Novel dolabellane-type diterpene alkaloids with lipid metabolism promoting activities from the seeds of Nigella sativa

Article abstract of DOI:10.1021/ol036239c

(Equation presented) Four new dolabellane-type diterpene alkaloids, nigellamines A₁ (1), A₂ (2), B₁ (3), and B ₂ (4), were isolated from the seeds of Nigella sativa. Their absolute stereostructures were determined on the basis of chemical and physicochemical evidence. Nigellamines A₁ (1), B₁ (3), and B₂ (4) were found to show potent lipid metabolism promoting activity in primary cultured mouse hepatocytes, and their activities were equivalent to that of a PPAR-α agonist, clofibrate.

Full text of DOI:10.1021/ol036239c

ORGANIC
LETTERS
2004
Vol. 6, No. 6
869-872
Novel Dolabellane-Type Diterpene
Alkaloids with Lipid Metabolism
Promoting Activities from the Seeds of
Nigella sativa
Toshio Morikawa, Fengming Xu, Yousuke Kashima, Hisashi Matsuda,
Kiyofumi Ninomiya, and Masayuki Yoshikawa*
Kyoto Pharmaceutical UniVersity, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan
shoyaku@mb.kyoto-phu.ac.jp
Received November 15, 2003 (Revised Manuscript Received February 12, 2004)
ABSTRACT
Four new dolabellane-type diterpene alkaloids, nigellamines A (1), A (2), B1 (3), and B2 (4), were isolated from the seeds of Nigella sativa.
1
2
Their absolute stereostructures were determined on the basis of chemical and physicochemical evidence. Nigellamines A (1), B1 (3), and B2
1
(4) were found to show potent lipid metabolism promoting activity in primary cultured mouse hepatocytes, and their activities were equivalent
to that of a PPAR-r agonist, clofibrate.
The Ranunculaceae annual plant Nigella satiVa L. is widely
cultivated in Arabian countries, and the seeds are commonly
called “black cumin”. The seeds of this plant have been used
as a food and spice and also prescribed in Egyptian folk
medicine for the treatment of asthma, flatulence, polio,
kidney stones, abdominal pain, etc.1
In the course of our characterization studies on bioactive
constituents from Egyptian medicinal herbs,2 four novel
dolabellane-type diterpene alkaloids named nigellamines A1
(1), A2 (2), B1 (3), and B2 (4) were isolated from the
methanolic extract of the seeds of N. satiVa. This paper deals
with the absolute stereostructure elucidation and lipid
metabolism promoting activities of nigellamines (1-4).
The seeds of N. satiVa (purchased in Egypt) were extracted
with methanol three times under reflux for 3 h. The
methanolic extract (17.4% from this natural medicine) was
partitioned into an EtOAc and water mixture to give an
EtOAc-soluble fraction (10.1%) and an aqueous phase
(7.3%). The EtOAc-soluble fraction was subjected to ordinary-
phase [n-hexanes-EtOAc (20:1-10:1-5:1-2:1-1:2) to
CHCl3-MeOH-H2O (20:3:1, lower layer 6:4:1) to MeOH]
and reversed-phase column chromatographies (MeOH-
H2O), and finally HPLC (YMC-Pack ODS-5-A, 250×20 mm
i.d., MeOH-H2O or CH3CN-H2O) to give nigellamines A1
(1, 0.0096% from the natural medicine), A2 (2, 0.0078%),
B1 (3, 0.0012%), and B2 (4, 0.0036%).
Nigellamine A1 (1) was isolated as a white powder with
negative optical rotation [[R]27D -23.4 (c ) 1.20, CHCl3)].
The positive-ion fast atom bombardment (FAB)-MS of 1
showed a quasimolecular ion peak at m/z 650 (M + H)+,
and the molecular formula C40H43NO7 of 1 was determined
(1) (a) Sayed, K. A.; Ross, S. A.; El Sohly, M. A.; Khalafalla, M. M.;
Abdel-Halim, O. B.; Ikegami, F. Saudi Pharm. J. 2000, 8, 175-181. (b)
Enomoto, S.; Asano, R.; Iwahori, Y.; Narui, T.; Okada, Y.; Singab, A. N.
B.; Okuyama, T. Biol. Pharm. Bull. 2001, 24, 307-310 and literature cited
therein.
