Isolation, synthesis, and neurite outgrowth-promoting activity of illicinin A from the flowers of Illicium anisatum

Article abstract of DOI:10.1016/j.tet.2009.08.015

Two new prenylated C₆-C₃ compounds, illicinin A (1) and (4S)-illicinone I (2), were isolated from the flowers of Illicium anisatum. The structures of the new compounds were elucidated by spectroscopic methods. The absolute structure of (4S)-illicinone I was determined by comparing its CD spectrum and specific rotation with those of (4S)-illicinone A (4). Illicinin A (1) and 4-allyl-2,6-dimethoxy-3-(3-methylbut-2-enyl)phenol (3) were found to exhibit neurite outgrowth-promoting activity at concentrations ranging from 0.1 to 10 μM in primary cultured rat cortical neurons. Illicinin A and its derivatives were synthesized for structure-activity relationship studies by employing sequential Stille reactions to introduce a prenyl and an allyl group to the benzene ring, thereby indicating that an allylphenyl moiety in the molecule of 1 is essential for its neurotrophic properties.

Full text of DOI:10.1016/j.tet.2009.08.015

Tetrahedron 65 (2009) 8354–8361
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Tetrahedron
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Isolation, synthesis, and neurite outgrowth-promoting activity of illicinin A from
the flowers of Illicium anisatum
Shigeki Takaoka a, Noriko Takaoka a, Yuka Minoshima a, Jian-Mei Huang b, Miwa Kubo a, Kenichi Harada a,
,
Hideaki Hioki a, Yoshiyasu Fukuyama a
*
a Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan
b Faculty of Pharmaceutical Sciences, Beijing University of Chinese Medicine, Beijing 100029, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 July 2009
Received in revised form 6 August 2009
Accepted 6 August 2009
Available online 11 August 2009
Two new prenylated C6–C3 compounds, illicinin A (1) and (4S)-illicinone I (2), were isolated from the
flowers of Illicium anisatum. The structures of the new compounds were elucidated by spectroscopic
methods. The absolute structure of (4S)-illicinone I was determined by comparing its CD spectrum and
specific rotation with those of (4S)-illicinone A (4). Illicinin A (1) and 4-allyl-2,6-dimethoxy-3-(3-
methylbut-2-enyl)phenol (3) were found to exhibit neurite outgrowth-promoting activity at concen-
trations ranging from 0.1 to 10 mM in primary cultured rat cortical neurons. Illicinin A and its derivatives
Keywords:
were synthesized for structure–activity relationship studies by employing sequential Stille reactions to
introduce a prenyl and an allyl group to the benzene ring, thereby indicating that an allylphenyl moiety
in the molecule of 1 is essential for its neurotrophic properties.
Illicium anisatum
Prenylated C6–C3 compound
Illicinin A
Ó 2009 Elsevier Ltd. All rights reserved.
(4S)-Illicinone I
Neurotrophic activity
Synthesis
Stille reaction
1. Introduction
anisatum as well as the synthesis and neurite outgrowth-pro-
moting activity of 1 and its analogues for structure–activity re-
In the course of our search for neurotrophic factor-like small
molecules in natural products, honokiol (5), a biaryl compound
with two allyl and two hydroxyl groups, has been shown to sig-
nificantly enhance neurite outgrowth promotion and neuronal
survival in primary cultured rat cortical neurons.1 A further study
on the relationship between neurite outgrowth-promoting activity
and the structures of honokiol and its analogues revealed that the
40-hydroxyl group and 5-allyl group in the structure of 5 are re-
sponsible for honokiol-mediated neurite outgrowth-promoting
activity.2 However, there remains the question of whether the
biaryl group in 5 is necessary for its activity. As part of our chemical
studies on the Illicium species,3 we first investigated the chemical
components of the flowers of Illicium anisatum, resulting in the
isolation of two new prenylated C6–C3 compounds 1 and 2 named
illicinin A and (4S)-illicinone I. The isolation of the allylphenol
compound illicinin A (1) may help us to answer the above question.
In this paper, we deal with the isolation and structures of
illicinin A (1) and (4S)-illicinone I (2) from the flowers of I.
lationship studies (Fig. 1).
O
9
9
7
7
1
2
5
4
1
OMe
OH
O
O
15
5
10
OMe
3
10
4
OMe
OMe
OMe
12
12
13
13
1
2
3
OH
O
4'
O
O
HO
5
5
4
* Corresponding author. Tel.: þ81 88 602 8435; fax: þ81 88 655 3051.
E-mail address: fukuyama@ph.bunri-u.ac.jp (Y. Fukuyama).
Figure 1. Prenylated C6–C3 compounds 1–4 isolated from the flowers of I. anisatum
and honokiol (5).
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.08.015

S. Takaoka et al. / Tetrahedron 65 (2009) 8354–8361
8355
2. Results and discussion
Compound 2 has the molecular formula, C16H22O3, as estab-
lished by HREIMS of its molecular ion peak at m/z 262. The IR
absorption band at 1667 cmꢀ1 revealed the presence of a conju-
gated carbonyl group. The 1H NMR and 13C NMR data of 2 were
similar to those of (4S)-illicinone A (4).5 However, the appearance
of two singlet signals at dH 3.77 and 3.11 (3H for each) and the
absence of the singlet signal at dH 5.87 (2H) present in 4 supposed
that 2 was a structure with two methoxy groups at the C-4 and C-
5 positions instead of the methylenedioxy group present in 4. The
respective HMBC correlation of the methoxy group at dH 3.11 and
3.77 with C-4 (dC 78.0) and C-5 (dC 172.8) supported the presumed
structure for 2. The CD spectrum of 2 showed the same negative
cotton effect at 280 nm as that of 4. The C-4 chiral center should
have the same S configuration as 4, and thus 2 was named (4S)-
illicinone I.
