Synthesis and evaluation of in vitro and in vivo trypanocidal properties of a new imidazole-containing nitrophthalazine derivative

Article abstract of DOI:10.1016/j.ejmech.2015.10.034

A series of new phthalazine derivatives (1-4) containing imidazole rings and functionalized with nitro groups in the benzene ring of the phthalazine moiety were prepared and identified on the basis of their MS, elemental analyses and bidimensional ¹H and ¹³C NMR data, and their trypanocidal activity was tested. The 8-nitrosubstituted compound (3) was more active in vitro against Trypanosoma cruzi and less toxic against Vero cells than the reference drug benznidazole, and showed a SI value that was 47-fold better than the reference drug in amastigote forms. It also remarkably reduced the infectivity rate in Vero cells and decreased the reactivation of parasitemia in immunodeficient mice. Ultrastructural alterations found in epimastigotes treated with 3 confirmed extensive cytoplasm destruction in the parasites, whereas histopathological analysis of the hearts of mice infected and treated with 3 resulted in a decrease in cardiac damage. Biochemical markers showed that livers, hearts, and kidneys of treated mice were substantially unaffected by the administration of 3, despite the presence of the potentially toxic nitro group. It was also found that this compound selectively inhibited the antioxidant parasite enzyme Fe-superoxide dismutase (Fe-SOD) in comparison with human CuZn-SOD, and molecular modeling suggested interaction with the H-bonding system of the iron-based moiety as a feasible mechanism of action against the enzyme.

Full text of DOI:10.1016/j.ejmech.2015.10.034

Accepted Manuscript
Synthesis and Evaluation of In Vitro and In Vivo Trypanocidal Properties of a New
Imidazole-Containing Nitrophthalazine Derivative
Francisco Olmo, Fernando Gómez-Contreras, Pilar Navarro, Clotilde Marín, María
J.R. Yunta, Carmen Cano, Lucrecia Campayo, David Martín-Oliva, María José
Rosales, Manuel Sánchez-Moreno
PII:
S0223-5234(15)30316-0
10.1016/j.ejmech.2015.10.034
EJMECH 8169
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 July 2015
Revised Date: 18 October 2015
Accepted Date: 20 October 2015
Please cite this article as: F. Olmo, F. Gómez-Contreras, P. Navarro, C. Marín, M.J.R. Yunta, C. Cano,
L. Campayo, D. Martín-Oliva, M.J. Rosales, M. Sánchez-Moreno, Synthesis and Evaluation of In
Vitro and In Vivo Trypanocidal Properties of a New Imidazole-Containing Nitrophthalazine Derivative,
European Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.10.034.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
GRAPHICAL ABSTRACT
A new nitrophthalazine derivative 3 was effective against T. cruzi parasites, inhibited the activity of
their Fe-SOD enzyme and did not show remarkable side-effects in mice.

ACCEPTED MANUSCRIPT
Synthesis and Evaluation of In Vitro and In Vivo
Trypanocidal Properties of a New Imidazole-Containing
Nitrophthalazine Derivative
a
b
c
Francisco Olmo , Fernando Gómez-Contreras *, Pilar Navarro , Clotilde
a
b
b
b
Marín , María J. R. Yunta , Carmen Cano , Lucrecia Campayo , David
d
a
a
Martín-Oliva , María José Rosales , Manuel Sánchez-Moreno *
a
Departamento de Parasitología, Instituto de Investigación Biosanitaria, Hospitales Universitarios de
Granada, Universidad de Granada, E-18071 Granada, Spain
b
Departamento de Química Orgánica, Facultad de Química, Universidad Complutense, E-28040
Madrid, Spain
c
Instituto de Química Médica, Centro de Química Orgánica M. Lora-Tamayo, CSIC, E-28006 Madrid,
Spain
d
Departamento de Biología Celular. Facultad de Ciencias. Universidad de Granada. Spain
Corresponding authors. Manuel Sánchez-Moreno: Phone: +34-958-242-369; Fax: +34-958-243-862;
Email: msanchem@ugr.es. Fernando Gómez-Contreras: Phone: +34-913-944-226; Fax: +34-913-944-
103; Email: fercon@ucm.es
Keywords: Alkylaminonitrophthalazines; imidazole; trypanocidal activity; Chagas acute and chronic
phases; Fe–SOD inhibition; histopathological analysis
ABSTRACT
A series of new phthalazine derivatives (1–4) containing imidazole rings and
functionalized with nitro groups in the benzene ring of the phthalazine moiety were
prepared and identified on the basis of their MS, elemental analyses and bidimensional
1
13
H- and C-NMR data , and their trypanocidal activity was tested. The 8-nitrosubstituted
compound (3) was more active in vitro against T. cruzi and less toxic against Vero cells
than the reference drug benznidazole, and showed a SI value that was 47-fold better than
the reference drug in amastigote forms. It also remarkably reduced the infectivity rate in
Vero cells and decreased the reactivation of parasitemia in immunodeficient mice.
Ultrastructural alterations found in epimastigotes treated with 3 confirmed extensive

ACCEPTED MANUSCRIPT
cytoplasm destruction in the parasites, whereas histopathological analysis of the hearts of
mice infected and treated with 3 resulted in a decrease in cardiac damage. Biochemical
markers showed that livers, hearts, and kidneys of treated mice were substantially
unaffected by the administration of 3, despite the presence of the potentially toxic nitro
group. It was also found that this compound selectively inhibited the antioxidant parasite
enzyme Fe–superoxide dismutase (Fe–SOD) in comparison with human CuZn–SOD, and
molecular modeling suggested interaction with the H-bonding system of the iron-based
moiety as a feasible mechanism of action against the enzyme.
1. Introduction
Trypanosomatid protozoa are the etiological agents of several major insect
transmitted parasitic illnesses such as Chagas disease (Trypanosoma cruzi), sleeping
sickness (Trypanosoma brucei subsp. rhodesiense and subsp. gambiense), and
leishmaniasis (Leishmania spp.). These infections are concentrated in the poorest areas of
the planet and are considered to be the three neglected tropical diseases (NTDs) with the
highest rates of death [1]. Among them, Chagas disease is endemic in 21 Latin American
countries and, although confined initially to the American continent, it is spreading to the
rest of the world, owing to increased migration rates and different transmission modes,
such as food, blood transfusions, organ transplants, and mother–newborn child infection
[
2]. Chagas disease has two phases: an acute stage, shortly after infection, and a chronic
stage, developing over many years. The acute phase may have very mild, or even no,
symptoms. After the acute phase, the disease goes into remission. Although the
symptoms tend to resolve themselves, the infection persists if it is untreated. During the
chronic phase, it may remain silent for decades, or even for life. However, about 30% of
those infected develop life-threatening heart and/or digestive disorders, causing
irreversible damage to the heart, esophagus, and colon [3].
Existing drug treatments for these diseases are far from satisfactory, and the
development of a vaccine remains a goal to be achieved. Current therapies are not
adequate, owing to several factors such as low therapeutic indexes, leading to high
toxicities and unacceptable side effects, the emergence of resistant parasites, and
difficulties in treatment compliance because of complex protocols and the high prices that
are unaffordable for the affected countries [4]. These drawbacks of the current therapies
make the search for new drugs an urgent requirement. Nevertheless, investment in the
development of new drugs against these diseases is not financially attractive for

ACCEPTED MANUSCRIPT
pharmaceutical companies. In recent years, this bleak picture is changing, thanks to
financial backing from nonprofit organizations and the involvement of public–private
partnerships [5].
The main drugs currently used to treat Chagas disease are two nitroaromatic
heterocycles: furane-based nifurtimox (NF) and imidazole-based benznidazole (BZN) [6].
Both drugs are effective in the acute phase of the disease, but their efficacy is low in the
more challenging chronic phase [7], although BZN exhibits a better safety and efficacy
profile than NF. Furthermore, these compounds have severe side effects including
anorexia, vomiting, peripheral polyneuropathy, and allergic dermopathy [8]. The presence
of nitro groups attached to heterocyclic rings suggests that these drugs act on the parasite
through nitro reduction with the intermediation of neurotensin receptor (NTR) enzymes.
Those reductions operate via nitroso and hydroxylamino intermediates, producing amines
that modify biomolecules. However, the mode of action remains under discussion [9]. The
high toxicity in humans is probably a result of oxidative or reductive tissue damage, which
is inseparably linked to their antiparasitic activity [4].
Assuming the necessity of new antichagasic drugs, many research groups are
involved in the design of molecules that are able to inhibit the performance of enzymes
essential for parasite growth and survival [10]. In that context, one of the main self-defense
strategies of T. cruzi is its highly active iron superoxide dismutase (Fe–SOD), which is an
very efficient shield for preventing any oxidative damage from the host, in combination with
peroxidases [11]. T. cruzi Fe–SOD is not present in mammals, which use CuZn–SOD or
Mn–SOD for the same purpose, so that molecules acting selectively over Fe–SOD could
be potential candidates for Chagas therapy [12]. On that basis, in previous work, we
synthesized phthalazine and benzo[g]phthalazine derivatives containing imidazole rings
linked to the pyridazine rings through flexible alkylamine arms (Fig. 1). Remarkably, most
of those compounds were more active in vitro against T. cruzi and less toxic against
mammal Vero cells than the reference drug BZN [13,14]. Furthermore, in vivo tests
performed with the phthalazine derivatives on acute-phase Chagas disease gave
parasitemia inhibition values better than those of BZN [14,15], and a decrease in the
reactivation of parasitemia was found in the chronic phase for immunodeficient mice [14].
Interestingly, trypanocidal features are clearly structure-dependant, as the 1,4-
bis(alkylamino) derivatives were always less active than their 1-alkylamino-4-chloro
counterparts. Simultaneously, these heterocyclic systems inhibited the antioxidant parasite
enzyme Fe–SOD, and the bis(alkylamino) derivatives were less effective inhibitors than
their monoalkylamino analogues, so that a relationship between anti-T. cruzi activity and

