After being isolated from natural sources, the importance of the C-3 hydroxyl group of betulinic and ursolic acids 1 and 2 was ascertained through usual acetylation (Scheme 1). The acetylated compounds numbered 1a and 2a were obtained in 98% yield. The coupling reaction of 1a and 2a with N-tert-butoxycarbonyl-1,4-bis(3-aminopropyl)piperazine occurred following activation of the carboxylic acid at C-17 with oxalyl chloride as previously described [10]. The corresponding derivatives 1b and 2b were then isolated in 80% yield. Treatment with 10% trifluoroacetic acid/dichloromethane removed the tert-butoxycarbonyl (Boc) to provide compounds 1c and 2c in 70% yield (Scheme 1). All compounds were fully characterised by spectroscopic data.

All compounds were evaluated in vitro against the P. falciparum chloroquine-sensitive 3D7 strain in comparison to chloroquine which IC₅₀ value was 29 nM. Compound 1 showed moderate activity (IC₅₀ 18 µM) and 2 was inactive (IC₅₀ 36 µM) (Figure 2, Table 1) which results are in agreement with those of the literature [6,17,18].

Acetylated derivatives 1a and 2a showed better antimalarial activity than their aglycones (IC₅₀ values of 4 and 14 µM, respectively) (Figure 2, Table 1). The highest values were obtained for the piperazinyl derivatives 1c (IC₅₀ 220 nM) and 2c (IC₅₀ 175 nM). Protected compounds 1b and 2b presented IC₅₀ values of 5 and 15 µM, respectively, demonstrating a lower capacity to reduce parasitemia in comparison to unprotected compounds. As shown in Figure 2, the piperazine group contributed to a significant improvement in activity as also reported [19].

The active derivatives 1a–c and 2c were evaluated for their cytotoxicity in human embryonic kidney cells (HEK293T) by monitoring the number of living cells at the same concentrations used to evaluate the antiplasmodial activity (100, 10, 1, 0.1, 0.01 and 0.001 µM). Many research groups have tried to find cytotoxicity activity from triterpenoids, including betulinic acid and their derivatives. Thibeault et al. presented cytotoxicity results of lupane-type triterpenoid glyceryl esters. Betulinic acid showed values of 18 to 57 µM in several cell lines [20].

On the other hand, the cytotoxicity of heterocyclic triteprenoids derived from betulin and betulinic acid was tested against seven tumor and two normal fibroblast cell lines showing activity values from 1.0 to 55.5 µmol/L [21]. The results displayed in Table 1 show that 1a was not cytotoxic at the tested concentrations, while the piperazinyl derivatives 1b, c and 2c showed IC₅₀ values of 4 µM. Moreover, the most active compounds 1c and 2c presented SI values of 18 and 23, respectively, being considered non-toxic.

Like most eukaryotic cells, malaria parasites have internal Ca²⁺ pools responsible for regulation of signalling processes during invasion, maturation and division that can be mobilised upon agonist stimulation in the plasma membrane [22]. The effect of compound 2c through modulation of one or more calcium pathways in P. falciparum (3D7) was demonstrated using the fluorescent calcium probe Fluo4/AM. This molecule was chosen because it has the highest selectivity index (23, Table 1).

The typical rise in cytosolic calcium in isolated parasites after addition of compound 2c (100 µM) and thapsigargin (THG) (10 µM) is presented in Figure 3a–c. Because THG has been known to modulate Ca²⁺ via inhibition of sarco-endoplasmatic reticulum Ca²⁺ ATPase (SERCA) [23], it was used as the control in this experiment. The control experiment (with methanol addition) showed clearly that the solvent did not elicit calcium increase within the parasites (Figure 3a). On the contrary, when THG was added after compound 2c, the calcium increase was greater than the conventional calcium mobilisation observed with the control one (Figure 3b). These results indicated that 2c can modulate parasite calcium homeostasis.

Infrared spectra (IR) were obtained using a PerkinElmer FT-IR System Spectrum BX instrument. ¹H and ¹³C-NMR spectra were recorded on Varian Inova 300 and Varian VNMRS 300 spectrometers using TMS as internal standard. Chemical shifts are reported in parts per million (δ). An UltrOTOF (BrukerDaltonics) mass spectrometer was used to obtain HR-EI-MS spectra. Melting points were determined using a Koffler instrument. All reactions were monitored by thin-layer chromatography on Merck silica plates GF using different solvent systems. Column chromatography was carried out on silica gel GF (Merck, 60–230 mesh) using gradient mixtures (CH₂Cl₂/methanol). All reagents were purchased as analytical grade. All solvents were dried and distilled prior use. Dichloromethane was dried over CaCl₂ for 24 h then was submitted to reflux.

