Synthetic single and double aza-scorpiand macrocycles acting as inhibitors of the antioxidant enzymes iron superoxide dismutase and trypanothione reductase in Trypanosoma cruzi with promising results in a murine model

Article abstract of DOI:10.1039/c4ra09866h

The anti-chagasic activity of a series of eleven derivatives of aza-scorpiand-like macrocycles, some of them newly synthesised, was assayed. The four compounds with the best selectivity indices in vitro were subjected to in vivo assays. Tests in a murine model of the acute phase of Chagas disease showed a two-fold reduction in parasitaemia compared to that with benznidazole. Furthermore, compounds 7 and 11, with 4-pyridine and phenanthroline substituents in the lateral chain, caused a remarkable decrease in parasitaemia reactivation during the chronic phase after inducing immunosuppression in mice. These activity studies were complemented by measuring their inhibitory effect towards the antioxidant parasite-specific enzymes, iron superoxide dismutase (Fe-SOD) and trypanothione reductase, the metabolites excreted after treatment and ultrastructural alterations. The ability of the selected macrocycles to complex with Fe(ii) and Fe(iii) was studied using potentiometric methods. Detailed molecular dynamics studies provided interesting pointers about the way in which the compounds approach and modify the active centre of Fe-SOD. The activity, low toxicity, stability, low cost of the starting materials and straightforward synthesis make these compounds appropriate molecules for the development of affordable anti-chagasic agents. This journal is

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Table of contents entry
Synthetic scorpiand-like azamacrocycles selectively inhibit SOD and TR enzymes of
Trypanosoma cruzi causing parasites death and increasing the mice survival after
infection and treatment.

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Synthetic
single
and
double
aza-scorpiand
macrocycles act as inhibitors of the antioxidant
enzymes iron superoxide dismutase and
trypanothione reductase in Trypanosoma cruzi with
Cite this: DOI: 10.1039/x0xx00000x
promising results in a murine model.
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
www.rsc.org/
a
b
a
b
b
F. Olmo, M. P. Clares, C. Marín, J. González, M. Inclán, C. Soriano, K.
c
a
Urbanová, R. Tejero, M. J. Rosales, R.L. Krauth-Siegel, M. Sánchez-Moreno,
d
a
e
a,*
b,*
and E. García-España.
The anti-chagasic activity of a series of eleven derivatives of aza-scorpiand-like macrocycles,
some of them newly synthesised, was assayed. The four compounds with the best selectivity
indices in vitro were subjected to in vivo assays. Tests in a murine model of the acute phase of
Chagas disease showed a two-fold reduction in parasitaemia compared to that with
benznidazole. Furthermore, compounds
7 and 11, with 4-pyridine and phenanthroline
substituents in the lateral chain, caused a remarkable decrease in parasitaemia reactivation
during the chronic phase after inducing immunosuppression in mice. These activity studies
were complemented by measuring their inhibitory effect towards the antioxidant parasite-
specific enzymes Fe-superoxide dismutase (Fe-SOD) and trypanothione reductase (TR), the
metabolites excreted after treatment and ultrastructural alterations. The ability of selected
macrocycles to complex with Fe(II) and Fe(III) was studied by potentiometric methods.
Detailed molecular dynamics studies provided interesting hints about the way in which the
compounds approach and modify the active centre of Fe-SOD. The activity, low toxicity,
stability, low cost of the starting materials and straightforward synthesis make these
compounds appropriate molecules for the development of affordable anti-chagasic agents.
6
better safety and efficacy profile. However, their efficacies are
7
very low during the chronic phase. Furthermore, these
Introduction
compounds have severe side effects, including anorexia,
8
vomiting, peripheral polyneuropathy and allergic dermopathy.
Chagas disease (CD) is a chronic, systemic, parasitic infection
1
caused by the protozoan Trypanosoma cruzi and is identified
by the WHO as one of the world’s 17 most neglected tropical
The presence of nitro groups attached to the heterocyclic rings
suggests that these drugs act on the parasite by nitro reduction,
providing reduced intermediates that covalently modify
biomolecules. However, their mode of action is currently under
2
diseases. This disease represents one of the main public health
concerns in 21 countries in Latin America where about 7-8
3
million people are currently infected with T. cruzi. Actually,
9
discussion. Their high toxicity in humans is probably the result
of oxidative or reductive tissue damage and is inextricably
CD is becoming an emerging health problem in non-endemic
areas, since the parasite has travelled outside of South and
Central America because of population migration (North
America, Pacific region and Europe). Thus, CD is becoming a
new worldwide epidemiological, economic, social and political
challenge. However, as reported by the WHO, whereas research
funding to combat HIV/AIDS, malaria and tuberculosis (TB)
has been considerably increased in the last years, that devoted
to attend other infectious diseases associated with poverty, such
5
linked to their antiparasitic activity. Recently, it has been
proposed that the toxicity of these drugs could be linked to their
microsomal nitroreduction to reactive metabolites inside the
1
0
liver. Therefore, existing drug treatments for CD are far from
satisfactory and the development of a vaccine is a goal yet to be
achieved. The discovery of low-cost new drugs circumventing
the drawbacks of conventional drugs seems to be an urgent
4
4
necessity.
as CD, has not been increased in the same manner.
Currently, the main drugs used to treat Chagas disease are
two nitroaromatic heterocycles: the furane-based nifurtimox
Research into antichagasic agents is mainly focused on
biochemical metabolic key pathways or crucial parasite-specific
enzymes. Sterol metabolism, kinetoplast DNA linked-sites,
5
and the imidazole-based benznidazole (Bz). Both drugs are
effective in the acute phase of the disease, although Bz shows a
trypanothione
reductase,
cysteine
proteinase,
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hypoxanthineguanine phospho-ribosyltransferase, dihydrofolate showed that the synthetic aza-scorpiand-like macrocyclic
reductase, and glyceraldehyde 3-phosphate dehydrogenase have derivatives are potentially promising agents for the treatment of
1
1
16
trypanosomatid infections. In the present work, we tested the
fed the main lines of investigation during last years.
