SEARCH

SEARCH BY CITATION

Keywords:

  • 3-alkylpyridine alkaloids;
  • Leishmania;
  • marine alkaloids

Abstract

  1. Top of page
  2. Abstract
  3. Experimental Section
  4. Results and Discussion
  5. Conclusions and Future Directions
  6. Acknowledgments
  7. References

A series of oxygenated analogues of marine 3-alkylpyridine alkaloids were synthesized, and their leishmanicidal activity was assayed. All compounds were prepared from 3-pyridinepropanol in few steps and in good yields. The key step for the synthesis of these compounds was a classic Williamson etherification under phase-transfer conditions. Besides toxicity in peritoneal macrophages, the compounds exhibited a significant leishmanicidal activity. Of twelve compounds tested, five showed a strong leishmanicidal activity against promastigote forms of Leishmania amazonensis and L. braziliensis with IC50 below 10 μm. Compounds 11, 14, 15, and 16 showed a strong leishmanicidal activity on intracellular amastigotes (IC50 values of 2.78; 0.27; 1.03, and 1.33 μm, respectively), which is unlikely to be owing to the activation of nitric oxide production by macrophages.

Leishmaniasis is one of the world’s most neglected tropical diseases, affecting largely the poorest of the poor, mainly in the developing countries (1). This disease is endemic in 88 countries, with an incidence of approximately 1.5–2 million new cases per year. Leishmaniasis is caused by several protozoa of the genus Leishmania, which display various clinical manifestations that range from the cutaneous to the visceral forms, depending on interactions between the parasite and the host immune response.

In Latin America, cutaneous leishmaniasis, also referred as American cutaneous leishmaniasis (ACL) or American tegumentary leishmaniasis, is caused by multiple and phylogenetically distinct Leishmania species (1–3). The clinical forms include localized, disseminated, diffuse, and mucocutaneous leishmaniasis (4–6). In addition, ACL is present with atypical lesions in patients with AIDS (1). About 90% of the ACL cases are observed in Brazil. In the last 20 years, this disease has increased to all Brazilian states (5). In Brazil, L. braziliensis has been related as the major causative agent of the ACL, and although this clinical form of leishmaniasis is a self-limited disease, some infected patients develop severe secondary manifestations as the mucosal or disseminated leishmaniasis (2,5,7). L. amazonensis has been associated with all clinical forms of leishmaniasis, including severe anergic diffuse cutaneous leishmaniasis, which is a rare clinical form that is very refractory to current chemotherapy (2,5,8).

The first line of drugs for all clinical manifestations of leishmaniasis, including ACL, is based on pentavalent antimonial compounds as sodium stiboglucanate Pentostam) or meglumine antimoniate (Glucantime). These drugs have been used for the treatment of ACL the last 50 years, but cause toxic effects, including myalgia, pancreatitis, cardiac arrhythmias, and hepatitis (9,10). Second-line drugs are amphotericin B and pentamidine, but these drugs are very toxic and cause adverse effects (7,9,10). Liposomal formulation of amphotericin B (Ambisome) is considered as highly effective, with low toxicity, but is more expensive thus limiting the therapeutic use in non-developed countries (9). Miltefosine has been pointed as the first oral treatment for leishmaniasis, and the advantages are safe and effective. The limitations include severe gastrointestinal toxicity and teratogenic action. In addition, miltefosine failed to treat the American species related to ACL (11). As a result, new drugs for the treatment of leishmaniasis still require improvement (12).

Natural products have been used in traditional medicine as therapies of several parasitic diseases, including leishmaniasis. Although the vast majority of natural products are derived from plants, from the 1970s, research involving marine organisms has shown the great potential of these products as a source of bioactive compounds, and several have been isolated or synthesized (13,14). A variety of marine sources, such as tunicates, red algae and sponges have shown to produce bioactive compounds with a wide spectrum of pharmacological actions including anti-inflammatory, antibacterial, anticoagulant, and antiviral properties (13,15). Many marine sponges serve as rich sources of alkaloids with significant antileishmanial potentials. The order Haplosclerida has shown to be a particularly rich source of bioactive alkaloids (16). Araguspongin C (Figure 1, compound 1), for example, that is isolated from a marine sponge Haliclona exigua, displays leishmanicidal activity against promastigotes, as well as amastigotes at 100 μg/mL concentrations (17). Nevertheless, 3-alkyl pyridine alkaloids have also been found in this order. Viscosaline (Figure 1, compound 2), a 3-alkyl pyridinium alkaloid that is isolated from Haliclona viscosoei, presents high antimicrobial activity against several bacteria (18). Theonelladin C (Figure 1, compound 3), an alkaloid structurally related to viscosaline and obtained from Theonella swinhoei, is an important antineoplastic alkaloid (19).

image

Figure 1.  Examples of bioactive marine alkaloids.

