Molecules 2014, 19, 5761-5776; doi:10.3390/molecules19055761 OPEN ACCESS molecules ISSN 1420-3049 www.mdpi.com/journal/molecules Article Antiparasitic Activity of Natural and Semi-Synthetic Tirucallane Triterpenoids from Schinus terebinthifolius (Anacardiaceae): Structure/Activity Relationships Thiago R. Morais 1, Thais A. da Costa-Silva 2, Andre G. Tempone 2, Samanta Etel T. Borborema 2, Marcus T. Scotti 3, Raquel Maria F. de Sousa 4, Ana Carolina C. Araujo 4, Alberto de Oliveira 4, Sérgio Antônio L. de Morais 4, Patricia Sartorelli 1 and João Henrique G. Lago 1,* 1 Instituto de Ciências Ambientais, Químicas e Farmacêuticas, Universidade Federal de São Paulo, Diadema, SP 09972270, Brazil 2 Centro de Parasitologia, Instituto Adolfo Lutz, São Paulo, SP 01246902, Brazil 3 Centro de Ciências Aplicadas e Educação, Universidade Federal da Paraíba, Rio Tinto, PB 58297000, Brazil 4 Instituto de Química, Universidade Federal de Uberlândia, Uberlândia, MG 38400902, Brazil * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +55-11-3319-3300; Fax: +55-11-4043-6428. Received: 12 March 2014; in revised form: 26 April 2014 / Accepted: 28 April 2014 / Published: 5 May 2014 Abstract: Leishmaniasis and Chagas are diseases caused by parasitic protozoans that affect the poorest population in the World, causing a high mortality and morbidity. As a result of highly toxic and long-term treatments, the discovery of novel, safe and more efficacious drugs is essential. In this work, the in vitro antiparasitic activity and mammalian cytotoxicity of three natural tirucallane triterpenoids, isolated from leaves of Schinus terebinthifolius (Anacardiaceae), and nine semi-synthetic derivatives were investigated against Leishmania (L.) infantum and Trypanosoma cruzi. Trypomastigotes of T. cruzi were the most susceptible parasites and seven compounds demonstrated a trypanocidal activity with IC values in the range between 15 and 58 µg/mL. Four 50 compounds demonstrated selectivity towards the intracellular amastigotes of Leishmania, with IC values in the range between 28 and 97 µg/mL. The complete characterization of 50 triterpenoids was afforded after thorough analysis of nuclear magnetic resonance (NMR) data as well as electrospray ionization mass spectrometry (ESI-MS). Additionally, structure-activity relationships were performed using Decision Trees. Molecules 2014, 19 5762 Keywords: Schinus terebinthifolius; leaves; tirucallane triterpenoids; Leishmania (L.) infantum; Trypanosoma cruzi; structure-activity 1. Introduction Specimens of Schinus terebinthifolius, known in Brazil as “aroeira-vermelha” or “aroeira-pimenteira”, are large trees that can reach 40 m height and 1–3 m in diameter [1,2]. In folk medicine, this plant has been used to treat ulcers, respiratory problems, wounds, rheumatism, gout, diarrhea, skin disease and arthritis, as well as antiseptic and anti-inflammatory [3]. In addition, decoctions of flowers, stems, leaves and fruits are used for the treatment of tumors and hanseniasis [4]. Previous chemical studies with leaves extracts of S. terebinthifolius have been carried out and fatty acids and terpenoids were isolated, especially tirucallane derivatives (masticadienoic acid and schinol) which have shown inhibitory activity on phospholipase A2 [5] and antifungal potential against Paracoccidioides brasiliensis [6]. Other compounds, such as phenolic derivatives (gallic acid, methyl and ethyl gallates) and flavonoids (trans-catechin, kaempferol, quercitrin, afzelin, myricetin, and myricetrin) were also isolated from the leaves and displayed antiradical and cytotoxic activities [7,8]. Chemical analysis of barks of S. terebinthifolius indicated the presence of