Structure-activity relationships of 1′S-1′-acetoxychavicol acetate for inhibitory effect on NO production in lipopolysaccharide-activated mouse peritoneal macrophages

Article abstract of DOI:10.1016/j.bmcl.2005.01.070

1′S-1′-Acetoxychavicol acetate from the rhizomes of Alpinia galanga inhibited nitric oxide (NO) production in lipopolysaccharide-activated mouse peritoneal macrophages with an IC₅₀ value of 2.3 μM. To clarify the structure-activity relationship of 1′S-1′- acetoxychavicol acetate, various natural and synthetic phenylpropanoids and synthetic phenylbutanoids were examined, and the following structural requirements were clarified. (1) The para or ortho substitution of the acetoxyl and 1-acetoxypropenyl groups at the benzene ring was essential. (2) The S configuration of the 1′-acetoxyl group was preferable. (3) The presence of the 3-methoxyl group and disappearance of the 2′-3′ double bond by hydrogenation reduced the activity. (4) The substitution of acetyl groups with propionyl or methyl groups reduced the activity. (5) Lengthening of the carbon chain between the 1′- and 2′-positions reduced the activity.

Full text of DOI:10.1016/j.bmcl.2005.01.070

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1949–1953
Structure–activity relationships of 10S-10-acetoxychavicol
acetate for inhibitory effect on NO production in
lipopolysaccharide-activated mouse peritoneal macrophages
Hisashi Matsuda, Shin Ando, Toshio Morikawa, Shinya Kataoka
and Masayuki Yoshikawa*
Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan
Received 17 December 2004; accepted 20 January 2005
Abstract—10S-10-Acetoxychavicol acetate from the rhizomes of Alpinia galanga inhibited nitric oxide (NO) production in lipopoly-
saccharide-activated mouse peritoneal macrophages with an IC50 value of 2.3 lM. To clarify the structure–activity relationship of
10S-10-acetoxychavicol acetate, various natural and synthetic phenylpropanoids and synthetic phenylbutanoids were examined, and
the following structural requirements were clarified. (1) The para or ortho substitution of the acetoxyl and 1-acetoxypropenyl groups
at the benzene ring was essential. (2) The S configuration of the 10-acetoxyl group was preferable. (3) The presence of the 3-methoxyl
group and disappearance of the 20–30 double bond by hydrogenation reduced the activity. (4) The substitution of acetyl groups with
propionyl or methyl groups reduced the activity. (5) Lengthening of the carbon chain between the 10- and 20-positions reduced the
activity.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
tective and antiallergic constituents from the rhizomes
of A. galanga and their structure activity relationships
as well as mechanism of action.2,3 In our continuing
studies on this natural medicine, we found that the aque-
ous acetone extract had a potent inhibitory effect on
lipopolysaccharide (LPS)-induced nitric oxide (NO)
production in mouse peritoneal macrophages
(IC50 = 7.3 lg/mL). From the aqueous acetone extract,
five known phenylpropanoids [10S-10-acetoxychavicol
acetate (1S, 2 .l3M), 10S-10-acetoxyeugenol acetate
(3S, 11 lM), trans-p-hydroxycinnamaldehyde (30,
20 lM), trans-p-hydroxycinnamyl acetate (31, 72 lM),
trans-p-coumaryl diacetate (33, 19 lM)], new three 8–
90 linked neolignans [galanganal (68 lM), galanganol
B (88 lM)], and a new sesquineolignan [galanganol C
(33 lM)] were isolated as active principles.4 Recently,
Ohata et al. reported that 1S from L. galanga strongly
inhibited the production of NO by preventing the acti-
vation of nuclear factor-jB (NF-jB), which regulates
inducible NO synthase in LPS- or interferon (IFN)-c-
treated murine macrophage-like RAW264 cells.5 How-
ever, the structure–activity relationship of 1S was not
clarified.
The inorganic free radical nitric oxide (NO) has been
implicated in physiological and pathological processes
such as vasodilation, nonspecific host defense, ischemia
reperfusion injury, and chronic or acute inflammation.
