Osteoclast-forming suppressing compounds, gargalols A, B, and C, from the edible mushroom Grifola gargal

Article abstract of DOI:10.1016/j.tet.2011.05.091

Three novel sterols, gargalols A-C (1-3), and four known ones were isolated from the edible mushroom Grifola gargal. The structures of 1-7 were determined or identified by the interpretation of spectroscopic data. Compounds 1-5 suppressed the formation of

Full text of DOI:10.1016/j.tet.2011.05.091

Tetrahedron 67 (2011) 6576e6581
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Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Osteoclast-forming suppressing compounds, gargalols A, B, and C, from the edible
mushroom Grifola gargal
,
,
Jing Wu a y, Jae-Hoon Choi b y, Miyuki Yoshida b, Hirofumi Hirai b, Etsuko Harada c, Kikuko Masuda d,
Tomoyuki Koyama d, Kazunaga Yazawa d, Keiichi Noguchi e, Kazuo Nagasawa f, Hirokazu Kawagishi a
,b,
*
a Graduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan
b Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529, Japan
c Iwadekingaku Laboratory, Tsu 514-0012, Japan
d Graduate School of Marine Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7, Konan, Minato-ku, Tokyo 108-8477, Japan
e Instrumentation Analysis Center, Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho, Koganei, Tokyo 184-8588, Japan
f Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 April 2011
Received in revised form 20 May 2011
Accepted 21 May 2011
Available online 30 May 2011
Three novel sterols, gargalols AeC (1e3), and four known ones were isolated from the edible mushroom
Grifola gargal. The structures of 1e7 were determined or identified by the interpretation of spectroscopic
data. Compounds 1e5 suppressed the formation of osteoclast without toxicity.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Mushroom
Grifola gargal
Suppression of the formation of osteoclast
Structure determination
1. Introduction
strong activity in the extract of the mushroom Grifola gargal, and
tried to isolate the active principles from the mushroom.
Osteoporosis is a serious health problem that predominantly
affects postmenopausal women and aged people leading to a high
risk of fracture. Osteoclasts are derived from the monocyte/macro-
phage cell lineage and are specialized cells responsible for the
breakdown of bone.1 The progressive bone loss is due to both an
increase in osteoclastic bone resorption and a decrease in osteo-
blastic bone formation.2 Therefore, substances, which can suppress
the formation of osteoclasts are candidates for therapy or can be
used as supplements or functional foods to prevent osteoporosis.
Recently, beneficial effects of natural products and their derivatives
that affect the process of bone remodeling, in particular bone re-
sorption, have been reported. For example, two sterols were isolated
as suppressive compounds from the edible mushroom Leccinum
extremiorientale by us.3 Earlier we have reported the isolation of
novel osteoclast-forming suppressing compounds, chaxines AeC
and some steroids, fromthe edible mushroom Agrocybe chaxingu.4e6
During further screening for the osteoclast-formation sup-
pressing effects of the extracts of various mushrooms, we found
G. gargal is an edible mushroom with a characteristic almond
flavor, collected and eaten by native people of southern Argentina
and Chile. The species has only been reported from the Nothofagus-
dominated forests of the area. Nutraceutical properties and phar-
macological potential of the mushroom have been studied; aqueous
extracts of the mushroom showed the anti-oxidant and anti-
inflammatory effects and the methanol extracts displayed a free
radical scavenging activity.7 Commercial production of the mush-
room has just started in Japan.8 Here we describe the isolation,
structural determination, and biological activity of compounds from
the mushroom.
2. Results and discussion
The dried fruiting bodies of G. gargal were extracted with hex-
ane, EtOAc and then with EtOH subsequently. Since EtOAc-soluble
fraction showed the strong suppressing activity against the for-
mation of osteoclast, this fraction was repeatedly subjected to
column chromatography, being guided by the result of the bioassay.
As a consequence, three novel compounds (1e3) and four known
ones were purified.
* Corresponding author. E-mail address: achkawa@ipc.shizuoka.ac.jp (H.
Kawagishi).
y
These authors contributed equally to this work.
