A series of nonsecosteroidal vitamin D receptor agonists for osteoporosis therapy

Article abstract of DOI:10.1016/j.bmc.2013.01.042

In an extension of our study on gamma hydroxy carboxylic acid analogs, we explored a series of nonsecosteroidal vitamin D receptor (VDR) agonists in which 1,3-diol of 1,25(OH)₂D₃ had been replaced by aryl acetic acid. These analogs s

Full text of DOI:10.1016/j.bmc.2013.01.042

Bioorganic & Medicinal Chemistry 21 (2013) 1823–1833
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A series of nonsecosteroidal vitamin D receptor agonists
for osteoporosis therapy
⇑
Hirotaka Kashiwagi , Yoshiyuki Ono, Masateru Ohta, Susumu Itoh, Fumihiko Ichikawa, Suguru Harada,
Satoshi Takeda, Nobuo Sekiguchi, Masaki Ishigai, Tadakatsu Takahashi
Research Division, Chugai Pharmaceutical Co., Ltd, 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
In an extension of our study on gamma hydroxy carboxylic acid analogs, we explored a series of nonse-
costeroidal vitamin D receptor (VDR) agonists in which 1,3-diol of 1,25(OH)2D3 had been replaced by aryl
acetic acid. These analogs showed very potent activity in vitro compared with 1,25(OH)2D3. An X-ray
analysis of 8d showed that the inserted phenyl ring well mimicked the folded methylene linker of the
gamma hydroxy carboxylic acid moiety but the carboxylic acid of 8d interacted with VDR in a different
manner from gamma hydroxy carboxylic acids. Through our in vivo screening in an osteoporosis rat
model using immature rats, we identified a potent active vitamin D3 analog, compound 7e. In mature rats
of the same model, compound 7e also showed good PK profiling and excellent ability to prevent bone
mineral density loss without severe hypercalcemia. Our nonsecosteroidal VDR agonist 7e (CH5036249)
could be a possible new drug candidate for treating osteoporosis in human.
Received 26 November 2012
Revised 16 January 2013
Accepted 18 January 2013
Available online 8 February 2013
Keywords:
Vitamin D3
VDR agonist
Nonsecosteroid
Osteoporosis
CH5036249
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
in bone mass than alfacalcidol and comparable calcium absorption
and parathyroid hormone levels in osteoporosis model rats.12,13 In
Osteoporosis is a disease related to a decrease of bone mineral
density (BMD), and the increased bone fragility in patients with
this disease often causes bone fractures.1 Especially in the case of
elderly postmenopausal women, bone fracture is linked to patients
becoming bedridden with long-term care requirement and suffer-
ing a remarkably decreased quality of life (QOL). Prevention and
treatment are important for improving the QOL of the aging popu-
lation worldwide.
In bone, calcium is the most important component, and calcium
deficiency, which is a major risk factor for osteoporosis is caused
by a decrease in vitamin D. Adequate intake of vitamin D and cal-
cium is recommended as a baseline therapy for prevention and as a
treatment for all osteoporosis patients. Evidence suggests that vita-
min D supplementation may have favorable effects on BMD and
even reduce the risk of fracture.2–10 Furthermore, alfacalcidol
a 12-month phase II study (218 patients), eldecalcitol dose-depen-
dently increased BMD in lumbar spine and hip and in a 3-year
phase III study (1054 patients), eldecalcitol, which has a safety pro-
file similar to alfacalcidol, was more efficacious in preventing ver-
tebral fracture in osteoporotic patients.14–17
Because the calcemic effect of VDR agonists generally limits the
dose it is possible to give in clinic, we were prompted to search for
novel chemical class of VDR agonist for osteoporosis therapy, and
discovering a potent VDR agonist without calcium effect is our ulti-
mate goal.
In 1999, Boehm et al. reported the first nonsecosteroidal VDR
agonist, LG190178 (4), and some groups since then have reported
its derivatives.18–24 We focused on compound 4 as a new chemical
class lead and conducted systematic SAR studies on the A part and
the side chain of 4 to characterize the analogs, identifying carbox-
ylic acid derivatives 5 and 6 as more potent nonsecosteroidal VDR
agonists than 1,25(OH)2D3.25–27 From our X-ray analysis, we noted
that a tight hydrogen-bonding network with VDR was made by the
A part of 5 and 6. However, these molecules have a lot of rotatable
bonds in the A part, and gamma hydroxy carboxylic acid was easily
cyclized to lactone in acidic conditions (Chart 1).
Then, by modeling based on X-ray conformation we attempted
to optimize the A part again to reduce the flexibility and to avoid
lactone formation by replacing the rotatable bonds with an aro-
matic ring. As a result, we identified a series of phenylacetic
acid-type derivatives with very potent VDR agonistic activity. We
(1a-hydroxyvitamin D3, 1), a prodrug of active vitamin D3 (calci-
triol, 1
a
,25-dihydroxyvitamin D3 [1 ,25(OH)2D3], 2) is an effica-
a
cious and safe medication for the prevention and treatment of
osteoporosis that has been used in clinics in Japan for more than
25 years. To improve upon this existing therapy, we pursued stud-
ies in secosteroidal vitamin D3 analogs and identified the analog
eldecalcitol (3).11 Eldecalcitol, which was approved in Japan as a
new osteoporosis treatment drug last year, shows greater increase
⇑
Corresponding author. Tel.: +81 550 87 8626; fax: +81 550 87 5326.
E-mail address: kashiwagihrt@chugai-pharm.co.jp (H. Kashiwagi).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.042

1824
H. Kashiwagi et al. / Bioorg. Med. Chem. 21 (2013) 1823–1833
R1
Side Chain
Side Chain
R1
R1
OH
R1
OR2
R1
OR2
R1
OH
R
25
H
OH
O
a
c
R
R1
H
OTf
11: R1 = Et, R2 = H
B
OH
O
O
3
HO
1
OH
9: R1 = Et, R2 = H
10: R1 = CF3, R2 = MOM
2
A ring
O
O
(S)
12: R1 = CF3, R2 = MOM
A part
R2
HO
b
14: R1 = Et
15: R1 = CF3
HOOC
OH
OH
4: LG190178
13: R1 = CF3, R2 = H
1: R1 = H, R2 = H; alfacalcidol
2: R1 = OH, R2 = H; calcitriol
3: R1 = OH, R2 = OCH2CH2CH2OH; eldecalcitol
5: R = Et
6: R = CF3
R1
R1 = Et
OH
16a: R3 = CO2Me, R4 = H, X = CH
16b: R3 = H, R4 = CO2Me, X = CH
16c: R3 = CH2CO2Me, R4 = H, X = CH
16d: R3 = CH2CO2Me, R4 = F, X = CH
16e: R3 = CH2CO2Me, R4 = H, X = N
R1
d or e
R1
R1 = Et
OH
R1
7a: R2 = COOH, R3 = H, X = CH
7b: R2 = H, R3 = COOH, X = CH
7c: R2 = CH2COOH, R3 = H, X = CH
7d: R2 = CH2COOH, R3 = F, X = CH
7e: R2 = CH2COOH, R3 = H, X = N
R
1 = CF3
X
17c: R3 = CH2CO2Me, R4 = H, X = CH
17d: R3 = CH2CO2Me, R4 = F, X = CH
17e: R3 = CH2CO2Me, R4 = H, X = N
R4
R3
R1 = CF3
X
8c: R2 = CH2COOH, R3 = H, X = CH
8d: R2 = CH2COOH, R3 = F, X = CH
8e: R2 = CH2COOH, R3 = H, X = N
R3
R1
OH
R1
R2
R1 = Et
7a: R3 = CO2H, R4 = H, X = CH
7b: R3 = H, R4 = CO2H, X = CH
7c: R3 = CH2CO2H, R4 = H, X = CH
7d: R3 = CH2CO2H, R4 = F, X = CH
7e: R3 = CH2CO2H, R4 = H, X = N
f
Chart 1. Structures of the secosteroids alfacalcidol (1), calcitriol (2), and eldecalc-
itol (3), and of the nonsecosteroidal analog LG190178 (4) and nonsecosteroidal
carboxylic acid analogs (5 to 7a–e and 8c–e). Parts labeled A of 5 and 6 (upper right)
show the structural correspondence to the A ring of secosteroids (upper left).
X
R1 = CF3
8c: R3 = CH2CO2H, R4 = H, X = CH
8d: R3 = CH2CO2H, R4 = F, X = CH
8e: R3 = CH2CO2H, R4 = H, X = N
R4
R3
also managed to combine the aryl acetic acid-type A parts and the
(E)-olefin side chain with two terminal trifluoromethyl (CF3)
groups, which had shown the most potent in vitro activity in our
previous study.21 As we expected, we found a series of potent
VDR agonists. The structure of one of these compounds, 8d, with
VDR was solved by X-ray, and the detailed interactions of the A
part and the side chain with VDR were confirmed. Furthermore,
we evaluated the preventative effect of these compounds on
BMD loss and serum calcium in an osteoporosis rat model using
immature and mature rats. The pharmacokinetic parameters of
7e were very good and it showed no signs of toxicity in a variety
of safety tests both in vitro and in vivo. As a result, we identified
compound 7e (CH5036249) as the most promising clinical
candidate.
Scheme 1. Reagents and conditions: (a) PhN(Tf)2, Et3N, rt, CH2Cl2, 98–99%; (b) TFA,
rt, CH2Cl2, 99%; (c) PdCl2(dppf), dppf, bis(pinacolato)diboron, AcOK, 110 °C, 1,4-
dioxane 65ꢀ90%; (d) aryl bromides or methyl 4-chloro-2-fluorophenylacetate,
Pd(OAc)2, SPhos, K3PO4, 100 °C, toluene–H2O, 42–92%; (e) methyl 2-(6-bromopyri-
din-3-yl)acetate, Pd(PPh3)4, K3PO4, 110 °C, DMF, 48–75%; (f) 1 M NaOH, rt, THF–
MeOH, 40–95%.
