Anti-diabetic and anti-parasitic properties of a family of luminescent zinc coordination compounds based on the 7-amino-5-methyl-1,2,4-triazolo[1,5-a]pyrimidine ligand

Article abstract of DOI:10.1016/j.jinorgbio.2020.111235

We report on the formation of a triazolopyrimidine derivative ligand, 7-amino-5-methyl-1,2,4-triazolo[1,5-a]pyrimidine (7-amtp), and a new family of coordination compounds based on this ligand and zinc as metal ion, synthesized by conventional routes. These materials possess different mononuclear structures, namely [ZnCl₂(7-amtp)₂] (1), [Zn(7-amtp)₂(H₂O)₄](NO₃)₂·2(7-amtp)·6H₂O (2) and [Zn(7-amtp)₂(H₂O)₄](SO₄)·1.5H₂O (3) derived from the use of different zinc (II) salts, in such a way that the counterions govern the crystallization to a large extent. These compounds present and show variable luminescent properties based on ligand-centred charge transfers which have been deeply studied by Time Dependent Density Functional Theory (TD-DFT) calculations. When these compounds are transferred to solution, preserving complex entities as corroborated by NMR studies, they present interesting anti-diabetic and anti-parasitic capabilities, with a comparatively higher selectivity index than other previously reported triazolopyrimidine-based materials. The results derived from in vivo experiments conducted in mice also confirm their promising activity as anti-diabetic drug being capable of dropping glucose levels after oral administration. Therefore, these new materials may be considered as excellent candidates to be further investigated in the field of luminescent coordination compounds with biomedical applications.

Full text of DOI:10.1016/j.jinorgbio.2020.111235

JournalofInorganicBiochemistry212(2020)111235
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Anti-diabetic and anti-parasitic properties of a family of luminescent zinc
coordination compounds based on the 7-amino-5-methyl-1,2,4-triazolo[1,5-
a]pyrimidine ligand
⁎
Ginés M. Esteban-Parraa, Eider San Sebastiánb, Javier Cepedab, Cristina Sánchez-Gonzálezc, ,
⁎
Lorenzo Rivas-Garcíac, Juan Llopisc, Pilar Arandac, Manuel Sánchez-Morenod, Miguel Quirósa, ,
⁎
Antonio Rodríguez-Diégueza,
a Dept. of Inorganic Chemistry, University of Granada, C/Severo Ochoa s/n, 18071 Granada, Spain
b Departmento de Quimica Aplicada, Facultad de Química, University of The Basque Country (UPV/EHU), 20018 San Sebastian, Spain
c Dept. of Physiology, University of Granada, Cartuja Campus, 18071 Granada, Spain
d Dept. of Parasitology, University of Granada, Avda. Severo Ochoa s/n, 18071 Granada, Spain
A R T I C L E I N F O
A B S T R A C T
Keywords:
We report on the formation of a triazolopyrimidine derivative ligand, 7-amino-5-methyl-1,2,4-triazolo[1,5-a]
pyrimidine (7-amtp), and a new family of coordination compounds based on this ligand and zinc as metal ion,
synthesized by conventional routes. These materials possess different mononuclear structures, namely [ZnCl2(7-
amtp)2] (1), [Zn(7-amtp)2(H2O)4](NO3)2·2(7-amtp)·6H2O (2) and [Zn(7-amtp)2(H2O)4](SO4)·1.5H2O (3) derived
from the use of different zinc (II) salts, in such a way that the counterions govern the crystallization to a large
extent. These compounds present and show variable luminescent properties based on ligand-centred charge
transfers which have been deeply studied by Time Dependent Density Functional Theory (TD-DFT) calculations.
When these compounds are transferred to solution, preserving complex entities as corroborated by NMR studies,
they present interesting anti-diabetic and anti-parasitic capabilities, with a comparatively higher selectivity
index than other previously reported triazolopyrimidine-based materials. The results derived from in vivo ex-
periments conducted in mice also confirm their promising activity as anti-diabetic drug being capable of
dropping glucose levels after oral administration. Therefore, these new materials may be considered as excellent
candidates to be further investigated in the field of luminescent coordination compounds with biomedical ap-
plications.
Zinc
7-Amino-5-methyl-1,2,4-triazolo[1,5-a]
pyrimidine ligand
Luminescence
Diabetes
Lesihmaniasis
Chagas
1. Introduction
drugs that can be administered orally for the treatment of diabetes, most
of them based on organic compounds [3–5]. As an alternative to this type
Diabetes mellitus (DM) is a group of metabolic disorders which raises
blood sugar levels over a prolonged period. This disease generates a huge
social and economic problem to the world society since DM has become
one of the most relevant health issues in the first world countries, with
422 million adults affected all over the world, and more than a half of
them living in these countries [1]. For this reason, a great quantity of
drugs are used for the treatment of diabetes to control plasma glucose
levels and achieve insulin-mimetic effects, in which each class of drug has
different mechanisms of action. Unfortunately, common anti-diabetic
drugs are associated with several adverse effects, so that the search for
new drugs that can fight this disease effectively with minimal side effects
is still an ongoing research field [2]. Recent studies aim to develop new
of materials, coordination compounds have shown promising activity as
hypoglycaemic agents for the pharmacotherapy of diabetes [6–7]. Among
them, compounds containing Zn (Zn2+) have been investigated on recent
studies using animal models. In particular, some clinical reports strongly
support the thought that Zn2+ deficiency occurs in diabetic conditions,
suggesting that Zn2+ supplementation will benefit or correct the diabetes-
induced Zn2+ status [8–9]. This cation plays essential structural roles in
many proteins and enzymes but also has shown insulin enhancing activity
in vivo [10]. Considering the different families of Zn2+ compounds syn-
thesized and found in literature, it may be envisaged that mononuclear
Zn2+ coordination compounds could present interesting anti-diabetic
properties [11–25].
⁎ Corresponding authors.
E-mail addresses: crissg@ugr.es (C. Sánchez-González), mquiros@ugr.es (M. Quirós), antonio5@ugr.es (A. Rodríguez-Diéguez).
https://doi.org/10.1016/j.jinorgbio.2020.111235
Received 27 April 2020; Received in revised form 7 August 2020; Accepted 14 August 2020
Availableonline23August2020
0162-0134/©2020PublishedbyElsevierInc.

G.M. Esteban-Parra, et al.