Among the trypanosomatid-specific enzymes, we have in vitro antiparasitic activity of 11 new polyaminic compounds
focused our attention on iron superoxide dismutase (Fe-SOD). and their toxicity against Vero cells. We compared the new
This enzyme, not found in mammals which have only compounds with Bz as a reference drug. Then, we chose the
manganese and copper/zinc superoxide dismutases (Mn and compounds with the best selectivity indices for the in vivo assay
Cu/Zn-SOD), plays an essential role in the defence of the of the trypanocidal activity tests in the acute and chronic phases
parasite against oxidation generated by the host immune using female BALB/c mice as an animal model. The effect of
1
2
system. Indeed, it has been shown that parasitic protozoan the compounds on the ultrastructure of T. cruzi was also studied
survival is closely related to the ability of Fe-SOD to evade by transmission electronic microscopy (TEM) experiments to
1
3
1
toxic free radical damage originated by their hosts. Because of confirm the type of damage caused to the parasite. H-NMR
the predominant role of the prostetic groups, interaction with analysis of the nature and percentage of excreted metabolites
Fe-SOD active sites could be an efficient way of deactivating was performed to obtain information about the inhibitory effect
the antioxidant effect of the enzyme.
of our compounds on the glycolytic pathway since this
Regarding this point, in the last few years, our research represents the prime energy source of the parasite. Moreover, in
group has designed a new family of polyamine compounds order to check for metabolic disturbances or abnormalities
consisting of a macrocyclic pyridinophane core substituted with associated with treatment, representative biochemical
1
4
side chains containing additional donor atoms (Figure 1).
These macrocycles are termed scorpiand ligands since the side
chain can fold towards the macrocyclic core following the good inhibitors of Fe-SOD, not only in T. cruzi, but also in
parameters were measured for all groups of mice.
Previously, we have demonstrated that these derivatives are
1
6b
1
6a
binding of target guest species such as protons, anions or metal Leishmania spp
.
An evaluation of their effectiveness as
1
4,15
ions.
putative inhibitors of Fe-SOD in relation to human CuZn-SOD
and also of trypanothione reductase is presented. Finally, an
exhaustive molecular dynamic study was carried out to
envisage the way in which these drugs approach and inhibit the
active centre of Fe-SOD.
Results and discussion
Chemistry.
Synthesis of the compounds
was accomplished as described previously
modification of the Richman-Atkins procedure.
.
The synthesis of
1
,
2
,
3
,
4
, and 11
1
4,17
following a
1
8
First,
pertosylated polyamine tris(2-aminoethyl)amine (tren) was
reacted with 2,6-bis(bromomethyl)pyridine in a 1:1 molar ratio
using K CO3 as the base in refluxing CH CN; this led to
2
3
tosylated compound 12. Detosylation was carried out with
HBr/HAc/PhOH, obtaining 12 as hydrobromide salt.
Compounds and 11 were obtained by reacting 12, in
its free amine form, with the corresponding carboxaldehydes
Scheme 1) in dry ethanol followed by in situ reaction with
a
1, 2, 3, 4
(
sodium borohydride. The compounds were finally precipitated
as hydrochloride salts.
Compound 13 was obtained by the reaction of pertosylated
polyamine 12 with N-(3-bromopropyl)phthalimide in a 1:1
molar ratio using K CO3 as the base in refluxing CH CN.
2
3
Deprotection and detosylation were carried out with hydrazine
monohydrate and HBr/HAc/PhOH, respectively, obtaining 13
1
as a tetrahydrobromide salt. Compounds 5, 6, 7 and 8 were
7a
obtained by reacting 13, in its free amine form, with the
corresponding carboxaldehydes (Scheme 1) in dry ethanol
followed by in situ reaction with sodium borohydride. The
compounds were finally precipitated as the hydrochloride
1
7a,19
salts.
The synthesis of
moles of 12 with one mole of pyridine-2,6-dicarboxaldehyde or
,10-phenanthroline-2,9-dicarboxaldehyde, respectively, in dry
ethanol, followed by in situ reduction with sodium borohydride.
The compounds were finally precipitated as their hydrochloride
9
and 10 was carried out by reacting two
1
Figure 1. Chemical formula of aza-scorpiand derivatives. The common core for
compounds 1-8 is depicted in the square.
1
7b
salts.
Recently, we have studied the in vitro action of several of
these compounds against Trypanosoma cruzi and two species of
Leishmania (L. infantum and L. braziliensis). The results
2
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N
N
O
N
R
1
-CHO
O
Br
HBr/HAc/PhOH
Reflux
N
N
N
N
N2H4
N
N
N
N
NaBH
4
N
N
K
2
CO
3
NTs
NTs
NTs
NTs
NTs
NTs
NH
NH
NH
NH
EtOH
EtOH
CH
3
CN
O
1
R =
NHTs
N
Ts
N
N
Ts
NH
2
NH
NH
2
R
1
O
R
2
(CHO)
2
HBr/HAc/PhOH
Reflux
N
EtOH
NaBH
4
N
N
N
R
2
R
3
-CHO
NaBH
EtOH
NH HN
N
N
N
N
4
N
N
N
R
3
=
NH
NH
NH
NH
2
R =
NH
NH
NH
NH
N
N
N
R
3
N
N
H
N
H
N
H
NH
2
Scheme 1. Synthetic route for the preparation of compounds 1-13.
An interesting point for this discussion and for the posterior
Degree of protonation of the compounds. An important molecular dynamics study is to know the protonation degree of
parameter that must be taken into account when dealing with the compounds at pH 7.4. Such data are plotted in Figure 2; it
polyamines is their acid-base behaviour. The protonation state can be seen that while all the simple non-enlarged aza-
of the amine groups in the macrocycle affects their interaction scorpiands
with other substrates since it defines their donor capabilities enlarged scorpiand molecules
and their behaviour as hydrogen bond donors or acceptors. of 3. Tritopic macrocycles
1
-
4
and 11 had protonation degrees of ca. 2, the
reached a protonation degree
and 10 were the most charged
5-9
9
Table S1 presents the protonation constants determined by compounds, reaching a protonation degree of almost 4 at this
potentiometric titrations using 0.15 M NaCl as ionic strength. pH. Previous NMR studies have shown that first two protons
As can be seen in the Table, the number of protonated species bind preferentially to the amino groups of the macrocyclic core
1
7b
corresponds with the number of secondary or primary amino of
groups present either in the macrocyclic core or in the arms. apart from the two secondary amino groups of the core, one of
1
-
4
and 11
.