2. Chemistry
The synthesis of compounds 7a–e and 8c–e is shown in
Scheme 1. Compounds 9 and 10 were prepared by the method re-
ported previously.25 The treatment of phenols with bis(trifluoro-
methanesulfonyl)amide and triethlamine at rt afforded triflates
11 and 12 in excellent yield (98% and 99%, respectively). The
methoxymethyl (MOM) group of 12 was deprotected with trifluo-
roacetic acid at rt in 99% yield. The triflates 11 and 13 were con-
verted to boronates 14 and 15 with bis(pinacolato)diboron in the
presence of palladium catalyst and potassium acetate at 110 °C
in moderate yields. Suzuki coupling of these boronates and aryl
bromides (methyl 3-bromobenzoate, methyl 4-bromobenzoate,
methyl 4-bromophenylacetate) or aryl chloride (methyl 4-chloro-
2-fluorophenylacetate) in the presence of palladium acetate with
2-dicyclohexylphosphino-20,60-dimethoxy-1,10-biphenyl (SPhos)
ligand afforded biphenyl esters 16a–d and 17c–d in moderate to
high yields. In the case of pyridine analogs, Suzuki coupling reac-
tions of boronates 14 and 15 with methyl 2-(6-bromopyridine-3-
yl)acetate were conducted at 110 °C using tetrakis(triphenylphos-
phine)palladium catalyst and gave biphenyl esters 16e and 17e
in moderate yields (48% and 75%, respectively). These esters 16a–
e and 17c–e were hydrolyzed to carboxylic acids 7a–e and 8c–e
in moderate to high yields by treatment of 1 M sodium hydroxide
solution and the following acidification process.
Figure 1. Superposition of modeled 7c and 6 in the crystal (PDB ID: 3W0C).
Compound 7c is depicted in blue and compound 6 is depicted in white.
3. Results and discussion
All synthesized compounds were evaluated in MG-63 cells by
reporter gene assay (RGA) (which contains vitamin D response ele-
ment [VDRE] sequence derived from mouse osteopontin promoter)
to measure vitamin D agonistic activity, and by osteocalcin produc-
tion activity to measure the bone formation activity of the func-
tional vitamin
D agonistic effect. VDRE RGA activity and
osteocalcin activity are represented by relative EC50 values based
on 1,25(OH)2D3 being assigned as 100% (a higher figure means
stronger activity).25–27

H. Kashiwagi et al. / Bioorg. Med. Chem. 21 (2013) 1823–1833
1825
His305
His397
2.7
2.9
Arg274
2.8
3.2
Figure 2. Model of compound 7c in VDR. Key hydrogen bonding interactions are shown by red dotted lines and each distance is given in angstrom (Å).
Table 1
VDRE reporter gene and osteocalcin induction activity of carboxylic acid analogs in the A part
Compound
R1
R2
R3
X
VDRE RGA activity (%)
OC activity (%)
R1
LG190178
1,25(OH)2D3
5
6.7
100
105
5
13
89
112
89
592
135
116
121
185
100
447
12
11
1044
508
OH
R1
Et
Et
Et
Et
Et
Et
CF3
CF3
CF3
CF3
7a
7b
7c
7d
7e
6
8c
8d
8e
COOH
H
CH2COOH
CH2COOH
CH2COOH
H
COOH
H
F
H
CH
CH
CH
CH
N
X
652
R3
3746
2233
1624
1848
R2
CH2COOH
CH2COOH
CH2COOH
H
F
H
CH
CH
N
For compound 5 and 6 to exhibit potent VDR agonistic activity,
it was very important for the carboxylic acid group to interact with
Arg274 by forming a salt bridge. X-ray analysis of compound 6
with VDR showed that the methylene linker in the A part was
folded like a ring without a linear form.27 We focused on this
folded conformation of the A-part moiety again and attempted to
replace the methylene linker in the A part with a new phenyl ring
to reduce flexibility. At the same time as inserting the new phenyl
ring, we determined the optimal position of the carboxylic acid
that binds to the ring.
Based on the crystallographic structure of compound 6 in com-
plex with VDR (PDB ID: 3W0C), we constructed a docking model of
the analog with a diethyl group in the side chain (Fig. 1). The com-
pounds 7a–c were manually docked into the crystal structure of
VDR using software MOLOC.28 These compounds were optimized
using the MAB force field with a fixed VDR structure.
Figure 2 shows the result of modeling compound 7c. In the
model, the newly introduced phenyl ring (replacing the methylene
linker in the A part of compound 6) was beautifully adjusted in
VDR without any steric repulsion to VDR protein. Next, to form
the salt bridge interaction with Arg274 that was shown in com-
pound 6, the carboxylic acid group was introduced to the para po-
sition on the newly introduced phenyl ring with a methylene (7c).
In our model, this carboxylic acid group was superposed excel-
lently with that of compound 6 and was expected to form a salt
bridge with an ideal length (Figs. 1 and 2). The directly linked car-
boxylic acids on the phenyl ring at para or meta position (com-
pounds 7a and 7b) were too far from Arg274 to form a salt
bridge interaction in this model.
Table 1 shows all the in vitro results of compounds 7a–e and
8c–e. In benzoic acid analogs, those substituted at both the meta
position 7a and the para position 7b showed very weak activity
in the VDRE RGA and osteocalcin assays, as expected from the
modeling with VDR. Because this activity was one-tenth of
1,25(OH)2D3, it seems these carboxylic acids could not form the
salt bridge with Arg274. On the other hand, compound 7c, which
had an acetic acid at para position on the new phenyl ring, showed
potent activity equivalent to 1,25(OH)2D3 in the VDRE reporter as-
say and, surprisingly, 10-fold more potent activity than
1,25(OH)2D3 in the osteocalcin induction assay. From these results,
the carboxylic acid group of 7c could form a good salt bridge with
Arg274 in the same manner as that of 5 and accordingly we consid-
ered that the rigid 4-phenylacetic acid analog was completely
mimicking the role of the folded carboxylic acid moiety in the A
part of compound 5. In our results, compound 7c showed potent
in vitro activity.
In a series of gamma hydroxy carboxylic acid analogs, the hy-
droxy group in the A-part had hydrogen-bonding interactions with

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H. Kashiwagi et al. / Bioorg. Med. Chem. 21 (2013) 1823–1833
log 8c was only 1.5-fold more potent than diethyl analog 7c in the
F1
VDRE reporter assay and twofold more potent in the osteocalcin
assay. From these results, we realized that there is a distinct differ-
ence in the A part between these gamma hydroxy carboxylic acid
analogs and the phenylacetic acid analogs, and that di-CF3 analogs
with phenylacetic acid analogs were weaker than we expected.
To confirm the detailed interactions of the phenylacetic acid
group and CF3 groups with VDR, we attempted to cocrystallize a
human VDR ligand-binding domain with a series of our com-
pounds, in accordance with a previously published method.25,27
From the crystallization screening, we obtained a cocrystal of 8d/
VDR. Figure 3 shows the superposition of the conformation of 8d
and 6 in each crystal. The side chain moieties of both compounds
were completely superposed but some differences appeared in
the A part. The carboxylic acid group in 8d was slightly off the po-
sition of that in 6, and the fluorine atom was located on the oppo-
site side to that we expected. The methyl group on the bisphenyl
core of the A part side in 8d was also located on the opposite side
to that in compound 6.
F2
F3
F6
F5
F4
F7
Figure 3. Superposition of binding conformations with VDR between compounds
8d and 6 in the crystal (PDB ID: 3W0Y and 3W0C). Compound 8d is depicted in
magenta and compound 6 is depicted in white. All fluorine atoms in 8d are labeled
from F1 to F7.
Figure 4 shows the overall structure of compound 8d which was
consistent with that of compound 6. The hydroxy group in the side
chain moiety interacted with His305 and His397. The diethyl moi-
ety between bisphenyl rings made a good CH–
p interaction with
Trp278. It should be noted that the newly introduced phenyl ring
in the A part fitted well, and the acetic acid moiety was placed near
the targeted Arg274. However, instead of making a salt bridge with
Arg274, this carboxylic acid interacted by hydrogen bondings with
Arg274, two crystal waters and the backbone nitrogen of Asp144.
The two crystal waters also made hydrogen bondings with
Arg274 and Tyr236 in the same manner as reported (Fig. 5).29
Ser278 and Tyr143 of VDR and contributed to increasing the po-
tency in vitro. To add an extra interaction with VDR to compound
7c, such as the hydroxy group in compound 5, we attempted to
either introduce a fluorine atom at 3 position on the newly intro-
duced phenyl ring or replace the phenyl ring with pyridine. As a re-
sult, fluorine analog 7d showed more potent activity than
1,25(OH)2D3 and pyridine analog 7e showed equivalent activity
to 1,25(OH)2D3 in the VDRE assay. In the osteocalcin assay, com-
pound 7d showed fivefold and 7e showed 6.5-fold more potent
activity than 1,25(OH)2D3. Although these compounds had potent
activity, they still had less than compound 7c. We think com-
pounds 7d and 7e were not gaining any additional hydrogen-bond-
ing interactions, in spite of having the A part located in the
hydrophilic pocket formed by Arg278, Tyr143, Ser278, Ser237,
and crystal water.
The newly introduced phenyl ring made a good
p–p interaction
with Phe150. The fluorine atom in the A part, labeled F7, was
placed on the opposite side to the hydroxy group of 6 and formed
van der Waals (vdW) interactions with the Cf and C
e atoms of
Phe150 and the Cd and C atoms of Tyr236, and a weak electro-
e
static interaction with the hydroxy group of Ser237. This fluorine
atom was also located close to (within 4 Å of) the carboxylic acid
moiety, which may have contributed to fixing the conformation
of this moiety by weak electrostatic interaction.
