RESUMEN
The binding of 25-hydroxy-[26,27-3H]vitamin D-3 and 25-hydroxy-[26,27-3H]vitamin D-2 to the vitamin D binding protein in the plasma of both rats and chicks has been studied. In the case of rats, sucrose density gradient analysis, competitive displacement, and Scatchard analysis demonstrate that 25-hydroxyvitamin D-3 and 25-hydroxyvitamin D-2 are bound equally well to the vitamin D binding protein. In contrast, 25-hydroxyvitamin D-2 is poorly bound, while 25-hydroxyvitamin D-3 is tightly bound to the vitamin D binding protein in chick plasma. On the other hand, the chick intestinal receptor binds 1,25-dihydroxyvitamin D-2 and 1,25-dihydroxyvitamin D-3 equally well with a KD of 7.10(-11) M for both compounds. These results strongly suggest that the failure of the plasma transport protein in chicks to bind the vitamin D-2 compounds may be responsible for their relative ineffectiveness in these animals.
Asunto(s)
Ergocalciferoles/análogos & derivados , Proteína de Unión a Vitamina D/sangre , 25-Hidroxivitamina D 2 , Animales , Calcifediol/sangre , Centrifugación por Gradiente de Densidad , Pollos , Ergocalciferoles/sangre , Masculino , Ratas , Especificidad de la EspecieRESUMEN
1 alpha, 2 alpha,25-Trihydroxy-19-norvitamin D3, 1 alpha, 2 beta,25-trihydroxy-19-norvitamin D3, and their alkoxy analogs were efficiently prepared in a convergent synthesis, starting with (-)-quinic acid and a Windaus--Grundmann type ketone. Configurations of the A-ring fragment substituents were determined by 1H,1H COSY 2D spectra and 1H NOE difference spectroscopy. The new analogs exhibited selective activity in stimulating intestinal calcium transport while having little or no activity in mobilizing bone calcium. They also showed HL-60-differentiating activity equal to or 10 times lower than that of 1 alpha,25-dihydroxyvitamin D3.
Asunto(s)
Calcitriol/análogos & derivados , Animales , Transporte Biológico , Calcitriol/síntesis química , Calcitriol/farmacología , Calcio/metabolismo , Diferenciación Celular/efectos de los fármacos , Humanos , Mucosa Intestinal/metabolismo , Intestinos/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Masculino , Ratas , Ratas Sprague-Dawley , Células Tumorales CultivadasRESUMEN
New highly active isomers of the natural hormone 1alpha, 25-dihydroxyvitamin D3 possessing an exomethylene group at the 2-position were prepared in a convergent manner, starting with (-)-quinic acid and the corresponding (20R)- and (20S)-25-hydroxy Grundmann ketones. These 2-methylene-19-norvitamins were efficiently converted to the 2-methyl and 2-hydroxymethyl derivatives, some of which exhibited pronounced in vivo biological activity. Configurations of the A-ring substituents were determined by 1H NOE difference spectroscopy as well as by spin decoupling experiments. It was established that the bulky methyl and hydroxymethyl substituents at C-2, due to their large conformational free energies, occupy mainly equatorial positions. Additionally, hydroxylation of the C(10)-C(19) double bond in 1alpha,25-(OH)2D3 was performed, resulting in 1alpha,19,25-trihydroxy-10,19-dihydrovitamin D3 derivatives in which the hydroxymethyl substituent at C-10, for steric reasons, is forced to occupy an axial position. In consequence, the vitamin D3 analogues were synthesized in which the 1alpha-hydroxy group, required for biological activity, is almost exclusively axially or equatorially oriented because of stabilization of the single A-ring chair conformations. The relative ability of the synthesized analogues to bind the porcine intestinal vitamin D receptor was assessed and compared with that of the natural hormone. It was established that vitamins possessing the axial orientation of the 1alpha-hydroxy substituent exhibit a significantly increased receptor binding affinity. Compounds with a 2-methylene substituent showed selective calcemic activity profiles, being extremely effective on bone calcium mobilization. 2alpha-Methyl-substituted vitamins proved to be much more active in vivo than the corresponding epimers with 2beta-configuration. All of the 2-substituted vitamins exhibited pronounced HL-60 differentiating activity, those 2alpha-substituted in the 20S-series being especially potent. The present studies imply that the axial orientation of the 1alpha-hydroxy group is necessary for biological activity of vitamin D compounds.
