## 4 Discussion  
The results of this study confirmed our initial assumption that 2,6,7-trihydroxy-xanthene-3-ones can form complexes with iron and copper. Based on the comparison between hematoxylin (mild competitive conditions, wherein the final concentration of the indicator was equimolar to that of copper) and BCS (the indicator was given in 20-fold excess), it is apparent that these xanthones can also chelate copper. In contrast to iron, however, the complexes are not stable. In the latter case, the iron complexes were stable under 20-fold excess of the indicator ferrozine. The 2-hydroxy-3-one fragment is probably responsible for the chelation, due the higher electron density of the oxo group. Interestingly, the 6,7-dihydroxy fragment and the mentioned 2-hydroxy-3-one site were clearly not able to bind iron or copper simultaneously, since the proposed complexes had a stoichiometry of 1:1 or 2:1, xanthone:iron. Further, the functional groups on the benzene ring at position 9 are not involved, since the 3','-dihydroxyderivative (**3**) was not more potent than the other compounds without this additional chelation site. This outcome was rather surprising since, for example, flavonoids possessing a benzene ring with a catechol substitution can bind more metals than those without this chelation site [9; 33]. The differences in iron chelation were apparently minor, and seemed to be more related to the possible influence of the substituents on entropic factors (solvation) rather than on the electron density of the xanthene core. In particular, the trifluoromethyl derivative **9** was the most potent chelator. On the other hand, the 2'-hydroxy-3'-methoxy (**2**) and 2'-chloro-6'-fluoro derivatives (**12**) decreased chelation. The metal-reducing effect was markedly dependent on the substituent as well. Figure 8: Effect of 4’-trifluoromethylomethyl derivative (**9**) on copper- and iron-induced red blood cell lysis. (**A**): The derivative afforded protection against 500 uM of copper. (**B**): The derivative caused an increase in red blood cell lysis, caused by 2.5 mM of iron. As regards the influence of the substitution taking place on the C(9) phenyl ring on iron chelation and Cu\({}^{2+}\) reduction, clear-cut relationships are impossible to find. Intuitive chemical reasoning supported by simple MM2 force field calculations (part 2.3, Figure 9) leads to the conclusion that the C(9) phenyl moiety cannot be coplanar with the xanthene scaffold, due to the unfavorable interaction of the C(2\({}^{\prime}\)) and C(6\({}^{\prime}\)) substituents (including hydrogen) with the xanthene hydrogens at C(1) and C(8). As a consequence, neither the C(9) phenyl nor its substituents can communicate with the xanthene moiety through resonance effects, but only through field effects. Furthermore, since the \(\alpha\)-hydroxy ketone fragment is responsible for chelation, it is reasonable to assume that only the field effect of the substituents at C(2\({}^{\prime}\)) and C(6\({}^{\prime}\)) (i.e., the closest ones to the chelation site) may have some influence on metal chelation and reduction. For iron chelation, a few notable relationships can be observed. Most interestingly, the 4\({}^{\prime}\)-dimethylamino phenyl derivative **8** appears to be the least effective chelator at low pH values. Similarly, the _ortho_-substituted phenols **2** and **12** are among the worst chelating compounds under most pH values, with the exception of pH 5.5, at which 2\({}^{\prime}\)-chloro-6\({}^{\prime}\)-fluorophenyl xanthone **12** became a better chelator than the 4\({}^{\prime}\)-dimethylamino derivative **8**. Thus, we can conclude that the low efficiency of the latter most likely resulted from the protonation of the dimethylamino group under acidic conditions, which boosted its electron-withdrawing properties through charge-formation [\({}^{+}\)N(CH\({}_{3}\))\({}_{2}\)]. Next, both phenol **2** and phenol **12** also bear substituents with a withdrawing (-I) effect (OH, OCH\({}_{3}\) or F, Cl, respectively).