JournalofInorganicBiochemistry212(2020)111235
As expected, a delicate rational design of the organic ligand to be
coordinated to the central cation plays a key role. In this line, several
ligands containing N atoms have demonstrated to be excellent and
versatile building blocks that, with the appropriate charge and multi-
connectivity patterns, produce, under conventional routes, multi-
dimensional coordination compounds with fascinating physical prop-
erties. In this respect, we previously reported the synthesis, structural
characterization and anti-diabetic evaluation of multidimensional co-
ordination compounds composed of this kind of ligands [26] and zinc
and vanadium ions. Of particular interest is the fact that the meta-
l–organic hybrid nature of these materials offers potentially limitless
arrangement types and topological architectures [27–28], reinforcing
their versatility of use.
new family of Zn2+ coordination complexes based on the novel tria-
zolopyrimidine derivative 7-amtp ligand, [ZnCl2(7-amtp)2] (1), [Zn(7-
amtp)2(H2O)4](NO3)2·2(7-amtp)·6H2O (2) and [Zn(7-amtp)2(H2O)4]
(SO4)·1.5H2O (3). 1–3 compounds are mononuclear entities in which 7-
amtp coordinates via N3 atom (see Scheme I for atom numbering) to
zinc ions. Luminescent measurements of compounds have been per-
formed along in vitro anti-parasitic activities and in vivo anti-diabetic
properties have been studied in the diabetic murine model STZ-CD1,
constituting, to the best of our knowledge, the first report on Zn-based
compounds as glucose lowering agents.
2. Results and discussion
In the last ten years, we and others have reported the use of different
triazolopyrimidine derivative ligands with specific antiparasitic activ-
ities, with a particular focus on the behaviour of the adenine-derivative
7-amino-1,2,4-triazolo[1,5-a]pyrimidine ligand (7-atp) [29–37]. Based
on these previous results, a new amine derivative triazolopyrimidine
ligand (7-amino-5-methyl[1,2,4]triazolo[1,5-a]pyrimidine; 7-amtp
hereafter) is designed and synthesized in this work (Scheme I). Ad-
ditionally, three coordination compounds based on 7-amtp ligand and
Zn2+ are synthesized and characterized, and their anti-diabetic activity
evaluated. In this line, it is worth it to mention that the antidiabetic
properties of trizolopyrimidine-derivative ligands have been previously
reported [38–42] but, to the best of our knowledge, this is the first time
where a potential anti-diabetic compound merges the biological activity
of this type of ligands with the insulin enhancing activity of the Zn2+
cation. (See Scheme II.)
2.1. Description of the structures
2.1.1. (7-amtp)·(H2O)
The free 7-amtp ligand crystallizes in the orthorhombic Pbca space
group with two crystallographically independent molecules in the
asymmetric unit, which also contains two crystallization water mole-
cules, one of them disordered between two positions. The crystal ar-
chitecture is mainly built by hydrogen bonds with water molecules
interacting between them (O…O distance, 2.648(3)/2.756(3) Å) and
acting as donor towards N3 and N4 atoms of the organic moiety (O…N
distances, 2.827(3)/2.850(3), 2.880(2) and 2.820(2) Å, see Fig. 1). The
amino groups also act as H-bond donors towards one of the water
molecules (N…O distance, 2.746(3)/2.784(3) Å) and to N1 and N3
atoms of neighbouring heterocycles (N…N distances, 2.876(2) and
3.009(2) Å). The planar aromatic moieties stack along the b axis (dis-
tance ~3.35 Å), each stack being built by symmetry equivalent mole-
cules, related by the b glide plane perpendicular to the a axis (Fig. S20).
Viewed from this direction, the two stacks alternate in a zig-zag-like
fashion along the c axis.
Equally important is the fact that, due to its extended aromaticity
and to the presence of several heteroatoms in the ring, this ligand
should be a good candidate to show luminescent properties, which
might be enhanced when coordinated to Zn2+ ions [43–45]. In this
sense, coordination compounds containing metal ions with closed shell
configuration, such as d10 metals (Zn2+ and Cd2+) have attracted ex-
tensive interest in recent decades, given their ability to render fasci-
nating structures [46–51]. This fact is a consequence of the absence of
ligand field constraints, associated with the d10 configuration of these
ions, which provides flexible coordination environments that can be
adapted to a wide variety of geometries. Hence, they allow fine tuning
of the structures and/or topologies. Moreover, the closed-shell config-
uration also possesses some additional advantages regarding the pho-
toluminescence (PL) properties [52–53], since the absence of potential
quenching processes derived from d–d transitions permits efficient lu-
minescent emission. This fact should allow us to develop multi-
functional materials with interesting luminescent and biological prop-
erties.
2.1.2. [ZnCl2(7-amtp)2] (1)
Compound 1 crystallizes in monoclinic P21/c space group, the
asymmetric unit being built by just one tetrahedral coordination entity
(Fig. 2). Bond lengths and angles are listed in the supplementary ma-
terial (Table S1). The tetrahedral coordination polyhedron involves two
chloride ions and two 7-amtp ligands coordinated to the metallic cation
through N3 nitrogen atom of the ring, which is the most usual co-
ordination mode of these kind of ligands. The corresponding bond
distances are 2.2395(5) and 2.2934(5) Å (ZneCl) and 2.015(2) and
2.021(2) Å (ZneN3). These units join to one another through hydrogen
bonding interactions between chlorine atoms of one complex and
amino groups of 7-amtp from neighbouring units (N…Cl distances.
3.344(2), 3.233(2) and 3.280(2) Å). The complexes are associated in
centrosymmetric couples via by π-stacking interaction (Fig. S21).
Therefore, we report herein the synthesis and characterization of a
Scheme I. Chemical structure of adenine, as well as 7-atp and 7-amtp ligands. Biochemical and IUPAC ring-numbering system has been employed for purine and
1,2,4-triazolo[1,5-a]pyrimidine ligands, respectively.
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G.M. Esteban-Parra, et al.
JournalofInorganicBiochemistry212(2020)111235
Scheme II. Synthesis procedure for 7-amtp.
2.1.4. [Zn(7-amtp)2(H2O)4](SO4)·1.5H2O (3)
2.1.3. [Zn(7-amtp)2(H2O)4](NO3)2·2(7-amtp)·6 H2O (2)
Compound 2 crystallizes in the triclinic P-1 space group. The moi-
eties present in the structure are shown in Fig. 3. Selected bond lengths
and angles are given in Table S2. The coordination entity of this com-
pound consists of a zinc cation located on an inversion centre, dis-
playing an octahedral coordination sphere with four water molecules in
the equatorial plane and two 7-amtp ligands coordinated through N3
nitrogen atoms occupying the positions of the reference axis; the N4
atom of this ligand accepts an H-bond from one of the coordinated
water molecules (O1W…N4A distance, 2.713(2) Å) closing a 6-member
pseudo-chelating ring. The bond distances in zinc coordination sphere
are 2.1471(14) Å for the organic ligand and 2.0650(13) and 2.1830(13)
Å for the water molecules. Another molecule of 7-amtp is present in the
second coordination sphere, interacting with that linked to the metal by
π stacking, with alternating coordinated and non-coordinated 7-amtp
moieties stacked along the b axis. The asymmetric unit is completed by
one nitrate ion that counterbalances the positive charge of zinc and
three non-coordinated water molecules. The later are assembled in
centrosymmetric groups of six molecules, with a R4(1) ring and two
pending molecules (Fig. S22), which are further linked to the co-
ordinated water molecules and to the anions. Likewise, the amino
groups of both ligands also act as H-bond donors towards the groups of
six interstitial water molecules and towards the N1 atom of the non-
coordinated 7-amtp.
