Structural development of non-secosteroidal vitamin D receptor (VDR) ligands without any asymmetric carbon

Article abstract of DOI:10.1016/j.bmc.2018.11.008

Non-secosteroidal VDR ligands without any assymmetric carbon were designed and synthesized based on the structure of the previously reported non-secosteroidal VDR agonist LG190178. The VDR-agonistic activity of all synthesized compounds was evaluated, and

Full text of DOI:10.1016/j.bmc.2018.11.008

Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structural development of non-secosteroidal vitamin D receptor (VDR)
ligands without any asymmetric carbon
⁎
Takashi Misawaa, , Genichiro Tsujia, Takeo Takahashia, Eiji Ochiaib, Ken-ichiro Takagib,
⁎
Kyohei Horieb, Shinji Kakudab, Midori Takimoto-Kamimurab, Masaaki Kuriharaa,c,
,
⁎
Yosuke Demizua,
a Division of Organic Chemistry, National Institute of Health Sciences, 3-25-26 Tonomachi, Kawasaki-ku, Kawasaki-shi, Kanagawa 210-9501, Japan
b Teijin Institute for Bio-Medical Research, Teijin Pharma Ltd., Tokyo 191-8512, Japan
c Department of Pharmaceutical Sciences, International University of Health and Welfare, 2600-1 Kitakanemaru, Otawara City, Tochigi 324-8501, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
Non-secosteroidal VDR ligands without any assymmetric carbon were designed and synthesized based on the
structure of the previously reported non-secosteroidal VDR agonist LG190178. The VDR-agonistic activity of all
synthesized compounds was evaluated, and 7b emerged as a potent agonist activity with an EC50 value of
9.26 nM. Moreover, a docking simulation analysis was also performed to determine the binding mode of 7b with
VDR-LBD.
Vitamin D receptor
Non-secosteroid
Asymmetric carbon
Docking study
1. Introduction
potent non-secosteroidal VDR ligand14 and the broad structural devel-
opment and the subsequent biological studies of LG190178 were per-
The vitamin D receptor (VDR) belongs to the family of nuclear re-
ceptors (NR) and is involved in several biological functions, such as
calcium and phosphorus homeostasis.1,2 The binding of 1α,25-dihy-
droxyvitamin D3 (1 α,25(OH)2D3: VD3), which is an endogenous VDR
ligand, to the ligand binding domain (LBD) of VDR can induce con-
formational changes as well as the formation of a homo- or hetero-
dimers with the retinoid X receptors (RXRs), thus subsequently pro-
moting the target gene transcription.3,4 The binding mode between 1
α,25(OH)2D3 and VDR-LBD has been previously revealed by X-ray
analysis.5 This study indicated that 1 α,25(OH)2D3 formed hydrogen
bonds with six amino acid residues, i.e., Tyr143, Ser233, Arg274,
Ser278, His305, and His397, resulting in the efficient transcriptional
activation of VDR. It has been also reported that the VDR ligands might
constitute promising therapeutic candidates for the treatment of several
diseases, such as cancers and autoimmune diseases.6–8 Up to date,
several VDR ligands have been described, as shown in Fig 1.9–11
formed.15–18 Previously, we demonstrated that YR301, which is an (2S,
2′R)-analog of LG190178, displayed the most potent VDR agonistic
activity among other isomers. Furthermore, the X-ray crystallography
structure of the complex between rat VDR-LBD and YR301 was solved
and revealed that the four amino acid residues (Ser233, Arg270,
His301, and His393) played an important role to form hydrogen bonds
with YR301, hence promoting its VDR transcriptional activity.19
Moreover, a structure-activity relationship (SAR) analysis was per-
formed based on the YR301 skeleton and demonstrated that YR335,
which features a longer alkyl chain than YR301 to form the hydrogen
bonds with Tyr143 or Ser274, exhibited similar VDR-agonistic activity
as that of YR301 (Fig. 2).20 However, reported VDR ligands such as
YR301 and YR335 contain two chiral centers at the side chains that are
necessary to form hydrogen bonds with the LBD of VDR. Therefore,
possible structural variations based on YR301 skeleton are limited and
the great efforts are generally required for the synthesis of such VDR
ligands. Previously, the VDR ligands without any chiral carbon have
been reported.21,22 We also reported that YR311 and YR313, which
have a chiral center only at the 2′-position, exhibited potent VDR
agonistic activity via the hydrogen bond formation with six amino acid
residues in analogy to 1 α,25(OH)2D3, indicating that the presence of
chiral centers at the side chain is not absolutely necessary.20 Based on
Although the significant therapeutic potential of some
1
α,25(OH)2D3 analogs has been reported,12,13 their application was
limited due to their secosteroid skeleton, which makes them chemically
unstable against the visible light, thus impending them to fully exert
their therapeutic action. Therefore, the VDR ligands based on a non-
secosteroidal skeleton are urgently needed. LG190178 was the first
⁎ Corresponding authors at: Division of Organic Chemistry, National Institute of Health Sciences, 3-25-26 Tonomachi, Kawasaki-ku, Kawasaki-shi, Kanagawa 210-
9501, Japan (M. Kurihara).
