In vitro activity of scorpiand-like azamacrocycle derivatives in promastigotes and intracellular amastigotes of Leishmania infantum and Leishmania braziliensis

Article abstract of DOI:10.1016/j.ejmech.2013.01.001

The activity of a family scorpiand-like azamacrocycles against Leishmania infantum and Leishmania braziliensis was studied using promastigotes, axenic and intracellular amastigotes forms. All the compounds are more active and less toxic than meglumine ant

Full text of DOI:10.1016/j.ejmech.2013.01.001

European Journal of Medicinal Chemistry 62 (2013) 466e477
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
In vitro activity of scorpiand-like azamacrocycle derivatives
in promastigotes and intracellular amastigotes of Leishmania
infantum and Leishmania braziliensis
Clotilde Marín a, M. Paz Clares b, Inmaculada Ramírez-Macías a, Salvador Blasco b,
Francisco Olmo a, Conxa Soriano c, Begoña Verdejo b, María José Rosales a,
**
,
a,
*
David Gomez-Herrera a, Enrique García-España b , Manuel Sánchez-Moreno
a Departamento de Parasitología, Facultad de Ciencias, Universidad de Granada, E-18071 Granada, Spain
b Instituto de Ciencia Molecular, Departamento de Química Inorgánica, Universidad de Valencia, E-46980 Paterna, Valencia, Spain
c Departamento de Química Orgánica, Universidad de Valencia, E-46100 Burjassot, Valencia, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
The activity of a family scorpiand-like azamacrocycles against Leishmania infantum and Leishmania
braziliensis was studied using promastigotes, axenic and intracellular amastigotes forms. All the com-
pounds are more active and less toxic than meglumine antimoniate (Glucantime). Moreover, the data on
infection rates and amastigotes showed that compounds P2Py, PN and P3Py are the most active against
both species of Leishmania. On the other hand, studies on the inhibitory effect of these compounds on
SOD enzymes showed that while the inhibition of the Fe-SOD enzyme of the promastigote forms of the
parasites is remarkable, the inhibition of human CuZn-SOD and Mn-SOD from Escherichia coli is negli-
gible. The ultrastructural alterations observed in treated promastigote forms confirmed that the com-
pounds having the highest activity were those causing the largest cell damage. The modifications
observed by 1H NMR, and the amounts of catabolites excreted by the parasites after treatment with the
compounds, suggested that the catabolic mechanism could depend on the structure of the side chains
linked to the aza-scorpiand macrocycles.
Received 15 November 2012
Received in revised form
26 December 2012
Accepted 3 January 2013
Available online 19 January 2013
Keywords:
Leishmania infantum
Leishmania braziliensis
Synthetic aza-scorpiand
Antileishmanial activity
SOD inhibition
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
parts (arms, legs, face), or the visceral form (VL, also known as kala-
azar) which is fatal if not treated in time.
Leishmaniasis is still one of the world’s most neglected diseases
which occurs in 98 countries, affecting largely the poorest of the
poor, mainly in developing countries. 350 Million people are con-
sidered at risk of contracting leishmaniasis, and over 2 million new
cases occur yearly [1]. The etiological agents are different species of
protozoa of the genus Leishmania, which are transmitted by Diptera
of the genus Phlebotomus in the Old World, and Lutzomyia in the
New World.
Surprisingly, leishmaniasis is the only tropical disease which is
being treated by nonleishmanial drugs [2]. The spectrum and effi-
cacy of available antileishmanial drugs is limited. The first-line
drugs used for treatment of leishmaniasis are pentavalent anti-
monial compounds (sodium stibogluconate or Pentostam and
meglumine antimoniate or Glucantime), while amphotericin B,
pentamidine and Paromomycin (aminosidine) are used as second-
line chemotherapy. Other alternative antileishmanial medicines are
miltefosine, azithromycin, allopurinol, dapsone, sitamaquine,
rifampicin or azole medicines: ketoconazole, fluconazole, itraco-
nazole. Combining drugs may bring significant advantages and
better therapeutic effects than each of the substances alone [2,3].
Although in the past decade the number of treatments has
increased, they have numerous drawbacks such as difficulty in
administration, length of treatment, toxicity, cost, availability
limited in disease endemic regions and increasing parasitic resist-
ance [4,5]. In addition, the efficacy of the substances differs dras-
tically depending on the clinical form of leishmaniasis, Leishmania
Leishmaniasis has a broad diversity of clinical forms: cutaneous
(CL), mucosal or muco-cutaneous (MCL) ulcers that although can
heal spontaneously produce disfiguring lesions on exposed body
* Corresponding author. Tel.: þ34 958 242 369; fax: þ34 958 243 862.
** Corresponding author. Tel.: þ34 963 544879; fax: þ34 96 354 3274.
E-mail addresses: cmaris@ugr.es (C. Marín), enrique.garcia-es@uv.es (E. García-
España), msanchem@ugr.es (M. Sánchez-Moreno).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.01.001