anthraquinones, xanthones and steroids [9]. Additionally, essential oils from leaves, flowers and fruits of S. terebinthifolius were also analyzed, and proved to be composed of mono- and sesquiterpenes. With regards to the evaluation of the biological activity, the volatile oils of fruits showed allelopathic, cytotoxic and trypanocidal activities, while the leaf oil showed cytotoxic activity [10–15]. Furthermore, previous work demonstrated anti-Leishmania amazonensis (promastigotes) activity of the aqueous and hydro-alcoholic extracts from leaves of S. terebinthifolius [16]. Considering other plants from the genus Schinus, the MeOH extract of the leaves and fruits of S. molle have also shown activity against L. infantum, Trypanosoma brucei, T. cruzi, and Plasmodium falciparum [17]. Leishmaniasis and Chagas’ disease are parasitic diseases caused by the protozoans Leishmania spp. and Trypanosoma cruzi, respectively. They are recognized by World Health Organization among the World’s most neglected diseases, affecting millions of people [18]. Considering the limited and highly toxic therapeutic arsenal, the study of alternative therapies is essential. In continuation of the investigation of bioactive compounds from Brazilian flora [19–22], the present study was undertaken to determine the effectiveness and cytotoxicity of three main compounds isolated from leaves extract of S. terebinthifolius (E- and Z-masticadienoic acids and Z-schinol) against promastigotes and intracellular amastigotes of Leishmania (L.) infantum, as well as trypomastigote forms of Trypanosoma cruzi. Aiming to establish relationships between the chemical structures and the antiparasitic activity, nine semi-synthetic tirucallane derivatives were obtained and tested against parasites and mammalian cells. 2. Results and Discussion Schinus terebinthifolius is a Brazilian plant that produces great amounts of tirucallane derivatives such as (Z)-masticadienoic (1) and (E)-masticadienoic (2) acids as well as (Z)-schinol (3). In this work, Molecules 2014, 19 5763 these compounds were isolated from leaves of this plant using several chromatographic techniques and their structures were confirmed by NMR and ESI-MS spectral analysis and comparison with data described in the literature [5,23]. The triterpenoids 1–3 were subjected to different reactions: reduction of carbonyl group at C-3, methylation of carboxyl group at C-27, acetylation of hydroxyl group at C-3 and hydrogenation of double bond at C-24, to afford nine derivatives (1a–c, 2a–d, 3a, and 3b–Figure 1), being 1c, 2c, 2d, 3a, and 3b new compounds. The structures of these compounds were confirmed by MS and 13C-NMR by the comparison of respective spectral data with those recorded for 1–3. In the case of carbonyl reduction (compounds 1a and 2a) the absence of a peak at δ 217 associated to the occurrence of a signal at range δ 79–76 indicates the presence of an oxymethine carbon at C-3. The 13C-NMR spectra of derivatives 2b, 2c and 3b showed additional peaks assigned to carbonyl (δ 170) and methyl (δ 20) carbons of acetyl group at C-3. The hydrogenated derivatives 1b, 1c and 2c showed two additional units in the ESI-MS mass spectra in comparison with 1 and 2b. This data, associated to the absence of peaks assigned to C-24 and C-25 at δ 146 and 126, respectively, indicated that the hydrogenation occurs exclusively at Δ24. Finally, structures of methyl esters 2d and 3a were confirmed by the presence of additional peaks at δ 52 in the respective 13C-NMR spectra. Figure 1. Structures of natural compounds 1–3 and semi-synthetic 1a–c, 2a–d, 3a, and 3b tirucallane triterpenoids. 