In vitro and in vivo trypanosomicidal activity of pyrazole-containing macrocyclic and macrobicyclic polyamines: Their action on acute and chronic phases of chagas disease

Article abstract of DOI:10.1021/jm2017144

The in vitro and in vivo anti-Trypanosoma cruzi activity of the pyrazole-containing macrobicyclic polyamine 1 and N-methyl- and N-benzyl-substituted monocyclic polyamines 2 and 3 was studied. Activity against both the acute and chronic phases of Chagas disease was considered. The compounds were more active against the parasite and less toxic against Vero cells than the reference drug benznidazole, but 1 and 2 were especially effective, where cryptand 1 was the most active, particularly in the chronic phase. The activity results found for these compounds were complemented and discussed by considering their inhibitory effect on the iron superoxide dismutase enzyme of the parasite, the nature of the metabolites excreted after treatment, and the ultrastructural alterations produced. A complementary histopathological analysis confirmed that the compounds tested were significantly less toxic to mammals than the reference drug and that 1 and 2 exhibited lower levels of damage than 3.

Full text of DOI:10.1021/jm2017144

Article
pubs.acs.org/jmc
In Vitro and in Vivo Trypanosomicidal Activity of Pyrazole-
Containing Macrocyclic and Macrobicyclic Polyamines: Their Action
on Acute and Chronic Phases of Chagas Disease
Manuel Sanchez-Moreno,*,† Clotilde Marín,† Pilar Navarro,*,‡ Laurent Lamarque,‡
́
Enrique García-Espana,*,§ Carlos Miranda,‡ Oscar Huertas,† Francisco Olmo,†
̃
Fernando Gomez-Contreras,∥ Javier Pitarch,§ and Francisco Arrebola⊥
́
†
⊥
Departamento de Parasitología, Facultad de Ciencias, and Departamento de Histología, Facultad de Medicina, Universidad de
Granada, E-18071 Granada, Spain
‡
́ ́
Instituto de Química Medica, Centro de Química Organica M. Lora-Tamayo, CSIC, E-28006 Madrid, Spain
§ Departamento de Química Inorgan
́
ica, Instituto de Ciencia Molecular, Universidad de Valencia, E-46980 Paterna (Valencia), Spain
∥
Departamento de Química Organ
́
ica, Facultad de Química, Universidad Complutense, E-28040 Madrid, Spain
S
* Supporting Information
ABSTRACT: The in vitro and in vivo anti-Trypanosoma cruzi activity
of the pyrazole-containing macrobicyclic polyamine 1 and N-methyl-
and N-benzyl-substituted monocyclic polyamines 2 and 3 was studied.
Activity against both the acute and chronic phases of Chagas disease was
considered. The compounds were more active against the parasite and
less toxic against Vero cells than the reference drug benznidazole, but 1
and 2 were especially effective, where cryptand 1 was the most active,
particularly in the chronic phase. The activity results found for these
compounds were complemented and discussed by considering their
inhibitory effect on the iron superoxide dismutase enzyme of the
parasite, the nature of the metabolites excreted after treatment, and the ultrastructural alterations produced. A complementary
histopathological analysis confirmed that the compounds tested were significantly less toxic to mammals than the reference drug
and that 1 and 2 exhibited lower levels of damage than 3.
years after infection, and many of them will suffer cardiac failure
and sudden death.9 The control and treatment of Chagas
disease constitutes a very large economic burden for Latin
American countries.
INTRODUCTION
■
Chagas disease (trypanosomiasis) is a potentially fatal parasitic
disease that is recognized by the World Health Organization
(WHO) as being one of the world’s most neglected tropical
diseases. It is widespread throughout Latin America1 and is
caused by a protozoan, Trypanosoma cruzi, which is primarily
transmitted by some species of blood-feeding triatomine
insects, mainly Triatoma infestans, Rhodius prolixus, and
Triatoma dimidiata.2 This disease can also be transmitted by
nonvectorial mechanisms, including blood transfusion or organ
donation,3 transfer from mother to child during pregnancy,4 or
even the ingestion of food contaminated by the parasite.5
Because of increasing international travel and immigration, this
disease has spread not only across all of Latin America, but
across the United Sates, Canada, Spain, Italy, and other
countries.6,7 The best estimates of Chagas disease cases in Latin
America report 10−14 million infected people. The initial,
acute infection is usually asymptomatic, where most patients do
not realize that they have been contaminated with the parasite;
this is because the parasite load is fairly small.8 The acute phase
can further evolve into a chronic stage, in which about 30% of
infected individuals develop severe cardiac, peripheral nervous
system, or digestive complications, sometimes within 10−30
The main drugs currently used to treat Chagas disease are
two nitroaromatic heterocycles: the furane-based nifurtimox
and the imidazole-based benznidazole (BZN).10 Both drugs are
effective in the acute phase of the disease, although
benznidazole shows a better safety and efficacy profile,11 but
the efficacy of both is very low in the chronic phase.12
Furthermore, these compounds have severe side effects,
including anorexia, vomiting, peripheral polyneuropathy, and
allergic dermopathy. The presence of nitro groups attached to
the heterocyclic rings suggests that these drugs act on the
parasite by nitro reduction, originating reduced intermediates
that covalently modify biomolecules, although its mode of
action is currently under discussion.13 Their high level of
toxicity against humans is probably a result of oxidative or
reductive tissue damage and is inextricably linked to their
Received: December 20, 2011
Published: March 26, 2012
© 2012 American Chemical Society
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Figure 1. Macrocyclic and macrobicyclic pyrazole-containing polyamines tested in this work.
Scheme 1. Preparation of the Pyrazole-Based Macrocycles
antiparasitic activity.10 Therefore, less toxic and more selective
drugs are urgently needed.6
metal ion or by changes in the coordination geometry could be
effective ways to deactivate its antioxidant features, and they
would presumably affect both the growth and survival of
parasite cells. Closely connected to this idea, in previous work
our research group synthesized 1,4-bis(alkylamino)benzo[g]-
phthalazine derivatives capable of forming dinuclear complexes
with transition metals; so far we have shown that, in the free
form, they are selective inhibitors of Fe-SOD and that they
simultaneously show remarkable in vitro and in vivo activity
against T. cruzi.16,17 We proposed that one feasible mechanism
One promising target for new drugs against Chagas disease is
iron superoxide dismutase (Fe-SOD). It has been shown that
parasitic protozoan survival is closely related to the ability of
some enzymes to evade toxic free-radical damage.14 One of the
main protective mechanisms involves Fe-SOD, a specific
enzyme of trypanosomatids that acts as a scavenger of
superoxide ions and hydroxyl radicals.15 Since prosthetic
groups are essential in all enzymatic processes, modifications
in the enzyme active site of Fe-SOD by dissociation of the
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a
Table 1. In Vitro Activity, Toxicity, and Selectivity Index for Compounds 1-4 against Extra- and Intracellular Forms of T. cruzi
d
IC50 (μM)
SI
epimastigote
axenic amastigote
intracellular amastigote
epimastigote
forms
axenic amastigote
forms
intracellular amastigote
forms
b
b
b
c
forms
forms
forms
Vero cells
BZN
15.9 1.1
1.3 0.1
1.3 0.3
16.5 3.2
46.4 7.4
18.9 1.5
7.2 2.2
8.8 1.8
10.0 2.9
20.8 3.5
23.3 4.6
6.2 0.8
6.5 1.6
13.8 2.5
13.6 0.9
149.1 13.5
178.5 21.3
195.4 11.7
21.5 3.3
0.8
0.7
0.6
1
2
3
4
114.7 (143)
137.3 (172)
11.8 (15)
0.5 (0.6)
20.7 (30)
20.3 (29)
19.5 (28)
1.0 (1.4)
24.0 (40)
27.5 (46)
14.2 (24)
1.1 (1.8)
18.4 3.3
a
b
The results are averages of three separate determinations. IC50 = the concentration required to give 50% inhibition, calculated by linear regression
c
d
analysis from the Kc values at the concentrations used (1, 10, 25, 50, and 100 μM). After 72 h of culture. Selectivity index = IC50 Vero cells
toxicity/IC50 activity of extracellular or intracellular forms of the parasite. In parentheses are: the number of times that the compound SI exceeded
the reference drug SI.
for their trypanosomicidal activity could be related to
competition for the metal ion of Fe-SOD.17
performed in order to gain some information about the
inhibitory effect of our compounds on the glycolytic pathway,
since this represents the prime energy source of the parasite.
Finally, a histopathological analysis was performed in order to
gain better insights into the toxicity levels obtained.
