Introduction

Leishmaniases are diseases caused by protozoan parasites that affect millions of people in more than 88 countries worldwide. These parasites are transmitted by female sand flies belonging to the genus Phlebotomus and Lutzomyia in the Old and New World, respectively. Leishmaniasis causes three main forms of clinical disease: (1) visceral leishmaniasis, the most severe form, is usually fatal if not treated and affects internal organs such as the liver, spleen and bone marrow; (2) mucocutaneous leishmaniasis, a chronic form, causes extensive destruction and disfiguration of the nasopharynx region; and (3) cutaneous leishmaniasis, the mildest form, is usually self-healing within a few months to years, causing scarring at the site of the lesion(s). First-line drugs include pentavalent antimony (Sbv) compounds, pentamidine or amphotericin B. All these drugs are administrated by injection and require clinical supervision or hospitalization because of the possibility of severe side effects. However, parasite resistance to Sbv drugs has resulted in the discontinued use of these compounds in some endemic regions for visceral leishmaniasis.1 Liposomal amphotericin B shows reduced toxicity, but is prohibitively expensive for use in less-developed countries. Recently, miltefosine, an alkylphospholipid derivative and the first orally administered drug, has been approved for use in India. However, the teratogenic effects of this drug prevent its use in pregnant women,2, 3 and parasite resistance is easily generated in the laboratory.4 As such, there is an urgent need for the development and testing of new compounds for the treatment of all clinical forms of leishmaniasis.

Betulin 1 (lup-20(29)-ene-3β,28-diol) is an abundant naturally occurring triterpene found in the plant kingdom (Figure 1). It is the principal extractive (up to 30% of dry weight) of the bark of white-barked birch trees (Betula sp.).5 This pentacyclic triterpene can be converted into betulinic acid 2,6 which has shown anti-inflammatory,7 antimalarial8 and especially cytotoxic activity against several tumor cell lines by inducing apoptosis in cells.9, 10 Some betulin derivatives have also shown remarkable anti-human immunodeficiency virus activity with new mechanisms of action.11, 12 Structure–activity relationship studies and pharmacological properties of betulin and its derivatives have been reviewed recently.13

Figure 1
figure 1

Chemical structures of betulin 1, betulinic acid 2, dihydrobetulinic acid 3, betulin aldehyde 4 and betulin heterocycloadduct between 3,28-di-O-acetyllupa-12,18-diene and 4-methylurazine 5.

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Previously, dihydrobetulinic acid 3 was examined as a new lead compound for anti-leishmanial therapy.14 It was shown that it targeted DNA topoisomerases I and II by preventing DNA cleavage and formation of an enzyme–DNA complex, which ultimately induced apoptosis in Leishmania donovani promastigotes and amastigotes in infected macrophages with an IC50 value of 2.6 and 4.1 μM, respectively. Parasitic burden in golden hamsters was reduced by 92% after a 6-week treatment with dihydrobetulinic acid 3 (10 mg kg−1 body weight). In another study, in which leishmanicidal inhibition activity of a plethora of natural products was screened, betulinic acid 2 isolated in small quantities from Betula platyphylla var. japonica was found to be weakly active against Leishmania major promastigotes, the extracellular form of the parasite, with an IC50 value of 88 μM.15 It was also noted that in triterpenes with ursane, oleanane or lupane skeletons, a carboxyl substituent was required for anti-leishmanial activity. In a related study, it was shown that a rare natural product, betulin aldehyde 4, obtained from Doliocarpus dentatus (Aubl.) showed in vitro activity against Leishmania amazonensis amastigotes in infected macrophages, reducing infection by 88% at 136 μM and by 58% at 68 μM.16 At these doses, 4 also showed some toxicity against peritoneal macrophages, with survival indices of 70 and 80%, respectively. Previously, we studied anti-leishmanial activity of heterocyclic betulin derivatives, in which the heterocycloadduct between 3,28-di-O-acetyllupa-12,18-diene and 4-methylurazine 5 was the most effective derivative with a GI50 value of 8.9 μM against L. donovani amastigotes.17 These results prompted us to investigate more closely the anti-leishmanial activity of 24 betulin derivatives that have been chemically modified in positions C-3, C-28 and C-20–C-29 of the lupane skeleton.

