Concise synthesis and characterization of novel seco-steroids bearing a spiro-oxetane instead of a metabolically labile C3-hydroxy group

Article abstract of DOI:10.1016/j.tetlet.2014.05.060

Two novel stereoisomeric analogues of 1,25-dihydroxyvitamin D₃ bearing a spiro-oxetane at the C3 position of the A-ring have been designed and synthesized in a convergent manner. The absolute configuration at the C1 position of the synthesized

Full text of DOI:10.1016/j.tetlet.2014.05.060

Tetrahedron Letters 55 (2014) 3805–3808
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Concise synthesis and characterization of novel seco-steroids bearing
a spiro-oxetane instead of a metabolically labile C3-hydroxy group
⇑
Toshie Fujishima , Tsutomu Suenaga, Takato Nozaki
Faculty of Pharmaceutical Sciences at Kagawa Campus, Tokushima Bunri University, Shido, Sanuki, Kagawa 769-2193, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Two novel stereoisomeric analogues of 1,25-dihydroxyvitamin D3 bearing a spiro-oxetane at the C3 posi-
tion of the A-ring have been designed and synthesized in a convergent manner. The absolute configura-
tion at the C1 position of the synthesized compounds was determined by the circular dichroism exciton
chirality method using the corresponding C1-allylic benzoates. The replacement of the C3-hydroxy group
with a spiro-oxetane provided an advantageous conformational preference for the parent seco-steroids,
which would facilitate the formation of a stable receptor complex.
Received 31 March 2014
Revised 22 April 2014
Accepted 14 May 2014
Available online 23 May 2014
Keywords:
Heterocycles
Hormone
Oxetane
Ó 2014 Elsevier Ltd. All rights reserved.
Steroid
Vitamin
The biologically active form of the seco-steroid,
1a,25-
dihydroxyvitamin D3 (1), plays a critical role in the regulation of
calcium homeostasis (Fig. 1).1 The major molecular target of 1 is
the vitamin D receptor (VDR), which belongs to the nuclear recep-
tor super-family and functions as a ligand-inducible transcription
factor.1a,b In contrast to other nuclear receptors, the VDR has no
subtypes and is ubiquitously expressed in many different tissue
types throughout the human body, which suggests that 1 could
be involved in many other biological activities besides its classical
role in calcium homeostasis.1c,d The results of recent studies have
also suggested that bone itself could be a target for therapeutic
agents designed to effect the immune system. The functions of 1
and several other seco-steroids, as well as the functions of the
VDR, have consequently become the focus of considerable research
interest of scientists in fields of drug discovery and pharmacology
because of their potential applications to the development of novel
therapeutic agents.1e,f
Figure 1. Structures of 1a,25-dihydroxyvitamin D3 (1) and the novel seco-steroids
having a spiro-oxetane at the C3 position (2a and 2b).
because of the lack of the B-ring, are critical to the formation of a
stable complex with the receptor.3 In contrast to the epimerization
of the 1a-hydroxy group in 1, which results in an approximately
The X-ray crystal structure of the VDR in complex with 1 has
been solved and revealed that each of the three hydroxy groups
in 1 made critical hydrogen bonding interactions to two of
the amino acid residues in the ligand-binding domain (LBD) of
500-fold reduction in the VDR binding affinity, epimerizaion at
the C3-position of 1 has been shown to have little impact on the
binding affinity. The C3-epimer of 1 was estimated to be 24% of
the affinity of 1 using chick intestinal VDR,4 which suggested that
the LBD of the VDR possessed an additional space in the vicinity of
the C3-position of the ligands to allow for the epimerization at this
position. This result occurred in direct contrast to the strict recog-
nition required at the C1-position. Epimerization at the C3-position
has also been reported to be involved in cell-specific metabolic
pathways, functions of which remain unclear.5 It has been
the VDR.2 In particular, the stereochemistries of the 1
a- and
3b-hydroxy groups (using steroidal numbering) in the A-ring of
the seco-steroid hormone, which rapidly inter-converts between
the two major chair conformations (i.e.,
a-form and b-form)
⇑
Corresponding author. Tel.: +81 87 894 5111; fax: +81 87 894 0181.