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.05.091

J. Wu et al. / Tetrahedron 67 (2011) 6576e6581
6577
28
22
21
18
26
20
17
27
19
13
14
11
1
4
10
OH
O
O
6
HO
HO
HO
HO
HO
O
HO
O
2
1
3
4
O
O
O
O
OH
O
O
6
HO
O
5
7
O
HO
O
Gargalol A (1) was purified as a white powder. Its molecular
formula was determined as C28H44O3 by HRESIMS m/z 451.3156
[MþNa]þ (calcd for C28H44NaO3, 451.3188), indicating the presence
of seven degrees of unsaturation in the molecule. The structure of 1
was elucidated by interpretation of NMR spectra including DEPT,
COSY, HMBC, and HMQC. The DEPT experiment indicated the
presence of six methyls, six methylenes, twelve methines, and four
quaternary carbons. In the NMR spectra of 1, typical signals of
a sterol, such as a side-chain olefine (C-22, dH 5.14, dd, J¼15.3,
6.7 Hz; dC 135.3: C-23, dH 5.20, dd, J¼15.3, 8.5 Hz; dC 132.2),
a hydroxymethine (C-3, dH 4.06, m; dC 62.7), four doublet methyls
(C-21, dH 1.00, d, J¼6.4 Hz; dC 21.0: C-26, 27, dH 0.80, d, J¼6.8 Hz,
0.82, d, J¼6.8 Hz; dC 19.6, 19.9: C-28, dH 0.89, d, J¼6.4 Hz; dC 17.6),
and two singlet methyls (C-18, dH 0.57, s; dC 12.2: C-19, dH 1.04, s; dC
21.1) were observed. The complete assignment of the protons and
carbons and the HMBC correlations were summarized in Table 1.
The structure including relative stereochemistry of 1 was con-
firmed by X-ray crystallography analysis (Fig. 1). In addition, the
absolute configuration of 1 was determined by circular dichroic
Compound 4 was purified as a white powder. Its molecular
formula was determined as C28H42O3 by HRESIMS m/z 425.3029
[MꢀH]ꢀ (calcd for C28H41O3, 425.3056). The 1H and 13C NMR data
of 4 were very similar to those of 3 (Table 1). The comparison of
the molecular formula of 4 with that of 3 indicates that 4 is
a dehydroxylated form of 3. The position of the missing hydroxy
was elucidated by the chemical shift of position 14 (dH 2.11; dC
48.8) and the HMBC correlations (Table 1). Compound 4 has been
previously reported as a product of thermal rearrangement of
ergosterol peroxide but its spectroscopic data have not been given
in the report.10 This is the first report of isolation of 4 from
a natural source. The absolute configuration of 4 was determined
by comparison of its specific rotation value with that reported
27
previously:10 compound 4, [
a
]
þ43.0 (c 1.10, CHCl3); reported
data, [
a
]
25 þ40.6 (c 1.10, CHCl3)D. As a result, the structure of 4 was
D
determined as shown.
The data of NMR, MS, IR and specific rotation of 5 were identical
with those of the compound that had been isolated from two kinds
of mushrooms, Pleurotus eryngii and Panellus serotinus and whose
exciton chirality method using its dinaphthoate (lmax
(
D3); 242
stereochemistry had been determined as 3
,14
-epoxy, by interpretation of the NOESY data.11 However,
X-ray crystallography analysis on a p-bromobenzoate of 5 in this
study indicated that 5 was 5 ,9 -epidioxy-8 ,14 -epoxy-(22E)-
ergosta-6,22-dien-3 -ol (Fig. 1).
b-ol, 5a,9a-epidioxy,
(þ20.4), 230 (ꢀ12.6) nm).9 As a result, the structure of 1 was de-
8a
a
termined to be 4b,5b-epoxy-(22E)-ergosta-7,22-dien-3b,6a-diol.
Gargalol B (2) was isolated as a white powder. Its molecular
formula was determined as C28H42O4 by HRESIMS m/z 441.2983
[MꢀH]ꢀ (calcd for C28H41O4, 441.3005) and the degree of unsatu-
ration of the compound was eight. The NMR data of 2 were similar
to those of 1 (Table 1), suggesting this compound was also a sterol.
The structure elucidation was accomplished in the same manner as
1 (Table 1). The DEPT experiment indicated the presence of six
methyls, eight methylenes, seven methines, and seven quaternary
carbons. The molecular formula, the HMBC correlations (Fig. 2 and
Table 1) and the chemical shifts indicated the presence of a hy-
droxyl (dH 3.93, m; dC 66.0), an enone (dC 197.8, 126.5, 172.4), and
a peroxide (dC 85.9, 85.8) at the positions of C-3, C-7/8/14, and C-5/
C-9. As a result, the planar structure of 2 was determined as shown.