2.1. Structure of new compounds 1 and 2
Compounds 1 and 2 were isolated from the methanol extract of
the flowers of I. anisatum along with the previously known sesqui-
terpenes pseudoanisatin4 and 6-deoxypseudoanisatin5 and the pre-
nylated C6–C3 compounds, (4S)-illicinone A (4),6 (2R,4S)-illicinone B,7
and 4-allyl-2,6-dimethoxy-3-(3-methyl-2-butenyl)phenol (3).7
Compound 1 has the molecular formula C16H20O3 as established
by high resolution (HR) EIMS (m/z 260.1446 [Mþ]), and its IR
spectrum revealed the presence of a benzene moiety at 1620 and
1478 cmꢀ1. The 1H NMR spectrum of 1 contained signals due to an
allyl group at dH 5.87 (1H, ddt, J¼17.6, 12.8, 6.3 Hz), 4.98 (1H, dd,
J¼17.6, 1.9 Hz), 4.97 (1H, dd, J¼12.8, 1.9 Hz), and 3.48 (2H, br d,
J¼6.3 Hz); a prenyl group at dH 5.29 (1H, t, J¼6.6 Hz), 3.24 (2H, d,
J¼6.6 Hz), 1.71 (3H, s), and 1.65 (3H, s); a methylenedioxy group at
dH 5.27 (2H, s); a methoxy group at dH 3.79 (3H, s); and an aromatic
singlet signal at dH 6.49 (1H, s). These spectral data suggested that 1
consisted of a pentasubstituted benzene ring with allyl, prenyl,
methylenedioxy, and methoxy groups. The disposition of the four
substituents was elucidated by analysis of the NOESY and HMBC
spectra (Fig. 2). The sole aromatic signal showed HMBC correlations
with the oxygenated carbons (C-1 and C-2) resonating at dC 147.8
and 135.6. Both the C-1 and C-2 carbons had additional HMBC
correlations with the methylenedioxy signal at dH 5.27, indicating
that the methylenedioxy group was located at the C-1 and C-2
positions. The NOE cross-peak between the aromatic and the H-7
methylene signals provided compelling evidence for the allyl group
being ortho to the sole aromatic proton. Sequential NOE correla-
tions of H-7 to the H-10 methylene of the prenyl group and the
H-10 to the methoxy signal indicated that the allyl, prenyl, and
methoxy groups were placed in order. An additional HMBC, shown
in Figure 2, confirmed the structure of 1 to be 1,2-methylenedioxy-
3-methoxy-4-prenyl-5-allylbenzene.
2.2. Syntheses of illicinin A (1) and its analogues
According to our biological studies using primary cultured rat
cortical neurons,1 illicinin A (1) and compound 3 significantly
promoted neurite outgrowth at concentrations ranging from
0.1 mM to 10 mM (Figs. 4 and 5). In order to further study their
structure–activity relationship, illicinin A and its analogues (1a–1d)
were synthesized (Fig. 3).
OH
OH
OH
OH
OMe
OMe
HO
1a
1b
O
O
O
O
6
7
O
O
OMe
OMe
1
1c
1d
10
3
Figure 3. Synthesized derivatives 1a–1d.
OMe
First, we explored the synthesis of illicinin A (1) based on
a palladium-catalyzed Stille reaction that sequentially introduced
an allyl and a prenyl group to the substituted benzene ring. The
synthetic route is shown in Scheme 1.
HMBC
NOESY
Figure 2. HMBC and NOESY correlations for 1.
EtO2C
EtO2C
O
HO2C
EtO2C
OH
OH
OH
O
O
d
c
a
b
OH
O
Br
OH
OH
OH
OH
6
7
8
gallic acid
EtO2C
Br
O
O
O
O
O
O
O
HO
e
f
O
Br
Br
OMe
OMe
OMe
OMe
9
10
11
1
Scheme 1. Reactions and conditions: (a) EtOH, H2SO4, reflux, 93%; (b) CH2I2, Cs2CO3, DMF, 120 ꢁC, 91%; (c) Br2, t-BuNH2, tol, rt, 80%; (d) MeI, K2CO3, acetone, 100%; (e) DIBAL$THF,
ꢀ78 ꢁC, 100%; (f) SOCl2, benzene, 70 ꢁC, then CH2]CHSnBu3, 10 mol % Pd(Ph3P)4, DMF, 70 ꢁC, 85%.

8356
S. Takaoka et al. / Tetrahedron 65 (2009) 8354–8361
The synthesis commenced with the esterification of commer-
those of natural 1, and thereby the structure of illicinin A was
established.
cially available gallic acid into the ethyl ester 6, the diol of which
was protected as dioxymethane (7).7 The ortho-position to the re-
sidual hydroxyl group was selectively brominated in the presence
of tert-butylamine8 to give the bromide 8 in 80% yield. After
methylation of the hydroxyl group in 8, the ester 9 was reduced
with DIBAL to give the alcohol 10, which was subjected to the first
palladium-catalyzed Stille reaction.9 Treatment of 10 with thionyl
chloride provided an unstable chloromethyl group, which was not
isolated and reacted with tributylvinyltin in the presence of
10 mol % Pd(Ph3P)4, giving rise to the desired ally compound 11 in
85% yield. However, no attempt to introduce the prenyl group using
the Stille reaction produced illicinin A (1). The poor reactivity of 11
toward Pd(0) was presumably due to the presence of an electron-
rich benzene. Hence, the order of the two kinds of Stille reactions
was reversed; i.e., employing the first Stille reaction using tribu-
tylprenyltin to the ester group-bearing compound 8. As expected,
upon treatment of 8 with tributylprenyltin in the presence of
Pd(Ph3P)4 the reaction proceeded smoothly to afford the prenyl-
substituted derivative (Scheme 2). The ethyl ester group in 12 was
reduced with LiAlH4 to produce the alcohol intermediate 13 in 63%
yield over two steps. Compound 13 was chlorinated with SOCl2, and
the resulting crude product 14 was reacted with tributylvinyltin in
the presence of Pd(Ph3P)4 to give rise to illicinin A (1) in two steps
and 78% overall yield. All spectroscopic data were identical with
Next, our attention was directed toward preparation of de-
rivatives 1a–1d to define their structure–activity relationship. At-
tempts to derive 1a from 1 have not been successful.10 The synthesis
of 1a essentially followed the same route as that used for 1 starting
from 1511 with a dioxydiphenylmethyl group as the protecting group
of the vicinal diol (Scheme 3). Stepwise bromination and methyla-
tion of 15 gave rise to 16, which was in turn reacted with tribu-
tylprenyltin in the presence of 10 mol % PdCl2dppf in DMFat 70 ꢁC to
afford the prenylated compound 17 in 56% yield. The ester group of
17 was reduced tothe alcohol 18, and then it was converted tobenzyl
chloride, which was directly subjected to the second Stille reaction
with tributylvinyltin in the presence of 10 mol % Pd(Ph3P)4, giving
rise to 19 in a quantitative yield. Finally, treatment of 19 with AcOH/
H2O in the ratio 2:1 at 110 ꢁC provided 1a in 23% yield along with 1b
in 27% yield. When the ratio of AcOH/H2O was changed to 5:1, the
sole product 1a was obtained in 57% yield.
Compound 1c was easily derived from
1 by catalytic
hydrogenation.
The regioisomer 1d, which carries a prenyl substituent at the C-
6 position of the gallic acid, was also synthesized from methylene-
2,3-di-O-methyl-4-O-protected gallic acid 20 as follows. Upon
treatment of compound 20 with bromine, bromination regiose-
lectively took place to give rise to 21. The methyl ester and bromide
groups were converted to an allyl group and a prenyl group using
successive Stille reactions with tributylvinyltin and allyltributyltin,
respectively, to produce compound 1d.