The promising results obtained from the open-chain ligands
mentioned above prompted us to focus our attention on the
series of macrocyclic compounds 1−4 (Figure 1). We had
previously proposed a macrobicyclic polyamine containing
three 3,5-disubstituted pyrazole rings with the structure 1,18,19
and two monocyclic dinuclear polyamines endowed with N-
Me20 (2) or N-Bn21 (3) substituted pyrazole rings have also
been described. Now we have synthesized these three
polyamines with a higher grade of purity in both the free and
hydrochloride forms, which, in the case of 2, required the
design of improved synthesis and purification procedures. In
addition, a more lipophilic pyrazole-containing dinuclear
polyamine with bulky octyl groups attached to the amine
nitrogens at the side chains (4), which had also been described
previously, was prepared again.22 These compounds were
selected because it is well-known that polyaminic macrocycles
including pyrazole moieties present a variety of possibilities as
chelating agents, since the pyrazole units can behave as
monodentate ligands through their sp2 nitrogens or as bridged
bis(monodentate) η1:η1 ligands through deprotonation.23 Some
of us previously showed that transition metals favor the
deprotonation of 1H-pyrazole rings at the polyamine macro-
cycle in aqueous solution and at physiological pH, forming
Cu(II) or Zn(II) dipyrazolate salts in which the nitrogens acted
as exobidentate ligands.19,24,25 On the other hand, X-ray
diffraction techniques showed that the N-Bn substituted sp2
nitrogens of the pyrazole rings in 3 behave as monodentate
ligands in the formation of Cu(II) or Zn(II) neutral dinuclear
complexes.19,25 Other diverse acyclic or cyclic polyamine
ligands that are good complexants of transition metals have
been used as functional mimics of SOD enzymes,26 and
modifying the homeostasis of essential metal ions through the
use of metal chelating compounds is a common practice in
inorganic medicinal chemistry.27
RESULTS AND DISCUSSION
■
Chemistry. The polyaminic macrocycles 1−4 (Figure 1)
were prepared as shown in Scheme 1. Following a procedure
previously reported by some of us,19 the cryptand containing
three 1H-pyrazole rings (1, mp 244−245 °C from H2O, 65%
yield) was obtained via the condensation of 1H-pyrazole-3,5-
dicarbaldehyde (5) with tris(2-aminoethyl)amine in methanol
and a further in situ reduction of the resulting Schiff base with
sodium borohydride. The monocyclic polyamine containing
two 1-benzyl-pyrazole units (3) was obtained in two separated
steps as also previously reported by us.21 In the first step,
dipodal [2 + 2] condensation of the previously described 1-
benzylpyrazole-3,5-dicarbaldehyde (7)21 with 1,5-diamine-3-
azapentane in acetonitrile afforded a Schiff base 8 containing
two imidazolidine rings located at the pyrazole side close to the
benzyl substituents [161−162 °C (EtOH), 55% yield]. After
this, the reduction of 8 in the presence of sodium borohydride/
ethanol led to the desired polyamine 3 [137−138 °C (toluene),
83% yield]. The related polyamine containing two 3,5-
disubstituted 1-methylpyrazole rings (2) had previously been
reported as a thick liquid.20 For this work we used an improved
method of synthesis and more accurate purification procedures,
and were able to isolate compound 2 as a crystalline white solid
[mp 133−135 °C (toluene)] in 70% yield. The new method
was performed in a unique reaction step via the condensation
of 1-methylpyrazole-3,5-dicarbaldehyde with 1,5-diamino-3-
azapentane and in situ treatment with sodium borohydride.
The polyamine macrocycle N-octyl-substituted at the side
chains (4) was obtained as previously described22 via the
condensation of 1H-pyrazole-3,5-dicarbaldehyde with 1,5-
diamino-3-octyl-3-azapentane in methanol and in situ treatment
with sodium borohydride. After careful chromatographic
purification, compound 4 was isolated as a pure syrup with Rf
0.54 (TLC, MeOH/30% aqueous NH4OH, v:v 10:1) with a
53% yield.
In this work, we evaluated the effectiveness of compounds
1−4 as selective inhibitors of Fe-SOD in relation to human
CuZn-SOD; their in vitro antiparasitic activity and toxicity
against Vero cells were tested and compared with values
obtained for the reference drug benznidazole (BZN), and on
the basis of the results obtained, their in vivo trypanosomicidal
activity on female BALB/c mice was measured both in the
acute and chronic phase. The effect of compounds 1−4 on the
ultrastructure of T. cruzi was also studied by transmission
electronic microscopy (TEM) experiments in order to confirm
the type of damage caused to the parasite cells. A NMR analysis
of the nature and percentage of the metabolites excreted was
Finally, further treatment of compounds 1−4 with HCl in
EtOH afforded the corresponding hydrochloride salts 1·8HCl
(mp 267−269 °C, 85% yield), 2·6HCl (mp 255−256 °C, 88%
yield), 3·6HCl (mp 247−249 °C, 85% yield), and 4·6HCl (mp
230−231 °C, 81% yield) as stable solids. Among them,
compounds 2·6HCl and 3·6HCl are described for the first time
in this work. Both compounds were unequivocally identified on
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Figure 2. Effect of pyrazole-containing polyamine derivatives 1−3 on the infection rate and growth of T. cruzi. (A) Rate of infection, (B) mean
○
▲
number of amastigotes per infected Vero cell, and (C) number of trypomastigotes in the culture medium: ( ) control; ( ) BZN; (Δ) compound 1;
●
( ) compound 2; (+) compound 3. Measured at IC25. Values are the means of three separate experiments.
1
the basis of their analytical data and H NMR and 13C NMR
spectroscopy. Assignment of the NMR signals was achieved
using bidimensional techniques (gHMQC and gHMBC). The
ESI mass spectra (positive mode) showed molecular ions
corresponding to the proposed structures (m/z 419.4 and 571.5
for 2 and 3, respectively).
We added the number of times that the compound SI exceeded
the benznidazole SI, in parentheses, since this value indicates
the in vitro potentiality of the test compounds with respect to
the reference drug.
If we consider the activity IC50 data shown in Table 1, it can
be seen that the 1H-pyrazole cryptand 1 and the 1-
methylpyrazole macrocyclic polyamine 2 were the most active
of the four compounds tested in the three assays performed.
Furthermore, they were much more active than benznidazole
against both the extra- and intracellular forms of the parasite.
Next in the activity ranking was the 1-benzylpyrazole derivative
3, whereas the lipophilic polyamine 4, N-octyl-substituted in
the side chains, was the least active in all cases. Regarding the
cytotoxicity evaluation against Vero cells, it was shown that
compounds 1−3 were substantially less toxic than the reference
drug (IC50 = 149.1, 178.5, and 195.4 μM, respectively, against
13.6 μM for benznidazole), and even 4 was slightly less toxic
(IC50 = 21.5 μM). Considering now the more illustrative
selectivity index values, compounds 1 and 2 were again the
most potentially interesting, since their SI values were 143, 30,
and 40 times higher than that of benznidazole in the case of 1,
and 172, 29, and 46 times higher in the case of 2. Compound 3
was less effective in all cases but, in spite of this, its SI was 15,
28, and 24 times higher than that of BZN, whereas 4 showed SI
values very close to those of the reference drug (0.6, 1.4, and
1.8). If we take into account the structural features of the four
compounds tested, the bulky and lipophilic macrocycle 4 was
clearly the least active of all. When comparing 2 and 3, the N-
In Vitro Anti-T. cruzi Evaluation. In the first step, we
evaluated the in vitro activity of compounds 1−4 in their
hydrochloride forms against T. cruzi epimastigotes and
amastigotes of an SN3 strain isolated from Colombian R.
prolixus. Since extracellular epimastigotes are not the developed
form of the parasite in vertebrate hosts, assays performed using
these are less indicative than assays that use intracellular
amastigotes.28 With this in mind, we prepared and tested
extracellular epimastigotes and extracellular axenic amastigotes,
and in a further step, we infected Vero cells with metacyclic
forms of T. cruzi that were transformed into amastigotes 1 day
after infection. We selected this cell line because it has shown to
be a very effective host for T. cruzi parasites.
The IC50 values obtained for the epimastigotes, axenic
amastigotes, and intracellular amastigotes for the test
compounds at concentrations of 1−100 μM are shown in the
first three columns of Table 1, which also includes the results
obtained from the reference drug benznidazole. Toxicity values
against mammalian Vero cells after 72 h of culture were also
calculated, and the selectivity indexes (SI = IC50 Vero cells
toxicity/IC50 activity of extracellular or intracellular forms of the
parasite) are also shown in the last three columns of Table 1.
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Figure 3. (A) In vitro inhibition (%) of Fe-SOD in T. cruzi epimastigotes by compounds 1−4 (activity 20.77 3.18 U/mg). (B) In vitro inhibition
of CuZn-SOD in human erythrocytes by compounds 1−4 (activity 23.36 4.21 U/mg). The values are the average of five separate determinations.
Differences between the activities of the control homogenate and those incubated with compounds 1−4 were obtained using the Newman−Keuls
test. IC50 = the concentration required to give 50% inhibition, which was calculated via linear regression analysis from the Kc values at the
concentrations used (1, 10, 25, 50, and 100 μM).
methyl-substituted macrocycle 2, with the less bulky substituent
attached to the pyrazole nitrogen, was clearly more effective
than the N-benzyl-substituted analog 3. In summary, the less
hindered compounds 1 and 2 appeared to be promising
candidates for in vivo assays. After considering the much poorer
results obtained from compound 4 and in accordance with the
usual protocol, all subsequent assays were exclusively
performed with the three compounds that showed the most
significant activity in these preliminary tests, with the only
exception being those related to the inhibition of parasitic and
human SOD enzymes.