E-mail addresses: misawa@nihs.go.jp (T. Misawa), mkurihara@iuhw.ac.jp (M. Kurihara), demizu@nihs.go.jp (Y. Demizu).
https://doi.org/10.1016/j.bmc.2018.11.008
Received 1 October 2018; Received in revised form 5 November 2018; Accepted 8 November 2018
0968-0896/©2018ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:Misawa,T.,Bioorganic&MedicinalChemistry,https://doi.org/10.1016/j.bmc.2018.11.008

T. Misawa et al.
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Fig. 1. Chemical structures of 1 α,25(OH)2D3 and other synthesized VDR ligands.
Fig. 2. Chemical structures of reported YR301 derivatives and designed compounds described in this study.
this knowledge, we herein designed and synthesized the achiral non-
secosteroidal VDR ligands to investigate the effects of the lack of a
chirality at 2′-position on the VDR transcriptional activity.
respectively. Furthermore, the phenol moiety of 5b was alkylated with
diethyl bromomalonate and the resulting ester 10 was then reduced to
target alcohol 11 using LiAlH4 (Scheme 1). Next, one of the two hy-
droxy groups of compound 1 was alkylated using 16,25 to give com-
pounds 12, whose free phenoxy group was further reacted either with
(S)-glycidol, or 1726 followed by deprotection of the benzyl group by
using Pd(OH)2/C under a H2 atmosphere to afford target compounds 13
and 14, respectively (Scheme 2).
2. Results and discussion
A series of YR301 derivatives with or without a chiral center were
designed and synthesized according to the synthetic routes were shown
in Schemes 1 and 2. Briefly, a one of the two hydroxy functionalities of
diphenylpentane compound 1, which was prepared as previously re-
ported,23 was protected with a benzyl group. Then, the remaining free
hydroxy group of 2 was alkylated using either isobutylene oxide or
ethyl bromoacetate followed by treatment with an ethyl Grignard re-
agent, to afford 4a and 4b, respectively. After deprotection of the
benzyl group using Pd/(OH)2 under a H2 atmosphere, the hydroxy
group of each compound was reacted with either (S)-glycidol or com-
pound 924 to afford the target molecules 6a–6b and 7a–7b,
We then evaluated the VDR-mediated transcriptional activity of the
synthesized compounds by determining both the EC50 and IC50 values
as well as their efficacies of the synthesized compounds using PGL3-hOs
(osteocalcin), pCDNA-hVDR, pCDNA-hRXR, and phRL-TK as previously
reported.20 HOS cells were incubated with the synthesized compounds
either in the absence or presence of 1α,25(OH)2D3 (3 nM). After in-
cubation, the cells were assayed for reporter gene activities. The acti-
vation ratio obtained with 3-nM 1,25α(OH)2D3 was defined as 100%.
The VDR-agonistic activity of the synthesized compounds was shown in
2

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Scheme 1. Reagents and conditions: (a) BnBr, K2CO3, acetone, rt.; (b) Ethyl bromoacetate, K2CO3, DMF, rt.; (c) Isobutylene oxide, NaH, DMF, rt. to 55 °C; (d)
EtMgBr, THF, 0 °C to rt.; (e) Pd(OH)2/C, H2 gas, MeOH, rt.; (f) (S)-glycidol, NaH, DMF, rt. to 60 °C; (g) 9, NaH, DMF, rt. to 100 °C; (h) 80% AcOH, rt.; (i) Diethyl
bromomalonate, K2CO3, DMF, 60 °C; (j) LiAlH4, THF, 0 °C to rt.
Table 1. At first, we examined the effects of a chiral center at the 2′-
position on the VDR agonistic activity. As a result, YR301 showed po-
tent a VDR agonistic activity similar to that of 1 α,25(OH)2D3, (1
α,25(OH)2D3, EC50 = 0.011 nM; YR301, EC50 = 0.040 nM), while 6a,
6b, and 13 exhibited the weak and moderate VDR-agonistic activities
(VDR-agonistic activity: 6a, EC50 = 163 nM; 6b, EC50 = 18 nM; 13,
EC50 = 41.7 nM), suggesting that the chirality at the 2′-position was not
absolutely necessary for VDR activation. Moreover, compounds 7a, 7b,
and 11, which have two ethyl groups, exhibited the higher VDR-
agonistic activity than other compounds (VDR-agonistic activity: 7a,
EC50 = 76.5 nM; 7b, EC50 = 9.26 nM; 11; EC50 = 17.2 nM), indicating
that the LBD of VDRs has a cavity to accommodate the ethyl group
around the 2′-position of YR compounds, thus a higher hydrophobicity
compared to other compounds could be responsible for the enhance-
ment of their VDR-agonistic activity. Furthermore, the shift of the hy-
droxy group from the 2′- to 3′-position decreased their VDR-agonistic
activity of the resulting compounds compared to YR301, indicating that
the hydroxy group at the 2′-position plays a pivotal role in formation of
Scheme 2. Reagents and conditions: (a) 16, K2CO3, DMF, 100 °C; (b) (S)-glycidol, NaH, DMF, rt. to 60 °C; (c) 17, K2CO3, DMF, 100 °C; (d) Pd(OH)2/C, H2 gas, MeOH,
rt.