Asunto(s)
Calcitriol/análogos & derivados , Animales , Unión Competitiva , Huesos/efectos de los fármacos , Huesos/metabolismo , Calcitriol/síntesis química , Calcitriol/química , Calcitriol/metabolismo , Calcitriol/farmacología , Calcio/metabolismo , Calcio de la Dieta/administración & dosificación , Diferenciación Celular/efectos de los fármacos , Línea Celular , Humanos , Técnicas In Vitro , Mucosa Intestinal/metabolismo , Masculino , Conformación Molecular , Ratas , Receptores de Calcitriol/metabolismo , Relación Estructura-Actividad , PorcinosRESUMEN
1 alpha, 25-dihydroxy-18-norvitamin D3 and 1 alpha, 25-dihydroxy-18,19-dinorvitamin D3 were prepared via Wittig-Horner coupling of 25-hydroxy-18-nor Grundmann type ketone with the corresponding A-ring phosphine oxides. Configuration at C-13 in the 18-nor Grundmann type alcohol (C,D-ring synthon), obtained by oxidative degradation of vitamin D3, was determined by 1H NMR spectroscopy and molecular mechanics calculations. Additional proof of the assigned trans-C/D-junction of the key intermediate 18-nor Grundmann type ketone follows from its chiroptical properties (circular dichroism data) and further chemical transformations. 1 alpha, 25-Dihydroxy-18-norvitamin D3 was found more potent than 1 alpha, 25-dihydroxyvitamin D3 in binding to the porcine intestinal vitamin D receptor (5-10x), in differentiation of HL-60 cells (5-10x), and in inhibition of HL-60 proliferation. 1 alpha, 25-Dihydroxy-18, 19-dinorvitamin D3 appeared equally active as 1 alpha, 25-dihydroxyvitamin D3 in these activities. In vivo, 1 alpha, 25-dihydroxy-18-norvitamin D3 was only slightly less active than 1 alpha, 25-dihydroxyvitamin D3 in intestinal calcium transport and bone calcium mobilization, while 1 alpha, 25-dihydroxy-18,19-dinorvitamin D3 showed activities 10 times lower. These studies imply that deletion of C-18 does not impair activity of analogs of 1 alpha, 25-dihydroxyvitamin D3.
Asunto(s)
Calcitriol/análogos & derivados , Animales , Transporte Biológico Activo , Huesos/metabolismo , Calcitriol/química , Calcitriol/metabolismo , Calcitriol/farmacología , Calcio/metabolismo , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Células HL-60 , Humanos , Mucosa Intestinal/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
Construction of 25-hydroxy-steroidal side chain substituted with iodine at C-22 was elaborated on a model PTAD-protected steroidal 5,7-diene and applied to a synthesis of (22R)- and (22S)-22-iodo-1alpha,25-dihydroxyvitamin D3. Configuration at C-22 in the iodinated vitamins, obtained by nucleophilic substitution of the corresponding 22S-tosylates with sodium iodide, was determined by comparison of their iodine-displacement processes and cyclizations leading to isomeric five-membered (22,25)-epoxy-1alpha-hydroxyvitamin D3 compounds. Also, 20(22)-dehydrosteroids have been obtained and their structures established by 1H NMR spectroscopy. When compared to the natural hormone, (E)-20(22)-dehydro-1alpha,25-dihydroxyvitamin D3 was found 4 times less potent in binding to the porcine intestinal vitamin D receptor (VDR) and 2 times less effective in differentiation of HL-60 cells. 22-Iodinated vitamin D analogues showed somewhat lower in vitro activity, whereas (22,25)-epoxy analogues were inactive. Interestingly, it was established that (22S)-22-iodo-1alpha,25-dihydroxyvitamin D3 was 3 times more potent than its (22R)-isomer in binding to VDR and four times more effective in HL-60 cell differentiation assay. The restricted mobility of the side chain of both 22-iodinated vitamin D compounds was analyzed by a systematic conformational search indicating different spatial regions occupied by their 25-oxygen atoms. Preliminary data on the in vivo calcemic activity of the synthesized vitamin D analogues indicate that (E)-20(22)-dehydro-1alpha,25-dihydroxyvitamin D3 and 22-iodo-1alpha,25-dihydroxyvitamin D3 isomers were ca. ten times less potent than the natural hormone 1alpha,25-(OH)2D3 both in intestinal calcium transport and bone calcium mobilization.
