Novel nonsecosteroidal vitamin D3 carboxylic acid analogs for osteoporosis, and SAR analysis

Article abstract of DOI:10.1016/j.bmc.2011.07.001

Novel vitamin D₃ analogs with carboxylic acid were explored, focusing on a nonsecosteroidal analog, LG190178, with a bisphenyl skeleton. From X-ray analysis of these analogs with vitamin D receptor (VDR), the carboxyl groups had very unique hyd

Full text of DOI:10.1016/j.bmc.2011.07.001

Bioorganic & Medicinal Chemistry 19 (2011) 4721–4729
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel nonsecosteroidal vitamin D3 carboxylic acid analogs for osteoporosis,
and SAR analysis
⇑
Hirotaka Kashiwagi , Yoshiyuki Ono, Kazuki Shimizu, Tsuyoshi Haneishi, Susumu Ito, Shigeyuki Iijima,
Takamitsu Kobayashi, Fumihiko Ichikawa, Suguru Harada, Hideki Sato, 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:
Novel vitamin D3 analogs with carboxylic acid were explored, focusing on a nonsecosteroidal analog,
LG190178, with a bisphenyl skeleton. From X-ray analysis of these analogs with vitamin D receptor
(VDR), the carboxyl groups had very unique hydrogen bonding interactions in VDR and mimicked
Received 23 May 2011
Revised 30 June 2011
Accepted 1 July 2011
Available online 8 July 2011
1a-hydroxy group and/or 3b-hydroxy group of 1a,25-dihydroxyvitamin D3. A highly potent analog, 6a,
with good in vitro activity and pharmacokinetic profiles was identified from an SAR study. Compound
6a showed significant prevention of bone loss in a rat osteoporosis model by oral administration.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Vitamin D3
VDR agonist
Nonsecosteroidal
Bisphenylmethane
Osteoporosis
1. Introduction
in the side chain of 3 worked like the 1-OH and 25-OH groups of 1,
respectively.17–19
1
a
,25-Dihydroxyvitamin D3, [1,25(OH)2D3], 1, is a known classic
Hydrogen bonding of the 2-OH group in the A-part of 3 with
Ser237 and Arg274 would be of especial interest, because Arg274
in VDR is an essential amino acid which binds the 1-OH group of
1, and indeed the lack of this interaction, such as in 25-OH vitamin
D3, decreased the binding affinity to VDR. However, the structure–
activity relationships (SAR) of the A-part of 2 is still unclear.
Here we describe the SAR of nonsecosteroidal carboxylic acid
derivatives focusing on an interaction of the A-part of the ligand
with Arg274. We attempted to introduce a carboxyl group with
methylene linker into the A-part of the bisphenyl skeleton (Fig. 1).
The SAR study revealed the carboxyl group and methylene linker
length significantly influence the VDR agonistic activity. As a result,
we successfully confirmed that our carboxyl group had a salt bridge
interaction to Arg274 in VDR from X-ray analysis of these
derivatives.
ligand of vitamin D receptor (VDR) which is synthesized in skin,
liver, and kidney and plays an essential role in calcium homeosta-
sis.1–3 It stimulates calcium absorption in intestine, regulates bone
formation and resorption, enhances calcium reabsorption in
kidney, and inhibits the synthesis and secretion of parathyroid hor-
mone (PTH) in the parathyroid gland.4 Physiological actions of 1 are
exhibited by binding to VDR and the consequent formation of a
complex with several co-factors which binds to vitamin D respon-
sive element (VDRE) in the promoter moiety of target genes
controlling the transcription.5–9
Over three decades, derivatizations of vitamin D3 have focused
on the secosteroidal skeleton. Chemical modifications have led to a
wide range of derivatives, especially around the side chain moiety
and the A ring moiety of secosteroidal vitamin D3 structure.
Accordingly, more potent VDR agonists than 1,25(OH)2D3 (1) have
been reported.10
We evaluated these analogs in the reporter gene activity of
VDRE and osteocalcin production activity as a VDR agonist in vitro.
Also, in vivo effects on bone and serum calcium levels were evalu-
ated using a rat osteoporosis model, which is commonly used in
the evaluation of the activity of vitamin D3 analogs.20,21
Meanwhile, there has been a growing interest in nonsecosteroi-
dal VDR agonists, since a bisphenyl compound, LG190178 (2),
showed characteristics of a vitamin D3 analog in vitro.11–16
Recently the crystal structure of YR301 (3), which is a stereoiso-
mer of 2, with VDR was reported and it has revealed that the second-
ary hydroxyl group in the A-part and the secondary hydroxyl group
2. Chemistry
The synthesis of compounds 4a–d and 6a is shown in Scheme 1.
Compound 7 was prepared from bisphenol by the method reported
⇑
Corresponding author.
E-mail address: kashiwagihrt@chugai-pharm.co.jp (H. Kashiwagi).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.07.001

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H. Kashiwagi et al. / Bioorg. Med. Chem. 19 (2011) 4721–4729
Side Chain
Side Chain
b-elimination. Pyridinium dichromate (PDC) oxidation of alcohol
9 afforded carboxylic acid 10. Reduction of ketone group of 10
using NaBH4 gave carboxylic acid 4b. Alkylation of phenol 7 with
tosylate 11 afforded lactone 12 and was then treated with NaBH4
to give alcohol 13. Compound 13 was hydrolyzed with 1 N NaOH
to afford gamma hydroxycarboxylic acid 6a in very good yield.
Carbon link side chain analogs were prepared from bisphenol
14, which was also reported by Boehm11 (Scheme 2). Treatment
of 14 with triflic anhydride and pyridine afforded mono triflate
15. Palladium-catalyzed Sonogashira coupling of triflate 15 with
16 gave acetylene 17 and the following hydrogenation gave phenol
18 in moderate yield. Alkylation of phenol group using K2CO3 and
alkyl halides and tosylate 11 afforded corresponding ester 19a–c
and lactone 20 in very good yield. Every ester or lactone was
hydrolyzed in the usual manner and carboxylic acid derivatives
5a–c and gamma hydroxycarboxylic acid 6b were obtained,
respectively.
OH
O
25
H
OH
H
A ring
O
3
HO
A part
OH
1
HO
OH
LG190178
OH
1 1α, 25-dihydroxyvitamin D3
2
OH
O
X
2'(R)
4a: X = O; R = CH2COOH
4b: X = O; R = (CH2)2COOH
4c: X = O; R = (CH2)3COOH
4d: X = O; R = (CH2)4COOH
5a :X = CH2; R = (CH2)4COOH
5b: X = CH2; R = (CH2)5COOH
5c: X = CH2; R = (CH2)6COOH
6a: X = O;
O
O
R
2(S)
HO
3. Results and discussion
OH
R = CH2CH(OH)(CH2)2COOH
6b: X = CH2;
3
YR301
All synthesized compounds were evaluated by reporter gene as-
say in MG-63 cells, which contained VDRE sequence derived from
mouse osteopontin promoter for vitamin D agonistic activity. The
osteocalcin production activity in MG-63 cells was evaluated to
measure bone formation activity of functional vitamin D agonistic
effect.22–24 VDRE reporter gene activity and osteocalcin activity are
represented by a relative EC50 value which is the EC50 value of
1,25(OH)2D3 assigned as 100% (a larger figure means stronger
activity).
At first, we examined the biological activity to see the impact of
linker length between carboxyl group and bisphenyl ring on VDR
agonistic activity (4a–d, Table 1).
