Strategies to inhibit steroid cytochrome P450 enzymes to benefit human health: development of steroid ligands for P450s 17A1, 19A1, and 8B1 to treat cancer and obesity

Abstract

Several human cytochrome P450 enzymes (P450s) found in steroid/oxysterol biosynthesis are therapeutic targets to treat disease. This review article describes current research strategies to develop various inhibitors of three steroid P450s (P450s 17A1, 19A1, and 8B1) in order to benefit human health. (i) P450 17A1 (17α-hydroxylase/17,20-lyase) activity involves the hydroxylation at C17 and cleavage of the 17-20 bond to yield androgens. Abiraterone and galeterone are steroid inhibitors of P450 17A1, which both bear heterocycles (pyridine and benzimidazole) at C17 of the steroid moiety, the location of the enzymatic activity of P450 17A1. (ii) P450 19A1, the enzyme also known as aromatase, catalyzes the cleavage of the C10-C19 bond of androgens to give estrogens. Exemestane, which has the steroid structure of an androgen possessing an exocyclic methylene at C6, is a successful inhibitor of P450 19A1 used to treat breast cancer. (iii) P450 8B1 is the oxysterol-12α-hydroxylase enzyme that catalyzes the hydroxylation of the C12 position of its steroid based substrates. The hydroxylation of the C12 position ultimately forms the bile acid, cholic acid, which has implications in obesity. Mice lacking the gene for the expression of P450 8B1 resist weight gain and the inhibition of P450 8B1 activity has been suggested as a potential treatment of obesity. Studies towards a rationally designed inhibitor of P450 8B1 are described. This research in medicinal chemistry combines expertise in both organic synthesis and biochemistry, with the goal to improve human health.

Fig. 1. (A) Non-steroidal P450 inhibitors to benefit human health: osilodrostat (1), orteronel (2), letrozole (3), anastrozole (4), ketoconazole (5), and fluconazole (6). (B) Steroid-based P450 inhibitors used to treat human diseases: abiraterone (7), galeterone (8), and exemestane (9). Shown in the figure is 2S,4R-(+)-ketoconazole but the compound is used as a racemic mixture.

Fig. 2. Cholesterol (10) is either (A) converted to pregnenolone (11) by P450 11A1 or (B) hydroxylated by P450 7A1 to yield 7α-hydroxycholesterol (13), which is converted to 7α-hydroxycholest-4-en-3-one (14), the oxysterol precursor to bile acids.

Fig. 3. The inhibition of certain steps in steroid metabolism can lead to therapeutic strategies. The side chain of cholesterol is cleaved by P450 11A1 to pregnenolone (11). Pregnenolone is converted to aldosterone (18), cortisol (22), testosterone (21), or estradiol (26). Inhibitors are employed in these pathways to treat disease including: (A) osilodrostat (1), an inhibitor of P450 11B1 and 11B2 to treat Cushing's disease, (B) abiraterone (7), which blocks P450 17A1 activity to treat prostate cancer, and (C) exemestane (9), which inhibits P450 19A1 to treat breast cancer.

Fig. 4. Cholesterol (10) is also converted to 7α-hydroxycholest-4-en-3-one (14), which is the substrate of P450 8B1. P450 8B1 catalyzes the 12α-hydroxylation of 7α-hydroxycholest-4-en-3-one (14) to yield 7α,12α-dihydroxycholest-4-en-3-one (27), the precursor of cholic acid (36). The subsequent 9 steps result in the formation of cholic acid (36). The inhibition of P450 8B1, the oxysterol 12α-hydroxylase enzyme, has been suggested as a strategy to treat obesity.

Fig. 5. Reactions catalyzed by P450 17A1, including 17α-hydroxylase and 17,20-lyase activities on 21-carbon steroid substrates.

Scheme 1. First synthesis of abiraterone (7) via Suzuki coupling of 17-enol triflate (37) with diethyl(3-pyridyl)borane (39).

Scheme 2. Large-scale synthesis of abiraterone acetate (40) via Suzuki coupling of 17-vinyl iodide 42 with diethyl(3-pyridyl)borane (39).

Scheme 3. Tosylhydrazone-based, halogen-free approach to abiraterone acetate (40) via direct coupling with 3-bromopyridine (45).

Scheme 4. Reversed-polarity Suzuki coupling using steroidal boronic acid 46 with 3-bromopyridine (45).

Fig. 6. Summary of the different methods to synthesize abiraterone (7).

Scheme 5. Synthesis of galeterone (8) from dehydroepiandrosterone (DHEA) acetate (37) by the Njar research group.

Scheme 6. Alternative proposal to synthesize galeterone (8) through an allylic carbonate intermediate 57.

Scheme 7. Alternative routes to synthesize galeterone from (A) a Buchwald–Hartwig cross-coupling reaction or (B) a Chan–Lam coupling reaction to form the C–N bond at C17.