Download figure to PowerPoint

The relatively simple structure of 3-alkyl pyridine alkaloids, viscosaline, and theonelladin C, the strong biological activity of these compounds, and our long-standing interest in the chemistry of sponge alkaloids prompted our investigation of the synthesis and biological evaluation of oxygenated analogues. In a recent work, some compounds of this type were assayed against Plasmodium falciparum (20). In this paper, some other analogues were tested against Leishmania species, and the results confirm the potential leishmanicidal activity of oxygenated marine alkaloids analogues.

Experimental Section

  1. Top of page
  2. Abstract
  3. Experimental Section
  4. Results and Discussion
  5. Conclusions and Future Directions
  6. Acknowledgments
  7. References

Chemistry

Reagents and solvents were purchased as reagent grade and used without further purification except of THF, which was distilled over sodium/benzophenone. NMR spectra were recorded on Bruker Avance DRX-200 or DRX-400 spectrometers. Chemical shifts are reported as δ (ppm) downfield from TMS, and the J values are reported in Hz. Column chromatography was performed with silica gel 60, 70–230 mesh (Merck). Compounds 5, 6, 7, 10, 12, 13, and 16 were synthesized as previously reported (20). The new salts 8, 9, 11, 14, and 15 were prepared and characterized as described below.

General procedure for the synthesis of salts 8 and 15

A solution of the pyridine derivative 7 or 13 (0.07 mmol) in methyl iodide (3.0 mL) was stirred at room temperature for 12 h. The excess of methyl iodide was removed under reduced pressure. The residue obtained was purified by column chromatography (SiO2, EtOAc and MeOH) to give salts 8 and 15, respectively, as oily products.

8 Yield 71%; oily product; 1H-NMR (200 MHz, CDCl3): 1.28–1.56 (m, 14H, H-11, H-12, H-13, H-14, H-15, H-16; H-17); 1.89–2.17 (m, 2H, H-8); 2.90–3.10 (m, 2H, H-7); 3.18–3.30 (m, 2H, H-18); 3.39–3.46 (m, 4H, H-9, H-10); 4.66 (s, 3H, CH3); 7.90–8.10 (m, 1H, H-5); 8.25–8.28 (m, 1H, H-4); 9.00–9.24 (m, 2H, H-2, H-6). 13C-NMR (50 MHz, CDCl3): 26.1; 26.6; 28.7; 29.0; 29.3; 29.3; 29.6; 30.0; 30.9; 49.3; 51.4; 68.9; 71.1 127.7; 143.0; 143.7; 144.89; 145.03.

15 Yield 100%; oily product; 1H-NMR (200 MHz, CDCl3): 1.28 (s, 9H, Boc); 1.40–1.60 (m, 12H, H-11, H-12, H-13, H-14, H-15, H-16); 1.93-2.03 (m, 4H, H-8, H-17); 2.53–2.59 (m, 2H, H-20); 2.97–3.14 (m, 2H, H-7); 3.17–3.20 (m, 2H, H-18); 3.39–3.46 (m, 6H, H-9, H-10, H-19); 3.67 (s, 3H, OCH3); 4.67 (s, 3H, CH3); 7.97–8.03 (m, 1H, H-5); 8.27 (d, J = 7.60 Hz, 1H, H-4); 9.00–9.20 (m, 2H, H-2, H-6). 13C-NMR (50 MHz, CDCl3): 26.1; 26.7; 29.3; 29.3; 29.5; 29.7; 28.4; 30.0; 43.2; 47.6; 49.4; 51.6; 68.9; 71.1; 79.5; 127.7; 143.0; 143.8; 144.9; 145.1; 155.3.

General procedure for the synthesis of salts 11 and 14

To a solution of 3-pyridinepropanol 4 or 13 (3.70 mmol) in methanol (4 mL), 1-chloro-2,4-dinitrobenzene (7.40 mmol) was added, and the mixture heated at reflux for 24 h. The mixture was then allowed to cool, and the solvent was removed under reduced pressure. The residue obtained was chromatographed on silica gel using methylene chloride/MeOH (80:20) to produce compounds 11 or 14, respectively.

