Evaluation of triterpenes derivatives in the viability of Leishmania amazonensis and Trichomonas vaginalis

Abstract

Trichomonas vaginalis and Leishmania spp. are protozoal species responsible for millions of cases of parasitic diseases worldwide. Considering the potential of natural products and the need for more effective and less toxic alternatives to treat trichomoniasis and leishmaniasis, this study aimed to evaluate the effect of two series of triterpenes derivatives with different modifications at C-3 and C-28 positions of the ursolic acid (UA) and betulinic acid (BA) against trophozoites of Trichomonas vaginalis and promastigotes forms of Leishmania (L.) amazonensis. The compounds modified just at C-3 were the most active. The 3β-acetyl betulinic acid (1b) reduced the trophozoites viability of T. vaginalis at 74%, followed by the 3-oxo ursolic acid and 3-oxo betulinic acid (3a and 3b) compounds (55% of reduction). The compound 3β-isobutyl ursolic acid (7a) inhibited the viability of L. amazonensis promastigotes by 55%. Therefore, analyzing the structure-activity relationship and the data of literature, it is possible to suppose that the inclusion of polar groups in the skeletons could improve the antiprotozoal activity. Overall, further studies are necessary to develop triterpenic derivatives with more powerful trichomonicidal and leishmanicidal properties.

Keywords:
Betulinic acid; Leishmania amazonensis; Semisynthesis; Trichomonas vaginalis; Ursolic acid

INTRODUCTION

Human pathogens, such as Trichomonas vaginalis and Leishmania spp., are unicellular eukaryotes (protists) representative of the supergroup Excavata that includes a few other parasites such as Trypanosoma and Giardia. These protozoa are responsible for a broad range of health diseases around the world, mainly in the developing countries (Kusdian, Gould, 2015).

Trichomonas vaginalis is a microaerophilic mucosal pathogen, which affects the human urogenital tract causing trichomoniasis, the most common non-viral sexually transmitted disease (STD) in the world (WHO, 2012). According to the World Health Organization (2012), there are about 276 million new cases of trichomoniasis annually worldwide. The parasite mainly affects the urogenital tract of both men and women, and it may cause asymptomatic infection or lead to urethritis or vaginitis. Studies have indicated several complications related to this disease, including amplification of HIV transmission, risk of low birth weight, and preterm delivery (Schwebke, Burgess, 2004). The Food and Drug Administration (FDA, USA) recommends the treatment for trichomoniasis with a few drugs of choice belonging to 5-nitroimidazole class, with metronidazole and tinidazole - the only two approved drugs. Besides the side effects observed during treatment, another important limitation regarding the nitroimidazoles administration is the emergence of metronidazole-resistant isolates estimated in 2.5 to 10%. The reliance on a single therapeutic class is problematic and alternative treatments are urgently needed (Klebanoff et al., 2001; Cudmore et al., 2004).

Leishmaniasis are neglected infectious diseases caused by protozoan belonging to the genus Leishmania, and are transmitted via the bites of infected female phlebotomines. There are three main forms of the disease: visceral, cutaneous, and mucocutaneous (Silveira, Lainson, Corbett, 2004; WHO, 2018). An estimated 700,000 to 1 million new cases and 20,000 to 30,000 deaths occur annually around the world, still being a serious disease in tropical and subtropical areas (WHO, 2018). There are more than 20 Leishmania species that are transmitted to humans, among them Leishmania (L.) amazonensis (Lainson, 2010). This species is the most widely distributed in Brazil and can cause mucocutaneous leishmaniasis, which ranges from small cutaneous nodules to gross mucosal tissue destruction (Silveira, 2009; Machado, Penna, 2012). The type of disease, parasite species, and the immunological status of the host defines the treatment of leishmaniasis. The pentavalent antimonials, pentamidine, amphotericin B, paromomycin, and miltefosine are the drugs available for disease treatment; however, they all have limitations such as high costs, specific toxicity, the emergence of resistance and the need for parenteral administration. In this scenario, the discovery and development of new effective drugs is imperative; nevertheless, leishmaniasis is one of the most neglected tropical diseases in terms of drug discovery (Singh et al., 2014; Rajasekaran, Chen, 2015).

Taking into account the need for more effective and safer alternatives to treat trichomoniasis and leishmaniasis, and the rich structural diversity of natural products, research with focus on the investigation of natural products with activity against these protozoa have increased. A recent review referring to the potential of natural and synthetic products with anti-trichomonal activity, demonstrated that terpenes, phenolic compounds, and alkaloids are promising potential compounds against T. vaginalis (Vieira et al., 2015). Likewise, it has been demonstrated for Leishmania, that quinones, alkaloids, terpenes, saponins, phenolic and their derivatives have shown antiparasitic properties and selective pharmacological properties modes of action (Singh et al., 2014).