The first three protons binding to 5-9 involve,
14
Protonation of the tertiary amines or pyridine groups was not the amine groups of the lateral chain.
observed, at least in the 2.0-11.0 pH range of the potentiometric
titrations. Only in the case of the two macrocycles containing a Interaction with iron. Since one of the potential targets of the
phenanthroline unit and of the macrocycle with a 4-pyridine studied compounds was the Fe-SOD of the parasites, it seemed
unit in their arms (10 11 and , respectively) was a further interesting to evaluate the affinity of these chelating ligands for
protonation step inferred. iron either in its II or III oxidation state. In Table S2, we
present estimates of the binding constants for and as
,
7
1
,
2
7
representatives of this series. As can be seen, all three
compounds form stable complexes with either Fe(II) or Fe(III);
the stability of the latter ones was remarkable and comparable
2
+
to that found for the Cu complexes previously described by
1
4,17,19
some of us.
However, the stability of the Fe(III)
complexes was not high enough to avoid the formation of
hydroxo species and of Fe(III) hydroxide at basic pH values.
On the other hand, Fe(III) systems might have been affected by
side oxidation of amine groups to imines and, therefore, the
stability constants are presented as tentative values. Anyway, in
view of the distribution diagrams collected in Figure 3 and S1,
it is clear that if the ligands approach the active site of Fe-SOD
quite closely, some competition might be established with the
primary ligands of the active centre for the capture of the metal
ions.
Figure 2. Protonation degree of the different compounds at pH 7.4.
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observed (see Electronic Supplementary Information Table S3).
This can probably be attributed to the protective effect that the
2
Mn complexes of these ligands exert over free radicals
+
2
0
generated in the oxidative metabolism.
Significant differences were found, however, in the
unspecific cytotoxicity against Vero cells and, of course, in the
selectivity indices (SI) resulting from these data. Despite this,
there was not a clear relationship between toxicity and
structure, and the more toxic compounds appeared to be the
double scorpiands (
large aromatic pyrene units (
9
,
10) and the single scorpiands containing
). Taking into account all the
4, 8
data included in Table 1, compounds 1, 2, 7 and 11 were the
best compounds, with SI values exceeding by 50 times or more
those of Bz, depending on the parasite stage. According to
some authors, only compounds with SI values exceeding 50
times the SI value of Bz are good enough to be considered
2
1
interesting as antiparasitic drugs.
Before starting the in vivo analysis of compounds
1, 2, 7
Figure 3. Distribution diagrams of the Fe(II)-
Biological Activity
1
systems.
and 11, an infectivity assay was performed. In such an assay,
which is an in vitro reproduction of the life cycle of the parasite
(see Electronic Supplementary Information, Infectivity section
and Figure S2), it can be observed that all four selected
In vitro trypanocidal evaluation
.
The in vitro activity of compounds decreased the infection rate, i.e. by 73, 68, 82 and
compounds
epimastigote
amastigote) forms of T. cruzi, as described in the Experimental consequently, the release of trypomastigote forms into the
1
−
11 was evaluated against extracellular 76%, respectively. In the same way, they decreased the number
(
and trypomastigote) and intracellular of amastigote forms per infected cell at rates higher than 50%;
(
Section. More indicative data were found with the assays media decreased to 60, 44, 80 and 69%.
performed on the intracellular amastigote forms, since
epimastigotes are not the fully developed parasitic forms in In vivo activity of scorpiand-like azamacrocyclic derivatives
.
vertebrate hosts. Finally, the compounds were tested against The promising results obtained for the in vitro tests prompted
blood trypomastigotes, since these parasitic forms, along with us to study the in vivo activity of compounds 1, 2, 7 and 11 in a
the amastigotes, are responsible for the chronic phase of CD.
The IC50 values obtained for the extracellular and intracellular
murine model (BALB/c mice).
The effectiveness of drugs currently in use against Chagas
forms are shown in the first three columns of Table 1, which disease varies widely between the acute and chronic phases.
includes the data obtained for the reference drug Bz. Toxicity Therefore, we decided to evaluate our compounds in both
values were calculated against mammalian Vero cells after 72 h phases. For acute phase experiments, we considered the first 40
of culture and the selectivity indices (SI, calculated as IC50 days after infection, whereas the effect on the chronic phase
Vero cells toxicity/IC50 activity of extracellular or intracellular was studied between days 40 and 120 after infection for those
forms of the parasite) are shown in the last three columns of compounds with high efficiency during the acute phase.
Table 1. The numbers in brackets show the SI of the compound
relative to that of Bz. This value indicates the in vitro potential vivo experiments performed. The intraperitoneal administration
of the test compounds with respect to the reference drug.
route was preferred to the intravenous procedure because
All compounds showed activity against the three parasite intraperitoneal treatment substantially reduces animal
Scheme S1 in the ESI file provides a picture of all the in
2
2
stages, and some of them were more active than Bz. Regarding mortality.
the cytotoxicity against Vero cells, the compounds were
substantially less toxic than the reference drug (see the IC50
values in Table 1). Considering the more illustrative selectivity
index, compounds
SI values 25 to 77-fold higher than those of Bz (Table 1). The
SI values of the other compounds were lower, indicating that
1, 2, 7, and 11 were of potential interest with
2+
they are less suitable. However, when we checked the Mn
complexes of compounds to and 11, a lower effect was
1
7
Table 1. In vitro activity, toxicity and selectivity index found for the aza-scorpiand-like macrocyclic derivatives on the extracellular and intracellular forms of T. cruzi.
