Part of the thesis of J.B.A. A preliminary account on part of this work has been published in abstract form (Planta Med. 2013, 79, 1129. ‡ This work is cordially dedicated to H. M. Schmidt, Havixbeck, the keeper and co-owner of the Buxus hedge under study, on the occasion of her 50th birthday. Abstract: Buxus sempervirens L. (European Box, Buxaceae) has been used in ethnomedicine to treat malaria. In the course of our screening of plant extracts for antiprotozoal activity, a CH 2 Cl 2 extract from leaves of B. sempervirens showed selective in vitro activity against Plasmodium falciparum (IC 50 = 2.79 vs. 20.2 µg/mL for cytotoxicity against L6 rat cells). Separation of the extract by acid/base extraction into a basic and a neutral non-polar fraction led to a much more active and even more selective fraction with alkaloids while the fraction of non-polar neutral constituents was markedly less active than the crude extract. Thus, the activity of the crude extract could clearly be attributed to alkaloid constituents. Identification of the main triterpene-alkaloids and characterization of * OPEN ACCESSMolecules 2014, 19 6185 the complex pattern of this alkaloid fraction was performed by UHPLC/+ESI-QTOF-MS analyses. ESI-MS/MS target-guided larger scale preparative separation of the alkaloid fraction was performed by 'spiral coil-countercurrent chromatography'. From the most active subfraction, the cycloartane alkaloid O-tigloylcyclovirobuxeine-B was isolated and evaluated for antiplasmodial activity which yielded an IC 50 of 0.455 µg/mL (cytotoxicity against L6 rat cells: IC 50 = 9.38 µg/mL). O-tigloylcyclovirobuxeine-B is thus most significantly responsible for the high potency of the crude extract.

Abstract:In the course of our ongoing screening of plants of the family Asteraceae for antiprotozoal activity, a CH 2 Cl 2 -extract from the flowering aerial parts of Achillea ptarmica L. (sneezewort yarrow) was found to be active in vitro against Trypanosoma brucei rhodesiense (IC 50 = 0.67 µg/mL) and Plasmodium falciparum (IC 50 = 6.6 μg/mL). Bioassay guided fractionation led to the isolation and identification of five alkamides from the most active fractions. Pellitorine and 8,9-Z-dehyropellitorine are the main components of the extract. Beside these olefinic acid amides, four alkamides with diene-diyne structures were isolated. All alkamides were tested for antiprotozoal activity in vitro. Pellitorine was the most active compound so far within this study against P. falciparum (IC 50 = 3.3 µg/mL), while 8,9-Z-dehydropellitorine was most active against T. b. rhodesiense (IC 50 = 2.0 µg/mL). The activity of pure pellitorine against Plasmodium is higher than that of the crude extract and thus explains the activity of the latter. None of the isolated alkamides, however, was as active against T. b. rhodesiense as the crude extract whose antitrypanosomal activity must therfore be due to a synergistic effect of the isolated compounds or to more active yet to be identified constituents.

As part of our research for new leads against human African trypanosomiasis (HAT), we report on a 3D-QSAR study for antitrypanosomal activity and cytotoxicity of aminosteroid-type alkaloids recently isolated from the African medicinal plant Holarrhena africana A. DC. (Apocynaceae), some of which are strong trypanocides against Trypanosoma brucei rhodesiense (Tbr), with low toxicity against mammalian cells. Fully optimized 3D molecular models of seventeen congeneric Holarrhena alkaloids were subjected to a comparative molecular field analysis (CoMFA). CoMFA models were obtained for both, the anti-Tbr and cytotoxic activity data. Model performance was assessed in terms of statistical characteristics (R2, Q2, and P2 for partial least squares (PLS) regression, internal cross-validation (leave-one-out), and external predictions (test set), respectively, as well as the corresponding standard deviation error in prediction (SDEP) and F-values). With R2 = 0.99, Q2 = 0.83 and P2 = 0.79 for anti-Tbr activity and R2 = 0.94, Q2 = 0.64, P2 = 0.59 for cytotoxicity against L6 rat skeletal myoblasts, both models were of good internal and external predictive power. The regression coefficients of the models representing the most prominent steric and electrostatic effects on anti-Tbr and for L6 cytotoxic activity were translated into contour maps and analyzed visually, allowing suggestions for possible modification of the aminosteroids to further increase the antitrypanosomal potency and selectivity. Very interestingly, the 3D-QSAR model established with the Holarrhena alkaloids also applied to the antitrypanosomal activity of two aminocycloartane-type compounds recently isolated by our group from Buxus sempervirens L. (Buxaceae), which indicates that these structurally similar natural products share a common structure–activity relationship (SAR) and, possibly, mechanism of action with the Holarrhena steroids. This 3D-QSAR study has thus resulted in plausible structural explanations of the antitrypanosomal activity and selectivity of aminosteroid- and aminocycloartane-type alkaloids as an interesting new class of trypanocides and may represent a starting point for lead optimization.

