Synthesis and structure-activity relationships of radicicol derivatives and WNT-5A expression inhibitory activity

Article abstract of DOI:10.1016/j.bmc.2009.04.060

WNT-5A, a secretory glycoprotein, is related to the proliferation of dermal papilla cells. To develop a hair-growth stimulant, we have been searching for inhibitors of WNT-5A expression. We identified radicicol (1) as an active compound, and synthesized s

Full text of DOI:10.1016/j.bmc.2009.04.060

Bioorganic & Medicinal Chemistry 17 (2009) 4622–4635
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and structure–activity relationships of radicicol derivatives
and WNT-5A expression inhibitory activity
a
b
b
b
a
Hideki Shinonaga a, , Toshiya Noguchi , Akiko Ikeda , Mari Aoki , Natsuko Fujimoto , Akira Kawashima
*
a Medicinal Chemistry Laboratories, Taisho Pharmaceutical Co., Ltd, 403, Yoshino-cho 1-chome, Kita-ku, Saitama-shi, Saitama 331-9530, Japan
b Molecular Function and Pharmacology Laboratories, Taisho Pharmaceutical Co., Ltd, 403, Yoshino-cho 1-chome, Kita-ku, Saitama-shi, Saitama 331-9530, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
WNT-5A, a secretory glycoprotein, is related to the proliferation of dermal papilla cells. To develop a hair-
growth stimulant, we have been searching for inhibitors of WNT-5A expression. We identified radicicol
(1) as an active compound, and synthesized several radicicol derivatives. Among them, 6,7-dihydro-10a-
hydroxy radicicol (31) was found to function as a new potent WNT-5A expression inhibitor with rela-
tively low toxicity and excellent stability.
Received 28 March 2009
Revised 26 April 2009
Accepted 28 April 2009
Available online 3 May 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Hair-growth
WNT-5A
Radicicol
SAR
1. Introduction
isolated 10 new radicicol analogues and 10 known radicicol ana-
logues from a culture broth of the fungus Pochonia chlamydosporia
Hair loss or hair thinning is a common complaint of people liv-
ing in today’s stressful world.1 Some drugs such as minoxidil2,3 and
finasteride,4 that counter hair loss have been developed, but their
therapeutic effects are limited. Consequently, a new drug is re-
quired that induces hair-growth through a new mechanism. Hu-
man hair follicles are made up of epithelial and mesenchymal
dermal cells such as keratinocytes, dermal papilla cells, fibroblasts,
and sebaceous cells; and the hair-growth cycle (hair cycle) is con-
trolled through interactions among these cells. The hair shaft is
formed by the proliferation and differentiation (keratinization) of
follicular keratinocytes. Dermal papillae regulate the proliferation,
differentiation, and apoptosis of these follicular keratinocytes,
which play a key role in controlling the hair cycle.5 Wingless-type
mouse mammary tumor virus integration-site family, member 5A
(WNT-5A) is a secretory glycoprotein that belongs to the WNT
family. WNTs are important intercellular signaling molecules that
regulate axis formation and organ formation during the fetal
stage.6,7 We have been studying molecules that regulate the prolif-
eration of dermal papilla cells to develop a hair-growth stimulant
and recently found that WNT-5A was highly expressed in the der-
mal papillae of depilated skin. A WNT-5A expression inhibitor pro-
motes the proliferation of dermal papilla cells,8 and by using such
an inhibitor against WNT-5A expression as a guide for bioassay, we
obtained radicicol (1)9–12 as a potently active compound. We have
var. chlamydosporia and reported the structures and WNT-5A
expression inhibitory activity of these compounds.13,14 In this
study, we report the synthesis of the derivatives of radicicol and
monocillin (radicicol dechlorinated analogue),15,16 their WNT-5A
expression inhibitory activity, the structure–activity relationships
(SARs), cytotoxicity of the compounds against dermal papilla cells,
and their chemical stability.
2. Results and discussion
2.1. Chemistry
To obtain radicicol (1) and monocillins [monicillin I (3), II (5),
and III (4)] as starting materials (Fig. 1), we cultured the strain
TF-0480, identified as Pochonia chlamydosporia, and purified the
resulting compounds using silica gel column chromatography, pre-
parative TLC and recrystallization.14
To study the SARs of the radicicol derivatives and their WNT-5A
expression inhibitory activity, some of the moieties in 1 were al-
tered; (i) the phenolic hydroxyl groups at C-14 and C-16, (ii) the
epoxide ring at C4–C5, (iii) the double bond at C6–C9, (iv) the car-
bonyl group at C-10, and (v) the halogen substituent at C-13
(Fig. 1). The reactions used for the transformation are shown in
Schemes 1–3.
Methylation and acetylation of the phenolic hydroxyl groups
(C-14 and C-16) in 1 produced dimethyl- (6) and diacetyl-deriva-
tives (7), respectively.
* Corresponding author. Tel.: +81 48 669 3109; fax: +81 48 652 7254.
E-mail address: h.shinonaga@po.rd.taisho.co.jp (H. Shinonaga).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.04.060

H. Shinonaga et al. / Bioorg. Med. Chem. 17 (2009) 4622–4635
4623
J4,5 = 3.0 Hz) and NOESY data. The NOE correlations between H-2/
H-4, H-4/H-8, H-3b/H-5, and H-5/H-8 indicated the relative config-
uration of 8 at C4-C5, together with the conformation of 1. The cyc-
lic carbonate-derivative (9) was obtained by treating 1 with
chlorosulfonyl isocyanate.18 The stereochemistry around the new
ring was determined by the NOESY spectrum exhibiting NOE cross
peaks between H3-1/H-4, H-4/H-6, and H-2/H-8.
1
4
OH
R
O
18
(ii)
6
15
O
(i)
HO
(v)
O
13
10
O
(iii)
(iv)
Treatment of 1 with 1 M hydrochloric acid in DMF produced
two chlorohydrin stereoisomers (10, 11) and two diol-derivatives
(12, 13) at the epoxide moiety of 1, as well as an unexpected prod-
uct (14) with a new dihydro-furan ring formed between C-5 and C-
8. The relative stereochemistries of these compounds (10–13) were
compared with known chlorohydrin derivative19 and assigned
based on 1H–1H coupling constants (10: J4,5 = 5.8 Hz, 12:
J4,5 = 6.1 Hz, 11: J4,5 = 3.0 Hz, 13: J4,5 = 3.0 Hz) and NOESY correla-
tions (10 and 12: H-5/H-6 and H-5/H-8, 11 and 13: H-4/H-5 and
H-5/H-9) for the NMR spectra of 10–13.
1: R=Cl (radicicol)
3: R=H (monocillin I)
OH
O
O
O
HO
R
O
(iii) Hydrogenation of 1 produced two dihydro-derivatives and
one tetrahydro-derivative (15) and three secondary alcohol deriv-
atives (17–19) were converted from the epoxide. The two dihydro-
derivatives were identified as 7,8-dihydo-derivative (2) and 6,9-
dihydro-derivative (16) based on their 2D NMR spectra. Recently,
compound 2 was reported as pochonin A, a natural product.20
6,7,8,9-Tetrahydro-13-dechloro derivatives 37 and 38 were syn-
thesized from 4 and 5 in the same manner.
2: R=Cl (pochonin A)
4: R=H (monocillin III)
OH
O
O
HO
O
The Michael addition of the
a,b,c,d-conjugated carbonyl moie-
ties to 1 by treatment with thiophenol21 or thioacetic acid22 pro-
duced 1,6-adducts (20–23) that transferred the double bond at
5: (monocillin II)
Figure 1. Radicicol analogues (1–5) and conversion plan of radicicol.
the b,c-position and formed a Z- or E-configuration (Scheme 2).
The stereochemistry of 1,6-adduct at the C-6 position was deduced
using the 1H–1H coupling constants (20: J4,5 = 2.4 Hz, J5,6 = 8.5 Hz,
22: J4,5 = 1.8 Hz, J5,6 = 7.9 Hz) and NOESY correlations (20 and 22:
H-2/H-4, H-4/H-6, H-6/H-7, H-6/H-8, and H-5/H-6) for the NMR
spectra of 20 and 22.
To examine the influence of substituent at C-6 on WNT-5A
activity, we investigated hydrogenation of the Michael addition
derivative (20). Hydrogenation of 20 was attempted using several
The epoxide ring at position C4–C5 was replaced with a thiox-
irane (8) and a cyclic carbonate (9) rings, respectively. The thioxi-
rane-derivative (8) was obtained by treating 1 with potassium
thiocyanate in the presence of indium(III) bromide in acetonitrile
(MeCN).17 The E-stereochemistry at the 4,5-thioxirane ring was as-
signed based on a 1H–1H coupling constants (8: J4,5 = 5.4 Hz, 1:
OR
16
O
O
O
14
RO
Cl
O
6: R=Me
7
: R=Ac
a or b
OH
Cl
O
OH
Cl
O
O
O
O
O
d
c
S
1
O
HO
HO
5
5
O
O
e
8
9
OH
Cl
O
OH
Cl
O
OH
O
O
O
O
R
R
HO
HO
HO
HO
HO
+
+
HO
O
Cl
O
O
O
10
11
: R=Cl
: R=Cl
13: R=OH
12: R=OH
14
Scheme 1. Reagents and conditions: (a) MeI, K2CO3, DMF, rt, 6.5 h, 75% (6); (b) Ac2O, pyridine, rt, 6.5 h, 98% (7); (c) KSCN, InBr3, MeCN, 60 °C, 8 h, 3% (8); (d) ClSO2NCO,
CH2Cl2, rt, 6 h, 20% (9); (e) 1 M HCl, 1,4-dioxane, rt, 0.5 h, 12% (10), 2% (11), 2% (12), 2% (13), 11% (14).

4624
H. Shinonaga et al. / Bioorg. Med. Chem. 17 (2009) 4622–4635
OH
O
OH
Cl
O
OH
O
O
O
O
O
O
O
a
1
+
+
HO
+
+
HO
HO
Cl
Cl
O
O
O
a'
2
15
16
OH
Cl
O
OH
O
OH
O
O
O
O
4
HO
HO
HO
HO
HO
HO
Cl
Cl
O
O
O
17
18
19
OH
O
OH
O
O
O
O
O
b or c
HO
HO
+
6
Cl
Cl
O
SR
O
SR
20
: R=Ph
22: R=Ac
21: R=Ph
23
: R=Ac
OH
O
d
O
O
HO
Cl
O
S
24
Scheme 2. Reagents and conditions: (a) H2, 5% Pd–C (PH, wet-type), AcOEt, rt, 3 h, 38% (2), 31% (15), 0.2% (16); (a0) same condition of (a), 1% (17), 4% (18), 29% (19); (b) PhSH,
TEA, DMF, 0 °C, 3 h, 72% (20), 8% (21); (c) AcSH, TEA, DMF, 0 °C, 16 h, 11% (22), 3% (23); (d) H2, Wilkinson’s catalyst, EtOH, rt, 34 h, 8% (24).
metal catalysts23 including palladium, platinum, nickel, and rho-
dium; As a result, hydrogenation was obtained using palladium(II)
acetate, platinum(IV) oxide, and Wilkinson’s catalyst.24,25 Hydro-
genation with palladium carbon, palladium 2 wt % on strontium
carbonate,26 Lindlar catalyst,27 and rhodium carbon was unsuc-
cessful. Treatment with Raney nickel28 resulted in an overreaction
that progressed to dehydrogenation and dechlorination. Moreover,
a dihydro-derivative was not observed during LC–MS analysis.
Hydrogenation of 20 by treatment with Wilkinson’s catalyst pro-
duced fewer byproducts than treatment with the other catalysts
(palladium[II] acetate and platinum[IV] oxide) and produced a
dihydro-derivative (24).
(iv) To examine the influence of the carbonyl group at the C-10
position on WNT-5A expression inhibitory activity, compound 1
was treated with hydroxylamine hydrochloride19 to produce a ste-
reoisomer mixture of a 10-oxime derivative (25) (Z:E = 1:0.7, as cal-
culated by integrating the 1H NMR signals) (Scheme 3). The
stereochemistry of 25 at C-10 was determined based on the results
of 13C NMR chemical shifts at C-11 (E-oxime exhibited a high-field
shift at about dC 29 [E-oxime], 36 ppm [Z-oxime])29,30 and a NOESY
experiment. Furthermore, we synthesized O-benzyloxime- and O-
methyloxime-derivativesandseparatedthemusingC18 HPLCto pro-
duce Z-isomers (26 and 28) and E-isomers (27 and 29), respectively.
A Luche reduction31,32 of compounds 1, 2, and 15 produced
mainly 1,2-reductive derivatives at C-10 (Scheme 3). The produc-
tion ratio of the stereoisomers were controlled by the presence
of the double bond (rigidity). The ratios of the two stereoisomers
derived from 1, 2, and 15 were 10:0, 9:1, and 4:6, respectively.
Two stereoisomers were separated using C18 HPLC; the com-
pounds eluted earlier, possessed good crystallinity, and the crystals
of 31 and 33 were grown in methanol.
X-ray diffraction analyses of 31 and 33 were performed, and the
crystal structure with the absolute configuration of 31 is shown in
Figure 2. The absolute stereochemistry of 31 was 1R, 4R, 5R, and
10R, and the hydroxyl group at C-10 in both compounds was in
the
a-configuration.
Compound 30, derived from 1, was identified as a 10-
a
-hydroxy
radicicol by comparing its NMR data with that of 31–34. Com-
pounds 32 and 34 were identified as 10b-hydroxy-derivatives
based on the difference in their NMR chemical shifts around C-
10, compared with those of 31 and 33.
To obtain several 10-hydroxy derivatives, compounds 35 and 36
were derived from 16 by Luche reduction (ratio, 4:6). Dechloro
derivatives 39, 40, and 41 were synthesized from 4 and 5 in the
same manner. Methylation of 31 produced monomethyl- (42)
and dimethyl-derivatives (43) at the phenolic hydroxyl groups.
Acetylation of 31 produced a triacetyl-derivative (44) (Scheme 3).
2.2. WNT-5A expression inhibitory activity and structure–
activity relationship
The inhibition of WNT-5A expression was measured using the
QuantiGene assay (Panomics), which is a signal amplification nu-
cleic acid probe assay used for direct quantification of cellular
mRNA.33 Cytotoxicity against dermal papilla cells was measured
TM
using the Alamar Blue assay (Biotium). The WNT-5A expression

H. Shinonaga et al. / Bioorg. Med. Chem. 17 (2009) 4622–4635
4625
OH
Cl
O
OH
O
OH
Cl
O
O
O
O
b or c
a
O
O
O
1
HO
HO
HO
+
10
Cl
N
N
RO
N
HO
OR
25
d
26
27
: R=Bn
: R=Bn
28 : R=Me
29 : R=Me
OH
O
O
OR3
O
O
O
HO
d
O
10
Cl
2
R2O
HO
10
Cl
R1O
30
6
f
42
: R1=R3=H, R2=Me
43 : R1=H, R2=R3=Me
31 : 10 α-OH
32
: 10 β-OH
(R1=R2=R3=H)
g
44
: R1=R2=R3=Ac
OH
O
OH
O
d
d
O
O
O
15
O
16
HO
HO
10
10
Cl
Cl
HO
6
6
HO
35
33
: 10α-OH
: 10 α−OH
36: 10β-OH
34 : 10 β-OH
OH
O
OH
O
O
e
O
d
O
O
4
5
HO
HO
10
O
HO
37
39
OH
O
OH
O
e
d
O
O
HO
HO
10
HO
O
38
40
: 10 α-OH
41: 10 β-OH
Scheme 3. Reagents and conditions: (a) HONH2ꢀHCl, pyridine, 40 °C, 2.5 h, 18% (25); (b) BnONH2ꢀHCl, pyridine, 40 °C, 5.5 h, 3% (26), 2% (27); (c) MeONH2ꢀHCl, pyridine, 40 °C,
5.5 h, 6% (28), 5% (29); (d) NaBH4, CeCl3ꢀ7H2O, MeOH, 0 °C, 5 min, 30% (30), 24% (31), 2% (32), 21% (33), 33% (34), 7% (35), 13% (36), 71% (39), 79% (40), 21% (41); (e) H2, 5% Pd–
C (PH, wet-type), AcOEt, rt, 1.5 h, 87% (37), 75% (38); (f) MeI, K2CO3, DMF, rt, 4.5 h, 18% (42), 40% (43); (g) Ac2O, pyridine, rt, 17 h, 88% (44).
inhibitory activity and cytotoxicity against dermal papilla cells of
the radicicol derivatives are summarized in Table 1. The SARs of
the radicicol derivatives and their WNT-5A expression inhibitory
activity and cytotoxicity were as follows (Fig. 3):
Most of the derivatives (11–14) were inactivated when the
epoxide ring was opened. Chlorohydrin (10) exhibited WNT-5A
activity, but reverted to radicicol (1) in the assay medium (phos-
phate buffer, pH 7.4). Compared with other ring-formed deriva-
tives, such as 8 and 9, the epoxide ring appeared to exhibit an
appropriate rigidity for a 14-membered macrolactone.
The Michael addition derivatives in the assay medium also re-
verted to radicicol, similar to chlorohydrin (10). To prevent this
retro reaction, Michael addition derivative (20) was reduced to
produce the 6a-thiophenyl-7,8,9-trihydro-derivative (24), which
was inactive. However, 6,7,8,9-tetrahydro-radicicol (15) exhibited
Figure 2. ORTEP structural plot of 31.
WNT-5A activity. Therefore, we suspect that not only the 14-mem-