In our previous report, a series of di-CF3 analogs in the side
chain moiety showed very potent VDR agonistic activity; especially
(E)-olefin analog 6 was the most potent VDR agonist.21 We at-
tempted to combine this potent side chain part with the newly
found phenyl acetic acid moieties in the A part. As a result, phenyl
acetic acid analog 8c showed 1.35-fold more potent activity than
1,25(OH)2D3 in the VDRE assay, and 22-fold more potent activity
in the osteocalcin assay. 3-Fluorophenyl acetic acid 8d and pyri-
dine acetic acid 8e also showed more potent activity than
1,25(OH)2D3: respectively, 1.16-fold and 1.21-fold in VDRE RGA
and 16-fold and 18-fold in the osteocalcin assay.
In the case of the A part of the gamma hydroxy carboxylic acid
analog, an (E)-olefin side chain with terminal di-CF3 group was
very attractive for increasing VDR agonistic activity.
In detail, all six fluorine atoms had tight hydrophobic interac-
tions with VDR residues; the two CF3 electron-withdrawing groups
increased the acidity of the hydroxy group in the side chain; the
free rotation of the olefin linker was limited by the methyl group
on the bisphenyl core; and the olefin itself was more hydrophobic
The fluorine interactions between the side chain and VDR were
almost the same as previously reported.27 All fluorine atoms (F1–
F6) had many vdW contacts with hydrophobic residue of VDR as
well as some electrostatic interactions. Within 4-Å-distance of
each fluorine, a total of 29 contacts (CH–F and NH–F interactions)
were observed.
In these results, phenylacetic acid derivatives showed equiva-
lent or more potent activity than 1,25(OH)2D3. However, the SAR
characteristics of these analogs were different from those of gam-
ma hydroxy carboxylic acid analogs. Conformational analysis by X-
ray of compounds 6 and 8d showed that the side chain parts were
completely superposed, the hydroxy group had hydrogen bonding
with two histidines, and the pattern of interactions of six fluorines
with hydrophobic amino acids was the same. On the other hand,
the newly introduced phenyl ring mimicked well the folded meth-
ylene linker in the A part of compound 6, even though the position
of the carboxylic acid group was not superposed completely. Con-
sequently, the carboxylic acid of compound 8d formed hydrogen-
bonding interactions with Arg274 and crystal water instead of
making the salt bridge interaction. Furthermore, phenylacetic acid
analogs did not have a substituent corresponding to the hydroxy
group in the A part of compound 5 or 6 that interacted with
Ser278 and Tyr143 by hydrogen bonding. This interaction corre-
sponded to the hydroxy group of 3-position of 1,25(OH)2D3, and
the lack of it could have resulted in a different chemical class from
the series of gamma hydroxy carboxylic acid analogs and would
therefore produce a different SAR.
than other linkers, which can be explained by the
p constant. We
think the total sum of these effects contributed to achieving the
superagonistic activity seen in compound 6, which was sixfold
more potent than 1,25(OH)2D3 in the VDRE reporter assay and
37-fold more potent in the osteocalcin assay. Compared to diethyl
analog 5, di-CF3 analog 6 was sixfold more potent in the VDRE re-
porter assay and eightfold more potent in the osteocalcin assay.
However, with phenylacetic acid analogs in the A part, di-CF3 ana-

H. Kashiwagi et al. / Bioorg. Med. Chem. 21 (2013) 1823–1833
1827
His305
His397
2.7
2.7
2.8
2.7
Arg274
2.7
2.5
2.8
Figure 4. Overall X-ray crystal structure of compound 8d (magenta) bound to VDR (cyan). Key hydrogen bonding interactions are shown by red dotted lines and each
distance is given in Å (PDB ID: 3W0Y).
Phe150
F7
2.8
2.5
3.0
Tyr236
2.7
2.7
2.8
Ser237
Arg274
Figure 5. Detailed hydrogen bonding network and fluorine interactions around the A part of compound 8d (magenta) bound to VDR (cyan). Key hydrogen-bonding
interactions are shown by red dotted lines and each distance is given in Å. Fluorine contacts within 4 Å of VDR residue atoms are shown by blue dotted lines. The fluorine
atom is labeled F7 (PDB ID: 3W0Y).
Next, we evaluated the potency of a series of compounds (7c,
7e, 8c, and 8d) in vivo using immature, osteoporosis rats (Table 2).
Female, 8-week-old, Sprague–Dawley rats were either ovariecto-
mized (OVX) to produce the estrogen-deficient condition observed
in postmenopausal women that promotes BMD loss, or sham-oper-
ated. The compounds (25 and 50 ng/kg of 7c, 10, 20 and 40 ng/kg of
7e, 10 and 30 ng/kg of 8c, and 20 and 60 ng/kg of 8d, per day) were
orally administered to OVX rats while only vehicle was adminis-
tered to the OVX-control and sham-operated groups. These doses
were determined from the serum calcium level after 4-day treat-
ment of normal rats by po administration of three dosages of each
compound. The dose at which around 11 mg/dL of serum calcium
level was observed was selected as the higher dose of this study
(data not shown). After five times per week administration for
4 weeks, BMD in the distal femur was measured by dual-energy
X-ray absorptiometry. Compound 7c dose-dependently prevented
BMD loss in osteoporosis rats, and the ratio to the vehicle group
was 42% at 25 ng/kg and 81% at 50 ng/kg. Normal serum calcium
level was maintained at the 25 ng/kg dosage but a rise in the level
of calcium was observed in the 50 ng/kg dosing group. Compound
7e not only prevented BMD loss, but also increased BMD, with the
ratios to the vehicle group of 122% at 10 ng/kg, 172% at 20 ng/kg,
and 198% at 40 ng/kg. In the 10 and 20 ng/kg groups, serum cal-
cium elevation was not observed but in the 40 ng/kg group, a clear
calcium elevation was observed. Compounds 8c and 8d also
showed dose-dependent BMD loss prevention at 10 and 30 ng/kg,

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H. Kashiwagi et al. / Bioorg. Med. Chem. 21 (2013) 1823–1833
Table 2
Oral once-daily treatment for 4 weeks prevented BMD loss in eight-week-old osteoporosis model rats
Compound
Dose (ng/kg)
Sham
Femoral distal BMD (mg/cm2)
Serum Ca (mg/dL)
Treated
Vehicle
Treated
D
BMDa (%)
Vehicle
D
Cab (%)
1,25(OH)2D3
20
80
320
25
50
10
20
40
10
30
20
60
166.1 2.6*
162.9 4.0*
133.6 5.2
134.9 2.7
150.9 4.3*
152.3 3.7*
148.7 2.2*
158.2 2.2*
143.7 3.3*
148.6 4.0*
151.3 2.2*
148.6 3.1
157.0 2.5*
144.4 3.1
150.3 3.3*
4
53
58
42
81
122
172
198
47
89
25
9.82 0.12
10.07 0.14
10.07 0.05
10.32 0.12*
10.37 0.08
11.20 0.12*
9.97 0.16
10.50 0.17
11.13 0.24*
10.33 0.16
10.98 0.21*
10.58 0.07*
11.32 0.15*
2.5
2.5
5.1
0.7
8.7
7c
7e
138.6 1.3
131.6 2.7
10.30 0.18
10.27 0.15
#
141.5 3.9
ꢀ2.9
2.2
8.4
0.6
6.9
3.0
8c
8e
159.1 2.5*
159.1 2.5*
139.4 3.0
139.4 3.0
10.27 0.08
10.27 0.08
55
10.2
All data show mean value SE (n = 6).
*
p <0.05 versus vehicle.
p = 0.06 versus vehicle.
#
a
D
D
BMD was calculated as follows: (treated BMDꢀvehicle BMD)/(sham BMDꢀvehicle BMD) ꢁ 100 (%).
b
Ca was calculated as follows: (treated Caꢀvehicle Ca)/(vehicle Ca) ꢁ 100 (%).
Table 3
tion due to normal growth. To confirm the ability to prevent
BMD loss without including bone formed by normal growth, we
evaluated the same osteoporosis model using 8-month-old rats.
We consider the osteoporosis shown in post menopausal women
is better reflected by the mature rats of this osteoporosis model
than by immature ones.
We evaluated the potency of 7e in mature rats of the osteopo-
rosis model (Fig. 6). Compound 7e was orally administered to five
groups (0.75, 1.5, 3.0, 6.0 and 12 ng/kg per day) of female, 8-
month-old, Sprague–Dawley rats, and vehicle was administered
to the OVX-control and sham-operated groups. After administra-
tion five times per week for 4 weeks, BMD in the distal femur
and lumbar spine was measured. BMD in the OVX-control group
was significantly reduced compared with the sham-operated
group. In rats in which osteoporosis had been caused by ovariec-
tomy, compound 7e dose-dependently prevented BMD loss in both
distal femur and lumbar spine without body weight loss. Normal
serum calcium level was maintained at the doses of 0.75–6.0 ng/
kg; however, in the highest (12 ng/kg) dosing group, a rise in cal-
cium level was observed.
Compound 7e was not the most potent one in vitro; however it
showed potent BMD effect in vivo. When speculating upon this dis-
crepancy, we noted that VDR agonists relate strongly to both bone
formation and bone resorption. Because we only evaluated bone
formation as one of the functional VDR agonistic effects by looking
at osteocalcin induction activity, we did not evaluate whether 7e
suppressive effect on bone resorption30 was more potent than
other compounds. Further evaluation will explain the discrepancy
between in vivo and in vitro effects of 7e on bone.
In further safety assessment, compound 7e (CH5036249)
showed negative results on the micro-AMES and micronucleus
tests in vitro, and also showed no concerns of toxicity except for
hypercalcemia in a 4-week repeated administration to normal rats
at the higher dosage of 108 ng/kg in vivo. CYP induction ability of
compound 7e at 10 nM concentration was negative for CYP1A and
weaker than 1,25(OH)2D3 for CYP3A.31 We consider our nonsecos-
teroidal VDR agonist CH5036249 to be a possible new drug candi-
date for the treatment of osteoporosis in human.