The betulinic acid was obtained from Platanus acerifolia. A voucher specimen (ICN 182537) is on deposit in the Botany Department Herbarium of Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil. The peel of Malus domestica was a byproduct from the juice industry company Tecnovin S.A., Vacaria, RS, Brazil, and was used to obtain the ursolic acid. Both powdered dried materials were separately subjected to reflux with ethanol 60% (250 mL, 2 h). Then, after removing the ethanol under vacuum, each residual aqueous phase was separately extracted with ethyl acetate, and the organic phase was evaporated to dryness to obtain a residue that was submitted to recrystallisation. The betulinic acid was recrystallized from methanol, while ursolic acid was recrystallized using acetonitrile. Betulinic acid (1). Yield 1.5%. Its structure was confirmed by spectroscopic methods which were in agreement with literature data [6,7]. Ursolic acid (2). Yield 4%. Its structure was confirmed by spectroscopic methods and literature data [10].

The ursolic acid derivatives were prepared using a semisynthetic route developed by Gnoatto and co-workers which details of the chemistry and structural elucidation were described previously [10]. Briefly, the acetylated ursolic acid 2a was submitted to a coupling reaction with N-Boc-bisaminopropylpiperazine in basic conditions to obtain 2b, and then the deprotected analogue 2c was obtained by removing the N-Boc-amino group. All structural assignments were compared with the published data [10]. The betulinic acid derivatives were obtained using an analogous route.

3-O-Acetylbetulinic acid (1a). Yield 98%. It was prepared from the corresponding aglycone 1 by usual acetylation [10].

N-{3-[4-(3-Aminopropyl)piperazinopropyl]terbutylcarbamate}-3-acetylbetulinamide (1b). Oxalyl chloride (0.15 mL, 1.7 mmol) was added to 1a (285 mg, 0.57 mmol) dissolved in CH₂Cl₂ (30 mL) at 0 °C, and the resulting solution was stirred under N₂ for 3 h. Subsequently, triethylamine (0.5 mL, 3.4 mmol) and then tert-butyl 3-[4-(3-aminopropyl)piperazinylpropyl]carbamate (510 mg, 1.7 mmol) were added to the reaction, which was then stirred for 24 h at room temperature. Next, water was added and the resulting product was extracted with CH₂Cl₂ (3 × 30 mL); the organic layers were combined, dried over Na₂SO₄ and evaporated under vacuum to afford 1b as a white solid. Yield: 80%; m.p. 129 °C; IR (ATR cm⁻¹): 3406 (NH); 2943 (C-H); 1777 (C=O acetyl); 1687 (C=O amide); 1555 (C=C); 1425 (C-O); 1380 (C-H); 1259 (C-O-C); 1176 (C-N). ¹H-NMR (CDCl₃, δ): 0.84 (s, 3H, H₂₆); 0.85 (s, 3H, H₂₇); 0.87 (s, 3H, H₂₅); 0.95 (s, 3H, H₂₃); 0.97 (s, 3H, H₂₄); 1.29 (d, 4H, H₁₁, H₁₂); 1.38 (m, 3H, H₆, H₉); 1.45 (s, 9H, H₃C-Boc); 1.55 (t, 4H, H₁₅, H₇); 1.65 (m, 6H, H₁₃, 2 × CH₂, H₅); 1.69 (s, 3H H₃₀); 1.76 (t, 4H, H₁₆, H₁) 1.97 (t, 3H, H₁₈, H₂); 2.04 (s, 3H, H₃C-COO-); 2.47 (m, 15H, H₁, 7 × CH₂); 3.19 (m, 2H, CH₂); 3.32 (m, 4H, 2 × CH₂); 4.48 (dd, 1H, H₃); 4.59 (brs,1H, H_29a); 4.75 (brs, 1H, H_29b); 5.36 (1H, NH); 6.86 (1H, NH). ¹³C-NMR (CDCl₃, δ): 14.55; 16.14; 16.22; 16.43; 18.17; 19.46; 20.94; 20.20; 23.68; 21.22 (H₃C-COO-); 25.57; 26.42; 28.41 (H₃C-Boc); 29.41; 30.91; 33.84; 34.93; 37.12; 37.58; 37.77; 38.39; 38.46; 39.55; 39.87; 40.76; 42.42; 46.68; 50.20; 50.57; 53.10; 53.45; 55.48 (4C); 56.73; 57.85; 75.80; 78.79 (Cq-Boc); 80.93; 109.23; 151.04; 156.0 (CO-Boc); 170.90 (H₃C-COO-); 176.14 (C-28). HRMS (ESI-MS, m/z); [M+H]⁺: Calcd. for C₄₇H₈₀N₄O₅: 781.1619; Found: 781.6175.