EtO2C
EtO2C
Br
O
O
OMe
O
O
a
b
O
O
OMe
HO
c
OH
2.3. Neurite outgrowth-promoting activity of illicinin A
and its analogues
8
13
12
O
We have continued to search for neurotrophic factor-like mol-
ecules from natural products, and some unique neurotrophic
compounds such as merrilactone A12 and jiadifenin13 were found in
Illicium merrillianum and I. jiadifengpi. Thus, the Illicum plants can
be regarded as an important source of novel neurotrophic com-
pounds. All compounds (1–4) isolated from the flowers of I. ani-
satum were evaluated for their neurite outgrowth-promoting
activity in primary cultured rat cortical neuron cells. As a result,
Cl
O
e
d
illicinin A (1)
O
OMe
O
OMe
1c
14
Scheme 2. Reactions and conditions: (a) Me2C]CHCH2SnBu3, 10 mol % Pd(Ph3P)4,
DMF; (b) LiAlH4, THF, rt, 63% (two steps); (c) SOCl2, benzene, 70 ꢁC; (d) CH2]CHSnBu3,
10 mol % Pd(Ph3P)4, DMF, 70 ꢁC, 78% (two steps); (e) H2, PtO2, CH2Cl2, 50%.
MeO2C
MeO2C
Br
MeO2C
a
O
O
O
O
O
O
Ph
Ph
b
c
d
Ph
Ph
Ph
Ph
gallic acid
OH
OMe
OMe
16
17
15
O
O
OH
OH
OH
OH
O
e
f
Ph
Ph
Ph
Ph
HO
O
OMe
OMe
1a
OMe
OMe
HO
19
1b
18
Br
MeO2C
MeO2C
j
MeO2C
O
O
O
O
O
O
g
O
O
i
h
gallic acid
OMe
OMe
OMe
1d
OMe
21
22
20
Scheme 3. Reactions and conditions: (a) MeOH, H2SO4, reflux, and then Ph2CCl2, 170 ꢁC, 97% (two steps); (b) Br2, t-BuNH2, tol, rt, and then MeI, K2CO3, acetone, reflux, 100%; (c)
10 mol % PdCl2dppf, DMF, 70 ꢁC, 56%; (d) LiAlH3, THF, rt, 100%; (e) SOCl2, benzene, 70 ꢁC, and then CH2]CHSnBu3, 10 mol % Pd(Ph3P)4, DMF, 70 ꢁC, 99% (two steps); (f) AcOH/H2O
(5:1), 110 ꢁC; (g) Me2SO4, Na2BO4, EtOH, rt, 72%, then CH2I2, Cs2CO3, DMF, 120 ꢁC, 91%; (h) Br2, Fe, CH2Cl2, 0 ꢁC, 73%; (i) Me2C]CHCH2SnBu3, 10 mol % Pd(Ph3P)4, DMF, 125 ꢁC, 54%; (j)
1. LiAlH4, THF, rt, 2. SOCl2, benzene, 70 ꢁC, 3. CH2]CHSnBu3, 10 mol % Pd(Ph3P)4, DMF, 70 ꢁC, 50% (three steps).

S. Takaoka et al. / Tetrahedron 65 (2009) 8354–8361
8357
illicinin A (1) and 4-allyl-2,6-dimethoxy-3- (3-methyl-2-butenyl)
phenol (3) showed neurite outgrowth-promoting activity at con-
centrations ranging from 0.1 M to 10 M as shown in Figures 4 and
5; whereas, 2 and 4 did not enhance neurite outgrowth at the same
concentrations.
According to our previous studies on honokiol (5) and its ana-
logues,1,2,14 it is supposed that the biaryl moiety in 5 may contribute
to its neurotrophic activity, because both o- and p-allyphenols, two
structural units of 5, are toxic against cultured neurons at 1 mM.
Although the neurotrophic activity of 1 is less potent than that of
honokiol, the present results suggested that an allylbenzene would
be the basic structure behind the neurite outgrowth-promoting
activity. In order to further understand the structure–activity re-
lationship, the neurotrophic properties of the derivatives 1a–1d
were evaluated by measuring the longest neurites among rat cor-
tical neurons cultured in the presence of each analogue. The neu-
ronal cells were cultured in Neurobasal Medium (NBM)
m
m
supplemented with B2715 and the compounds (1
mM) for 6 d and
were then visualized after being stained with anti-MAP 2 antibody.
The longest neurite enhanced by each compound was calculated.
The results are summarized in Figure 5. Among the screened
compounds, 1c decreased neurite outgrowth, and the others were
comparable with illicinin A (1) (Fig. 5). These results indicate that
an allyl function in 1 is essential for preserving neurotrophic ac-
tivity. Among the active analogues, the extent of the activities of 1a,
1b, and 3, which posses 1,2-free diol group slightly increased, im-
plying that the presence of two vicinal oxy groups is important for
neurotrophic activity, but their methylenedioxy forms were found
to have nothing to do with the enhancement of its activity. In ad-
dition, the presence and the position of the prenyl group in 1 were
found not to play an important role in neurotrophic activity.
a
3. Summary
We have isolated two new prenylated C6–C3 compounds from
the flowers of I. anisatum, and the aromatic compound illicinin A (1)
has been found to promote neurite outgrowth in primary cultured
rat cortical neurons. The structure of 1 has been confirmed by its
total synthesis using two sequential Stille reactions to append allyl
and prenyl groups onto its gallic acid ring. Illicinin A is the first
example of a simple allylphenol derivative with neurotrophic
properties and appears to be promising as a seed compound for the
development of low-molecular neurotrophic factor-like com-
pounds16 via addition of a prenyl group to a variety of functional
groups based on structure–activity studies. Additional structure–
activity and mechanism studies of 1 are in progress and will be
reported in due course.
b
4. Experimental
4.1. General information
Melting point was measured on a MPJ-2 instrument (un-
corrected). Optical rotations were assessed on a Jasco DIP-1000
digital polarimeter. IR and UV spectra were measured on a Jasco FT-
IR 5300 and a Shimadzu UV-300 spectrophotometer, respectively.
CD spectra were measured on a Jasco J-725 instrument. NMR
spectra were recorded on Varian Unity 200, Mercury 300, Unity
600, and JEOL JNM-ECP 400, JNM-GX 400 instruments. Chemical
321
319
**
307
350
300
250
200
150
100
50
287
c
255
251
shifts are given as
d (ppm) with TMS as an internal standard. The
MS spectra were recorded on a JEOL AX-500 instrument. Column
chromatography was carried out on Merck silica gel 60 (70–230
mesh and 230–400 mesh), Wakogel C-300, or Sephadex LH-20. All
organic layers were dried over anhydrous MgSO4.
0
Control
bFGF
0.1
1.0
10
0.01
(0.5 % EtOH) (40 ng/mL)
4.2. Extraction and isolation
(µM)
The flowers of I. anisatum were collected in Tokushima, Japan
and a voucher specimen (1742FR) is deposited in the Institute of
Pharmacognosy (TBU). The dried material (1 kg) was crushed and
extracted with MeOH at room temperature. The MeOH extract
(137 g) mixed with Celite was subjected to silica gel column chro-
matography by elution with hexane, hexane/CH2Cl2 (1:1), CH2Cl2,
CH2Cl2/EtOAc (1:1), EtOAc, and EtOAc/MeOH (7:3) to give frs. 1–6.