ACCEPTED MANUSCRIPT
Fe–SOD inhibition was proposed by our group. Molecular modelling studies suggested
that these highly complexing polyaminic heterocycles could affect Fe–SOD performance
by interacting with the H-bonding amino-acid system of the iron-based moiety.
Although the presence of a nitro group in a compound often (but not always)
increases toxicity issues in the host, many nitroaromatic drugs have been used to treat
parasitic infections, and compounds like nifurtimox, BZN, megazol, fexinidazole, and
furaltadone are nitroheterocycles that specifically exhibit, as do other nitrocompounds,
activity against T. cruzi and other trypanosomatids [16]. As noted above, their antiparasitic
activity seems to be closely related to their ability to generate radical anions or reduced
reactive species that increase oxidative stress in the parasite [17]. Many of the toxic
effects observed after the administration of nitro drugs were found after prolonged
treatment, but not with regimens of shorter duration. The potential of drugs like
fexinidazole has encouraged researchers to further investigate the chemotherapeutic
potential of nitro drugs for trypanosomatid diseases [18]. In this article, we report the
antichagasic properties of a new series of imidazole-containing nitrophthalazine
derivatives, in which the nitro group is located in two different positions with respect to the
alkylamino side chain (Fig. 2). This allows us to evaluate whether different nitro locations
also cause a structure–activity relationship and how is it related to Fe–SOD enzyme
inhibition. We only deal with monoalkylamino-substituted compounds, as they have proved
to be more active than their bis(alkylamino)-substituted analogs.
The antimicrobial properties were evaluated, first, in vitro against epimastigote,
amastigote, and trypomastigote forms of T. cruzi, and Vero cells were used for the
determination of unspecific cytotoxicity. The ability of the compounds to interfere with Fe–
SOD was measured and structure–activity relationships were considered by performing
molecular modelling of the enzyme–compound interaction. In vivo trypanocidal activities of
the most active compounds were studied using a murine model from acute to chronic
phases of Chagas, and the toxicity induced in the host was measured by analyzing
different biochemical parameters in markers of the kidney, heart, and liver profiles.
Ultrastructural alterations in parasites treated with the compound were investigated by
transmission electron microscopy (TEM) in order to evaluate the damage caused.

ACCEPTED MANUSCRIPT
2
. Experimental section
2
.1. Chemistry
The two starting imidazole-containing amines, 3-(imidazol-1-yl)propylamine and 2-
imidazol-4-yl)ethylamine (histamine), were purchased from Sigma–Aldrich and used
(
without further purification. 3-Nitrophthalic acid was also obtained from Sigma–Aldrich.
Solvents were dried using standard techniques [19]. 3-Nitrophthalic anhydride was
prepared by refluxing 3-nitrophthalic acid in acetic anhydride for 1 h [20]. All of the
reactions were monitored using thin-layer chromatography (TLC) on precoated aluminum
sheets of silica gel 60PF254 (Merck, layer thickness 0.2 mm). Compounds were detected
using UV light (254 nm). Chromatographic separations were performed on columns in the
indicated solvent system using flash chromatography on silica gel (particle size 0.040-
0
.063 mm). Melting points were determined in a Gallenkampf apparatus and were
1
uncorrected. H NMR spectra were recorded on a Bruker DPX 300 MHz and an AVIII at
13
7
00 MHz, whereas C NMR spectra were recorded at 75 MHz at room temperature
employing DMSO-d as the solvent. Chemical shifts were reported in ppm (δ scale) from
6
1
13
tetramethylsilane (TMS). All assignments were performed on the basis of H– C
heteronuclear single quantum coherence (gHSQC, spectra included in the Supplementary
material file) and heteronuclear multiple-bond coherence (gHMBC) experiments. IR
spectra were recorded on a Bruker alpha spectrometer with a DTGS detector at room
−1
temperature, furnished with ATR (attenuated total reflection), in the 7500−360 cm range.
Electrospray mass spectra were recorded with Bruker Esquire-LC apparatus. Elemental
combustion analyses were performed in a Perkin–Elmer 2400-CHN instrument by the CAI
of Microanalysis, Universidad Complutense, Madrid, Spain. Analyses indicated by the
symbols of the elements or functions were within ± 0.4 % of the theoretical values and are
shown in the Supplementary material file.
2
.1.1 Synthesis of 5-nitrophthalhydrazide
The original synthesis described by Drew and Garwood [20] was modified as
follows: 3-nitrophthalic anhydride (4 g, 0.02 mmol) was dissolved in boiling ethylene glycol
30 mL). After that, 80% hydrazine hydrate (1.04 g, 0.02 mmol) was added drop-wise and
(
the mixture was refluxed for 1 h. When the resulting solution was cooled to room
temperature, a yellow solid was formed. The precipitate was filtered to give 3.28 g (79%)
Beilstein 24 373
of 5-nitrophthalhydrazide; mp > 300 ºC. (literature value mp > 300 ºC).

ACCEPTED MANUSCRIPT
2
.1.2 Synthesis of 1,4-dichloro-5-nitrophthalazine
A solution of 5-nitrophthalhydrazide (1.0 g, 4.8 mmol) in phosphorous oxychloride
13 mL) and N,N-dimethylaniline (1.2 mL) was heated at 110 ºC for 1 h. After cooling to
(
room temperature, the solvent was evaporated under vacuum and the obtained residue
was poured into ice and the mixture was stirred at 0-15 ºC until a crystalline solid was
formed. The precipitate was filtered, washed with cold water, dried in vacuo, and purified
by flash column chromatography (toluene/ethyl acetate, v/v: 2/1). The fraction of R = 0.53
f
afforded 580 mg of a solid, which was identified as the title compound (50% yield), mp
-1
1
1
76–178ºC. IR (KBr) νmax = 3109, 2925, 2856, 1643, 1574, 1517, 758 cm . H NMR
(
DMSO-d ) δ: 8.71 (d, 1H, J = 8.7 Hz, H-6), 8.68 (d, 1H, J = 8.7 Hz, H-8), 8.42 (t, 1H, H-7)
6
13
ppm. C NMR (DMSO-d ): δ 154.8 (C-1), 148.8 (C-4), 145.0 (C-7), 135.6 (C-5), 130.2 (C-
6
+
6
2
), 129.8 (C-8), 127.7 (C-8a), 117.7 (C-4a) ppm. MS-ESI (MeOH): m/z (%), 243 (MH , 43),
25 (11), 157 (100).
2
.1.3 Synthesis of 1 and 2 (Method A)
A solution of 1,4-dichloro-5-nitrophthalazine (0.952 g, 3.9 mmol), 3-(imidazol-1-
yl)propylamine (0.976 g, 7.8 mmol), and triethylamine (2 mL) in toluene (20 mL) was
heated at 85 ºC for 2 h. After cooling to room temperature, the solvent was removed under
reduced pressure. The residue was purified by flash column chromatography
(toluene/ethyl acetate/ethanol, v/v: 0.5/1/0.5). The appropriate fractions (monitored by TLC
with the same eluent mixture composition) were combined to give three alkylamination
products. The fraction with R = 0.45 was discarded as an hydroxilated subproduct. The
f
fraction with R = 0.56 afforded 195 mg (15% yield) of an orange solid, which was identified
f
as 1-[3-(imidazol-1-yl)propylamino]-4-chloro-8-nitrophthalazine (1), mp 164−165 °C . IR
-1 1
(
KBr) νmax: 3417, 2937, 1645, 1534, 1513, 1302, 1081 cm . H-NMR (DMSO-d ) δ: 8.54 (d,
6
1
H, J = 7.6 Hz, H-7), 8.43 (d, 1H, J = 8.3 Hz, H-5), 8.17 (t, 1H, J = 8.0 Hz, H-6), 7.64 (s,
H, H-2’), 7.20 (s, 1H, H-5’), 6.90 (s, 1H, H-4’), 6.79 (t, 1H, J = 5.1 Hz, NH-C1), 4.08 (t, 2H,
1
13
J = 6.9 Hz, Hγ), 3.41 (m, 2H, Hα), 2.09 (m, 2H Hβ). C-NMR (DMSO-d ) δ: 151.34 (C-1),
6
1
46.23 (C-8), 144.41 (C-4), 137.73 (C-2’), 133.60 (C-6), 130.16 (C-7), 129.97(C-5), 128.85
(C-4’), 127.46 (C-4a), 119.78 (C-5’), 111.32 (C-8a), 44.23 (Cγ), 39.41 (Cα), 29.86 (Cβ).
+
MS-ESI (MeOH) m/z (%): 333 (MH , 100). Anal. C H ClN O (C, H, N). The fraction with
14
13
6
2
R = 0.20 afforded 275 mg (21% yield) of a yellow solid which was identified as 1-[3-
f
(imidazol-1-yl)propylamino]-4-chloro-5-nitrophthalazine (2), mp > 211 °C (dec). IR (KBr)
-1 1
νmax: 3280, 2954, 1654, 1539, 1519, 1015 cm . H-NMR (DMSO-d ) δ: 8.62 (d, 1H, J = 8.0
6
Hz H-6), 8.41 (d, 1H, J = 7.5 Hz, H-8), 8.16 (t, 1H, J = 8.1 Hz, H-7), 8.09 (t, 1H, J = 5.1 Hz,