3

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Table 1
development targeting the hydrogen bond formation with Tyr143
VDR-agonistic activity of the synthesized compounds.
might guide the development of more potent achiral VDR ligands.
Comp.
VDR-agonistic activity EC50 (nM) (Max efficacy %)a
4. Experimental
1 α,25(OH)2D3
0.011
YR301
6a
0.040
4.1. Chemistry
163 (67)
18 (90)
6b
4.1.1. General
7a
76.5 (91)
9.26 (1 0 0)
17.2 (1 0 0)
41.7 (1 0 0)
76.9 (91)
7b
All chemical reagents and solvents were obtained from Sigma-
Aldrich, Tokyo Chemical Industry Co. LTD. and Fujifilm Wako Pure
Chemical Co. LTD. and were used without purification. Analytical TLC
was carried out using Merck silica gel 60 F254 pre-coated plates and
visualized using a 254 nm UV lamp, phosphomolybdic acid, p-ani-
saldehyde or ninhydrin staining. Column chromatography was per-
formed with silica gel (spherical, neutral) purchased from Kanto
Chemical. 1H and 13C NMR spectra were obtained on a Varian AS 400
Mercury spectrometer (400 MHz for 1H and 100 MHz for 13C). Chemical
shifts are expressed as ppm downfield from a solvent residual peak or
internal standard tetramethylsilane (TMS). FT-IR spectra were recorded
on a JASCO FT/IR-4100 spectrometer at 1 cm−1 resolution, with an
average of 128 scans used for the solution (CDCl3) method and a
0.1 mm path length for NaCl cells. Mass spectra were recorded with
SHIMADZU LCMS-IT-TOF spectrometer.
11
13
14
a
Max efficacy treated with 1 α,25(OH)2D3 is defined as 100%.
the hydrogen bond with the VDR-LBD. Although the VDR agonistic
activities of the synthesized compounds were lowered compared to that
of YR301, the developed achiral non-secosteroidal VDR ligand 7b no-
tably displayed an EC50 value = 9.26 nM.
Finally, we performed a computational docking study to investigate
the binding mode of 7b to VDR, since it can be useful to explain how
asymmetric VDR ligands exert their VDR-agonistic activity. The X-ray
co-crystal structure of VDR bound to YR301 was obtained from the
Protein Data Bank (PDB: 2ZFX)27 and used in this docking study. The
docking study of 7b was conducted via a conformational search analysis
using MOE. As shown in Fig. 3, YR301 formed six hydrogen bonds with
four amino acid residues, specifically, the 2′-OH was H-bonded to
His301 and His393, the 2-OH was to Arg270 and Ser233, and the 1-OH
was directly H-bonded to Arg270, while being bound to Arg270 via a
water molecule-mediated H-bond. On the other hand, ligand 7b formed
hydrogen bonds with 5 amino acid residues (Ser233, Arg270, Ser274,
His301, and His393) occurring in the LBD of VDR. Compound 7b
formed the hydrogen bonds with His301 and His 393 analogously to
YR301 did, while the 2-OH of 7b formed the three hydrogen bonds with
three amino acids, Ser233, Arg270, and Ser274. Therefore, the less
hydrogen bonds were assumed to be responsible for the lower VDR-
agonistic activity observed for 7b compared to that of YR301.
4.1.2. 4-(3-(4-(benzyloxy)-3-methylphenyl)pentan-3-yl)-2-methylphenol
(2)
To a solution of 1 (580 mg, 2.4 mmol) in acetone (5 mL) was added
K2CO3 (279 mg, 2.20 mmol), followed by benzyl bromide (238 μL,
2.00 mmol). After being stirred at room temperature for 7 h, the reac-
tion mixture was quenched with water, and extracted with EtOAc three
times. The combined organic extracts were dried over Na2SO4, filtered,
concentrated under reduced pressure. The residue was purified by silica
gel column chromatography (Hexanes/EtOAc = 20:1 to 10:1) to give 2
as a colorless oil (346 mg, 45%).
1H NMR (CDCl3) δ7.62–7.31 (m, 5H), 6.97–6.87 (m, 4H), 6.78 (d,
J = 8.4 Hz, 1H), 6.65 (d, J = 8.4 Hz, 1H), 5.04 (s, 2H), 4.58 (s, 1H),
2.23 (s, 3H), 2.20 (s, 3H), 2.07–2.00 (m, 4H), 0.61 (t, J = 7.2 Hz, 6H);
13C NMR (CDCl3) δ 154.6, 151.3, 141.2, 140.9, 137.7, 130.6, 128.4,
127.6, 127.2, 126.7, 126.1, 125.8, 122.4, 114.0, 110.3, 69.8, 48.4,
3. Conclusion
29.3, 16.6, 16.1, 8.46; ESI-MS (positive) : m/z 397.3 [M+Na]+
.