As expected, the carboxylic acid derivatives exhibited VDR ago-
nistic activity. In addition, we found that there is an optimal linker
length (4d and 5a), which showed more potent activity than
R = CH2CH(OH)(CH2)2COOH
Figure 1. Structures of secosteroids 1,25(OH)2D3 (1), nonsecosteroidal analog
LG190178 (2) and YR301 (3), and novel nonsecosteroidal carboxylic acid derivatives
(4a–d, 5a–c, 6a, and 6b). Part labeled A of 2 (upper right) shows the structural
correspondence to the A ring of secosteroids (upper left).
by Boehm.11 Alkylation of 7 with K2CO3 and various alkylbromides
gave corresponding ester (8a, 8c, and 8d) in moderate yields.
Hydrolysis of ester and the following reduction of ketone using
NaBH4 gave carboxylic acid analogs in good yields (4a, 4c, and 4d).
Compound 4b was prepared from alcohol 9, which was ob-
tained from the alkylation of phenol 7 using 3-bromo-1-propanol.
In the case of direct alkylation of phenol 7 using 3-bromopropio-
noic acid ester, no product was obtained because of dominant
OH
O
O
O
a
b
8a: R = CH2COOEt
8c: R = (CH2)3COOEt
8d: R = (CH2)4COOMe
4a: R = CH2COOH
4c: R = (CH2)3COOH
4d: R = (CH2)4COOH
O
O
R
R
O
O
OH
O
O
O
c
e
7
9: R = CH2OH
4b
d
10: R = COOH
O
O
OH
R
COOH
OTs
11
X
OH
f
O
O
O
h
O
12: X = O
g
6a
13: X = OH and H
O
O
HOOC
OH
O
O
Scheme 1. Reagents and conditions: (a) K2CO3, Br(CH2)nCO2R, 70 °C, DMF, 31–52%; (b) (1) 1 N NaOH, 60 °C, MeOH; (2) NaBH4, room temperature, MeOH–THF, 74–88% (for
two steps); (c) K2CO3, 3-bromo-1-propanol, 50 °C DMF, 50%; (d) PDC, room temperature, DMF, 9.5%; (e) NaBH4, room temperature, MeOH, 67%; (f) K2CO3, 11, 70 °C, DMF, 49%;
(g) NaBH4, room temperature, MeOH, 41%; (h) 1 N NaOH, room temperature, MeOH–THF, 74%.

H. Kashiwagi et al. / Bioorg. Med. Chem. 19 (2011) 4721–4729
4723
OH
OH
OH
16
R
c
b
14: R = OH
15: R = OTf
17
18
a
OH
OH
OH
OH
OH
d
e
19a: R’ = (CH2)4COOMe
19b: R’ = (CH2)5COOEt
19c: R’ = (CH2)6COOEt
*
20: R’ =
O
5a: R’ = (CH2)4COOH
5b: R’ = (CH2)5COOH
5c: R’ = (CH2)6COOH
O
O
R'
R'
*
6b: R’ =
HOOC
OH
O
Scheme 2. Reagents and conditions: (a) Tf2O, pyridine, 0 °C to room temperature, CH2Cl2, 37%; (b) 16, CuI, Pd(PPh3)4, Et3N, 80 °C, CH3CN, 34%; (c) 10% Pd/C, H2, room
temperature, ethyl acetate, 98%; (d) K2CO3, Br(CH2)nCO2R or 11, 70 °C, DMF, 78–88%; (e) 1 N NaOH, 60 °C, MeOH, 70–84%.
Table 1
VDRE reporter gene activity and osteocalcin induction activity of carboxylic acid analogs
R1
O
X
OH
Compound
R1
X
Ratio of VDRE reporter activity# (%)
Ratio of osteocalcin activity# (%)
4a
4b
4c
4d
5a
5b
5c
6a
HOOC–CH2–
HOOC–(CH2)2–
HOOC–(CH2)3–
HOOC–(CH2)4–
HOOC–(CH2)4–
HOOC–(CH2)5–
HOOC–(CH2)6–
(S)–HOOC–(CH2)2CH(OH)CH2–
(S)–HOOC–(CH2)2CH(OH)CH2–
HO–CH2CH(OH)CH2–
O
O
O
O
CH2
CH2
CH2
O
CH2
O
1.4
6.2
2.6
19
31
16
2.2
20
52
6.7
100
<13
49
<13
205
368
168
9.3
484
68
185
100
6b
LG190178
1,25(OH)2D3
#
1,25(OH)2D3 ratio of EC50. 1,25(OH)2D3 was assigned to 100% (a larger figure means stronger activity).
LG190178 in the VDRE reporter gene and osteocalcin assays. The
shorter methylene analogs (4a–c) exhibited weaker activities than
4d, but the activities were not proportional to the linker length in
either VDRE reporter gene activity or osteocalcin activity. The long-
er five-methylene linker analog (5b) showed moderate activities;
however, a six-methylene linker analog (5c) dramatically reduced
both activities. Introduction of a hydroxyl group into the gamma
position of the carboxyl group in 4d and 5a maintained efficacy
in VDRE reporter gene activity and osteocalcin activity (6a and 6b).
To confirm the detailed interaction of a carboxyl group with
VDR, we cocrystallized the ligand-binding domain for human
VDR with 4a, 4d and 6b in accordance with a method previously
published by Moras et al.25,26 (Figs. 2–4). The X-ray structure,
determined to a resolution of 2.0 Å, shows the overall structure
of 4d was consistent with that of YR301 (Fig. 2). The hydroxyl
group in the side chain moiety interacted with His305 and
His397 and these interactions were seen in the crystal structures
of 4a and 6b with VDR in the same manner. The diethyl moiety be-
also has a hydrogen bonding interaction with Ser237. However 4d
has no hydrogen bonding interaction with Ser278 and Tyr134 in
the way that the 3-OH group of 1,25(OH)2D3 interacts with them.
On the other hand, shorter methylene linker analog 4a had another
pattern of interaction (Fig. 3). The carboxyl group of 4a has hydro-
gen bonding interactions with Ser278 and Tyr134; however, with
Ser237 and Arg274, 4a shows an indirect hydrogen bonding inter-
action via crystal water. In the case of 6b, the carboxyl group has
the same hydrogen bonding interactions with Ser237 and Arg274
of VDR as 4d and the additional gamma hydroxyl group makes
indirect hydrogen bonding interactions with Ser278, Tyr134 and
Arg274 via crystal water (Fig. 4).
From these results, the interactions of a ligand with Ser237 and
Arg274 might contribute to enhanced VDR agonistic activity. In
VDRE reporter gene activity, 4d and 6b, which had tight binding to
Ser 237 and Arg274, indicated stronger activity than 4a which had
no direct binding to Ser237 and Arg274 and showed weak activity.
On the other hand, 6a and 6b, which could have interactions with
Ser278 and Tyr134 via crystal water, showed similar VDRE reporter
gene activity to corresponding analogs (4d and 5a). It also suggested
that the indirect hydrogen bonding interactions with Ser278 and
Tyr134 could not be associated with VDR agonistic activity.
tween bisphenyl rings made a good CH–p interaction with Trp278.
The diethyl moieties were buried in the hydrophobic pocket
formed by Leu227, Val234, Leu404 and Val418. It should be noted
that the carboxyl group of 4d forms a salt bridge with Arg274 and

4724
H. Kashiwagi et al. / Bioorg. Med. Chem. 19 (2011) 4721–4729
Figure 4. X-ray crystal structure of compound 6b (magenta) bound to VDR (green).
Key hydrogen bonding interactions are shown by red dotted lines and each distance
is indicated in Å. (PDB: 3AZ3).
gene and osteocalcin activity could not be explained by the inter-
actions between VDR and its ligand alone. It might be better to
consider the interactions between the VDR ligand complex and
other co-factors. For the transcriptional activation of VDR, it is re-
quired that the AF-2 transactivation motif of VDR interacts with
several types of cofactor such as VDR interacting proteins
(DRIPs).27
In the side chain part, LG190178 was originally reported as a
racemic mixture.11 However, Hakamata et al.17 separated each iso-
mer of the side chain and reported (R) isomer had stronger VDR
agonistic activity than (S) isomer. The compounds reported here
are all racemic mixtures of the side chain hydroxyl group because
we used a comparison to LG190178 to evaluate the effect of spe-
cific modifications to the A-part. We expect (R) isomer of the side
chain could exhibit stronger VDR agonistic activity and will discuss
a detailed SAR analysis of these and other hydroxyl group configu-
rations of the side chain part at another time.
