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

Tu M Ho; Francis K Yoshimoto

doi:10.1039/d5md00954e

. 2025 Dec 29. Online ahead of print. doi: 10.1039/d5md00954e

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

Tu M Ho ^a, Francis K Yoshimoto ^a,^✉

PMCID: PMC12809264 PMID: 41551023

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.

Several human cytochrome P450 enzymes (P450s) found in steroid/oxysterol biosynthesis are therapeutic targets to treat disease.

1. Overview of steroidogenic cytochrome P450s

Cytochrome P450 enzymes (P450s) are a superfamily of 57 enzymes in humans that catalyze diverse oxidative transformations. Because of their essential functions in steroid and sterol metabolism, several P450 enzymes have become attractive drug targets, with inhibitors already developed for endocrine, oncologic, and metabolic diseases.^1,2 This review article mainly focuses on the following three steroidogenic cytochromes P450: P450 17A1 (17α-hydroxylase/17,20-lyase), P450 19A1 (aromatase), and P450 8B1 (oxysterol-12α-hydroxylase).

The inhibition of certain cytochrome P450s has already provided clinical benefit to treat human diseases. For instance, P450 11B1 (11β-hydroxylase) has been inhibited with osilodrostat (Fig. 1, 1) to treat Cushing's disease,^3–5 while orteronel (2) is being investigated for the treatment of prostate cancer through the inhibition of P450 17A1 (17α-hydroxylase/17,20-lyase).⁶ Non-steroidal aromatase inhibitors such as letrozole (3) and anastrozole (4)⁷ have become mainstays in hormone-dependent breast cancer therapy.^8,9 In addition, non-human P450s such as P450 51A1 from Candida albicans have been targeted by antifungal agents such as ketoconazole (5) and fluconazole (6), both heterocyclic compounds inhibit P450 51A1 (lanosterol 14α-demethylase) activity.^10–12 In addition to non-steroidal inhibitors (Fig. 1A), several steroid-based compounds have been developed to block cytochrome P450 enzymes (Fig. 1B). For example, abiraterone (Fig. 1, 7) is a selective inhibitor of P450 17A1 that is FDA-approved for the treatment of androgen-dependent prostate cancer.^13,14 Galeterone (8) is another steroid inhibitor with a heterocycle at C17 that blocks the activity of P450 17A1. The presence of the heterocycle in the ligand results in the displacement of the axial water molecule in the resting state of the enzyme and density functional theory (DFT) calculations have suggested an exothermic reaction of 5.3 kcal mol⁻¹ upon ligand binding.¹⁵ While other intermolecular interactions such as hydrogen bonding,¹⁶ π–π,¹⁷ cation–π,¹⁸ and CH–π¹⁹ interactions (interaction energies of −0.5 to −4 kcal mol⁻¹) between the ligand and protein are possibilities, the Lewis base–Lewis acid interaction between the heterocyclic nitrogen lone pair and the iron active site seem to be the common motif for P450 inhibitor design. Benefits of using a heterocycle on the ligand that targets the iron center of the P450 enzymes are the strength of the stabilization of energy that results and also the fact that this interaction directly targets the active site of the protein, where catalysis occurs at the iron center. However, cation–π interactions have been suggested to be an additive effect in stabilizing the inhibitor in the active site of the P450 protein (cf. quinoline-4-carboxamide with P450 2C9).^20,21 Hydrogen bonding interactions are also commonly found between the ligand and the P450 enzyme,²² but these interactions do not distinguish between an inhibitor and a substrate. For instance, abiraterone has a hydrogen bonding interaction with asparagine-202 (N202) of P450 17A1, which is the same interaction found between the 3-hydroxy group of the steroid substrate, pregnenolone, and P450 17A1.

In contrast, exemestane (9), which lacks a heterocycle, is a steroid inhibitor of P450 19A1 (aromatase) and is clinically approved for the treatment of estrogen-dependent breast cancer.^23,24 Previous studies has suggested that exemestane is a mechanism-based inhibitor, where the catalytic activity of P450 19A1 onto exemestane causes an irreversible inhibition of the enzyme^25,26 in a time dependent manner.

Understanding the biochemical pathways and the enzymes involved in the production of steroids facilitates the development of therapeutic treatments for human disease. For instance, treatment of androgen-dependent prostate cancer patients with abiraterone may be beneficial to shut down P450 17A1 activity to minimize the production of androgens – however, by inhibiting P450 17A1 activity, glucocorticoids (e.g. cortisol) are also depleted because P450 17A1 is found relatively upstream in the biosynthesis of glucocorticoids, androgens, and estrogens. Therefore, in addition to abiraterone, patients must also take prednisolone to replace the loss of corticosteroids when P450 17A1 activity is blocked.

Cholesterol (10) is the common biosynthetic precursor for steroid hormones (i.e. glucocorticoids, mineralocorticoids, androgens, and estrogens) and bile acids^27,28 (e.g. cholic acid, chenodeoxycholic acid). Steroid biosynthesis begins with the cleavage of the side chain of cholesterol at C20–C22 by P450 11A1 (Fig. 2A) to yield isocaproaldehyde (12) and pregnenolone (11),²⁹ the first 21-carbon steroid hormone precursor. Alternatively, cholesterol is hydroxylated by P450 7A1 (Fig. 2B) to yield 7α-hydroxycholesterol (13) then oxidized at C3 and isomerized by 3β-hydroxysteroid dehydrogenase (3β-HSD) to give 7α-hydroxycholest-4-en-3-one (14).

As described below, pregnenolone is converted to mineralocorticoids (aldosterone), glucocorticoids (cortisol), androgens (testosterone), and estrogens (estradiol) (Fig. 3).

• Pregnenolone (11) to aldosterone (18): pregnenolone (11) is the substrate of 3β-hydroxysteroid dehydrogenase (3β-HSD) to yield progesterone (Fig. 3, 15). The enzyme has a broad steroid substrate scope and generally converts its substrates that have the 3β-hydroxy-Δ^5,6-steroid backbone to the 3-keto-Δ⁴-steroid moiety. Progesterone (15) undergoes 21-hydroxylation to deoxycorticosterone (16) by P450 21A2. Interestingly, P450 21A2 deficiency is related to a clinical disorder: congenital adrenal hyperplasia (CAH), which is a reminder that enzymatic activity does not always need to be inhibited for maintaining homeostasis. Deoxycorticosterone (16) is converted to corticosterone (17) through 11β-hydroxylation by both P450s 11B1 and 11B2, the targets of osilodrostat (1) (Fig. 3A). P450s 11B1 and 11B2 have 93% sequence identity but only P450 11B2 oxidizes the C18 methyl of corticosterone (17) to the C18-aldehyde to form aldosterone (18).³⁰

• Progesterone (15) to cortisol (22): progesterone (15) is also a substrate for P450 17A1 and is converted to 17α-hydroxyprogesterone (20).³¹ P450 21A2 catalyzes the 21-hydroxylation of 17α-hydroxyprogesterone (20) to yield 11-deoxycortisol (21). Alternatively, P450 17A1 catalyzes the hydroxylation of pregnenolone to yield 17α-hydroxypregnenolone (Fig. 3, 11 to 19). In humans, 17α-hydroxypregnenolone (19) is a good 17,20-lyase substrate for P450 17A1, but this intermediate (19) is also a substrate for 3β-HSD, which converts 17α-hydroxypregnenolone (19) to 17α-hydroxyprogesterone (20). 17α-Hydroxyprogesterone (20) can be further hydroxylated by P450 21A2 to yield 11-deoxycortisol (21). 11-Deoxycortisol (21) is converted to cortisol (22) by P450 11B1, another target of osilodrostat (1).

