Exploration and Characterization of the Antimalarial Activity of Pyrimidine-2,4-Diamines for which Resistance is Mediated by the ABCI3 Transporter

Abstract

The spread of drug-resistant Plasmodium strains is diminishing the effectiveness of current antimalarials, highlighting the importance of discovering new therapeutics with novel targets. A screen of the Jumpstarter library against P. falciparum identified W482 with a pyrimidine-2,4-diamine scaffold. Structure-activity relationships reveal the importance of the pyrimidine core and its endocyclic nitrogen, while alternative amines are tolerated in the 4-position. Bulky and hydrophobic carboxamides or substituted phenyl ureas display the most potent antiplasmodial activity. Resistance selection and whole genome sequencing reveal an amplification of the gene encoding the ABCI3 transporter protein W482-resistant parasites. W482 is found to exhibit greater activity against parasites with reduced expression of ABCI3, confirming that resistance is related to the transporter. W482 arrests asexual parasites at the ring to trophozoite transition stage and exhibits a fast-killing profile with a lag phase of 24 h. Improving the antiparasitic activity alongside metabolic stability and solubility remains a challenge in the future development of the pyrimidine-2,4-diamine class.

Keywords: Plasmodium; ABCI3 transporter; antimalarial; malaria; pyrimidine‐2,4‐diamine.

Figure 1

Activity against asexual P. falciparum 3D7 parasites of hit compounds with the pyrimidine‐2,4‐diamine scaffold from screens of the Janssen Jumpstarter library and the GSK TCAMS library.

Figure 2

Summary of SAR.

Scheme 1

Synthesis of 4‐substituted amine analogs. Reagents and conditions: a) (i) oxalyl chloride, DCM, cat. DMF, 20 °C, 1 h; (ii) tert‐butyl (4‐aminophenyl)carbamate, Et₃N, DCM, 20 °C, 2 h; b) TFA, DCM, 20 °C, 2 h; c) aliphatic amine, DIPEA, DMF, 20 °C, 18 h; d) 58, tert‐butanol, reflux, 18 h. R = aliphatic amine substituents shown in Table 1.

Scheme 2

Synthesis of 6‐substituted analogs. Reagents and conditions: a) dimethylamine, DIPEA, DMF, 20 °C, 18 h; b) 58, tert‐butanol, reflux, 18 h. R = substituents shown in Table 2.

Scheme 3

Synthesis of analogs with alternate heterocycles. Reagents and conditions: a) dimethylamine, DIPEA, DMF, 20 °C, 18 h; b) 58, tert‐butanol, reflux, 18 h for 16–18 or 58, G3‐Pd‐xantphos, Cs₂CO₃, 1,4‐dioxane, µW, 120 ºC, 1 h for 19–22. Analog variations are shown in Table 3.

Scheme 4

Synthesis of analogs with central ring modifications. Reagents and conditions: a) 4‐nitro‐aniline, T3P, DIPEA, DCE, µW, 130 ºC, 1 h; b) (i) Pd/C, H₂, methanol, 20 °C, 1 h; (ii) 59, tert‐butanol, reflux, 18 h. c) 5‐nitropyridin‐2‐amine, tert‐butanol, reflux, 18 h; d) (i) Pd/C, H₂, methanol, 20 °C, 1 h; (ii) 3‐cyclohexylpropanoic acid, T3P, DIPEA, DCE, µW, 130 ºC, 1 h. Analog variations are shown in Table 4.

Scheme 5

Synthesis of analogs with carboxamide variations. Reagents and conditions: a) Boc‐piperazine, DIPEA, DMF, 20 °C, 18 h; b) (i) 4‐nitroaniline, G3‐Pd‐xantphos, Cs₂CO₃, 1,4‐dioxane, µW, 120 ºC, 1 h; (ii) Pd/C, H₂, methanol, 20 °C, 1 h; c) (i) oxalyl chloride, cat. DMF, DCM, 20 °C, 1 h; (ii) Et₃N, DCM, 20 °C, 2 h; d) 4N HCl, dioxane, 20 °C, 18 h; e) (i) 4‐nitrophenyl chloroformate, THF, 20 °C, 30 min; (ii) 4‐fluoroaniline, Et₃N, THF, 20 °C, 20 h.

Figure 3

The activity of W482 (1) against W482‐selected populations and their 3D7 parental. The data are mean and SD from 3 independent 72 h Pf LDH assays.

Figure 4

Sensitivity of PfABCI3‐HAreg parasites to growth inhibition by chloroquine (a), MMV020746 (b), and W482 (1) (c). Data for parasites in which pfabci3‐ha mRNA was reduced (+ GlcN) are shown in red. GlcN (5 mM) was added to the parasites 48 h prior to the assay and maintained throughout the 72 h assay. The data for parasites expressing a normal level of PfABCI3‐HA (‐GlcN) are shown in black. The data in all panels are from four independent experiments performed on different days. The Table shows IC₅₀ values (mean ± SEM, n = 4) derived from the experiments shown in a–c. Parasites that were treated with or without GlcN (5 mM) were tested concurrently in each assay.

Figure 5

a) Stage of arrest assay showing that W482 (1) blocks ring‐trophozoite transition in the asexual stage of P. falciparum. Ring‐stage parasites were exposed to 10 × EC₅₀ of W482 (1) (1.4 µM) for 60 h, with samples taken every 12 h for Giemsa‐stained blood smears (a) and b) parasitemia quantification via SYBR Green staining and flow cytometry Data points represent the average of three technical replicates for parasitemia. Replicates of Giemsa‐stained blood smears can be found in Figure S4 (Supporting Information). c) Parasite reduction ratio assay conducted at 10 × EC₅₀ of W482 (1). Data represent means and SD of P. falciparum 3D7 parasite survival measuring 3 independent replicates by two‐color flow cytometry using CFDA‐SE and Hoescht 33,342 staining.