Figure 2. X-ray crystal structure of compound 4d (blue) bound to VDR (green). Key
hydrogen bonding interactions are shown by red dotted lines and each distance is
indicated in Å. (a) Overall structure of compound 4d bound to VDR. (b) Detailed
hydrogen bonding network around A-part of compound 4d. (PDB: 3AZ2).
Next, we assessed pharmacokinetic parameters in carboxylic
acid derivatives (4d and 6a) in rat at the oral and intravenous sin-
gle dosage of 100 lg/kg (Table 2). 3-Hydroxypentanoic acid 6a ap-
pears substantially better than pentanoic acid 4d, with an
especially dramatic improvement in total clearance in iv adminis-
tration (3054 mL/h/kg for 4d vs 88 mL/h/kg for 6a). The significant
improvement in clearance suggests that the gamma hydroxyl
group of 6a would stabilize the A-part from metabolic oxidation.
This enhancement in pharmacokinetic properties, along with its
excellent in vitro efficacy, led us to choose 6a for an in vivo disease
model study.
We evaluated the effect on the prevention of bone mineral den-
sity (BMD) loss of 6a in osteoporosis model rats (Fig. 5). Female
Sprague–Dawley rats, 8 weeks old, were either ovariectomized
(OVX) to produce the estrogen-deficient condition which promotes
BMD loss observed in postmenopausal women or were sham-oper-
ated. Compound 6a was orally administered to three groups of
OVX rats (0.22 lg/kg, 0.67 lg/kg and 2.0 lg/kg per day). Both the
OVX-control and sham-operated groups were administered only
vehicle. After daily administration for 4 weeks, BMD in the distal
femur was measured by dual-energy X-ray absorptiometry. BMD
in the OVX-control group was significantly reduced compared with
the sham-operated group (Fig. 5). Compound 6a dose-dependently
Figure 3. X-ray crystal structure of compound 4a (yellow) bound to VDR (green).
Key hydrogen bonding interactions are shown by red dotted lines and each distance
is indicated in Å. (PDB: 3AZ1).
In an osteocalcin assay, 4a showed 14-fold less potency than
LG190178 but the potency of 4d to it was similar. Although 6b,
which had the most potent VDRE reporter gene activity, showed
only one-third the activity of LG190178, 6a showed the most po-
tent osteocalcin activity. This discrepancy between VDRE reporter
prevented BMD loss caused by ovariectomy. The 0.67 lg/kg group
of 6a maintained the equivalent level of BMD to that of the sham-
operated group without the body weight loss or hypercalcemia
shown by the highest (2.0
lg/kg) administration group. These
two in vivo results, the prevention of BMD loss and the elevation

H. Kashiwagi et al. / Bioorg. Med. Chem. 19 (2011) 4721–4729
4725
Table 2
once-daily oral administration. Further modification from 6a is
ongoing and the SAR results will be published.
Pharmacokinetic parameters of 4d and 6a
4d
6a
po
iv
po
iv
5. Experimental
F (%)
33
13
T1/2 (h)
3.1
3.6
10.9
3.8
9350
1.3
8.0
10.6
5.1. Chemistry: General
Cmax (ng/mL)
AUCinf (ng/mLÃh)
MRT (h)
11.6
151.1
12.3
691
32.9
0.5
3054
1151.6
9.2
88
Purchased reagents and solvents were used without further
purification unless otherwise noted. 1H and 13C NMR spectra were
carried out on VARIAN 400-MR spectrophotometers; chemical
shifts are reported in parts per million (ppm) downfield from that
of internal tetramethylsilane (TMS). Mass spectrophotometry was
measured with a Waters ZQ2000 electrospray ionization (ESI) sys-
tem. High-resolution mass spectra (HRMS) were recorded on Ther-
mo 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).
CLt (mL/h/kg)
Oral and intravenous administration (100
lg/kg) in rat.
a
170
*
160
*
5.1.1. (4-{1-[4-(3,3-Dimethyl-2-oxo-butoxy)-3-methyl-phenyl]-
1-ethyl-propyl}-2-methyl-phenoxy)-acetic acid ethyl ester (8a)
To a solution of 7 (32.3 mg, 0.084 mmol) in DMF (1.0 mL) were
added K2CO3 (23 mg, 0.169 mmol) and ethyl bromoacetate (28 mg,
0.169 mmol). The mixture was stirred at 70 °C overnight. Then the
mixture was diluted with AcOEt, washed with water and brine,
dried over anhydrous MgSO4, and concentrated. The obtained res-
idue was purified by silicagel chromatography (n-hexane/AcOEt =
100:0 to 20:80) to give 8a (20.6 mg, 52%) as a colorless oil. 1H
NMR (CDCl3) d: 0.58 (6H, t, J = 7.3 Hz), 1.25 (9H, s), 1.29 (3H, t,
J = 7.1 Hz), 2.01 (4H, q, J = 7.2 Hz), 2.22 (3H, s), 2.23 (3H, s), 4.26
(2H, q, J = 7.1 Hz), 4.60 (2H, s), 4.83 (2H, s), 6.49 (1H, d,
J = 8.4 Hz), 6.57 (1H, d, J = 9.2 Hz), 6.87–6.92 (4H, m). 13C NMR
(CDCl3) d: 210.0, 169.3, 153.9, 153.8, 141.7, 141.4, 130.8, 130.7,
126.1, 126.0, 126.0, 125.9, 110.2, 110.1, 69.6, 65.8, 61.1, 48.4,
43.2, 29.2, 26.3, 16.6, 16.5, 14.1, 8.4. MS (ESI positive): 486
(M+NH4)+.
*
150
140
130
Sham Vehicle
0.22
μg/kg
0.67
μg/kg
2.0
μg/kg
b
13
*
12
11
10
9
8
5.1.2. 4-(4-{1-[4-(3,3-Dimethyl-2-oxo-butoxy)-3-methyl-
phenyl]-1-ethyl-propyl}-2-methyl-phenoxy)-butyric acid ethyl
ester (8c)
7
6
The yield was 46%. Colorless oil. 1H NMR (CDCl3) d: 0.59 (6H, t,
J = 7.3 Hz), 1.26 (9H, s), 1.26 (3H, t, J = 7.1 Hz), 2.01 (4H, q,
J = 7.3 Hz), 2.09–2.15 (2H, m), 2.15 (3H, s), 2.24 (3H, s), 2.54 (2H,
t, J = 7.3 Hz), 3.98 (2H, t, J = 6.1 Hz), 4.15 (2H, q, J = 7.2 Hz), 4.84
(2H, s), 6.50 (1H, d, J = 8.3 Hz), 6.67 (1H, d, J = 8.8 Hz), 6.89–6.94
(4H, m). 13C NMR (CDCl3) d: 210.1, 173.4, 154.5, 153.9, 141.5,
140.5, 130.7, 130.4, 126.0, 125.9, 125.4, 110.1, 109.6, 69.6, 66.4,
60.4, 48.3, 43.2, 30.9, 29.2, 26.3, 24.8, 16.7, 16.5, 14.2, 8.4. MS
(ESI positive): 514 (M+NH4)+.
5
Sham
Vehicle
0.22
μg/kg
0.67
μg/kg
2.0
μg/kg
Figure 5. (a) Oral once-daily treatment of 6a prevented BMD loss in osteoporosis
model rats. (b) Serum calcium concentration in osteoporosis model rat. ⁄P <0.05
versus vehicle.
of serum calcium concentration, were typical VDR agonistic actions
which were reported in in vivo evaluations of secosteroidal vita-
min D3 analogs.20,21
5.1.3. 5-(4-{1-[4-(3,3-Dimethyl-2-oxo-butoxy)-3-methyl-phen
yl]-1-ethyl-propyl}-2-methyl-phenoxy)-pentanoic acid methyl
ester (8d)
4. Conclusion
The yield was 31%. Colorless oil. 1H NMR (CDCl3) d: 0.58 (6H, t,
J = 7.3 Hz), 1.25 (9H, s), 1.81–1.86 (4H, m), 2.01 (4H, q, J = 7.3 Hz),
2.15 (3H, s), 2.24 (3H, s), 2.41 (2H, t, J = 7.1 Hz), 3.67 (3H, s), 3.94
(2H, t, J = 5.7 Hz), 4.83 (2H, s), 6.49 (1H, d, J = 8.4 Hz), 6.66 (1H, d,
J = 8.4 Hz), 6.87–6.94 (4H, m). 13C NMR (CDCl3) d: 210.0, 174.0,
154.6, 153.9, 141.6, 140.4, 130.7, 130.4, 126.0, 126.0, 125.9,
125.4, 110.1, 109.6, 69.6, 67.0, 51.5, 48.3, 43.2, 33.7, 29.3, 28.8,
26.3, 21.8, 16.6, 16.5, 8.4. MS (ESI positive): 514 (M+NH4)+.
Novel nonsecosteroidal vitamin D3 agonists were explored.
They have carboxylic acid instead of 1,3-diol of 1,25(OH)2D3 and
compounds 4d and 5a were identified as the optimized linker
length analogs. Additional hydroxyl group in gamma position of
the carboxyl group in 4d and 5a significantly improved metabolic
stability. X-ray structure of 6b exhibited the carboxyl group tightly
interacting with Ser237 and Arg274 and the gamma hydroxyl
group interacting with Ser278 and Tyr134 via crystal water.
Through our SAR studies, compound 6a was identified as the opti-
mal compound showing excellent in vitro potency and pharmaco-
kinetics. In a rat osteoporosis model, 6a was confirmed to have
significant prevention of bone loss without hypercalcemia via
5.1.4. (4-{1-Ethyl-1-[4-(2-hydroxy-3,3-dimethyl-butoxy)-3-
methyl-phenyl]-propyl}-2-methyl-phenoxy)-acetic acid (4a)
To a solution of 8a (10.8 mg, 0.023 mmol) in MeOH (1.0 mL)
was added 1 N NaOH (0.115 mL, 0.115 mmol). The mixture was

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H. Kashiwagi et al. / Bioorg. Med. Chem. 19 (2011) 4721–4729
stirred at 60 °C for 30 min. After the mixture was poured into 1 N
HCl, the products were extracted with CH2Cl2. The extracts were
dried over anhydrous MgSO4, filtered, and concentrated. The ob-
tained residue was dissolved in MeOH (0.5 mL) and THF (0.5 mL).
To the solution was added NaBH4 (2.6 mg, 0.069 mmol). The mix-
ture was stirred at room temperature for 1 h. Then the mixture
was poured into sat. NH4Cl aq solution, and the products were ex-
tracted with CH2Cl2. The extracts were dried over anhydrous
MgSO4, filtered, and concentrated. The obtained residue was puri-
fied preparative TLC (10% MeOH/CHCl3) to give 4a (8.5 mg, 84%) as
a colorless oil. 1H NMR (CDCl3) d: 0.58 (6H, t, J = 7.2 Hz), 1.01 (9H,
s), 2.11 (4H, q, J = 7.2 Hz), 2.16 (3H, s), 2.19 (3H, s), 3.71 (1H, dd,
J = 8.8, 2.5 Hz), 3.85 (1H, t, J = 8.9 Hz), 4.08 (1H, dd, J = 9.2,
2.5 Hz), 4.59 (2H, s), 6.59 (1H, d, J = 8.2 Hz), 6.69 (1H, d,
J = 8.4 Hz), 6.86–6.96 (4H, m). 13C NMR (CDCl3) d: 173.2, 154.3,
153.3, 142.3, 140.9, 130.9, 130.6, 126.2, 126.1, 126.0, 125.5,
110.6, 110.0, 77.2, 69.1, 65.6, 48.4, 33.5, 29.2, 26.0, 16.6, 16.5,
8.4. HRMS (ESI negative): Calcd for C27H37O5 441.2646. Found:
441.2636 (MÀH)À.
5.1.8. 3-(4-{1-[4-(3,3-Dimethyl-2-oxo-butoxy)-3-methyl-
phenyl]-1-ethyl-propyl}-2-methyl-phenoxy)-propionic acid
(10)
To a solution of 9 (44 mg, 0.100 mmol) in DMF (1.0 mL) was
added PDC (113 mg, 0.300 mmol). The mixture was stirred at room
temperature for three days. Then the mixture was poured into 1 N
HCl and the products were extracted with AcOEt. The extracts were
washed with water and brine, dried over anhydrous MgSO4, and
concentrated. The obtained residue was purified by preparative
TLC (n-hexane/AcOEt = 1:1) to give 10 (4.3 mg, 9.5%) as a colorless
oil. 1H NMR (CDCl3) d: 0.58 (6H, t, J = 7.2 Hz), 1.25 (9H, s), 2.01 (4H,
q, J = 7.3 Hz), 2.13 (3H, s), 2.24 (3H, s), 2.85 (2H, t, J = 6.1 Hz), 4.23
(2H, t, J = 6.1 Hz), 4.83 (2H, s), 6.49 (1H, d, J = 8.4 Hz), 6.70 (1H, d,
J = 8.6 Hz), 6.89–6.96 (4H, m). 13C NMR (CDCl3) d: 210.1, 175.6,
154.0, 153.9, 141.5, 141.1, 130.7, 130.5, 126.1, 126.0, 125.9,
125.6, 110.1, 110.0, 69.6, 63.1, 48.4, 43.2, 34.4, 29.2, 26.3, 16.6,
16.4, 8.4. MS (ESI positive): 472 (M+NH4)+.
5.1.9. 3-(4-{1-Ethyl-1-[4-(2-hydroxy-3,3-dimethyl-butoxy)-3-
methyl-phenyl]-propyl}-2-methyl-phenoxy)-propionic acid
(4b)
5.1.5. 4-(4-{1-Ethyl-1-[4-(2-hydroxy-3,3-dimethyl-butoxy)-3-
methyl-phenyl]-propyl}-2-methyl-phenoxy)-butyric acid (4c)
The yield was 74%. Colorless oil. 1H NMR (CDCl3) d: 0.59 (6H, t,
J = 7.2 Hz), 1.01 (9H, s), 2.02 (4H, q, J = 7.3 Hz), 2.09–2.15 (2H, m),
2.15 (3H, s), 2.17 (3H, s), 2.61 (2H, t, J = 7.2 Hz), 3.71 (1H, dd,
J = 8.8, 2.5 Hz), 3.85 (1H, t, J = 9.0 Hz), 3.99 (2H, t, J = 5.9 Hz), 4.09
(1H, dd, J = 9.2, 2.5 Hz), 6.67 (2H, d, J = 8.6 Hz), 6.69 (2H, d,
J = 8.6 Hz), 6.88–6.97 (4H, m). 13C NMR (CDCl3) d: 178.1, 154.4,
154.2, 141.2, 140.7, 130.6, 130.5, 126.1, 126.0, 125.5, 125.4,
110.0, 109.6, 77.2, 69.1, 66.2, 48.4, 33.5, 30.5, 29.3, 26.0, 24.6,
The yield was 67%. Colorless oil. 1H NMR (CDCl3) d: 0.59 (6H, t,
J = 7.2 Hz), 1.01 (9H, s), 2.02 (4H, q, J = 7.2 Hz), 2.13 (3H, s), 2.17
(3H, s), 2.85 (2H, t, J = 6.3 Hz), 3.71 (1H, dd, J = 8.8, 2.5 Hz), 3.85
(1H, t, J = 8.9 Hz), 4.09 (1H, dd, J = 9.1, 2.4 Hz), 4.23 (2H, t,
J = 6.2 Hz), 6.69 (1H, d, J = 8.4 Hz), 6.71 (1H, d, J = 8.2 Hz), 6.88–6.97
(4H, m). 13C NMR (CDCl3) d: 175.0, 154.2, 154.0, 141.1, 141.1,
130.6, 130.5, 126.1, 126.0, 125.7, 125.4, 110.0, 110.0, 77.2, 69.1,
63.1, 48.4, 34.3, 33.5, 29.2, 26.0, 16.6, 16.4, 8.4. HRMS (ESI negative):
Calcd for C28H39O5 455.2803. Found: 455.2799 (MÀH)À.