(2) (a) Yoshikawa, M.; Xu, F.; Morikawa, T.; Ninomiya, K.; Matsuda,
H. Bioorg. Med. Chem. Lett. 2003, 13, 1045-1049. (b) Yoshikawa, M.;
Morikawa, T.; Xu, F.; Ando, S.; Matsuda, H. Heterocycles 2003, 60, 1787-
1792 and literature cited therein.
10.1021/ol036239c CCC: $27.50 © 2004 American Chemical Society
Published on Web 02/27/2004

by high-resolution MS measurement.3 The IR (KBr) spectrum
of 1 showed absorption bands at 1717, 1647, 1636, 1592,
1541, 1509, 1420, 1277, 1111, 1069, 1026, 947, 756, and
712 cm-1 ascribable to ester carbonyl, olefin, and ether
functions and aromatic rings. In the UV spectrum of 1 (in
MeOH), absorption maxima were observed at 226 (log ꢀ
NOESY experiment on 1a, in which NOE correlations were
observed between the following proton pairs: H-2 and H-11,
H3-16; H-3 and H-7, H-15b; Hâ-5 and Hâ-6, H-7; HR-6 and
H3-17; Hâ-6 and H-7; H-7 and Hâ-9, H-15b; HR-9 and H-10,
H3-17; H-10 and H-11; H2-14 and H2-15 (Figure 2). Finally,
1
4.61) and 264 (3.73) nm. The H and 13C NMR (CDCl3,
Table 1) spectra of 1 showed signals assignable to four
methyls, a methylene, and three methine bearing the oxygen
function, an olefin, two benzoyl groups, and a nicotinoyl
group together with five methylenes (H2-5, 6, 9, 13, 14), a
methine (H-11), and five quaternary carbons (C-1, 4, 8, 12,
18). Treatment of 1 with 0.1% sodium methoxide (NaOMe)-
MeOH at room temperature furnished the desacyl derivative
nigellanol A (1a)4,5 together with methyl nicotinate and
methyl benzoate, which were identified by HPLC analysis.6
The dolabellane-type diterpene structure and positions of acyl
groups in 1 were constructed on the basis of various NMR
1
experiments.7 Thus, the H-1H COSY experiments on 1
indicated the presence of four partial structures in bold lines
as shown in Figure 1 (C-2-3, C-5-7, C-9-11, C-13-14).
Figure 2. NOE correlations of 1a and 3b.
the absolute stereostructure of 1 was determined by applica-
tion of the CD excitation chirality method for an allylic
benzoate.8 Thus, 1 was treated with 0.1% NaOMe-MeOH
at 0 °C to give the partial desacylated derivatives including
2-O-benzoylnigellanol A (1b).9 Compound 1b showed a
negative Cotton effect [246 nm (∆ꢀ -1.31) in MeOH], which
indicated the absolute configuration at the 2-position in 1b
to be S. On the basis of this evidence, the absolute
stereostructure of 1 was determined.
Nigellamine A2 (2),10 a white powder, [R]27D -24.2 (c )
1.00, CHCl3), C39H42N2O7, and its IR and UV spectra were
very similar to those of 1. The proton and carbon signals in
the 1H and 13C NMR (CDCl3, Table 1) spectra7 of 2 indicated
the presence of nigellanol A part, two nicotinoyl groups, and
a benzoyl group. The positions of the acyl groups in 2 were
determined by HMBC experiment as shown in Figure 1.
Treatment of 2 with 0.1% NaOMe-MeOH at room tem-
Figure 1. 1H-1H COSY and HMBC correlations of 1-4.
In the HMBC experiment of 1, long-range correlations were
observed between the following proton and quaternary carbon
pairs: H-2, 11, H2-15, and C-1; H-2, H2-5, H3-16, and C-4;
H-7, H2-9, H3-17, and C-8; H-11, H2-13, H3-19, 20, and C-12;
H3-19, 20 and C-18; H-2, H-6′, and C-7′; H-10, H-6′′, and
C-7′′; H2-15, H-2′′′,6′′′, and C-7′′′ (Figure 1). Next, the
relative stereostructures of 1 and 1a were confirmed by
(6) Methyl nicotinate (i) and methyl benzoate (ii) were identified by
HPLC analysis through comparison with standard samples obtained by
diazomethane methylation of commercial nicotinic acid and benzoic acid.
