibition in the Leishmania parasites and in
human erythrocytes
Since we had previously found that the anti-T. cruzi activity of
compounds 1, 2, 8 and 9 was always accompanied by inhibitory
activity on the antioxidant enzyme Fe-SOD,28 we tested the
effects of those compounds on Fe-SOD isolated from
Table 3 Variation in the height of the peaks corresponding to catabolites excreted by Leishmania spp. promastigotes in the presence of
compounds aza-scorpiand like macrocycles (1, 2, 8 and 9) with respect to the control testa
Leishmania infantum
Leishmania braziliensis
Leishmania donovani
Comp.
Lac
Ala
A
S
Lac
Ala
A
S
Lac
Ala
A
S
1
2
8
9
+4%
+46%
+3%
+11%
+42%
+11%
+38%
+10%
+22%
+4%
+12%
+41%
+11%
+3%
ꢁ11%
0%
+7%
+39%
ꢁ10%
0%
+7%
+24%
ꢁ11%
ꢁ13%
ꢁ1%
ꢁ7%
ꢁ1%
+6%
+6%
+13%
ꢁ13%
ꢁ13%
ꢁ20%
ꢁ20%
ꢁ40%
ꢁ35%
ꢁ45%
+41%
+35%
+53%
+65%
ꢁ36%
ꢁ37%
ꢁ34%
ꢁ37%
+39%
+4%
ꢁ1%
a
A: acetate; Ala: L-alanine; Lac: D-lactate; S, succinate; (ꢁ): peak inhibition; (+): peak increasing.
17452 | RSC Adv., 2016, 6, 17446–17455
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L. infantum, L. braziliensis and L. donovani over a range of are able to get closer to the active centre of the enzyme, as is the
concentrations from 0.1 to 100 mM. We used promastigote case of compounds 1 and 2, so they might produce greater
forms of both species, which excrete Fe-SOD when cultured in inhibition.16,17
a medium lacking inactive FBS.33 The inhibition data obtained
are shown in Fig. 9, graphs (B), (C) and (D), and the corre-
sponding IC50 values are included for easier evaluation of the
graphs displayed. For comparison, graph (A) of Fig. 9 shows the
Experimental
Chemistry
The ESI† contains the synthetic procedure and the character-
effects of the same compounds on CuZn-SOD obtained from
isation data (NMR, mass spectrometry and elemental micro-
human erythrocytes. The most remarkable result was the
signicant inhibitory effect on Fe-SOD found for the highly analysis) of all the compounds. Details of the potentiometric
titrations, calculation of acid–base and stability constants are
also included in the ESI.†
leishmanicidal compounds 1 and 2 in the three species tested,
whereas their inhibition of human CuZn-SOD was clearly lower.
If we consider the IC50 calculated for L. infantum, inhibition of
Fe-SOD by compounds 2 and 1 was respectively 5.3- and 3.2-fold
higher than inhibition of CuZn-SOD, with respective values of
10.6-, and 4.2-fold higher in the case of L. braziliensis and 8.5-
and 2.4-fold higher values in the case of L. donovani. All these
data seem to conrm some kind of relation between parasiti-
cidal activity and inhibition of Fe-SOD, in accordance with
results described in previous work.28 Fe-SOD inhibition could
also, at another level, be related to the catabolic changes dis-
cussed above, since a mitochondrial malfunction, originating
from the redox stress produced by inhibition of the
mitochondrion-resident Fe-SOD enzyme,32 should result in
severe alteration of pyruvate metabolism and a consequent
decrease in the production of succinate.
It should be noted that, as mentioned previously, these
compounds not only showed greater alterations in glucose
catabolism but they also led to greater levels of Fe-SOD inhibi-
tion. Because the Fe-SOD present in mitochondria is an essen-
tial part of the antioxidant protective response of the parasite,
its inhibition would be related to a decrease in the rate of
survival for the parasite. Because of this fact, it is hypothesized
that compounds having a quinoline nitrogen on the side chain
Parasite strain and culture
Promastigote forms of L. infantum (MCAN/ES/2001/UCM-10),
L. braziliensis (MHOM/BR/1975/M2904) and L. donovani
(MHOM/PE/84/LC26) were cultured in vitro in medium
trypanosomes liquid (MTL) supplemented with 10% inactive
ꢃ
foetal calf serum (FCS) kept in an air atmosphere at 28 C, in
Roux asks (Corning, USA) with a surface area of 75 cm2,
21
´
according to the methodology described by Gonzalez et al.
