Synthesis of 2α-heteroarylalkyl active vitamin D3 with therapeutic effect on enhancing bone mineral density in vivo

Article abstract of DOI:10.1021/ml400098w

2α-Heteroarylethyl-1α,25-dihydroxyvitamin D₃ analogues, which were designed to form a hydrogen bond between Arg274 of human vitamin D receptor (hVDR) and a nitrogen atom of the heteroaromatic ring at the 2α-position, were synthesized. Among them, 2α-[2-(tetrazol-2-yl) ethyl]-1α,25-dihydroxyvitamin D₃ showed higher osteocalcin promoter transactivation activity in human osteosarcoma (HOS) cells and a greater therapeutic effect in ovariectomized (OVX) rats, osteoporosis model animals, on enhancing bone mineral density than those of active vitamin D ₃. X-ray cocrystallographic analysis of the hVDR-ligand complex confirms that the new hydrogen bond formation stabilized the complex.

Full text of DOI:10.1021/ml400098w

Letter
pubs.acs.org/acsmedchemlett
Synthesis of 2α-Heteroarylalkyl Active Vitamin D3 with Therapeutic
Effect on Enhancing Bone Mineral Density in Vivo
Miki Matsuo,† Asami Hasegawa,† Masashi Takano,† Hiroshi Saito,‡ Shinji Kakuda,‡ Takayuki Chida,‡
Ken-ichiro Takagi,‡ Eiji Ochiai,‡ Kyohei Horie,‡ Yoshifumi Harada,‡ Midori Takimoto-Kamimura,‡
Kazuya Takenouchi,‡ Daisuke Sawada,† and Atsushi Kittaka*,†
†Faculty of Pharmaceutical Sciences, Teikyo University, Itabashi-ku, Tokyo 173-8605, Japan
‡Teijin Institute for Bio-medical Research, Teijin Pharma Ltd., Hino-shi, Tokyo 191-8512, Japan
S
* Supporting Information
ABSTRACT: 2α-Heteroarylethyl-1α,25-dihydroxyvitamin D3 analogues, which were
designed to form a hydrogen bond between Arg274 of human vitamin D receptor
(hVDR) and a nitrogen atom of the heteroaromatic ring at the 2α-position, were
synthesized. Among them, 2α-[2-(tetrazol-2-yl)ethyl]-1α,25-dihydroxyvitamin D3
showed higher osteocalcin promoter transactivation activity in human osteosarcoma
(HOS) cells and a greater therapeutic effect in ovariectomized (OVX) rats,
osteoporosis model animals, on enhancing bone mineral density than those of active
vitamin D3. X-ray cocrystallographic analysis of the hVDR-ligand complex confirms
that the new hydrogen bond formation stabilized the complex.
KEYWORDS: Vitamin D, vitamin D receptor, X-ray cocrystallographic analysis, osteocalcin, bone mineral density,
in vivo antiosteoporotic effect
a heteroaromatic ring on binding to the hVDR instead of the
OH group and on biological activities in vitro and in vivo.
We designed 2α-[2-(tetrazol-2-yl)ethyl]-1α,25(OH)2D3 (1a)
and the related compounds 1b−f since the number of atoms
from the 2α-position to the terminal oxygen of O1C3 is four,
and compound 1a, for example, also consists of four atoms to
the nitrogen that could coordinate to Arg274, but only 1f has
five to the nitrogen atom (Figure 1).