In order to achieve more accurate information concerning
the in vitro activity of compounds 1−3, the propagation of the
parasite in Vero cells was studied by measuring the infection
rates and the average number of amastigotes and trypomasti-
gotes present during a 10-day treatment period. Vero cells were
incubated for 2 days and then infected with metacyclic forms of
T. cruzi. The cells were invaded and progressive morphological
conversion to the amastigote forms took place. During days 1−
10 the rate of host cell infection gradually increased: 92%
percent cells were infected on day 10 (Figure 2A). The test was
performed again in the presence of each of the three
compounds assayed and also in the presence of the reference
drug. It was found that the infection rate decreased in all cases
with respect to the control by the end of the test, but not to the
same extent. The decrease in the infection rate for compounds
1−3 was always greater than 50%, making them much more
effective than benznidazole (23%). Compounds 1 and 2
showed an especially remarkable behavior, with decreases in the
infection rate of 74% and 80% respectively, when compared to
the control.
The average number of amastigotes increased to a peak on
the 6th day of culture and decreased thereafter (Figure 2B);
this was because the rupture of Vero cells released amastigotes,
which were further transformed into trypomastigotes. The
results obtained agreed with those mentioned above for the
infection rates. At the end of treatment, the three test
compounds had significantly decreased the number of
amastigotes present in the cells by 50%, 42%, and 26%,
respectively, for 1, 2, and 3, whereas BZN only showed a
reduction of 13%. As before, compounds 1 and 2 were clearly
more efficient than the bulky N-substituted 3.
Regarding the number of trypomastigotes found in the
medium (Figure 2C), a maximum was reached in the control
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on day 10 (7.3 × 103), but this value was substantially reduced
in the presence of compounds 1 and 2 (70% and 64%,
respectively) and was even further reduced in the presence of 3
(34%) and the reference drug (46%). In summary, the Vero
cells propagation data showed results that are in accordance
with those described for intra- and extracellular forms of T.
cruzi.
mouse died in any of our experiments performed either with
the control or with compounds 1−3 at the concentrations used
(1 mg/kg). However, the survival percentage for the mice
treated with BZN was only about 80%. The female mice were
inoculated with trypomastigotes using the method described in
the Experimental Section, and treatment with the compounds
to assay began via the ip route 5 days postinfection and was
maintained for 5 days. A group of mice treated in the same
manner but only using vehicle (control) was also included.
During the study of acute phase activity, the level of parasitemia
was determined every 2 days.
Inhibitory Effect on the T. cruzi Fe-SOD Enzyme. Since
we had previously found that some benzo[g]phthalazine
derivatives are able to complex metal ions and show remarkable
inhibitory activity on the antioxidant enzyme Fe-SOD of T.
cruzi,16,17 compounds 1−4 were tested in order to estimate
their potential to bind in the active site, thereby inhibiting the
enzyme. We used epimastigote forms of T. cruzi, which were
shown to excrete Fe-SOD when cultured in a medium lacking
inactive fetal bovine serum,29 and a range of drug
concentrations from 1 to 100 μM was applied in each case.
The results obtained are shown in Figure 3A, and the
corresponding IC50 values were calculated and are included in
the same figure. Significant inhibitory values of Fe-SOD activity
were found for all compounds tested. Compounds 1−3 showed
98% inhibition at the 50 μg/mL dose, and even the less active
N-octyl-substituted polyamine 4 achieved 65% inhibition at the
same concentration. Macrocycles 1−3 showed between 70%
and 90% inhibition when the 25 μg/mL dose was used, and the
N-methyl-substituted macrocycle 2 reached a level of inhibition
of 75% at a concentration as low as 10 μg/mL. Figure 3A
clearly shows that at lower doses compound 2 was much more
effective than 1 and 3, whereas progressively increasing the
concentration of the drugs led to a similar effectiveness of 1 and
2 at first and then 1−3, later on. It was also found that the IC50
values were more significant in the case of compound 2.
The design of an effective drug able to inhibit the parasite Fe-
SOD requires the inhibition of human SOD to occur to a lower
extent; therefore, we have also assayed the effect of compounds
1−4 on Cu/Zn-SOD of human erythrocytes (Figure 3B). The
results obtained show that the inhibition percentages for human
CuZn-SOD were very small for both the higher and lower
dosages, with IC50 values reaching 274.1, 96.6, 154.6, and 89.2
μM for 1, 2, 3, and 4, respectively, in sharp contrast with the
values calculated in part A. All of the componds tested showed
remarkable selectivity in the inhibition of Fe-SOD/CuZn-SOD,
but the N-methyl-substituted polyamine 2, with an IC50 value
23 times higher in the case of Fe-SOD, appeared to be the most
effective and most selective compound for Fe-SOD inhibition
with respect to human SOD. These results enhance the interest
in the potential antiparasitical activity of the macrocyclic
polyamines studied in this work and could agree with the
hypothesis of some kind of interaction with the iron atom of
SOD being one of the feasible mechanisms involved in this
activity.
Figure 4 shows the number of trypomastigotes against days
of treatment until day 30. On the day of the maximum parasitic
Figure 4. Parasitemia in the murine model of acute Chagas disease:
○
▲
control ( ) mice and mice that received 1 mg/kg doses of BZN ( ),
●
compound 1 (Δ), compound 2 ( ), and compound 3 (+).
burden (day 14 after infection) all of the test compounds
greatly reduced the number of trypomastigotes. Furthermore, it
was shown that compounds 1 and 2 reduced the level of
parasitemia on day 30 by 53% and 39%, respectively. These
values were significantly greater than that found for the
reference drug (26%). However, the N-benzyl-substituted
polyamine 3 was less effective than benznidazole (16%).
Concerning activity in the chronic phase, Table 2 shows the
differences found in the level of anti-T. cruzi antibodies between
Table 2. Differences in the Level of Anti-T. cruzi Antibodies
between Days 40 and 120 Postinfection for Compounds 1−3
and BZN, Expressed in Absorbance Units (abs)
a
b
compound
ΔA
control (untreated)
+0.206
+0.116
−0.437
−0.291
+0.008
benznidazole
1
2
3
In Vivo Anti-T. cruzi Activity. The good results obtained
in the in vitro tests prompted us to study the in vivo activity of
compounds 1−3 on female BALB/c mice. Since the
effectiveness of drugs currently in use against Chagas disease
varies widely from the acute to the chronic phase, we decided
to evaluate the impact of our compounds on both phases. For
the acute phase experiments, we considered the first 30 days
after infection, whereas the effect on the chronic phase was
studied between days 40 and 120 after infection. The
intraperitoneal doping route was preferred to the intravenous
procedure because it is well-known that intraperitoneal
treatment substantially reduces animal mortality.30 In fact, no
a
1 mg/kg/day, intraperitoneal route, administered for 5 days (see
b
Experimental Section). ΔA = (absorbance at 490 nm, day 120
postinfection) − (absorbance at 490 nm, day 40 postinfection).
days 40 and 120 postinfection. It was shown that compounds 1
and 2 significantly reduced the levels of antibodies with respect
to the control, where the effect of compound 1 was much more
consistent. Compound 3 showed a lower performance, but it
was still better than that of BZN. The order of in vivo activity
was as follows: 1 > 2 ≫ 3 > BZN. It should be noted that the
bicyclic macrocycle 1 was the most effective against both the
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acute and chronic phases of Chagas disease. A certain
relationship between the structural features of the compounds
tested and the trypanosomicidal activity observed can be
tentatively proposed if we consider that all of the compounds
contain polyaminic moieties that are easy to protonate. We
determined the protonation constants of compound 2 at 298.1
K in 0.15 M NaCl and the results obtained were compared to
the constants previously calculated for 1 and 3.19,21,31 Plots of
the average protonation degree vs pH were calculated from the
protonation constants and are collected in the Supporting
Information (Table S1 and Figure S1). Figure 5 shows a bar
We were interested in evaluating the effect of the macrocyclic
polyamines on metabolite excretion; thus, the 1H NMR spectra
obtained from the trypanosomatids treated with compounds
1−4 (Figure 2S in the Supporting Information) were recorded
and compared with those obtained from cell-free medium
(control) 4 days after inoculation with the T. cruzi strain. In the
control experiment, the major metabolites excreted by T. cruzi
were acetate and succinate, with smaller percentages of L-
alanine, L-lactate, and ethanol, in agreement with the data in the
literature.34 When the trypanosomatids were treated with
compounds 1−3, the excretion of some of these catabolites was
neatly altered at the dosage used, whereas the presence of
benznidazole did not lead to significant alterations in fuel
metabolism. Variation percentages in the height of the signals
corresponding to the significant catabolites are shown in Table
3. It can be seen that the excretion of both succinate and acetate
Table 3. Variations in the Percentages of Catabolic End
Products Excreted by T. cruzi Epimastigotes in the Presence
of Compounds 1−3 and Benznidazole Compared to the
a
Control
compd
Suc
Ac
Lac
Ala
Et
1
−7
−70
−19
=
−22
−15
−23
+6
=
=
=
=
=
=
=
=
=
2
−58
=
3
BZN
=
Figure 5. Bar plot of the degree of protonation of compounds 1−3 at
pH = 7.4.