However, since any significant NOE was not observed, the stereo-
chemistry of 2 could not be determined.
a
a
b
b
b
Based on the comparison of the spectroscopic data for 6 and 7
with those reported in previous papers,12,13 6 and 7 were identified
as
3b,5a-dihydroxy-(22E)-ergosta-7,22-diene-6-one and 3b-hy-
droxy-(22E)-ergosta-5,8,22-triene-7-one, respectively.
Compounds 1e7 were evaluated in the osteoclast-forming as-
say. The assay is based on the principle that osteoclast-like multi-
nucleated cells can be formed in vitro from co-cultures of mouse
bone marrow cells and osteoblastic cells by treatment with osteo-
tropic factors. By adding suppressive agents, the formation of os-
teoclast is inhibited during the differentiation. As shown in Fig. 3, 2
and 4 inhibited osteoclast formation at lower concentration
(0.78 mg/mL) than the other compounds, and 5 exhibited the ac-
tivity dose-dependently and the activity was the strongest among
Gargalol C (3) was purified as a white powder. Its molecular
formula was determined as C28H42O4 by HRESIMS m/z 465.2877
[MþNa]þ (calcd for C28H42NaO4, 465.2890) and the degree of
unsaturation of the compound was eight. The NMR data of 3 were
similar to those of 1 and 2 (Table 1). The molecular formula, the
HMBC correlations (Fig. 2 and Table 1) and the chemical shifts in-
dicated the presence of two hydroxyls (C-3, dH 3.97, m; dC 68.7: C-
14, dC 80.8), an epoxide (C-5, dC 65.6: C-6, dH 3.34, s; dC 62.3), and an
enone (C-7, dC 200.2; C-8, dC 133.0; C-9, dC 157.0). Since an NOE was
observed between H-6 and H-19 in the NOE difference and NOESY
experiments, the relative configuration at C-5, C-6, and C-19 was
determined as shown. However the stereochemistry of the other
parts remains unknown.
all the compounds. On the other hand, compounds 6 and 7 signif-
icantly showed cytotoxicity even at 1.56 mg/mL (data not shown).
The structureeactivity relationship and the mode of action of the
compounds remain unsolved.
3. Experimental
3.1. General
1H NMR spectra (one- and two-dimensional) were recorded on
a JEOL lambda-500 spectrometer at 500 MHz, while 13C NMR
spectra were recorded on the same instrument at 125 MHz. The
HRESIMS spectra were measured on
a JMS-T100LC mass

6578
J. Wu et al. / Tetrahedron 67 (2011) 6576e6581
Table 1
1H and 13C NMR data for 1e4 (in CDCl3)
Position
1
2
dH (multiplicity, J in Hz)
dC
25.0
HMBC correlation
dH (multiplicity, J in Hz)
dC
28.2
HMBC correlation
1
2
3
4
5
6
7
8
1.23 (m), 1.42 (m)
1.44 (m), 1.62 (m)
4.06 (m)
3.76 (m)
d
C-2, 3, 5, 9, 10
C-1, 3, 4, 10
C-1
C-2, 3, 5, 6
d
1.42 (m), 2.04 (m)
1.45 (m), 1.99 (m)
3.93 (m)
1.45 (m), 2.13 (m)
d
C-2, 3, 5, 10
C-3, 10
d
26.5
62.7
58.0
70.2
65.2
119.1
142.2
41.7
35.6
22.6
39.1
43.9
55.1
22.3
31.4
66.0
37.3
85.9
50.4
197.8
126.5
85.8
51.4
23.6
33.0
46.0
172.4
31.0
C-2, 3, 5, 10
d
4.61 (br s)
5.19 (m)
d
C-4, 5, 7, 8
C-5, 14
d
2.46 (d, 19.2), 2.56 (d, 19.2)
d
C-4, 5, 7, 8, 10
d
d
d
9
2.17 (m)
d
C-7, 8, 10, 11, 14, 19
d
d
d
10
11
12
13
14
15
d
d
1.41(m), 1.52 (m)
1.27 (m), 2.02 (m)
d
C-8, 9, 12, 13
C-9, 11, 13, 14, 17, 18
d
1.93 (m), 1.98 (m)
1.45 (m), 1.96 (m)
d
C-8, 9, 10, 12, 13
C-13, 14, 17
d
1.88 (m)
1.43(m), 1.53 (m)
C-7, 8, 13, 15, 18
C-8, 13, 14, 16
d
d
2.74 (dd, 21.5, 8.9)
2.98 (ddd, 21.5, 9.2, 9.2)
1.45 (m), 1.79 (m)
1.33 (m)
0.90 (s)
1.05 (s)
2.13 (m)
1.02 (d, 6.7)
5.19 (m)
5.22 (m)
1.85 (m)
C-8, 13, 14, 16, 17
16
17
18
19
20
21
22
23
24
25
26
27
28
1.27 (m), 1.74 (m)
1.27 (m)
0.57 (s)
1.04 (s)
2.01 (m)
1.00 (d, 6.4)
5.14 (dd, 15.3, 6.7)
5.20 (dd, 15.3, 8.5)
1.84 (m)
28.1
55.9
12.2
21.1a
40.4
21.0a
135.3
132.2
42.8
33.1
19.6
19.9
17.6
C-13, 15, 17
27.7
54.8
17.6
16.0
38.5
21.4
132.9
134.6
42.9
33.3
20.0
19.6
17.5
C-13, 14, 17
C-12, 13, 16, 20
C-13, 14, 17
C-1, 5, 9, 10
C-17, 22, 23
C-13, 16, 18, 22
C-12, 13, 14, 17
C-1, 5, 9, 10
C-17, 21, 22, 23
C-17, 20, 22
C-20, 21, 23, 24
C-20, 22, 24, 25, 28
C-22, 23, 25, 26, 28
C-23, 24, 26, 27
C-24, 25, 27
C-17, 20, 22
C-20, 21, 23, 24
C-20, 22, 24, 25
C-22, 23, 25, 26, 27, 28
C-23, 24, 26, 27
C-24, 25, 27
1.45 (m)
1.45 (m)
0.80 (d, 6.8)
0.82 (d, 6.8)
0.89 (d, 6.4)
0.80 (d, 6.7)
0.82 (d, 6.7)
0.90 (d, 6.7)
C-24, 25, 26
C-23, 24, 25
C-24, 25, 26
C-23, 24, 25
Position
3
4
dH (multiplicity, J in Hz)
dC
HMBC correlation
dH (multiplicity, J in Hz)
dC
HMBC correlation
1
2
3
4
5
6
7
8
1.69 (m), 1.91 (m)
1.69 (m), 2.06 (m)
3.97 (m)
1.52 (m), 2.27 (m)
d
28.9
30.3
68.7
38.3
65.6
62.3
200.2
133.0
157.0
40.7
23.0
29.8
44.8
80.8
35.4
26.0
44.8
16.4
23.0
39.6
21.0
134.8
132.8
42.8
33.1
19.6
19.9
17.5
C-2, 3, 5, 9, 10
C-3, 4, 10
d
19 1.70 (m), 1.84 (m)
1.70 (m), 2.02 (m)
3.93 (m)
1.49 (m), 2.24 (m)
d
30.4
30.6
68.4
38.2
64.5
62.4
196.7
128.7
158.0
40.5
25.6
35.6
42.1
48.8
24.3
29.4
53.3
11.5
24.1
40.3
21.0
135.3
132.2
42.8
33.0
19.6
19.9
17.5
C-3, 5, 9, 10, 19
C-4, 10
C-2, 4
C-3, 5, 6, 10
d
C-2, 3, 5, 6, 10
d
3.34 (s)
d
C-4, 5, 7, 8
d
3.25 (s)
d
C-4, 5, 7, 10
d
d
d
d
d
9
d
d
d
d
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
d
d
d
d
2.10 (m), 2.14 (m)
1.47 (m), 1.69 (m)
d
C-8, 9, 12, 13
C-9, 11, 13, 18
d
2.21 (m), 2.23 (m)
1.45 (m), 1.96 (m)
d
C-8, 9, 13
C-9, 11, 13, 14, 17, 18
d
d
d
2.11 (m)
C-8, 9, 13, 15
C-8, 14
C-13, 14, 15, 17
C-12, 13, 14, 18, 20, 21, 22
C-12, 13, 14, 17
C-5, 9, 10
1.74 (m), 1.89 (m)
1.38 (m), 1.47 (m)
1.44 (m)
0.93 (s)
1.21 (s)
C-8, 13, 14, 16, 17
C-13, 14, 15, 17, 20
C-16, 22
C-12, 13, 14, 17
C-1, 5, 9, 10
C-22, 23
C-17, 20, 22
C-20, 21, 23, 24
C-20, 22, 24, 25, 28
C-22, 23, 25, 26, 27, 28
C-23, 24, 26, 27, 28
C-24, 25, 27
C-24, 25, 26
C-23, 24, 25
1.80 (m), 1.95 (m)
1.33 (m), 1.74 (m)
1.10 (m)
0.55 (s)
1.21 (s)
2.06 (m)
1.99 (m)
d
1.01 (d, 6.5)
5.11 (dd, 15.3, 6.7)
5.20 (dd, 15.3, 7.6)
1.83 (m)
0.99 (d, 7.3)
5.12 (dd, 15.9, 7.3)
5.19 (dd, 15.9, 7.3)
1.80 (m)
C-17, 20, 22
C-17, 20, 21, 24
C-20, 24, 25, 28
C-22, 23, 26, 27
C-23, 24, 26, 27
C-24, 25, 27
C-24, 25, 26
C-23, 24, 25
1.45 (m)
1.44 (m)
0.79 (d, 7.0)
0.81 (d, 7.0)
0.88 (d, 7.0)
0.78 (d, 7.3)
0.79 (d, 7.3)
0.88 (d, 7.3)
a
Interchangeable.