Fig. 7. (A) P450 17A1 with abiraterone (PDB ID: 3RUK). A hydrogen bond between N202 (asparagine-202) of P450 17A1 and the C3-hydroxy of abiraterone is shown with a distance of 2.7 Å. The pyridine nitrogen of abiraterone is 2.0 Å away from the iron active site of P450 17A1. (B) P450 17A1 with galeterone bound (PDB ID: 3SWZ). Structures of P450 17A1 bound with (C) pregnenolone (PDB ID: 4NKW) and (D) 17α-hydroxypregnenolone (PDB ID: 4NKZ) in the active site. Red “+” signs in Fig. 7C and D are water molecules.

Fig. 8. Reaction catalyzed by P450 19A1 aromatase converting androgens to estrogens in 3 steps: the first two steps involve hydroxylation at C19 then the third step results in the cleavage of the C10–C19 bond to release formic acid and estrogen (estrone and estradiol from androstenedione and testosterone, respectively).

Scheme 8. Synthesis of exemestane (9) from 17β-hydroxy-androsta-1,4-diene-3-one (62).

Scheme 9. Synthesis of exemestane (9) from androstenedione (24) using 6-methylenation followed by DDQ dehydrogenation of C1. DDQ: 2,3-dichloro-5,6-dicyano-1,4-benzoquinone.

Scheme 10. Synthesis of exemestane (9) from androstenedione (27) through a tribromide intermediate 65. pTsOH: para-toluenesulfonic acid; TEOF: triethylorthoformate; DABCO: 1,4-diazabicyclo[2.2.2]octane, DMF: dimethylformamide.

Scheme 11. Synthesis of exemestane (9) from testosterone (25). pTsOH: para-toluenesulfonic acid; TEOF: triethylorthoformate; BSTFA: bis(trimethylsilyl)trifluoroacetamide; TfOH: trifluoromethanesulfonic acid (triflic acid).

Scheme 12. Hexacyclic products 70 and 71 formed when DDQ and p-chloranil were used to dehydrogenate C1 of the steroid precursor 64.

Fig. 9. Exemestane (9) bound to P450 19A1 (PDB ID: 3S7S). (A) Serine-478 (S478) of P450 19A1 interacts with the exocyclic methylene of exemestane with a distance of 4.78 Å, suggesting a possible nucleophilic attack of the residue of the electrophilic site of exemestane. Aspartate-309 (D309) hydrogen bonds to the C3-ketone oxygen of exemestane (9) – the distance is 3.0 Å between the aspartate and the C3-oxygen. The amide nitrogen backbone of Met-374 hydrogen bonds to the C17-ketone of exemestane with a distance of 2.7 Å. (B) The nitrogen atoms in the porphyrin ring of P450 19A1 are in close proximity to the C1 position of exemestane, a potential Michael acceptor.

Fig. 10. Potential exemestane (9) inhibition of P450 19A1 through heme adduction at the electrophilic C1 position of exemestane. The illustration above shows a possible heme adduct formation through nucleophilic attack of the porphyrin nitrogen lone pair from P450 19A1 at C1 of exemestane.

Fig. 11. Reaction catalyzed by P450 8B1, giving rise to cholic acid (36). See Fig. 4 for the 9 steps to cleave C24–C25 (14 to 72 and 27 to 36).

Scheme 13. Synthesis of C12-pyridine containing steroids 77, 78, and 79 from dehydroepiandrosterone (23).

Fig. 12. (A) P450 8B1 bound to a C12-pyridyl steroid (compound 78) (PDB ID: 8EOH). (B) Hydrogen bonding interactions between (i) W281 of P450 8B1 and the 3-keto-oxygen on compound 78 (4.3 Å), and (ii) Y102 of P450 8B1 and the pyridine nitrogen of the steroid compound 78 (2.6 Å) (PDB ID: 8EOH). (C) P450 8B1 bound to (S)-tioconazole (PDB ID: 7LYX) – the hydrogen bond between D210 and W281 is shown (3.2 Å). (D) Y102 of P450 8B1 is hydrogen bonding to a glycerol molecule (2.4 Å) (PDB ID: 7LYX).

Fig. 13. Cartoon representation of the crystal structures of P450 8B1 with (A) C12-pyridyl steroid 78 bound (PDB 8EOH) and (B) (S)-tioconazole (80) bound along with a glycerol molecule (81) that hydrogen bonds to Y102 (tyrosine-102) (PDB 7LYX).

Scheme 14. Steroids with a pyridine fused at C11 and C12 were synthesized (82, 83, 84, 85, 86, and 87) to potentially test the hypothesis of the tryptophan-281 ceiling in P450 8B1.

Tu M. Ho

Francis K. Yoshimoto