Abstract

Objectives

To evaluate the in vitro leishmanicidal activity of imidazole-based (14) and pyrazole-based (56) benzo[g]phthalazine derivatives against Leishmania infantum and Leishmania braziliensis.

Methods

The in vitro activity of compounds 16 was assayed on extracellular promastigote and axenic amastigote forms, and on intracellular amastigote forms of the parasites. Infectivity and cytotoxicity tests were performed on J774.2 macrophage cells using meglumine antimoniate (Glucantime) as the reference drug. The mechanisms of action were analysed by iron superoxide dismutase (Fe-SOD) and copper/zinc superoxide dismutase (CuZn-SOD) inhibition, metabolite excretion and transmission electronic microscopy (TEM).

Results

Compounds 16 were more active and less toxic than meglumine antimoniate. Data on infection rates and amastigote mean numbers showed that 2, 4 and 6 were more active than 1, 3 and 5 in both L. infantum and L. braziliensis. The inhibitory effect of these compounds on the antioxidant enzyme Fe-SOD of promastigote forms of the parasites was remarkable, whereas inhibition of human CuZn-SOD was negligible. The ultrastructural alterations observed in treated promastigote forms confirmed the greater cell damage caused by the most active compounds 2, 4 and 6. The modifications observed by 1H-NMR in the nature and amounts of catabolites excreted by the parasites after treatment with 16 suggested that the catabolic mechanisms could depend on the structure of the side chains linked to the benzo[g]phthalazine moiety.

Conclusions

All the compounds assayed were active in vitro against the two Leishmania species and were less toxic against mammalian cells than the reference drug, but the monosubstituted compounds were significantly more effective and less toxic than their disubstituted counterparts.

Introduction

Leishmaniasis is the name assigned to a group of diseases caused by kinetoplastid protozoan parasites of the genus Leishmania. Its impact on public health is evident when considering the fast expansion of endemic zones over recent years. It is now endemic in 98 countries, mainly in the New World, but also in Europe and Asia. More than 350 million people are at risk, and 2 million new cases arise every year, with an annual mortality rate higher than 60 000, a number that is only surpassed by malaria among parasitic diseases.1,2 In fact, leishmaniasis is placed ninth in the WHO global analysis of the most severe infectious diseases. In recent times, it has been reported that coinfection of HIV with leishmaniasis in immunocompromised hosts is a new factor that is increasing the endemic areas all around the world.3

Leishmaniasis appears as three major clinical forms in humans: (i) visceral, the most severe and life-threatening form; (ii) cutaneous, originating as nodules and ulcers that may persist for years; and (iii) mucocutaneous, causing permanent lesions in the mouth, nose or genital mucosa.4 They are produced by multiple and phylogenetically distinct species. Leishmania infantum is considered the main aetiological agent of visceral leishmaniasis in southern Europe. It uses dogs as the reservoir and affects mainly children between 1 and 4 years old, although since the advent of HIV infection and increased use of immunosuppression for transplants and chemotherapy, nearly half of the new cases are now in adults.5–7 Another significant species, derived from the subgenus Viannia, is Leishmania braziliensis, which occurs mainly in the Andean countries and the Amazonian basin and causes cutaneous and mucocutaneous leishmaniasis.1,2,8 Those two species were selected by us as representative targets for the biological assays performed in the present work.

The design of new drugs active against the different forms of these parasitic diseases, especially visceral leishmaniasis, is an urgent necessity. To date, no effective vaccination is available, and diagnostic tools are not specific due to deficient vector control measures.9,10 Physicians rely mainly on chemotherapy as the primary weapon to fight all the clinical manifestations mentioned above. However, the drugs in use until now are largely ineffective or cause a multitude of severe toxic side effects and, furthermore, the parasitic organisms rapidly develop strong drug resistance, decreasing their sensitivity to these drugs.11 For more than 50 years, heavy metal derivatives, mainly pentavalent antimonials, have been used as the standard drugs for the treatment of leishmaniasis. Among them, the most representative are sodium stibogluconate (Pentostam) and meglumine antimoniate (Glucantime). It is supposed, but not verified, that they act by inhibiting ATP synthesis. However, antimonials cause many toxic effects, including nausea, vomiting, diarrhoea, skin eruptions, dizziness, cardiac arrhythmia, hypotension, hepatitis and pancreatitis. Furthermore, drug administration is difficult, low dosages favour resistance in the parasite and higher dosages are disturbingly toxic.12,13 Other types of drugs have been tested recently. Pentamidines are only effective against cutaneous leishmaniasis and, although better tolerated, they cause diabetes mellitus at high doses. Fluconazole, amphotericin B and miltefosine are not useful against visceral leishmaniasis and, in spite of their milder toxicity, effectiveness against other forms of the disease is limited.12,13 In summary, more effective, less expensive and less toxic drugs are needed.

Antioxidant enzymes count among the main defence mechanisms for the survival of Leishmania parasites. They protect the parasite against the toxic oxygen intermediates and nitrogen species produced during mitochondrial respiration that damage proteins, lipids and DNA, leading to cell death.14 One of the most important is superoxide dismutase (SOD), which is localized in the mitochondria and is responsible for the dismutation of radical anion peroxide into oxygen peroxide and molecular oxygen. Although there are several types of SOD, they can be differentiated by the nature of the metal ion co-factors (i.e. Cu2+ and Zn2+ in human SOD). Interestingly, the only type of SOD present in trypanosomatids is iron SOD (Fe-SOD).15 The enzyme appears as a dimeric protein, and the coordination geometry of the active Fe site is a distorted trigonal bipyramidal arrangement, whose axial ligands are His43 and a solvent molecule, and whose in-plane ligands are His95, Asp195 and His199.16 Studies performed on the role of this enzyme in Leishmania species confirm that it is an essential defence line in host–parasite relationships. Since Fe-SOD is not present in humans, it may be considered an attractive target for leishmaniasis drug therapy.17