In this study, we designed and synthesized a set of the achiral non-
secosteroidal VDR ligands based on the structure of LG190178, and
evaluated their VDR-agonistic activity by reporter luciferase assay.
Moreover, we performed a docking study analysis to reveal the binding
4.1.3. Ethyl
2-(4-(3-(4-(benzyloxy)-3-methylphenyl)pentan-3-yl)-2-
methylphenoxy)acetate (3)
To a suspension of 2 (196 mg, 0.52 mmol) and K2CO3 (109 mg,
0.80 mmol) in DMF (5 mL) was added ethyl bromoacetate (86 μL,
0.78 mmol), and the reaction mixture was stirred at 50 °C for 19 h. After
cooling to room temperature, the reaction mixture was poured into 1 M
HCl, and extracted with Et2O three times. The combined organic ex-
tracts were dried over Na2SO4, filtered, concentrated under reduced
pressure. The residue was purified by silica gel column chromatography
(Hexanes/EtOAc = 10 : 1) to give 3 as a colorless oil (214 mg, 89%).
1H NMR (CDCl3) δ 7.45–7.28 (m, 5H), 6.96–6.91 (m, 4H), 6.76 (d,
mode of the synthesized compounds to VDR-LBD. As
a result,
Compound 7b was successfully identified as the optimal achiral non-
secosteroidal VDR ligand among these compounds. Moreover, the
docking study analysis revealed that the number of hydrogen bonds of
7b with VDR was reduced compared to that of 1 α,25(OH)2D3 with
VDR-LBD, hence leading to a lower VDR agonistic activity of 7b com-
pared to that of 1 α,25(OH)2D3. Despite the decrease in VDR-agonistic
activity observed for this series of compounds, further structural
Ser274
His301
Tyr143
Ser233
His393
Arg270
Fig. 3. Computational model of the Vitamin D receptor ligand binding domain with YR301 and 7b. YR301 and 7b are colored in orange and grey, respectively.
4

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J = 8.4 Hz, 1H), 6.57 (d, J = 8.8 Hz, 1H), 5.03 (s, 2H), 4.59 (s, 2H),
4.59 (q, J = 14.4 Hz, 2H), 2.23 (s, 3H), 2.22 (s, 3H), 2.02 (q,
J = 14.4 Hz, 4H), 1.27 (t, J = 7.2 Hz, 3H), 0.59 (t, J = 7.6 Hz, 6H)
4.1.7.1. Yield 47%, colorless oil. IR (neat): ν = 3392, 2966, 2936, 2878,
2370, 2363, 2332, 1504, 1457, 1275, 1259, 1135, 1046 cm−1 1H NMR
(CDCl3) δ 6.97–6.89 (m, 4H), 6.71 (d, J = 3.6 Hz, 1H), 6.69 (d,
J = 3.2 Hz, 2H), 4.15–4.10 (m, 1H), 4.04 (d, J = 6.0 Hz, 2H),
3.86–3.76 (m, 2H), 3.80 (s, 2H), 2.60 (d, J = 5.2 Hz, 1H), 2.18 (s,
3H), 2.17 (s, 3H), 2.08 (br. s, 1H), 2.02 (q, J = 13.2 Hz, 4H), 1.70 (br. s,
1H), 1.66 (q, J = 14.8 Hz, 4H), 0.93 (t, J = 7.6 Hz, 6H), 0.59 (t,
J = 7.2 Hz, 6H); 13C NMR (CDCl3) δ 154.3, 154.0, 141.4, 140.9,
130.6, 130.5, 125.5, 125.3, 110.0, 109.9, 74.1, 72.3, 70.4, 69.2, 63.9,
48.4, 29.3, 28.6, 16.6, 8.4, 7.8; [HR-ESI(+)] m/z calcd for C28H42O5 [M
+Na]+ 481.2924; found 481.2957.
4.1.4. 1-(4-(3-(4-(Benzyloxy)-3-methylphenyl)pentan-3-yl)-2-
methylphenoxy)-2-methylpropan-2-ol (4a)
To a stirred solution of 2 (57 mg, 0.15 mmol) in DMF (3 mL) was
added NaH (60% oil suspension, 9.1 mg, 0.23 mmol) under argon at-
mosphere. After being stirred at room temperature for 30 min, the re-
action mixture was added isobutylene oxide (270 μL, 2.92 mmol). The
reaction mixture was stirred at 55 °C for 15 h, then quenched with
water and concentrated under reduced pressure. The residue was pur-
ified by silica gel column chromatography (Hexanes/EtOAc = 4:1) to
give 4a as a colorless oil (56 mg, 86%).
