In this study, semisynthetic compounds were obtained, derived from natural sources, with potential utility as anti-malarial agents, using a simple and inexpensive strategy.
The triterpenes BA and UA exhibited moderate anti-malarial activity in vitro[12, 22]. Both compounds were obtained from natural sources with good yields. This work reported for the first time the semisynthesis of 18 BA and UA derivatives with ester substituents at C-3, designed to improve anti-malarial activity, reduce cytotoxicity and search for new targets. These derivatives were obtained using a single-step, inexpensive synthesis adequate for industrial-scale processing. The anti-malarial activities of the synthesized compounds were in the range of 5 and >100 μM against CQ-sensitive P. falciparum 3D7, with three compounds exhibiting good activity (1e, 1f and 2e). These three derivatives possessed a four-carbon side-chain and had anti-malarial activity three to five times greater than that of BA and UA. Some reports have reported a change in activity after modification on the triterpene carbon 3. The 3-O-acetyl ester of ursolic acid exhibited enhanced activity against CQ-resistant P. falciparum FcB1 (IC50 = 24.93 μM) as compared with UA (IC50 = 52.93 μM) . Interestingly, the 3-O-acetyl ester of oleanolic acid, an isomer of UA, was also active against a multidrug-resistant P. falciparum K1 strain . In another study, the 3-O-acetyl ester of lupeol containing a functional ester group at C-3 was more active than its precursor against CQ-resistant P. falciparum FCR-3 . In view of these findings, modifications at the C-3 position of BA and UA were designed to evaluate the influence of side-chain length and presence of polar or lipophilic groups (such as carboxylic acids, aromatic rings or halogens) on their ability to impair parasite growth. In these series, acylated derivatives with shorter side-chains (1e, 1f and 2e) had improved anti-malarial activity (IC50 = 5, 8 and 7 μM, respectively). It was also obtained information on the presence of a second carboxylic acid group, as in derivatives 1d (IC50 = 70 μM), 1g (IC50 = 66 μM) and 2g (IC50 = 58 μM); and the presence of a halogen, such as chlorine and fluorine, as in derivatives 1h (IC50 = 22 μM), 1i (IC50 >100 μM), 2h (IC50 = 58 μM) and 2i (IC50 >100 μM). These functional groups did not potentiate anti-malarial action.
Comparison of derivatives with the same substituents derived from series BA and series UA demonstrated that BA derivatives are generally more active against P. falciparum 3D7 strain than UA derivatives. The most active derivatives synthesized, 1e, 1f and 2e, did not show any cytotoxicity against HEK293T cells at the tested concentration of 100 μM. This excellent result justifies potential in vivo experiments with these compounds in future. Follow-up studies with non-cytotoxic compounds are important, as adverse effects such as hypoglycaemia, cardiotoxicity, and gastrointestinal discomfort have been described with the anti-malarials: QN, halofantrine, and CQ/proguanil .
The molecular mechanism of action of triterpenes such as BA and UA is still poorly understood. Previous research has suggested that the mechanism of action of UA and its derivatives could be similar to that of CQ, i.e. inhibition of β-haematin formation [13, 25]. It was chose to assess this mechanism of action and another mechanism that has been suggested for an important anti-malarial agent, atovaquone: an inhibitory effect on ΔΨm. The ΔΨm consists of chemical and electrical components generated by electron transport chain enzymes, and its determination is widely used to characterize cellular metabolism, viability and apoptosis . The ΔΨm assay was performed for the more active derivatives, 1e, 1f and 2e, by accumulation of the lipophilic cationic fluorescent probe DiOC6(3). When DiOC6(3) is incubated with iRBC, it diffuses into cells and concentrates several orders of magnitude into negative-inside mitochondria. Probe accumulation into parasite mitochondria is dependent on the presence of a membrane potential, collapse of which will result in diffusion of the probe out of the mitochondria, resulting in signal dissipation. The tested derivatives did not exhibit histogram displacement, as observed after incubation with CCCP, indicating dissipation of the membrane potential and abolition of probe accumulation. This experiment showed that, unlike CCCP, the tested compounds were not able to collapse ΔΨm (Figure 3).
Derivatives 1e, 1f and 2e were also evaluated for inhibition of β-haematin formation, and displayed less inhibitory activity than that of CQ. This assay is based on the ability of Plasmodium to use haemoglobin as a source of amino acids, resulting in the formation of potentially toxic ferriprotoporphyrin IX. CQ and other anti-malarial drugs act by inhibiting ferriprotoporphyrin IX detoxification through haem polymerization. The action of these anti-malarial drugs on β-haematin formation takes place during the intra-erythrocytic phase of the parasite, within the food vacuole. The parasite converts haem into the malarial pigment haemozoin . Derivatives 1e and 2e derivatives exhibited similar inhibition percentages (25% and 26% respectively), whereas 1f displayed less inhibitory activity (14%) than the other tested derivatives. Thus, it was observed that modification of carbon 3 plays an important role in the inhibition of β-haematin formation.