Chemical transformation and biological activities of ambrein, a major product of ambergris from Physeter macrocephalus (Sperm Whale)

Article abstract of DOI:10.1021/np068029p

Ten new derivatives (2-11) of ambrein (1), isolated from ambergris, were prepared by chemical transformation. Oxidation and/or cyclization were effected by reactions with selenium oxide or p-toluenesulfonyl chloride or with the use of shortwave UV light.

Full text of DOI:10.1021/np068029p

J. Nat. Prod. 2007, 70, 147-153
147
Chemical Transformation and Biological Activities of Ambrein, a Major Product of Ambergris
from Physeter macrocephalus (Sperm Whale)
Ya-Ching Shen,*,† Shi-Yie Cheng,‡ Yao-Haur Kuo,§ Tsong-Long Hwang, Michael Y. Chiang,| and Ashraf Taha Khalil†
School of Pharmacy, College of Medicine, National Taiwan UniVersity, Taipei, Taiwan 100, Republic of China, Institute of Marine
Biotechnology and Resources, National Sun Yat-sen UniVersity, 70 Lien-Hai Road, Kaohsiung, Taiwan 80424, Republic of China, National
Research Institute of Chinese Medicine, Taipei, Taiwan, Republic of China, Graduate Institute of Natural Products, Chang Gung UniVersity,
Taoyuan 333, Taiwan, Republic of China, and Department of Chemistry, National Sun Yat-sen UniVersity, Taiwan, Republic of China
ReceiVed June 26, 2006
Ten new derivatives (2-11) of ambrein (1), isolated from ambergris, were prepared by chemical transformation. Oxidation
and/or cyclization were effected by reactions with selenium oxide or p-toluenesulfonyl chloride or with the use of
shortwave UV light. The structures of 2-12 were elucidated by spectroscopic analysis, with the structure and relative
configuration of 9 confirmed by single-crystal X-ray crystallography. The cytotoxic activities of 1-12 were investigated
against human liver carcinoma (Hepa59T/VGH), colon adenocarcinoma (WiDr), lung carcinoma (A-549), and human
breast adenocarcinoma (MCF-7) cell lines. The anti-inflammatory activities of 1-11, in terms of the inhibition of human
neutrophil function, were also evaluated.
Ambergris is a physiological product found either in the intestines
of the sperm whale, Physeter macrocephalus L. (Physeteridae), or
as a waste product discarded into the sea. It occurs in gray or brown
waxy masses, associated with the squid on which the whales feed.1,2
Since ancient times, ambergris has been one of the most highly
valued perfumery materials and is also used in tradititional medicine
to treat migraine headaches, rheumatism, and nervous diseases, and
as an aphrodisiac.3 Ambrein, a triterpene alcohol (1), is the major
constituent of ambergris, used as a perfume additive for the tenacity
or persistence of its odor,4 and has been reported to possess anti-
inflammatory, antinociceptive, and aphrodisiac activities.5 The anti-
inflammatory properties of ambrein (1) suggest the possible
interaction of this compound and prostaglandins in the mediation
of pain.6 Oxidative degradation of ambrein (1), as a result of
exposure of ambergris to air and sunlight, is responsible for the
organoleptic properties of this compound, as it leads to formation
of the odorous compound Ambrox (13), with wide industrial
applications in perfumery.7 In view of the medicinal potential of
ambrein, we have prepared 10 new derivatives (2-11), in addition
to the known compound 12. Allylic oxidation is a useful tool for
preparation of many pharmaceuticals, dyes, agricultural products,
and polyester monomers.8 Compounds 2-8 were produced as
products of SeO2-catalyzed allylic oxidation and benzoylation, while
9 and 10 are products of cyclization with toluenesulfonyl chloride
(p-TsCl), and 11 and 12 are products of photooxidation by UV
light (δ 254 nm). The structures of 2-11 were elucidated through
spectroscopic data analysis, and single-crystal X-ray crystallography
in the case of 9. The cytotoxic activities of 1-12 were investigated
against four human cancer cell lines. In addition, the anti-
inflammatory activities of 1-11 were tested on elastase release by
inhibition of human neutrophils.
13C NMR spectrum displayed resonances for one oxygenated
quaternary carbon (δC 74.3), an olefinic CH (δC 124.7), a quaternary
olefinic carbon (δC 135.9), and an exomethylene carbon (δC 108.9).
The identity of 1 was confirmed as E-ambrein through mass
spectrometry (elemental formula C30H52O) and from its NMR
spectroscopic data, which were consistent with literature values.9
Treatment of ambrein (1) with selenium dioxide in dioxane at room
temperature (Scheme 1),10,11 followed by chromatographic separa-
tion, afforded four selective oxidation products: 15R-hydroxyam-
brein (2) (7.5%), 15â-hydroxyambrein (3) (7.6%), 27-hydroxyam-
brein (4) (4.9%), and 15-oxoambrein (5) (0.75%).
Compounds 2 and 3 were both assigned the same molecular
formula, C30H52O2. The NMR spectroscopic data of 2 and 3 were
quite similar to those of ambrein (1) except for the replacement of
the C-15 methylene group by two oxymethines, resonating at δH
3.91, δC 75.1 in 2 and at δH 4.48, δC 67.7 in 3. In each case, H-15
was 3J-correlated with C-13, C-17, and C-27, confirming hydroxy-
lation at C-15. The difference in the chemical shifts of H-15 and
C-15 suggested that 2 and 3 possess different configurations at C-15.
Mosher’s method for the determination of the configuration of
secondary alcohols was applied to 2 and 3 in order to distinguish
between these two compounds.12 In the case of 2, the differences
between the (R)-MTPA and (S)-MTPA esters (∆δ ) δS - δR) are
summarized in Figure 1a and indicated the S-configuration at C-15.
On the other hand, the values for (∆δ ) δS - δR) for 3 were
indicative of the R-configuration at C-15 (Figure 1b).
Compound 4 exhibited a molecular formula of C30H52O2, as
determined by HRESIMS (m/z 427.3943). Its NMR data were
similar to those of ambrein (1), but with the disappearance of the
C-27 methyl group signal and its replacement by an oxymethylene
signal at δC 66.3 in 4. The oxymethylene protons at δH 4.03 (2H,
s, H-27) showed HMBC correlations with the quaternary carbon
at δ 139.5 (C-14), the olefinic methine carbon at δC 126.8 (C-13),
and the methylene carbon at δC 30.7 (C-15), thus confirming
hydroxylation at C-27. Compound 4 was assigned the structure 27-
hydroxyambrein (yield 4.9%).
The FABMS of 5 revealed a [M + Na]+ ion peak at m/z 465,
corresponding to the molecular formula C30H50O2. The 13C NMR
data of 5 displayed a low-field carbonyl signal at δC 201.8, in
addition to 11 CH2 signals, instead of 12 as in 1. This carbonyl
was 3J-correlated to an olefinic proton at δH 6.69 (H-13), a methyl
signal at δH 1.76 (H-27), and a methine at δH 2.65 (H-17), and this
was accompanied by a downfield shift of H-13 (∆δH 1.53), H-16
(∆δH ) 1.13 ppm), and C-16 (∆δC ) 10.7 ppm), in comparison to
Results and Discussion
The 1H NMR spectrum of 1 revealed the presence of signals for
seven methyls, one olefinic CH (δH 5.16, t, J ) 6.4 Hz, H-13),
and two exomethylene singlets at δH 4.75 and 4.54 (H2-30). The
* Author to whom correspondence should be addressed. Tel: 886-02-
3123456, ext. 2226. Fax: 886-02-23919098. E-mail: ycshen@ntu.edu.tw.
† National Taiwan University.
‡ Institute of Marine Biotechnology and Resources, National Sun Yat-
Sen University.
§ National Research Institute of Chinese Medicine.
Graduate Institute of Natural Products, Chang Gung University.
| Department of Chemistry, National Sun Yat-sen University.
10.1021/np068029p CCC: $37.00
© 2007 American Chemical Society and American Society of Pharmacognosy
Published on Web 01/19/2007