In vitro activity assays
The compounds to be tested were rst dissolved in dime-
thylsulfoxide (DMSO, Panreac, Barcelona, Spain) at a concen-
tration of 0.1% and then assayed for toxicity and inhibitory
effects on parasite and mammalian cells growth as previously
described.21
Cell culture and cytotoxicity tests
The macrophage line J774.2 [European collection of cell
cultures (ECACC) number 91051511] was derived in 1968 from
a tumour in a female BALB/c mouse. The macrophages were
grown in minimal essential medium (MEM) plus glutamine
(2 mM) and 20% inactive FCS, with a humidied atmosphere of
95% air and 5% CO2 at 37 ꢃC. The cytotoxicity testing on
macrophages was performed by ow cytometric analysis
according to a method previously described.33 The percentage of
viable cells was calculated with respect to the control culture.
The IC50 was calculated using linear regression analysis from
the Kc values of the concentrations employed.
Promastigote assay: extracellular forms
The compounds were dissolved in the culture medium to give
nal concentrations of 100, 50, 25, 10 and 1 mM. The effects of
each compound against the promastigote forms at the different
concentrations were tested at 72 h using a Neubauer haemo-
cytometric chamber. The leishmanicidal effect was expressed as
the IC50 value, i.e. the concentration required to result in 50%
inhibition, calculated by linear regression analysis from the Kc
values of the concentrations employed.
Fig. 9 (A) In vitro inhibition of CuZn-SOD in human erythrocytes by
compounds 1, 2, 8 and 9. (B–D) In vitro inhibition (%) of Fe-SOD of
Leishmania infantum, Leishmania braziliensis and Leishmania dono-
vani promastigotes by compounds 1, 2, 8 and 9. Values are the average
of three separate determinations. Differences between the activities of
the control homogenate and those incubated with the tested
compounds were obtained according to the Newman–Keuls test. IC50
was calculated by linear regression analysis from the Kc values at the
concentrations employed (1, 10, 25, 50 and 100 mM).
Amastigote assay: intracellular forms
J774.2 macrophage cells were grown and seeded at a density of
1 ꢀ 104 cells per well in 24-well microplates (Nunc) with
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rounded coverslips on the bottom and cultured for 2 days. The
adherent macrophages were then infected with promastigotes
of L. infantum, L. braziliensis and L. donovani in the stationary
growth phase, at a ratio of 10 : 1 and maintained for 24 h at
37 ꢃC in air containing 5% CO2. Non-phagocytosed parasites
were removed by washing, and the infected cultures were
incubated with the testing compounds (concentrations ranging
from 1 to 100 mM) and then cultured for 72 h in MEM plus
glutamine (2 mM) and 20% inactive FCS. Compound activity
was determined from the percentage reductions in amastigote
number in treated versus untreated cultures in methanol-xed
and Giemsa-stained preparations. Values are the means of
three separate determinations.22
Fe-SOD enzymatic inhibition
Parasites were collected in the logarithmic growth phase by
centrifugation (400g for 10 min at room temperature). The
pellet obtained aer centrifugation, was resuspended in 3 mL
of STE buffer (0.25 M sucrose, 25 mM Tris–HCl, 1 mM EDTA,
pH 7.8) and the cells were lysed by three cycles of sonication
for 30 s each at 60 W. The sonicated homogenate was
centrifuged at 1500g for 5 min at 4 ꢃC, and the pellet was
washed three times in ice-cold STE buffer. This fraction was
centrifuged (2500g for 10 min at 4 ꢃC) and the supernatant
was collected. Then, the supernatant was subjected to ice-
cold ammonium sulphate precipitation between 35 and
85% salt concentration and the resulting precipitate was
dissolved in 2.5 mL of distilled water and desalted by chro-
matography in Sephadex G-25 column (GE Healthcare Life
Sciences®, PD 10 column) previously equilibrated with 2 mL
of distilled water, taking it up to a nal volume of 3.5 mL.36
The protein content was quantied using the Sigma Bradford
test, which uses bovine serum albumin (BSA) as a standard.37
Iron and copper–zinc superoxide dismutases activities were
determined using a previously described method38 that
measures the reduction in nitroblue tetrazolium (NBT) by
superoxide ions. According to the protocol, 845 mL of stock
solution [3 mL of L-methionine (300 mg, 10 mLꢁl), 2 mL of
NBT (1.41 mg, 10 mLꢁ1) and 1.5 mL of Triton X-100 1% (v/v)]
were added into each well, along with 30 mL of the parasite
homogenate fraction, 10 mL of riboavin (0.44 mg, 10 mLꢁl),
and an equivalent volume of the different concentrations of
the compounds being tested. Seven different concentrations
were used for each agent, from 0.1 to 100 mM. In the control
experiment the volume was made up to 1000 mL with 50 mM
potassium phosphate buffer (pH 7.8, 3 mL), and 30 mL of the
parasite homogenate fraction were added to the mixtures
containing the compounds. Then, the absorbance (A0) was
measured at 560 nm in a UV spectrophotometer. Aerward,
each well was illuminated with UV light for 10 min under
constant stirring and the absorbance (A1) was measured
again. The human CuZn-SOD and substrates used in these
assays were obtained from Sigma Chemical Co. The resulting
data were analysed using the Newman–Keuls test.