In recent years our research group has designed new heterocyclic systems with general structures I and II (Figure 1). These compounds contain a nucleus of benzo[g]phthalazine functionalized in the 1 and 4 positions of the pyridazine ring with flexible alkylamino side chains. The alkylamino substituents vary in nature and length. Series I exhibit terminal sp3 amino groups or heterocyclic rings endowed with sp2 nitrogen atoms, whereas series II is characterized by the presence of a terminal OH group. Both models have shown complexing ability for transition metals, but the complex structure strongly depends on the side chain terminal functionalization: the sp2 or sp3 nitrogens of podands I are actively involved in complexation, giving rise to monopodal dinuclear complexes, whereas podands II afford tripodal dinuclear complexes in which the terminal hydroxy groups are not involved.18 The antiparasitic activity of both groups of compounds has been evaluated in vitro against Trypanosomacruzi epimastigotes and amastigotes, and good activities were found for the type I compounds, especially in the case of sp2 nitrogens, while the type II compounds were clearly less active. Furthermore, structures with terminal nitrogens inhibited the action of the Fe-SOD enzyme much better than one with terminal OH groups. Human copper/zinc SOD (CuZn-SOD) inhibition was negligible in any case. We concluded that, in all cases tested, the complexing ability could be related in some way to Fe-SOD inhibition and, consequently, to trypanosomicidal activity.18

Figure 1.

Schematic models of 1,4-bis(alkylamino)benzo[g]phthalazine derivatives functionalized with amino, pyridine or hydroxy groups at the end of the side chains.

Figure 1.

Schematic models of 1,4-bis(alkylamino)benzo[g]phthalazine derivatives functionalized with amino, pyridine or hydroxy groups at the end of the side chains.

With the aim of improving the biological results described above, new 1-alkylamino-4-chloro- and 1,4-dialkylaminobenzo[g]phthalazine derivatives with general structures III and IV (Figure 2) were prepared.19,20 The side chains were functionalized with terminal imidazole (compounds 14, Figure 3) or pyrazole (compounds 56) rings. Both pentagonal heterocycles are endowed with electron-donating sp2 nitrogens, which suggests excellent complexing properties against transition metals, and the imidazole derivatives had previously been shown to be active against trypanosomatids.12 Activity tests performed in vitro and in vivo with T. cruzi demonstrated that all compounds 16 were effective against both the acute and chronic phases of Chagas’ disease. Antiparasitic activity in vitro was enhanced with respect to series I and II. Moreover, the new compounds were more active and less toxic than the reference drug (benznidazole). Among them, the monosubstituted derivatives 2, 4 and 6 gave better results both in terms of activity and the absence of toxicity than their disubstituted analogues. In accordance with our expectations, excellent values of inhibition of Fe-SOD activity were also obtained, whereas the effect on human CuZn-SOD was negligible.19,20

Figure 2.

Schematic models of 1-alkylamino-4-chloro- and 1,4-bis(alkylamino)benzo[g]phthalazine derivatives functionalized with imidazole or pyrazole rings at the end of the side chains.

Figure 2.

Schematic models of 1-alkylamino-4-chloro- and 1,4-bis(alkylamino)benzo[g]phthalazine derivatives functionalized with imidazole or pyrazole rings at the end of the side chains.

Figure 3.

Benzo[g]phthalazine derivatives tested against Leishmania infantum and Leishmania braziliensis in this study.

Figure 3.

Benzo[g]phthalazine derivatives tested against Leishmania infantum and Leishmania braziliensis in this study.

Taking into account the essential role played by the Fe-SOD enzyme in both Leishmania and T. cruzi parasites, we considered it to be of great interest to study the activity of compounds 16 against L. infantum and L. braziliensis (promastigote and amastigote forms), as representative species causing visceral and cutaneous leishmaniasis, respectively. In this work, their antiproliferative activity and unspecific mammalian cytotoxicity in the species considered were evaluated in vitro, and these measures were complemented by infectivity assays on J774.2 macrophage cells. Furthermore, the treated parasites were submitted to a thorough study of the possible mechanisms of action of the compounds assayed, as follows: (i) inhibition of the parasitic Fe-SOD and human CuZn-SOD enzymes was tested and compared; (ii) an 1H-NMR study concerning the nature and percentage of metabolite excretion was performed in order to obtain information on the inhibitory effect of 16 on the glycolytic pathway, since it represents the primary source of energy for the parasite; and (iii) alterations caused in the cell ultrastructure of the parasites were recorded using transmission electronic microscopy (TEM).

Materials and methods

Chemistry

The starting amines were 2-(imidazol-4-yl)-ethylamine (histamine), 3-(imidazol-1-yl)propylamine and 3-aminopyrazole and were purchased from Sigma-Aldrich and used without further purification. 1,4-Dichlorobenzo[g]phthalazine was obtained from 2,3-naphthalene-dicarboxylic acid as previously reported by our group.21 Solvents were dried using standard techniques.22 All the reactions were monitored using thin-layer chromatography (TLC) on precoated aluminium sheets of silica gel 60F254. Compounds were detected with UV light (245 nm). Chromatographic separations were performed on columns using flash chromatography on silica gel (particle size 0.040–0.063 mm) or standard techniques on basic aluminium oxide. Melting points were determined in a Reichert–Jung hot-stage microscope. 1H- and 13C-NMR spectra were recorded with Varian Unity XL-300, Varian Unity Inova-400 or Varian Unity 500 spectrometers at room temperature employing CDCl3, CD3OD or DMSO-d6 as solvents. Chemical shifts were recorded in ppm from tetramethylsilane (Δ scale). Infrared (IR) spectra were recorded on a Perkin Elmer 681 spectrometer. Electrospray mass spectra were recorded with a Hewlett-Packard 1100 MSD apparatus and fast atomic bombardment mass spectra with a VG Autospec spectrometer using an m-nitrobenzyl alcohol matrix. Elemental analyses were provided by the Departamento de Análisis, Centro de Química Orgánica ‘Manuel Lora Tamayo’, CSIC, Madrid, Spain.