he pleiotropically hormonal active metabolite form of
secosteroid vitamin D3, 1α,25-dihydroxyvitamin D3
T
[1α,25(OH)2D3], exerts its biological activity via binding and
modulation of the vitamin D receptor (VDR), a member of the
nuclear receptor superfamily of transcriptional regulators.1
1α,25(OH)2D3 plays pivotal roles in maintaining calcium and
phosphorus homeostasis and bone remodeling. 1α,25(OH)2D3
also acts as a regulator in a wide range of fundamental
physiological processes including cell growth and differ-
entiation, immune function, embryonic development, and
inflammatory reactions.2,3 One of the most important factors
when 1α,25(OH)2D3 binds to the VDR is hydrogen bond
formation between the 1α-OH group and the Arg274 residue of
the hVDR.4
The ligand binding domain (LBD) of the hVDR contains
water molecules from the A-ring anchoring moiety to the
surface of the protein, called a water channel, to stabilize the
VDR-[1α,25(OH)2D3] complex by forming hydrogen bond
networks.5 X-ray crystallographic analyses of the VDR-[2α-(3-
hydroxypropyl)-1α,25(OH)2D3 (O1C3)] and VDR-[2α-(3-
hydroxypropoxy)-1α,25(OH)2D3 (O2C3)] complexes have
clearly demonstrated that the terminal hydroxy group of both
synthetic ligands forms a hydrogen bond with Arg274 and
replaces one of the water molecules in the LBD of the hVDR to
stabilize the complex;5 therefore, O1C3 and O2C3 showed 3-
and 1.8-times greater binding affinity for the VDR than the
natural hormone, respectively.6−8 Here we studied the effects of
Figure 1. Structures of active vitamin D3 and 2α-substituted synthetic
analogues, O1C3, O2C3, and 1a−f.
Received: March 8, 2013
Accepted: May 28, 2013
© XXXX American Chemical Society
A
dx.doi.org/10.1021/ml400098w | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
Synthesis of the A-ring precursors enyne 2a,b is shown in
Scheme 1. Previously, we reported the preparation of methyl 3-
Other azols, 1,2,4-triazole, 1,2,3-triazole, and imidazole, were
also attached to enyne unit 10 through SN2 reactions (Scheme
2). Alkylation of 1,2,3-triazole gave two products, and 2-N-alkyl
a
Scheme 1. Synthesis of A-Ring Precursors 2a,b
Scheme 2. Preparation of A-Ring Precursors 2c−f from
a
Enyne Alcohol 10
a
Reagents and conditions: (a) DIAD, PPh3, 1,2,4-triazole, THF, 94%.
(b) DIAD, PPh3, 1H-1,2,3-triazole, THF, 2d 85% and 2e 15%. (c)
MsCl, Et3N, CH2Cl2, 91%. (d) NaH, imidazole, THF, 71%.
triazole 2d was the main product. For imidazole, it was
necessary to produce imidazole anion by NaH since the N-
alkylation reaction failed under the Mitsunobu conditions.
As shown in Scheme 3, the CD-ring bromoolefin 1214 and
enynes 2a−f were coupled under Trost coupling conditions15
a
Reagents and conditions: (a) TBSOTf, 2,6-lutidine, CH2Cl2, 85%.
a
Scheme 3. Trost Coupling between 12 and 2a−f
(b) 9-BBN, THF, and then, H2O2, aq. NaOH, 92%. (c) PivCl, py,
CH2Cl2, 95%. (d) NBS, BaCO3, CCl4, 87%. (e) Zn powder,
NaBH3CN, 1-PrOH−H2O (5:1), 70%. (f) TsCl, py, 96%. (g)
TBAF, THF, 80%. (h) BuLi, TMS-acetylene, BF3·OEt2, THF, 78%.
(i) K2CO3, MeOH, 71%. (j) TBSOTf, 2,6-lutidine, CH2Cl2, 93%. (k)
DIBAL-H, CH2Cl2, 92%. (l) DIAD, PPh3, 1H-tetrazole, THF, 2a 81%
and 2b 19%.
deoxy-3-C-ethenylaltropyranoside 3 from methyl α-D-gluco-
side,9 and 3 was converted to alcohol 4 via a hydroboration−
oxidation reaction after TBS protection at the 2-OH group of 3.
