Synthesis of dl-standishinal and its related compounds for the studies on structure-activity relationship of inhibitory activity against aromatase

Article abstract of DOI:10.1016/j.bmc.2007.01.031

dl-Standishinal (1), an aromatase inhibitor isolated from Thuja standishii, was synthesized in 15 steps from p-formylanisole via aldol reaction of 12-hydroxy-6,7-secoabieta-8,11,13-trien-6,7-dial (2). In the present study, we found that the aldol condensa

Full text of DOI:10.1016/j.bmc.2007.01.031

Bioorganic & Medicinal Chemistry 15 (2007) 2736–2748
Synthesis of DL-standishinal and its related compounds for the
studies on structure–activity relationship of inhibitory
activity against aromatase
Takahiro Katoh,a Taichi Akagi,a Chie Noguchi,a Tetsuya Kajimoto,a Manabu Node,a,*
b
b
b
´
Reiko Tanaka, Manabu Nishizawa (nee Iwamoto), Hironori Ohtsu,
Noriyuki Suzukic and Koichi Saitoc
aDepartment of Pharmaceutical Manufacturing Chemistry, 21st Century COE Program, Kyoto Pharmaceutical University,
1 Shichono-cho, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan
bLaboratory of Medicinal Chemistry, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki,
Osaka 569-1094, Japan
cEnviromental Health Science Laboratory, Sumitomo Chemical Co., Ltd., 1-98, Kasugadenaka 3-chome, Konohana-ku,
Osaka 554-8558, Japan
Received 29 November 2006; revised 17 January 2007; accepted 18 January 2007
Available online 20 January 2007
Abstract—DL-Standishinal (1), an aromatase inhibitor isolated from Thuja standishii, was synthesized in 15 steps from p-formylan-
isole via aldol reaction of 12-hydroxy-6,7-secoabieta-8,11,13-trien-6,7-dial (2). In the present study, we found that the aldol
condensation of 2 proceeded in excellent yield with the protonic catalyst such as d-camphorsulfonic acid in CH2Cl2. Moreover,
structure-activity relationship of 1 and its related compounds was studied and it was revealed that the isomers having cis-configuration
on the A/B-ring generally exhibited more potent inhibitory activities against aromatase than those with trans-configuration.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
have already been reported,7–18 one of the authors found
that a family of diterpenes, such as standishinal (1) iso-
lated from Thuja standishii (Gord.) Carr. (Cupressa-
ceae), is another example of the inhibitors.19–21
Standishinal (1) is not only a promising lead candidate
of anti-breast cancer agents but it also has an unusual
6(7 ! 11)abeo-abietane skeleton. Although several
synthetic approaches to natural products having the
abeo-abietane skeleton such as dichroanals and taiwan-
quinolines were reported in recent times,22–26 their meth-
ods were not applicable to the synthesis of 1 with the A/
B trans-configuration. On the other hand, Tanaka
succeeded in the chemical conversion from 12-hy-
droxy-6,7-secoabieta-8,11,13-trien-6,7-dial (2), another
component of the same plant source, to 1 by Lewis acid
(BF3ÆEt2O)-catalyzed aldol reaction (Fig. 1).19 On the
basis of these backgrounds, we would like to report
the first total synthesis of DL-1 by using the acid-cata-
lyzed aldol reaction of 2 as a key step. Moreover, struc-
ture-activity relationship of 1 and its derivatives
obtained during the synthetic study of 1 would also be
discussed.
Breast cancer is an aromatase-dependent cancer and has
been the major cause of death of women in recent
times.1 Since the aromatase catalyzes the conversion of
androgens into estrogens, inhibitors against the enzyme
are considered to be valuable as therapeutic agents in
the treatment of breast cancer. Both steroidal and
non-steroidal compounds are known as aromatase
inhibitors, that is, exemestane2 is an example of the
former, while fadrozole,3 anastrozole,4 and letrozole5 re-
leased as second and third generations of marketed med-
icines belong to the latter. Recent reports demonstrate
that the third generation of aromatase inhibitors is more
effective than tamoxifen in postmenopausal breast can-
cer patients6, so aromatase inhibitors are mainly used
today for the treatment of advanced breast cancer of
postmenopausal women. While many kinds of inhibitors
Keywords: Standishinal; Aromatase; Inhibitor; abeo-abietane.
*
Corresponding author. Tel.: +81 75 595 4639; fax: +81 75 595
4775; e-mail: node@mb.kyoto-phu.ac.jp
0968-0896/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.01.031

T. Katoh et al. / Bioorg. Med. Chem. 15 (2007) 2736–2748
2737
OR
OHC
OHC
H
OH
OH
CHO
H
H
OH
(-)-standishinal (1)
12-hydroxy-6,7-secoabieta-
ferruginol (3): R = H
8,11,13-triene-6,7-dial (2)
O-methyl ferruginol (4): R = Me
Figure 1. Structures of standishinal (1) and its related compounds.
2. Results and discussion
OMe
CHO
OMe
From a viewpoint of biosynthesis, the dialdehyde (2)
could be derived from ferruginol (3) by the oxidative
cleavage of the C–C bond between C-6 and C-7. Thus,
ferruginol (3) or ferruginyl methyl ether (4) was chosen
as an intermediate in the synthesis of 1. Synthesis of 3
has been reported by several groups to date.27–32
Ramana’s group developed a domino reaction of anisole
with (2,6,6-trimethylcyclohex-1-enyl)acetic acid, which
was easily prepared from citral, to attain a facile synthe-
sis of 3.28 Ogasawara and his colleagues also found a
novel single-step reaction for constructing hexahydroph-
enanthrene, which was facilely converted to (+)-3.29
King’s and Tada’s groups succeeded in short-step total
synthesis of 3 (10 steps), however, the overall yields were
lower than 10%.30,31 Tada and his colleagues, further-
more, applied the method to the synthesis of (+)-3 using
optical resolution by deriving to esters with (R)-binaph-
thol.31 On the other hand, Pan and his colleague also
reported the synthesis of optically active ferruginyl
methyl ether (4) in seven steps; however, it required a
chiral aldehyde which had to be prepared by taking four
steps from a commercially available substrate.32 Thus,
we started the synthesis of 1 with the preparation of fer-
ruginyl methyl ether (4) based on the King’s method30
since the King’s synthetic route can be easily modified
for the asymmetric synthesis of 3 by taking advantage
of our recent study on the synthesis of (ꢀ)-podocarpic
acid using lipase-catalyzed reaction.33 Two routes were
herein applicable to prepare 4, that is, one route ad-
vanced annulation of the B-ring followed by introduc-
tion of isopropyl group into the C-ring (Route A,
King’s method), and the other adopted B-ring’s cycliza-
tion of the substrate having isopropyl group on the C-
ring (Route B, modified King’s method) (Scheme 1).
OMe
Route B
Route A
OMe
OMe
OMe
H
H
4
Scheme 1. Synthetic strategy of O-methyl ferruginyl ether (4).
is, 3-isopropyl-4-methoxybenzaldehyde (12) prepared
from o-isopropylanisole (13) with dichloromethoxyme-
thane and tin(IV) chloride was derived to dibromoolefin
(14), which was successively treated with n-butyllithium
and 2,2,6-trimethylcyclohexanone to afford propargyl
alcohol (15). The triple bond of 15 was reduced by
hydrogenation to afford tertiary alcohol (16). Showing
a good contrast to the acid-catalyzed cyclization of 8,
treatment of 16 with Eaton’s reagent afforded a mixture
of 4 and its epimer at C-5 (17) (Scheme 2). The structure
of 17 was secured mainly by the d value of the signal as-
signed to the C4-a methyl group, which should appear
1
around at d 0.4 ppm in the H NMR spectra.32,35
The differences in the stereochemistry of the A/B ring on
cyclization 8 and 16 could be explained by the difference
in the rate of protonation on the methoxy group.
Namely, in the cyclization of 8, protonation on the
methoxy group occurred faster than successive proton-
ation on the double bond produced by dehydration of
the hydroxyl group at C-5, and the latter protonation
generated tertiary carbocation. Once the carbocation
was formed, intramolecular Friedel–Crafts alkylation
proceeded at the meta-position of the methoxy group
due to the decrease in the electron density of the aromat-
ic ring. On the other hand, in the cyclization of 16,
protonation hardly occurred on the methoxyl group
due to the bulkiness of the neighboring iso-propyl
group, therefore, the para-position of the methoxy
group reacted with the tertiary carbocation resulting
from dehydration of the hydroxyl group at C-5 and suc-
cessive protonation. Since the six-membered ring was
First, p-methoxybenzaldehyde (5) was derived to di-
bromoolefin (6) by Corey–Fuchs’s method34 and 6 was
converted to lithium acetylide which was treated with
2,2,6-trimethylcyclohexanone to afford propargyl alco-
hol (7) according to the King’s method (Route A).
The triple bond of 7 was reduced by catalytic hydroge-
nation on Pd–C, and obtained tertiary alcohol (8) was
cyclized with Eaton’s reagent (P2O5 in CH3SO3H) to
afford tricyclic compound (9). Friedel–Crafts acylation
of 9 with acetyl chloride in the presence of aluminum
chloride gave 10, which was reacted with methylmagne-
sium bromide to give 11. Hydrogenolysis of 11 afforded
4
32 in excellent yield. Next, Route B was attempted, that

