Design, synthesis and X-ray crystallographic study of new nonsecosteroidal vitamin D receptor ligands

Article abstract of DOI:10.1016/j.bmcl.2011.08.047

We designed and synthesized nonsecosteroidal vitamin D receptor (VDR) ligands that formed H-bonds with six amino acid residues (Tyr143, Ser233, Arg270, Ser274, His301 and His393) of the VDR ligand-binding domain. The ligand YR335 exhibited potent transcriptional activity, which was comparable to those of 1α,25-dihydroxyvitamin D₃ and YR301. The crystal structure of the complex formed between YR335 and the VDR ligand-binding domain was solved, which revealed that YR335 formed H-bonds with the six amino acid residues mentioned above.

Full text of DOI:10.1016/j.bmcl.2011.08.047

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6104–6107
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and X-ray crystallographic study of new
nonsecosteroidal vitamin D receptor ligands
Yosuke Demizu a, Takeo Takahashi a, Fumiya Kaneko a, Yukiko Sato a, Haruhiro Okuda b, Eiji Ochiai b,
Kyohei Horie b, Ken-ichiro Takagi b, Shinji Kakuda b, Midori Takimoto-Kamimura b, Masaaki Kurihara a,
⇑
a Division of Organic Chemistry, National Institute of Health Sciences, 1-18-1, Kamiyoga, Setagaya, Tokyo 158-8501, Japan
b Teijin Institute for Bio-medical Research, Teijin Pharma Ltd, Tokyo 191-8512, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We designed and synthesized nonsecosteroidal vitamin D receptor (VDR) ligands that formed H-bonds
with six amino acid residues (Tyr143, Ser233, Arg270, Ser274, His301 and His393) of the VDR ligand-
binding domain. The ligand YR335 exhibited potent transcriptional activity, which was comparable to
those of 1a,25-dihydroxyvitamin D3 and YR301. The crystal structure of the complex formed between
YR335 and the VDR ligand-binding domain was solved, which revealed that YR335 formed H-bonds with
Received 6 June 2011
Revised 5 August 2011
Accepted 10 August 2011
Available online 17 August 2011
the six amino acid residues mentioned above.
Keywords:
Ó 2011 Elsevier Ltd. All rights reserved.
Vitamin D receptor
Nonsecosteroidal ligand
X-ray crystallographic analysis
1
a
,25-Dihydroxyvitamin D3 (1
a
,25(OH)2D3), the physiologically
the rat VDR LBD at a resolution of 2.0 Å and revealed that YR301
formed H-bonds with Ser233, Arg270, His301 and His393, but
not with Tyr143 or Ser274.12 YR301 formed an H-bond network
containing just four residues (Fig. 2B). We therefore attempted to
design nonsecosteroidal ligands with the ability to bind with all
six residues of the VDR.
We designed and synthesized nonsecosteroidal ligands (Fig. 3).
Docking studies predicted that their hydroxyl groups (red spheres)
formed H-bonds with Tyr143 and Ser274. A docking simulation
was performed by carrying out a conformational search for ligands
of the VDR LBD using MacroModel. The lowest energy conformer
was used in the docking model.13 Synthetic schemes for each ligand
are shown in Scheme 1. Compound 1 was synthesized from a ketone
via (R)-CBS-oxazaborolidine-catalyzed (CBS; Corey–Baski–Shibata)
asymmetric borane reduction, as reported previously.14 Compound
1 was then treated with mesylate 3, 5 or 9, or epoxide 7 to give the
ligands through deprotection. YR333 and YR334 are diastereomers
and have not been differentiated. YR335, an (R,R)-isomer, was
synthesized from (2S)-butane-1,2,4-triol.15 YR336, the diastereo-
mer of YR335, was determined to be an (S,R)-isomer.
active form of vitamin D, regulates various biological events
including calcium and phosphorus homeostasis and cell differenti-
ation (Fig. 1).1,2 The biological responses of 1
a,25(OH)2D3 are med-
iated by the vitamin D receptor (VDR), which is a member of the
nuclear receptor superfamily and acts as a ligand-dependent gene
transcription factor in combination with co-activators.3,4
1a,25
(OH)2D3 has significant therapeutic potential for the treatment of
osteoporosis, various types of rickets, secondary hyperparathyroid-
ism, psoriasis, autoimmune diseases, and cancer; however, therapy
using 1
mia. Therefore, it is necessary to develop new VDR ligands. In 2000,
Moras et al. revealed the binding mode between 1 ,25(OH)2D3 and
the VDR ligand-binding domain (VDR LBD) using X-ray analysis.5
Their results showed that 1 ,25(OH)2D3 formed H-bonds with six
a,25(OH)2D3 is limited because it often causes hypercalce-
a
a
amino acid residues, Tyr143, Ser237, Arg274, Ser278, His305 and
His397 (Fig. 2A).
The discovery of LG190178, the first nonsecosteroidal VDR ago-
nistic ligand was significant,6 and many subsequent studies of the
nonsecosteroidal ligands of the VDR have been performed.7–10 We
have also reported that the (2S,20R)-analog (YR301) of LG190178 is
a major active isomer (Fig. 1).11 Moreover, we succeeded in solving
the crystal structure of the complex formed between YR301 and
Next, the ligands were examined using a transactivation assay
involving the human osteocalcin promoter.16 The results are sum-
marized in Table 1. YR335 exhibited potent transcriptional activity,
which was comparable to those of 1a,25-dihydroxyvitamin D3 and
YR301. Most of the ligands except YR311 showed moderate activ-
ity. YR336, the diastereomer of YR335, displayed activity that was
two orders of magnitude lower than that of YR335.
The crystal structure of the LBD of the complex formed between
the rat VDR and YR335 was solved to a resolution of 1.8 Å and is
⇑
Corresponding author. Tel.: +81 3 3700 1141; fax: +81 3 3707 6950.
E-mail address: masaaki@nihs.go.jp (M. Kurihara).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.08.047

