Discovery of Potent Antimalarial Type II Kinase Inhibitors with Selectivity over Human Kinases

Abstract

While progress has been made in the effort to eradicate malaria, the disease remains a significant threat to global health. Acquired resistance to frontline treatments is emerging in Africa, urging a need for the development of novel antimalarial agents. Repurposing human kinase inhibitors provides a potential expedited route given the availability of a diverse array of kinase-targeting drugs that are approved or in clinical trials. Phenotypic screening of a library of type II human kinase inhibitors identified compound 1 as a lead antimalarial, which was initially developed to target human ephrin type A receptor 2 (EphA2). Here, we report a structure-activity relationship study and lead optimization of compound 1, which led to compound 33, with improved antimalarial activity and selectivity.

Figure 1.

Compound 1 prevents blood-stage malaria infection when applied prophylactically (A, C) and cures infection when given therapeutically (B, D) at 15 and 50 mg/kg. For A and C, mice (infected and untreated control group) were pretreated with vehicle alone or given a single 15 or 50 mg/kg dose of a control compound, GNF179, or compound 1 via IV injection in the vehicle. After 6 h groups of 5 mice were infected with 10⁵ P. berghei^Luc sporozoites suspended in DMEM media via IV injection. For B and D, mice were infected with 10⁷ fresh P. berghei^Luc iRBCs suspended in cryopreservation solution via IP infection and then treated afterward at 1, 2, 3, and 4 days post-infection via IV injection in the standard Peter’s test using compounds described above. Parasitemia was determined by flow cytometry at the indicated days for A and B. Survival is shown in C and D.

Figure 2.

Killing profile of compound 1 in blood-stage P. falciparum. Fast-acting DHA and slow-acting atovaquone are controls. (A–C) Flow-cytometry-based assay evaluated parasite viability after treatment for 12 (A), 24 (B), and 48 (C) hours. Graphs depict the average of 3 biological replicates ± SEM (D) P. falciparum parasite reduction ratio of both compounds 1 and 16 with the accompanying table. The graph presents data from 2 biological replicates ± SEM. The lag phase is the time before the maximal killing rate. PRR (parasite reduction ratio) is the reduction of viable parasites over one life cycle. 99.9% PCT (parasite clearance time) is the time required to clear the initial parasite load by 3-log units.

Figure 3.

Stage specific activity of compound 1. (A–D) Flow cytometry to detect DNA replication and Giemsa-stained thin blood smears to monitor morphology, with treatment at 6 HPI (A), 18 HPI (B), 30 HPI (C), and 42 HPI (D). Histograms representative of 3 biological replicates. (E) Stage specific EC₅₀ of compound 1 confirms increased potency in early asexual stage. Graph reflects 3 biological replicates ± SEM.

Figure 4.

Compounds 1 and 16 are effective in artemisinin and piperaquine resistant isolates. Parasite lines are resistant (−R) or sensitive (−S) to artemisinin (ART) or piperaquine (PPQ). (A) Ring Stage Survival Assay and (B) Piperaquine Survival Assay graphs reflect average % survival ± SEM of 3 biological replicates. (C, D) EC₅₀ determination, graphs depict average ± SEM of 3 biological replicates.

Figure 5.

Compound 1 maintains potency in Ugandan field isolates. Compound 1 was evaluated against P. falciparium ex vivo isolates from patients in the Ugandan towns of Patongo, Tororo, and Busiu. Ring stage Dd2 and 3D7 were also evaluated for comparison with lab-adapted strains. Graph shows individual EC₅₀ values of ex vivo isolates and average of Dd2 and 3D7 EC₅₀ values from biological triplicates.

Figure 6.

Compound 1 displays no cross-resistance in P. falciparum strains resistant to known antiplasmodials. Results displayed are the average of 2 biological replicates ± SEM.

Scheme 1.. Synthesis of Compounds 1, 6, and 8–16

^aReagents and conditions: (a) 3-chloro-5-nitrobenzoic acid, HATU, DIPEA, DMF, 25 °C, 1 h; (b) Fe, NH₄Cl, EtOH/H₂O, 80 °C, 5 h; (c) acid or acyl chloride, coupling condition.

Scheme 2.. Synthesis of Compounds 7, 17–23

^aReagents and conditions: (a) Triphosgene, 1-methylpiperazine, DIPEA, CH₂Cl₂, 0–25 °C (b) 17: 3-bromo-5-(thiophen-2-yl) pyridine, K₃PO₄, t-BuBrettPhos Pd G3, t-BuBrettPhos, 2Me-THF. 80 °C 18: 5-(thiophen-2-yl)nicotinaldehyde, NaBH₃CN, AcOH, 25 °C, 2 h; (c) 3-chloro-5-nitrobenzoic acid, HATU, DIPEA, DMF, 25 °C; (d) Fe, NH₄Cl, EtOH/H₂O, 75 °C, 3 h; (e) 5-(thiophen-2-yl)nicotinoyl chloride, DIPEA, DMF, 25 °C. (f) NaOH, THF/MeOH/H₂O, 25 °C.

Scheme 3.. Synthesis of Compounds 2–5, 24–33

^aReagents and conditions: (a) 5-nitrobenzoic acid derivatives, HATU, DIPEA, DMF, 25 °C, 12 h; (b) Fe, NH₄Cl, EtOH/H₂O, 75 °C, 2 h; (c) acid or acyl chloride, coupling condition.