a
Suc, succinate; Ac, acetate; Lac, L-lactate; Ala, L-alanine; Et, ethanol;
−, peak inhibition; +, peak increasing; =, peak modification
undetected.
plot in which the degree of protonation of the three
compounds at physiological pH (7.4) is represented. It is
evident from this figure that the order of protonation was 1 > 2
> 3, so that the protonation ability could, in some way, be
conditioning the in vivo activity of these compounds.
was inhibited by all three compounds. The excretion of acetate
was moderately inhibited from 15% to 23%, which was very
similar for all three compounds tested. However, greater
variations were found in the inhibition of succinate, the level of
which was low for the cryptand 1, moderate for the N-benzyl-
substituted polyamine 3, and surprisingly high (70%) for the N-
methyl-substituted polyamine 2, which was also the only
compound that inhibited the formation of lactate (58%). Since
the less bulky compound 2 showed greater alterations to the
level of glucose metabolism of the parasite, and taking into
account the relevant role of mitochondria in the formation of
the catabolic end products,35 it could be possible that its smaller
size allows it to more easily penetrate the cristae (tubular
invaginations of the inner mitochondrial membrane)36 leading
to subsequent changes in the metabolic pathway. Regarding this
point, it should be noted that 2 not only showed greater
alterations in glucose catabolism but also that it led to greater
levels of Fe-SOD inhibition. Since the Fe-SOD present in
mitochondria is an essential part of the whole enzyme excreted
by epimastigotes in the inhibition tests,37 this fact would agree
with the hypothesis above that this less bulky compound might
have a greater ability to pass through the mitochondrial
membrane.
Other Studies on the Mechanism of Action. In order to
obtain further information about the action mechanisms of
compounds 1−3 on the parasite, we performed the following
experiments.
Metabolite Excretion Effect. Trypanosomatids depend on
the carbon sources present in their hosts for their energy
metabolism. Most of them, including T. cruzi, mainly use
glucose as their energy source, which is abundant in the fluids
of vertebrate hosts. At the end of the oxidation process glucose
should be totally degraded to CO2, but the parasite is not able
of this under aerobic conditions and excretes a large part of its
carbon skeleton into the medium as fermented metabolites at
variable grades of oxidation.32 1H NMR spectra enable us to
determine the fermented metabolites excreted by the
trypanosomatid during its in vitro culture, and the use of this
technique when a drug has been added to the parasite may
provide us with valuable information regarding the metabolic
changes induced by the drug. The most significant of the
metabolites excreted by T. cruzi is succinate, the main role of
which could be maintenance of the glycosomal redox balance,
since it provides two oxidoreductase enzymes that allow
reoxidation of the NADH produced in the glycolytic pathway.
The fermentation of succinate is favored because it only
requires half the amount of phosphoenolpyruvate (PEP)
produced to maintain the NAD+/NADH balance. The
remaining pyruvate is converted inside the mitochondria33
mainly in succinate but also into other major catabolites: in T.
cruzi these metabolites are acetate, L-lactate, L-alanine, and, to a
lesser extent, ethanol.
Ultrastructural Alterations. The morphological alterations
of T. cruzi epimastigotes caused by compounds 1−3 were
analyzed with transmission electron microscopy (TEM), in
order to determine whether or not the post-treatment
modifications in the parasite cells could provide more detailed
evidence about the way in which the compounds tested affected
parasite survival. The most significant variations compared to
the control cells are shown in Figure 6. All three compounds
caused substantial damage to the parasite cells, and as a rule,
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indentations and undulations. The presence of electron-dense
particles similar to those mentioned in the case of 2 was also
observed. Comparing the results obtained for both compounds,
the most significant difference found was that modifications in
the structure of the membranes were much more substantial for
polyamine 2, again in accordance with the lower effectiveness
shown by 3 in the in vivo activity assays.
In summary, a wide range of ultrastructural alterations to the
epimastigote forms of T. cruzi treated with compounds 1−3
was found. These alterations, which mainly took place at the
mitochondrial and cytoplasmic levels, could be related to the
metabolic changes mentioned above concerning the production
of succinate and acetate, which might originate from a
disturbance in the enzymes involved in pyruvate metabolism
inside the cells.
Histopathological Analysis. In order to gain further
insights into the level of toxicity induced by our compounds on
the liver, a key organ in many vital functions, we performed a
histopathological analysis on female BALB/c mice infected with
the parasite and treated with compounds 1−3. The results
obtained are summarized in Table 4 and shown graphically in
Table 4. Degrees of Histopathological Alteration Found in
the Liver of Mice Infected with T. cruzi: Untreated and
Treated with 5 mg/kg of Benznidazole or Compounds 1−3,
after 120 dpi
Figure 6. Ultrastructural alterations shown by TEM in epimastigotes
of T. cruzi treated with compounds 1−3. (Plate C) Control parasite
showing organelles with their characteristic features, such as
mitochondrion (M), glycosomes (G), microtubules (MT), vacuoles
(V), and flagellum (F); bar = 0.583 μm. (Plate 1) Epimastigotes of T.
cruzi treated with compound 1 with intense vacuolization (V) and very
electron dense (arrow) and distorted shapes (DO); bar = 1.590 μm.
(Plate 2) Epimastigotes of T. cruzi treated with compound 2, with
vacuolization (V), cytoplasm full of reservosomes (R), and altered
nucleus (N); bar = 0.350 μm. (Plate 3) Epimastigotes of T. cruzi
treated with compound 3, with vacuolization (V), a distorted shape
(DO), and electron-dense sectors (arrow); bar = 1.000 μm.
a
damage level
histopathological
b
alterations
control − control +
BZN
++
1
2
3
granulomas with
limphocitic
infiltration
+
+++++
+
+
++
delocalized hepatic
destruction
0
+
0
++++
+++
++++
+
0
+
0
blood vessels with
thickened walls
0
0
++ ++
lymphocytic
++++
+
0
++
infiltration in the
portal tracts
extensive cytoplasmic vacuolization was observed in all cases.
However, cryptand 1 was clearly the most effective (second
panel in Figure 6) because it completely altered the
morphology of the protozoan cells which, after treatment,
showed extremely electron-dense vesicles within both the
cytoplasm and the nucleus. The shape of most of the parasites
was so distorted that their morphology was completely
unrecognizable, and they were also filled with empty vacuoles
or membranous structures. Only very few parasites showed a
normal appearance, and many were dead. These results were
consistent with the predominant in vivo activity previously
found for 1 compared to the monocyclic polyamines. Although
the epimastigotes treated with 2 and 3 clearly showed less
damaged, they also presented various morphological alterations
that differed depending on the nature of the compound tested.
Treatment with the N-methyl-substituted polyamine 2 (third
panel in Figure 6) reslted in the appearance of reservosomes
inside the cytoplasm of the trypanosomatids, and in many cases
the nucleus was unrecognizable, presenting a flabby aspect and
discontinuous membrane. Many parasites showed extremely
electron-dense vesicle-like structures reminiscent of waste
products. In turn, the N-benzyl-substituted polyamine 3 (fourth
panel in Figure 6) induced strong vacuolization of the
epimastigotes; furthermore, the vacuoles were occasionally
associated, occupying the entire cytoplasm. Sometimes the
shape of the parasites was distorted, showing abnormal
interstitial hemorrhage
0
0
+++++
+++
++
0
0
++
+
hepatic destruction in
the portal tracts
+++++
++
+
necrotic cells in the
portal tracts
0
++++
+++
+
+
0
a
Damage level: + mild; ++ light; +++ moderate; ++++ abundant;
b
+++++ severe. Sanchez-Moreno et al.17b
Figure 3S, which can be found in the Supporting Information.
We found that mice that were infected but not treated
developed severe alterations (control + column) after 120 days
compared to uninfected mice (control − column). Those
modifications were undoubtedly due to the action of the
parasite during the chronic phase. The most remarkable
alterations were lymphoyitic infiltration with the formation of
many microgranulomas (+++++), delocalized hepatic destruc-
tion (++++), blood vessels with thickened walls (+++),
lymphocitic infiltration in the portal tracts (++++), and
interstitial hemorrhage (+++++). The presence of many
necrotic cells in the portal tracts was also detected (++++).
In a previous assay17b we found that treatment of parasitized
mice with the reference drug (BZN) reduced the formation of
granulomas (++), interstitial hemorrhage (++), thickening of
blood vessels, and, to a lesser extent, the presence of necrotic
cells (+++), due to the lower number of parasites remaining
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hexaene (1). Pyrazoledicarbaldehyde21 (372 mg, 3.0 mmol) was
dissolved in warm methanol (180 mL) and added dropwise
during 2 h under argon to a stirred solution of tris(2-
aminoethyl)amine (292 mg, 2.0 mmol) in methanol (120 mL).