• Pregnenolone (11) to testosterone (24) to estradiol (26): when pregnenolone (11) is converted to 17α-hydroxypregnenolone (19)³² then to dehydroepiandrosterone (23) by P450 17A1. The latter reaction is promoted by cytochrome b₅ (b₅).³¹ Cytochrome b₅ is a protein known to interact with P450 17A1 and stimulates^33–36 the C17–C20 cleavage step. Dehydroepiandrosterone (23) is converted to androstenedione (24) by 3β-HSD. Subsequently, the 17-ketone of androstenedione (24) is reduced by 17β-hydroxysteroid dehydrogenase (17β-HSD)³⁷ to yield testosterone (25), which is also a substrate for P450 19A1 that converts testosterone to estradiol (26). P450 17A1 and P450 19A1 are targets of abiraterone (7) (Fig. 3B) and exemestane (9) (Fig. 3C), respectively.

Cholesterol (10) to cholic Acid (36) (Fig. 4): cholesterol (10) can also be converted to the substrate of P450 8B1, 7α-hydroxycholest-4-en-3-one (14) through the action of two enzymes: P450 7A1 and 3β-HSD (Fig. 2B). P450 8B1 is the oxysterol-12α-hydroxylase, which converts 7α-hydroxycholest-4-en-3-one (14) to 7α,12α-dihydroxycholest-4-en-3-one (27).

The subsequent 9 steps involve the conversion of 7α,12α-dihydroxycholest-4-en-3-one (27) to cholic acid (36),^28,38–40 a primary bile acid implicated in obesity. Step 1 of 9 (27 to 28): this process begins with reduction of the Δ⁵ to 5β-reduced steroid backbone by 3-oxo-5β-steroid-4-dehydrogenase (AKR1D1,⁴¹ aldo-ketoreductase family 1 member D1). Step 2 of 9 (28 to 29): subsequent reduction of the 3-ketone⁴² to the 3α-hydroxy group is catalyzed by 3α-hydroxysteroid dehydrogenase. Step 3 of 9 (29 to 30): oxidation at C26 by P450 27A1 (ref. 43) three consecutive times yields the carboxylic acid product at C26. Step 4 of 9 (30 to 31): the free carboxylic acid is conjugated as the thioester^44,45 of CoA by SLC27A5 (solute carrier family 27 member 5). Step 5 of 9 (31 to 32): epimerization of the C25 center by AMACR (alpha-methyl acylCoA-racemase) follows to yield the epimerized alpha methyl chiral carbon center at C25.^46–48 Step 6 of 9 (32 to 33): oxidation at C24 is catalyzed by ACOX2 (acyl-CoA oxidase 2).⁴⁹ Step 7 of 9 (33 to 34): oxidation of the C24-alcohol is catalyzed by HSD17B4 (hydroxysteroid dehydrogenase 17B4, D-bifunctional protein),²⁷ which yields the C24-ketone.⁵⁰ Step 8 of 9 (34 to 35): the C24–C25 bond is cleaved by the SCP2 (sterol carrier protein 2, peroxisomal thiolase 2)^51,52 to release propionyl-CoA and 3,7,12-trihydroxycholanoyl-CoA. Step 9 of 9 (35 to 36): acyl-CoA thioesterase hydrolyzes the thioester bond to release the free carboxylic acid at C24.⁵³

More recently, inhibition of P450 8B1 has been proposed as a therapeutic strategy to combat obesity and metabolic disease (Fig. 4).⁵⁴ Research in our laboratory was focused on developing selective small-molecule inhibitors of P450 8B1 to explore this therapeutic potential.^55,56

To highlight the potency of representative steroid based inhibitors, Table 1 summarizes IC₅₀ values (inhibitor concentration at which 50% of enzyme activity is suppressed) for key compounds targeting P450 17A1 and 19A1. Gonzalez and Guengerich³¹ determined the IC₅₀ value of S-orteronel to be 1.5 μM on P450 17A1 hydroxylation of pregnenolone to 17α-hydroxypregnenolone using purified human enzyme (Table 1, entry 1). In this set of experiments, the researchers also determined the inhibition properties of (S)-orteronel on the lyase activity of P450 17A1 in the conversion of 17α-hydroxypregnenolone to dehydroepiandrosterone. Furthermore, R-orteronel had a higher IC₅₀ values of 6.4 μM and 2.8 μM on P450 17A1 activities converting pregnenolone to 17α-hydroxypregnenolone and 17α-hydroxypregnenolone to dehydroepiandrosterone, respectively. The research labs of Scott and Aube reported the IC₅₀ values of abiraterone and galeterone on P450 17A1-catalyzed hydroxylation of progesterone to 17α-hydroxyprogesterone to be 4.9 and 28 nM, respectively,⁵⁷ using purified human protein (Table 1, entries 2 and 3).

Table 1. Inhibitors of P450 17A1 and 19A1 with IC₅₀ values.

Entry	Enzyme	Inhibitor	IC₅₀	Reference
1	P450 17A1	(S)-Orteronel (2)	1.5 μM^a	31
2	P450 17A1	Abiraterone (7)	4.9 nM^b	57
3	P450 17A1	Galeterone (8)	28 nM^b	57
4	P450 19A1	Exemestane (9)	1.3 μM	58
5	P450 19A1	Letrozole (3)	2 nM	60
6	P450 19A1	Anastrozole (4)	1.0 μM	63

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Purified P450 17A1 enzyme was used and the 17-hydroxylation of pregnenolone was monitored. For comparison with entries 2 and 3, (S)-orteronel inhibited P450 17A1-catalyzed 17-hydroxylation of progesterone with an IC₅₀ value of 0.32 μM.

Purified P450 17A1 enzyme was used and 17-hydroxylation of progesterone was monitored.

Exemestane had a reported IC₅₀ value of 1.3 μM for the aromatization of androstenedione using S9 fractions (supernatant centrifuged at 9000 × g) of HEK293 (human embryonic kidney 293) cells overexpressing recombinant P450 19A1 (aromatase).⁵⁸ The same research group reported an IC₅₀ of 0.92 μM in a separate published study.⁵⁹ Letrozole has been reported to have an IC₅₀ value of 2 nM against P450 19A1 activity – researchers in this particular study used a DBF (dibenzylfluorescein) fluorometric substrate and a commercially available Gentest kit, which uses a recombinant human aromatase enzyme.⁶⁰ Another study reported letrozole to have an IC₅₀ value of 0.7 nM (ref. 61) when a tritiated water release assay was employed using [1β-³H]-androst-4-en-3,17-dione as the substrate in the presence of JEG-3 cells, which was used as the source of enzyme. JEG-3 cells⁶² are derived from human choriocarcinoma, a type of cancer originating from trophoblastic cells in placenta. For the determination of the IC₅₀ value of anastrozole on P450 19A1 to be 1.3 μM. In this study,⁶³ the researchers reported the use of BD Supersome P450 19A1 microsomal proteome as the enzyme source, and an activity-based probe (ABP) based on the scaffold of 2-ethynylnaphthalene.