Figure 4. Neurotrophic effect of 1 in primary cultured cortical neurons. After the rat
cortical neurons (9000 cells cmꢀ2) had been cultured for 6 d in the presence of 0.5%
ethanol and illicinin A (1), the neurons were stained with anti-MAP 2 as described in
the text; (a): morphology of the neurons in the presence of 0.5% ethanol as a vehicle
control; (b): morphology of the neurons in the presence of 1 (1
mM); (c): quantitative
analysis of neurite length ( m). bFGF (40 ng/mL) was used as a positive control. The
m
data are expressed as meansꢂSE (n¼80); Student’s t-test; *: P<0.05, **: P<0.01 versus
control.

8358
S. Takaoka et al. / Tetrahedron 65 (2009) 8354–8361
Figure 5. A comparison of the neurite length of rat cortical neurons induced by 1 and its analogues 1a–1d at 1 mM. In each group, the mean length of the primary dendrite-like
processes was determined in 80 neurons selected from random fields. *: P<0.05; **: P<0.01; ***: P<0.001 versus control. Data presented here were derived from one of two
repeated experiments with similar results.
Fr. 1 (5.0 g) was further chromatographed on a silica gel eluted with
CHCl3, EtOAc, and MeOH gradiently to give frs. 7–19. Fr. 9 was
separated by column chromatography on a Sephadex LH-20 col-
umn to yield frs. 20–22. Illicinin A (1, 2.1 mg) and 3 (10 mg) were
obtained from fr. 21. Fr. 15 was separated by column chromato-
graphy on a Sephadex LH-20 eluted with MeOH to yield frs 23–32.
(4S)-Illicinone I (2, 2.4 mg) and 4 (20 mg) were obtained from fr. 30
using a chromatographic method (silica gel, hexane/CHCl3, 1:5).
cooled to room temperature. After the ethanol had evaporated,
water was added. The aqueous solution was extracted with EtOAc.
The combined organic layers were washed with H2O and brine,
dried, and concentrated in vacuo to give 6 (19.4 g) as a white solid
(from hexane, mp 148–149 ꢁC). 1H NMR (200 MHz, CD3OD)
d: 7.04
(2H, s), 4.86 (3H, br s), 4.26 (2H, q, J¼7.1 Hz),1.33 (3H, t, J¼7.1 Hz). 13C
NMR (50 MHz, CD3OD) d: 168.6,146.5,139.7,121.8,110.0, 61.7, 14.6. IR
(cmꢀ1): 3369, 1682. EIMS m/z (rel int.): 198 (54), 170 (19), 153 (100).
HREIMS: Mþ calcd for C9H10O5: 198.0528, found: 198.0524.
4.2.1. Illicinin A (1). Colorless oil. EIMS m/z (rel int): 260 (66), 245
(17), 204 (100). HREIMS: Mþ calcd for C16H20O3: 260.1413, found:
4.3.2. 7-Hydroxy-benzo[1,3]dioxole-5-carboxylic acid ethyl ester
(7). To a solution of 6 (200 mg, 1.0 mmol) in dry DMF (10 mL) were
added Cs2CO3 (362 mg, 1.01 mmol) and CH2I2 (0.9 mL, 1.0 mmol).
The mixture was stirred at 120 ꢁC for 1 h and then cooled to room
temperature. The resulting mixture was poured into water. The
aqueous layer was extracted with ether. The ether layer was se-
quentially washed with water, 20% Cu(NO3)2 solution, and brine
then dried, filtered, and evaporated. The obtained residue was
purified by column chromatography on silica gel eluted with hex-
ane/EtOAc (4:1) to give 7 (191 mg, 91%) as a white powder (from
260.1446. UV (EtOH) lmax nm (
3
): 287 (2900), 225 (11,400). FTIR
(cmꢀ1): 1620, 1478. 1H NMR (400 MHz, C6D6)
d
: 6.49 (1H, s, H-6),
5.87 (1H, ddt, J¼17.6, 12.8, 6.3 Hz, H-8), 5.29 (1H, t, J¼6.6 Hz, H-11),
5.27 (2H, s, H-15), 4.98 (1H, dd, J¼17.6, 1.9 Hz, H-9), 4.97 (1H, dd,
J¼12.8, 1.9 Hz, H-9), 3.79 (3H, s, MeO), 3.48 (2H, d, J¼6.3 Hz, H-7),
3.24 (2H, br d, J¼6.6 Hz, H-10), 1.71 (3H, s, H-13), 1.65 (3H, s, H-14).
13C NMR (100 MHz, C6D6)
d: 147.8 (C-1), 142.3 (C-3), 137.7 (C-8),
135.6 (C-2), 132.2 (C-5), 130.5 (C-12), 125.6 (C-4), 124.5 (C-11), 115.5
(C-9), 104.6 (C-6), 100.5 (C-15), 59.3 (MeO), 37.4 (C-7), 25.9 (C-13),
25.8 (C-10), 17.9 (C-14).
hexane, mp 100–102 ꢁC). 1H NMR (200 MHz, CDCl3)
d: 7.40 (1H, s),
7.14 (1H, s), 6.50 (1H, br s), 6.04 (2H, s), 4.34 (2H, q, J¼7.0 Hz), 1.37
21.5
4.2.2. (4S)-Illicinone I (2). Colorless oil. [
a
]
þ9.1 (c 0.23, EtOH).
(3H, t, J¼7.0 Hz). 13C NMR (50 MHz, CDCl3)
d: 166.6, 148.9, 139.4,
D
CD (EtOH): D3 (287 nm) ꢀ2.3. EIMS m/z (rel int): 262 (Mþ, 23), 194
138.6, 124.6, 114.2, 103.0, 102.3, 61.3, 14.2. IR (cmꢀ1): 3404, 1688.
EIMS m/z (rel int.): 210 (67), 165 (100), 137 (17). HREIMS: Mþ calcd
for C10H10O5: 210.0528, found: 210.0519. Anal. Calcd for C10H10O5:
C, 57.14; H, 4.80. Found: C, 57.31; H, 4.90.