Pharmacokinetic parameters of 7e after intravenous and oral administration at
100 lg/kg in rats (n = 3)
iv
po
F (%)
T1/2
95.1
8.5 1.4
17.6 3.3
54.0 8.2
1648 195
28.3 4.7
Cmax (ng/mL)
AUCinf (ng/mL⁄h)
MRT (h)
Vss (mL/kg)
CLt (mL/h/kg)
1128 203
11.2 2.2
990 16
90.6 16.2
and at 20 and 60 ng/kg respectively. Lower dose groups showed
the normal range of serum calcium level; however, in higher dose
groups, a rise in serum calcium was observed for both compounds
8c and 8d. From these results, we confirmed the effect of prevent-
ing BMD loss in all the tested compounds in osteoporosis rats.
Especially in lower dosing groups, all compounds showed moder-
ate to potent effect while maintaining serum calcium in the normal
range. Surprisingly, the BMD levels of compound 7e at the doses of
10 and 20 ng/kg were higher than those of the sham group, with-
out raising the serum calcium. This in vivo efficacy of 7e was
clearly more potent than that of 1,25(OH)2D3.
From the efficacy in the osteoporosis model using immature
rats and the pharmacokinetic profiles, we selected the pyridine
derivative 7e for further evaluation. Table 3 shows the pharmaco-
kinetic parameters in male rat at the single dosage of 100 lg/kg via
intravenous and oral administration. Compound 7e shows excel-
lent bioavailability (F = 95%), a favorable half-life (T1/2 = 17.6 h), a
long mean residence time (MRT = 28.3 h), and small total clearance
(CLt = 91 mL/h/kg).
Using 8-week-old rats of the osteoporosis model was very con-
venient for screening the compounds, but in immature rats the ef-
fect of preventing BMD loss in an estrogen-deficient condition may
have been affected by an increase in BMD caused by bone forma-
tion due to normal growth. To confirm the ability to prevent
BMD loss without including bone formed by normal growth, we
evaluated the same osteoporosis model using 8-month-old rats.
We consider the osteoporosis shown in post menopausal women
is better reflected by the mature rats of this osteoporosis model
than by immature ones.
4. Conclusion
Using 8-week-old rats of the osteoporosis model was very con-
venient for screening the compounds, but in immature rats the ef-
fect of preventing BMD loss in an estrogen-deficient condition may
have been affected by an increase in BMD caused by bone forma-
A series of nonsecosteroidal VDR agonists were explored in
an extension of our study of gamma hydroxy carboxylic acid
analogs. They have aryl acetic acid instead of 1,3-diol of
1,25(OH)2D3 and showed more potent activity in vitro than

H. Kashiwagi et al. / Bioorg. Med. Chem. 21 (2013) 1823–1833
1829
were carried out on VARIAN 400-MR spectrophotometers; chem-
ical shifts are reported in parts per million (ppm) downfield
from that of internal tetramethylsilane (TMS). Mass spectropho-
tometry was measured with a Waters ACQUITY SQD electrospray
ionization (ESI) system. High-resolution mass spectra (HRMS)
were recorded on Thermo Fisher Scientific LTQ Orbitrap XL
(ESI) instruments. Chromatographic purification was carried out
using Merck silica gel 60 (column) or Merck silica gel 60 PF254
(preparative TLC).
210
200
190
180
170
160
150
140
130
120
(a)
*
*
*
5.1.1. Trifluoromethanesulfonic acid 4-{1-Ethyl-1-[4-((E)-3-
ethyl-3-hydroxy-1-pentenyl)-3-methylphenyl]propyl}-2-
methylphenyl ester (11)
Sham
Vehicle 0.75
1.5
3.0
6.0
12
ng/kg ng/kg
ng/kg ng/kg ng/kg
To a solution of 9 (100 mg, 0.263 mmol) in CH2Cl2 (3 mL) were
added
N-phenylbis(trifluoromethanesulfonimide)
(122 mg,
(b)
200
190
180
170
160
150
140
130
120
0.342 mmol) and Et3N (0.072 mL, 0.526 mmol), and the mixture
was stirred at rt overnight. The mixture was concentrated and
the obtained residue was purified by preparative TLC (n-hexane/
AcOEt = 4:1) to afford 11 (132 mg, 98%) as colorless oil. 1H NMR
(CDCl3) d: 0.62 (6H, t, J = 7.2 Hz), 0.92 (6H, t, J = 7.4 Hz), 1.64 (4H,
q, J = 7.4 Hz), 2.08 (4H, q, J = 7.2 Hz), 2.32 (6H, s), 6.03 (1H, d,
J = 16.0 Hz), 6.76 (1H, d, J = 16.0 Hz), 6.90–6.94 (2H, m), 7.05 (1H,
dd, J = 8.7, 1.9 Hz), 7.09–7.12 (2H, m), 7.32 (1H, d, J = 7.8 Hz). 13C
NMR (CDCl3) d: 149.1, 146.6, 146.2, 136.2, 134.7, 133.8, 131.6,
129.7, 129.5, 129.4, 127.3, 125.8, 125.7, 125.0, 120.2, 76.0, 49.3,
33.2, 29.0, 20.1, 16.5, 8.3, 7.8. MS (ESI positive): 535 (M+Na)+.
*
*
*
*
Sham
Vehicle
0.75
1.5
3.0
6.0
12
ng/kg ng/kg
ng/kg ng/kg ng/kg
5.1.2. Trifluoromethanesulfonic acid 4-{1-ethyl-1-[3-methyl-4-
((E)-4,4,4-trifluoro-3-methoxymethoxy-3-trifluoromethyl-1-
butenyl)phenyl] propyl}-2-methylphenyl ester (12)
12.0
11.0
10.0
9.0
(c)
The yield was 99%. Colorless oil. 1H NMR (CDCl3) d: 0.62 (6H, t,
J = 7.2 Hz), 2.08 (4H, q, J = 7.2 Hz), 2.32 (6H, s), 3.51 (3H, s), 4.97
(2H, s), 6.08 (1H, d, J = 16.6 Hz), 6.96–7.00 (2H, m), 7.01–7.08
(2H, m), 7.11 (1H, d, J = 8.4 Hz), 7.33–7.38 (2H, m). 13C NMR (CDCl3)
d: 149.2, 148.7, 146.4, 137.6, 135.9, 131.6, 130.1, 129.8, 127.3,
126.2, 125.5, 123.8, 121.0, 120.4, 120.2, 117.0, 115.2, 93.3, 56.4,
49.6, 29.0, 19.9, 16.6, 8.3. MS (ESI positive): 654 (M+NH4)+.
*
*
8.0
7.0
6.0
5.1.3. Trifluoromethanesulfonic acid 4-{1-Ethyl-1-[3-methyl-4-
((E)-4,4,4-trifluoro-3-hydroxy-3-trifluoromethyl-1-
butenyl)phenyl] propyl}-2-methylphenyl ester (13)
Sham
Vehicle
0.75
ng/kg ng/kg
1.5
3.0
6.0
12
ng/kg ng/kg ng/kg
To a solution of 12 (2.41 g, 3.79 mmol) in CH2Cl2 (20 mL) was
added trifluoroacetic acid (2.0 mL) and the mixture was stirred at rt
for 30 min. The reaction mixture was concentrated and purified with
silicagel chromatography (n-hexane/AcOEt = 100:0 to 70:30) to give
13 (2.24 g, 99%) as a colorless oil. 1H NMR (CDCl3) d: 0.62 (6H, t,
J = 7.1 Hz), 2.10 (4H, q, J = 7.1 Hz), 2.32 (3H, s), 2.35 (3H, s), 6.12 (1H,
d, J = 15.8 Hz), 6.96–7.00 (2H, m), 7.05 (1H, dd, J = 8.6, 2.2 Hz), 7.09
(1H, s), 7.12 (1H, d, J = 8.6 Hz), 7.37 (1H, d, J = 8.8 Hz), 7.41 (1H, d,
J = 15.8 Hz). 13C NMR (CDCl3) d: 148.9, 148.8, 146.4, 135.8, 135.0,
131.7, 131.4, 130.1, 129.8, 127.3, 126.1, 125.6, 123.9, 121.1, 120.3,
120.2, 117.1, 49.5, 29.0, 19.9, 16.5, 8.2. MS (ESI positive): 593 (M+H)+.
Figure 6. Oral once-daily treatment of compound 7e prevented BMD loss in distal
femur (a) and lumbar spine (b) in 8-month-old osteoporosis model rats. (c) Serum
calcium concentration in osteoporosis model rats. All data are the mean value SE
(n = 8). ⁄p <0.05 versus vehicle.
1,25(OH)2D3. An X-ray analysis of 8d showed that the substitut-
ing phenyl ring mimicked well the folded methylene linker of
gamma hydroxy carboxylic acid moiety but the carboxylic acid
of 8d interacted with VDR in
a different pattern from the
one in gamma hydroxy carboxylic acids. Through our in vivo
screening using immature rats of an osteoporosis model, a po-
tent active vitamin D3 analog, compound 7e, was identified.
Compound 7e also showed excellent ability to prevent BMD loss
in mature rats of the osteoporosis model, without severe hyper-
calcemia and with good PK profiling. Our nonsecosteroidal VDR
agonist 7e (CH5036249) could be a possible new drug candidate
for the treatment of osteoporosis in humans.
5.1.4. (E)-3-Ethyl-1-(4-{1-ethyl-1-[3-methyl-4-(4,4,5,5-
tetramethyl-[1,3,2]dioxaborolan-2-yl) phenyl] propyl}-2-
methylphenyl)-1-penten-3-ol (14)
To a solution of 11 (1.6 g, 3.1 mmol) in 1,4-dioxane (20 mL) were
added
1,10-Bis(diphenylphosphino)-ferrocene
(dppf)
(104 mg,
0.19 mmol), KOAc (0.919 g, 9.4 mmol), Bis(pinacolato)diborone (1.03 g,
4.1 mmol), and [1,1-bis(diphenylphosphino)-ferrocene]dichloropalla-
dium(II) (PdCl2dppf), complex with CH2Cl2 (1:1) (0.15 g, 0.19 mmol).
The mixture was stirred at 80 °C overnight under nitrogen atmosphere.
The mixture was poured into H2O and products were extracted with
CH2Cl2. TheextractsweredriedoveranhydrousMgSO4 andconcentrated.
The obtained residue was chromatographed on silica gel (n-hexane/
AcOEt = 10:1) to afford 14 (1.0 g, 65%) as a colorless oil. 1H NMR (CDCl3)
5. Experimental
5.1. Chemistry: general
Purchased reagents and solvents were used without further
purification unless otherwise noted. 1H and 13C NMR spectra

1830
H. Kashiwagi et al. / Bioorg. Med. Chem. 21 (2013) 1823–1833
d: 0.60 (6H, t, J = 7.3 Hz), 0.91 (6H, t, J = 7.3 Hz), 1.32 (12H, s), 1.63 (4H, q,
J = 7.3 Hz), 2.08 (4H, q, J = 7.3 Hz), 2.29 (3H, s), 2.48 (3H, s), 6.01 (1H, d,
J = 15.8 Hz), 6.74 (1H, d, J = 16.0 Hz), 6.91–7.01 (4H, m), 7.28 (1H, d,
J = 8.2 Hz), 7.64 (1H, d, J = 7.6 Hz). 13C NMR (CDCl3) d: 151.4, 147.5,
144.0, 135.8, 135.2, 134.4, 133.3, 130.0, 129.6, 125.9, 125.9, 124.8,
124.7, 83.2, 75.9, 49.6, 33.3, 29.0, 24.9, 22.5, 20.2, 8.4, 7.9. MS (ESI posi-
tive): 508 (M+H)+.
q, J = 7.3 Hz), 2.23 (3H, s), 2.34 (3H, s), 3.67 (2H, s), 3.73 (3H, s), 6.03
(1H, d, J = 15.9 Hz), 6.76 (1H, d, J = 15.9 Hz), 6.97–7.03 (3H, m),
7.05–7.10 (2H, m), 7.30 (4H, s), 7.33 (1H, d, J = 8.1 Hz). 13C NMR
(CDCl3) d: 172.2, 147.6, 147.5, 140.8, 138.3, 135.9, 134.4, 134.1,
133.3, 132.1, 129.9, 129.5, 129.1, 128.8, 126.0, 125.9, 125.7,
124.9, 76.0, 52.1, 49.3, 40.9, 33.3, 29.1, 20.8, 20.3, 8.5, 7.9. MS
(ESI negative): 511 (MꢀH)ꢀ.
5.1.5. (E)-4-(4-{1-Ethyl-1-[3-methyl-4-(4,4,5,5-tetramethyl-
[1,3,2]dioxaborolan-2-yl) phenyl] propyl}-2-methylphenyl)-
1,1,1-trifluoro-2-trifluoromethyl-3-buten-2-ol (15)
5.1.10. (40-{1-Ethyl-1-[4-((E)-3-ethyl-3-hydroxy-1-pentenyl)-3-
methylphenyl]propyl}-3-fluoro-20-methylbiphenyl-4-yl)acetic
acid methyl ester (16d)
The yield was 90%. Colorless oil. 1H NMR (CDCl3) d: 0.61 (6H, t,
J = 7.2 Hz), 1.32 (12H, s), 2.09 (4H, q, J = 7.2 Hz), 2.31 (3H, s), 2.48
(3H, s), 6.08 (1H, d, J = 16.0 Hz), 6.93–7.02 (4H, m), 7.33 (1H, d,
J = 7.6 Hz), 7.38 (1H, d, J = 16.4 Hz), 7.64 (1H, d, J = 7.6 Hz). 13C
NMR (CDCl3) d: 151.0, 149.8, 144.1, 135.5, 135.3, 135.1, 130.9,
130.3, 130.2, 129.6, 128.7, 126.2, 126.2, 125.3, 125.2, 124.6, 116.6,
83.3, 49.8, 28.9, 24.8, 22.5, 19.9, 8.3. MS(ESIpositive): 588(M+NH4)+.
The yield was 76%. Colorless oil. 1H NMR (CDCl3) d: 0.65 (6H, t,
J = 7.1 Hz), 0.92 (6H, t, J = 7.4 Hz), 1.64 (4H, q, J = 7.4 Hz), 2.11 (4H,
q, J = 7.0 Hz), 2.24 (3H, s), 2.34 (3H, s), 3.71 (2H, s), 3.74 (3H, s), 6.03
(1H, d, J = 16.0 Hz), 6.76 (1H, d, J = 16.0 Hz), 6.97–7.09 (7H, m), 7.28
(1H, d, J = 7.4 Hz), 7.33 (1H, d, J = 8.0 Hz). 13C NMR (CDCl3) d: 171.3,
159.3, 148.0, 147.5, 143.3, 137.3, 135.9, 134.5, 134.1, 130.8, 130.0,
129.9, 128.9, 126.0, 125.8, 125.7, 125.2, 125.1, 124.9, 119.2, 116.4,
76.0, 52.2, 49.3, 34.1, 33.3, 29.1, 20.8, 20.3, 8.5, 7.9. MS (ESI nega-
tive): 529 (MꢀH)ꢀ.
5.1.6. General procedure for Suzuki coupling reaction of
boronate 14 and 15
To a solution of boronate (0.037 mmol) in toluene (1.5 mL) and H2O
(0.15 mL) were added arylhalide (methyl 3-bromobenzoate, methyl 4-
bromobenzoate, methyl (4-bromophenyl)-acetate, or methyl (4-chloro-
5.1.11. (40-{1-Ethyl-1-[3-methyl-4-((E)-4,4,4-trifluoro-3-
hydroxy-3-trifluoromethyl-1-butenyl)phenyl]propyl}-20-
methylbiphenyl-4-yl)acetic acid methyl ester (17c)
2-fluorophenyl)-acetate)
(0.055 mmol),
Pd(OAc)2
(0.8 mg,
The yield was 92%. Colorless oil. 1H NMR (CDCl3) d: 0.66 (6H, t,
J = 7.3 Hz), 2.13 (4H, q, J = 7.2 Hz), 2.22 (3H, s), 2.34 (3H, s), 3.66
(2H, s), 3.71 (3H, s), 6.10 (1H, d, J = 16.1 Hz), 7.00 (1H, dd, J = 8.1,
1.7 Hz), 7.03–7.10 (4H, m), 7.29 (4H, d, J = 2.0 Hz), 7.36 (1H, d,
J = 8.8 Hz), 7.39 (1H, d, J = 15.6 Hz). 13C NMR (CDCl3) d: 172.2,
149.9, 147.1, 140.7, 138.5, 135.5, 134.3, 132.8, 132.1, 130.9, 130.2,
129.9, 129.5, 129.1, 128.8, 126.3, 125.6, 125.4, 121.2, 116.5, 52.1,
49.5, 40.8, 29.0, 20.8, 20.0, 8.4. MS (ESI negative): 591 (MꢀH)ꢀ.
0.0037 mmol), 2-dicyclohexylphosphino-20,60-dimethoxy-1,10-biphenyl
(SPhos) (3.0 mg, 0.0073 mmol), and K3PO4 (23 mg, 0.110 mmol) and
the mixture was stirred at 100 °C for 1.5 h under nitrogen atmosphere.
The mixture was poured into satd NaHCO3 aq solution and the products
were extracted with CH2Cl2. The extracts were dried over anhydrous
MgSO4 and concentrated. The obtained residue was purified by prepara-
tive TLC (n-hexane/AcOEt = 4:1) to give ester.
5.1.7. 40-{1-Ethyl-1-[4-((E)-3-ethyl-3-hydroxy-1-pentenyl)-3-
methylphenyl] propyl}-20-methylbiphenyl-4-carboxylic acid
methyl ester (16a)
5.1.12. (40-{1-Ethyl-1-[3-methyl-4-((E)-4,4,4-trifluoro-3-
hydroxy-3-trifluoromethyl-1-butenyl)phenyl]propyl}-3-fluoro-
20-methylbiphenyl-4-yl)acetic acid methyl ester (17d)
The yield was 67%. Colorless oil. 1H NMR (CDCl3) d: 0.66 (6H, t,
J = 7.2 Hz), 0.92 (6H, t, J = 7.5 Hz), 1.65 (4H, q, J = 7.5 Hz), 2.13 (4H,
q, J = 7.3 Hz), 2.23 (3H, s), 2.34 (3H, s), 3.94 (3H, s), 6.03 (1H, d,
J = 15.9 Hz), 6.76 (1H, d, J = 15.9 Hz), 6.96–7.02 (6H, m), 7.05 (1H,
dd, J = 7.7, 2.1 Hz), 7.08–7.12 (3H, m), 7.33 (1H, d, J = 7.8 Hz), 7.41
(2H, d, J = 8.6 Hz), 8.07 (2H, d, J = 8.6 Hz). 13C NMR (CDCl3) d:
167.2, 148.2, 147.5, 146.9, 137.7, 135.9, 134.5, 134.0, 133.4,
130.1, 129.9, 129.5, 129.4, 129.3, 128.9, 126.0, 125.9, 125.8,
124.9, 76.0, 52.1, 49.4, 33.3, 29.1, 20.7, 20.3, 8.5, 7.9. MS (ESI neg-
ative): 497 (MꢀH)ꢀ.