4626
H. Shinonaga et al. / Bioorg. Med. Chem. 17 (2009) 4622–4635
Table 1
and that the influence of these two substituents on conformation is
important for WNT-5A expression inhibitory activity. The chemical
stabilities of the 10a-hydroxy-derivatives 30, 31, 33, and 35 dif-
fered. Compound 31 was more stable than 30, 33, and 35 under
acidic (pH 2) or neutral conditions (pH 7.4 in phosphate buffer sal-
ine) (data not shown).
WNT-5A expression inhibitory activities and cytotoxicities against dermal papilla
cells of radicicol (1) and radicicol derivatives (2–44)
a
a
Compound
IC50
(
l
M)
TC50
(l
M)
Compound
IC50
(
l
M)
TC50 (lM)
WNT-5A
WNT-5A
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
0.19
2.64
1.93
9.43
7.36
3.76
0.30
9.13
15.71
13.28
>50
>50
>50
>50
2.24
7.69
>50
>50
>50
15.35
57.32
2.90
28.87
17.62
8.40
29.09
28.23
28.11
41.98
N.T.
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
0.50
>50
>100
>50
89.45
65.61
42.13
The 10
good balance between WNT-5A activity and cytotoxicity. The crys-
tallinity, solubility and chemical stability of 10 -hydroxy-deriva-
a-hydroxy-derivatives were new compounds with a
0.26
0.51
0.06
0.15
1.22
2.07
1.89
>100
5.89
>100
>50
>50
14.95
>50
13.24
>50
>50
a
tive (31) make it a promising compound. Therefore, we believe
that the radicicol substituent at C-10 and the adjacent olefins are
important regions for WNT-5A activity and chemical stability.
Radicicol (monorden) exhibits antibiotic,9 antitumor,34 and
antimalarial35 activities. Recently, radicicol oxime derivatives have
been reported as antitumor medications, based on their inhibitory
actions on tyrosine kinase and heat shock protein 90 (HSP90).19,36
Oxime derivatives is stable and efficacious in vivo for improving
the instability that caused by the presence of the epoxide and
>100
>100
>100
>100
>100
>100
>100
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
>100
>100
N.T.
N.T.
N.T.
53.36
a c,d-conjugated carbonyl moieties.
,b,
>100
Yang et al.37 synthesized cycloproparadicicol, in which the epox-
N.T.
N.T.
N.T.
N.T.
N.T.
ide at C4–C5was replacedwith cyclopropyl, to improvethechemical
stability, and this compound inhibited HSP90 (IC50 160 nM).
Pochonin A, a naturally occurring 6,7-dihydro-radicicol,20 also
exhibited HSP90 affinity (IC50 90 nM) and was more stable than
radicicol (1).38
0.13
0.27
2.34
>100
>100
>100
>50
>100
N.T.: not tested.
a
Turbyville et al.39 reported that the structure–activity relation-
ship (SAR) and chemical stability of radicicol derivatives and
HSP90 inhibitory activity indicate that an sp2-hybridized carbon
at C-10, steric strain substituents at C4–C5 (oxirane, cyclopropyl,
and double bond) and a macrocyclic system were important factors
for activity, however, the Cl atom at C-13 was not essential for
HSP90 activity and enhanced cytotoxicity. Hellwig et al.20 reported
the SARs of radicicol and radicicol analogues, including pochonins
A–F, and antiviral activity. The epoxide ring at C4–C5 is important
for antiviral activity, but a double bond at the same position pro-
duces an inactive compound and the Cl atom at C-13 is not essen-
tial. Compared with the SARs necessary for inhibition of WNT-5A
expression, these SARs are similar to that of WNT-5A expression
inhibitory activity except for importance of the Cl atom at C-13.
TC50: half maximal toxic concentration.
alkyl: inactive
acyl: active
open: inactive
-O- -S-, -O(CO)O- : weak
OH
Cl
O
15
O
O
4
HO
13
10
6
O
Cl
H: toxic
chemical stability :
6, 7-dihydro > 6,9-dihydro = 6,7,8,9-tetrahydro
α-OH, N-OR: active, low toxic
β-OH : inactive
3. Conclusion
Figure 3. Structure–activity relationships (SARs) of radicicol derivatives and WNT-
5A expression inhibitory activity.
To develop a hair-growth stimulant, we have been searching for
radicicol analogues or derivatives for a new inhibitor against WNT-
5A expression. As a result, we synthesized and selected the 6,7-
bered macrolactone conformation formed from a double bond (C6–
C9) and epoxide (C4–C5) but also the presence or size of the sub-
stituents in the macrolactone contributed to the inhibition of
WNT-5A expression.
dihydro-10a-hydroxy derivative 31 from amongst the radicicol
derivatives, which exhibited moderate inhibition against WNT-5A
expression and no cytotoxicity against dermal papilla cells. We
confirmed that active compounds with an inhibiting effect on
WNT-5A expression also exerted dermal papilla cell proliferation
The chemical stabilities of hydrogenated radicicol derivatives
(2, 15, and 16) differed; compound 2 was stable under acidic and
in long-term storage conditions, but 15 and 16 were unstable un-
der these conditions. These results indicated that the conjugation
between the carbonyl at C-10 and the double bond is important
for WNT-5A expression inhibitory activity and chemical stability.
10-Oxime derivatives (25–29) retained their ability to inhibit
WNT-5A expression and exhibited lower cytotoxicity against der-
mal papilla cells. In case of the O-benzyloxime-derivative, the E-
isomer (27) was more potent than the Z-isomer (26); however,
the Z-isomer (28) of the O-methyloxime-derivative was more po-
tent than the E-isomer (29).
activity (data report in another article). 10-Oxime- or 10a-hydro-
xy-radicicol derivatives exhibited a good active profile in vitro.
The new radicicol derivative 31 exhibits a moderate solubility
and chemical stability. Therefore, in future, we plan to perform a
sequential study on the in vivo trichogenous activity of 31 and will
report the results of this study in another article.
4. Experimental
4.1. General experimental procedures
10
activity and no cytotoxicity, however, 10b-hydroxy-derivatives
did not inhibit WNT-5A expression. The 10 -hydroxy-derivatives
31 and 39 differed in the presence of a Cl atom at C-13, and com-
pound 31 was 7-fold more active than 39. These results suggested
that the Cl atom at the C-13 and C-10 substituents were very close
a-Hydroxy-derivatives (30, 31, and 33) exhibited inhibitory
1H and 13C nuclear magnetic resonance (NMR) spectra were re-
corded at 500 MHz using a JEOL Alpha 500 or Lambda 500 or ECA
500 spectrometer. The coupling constants (J) are given in hertz
(Hz), and the abbreviations s, d, t, q, br, and m refer to singlet, dou-
blet, triplet, quartet, broad, and multiplet peaks, respectively. All
a

H. Shinonaga et al. / Bioorg. Med. Chem. 17 (2009) 4622–4635
4627
assignments were based on 1H–1H correlation spectroscopy
(COSY), heteronuclear single- or multiple-quantum coherence
(HSQC or HMQC, respectively), heteronuclear multiple-bond corre-
lation (HMBC), and nuclear overhauser effect spectroscopy
(NOESY) methods. Chemical shifts are reported in parts per million
(ppm), with the solvent peaks used as an internal standard. Elec-
tron impact (EI) and electrospray ionization (ESI) mass spectra
were obtained using a JEOL JMS-SX102 spectrometer and Micro-
mass Platform LC mass spectrometer, respectively. Melting points
were determined using a MP-500D micro-melting point apparatus
(Yanaco). Optical rotations were measured using an AUTOPOL V
digital polarimeter (Rudolph Research Analytical). UV spectra were
recorded using a V-520 UV/Vis spectrophotometer (JASCO). IR
spectra were measured using a Spectrum One FT-IR spectrometer
(PerkinElmer). The reactions monitored by TLC and HPLC analysis
(system 1). For TLC analysis, we used Merck Silica Gel 60F254
thin-layer plates, UV light as the visualizing agent and 10% metha-
nolic phosphomolybdic acid and heat as the developing method.
The HPLC analysis system (system 1); LC1100 (Agilent) was oper-
ated with the following conditions: column, YMC-Pack ODS-AM,
4.2.2. Cytotoxicity against dermal papilla cells
Dermal papilla cells were sowed in a 96-well plate to yield a
density of 5 ꢁ 103 cells/well and cultured 16 h in MEM containing
12% FBS. The medium was then replaced with a medium to which
no compounds had been added or a medium that contained the
test compounds and was cultured for an additional 24 h. The
medium was then replaced with a medium containing 10% Ala-
TM
mar Blue (Wako Pure Chemical Industries) and the culture was
continued for an additional 4 h. Finally, the fluorescence intensity
(Ex 544 nm, Em 590 nm) was measured using a WALLAC 1420
ARVOsx.
4.3. Preparation and characteristics of starting materials:
radicicol (1), monocillin I (3), monocillin III (4), and monocillin
II (5)
These compounds were obtained by fungal fermentation in our
laboratory,14 and their structures were confirmed using spectral
data described in the previous studies.11,16,20
3
l
m, 4.6 ꢁ 100 mm (YMC Co.); eluent A, H2O + 0.001% trifluoro-
4.3.1. Radicicol (1)
acetic acid (TFA); eluent B, acetonitrile (MeCN); gradient, 15–85%
B, 30 min; flow rate, 1.0 mL/min; temperature, 40 °C. LC–MS sys-
tem (system 2): LC/MSD (Agilent) was operated in the ESI positive
and negative mode with the following conditions: column, YMC-
Colorless crystal; mp 181–183 °C, lit.:10 195 °C; ½a D20
ꢂ
+194.6 (c
1.00, chloroform), lit.:10
anol) kmax (log e) 215 (4.58), 266 (4.34), and 308 sh (3.92) nm; IR
[a] +216 (c 1.00, chloroform); UV (meth-
D
(KBr) mmax 3418, 2988, 1706, 1655, 1607, 1438, 1360, 1306, 1244,
1110, 1043, 982, 925, 848, 732, 712, 670, 602 cmꢃ1; HPLC, tR
8.10 min (system 1); 1H NMR (chloroform-d/methanol-d4,
500 MHz) d 7.48 (1H, dd, J = 15.9, 9.8 Hz, H-8), 6.45 (1H, s, H-15),
6.15 (1H, t, J = 9.8 Hz, H-7), 6.05 (1H, d, J = 15.9 Hz, H-9), 5.78
(1H, dd, J = 9.8, 3.0 Hz, H-6), 5.41 (1H, ddq, J = 3.7, 3.7, 6.7 Hz, H-
2), 4.51 (1H, d, J = 16.5 Hz, H-11a), 3.86 (1H, d, J = 16.5 Hz, H-
11b), 3.19 (1H, br s, H-5), 2.94 (1H, dt, J = 9.6, 3.0 Hz, H-4), 2.33
(1H, dt, J = 15.3, 3.7 Hz, H-3a), 1.83 (1H, ddd, J = 15.3, 9.6, 3.7 Hz,
H-3b), 1.48 (3H, d, J = 6.7 Hz, H3-1); 13C NMR (chloroform-d/meth-
anol-d4, 125 MHz) d 199.1 (s, C-10), 168.9 (s, C-18), 161.2 (s, C-16),
158.2 (s, C-14), 139.6 (d, C-8), 135.7 (s, C-12), 135.0 (d, C-6), 130.5
(d, C-9), 130.1 (d, C-7), 116.1 (s, C-13), 108.0 (s, C-17), 103.4 (d, C-
15), 71.2 (d, C-2), 56.1 (d, C-4), 55.8 (d, C-5), 46.3 (t, C-11), 36.3 (t,
C-3), 18.5 (q, C-1); ESI-MS (neg.) m/z (%) 363.0 (100, [MꢃH]ꢃ),
365.0 (40); HRESI-MS m/z 363.0647 (calcd for C18H16Cl1O6
Pack ODS-AM,
3
l
m, 4.6 ꢁ 100 mm (YMC Co.); eluent A,
H2O + 5 mM HCOONH4 + 5 mM HCOOH; eluent B, MeCN + 5 mM
HCOOH; gradient, 15–85% B, 30 min; flow rate, 1.0 mL/min; tem-
perature, 40 °C. MS parameters: scan range, 100–1200; capillary
voltage, 3000 V; fragmentor voltage, 100 V. The following materials
were used to separate the compounds: silica gel column chromatog-
raphy, Wako Pure Chemical C-200; preparative thin layer chroma-
tography, Merck silica gel plate 60F254 (layer thickness; 0.5 mm);
gel filtration, Sephadex LH-20 (Pharmacia Co.). Preparative HPLC
was performed using a Waters M600 system as follows: preparative
HPLC system, Waters M600; column, YMC-Pack Pro C18 AS-343,
5
l
m, 20 ꢁ 250 mm; flow rate, 10 mL/min; UV detection, 254 nm;
temperature, 40 °C; eluent, MeCN/H2O (+0.25% acetic acid).
4.2. Biological assays
[MꢃH]ꢃ, 363.0635,
D +1.2 mmu).
4.2.1. Inhibition of WNT-5A expression (quantification of
mRNA using the QuantiGene method)
4.3.2. Monocillin I (3)
Human dermal papilla cells, which were a gift from Dr. S. Arase
(University of Tokushima Faculty of Medicine), were cultured in
MEM (Invitrogen) containing 12% fetal bovine serum (FBS). The
dermal papilla cells in the fifth subculture generation were sowed
in a 96-well plate at a density of 1 ꢁ 104 cells/well and cultured
overnight. The medium was then replaced with one that contained
the test compounds or one that did not contain any test com-
pounds, and was cultured for an additional 24 h. After 24 h, the
amount of WNT-5A mRNA was measured using a QuantiGene High
Volume Kit (Bayer Medical) by the branched DNA (bDNA) signal
amplification method.33 In accordance with the manufacturer’s
protocol, the cells were lysed with a lysis mixture and the lysis
solution was added to the capture plate.
Colorless oil; ½a D20
ꢂ
+6.9 (c 1.00, acetone); UV (methanol) kmax
(log e) 217 (4.13), 262 (3.98), and 294 sh (3.67) nm; IR (KBr) mmax
3388, 2980, 2941, 1708, 1651, 1625, 1588, 1504, 1451, 1384,
1346, 1311, 1262, 1209, 1169, 1121, 1099, 1044, 1027, 993,
924, 850, 802, 754, 718, 693, 667, 618, 520 cmꢃ1; HPLC, tR
8.34 min (system 1); 1H NMR (acetone-d6, 500 MHz) d 10.94
(1H, br s, 16-OH), 9.16 (1H, br s, 14-OH), 7.83 (1H, dd, J = 16.1,
11.3 Hz, H-8), 6.30 (1H, dd, J = 11.3, 10.7 Hz, H-7), 6.29 (1H, d,
J = 2.7 Hz, H-15), 6.28 (1H, d, J = 2.7 Hz, H-13), 5.97 (1H, d,
J = 16.1 Hz, H-9), 5.83 (1H, dd, J = 10.7, 2.7 Hz, H-6), 5.49 (1H,
ddq, J = 3.7, 3.7, 6.7 Hz, H-2), 4.97 (1H, d, J = 14.0 Hz, H-11a),
3.56 (1H, d, J = 14.0 Hz, H-11b), 3.32 (1H, q, J = 2.7 Hz, H-5), 3.12
(1H, dt, J = 8.5, 2.7 Hz, H-4), 2.44 (1H, ddd, J = 14.9, 3.7, 2.7 Hz,
H-3a), 1.85 (1H, ddd, J = 14.9, 8.5, 3.7 Hz, H-3b), 1.60 (3H, d,
J = 6.7 Hz, H3-1); 13C NMR (acetone-d6, 125 MHz) d 198.9 (s, C-
10), 170.4 (s, C-18), 165.5 (s, C-16), 162.9 (s, C-14), 141.6 (d, C-
8), 140.3 (s, C-12), 137.2 (d, C-6), 131.7 (d, C-9), 130.6 (d, C-7),
110.1 (d, C-13), 106.0 (a, C-17), 102.8 (d, C-15), 72.0 (d, C-2),
56.0 (d, C-4), 55.7 (d, C-5), 43.9 (t, C-11), 36.9 (t, C-3), 18.9 (q,
C-1); ESI-MS (neg.) m/z (%) 329.1 (100, [MꢃH]ꢃ), 330.1 (20);
HRESI-MS m/z 329.1016 (calcd for C18H17O6 [MꢃH]ꢃ, 329.1025,
Next, a set of probes specific to WNT-5A8 was added and the
reaction was allowed to proceed at 53 °C for 20 h. After the plate
was washed using 0.1 ꢁ SSC (3.0 M NaCl and 0.3 M sodium citrate)
containing 0.03% lauryl sulfate, an amplification probe that con-
tained bDNA was added and allowed to react at 46 °C for 1 h. After
the plate was washed, a probe labeled with alkaline phosphatase
was added and allowed to react at 46 °C for 1 h. After the plate
was washed, the Lumi-Phos Plus substrate was added and the reac-
tion was allowed to proceed at 46 °C for 30 min. Luminescence was
measured using a WALLAC 1420 ARVOsx.
D
ꢃ0.9 mmu).