[tR (i) 5.58, (ii) 15.80 min, detection: UV (254 nm), column: YMC-Pack
ODS-5-A, 250 × 4.6 mm i.d., mobile phase: MeOH-H2O (60: 40, v/v);
flow rate 0.7 mL/min].
(7) The 1H and 13C NMR spectra of 1-4 were assigned with the aid of
1H-1H and 13C-1H COSY, DEPT, and HMBC experiments.
(8) Harada, N.; Iwabuchi, J.; Yokota, Y.; Uda, H.; Nakanishi, K. J. Am.
Chem. Soc. 1981, 103, 5590-5591.
(3) 1: high-resolution positive-ion FAB-MS calcd for C40H44NO7 (M
+ H)+ 650.3118, found 650.3123; CD (MeOH, ∆ꢀ) -1.63 (259 nm).
(4) 1a: high-resolution FAB-MS calcd for C20H32O4Na (M + Na)+
359.2198, found 359.2202; 1H NMR (CD3OD) δ 1.36, 1.70, 1.74 (3H each,
all s, H3-17, 19, 20), 1.51 (1H, dd, J ) 12.8, 13.8 Hz, Hâ-9), 1.62 (3H, d,
J ) 1.2 Hz, H3-16), 1.72 (1H, m, HR-6), 1.83 (1H, m, Hâ-6), 2.03 (1H, m,
Hâ-13), 2.12 (1H, m, HR-13), 2.19 (1H, dd, J ) 5.5, 13.8 Hz, HR-9), 2.27
(1H, m, HR-5), 2.29 (1H, br s, H-11), 2.32 (1H, m, Hâ-14), 2.37 (1H, ddd,
J ) 5.2, 12.6, 12.6 Hz, Hâ-5), 2.46 (1H, m, HR-14), 2.96 (1H, d, J )
ca.10 Hz, H-7), 3.90 (1H, d, J ) 10.4 Hz, H-2), 3.94, 4.00 (1H each, both
d, J ) 11.0 Hz, H2-15), 3.99 (1H, br dd, J ) ca. 6, 13 Hz, H-10), 5.37 (1H,
dd, J ) 1.2, 10.4 Hz, H-3); positive-ion FAB-MS m/z 359 (M + Na)+.
(5) Spartan (version ’02, Wavefunction, Inc., Irvine, CA) was used to
build and optimize the conformations of 1a and 3b (Figure 2) using MOPAC
(AM1) program. Those comformations were also supported by the NOE
correlations in the NOESY experiments, respectively.
(9) 1b: 1H NMR (CDCl3): δ 1.40, 1.70, 1.76 (3H each, all s, H3-17,
20, 19), 1.64 (1H, dd, J ) 12.8, 13.8 Hz, Hâ-9), 1.67 (1H, m, HR-6), 1.72
(1H, m, Hâ-14), 1.79 (3H, d, J ) 0.9 Hz, H3-16), 1.93 (1H, m, Hâ-6), 2.28
(1H, m, HR-14), 2.30 (2H, m, H2-5), 2.34 (2H, m, H2-13), 2.37 (1H, dd, J
) 5.2, 13.8 Hz, HR-9), 2.48 (1H, br s, H-11), 2.92 (1H, br d, J ) ca. 9 Hz,
H-7), 4.11 (1H, m, H-10), 4.14, 4.26 (1H each, both d, J ) 11.3 Hz, H2-
15), 5.28 (1H, dd, J ) 0.9, 10.4 Hz, H-3), 5.32 (1H, d, J ) 10.4 Hz, H-2),
7.45 (2H, dd, J ) 7.6, 8.3 Hz, H-3′, 5′), 7.57 (1H, tt, J ) 1.2, 7.6 Hz,
H-4′), 8.03 (2H, dd, J ) 1.2, 8.3 Hz, H-2′, 6′).
(10) 2: high-resolution FAB-MS calcd for C39H43N2O7 (M + H)+
651.3071, found 651.3065; CD (MeOH, ∆ꢀ) -1.42 (256 nm); UV (MeOH,
log ꢀ) 220 (4.50), 264 (3.82) nm; IR (KBr) 1717, 1647, 1636, 1592, 1541,
1509, 1456, 1279, 1109, 1024, 947, 741, 703 cm-1; Positive-ion FAB-MS
m/z 651 (M + H)+.