After stirring overnight at room temperature, NaBH4 (340 mg,
9.0 mmol) was added portionwise, and after 2 h the solvent was
evaporated to dryness under reduced pressure. The residue was
directly recrystallized from water, and the obtained crystals,
after drying under vacuum at 80 °C over phosphorus pentoxide,
were shown to be the title compound as the monohydrate (381
mg, 65%). Mp: 244−245 °C (lit. mp: 245 °C19). 1H NMR (400
MHz, CD3OD): δ 6.24 (s, 3H, H-4), 3.88 (s, 12H, H-6), 2.90−
2.93 (m, 12H, H-7), 2.78−2,81 (m, 12H, H-8) ppm. 13C NMR
(100 MHz, CD3OD): δ 147.92 (br s, C-3, C-5), 103.28 (C-4),
54.86 (C-8), 48.54 (C-7), 46.47 (C-6) ppm. MS (FAB) m/z
(%): 569 (MH+, 60). Anal. (C27H48N14·H2O) C, H, N.
after drug action. On the contrary, the level of delocalized
hepatic destruction was unchanged and very severe hepatic
destruction was found in the portal tracts (+++++), caused by
the BZN toxicity.
After treatment of the infected mice with polyamines 1−3 it
was shown that the alterations, as a whole, were not as
significant as those mentioned above for BZN, indicating a
remarkably lower level of toxicity for the three compounds
tested at 120 dpi. Compounds 1 and 2, which were the most
active in vivo, generally showed lower levels of damage than
compound 3. In both cases all of the aforementioned
alterations were nonexistent (0) or mild (+), with the only
exceptions being hepatic destruction in the portal tracts (1) and
blood vessels thickening (2), which only occurred to a light
degree (++). A strong reduction in the levels of hepatic
destruction (both delocalized and in portal tracts) compared to
BZN was especially noteworthy for compounds 1 and 2. Even
compound 3 showed much better results than BZN regarding
the hepatic destruction (mild or none at all).
From the results of this work, we conclude that the
macrocyclic polyamines 1 and 2 showed remarkable in vitro
and in vivo trypanosomicidal activity, being especially active
against both the acute and chronic phases of Chagas disease.
These compounds also showed a much lower level of toxicity
than benznidazole, as shown by the Vero cell and
histopathological studies, and they were almost inactive against
human SOD but active against the Fe-SOD of the parasite. On
this basis, we believe that both compounds fulfill the
requirements needed in order to perform a more detailed
study of the nature of the mechanisms involved in their activity
patterns, and furthermore, that they are serious candidates for
studying parasitical activity at a higher level.
[1]·8HCl. A mixture of compound 1 (293.4 mg, 0.5 mmol) and 1 M
aqueous hydrochloric acid (4 mL) was stirred for 12 h. After addition
of ethanol (50 mL), the crystallized white solid was isolated by
filtration and dried over phosphorus pentoxide at 60 °C. Yield: 381 mg
19
1
(85%). Mp: 267−269 °C (lit. mp: 266−269 °C ). H NMR (500
MHz, D2O): δ 6.59 (s, 3H, H-4), 4.27 (s, 12H, H-6), 3.31 (t, 12H, H-
7), 2.28 (t, 12H, H-8) ppm. 13C NMR (125 MHz, D2O): δ 140.32 (C-
3, C-5), 109.88 (C-4), 51.25 (C-8), 46.24 (C-7), 44.58 (C-6) ppm.
Anal. (C27H48N14·8HCl·2H2O) C, H, N, Cl.
13,26-Dimethyl-3,6,9,12,13,16,19,22,25,26-decaazatricyclo-
[22.2.1.111,14]octacosa-1(27),11,14(28),24-tetraene (2).20 A solution
of 1-methyl-3,5-pyrazoledicarbaldehyde (6) (276 mg, 2 mmol) in 10
mL of MeCN was added in a dropwise manner, under argon, to a
constantly stirred solution of freshly distilled 1,5-diamino-3-azapentane
(206 mg, 2 mmol) in 20 mL of the same solvent. After stirring
overnight at room temperature, MeOH (40 mL) and sodium
borohydride (304 mg, 8 mmol) were added, and after 2 h, the
solvent was evaporated to dryness under reduced pressure. Then, 50
mL of CHCl3 was added, and after stirring for 2 h, the resulting white
precipitate (borosodic salts) was filtered and collected. The filtrate was
concentrated and the residue was purified by flash column
chromatography on silica gel (MeOH/30% aqueous NH4OH, 49:1
to 46:4). The fractions containing the product of Rf = 0.5 (TLC,
MeOH/30% aqueous NH4OH, 4:1) were combined and evaporated
to dryness, and the residue was recrystallized from toluene (8 mL) to
give a white solid which was dried over P2O5 at 60 °C for 12 h
affording 295 mg (70% yield) of 2. Mp: 133−135 °C (lit.: thick oil).20
1H NMR (CDCl3, 400 MHz): δ 6.06 (s, 2H, H-4), 3.75 (s, 6H, N−
CH3), 3.72 (s, 4H, H-6′), 3.71 (s, 4H, H-6), 2.75 (m, 16H, H-7, H-7′,
H-8, H-8′), 2.19 (br s, 6H, NH) ppm. 13C NMR (CDCl3, 100 MHz):
δ 149.86 (C-3), 141.70 (C-5), 104.01 (C-4), 49.16, 49.01, 48.97 (C-7,
C-7′, C-8, C-8′), 47.18 (C-6), 44.44 (C-6′), 36.06 (N−CH3) ppm. MS
(ESI+, MeOH) m/z (%): 441.4 (M + Na)+, 419.5 (MH)+. Anal.
(C20H38N10) C, H, N.
[2]·6HCl. A mixture of 2 (209 mg, 0.5 mmol) and 1 M aqueous
hydrochloric acid (4 mL) was stirred for 12 h. After the addition of
ethanol (50 mL), a white solid was formed, filtered off, and dried over
phosphorus pentoxide at 60 °C for 48 h. Yield: 296 mg (88%). Mp:
255−256 °C. 1H NMR (D2O, 500 MHz): δ 6.68 (s, 2H, H-4), 4.39 (s,
4H, H-6′), 4.21 (s, 4H, H-6), 3.79 (s, 6H, N−CH3), 3.36 (m, 16H, H-
7, H-7′, H-8, H-8′) ppm. 13C NMR (D2O, 125 MHz): δ 142.27 (C-3),
135.75 (C-5), 111.12 (C-4), 45.24, 45.14 (C-8, C-8′), 45.01 (C-6),
43.56, 43.00 (C-7, C-7′), 42.34 (C-6′), 38.00 (N−CH3) ppm. MS
(ESI+, H2O/HCOOH): m/z 419.4 (MH − 6HCl)+. Anal.
(C20H38N10·6HCl·2H2O) C, H, N, Cl.
EXPERIMENTAL SECTION
■
Chemistry. 3,5-Dimethylpyrazole, tris-2-(aminoethyl)amine, and
1,5-diamino-3-azapentane were purchased from commercial sources
(Sigma-Aldrich) and used without further purification. 1,5-Diamino-3-
octylazapentane is not commercial and was obtained from 1,5-
diamino-3-azapentane.22 1H-3,5-pyrazoledicarbaldehyde and 1-benzyl-
3,5-dicarbaldehyde were obtained from 3,5-dimethylpyrazole accord-
ing to a procedure previously described by our research group.21 1-
Methyl-3,5-pyrazoledicarbaldehyde was obtained from diethyl 1-
methyl-3,5-pyrazoledicarboxylate as described by Kumar et al.20 The
solvents were dried using standard techniques.38 All reactions were
monitored by thin-layer chromatography (TLC) using DC-Alufolien
Silica gel 60PF254 (Merck; layer thickness, 0.2 mm). The compounds
were detected using UV light (254 nm), iodine, or phosphomolybdic
acid. Melting points were determined using a Reichert-Jung hot-stage
microscope and are uncorrected. The H and 13C NMR spectra were
1
recorded on Varian Inova-300, Varian Inova-400, and Varian Unity-
500 spectrometers. The chemical shifts are reported in parts per
million (ppm) from tetramethylsilane (TMS), but they were measured
against the solvent signals. All assignments were made on the basis of
1H−13C heteronuclear multiple quantum coherence experiments
(GHMQC and GHMBC). The electrospray ionization (ESI) mass
spectra were registered on a Hewlett-Packard 1100 MSD spectrometer
in the electrospray positive mode. The fast atom bombardment (FAB)
mass spectra were obtained on a VG AutoSpec spectrometer using a
m-nitrobenzyl alcohol matrix. Elemental analyses were performed by
́
the Departamento de Analisis, Centro de Quimica Organica “Manuel
́ ́
Lora Tamayo”, CSIC, Madrid, Spain. Elemental analysis was used to
ascertain a purity higher than 95% for all of the biologically tested
compounds.