ACCEPTED MANUSCRIPT
NH-C1), 7.76 (s, 1H, H-2’), 7.26 (s, 1H, H-5’), 6.94 (s, 1H, H-4’), 4.12 (t, 2H, J = 6.9 Hz,
13
Hγ), 3.54 (m, 2H, H
α
), 2.15 (m, 2H H
β). C-NMR (DMSO-d ) δ: 153.72 (C-1), 145.32 (C-5),
6
1
1
45.23 (C-4), 137.76 (C-2’), 133.05 (C-7), 128.42 (C-4’), 128.22 (C-8), 127.31 (C-6),
21.28 (C-8a), 119.97 (C-5’), 116.66 (C-4a), 44.53 (Cγ), 39.09 (Cα), 30.01 (Cβ). MS-ESI
+
(
MeOH) m/z (%): 333 (MH , 100). Anal. C H ClN O ·0.5AcOEt (C, H, N).
14
13
6
2
2
.1.4 Synthesis of 3 and 4 (Method B)
A solution of 1,4-dichloro-5-nitrophthalazine (0.733 g, 3.0 mmol), 2-(1H-imidazol-4-
yl)ethanamine (0.666 g, 6.0 mmol), and triethylamine (1.5 mL) in toluene (35 mL) was
heated at 100 ºC for 5 h. After cooling to room temperature, the solvent was removed
under reduced pressure. The residue was purified by flash column chromatography
(toluene/ethyl acetate/ethanol, v/v: 0.5/1/0.5). The appropriate fractions (monitored by TLC
with the same eluent mixture composition) were combined to give three alkylamination
products. The fraction with R = 0.27 was discarded as an hydroxilated subproduct. The
f
fraction with R = 0.51 afforded 86 mg (9% yield) of a yellow solid, which was identified as
f
1
-[2-(imidazol-4-yl)ethylamino]-4-chloro-8-nitrophthalazine (3), mp 133−134 °C . IR (KBr)
-1 1
νmax: 3405, 2925, 1723, 1562, 1214 cm . H-NMR (DMSO-d ) δ: 8.49 (d, 1H, J = 8.3 Hz,
6
H-7), 8.43 (d, 1H, J = 7.8 Hz, H-5), 8.16 (t, 1H, J = 8.2 Hz, H-6), 7.63 (s, 1H, H-2’), 6.90 (s,
13
1
H, H-5’), 6.79 (brs, 1H, NH-C1), 3.71 (m, 2H, H
α), 2.85 (t, 2H, J = 7.0 Hz, Hβ). C-NMR
(
DMSO-d ) δ: 151.02 (C-1), 145.98 (C-8), 144.27 (C-4), 135.08 (C-2’), 135.00 (C-4’),
6
1
4
33.41(C-6), 129.87 (C-5), 128.83 (C-7), 127.14 (C-4a), 116.39 (C-5’), 110.98 (C-8a),
+
2.68 (C
α
), 25.96 (C
β
). MS-ESI (MeOH) m/z (%): 319 (MH , 100). Anal.
C H ClN O ·0.5H O (C, H, N). The fraction with R = 0.16 afforded 241 mg (25% yield)
13
11
6
2
2
f
of a yellow solid, which was identified as 1-[2-(imidazol-4-yl)ethylamino]-4-chloro-5-
nitrophthalazine (4), mp > 322 °C (dec). IR (KBr) νmax: 3252, 2925, 2854, 1544, 1460, 1413
-1 1
cm . H-NMR (DMSO-d ) δ: 8.60 (d, 1H, J = 8.1 Hz, H-6), 8.41 (d, 1H, J = 7.5 Hz, H-8),
6
8
1
.21 (t, 1H, J = 5.1 Hz, NH-C1), 8.16 (t, 1H, J = 8.1 Hz, H-7), 7.57 (s, 1H, H-2’), 6.87 (s,
13
H, H-5’), 3.80 (m, 2H, Hα), 2.96 (t, 2H, J = 7.2 Hz, Hβ). C-RMN (DMSO-d ) δ: 153.61
6
(C-1), 145.33 (C-4, C-5), 137.59 (C-4’), 135.18 (C-2’), 133.10 (C-7), 129.94 (C-5’), 128.19
(C-8), 127.21 (C-6), 121.28 (C-8a), 116.70 (C-4a), 42.07 (Cα), 26.26(Cβ). MS-ESI (MeOH)
+
m/z (%): 319 (MH , 100). Anal. C H ClN O (C, H, N).
14
13
6
2

ACCEPTED MANUSCRIPT
2
2
.2. Parasite strain and culture
.2.1 Epimastigote and trypomastigote forms
Epimastigote forms of T. cruzi SN3 strain (IRHOD/CO/2008/SN3) isolated from
domestic Rhodnius prolixus from Guajira (Colombia) [21] were cultivated in vitro in
trypanosomes liquid (MTL) with 10% inactivated fetal bovine serum and were kept in an air
2
atmosphere at 28 °C , in Roux flasks (Corning, USA) w ith a surface area of 75 cm ,
according to a previously described methodology [22]. The transformation of epimastigote
to metacyclic forms was achieved by metacyclogenesis induced by culturing a 5-day-old
culture of epimastigote that was harvested by centrifugation at 7000 g for 10 min at 10 ºC.
8
The parasites were then incubated for 2 h at 28ºC at a density of 5 × 10 cells/mL in TAU
medium (190 mM NaCl, 17 mM KCl, 2 mM MgCl , 2 mM CaCl , 8 mM phosphate buffer,
2
2
pH 6.0). Thereafter, the parasites were incubated in a 1:100 dilution (final epimastigote
6
forms concentration: 5 × 10 cells/mL) for 96 h at 28ºC in TAU3AAG medium (TAU
supplemented with 10 mM L-proline, 50 mM L-sodium glutamate, 2 mM
aspartate and 10 mM -glucose) in 25 mL culture flasks with a layer of culture medium
that was not more than 1 cm in depth [23].
L-sodium
D
2
.2.2 Cell culture and cytotoxicity tests
Vero cells (EACC number 84113001), originally obtained from monkey kidneys,
were grown in RPMI (Gibco) supplemented with 10% inactivated fetal bovine serum in a
humidified 95% air/5% CO atmosphere at 37ºC for 2 days. The cytotoxicity test for Vero
2
cells was performed according to a previously described methodology [22]. After 72 h of
treatment, the cell viability was determined by flow cytometry. Thus, 100 ꢀL per well of
propidium iodide solution (100 mg/mL) was added and incubated for 10 min at 28 °C in
darkness. Afterwards, 100 ꢀL per well of fluorescein diacetate (100 ng/mL) was added and
incubated under the same conditions. Finally, the cells were recovered by centrifugation at
4
00 g for 10 min and the precipitate was washed with phosphate-buffered saline (PBS).
Flow cytometric analysis was performed with a FACSVantage flow cytometer (Becton
Dickinson). The percentage viability was calculated in comparison with the control culture.
The IC50 was calculated using linear regression analysis from the K values of the
c
concentrations employed (0.1–100 ꢀM).

ACCEPTED MANUSCRIPT
2
2
.3. In vitro activity assays
.3.1 Epimastigote assays (extracellular forms)
The obtained compounds and the reference drug (BZN) were dissolved in DMSO
(Panreac, Barcelona, Spain) at a concentration of 0.01% (v/v) and assayed as nontoxic
and without inhibitory effects on parasite growth, according to a reported procedure [22].
The compounds were added to the culture medium at dosages of 100, 50, 25, 10, l, 0.5,
0
.25, and 0.1 ꢀM. The effects of each compound against epimastigote forms of T. cruzi
were tested at 72 h using a Neubauer hemocytometric chamber. The antitrypanosomatid
effect is expressed as the IC , that is, the concentration required to give 50% growth
50
inhibition, calculated by linear regression analysis from the K values of the concentrations
c
employed.
2
.3.2. Amastigote assays (intracellular forms)
Vero cells were grown as described above in RPMI medium (Gibco). Cells were
4
seeded at a density of 1 × 10 cells per well in 24-well microplates (Nunc) with rounded
coverslips on the bottom and cultured for 2 days. Afterwards, the cells were infected in
vitro with metacyclic forms at a ratio of 10:1 over 12 h. The nonphagocytosed parasites
were removed by washing, and then the drugs (at 100, 50, 25, 10, 1, 0.5, 0.25, and 0.1
ꢀ
M) were added. Vero cells with the drugs were incubated for 72 h at 37 °C in 5% CO 2.
The drug activity was determined on the basis of the number of amastigote forms in the
treated and untreated cultures in methanol-fixed and Giemsa-stained preparations. The
number of amastigote forms was determined by analyzing 200 host cells distributed in
randomly chosen microscopic fields. The antitrypanosomatid effect is expressed as the
IC .
50
2
.4. SOD inhibition studies
Epimastigote T. cruzi, cultured as described above, were collected by
centrifugation. The pellet was suspended in 3 mL of STE buffer (0.25 M sucrose, 25 mM
Tris–HCl, 1 M EDTA, pH 7.8) and the cells were disintegrated through three cycles of
ultrasonication, each for 30 s at 60 W. The homogenate was centrifuged for 5 min at 1500
g and 4º C, and the pellet was washed three times in ice-cold STE buffer. This fraction
was centrifuged (2500 g for 10 min at 4 ºC) and the supernatant was collected. The protein
concentrations in the supernatant were determined using the Bradford method [24]. The

