Relationship between antimalarial activity and heme alkylation for spiro- and dispiro-1,2,4-trioxolane antimalarials

Abstract

The reaction of spiro- and dispiro-1,2,4-trioxolane antimalarials with heme has been investigated to provide further insight into the mechanism of action for this important class of antimalarials. A series of trioxolanes with various antimalarial potencies was found to be unreactive in the presence of Fe(III) hemin, but all were rapidly degraded by reduced Fe(II) heme. The major reaction product from the heme-mediated degradation of biologically active trioxolanes was an alkylated heme adduct resulting from addition of a radical intermediate. Under standardized reaction conditions, a correlation (R2 = 0.88) was found between the extent of heme alkylation and in vitro antimalarial activity, suggesting that heme alkylation may be related to the mechanism of action for these trioxolanes. Significantly less heme alkylation was observed for the clinically utilized artemisinin derivatives compared to the equipotent trioxolanes included in this study.

FIG. 1.

Structures of artemisinin and trioxolane 1 (OZ277 as tosylate salt).

FIG. 2.

Structures of spiro and dispiro-1,2,4-trioxolanes. Refer to Table 1 for definitions of R, X, and R′.

FIG. 3.

Kinetic profiles of the percentage of heme remaining versus time for trioxolanes 2 (solid line), 3 (dashed line), and 4 (dotted line) in 50% ACN-H₂O at 20°C in the presence of excess sodium dithionite under argon.

FIG. 4.

Visible spectra of unreacted Fe(II) heme (dotted line) and reaction product (solid line) from reaction of heme (10 μM) with trioxolane 12 (10 μM) in 50% ACN-water with excess dithionite under argon.

FIG. 5.

Proposed reaction mechanism for alkylation of Fe(II) heme by trioxolanes. For clarity, only alkylation at the β meso position is shown for the heme adduct 23 (m/z 782.3); the product is likely to be a mixture of isomeric adducts (21).

FIG. 6.

Relationship between heme alkylation and in vitro antimalarial activity against P. falciparum (4, 6, 7, 30-33) for neutral and basic trioxolanes (filled circles), acidic and anionic trioxolanes (open circles), and artemisinin derivatives (open triangles). (A) Relative loss of heme. (B) Heme adduct A₄₇₂ (or A₄₆₇ for the artemisinin derivatives). The solid lines represent the regression lines for the neutral and basic trioxolanes.