Antiprotozoal Nor-Triterpene Alkaloids from Buxus sempervirens L.

Abstract

Malaria and human African trypanosomiasis (HAT; sleeping sickness) are life-threatening tropical diseases caused by protozoan parasites. Due to limited therapeutic options, there is a compelling need for new antiprotozoal agents. In a previous study, O-tigloylcyclovirobuxeine-B was recovered from a B. sempervirens L. (common box; Buxaceae) leaf extract by bioactivity-guided isolation. This nor-cycloartane alkaloid was identified as possessing strong and selective in vitro activity against the causative agent of malaria tropica, Plasmodium falciparum (Pf). The purpose of this study is the isolation of additional alkaloids from B. sempervirens L. to search for further related compounds with strong antiprotozoal activity. In conclusion, 25 alkaloids were obtained from B. sempervirens L., including eight new natural products and one compound first described for this plant. The structure elucidation was accomplished by UHPLC/+ESI-QqTOF-MS/MS and NMR spectroscopy. The isolated alkaloids were tested against Pf and Trypanosoma brucei rhodesiense (Tbr), the causative agent of East African sleeping sickness. To assess their selectivity, cytotoxicity against mammalian cells (L6 cell line) was tested as well. Several of the compounds displayed promising in vitro activity against the pathogens in a sub-micromolar range with concurrent high selectivity indices (SI). Consequently, various alkaloids from B. sempervirens L. have the potential to serve as a novel antiprotozoal lead structure.

Identification of Antiprotozoal Compounds from Buxus sempervirens L. by PLS-Prediction

“…This might suggest that the antitrypanosomal activity of these four compounds could be due to an unspecific cytotoxic effect rather than a selective impact on the parasites. However, the cytotoxicity of compound 11 (3.55 min: 415.375 m/z) against the same L6 cell line was already tested in our previous study [7] and it was much lower than the antitrypanosomal activity (IC50 value against Tbr of 1.5 µM vs. 35.5 µM for cytotoxicity against L6 cells; SI: 24). With some caution, this result may probably be extrapolated to the other three compounds dominating the PLS model for cytotoxicity.…”

“…Cyclomicrophylline-A (4) (3.09 min: 445.388 m/z) was also found as an active compound in the model against Pf (see Section 2.2.2 above, Figure 2, Table 3) and had already demonstrated activity in the in vitro assay against Tbr with an IC 50 value of 2.3 µM [7]. In addition, compound 11 (3.55 min: 415.375 m/z) could be assigned (Figures S28-S30, Supplementary Materials) to cyclovirobuxeine-B, which had also been isolated and displayed promising antitrypanosomal activity (IC 50 : 1.5 µM) in our previous study [7]. These findings, as in the case of the antiplasmodial model above, corroborate the validity of the calculated model.…”

“…The finding of these two compounds with already proven antiplasmodial activity can be taken as evidence for the validity of the calculated PLS model. Based on the MS chromatograms and the fragmentation pattern, as well as by identity of the retention times with samples of the authentic, isolated alkaloids [7], com-pounds 1 (4.98 min: 497.416 m/z) (Figures S2 and S3, Supplementary Materials) and 4 (3.09 min: 445.388 m/z) (Figures S9-S11, Supplementary Materials) were identified as Otigloylcyclovirobuxeine-B (1) and cyclomicrophylline-A (4), respectively, both isolated in our previous study and proven to be highly active against Pf in the in vitro assay (IC 50 : 1.05 µM (1); IC 50 : 1.76 µM (4)) [7]. The finding of these two compounds with already proven antiplasmodial activity can be taken as evidence for the validity of the calculated PLS model.…”

“…The structure, thus determined from the mass spectral data (Figure 2), could not be found in the literature, so that we assume, to the best of our knowledge, that compound 2 is a new natural product. It should be noted that the stereochemistry of 2, which is not accessible on grounds of the mass spectra alone, is assumed in analogy with related compounds of known configuration (e.g., reported in [7]). The generic name of the new compound, O-tigloylcyclomicrophylline-D, was chosen based on the existing classification for Buxus-alkaloids [15,16].…”

“…In our previous study, nor-triterpene alkaloids of the nor-cycloartane type from B. sempervirens L. displayed promising in vitro antiprotozoal activity and selectivity [7]. In the mentioned study, 25 alkaloids were isolated in a systematic manner from the aerial parts of B. sempervirens and tested for their antiprotozoal activity; several constituents with high activity against Pf and/or Tbr were isolated besides compounds with medium or low levels of activity.…”