New 1α,25-dihydroxy-19-norvitamin D3 analogs with a frozen A-ring conformation

Article abstract of DOI:10.1016/j.jsbmb.2010.03.011

We have recently described the synthesis of 1α,25-dihydroxy-19-norvitamin D₃ analogs 2 and 3, possessing an additional ring connecting their 3β-oxygen and C-2. Such structural constrains prevent the A-ring conformational flexibility and the ana

Full text of DOI:10.1016/j.jsbmb.2010.03.011

Journal of Steroid Biochemistry & Molecular Biology 121 (2010) 46–50
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New 1␣,25-dihydroxy-19-norvitamin D3 analogs with a frozen A-ring
conformationଝ
Agnieszka Glebockaa,b, Rafal R. Sicinskia,b, Lori A. Pluma, Hector F. DeLucaa,∗
a Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706, USA
b Department of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
We have recently described the synthesis of 1␣,25-dihydroxy-19-norvitamin D3 analogs 2 and 3, possess-
ing an additional ring connecting their 3␤-oxygen and C-2. Such structural constrains prevent the A-ring
conformational flexibility and the analogs exist exclusively in the ␣-chair form with their 1␣-hydroxy
groups fixed in the axial position. The analogs bind very poorly to vitamin D receptor and are devoid of
transcriptional activity. Rather unexpectedly, when tested in vivo in rats, they exhibited calcemic response
significantly delayed compared to 1␣,25-dihydroxyvitamin D3 (1). Such a response might be due to the
metabolic conversion (ether cleavage?) of these compounds in the living organisms. It was therefore of
interest to obtain and evaluate biologically the analogous compounds having an additional ring of purely
hydrocarbon nature. Such analog 4 of 1␣,25-dihydroxy-19-norvitamin D3, characterized by the presence
of an equatorial 1␣-hydroxy group (␤-chair form), has been synthesized by us and tested biologically.
The geometrical isomer 5 having a fixed 3␤-hydroxy group was also obtained. These compounds were
formed in the Julia coupling of the sulfone derived from the Grundmann ketone, and the A-ring fragment
prepared in the multi-step synthesis from the (−)-quinic acid. Contrary to its counterpart 5, the analog 4
retained some affinity to vitamin D receptor.
Received 30 October 2009
Received in revised form 8 February 2010
Accepted 1 March 2010
Keywords:
Vitamin D analogs
19-Norvitamin D3
Vitamin D receptor
VDR binding
Cellular HL-60 differentiation
© 2010 Elsevier Ltd. All rights reserved.
1. Introduction
have synthesized the 19-norvitamin D analogs 2 and 3 in which
the ring A could exist only in the ␣-chair form [9,10]. Although
The importance of multiple physiological processes controlled
by 1␣,25-dihydroxyvitamin D3 (1␣,25-(OH)2D3, 1; Fig. 1), the hor-
monally active metabolite of vitamin D3, has stimulated a very
intensive research directed towards its biology [1,2] and chem-
istry [3]. Since its discovery, an intriguing problem has attracted
the attention of researchers [4–6]. Which A-ring conformer of the
vitamin D compound is required for binding to the vitamin D recep-
tor (VDR)? In 1974, Norman hypothesized that the ␤-chair form
(Fig. 2a) and, consequently, an equatorial orientation of the 1␣-
hydroxy group is needed for the formation of a complex between
1␣,25-(OH)2D3 and VDR [7]. Almost four decades later, the X-ray
crystallographic studies of a complex of the natural hormone 1 and
hVDR mutant supported this assumption [8]. To exclude the pos-
sibility of chair inversion occurring in the physiological milieu, we
these compounds had very poor binding affinities to VDR, they both
showed surprisingly high calcemic activity in vivo, suggesting a pos-
sible cleavage of their dihydropyran (dihydrofuran) ring. Therefore,
it seemed to be of interest to synthesize the related compound 4
possessing its ring A in the ␤-chair conformation, prevented from
an interconversion to the alternative form. It could be expected
that the hydrocarbon nature of the attached cyclopentene ring will
considerably increase the stability of such a vitamin D analog in the
living organisms.
2. Materials and methods
2.1. Preparation of the 1˛,25-dihydroxy- and
3ˇ,25-dihydroxy-19-norvitamin D3 analogs 4 and 5
The vitamin D analogs 4 and 5 were synthesized at the Depart-
ment of Biochemistry, University of Wisconsin-Madison and at
the Department of Chemistry, University of Warsaw, according
to the synthetic route presented in Scheme 1. The spectroscopic
and analytical data of all obtained compounds confirmed the
ଝ
Special issue selected article from the 14th Vitamin D Workshop held at Brugge,
Belgium on October 4–8, 2009.
∗
Corresponding author. Tel.: +1 608 262 1620; fax: +1 608 262 7122.
E-mail address: deluca@biochem.wisc.edu (H.F. DeLuca).
0960-0760/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jsbmb.2010.03.011