(57), 67 (100). HREIMS: Mþ calcd for C16H22O3: 262.1569, found:
262.1579. UV (EtOH) lmax nm (
3
): 236 (7100); IR (cmꢀ1): 1667, 1638,
1611. 1H NMR (600 MHz, CDCl3)
d
: 6.22 (1H, t, J¼1.4 Hz, H-3), 5.89
(1H, ddt, J¼17.0, 10.4, 6.6 Hz, H-8), 5.68 (1H, s, H-6), 5.09 (1H, dd,
J¼10.4, 1.9 Hz, H-9), 5.08 (1H, dd, J¼17.0, 1.9 Hz, H-9), 4.81 (1H,
ddqq, J¼8.2, 7.1, 1.1, 0.5 Hz, H-11), 3.77 (3H, s, MeO-5), 3.11 (3H, s,
MeO-4), 3.09 (2H, m, H-7), 2.66 (1H, dd, J¼13.7, 8.2 Hz, H-10), 2.45
(1H, dd, J¼13.7, 7.1 Hz, H-10), 1.62 (3H, d, J¼0.5 Hz, H-14), 1.55 (3H,
4.3.3. 6-Bromo-7-hydroxy-benzo[1,3]dioxole-5-carboxylic acid ethyl
ester (8). To a solution of tert-butylamine (0.5 mL, 4.8 mmol) in
anhydrous toluene was added bromine (0.1 mL, 2.4 mmol) in
10 min at ꢀ78 ꢁC under an argon atmosphere. After the mixture
had been stirred for 1 h, a solution of 7 (500 mg, 2.4 mmol) in
CH2Cl2 was added at ꢀ78 ꢁC in 1 h. The reaction mixture was stirred
at room temperature for 4 h and then concentrated in vacuo. The
residue was purified by chromatography on silica gel (CHCl3/EtOAc,
9:1) to give 8 (547.7 mg, 80%) as a colorless plate crystal (from
d, J¼1.1 Hz, H-13). 13C NMR (150 MHz, CDCl3)
d: 186.9 (C-1), 172.8
(C-5), 141.8 (C-3), 140.0 (C-2), 136.0 (C-12), 135.1 (C-8), 116.8 (C-9),
116.3 (C-11), 104.9 (C-6), 78.0 (C-4), 55.9 (MeO-5), 52.6 (MeO-4),
37.2 (C-10), 32.8 (C-7), 25.9 (C-14), 17.9 (C-13).
4.3. Syntheses of illicinin A and its analogues
hexane, mp 79–80 ꢁC). 1H NMR (300 MHz, CDCl3)
d: 7.09 (1H, s),
6.09 (2H, s), 4.36 (2H, q, J¼7.0 Hz), 1.39 (3H, t, J¼7.0 Hz). 13C NMR
4.3.1. 3,4,5-Trihydrobenzoic acid ethyl ester (6). To a stirred solution
of gallic acid (20 g) in EtOH (500 mL) was added 5 mL of condensed
sulfuric acid. The mixture was stirred at 90 ꢁC for 10 h and then
(75 MHz, CDCl3) d: 165.1, 147.8, 137.5, 137.3, 124.5, 105.4, 104.6,
102.3, 61.6,14.2. IR (KBr) (cmꢀ1): 3439,1720. EIMS m/z (rel int.): 290
(88), 288 (88), 243 (100), 245 (97). HREIMS: Mþ calcd for

S. Takaoka et al. / Tetrahedron 65 (2009) 8354–8361
8359
C10H9O5Br: 287.9633, found: 287.9638. Anal. Calcd for C10H9O5Br:
C, 41.55; H, 3.14. Found: C, 41.34; H, 2.94.
with brine, dried, filtered, and concentrated in vacuo. The resulting
residue was purified by column chromatography on silica gel eluted
with hexane/EtOAc (2:1) to give 13 (833 mg, 63% yield for two steps)
as a white powder (from hexane, mp 61–63 ꢁC). IR (cmꢀ1): 3370. 1H
4.3.4. 6-Bromo-7-methoxybenzo[1,3]dioxole-5-carboxylic acid ethyl
ester (9). To a solution of 8 (733 mg, 2.5 mmol) in acetone were
added K2CO3 (3.2 g, 22.8 mmol) and CH3I (1.5 mL, 10.1 mmol). The
reaction mixture was stirred for 2 h and then filtered and evapo-
rated to give 9 (765 mg, 100%) as a yellow oil. 1H NMR (200 MHz,
NMR (300 MHz, CDCl3)
J¼6.9 Hz), 4.52 (2H, s), 3.93 (3H, s), 3.32 (2H, d, J¼6.9 Hz),1.76 (3H, s),
1.67 (3H, s). 13C NMR (75 MHz, CDCl3)
: 147.2, 141.7, 136.6, 132.8,
d: 6.60 (1H, s), 5.87 (2H, s), 5.04 (1H, t,
d
131.6, 125.6, 123.7, 103.5, 100.9, 63.3, 59.8, 25.7, 24.7, 17.9. EIMS m/z
(rel int.): 250 (100), 232 (41), 217 (90), 194 (90), 165 (21). HREIMS:
calcd 250.1205 for C14H18O4, found 250.1193. Anal. Calcd for
C14H18O4: C, 67.18; H, 7.25. Found: C, 66.11; H, 7.33.
CDCl3)
d
: 7.00 (1H, s), 6.04 (2H, s), 4.36 (2H, q, J¼7.1 Hz), 4.02 (3H, s),
1.39 (3H, t, J¼7.1 Hz). IR (cmꢀ1): 1726. EIMS m/z (rel int.): 304 (100),
302 (100), 276 (17), 274 (19), 259 (91), 257 (95). HREIMS: Mþ calcd
for C11H11O5Br: 301.9790, found: 301.9784.
4.3.9. Synthesis of illicinin
A (1). Thionyl chloride (0.2 mL,
4.3.5. (6-Bromo-7-methoxybenzo[1,3]dioxole-5-yl)methanol
(10). To a solution of 9 (240 mg, 0.8 mmol) in THF (10 mL) was
added i-Bu2AlH (1.1 M solution in hexane, 1.6 mmol) at ꢀ78 ꢁC, and
the reaction mixture was stirred for 1 h. Water was carefully added,
and the aqueous solution was extracted with EtOAc. The combined
organic layers were washed with brine, dried, filtered, and evapo-
rated to give the residue, which was purified by column chroma-
tography on silica gel (hexane/EtOAc, 2:1) to give 10 (208 mg, 100%)
as a white powder (from hexane, mp 61–63 ꢁC). IR (cmꢀ1): 3370. 1H
2.4 mmol) was added dropwise to a solution of 13 (200 mg,
0.8 mmol) in benzene. The mixture was stirred at 70 ꢁC for 1.5 h.
After being cooled down to room temperature, the resulting mix-
ture was concentrated in vacuo to give a crude product, to which
tributylvinyltin (0.2 mL, 2.4 mmol), dried DMF (20 mL), and
Pd(Ph3P)4 (50 mg, 0.08 mmol) were added successively. The re-
action mixture was stirred at 70 ꢁC for 1.5 h and cooled to room
temperature. The resulting mixture was poured into water, filtered,
and extracted with ether. The organic layer was sequentially
washed with water, 20% Cu(NO3)2 solution, water, and brine and
then dried, filtered, and evaporated. The residue was purified by
column chromatography on silica gel eluted with hexane/EtOAc
(9:1) to give 1 (163 mg, 78%), the spectral data of which were
identical to those of natural 1.
