‘Slow-binding’ sixth-ligand inhibitors of cytochrome P-450 aromatase. Studies with 19-thiomethyl- and 19-azido-androstenedione

Abstract

The progress curves for the inhibition of aromatase by 19-thiomethylandrostenedione and 19-azidoandrostenedione were found to be non-linear where the extent of inhibition increased with time. Further experiments enabled these compounds to be classified as 'slow-binding' inhibitors of aromatase. The phenomenon was attributed to the formation of an initial E.I complex that rearranged to another species (E.I*) in which the interaction between the enzyme and inhibitor had been maximized, giving rise to tighter binding. When 19-thiomethylandrostenedione was used as the inhibitor the t0.5 (half-time) for the dissociation of E.I* was calculated to be 12.6 min with Ki and Ki* values of 2.4 and 1.4 nM respectively. In the case of 19-azidoandrostenedione, the two separate dissociation constants were not determined, and a single Ki value of 5 nM was obtained. The conclusions drawn from kinetic studies were confirmed by absorption spectrometry, when time-dependent formation of complexes between aromatase and either 19-thiomethylandrostenedione or 19-azidoandrostenedione were observed by the formation of 'Type II' spectra. The two complexes respectively had maxima at 429 and 418 nm. The spectral data suggested that the two inhibitors interact with the haem iron of aromatase, forming hexaco-ordinated species for which structural models are presented.

A review of mechanistic studies on aromatase (CYP19) and 17α-hydroxylase-17,20-lyase (CYP17)

“…We reasoned that such compounds should be strong and specific aromatase inhibitors. Accordingly, two steroids, 19-thiomethyl-and 19-azido-androstenedione (29 and 30, Scheme 8) fulfilling these criteria were discovered in 1985 [86,87] and their mode of inhibition established by kinetics and spectroscopic studies [88]. These compounds, 19-thiomethyland 19-azido-androstenedione had K i values of 1 × 10 −9 and 5 × 10 −9 M, respectively, and interacted with aromatase as shown in 31 and 32 (Scheme 8).…”

Novel 10β-aziridinyl steroids; inhibitors of aromatase

“…Furthermore, the 19R-compounds 1 and 3 show4 spectroscopic evidence for coordination of the epoxide or thiirane heteroatoms with the enzyme's heme iron. Consequently, we embarked on the synthesis of the hitherto undescribed aziridinyl steroids 17, 18, 19 and 20 (Scheme 2 and 3) thinking that they might show strong coordination of the aziridine nitrogen to the heme iron of aromatase. We also considered the possibility that, because of the reactivity of the aziridine ring,' these aziridinyl steroids might react covalently at the active site of the enzyme, resulting in irreversible enzyme inhibition.…”

“…We also considered the possibility that, because of the reactivity of the aziridine ring,' these aziridinyl steroids might react covalently at the active site of the enzyme, resulting in irreversible enzyme inhibition. These compounds are also especially interesting because attempts to prepare steroidal 19-primary amino-4-en-3-ones have been unsuccessful, although 19-amido 6-8 and 19tertiary amino steroids have been described. This paper describes the synthesis of novel aziridinyl steroids, and their evaluation as inhibitors of microsomal aromatase.…”

In vitro inhibition of the estrogen biosynthesis enzyme system with new steroid derivatives

“…Taking into account the aromatase inhibitory effect of 19-azido-4-androstene-3,17-dione [Wright et al 1991] and certain P45017α inhibitor abiraterone analogs [Brossard et al 2013], we performed tests whether our 17-azido compounds are capable of inhibiting this enzyme. Incubations with high concentrations (10 µM and 50 µM) resulted in RC-s similar to the non-inhibited controls.…”

“…The neuroactive 17αand 17β-azido-5β-androstan-3α-ol were studied by Vidrna et al [Vidrna et al 2011]. hormone metabolism [Wright et al 1991]. They have found reversible inhibitor binding and hypothesized that the first nitrogen atom in the azide group (attached directly to the C-19 carbon) is most likely to be involved in coordination to the heme iron of aromatase.…”