ABSTRACT
As part of our program on synthesis of labeled vitamin D metabolites and analogs, we describe here an efficient and versatile synthetic approach to 28,28,28-trideutero- 25-hydroxydihydrotachysterol2 where isotopic labeling was incorporated stereoselectively in the last step of the synthesis. This deuterated compound will allow the study this analog in vitro or in vivo and to measure AT10-like compounds in serum by LC-MS/MS.
Subject(s)
Dihydrotachysterol/analogs & derivatives , Dihydrotachysterol/analysis , Dihydrotachysterol/chemistry , Vitamin D/analogs & derivatives , Vitamin D/metabolism , Deuterium/chemistry , Dihydrotachysterol/chemical synthesis , Staining and Labeling , Vitamin D/chemistryABSTRACT
Dihydrotachysterol (DHT), a reduced vitamin D analog in which the A-ring has been rotated through 180 degrees is a biologically active molecule which can be used to study the structural requirements for the calcemic and cell differentiating properties of the vitamin D hormone, 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25-(OH)2D3), as well as to investigate the specificity of the enzyme systems that catalyze the formation of this hormone. In this study we showed that dihydrotachysterol was metabolized in vivo into a significant polar metabolite observed on straight-phase high performance liquid chromatography (HPLC) which subsequently split into two peaks on reverse-phase HPLC. These two metabolites were identified by HPLC and gas chromatography-mass spectrometry techniques as 1 alpha,25-(OH)2DHT and 1 beta,25-(OH)2DHT. This pair of metabolites was formed from either DHT2 or DHT3. Standard 1 alpha,25-(OH)2DHTs were generated in vitro from chemically synthesized 1-hydroxydihydrotachysterol precursors using a liver hepatoma cell system. Both 1 alpha,25-(OH)2D2 and 1 alpha,25-(OH)2DHT3 showed a binding affinity to the mammalian vitamin D receptor only 50-100 less than 1 alpha,25-(OH)2D3 whereas 1 beta,25-(OH)2DHTs showed poor binding. On the other hand 1 beta,25-(OH)2DHT3 bound to the rat vitamin D transport protein (DBP) with stronger affinity than did 1 alpha,25-(OH)2DHT3. When tested in a COS-1 cell transfection assay system using a rat osteocalcin vitamin D responsive element coupled to a growth hormone reporter gene, 1 alpha,25-(OH)2DHT3 showed a biological activity only 10 times lower than 1 alpha,25-(OH)2D3. It is therefore suggested that 1 alpha,25-(OH)2DHT probably represents the metabolite of DHT responsible for some of its in vivo effects although we cannot rule out in vivo effects of other metabolites identified. Our studies suggest that 1 alpha,25-dihydroxylated DHTs represent a promising novel group of vitamin D analogs worthy of study for cell differentiation as well as calcemic properties.
Subject(s)
Dihydrotachysterol/analogs & derivatives , Dihydrotachysterol/metabolism , Vitamin D-Binding Protein/metabolism , Animals , Biotransformation , Carcinoma, Hepatocellular , Cell Line , Chromatography, High Pressure Liquid , Dihydrotachysterol/chemical synthesis , Dihydrotachysterol/chemistry , Gas Chromatography-Mass Spectrometry , Growth Hormone/metabolism , Humans , Liver Neoplasms , Magnetic Resonance Spectroscopy , Molecular Structure , Rats , Stereoisomerism , Transfection , Tritium , Tumor Cells, CulturedABSTRACT
Dihydrotachysterol2 (DHT2) is a synthetic analogue of vitamin D2. DHT2 is used extensively in the treatment of renal osteodystrophy and hypoparathyroidism. It is equally efficacious as 1 alpha,25-dihydroxyvitamin D3 and 1 alpha-hydroxyvitamin D3. Moreover, it offers interesting therapeutical advantages and it is surprising that until recently little was known of its metabolism and sites of action. This paper deals with studies on the pharmacology of DHT2 in rats. Following the synthesis of [3H]DHT2 and oral administration, evidence was obtained that DHT2 is metabolized extensively; three of the major metabolites could be identified as 25-hydroxy-DHT2, 1 alpha,25- and 1 beta,25-dihydroxy-DHT2.
Subject(s)
Dihydrotachysterol , Animals , Chemical Phenomena , Chemistry , Dihydrotachysterol/chemical synthesis , Dihydrotachysterol/pharmacokinetics , Dihydrotachysterol/pharmacology , Kinetics , Rats , TritiumABSTRACT
Dihydrotachysterol2 (DHT2), a 5,6-trans derivative of vitamin D2, is very successfully used in the treatment of hypoparathyroidism and renal osteodystrophy. However, the metabolism and the action of DHT2 are poorly understood. Investigations into metabolism of DHT2 start at the synthesis of the radioactively labelled compound. This paper deals with a two-step synthesis of [10S(19)-3H]-dihydrotachysterol2 from vitamin D2. Vitamin D2 can be converted into 5,6-trans vitamin D2 by iodination under irradiation and by triplet-sensitized isomerization. The first method led to unwanted side-reaction products which were difficult to separate from 5,6-trans vitamin D2. Triplet-sensitized isomerization yielded merely 5,6-trans vitamin D2 which could be separated from the residual starting material by chromatography on silica gel and recrystallization. [10S](19)-3H]-Dihydrotachysterol2 with a specific radioactivity of 56 kCi/mol was prepared from 5,6-trans vitamin D2 via partial, homogeneous catalytic reduction of the 10S(19) double bond with tritium gas. It was purified by high-performance liquid chromatography (h.p.l.c.) and characterized by chromatography and ultra-violet absorption spectrophotometry. The biological usefulness of the material was demonstrated in rats following intragastric administration. Blood was collected after 24 and 48 h and fractionation of serum lipids on h.p.l.c. showed 5 peaks of radioactivity.