Compound 3 crystallizes in triclinic P-1 space group. There are two
crystallographically independent but chemically equivalent [Zn(7-
amtp)2(H2O)4]2+ cations, both placed on inversion centres and dis-
playing very similar fragments to that described for compound 2: a zinc
cation coordinated to two N3-coordinated 7-amtp ligands in the re-
ference axis and four water molecules coordinated in the plane per-
pendicular to the reference axis. One of the cations is disordered be-
tween two positions, one of them is rotated ~30° around the NeZneN
axis from the other, the disorder thus affecting the coordinated water
molecules. A sulphate anion balancing the charge and two crystal-
lization water molecules (one of them with half occupancy close to an
inversion centre) complete the structure, a view of the moieties being
depicted in Fig. 4. Bond distances in the coordination entity range from
2.08 to 2.15 Å for ZneO bonds whereas ZneN distances are 2.145(2)
and 2.208(2) Å. The supramolecular structure is governed by hydrogen
bond interactions, with a complex network further complicated by the
disorder in both coordinated and non-coordinated water molecules. The
disordered complex shows a pseudo-chelating ring analogous to that
described for compound 2 whereas, for the other one, the interaction is
indirect through a non-coordinated water molecule (coordinated H2O…
non-coordinated H2O…N4). A noteworthy interaction is that taking
place between 7-amtp moieties related by inversion centres and invol-
ving two NH2…N1 H-bonds (N…N distances, 3.022(3) Å for ligand A
Fig. 1. Fragment of the crystal structure of 7-amtp ligand showing most relevant hydrogen bonding interactions. Colour code: carbon, grey; hydrogen, white;
nitrogen, blue; oxygen, red. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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G.M. Esteban-Parra, et al.
JournalofInorganicBiochemistry212(2020)111235
Fig. 2. Perspective view of mononuclear coordination compound 1. Hydrogen atoms have been omitted for clarity. Colour code: Zinc, light steel blue; nitrogen, blue;
carbon, grey; chlorine, green. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. Perspective view of compound 2. Hydrogen atoms have been omitted for clarity. Colour code: zinc, light steel blue; oxygen, red; nitrogen, blue; carbon, grey.
(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
and 2.951(3) Å for ligand B) this interaction links the complex cations
forming rows in the [001] and [011] directions. The anions are also
involved in the H-bond network, acting as acceptors for water mole-
cules and amino groups. All organic moieties are roughly perpendicular
to the 110 direction but no π-stacking interactions are clearly defined.
signals, apart from the one corresponding to the methanol
(δMeOD 47.38 ppm, which acts again as internal patron:
=
δCH₃ = 22.97 ppm, δC6 = 90.32, δC7 = 149.32, δC3a = 153.58,
δ
C2 = 155.26 and δC5 = 164.49 ppm.
The results, which only show the expected signals, confirm the
purity of the samples.
2.2. NMR studies
2.3. Stability studies
In order to determine the purity of the synthesized ligand and
whether the crystallized complexes are representative for the sample in
solution, 1H and 13C NMR studies were carried. The results of these
assays are shown in the figures below.
To determine the stability of the complexes, 1H NMR spectra (Figs.
S12–S19) have been recorded for free 7-amtp and the tree zinc com-
plexes using the culture medium of the parasites and 10% D2O as sol-
vent to check the stability of the compounds in solution. All spectra
have been recorded twice: i) just after the preparation of the solutions
and ii) after 72 h, which corresponds to the longest time that the
compounds remain in the solution for biological studies.
1
Fig. 5 shows the H NMR spectrum for the 7-amtp ligand, carried
out in deuterated methanol. Three signals, apart from the two corre-
sponding to the solvent (δCHD₂ = 3.32 ppm y δOH = 4.86 ppm) that acts
as internal patron, are shown. The signals correspond to the methyl
group (δCH₃ = 2.49 ppm), C6 carbon (δC6 = 6.28 ppm) and C2 carbon
(δC2 = 8.29 ppm). The amino group signal does not appear because its
protons are involved in hydrogen bonding interactions and switch with
the solvent.
The free ligand spectra show the expected three signals corre-
sponding to the methyl group (δCH₃ = 2.32 ppm), C6 carbon
(δC6 = 6.17 ppm) and C2 carbon (δC2 = 8.21 ppm) and the signal of the
solvent (δ = 4.70 ppm) that acts as internal patron. The spectrum re-
mains practically identical after 72 h.
The 13C NMR spectrum is shown in Fig. 6. It shows six different
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G.M. Esteban-Parra, et al.
JournalofInorganicBiochemistry212(2020)111235
Fig. 4. Perspective view of compound 3. Hydrogen atoms have been omitted for clarity. Colour code: zinc, light steel blue; oxygen, red; nitrogen, blue; carbon, grey;
sulphur, yellow. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
In the case of compound 1, the three peaks are slightly shifted with
respect to the free ligand (δCH₃ 2.38 ppm), C6 carbon
(δC6 = 6.29 ppm) and the C2 carbon signal is now divided in two
signals (δC2 = 8.25 and 8.37 ppm), and remains unaltered after 72 h.
Regarding compounds 2 and 3, both compounds present very similar
spectra, that are practically identical to the free 7-amtp, except for the
=
Fig. 5. 1H NMR spectrum for 7-amtp.
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G.M. Esteban-Parra, et al.
JournalofInorganicBiochemistry212(2020)111235
Fig. 6. 13C NMR spectrum for 7-amtp.
presence of a new peak centred at 7.32 ppm in compound 2 and
7.36 ppm in compound 3. As in the other cases, both spectra remain
practically unaltered after 72 h in solution.
performance of these compounds to evaluate their potential applica-
tions on biomedicine. The emission spectra of 7-amtp and 1–3 at room
temperature are shown in Fig. 7. Free 7-amtp ligand shows an intense
emission band, centred at about 418 nm, and a much weaker band at
around 720 nm, upon excitation at λ = 308 nm. The emission spectrum
of mononuclear compound 1 shows a weak displacement in the first
band, centred at 406 nm, and also reveals a new and less intense band
centred at ca. 540 nm. Interestingly, the PL behaviour of compounds 2
and 3 (though similar to each other) differ substantially from that of 1.
In this line, the intensity of the more energetic band, centred now at ca.
410 nm, drops substantially and the less energetic band is now equally
significant. The latter band, centred at ca. 480 nm and 490 nm re-
spectively for 2 and 3, is therefore significantly blue-shifted with re-
spect to the equivalent signal of compound by 1.
Those results suggest that, even if there is a dissociation equilibrium
in aqueous solution, all compounds remain, at least, their complex
identity (which makes them suitable for biological assays in that kind of
media.
