Pyridine indole hybrids as novel potent CYP17A1 inhibitors

Abstract

Prostate cancer (PCa) is one of the most prevalent malignancies affecting men worldwide, and androgen deprivation therapy (ADT) is a primary treatment approach. CYP17A1 inhibitors like abiraterone target the steroidogenic pathway to reduce androgen levels, but their clinical efficacy is limited by drug resistance and adverse effects. This study reports the synthesis and evaluation of novel CYP17A1 inhibitors derived from a previously identified hit compound. Several analogs were synthesised, including an unexpected di-cyano derivative, which demonstrated increased potency against CYP17A1 compared to abiraterone. Biological assays revealed that these compounds significantly inhibited CYP17A1 enzymatic activity and altered steroid biosynthesis. Among the newly synthesised inhibitors, compound 11 showed the highest potency (IC₅₀ = 4 nM) and the related compound 14 presented a template for further development. A combined docking and molecular dynamics approach was used to identify the possible target binding modes of the compounds.

Keywords: CYP17A1; enzyme inhibition; inhibitors; prostate cancer.

Graphical abstract

Figure 1.

Adrenal steroidogenesis. The steroidogenic pathway in the adrenal cortex as shown, highlights the roles of the different zones: the Zona glomerulosa, the outermost layer, responsible for synthesising aldosterone, a mineralocorticoid: the Zona fasciculata, middle of the adrenal cortex, this zone primarily produces glucocorticoids, such as cortisol. The zona fasciculata is regulated by the hypothalamic-pituitary-adrenal (HPA) axis via the secretion of adrenocorticotropic hormone (ACTH) from the pituitary. The Zona reticularis, the innermost layer of the adrenal cortex is responsible for the production of adrenal androgens, including dehydroepiandrosterone (DHEA), DHEA sulphate (DHEA-S), and testosterone. These hormones can be converted into more potent androgens, dihydrotestosterone (DHT) or oestrogens in peripheral tissues. While not regulated directly by the HPA axis, adrenal androgen production is influenced by ACTH to a lesser extent than glucocorticoids. In prostate cancer, adrenal androgens—along with those produced by the testes—are converted to DHT. DHT binds to androgen receptor, mediates gene transcription, and leads to cancer cell proliferation, increasing PSA (Prostate-Specific Antigen) levels, and contributing to the development of cancer cell immortality. Steroidogenesis involves several key enzymes, including P450scc (Cholesterol side-chain cleavage enzyme, CYP11A1), P450c17 (17α-hydroxylase/17,20-lyase, CYP17A1), P450c21 (21-hydroxylase, CYP21A2), P450cAS (Aldosterone synthase, CYP11B2), P450c11 (11β-hydroxylase, CYP11B1), 3β-HSD (3β-hydroxysteroid dehydrogenase), SULT2A1 (Sulfotransferase), AKR1C3 (Aldo-keto reductase family 1, member C3), and cholesterol transport protein StAR (Steroidogenic acute regulatory protein).

Figure 2.

Structures of abiraterone and hit compound 1.

Scheme 1.

Proposed mechanism of a “+24” compound formation.

Scheme 2.

General reaction scheme of the synthesis of compounds 1–11. Compounds 1/10 and 5/11 were produced in a single reaction and separated chromatographically. Other compounds were isolated as a single compound. Yields are provided for individual compounds in parentheses.

Scheme 3.

Synthesis of compounds 12–15. 12 Pd(OAc)₂, SPhos, K₃PO₄, dioxane, reflux; 13 Pd(PPh₃)₄, K₂CO₃, dioxane, 60 °C, 12 h; 14 Pd(PPh₃)₄, K₂CO₃, dioxane, 60 °C, 12 h; 15 Pd(OAc)₂, XPhos, K₃PO₄, dioxane, reflux. Yields are provided for individual compounds in parentheses.

Figure 3.

The activity of CYP17A1 evaluated in H295R cells treated with the compounds. (A) shows the impact of the compounds on the 17α-hydroxylase and 17,20-lyase activity, with DMSO as a negative control and abiraterone (ABT) as a positive control. The data indicate the percentage of activity remaining relative to the control. (B) presents the dose-response curves for inhibition of 17,20-lyase activity. Values are presented as mean ± standard deviation from three independent experiments. Statistical significance was determined using [Two-way ANNOVA, Sidak post-hoc test], with a p-values <0.05 considered significant. **p < 0.001, ^****p < 0.0001.

Figure 4.

Steroid profile for selected compounds (1, 5, 11, 14) and abiraterone (ABT) compared to DMSO control. (A) shows the changes in androgen-related steroid metabolites in response to treatment with DMSO, compounds 1, 5, 11, 14 at 10 µM, and ABT. Each bar represents the average ± SD metabolite level for each treatment as a % of the control. (B) Shows the percentage activity of CYP17A1’s 17α-hydroxylase and combined with 17,20-lyase function, calculated as ratios, normalised to DMSO-treated cells (100%).

Figure 5.

Cytotoxicity assessment of various compounds in VCaP and LNCaP prostate cancer cell lines using the resazurin assay. The heatmap displays cell viability following treatment with 10 µM of each compound at 24 and 48 h. DMSO served as a negative control, while abiraterone (ABT) was used as a positive control. Results from three independent experiments are presented for each compound.

Figure 6.

Impact of selected compounds on cell migration in the wound healing assay showing quantitative analysis of wound closure. Data are expressed as the pixels from the image of wound area remaining open after 24 h, with DMSO as the control. Treatment with 11 and 14 significantly inhibited cell migration, as evidenced by a larger remaining wound area compared to the control. The data indicates the relative efficacy of each compound in affecting cell migration. Values are presented as mean ± standard deviation from three independent experiments. Statistical significance was determined using One-way ANNOVA, [Dunnet’s post-hoc test], with a p-values <0.05 considered significant. **p < 0.01.

Figure 7.

GOLD predicted binding modes to CYP17A1. (A) 1 (C-atoms cyan) and 10 (C-atoms white) from GOLD docking without constraints. Only contacts between 1 and CYP17A1 are shown. (B) 5 (C-atoms cyan) and 11 (C-atoms white) from GOLD docking without constraints. Only contacts between 5 and CYP17A1 are shown. (C) 1 (C-atoms cyan) and 5 (C-atoms white) from GOLD docking with constraints. Only contacts between 1 and CYP17A1 are shown. (D) 10 (C-atoms cyan) and 11 (C-atoms white) from GOLD docking with constraints. Only contacts between 10 and CYP17A1 are shown.

Figure 8.

Binding of 14 to CYP17A1. The surface of Asn202, Ile205, Ile206, and Leu209 on Helix F and Gly301, Ala302, and Thr306 on Helix I are the only residues in direct contact with compound 14. The dotted lines illustrate the shape of the contact between compound 14 and the surfaces of Asn202, Ile205, Ile206, and Leu209 in Helix F and Gly301, Ala302, and Thr306 in Helix I. The arrows indicate space not occupied by 14 and potential space for substituents.