In our ongoing study to evaluate the antiprotozoal activity of alkamides from Asteraceae, a dichloromethane extract from the roots of Anacyclus pyrethrum L. showed a moderate in vitro activity against the NF54 strain of Plasmodium falciparum and against Leishmania donovani (amastigotes, MHOM/ET/67/L82 strain). Seven pure alkamides and a mixture of two further alkamides were isolated by column chromatography followed by preparative high performance liquid chromatography. The alkamides were identified by mass- and NMR-spectroscopic methods as tetradeca-2E,4E-dien-8,10-diynoic acid isobutylamide (anacycline, 1), deca-2E,4E-dienoic acid isobutylamide (pellitorine, 2), deca-2E,4E,9-trienoic acid isobutylamide (3), deca-2E,4E-dienoic acid 2-phenylethylamide (4), undeca-2E,4E-dien-8,10-diynoic acid isopentylamide (5), tetradeca-2E,4E,12Z-trien-8,10-diynoic acid isobutylamide (6), and dodeca-2E,4E-dien acid 4-hydroxy-2-phenylethylamide (7). Two compounds—undeca-2E,4E-dien-8,10-diynoic acid 2-phenylethylamide (8) and deca-2E,4E-dienoic acid 4-hydroxy-2-phenylethylamide (9)—were isolated as an inseparable mixture (1:4). Compounds 3, 4, and 5 were isolated from Anacyclus pyrethrum L. for the first time. While compounds 4 and 5 were previously known from the genus Achillea, compound 3 is a new natural product, to the best of our knowledge. All isolated alkamides were tested in vitro for antiprotozoal activity against Plasmodium falciparum, Trypanosoma brucei rhodesiense, Trypanosoma cruzi, and Leishmania donovani and for cytotoxicity against L6 rat skeletal myoblasts.

As part of our research for new leads against human African trypanosomiasis (HAT), we report on a 3D-QSAR study for antitrypanosomal activity and cytotoxicity of aminosteroid-type alkaloids recently isolated from the African medicinal plant Holarrhena africana A. DC. (Apocynaceae), some of which are strong trypanocides against Trypanosoma brucei rhodesiense (Tbr) with low toxicity against mammalian cells. Fully optimized 3D molecular models of seventeen congeneric Holarrhena alkaloids were subjected to a comparative molecular field analysis (CoMFA). CoMFA models were obtained for both, the anti-Tbr and cytotoxic activity data. Model performance was assessed in terms of statistical characteristics (R2, Q2 and P2 for partial least squares (PLS) regression, internal cross-validation (leave-one-out) and external predictions (test set), respectively, as well as the corresponding SDEP and F-values). With R2=0.99, Q2=0.83 and P2=0.79 for anti-Tbr activity and R2=0.94, Q2=0.64, P2=0.59 for cytotoxicity against L6 rat skeletal myoblasts, both models were of good internal and external predictive power. The regression coefficients of the models representing the most prominent steric and electrostatic effects on anti-Tbr and for L6 cytotoxic activity were translated into contour maps and analyzed visually, allowing suggestions for possible modification of the aminosteroids to further increase the antitrypanosomal potency and selectivity. Very interestingly, the 3D-QSAR model established with the Holarrhena alkaloids also applied to the antitrypanosomal activity of two aminocycloartane-type compounds recently isolated by our group from Buxus sempervirens L. (Buxaceae), which indicates that these structurally similar natural products share a common SAR and, possibly, mechanism of action with the Holarrhena steroids. This 3D-QSAR study has thus resulted in plausible structural explanations of the antitrypanosomal activity and selectivity of aminosteroid- and aminocycloartane-type alkaloids as an interesting new class of trypanocides and may represent a starting point for lead optimization.