13,26-Dibenzyl-3,6,9,12,13,16,19,22,25,26,-decaazapentacyclo-
[22.2.1.111,14.02,6.015,19]octacosa-1(27),9,11,14(28),22,24-hexaene
(8). A solution of 1-benzyl-3,5-pyrazoledicarbaldehyde (7) (642 mg,
3.0 mmol) in 30 mL of MeCN was added dropwise to a stirring
solution of diethylenetriamine (310 mg, 3.0 mmol) in 30 mL of the
same solvent. After the mixture was stirred overnight, the Schiff base
imidazolidine 8 separated as a thick oil, which solidified after
scratching to give a white solid that was crystallized from EtOH
(464 mg, 55%). Mp: 161−162 °C (lit. mp: 160−162 °C21). 1H NMR
Preparation of the Macrocyclic Polyamines 1−4.
1,4,7,8,11,14,17,20,21,24,29,32,33,36-Tetradecaazapentacyclo-
[12.12.126,9.119,22.131,34]hentetraconta-6,9(41),19(40),21,31,34(39)-
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(CDCl3. 400 MHz): δ 8.35 (s, 1H, H-6), 8.31(s, 1H, H-6′), 7.72, s,
1H, H-4), 7.23, (s, 1H, H-4′), 5.55 (d, 1H, HA-BnCH2), 5.54 (d, 1H,
HB-BnCH2), 5.47 (d, 1H, HA-Bn′CH2), 5.40 (d, 1H, HB-Bn′CH2), 4.17
(s, 1H, H-9), 4.32 (s, 1H, H-9′), 3.78 (m, 1H, HA-7), 3.54 (m, 1H, HB-
7), 3.67 (m, 1H, HA-7′), 3.49 (m, 1H, HB-7′), 3.05, (m, 1H, HA-8),
2.51 (m, 1H, HB-8), 3.01 (m, 1H, HA-8′), 2.57 (m, 1H, HB-8′), 3.22(m,
1H, HA-10), 3.17 (m, 1H, HB-10), 3.18, (m, 1H, HA-10′), 3.12 (m, 1H,
HB-10′), 3.47 (m, 1H, HA-11), 2.34 (m, 1H, HB-11), 3.33 (m, 1H, HA-
11′), 2.37 (m, 1H, HB-11′) ppm. 13C NMR (CDCl3, 100 MHz): δ
150.18, 149.99 (C-3, C-3′), 144,70, 145.76 (C-5, C-5′), 105.66, 105.22
(C-4, C-4′), 155.62, 155.37 (C-6, C-6′), 74.78, 74.69 (C-9, C-9′),
59.24, 59.52 (C-7, C-7′), 54.04, 54.34 (C-8, C-8′), 45.18, 45.62 (C-10,
C-10′), 52.35, 52.80 (C-11, C-11′), 53.44, 53.42 (C-BnCH2, C-
Bn′CH2), 137.10, 137.94 (C-Bn ipso, C-Bn′ ipso), 126.73, 126.61 (C-
Bn m-, C-Bn′ m-),128.68, 128.60 (C-Bn o-, C-Bn′ o-), 127.61, 127.49
(C-Bn p-, C-Bn′ p-) ppm. MS (FAB) m/z (%): 563 (MH+, 54). Anal.
(C32H38N10) C, H, N.
13,26-Dibenzyl-3,6,9,12,13,16,19,22,25,26-decaazatricyclo-
[22.2.1.111,14]octacosa-1(27),11,14(28),24-tetraene (3). To a stirred
suspension of Schiff base 8 (450 mg, 0.8 mmol) in 20 mL of EtOH
was added solid NaBH4 (302 mg, 8.0 mmol) portionwise. The
reduction was carried out at room temperature for 2 h. The solvent
was then evaporated, and after addition of 20 mL of H2O, the pH was
adjusted to 9 by addition of 5% aqueous HCl. The solution was
extracted with CHCl3, and the organic layer was dried (Na2SO4) and
evaporated to yield a white solid, which was crystallized from toluene
(378 mg, 83%). Mp: 137−138 °C (lit. mp: 136−138 °C21). 1H NMR
(D2O, pH = 12, 400 MHz): δ 7.15 (m, 6H, H-Bn m-, p-), 6.97 (m, 4H,
H-Bn o-), 6.17 (s, 2H, H4), 5.17 (s, 4H, H-BnCH2), 3.57 (s, 8H, H-6,
H-6′, 2.41 (s, 8H, H-7, H-7′), 2.32 (t, 4H, H-8), 2.23 (t, 4H, H-8′)
ppm. 13C NMR (D2O, pH 12, 75 MHz): δ 150.75 (C-3), 143.70 (C-
5), 138.03 (C-Bn ipso), 130.02 (C-Bn o-), 127.64 (C-Bn m-), 129.05
(C-Bn p-), 106.34 (C-4), 53.20 (C-BnCH2), 47.14 (C-7, C-7′), 48.15
(C-8), 46.73 (C-8′), 45.90 (C-6), 43.42 (C-6′) ppm. MS (FAB) m/z
(%): 571 (MH+, 69). Anal. (C32H46N10) C, H, N.
[3]·6HCl. A mixture of 3 (285 mg, 0.5 mmol) and 1 M aqueous
hydrochloric acid (4 mL) was stirred for 12 h. After the addition of
ethanol (50 mL), a white solid was formed, filtered off, and dried over
phosphorus pentoxide at 60 °C for 48 h. Yield: 350 mg (85%). Mp:
247−249 °C. 1H NMR (D2O, 400 MHz): δ 7.28 (m, 6H, H-Bn m-, H-
Bn p-), 7.01 (m, 4H, H-Bn o-) 6.78 (s, 2H, H-4), 5.40 (s, 4H, H-
BnCH2) 4.35 (s, 4H, H-6′), 4.26 (s, 4H, H-6), 3.35−3.40 (m, 8H, H-
7′, H-8′) 3.30−3.35 (m, 8H, H-7, H-8) ppm. 13C NMR (CDCl3, 75
MHz): δ 143.22 (C-3), 136.21, 136.82 (C-5, C-Bn ipso: interchange-
able signals), 130.44 (C-Bn m-), 129.70 (C-Bn p-), 128.10 (C-Bn o-),
111.23 (C-4), 54.58 (C-BnCH2), 45.30 (C-6), 45.20 (C-8′), 45.14 (C-
7′) 43.75, 43.19 (C-7, C-8: interchangeable signals), 42.56 (C-6′) ppm.
MS (ESI+, H2O/HCOOH) m/z positive mode (%): 571.5 (MH −
6HCl)+. Anal. (C32H46N10·6HCl·2H2O) C, H, N, Cl.
6,19-Dioctyl-3,6,9,12,13,16,19,22,25,26-decaazatricyclo-
[22.2.1.111,14]octacosa-1(27),11,14(28),24-tetraene (4). 3,5-Pyrazole-
dicarbaldehyde (372 mg, 3 mmol) was dissolved in hot methanol (180
mL). This solution was then cooled to room temperature and added
dropwise under argon to a stirred solution of 1,5-diamine-3-octyl-3-
azapentane22 (646 mg, 3 mmol) in methanol (300 mL). The reaction
was monitored by TLC (Cl3CH/MeOH 10:1), and when it was
complete (ca. 12 h), NaBH4 (681 mg, 18 mmol) was added
portionwise. After 2 h of reaction, the solvent was evaporated to
dryness under reduced pressure. The residual syrup was purified by
flash column chromatography on silica gel (MeOH/30% aqueous NH3
48:2). The free macrocycle was obtained as a colorless syrup. TLC: Rf
= 0.54, MeOH/30% aqueous NH4OH 10:1. Yield: 517 mg (53%). 1H
NMR (CDCl3, 400 MHz): δ 6.11(s, 2H, H-4), 3.81 (s, 8H, H-6), 2.84
(m, 8H, H-7), 2.65 (m, 8H, H-8), 2.48 (m, 4H, H-1 octyl), 1.41 (m,
4H, H-2 octyl), 1.21 (m, 20H, H-3 octyl, H-4 octyl, H-5 octyl, H-6
octyl, H-7 octyl), 0.83 (t, 6H, H-8 octyl) ppm. 13C NMR (CDCl3, 100
MHz): δ 145.67 (C-3, C-5), 102.47 (C-4). 54.18 (C-1 octyl), 51.29
(C-8), 46.98 (C-7), 45.30 (C-6), 31.79 (C-6 octyl), 29.49, 29.25,
27.51, 25.88 (C-2 octyl, C-3 octyl, C-4 octyl, C-5 octyl), 22.6 (C-7
octyl), 14.1 (C-8 octyl) ppm. MS (ESI+, MeOH) m/z (%): 616
(MH+). Anal. (C34H66N10·2H2O) C, H, N.