N-{3-[4-(3-aminopropyl)piperazinyl]propyl}-3-acetylbetulinamide (1c). The N-Boc-amino protecting group of compound 1b was removed using trifluoroacetic acid 10%/dichloromethane mixture according to the literature [10], resulting in the deprotected analogue 1c. Yield 70%; m.p. 120 °C. Its spectroscopic data were similar to those of 1b, excepting for the absence of the N-Boc-amino signals. HRMS (ESI-MS, m/z), [M+H]⁺: Calcd. for C₄₀H₇₂N₄O₃: 681.0461; Found: 681.5712.

The chloroquine-sensitive 3D7 strain of P. falciparum was grown in RPMI (Gibco/Life Technologies, Grand Island, NY, USA) containing 37.5 mM HEPES, supplemented with 7 mM D-glucose, 6 mM NaOH, 25 mL gentamicin sulphate, 2 mM L-glutamine and 10% human serum albumin. The strains were maintained in human erythrocytes in a gas mixture containing 5% O₂, 5% CO₂ and 90% N₂ [24]. The culture was synchronised by 10% sorbitol treatment [25]. The antimalarial activity was measured using the microdilution technique in vitro. Red cells with parasitemia between 1–2% and 2% hematocrit were incubated with the compounds 1, 1a–c and 2, 2a–c for 48 h. Samples were dissolved in water, dimethylsulfoxide (DMSO) or methanol and serially diluted with the culture medium. To evaluate parasitemia, Giemsa staining and flow cytometric analysis were used. The concentration of the drug required to inhibit parasite growth was determined by comparing the fluorescence incorporated into the treated and untreated cultures (control). The concentration causing 50% inhibition (IC₅₀) was determined from the drug concentration response curve. Methanol and DMSO were used at the maximum concentration of 0.1% and did not inhibit parasite growth.

Human embryonic kidney (HEK293T) cells were cultured in surface area 75 cm² (Greiner Bio-One, Frickenhausen, Germany) vented tissue culture flasks at 37 °C in a humidified atmosphere containing 5% CO₂ in Dulbecco’s modified essential medium (Gibco/Life Technologies, Grand Island, NY, USA) supplemented with 10% (v/v) foetal bovine serum, 100 U/mL penicillin and 100 µg/mL streptomycin. After 24 h, 50 µl of each drug was added with 0.5% DMSO or methanol to a single well. The cells were incubated with the test drugs for an additional 24 and 48 h and then harvested with trypsin, gently pelleted by centrifugation and re-suspended in PBS. The cells were stained with dihydroethidium solution (10 mg/mL in phosphate buffered saline), gently vortexed and incubated for 40 min at 37 °C in the dark. Ten thousand gated events were acquired for each sample (FACSCalibur BD and CellQuest software was used for analysis).

Erythrocytes (RBCs) infected by P. falciparum (3D7) at the trophozoite stage were lysed with 10 mg/mL saponin (in phosphate-buffered saline) in the presence of 20 µg/mL protease inhibitors (pepstatin A, leupeptin, antipain and chymostatin) and benzamidine (0.5 mmol/L). Erythrocyte membranes were then removed through centrifugation (8700 rpm for 10 min at 4 °C). The parasites were washed three times in buffer M (116 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO₄, 5.5 mM D-glucose, 50 mM MOPS, pH 7.3) with CaCl₂ (2 mM). The parasites were then re-suspended in the same buffer with Fluo4-AM (5 μM) probenecid (1.8 mM), an inhibitor of organic anion transport (to prevent dye sequestration and release), 20 mg/mL protease inhibitors (pepstatin A, leupeptin, antipain and chymostatin) and benzamidine (0.5 mmol/L) for 50 min at 37 °C. The parasite suspension was then washed three times with buffer M. Spectrofluorimetric measurements were performed in a Shimadzu RF-5301 PC instrument at 37 °C with isolated P. falciparum (3D7) parasites (10⁸ cells mL⁻¹) incubated with MOPS buffer in a 1 mL cuvette. Compound 2c (100 μM) and THG (10 μM) were added during time course experiments, and excitation/emission wavelengths were adjusted to 505/530 nm for Fluo4-AM. Methanol (0.05%) was used as a control. Addition of the detergent digitonin induced the maximum increase in calcium that was abolished by addition of the calcium chelator EGTA.

Student’s test was chosen for comparisons between two groups, whereas repeated ANOVA measures were used for comparisons among more than two groups. The results are expressed as the mean ± SEM of at least three individual experiments. P < 0.05 was considered a statistically significant difference. GraphPad Prism software (San Diego, CA) was used for all statistical tests.