Y. Demizu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6104–6107
6105
25
OH
H
C
D
H
(S)
(R)
HO
O
O
OH
OH
A
1
3
1α,25(OH)2D3
YR301
HO
OH
2
Figure 1. Chemical structures of 1a,25(OH)2D3 and YR301.
Figure 2. H-bond networks detected in the X-ray structures of the complexes (a) between the VDR LBD and 1
a
,25(OH)2D3, and (b) between the VDR LBD and YR301.
OH
HO
O
O
HO
HO
O
O
OH
OH
YR313
YR311
HO
HO
HO
O
O
HO
O
O
OH
OH
OH
OH
YR333 (faster retention time), YR334 (slower retention time)
HO
HO
(R)
(R)
HO
O
(R)
O
HO
O
(S)
O
OH
OH
YR335
YR336
Figure 3. Chemical structures of the synthesized nonsecosteroidal ligands.

6106
Y. Demizu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6104–6107
HO
O
OH
1
1) DHP, -TSA
p
2) LiAlH4
OMs
OH
1
1) K2CO3,
3) NaH, BnBr
EtO2C
CO2Et
YR311
BnO
O
OBn
4)
p
-TSA
2) Pd/C, H2
3
2
5) MsCl, Et3N
61%
45%
1) acetone,
p
-TSA
1) K2CO3, 1
OMs
HO
HO
OH
YR313
2) MsCl, Et3N
2) AcOH, H2O
O
4
5
95%
75%
1) NaH, BnBr
1) K2CO3, 1
OBn
OBn
OH
OH
O
YR333 and YR334
2)
m-CPBA
2) Pd/C, H2
3) Chiral HPLC using
IA column
86%
6
7
33%
1) NaH, BnBr
2) MsCl, Et3N
50%
1) K2CO3, 1
OH
OBn
OBn
HO
HO
BnO
YR335 and YR336
2) Pd/C, H2
OH
OMs
3) Chiral HPLC using
IA column
8
9
43%
1
1) K2CO3,
1) NaH, BnBr
2) MsCl, Et3N
53%
OH
YR335
BnO
2) Pd/C, H2
51%
OMs
OH
(2S)-butane-1,2,4-triol [(
S
)-8]
(S
)-9
Scheme 1. Synthesis of ligands.
Table 1
Transcriptional activity of ligands16
H-bonds with the six amino acid residues shown in Figure 4.
YR301 formed six H-bonds with four amino acid residues, that is
to say, the 20-OH was H-bonded to His301 and His393, the 2-OH
was H-bonded to Arg270 and Ser233, and the 1-OH was directly
H-bonded to Arg270 and was bound to Arg270 via a water mole-
cule-mediated H-bond (Fig. 2B).
Although all of the designated ligands were able to form addi-
tional H-bonds with Ser274 and Tyr143 in the docking simulation,
as shown in Figure 3, except for YR335, none of them displayed po-
tent activity. Therefore, it is highly probable that even if the desig-
nated ligands formed H-bonds with the desired amino acid
residues, their steric energies were not lowered and their activities
were not increased.
In conclusion, we designed and synthesized nonsecosteroidal
VDR ligands that formed H-bonds with six amino acid residues
(Tyr143, Ser233, Arg270, Ser274, His301 and His393) of the VDR
LBD. The ligand YR335 showed potent transcriptional activity,
which was comparable to that of YR301. Furthermore, to under-
stand the strong activity of YR335, the crystal structure of the com-
plex formed between YR335 and the rat VDR LBD was solved,
which revealed that YR335 formed H-bonds with the six amino
acid residues mentioned above.
Ligands
Transcriptional EC50 (nM)
YR311
YR313
YR333
YR334
YR335
YR336
YR301
19.4
1.11
0.92
1.25
0.06
1.29
0.04
0.01
1a,25(OH)2D3
shown in Figure 4. The crystal structure of the complex formed
between YR335 and the VDR ligand-binding domain was solved
to reveal that YR335 formed H-bonds with six amino acid residues
(Tyr143, Ser233, Arg270, Ser274, His301 and His393).17 Contrary
to YR301, YR335 was confirmed to form H-bonds with Ser278
and Tyr143.
Although YR335 was able to form additional H-bonds with
Ser274 and Tyr143, its transcriptional activity was almost the same
as that of YR301. This was probably because YR335 and YR301
contained the same number of H-bonds and that the stabilization
energy of YR335 was similar to that of YR301. YR335 formed six