870
Org. Lett., Vol. 6, No. 6, 2004

a
Table 1. 1H (500 MHz) and 13C NMR (125 MHz) Data of Nigellamines A1 (1), A2 (2), B1 (3), and B2 (4) in CDCl3
1
2
3
4
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
1
2
3
4
5R
5â
56.5
73.1
56.6
73.7
54.8
54.9
74.0
122.0
142.2
38.1
5.45 d (10.4)
5.74 dd (0.9, 10.4)
5.48 d (10.3)
5.57 d (10.4)
73.4 5.59 d (10.7)
122.4 5.79 dd (0.9, 10.7)
141.7
38.1 2.36 br d (ca. 13)
2.47 ddd
123.8 5.73 d (10.3)
139.8
37.9
22.8
123.4 5.78 dd (0.9, 10.4)
140.3
37.9
22.8
2.39 br d (ca. 13)
2.47 ddd
2.38 m
2.48 ddd
2.35 br d (ca. 13)
2.47 ddd
(4.9, 12.9, 12.9)
1.72 m
2.01 m
(5.2,12.8,12.8)
1.65 m
2.02 d-like
3.07 br d (ca. 9)
(4.9, 12.9, 12.9)
1.67 m
2.00 m
(4.9, 12.9, 12.9)
23.0 1.66 m
6R
6â
7
23.0
2.02 m
3.08 br d (ca. 10)
65.5
58.6
42.0
65.4
58.6
42.0
2.99 br d (ca. 10)
66.9 2.99 br d (ca. 10)
58.8
66.9
58.7
41.0
8
9R
9â
10
11
12
13
2.59 dd (5.5, 13.8)
1.62 dd (12.5, 13.8)
5.70 br dd (ca. 6.13)
2.72 br s
2.59 dd (5.5, 13.7)
1.61 dd (12.5, 13.7)
5.70 br dd (ca. 6, 13)
2.71 br s
2.72 dd (5.8, 13.7)
1.59 dd (12.2, 13.7)
5.92 br dd (ca. 6, 12)
2.85 br s
41.0 2.71 dd (5.8, 13.4)
1.58 dd (12.8, 13.4)
74.0 5.92 br dd (ca. 6, 13)
51.8 2.84 br s
145.9
128.8 5.81 br s
38.3 2.74 dd (3.0, 17.0)
2.79 br d (ca. 17)
66.3 5.11 d (11.0)
5.28 d (11.0)
17.3 1.94 d (0.9)
17.2 1.52 s
82.5
*26.1 *1.49 s
*27.2 *1.51 s
129.9
129.7 9.09 br s
128.1
75.4
48.1
135.8
28.3
31.4
75.4
48.1
135.6
28.2
31.4
73.9
51.8
145.9
128.8
38.4
2.31 m (2H)
2.36 m (2H)
2.34 m
2.23 m
4.90 d (11.0)
5.31 d (11.0)
1.87 s
5.79 br s
2.78 br s (2H)
14R 2.34 m
14â 2.20 m
15
4.93 d (11.0)
67.2
67.0
5.12 d (11.0)
5.26 d (11.0)
1.94 d (0.9)
1.52 s
66.2
5.29 d (11.0)
1.87 d (0.9)
1.54 s
16
17
18
19
20
1′
2′
3′
4′
5′
16.5
18.4
127.1
22.6
21.8
130.0
16.7
18.4
127.3
22.6
21.8
125.9
17.3
17.2
82.6
*26.0
*27.2
125.9
150.9
1.53 s
1.66 s
1.88 s
1.67 s
1.88 s
*1.48 s
*1.51 s
7.84 dd (1.3, 8.3)
7.13 dd (7.6, 8.3)
7.44 tt (1.3, 7.6)
7.13 dd (7.6, 8.3)
7.84 dd (1.3, 8.3)
129.7 9.11 br s
128.1
132.8 8.65 br d (ca. 5)
128.1 6.98 dd (4.9, 8.0)
129.7 7.96 ddd
(1.8, 1.8, 8.0)
151.0 7.81 dd (1.3, 8.3)
7.12 dd (7.6, 8.3)
153.3 7.44 tt (1.3, 7.6)
122.9 7.12 dd (7.6, 8.3)
136.9 7.81 dd (1.3, 8.3)
132.9 8.66 br s
128.1 6.96 dd (4.9, 8.0)
129.7 7.93 ddd
(1.8, 1.9, 8.0)
153.2
122.9
137.0
6′
7′
1′′
2′′
4′′
166.5
125.9
165.1
125.8
166.5
126.0
165.2
125.8
150.8
153.6
123.6
9.24 br d (ca. 2)
8.78 br d (ca. 5)
7.41 dd (4.9, 8.0)
150.8 9.24 br s
153.6 8.79 br d (ca. 4)
123.5 7.42 m
150.8 9.21 br s
153.7 8.78 br s
123.6 7.42 dd (4.9, 8.0)
150.8 9.20 br s
153.6 8.74 br s