22 24 21 26 20 25 18 23 12 19 11 13 17 27 16 14 15 1 9 8 2 10 28 3 5 7 4 6 30 29 1 R = O; R = H; Δ 24 Z 1 2 1a R = OH; R = H; Δ 24 Z 1 2 1b R = OH; R = H 1 2 1c R = O; R = H 1 2 2 R = O; R = H; Δ 24 E 1 2 2a R = OH; R = H; Δ 24 E 1 2 2b R = OAc; R = H; Δ 24 E 1 2 2c R = OAc; R = H 1 2 2d R = O; R = Me; Δ 24 E 1 2 3 R = OH; R = H; Δ 24 Z 1 2 3a R = OH; R = Me; Δ 24 Z 1 2 3b R = OAc; R = Me; Δ 24 Z 1 2 Molecules 2014, 19 5764 The antileishmanial and antitrypanosomal activities of three natural and nine semi-synthetic derivatives of tirucallane triterpenoids were evaluated against L. (L.) infantum and T. cruzi. According to the colorimetric assay of MTT and light microscopy, seven compounds killed 100% of trypomastigote forms of T. cruzi at the highest tested concentration, resulting in IC values in the 50 range of 15.75 to 58.36 µg/mL (Table 1). Table 1. Antiparasitic (antileishmanial and antitrypanosomal) and cytotoxic effects of natural compounds 1–3, semi-synthetic 1a–c, 2a–d, 3a, and 3b and standards. IC (g/mL) a CI95% CC (μg/mL) b CI95% SI 50 50 L. infantum L. infantum T. cruzi Compounds NCTC AMAc TRYd Promastigotes Amastigotes Trypomastigotes 1 NA 66.51 (47.68–92.79) NA >200 >3 – 1a NA NA 20.18 (16.70–24.39) 69.50 (64.01–75.45) – 3.4 1b NA NA 17.64 (15.97–19.50) 76.39 (70.02–83.33) – 4.3 1c NA NA NA >200 – – 2 NA 64.90 (41.48–101.50) 15.75 (9.80–25.30) 96.48 (77.38–120.30) 1.5 6.1 2a NA 97.59 (89.82–106.00) 29.59 (25.61–34.18) 95.49 (65.22–139.80) 1.0 3.2 2b NA NA 58.36 (42.82–79.55) 69.31 (38.74–82.64) – 1.1 2c NA NA 49.20 (41.69–58.05) 57.78 (56.10–59.52) – 1.2 2d NA NA NA >200 – – 3 57.82 (54.01–61.91) 28.95 (19.87–42.16) 16.28 (8.94–29.60) 69.50 (64.01–75.45) 2.4 4.3 3a NA NA NA >200 – – 3b NA NA NA >200 – – miltefosine 6.87 7.25 – 49.72 – – benznidazole – – 114.68 – – – IC : 50% inhibitory concentration; CC : 50% cytotoxic concentration (mammalian cells); NA: not active; 50 50 CI95%: 95% Confidence Interval; SI AMA: selectivity index amastigotes (CC mammalian cells/IC 50 50 Leishmania amastigotes); SI TRY: selectivity index trypomastigotes (CC mammalian cells/IC 50 50 trypomastigotes). Despite the lack of activity of compound 1, the semi-synthetic derivatives 1a and 1b demonstrated antitrypanosomal activity, with IC values of 20.18 and 17.64 µg/mL, respectively. In addition, an 50 enhanced mammalian toxicity was also observed. Otherwise, compound 2, the most effective against the trypomastigotes (IC 15.75 µg/mL), demonstrated a higher activity when compared to the 50 semi-synthetic derivatives 2a, 2b and 2c, which showed moderate inhibitory effects and IC values 50 between 29.59 and 58.36 µg/mL. Considering the 95% confidence intervals, the mammalian toxicity of 2a, 2b and 2c was comparable to that of the natural prototype 2. Likewise, the natural compound 3, which showed an IC value of 16.28 µg/mL against trypomastigotes, also rendered less effective 50 derivatives after methylation at C-27 (3a) and acetylation at C-3 (3b), although the derivatization resulted in no more cytotoxic compounds when compared to the natural compound 3. Benznidazole was used as a standard drug and gave an IC value of 114.68 µg/mL [21]. Considering the relation 50 between the antiparasitic activity and mammalian cytotoxicity, given by the selectivity index (CC /IC ), compounds 2, 3 and the semi-synthetic derivative 1b demonstrated the highest indexes, 50 50 ranging from 4 to 6. According to the Food and Drug Administration guidance for the development of Molecules 2014, 19 5765 drugs [24], it is desirable to have a high selectivity index giving maximum activity with minimal cell toxicity. Among the twelve tested compounds, only the natural prototype 3 showed effectiveness against L. infantum promastigotes, with an IC