Development of Chemical Entities Endowed with Potent Fast-Killing Properties against Plasmodium falciparum Malaria Parasites

Abstract

One of the attractive properties of artemisinins is their extremely fast-killing capability, quickly relieving malaria symptoms. Nevertheless, the unique benefits of these medicines are now compromised by the prolonged parasite clearance times and the increasing frequency of treatment failures, attributed to the increased tolerance of Plasmodium falciparum to artemisinin. This emerging artemisinin resistance threatens to undermine the effectiveness of antimalarial combination therapies. Herein, we describe the medicinal chemistry efforts focused on a cGMP-dependent protein kinase (PKG) inhibitor scaffold, leading to the identification of novel chemical entities with very potent, similar to artemisinins, fast-killing potency against asexual blood stages that cause disease, and activity against gametocyte activation that is required for transmission. Furthermore, we confirm that selective PKG inhibitors have a slow speed of kill, while chemoproteomic analysis suggests for the first time serine/arginine protein kinase 2 (SRPK2) targeting as a novel strategy for developing antimalarial compounds with extremely fast-killing properties.

Chart 1. Structures of Artemisinin-Based Drugs and Compounds with Potent Inhibitory Activity against PKG (2–6)

Scheme 1. Synthetic Procedure Followed for the Preparation of the Thiazole Derivatives 6–8 and 19–57

Reagents and conditions: (i) LDA, THF, −78 °C to rt, (ii) SO₂Cl₂, CHCl₃, 0 °C to rt, (iii) EtOH, reflux, (iv) Oxone, MeOH:H₂O, rt, (v) NaH, THF, rt or iPrOH, catalytic HCl/dioxane, 120 °C or DMSO, 100 °C (depending on amine’s basicity), (vi) Pd₂(dba)₃, XantPhos, t-BuOK, toluene, reflux, (vii) LiOH·H₂O, THF:EtOH:H₂O, reflux.

Scheme 2. Synthetic Procedure Followed for the Preparation of the Thiazole Derivatives 9–18

Reagents and conditions: (i) EtOH, reflux, (ii) CuCl₂, t-BuONO, CH₃CN, rt, (iii) morpholine or pyrrolidine, THF, rt, (iv) m-CPBA, CH₂Cl₂, rt, (v) 1-Boc-4-(4-aminophenyl)piperazine, TFA, iPrOH, reflux, (vi) TFA or 4 N HCl/dioxane, iPrOH, reflux, (vii) RCOCl or RSO₂Cl, Et₃N, CH₂Cl₂, or HATU, DIPEA, DMF.

Scheme 3. Synthetic Route Used for the Preparation of the Thiazole Derivatives 58–60

Reagents and conditions: (i) chloroacetaldehyde, acetone, reflux, (ii) NBS, DMF, rt, (iii) LDA, −78 °C to rt, (iv) Pd(OAc)₂, XPhos or cataCXiumA, CsCO₃, THF:H₂O or toluene:H₂O, reflux, (v) NIS, catalytic CF₃COOH, CH₃CN, rt or I₂, n-BuLi, THF, −78 °C to rt, (vi) Pd(PPh₃)₄, CuI, DMF, 70 °C, (vii) Pd₂(dba)₃, XantPhos, t-BuOK, toluene, reflux, (viii) 4 M HCl/dioxane, dioxane, rt.

Figure 1

PRR study with compounds 28A and 31. Artesunate (fast rate of killing), pyrimethamine (moderate rate of killing), and atovaquone (slow rate of killing) have also been included.

Figure 2

PRR study with compounds 5 and 28A. Artesunate (fast rate of killing), pyrimethamine (moderate rate of killing), and atovaquone (slow rate of killing) have also been included.

Figure 3

PRR study with compounds 59 and 60. Artesunate (fast rate of killing), pyrimethamine (moderate rate of killing), and atovaquone (slow rate of killing) have also been included.

Figure 4

Chemoproteomics profiling of compounds 31 and 50. (a) Both compounds were profiled on Kinobeads, which represent a combination of immobilized promiscuous ATP-competitive kinase inhibitors, in a P. falciparum protein extract. A total of 54 P. falciparum kinases were analyzed. The concentration of the “free” compounds used for competition, compound 50 and compound 31, was between 0.08 and 20 μM over six samples. (b) Compounds 50 and 31 were profiled with a bead matrix generated by immobilizing either compound 50 or compound 31 to beads, and competed with the respective “free” analogue over six concentrations between 0.08 and 20 μM. The Heatmaps show the protein kinases affected by any of the two compounds in two independent experiments, respectively. The values shown are apparent pK_d values (blue: decreasing apparent pK_d values; white: no competition; gray: protein not identified).