The OH group of 4, protected by a pivaloyl group followed by
our usual protocol, gave enyne 10 in good yield.10 Briefly, NBS
treatment of benzylidene acetal gave bromide 5, which reacted
with activated zinc powder in the presense of NaBH3CN to
provide alcohol 6. The alcohol was converted to epoxide 7
through sulfonylation of the primary alcohol followed by TBAF
treatment. Ethynylation of 7 using lithium TMS-acetylide in the
presence of BF3·OEt2 in THF and subsequent solvolysis in
K2CO3/MeOH supplied enyne 9.11 Bis-O-silylation with
TBSOTf/2,6-lutidine followed by hydride reduction of the
ester part afforded 10.10 Mitsunobu reaction between 10 and
1H-tetrazole gave the desired protected enynes 2a and 2b in
81% and 19% yields, respectively. When comparing 1H NMR12
and 13C NMR13 chemical shifts of appropriate methylene and
methyne CHs of 2a and 2b, these two isomers were
distinguishable, i.e., methylene protons of 2-N-alkyl substituted
tetrazole 2a appeared in a lower field (4.68−4.82 ppm) than
those of the corresponding 1-N-alkyl substituted tetrazole 2b
(4.47−4.64 ppm). Instead, the methyne proton of 2a appeared
a
Reagents and conditions: (a) Pd(PPh3)4, Et3N−toluene (1:1), reflux.
(b) TBAF, THF, for two steps: 1a 40%, 1b 37%, 1c 29%, 1d 27%, 1e
49%, and 1f 48%.
followed by deprotection of O-silyl groups using TBAF to give
the new 2α-heteroarylalkyl vitamin D3 analogues 1a−f,
respectively.
The preliminary biological activities, hVDR binding affinity
and osteocalcin promoter transactivation activity in human
osteosarcoma (HOS) cells, of the new compounds 1a−f were
evaluated (Table 1). Among them, 2α-[2-(tetrazol-2-yl)ethyl]-
Table 1. hVDR Binding Affinity and Osteocalcin Promoter
Transactivation Activity in HOS Cells
relative hVDR
binding affinity (%)
osteocalcin transactivation activity in
HOS/SF cells (EC50, nM)
compd
1α,25(OH)2D3
100
64
<1
4
0.026
0.010
0.29
0.15
0.044
0.21
0.68
−
1a
1b
1c
1d
1e
1f
1
12
<1
2
in a higher field (8.46 ppm) than that of 2b (8.56 ppm) in H
NMR; the methylene and methyne carbons of 2a (53.0 and
152.6 ppm) appeared in a lower field than those of 2b (48.2
and 142.1 ppm) in 13C NMR, respectively.12,13
13
1
B
dx.doi.org/10.1021/ml400098w | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
1α,25(OH)2D3 (1a) showed potent binding affinity for hVDR
and greater transactivation activity (EC50 9.8 pM) than that of
the natural hormone, 1α,25(OH)2D3. Analogue 1d showed
moderate hVDR binding affinity and almost the same level of
transactivation activity as 1α,25(OH)2D3. Compounds 1b, 1e,
and 1f showed no effective binding affinity for hVDR, just like
2α-phenethyl-1α,25(OH)2D3 (13, R = Ph in Scheme 3).16
Effects of the 2-N-alkylated tetrazole ring of 1a on the in
vitro biological results were strong, and next we tested the in
vivo therapeutic effect of 1a using an ovariectomized (OVX) rat
as an osteoporosis model animal. Twelve-week-old Sprague−
Dawley female rats were ovariectmized and fed a normal diet
containing 1.0% Ca ad libitum for 4 weeks. The rats were then
administrated 1a at doses of 0.007 μg/kg/day and 0.02 μg/kg/
day, 5 times a week for 4 weeks. Twenty-four hours after the
final administration, blood was collected under anesthesia, the
rats were euthanized, and bone mineral density (BMD) of the
spine (L4−L5) bone mass was measured by dual X-ray
absorption meter. The results of BMD and serum Ca density
are shown in Tables 2 and 3. The newly synthesized analogue
Figure 2. X-ray studies on hVDR LBD−1a complex. The A-ring part is
magnified.
Alkylated tetrazole ring might have an equilibrium relationship
between nitrogen and carbon atoms against Arg274 by free
rotation of the 1-N-alkylated single bond in the solution. The
dynamic difference in solution might reflect the lower binding
affinity of the 1-N-alkylated tetrazole ring.