[4]·6HCl. A mixture of compound 4 (326 mg, 0.5 mmol) and 1 M
aqueous HCl (3 mL) was stirred for 12 h. After addition of EtOH (50
mL), the solvent was evaporated to dryness. Next, 30 mL of EtOH was
added, and the resulting solution was stirred for 1 h. A white solid was
formed, filtered off, and dried over phosphorus pentoxide at 60 °C for
48 h. Yield: 337 mg (81%). Mp: 230−231 °C (lit. mp:22 228−231
°C). 1H NMR (D2O, 400 MHz): δ 6.57 (s, 2H, H-4), 4.22 (s, 8H, H-
6), 3.36 (m, 8H, H-8), 3.30 (m, 8H, H-7), 3.04 (t, 4H, H-1 octyl), 3.22
(m, 4H, H-2 octyl), 1.06 (m, 10H, H-3 octyl, H-4 octyl, H-5 octyl, H-6
octyl, H-7 octyl), 0.61 (t, 6H H-8 octyl) ppm. 13C NMR (D2O, 100
MHz): δ 139.68 (C-3, C-5), 110.18 (C-4), 55.52 (C-1 octyl), 49.87
(C-8), 43.89 (C-6), 41.81 (C-7), 32.11 (C-6 octyl), 29.33 (C-4 octyl,
C-5 octyl), 26.73 (C-3 octyl), 23.97 (C-2 octyl), 23.13 (C-7 octyl),
14.55 (C-8 octyl) ppm. MS (ESI+, H2O): m/z 616 ([MH − 6HCl]+).
Anal. (C34H66N10·6HCl) C, H, N.
Parasite Strain, Culture. T. cruzi SN3 strain of IRHOD/CO/
2008/SN3 was isolated from domestic R. prolixus, the biological origin
of which is Guajira, Colombia.39 Epimastigote forms were grown in
axenic Grace’s insect medium (Gibco) supplemented with 10%
inactivated fetal bovine serum (FBS) at 28 °C in tissue-culture flasks.
In order to obtain the parasite suspension for the trypanocidal assay,
the epimastigote culture (in the exponential growth phase) was
concentrated by centrifugation at 400g for 10 min and the number of
flagellates was counted in a hemocytometric chamber.
Transformation of Epimastigotes to the Metacyclic Form.
Metacyclogenesis was induced by culturing the parasites at 28 °C in
modified Grace’s medium (Gibco) for 12 days, as described
previously.40 Twelve days after cultivation at 28 °C, metacyclic
forms were counted in a Neubauer hemocytometric chamber. The
proportion of metacyclic forms was around 40% at this stage.
Cell Culture and Cytotoxicity Tests. Vero cells (Flow) were
grown in RPMI (Gibco), supplemented with 10% inactivated fetal
bovine serum, in a humidified 95% air, 5% CO2 atmosphere at 37 °C
for 2 days. For the cytotoxicity testing, cells were placed in 25 mL
Colie-based bottles (Sterling) and centrifuged at 100g for 5 min. The
culture medium was removed, and fresh medium was added to a final
concentration of 1 × 105 cells/mL. This cell suspension was
distributed in a culture tray (with 24 wells) at a rate of 100 μL/well
and incubated for 2 days at 37 °C in a humidified atmosphere enriched
with 5% CO2. The medium was removed, and fresh medium was
added together with each test compound (at concentrations of 100,
50, 25, 10, and 1 μM). After 72 h of treatment, cell viability was
determined by flow cytometry according to a methodology described
by us.41
In Vitro Activity Assays: Extracellular Forms. Epimastigote
Assay. Epimastigotes were collected in the exponential growth
phase and distributed in culture trays (with 24 wells) at a final
concentration of 5 × 104 parasites/well. The compounds to be
tested and benznidazole were dissolved in medium trypano-
somes liquid (MTL) and were tested at 100, 50, 25, 10, and 1
μM. The effects of the different concentrations of each
compound against the epimastigotes were tested for 72 h
using a Neubauer hemocytometric chamber. The trypanocidal
effect is expressed as IC50 values, i.e., the concentration required
to result in 50% inhibition, as calculated by linear-regression
analysis from the Kc values of the concentrations used.
in Vitro Activity Assays: Intracellular Forms. Axenic
Amastigotes Assay. Axenic amastigotes of T. cruzi, were cultured
following the methodology described previously by Moreno et
al.42 Thus, the epimastigote transformation to amastigotes was
achieved after 3 days of culture in M199 medium (Invitrogen,
Leiden, The Netherlands) supplemented with 10% heat-
inactivated FCS, 1 g/L β-alanine, 100 mg/L L- asparagine,
200 mg/L saccharose, 50 mg/L sodium pyruvate, 320 mg/L
malic acid, 40 mg/L fumaric acid, 70 mg/L succinic acid, 200
mg/L α-ketoglutaric acid, 300 mg/L citric acid, 1.1 g/L sodium
bicarbonate, 5 g/L MES, 0.4 mg/L hemin, and 10 mg/L
gentamicine, pH 5.4 at 37 °C. The effect of each compound
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Journal of Medicinal Chemistry
Article
against the axenic amastigotes was tested for 48 h using a
Neubauer hemocytometric chamber. The trypanocidal effect is
expressed as IC50 values, i.e., the concentration required to
result in 50% inhibition, as calculated by linear-regression
analysis from the Kc values of the concentrations used.
were divided into the following groups: (1) uninfected (not infected
and not treated), (2) untreated (infected with T. cruzi but not treated),
(3) uninfected [not infected but treated with 1 mg/kg body weight/
day, for 5 consecutive days (5−10 days postinfection) via the
intraperitoneal route],46 and (4) treated [infected and treated for 5
consecutive days (5−10 days postinfection) with the test compounds
and benznidazole]. This animal experiment was performed with the
approval of an ethical committee of the University of Granada.
Treatments were started 5 days after the animals were infected. The
compounds were administered in a similar way as explained above and
at the same concentrations. A blood sample (5 μL) drawn from the
mandibular vein of each treated mouse was taken and diluted at a ratio
of 1:15 with 50 μL of 0.1 M citrate buffer:citric acid, 0.1 M sodium
citrate, and 20 μL of lysis buffer at pH 7.2. The parasites were counted
using an inmersion objetive. The number of metacyclic forms of T.
cruzi in the bloodstream was recorded every 2 days from 5 to 30 days
postinfection. The number of metacyclic forms was expressed as
parasites/mL. Circulating anti-T. cruzi antibodies on days 40 and 120
postinfection were quantitatively evaluated using an enzyme-linked
immunoassay. The blood, diluted to 1:50 in PBS, was reacted with an
antigen composed of an excreted Fe-SOD of T. cruzi epimastigotes.
The results were expressed as the ratio of the absorbance of each
sample at 490 nm to the cutoff value. The cutoff point for each
reaction was the mean of the values determined for the negative
controls plus 3 times the standard deviation.47
Amastigotes Assay. Vero cells were cultured in RPMI medium in a
humidified 95% air and 5% CO2 atmosphere at 37 °C. The cells were
seeded at a density of 1 × 104 cells/well in 24-well microplates (Nunc)
with rounded coverslips on the bottom and then cultivated for 2 days.
Afterward, adhered Vero cells were infected in vitro with metacyclic
forms of T. cruzi, at a ratio of 10:1 and maintained for 24 h at 37 °C in
5% CO2 in air. The extracellular parasites were removed by washing,
and the infected cultures were incubated with the compounds (1, 10,
25, 50, and 100 μM concentrations) and cultured for 72 h in RPMI
and 10% inactivated fetal bovine serum. The activity of the compounds
was determined from the percentage reduction in the number of
amastigotes in the treated and untreated cultures in methanol-fixed
and Giemsa-stained preparations. The values are the means of four
separate determinations.43 The trypanocidal effect was expressed as
IC50 values.
Infectivity Assay. The Vero cells were cultured in RPMI medium
as described above. Afterward, the cells were infected in vitro with
metacyclic forms of T. cruzi at a ratio of 10:1. The test compounds
(IC25 concentrations) were added immediately after infection and
incubated for 12 h at 37 °C in a 5% CO2 atmosphere. The extracellular
parasites and the test compounds were removed by washing and the
infected cultures were grown for 10 days in fresh medium. Fresh
culture medium was added every 48 h. The activity of each compound
tested was determined from the percentage of infected cells and the
number of amastigotes per infected cell in treated and untreated
cultures in the methanol-fixed and Giemsa-stained preparations. The
percentage of infected cells and the mean number of amastigotes per
infected cell were determined by analyzing more than 100 host cells
distributed throughout randomly chosen microscopic fields. The
values are the means of four separate determinations. The number of
trypomastigotes in the medium was determined as previously
described.40
SOD Enzymatic Inhibition. The parasites cultured as described
above were centrifuged. 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
disrupted by three cycles of sonic disintegration, 30 s each at 60 V.