C. Marín et al. / European Journal of Medicinal Chemistry 62 (2013) 466e477
467
species and geographical region [6]. Thus, there is still a continuing
need for new chemical substances with a leishmanicidal effect, for
optimized delivery systems and newer cost effective drugs.
Thus, the development of new leishmanicidal drugs is an
important goal. In this respect, several compounds including syn-
thetic ones, or natural products extracted from plants or marine
sources, have shown different degrees of efficacy in the treatment
of experimental leishmaniasis [7e9].
In relation to this matter, our research group has designed in
recent years a new family of polyamine compounds that consists of
a macrocyclic pyridinophane core appended with lateral chains
containing additional donor atoms; the structures are shown in Fig.1.
These macrocycles are termed scorpiand ligands since the lateral
chain can fold towards the macrocyclic core, following the binding of
target guest species such as protons, anions or metal ions [10].
The aim of the present work is to identify new candidates for
designing potent, selective, and low toxicity anti-trypanosomatids.
We have evaluated the antiproliferative in vitro activity against
Leishmania infantum and Leishmania braziliensis (promastigote and
amastigote forms). Unspecific mammal cytotoxicity of the most active
compounds was evaluated in vitro, and the less toxic derivatives were
submitted to a more thorough study of their possible action mecha-
nisms. Furthermore, a nuclear magnetic resonance spectroscopy (1H
NMR) study was performed in order to gain details regarding its na-
ture and the percentage of metabolites excreted causing inhibitory
effects of the glycolic pathway, since it represents the primary source
of energy for the parasite. Finally, the effects of the compounds on the
ultra-structure of Leishmania spp. were considered on the basis of
transmission electron microscopy studies (TEM).
2. Materials and methods
2.1. Synthesis
The synthesis of the compounds was performed as depicted in
Scheme 1 [10,11].
2.1.1. 6-(2-Aminoethyl)-3,6,9-triaza-1-(2,6)-pyridinecyclodecaphane
(P$3HBr)
Tris-2-(aminoethyl)amine (6 g, 41.7 mmol) was dissolved in
400 mL of THF adding 100 mL of an aqueous solution of K2CO3 (17 g,
125 mmol). TsCl (125 mmol) dissolved in 100 mL of THF was added
dropwise for an hour and the stirring was kept for 24 h. The organic
phase was taken to dryness and the residue was suspended in
refluxing ethanol to give a white solid. The solid was filtered and
washed with EtOH (yield 63%). mp 60e62 ꢀC. Tris[2-(N-p-tol-
ylsulfonylaminoethyl)]amine (TrenTs). 1H NMR (CDCl3, 300 MHz):
dH (ppm): 2.41 (s, 9H), 2.47 (t, J ¼ 5 Hz, 6H), 2.91 (t, J ¼ 6 Hz, 3H),
2.92 (t, J ¼ 6 Hz, 3H), 5.94 (s, 3H), 7.0 (d, J ¼ 8 Hz, 6H), 7.80
Scheme 1. General procedure for the synthesis of P, PN, P2Py and P3Py.
(d, J ¼ 8 Hz, 6H). 13C NMR (CDCl3, 75.43 MHz): dC (ppm): 21.9, 41.2,
54.5, 127.7, 130.2, 137.2, 143.8.
TrenTs (5.7 mmol) and K2CO3 (57.0 mmol) were dissolved in dry
CH3CN (50 mL). 2,6-Bis(bromomethyl)pyridine (5.7 mmol) dissolved
in dry CH3CN (30 mL) was slowly added. The mixture was refluxed
under a nitrogen atmosphere for 20 h. The solid obtained was filtered,
washed with dry CH3CN, and then it was taken to dryness, getting an
oil that was refluxed in ethanol to give a white solid (yield 51.4%). mp
151e153 ꢀC. 6-[2-(p-Tolylsulfonylamino)ethyl]-3,6,9-triaza-3, 9-p-
tolylsulfonyl-1-(2,6)-pyridinecyclodecaphane (PytrenTs). 1H NMR
(CDCl3, 300 MHz): dH (ppm): 2.31 (t, J ¼ 7 Hz, 4H), 2.36 (t, J ¼ 7 Hz,
2H), 2.41 (s, 3H), 2.45 (s, 6H), 2.87e2.81 (m, 2H), 3.06 (t, J ¼ 7 Hz, 4H),
4.31 (s, 4H), 5.05 (t, J ¼ 6 Hz, 1H), 7.36e7.28 (m, 8H), 7.75e7.68 (m,
7H). 13C NMR (CDCl3, 75.43 MHz): dC (ppm): 21.9, 40.9, 45.9, 52.2,
54.2, 55.1, 124.1, 127.4, 130.2, 136.1, 137.1, 139.2, 143.9, 144.1, 155.5.
PytrenTs (6.75 mmol), HBr/HAc (250 mL) and PhOH (257 mmol),
were refluxed for 24 h. The solid obtained was filtered washing
with EtOH/CH2Cl2 (1:1) (yield 65.7%). mp 201e203 ꢀC. 1H NMR
(D2O, 300 MHz): dH (ppm): 7.95 (t, J ¼ 8 Hz, 1H), 7.44 (d, J ¼ 8 Hz,
2H), 4.63 (s, 4H), 3.29e3.21 (m, 6H), 3.02 (t, J ¼ 7 Hz, 2H), 2.92 (t,
J ¼ 5 Hz, 4H). 13C NMR (D2O, 75.43 MHz): dC (ppm): 30.5, 35.6, 46.2,
49.8, 50.9, 52.3, 122.5, 140.1, 149.2. Anal. Calcd. for C13H23N5$4HBr:
C, 27.25; H, 4.74; N, 12.22. Found: C, 27.94; H, 5.11; N, 11.86.
Fig. 1. Drawing of the compounds.