Y. Demizu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6104–6107
6107
Figure 4. H-bond network formed between the VDR LBD and YR335 in the X-ray structure.
15. Spectroscopic data for YR335: Colorless oil; ½a D22
ꢀ
= ꢁ13.5 (c 1.5, CHCl3); 1H
Acknowledgment
NMR (400 MHz, CDCl3) d 6.91–6.96 (m, 4H), 6.79 (d, J = 8.4 Hz, 1H), 6.69 (d,
J = 8.0 Hz, 1H), 4.55 (m, 1H), 4.12 (m, 1H), 3.70–3.90 (m, 6H), 2.46 (br s, 1H),
2.18 (s, 3H), 2.16 (s, 3H), 1.97–2.06 (m, 8H), 1.00 (s, 9H), 0.59 (t, J = 7.2 Hz, 6H);
13C NMR (100 MHz, CDCl3) d 154.5, 153.5, 141.7, 141.3, 131.0, 130.8, 126.5,
126.4, 125.7, 112.4, 110.3, 77.0, 76.5, 69.4, 64.7, 64.4, 59.4, 48.6, 34.3, 33.8,
29.5, 26.3, 25.6, 17.0, 16.9, 8.7; ESI(+)-MS m/z 495 [M+Na]+.
This study was partly supported by a Kaneka Award for Syn-
thetic Organic Chemistry, Japan.
16. The human osteocalcin gene promoter fragment ꢁ838/+10 was cloned into the
pGL3 reporter plasmid (Promega), and the human VDR and RXR genes were
cloned into the pCDNA3 expression vector (Invitrogen). Hos cells were
maintained in phenol red free DMEM (Invitrogen) containing 10% FCS
(Invitrogen). Prior to the transfection, the cells were plated in a 96-well
plates at a density of 400,000 cells per well in Opti-MEM (Invitrogen). The cells
were then transfected with human osteocalcin reporter vector (pGL3-hOc:
100 ng/well), the human VDR and RXR expression vectors (pCDNA-hVDR and
pCDNA-hRXR, respectively, 10 ng/well) and phRL-TK (Promega: 25 ng/well)
References and notes
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using 50
transfection, they were treated with ethanol vehicle and various
concentrations of the VDR ligands (from 10 pM to 10 M). Luciferase activity
ll of Lipofectamine 2000 reagent (Invitrogen). Three hours after the
l
was quantitated the next day using a luminometer (Berthold) and the Dual-Glo
luciferase assay reagent (Promega).
17. VDR-LBD protein had the same structure and was purified and complexed by
the same protocol as described in reference 12. Its crystal structure has been
deposited in the RCSB under PDB code: 3AUN. The initial crystals of the VDR
LBD complexed with YR335 were obtained under the following crystallization
conditions: 100 mM MOPS (pH 7.0), 200 mM ammonium citrate, 20% PEG
4000, and 4% 2-propanol. The crystals were cryoprotected in 30% glycerol and
cooled at 79 K, and X-ray data were collected using SPring-8 beamline BL41XU.
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was achieved with MOLREP19 from CCP4 (Collaborative Computational Project,
No. 4, 1994), and the structure was built using COOT20 and refined using
REFMAC.21 Crystal data: spacer group: P21212, a = 44.24, b = 47.33,
c = 136.52 Å, data collection: SPring-8, beamline BL41XU, wavelength:
1.000 Å, resolution = 50.00–1.80 Å, total number of reflections = 185203,
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Takimoto-Kamimura, M. Acta Cryst. 2008, F64, 970.
unique
reflection = 27038,
Rmerge = 0.046,
completeness = 98.6%,
multiplicity = 3.7, average (I/
r(I)) = 41.8, Rfactor = 19.6%, Rfree = 23.7%.
13. Docking models of the ligands bound to the VDR-LBD were constructed via a
conformational search using MacroModel. AMBER⁄ was used as the force field,
and Mixed MCMM/LowMode was used as the conformational search method.
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Kittaka, A.; Kato, S.; Okuda, H.; Kurihara, M. Lett. Org. Chem. 2011, 8, 43.
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