ACCEPTED MANUSCRIPT
Fe–SOD and CuZn–SOD activities were determined using the method described by Beyer
and Fridovich [25], which measures the reduction of nitrobluetetrazolium (NBT) by
superoxide ions. Into each cuvette, 845 ꢀL of the stock solution [3 mL of L-methionine (300
−1
−1
mg, 10 mL ), 2 mL of NBT (1.41 mg, 10 mL ) and 1.5 mL of Triton X-100 1% (v/v)] was
added, together with 30 ꢀL of the parasite homogenate fraction, 10 ꢀL of riboflavin (0.44
−1
mg, 10 mL ) and an equivalent volume of the of the compound solution. Five different
concentrations were used for each compound (1–100 ꢀM). In the control experiment, the
volume was made up to 1000 ꢀL with 50 mM potassium phosphate buffer (pH 7.8),
whereas 30 ꢀL of the parasite homogenate fraction was added to mixtures containing the
compounds. Then, the absorbance (A ) was measured at 560 nm in a spectrophotometer.
0
Every cuvette was illuminated with a fixed lamp light for 10 min under constant stirring and
the absorbance (A ) was measured. CuZn–SOD (isolated from human erythrocites),
1
coenzymes, and substrates used in these assays are commercial products and were
purchased from Sigma Chemical Co. The obtained data were analyzed using the
Newman–Keuls test [25].
2
.5.Molecular modeling
Molecular-modeling studies were carried out using the AMBER method
implemented in Hyperchem 8.0 package [26], modified by the inclusion of appropriate
parameters [27]. Starting structures for compounds 1–4 were built by using Hyperchem
capabilities. Its geometry was minimized to a maximum energy gradient of 0.1 kcal/mol
with the AMBER force field, using the Polak–Ribiere (conjugate gradient) minimizer, and
the simulated annealing procedure was used to cover all conformational space. The most
stable extended geometry was always used in all calculations of the interaction with the
enzyme. To mimic the conditions used in the activity measurements, that is, water as the
solvent, all calculations were carried out in vacuo with a distance-dependent dielectric
constant value [28]. Charge assignations for all atoms was done by means of ab initio
calculations using the STO-3G basis set, as it is compatible with the AMBER force field,
prior to energy minimization using AMBER. The T. cruzi Fe–SOD enzyme structure was
obtained from the Brookhaven protein data bank (2gpc) and its energy minimized in the
same way. Interaction studies were performed, starting from structures with the compound
positioned in the border of the enzyme cavity. Entering the cavity was forced using a
restraint for the N–Fe distance, slowly decreasing this distance and letting the complex

ACCEPTED MANUSCRIPT
achieve the minimum energy conformation with no restraints, for all the small driving steps,
using the same conditions mentioned above.
2
.6. Ultrastructural alterations
5
Epimastigote forms of T. cruzi were cultured at a density of 5 × 10 cells/mL in each
corresponding medium containing the compounds tested at their IC concentrations. After
25
7
2 h, these cultures were centrifuged at 400 g for 10 min and the pellets produced were
washed in PBS and then mixed with 2% (v/v) paraformaldehyde/glutaraldehyde in 0.05 M
cacodylate buffer (pH 7.4) for 4 h at 4 ºC. Following this, the pellets were prepared for
TEM study using a previously described technique [29].
2
.7. Infection assays
Vero cells were grown under the same conditions expressed over 2 days.
Afterwards, the cells were infected in vitro with metacyclic forms of T. cruzi at a ratio of
0:1. The drugs (IC25 concentrations) were added immediately after infection and were
1
incubated for 12 h at 37 °C in 5% CO . The nonphagocytosed parasites and the drugs
2
were removed by washing, and then the infected cultures were grown for 10 days in fresh
medium. Fresh culture medium was added every 48 h. The drug activity was determined
from the percentage of infected cells, the number of amastigote forms per infected cell,
and the number of trypomastigote forms in the medium [15], in treated and untreated
cultures, as well as in methanol-fixed and Giemsa-stained preparations. The percentage of
infected cells and the mean number of amastigote forms per infected cell were determined
by analyzing 200 host cells distributed in randomly chosen microscopic fields every 48 h.
The number of trypomastigote forms in the medium was determined using a Neubauer
hemocytometric chamber every 48 h.
2
2
.8. In vivo trypanocidal activity assays
.8.1. Mice infection and treatment
This experiment was 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 female albino mice
(6−8 weeks old, 25−30 g weight) maintained under a 12 h dark/light cycle (lights on at

ACCEPTED MANUSCRIPT
0
7:30 h) at a temperature of 22 ± 3 °C and provided with water and standard chow ad
3
libitum, were inoculated through the intraperitoneal route with 5 × 10 blood trypomastigote
forms of T. cruzi per mouse obtained from previously infected mice blood. The animals
were divided as follows: I positive control group (mice infected but not treated) and II study
group (mice infected and treated with the compounds under study). The administration of
the tested compounds was initiated on the fifth day of infestation once the infection was
confirmed, and doses of 25 mg/kg body weight were administered every two days until day
1
3 pi through the intraperitoneal route. Peripheral blood was obtained from the mandibular
vein of each mouse (5 ꢀL samples) and dissolved in 495 ꢀL of PBS solution at a 1:100
dilution. The circulating parasite numbers were quantified with a Neubauer’s chamber for
counting blood cells. This counting was performed every 3 days over 40 days (acute
phase). The number of bloodstream forms was expressed as parasites/mL.
2
.8.2. Cyclophosphamide-induced immunosuppression and assessment of the cure
After day 60, the mice entered the chronic phase of the experiment where
parasitemia continued decreasing its levels progressively, independently of the treatment.
So, on day 150, the parasitemia was checked and found to be undetectable by fresh blood
microscopic examination; then, the mice received four intraperitoneal doses of 200
mg/kg·w of body mass cyclophosphamide monohydrate (CP) (ISOPAC®) on alternate
days, as previously described [30]. The efficacy of such an immunosuppression procedure
to assess cryptic infection was verified by the high parasitemias under microscopic
examination and/or mortality close to the 100% of chronically untreated mice, having
received the immunosuppression treatment. Within 1 week after the last CP injection,
parasitemia was evaluated according to the procedure described for the acute phase to
quantify the presence of blood trypomastigote forms as a reactivation rate. Finally, the
mice were bled out under gaseous anesthesia (CO ) through a heart puncture and the
2
blood was collected. Blood was incubated for 2 h at 37 ºC and then over night at 4º C in
order to allow clotting and to obtain the serum from the samples after centrifuging the
supernatant twice at 1000 and 2700 g, consecutively. The serum was divided into aliquots
and used for ELISA and biochemical analyses, as explained below. Hearts were harvested
and immediately flushed free of blood by gentle infusion of 10 mL of prewarmed PBS
through the left ventricle [31] in order to avoid contamination of collected tissue with blood
parasites. Afterwards, they were frozen at –80 ºC and stored until they were used for DNA
extraction.

ACCEPTED MANUSCRIPT
2
.9. ELISA tests
Fe–SOD excreted from the parasite, cultured, and processed, as described by
Lopez-Céspedes et al. [32], was used as the antigen fraction. The partially purified protein
fraction was coated onto polystyrene microtiter plates (Nunc, Denmark) as 1.5 ꢀg in
carbonate buffer (pH 8.2) for 2 h at 37 °C . The ant igen remaining on the plate was
eliminated by washing three times with 0.05% PBS-Tween 20 (washing buffer). Free
adsorption sites were taken by incubation (2 h at 37 °C ) with blocking buffer [0.2% PBS-
Tween 20, 1% bovine serum albumin (BSA)]. After the mixture was washed, as described
previously, the plates were incubated (45 min at 37 °C ) with a serum dilution of 1:80 in
washing buffer. After the second wash, the plates were incubated in darkness for 20 min
with 100 ꢀL of an enzyme-conjugated antibody (anti-IgG peroxidase) at a dilution of
1
:1000. The enzyme reaction was developed with the chromogenic substrate o-
phenylenediamine dihydrochloride (OPD, Sigma) and 10 ꢀL of 30% H O per 25 mL for 20
2
2
min in the dark. The reaction was stopped by the addition of 50 ꢀL of HCl (3 M). The
absorbance was read at 492 nm in a microplate reader (Sunrise, TECAN). All of the
samples were analyzed in triplicate in polystyrene microtiter plates. Mean and standard
deviations of the optical densities of the negative control sera were used to calculate the
cutoff value.
2
.10. DNA extraction and qualitative PCR
Hearts were thawed and then grinded using a Potter-Elvehjem to follow the
purification procedure using a Wizard® Genomic DNA Purification Kit (Promega). PCR
was performed with two primers designed in our laboratory [33], based on the published
sequence of the enzyme SOD T. cruzi CL Brenner (GenBank accession No. XM_808937),
which amplifies a fragment of approximately 300 bp, which belong to the SOD gene b of T.
cruzi. PCR was performed in a total volume of 20 ꢀL. The reaction mixture included 1 ꢀL
of DMSO, 200 nM of iSODd, 200 nM of iSODr, 10 mM Tris-HCl (pH 9.0), 1.5 mM MgCl2,
5
0 mM KCl, 0.01% gelatin, 0.1% triton X-100, 100 mM of each dNTP, 0.5 U of Taq DNA
polymerase, 0.5–1 ꢀg of DNA and HPLC water to a final volume of 20 ꢀL. The
amplification was performed in a Thermal Cycler TM MyCycler (BioRad) with the following
routine temperature: 95 ºC/3 min, 30 cycles of 95 °C/30 s, 55.5 ºC/45 s, 72 ºC/30 s, and 72
ºC/7 min. Next, the amplification products were subjected to electrophoresis on 1.6%
agarose gel containing 1:10000 of GelRed™ Nucleic Acid Gel Stain, for 90 min at 90 V.