Infectivity assay
Adherent macrophage cells grown as described above were
infected in vitro with promastigote forms of L. infantum,
L. braziliensis or L. donovani, at a ratio of 10 : 1. The compounds
to be tested (IC25 concentrations) were added immediately aer
infection, and incubated for 12 h at 37 ꢃC in 5% CO2. Non-
phagocytosed parasites and compounds were removed by
washing, and then the infected cultures were grown for 10 days
in fresh medium. Cultures were washed every 48 h and fresh
culture medium was added. Compound activity was determined
on the basis of both the percentage of infected cells and the
number of amastigotes per infected cell in treated and
untreated cultures in methanol-xed and Giemsa-stained
preparations. The percentage of infected cells and the mean
number of amastigotes per infected cell were determined by
analysing more than 200 host cells distributed in randomly
chosen microscopic elds. Values are the means of three
separate determinations.
Ultrastructural alterations
The parasites were cultured at a density of 5 ꢀ 105 cells per mL
in the corresponding medium, each of which contained
a compound to be tested at the IC25 dose. Aer 72 h, the cultures
were centrifuged at 400g for 10 min, and the pellets produced
were washed in phosphate-buffered saline (PBS) and then
incubated with 2% (v/v) p-formaldehyde/glutaraldehyde in
ꢃ
0.05 M cacodylate buffer (pH 7.4) for 24 h at 4 C. The pellets
were then prepared for Transmission Electron Microscopy
(TEM) using a technique previously described.34
Conclusions
The compounds 1, 2, 8 and 9 were identied as hit-to-lead
according to their leishmanicidal properties. The effect at the
Metabolite excretion
Cultures of L. infantum, L. braziliensis and L. donovani promas- ultrastructure level, the selected compounds were responsible
tigotes (initial concentration 5 ꢀ 105 cells per mL) received the as vacuolization, disintegration of the cell cytoplasm and
IC25 dose of each compound (except for control cultures). Aer rupture of the parasites. These cellular effects were also
ꢃ
incubation for 72 h at 28 C the cells were centrifuged at 400g consistent with the changes in the excretion products prole of
for 10 min. The supernatants were collected to determine the parasites treated. Probably, the main target is the Fe-SOD for
excreted metabolites by 1H NMR, and chemical shis were compounds 1 and 2 in the three species tested. On this basis, we
expressed in ppm, using sodium 2,2-dimethyl-2-silapentane-5- think that they fulll the requirements needed to justify a more
sulphonate as the reference signal. The chemical displace- detailed investigation as to the nature of the mechanisms
ments used to identify the respective metabolites were consis- involved in their patterns of activity, and that they could be
tent with those described previously by some of the co- considered candidates for studying antiparasitic activity at
authors.35
a higher level.
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´
´
R. L. Krauth-Siegel, M. Sanchez-Moreno and E. Garcıa-
Acknowledgements
˜
Espana, RSC Adv., 2014, 4, 65108–65120.
´
´
19 M. Inclan, M. T. Albelda, J. C. Frıas, S. Blasco, B. Verdejo,
Financial support by the Spanish MINECO and FEDER (Projects
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C. Serena, C. Salat-Canela, M. I. Dıaz, A. Garcıa-Espana
Consolider-Ingenio,
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