a
b
IC (µM)
SI
5
0
Toxicity
IC Vero
Cell (µM)
50
Epimastigote Amastigote Trypomastigote
forms
Epimastigote Amastigote Trypomastigote
forms
Compound
forms
forms
forms
forms
Bz
1
2
3
4
16.2±0.8
3.3±0.5
4.2±0.7
16±2
23.6±0.7
6±1
3.2±0.5
13±2
4.2 ±0.3
22.6±0.8
5±2
5±1
22±3
7.4±0.7
13.6± 0.9
187±14
146± 9
214±15
83± 5
0.8
0.6
0.6
57 (71)
35 (44)
13 (16)
26 (33)
31 (52)
46 (77)
16 (27)
20 (33)
37 (62)
29 (48)
10 (17)
11 (18)
3.2±0.3
4
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ARTICLE
5
6
7
8
9
18.0±0.5
16±1
3.3±0.2
29±2
4.8±0.5
4.1±0.6
7.9±0.6
14±1
33±2
131±13
88±6
98±7
84±10
76±5
94±6
7 (9)
6 (8)
30 (38)
3 (4)
16 (20)
23 (29)
26 (33)
9 (15)
10 (17)
35 (58)
3 (5)
15 (25)
26 (43)
39 (65)
4 (7)
3 (5)
15 (25)
4 (7)
8 (13)
10 (17)
17 (28)
8.4±0.7
2.8±0.5
25±3
5.1±0.4
3.6±0.1
5.2±0.3
27±2
6.5±0.4
19.4±0.8
9±1
9.2±0.8
12±1
1
1
0
1
202±11
a
The values are the average of three separate determinations. IC50 = the concentration required to give 50% inhibition, calculated by linear regression analysis from the
b
values at the concentrations employed (1, 10, 25, 50 and 100 µM). Selectivity index =IC50 Vero cells/IC50 extracellular or intracellular form of the parasite. In brackets:
number of times that compound exceeds the reference drug SI (for the extracellular and intracellular forms of T. cruzi
)
In fact, none of the animals died in any of our experiments
performed either with the control or with 1, 2, 7 and 11 at the
concentrations used (5 mg/kg of body mass). Female mice were
inoculated with trypomastigotes, as described in the
experimental section. Treatment, via the intraperitoneal route (i.
p.), with the compounds of interest began 5 days post-infection
(
pi) and was maintained for an additional 5 days. Another
group of mice was treated in the same manner, but with only
the vehicle (control). During the study of acute phase activity,
the levels of parasitaemia were determined every 2 days. Figure
4
shows the number of trypomastigote forms found in the blood
until day 40 pi. On the days of maximum parasitic burden (peak
days, 16-22 days pi) all compounds greatly reduced the number
of circulating parasites. Furthermore, this reduction lasted until
day 40 and was more significant for the groups treated with the
compounds under study than for those treated with Bz. The
order of in vivo activity towards the trypomastigote forms in the
acute phase was:
7 > 11 > 2 >> 1 > Bz.
Figure 4 Parasitaemia in the murine model of acute Chagas disease (A and B): (-
-) Control and dose receiving 5 mg/kg of body mass dose of: (- -) Bz; in A: (-
-) compound ; (-ꢀ-) compound ; in B: (- -) compound ; (-ꢀ-) compound 11
All compounds were administered by the i.p. route. Grey shading represents the
treatment period. Values are the means of six mice +/- standard deviation.
▲
ꢀ
ꢀ
1
2
ꢀ
7
.
Figure 5 Immunosuppression experiment in the mouse model. (A) Reactivation
of parasitaemia after immunosuppression. (
Values are the means of three separate experiments and error bars represent the
mean +/- standard deviation. ( ) Polymerase chain reaction (PCR) analysis of
B) Total IgG levels of anti-T. cruzi.
C
heart tissue at the day 120 after infection. All mice from lane 3 to 10 were
infected.
Compounds
between 30 and 63% of the parasites survived the treatment
Figure 4A), while and 11 displayed the highest activity
during the acute phase (parasitaemia reduction greater than
0% by the end of the acute phase), as shown in Figure 4B. So,
in view of these results, the group of mice treated with
1 and 2 showed moderate activity, since
(
7
ELISA assays were used as an alternative method for
verifying the effectiveness of these two compounds in the
8
chronic
immunosuppression cycle. According to the literature, IgG
titres in Balb/c become stable during the chronic phase of the
phase
after
being
challenged
with
an
compounds
conditions.
The
7
and 11 were kept until day 120 pi under the same
2
3
disease; this was confirmed in Figure 5B, which shows the
anti-T. cruzi IgG levels. The IgG levels of the control group
increased by about 40% due to the presence of parasites in the
bloodstream after immunosuppression. However, the
experiment was accomplished by an
immunosuppression test on the treated and control mice at day
20 pi (late chronic phase, in which there are no parasites
1
remaining in the bloodstream). Figure 5A shows the
reactivation of parasitaemia after immunosuppression. The
control group recovered its initial parasitaemia almost entirely,
thus giving a reactivation of 100%. In contrast, mice treated
with 11 showed a parasitaemia load of 60% relative to their
differences
immunosuppressed groups were smaller in the case of 11 and
not significant for those treated with . This means that the
between
immunosuppressed
and
non-
7
parasite-specific IgG level was not higher than the remaining
amount of total unspecific level of IgG caused by the
hypergammaglobulinaemia characteristic of infection with
previous burden, while mice treated with
lower reactivation of only 50%.
7 displayed an even
2
3,24
Trypanosoma cruzi
.
Finally, Figure 5C shows the PCR
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results after necropsy. The hearts were ground and underwent transformation of PEP in pyruvate in the presence of pyruvate
2
4a
total DNA extraction and amplification of a fragment within the kinase or pyruvate phosphate dikinase. Therefore, it seems
parasite SOD-gene. The hearts of control animals confirmed the possible that and 11 interact with pyruvate kinase enzymes,
presence of parasites, while the hearts of mice treated with thereby modifying the glucose metabolism of the parasite at the
were relatively cleared from parasites (25% of the control value pyruvate stage. On the other hand, it is interesting to note that
thus confirming the degree of curative effect of the decrease in succinate observed with and 11 could also be
We can conclude that the limited effectiveness of , the best related to dysfunction of the mitochondria, due to the redox
compound found in this study, was due to the low dosage used stress produced by inhibition of the mitochondrion-resident Fe-
7
7
7
.
7
7
2
8
during the acute phase. Hence, we must take into consideration SOD enzyme. This should result in a decrease in pyruvate
future experiments where we will increase the dosage and also metabolism and a consequent decrease in succinate produced in
modify the schedule of treatment for better exposure of the mitochondria. All these data point out that the severe
compound in the bloodstream. This increase in compound modifications generated in organelles like glycosomes or
administration should not result in enhanced toxicity. Indeed, mitochondria by
7 and 11 is the ultimate reason for the
according to the data shown in Table S4, all the biochemical alterations observed in the excretion products of T. cruzi
parameters tested regarding kidney, heart and liver profiles
were maintained after compound administration; only the uric
.
acid values showed an insignificant decrease of 10% (see the
electronic supplementary information).
Molecular Mechanisms of Action
Metabolite excretion study. Since trypanosomatids are unable
to completely degrade glucose to CO , they excrete a
2
considerable portion of its hexose skeleton as partially oxidised
fragments in the form of fermented metabolites, whose nature
and percentage depend on the pathway used for glucose
2
5
metabolism. The catabolism products in Trypanosoma cruzi
were acetate and succinate, with smaller percentages of L-
2
6
alanine and D-lactate, in agreement with data in the literature.
