MMV Project of the Year award - 2003

4(1H)-pyridones

The interest of GlaxoSmithKline (GSK) in 4(1H)-pyridones goes back to the 1980s when, in a programme carried out at Wellcome (one of the GSK heritage companies) on electron transport inhibitors as antiparasitic agents, a novel family of 4(1H)-pyridones, analogues of clopidol, was seen to show both in vitro and in vivo antimalarial activity. This new class of compounds selectively inhibited oxygen consumption in isolated mitochondria of Plasmodium falciparum. The compounds were also potent inhibitors of erythrocyte parasitization by strains of P. falciparum having sensitivity and resistance to atovaquone, chloroquine and pyrimethamine. They were efficacious in malaria mouse models of atovaquone-sensitive and atovaquone-resistant Plasmodium yoelii.

Building on these discoveries and following the strategy designed by GSK’s Diseases of the Developing World (DDW) initiative, researchers at the GSK Centre for Diseases of the Developing World at Tres Cantos in Spain, in partnership with MMV, advanced this promising class of antimalarial drugs to the selection of a candidate for drug development. Thus the new 4(1H)-pyridone derivative GW844520 was selected.

4(1H)-pyridone derivatives are selective inhibitors of plasmodial mitochondrial function. P. falciparum uses little oxygen and generates ATP predominantly, if not exclusively, by anaerobic fermentation in the cytoplasm. Mitochondria from Plasmodium spp. play additional roles compared with mammalian mitochondria (in pyrimidine and haem synthesis, for example). Some of these reactions need the mitochondrial electron transport chain as a sink for reducing equivalents eliminated from substrates. The electron transport chain also contributes to the electrochemical proton gradient across the inner mitochondrial membrane, and this in turn provides the energy for the transport of metabolic intermediates and macromolecules.

Atovaquone blocks electron transport by inhibiting cytochrome b, a critical element in the ubiquinone:cytochrome c oxidoreductase complex, also known as respiratory complex III, or cytochrome bc1 complex. The (41H)-pyridone derivative GW844520 was tested on isolated mitochondria side by side with atovaquone. It displayed the same pattern of inhibition of individual redox reactions, providing strong circumstantial evidence that the site of action of 4(1H)-pyridone is respiratory complex III.

Pyridones show no cross-resistance with atovaquone. Results obtained from P. falciparum mutants (having diminished sensitivity to pyridone derivative GW844520) point to cytochrome b as the molecular target for the 4(1H)-pyridones, and more specifically to the same protein domain that also contributes binding sites for atovaquone. Importantly, the binding site is probably adjacent but it is clearly not completely overlapping with that of atovaquone, as none of the four tested mutants display cross-resistance.

A chemical programme was carried out starting from the clopidol molecule. The programme explored substitutions at different positions and established structural activity relationships (SAR). One of the most successful classes of 4(1H)-pyridone derivatives is represented by the derivatives substituted at position 5 with lipophilic substitutes, particularly the ary-oxy-aryl substituted derivatives. Optimization of this series led to GW844520, which has been recently selected as a candidate for clinical development. GW844520 was prepared following a straightforward synthetic route involving commercially available reagents and the use of tractable and robust intermediates.

Compounds from this class of 4(1H)-pyridone derivatives, particularly candidate GW844520, exhibited excellent in vitro activity against the
key pathogen, P. falciparum. Activity was also demonstrated against organisms resistant to marketed compounds such as atovaquone, chloroquine, pyrimethamine and mefloquine. The selectivity ratio in vitro, studied in terms of mitochondrial function and whole cell inhibition (activity against P. falciparum vs. cytotoxicity), was generally around 1000. In vitro data (membrane depolarization assay) against P. yoelii, the species used as a surrogate of P. falciparum in in vivo studies, correlated with those obtained with P. falciparum. The activity of the combination of GW844520 with most antimalarial agents in clinical use, and other antimicrobial drugs, was investigated in vitro against three strains of P. falciparum. All combinations investigated were additive. No antagonistic effects were observed with any of the combinations tested.

GW844520 showed a low frequency of spontaneous resistance (<10-8) against P. falciparum tested in vitro. In addition, in vivo studies were carried out, designed to maximize the likelihood of finding resistant isolates in CD1 immunocompetent and SCID beige immunodeficient mice. No resistant isolates of P. yoelii were selected from animals treated with GW844520 at various doses. However, atovaquone used as positive control showed selection of resistant parasites at the same doses.

In vivo efficacy of GW844520 was demonstrated in mouse models of infection with P. yoelii and P. falciparum, including the standard 4-day dosing model and single dose, via oral administration. GW844520 was well tolerated in a 4-day mouse toxicity study at doses of up to 1000 mg/kg per day, and showed a good therapeutic window. GW844520 proved negative in bacterial and mammalian mutagenicity screening assays.

GW844520 has low total clearance (all species) and a long elimination half-life in both monkey and dog. Oral bioavailability following administration as a solution was high in all species (51-100%). Administering GW844520 with reduced particle size resulted in a considerable increase in oral exposure that is adequate for progression into safety assessment studies.

GW844520 exhibits many of the features desirable in a drug development candidate for treating uncomplicated P. falciparum malaria: potent antimalarial activity combined with apparently low resistance selection pressure, and, within the limitations of the studies outlined here, low toxicity. The lack of antagonism or cross-resistance with existing drugs allows some flexibility in its use in combination therapy. The synthesis of GW844520 on a multigram scale has proved relatively straightforward and the compound exhibits good solid state stability – these being important aspects in the development of an affordable antimalarial drug that is also suitable for endemic areas. While there appear to be some similarities in the mode of action of GW844520 and atovaquone, the lack of cross-resistance and a dramatically lower propensity for the generation of resistance differentiates 4(1H) -pyridones from atovaquone. Depending on the outcome of formal toxicity studies, it is hoped that the first studies in humans will start in 2005.