NO is produced by the oxidation of L-arginine catalyzed
by NO synthase (NOS). In the NOS family, inducible
NOS is particularly well known to be involved in the
pathological overproduction of NO.1
The Zingiberaceae plant Alpinia (A.) galanga SWARTZ
(syn. Languas galanga STUNZ) is widely cultivated in
South and Southeast Asian countries. The rhizomes of
this plant are extensively used as a spice or ginger sub-
stitute for flavoring foods, and also as a stomathic in
traditional Chinese medicine, or as a carminative,
antiflatulent, antifungal, and anti-itching agents in tradi-
tional Thai medicine. We previously reported gastropro-
Keywords: 10S-10-Acetoxychavicol acetate; Structure–activity relation-
ship; Nitric oxide production; Phenylpropanoid; Lipopolysaccharide;
Macrophage.
*
In the present study, we describe the structure–activity
relationship of 1S for the inhibition of NO production,
Corresponding author. Tel.: +81 75 595 4633; fax: +81 75 595
4768; e-mail: shoyaku@mb.kyoto-phu.ac.jp
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.01.070

1950
H. Matsuda et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1949–1953
making comparisons with the activities of various natu-
ral and synthetic related compounds.
chavicol acetate (16). Compounds 1,8 1R,9 5S,10 6,8 7–
14,11–18 21S,19 22S,20 23,8 and 24–2921–26 were synthe-
sized by Grignard reaction and then acylation, methyla-
tion or hydrogenation from benzaldehyde and ortho-,
meta-, and para-hydroxybenzaldehydes as shown in
Scheme 1.
2. Results and discussion
2.1. Compounds isolated from the rhizomes of A. galanga
2.3. Inhibitory effects of phenylpropanoids from
A. galanga and related compounds on NO production
in LPS-activated mouse peritoneal macrophages and
the structural requirements for the activity
10S-10-Acetoxychavicol acetate (1S, 1.10% from the
dried rhizome), 10S-10-hydroxychavicol acetate (2S,
0.048%), 10S-10-acetoxyeugenol acetate (3S, 0.038%),
chavicol b-D-glucopyranoside (17, 0.023%), methyleuge-
nol (20, 0.0006%), trans-p-hydroxycinnamaldehyde (30,
0.028%), trans-p-hydroxycinnamyl acetate (31, 0.021%),
trans-p-coumaryl alcohol (32, 0.052%), and trans-p-
coumaryl diacetate (33, 0.015%) were isolated from
dried rhizomes of A. galanga as described previously.2–4
Among the phenylpropanoids from A. galanga, 10S-10-
acetoxychavicol acetate (1S) and 10S-10-acetoxyeugenol
acetate (3S) showed inhibitory activity with respective
IC50 values of 2.3 and 11 lM and no cytotoxic effect
(Table 1). The inhibitory activity of 1S was stronger
than those of two NOS inhibitors [NG-monomethyl-
L-arginine (L-NMMA, IC50 = 57 lM) and guani-
dinoethyldisulfide (GED, 7.4 lM)] and an inhibitor of
NF-jB activation [caffeic acid phenethyl ester (CAPE,
15 lM)].4
2.2. Related natural and synthetic compounds6
(S)-a-Vinylbenzyl alcohol (4S) was purchased from
Fluka Co. Ltd. Eugenol (19), and p-methoxycinnamic
acid (38) were from Wako Pure Chemical. trans-Cin-
namic acid (34), trans-o-coumaric acid (35), and caffeic
acid (37) were from Nacalai Tesque. trans-m-Coumaric
acid (36) was from Tokyo Kasei Kogyo. 3,4-Dimethoxy-
cinnamic acid (39) and 3,5-dimethoxy-4-hydroxycin-
