Identification of a metabolically stable triazolopyrimidine-based dihydroorotate dehydrogenase inhibitor with antimalarial activity in mice

Abstract

Plasmodium falciparum causes 1-2 million deaths annually. Yet current drug therapies are compromised by resistance. We previously described potent and selective triazolopyrimidine-based inhibitors of P. falciparum dihydroorotate dehydrogenase (PfDHODH) that inhibited parasite growth in vitro; however, they showed no activity in vivo. Here we show that lack of efficacy against P. berghei in mice resulted from a combination of poor plasma exposure and reduced potency against P. berghei DHODH. For compounds containing naphthyl (DSM1) or anthracenyl (DSM2), plasma exposure was reduced upon repeated dosing. Phenyl-substituted triazolopyrimidines were synthesized leading to identification of analogs with low predicted metabolism in human liver microsomes and which showed prolonged exposure in mice. Compound 21 (DSM74), containing p-trifluoromethylphenyl, suppressed growth of P. berghei in mice after oral administration. This study provides the first proof of concept that DHODH inhibitors can suppress Plasmodium growth in vivo, validating DHODH as a new target for antimalarial chemotherapy.

Figure 1. Species-variable inhibitor binding site of PfDHODH

A. Structure of the DHODH inhibitor 2-cyano-3-hydroxy-N-[4-(trifluoromethyl)phenyl]-2-butenamide (A77 1726) 1. B. Amino acid residues within 4.2 Å of the co-crystallized inhibitor 1 are shown. The inhibitor (pink) is displayed showing the Van der Waals surface, the FMN cofactor (yellow) and orotate (pink) are displayed as sticks. Residues in grey are conserved between PfDHODH and hDHODH, while residues in green are variable. The two amino acid residues that differ between PfDHODH and PbDHODH are indicated by *. The figure was generated using PyMol from the file 1TV5.pdb.

Figure 2. Plasma exposure of 2 and 3 after repeat dosing in mice

Control mice (designated C) were administered blank vehicle for 4 days, followed by a single oral dose of 2 (panel A) or 3 (panel B) at 50 mg/kg (n=3 mice per time point) on day 5. For the repeat dosing groups, an oral dose of 50 mg/kg was administered 1x daily using two schedules: a) compound was administered on days 3, 4 and 5 with vehicle administered on days 1 and 2, or b) compound was administered on days 1–5. Plasma concentrations were measured at 30 and 240 min after the day 5 dosing for all groups.

Figure 3. Comparison of the series activity on PfDHODH vs P. falciparum whole cell assays

The log of the PfDHODH IC₅₀ data are plotted versus the log of the P. falciparum EC₅₀ data (in micromolar). The plotted data include compounds 2, 3, 12, 13, 16, 18, 21, 24, 27, 36, 40, 42 and 47 described in Table 1 and those previously reported from the series (compounds 8–12 and 14–17 from 22). Compounds for which solubility limits prevented the quantitative determination of either the IC₅₀/EC₅₀ have been left off the plot. Data were fitted by linear regression analysis where r² = 0.73, slope = 0.78 ± 0.087.

Figure 4. In vivo mouse efficacy studies for compound 21

A. Plasma concentrations after a single oral dose (20 mg/kg or 50 mg/kg) of 21 in mice. Peak plasma concentrations were 7.9 and 27.6 µM (2.3 and 8.1 µg/mL) and the apparent half-life was 1.8 h and 3 h at 20 mg/kg and 50 mg/kg, respectively. B. Efficacy in the standard P. berghei mouse model. Mice were infected with parasites on day 0, and compound 21 dosing began on day 1 after the presence of parasites was established. Dosing was by the oral route at either 50 mg/kg q.d. for 4 days (squares, solid lines) or 50 mg/kg b.i.d. for 4 days (triangles, solid lines) (days 1–4). No drug control data for each group is displayed with a broken line.

Scheme 1

Synthetic strategy for the compounds 8–47a. ^a Reagents and conditions:(i) CH₃COCH₂CO₂C₂H₅, AcOH, 3.5 h, reflux, 55%.; (ii) POCI₃, 45 min., reflux, 58%.; (iii) Ar-NH₂, EtOH, 2–30 h, room temp., 80–87%.