NMR (300 MHz, CDCl3)
d
: 6.73 (1H, s), 5.96 (2H, s), 4.63 (2H, s), 4.02
: 148.7, 140.3, 136.7, 134.1, 106.6,
(3H, s). 13C NMR (75 Hz, CDCl3)
d
103.3, 101.6, 65.2, 60.1. EIMS m/z (rel int.): 262 (Mþ, 91), 260 (Mþ,
100), 245 (21), 243 (21), 181 (52). HREIMS: Mþ calcd for C9H9O4Br:
259.9684, found: 259.9672. Anal. Calcd for C9H9O4Br: C, 41.41; H,
3.47. Found: C, 41.76; H, 3.70.
4.3.10. 7-Hydroxy-2,2-diphenylbenzo[1,3]dioxole-5-carboxylic acid
ethyl ester (15). Concd sulfuric acid (7.5 mL) was added to a solu-
tion of gallic acid (50 g) in MeOH (500 mL). The reaction mixture
was stirred at 90 ꢁC for 10 h and cooled to room temperature. The
solvent was removed in vacuo, and water was added. After
the aqueous solution had been extracted three times with EtOAc,
the combined organic layers were washed with water and brine,
dried, and concentrated in vacuo to give methyl ester (46 g, 93%
yield). The methyl ester derivative (24.7 g, 134 mmol) was added to
dichlorodiphenylmethane (25 mL, 134 mmol). The reaction mix-
ture was stirred at 170 ꢁC for 10 min. After being cooled to room
temperature, the resulting mixture was added to benzene, and the
organic layer was washed with water and brine, dried, and con-
centrated in vacuo to give 15 (45.3 g, 97% yield) as a solid. 1H NMR
4.3.6. 6-Allyl-5-bromo-4-methoxybenzo[1,3]dioxole (11). To a solu-
tion of 10 (240 mg, 0.9 mmol) in benzene (10 mL) was added thi-
onyl chloride (0.1 mL, 1.2 mmol), and the reaction mixture was
stirred at 70 ꢁC for 1.5 h. After being cooled down to room tem-
perature, the solvent was removed under reduced pressure. To the
residue, tributylvinyltin (0.3 mL, 0.9 mmol), dry DMF (20 mL), and
Pd(Ph3P)4 (100 mg, 0.09 mmol) were added successfully, and the
mixture was stirred at 70 ꢁC for 1.5 h. The cooled reaction mixture
was diluted with water and filtered. The resulting filtrate was
extracted with ether. The organic layer was washed with 20%
Cu(NO3)2 solution, water, and brine, before being filtered and dried.
The organic layer was evaporated to give the residue, which was
purified by column chromatography on silica gel (hexane/EtOAc,
7:1) to yield 11 (211 mg, 85%) as a yellow oil. 1H NMR (300 MHz,
(200 MHz, CDCl3) d: 7.47–7.54 (4H, m), 7.37–7.39 (6H, m), 7.30 (1H,
CDCl3)
d
: 6.49 (1H, s), 5.94 (2H, s), 5.91 (1H, ddt, J¼19.2, 12.9,
d, J¼1.4 Hz), 7.21 (1H, d, J¼1.4 Hz), 5.17 (1H, br s), 3.85 (3H, s).
6.6 Hz), 5.10 (1H, dd, J¼19.2, 1.6 Hz), 5.06 (1H, dd, J¼12.9, 1.6 Hz),
4.20 (3H, s), 3.40 (2H, d, J¼6.6 Hz). EIMS m/z (rel int.): 272 (100),
270 (100), 245 (40), 243 (40), 133 (75). HREIMS: Mþ calcd for
C11H11O3Br: 269.9891, found: 269.9884.
4.3.11. 6-Bromo-7-methoxy-2,2-diphenylbenzo[1,3]dioxole-5-carb-
oxylic acid ethyl ester (16). Compound 16 (1.28 g, 100%) was syn-
thesized from 15 (1 g, 2.9 mmol) according to the same procedure
as described for the preparation of 8 from 7. Colorless prism; mp
4.3.7. 7-Methoxy-6-(3-methylbut-2-enyl)benzo[1,3]dioxole-5-carb-
oxylic acid ethyl ester (12). To a solution of 8 (1.5 g, 5.2 mmol) and
tributyl(3-methylbut-2-enyl)tin (5.6 g, 20.8 mmol) in anhydrous
DMF (30 mL) was added Pd(Ph3P)4 (644 mg, 0.5 mmol) under an
argon atmosphere. The mixture was stirred at 125 ꢁC for 12 h and
then cooled to room temperature. The resulting mixture was
poured into water and extracted with ether. The ether layer was
sequentially washed with water, 20% Cu(NO3)2 solution, water, and
brine and then dried, filtered, and evaporated. The residue was
purified by column chromatography on silica gel eluted with hex-
ane/EtOAc (4:1) to give 12 (890 mg) as an oil.
77–79 ꢁC. IR (cmꢀ1): 1732. 1H NMR (200 MHz, CDCl3)
d
: 7.52–7.56
(4H, m), 7.37–7.40 (6H, m), 7.08 (1H, s), 4.09 (3H, s), 3.87 (3H, s). 13C
NMR (50 MHz, CDCl3) : 166.3, 147.9, 140.8, 140.1, 139.4, 129.6,
d
128.5, 126.3, 119.1, 109.0, 105.6, 60.4, 52.4. EIMS m/z (rel int.): 442
(98), 440 (99), 411 (21), 409 (23), 365 (100), 363 (99), 165 (56).
HREIMS: Mþ calcd for C22H17O5Br: 440.0259, found: 440.0237.
Anal. Calcd for C22H17O5Br: C, 59.88; H, 3.88. Found: C, 59.90; H, 4.0.
4.3.12. 7-Methoxy-6-(3-mehtylbut-2-enyl)-2,2-diphenylbenzo[1,3]-
dioxole-5-carboxylic acid ethyl ester (17). Compound 17 (59 mg,
56%) was synthesized from 16 (100 mg, 0.2 mmol) according to the
same procedure as described for the preparation of 12. Yellow oil. IR
4.3.8. [7-Methoxy-6-(3-methylbut-2-enyl)benzo[1,3]dioxol-5-yl]me-
thanol (13). To a solution of 12 (890 mg, 3 mmol) inTHF (30 mL) was
added LiAlH4 (160 mg, 4.2 mmol). The reaction mixture was stirred
at room temperature for 1 h. The excess LiAlH4 was decomposed by
adding a mixture of EtOAc and water. The organic layer was washed
(cmꢀ1): 1723. 1H NMR (300 MHz, CDCl3)
d: 7.52–7.59 (4H, m), 7.31–
7.47 (6H, m), 7.14 (1H, s), 5.12 (1H, t, J¼6.6 Hz), 4.01 (3H, s), 3.81 (3H,
s), 3.63 (2H, d, J¼6.6 Hz), 1.75 (3H, s), 1.66 (3H, s). 13C NMR (75 MHz,
CDCl3) d: 167.6, 146.5, 141.6, 139.7, 131.4, 131.2, 130.0, 129.3, 128.3,
126.3, 123.5, 117.8, 105.1, 59.9, 51.9, 25.9, 25.8, 17.9. EIMS m/z (rel

8360
S. Takaoka et al. / Tetrahedron 65 (2009) 8354–8361
int.): 430 (68), 398 (43), 387 (67), 375 (100), 359 (20), 321 (23).