Considering the potential of the natural products and that research of therapeutic alternatives for parasitic diseases are needed, this study aimed to evaluate the activity of two series of semisynthetic derivatives with different modifications at C-3 and C-28 positions of the triterpenes ursolic acid and betulinic acid against trophozoites of T. vaginalis and promastigotes forms of Leishmania (L.) amazonensis.

MATERIAL AND METHODS

Ursolic and betulinic acids extraction

Betulinic acid (BA) was obtained from barks of Platanus acerifolia, collected in Bento Gonçalves, RS, Brazil (29°10’40.43”S 51°34’2.2”W). Ursolic acid (UA) was isolated from apple pomace (Malus domestica); a by-product of juice manufacture Tecnovin Ltd., Bento Gonçalves, RS, Brazil (Cargnin, Gnoatto, 2017). The triterpenes identities were confirmed by spectroscopic comparison with analytical standard and related literature (Tkachev et al., 1994).

Semisynthesis of UA and BA series

Briefly, compounds modified at C-3 position were prepared by the reaction of UA or BA with the appropriate anhydride or oxidizing agent, as previously described (Gnoatto et al., 2008b; Silva et al., 2013). Derivatives with ester or ketone substituents at C-3 were then submitted to another reaction for modifications at C-28, and methyl and imidazole ring at C-28 were incorporated (Scheme 1). The derivatives identities were confirmed by analysis of spectroscopic data (IR, ¹H-NMR and ¹³C-NMR spectra and Mass spectra - LC-MS), and compared with data of literature (Santos et al., 2009; Leal et al., 2012; Silva et al., 2013).

SCHEME 1
Synthesis of derivatives 1a-8b. For the compounds 1a,1b; 3a,3b; 5a,5b; 7a,7b, the UA and BA were submitted separately at followed reactions: (a) dichloromethane, acetic anhydride, pyridine, rt., 24h; (b) acetone, Jones Reagent at 0 °C, rt., 3h; (c) butyric anhydride, DMAP, rt., 24h; (d) isobutyric anhydride, DMAP, rt., 24h. For the compounds 2a, 2b; 4a, 4b; 6a, 6b; 8a, 8b, the synthetic route followed this condition: (e) dichloromethane, oxalyl choride, N₂ atmosphere, 0 °C, 3h. - trimethylamine, 0 °C - imidazole, rt., 24h.

Biological evaluation

In vitro anti-Trichomonas vaginalis activity

Trichomonas vaginalis isolate (ATCC 30236) were cultured axenically in vitro in trypticase-yeast extract-maltose (TYM) medium (pH 6.0), supplemented with 10% heat-inactivated bovine serum (HIBS), and incubated at 37 °C (Diamond, 1957). Organisms in the logarithmic phase of growth and exhibiting more than 95% viability and normal morphology were harvested, centrifuged, washed and resuspended in fresh TYM medium. To perform the screening assay, the compounds were solubilized in dimethyl sulfoxide (DMSO) (0.6%) at final concentrations of 100 µM. The trophozoites were incubated at a density of 2.0×10⁵ trophozoites/mL, at 37 °C for 24 h. The T. vaginalis viability was determined by counting in hemocytometer using trypan blue as exclusion dye. A negative control with trophozoites maintained in TYM medium, a control of the vehicle, and a positive control (metronidazole 8.0 µM) were carried out. The results were expressed as the percentage of living parasites after 24 h of the incubation period considering motility and normal morphology (percentage of living organisms compared to negative control).

In vitro anti-Leishmania amazonensis activity

Promastigotes Leishmania (Leishmania) amazonensis (IFLA/67/BR/PH8) were cultured in vitro at 23 °C in RPMI medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 20 mM Hepes (N-2-ydroxyetrylpiperazine-N’-2-ethanesulfonic acid), and 50 mg/mL of gentamycin and subcultured every 96 h. The anti-L. amazonensis activity of compounds was determined by measure of viability of promastigotes forms by MTT assay (Pal et al., 2011), with modifications. For these experiments, promastigotes in stationary phase were seeded at 1.0×10⁶ parasite/100µL/well in 96-well plates in complete RPMI medium with compounds at final concentration of 100 µM, solubilized in DMSO (0.6%). Pentamidine (9.0 µM) was used as reference anti-leishmanial agent. Promastigotes were incubated at 23 ºC for 72 h. Afterwards, 10 µL of a MTT solution (5 mg/mL in phosphate buffered saline - PBS) was added to each well and incubated for further 4 h at 23 °C. Subsequently, 100 µL of DMSO was added to each well and was incubated for 1 h at room temperature. The optical density (OD) was measured at 540 nm. The results are expressed as percentage of viable promastigotes, compared with controls.