namic acid (40) were from Sigma. Demethyleugenol
(18)7 was isolated from the leaves of Piper betle. Chavi-
col (15) was obtained by hydrolysis of chavicol b-D-
glucopyranoside (17) and acetylation of 15 afforded
To clarify the structure–activity relationships of the
active phenylpropanoids 1S and 3S, we examined the
inhibitory effects of natural and synthetic phenylpro-
panoids (1–26, 30–40) and synthetic phenylbutanoids
(27–29) on the NO production. 10S-10-Hydroxychavicol
acetate (2S) lacking the 10-acetyl group and chavicol
acetate (16) lacking the 10-acetoxyl group were less
active than 1S [1S (IC50 = 2 .3lM) > 16 (65 lM) > 2S
3'
2'
HO
AcO
CHO
iv)
i), ii), iii)
OH
OH
OAc
41 (para), 42 (meta), 43 (ortho)
6, 10, 13
1, 12, 14
i), ii), iv), iii)
vii)
vi)
viii)
HO
H3CO
AcO
PrO
HO
AcO
HO
AcO
v)
iv)
iv)
OH
5S
21S
22S
4S
OCH3
OCH3
OPr
11
7
8
9
HO
AcO
AcO
4'
R3O
3'
: R1 = H, R2 = OAc, R3 = Ac
: R1 = R3 = H, R2 =OH
: R1 =H , R2 = OPr, R3 =Pr
: R1 = OAc, R2 = H, R3 = Ac
23
24
25
26
R1
i), ix), iii)
iv)
v)
v)
1, 6, 9, 14
OH
OAc
OAc
R2
27
28
29
Scheme 1. Reagents and conditions: (i) t-butyldimethylchlorosilane, imidazole/pyridine, rt; (ii) vinylmagnesium bromide/THF, 0 ꢁC; (iii)
tetrabutylammonium fluoride/THF, rt; (iv) Ac2O/pyridine, rt; (v) H2, 5% Pd–C/MeOH, rt; (vi) propionylchloride/pyridine, rt; (vii) CH3I, K2CO3/
DMF, rt; (viii) CH3I, NaH/DMF, rt; (ix) allylmagnesium bromide/THF, 0ꢁC.

H. Matsuda et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1949–1953
1951
Table 1. Effects of phenylpropanoids from the rhizomes of A. galanga and related natural and synthetic compounds on NO production in LPS-
activated mouse peritoneal macrophages-1
R1
R2
R3
R4
IC50 (lM)
10-Acetoxychavicol acetate (1)
H
H
OAc
OAc
OAc
OAc
OAc
H
OAc
OAc (S)
OAc (R)
OH (S)
OAc (S)
OH (S)
OAc (S)
OH
4.9
10S-10-Acetoxychavicol acetate (1S)
H
H
2
8.2
.3
10R-10-Acetoxychavicol acetate (1R)
10S-10-Hydroxychavicol acetate (2S)
10S-10-Acetoxyeugenol acetate (3S)
4S
H
H
H
H
>100 (46%)
11
H
OCH3
H
H
>100 (19%)
>100 (20%)
>100 (À1%)
>30 (À3%)a
>100 (22%)
30
5S
6
H
H
H
H
H
OH
OCH3
OCH3
OPr
H
7
H
H
OH
8
H
H
OCH3
OPr
OH
9
10
H
H
H
OH
OH
OAc
H
>100 (1%)
>100 (25%)
>100 (20%)
>100 (0%)
3.2
11
12
H
H
OAc
OAc
OH
H
H
13
14
OH
OAc
H
H
H
H
OAc
H
Chavicol (15)
H
OH
OAc
O-Glc
OH
OH
OCH3
>100 (43%)
65
Chavicol acetate (16)
Chavicol b-D-glucopyranoside (17)
Demethyleugenol (18)
Eugenol (19)
H
H
H
H
H
H
>100 (6%)
15
H
OH
OCH3
OCH3
H
H
H
>100 (15%)
>100 (1%)
Methyleugenol (20)
H
H
R1
R2
R3
IC50 (lM)
21S
22S
23
H
H
H
H (S)
Ac (S)
Ac
>100 (À4%)
H
>100 (25%)
10
>100 (11%)
H
OAc
OH
OPr
H
24
25
H
H
Pr
H
OAc
59
99
26
Ac
Values in parentheses represent the inhibition (%) at 100 lM (N = 4).
a Cytotoxic effect was observed at 100 lM. Bioassay methods for inhibitory activity in LPS-activated mouse peritoneal macrophages were described
previously.27
(>100 lM)]. In addition, compound 5S lacking the 4-
acetoxyl group was inactive. Compound 14 (3.2 lM)
with the 2and 1 -acetoxyl groups showed equipotent
the racemate (1)[1S (2.3 lM) > 1 (4.9 lM) > 1R
(8.2 lM)]. This result suggested that the S configuration
of the 10-acetoxyl group was preferable for the activity.