4628
H. Shinonaga et al. / Bioorg. Med. Chem. 17 (2009) 4622–4635
4.3.3. Monocillin III (4)
8.6, 3.7 Hz, H-3b), 1.50 (3H, d, J = 6.7 Hz, H3-1); 13C NMR (chloro-
Colorless crystal; mp 197–200 °C, lit.:16 204–205 °C; ½a D20
ꢂ
ꢃ0.8
form-d, 125 MHz) d 196.4 (s, C-10), 166.0 (s, C-18), 156.9 (s, C-
16), 156.3 (s, C-14), 138.6 (d, C-8), 135.6 (d, C-6), 132.4 (s, C-12),
131.0 (d, C-9), 130.2 (d, C-7), 117.9 (s, C-17), 115.5 (s, C-13), 95.5
(d, C-15), 70.2 (d, C-2), 56.4 (q, 14-OMe), 56.3 (q, 16-OMe), 55.6
(d, C-4), 55.5 (d, C-5), 45.2 (t, C-11), 37.3 (t, C-3), 18.6 (q, C-1);
ESI-MS m/z 415 [M+Na]+ as C20H21ClO6Na.
(c 1.00, acetone); UV (methanol) kmax (log e) 217 (4.49), 264
(4.00), and 304 (3.72) nm; IR (KBr) mmax 3613, 3418, 2985, 1674,
1643, 1621, 1584, 1493, 1464, 1440, 1418, 1389, 1358, 1342,
1312, 1260, 1217, 1196, 1175, 1143, 1100, 1078, 1059, 1036,
1021, 986, 922, 896, 879, 850, 805, 738, 585, 556, 502 cmꢃ1; HPLC,
tR 7.51 min (system 1); 1H NMR (acetone-d6, 500 MHz) d 11.91 (1H,
br s, 16-OH), 9.23 (1H, br s, 14-OH), 6.91 (1H, ddd, J = 15.8, 11.0,
4.6 Hz, H-8), 6.27 (1H, d, J = 2.4 Hz, H-15), 6.24 (1H, d, J = 2.4 Hz,
H-13), 6.02 (1H, d, J = 15.8 Hz, H-9), 5.18 (1H, m, H-2), 4.65 (1H,
d, J = 17.7 Hz, H-11a), 3.65 (1H, d, J = 17.7 Hz, H-11b), 2.80 (1H,
ddd, J = 5.5, 4.0, 2.7 Hz, H-4), 2.51 (1H, dt, J = 9.4, 2.7 Hz, H-5),
2.47 (1H, dd, J = 8.8, 4.6 Hz, H-7a), 2.29 (1H, m, H-7b), 2.27 (1H,
m, H-6a), 2.06 (1H, dt, J = 16.1, 4.0 Hz, H-3a), 1.63 (1H, ddd,
J = 16.1, 5.5, 4.0 Hz, H-3b), 1.37 (3H, d, J = 6.7 Hz, H3-1), 1.19 (1H,
m, H-6b); 13C NMR (acetone-d6, 125 MHz) d 196.9 (s, C-10),
171.9 (s, C-18), 167.1 (s, C-16), 163.4 (s, C-14), 148.6 (d, C-8),
141.3 (s, C-12), 131.9 (d, C-9), 113.9 (d, C-13), 105.9 (s, C-17),
102.8 (d, C-15), 72.5 (d, C-2), 57.0 (d, C-5), 55.7 (d, C-4), 48.2 (t,
C-11), 37.0 (t, C-3), 31.8 (t, C-6), 29.8 (t, C-7), 18.1 (q, C-1); ESI-
MS (neg.) m/z (%) 331.0 (100, [MꢃH]ꢃ), 332.0 (20); HRESI-MS m/z
4.4.2. Radicicol diacetate (7)
Acetic anhydride (4 mL) was added to a solution of 1 (15.3 mg,
0.042 mmol) in pyridine (1.5 mL), and the mixture was stirred for
6.5 h at room temperature. The reaction mixture was poured over
ice water and extracted with ethyl acetate. The organic layer was
washed with brine, dried over sodium sulfate, evaporated in vacuo
and purified using preparative TLC (developing solvent, chloro-
form–methanol = 95:5) to produce compound 7 (18.5 mg, 98.4%).
Compound 7: colorless oil; HPLC, tR 11.35 min (system 1); 1H
NMR (chloroform-d, 500 MHz) d 7.45 (1H, dd, J = 16.5, 11.0 Hz, H-
8), 7.02 (1H, s, H-15), 6.12 (1H, t, J = 11.0 Hz, H-7), 6.03 (1H, d,
J = 16.5 Hz, H-9), 5.74 (1H, dd, J = 11.0, 3.0 Hz, H-6), 5.37 (1H, m,
H-2), 4.04 (1H, d, J = 15.9 Hz, H-11a), 3.91 (1H, d, J = 15.9 Hz, H-
11b), 3.49 (1H, dd, J = 3.7, 3.0 Hz, H-5), 2.99 (1H, dt, J = 8.5,
3.7 Hz, H-4), 2.38 (1H, ddd, J = 14.6, 3.7, 3.0 Hz, H-3a), 2.31 (3H,
s, 16-OCOMe), 2.24 (3H, s, 14-OCOMe), 1.54 (1H, ddd, J = 14.6,
8.5, 3.7 Hz, H-3b), 1.51 (3H, d, J = 6.7 Hz, H3-1); 13C NMR (chloro-
form-d, 125 MHz) d 195.6 (s, C-10), 168.1 (s, C-18), 167.5 (s, 14-
OCOMe), 163.8 (s, 16-OCOMe), 148.7 (s, C-16), 146.6 (s, C-14),
139.1 (d, C-8), 136.1 (d, C-6), 133.6 (s, C-12), 130.6 (d, C-9), 129.9
(d, C-7), 126.5 (s, C-15), 126.3 (s, C-13), 117.8 (d, C-15), 70.9 (d,
C-2), 55.4 (d, C-4), 55.0 (d, C-5), 45.1 (t, C-11), 37.1 (t, C-3), 20.7
(q, 14-OCOMe), 20.6 (q, 16-OCOMe), 18.6 (q, C-1); ESI-MS m/z
471 [M+Na]+ as C22H21ClO8Na.
331.1188 (calcd for C18H19O6 [MꢃH]ꢃ, 331.1182,
D +0.6 mmu).
4.3.4. Monocillin II (5)
Colorless crystal; mp 188–191 °C, lit.:16 198–200 °C; ½a D20
ꢂ
+23.7
(c 0.43, acetone); UV (methanol) kmax (log e) 224 (4.60), 262 (4.18),
and 302 (3.90) nm; IR (KBr) mmax 3313, 2944, 1674, 1646, 1612,
1594, 1498, 1485, 1460, 1442, 1414, 1366, 1332, 1317, 1258,
1216, 1173, 1140, 1108, 1036, 979, 924, 892, 860, 823, 790, 728,
691, 625, 544 cmꢃ1; HPLC, tR 11.71 min (system 1); 1H NMR
(DMSO-d6, 500 MHz) d 10.27 (1H, br s, 16-OH), 9.92 (1H, br s,
14-OH), 6.64 (1H, ddd, J = 16.1, 7.6, 6.4 Hz, H-8), 6.20 (1H, d,
J = 2.4 Hz, H-15), 6.09 (1H, d, J = 2.4 Hz, H-13), 5.86 (1H, d,
J = 16.1 Hz, H-9), 5.31 (1H, ddd, J = 15.2, 7.6, 3.0 Hz, H-4), 5.29
(1H, ddd, J = 15.2, 7.0, 4.3 Hz, H-5), 5.09 (1H, m, H-2), 3.90 (1H, d,
J = 15.2 Hz, H-11a), 3.50 (1H, d, J = 15.2 Hz, H-11b), 2.39 (1H, dt,
J = 14.0, 3.0 Hz, H-3a), 2.25–2.13 (3H, m, H-3b, H-6a and H-7a),
2.12 (1H, m, H-7b), 2.04 (1H, m, H-6b), 1.23 (3H, d, J = 6.4 Hz, H3-
1); 13C NMR (DMSO-d6, 125 MHz) d 196.6 (s, C-10), 168.4 (s, C-
18), 160.2 (s, C-16), 159.5 (s, C-14), 148.2 (d, C-8), 136.6 (s, C-
12), 131.2 (d, C-5), 129.7 (d, C-9), 128.3 (d, C-4), 110.3 (s, C-17),
108.8 (d, C-13), 101.4 (d, C-15), 71.0 (d, C-2), 45.2 (t, C-11), 37.9
(t, C-3), 30.4 (t, C-6), 30.3 (t, C-7), 19.2 (q, C-1); ESI-MS (neg.) m/z
(%) 315.1 (100, [MꢃH]ꢃ), 316.1 (20); HRESI-MS m/z 315.1228
4.4.3. 4,5-Thioxirane radicicol derivative (8)
Indium(III) bromide (48 mg, 0.14 mmol, 0.1 equiv) and potas-
sium thiocyanate (125 mg, 1.29 mmol, 1.3 equiv) were added to a
solution of 1 (366 mg, 1.00 mmol) in MeCN (12 mL) at room tem-
perature. The mixture was stirred for 8 h at 60 °C and then cooled
to room temperature. The reaction mixture was diluted with water
and extracted with ethyl acetate. The organic layer was washed
with brine, dried over sodium sulfate, evaporated in vacuo and
purified using preparative HPLC (eluent, MeCN–H2O = 40:60), and
was further purified using preparative TLC (developing solvent,
chloroform–methanol = 9:1) to produce compound 8 (12.2 mg,
3.0%). Compound 8: colorless oil; HPLC, tR 11.68 min (system 1);
1H NMR (acetone-d6, 500 MHz) d 7.42 (1H, dd, J = 16.0, 9.2 Hz, H-
8), 6.57 (1H, s, H-15), 6.15 (1H, dd, J = 10.7, 9.2 Hz, H-7), 6.10
(1H, d, J = 16.0 Hz, H-9), 5.91 (1H, dd, J = 10.7, 4.6 Hz, H-6), 5.36
(1H, m, H-2), 4.45 (1H, d, J = 16.0 Hz, H-11a), 4.40 (1H, d,
J = 16.0 Hz, H-11b), 3.55 (1H, dd, J = 5.4, 4.6 Hz, H-5), 3.29 (1H, dt,
J = 7.6, 5.4 Hz, H-4), 2.46 (1H, ddd, J = 15.3, 5.4, 2.3 Hz, H-3a),
2.11 (1H, ddd, J = 15.3, 7.6, 6.9 Hz, H-3b), 1.46 (3H, d, J = 6.1 Hz,
H3-1); 13C NMR (acetone-d6, 125 MHz) d 195.7 (s, C-10), 169.3 (s,
C-18), 159.8 (s, C-16), 157.7 (s, C-14), 138.8 (d, C-8), 136.6 (d, C-
6), 135.9 (s, C-12), 132.6 (d, C-9), 128.8 (d, C-7), 116.5 (s, C-17),
111.1 (s, C-13), 103.5 (d, C-15), 74.1 (d, C-2), 43.6 (t, C-11), 43.2
(t, C-3), 40.5 (d, C-4), 40.4 (d, C-5), 20.2 (q, C-1); ESI-MS m/z 379
[MꢃH]ꢃ as C18H16ClO5S.
(calcd for C18H19O5 [MꢃH]ꢃ, 315.1232,
D
ꢃ0.4 mmu).
4.4. Preparation and characteristics of radicicol derivatives
4.4.1. Radicicol dimethyl ether (6)
Potassium carbonate (3 mg, 0.022 mmol) and methyl iodide
(4 mL, 64.2 mmol) were added to a solution of radicicol (1)
(19.0 mg, 0.052 mmol) in N,N-dimethylformamide (1 mL), and the
mixture was stirred for 6.5 h at room temperature. The reaction
mixture was diluted with water and extracted with ethyl acetate.
The organic layer was washed with brine, dried over sodium sulfate,
evaporated in vacuo and purified using preparative TLC (developing
solvent, chloroform–methanol = 95:5) to produce compound 6
(15.3 mg, 74.8%). Compound 6: colorless oil; HPLC, tR 11.27 min
(system 1); 1H NMR (chloroform-d, 500 MHz) d 7.48 (1H, dd,
J = 15.9, 10.4 Hz, H-8), 6.45 (1H, s, H-15), 6.12 (1H, t, J = 10.4 Hz,
H-7), 6.07 (1H, d, J = 15.9 Hz, H-9), 5.69 (1H, dd, J = 10.4, 4.3 Hz,
H-6), 5.35 (1H, m, H-2), 3.95 (1H, d, J = 15.9 Hz, H-11a), 3.89 (3H,
s, 16-OMe), 3.83 (3H, s, 14-OMe), 3.75 (1H, d, J = 15.9 Hz, H-11b),
3.40 (1H, dd, J = 4.3, 1.8 Hz, H-5), 3.02 (1H, dt, J = 8.6, 1.8 Hz, H-4),
2.40 (1H, ddd, J = 14.6, 3.7, 1.8 Hz, H-3a), 1.61 (1H, ddd, J = 14.6,
4.4.4. 4,5-Carbonate radicicol derivative (9)
Chlorosulfonyl isocyanate (0.5 mL, 5.74 mmol, 2.2 equiv) was
added to a solution of 1 (930 mg, 2.55 mmol) in dichloromethane
(35 mL), and the mixture was stirred for 6 h at room temperature.
The reaction mixture was diluted with water and extracted with
ethyl acetate. The organic layer was washed with brine, dried over
sodium sulfate, evaporated in vacuo and purified using preparative