123.7 7.43 dd (4.9, 8.0)
5′′
6′′
8.31 ddd (1.9, 2.2, 8.0) 137.1 8.31 ddd (1.8, 1.8, 7.9) 137.1 8.32 ddd (1.8, 1.9, 8.0) 137.4 8.31 ddd (1.8, 1.9, 8.0) 137.4
7′′
164.4
130.3
164.4
130.1
165.1
130.2
165.1
130.1
129.9
128.6
133.3
128.6
129.9
166.4
1′′′
2′′′
3′′′
4′′′
5′′′
6′′′
7′′′
8.14 dd (1.3, 8.3)
7.39 dd (7.6, 8.3)
7.58 tt (1.3, 7.6)
7.39 dd (7.6, 8.3)
8.14 dd (1.3, 8.3)
130.1 8.10 br d (ca. 8)
128.4 7.39 dd (7.8, 7.9)
133.1 7.58 br t (ca. 8)
128.4 7.39 dd (7.8, 7.9)
130.1 8.10 br d (ca. 8)
166.6
129.9 8.14 dd (1.3, 8.3)
128.5 7.40 dd (7.7, 8.3)
133.3 7.60 tt (1.3, 7.7)
128.5 7.40 dd (7.7, 8.3)
129.9 8.14 dd (1.3, 8.3)
166.5
130.1 8.11 dd (1.3, 8.3)
128.5 7.42 dd (7.6, 8.3)
133.1 7.60 tt (1.3, 7.6)
128.5 7.42 dd (7.6, 8.3)
130.1 8.11 dd (1.3, 8.3)
166.6
a Entries marked with an asterisk may be interchangeable within the same column.
perature furnished 1a, methyl nicotinate, and methyl ben-
Nigellamine B1 (3), a white powder, [R]27 +20.2 (c )
D
zoate,6 so that the absolute stereostructure of 2 was elucidated
as the same as that of 1. Moreover, treatment of 2 with 0.1%
NaOMe-MeOH at 0 °C gave the partial desacylated
derivatives including 2-O-nicotinoylnigellanol A (2a).11 The
CD spectrum of 2a showed a negative Cotton effect [264
nm (∆ꢀ -1.76) in MeOH]. This evidence suggested that
allylic benzoate rule is also applicable to the allyl nicotinate
moiety.
0.80, CHCl3), C40H43NO9, showed a quasimolecular ion peak
at m/z 682 (M + H)+ in positive-ion FAB-MS.12 The IR
spectrum of 3 showed absorption bands at 1725, 1653, 1592,
1541, 1507, 1420, 1281, 1111, 1024, 943, 741, and 702 cm-1
ascribable to ester carbonyl, olefin, and ether functions and
aromatic rings. In the UV spectrum of 3 (measured in
MeOH), absorption maxima were observed at 227 (log ꢀ
1
4.72) and 264 (3.91) nm. The H and 13C NMR (CDCl3,
Org. Lett., Vol. 6, No. 6, 2004
871

Table 1) spectra of 3 showed signals assignable to four
methyls, a methylene and three methine bearing the oxygen
function, two olefins, a nicotinoyl, two benzoyl groups6
together with four methylenes, a methine, and five quaternary
carbons. Treatment of 3 with triphenylphosphine (PPh3) gave
the 18-hydroxyl derivative (3a).13 The 18-carbon signal in
the 13C NMR spectrum of 3a (δC 71.5) was observed at a
higher field than that of 3 (δC 82.5). Treatment of 3a with
0.1% NaOMe-MeOH at room temperature gave the desacyl
derivative (3b).5,14 The relative stereostructures of 3, 3a, and
3b were confirmed by NOESY experiment on 3b, in which
NOE correlations were observed as shown in Figure 2.