spectrometer. CD spectrum was recorded on a JASCO J-820 spec-
tropolarimeter. A JASCO grating infrared spectrophotometer was
used to record the IR spectra. The specific rotation values were
measured by using a JASCO DIP-1000 polarimeter. HPLC separa-
tions were performed with a JASCO Gulliver system using reverse-
phase HPLC columns (Develosil C30-UG-15/30, Nomura chemical
Co., Ltd., Japan; Wakosil-II 5C18 HG Hrep, Wako, Japan; Capcell PAK
C18 AQ, Shiseido, Japan; COSMOSIL Cholester water, Nacalai tesque,
Japan; Phenomenex Luna PFP (2), Shimadzu GLC Ltd., Japan) and
a normal-phase HPLC column (Senshu PAK AQ, Senshu scientific
Co., Ltd., Japan). Silica gel plate (Merck F254) and silica gel 60N
(Merck 100e200 mesh) were used for analytical TLC and for flash
column chromatography, respectively.
3.2. Fungus materials
A voucher specimen of the organism is located in Iwadekingaku
laboratory.

J. Wu et al. / Tetrahedron 67 (2011) 6576e6581
6579
Fig. 1. ORTEP drawings of 1 and p-bromobenzoate of 5 with ellipsoids at the 50% probability level. Hydrogen atoms are shown as small spheres of arbitrary radii.
was fractionated by silica gel flash column chromatography
(hexane/EtOAc 9:1; CH2Cl2; CH2Cl2/acetone 8:2; acetone; EtOH
and MeOH/H2O 9:1, 2.0 L each) to obtain 28 fractions (fractions
1e28). Fraction 17 (296 mg) was separated by reverse-phase HPLC
(Wakosil-II 5C18 HG Hrep, 95% MeOH) to afford compound 5
(11.1 mg). Fraction 18 (3.6 g) was further separated by reverse-
phase HPLC (Develosil C30-UG-15/30, 90% MeOH), affording 57
fractions (fractions 18-1 to 18-57). Compounds 1 (2.3 mg), 4
(13.9 mg), and 7 (2.0 mg) were obtained from fraction 18-49
(75.7 mg) by normal-phase HPLC (Senshu Pak AQ, EtOAc/CHCl3
1:9). Compound 2 (2.9 mg) was purified from fraction 18-33
(46.5 mg) by normal-phase HPLC (Senshu PAK AQ, CHCl3). Fraction
18-21 (16.1 mg) was further separated by reverse-phase HPLC
(Capcell PAK C18 AQ, 80% MeOH) to obtain compound 3 (2.9 mg).
Fractions 18e45 (53.4 mg) was separated by normal-phase HPLC
(Senshu Pak AQ, EtOAc/CHCl3 1:9) and reverse-phase HPLC
(COSMOSIL Cholester water, 90% MeOH) to give compound 6
(4.0 mg).
3.3.1. Gargalol A (1). White powꢀd1er; [
a
]
28 þ19 (c 0.29, CHCl3); mp
D
190e192 ꢁC; IR (neat): 3398 cm
;
1H and 13C NMR, see Table 1;
ESIMS m/z 451 [MþNa]þ; HRESIMS m/z 451.3158 [MþNa]þ (calcd
for C28H44NaO3, 451.3188).
