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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 _{50} values in the range between 15 and 58 µg/mL. Four compounds demonstrated selectivity towards the intracellular amastigotes of _{50} values in the range between 28 and 97 µg/mL. The complete characterization of 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.

Specimens of

Previous chemical studies with leaves extracts of

Leishmaniasis and Chagas’ disease are parasitic diseases caused by the protozoans

^{13}C-NMR by the comparison of respective spectral data with those recorded for ^{13}C-NMR spectra of derivatives ^{2}^{4}. Finally, structures of methyl esters ^{13}C-NMR spectra.

Structures of natural compounds

The antileishmanial and antitrypanosomal activities of three natural and nine semi-synthetic derivatives of tirucallane triterpenoids were evaluated against _{50} values in the range of 15.75 to 58.36 µg/mL (

Antiparasitic (antileishmanial and antitrypanosomal) and cytotoxic effects of natural compounds

IC_{50} (g/mL) ^{a} CI95% |
CC_{50} (μg/mL) ^{b} CI95% |
SI | ||||

Compounds | NCTC | AMA^{c} |
TRY^{d} |
|||

Promastigotes | Amastigotes | Trypomastigotes | ||||

NA | 66.51 (47.68–92.79) | NA | >200 | >3 | – | |

NA | NA | 20.18 (16.70–24.39) | 69.50 (64.01–75.45) | – | 3.4 | |

NA | NA | 17.64 (15.97-19.50) | 76.39 (70.02-83.33) | – | 4.3 | |

NA | NA | NA | >200 | – | – | |

NA | 64.90 (41.48–101.50) | 15.75 (9.80–25.30) | 96.48 (77.38–120.30) | 1.5 | 6.1 | |

NA | 97.59 (89.82–106.00) | 29.59 (25.61–34.18) | 95.49 (65.22–139.80) | 1.0 | 3.2 | |

NA | NA | 58.36 (42.82–79.55) | 69.31 (38.74–82.64) | – | 1.1 | |

NA | NA | 49.20 (41.69–58.05) | 57.78 (56.10–59.52) | – | 1.2 | |

NA | NA | NA | >200 | – | – | |

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 | |

NA | NA | NA | >200 | – | – | |

NA | NA | NA | >200 | – | – | |

6.87 | 7.25 | – | 49.72 | – | – | |

– | – | 114.68 | – | – | – |

IC_{50}: 50% inhibitory concentration; CC_{50}: 50% cytotoxic concentration (mammalian cells); NA: not active_{50} mammalian cells/IC_{50} _{50} mammalian cells/IC_{50} trypomastigotes).

Despite the lack of activity of compound _{50} values of 20.18 and 17.64 µg/mL, respectively. In addition, an enhanced mammalian toxicity was also observed. Otherwise, compound _{50} 15.75 µg/mL), demonstrated a higher activity when compared to the semi-synthetic derivatives _{50} values between 29.59 and 58.36 µg/mL. Considering the 95% confidence intervals, the mammalian toxicity of _{50} value of 16.28 µg/mL against trypomastigotes, also rendered less effective derivatives after methylation at C-27 (_{50} value of 114.68 µg/mL [_{50}/IC_{50}), compounds

Among the twelve tested compounds, only the natural prototype _{50} value of 57.82 µg/mL. The modifications of the semi-synthetic tirucallane triterpenoid derivatives (compounds _{50} values in the 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 [_{50} values of 96 and 69 µg/mL, respectively. Considering the selectivity index, compound _{50}) of 49.72 µg/mL and resulted in a SI of 7. Miltefosine was used as a standard drug against promastigotes and amastigotes, with IC_{50} values of 6.87 and 7.25 µg/mL, respectively [

The decision tree (DT) model (

Triterpenoids ^{24}, 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

Decision Trees (DT) generated for the set of triterpenoids with antiparasitic activity. (

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).

NCTC | |||||||||
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Compounds | Experimental | Train | Validation | Experimental | Train | Validation | Experimental | Train | Validation |

I | I | I | A | A | A | I | I | I | |

A | A | A | A | A | A | I | I | I | |

A | A | A | A | I | I | I | I | I | |

A | A | A | I | A | A | A | A | A | |

A | A | A | I | I | I | A | A | I | |

A | A | A | I | I | I | I | I | I | |

I | I | A | I | I | I | I | I | A | |

A | A | A | A | A | A | A | A | A | |

I | I | I | I | I | I | I | I | A | |

A | A | A | I | I | I | I | I | I | |

A | A | A | I | I | I | I | I | I | |

A | A | A | I | I | I | I | I | I |

Cytotoxicity of triterpenoids was also related to the difference of hydrophobic values (