In addition, the substitution of acetyl groups with propi-
onyl or methyl groups reduced the activity, and the sub-
stitution pattern of the acetoxyl and 1-acetoxylpropenyl
groups at the benzene ring should be ortho or para for
the activity (Table 2).
0
activity to 1 (4.9 lM) with the 4 and 10-acetoxyl groups,
but compound 12 (>100 lM) with the 3 and 10-acetoxyl
groups lacked activity. Substitution of the acetyl
groups of 1 with propionyl groups reduced the activity,
and the substitution with methyl groups resulted in a
complete loss of activity [1 (4.9 lM) > 9 (30 lM) > 8
(>100 lM)]. On the other hand, 10S-10-acetoxyeugenol
acetate (3S, 11 lM) and demethyleugenol (18, 15 lM)
showed substantial activity but were weaker than 1S,
although 3S has the 4 and 10S-acetoxyl groups and 18
lacked the 4-acetyl and 10-acetoxyl groups. These find-
ings suggested that the 4- and 10-acetoxyl groups of 1S
were essential for strong activity except for 18, and the
3-methoxyl group reduced the activity. As to the rela-
tion between the absolute stereostructure of 1S and
the activity, 1S showed stronger activity than 1R and
Next, contribution of the 20–30 double bond of 1 to the
activity was investigated. The dihydroderivatives of 1, 9,
and 14 were less active than 1, 9, and 14 [1 (4.9 lM) > 23
(10 lM); 9 (30 lM) > 25 (59 lM); 14 (3.2 lM) > 26
(99 lM)].
On the other hand, phenylpropanoids (32, 34–40) with
the 10–20 double bond did not inhibit the production
of NO either. However, 30 (20 lM) with the 4-hydroxyl
and 20-aldehyde groups, 31 (72 lM) with the 4-hydroxyl

1952
H. Matsuda et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1949–1953
Table 2. Effects of phenylpropanoids from the rhizomes of A. galanga and related natural and synthetic compounds on NO production in LPS-
activated mouse peritoneal macrophages-2
30–40
R1
R2
IC50 (lM)
27
28
29
C@C
C@C
CAC
H
H
>100 (13%)
Ac
Ac
Ac
Ac
9.2
47
R1
R2
R3
R4
R5
IC50 (lM)
trans-p-Hydroxycinnamaldehyde (30)
trans-p-Hydroxycinnamyl acetate (31)
trans-p-Coumaryl alcohol (32)
trans-p-Coumaryl diacetate (33)
trans-Cinnamic acid (34)
H
H
H
H
H
OH
H
H
H
H
H
H
OH
OH
OH
OAc
H
H
CHO
20
72
H
H
CH2OAc
CH2OH
CH2OAc
COOH
COOH
COOH
COOH
COOH
COOH
COOH
H
H
>100 (24%)
19
H
H
H
H
>100 (15%)
>100 (7%)
>100 (3%)
>100 (15%)
>100 (27%)
>100 (1%)
>100 (27%)
trans-o-Coumaric acid (35)
trans-m-Coumaric acid (36)
Caffeic acid (37)
H
H
H
OH
OH
H
H
H
OH
OCH3
OCH3
OH
H
p-Methoxycinnamic acid (38)
3,4-Dimethoxycinnamic acid (39)
3,5-Dimethoxy-4-hydroxycinnamic acid (40)
H
OCH3
OCH3
H
OCH3
Refer to footnotes of Table 1.
and 30-acetoxyl groups, and 33 (19 lM) with the 4- and
30-acetoxyl groups showed moderate levels of activity.
Phenylbutanoid 28 with the 4- and 10-acetoxyl groups
and the 30–40 double bond was also active, but less active
than 1, and the dihydroderivative of 29 was less active
than 28 [1 (4.9 lM) > 28 (9.2 lM) > 29 (47 lM)]. These
results suggested that the hydrogenation of the 20–30
double bond of 1 and the 30–40 double bond of 28 and
lengthening of the carbon chain between the 10- and
20-positions in 1 reduced the activity.
