Demethylation, of lanosterol

C]-(35)-2,3-epoxysqualene and its racemate have been prepared by two routes in a metabolically non-labile position relative to the demethylation of lanosterol to cholesterol (equation 70 and 71). The racemic [24,30-14C]-2,3-epoxysqualene, 192, has been obtained163 by condensation of (35, 3/ )-2,3-epoxytrisnorsqualene aldehyde 193 with freshly prepared 14C-labelled isopropylidenephosphorane, 194 (equation 70). 

Figure 22-8 Steps in the demethylation of lanosterol. The most frequent sequence, labeled [1], begins with demethylation at C-14 by the action of a cytochrome P450 and is followed [2] by the successive demethylation of the 0C-CH3 and (S-CH3 at C-4 by an NADH-dependent oxygenase.

Fig. 12. Deformylation of C(19) 14a-demethylation of lanosterol 64 side chain cleavage of cholesterol 66...

Except for aromatization, the 14a-demethylation of lanosterol 64 seems to operate by the same sequence of events in order to remove the angular C(14) methyl group with concomitant introduction of the C(14) double bond to furnish 65 (Fig. 12). 

Mode of action Ketoconazole interacts with C-14 a-demethylase (a cytochrome P-450 enzyme) to block demethylation of lanosterol to ergosterol, the principal sterol of fungal membranes Figure 34.4). This inhibition disrupts membrane function and increases permeability. Ketoconazole acts in an additive manner with flucytosine against Candida, but antagonizes amphotericin B s antifungal activity. 

In our laboratory, the major sterol biosynthesised in untreated extracts was the triene (VIII) (1J), rather than ergosterol Itself which is, of course, the end product of the pathway in intact cells. It should be noted that VIII arises directly from 14-demethylation of lanosterol. In the presence of 0.1 yM prochloraz (or even 0.01 pM prochloraz in some experiments) the concentration of VIII was significantly reduced, while the level of lanosterol Increased (4) indicating clearly that prochloraz inhibited 14-demethylation. At higher fungicide concentrations both the triene and ergosterol were totally absent and only lanosterol was present. 

Since ergosterol is used in the formation of the leishmanial cell membrane, inhibition of ergosterol biosynthesis has been considered as a useful target for chemotherapeutic attack. Allylamines (eg. terbinafine) and imidazole antifungals (eg. ketoconazole) have been found to interfere with different steps in the biosynthetic pathway of C28 sterols in leishmania and fungi. Allylamines inhibit the microsomal squalene 2,3-epoxidase and, therefore, inhibit the synthesis of squalene epoxide, the precursor of lanosterol. Imidazoles, on other hand, inhibit cytochrome P-450 dependent C-14 demethylation of lanosterol leading to decreased or no synthesis of ergosterol [30]. 

As indicated, the oxidative demethylation of lanosterol in rat liver preparations is inhibited by CO. However, if 32-hydroxylanosterol is used as substrate, CO no longer inhibits. This indicates that in animals cytochrome P-450 catalyzes only the first oxidation and that other cytochromes are used in the subsequent steps. Apparently, the yeast and liver systems are different [5]. 

Figure 6.42. A possible mechanism for the final step in the 14a-demethylation of lanosterol that employs the isolated Baeyer-Villiger rearrangement product. Radical decomposition of the peroxyhemiacetal intermediate may also lead to the observed demethylated product.

J.L. Gaylor (1986). Oxidative demethylation of lanosterol in cholesterol biosynthesis Accumulation of sterol intermediates. J. Lipid Res. 27, 1-10. 

Aoyama, Y., Y. Yoshida, S. Hata, T. Nishino, and H. Katsuki (1983). Buthiobate A potent inhibitor for yeast cytochrome P450 catalysing 14a-demethylation of lanosterol. Biochem. Biophys. Res. Commun. 115,642-647. 

A) It is highly effective in treatment of aspergillosis It does not penetrate the blood-brain barrier Its oral bioavailability is less than that of ketoconazole It inhibits demethylation of lanosterol It is a potent inhibitor of hepatic drug-metabolizing enzymes... 

Inhibition of Demethylation of Lanosterol at C-14 and Other Effects on Lipid Metabolism ... 

The cytochrome P450-catalyzed cleavage of a carbon-carbon (C-C) bond has long been of interest because of the key role this transformation plays in the biosynthesis of cholesterol and all the sterol hormones derived from it. These reactions include the 14a-demethylation of lanosterol by CYP51, truncation of the cholesterol side chain... 

Scheme 12.5 Demethylation of lanosterol (16) to 4,4-dimethyl-5a-cholesta-8,14,24-diene-3 3-ol (19) catalyzed by lanosterol 14a-demethylase (CYP51).

Another example for the cleavage of C-C bonds via multiple substrate oxidations is the demethylation of lanosterol to a precursor of cholesterol, 4,4-dimethyl-5a-cholesta-8,14,24-diene-3p-ol, catalyzed by a lanosterol 14a-demethylase (CYP51) [39]. The mechanism includes three steps and proceeds via initial hydroxylation of the C14 methyl group (corresponds to C32 hydroxylation), followed by further oxidation of the alcohol to the aldehyde. Finally, acyl cleavage occurs, leading to the formation of a double bond in the steroid (Scheme 5.8). 

Scheme 5.8 Demethylation of lanosterol via multistep oxidations catalyzed by CYP51.

This response has been attributed to a decreased rate of HMG-CoA reductase synthesis (28-30) and in some instances to an increase in enzyme degradation (28-30). Other oxysterols are known to depress the rate of cholesterol synthesis from lanosterol in rat liver homogenates and may inhibit the 14-demethylation of lanosterol (31-33). 

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