H. Shinonaga et al. / Bioorg. Med. Chem. 17 (2009) 4622–4635
4629
TLC (developing solvent, n-hexane–acetone = 1:1), and was further
purified using preparative HPLC (eluent, MeCN–H2O = 45:55) to
produce compound 9 (189.8 mg, 19.5%). Compound 9: colorless
oil; HPLC, tR 8.04 min (system 1); 1H NMR (acetone-d6, 500 MHz)
d 9.98 (1H, br s, 16-OH), 9.60 (1H, br s, 14-OH), 7.53 (1H, dd,
J = 15.9, 10.4 Hz, H-8), 6.58 (1H, s, H-15), 6.44 (1H, dd, J = 11.0,
10.4 Hz, H-7), 6.13 (1H, d, J = 15.9 Hz, H-9), 5.98 (1H, dd, J = 11.0,
7.3 Hz, H-6), 5.59 (1H, m, H-5), 5.56 (1H, m, H-2), 4.75 (1H, dt,
J = 6.7, 3.7 Hz, H-4), 4.36 (1H, d, J = 16.5 Hz, H-11a), 4.01 (1H, d,
J = 16.5 Hz, H-11b), 2.42 (1H, ddd, J = 16.5, 6.7, 3.0 Hz, H-3a), 2.30
(1H, ddd, J = 16.5, 6.1, 3.7 Hz, H-3b), 1.54 (3H, d, J = 6.1 Hz); 13C
NMR (acetone-d6, 125 MHz) d 196.9 (s, C-10), 168.3 (s, C-18),
160.0 (s, C-16), 157.9 (s, C-14), 154.3 (s, C-10; –O–CO–O–), 138.3
(d, C-8), 136.2 (s, C-12), 134.6 (d, C-6), 133.2 (d, C-9), 131.1 (d, C-
7), 115.5 (s, C-13), 111.1 (s, C-17), 103.9 (d, C-15), 80.1 (d, C-4),
77.7 (d, C-5), 70.4 (d, C-2), 45.6 (t, C-11), 36.5 (t, C-3), 19.8 (q, C-
1); ESI-MS m/z 407 [MꢃH]ꢃ as C19H16ClO8.
1.8 Hz, H-3a), 1.87 (1H, ddd, J = 15.3, 9.2, 3.7 Hz, H-3b), 1.42 (3H,
d, J = 6.7 Hz, H3-1); 13C NMR (methanol-d4, 125 MHz) d 200.2 (s,
C-10), 168.4 (s, C-18), 156.9 (s, C-16), 156.7 (s, C-14), 142.9 (d, C-
8), 142.4 (d, C-6), 134.7 (s, C-12), 131.5 (d, C-9), 130.0 (d, C-7),
116.6 (s, C-17), 114.3 (s, C-13), 103.8 (d, C-15), 72.0 (d, C-5), 71.9
(d, C-4), 71.2 (d, C-2), 45.8 (t, C-11), 37.8 (t, C-3), 19.4 (q, C-1);
ESI-MS m/z 381 [MꢃH]ꢃ as C18H18ClO7.
4.4.5.4. 4,5-Diol derivative (13).
Colorless oil; HPLC, tR
4.57 min (system 1); 1H NMR (methanol-d4, 500 MHz) d 7.36
(1H, dd, J = 15.9, 10.4 Hz, H-8), 6.44 (1H, s, H-15), 6.12 (1H, t,
J = 10.4 Hz, H-7), 5.96 (1H, d, J = 15.9 Hz, H-9), 5.94 (1H, dd,
J = 10.4, 7.3 Hz, H-6), 5.45 (1H, m, H-2), 4.56 (1H, m, H-5), 4.53
(1H, d, J = 15.9 Hz, H-11a), 3.87 (1H, d, J = 15.9 Hz, H-11b), 3.64
(1H, dt, J = 9.8, 3.0 Hz, H-4), 2.10 (1H, ddd, J = 14.6, 9.2, 3.0 Hz, H-
3a), 1.83 (1H, ddd, J = 14.6, 9.8, 3.0 Hz, H-3b), 1.44 (3H, d,
J = 6.1 Hz, H3-1); 13C NMR (methanol-d4, 125 MHz) d 199.5 (s, C-
10), 168.7 (s, C-18), 158.9 (s, C-16), 158.2 (s, C-14), 143.7 (d, C-
6), 140.6 (d, C-8), 135.8 (s, C-12), 131.3 (d, C-9), 127.2 (d, C-7),
115.9 (s, C-17), 113.0 (s, C-13), 104.0 (d, C-15), 72.7 (d, C-4), 71.2
(d, C-2), 69.5 (d, C-5), 44.5 (t, C-11), 39.3 (t, C-3), 21.0 (q, C-1);
ESI-MS m/z 381 [MꢃH]ꢃ as C18H18ClO7.
4.4.5. Chlorohydrin (10), (11) and diol (12), (13) derivatives
One-molar hydrochloric acid (2 mL) was added to a solution of
1 (611 mg, 1.67 mmol) in 1,4-dioxane (8 mL), and the mixture was
stirred for 30 min at room temperature. The reaction mixture was
neutralized with 1 M sodium hydroxide, evaporated in vacuo and
purified using preparative HPLC (eluent, MeCN–H2O = 35:65) to
produce compounds 10 (83.2 mg, 12.4%), 11 (10.4 mg, 1.5%), 12
(10.7 mg, 1.7%), and 13 (9.9 mg, 1.5%).
4.4.6. Compound (14)
One-molar hydrochloric acid (12 mL) was added to a solution of
1 (930 mg, 2.55 mmol) in 1,4-dioxane (14 mL), and the mixture
was stirred for 2 h at room temperature. The reaction mixture
was diluted with water and extracted with ethyl acetate. The or-
ganic layer was washed with brine, dried over sodium sulfate,
and concentrated to produce a colorless oil. The crude oil was puri-
fied using preparative TLC (developing solvent, chloroform–meth-
anol–n-hexane = 5:1:5), and was further purified using preparative
HPLC (eluent, MeCN–H2O = 30:70 to 40:60, gradient) to produce
compound 14 (103.7 mg, 10.6%). Compound 14: colorless oil;
HPLC, tR 5.21 min (system 1); 1H NMR (DMSO-d6, 500 MHz) d
10.47 (1H, br s, 16-OH), 9.97 (1H, s, 14-OH), 6.50 (1H, s, H-15),
6.00 (1H, dt, J = 6.1, 1.8 Hz, H-6), 5.89 (1H, dt, J = 6.1, 1.8 Hz, H-7),
5.20 (1H, m, H-2), 5.01 (1H, m, H-8), 4.88 (1H, d, J = 6.7 Hz, OH-
4), 4.21 (1H, dt, J = 9.8, 1.8 Hz, H-5), 3.90 (1H, d, J = 18.3 Hz, H-
11a), 3.71 (1H, d, J = 18.3 Hz, H-11b), 3.20 (1H, m, H-4), 2.76 (1H,
dd, J = 14.6, 4.9 Hz, H-9a), 2.37 (1H, dd, J = 14.6, 5.5 Hz, H-9b),
2.16 (1H, m, H-3a), 1.44 (1H, ddd, J = 14.6, 7.9, 3.0 Hz, H-3b), 1.26
(3H, d, J = 6.7 Hz, H3-1); 13C NMR (DMSO-d6, 125 MHz) d 203.8 (s,
C-10), 166.4 (s, C-18), 154.7 (s, C-16), 154.4 (s, C-14), 130.2 (s, C-
12), 130.0 (d, C-7), 129.3 (d, C-6), 115.3 (s, C-13), 111.7 (s, C-17),
102.4 (d, C-15), 88.4 (d, C-5), 83.3 (d, C-8), 70.3 (d, C-4), 69.7 (d,
C-2), 48.3 (t, C-11), 45.1 (t, C-9), 37.6 (t, C-3), 19.2 (q, C-1); ESI-
MS m/z 381 [MꢃH]ꢃ; HREI-MS m/z 382.0813 (calcd for C18H18Cl1O7
4.4.5.1. Chlorohydrin derivative (10) (pochonin C;20 syn-
thetic).
Colorless oil; HPLC, tR 5.72 min (system 1); 1H NMR
(methanol-d4, 500 MHz) d 7.21 (1H, dd, J = 16.1, 11.3 Hz, H-8),
6.46 (1H, s, H-15), 6.17 (1H, dd, J = 11.3, 10.0 Hz, H-7), 5.95 (1H,
d, J = 16.1 Hz, H-9), 5.75 (1H, t, J = 10.0 Hz, H-6), 5.38 (1H, m, H-
2), 5.12 (1H, dd, J = 10.0, 5.8 Hz, H-5), 4.21 (1H, d, J = 16.2 Hz, H-
11a), 3.99 (1H, ddd, J = 9.1, 5.8, 2.4 Hz, H-4), 3.66 (1H, d,
J = 16.2 Hz, H-11b), 2.03 (1H, ddd, J = 15.2, 7.0, 2.4 Hz, H-3a), 1.89
(1H, ddd, J = 15.2, 9.1, 3.4 Hz, H-3b), 1.42 (3H, d, J = 6.4 Hz, H3-1);
13C NMR (methanol-d4, 125 MHz) d 199.8 (s, C-10), 168.1 (s, C-
18), 157.0 (s, C-16), 156.9 (s, C-14), 141.1 (d, C-8), 138.1 (d, C-6),
134.6 (s, C-12), 132.6 (d, C-9), 130.9 (d, C-7), 116.3 (s, C-17),
114.3 (s, C-13), 103.9 (d, C-15), 72.6 (d, C-4), 70.9 (d, C-2), 60.6
(d, C-5), 45.7 (t, C-11), 38.2 (t, C-3), 19.3 (q, C-1); ESI-MS m/z 399
[MꢃH]ꢃ; HRESI-MS m/z 423.0736 (calcd for C18H18Cl2O6Na
[M+Na]+, 423.0378,
D
ꢃ0.2 mmu).
4.4.5.2. Chlorohydrin derivative (11).
Colorless oil; HPLC, tR
7.22 min (system 1); 1H NMR (methanol-d4, 500 MHz) d 7.18 (1H,
dd, J = 15.9, 9.8 Hz, H-8), 6.46 (1H, s, H-15), 6.14 (1H, t, J = 9.8 Hz,
H-7), 6.02 (1H, t, J = 9.8 Hz, H-6), 6.00 (1H, d, J = 15.9 Hz, H-9),
5.27 (1H, m, H-2), 5.00 (1H, dd, J = 9.8, 3.0 Hz, H-5), 4.73 (1H, d,
J = 15.3 Hz, H-11a), 3.88 (1H, dt, J = 9.8, 3.0 Hz, H-4), 3.76 (1H, d,
J = 15.3 Hz, H-11b), 2.12 (1H, ddd, J = 14.0, 9.8, 3.0 Hz, H-3a), 1.96
(1H, ddd, J = 14.0, 10.4, 3.0 Hz, H-3b), 1.46 (3H, d, J = 6.1 Hz, H3-
1); 13C NMR (methanol-d4, 125 MHz) d 198.7 (s, C-10), 168.5 (s,
C-18), 158.6 (s, C-16), 157.8 (s, C-14), 139.1 (d, C-6), 138.6 (d, C-
8), 135.4 (s, C-12), 131.7 (d, C-9), 127.6 (d, C-7), 115.7 (s, C-17),
113.8 (s, C-13), 104.1 (d, C-15), 72.1 (d, C-4), 71.0 (d, C-2), 60.4
(d, C-5), 44.5 (t, C-11), 40.6 (t, C-3), 20.8 (q, C-1); ESI-MS m/z 399
[MꢃH]ꢃ as C18H17Cl2O6.
[M]+, 382.0819,
D
ꢃ0.6 mmu).
4.4.7. Hydrogenation of radicicol (1) (i)
Five-percent palladium–carbon (PH, wet-type, Kawaken Fine
Chemicals Co.) (255 mg) was added to a solution of 1 (10.8 g,
29.6 mmol) in ethyl acetate (140 mL). After hydrogenation for 3 h
at room temperature under atmospheric pressure, the catalyst
was removed by filtration and the filtrate was concentrated in va-
cuo. The residue was purified using silica gel column chromatogra-
phy (eluent, n-hexane–ethyl acetate = 3:2) to produce compounds
15 (3.43 g, 31.4%), 2 (4.14 g, 38.1%) and a semi-pure fraction
(500 mg) containing compound 2. The semi-pure fraction was
purified using preparative HPLC (eluent, MeCN–H2O = 38:62) to
produce compound 2 (177.8 mg, 1.7%) and 16 (20.7 mg, 0.2%).
4.4.5.3. 4,5-Diol derivative (12).
Colorless oil; HPLC, tR
3.71 min (system 1); 1H NMR (methanol-d4, 500 MHz) d 7.30
(1H, dd, J = 15.9, 11.0 Hz, H-8), 6.41 (1H, s, H-15), 6.14 (1H, t,
J = 11.0 Hz, H-7), 5.90 (1H, d, J = 15.9 Hz, H-9), 5.73 (1H, dd,
J = 11.0, 8.5 Hz, H-6), 5.40 (1H, m, H-2), 4.78 (1H, dd, J = 8.5,
6.1 Hz, H-5), 4.18 (1H, d, J = 15.9 Hz, H-11a), 3.80 (1H, m, H-4),
3.63 (1H, d, J = 15.9 Hz, H-11b), 1.95 (1H, ddd, J = 15.3, 6.7,
4.4.7.1. 6,7,8,9-Tetrahydroradicicol (15).
153–155 °C; UV (methanol) kmax (log ) 215 (4.40), 259 (3.89), and
310 (3.83) nm; IR (KBr) mmax 3120, 2940, 1714, 1665, 1609, 1578,
Colorless solid; mp
e

4630
H. Shinonaga et al. / Bioorg. Med. Chem. 17 (2009) 4622–4635
1487, 1467, 1436, 1417, 1370, 1354, 1340, 1309, 1264, 1228, 1177,
1146, 1115, 1070, 1053, 1036, 1023, 974, 930, 874, 837, 782, 695,
669, 632, 606, 549 cmꢃ1; HPLC, tR 8.57 min (system 1); 1H NMR
(chloroform-d/methanol-d4, 500 MHz) d 6.37 (1H, s, H-15), 5.15
(1H, m, H-2), 4.25 (1H, d, J = 18.3 Hz, H-11a), 4.02 (1H, d,
J = 18.3 Hz, H-11b), 2.68 (1H, m, H-4), 2.53 (1H, dt, J = 8.8, 3.0 Hz,
-5), 2.34 (2H, m, H2-9), 2.04 (1H, ddd, J = 15.5, 5.5, 4.3 Hz, H-3),
1.92 (1H, ddd, J = 14.0, 7.0, 3.0 Hz, H-6a), 1.77 (1H, ddd, J = 15.5,
6.4, 3.0 Hz, H-3b), 1.64 (1H, m, H-8a), 1.41 (2H, m, H-7a and H-
8b), 1.36 (1H, m, H-7b), 1.32 (3H, d, J = 6.4 Hz, H3-1), 1.02 (1H, m,
H-6b); 13C NMR (chloroform-d/methanol-d4, 125 MHz) d 208.0 (s,
C-10), 169.6 (s, C-18), 162.2 (s, C-16), 158.2 (s, C-14), 135.6 (s, C-
12), 115.6 (s, C-13), 106.4 (s, C-17), 103.1 (d, C-15), 70.9 (d, C-2),
57.6 (d, C-5), 54.8 (d, C-4), 46.5 (t, C-11), 40.3 (t, C-9), 36.1 (t, C-
3), 30.8 (t, C-6), 23.2 (t, C-7), 22.1 (t, C-8), 18.6 (q, C-1); ESI-MS
m/z 367 [MꢃH]ꢃ; HREI-MS m/z 368.1021 (calcd for C18H21Cl1O6
pounds 17 (17.4 mg, 1.4%), 18 (49.4 mg, 4.1%), and 19
(347.4 mg, 28.7%).
4.4.8.1. 5,6,9-Trihydro-4
a-hydroxyradicicol (17).
Colorless
oil; HPLC, tR 6.37 min (system 1); 1H NMR (chloroform-d,
500 MHz) d 6.53 (1H, s, H-15), 5.60 (1H, ddd, J = 15.2, 7.6, 7.0 Hz,
H-7), 5.47 (1H, dt, J = 15.2, 7.6 Hz, H-8), 5.33 (1H, m, H-2), 4.59
(1H, d, J = 18.3 Hz, H-11a), 4.48 (1H, d, J = 18.3 Hz, H-11b), 3.78
(1H, ddd, J = 6.7, 6.4, 5.2 Hz, H-4), 3.16 (1H, dd, J = 12.5, 7.6 Hz,
H-9a), 3.04 (1H, dd, J = 12.5, 7.6 Hz, H-9b), 2.20 (2H, m, H2-6),
1.89 (1H, ddd, J = 14.9, 7.6, 6.4 Hz, H-3a), 1.79 (1H, ddd, J = 14.9,
6.7, 3.0 Hz, H-3b), 1.63 (2H, m, H2-5), 1.40 (3H, d, J = 6.4 Hz, H3-
1); 13C NMR (chloroform-d, 125 MHz) d 205.0 (s, C-10), 168.0 (s,
C-18), 161.2 (s, C-16), 156.0 (s, C-14), 135.8 (s, C-12), 135.2 (d, C-
7), 123.5 (d, C-8), 115.2 (s, C-13), 108.5 (s, C-17), 103.7 (d, C-15),
71.7 (d, C-2), 66.4 (d, C-4), 46.4 (t, C-9), 45.1 (t, C-11), 42.1 (t, C-
3), 35.3 (t, C-5), 28.0 (t, C-6), 20.6 (q, C-1); ESI-MS m/z 367
[MꢃH]ꢃ as C18H20ClO6.
[M]+, 368.1027,
D
ꢃ0.6 mmu).
4.4.7.2.
thetic).
6,7-Dihydroradicicol (2) (pochonin A;20 syn-
Colorless crystal; mp 208–210 °C; ꢃ15.3 (c
½
a 2D0
ꢂ
4.4.8.2. 5,8,9-Trihydro-4
a-hydroxyradicicol (18).
Colorless
1.00, chloroform); UV (methanol) kmax (log
e
) 220 (4.58), 261
oil; HPLC, tR 6.62 min (system 1); 1H NMR (chloroform-d,
500 MHz) d 6.50 (1H, s, H-15), 5.50–5.41 (3H, m; small coupling,
H-2, H-6 and H-7), 4.38 (1H, d, J = 17.7 Hz, H-11a), 4.24 (1H, d,
J = 17.7 Hz, H-11b), 3.60 (1H, ddd, J = 6.7, 6.4, 5.8 Hz, H-4), 2.73
(1H, ddd, J = 13.4, 8.8, 3.0 Hz, H-9a), 2.40 (1H, m, H-8a), 2.32 (1H,
m, H-9b), 2.29 (1H, m, H-5a), 2.20 (1H, m, H-8b), 2.04 (1H, m, H-
5b), 1.86 (2H, m, H2-3), 1.42 (3H, d, J = 6.4 Hz, H3-1); 13C NMR
(chloroform-d, 125 MHz) d 207.8 (s, C-10), 168.8 (s, C-18), 161.9
(s, C-16), 156.7 (s, C-14), 136.1 (s, C-12), 131.1 (d, C-7), 126.5 (d,
C-6), 115.1 (s, C-13), 107.8 (s, C-17), 103.8 (d, C-15), 71.2 (d, C-
2), 69.3 (d, C-4), 47.4 (t, C-11), 41.4 (t, C-9), 41.1 (t, C-3), 34.6 (t,
C-5), 22.4 (t, C-8), 20.3 (q, C-1); ESI-MS m/z 367 [MꢃH]ꢃ as
C18H20ClO6.
(3.97), and 315 (3.95) nm; IR (KBr) mmax 3409, 2986, 2936, 1703,
1648, 1601, 1580, 1490, 1450, 1423, 1382, 1355, 1311, 1245,
1147, 1104, 1036, 969, 886, 847, 803, 784, 705, 670, 639 cmꢃ1
;
HPLC, tR 7.87 min (system 1); 1H NMR (chloroform-d/methanol-
d4, 500 MHz) d 6.85 (1H, ddd, J = 15.9, 11.0, 4.3 Hz, H-8), 6.48
(1H, s, H-15), 6.05 (1H, d, J = 15.9 Hz, H-9), 5.21 (1H, m, H-2),
4.40 (1H, d, J = 18.3 Hz, H-11a), 4.27 (1H, d, J = 18.3 Hz, H-11b),
2.74 (1H, dt, J = 4.3, 2.4 Hz, H-4), 2.52 (1H, dt, J = 9.8, 2.4 Hz, H-5),
2.48 (1H, m, H-7a), 2.35 (1H, ddd, J = 14.0, 7.3, 2.4 Hz, H-6a), 2.23
(1H, m, H-7b), 1.97 (1H, ddd, J = 15.9, 4.9, 2.4 Hz, H-3a), 1.81 (1H,
dt, J = 15.9, 4.3 Hz, H-3b), 1.21 (1H, m, H-6b), 1.41 (3H, d,
J = 6.1 Hz, H3-1); 13C NMR (chloroform-d/methanol-d4, 125 MHz)
d 197.0 (s, C-10), 170.3 (s, C-18), 163.4 (s, C-16), 158.8 (s, C-14),
148.0 (d, C-8), 136.1 (s, C-12), 130.1 (d, C-9), 116.3 (s, C-13),
106.3 (s, C-17), 103.5 (d, C-15), 72.1 (d, C-2), 57.6 (d, C-5), 56.0
(d, C-4), 45.1 (t, C-11), 36.5 (t, C-3), 30.9 (t, C-6), 29.2 (t, C-7),
17.8 (q, C-1); ESI-MS m/z 365 [MꢃH]ꢃ; HRESI-MS m/z 365.0789
4.4.8.3. 5,6,7,8,9-Pentahydro-4a-hydroxyradicicol (19).
Col-
orless oil; HPLC, tR 7.03 min (system 1); 1H NMR (chloroform-d,
500 MHz) d 6.54 (1H, s, H-15), 5.47 (1H, m, H-2), 4.51 (1H, d,
J = 17.7 Hz, H-11a), 4.33 (1H, d, J = 17.7 Hz, H-11b), 3.71 (1H, ddd,
J = 6.7, 6.1, 5.5 Hz, H-4), 2.58 (1H, ddd, J = 15.9, 8.5, 3.0 Hz, H-9a),
2.32 (1H, ddd, J = 15.9, 9.2, 3.7 Hz, H-9b), 1.95 (1H, ddd, J = 15.3,
7.3, 5.5 Hz, H-3a), 1.84 (1H, ddd, J = 15.3, 6.7, 3.0 Hz, H-3b), 1.69
(1H, m, H-8a), 1.60 (2H, m, H-5a and H-8b), 1.46 (2H, m, H-6a
and H-7a), 1.44 (3H, d, J = 6.7 Hz, H3-1), 1.32 (1H, m, H-5b), 1.24
(2H, m, H-6b and H-7b); 13C NMR (chloroform-d, 125 MHz) d
207.9 (s, C-10), 168.3 (s, C-18), 161.7 (s, C-16), 156.3 (s, C-14),
136.2 (s, C-12), 115.3 (s, C-13), 107.9 (s, C-17), 103.8 (d, C-15),
71.4 (d, C-2), 66.4 (d, C-4), 46.5 (t, C-11), 42.3 (t, C-3), 40.6 (t, C-
9), 34.9 (t, C-5), 25.2 (t, C-7), 22.8 (t, C-6), 22.0 (t, C-8), 20.3 (q,
C-1); ESI-MS m/z 369 [MꢃH]ꢃ as C18H22ClO6.
(calcd for C18H18Cl1O6 [MꢃH]ꢃ, 365.0792,
D
ꢃ0.3 mmu).
4.4.7.3. 6,9-Dihydroradicicol (16).
Colorless oil; HPLC, tR
7.40 min (system 1); 1H NMR (chloroform-d/methanol-d4,
500 MHz) d 6.38 (1H, s, H-15), 5.40 (1H, ddd, J = 15.3, 7.9, 7.3 Hz,
H-8), 5.28 (1H, ddd, J = 15.3, 7.3, 3.7 Hz, H-7), 5.18 (1H, m, H-2),
4.22 (1H, d, J = 18.3 Hz, H-11a), 4.06 (1H, d, J = 18.3 Hz, H-11b),
3.14 (1H, dd, J = 12.2, 7.9 Hz, H-9a), 2.81 (1H, dd, J = 12.2, 7.3 Hz,
H-9b), 2.69 (1H, dt, J = 9.2, 3.0 Hz, H-4), 2.60 (1H, dt, J = 7.9,
3.0 Hz, H-5), 2.53 (1H, ddd, J = 14.0, 7.9, 3.7 Hz, H-6a), 2.10 (1H,
ddd, J = 14.6, 4.9, 3.0 Hz, H-3a), 1.66 (2H, m, H-3b and 6b), 1.31
(3H, d, J = 6.7 Hz, H3-1); 13C NMR (chloroform-d/methanol-d4,
125 MHz) d 205.6 (s, C-10), 169.1 (s, C-18), 161.0 (s, C-16), 157.8
(s, C-14), 135.2 (s, C-12), 129.2 (d, C-7), 125.9 (d, C-8), 115.6 (s,
C-13), 107.6 (s, C-17), 103.1 (d, C-15), 71.5 (d, C-2), 58.2 (d, C-5),
53.7 (d, C-4), 45.8 (t, C-9), 45.4 (t, C-11), 36.2 (t, C-3), 33.8 (t, C-
6), 18.9 (q, C-1); ESI-MS m/z 365 [MꢃH]ꢃ as C18H18ClO6.
4.4.9. Michael addition of radicicol (1) (i)
Triethylamine (220 lL, 1.57 mmol) and thiophenol (500 mg,
4.54 mmol, 1.39 equiv) were added to a solution of 1 (1190 mg,
3.26 mmol) in N,N-dimethylformamide (10 mL), and the solution
was stirred for 3 h at 0 °C. The reaction mixture was diluted with
dilute hydrochloric acid and extracted with ethyl acetate. The or-
ganic layer was washed with brine, dried over sodium sulfate,
evaporated in vacuo and purified using preparative HPLC (eluent,
MeCN–H2O = 58:42) to produce compounds 20 (1109.2 mg,
71.6%) and 21 (126.9 mg, 8.2%).
4.4.8. Hydrogenation of radicicol (1) (ii)
Five-percent palladium–carbon (PH, wet-type) was added to a
solution of 1 (11.9 g, 32.6 mmol) in ethyl acetate (160 mL). After
hydrogenation for 3 h at room temperature under atmospheric
pressure, the catalyst was removed by filtration and the filtrate
was concentrated in vacuo. The residue was purified using silica
gel column chromatography (eluent, n-hexane–ethyl ace-
tate = 3:2 to 1:1) to produce compounds 15, 2, and a semi-pure
fraction (936 mg). The semi-pure fraction was purified using pre-
parative HPLC (eluent, MeCN–H2O = 34:66) to produce com-
4.4.9.1. 7,8-(Z)-9-Hydro-6
a-thiophenylradicicol (20).
Color-
less solid; mp 219–222 °C; HPLC, tR 13.00 min (system 1); 1H NMR
(DMSO-d6, 500 MHz) d 10.39 (1H, s, 16-OH), 9.91 (1H, s, 14-OH),
7.40 (2H, t, J = 7.3 Hz, H-20), 7.31 (2H, t, J = 7.3 Hz, H-30), 7.25 (1H,
t, J = 7.3 Hz, H-40), 6.48 (1H, s, H-15), 5.84 (1H, dt, J = 15.3, 7.9 Hz,