E-mail address: tofu@kph.bunri-u.ac.jp (T. Fujishima).
http://dx.doi.org/10.1016/j.tetlet.2014.05.060
0040-4039/Ó 2014 Elsevier Ltd. All rights reserved.

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T. Fujishima et al. / Tetrahedron Letters 55 (2014) 3805–3808
Four-membered cyclic ethers, which are otherwise known as
oxetanes, can act as surrogates for ‘deeper’ carbonyl groups with
high hydrogen avidity, without the liability associated with the
inherent high reactivity of the carbonyl group.6 The nominal anal-
ogy of oxetanes to carbonyl groups has been demonstrated, and the
former can be beneficial when a larger volume of occupancy and
deeper oxygen placement are required in the receptor pocket.6a,b
In view of our previous results with the C2-modified analogues,7
it was envisaged that the replacement of the 3b-hydroxy group
at the C3 position of the A-ring of 1 with a spiro-oxetane would
generate novel VDR ligands and eliminate the problems associated
with the epimerization of the C3 stereo-center. The spiro-oxetane
would also fill the space within the binding site with its hydro-
philic oxygen atom and two hydrophobic methylene moieties.
Herein, we report the concise synthesis and structural analysis of
two novel seco-steroids (2a and 2b) bearing a spiro-oxetane at
their C3 position.
The novel VDR ligands (2a and 2b) were synthesized according
to the convergent method pioneered by Trost et al.8 The requisite
A-ring enyne precursor 11 was synthesized from the symmetrical
diol 3,3-bis(2-hydroxyethyl)oxetane (4),6a,9 which was prepared
from 3-oxetanone (3) in four steps (Scheme 1). The protection of
one of the hydroxy groups in 4 with a p-methoxybenzyl (PMB)
group provided alcohol 5 in 66% yield. Subsequent Swern oxidation
of the remaining primary alcohol in 5 gave the corresponding alde-
hyde 6 in 95% yield, which was then treated with vinylmagnesium
bromide to afford an enatiomeric mixture of the allylic alcohols
( )-7 in 89% yield. Protection of the secondary alcohol in ( )-7 with
tert-butyldimethylsilyl chloride (TBSCl) gave the corresponding
TBS ether ( )-8 in 88% yield, which was treated with 2,3-
dichloro-5,6-dicyano-p-benzoquinone (DDQ) to remove the PMB
group and afford the primary alcohol ( )-9 in 99% yield. Swern oxi-
dation of alcohol ( )-9 gave the corresponding aldehyde ( )-10 in
96% yield, which was converted to the requisite A-ring synthon
according to the Corey–Fuchs method10 via the corresponding
dibromide, which was subsequently treated with n-butyl lithium
to give the enyne precursor ( )-11 in 80% yield over the two steps,
without affecting the oxetane structure.
Scheme 1. Synthesis of the A-ring enyne precursor 11.
Scheme 2. Synthesis of the spirocyclic seco-steroids 2a and 2b.
suggested that the corresponding 3-keto compounds could also
participate in this epimerization process.5 With all of this in mind,
it was envisaged that the modification of the seco-steroids at the C3
position would generate important compounds that could serve as
tools to shed lights on the structure-function relationships of the
VDR.