2.4. Photoluminescence studies
Solid state photoluminescence measurements were performed on
polycrystalline samples of compounds 1–3 and free 7-amtp ligand with
the aim of getting a representative characterization of the emissive
The PL emission properties of these type of complexes is commonly
assigned to intraligand π ← π* electronic transitions. This may be
checked by means of Time Dependent Density Functional Theory (TD-
DFT) calculations. TD-DFT calculated emission spectrum of the free 7-
amtp and the graphical representation of the molecular orbitals (MO)
involved in the emission process are shown in Fig. 8, where the main
band computed at 403 nm arises from electronic relaxation processes
occurring between two sets of molecular orbitals of mixed-nature and
centred all over the ligand scaffold. In this sense, the large green ver-
tical lines at 401 and 424 nm, together with the vibrationally related
and signal overlapping shorter green vertical lines, represent the energy
of the most relevant electronic transitions occurring between the first
computed excited singlet-state S1 and the ground S0 state. Represented
MOs indicate that the promoted electron finally drops from the lowest
unoccupied molecular orbital (LUMO) to both the highest occupied
molecular orbital (HOMO) (424 nm) as well as to the HOMO-1
(401 nm) Additional data on the computed electronic transitions are
collected in Table 1. Interestingly, as observed in the optimized S1
singlet state excited geometry of 7ampt represented in Fig. 8, an evident
Fig. 7. Room temperature solid state emission spectra of 7-amtp (solid line),
compound 1 (dashed line), 2 (circles) and 3 (crosses) upon sample excitation at
λ
ex = 308 nm.
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G.M. Esteban-Parra, et al.
JournalofInorganicBiochemistry212(2020)111235
orbital). Electrons reach the latter orbital upon undergoing an emissive
relaxation from the 7-amtp-ligand centred LUMO +3 and LUMO +4
molecular orbitals, respectively. Both MOs are involved in the two
mayor contributions identified in this region. In this sense, the H-1 ←
L + 3 transition is responsible for the 532 nm line, whereas the 589 nm
line arises from an H-1 ← L + 4 electronic relaxation process.
Computed emission spectrum of 2 (Fig. 9c), again in very good
agreement with the experimental data, revealed a single and rather
wide maximum centred at ca. 453 nm, followed by a slowly decaying
shoulder centred in ca. 489 nm. Both signals arise from two mayor
transitions at 479 nm and 509 nm (green and blue vertical large lines,
see Table 1), respectively, as well as from multiple vibrationally related
less significant transitions (shorter green and blue vertical lines). As
indicated in the diagram depicted Fig. 9d), the observed PL behaviour is
understood on the basis of a complex inter-ligand electronic relaxation
process occurring between the LUMO +21 and the HOMO; on the
contrary, the mentioned shoulder at ca. 489 nm is actually derived from
a major electronic transition occurring at 509 nm (large blue vertical
line in Fig. 9c) as well as the related vibrational levels (shorter blue
vertical lines). These electronic transitions have a complex origin, since
both metal and ligand contributions are observed in the LUMO +23
identified as the one releasing the electrons responsible for the men-
tioned emission shoulder.
Fig. 8. Experimental (solid line) and TD-DFT computed (dashed line) emission
spectrum of 7-amtp. Green vertical lines identify the computed main transitions
responsible for the corresponding maximum (see Table 1). (For interpretation
of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
2.5. Anti-parasitic assays
Table 1
Major electronic transitions computed to be responsible for the emission spectra
7-amtp derivative and three obtained zinc (II) complexes were as-
sayed against extracellular forms of L. infantum, L. braziliensis and T.
cruzi. Additionally, cytotoxicity studies were carried out over macro-
phages and Vero cells, which are host cells for Leishmania spp. and T.
cruzi, respectively. The results of in vitro assays are shown in Table 2.
The 7-amtp derivative shows a quite similar antiproliferative ac-
tivity compared to the observed for all previously studied triazolopyr-
imidine derivatives [54], with SI that are a little better than the values
for the reference drugs. On the other hand, we can see that all com-
pounds show a remarkable activity against all parasites, especially
compound 1 against L. braziliensis and compound 2 against T. cruzi,
whose IC50 values are below the lowest concentration studied. Ad-
ditionally, all compounds show a cytotoxicity that is a magnitude order
higher than reference drugs. This fact united to the great anti-
proliferative activity results on high selectivity indexes, which are 6
times better than reference drugs in the less effective situations and
more than three hundred in the best assays. Additionally, all com-
pounds present SI values that are rather higher than the obtained for
similar zinc compound previously obtained [55,56]. These data show
that all compounds and especially compound 1 are promising prodrugs
to continue studying them in further in vitro and in vivo assays to
conclude their potential use as antiparasitic therapeutic agents.
of 7-amtp, 1 and 2.
Emiss. (nm)
Main trans.
Symm.
Osc. st
7-amtp
401
424
386
448
532
589
479
509
H-1 ← L (90%)
Singlet
Singlet
Singlet
Singlet
Singlet
Singlet
Singlet
Singlet
0.0634
0.0579
0.0012
0.0011
0.0065
0.0051
0.0452
0.0419
H ← L (95%)
1
H-1 ← L (91%)
H-1 ← L (90%)
H-1 ← L + 4 (90%)
H-1 ← L + 3 (90%)
H ← L + 23 (90%)
H ← L + 21 (95%)
2
out-of-plane twist of the amine substituent is forced upon electron ab-
sorption and subsequent vibrational relaxation of the ligand, yielding
an S1 excited state where the N atom of the amine substituent is posi-
tioned up to 76° out of the plane generated by the aromatic portion of
the ligand. Notwithstanding the fact that the use of no geometrical
constrains in the emission calculations may permit larger conforma-
tional adjustments in the molecules than the ones permitted in a closely
packed crystal of 7ampt, the mentioned twist might still occur in the
sample, thought its extent is difficult to measure.
The same type of calculations carried out on 1 yielded the compu-
tational emission spectrum shown in Fig. 9a (dashed line), which is,
again, in very good agreement with the experimental data.
2.6. In vivo assays
Characterized by one wide band centred at ca. 381 nm, and a
weaker one at ca. 566 nm, both computed bands actually result from
electron relaxation processes occurring from two distinct but en-
ergetically low-lying singlet excited states, S1 and S2. The former ex-
cited state geometry reveals a significant out-of-plane twist of the amine
group in 7ampt, resembling S1 in the free 7ampt ligand, whereas the
latter (S2) does not. As derived from the diagrams shown in Fig. 9b, the
more energetic emission can be described as a ligand-to-ligand charge
transfer (LLCT) process happening in the thermally relaxed S1 species,
and can be ascribed to an electron decay from a 7-amtp ligand centred
large LUMO orbital to the as well 7-amtp ligand centred HOMO-1
molecular orbital. The less energetic emission band, also a LLCT pho-
toluminescent process, differs substantially from the one just described,
as the HOMO-1 molecular orbital receiving the relaxing electron was
computed to be centred in the chloride ligand (presumably 3p atomic
The essays were performed in STZ-CD1 mice with severe hy-
perglycaemia induced as indicated in the experimental section. The test
of the oral glucose tolerance (Fig. 10) showed different types of hy-
poglycaemic effects exerted by the above characterized Zn compounds.