148 Journal of Natural Products, 2007, Vol. 70, No. 2
Shen et al.
Scheme 1
1. These findings indicated the presence of a carbonyl at C-15,
and consequently 5 was identified as 15-oxoambrein.
of 9, C30H50O, was determined from HRESIMS and NMR data.
The calculated six degrees of unsaturation suggested the formation
of an extra ring, relative to 1 (C30H52O). The oxymethine proton at
δH 4.12 (dd, J ) 9.9, 1.2 Hz, H-13) showed HMBC correlations
with the methylene carbon at δC 15.9 (C-12) and the methyl carbon
at δC 12.4 (C-27). The olefinic proton at δH 5.34 (t, J ) 6.6 Hz,
H-15) had HMBC correlations with C-16 and C-27, in addition to
a NOESY correlation with H-17. These findings, together with
COSY NMR spectoscopic data (Figure 2), led to the conclusion
that reaction with TsCl introduced an ether linkage between C-8
and C-13, thus forming a tetrahydropyran ring that was associated
with rearrangement of the double-bond position from C-13 and C-14
to C-14 and C-15. The NOESY correlation between H-13 and H-26
implied a â-orientation. A single-crystal X-ray diffraction analysis
The benzoyl esters of 2-4 were prepared by reaction with
benzoyl chloride to yield 6-8, respectively, as shown in Scheme
1. The NMR spectra revealed the presence of characteristic benzoyl
group signals (Experimental Section). In addition, benzoylation
induced significant downfield shifts of H-15 (∆δH ) 1.82 ppm in
6 and ∆δH ) 0.90 ppm in 7) and H-27 (∆δH ) 0.22 ppm). The
structures of these derivatives were confirmed by the HMBC
correlations of the benzoyl carbonyl carbon signal in each case with
H-15 (in 6 and 7) or H-27 (in 8).
Reaction of 3 with p-toluenesulfonyl chloride (TsCl)2,13 afforded
the cyclized products 9 and 10 (Scheme 2). A preferential SN2
reaction proceeded through a nucleophilic attack that involved
inversion of the orientation at C-15 in 10.14 The molecular formula

