Leishmaniasis is caused by the protozoan parasite Leishmania spp. and affects 12 million people worldwide, with 50 000 deaths annually attributed to it. Leishmania spp. relies on ergosterol for survival and an important step in its biosynthesis is catalysed by CYP710C1. CYP710C1, found in Leishmania spp., belongs to the superfamily of cytochromes P450 and introduces a double bond at the C22 position in a sterol precursor to form ergosterol. P450 catalysed sterol dehydrogenation is less common compared with the mono-oxygenations catalysed by most P450s. The natural substrate(s) of CYP710C1 and the mechanism of Δ22 dehydrogenation in ergosterol biosynthesis are unknown. Hence, a range of potential substrates and chemical probes were prepared to study CYP710C1. It was envisioned that this would lead us to the design of specific inhibitors for CYP710C1 which could potentially be used as anti-leishmaniasis chemotherapeutics.
The synthesis of two potential substrates for CYP710C1, ergosta-5,7-dien-3β-ol (1) and ergosta-5,7-24(28)-trien-3β-ol (2) was investigated. A synthetic route established in literature was adapted to prepare 1 and an extra step utilising the characteristic homoannular Δ5,7 diene system of the sterol precursor was introduced to isolate the desired target efficiently. A new synthetic pathway was developed to synthesise 2 and this pathway is envisioned to be more versatile and practical in generating a variety of substrate analogues. This is particularly important for enzymatic studies to investigate the flexibility of the active site of CYP710C1 in binding various substrate analogues. A deuterium labelling study was initiated to examine the dehydrogenation mechanism of CYP710C1. A range of methods were explored to incorporate deuterium regio- and/or stereospecifically into 1.
A deuterated analogue of 1 was synthesised via reductive reactions with deuterated reducing agents. Regiospecific deuteration was achieved through Barton-McCombie deoxygenation and the compound will be a useful chemical probe for mechanistic studies of CYP710C1. In addition, the synthetic methodologies developed for deuterium incorporation into 1 provides an excellent alternate route to synthesise 1 if a non-deuterated reducing agent was employed.
The synthesis of a series of inhibitors for CYP710C1, CYP125A1 and CYP142A1 were explored. CYP125A1 and CYP142A1 are P450s in Mycobacterium tuberculosis that catalyse the ω-hydroxylation of cholesterol. It is known that azole drugs are common inhibitors for P450s and therefore, presumably they should work against these enzymes, as observed for other P450s. Conventional azole drugs that do not possess structural resemblance to the substrates may lack specificity in action and therefore it was postulated that by having the azole component attached to the substrate core, an increase in efficacy of the inhibitor would be observed. Hence, imidazole derivatives with ergosterol, cholesterol and lanosterol cores were synthesised via the displacement of the corresponding mesylate with imidazole. The imidazole group was positioned at C22 and C24 in the steroidal side chain to examine the flexibility of the active site in accepting different aliphatic chain lengths. Two other sterol inhibitors that bear a terminal acetylene or a geminal difluoromethyl group in the side chain were also synthesised as potential inhibitors for CYP125A1 and CYP142A1. The binding affinities of the inhibitors for the enzymes were then investigated. The imidazole derivatives of sterols exhibited low selectivity although they bound tightly to the enzymes. These inhibitors would be useful for preliminary inhibition studies but they would not be good specific inhibitors. Further investigation is required to confirm the potential of the acetylenic sterol and the geminal difluoromethyl sterol as irreversible inhibitors for CYP142A1 and CYP125A1.