2738
Route A
T. Katoh et al. / Bioorg. Med. Chem. 15 (2007) 2736–2748
OMe
OMe
OMe
X
R
H3C
OH
OMe
f
e
d
d
b, c
68
4
90
97
99
92
CH3
CH3
H
OH
H
OMe
5: R = CHO
6: R = C=CBr2 (99
10: X = O
7
8
9
a
g
)
11: X = CH3, OH (98
)
Route B
OMe
OMe
H3C
MeO
R
OH
OMe
OMe
h
b, c
80
e
d
+
4
CH3
CH3
95
99
58
OH
H
15
13
12: R = CHO
14: R = CH=CBr2 (99
17
16
a
)
37
Scheme 2. Synthesis of O-methyl ferruginyl ether (4). Reagents: (a) CBr4, Ph3P; (b) n-BuLi; (c) 2,2,6-trimethylcyclohexanone; (d) Pd–C/H2;
(e) Eaton’s reagent; (f) AcCl, AlCl3; (g) MeMgBr; (h) SnCl4, Cl2CHOMe.
formed as an intermediate in the reaction of 8 with Ea-
ton’s reagent, trans-fused decaline system was produced
prior to cis-fused decaline. Meanwhile, a five-membered
ring was formed fast in the reaction of 16, thus, sponta-
neous dienone–phenol rearrangement afforded both
cis- and trans-isomers (Scheme 3).
explained by the steric repulsion between the methyl
group at C-4 and newly generated hydroxyl group.
Dehydration of 20 with p-toluenesulfonic acid was at-
tained in refluxed benzene to give 22, of which methyl
ether was deprotected with odorless dodecanethiol and
n-butyllithium to afford 6,7-dehydroferruginol (23)
(Scheme 4).37,38
Next, ferruginyl methyl ether (4) was transformed to the
dialdehyde (2) according to Cheng’s procedure.36 Oxida-
tion of 4 with chromium trioxide in acetic acid afforded
O-methyl 7-oxo-ferruginyl ether (19), which was reduced
with sodium borohydride to give alcohol (20). Interest-
ingly, oxidation of a mixture of 4 and 17 with chromium
trioxide afforded 19 and its epimer (21), which remained
intact under the reduction condition with sodium
borohydride. The resistance of 21 to the reduction was
Since ozonolysis of the double bond at C6–C7 in 23
failed to afford 2, 6,7-dehydroferruginyl methyl ether
(22) was employed for the ozonolysis. In spite of satis-
factory yield in the ozonolysis to afford dialdehyde 24,
deprotection of the methyl ether with lithium n-dodecyl-
thiolate did not succeed in obtaining 2 at all. When ace-
tate (25) was exposed to the ozonolysis and successive
deprotection with potassium carbonate, both dialdehyde
Route A
+
O
Me
+
O
H
Me
H
OMe
OMe
+
H
H
OH
9
8
Route B
+
OMe
OMe
OMe
OMe
OMe
+
fast
OH
H
H
H
16
+
4
17
Scheme 3. Mechanism of acid-catalyzed cyclization of the B-ring.

T. Katoh et al. / Bioorg. Med. Chem. 15 (2007) 2736–2748
2739
OMe
OR
OMe
OMe
c
a
b
99
76
O
OH
H
H
H
H
H
4
19
20
22: R = Me (99
23: R = H (100
)
)
OMe
OMe
a
b
20 (42 ) + 21 (50
)
90
O
H
4 + 17
19 + 21
Scheme 4. Transformation of compounds 4 and 17 to compounds 20–23. Reagents: (a) CrO3; (b) AcOH; (c) p-TsOH; (d) Dod-SH, n-BuLi.
Table 1. Ozonolysis of abieta-6,8,11,13-tetraene derivatives and successive deprotection
OR
OR
Ozonolysis
Deprotection
2
O3 then PPh3
CH2Cl2
CHO
CHO
H
H
Entry
Starting material
R
Ozonolysis
Deprotection
Product
Yield (%)
Condition
—
Yield (%)
1
2
3
4
23
22
25
27
H
2
24
26
28
Trace
70
85
Me
Ac
Dod-SLi/HMPA, 100 °C, 1 h
K2CO3/MeOH, rt, 5 h
0
77
91
TBS
89
TBAF/THF, 0 °C, 30 min
intermediate (26) and 2 were obtained in good yield
(Table 1). Finally, the reaction of the ferruginol deriva-
tive (27) protected with tert-butyldimethylsilyl group
gave the highest overall yield to afford 2 via 28 (Table
1, entry 4). The obtained dialdehyde (2) was not convert-
ed to 1 with any Lewis acid such as AlCl3, and SnCl4
while faintly attainable with BF3ÆEt2O and well proceed-
ed by protic acid-catalyzed aldol reaction, especially
with D-camphorsulfonic acid in CH2Cl2, in excellent
yield (84%) (Table 2). All of the spectral data of 1
coincided with those in the literature.
intermediates with A/B cis-configuration, the cis-isomer
(21) was reduced with lithium aluminum hydride to af-
ford 29, which was derived to a dehydrated compound
30 by treatment with p-toluenesulfonic acid in benzene
under a refluxed condition. After cleavage of the methyl
ether in 30 with dodecanethiol in the presence of butylli-
thium, a newly generated hydroxyl group was reprotect-
ed with tert-butyldimethylsilyl group to afford 31. The
double bond of 31 was oxidatively cleaved by ozonolysis
to give dialdehyde (32), of which the protecting group
(TBS) was removed with TBAF to yield 33 (Scheme 5).
In order to compare the inhibitory activities of the
synthetic intermediates of 1 with the corresponding
Finally, evaluation of the some obtained compounds (1,
2, 4, 17, 19, 21, 30, and 33) for aromatase inhibitory
Table 2. Acid-catalyzed aldol reaction from 2 to DL-l
2
dl-1
Entry
Reagent/Solvent
Temperature
Time (h)
Yield (%)
1
2
3
4
5
6
7
BF3Et2O (1.2 equiv)/CH2Cl2
AlCl3 (3.2 equiv)/CH2Cl2
SnCl4 (1.2 equiv)/CH2Cl2
MsOH (1.2 equiv)/CH2Cl2
p-TsOH (1.2 equiv)/CH2Cl2
(+)-CSA (1.2 equiv)/CH2Cl2
(+)-CSA (1.2 equiv)/Et2O
ꢀ78 to 0°C
4
4
8
—
—
—
36
84
8
ꢀ10 °C
ꢀ78 °C to rt
23
30
1
ꢀ10 °C to rt
rt
rt
rt
8.5
66

2740
T. Katoh et al. / Bioorg. Med. Chem. 15 (2007) 2736–2748
OMe
OR
OHC
e
a
b
OR
21
93
84
84
CHO
OH
H
H
H
29
32: R = TBS
33: R = H (91
30: R = Me
31: R = TBS (92
f
c, d
)
)
Scheme 5. Chemical conversion from compound 21 to dial 33. Reagents: (a) LiAlH4; (b) p-TsOH, benzeneNaBH4; (c) n-BuLi, Dod-SH; (d) TBSCl,
imidazole; (e) O3 then PPh3; (f) TBAF.
Table 3. Aromatase inhibitory activities of synthesized compounds
Compound
Inhibitiona (%)
300 nM
1 lM
3 lM
10 lM
**
**
Ketoconazole
Letrozole
81.7 2.2**
89.3 1.9**
1.1 5.0
5.9 7.4
<0
84.2 3.5**
91.1 6.4**
10.3 10.0
<0
87.5 3.2**
83.8 3.9**
6.7 6.5
87.9 5.1
92.5 6.0
Compound 1
Compound 2
Compound 19
Compound 4
Compound 33
Compound 30
Compound 21
Compound 17
23.7 10.6*
9.2 11.5
3.3 8.0
6.7 5.1
18.1 10.9*
19.8 15.4
4.8 11.5
9.0 9.9
2.2 3.8
4.1 9.4
**
**
**
22.2 6.2
24.5 7.5
24.9 4.3**
18.6 10.0*
23.9 7.9**
18.4 7.7**
25.1 2.6
19.8 6.9**
19.5 8.6*
19.7 8.7*
16.2 2.8**
20.7 10.0*
15.2 8.0*
19.7 5.8**
28.7 17.5*
15.9 5.9**
a Aromatase inhibitory activity was evaluated by measuring a highly fluorescent product released from 3 lM methoxy-4-trifluoromethyl-coumarin
(MFC) at 37 °C for 30 min, in the presence of inhibitors. The blank was realized without adding the human recombinant aromatase enzyme. The
results are expressed as % of inhibition to a standard control which was incubated with the human recombinant aromatase enzyme and without
inhibitor. Values represent means SD of n = 4 experiments. Asterisks indicate significant differences from a standard control using Student t test.
* p < 0.05.
** p < 0.01.
activity was performed using a fluorometric substrate
and human recombinant aromatase expressed in insect
cells with baculovirus system. As shown in Table 3,
two typical inhibitors, ketoconazole and letrozole,
inhibited aromatase activity significantly (p < 0.01),
showing this assay for evaluating aromatase inhibitors
to be sensitive and reliable. The maximum inhibition
with ketoconazole (10 lM) or letrozole (10 lM) was
87.9% or 92.5%, respectively, relative to solvent control.
Under the same conditions, no significant (p < 0.05)
inhibition was found with the compounds 2 and 4
(300 nM–10 lM). In addition, only weak inhibitory
activities were observed with compounds 1 and 19, of
which the configurations on the A-ring are trans as same
as 2 and 4. Being a good contrast with these results,
compounds 17, 21, 30, and 33 having cis-configuration
on the A ring showed the significant inhibitory effects
(Fig. 2).
3. Conclusion
Aromatase Inhibition
100
90
In conclusion, we have succeeded in the first total syn-
thesis of DL-standishinal (1) and found that the structur-
ally related compounds of 1 having the cis-configuration
on the A-ring showed more potent inhibitory activities
than those with the trans-configuration against aroma-
tase, of which the excess activity could cause breast
cancer.
80
ketoconazole
70
letrozole
60
50
40
30
20
10
0
1
19
33
30
21
17
4. Experimental
4.1. General
-6.5
10
Concentration (M)
10-6.0
10-5.5
10-5.0
Infrared (IR) spectra were recorded on a Shimadzu
FTIR-8300 diffraction grating infrared spectrophotome-
ter and H NMR spectra were obtained on a Varian
1
Figure 2. Aromatase inhibition.