The initial preparation and identification of compounds 16 has been described previously by our group, but was carried out again in sufficient quantities and purity for performing all the biological assays described in this work. The syntheses followed the procedures established by us,19,20 so only the amounts employed and the yields obtained are indicated below.

Preparation of the imidazole-based compounds 1 and 2

A solution of 1,4-dichlorobenzo[g]phthalazine (1.54 g, 6.24 mM), 2-(imidazol-4-yl)ethylamine (2.77 g, 25.0 mM) and triethylamine (1.90 g, 19 mM) in xylene (200 mL) was heated at 120–130°C for 10 h. Standard work up of the reaction mixture19 afforded 0.52 g (22% yield) of 1,4-bis[2-(imidazol-4-yl)ethylamino]benzo[g]phthalazine [1, melting point (mp) 124–125°C, mass spectra fast atom bombardment (MS FAB): 399 [M+ + 1]] and 1.32 g (63% yield) of 1-[2-(imidazol-4-yl)ethylamino]-4-chlorobenzo[g]phthalazine (2, mp 243–245°C, MS FAB 323 [M+]).

Preparation of the imidazole-based compounds 3 and 4

A solution of 1,4-dichlorobenzo[g]phthalazine (1.60 g, 6.44 mM), 3-(imidazol-1-yl)propylamine (3.10 g, 26.0 mM) and triethylamine (1.90 g, 19 mM) in xylene (200 mL) was heated at 120–130°C for 10 h. Standard work up of the reaction mixture19 afforded 0.81 g (29% yield) of 1,4-bis[3-(imidazol-1-yl)propylamino]benzo[g]phthalazine (3, mp 120–123°C, MS FAB: 427 [M+ + 1]) and 1.01 g (49% yield) of 1-[3-(imidazol-1-yl)propylamino]-4-chloro-benzo[g]phthalazine (4, mp 215–217°C, MS FAB 337 [M+]).

Preparation of the pyrazole-based compounds 5 and 6

A solution of 1,4-dichlorobenzo[g]phthalazine (2.24 g, 8.96 mM), 3-aminopyrazole (1.64 g, 19.60 mM) and triethylamine (4.80 g, 48 mM) in xylene (250 mL) was heated at 120–130°C for 10 h. Standard work up of the reaction mixture20 afforded 0.60 g (18% yield) of 1,4-bis(3′-pyrazolylamino)benzo[g]phthalazine in the monohydrochloride form [5 · HCl, mp 284–286°C, electrospray ionization mass spectra (ESI-MS) positive mode: 343 [MH-Cl]+] and 0.40 g (16% yield) of free 4-chloro-1-(3′-pyrazolylamino)benzo[g]phthalazine (6, mp 232–234°C, ESI-MS positive mode 296 [MH]+).

Parasite strain and culture

L. infantum (MCAN/ES/2001/UCM-10) and L. braziliensis (MHOM/BR/1975/M2904) were cultured in vitro in medium trypanosomes liquid (MTL) together with 10% inactive fetal 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 by González et al.23

SOD enzymatic inhibition

Parasites cultured as described above were suspended (0.5–0.6 g/mL) in 3 mL of STE buffer 1 (0.25 M sucrose, 25 mM Tris–HCl, 1 M EDTA, pH 7.8) and disrupted by three cycles of sonic disintegration, for 30 s each at 60 V. The sonicated homogenate was centrifuged at 1500 g 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 (2500 g 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 (9000 g for 20 min at 4°C), redissolved 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 (Pharmacia, PD 10), previously balanced 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.

Fe-SOD activity was determined by NAD(P)H oxidation according to Paoletti and Mocali.24 One unit was the amount of enzyme required to inhibit the rate of NAD(P)H reduction by 50%. The CuZn-SOD from human erythrocytes used in these assays was obtained from Boehringer (Mannheim), while all the coenzymes and substrates came from Sigma Chemical Co. Data obtained were analysed according to the Newman–Keuls test.

In vitro activity assays: extracellular forms

Promastigote assay

Compounds 16 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 González et al.23 The compounds were dissolved in the culture medium at concentrations of 100, 50, 25, 10 and 1 μM. The effects of each compound against the promastigote forms and its concentrations were tested at 72 h using a Neubauer haemocytometric chamber. The leishmanicidal 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.

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 González et al.23 After 72 h of treatment, cell viability was determined by flow cytometry. Thus, 100 μL/well of propidium iodide solution (100 μg/mL) were added and incubated for 10 min at 28°C in darkness. Afterwards, 100 μL/well of fluorescein diacetate (100 ng/mL) was added and incubation was performed under the conditions described above. Finally, the cells were recovered by centrifugation at 400 g for 10 min and the precipitate was washed with PBS. Flow cytometric analysis was performed with a FACSVantage™ 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.

Infectivity assay

J774.2 macrophage cells were grown in minimal essential medium (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 microplates (Nunc) with rounded coverslips on the bottom and cultured for 2days. 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 incubation was performed for 12 h at 37°C in 5% CO2. Non-phagocytosed 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 analysing more than 200 host cells distributed in randomly chosen microscopic fields. Values are the means of three separate determinations.