Compound 1 did not avoid the increase of the glycaemic peak 30 min
after glucose oral administration, but reduced significantly glycaemia
after 60 min in comparison with untreated diabetic mice, and achieved
the normalization of blood glucose levels, approaching the glucose le-
vels to that found in healthy mice at the end of the test. Compound 2
yielded the best anti-diabetic properties of the three compounds, be-
cause mice treated with compound 2 maintained the lower blood glu-
cose levels during all the test. 30 min after glucose oral administration
mice treated with compound 2 showed glucose levels statistically equal
to healthy mice. On the other hand, compound 3 exhibited a blood
glucose lowering effect consisting in preventing the glycaemic peak
7

G.M. Esteban-Parra, et al.
JournalofInorganicBiochemistry212(2020)111235
Fig. 9. Experimental (solid line) and computed (dashed line) emission spectra of compound 1 (a) and 2 (c). Main transitions in the computed spectrum are identified
by large vertical green and blue lines, respectively, whereas vibrationally related transitions are depicted by shorted lines; (b) Graphical representation of the MOs in
S1 and S2 of compound 1 (b) and 2 (d) involved in the main electronic transitions. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
caused by the oral glucose load. However, unlike compounds 1 and 2,
compound 3 failed to normalize glucose levels and bring them closer to
the levels found in healthy mice. None of those effects is observed for
the free ligand that, far from exerting an anti-diabetic effect, exerted a
hyperglycaemic effect from the beginning until the end of the test.
The results obtained in vivo in the present study show that the
tested compounds display interesting potential as anti-diabetic drugs
[57]. Compound 2 shows an interesting effect 30 min after oral glucose
overload and manages to reach blood glucose levels of healthy mice,
but does not avoid a glycaemic peak of 37% of the baseline at 15 min.
Meanwhile, compound 3 fails to normalize blood glucose at the end of
the test, but prevents the trigger effect allowing an increase in gly-
caemia of only 13%. The synergistic effect of the simultaneous com-
bined administration of the two compounds could cover the two tar-
gets: avoid the postprandial glycaemic peak and normalize glucose
levels to those of the state of health. Further studies will be needed to
better understand the mechanisms underlying the effects of the syn-
thetized compounds.
Table 2
In vitro activity of 7-amtp and zinc (II) complexes and reference drugs against promastigote forms of Leishmania spp., epimastigote forms of Trypanosoma cruzi, J774.2
macrophages and Vero cells after 72 h of incubation at 37 °C.
Compound
IC50
SD (μM)a
SIb
L. inf.
L. brazi.
T. cruzi
Macrophages J774.2
Vero cells
–
L. inf.
L. brazi.
T. cruzi
Glucantime®
18.0
–
0.6
2.6
25.6
–
1.6
1.1
–
15.2
–
1.3
0.8
0.6
–
Benznidazole®
24.2
35.6
8.4
1.9
2.8
13.6
0.9
–
–
0.9
7-amtp
32.9
9.0
13.4
< 1
7.5
5.3
147.9
218.1
144.7
179.3
14.0
394.3
288.5
302.6
406.6
31.5
23.1
24.2
32.5
5.3 (7)
24.3 (30)
5.0 (6)
8.9 (11)
13.1 (22)
> 218.1 (363)
19.4 (32)
33.8 (56)
11.1 (12)
34.3 (38)
> 302.6 (336)
39.1 (43)
1
2
3
0.7
0.7
17.4
11.6
14.3
29.1
20.1
2.3
1.6
0.6
0.4
< 1
10.4
0.8
The results presented are averages of three separate determinations.
a
The concentration required to obtain 50% inhibition, calculated through a linear regression analysis from the Kc values at the concentration employed (1, 10, 25
and 100 μM for promastigote and epimastigote forms of Leishmania spp. and Trypanosoma cruzi respectively and 50, 100, 200 and 400 μM for host cells).
b
Selectivity index (SI) = IC50 against J774.2 macrophages or Vero cells / IC50 parasite (promastigote or epimastigote forms respectively).
8

G.M. Esteban-Parra, et al.
JournalofInorganicBiochemistry212(2020)111235
Fig. 10. Oral glucose tolerance test for control (C), diabetic untreated (D), and diabetic mice treated with the ligand (DL) or Zn compound 1 (DC1), 2 (DC2) or 3
(DC3). Data are presented as mean SD. p < 0.05.
To the best of our knowledge, this is the first time that Zn-based
compounds behaving as glucose lowering agents have been tested in
diabetic murine model STZ-CD1, proving that it is an adequate model to
validate the effectiveness of new designed drugs. This murine model of
low size and body weight presents the advantage of requiring the
synthesis of a lower amount of drugs and responds in a sensitive way to
the administration of drugs related to hydrocarbon metabolism, this
being more versatile that the use of Wistar rats [58–62].
proposed mechanisms of action are summarized. Zn compounds have
been shown to increase the activity of glycolytic enzymes such as
phosphofructokinase (PFK) and pyruvate kinase (PK). Furthermore, in
zinc-treated cells a progressive activation of ERK2/MAPK1 (Mitogen-
activated protein kinase-1) was observed. Zinc is reversible inhibitor of
α-glucosidase activity in the gut. In skeletal muscles Zinc-α2-glyco-
protein promotes the phosphorylation of AMP-activated protein kinase
(AMPKα) and increases cellular glucose transporter type 4 (GLUT4)
protein. Zinc stimulate the phosphorylation of the IR-β (insulin re-
ceptor) subunit, and inhibits glycogen synthase kinase-3 (GSK-3β),
which is a phosphorylating and an inactivating agent of glycogen syn-
thase, with resultant increase in glycogen synthesis. Zinc-α2-glycopro-
teins increase the expression of the lipolytic enzymes, adipose trigly-
ceride lipase and hormone-sensitive lipase in the white adipose tissue
[63].
2.7. Comparative studies with previous zinc (II) complexes containing 7-atp
Due to the great similarity between 7-atp and 7-amtp, we decided to
compare synthesized compounds with the previously reported com-
plexes of zinc (II) containing 7-atp. To our knowledge, three complexes
had been previously obtained with the mentioned ligand and ion,
containing the auxiliary ligands bipyrimidine (bpym), malonate (mal)
and 1,3-propanediamine (tn): [Zn2(μ-7-atp)2(μ-mal)2(H2O)2] [31], {[Zn
(μ-7-atp)(tn)](ClO4)}n [36], [Zn2(7-atp)4(μ-bpym)(H2O)4](ClO4)4·2(7-
atp) [56].