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C. Marín et al. / European Journal of Medicinal Chemistry 62 (2013) 466e477
2.1.2. 6-[4-(1-Naphthyl)-3-azabutyl]-3,6,9-triaza-1-(2,6)-
pyridinecyclodecaphane (PN$3HCl)
2.94 (t, J ¼ 5 Hz, 4H), 3.11 (t, J ¼ 7 Hz, 2H), 3.30 (t, J ¼ 5 Hz, 4H), 3.48 (t,
J ¼ 7 Hz, 2H), 4.64 (s, 4H), 4.62 (s, 2H), 7.45 (d, J ¼ 8 Hz, 2H), 7.96 (t,
J ¼ 8 Hz, 1H), 8.20 (dd, J1 ¼ 8 Hz, J2 ¼ 6 Hz, 1H), 8.83 (d, J ¼ 8 Hz, 1H),
8.91 (d, J ¼ 6 Hz, 1H), 9.05 (d, J ¼ 2 Hz,1H).13C NMR (D2O, 75.43 MHz):
dC (ppm): 43.9, 45.8, 47.7, 49.5, 50.4, 50.8, 122.1, 128.0, 129.5, 131.1,
142.5,148.7,148.8. Anal. Calcd. for C19H28N6$3HCl: C, 50.68; H, 6.94; N,
18.75. Found: C, 50.8; H, 7.0; N, 18.8.
6-(2-Aminoethyl)-3,6,9-triaza-1-(2,6)-pyridinecyclodecaphane
(P) (1.43 g, 5.73 mmol) and 1-naphthaldehyde (0.90 g, 5.73 mmol)
were dissolved in 100 mL of dry ethanol and stirred at room tem-
perature for 2 h. Then, sodium borohydride (2.2 g, 58 mmol) was
added, and the bulk was stirred for 2 h more. The solution was vacuum
evaporated and extracted with CHCl3/H2O (6 ꢁ 100). The organic
phase was taken to dryness and redissolved in dry ethanol. The hy-
drochloride salt of the product was precipitated by adding a con-
centrated hydrochloride acid solution (yield 65%). mp 207e209 ꢀC. 1H
NMR (D2O, 300 MHz): dH (ppm): 2.91 (t, J ¼ 5 Hz, 4H), 3.09 (t, J ¼ 6 Hz,
2H), 3.25 (t, J ¼ 6 Hz, 4H), 3.43 (t, J ¼ 8 Hz, 2H), 4.62 (s, 4H), 4.82 (s, 2H),
7.45 (d, J ¼ 8 Hz, 2H), 7.58e7.75 (m, 4H), 7.96 (t, J ¼ 8 Hz, 1H), 8.07 (t,
J ¼ 7 Hz, 2H), 8.14 (d, J ¼ 8 Hz, 1H). 13C NMR (D2O, 75.43 MHz): dC
(ppm): 43.1, 46.2, 48.8, 49.7, 50.8, 51.2, 122.5, 122.8, 125.9, 127.1, 127.8,
129.5,129.9,131.0,132.3,140.1. Anal. Calcd. for C24H31N5$3HCl$1.5H2O:
C, 54.81; H, 7.09; N, 13.31. Found: C, 55.10; H, 7.42; N, 13.38.
The synthesis of Pal$5HBr was accomplished as illustrated in
Scheme 2 following Ref. [12].
2.1.5. 6-(3,7-Diazaheptyl)-3,6,9-triaza-1-(2,6)-pyridinecyclodecaphane
(Pal$5HBr)
6-[2-(p-Tolylsulfonylamine)ethyl]-3,6,9-triaza-3,
9-p-tol-
ylsulfonyl-1-(2,6)-pyridinecyclodecaphane (PytrenTs) (2.59 g,
3.64 mmol), and K2CO3 (1.51 g, 10.93 mmol) were suspended with
300 mL of CH3CN. To this mixture N-(3-bromopropyl)phthalimide
(1.19 g, 4.14 mmol) in 150 mL of CH3CN was added, then it was
refluxed for 48 h and filtered off. The solution was vacuum evapo-
rated to dryness and the residue was suspended in refluxing ethanol
to give a white solid (yield 98%). 1H NMR (CDCl3, 300.13 MHz) dH
(ppm): 1.74e1.84 (m, 2H), 2.26e2.39 (m, 11H), 2.55 (t, 2H, J ¼ 7 Hz),
2.91 (t, 2H, J ¼ 7 Hz), 3.03e3.10 (m, 8H), 3.60e3.67 (m, 2H), 4.26
(s, 4H), 7.19e7.29 (m, 10H), 7.61e7.80 (m, 9H). This solid (2.75 g,
3.57 mmol) was treated with hydrazine hydrate 85% (3 mL) and
ethanol (500 mL) and the mixture was refluxed for 24 h, then the
resulting solid was filtered off. The solution was vacuum evaporated
to give an oil (yield 90%). 1H NMR (CDCl3, 300.13 MHz): dH (ppm):
1.50e1.61 (m, 2H), 2.33e2.42 (m, 12H), 2.52 (t, 2H, J ¼ 7 Hz), 2.70
(t, 2H, J ¼ 7 Hz), 2.96e3.10 (m, 8H), 4.30 (s, 4H), 7.18e7.30 (m, 8H),
7.60e7.70 (m, 7H). 13C NMR (CDCl3, 75.43 MHz): dC (ppm): 21.7, 21.8,
32.2, 39.0, 44.7, 47.2, 47.3, 51.9, 54.7, 124.1, 127.4, 127.4, 130.0, 130.1,
136.0, 136.4, 138.9, 143.5, 143.8, 155.2.
2.1.3. 6-[4-(2-Pyridyl)-3-azabutyl]-3,6,9-triaza-1-(2,6)-pyridine-
cyclodecaphane (P2Py$3HCl)
The same procedure as for the synthesis of PN was applied, but
using 2-pyridinecarbaldehyde instead of 1-naphthaldehyde (yield
65.7%). mp 208e210 ꢀC. 1H NMR (D2O, 300 MHz): dH (ppm): 2.93 (t,
J ¼ 5 Hz, 4H), 3.13 (t, J ¼ 8 Hz, 2H), 3.28 (t, J ¼ 5 Hz, 4H), 3.45 (t, J ¼ 8 Hz,
2H) 4.57 (s, 2H), 4.64 (s, 4H), 7.45 (d, J ¼ 8 Hz, 2H), 7.75 (ddd,
J1 ¼ J2 ¼ 8 Hz, J3 ¼ 1 Hz, 1H), 7.83 (d, J ¼ 8 Hz, 1H), 7.96 (t, J ¼ 8 Hz,1H),
8.25 (ddd, J1 ¼ J2 ¼ 8 Hz, J3 ¼ 2 Hz,1H), 8.72 (dd, J1 ¼5 Hz, J2 ¼1 Hz,1H).
13C NMR (D2O, 75.43 MHz): dC (ppm): 43.9, 46.2, 49.8, 50.0, 50.8, 51.2,
122.5, 126.5, 126.6, 140.1, 143.3, 146.8, 147.6, 149.2. Anal. Calcd. for
C19H28N6$3HCl: C, 50.68; H, 6.94; N,18.75. Found: C, 50.3; H, 7.1; N,18.5.
2.1.4. 6-[4-(3-Pyridyl)-3-azabutyl]-3,6,9-triaza-1-(2,6)-pyridine-
cyclodecaphane (P3Py$3HCl)
The same procedure as for the synthesis of P2Py was applied, but
using 3-pyridinecarbaldehyde instead of 2-pyridinecarbaldehyde
(yield 75.8%). mp 210e215 ꢀC. 1H NMR (D2O, 300 MHz): dH (ppm):
Pytren-alTs (6.75 mmol) was suspended with PhOH (257 mmol)
and HBr/HAc 33% (250 mL) and the mixture was subjected to reflux for
24 h. The solid obtained was filtered washing with EtOH/CH2Cl2 (1:1)
to give Pal$5HBr (yield 63%). mp 207e209ꢀC.1HNMR(D2O, 300 MHz):
dH (ppm): 1.99e2.10 (m, 2H), 2.80e2.90 (m, 4H), 2.95e3.06 (m, 4H),
Scheme 2. General procedure for the synthesis of Pal.