In vitro activity assays: intracellular forms

Axenic amastigote assay

Axenic amastigote forms of Leishmania spp. were cultured following the methodology described previously by Moreno et al.25 Thus, promastigote transformation to amastigotes was achieved after 3days 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 sucrose, 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 2-(N-morpholino)ethanesulfonic acid (MES), 0.4 mg/L haemin and 10 mg/L gentamicin, pH 5.4, at 37°C. The effect of each compound against axenic amastigote forms was tested at 48 h using a Neubauer haemocytometer.

Amastigote assay

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 μM) and then cultured for 72 h in MEM plus glutamine (2 mM) and 20% inactive FCS. Compound activity was determined from the percentage reductions in amastigote number in treated and untreated cultures in methanol-fixed and Giemsa-stained preparations. Values are the means of three separate determinations.23

Metabolite excretion

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 400 g for l0 min. The supernatants were collected to determine the excreted metabolites by 1H-NMR, and chemical shifts were expressed in 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 by Fernández-Becerra et al.26

Ultrastructural alterations

The parasites were cultured at a density of 5 × 105 cells/mL in the corresponding medium, each of which contained the compounds tested at the IC25 concentration. After 96 h, the cultures were centrifuged at 400 g 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 4°C. The pellets were then prepared for TEM employing the technique of González et al.23

Results and discussion

As explained above, previous studies have indicated that benzo[g]phthalazine derivatives 16 may be considered to be prospective chemotherapeutic drugs in the treatment of diseases caused by members of the Trypanosomatidae.19,20 We comment now on the results obtained concerning the toxic activity of these compounds against two species of Leishmania (L. infantum and L. braziliensis).

SOD enzymatic inhibition in parasites and human erythrocytes

The marked inhibitory activity of compounds 16 on the essential antioxidant enzyme Fe-SOD of T. cruzi20,21 prompted us to assay their effect on the two Leishmania species at a range of concentrations from 1 to 100 μM. We used promastigote forms of L. infantum and L. braziliensis, which excrete Fe-SOD when cultured in a medium lacking inactive FCS.24 Inhibition data obtained are shown in Figure 4(a and b), and the corresponding IC50 values are included in order to make the interpretation of results easier. For comparison, Figure 4(c) shows the effect of the same compounds on CuZn-SOD obtained from human erythrocytes. The most remarkable result was that Fe-SOD activity was significantly inhibited by the six tested compounds, whereas inhibition of human CuZn-SOD was consistently lower, with IC50 values ranging from 120.0 to 350.3 μM. Different behaviour was observed between the two Leishmania species assayed. The pyrazole-based derivatives 5 and 6 were much more effective against L. infantum than the imidazole-based compounds, with 6 about 12-fold more inhibitory than 1 and 4, and about 25- and 60-fold more inhibitory than 2 and 3, respectively. The negligible inhibitory effect of the monosubstituted derivative 6 against human SOD should also be noted (IC50 350.3 μM).

Figure 4.

In vitro inhibition (%) of Fe-SOD in (a) Leishmania infantum and (b) Leishmania braziliensis promastigotes for compounds 16. (c) In vitro inhibition of CuZn-SOD in human erythrocytes for compounds 16. Values are means of three separate determinations. Differences between the activities of the control homogenate and those incubated with compounds 16 were analysed with the Newman–Keuls test. aIC50 was calculated by linear regression analysis from the Kc values at the concentrations employed (1, 10, 25, 50 and 100 μM).

Figure 4.

In vitro inhibition (%) of Fe-SOD in (a) Leishmania infantum and (b) Leishmania braziliensis promastigotes for compounds 16. (c) In vitro inhibition of CuZn-SOD in human erythrocytes for compounds 16. Values are means of three separate determinations. Differences between the activities of the control homogenate and those incubated with compounds 16 were analysed with the Newman–Keuls test. aIC50 was calculated by linear regression analysis from the Kc values at the concentrations employed (1, 10, 25, 50 and 100 μM).

In the case of L. braziliensis, differences in the activity of the six tested compounds were much less marked, since the most effective compound (1) was only about 5-fold more inhibitory than the least active one (3). The disubstituted derivative 3 had the worst IC50 against both L. infantum and L. braziliensis. We conclude that the IC50 values could support some kind of interaction of these benzo[g]phthalazine derivatives with Fe-SOD active sites, especially on the basis of the results described for L. infantum: the increased system rigidity in compounds 5 and 6 and the presence of pyrazole rings with well-known complexing ability should allow a cooperative action of both the pyridazine and pyrazole nitrogens in metal complexation and should favour enzyme inhibition.

In vitro antileishmanial evaluation

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 indicative of the potential leishmanicidal activity of the compounds tested. Consequently, a preliminary test using extracellular promastigote 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.23 Besides extracellular promastigotes, we also prepared extracellular axenic amastigote forms of both parasites according to the procedure described by Moreno et al.25 Intracellular assays were performed by infecting macrophage cells with promastigotes, which transformed into amastigotes within 1day after infection. Tables 1 and 2 show the IC50 values obtained after 72 h of exposure when compounds 16 were assayed in promastigote forms, axenic amastigote forms and intracellular amastigote forms of L. infantum and L. braziliensis. Values for the reference drug, meglumine antimoniate, are included in all cases for comparison.

Table 1.

