We have previously shown that hyperforin is a potent multi-target antiangiogenic compound 
. This observations adds to the antimetastasic effect previously reported and was confirmed by other authors, using stable salts of the bioactive compound 
. In the present study, we have used dicyclohexylammonium hyperforinate (hyperforin-DCHA, compound (1
) in ) as a stable form of hyperforin maintaining its bioactivity. In fact, our results with compound (1
) as a positive control compound show similar results to those published for the free acid form at slightly lower concentrations, as expected for a stabilized form of the compound 
Hyperforin instability is due to the contemporary presence of fastly reacting functional groups: an enolized β-diketone moiety, apparently present in solution as 7-hydroxy, 9-keto tautomer due to the formation of a hydrogen bonding between the ketone in position 1 and the 7-hydroxy group, and the close proximity of this latter to the double bond of the 6- prenyl group. In addition, carbon 8 is strongly nucleophilic, and easily oxidized. Both these characteristics induce a fast reactivity toward oxidizing agents, including light, and lead to unexpected derivatives, some of which also accumulate in the extracts, like compounds (2
) and (3
. One of the major degradation routes for hyperforin is the formation of furan derivatives by mutual oxidative interaction of the enol moiety and the prenyl chains, irreversibly blocking the 7-hydroxy in an ether linkage 
. In compound (3
), a hemiacetal species is formed by the introduction of an electrophilic oxygen at C8, which in situ
reacts with the spatially faced carbonyl group at C1.
) and (3
) were chosen among the different oxidized derivatives to be investigated for their antiangiogenic potential. They represent very stable hyperforin derivatives, where the overall molecular structure is preserved but the enolized β-diketone functionality has collapsed to form furan rings. In previous works, compounds (2
) and (3
) have shown to be less active than hyperforin in vitro
as inhibitors of synaptosomal serotonin reuptake, but they had a comparable effect as growth inhibitors of P. falciparum
cultures, although showing less toxicity 
. Oxidized hyperforin derivatives (2
) and (3
) have also shown to be equally or more potent than hyperforin as inhibitors of 5-lipooxygenase activity 
. In addition, furohyperforins are also reported to potently inhibit CYP3A4 enzyme activity 
thus inferring the enolized β-diketone moiety a significant role in modulating many kinds of activities. Our results herein presented altogether show that these compounds behave as much less potent antiangiogenic compounds than hyperforin. This is evidenced by the limited activity shown in all the panel of tests used.
The role of the four carbonyl groups, functionalities that could be involved in hydrogen bondings with enzymes active sites, was also investigated. The reaction of hyperforin with different reducing agents produced compounds (4
) to (8
. Compounds (4
) to (7
) are formally 7-deoxohyperforins, where the C1-C10 non enolizable β-diketone moiety survived to reduction, like in compound (4
), or was partially reduced to a 10-oxymethine (compounds 5
) or totally reduced to the 1,10 diol (compound 7
). Interestingly enough, compounds (4
) to (7
) have no relevant effects as anti-angiogenic compounds. In these cases the molecules loose a number of intra and inter molecular bondings, while modifying the relative spatial distribution of the oxygenated functions. All of them are much less active than hyperforin, but we should stress that compound (5
) is the worst, with IC50 and MIC values much higher than the other tested compounds.
The most relevant activities (equal or slightly more potent than those exhibited by hyperforin-DCHA) were observed on compound (8
), formally a tetrahydrohyperforin, whose enolized β- diketone moiety is reversed with respect to the natural product (9-OH, 7-keto versus 7-OH, 9-keto). This is due to the formation of a strong intramolecular hydrogen bond between the donor 9-OH group and the acceptor hydroxyl at 10 position, which also draws the stereochemical control of the reaction, only producing the 10S
stereoisomer. Apparently, compound (8
) is particularly stable if compared to hyperforin and this can be attributed to the strong intramolecular hydrogen bonding that produces orthorombic crystals 
Altogether, the results discussed above indicate that only compound (8), namely, tetrahydrohyperforin exhibits antiangiogenic effects similar to those shown by hyperforin (compound 1). To proceed further, we decided to focus our additional experiments on these two compounds and an additional one (compound 9): the satured compound octahydrohyperforin (), obtained by catalytic hydrogenation of hyperforin. This compound is devoid of the rapid oxidative degradation due to the presence of prenyl double bonds in hyperforin, it appears to be a stable derivative and it is endowed of increased lipophilicity. In all the tested in vitro assays, octahydrohyperforin behaved as an inhibitor more potent than hyperforin. Furthermore, its stronger antiproliferative effects on BAEC as compared with non-endothelial cells suggest that octahydrohyperforin is more specific for endothelial cells than hyperforin itself. Finally, octahydrohyperforin also behaves as the most potent inhibitor in an in vivo Matrigel plug assay of angiogenesis.
In conclusion, we can assert that the enolized β-dicarbonyl system is peculiar for the biological activity of hyperforin as an anti-angiogenic compound, whichever tautomer is present in solution, since the products devoid of this functionality are inactive or less active. Apparently the C1 and C10 carbonyl groups and the prenyl double bonds are not essential to maintain the activity, as shown by the behavior of compounds (8) and (9). Altogether, our results identify tetrahydrohyperforin and octahydrohyperforin as two new potent inhibitors of angiogenesis and unveil the central role played by the enolized β-dicarbonyl system in the anti-angiogenic effect of hyperforin. On the one hand, these data could be useful for the rational design and chemical synthesis of more effective hyperforin derivatives as anti-angiogenic drugs. On the other hand, the potential of tetrahydrohyperforin and octahydrohyperforin as antiangiogenic compounds deserves to be studied more in depth, including a molecular characterization of their effects on specific targets. Future experimental efforts in both directions seem to be warranted.