The A-ring enyne precursor ( )-11 was then coupled to the
CD-ring portion 128 in the presence of tetrakis(triphenylphos-
phine)palladium, followed by deprotection with tetra-n-buthylam-
monium fluoride (TBAF). These reactions proceeded smoothly to
afford a mixture of the novel seco-steroids (2a and 2b) bearing a
spirocyclic oxetane at the C3-position in 66% yield over the two
Table 1
a
1H NMR spectral data for the spirocyclic seco-steroids 2a and 2b and the corresponding C1-benzoates 13a and 13b in CDCl3
Positionsb
2a
2b
13a
13b
1
4.05 m
4.16 m
5.52 dd (6.5, 4.0)
2.28 dd (13.3, 6.5)
2.22 dd (13.3, 4.0)
4.57 d (6.0)
4.46 d (6.0)
4.55 d (6.0)
4.37 d (6.0)
2.76 d (13.1)
2.63 d (13.1)
6.48 d (11.3)
5.89 d (11.3)
5.04 s
5.56 dd (6.2, 3.9)
2.20 dd (13.4, 3.9)
2.29 dd (13.4, 6.2)
4.56 d (6.0)
4.38 d (6.0)
4.59 d (6.0)
4.45 d (6.0)
2.62 d (13.2)
2.77 d (13.2)
6.46 d (11.0)
5.87 d (11.0)
5.07 s
2a
1.79 dd (12.4, 9.5)
2.35 ddd (12.4, 4.3, 1.3)
4.53 d (6.0)
4.39 d (6.0)
4.45 d (6.0)
2.17 dd (12.9, 4.0)
1.97 dd (12.9, 8.1)
4.48 d (5.9)
4.35 d (5.9)
4.60 d (6.0)
4.38 d (6.0)
2.58 d (13.2)
2.62 d (13.2)
6.43 d (11.3)
5.95 d (11.3)
4.97 s
2b
Oxetane
Oxetane
Oxetane
Oxetane
3
3
3b2
3b4
a
a
2c
4
4.36 d (6.0)
4a
2.51 d (13.1)
2.65 d (13.1)
6.40 d (11.2)
5.96 d (11.2)
4.98 t (1.7)
4b
6
7
19E
19Z
5.32 t (1.7)
5.28 s
5.37 s
5.39 s
a
Assignments were based on data from COSY, HSQC, and NOESY experiments: Chemical shifts (d) have been expressed in ppm and the coupling constants (J) expressed in
Hz.
b
Steroidal numbering system was used.
c
The two oxetane protons (3
a
2 and 3
a
4) attached to the same carbon atom at the C3
a
position were denoted as ‘3
a’. The proton projecting toward the C2 position, which
was confirmed to be in the vicinity of the C2 position by a NOESY experiment, was denoted ‘3
a2’, and the other proton was denoted ‘3a4’. The remaining two oxetane protons
(3b2 and 3b4) were defined in the same way.

T. Fujishima et al. / Tetrahedron Letters 55 (2014) 3805–3808
3807
steps (Scheme 2). The resulting C1-epimeric mixture of 2a and 2b
was separated by HPLC, and the presence of the spiro-oxetane
structure in both stereoisomers 2a (more polar isomer) and 2b
(less polar isomer) was confirmed by the 1H NMR analysis.7 The
1H NMR spectra of compounds 2a and 2b showed four distinct
doublets with geminal coupling constants in the range of 5.9–
6.0 Hz, which indicated the presence of a slightly restricted four-
membered ring (Table 1).6
The absolute configurations at the C1 positions of 2a and 2b
were determined by the circular dichroism (CD) exciton chirality
method using the benzoates of the allylic alcohols.11 The CD allylic
benzoate method has been successfully applied to allylic alcohols
bearing an exomethylene double bond,11b,c as well as to alcohols
containing additional chromophores in their parent structures.11d
Based on these reports, it was envisaged that this method would
be suitable to determine the stereochemistry of the C1-epimeric
seco-steroids 2a and 2b, which had a C(10)-19 exomethylene moi-
ety in their A-ring. The C1 hydroxy groups in 2a and 2b were trea-
ted with benzoyl chloride and 4-dimethylaminopyridine (DMAP)
to give the corresponding allylic benzoates 13a and 13b, respec-
tively, in 72% yield in both cases (Scheme 3). The CD spectra of
the benzoates 13a and 13b exhibited complementary signs of their
Cotton effect, whereas the CD spectra of the parent compounds 2a
and 2b showed no significant exciton chirality (see the Supplemen-
tary data). The differential CD spectra of the benzoate 13a and 2a
showed a positive Cotton effect at 242 nm, which suggested a
clockwise relationship between the C1 benzoate and the other
chromophore, whereas those of the benzoate 13b and 2b showed
a negative Cotton effect at 241 nm, which suggested a counter-
clockwise relationship between the two chromophores (Fig. 2).