ACCEPTED MANUSCRIPT
2
.11. Toxicity tests through clinical chemistry measurements
A fraction of the obtained serum was sent to the Biochemical Service at the
University of Granada, where a series of parameters were measured according to the
commercial kits acquired from Cromakit® by BS-200 Chemistry Analyzer (Shenzhen
mindray, Biomedical electronics co., Ltd.). With the levels obtained for different populations
of sera, biochemical studies in mice uninfected, infected and in the acute phase, or
infected in the chronic phase were performed on 15 individuals. And 6 infected individuals
were treated with compound 3 on day 120m pi. n = 15, n = 6), we calculated the mean
value and standard deviation. Finally, we also calculated the confidence interval for the
mean normal populations based on a confidence level of 95% [100 × (1–α) = 100 ×
(1.05)%]. The obtained ranges are shown in Table 3, which allows the comparison and
analysis of the sera studied in this work.
3
. Results and discussion
3
.1 Synthesis and identification
The starting 1,4-dichloro-5-nitrophthalazine material was prepared from 3-
nitrophthalic acid, as shown in Scheme 1, by successive treatment with acetic anhydride,
an equivalent amount of aqueous hydrazine hydrate in ethylenglycol, and phosphorous
oxychloride/N,N-dimethylaniline. In the last step, conditions related to those previously
established by our group for the synthesis of 1,4-dichloro-5-nitrobenzo[g]phthalazine [34]
were used, and the dichloro derivative was obtained in a 50% yield. Alkylamino-substituted
derivatives 1 and 2 were obtained with respective yields of 15 and 21% through
nucleophilic substitution of the chlorine atom attached to the C-1 or C-4 positions,
respectively, of 1,4-dichloro-5-nitrophthalazine with 3-(imidazol-1-yl)propylamine by
heating in toluene at 85ºC for 2 h, using triethylamine as the chlorine acceptor (method A).
Two equivalents of the imidazoleamine were used because that reactant partially
decomposes under the reaction conditions required for achieving substitution of the
chlorine atom. In contrast with previous related reactions from 1,4-dichlorophthalazine, the
formation of the bis(alkylamino)-substituted derivatives was not detected, owing to the
deactivating effect of the nitro group on the phthalazine system. However, substitution by
hydroxy groups gave side products that were discarded. When the same conditions were
used for the preparation of 3 and 4 with 2-(1H-imidazol-4-yl)ethanamine as the

ACCEPTED MANUSCRIPT
aminoalkylating agent, compound 3 was not obtained; therefore, different reaction
conditions were tested. Finally, increasing the reaction time and temperature to 5 h and
1
00ºC, respectively, (method B) allowed the isolation of both 3 and 4 in respective yields
of 9 and 25%. Isolation of 1–4 from the crude reaction mixtures was performed by column
flash chromatography with a toluene/ethyl acetate/ethanol mixture of increasing polarity.
All of the newly synthesized compounds were unequivocally identified by their
1
13
analytical, electrospray ionization mass spectra (MS-ESI), IR, H NMR, and C NMR
spectroscopic data, as shown in the Experimental Section. Scheme 1 displays the
molecular ions obtained from the electrospray mass spectra, which, in all cases, agree
1
13
with the proposed structures. For an accurate assignment of the signals in the H and C
NMR spectra, which were registered in deuterated dimethylsulfoxide (DMSO-d6),
heteronuclear simple quantum coherence (gHSQC) and heteronuclear multiple bond
coherence (gHMBC) experiments were performed. Registering those bidimensional
13
spectra was very useful, since it allowed us to identify the C chemical shifts of every
carbon atom, which was crucial for differentiating between the two isomers of each pair of
compounds.
1
The H NMR spectra of all of the alkylamino-substituted products showed signals
corresponding to the three aromatic protons of the phthalazine system as the two
expected doublets and one triplet, whereas the two protons of the imidazole ring appeared
as singlets and the integration lines confirmed that they were, in all cases,
1
monoalkylamino-substituted compounds. However, H NMR data did not allow us to
assign the position of the nitro group in the obtained pairs of isomers. The only relevant
difference was a significant deshielding of the NH signal attached to C-1 in compound 2
with respect to 1 (+1.3 ppm) and 4 with respect to 3 (+1.4 ppm). When comparing the NH
shifts with those of compounds IV [14], which are devoid of the nitro group, 2 and 4
exhibited very similar values, suggesting that the nitro substituent could be attached to C-
5
, that is, far from the alkylamino substituent and unable to interact with the NH group, but
this was not conclusive. The search for a NOE effect between the NH substituent and the
H-8 atom of compound 4 did not give any clear evidence of intramolecular interaction.
13
Comparison of the theoretical and experimental C spectra of the pairs of isomers was
more informative, as remarkable differences were found between the calculated spectra of
the phthalazine moiety of the 5-nitro and 8-nitro isomers. As expected, the most significant
were those concerning quaternary C-4a and C-8a, located in α and β positions with respect
to the carbons supporting the nitro group. [35,36] Table 1 shows the theoretical shift
differences found for those carbon atoms between the 5-NO and 8-NO isomers in
2
2

ACCEPTED MANUSCRIPT
comparison with the experimental ones, corresponding to the pairs of compounds 1–2 and
–4. It can be seen that the obtained results fit with the assignment proposed previously
3
and that shielding or deshielding of the carbon atoms followed the same pattern in all
cases, so that the 5-nitro disposition can be ascribed to compounds 2 and 4.
3
.2 In vitro trypanocidal evaluation
In order to obtain preliminary information, the in vitro activity of compounds 1–4 was
evaluated against epimastigote, amastigote, and trypomastigote forms of T. cruzi as
described in the Experimental Section. Epimastigote extracellular forms are the most
commonly used, owing to easier handling, but tests performed on intracellular amastigotes
are more indicative, as extracellular forms are not the developed form of the parasite in
vertebrate hosts [37]. Therefore, Vero cells were infected with metacyclic forms of T. cruzi,
which were transformed into amastigotes within 1 day of infection and treated with
compounds 1–4. Their activity against blood trypomastigotes was also tested, as these
forms of the parasite are responsible for the chronic phase of Chagas disease. Table 2
shows the IC values obtained after 72 h of exposure and at 1–100 ꢀM concentrations
50
when compounds 1–4 were assayed against the three parasite forms mentioned above.
Values obtained with the reference drug BZN were included in all cases for comparison. It
was shown that compound 3 was the most active of the four compounds tested for the
three parasite forms assayed, being the best result obtained for intracellular amastigote
forms, with an IC of 4.0 ꢀM, although activity values were homogeneous for the three
50
forms, as they ranged between 4.0 ꢀM and 6.1 ꢀM. Furthermore, compound 3 was more
effective than the reference drug in all cases. It is also interesting to note that
nitrocompound 3 was more active against amastigote forms than its unsubstituted analog
described in reference [14] ( IC 4.0±0.3 µM against 9.4±1.2 µM). On the other hand, the
50
cytotoxicity of compounds 1–4 was evaluated using mammalian Vero cells as the cellular
model (also in Table 2). Interestingly, toxicity data against Vero cells after 72 h of culture
showed that all of the assayed phthalazine derivatives were substantially less cytotoxic
than BZN, despite the presence of the a priori conflictive nitro group in all of the structures
considered. Citotoxicity values ranged from 112.9 to 298.6 ꢀM, which are in sharp contrast
to the value obtained for BZN (13.6 ꢀM).
From the in vitro data reported above, more informative selectivity index (SI) values
could be obtained and are shown in the last columns of Table 2. Good results were found
for all of the four compounds in all parasite forms tested when the number of times that the

ACCEPTED MANUSCRIPT
SI of each compound exceeded the SI of BZN (in brackets) were calculated, with SI values
exceeding those of BZN between 12 and 47 times. The best comparative results (29-, 20-,
4
7-, and 21-fold for compounds 1, 2, 3, and 4, respectively) were obtained against the
most significant intracellular amastigote forms. On the other hand, it was confirmed that
the 8-nitrophthalazine derivative (3) was the most effective among the compounds tested,
as it showed the best SI values against the three forms, being 47 times more selective
than BZN when assayed against amastigote forms, whereas the other three compounds
exhibited lower and more homogeneous SI numbers, ranging between 20 and 29 times
more selective than the reference drug.
3
.3 Inhibitory effect on the T. cruzi Fe–SOD enzyme.
As mentioned above, the antiparasitic activity of other imidazole-containing
pyridazine derivatives was previously related to their inhibitory activity against the Fe–SOD
enzyme of the parasite, which could be considered as a potential target for the design of
new molecules that are able to act in the iron-moiety environment [13-15]. In order to
obtain more evidence, the effects of compounds 1–4 on T. cruzi Fe–SOD were tested at
concentrations ranging from 1 to 100 ꢀM. Epimastigote forms of T. cruzi were used, which
excrete Fe–SOD when cultured in a medium lacking inactive fetal calf serum (FCS) [38].
The obtained inhibition data are shown in Fig. 3A, and the corresponding IC values are
50
included in parentheses to make the interpretation of the results easier. For comparison,
Fig. 3B shows the effect of the same compounds on commercial CuZn–SOD isolated from
human erythrocytes. All four compounds were active against Fe–SOD. However, when
looking at Fig. 3A, it can be seen that the most trypanosomicidal compound (3) inhibited
the enzyme activity to a great extent, whereas the inhibitory activities of compounds 1, 2,
and 4 were visibly lower. The graph shows that 8-nitro derivative 3 reached 100%
inhibition of Fe–SOD at a concentration of 100
ꢀM and inhibited Fe–SOD by more than
6
0% at 50 ꢀM, whereas the other three compounds stayed below 50% inhibition even at a
concentration of 100 ꢀM. Interestingly, the effect of compound 3 on human CuZn–SOD
was substantially lower than in the case of Fe–SOD, as the inhibitory activity scarcely
reached 30% at a concentration of 100 ꢀM. That selectivity could result in potential activity
against Chagas disease with scarce side-effects in human organs. Concerning the IC50
data, values higher than 100 ꢀM were obtained in all cases, with the remarkable exception
of compound 3, which gave an IC50 of 22 ꢀM against Fe–SOD, being al least five times
more inhibitory than the other compounds, whereas an IC50 > 100 ꢀM was calculated