In summary, we synthesized 2α-heteroarylalkyl-1α,25-dihy-
droxyvitamin D3 analogues 1a−f for the first time and tested
their biological activity. Among them, we found that 1a showed
higher osteocalcin promoter transactivation activity in HOS
cells and a greater therapeutic effect in vivo in OVX rats on
enhancing bone mineral density without hypercalcemic side
effects than those of the natural hormone. X-ray cocrystallo-
graphic analysis of the hVDR LBD−1a complex confirmed the
new hydrogen bond formation between the nitrogen atom of
the tetrazole ring and Arg274. We believe that 2α-
heteroarylalkyl active vitamin D analogues would be potent
candidates for osteoporosis chemotherapy.
Table 2. Therapeutic Effects of 1a Using Ovariectomized
(OVX) Rat Model
dose
(μg/kg/day)
BMD
serum Ca
(mg/dL)
(g/cm2)
control (sham)
control (OVX)
1a (OVX)
0.231 0.015
0.209 0.011
0.214 0.012
0.223 0.017
9.71 0.27
9.25 0.11
9.41 0.20
9.66 0.25
0.007
0.02
1a (OVX)
Table 3. Therapeutic Effects of 1α,25(OH)2D3 Using
Ovariectomized (OVX) Rat Model
dose
(μg/kg/day)
BMD
serum Ca
(mg/dL)
(g/cm2)
control (sham)
control (OVX)
0.245 0.010
0.209 0.019
0.218 0.010
9.72 0.40
9.14 0.24
10.14 0.37
ASSOCIATED CONTENT
* Supporting Information
Detailed experimental procedures and spectral data for all new
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
S
1α,25(OH)2D3
(OVX)
0.025
0.1
1α,25(OH)2D3
(OVX)
0.228 0.013
10.35 0.27
AUTHOR INFORMATION
Corresponding Author
*(A.K.) Tel: (+81) 3 3964 8109. Fax: (+81) 3 3964 8117. E-
mail: akittaka@pharm.teikyo-u.ac.jp.
■
1a (Table 2) showed an increase of BMD at low doses of 0.007
and 0.02 μg/kg/day without the significant side effect of
increased serum calcium, i.e., hypercalcemia compared to
1α,25(OH)2D3 (Table 3).
Funding
Next, we studied the crystal structure of the complex
between truncated hVDR LBD and 1a to see new interactions
of the tetrazole ring and amino acid residues of the LBD
(Figure 2).17 The hVDR LBD−1a complex was superimposed
on the hVDR LBD−1α,25(OH)2D3 complex with an root-
mean-square deviation (rmsd) of 0.45 Å on all Cα atoms.
Clearly, one of the nitrogen atoms of the 2-N-alkylated
tetrazole ring formed a direct hydrogen bond with Arg274
(2.99 Å distance), which was also shown in the hVDR LBD−
O1C3 complex between the 2α-terminal OH group of O1C3
and Arg274 to stabilize the complex after missing two water
molecules from the original hVDR LBD−1α,25(OH)2D3
complex.5 Although we were also able to analyze the crystal
structure of the hVDR LBD−1b complex and found that
ligands 1a and 1b were superimposable in the hVDR (data not
shown), it was difficult to explain why 1a showed much higher
binding affinity than 1b from the crystal structures. 1-N-
This work was supported in part by a Grant-in-Aid from the
Ministry of Education, Culture, Sports, Science and Technol-
ogy (No. 23790021 to M.T.) as well as Grants-in-Aid from the
Japan Society for the Promotion of Science (No. 23590015 to
D.S. and No. 24590021 to A.K.).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Ms. Junko Shimode and Ms. Miki Takahashi (Teikyo
University) for the spectroscopic measurements.
■
ABBREVIATIONS
■
VDR, vitamin D receptor; LBD, ligand binding domain; OVX,
ovariectomized; BMD, bone mineral density; TBS, tert-
butyldimethylsilyl; 9-BBN, 9-borabicyclo[3.3.1]nonane; PivCl,
C
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ACS Medicinal Chemistry Letters
Letter
(17) The Protein Data Bank accession numbers for the coordinates
of the structures of the VDR complex with 1a and 1b reported in this
letter are 4ITE and 4ITF, respectively.
pivaloyl chloride; NBS, N-bromosuccinimide; TBAF, tetrabu-
tylammonium fluoride; TMS, trimethylsilyl; DIBAL-H, diiso-
butylaluminium hydride; DIAD, diisopropyl azodicarboxylate
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