Scientific Research from the Ministry of Education,
Culture, Sports, Science and Technology of Japan.
References and notes
1. Salerno, L.; Sorrenti, V.; Di Giacomo, C.; Romeo, G.;
Siracusa, M. A. Curr. Pharm. Des. 2002, 8, 177–200.
2. Matsuda, H.; Pongpiriyadacha, Y.; Morikawa, T.; Ochi,
M.; Yoshikawa, M. Eur. J. Pharmacol. 2003, 471, 59–67.
3. Matsuda, H.; Morikawa, T.; Managi, H.; Yoshikawa, M.
Bioorg. Med. Chem. Lett. 2003, 13, 3197–3202.
4. Morikawa, T.; Ando, S.; Matsuda, H.; Kataoka, S.;
Muraoka, O.; Yoshikawa, M. Chem. Pharm. Bull., in press.
5. Ohata, T.; Fukuda, K.; Murakami, A.; Ohigashi, H.;
Sugimura, T.; Wakabayashi, K. Carcinogenesis 1998, 19,
1007–1012.
In conclusion, the structural requirements of 10S-10-acet-
oxychavicol acetate (1S) and 10S-10-acetoxyeugenol ace-
tate (3S) from the rhizomes of A. galanga for activity to
inhibit production of NO were clarified as follows. (1)
The para or ortho substitution of the acetoxyl and 1-
acetoxylpropenyl group at the benzene ring was essen-
tial. (2) The S configuration of the 10-acetoxyl group
was preferable. (3) The presence of the 3-methoxyl
group and disappearance of the 20–30 double bond by
hydrogenation reduced the activity. (4) The substitution
of acetyl groups with propionyl or methyl groups
reduced the activity. (5) Lengthening of the carbon
chain between the 10- and 20-positions reduced the
activity.
6. The details will be presented in a full paper.
7. Lee-Chen, S.-F.; Chen, C.-L.; Ho, L.-Y.; Hsu, P.-C.;
Chang, J.-T.; Sun, C.-M.; Chi, C.-W.; Liu, T.-Y. Muta-
genesis 1996, 11, 519–523.
8. Kondo, A.; Ohigashi, H.; Murakami, A.; Suratwadee, J.;
Koshimizu, K. Biosci. Biotech. Biochem. 1993, 57, 1344–
1345.
9. Compound 1R was purified from 18 using HPLC [column:
CHIRALCEL OF (250 · 4.6 mm i.d., Daicel Chemical
Ind., Ltd); detection: UV (254 nm); mobile phase: isopro-
panol/hexane (1:90, v/v); flow rate 1.0 mL/min; tR 1R:
21
22
33.6 min {½aꢀD +57.1 (c 0.28, EtOH)}, 1S: 36.6 min {½aꢀD
Acknowledgements
À56.5 (c 1.00, EtOH)} (ca. 1:1)].
10. Compound 5S: colorless oil. High resolution EI-MS: calcd
for C11H12O2 (M+), 176.0837. Found: 176.0830. 1H NMR
(270 MHz, CDCl3): d 2.04 (3H, s), 5.18 (2H, m), 6.02 (1H,
This research was supported by the 21st COE Program,
Academic Frontier Project, and Grand-in Aid for

H. Matsuda et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1949–1953
1953
m), 6.19 (1H, d, J = 6.0 Hz), 7.17–7.28 (5H, m). EI-MS
m/z (%): 176 (M+, 4), 83 (100).
11. Greer, A.; Vassilikogiannakis, G.; Lee, K.-C.; Koffas, T.
S.; Nahm, K.; Foote, C. S. J. Org. Chem. 2000, 65, 6876–
6878.
(1H, ddd, J = 5.6, 10.6, 17.2Hz), 6.44 (1H, d, J = 5.6 Hz),
7.07 (1H, br d, J = ca. 8 Hz), 7.23 (1H, br dd, J = ca. 8,
8 Hz), 7.33 (1H, br dd, J = ca. 8, 8 Hz), 7.43 (1H, br d,
J = ca. 8 Hz). EI-MS m/z (%): 234 (M+, 3), 132(100).
