Structure-guided lead optimization of triazolopyrimidine-ring substituents identifies potent Plasmodium falciparum dihydroorotate dehydrogenase inhibitors with clinical candidate potential

Abstract

Drug therapy is the mainstay of antimalarial therapy, yet current drugs are threatened by the development of resistance. In an effort to identify new potential antimalarials, we have undertaken a lead optimization program around our previously identified triazolopyrimidine-based series of Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) inhibitors. The X-ray structure of PfDHODH was used to inform the medicinal chemistry program allowing the identification of a potent and selective inhibitor (DSM265) that acts through DHODH inhibition to kill both sensitive and drug resistant strains of the parasite. This compound has similar potency to chloroquine in the humanized SCID mouse P. falciparum model, can be synthesized by a simple route, and rodent pharmacokinetic studies demonstrated it has excellent oral bioavailability, a long half-life and low clearance. These studies have identified the first candidate in the triazolopyrimidine series to meet previously established progression criteria for efficacy and ADME properties, justifying further development of this compound toward clinical candidate status.

Fig. 1

Structures of select triazolopyrimidines

Fig. 2

P. falciparum in vitro growth curves and genetic rescue with yeast DHODH for 38. Data were collected in human serum and were fitted to the 4 parameter dose response equation in Graphpad Prism to obtain EC₅₀ values. 38 (P. falciparum 3D7; EC₅₀ = 0.019 μM (0.016–0.022)); 3 (P. falciparum 3D7; EC₅₀ = 1.1 μM (1.1–1.2)); 38 (P. falciparum D10; EC₅₀ = 0.086 μM (0.066–0.11)); 38 (P. falciparum D10 transfected with yeast DHODH; EC₅₀ >20 μM). Values in parenthesis represent the 95% confidence interval. Addition of proguanil did not restore sensitivity to the D10_yeast DHODH cell line (EC₅₀ >20 μM; data not shown).

Fig. 3

X-ray structure determination of PfDHODH in complex with 37. A. Structural alignment of PfDHODH_Δ384-413-37 (green) in comparison to PfDHODH bound with 2 (pdb 3I6R) (pink). Residues within 4Å of the bound inhibitor are displayed. L-orotate and the FMN cofactor are also displayed. B. Van der Waals surface representation of PfDHODH bound with 37. The enzyme surface is shown in pink, and orotate, FMN and 37 are shown in green as sticks. Structures were displayed using PyMol.

Fig. 3

X-ray structure determination of PfDHODH in complex with 37. A. Structural alignment of PfDHODH_Δ384-413-37 (green) in comparison to PfDHODH bound with 2 (pdb 3I6R) (pink). Residues within 4Å of the bound inhibitor are displayed. L-orotate and the FMN cofactor are also displayed. B. Van der Waals surface representation of PfDHODH bound with 37. The enzyme surface is shown in pink, and orotate, FMN and 37 are shown in green as sticks. Structures were displayed using PyMol.

Fig. 4

Pharmacokinetic analysis of A) 38 and B) 37 after oral dosing in mice (n=1 mouse per time point). Plots represent plasma concentration versus time after a single dose. Dose levels and route are shown in the graph legend.

Fig. 5

Pharmacokinetic analysis of A) 38 and B) 37 after oral (PO) or IV dosing in rats (n=2 rats per dose route). Plots represent plasma concentration versus time after a single dose. Dose levels and route are shown in the graph legend.

Fig. 6

Efficacy of 38 in SCID mice infected with P. falciparum after oral dosing. Mice were infected with parasites on day zero and a single oral dose/day was given on days 3, 4, 5 and 6. The limit of detection of the assay is 0.01% parasitemia. Dose levels are shown in the graph legend.

Scheme 1

Synthesis of the triazolopyrimidine compounds 11–55, 100 General conditions. (i) EtOH, reflux 5 h, over night at RT; (ii) 9a–9g, 9j, 9k, 9m–9o: 1,4 dioxane, DMF, requisite acid chloride, reflux O/N or 9h, 9i; NaEtO, EtOH, 80°C, 30 min, appropriate difluoropropanoate or difluorobutanoate, 30 min, RT, (1.5 – 3) h, 80°C; (R defined in Table 1) (iii) 9l, 9p AcOH, reflux, 8 h; (iv) reflux in POCl₃; (v) requisite aniline (R₁ and R₂ defined in Table 1), EtOH, reflux 1–3 h.

Scheme 2

Synthesis of the triazolopyrimidine compounds 66–84 General conditions. (i) POCl₃, reflux, 10 h; (ii) requisite aniline (R₁ and R₂ defined in Table 1), EtOH, RT; (iii) AcOH, RT, H₂O₂, Na₂WO₄ (cat), 50°C; (iv) R₃OH (R₃ defined in Table 1), NaH, THF, microwave, 120°C, (0.5 – 1) h

Scheme 3

Synthesis of triazolopyrimidines 89 – 97 Reagents and conditions. (i) amine in MeOH or THF, MW, 120°C, 30–45 min, (40 – 70%).

Scheme 4

Synthesis of triazolopyrimidines 98, 99 and 101 Reagents and conditions. (i) MeOH, H-cube hydrogenation, 50°C, 10% Pd/C, (11 – 12%)