Total synthesis of biologically active 20S-hydroxyvitamin D3

Abstract

A total synthetic strategy of 20S-hydroxyvitamin D3 [20S-(OH)D3] involving modified synthesis of key intermediates 7 and 12, Grignard reaction to stereoselectively generate 20S-OH and Wittig-Horner coupling to establish D3 framework, was completed in 16 steps with an overall yield of 0.4%. The synthetic 20S-(OH)D3 activated vitamin D receptor (VDR) and initiated the expression of downstream genes. In addition, 20S-(OH)D3 showed similar inhibitory potency as calcitriol [1,25(OH)2D3] on proliferation of melanoma cells.

Keywords: 20S-Hydroxyvitamin D3; PCR; Reporter assay; Thymidine incorporation; Total chemical synthesis; VDR activation.

Fig. 1

Marketed vitamin D analogs and noncalcemic 20S-(OH)D3

Fig. 2

Preferred conformation of the intermediate (compound 12) calculated with density function theory with 6–31G** baseset. “Front” side attack by the bulky Grignard agent 13 to form 20R isomer of 14 is prohibited due to the steric hindrance from the 18-methyl and other moieties in compound 12, while the “back” side attack to form 20S isomer is strongly favored.

Fig. 3

20S-(OH)D3 stimulates mRNA levels of VDR and CYP24A1 at 6 h (A) and 24 h (B). HaCaT cells were treated for 6 h with 100 nM of 20S-(OH)D3, 1,25(OH)₂D3 or DMSO only (solvent) as a control. The mRNA was isolated and qPCR performed using specific primers for VDR and CYP24A1 genes. Data are presented as mean ± SE (n = 3, * p < 0.05, *** p < 0.005, **** p < 0.0001).

Fig. 4

Anti-proliferative effects of 20S-(OH)D3 and 1,25(OH)₂D3 on human SKEML-188 melanoma cells. Cells were treated with 20S-(OH)D3, 1,25(OH)₂D3 or DMSO (solvent) as a control to assess their inhibitory effects on DNA synthesis. Data are presented as mean ± SE (n = 3, * p < 0.05, ** p < 0.01, *** p < 0.005).

Fig. 5

20S-(OH)D3 activates luciferase activity in VDRE-LUC transduced Jurkat and HaCaT cells. Cells were treated for 24 h with 100 nM 20S-(OH)D3, 1,25(OH)₂D3 or DMSO as a control. Data are presented as mean ± SE (n = 3, * p < 0.05, ** p < 0.01).

Scheme 1

Retrosynthetic analysis of 20S-(OH)D3

Scheme 2

Synthesis of intermediate 7 Reagents: (a): KMnO₄, EtOH-H₂O; 65%; (b): i. Pb(OAc)₄, Na₂CO₃, DCM; ii. Red-Al, benzene; 43% for 3 and 86% for 4 in two steps; (c): TBSCl, Imidazole, DCM; 88%; (d): TBAF, THF; 78%; (e): i. n-BuLi, p-TsCl, THF; ii. n-BuLi, Ph₂PH, THF; iii. H₂O₂, H₂O, DCM; 59% in three steps.

Scheme 3

Synthesis of intermediate 12 Reagents: (a): Ac₂O, Pyr, DMAP, DCM; 92%; (b): OsO₄, NMO, Acetone/t-BuOH; 67%; (c): NaIO₄, H₂O-THF; 66% (d): Morpholine, p-TsOH, PhH, reflux; (e): KMnO₄/Al₂O₃, Acetone; 70% in two steps.

Scheme 4

Synthesis of 20S-(OH)D3 Reagents: (a): Mg. THF; (b): THF;80% (c): PDC, DCM; 89%; (d): EOMCl, DMAP, TEA, DCM; 90%; (e): 7, PhLi, THF; 53%; (f): CSA, DCM-MeOH; 37%.