A. Glebocka et al. / Journal of Steroid Biochemistry & Molecular Biology 121 (2010) 46–50
47
Fig. 1. Chemical structure of 1␣,25-dihydroxyvitamin D3 (calcitriol, 1) and its analogs.
assigned structures. The details of synthesis will be reported else-
2.2.3. Transcriptional assay
where.
The transcriptional activity was measured in ROS 17/2.8
(bone) cells that were stably transfected with the 24-hydroxylase
(24OHase) gene promoter upstream of the luciferase reporter gene
[13]. Cells were given a range of doses. Sixteen hours after dosing,
the cells were harvested and luciferase activities were measured
using a luminometer. Each experiment was performed twice, each
time in duplicate.
2.2. In vitro studies
2.2.1. Measurement of binding to the rat recombinant vitamin D
receptor
The procedure for obtaining the purified full-length recombi-
nant rat receptor used in the binding studies will be reported in
detail elsewhere. The competition binding assays were performed
using 1␣,25-(OH)2[26,27-3H]D3 as described previously [11]. The
experiment was in duplicate.
3. Results and discussion
3.1. Chemical synthesis of 4 and 5
2.2.2. Measurement of cellular differentiation
The known cyclic diacetal 6 (Scheme 1), derived from the com-
mercially available (−)-quinic acid [14], was subjected to reaction
with iodine and triphenylphosphine, resulting in the formation of a
single product 7 with an inverted configuration at the stereogenic
center. The addition of the radical, generated from the iodide 7
and tributylstannane, to acrylonitrile provided compound 8 with
an equatorial 2ꢀ-cyano-ethyl substituent. The nitrile 8 was further
Human promyelocytic leukemia HL-60 cells (obtained from
ATTC) were plated at 1.2 × 105 cells/mL and incubated. Eighteen
hours after plating the compounds tested were added, and after
four days the cells were harvested and the nitro blue tetrazolium
(NBT) reduction assay was performed. This method is described in
detail elsewhere [12].
Fig. 2. Conformational equilibrium in ring A in 1␣-hydroxyvitamin D analogs (a) and the A-ring conformation of the synthesized analogs 4 (b) and 5 (c).

48
A. Glebocka et al. / Journal of Steroid Biochemistry & Molecular Biology 121 (2010) 46–50
Scheme 1. (a) (MeCO)2, CH(OMe)3, CSA, MeOH, 85%; (b) I2, Ph3P, Im, PhMe, 54%; (c) CH2 CHCN, AIBN, PhMe, Bu3SnH, 67%; (d) DIBALH, PhMe, 33%; (e) (PPh3)3RhCl, Ph3P,
Me3SiCHN2, i-PrOH, THF, 21%; (f) TFA, H2O, 58%; (g) t-BuMe2SiCl, Im, DMF, 50%; (h) Dess-Martin periodinane, CH2Cl2, 88%; (i) Ph3P+CH3 Br−, n-BuLi, THF, 60%; (j) 15, PhMe,
82%; (k) LiAlH4, THF, 81%; (l) NaIO4, MeOH, 95%; (m) 19, LiHMDS, THF; (n) n-Bu4NF, Et3N, THF, 26% (over two steps).
reduced to the aldehyde 9 which was subsequently methyle-
nated using the rhodium catalyzed process developed by Lebel and
Paquet [15]. The formed terminal alkene 10 was then converted in
several steps into the diolefin 14 which underwent smoothly the
ring-closing metathesis carried out in the presence of the 2nd gen-
eration Grubbs catalyst 15 [16]. The obtained bicyclic hydroxy ester
16 was transformed into the desired A-ring fragment, protected
hydroxycyclohexanone 18. This ketone was then subjected to the
modified Julia olefination with the known thiazoline sulfone 19 [17]
and lithium bis(trimethylsilyl)amide. Removal of the silyl protect-

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.
ing groups in the obtained products gave the expected mixture of
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-
former with the secondary hydroxy group (1␣ or 3␤, respectively)
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.
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