11 Yield 100%; oily product; 1H-NMR (200 MHz, CDCl3): 2.04 (qn, J8,9 = J8,7 = 7,00 Hz, 2H, H-8); 3.09 (t, J = 7.00 Hz, 2H, H-7); 3.70 (t, J = 7.00 Hz, 2H, H-9); 8.23–8.34 (m, 2H, H-5 e H-6′); 8.85 (d, J = 8.20 Hz, 1H, H-4); 8.95 (dd, J5,6 = 8,70 Hz, J5′,3′ = 2,50 Hz, 1H, H-5′); 9.04 (d, J6,5 = 6,20 Hz, 1H, H-6); 9.11 (s, 1H, H-2); 9.38 (d, J3′,5′ = 2,50 Hz, 1H, H-3′). 13C-NMR (50 MHz, CDCl3): 30.2; 33.3; 61.9; 124.2; 129.5; 132.1; 132.6; 140.2; 144.5; 145.6; 145.9; 150.7; 151.1.

14 Yield 95%; oily product; 1H-NMR (200 MHz, CDCl3): 1.32 (s, 9H, Boc); 1.40–1.55 (m, 14H, H-11, H-12, H-13, H-14, H-15, H-16, H-17); 2.03–2.10 (m, 2H, H-8); 2.56 (t, J20,19 = 6,80 Hz, 2H, H-20); 3,10 (t, J7,8 = 7,50 Hz, 2H, H-7); 3.20 (t, J18,17 = 7,10 Hz, 2H, H-18); 3.41–3.55 (m, 6H, H-9, H-10, H-19); 3.67 (s, 3H, OCH3); 8.29–8.37 (m, 2H, H-5 e H-6′); 8.84–8.95 (m, 2H, H-4 e H-5′); 9.20 (d, J6,5 = 5,20 Hz, 1H, H-6);); 9.24–9.32 (m, 2H, H-2 e H-3′). 13C-NMR (200 MHz, CDCl3): 27.3; 27.8; 30.4; 30.5; 30.6; 30.7; 30.8; 31.2; 28.8; 34.0; 34.0; 44.5; 48.7; 52.3; 70.2; 72.1; 81.1; 123.2; 129.0; 131.2; 132.7; 140.2; 144.6; 144.7; 145.5; 146.5; 150.2; 151.1; 157.1; 174.0.

Synthesis of salt 9

A solution of 7 (0.0533 g, 0.17 mmol) in benzyl chloride (0.03 mL, 0.26 mmol) was heated at 90 °C for 2 h. The mixture was allowed to cool, and the excess amount of benzyl chloride was removed under reduced pressure to give the desired product 9 as yellow oil. Yield 93%; oily product; 1H-NMR (200 MHz, CDCl3): 1.28–1.30 (m, 12H, H-12, H-13, H-14, H-15, H-16; H-17); 1.49–1.62 (m, 2H, H-11); 1.95–1.98 (m, 2H, H-8); 2.93 (t, J7,8 =7,10 Hz, 2H, H-7); 3.22–3.41 (m, 6H, H-9, H-10, H-18); 6.27 (s, 2H, CH2Ph); 7.30–7.69 (m, 5H, Ph); 7.94–7.96 (m, 1H, H-5); 8.17 (d, J4,5 = 7,40 Hz, 1H, H-4); 9.42 (s, 1H, H-2); 9.54–9.56 (m, 1H, H-6). 13C-NMR (50 MHz, CDCl3): 26.0; 26.6; 28.7; 29,0; 29.2; 29.3; 29.5; 29.6; 30.0; 51.4; 64.0; 68.8; 71.0; 127.7; 129.4; 129.5; 129.7; 133.3; 142.8; 143.7; 144.3; 144.8.

Pharmacology

Antileishmanial assay

Parasites and culture cell.  Promastigotes of L. amazonensis (IFLA/Br/67/PH8) were cultured in Warren’s medium (brain heart infusion – BHI– plus hemin and folic acid), and promastigotes of L. braziliensis (MHOM/Br/75/M2903) were cultured in BHI medium (brain heart infusion – plus l-glutamin), both supplemented with 10% fetal bovine serum at 24 °C. Fetal bovine serum was purchased from Cultilab (Campinas, São Paulo, Brazil); brain heart infusion (BHI) from Himédia (Mumbai, Indian); and hemin and folic acid were purchased from Sigma Chemical Co (St. Louis, MO, USA).

Promastigotes assay.  The antileishmanial activity was determined by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) method based on tetrazolium salt reduction by mitochondrial dehydrogenases (21,22). Briefly, promastigotes from a logarithmic phase culture were suspended to yield 2 million cells/mL (L. amazonensis) or 3 million cells/mL (L. braziliensis) after Neubauer chamber counting. The test was performed in 96-well microtiter plates maintained at 24 °C. The analysis was made in duplicate. Parasites were exposed to increasing concentration of the compound (at minimum six serial dilutions) for 72 h at 24 °C. Controls containing 0.5% DMSO and medium alone were also included. Absorbance was measured at 570 nm (Multiskan MS microplate reader; LabSystems Oy, Helsink, Finland). The results are expressed as the concentration-inhibiting parasite growth by 50% (IC50) after a 3-day incubation period. IC50 values were obtained by using GraFit version 5 software (Erithacus Software Ltd., Horley, UK) and expressed in μm ± S.D. Amphotericin B deoxycholate (supplied by Cristália, São Paulo, Brazil) was used as the reference standard.