Furthermore, structural biology has played an important role in guiding the inhibitor design strategy through a rational approach. Crystal structures of P450 enzymes bound to inhibitors and substrates (Table 2) illustrate the molecular basis of selectivity and potency, ranging from P450 17A1 with abiraterone to P450 191 with exemestane, and more recently, P450 8B1 bound to pyridine-containing steroids. Out of the 57 human cytochrome P450 enzymes, 50 are microsomal proteins^64,65 (i.e. they are bound to the endoplasmic reticulum membrane) and 7 are mitochondrial.⁶⁶ P450s 17A1, 19A1, and 8B1 are microsomal proteins and have been historically difficult to crystallize. Recently several research groups successfully solved the structures of these proteins: P450 17A1,^67,68 P450 19A1,^69–72 and P450 8B1 (ref. 56 and 73) – also see Table 2 for PDB IDs. The structures of P450 17A1 have been solved both with substrates⁶⁸ and inhibitors⁶⁷ bound by the Scott Research Laboratory. The structures of P450 17A1 with the substrates were obtained from the A105L mutant (alanine to leucine at position 105). The mutation was likely required to stabilize the ligand in the active site⁷⁴ (human P450 17A1 hydroxylates progesterone at C16 and C17 in a 1 : 3 ratio while the site directed mutant A105L⁷⁵ changes this regioselectivity to 1 : 10). The crystal structures of P450 19A1 with its substrates⁷² and inhibitors⁷⁰ have also been solved by the Ghosh Research Laboratory. The crystal structure can help in elucidating how the inhibitors block enzymatic activity (see Fig. 9 showing the structure of P450 19A1 with exemestane below). Similarly, the Scott Research Group has also solved the structures of P450 8B1 with (S)-tioconazole and a steroid analog containing a pyridine ring at C12 (Fig. 12). These structures have provided valuable information in guiding future efforts to design a selective inhibitor for P450 8B1.

Table 2. Crystal structures of P450 17A1, P450 19A1, and P450 8B1 with various ligands bound.

Entry	Protein	PDB ID	Ligand	Reference
1	P450 17A1	3RUK	Abiraterone (7)	67
2	P450 17A1	3SWZ	Galeterone (8)	67
3	P450 17A1^a	4NKW	Pregnenolone (11)	68
4	P450 17A1^a	4NKX	Progesterone (15)	68
5	P450 17A1^a	4NKZ	17α-Hydroxypregnenolone (19)	68
6	P450 17A1^a	4NKY	17α-Hydroxyprogesterone (20)	68
7	P450 19A1	3S7S	Exemestane (9)	70
8	P450 19A1	5JL9	Androstenedione (24)	71
9	P450 19A1	5JKW	Testosterone (25)	71
10	P450 8B1	7LYX	(S)-Tioconazole (80)	73
11	P450 8B1	8EOH	12-Pyridyl-steroid (78)	56

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P450 17A1 A105L mutant was used. The amino acid residue alanine-105 was mutated to leucine-105 to enable crystallization with the substrates.⁷⁵

2. Synthesis of steroidal inhibitors of cytochrome P450 17A1

2.1. Synthesis of abiraterone and galeterone

Cytochrome P450 17A1 is an enzyme that catalyzes two reactions at the C17 position on steroidal scaffolds: 17α-hydroxylation of 21 carbon steroids and C17–C20 bond scission of its hydroxylated products. First, it converts pregnenolone (Fig. 5, 11) and progesterone (15) into their corresponding 17-hydroxy derivatives, 19 and 20 through a regio- and stereoselective hydroxylation. These intermediates are substrates for a 17,20-lyase reaction, also catalyzed by 17A1, to yield 19-carbon steroids, dehydroepiandrosterone (23) and androstenedione (24) – the lyase reaction is significantly promoted by cytochrome b₅ (b₅) (20 to 24, 0.019 s⁻¹ and 0.00031 s⁻¹ (61 fold), with and without b₅; 19 to 23, 0.071 s⁻¹ and 0.0059 s⁻¹ (12 fold), with and without b₅).³¹

Androstenedione (24) is converted to testosterone (25) by 17β-HSD. Because the lyase products of P450 17A1 (23 and 24) are precursors of testosterone (25), P450 17A1 plays a central role in androgen biosynthesis.

In prostate cancer, androgens such as testosterone (25) drive tumor growth, and P450 17A1 is the key enzyme responsible for producing these androgen precursors. When P450 17A1 activity is inhibited by drugs such as abiraterone (7) or galeterone (8), the androgen sources are cut off, depriving prostate cancer cells of essential growth signals.

2.1.1. Synthesis of abiraterone through a Suzuki cross-coupling: pyridylborane

Among the clinically approved P450 17A1 inhibitors, abiraterone is of particular interest due to its strong potency toward the inhibitory activity. The first reported synthesis of abiraterone in 1997 (Scheme 1, 7)⁷⁶ began with dehydroepiandrosterone 3-acetate (37), which was converted to the corresponding 17-enol triflate 38 using trifluoromethanesulfonic (triflic) anhydride and a bulky base. The key step was a palladium-catalyzed Suzuki coupling between triflate 38 and diethyl(3-pyridyl)borane (39), which introduced the heteroaryl substituent at C17 in 84% and resulted in abiraterone acetate (40). Hydrolysis of the C3 acetate of 40 afforded abiraterone (7), giving the overall yield of 38% over three steps. While this strategy provided the first route to abiraterone, it had notable drawbacks, including the use of toxic and costly triflate reagents and the need for chromatography to remove a byproduct from the triflation step (specifically, others¹⁴ have reported 7-(N-phenylamino)androsta-5,16-dien-3β-yl acetate could not be removed).