value of 57.82 µg/mL. The modifications of the semi-synthetic 50 tirucallane triterpenoid derivatives (compounds 1a–1c, 2a–2d, 3a and 3b) showed no improvement of the antileishmanial effectiveness against the extracellular forms of L. infantum. However, when tested against the intracellular amastigotes, four compounds (1, 2, 2a and 3) resulted in IC values in the 50 range of 28.95 to 97.59 µg/mL. This effect could be ascribed to a possible macrophage activation, which could also have contributed to an oxygen burst and up-regulation of cytokines by host cells [25]. The possible immunomodulatory effect of these compounds may be investigated in future assays. Similarly, the pentavalent antimonial glucantime, the main clinical drug in use for leishmaniasis has also shown no effectiveness against the extracellular forms of the parasite, and its antiparasitic activity has been attributed to host cell activation [26]. Except for compound 1, which showed no mammalian toxicity up to the highest tested concentration (>200 µg/mL), compounds 2 and 3 resulted in CC 50 values of 96 and 69 µg/mL, respectively. Considering the selectivity index, compound 1 was the most promising candidate without toxicity to NCTC cells, showing a value higher than 3. Miltefosine showed a 50% cytotoxic concentration (CC ) of 49.72 µg/mL and resulted in a SI of 7. Miltefosine was 50 used as a standard drug against promastigotes and amastigotes, with IC values of 6.87 and 50 7.25 µg/mL, respectively [27]. The decision tree (DT) model (Figure 2) selected with the aim of establishing relationships between chemical structures and antitrypanosomal activity of compounds 1a–c, 2a–d, 3a and 3b used the DD5 descriptor. This molecular descriptor quantifies the variation between the maximum hydrophobic volume obtained upon variation of ligand conformation and the hydrophobic volume of the imported three dimensional structure into Volsurf at a Molecular Interaction Fields (MIF) energy value of −1.0 kcal/mol [28]. Triterpenoids 1, 1c and 2d displayed equal or lower DD5 values than 0.125. Thus, it is possible to infer that compounds with a carbonyl group at C-3 with Z configuration at Δ24, with an ester group at C-27, or without a double bond between C-24/C-25 showed lower differences of hydrophobic volumes and are inactive against the T. cruzi. The DT model predicts for all 12 compounds (100%) of antitrypanosomal activity for the training set and 11 compounds (92.9%) for internal validation (Table 2). The obtained data also suggested that the stereochemistry of hydroxyl group at C-3 to triterpenoids 3 and 1a was determinant to antileishmanial activity (promastigote and amastigote forms of L. infantum) since these compounds displayed the same planar structure but different configuration. However, using a DT model with only one descriptor as DD8 (differences of the hydrophobic volumes at energy level −1.6 kcal/mol) it was possible to predict activity against L. infantum amastigotes for 10 compounds (83.3%) of the training set and internal validation (Table 2) [29]. The data showed that compounds with higher values of DD8 than 0.125 were active. Triterpenoids with carbonyl or hydroxyl at C-3, double bond at C-24, and carboxylic acid at C-27, showed higher values of DD8 (compounds 1, 1a, 2 and 3). It is important to highlight that the prediction activity error of compound 1a was due to DD8, which is a three dimensional descriptor that does not encode directly the stereochemistry but the difference of 3D conformations of the compounds. Molecules 2014, 19 5766 Figure 2. Decision Trees (DT) generated for the set of triterpenoids with antiparasitic activity. (A) The compounds with higher values of DD5 (differences of the hydrophobic volumes at energy level of −1.0 kcal/mol) than 0.125 were active against T. cruzi trypomastigotes. (B) Compounds with higher values of DD8 (differences of the hydrophobic volumes at energy level of −1.6 kcal/mol) than 0.125 were active against L. infantum amastigotes. (C) Triterpenoids with DD4 (differences of the hydrophobic volumes at energy level of −0.8 kcal/mol) values higher than 0.8125 or DD5 values lower or equal to 0.25 were not cytotoxic against NCTC. The numbers in brackets show the number of compounds correctly classified/incorrectly as active (A) or inactive (I). Table 2. Comparison of experimental antileishmanial, antitrypanosomal and cytotoxic activities with data predicted by Decision tree models for the training set and internal cross validation (leave-one-out). T. cruzi (Trypomastigotes) L. infantum (Amastigotes) NCTC Compounds Experimental Train Validation Experimental Train Validation Experimental Train Validation 1 I I I A A A I I I 2 A A A A A A I I I 2a A A A A I I I I I 1a A A A I A A A A A 1b A A A I I I A A I 2b A A A I I I I I I 2d I I A I I I I I A 3 A A A A A A A A A 1c I I I I I I I I A 2c A A A I I I I I I 3a A A A I I I I I I 3b A A A I I I I I I Cytotoxicity of triterpenoids was also related to the difference of hydrophobic values (Figure 2) once descriptors DD4 and DD5 were selected and compounds with higher values than 0.8125 to DD4 (differences of the hydrophobic volumes at energy level of −0.8 kcal/mol) or lower or equal values than 0.25 to DD5 were not cytotoxic against NCTC. DT model predicted 100% of cytotoxicity of the training set and 75% of internal validation (Table 2). Therefore, the cytotoxic compounds displayed Molecules 2014, 19 5767 hydroxyl group at C-3 and Z configuration or absence of double bond between C-24 and C-25 with carboxylic acid at C-27 (compounds 1a, 1b and 3). 3. Experimental 3.1. General Experimental Procedures All solvents used were of analytical grade and purchased from CAAL (São Paulo, Brazil). Silica gel (230–400 mesh, Merck, Darmstadt, Germany) and Sephadex LH-20 (Aldrich, St. Louis, MO, USA) were used for column chromatographic separation, while silica gel 60 F (Merck) was used for 254 analytical TLC (0.25 mm). 1H and 13C spectra were recorded, respectively, at 300 and 75 MHz in a Bruker Ultrashield 300 Advance III spectrometer. CDCl (Aldrich) was used as solvent and the 3 residual peak of the non-deuterated solvent as internal standard. Chemical shifts (δ) are reported in ppm and coupling constant (J) in Hz. ESI-MS were measured with a Platform II mass spectrometer (Micromass, MA, USA), operating in negative mode. 3.2. Plant Material Leaves of S. terebinthifolius were randomly collected from an individual tree at Mogi-Guaçu region (São Paulo State, Brazil) on February/2010 by Dr. Maria Claudia Marx Young from Instituto de Botânica (São Paulo, Brazil), where a reference specimen (SP272591) was deposited. 3.3. Extraction and Isolation of Natural Triterpenoids 1–3 The leaves of S. terebinthifolius were dried at 40 °C during 7 days. After grinding, the plant material (1 kg) was extracted with n-hexane (10 × 1 L) for removal of fatty material. The remaining material was submitted to an exhaustive extraction with EtOH at room temperature using an accelerated solvent extractor system (Dionex ASE-350). Distillation of the solvent under reduced pressure yielded the crude EtOH extract, which was partitioned between EtOH-H O 1:2 (500 mL) and hexane (4 × 250 mL) to afford 2 22 g of n-hexane phase after solvent removal under reduced pressure. Thus, part of the n-hexane phase (20 g) was subjected to fractionation by silica gel column chromatography and