Synthesis and evaluation of vitamin D receptor-mediated activities of cholesterol and vitamin D metabolites

Abstract

A systematic study with phase 1 and phase 2 metabolites of cholesterol and vitamin D was conducted to determine whether their biological activity is mediated by the vitamin D receptor (VDR). The investigation necessitated the development of novel synthetic routes for lithocholic acid (LCA) glucuronides (Gluc). Biochemical and cell-based assays were used to demonstrate that hydroxylated LCA analogs were not able to bind VDR. This excludes VDR from mediating their biological and pharmacological activities. Among the synthesized LCA conjugates a novel VDR agonist was identified. LCA Gluc II increased the expression of CYP24A1 in DU145 cancer cells especially in the presence of the endogenous VDR ligand 1,25(OH)2D3. Furthermore, the methyl ester of LCA was identified as novel VDR antagonist. For the first time, we showed that calcitroic acid, the assumed inactive final metabolite of vitamin D, was able to activate VDR-mediated transcription to a higher magnitude than bile acid LCA. Due to a higher metabolic stability in comparison to vitamin D, a very low toxicity, and high concentration in bile and intestine, calcitroic acid is likely to be an important mediator of the protective vitamin D properties against colon cancer.

Keywords: 1,25(OH)(2)D(3); CYP24A1; Calcitroic acid; DU145; Lithocholic acid; Metabolites; Vitamin D receptor.

Figure 1. Summary of the potential natural VDR ligands

Figure 2

A) CYP24A1 gene regulation by VDR ligands (7.5 μM) in DU145 cells after 18 hours. Stars represent P < 0.001 (***) compared to vehicle DMSO. B) CYP24A1 gene regulation by VDR ligands (7.5 μM) in DU145 in the presence of 20 nM 1,25(OH)₂D₃ after 18 hours. Stars represent P < 0.001 (***) compared to 1,25(OH)₂D₃. All values represent standard errors of mean calculated from three biological independent experiments performed in triplicate. All significance values were determined using ANOVA (Dunnett’s comparison test).

Scheme 1. Conversion of LCA to LCA acetate and SLCA

i) Pyridine, acetyl chloride, cat. 4-DMAP, rt, 1 h; ii) sulfuric acid, acetic anhydride, pyridine, 50-55°C, 30 min. iii) 25% ammonia in water, 0°C, 15 min.

Scheme 2. Synthetic scheme for converting LCME to LCA O-glucuronide I and II

i) Dry benzene, CdCO₃, acetobromo-α-D-glucuronic acid methyl ester, reflux, 5 h; ii) MeOH, 1M NaOH, rt, 4 h followed by 5% HCl; iii) TBDMSCl, DMF, imidazole, rt; iv) THF, 2M NaOH, 75°C, 24 h followed by 2M HCl; v) pyridine, CDI, reflux, 3 h; vi) a) NaH, tetrabutylammonium glucuronate, 50°C, 4-5 h; b) acetic acid; vii) THF, tetrabutylammonium fluoride, 40°C, H₂O.

Scheme 3. Tetrabutylammonium salt (1) formation of glucuronic acid

i) MeOH, 40°C, 1 h.