Asunto(s)
Calcitriol/análogos & derivados , Animales , Calcitriol/síntesis química , Calcitriol/farmacología , Diferenciación Celular/efectos de los fármacos , Células HL-60 , Humanos , Técnicas In Vitro , Espectroscopía de Resonancia Magnética , Conformación Molecular , Receptores de Calcitriol/metabolismo , Estereoisomerismo , Relación Estructura-Actividad , TermodinámicaRESUMEN
Synthesis of a C-24-epimeric mixture of 25-hydroxy-[26,27-3H]vitamin D2 and a C-24-epimeric mixture of 1,25-dihydroxy-[26,27-3H]vitamin D2 by the Grignard reaction of the corresponding 25-keto-27-nor-vitamin D2 and 1 alpha-acetoxy-25-keto-27-nor-vitamin D3 with tritiated methyl magnesium bromide is described. Separation of epimers by high-performance liquid chromatography afforded pure radiolabeled vitamins of high specific activity (80 Ci/mmol). The identities and radiochemical purities of 25-hydroxy-[26,27-3H[vitamin D2 and 1,25-dihydroxy-[26,27-3H]vitamin D2 D2 were established by cochromatography with synthetic 25-hydroxyvitamin D2 or 1,25-dihydroxyvitamin D2. Biological activity of 25-hydroxy-[26,27-3H]vitamin D2 was demonstrated by its binding to the rat plasma binding protein for vitamin D compounds, and by its in vitro conversion to 1,25-dihydroxy-[26,27-3H]vitamin D2 by kidney homogenate prepared from vitamin D-deficient chickens. The biological activity of 1,25-dihydroxy-[26,27-3H]vitamin D2 was demonstrated by its binding to the chick intestinal receptor for 1,25-dihydroxyvitamin D3.
Asunto(s)
Ergocalciferoles/análogos & derivados , 25-Hidroxivitamina D 2 , Animales , Unión Competitiva , Ergocalciferoles/síntesis química , Ergocalciferoles/aislamiento & purificación , Ergocalciferoles/metabolismo , Técnicas In Vitro , Riñón/metabolismo , Unión Proteica , Estereoisomerismo , Tritio , Deficiencia de Vitamina D/metabolismo , Proteína de Unión a Vitamina D/metabolismoRESUMEN
The biological activity of 1,25-dihydroxyvitamin D2 [1,25(OH)2D2] and 24-epi-1,25-dihydroxyvitamin D2 [24-epi-1,25(OH)2D2] has been determined in vitamin D-deficient rats. The biological effectiveness of 1,25(OH)2D2 is equal to that reported previously for 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] (15) in intestinal calcium transport, mineralization of bone, mobilization of bone calcium, and elevation of plasma inorganic phosphorus of rachitic rats. However, 24-epi-1,25(OH)2D2 is at best one-half as active as 1,25(OH)2D2 in stimulating intestinal calcium transport and in the mineralization of rachitic bone. The 24-epi-1,25(OH)2D2 is one-third as active as 1,25(OH)2D3 in binding to the chick intestinal receptor for 1,25(OH)2D3. Thus receptor discrimination may account for the twofold difference in intestinal calcium transport activity. 24-Epi-1,25(OH)2D2 appeared inactive in in vivo mobilization of bone calcium or bone phosphorus. On the other hand, in fetal rat bone in culture, the epi compound was only five times less active than 1,25(OH)2D2 in inducing resorption. Short-term experiments on bone mineral mobilization in vivo show that the 24-epi-1,25(OH)2D2 does induce bone calcium mobilization but that its activity in this respect is short lived. It is suggested that 24-epi-1,25(OH)2D2 and, as a result, it shows preferential activity on intestine whose response to a single dose of 1,25(OH)2D2 remains for several days, whereas the short-lived bone system does not remain stimulated during the 24-h period between doses.
