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(NADH): ubiquinone oxidoreductase in complex I, which is a membrane-bound protein of the mitochondrial electron transport system13-15 and the ubiquinone-linked NADH oxidase in the plasma membranes of cancerous cells.17 Annonaceous acetogenins are now considered as the most potent, effective in nanomolar concentrations, among the diverse inhibitors of mitochondrial complex I.16,122,138 While no work has been conducted on the molecular inhibitory mechanisms of these compounds, the work of Shimada et al.43,44 and Miyoshi et al.45 using liposomal membranes and the submitochondrial vesicle particles, respectively, may, on one hand, explain and verify our structure–activity relationship profiles and, on the other hand, drastically challenge our understanding of the significance of the relative and absolute stereochemistries of the THF or THP ring systems. Studying the conformations of the Annonaous acetogenins within liposomes made of dimyristoylphosphatidylcholine (DMPC) and on the basis of 1H NMR intermolecular nuclear Overhauser effects (NOEs), differential calorometric scanning data, and DMPC–Mn2+ peak-broadening studies, Shimada et al. found that the Annonaceous acetogenins, containing either mono-, adjacent bis-, or nonadjacent bis-THF ring moieties, have their THF ring moieties residing at the interfacial regions of the lipid membranes.43,44 It was concluded that the THF group-(s) serve as a hydrophilic anchor in the lipid membranes. It was also found that the position of the THF ring anchor along the acetogenin chain determines the depths of penetration of the lactone functional group within the lipid bilayer. Thus, the lactone ring, tethered to spacer moieties of different lengths, penetrates the lipid membrane to different depths, acts directly with the protein receptor site-(s), and adapts to the geometry of specific cell types. This would seem to explain cell type selectivities as seen with several of these compounds.34

In a study to determine the essential structural factors, Miyoshi et al. used 22 representative acetogenin compounds to conduct a mechanistic and structural activity study against submitochondrial particles.45 They also studied the three-dimensional structure of the bis-THF ring moieties of the different possible stereoisomers optimized by quantum chemical calculations (MNDO-AM1). Implementation of COMPASS (COMmon geometrical Pattern Search System) and molecular field fitting for all combinations of the stable conformations of each molecule, in this work led to several important conclusions. First, the inhibition mechanism of Annonaceous acetogenins was found to be noncompetitive against exogenous ubiquinones; this conclusion is not in agreement with the results of Friedrich et al.95 and Delgi Espositi et al.96 where Annonaceous acetogenins were concluded to bind competitively with respect to the ubiquinone at complex I. Second, while both rotenone and the Annonaceous acetogenins do bind noncompetitively against exogenous ubiquinone at complex I, they have different binding sites; however, most Annonaceous acetogenins (except rolliniastatin-2 and cherimolin-1) and rotenone were found to be mutually exclusive inhibitors.96,136 Third, stereochemistry surrounding the THF rings, surprisingly, seems to be much less important for activity at the enzyme level than in previous bioassays; this was corroborated by an exhaustive conformational space search analysis that indicated that the model compounds, with different stereochemical arrangements around the THF moieties, were in fairly good superimposition. Fourth, like the findings of González et al.122,137 and Gallardo et al.,138 proper length and flexibility of the alkyl spacer moiety, which links the THF and the α,β-unsaturated γ-lactone ring moieties, were essential for potent activity.45 Indeed, as Miyoshi et al. stated, if the THF rings serve as an anchor at the interface of the membrane, the stereochemical differences within the THF rings of the acetogenins should not make much difference in their bioactivity profiles. This work is in good agreement with the earlier results obtained by Shimada et al.43,44 and with several of our earlier SAR studies in isolated mitochondrial whole cells, and insects.21,22,71,97-99

ated γ-lactone ring moieties, were essential for potent activity.[45] Indeed, as Miyoshi et al. stated, if the THF rings serve as an anchor at the interface of the membrane, the stereochemical differences within the THF rings of the acetogenins should not make much difference in their bioactivity profiles. This work is in good agreement with the earlier results obtained by Shimada et al.[43,44] and with several of our earlier SAR studies in isolated mitochondrial whole cells, and insects.[21,22,71,97-99]

While the acetogenins are not ionophoric in living cells,100 several investigators have studied the ion complexation ability of the Annonaceous acetogenins.288,101 There is no evidence that Ca2+ or Mg2+ complexation contribute to the potent cytotoxic or pesticidal activities of these compounds. Some researchers have proposed, with little justification, that complexation with the Fe–S cluster, in the proven protein target, may contribute to their activity; yet, the same researchers studied Ca2+ and Mg2+ complexation of acetogenins in organic solvents. Investigations of Fe2+− and FeS−acetogenin complexation in aqueous media are suggested.

The door is now open for further studies of the molecular inhibitory mechanism of Annonaceous acetogenins against their protein targets, i.e., mitochondrial NADH ubiquinone oxidoreductase and plasma membrane NADH oxidase. Regardless of their future utility as chemotherapeutic, pesticidal, antimicrobial, etc. agents, they should become, at the least, useful tools, in the future, to probe the biochemistry of these active sites.

On the basis of the recent work of Miyoshi et al.,45 Oberlies et al.,21 He et al.,97 Alali et al.,22 Landolt et al.,98 Gu et al.,71 Alfonso et al.,99 and Delgi Esposti et al.,96 the following generalizations can be stated concerning the SARs of the Annonaceous acetogenins: (1) The bis-adjacent-THF acetogenins are the most potent among this family; the nonadjacent bis-THF compounds are, in general but not always, superior to the mono-THF compounds, which, in turn, are more potent than the nonring THF acetogenins. (2) The α,β-unsaturated γ-lactone at the end of the chain is crucial for activity. (3) If all other structural features are identical, the shorter C-35 acetogenins are more potent than the C-37 compounds. (4) The spacer, i.e., the distance between the OH-flanked THF and the γ-lactone, is critical to the potency and selectivity of the acetogenins; e.g., a 13-carbon space in the OH-flanked mono- and bis-THF compounds is optimum for activity. (5) Neither the 4-OH group nor the 10-OH group is essential for activity. (6) Three hydroxyl groups, two flanking the THF ring(s) and another somewhere in the long hydrocarbon chain, provide both the optimal position and polarity needed for the most potent activity, and beyond the tetra-hydroxylated acetogenins the activity drops drastically. (7) A ketone instead of a hydroxyl functional group decreases the activity. (8) Ketolactone acetogenins are still active but usually less active and more selective than their parent compounds; they may have different space group distances for optimal activity than their parent compounds, but this point needs further study. (9) The THP ring compounds are as active as the THF compounds and have the same mechanisms of action.

In their recent SAR study of Annonaceous acetogenins in purified rat liver mitochondria,99 Alfonso et al. noted that the activity of the nonadjacent bis-THF ring acetogenins depends on the distance between the two THF rings; i.e., the activity decreases to that of a mono-THF ring acetone.