The sonicated homogenate was centrifuged at 1500g for 5 min at 4 °C,
and the pellet was washed three times in ice-cold STE buffer. This
fraction was centrifuged (2500g for 10 min at 4 °C) and the
supernatant was collected. The protein concentrations were
determined using the Bradford method.44 Iron and copper−zinc
superoxide dismutases (Fe-SOD and CuZn-SOD) activities were
determined using the method described by Beyer and Fridovich,45
which measures the reduction in nitroblue tetrazolium (NBT) by
superoxide ions. Into each bucket was added 845 μL of stock solution
[3 mL of L-metionine (300 mg, 10 mL−l), 2 mL of NBT (1.41 mg, 10
mL−1) and 1.5 mL of Triton X-100 1% (v/v)], along with 30 μL of the
parasite homogenate fraction, 10 μL of riboflavine (0.44 mg, 10 mL−l),
and an equivalent volume of the different concentrations of the
compounds tested. Five different concentrations were used for each
product: 1, 2.5, 5, 12.5, and 25 μM (equivalent to 5, 12.5, 25, 62, and
125 μL, respectively, of the stock solution). In the control experiment
the volume was made up to 1000 μL with 50 mM potassium
phosphate buffer (pH 7.8, 3 mL), whereas 30 μL of the parasite
homogenate fraction was added to the mixtures containing the
compounds. Then, the absorbance (A0) was measured at 560 nm in a
spectrophotometer. Following this, each bucket was illuminated with
UV light for 10 min under constant stirring and the absorbance (A1)
was measured. The human CuZn-SOD, coenzymes and substrates
used in these assays were obtained from Sigma Chemical Co. The data
obtained were analyzed using the Newman−Keuls test.
Metabolite Excretion. Cultures of T. cruzi epimastigotes (initial
concentration 5 × 105 cells/mL) received IC25 of the compounds
(except for the control cultures). After incubation for 96 h at 28 °C,
the cells were centrifuged at 400g for 10 min. The supernatants were
1
collected in order to determine the excreted metabolites through H
NMR, and the chemical shifts were expressed in parts per million
(ppm), using sodium 2,2-dimethyl-2-silapentane-5-sulfonate as the
reference signal. The chemical displacements used to identify the
respective metabolites were consistent with those described by us.48
Ultrastructural Alterations. The parasites were cultured at a
density of 5 × 105 cells/mL in each corresponding medium containing
the compounds tested at the concentration of IC25. After 96 h, these
cultures were centrifuged at 400g for 10 min, and the pellets produced
were washed in PBS and then mixed with 2% (v/v) p-formaldehyde/
glutaraldehyde in 0.05 M cacodylate buffer (pH 7.4) for 4 h at 4 °C.
Following this, the pellets were prepared for transmission electron
microscopy study using a technique described by us.43
Histopathological Analysis. The liver was extracted from each
mouse 120 days postinfection, cut longitudinally, rinsed in ice-cold
PBS, and fixed in 10% buffered formalin. The tissues were dehydrated
and embedded in paraffin. Sections were cut at a thickness of 4−5 μm
and stained with hematoxylin−eosin (H&E) and Masson’s trichro-
mestaining protocol (Trichrome). The slides were coded for a blinded
analysis. Histological examinations were performed using a conven-
tional light microscope; each slide was visualized in at least 30 fields
(total magnifications: ×40, ×100, ×200, and ×400). The histological
alterations were given a score of 0 (−) to 5 (+++++), with 0
representing a complete absence of alterations and 5 representing the
most severe alterations.
ASSOCIATED CONTENT
* Supporting Information
■
S
Details on the combustion analysis, the protonation constants
and the degrees of protonation at pH 2−12, the NMR spectra
obtained from the metabolite excretion studies, and details of
the histopathological analysis. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
In Vivo Trypanosomicidal Activity Assay. Groups of three
BALB/c female mice (6−8 weeks old; 20−25 g), maintained under
standard conditions, were infected (bloodstream) with 1 × 105
metacyclic forms of T. cruzi via the intraperitoneal route. The animals
*M.S.-M.: phone, +34-958-242-369; fax: +34-958-243-862; e-
mail: msanchem@ugr.es. P.N.: phone: +34-91-258-7572; fax:
+34-91-564-4853; e-mail: iqmnt38@iqm.csic.es. E.G.-E.:
4241
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Article
(11) Viotti, R.; Vigliano, C.; Lococo, B.; Alvarez, M. G.; Petti, M.;
Bertocchi, G.; Armenti, A. Side effects of benznidazole as treatment in
chronic Chagas disease: Fears and realities. Expert Rev. Anti. Infect.
Ther. 2009, 7, 157−163.
phone: +34-963-544879; fax: +34-96-354-3274; e-mail:
Enrique.Garcia-Es@uv.es.
Notes
The authors declare no competing financial interest.
(12) Schofield, C. J.; Jannin, J.; Salvatella, R. The future of Chagas
disease control. Trends Parasitol. 2006, 22 (12), 583−588.
(13) (a) Maya, J. D.; Cassels, D. K.; Iturriaga-Vasquez, P.; Ferreira, J.;
ACKNOWLEDGMENTS
■
Faundez, M.; Galanti, N.; Ferreira, A.; Morello, A. Mode of action of
́
The authors thank the MCINN Projects: Consolider Ingenio
CSD2010-00065 and CTQ2009-14288-C04-01, the FEDER
funds and Generalitat Valenciana PROMETEO 2011/008, the
Santander-Universidad Complutense Research Program GR58/
08-921371-891, and the Spanish MEC Project CGL2008-
03687-E/BOS for financial support. J.P. thanks MCINN for a
predoctoral fellowship. We are also grateful to the NMR and
Elemental Analysis Services of the Centro Nacional de Quimica
Organica Manuel Lora-Tamayo (C.S.I.C) and also to the
Transmission-Electron Microscopy and Nuclear Magnetic
Resonance Spectroscopy Services of the CIC-University of
Granada.
natural and synthetic drugs against Trypanosoma cruzi and their
interaction with the mammalian host. Comp. Biochem. Physiol. A Mol.
Integr. Physiol. 2007, 146 (4), 601−620. (b) Wilkinson, S. R.; Bot, C.;
Kelly, J. M.; Hall, B. S. Trypanocidal activity of nitroaromatic
prodrugs: Current treatments and future perspectives. Curr. Top. Med.
Chem. 2011, 11 (16), 2072−2084.
(14) (a) Mehlotra, R. K. Antioxidant defense mechanisms in parasitic
protozoa. Crit. Rev. Microbiol. 1996, 22 (4), 295−314. (b) Atwood, J.
A.; Weatherly, D. B., 3rd; Minning, T. A.; Bundy, B.; Cavola, C.;
Opperdoes, F. R.; Orlando, R.; Tarleton, R. L. The Trypanosoma cruzi
proteome. Science 2005, 309, 473−476.
(15) Ghosh, S.; Goswami, S.; Adhya, S. Role of superoxide dismutase
in survival of Leishmania within the macrophage. Biochem. J. 2003, 369,
447−452.
ABBREVIATIONS USED
■
(16) Rodriguez-Ciria, M.; Sanz, A. M.; Yunta, M. J. R.; Gomez-
Contreras, F.; Navarro, P.; Sanchez −Moreno, M.; Boutaleb-Charki,
SOD, superoxide dismutase; WHO, World Health Organiza-
tion; BZN, benznidazole; TEM, transmission electron micros-
copy; HMQC, heteronuclear single quantum coherence
experiment; HMBC, heteronuclear multiple bond coherence
experiment; ESI, electrospray ionization; SI, selectivity index;
dpi, days postinfection; TLC, thin-layer chromatography; TMS,
tetramethylsilane; MEM, minimal essential medium; NAD(P)-
H, nicotinamide adenine dinucleotide phosphate; PEP,
phosphoenolpyruvate; FAB, fast atom bombardment; FBS,
fetal bovine serum; MTL, medium trypanosomes liquid;
EDTA, ethylendiaminotetraacetic acid; MES, 4-morpholinoe-
thanesulfonic acid; NBT, nitroblue tetrazolium; PBS, phos-
phate-buffered saline solution
S.; Osuna, A.; Castineiras, A.; Pardo, M.; Cano, C.; Campayo, L. 1,4-
̃
Bis(alkylamino)benzo[g]phthalazines able to form complexes of
Cu(II) which as free ligands behave as SOD inhibitors and show
efficient in vitro activity against Trypanosoma cruzi. Bioorg. Med. Chem.
2007, 15, 2081−2091.
́
(17) (a) Sanz, A. M.; Gomez-Contreras, F.; Navarro, P.; Sanchez-
Moreno, M.; Boutaleb-Charki, S.; Campuzano, J.; Pardo, M.; Osuna,
A.; Cano, C.; Yunta, M. J. R.; Campayo, L. 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. 2008, 51 (6), 1962−1966. (b) Sanchez-Moreno, M.; Sanz, A.
M.; Gomez-Contreras, F.; Navarro, P.; Marín, C.; Ramírez-Macías, I.;
Rosales, M. J.; Olmo, F.; García-Aranda, I.; Campayo, L; Cano, C.;
Arrebola, F.; Yunta, M. J. R. In vivo trypanosomicidal activity of
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