Fourthermore, the partial desacyl derivatives including the
10,15-desacyl derivative (3c) were obtained by treatment of
3a with 0.1% NaOMe-MeOH at 0 °C.15 Compound 3c
showed a negative Cotton effect [229 nm (∆ꢀ -2.20) in
MeOH], which indicated the absolute configuration at the
2-position in 3c to be S. Consequently, the absolute stereo-
structure of 3 was determined.
(CDCl3) and 13C NMR (Table 1) spectra7 of 4 indicated the
presence of the same functional groups as 3, except for the
signals due to an acyl group.6 The positions of the acyl
groups in 4 were clarified by the HMBC experiment as
shown in Figure 1. Treatment of 4 with PPh3 furnished the
18-hydroxyl derivative (4a),17 and successive treatment of
4a with 0.1% NaOMe-MeOH at room temperature gave
3b, so that the absolute stereostructure of 4 was determined
to be the same as that of 3. Previously, several dolabellane-
type diterpenes were isolated from the marine soft coral,
brown algae, or liverwort;18 however, the isolation reports
of this type of diterpenes from the higher plants were very
rare. Until now, only one plant material has been reported
on the aerial part of Chrozophora oblique.19 Furthermore,
these dolabellane-type diterpenes are the first known to have
a nicotinic acid and/or hydroperoxyl group at the 18-position.
Effects of nigellamines (1-4) on stored triglyceride in
primary cultured mouse hepatocytes were examined,20 and
1 [inhibition (%) at 0.1 µM: 64 ( 4], 3 (70 ( 2%), and 4
(79 ( 2%) were found to show potent reduction of
triglyceride levels. Their activities were equivalent to that
of a hypolipidemic medicine, clofibrate (64 ( 5%).
Nigellamine B2 (4),16 colorless fine crystals (mp 139.5-
141.5 °C from MeOH), [R]27 +20.0 (c ) 1.10, CHCl3),
D
C39H42N2O9 and its IR and UV spectra were very similar to
1
those of 3. The proton and carbon signals in the H NMR
Supporting Information Available: 1H and 13C NMR
spectra of nigellamines (1-4) and experimental procedures.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(11) 2a: high-resolution EI-MS calcd for C26H35NO5 (M+) 441.2515,
found 441.2524; 1H NMR (CD3OD) δ 1.40, 1.73, 1.81 (3H each, all s,
H3-17, 20, 19), 1.62, 2.40 (1H each, both m, H2-9), 1.76, 1.87 (1H each,
both m, H2-6), 1.77 (3H, d, J ) 0.9 Hz, H3-16), 2.24-2.35 (4H, m, H2-13,
14), 2.48, 2.53 (1H each, both m, H2-5), 2.49 (1H, br s, H-11), 3.01 (1H,
d, J ) ca. 9 Hz, H-7), 4.04, 4.22 (1H each, both d, J ) 11.6 Hz, H2-15),
4.05 (1H, br dd, J ) ca. 6, 12 Hz, H-10), 5.39 (1H, d, J ) 10.7 Hz, H-2),
5.46 (1H, br d, J ) ca. 11 Hz, H-3), 7.59 (1H, dd, J ) 4.5, 7.6 Hz, H-5′),
8.40 (1H, br s, J ) ca. 8 Hz, H-6′), 8.76 (1H, br s, H-4′), 9.13 (1H, br s,
H-2′); EI-MS m/z 441 (M+, 2), 423 (M+ - H2O, 25), 121 (100).
(12) 3: high-resolution FAB-MS calcd for C40H44NO9 (M + H)+
682.3016, found 682.3013; CD (MeOH, ∆ꢀ) +4.98 (217 nm), -2.18 (236
nm); positive-ion FAB-MS m/z 682 (M + H)+.
OL036239C
(16) 4: high-resolution FAB-MS calcd for C39H43N2O9 (M + H)+
683.2969, found 683.2965; CD (MeOH, ∆ꢀ) +6.03 (221 nm), -0.64 (255
nm); UV (MeOH, log ꢀ): 221 (4.71), 264 (4.09) nm; IR (KBr) 1717, 1647,
1636, 1592, 1541, 1509, 1456, 1281, 1113, 1026, 941, 743, 714 cm-1
;
positive-ion FAB-MS m/z 683 (M + H)+.
