Triaminopyrimidine is a fast-killing and long-acting antimalarial clinical candidate

Abstract

The widespread emergence of Plasmodium falciparum (Pf) strains resistant to frontline agents has fuelled the search for fast-acting agents with novel mechanism of action. Here, we report the discovery and optimization of novel antimalarial compounds, the triaminopyrimidines (TAPs), which emerged from a phenotypic screen against the blood stages of Pf. The clinical candidate (compound 12) is efficacious in a mouse model of Pf malaria with an ED99 <30 mg kg(-1) and displays good in vivo safety margins in guinea pigs and rats. With a predicted half-life of 36 h in humans, a single dose of 260 mg might be sufficient to maintain therapeutic blood concentration for 4-5 days. Whole-genome sequencing of resistant mutants implicates the vacuolar ATP synthase as a genetic determinant of resistance to TAPs. Our studies highlight the potential of TAPs for single-dose treatment of Pf malaria in combination with other agents in clinical development.

Figure 1. Synthetic schemes and reaction conditions for compounds 7, 8, 9 and 12.

(A) Pyridine, microwave, 150 °C, 45 min. (B) (i) POCl₃, reflux, 6 h (ii) sodium carbonate, di-tert-butyl dicarbonate, room temperature, 16 h. (C) N,N-Diisopropylethylamine (DIPEA), ethanol, microwave, 110 °C, 1 h. (D) (i) Potassium tert-butoxide, 2,2′-bis(diphenylphosphino)-1,1'-binaphthyl (BINAP), pd₂(dba)₃, toluene, reflux, 12 h. (E) HCl (4 N) in dioxane, 15–30 min. (F) Compound 9, DIPEA, dichloromethane, formaldehyde (HCHO), sodium cyanoborohydride, 15 min.

Figure 2. Evolution of clinical candidate compound 12 and its in vitro and in vivo killing kinetics profile.

(a) Schematic of medicinal chemistry optimization and identification of clinical candidate 12. (b) In vitro parasite reduction ratio (PRR) for compound 12. The graph shows change in the number of viable parasites over time after exposure of Pf 3D7A to atovaquone ([cirf ]), chloroquine (▪), pyrimethamine (▴) or compound 12 (▾) at a concentration (conc.) equal to 10 times their respective IC₅₀s. (c) Percentage parasitemia in peripheral blood of mice infected with Pf3D7^0087/N9 (n=2 per group) after treatment with compound 12 at 10 (□), 20 (▴), 40 (○) or 80 (Δ) mg kg⁻¹ or with vehicle (▾). Dotted line indicates the lower limit of quantification (LLOQ) for % parasitemia estimation. (d) Blood concentration versus time profile for compound 12 in infected mice is depicted after the first dose of 10 ([cirf ]), 20 (□), 40 (▴) or 80 (Δ) mg kg⁻¹. Dotted line indicates the Pf IC₅₀. Two mice were used per dose group. (e) Predicted (lines) and observed (symbols) change in the total parasite burden in infected mice in the Pf/SCID model after treatment with compound 12 at 10 (Δ), 20 (▴), 40 (♦) or 80 (□) mg kg⁻¹ or with vehicle (○). SCID, severe combined immunodeficient.

Figure 3. Improvement in safety margins results in the nomination of compound 12 as a clinical candidate.

(a) Secondary pharmacology selectivity plot for compound 2. Selectivity ratio for AChE, α_1A and M₂ (IC₅₀ against targets (♦)/blood C_min at ED₉₀ in the Pf/SCID model (solid vertical line) was <1. Free plasma concentration range achieved in rat (dotted lines; C_min and C_max) and guinea pig (dashed line; C_min and C_max) in vivo illustrates targets covered during toxicity studies, and that selectivity against all targets was less than our target of >100-fold (long dash). (b) Secondary pharmacology selectivity plot for compound 12. Blood C_min at ED₉₀ in the Pf/SCID model (solid line) is significantly lower than that of compound 2. Success in achieving desired in vitro selectivity illustrated as all off-target potencies (♦) sit to the right of our selectivity target: >100-fold (long dash). Free plasma concentration range achieved in rat (dotted lines; C_min and C_max) and guinea pig (dashed line; C_min and C_max) in vivo illustrates reduced pharmacological coverage during toxicity studies compared with compound 2, which, in turn, translated into an improved safety margin for compound 12.