3. Conclusion
In summary, the novel 7-amino-5-methyl-1,2,4-triazolo[1,5-a]pyr-
imidine ligand and new family of zinc coordination compounds have
been synthesized and characterized by single crystal X-ray diffraction:
[ZnCl2(7-amtp)2] (1), [Zn(7-amtp)2(H2O)4]·(NO3)2·(7-amtp)2·6H2O (2)
and [Zn(7-amtp)2(H2O)4]SO42·1,54H2O (3). They are mononuclear
compounds in which 7-amine-5-methyl-1,2,4-triazolo[1,5-a]pyrimidine
coordinates by N3 atom to zinc ions. Solid state photoluminescence
spectra of compounds 1–3 showed the relevance of the excellent lu-
minescence afforded by the 7-amtp ligand, which is modulated ac-
cording to the conformation acquired in the complex. An exhaustive
computational study with TD-DFT theory, permits identifying the mo-
lecular orbitals (with high ligand contribution) involved in the excita-
tion and emission, concluding that both first- (S1) and second (S2) ex-
cited states take part in the luminescence process. In particular, under
excitation with ultraviolet light, all compounds share the occurrence of
two bands with blue and green emission, the first of which arises from
an intraligand charge transfer whereas the second one has a more
complex origin involving a ligand-to-metal charge transfer.
Regarding the structure, the three complexes presented in this work
are mononuclear and discrete entities, with tetrahedral and octahedral
structures, for compounds (1) in the first case and (2) and (3) for the
second one. However, the compounds with 7-atp containing bipyr-
imidine and malonate [31,56] are both dinuclear entities, and the one
that contains 1,3-propanediamine [36] forms chains. In fact, only the
compound with bpym shows the same coordination way for the ligand,
it is monodentate through N3, whereas the others show a bridge mode,
through N3 and N4 in the case of the compound with malonate, and
through N3 and through N2, N4 and the amino nitrogen in the chain-
forming complex. However, the luminescence properties observed in
the three compounds, however, are quite similar to the observed for the
two compounds that were analysed in that way [31,36].
Zinc compounds have shown effectiveness in the treatment of type 1
and type 2 diabetes by acting at different levels: for their antioxidant
properties and acting on numerous intracellular enzymes involved in
lipid and glucidic metabolism. Among others, some of the main
9

G.M. Esteban-Parra, et al.
JournalofInorganicBiochemistry212(2020)111235
These materials also possess interesting anti-parasitic and anti-dia-
betic capabilities. On the one hand, all compounds show a remarkable
activity against all parasites, especially compound 1 against L. brazi-
liensis and compound 2 against T. cruzi, whose IC50 values are below the
lowest concentration studied. When dissolved in the culture medium of
the parasites, the complexes are preserved (though compounds 2 and 3
are partially dissociated) as confirmed by NMR experiments. This fact
makes the compounds very suitable for further antiparasitic studies,
both in vitro and in vivo, to determine their applicability as drugs. On
the other hand, compounds display interesting potential as anti-diabetic
drugs due to compound 2 shows an interesting effect 30 min after oral
glucose overload and manages to reach blood glucose levels of healthy
mice, meanwhile compound 3 fails to normalize blood glucose at the
end of the test, but prevents the trigger effect allowing an increase in
glycaemia of only 13%. Moreover, to the best of our knowledge, this is
the first time that Zn-based compounds behaving as glucose lowering
agents have been tested in diabetic murine model STZ-CD1, proving
that it is an adequate model to validate the effectiveness of new de-
signed drugs. With all of the above in mind, we have demonstrated the
capacity of this new ligand to form coordination compounds, making of
them excellent candidates to be further investigated as luminescent
probes with biomedical potential applications.
4.3. Synthesis of [ZnCl2(7-amtp)2] (1)
A solution of 2 mmol (0.330 g) of 7-amtp in 15 mL of water was
prepared with stirring and soft warming. Once the ligand was totally
dissolved, a solution of ZnCl2 (2 mmol, 0.389 g) was added. The re-
sulting solution was left at room temperature. After 48 h, yellow pris-
matic crystals suitable for XRD measurements appeared and were col-
lected by vacuum filtration. Yield: 84%, based on Zn. Anal. calcd. for
C
12H14N10Cl2Zn: C, 33.16; H, 3.25; N, 32.23. Found: C, 33.12; H, 3.08;
N, 32.30%. IR: 1498 and 1597 cm−1 (ʋN-H(flex)), 1562 cm−1 (ʋpy),
1645 cm−1 (ʋtp), 3350 and 3457 cm−1 (ʋN-H(tens)).
4.4. Synthesis of [Zn(7-amtp)2(H2O)4](NO3)2·2(7-amtp)·6H2O (2)
A solution of 2 mmol (0.300 g) of 7-amtp in 15 mL of water was
prepared as previously described. Then, a solution of Zn(NO3)2
(2 mmol, 0.415 g) in the same solvent was added to the ligand solution.
After 24 h at room temperature, pale yellow prismatic crystals suitable
for XRD measurements appeared and were collected by vacuum filtra-
tion. Yield: 84%, based on Zn. Anal. calcd. for C24H48N22O16Zn: C,
29.84; H, 5.01; N, 31.89. Found: C, 29.89H, 4.92; N, 31.81%. IR:
1298 cm−1 (ʋNO₃), 1504 and 1605 cm−1 (ʋN-H(flex)), 1579 cm−1 (ʋpy
and 1639 cm−1 (ʋtp).
)
4. Experimental section
4.5. Synthesis of [Zn(7-amtp)2(H2O)4]SO4·1,5H2O (3)
4.1. Materials and physical measurements
A solution of ZnSO4 (2 mmol, 0.321 g) in water was added over a
solution of 2 mmol (0.296 g) of 7-amtp in 15 mL of the same solvent.
The solution was left at room temperature during 72 h. Finally, light
yellow prismatic crystals suitable for XRD measurements appeared and
were collected by vacuum filtration. Yield: 82%, based on Zn. Anal.
Calcd. for C12H25N10O9.5SZn: C, 25.79; H, 4.51; N, 25.06. Found: C,
25.98; H, 4.72; N, 25.12%. IR: 1090 cm−1 (ʋSO₄), 1495 and 1591 cm−1
(ʋN-H(flex)), 1583 cm−1 (ʋpy) and 1653 cm−1 (ʋtp).
All reagents were obtained from commercial sources and used as
received. Elemental analyses were carried out at the “Centro de
Instrumentación Científica” of the University of Granada on a THERMO
SCIENTIFIC analyser model Flash 2000. The IR spectra on powdered
samples were recorded with a BRUKER TENSOR 27 FT-IR and OPUS
data collection program. The UV spectra in solution were collected on
an Agilent Technologies Cary 100 Spectrophotometer. Powder DRX
data were collected on a Bruker D2 Phaser diffractometer with mono-
4.6. Crystallographic refinement and structure solution
chromated CuKα radiation (λ
= 1.5405 Å) over the range
5 < 2θ < 35°. Thermal behaviour (thermogravimetry – TG – and
differential scanning calorimetry – DSC) was studied under an air flow
in Shimadzu TGA-50 and Shimadzu DSC-50 equipments at heating rates
of 20 and 10 °C min−1 respectively. NMR spectra were collected with a
high definition 500 MHz NMR spectrometer BRUKER Avance NEO
using MeOD as solvent. Those used to analyse the stability of the
complexes in solution, were carried in the same spectrometer, using
medium trypanosome liquid (MTL) + 10% D2O as solvent.