2.1.2. Synthesis of abiraterone through a 17-vinyl iodide

While the first report described the synthesis of abiraterone itself for biological evaluation, later studies reported on the synthesis of abiraterone acetate (40),^77,78 which serves as the clinically used prodrug (the acetate is hydrolyzed in vivo by esterases). Potter and co-workers subsequently reported⁷⁶ on a modified synthesis of abiraterone using a different steroid cross coupling partner (Scheme 2). A palladium-catalyzed Suzuki cross-coupling strategy was employed; however, instead of the 17-enol triflate, the coupling partner was 17-vinyl iodide 42, which was introduced to avoid triflation and simplify precursor preparation. To access the 17-vinyl iodide, dehydroepiandrosterone (DHEA, 23) was condensed with hydrazine monohydrate to give hydrazone 41 in 94%, followed by iodination with I₂ and tetramethylguanidine to afford the 17-vinyl iodide 42 in 83%. The Suzuki coupling reaction between vinyl iodide 42 and borane 39, however, was less efficient than the original route due to the formation of bis-steroidal 43. As a result, abiraterone acetate (40) was obtained in only 36% yield after acetylation, primarily due to difficulties in crystallizing. The use of a 17-vinyl bromide⁷⁹ for the Suzuki cross-coupling has also been reported by treating the 17-hydrazone 41 with N-bromosuccinimide (NBS) and 1,1,3,3-tetramethylguanidine (TMG).¹⁴

2.1.3. Synthesis of abiraterone through a modified Heck cross coupling (tosylhydrazone)

The synthetic route to abiraterone acetate (40) was improved in a later report in Heterocycles,⁸⁰ which used tosylhydrazine to react with the C17 ketone (Scheme 3). Dehydroepiandrosterone (23) was condensed with tosylhydrazine to prepare tosylhydrazone 44, which was more stable and isolable. The resulting tosylhydrazone 44 can undergo palladium-catalyzed cross-coupling directly with 3-bromopyridine (45) without the iodination step, giving abiraterone (7) in 63%. Because this route did not use the common Suzuki coupling like previous studies, there was no presence of toxic reagents such as triflic anhydride or iodide. Overall, abiraterone acetate (40) was obtained in three steps in 30% yield from dehydroepiandrosterone.

2.1.4. Synthesis of abiraterone through a steroidal borane

In contrast to earlier syntheses that relied on pyridyl boranes and steroidal electrophiles, another strategy reversed the polarity of the Suzuki coupling (Scheme 4).⁸¹ Specifically, the steroid 46 containing a boronic acid at C17 was generated via lithiation–borylation of the 3-TBS protected version of vinyl iodide 42 using n-BuLi and B(OEt)₃, followed by aqueous hydrolysis. By placing the boron functionality on the steroid instead of the pyridine and employing 3-bromopyridine (47) as the Suzuki coupling partner, this strategy avoids the instability of pyridyl boronic acids, which often undergo protodeboronation and decompose to pyridine, and minimized the formation of bis-steroidal dimers commonly encountered in earlier vinyl iodide approaches.⁷⁶ Furthermore, the steroidal boronic acid (46) was less volatile and more practical for isolation and scale-up. Direct organolithium addition onto the C17-ketone of dehydroepiandrosterone-3-OTBS ether has been reported in a patent, where 3-bromopyridine underwent halogen–metal exchange with n-BuLi and treated with the steroid. The resulting 17-hydroxy group was converted to the mesylate and underwent elimination.¹⁴

Fig. 6 shows the summary of the different methods to synthesize abiraterone to incorporate the pyridine at C17 of the steroid. The use of vinyl triflate 38, vinyl iodide 42, vinyl boronic acid 46, and vinyl bromide 47 at C17 show the utility of the Suzuki cross coupling method while the tosylhydrazone 44 also serves as a cross-coupling partner with 3-bromopyridine (45). In addition, Table 3 summarizes some of the reported methods to synthesize abiraterone through the different approaches to form the C17–pyridine bond. All of the reactions listed use a palladium-catalyzed cross coupling method to form the C17–pyridine bond.

Table 3. Summary of different syntheses of abiraterone (7) to form the bond at C17.

Entry	Key C17–pyridine bond forming step	Reference
1	17-Enol triflate 38 and 3-pyridyl-diethylborane (39) (Suzuki)	13
2	17-Vinyl iodide 42 with 3-pyridyl-diethylborane (39) (Suzuki)	76, 82
3	17-Tosylhydrazone 44 with 3-bromopyridine (45)	80
4	17-Vinyl boronic acid 46 with 3-bromopyridine (45) (Suzuki)	14, 83
5	17-Vinyl bromide 47 with 3-pyridyl-diethylborane (39) (Suzuki)	79

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2.1.5. Synthesis of galeterone via addition elimination of benzimidazole at C17

In addition to the potent inhibitor, abiraterone, galeterone⁸⁴ (8) is a moderate inhibitor towards P450 17A1 (see Table 1). The first synthesis of galeterone was published in 1998.⁸⁵ Pharmacologically, galeterone has a dual mechanisms to treat prostate cancer: P450 17A1 inhibition (IC₅₀ = 28 nM (ref. 57) – where IC₅₀ is the concentration of galeterone to inhibit the activity of P450 17A1 by 50%) and androgen receptor (AR) binding (EC₅₀ = 405 nM – where EC₅₀ is the concentration of galeterone required to displace tritiated R1881 (methyltrienone,⁸⁶ a stable synthetic androgen) from the androgen receptor).⁸⁷

To begin the synthesis of galeterone (Scheme 5), dehydroepiandrosterone acetate (37) underwent a Vilsmeier–Haack reaction through heating with POCl₃ and DMF in CHCl₃ to yield 17-chloro-16-formyl derivative 48 in 77%. During this reaction, deformylation product of 48, 3β-acetoxy-17-chloroandrost-5,16-diene (49), was also observed (11%) due to the instability of 48 under acidic and heating conditions. The nucleophilic vinylic addition–elimination occurred between the C17-chloro intermediate 48 and the nitrogen of benzimidazole 50 in the presence of potassium carbonate, resulted in 51 in 89%. In this substitution reaction, the presence of the 16-formyl group is required to stabilize the C16 carbanion intermediate.⁸⁸ Subsequently, the formyl group at C16 of 51 was deformylated through refluxing with catalytic palladium on activated charcoal⁸⁹ in benzonitrile to give 52 in 93%. The final step in the synthetic route was the hydrolysis of the acetate group at C3, which afforded galeterone (8) in four steps from dehydroepiandrosterone acetate (37).

2.1.6. Potential alternative routes to incorporate the benzimidazole of galeterone

Considering that abiraterone (7) and galeterone (8) both target P450 17A1 to block androgen production, in comparison to abiraterone synthesis, the benefit of the synthesis of galeterone could be the key step to incorporate the heterocycle at C17. For abiraterone, a Suzuki cross-coupling reaction is generally employed at the C17 position to introduce the pyridine ring (Fig. 6 and Table 3) while in the synthesis of galeterone (8), the benzimidazole heterocycle is incorporated through a nucleophilic addition followed by elimination. Although in galeterone, a deformylation step required the use of Pd/C catalyst, the use of expensive Pd catalysts such as the ones used in the various Suzuki cross-coupling reactions reported for the syntheses of abiraterone are avoided.

Indeed, reactions involving C–N bond formation from benzimidazole and a vinyl halide coupling partner have been scarce due to the lack of methods to efficiently substitute at the nitrogen of benzimidazoles. Future directions to improve the yield for the synthesis of galeterone could involve providing alternative routes distinguishable from the original synthesis (Scheme 5, 37 to 8) to form the bond between the nitrogen of benzimidazole and the C17-position of the steroid directly. One potential method could involve an iridium catalyzed C–N bond formation between an allylic carbonate and benzimidazole in the presence of stoichiometric base.⁹⁰ This strategy would involve the synthesis of a steroid containing an allylic carbonate in the D-ring (Scheme 6, 57). Beginning with dehydroepiandrosterone-3-TBS ether (53), the formation of the silyl enol ether at C17 using TESOTf in dichloromethane at −78 °C, followed by Saegusa–Ito oxidation conditions would afford the 5,16-dien-17-one 54. Nucleophilic epoxidation with NaOH/H₂O₂ would yield the epoxyketone 55. Wharton reaction of the 15,16-epoxyketone 55 would yield the allylic alcohol (56), which could be treated with methylchloroformate to form the allylic carbonate 57. An iridium-catalyzed C–N bond formation with benzimidazole would follow and deprotection of the TBS at C3 under acidic conditions could yield galeterone (8).

