Iron(III) Protoporphyrin IX Complexes of the Antimalarial <i>Cinchona</i> Alkaloids Quinine and Quinidine

The antimalarial properties of the <i>Cinchona</i> alkaloids quinine and quinidine have been known for decades. Surprisingly, 9-epiquinine and 9-epiquinidine are almost inactive. A lack of definitive structural information has precluded a clear understanding of the relationship between molecular structure and biological activity. In the current study, we have determined by single crystal X-ray diffraction the structures of the complexes formed between quinine and quinidine and iron­(III) protoporphyrin IX (Fe­(III)­PPIX). Coordination of the alkaloid to the Fe­(III) center is a key feature of both complexes, and further stability is provided by an intramolecular hydrogen bond formed between a propionate side chain of Fe­(III)­PPIX and the protonated quinuclidine nitrogen atom of either alkaloid. These interactions are believed to be responsible for inhibiting the incorporation of Fe­(III)­PPIX into crystalline hemozoin during its <i>in vivo</i> detoxification. It is also possible to rationalize the greater activity of quinidine compared to that of quinine

The Single Crystal X‑ray Structure of β‑Hematin DMSO Solvate Grown in the Presence of Chloroquine, a β‑Hematin Growth-Rate Inhibitor

Single crystals of solvated β-hematin were grown from a DMSO solution containing the antimalarial drug chloroquine, a known inhibitor of β-hematin formation. In addition, a kinetics study employing biomimetic lipid–water emulsion conditions was undertaken to further investigate the effect of chloroquine and quinidine on the formation of β-hematin. Scanning electron microscopy shows that the external morphology of the β-hematin DMSO solvate crystals is almost indistinguishable from that of malaria pigment (hemozoin), and single crystal X-ray diffraction confirms the presence of μ-propionato coordination dimers of iron­(III) protoporphyrin IX. The free propionic acid functional groups of adjacent dimers hydrogen bond to included DMSO molecules, rather than forming carboxylic acid dimers. The observed exponential kinetics were modeled using the Avrami equation, with an Avrami constant equal to 1. The decreased rate of β-hematin formation observed at low concentrations of both drugs could be accounted for by assuming a mechanism of drug adsorption to sites on the fastest growing face of β-hematin. This behavior was modeled using the Langmuir isotherm. Higher concentrations of drug resulted in decreased final yields of β-hematin, and an irreversible drug-induced precipitation of iron­(III) protoporphyrin IX was postulated to account for this. The model permits determination of the equilibrium adsorption constant (<i>K</i>_ads). The values for chloroquine (log <i>K</i>_ads = 5.55 ± 0.03) and quinidine (log <i>K</i>_ads = 4.92 ± 0.01) suggest that the approach may be useful as a relative probe of the mechanism of action of novel antimalarial compounds

The Effects of Quinoline and Non-Quinoline Inhibitors on the Kinetics of Lipid-Mediated β‑Hematin Crystallization

The throughput of a biomimetic lipid-mediated assay used to investigate the effects of inhibitors on the kinetics of β-hematin formation has been optimized through the use of 24-well microplates. The rate constant for β-hematin formation mediated by monopalmitoyl-<i>rac</i>-glycerol was reduced from 0.17 ± 0.04 min^–1 previously measured in Falcon tubes to 0.019 ± 0.002 min^–1 in the optimized assay. While this necessitated longer incubation times, transferring aliquots from multiple 24-well plates to a single 96-well plate for final absorbance measurements actually improved the overall turnaround time per inhibitor. This assay has been applied to investigate the effects of four clinically relevant antimalarial drugs (chloroquine, amodiaquine, quinidine, and quinine) as well as several short-chain 4-aminoquinoline derivatives and non-quinoline (benzamide) compounds on the kinetics of β-hematin formation. The adsorption strength of these inhibitors to crystalline β-hematin (<i>K</i>_ads) was quantified using a theoretical kinetic model that is based on the Avrami equation and the Langmuir isotherm. Statistically significant linear correlations between lipid-mediated β-hematin inhibitory activity and <i>K</i>_ads values for quinoline (<i>r</i>² = 0.76, <i>P</i>-value = 0.0046) and non-quinoline compounds (<i>r</i>² = 0.99, <i>P</i>-stat = 0.0006), as well as between parasite inhibitory activity (D10) and <i>K</i>_ads values for quinoline antimalarial drugs and short-chain chloroquine derivatives (<i>r</i>² = 0.64, <i>P</i>-value = 0.0098), provide a strong indication that drug action involves adsorption to the surface of β-hematin crystals. Independent support in this regard is provided by experiments that spectrophotometrically monitor the direct adsorption of antimalarial drugs to preformed β-hematin