16.6, 16.5, 8.4. HRMS (ESI negative): Calcd for
C29H41O5
469.2959. Found: 469.2965 (MÀH)À.
5.1.10. (S)-5-(4-{1-[4-(3,3-Dimethyl-2-oxo-butoxy)-3-methyl-
phenyl]-1-ethyl-propyl}-2-methyl-phenoxymethyl)-dihydro-
furan-2-one (12)
5.1.6. 5-(4-{1-Ethyl-1-[4-(2-hydroxy-3,3-dimethyl-butoxy)-3-
methyl-phenyl]-propyl}-2-methyl-phenoxy)-pentanoic acid
(4d)
The yield was 49%. Colorless oil. 1H NMR (CDCl3) d: 0.58 (6H, t,
J = 7.3 Hz), 1.25 (9H, s), 2.01 (4H, q, J = 7.3 Hz), 2.15 (3H, s), 2.23
(3H, s), 2.28–2.36 (1H, m), 2.39–2.49 (1H, m), 2.52–2.61 (1H, m),
2.72–2.82 (1H, m), 4.06 (1H, dd, J = 10.3, 3.4 Hz), 4.17 (1H, dd,
J = 10.4, 3.3 Hz), 4.83 (2H, s), 4.85–4.91 (1H, m), 6.49 (1H, d,
J = 8.2 Hz), 6.66 (1H, d, J = 8.4 Hz), 6.86–6.97 (4H, m). 13C NMR
(CDCl3) d: 210.0, 177.2, 154.0, 153.9, 141.5, 141.4, 130.7, 130.7,
126.2, 126.0, 125.9, 125.5, 110.1, 109.8, 77.9, 69.5, 69.3, 48.4,
43.2, 29.2, 28.3, 26.3, 24.1, 16.6, 8.4. MS (ESI positive): 498
(M+NH4)+.
The yield was 88%. Colorless oil. 1H NMR (CDCl3) d: 0.59 (6H, t,
J = 7.3 Hz), 1.01 (9H, s), 1.84–1.87 (4H, m), 2.01 (4H, q, J = 7.3 Hz),
2.15 (3H, s), 2.17 (3H, s), 2.45–2.47 (2H, m), 3.71 (1H, dd, J = 8.8,
2.5 Hz), 3.85 (1H, t, J = 8.9 Hz), 3.94–3.96 (2H, m), 4.09 (1H, dd,
J = 9.2, 2.5 Hz), 6.66 (2H, d, J = 8.6 Hz), 6.69 (2H, d, J = 8.6 Hz),
6.88–6.98 (4H, m).13C NMR (CDCl3) d: 179.1, 154.6, 154.2, 141.3,
140.4, 130.6, 130.4, 126.1, 126.0, 125.4, 125.4, 110.0, 109.6, 77.3,
69.1, 67.0, 48.3, 33.6, 33.5, 29.3, 28.7, 26.0, 21.5, 16.6, 16.5, 8.4.
HRMS (ESI negative): Calcd for
C30H43O5 483.3116. Found:
5.1.11. (S)-5-(4-{1-Ethyl-1-[4-(2-hydroxy-3,3-dimethyl-butoxy)-
3-methyl-phenyl]-propyl}-2-methyl-phenoxymethyl)-dihydro-
furan-2-one (13)
483.3111 (MÀH)À.
The yield was 41%. Colorless oil. 1H NMR (CDCl3) d: 0.59 (6H, t,
J = 7.2 Hz), 1.01 (9H, s), 2.02 (4H, q, J = 7.2 Hz), 2.15 (3H, s), 2.17
(3H, s), 2.27–2.36 (1H, m), 2.39–2.49 (1H, m), 2.51–2.61 (1H, m),
2.72–2.82 (1H, m), 3.70 (1H, dd, J = 8.7, 2.4 Hz), 3.85 (1H, t,
J = 8.9 Hz), 4.04–4.11 (2H, m), 4.17 (1H, dd, J = 10.4, 3.3 Hz),
4.85–4.91 (1H, m), 6.66 (1H, d, J = 8.4 Hz), 6.69 (1H, d, J = 8.4 Hz),
6.88–6.97 (4H, m). 13C NMR (CDCl3) d: 177.2, 154.3, 153.8, 141.6,
141.0, 130.7, 130.6, 126.1, 126.1, 125.5, 125.5, 110.0, 109.8, 77.9,
77.2, 69.3, 69.1, 48.4, 33.5, 29.2, 28.3, 26.0, 24.1, 16.6, 16.6, 8.4. MS
(ESI positive): 505 (M+Na)+.
5.1.7. 1-(4-{1-Ethyl-1-[4-(3-hydroxy-propoxy)-3-methyl-
phenyl]-propyl}-2-methyl-phenoxy)-3,3-dimethyl-butan-2-one
(9)
To a solution of 7 (76 mg, 0.199 mmol) in DMF (2.0 mL) were
added K2CO3 (54.9 mg, 0.397 mmol) and 3-bromo-1-propanol
(55.2 mg, 0.397 mmol). The mixture was stirred at 50 °C overnight.
Then the mixture was poured into sat. NH4Cl aq solution and the
products were extracted with AcOEt. The extracts were washed
with water and brine, dried over anhydrous MgSO4, and concen-
trated. The obtained residue was purified by preparative TLC
(n-hexane/AcOEt = 1:1) to give 9 (44.2 mg, 50%) as a colorless oil.
1H NMR (CDCl3) d: 0.59 (6H, t, J = 7.2 Hz), 1.25 (9H, s), 1.98–2.13
(6H, m), 2.16 (3H, s), 2.24 (3H, s), 3.89 (2H, t, J = 5.8 Hz), 4.11
(2H, t, J = 5.8 Hz), 4.83 (2H, s), 6.49 (1H, d, J = 8.4 Hz), 6.71 (1H, d,
J = 8.4 Hz), 6.89–6.95 (4H, m). 13C NMR (CDCl3) d: 210.2, 154.4,
153.9, 141.5, 140.8, 130.7, 130.5, 126.1, 126.0, 125.9, 125.2,
110.1, 109.6, 69.5, 66.0, 61.1, 48.3, 43.2, 32.0, 29.2, 26.3, 16.6,
16.6, 8.4. MS (ESI positive): 463 (M+Na)+.
