In contrast, these two groups were fairly equivalent with respect to in vitro potency within the triazolopyrimidines,26 though clearly in that case SF5-aniline provided better plasma exposure and more potent in vivo activity

In contrast, these two groups were fairly equivalent with respect to in vitro potency within the triazolopyrimidines,26 though clearly in that case SF5-aniline provided better plasma exposure and more potent in vivo activity. Several potent analogues were identified, including a compound that showed in vivo antimalarial activity. The isoxazolopyrimidines were more rapidly metabolized than their triazolopyrimidine counterparts, and the pharmacokinetic data were not consistent with the goal of a single-dose treatment for malaria. Introduction Malaria remains one of the most significant infectious diseases worldwide, with PF-04217903 methanesulfonate people in 91 countries at risk for infection by the parasite that is the causative agent of the disease.1?4 The WHO reported 216 million cases in 2016 with nearly 0.5 million deaths.5 Five species cause malaria in humans, but most deaths are caused by ATP4, and DSM265 (1)19 (Figure ?Figure11), an inhibitor of dihydroorotate dehydrogenase (DHODH). A number of additional compounds are either in phase I clinical development or undergoing preclinical testing.3,13,14 The need to introduce all new agents as combination therapies to protect against resistance, as well as the requirement to provide compounds efficacious against several species, and for use in both treatment and prevention, requires a robust pipeline to meet clinical needs.20 Additionally, the emphasis on identifying compounds with pharmacokinetic properties that can support a single-dose cure to improve patient compliance increases the challenge of identifying compounds with the required properties. Open in a separate window Figure 1 Structures of DHODH is a mitochondrial enzyme that utilizes flavin mononucleotide (FMN) and coenzyme Q (CoQ) to catalyze the oxidation of dihydroorotate to orotic acid. We used a target-based high-throughput screening approach to identify the inhibitors of DHODH (DHODH and as a single dose.21?24 Subsequently, we also identified additional compounds from the triazolopyrimidine series with improved physicochemical properties (e.g., 2)22 (Figure ?Figure11). In our search to identify additional chemical scaffolds with the DHODH inhibitory PF-04217903 methanesulfonate activity, which could be progressed for the treatment of malaria, we uncovered an isoxazolopyrimidine scaffold that is closely related to the triazolopyrimidine series (e.g., 1 and 2). The initial isoxazolopyrimidine hit 3 (Figure ?Figure11 and Table 1) was identified in a phenotypic screen of the GSK compound library28 and was later found to inhibit DHODH. To determine if the isoxazolopyrimidine series had unique properties relative to the triazolopyrimidine series, we undertook a hit-to-lead expansion, identifying several potent and selective analogues. Table 1 StructureCActivity Relationshipa Open in a separate window Open in a separate window aThe IC50 and EC50 PF-04217903 methanesulfonate data of DHODH and were collected in triplicate across the full dose range; the 95% confidence interval of the fit is provided in parenthesis. For compound 3, error represents the standard deviation for four replicates. bAlbumax-based media were used to collect the indicated data. Data for 1 and 2 were previously reported.31,32 Results and Discussion Identification of the Isoxazolopyrimidine Series The lead isoxazolopyrimidine (3; Table 1) was identified in a large-scale phenotypic screen for antimalarial compounds; the dataset for this full screen has previously been made public.28 The compound 3 was subsequently identified as a DHODH inhibitor with moderate potency against both the enzyme and the parasite in whole-cell assays (Table 1). The identification of the target was based on structural similarity to the triazolopyrimidine Rabbit polyclonal to CDKN2A series (Figure ?Figure11), and on secondary screening assays, including the screening of a transgenic parasite line that expresses cytoplasmic yeast DHODH29 and is therefore resistant to inhibitors that target mitochondrial-type DHODHs such as DHODH. Chemistry To improve the potency of 3 and to build in the characteristics required for good in vivo activity, we undertook a hit-to-lead expansion of the series. Compound 3 showed some disconnect between the enzyme and parasite data, suggesting potential permeability issues, as potency on the parasite was less than that on the enzyme (Table 1). It was also rapidly metabolized in mouse liver microsomes (Table 2) and showed poor exposure when dosed orally in mice (and improved the aqueous solubility (e.g., Figure ?Figure11; 2).31 Thus, we systematically incorporated each of these substitutions into the isoxazolopyrimidine series. In the triazolopyrimidines, substantial increases in potency were achieved by substituting the C2 carbon with linear fluorohydrocarbons (e.g., CF2CH3 of 1 1);26 however, as this position was not available for substitution in the isoxazolopyrimidines, we also tested the effect of replacing the isoxazolopyrimidine ring methyls with CF3 on the potency and metabolic stability. Because of the proximity of the C2 carbon to the hydroxyl group of Y528 in the and evaluated for activity against the 3D7 parasites in whole-cell assays (Table 1). Similar to the triazolopyrimidine series,26,32 the best potency on both 3D7 parasites was observed for compounds containing SF5-aniline (14 and 32) and those with tetrahydro-2-naphthyl amines (9 and 11). The SF5-aniline group was 2C5-fold more potent within this series than the CF3-aniline group (14 vs 15; 32 vs.

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