X-ray data collection of suitable single crystals of compounds were
done at 100(2) K on a Bruker VENTURE area detector equipped with
graphite monochromated Mo-Kα radiation (λ = 0.71073 Å) by ap-
plying the ω-scan method. The data reduction were performed with the
APEX2 [65] software and corrected for absorption using SADABS [66].
Crystal structures were solved by direct methods using the SIR97 pro-
gram [67] and refined by full-matrix least-squares on F2 including all
reflections using anisotropic displacement parameters by means of the
SHELXL program (version 2018/3) [68]. One of the water molecules in
the structure of the ligand have been disordered between two equally
populated positions. Likewise, the water molecules belonging to one of
the complexes in compound 3 have been disordered between two po-
sitions with relative occupancies 0.6 and 0.4 and an interstitial water
molecule in this compound is also disordered between two nearby po-
sitions related by an inversion centre. Hydrogen atoms belonging to the
heterocycle were included in ideal positions riding on their parent
atoms where those belonging to water molecules were located in
Fourier difference maps and refined with fixed OeH distances (0.84 Å)
with the exception of those belonging to the disordered water molecules
in compound 3 that were not located or introduced. An isotropic
thermal displacement parameter 1.2 times or 1.5 times those of their
parent atoms was used for H-atoms. Details of the structure determi-
nation and refinement of compounds are summarized in Table S1.
Crystallographic data for the structures reported in this paper have been
deposited at the Crystallography Open Database (COD) with reference
numbers 3000220-23 and at the Cambridge Crystallographic Data
Centre (deposition nos. 1893521–24). The files may be directly down-
loaded from COD website (http://www.crystallography.net) or ob-
tained free of charge on application to the CCDC Director, 12 Union
4.2. Synthesis of 7-amino-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidine (7-
amtp·H2O)
The triazolopyrimidinic derivative used as
a
ligand was
prepared following the method described by Makisumi [64], in which
29 mmol (0.5 g) of 7-hydroxy-5-methyl-1,2,4-triazolo[1,5-a]pyrimidine
(HmtpO) were added on a round flask with 10 mL of phosphoryl
chloride and put in reflux for 90 min, while the mixture turned dark
orange. After this time, solution is cooled up to room temperature and
the mixture is basified with sodium hydrogen carbonate until there was
no visible reaction. Then, the solution is extracted with di-
chloromethane and the chlorated intermediate is collected by using a
rotoevaporator. Obtained product (7-chloro-5-methyl-1,2,4-triazolo
[1,5-a]pyrimidine) was putted in a excess of commercial ammonia so-
lution and magnetically stirred during an hour. After this time, yellow
crystals of amino derivative (7-amtp) were obtained, some of them
suitable for XRD measures. Yield: 73%, based on HmtpO. Anal. Calcd.
C6H9N5O: C, 43.11; H, 5.43; N, 41.89. Found: C, 43.22; H, 5.39; N,
42.01. IR: 1483 cm−1 (ʋN-H(flex)), 1573 cm−1 (ʋpy), 1660 cm−1 (ʋtp),
3096 cm−1 (ʋO-H) and 3300 cm−1 (ʋN-H(tens)).
10

G.M. Esteban-Parra, et al.
JournalofInorganicBiochemistry212(2020)111235
Road, Cambridge, CB2 1EZ, U.K. (Fax: +44-1223-335033; e-mail:
deposit@ccdc.cam.ac.uk or http://www.ccdc.cam.ac.uk).
3500 cells/well for Vero cells) to a volume of 100 μL/well and then
were incubated at 37 °C with 5% CO2 during 24 h. The complexes so-
lutions were prepared in advance corresponding to the average growing
cells (RPMI 10% FBS for Vero cells and MEM + Glut 20% FBS for
macrophages) at the double of the highest concentration to be tested.
The solutions were performed in a sterile bath with the different
channels, by adding 100 μL of complex solution or medium (only
adding medium in the control wells) to the corresponding well. After
that, the plate was incubated at 37 °C with 5% CO2 for 48 h. Two days
after, 20 μL of Alamar Blue dye (10% of the volume of the well) were
added to each well and incubated at 37 °C with 5% CO2 during another
day. The whole incubation time once the products were added was
72 h, coinciding with the screening period to have comparable se-
lectivity index (SI) results. Finally, the plate was read with an ELISA
reader with Alamar Blue.
4.7. Luminescence measurement
A Varian Cary-Eclipse fluorescence spectrofluorimeter was used to
obtain the fluorescence spectra. The spectrofluorimeter was equipped
with a xenon discharge lamp (peak power equivalent to 75 kW),
Czerny–Turner monochromators, and an R-928 photomultiplier tube
which is red sensitive (even 900 nm) with manual or automatic voltage
control using the Cary Eclipse software. The photomultiplier detector
voltage was 700 V and the instrument excitation and emission slits were
set at 5 and 5 nm, respectively.
4.8. TD-DFT calculations
The PL spectra of complexes 1 and 2 (3 was omitted due to simi-
larity with 2) and free 7-amtp ligand were calculated computationally
by means of TD-DFT using the Gaussian 09 package [69]. using the
CAM-Becke three parameter hybrid functional with the non-local cor-
relation functional of Lee-Yang-Parr (B3LYP) [70–72] along with 6-
311G++(d,p) basis set [73] was adopted for all atoms but for the
central zinc cations. Instead, the LANL2DZ [74–76] basis set along with
the corresponding effective core potential (ECP) was used for metal
atoms. The latter strategy has proven successful in describing lumi-
nescence performance of zinc based coordination compounds [77,78].
The 200 lowest excitation and emission energies were calculated by the
TD-DFT method. Results were analysed with GaussSum program
package [79] and molecular orbitals plotted using GaussView 5 [80].
4.11. In vivo assays
Forty-eight female CD1 mice (31.2
g
body weight and
130
38 mg/dL fasting glycaemia at the beginning of the experi-
mental period) were randomly distributed into 6 groups of 8 animals
each. In 5 groups, type I diabetes was induced by the pharmacological
administration on consecutive days of two doses of 70 mg/kg body
weight of streptozotocin (STZ) as diabetogenic agent [82]. After 7 days,
mice shown significant hyperglycaemia (301
65 mg/dL). The ex-
perimental groups are described as follows: a) control group: 8 healthy
mice; b) diabetic untreated group: 8 STZ diabetic mice; c) diabetic
group treated with the ligand: 8 STZ diabetic mice treated with 7-amtp;
d) diabetic group treated with compound 1: 8 STZ diabetic mice treated
with compound 1 as glucose lowering agent; e) diabetic group treated
with compound 2: 8 STZ diabetic mice treated with compound 2 as
glucose lowering agent; and f) diabetic group treated with compound 3:
8 STZ diabetic mice treated with compound 3 as glucose lowering
agent. The mice were fed control chow diet and were given drinking
water ad libitum throughout the experimental period.
