Synthesis and biological evaluation of steroidal derivatives bearing a small ring as vitamin D receptor agonists

Article abstract of DOI:10.1016/j.bmcl.2017.05.089

A novel series of 3-ketolithocholic acid derivatives as well as estrone derivatives bearing a small ring for the conformational fixation of the side chain were synthesized by using a catalytic [2+2] cycloaddition and a ring-contraction rearrangement. The steroidal derivatives were evaluated for transcriptional activation of vitamin D receptor by luciferase reporter assays. Among them, two estrone derivatives showed a higher efficacy of the transactivation of vitamin D receptor than 3-ketolithocholic acid, and the small ring moieties were found to be important for the efficacy.

Full text of DOI:10.1016/j.bmcl.2017.05.089

Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of steroidal derivatives bearing a
small ring as vitamin D receptor agonists
a
a
b
b
c
Norihito Arichi , Shinichi Fujiwara , Michiyasu Ishizawa , Makoto Makishima , Duy H. Hua ,
a
a
a,
⇑
Ken-ichi Yamada , Yousuke Yamaoka , Kiyosei Takasu
a
Graduate School of Pharmaceutical Sciences, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan
Division of Biochemistry, Department of Biomedical Sciences, School of Medicine, Nihon University, Itabashi-ku, Tokyo 173-8610, Japan
Department of Chemistry, Kansas State University, Manhattan, KS 66506-0401, USA
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of 3-ketolithocholic acid derivatives as well as estrone derivatives bearing a small ring for
the conformational fixation of the side chain were synthesized by using a catalytic [2+2] cycloaddition
and a ring-contraction rearrangement. The steroidal derivatives were evaluated for transcriptional acti-
vation of vitamin D receptor by luciferase reporter assays. Among them, two estrone derivatives showed
a higher efficacy of the transactivation of vitamin D receptor than 3-ketolithocholic acid, and the small
ring moieties were found to be important for the efficacy.
Received 11 May 2017
Revised 30 May 2017
Accepted 31 May 2017
Available online xxxx
Keywords:
Steroids
Ó 2017 Published by Elsevier Ltd.
Vitamin D receptor
Cyclopropanes
Cyclobutanes
[
2+2] cycloaddition
Calcitriol, which is the active form of vitamin D3, modulates a
small ring carbocycles have been used to restrict a rotatable side
chain because the practical synthetic methods are still lacking.7
We recently developed a strategy for construction of a cyclobutane
or a cyclopropane ring as a rigid structural unit on the D-ring of a
steroidal backbone by using a stereoselective catalytic [2+2]
broad spectrum of biological functions such as bone homeostasis,
immunity, cellular growth, and differentiation through binding to
the vitamin D nuclear receptor (VDR) (Fig. 1).1 The derivatives of
calcitriol are effective in the treatment of osteoporosis and psoria-
2
sis. However, their therapeutic use is limited because of their sev-
cycloaddition and a stereospecific ring-contraction rearrange-
1
c
8–10
ere side effects such as hypercalciuria and hypercalcemia. It was
found that lithocholic acid (LCA), which is a secondary bile acid,
and its metabolite, 3-ketolithocholic acid (3-keto LCA), also bind
to VDR and exhibit the agonistic activities although their
ment.
We became interested in their biological activity as
VDR agonists. In addition, we envisaged that we would apply the
synthetic strategy to the synthesis of a new class of 3-keto LCA
derivatives bearing a small ring for conformational regulation of
the carbon side chain. Herein, we report synthesis of the 3-keto
LCA derivatives and biological evaluation of the derivatives as well
as estrone derivatives as VDR agonists.
3
structures fundamentally differ from that of calcitriol. Lithocholic
acid derivatives such as LCA acetate and LCA propionate act as
selective VDR agonists with greater potency than LCA, and these
4
derivatives can activate VDR without inducing hypercalcemia.
Our synthesis of 3-keto LCA derivatives commenced from com-
mercially available 4-androstene-3,17-dione, which was subjected
to hydrogenation in 4-methylpydirine as a solvent and separation
by recrystallization to give 1 in 74% yield as a single diastereomer
The structure of the complexes of the ligand-binding domain of
VDR with LCA derivatives were solved by X-ray crystallography,
which revealed that LCA and its derivatives bind to the same bind-
ing pocket as calcitriol, but in the opposite orientation.5
11
(Scheme 1). Selective protection of the less hindered A-ring car-
Conformational fixation by introduction of a structurally rigid
moiety such as an unsaturated bond or a ring structure is a com-
mon strategy in drug development to increase biological activity
and/or reduce side effects.6 However, only a limited number of