(17) 4a: 1H NMR (CDCl3) δ 1.46, 1.51, 1.52 (3H each, all s, H3-19,
20, 17), 1.66 (1H, m, HR-6), 1.69 (1H, dd, J ) 12.8, 13.4 Hz, Hâ-9), 1.97
(3H, d, J ) 0.9 Hz, H3-16), 2.02 (1H, m, Hâ-6), 2.37 (1H, br d, J ) ca. 13
Hz, HR-5), 2.48 (1H, ddd, J ) 4.9, 12.8, 12.8 Hz, Hâ-5), 2.56 (1H, dd, J
) 5.8, 13.4 Hz, HR-9), 2.65 (2H, br s, H2-14), 2.88 (1H, br s, H-11), 2.99
(1H, br d, J ) ca. 10 Hz, H-7), 5.15, 5.28 (1H each, both d, J ) 11.0 Hz,
H2-15), 5.54 (1H, br s, H-13), 5.55 (1H, d, J ) 10.7 Hz, H-2), 5.80 (1H,
dd, J ) 0.9, 10.7 Hz, H-3), 6.29 (1H, br dd, J ) ca. 6, 13 Hz, H-10), 6.95
(1H, dd, J ) 4.9, 8.0 Hz, H-5′), 7.42 (2H, dd, J ) 7.6, 8.3 Hz, H-3′′′, 5′′′),
7.46 (1H, dd, J ) 4.9, 8.0 Hz, H-5′′), 7.60 (1H, tt, J ) 1.2, 7.6 Hz, H-4′′′),
7.92 (1H, ddd, J ) 1.8, 1.9, 8.0 Hz, H-6′), 8.11 (2H, dd, J ) 1.2, 8.3 Hz,
H-2′′′, 6′′′), 8.35 (1H, ddd, J ) 1.8, 1.9, 8.0 Hz, H-6′′), 8.66 (1H, br d, J
) ca. 5 Hz, H-4′), 8.82 (1H, br d, J ) ca. 5 Hz, H-4′′), 9.09 (1H, br s,
H-2′), 9.26 (1H, br s, H-2′′).
(13) 3a: 1H NMR (CDCl3) δ 1.46, 1.51, 1.52 (3H each, all s, H3-19,
20, 17), 1.67 (1H, m, HR-6), 1.70 (1H, dd, J ) 12.4, 13.5 Hz, Hâ-9), 1.97
(3H, d, J ) 1.1 Hz, H3-16), 2.01 (1H, m, Hâ-6), 2.36 (1H, m, HR-5), 2.45
(1H, ddd, J ) 4.9, 12.9, 12.9 Hz, Hâ-5), 2.55 (1H, dd, J ) 5.7, 13.5 Hz,
HR-9), 2.66 (2H, br s, H2-14), 2.88 (1H, br s, H-11), 2.99 (1H, br d, J )
ca.10 Hz, H-7), 5.16, 5.27 (1H each, both d, J ) 11.0 Hz, H2-15), 5.53
(1H, br s, H-13), 5.51 (1H, d, J ) 10.3 Hz, H-2), 5.79 (1H, dd, J ) 1.1,
10.3 Hz, H-3), 6.29 (1H, br dd, J ) ca. 6, 12 Hz, H-10), 7.09 (2H, dd, J
) 7.6, 8.3 Hz, H-3′, 5′), 7.40 (2H, dd, J ) 7.6, 8.3 Hz, H-3′′′, 5′′′), 7.42
(1H, tt, J ) 1.3, 7.6 Hz, H-4′), 7.43 (1H, dd, J ) 4.9, 8.0 Hz, H-5′′), 7.60
(1H, tt, J ) 1.3, 7.6 Hz, H-4′′′), 7.80 (2H, dd, J ) 1.3, 8.3 Hz, H-2′, 6′),
8.14 (2H, dd, J ) 1.3, 8.3 Hz, H-2′′′, 6′′′), 8.34 (1H, ddd, J ) 1.9, 1.9, 8.0
Hz, H-6′′), 8.78 (1H, br d, J ) ca. 5 Hz, H-4′′), 9.24 (1H, br s, H-2′′).
(14) 3b: 1H NMR (CDCl3) δ 1.33, 1.45, 1.52 (3H each, all s, H3-17,
19, 20), 1.49 (1H, dd, J ) 12.5, 13.5 Hz, Hâ-9), 1.61 (1H, m, HR-6), 1.69
(3H, d, J ) 0.9 Hz, H3-16), 1.92 (1H, m, Hâ-6), 2.28 (1H, m, HR-5), 2.37
(1H, ddd, J ) 4.6, 12.2, 12.2 Hz, Hâ-5), 2.35 (1H, dd, J ) 5.5, 13.5 Hz,
HR-9), 2.50 (1H, br s, H-11), [2.65 (1H, br d, J ) ca. 18 Hz), 2.79 (1H,
dd, J ) 3.4, 17.4 Hz), H2-14], 2.83 (1H, br d, J ) ca.10 Hz, H-7), 4.05
(1H, d, J ) 10.7 Hz, H-2), 4.16, 4.20 (1H each, both d, J ) 11.0 Hz,
H2-15), 4.51 (1H, br dd, J ) ca. 6, 13 Hz, H-10), 5.48 (1H, dd, J ) 0.9,
10.7 Hz, H-3), 5.75 (1H, br s, H-13).
(18) Costantino, V.; Fattorusso, E.; Mangoni, A.; Rosa, M. D.; Ianaro,
A.; Aknin, M.; Gaydou, E. M. Eur. J. Org. Chem. 1999, 227-230.
(19) Mohamed, K. M.; Ohtani, K.; Kasai, R.; Yamasaki, K. Phytochem-
istry 1994, 37, 495-500.
(20) (a) Hepatocytes were isolated from male ddY mice (ca. 40 g) using
the collagenase perfusion method.20b A cell suspension of 8 × 104 cells in
200 µL William’s E medium containing fetal calf serum (FCS, 10%),
penicillin (100 units/µL), and streptomycin (100 µg/mL) was incubated on
a 48-well tissue culture plate, and pre-cultured for 2 h at 37 °C under a 5%
CO2 atmosphere. An aliquot (200 µL) of the medium containing test sample
was added to the each well, and the cells were cultured for 20 h. The plate
was centrifuged (2000 rpm, 4 °C, 10 min), the supernatant was removed,
and 120 µL of distilled water was added to each well. The hepatocytes
were broken with sonication, and the suspension was centrifuged. To
determine the concentration of triglyceride in the supernatant using a
commercial kit (Triglyceride G-test wako), each test compound was
dissolved in DMSO, and the solution was added to the medium (final DMSO
concentration was 0.5%). (b) Seglen P. O. Methods Cell Biol. 1976, 13,
29-83.
(15) 3c: 1H NMR (CDCl3): δ 1.39, 1.50, 1.53, 1.86 (3H each, all s,
H3-17, 19, 20, 16), 1.66 (1H, m, HR-6), 1.70 (1H, dd, J ) 12.8, 13.4 Hz,
Hâ-9), 1.94 (1H, m, Hâ-6), 2.29 (1H, m, HR-5), 2.33 (1H, ddd, J ) 5.2,
12.8, 12.8 Hz, Hâ-5), 2.43 (1H, dd, J ) 5.2, 13.4 Hz, HR-9), 2.23 (1H, dd,
J ) 3.1, 17.6 Hz, HR-14), 2.73 (1H, br s, H-11), 2.86 (1H, br d, J ) ca.10
Hz, H-7), 2.93 (1H, br d, J ) ca. 18 Hz, Hâ-14), 4.11, 4.29 (1H each, both
d, J ) 11.6 Hz, H2-15), 4.67 (1H, br dd, J ) ca. 5, 13 Hz, H-10), 5.32 (1H,
d, J ) 10.4 Hz, H-3), 5.34 (1H, d, J ) 10.4 Hz, H-2), 5.72 (1H, br s,
H-13), 7.44 (2H, dd, J ) 7.6, 8.3 Hz, H-3′, 5′), 7.57 (1H, tt, J ) 1.2, 7.6
Hz, H-4′), 8.01 (2H, dd, J ) 1.2, 8.3 Hz, H-2′, 6′).
872
Org. Lett., Vol. 6, No. 6, 2004