ACCEPTED MANUSCRIPT
against CuZn–SOD. Therefore, the highest antipararasitic activity was in correlation with
the highest inhibitory activity of Fe–SOD, as found previously for other series of azole-
containing phthalazine derivatives.
3
.4 Molecular modeling study
In previous work with related phthalazine derivatives [14,39], we found some
correlation between the experimental Fe–SOD inhibitory activity and theoretical
perturbation of the geometry at the enzyme core, both in T. cruzi and in Leishmania
parasites. Therefore, a tentative molecular modeling study on the mode of interaction with
the enzyme was performed. The core of an Fe–SOD enzyme monomer contains an iron
atom as a five-coordinated active site ligated by three histidine units, an aspartate group,
-
and an axial H O or HO ligand supported by an essential H-bonding network, comprised
2
of amino acids [40]. Specifically, the coordinated solvent engages in H bonds with the
Asp156-ligand and with the conserved active site Gln69 [41] (Fig. 4A). Several studies
have emphasized the importance of those ligands and its H-bonding partners in tuning the
antioxidant activity of Fe–SOD [42]. The coordinated solvent acts as a proton
donor/acceptor, thereby facilitating the release of peroxide generated in the reduction of
the substrate [41], and it also plays a critical role in modulating the reduction potential of
the iron center [42]. It is supposed that the protonation state is strongly influenced by H
bonding with Gln69 and, in turn, H bonds to a network including Tyr34, Asn72, and
Trp122, among others. On that basis, it has been proposed that enzyme inhibition could
take place by modification of the H-bonding system induced by irruption of an external
compound [43].
The ability of related benzo[g]phthalazine derivatives to modify the H-bonding
network at the active site of the Fe–SOD enzyme (obtained from the Brookhaven protein
data bank 2gpc entry) had been tested previously using the Amber forcefield implemented
in Hyperchem 8.0 [14,39]; the methodology used is specified in the Experimental Section.
Fig. 4A shows the active site of the enzyme, with the H-bonding system outlined above
displayed as dashed lines and the solvent ligand in the foreground linked to the iron atom.
The key interactions with Gln69 and Asp156 are clearly seen. The highly inhibitory 8-
nitrophthalazine derivative 3 was then buried into one monomer with the imidazole ring
pointing towards the metal ion, and the energetically most favored disposition was
identified (Fig. 4B). As a consequence of the approach of the molecule, the amino acids
surrounding the iron atom were displaced, apart from their initial positions and the H-

ACCEPTED MANUSCRIPT
bonding pattern appeared remarkably distorted. The three histidine molecules and Tyr34
-
were especially affected, but the most relevant point was that the H bonds of the H O/HO
2
ligand with Gln69 and Asp156 were missing, as both amino acids moved towards the rear
of the active center, as seen in the Figure and, consequently, the hydroxyl ligand remained
too far away for creating stable interactions with them. In fact, the H –O and OAsp–Hlig
Gln
lig
distances increased from 2.30 and 2.32 Å to 3.61 and 4.57 Å, respectively. In a further
step, the same molecular modeling treatment was given to the less inhibitory isomer 4
(Fig. 4C). It was found that the presence of the molecule also disturbed the H-bonding
network surrounding the active center, but, interestingly, the hydrogen bond between
-
Gln69 and the H O/HO ligand was maintained with a calculated H –O distance of 2.52
2
Gln
lig
Å, whereas that corresponding to the OAsp–H disappeared, as the aspartate oxygen was
lig
now located very far from the ligand’s hydrogen atom, at a distance of 5.40 Å. We think
that these theoretical calculations could explain, in part, the remarkable inhibitory effect
found experimentally for compound 3.
Both the trypanocidal activity and Fe–SOD inhibitory properties explained above
supported the convenience of carrying out more detailed in vitro and in vivo assays with
the 8-nitrophthalazine derivative 3 in order to obtain more information about its mode of
action and its apparent advantages with respect to BZN. In accordance with the usual
working procedure, compounds 1, 2, and 4 were not tested in further studies, owing to the
less significant SI results obtained.
3
.5 Infectivity assay
To gain better insight into the trypanocidal activity of compound 3, its effect on the
infectivity and intracellular replication of T. cruzi amastigotes was subsequently
determined. The propagation of the parasite in Vero cells was evaluated by measuring the
infection rates and the average number of amastigotes and trypomastigotes present during
a 10-day treatment period. Vero cells were cultured for 2 days and then infected with
epimastigote forms in the stationary phase. The parasites invaded the cells and underwent
morphological conversion to amastigotes within 2 days of infection. The IC was used as
25
the test dosage and BZN was the reference drug. On day 8, the rate of host-cell infection
reached its maximum in the control experiment. When 8-nitrophthalazine derivative 3 was
added to the infected Vero cells, a remarkable decrease in host-cell infection rate, which
reached 95% on day 8 and remained stationary until day 10, was observed, making
compound 3 much more effective than the reference drug, which only afforded a mere

ACCEPTED MANUSCRIPT
2
0% reduction on day 10 (Fig. 5A). The measure of the mean number of amastigotes per
infected Vero cell (Fig. 5B) led to similar results; at the end of the treatment, the
nitrophthalazine derivative had decreased the number of amastigotes present in the cells
by 74%, whereas BZN only allowed a poor reduction of 15%. Regarding the number of
trypomastigote forms released in the medium (Fig. 5C), the inhibition values were
generally less significant. Even so, compound 3 reached 55% reduction and almost
doubled the inhibitory effect of BZN (30%), which remained less effective. Ultimately,
infectivity tests confirmed the in vitro antiparasitic activity shown in Table 2 for compound
3
, as well as its higher efficacy with respect to the reference drug.
3
.6 Ultrastructural alterations
The remarkable trypanosomicidal activity shown by 8-nitrophthalazine derivative 3
should cause important damage in the parasite cells. Therefore, the morphological
alterations created in T. cruzi epimastigotes were analyzed using TEM in order to obtain
some information about the way the cell structure was affected. The most significant
variations compared to the control cells are shown in Fig. 6 for parasites treated with
compound 3. As expected, highly significant alterations were found. Some parasites died
(second panel in Fig. 6) and deep modifications were evident in most of the rest. The
shape of many parasites was heavily distorted, as they were very swollen with cytoplasms
filled with empty vacuoles and other vacuoles containing large amounts of electron-dense
material (third panel in Fig. 6). Some mitochondria were also swollen and devoid of crypts,
looking like vacuoles filled with a more dense content than that of lipidic ones. Others were
filled with smaller vacuoles, and kinetoplasts were very swollen and disorganized. The
number of ribosomes present in treated epimastigotes was much lower than that found in
the control cultures (third panel in Fig. 6). These findings, regarding essential modifications
in the composition of the parasite cells, are consistent with the significant trypanocidal
activity data found for compound 3.
3
.7 In vivo anti-T. cruzi activity in Balb/c female albino mice.
As compound 3 showed remarkable SI values with respect to BNZ in the in vitro
experiments and was the best inhibitor of the parasitic Fe–SOD enzyme, it was selected
for further in vivo studies in the chosen murine model. The trypanocidal activity during the
acute phase of Chagas disease [until 40 days post infection (pi)] was first investigated.

ACCEPTED MANUSCRIPT
3
Female Balb/c mice were inoculated with 5 x 10 blood trypomastigotes obtained from a
mouse previously infected. We opted for the intraperitoneal doping route, which usually
leads to lower mortality rates than the intravenous procedure [44], and 125 mg of
compound 3/kg of body mass (mpk) were administered in five doses of 25 mpk every two
days from the fifth day of infestation until day 13 pi, according to the pharmacokinetic of
the compound (data not shown). Parasite counting was performed on 5 µL blood samples
obtained from the mandibular vein. Interestingly, none of the animals treated with
compound 3 died during the treatment, despite the mammalian toxicity typically ascribed to
the nitro functional group in antiparasitic drugs. Fig. 7A shows the effect caused by
nitrophthalazine derivative 3 on the parasitemia levels in blood during the acute phase of
Chagas disease (0–40 days pi). A reduction of parasitemia in mice treated with 3 was
evident as early as the beginning of the treatment (day 5 pi), and was maintained, not only
until the end of treatment, but to the end of the acute phase. Reduction of parasitemia was
not found in mice treated with BZN under the same conditions. The next step was to
evaluate the behavior of 3 in the chronic phase, in which the positive effect of the current
drugs is under much discussion [45]. From day 120 pi, parasitemia remaining in the blood
is virtually undetectable and the parasite is nested inside the target organs in the
amastigote form. A major problem during the treatment of Chagas disease is the high
reactivation ability of the parasitemia in immunocompromised individuals cured with
apparent success. It has been shown that apparently cured patients who had been
submitted to kidney or liver transplantation, diagnosed with AIDS, or treated with
anticancer chemotherapy, experimented Chagas reactivation with a very aggressive
clinical course [46]. In order to obtain information about this matter, the mice treated as
described above were kept until day 120 pi and were subsequently divided into two
subgroups, one of which was maintained under the same conditions without further
treatment, whereas the other was subjected to three successive cycles of
immunosuppression with cyclophosphamide monohydrate, as described in the Material
and methods Section [47]. That treatment caused the reactivation of parasitemia, which
was controlled previously by the immune system of the mouse. Blood samples were
extracted to determine the parasitemia levels of treated mice in comparison with the
corresponding non-immunosuppressed subgroup of mice. As reactivation is proportional to
the survival rate of the parasites, the effect caused by compound 3 could be estimated and
is depicted in Fig. 7B in the form of an illustrative tridimensional graph. Parasitemia
reactivation reached 80.3% with respect to the acute phase in the control group of infected
and untreated mice, whereas it decreased to 48.5% in the treated mice group, revealing a

