Selective cleavage of an unactivated C-C bond is chemically difficult. However, the highly specialized steroidogenic cytochromes P450 aromatase and lanosterol-14a-demethylase accomplish this difficult transformation with relative ease. In the case of aromatase, androstenedione (72) is converted to estrone (73) by three sequential oxidations of the C-19 methyl group, culminating in the elimination of formic acid and the aromatization of the A ring (eqn ). While the first two steps (sequential hydroxylations of C-19 to form a hydrated aldehyde) are well understood, the final oxidation leading to loss of formic acid and aromatization is not. An attractive possibility suggested by Akhtar et al.111 is that an O2-derived peroxide might be involved in the final C-C bond cleavage step.
To investigate this possibility, Vaz etal.112 used cyclohexanecarboxaldehyde (74) as a simple model of the C-19 aldehyde of androstendione. Upon treatment with CYP2B4, NADPH, and cytochrome P450 reductase, the aldehyde-cyclohexyl ring C-C bond of 74 was cleaved, forming cyclohexene (75) and formic acid (eqn ). The reaction was supported if hydrogen peroxide replaced NADPH and cytochrome P450 reductase, but was not supported if other oxidants such as iodosobenzene, m-perchlorobenzene, and cumyl hydroperoxide were used. The authors propose that an O2-derived heme-iron-bound peroxide attacks the carbonyl carbon, to form an enzyme-bound peroxyhemiacetal-like intermediate (76). The intermediate rearranges either by a concerted or sequential mechanism, to yield the observed products.
In a subsequent paper, Roberts etal.113 found that CYP2B4 would selectively deformylate a number of simple a- or b-branched-chain, but not normal-chain, aldehydes, to generate alkenes. Still later, Vaz and co-workers114-117 provided strong evidence that cytochrome P450 heme FeO2 , or FeO2H2 +, rather than FeO3 +, is the active oxidant in deformylation reactions.
The oxidation of terminal aryl alkynes by cytochrome P450 forms the corresponding substituted aryl acetic acid. Early on it was shown118 that if the alkyne hydrogen atom of 4-ethynylbiphenyl was replaced with deuterium (77), it would be quantitatively retained on the a-carbon of the acid metabolite (79) (Scheme 7). The reaction proceeds by heme FeO3 + addition to the terminal acetylenic carbon, with concerted migration of the hydrogen atom to the adjacent carbon atom, leading to the formation of a substituted ketene (78). The reactive ketene is hydrolyzed, to generate the final product (79). If heme FeO3 + adds to the inner carbon of the acetylene group, the reaction takes an entirely different course. This latter reaction pathway leads to alkylation of a heme nitrogen, and destruction of the
5.05.2.1.5 Oxidation at a heteroatom
5.05.2.1.5.1 Mechanism for oxidation at a nitrogen atom
It is clear from the literature that while N-oxides and N-hydroxylated compounds are observable metabolites, they are minor relative to the products of N-dealkylation. They appear to only become more significant when N-dealkylation is not an option.57 A caveat, however, is that N-hydroxy compounds are not all that stable, particularly in vivo, where they can be reduced back to the amine or further oxidized to even less stable compounds. The degree to which they contribute to nitrogen metabolism may be underestimated because of their relative instability.
N-Hydroxylation is most frequently found occurring with primary and, to a lesser extent, secondary alkyl and aryl amines or with aryl amides. For example, the b-phenethylamine phentermine (80),121 the arylamine dapsone (82),122 and the aryl amide 2-acetoaminofluorene (84)123 are all oxidized, to yield significant amounts of the corresponding N-hydroxy metabolite (eqns -).
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