C. Marín et al. / European Journal of Medicinal Chemistry 62 (2013) 466e477
469
3.08e3.30 (m, 8H), 4.55(s, 4H), 7.36 (d, J ¼ 7.8 Hz, 2H), 8.01 (t, J ¼ 7.8 Hz,
1H). 13C NMR (D2O, 75.43 MHz): dC (ppm): 24.2, 36.8, 43.6, 45.4, 46.2,
49.8, 50.8, 51.2, 122.5, 140.1, 149.2. Anal. Calcd. for
C16H32N6$5HBr$2H2O: C, 25.68; H, 5.03; N, 10.86. Found: C, 25.66; H,
5.52; N, 11.22.
(Nunc) with rounded cover slips on the bottom and cultured for 2
days. The cells were then infected in vitro with promastigote forms
of L. infantum or L. braziliensis at a ratio of 10:1. The drugs (IC25
concentrations) were added immediately after infection and in-
cubation was performed for 12 h at 37 ꢀC in 5% CO2. Non-
phagocytised parasites and drugs were removed by washing, and
then the infected cultures were grown for 10 days in fresh medium.
Cultures were washed every 48 h and fresh culture medium was
added. Drug activity was determined on the basis of both the
percentage of infected cells and the number of amastigotes per
infected cell in treated and untreated cultures in methanol-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 200 host cells distributed in
randomly chosen microscopic fields. Values are the means of three
separate determinations.
2.2. Parasite strain and culture
L. infantum (MCAN/ES/2001/UCM-10) and L. braziliensis
(MHOM/BR/1975/M2904) were cultured in vitro in medium try-
panosomes liquid (MTL) together with 10% inactive foetal calf
serum (FCS) kept in an air atmosphere at 28 ꢀC, in Roux flasks
(Corning, USA) with a surface area of 75 cm2, according to the
methodology described in Ref. [13].
2.3. Superoxide dismutase enzymatic inhibition
Parasites cultured as described above were suspended (0.5e
0.6 g/mL) in 3 mL of STE buffer 1 (0.25 M sucrose, 25 mM Trise
HCl, 1 M EDTA, pH 7.8) and disrupted by three cycles of sonic dis-
integration, for 30 s each at 60 W. The sonicated homogenate was
centrifuged at 1500g for 10 min at 4 ꢀC, and the pellet was washed
three times with ice-cold STE buffer 1, giving a total supernatant
fraction of 9 mL. This fraction was centrifuged (2500g for 10 min at
4 ꢀC), the supernatant was collected and solid ammonium sulphate
was added. The protein fraction, which precipitated between 35%
and 85% salt concentration, was centrifuged (9000g for 20 min at
2.5. Cell culture and cytotoxicity tests
The macrophage line J774.2 [European collection of cell cultures
(ECACC) number 91051511] was derived in 1968 from a tumour in
a female BALB/c mouse. The cytotoxicity test on macrophages was
performed according to the method described by Ref. [15]. After
72 h of treatment with 100, 50, 25, l0, and 1 mM of product, cell
viability was determined by flow cytometry. Thus, 100 mL/well of
propidium iodide solution (100 mg/mL) was added and incubated
for 10 min at 28 ꢀC in darkness. Afterwards, 100 mL/well of fluo-
rescein diacetate (100 ng/mL) was added and incubation was per-
formed under the conditions described above. Finally, the cells were
recovered by centrifugation at 400g for 10 min and the precipitate
was washed with PBS. Flow cytometric analysis was performed
with a FACS Vantage TM flow cytometer (Becton Dickinson). The
percentage viability was calculated with respect to the control
culture. The IC50 was calculated using linear regression analysis
from the Kc values of the concentrations employed.
4
ꢀC), dissolved in 2.5 mL of 20 mM potassium phosphate buffer
(pH 7.8) containing 1 mM EDTA (buffer 2) and dialysed on
a Sephadex G-25 column (GE Healthcare, PD 10), previously bal-
anced with buffer 2, bringing it to a final volume of 3.5 mL (fraction
of the homogenate). The protein concentrations were determined
by the Bradford method (SigmaeAldrich, St. Louis, M.O.), using
albumen from bovine serum as standard.
Iron superoxide dismutase (Fe-SOD) activity was performed by
spectrometric measurement of NBT-u.v. light oxidation according to
the method described by Beyer and Fridovich [14] which measures
the reduction of NBT by superoxide ions. One unit was the amount
of enzyme required to inhibit the rate of NBT reduction by 50%. The
manganese superoxide dismutase (Mn-SOD) and copperezinc su-
peroxide dismutase (CuZn-SOD) from Escherichia coli and human
erythrocytes, respectively, used in these assays were obtained from
Boehringer (Mannheim), while all the coenzymes and substrates
came from SigmaeAldrich. Data obtained were analyzed according
to the NewmaneKeuls test.
2.6. In vitro activity assays: intracellular forms
2.6.1. Axenic amastigote assay
Axenic amastigote forms of Leishmania spp. were cultured
following the methodology described previously in Ref. [16]. Thus,
promastigote transformation to amastigotes was achieved after 3
days of culture in M199 medium (Invitrogen, Leiden, The Neth-
erlands) supplemented with 10% heat-inactivated FCS, 1 g/L
b
-alanine, 100 mg/L
sodium pyruvate, 320 mg/L malic acid, 40 mg/L fumaric acid,
70 mg/L succinic acid, 200 mg/L -ketoglutaric acid, 300 mg/L
L-asparagine, 200 mg/L sucrose, 50 mg/L
2.4. In vitro activity assays: extracellular forms
a
citric acid, 1.1 g/L sodium bicarbonate, 5 g/L 2-(N-morpholino)
ethanesulfonic acid (MES), 0.4 mg/L haemin and 10 mg/L genta-
micin, pH 5.4, at 37 ꢀC. The effect of each compound against
axenic amastigote forms was tested at 48 h using a Neubauer
haemocytometer chamber.
2.4.1. Promastigote assay
The compounds were dissolved in DMSO (Panreac, Barcelona,
Spain) at a concentration of 0.1% and were assayed as non-toxic and
without inhibitory effects on parasite growth, according to Ref. [13].
The compounds were dissolved in the culture medium at concen-
trations of 100, 50, 25, 10 and 1
mM. The effects of each compound
2.6.2. Amastigote assay
against the promastigote forms and their concentrations were tested
at 72 h using a Neubauer haemocytometric chamber. The leishma-
nicidal effect was expressed as the IC50 value, i.e. the concentration
required to result in 50% inhibition, calculated by linear regression
analysis from the Kc values of the concentrations employed.
Adherent macrophage cells were infected with promastigotes
in the stationary growth phase of Leishmania spp. at a ratio of
10:1 and maintained for 24 h at 37 ꢀC in air 5% CO2. Non-
phagocytosed parasites were removed by washing, and the
infected cultures were incubated with the compounds (1, 10, 25,
50 and 100 mM) and then cultured for 72 h in MEM plus gluta-
2.4.2. Infectivity assay
mine (2 mM) and 20% inactive FCS. Compound activity was
determined from the percentage reductions in amastigote num-
bers in treated and untreated cultures in methanol-fixed and
Giemsa-stained preparations. Values are the averages of three
separate determinations [13].
J774.2 macrophage cells were grown in minimal essential me-
dium (MEM) plus glutamine (2 mM) and 20% inactive FCS, in
a humidified atmosphere of 95% air and 5% CO2 at 37 ꢀC. Cells were
seeded at a density of 1 ꢁ 104 cells/well in 24-well micro plates