An even simpler alternative to introduce the heterocycle at C17 for the synthesis of galeterone may involve a Buchwald–Hartwig cross-coupling⁹¹ between a vinyl iodide at C17 of the steroid 42 and benzimidazole (Scheme 7A). Similarly, a Chan–Evans–Lam coupling⁹² could be employed between a C17-boronic acid intermediate 46 and benzimidazole would give galeterone-3-TBS ether (58), which could be deprotected with TBAF in THF to yield galeterone (8) (Scheme 7B). Both the vinyl iodide (Scheme 2) and the 17-boronic acid (Scheme 4) have been synthesized in the studies related to abiraterone. It is unclear if these reactions have been attempted in the past, but alternative methods would be worthwhile to provide complementary routes to access galeterone.

Furthermore, benzimidazole derivatives are more commonly made de novo from condensing a 1,2-diaminobenzene derivative and an aldehyde source.⁹³ However, in the case of galeterone, the challenge could be to synthesize the diamino benzene derivative bearing the steroid, which would make this approach not as attractive as the cross-coupling methods presented above.

2.2. Crystal structures of P450 17A1 with abiraterone and galeterone

The crystal structures of the complex of P450 17A1 with abiraterone (PDB: 3RUK) and P450 17A1 with galeterone (PDB: 3SWZ) are shown in Fig. 7A and B. Not only is the active iron site of the enzyme is bound to the C17 pyridine of the inhibitor, but also, a hydrogen bonding interaction between the asparagine-202 (N202) residue of P450 17A1 and the C3-hydroxy moiety of abiraterone (Fig. 7A). Similarly, the N3 of the benzimidazole moiety of galeterone coordinates to the iron active site of P450 17A1 and the C3-hydroxy group hydrogen bonds to N202 of the protein (Fig. 7B). Fig. 7C and D show the active sites of P450 17A1 bound to its endogenous substrates, pregnenolone (11) and 17α-hydroxypregnenolone (19). The N202 residue of P450 17A1 consistently shows a hydrogen bond with the C3-hydroxy group of each steroid ligand supporting the successful design of the inhibitor based on the substrate backbone (steroid backbone).

2.3. Cytochrome P450 19A1 (aromatase) inhibition to treat breast cancer

Cytochrome P450 19A1 (aromatase) is the enzyme that catalyzes the aromatization of the steroid A-ring (Fig. 8), converting 19-carbon androgens (e.g. androstenedione (24) and testosterone (25)) into 18-carbon estrogens (e.g. estrone (60) and estradiol (26)) through 3 sequential oxidation steps at the C19 methyl group.^94,95 The first two steps involve hydroxylation at C19 to form a gem-diol intermediate (59 or 61), which undergoes C10–C19 bond scission to release the estrogen product (60 or 26). Because certain breast and ovarian cancers rely on estrogens to grow, the direct inhibition of P450 19A1 has been used to treat these estrogen-dependent cancers.

2.3.1. Synthesis of exemestane

Among P450 19A1 inhibitors (i.e. letrozole, anastrozole, and exemestane), exemestane (9) is notable for its steroid backbone, which closely resembles the natural substrate of aromatase. This structural mimicry of the substrate with a potential electrophilic site at C6 enables irreversible, mechanism-based inhibition, making it distinguishable from non-steroidal reversible inhibitors such as letrozole (3) and anastrozole (4).⁹⁶ The first synthesis of exemestane was published in 1993,⁹⁷ which began from 17β-hydroxy-androsta-1,4-diene-3-one (Scheme 8, 62). The methylene group at C6 was introduced through a Mannich reaction with paraformaldehyde (PFA), dimethylamine, and isoamyl alcohol to yield 63 in 35%. The C17-hydroxy of 63 was oxidized using the Jones reagent to yield exemestane (9) in two steps with 30% overall yield.

In 2002,⁹⁸ a review article published by Lombardi on exemestane synthesis reported a strategy to prepare this inhibitor from androstenedione (24) by introducing the 6-methylene group followed by 1,2-dehydrogenation (Scheme 9). The synthesis employed a known method using POCl₃ and diethoxymethane to introduce the exocyclic methylene group at the C6 position,⁹⁹ giving the intermediate 64 in 60%. The subsequent step involves the dehydrogenation of C1 using 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) to furnish exemestane (9) in 40%.¹⁰⁰

Another notable synthetic route to access exemestane (9)⁹⁸ involved the introduction of the alkene at C1 through bromination at C2 followed by elimination. Specifically, androstenedione (Scheme 10, 24) underwent a Mannich reaction¹⁰¹ with triethylorthoformate (TEOF) and p-toluenesulfonic acid (pTsOH), followed by the treatment with N-methylaniline and aqueous formaldehyde. This condition was slightly different compared with the 1993 study⁹⁷ where PFA, dimethylamine, and isoamyl alcohol were used. The 6-methylene intermediate 64 was obtained in 73%, which was then brominated with Br₂ in HBr to afford tribromide 65 in 84%. A debromination was achieved in two steps using sodium iodide in acetone to yield monobromide 66, followed by elimination of the C2-bromide with 1,4-diazabicyclo[2.2.2]octane (DABCO) in N,N-dimethylformamide, giving exemestane (9) in 47%.

Given the low yield of the DDQ reaction to dehydrogenate the C1 position on the steroid backbone (Scheme 9, 64 to 9), the Bermejo group¹⁰² developed an efficient 1,2-dehydrogenation method using chloranil, bis(trimethylsilyl)trifluoroacetamide (BSTFA) and catalytic amount of p-toluenesulfonic acid (pTsOH). Specifically, testosterone (Scheme 11, 25) underwent a Mannich reaction to give the 6-methylene intermediate 67 in 89%, which was subsequently acetylated to the 17-acetate 68 in 99% yield. The key reaction to desaturate the C1 position involved the treatment of 68 with chloranil, BSTFA, and TfOH, affording Δ^1,4-3-keto steroid 69 and improved the isolated yield of from 40% to 85%. The C17-acetate was hydrolyzed with NaOH in MeOH to yield the 17-hydroxy group (69 to 63, 95% yield). Subsequent oxidation of the C17 alcohol with Jones reagent gave exemestane (63 to 9, 100% yield). This sequence gave exemestane in 71% overall yield from testosterone (25).