The detection of large amounts of succinate as a major end Figure 6. Variation percentages in the height of the peaks corresponding to
product is a usual feature, because it relies on glycosomal redox metabolites excreted by T. cruzi epimastigotes in the presence of aza-scorpiand-
like macrocycles derivatives at their IC25 compared to a control sample. Values
balance, enabling re-oxidation of NADH produced in the
glycolytic pathway. Succinic fermentation requires only half of
are the means of three separate experiments and error bars represent the standard
deviation.
the phosphoenolpyruvate (PEP) produced to maintain
NAD+/NADH balance. The remaining pyruvate is converted
inside the mitochondrion and the cytosol into acetate, D-lactate,
L-alanine, or ethanol according to the degradation pathway
Ultrastructural alterations. The trypanocidal activity shown by
, 2, 7 and 11 should lead to relevant damage to parasite cells.
Therefore, we performed a transmission electron microscopy
TEM) study on the epimastigote forms of T. cruzi treated with
, 2, 7 and 11. As expected, significant morphological
alterations were observed when compared with untreated
control cells (Figure 7). Without any doubt, the most effective
compounds came out to be and 11, which caused considerable
disturbances in many parasites. Compound (Figure 7B)
mainly caused disorganisation in the plasma membrane,
enormous vacuoles inside, and mitochondria were found to
swollen and expanded. The nuclei were also swollen and
vacuolated, and in some parasites, the cytoplasm was full of
1
2
7
followed by each species.
To obtain some information about the effect of the tested
compounds on parasite glucose metabolism, we registered the
(
1
1
H NMR spectra of T. cruzi epimastigote forms after treatment
with 1, 2, 7 and 11. The final excretion products were identified
qualitatively and quantitatively (spectra not shown). The results
were compared with those obtained from parasites maintained
in a cell-free medium (control) for 96 hours. The characteristic
presence of acetate, succinate, D-lactate and L-alanine was
confirmed in the control experiments. As expected, succinate
and acetate were the most abundant end products identified.
However, after treatment of the parasites with the selected
compounds, the excretion of catabolites was substantially
altered at the dosages employed. Figure 6 shows the changes
observed compared to the control. Remarkable differences in
the catabolic pathway were observed, depending on the
compound used, which seemed to be connected with the
trypanocidal activity described above. The main features found
were substantial decreases in succinate and lactate and, to a
7
1
reservosomes. More drastic effects were caused by 2, as can be
appreciated in Figure 7C, where some epimastigote forms were
totally swollen with giant vacuoles which filled almost all the
cytoplasm. Moreover, the mitochondria were disorganised and
swollen, with small crystals and a less electron dense zone than
usual that could be related to a loss of function. The parasites
treated with compound 11 (Figure 7E) were totally disturbed,
showing an empty cytoplasm, broken plasma membrane and
some organelles were unrecognisable. Some similar alterations
lesser extent, alanine. The most active compounds
decreased succinate, D-lactate and L-alanine by -28.4%, -
9.7% and -22.1% and -28.4%, -27.0% and -23.7%,
respectively.
All these data can be interpreted on the basis of a change in
the succinate, D-lactate, L-alanine pathways occurring in the
presence of the most active compounds and 11. It is well-
known that D-lactate and L-alanine originate from the
7 and 11
occurred when the epimastigote forms were treated with
(
density was decreased in the nucleus. Mitochondria were
swollen and microtubules were discontinuous along the
cytoplasm.
7
Figure 7D), where ribosomes were very few and the electron
3
7
6
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Inhibitory effect on the T. cruzi Fe-SOD enzyme. These
results prompted us to evaluate the inhibitory effect of
and 11 on Fe-SOD activity. The results are shown in Figure 8.
Significant inhibitory values of Fe-SOD activity were found for
the four compounds tested. Compounds and 11 showed
B
F
1, 2, 7
N
7
values close to 100% inhibition at 25-50 µM with IC50 values
ranging from 4.2 to 6.6 µM. The design of an effective drug
able to inhibit the parasite Fe-SOD without inhibiting human
SOD is an interesting goal. Therefore, we also assayed the
effect of these compounds on Cu/Zn-SOD from human
erythrocytes. The results show that the inhibition percentages
for human CuZn-SOD were lower than for Fe-SOD. The lead
M
V
K
V
M
V
A
B
C
MT
compounds 7 and 11 reached IC50 values of 98.0 and 55.8 µM
for Cu-Zn-SOD, respectively.
M
It should be noted that, as mentioned above, these
compounds not only showed greater alterations in glucose
catabolism but they also led to greater levels of Fe-SOD
inhibition. Since the Fe-SOD present in mitochondria is an
essential 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. According to this fact, we
hypothesise that compounds having a pyridine nitrogen on the
side chain are able to get closer to the active centre of the
N
R
M
E
N
D
enzyme, as is the case of compound
greater inhibition. Modelling studies were carried out to obtain
clues about this point.
7, so they might produce a
Figure 7. Ultrastructural alterations shown by TEM in epimastigotes of T. cruzi
treated with aza-scorpiand-like macrocycles and 11 , control parasite
showing typical organelles such as nucleus (N), mitochondria (M), glycosomes
G), microtubules (MT), vacuoles (V), reservosomes (R), kinetoplast (K) and
flagellum (F); , treated with aza-scorpiand-like macrocycles , treated with
the compound , treated with compound and , treated with compound 11, at
the IC25 concentration. Scale bar = 1µm.
1
,
2
,
7
. A
(
B
1; C
2
;
D
7
E
Figure 8. In vitro inhibition (%) of CuZn-SOD from human erythrocytes for compounds (activity 23.36/4.21 U/mg) and Fe-SOD from T. cruzi epimastigotes for
compounds (activity 20.77/3.18 U/mg). x-axis represent the compound concentrations given for each enzyme. Enzyme concentration were kept constant in all assays.