4.9. Antiproliferative assays
Promastigote forms of L. infantum (MCAN/ES/2001/UCM-10), L.
braziliensis (MHOM/BR/1975/M2904), and epimastigote forms of
Trypanosoma cruzi (IRHOD/CO/2008/SN3) were cultivated in vitro in
medium trypanosome liquid (MTL) [Hank's Balanced Salt Solution
(HBSS) (Gibco), NaHCO3, lactalbumin, yeast extract, bovine hae-
moglobin and antibiotics] with 10% inactive fetal bovine serum and
were kept in an air atmosphere at 28 °C, in Roux flasks (Corning, USA)
with a surface area of 75 cm2, according to the methodology described
by Gonzalez et al. [81] The screening of extracellular forms of parasites
was carried out using 24-well plates with MTL medium and 5 × 104
parasites per well. The products were tested at 1, 10, 25 and 50 μM,
prepared from mother aqueous solutions of the compounds, leaving
some wells without drugs as control, and were incubated at 28 °C
during 72 h before the parasite final count.
The Zn compounds were administered at the dose of 15 mg Zn/kg
[83] body weight as dissolved in water (extemporary preparation)
using oral gavages (1 ≤ 100 μM) in volumes of 0.1 ml [84,34]. The oral
glucose tolerance test was performed obtaining peripheral blood from
the tail vein of the mice as described previously [57]. The blood glucose
levels were analysed by the use of a glucometer (Accucheck Aviva,
Roche).
All the animals were group-housed in metabolism cages. The cages
were located in a well ventilated, temperature-controlled room
(21
2 °C) with relative humidity ranging from 40 to 60%, and a
light–dark period of 12 h. All the experiments were carried out in ac-
cordance with Directional Guides Related to Animal Housing and Care
(European Council Community, 1986) and all procedures were ap-
proved by the Animal Experimentation Ethics Committee of the
University of Granada.
4.10. Cell culture and cytotoxicity test
The cytotoxicity tests for macrophages and Vero cells were per-
formed at the Cell Experiment Unit in the “Centro de Instrumentación
Científica” of the University of Granada, according to the methodology
described below. The experiment was carried out in 96-well plates to be
measured in the ELISA reader. The growth inhibition of mammalian
cells was studied using macrophages for the three strains of Leishmania
spp. and Vero cells for T. cruzi. J774.2 macrophages (European
Collection of Cell Culture – ECACC – number 91051511), which were
originally obtained from a tumour in a female BALB/c rat in 1968, were
grown in a minimum essential medium (MEM) plus glutamine (2 mM)
and supplemented with 20% inactivated fetal bovine serum (FBS). Vero
cells (Flow) were grown in Roswell Park Memorial Institute medium
(RPMI), which was supplemented with 10% inactivated fetal bovine
serum. Both cell cultures were incubated in a humidified 95% air, 5%
CO2 atmosphere at 37 °C for several days.
Declaration of competing interest
There is not any conflict of interest.
Acknowledgements
Financial support was given by Junta de Andalucía (Spain) (project
number FQM-195, FQM-394 and FQM-1484) and the Spanish Ministry
of Science, Innovation and Universities (MCIU/AEI/FEDER, UE)
(PGC2018-102052-A-C22 and PGC2018-102052-B-C21) (University
Faculty Training Plan – FPU Grants). The authors thank for technical
and human support provided by SGIker of UPV/EHU and European
funding (ERDF and ESF).
The products were tested at 50, 100, 200 and 400 μM. First, the cells
were sowed in a 96-well plate (2500 cells/well for macrophages and
11

G.M. Esteban-Parra, et al.
JournalofInorganicBiochemistry212(2020)111235
Abbreviations
[21] P.T. Snee, R.C. Somers, G. Nair, J.P. Zimmer, M.G. Bawendi, D.G. Nocera, J. Am.
Chem. Soc. 128 (2006) 13320.
[22] J.-S. Hu, Y.-J. Shang, X.-Q. Yao, L. Qin, Y.-Z. Li, Z.-J. Guo, H.-G. Zheng, Z.-L. Xue,
Cryst. Growth Des. 10 (2010) 2676.
7-atp 7-amino[1,2,4]triazolo[1,5-a]pyrimidine
7-amtp 7-amino-5-methyl [1,2,4]triazolo[1,5-a]pyrimidine
tn 1,3-propanediamine
[23] A.B. Caballero, A. Rodriguez-Dieguez, E. Barea, M. Quiros, J.M. Salas,
CrystEngComm 12 (2010) 3038–3045.
[24] A.J. Calahorro, A. Peñas-Sanjuan, M. Melguizo, D. Fairen-Jimenez, G. Zaragoza,
B. Fernández, A. Salinas-Castillo, A. Rodríguez-Diéguez, Inorg. Chem. 52 (2013)
546–548.
bpym 2,2′-bipyrimidine
mal malonate
[25] Z. Xia, Q. Wei, Q. Yang, C. Qiao, S. Chen, CrystEngComm 15 (2013) 86.
[26] B. Fernández, A. Gómez-Víchez, C. Sánchez-González, J. Bayón, E. San Sebastian,
S. Gómez-Ruiz, C. López-Chaves, P. Aranda, J. Lllopis, A. Rodríguez-Diéguez, New
J. Chem. 40 (2016) 5387–5393.
HmtpO 7-hydroxy-5-methyl[1,2,4]triazolo[1,5-a]pyrimidine
AMPKα AMP-activated protein kinase
ERK2/MAPK1 mitogen-activated protein kinase-1
FBS fetal bovine serum
[27] V. Guillerm, D. Kim, J.F. Eubank, R. Luebke, X. Liu, K. Adil, M.S. Lah, M. Eddaoudi,
Chem. Soc. Rev. 43 (2014) 6141–6172.
GLUT4 glucose transporter type 4
GSK-3β glycogen synthase kinase-3
[28] M. Eddaoudi, D.F. Sava, J.F. Eubank, K. Adil, V. Guillerm, Chem. Soc. Rev. 44
(2015) 228–249.
[29] J.M. Mendez-Arriaga, I. Oyarzabal, G. Escolano, A. Rodriguez-Dieguez, M. Sanchez-
Moreno, J.M. Salas, J. Inorg. Biochem. 180 (2018) 26–32.
HBSS
Hank's Balanced Salt Solution
HOMO highest occupied molecular orbital
IC inhibitory concentration
[30] A. Dominguez-Martin, M. Brandi-Blanco, A. Matilla-Hernandez, H. El Bakkali,
V.M. Nurchi, J.M. Gonzalez-Perez, A. Castineiras, J. Niclos-Gutierrez, Coord. Chem.
Rev. 257 (2013) 2814–2838.
IC50 half maximal inhibitory concentration
IR-β insulin receptor
[31] A.B. Caballero, A. Rodriguez-Dieguez, L. Lezama, J.M. Salas, Cryst. Growth Des. 12
(2012) 3583–3593.
LLCT ligand-to-ligand charge transfer
LUMO lowest unoccupied molecular orbital
MEM Minimal Essential Medium
MTL Medium Trypanosome Liquid
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