Antiplasmodial and Cytotoxic Activity of Natural Bisbenzylisoquinoline Alkaloids Cindy K. Angerhofer, † He ´le `ne Guinaudeau,* ,‡ Varima Wongpanich, † John M. Pezzuto, † and Geoffrey A. Cordell † Program for Collaborative Research in the Pharmaceutical Sciences, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, College of Pharmacy, Chicago, Illinois, and Department of Pharmacognosy, Faculty of Pharmacy, University of Angers, France Received April 13, 1998 As part of an ongoing collaborative effort to discover new antimalarial agents from natural sources, we have tested 53 bisbenzylisoquinoline alkaloids for cytotoxicity against cultured mammalian cells and for antiplasmodial activity against chloroquine-sensitive and chloroquine-resistant clones of Plasmodium falciparum. The isolates from Cyclea barbata, Stephania pierrei, Stephania erecta, Pachygone dasycarpa, Cyclea atjehensis, Hernandia peltata, Curare candicans, Albertisia papuana, and Berberis valdiviana exhibited a wide range of biological potencies in antiplasmodial assays, and the majority exhibited some degree of cytotoxicity against human KB cells. More than half of the compounds tested, however, showed selective antiplasmodial activity, with >100-fold greater toxicity toward one or both of the P. falciparum clones, relative to cultured mammalian cells. The most selective alkaloids were (-)-cycleanine (40), (+)- cycleatjehine (50), (+)-cycleatjehenine (49), (+)-malekulatine (3), (-)-repandine (13), and (+)-temuconine (2). As a result of these studies, relationships between the structures, the stereochemistry, and the substitution patterns of these alkaloids and their in vitro antiplasmodial and cytotoxic activities are beginning to emerge. Bisbenzylisoquinolines are a large and diverse group of natural alkaloids that occur in many plant species, par- ticularly in members of the Menispermaceae, Berberi- daceae, Ranunculaceae, Annonaceae, and Monimiaceae. 1-4 Many of the plants that contain these compounds enjoy a folkloric reputation as medicinals in various cultures. 1-4 Recently, bisbenzylisoquinoline alkaloids have been widely demonstrated to possess a number of interesting and potent biological activities, including cytotoxicity and/or antiplas- modial activity. 1-4 Classically, these dimers can be divided into three categories: biscoclaurines, coclaurine-reticu- lines, and bisreticulines. The two moieties are usually bound by one diaryl ether bridge or more, although carbon-carbon bridges or a methylene-oxy bridge may be present. The bisbenzylisoquinoline alkaloids are classified according to the nature, the number, and the attachment point of the bridges. In each subgroup, the alkaloids differ by the nature of their oxygenated substituents, the degree of unsaturation of the heterocyclic rings, and the stereo- chemistry of their two chiral centers, C-1 and C-1′. The diversity of pharmacological effects observed within this group of molecules is obviously a function of differences in chemical structures; however, convincing structure-activ- ity relationships had not been developed previously for the bisbenzylisoquinoline alkaloids. Over the past several years, we have explored this class of complex alkaloids in considerable spectroscopic detail, as well as evaluating their biologic potential to serve as new antimalarial agents. Through bioassay-directed frac- tionation, we have isolated a variety of known and novel bisbenzylisoquinoline alkaloids from several plants in the Menispermaceae, including Stephania erecta, 5 Stephania pierrei, 6 Cyclea barbata, 7-9 and Pachygone dasycarpa. 10 In addition, a number of bisbenzylisoquinoline alkaloids that had been isolated in the course of phytochemical studies of other species, such as Cyclea atjehensis 11,12 (Guinaudeau and Ovono, unpublished result), Curare candicans, 13 Coc- culus pendulus (Menispermaceae), 14 Hernandia peltata (Hernandiaceae), 15 and Berberis valdiviana (Berberidace- ae) 16 were available for investigation. Applying identical methodologies, these compounds have been analyzed for cytotoxicity toward mammalian cells, as well as for anti- plasmodial activity with chloroquine-sensitive and chloro- quine-resistant, mefloquine-sensitive clones of Plasmodium falciparum. This approach is used to determine antima- larial potency as well as selectivity. Compilation of the results obtained with 53 bisbenzylisoquinoline alkaloids has facilitated the analysis of structure-activity relation- ships wherein the goal is to define the structural features that might be responsible for selective antiplasmodial activity. Results and Discussion Of the bisbenzylisoquinoline alkaloids examined in this study (1-53), only three, (-)-isocuricycleatjine (45), (-)- dehydroisocuricycleatjenine (47), and (+)-tubocurarine chlo- ride (48) failed to show significant in vitro antiplasmodial activity against either of the P. falciparum clones tested (Table 1). Seven additional compounds (15, 19, 38, 43, 44, 46, 53) exhibited weak activity, with antiplasmodial IC 50 values of 1000-2400 nM in at least one of the clones. The remaining 43 compounds were determined to have IC 50 values of <1000 nM against both D6 and W2 clones, and of these, 27 demonstrated potent activity of <200 nM with at least one of the clones. To further analyze the antima- larial potential of these bisbenzylisoquinoline alkaloids, all compounds were evaluated for cytotoxicity with human epidermoid carcinoma (KB) cells. Many compounds have been reported in the literature as “antimalarials” on the basis of in vitro data against malarial parasites. Although completely valid, these data * To whom correspondence should be addressed: Department of Phar- macognosy, Faculty of Pharmacy, University of Angers, 16 boulevard Danviers, 49045 Angers Cedex, France. Tel.: 33(0) 2 41 22 66 63. Fax: 33- (0) 2 41 48 67 33. † University of Illinois at Chicago. ‡ University of Angers, France. 59 J. Nat. Prod. 1999, 62, 59-66 10.1021/np980144f CCC: $18.00 © 1999 American Chemical Society and American Society of Pharmacognosy Published on Web 10/31/1998