Syntheses of stable isotope-labeled 6β-hydroxycortisol, 6β-hydroxycortisone, and 6β-hydroxytestosterone

Abstract A method is described for the preparation of two types of multi-labeled 6β-hydroxycortisol containing either five deuterium atoms at C-19 methyl and C-1 methylene or four 13 C atoms at C-1, C-2, C-4, and C-19 in addition to the five deuterium atoms for use as analytical internal standards for gas chromatography-mass spectrometry (GC-MS). BMD derivatives of [1,1,19,19,19- 2 H 5 ]cortisone and [1,2,4,19- 13 C 4 ,1,1,19,19,19- 2 H 5 ]cortisone (cortisone- 2 H 5 -BMD and cortisone- 13 C 4 , 2 H 5 -BMD) were first synthesized via indan synthon method starting from optical active 11-oxoindanylpropionic acid and labeled isopropenyl anion ([1,1,3,3,3- 2 H 5 ]- or [1,3- 13 C 2 ,1,1,3,3,3- 2 H 5 ]isopropenyl anion). The labeled isopropenyl anion was prepared from commercially available [1,1,1,3,3,3- 2 H 6 ]- or [1,3- 13 C 2 ,1,1,1,3,3,3- 2 H 6 ]acetone. Ultraviolet (UV) irradiated autoxidation at C-6 position of 3-ethyl-3,5-dienol ether derivatives of the labeled cortisone-BMDs gave 6β-hydroxy-[1,1,19,19,19- 2 H 5 ]cortisone-BMD and 6β-hydroxy-[1,2,4,19- 13 C 4 ,1,1,19,19,19- 2 H 5 ]cortisone-BMD, respectively, as a mixture of 6β- and 6α-epimers in a ratio of 4:1. Separation of 6β- and 6α-epimers by thin-layer chromatography (TLC) and subsequent hydrolysis of the BMD group at C-17 gave pure labeled 6β-hydroxycortisone. After protecting the keto group at C-3 of the labeled 6β-hydroxycortisone-BMD as semicarbazone, reduction of 11-keto group with NaBH 4 and subsequent removal of the C-3 and C-17 protecting groups gave 6β-hydroxy-[1,1,19,19,19- 2 H 5 ]cortisol (6β-hydroxycortisol- 2 H 5 ) and 6β-hydroxy-[1,2,4,19- 13 C 4 ,1,1,19,19,19- 2 H 5 ]cortisol (6β-hydroxycortisol- 13 C 4 , 2 H 5 ), respectively, as a mixture of 6β- and 6α-epimers (6β:6α=4.4:1). The isotopic compositions of 6β-hydroxycortisol- 2 H 5 and 6β-hydroxycortisol- 13 C 4 , 2 H 5 were 90.9 and 92.1 at.%, respectively. Furthermore, 6β-hydroxy-[1α,16,16,17α- 2 H 4 ]testosterone was synthesized by the UV irradiated autoxidation at C-6 position of 3-ethyl-3,5-dienol ether derivative of deuterium-labeled testosterone ([1α,16,16,17α- 2 H 4 ]testosterone) obtained by using catalytic deuteration and hydrogen–deuterium exchange reactions.