In vitro activity, toxicity and selectivity index for imidazole-based (14) and pyrazole-based (56) benzo[g]phthalazine derivatives in extra- and intracellular forms of L. infantum

Compounds Activity IC50 (μM)a
 
cMacrophage toxicity IC50 Selectivity indexb
 
Promastigote forms Axenic amastigote forms Intracellular amastigote forms Promastigote forms Axenic amastigote forms Intracellular amastigote forms 
Glucantime 18.0 ± 3.1 30.0 ± 2.7 24.2 ± 2.6 15.20 ± 1.3 0.8 0.5 0.6 
15.3 ± 1.7 16.2 ± 3.0 24.2 ± 2.6 306.9 ± 16.7 20 (25) 19 (38) 13 (22) 
12.6 ± 1.8 14.3 ± 1.4 20.4 ± 1.1 467.8 ± 20.5 37 (46) 33 (66) 23 (38) 
15.4 ± 3.3 17.7 ± 2.0 21.2 ± 3.6 98.9 ± 8.9 6 (7) 6 (12) 5 (8) 
20.1 ± 3.2 31.3 ± 4.2 46.9 ± 6.6 289.7 ± 11.4 14 (17) 9 (18) 6 (10) 
17.3 ± 2.2 28.4 ± 1.5 26.5 ± 2.9 269.8 ± 13.7 16 (20) 9 (18) 10 (17) 
10.0 ± 0.7 11.5 ± 0.9 20.3 ± 1.2 299.9 ± 17.9 30 (37) 26 (52) 15 (25) 
Compounds Activity IC50 (μM)a
 
cMacrophage toxicity IC50 Selectivity indexb
 
Promastigote forms Axenic amastigote forms Intracellular amastigote forms Promastigote forms Axenic amastigote forms Intracellular amastigote forms 
Glucantime 18.0 ± 3.1 30.0 ± 2.7 24.2 ± 2.6 15.20 ± 1.3 0.8 0.5 0.6 
15.3 ± 1.7 16.2 ± 3.0 24.2 ± 2.6 306.9 ± 16.7 20 (25) 19 (38) 13 (22) 
12.6 ± 1.8 14.3 ± 1.4 20.4 ± 1.1 467.8 ± 20.5 37 (46) 33 (66) 23 (38) 
15.4 ± 3.3 17.7 ± 2.0 21.2 ± 3.6 98.9 ± 8.9 6 (7) 6 (12) 5 (8) 
20.1 ± 3.2 31.3 ± 4.2 46.9 ± 6.6 289.7 ± 11.4 14 (17) 9 (18) 6 (10) 
17.3 ± 2.2 28.4 ± 1.5 26.5 ± 2.9 269.8 ± 13.7 16 (20) 9 (18) 10 (17) 
10.0 ± 0.7 11.5 ± 0.9 20.3 ± 1.2 299.9 ± 17.9 30 (37) 26 (52) 15 (25) 

Results are means of three separate determinations.

aIC50 was calculated by linear regression analysis from the Kc values at concentrations employed (1, 10, 25, 50 and 100 μM).

bSelectivity index (SI) is IC50 macrophage toxicity/IC50 activity in extracellular or intracellular forms of the parasite. The figure shown in parentheses is the fold increase in the SI of the compound compared with that of the reference drug.

cAgainst J774.2 macrophages after 72 h of culture.

Table 2.

In vitro activity, toxicity and selectivity index for imidazole-based (14) and pyrazole-based (56) benzo[g]phthalazine derivatives in extra- and intracellular forms of L. braziliensis

Compounds Activity IC50 (μM)a
 
cMacrophage toxicity IC50 Selectivity indexb
 
Promastigote forms Axenic amastigote forms Intracellular amastigote forms Promastigote forms Axenic amastigote forms Intracellular amastigote forms 
Glucantime 25.6 ± 1.6 31.1 ± 3.0 30.4 ± 6.1 15.20 ± 1.3 0.6 0.5 0.5 
30.5 ± 1.6 43.8 ± 7.7 68.10 ± 6.2 306.9 ± 16.7 10 (17) 7 (14) 5 (10) 
10.8 ± 0.4 12.3 ± 2.3 26.2 ± 0.9 467.8 ± 20.5 43 (72) 38 (76) 18 (36) 
22.4 ± 2.6 47.5 ± 6.4 20.9 ± 2.1 98.9 ± 8.9 4 (7) 2 (4) 5 (10) 
27.2 ± 1.1 32.4 ± 7.1 37.9 ± 0.1 289.7 ± 11.4 11 (18) 9 (18) 8 (16) 
21.3 ± 0.9 20.7 ± 3.7 23.3 ± 0.5 269.8 ± 13.7 13 (22) 13 (26) 12 (24) 
12.9 ± 1.3 21.9 ± 4.4 19.9 ± 1.4 299.9 ± 17.9 23 (38) 14 (28) 15 (30) 
Compounds Activity IC50 (μM)a
 
cMacrophage toxicity IC50 Selectivity indexb
 
Promastigote forms Axenic amastigote forms Intracellular amastigote forms Promastigote forms Axenic amastigote forms Intracellular amastigote forms 
Glucantime 25.6 ± 1.6 31.1 ± 3.0 30.4 ± 6.1 15.20 ± 1.3 0.6 0.5 0.5 
30.5 ± 1.6 43.8 ± 7.7 68.10 ± 6.2 306.9 ± 16.7 10 (17) 7 (14) 5 (10) 
10.8 ± 0.4 12.3 ± 2.3 26.2 ± 0.9 467.8 ± 20.5 43 (72) 38 (76) 18 (36) 
22.4 ± 2.6 47.5 ± 6.4 20.9 ± 2.1 98.9 ± 8.9 4 (7) 2 (4) 5 (10) 
27.2 ± 1.1 32.4 ± 7.1 37.9 ± 0.1 289.7 ± 11.4 11 (18) 9 (18) 8 (16) 
21.3 ± 0.9 20.7 ± 3.7 23.3 ± 0.5 269.8 ± 13.7 13 (22) 13 (26) 12 (24) 
12.9 ± 1.3 21.9 ± 4.4 19.9 ± 1.4 299.9 ± 17.9 23 (38) 14 (28) 15 (30) 

Results are means of three separate determinations.

aIC50 was calculated by linear regression analysis from the Kc values at concentrations employed (1, 10, 25, 50 and 100 μM).

bSelectivity index (SI) is IC50 macrophage toxicity/IC50 activity in extracellular or intracellular forms of the parasite. The figure shown in parentheses is the fold increase in the SI of the compound compared with that of the reference drug.

cAgainst J774.2 macrophages after 72 h of culture.

