African trypanosomiasis: Synthesis & SAR enabling novel drug discovery of ubiquinol mimics for trypanosome alternative oxidase

Abstract

African trypanosomiasis is a parasitic disease affecting 5000 humans and millions of livestock animals in sub-Saharan Africa every year. Current treatments are limited, difficult to administer and often toxic causing long term injury or death in many patients. Trypanosome alternative oxidase is a parasite specific enzyme whose inhibition by the natural product ascofuranone (AF) has been shown to be curative in murine models. Until now synthetic methods to AF analogues have been limited, this has restricted both understanding of the key structural features required for binding and also how this chemotype could be developed to an effective therapeutic agent. The development of 3 amenable novel synthetic routes to ascofuranone-like compounds is described. The SAR generated around the AF chemotype is reported with correlation to the inhibition of T. b. brucei growth and corresponding selectivity in cytotoxic assessment in mammalian HepG2 cell lines. These methods allow access to greater synthetic diversification and have enabled the synthesis of compounds that have and will continue to facilitate further optimisation of the AF chemotype into a drug-like lead.

Keywords: Antiprotozoal agents; Medicinal chemistry; Oxidoreductase; Synthesis design.

Graphical abstract

Fig. 1

Current HAT treatments.

Fig. 2

Ascofuranone and previous synthetic starting points.

Scheme 1

Lewis acid catalysed synthesis: a) NEt₃, AcCl, CH₂Cl₂, 68%; b) geranyl bromide, K₂CO₃, KI, DMF, 50%; c) Florisil, toluene, 28%; d) NaOH, THF/MeOH, 97%.

Scheme 2

Ortho-lithiation syntheses: a) NaH, MOMCl, DMF, 93%; b) 1) n-BuLi, geranyl bromide, THF 2) n-BuLi, DMF, 48%; c) n-BuLi, bromooctane, THF, 0 °C, 95%; d) ethylene glycol, mw 160 °C, 98%; e) POCl₃, DMF, 96%; f) SO₂Cl₂, CH₂Cl₂, quant.

Scheme 3

Diversifiable synthesis: a) NaH, ^tBu-acrylate, DMF, 80 °C, 95%, b) octanal, l-proline, NaBH₃CN, EtOH, quant; c) SO₄Me₂, NaH, THF, 95%; d) LDA, methyl formate, THF, −78 °C, 64%; e) DDQ, CH₂Cl₂, 84%.

Fig. 3

SDS-PAGE and Western analysis of rTAO with Quick Coomassie stain (Generon QC154201/008). 1 Molecular weight markers, 2 Concentrated purified rTAO protein, 3 WB 100 ng protein load. Polyclonal Ab from Agrisera (raised against plant AOX1/AOX2 (AS04 054) >70% homology to TAO).

Fig. 4

Michalis-Menten kinetics of rTAO. Data plotted is the mean of 3 independent experiments each with triplicate points for each condition. The error plotted is the SEM of the 3 independent experiments. The relative background turnover of substrate without enzyme was subtracted from each condition internally generated within each experiment in triplicate. 3 nM of purified rTAO was used. Graph generated in GraphPad Prism^®.

Fig. 5

IC₅₀ data for colletochlorin B from 4 parameter logistic regression of 10 point inhibitory concentration data. Data from 3 independent experiments reported concentrations run in duplicate per experiment, SEM plotted of experiments. 3 nM of purified rTAO was used.

Fig. 6

IC₅₀ data for concentration of colletochlorin B required to inhibit 50% growth of T. b. brucei Lister427, following incubation with compound for 48 h. Growth inhibition was measured by quantifying ATP levels. 4 parameter logistic regression of 10 point inhibitory concentration data. Data from 3 independent experiments reported concentrations run in duplicate per experiment, SEM plotted of experiments.

Fig. 7

Michalis-Menten inhibitor kinetics of rTAO, in presence of increasing concentration of inhibitor CCB. Data plotted is the mean of 3 independent experiments each with triplicate points for each condition. The error plotted is the SEM of the 3 independent experiments. The relative background turnover of substrate without enzyme was subtracted from each condition internally generated within each experiment in triplicate. 2 nM of purified rTAO was used. Graph generated in GraphPad Prism^®.

Fig. 8

Dimethoxy analogue.