Synthesis of halogenated pregnanes, mechanistic probes of steroid hydroxylases CYP17A1 and CYP21A2

Abstract

The human steroidogenic cytochromes P450 CYP17A1 (P450c17, 17α-hydroxylase/17,20-lyase) and CYP21A2 (P450c21, 21-hydroxylase) are required for the biosynthesis of androgens, glucocorticoids, and mineralocorticoids. Both enzymes hydroxylate progesterone at adjacent, distal carbon atoms and show limited tolerance for substrate modification. Halogenated substrate analogs have been employed for many years to probe cytochrome P450 catalysis and to block sites of reactivity, particularly for potential drugs. Consequently, we developed efficient synthetic approaches to introducing one or more halogen atom to the 17- and 21-positions of progesterone and pregnenolone. In particular, novel 21,21,21-tribromoprogesterone and 21,21,21-trichloroprogesterone were synthesized using the nucleophilic addition of either bromoform or chloroform anion onto an aldehyde precursor as the key step to introduce the trihalomethyl moieties. When incubated with microsomes from yeast expressing human CYP21A2 or CYP17A1 with P450-oxidoreductase, CYP21A2 metabolized 17-fluoroprogesterone to a single product, whereas incubations with CYP17A1 gave no products. Halogenated steroids provide a robust system for exploring the substrate tolerance and catalytic plasticity of human steroid hydroxylases.

Figure 1

Halogenated steroid substrates (1–18) synthesized for incubation with steroidogenic cytochromes P450, CYP17A1 and CYP21A2.

Figure 2

Explanation of C-17 stereochemistry of 21-F₃-pregnenolone – AB ring system omitted for clarity.

Figure 3

In vitro studies of 17-fluoroprogesterone as a substrate for CYP21A2. HPLC traces of incubations: (A) 17-fluoroprogesterone (1) incubation with CYP21A2, (B) 17-fluoroprogesterone (1) incubation with CYP21A2 and ketoconazole, (C) progesterone incubation with CYP21A2, (D) progesterone incubation with CYP21A2 and ketoconazole. The formation and migration of compound F1 is consistent with 17-fluoro-21-hydroxyprogesterone. Conversion of 17-fluoroprogesterone to F1 was inhibited 65% by pre-incubation with ketoconazole, and conversion of progesterone to 11-deoxycorticosterone was inhibited 40% by ketoconazole using the same conditions. The peak at 30 min is due to the change in solvent gradient.

Scheme 1

Synthesis of 21-F₃-progesterone via Zard methodology:

Scheme 2

21-Cl₃-pregnenolone-3-trichloroacetate (9) synthesis using trichloroacetic anhydride:

Scheme 3

21-trihaloprogesterone syntheses using haloform:

Scheme 4

17-haloprogesterone syntheses:

Scheme 5

21-monobromoprogesterone synthesis:

Scheme 6

16,17-dehydro-21-fluoroprogesterone synthesis: