Development of novel lithocholic acid derivatives as vitamin D receptor agonists

Article abstract of DOI:10.1016/j.bmc.2019.07.003

Lithocholic acid (2) was identified as the second endogenous ligand of vitamin D receptor (VDR), though its binding affinity to VDR and its vitamin D activity are very weak compared to those of the active metabolite of vitamin D₃, 1α,25-dihydroxyvitamin D₃ (1). 3-Acylated lithocholic acids were reported to be slightly more potent than lithocholic acid (2) as VDR agonists. Here, aiming to develop more potent lithocholic acid derivatives, we synthesized several derivatives bearing a 3-sulfonate/carbonate or 3-amino/amide substituent, and examined their differentiation-inducing activity toward human promyelocytic leukemia HL-60 cells. Introduction of a nitrogen atom at the 3-position of lithocholic acid (2) decreased the activity, but compound 6 bearing a 3-methylsulfonate group showed more potent activity than lithocholic acid (2) or its acylated derivatives. The binding of 6 to VDR was confirmed by competitive binding assay and X-ray crystallographic analysis of the complex of VDR ligand-binding domain (LBD) with 6.

Full text of DOI:10.1016/j.bmc.2019.07.003

Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of novel lithocholic acid derivatives as vitamin D receptor
agonists
a
b
b,⁎
a
a
c
Hiroyuki Masuno , Yuko Kazui , Aya Tanatani , Shinya Fujii , Emiko Kawachi , Teikichi Ikura ,
c
d
a,⁎
Nobutoshi Ito , Keiko Yamamoto , Hiroyuki Kagechika
a
Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan
Department of Chemistry, Faculty of Science, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan
Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
Showa Pharmaceutical University, 3-3165 Higashi-Tamagawagakuen, Machidashi, Tokyo 194-8543, Japan
b
c
d
A R T I C L E I N F O
A B S T R A C T
Keywords:
Lithocholic acid (2) was identified as the second endogenous ligand of vitamin D receptor (VDR), though its
Vitamin D
binding affinity to VDR and its vitamin D activity are very weak compared to those of the active metabolite of
Nuclear receptor
Lithocholic acid
Cell differentiation
vitamin D
3
, 1α,25-dihydroxyvitamin D (1). 3-Acylated lithocholic acids were reported to be slightly more
3
potent than lithocholic acid (2) as VDR agonists. Here, aiming to develop more potent lithocholic acid deriva-
tives, we synthesized several derivatives bearing a 3-sulfonate/carbonate or 3-amino/amide substituent, and
examined their differentiation-inducing activity toward human promyelocytic leukemia HL-60 cells.
Introduction of a nitrogen atom at the 3-position of lithocholic acid (2) decreased the activity, but compound 6
bearing a 3-methylsulfonate group showed more potent activity than lithocholic acid (2) or its acylated deri-
vatives. The binding of 6 to VDR was confirmed by competitive binding assay and X-ray crystallographic analysis
of the complex of VDR ligand-binding domain (LBD) with 6.
1
. Introduction
and Ser274, and indirectly with Ser233 and Arg270 via a water mole-
cule. These four amino acid residues interact with the two hydroxyl
Vitamin D
3
plays important roles in many physiological processes,
groups of 1α,25(OH)
2
D
3
(1) in the complex with 1α,25(OH)
2
D (1).
3
including calcium and phosphate homeostasis, bone metabolism, and
The 25-hydroxyl group of 1α,25(OH)
2
D (1) forms hydrogen bonds
3
1
immune regulation. Its active metabolite, 1α,25-dihydroxyvitamin D
3
with His301 and His393, which are involved in hydrogen bonding di-
rectly with the acetyl group of lithocholic acid 3-acetate (3a), and in-
directly with the 3-hydroxyl group of lithocholic acid (2) via a water
molecule. Thus, introduction of an acetyl group at the 3-position of
lithocholic acid (2) alters the interactions with the amino acid residues
of VDR LBD, which presumably causes the increase of the vitamin D
activity of lithocholic acid 3-acetate (3a). Therefore, in this study, we
designed and synthesized several lithocholic acid derivatives with
various polar substituents at the 3-position, and examined their vitamin
D activities (Fig. 2).
[
1α,25(OH)
2
D , 1] (Fig. 1), is an endogenous agonist of vitamin D
3
1
nuclear receptor (VDR), and induces increased expression of target
genes.
In 2001, Makishima et al. identified lithocholic acid (2), a bile acid
formed from chenodeoxycholate, as the second endogenous agonist of
2
VDR. Although the role of lithocholic acid in VDR functions, as well as
3
potential clinical application, has been investigated, its potency as a
VDR ligand is very low compared to that of 1α,25(OH)
2
D (1). Some 3-
3
acylated lithocholic acid derivatives, such as 3-acetate (3a) and 3-
propionate (3b), were more potent than lithocholic acid (2) itself, but
4
,5
their vitamin D activities were still low.
2. Results and discussion
We previously studied the binding of lithocholic acid (2) and li-
4
thocholic acid 3-acetate (3a) by means of docking study and X-ray
2.1. Synthesis
6
crystallographic analysis of the complex of VDR ligand binding domain
(
LBD) with these ligands. The crystal structures indicated that the
Various polar groups were introduced at the 3-hydroxyl group of
carboxy group of 2 and 3a forms hydrogen bonds directly with Tyr143
lithocholic acid (2), as shown in Scheme 1. Lithocholic acid (2) was
⁎
Corresponding authors.
E-mail addresses: tanatani.aya@ocha.ac.jp (A. Tanatani), kage.chem@tmd.ac.jp (H. Kagechika).
https://doi.org/10.1016/j.bmc.2019.07.003
Received 4 June 2019; Received in revised form 1 July 2019; Accepted 3 July 2019
0968-0896/ © 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Hiroyuki Masuno, et al., Bioorganic & Medicinal Chemistry, https://doi.org/10.1016/j.bmc.2019.07.003

H. Masuno, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
concentrations above 1 μM, while compound 6 showed moderate po-
tency to activate VDR, and the potency of 6 was similar to that of the 3-
acetate 3a. Competitive binding experiments showed that compound 6
binds to VDR, and its binding affinity was higher than that of the
control compound, 3-propionate 3b that was most among three known
5
compounds 2, 3a and 3b (Fig. 5). These results indicate that com-
pound 6 is a VDR agonist, and induces differentiation of HL-60 cells.
2.3. X-ray crystallographic analysis
In order to clarify the VDR-binding mode of lithocholic acid deri-
Fig. 1. Structures of activated vitamin D
3
(1α,25(OH)
2
D , 1), lithocholic acid
3
vatives, we carried out X-ray crystallographic analysis of the complex of
rat VDR LBD (residues 116–423, Δ165–211) with compound 6 ac-
cording to the method reported in our previous study. A synthetic
peptide containing the target sequence of the coactivator MED1
(
2), and lithocholic acid 3-acylates (3).
(
mediator of RNA polymerase II transcription subunit 1, also known as
ARC205 or DRIP205) was included in the crystallization solution of
VDR LBD and compound 6.
The overall structure of VDR LBD in the ternary complex, de-
termined at 1.8 Å resolution (Table 1), was nearly identical to that in
the complex with lithocholic acid (2) or 3-acetate (3a) (Fig. 6a). Com-
pound 6 is located in the ligand-binding pocket, which is common to
proteins in the nuclear receptor superfamily, in a similar manner to
1
α,25(OH)
2
D (1) and lithocholic acid (2) (Fig. 6b). The carboxy group
3
of compound 6 formed hydrogen bonds with Tyr143 in helix 1 and
Ser274 in helices 4/5, both of which form hydrogen bonds with the 3-
Fig. 2. Structures of lithocholic acid derivatives.
hydroxyl group on the cyclohexane ring of 1α,25(OH)
2
D (1) in the
3
crystal. The carboxy group of 6 also formed hydrogen bonds via a water
molecule with Arg270 in helices 4/5 and Ser233 in helix 3. In the case
of the complex of VDR LBD with 1α,25(OH)
2
D (1), these amino acid
3
converted to benzyl ester 10, which was reacted with acyl chloride,
methyl chlorocarbonate, or methanesulfonyl chloride, followed by re-
moval of the benzyl group to afford compounds 4–6, respectively.
Compounds 7–9 bearing a 3-nitrogen functionality were synthesized
as shown in Scheme 2. Mitsunobu reaction of methyl lithocholate (14)
with diethyl azodicarboxylate (DEAD), triphenylphosphine, and formic
acid afforded 3β-formate 15. Treatment of 15 with sodium methoxide
in methanol gave methyl 3-epi-lithocholate (16). Further Mitsunobu
reaction of 16 with phthalimide as a nucleophile, followed by treatment
with hydrazine, afforded 3α-amino derivative 18 as the pure isomer,
residues interacted directly with the 1-hydroxyl group of 1. The inter-
actions of the carboxyl group of compound 6 with VDR amino acid
residues are similar to those of lithocholic acid (2), 3a and 3b. In
contrast to the direct interaction of two hydroxyl groups on the cyclo-
hexane ring of 1α,25(OH) D (1) with four amino acid residues, two of
2
3
them formed hydrogen bonds with the lithocholic acid derivatives via a
water molecule, which may be one of the reason for the weaker vitamin
D activity of the derivatives, compared to 1.
The 3-methanesulfonyl group of 6 also interacted with amino acid
residues of VDR. Thus, one of the oxygen atoms of the 3-methane-
sulfonyl group formed hydrogen bonds with His301 in helix 6 and
His393 in helix 11, though there was no significant interaction of the
other oxygen atom with VDR. These histidine residues form hydrogen
1
deduced by H NMR. Acylation or sulfonylation of 18 gave amide 19
and sulfonamide 20, respectively. Finally, compounds 7–9 were ob-
tained by basic hydrolysis of 18, 19, and 20, respectively.
bonds with the 25-hydroxyl group of 1α,25(OH)
2
3
D (1) or the carbonyl
2.2. Biological evaluation
group of the 3-substituent of 3 in the crystal structures. In the complex
of VDR with 1α,25(OH)
2
D (1), the two histidines are located at similar
3
The vitamin D activity of the synthesized lithocholic acid deriva-
distances from the 25-hydroxyl group (less than 3.0 Å between the ε-
nitrogen atom of histidine and the oxygen atom of the hydroxyl group).
In the case of 3-propionate 3b, the distances from the carbonyl oxygen
atom are 3.80 Å and 3.31 Å for His301 and His393, respectively, which
are similar to those (3.99 Å for His301 and 3.30 Å and His393) in the
complex of VDR with 6.
tives was evaluated in terms of cell differentiation-inducing activity
7
toward human acute promyelocytic leukemia cell line HL-60. HL-60
cell differentiation was determined by measuring the ratio of nitroblue
tetrazolium (NBT)-positive cells in the concentration range of
−
7
−5
1
0
M–10 M test compound. Under these assay conditions, litho-
cholic acid (2) did not induce differentiation of HL-60 cells at con-
−
5
centrations below 10 M (data not shown), while the acetate 3a
3. Conclusion
−
5
showed differentiation-inducing activity only at 10 M (Fig. 3). Acy-
lates 4a/b and carbonate 5 were inactive, but the methanesulfonate 6
We designed and synthesized lithocholic acid derivatives bearing
various 3-substituents as candidate VDR agonists. Compounds bearing
nitrogen at the 3-position exhibited weaker activity than the corre-
sponding oxygen analogs. Among the synthesized compounds, com-
pound 6 bearing 3-methanesulfonate showed the most potent activity in
HL-60 cell differentiation assay. Compound 6 showed VDR transacti-
vation and binding ability. Further, we determined the crystal structure
of the complex of rVDR LBD with 6, which indicated that direct hy-
drogen bond formation of the functional group at the 3-position of li-
thocholic acid derivatives contributes to the higher potency, compared
with lithocholic acid (2). The compounds developed in this study and
−
6
was more active than 3a, showing an IC50 of 1.2 × 10 M. Among the
compounds with a nitrogen-containing functionality, only compound 9
showed differentiation-inducing activity, but its potency was lower
than that of the corresponding oxygen-containing derivatives (3a vs 8
or 6 vs 9). Thus, 3-methanesulfonate 6 was the most active analog of
lithocholic acid (2) among the synthesized compounds.
Next, the VDR transactivation ability of selected compounds was
examined by means of luciferase reporter gene assay (Fig. 4). Com-
pounds 3 were used as the control, since the VDR transactivation ability
5
of compound 2 was very low. Compound 4a showed activity only at
2

H. Masuno, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
Scheme 1. Synthesis of compounds 4–6. Reagents and conditions: (a) benzyl bromide, K
2
CO
3
, DMF; (b) R-Cl, pyridine, 0 °C; (c) H , Pd-C, ethanol; (d) TFA.
2
the SAR information should be helpful in the development of new-
generation nonsecosteroidal VDR ligands with high potency.
(δ-63 ppm) was used as external standard in 19F NMR spectra. Data are
reported as follows: chemical shift, multiplicity (s, singlet; d, doublet; t,
triplet; q quartet; br, broad; and m, multiplet), coupling constants (Hz),
and integration. Mass spectra were collected on a Bruker Daltonics
micrO TOF focus-II in the positive ion mode.
4
. Experimental
4
.1. General
4
.2. Synthesis
All reagents were purchased from Sigma-Aldrich Chemical Co.,
Tokyo Kasei Kogyo Co., Wako Pure Chemical Industries, and Kanto
Chemical Co., Inc. Silica gel for column chromatography was purchased
4.2.1. Compound 4a
A solution of lithocholic acid (2, 110 mg, 0.30 mmol) in tri-
fluoroacetic acid (2 ml) was stirred at room temperature for 4 h, then
aqueous sodium bicarbonate was added, and the reaction mixture was
extracted with ethyl acetate. The organic layer was washed with aqu-
eous sodium bicarbonate, and water, dried over magnesium sulfate, and
evaporated. The residue was chromatographed on silica gel (ethyl
acetate/hexane 1:9) to give 4a (110 mg, 80%). Colorless Powder
1
from Kanto Chemical Co., Inc. H NMR spectra were recorded on at
6
00 MHz on a Bruker AVANCE 600 spectrometer or at 500 MHz on a
Bruker AVANCE 500 spectrometer or at 400 MHz on a Bruker AVANCE
1
3
4
00 spectrometer. C NMR spectra were recorded on at 125 MHz on a
Bruker AVANCE 500 spectrometer. Chemical shifts are reported in ppm
1
9
as δ values from tetramethylsilane. F NMR spectra were recorded on
at 376.5 MHz on a Bruker AVANCE 400 spectrometer. Trifluorotoluene
(ethanol/H
2
O); mp 177–178 °C; 1H NMR (400 MHz, CDCl
) δ 4.93 (m,
3
Scheme 2. Synthesis of compounds 7–9. Reagents and conditions: (a) AcCl, MeOH; (b) DEAD, PPh
3
, HCOOH, THF; (c) NaOMe, MeOH; (d) DEAD, PPh
3
, phthalimide,
toluene; (e) 1) hydrazine monohydrate, MeOH, 2) HCl aq; (f) NaOH aq, MeOH; (g) R-Cl, Et
3
N, CH Cl
2
2
.
3

H. Masuno, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
Fig. 3. HL-60 cell differentiation-inducing activity of
lithocholic acid derivatives. (a) ●: 1α,25(OH)
2
D (1),
3
○
1
: 3a, □: 3b, △: 4a, ▲: 4b, ◊: 5, ■: 6; (b) ●:
α,25(OH) (1), ◊: 7, △: 8, ▲: 9.
2 3
D
Table 1
Data collection and refinement statistics for crystal structure of VDR LBD/
coactivator peptide/compound 6 complex. Values in parentheses are for the
highest-resolution shell.
Data Collection
Unit cell dimensions
a (Å)
153.9
43.7
41.8
96.1
C2
b (Å)
c (Å)
β (deg)
Space group
Resolution (Å)
Completeness (%)
Redundancy
No of unique reflections
Average I/σ(I)
CC1/2
50–1.8
98.6
3.5
(1.83–1.80)
(91.9)
(2.9)
25,497
16.3
(1198)
(1.3)
(0.760)
Refinement
R-cryst (%)
21.3
25.7
0.010
1.41
R-free (%)
RMS bond length (Å)
RMS bond angles (deg)
Atoms
Fig. 4. VDR transactivation ability of lithocholic acid derivatives. ●:
Protein
1984
57
1
α,25(OH)
2
3
D (1), □: 3a, ▲: 3b, ×: 4a, ○: 6.
Ligand and water
1
1
H), 2.40 (ddd, 1H, J = 5.2, 10.2, 15.6 Hz), 2.26 (ddd, 1H, J = 6.4, 9.4,
13
5.8 Hz), 0.95 (s, 3H), 0.92 (d, 3H, J = 6.4 Hz), 0.65 (s, 3H). C NMR
) δ 180.9, 156.99 (q, JCF = 41.7 Hz), 114.6 (q,
CF = 286.2 Hz), 79.4, 56.4, 55.9, 42.7, 41.9, 40.4, 40.0, 35.7, 35.3,
(
100 MHz, CDCl
3
J
3
1
4.7, 34.5, 31.6, 31.1, 30.7, 28.1, 26.9, 26.2, 26.1, 24.1, 23.2, 20.8,
19
8.2, 12.0. F NMR (376.5 MHz, CDCl
3
) δ-75.6. HRMS-ESI (m/z):
−
[
M−H] calcd for C26
H
38
F
3
O 471.2728; found, 471.2726.
4
4
.2.2. Compound 10
Potassium carbonate (810 mg, 5.86 mmol) was added to a solution
of lithocholic acid (2.00 g, 5.31 mmol) in DMF (14 ml). After 30 min,
benzyl bromide (950 μl, 7.99 mmol) was added, and resulting solution
was stirred at room temperature for 3 h. Ethyl acetate and water were
added to the reaction mixture. The organic layer was washed with
water, dried over magnesium sulfate, and evaporated. The residue was
chromatographed on silica gel (ethyl acetate/hexane 1:9 to 1:1) to give
compound 10 (2.22 g, 89%). Colorless powder (ethyl acetate/hexane;
1
-11 -10 -9
-8
-7 -6
-5
-4
mp 123–124 °C. H NMR (400 MHz, CDCl
3
) δ 7.34 (m, 5H), 5.11 (ABq.
2
H, J = 12.3 Hz), 3.62 (m, 1H), 2.40 (ddd, J = 15.4, 9.9, 5.1 Hz), 2.27
Concentration (log, M)
(
ddd 1H, J = 15.4, 8.9, 6.5 Hz), 0.91 (s, 3H), 0.49 (d, 3H, J = 6.5 Hz),
Fig. 5. VDR-binding ability of lithocholic acid derivatives. ●: 1α,25(OH)
1), ▲: 3b, ○: 6.
D
2 3
13
0
1
.62 (s, 3H). C NMR (100 MHz, CDCl
3
) δ 174.1, 136.1, 128.5, 128.2,
(
28.1, 71.8, 66.1, 56.5, 55.9, 42.7, 42.1, 40.4, 40.1, 36.4, 35.8, 35.3,
4

H. Masuno, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
Fig. 6. Crystal structure of the complex of VDR with 6. (a) Overall structure, and (b) VDR binding feature of 6, compared with those of (c) 1α,25(OH)
2
3
D (1) (PDB:
RK3)9 and (d) lithocholic acid (2) (PDB: 3W5P) .
6
1
3
4.5, 31.3, 31.0, 30.5, 28.2, 27.2, 26.4, 24.2, 23.4, 20.8, 18.2, 12.0.
7%). 1H NMR (400 MHz, CDCl
) δ 7.33 (m, 5H), 5.18 (ABq, 2H,
3
+
HRMS-ESI (m/z): [M+Na] calcd for C31
H
46NaO
3
489.3339; found,
J = 12.4 Hz), 4.58 (m, 1H), 3.76 (s, 3H), 2.40 (ddd, 1H, J = 5.0, 9.9,
15.0 Hz), 2.27 (ddd, 1H, J = 6.8, 9.2, 15.6 Hz), 0.93 (s, 3H), 0.90 (d,
4
89.3338.
1
3
1
H, J = 6.3 Hz), 0.61 (s, 3H). C NMR (100 MHz, CDCl ) δ 174.0,
3
4.2.3. Compound 11
155.2, 136.1, 128.5, 128.2, 128.1, 78.3, 66.0, 56.4, 55.9, 54.4, 42.7,
4
-Dimethylaminopyridine (13 mg, 0.11 mmol) and benzoyl chloride
41.8, 40.3, 40.1, 35.7, 35.3, 34.9, 34.5, 32.1, 31.2, 30.9, 28.1, 27.0,
+
(
20 μl, 0.22 mmol) were added to a solution of 10 (50 mg, 0.11 mmol)
26.5, 26.2, 24.1, 23.2, 20.8, 18.2, 12.0. HRMS-ESI (m/z): [M+Na]
in pyridine (0.5 ml) at 0 °C. The mixture was stirred at room tempera-
ture for 24 h, then aqueous hydrochloric acid was added, and the whole
was extracted with ethyl acetate. The organic layer was washed with
aqueous sodium bicarbonate, and water, dried over magnesium sulfate,
and evaporated. The residue was chromatographed on silica gel (ethyl
calcd for C33
H
48NaO 547.3394; found, 547.3390.
5
4.2.5. Compound 13
Methanesulfonyl chloride (35 μl, 0.46 mmol) was added to a solu-
tion of 10 (43 mg, 0.091 mmol) in pyridine (0.5 ml) at 0 °C. After 2 h,
the mixture was poured into water, and extracted with ethyl acetate.
The organic layer was washed with water, dried over magnesium sul-
fate, and evaporated. The residue was chromatographed on silica gel
acetate/hexane 1:19 to 1:10) to give 11 (36 mg, 64%), together with
1
unreacted 10 (18 mg, 35%). H NMR (400 MHz, CDCl
3
) δ 8.05 (m, 2H),
7
5
.54 (tt, J = 1.4, 7.4 Hz, 1H), 7.43 (t, J = 7.6 Hz, 2H), 7.35 (m, 6H),
1
.13 (d, J = 12.4 Hz, 2H), 5.09 (d, 2H, J = 12.3 Hz), 4.97 (m, 1H), 2.40
(ethyl acetate/hexane 1:9) to give 13 (50 mg, 99%). H NMR (400 MHz,
(
ddd, 1H, J = 5.1, 10.0, 15.3 Hz), 2.27 (ddd, 1H, J = 6.6, 9.1, 15.6 Hz),
CDCl ) δ 7.36 (m, 5H), 5.11 (ABq, 2H, J = 12.3 Hz), 4.65 (m, 1H), 3.00
3
1
3
0
.96 (s, 3H), 0.91 (d, 3H, J = 6.1 Hz), 0.63 (s, 3H). C NMR (100 MHz,
(s, 3H), 2.40 (ddd, 1H, J = 5.0, 9.9, 15.0 Hz), 2.27 (ddd, 1H, J = 6.8,
13
CDCl
3
) δ 174.1, 166.1, 136.1, 132.6, 130.9, 129.5, 128.5, 128.2, 128.1,
9.2, 15.6 Hz), 0.93 (s, 3H), 0.90 (d, 1H, J = 6.3 Hz), 0.62 (s, 3H).
C
7
3
5.0, 66.1, 56.5, 56.0, 42.7, 41.9, 40.5, 40.1, 35.8, 35.3, 35.1, 34.6,
NMR (100 MHz, CDCl ) δ 174.0, 136.1, 128.5, 128.2, 128.1, 82.9, 66.1,
3
2.4, 31.3, 31.0, 28.2, 27.0, 26.7, 26.3, 24.2, 23.4, 20.9, 18.3, 12.0.
56.4, 55.9, 42.7, 42.1, 40.4, 40.0, 38.8, 35.7, 35.3, 35.0, 34.4, 33.3,
+
HRMS-ESI (m/z): [M+Na] calcd for C38
H
50NaO 593.3601; found,
4
31.2, 30.9, 28.1, 27.8, 26.8, 26.2, 24.1, 23.1, 20.8, 18.2, 12.0. HRMS-
5
4
1
93.3599.
ESI (m/z): [M+Na]+ calcd for
C
32
H
48NaO S 567.3115; found,
5
5
67.3109.
.2.4. Compound 12
Methyl chloroformate (17 μl, 0.22 mmol) was added to a solution of
4.2.6. Compound 4b
0 (51 mg, 0.11 mmol) in pyridine (0.5 ml) at 0 °C, and the mixture was
A suspension of 11 (85 mg, 0.15 mmol) and 10% palladium on
carbon (16 mg) in methanol (1 ml) and formic acid (0.02 ml) was
stirred in a hydrogen atmosphere for 15 h. The reaction mixture was
filtered on Celite, and the filtrate was evaporated. The residue was
chromatographed on silica gel (ethyl acetate/hexane 1:4) to give 4b
stirred at room temperature for 7 h. Then, methyl chloroformate
(
240 μl, 3.1 mmol) was added, and the mixture was stirred overnight.
After addition of methanol, the solvent was removed in vacuo. The
residue was chromatographed on silica gel (ethyl acetate/hexane 1:99
to 1:19) to give 12 (39 mg, 68%), together with unreacted 10 (4 mg,
1
(63 mg, 88%). Colorless Powder (methanol); mp 100–102 °C; H NMR
5

H. Masuno, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
(
400 MHz, CDCl
3
) δ 8.05 (m, 2H), 7.54 (t, J = 7.6 Hz, 1H), 7.43 (t,
56.2, 51.7, 43.0, 40.4, 40.1, 37.5, 35.8, 35.6, 35.1, 31.3, 31.2, 30.8,
J = 7.6 Hz, 2H), 4.98 (m, 1H), 2.40 (ddd, 1H, J = 5.2, 10.3, 15.6 Hz),
30.8, 28.4, 26.6, 26.3, 25.2, 24.4, 24.0, 21.3, 18.5, 12.3. HRMS-ESI (m/
2
3
1
4
2
.26 (ddd, 1H, J = 6.3, 9.6, 15.8 Hz), 0.96 (s, 3H), 0.93 (d, J = 6.4 Hz,
z): [M+Na]+ calcd for C26
H
42NaO 411.2975; found 441.2964.
4
1
3
H), 0.66 (s, 3H). C NMR (100 MHz, CDCl ) δ 180.2, 166.1, 132.7,
3
30.9, 129.5, 128.2, 77.3, 77.0, 76.7, 75.0, 56.5, 56.0, 42.7, 41.9, 40.5,
0.1, 35.8, 35.3, 35.1, 34.6, 32.3, 30.9, 30.7, 28.2, 27.0, 26.7, 26.3,
4
.2.11. Compound 16
Sodium methoxide (155 mg) was added to a solution of 15 (699 mg,
+
4.2, 23.3, 20.9, 18.2, 12.0. HRMS-ESI (m/z): [M+Na] calcd for
1
.67 mmol) in methanol (150 ml). After 2 h, the reaction mixture was
C
31
H
44NaO 503.3132; found, 503.3133.
4
poured into 2 M hydrochloric acid. The resulting precipitate was wa-
shed with water, and dissolved in ethanol and ethyl acetate. The solu-
tion was filtered, and the filtrate was evaporated. The residue was
chromatographed on silica gel (ethyl acetate/hexane 1:4 to 1:1) to af-
4
.2.7. Compound 5
A suspension of 12 (85 mg, 0.15 mmol) and 10% palladium on
carbon (16 mg) in ethanol (2 ml) was stirred in a hydrogen atmosphere
for 1 h. The reaction mixture was filtered on Celite, and the filtrate was
evaporated. The residue was chromatographed on silica gel (ethyl
1
ford 16 (606 mg, 93%). H NMR (600 MHz, CDCl
3
) δ 4.10 (brs, 1H),
3
.66 (s, 3H), 2.35 (ddd, 1H, J = 14.8, 9.9, 4.9 Hz), 2.21 (ddd, 1H,
J = 15.9, 9.9, 6.6 Hz), 0.95 (s, 3H), 0.91 (d, 3H, J = 6.1 Hz), 0.64 (s,
acetate/hexane 1:9 to 1:4) to give 5 (63 mg, 88%). Colorless Powder
13
3
4
2
H). C NMR (126 MHz, CDCl
3
) δ 175.0, 67.4, 56.8, 56.2, 51.7, 43.0,
1
(
ethanol); mp 200–202 °C; H NMR (400 MHz, CDCl
3
) δ 4.58 (m, 1H),
0.4, 39.9, 36.8, 35.8, 35.6, 35.3, 33.7, 31.3, 31.2, 30.1, 28.4, 28.1,
3
.76 (s, 3H,), 2.26 (ddd, 1H, J = 6.5, 9.6, 15.8 Hz), 2.40 (ddd, 1H,
+
6.9, 26.5, 24.4, 24.1, 21.3, 18.5. HRMS-ESI (m/z): [M+Na] calcd for
J = 5.1, 10.1, 15.4 Hz), 0.93 (s, 3H), 0.92 (d, 1H, J = 7.1 Hz), 0.64 (s,
C
25
H
42NaO 413.3026; found 413.3026.
3
1
3
3
1
4
2
H). C NMR (100 MHz, CDCl ) δ 180.4, 167.7, 155.3, 132.4, 130.8,
3
28.8, 78.4, 77.3, 77.0, 76.7, 68.1, 56.4, 55.9, 54.4, 42.7, 41.8, 40.3,
4
.2.12. Compound 17
0.1, 38.7, 35.7, 35.3, 34.9, 34.5, 32.1, 31.0, 30.7, 30.3, 28.9, 28.1,
Triphenylphosphine (379 mg, 1.4 mmol), phthalimide (231 mg,
.6 mmol), and DEAD (2.2 M solution in toluene, 620 μl, 1.4 mmol)
7.0, 26.5, 26.2, 24.1, 23.7, 23.2, 22.9, 20.8, 18.2, 14.0, 12.0, 10.9.
1
+
HRMS-ESI (m/z): [M+Na] calcd for C26
H
42NaO 457.2924; found,
5
were added to a solution of 16 (281 mg, 0.72 mmol) in toluene (4 ml).
After 2.5 h, the reaction mixture was poured into water, and extracted
with ethyl acetate. The organic layer was washed with water, dried over
sodium sulfate, and evaporated. The residue was chromatographed on
4
57.2920.
4
.2.8. Compound 6
A suspension of 12 (19 mg, 0.035 mmol) and 10% palladium on
silica gel (ethyl acetate/hexane 1:19 to 1:9) to afford 17 (218 mg, 59%).
carbon (17 mg) in ethanol (1 ml) was stirred in a hydrogen atmosphere
for 1 h. The reaction mixture was filtered on Celite, and the filtrate was
evaporated. The residue was chromatographed on silica gel (ethyl
1
H NMR (400 MHz, CDCl ) δ 7.80 (dd, J = 5.4, 2.9 Hz), 7.69 (dd,
3
J = 5.6, 3.2 Hz), 4.18 (m, 1H), 3.67 (s, 3H), 2.73 (q, 1H, J = 12.7 Hz),
2
3
1
5
3
.35 (m, 2H), 2.22 (ddd, 1H, J = 16.1,9.8,7.0 Hz), 0.97 (s, 3H), 0.92 (d,
acetate/hexane 1:9 to 1:4) to give 6 (11 mg, 70%). Colorless Powder
13
H, J = 6.3 Hz), 0.66 (s, 3H). C NMR (126 MHz, CDCl ) δ 175.1,
3
1
(
ethanol); mp 156–157 °C; H NMR (400 MHz, CDCl
3
) δ 4.65 (m, 1H),
68.8, 168.8, 134.0, 134.0, 132.3, 132.3, 123.2, 123.2, 56.6, 56.0,
1.7, 51.5, 43.3, 43.0, 40.7, 40.3, 36.8, 36.0, 35.6, 34.8, 31.2, 31.2,
3
.00 (s, 3H), 2.40 (ddd, 1H, J = 5.0, 9.9, 15.0 Hz), 2.27 (ddd, 1H,
J = 6.8, 9.2, 15.6 Hz), 0.93 (s, 3H), 0.92 (d, 1H, J = 6.1 Hz), 0.64 (s,
0.0, 28.4, 27.3, 26.5, 24.5, 24.4, 23.7, 21.1, 18.5, 12.3. HRMS-ESI (m/
1
3
3
4
2
H). C NMR (100 MHz, CDCl ) δ 180.0, 83.0, 56.4, 55.9, 42.7, 42.1,
3
+
z): [M+Na] calcd for C33
H
45NNaO 542.3241; found 542.3220.
4
0.4, 40.0, 38.8, 35.7, 35.3, 35.0, 34.4, 33.3, 30.9, 30.7, 28.1, 27.8,
+
6.8, 26.2, 24.1, 23.1, 20.8, 18.2, 12.0. HRMS-ESI (m/z): [M+Na]
4
.2.13. Compound 18
calcd for C25
H42NaO S 477.2645; found, 477.2651.
5
Hydrazine monohydrate (250 μl, 5.0 mmol) was added to a solution
of 17 (520 mg 1.00 mmol) in methanol (25 ml). The mixture was heated
at reflux for 5.5 h, poured into brine, and extracted with di-
chloromethane. The organic layer was washed with aqueous sodium
hydrogen carbonate, dried over sodium sulfate, and evaporated. The
residue was dissolved in diethyl ether, and 1 M HCl was added. The
4
.2.9. Compound 14
Acetyl chloride (50 μl) was added to a solution of lithocholic acid (2,
4
97 mg, 1.32 mmol) in methanol (5 ml). The mixture was stirred for 4 h
at room temperature, then poured into water. The resulting precipitate
was taken up in ethanol. The solution was filtered, and the filtrate was
evaporated. The residue was recrystallized from hexane to give 14
1
precipitate was collected to afford 18 (372 mg, 87%). H NMR
1
(600 MHz, CDCl ) δ 8.30 (s, 3H), 3.66 (s, 3H), 3.18 (brs, 1H), 2.36 (ddd,
(
438 mg, 85%). H NMR (400 MHz, CDCl
3
) δ 3.66 (s, 3H), 3.61 (m, 1H),
3
1
H, J = 15.1, 10.3, 4.8 Hz), 2.21 (ddd, 1H, J = 15.1, 10.3, 6.8 Hz), 0.91
2
6
.35 (ddd, 1H, J = 15.4, 10.4, 5.0 Hz), 2.21 (ddd, 1H, J = 15.4, 9.9,
13
13
(s, 3H), 0.90 (d, 3H, J = 6.2 Hz), 0.63 (s, 3H). C NMR (150 MHz,
.6 Hz), 0.91 (s, 3H), 0.90 (d, 3H, J = 6.5 Hz,), 0.64 (s, 3H C NMR
CDCl
3
) δ 174.77, 55.07, 55.77, 51.95, 51.47, 42.66, 42.22, 40.30,
(
150 MHz, CDCl
3
) δ 174.79, 71.86, 56.47, 55.92, 51.47, 47.71, 42.07,
3
2
9.80, 35.80, 35.35, 35.10, 34.56, 31.63, 31.09, 30.97, 28.13, 26.79,
4
2
0.41, 40.15, 36.44, 35.83, 35.36, 35.33, 34.56, 31.04, 30.98, 30.53,
+
6.25, 24.20, 23.39, 20.85, 18.27, 12.01. HRMS-ESI (m/z): [M+H]
8.18, 27.17, 26.40, 24.20, 23.36, 20.81, 18.25, 12.03. HRMS-ESI (m/
+
calcd for C
H
25 44
NO 390.3367; found 390.3356.
z): [M+Na] calcd for C25
H
42NaO
3
413.3026; found 413.3026.
2
4
.2.10. Compound 15
4.2.14. Compound 7
Triphenylphosphine (79 mg, 0.30 mmol), formic acid (12 μl,
.3 mmol), and DEAD (2.2 M solution in toluene, 140 μl, 0.30 mmol)
2 M aqueous sodium hydroxide (1 ml) was added to a solution of 18
(40 mg, 0.093 mmol) in methanol (3 ml). The mixture was stirred at
0 °C for 1 h, and at room temperature for 8 h, then poured into 2 M
hydrochloric acid. The resulting precipitate was dissolved in methanol.
0
were successively added to a solution of compound 14 (50 mg
0
.13 mmol) in toluene (1 ml). The mixture was stirred at 0 °C for 1 h,
and at room temperature for 17 h, then poured into aqueous sodium
bicarbonate, and extracted with ethyl acetate. The organic layer was
washed with water, dried over sodium sulfate, and evaporated. The
The solution was filtered, and the filtrate was evaporated to give 7
1
(27 mg, 77%). H NMR (600 MHz, CD
3
OD) δ 3.07 (m, 1H), 2.18 (ddd,
1H, J = 13.7, 10.3, 4.8 Hz), 2.02 (m, 2H), 0.97 (s, 3H), 0.92 (d,
1
3
residue was chromatographed on silica gel (ethyl acetate/hexane 1:9)
3H,J = 6.2 Hz), 0.67 (s, 3H). C NMR (126 MHz, CD OD) δ 183.59,
3
1
to give 15 (37 mg, 70%). H NMR (400 MHz, CDCl
3
) δ 8.06 (s, 1H), 5.22
58.11, 57.77, 52.49, 44.03, 43.47, 41.96, 41.67, 37.35, 37.28, 36.25,
(
(
brs, 1H), 3.66 (s, 3H), 2.34 (ddd, 1H, J = 15.2, 10.0, 5.2 Hz), 2.21
36.16, 35.74, 34.12, 32.76, 29.41, 28.11, 27.64, 27.03, 25.39, 23.88,
+
ddd, 1H, J = 16.8, 10.4, 6.8 Hz), 0.96 (s, 3H), 0.91 (d, 3H, J = 6.4 Hz),
22.07, 19.11, 12.62. HRMS-ESI (m/z): [M+H] calcd for C24
H42NO
2
0
.65 (s, 3H). 13C NMR (126 MHz, CDCl
3
) δ 175.0, 161.1, 71.2, 56.8,
376.3210; found 376.3199.
6

H. Masuno, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
4
.2.15. Compound 19
4.3. HL-60 cell differentiation assay7
Acetic anhydride (200 μl) was added to a solution of 18 (68.5 mg,
.16 mmol) in dichloromethane (1.5 ml) and pyridine (0.5 μl). The
0
HL-60 cells were cultured in RPMI-1640 medium supplemented
mixture was stirred at room temperature for 22.5 h, poured into water,
and extracted with dichloromethane. The organic layer was washed
with water, dried with sodium sulfate, and evaporated. The residue was
with 5% FBS and penicillin G and streptomycin at 37 °C under 5% CO
2
4
in air. The cells were diluted to 8.0 × 10 cells/mL with RPMI-1640
(5% FBS), and an ethanol solution of a test compound was added to give
−9
−6
recrystallized from ethyl acetate to afford 19 (36 mg, 53%). Colorless
10 –10 M final concentration. Control cells were treated with the
1
prisms (ethyl acetate); mp 208 °C; H NMR (600 MHz, CDCl
3
) δ 5.29 (br
same volume of ethanol alone. 1α,25(OH)
2
D was always assayed at the
3
d, J = 7.6 Hz, 1H), 3.66 (s, 3H), 3.78 (m, 1H), 2.35 (ddd, 1H, J = 15.8,
0.3, 5.5 Hz), 2.22 (ddd, 1H, J = 15.1, 9.6, 6.2 Hz), 1.95 (s, 1H), 0.93
same time as a positive control. The cells were incubated at 37 °C under
1
5% CO in air for 4 days. The percentage of differentiated cells was
2
1
3
(
s, 3H), 0.91 (d, 3H, J = 6.2 Hz), 0.64 (s, 3H). C NMR (150 MHz,
determined by nitro-blue tetrazolium (NBT) reduction assay. Cells were
incubated at 37 °C for 20 min in RPMI-1640 (5% FBS) and an equal
volume of phosphate-buffered saline (PBS) containing NBT (0.2%) and
12-O-tetradecanoylphorbol 13-acetate (TPA; 200 ng/mL). The percen-
tage of cells containing blue-black formazan was determined in a
minimum of 200 cells.