5.1.12. (S)-5-(4-{1-Ethyl-1-[4-(2-hydroxy-3,3-dimethyl-butoxy)-
3-methyl-phenyl]-propyl}-2-methyl-phenoxy)-4-hydroxy-
pentanoic acid (6a)
The yield was 74%. Colorless oil. 1H NMR (CD3OD) d: 0.58 (6H, t,
J = 7.2 Hz), 1.00 (9H, s), 1.79–1.89 (1H, m), 1.93–2.00 (1H, m), 2.03
(4H, q, J = 7.2 Hz), 2.14 (3H, s), 2.15 (3H, s), 2.37 (2H, td, J = 7.3,
3.1 Hz), 3.61 (1H, dd, J = 7.8, 2.9 Hz), 3.84–3.91 (3H, m),

H. Kashiwagi et al. / Bioorg. Med. Chem. 19 (2011) 4721–4729
4727
3.93–3.97 (1H, m), 4.11 (1H, dd, J = 10.0, 2.9 Hz), 6.75 (2H, dd,
J = 8.4, 2.2 Hz), 6.84 (2H, dd, J = 5.5, 2.0 Hz), 6.95 (2H, dt, J = 8.4,
2.4 Hz). 13C NMR (CD3OD) d: 181.1, 154.8, 154.7, 140.6, 140.6,
130.1, 130.1, 125.7, 125.7, 125.3, 109.7, 77.2, 71.7, 70.0, 69.4,
47.8, 34.0, 33.6, 30.0, 28.7, 25.2, 15.4, 15.3, 7.3. HRMS (ESI nega-
tive): Calcd for C30H43O6 499.3065. Found: 499.3060 (MÀH)À.
J = 9.8 Hz), 3.67 (3H, s), 3.94 (2H, t, J = 5.6 Hz), 6.66 (1H, d,
J = 8.4 Hz), 6.92–6.94 (4H, m), 7.02 (1H, d, J = 7.8 Hz). 13C NMR
(CDCl3) d: 174.0, 154.6, 146.4, 140.3, 137.2, 134.8, 130.4, 129.8,
127.8, 126.1, 125.7, 125.4, 109.6, 80.0, 67.0, 51.5, 48.6, 35.0,
33.7, 32.0, 30.4, 29.2, 28.8, 25.6, 21.8, 19.7, 16.5, 8.5. MS (ESI
positive): 519 (M+Na)+.
5.1.13. Trifluoromethanesulfonic acid 4-[1-ethyl-1-(4-hydroxy-
3-methyl-phenyl)-propyl]-2-methyl-phenyl ester (15)
5.1.17. 6-(4-{1-Ethyl-1-[4-(3-hydroxy-4,4-dimethyl-pentyl)-3-
methyl-phenyl]-propyl}-2-methyl-phenoxy)-hexanoic acid
ethyl ester (19b)
To a solution of 14 (9.0 g, 31.6 mmol) in CH2Cl2 (300 mL) were
added pyridine (3.0 mL, 37.2 mmol) and trifluoromethanesulfonic
anhydride (5.7 mL, 34.7 mmol) at 0 °C and the mixture was stirred
for 1 h. The mixture was diluted with AcOEt, washed with sat.
NaHCO3 aq solution and brine, dried over anhydrous MgSO4, and
concentrated. The obtained residue was chromatographed on silica
gel (n-hexane/AcOEt = 90:10 to 0:100) to afford 15 (4.9 g, 37%) as a
colorless oil. 1H NMR (CDCl3) d: 0.60 (6H, t, J = 7.3 Hz), 2.03 (4H, q,
J = 7.3 Hz), 2.20 (3H, s), 2.31 (3H, s), 4.78 (1H, br s), 6.67 (1H, d,
J = 8.2 Hz), 6.82–6.86 (2H, m), 7.02–7.11 (3H, m). 13C NMR (CDCl3)
The yield was 78%. Colorless oil. 1H NMR (CDCl3) d: 0.60 (6H, t,
J = 7.2 Hz), 0.89 (9H, s), 1.25 (3H, t, J = 7.1 Hz), 1.39–1.56 (3H, m),
1.62–1.83 (4H, m), 1.84–1.92 (1H, m), 2.03 (4H, q, J = 7.4 Hz),
2.15 (3H, s), 2.25 (3H, s), 2.33 (2H, t, J = 7.4 Hz), 2.55–2.58 (1H,
m), 2.82–2.90 (1H, m), 3.24 (1H, dd, J = 10.3, 1.3 Hz), 3.92 (2H, t,
J = 6.3 Hz), 4.12 (2H, q, J = 7.2 Hz), 6.66 (1H, d, J = 8.4 Hz),
6.91–6.95 (4H, m), 7.02 (1H, d, J = 7.6 Hz). 13C NMR (CDCl3) d:
173.7, 154.7, 146.4, 140.2, 137.2, 134.7, 130.4, 129.8, 127.8,
126.1, 125.7, 125.4, 109.7, 79.9, 67.3, 60.2, 48.6, 35.0, 32.4, 32.0,
30.4, 29.2, 29.1, 25.8, 25.6, 24.7, 19.6, 16.5, 14.2, 8.5. MS (ESI posi-
tive): 547 (M+Na)+.
d: 151.6, 149.5, 146.2, 139.9, 131.6, 130.5, 129.5, 127.3, 126.6,
122.9, 120.1, 117.0, 114.2, 48.9, 29.2, 16.6, 16.0, 8.3. MS (ESI nega-
tive): 415 (MÀH)À.
5.1.18. 7-(4-{1-Ethyl-1-[4-(3-hydroxy-4,4-dimethyl-pentyl)-3-
methyl-phenyl]-propyl}-2-methyl-phenoxy)-heptanoic acid
ethyl ester (19c)
5.1.14. 4-{1-Ethyl-1-[4-(3-hydroxy-4,4-dimethyl-pent-1-ynyl)-
3-methyl-phenyl]-propyl}-2-methyl-phenol (17)
To a solution of 15 (190 mg, 0.46 mmol) in MeCN (2.3 mL) were
added 4,4-Dimethyl-pent-1-yn-3-ol (16) (103 mg, 0.92 mmol),
Pd(PPh3)4 (53 mg, 0.046 mmol), CuI (9 mg, 0.046 mmol), and Et3N
(0.192 mL, 1.38 mmol). The mixture was stirred in a sealed tube
at 80 °C for 3 h. The mixture was poured into KHSO4 aq solution
and the products were extracted with AcOEt. The extracts were
dried over anhydrous Na2SO4, filtered, and concentrated. The ob-
tained residue was purified by silica gel chromatography (n-hex-
ane/AcOEt = 100:0 to 84:16) to give 17 (59 mg, 34%) as a pale
brown form. 1H NMR (CDCl3) d: 0.59 (6H, t, J = 7.3 Hz), 1.07 (9H,
s), 2.03 (4H, q, J = 7.2 Hz), 2.19 (3H, s), 2.38 (3H, s), 4.26 (1H, d,
J = 6.1 Hz), 4.58 (1H, d, J = 1.6 Hz), 4.88 (1H, s), 6.65 (1H, d,
J = 8.0 Hz), 6.83 (1H, d, J = 2.3 Hz), 6.94 (1H, dd, J = 8.0, 1.8 Hz),
7.00 (1H, s), 7.28 (1H, d, J = 8.0 Hz). 13C NMR (CDCl3) d: 151.6,
149.7, 140.2, 139.2, 131.3, 130.6, 129.1, 126.5, 125.5, 122.8,
119.3, 114.0, 92.1, 84.9, 72.0, 49.0, 36.1, 29.0, 25.4, 21.1, 16.1,
8.3. MS (ESI positive): 401 (M+Na)+.
The yield was 83%. Colorless oil. 1H NMR (CDCl3) d: 0.60 (6H, t,
J = 7.3 Hz), 0.89 (9H, s), 1.25 (3H, t, J = 7.1 Hz), 1.46–1.54 (5H, m),
1.62–1.69 (2H, m), 1.76–1.83 (3H, m), 2.04 (4H, q, J = 7.3 Hz), 2.15
(3H, s), 2.25 (3H, s), 2.30 (2H, t, J = 7.5 Hz), 2.52–2.59 (1H, m), 2.82–
2.90 (1H, m), 3.25 (1H, dd, J = 10.6, 1.4 Hz), 3.91 (2H, t, J = 6.4 Hz),
4.13 (2H, q, J = 7.2 Hz), 6.67 (1H, d, J = 8.4 Hz), 6.90–6.95 (4H, m),
7.02 (1H, d, J = 7.6 Hz). 13C NMR (CDCl3) d: 173.8, 154.7, 146.4,
140.1, 137.2, 134.7, 130.4, 129.8, 127.8, 126.0, 125.7, 125.4, 109.7,
79.9, 67.5, 60.2, 48.6, 35.0, 34.2, 32.0, 30.4, 29.2, 29.2, 28.9, 25.9,
25.6, 24.7, 19.6, 16.5, 14.2, 8.5. MS (ESI positive): 561 (M+Na)+.