H. Shinonaga et al. / Bioorg. Med. Chem. 17 (2009) 4622–4635
4631
H-8), 5.55 (1H, dd, J = 15.3, 8.5 Hz, H-7), 5.08 (1H, m, H-2), 3.84
(2H, m, H2-11), 3.61 (1H, t, J = 8.5 Hz, H-6), 3.05 (1H, dd, J = 8.5,
2.4 Hz, H-5), 2.99 (2H, m, H2-9), 2.94 (1H, ddd, J = 8.5, 4.3, 2.4 Hz,
H-4), 2.24 (1H, ddd, J = 14.0, 9.8, 4.3 Hz, H-3a), 1.31 (1H, ddd,
J = 14.0, 8.5, 2.4 Hz, H-3b), 1.22 (3H, d, J = 6.7 Hz, H3-1); 13C NMR
(DMSO-d6, 125 MHz) d 203.1 (s, C-10), 166.9 (s, C-18), 154.6 (s,
C-16), 154.0 (s, C-14), 133.5 (s, C-10), 132.0 (s, C-12), 131.2 (2d,
C-20), 130.6 (d, C-7), 128.9 (2d, C-30), 127.2 (d, C-8), 126.9 (d, C-
40), 115.0 (s, C-13), 112.0 (s, C-15), 102.2 (d, C-15), 70.3 (d, C-2),
59.3 (d, C-5), 54.6 (d, C-4), 52.2 (d, C-6), 45.1 (t, C-9), 43.5 (t, C-
11), 37.3 (t, C-3), 20.0 (q, C-1); ESI-MS m/z 473 [MꢃH]ꢃ as
C24H22ClO6S.
203.8 (s, C-10), 193.9 (s, C-10), 167.2 (s, C-18), 154.7 (s, C-16),
154.4 (s, C-14), 132.5 (s, C-12), 126.7 (d, C-8), 126.1 (d, C-7),
114.6 (s, C-13), 112.0 (s, C-17), 102.3 (d, C-15), 70.2 (d, C-2), 58.8
(d, C-5), 52.9 (d, C-4), 44.2 (t, C-11), 40.9 (d, C-6), 40.4 (t, C-9),
37.8 (t, C-3), 30.6 (q, C-20), 20.3 (q, C-1); ESI-MS m/z 439 [MꢃH]ꢃ
as C20H20ClO7S.
4.4.11. Dehydrogenation of compound 20
Wilkinson0s catalyst (chlorotris [triphenylphosphine rhodium
(I)]) (453 mg, 0.48 mmol, 0.20 equiv) was added to a solution of
20 (1090 mg, 2.29 mmol) in ethanol (40 mL). After hydrogenation
for 34 h at room temperature under atmospheric pressure, the cat-
alyst was removed by filtration and the filtrate was concentrated in
vacuo. The residue was purified using silica gel column chromatog-
raphy (eluent, n-hexane–ethyl acetate = 4:1), then further purified
using preparative HPLC (eluent, MeCN–H2O = 60:40) to produce
compound 24 (84.0 mg, 7.7%).
4.4.9.2. 7,8-(E)-9-Hydro-6
a
-thiophenylradicicol (21).
Color-
less oil; HPLC, tR 13.19 min (system 1); 1H NMR (DMSO-d6,
500 MHz) d 10.36 (1H, br s, 16-OH), 9.90 (1H, s, 14-OH), 7.43
(2H, d, J =7.3 Hz, H-20), 7.31 (2H, m, H-30), 7.29 (1H, m, H-40),
6.48 (1H, s, H-15), 5.62 (1H, dt, J = 10.4, 6.7 Hz, H-8), 5.58 (1H, t,
J = 10.4 Hz, H-7), 4.90 (1H, m, H-2), 3.92 (2H, m, H2-11), 3.78 (1H,
dd, J = 10.4, 8.8 Hz, H-6), 3.06 (1H, dd, J = 18.3, 10.4 Hz, H-9a),
2.97 (1H, dd, J = 8.8, 1.8 Hz, H-5), 2.85 (1H, dd, J = 18.3, 6.7 Hz, H-
9b), 2.76 (1H, m, H-4), 2.16 (ddd, J = 14.3, 11.3, 3.0 Hz, H-3a),
1.22 (1H, m, H-3b), 1.20 (3H, d, J = 6.1 Hz, H3-1); 13C NMR
(DMSO-d6, 125 MHz) d 202.5 (s, C-10), 166.8 (s, C-18), 154.7 (s,
C-16), 154.0 (s, C-14), 133.4 (2d, C-20), 132.6 (s, C-10), 131.3 (s, C-
12), 130.1 (d, C-7), 129.1 (2d, C-30), 128.0 (d, C-40), 124.0 (d, C-8),
115.5 (s, C-13), 111.9 (s, C-17), 102.4 (d, C-15), 70.6 (d, C-2), 58.6
(d, C-5), 56.2 (d, C-4), 49.0 (d, C-6), 43.2 (t, C-11), 42.3 (t, C-9),
37.6 (t, C-3), 20.3 (q, C-1); ESI-MS m/z 473 [MꢃH]ꢃ as C24H22ClO6S.
4.4.11.1. 7,8,9-Trihydro-6a-thiophenylradicicol (24).
Color-
less solid; mp 169–172 °C; HPLC, tR 13.62 min (system 1); 1H NMR
(DMSO-d6, 500 MHz) d 10.46 (1H, br s, 16-OH), 10.00 (1H, br s, 14-
OH), 7.45 (2H, d, J = 7.3 Hz, H-20), 7.32 (2H, t, J = 7.3 Hz, H-30), 7.26
(1H, t, J = 7.3 Hz, H-40), 6.50 (1H, s, H-15), 5.07 (1H, m, H-2), 4.05
(1H, d, J = 18.6 Hz, H-11a), 3.90 (1H, d, J = 18.6 Hz, H-11b), 2.86
(1H, br s, H-4), 2.85 (1H, br s, H-5), 2.76 (1H, dt, J = 8.8, 5.2 Hz,
H-6), 2.42 (2H, m, H2-9), 2.21 (1H, ddd, J = 14.0, 10.4, 3.6 Hz, H-
3a), 1.78 (1H, m, H-8a), 1.70 (1H, m, H-8b), 1.62 (1H, m, H-7a),
1.41 (1H, m, H-7b), 1.31 (1H, ddd, J = 14.0, 8.5, 2.4 Hz, H-3b), 1.24
(3H, d, J = 6.1 Hz, H3-1); 13C NMR (DMSO-d6, 125 MHz) d 206.7 (s,
C-10), 167.4 (s, C-18), 155.1 (s, C-16), 154.9 (s, C-14), 133.7 (s, C-
10), 132.3 (s, C-12), 132.2 (2d, C-20), 129.1 (2d, C-30), 127.3 (d, C-
40), 114.8 (s, C-13), 112.1 (s, C-17), 102.5 (d, C-15), 70.7 (d, C-2),
60.8 (d, C-5), 56.2 (d, C-4), 49.3 (d, C-6), 44.9 (t, C-11), 39.9 (t, C-
9), 37.2 (t, C-3), 30.3 (t, C-7), 21.3 (t, C-8), 20.1 (q, C-1); ESI-MS
m/z 475 [MꢃH]ꢃ as C24H24ClO6S.
4.4.10. Michael addition of radicicol (1) (ii)
Triethylamine (56 lL, 0.40 mmol) and thioacetic acid (163 mg,
2.14 mmol, 2.06 equiv) were added to a solution of 1 (378 mg,
1.04 mmol) in N,N-dimethylformamide (6 mL), and the solution
was stirred for 16 h at 0 °C. The reaction mixture was diluted with
dilute hydrochloric acid, and extracted with ethyl acetate. The or-
ganic layer was washed with brine, dried over sodium sulfate,
evaporated in vacuo and purified using preparative HPLC (eluent,
MeCN–H2O = 35:65) to produce compounds 22 (49.0 mg, 10.7%)
and 23 (15.7 mg, 3.4%).
4.4.12. 10-Oxime radicicol (25)
Hydroxylamine hydrochloride (180 mg, 2.59 mmol, 4.46 equiv)
was added to a solution of 1 (210 mg, 0.58 mmol) in pyridine
(4 mL). The mixture was stirred for 2.5 h at 40 °C and cooled to
room temperature. The mixture was diluted with water, extracted
with ethyl acetate, dried over sodium sulfate and concentrated to
produce a yellowish residue. The residue was purified using pre-
parative TLC (developing solvent, chloroform–methanol = 90:10)
to produce compound 25 (40.0 mg, 18.3%, Z-isomer: E-iso-
mer = 1:0.7). Compound 25: colorless oil; HPLC, tR 5.86 min (sys-
tem 1); 1H NMR (methanol-d4, 500 MHz) (major isomer) d 7.22
(1H, dd, J = 15.9, 11.0 Hz, H-8), 6.83 (1H, d, J = 15.9 Hz, H-9), 6.43
(1H, s, H-15), 6.16 (1H, m, H-7), 5.56 (1H, dd, J = 10.4, 3.0 Hz, H-
6), 5.29 (1H, m, H-2), 3.90 (1H, d, J = 15.9 Hz, H-11a), 3.79 (1H, d,
J = 15.9 Hz, H-11b), 3.34 (1H, m, H-5), 3.01 (1H, ddd, J = 9.2, 3.7,
1.8 Hz, H-4), 2.41 (1H, m, H-3a), 1.57 (1H, m, H-3b), 1.52 (3H, d,
J = 6.7 Hz, H3-1); 13C NMR (methanol-d4, 125 MHz) d 169.0 (s, C-
18), 157.1 (s, C-16), 156.7 (s, C-14), 154.8 (s, C-10), 135.5 (s, C-
12), 132.8 (2d, C-6 and C-8), 132.5 (d, C-7), 121.9 (d, C-9), 116.2
(s, C-13), 114.6 (s, C-17), 103.4 (d, C-15), 72.3 (d, C-2), 57.0 (d, C-
4), 56.6 (d, C-5), 38.1 (t, C-3), 36.2 (t, C-11), 18.6 (q, C-1); ESI-MS
m/z 378 [MꢃH]ꢃ; HRESI-MS m/z 402.0725 (calcd for
4.4.10.1. 7,8-(Z)-9-Hydro-6
a
-thioacetylradicicol (22).
Col-
orless oil; HPLC, tR 10.46 min (system 1); 1H NMR (DMSO-d6,
500 MHz) d 10.37 (1H, s, 16-OH), 9.91 (1H, s, 14-OH), 6.48 (1H, s,
H-15), 5.91 (1H, ddd, J = 15.3, 7.9, 7.3 Hz, H-8), 5.63 (1H, dd,
J = 15.3, 7.9 Hz, H-7), 5.10 (1H, m, H-2), 3.87 (2H, m, H2-11), 3.76
(1H, t, J = 7.9 Hz, H-6), 3.11 (1H, dd, J = 7.9, 1.8 Hz, H-5), 3.07 (1H,
dd, J = 15.3, 7.3 Hz, H-9a), 3.00 (2H, m, H-4 and H-9b), 2.35 (3H,
s, H3-20), 2.23 (1H, ddd, J = 14.6, 10.4, 3.7 Hz, H-3a), 1.30 (1H,
ddd, J = 14.6, 8.5, 2.3 Hz, H-3b), 1.22 (3H, d, J = 6.1 Hz, H3-1); 13C
NMR (DMSO-d6, 125 MHz) d 203.0 (s, C-10), 193.6 (s, C-10), 166.9
(s, C-18), 154.6 (s, C-16), 153.9 (s, C-14), 132.0 (s, C-12), 130.0 (d,
C-7), 127.2 (d, C-8), 115.0 (s, C-13), 112.1 (s, C-17), 102.2 (d, C-
15), 70.2 (d, C-2), 58.0 (d, C-5), 55.3 (d, C-4), 48.1 (d, C-6), 45.2
(t, C-9), 43.4 (t, C-11), 37.3 (t, C-3), 30.6 (q, C-20), 20.1 (q, C-1);
ESI-MS m/z 439 [MꢃH]ꢃ as C20H20ClO7S.
4.4.10.2. 7,8-(E)-9-Hydro-6
a
-thioacetylradicicol (23).
Col-
orless oil; HPLC, tR 10.79 min (system 1); 1H NMR (DMSO-d6,
500 MHz) d 10.39 (1H, s, 16-OH), 9.87 (1H, s, 14-OH), 6.49 (1H, s,
H-15), 5.67 (1H, dt, J = 11.0, 4.9 Hz, H-8), 5.17 (1H, dd, J = 11.0,
10.4 Hz, H-7), 5.13 (1H, m, H-2), 4.80 (1H, d, J = 10.4 Hz, H-6),
3.98 (2H, m, H2-11), 3.71 (1H, dd, J = 18.3, 11.0 Hz, H-9a), 3.20
(1H, dd, J = 18.3, 4.9 Hz, H-9b), 3.06 (1H, br s, H-5), 2.58 (1H, m,
H-4), 2.38 (3H, s, H3-20), 2.08 (1H, m, H-3a), 1.30 (1H, m, H-3b),
1.22 (3H, d, J = 6.1 Hz, H3-1); 13C NMR (DMSO-d6, 125 MHz) d
C18H18Cl1N1O6Na [M+Na]+, 402.0720,
D +0.5 mmu).
4.4.13. Preparation of 10-O-benzyloxime radicicol
O-Benzylhydroxylamine hydrochloride (1338 mg, 8.70 mmol,
2.36 equiv) was added to a solution of 1 (1344 mg, 3.69 mmol) in
pyridine (10 mL). The mixture was stirred for 5.5 h at 40 °C and
cooled to room temperature. The mixture was diluted with water,
extracted with ethyl acetate, dried over sodium sulfate and con-