2.3.2. C1-dehydrogenation with DDQ and p-chloranil gave [4 + 2] hexacyclic products

The use of DDQ and p-chloranil to dehydrogenate the C1 position of 6-methylenandrost-4-ene-3,17-dione, the exocyclic methylene precursor gave hexacyclic products (Scheme 12, 64 to 70 or 64 to 71).¹⁰³ With p-chloranil, a double dehalogenation occurred after the initial [4 + 2] reaction to yield the aromatic ring or with DDQ, a dehydrogenation occurred after the [4 + 2] reaction. The formation of the undesired byproduct was dependent on the solvent – where polar protic solvents, aromatic solvents, and polar aprotic solvents gave rise to trace, 3–10% yields, and 12–36% yields, respectively.

2.3.3. Synthesis of isotope-labeled exemestane

In the pharmaceutical industry, the use of isotopically labeled drugs are important for elucidating the metabolic pathway using mass spectrometry and radiolabeled compounds (e.g.¹⁴C- and ³H-). The benefit of radiolabeled pharmaceuticals offers a sensitive method to detect the metabolites after in vitro incubation with liver microsomes (S9 fraction, the supernatant after centrifuging liver homogenates at 9000 × g). The use of ¹⁴C- as the isotope is probably more common due to the introduction of a significant kinetic isotope effect when a tritium atom is introduced into the compound (the carbon–tritium bond is stronger than the carbon–deuterium bond, which is stronger than the carbon–hydrogen bond due to the increased reduced mass with the heavier hydrogen isotope (reduced mass = m₁m₂/(m₁ + m₂), m₁ = mass of carbon, m₂ = mass of hydrogen isotope)). This change in bond strength (aliphatic C–H bond ∼ 100 kcal mol⁻¹vs. aliphatic C–D bond ∼ 101 kcal mol⁻¹) can result in “metabolic switching”, a phenomenon where cytochrome P450 enzymes could hydroxylate the substrate at a different position of the deuterated substrate due to the stronger C–D bond, leading to alternative regioselectively hydroxylated metabolites.¹⁰⁴ On the other hand, the change in the reduced mass is not as significant when the carbon isotope is incorporated (¹⁴C from ¹²C vs.³H from ¹H).

Moreover, radiolabeled compounds offer a uniform sensitivity through β-RAM emission as opposed to mass spectrometry methods, which rely on how ionizable the different compounds are to be detected. Considering the benefits of isotopically labeled pharmaceuticals to study the drug metabolism pharmacokinetic parameters of small molecule medicines, a highlight of exemestane synthesis is featured, where the researchers used [¹³C]-formaldehyde to introduce the exocyclic methylene of exemestane.¹⁰⁵ Although ¹³C-formaldehyde was used instead of ¹⁴C-formaldehyde in this study, after successful synthesis of the ¹³C-labeled exemestane, the same procedures could be employed to access ¹⁴C-labeled exemestane.

2.4. Crystal structure of P450 19A1 with exemestane

Fig. 9 shows the crystal structure of P450 19A1 bound to exemestane (9). The crystal structure shows serine-478 (S478) of P450 19A1 in close to the exocyclic methylene carbon of exemestane (4.6 Å) (Fig. 9A). Furthermore, previous studies^106,107 have suggested that metabolites of exemestane also have potent anticancer properties. One particular report indicated that the 1α,2α-epoxidized metabolite of exemestane had an IC₅₀ of 810 nM against human placental aromatase.¹⁰⁶ However, this value was nearly 20-fold higher (less potent) than the parent exemestane with an IC₅₀ of 42 nM (ref. 98) against human placental aromatase. However, this difference in inhibition properties suggest the importance of the presence of the double bond at C1 for exemestane. In fact, a careful analysis of the crystal structure shows the heme nitrogen atoms are in close proximity of the C1 position of exemestane (4 Å) (Fig. 9B), suggesting a potential aza-Michael addition of the porphyrin nitrogen onto the C1 of exemestane to potentially irreversibly inhibit P450 19A1. There have been prior studies of ligands crosslinking with the heme of P450 enzymes through mechanism based inactivation (e.g. ethinylestradiol and P450 3A4)^108,109 and exemestane could potentially be inhibiting P450 in a similar fashion where the small molecule forms a heme adduct. Exemestane likely does not need to undergo metabolic activation since exemestane already possesses an electrophilic site a C1. The presence of the double bond at C1 was important for time-dependent inhibition properties⁹⁸ of P450 19A1. Exemestane and 1,4-androstadienedione both had time-dependent inhibition with t_1/2 of 13.9 and 24.1 min and K_i of 26 and 92 nM, respectively. In contrast, 6-methylenandrostenedione showed no time dependent inhibition of P450 19A1, which further suggests the inhibitory role of the C1 olefin in exemestane.

The crystal structure of exemestane with P450 19A1, the relatively short distance (4 Å) of one of the nitrogen atoms in the porphyrin and the electrophilic C1 position of exemestane (Fig. 9B) supports a potential covalent adduct formation of the heme onto exemestane (Fig. 10). The nitrogen lone pair of the porphyrin ring could attack the activated C1 position of exemestane after activation of the enone from the hydrogen bonding between D309 of P450 19A1 and the C3-oxygen of exemestane (Fig. 9A, 3 Å distance between D309 and C3 oxygen of exemestane).

2.5. Synthesis of a potential steroid inhibitor of cytochrome P450 8B1

Cytochrome P450 8B1 (P450 8B1) is the oxysterol 12α-hydroxylase enzyme that hydroxylates 7α-hydroxycholestenone (14) to yield 7α,12α-dihydroxycholestenone (27), which leads to cholic acid (Fig. 11). Cholic acid (36) is the bile acid that enhances absorption of cholesterol and other fat-soluble nutrients. The bile acid that increases from the absence of P450 8B1 activity is chenodeoxycholic acid (CDCA) (72), which has been shown to decrease dietary cholesterol absorption.^110–112

Potential P450 8B1 inhibitors were synthesized and published in 2022 and 2023 (Scheme 13).^55,56 In these studies, a pyridine ring was incorporated at C12 of the steroid, the hydroxylation site of P450 8B1. The design of these inhibitors followed a similar strategy for the synthesis of the inhibitor of P450 17A1, where a pyridine was incorporated at C17, the site of enzymatic hydroxylation. The synthetic route began with dehydroepiandrosterone (DHEA) (Scheme 13, 23), which was converted through fours steps (i.e. (i) TBS protection at C3, (ii) imine formation for the Schonecker oxidation, (iii) ketalization at C17, and (iv) oxidation of the C12-alcohol to the ketone) to give intermediate 73. To prepare the coupling partner, the ketone 73 was condensed with hydrazine monohydrate to yield C12-hydrazone 74, followed by iodination with triethylamine and I₂ in THF and subsequent removal of the protecting groups at C3 and C17 with HCl in THF to afford C12-vinyl iodide 75 in 66% after two steps. Suzuki cross-coupling reaction between vinyl iodide 75 and 3-pyridylboronic acid (76) gave 77 in 71%. The analog 78 was prepared by oxidizing 77 under Oppenauer conditions with Al(OiPr)₃ and N-methylpiperidone in refluxing toluene to afford the 3-keto-Δ⁴ product. The resulting diketone 78 was reduced with NaBH₄ in CH₃OH to yield the 3,17-diol product, 79. The 3-keto-Δ⁴ steroid 78 crystallized with P450 8B1 (Fig. 12, PDB: 8EOH).⁵⁶

