Property and Activity Refinement of Dihydroquinazolinone-3-carboxamides as Orally Efficacious Antimalarials that Target PfATP4

Abstract

To contribute to the global effort to develop new antimalarial therapies, we previously disclosed initial findings on the optimization of the dihydroquinazolinone-3-carboxamide class that targets PfATP4. Here we report on refining the aqueous solubility and metabolic stability to improve the pharmacokinetic profile and consequently in vivo efficacy. We show that the incorporation of heterocycle systems in the 8-position of the scaffold was found to provide the greatest attainable balance between parasite activity, aqueous solubility, and metabolic stability. Optimized analogs, including the frontrunner compound S-WJM992, were shown to inhibit PfATP4-associated Na⁺-ATPase activity, gave rise to a metabolic signature consistent with PfATP4 inhibition, and displayed altered activities against parasites with mutations in PfATP4. Finally, S-WJM992 showed appreciable efficacy in a malaria mouse model and blocked gamete development preventing transmission to mosquitoes. Importantly, further optimization of the dihydroquinazolinone class is required to deliver a candidate with improved pharmacokinetic and risk of resistance profiles.

Figure 1

Structures of the antimalarial clinical candidates and the dihydroquinazolinone antimalarial class which target PfATP4.

Scheme 1. General Synthesis of the Dihydroquinazolinone-3 Carboxamide Analogs

Reagents and Conditions: (i) R′NH₂, TCFH, NMI, MeCN; (ii) α-ketoglutaric acid, AcOH, 80 or 135 °C; (iii) R″NH₂, HBTU, DIPEA, THF, 21 or 60 °C; (iv) R″NH₂, TCFH, NMI, MeCN

Scheme 2. Synthesis of 8-Substituted Analogs

Reagents and Conditions: (i) R′B(OH)₂, Pd(dppf)Cl₂ (5–10 mol %), K₃PO₄, dioxane/H₂O, 110 °C, 1 h; Or aliphatic or aryl amine (R′), XantPhos-Pd-G3, Cs₂CO₃, dioxane 120 °C, 1 h.

Figure 2

Summary of the structure activity-property relationship.

Figure 3

Analog S-WJM992 10ahb inhibits Na⁺-ATPase activity in membranes prepared from isolated P. falciparum parasites more potently than the less active isomer R-WJM992 10aha. (A) The effects of analogs 10aha (2 μM) and 10ahb (2 μM), DHA (50 nM; negative control) and 0.2% v/v DMSO (solvent-only control) on ATPase activity in membranes prepared from 3D7 parasites, under high-[Na⁺] conditions (152 mM Na⁺) and low-[Na⁺] conditions (2 mM Na⁺; arising from the addition of 1 mM Na₂ATP) in the presence and absence of KAE609 1 (50 nM). The symbols show the data from individual experiments and the bars show the mean (+SEM) from 4 independent experiments performed with different membrane preparations. The data show the P_i produced as a percentage of that measured in the 152 mM Na⁺ control. In individual experiments, the P_i produced in the 152 mM Na⁺ control varied from 72 to 139 nmol per mg (total) protein per min. The (prenormalized) data for different compounds and conditions were compared using repeated measures one-way ANOVA with a Geisser-Greenhouse correction and post hoc Tukey test. For comparisons between different conditions for the same test compound (or for the control), significant differences are shown with black asterisks. For comparisons between the control and a compound under the same test condition, a significant difference is shown with a colored asterisk (in this case, a gray asterisk, as the only significant difference was between the control and analog 10ahb in the 152 mM Na⁺ condition). *P < 0.05, **P < 0.01. (B) Potency of analog 10aha (black circles) and 10ahb (red circles) against PfATP4-associated ATPase activity in membranes prepared from 3D7 parasites. The data are the mean (±SEM) obtained from four independent experiments performed with different membrane preparations, with the exception of the highest two concentrations of 10aha, for which data are from three independent experiments. (C,D) Potency of analogs 10aha (black; C) and 10ahb (red; D) against PfATP4-associated ATPase activity in membranes prepared from Dd2-Polδ parasites (closed triangles) and Dd2-Polδ-PfATP4^G358S parasites (open triangles). The data are the mean (shown + or – SEM) from four (C) or three (D) independent experiments. In (B–D), PfATP4-associated ATPase activity was calculated by subtracting the P_i production measured in the low-[Na⁺] (2 mM) condition from that measured in high-[Na⁺] (152 mM) in the presence of each of the different concentrations of analogs 10aha and 10ahb and is expressed as a percentage of that obtained for the high-[Na⁺] (152 mM) control.

Figure 4

Metabolomic signature of RS-WJM992 10ah reflects that of KAE609 1. (A) Principal component analysis of the global metabolite profiles showing overlap of 10ah with KAE609 1. (B) Heatmap analysis of the relative abundance of all metabolites revealed that both 10ah and KAE609 induce widespread metabolic disruption. (C) Pearson correlation of the average log₂ fold-change for all peptides significantly perturbed by 10ah and KAE609 (p < 0.05 and fold-change ≥1.5 or ≤0.67). Approximately 43% of perturbed peptides were mapped to the sequence of hemoglobin. (D) Significant perturbations to pyrimidine biosynthesis and nucleotide metabolites (p < 0.05 and fold-change ≥1.5 or ≤0.67). (E) Significant perturbations to central carbon metabolites (p < 0.05 and fold-change ≥1.5 or ≤0.67). For (D,E) data represents the log₂ fold-change of treated samples expressed relative to the average of the untreated control (n = 3–4).

Figure 5

Activity of R-WJM992 10aha and S-WJM992 10ahb in a standard membrane feeding assay. Oocyst counts and intensity per each midgut dissected from Anopheles stephensi mosquitoes 7 days post the blood meal infected with P. falciparum NF54 stage V gametocytes treated with compound at the indicated concentration. Red bars indicate average oocyst intensity. Numbers indicate the total number of mosquito midguts dissected per treatment group. Infection prevalence indicates the percentage of mosquitoes that were infected. Transmission intensity (oocyst burden) was compared between each group and the vehicle control in a pairwise fashion and significance was tested using the Wilcoxon rank sum test. **P < 0.001; *P < 0.05. Repeat experiments are shown in Figure S13.

Figure 6

Activity of RS-WJM992 10ah in a P. falciparum 4 day humanized NOD-scid IL-2Rγ^null mouse model. 10ah was dosed orally at 25 mg/kg q.d. for 4 days (indicated by blue arrows) while chloroquine was dosed orally at 10 mg/kg q.d. for 2 days (indicated by brown arrows). Parasitemia is expressed as the % of P. falciparum-infected human erythrocytes and was measured at each time point by flow cytometry using TER-119-phycoerythrine and SYTO-16 staining. Parasitemia below 0.005% could not be quantified, indicated by the dotted line.