19. Compound 21S was identified by comparison of its
physical data with that of commercially obtained sample
(Fluka Co. Ltd).
20. (a) Horiuchi, K.; Kobashi, K.; Nagata, H.; Satoh, T.;
Suemitsu, R. Biosci. Biotech. Biochem. 1994, 58, 1330–
1331; (b) Compound 22S: colorless oil. High resolution
EI-MS: calcd for C11H14O2 (M+), 178.0994. Found:
178.0989. 1H NMR (270 MHz, CDCl3): d 0.88 (3H, t,
J = 7.3 Hz), 1.87 (2H, m), 2.07 (3H, s), 5.66 (1H, t,
J = 7.0 Hz), 7.25–7.37 (5H, m). EI-MS m/z (%): 178 (M+,
9), 118 (100).
12. Compound 8: colorless oil. High resolution EI-MS: calcd
for C11H14O2 (M+), 178.0994. Found: 178.0998. 1H NMR
(270 MHz, CDCl3): d 3.30, 3.80 (3H each, both s), 4.57
(1H, d, J = 6.6 Hz), 5.18 (1H, br d, J = ca. 10 Hz), 5.25
(1H, br d, J = ca. 17 Hz), 5.93 (1H, ddd, J = 6.6, 10.4,
17.0 Hz), 6.88, 7.24 (2H, d, J = 8.6 Hz). EI-MS m/z (%):
178 (M+, 3), 83 (100).
13. Compound 9: colorless oil. High resolution EI-MS: calcd
for C15H18O4 (M+), 262.1205. Found: 262.1196. 1H NMR
(270 MHz, CDCl3): d 1.15 (3H, t, J = 7.6 Hz), 1.26 (3H, t,
J = 7.4 Hz), 2.38 (2H, q, J = 7.4 Hz), 2.58 (2H, q,
J = 7.6 Hz), 5.24 (1H, br d, J = ca. 10 Hz), 5.29 (1H, br
d, J = ca. 17 Hz), 5.98 (1H, ddd, J = 5.8, 10.4, 16.7 Hz),
6.28 (1H, d, J = 5.8 Hz), 7.07, 7.36 (2H each, both d,
J = 8.6 Hz). EI-MS m/z (%): 262 (M+, 4), 206 (50), 150
(72), 132 (100).
21. Pohl, L. R.; Haddock, R.; Garland, W. A.; Trager, W. F.
J. Med. Chem. 1975, 18, 513–519.
22. Compound 25: colorless oil. High resolution EI-MS: calcd
for C15H20O4 (M+), 264.1361. Found: 264.1357. 1H NMR
(270 MHz, CDCl3): d 0.88 (3H, t, J = 7.3 Hz), 1.13 (3H, t,
J = 7.7 Hz), 1.26 (3H, t, J = 7.3 Hz), 1.85 (2H, m), 2.35
(2H, q, J = 7.3 Hz), 2.58 (2H, q, J = 7.7 Hz), 5.68 (1H, t,
J = 7.0 Hz), 7.05, 7.32(2H each, both d, J = 8.4 Hz). EI-
MS m/z (%): 264 (M+, 4), 208 (17), 134 (100).
23. Pedrocchi-Fantoni, G.; Servi, S. J. Chem. Soc., Perkin
Trans. 1 1992, 1029–1033.
24. Wada, M.; Ohki, H.; Akiba, K. Bull. Chem. Soc. Jpn.
1990, 63, 1738–1747.
14. Compound 10: colorless oil. High resolution EI-MS: calcd
1
for C9H10O2 (M+), 150.0681. Found: 150.0687. H NMR
(270 MHz, CDCl3): d 5.03 (1H, d, J = 6.1 Hz), 5.11 (1H,
br d, J = ca. 10 Hz), 5.26 (1H, br d, J = ca. 17 Hz), 5.99
(1H, ddd, J = 6.1, 10.4, 17.1 Hz), 6.66 (1H, ddd, J = 2.1,
2.5, 8.1 Hz), 6.81 (2H, m), 7.13 (1H, dd, J = 8.1, 8.1 Hz).