If we consider now the results displayed in Table 1 for activity against L. infantum, the leishmanicidal activity in both extra- and intracellular forms was similar or, in most cases, higher than that found for meglumine antimoniate, with the best results those of compounds 2 and 6. Only the imidazole derivative 4 exhibited lower activity than meglumine antimoniate in the three forms assayed. More interesting are the toxicity data, since all six compounds tested were found to be much less toxic for macrophages than the reference drug. Thus, compound 2 was 31-fold less toxic than meglumine antimoniate, and even the most toxic among them, the disubstituted derivative 3, was 6.5-fold more benign. Toxicity values substantially influence the more informative selectivity index (SI) data, and best values were again obtained for the monosubstituted compounds 2 and 6, with SI exceeding those of the reference drugs by 46-, 66- and 38-fold in the case of 2, and by 37-, 52- and 25-fold for 6. It should be noted that, when comparing the SI of every pair of compounds with the same type of side chain (i.e. 1 and 2, 3 and 4, 5 and 6), the monosubstituted derivatives always gave better results than their disubstituted analogues, as happened in the T. cruzi tests.20 The same behaviour was found when considering the macrophage toxicity of the three pairs, since the monosubstituted compounds were always less toxic than their counterparts.

Very similar conclusions can be extracted from the L. braziliensis results shown in Table 2. The monosubstituted compounds 2 and 6 again gave the best SI results in the three assays performed, with values exceeding those of the reference drugs 72-, 76- and 36-fold in the case of 2, and 38-, 28- and 30-fold for 6. It can also be seen that the three monosubstituted compounds 2, 4 and 6 had a better SI than their respective disubstituted pairs 1, 3 and 5. The imidazole-based derivative 2 had the lowest toxicity and highest SI values in both L. infantum and L. braziliensis.

Going one step further in the activity study, the effects of the compounds on the infectivity and intracellular replication of the amastigote forms were determined. Macrophages were cultured and infected with promastigotes in the stationary phase. The parasites invaded the cells and underwent morphological conversion to amastigotes within 1 day after infection. On day 10, the rate of host cell infection reached its maximum (control experiment).

For these assays we used the IC25 of each product as the test concentration, with meglumine antimoniate as the reference drug. As shown in Figure 5, when the imidazole compounds 14 (Figure 5a) and the pyrazole derivatives 5 and 6 (Figure 5b) were added to macrophages infected with L. infantum promastigotes, the infection rate significantly decreased with respect to the control in all the compounds tested, and five of them were also more effective than meglumine antimoniate, with the disubstituted 3 as the only exception (56% compared with 60% for the reference drug). The three monosubstituted derivatives 2, 4 and 6 (81%, 78% and 76%, respectively, in terms of infectivity decrease) were substantially more active than their disubstituted analogues. On the other hand, data on the mean number of amastigotes per infected macrophage cells (Figure 5c and d) led to similar but even more compelling conclusions: all six compounds were much more effective than meglumine antimoniate (only a 22% decrease), and the three monosubstituted compounds were clearly more active than the disubstituted ones (67%, 61% and 47% for 2, 4 and 6 compared with 32%, 46% and 32% for 1, 3 and 5, respectively).

Figure 5.

Effects of the imidazole-based (14) and pyrazole-based (56) benzo[g]phthalazine derivatives on the infection and growth rates of Leishmania spp. (a, b) Rate of infection of L. infantum. (c, d) Mean number of amastigotes per infected J774 A.2 macrophage for L. infantum (at IC25). Values are means of three separate experiments. Glucant., Glucantime (meglumine antimoniate).

Figure 5.

Effects of the imidazole-based (14) and pyrazole-based (56) benzo[g]phthalazine derivatives on the infection and growth rates of Leishmania spp. (a, b) Rate of infection of L. infantum. (c, d) Mean number of amastigotes per infected J774 A.2 macrophage for L. infantum (at IC25). Values are means of three separate experiments. Glucant., Glucantime (meglumine antimoniate).

The same experiment was performed with L. braziliensis, and the results obtained concerning infection rates (Figure 6a and b) and amastigote numbers (Figure 6c and d) are shown in Figure 6. In both cases, all six compounds were more effective than meglumine antimoniate, and also in both cases the three monosubstituted compounds were found to be more active than the disubstituted ones. The infectivity rates calculated from Figure 6 were 2 (82%) > 4 (78%) > 6 (70%) ≫ 1 (57%) > 3 (51%) > 5 (47.13%) > meglumine antimoniate (40%), and the decreases in amastigote number were 2 (74%) > 6 (65%) > 4 (64%) > 3 (60%) > 1 (57%) > 5 (51%)>meglumine antimoniate (47%).

Figure 6.

Effects of the imidazole-based (14) and pyrazole-based (56) benzo[g]phthalazine derivatives on the infection and growth rates of Leishmania spp. (a, b) Rate of infection of L. braziliensis. (c, d) Mean number of amastigotes per infected J774 A.2 macrophage for L. braziliensis (at IC25). Values are means of three separate experiments. Glucant., Glucantime (meglumine antimoniate).

Figure 6.