Differences between the activities of the control homogenate and those ones incubated with compounds were obtained according to the Newman-Keuls test. Values in
the legend represent the IC50 = the concentration required to give 50% inhibition was calculated by linear regression analysis from the Kc values at the concentrations
employed (0.1-100 mM).. IC50 values for Cu/Zn SOD = 123 µM; 172 µM; 98 µM and 56 µM, respectively for Comp 1, 2. 7, and 11. IC50 values for Fe SOD = 13 µM;
4
2 µM; 4 µM and 7 µM, respectively for Comp 1, 2. 7, and 11. Values are the average of three separate determinations and error bars represent the standard deviation
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Figure 9. A) Representation of the active centre and residues forming the funnel
gateway of Fe-SOD Trypanosoma cruzi protein PDB_ID 2gpc. Relevant
hydrogen bond connections are presented as dotted lines. B) Cartoon representing
the superimposition of the original 2gpc protein (rainbow colours) (only one
Modelling studies. In order to learn about possible ways in
which the compounds yielding the best trypanocidal activity
inhibit the active centre of Fe-SOD, we carried out a molecular chain) over the molecular dynamics minimum energy structure (pale blue) of the
protein with compound 11 (magenta). Original residues have the corresponding
rainbow colours while final dynamics residues are pale brown. Helices I are
represented on the left.
dynamics study in which the compounds were approached to
Fe-SOD T. cruzi protein PDB_ID 2gpc. As commented with
more detail in the experimental section, a distance restraint was
applied between the heavy atoms of the active site residues and
The molecular dynamics studies with compounds 1, 2, 7
-
6 -1/6
the heavy atoms of the compounds, using <r > average of all
interaction distances to atoms of the groups. After the
equilibration stage at 300 K, a total of 10 ns molecular
dynamics was performed. Finally, the five minimum energy
conformers were selected from the trajectory and minimised by
removing all the restrains.
and 11 revealed profound changes in the active site
environment. Although all the compounds examined displaced
the gateway amino acids H31 and Y35, two different
approaching orientations were identified. For
quinoline and phenanthroline subunits at the arm lie towards
the exterior of the funnel while the macrocyclic core
approaches the active centre, while for the macrocycle is
1, 2 and 11, the
T. cruzi Fe-SOD is a dimeric protein with the polypeptidic
chains constituted by seven helical regions and three anti-
7
placed on the other way around, extending its lateral chain
containing the 4-pyridine units towards the metal site and
leaving the macrocyclic core oriented outwards. Although in
none of these examples do the donor atoms of the inhibitors
bind directly to the metal ion, they produce drastic changes in
the hydrogen bond network around the active site at the time
that they significantly alter helix 1 (see Figure 9B and Figure
S4). Interestingly, the molecules approach the active site by
taking advantage of a disrupted turn in the helix. The
interaction of the compounds with the proteins pushes back the
upper part of helix 1. As expected, all the compounds remove
the hydrogen bond network formed by Y35 and Q71 and are
involved in hydrogen bonding either with them or/and with
2
9
parallel β-strands. The substrates approach the active site
through a funnel in which H31 and Y35 serve as the gateway
and restrict the access of small molecules towards the active
site (Figure 9). The metal ion is coordinated in trigonal
bipyramidal geometry to H27, H75, D160 and H164 and to a
water molecule or a hydroxide anion. A hydrogen bond
network involving the gateway residues Y35 and Q71 and the
water molecule or hydroxide ion directly bound to the metal
facilitate proton exchange between the active centre and the
3
0
exterior of the protein. Apart from this, residues E163 and
Y167 extend towards the surface of the protein and help
stabilise the dimer through hydrogen bonding with E163 and
3
0,31
H31 of the other unit, respectively
electronic supplementary materials).
(see Figure S3,
H31. For instance, while
Q71, 11 establishes hydrogen bonds with H31 and Q71 (Figure
S4).
In its turn,
H164, which forms part of the first coordination sphere of the
metal ion lying the 4-quinoline unit parallel to the shifted Y35
of the gateway. Finally, in the case , hydrogen bonds between
the inhibitor and Y35 and H31 are observed while Q72 does not
participate in the hydrogen bonding network. Additionally,
1 forms hydrogen bonds with Y35 and
2
makes hydrogen bonds with Q71 and also with
A
7
7
hydrogen also bonds to a leucine residue (L26). One surprising
and very interesting result regards the way in which these
molecules fit and close the funnel of the enzyme. As seen in the
surface map of Figure 10, 11 (phenanthroline) completely
occludes the funnel, preventing any other molecule from
approaching the active site. The same event, although not to
such a large extent, occurs for the other compounds (see Figure
S5 in the ESI).
Trypanothione reductase (TR) inhibition assay. The best
compounds were selected to evaluate their effects on T. cruzi
trypanothione reductase (TR). All four compounds proved to be
inhibitors of TR (Figure 11). At fixed concentrations of 40 and
B
1
disulphide (TS ), the degree of inhibition varied between 14
00 µM for both the inhibitor and the substrate trypanothione
2
and 89%. The most active in vivo trypanocidal compound
the lowest inhibitory activity (lower than 50%), which renders
TR unlikely as a potential target. The other three compounds
and 11 caused 74 to 89% inhibition when 100 µM of the
7 had
1
,
2
compound and 40 µM of TS were applied, indicating that they
2
are inhibitors of the enzyme. Notably, no significant difference
(
around 10%) in the degree of inhibition at the two TS2
concentrations was observed. This strongly indicates that these
compounds do not act as pure competitive inhibitors.
8
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values were 16 µM and >200 µM (Figure 12). In any case, a
higher affinity of the compound for the free enzyme compared
to the enzyme-substrate complex was observed, as can be
inferred from the calculated Ki < Ki’ values.
Figure 10. Solvent accessible surface of (A) original 2gpc protein and (B) 2gpc
and minimised compound 11 (light brown) showing the occlusion of the funnel.
H31 and Y35 are presented in magenta and blue, respectively.
Figure 12. Lineweaver−Burk plots for the inhibition of TcTR by compound
(B), and 11 (C). The assays contained 100 ꢁM NADPH and none, 20 µM, and
0 µM fixed concentrations of the inhibitor The TS concentration was varied
between 20, 40, 80, 100 and 200 µM.
1(A),
2
6
2
In silico pharmacokinetic evaluation. Compounds
, which showed the best trypanocidal activity, were also
submitted to an in silico pharmacokinetic properties evaluation.
To evaluate the absorption rate, necessary for oral
1, 2, 7 and
1
1
Figure 11. Inhibition of TcTR by compounds 1, 2, 7 and 11. The activity was administration, we analysed the number of free rotatable bonds
measured following the NADPH consumption at 340 nm as described in the (n-ROTB) and Lipinski’s “rule of five” for the lead compounds.