Amastigotes assay.  Concerning the amastigotes in vitro model, inflammatory macrophages were obtained from BALB/c mice previously inoculated intraperitoneally with 2 mL of 3% thioglycollate medium (Sigma) (22,23). Briefly, peritoneal macrophages were plated at 2 × 106 cells/mL on coverslips (13 mm diameter) previously arranged in a 24-well plate in RPMI 1640 medium supplemented with 10% inactivated FBS and allowed to adhere for 24 h at 37 °C in 5% CO2. Adherent macrophages were infected with L. amazonensis (IFLA/Br/67/PH8) promastigotes in the stationary growth phase using a ratio of 1:5 at 33 °C for 3 h. Non-internalized promastigotes were eliminated, and solutions of tested compounds were added in different concentrations and maintained at 33 °C in 5% CO2 for 72 h. Slides were fixed and stained with Giemsa for parasite counting (optical microscopy, 1000× magnification). The parasite burden was evaluated by counting the intracellular parasite and uninfected and infected macrophages (at minimum 100 cells infected) in treated and untreated cultures. The results were expressed as the number of amastigotes for 200 macrophages.

Cytotoxicity against mammalian cells

Mouse peritoneal macrophages were used for cytotoxicity assay. The cells were incubated with the compounds in a serial dilution, in duplicate at each concentration. The viability of the macrophages was determined with the MTT assay and was confirmed by comparing the control group morphology via light microscopy (21–23). Dose–response curves were plotted (values expressed as percentage of control optical density), and CC50 values (50% cytotoxicity concentration) were obtained by using GraFit version 5 software (Erithacus Software Ltd., Horley, UK).

Nitric oxide (NO) production

NO production was determined in an aliquot (50 μL) of the supernatants of L. amazonensis macrophages after 48 h in the presence of the compounds. The assay was performed as described by Green et al., 1982 (24). Briefly, 50 μL of Griess reagent: 1% sulfanilamide in 2.5% of H3PO4 and 0.1% N-1-diidrocloreto de naftiletilenodiamina in 2.5% H3PO4 (v/v) was added to 50 μL of each sample in a 96-well microplate. Blank reference and standard curve were determined. The absorbance was measured at 540 nm using a microplate reader (Multiskan MS microplate reader, LabSystems Oy, Helsink, Finland). Nitrite content was quantified by extrapolation from sodium nitrite standard curve in each experiment. All the assays were carried out in duplicate. IFN-γ at 0.9 ng/mL and 1 μg/mL LPS (Escherichia coli J5 – from Sigma-Aldrich) were used as positive control.

Statistical analysis

For promastigote forms of Leishmania and cytotoxicity against macrophages, the IC50 and CC50 values were measured at 5% significance level (p < 0.05, CI 95%), and calculated using a nonlinear regression curve, by using GraFit Version 5 software (Erithacus Software Ltd., Horley, UK). For amastigote forms of L. amazonensis, the IC50 values were measured at 5% significance level (p < 0.05, CI 95%), calculated by Litchtfiet and Wilcoxon method using the Probit, and the graphs were plotted by the program GraphPad Prism 4 (GraphPad Software, San Diego, CA, USA). One-way anova was applied to compare all the groups. Dunnett post hoc test was applied to compare all the groups with the control group. Differences were regarded as significant when p < 0.0001 (***) and p < 0.001 (**).

Results and Discussion

  1. Top of page
  2. Abstract
  3. Experimental Section
  4. Results and Discussion
  5. Conclusions and Future Directions
  6. Acknowledgments
  7. References

Chemistry

Compounds 5, 6, 7, 10, 12, 13, and 16 were synthesized as previously reported (20). The synthesis of the new 3-alkylpyridine compounds 8, 9, 11, 14, and 15 is depicted in Scheme 1.

image

Figure  Scheme 1: .  Synthesis of the tested oxygenated marine analogues.

Download figure to PowerPoint

The Zincke′s salts (25) 11 and 14 were easily prepared from 3-pyridine propanol and 13, respectively, by treatment with 1 equivalent of 1-chloro-2,4-dinitrobenzene under methanol reflux. The pyridinium salts 8 and 15 were prepared by alkylation with methyl iodide under standard conditions. Similarly, benzyl salt 9 was prepared by 7 with excess benzyl chloride.