Results and Discussion

We found that betulin 1 (isolated from Betula sp.) has moderate anti-leishmanial activity against L. donovani axenic amastigotes, showing 35% inhibition at 50 μM in a microplate assay (Table 1). Acetylation, esterification or etherification of the hydroxy groups at C-3 or C-28 in most cases retained anti-leishmanial activity. We observed that 28-O-Cinnamoylbetulin 6 was totally inactive and 28-O-nicotinoylbetulin 7, 28-O-tetrahydropyranylbetulin 8, 28-O-chrysanthemoylbetulin 9 and betulinyl-28-O-carboxymethoxycarvacrolate 10 were only slightly active. Only 28-O-(N-acetylanthraniloyl)betulin 11 and 28-O-bromoacetylbetulin 12 showed improved anti-leishmanicidal activity (59 and 86% inhibition at 50 μM, respectively), compared with 1. In addition, 3-O-acetylbetulin 13 had similar anti-leishmanial inhibition activity compared with the starting material betulin 1, whereas 3,28-di-O-acetylbetulin 14 and 3,28-di-O-levulinoylbetulin 15 were totally inactive.

Table 1 Anti-leishmanial activities at 50 μM on microplate assay and GI50 values for the most potent synthetic betulin derivatives against Leishmania donovan i axenic amastigotes
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Oxidation of 1 seems to have a beneficial effect on anti-leishmanial activity. Betulin aldehyde 4 displayed improved anti-leishmanial activity with a 64% inhibition at 50 μM. Betulinic acid 2 possessed moderate anti-leishmanial activity with a 40% inhibition at 50 μM. 28-O-Acetyl-3-oxobetulin 16 and betulonic aldehyde 17 showed moderate anti-leishmanial activity similar to the starting material 1, but betulonic acid 18 had remarkable anti-leishmanial activity with a 98% inhibition at 50 μM. Reduction of the carbon–carbon double bond of betulonic acid 18 to the corresponding dihydrobetulonic acid 19 decreased anti-leishmanial activity to 72% at 50 μM. Furthermore, methylation of betulonic acid 18 to methyl betulonate 20 decreased the inhibition activity at 50 μM to 40%. L-aspartyl amide of betulonic acid 21 showed reduced leishmanicidal activity compared with betulonic acid 18, with a 69% inhibition at 50 μM. Vanillyl betulonate 22 was totally inactive.

Removal of the C-3 hydroxy group of 1 resulted in 3-deoxy-2,3-didehydrobetulin 23, the anti-leishmanial activity of which diminished to 13% at 50 μM. Oxime derivatives 24 and 25 showed good leishmanicidal activities at 50 μM, with 69 and 73% inhibition, respectively. Moreover, betulin derivative 26 with a nitrile group at C-28 showed good anti-leishmanial activity with a 63% inhibition at 50 μM.

Derivatives (12, 18, 19, 21 and 25) that showed the best anti-leishmanial activity on microplate assay at 50 μM against L. donovani axenic amastigotes were selected for further investigations: GI50 values, cytotoxicity to the macrophage cell line THP-1 and anti-leishmanial activity against the L. donovani-infected macrophage cell line THP-1 were evaluated. Betulonic acid 18 showed the best GI50 value of 14.6 μM on microplate assay against L. donovani axenic amastigotes, followed by L-aspartyl amide derivative 21 and oxime derivative 25, with GI50 values of 21.2 and 22.8 μM, respectively (Table 1). 28-O-Bromoacetylbetulin 12 and dihydrobetulonic acid 19 had moderate GI50 values of 34.9 and 56.0 μM, respectively. Cytotoxicity of derivatives 12, 18, 19, 21 and 25 was tested against the macrophage cell line THP-1 at concentrations of 50, 25 and 12.5 μM (Table 2). Betulonic acid 18 showed cytotoxicity against the THP-1 cell line at all test concentrations. Dihydrobetulonic acid 19 and oxime derivative 25 showed cytotoxicity against the THP-1 cell line at 50 and 25 μM, but at 12.5 μM concentration, cytotoxicity of 19 and 25 was reduced to 22.0 and 13.6%, respectively. L-aspartyl amide derivative 21 and 28-O-bromoacetylbetulin 12 were nontoxic to macrophage cell line THP-1 at all test concentrations.