eluted with increasing amount of EtOAc in n-hexane to afford 37 fractions (125 mL each). After analysis by TLC, these fractions were pooled into eight groups (A-H). Group E (1,001 mg) was subjected to fractionation on a Sephadex LH-20 column (3 × 50 cm–flow 1.0 mL/min) and eluted with MeOH, yielding 30 fractions (3 mL each), which were pooled into six groups after analysis by TLC (E1 to E6). Groups E2 (310 mg) and E3 (500 mg) consisted of 2 and 1, respectively. Group F (1800 mg) was subjected to separation on a Sephadex LH-20 column (3 × 60 cm–flow 0.7 mL/min) and eluted with MeOH, yielding 40 fractions (3 mL each), which, after monitoring by TLC, were pooled into four groups (C1 to C4). Group C2 (1020 mg) was subjected to fractionation on a Sephadex LH-20 column (3 × 50 cm–flow 1.0 mL/min) eluted with MeOH, yielding 27 fractions (3 mL each), which were pooled into four groups (C2/1 to C-2/4) after analysis by TLC. Purification of group C2/3 (580 mg) by silica gel column chromatography (3 × 40 cm), using increasing amounts of EtOAc in hexane, allowed the isolation of 3 (90 mg). Molecules 2014, 19 5768 3-Oxotirucalla-7,24Z-dien-27-oic acid (Z-masticadienoic acid, 1). Amorphous solid. ESI-MS m/z 453 [M−H]−; 1H-NMR (CDCl ), δ/ppm: 6.02 (t, J = 6.0 Hz, H-24), 5.30 (t, J = 3.0 Hz, H-7), 2.77 (td, 3 J = 15.1 and 6.0 Hz, H-3), 2.56 (m, H-23), 2.27 (m, H-5), 2.23 (m, H-2), 2.09 (m, H-6), 1.97 (m, H-16), 1.90 (s, CH -26), 1.89 (m, H-9), 1.57 (m, H-22), 1.54 (m, H-20), 1.52 (m, H-15), 1.50 (m, 3 H-12), 1.47 (m, H-17), 1.41 (m, H-1), 1.12 (s, CH -28), 1.05 (s, CH -30), 1.00 (s, CH -29), 0.89 (m, 3 3 3 H-11), 0.88 (d, J = 6.0 Hz, CH -21), 0.87 (s, CH -18), 0.77 (s, CH -19). 13C-NMR (CDCl ), δ/ppm: 3 3 3 3 217.1 (C-3), 173.4 (C-27), 146.2 (C-24), 146.1 (C-8), 126.5 (C-25), 117.8 (C-7), 52.9 (C-17), 51.2 (C-14), 48.6 (C-5), 48.4 (C-9), 47.9 (C-4), 43.5 (C-13), 38.5 (C-1), 36.1 (C-20), 35.7 (C-10 and C-15), 35.0 (C-2), 34.1 (C-12), 33.8 (C-22), 28.2 (C-16), 27.4 (C-28), 27.3 (C-29), 26.9 (C-23), 25.4 (C-30), 24.5 (C-6), 20.6 (C-26), 18.3 (C-18), 18.2 (C-11), 18.0 (C-21), 13.0 (C-19). 3-Oxotirucalla-7,24E-dien-27-oic acid (E-masticadienoic acid, 2). Amorphous solid. ESI-MS m/z 453 [M−H]−; 1H-NMR (CDCl ), δ/ppm: 6.06 (t, J = 6.9 Hz, H-24), 5.30 (m, H-7), 2.77 (td, J = 15.0 and 3 6.0 Hz, H-2), 2.56 (m, H-23), 2.24 (m, H-2), 2.10 (m, H-6), 2.09 (m, H-5), 2.03 (m, H-16), 2.00 (m, H-9), 1.92 (s, CH -26), 1.73 (m, H-15), 1.60 (m, H-12), 1.57 (m, H-11), 1.49 (m, H-1), 1.47 (m, H-20), 3 1.42 (m, H-17), 1.30 (m, H-22), 1.12 (s, CH -19), 1.04 (s, CH -29), 1.00 (s, CH -28 and CH -30), 0.89 3 3 3 3 (d, J = 6.0 Hz, CH -21), 0.81 (s, CH -18). 13C-NMR (CDCl ), δ/ppm: 217.1 (C-3), 173.1 (C-27), 145.6 3 3 3 (C-24), 145.8 (C-8), 126.7 (C-25), 117.9 (C-7), 52.2 (C-17), 51.1 (C-14), 52.8 (C-5), 47.8 (C-4), 43.5 (C-13), 48.5 (C-9), 38.5 (C-1), 36.0 (C-20), 35.0 (C-23), 34.9 (C-10), 34.6 (C-2), 34.0 (C-15), 33.6 (C-12), 26.0 (C-22), 28.2 (C-16), 27.4 (C-30), 24.5 (C-28), 24.3 (C-6), 21.9 (C-18), 21.6 (C-29), 18.2 (C-11), 18.1 (C-21), 12.7 (C-19), 11.9 (C-26). 3α-Hydroxytirucalla-7,24Z-dien-27-oic acid (Z-schinol, 3). Amorphous solid. ESI-MS m/z 455 [M−H]−; 1H-NMR (CDCl ), δ/ppm: 6.07 (t, J = 6.3 Hz, H-24), 5.28 (m, H-7), 3.46 (dd, J = 10.2 and 3 5.4 Hz, H-3), 2.50 (m, H-9), 2.46 (m, H-23), 2.05 (s, CH -26), 2.04 (m, H-2), 1.97 (m, H-16), 1.95 (s, 3 CH -30), 1.91 (m, H-5), 1.90 (m, H-6), 1.59 (m, H-12), 1.56 (m, H-11), 1.46 (m, H-1), 1.43 (m, H-15), 3 1.42 (m, H-17), 1.39 (m, H-20), 1.37 (m, H-22), 0.93 (s, CH -28), 0.90 (s, CH -29), 0.89 (d, 3 3 J = 6.0 Hz, CH -21), 0.83 (s, CH -18), 0.77 (s, CH -19). 