4632
H. Shinonaga et al. / Bioorg. Med. Chem. 17 (2009) 4622–4635
centrated to produce a colorless oil. The residue was purified using
preparative HPLC (eluent, MeCN–H2O = 55:45) to produce com-
pounds 26 (538.6 mg, 31.2%) and 27 (271.2 mg, 15.1%).
4.4.14.2. 10-(E)-O-methyloxime radicicol (29).
Colorless oil;
HPLC, tR 10.98 min (system 1); 1H NMR (acetone-d6, 500 MHz) d
9.82 (1H, br s, 16-OH), 9.18 (1H, br s, 14-OH), 7.14 (1H, dd,
J = 16.0, 10.7 Hz, H-8), 6.55 (1H, s, H-15), 6.16 (1H, d, J = 16.0 Hz,
H-9), 6.15 (1H, t, J = 10.7 Hz, H-7), 5.47 (1H, dd, J = 10.7, 3.0 Hz,
H-6), 5.35 (1H, m, H-2), 4.58 (1H, d, J = 16.0 Hz, H-11a), 3.95 (3H,
s, OMe), 3.70 (1H, d, J = 16.0 Hz, H-11b), 3.22 (1H, br s, H-5), 2.95
(1H, ddd, J = 9.2, 3.0, 1.5 Hz, H-4), 2.43 (1H, dt, J = 14.5, 3.0 Hz, H-
3a), 1.62 (1H, ddd, J = 14.5, 9.2, 3.8 Hz, H-3b), 1.54 (3H, d,
J = 6.1 Hz, H3-1); 13C NMR (acetone-d6, 125 MHz) d 168.3 (s, C-
18), 158.4 (s, C-16), 157.2 (s, C-14), 155.0 (s, C-10), 137.0 (s, C-
12), 132.0 (d, C-7), 131.3 (d, C-8), 130.5 (d, C-6), 130.0 (d, C-9),
114.0 (s, C-13), 113.9 (s, C-17), 103.4 (d, C-15), 72.0 (d, C-2), 62.3
(q, OMe), 56.1 (d, C-4), 55.9 (d, C-5), 37.4 (t, C-3), 28.4 (t, C-11),
18.4 (q, C-1); ESI-MS m/z 392 [MꢃH]ꢃ as C19H19ClNO6.
4.4.13.1. 10-(Z)-O-benzyloxime radicicol (26).
Colorless oil;
HPLC, tR 13.52 min (system 1); 1H NMR (acetone-d6, 500 MHz) d
9.73 (1H, s, 16-OH), 9.12 (1H, s, 14-OH), 7.41 (2H, d, J = 6.9 Hz, H-
30), 7.34 (2H, dd, J = 7.6, 6.9 Hz, H-40), 7.29 (1H, d, J = 7.6 Hz, H-50),
7.25 (1H, dd, J = 16.0, 11.5 Hz, H-8), 6.78 (1H, d, J = 16.0 Hz, H-9),
6.54 (1H, s, H-15), 6.20 (1H, dd, J = 11.5, 10.7 Hz, H-7), 5.58 (1H, dd,
J = 10.7, 3.8 Hz, H-6), 5.35 (1H, m, H-2), 5.17 (2H, s, H2-10), 4.13
(1H, d, J = 16.0 Hz, H-11a), 4.00 (1H, brd, J = 16.0 Hz, H-11b), 3.29
(1H, br s, H-5), 3.02 (1H, dt, J = 8.4, 2.3 Hz, H-4), 2.44 (1H, ddd,
J = 14.5, 3.8, 2.3 Hz, H-3a), 1.65 (1H, ddd, J = 14.5, 8.4, 3.8 Hz, H-
3b), 1.55 (3H, d, J = 6.1 Hz, H3-1); 13C NMR (acetone-d6, 125 MHz) d
168.1 (s, C-18), 158.2 (s, C-16), 157.1 (s, C-14), 154.4 (s, C-10),
139.4 (s, C-20), 136.6 (s, C-12), 132.9 (2d, C-6 and C-8), 132.1 (d, C-
7), 129.0 (2d, C-40), 128.8 (2d, C-30), 128.4 (d, C-50), 121.8 (d, C-9),
115.2 (s, C-13), 113.9 (s, C-17), 103.5 (d, C-15), 76.6 (t, C-10), 71.9
(d, C-2), 56.0 (d, C-4), 55.6 (d, C-5), 37.4 (t, C-3), 35.7 (t, C-11), 18.7
(q, C-1); ESI-MS m/z 468 [MꢃH]ꢃ as C25H23ClNO6.
4.4.15. Luche reduction of radicicol (1)
A mixture of 1 (91.5 mg, 0.25 mmol) and cerium(III) chloride
heptahydrate (88 mg, 0.24 mmol, 0.96 equiv) in methanol (5 mL)
was stirred for 10 min at room temperature. Sodium borohydride
(60 mg, 1.59 mmol, 6.36 equiv) was added and the reaction mix-
ture was stirred for 5 min at 0 °C. After stirring, the reaction mix-
ture was diluted with water and extracted with ethyl acetate and
the organic layer was dried over sodium sulfate and concentrated
to produce a colorless oil. The crude oil was purified using prepara-
tive TLC (developing solvent, chloroform–methanol = 90:10) to
produce compound 30 (27.2 mg, 29.6%).
4.4.13.2. 10-(E)-O-benzyloxime radicicol (27).
Colorless oil;
HPLC, tR 13.71 min (system 1); 1H NMR (acetone-d6, 500 MHz) d
9.88 (1H, s, 16-OH), 9.21 (1H, s, 14-OH), 7.45 (2H, d, J = 6.9 Hz,
H-30), 7.36 (2H, dd, J = 7.6, 6.9 Hz, H-40), 7.30 (1H, t, J = 7.6 Hz, H-
50), 7.14 (1H, dd, J = 16.0, 10.7 Hz, H-8), 6.54 (1H, s, H-15), 6.17
(1H, d, J = 16.0 Hz, H-9), 6.15 (1H, t, J = 10.7 Hz, H-7), 5.47 (1H,
dd, J = 10.7, 3.8 Hz, H-6), 5.35 (1H, m, H-2), 5.22 (2H, s, H2-10),
4.65 (1H, d, J = 16.0 Hz, H-11a), 3.76 (1H, d, J = 16.0 Hz, H-11b),
3.22(1H, m, H-5), 2.94 (1H, m, H-4), 2.42 (1H, dt, J = 14.5, 3.8 Hz,
H-3a), 1.62 (1H, ddd, J = 14.5, 9.9, 4.6 Hz, H-3b), 1.53 (3H, d,
J = 6.1 Hz, H3-1); 13C NMR (acetone-d6, 125 MHz) d 168.4 (s, C-
18), 158.6 (s, C-16), 157.1 (s, C-14), 155.4 (s, C-10), 138.9 (s, C-
20), 137.0 (s, C-12), 132.0 (d, C-7), 131.5 (d, C-8), 130.6 (d, C-6),
130.0 (d, C-9), 129.1 (2d, C-30), 129.0 (2d, C-40), 128.6 (d, C-50),
114.3 (s, C-13), 113.8 (s, C-17), 103.4 (d, C-15), 77.1 (t, C-10), 72.0
(d, C-2), 56.1 (d, C-4), 55.9 (d, C-5), 37.4 (t, C-3), 28.8 (t, C-11),
18.4 (q, C-1); ESI-MS m/z 468 [MꢃH]ꢃ as C25H23ClNO6.
4.4.15.1. 10
a
-Hydroxyradicicol (30).
Colorless oil; HPLC, tR
6.43 min (system 1); 1H NMR (methanol-d4, 500 MHz) d 6.35
(1H, s, H-15), 5.90-5.78 (3H, m, H-7, H-8 and H-9), 5.31 (1H, dd,
J = 11.3, 4.9 Hz, H-6), 5.21 (1H, m, H-2), 4.52 (1H, ddd, J = 10.4,
5.5, 4.0 Hz, H-10), 3.73 (1H, dd, J = 12.8, 10.4 Hz, H-11a), 3.28
(1H, m, H-5), 3.18 (1H, dd, J = 12.8, 4.0 Hz, H-11b), 2.97 (1H, dt,
J = 7.6, 3.0 Hz, H-4), 2.50 (1H, ddd, J = 16.1, 7.6, 3.0 Hz, H-3a),
1.95 (1H, ddd, J = 16.1, 5.5, 3.0 Hz, H-3b), 1.41 (3H, d, J = 6.7 Hz,
H3-1); 13C NMR (methanol-d4, 125 MHz) d 171.2 (s, C-18), 162.8
(s, C-16), 159.0 (s, C-14), 139.1 (d, C-8), 139.1 (s, C-12), 130.7 (d,
C-7), 129.3 (d, C-6), 127.4 (d, C-9), 115.9 (s, C-13), 108.6 (s, C-
17), 103.5 (d, C-15), 72.7 (d, C-10), 70.3 (d, C-2), 57.2 (d, C-4),
56.0 (d, C-5), 40.0 (t, C-11), 36.0 (t, C-3), 20.1 (q, C-1); ESI-MS m/
z 731 [2 M-H]-; HRESI-MS m/z 389.0757 (calcd for C18H19Cl1O6Na
4.4.14. Preparation of 10-O-methyloxime radicicol
O-Methylhydroxylamine hydrochloride (1338 mg, 16.02 mmol,
8.52 equiv) was added to a solution of 1 (685 mg, 1.88 mmol) in
pyridine (6 mL). The mixture was stirred for 5.5 h at 40 °C and
cooled to room temperature. The mixture was diluted with water,
extracted with ethyl acetate, dried over sodium sulfate and con-
centrated to produce a colorless oil. The residue was purified using
preparative HPLC (eluent, MeCN–H2O = 40:60) to produce com-
pounds 28 (144.6 mg, 19.9%) and 29 (107.1 mg, 14.4%).
[M+Na]+, 389.0768,
D
ꢃ1.1 mmu).
4.4.16. Luche reduction of compound 2
A mixture of 2 (602 mg, 1.64 mmol) and cerium(III) chloride
heptahydrate (2.14 g, 5.74 mmol, 3.5 equiv) in methanol (13 mL)
was stirred for 30 min at room temperature. Sodium borohydride
(180 mg, 4.76 mmol, 2.9 equiv) was added and the reaction mix-
ture was stirred for 5 min at 0 °C. After stirring, the reaction mix-
ture was diluted with water, extracted with ethyl acetate and the
organic layer was dried over sodium sulfate and concentrated to
produce a colorless oil. The crude oil was purified using preparative
TLC (developing solvent, chloroform–methanol = 93:7) and further
purified using preparative HPLC (eluent; MeCN–H2O = 35:65) to
produce compounds 31 (144.9 mg, 24.0%) and 32 (13.8 mg, 2.3%).
4.4.14.1. 10-(Z)-O-methyloxime radicicol (28).
Colorless oil;
HPLC, tR 10.65 min (system 1); 1H NMR (acetone-d6, 500 MHz) d
9.65 (1H, br s, 16-OH), 9.10 (1H, br s, 14-OH), 7.19 (1H, dd,
J = 16.0, 11.5 Hz, H-8), 6.65 (1H, d, J = 16.0 Hz, H-9), 6.51 (1H, s,
H-15), 6.15 (1H, dd, J = 11.5, 10.7 Hz, H-7), 5.54 (1H, dd, J = 10.7,
3.8 Hz, H-6), 5.32 (1H, m, H-2), 4.06 (1H, d, J = 16.0 Hz, H-11a),
3.95 (1H, d, J = 16.0 Hz, H-11b), 3.84 (3H, s, OMe), 3.26 (1H, dd,
J = 3.8, 1.5 Hz, H-5), 2.98 (1H, ddd, J = 8.4, 3.0, 1.5 Hz, H-4), 2.43
(1H, ddd, J = 14.5, 3.8, 3.0 Hz, H-3a), 1.62 (1H, ddd, J = 14.5, 8.4,
3.8 Hz, H-3b), 1.53 (3H, d, J = 6.1 Hz, H3-1); 13C NMR (acetone-d6,
125 MHz) d 168.0 (s, C-18), 158.1 (s, C-16), 157.0 (s, C-14), 153.8
(s, C-10), 136.6 (s, C-12), 132.8 (d, C-8), 132.7 (d, C-6), 132.1 (d,
C-7), 121.6 (d, C-9), 115.1 (s, C-13), 114.0 (s, C-17), 103.5 (d, C-
15), 71.9 (d, C-2), 62.0 (q, OMe), 56.0 (d, C-4), 55.6 (d, C-5), 37.4
(t, C-3), 35.7 (t, C-11), 18.7 (q, C-1); ESI-MS m/z 392 [MꢃH]ꢃ as
C19H19ClNO6.
4.4.16.1. 6,7-Dihydro-10
a
ꢂ
-hydroxyradicicol (31).
ꢃ109.8 (c 1.00, acetone); UV (meth-
Colorless
crystal; mp 210–213 °C; ½a D20
anol) kmax (log e) 221 (4.45), 264 (3.93), and 314 (3.79) nm; IR (KBr)
mmax 3414, 3166, 2983, 2933, 1678, 1645, 1604, 1579, 1490, 1455,
1439, 1414, 1385, 1364, 1342, 1310, 1246, 1198, 1119, 1109, 1078,
1017, 978, 919, 876, 845, 810, 785, 705, 672, 634, 619 cmꢃ1; HPLC,
tR 6.46 min (system 1); 1H NMR (methanol-d4, 500 MHz) d 6.32
(1H, s, H-15), 5.38 (1H, ddd, J = 15.5, 12.8, 4.9 Hz, H-8), 5.28 (1H,
dd, J = 15.5, 5.2 Hz, H-9), 5.07 (1H, m, H-2), 4.33 (1H, m, H-10),