bonyl group, followed by treatment with tert-butyldimethylsilyl
trifluoromethanesulfonate (TBSOTf), delivered silyl enol ether 3.
EtAlCl -catalyzed [2+2] cycloaddition of 3 with hexafluoroiso-
2
propyl (HFIP) acrylate was carried out at different temperatures.
When the reaction was performed at À78 °C, kinetic product
trans-4 was selectively obtained along with cis-4 (trans:cis = 96:4)
as a minor diastereomer. Reduction of the resulting diastereomeric
⇑
Corresponding author.
E-mail address: kay-t@pharm.kyoto-u.ac.jp (K. Takasu).
http://dx.doi.org/10.1016/j.bmcl.2017.05.089
960-894X/Ó 2017 Published by Elsevier Ltd.
0
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N. Arichi et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
mixture
4 by sodium bis(2-methoxyethoxy)aluminumhydride
(
Red–Al), followed by separation of the products on silica gel col-
umn chromatography, gave trans-5 as a pure diastereomer in 68%
yield for two steps. The [2+2] cycloaddition at room temperature
afforded thermodynamically more stable cis-4 exclusively, which
1
2
was reduced to cis-5 with Red–Al.
With the cyclobutane scaffolds constructed, we next installed a
carbon side chain on the small ring (Scheme 2). Oxidation of trans-
5
by tetrapropylammonium perruthenate (TPAP), followed by Hor-
ner-Wadsworth-Emmons reaction, gave trans-7. Reduction of the
resulting conjugated double bond by treatment with magnesium
in methanol, followed by removal of the thioacetal with N-chloro-
1
3,14
succinimide (NCS), provided trans-8.
using HF and saponification with LiOH furnished trans-9. Ester cis-
was also synthesized from cis-5 by the same procedure. When
Removal of the TBS group
8
cis-8 was treated with tetrabutylammonium fluoride (TBAF) to
remove the TBS group, simultaneous hydrolysis of the ester moiety
was observed to afford cis-9 in 67% yield.
Fig. 1. Chemical structures of calcitriol, LCA, LCA acetate, LCA propionate, and 3-
keto LCA.
Scheme 1. Construction of a cyclobutane ring on the steroidal D-ring.
Scheme 2. Synthesis of 3-keto LCA analogs bearing a cyclobutane ring.
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N. Arichi et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
3
Scheme 3. Synthesis of 3-keto LCA analogs bearing a spirocyclopropane ring.
Our attention turned toward synthesis of 3-keto LCA derivatives
bearing a spirocyclic cyclopropane ring (Scheme 3). Cleavage of the
TBS group of trans-5, followed by acetyl protection of the primary
alcohol, gave trans-11. Ring-contraction rearrangement of trans-11
was performed using thionyl chloride and triethylamine to give
mide reduction,15 and removal of the thioacetal followed by
hydrolysis afforded (20R)-17. The synthesis of (20S)-17 was also
accomplished from cis-5 by the same procedure.
The synthesized LCA derivatives as well as estrone derivatives
trans-18, cis-18, (20R)-19 and (20S)-19, which were synthesized
8
b
(
1
20R)-12 in 49% yield as a 7:3 mixture of diastereomers at the C-
6 stereogenic center. Removal of the acetyl group and dechlorina-
tion were carried out in one-pot with NaBH in DMSO at 130 °C to
previously,
were evaluated at various concentrations to
determine transactivation of VDR by luciferase reporter assays
(Fig. 2A). Among them, only estrone derivatives trans-18 and
(20R)-19 showed a slightly higher efficacy, maximum effect, of
the transcriptional activity of VDR than 3-keto LCA. The
diastereomers cis-18 and (20S)-19 activated VDR less effectively
than 3-keto LCA. Unfortunately, as seen in trans-9, cis-9, (20R)-17
and (20S)-17, installation of the small ring onto the D-ring of 3-
4
give alcohol (20R)-13. TPAP oxidation of (20R)-13 and the succes-
sive olefination provided (20R)-15. The reduction of the resulting
double bond was unsuccessful using magnesium and methanol,
which resulted in a simultaneous ring opening of the cyclopropane
ring. However, the chemoselective reduction was achieved by dii-
Fig. 2. (A) Transactivation of VDR by estrone derivatives or 3-keto LCA derivatives. (B) Comparison of the activity of estrone derivatives with or without a small ring. (C)
Chemical structures of estrone derivatives.
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N. Arichi et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
keto LCA resulted in a significant loss of the activity. To examine
the effect of the small rings of trans-18 and (20R)-19 on the
transcriptional activity, estrone derivative 20 without a small
ring was also evaluated (Fig. 2B). The reduction in the activity at
higher concentrations results from cytotoxicity. Whereas 20
showed a higher potency (EC50 = 0.85 mM) than trans-18 and
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A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2017.05.
0
89.
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