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_{254} (Merck) was used for analytical TLC (0.25 mm). ^{1}H and ^{13}C spectra were recorded, respectively, at 300 and 75 MHz in a Bruker Ultrashield 300 Advance III spectrometer. CDCl_{3} (Aldrich) was used as solvent and the residual peak of the non-deuterated solvent as internal standard. Chemical shifts (δ) are reported in ppm and coupling constant (

Leaves of

The leaves of _{2}O 1:2 (500 mL) and hexane (4 × 250 mL) to afford 22 g of

^{−}; ^{1}H-NMR (CDCl_{3}), δ/ppm: 6.02 (t, _{3}-26), 1.89 (m, H-9), 1.57 (m, H-22), 1.54 (m, H-20), 1.52 (m, H-15), 1.50 (m, H-12), 1.47 (m, H-17), 1.41 (m, H-1), 1.12 (s, CH_{3}-28), 1.05 (s, CH_{3}-30), 1.00 (s, CH_{3}-29), 0.89 (m, H-11), 0.88 (d, _{3}-21), 0.87 (s, CH_{3}-18), 0.77 (s, CH_{3}-19). ^{13}C-NMR (CDCl_{3}), δ/ppm: 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).

^{−}; ^{1}H-NMR (CDCl_{3}), δ/ppm: 6.06 (t, _{3}-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), 1.42 (m, H-17), 1.30 (m, H-22), 1.12 (s, CH_{3}-19), 1.04 (s, CH_{3}-29), 1.00 (s, CH_{3}-28 and CH_{3}-30), 0.89 (d, _{3}-21), 0.81 (s, CH_{3}-18). ^{13}C-NMR (CDCl_{3}), δ/ppm: 217.1 (C-3), 173.1 (C-27), 145.6 (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).

^{−}; ^{1}H-NMR (CDCl_{3}), δ/ppm: 6.07 (t, _{3}-26), 2.04 (m, H-2), 1.97 (m, H-16), 1.95 (s, CH_{3}-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), 1.42 (m, H-17), 1.39 (m, H-20), 1.37 (m, H-22), 0.93 (s, CH_{3}-28), 0.90 (s, CH_{3}-29), 0.89 (d, _{3}-21), 0.83 (s, CH_{3}-18), 0.77 (s, CH_{3}-19). ^{13}C-NMR (CDCl_{3}), δ/ppm: 173.6 (C-27), 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).

To a solution of _{4} (42 mg) in small portions and carefully. The reaction mixture was stirred overnight at room temperature. After addition of H_{2}O, the solvent was partially evaporated under reduced pressure. The residue was extracted with EtOAc (3 × 25 mL) and the combined organic layers were dried over Na_{2}SO_{4}, filtered and concentrated. Purification by silica gel chromatography (hexane/EtOAc 7:3) afforded

In a high-pressure reactor (stainless steel), was added _{2} (20 atm), the mixture was stirred for 3 h at 100 °C. Then, the product was dissolved in CH_{2}Cl_{2} and the catalyst removed by filtration over a bed of Celite. Purification by silica gel chromatography (hexane/EtOAc 9:1) afforded

A solution of _{2}O (5 mL), the residue was extracted with CH_{2}Cl_{2} (3 × 25 mL). The organic layer was dried over Na_{2}SO_{4}, filtered and concentrated. Purification by silica gel chromatography with hexane/EtOAc (7:3) afforded

To a solution of KOH (1.7 g) in H_{2}O (2.3 mL) and EtOH (8.3 mL) was added a solution of Diazald (7.2 g) dissolved in Et_{2}O (80 mL). This mixture was heated and the product distillated to afford ether solution of diazomethane (0.75 g). Immediately, an excess of diazomethane was added to _{2}SO_{4}, filtered and concentrated under reduced pressure. Purification by silica gel chromatography (hexane/EtOAc 9:1) afforded compounds

^{−}; ^{13}C-NMR (CDCl_{3}), δ/ppm: 169.2 (C-27), 146.5 (C-24), 144.0 (C-8), 127.3 (C-25), 118.5 (C-7), 79.3 (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).

^{−}; ^{13}C-NMR (CDCl_{3}), 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 (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).

^{−}; ^{13}C-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).

^{−}; ^{13}C-NMR (CDCl_{3}), δ/ppm: 173.1 (C-27), 146.0 (C-8), 145.6 (C-24), 126.7 (C-25), 117.8 (C-7), 76.4 (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).

