Discovery of antiplasmodial pyridine carboxamides and thiocarboxamides

Abstract

Malaria continues to be a significant burden, particularly in Africa, which accounts for 95% of malaria deaths worldwide. Despite advances in malaria treatments, malaria eradication is hampered by insecticide and antimalarial drug resistance. Consequently, the need to discover new antimalarial lead compounds remains urgent. To help address this need, we evaluated the antiplasmodial activity of twenty-two amides and thioamides with pyridine cores and their non-pyridine analogues. Twelve of these compounds showed in vitro anti-proliferative activity against the intraerythrocytic stage of Plasmodium falciparum, the most virulent species of Plasmodium infecting humans. Thiopicolinamide 13i was found to possess submicromolar activity (IC₅₀ = 142 nM) and was >88-fold less active against a human cell line. The compound was equally effective against chloroquine-sensitive and -resistant parasites and did not inhibit β-hematin formation, pH regulation or PfATP4. Compound 13i may therefore possess a novel mechanism of action.

Keywords: Amides; Antiplasmodial; Malaria; P. falciparum; Pyridine; Thioamides.

Graphical abstract

Fig. 1

Antimalarial agents: Artemisinin (1), Quinine (2), Mefloquine (3), Chloroquine (4), Methylene blue (5), Proguanil (6), Halofantrine (7), Arterolane (8), 2-acetylpyridine-4-phenyl-3-thiosemicarbazone (9). Quinoline, amide, thioamide and pyridine moieties are shown in red.

Scheme 1

a) EDCI (HOBt), DCM/DMF, rt, 1–6 days; b) (COCl)₂, ArCOOH, DMF, 0 °C then amine, DCM, Et₃N, rt, 16–18 h; c) Et₃N or pyr, DCM/PhMe, ArCOCl, 0 °C then amine rt, 18 h.

Scheme 2

a) conc. HCl, THF, 12j (81%); b) NaCNBH₃, AcOH; c) Boc₂O, THF/H₂O (1:1), 12l (44% over two steps).

Scheme 3

Thionation of amides 12a-l using LR. ^a3 mol. equiv. LR, reflux, PhMe, 24 h; ^b0.6 mol. equiv. LR, reflux, PhMe, 24 h; ^c1.2 mol. equiv. LR, reflux, PhMe, 24 h; ^d0.6-1 mol. equiv. LR, MW, PhMe, 0.5 h.

Fig. 2

Effect of thiocarboxamide 13i on the proliferation of intraerythrocytic stage P. falciparum (strain 3D7) and HFF cells in vitro over 96 h. The data are from three independent experiments and error bars represent standard error of the mean (SEM). Where not visible, error bars are smaller than the symbol.

Fig. 3

Compound 13i does not affect parasite [Na⁺]_cytor pH_cyt. (A,B) Isolated 3D7 parasites loaded with SBFI were exposed to 13i (purple; 1 μM or 10 μM), solvent alone (black; DMSO control), or cipargamin (blue; 50 nM; positive control for PfATP4 inhibition). (C,D) BCECF-loaded 3D7 trophozoites were exposed to 13i (purple; 1 μM or 10 μM), cipargamin (blue; 50 nM; positive control for PfATP4 inhibition), MMV007839 (pink; 2 μM; positive control for PfFNT inhibition), CCCP (grey; 100 nM; protonophore), concanamycin A (green; 100 nM; positive control for V-type H⁺-ATPase inhibition) or solvent alone (black; DMSO control). Panels A and C show representative traces from a single Na⁺ (A) or pH (C) experiment. Panels B and D show data averaged from three independent experiments performed on different days in which all compounds/concentrations were tested concurrently. The bars and error bars show the mean + SEM and the symbols show the data from each individual experiment. The data for each compound and concentration were compared to those for the DMSO control using one-way ANOVA with Dunnett’s multiple comparisons test, with the p values shown on the figure. The final [Na⁺]_cyt (B) and pH_cyt (D) reached were averaged from the data points obtained 85–90 min and 55–60 min after parasites were first exposed to the compound/solvent, respectively. The DMSO concentration was 0.1% v/v in pH experiments and 0.1–0.2% v/v in Na⁺ experiments.