5.1.19. (S)-5-(4-{1-ethyl-1-[4-(3-hydroxy-4,4-dimethyl-pentyl)-
3-methyl-phenyl]-propyl}-2-methyl-phenoxymethyl)-dihydro-
furan-2-one (20)
The yield was 88%. Colorless oil. 1H NMR (CDCl3) d: 0.59 (6H, t,
J = 7.2 Hz), 0.89 (9H, s), 1.45–1.55 (1H, m), 1.75–1.83 (1H, m), 2.04
(4H, q, J = 7.2 Hz), 2.15 (3H, s), 2.25 (3H, s), 2.28–2.36 (1H, m),
2.39–2.49 (1H, m), 2.51–2.61 (1H, m), 2.65–2.80 (1H, m), 2.81–
2.90 (1H, m), 3.24 (1H, dd, J = 10.4, 1.4 Hz), 4.06 (1H, dd, J = 10.3,
3.4 Hz), 4.16 (1H, dd, J = 10.3, 3.4 Hz), 4.87–4.90 (1H, m), 6.66
(1H, d, J = 8.6 Hz), 6.80–6.98 (4H, m), 7.02 (1H, d, J = 8.6 Hz). 13C
NMR (CDCl3) d: 177.2, 153.8, 146.2, 141.5, 137.3, 134.8, 130.7,
129.8, 127.8, 126.2, 125.6, 125.5, 109.8, 79.9, 77.9, 69.3, 48.7,
35.0, 32.0, 30.4, 29.1, 28.3, 25.6, 24.1, 19.7, 16.6, 8.4. MS (ESI posi-
tive): 503 (M+Na)+.
5.1.15. 4-{1-Ethyl-1-[4-(3-hydroxy-4,4-dimethyl-pentyl)-3-
methyl-phenyl]-propyl}-2-methyl-phenol (18)
To a solution of 17 (99 mg, 0.262 mmol) in AcOEt (2 mL) was
added 10% Pd/C (10 mg) and the mixture was stirred at room tem-
perature for 30 min under hydrogen atmosphere. The mixture was
filtered through celite and concentrated to give 18 (98 mg, 98%) as
a colorless form. 1H NMR (CDCl3) d: 0.60 (6H, t, J = 7.3 Hz), 0.89 (9H,
s), 1.46–1.57 (1H, m), 1.76–1.84 (1H, m), 2.03 (4H, q, J = 7.6 Hz),
2.19 (3H, s), 2.24 (3H, s), 2.52–2.59 (1H, m), 2.85–2.89 (1H, m),
3.27 (1H, dd, J = 10.6, 1.6 Hz), 5.26 (1H, br s), 6.64 (1H, d,
J = 8.4 Hz), 6.85 (1H, dd, J = 8.3, 2.2 Hz), 6.90–6.94 (3H, m), 7.02
(1H, d, J = 8.4 Hz). 13C NMR (CDCl3) d: 151.4, 146.4, 140.8, 137.2,
134.8, 130.6, 129.8, 127.8, 126.6, 125.6, 122.6, 114.0, 80.1, 48.6,
35.0, 32.0, 30.4, 29.2, 25.7, 19.6, 16.1, 8.5. MS (ESI positive): 405
(M+Na)+.
5.1.20. 5-(4-{1-Ethyl-1-[4-(3-hydroxy-4,4-dimethyl-pentyl)-3-
methyl-phenyl]-propyl}-2-methyl-phenoxy)-pentanoic acid
(5a)
The yield was 84%. Colorless oil. 1H NMR (CDCl3) d: 0.60 (6H, t,
J = 7.2 Hz), 0.89 (9H, s), 1.49–1.54 (1H, m), 1.76–1.82 (1H, m),
1.84–1.86 (4H, m), 2.04 (4H, q, J = 7.3 Hz), 2.16 (3H, s), 2.26 (3H,
s), 2.45–2.47 (2H, m), 2.52–2.59 (1H, m), 2.83–2.90 (1H, m), 3.26
(1H, dd, J = 10.4, 1.4 Hz), 3.94–3.96 (2H, m), 6.67 (1H, d,
J = 8.4 Hz), 6.90–6.96 (4H, m), 7.02 (1H, d, J = 7.8 Hz). 13C NMR
(CDCl3) d: 179.0, 154.6, 146.4, 140.3, 137.2, 134.8, 130.4, 129.8,
127.8, 126.1, 125.7, 125.4, 109.6, 80.1, 67.0, 48.6, 35.0, 33.6, 32.0,
30.4, 29.2, 28.7, 25.6, 21.5, 19.7, 16.5, 8.5. HRMS (ESI negative):
Calcd for C31H45O4 481.3323. Found: 481.3325 (MÀH)À.
5.1.16. 5-(4-{1-Ethyl-1-[4-(3-hydroxy-4,4-dimethyl-pentyl)-3-
methyl-phenyl]-propyl}-2-methyl-phenoxy)-pentanoic acid
methyl ester (19a)
The yield was 78%. Colorless oil. 1H NMR (CDCl3) d: 0.60 (6H, t,
J = 7.2 Hz), 0.89 (9H, s), 1.45–1.55 (1H, m), 1.76–1.86 (5H, m),
2.04 (4H, q, J = 7.3 Hz), 2.16 (3H, s), 2.26 (3H, s), 2.41 (2H, t,
J = 7.1 Hz), 2.54–2.58 (1H, m), 2.82–2.90 (1H, m), 3.25 (1H, d,

4728
H. Kashiwagi et al. / Bioorg. Med. Chem. 19 (2011) 4721–4729
5.1.21. 6-(4-{1-Ethyl-1-[4-(3-hydroxy-4,4-dimethyl-pentyl)-3-
methyl-phenyl]-propyl}-2-methyl-phenoxy)-hexanoic acid (5b)
The yield was 76%. Colorless oil. 1H NMR (CDCl3) d: 0.60 (6H, t,
J = 7.2 Hz), 0.89 (9H, s), 1.46–1.59 (3H, m), 1.69–1.84 (5H, m), 2.04
(4H, q, J = 7.3 Hz), 2.15 (3H, s), 2.25 (3H, s), 2.39 (2H, t, J = 7.4 Hz),
2.52–2.59 (1H, m), 2.82–2.90 (1H, m), 3.26 (1H, d, J = 9.4 Hz), 3.93
(2H, t, J = 6.2 Hz), 6.67 (1H, d, J = 8.4 Hz), 6.90–6.96 (4H, m), 7.02
(1H, d, J = 7.8 Hz). 13C NMR (CDCl3) d: 179.2, 154.7, 146.4, 140.2,
137.2, 134.7, 130.4, 129.8, 127.8, 126.1, 125.7, 125.4, 109.7, 80.1,
67.3, 48.6, 35.0, 33.9, 32.0, 30.4, 29.2, 29.1, 25.7, 25.6, 24.4, 19.7,
16.5, 8.5. HRMS (ESI negative): Calcd for C32H47O4 495.3480.
Found: 495.3480 (MÀH)À.
5.3. Osteocalcin induction assay
MG-63 cells were plated at 2 Â 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 10À7 to 10À11 mol/L with 0.1% DMSO), and incubated
for 8 h. After changing culture medium to fresh one, the cells were
incubated for an additional four days, and then supernatant was
collected and stored at À80 °C.