4.1.8. 2-((4-(3-(4-(2-Hydroxy-2-methylpropoxy)-3-methylphenyl)pentan-
3-yl)-2-methylphenoxy)methyl)propane-1,3-diol (7a)
1H NMR (CDCl3) δ 7.52–7.31 (m, 5H), 6.99–6.89 (m, 4H), 6.78 (d,
J = 8.0 Hz, 1H), 6.70 (d, J = 8.0 Hz, 1H), 3.78 (s, 2H), 3.04 (s, 2H),
2.23 (s, 3H), 2.19 (s, 3H), 2.08–1.99 (m, 4H), 1.29 (s, 3H), 1.18 (s, 3H),
0.87 (br. s, 1H), 0.59 (t, J = 7.2 Hz, 6H); 13C NMR (CDCl3) δ 154.6,
154.2, 141.2, 140.9, 137.7, 130.6, 130.5, 128.4, 127.6, 127.1, 126.3,
126.2, 126.1, 125.8, 125.4, 110.3, 110.0, 75.8, 70.2, 69.8, 48.4, 29.3,
To a solution of 5a (71 mg, 0.20 mmol) in DMF (3 mL) was added
NaH (60% oil suspension, 12 mg, 0.30 mmol) under argon atmosphere.
After being stirred at room temperature for 30 min, the reaction mix-
ture was added 9 (45 mg, 0.20 mmol). The reaction mixture was stirred
at 100 °C for 15 h, then cooled to room temperature, quenched with
water and concentrated under reduced pressure. The residue was pur-
ified by silica gel column chromatography (Hexanes/EtOAc = 10:1) to
give a colorless oil.
26.2, 16.7, 16.6, 8.47; ESI-MS (positive):m/z 469.4 [M+Na]+
.
4.1.5. 3-((4-(3-(4-(Benzyloxy)-3-methylphenyl)pentan-3-yl)-2-
The obtained intermediate (78 mg, 0.16 mmol) was dissolved in
80% aqueous AcOH (2 mL), and the reaction mixture was stirred at
room temperature for 3 h. The reaction mixture was concentrated under
reduced pressure, and the residue was purified by silica gel column
chromatography (Hexanes/EtOAc = 2:1–0:1) to give 7a as a colorless
oil (36 mg, 50%). The obtained product was further purified by HPLC
(Hexanes/2-propanol = 3:1).
methylphenoxy)methyl)pentan-3-ol (4b)
To a stirred solution of 3 (214 mg, 0.46 mmol) in THF (3.5 mL) was
added EtMgBr (4 M in THF, 4.0 mL, 1.60 mmol) at 0 °C under argon
atmosphere. After completion of the addition, the reaction mixture was
stirred at room temperature for 30 min, then quenched with 1 M HCl
and extracted with Et2O three times. The combined organic extracts
were dried over Na2SO4, filtered, concentrated under reduced pressure.
The residue was purified by silica gel column chromatography
(Hexanes/EtOAc = 10:1) to give 4b as a colorless oil (178 mg, 80%).
1H NMR (CDCl3) δ 7.45–7.30 (m, 5H), 6.97–6.91 (m, 4H), 6.77 (d,
J = 8.0 Hz, 1H), 6.69 (d, J = 8.4 Hz, 1H), 5.03 (s, 2H), 3.80 (s, 2H),
2.22 (s, 3H), 2.18 (s, 3H), 2.07 (s, 1H), 2.03 (q, J = 14.0 Hz, 4H),
1.69–1.63 (m, 4H), 0.93 (t, J = 7.6 Hz, 6H), 0.60 (t, J = 7.2 Hz, 6H).
IR (neat): ν = 3852, 3799, 3749, 3734, 3710, 3688, 3648, 3627,
3566, 3360, 3005, 2988, 2362, 2355, 2340, 1716, 1505, 1472, 1260,
1136, 1014, 897 cm−1 1H NMR (CDCl3) δ 6.98–6.90 (m, 4H), 6.72 (d,
J = 8.4 Hz, 1H), 6.68 (d, J = 8.4 Hz, 1H), 4.10 (d, J = 5.6 Hz, 2H), 3.96
(d, J = 5.6 Hz, 4H), 3.77 (s, 2H), 2.29 (br. s, 1H), 2.27–2.23 (m, 1H),
2.20 (s, 3H), 2.18 (br. s, 1H), 2.15 (s, 3H), 2.02 (q, J = 14.8 Hz, 4H),
1.35 (s, 6H), 1.25 (br. s, 1H), 0.60 (t, J = 7.2 Hz, 4H); 13C NMR (CDCl3)
δ 154.6, 154.5, 154.5, 141.4, 130.9, 125.8, 125.6, 110.3, 110.0, 76.1,
70.6, 67.4, 63.7, 48.8, 43.0, 29.6, 26.6, 17.0, 16.9, 8.8; ESI-MS (posi-
4.1.6. (S)-3-(4-(3-(4-(2-Hydroxy-2-methylpropoxy)-3-methylphenyl)
pentan-3-yl)-2-methylphenoxy)propane-1,2-diol (6a)
tive):m/z 467.2 [M+Na]+
.