EI-MS m/z (%): 150 (M+, 34), 132(100).
15. Compound 11: colorless oil. High resolution EI-MS: calcd
for C11H12O3 (M+), 192.0786. Found: 192.0791. 1H NMR
(270 MHz, CDCl3): d 2.12 (3H, s), 5.24 (1H, br d, J = ca.
10 Hz), 5.29 (1H, br d, J = ca. 17 Hz), 5.98 (1H, ddd,
J = 5.9, 10.4, 17.1 Hz), 6.19 (1H, d, J = 5.9 Hz), 6.77 (1H,
ddd, J = 2.0, 2.6, 8.1 Hz), 6.83 (1H, dd, J = 2.0, 2.0 Hz),
6.91 (1H, br d, J = ca. 8 Hz), 7.21 (1H, dd, J = 7.7,
8.1 Hz). EI-MS m/z (%): 192(M +, 31), 132(100).
16. Compound 12: colorless oil. High resolution EI-MS: calcd
for C13H14O4 (M+), 234.0892. Found: 234.0888. 1H NMR
(270 MHz, CDCl3): d 2.11, 2.29 (3H each, both s), 5.26
(1H br d, J = ca. 10 Hz), 5.31 (1H, br d, J = ca. 17 Hz),
5.97 (1H, ddd, J = 5.9, 10.2, 16.8 Hz), 6.26 (1H, d,
J = 5.9 Hz), 7.04 (1H, br d, J = ca. 8 Hz), 7.09 (1H, br
s), 7.21 (1H, br d, J = ca. 8 Hz), 7.35 (1H, dd, J = 7.7,
7.9 Hz). EI-MS m/z (%): 234 (M+, 9), 150 (100).
25. Compound 28: colorless oil. High resolution EI-MS: calcd
for C14H16O4 (M+), 248.1049. Found: 248.1040. 1H NMR
(270 MHz, CDCl3): d 2.05, 2.27 (3H each, both s), 2.58
(2H, m), 5.05, 5.08 (1H each, both d-like), 5.67 (1H, m),
5.80 (1H, t, J = 6.3 Hz), 7.06, 7.34 (2H, d, J = 8.7 Hz). EI-
MS m/z (%): 248 (M+, 1), 146 (100).
26. Compound 29: colorless oil. High resolution EI-MS: calcd
for C14H18O4 (M+), 250.1205. Found: 250.1214. 1H NMR
(270 MHz, CDCl3): d 0.91 (3H, t, J = 7.3 Hz), 1.29, 1.80
(2H each, both m), 2.05, 2.28 (3H each, both s), 5.74 (1H,
t, J = 6.3 Hz), 7.05, 7.33 (2H, d, J = 8.6 Hz). EI-MS m/z
(%): 250 (M+, 5), 123 (100).
27. (a) Morikawa, T.; Tao, J.; Ando, S.; Matsuda, H.;
Yoshikawa, M. J. Nat. Prod. 2003, 66, 638–645; (b) Tao,
J.; Morikawa, T.; Ando, S.; Matsuda, H.; Yoshikawa, M.
Chem. Pharm. Bull. 2003, 51, 654–662; (c) Abdel-
Halim, O. B.; Morikawa, T.; Ando, S.; Matsuda, H.;
Yoshikawa, M. J. Nat. Prod. 2004, 67, 1119–1124; (d)
Matsuda, H.; Morikawa, T.; Ando, S.; Oominami, H.;
Murakami, T.; Kimura, I.; Yoshikawa, M. Bioorg. Med.
Chem. 2004, 12, 3037–3046, and references cited
therein.
17. Talley, J. J.; Evans, I. A. J. Org. Chem. 1984, 49, 5267–
5269.
18. Compound 14: colorless oil. High resolution EI-MS: calcd
for C13H14O4 (M+), 234.0892. Found: 234.0888. 1H NMR
(270 MHz, CDCl3): d 2.07, 2.30 (3H each, both s), 5.25 (1H,
br d, J = ca. 10 Hz), 5.27 (1H, br d, J = ca. 17 Hz), 6.00