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C. Marín et al. / European Journal of Medicinal Chemistry 62 (2013) 466e477
2.7. Metabolite excretion
which also include the results obtained for the reference drug
meglumine antimoniate (Glucantime). Toxicity values against
J774.2 macrophages cells were also calculated, and the selectivity
indexes (SI, IC50 macrophage cells toxicity/IC50 activity of extrac-
ellular or intracellular forms of the parasite), also shown in the last
three columns of Tables 2 and 3. The number of times that the
compound SI exceeded the Glucantime SI, is shown in brackets,
since this value is very illustrative of the in vitro potentiality of the
compounds tested with respect to the reference drug.
Tables 2 and 3 show the IC50 values obtained after 72 h of
exposure, when compounds were assayed against the different
parasite forms studied. Values for the reference drug, Glucantime,
are included in all cases for comparison.
Cultures of L. infantum and L. braziliensis promastigotes (initial
concentration 5 ꢁ 105 cells/mL) received the IC25 of the compounds
(except for control cultures). After incubation for 96 h at 28 ꢀC the
cells were centrifuged at 400g for l0 min. The supernatants were
collected to determine the excreted metabolites by 1H NMR, and
chemical shifts were expressed in parts per million (ppm), using
sodium 2,2-dimethyl-2-silapentane-5-sulphonate as the reference
signal. The chemical displacements used to identify the respective
metabolites were consistent with those described in Ref. [17].
2.8. Ultrastructural alterations
If we consider now the results displayed in Table 2 for activity
against L. infantum, the leishmanicidal activity in both extra- and
intracellular forms was similar or, in most cases, higher than founded
for Glucantime, the best results were achieved by compounds P,
P2Py, PN and P3Py. However, when we have checked the MnII
complexes of these compounds (P*, P2Py*, PN*, P3Py* and Pal*) an
opposite effect was observed. Such an effect can be ascribed to the
protective effect that has been described for MnII complexes of aza-
scorpiand ligands [18]. More interesting are the toxicity data, since
all compounds tested were found to be much less toxic for macro-
phages than the reference drug. In general, all the compounds were
from 14 to 30-fold less toxic than Glucantime. Toxicity values sub-
stantially influence the more informative selectivity index (SI) data,
and best values were again obtained for the compounds P2Py, PN
and P3Py, with SI exceeding those of the reference drugs by 37-, 32-
and 30-fold in the case of P2Py, by 49-, 29, -27-fold for PN, and by
34-, 39-, 57-fold for P3Py.
Very similar conclusions are extracted from the L. braziliensis
results shown in Table 3. The compounds P2Py, PN and P3Py, and in
the same way the P gave the best SI results in the three assays per-
formed, with values exceeding those of the reference drugs 37-, 32-,
30-fold in the case of P2Py, 49-, 29- and 27-fold for PN, 34-, 39- and
57-fold for the compound P3Py and for the P was 30-, 29- and 29-
fold, respectively. It was also observable that the compounds P2Py,
PN, P3Py and P had a better SI than the rest of compound assayed.
The aza-scorpiand like macrocyclic derivatives P3Py had the lowest
toxicity and highest SI values in both L. infantum and L. braziliensis.
Different authors have claimed that compounds having a SI
values 20-fold higher than the reference are to be considered in
possession of leishmanicidal properties [19]. This requirement is
satisfied by compounds P2Py, PN and P3Py, in the case of
L. infantum and also the compound P in the case of L. braziliensis.
The parasites invaded the cell and were converted to amastigotes
within one day after infection and the rate of host-cell infection
reached its maximum on day 10 (control experiment). The propa-
gation of the parasite in J774.2 macrophages was studied by
The parasites were cultured at a density of 5 ꢁ 105 cells/mL in
MTL medium, each of which contained the compounds tested at
the IC25 concentration. After 96 h, the cultures were centrifuged at
400g for 10 min, and the pellets produced were washed in PBS and
then incubated with 2% (v/v) p-formaldehyde/glutaraldehyde in
0.05 M cacodylate buffer (pH 7.4) for 2 h at 48 ꢀC. The pellets were
then prepared for TEM employing the technique of Ref. [13].
3. Results
3.1. Chemistry
The polyamine macrocycles P, PN, P2Py and P3Py (Fig. 1) were
prepared as shown in Scheme 1 by a procedure previously
reported by some of us [10,11]. The synthesis of the ligands was
achieved following a modification of the RichmaneAtkins proce-
dure by reaction of the pertosylated polyamine tren with 2,6-
bis(bromomethyl)pyridine in 1:1 molar ratio using K2CO3 as a base
in refluxing CH3CN. Detosylation is carried out with HBr/HAc, and
the final product (P) is obtained as the hydrobromide salt. Com-
pounds PN, P2Py and P3Py are obtained by reacting P in its free
amine form with 1-naphthaldehyde, 2 or 3-pyridinecarbaldehyde,
respectively, in dry ethanol followed by in situ reaction with sodium
borohydride and precipitation as hydrochloride salts. The polyamine
Pal was obtained by reaction of the pertosylated polyamine P with
N-(3-bromopropyl)phthalimide in 1:1 molar ratio using K2CO3 as
a base in refluxing CH3CN (Scheme 2) [12]. Deprotection and deto-
sylation were carried out with hydrazine and HBr/HAc respectively,
and the final product (Pal) was obtained as its hydrobromide salt.
A property that could be affecting the in vivo activity of these
compounds is their actual protonation degree at the physiological
pH of 7.4. We have determined by pH-metric titration the basicity
constants of the polyamines used in this study [10,11].
Among the compounds studied, Pal is the most basic since it
possesses an additional secondary nitrogen atom in the tail with
respect to P, while PN is the less basic likely due to the hydrophobic
environment generated by the naphthalene spacer. The basicity of
P2Py and P3Py is close basicity although slightly higher for the former.
These constants allow for deriving the average protonation de-
gree at any pH (see bottom file of Table 1) shows such a represen-
tation for pH 7.4. While, as expected, Pal is the compound having
the highest charge, P3Py is however the one with a lower net
charge at pH 7.4. As below discussed P3Py is also the compound
showing the most promising leishmanicidal activity.
Table 1
Stepwise basicity constants for compounds P, PN, P2Py, P3Py and Pal determined in
0.15 M NaClO4 at 298.1 K.
Reaction
Pa
PNa
P2Pyb
P3Pyb Palc
L þ H $ LHd
HL þ H $ LH2
H2L þ H $ LH3
H3L þ H $ LH4
log bc
10.19 (6)e 10.01 (1) 10.21 (1) 9.84 (5) 10.13 (1)
9.19 (3) 8.71 (1) 8.84 (1) 8.52 (8) 9.40 (1)
7.94 (4) 7.27 (1) 7.39 (2) 6.8 (1) 8.27 (1)
3.12 (2) 3.4 (1) 7.02 (1)
27.32
25.99
29.56 (2) 28.6 (1) 34.84 (1)
3.2. In vitro antileishmanial evaluation
Mean nꢀ protons at pH ¼ 7.4 2.7
2.4
2.5 2.0 3.2
a
Taken from Ref. [7].
Taken from Ref. [8].
This work.
Charges omitted.
The IC50 values obtained for the promastigote, axenic amasti-
gote and amastigote forms of the L. infantum and L. braziliensis for
the tested compounds at concentrations ranging from 1 to 100
shown in the first three columns of Tables 2 and 3, respectively,
b
c
d
mM,
e
Values in parenthesis are standard deviations in the last significant figures.