To a solution of 4a (99 mg, 0.24 mmol) in MeOH (5 mL) was added Pd
(OH)2 (20 mg), and stirred at room temperature under H2 gas atmosphere
for 1.5 h. The reaction mixture was filtered through Celite to remove a
catalyst, and the filtrate was concentrated under reduced pressure to a
colorless oil 5a, which was used in next reaction without further pur-
ification. To a solution of 5a (78 mg, 0.23 mmol) in DMF (3 mL) was
added NaH (60% oil suspension, 15 mg, 0.38 mmol) under argon atmo-
sphere. After being stirred at room temperature for 30 min, the reaction
mixture was added (S)-glycidol (30 μL, 0.45 mmol). The reaction mixture
was stirred at 60 °C for 16 h, then cooled to room temperature, quenched
with water and concentrated under reduced pressure. The residue was
purified by silica gel column chromatography (Hexanes/EtOAc = 3:1 to
1:1) to give 6a as a colorless oil (23 mg, 22%). The obtained product was
further purified by HPLC (Hexanes/2-propanol = 4:1).
4.1.9. 2-((4-(3-(4-(2-Ethyl-2-hydroxybutoxy)-3-methylphenyl)pentan-3-
yl)-2-methylphenoxy)methyl)propane-1,3-diol (7b)
7b was prepared by a similar procedure for synthesis of 7a de-
scribed above. The intermediate (colorless oil) was obtained by pur-
ification with silica gel column chromatography (Hexanes/
EtOAc = 4:1). 7b was obtained by purification with silica gel column
chromatography (Hexanes/EtOAc = 2:1 to 0:1).
4.1.9.1. Yield 30%, colorless oil. The obtained product was further
purified by HPLC (Hexanes/2-propanol = 3:1).
IR (neat):ν = 3648, 3390, 3004, 2988, 2372, 2361, 2338, 1505,
1457, 1275, 1260, 1136, 1041, 896, 804 cm−1 1H NMR (CDCl3) δ
6.97–6.89 (m, 4H), 6.72 (d, J = 8.4 Hz, 1H), 6.69 (d, J = 8.4 Hz, 1H),
5.74 (br. s, 1H), 4.09 (d, J = 8.8 Hz, 2H), 3.96 (t, J = 4.8 Hz, 4H), 3.80
(s, 2H), 2.28–2.24 (m, 1H), 2.18 (s, 3H), 2.15 (s, 3H), 2.13 (br. s, 1H),
2.07–1.99 (m, 4H), 1.70–1.63 (m, 4H), 1.43 (br. s, 1H), 0.93 (t,
J = 7.2 Hz, 6H), 0.59 (t, J = 7.2 Hz, 6H); 13C NMR (CDCl3) δ 154.5,
154.5, 141.3, 141.2, 130.8, 130.7, 125.7, 125.5, 110.2, 109.9, 74.3,
72.5, 67.4, 63.6, 48.6, 42.9, 29.5, 28.9, 16.9, 8.7, 8.0; [HR-ESI(+)] m/z
calcd for C297H44O5 [M+Na]+ 495.3081; found 495.3066.
IR (neat): ν = 3342, 2969, 2367, 2363, 2332, 1504, 1457, 1276,
1260, 1137, 1042 cm−1 1H NMR (CDCl3) δ 7.00–6.90 (m, 4H), 6.69 (t,
J = 9.2 Hz, 2H), 4.11 (br. s, 1H), 4.04 (t, J = 4.4 Hz, 2H), 3.82 (d,
J = 19.2 Hz, 1H), 3.77 (s, 2H), 2.55 (br. s, 1H), 2.25 (s, 1H), 2.20 (s,
3H), 2.17 (s, 3H), 1.35 (s, 6H), 1.25 (s, 4H), 1.19 (br. s, 1H), 0.59 (t,
J = 7.6 Hz, 6H); 13C NMR (CDCl3) δ 141.5, 141.0, 130.7, 130.6, 126.3,
126.2, 125.5, 125.4, 110.1, 110.0, 75.8, 70.4, 70.3, 69.2, 63.9, 48.4,
29.3, 26.2, 16.6, 8.4; ESI-MS (positive):m/z 453.3 [M+Na]+
.
4.1.10. Diethyl
2-(4-(3-(4-(2-ethyl-2-hydroxybutoxy)-3-methylphenyl)
4.1.7. (S)-3-(4-(3-(4-(2-ethyl-2-hydroxybutoxy)-3-methylphenyl)pentan-
3-yl)-2-methylphenoxy)propane-1,2-diol (6b)
pentan-3-yl)-2-methylphenoxy)malonate (10)
To a solution of 5b (80 mg, 0.17 mmol) in DMF (5 mL) was added
K2CO3 (37 mg, 0.26 mmol), followed by diethyl bromomalonate (34 μL,
0.20 mmol), and the reaction mixture was stirred at 100 °C for 22 h.
6b was prepared by a similar procedure for synthesis of 6a de-
scribed above using 5b as an intermediate.
5

T. Misawa et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
After cooling to room temperature, the reaction mixture was added 1 M
HCl, and extracted with EtOAc three times. The combined organic ex-
tracts were dried over Na2SO4, filtered, concentrated under reduced
pressure. The residue was purified by silica gel column chromatography
(Hexanes/EtOAc = 4:1) to give 10 as a colorless oil (103 mg, quant.).