^{−}; ^{13}C-NMR (CDCl_{3}), δ/ppm: 171.1 (C-27), 171.0 (C=O), 146.0 (C-24), 145.7 (C-8), 126.6 (C-25), 117.7 (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).

^{−}; ^{13}C-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).

^{−}; ^{13}C-NMR (CDCl_{3}), 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 (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).

^{−}; ^{13}C-NMR (CDCl_{3}), δ/ppm: 168.6 (C-27), 145.8 (C-8), 144.1 (C-24), 126.4 (C-25), 117.8 (C-7), 79.3 (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).

^{−}; ^{13}C-NMR (CDCl_{3}), δ/ppm: 171.0 (C-27), 168.6 (C=O), 145.9 (C-8), 144.1 (C-24), 126.4 (C-25), 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).

BALB/c mice and Golden hamsters (

Isolated promastigotes of _{2}-humidified incubator [

Peritoneal macrophages were collected from the peritoneal cavity of female BALB/c mice by washing with RPMI-1640 without phenol red, supplemented with 10% FBS. NCTC (clone 929) cells were maintained in RPMI-1640 (without phenol red and supplemented with 10% FBS) at 37 °C in a humidified atmosphere containing 5% CO_{2} [

To determine the 50% inhibitory concentration (IC_{50}) against ^{6}/well with a final volume of 150 μL. The tested compounds were incubated in a range concentration of 100 to 0.78 μg/mL for 48 h at 24 °C. The viability of promastigotes was verified by motility and morphology using light microscopy and by the colorimetric MTT assay [_{50} values lower than 100 µg/mL were considered not active [

Peritoneal macrophages were obtained as described previously and ^{5} cells per well in Nunc™ 16-well slide chambers (Aldrich, St. Louis, MO, USA) for 24 h at 37 °C in a 5% CO_{2}-humidified incubator. Amastigotes were added to macrophages at 10:1 ratio (amastigotes:macrophage) and incubated for 24 h. Non-internalized parasites were removed by washing twice with medium and the cells were then incubated with compounds in a range concentration of 100 to 0.78 μg/mL for 120 h at 37 °C in 5% CO_{2}, using miltefosine as standard drug. At the end of the assay, the cells were fixed in methanol, stained with Giemsa and observed under a light microscope to determine the number of infected macrophages out of 400 cells [

Compounds _{50}) as described above for the antileishmanial assay. Free trypomastigotes obtained from LLC-MK2 cultures were counted in a Neubauer hemocytometer and seeded at 1 × 10^{6}/well in 96-well microplates. The tested compounds were incubated in a range concentration of 100 to 0.78 μg/mL for 24 h at 37 °C in a 5% CO_{2} humidified incubator, using benznidazole as standard drug. The viability of the trypomastigotes was verified by the MTT assay as described above [

The 50% cytotoxic concentration (CC_{50}) was determined in NCTC clone 929 cells. NCTC cells were seeded at 6 × 10^{4} cells/well in 96-well microplates at 37 °C in a 5% CO_{2}. The mammalian cells were incubated with tested compounds to the highest concentration of 200 μg/mL for 48 h at 37 °C, using miltefosine as standard drug. The viability of the cells was determined by MTT assay at 570 nm [_{50} NCTC cells/IC_{50} parasites.

The data obtained represent the mean and standard deviation of duplicate samples from at least three independent assays. The IC_{50 }and CC_{50 }values were calculated using sigmoid dose-response curves in Graph Pad Prism 5.0 software (GraphPad Software, San Diego, CA, USA).

The structures were drawn using the Marvin Sketch v. 6.1.4 [

Knime 2.7.1 software (KNIME 2.7.1 the Konstanz Information Miner Copyright, 2003–2013) [

The results presented herein indicate that natural tirucallane triterpenoids

The authors would like to thank the CNPq (300546/2012-2 and 471458/2012-0), FAPESP (2011/51739-0, 2012/18756-1 and 2013/16320-4) and FAPEMIG for providing financial support and fellowships. The authors are grateful to Diogo Oliveira-Silva (UNIFESP) for the ESI-MS measurements. This publication is part of the activities of the Research Network Natural Products against Neglected Diseases (ResNetNPND):

Conceived and designed the experiments: AGT, MTS, JHGL. Performed the experiments: TRM, TACS, AGT, SETB, MTS, RMFS, ACCA, AO, JHGL. Analyzed the data: TRM, TACS, AGT, SETB, MTS, RMFS, AO, SALM, PS, JHGL. Contributed reagents/materials/analysis tools: AGT, SETB, MTS, AO, SALM, PS, JHGL. Wrote the paper: AGT, MTS, JHGL.

The authors declare no conflict of interest.