## 6 Biological Activity of the Hapalindole-Type Alkaloids
### Biochemical, Molecular and Cellular Bioactivity  
Aside from their antimicrobial activity, subsequent studies have suggested a wide range of biological activity at the biochemical, molecular and cellular level that is, likewise, relevant to both the potential of the hapalindole-type alkaloids as drug leads, and to their possible role as toxins. At the cellular level, several of the indole alkaloids have been shown to be cytotoxic. In addition to antimicrobial activity, several of the hapalindoles (including A, C, E, H-J, U and X) were shown to be cytotoxic to both normal mammalian (_i.e._, Vero) and various cancer cell lines [34; 62]. Similar to antimicrobial activity, tetracyclic hapalindole A and J--and the unusual X--were particularly active in these studies, as was hapalindole H (although it was not comparatively tested for antimicrobial activity in the same study), whereas others (_i.e._, I and U) were only moderately and/or variably cytotoxic [34]. The difference in cytotoxicity between, for example, hapalindole H and U is perhaps notable as they differ only in relative stereochemistry at C-10/11/15, and suggest a stereoselectivity of this biological activity. Similarly, in a prior study, the tricyclic variant, 12-_epi_-hapalindole E, was found to be moderately cytotoxic to a mouse myeloma cell line [62]. In addition, some oxidized hapalindoles (e.g., hapalonamide H, anhydrohapaloxindole A) shared cytotoxicity equivalent to the hapalindoles, whereas others (_i.e._, 13-hydroxydechlorofontonamide) did not [34]. This finding is intriguing as both active and inactive variants (e.g., hapalonamide H and 13-hydroxydechlorofontonamide, respectively) include representatives which lack an intact indole ring. Both tetra- and pentacyclic ambiguine isonitriles including numerous congeners (A-C, E, F, I, K-O), as well as the unique ambiguine G nitrile, were also found to be cytotoxic to a mammalian cell line [37; 42], whereas ambiguine D isonitrile was shown to inhibit mitosis in plant cells [66]. In a recent study, it was specifically shown that ambiguine I isonitrile induced apoptosis in the MCF-7 breast cancer cell line, and led to arrest of cells in G1 phase [65]. Notably, the chemically related fischambiguines A and B were effectively not found to be cytotoxic ([PERSON] _et al._, 2010), although further investigation is clearly needed. Interestingly, however, given the considerable deviation in carbon backbone, the fischerinololes were found to be cytotoxic, and fischerinolde L, in particular, was shown to be as equally cytotoxic (to both normal and cancer cells) as hapalindole-type alkaloids [44]. Arguably, the sub-class which has received the most attention in terms of cytotoxicity, particularly in relation to potential as anticancer drug, has been the welwitindolinones, and specifically welwitindolinone C isothicyanate and its \(N\)-methyl derivative--or _welwistatin_--that were found to inhibit cell proliferation in various cancer cell lines, and moreover, to circumvent (as discussed below) multidrug resistance [67; 68]. As a result of this compelling biological activity, considerable attention has been devoted, in particular, to synthetic studies of the welwistatin and other welwitindolinones [47]. With respect to biochemical or molecular underpinnings of the pharmacology of the hapalindole-type alkaloids, several possible mechanisms have, in fact, been identified (Figure 16). In a study of ambiguine I, for example, it was shown that observed induction of apoptosis and cell-cycle arrest could be specifically attributed to the inhibition of pathways involving the transcription factor, NF-\(\kappa\)B, which has a well-established role in regulating multiple pathways involved in cancer progression including suppression of apoptosis [65]. It was generally shown in these studies that apoptosis occurred by way of a caspase-independent mechanism, and involved mitochondrial dysfunction including elevated levels of reactive oxygen species (ROS).