The yield was 61%. Colorless oil. 1H NMR (CDCl3) d: 0.65 (6H, t,
J = 7.2 Hz), 2.12 (4H, q, J = 7.2 Hz), 2.23 (3H, s), 2.36 (3H, s), 3.71
(2H, s), 3.74 (3H, s), 6.10 (1H, d, J = 15.8 Hz), 6.99–7.09 (7H, m),
7.28 (1H, d, J = 7.8 Hz), 7.37 (1H, d, J = 8.4 Hz), 7.39 (1H, d,
J = 16.0 Hz). 13C NMR (CDCl3) d: 171.4, 159.3, 149.9, 147.7, 143.3,
143.2, 137.4, 135.6, 135.1, 134.2, 130.9, 130.8, 130.2, 130.0,
129.0, 126.3, 125.7, 125.5, 125.1, 125.1, 119.4, 116.4, 116.4, 52.3,
49.5, 34.1, 29.0, 20.8, 20.0, 8.4. MS (ESI negative): 609 (MꢀH)ꢀ.
5.1.13. [6-(4-{1-Ethyl-1-[4-((E)-3-ethyl-3-hydroxy-1-pentenyl)-
3-methylphenyl]propyl}-2-methylphenyl)pyridin-3-yl]acetic
acid methyl ester (16e)
5.1.8. 40-{1-Ethyl-1-[4-((E)-3-ethyl-3-hydroxy-1-pentenyl)-3-
methylphenyl] propyl}-20-methylbiphenyl-3-carboxylic acid
methyl ester (16b)
To a solution of 14 (50 mg, 0.102 mmol) in DMF (1 mL) were
added
methyl
2-(6-bromopyridin-3-yl)acetate
(38.0 mg,
The yield was 51%. Colorless oil. 1H NMR (CDCl3) d: 0.67 (6H, t,
J = 7.3 Hz), 0.93 (6H, t, J = 7.5 Hz), 1.65 (4H, q, J = 7.5 Hz), 2.13 (4H,
q, J = 7.3 Hz), 2.22 (3H, s), 2.34 (3H, s), 3.92 (3H, s), 6.03 (1H, d,
J = 15.9 Hz), 6.77 (1H, d, J = 15.9 Hz), 6.98–7.02 (2H, m), 7.05 (1H,
dd, J = 7.9, 1.8 Hz), 7.08–7.11 (2H, m), 7.33 (1H, d, J = 8.1 Hz), 7.46
(1H, t, J = 7.7 Hz), 7.53 (1H, d, J = 7.6 Hz), 7.99 (1H, d, J = 7.6 Hz),
8.02 (1H, s). 13C NMR (CDCl3) d: 167.2, 148.0, 147.6, 142.2, 137.7,
135.9, 134.5, 134.1, 133.8, 133.4, 130.5, 130.0, 129.9, 129.6,
129.0, 128.1, 127.7, 126.0, 125.9, 125.8, 124.9, 76.0, 52.1, 49.3,
33.3, 29.1, 20.7, 20.3, 8.5, 7.9. MS (ESI positive): 521 (M+H)+.
0.165 mmol), tetrakis(triphenylphosphine)palladium(0) (23.1 mg,
0.020 mmol), and K3PO4 (63.9 mg, 0.301 mmol). The mixture was
stirred at 110 °C for 30 min under nitrogen atmosphere. The mix-
ture was poured into satd NaHCO3 aq solution and the products
were extracted with AcOEt. The extracts were washed with water
and brine, dried over anhydrous MgSO4 and concentrated. The ob-
tained residue was purified by preparative TLC (n-hexane/
AcOEt = 2:1) to give 16e (24.6 mg, 48%) as a colorless oil. 1H NMR
(CDCl3) d: 0.65 (6H, t, J = 7.2 Hz), 0.92 (6H, t, J = 7.4 Hz), 1.64 (4H,
q, J = 7.4 Hz), 2.11 (4H, q, J = 7.0 Hz), 2.32 (3H, s), 2.32 (3H, s),
3.68 (2H, s), 3.75 (3H, s), 6.02 (1H, d, J = 16.0 Hz), 6.75 (1H, d,
J = 16.0 Hz), 6.96–7.01 (2H, m), 7.05–7.10 (2H, m), 7.27 (1H, d,
J = 7.0 Hz), 7.31 (1H, d, J = 7.8 Hz), 7.39 (1H, d, J = 8.2 Hz), 7.69
(1H, dd, J = 8.0, 2.2 Hz), 8.57 (1H, s). 13C NMR (CDCl3) d: 171.4,
159.0, 149.6, 148.7, 147.6, 137.0, 136.9, 135.9, 134.8, 134.5,
133.4, 130.5, 130.1, 129.0, 127.2, 126.0, 125.9, 124.8, 123.8, 76.0,
5.1.9. (40-{1-Ethyl-1-[4-((E)-3-ethyl-3-hydroxy-1-pentenyl)-3-
methylphenyl]propyl}-20-methylbiphenyl-4-yl)acetic acid
methyl ester (16c)
The yield was 42%. Colorless oil. 1H NMR (CDCl3) d: 0.66 (6H, t,
J = 7.2 Hz), 0.92 (6H, t, J = 7.5 Hz), 1.65 (4H, q, J = 7.5 Hz), 2.12 (4H,

H. Kashiwagi et al. / Bioorg. Med. Chem. 21 (2013) 1823–1833
1831
52.3, 49.4, 38.0, 33.3, 29.1, 20.6, 20.3, 8.4, 7.9. MS (ESI positive):
5.1.19. (40-{1-Ethyl-1-[4-((E)-3-ethyl-3-hydroxy-1-pentenyl)-3-
514 (M+H)+.
methylphenyl]propyl}-3-fluoro-20-methylbiphenyl-4-yl)acetic
acid (7d)
5.1.14. [6-(4-{1-Ethyl-1-[3-methyl-4-((E)-4,4,4-trifluoro-3-
hydroxy-3-trifluoromethyl-1-butenyl)phenyl]propyl}-2-
methylphenyl)pyridin-3-yl]acetic acid methyl ester (17e)
The yield was 75%. Colorless oil. 1H NMR (CDCl3) d: 0.64 (6H, t,
J = 7.1 Hz), 2.11 (4H, q, J = 7.0 Hz), 2.28 (3H, s), 2.28 (3H, s), 3.60
(2H, s), 3.72 (3H, s), 6.05 (1H, d, J = 16.2 Hz), 6.98–7.06 (4H, m),
7.23 (1H, dd, J = 8.0, 2.2 Hz), 7.30 (1H, dd, J = 8.0, 3.3 Hz), 7.36
(1H, d, J = 16.2 Hz), 7.38 (1H, d, J = 7.8 Hz), 7.70 (1H, d, J = 7.6 Hz),
8.54 (1H, s). 13C NMR (CDCl3) d: 171.3, 158.7, 150.5, 150.0, 149.6,
149.3, 140.8, 139.7, 139.5, 135.5, 134.8, 131.1, 130.3, 130.2,
129.0, 129.0, 127.9, 126.1, 125.7, 125.4, 124.1, 124.0, 117.0, 52.4,
49.5, 37.9, 28.9, 20.5, 19.9, 8.3. MS (ESI positive): 594 (M+H)+.
The yield was 79%. Colorless oil. 1H NMR (CDCl3) d: 0.65 (6H, t,
J = 7.1 Hz), 0.92 (6H, t, J = 7.4 Hz), 1.64 (4H, q, J = 7.3 Hz), 2.11 (4H,
q, J = 7.2 Hz), 2.23 (3H, s), 2.33 (3H, s), 3.75 (2H, s), 6.02 (1H, d,
J = 16.0 Hz), 6.76 (1H, d, J = 16.0 Hz), 6.96–7.09 (7H, m), 7.29 (1H,
d, J = 7.8 Hz), 7.32 (1H, d, J = 8.0 Hz). 13C NMR (CDCl3) d: 175.9,
159.4, 148.0, 147.5, 143.6, 137.2, 135.8, 134.5, 134.1, 130.9,
130.0, 129.9, 128.9, 126.0, 125.9, 125.8, 125.2, 125.2, 124.9,
118.6, 116.4, 76.1, 49.3, 33.9, 33.3, 29.1, 20.8, 20.3, 8.5, 7.9. HRMS
(ESI negative): Calcd for C34H40FO3 515.2956. Found: 515.2956
(MꢀH)ꢀ.
5.1.20. [6-(4-{1-Ethyl-1-[4-((E)-3-ethyl-3-hydroxy-1-pentenyl)-
3-methylphenyl]propyl}-2-methylphenyl)pyridin-3-yl]acetic
acid (7e)
5.1.15. General procedure for hydrolysis of ester group of 16a–e,
17c–e
The yield was 65%. Colorless oil. 1H NMR (CDCl3) d: 0.65 (6H, t,
J = 7.2 Hz), 0.92 (6H, t, J = 7.4 Hz), 1.64 (4H, q, J = 7.4 Hz), 2.11 (4H,
q, J = 7.2 Hz), 2.30 (3H, s), 2.32 (3H, s), 3.70 (2H, s), 6.02 (1H, d,
J = 15.8 Hz), 6.75 (1H, d, J = 16.0 Hz), 6.97–7.01 (2H, m), 7.05–7.09
(2H, m), 7.26 (1H, d, J = 7.2 Hz), 7.31 (1H, d, J = 7.8 Hz), 7.39 (1H,
d, J = 8.0 Hz), 7.71 (1H, d, J = 7.4 Hz), 8.59 (1H, s). 13C NMR (CDCl3)
d: 174.3, 158.8, 149.4, 148.9, 147.5, 137.3, 136.7, 134.8, 134.5,
133.4, 130.4, 130.0, 129.0, 128.8, 126.0, 125.8, 125.5, 124.8,
124.0, 76.0, 49.4, 37.7, 33.3, 29.1, 20.6, 20.3, 8.4, 7.9. HRMS (ESI
To a solution of ester (0.028 mmol) in THF (1.5 mL) and MeOH
(1.5 mL) was added 1 N NaOH (0.084 mL, 0.084 mmol) and the
mixture was stirred at rt overnight. The mixture was poured into
satd NH4Cl aq solution and the products were extracted with
CH2Cl2. The extracts were dried over anhydrous MgSO4 and con-
centrated. The obtained residue was purified by preparative TLC
(CHCl3/MeOH = 10:1) to afford carboxylic acid.