Asunto(s)
Resorción Ósea/efectos de los fármacos , Calcitriol/farmacología , Calcio/metabolismo , Ergocalciferoles/análogos & derivados , Absorción Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Receptores de Esteroides/metabolismo , Animales , Pollos , Ergocalciferoles/metabolismo , Ergocalciferoles/farmacología , Cinética , Masculino , Ratas , Receptores de Calcitriol , Valores de Referencia , Deficiencia de Vitamina D/metabolismoRESUMEN
In vitro incubation of 24-epi-25-hydroxyvitamin D2 with chicken kidney homogenate produced several compounds, one of which had an affinity equal to that of 1,25-dihydroxyvitamin D2 for the chick intestinal receptor. The affinity of 24-epi-1,25-dihydroxyvitamin D2 for the same receptor was found to be half that of 1,25-dihydroxyvitamin D2. The unknown compound was produced only when homogenate was prepared from pooled kidneys taken from both vitamin D deficient and replete chickens. The compound has been tentatively identified as 1,25-dihydroxy-22-dehydro-26-homovitamin D3 by ultraviolet absorption spectrophotometry and mass spectrometry. Chemical synthesis of 1,25-dihydroxy-22-dehydro-26-homovitamin D3 provided additional evidence for the structure. Administration of this 26-homologue of 1,25-dihydroxyvitamin D3 at the dose level of 650 pmol/rat stimulated bone calcium mobilization in the hypocalcemic rat equal to that of 1,25-dihydroxyvitamin D3. Thus, this paper demonstrates unique methyl migration on the side chain of 24-epi-1,25-dihydroxyvitamin D3 to form a more biologically potent analogue.
Asunto(s)
Calcitriol/análogos & derivados , Ergocalciferoles/metabolismo , Riñón/metabolismo , Animales , Unión Competitiva , Calcitriol/biosíntesis , Calcitriol/aislamiento & purificación , Pollos , Mucosa Intestinal/metabolismo , Cinética , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Receptores de Calcitriol , Receptores de Esteroides/metabolismo , Espectrofotometría UltravioletaRESUMEN
In vitro incubation of all-trans-retinol (atROL) with kidney homogenate from vitamin A-deficient and retinoic acid-supplemented (VAD-RAS) female rats produces a new retinol metabolite. Reverse-phase (RP) and normal-phase (NP) high-performance liquid chromatography (HPLC) analysis showed that this metabolite coelutes with the unknown all-trans-retinol (atROL) metabolite previously found in the day 10 conceptus and kidneys of vitamin A-deficient rats maintained on all-trans-retinoic acid (VAD-RA) and given 2 microg of [3H]atROL. Normal-phase (NP) HPLC purification of the metabolite collected from a RP HPLC column further separated the radiolabeled material into two components. The two isolated compounds have identical or very similar spectroscopic properties. Their nuclear magnetic resonance (1H NMR) and mass spectra (MS) indicated that they are isomers. Spectroscopic studies of the metabolites and their derivatives showed that they are nine-carbon fragments resulting from an oxidative cleavage of the side chain of atROL. The cleavage occurs at C-9, and the product is then oxidized to a keto group. The primary hydroxy group from atROL is preserved in the metabolite. A sulfide bridge is formed between C-11 and C-14, which interrupts the conjugation. The formation of the new metabolites, possessing a 2,5-dihydrothiophene ring, is catalyzed by an enzyme(s) located in the cytosolic fraction of kidneys. The process represents a new retinol metabolic pathway; however, its biological significance is unknown.