CDCl
3
) δ 174.78, 169.12, 56.66, 56.06, 51.49, 49.44, 42.75, 42.30,
4
2
0.58, 40.22, 35.78, 35.38, 34.55, 33.70, 31.08, 31.01, 28.20, 27.98,
6.97, 26.41, 24.18, 23.64, 23.53, 20.81, 18.27, 12.04. HRMS-ESI (m/
+
z): [M+H] calcd for C27
H
46NO 432.3472; found 432.3461.
3
4
.2.16. Compound 20
4
.4. Luciferase reporter assay
Methanesulfonyl chloride (300 μl) was added to a solution of 18
(
69 mg, 0.16 mmol) in dichloromethane (1.5 ml) and pyridine (0.5 μl).
COS-7 cells were grown in Dulbecco’s modified Eagle’s medium
The mixture was stirred at room temperature for 17 h, poured into
water, and extracted with dichloromethane. The organic layer was
washed with water, dried over sodium sulfate, and evaporated. The
residue was chromatographed on silica gel (ethyl acetate/hexane 1:4 to
(
DMEM) supplemented with 5% fetal calf serum (FCS). Cells were
4
seeded on 24-well plates at a density of ca. 2 × 10 per well. After 24 h,
cells were transfected with a reporter plasmid containing three copies
of the mouse osteopontin VDRE (5-GGTTCAcgaGGTTCA, SPPx3-TK-
LUC), a hVDR expression plasmid (pCMX-hVDR), and the internal
control plasmid containing sea pansy luciferase expression construct
2
1
:3) to afford 20 (58 mg, 77%). Colorless prisms (ethyl acetate); mp
1
55 °C; H NMR (600 MHz, CDCl
3
) δ 4.13 (m, 1H), 3.66 (s, 3H), 3.32
(
m, 1H), 2.35 (ddd, 1H, J = 15.1, 9.6, 4.8 Hz), 2.22 (ddd,1H, J = 15.8,
(
pRL-CMV) by the lipofection method as described previously. After 4 h
9
.6, 6.2 Hz), 1.96 (dt, 1H, J = 13.8, 2.7 Hz), 0.93 (s, 3H), 0.91 (d, 3H,
13
incubation, the medium was replaced with fresh DMEM containing 1%
FCS (HyClone, UT). The next day, the cells were treated with the in-
J = 6.9 Hz), 0.64 (s, 3H). C NMR (150 MHz, CDCl
3
) δ 174.76, 56.48,
5
3
2
5.96, 54.12, 51.49, 42.72, 42.57, 42.25, 40.51, 40.09, 35.86, 35.74,
dicated concentration of 1α,25(OH)
2
D (1) or lithocholic acid deriva-
3
5.35, 35.07, 34.40, 31.04, 30.98, 29.35, 28.16, 26.92, 26.31, 24.15,
tives, or ethanol vehicle, and cultured for 24 h. Cells in each well were
harvested with cell lysis buffer, and the luciferase activity was mea-
sured with a luciferase assay kit (Tokyo Ink, Inc., Japan) according to
the manufacturer’s instructions. Transactivation measured in terms of
luciferase activity was normalized with the internal control. All ex-
periments were done in triplicate.
+
3.45, 20.80, 18.26, 12.03. HRMS-ESI (m/z): [M+Na] calcd for
C
26
H
45NNaO S 490.2967; found 490.2951.
4
4.2.17. Compound 8
2
M aqueous hydroxide (4 ml) was added to a solution of 19 (19 mg,
0
.045 mmol) in methanol (12 ml). The mixture was stirred at room
4
.5. Competitive binding assay
temperature for 3.5 h, then poured into 2 M hydrochloric acid, and
extracted with dichloromethane. The organic layer was washed with
water, dried over sodium sulfate, and evaporated. The residue was re-
crystallized from ethyl acetate to yield compound 8 (13 mg, 69%).
8
Binding affinity was investigated according to our previous report.
Bovine thymus VDR purchased from Yamasa Biochemical (Choshi, Chiba,
Japan) was dissolved in 0.05 M phosphate buffer (pH 7.4) containing
1
Colorless prisms (ethyl acetate); mp 268 °C; H NMR (400 MHz, CD
3
OD)
0
.3 M KCl and 5 mM dithiothreitol just before use. The receptor solution
δ 4.98 (br s, 1H), 3.70–3.63 (m, 1H), 3.32 (s, 3H), 2.33 (ddd, J = 15.2,
3
(
500 μl) in an assay tube was incubated with 0.072 nM [ H]1α,25-
1
8
0
5
2
0, 5.2 Hz, 1H), 2.20 (ddd, J = 15.2, 8.4, 6.8 Hz, 1H), 2.04 (dt, J = 12,
.4 Hz, 1H), 1.99–1.02 (m, 25H), 0.98 (s, 3H), 0.96 (d, J = 6.4 Hz, 3H),
(OH)
2 3
D , together with graded amounts of each vitamin D analog or
3
1
3
vehicle for 19 h at 4 °C. Bound and free [ H] 1α,25(OH)
2
D (1) were
3
.71 (s, 3H) . C-NMR (126 MHz, CD OD) δ 179.0, 172.5, 58.2, 57.7,
3
separated on dextran-coated charcoal for 20 min at 4 °C. The assay tubes
were centrifuged at 1000g for 10 min. The radioactivity of the super-
natant was counted. These experiments were done in duplicate.
1.1, 50.5, 44.1, 42.0, 41.7, 37.4, 37.2, 36.9, 35.8, 34.4, 32.7, 32.6,
9.4, 28.6, 28.4, 27.8, 25.4, 24.2, 22.9, 22.1, 18.9, 12.6. HRMS-ESI (m/
+
z): [M+H] calcd for C26
H
44NO 418.3316. Found 418.3308.
3
4
.6. X-ray crystallographic analysis
4
.2.18. Compound 9
2
M aqueous hydroxide (7 ml) was added to a solution of compound
Crystals of VDR complexes were prepared according to the method
9
2
0 (24 mg, 0.050 mmol) in methanol (21 ml). The mixture was stirred
of Vanhooke et al. with some modifications. The rat VDR LBD (re-
sidues 116–423, Δ165–211) was cloned as an N-terminal His6-tagged
fusion protein into the pET14b expression vector and overproduced in
Escherichia coli C41. The cells were grown at 37 °C in LB medium (in-
cluding ampicillin 100 mg/L) and subsequently induced for 6 h with
15 µM isopropyl-β-D-thiogalactopyranoside (IPTG) at 23 °C. The pur-
ification procedure included affinity chromatography on a Ni-NTA
column, followed by dialysis and cation-exchange chromatography (SP-
Sepharose). After tag removal by thrombin digestion, protease was re-
moved by filtration through a HiTrap benzamidine column and the
protein was further purified by gel filtration on a Super-dex200 column.