2.6. Crystal structure of P450 8B1 with C12-pyridine containing steroid

None of the C12-pyridine containing steroid analogs bound to the active site of wild-type P450 8B1 as determined by binding titration experiments with UV-vis spectrophotometry. However, the C12-pyridine containing steroids did indeed reach the active site of the W281F site-directed mutant enzyme (tryptophan-281 was mutated to a phenylalanine residue),⁵⁶ which was indicated by yielding a type II-binding difference spectrum in binding titrations with UV-vis spectrophotometry. Despite the inability of the C12-pyridine containing steroids to bind to the iron active site of P450 8B1, a crystal structure was successfully obtained with compound 69 and wild-type P450 8B1 (Fig. 12A). The nitrogen of the pyridine moiety of compound 69 hydrogen bonds to tyrosine-102 (Y102) of the B′-helix of the enzyme (Fig. 12B, 2.6 Å). The crystal structure of P450 8B1 bound to (S)-tioconazole (PDB ID: 7LYX)⁷³ shows that the tryptophan-281 (W281) residue in the I-helix hydrogen bonds with aspartate-210 (D210) in the F-helix (Fig. 12C, 3.2 Å), this hydrogen bonding interaction results in a ceiling with a height of 8.2 Å from the iron active site to W281. Additionally, a glycerol molecule is hydrogen bonding to Y102 when tioconazole is bound to P450 8B1 (Fig. 12D, 2.4 Å). In contrast, P450 8B1 with the C12-pyridyl steroid 78 bound has an 8.6 Å ceiling from the active iron center to W281 of the I-helix, which limits the size of the ligand that could “fit” for hydroxylation. P450 enzymes have twelve α-helices (A–L) and the I-helix is where the substrate binds to the protein for catalysis; while the F–G helices together make the a loop the functions like a “lid” to hold the substrate in place. Since the F–G loop limits the height of the ligand, our future proposals for inhibitor synthesis involved a design of a steroid containing a heterocycle in the C-ring that could be “shorter” between C5 and the pyridine ring. The pyridine containing steroids that were accessed from Suzuki cross coupling between the vinyl iodide at C12 and the 3-pyridylboronic acid unit had a “height” of 9 Å between C5 and the top of the pyridine ring. Despite these unsuccessful efforts to synthesize a P450 8B1 inhibitor that directly binds to the active site of the enzyme, the insightful results from protein crystallography provide new direction for future studies to develop a selective P450 8B1 inhibitor.

In order to present a perspective with chemical structures, a cartoon representation is shown in Fig. 13 where either the C12-pyridyl steroid 78 is bound to P450 8B1 (Fig. 13A) or (S)-tioconazole is bound to P450 8B1 (Fig. 13B). The tyrosine-102 (Y102) residue is shown hydrogen bonding to the pyridine nitrogen of the steroid ligand 78 while in the case of (S)-tioconazole, a glycerol molecule (81) is hydrogen bonding to Y102. Furthermore, the key tryptophan-281 (W281) residue is hydrogen bonding to the C3-ketone of the steroid ligand (Fig. 13A) while in the (S)-tioconazole, W281 is hydrogen bonding to aspartate-210 (D210) of the F–G loop.

2.7. Future studies related to P450 8B1 inhibitor synthesis

Based on the information gathered from the crystal structure of P450 8B1 with the C12-pyridine containing steroid 78 (Fig. 12), a new steroid analog was synthesized in our laboratory (Scheme 14). Due to the tryptophan-281 (W281) of the I-helix in P450 8B1, a “ceiling” of 8.6 Å is present above the iron active site (Fig. 12B). In order to fit into the active site of P450 8B1, a pyridine was fused at C11 and C12 of the steroid, which gave rise to a steroid with a length of 6.6 Å (ref. 113) from the C5 carbon to the pyridine ring. To synthesize this derivative, ketone 73 was treated with propargylamine in the presence of CuCl₂ and O₂ in EtOH to directly yield a pyridine derivative.¹¹⁴ Acidic workup with HCl followed by wash with aqueous NaOH gave the 3-hydroxy product 82. Oxidation of the C3-hydroxy group with Dess–Martin periodinane gave the 3-keto-Δ⁵ product (82 to 83), which was isomerized to the 3-keto-Δ⁴ steroid and deprotected at C17 (17-ketal to the 17-ketone) after refluxing with HCl in THF/H₂O to give diketone 84. Treatment of diketone 84 with NaBH₄ in MeOH gave the 3,17-diol product 85. In addition, ketal 82 was deprotected with HCl in THF/H₂O to yield the ketone 86, which was reduced with NaBH₄ in MeOH to yield Δ⁵-3,17-diol 87. Future research efforts will focus on heterocycle-containing steroids at the C-ring to selectively target the iron active site of P450 8B1.

3. Conclusion

In summary, due to the production of steroid hormones and their various roles in human health, the ability to block the activity of the enzymes that produce these hormones can lead to better treatments of diseases (Table 4). As more biochemical discoveries are made through basic research efforts (e.g. discovering previously unknown enzymatic activities and identifying new agonists/antagonists for hormone receptors), more opportunities for medicinal chemists arise to target and block the activity of specific proteins. In this regard, our research laboratory chose to focus on the development of inhibitors of cytochrome P450 8B1 to treat obesity and cardiovascular health. The evolution of the syntheses in collaboration with the laboratories of other research expertise show how interdisciplinary research in organic synthesis and enzymology can lead to impactful and positive results that will benefit human health.

Table 4. List of P450 enzyme resulting in hormone production relevant to disease.

Entry	Enzyme	Steroid hormone^a	Disease	Inhibitor
1	P450 17A1	Testosterone	Prostate cancer	Abiraterone
2	P450 19A1	Estrogen	Breast cancer	Exemestane
3	P450 8B1	Cholic acid	Obesity	Under development

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The direct enzymatic product may be a biosynthetic precursor to the final steroid hormone (i.e. testosterone is the androgen steroid hormone that comes from the activity of P450 17A1, which converts pregnenolone to 17α-hydroxypregnenolone to dehydroepiandrosterone – see Fig. 2 for structures).

Although the inhibition of P450 17A1 and P450 19A1 have been thoroughly studied and successful inhibitors have been developed, new problems continue to arise. One major problem is that cancer commonly undergoes mutations and undergo drug resistance¹¹⁵ mechanisms. For instance, although aromatase inhibitors block the production of estrogens so that estrogen receptors cannot be activated by the endogenous hormones, some cancers have point mutations in the estrogen receptor (ER) so that it remains constitutively active and there may not be a need for a ligand to activate the receptor. Single point mutations in the ligand binding domain of the estrogen receptor α have been observed where a tyrosine at position 537 is substituted with an alanine or serine residue,¹¹⁶ and this specific mutant resulted in a functional mutation that caused a ligand independent ER transcriptional activity. Similar observations have been made with the androgen receptors^117–120 in castration-resistant prostate cancer. Therefore, efforts to develop a drug that could simultaneously target two different protein targets by both blocking the P450 enzyme that produces the hormone and also targeting the receptor in novel binding regions, could potentially be a valuable improvement in treatment of cancer.