Pharmacology

Table 1 shows the leishmanicidal effect of alkaloids in promastigote forms of L. amazonensis and L. braziliensis. Of twelve compounds tested, five showed significant leishmanicidal activity with IC50 below 10 μm. Compounds 8 and 9 were the most active in both Leishmania species with IC50 ranging from 0.46 to 1.92 μm. Both compounds have a terminal azide group and the same alkyl chain length. In comparison with the azide compound 7, the quaternization of the pyridine nitrogen seems to increase significantly the leishmanicidal activity of these compounds. Apparently, neither the chemical features of the agent used to alkylate the pyridine ring nor the type of counter-ion influence the activity in both Leishmania species. The salts 14, 15, and 16 containing a lateral chain with terminal ester group, similarly to natural viscosaline alkaloid, were also very active. In general, all pyridinium salts were more active than the corresponding pyridine precursors (7, 12, and 13). Another evidence that suggests that the azide group is important for the activity comes from the fact that compound 7 was among the most active pyridine compound derivatives.

Table 1.   Effect of 3-alkylpyridine marine alkaloid analogues in promastigotes of Leishmania species, macrophages and selectivity index
CompoundsIC50m)aCC50m)bSIc
L. a. (A) L. b. (B)Macrophages (C)C/AC/B
  1. IC50 values are the average of three independent experiments ± standard errors of the mean.

  2. aValues of inhibitory concentration of 50% of parasites (IC50): promastigotes of L. amazonensis (L. a.) and L. braziliensis (L. b).

  3. bCC50 values (50% cytotoxicity concentration) on macrophages.

  4. cSI, selectivity index (CC50 of macrophages/IC50 of promastigote forms of L. amazonensis-A or L. braziliensis-B).

  5. dAmb (amphotericin B) was used as reference drug.

5>87.00>87.00>100.00
6>87.00>87.00>100.00
714.95 ± 0.3127.61 ± 0.481.11 ± 0.240.070.04
81.07 ± 0.021.92 ± 0.250.37 ± 0.050.300.20
91.09 ± 0.060.46 ± 0.03<0.098<0.10<0.20
10 >87.00>87.0035.48 ± 0.47
11 23.92 ± 0.8034.17 ± 1.65>100.00>4.20>3.00
12 >87.00>87.009.00 ± 0.13
13 >87.00>87.00>100.00
14 4.53 ± 0.205.88 ± 0.867.93 ± 0.241.701.30
15 11.41 ± 0.182.61 ± 0.1910.53 ± 0.050.904.00
16 2.87 ± 0.174.64 ± 0.2415.75 ± 1.915.503.40
Amb d 0.15 ± 0.0090.4 ± 0.06>1.00>6.70>2.50

Variation in species sensitivity has been demonstrated in vitro with several Leishmania species, including pentavalent antimonials and amphotericin B, which are considered as reference drugs for leishmaniasis (26,27). This fact is relevant because it can reflect implications on the clinical outcome. Interestingly, the marine alkaloid 3-alkylpyridinium analogues showed similar activity in both Leishmania species.

With respect to cytotoxicity effects on macrophages, the compounds showed a significant toxicity against murine peritoneal macrophages, and only three compounds showed selectivity index above one (Table 1). The compounds with better antipromastigote activity (8 and 9) exhibited a strong toxicity against macrophages. It is interesting to point that these compounds exhibited lower in vitro toxicity against the human lungs fibroblast with IC50 above 50 μm (20). Data from our laboratory has indicated that peritoneal macrophages are more sensible in vitro assays to several compounds tested, including mercaptopurine derivatives and aminoquinoline derivatives, than cultured macrophages as J744A1 cells lines.

Vermeersch et al. (28) recently recommended that the intracellular amastigote model could be employed as the ‘gold standard’ for antileishmanial in vitro evaluation. Amastigote forms are the stage of parasite found in the mammalian host and responsible for the human disease. So, we used L. amazonensis amastigotes as a model to demonstrate the leishmanicidal activity of compounds in the intracellular parasite stage because this model is well established in our laboratory. In this assay, the macrophages were efficiently infected (controls = 60% of infected macrophages, with five parasites/infected cell). Compounds with significant L. amazonensis antipromastigotes activity (IC50 < 25 μm) and selectivity index above one or near one were used for this assay (Table 1). Regarding this, only compounds 11, 14, 15, and 16 were tested in amastigotes model, and the results are shown in Table 2 and Figure 2. In a recent paper, the authors showed that glucantime, which is one of the first-choice drugs for the treatment of leishmaniasis, exhibited a selectivity index of <1.0 (ratio macrophages and promastigotes of L. braziliensis and L. infantum) (29).

