Design, Synthesis, and Evaluation of Niclosamide Analogs as Therapeutic Agents for Enzalutamide-Resistant Prostate Cancer

Abstract

Niclosamide effectively downregulates androgen receptor variants (AR-Vs) for treating enzalutamide and abiraterone-resistant prostate cancer. However, the poor pharmaceutical properties of niclosamide due to its solubility and metabolic instability have limited its clinical utility as a systemic treatment for cancer. A novel series of niclosamide analogs was prepared to systematically explore the structure-activity relationship and identify active AR-Vs inhibitors with improved pharmaceutical properties based on the backbone chemical structure of niclosamide. Compounds were characterized using ¹H NMR, ¹³C NMR, MS, and elemental analysis. The synthesized compounds were evaluated for antiproliferative activity and downregulation of AR and AR-V7 in two enzalutamide-resistant cell lines, LNCaP95 and 22RV1. Several of the niclosamide analogs exhibited equivalent or improved anti-proliferation effects in LNCaP95 and 22RV1 cell lines (B9, IC₅₀ LNCaP95 and 22RV1 = 0.130 and 0.0997 μM, respectively), potent AR-V7 down-regulating activity, and improved metabolic stability. In addition, both a traditional structure-activity relationship (SAR) and 3D-QSAR analysis were performed to guide further structural optimization. The presence of two -CF₃ groups of the most active B9 in the sterically favorable field and the presence of the -CN group of the least active B7 in the sterically unfavorable field seem to make B9 more potent than B7 in the antiproliferative activity.

Keywords: androgen receptor variant 7 (AR-V7) down-regulators; enzalutamide-resistant prostate cancer; niclosamide analogs; structure–activity relationship (SAR).

Figure 1

Niclosamide and other non-nonsteroidal AR competitive inhibitors.

Figure 2

Design of compounds (A–C).

Scheme 1

Synthesis route of compounds A1–A20, B1, B3–B16 and C1–C5. Reactants and reaction conditions: (a) thionyl chloride, DMF (cat.), anhydrous THF, 0 °C- r.t.; (b) substituted anilines, DMAP (cat.), anhydrous THF, and 0 °C- r.t.

Scheme 2

Synthesis of compound B2. Reactants and reaction conditions: (a) reduced iron powder, HCl (aq.), and MeOH/EtOH (1/2, v/v).

Scheme 3

Synthesis of compound C6–C8. Reactants and reaction conditions: (a) acetic anhydride or trifluoromethanesulfonic anhydride, pyridine, anhydrous DCM, and 0 °C- r.t.

Figure 3

Downregulation of AR and AR–V7 expression in LNCaP95 cells by niclosamide and B1.

Figure 4

Downregulation of AR and AR–V7 expression in 22RV1 cells by niclosamide and compound B1.

Figure 5

Downregulation of AR and AR–V7 expression in LNCaP95 and 22RV1 cells by niclosamide, compounds B3, B9, and B14.

Figure 6

SAR study on niclosamide analogs.

Figure 7

Actual vs. predicted activities for the training (blue squares) and test (red triangles) set compounds obtained from the field-based QSAR. The residual line limits of ±0.35 are shown by dotted-dash lines.

Figure 8

Contour mapping of the filed-based QSAR for the most active (B9) and least active (B7) compounds in 22RV1. (a) Steric: green (favored) and yellow (unfavored), (b) electrostatic: blue (+) and red (−), (c) hydrophobic: yellow (favored) and white (unfavored), (d) H-bond donor: blue-violet (favored) and cyan (unfavored), and (e) H-bond acceptor: red (favored) and magenta (unfavored).