The frozen supernatant was thawed slowly at room tempera-
ture and Gla-type osteocalcin EIA kit (Takara Bio. Inc., Tokyo, Ja-
pan) was used to measure osteocalcin. Absorbance at 450 nm
was measured on a plate reader (Model 3550, Bio–Rad Laborato-
ries, CA, USA) and each concentration was calculated by compari-
5.1.22. 7-(4-{1-Ethyl-1-[4-(3-hydroxy-4,4-dimethyl-pentyl)-3-
methyl-phenyl]-propyl}-2-methyl-phenoxy)-heptanoic acid
(5c)
The yield was 70%. Colorless oil. 1H NMR (CDCl3) d: 0.60 (6H, t,
J = 7.2 Hz), 0.89 (9H, s), 1.39–1.56 (5H, m), 1.64–1.71 (2H, m), 1.75–
1.84 (3H, m), 2.04 (4H, q, J = 7.3 Hz), 2.15 (3H, s), 2.25 (3H, s), 2.37
(2H, t, J = 7.5 Hz), 2.54–2.57 (1H, m), 2.82–2.90 (1H, m), 3.26 (1H,
dd, J = 10.4, 1.6 Hz), 3.92 (2H, t, J = 6.4 Hz), 6.67 (1H, d, J = 8.4 Hz),
6.90–6.95 (4H, m), 7.02 (1H, d, J = 7.8 Hz). 13C NMR (CDCl3) d:
179.2, 154.7, 146.4, 140.2, 137.2, 134.7, 130.4, 129.8, 127.8,
126.0, 125.7, 125.4, 109.7, 80.0, 67.5, 48.6, 35.0, 33.8, 32.0, 30.4,
29.2, 29.2, 28.8, 25.9, 25.6, 24.6, 19.7, 16.5, 8.5. HRMS (ESI nega-
tive): Calcd for C33H49O4 509.3636. Found: 509.3627 (MÀH)À.
son with standards using
lplate Manager I software (Bio–Rad
Laboratories). The EC50 values were determined and the inductive
activity was calculated as the ratio of the EC50 value of the com-
pounds to that of 1,25(OH)2D3.
5.4. Bone mineral density and serum calcium evaluation in the
ovariectomized rats
Sham-operated (n = 8) and OVX eight-week-old female Spra-
gue–Dawley (Crl:CD(SD)) rats (Charles River Japan Inc.) were used.
One day after the surgery, OVX rats were divided into four groups
(n = 8), and were orally administered compound 6a (0.22, 0.67,
5.1.23. 5-(4-{1-Ethyl-1-[4-(3-hydroxy-4,4-dimethyl-pentyl)-3-
methyl-phenyl]-propyl}-2-methyl-phenoxy)-4(S)-hydroxy-
pentanoic acid (6b)
2.0 lg/kg, five times per week) or vehicle (medium-chain triglycer-
ide (MCT), 1 mL/kg, five times per week) for 4 weeks. 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 centrifugation of the blood samples and was stored at À20 °C.
The right femur was stored in 70% ethanol at 4 °C. Serum calcium
concentration was measured by an autoanalyzer (Hitachi 7170, To-
kyo, Japan). The BMD in distal femur was measured by dual-energy
X-ray absorptiometry (DCS-600-EX, Aloka, Japan).
The yield was 76%. Colorless oil. 1H NMR (CD3OD) d: 0.58 (6H, t,
J = 7.3 Hz), 0.87 (9H, s), 1.43–1.54 (1H, m), 1.69–1.89 (2H, m), 1.92–
2.01 (1H, m), 2.05 (4H, q, J = 7.8 Hz), 2.14 (3H, s), 2.23 (3H, s), 2.34–
2.40 (2H, m), 2.50–2.57 (1H, m), 2.82–2.91 (1H, m), 3.15 (1H, dd,
J = 10.4, 1.4 Hz), 3.88–3.91 (2H, m), 3.92–4.00 (1H, m), 6.75 (1H,
d, J = 8.6 Hz), 6.84 (1H, d, J = 2.0 Hz), 6.88–6.96 (3H, m), 7.01 (1H,
d, J = 7.8 Hz). 13C NMR (CDCl3) d: 181.2, 154.7, 146.2, 140.5,
137.4, 134.4, 130.1, 129.5, 127.7, 125.7, 125.3, 125.2, 109.8, 78.9,
71.8, 70.0, 47.8, 34.5, 34.0, 31.9, 30.0, 29.9, 28.7, 24.9, 22.8, 18.3,
15.3, 7.3. HRMS (ESI negative): Calcd for C31H45O5 497.3272.
Found: 497.3278 (MÀH)À.
5.5. Crystallographic structure analysis
Protein sample preparation and crystallization were based on
the methods by Moras et al.24,25 X-ray diffraction data collection
and data processing were carried out by generally accepted meth-
ods. After brief soaking in the buffer containing the 30% glycerol,
0.6 M ammonium sulfate and 0.1 M MES (pH6.0), crystals were
trapped in the fiber loops and flash-cooled with liquid nitrogen.
Crystals were stored and transported in the dry shipper. Data
5.2. VDRE reporter gene assay
MG-63 cells were plated at 2 Â 103 cells/200
lL/well in a 96-
well white cell-culture plate and were incubated at 37 °C in 5%
CO2 incubator for 24 h. Then, MG-63 cells were cotransfected with
0.05
tained three repeats of the VDRE sequence from mouse osteopon-
tin promoter and 0.001 of pRL–SV40 vector (Promega
lg of the pGV2–basic/VDRE–luciferase vector which con-
lg
Table 3
Corporation, WI, USA) using Lipofectamine (Invitrogen). The cells
were added to minimum essential medium (MEM) containing 5%
fetal bovine serum treated with dextran-coated charcoal (DCC–
FBS) and were incubated for 8 h. The cells were treated with the se-
rial diluted compounds (final concentrations were 10À7 to
10À11 mol/L with 0.1% DMSO) and were incubated for an additional
three days. After removing the supernatants, the cells were lysed in
cell-lysis buffer and luciferase activity was measured by DLR™ As-
say System (Promega Corporation, WI, USA) and the luminescence
was detected by Wallac ALVO SX 1420 multi-label counter (Perkin-
Elmer, Inc., MA, USA). The half maximal effective concentrations
(EC50) were determined and the inductive activity was calculated
as the ratio of the EC50 value of the compounds to that of
1,25(OH)2D3 (Solvay Pharmaceuticals, Weesp, The Netherlands)
which was used as a positive control.
Crystal and diffraction data of VDR with compound 4a, 4d, and 6b
4a
4d
6b
Wavelength (Å)
Cell (Å)
1.0
1.0
1.0
(a)
(b)
(c)
44.6
51.7
131.6
2.1
44.5
51.8
132.4
2.0
44.3
52.2
132.0
1.9
Resolution (Å)
Completeness (last shell) (%)
Rsym (last shell) (%)
I/Sigma (last shell)
Rcryst
99.3 (98.9) 93.7 (88.6) 89.6 (80.0)
7.4 (25.3)
6.1 (2.7)
19.5
7.7 (17.4)
6.0 (3.8)
18.8
6.9 (18.2)
6.7 (3.6)
22.6
0.010
0.99
rmsd bond length (Å)
rmsd bond angles (deg)
0.010
0.96
0.011
1.18
No. of non-hydrogen protein atoms 1993
No. of water molecules 235
1993
298
1993
148

H. Kashiwagi et al. / Bioorg. Med. Chem. 19 (2011) 4721–4729
4729
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Acknowledgments
The authors thank Ms. Hitomi Suda of Chugai Pharmaceutical
Co., Ltd for HRMS measurements of the compounds, and Dr. Hiro-
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