HREIMS: calcd for C27H26O5: 430.1780, found: 430.1785.
and was then acidified with 2 M HCl and extracted with EtOAc. The
combined organic layers were washed with water and brine, dried,
filtered, and concentrated. The residue was purified by column chro-
matography on silica gel (benzene/ethyl acetate/CH3CO2H, 8:2:0.5) to
a methyl ester (3.87 g, 72%), which was produced as a solid; mp 112–
113 ꢁC (fromhexane). IR (cmꢀ1): 3374,1696.1HNMR(300 MHz,CD3OD)
4.3.13. [7-Methoxy-6-(3-methylbut-2-enyl)-2,2-diphenylbenzo[1,3]-
dioxole-5-yl]methanol (18). Compound 18 (374 mg, 100%) was
prepared from 17 (400 mg, 0.9 mmol) according to the method
described for the preparation of 13. Yellow oil. IR (cmꢀ1): 3428.1H
d
: 7.16 (1H, d, J¼1.9 Hz), 7.12 (1H, d, J¼1.9 Hz), 4.88 (2H, s), 3.81 (3H, s),
NMR (300 MHz, CDCl3)
d: 7.54–7.59 (4H, m), 7.31–7.39 (6H, m), 6.67
3.79 (3H, s). EIMS m/z(rel int.): 198(78),183(5),167(100),153(10). Anal.
Calcd for C9H10O5: C, 54.55; H, 5.09. Found: C, 54.26; H ,4.98. The
monomethyl ester (1.5 g, 7.58 mmol) was treated in a manner similar to
that described for the preparation of 4. The resulting product was pu-
rified by column chromatography on silica gel eluted with benzene/
EtOAc/AcOH (9:1:0.5) to afford 20 (1.45 g, 91%) as awhite powder (from
(1H, s), 5.04 (1H, t, J¼6.9 Hz), 4.53 (2H, d, J¼5.8 Hz), 4.04 (3H, s),
3.33 (2H, d, J¼6.9 Hz), 1.75 (3H, s), 1.66 (3H, s). 13C NMR (75 MHz,
CDCl3) d: 167.6, 146.5, 141.6, 139.7, 131.4, 131.2, 130.0, 129.3, 128.3,
126.3, 123.5, 117.9, 105.1, 59.9, 51.9, 25.9, 17.9. EIMS m/z (rel int.):
402 (9), 384 (100), 369 (44), 307 (39), 182 (25). HREIMS: Mþ calcd
for C26H26O4: 402.1831, found: 402.1870.
hexane, mp 70–71 ꢁC). 1H NMR (300 MHz, CDCl3)
d: 7.33 (1H, d,
J¼1.1 Hz), 7.21 (1H, d, J¼1.1 Hz), 6.06 (2H, s), 3.94 (3H, s), 3.89 (3H, s).13C
4.3.14. 6-Allyl-4-methoxy-5-(3-methylbut-2-enyl)-2,2-diphenylbenzo-
[1,3]dioxole (19). Compound 19 (129 mg, 99%) was prepared from 18
(130 mg, 0.32 mmol) according to the same procedure described for the
NMR (75 MHz, CDCl3) d: 166.4, 148.6, 143.3, 139.4, 124.4, 109.9, 103.9,
102.3, 56.6, 52.2. IR (KBr) (cmꢀ1): 1729. EIMS m/z (rel int.): 210 (82),179
(100), 151 (18), 135 (3). HREIMS: Mþ calcd for C10H10O5: 210.0529,
found: 210.0518. Anal. Calcd for C10H10O5: C, 57.14; H, 4.80. Found: C,
49.73; H, 3.95.
preparation of 1 from 13. Yellow oil. 1H NMR (300 MHz, CDCl3)
d: 7.55–
7.59 (4H, m), 7.32–7.36 (6H, m), 6.45 (1H, s), 5.88 (1H, ddt, J¼16.8, 12.1,
6.3 Hz), 5.01 (1H, t, J¼6.3 Hz), 5.01 (1H, dd, J¼16.8,1.4 Hz), 4.96 (1H, dd,
J¼12.1, 1.4 Hz), 4.02 (3H, s), 3.25 (4H, d, J¼6.3 Hz), 1.73 (3H, s), 1.65 (3H,
4.3.18. 4-Bromo-7-methoxybenzo[1,3]dioxole-5-carboxylic acid methyl
ester (21). A mixture of 20 (3.12 g, 14.86 mmol) and Fe (320 mg) was
suspended in CH2Cl2 (45 mL). Upon cooling to ꢀ78 ꢁC, bromine
(0.73 mL, 14.8 mmol) was added. The reaction mixture was warmed
to 0 ꢁC and stirred for 4 h. The organic layer was washed with satd
Na2S2O3 solution, water, and brine, and then dried and filtered. The
solvent was removed under a vacuum. The residue was purified by
columnchromatographyonsilicageleluted withbenzenetoafford21
(3.11 g, 73% yield) as a needle crystal (from hexane, mp 104–105 ꢁC).
s). 13C NMR (75 MHz, CDCl3)
d: 146.7, 141.4, 140.5, 137.3, 135.0, 131.7,
130.9, 129.0, 128.2, 126.4, 125.2, 123.5, 116.5,115.5, 104.1, 59.8, 37.2, 25.7,
25.1,17.9. EIMS m/z (rel int.): 412 (100), 398 (15), 370 (15), 357 (61), 335
(21). HREIMS: Mþ calcd for C28H28O3: 412.2038, found: 412.2034.
4.3.15. 5-Allyl-3-methoxy-4-(3-methylbut-2-enyl)benzene-1,2-diol (1a)
and 5-allyl-4-(3-hydroxy-3-methylbutyl)benzene-1,2,3-triol (1b). A
mixture of 14 (100 mg) and 5 mL of acetic acid/H2O (2:1) was stirred at
110 ꢁC for 1 h. After being cooled to room temperature, the resulting
mixture was concentrated in vacuo and purified by column chroma-
tography on silica gel eluted with CHCl3/EtOAc (4:1) to give 1a (23%
yield) and 1b (27% yield). Compound 1a: yellow oil. IR (cmꢀ1): 3416. 1H
IR (cmꢀ1): 1725. 1H NMR (300 MHz, CDCl3)
3.95 (3H, s), 3.93 (3H, s). 13C NMR (75 MHz, CDCl3)
d
: 7.27 (1H, s), 6.15 (2H, s),
d: 165.4, 148.0,
142.2,138.3,124.2,112.4,102.4, 94.4, 56.7, 52.2. EIMS m/z (relint.):290
(98), 288 (99), 259 (99), 257 (100), 210 (25), 179 (27). HREIMS: Mþ
calcd for C10H9O5Br: 287.9634, found: 287.9620. Anal. Calcd for
C10H9O5Br: C, 41.55; H, 3.14. Found: C, 47.26; H, 3.95.