Effects of the imidazole-based (14) and pyrazole-based (56) benzo[g]phthalazine derivatives on the infection and growth rates of Leishmania spp. (a, b) Rate of infection of L. braziliensis. (c, d) Mean number of amastigotes per infected J774 A.2 macrophage for L. braziliensis (at IC25). Values are means of three separate experiments. Glucant., Glucantime (meglumine antimoniate).

If we now compare the results obtained for both Leishmania species, it can be concluded that, as happened with T. cruzi, the monosubstituted derivatives were clearly the most active, and they were also much less toxic against the host than meglumine antimoniate. The imidazole-based compounds 2 and 4 seemed to be more effective than the pyrazole-based 6, with 2 as the most active and also the least toxic of all.

Ultrastructural alterations

The remarkable leishmanicidal activity shown by compounds 16 should induce important damage to parasite cells. Therefore, we performed a TEM study on promastigote forms of L. infantum and L. braziliensis cultured in medium containing the compounds tested at their IC25 concentrations. As expected, significant morphological alterations were observed compared with untreated control cells. Figure 7 shows the structural features obtained from control and treated cells of L. infantum. Treatment with meglumine antimoniate resulted in morphological alterations in the promastigotes, which exhibited reduced size and shape abnormalities, presenting in many cases small, strongly electron-dense vesicles. In turn, all six compounds tested led to major morphological changes. The most frequent structural modifications consisted of intense vacuolization (Figure 7, see 1, 2, 3, 4 and 5), very significant size reduction, distortions in the appearance of the parasite, which in many cases adopted a star-shaped form (see 2, 5 and 6; marked with an arrow), and breaking of the cytoplasmic membrane (6). The cytoplasm was poorly electron dense, and usually had a granular appearance (see 1), whereas kinetoplasts and nucleus showed the same flabby aspect as the cytoplasm itself. As a confirmation of the infection rate data commented on above, the monosubstituted derivatives 2, 4 and 6 exhibited the highest numbers of dead and shape-distorted parasites. Thus, although some of the parasites treated with 2 exhibited a normal morphology, many promastigotes were found to be dead and many others were vacuolated, poorly electron dense or star-shaped, and a high number of glycosomes was observed. Many dead and vacuolated promastigotes were also found after treatment with 4. However, the most harmful compound seemed to be the pyrazole-based monosubstituted derivative 6, since it caused the death of most of the parasites, and the remaining parasites appeared to be distorted and of a smaller size than normal.

Figure 7.

Ultrastructural alterations observed by TEM in L. infantum treated with meglumine antimoniate and compounds 16. Control, control parasite (bar 1 μm); Gluc, modifications after treatment with meglumine antimoniate (Glucantime) (bar 2.33 μm). 1, 2, 3 (bar 1 μm), 4 (bar 2.33 μm), 5 (bar 1 μm) and 6 (bar 1.59 μm), promastigotes treated with the respective compounds. N, nucleus; M, mitochondrion; V, vacuole; F, flagellum; G, glycosome; K, kinetoplast; D, dead parasites; Ve, electron-dense vesicles. Single arrows indicate distorted promastigotes.

Figure 7.

Ultrastructural alterations observed by TEM in L. infantum treated with meglumine antimoniate and compounds 16. Control, control parasite (bar 1 μm); Gluc, modifications after treatment with meglumine antimoniate (Glucantime) (bar 2.33 μm). 1, 2, 3 (bar 1 μm), 4 (bar 2.33 μm), 5 (bar 1 μm) and 6 (bar 1.59 μm), promastigotes treated with the respective compounds. N, nucleus; M, mitochondrion; V, vacuole; F, flagellum; G, glycosome; K, kinetoplast; D, dead parasites; Ve, electron-dense vesicles. Single arrows indicate distorted promastigotes.

In a similar way, Figure 8 compares the alterations produced by meglumine antimoniate and compounds 16 in L. braziliensis promastigotes. The results obtained are very closely related to those commented on above for L. infantum. The presence of meglumine antimoniate led to a high percentage of parasites with many electron-dense vesicles that may well have been parasite excretion vesicles. Treatment with any of the six compounds led to a remarkable number of dead parasites, and many others were swollen or presented an unrecognizable and flabby cytoplasm, full of vacuoles and exhibiting a broken cytoplasmic membrane. As observed for L. infantum, and again in accordance with the measured infection rates, the monosubstituted derivatives 2, 4 and 6 seemed to be more harmful than their disubstituted analogues, with a higher amount of dead and shape-distorted parasites.

Figure 8.

Ultrastructural alterations observed by TEM in L. braziliensis treated with meglumine antimoniate and compounds 16. Control, control parasite (bar 1 μm); Gluc, modifications after treatment with meglumine antimoniate (Glucantime) (bar 1.59 μm). 1 and 2 (bar 2.33 μm), 3, 4 and 5 (bar 1 μm) and 6 (bar 1.59 μm), promastigotes treated with the respective compounds. N, nucleus; M, mitochondrion; V, vacuole; F, flagellum; G, glycosome; K, kinetoplast; D, dead parasites; Ve, electron-dense vesicles. Single arrows indicate distorted promastigotes.

Figure 8.

Ultrastructural alterations observed by TEM in L. braziliensis treated with meglumine antimoniate and compounds 16. Control, control parasite (bar 1 μm); Gluc, modifications after treatment with meglumine antimoniate (Glucantime) (bar 1.59 μm). 1 and 2 (bar 2.33 μm), 3, 4 and 5 (bar 1 μm) and 6 (bar 1.59 μm), promastigotes treated with the respective compounds. N, nucleus; M, mitochondrion; V, vacuole; F, flagellum; G, glycosome; K, kinetoplast; D, dead parasites; Ve, electron-dense vesicles. Single arrows indicate distorted promastigotes.

