A series of analogs of 1α,25-dihydroxyvitamin D3[1,25(OH)2D3] with alkyl substitutions in 26- and 27-positions were tested for calcium (Ca) regulating activity. The potencies of dialkyl analogs in stimulating bone resorption in neonatal mouse calvaria cultures were the highest in 1α,25-dihydroxy-26,27-dimethylvitamin D3[1,25(OH)2-(Me)2D3], followed by 1,25(OH)2D3, 1α,25-dihydroxy-26,27-diethylvitamin D3[1,25(OH)2(Et)2D3], and 1α,25-dihydroxy-26,27-dipropylvitamin D3[1,25(OH)2(Pr)2D3] in that order. A similar order of potential regarding formation of osteoclast-like cells in mouse bone marrow cell cultures and on bone Ca mobilization with long-term vitamin D-deficient rats was observed in the same series. The relative potencies of 1,25(OH)2D3, 1,25(OH)2(Me)2D3, 1,25(OH)2(Et)2D3, and 1,25(OH)2(Pr)2D3 in competing with 1,25(OH)2D3 for binding to chick intestinal cytosol receptors were 1:1:0.16:0.036. A similar order of potential in case of intestinal Ca transport in situ was observed in the same series. The potencies of dialkyl analogs in competing with 25-hydroxy-vitamin D3 for binding to rat serum vitamin D binding protein were much lower than that of 1,25(OH)2D3. Effect of 1,25(OH)2(Me)2D3 on osteopenia in rats induced by ovariectomy and right sciatic neurotomy was higher than that of 1,25(OH)2D3. From these results, the lengthening by one carbon at 26- and 27-positions was shown to maintain the Ca regulatory activity of 1,25(OH)2D3.

To determine the possibility that methyl substitution in 26- and 27-positions of 24R,25-dihydroxyvitamin D3 [24,25(OH)2D3] alters activities of the original compound, the effects of 24,25(OH)2D3 on calcium (Ca) regulating activity were compared with those of its methyl analog [24,25(OH)2(CH3)2D3] in addition to 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3]. 24,25(OH)2D3 at 10-6 M and 24,25(OH)2(CH3)2D3 at 10-7 M and above significantly stimulated both bone resorption in neonatal mouse calvaria cultures and formation of osteoclast-like multinucleated cells (MNC) in mouse bone marrow cultures. A stimulative effect of 1,25(OH)2D3 on bone resorption and MNC formation was recognized in very low concentrations (10-11 M and above). Although a potency of 24,25(OH)2(CH3)2D3 in stimulating bone calcium (Ca) mobilization and intestinal Ca transport was higher than that of 24,25(OH)2D3, the potencies of both compounds were similar to that of 1,25(OH)2D3 unlike in vitro experiments. As 1,24R,25-trihydroxy-26,27-dimethylvitamin D3 showed almost the same effect as 24,25(OH)2(CH3)2D3, the dihydroxy form is suggested to be hydroxylated at 1α position and converted to trihydroxy form in vitamin D-deficient rats. From these results, methyl substitution in 26- and 27-position of 24,25(OH)2D3 was found to elevate Ca regulating activity of the original compound. In addition, it is suggested that the basis for a similarity in potency between 1,25(OH)2D3 and 24,25(OH)2D3 or its dimethyl analog in vitamin D-deficient rats is likely the result of 1 α-hydroxylation.

Two fluoro analogs of 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], 24a-homo-24,24-difluoro-1α,25-dihydroxyvitamin D3 [24aF2-homo-1,25(OH)2D3], and 26,27-dimethyl-24,24-difluoro-1α,25-dihydroxyvitamin D3 [24F2-1,25(OH)2(Me)2D3] were examined for calcium (Ca)-regulating activity. The objective of the present study was to determine whether or not fluoro substitution at 24-position would alter activities of the original compounds, that is, 26,27-dimethyl 1α, 25-dihydroxyvitamin. D3[1,25(OH)2 (Me)2D3] and 24-homo-1α,25-dihydroxyvitaminD3[24homo-1,25(OH)2D3], respectively. The relative activities of 24aF2-homo-1,25(OH)2D3, 24F2-1,25(OH)2(Me)2D3, and 1,25(OH)2D3 in competing with 1,25(OH)2D3 for binding to chick intestinal cytosol receptor were 0.28:0.5:1.0. The relative potencies of the same series of compounds in competition for the vitamin D-deficient rat serum binding sites were 0.04:0.15:1. Bone-resorbing activities of two fluoro analogs in cultures of neonatal mouse parietal bones were more potent than that of 1,25(OH)2D3. Similar results were recognized in stimulating activities of osteoclast-like cell formation. Responses of two fluoro analogs to intestinal Ca absorption were similar to that of 1,25(OH)2D3. The potencies of 1,25(OH)2D3. and its fluoro analogs in bone Ca mobilization were the highest with 1,25(OH)2D3. followed by 24F2 1,25(OH)2(Me)2D3 and 24aF2-homo-1,25(OH)2D3, in that order. From these results and the data of Paulson et al. [24], fluoro substitution in 24-position of 1,25(OH)2D3. apparently does not alter their activities, hence, the fluoro substitution at 24-position of 1,25(OH)2D3. and the elongation of side chain of 1,25(OH)2D3. may not intensify Ca-regulating activity.