3.3.2. Gargalol B (2). White powder; [
a
]
27 þ47 (c 0.26, CHCl3); mp
D
Fig. 2. 2D NMR correlations of 2 and 3.
59e60 ꢁC; IR (neat): 3348, 1683 cmꢀ1; 1H and 13C NMR, see Table 1;
ESIMS m/z 441 [MꢀH]ꢀ; HRESIMS m/z 441.2983 [MꢀH]ꢀ (calcd for
C28H41O4, 441.3005).
3.3. Extraction and isolation
Powder of the air-dried fruiting bodies of G. gargal (8.78 kg)
was successively extracted with hexane (5 L, twice), EtOAc (5 L,
twice) and then EtOH (5 L, twice). The EtOAc-soluble part (90.1 g)
3.3.3. Gargalol C (3). White powder; [
a
]
24 þ118 (c 0.11, CHCl3); mp
D
194e196 ꢁC; IR (neat): 3435, 1658 cmꢀ1; 1H and 13C NMR, see Table
Fig. 3. Inhibitory activity of 1e7 against osteoclast formation. Closed and open columns indicate cell viability and osteoclast formation, respectively. TRAP-positive multinucleated
cells that had more than three nuclei were counted. Cell viability was determined by MIT assay. Data are the meanꢂSE of two cultures (*P<0.05 vs control using Student’s t-test).

6580
J. Wu et al. / Tetrahedron 67 (2011) 6576e6581
1; ESIMS m/z 465 [MþNa]þ; HRESIMS m/z 465.2877 [MþNa]þ
0.558 mmꢀ1, F(000)¼944. The size of the crystal used for measure-
ments was 0.60ꢃ0.25ꢃ0.05 mm. Diffraction data were collected on
a Rigaku R-AXIS-RAPID diffractometer with imaging plate detector.
(calcd for C28H42NaO4, 465.2890).
3.3.4. Compound 4. White powder; [
a
]
27 þ43.0 (c 1.10, CHCl3); mp
45,484 reflections were collected in the range 4.70<q<68.25, of
206e207 ꢁC; IR (neat): 3392, 1653 cmꢀ1D; 1H and 13C NMR, see Table
1; ESIMS m/z 425 [MꢀH]ꢀ; HRESIMS m/z 425.3029 [MꢀH]ꢀ (calcd
for C28H41O3, 425.3056).
which 2639 unique (Rint¼0.0424) reflections. The structure was re-
fined by full-matrix least-squares procedure on F2 values using all
unique reflections. The final R indices were R(F)¼0.0348, wR(F2)¼
0.0960 (2512 reflections with I>2
s
(I)) with goodness-of fit¼1.028.
27
3.3.5. Compound 5. White powder; [
a
]
þ4.5 (c 1.10, CHCl3); mp
p-Bromobenzoate of 5: C35H45BrO5, M¼625.62, monoclinic,
D
152e154 ꢁC; IR (neat): 3425 cmꢀ1; ESIMS m/z 465 [MþNa]þ;
a¼6.4110(10) A, b¼9.373(2) A, c¼26.057(6) A, b¼95.832(12)ꢁ,
ꢀ ꢀ ꢀ
HRESIMS m/z 465.2961 [MþNa]þ (calcd for C28H42NaO4, 465.2981);
V¼1557.7(5) A , T¼95 K, space group P21, Z¼2,
l
¼0.8000 A,
3
ꢀ
ꢀ
1H NMR (500 MHz, CDCl3):
d
0.80 (H-26), 0.82 (H-27), 0.90 (H-28),
m
(
l
¼0.80)¼1.798 mmꢀ1, F(000)¼660. The size of the crystal used
0.91 (H-18), 0.98 (H-21), 1.11 (H-19), 1.35 (H-1a), 1.44 (H-16a), 1.45
(H-12a, H-25), 1.46 (H-17), 1.50 (H-2a), 1.53 (H-11a), 1.56 (H-12b),
1.64 (H-4a), 1.65 (H-15a), 1.67 (H-16b), 1.70 (H-11b), 1.84 (H-24),
1.88 (H-1b), 1.92 (H-2b), 2.00 (H-15b), 2.11 (H-20), 2.20 (H-4b), 3.99
(H-3), 5.15 (H-22), 5.21 (H-23), 5.53 (H-7), 5.85 (H-6); 13C NMR
for measurements was 0.10ꢃ0.05ꢃ0.01 mm. Diffraction data were
collected at PF-AR NW12A beamline (Tsukuba, Japan), with ADSC
Quantum 210r CCD detector. 11,915 reflections were collected in the
range 1.76<
q<25.01, of which 3561 unique (Rint¼0.096) reflections.