Table 2.   Effect of the compounds on intracellular amastigotes of L. amazonensis and selectivity index
CompoundsIC50m)a (95% CI)SIb
  1. CI, confidence interval. Peritoneal macrophages previously infected with L. amazonensis promastigotes in the stationary growth phase were exposed to the compounds for 72 h.

  2. aResults from two assays in duplicate are shown as IC50 values in μm.

  3. bSI, selectivity index (CC50 of macrophages/IC50 of amastigotes of L. amazonensis).

  4. cAmB (amphotericin B) was used as reference drug.

11 2.78 (1.31–5.91)>36
14 0.27 (0.13–0.58)29
15 1.03 (0.45–2.38)10
16 1.33 (1.03–1.72)12
AmB c 0.18 (0.12–0.26)>5.55
image

Figure 2.  Effect of 3-alkylpyridine marine alkaloid analogues on L. amazonensis interiorized in peritoneal macrophage cells. Peritoneal macrophages previously infected with L. amazonensis promastigotes in the stationary growth phase were exposed to the compounds for 72 h. Results from two assays in duplicate and were expressed as the number of amastigotes for 200 macrophages. All results were significant (***p < 0.0001).

Download figure to PowerPoint

Figure 2 shows the effect of these compounds after L. amazonensis–macrophage interaction. All compounds showed a significant effect against the amastigote forms; however, this activity showed a weak dose dependency. The IC50 values for all compounds were below 3 μm, and compound 14 showed the best IC50 values (0.27 μm) as demonstrated in Table 2. It is interesting to note that the compounds were at minimum ten times less toxic to host cells, and compound 14 that showed the best activity against amastigotes of L. amazonensis was 29-fold more selective against the parasite. Furthermore, the IC50 values against amastigote forms were better than the IC50 values for promastigotes. In a recent paper, Muylder et al. (30) established a cut-off regarding the specificity of compounds between these two stages of the parasite Leishmania. Specificity index, given by the ratio between promastigote IC50 and amastigote IC50, established that values above two define compounds as more active against the intracellular amastigote stage; compounds with values below 0.4 indicate compounds more active against promastigotes; while compounds with values between 0.4 and 2 are considered active against both stages (30). Regarding this, the specificity index of the compounds tested in this work showed that all the values are found above 2 (8.60, 16.80, 11.08, and 2.16 for the compounds 11, 14, 15, and 16, respectively), reinforcing the specificity of the compounds in intracellular amastigotes. As a matter of fact, the compound 14, the more active in amastigote forms proved to be more specific also, being 16.80 times more destructive to the intracellular stage of Leishmania.

These results are very interesting because although promastigote forms of the genus Leishmania can be used for screenings of compounds, amastigote forms are responsible for all clinical manifestations in humans. Despite the more complex and labor-intensive protocol, the intracellular amastigote model has been cited as the golden standard for in vitro Leishmania drug discovery research (9,28). The results showed in this work reinforce the potential leishmanicidal activity of the oxygenated analogues of marine 3-alkylpyridine alkaloids.

Macrophages are considered major host cells for Leishmania, and the immunological response against this parasite include phagocytosis and generate reactive nitrogen products as nitric oxide (NO) (31). Because of the good activity shown by the compounds against amastigote forms of L. amazonensis, it was interesting to investigate whether this activity could involve some modulator mechanism of cellular response. However, in general, none of the 3-alkylpyridine marine alkaloid analogues induced significant nitrite production in the culture medium in all concentrations used compared with untreated controls (data not shown). So, the leishmanicidal effect observed on amastigote forms is unlikely to be attributed activation of NO production by macrophages. Immunomodulatory properties of marine compounds have been described along with their inhibitory activity on NO production (32). Alternately, there can be another modulator mechanism of cellular response involved or the compounds can act directly on the parasites.

Conclusions and Future Directions

  1. Top of page
  2. Abstract
  3. Experimental Section
  4. Results and Discussion
  5. Conclusions and Future Directions
  6. Acknowledgments
  7. References

In summary, we herein show that some very simple 3-alkylpyridine marine alkaloid analogues exhibit a marked leishmanicidal activity in both promastigote and amastigote forms of Leishmania. Among the compounds synthesized, those containing a terminal azide moiety at the side chain and/or a quaternarized nitrogen exhibited higher potency. Therefore, such compounds are promising for developing a new class of leishmanicidal agents and require further investigation.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Experimental Section
  4. Results and Discussion
  5. Conclusions and Future Directions
  6. Acknowledgments
  7. References

We are grateful to Dr. Ana Paula Ferreira (UFJF) for a sample of IFN-γ and to FAPEMIG, CAPES, CNPq and BIC/UFJF for the financial support.