ACCEPTED MANUSCRIPT
significant effect on the chronic course of infection. The result was confirmed by evaluating
the immunological status of mice involved in the experiment. The detection of total
immunoglobuline G (IgG) allows evaluation of the immune status of the mice, as IgG
indicates the level of protection that should be attributed to the tested compounds, as well
as the innate protection that mice have naturally. According to the previous reports, IgG
titres in Balb/c become stable during the chronic phase of the disease [48]. The enzyme-
linked immunosorbent assay (ELISA) was used to detect the IgG levels [34], and the
antigen source was the Fe–SOD enzyme isolated in our laboratory. The control IgG levels
followed the expected pattern for nonspecific hypergammaglobulinaemia at the beginning
of infection and remained stable when the infection became chronic. After the
immunosuppression treatment, parasitemia reactivation induced a response with
increased levels of specific IgG isotypes. Fig. 7C shows that reactivation is lower in mice
treated with compound 3 than in untreated mice, confirming the ability of 3 to reduce
parasitemia. However, reactivation had found to be better in its unsubstituted analog [14],
maybe due to the different pharmacokinetic properties of both molecules. Lastly, Fig. 7D
represents the results obtained from a qualitative polymerase chain reaction (PCR) assay
[
49] performed after necropsy for the evaluation of the parasitic load in cardiac tissue of
mice in both the control and treated groups. The hearts were ground and used for total
DNA extraction and amplification of a fragment of 278 bp, belonging to SOD gene b of T.
cruzi. The hearts of control animals confirmed the presence of parasites, whereas those of
mice treated with 3 (lanes 9–14 in Fig. 7D) were free of parasites in two of the six cases
studied, indicating a 33% percentage of curing. Therefore, the PCR assay also indicated
partial effectivity of compound 3 at the administered dose.
3
.8 Histopathological alterations
As discussed above, interactions between the parasite and the target organs of the
guest generate serious damage at morphological and structural levels. Furthermore, the
action of the immune system against the infection may cause huge cell distruction inside
the organ, as the parasite is in an intracellular location and its elimination is inextricably
linked to obliteration of the related cell. As a consequence, we evaluated the damage
caused by the parasite in the heart of infected mice during the chronic phase of the
disease in both the control and treated groups. The first column of Table 3 lists the most
common traces of histopathological cardiac damage, corresponding to a T. cruzi infection.
Columns 2 and 3 display the results observed after examination of hearts of untreated

ACCEPTED MANUSCRIPT
mice and 3-treated mice 120 days pi, respectively. From the comparison of the data
obtained with both groups, it can be seen that treatment with the nitrophthalazine
derivative results in a decrease in cardiac damage, as inflammatory infiltrates change from
moderate to mild, subepicardial fibrosis is reduced from severe to mild, and degeneration
of cardiomyocites declines from abundant to light. This improvement in histopathological
heart alterations after the administration of 3 is confirmed by images obtained after
necropsy, which are included in Fig. 8. Panels A and B show the condition of the organ in
an infected control mouse using two different staining methods. Panels C and D display
alterations found in an infected mouse treated with compound 3. Strong subepicardial
fibrosis and cardiomyocite degeneration are observed in panels A and B, whereas panels
C and D indicate a situation with scarce or even nonexistent fibrosis symptoms. All of
these data confirm the beneficial effect of the administration of compound 3 on the infected
organ during the chronic stage of the disease.
3
.9 Biochemical parameter alterations
It is known that the presence of the nitro group in molecules with potential anti-T.
cruzi properties often results in harmful side effects, affecting essential organs of the
guest. For that reason, we studied the biochemical kidney, heart, and liver profiles in mice
treated with the nitrophthalazine derivative 3 in order to evaluate the toxicity of this
compound after its administration and further metabolization. Later, serum samples were
collected during necropsy (final day of the experiment) to determine whether some
irreversible toxicity or chronic damage could be detected at that stage in the above
mentioned organs. Table 4 shows the most significant marker profiles obtained for the
three organs in three groups of mice: uninfected, infected and acute phase, or infected in
the chronic phase. Significant alterations in the parameters range of the two infected
groups are clearly seen with respect to the uninfected mice. The last file displays how the
markers profiles in the chronic phase are affected in mice treated with compound 3. It is
shown that there are no relevant changes in either the kidney or liver markers tested and,
therefore, those organs are not damaged by the administration of 3, even at the relatively
high dose used in the assays. Concerning heart markers, mild variations in the CK-MB
enzyme levels (with an increase lower than 30%) and in the LDH value (with a decrease of
about 10%) are indicative that curing was not complete, as deduced from the
histopathological heart images of Figure 8. So it seems that compound 3 does not cause
relevant toxicity in the key organs considered at the administered doses.

ACCEPTED MANUSCRIPT
We conclude that the introduction of nitro groups into alkylaminophthalazine
derivatives functionalized with imidazole rings allowed us to obtain a new compound with
better in vitro selectivity-index values against T. cruzi than BZN. That compound also
exhibited selective inhibitory ability against the Fe–SOD enzyme and, causing great
damage to the parasite cells, does not result in significant alterations in the key organs of
mice tested after its administration in the chronic stage of Chagas disease.
Acknowledgements
The authors thank the MCINN Projects Consolider Ingenio CSD2010-00065 and
CTQ2009-14288-C04-01 for financial support. We are also grateful to the NMR and
Elemental Analysis C.A.I.s of the Universidad Complutense (Madrid), and also to the
transmission electron microscopy and nuclear magnetic resonance spectroscopy services
of the CIC-University of Granada. F.O. is grateful for a FPU grant from the Ministry of
Education of Spain.
References
[
1] (a) A. Cavalli, M. L. Bolognesi, Neglected Tropical Diseases: multi-target-directed
ligands in the search for novel lead candidates against Trypanosoma and Leishmania.
J. Med. Chem. 52 (2009) 7339–7359.
(b) S. Espuelas, D. Plano, P. Nguewa, M. Font, J. A. Palop, J. M. Irache, C.
Sanmartín, Innovative lead compounds and formulation strategies as newer
kinetoplastid therapies, Curr. Med. Chem. 19 (2012) 4259–4288.
[
[
[
[
2]
WHO,
http://www.who.int/mediacentre/fact sheets/fs340/en/
3] A. Rassi- Jr., A. Rassi, Marin Nieto, J. A.Chagas, disease. Lancet 375 (2010) 1388-
402.
Chagas
disease:
Fact
Sheet
Nº
349,
March
(2014),
1
4] J. A Urbina, Specific chemotherapy of Chagas disease: relevance, current limitations
and new approaches, Acta Trop. 115 (2010) 55–68.
5] J. C. Dujardin, D. González-Pacanowska, S. L. Croft, O. F. Olesen, G. F. Späth,
Collaborative actions in anti-trypanosomatid chemotherapy with partners from disease
endemic areas, Trends Parasitol. 26 (2010) 395–403.
[
6] P. M. M. Guedes, G. K. Silva, F. R. S. Gutierrez, J. S. Silva, Current status of Chagas
disease chemotherapy. Expert Rev. Anti-Infect. Ther. 9 (2011) 609–620.

ACCEPTED MANUSCRIPT
[
[
7] R. Viotti, C. Vigliano, B. Lococo, M. G. Alvarez, M. Petti, G. Bertocchi, A. Armenti, Side
effects of Bnz as treatment in chronic Chagas disease: fears and realities. Expert Rev.
Anti-Infe 7 (2009) 157-163.
8] (a) C. J. Schofield, J. Jannin, R. Salvatella, The future of Chagas disease control.
Trends Parasitol. 22 (2006) 583-588; (b) J. D. Maya, D. K Cassels, P. Iturriaga-
Vasquez, Mode of action of natural and synthetic drugs against Trypanosoma cruzi
and their interaction with the mammalian host. Comp. Biochem. Phys. A 146 (2007)
6
01-620.
[
9] S. R. Wilkinson, C. Bot, J. M. Kelly, B. S. Hall, Trypanocidal activity of nitroaromatic
prodrugs: current treatments and future perspectives, Curr. Top. Med. Chem. 11 (2011)
2
072-2084.
[
[
[
[
10] M. N. Soeiro, S. L. de Castro, Trypanosoma cruzi targets for new chemotherapeutic
approaches, Expert Opin, Ther. Targets 13(1) (2009) 105-121.
11] A. F. Miller, Superoxide dismutases: active sites that save, but a protein that kills,
Curr. Opin. Chem. Biol. 8 (2004) 162-168.
12] J. F Turrens, Oxidative stress and antioxidant defences: a target for the treatment of
diseases caused by parasitic protozoa, Mol. Asp. Med. 25 (2004) 211-220.
13] A. M. Sanz, F. Gómez-Contreras, P. Navarro, M. Sánchez-Moreno, S. Boutaleb-
Charki, J. Campuzano, M. Pardo, A. Osuna, C. Cano, M. J. R. Yunta, L. Campayo,
Efficient inhibition of Fe-SOD and of Trypanosoma cruzi growth by
benzo[g]phthalazine derivatives functionalized with one or two imidazole rings, J. Med.
Chem. 51 (2008) 1962-1966.
[
[
[
14] M. Sánchez-Moreno, F. Gómez-Contreras, P. Navarro, C. Marín, F. Olmo, M. J. R.
Yunta, A. M. Sanz, M. J. Rosales, C. Cano, L. Campayo, Phthalazine derivatives
containing imidazole rings behave as Fe-SOD inhibitors and show remarkable anti-T.
cruzi activity in immunodeficient-mouse mode of infection, J. Med. Chem. 55 (2012)
9
900-9913.
15] M. Sánchez-Moreno, A. M. Sanz, F. Gómez-Contreras, P. Navarro, C. Marín, I.
Ramírez-Macias, M. J. Rosales, F. Olmo, I. García-Aranda, L. Campayo, C. Cano, F.
Arrebola, M. J. R. Yunta, In vivo trypanosomicidal activity of imidazole- or pyrazole-
based benzo[g]phthalazine derivatives against acute and chronic phases of Chagas
disease, J. Med. Chem. 54 (2011) 970-979.
16] S. Patterson, S. Wyllie, Nitro drugs for the treatment of trypanosomatid diseases: past,
present, and future prospects, Trends Parasitol. 30 (2014) 289-298.