C. Marín et al. / European Journal of Medicinal Chemistry 62 (2013) 466e477
471
Table 2
In vitro activity, toxicity and selectivity index found for the scorpiand-like azamacrocycle derivatives on extracellular and intracellular forms of Leishmania infantum.
Compounds
Activity IC50
Extracellular
promastigote
forms
(
m
M)a
Toxicity
J774.2
SIc
Axenic
amastigote
forms
Intracellular
amastigote
forms
Extracellular
promastigote
forms
Axenic
Intracellular
macrophages
amastigote
forms
amastigote
forms
IC50b
Glucantime
P
P*
P2py
P2py*
PN
18.0
18.9
40.7
15.5
21.2
21.7
38.2
18.8
40.8
24.2
27.0
31.0
29.2
40.4
25.4
77.8
28.1
45.0
15.9
33.7
29.8
33.6
30.8
25.6
30.7
19.6
50.3
17.4
60.1
6.3
15.2
231.6
218.6
254.0
275.7
365.3
387.9
467.8
223.9
220.4
258.1
0.8
12.2 (15)
5.4 (7)
16.4 (20)
13.0 (16)
18.8 (23)
10.2 (13)
24.9 (31)
5.5 (7)
0.5
0.5
8.0 (16)
5.4 (11)
10.0 (20)
3.5 (7)
13.0 (26)
8.6 (17)
29.4 (59)
6.6 (13)
7.4 (15)
7.7 (15.4)
9.0 (18)
7.1 (14)
13.0 (26)
5.5 (11)
21.0 (42)
6.5 (13)
74.3 (149)
4.5 (9)
PN*
P3py
P3py*
Pal
50.1
44.9
61.6
9.1 (11)
9.6 (12)
4.9 (10)
4.2 (8)
Pal*
Results are averages of four separate determinations.
a
IC50 ¼ the concentration required to give 50% inhibition, calculated by linear regression analysis from the Kc values at concentrations employed (1, 10, 25, 50 and 100
mM).
b
c
Towards J774.2 macrophage after 72 h of culture.
Selectivity index ¼ IC50 macrophage/IC50 extracellular and intracellular form of parasite. In brackets: number of times that compound SI exceeds the reference drug SI.
measuring the infection rates and the average number of amasti-
gotes presented during a 10-day treatment period in L. infantum,
where the rate of host-cell infection gradually increased: values
close to 62% percent cells were infected on day 10 (Fig. 2A). The IC25
of each of the products was used as the test dosage in these
assays and Glucantime was the reference drugs. The decrease in
the infection rate for the compounds P2Py, PN and P3Py was
always greater than 62%, making them much more effective than
Glucantime (24%). Compound P3Py showed an especially remark-
able behaviour by dropping the infection rate an 84%, close but
minor is the effectiveness of compounds P2Py and PN with
a reduction of 77% and 74% respectively, when were compared
against the control. The average number of amastigotes increased to
a peak on the 10th day of culture (Fig. 2B); at the end of treatment,
the three compounds tested had significantly decreased the number
of amastigotes present in the cells by 67%, 71%, and 77% respectively
for P2Py, PN and P3Py, whereas Glucantime only showed a reduc-
tion of 30%. The same experiment was performed with L. braziliensis,
and the results obtained concerning infection rates (Fig. 3A) and
amastigote numbers (Fig. 3B) are shown in Fig. 3. Those results were
similar to the ones obtained by L. infantum, with the exception of the
inclusion of P compound. Nevertheless, the best result was provided
by the compound P3Py which was the most effective reducing
the inhibition capacity and intracellular replication, against both
Leishmania spp.
3.3. Studies on the mechanism of action
We performed several experiments to elucidate a possible mech-
anism of action for the aza-scorpiand like macrocyclic derivatives,
which showed a SI number 20 times higher. Thus, the compounds
selected in the case of L. infantum were P2Py, PN and P3Py, while in
the case of L. braziliensis (P, P2Py, PN and P3Py).
3.3.1. SOD enzymatic inhibition in parasites and human
erythrocytes
These results prompted us to evaluate the inhibitory effect of the
P, P2Py, PN and P3Py compounds on SOD activity to test their
potential as competitors for the metallic ions of the enzyme. We
used promastigote forms of Leishmania spp., which were shown to
excrete Fe-SOD when cultured in a medium lacking inactive foetal
bovine serum [20], and a range of drug concentrations from 0.1 to
50
mM was applied in each case.
The results obtained shown in Fig. 4, and the corresponding IC50
values were calculated and are included in the same figure. Signifi-
cant inhibitory values of Fe-SOD activity were found for all com-
pounds tested (Fig. 4C and D). In general, all the compounds
significantly inhibited the Leishmania spp. Fe-SOD activity. Dis-
tinguishing, the compound P3Py which showed a 100% inhibition at
the 5
mM and 12.5 mM dose for L. infantum and L. braziliensis,
respectively (Fig. 4C and D). The design of an effective drug able to
Table 3
In vitro activity, toxicity and selectivity index found for the scorpiand-like azamacrocycle derivatives on extracellular and intracellular forms of Leishmania braziliensis.
Compounds
Activity IC50
Extracellular
promastigote
forms
(
m
M)a
Toxicity
J774.2
SIc
Axenic
amastigote
forms
Intracellular
amastigote
forms
Extracellular
promastigote
forms
Axenic
amastigote
forms
Intracellular
amastigote
forms
macrophages
IC50b
Glucantime
P
P*
P2py
P2py*
PN
25.6
11.8
32.8
11.3
12.4
12.3
29.5
23.0
21.2
15.5
25.5
30.9
16.0
54.3
16.0
26.7
24.9
59.2
23.9
41.6
31.4
42.6
19.7
10.0
30.4
10.4
16.4
16.6
28.9
10.3
47.0
19.1
77.8
15.20
231.6
218.6
254.0
275.7
365.3
387.9
467.8
223.9
220.4
258.1
0.6
0.5
14.5 (29)
4.0 (8)
0.8
23.2 (29)
7.3 (9)
24.4 (30)
16.8 (21)
22.0 (27)
13.4 (17)
45.4 (57)
4.8 (6)
19.6 (30)
6.8 (11)
22.5 (37)
22.2 (37)
29.7 (49)
13.1 (22)
20.3 (34)
10.6 (18)
14.2 (24)
10.1 (17)
15.9 (32)
10.3 (20)
14.7 (29)
6.6 (13)
19.6 (39)
5.4 (11)
7.0 (14)
6.1 (12)
PN*
P3py
P3py*
Pal
11.5 (14)
3.3 (4)
Pal*
Results are averages of four separate determinations.
a
IC50 ¼ the concentration required to give 50% inhibition, calculated by linear regression analysis from the Kc values at concentrations employed (1, 10, 25, 50 and 100
mM).
b
c
Towards J774.2 macrophage after 72 h of culture.
Selectivity index ¼ IC50 macrophage/IC50 extracellular and intracellular form of parasite. In brackets: number of times that compound SI exceeds the reference drug SI.

472
C. Marín et al. / European Journal of Medicinal Chemistry 62 (2013) 466e477
Fig. 2. Effect of scorpiand-like azamacrocycle derivatives on the infection rate and
growth of L. infantum: (A) rate of infection; (B) mean number of amastigotes per
infected macrophage cell: (:) control; (B) Glucantime; (C) P2Py; (þ) PN; (-) P3Py
measured at IC25. Values are the means of three separate experiments.
Fig. 3. Effect of scorpiand-like azamacrocycle derivatives on the infection rate and
growth of L. braziliensis: (A) rate of infection; (B) mean number of amastigotes per
infected macrophage cell: (:) control; (B) Glucantime; (C) P2Py; (þ) PN; (-) P3Py
measured at IC25. Values are the means of three separate experiments.
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 on Mn-SOD and Cu/Zn-SOD of human erythrocytes
(Fig. 4A and B). The results obtained showed that the inhibition
percentages for human CuZn-SOD and Mn-SOD were very small for
both the higher and the lower dosages, IC50 values 381.1,178.5, 408.0
treated cells showed that the three compounds: P2Py, PN and
P3Py significantly altered the metabolites excreted by L. infantum.
When the promastigote forms of L. infantum were treated with
compound P2Py at IC25 doses, the excretion of the catabolites
succinate was clearly altered (Fig. 5B and Table 4) and a new peak,
which was subsequently identified as ethanol, appeared in the
spectrum. All excreted metabolites presented a reduction when PN
product was added to the culture medium (Fig. 5C and Table 4)
although significant ethanol excretion increase was observed. After
and 641.0
pounds P, P2Py, PN and P3Py against L. infantum (Fig. 4A) values
111.4, 40.1, 58.3 and 63.6 M were obtained on CuZn-SOD from hu-
mM were reached on Mn-SOD from E. coli and for com-
m
man, for the tested compounds against L. braziliensis (Fig. 4B).
addition of product P3Py, succinate, D-lactate and L-alanine excre-
3.3.2. Metabolite excretion effect
tions were reduced (Fig. 5D and Table 4), an increase on ethanol
production could also be observed. When the test was ran against
Leishmania species have a high rate of glucose consumption,
which results in the acidification of the culture medium due to the
incomplete oxidation of glucose, they excrete into the medium
a considerable part of the hexose skeleton as partially oxidized
fragments in the form of fermented metabolites, although their
nature and percentage depend on the pathway used for glucose
metabolism by each of the species considered [21,22]. 1H NMR
spectroscopy enables the identification of the fermented metabo-
lites excreted by the parasites during in vitro culture. Fig. 5A shows
the spectrum produced by cell-free medium four days after inocu-
lation with L. infantum. The peaks that correspond to the major
metabolites produced and excreted during growth were apparent
when these spectrums were compared versus fresh medium
(spectra not shown). L. infantum excretes mainly succinate and ac-
L. braziliensis in fresh medium succinate, acetate and both
D-lactate
and -alanine were excreted, also a significant amount of ethanol
L
(Fig. 6A). After the treatment of cultures with products: P, P2Py, PN,
and P3Py; all compounds exhibited similar behaviour comparing
with L. infantum but with some exceptions, such as, the ethanol
outputs were clearly decreased (P2Py > P3Py [ PN). Regarding
the D-lactate, the only compound that showed an increase effect
was P3Py (Fig. 6 and Table 4).
3.3.3. Ultrastructural alterations
TEM revealed substantial morphological alterations in Leishma-
nia spp. promastigotes after treatment with the newly aza-scorpiand
like macrocyclic derivatives compared with the control sample
(Figs. 7 and 8). All of the tested compounds (P2Py, PN, and P3Py)
induced alterations in L. infantum promastigotes, but the product
etate as its major metabolites and in lower amounts
D-lactate and L-
alanine (Fig. 6A). The 1H NMR spectra of the medium from drug-