References

  1. Top of page
  2. Abstract
  3. Experimental Section
  4. Results and Discussion
  5. Conclusions and Future Directions
  6. Acknowledgments
  7. References
  • 1
    World Health Organization (WHO). Leishmaniasis. Available at: http://www.who.int/leishmaniasis/en/ (accessed September 20, 2011).
  • 2
    Silveira F.T., Lainson R., De Castro Gomes C.M., Laurenti M.D., Corbett C.E. (2009) Immunopathogenic competences of Leishmania (V.) braziliensis and L. (L.) amazonensis in American cutaneous leishmaniasis. Parasite Immunol;31:423431.
  • 3
    Amato V.S., Tuon F.F., Siqueira A.M., Nicodemo A.C., Neto V.A. (2007) Treatment of mucosal leishmaniasis in Latin America: systematic review. Am J Trop Med Hyg;77:266274.
  • 4
    Larson E.E., Marsden P.D. (1987) The origin of espundia. Trans R Soc Trop Med Hyg;81:880885.
  • 5
    Brasil (2010) Manual de Vigilância da Leishmaniose Tegumentar Americana, 3rd edn. Brasília (DF): Ministério da Saúde.
  • 6
    Silveira F.T., Lainson R., Corbett C.E. (2004) Clinical and immunopathological spectrum of American cutaneous leishmaniasis with special reference to the disease in Amazonian Brazil: a review. Mem Inst Oswaldo Cruz;99:239251.
  • 7
    Oliveira L.F., Schubach A.O., Martins M.M., Passos S.L., Oliveira R.V., Marzochi M.C., Andrade C.A. (2011) Systematic review of the adverse effects of cutaneous leishmaniasis treatment in the New World. Acta Trop;118:8796.
  • 8
    Barral A., Pedral-Sampaio D., Grimaldi Junior G., Momen H., McMahon-Pratt D., Jesus A.R., Badaró R., Barral-Netto M., Carvalho E.M., Jhonson W.D. (1991) Leishmaniasis in Bahia, Brazil: evidence that Leishmania amazonensis produces a wide spectrum of clinical disease. Am J Trop Med Hyg;44:536546.
  • 9
    Tempone A.G., Martins de Oliveira C., Berlinck R.G. (2011) Current approaches to discover marine antileishmanial natural products. Planta Med;77:572585.
  • 10
    Richard J.V., Werbovetz K.A. (2010) New antileishmanial candidates and lead compounds. Curr Opin Chem Biol;14:19.
  • 11
    Soto J., Arana B.A., Toledo J., Rizzo N., Vega J.C., Diaz A., Luz M., Gutierrez P., Arboleda M., Berman J.D., Junge K., Engel J., Sindermann H. (2004) Miltefosine for new world cutaneous leishmaniasis. Clin Infect Dis;38:12661272.
  • 12
    Avlonitis N., Lekka E., Detsi A., Koufaki M., Calogeropoulou T., Scoulica E., Siapi E., Kyrikou I., Mavromoustakos T., Tsotinis A., Golic Grdadolink S., Makriyannis A. (2003) Antileishmanial ring-substituted ether phospholipids. J Med Chem;46:755767.
  • 13
    Cutignano A., Tramice A., De Caro S., Villani G., Cimino G., Fontana A. (2003) Biogenesis of 3-alkylpyridine alkaloids in the marine mollusc Haminoea Orbignyana. Angew Chem Int Ed;42: 26332636.
  • 14
    Mayer A.M.S., Rodríguez A.D., Berlinck R.G.S., Hamann M.T. (2009) Marine pharmacology in 2005–6: marine compounds with anthelmintic, antibacterial, anticoagulant, antifungal, anti-inflammatory, antimalarial, antiprotozoal, antituberculosis, and antiviral activities; affecting the cardiovascular, immune and nervous systems, and other miscellaneous mechanisms of action. Biochim Biophys Acta;1790:283308.
  • 15
    Gupta L., Talwar A., Nishi Palne S., Gupta S., Chauhan P.M. (2007) Synthesis of marine alkaloid: 8,9-dihydrocoscinamide B and its analogues as Novel class of antileishmanial agents. Bioorg Med Chem Lett;17:40754079.
  • 16
    Almeida A.M.P., Berlinck R.G.S., Hajdu E. (1997) Alcalóides alquilpiridínicos de esponjas marinhas. Quim Nova;20:170185.
  • 17
    Dube A., Singh N., Saxena A., Lakshmi V. (2007) Antileishmanial potential of a marine sponge, Haliclona exigua against experimental visceral leishmaniasis. Parasitol Res;101:317324.