The structure was refined by full-matrix least-squares procedure on
F2 values using all unique reflections. The final R indices were R(F)¼
0.0831, wR(F2)¼0.1612 (all reflections) with goodness-of fit¼1.068.
Crystallographic data for 1 and p-bromobenzoate of 5 have been
deposited at The Cambridge Crystallographic Data Centre and allo-
cated the deposition number, CCDC 791830 and 791831, respectively.
The data can be obtained free of charge via www.ccdc.cam.ac.uk/
products/csd/request.
(125 MHz, CDCl3): d 15.5 (C-18), 15.6 (C-19), 17.6 (C-28), 19.6 (C-26),
19.7 (C-11), 19.9 (C-27), 21.0 (C-21), 26.5 (C-15), 27.2 (C-16), 27.6 (C-
1), 30.8 (C-2), 33.1 (C-25), 33.3 (C-12), 35.6 (C-4), 39.1 (C-20), 40.2
(C-13), 42.8 (C-24), 50.5 (C-10), 55.7 (C-17), 63.8 (C-8), 66.0 (C-3),
75.2 (C-14), 85.8 (C-5), 86.8 (C-9),128.7 (C-7),132.8 (C-23),134.9 (C-
22), 135.6 (C-6).
3.4. Preparation of dinaphthoate of 1
3.7. Bioassay
Compound 1 (1.0 mg, 2.3
chloride (8.1 mg, 42.5 mol) and 4-N,N-dimethylaminopyridine
(2.5 mg, 20.5 mol) in pyridine (50
L) at 65 ꢁC for 3 days. The
mmol) was stirred with 2-naphthoyl
m
The stromal/osteoblastic cells, UAMS-32, were cultured in
a-
m
m
minimal essential medium ( -MEM) (ICN Biomedicals, Inc.) con-
a
resulting mixture was evaporated to dryness under reduced pressure
and then separated by reverse-phase HPLC (Phenomenex Luna PFP
(2), 99% CH3CN) to give a dinaphthoate of 1 (0.41 mg). Dinaphthoate
taining 10% fetal bovine serum (FBS) for a week. The cells were
detached from the culture dishes by using trypsineEDTA, suspended
in a-MEM containing 10% FBS and used for the co-culture as osteo-
of 1. CD (0.00222 M, CH3CN) lmax
(
D3) 242 (þ20.4), 230 (ꢀ12.6) nm;
blastic cells. Bone marrow cells were isolated from mice as described
previously.14 Femoral and tibiae bone marrow cells were collected
from 5-week-old mice, which had been killed by cervical dislocation.
The tibiae and femora were removed and dissected free of adhering
tissues. The bone ends were removed and the marrow cavities were
flushed by slowly injecting media with a 26-gauge needle. The os-
teoblastic cells and bone marrow cells collected and washed to be
used in the co-culture subsequently. Osteoclasts were prepared from
a co-culture system as previously described.15 The osteoblastic cells
(1.0ꢃ104 cells/well) were co-cultured with bone marrow cells
1H NMR (500 MHz, in CDCl3):
d
7.49e8.62 (14H, aromatic naphthoate
protons), 6.15(1H, br, s, H-6), 5.59(1H, m, H-3), 5.13e5.28(3H, m, H-7,
22, 23), 4.00 (1H, d, 4.0, H-4), 2.37 (1H, m, H-9), 1.22e2.10 (17H, m,
H-1, 2,11,12,14,15,16,17, 20, 24, 25),1.27 (3H, s, H-19),1.03 (3H, d, 6.7,
H-21), 0.90 (3H, d, 7.0, H-28), 0.82 (3H, d, 7.0, H-27), 0.81 (3H, d, 7.0,
H-26), 0.63 (3H, s, H-18); ESIMS m/z 759 [MþNa]þ.
3.5. Preparation of p-bromobenzoate of 5
Compound 5 (2.0 mg) was dissolved in 0.5 mL anhydrous pyri-
dine in a 4 mL vial, and p-bromobenzoyl chloride (10.4 mg) was
added to the solution. After stirring at 50 ꢁC for 2 days, the reaction
mixture was evaporated to dryness under reduced pressure and
then separated by normal-phase HPLC (Senshu PAK AQ, hexane/
CHCl3 8:2) to give a p-bromobenzoate (1.2 mg) of 5.