1H NMR (CDCl3) δ 6.94–6.87 (m, 4H), 6.70–6.60 (m, 2H), 5.13 (s,
1H), 4.49–4.26 (m, 4H), 3.80 (s, 2H), 2.28 (s, 3H), 2.18 (s, 3H), 2.07
(br. s, 1H), 2.05–1.99 (m, 4H), 1.70–1.64 (m, 4H), 1.31–1.26 (m, 6H),
0.94 (t, J = 7.6 Hz, 6H), 0.58 (t, J = 12.4 Hz, 6H).
140.9, 131.0, 126.5, 126.4, 109.9, 70.8, 69.5, 65.5, 64.3, 48.7, 45.7,
39.4, 29.8, 15.4, 8.8; [HR-ESI(+)] m/z calcd for C27H40O5 [M+Na]+
467.2768; found 467.2812.
4.1.14. 4-(4-(3-(4-((2,2-Dimethyl-1,3-dioxan-5-yl)methoxy)-3-
methylphenyl)pentan-3-yl)-2-methylphenoxy)-2-methylbutan-2-ol (14)
To a suspension of 12 (27 mg, 0.07 mmol) and K2CO3 (17 mg,
0.12 mmol) in DMF (5 mL) was added 17 (80 mg, 0.22 mmol), and the
reaction mixture was stirred at 100 °C for 72 h. After cooling to room
temperature, the reaction mixture was poured into 1 M HCl, and ex-
tracted with Et2O three times. The combined organic extracts were
dried over Na2SO4, filtered, concentrated under reduced pressure. The
residue was purified by silica gel column chromatography (Hexanes/
EtOAc = 10:1) to give a colorless oil (65 mg). This obtained product
was dissolved in MeOH (4 mL), and added Pd(OH)2/C (26 mg). The
reaction mixture was stirred at room temperature under H2 gas atmo-
sphere for 1 h. The reaction mixture was filtered through Celite to re-
move a catalyst, and the filtrate was concentrated under reduced
pressure. The residue was purified by silica gel column chromatography
(Hexanes/EtOAc = 1:1–0:1) to give 14 (17 mg, 40%) as a colorless oil.
The obtained product was further purified by HPLC (Hexanes/2-pro-
panol = 3:1).
4.1.11. 2-(4-(3-(4-(2-Ethyl-2-hydroxybutoxy)-3-methylphenyl)pentan-3-
yl)-2-methylphenoxy)propane-1,3-diol (11)
To a stirred solution of LiAlH4 (35 mg, 0.93 mmol) in THF (5 mL) at
0 °C was slowly added dropwise a solution of 10 (103 mg, 0.19 mmol)
in THF (2 mL). After completion of the addition, the reaction mixture
was gradually warm up room temperature, and stirred for 3.5 h. The
reaction mixture was filtered through Celite, and the filtrate was con-
centrated under reduced pressure. The residue was purified by silica gel
column chromatography (Hexanes/EtOAc = 5:1) to give 11 (11 mg,
11%) as a colorless oil. The obtained product was further purified by
HPLC (Hexanes/2-propanol = 5:1).
IR (neat):ν = 3851, 3733, 3647, 3005, 2989, 2359, 2341, 1682,
1651, 1506, 1456, 1260, 896 cm−1 1H NMR (CDCl3) δ 6.96–6.90 (m,
4H), 6.69 (m, 4H), 6.69 (d, J = 8.8 Hz, 1H), 6.66 (d, J = 8.0 Hz, 1H),
4.11 (m, 1H), 3.93 (d, J = 9.2 Hz, 1H), 3.80 (s, 2H), 3.76 (d, J = 9.2 Hz,
1H), 2.32 (s, 1H), 2.18 (s, 6H), 2.02 (q, J = 15.2 Hz, 4H), 1.95 (br. s,
1H), 1.67 (q, J = 17.6 Hz, 4H), 1.20 (br. s, 1H), 0.94 (t, J = 7.6 Hz, 4H)
,0.60 (t, J = 7.2 Hz, 6H); 13C NMR (CDCl3) δ 154.5, 154.4, 141.4,
141.2, 130.8, 130.7, 125.7, 125.6, 110.3, 110.2, 74.3, 73.4, 72.5, 66.7,
48.6, 29.6, 29.5, 28.9, 28.8, 16.8, 8.7, 8.7, 8.0, 8.0; [HR-ESI(+)] m/z
calcd for C28H42O5 [M+Na]+ 481.2924; found 481.2920.
IR (neat): ν = 3852, 3743, 3733, 3646, 3627, 3004, 2988, 2362,
2353, 2325, 1651, 1557, 1506, 1472, 898 cm−1 1H NMR (CDCl3) δ
6.97–6.91 (m, 4H), 6.73 (d, J = 8.8 Hz, 2H), 4.17 (t, J = 6.0 Hz, 2H),
4.10 (d, J = 5.6 Hz, 2H), 3.96 (t, J = 5.6 Hz, 4H), 2.74 (br. s, 1H),
2.23–2.22 (m, 1H), 2.17 (s, 6H), 2.15 (br. s, 1H), 2.12 (t, J = 5.2 Hz,
2H), 2.05–1.99 (m, 4H), 1.58 (br. s, 1H), 1.32 (s, 6H), 0.66 (t,
J = 7.2 Hz, 6H); 13C NMR (CDCl3) δ 154.5, 141.3, 130.8, 126.4, 126.3,
109.9, 109.8, 70.7, 67.4, 65.4, 63.6, 48.6, 42.9, 41.8, 38.2, 29.7, 29.5,
17.0, 16.8, 8.7, 8.4; [HR-ESI(+)] m/z calcd for C27H40O5 [M+Na]+
481.2924; found 481.2968.