13C-NMR (CDCl ), δ/ppm: 173.6 (C-27), 3 3 3 3 146.1 (C-24), 146.0 (C-8), 125.7 (C-25), 117.8 (C-7), 76.4 (C-3), 53.3 (C-17), 51.6 (C-14), 48.6 (C-9), 44.5 (C-5), 43.9 (C-13), 37.4 (C-4), 36.4 (C-2), 36.2 (C-1 and C-20), 36.1 (C-22), 35.6 (C-10), 34.7 (C-12), 34.0 (C-15), 28.2 (C-16 and C-28), 27.4 (C-30), 26.9 (C-23), 24.3 (C-6), 22.5 (C-29), 21.4 (C-18), 20.6 (C-26), 18.7 (C-21), 18.5 (C-11), 13.0 (C-19). 3.4. Preparation of Semi-Synthetic Compounds 3.4.1. Reduction of the Carbonyl Group at C-3 (Compounds 1 and 2) To a solution of 1 (100 mg) or 2 (50 mg) dissolved in MeOH (10 mL) was added NaBH (42 mg) in 4 small portions and carefully. The reaction mixture was stirred overnight at room temperature. After addition of H O, the solvent was partially evaporated under reduced pressure. The residue was 2 extracted with EtOAc (3 × 25 mL) and the combined organic layers were dried over Na SO , filtered 2 4 and concentrated. Purification by silica gel chromatography (hexane/EtOAc 7:3) afforded 1a (41 mg) or 2a (46 mg). Molecules 2014, 19 5769 3.4.2. Hydrogenation of Δ24 (Compounds 1, 1a, 2b) In a high-pressure reactor (stainless steel), was added 1 (20 mg) or 1a (20 mg) or 2b (15 mg) and Ni-Raney catalyst (50 mg). After addition of H (20 atm), the mixture was stirred for 3 h at 100 °C. 2 Then, the product was dissolved in CH Cl and the catalyst removed by filtration over a bed of 2 2 Celite. Purification by silica gel chromatography (hexane/EtOAc 9:1) afforded 1c (15 mg) or 1b (14 mg) or 2c (9 mg). 3.4.3. Acetylation of Hydroxyl Group at C-3 (Compounds 2a and 3a) A solution of 2a (50 mg) or 3a (15 mg) was dissolved in pyridine (4 mL) and cooled to 0 °C. Acetic anhydride (2 mL) was added and was stirred overnight at room temperature. Excess of reagents were removed under reduced pressure. After addition of H O (5 mL), the residue was 2 extracted with CH Cl (3 × 25 mL). The organic layer was dried over Na SO , filtered and 2 2 2 4 concentrated. Purification by silica gel chromatography with hexane/EtOAc (7:3) afforded 2b (35 mg) or hexane/EtOAc (95:5) afforded 3b (10 mg). 3.4.4. Methylation of Carboxylic Acid (Compounds 2 and 3) To a solution of KOH (1.7 g) in H O (2.3 mL) and EtOH (8.3 mL) was added a solution of 2 Diazald (7.2 g) dissolved in Et O (80 mL). This mixture was heated and the product distillated to 2 afford ether solution of diazomethane (0.75 g). Immediately, an excess of diazomethane was added to 2 (70 mg) or 3 (25 mg). The organic layer was dried over Na SO , filtered and concentrated under 2 4 reduced pressure. Purification by silica gel chromatography (hexane/EtOAc 9:1) afforded compounds 2d (50 mg) or 3a (21 mg). 3ξ-Hydroxytirucalla-7,24Z-dien-27-oic acid (1a). Amorphous solid. ESI-MS m/z 455 [M−H]−; 13C-NMR (CDCl ), δ/ppm: 169.2 (C-27), 146.5 (C-24), 144.0 (C-8), 127.3 (C-25), 118.5 (C-7), 79.3 3 (C-3), 53.3 (C-17), 51.6 (C-14), 49.1 (C-9), 44.8 (C-5), 43.9 (C-13), 37.7 (C-4), 36.5 (C-20), 36.1 (C-22), 35.1 (C-10), 34.4 (C-15), 34.3 (C-12), 31.7 (C-1), 28.6 (C-16), 28.2 (C-28), 27.5 (C-30), 26.9 (C-2), 26.1 (C-23), 24.3 (C-6), 22.0 (C-29 and C-18), 20.9 (C-26), 18.4 (C-21 and C-11), 13.2 (C-19). 3ξ-Hydroxytirucall-7-en-27-oic acid (1b). Amorphous solid. ESI-MS m/z 457 [M−H]−; 13C-NMR (CDCl ), δ/ppm: 181.5 (C-27), 146.1 (C-8), 117.8 (C-7), 76.3 (C-3), 53.0 (C-17), 51.2 (C-14), 48.6 3 (C-9), 44.5 (C-5), 43.4 (C-13), 39.2 (C-25), 37.4 (C-4), 36.0 (C-20), 35.8 (C-24), 35.0 (C-10), 34.9 (C-22), 34.7 (C-12), 34.0 (C-15), 31.2 (C-1), 28.2 (C-16), 27.7 (C-23 and C-28), 25.3 (C-2), 24.0 (C-6), 21.9 (C-18), 21.8 (C-29), 21.6 (C-30), 18.3 (C-11), 17.9 (C-21), 17.0 (C-26), 12.9 (C-19). 