NMR (300 MHz, CD3OD)
d
: 6.39 (1H, s), 5.89 (1H, ddt, J¼16.5, 12.4,
6.3 Hz), 5.00 (1H, dd, J¼16.5, 1.9 Hz), 4.98 (1H, m), 4.97 (1H, dd, J¼12.4,
1.9 Hz), 3.72 (3H, s), 3.24 (4H, d, J¼6.3 Hz), 1.74 (3H, s), 1.66 (3H, s). 13C
NMR (75 MHz, CD3OD)
d: 147.9, 145.2, 139.2, 137.6, 131.3, 130.1, 125.7,
4.3.19. 7-Methoxy-4-(3-methylbut-2-enyl)benzo[1,3]dioxole-5-carb-
oxylic acid methyl ester (22). Compound 22 was prepared through
the same procedure as described for the preparation of 17. Compound
21 (280 mg) gave 22 (146 mg, 54% yield) as a colorless oil. IR (cmꢀ1):
125.5, 115.2, 113.4, 61.0, 37.7, 25.9, 25.9, 18.0. EIMS m/z (rel int.): 248
(77), 233 (16), 205 (28), 192 (100), 159 (46). HREIMS: Mþ calcd for
C15H20O3: 248.1412, found: 248.1398.
Compound 1b: yellow oil. IR (cmꢀ1): 3412. 1H NMR (300 MHz,
1729.1H NMR (300 MHz, CDCl3)
d
: 7.22 (1H, s), 6.04 (2H, s), 5.20(1H, t,
J¼6.9 Hz), 3.90 (3H, s), 3.86 (3H, s), 3.60 (2H, d, J¼6.9 Hz),1.68 (3H, s),
1.67 (3H, s).13C NMR (75 MHz, CDCl3)
: 167.2,147.7,141.1,138.1,132.1,
CD3OD)
d
: 6.38 (1H, s), 5.90 (1H, ddt, J¼16.5, 12.6, 6.3 Hz), 4.99 (1H,
dd, J¼16.5, 1.9 Hz), 4.96 (1H, dd, J¼12.6, 1.9 Hz), 3.78 (3H, s), 3.24
(2H, d, J¼6.3 Hz), 2.58 (2H, m), 1.57 (2H, m), 1.25 (6H, s). EIMS m/z
(rel int.): 266 (9), 248 (54), 233 (8), 192 (100), 177 (22), 161 (38).
HREIMS: Mþ calcd for C15H22O4: 266.1518, found: 266.1542.
d
123.0, 122.3, 111.3, 101.9, 56.6, 51.9, 25.7, 17.9, 17.5. EIMS m/z (rel int.):
278 (Mþ,100), 246 (75), 235 (55), 231 (79), 223 (63), 207 (29).
HREIMS: Mþ calcd for C15H18O5: 278.1142, found: 278.1159.
4.3.16. 4-Methoxy-5-(3-methylbutyl)-6-propylbenzo[1,3]dioxole
(1c). A solution of illicinin A (1) (13.6 mg, 0.05 mmol) in CH2Cl2
(5 mL)wasstirredoverPtO2 (5 mg)underanatmosphereofhydrogen
for 12 h. The resulting mixture was purified by column chromato-
graphy on silica gel (hexane/EtOAc, 9:1) to give 1c (6.8 mg, 50%) as
4.3.20. 5-Allyl-7-methoxy-4-(3-methylbut-2-enyl)benzo[1,3]dioxole
(1d). Compound 1d (68 mg, 50% yield in three steps) was prepared
from 22 (148 mg, 0.47 mmol) according to the same procedures
used for the preparation of illicinin A (1) from 12. Yellow oil. 1H
NMR (300 MHz, CDCl3) d: 6.39 (1H, s), 5.90 (1H, m), 5.36 (2H, m),
ayellowoil.1HNMR (200 MHz, CDCl3)
d: 6.40(1H, s), 5.86(2H, s), 3.96
5.11 (1H, m), 5.06 (1H, m), 4.99 (1H, m), 3.51 (3H, s), 3.47 (2H, d,
(3H, s), 2.43–2.56 (4H, m), 1.50–1.66 (1H, m), 1.26–1.50 (4H, m), 0.95
J¼6.2 Hz), 3.39 (2H, d, J¼7.0 Hz), 1.76 (3H, s), 1.67 (3H, s). 13C NMR
(6H, d, J¼6.6 Hz), 0.87–1.00 (3H, m). 13C NMR (50 MHz, CDCl3)
d
:
(75 MHz, CDCl3) d: 147.3, 141.7, 137.4, 132.4, 131.9, 122.4, 115.6, 115.4,
146.7,141.6,134.7,134.3,126.4,103.7,100.5, 59.6, 40.2, 35.0, 29.7, 24.8,
24.1, 22.5, 14.2. EIMS m/z (rel int.): 264 (38), 208 (13), 207 (100), 179
(20). HREIMS: Mþ calcd for C16H24O3: 264.1714, found: 264.1697.
108.7, 101.2, 93.0, 56.6, 36.7, 25.6, 24.9, 17.8. EIMS m/z (rel int.): 260
(Mþ, 94), 245 (21), 204 (100), 203 (53), 173 (30). HREIMS: Mþ calcd
for C16H20O3: 260.1412, found: 260.1388. Anal. Calcd for C16H20O3:
C, 67.18; H, 7.25. Found: C, 67.89; H, 7.65.
4.3.17. 7-Methoxybenzo[1,3]dioxole-5-carboxylic acid methyl ester
(20). An aqueous solution of Na2BO4$10H2O (10.36 g, 27.17 mmol) was
slowly added to a solution of gallic acid methyl ester (5 g, 27.2 mmol) in
EtOH (12.5 mL) for 30 min. After being stirred for 30 min, K2CO3 (3.75 g,
27.17 mmol) and (CH3)2SO4 (2.57 mL, 27.17 mmol) were added. The
reaction mixture continued to be stirred at room temperature for 15 h,
4.4. Cell culture procedure for neurite outgrowth-promoting
activity screening1,16
All operations were carried out under sterile conditions. The
neuronal cells were separated from the cerebral hemispheres of

S. Takaoka et al. / Tetrahedron 65 (2009) 8354–8361
8361
fetal 18 days SD rats (Japan SLC, Inc.), suspended in 10% FBS/DMEM,
and then seeded at 9000 cells cmꢀ2 into poly-
-lysine-coated 24
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We thank Dr. M. Tanaka and Ms. Y. Okamoto (TBU) for taking the
600 MHz NMR and mass spectra measurements. This work was
supported by the Open Research Fund from the Promotion and
Mutual Corporation for Private Schools of Japan.