4.1.12. 4-(3-(4-(3-Hydroxy-3-methylbutoxy)-3-methylphenyl)pentan-3-
yl)-2-methylphenol (12)
5. Biology
To a suspension of 1 (1.00 g, 3.50 mmol) and K2CO3 (0.58 g,
4.22 mmol) in DMF (20 mL) was added 16 (0.64 g, 3.50 mmol), and the
reaction mixture was stirred at 100 °C for 15 h. After cooling to room
temperature, the reaction mixture was poured into 1 M HCl, and ex-
tracted with Et2O three times. The combined organic extracts were
dried over Na2SO4, filtered, concentrated under reduced pressure. The
residue was purified by silica gel column chromatography (Hexanes/
EtOAc = 5:1) to give 12 as a colorless oil (68 mg, 5%).
5.1. Cell culture
Human osteosarcoma Hos cells maintained in Dulbecco’s modified
Eagle’s medium (DMEM) cotanining 10% fetal bovine serum (FBS) and
100 μg/mL of kanamycin with 10 μg/ml insulin (Sigma, St. Louis, MO,
USA). Cells were treated with various concentrations of the compounds
for 6 h.
1H NMR (CDCl3) δ 6.94–6.90 (m, 4H), 6.72 (s, 1H), 6.70 (s, 1H),
4.15 (t, J = 8.0 Hz, 2H), 2.75 (br. s, 1H), 2.16 (s, 6H), 2.06 (q,
J = 14.4 Hz, 4H), 2.00 (t, J = 8.0 Hz, 2H), 1.63 (br. s, 1H), 0.59 (t,
J = 7.2 Hz, 6H).
5.2. Reporter gene Luciferase assay
The human osteocalcin gene promoter fragment -838/+10 was
cloned into pGL3receptor 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)
using 50 μL of Lipofectamine 2000 reagent (Invitrogen). Three hours
after the transfection, they were treated with ethanol vehicle and var-
ious concentrations of the VDR ligands (from 10 pM to 10 μM).
Luciferase activity was quantitated the next day using a luminometer
(Berthold) and the Dual-Glo luciferase assay reagent (Promega).
4.1.13. (S)-3-(4-(3-(4-(3-Hydroxy-3-methylbutoxy)-3-methylphenyl)
pentan-3-yl)-2-methylphenoxy)propane-1,2-diol (13)
To a suspension of 12 (68 mg, 0.18 mmol) and K2CO3 (37 mg,
0.54 mmol) in acetone (6 mL) was added (S)-glycidol (36 μL,
0.27 mmol), and the reaction mixture was stirred at 60 °C for 21 h. After
cooling to room temperature, the reaction mixture was poured into 1 M
HCl, and extracted with EtOAc three times. The combined organic ex-
tracts were dried over Na2SO4, filtered, concentrated under reduced
pressure. The residue was purified by silica gel column chromatography
(Hexanes/EtOAc = 1:1) to give 13 as a colorless oil (2.8 mg, 3%). The
obtained product was further purified by HPLC (Hexanes/2-pro-
panol = 3:1).
IR (neat): ν = 3743, 3342, 3005, 2988, 2370, 2362, 2353, 1652,
1505, 1473, 1260, 1136, 1040, 897 cm−1 1H NMR (CDCl3) δ 6.96 (d,
J = 8.4 Hz, 2H), 6.91 (d, J = 7.6 Hz, 2H), 6.71 (t, J = 9.2 Hz, 2H), 4.17
(t, J = 6.0 Hz, 2H), 4.12–4.10 (m, 1H), 4.04 (d, J = 4.8 Hz, 2H),
3.86–3.77 (m, 2H), 2.74 (br. s, 1H), 2.56 (br. s, 1H), 2.17 (s, 3H), 2.15
(s, 3H), 2.03 (q, J = 14.4 Hz, 4H), 2.01 (t, J = 8.0 Hz, 2H), 1.55 (br. s,
1H), 1.32 (s, 6H), 0.59 (t, J = 7.2 Hz, 6H); 13C NMR (CDCl3) δ 158.5,
Acknowledgement
This work was supported in part by grants from AMED under Grant
Number JP18mk0101120, the Japan Society for the Promotion of
Science (KAKENHI, Grants 15K18905, 18K14880 to T.M., 17k08385 to
6

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Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Y.D.), TERUMO FOUNDATION for life sciences and ARTS (to T.M. and
Y.D.), Takeda Science foundation (to T.M., and Y.D.), and The Naito
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