Obesity is another health problem that has a complex cell signaling mechanism¹²¹ and a variety of different protein targets to treat the disease¹²² (glucagon like peptide-1 (GLP-1) receptor, angiopoetin-like proteins,¹²³ dipeptidyl peptidase,¹²⁴ receptor-interacting protein-140,¹²⁵ glucose-dependent insulinotropic polypeptide receptor (GIPR),¹²⁶ kinases,¹²⁷ G-protein coupled receptor-75,¹²⁸ and others).¹²⁹ The development of treatments for obesity have been pursued since the 1930s. The recent advances involve GLP-1 receptor agonists¹³⁰ such as semaglutide and liraglutide with unwanted side effects¹³¹ such as acute pancreatitis.¹³² Moreover, bile acids such as cholic acid and chenodeoxycholic acid play important roles in liver health. For instance, the farnesoid X receptor (FXR)^133,134 and G-protein bile acid coupled receptor-1 (GPBAR-1)^135–137 bind to bile acids and can regulate the health of the digestive system. FXR is activated by CDCA (EC₅₀ = 10 μM).¹³⁸ Future endeavors could be related to the development of a small molecule that could selectively inhibit P450 8B1 to regulate bile acid composition, and, in turn, be exploited to treat obesity.

Conflicts of interest

There is no conflict of interest to declare.

Abbreviations

Cytochrome P450 17A1: P450 17A1, 17α-hydroxylase or 17/20-lyase
Cytochrome P450 19A1: P450 19A1, aromatase
Cytochrome P450 8B1: P450 8B1, oxysterol-12α-hydroxylase
Cytochrome P450 11B1: 11β-Hydroxylase
Cytochrome P450 11B2: P450 11B2, aldosterone synthase
NBS: N-Bromosuccinimide
TMG: Tetramethylguanidine
3β-HSD: 3beta-hydroxysteroid dehydrogenase
17β-HSD: 17beta-hydroxysteroid dehydrogenase

Acknowledgments

We are sincerely grateful to the past research efforts of students in this laboratory and research collaborators that helped progress our work on developing cytochrome P450 8B1 inhibitors. The work in our laboratory was funded by the Max and Minnie Tomerlin Voelcker Fund. Francis K. Yoshimoto, PhD held a Voelcker Fund Young Investigator Award from the Max and Minnie Tomerlin Voelcker Fund. We thank the reviewers for their expertise, valuable feedback, and kind suggestions that helped us improve the quality of this manuscript.

Biographies

Biography

Dr. Tu Ho received her B.S. in Biochemistry in 2021 and earned her Ph.D. in Chemistry from the University of Texas at San Antonio (UTSA) in 2025. Her doctoral research focused on the multi-step synthesis of biologically relevant small molecules, with an emphasis on steroidal scaffolds and structure–activity relationship studies. She has developed synthetic routes to endogenous hormones, bioactive natural products, and heterocycle-containing steroid analogs, including a mild strategy to incorporate a 14β-hydroxy group in the steroid backbone and the design of pyridine-based inhibitors targeting cytochrome P450 8B1. Her work has been published in Steroids (2023) and has supported collaborations aimed at elucidating enzyme mechanisms and advancing therapeutic discovery. She is broadly interested in applying synthetic organic chemistry to medicinal and pharmaceutical research, including active pharmaceutical ingredient (API) development and chemical process optimization.

Biography

Dr. Yoshimoto received his undergraduate research training in organic synthesis in the laboratory of Professor Richmond Sarpong at UC Berkeley where he obtained his B.S. in Chemistry and B.A. in Linguistics in 2005. He obtained his Ph.D. training first at UT Southwestern and then at University of Michigan where he synthesized steroid analogs to study the C–H hydroxylation mechanisms of steroidogenic cytochromes P450 17A1 and 21A2 in the lab of Professor Richard J. Auchus. After defending his Ph.D. in 2012, he continued his training as a postdoctoral researcher in the lab of Professor F. Peter Guengerich at Vanderbilt University (2012–2016), where he elucidated the mechanisms of the C–C bond cleavage steps of steroidogenic cytochromes P450 19A1, 17A1, and 11A1. He began his independent research career at the University of Texas at San Antonio and he is most proud of the students he trained in his lab, who have continued to pursue their own scientific research careers.

Data availability

Data describing the synthesis of steroids with a pyridine fused at C11 and C12 (Scheme 14) are available at ChemRxiv and can be found at: https://doi.org/10.26434/chemrxiv-2025-6l4pf.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Data describing the synthesis of steroids with a pyridine fused at C11 and C12 (Scheme 14) are available at ChemRxiv and can be found at: https://doi.org/10.26434/chemrxiv-2025-6l4pf.

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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

Tu M Ho

Francis K Yoshimoto

Abstract

1. Overview of steroidogenic cytochrome P450s

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.

Table 1. Inhibitors of P450 17A1 and 19A1 with IC50 values.

Table 2. Crystal structures of P450 17A1, P450 19A1, and P450 8B1 with various ligands bound.

2. Synthesis of steroidal inhibitors of cytochrome P450 17A1

2.1. Synthesis of abiraterone and galeterone

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

2.1.1. Synthesis of abiraterone through a Suzuki cross-coupling: pyridylborane

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

2.1.2. Synthesis of abiraterone through a 17-vinyl iodide

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

2.1.3. Synthesis of abiraterone through a modified Heck cross coupling (tosylhydrazone)

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

2.1.4. Synthesis of abiraterone through a steroidal borane

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).

Table 3. Summary of different syntheses of abiraterone (7) to form the bond at C17.

2.1.5. Synthesis of galeterone via addition elimination of benzimidazole at C17

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

2.1.6. Potential alternative routes to incorporate the benzimidazole of galeterone

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.

2.2. Crystal structures of P450 17A1 with abiraterone and galeterone

2.3. Cytochrome P450 19A1 (aromatase) inhibition to treat breast cancer

2.3.1. Synthesis of exemestane

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).

2.3.2. C1-dehydrogenation with DDQ and p-chloranil gave [4 + 2] hexacyclic products

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

2.3.3. Synthesis of isotope-labeled exemestane

2.4. Crystal structure of P450 19A1 with exemestane

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.

2.5. Synthesis of a potential steroid inhibitor of cytochrome P450 8B1

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).

2.6. Crystal structure of P450 8B1 with C12-pyridine containing steroid

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).

2.7. Future studies related to P450 8B1 inhibitor synthesis

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.

3. Conclusion

Table 4. List of P450 enzyme resulting in hormone production relevant to disease.

Conflicts of interest

Abbreviations

Acknowledgments

Biographies

Biography

Tu M. Ho.

Biography

Francis K. Yoshimoto.

Data availability

References

Associated Data

Data Availability Statement

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Table 1. Inhibitors of P450 17A1 and 19A1 with IC₅₀ values.