3-Oxotirucall-7-en-27-oic acid (1c). Amorphous solid. ESI-MS m/z 455 [M−H]−; 13C-NMR (CDCl ), 3 δ/ppm: 217.0 (C-3), 182.4 (C-27), 146.0 (C-8), 117.8 (C-7), 53.0 (C-5), 52.3 (C-17), 51.2 (C-14), 48.5 (C-9), 47.9 (C-4), 43.5 (C-13), 38.5 (C-1), 35.8 (C-20 and C-24), 35.7 (C-25), 35.0 (C-10 and C-22), 34.9 (C-2), 34.0 (C-15), 33.7 (C-12), 28.2 (C-16), 27.4 (C-23), 24.5 (C-6 and C-28), 24.1 (C-30), 22.0 (C-18), 21.6 (C-29), 18.3 (C-21 and C-11), 17.0 (C-26), 12.8 (C-19). Molecules 2014, 19 5770 3ξ-Hydroxytirucalla-7,24E-dien-27-oic acid (2a). Amorphous solid. ESI-MS m/z 455 [M−H]−; 13C-NMR (CDCl ), δ/ppm: 173.1 (C-27), 146.0 (C-8), 145.6 (C-24), 126.7 (C-25), 117.8 (C-7), 76.4 3 (C-3), 52.2 (C-17), 51.6 (C-14), 48.6 (C-9), 44.5 (C-5), 43.9 (C-13), 37.4 (C-4), 36.4 (C-2), 36.1 (C-1), 36.0 (C-20), 35.6 (C-10), 35.0 (C-23), 34.7 (C-12), 34.0 (C-15), 28.2 (C-16), 27.4 (C-30), 26.0 (C-22), 24.5 (C-28), 24.3 (C-6), 21.9 (C-18), 21.6 (C-29), 18.5 (C-11), 18.1 (C-21), 12.7 (C-19), 11.9 (C-26). 3ξ-Acetoxytirucalla-7,24E-dien-27-oic acid (2b). Amorphous solid. ESI-MS m/z 497 [M−H]−; 13C-NMR (CDCl ), δ/ppm: 171.1 (C-27), 171.0 (C=O), 146.0 (C-24), 145.7 (C-8), 126.6 (C-25), 117.7 3 (C-7), 81.1 (C-3), 52.8 (C-17), 51.2 (C-14), 50.7 (C-5 and C-9), 43.5 (C-13), 37.8 (C-4), 36.8 (C-20), 36.0 (C-1 and C-22), 34.8 (C-10), 34.6 (C-15), 33.9 (C-12), 28.2 (C-16), 27.2 (C-28), 27.0 (C-23), 24.2 (C-2), 23.7 (C-6), 21.8 (C-18 and C-30), 21.4 (C-29), 21.0 (Me), 18.2 (C-21 and C-11), 13.1 (C-19), 11.9 (C-26). 3ξ-Acetoxytirucall-7-en-27-oic acid (2c). Amorphous solid. ESI-MS m/z 499 [M−H]−; 13C-NMR (CDCl ), 3 δ/ppm: 171.1 (C-27), 170.9 (C=O), 145.9 (C-8), 117.6 (C-7), 81.2 (C-3), 53.0 (C-17), 51.1 (C-14), 50.8 (C-5 and C-9), 43.5 (C-13), 37.8 (C-4 and C-25), 36.0 (C-1 and C-20), 35.9 (C-24), 34.8 (C-10 and C-22), 34.7 (C-15), 34.0 (C-12), 28.2 (C-16), 27.4 (C-28), 27.3 (C-23), 24.2 (C-2), 23.8 (C-6), 21.9 (C-18), 21.4 (C-29), 21.0 (Me), 18.3 (C-11), 18.1 (C-21 and C-30), 17.0 (C-26), 13.1 (C-19). Methyl 3-oxotirucalla-7,24E-dien-27-oate (2d). Amorphous solid. ESI-MS m/z 467 [M−H]−; 13C-NMR (CDCl ), δ/ppm: 216.9 (C-3), 168.6 (C-27), 145.9 (C-8), 144.1 (C-24), 126.5 (C-25), 117.8 (C-7), 52.9 3 (C-5), 52.8 (OMe), 52.3 (C-17), 51.2 (C-14), 48.5 (C-9), 47.9 (C-4), 43.5 (C-13), 38.5 (C-1), 36.0 (C-20), 35.7 (C-23), 35.0 (C-10), 34.9 (C-2), 34.1 (C-15), 33.6 (C-12), 28.2 (C-16), 26.7 (C-22), 27.4 (C-30), 24.5 (C-28), 24.4 (C-6), 22.0 (C-18), 21.6 (C-29), 18.3 (C-21), 18.2 (C-11), 12.8 (C-19 and C-26). Methyl 3α-hydroxytirucalla-7,24Z-dien-27-oate (3a). Amorphous solid. ESI-MS m/z 469 [M−H]−; 13C-NMR (CDCl ), δ/ppm: 168.6 (C-27), 145.8 (C-8), 144.1 (C-24), 126.4 (C-25), 117.8 (C-7), 79.3 3 (C-3), 52.9 (C-5), 52.2 (OMe), 51.1 (C-14), 50.6 (C-17), 48.9 (C-9), 43.6 (C-13), 37.2 (C-4), 36.1 (C-1), 35.7 (C-10 and C-20), 35.6 (C-23), 34.9 (C-2 and C-12), 34.0 (C-15), 28.2 (C-16), 27.7 (C-28), 27.3 (C-30), 26.7 (C-22), 23.9 (C-6), 21.9 (C-18 and C-29), 20.7 (C-26), 18.2 (C-11), 18.1 (C-21), 13.1 (C-19). Methyl 3α-acetoxytirucalla-7,24Z-dien-27-oate (3b). Amorphous solid. ESI-MS m/z 513 [M−H]−; 13C-NMR (CDCl ), δ/ppm: 171.0 (C-27), 168.6 (C=O), 145.9 (C-8), 144.1 (C-24), 126.4 (C-25), 3 117.6 (C-7), 81.1 (C-3), 52.9 (C-5), 52.8 (OMe), 51.2 (C-17 and C-14), 48.8 (C-9), 47.6 (C-4), 43.5 (C-13), 38.1 (C-1), 36.0 (C-20), 35.7 (C-23), 34.8 (C-10 and C-2), 34.0 (C-15), 33.7 (C-12), 28.2 (C-16), 27.6 (C-30), 26.7 (C-22), 24.2 (C-6 and C-28), 21.9 (C-18), 21.3 (C-29), 20.7 (C-26), 20.1 (Me), 18.2 (C-11), 18.1 (C-21), 13.1 (C-19). 3.5. Bioassay Procedures BALB/c mice and Golden hamsters (Mesocricetus auratus) were supplied by the animal breeding facility at the Instituto Adolfo Lutz, São Paulo, Brazil and maintained in sterilized cages under a controlled environment, receiving water and food ad libitum. Animal procedures were performed with