In vitro cytotoxicity assay

Cell viability was determined by a colorimetric method (Mosmann, 1983), using VERO (African Green Monkey Kidney, ATCC CCL-81) cells and MTT reagent (Sigma-Aldrich, USA). Briefly, VERO cells were cultured in RPMI 1640 (Sigma) supplemented with 10% FBS, 20 mmol/L Hepes, and 50 mg/mL of gentamycin. Experiments were performed in 96-well microtiter plates, where a suspension of 1.0×10⁴ cells per well was incubated in a humidified atmosphere with 5% CO₂ at 37 °C. After 24 h of cell adhesion, the cells were treated with test compounds at concentrations ranging from 400 to 6.25 µM, dissolved in DMSO (0.5%). Next 24, 48 and 72 h of incubation, stock MTT solution (5 mg/mL in PBS) was added to each well and were incubated for more 2 h at 37 °C. Subsequently, 100 µL of DMSO was added to dissolve the insoluble purple formazan, and the OD of each well was measured at 570 nm. Three wells per dose were analyzed for each sample in three different experiments, and the results were expressed as the percentage of viable cells in comparison to negative control (untreated cells). The cytotoxicity of each test compound was expressed as CC₅₀, the cytotoxic concentration of sample that inhibited cell growth by 50%.

Statistical analysis

All analyses were accomplished in triplicate and results were expressed as mean ± standard deviation (SD). The data were subjected to one-way analysis of variance (ANOVA) and P-values below 0.05 were regarded as significant. Dunnett’s multiple comparisons test was used to identify significant differences between means among the different treatments (GraphPad Prism Software).

RESULTS AND DISCUSSION

The worldwide incidence rates of infection by Leishmania spp. and T. vaginalis are startling. Leishmaniasis and trichomoniasis are not notifiable diseases in all the countries where these diseases are endemic and any surveillance system is available to detect resistance. Therefore, a substantial number of cases of these diseases are underestimated, leading to neglected parasitic infection status (Choffnes, Relman, 2011).

Previously, our research group evaluated the potential of some semisynthetic derivatives of pentacyclic triterpenes ursolic acid (UA) and betulinic acid (BA) against Leishmania spp. and Trichomonas vaginalis species. Seven UA derivatives were evaluated against the promastigote forms of L. amazonensis and the N-{3-[4-(3-(Bis(4-hydroxybenzyl)amino)propyl)piperazinyl]propyl}-3-O-acetylursolamide (Figure 1) was the most active compound with EC₅₀ = 10 µM (Gnoatto et al., 2008a). Against T. vaginalis, among the six derivatives tested, the N-{3-[4-(3-aminopropyl)piperazinyl]propyl}-3-acetylbetulinamide (Figure 1) was active, with a MIC value of 91.2 µM (Innocente et al., 2014). Both active compounds have an acetyl group at C-3 and a piperazinyl at C-28 positions. Hence, in this study, we have evaluated the activity of different derivatives of UA and BA against these important neglected protozoa species.

FIGURE 1
Compounds previously tested: 1. N-{3-[4-(3-(Bis(4-hydroxybenzyl)amino)propyl)piperazinyl]propyl}-3-Oacetylursolamide; 2. N-{3-[4-(3-aminopropyl)piperazinyl]propyl}-3-acetylbetulinamide; 3. 3β,6β,16β-trihydroxylup-20(29)-ene; 4. 3β,6β,16β-trihydroxylup-20(29)-ene derivative, with 2 acetyl groups.

Two series of ursolic acid and betulinic acid derivatives have been designed and semisynthetized by modifications at C-3 and C-28 positions, generating sixteen derivatives (Figure 2). When they were evaluated against T. vaginalis (Figure 3), at 100 µM, it was observed that the compound 1b, which is acetylated at C-3, reduces the parasite viability at 74%, followed by the compounds oxidized at C-3 (3a and 3b) (55% of viability reduction). The modifications at C-28 (2a, 2b; 4a, 4b; 6a, 6b; 8a, 8b) were especially not favorable for the anti-T. vaginalis activity. In the analysis of EC₅₀ of some active compounds, betulin presented an EC₅₀ value of the 60 µM, and the compounds 1b and 5b, 30 µM and 50 µM, respectively.