Experimental Section. The control used to obtain 100% of activity was assayed
without an inhibitor but rather the same amount of water used to dissolve the
compounds. The values are average of two independent measurements Error bars
show the standard deviation.
Lipinski’s descriptors evaluate the molecular properties for
drug pharmacokinetics in the human body, especially for oral
absorption. The rule states that the most “druglike” molecules
present clogP ≤ 5, molecular weight ≤ 500, number of hydrogen
bond acceptors (HBA) ≤ 10, and hydrogen bond donors (HBD)
3
3
Trypanothione reductase kinetic study. To obtain insight into ≤ 5. Lipinski’s molecular descriptors and the predicted data of
the molecular mode of action, the inhibitor constants and the some ADMET properties of
(which share the same
type of inhibition were determined using Lineweaver−Burk predicted values),
and 11 are summarised in Table 2. The
plots, which revealed that all three compounds and 11 predominant species at pH 7.4 (H L , for
behave as mixed type non-competitive inhibitors. Clearly, the H L for
1, 2
7
2
+
1
,
2
1
,
2
and 11 and
and 11
lines cut in the double reciprocal plot above the x-axis. In the showed acceptable n-ROTB values (≤10) and fulfilled
case of , Ki and Ki’ values of 4 and 53 ꢁM, respectively, were Lipinski’s rule of five. In addition, ADMET (absorption,
obtained. Compound was slightly less efficient giving Ki and distribution, metabolism, excretion and toxicity) properties
Ki’ values of 8 and 114 µM. Finally, for 11, the Ki and Ki’ were calculated using admetSAR (http://www.admetexp.org/),
2
3+
7
) was chosen for the evaluation. 1, 2, 7
3
3
2
1
2
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a freely accessible web-based application. The predicted data potential candidates to enter into clinical trials. Compounds
4
7
for BBB (blood−brain barrier) penetration and HIA (human and 11 selectively inhibit the Fe-SOD enzyme of the parasites.
intestinal absorption) were positive for all the tested Molecular dynamics studies indicated that these compounds
compounds. Caco-2 cell permeability was negative in all cases, block the access of substrates to the active site while they break
but with a moderate probability value. In the case of the hydrogen bond pattern around the active site. Moreover,
metabolism, various cytochromes P450 (CYP) were evaluated, compound
showing similar patterns for all the compounds. In terms of standard dosage of 5 mg/kg body mass. As shown by the NMR
toxicity, it was found that none of the compounds showed data, another mechanism of action of this series of compounds
mutagenic toxicity with respect to the AMES test or may be related to energetic mechanisms. On the other hand,
7 showed an in vivo cure rate of 25% at a safe
1
carcinogenic effects.
and 2 behaved as very effective trypanothione reductase (TR)
inhibitors. The damage to the defence system of parasites
produced by these compounds was reflected in the TEM
ultrastructural analysis by high vacuolisation. Moreover, the
compounds of choice displayed low in vivo toxicity, as
Conclusions
The trypanocidal properties of a family of simple and double reflected by the fulfilment of the ADMET rules. Finally, it
aza-scorpiand compounds, some of them newly synthesised, deserves to be mentioned that, due to their different
were examined both in vivo and in vitro. The experiments mechanisms of action, combined therapies should be further
allowed us to select compounds displaying improved efficiency considered to obtain improved efficiency.
A
patent
and lower toxicity than the reference drug. Parallel studies were (P201330699) has already been filed for this family of
carried out to establish the mechanism of action. Following this compounds.
series of experiments, four compounds were selected as
Table 2. Oral bioavailability, molecular properties and predicted ADMET properties of compounds 1, 2, 7 and 11.
Compounds 1& 2
Compound 7
Compound 11
Result
Prob. (%)
Result
Prob. (%)
Result
Prob. (%)
Lipinski Molecular Descriptors
HBA (≤10)
4
3
4
4
5
3
HBD (≤5)
clogP (≤5)
2.62
393.56
5
1.52
400.59
9
2.64
444.61
5
MW (≤500)
n-ROTB (≤10)
Absorption
BBB
+
+
-
87.9
72.2
61.6
+
+
-
67.7
74.1
65.9
+
+
-
87.9
72.2
61.6
HIA
Caco-2
Metabolism
CYP450 2C9 substrate
CYP450 2D6 substrate
CYP450 3A4 substrate
CYP450 1A2 inhibitor
CYP450 2C9 inhibitor
CYP450 2D6 inhibitor
CYP450 2C19 inhibitor
CYP450 3A4 inhibitor
Toxicity
NS
S
90.2
63.0
77.0
89.5
96.0
69.5
95.1
90.0
NS
S
89.8
55.9
73.5
91.8
95.4
51.0
96.7
93.7
NS
S
90.2
63.1
77.0
89.5
96.0
69.5
95.1
90.0
NS
NI
NI
NI
NI
NI
NS
NI
NI
NI
NI
NI
NS
NI
NI
NI
NI
NI
AMES toxicity
Carcinogens
-
-
68.6
93.2
-
-
66.4
93.9
-
-
68.6
93.2
a
n-ROTB, number of rotatable bonds; HBA, number of hydrogen bond acceptors; HBD, number of hydrogen bond donors; clogP, logarithm of compound partition
coefficient between n-octanol and water; MW, molecular weight; BBB, blood-brain barrier; HIA, human intestinal absorption; S, substrate; NS, nonsubstrate; I,
inhibitor; NI, noninhibitor.
1
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Studies on the mechanism of action:
Metabolite excretion study. Cultures of T. cruzi epimastigotes
Experimental
Chemistry
5
initial concentration 5 × 10 cells/mL) received IC25
(
concentrations of the compounds under study (except for
control cultures). After incubation for 96 h at 28°C, the cells
were centrifuged at 400 g for l0 min. The supernatants were
The ESI contains the synthetic procedure for compounds
4
2 and
prepared for the first time and the characterisation data
(
NMR, mass spectrometry and elemental microanalysis) of all
1
collected to determine the excreted metabolites using H NMR.
Chemical shifts were expressed in parts per million (ppm,
the compounds. Details of the potentiometric titrations,
calculation of acid-base and stability constants and molecular
modelling studies are also included in the ESI.