  • 18
    Volk C.A., Kock M. (2004) Viscosaline: new 3-alkyl pyridinium alkaloid from the Arctic sponge Haliclona viscosa. Org Biomol Chem;2:18271830.
  • 19
    Kobayashi J., Murayama T., Ohizumi Y. (1989) Theonelladins A∼D, novel antineoplasic pyridine alkaloids from the okinawan marine sponge Theonella-swinhoei. Tetrahedron Lett;30:833.
  • 20
    Hilário F.F., de Paula R.C., Silveira M.L.T., Viana G.H.R., Alves R.B., Pereira J.R.C.S., Silva L.M., de Freitas R.P., de Pilla Varotti F. (2011) Synthesis and evaluation of antimalarial activity of oxygenated 3-alkylpyridine marine alkaloid analogues. Chem Biol Drug Des;78:477482.
  • 21
    Mossman T. (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Meth;65:5558.
  • 22
    Carmo A.M.L., Silva F.M.C., Machado P.A., Fontes A.P., Pavan F.R., Leite C.Q., Leite S.R., Coimbra E.S., Da Silva A.D. (2011) Synthesis of 4-aminoquinoline analogues and their platinum(II) complexes as new antileishmanial and antitubercular agents. Biomed Pharmacother;65:204209.
  • 23
    de Carvalho G.S., Machado P.A., de Paula D.T., Coimbra E.S., da Silva A.D. (2010) Synthesis, cytotoxicity, and antileishmanial activity of N,N’-disubstituted ethylenediamine and imidazolidine derivatives. ScientificWorldJournal;10:17231730.
  • 24
    Green L.C., Wagner D.A., Glogwski J., Skipper P.L., Wishnok J.S., Tannenbaum S.R. (1982) Analysis of nitrate, nitrite, and nitrate in biological fluids. Anal Bioch;126:131138.
  • 25
    Viana G.H., Santos I.C., Alves R.B., Gil L., Marazano C., Freitas-Gil R.P. (2005) Microwave-promoted synthesis of chiral pyridinium salts. Tetrahedron Lett;46:77737776.
  • 26
    Escobar P., Sangeeta M., Marques C., Croft S.L. (2002) Sensitivities of Leishmania species to hexadecylphosphocholine (miltefosine), ET-18-OCH3 (edelfosine) and amphotericin B. Acta Trop;81:151157.
  • 27
    Coimbra E.S., Carvalhaes R., Grazul R.M., Machado P.A., De Souza M.V., Da Silva A.D. (2010) Synthesis, cytotoxicity and antileishmanial activity of some N-(2-(indol-3-yl)ethyl)-7-chloroquinolin-4-amines. Chem Biol Drug Des;75:628631.
  • 28
    Vermeersch M., da Luz R.I., Tote K., Timmermans J.P., Cos P., Maes L. (2009) In vitro susceptibilities of Leishmania donovani promastigote and amastigote stages to antileishmanial reference drugs: practical relevance of stage-specific differences. Antimicrob Agents Chemother;53:38553859.
  • 29
    Sánchez-Moreno M., Gómez-Contreras F., Navarro P., Marín C., Ramírez-Macías I., Olmo F., Sanz A.M., Campayo L., Cano C., Yunta M.J.R. (2011) In vitro leishmanicidal activity of imidazole- or pyrazole-based benzo[g]phthalazine derivatives against Leishmania infantum and Leishmania braziliensis species. J Antimicrob Chemother;67:387397.
  • 30
    Muylder G., Ang K.K.H., Chen S., Arkin M.R., Engel J.C., Mckerrow J.H. (2011) A Screen against Leishmania intracellular amastigotes: comparison to a promastigote screen and identification of a host cell-specific hit. PLoS Negl Trop Dis;5:e1253.
  • 31
    Evans T.G., Thai L., Granger D.L., Hibbs J.B. Jr (1993) Effect of in vivo inhibition of nitric oxide production in murine leishmaniasis. J Immunol;151:907915.
  • 32
    Mayer A.M., Rodríguez A.D., Berlinck R.G., Fusetani N. (2011) Marine pharmacology in 2007–8: marine compounds with antibacterial, anticoagulant, antifungal, anti-inflammatory, antimalarial, antiprotozoal, antituberculosis, and antiviral activities; affecting the immune and nervous system, and other miscellaneous mechanisms of action. Comp Biochem Physiol C Toxicol Pharmacol;153:191222.