FIGURE 2
Semisynthetic derivatives of UA and BA series.

FIGURE 3
Anti-T. vaginalis activity of UA and BA derivatives. Each bar represents the mean ± standard deviation of at least three independent experiments. Statistically significant differences (p ≤ 0.05) between treated and non-treated groups are indicated by (*).

Among the derivatives tested against L. amazonensis, at concentration of the 100 µM, three compounds presented reductions of viability below 50% (7a, 7b, betulin) (Figure 4). For these compounds, the EC₅₀ values were evaluated; betulin presented an EC₅₀ value of the 72.2 µM, and the compounds 7a and 7b, 85.7 µM and 119.4 µM, respectively. Differently from what was reported by Gnoatto et al. (2008b), who found a significant UA anti-promastigote activity (EC₅₀ = 20 µM) in the tested conditions, UA was not active against L. amazonensis promastigotes. Considering the concentration tested, data is in agreement with Peixoto et al. (2011), which showed anti-L. amazonensis activity for UA in concentration higher than 100 µM (EC₅₀ = 360.3 µM). Although the extraction of UA is simple and inexpensive, since the raw material is obtained from residue of apple juice manufacture (Cargnin, Gnoatto, 2017), the EC₅₀ value is outlying of the range considered acceptable in the development of new semisynthetic drug.

FIGURE 4
Anti-L. amazonensis activity of UA and BA derivatives. Each bar represents the mean ± standard deviation of at least three independent experiments. Statistically significant differences (p ≤ 0.05) between treated and non-treated groups are indicated by (*).

The triterpene BA presented a carboxylic acid at C-28 and betulin, another triterpene with a lupane skeleton, presented an alcohol group at position C-28 (R₂). The leishmanicidal activity of betulin was evaluated and it was more active than compounds of BA series, with reduction of promastigote forms of 68.7% (Figure 4). The activity was more than 20% higher than the most active compound of BA series. Therefore, taking into account the structure-activity relationship, the hydroxyl groups in triterpenes skeleton seem to be important for anti-leishmanial activity. Corroborating with this hypothesis, previous studies revealed that lupane-triterpene (3β,6β,16β-triidroxilup-20(29)-ene) (Figure 1) isolated from Combretum leprosum fruit extracts, exhibited a significant anti-leishmanial activity against L. amazonensis promastigotes, with an EC₅₀ of 7.2 µM, and is also effective in eliminating the L. (L.) amazonensis intracellular amastigotes at 109 µM (Teles et al., 2011; Teles et al., 2015). Moreover, a synthetic derivative with two hydroxyl groups replaced by acetyl (Figure 4), became inactive (Teles et al., 2011).

Considering the evaluation of the in vitro cytotoxicity of these compounds on mammalian cells (VERO cells) at 24, 48 and 72 h, the UA presented cytotoxic effect of CC₅₀ 200, CC₅₀ 101.64, and CC₅₀ 99.91 µM, respectively. The other compounds tested were not cytotoxic (CC₅₀> 400 µM). Even though the compounds were not cytotoxic in this cellular model, some structure modifications are necessary to improve the activity profile towards parasites.

Therefore, further studies are required to develop UA and BA derivatives with more potent leishmanicidal and trichomonicidal properties. Nevertheless, in the test conditions, the derivatives investigated did not exhibit pronounced activity against T. vaginalis and L. amazonensis, these preliminary results showed that, based on structure-activity relationship, triterpenes could be a source of novel anti-leishmanial and anti-trichomonas agents. Hence, the delineation of additional semisynthetic modifications, especially including polar groups, such as hydroxyl groups, in the skeletons could improve the activity of these compounds.

ACKNOWLEDGMENTS

The authors are grateful to the Brazilian agencies CAPES and CNPq for the work financial support and students´ fellowships and LRNANO for spectra data. The authors are also grateful to Programa de Pós-Graduação em Ciências Farmacêuticas - UFRGS, Malaria and Leishmaniasis Bioassays Platform - Fiocruz, Rondônia, and Instituto Nacional e Epidemiologia na Amazônia Ocidental - INCT-EpiAmO, Porto Velho, RO. T.T. thanks CNPq for research fellowship (grant 307447/2014-6).

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Publication Dates

• Publication in this collection
  20 Dec 2019
• Date of issue
  2019

History

• Received
  13 Aug 2017
• Accepted
  10 July 2018