Optimization of 2,3-Dihydroquinazolinone-3-carboxamides as Antimalarials Targeting PfATP4

Abstract

There is an urgent need to populate the antimalarial clinical portfolio with new candidates because of resistance against frontline antimalarials. To discover new antimalarial chemotypes, we performed a high-throughput screen of the Janssen Jumpstarter library against the Plasmodium falciparum asexual blood-stage parasite and identified the 2,3-dihydroquinazolinone-3-carboxamide scaffold. We defined the SAR and found that 8-substitution on the tricyclic ring system and 3-substitution of the exocyclic arene produced analogues with potent activity against asexual parasites equivalent to clinically used antimalarials. Resistance selection and profiling against drug-resistant parasite strains revealed that this antimalarial chemotype targets PfATP4. Dihydroquinazolinone analogues were shown to disrupt parasite Na⁺ homeostasis and affect parasite pH, exhibited a fast-to-moderate rate of asexual kill, and blocked gametogenesis, consistent with the phenotype of clinically used PfATP4 inhibitors. Finally, we observed that optimized frontrunner analogue WJM-921 demonstrates oral efficacy in a mouse model of malaria.

Figure 1

Known PfATP4 inhibitors and the hit compound class, which is the focus of this research. The biological activity of the two hit compounds is shown.

Scheme 1. Synthesis of the Benzamide Intermediate

Reagents and Conditions: (i) CDI, THF, 0 °C, then 8 M MeNH₂·HCl, 21 °C; (ii) RNH₂ or RNH₂·HCl, EDCI·HCl, HOBt, DIPEA, DMF, 0–21 °C; and (iii) MeNH₂·HCl, HATU, DIPEA, DMF, 21 °C.

Scheme 2. Synthesis of the 4-Trifluoromethoxy Benzamide Intermediate

Reagents and Conditions: (i) CuCN, DMF, 150 °C, 1 h, then PhCH₃, 110 °C, 1 h, 74%; (ii) 55% H₂SO₄, 100 °C, 15 h, 49%; (iii) MeNH₂·HCl, EDCI·HCl, HOBt, DIPEA, DMF, 0–21 °C, 55%; and (iv) Fe, NH₄Cl, MeOH/H₂O (4:1), 50 °C, 85%.

Scheme 3. Synthesis of the 1,5-Dioxo-dihydropyrrolo[1,2-a]quinazoline-3a-carboxylic Acid Scaffold and Analogues

Reagents and Conditions: (i) α-Ketoglutaric acid, AcOH, 80 or 118 °C; (ii) R″NH₂, HATU or PyBOP, DIPEA, THF, 21 or 60 °C; and (iii) R′NH₂, TCFH, NMI, MeCN/DMF (1:1), 21 °C.

Scheme 4. Synthesis of the 8-Substituted Quinazolinone Analogues using a Suzuki–Miyaura Reaction

Reagents and Conditions: (i) RB(OH)₂, Pd(dppf)Cl_2·CH₂Cl₂ (5–10 mol%), K₃PO₄, PhCH₃/H₂O (9:1), μW, 120 °C, 30 min.

Figure 2

Summary of the structure and activity relationship.

Figure 3

Structure of PfATP4 showing mutations found in 49-resistant clones (purple) and KAE609 and SJ733-resistant strains (orange) used in this study. Other reported PfATP4 mutations that confer resistance to SJ733 and KAE609^, are shown in gray. The homology model of PfATP4 was created using the crystal structure of the rabbit SERCA pump (PDB 2C88), as previously described.^, The predicted transmembrane region of PfATP4 is shown by the dashed line.

Figure 4

Stage of asexual arrest and killing rate of selected compounds. (A) Parasite morphology was determined by Giemsa-stained blood smears at 10 × EC₅₀ of each compound after treatment of ring-stage parasites. Compounds cause vacuolated parasites consistent with the morphology observed with the PfATP4 inhibitor KAE609. (B) Flow cytometry of SYBR green-stained infected RBCs determined that compounds arrest parasites at the early trophozoite stage. Data points represent the mean of three technical replicates. (C) PfATP4 inhibitors have a fast-to-moderate rate of parasite kill in a viability assay measured by flow cytometry of staining erythrocytes pre-labelled with CFSE and parasites stained with Hoechst. Compounds were used at 10 × EC₅₀. Data points represent the mean of three technical replicates.

Figure 5

Evaluation of 70 and 71 in Na⁺ (A) and pH (B) assays for the detection of PfATP4 inhibition. (A) Fluorescence measurements were performed with isolated SBFI-loaded 3D7 trophozoites suspended at 37 °C at pH 7.1 physiological saline solution containing either 70 or 71 (at concentrations of 1 or 0.2 μM) or cipargamin (50 nM; positive control for PfATP4 inhibition) or DMSO (0.1% v/v; solvent control). (B) Isolated BCECF-loaded 3D7 trophozoites (that prior to the measurements had been ATP-depleted through incubation in glucose-free saline) were suspended in a pH 7.1 saline solution containing glucose, concanamycin A (100 nM), and either 70 (5 μM), 71 (5 μM), cipargamin (50 nM), or DMSO (0.1% v/v; solvent control) at 37 °C and the fluorescence was recorded immediately. The data are from a single experiment, representative of two similar independent experiments.

Figure 6

Activity of 71 (WJM-921) (and 70) in a P. berghei 4-day mouse model. To infect mice, P. berghei parasites (2 × 10⁷ parasites) were injected into the tail vein. Compounds were administered q.d. (A) 40 mg/kg and (B) 80 mg/kg by p.o. in a HPMC vehicle 2 h after infection (day 0) and then on days 1, 2, and 3. On days 2, 3, and 4, blood smears were taken, and parasitemia was evaluated. Chloroquine (CQ) (10 mg/kg) was used as a positive control. Error bars represent SD. The statistical test was performed using two-way ANOVA. (C) Bioanalysis of 71 (and 70) concentration in plasma after the first 40 mg/kg dose.