The vicinal coupling constants between the protons at the C1 and
C2 positions of 13a and 13b were 6.5 and 6.2 Hz, respectively,
which suggested that the axial orientation of the C1-benzoyloxy
groups was favored over the equatorial orientation in both cases.3
These results were in agreement with the distinctive Cotton effect
results observed in the CD spectra.11 Taken together, these data
Figure 2. Differential CD spectra of the C1-benzoates (13a and 13b) and their
corresponding parents (2a and 2b) in ethanol. The concentration of each compound
was adjusted to 83
lM by using an e value of 18,000 at 265 nm, which was obtained
by UV spectroscopy.
equatorial in both cases for the A-ring conformational equilibrium,
compared with the corresponding parent compounds (Fig. 3).3 In
the case of 2a, a long range four-bond COSY correlation was
observed between one of the oxetane protons (H-3b2) and the pro-
ton (H-4b) at the C4 position, as shown in Figure 3. This correlation
suggested that the oxetane moiety was particularly rigid and effec-
tively planar, and that the oxetane structure would therefore allow
for an eclipsing interaction between the two rings.6 Based on these
results, it was envisaged that the positioning of the oxetane struc-
ture within the LBD of VDR would offer specific advantages in
terms of its interaction with the Ser-278 residue, which would
have an impact on the 3-epimer of 1.5 The placement of the oxe-
tane within the LBD would also give rise to a new hydrophobic
interaction with the Tyr-147 residue, as well as an additional
hydrophilic interaction with the Glu-277 residue.5
confirmed that the more polar isomer 2a possessed the 1a-config-
uration, whereas the less polar isomer 2b possessed the 1b-
configuration.
The seco-steroids 2a and 2b were subjected to 2D NMR analyses
to identify all the protons in the A-ring, including the four protons
belonging to the spiro-oxetane at the C3 position (Table 1).3,12 The
vicinal coupling constants between the protons at the C1 and C2
positions confirmed that the C1 hydroxy group preferred to be
Figure 3. The A-ring conformational structures of compounds 2a and 2b. The
numbers in the figure represent the equilibrium ratios, which were deduced from
the NMR analysis at 3.5 mM in CDCl3 at 27 °C.3 NOESY correlations are expressed
with arrows. Long range 4J couplings observed by COSY have been designated in a
red or blue color.
Scheme 3. Synthesis of the C1-allylic benzoates 13a and 13b and the conforma-
tions required to elicit the observed distinctive Cotton effects.

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T. Fujishima et al. / Tetrahedron Letters 55 (2014) 3805–3808
I.; Haussler, C. A.; Hsieh, D.; Hsieh, J.-C.; Jurutka, P. W. Calcified Tissue Int. 2013,
In conclusion, we have generated two novel seco-steroids (2a
92, 77; (c) Bikle, D. J. Clin. Endcrinol. Metab. 2009, 96, 26; (d) Wang, Y.; Zhu, J.;
DeLuca, H. F. Archiv. Biochem. Biophys. 2012, 523, 123; (e) Norman, A. W.
Endocrinology 2006, 147, 5542; (f) Plum, L. A.; DeLuca, H. F. Nat. Rev. Drug Disc.
2010, 9, 941.
and 2b) bearing a spiro-oxetane structure at the C3 position of their
A-rings, instead of the 3b-hydroxy group. A convergent synthetic
strategy involving the use of a palladium catalyst was developed
to provide access to these novel seco-steroids. The enyne precursor
11, which was required for the construction of the A-ring, was
prepared in excellent yield from 4 according to an eight-step
procedure. This sequence demonstrated that the oxetane structure
is robust and tolerant of a variety of synthetic conditions. The abso-
lute configuration at the C1 position of the synthesized compounds
(2a and 2b) was determined by CD from the corresponding allylic
benzoates (13a and 13b) using the exciton chirality method, which
revealed that the signs of the Cotton effects were complementary to
each other. Substitution of the C3-hydroxy group with a spiro-
oxetane conferred specific advantages of the A-ring structure and
conformational preferences for the parent seco-steroids, which
would enhance the formation of a stable ligand-receptor complex.
2. Rochel, N.; Wurtz, J. M.; Mitschler, A.; Klaholz, B.; Moras, D. Mol. Cell. 2000, 5,
173.
3. The A-ring of 1 and its related seco-steroids adopt two chair conformations, a-
form and b-form, and the conformational equilibrium can be estimated by
using the vicinal coupling constants between protons at the C3 and C4
positions (Jax,ax = 11.1 Hz, Jeq,eq = 2.7 Hz): (a) Okamura, W. H.; Norman, A. W.;
Wing, R. M. Proc. Natl. Acad. Sci. U.S.A. 1974, 71, 4194; (b) Eguchi, T.; Ikekawa, N.