The purity and homogeneity of the rVDR LBD were assessed by SDS-
PAGE.
at room temperature for 2 h, poured into 2 M hydrochloric acid, and
extracted with dichloromethane. The organic layer was washed with
water, dried over sodium sulfate, and evaporated. The residue was re-
crystallized from ethyl acetate to afford 9 (8 mg, 37%). Colorless prisms
1
(
ethyl acetate); mp 160–161.5 °C; H NMR (600 MHz, CDCl
3
,) δ 4.13
(
m, 1H), 3.332 (m, 1H), 2.35 (ddd,1H, J = 15.1, 9.6, 4.8 Hz), 2.22
ddd, 1H, J = 15.8, 9.6, 6.2 Hz), 1.96 (dt, 1H, J = 13.8, 2.7 Hz), 0.93 (s,
(
1
3
3
H), 0.91 (d, 3H, J = 6.9 Hz), 0.64 (s, 3H). C NMR (150 MHz,
CDCl
3
x) δ 178.51, 56.51, 55.99, 54.09, 42.75, 42.58, 42.25, 40.52,
4
2
0.11, 35.88, 35.75, 35.34, 35.06, 34.40, 30.75, 30.71, 29.36, 28.17,
6.93, 26.32, 24.17, 23.46, 20.81, 18.25, 12.04. HRMS-ESI (m/z): [M
+
+
Na] calcd for C25
H
43NNaO S 476.2810. Found 476.2823.
4
7

H. Masuno, et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
Purified rVDR LBD solution was concentrated to about 0.75 mg/mL
Foundation, the Cosmetology Research Foundation, and the Tokyo
by ultrafiltration. To an aliquot (800 µl) of the protein solution was
added a ligand (ca. 10 equiv), then the solution was further con-
centrated to about 1/8, and a solution (25 mM Tris-HCl, pH 8.0; 50 mM
Biochemical Research Foundation.
Appendix A. Supplementary data
NaCl; 10 mM DTT; 0.02% NaN
3
)
of coactivator peptide
(
H
2
N-KNHPMLMNLLKDN-CONH
2
) derived from DRIP205 was added.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmc.2019.07.003.
This solution of VDR/ligand/peptide was allowed to crystallize by the
vapor diffusion method using a series of precipitant solutions con-
taining 0.1 M MOPS–NaOH (pH 7.0), 0.1–0.4 M sodium formate,
References
1
2–22% (w/v) PEG4000, and 5% (v/v) ethylene glycol. Droplets for
crystallization were prepared by mixing 2 μl of complex solution and
1. Feldman D, Glorieux FH, Pike JW, eds. Vitamin D. 2nd ed. Elsevier Academic Press;
2
005.
1
μl precipitant solution, and droplets were equilibrated against 500 μl
2
.
Makishima M, Lu TT, Xie W, et al. Vitamin D receptor as an intestinal bile acid sensor.
precipitant solution at 20 °C.
Science (80-). 2002;296:5571.
Prior to diffraction data collection, crystals were soaked in a cryo-
protectant solution containing 0.1 M MOPS-NaOH (pH 7.0), 0.1–0.4 M
sodium formate, 15–20% PEG4000, and 17–20% ethylene glycol.
Diffraction data sets were collected at 100 K in a stream of nitrogen gas
at beamline BL-6A of KEK-PF (Tsukuba, Japan). Reflections were re-
corded with an oscillation range per image of 1.0°. Diffraction data
were indexed, integrated, and scaled using the program HKL2000 (HKL
Research Inc., USA). The structures were solved by molecular replace-
3. Nehring JA, Zierold C, DeLuca HF. Lithocholic acid can carry out in vivo functions of
vitamin D. Proc Natl Acad Sci USA. 2007;104:10006–10009.
4
.
Adachi R, Honma Y, Masuno H, et al. Selective activation of vitamin D receptor by
lithocholic acid acetate, a bile acid derivative. J Lipid Res. 2005;46:46–57.
5. Ishizawa M, Matsunawa M, Adachi R, et al. Lithocholic acid derivatives act as se-
lective vitamin D receptor modulators without inducing hypercalcemia. J Lipid Res.
2
008;49:763–772.
6
.
Masuno H, Ikura T, Morizono D, et al. Crystal structures of complexes of vitamin D
receptor ligand-binding domain with lithocholic acid derivatives. J Lipid Res.
2013;54:2206–2213.
1
0
7. Mangelsdorf DJ, Koeffler HP, Donaldson CA, Pike JW, Haussler MR. 1,25-
Dihydroxyvitamin D3-induced differentiation in a human promyelocytic leukemia
cell line (HL-60): receptor-mediated maturation to macrophage-like cells. J Cell Biol.
1984;98:391–398.
ment with the program Phaser in the Phenix using rat VDR LBD co-
ordinates (PDB code: 2ZLC), and finalized sets of atomic coordinates
were obtained after iterative rounds of model modification with the
1
1
12
8. Fujii S, Masuno H, Taoda Y, et al. Boron cluster-based development of potent non-
secosteroidal vitamin D receptor ligands: direct observation of hydrophobic inter-
action between protein surface and carborane. J Am Chem Soc.
2011;133:20933–20941.
program COOT and refinement with REFMAC . The coordinates and
structure factors have been deposited to Protein Data Bank (Entry ID:
6
K5O).
9
.
Vanhooke JL, Benning MM, Bauer CB, Pike JW, DeLuca HF. Molecular structure of
the rat vitamin D receptor ligand binding domain complexed with 2-carbon-sub-
stituted vitamin D 3 hormone analogues and a LXXLL-containing coactivator peptide.
Biochemistry. 2004;43:4101–4110.
Acknowledgments
1
1
1
0. Adams PD, Afonine PV, Bunkoczi G, et al. PHENIX: a comprehensive python-based
system for macromolecular structure solution. Acta Crystallogr D. 2010;66:213–221.
1. Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of coot. Acta
Crystallogr D. 2010;66:486–501.
This work was partly supported by JSPS KAKENHI Grant No.
1
5K08018 (to HM), 16K08318 (to AT) 17H03997 (to SF), JSPS Core-to-
Core Program, A. Advanced Research Networks, and MEXT Platform for
Drug Discovery, Informatics, and Structural Life Science, Japan. A part
of this research is based on the Cooperative Research Project of
Research Center for Biomedical Engineering. A.T. thanks The Naito
2. Murshudov GN, Skubák P, Lebede AA, et al. (2011) REFMAC5 for the refinement of
macromolecular crystal structures. Acta Crystallogr D. 2011;67:355–367.
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