Metabolite excretion

It is well known that trypanosomatids are unable to degrade glucose completely 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 considered.27,28 The final products of glucose catabolism in Leishmania are usually CO2, succinate, acetate, l-lactate, pyruvate, l-alanine and ethanol.29 Succinate is especially relevant since its main role is in maintaining the glycosomal redox balance, allowing the reoxidation of NADH produced in the glycolytic pathway. Succinic fermentation has the advantage of requiring only half of the phosphoenolpyruvate produced to maintain the NAD+/NADH balance, and the remaining pyruvate is converted inside the mitochondrion and the cytosol into acetate, l-lactate, l-alanine or ethanol according to the degradation pathway followed by each species.30

In order to obtain information concerning the effects of the tested compounds on glucose metabolism in the parasites, we recorded the 1H-NMR spectra of promastigotes from L. infantum and L. braziliensis after treatment with compounds 16, and the final excretion products were identified qualitatively and quantitatively. The results were compared with those found for promastigotes maintained in a cell-free medium (control) for 4 days after inoculation with the parasite. The characteristic presence of acetate, l-lactate and succinate was confirmed in control experiments performed in both species. However, noteworthy differences between them were the extensive presence of pyruvate and also the appearance of ethanol among the catabolites excreted by L. braziliensis, whereas these two products were not detected in L. infantum promastigotes. After treatment of the parasites with compounds 16, the excretion of the catabolites was clearly altered at the concentrations employed. The 1H-NMR obtained in all the tests performed are shown in Figures S1 (L. infantum) and S2 (L. braziliensis) (available as Supplementary data at JAC Online). Table 3 displays the modifications observed in the height of the spectrum peaks corresponding to the most representative final excretion products. From a careful examination of the data it would appear that there were marked differences in the catabolic pathway depending on the structure of the side chains linked to the benzo[g]phthalazine moiety. The pair of compounds 1/2, with imidazole rings linked through the C-4 carbon atom to a flexible aliphatic chain, gave rise to a decrease in the excretion of acetate and succinate, whereas the pair 5/6, containing pyrazole rings bonded directly to the benzo[g]phthalazine system through an exocyclic nitrogen, caused a sharp increase in the production of both catabolites. The variations originated by the pair 3/4 (with imidazole rings linked to the flexible aliphatic chain through the heterocyclic N-1 nitrogen and consequently lacking the NH group present in 1/2) were clearly less significant than those found for the other compounds tested. All these data could be interpreted on the basis of a change in glucose catabolism according to the structure of the respective side chains. The severe damage caused in parasite organelles such as glycosomes or mitochondria by these compounds should be related to the alterations observed in the final products of catabolism. Another point that should be taken into account is the fact that the pairs 1/2 and 5/6 contain five-membered heterocyclic rings with an acidic NH group, which is not present in 3/4. Therefore, differences in basicity could be responsible for the smaller variations found in the final catabolism products of 3/4 with respect to the rest of the compounds, since it is known that modifications in pH may affect metabolism in Leishmania species.31

Table 3.

Variation in the height of the peaks corresponding to catabolites excreted by L. infantum and L. braziliensis promastigote forms in the presence of imidazole-based (14) and pyrazole-based (56) benzo[g]phthalazine derivatives with respect to the control test

Compound L. infantum
 
L. braziliensis
 
Ac Suc Lac Et Ac Suc Lac Et Pyr 
−20% −19% +70% ↑ +9% −58% 
−11% −46% −50% ND −100% −14% −62% 
−11% −12% ND −12% −14% 
+9% +11% ↑ −60% −22% −11% −44% 
+22% +63% −14% ND −42% −52% −64% ND −100% 
+32% +85% +30% ND −64% −8% −27% +100% −100% 
Compound L. infantum
 
L. braziliensis
 
Ac Suc Lac Et Ac Suc Lac Et Pyr 
−20% −19% +70% ↑ +9% −58% 
−11% −46% −50% ND −100% −14% −62% 
−11% −12% ND −12% −14% 
+9% +11% ↑ −60% −22% −11% −44% 
+22% +63% −14% ND −42% −52% −64% ND −100% 
+32% +85% +30% ND −64% −8% −27% +100% −100% 

Ac, acetate; Suc, succinate; Lac, l-lactate; Et, ethanol; Pyr, pyruvate. (–) peak inhibition; (+) peak increasing; (=) no difference detected; (ND) peak undetected; (↑) peak undetected in control.

The behaviour of the L. braziliensis parasites in the presence of 16 was much more uniform than that described above for L. infantum. Variations in the final catabolism products did not seem to be dependent on the structural aspects of the compounds assayed. A decrease in the formation of acetate, succinate, l-lactate and especially pyruvate was observed for most of the test compounds. In fact, the presence of pyruvate was not detected at all in the pair 5/6. Taking into account the high grade of vacuolization and cell death found in L. braziliensis when performing the ultrastructural study commented on above, it is likely that such considerable cell and organelle degeneration could be the cause of the inhibition found in the production of the typical excretion products of this species.

Funding

This work was supported by the Spanish Ministerio de Ciencia e Innovacion (CTQ2009-14288-C04-01 and Consolider Ingenio CSD2010-00065), and also by the Santander-Universidad Complutense Research Programme (GR35/10-A-921371).

Transparency declarations

None to declare.

Supplementary data

Figures S1 and S2 are available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).

Acknowledgements

We thank the RMN and Microanalisis C.A.I.s of the Universidad Complutense and the Departamento de Analisis del Centro Nacional de Quimica Organica Manuel Lora-Tamayo (C.S.I.C) for analyses of the purity of the compounds tested. We are also grateful to the transmission electron microscopy and nuclear magnetic resonance spectroscopy services of the CIC-University of Granada for their contribution to the studies on ultrastructural alterations and catabolism.

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Supplementary data