δ
scale), using sodium 2,2-dimethyl-2-silapentane-5-sulphonate
as the reference signal. The chemical displacements used to
identify the respective metabolites were consistent with those
Biological activity – in vitro assays
3
5
described previously.
The parasites and mammalian cells used are described in the
electronic supplementary information. The procedures to test
the different compounds against all parasite forms and
mammalian cells individually as well as the infectivity assay,
which represents a reproduction of the life cycle in vitro, are
also provided.
Ultrastructural alterations. The epimastigotes of T. cruzi were
5
cultured at a density of 5 × 10 cells/mL in corresponding
medium containing the compounds tested at their IC25
concentrations. After 72 h, these cultures were centrifuged at
4
00 g for 10 min, and the pellets were washed with PBS and
then mixed with 2% (v/v) paraformaldehyde/glutaraldehyde in
.05 M cacodylate buffer (pH 7.4) for 4 h at 4ºC. Following
this, the pellets were prepared for transmission electron
microscopy (TEM) study using previously described
Biological activity – in vivo assays
Mouse infection and treatment
performed using the rules and principles of the international
guide for biomedical research in experimental animals and with
the approval of the ethical committee of the University of
Granada, Spain. Groups of six BALB/c albino female mice
0
. This experiment was
a
3
6
technique.
Superoxide dismutase (SOD) inhibition studies. The protocol
to obtain the enzyme from the culture and the experimental
measure of the inhibition assays are described in the ESI.
(
dark/light cycle (lights on at 07:30 h) at a temperature of 22 ± 3
6−8 weeks old, 25−30 g weight), maintained under a 12-h
◦
C and provided with water and standard chow ad libitum, were
5
inoculated via the intraperitoneal route with 5 x 10 blood
trypomastigotes of T. cruzi obtained from previously infected
Trypanothione
Recombinant trypanothione reductase from Trypanosoma cruzi
reductase
(TR)
inhibition
studies.
mice blood. The animals were divided as follows:
control group (mice infected but not treated); II, study group
mice infected and treated with the compounds under study).
I, positive
3
TcTR) was prepared following a published procedure. TS2
7
(
3
8
was generated enzymatically as described previously. TR
activity was measured in a total volume of 1 mL of 40 mM
HEPES, 1 mM EDTA, pH 7.5, in the presence of 100 ꢁM
(
The administration of the tested compounds began on the
seventh day of infestation once the infection was confirmed,
and doses of 25 mg/kg body weight per day were administered
for 5 consecutive days (7−12 days post-infection) by the
intraperitoneal route. Peripheral blood was obtained from the
mandibular vein of each mouse (5 ꢁL samples) and dissolved in
NADPH and varying concentrations of TS (20, 40, 80, 100 and
2
2
00 µM) and/or inhibitor (0, 28 and 100 µM). The absorption
decrease due to NADPH oxidation was followed at 340 nm and
5°C. Stock solutions of the inhibitors were prepared in water
according to their solubility.
2
4
95 ꢁL of a PBS solution at a dilution of 1:100. The circulating
The K and K ′ -values were determined using the following
parasite numbers were quantified with a Neubauer chamber for
counting blood cells. This counting was performed every 3 days
for 40 days (acute phase). The number of bloodstream forms
found was expressed as parasites/mL. From this point onwards,
the disease was considered chronic. The assays that follow are
detailed in the ESI, where cyclophosphamide-induced
immunosuppression and the assessment of cure are included.
i
i
3
2
equations:
Toxicity tests by clinical chemistry measurements. A fraction
of the serum, obtained as shown above, was sent to the
Biochemical Service at the University of Granada where a
series of parameters was measured according to commercial
kits acquired from Cromakit® using a BS-200 Chemistry
Analyzer. Shenzhen mindray. Bio-medical electronics co.,
LTD. With the levels obtained for different populations of sera
The ability of the compounds to induce the oxidase activity of
TR was measured in the presence of 100 ꢁM NADPH, with 40
and 100 ꢁM of the compound in a total volume of 1 mL and
about 2 U/ml of TR. Under these conditions, spontaneous
NADPH oxidation results in a minimal absorption decrease.
This activity was not accelerated in the presence of the
compounds, ruling out that they act as subversive substrates
(
n = 15, n = 6), we calculated the mean value and standard
deviation. Finally, we also calculated confidence intervals for
the mean normal populations based on a confidence level of
5% (100 × (1-α) = 100x (1-.05)%). The ranges obtained are
9
shown in Table S4, which allows for the comparison and
analysis of the sera studied in this work.
(
data not shown).
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1
4. B. Verdejo, A. Ferrer, S. Blasco, C.E. Castillo, J. González, J.
Latorre, M. A. Máñez, M. G. Basallote, C. Soriano, E. García-
España, Inorg. Chem. 2007, 46, 5707- 5719.
5. V. Amendola, L. Fabbrizzi, C. Mangano, P. Pallavicini, Acc.
Chem. Res. 2001, 34, 488-493.
Acknowledgements
Financial support for this study was received from the Spanish
Ministry of Economy and Competitiveness (MINECO),
CONSOLIDER-INGENIO
2010
CSD2010-00065
and
Generalitat Valenciana PROMETEO 2011/008. F.O. is grateful
for an FPU Grant from the Ministry of Education of Spain and
he also thanks Natalie Dirdjaja and Dr Alejandro Leroux,
Heidelberg, for their support with the kinetic analysis of TR.
16. (a) C. Marín, M.P. Clares, I. Ramírez-Macías, S. Blasco, F.
Olmo, C. Soriano, B. Verdejo, M.J. Rosales, D. Gómez-
Herrera, E. García-España, M. Sánchez- Moreno, Eur. J. Med.
Chem. 2013, 62, 466-477. (b) F. Olmo, C. Marín, M. P. Clares,
S. Blasco, M. T. Albelda, C. Soriano, R. Gutiérrez-Sánchez, F.
Arrebola-Vargas, E. García-España, M. Sánchez-Moreno, Eur.
J. Med. Chem. 2013, 70, 189-198.
aNotes and references
Departamento de Parasitología, Instituto de Investigación Biosanitaria
ibs. Granada. Universidad de Granada, Granada, Spain. e-mail:
msanchem@ugr.es
Instituto de Ciencia Molecular, Departamento de Química Inorgánica,
Universidad de Valencia, E-46980 Paterna (Valencia), Spain. e-mail:
enrique.garcia-es@uv.es
1
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