ACCEPTED MANUSCRIPT
[
[
17] R. B. Oliveira, A. P. Passos, R. O. Alves, A. J. Romanha, M. A. F. Prado, J. D. de
Souza jr., R.J. Alves, In vitro evaluation of the activity of aromatic nitrocompounds
against, Trypanosoma cruzi. Mem. Inst. Oswaldo Cruz 98(1) (2003) 141-144.
18] (a) M. V. Papadopoulou, W. D. Bloomer, H. S. Rosenzweig, S. R. Wilkinson, M.
Kaiser, Novel nitro(triazole/imidazole)-based heteroarylamides/sulfonamides as
potential antitrypanosomal agents, Eur. J. Med. Chem. 87 (2014) 79-88; (b) M. V.
Papadopoulou, W. D. Bloomer, G. I. Lepesheva, H. S. Rosenzweig, M. Kaiser, B.
Aguilera-Venegas, S.R. Wilkinson, E. Chatelain, J. R. Ioset, Novel 3-nitrotriazole-
based amides and carbinols as bifunctional antichagasic agents, J. Med. Chem. 58
(2015) 1307-1319; (c) B. Muro, F. Reviriego, P. Navarro, C. Marín, I. Ramírez-Macías,
M. J. Rosales, M. Sánchez-Moreno, V. J. Arán, New perspectives on the synthesis
and antichagasic activity of 3-alkoxy-1-alkyl-5-nitroindazoles, 74 (2014) 124-134.
19] D. D. Perrin, W. L. F. Armarego, D. R. Perrin, Purification of Laboratory Chemicals;
Pergamon Press: Oxford (1980).
[
[
20] H. D. K. Drew, R. F. Garwood, Chemiluminiscent organic compounds. Part VII.
Substituted phthalazine-1,4-diones. Effect of the substituent on the luminescent
power, J. Chem. Soc. (1939) 836-838.
[
[
21] J. Téllez-Meneses, A. M. Mejía-Jaramillo, O. Triana-Chávez, Biological
characterization of Trypanosoma cruzi stocks from domestic and sylvatic vectors in
Sierra Nevada of Santa Marta, Colombia, Acta Trop. 108 (2008) 26-34.
22] 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, Scorpiand-like
azamacrocycles prevent the chronic establishment of Trypanosoma cruzi in a murine
model, Eur. J. Med. Chem. 70 (2013) 189-198.
[
[
23] J. Cardoso, M. J. Soares, In vitro effects of citral on Trypanosoma cruzi
metacyclogenesis, Mem. Inst. Oswaldo Cruz 5 (2010) 1026-32.
24] M. M. Bradford, A refined and sensitive method for the quantification of microgram
quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72
(1976) 248-254.
[
[
25] W. F. Beyer, I. Fridovich, Assaying for superoxide dismutase activity: Some large
consequences of minor changes in conditions, Anal. Biochem. 161 (1987) 559-566.
26] D. A. Case, T. E. Cheatham, T. Darden, H. Gohlke, R. Luo, K. M. Merz, A. Onufriev,
C. Simmerling, B. Wang, R. J. Woods, The Amber biomolecular simulation programs,
J. Comput Chem. 26 (2005) 1668-1688.

ACCEPTED MANUSCRIPT
[
[
27] C. Miranda, F. Escartí, L. Lamarque, M. J. R. Yunta, P. Navarro, E. García-España,
M. L. Jimeno, New 1H-pyrazolecontaining polyamine receptors able to complex L-
glutamate in water at physiological pH values, J. Am Chem Soc. 126 (2004) 823-833.
28] F. Reviriego, P. Navarro, E. García-España, M. T. Abelda, J. C. Frías, A. Domenech,
M. J. R. Yunta, R. Costa, E. Ortí, Diazatetraester 1H-pyrazole crowns as fluorescent
chemosensors for AMPH, METH, MDMA (ecstasy), and dopamine, Organic Letters 10
(2008) 5099-5102.
[
29] I. Ramírez-Macías, C. Marín, R. Chahboun, I. Messouri, F. Olmo, M. J. Rosales, R.
Gutierrez-Sánchez, E. Alvarez-Manzaneda, M. Sánchez-Moreno, In vitro and in vivo
studies of the trypanocidal activity of four terpenoid derivatives against Trypanosoma
cruzi, Am. J. Trop. Med. Hyg. 87 (2012) 481-488.
[
[
[
30] S. Cencig, N. Coltel, C. Truyens, Y. Carlier, Parasitic loads in tissues of mice infected
with Trypanosoma cruzi and treated with AmBisome, PLoS Negl. Trop. Dis. 5 (2011)
e1216.
31] X. Ye, J. Ding, X. Zhou, G. Chen, S. F. Liu, Divergent roles of endotelial NF-kappaB in
multiple organ injury and bacterial clearance in mouse models of sepsis. J. Exp. Med.
2
05 (2008) 1303–1315.
32] A. Lopez-Céspedes, E. Villagran, K. Briceño-Alvarez, J. A. de Diego, H. L.
Hernandez-Montiel, C. Saldaña, M. Sánchez-Moreno, C. Marín, Trypanosoma cruzi:
seroprevalence detection in suburban population of Santiago de Queretaro (Mexico),
Sci. World J. (2012) Article ID 363595, 6 pages. Doi:10.1100/2012/914129.
[
33] F. Olmo, J. Escobedo-Ortega, P. Palma, M. Sánchez-Moreno, A. Mejía-Jaramillo, O.
Triana, C. Marín, Specific primers design based on the superoxide dismutase b gene
for Trypanosoma cruzi as a screening tool: Validation method using strains from
Colombia classified according to their discrete typing unit, Asian Pac. J. Trop. Med.
7
(11) (2014) 854-859.
[
[
34] L. Campayo, B. Jiménez, T. Manzano, P. Navarro, Diazapolycyclic compounds XXV.
Improved synthesis of 6-substituted 2,3-dihydrobenzo[g]phthalazine-1,4-dione
derivatives, Synthesis (1985) 197-200.
35] F. Reviriego, P. Navarro, A. Domenech, E. García-España, Effective complexation of
psychotropic phenethylammonium salts from a disodium dipyrazolate salt of macrocyclic
structure, J. Chem. Soc. Perkin Trans. 2 (2002) 1634-1638.
[
36] V.J. Arán, M. Kumar, J. Molina, L. Lamarque, P. Navarro, E. García-España, J. A. Ramírez, S.
V. Luis, B. Escudero, Synthesis and protonation behavior of 26-membered oxaaza and
polyaza macrocycles containing two heteroaromatic units of 3,5-disubstituted pyrazole or 1-

ACCEPTED MANUSCRIPT
1
13
benzylpyrazole. A potentiometric and H and C NMR study, J. Org. Chem. 64(17) (1999)
135-6146.
6
[
[
37] A. González-Coloma, M. Sánchez-Moreno, In vitro activity of C20-diterpenoid alkaloid
derivatives in promastigotes and intracellular amastigotes of Leishmania infantum, Int.
J. Antimicrob. Agents 25 (2005) 136-141.
38] M. E. Villagran, C. Marín, I. Rodríguez-González, J. A. de Diego, M. Sánchez-Moreno,
Use of an iron superoxide dismutase excreted by T. cruzi in the diagnosis of Chagas
disease: Seroprevalence in rural zones of the state of Queretaro, Mexico, Am. J. Trop.
Med. Hyg. 73 (2005) 510–516.
[
39] P. Navarro, M. Sánchez-Moreno, C. Marín, E. García-España, F. Ramírez-Macías, F.
Olmo, M. J. Rosales, F. Gómez-Contreras, M. J. R. Yunta, R. Gutierrez-Sánchez, In
vitro leishmanicidal activity of pyrazole-containing polyamine macrocycles which inhibit
the Fe-SOD enzyme of Leishmania infantum and Leishmania.braziliensis species,
Parasitology 141 (2014) 1031-1043.
[
[
[
40] A. F. Miller, D. L. Sorkin, K. Padmakumar, Anion binding properties of reduced and
oxidized iron-containing superoxide dismutase reveal no requirement for Tyrosine 34,
Biochemistry 44 (2005) 5969-5981.
41] E. Yikilmaz, D. W. Rodgers, A. F. Miller, The crucial importance of chemistry in the
structure-function link: manipulating hydrogen bonding in iron-containing superoxide
dismutase, Biochemistry 45 (2006) 1151-1161.
42] (a) C. K. Vance, A. F. Miller, Specificity and phenetic relationships of iron- and
manganese-containing superoxide dismutases on the basis of structure and sequence
comparisons, Biochemistry 40 (2001) 13079-13087; (b) E. Yikilmaz, J. Xie, T. C.
Brunold, A. F. Miller, Hydrogen-bond-mediated tuning of the redox potential of the
non-heme Fe site of superoxide dismutase, J. Am. Chem. Soc. 124 (2002) 3482-3483;
(c) C. K. Vance, A. F. Miller, A simple proposal that can explain the inactivity of metal-
substituted superoxide dismutases, J. Am. Chem. Soc. 120 (1998) 461-467; (d) W. C.
Stallings, A. L. Metzger, K. A. Pattridge, J. A. Fee, M. L. Ludwig, Structure-function
relationships in iron and manganese superoxide dismutases, Free Radic. Res.
Commun. 12-13 (1991) 259-268; (e) W. G. Han, T. Lovell, L. Noodleman, Coupled
redox potentials in manganese and iron superoxide dismutases from reaction kinetics
and density functional/electrostatics calculation, Inorg. Chem. 41 (2002) 205-218.
43] I. G. Muñoz, J. F. Moran, M. Becana, G. Montoya, The crystal structure of a
eukaryotic iron superoxide dismutase suggests intersubunit cooperation during
catalysis, Protein Sci. 14 (2005) 387–394.
[