T. Katoh et al. / Bioorg. Med. Chem. 15 (2007) 2736–2748
2741
Unity INOVA 400 spectrometer with tetramethylsilane
as an internal standard. 13C NMR spectra were obtained
on a Varian Unity INOVA 400 spectrometer with
CDCl3 as an internal standard. Mass spectra (MS) were
determined on a JEOL JMS-SX 102 A QQ or a JEOL
JMS-GC-mate mass spectrometer. Specific rotations
were recorded on a Horiba SEPA-200 automatic digital
polarimeter. Wakogel C-200 (silica gel) (100–200 mesh,
Wako) was used for open column chromatography.
Flash column chromatography was performed by using
Silica Gel 60N (Kanto Chemical Co., Inc.) as a solid
support of immobile phase. Kieselgel 60 F-254 plates
(Merck) were used for thin layer chromatography
(TLC). Preparative TLC (PTLC) was conducted with
Kieselgel 60 F-254 plate (0.25 mm, Merck) or Silica gel
60 F-254 plate (0.5 mm, Merk). Unless purification with
silica gel gave compound being pure enough, the com-
pounds were further treated with a recycle HPLC (JAI
LC-908) on GPC column (JAIGEL 1H and 2H). In
the case it is possible, diastereomeric mixtures were also
separated by a recycle HPLC (JAI LC-908) on silica gel
column (Kusano Si-10) after the purification mentioned
above.
saturated aqueous solution of ammonium chloride,
extracted with ethyl acetate. The organic layer was suc-
cessively washed with 5% aqueous solution of sodium
thiosulfate and saturated aqueous solution of sodium
chloride, dried over MgSO4, and condensed in vacuo.
The residue was purified by silica gel column chromatog-
raphy (hexane/ethyl acetate = 15:1) to afford 7 (6.33 g,
68%) as colorless crystals; mp: 68–69 °C (petroleum
ether); 1H NMR (400 MHz, CDCl3) d: 1.05 (3H, s), 1.10
(3H, d, J = 6.4 Hz), 1.18 (3H, s), 1.32–1.48 (3H, m),
1.49–1.55 (1H, m), 1.57–1.63 (1H, m), 1.65–1.72 (1H,
m), 1.94 (1H, s), 1.89–1.97 (1H, m), 3.81 (3H, s), 6.82–
6.86 (2H, m), 7.36–7.39 (2H, m); 13C NMR (100 MHz,
CDCl3) d: 16.6, 19.9, 21.3, 27.0, 32.9, 37.1, 38.2, 39.2,
55.3, 79.0, 87.0, 88.3, 113.9 (2C), 115.3, 133.0 (2C),
159.5; IR (KBr): 3535, 2978, 2953, 2930, 2359, 1605,
1510, 1452, 1292, 1246 cmꢀ1; MS (70 eV) m/z: 272
(M+), 239, 201, 159, 121; HRMS calcd for C18H24O2
(M+): 272.1776, found: 272.1774; Anal. Calcd for
C18H24O2: C, 79.37; H, 8.88. Found: C, 79.26; H, 8.70.
4.4. 2,2,6-Trimethyl-1-[20-(4-methoxyphenyl)ethyl]cyclo-
hexanol (8)
4.2. b-Dibromo-4-methoxystyrene (6)
10% Pd–C (ca. 1.0 g) was added to a solution of 7
(6.08 g, 22.3 mmol) in ethanol (80 ml), and the mixture
was stirred for 6 h at room temperature under hydrogen
atmosphere. After the reaction, the mixture was filtered
through Hyflo Super-Cel which was washed with ethyl
acetate, and the filtrate was condensed in vacuo. The
residue was purified by silica gel column chromatogra-
phy (hexane/ethyl acetate = 8:1) to afford 8 (6.12 g,
Carbon tetrabromide (54.5 g, 0.16 mmol) was added to
a solution of triphenylphosphine (86.2 g, 0.33 mol) in
CH2Cl2 at 0 °C. After stirring the mixture for 15 min,
4-methoxybenzaldehyde (5) (10 mL, 82.2 mmol) was
added dropwise, and then the reaction mixture was stir-
red for another 30 min at room temperature. After the
reaction, the reaction mixture was poured into ice water
and extracted with chloroform. The organic layer was
successively washed with 5% aqueous solution of sodi-
um thiosulfate and saturated aqueous solution of sodi-
um chloride, dried over MgSO4, and condensed in
vacuo. The residue was purified by silica gel column
chromatography (hexane/ethyl acetate = 10:0, 30:1,
15:1, and then 10:1) to afford 6 (24.0 g, 99%) as pale yel-
low crystals; mp: 35–36 °C (n-hexane); 1H NMR
(400 MHz, CDCl3) d: 3.81 (3H, s), 6.87–6.91 (2H, m),
7.40 (1H, s), 7.48–7.52 (2H, m); 13C NMR (100 MHz,
CDCl3) d: 55.3, 87.2, 113.8 (2C), 127.8, 129.9 (2C),
136.3, 159.6; IR (CHCl3): 3029, 3009, 2955, 2839,
1607, 1510, 1248, 1034 cmꢀ1; MS (70 eV) m/z: 294
(M++4), 292 (M++2), 290 (M+), 279, 277, 275, 132;
HRMS calcd for C9H8Br2 (M+): 289.8941, found:
289.8939; Anal. Calcd for C9H8Br2: C, 37.02; H, 2.76.
Found: C, 36.90, H, 2.89.
1
99%) as a colorless oil; H NMR (400 MHz, CDCl3)
d: 0.99 (3H, s), 1.009 (3H, d, J = 7.0 Hz), 1.012 (3H,
s), 1.20 (1H, s), 1.14–1.23 (1H, m), 1.31–1.39 (2H, m),
1.41–1.50 (2H, m), 1.56–1.64 (1H, m), 1.73–1.85 (2H,
m), 1.92 (1H, ddq, J = 9.5, 7.0, 4.2 Hz), 2.58–2.71 (2H,
m), 3.79 (3H, s), 6.82–6.86 (2H, m), 7.12–7.16 (2H, m);
13C NMR (100 MHz, CDCl3) d: 16.1, 20.3, 23.1, 25.2,
30.4, 30.8, 33.2, 37.68 (2C), 37.74, 39.4, 55.3, 113.8
(2C), 129.2 (2C), 135.5, 157.7; IR (CHCl3): 3007, 2959,
2936, 1611, 1512, 1464, 1240, 1177 cmꢀ1; MS (70 eV)
m/z: 276 (M+), 258, 134, 121; HRMS calcd for
C18H28O2 (M+): 276.2089, found: 276.2090.
4.5. 12-Methoxypodocarpa-8,11,13-triene (9)
Eaton’s reagent (12 g) was added to 8 (5.98 g, 21.6 mmol)
and the mixture was stirred for 4 h at room temperature.
After the reaction, the reaction mixture was diluted with
distilled water, neutralized with aqueous solution of 1 M
NaOH, and extracted with ethyl acetate. The organic
layer was successively washed with saturated aqueous
solution of sodium bicarbonate and saturated aqueous
solution of sodium chloride, dried over MgSO4, and con-
densed in vacuo. The residue was purified by silica gel
column chromatography (hexane/ethyl acetate = 40:1)
4.3. 2,2,6-Trimethyl-1-(4-methoxyphenylethynyl)cyclo-
hexanol (7)
n-Butyllithium (2.71 M in hexane, 25.4 ml, 68.7 mmol)
was added dropwise into a solution of 6 (10.0 g,
34.4 mmol) in THF (140 ml) at ꢀ78 °C, and the mixture
was stirred for 1 h and stirred for another 1 h at room
temperature. After cooling the reaction mixture at
ꢀ78 °C again, a solution of 2,2,6-trimethylcyclohexa-
none (5.06 g, 36.1 mmol) in THF (30 ml) was added
to the reaction mixture, the mixture was stirred for
1 h. After the reaction, the mixture was poured into
1
to afford 9 (5.01 g, 90 %) as a colorless oil; H NMR
(400 MHz, CDCl3) d: 0.93, 0.95, 1.19 (each 3H, s),
1.21–1.50 (4H, m), 1.57–1.80 (3H, m), 1.83–1.89 (1H,
m), 2.23–2.26 (1H, m), 2.74–2.91 (2H, m), 3.77 (3H, s),
6.66 (1H, dd, J = 8.4, 2.7 Hz), 6.81 (1H, d, J = 2.7 Hz),
6.96 (1H, d, J = 8.4 Hz); 13C NMR (100 MHz, CDCl3)