Bioorg. Chem. 1990, 18, 19; (c) Eguchi, T.; Kakimura, K.; Ikekawa, N. Bioorg.
Chem. 1991, 19, 327.
4. (a) Norman, A. W.; Bouillon, R.; Farach-Carson, M. G.; Bishop, J. E.; Zhou, L.-X.;
Nemere, I.; Zhao, J.; Muralidharan, K. R.; Okamura, W. H. J. Biol. Chem. 1993,
268, 20022; (b) Fujishima, T.; Konno, K.; Nakagawa, K.; Kurobe, M.; Okano, T.;
Takayama, H. Bioorg. Med. Chem. 2000, 8, 123.
5. Molnár, F.; Sigüeiro, R.; Sato, Y.; Araujo, C.; Schuster, I.; Antony, P.; Peluso, J.;
Muller, C.; Mouriño, A.; Moras, D.; Rochel, N. PLoS One 2011, 6, e18124.
6. (a) Wuitschik, G.; Rogers-Evans, M.; Buckl, A.; Bernasconi, M.; Märki, M.; Godel,
T.; Fischer, H.; Wagner, B.; Parrilla, I.; Schuler, F.; Schneider, J.; Alker, A.;
Schweizer, W. B.; Müller, K.; Carreira, E. M. Angew. Chem., Int. Ed. 2008, 47,
4512; (b) Wuitschik, G.; Carreira, E. M.; Wagner, B.; Fischer, H.; Parrilla, I.;
Schuler, F.; Rogers-Evans, M.; Müller, K. J. Med. Chem. 2010, 53, 3227; (c)
Burkhard, J. A.; Guérot, C.; Knust, H.; Roger-Evans, M.; Carreira, E. M. Org. Lett.
2010, 12, 1944.
7. Fujishima, T.; Nozaki, T.; Suenaga, T. Bioorg. Med. Chem. 2013, 21, 5209.
8. (a) Trost, B. M.; Dumas, J.; Villa, M. J. Am. Chem. Soc. 1992, 114, 9836; (b) Trost,
B. M.; Dumas, J. J. Am. Chem. Soc. 1992, 114, 1924.
9. (a) Allen, Jennifer R.; Chen, Jian J.; Frohn, Michael J.; Hu, Essa; Liu, Qingyian;
Pickrell, Alexander J.; Rumfelt, Shannon; Rzasa, Robert M.; Zhong, Wenge. PCT
Int. Appl. WO 2011143365 A1 20111117, 2011.; (b) Loy, Rebecca N.; Jacobsen,
Eric N. J. Am. Chem. Soc. 2009, 131, 2786.
Acknowledgement
This work was supported in part by a Grant-in-Aid for a scien-
tific research (Grant number 23590142) from the Ministry of Edu-
cation, Culture, Sports, Science, and Technology in Japan.
Supplementary data
10. (a) Corey, E. J.; Fuchs, P. L. Tetrahedron Lett. 1972, 13, 3769; (b) Acharya, H. P.;
Kobayashi, Y. Tetrahedron Lett. 2004, 45, 1202; (c) Yang, F. Y.; Newsome, J. J.;
Curran, D. P. J. Am. Chem. Soc. 2006, 128, 14200.
11. (a) Harada, N.; Iwabuchi, J.; Yokota, Y.; Uda, H.; Nakanishi, K. J. Am. Chem. Soc.
1981, 103, 5590; (b) Harada, N.; Iwabuchi, J.; Yokota, Y.; Uda, H.; Ochi, M. J.
Chem. Soc., Chem. Commun. 1984, 1220; (c) Fukushi, Y.; Yajima, C.; Mizutani, J.;
Tahara, S. Phytochemistry 1998, 49, 593; (d) Wei, H.; Itoh, T.; Kinoshita, M.;
Kotoku, N.; Aoki, S.; Kobayashi, M. Tetrahedron 2005, 61, 8054.
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.tetlet.2014.
05.060.
References and notes
1. (a)Vitamin D; Feldman, D., Wesley Pike, J., Adam, J. S., Eds., 3rd ed.; Elsevier
12. Mizhiritskii, M. D.; Konstantionovskii, L. E.; Vishkautsan, R. Tetrahedron 1996,
52, 1239.
Academic Press: Burlington, 2011; (b) Haussler, M. R.; Whitfield, G. K.; Kaneko,