A Methylene Group on C‑2 of 24,24-Difluoro-19-nor-1α,25-dihydroxyvitamin D₃ Markedly Increases Bone Calcium Mobilization in Vivo

Four side chain fluorinated analogues of 1α,25-dihydroxy-19-norvitamin D have been prepared in convergent syntheses using the Wittig–Horner reaction as a key step. Structures and absolute configurations of analogues <b>3</b> and <b>5</b> were confirmed by X-ray crystallography. All analogues showed high potency in HL-60 cell differentiation and vitamin D-24-hydroxylase (24-OHase) transcription as compared to 1α,25-dihydroxyvitamin D₃ (<b>1</b>). Most important is that all of the 20<i>S</i>-configured derivatives (<b>4</b> and <b>6</b>) had high bone mobilizing activity in vivo. However, in the 20<i>R</i> series, a 2-methylene group was required for high bone mobilizing activity. A change in positioning of the 20<i>R</i> molecule in the vitamin D receptor when the 2-methylene group is present may provide new insight into the molecular basis of bone calcium mobilization induced by vitamin D

Synthesis and Biological Activity of 2‑Methylene Analogues of Calcitriol and Related Compounds

In an attempt to prepare vitamin D analogues that are superagonists, (20<i>R</i>)- and (20<i>S</i>)-isomers of 1α-hydroxy-2-methylene­vitamin D₃ and 1α,25-dihydroxy-2-methylene­vitamin D₃ have been synthesized. To prepare the desired A-ring dienyne fragment, two different approaches were used, both starting from the (−)-quinic acid. The obtained derivative was subsequently coupled with the C,D-ring enol triflates derived from the corresponding Grundmann ketones, using the Sonogashira reaction. Moreover, (20<i>R</i>)- and (20<i>S</i>)-1α,25-dihydroxy-2-methylene­vitamin D₃ compounds with an (5<i>E</i>)-configuration were prepared by iodine catalyzed isomerization. All four 2-methylene analogues of the native hormone were characterized by high in vitro activity. As expected, the 25-desoxy analogues were much less potent. Among the synthesized compounds, two of them, 1α,25-dihydroxy-2-methylene­vitamin D₃ and its C-20 epimer, were found to be almost as active as 2-methylene-19-nor-(20<i>S</i>)-1α,25-dihydroxyvitamin D₃ (2MD) on bone but more active in intestine

Synthesis and Biological Activity of 25-Hydroxy-2-methylene-vitamin D₃ Analogues Monohydroxylated in the A‑ring

The 20<i>R</i>- and 20<i>S</i>-isomers of 25-hydroxy-2-methylene-vitamin D₃ and 3-desoxy-1α,25-dihydroxy-2-methylene-vitamin D₃ have been synthesized. Two alternative synthetic routes were devised for preparation of the required A-ring synthons, starting from the chiral compound derived from the (−)-quinic acid and, alternatively, from the commercially available achiral precursor, monoprotected 1,4-cyclohexanedione. The A-ring dienynes were coupled by the Sonogashira process with the respective C,D-ring fragments, the enol triflates derived from the protected (20<i>R</i>)- or (20<i>S</i>)-25-hydroxy Grundmann ketones. All four compounds possessed significant <i>in vivo</i> activity on bone calcium mobilization and intestinal calcium transport. The presence of a 2-methylene group increased intestinal calcium transport activity of all four analogues above that of the native hormone, 1α,25-dihydroxyvitamin D₃. In contrast, bone calcium mobilization was equal to that produced by 1α,25-dihydroxyvitamin D₃ in compounds having a (20<i>S</i>)-configuration or diminished to one-tenth that of 1α,25-dihydroxyvitamin D₃ in compounds with a (20<i>R</i>)-configuration

qPCR primers.

<p>qPCR primers.</p

Effect of 1, 2, 3, 5, or 7 doses of 2MbisP, CAGE-3 or atRA on AREG, EPGN, EREG, and HB-EGF mRNA expression in skin.

<p>AREG, EPGN, EREG and HB-EGF mRNAs were analyzed by RT-PCR in skin taken 4 h after receiving the final dose of vehicle, 2MbisP (690 nmol/kg), CAGE-3 (0.25 nmol/kg), or atRA (224 nmol/kg). The data are expressed relative to the vehicle-treated group (treatment/vehicle). Significant differences from the vehicle group at each respective dose are indicated by an asterisk, *<i>P</i>≤0.05 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188887#pone.0188887.s005" target="_blank">S5 Table</a>).</p

Structure of 1α,25(OH)₂D₃ compared to the 2-methylene-19-nor analogs, 2MbisP and CAGE-3.

<p>Structure of 1α,25(OH)₂D₃ compared to the 2-methylene-19-nor analogs, 2MbisP and CAGE-3.</p

EGFR ligand mRNA after 7 topical doses of 2MbisP, CAGE-3 or atRA.

<p>EGFR ligand mRNAs were analyzed by RT-PCR in skin taken 4 h after receiving the final dose of vehicle, 2MbisP (690 nmol/kg), CAGE-3 (0.25 nmol/kg), or atRA (224 nmol/kg) and the data are expressed relative to the respective vehicle-treated group (treatment/vehicle).</p

Dose response for 2MbisP, CAGE-3 and atRA on utricle area and epidermal thickness.

<p>Utricle area and epidermal thickness were measured in H&E stained tissue sections after the application of 7 doses of vehicle or varying doses of 2MbisP (A, B), CAGE-3 (C, D), or atRA (E, F). Significant differences from the vehicle group at each respective dose are indicated by an asterisk, *<i>P</i>≤0.05 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188887#pone.0188887.s002" target="_blank">S2 Table</a>).</p

qPCR primers.

<p>qPCR primers.</p

Effect of 2MbisP and atRA on utricle size and epidermal thickness after 2, 7 and 21 topical doses.

<p>(A) Utricle area and (B) epidermal thickness was measured in H&E stained tissue sections taken from mice receiving vehicle, 2MbisP (690 nmol/kg), or atRA (224 nmol/kg). The data are expressed as percent of vehicle. Significant differences from the vehicle group at each dosing time point are indicated by an asterisk, *<i>P</i>≤0.05 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188887#pone.0188887.s001" target="_blank">S1 Table</a>).</p