Table 2 Cytotoxicity of the most potent synthetic betulin derivatives on macrophage cell line THP-1
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Finally, anti-leishmanial activity of compounds 12, 19, 21 and 25 was tested against L. donovani-infected macrophage cell line THP-1, with concentrations that showed <30% cytotoxicity to the THP-1 cell line (Table 3). In all cases, anti-leishmanial activity was reduced when compared with that in the corresponding microplate assay with L. donovani axenic amastigotes. L-aspartyl amide derivative 21 and 28-O-bromoacetylbetulin 12 showed good anti-leishmanial activity at 50 μM, inhibiting 53 and 56% of the intracellular parasites, respectively (compared with 69 and 86% inhibition using axenic amastigotes in the microplate assay, respectively). At 25 μM, 28-O-bromoacetylbetulin 12 still had the best activity of the compounds examined showing 34% inhibition, dihydrobetulonic acid 19 and L-aspartyl amide derivative 21 were only weakly active at this concentration. Finally, at 12.5 μM concentration, oxime derivative 25 showed the best anti-leishmanial activity with a 52% inhibition, whereas L-aspartyl amide derivative 21 was totally inactive and the rest showed only weak activity.

Table 3 Anti-leishmanial activities of the most potent synthetic betulin derivatives against macrophage cell line THP-1 infected with Leishmania donovan i
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We have shown that by simple chemical modification, anti-leishmanial activity of ubiquitous naturally occurring triterpene, betulin, can be improved considerably. It is possible to derive relatively potent anti-leishmanial compounds with low micromolar GI50 values. In general, carbonyl or carboxyl groups at C-3 or C-28 have a beneficial effect in anti-leishmanial inhibition activity, and these compounds can be regarded as significant lead molecules for further improvement and optimization. Further studies are required to develop more potent betulin derivatives with leishmanicidal properties, and with no toxicity in macrophage cell lines or in human host cells. Moreover, thorough early ADME, biological mechanism and animal studies are required to evaluate anti-leishmanial activity in vivo.

Experimental Section

Chemical syntheses of betulin derivatives screened in this study for anti-leishmanial activity are described in detail elsewhere.18 Anti-leishmanial activities of betulin derivatives were screened using a fluorescent viability microplate assay with L. donovani (MHOM/SD/1962/1S-Cl2d) axenic amastigotes and alamarBlue (resazurin, AbD Serotec, Oxford, UK) as described previously.19, 20, 21 Initial screening was carried out by assessing the inhibition of amastigote growth at 50 μM of betulin derivative. All compounds were tested at least twice in triplicate. Complete medium, both with and without dimethyl sulfoxide, was used as negative controls (0% inhibition of amastigote growth). The most potent betulin derivatives from initial screening were selected for further investigation. For these compounds, the GI50 value (concentration for 50% growth inhibition) was also determined, as well as screening for activity on infected macrophages. The latter assay was carried out as previously described using the retinoic acid-treated human macrophage cell line THP-1 infected with L. donovani expressing the luciferase gene (Ld:pSSU-int/LUC) at a 3:1 parasite:macrophage ratio.17,22 Compounds (at 50, 25 and 12.5 μM) to be tested were added for 48 h, and luminescence was determined after adding a luciferase substrate and measuring in a microplate reader. Amphotericin B was included as a positive control on each plate and resulted in >90% inhibition at 1 μM. The effect of compounds on THP-1 cells alone was assessed using the alamarBlue viability assay.