5.1.16. 40-{1-Ethyl-1-[4-((E)-3-ethyl-3-hydroxy-1-pentenyl)-3-
methylphenyl] propyl}-20-methylbiphenyl-4-carboxylic acid
(7a)
negative): Calcd for C33H40NO3 498.3003. Found: 498.3016
(MꢀH)ꢀ.
The yield was 60%. Colorless oil. 1H NMR (CDCl3) d: 0.67 (6H, t,
J = 7.2 Hz), 0.93 (6H, t, J = 7.5 Hz), 1.66 (4H, q, J = 7.5 Hz), 2.13 (4H,
q, J = 7.2 Hz), 2.25 (3H, s), 2.34 (3H, s), 6.03 (1H, d, J = 15.9 Hz), 6.77
(1H, d, J = 16.1 Hz), 6.97–7.02 (2H, m), 7.06 (1H, dd, J = 7.9, 1.8 Hz),
7.09–7.13 (2H, m), 7.34 (1H, d, J = 7.8 Hz), 7.45 (2H, d, J = 8.6 Hz),
8.15 (2H, d, J = 8.6 Hz). 13C NMR (CDCl3) d: 171.6, 148.4, 147.7,
147.5, 137.6, 135.9, 134.5, 134.1, 133.4, 130.1, 129.9, 129.9,
129.5, 128.9, 127.4, 126.0, 125.9, 125.8, 124.9, 76.1, 49.4, 33.3,
29.1, 20.8, 20.3, 8.5, 7.9. HRMS (ESI negative): Calcd for C33H39O3
483.2905. Found: 483.2901 (MꢀH)ꢀ.
5.1.21. (40-{1-Ethyl-1-[3-methyl-4-((E)-4,4,4-trifluoro-3-
hydroxy-3-trifluoromethyl-1-butenyl)phenyl]propyl}-20-
methylbiphenyl-4-yl)acetic acid (8c)
The yield was 78%. Colorless oil. 1H NMR (CDCl3) d: 0.65 (6H, t,
J = 7.1 Hz), 2.11 (4H, q, J = 7.3 Hz), 2.21 (3H, s), 2.35 (3H, s), 3.68
(2H, s), 6.08 (1H, d, J = 16.1 Hz), 6.99 (1H, dd, J = 8.1, 1.7 Hz),
7.01–7.04 (2H, m), 7.04–7.09 (2H, m), 7.29 (4H, d, J = 2.0 Hz),
7.36 (1H, d, J = 7.8 Hz), 7.37 (1H, d, J = 16.1 Hz). 13C NMR (CDCl3)
d: 177.6, 150.0, 147.2, 141.0, 138.4, 135.6, 135.2, 134.3, 132.2,
131.4, 130.8, 130.2, 129.9, 129.6, 129.1, 129.0, 126.3, 125.6,
125.4, 121.4, 116.4, 49.5, 40.7, 29.0, 20.8, 20.0, 8.4. HRMS (ESI neg-
ative): Calcd for C32H31F6O3 577.2183. Found: 577.2195 (M–H)ꢀ.
5.1.17. 40-{1-Ethyl-1-[4-((E)-3-ethyl-3-hydroxy-1-pentenyl)-3-
methylphenyl] propyl}-20-methylbiphenyl-3-carboxylic acid
(7b)
5.1.22. (40-{1-Ethyl-1-[3-methyl-4-((E)-4,4,4-trifluoro-3-
hydroxy-3-trifluoromethyl-1-butenyl)phenyl]propyl}-3-fluoro-
20-methylbiphenyl-4-yl)acetic acid (8d)
The yield was 61%. Colorless oil. 1H NMR (CDCl3) d: 0.67 (6H, t,
J = 7.2 Hz), 0.93 (6H, t, J = 7.5 Hz), 1.66 (4H, q, J = 7.6 Hz), 2.13 (4H,
q, J = 7.3 Hz), 2.24 (3H, s), 2.34 (3H, s), 6.03 (1H, d, J = 15.9 Hz), 6.77
(1H, d, J = 15.9 Hz), 6.99–7.02 (2H, m), 7.06 (1H, dd, J = 7.9, 1.8 Hz),
7.08–7.13 (2H, m), 7.34 (1H, d, J = 8.6 Hz), 7.50 (1H, t, J = 7.7 Hz),
7.59 (1H, d, J = 7.8 Hz), 8.07 (1H, d, J = 7.6 Hz), 8.10 (1H, s). 13C
NMR (CDCl3) d: 171.6, 148.1, 147.6, 142.4, 137.6, 135.9, 134.6,
134.5, 134.2, 133.4, 131.1, 130.1, 129.9, 129.1, 129.0, 128.3,
128.2, 126.0, 125.9, 125.8, 124.9, 76.1, 49.4, 33.3, 29.1, 20.7, 20.3,
8.5, 7.9. HRMS (ESI negative): Calcd for C33H39O3 483.2905. Found:
483.2904 (MꢀH)ꢀ.
The yield was 95%. Colorless oil. 1H NMR (CDCl3) d: 0.65 (6H, t,
J = 7.0 Hz), 2.12 (4H, q, J = 6.9 Hz), 2.23 (3H, s), 2.36 (3H, s), 3.75
(2H, s), 6.09 (1H, d, J = 15.8 Hz), 6.99–7.10 (7H, m), 7.29 (1H, d,
J = 7.8 Hz), 7.37 (1H, d, J = 7.8 Hz), 7.38 (1H, d, J = 16.4 Hz). 13C
NMR (CDCl3) d: 176.2, 159.4, 149.9, 147.7, 143.5, 143.5, 137.4,
135.6, 135.2, 134.2, 130.9, 130.9, 130.8, 130.2, 130.0, 129.0,
126.3, 125.7, 125.5, 125.2, 125.2, 118.8, 116.4, 116.2, 49.5, 34.0,
29.0, 20.8, 20.0, 8.4. HRMS (ESI negative): Calcd for C32H30F7O3
595.2078. Found: 595.2090 (MꢀH)ꢀ.
5.1.18. (40-{1-Ethyl-1-[4-((E)-3-ethyl-3-hydroxy-1-pentenyl)-3-
methylphenyl]propyl}-20-methylbiphenyl-4-yl)acetic acid (7c)
The yield was 76%. Colorless oil. 1H NMR (CDCl3) d: 0.66 (6H, t,
J = 7.2 Hz), 0.92 (6H, t, J = 7.5 Hz), 1.65 (4H, q, J = 7.5 Hz), 2.12 (4H,
q, J = 7.3 Hz), 2.23 (3H, s), 2.33 (3H, s), 3.70 (2H, s), 6.02 (1H, d,
J = 15.9 Hz), 6.76 (1H, d, J = 16.1 Hz), 6.98–7.03 (3H, m), 7.05–7.10
(2H, m), 7.31 (4H, d, J = 1.7 Hz), 7.33 (1H, d, J = 7.8 Hz). 13C NMR
(CDCl3) d: 176.8, 147.6, 147.5, 141.0, 138.3, 135.8, 134.4, 134.1,
133.3, 131.4, 129.9, 129.6, 129.0, 128.9, 126.0, 125.9, 125.7, 124.9,
76.1, 49.3, 40.6, 33.3, 29.1, 20.8, 20.3, 8.5, 7.9. HRMS (ESI negative):
Calcd for C34H41O3 497.3061. Found: 497.3051 (MꢀH)ꢀ.
5.1.23. [6-(4-{1-Ethyl-1-[3-methyl-4-((E)-4,4,4-trifluoro-3-
hydroxy-3-trifluoromethyl-1-butenyl)phenyl]propyl}-2-
methylphenyl)pyridin-3-yl]acetic acid (8e)
The yield was 40%. Colorless oil. 1H NMR (CDCl3) d: 0.64 (6H, t,
J = 7.1 Hz), 2.12 (4H, q, J = 14.5 Hz), 2.24 (3H, s), 2.29 (3H, s), 3.63
(2H, s), 6.06 (1H, d, J = 15.8 Hz), 6.98–7.06 (4H, m), 7.29–7.32
(2H, m), 7.34–7.37 (2H, m), 7.72 (1H, dd, J = 8.1, 1.7 Hz), 8.52
(1H, s). 13C NMR (CDCl3) d: 174.5, 158.0, 150.5, 149.7, 148.9,
148.6, 138.1, 136.0, 135.6, 135.1, 134.9, 131.0, 130.2, 129.1,
128.9, 128.1, 128.0, 126.2, 125.7, 125.5, 125.4, 124.4, 117.0, 49.5,

1832
H. Kashiwagi et al. / Bioorg. Med. Chem. 21 (2013) 1823–1833
37.9, 28.8, 20.4, 19.9, 8.3. HRMS (ESI negative): Calcd for
C
5.5. Bone mineral density and serum calcium evaluation in the
ovariectomized eight-month-old rats
31H30F6O3 578.2124. Found: 578.2127 (MꢀH)ꢀ.