2742
T. Katoh et al. / Bioorg. Med. Chem. 15 (2007) 2736–2748
d: 19.1, 19.3, 21.6, 24.7, 19.5, 33.3, 33.5, 38.0, 38.8, 41.6,
50.3, 55.2, 110.1, 110.7, 127.5, 129.7, 151.4, 157.6; IR
(CHCl3): 2930, 2361, 1609, 1574, 1502, 1491,
1252 cmꢀ1; MS (70 eV) m/z: 258 (M+), 243, 227, 187,
173, 161, 147; HRMS calcd for C18H26O (M+):
258.1948, found: 258.1985.
(50 ml), and the mixture was stirred at 50 °C for 8 h un-
der hydrogen atmosphere. After the reaction, the mix-
ture was filtered through Hyflo Super-Cel which was
washed with ethyl acetate, and the filtrate was con-
densed in vacuo. An aqueous solution of the residue
was neutralized with an aqueous solution of sodium
bicarbonate and extracted with ethyl acetate. The organ-
ic layer was washed with saturated aqueous solution of
sodium chloride, dried over MgSO4, and condensed in
vacuo. The residue was purified by silica gel column
chromatography (hexane/ethyl acetate = 100:1) to afford
4.6. 13-Acetyl-12-methoxypodocarpa-8,11,13-triene (10)
Aluminum chloride (5.59 g, 41.9 mmol) and acetyl chlo-
ride (3.18 ml, 44.7 mmol) were added to a solution of 9
(4.81 g, 18.6 mmol) in CH2Cl2 at ꢀ10 °C, and the reac-
tion mixture was stirred for 2 h at ꢀ5 °C. After the reac-
tion, the reaction mixture was poured into ice water and
extracted with ethyl acetate. The organic layer was suc-
cessively washed with saturated aqueous solution of
sodium bicarbonate and saturated aqueous solution of
sodium chloride, dried over MgSO4, and condensed in
vacuo. The residue was purified by silica gel column
chromatography (hexane/ethyl acetate = 10:1) to afford
1
4 (4.73 g, 92%) as a colorless oil; H NMR (400 MHz,
CDCl3) d: 0.92, 0.94, 1.17 (each 3H, s), 1.19 (6H, d,
J = 7.0 Hz), 1.15–1.25 (1H, m), 1.32–1.51 (3H, m),
1.58–1.78 (3H, m), 1.83–1.89 (1H, m), 2.23–2.27 (1H,
m), 2.73–2.89 (2H, m), 3.21 (1H, septet, J = 7.0 Hz),
3.79 (3H, s), 6.72, 6.48 (each 1H, s); 13C NMR
(100 MHz, CDCl3) d: 19.2, 19.3, 21.6, 22.7, 22.9, 24.8,
26.4, 29.8, 33.3, 33.4, 37.8, 38.9, 41.7, 50.5, 55.6, 106.5,
126.4, 126.9, 134.1, 148.0, 155.0; IR (CHCl3): 3001,
2963, 2930, 2868, 1711, 1611, 1499, 1463, 1364,
1250 cmꢀ1; MS (70 eV) m/z: 300 (M+), 285, 257, 243,
229, 215, 203, 189, 163; HRMS calcd for C21H32O
(M+): 300.2453, found: 300.2457.
10 (5.73 g, 97%) as
a
colorless oil; 1H NMR
(400 MHz, CDCl3) d: 0.93, 0.95, 1.20 (each 3H, s),
1.15–1.32 (2H, m), 1.39–1.52 (2H, m), 1.61–1.80 (3H,
m), 1.85–1.91 (1H, m), 2.24–2.28 (1H, m), 2.58 (3H, s),
2.67–2.94 (2H, m), 3.87 (3H, s), 6.83 (1H, s), 7.45 (1H,
s); 13C NMR (100 MHz, CDCl3) d: 18.9, 19.2, 21.7,
24.5, 29.2, 31.8, 33.2, 33.5, 38.5, 38.8, 41.5, 50.0, 55.4,
107.5, 125.5, 127.5, 130.9, 156.4, 157.2, 199.5; IR
(CHCl3): 3007, 2930, 2868, 1668, 1605, 1495, 1464,
1404, 1267, 1240 cmꢀ1; MS (70 eV) m/z: 300 (M+),
257, 243, 149; HRMS calcd for C20H28O2 (M+):
300.2089, found: 300.2094.
4.9. 3-Isopropyl-4-methoxybenzaldehyde (12)
Tin (IV) chloride (27.9 g, 0.11 mol) and dichloro-
methoxymethane (16.4 g, 0.14 mol) were added to a
solution of 2-isopropylanisole (13) (10.7 g, 71.3 mmol)
in CH2Cl2 (160 ml) at 0 °C, and the mixture was stirred
for 1 h. After the reaction, the reaction mixture was
poured into ice water and extracted with chloroform.
The organic layer was washed with saturated aqueous
solution of sodium chloride, dried over MgSO4, and
condensed in vacuo. The residue was purified by silica
gel column chromatography (hexane/ethyl acetate = 6:1)
4.7. 12-Methoxyabieta-8,11,13-trien-15-ol (11)
Methyl magnesium bromide (3.0 M in diethyl ether,
12.2 ml, 36.5 mmol) was added to a solution of 10
(5.48 g, 18.2 mmol) in THF (200 ml) at 0 °C and the reac-
tion mixture was stirred for 4 h. After the reaction, the
reaction mixture was poured into saturated aqueous
solution of ammonium chloride and extracted with ethyl
acetate. The organic layer was washed with saturated
aqueous solution of sodium chloride, dried over MgSO4,
and condensed in vacuo. The residue was purified by sil-
ica gel column chromatography (hexane/ethyl ace-
tate = 7:1) to afford 11 (5.66 g, 98%) as a colorless oil;
1H NMR (400 MHz, CDCl3) d: 0.93, 0.95, 1.19 (each
3H, s), 1.18–1.25 (1H, m), 1.30–1.34 (1H, m), 1.38–1.54
(2H, m), 1.57 (3H, s), 1.58 (3H, s), 1.61–1.65 (1H, m),
1.66–1.82 (2H, m), 1.84–1.90 (1H, m), 2.22–2.25 (1H,
m), 2.72–2.89 (2H, m), 3.87 (3H, s), 4.22 (1H, s), 6.78
(1H, s), 6.92 (1H, s); 13C NMR (100 MHz, CDCl3) d:
19.1, 19.3, 21.6, 24.7, 29.7, 29.8, 29.9, 33.3, 33.4, 37.8,
38.8, 41.6, 50.3, 55.3, 72.2, 107.3, 126.2, 127.2, 132.9,
149.7, 155.0; IR (CHCl3): 3533, 3007, 2968, 2930, 2359,
1612, 1501, 1464, 1385, 1369, 1244 cmꢀ1; MS (70 eV)
m/z: 316 (M+), 302, 301, 285, 243, 201, 161; HRMS calcd
for C21H32O2 (M+): 316.2402, found: 316.2403.
1
to afford 12 (12.1 g, 95%) as a pale yellow oil; H NMR
(400 MHz, CDCl3) d: 1.24 (6H, d,J = 7.0 Hz), 3.33 (1H,
septet, J = 7.0 Hz), 3.92 (3H, s), 6.95 (1H, d, J = 8.4 Hz),
7.71 (1H, dd, J = 8.4, 2.0 Hz), 7.77 (1H, d, J = 2.0 Hz),
10.46 (1H, s); 13C NMR (100 MHz, CDCl3) d: 22.4,
26.7, 55.6, 110.0, 127.2, 129.7, 130.5, 137.9, 162.0,
191.3; IR (CHCl3): 3030, 3018, 3007, 2966, 2841, 2741,
1686, 1599, 1580, 1502, 1493, 1462, 1261, 1171 cmꢀ1
;
MS (70 eV) m/z: 178 (M+), 163, 149; HRMS calcd for
C11H14O2 (M+): 178.0094, found: 178.0092.
4.10. b-Dibromo-3-isopropyl-4-methoxystyrene (14)
A solution of 12 (12.0 g, 67.4 mmol) in CH2Cl2 (50 ml)
was added to a mixture prepared by adding carbon tet-
rabromide (44.7 g, 0.13 mol) into a solution of triphe-
nylphosphine (70.7 g, 0.27 mol) in CH2Cl2 (200 ml) at
0 °C. After stirring the reaction mixture for 2.5 h at
room temperature, the reaction mixture was poured into
ice water and extracted with chloroform. The organic
layer was successively washed with 5% aqueous solution
of sodium thiosulfate and saturated aqueous solution of
sodium chloride, dried over MgSO4, and condensed in
vacuo. The residue was purified by silica gel column
chromatography (hexane) to afford 14 (23.1 g, 99%) as
a colorless oil; 1H NMR (400 MHz, CDCl3) d: 1.20
4.8. O-Methyl ferruginyl ether (4)
10% Pd–C (ca. 578 mg) was added to a solution of 11
(5.39 g, 17.0 mmol) in methanol (50 ml) and acetic acid

T. Katoh et al. / Bioorg. Med. Chem. 15 (2007) 2736–2748
2743
(6H, d, J = 6.9 Hz), 3.31 (1H, septet, J = 6.9 Hz), 3.84
(3H, s), 6.82–6.84 (1H, m), 7.39–7.42 (3H, m); 13C
NMR (100 MHz, CDCl3) d: 22.6 (2C), 26.2, 55.4, 86.7,
110.0, 126.6, 127.0, 127.5, 136.8, 136.9, 156.9; IR
(CHCl3): 3032, 3007, 2964, 2870, 1603, 1497, 1464,
1250, 1032 cmꢀ1; MS (70 eV) m/z: 334 (M++2), 319,
304, 238, 174, 159, 144; HRMS calcd for C12H14Br2O
(M+): 331.9411, found: 331.9415.
20.3, 22.7 (2C), 23.2, 25.2, 26.8, 30.6, 30.8, 33.2, 37.7
(2C), 39.4, 55.5, 76.8, 110.4, 126.0, 126.2, 135.2, 136.9,
155.0; IR (CHCl3): 3612, 3007, 2963, 2870, 1497, 1464,
1244, 1171, 1034 cmꢀ1; MS (70 eV) m/z: 318 (M+),
300, 176, 163; HRMS calcd for C21H34O2 (M+):
318.2559, found: 318.2560.
4.13. 12-Methoxy-5b-abieta-8,11,13-triene (17) and
ferruginyl methyl ether (4)
4.11. 2,2,6-Trimethyl-1-(3-isopropyl-4-methoxyphenyl-
ethynyl)cyclohexanol (15)
Eaton’s reagent (3 g) was added to 16 (1.58 g,
4.96 mmol) and the mixture was stirred for 15 min at
room temperature. After the reaction, the reaction mix-
ture was poured into ice water. The aqueous solution
was neutralized with an aqueous solution of 1 M NaOH
and extracted with ethyl acetate. The organic layer was
successively washed with a saturated aqueous solution
of sodium bicarbonate and a saturated aqueous solution
of sodium chloride, dried over MgSO4, and condensed
in vacuo. The residue was purified by silica gel column
chromatography (hexane) to afford 4 (545 mg, 37%)
and 17 (870 mg, 58%) as a colorless oil; 1H NMR
(400 MHz, CDCl3) d: 0.39, 0.93, 1.171 (each 3H, s),
1.177 (3H, d, J = 6.9 Hz), 1.184 (3H, d, J= 6.9 Hz),
1.24–1.53 (6H, m), 1.90–1.97 (1H, m), 2.11–2.21 (1H,
m), 2.33–2.37 (1H, m), 2.72–2.85 (2H, m), 3.22 (1H, sep-
tet, J = 6.9 Hz), 3.80 (3H, s), 6.74 (1H, s), 6.79 (1H, s);
13C NMR (100 MHz, CDCl3) d: 18.4, 19.4, 22.86,
22.89, 23.0, 25.7, 26.4, 32.7, 34.2, 34.5, 37.3, 38.2, 43.0,
50.3, 55.6, 106.4, 126.2, 128.9, 133.8, 141.6, 154.8; IR
(CHCl3): 2961, 2937, 2864, 1612, 1502, 1464,
1244 cmꢀ1; MS (70 eV) m/z: 300 (M+), 285, 257, 243,
215, 203, 189, 163; HRMS calcd for C21H32O (M+):
300.2453, found: 300.2450.
n-Butyllithium (2.64 M in hexane, 7.12 ml, 18.8 mmol)
was added to a solution of 14 (3.14 g, 9.40 mmol) in
THF (40 ml) at ꢀ78 °C, and the mixture was stirred
for 1 h with keeping the temperature and for another
1 h at room temperature. After cooling the mixture at
ꢀ78 °C again, a solution of 2,2,6-trimethylcyclohexa-
none (1.45 g, 10.3 mmol) in THF (10 ml) was added to
the reaction mixture. Stirring at ꢀ78 °C for 2 h, the reac-
tion mixture was poured into saturated aqueous solu-
tion of ammonium chloride and extracted with ethyl
acetate. The organic layer was successively washed with
5% aqueous solution of sodium thiosulfate and saturat-
ed aqueous solution of sodium chloride, dried over
MgSO4, and condensed in vacuo. The residue was puri-
fied by silica gel column chromatography (hexane/ethyl
acetate = 20:1) to afford diastereomixture of 15 (2.37 g,
1
80%) as a colorless oil; H NMR (400 MHz, CDCl3)
d: 1.05 (3H, s), 1.11 (3H, d, J = 6.4 Hz), 1.19 (3H, s),
1.20 (6H, d, J = 6.9 Hz), 1.18–1.21 (1H, m), 1.33–1.38
(1H, m), 1.40–1.55 (2H, m), 1.57–1.64 (1H, m), 1.67–
1.74 (1H, m), 1.94 (1H, s), 1.89–1.99 (1H, m), 3.28
(1H, septet, J = 6.9 Hz), 3.83 (3H, s), 6.76–6.78 (1H,
m), 7.25–7.28 (2H, m); 13C NMR (100 MHz, CDCl3)
d: 16.7, 19.9, 21.3, 22.5 (2C), 26.7, 27.0, 32.9, 37.1,
38.2, 39.2, 55.4, 79.1, 87.6, 87.7, 110.1, 115.0, 129.4,
130.3, 137.1, 156.9; IR (CHCl3): 3609, 3007, 2964,
2220, 1603, 1495, 1464, 1250, 1175, 1032 cmꢀ1; MS
(70 eV) m/z: 314 (M+), 299, 296, 281, 257, 243, 229,
215, 163; HRMS cal;cd for C21H30O2 (M+): 314.2246,
found: 314.2247.
4.14. O-Methyl sugiyl ether (19)
A solution of chromium trioxide (168 mg, 1.68 mmol) in
acetic acid (5 ml) was added to a solution of 4 (420 mg,
1.40 mmol) in acetic acid (15 ml), and the mixture was
stirred for 2 h at room temperature. After the reaction,
the reaction mixture was poured into ice water. The
aqueous solution was neutralized with an aqueous solu-
tion of 1 M NaOH and extracted with ethyl acetate. The
organic layer was successively washed with saturated
aqueous solution of sodium bicarbonate and saturated
aqueous solution of sodium chloride, dried over MgSO4,
and condensed in vacuo. The residue was purified by sil-
ica gel column chromatography (hexane/benzene = 1:2)
to afford 19 (332 mg, 76%) as colorless crystals; mp:
4.12. 2,2,6-Trimethyl-1-[20-(3-isopropyl-4-methoxyphe-
nyl)ethyl]cyclohexanol (16)
10% Pd–C (ca. 20 mg) was added to a solution of 15
(84.2 mg, 0.27 mmol) in methanol (3 ml) and the mix-
ture was stirred for 2 h at room temperature under
hydrogen atmosphere. After the reaction, the reaction
mixture was filtered through Hyflo Super-Cel that was
successively washed with ethyl acetate. The filtrate was
condensed in vacuo and the residue was purified by sil-
ica gel column chromatography (hexane/ethyl ace-
tate = 10:1) to afford 16 (85 mg, 99%) as a colorless
1
132–133 °C (MeOH); H NMR (400 MHz, CDCl3) d:
0.93, 1.00 (each 3H, s), 1.19 (3H, d, J = 6.9 Hz), 1.22
(3H, d, J = 6.9 Hz), 1.25 (3H, d, J = 0.5 Hz), 1.23–1.31
(1H, m), 1.51–1.61 (2H, m), 1.66–1.72 (1H, m), 1.74–
1.83 (1H, m), 1.88 (1H, dd, J = 13.5, 4.2 Hz), 2.28–
2.32 (1H, m), 2.60 (1H, dd, A part of AB, J = 18.1,
13.5 Hz), 2.68 (1H, dd, B part of AB, J = 18.1,
4.2 Hz), 3.24 (1H, septet, J = 6.9 Hz), 3.89 (3H, s),
6.75 (1H, s), 7.89 (1H, s); 13C NMR (100 MHz, CDCl3)
d: 18.9, 21.4, 22.4, 22.5, 23.2, 26.5, 32.6, 33.3, 36.0, 38.0,
38.3, 41.3, 49.6, 55.4, 104.4, 124.1, 125.6, 135.2, 156.4,
161.6, 198.5; IR (CHCl3): 3483, 3005, 2964, 2932,
1663, 1597, 1562, 1495, 1277, 1261 cmꢀ1; MS (70 eV)
1
oil; H NMR (400 MHz, CDCl3) d: 0.99, 1.017 (each
3H, s), 1.018 (3H, d, J = 7.0 Hz), 1.22 (6H, d,
J = 6.9 Hz), 1.23 (1H, s), 1.19–1.23 (1H, m), 1.34–1.39
(2H, m), 1.44–1.50 (2H, m), 1.57–1.63 (1H, m), 1.76–
1.85 (2H, m), 1.89–1.96 (1H, m), 2.58–2.70 (2H, m),
3.30 (1H, septet, J = 6.9 Hz), 3.81 (3H, s), 6.78 (1H, d,
J = 8.2 Hz), 7.01 (1H, dd, J = 8.2, 2.3 Hz), 7.04 (1H, d,
J = 2.3 Hz); 13C NMR (100 MHz, CDCl3) d: 16.1,

2744
T. Katoh et al. / Bioorg. Med. Chem. 15 (2007) 2736–2748
m/z: 314 (M+), 299, 257, 177, 149; HRMS calcd for
C21H30O2 (M+): 314.2246, found: 314.2241; Anal. Calcd
for C21H30O2: C, 80.21; H, 9.62. Found: C, 80.11; H,
9.65.
4.18. 12-Methoxy-5b-abieta-8,11,13-trien-7-one (21)
Colorless crystals; mp: 152–154 °C; 1H NMR
(400 MHz, CDCl3) d: 0.31, 0.94 (each 3H, s), 1.21
(3H, d, J = 6.8 Hz), 1.22 (3H, d, J = 7.0 Hz), 1.29 (3H,
s), 1.31–1.39 (2H, m), 1.41–1.52 (3H, m), 1.82–1.84
(1H, m), 2.43–2.47 (1H, m), 2.76 (1H, d, A part of
AB, J = 19.0 Hz), 2.96 (1H, dd, B part of AB,
J = 19.0, 7.0 Hz), 3.24 (1H, septet, J = 7.0 Hz), 3.91
(3H, s), 6.73 (1H, s), 7.90 (1H, s); 13C NMR
(100 MHz, CDCl3) d: 19.2, 22.4, 22.5, 22.8, 26.6, 31.9,
34.5, 35.5, 36.1, 37.5, 37.9, 42.5, 52.2, 105.1, 125.7,
126.3, 135.0, 149.5, 161.7, 197.6; IR (CHCl3): 3462,
3007, 2964, 2936, 2868, 1657, 1597, 1560, 1499, 1271,
1254 cmꢀ1; MS (70 eV) m/z: 314 (M+), 299, 257, 149;
HRMS calcd for C21H30O2: 314.2246, found: 314.2247.
4.15. 12-Methoxyabieta-8,11,13-trien-7b-ol (20)
A solution of sodium borohydride (3 mg, 0.08 mmol) in
methanol (0.5 ml) was added to a solution of 19 (21 mg,
0.07 mmol) in methanol (1 ml) and the mixture was stir-
red for 13 h at room temperature. After the reaction, the
reaction mixture was diluted with water and extracted
with diethyl ether. The organic layer was washed with
saturated aqueous solution of sodium chloride, dried
over MgSO4, and condensed in vacuo. The residue
was purified by preparative thin layer chromatography
(hexane/ethyl acetate = 5:1) to afford 20 (21 mg, 99%)
as colorless crystals; mp: 128–130 °C (hexane); 1H
NMR (400 MHz, CDCl3) d: 0.94, 0.95 (each 3H, s),
1.19, 1.21 (each 3H, d, J = 6.8 Hz), 1.14–1.25 (1H, m),
1.28 (3H, s), 1.33–1.42 (2H, m), 1.46–1.51 (1H, m),
1.57–1.68 (2H, m), 1.70–1.81 (2H, m), 2.22–2.28 (2H,
m), 3.24 (1H, septet, J = 6.8 Hz), 3.80 (3H, s), 4.48
(1H, br s), 6.68 (1H, s), 7.33 (1H, s); 13C NMR
(100 MHz, CDCl3) d: 19.2, 21.5, 22.68, 22.72, 25.2,
26.6, 30.4, 33.2 (2C), 38.5, 38.9, 41.3, 49.4, 55.4, 71.0,
106.0, 124.9, 129.9, 134.8, 148.4, 156.3; IR (CHCl3):
3591, 3442, 3003, 2961, 2932, 2868, 1609, 1568, 1501,
1464, 1248 cmꢀ1; MS (70 eV) m/z: 316 (M+), 273, 241,
192, 177; HRMS calcd for C21H32O2 (M+): 316.2402,
found, 316.2399; Anal. Calcd for C21H32O2: C, 79.70;
H, 10.19. Found: C, 79.69; H, 10.10.
4.19. 6,7-Dehydroferruginyl methyl ether (22)
p-Toluenesulfonic acid (0.23 g, 1.21 mmol) was added to a
solution of 20 (3.83 g, 12.1 mmol) in benzene, and the mix-
ture was refluxed for 1 h. After the reaction, the reaction
mixture was poured into a saturated aqueous solution of
sodium bicarbonate and extracted with ethyl acetate. The
organic layer was washed with a saturated aqueous solu-
tion of sodium chloride, dried over MgSO4, and condensed
invacuo. The residue waspurified by silica gel columnchro-
matography (hexane/ethyl acetate = 50:1) to afford 22
(3.60 g, 99%) as a colorless oil; 1H NMR (400 MHz,
CDCl3) d: 0.97 (3H, s), 1.03 (3H, d, J = 0.5 Hz), 1.05 (3H,
s), 1.17 (3H, d, J = 6.9 Hz), 1.21 (3H, d, J = 6.9 Hz),
1.19–1.27 (1H, m), 1.50–1.55 (1H, m), 1.58–1.84 (3H, m),
2.10 (1H, dd, J = 3.1, 2.6 Hz), 2.15–2.18 (1H, m), 3.25
(1H, septet, J = 6.9 Hz), 3.83 (3H, s), 5.89 (1H, dd,
J = 9.5, 2.6 Hz), 6.50 (1H, dd, J = 9.5, 3.1 Hz), 6.69 (1H,
s), 6.90 (1H, s); 13C NMR (100 MHz, CDCl3) d: 19.0,
20.1, 22.5 (2C), 22.9, 26.3, 32.6, 32.9, 36.1, 37.9, 41.1,
51.1, 55.6, 104.7, 124.3, 125.8, 127.3, 127.4, 133.8, 147.0,
156.3; IR (CHCl3): 3003, 2961, 2930, 2868, 1603, 1556,
1495, 1464, 1258 cmꢀ1; MS (70 eV) m/z: 298 (M+), 283,
227, 216, 199, 173; HRMS calcd for C21H30O (M+):
298.2297, found: 298.2296.
4.16. Oxidation of ferruginyl methyl ether (4) and 17
A solution of chromium trioxide (0.37 g, 3.72 mmol) in
acetic acid (10 ml) was added to a solution of 4 and 17
(932 mg, 3.10 mmol) in acetic acid (20 ml), and the mix-
ture was stirred for 4 h at room temperature. After the
reaction, the reaction mixture was poured into ice water.
The aqueous solution was neutralized with an aqueous
solution of 1 M NaOH and extracted with ethyl acetate.
The organic layer was successively washed with saturated
aqueous solution of sodium bicarbonate and saturated
aqueous solution of sodium chloride, dried over MgSO4,
and condensed in vacuo. The residue was purified by sil-
ica gel column chromatography (hexane/benzene = 1:2)
to afford a mixture of 19 and 21 (880 mg, 90 %).
4.20. 6,7-Dehydroferruginol (23)
n-Butyllithium (2.71 M in hexane, 41.9 ml, 0.11 mol)
was added to a solution of dodecanethiol (27.2 ml,
0.11 mol) in HMPA (50 ml) and the mixture was stirred
for 30 min at room temperature. To the mixture 22
(3.39 g, 11.4 mmol) was added and the reaction mixture
was stirred for 5 h at 100 °C. After the reaction, the
reaction mixture was poured into 1 M hydrochloric acid
and extracted with diethyl ether. The organic layer was
successively washed with distilled water and saturated
aqueous solution of sodium chloride, dried over MgSO4,
and condensed in vacuo. The residue was purified by
silica gel column chromatography (hexane/ethyl ace-
tate = 15:1) to afford 23 (3.23 g, 100%) as a colorless
oil; 1H NMR (100 MHz, CDCl3) d: 0.96, 1.01, 1.04
(each 3H, s), 1.23 (3H, d, J = 6.9 Hz), 1.26 (3H, d,
J = 6.9 Hz), 1.25–1.29 (1H, m), 1.49–1.78 (5H, m),
2.05–2.10 (1H, m), 3.13 (1H, septet, J = 6.9 Hz), 4.69
(1H, s), 5.87 (1H, dd, J = 9.5, 2.7 Hz), 6.49 (1H, dd,
4.17. Reduction of a mixture of 19 and 21
A solution of sodium borohydride (5 mg, 0.14 mmol)
was added to a solution of 19 and 21 (35.7 mg,
0.11 mmol) in methanol (1 ml) at 0 °C and the mixture
was stirred for 13 h at room temperature. After the reac-
tion, the reaction mixture was diluted with water and
extracted with diethyl ether. The organic layer was
washed with saturated aqueous solution of sodium chlo-
ride, dried over MgSO4, and condensed in vacuo. The
residue was purified by preparative thin layer chroma-
tography (hexane/ethyl acetate = 8:1) to afford 20
(15.2 mg, 42%) and 21 (17.9 mg, 50%), which is recov-
ered as an intact substrate.

T. Katoh et al. / Bioorg. Med. Chem. 15 (2007) 2736–2748
2745
J = 9.5, 3.1 Hz), 6.58, 6.89 (each 1H, s); 13C NMR
4.23. 12-Acetoxy-6,7-secoabieta-8,11,13-trien-6,7-dial
(26)
(100 MHz, CDCl3) d: 19.0, 20.1, 22.4, 22.5, 22.8, 26.7,
32.6, 32.8, 36.0, 37.6, 41.0, 50.9, 109.5, 124.5, 126.3,
127.6, 130.9, 147.5, 152.1; IR (CHCl3): 3599, 3341,
Ozone gas was passed through a solution of 25 (300 mg,
0.92 mmol) in CH2Cl2 (30 ml) at ꢀ78 °C until pale blue
color of the solution maintained. After the reaction, ex-
cess amount of ozone was released by bubbling nitrogen
gas. To the reaction mixture, a solution of triphenyl-
phosphine (0.48 g, 1.84 mmol) in CH2Cl2 (10 ml) was
added and the mixture was stirred for 1 h at ꢀ78 °C
and for another 1 h at room temperature. The organic
solvent of the reaction mixture was evaporated and the
residue was purified by silica gel column chromatogra-
phy (hexane/ethyl acetate = 5:1) to afford 26 (283 mg,
3003, 2963, 2930, 2870, 1730, 1605, 1460, 1265 cmꢀ1
;
MS (70 eV) m/z: 284 (M+), 213, 202, 185, 159; HRMS
calcd for C20H8O (M+): 284.2140, found: 284.2137.
4.21. 12-Methoxy-6,7-secoabieta-8,11,13-trien-6,7-dial
(24)
Ozone gas was passed through a solution of 22 (102 mg,
0.34 mmol) in CH2Cl2 (20 ml) at ꢀ78 °C until pale blue
color of the solution maintained. After the reaction, ex-
cess amount of ozone was released by bubbling nitrogen
gas. To the reaction mixture, a solution of triphenyl-
phosphine (0.18 g, 0.68 mmol) in CH2Cl2 (5 ml) was
added and the mixture was stirred for 1 h at ꢀ78 °C
and for another 2 h at room temperature. The organic
solvent of the reaction mixture was evaporated and the
residue was purified by silica gel column chromatogra-
phy (hexane/ethyl acetate = 8:1) to afford 24 (78.9 mg,
1
81%) as a pale yellow oil; H NMR (400 MHz, CDCl3)
d: 0.58 (3H, s), 0.96 (6H, s), 1.23 (3H, d, J = 6.8 Hz),
1.24 (3H, d, J = 6.8 Hz), 1.25–1.29 (1H, m), 1.39–1.52
(2H, m), 1.65–1.85 (2H, m), 2.37 (3H, s), 2.30–2.39
(1H, m), 2.91 (1H, d, J = 4.7 Hz), 3.03 (1H, septet,
J = 6.8 Hz), 7.16 (1H, s), 7.92 (1H, s), 9.93 (1H, d,
J = 4.7 Hz), 10.73 (1H, s); 13C NMR (100 MHz, CDCl3)
d: 19.1, 21.0, 22.60 (2C), 22.63 (2C), 27.2, 29.8, 33.6,
36.9, 37.2, 40.2, 66.0, 121.6, 132.1, 133.1, 138.6, 149.4,
151.9, 169.0, 191.5, 205.2; IR (CHCl3): 2964, 2936,
2359, 1757, 1715, 1682, 1371, 1190, 1171 cmꢀ1; MS
(70 eV) m/z: 358 (M+), 301, 245, 203, 189; HRMS calcd
for C22H30O4 (M+): 358.2144, found: 358.2139.
1
70%) as a pale yellow oil; H NMR (400 MHz, CDCl3)
d: 0.67, 1.01 (each 3H, s), 1.16–1.22 (1H, m), 1.22, 1.23
(each 3H, d, J = 7.0 Hz), 1.54 (3H, s), 1.63–1.70 (1H, m),
1.73–1.87 (3H, m), 2.33–2.39 (1H, m), 3.12 (1H, d,
J = 4.2 Hz), 3.26 (1H, septet, J = 7.0 Hz), 3.93 (3H, s),
6.99, 7.83 (each 1H, s), 9.89 (1H, d, J = 4.2 Hz), 10.55
(1H, s); 13C NMR (100 MHz, CDCl3) d: 19.4, 22.2,
22.3, 26.4, 30.4, 33.5, 37.6, 40.8, 55.4 (2C), 65.0, 77.2,
101.2, 109.1, 127.5, 133.0, 134.9, 151.0, 160.7, 191.4,
205.6; IR (CHCl3): 2963, 2936, 2872, 2361, 2340, 1715,
1670, 1597, 1464, 1254 cmꢀ1; MS (70 eV) m/z: 330
(M+), 301, 287, 217, 161; HRMS calcd for C21H30O3
(M+): 330.2195, found: 330.2194.
4.24. 12-(tert-Butyldimethylsilyloxy)abieta-6,8,11,13-
tetraene (27)
tert-Butyldimethylsilyl chloride (0.30 g, 1.97 mmol) and
imidazole (0.27 g, 3.93 mmol) were added to a solution
of 23 (280 mg, 0.98 mmol) in DMF (10 ml) and the mix-
ture was stirred for 19 h at room temperature. After the
reaction, the reaction mixture was poured into a saturated
aqueous solution of ammonium chloride and extracted
with diethyl ether. The organic layer was washed with a
saturated aqueous solution of sodium chloride, dried over
MgSO4, and condensed in vacuo. The residue was puri-
fied by silica gel column chromatography (hexane/ethyl
acetate = 50:1) to afford 27 (383 mg, 98%) as a colorless
oil; 1H NMR (400 MHz, CDCl3) d: 0.23, 0.24, 0.96 (each
3H, s), 1.02 (12H, s), 1.04 (3H, s), 1.16 (3H, d, J = 6.9 Hz),
1.20 (3H, d, J = 6.9 Hz), 1.19–1.27 (1H, m), 1.50–1.60
(2H, m), 1.65–1.78 (2H, m), 2.04–2.09 (1H, m), 2.09
(1H, dd, J = 3.1, 2.7 Hz), 3.24 (1H, septet, J = 6.9 Hz),
5.87 (1H, dd, J = 9.5, 2.7 Hz), 6.49, 6.88 (1H, s); 13C
NMR (100 MHz, CDCl3) d: ꢀ4.1, ꢀ3.9, 18.3, 19.1,
20.2, 22.5, 22.7, 23.0, 25.9 (3C), 26.2, 32.6, 32.8, 36.0,
37.6, 41.1, 51.0, 112.4, 124.3, 126.2, 127.5, 135.6, 146.7,
152.2; IR (CHCl3): 3001, 2961, 2930, 2862, 1603, 1551,
1493, 1275, 1256, 1175 cmꢀ1; MS (70 eV) m/z: 398 (M+),
383, 341, 316, 273, 257, 153; HRMS calcd for C26H42OSi
(M+): 398.3005, found 398.3004.
4.22. 12-Acetoxyabieta-6,8,11,13-tetraene (25)
Acetic anhydride (0.37 ml, 3.90 mmol) and DMAP
(16 mg, 1.30 mmol) were added to a solution of 23
(370 mg, 1.30 mmol) in pyridine (10 ml) and the mixture
was stirred for 6 h at room temperature. After the reac-
tion, the reaction mixture was diluted with ice water, the
pH value of the aqueous solution was adjusted at 4.0
with 1 M hydrochloric acid, and extracted with ethyl
acetate. The organic layer was washed with saturated
aqueous solution of sodium chloride, dried over MgSO4,
and condensed in vacuo. The residue was purified by
silica gel column chromatography (hexane/ethyl ace-
tate = 20:1) to afford 25 (416 mg, 98%) as a colorless
1
oil; H NMR (400 MHz, CDCl3) d: 0.96, 1.038, 1.042
(each 3H, s), 1.17 (3H, d, J = 6.9 Hz), 1.21 (3H, d,
J = 6.9 Hz), 1.19–1.26 (1H, m), 1.49–1.77 (4H, m),
2.05–2.08 (1H, m), 2.13 (1H, dd, J = 3.1, 2.7 Hz), 2.31
(3H, s), 2.94 (1H, septet, J = 6.9 Hz), 5.99 (1H, dd,
J = 9.5, 2.7 Hz), 6.53 (1H, dd, J = 9.5, 3.1 Hz), 6.76,
6.97 (each 1H, s); 13C NMR (100 MHz, CDCl3) d:
18.9, 20.2, 22.5, 22.8, 23.1, 27.1, 32.5, 32.8, 35.9, 37.7,
40.9, 50.7, 116.1, 116.2, 124.5, 127.1, 130.1, 131.1,
136.9, 147.1, 169.8; IR (CHCl3): 2963, 2930, 2361,
2341, 1751, 1489, 1458, 1371, 1190 cmꢀ1; MS (70 eV)
m/z: 326 (M+), 284, 269, 202, 185, 159; HRMS calcd
for C22H30O2 (M+): 326.2246, found: 326.2244.
4.25. 12-(tert-Butyldimethylsilyloxy)-6,7-secoabieta-
8,11,13-trien-6,7-dial (28)
Ozone gas was passed through a solution of 27 (3.49 g,
8.77 mmol) in CH2Cl2 (200 ml) at ꢀ78 °C until pale blue
color of the solution maintained. After the reaction, ex-
cess amount of ozone was released by bubbling nitrogen

2746
T. Katoh et al. / Bioorg. Med. Chem. 15 (2007) 2736–2748
gas. To the reaction mixture, a solution of triphenyl-
phosphine (4.60 g, 17.5 mmol) in CH2Cl2 (20 ml) was
added and the mixture was stirred for 1 h at ꢀ78 °C
and for another 1 h at room temperature. The organic
solvent of the reaction mixture was evaporated and the
residue was purified by silica gel column chromatogra-
phy (hexane/ethyl acetate = 20:1) to afford 28 (3.37 g,
for C20H28O3: C, 75.91; H, 8.92. Found: C, 75.68; H,
8.85.
4.27. DL-Standishinal (1)
(+)-Camphorsulfonic acid (17 mg, 0.073 mmol) was
added to a solution of 2 (19 mg, 0.061 mmol) in CH2Cl2
(3 ml) and the mixture was stirred for 8 h at room tem-
perature. After the reaction, the reaction mixture was
extracted with ethyl acetate and the organic layer was
successively washed with a saturated aqueous solution
of sodium bicarbonate and a saturated aqueous solution
of sodium chloride, dried over MgSO4, and condensed
in vacuo. The residue was purified by silica gel prepara-
tive TLC (hexane/ethyl acetate = 4:1) to afford DL-1
(16 mg, 84%) as colorless crystals; mp: 187–189 °C (hex-
1
89%) as a colorless oil; H NMR (400 MHz, CDCl3)
d: 0.30, 0.68, 1.00 (each 3H, s), 1.04 (9H, s), 1.22, 1.23
(3H, d, J = 7.0 Hz), 1.48 (3H, s), 1.51–1.57 (1H, m),
1.62–1.74 (2H, m), 1.78–1.82 (2H, m), 2.27–2.34 (1H,
m), 3.13 (1H, d, J = 4.0 Hz), 3.26 (1H, septet,
J = 7.0 Hz), 6.94, 7.83 (each 1H, s), 9.88 (1H, d,
J = 4.0 Hz), 10.50 (1H, s); 13C NMR (100 MHz, CDCl3)
d: ꢀ4.0 (2C), 18.3, 19.3, 22.45, 22.53, 25.7 (3C), 26.3,
27.7, 28.5, 30.3, 33.5, 37.3, 37.4, 40.4, 64.6, 117.3,
128.2, 134.0, 137.0, 150.3, 157.7, 191.5, 205.5; IR
(CHCl3): 2962, 2932, 1717, 1672, 1595, 1497,
1259 cmꢀ1; MS (70 eV) m/z: 430 (M+), 401, 373, 345,
317, 249, 221, 179; HRMS calcd for C26H42O3Si (M+):
430.2903, found: 430.2905.
1
ane/chloroform); H NMR (400 MHz, CDCl3) d: 1.17,
1.19 (each 3H, s), 1.24, 1.25 (each 3H, d, J = 6.9 Hz),
1.26 (3H, s), 1.21–1.26 (1H, m), 1.51–1.55 (1H, m),
1.82 (1H, d, J = 10.2 Hz), 1.61–1.91 (3H, m), 2.10 (1H,
d, J = 8.1 Hz), 2.44–2.48 (1H, m), 3.27 (1H, septet,
J = 6.9 Hz), 5.40 (1H, dd, J = 10.2, 8.1 Hz), 7.69 (1H,
s), 8.26 (1H, s), 10.21 (1H, s); 13C NMR (100 MHz,
CDCl3) d: 19.9, 22.0, 22.3, 22.4, 23.9, 26.5, 33.4, 38.4,
41.0, 46.7, 67.2, 75.5, 124.5, 127.2, 128.5, 134.5, 153.9,
157.3, 189.9; IR (KBr): 3450, 3252, 2962, 2932, 2870,
4.26. 12-Hydroxy-6,7-secoabieta-8,11,13-trien-6,7-dial
(2)
4.26.1. Preparation from 26. Potassium carbonate
(31 mg, 0.22 mmol) was added to a solution of 26
(40 mg, 0.11 mmol) in methanol (1 ml) and the mixture
was stirred for 5 h at room temperature. After the reac-
tion, the reaction mixture was diluted with water. The
aqueous solution was neutralized with 1 M hydrochloric
acid and extracted with ethyl acetate. The organic layer
was washed with a saturated aqueous solution of
sodium chloride, dried over MgSO4, and condensed in
vacuo. The residue was purified by preparative TLC
(hexane/ethyl acetate = 4:1) to afford 2 (27.0 mg, 77%)
as colorless crystals.
1664, 1612, 1580, 1479, 1466, 1261, 1171, 1103 cmꢀ1
;
MS (70 eV) m/z: 316 (M+), 298, 283, 269, 255, 229,
213, 199, 171; HRMS calcd for C20H28O3 (M+):
316.2038, found: 316.2039; Anal. Calcd for C20H28O3:
C, 75.91; H, 8.92. Found: C, 76.03; H, 8.83.
4.28. Reduction of 21
A solution of compound 21 (51.3 mg, 0.163 mmol) in
tetrahydrofuran (1.0 ml) was added to a suspension of
lithium aluminum hydride (1.24 mg, 0.327 mmol) in
tetrahydrofuran (1.0 ml) and the reaction mixture was
stirred for 1 h at 0 °C and for another 1 h at room
temperature. The reaction was quenched by adding a
saturated aqueous solution of sodium sulfate and the
mixture was filtered through CeliteÒ. The filtrate was
extracted with diethyl ether and the organic layer was
washed with a saturated aqueous solution of sodium
chloride, dried over sodium sulfate, and condensed in
vacuo. The residue was purified by preparative silica
gel thin layer chromatography (hexane/ethyl ace-
tate = 5:1) to afford 29 (43.3 mg, 84%). 1H NMR
(400 MHz, CDCl3) d: 0.47, 1.00 (each 3H, s), 1.21
(6H, d, J = 7.0 Hz), 1.24 (3H, s), 2.10 (1H, m), 2.54
(1H, ddd, J = 6.6, 7.9, 14.3 Hz), 3.27 (1H, septet,
J = 7.0 Hz), 3.82 (3H, s, OMe), 4.81 (1H, br q,
J = 6.6 Hz), 6.72, 7.37 (each 1H, s).
4.26.2. Preparation from 28. tert-Butylammonium fluo-
ride (1.0 M in THF, 0.25 ml, 0.25 mmol) was added to
a solution of 28 (90 mg, 0.21 mmol) in THF (2 ml) at
0 °C and the mixture was stirred for 30 min at room
temperature. After the reaction, the reaction mixture
was poured into a saturated aqueous solution of ammo-
nium chloride and extracted with ethyl acetate. The
organic layer was washed with a saturated aqueous solu-
tion of sodium chloride, dried over MgSO4, and con-
densed in vacuo. The residue was purified by silica gel
column chromatography (hexane/ethyl acetate = 4:1)
to afford 2 (60 mg, 90%) as colorless crystals; mp: 195–
197 °C; 1H NMR (400 MHz, CDCl3) d: 0.70, 1.01 (each
3H, s), 1.26. 1.27 (each 3H, d, J = 6.9 Hz), 1.50 (3H, s),
1.50–1.83 (5H, m), 2.28–2.34 (1H, m), 3.13 (1H, d,
J = 3.8 Hz), 3.18 (1H, septet, J = 6.9 Hz), 6.34 (1H, br
s), 6.96, 7.84 (each 1H, s), 9.86 (1H, d, J = 3.8 Hz),
10.47 (1H, s); 13C NMR (100 MHz, CDCl3) d: 19.3,
22.20, 22.23, 26.7, 27.4, 28.1, 30.6, 33.5, 37.4, 37.6,
40.4, 64.6, 114.8, 127.9, 132.4, 134.6, 151.1, 157.9,
191.6, 206.2; IR (KBr): 3352, 2959, 2934, 1701, 1661,
1593, 1570, 1261, 1173 cmꢀ1; MS (70 eV) m/z: 316
(M+), 298, 283, 255, 203, 171; HRMS calcd for
C20H28O3 (M+): 316.2038, found: 316.2039; Anal. Calcd
4.29. Dehydration of 29
A solution of compound 29 (33.1 mg, 0.105 mmol) and
p-toluenesulfonic acid (2.0 mg) in benzene (1.0 ml) was
stirred under refluxed condition for 1 h. The reaction
mixture was then poured into a saturated aqueous solu-
tion of sodium bicarbonate and the mixture was extract-
ed with ethyl acetate. The organic layer was washed with
a saturated aqueous solution of sodium chloride, dried

T. Katoh et al. / Bioorg. Med. Chem. 15 (2007) 2736–2748
2747
over sodium sulfate, and condensed in vacuo. The resi-
due was purified by preparative silica gel thin layer chro-
matography (hexane) to afford 30 (29.0 mg, 93%) as a
colorless oil; 1H NMR, (400 MHz, CDCl3) d: 0.35,
0.88, 1.09 (each 3H, s), 1.192, 1.194 (each 3H, d,
J = 6.9 Hz), 1.78 (1H, d, J = 6.3 Hz), 2.40 (1H, br d,
J = 12.1 Hz), 3.24 (1H, septet, J = 6.9 Hz), 3.84 (3H, s,
OMe), 5.84 (1H, dd, J = 6.3, 9.7 Hz), 6.45 (1H,
J = 9.7 Hz), 6.75, 6.83 (each 1H, s); 13C NMR
(100 MHz, CDCl3) d: 19.3, 20.5, 22.7, 22.8, 26.4, 31.2,
33.4, 35.1, 35.5, 37.0, 42.1, 52.5, 55.7, 105.5, 124.7,
125.9, 127.3, 128.1, 133.5, 141.5, 156.3; IR (CHCl3)
until pale blue color of the solution maintained. After
the reaction, excess amount of ozone was released by
bubbling nitrogen gas. To the reaction mixture, a solu-
tion of triphenylphosphine in CH2Cl2 was added and
the mixture was stirred for 1 h at ꢀ78 °C and for anoth-
er 1 h at room temperature. The organic solvent of the
reaction mixture was evaporated and the residue was
purified by silica gel column chromatography (hexane/
1
ethyl acetate = 15:1) to afford 32 (170.1 mg, 84%). H
NMR (400 MHz, CDCl3) d: 0.276, 0.278 (each 3H, s),
0.89 (3H, s), 1.02 (9H, s), 1.18, 1.20 (each 3H, d,
J = 6.9 Hz), 1.28, 1.67 (each 3H, s), 2.28 (1H, dt,
J = 4.2, 12.5 Hz), 3.11 (1H, d, J = 5.6 Hz), 3.20 (1H, sep-
tet, J = 6.9 Hz), 6.81, 7.79 (each 1H, s), 9.36 (1H, d,
J = 5.6 Hz), 10.43 (1H, s); 13C NMR (100 MHz, CDCl3)
d: 18.3, 19.0, 22.2, 22.6, 25.7, 26.3, 28.7, 30.2, 31.4, 34.0,
35.1, 36.1, 40.2, 64.3, 116.1, 128.8, 136.9, 149.7, 157.8,
191.6, 204.2; IR (CHCl3): 3032, 2961, 2932, 2862,
1715, 1595, 1267, 1258, 1232, 1221, 1204 cmꢀ1; MS
(70 eV) m/z: 430 (M+), 401, 387, 373, 300, 285, 243;
HRMS calcd for C26H42O3Si (M+): 430.2903, found:
430.2897.
3007, 2961, 2936, 2864, 1499, 1464, 1248, 1057 cmꢀ1
;
MS (70 eV) m/z: 298 (M+), 298, 283, 241, 227, 216,
199, 173; HRMS calcd for C21H30O (M+): 298.2297,
found: 298.2290.
4.30. Conversion of 30 to 31
2.71 M solution of n-butyllithium in hexane (1.92 ml,
5.21 mmol) was added to a solution of dodecanethiol
(1.25 ml, 5.21 mmol) in HMPA (8.0 ml) and the mixture
was stirred for 1 h at 0 °C. The mixture was then added
to compound 29 (155.6 mg, 0.521 mmol) at room tem-
perature and the reaction mixture was stirred for 1.5 h
at 100 °C. The reaction was quenched by adding a satu-
rated aqueous solution of ammonium chloride and 1 M
hydrochloric acid and the mixture was extracted with
diethyl ether. The organic layer was washed with a sat-
urated aqueous solution of sodium chloride, dried over
sodium sulfate, and condensed in vacuo. The residue
was purified by preparative silica gel column chromato-
graphy (hexane/ethyl acetate = 25:1) to afford demethy-
lated derivative (146.1 mg, 99%), to which the solution
in dimethylformamide (5.0 ml), tert-butyldimethylsilyl
chloride (154.8 mg, 1.03 mmol), and imidazole
(139.9 mg, 2.05 mmol) were successively added. After
stirring the reaction mixture for 10 h at room tempera-
ture, a saturated aqueous solution of ammonium chlo-
ride was added. The mixture was extracted with
diethyl ether and the organic layer was washed with a
saturated aqueous solution of sodium chloride, dried
over sodium sulfate, and condensed in vacuo. The resi-
due was purified by preparative silica gel column chro-
matography (hexane/ethyl acetate = 50:1) to afford 31
4.32. Desilylation of 32
A solution of TBAF in tetrahydrofuran was added to a
solution of 32 (170.1 mg, 0.395 mmol) in tetrahydrofu-
ran (4.0 ml) and the mixture was stirred for 30 min at
room temperature. A saturated aqueous solution of
ammonium chloride was added to the reaction mixture,
which was extracted with ethyl acetate. The organic
layer was washed with a saturated aqueous solution of
sodium chloride, dried over sodium sulfate, and con-
densed in vacuo. The residue was purified by preparative
silica gel column chromatography (hexane/ethyl ace-
tate = 4:1) to afford 33 (113.1 mg, 91%). 1H NMR
(400 MHz, CDCl3) d: 0.90 (3H, s), 1.23, 1.25 (each
3H, d, J = 6.9 Hz), 1.28, 1.67 (each 3H, s), 2.33 (1H,
dt, J = 4.0, 12.6 Hz), 3.11 (1H, septet, J = 6.9 Hz), 3.14
(1H, d, J = 5.6 Hz), 5.81 (1H, br d, J = 10.4 Hz), 6.82,
7.80 (each 1H, s), 9.39 (1H, d, J = 5.6 Hz), 10.42 (1H,
s); 13C NMR (100 MHz, CDCl3) d: 18.9, 22.0, 22.3,
26.7, 28.6, 30.2, 31.2, 34.1, 35.1, 36.1, 40.2, 64.3, 113.3,
128.7, 132.2, 133.6, 150.5, 157.8, 191.5, 205.1; IR
(KBr): 3140, 2957, 2870, 1717, 1647, 1585, 1570, 1269,
1171 cmꢀ1; MS (70 eV) m/z: 316 (M+), 298, 283, 255,
242, 229, 109; HRMS calcd for C20H28O3 (M+):
316.2038, found: 316.2036; Anal. Calcd for C20H28O3:
C, 75.91; H, 8.92. Found: C, 75.91; H, 8.75.
(188.1 mg, 92%) as
a
colorless oil; 1H NMR
(400 MHz, CDCl3) d: 0.21, 0.24, 0.35, 0.88 (each 3H,
s), 1.02 (9H, s), 1.06 (3H, s), 1.17, 1.18 (each 3H, d,
J = 6.9 Hz), 1.77 (1H, d, J = 6.2 Hz), 2.30 (1H, br d,
J = 12.3 Hz), 3.23 (1H, septet, J = 6.9 Hz), 5.83 (1H,
dd, J = 6.7, 9.7 Hz), 6.45 (1H, d, J = 9.7 Hz), 6.67,
6.82 (each 1H, s); 13C NMR (100 MHz, CDCl3) d:
18.3, 19.1, 20.5, 22.9, 23.0, 25.9, 26.2, 31.3, 33.4, 35.1,
35.5, 36.6, 41.9, 52.4, 113.3, 124.6, 126.1, 127.8, 128.1,
135.3, 141.3, 152.1; IR (CHCl3): 3032, 2961, 2932,
2862, 1495, 1271, 1236, 1200 cmꢀ1; MS (70 eV) m/z:
398 (M+), 383, 316; HRMS calcd for C26H42OSi (M+):
398.3005, found: 398.3001.
4.33. Aromatase inhibitory assay
Aromatase inhibitory assays were carried out using the
CYP19/ Methoxy-4-trifluoromethylcoumarin (MFC)
High Throughput Inhibitory Screening Kit (BD Gen-
test, Woburn, MA, USA). MFC, the fluorometric sub-
strate, is rapidly converted by human recombinant
aromatase expressed in insect cells using the baculovirus
systems to the highly fluorescent product. Positive con-
trol inhibitors for aromatase, ketoconazole and letroz-
ole, were purchased from Wako Pure Chemical
Industries, Ltd (Osaka, Japan) and LKT Laboratories,
Inc. (St. Paul, MN), respectively.
4.31. Ozonolysis of 31
Ozone gas was passed through a solution of 31
(188.1 mg, 0.472 mmol) in CH2Cl2 (20 ml) at ꢀ78 °C

2748
T. Katoh et al. / Bioorg. Med. Chem. 15 (2007) 2736–2748
13. Recanatini, M.; Bisi, A.; Cavalli, A.; Belluti, F.; Gobbi, S.;
Rampa, A.; Valenti, P.; Palzer, M.; Palusczak, A.;
Hartmann, R. W. J. Med. Chem. 2001, 44, 672.
14. Su, B.; Diaz-Cruz, S.; Landini, S.; Brueggemeier, R. W.
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15. Jacobs, C.; Frotscher, M.; Dannhardt, G.; Hartmann, R.
W. J. Med. Chem. 2000, 43, 1841.
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T.; Kase, S.; Furukawa, T.; Takahashi, M.; Fukuda, S.;
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The NADPH-cofactor buffer for dilution of compounds
and enzyme–substrate mixture were prepared according
to the manufacturer’s instruments. The test compounds
and positive control inhibitors (dissolved in acetone)
were diluted serially in 1:3 ratio by the NADPH-cofac-
tor buffer in 96-well microplates. The reaction was initi-
ated by the addition of prewarmed enzyme–substrate
mixture and incubated for 30 min at 37 °C. Changes in
fluorescence value were measured with a multifunctional
microplate detection system ULTRA with suitable
409 nm excitation filters and 530 nm emission interfer-
ence (Tecan Japan Co. Ltd, Kanagawa, Japan).
The result obtained under the condition (Table 3) is also
viewed in Figure 2.
Acknowledgments
22. Banerjee, M.; Mukhopadhyay, R.; Achari, B.; Banerjee,
A. K. J. Org. Chem. 2006, 71, 2787.
23. McFadden, R. M.; Stolz, B. M. J. Am. Chem. Soc. 2006,
128, 7738.
24. Liang, G.; Xu, Y.; Seiple, I. B.; Trauner, D. J. Am. Chem.
Soc. 2006, 128, 11022.
25. Fillion, E.; Fishlock, D. J. Am. Chem. Soc. 2005, 127,
13144.
This research was financially supported in part by Fron-
tier Research Program and the 21st Century Center of
Excellence Program ‘Development of Drug Discovery
Frontier Integrated from Tradition to Proteome’ of
the Ministry of Education, Culture, Sport and Technol-
ogy, Japan.
26. Iwamoto, M.; Ohtsu, H.; Tokuda, H.; Nishino, H.;
Matsunaga, S.; Tanaka, R. Bioorg. Med. Chem. 2001, 9,
1911.
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