C. Marín et al. / European Journal of Medicinal Chemistry 62 (2013) 466e477
473
Fig. 4. (A) In vitro inhibition (%) of Mn-SOD from E. coli for the scorpiand-like azamacrocycle derivatives. (B) In vitro inhibition (%) of CuZn-SOD from human erythrocytes for the
scorpiand-like azamacrocycle derivatives. In vitro inhibition (%) of Fe-SOD in (C) L. infantum and (D) L. braziliensis promastigotes for the scorpiand-like azamacrocycle derivatives.
Values are means of three separate determinations. Differences between the activities of the control homogenate and those incubated with compounds were analyzed with the
NewmaneKeuls test. An IC50 was calculated by linear regression analysis from the Kc values at the concentrations employed (0.1e50 mM).
P2Py was one of the most effective products inducing the parasites
to death (Fig. 7-2), those that were not dead were high electro
density, suffering from size reduction and corrugated citoplasmatic
membrane surface. In all treated parasites an increase on the num-
ber of glycosomes was observed in comparison with the control
cultures. On parasites treated with PN product (Fig. 7-3) a high
number of strongly electro-dense vesicles was observed. Promasti-
gote forms treated contained a higher number of vesicles showing
death-like aspect. Majority of the parasites treated with those
products presented large vacuoles on their cytoplasm, most of those
were empty and in some others the kinetoplast or the mitochondria
were swollen. Equally very effective was the P3Py compound as can
be observed on Fig. 7-4, were the massive alterations on that pro-
mastigote sample were produced and are extendable to all parasites
treated. As could be observed, the cytoplasm was disorganized, both
mitochondria and the kinetoplast were swollen and some areas of
the citoplasmatic membrane were disrupted. When the effects of the
products P, PN, P2Py and P3Py were tested against L. braziliensis
using MET microscopy many structural alterations were observed
shown on Fig. 8. Only PN product induced a very few alterations
(photos not shown), while the remaining products were very
effective when compared with the control. On Fig. 8-2, the full ac-
tivity of Py products is shown were promastigotes treated were all
dead, presenting all altered and almost empty cytoplasm. Some
electro-dense vesicles were observed in both dead and living para-
sites. On cultures treated using P2Py product (Fig. 8-3) the super-
natant was full with cellular rests from the disruption of parasites,
although product P2Py was not as effective as product P, severe al-
terations on L. braziliensis promastigotes were induced, therefore
most of the parasites were swollen and with very few of the cyto-
plasm content, presenting high numbers of empty vacuoles.
with P3Py product presented the cytoplasm full of lipid nature
small size vacuoles and some other empty ones, both the flagellum
and the flagellum pocket seem to be disorganized, many nature free
flagellum were observed on the supernatant.
4. Discussion
The present results document the leishmanicidal effect of new
synthetic aza-scorpiand like macrocycles. Most studies on the
in vitro biological activity of new compounds against Leishmania
spp. are performed on promastigote forms because it is much easier
to work with these forms in vitro. However, since extracellular
forms are not the developed forms of the parasite in vertebrate
hosts, evaluations made with extracellular forms are merely indi-
cative of the potential leishmanicidal activity of the compounds
tested. Consequently, a preliminary test using extracellular pro-
mastigote forms should always be complemented by a subsequent
evaluation using intracellular forms (amastigotes in vertebrate host
cells) for a better understanding of the activity results obtained
[13]. Besides extracellular promastigotes, we also prepared
extracellular axenic amastigote forms of both parasites according to
the procedure described by Ref. [16]. Intracellular assays were
performed by infecting macrophage cells with promastigotes,
which transformed into amastigotes within 1 day after infection.
After taking in consideration the IC50 data shown in Tables 2 and
3, it can be seen that the P, P2Py, PN and P3Py were the most active
of the 10 compounds tested in the three assays performed and
against two Leishmania spp. studied (L. infantum and L. braziliensis).
Furthermore, they were much more active than Glucantime against
both the extra- and intracellular forms of the parasite. Although the
rest of compounds even showed some interesting activity, they
were less active in all cases. Regarding the cytotoxicity evaluation
against macrophage cells, it was shown that almost all the com-
pounds were substantially less toxic than the reference drug with
Also very effective was P3Py compound (Fig. 8, plate 4) that
produced great alterations either on the cytoplasm at its ultra-
structural level neither on the nuclear structure. Parasites treated

474
C. Marín et al. / European Journal of Medicinal Chemistry 62 (2013) 466e477
Fig. 5. 1H NMR spectra showing metabolites excrete by promastigote forms of
L. infantum untreated (A), L. infantum treated with compounds P2Py (B), L. infantum
treated with compounds PN (C) and L. infantum treated with P3Py (D), IC25 dosage. Lac,
D-lactate; Ala. L-alanine; Ac, acetate; Suc, succinate; Eth, ethanol; Pyr, pyruvate; Gly,
glycerol and DMSO, dimethyl sulfoxide.
Fig. 6. 1H NMR spectra showing metabolites excrete by promastigote forms of
L. braziliensis untreated (A), L. braziliensis treated with P (B), L. braziliensis treated with
P2Py (C) and L. braziliensis treated with PN (D), L. braziliensis treated with P3Py (E) IC25
dosage. Lac, D-lactate; Ala. L-alanine; Ac, acetate; Suc, succinate; Eth, ethanol; Pyr,
pyruvate; Gly, glycerol and DMSO, dimethyl sulfoxide.