Sham-operated (n = 8) or ovariectomized, eight-month-old, fe-
male, Sprague–Dawley (Crj: CD (SD)) rats (Charles River Japan,
Inc.) were used. One day after the surgery, the OVX rats were divided
into six groups (n = 8), and compound 7e (0.75, 1.5. 3.0, 6.0, 12 ng/kg,
five times per week) or vehicle (medium-chain triglyceride (MCT),
1 mL/kg, five times per week) was orally administered for
4 weeks.13,32 Vehicle-administered OVX rats and sham-operated
rats served as controls. After 24 h from the last administration, blood
was drawn from the abdominal aorta under ether anesthesia. Serum
was collected after the blood sample had been centrifuged and was
stored at ꢀ20 °C. The right femur and lumbarvertebrae (L2–L5)were
stored in 70% ethanol at 4 °C. Serum calcium concentrations were
measured by an autoanalyzer (Hitachi 7170, Tokyo, Japan). BMD in
the distal femur and lumbar spine was measured by dual-energy
X-ray absorptiometry (DCS-600-EX, Aloka, Japan).
5.2. VDRE reporter gene assay
MG-63 cells were plated at 2 ꢁ 103 cells/200
lL/well in a 96-
well white cell-culture plate and were incubated at 37 °C in 5%
CO2 for 24 h. Then, the MG-63 cells were cotransfected with
0.05
three repeats of the VDRE sequence from mouse osteopontin pro-
moter and 0.001 g of pRL-SV40 vector (Promega Corporation, WI,
lg of the pGV2-basic/VDRE-luciferase vector which contained
l
USA) using Lipofectamine (Invitrogen). The cells were added to
minimum essential medium (MEM) containing 5% fetal bovine ser-
um treated with dextran-coated charcoal (DCC-FBS) and were
incubated for 8 h. The cells were treated with the serial diluted
compounds (final concentrations were 10ꢀ7 to 10ꢀ11 mol/L with
0.1% DMSO) and were incubated for an additional 3 days. After
removing the supernatants, the cells were lysed in cell-lysis buffer
and luciferase activity was measured by DLR™ Assay System (Pro-
mega Corporation, WI, USA) and the luminescence was detected by
Wallac ALVO SX 1420 multi-label counter (PerkinElmer, Inc., MA,
USA). The half maximal effective concentrations (EC50) were deter-
mined and the inductive activity was calculated as the ratio of the
EC50 value of the compounds to that of 1,25(OH)2D3 (Solvay Phar-
maceuticals, Weesp, The Netherlands), which was used as a posi-
tive control.
5.6. Pharmacokinetic study
Compound 7e in a dosing solution of ethanol/Tween20/saline
(10:2:88) or ethanol/MCT (1:9) was administered to male Spra-
gue–Dawley rats (8 weeks old, Orient Bio, Co.) under a fed condition
at 100 lg/kg intravenously or orally, respectively (n = 3). Blood sam-
ples were collected from the jugular vein and immediately centri-
fuged to obtain plasma samples. The compound in plasma was
extracted with diethyl ether, and plasma concentration of the com-
pound was measured by an API3000 tandem mass spectrometer (AB
Sciex, Foster city, CA, USA) equipped with an Agilent 1100 liquid
chromatography (Agilent Technologies, Santa Clara, CA, USA).
5.3. Osteocalcin induction assay
MG-63 cells were plated at 2 ꢁ 103 cells/well in 200
lL of ser-
um-free MEM in a 96-well plate and were incubated at 37 °C in
5% CO2 incubator for 24 h. After washing cells with 5% DCC-FBS/
MEM (culture medium), the cells were added to culture medium
and treated with the serial diluted compounds (final concentra-
tions were 10ꢀ7 to 10ꢀ11 mol/L with 0.1% DMSO), and incubated
for 8 h. After changing the culture medium to a fresh one, the cells
were incubated for an additional 4 days, and then supernatant was
collected and stored at ꢀ80 °C.
5.7. Crystallographic structure analysis
Protein sample preparation and crystallization were based on
the methods by Moras and co-workers33,34 X-ray diffraction data
collection and data processing were carried out by generally ac-
cepted methods. After brief soaking in the buffer containing 30%
glycerol, 0.6 M ammonium sulfate and 0.1 M MES (pH6.3), crystals
were trapped in the fiber loops and flash-cooled with liquid nitro-
gen. Crystals were stored and transported in a dry shipper. Data
collections were carried out at the synchrotron beamline BL32B2
of the Spring-8 facility in Hyogo, Japan, operated by Pharmaceuti-
cal Consortium for Protein Structure Analysis. Crystals were kept
frozen during data collection with a vapor stream from liquid
nitrogen. X-ray diffraction data was collected with the R-AXIS V
imaging plate area detector. The crystals were isomorphous with
the space group P212121 reported by Moras and co-workers33,34
for the 1,25(OH)2D3–VDR complex. Data were processed with
HKL2000. The initial structure model for the refinement was con-
structed by removing all the water and the ligand atoms in the
1,25(OH)2D3–VDR complex structure in the Protein Databank
(PDB ID: 1db1). A rigid body refinement was followed by simulated
annealing using the CNX. Electron density in the ligand-binding
pocket clearly and unambiguously showed the ligand conforma-
tion. After fitting the ligand model into the electron density, struc-
ture refinement was continued with the software autoBUSTER and
the water molecules were placed automatically. The data collec-
tion and the refinement statistics are summarized in Table 4.
The frozen supernatant was thawed slowly at rt and Gla-type
osteocalcin EIA kit (Takara Bio. Inc., Tokyo, Japan) was used to mea-
sure osteocalcin. Absorbance at 450 nm was measured on a plate
reader (Model 3550, Bio-Rad Laboratories, CA, USA) and each con-
centration was calculated by comparison with standards using
lplate Manager I software (Bio-Rad Laboratories). The EC50 value
was determined and the inductive activity was calculated as the
ratio of the EC50 value of the compounds to that of 1,25(OH)2D3.
5.4. Bone mineral density and serum calcium evaluation in the
ovariectomized 8-week-old rats
Sham-operated (n = 6) and ovariectomized eight-week-old fe-
male Sprague–Dawley (Crj: CD (SD)) rats (Charles River Japan, Inc.)
were used. One day after the surgery, the OVX rats were divided into
each groups (n = 6), and were given compoundsorally (25 and 50 ng/
kg for 7c, 10, 20, and 40 ng/kg for 7e, 10 and 30 ng/kg for 8c, or 20 and
60 ng/kg for 8d, five times per week) or vehicle (medium-chain tri-
glyceride (MCT), 1 mL/kg, five times per week) for 4 weeks. Vehi-
cle-administered OVX rats and sham-operated rats served as
controls. After 24 h from the last administration, blood was drawn
from the abdominal aorta under ether anesthesia. Serum was col-
lected after the blood sample had been centrifuged and was stored
at ꢀ20 °C. The right femur was stored in 70% ethanol at 4 °C. Serum
calcium concentrations were measured by an autoanalyzer (Hitachi
7170, Tokyo, Japan). The BMD in distal femur was measured by dual-
energy X-ray absorptiometry (DCS-600-EX, Aloka, Japan).
5.8. Modeling
The compounds were manually docked in the crystal structure
of VDR (PDB ID: 3W0C). The conformations of compounds were
optimized using the MAB force field as implemented in the pro-
gram MOLOC28 with the VDR structure fixed.

H. Kashiwagi et al. / Bioorg. Med. Chem. 21 (2013) 1823–1833
9. Hayashi, Y.; Fujita, T.; Inoue, T. J. Bone Miner. Metab. 1992, 10, 50.
1833
Table 4
10. Orimo, H.; Shiraki, M.; Hayashi, Y.; Hoshino, T.; Onaya, T.; Miyazaki, S.;
Kurosawa, H.; Nakamura, T.; Ogawa, N. Calcif. Tissue Int. 1994, 54, 370.
11. Okano, T.; Tsugawa, N.; Masuda, S.; Takeuchi, A.; Kobayashi, T.; Nishii, Y.
Contrib. Nephrol. 1991, 91, 116.
12. Tsurukami, H.; Nakamura, T.; Suzuki, K.; Sato, K.; Higuchi, Y.; Nishii, Y. Calcif.
Tissue Int. 1994, 54, 142.
13. Uchiyama, Y.; Higuchi, Y.; Takeda, S.; Masaki, T.; Shira-Ishi, A.; Sato, K.;
Kubodera, N.; Ikeda, K.; Ogata, E. Bone 2002, 30, 582.
14. Matsumoto, T.; Miki, T.; Hagino, H.; Sugimoto, T.; Okamoto, S.; Hirota, T.;
Tanigawara, Y.; Hayashi, Y.; Fukunaga, M.; Shiraki, M.; Nakamura, T. J. Clin.
Endocrinol. Metab. 2005, 90, 5031.
15. Matsumoto, T.; Ito, M.; Hayashi, Y.; Hirota, T.; Tanigawara, Y.; Sone, T.;
Fukunaga, M.; Shiraki, M.; Nakamura, T. Bone 2011, 49, 605.
16. Sanford, M.; McCormack, P. L. Drugs 2011, 71, 1755.
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E. A. Chem. Biol. 1999, 6, 265.
Crystal and diffraction data of VDR with compound 8d
8d
Wavelength (Å)
Cell (Å)
(a)
(b)
(c)
1.0
45.2
51.1
132.7
2.0
90.4 (90.7)
8.8 (32.8)
17.5 (18.6)
20.9 (23.9)
0.010
Resolution (Å)
Completeness (last shell) (%)
Rsym (last shell) (%)
Rcryst (last shell) (%)
Rfree (last shell) (%)
Rmsd bond length (Å)
Rmsd bond angles (°)
No. of non-hydrogen protein atoms
No. of water molecules
0.99
1993
286
19. Swann, S. L.; Bergh, J.; Farach-Carson, M. C.; Ocasio, C. A.; Koh, J. T. J. Am. Chem.
Soc. 2002, 124, 13795.
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2060.
Acknowledgements
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The authors thank Ms. Hitomi Suda of Chugai Pharmaceutical
Co., Ltd, for HRMS measurements of the compounds and Editing
Services of Chugai Pharmaceutical Co., Ltd, for proofreading the
manuscript.
References and notes
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