(2.0ꢃ107 cells/well) in
a-MEM containing 10% FBS in 96-well plates
(Corning Inc.). The culture volume was made up to 200
m
L per well
with
a
-MEM supplemented with 10% FBS in the presence of 10ꢀ8 M
1a
,25(OH)2D3 (Biomol) and 10ꢀ6 M PGE2, with or without a sample.
All cultures were maintained at 37 ꢁC in a humidified atmosphere
containing 5% CO2 in air. Three-quarter of mediumwas changed after
co-culture for 3 days. After the cultivation, the adherent cells were
fixed with 10% formaldehyde in 10 mM phosphate-buffered saline
(pH 7.4) for 20 min. After being treated with 95% ethanol for 1 min,
the well surface was dried and treated with the TRAP staining solu-
tion [0.1 M sodium acetate buffer (pH 5.0) containing 50 mM sodium
tartrate, 0.1 mg/mL naphthol AS-MX phosphate (Sigma chemical
Co.), and 1 mg/mL fast red violet LB salt (Sigma chemical Co.)] for
30 min. The TRAP-positive multinucleated cells were then counted
under a microscope. Cell viability was evaluated using a 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
(Sigma chemical Co.) assay. After the culture, cells were treated with
1 mg/mL MTT for 2 h, then precipitated dye was solubilized into di-
methyl sulfoxide, and the absorbance was measured at 570 nm.
p-Bromobenzoate of 5. ESIMS m/z 647 [MþNa]þ; HRESIMS m/z
647.2328 [MþNa]þ (calcd for C35H45BrNaO5, 647.2348). 1H NMR
(500 MHz, in CDCl3):
d
0.80 (3H, d, J¼6.7 Hz, H-26), 0.81 (3H, d,
J¼7.0 Hz, H-27), 0.89 (3H, d, J¼6.7 Hz, H-28), 0.91 (3H, s, H-18), 0.98
(3H, d, J¼6.6 Hz, H-21), 1.14 (3H, s, H-19), 1.35 (1H, m, H-1),
1.45e1.60 (8H, m, H-2, H-11, H-12, H-16, H-17, H-25),1.65 (1H, m, H-
15), 1.70 (1H, m, H-11), 1.84 (1H, m, H-24), 1.88 (1H, m, H-1), 1.92
(1H, m, H-2), 2.00 (1H, m, H-15), 2.11 (1H, m, H-20), 2.20 (1H, m, H-
4), 5.17 (1H, dd, J¼15.4, 8.4 Hz, H-22), 5.24 (1H, dd, J¼15.4, 7.7 Hz,
H-23), 5.29 (1H, m, H-3), 5.56 (1H, d, J¼9.5 Hz, H-7), 5.87 (1H, d,
J¼9.5 Hz, H-6), 7.56 (2H, d, J¼8.5 Hz, COC6H4ep-Br), 7.86 (2H, d,
J¼8.5 Hz, COC6H4ep-Br).
3.6. X-ray crystallography analysis
3.8. Statistical analysis
Crystal data for 1: C28H44O3, M¼428.63, orthorhombic,
3
ꢀ
ꢀ
ꢀ
ꢀ
a¼7.71614(18) A, b¼8.53412(18) A, c¼37.6177(8) A, V¼2477.14(9) A ,
Data thus collected were analyzed statistically using Student’s t-
test to determine significant difference in the data among the
ꢀ
T¼193 K, space group P212121, Z¼4,
l
¼1.54187 A,
m(Cu Ka)¼

J. Wu et al. / Tetrahedron 67 (2011) 6576e6581
6581
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groups. P values less than 0.05 were considered significant. The
values are expressed as meanꢂSE.
Acknowledgements
8. Kawade, M.; Harada, E.; Nishioka, H.; Meguro, S. Mushroom Sci. Biotechnol.
2009, 17, 75e79 (in Japanese).
We thank V.K. Deo (Shizuoka University) and M. Hasimoto
(Hirosaki University) for valuable discussion.
9. Harada, N.; Nakanishi, K. Circular Dichroic Spectroscopy-Exciton Coupling in
Organic Stereochemistry; University Science Books: Mill Valley, CA, 1983.
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2002, 50, 551e553.
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