A. Glebocka et al. / Journal of Steroid Biochemistry & Molecular Biology 121 (2010) 46–50
49
Fig. 5. Transcriptional activity of 1␣,25-(OH)2D3 (1) and the synthesized analogs
4 and 5. Transcriptional assay was carried out in rat osteosarcoma cells stably
transfected with a 24-hydroxylase gene reporter plasmid. Each experiment was
performed twice, each time in duplicate.
Fig. 3. Competitive binding of 1␣,25-(OH)2D3 (1) and the synthesized analogs 4 and
5 to the rat recombinant vitamin D receptor. This experiment was carried out twice,
each time in duplicate.
two 19-norvitamin D analogs 4 and 5 which were purified and sep-
arated by reversed-phase HPLC. 1H NMR spectra of both vitamins
confirmed that their ring A is fixed in the single chair conformation
(Fig. 2b and c).
cell differentiation. These data are consistent with the transcrip-
tion results. In the reporter cell assay, the activity of the vitamin 4
is reduced 50 times compared to the native hormone, whereas its
counterpart 5 is less active by four orders of magnitude (Fig. 5).
The results of binding studies show that the vitamin 4 with an
equatorial 1␣-hydroxyl has an affinity for VDR of one order of mag-
nitude higher than the previously synthesized analog 3 despite
the fact that the latter one can form additional hydrogen bonds
involving the oxygen atom from its furan ring as an acceptor. This
confirms that the ligand-binding pocket of the VDR easily accom-
modates vitamin D analogs possessing their A ring in the -chair
form. Because vitamin 4 competes for the VDR binding at 10 nM, in
vivo activity is expected and these studies are in progress.
3.2. Biological evaluation of the synthesized analogs 4 and 5
Due to the presence of an exocyclic double bond being a part
of an additional five-membered ring [18], the cyclohexane ring A
in the synthesized analogs 4 and 5 exists as a “frozen” chair con-
assuming only an equatorial orientation. First, an ability of both
vitamins to bind the rat recombinant VDR has been assessed. Ana-
log 4, characterized by a bridge between C-2 and C-3, is less able
to bind to VDR by two orders of magnitude compared to the native
hormone (Fig. 3). Also, it has been found that the vitamin 4 is able
to stimulate differentiation of promyleocytic leukemia cells into
monocytes, being 15 times less potent than 1␣,25-(OH)2D3 in the
HL-60 assay (Fig. 4). The isomeric compound 5, with a hydrocarbon
bridge connecting C-1 and C-2, is practically devoid of any binding
affinity to the receptor and it also lacks activity in promoting cancer
Acknowledgements
The work was supported in part by funds from the Wisconsin
Alumni Research Foundation. Special thanks are addressed to Jean
Prahl and Jennifer Vaughan for carrying out the in vitro studies.
References
[1] D. Feldman, J.W. Pike, F.H. Glorrieux (Eds.), Vitamin D, second edition, Elsevier
Academic Press, Burlington, 2005.
[2] T. Suda, T. Shinki, N. Takahashi, The role of vitamin D in bone and intestinal cell
differentiation, Annu. Rev. Nutr. 10 (1990) 195–211.
[3] R. Bouillon, W.H. Okamura, A.W. Norman, Structure–function relationships in
the vitamin D endocrine system, Endocr. Rev. 16 (1995) 200–257.
[4] W.H. Okamura, J.A. Palenzuela, J. Plumet, M.M. Midland, Vitamin D: structure-
function analyses and the design of analogs, J. Cell. Biochem. 49 (1992) 10–18.
[5] O. Hofer, H. Kählig, W. Reischl, On the conformational flexibility of vitamin D,
Monatsh. Chem. 124 (1993) 185–198.
[6] W.H. Okamura, M.M. Midland, M.W. Hammond, N.A. Rahman, M.C. Dormanen,
I. Nemere, A.W. Norman, Chemistry and conformation of vitamin D molecules,
J. Steroid. Biochem. Mol. Biol. 53 (1995) 603–613.
[7] W.H. Okamura, A.W. Norman, R.M. Wing, Vitamin D: concerning the relation-
ship between molecular topology and biological function, Proc. Natl. Acad. Sci.
U.S.A. 71 (1974) 4194–4197.
[8] N. Rochel, J.M. Wurtz, A. Mitschler, B. Klaholz, D. Moras, The crystal structure
of the nuclear receptor for vitamin D bound to its natural ligand, Mol. Cell 5
(2000) 173–179.
[9] R.R. Sicinski, A. Glebocka, L.A. Plum, H.F. DeLuca, Design, synthesis, and biolog-
ical evaluation of a 1␣,25-dihydroxy-19-norvitamin D3 analogue with a frozen
A-ring conformation, J. Med. Chem. 50 (2007) 6154–6164.
[10] A. Glebocka, K. Sokolowska, R.R. Sicinski, L.A. Plum, H.F. DeLuca, New 1␣,
25-dihydroxy-19-norvitamin D3 compounds constrained in a single A-ring
conformation: synthesis of the analogues by ring-closing metathesis route and
their biological evaluation, J. Med. Chem. 52 (2009) 3496–3504.
[11] M.C. Dame, E.A. Pierce, J.M. Prahl, C.E. Hayes, H.F. DeLuca, Monoclonal antibod-
ies to the porcine intestinal receptor for 1, 25-dihydroxyvitamin D3: interaction
with distinct receptor domains, Biochemistry 25 (1986) 4523–4534.
Fig. 4. Differentiation activity of 1␣,25-(OH)2D3 (1) and the synthesized analogs 4
and 5. Differentiation state was determined by measuring the percentage of cells
reducing nitro blue tetrazolium (NBT).