ABSTRACT
The three-step conversion of vitamin D analogs to 6-oxo-3,5-cyclovitamin D derivatives followed by reduction with a tritide or deuteride reagent and subsequent cycloreversion gives 6-tritio(deutero)vitamin D derivatives and corresponding 5,6-trans-analogs. The method is general and affords the 6-labeled-vitamin D analogs in approximately 20% overall yield.
Subject(s)
Vitamin D/analogs & derivatives , Cholecalciferol , Deuterium , Ergocalciferols , Isotope Labeling/methods , TritiumABSTRACT
The first direct chemical synthesis of radiolabeled 1 alpha,25-dihydroxyvitamin D3 is reported. Unlike all previous syntheses, the new approach does not rely on enzymatic 1 alpha-hydroxylation of radiolabeled precursors. Rather, isotope is introduced in the last synthetic step by reaction of [3H]-methylmagnesium bromide with methyl 1 alpha-hydroxy-26,27-dinorvitamin D3-25-carboxylate to give 1 alpha,25-dihydroxy-[26,27-3H]vitamin D3 with a specific activity of 160 Ci/mmol. Mass spectroscopy confirmed that the radiohormone consists of a single isomer with six tritium atoms bound to carbons 26 and 27. Synthetically produced 1 alpha,25-dihydroxy[26,27-3H]vitamin D3 is indistinguishable from 1 alpha,25-dihydroxy-[26,27-3H]vitamin D3 obtained from the enzymatic 1 alpha-hydroxylation of 25-hydroxy[26,27-3H]vitamin D3 (160 Ci/mmol) by high-pressure liquid chromatography analysis and in the competitive binding assay using chick intestinal cytosol as the receptor source. Equilibrium dissociation constant measurements with the high specific activity radiohormone indicate a Kd of 8.2 x 10(-11) M for the chick intestinal cytosol 1 alpha,25-dihydroxyvitamin D3 receptor--a value considerably lower than the constants in the range of (1-5) x 10(-9) M previously reported.
Subject(s)
Dihydroxycholecalciferols/chemical synthesis , Hydroxycholecalciferols/chemical synthesis , Receptors, Drug/metabolism , Animals , Binding, Competitive , Calcitriol , Chickens , Cytosol/metabolism , Dihydroxycholecalciferols/metabolism , Duodenum/metabolism , Intestinal Mucosa/metabolism , Isotope Labeling/methods , Kinetics , Magnetic Resonance Spectroscopy , Male , Mass Spectrometry , TritiumSubject(s)
Cholecalciferol/pharmacology , Ergocalciferols/pharmacology , Sitosterols/chemical synthesis , Animals , Biological Transport/drug effects , Bone and Bones/drug effects , Bone and Bones/metabolism , Calcium/metabolism , Dose-Response Relationship, Drug , Intestinal Absorption/drug effects , Male , Rats , Sitosterols/pharmacology , Vitamin D Deficiency/metabolismSubject(s)
Bone and Bones/metabolism , Calcium/metabolism , Cholecalciferol/pharmacology , Intestinal Absorption/drug effects , Intestinal Mucosa/metabolism , Animals , Bone and Bones/drug effects , Cholecalciferol/chemical synthesis , Intestinal Mucosa/drug effects , Male , Mass Spectrometry , Rats , Spectrophotometry, Ultraviolet , Vitamin D Deficiency/metabolismSubject(s)
Calcium/metabolism , Hydroxycholecalciferols/pharmacology , Animals , Binding, Competitive , Biological Transport/drug effects , Bone and Bones/metabolism , Calcification, Physiologic/drug effects , Cholecalciferol/pharmacology , Dose-Response Relationship, Drug , Duodenum/physiology , Kidney/physiology , Male , Phosphorus/metabolism , Rats , Rickets/drug therapy , Structure-Activity RelationshipABSTRACT
1alphaHydroxylated-25-masked-vitamin D3 and analogs were synthesized as probes to help evaluate the role of 25-hydroxyl group in hormone-receptor interactions of 1alpha,25-dihydroxyvitamin D3 (13a). Synthetic work on model systems showed that the steroidal 25-hydroxyl group could be easily fluorinated in high yield with diethylaminosulfur trifluoride. Treatment of 25-fluoro compound with acetic acid resulted in both elimination and displacement of fluorine by acetate. The desired 1alpha-hydroxy-25-fluoro-vitamin D3 (14b) was obtained efficiently by fluorination and subsequent deacetylation of 1alpha,25-dihydroxyvitamin D3 1,3-diacetate (13b). Also obtained was a mixture of 1alpha-hydroxyvitamin D3-24- and 25-enes (15b). Both 14b and 15b were 300-400 times less active than 13a in the chick intestinal cytosol protein binding assay, making these analogs similar in potency to 1alpha-hydroxyvitamin D3 in vitro. The essentially equivalent activity of 14b and 15b with 1alpha-hydroxyvitamin D3 indicates that in the absence of a 25-hydroxyl group some alterations to the side chain carbons of 13a may be tolerated without further weakening analog-protein interactions. The fluoroanalog 14b was also about 250 times less potent than 13a in stimulating bone resorption in vitro. These compounds should prove to be valuable tools in aiding understanding of the salient structural features of the vitamin D3 metabolites.
Subject(s)
Hydroxycholecalciferols/chemical synthesis , Animals , Biological Assay , Bone and Bones/drug effects , Bone and Bones/metabolism , Calcium/metabolism , Carrier Proteins/metabolism , Chickens , Cytosol/metabolism , Fetus , Hydroxycholecalciferols/metabolism , Hydroxycholecalciferols/pharmacology , Intestinal Mucosa/metabolism , RatsABSTRACT
Chemically synthesized 1 alpha-hydroxy-25-fluorovitamin D3 was compared to 1,25-dihydroxyvitamin D3 for potency in the chick intestinal cytosol-binding protein assay, induction of intestinal calcium transport, mobilization of calcium from bone, and epiphyseal plate calcification in the rat. The 25-fluorinated analogue causes 50% displacement of 1,25-dihydroxy[23,24-3H]D3 at 1.8 X 10(-8) M in the competitive protein-binding assay, whereas only 5.6 X 10(-11) M of unlabeled 1,25-dihydroxyvitamin D3 is needed for equal competition. This 315-fold difference between and 1 alpha-hydroxy-25-fluorovitamin D3 indicates that the fluoro analogue is about equipotent with 1 alpha-hydroxyvitamin D3 in the protein-binding assay. However, 1 alpha-hydroxy-25-fluorovitamin D3 is 1/50 as active as 1,25-dihydroxyvitamin D3 in vivo in the stimulation of intestinal calcium transport and bone calcium mobilization in vitamin D deficient rats on a low-calcium diet. Likewise, 1 alpha-hydroxy-25-fluorovitamin D3 is about 40 times less active than 1,25-dihydroxyvitamin D3 in inducing endochondrial calcification in rachitic rats. No selective actions of 1alpha-hydroxy-25-fluorovitamin D3 were noted. Since the 25 position of the analogue is blocked by a fluorine atom, it appears that 25-hydroxylation of 1 alpha-hydroxylated vitamin D compounds in vivo is not an obligatory requirement for appreciable vitamin D activity.
