Stereoselective Synthesis of 24-Fluoro-25-Hydroxyvitamin D3 Analogues and Their Stability to hCYP24A1-Dependent Catabolism

Abstract

Two 24-fluoro-25-hydroxyvitamin D3 analogues (3,4) were synthesized in a convergent manner. The introduction of a stereocenter to the vitamin D3 side-chain C24 position was achieved via Sharpless dihydroxylation, and a deoxyfluorination reaction was utilized for the fluorination step. Comparison between (24R)- and (24S)-24-fluoro-25-hydroxyvitamin D3 revealed that the C24-R-configuration isomer 4 was more resistant to CYP24A1-dependent metabolism than its 24S-isomer 3. The new synthetic route of the CYP24A1 main metabolite (24R)-24,25-dihydroxyvitamin D3 (6) and its 24S-isomer (5) was also studied using synthetic intermediates (30,31) in parallel.

The First Convergent Synthesis of 23,23-Difluoro-25-hydroxyvitamin D3 and Its 24-Hydroxy Derivatives: Preliminary Assessment of Biological Activities

“…The CD-rings (9)(10)(11) were synthesized from Inhoffen-Lythgoe diol (12), and the introduction of the difluoro unit to the C23 position was performed using a difluorination reaction of 19 with DAST [21][22][23]. Stereoselective introduction of the hydroxy group to the C24 position was achieved using Sharpless asymmetric dihydroxylation of 28 [24,25]. The A-ring phosphine oxide (14) was prepared from vitamin D 3 [26].…”

In Silico Prediction of the Metabolic Resistance of Vitamin D Analogs against CYP3A4 Metabolizing Enzyme

“…The membrane fraction prepared from the recombinant Escherichia coli cells that expressed h CYP24A1 [ 25 , 26 ] was used to examine the metabolism of 1,25Ds, as previously done for the 1,25D2 analogs. The HPLC metabolic profiles of PRI-1901 and PRI-2205 are shown in Figure 8 .…”

“…CYP24A1-mediated degradation of the two analogs of 1,25D3 (PRI-1901 and PRI-2205) was determined using recombinant human CYP24A1 ( h CYP24A1) as described previously [ 25 , 26 ]. The reaction mixture containing 2.0 μM bovine adrenodoxin, 0.2 μM bovine adrenodoxin reductase, 20 nM CYP24A1, 5 µM 1,25D3 analog, 1 mM NADPH, 100 mM Tris-HCl (pH 7.4), and 1 mM EDTA in total volume of 100 mL, was incubated at 37 °C for 15 min.…”

“…The residue was dissolved in acetonitrile and centrifuged at 20,000× g for 15 min. The supernatant (applied volume 40 μL) was submitted to HPLC Capcell-Pak C18 UG120 4.6 mm × 250 mm column (Phenomenex, Tokyo, Japan) at a flow rate of 1 mL/ according to previously described conditions [ 25 , 26 ]. Samples were eluted by linear gradients of water-acetonitrile 20–100% solution (0–25 min) followed by 100% acetonitrile (25–40 min).…”

“…The expression of CYP24A1 mRNA is used as a measure of the transactivating activity of a vitamin D analog. We have already determined the metabolic conversion of 1,25D analogs to CYP24A1 using the membrane fraction from recombinant Escherichia coli cells that expressed h CYP24A1 [ 25 , 26 ]. The crystal structure of rat recombinant CYP24A1 (Δ2-32, S57D mutant) has been reported (SSRL BL9-2 and BL12-2) [ 27 , 28 ], but the structure of the native full-length human CYP24A1 is still lacking.…”