H. Shinonaga et al. / Bioorg. Med. Chem. 17 (2009) 4622–4635
4633
3.63 (1H, dd, J = 12.5, 9.4 Hz, H-11a), 3.23 (1H, dd, J = 12.5, 5.5 Hz,
H-11b), 2.85 (1H, dt, J = 6.7, 3.0 Hz, H-4), 2.50 (1H, dt, J = 9.1,
3.0 Hz, H-5), 2.27 (1H, ddd, J = 15.5, 6.7, 3.0 Hz, H-3a), 2.16 (1H,
m, H-7a), 2.01 (1H, m, H-6a), 1.95 (1H, m, H-7b), 1.85 (1H, ddd,
J = 15.5, 5.5, 2.4 Hz, H-3b), 1.35 (3H, d, J = 6.7 Hz, H3-1), 1.19 (1H,
m, H-6b); 13C NMR (methanol-d4, 125 MHz) d 171.8 (s, C-18),
163.0 (s, C-16), 159.2 (s, C-14), 140.0 (s, C-12), 133.2 (d, C-9),
129.1 (d, C-8), 115.9 (s, C-13), 109.3 (s, C-17), 103.4 (d, C-15),
71.8 (d, C-10), 70.9 (d, C-2), 59.6 (d, C-5), 58.3 (d, C-4), 40.2 (t, C-
11), 37.0 (t, C-3), 31.3 (t, C-6), 29.5 (t, C-7), 20.2 (q, C-1); ESI-MS
m/z 367 [MꢃH]ꢃ; HRESI-MS m/z 367.0952 (calcd for C18H20Cl1O6
1.94 (1H, m, H-6a), 1.57 (1H, m, H-7a), 1.50 (1H, m, H-8a), 1.40
(3H, d, J = 6.1 Hz, H3-1), 1.38 (2H, m, H-3b and H-9a), 1.35 (2H,
m, H-7b and H-8b), 1.29 (1H, m, H-6b); 13C NMR (methanol-d4,
125 MHz) d 169.8 (s, C-18), 156.8 (s, C-16), 156.6 (s, C-14), 138.5
(s, C-12), 115.9 (s, C-13), 114.6 (s, C-17), 102.9 (d, C-15), 72.3 (d,
C-10), 72.2 (d, C-2), 59.1 (d, C-5), 57.1 (d, C-4), 39.2 (t, C-3), 37.8
(t, C-11), 35.1 (t, C-9), 30.2 (t, C-6), 24.6 (t, C-7), 23.5 (t, C-8),
20.5 (q, C-1); ESI-MS m/z 369 [MꢃH]ꢃ as C18H22ClO6.
4.4.18. Luche reduction of compound 16
A mixture of 16 (153 mg, 0.42 mmol) and cerium(III) chloride
heptahydrate (213 mg, 0.57 mmol, 1.36 equiv) in methanol
(6 mL)/THF (2 mL) was stirred for 30 min at room temperature. So-
dium borohydride (79 mg, 2.1 mmol, 4.97 equiv) was added, and
the reaction mixture was stirred for 5 min at 0 °C. After stirring,
the reaction mixture was diluted with water, extracted with ethyl
acetate and the organic layer was dried over sodium sulfate and
concentrated to produce a colorless oil. The crude oil was purified
using silica gel column chromatography (eluent, n-hexane–ethyl
acetate = 3:2) and further purified using preparative HPLC (eluent,
MeCN–H2O = 35:65) to produce compounds 35 (11.3 mg, 7.3%) and
36 (19.8 mg, 12.9%).
[MꢃH]ꢃ, 367.0948,
D +0.4 mmu).
4.4.16.2. 6,7-Dihydro-10b-hydroxyradicicol (32).
Colorless
oil; HPLC, tR 6.11 min (system 1); 1H NMR (methanol-d4,
500 MHz) d 6.41 (1H, s, H-15), 5.64 (1H, dt, J = 15.5, 6.1 Hz, H-8),
5.37 (1H, dd, J = 15.5, 4.0 Hz, H-9), 4.93 (1H, m, H-2), 4.43 (1H,
m, H-10), 3.30 (1H, dd, J = 12.8, 9.8 Hz, H-11a), 3.25 (1H, dd,
J = 12.8, 5.5 Hz, H-11b), 2.92 (1H, dt, J = 8.5, 3.0 Hz, H-5), 2.86
(1H, dd, J = 5.8, 3.0 Hz, H-4), 2.20 (1H, m, H-7a), 2.17 (1H, m, H-
3a), 2.05 (1H, m, H-3b), 2.03 (1H, m, H-6a), 2.02 (1H, m, H-7b),
1.35 (3H, d, J = 6.1 Hz, H3-1), 1.27 (1H, m, H-6b); 13C NMR (metha-
nol-d4, 125 MHz) d 169.5 (s, C-18), 158.2 (s, C-16), 157.3 (s, C-14),
138.5 (s, C-12), 133.4 (d, C-9), 129.7 (d, C-8), 114.9 (s, C-13), 114.7
(s, C-17), 103.3 (d, C-15), 70.6 (d, C-10), 69.5 (d, C-2), 57.6 (d, C-5),
56.8 (d, C-4), 39.7 (t, C-11), 36.8 (t, C-3), 31.2 (t, C-6), 28.8 (t, C-7),
20.4 (q, C-1); ESI-MS m/z 367 [MꢃH]ꢃ; HRESI-MS m/z 391.0932
4.4.18.1. 6,9-Dihydro-10a-hydroxyradicicol (35).
Colorless
oil; HPLC, tR 7.06 min (system 1); 1H NMR (chloroform-d/metha-
nol-d4, 500 MHz) d 6.26 (1H, s, H-15), 5.52 (1H, dt, J = 15.3,
7.9 Hz, H-8), 5.35 (1H, dt, J = 15.3, 7.3 Hz, H-7), 5.22 (1H, m, H-2),
3.93 (1H, m, H-10), 3.23 (1H, dd, J = 14.0, 10.4 Hz, H-11a), 3.01
(1H, dt, J = 9.2, 2.4 Hz, H-4), 2.85 (1H, ddd, J = 5.5, 3.0, 2.4 Hz, H-
5), 2.79 (1H, dd, J = 14.0, 3.0 Hz, H-11b), 2.50 (1H, ddd, J = 14.0,
7.3, 3.0 Hz, H-6a), 2.19 (1H, m, H-3a), 2.16 (2H, m, H2-9), 2.01
(1H, m, H-6b), 1.37 (1H, ddd, J = 14.6, 9.2, 2.4 Hz, H-3b), 1.29 (3H,
d, J = 6.7 Hz, H3-1); 13C NMR (chloroform-d/methanol-d4,
125 MHz) d 169.4 (s, C-18), 157.7 (s, C-16), 155.8 (s, C-14), 138.1
(s, C-12), 129.8 (d, C-8), 127.6 (d, C-7), 113.7 (s, C-13), 111.8 (s,
C-17), 102.0 (d, C-15), 70.5 (d, C-2), 70.1 (d, C-10), 59.4 (d, C-5),
54.6 (d, C-4), 39.4 (t, C-9), 37.1 (t, C-3), 36.4 (t, C-11), 33.1 (t, C-
6), 19.3 (q, C-1); ESI-MS m/z 367 [MꢃH]ꢃ as C18H20ClO6.
(calcd for C18H21Cl1O6Na [M+Na]+, 391.0924,
D +0.8 mmu).
4.4.17. Luche reduction of compound 15
A mixture of 15 (26.5 mg, 0.072 mmol) and cerium(III) chloride
heptahydrate (100 mg, 0.27 mmol, 3.8 equiv) in methanol (5 mL)
was stirred for 30 min at room temperature. Sodium borohydride
(60 mg, 1.59 mmol, 22.0 equiv) was added and the reaction mix-
ture was stirred for 5 min at 0 °C. After stirring, the reaction mix-
ture was diluted with water, extracted with ethyl acetate and the
organic layer was dried over sodium sulfate and concentrated to
produce a colorless oil. The crude oil was purified using preparative
TLC (developing solvent, chloroform–methanol = 94:6) to produce
compounds 33 (5.7 mg, 21.2%) and 34 (8.8 mg, 32.7%).
4.4.18.2. 6,9-Dihydro-10b-hydroxyradicicol (36).
Colorless
oil; HPLC, tR 6.51 min (system 1); 1H NMR (chloroform-d/metha-
nol-d4, 500 MHz) d 6.21 (1H, s, H-15), 5.53 (1H, ddd, J = 15.3,
10.4, 4.9 Hz, H-8), 5.25 (1H, m, H-2), 5.17 (1H, ddd, J = 15.3, 9.8,
4.9 Hz, H-7), 4.05 (1H, m, H-10), 2.88 (1H, dd, J = 4.3, 2.4 Hz, H-
5), 2.83 (1H, dt, J = 14.0, 2.4 Hz, H-11a), 2.81 (1H, m, H-4), 2.66
(1H, dd, J = 14.0, 11.0 Hz, H-11b), 2.45 (1H, m, H-6a), 2.40 (2H, m,
H-6b and H-9a), 2.07 (1H, ddd, J = 14.0, 10.4, 3.0 Hz, H-3a), 1.90
(1H, m, H-9b), 1.27 (1H, ddd, J = 14.0, 6.7, 1.8 Hz, H-3b), 1.22 (3H,
d, J = 6.7 Hz, H3-1); 13C NMR (chloroform-d/methanol-d4,
125 MHz) d 169.8 (s, C-18), 155.1 (s, C-16), 154.9 (s, C-14), 136.1
(s, C-12), 129.8 (d, C-8), 127.5 (d, C-7), 114.4 (s, C-13), 112.7 (s,
C-17), 102.0 (d, C-15), 69.9 (d, C-2), 67.7 (d, C-10), 59.4 (d, C-5),
54.4 (d, C-4), 38.8 (t, C-9), 38.2 (t, C-3), 37.0 (t, C-11), 32.7 (t, C-
6), 20.1 (q, C-1); ESI-MS m/z 367 [MꢃH]ꢃ as C18H20ClO6.
4.4.17.1. 6,7,8,9-Tetrahydro-10
orless solid; mp 173–175 °C; IR (KBr)
a
-hydroxyradicicol (33).
Col-
m
max 3503, 3146, 2982, 2940,
1639, 1608, 1598, 1530, 1495, 1455, 1421, 1376, 1356, 1312, 1288,
1264, 1240, 1196, 1178, 1145, 1107, 1062, 1040, 1026, 927, 902,
855, 801, 774, 747, 735, 701, 672, 636, 608 cmꢃ1; HPLC, tR
6.87 min (system 1); 1H NMR (methanol-d4, 500 MHz) d 6.38
(1H, s, H-15), 5.20 (1H, m, H-2), 3.88 (1H, m, H-10), 3.21 (2H, m,
H2-11), 3.03 (1H, dt, J = 7.3, 2.4 Hz, H-4), 2.71 (1H, dt, J = 9.4,
2.4 Hz, H-5), 2.36 (1H, ddd, J = 15.5, 7.3, 2.4 Hz, H-3a), 2.09 (1H,
ddd, J = 14.3, 4.3, 2.4 Hz, H-6a), 1.74 (1H, ddd, J = 15.5, 7.3, 2.4 Hz,
H-3b), 1.57 (1H, m, H-9a), 1.52 (2H, m, H2-7), 1.48 (1H, m, H-9b),
1.45 (3H, d, J = 6.4 Hz, H3-1), 1.43 (2H, m, H2-8), 1.11 (1H, m, H-
6b); 13C NMR (methanol-d4, 125 MHz) d 171.1 (s, C-18), 160.1 (s,
C-16), 158.0 (s, C-14), 139.7 (s, C-12), 115.3 (s, C-13), 112.3 (s, C-
17), 103.0 (d, C-15), 72.8 (d, C-10), 71.4 (d, C-2), 60.5 (d, C-5),
58.2 (d, C-4), 39.9 (t, C-11), 38.0 (t, C-3), 36.8 (t, C-9), 32.2 (t, C-
6), 25.8 (t, C-8), 25.2 (t, C-7), 20.0 (q, C-1); ESI-MS m/z 369
[MꢃH]ꢃ as C18H22ClO6.
4.4.19. Hydrogenation of monocillin III (4)
Five-percent palladium–carbon (PH, wet-type) (30 mg) was
added to a solution of 4 (154 mg, 0.46 mmol) in ethyl acetate
(5 mL). After hydrogenation for 1.5 h at room temperature under
atmospheric pressure, the catalyst was removed by filtration and
the filtrate was concentrated in vacuo. The residue was purified
using preparative TLC (developing solvent, chloroform–metha-
nol = 95:5) to produce compound 37 (monocillin V16) (134.8 mg,
87.0%).
4.4.17.2. 6,7,8,9-Tetrahydro-10b-hydroxyradicicol (34).
Col-
orless oil; HPLC, tR 6.07 min (system 1); 1H NMR (methanol-d4,
500 MHz) d 6.39 (1H, s, H-15), 5.12 (1H, m, H-2), 3.82 (1H, m, H-
10), 3.15 (1H, dd, J = 13.4, 7.2 Hz, H-11a), 2.98 (1H, dd, J = 13.4,
5.5 Hz, H-11b), 2.88 (1H, dt, J = 7.3, 2.4 Hz, H-4), 2.81 (1H, dt,
J = 7.3, 2.4 Hz, H-5), 2.37 (1H, ddd, J = 14.0, 7.3, 3.7 Hz, H-3a),
Monocillin V (37 ) (synthetic): colorless oil; HPLC, tR 7.65 min
(system 1); 1H NMR (acetone-d6, 500 MHz)
d 6.28 (1H, d,