C. Marín et al. / European Journal of Medicinal Chemistry 62 (2013) 466e477
475
Table 4
Variation in the height of the peaks corresponding to catabolites excreted by L. infantum and L. braziliensis promastigote forms in the presence of scorpiand-like azamacrocycle
derivatives with respect to the control test.
Compounds
Leishmania infantum
Leishmania braziliensis
Ac
Suc
Lac
Ala
Et
Ac
Suc
Lac
Ala
Et
P
nd
nd
nd
nd
nd
18%
22%
6%
5%
6%
P2py
PN
P3py
ꢂ19%
ꢂ6%
1%
ꢂ59%
ꢂ3%
ꢂ36%
13%
12%
10%
110%
28%
ꢂ46%
ꢂ78%
0%
0%
19%
8%
0%
10%
ꢂ71%
ꢂ6%
ꢂ59%
ꢂ44%
ꢂ18%
8%
2%
ꢂ38%
ꢂ12%
ꢂ13%
ꢂ47%
Ac, acetate; Suc, succinate; Lac,
D
-lactate; Et, ethanol; Ala,
L-alanine. (ꢂ) peak inhibition; (þ) peak increasing; (¼) no difference detected; (nd) no determined.
IC50 values over 200
m
M, while against Glucantime it was 15.2
m
M.
extracellular forms of Leishmania spp. Attending to other studies
to elucidate the possible mechanisms of action, it should be
noted that these compounds not only showed greater alterations
in glucose catabolism, but also that it led to greater levels of Fe-
SOD inhibition. Since the Fe-SOD is a specific chemotherapy
target also present in mitochondrial localization, this fact would
agree with the hypothesis above that this less bulky compound
might have a greater ability to pass through the mitochondrial
membrane.
Considering now the more illustrative selectivity index values,
compounds P, P2Py and P3Py were again the most potentially
interesting, since their SI values were >20 times higher than that
one of Glucantime, both for L. infantum as for L. braziliensis, and
according to some authors [16], only those which the SI value is
over 20 are good enough to be consider interesting as antiparasitic.
The propagation in macrophage J774.2 cells data showed re-
sults that are in accordance with those described for intra- and
Fig. 7. Ultrastructural alterations by TEM in promastigotes of L. infantum treated with PN, P2Py and P3Py products. Plate 1. Control parasite of L. infantum showing organelles with
their characteristic aspect, such as nucleus (N), mitochondrion (M), glycosomes (G), microtubules (MT), flagellum (F), kinetoplast (K) and vacuoles (V) (bar ¼ 1.000 m). Plate 2.
Promastigotes of L. infantum treated with PN showed high-density vesicles (VE) and kinetoplast (K) and swollen mitochondria (M) (bar ¼ 1.590 m). Plate 3. L. infantum treated with
P2Py with glycosomes (G), high electro-dense (arrow) and corrugated citoplasmatic membrane (MB) (bar ¼ 1.590 m). Plate 4. Parasites treated with P3Py with mitochondrion
(M) and swollen kinetoplast (K) (bar ¼ 0.350 m).
m
m
m
m

476
C. Marín et al. / European Journal of Medicinal Chemistry 62 (2013) 466e477
Fig. 8. Ultrastructural alterations by TEM in promastigotes of L. braziliensis treated with P, P2Py and P3Py products. Plate 1. Control parasite of L. braziliensis showing organelles with
their characteristic aspect, such as nucleus (N), mitochondrion (M), glycosomes (G) and vacuoles (V) (bar ¼ 1.000 m). Plate 2. Promastigotes of L. braziliensis treated with P dead
(arrow) with high-density vesicles (VE) (bar ¼ 1.590 m). Plate 3. L. braziliensis treated with P2Py with remaining fragments of destroyed parasites (arrow), empty vacuoles (V) and
corrugated citoplasmatic membrane (MB) (bar ¼ 1.590 m). Plate 4. Parasites treated with P3Py with large empty vacuoles (V), large number of granulites (G) and altered flagellum
pocket (BF) (bar ¼ 0.583 m).
m
m
m
m
It is well known that trypanosomatids are unable to completely
degrade glucose to CO2 under aerobic conditions. As a consequence,
they excrete into the medium a considerable part of the hexose
skeleton as partially oxidized fragments in the form of fermented
metabolites, although their nature and percentage depend on the
pathway used for glucose metabolism by each of the species con-
sidered [21,22]. The final products of glucose catabolism in Leish-
The results were compared with those found for promastigotes
maintained in a cell-free medium (control) for four days after the
inoculation with the parasite. The characteristic presence of acetate,
D-lactate, and succinate was confirmed in the control experiments
performed on all two species. However, noteworthy differences
between them were reduction in the excretion of ethanol by
L. braziliensis, whereas ethanol was not detected in L. infantum
promastigotes. When parasites (L. braziliensis) were treated with the
compound P, no significant effect was found. The other compound
tested, P2py, showed a significant decrease of acetate in L. brazil-
iensis, whereas no variations were found in the case of L. infantum.
The cause of this metabolic change appears to be an action on en-
zymes acetate: succinate CoA transferase and succinyl-CoA synthe-
tase directly involved in mitochondrial production of acetate from
acetyl-CoA [25]. Another metabolite, which appears significantly
reduced in both species is the succinate, so it seems that the glyco-
somal enzymes that are involved in the succinate production must
be disrupted. Finally in L. braziliensis a remarkable decrease in the
output of ethanol was found also, which means that the only path-
way properly working that keep the parasite in optimum level of
energy to grow, is the way to use alanine in the cytosol, being
both glycosomal and mitochondrial pathways altered [26]. Those
mania are usually CO2, succinate, acetate, D-lactate, L-alanine, and
ethanol [23]. Among them, succinate specially is relevant since its
main role is in maintaining the glycosomal redox balance, allowing
the reoxidation of NADH produced in the glycolytic pathway. Suc-
cinic fermentation has the advantage of requiring only half of the
phosphoenolpyruvate produced to maintain the NADþ/NADH bal-
ance, and the remaining pyruvate is converted inside the mito-
chondrion and the cytosol into acetate,
D-lactate, L-alanine, or
ethanol according to the degradation pathway followed by each
species [24].
In order to obtain some information concerning the effect of the
tested compounds on metabolism of glucose in the parasites, we
registered the 1H NMR spectra of promastigotes from L. infantum and
L. braziliensis after treatment with compounds studied and the final
excretion products were identified qualitatively and quantitatively.

C. Marín et al. / European Journal of Medicinal Chemistry 62 (2013) 466e477
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[26]. Regarding to the compound P3py, both species were inhibited
in somehow in their glycosomal enzymatic clusters, so the subsis-
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decreasing its excretion to the medium with respect to control. On
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slightly increased in the culture medium, this suggests that despite
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with some step of tricarboxylic cycle (TCA) preventing the parasite
uses this fundamental way and having to resort to means of com-
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glucose. Another mechanism of action could be postulated for this
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transformed into alanine in an amount sufficient for it to appear in
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They also showed a wide range of ultrastructural alterations to
the promastigotes forms of Leishmania spp. treated with com-
pounds were 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.
Thus, these in vitro results show that the synthetic aza-scorpiand
like macrocyclic derivatives are potentially promising agents for the
treatment of Leishmania infection. Further in vivo studies are war-
ranted to further evaluate this potential, actually a patent about this
family of compound has been already filled (P201132035).
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Acknowledgements
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Moreno, Use of an iron superoxide dismutase excreted by Trypanosoma cruzi
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of Queretaro, Mexico, Am. J. Trop. Med. Hyg. 73 (2005) 510e516.
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of glycosomes in trypanosomatids, Biochem. Biophys. Acta 1763 (2006)
1463e1477.
Financial support by the Spanish Ministerio de Ciencia e Inno-
vación and FEDER funds of the E.U. (Projects CTQ2009-14288-CO4-
01 and CONSOLIDER INGENIO 2010 CSD2010-00065), Generalitat
Valenciana (PROMETEO 2011/008) is gratefully acknowledged.
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