4634
H. Shinonaga et al. / Bioorg. Med. Chem. 17 (2009) 4622–4635
J = 2.4 Hz, H-15), 6.24 (1H, d, J = 2.4 Hz, H-13), 5.21 (1H, m, H-2),
4.32 (1H, d, J = 17.4 Hz, H-11a), 3.70 (1H, d, J = 17.4 Hz, H-11b),
2.78 (1H, ddd, J = 5.8, 4.0, 2.4 Hz, H-4), 2.59 (1H, dt, J = 9.1,
2.4 Hz, H-5), 2.49 (2H, m, H2-9), 2.14 (1H, ddd, J = 15.5, 5.8,
2.4 Hz, H-3a), 1.96 (1H, m, H-6a), 1.87 (1H, ddd, J = 15.5, 4.0,
2.4 Hz, H-3b), 1.62 (1H, m, H-8a), 1.52 (1H, m, H-8b), 1.49 (2H,
m, H2-7), 1.40 (3H, d, J = 6.4 Hz, H3-1), 1.08 (1H, m, H-6b); 13C
NMR (acetone-d6, 125 MHz) d 207.7 (s, C-10), 171.5 (s, C-18),
166.1 (s, C-16), 163.2 (s, C-14), 140.7 (s, C-12), 113.6 (d, C-13),
106.4 (s, C-17), 102.8 (d, C-15), 71.2 (d, C-2), 57.7 (d, C-5), 55.4
(d, C-4), 50.5 (t, C-11), 40.8 (t, C-9), 37.0 (t, C-3), 31.8 (t, C-6),
24.2 (t, C-7), 22.9 (t, C-8), 19.4 (q, C-1); ESI-MS m/z 333 [MꢃH]ꢃ;
HRESI-MS m/z 357.1324 (calcd for C18H22O6Na [M+Na]+,
4.4.22. Luche reduction of monocillin II (5)
A mixture of 5 (233 mg, 0.74 mmol) and cerium(III) chloride
heptahydrate (754 mg, 2.02 mmol, 2.73 equiv) in methanol
(5 mL)/THF (2 mL) was stirred for 10 min at room temperature. So-
dium borohydride (236 mg, 6.24 mmol, 8.43 equiv) was added, and
the reaction mixture was stirred for 5 min at 0 °C. After stirring, the
reaction mixture was diluted with water, extracted with ethyl ace-
tate and the organic layer was dried over sodium sulfate and con-
centrated to produce a colorless oil. The crude oil was purified
using preparative HPLC (eluent, MeCN–H2O = 45:55) to produce
compounds 40 (184.6 mg, 78.7%) and 41 (49.3 mg, 21.0%).
4.4.22.1. 10
a
-Hydroxymonocillin II (40).
Colorless oil;
357.1314,
D
+1.0 mmu).
HPLC, tR 10.37 min (system 1); 1H NMR (acetone-d6, 500 MHz) d
6.50 (1H, d, J = 2.4 Hz, H-13), 6.23 (1H, d, J = 2.4 Hz, H-15), 5.43
(1H, ddd, J = 15.3, 9.2, 7.3 Hz, H-4), 5.35 (1H, ddd, J = 15.3, 11.0,
5.5 Hz, H-8), 5.33 (1H, dd, J = 15.3, 4.3 Hz, H-9), 5.22 (1H, m, H-
2), 5.20 (1H, ddd, J = 15.3, 6.1, 3.7 Hz, H-5), 4.17 (1H, m, H-10),
3.72 (1H, dd, J = 13.4, 7.3 Hz, H-11a), 2.85 (1H, dd, J = 13.4,
6.1 Hz, H-11b), 2.67 (1H, ddd, J = 15.3, 7.3, 3.7 Hz, H-3a), 2.29
(1H, ddd, J = 15.3, 9.2, 6.7 Hz, H-3b), 2.12 (2H, m, H2-6), 2.09 (1H,
m, H-7a), 1.94 (1H, m, H-7b), 1.39 (3H, d, J = 6.7 Hz, H3-1); 13C
NMR (acetone-d6, 125 MHz) d 172.4 (s, C-18), 166.3 (s, C-16),
162.8 (s, C-14), 145.5 (s, C-12), 135.0 (d, C-9), 133.9 (d, C-5),
128.3 (d, C-8), 126.4 (d, C-4), 112.1 (d, C-13), 105.9 (s, C-17),
102.0 (d, C-15), 75.0 (d, C-10), 73.3 (d, C-2), 43.4 (t, C-11), 37.9
(t, C-3), 31.9 (t, C-6), 31.0 (t, C-7), 19.5 (q, C-1); ESI-MS m/z 317
[MꢃH]ꢃ; HRESI-MS m/z 341.1378 (calcd for C18H22O5Na [M+Na]+,
4.4.20. Hydrogenation of monocillin II (5)
Five-percent palladium–carbon (PH, wet-type) (42 mg) was
added to a solution of 5 (211 mg, 0.67 mmol) in ethyl acetate
(11 mL). After hydrogenation for 1.5 h at room temperature under
atmospheric pressure, the catalyst was removed by filtration and
the filtrate was concentrated in vacuo. The residue was purified using
preparative TLC (developing solvent, chloroform–methanol = 95:5)
to produce compound 38 (nordinone15) (159.7 mg, 74.7%).
Nordinone ( 38) (synthetic): colorless oil; HPLC, tR 13.00 min
(system 1); 1H NMR (acetone-d6, 500 MHz)
d 6.30 (1H, d,
J = 2.4 Hz, H-15), 6.19 (1H, d, J = 2.4 Hz, H-13), 5.15 (1H, m, H-2),
4.61 (1H, d, J = 17.7 Hz, H-11a), 3.79 (1H, d, J = 17.7 Hz, H-11b),
2.77 (1H, ddd, J = 18.3, 11.0, 3.7 Hz, H-9a), 2.54 (1H, ddd, J = 18.3,
5.5, 3.7 Hz, H-9b), 1.85 (1H, m, H-3a), 1.80 (1H, m, H-8a), 1.53
(1H, ddd, J = 9.2, 7.3, 2.4 Hz, H-3b), 1.43 (1H, m, H-7a), 1.35–1.32
(7H, m, H-4a, H2-5, H2-6, H-7b and H-8b), 1.33 (3H, d, J = 6.7 Hz,
H3-1), 1.31 (1H, m, H-4b); 13C NMR (acetone-d6, 125 MHz) d
207.0 (s, C-10), 172.2 (s, C-18), 166.4 (s, C-16), 163.0 (s, C-14),
140.9 (s, C-12), 113.8 (d, C-13), 106.3 (s, C-17), 102.6 (d, C-15),
74.4 (d, C-2), 50.5 (t, C-11), 39.4 (t, C-9), 35.2 (t, C-3), 27.4 (t, C-
5), 24.6 (t, C-7), 23.8 (t, C-6), 23.3 (t, C-4), 22.4 (t, C-8), 21.2 (q,
C-1); ESI-MS m/z 319 [MꢃH]ꢃ; HRESI-MS m/z 343.1520 (calcd for
341.1365,
D +1.3 mmu).
4.4.22.2. 10b-Hydroxymonocillin II (41).
Colorless oil; HPLC,
tR 9.55 min (system 1); 1H NMR (acetone-d6, 500 MHz) d 6.53 (1H,
d, J = 2.4 Hz, H-13), 6.24 (1H, d, J = 2.4 Hz, H-15), 5.43 (1H, ddd,
J = 15.9, 7.3, 6.7 Hz, H-8), 5.39 (1H, ddd, J = 15.9, 7.3, 6.1 Hz, H-4),
5.28 (1H, ddd, J = 15.9, 8.5, 6.1 Hz, H-5), 5.27 (1H, m, H-9), 5.23
(1H, m, H-2), 4.25 (1H, m, H-10), 3.25 (1H, dd, J = 15.3, 5.5 Hz, H-
11a), 3.23 (1H, dd, J = 15.3, 5.5 Hz, H-11b), 2.56 (1H, ddd, J = 14.0,
6.1, 3.7 Hz, H-3a), 2.29 (1H, ddd, J = 14.0, 7.3, 6.1 Hz, H-3b), 2.12
(2H, m, H2-6), 2.02 (2H, m, H2-7), 1.37 (3H, d, J = 6.1 Hz, H3-1);
13C NMR (acetone-d6, 125 MHz) d 171.2 (s, C-18), 164.6 (s, C-16),
162.2 (s, C-14), 144.4 (s, C-12), 134.5 (d, C-9), 133.3 (d, C-5),
130.6 (d, C-8), 127.0 (d, C-4), 112.6 (d, C-13), 107.4 (s, C-17),
102.0 (d, C-15), 73.4 (d, C-10), 72.8 (d, C-2), 42.4 (t, C-11), 38.3
(t, C-3), 32.2 (t, C-6), 31.1 (t, C-7), 19.5 (q, C-1); ESI-MS m/z 317
[MꢃH]ꢃ; HRESI-MS m/z 341.1362 (calcd for C18H22O5Na [M+Na]+,
C18H24O5Na [M+Na]+, 343.1521,
D
ꢃ0.1 mmu).
4.4.21. Luche reduction of monocillin III (4)
A mixture of 4 (191 mg, 0.57 mmol) and cerium(III) chloride
heptahydrate (641 mg, 1.72 mmol, 3.02 equiv) in methanol
(10 mL)/THF (4 mL) was stirred for 10 min at room temperature.
Sodium borohydride (216 mg, 5.71 mmol, 10.02 equiv) was added,
and the reaction mixture was stirred for 5 min at 0 °C. After stir-
ring, the reaction mixture was diluted with water, extracted with
ethyl acetate and the organic layer was dried over sodium sulfate
and concentrated to produce a colorless oil. The crude oil was puri-
fied using preparative HPLC (eluent, MeCN–H2O = 30:70) to pro-
duce compound 39 (137.3 mg, 71.4%).
341.1365,
D
ꢃ0.3 mmu).
4.4.23. Methylation of compound 31
Potassium carbonate (3 mg, 0.022 mmol) and methyl iodide
(0.5 mL, 8.0 mmol) were added to a solution of 31 (34 mg,
0.09 mmol) in N,N-dimethylformamide (2 mL), and the mixture
was stirred for 4.5 h at room temperature. The reaction mixture
was diluted with water and extracted with ethyl acetate. The or-
ganic layer was washed with brine, dried over sodium sulfate,
evaporated in vacuo and purified using preparative HPLC (eluent,
MeCN–H2O = 38:62) to produce compounds 42 (6.4 mg, 18.1%)
and 43 (14.5 mg, 39.6%).
4.4.21.1. 10
a-Hydroxymonocillin III (39)
.
Colorless oil;
HPLC, tR 6.25 min (system 1); 1H NMR (methanol-d4, 500 MHz) d
6.30 (1H, d, J = 2.4 Hz, H-13), 6.20 (1H, d, J = 2.4 Hz, H-15), 5.54
(1H, ddd, J = 15.9, 4.9, 1.2 Hz, H-8), 5.43 (1H, dd, J = 15.9, 4.9 Hz,
H-9), 5.16 (1H, m, H-2), 4.13 (1H, m H-10), 3.77 (1H, dd, J = 12.2,
7.9 Hz, H-11a), 2.91 (1H, ddd, J = 6.1, 3.7, 2.4 Hz, H-4), 2.74 (1H,
dd, J = 12.2, 6.7 Hz, H-11b), 2.57 (1H, dt, J = 9.2, 2.4 Hz, H-5), 2.25
(1H, m, H-7a), 2.13 (1H, m, H-6a), 2.11 (2H, m, H2-3), 2.05 (1H,
m, H-7b), 1.40 (3H, d, J = 6.1 Hz, H3-1), 1.23 (1H, ddt, J = 14.0, 9.2,
3.7 Hz, H-6b); 13C NMR (methanol-d4, 125 MHz) d 172.6 (s, C-18),
166.4 (s, C-16), 163.5 (s, C-14), 144.6 (s, C-12), 133.3 (d, C-9),
129.7 (d, C-8), 113.3 (d, C-13), 106.2 (s, C-17), 102.5 (d, C-15),
74.9 (d, C-10), 70.5 (d, C-2), 58.9 (d, C-5), 58.3 (d, C-4), 43.2 (t, C-
11), 36.9 (t, C-3), 31.8 (t, C-6), 29.4 (t, C-7), 19.9 (q, C-1); ESI-MS
m/z 333 [MꢃH]ꢃ as C18H21O6.
4.4.23.1. 6,7-Dihydro-10
(42).
a-hydroxyradicicol monomethyl ether
Colorless oil; HPLC, tR 9.90 min (system 1); 1H NMR
(acetone-d6, 500 MHz) d 6.58 (1H, s, H-15), 5.53 (1H, dddd,
J = 15.2, 6.7, 4.3, 1.8 Hz, H-8), 5.36 (1H, dd, J = 15.2, 4.3 Hz, H-9),
5.16 (1H, m, H-2), 4.43 (1H, m), 3.94 (3H, s, 14-OMe), 3.89 (1H,
dd, J = 12.5, 9.8 Hz, H-11a), 3.35 (1H, dd, J = 12.5, 6.1 Hz, H-11b),
2.83 (1H, m, H-4), 2.50 (1H, dt, J = 2.4, 1.8 Hz, H-5), 2.33 (1H,
ddd, J = 15.8, 7.0, 3.0 Hz, H-3a), 2.21 (1H, m, H-7a), 2.06 (1H, m,

H. Shinonaga et al. / Bioorg. Med. Chem. 17 (2009) 4622–4635
4635
TURETM program package. Crystallographic data (excluding structure
factors) have been deposited at the Cambridge Crystallographic
Data Centre (CCDC) and allocated the deposition number CCDC
724738. Copies of the data can be obtained free of charge by apply-
ing to the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44-
[0]1223-336033 or e-mail: deposit@ccdc.cam.ac.uk).
H-6a), 2.00 (1H, m, H-7b), 1.98 (1H, m, H-3b), 1.43 (3H, d,
J = 6.7 Hz, H3-1), 1.22 (1H, dt, J = 17.0, 9.1 Hz, H-6b); 13C NMR (ace-
tone-d6, 125 MHz) d 171.8 (s, C-18), 164.5 (s, C-16), 160.4 (s, C-14),
140.4 (s, C-12), 133.4 (d, C-9), 127.9 (d, C-8), 116.4 (s, C-13), 108.0
(s, C-17), 100.2 (d, C-15), 70.7 (d, C-2), 70.6 (d, C-10), 58.3 (d, C-5),
57.2 (d, C-4), 56.9 (q, 14-OMe), 40.3 (t, C-11), 36.5 (t, C-3), 31.1 (t,
C-6), 29.3 (t, C-7), 20.2 (q, C-1); ESI-MS m/z 405 [M+Na]+; HRESIMS
m/z 405.1098 (calcd for C19H23Cl1O6Na [M+Na]+, 405.1081,
D
Acknowledgments
+1.7 mmu).
We would like to thank Dr. K. Kitamura and Dr. T. Nakao (Taisho
Pharmaceutical Co., Ltd) for their excellent advice and cooperation
throughout this investigation and Dr. S. Arase (University of Toku-
shima Faculty of Medicine) for supplying dermal papilla cells. We
also thank their colleagues, Dr. K. Matsumoto, A. Higuchi, Dr. A.
Okada, and Dr. Y. Nozawa, for recording the NMR, MS, X-ray, and
LC–MS data, and Y. Kawamura for the large-scale fermentation of
the radicicol.
4.4.23.2.
(43).
6,7-Dihydro-10
a-hydroxyradicicol dimethyl ether
Colorless oil; HPLC, tR 8.90 min (system 1); 1H NMR
(acetone-d6, 500 MHz) d 6.75 (1H, s, H-15), 5.58 (1H, m, H-8),
5.52 (1H, dd, J = 15.9, 6.7 Hz, H-9), 5.18 (1H, m, H-2), 4.49 (1H,
m, mH-10), 3.94 (3H, s, 16-OMe), 3.84 (3H, s, 14-OMe), 3.28 (1H,
dd, J = 13.4, 6.7 Hz, H-11a), 2.83 (1H, ddd, J = 8.6, 3.7, 1.8 Hz, H-
4), 2.73 (1H, dd, J = 13.4, 5.5 Hz, H-11b), 2.69 (1H, dt, J = 9.2,
1.8 Hz, H-5), 2.31 (1H, ddd, J = 14.6, 6.7, 3.7 Hz, H-3a), 2.20–2.10
(3H, m, H-6a and H2-7), 1.46 (3H, d, J = 6.7 Hz, H3-1), 1.40 (1H,
ddd, J = 14.6, 8.6, 3.0 Hz, H-3b), 1.09 (1H, m, H-6b); 13C NMR (ace-
tone-d6, 125 MHz) d 167.2 (s, C-18), 157.4 (s, C-16), 156.9 (s, C-14),
136.1 (s, C-12), 135.7 (d, C-9), 130.2 (d, C-8), 119.9 (s, C-17), 115.1
(s, C-13), 96.5 (d, C-15), 73.0 (d, C-10), 71.5 (d, C-2), 58.0 (d, C-5),
56.8 (q, 14-OMe), 56.7 (q, 16-OMe), 56.1 (d, C-4), 40.8 (t, C-11),
39.0 (t, C-3), 33.3 (t, C-6), 28.7 (t, C-7), 19.8 (q, C-1); ESI-MS m/z
419 [M+Na]+; HRESI-MS m/z 419.1243 (calcd for C20H25Cl1O6Na
References and notes
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D +0.6 mmu).
4.4.24. Acetylation of compound 31
Acetic anhydride (3 mL) was added to a solution of 31 (55 mg,
0.15 mmol) in pyridine (1 mL), and the mixture was stirred for
17 h at room temperature. The reaction mixture was poured ice
water and extracted with ethyl acetate. The organic layer was
washed brine, dried over sodium sulfate, evaporated in vacuo
and purified using preparative HPLC (eluent, MeCN–H2O = 60:40)
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14. Shinonaga, H.; Kawamura, Y.; Ikeda, A.; Aoki, M.; Sakai, N.; Fujimoto, N.;
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4.4.24.1.
(44).
6,7-Dihydro-10
a
-hydroxyradicicol
triacetate
Colorless oil; HPLC, tR 12.59 min (system 1); 1H NMR
(acetone-d6, 500 MHz) d 7.16 (1H, s, H-15), 5.74 (1H, dt, J = 14.6,
6.7 Hz, H-8), 5.55 (1H, m, H-10), 5.53 (1H, dd, J = 14.6, 7.3 Hz, H-
9), 5.30 (1H, m, H-2), 3.51 (1H, dd, J = 14.0, 7.9 Hz, H-11a), 3.05
(1H, dd, J = 14.0, 4.9 Hz, H-11b), 2.88 (1H, ddd, J = 7.9, 3.7, 2.4 Hz,
H-4), 2.71 (1H, dt, J = 9.2, 2.4 Hz, H-5), 2.34 (3H, s, 16-OCOMe),
2.30 (1H, ddd, J = 15.3, 7.9, 3.7 Hz, H-3a), 2.23 (3H, s, 14-OCOMe),
2.23–2.13 (3H, m, H-6a and H2-7), 1.94 (3H, s, 10-OCOMe), 1.48
(1H, ddd, J = 15.3, 7.9, 3.0 Hz, H-3b), 1.47 (3H, d, J = 6.7 Hz, H3-1),
1.15 (1H, m, H-6b); 13C NMR (acetone-d6, 125 MHz) d 169.9 (s,
10-OCOMe), 168.7 (s, 14-OCOMe), 168.4 (s, 16-OCOMe), 165.5 (s,
C-18), 149.2 (s, C-16), 147.6 (s, C-14), 136.1 (s, C-12), 134.1 (d, C-
8), 130.0 (d, C-9), 128.9 (s, C-17), 126.0 (s, C-13), 118.6 (d, C-15),
74.7 (d, C-10), 72.8 (d, C-2), 57.7 (d, C-5), 56.2 (d, C-4), 38.9 (t, C-
3), 37.5 (t, C-11), 32.9 (t, C-6), 28.0 (t, C-7), 20.9 (q, 10-OCOMe),
20.6 (q, 14-OCOMe), 20.5 (q, 16-OCOMe), 19.8 (q, C-1); ESI-MS m/
z 517 [M+Na]+; HRESI-MS m/z 517.1254 (calcd for C24H27Cl1O9Na
26. Woodward, R. B.; Sondheimer, F.; Taub, D.; Heusler, K.; McLamore, W. M. J. Am.
Chem. Soc. 1952, 74, 4223.
27. Büchi, G.; Wüest, H. Helv. Chim. Acta 1971, 54, 1767.
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Shiotsu, Y.; Akinaga, S.; Murakata, C. J. Med. Chem. 2003, 46, 2534.
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[M+Na]+, 517.1241,
D +1.3 mmu).
¯
35. Tanaka, Y.; Kamei, K.; Otoguro, K.; Omura, S. J. Antibiot. 1999, 52, 880.
36. Soga, S.; Neckers, L. M.; Schulte, T. W.; Shiotsu, Y.; Akasaka, K.; Narumi, H.;
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4.5. X-ray crystal analysis of compound 31
A single crystal of 31 was grown by crystallization from meth-
anol at room temperature. X-ray intensity data was collected on
a R-AXIS RAPID II diffractometer (Rigaku Co.) equipped with graph-
ite monochromatized Cu K radiation. The structure was solved by
a
direct methods and refined anisotropically using the CRYSTAL STRUC-