Chemical Transformation and Biological ActiVities of Ambrein
Journal of Natural Products, 2007, Vol. 70, No. 2 149
Figure 3. Perspective drawing of the X-ray structure of 9.
Figure 4. Key HMBC (arrows), NOESY (curves), and COSY (bold
lines) correlations of 10.
Scheme 3
Figure 1. ∆δ values obtained for the MTPA esters of (a) 2 and
(b) 3.
Figure 2. Selected HMBC (arrows), NOESY (curves), and COSY
(bold lines) correlations of 9.
Scheme 2
the presence of an oxygen bridge between C-8 and C-15. This
cyclization afforded the E-form of an eight-membered tetrahydro-
oxocine ring, which was supported by the COSY NMR spectrum
(Figure 4), and proved connectivities between H-11, H-12, and H-13
and between H-15, H-16, and H-17. The strong NOESY correlation
between H-15 and the C-26 methyl protons was consistent with
the â-configuration of H-15.
Photooxidation of 1 using UV light (254 nm)15 afforded 11 and
the degradation product 129 (Scheme 3). The NMR data of 11
(molecular formula C30H52O2) indicated the absence of olefinic
signals attributable to C-13 and C-14 in 1 and revealed the presence
of an oxymethine at δH 3.38 (dd, J ) 9.9, 2.0 Hz, H-13) and δC
75.2 (C-13), in addition to an oxygenated quaternary carbon at δC
74.9 (C-14). The oxymethine signal at δH 3.38 was correlated to
C-8, C-15, and C-27 in the HMBC spectrum. In addition, the H-27
protons exhibited a HMBC correlation with the oxyquaternary
carbon at δC 74.9 (C-14), confirming hydroxylation at C-14 and
formation of a tetrahydropyran ring as in 9. The strong NOESY
(Figure 3) was used to confirm the assignment of the structure 9,
which possesses an unusual ring C in the molecule.
Compound 10 exhibited the same molecular formula as 9, but
different NMR data, indicating a different cyclization pattern. The
oxymethine proton at δH 3.96 (dd, J ) 9.2, 0.9 Hz, H-15) exhibited
HMBC correlations with C-13, C-16, and, notably, C-8, suggesting

150 Journal of Natural Products, 2007, Vol. 70, No. 2
Shen et al.
Table 1. Cytotoxicity of Compounds 1-12 against Human
Tumor Cells (ED50, µg/mL)a
Pharmacia Biotech AB, Uppsala, Sweden) was used for separation.
S-(+)-R-Methoxy-R-trifluoromethylphenylacetyl chloride and R-(-)-
R-methoxy-R-trifluoromethylphenylacetyl chloride (MTPA esters) were
obtained from ACROS Organics (Geel, Belgium).
Animal Material. Ambergris, a secretion from the sperm whale
Physeter macrocephalus L., was purchased from a local drug store in
Kaohsiung, Taiwan, in June 2001. A voucher specimen (Pm-3) was
deposited at the School of Pharmacy, National Taiwan University,
Taipei, Taiwan.
Extraction and Isolation of Ambrein (1). The ambergris sample
(20 g) was dissolved in n-hexane and applied to the top of a silica gel
column (230-400 mesh), eluted with n-hexane-EtOAc (10:1), to give
four fractions (F-1 to F-4). Fraction F-4 was purified on a silica gel
column using n-hexane-EtOAc (50:1) to yield ambrein (1, 4.8 g, yield
24.2%).
compound
hepa59T/VGHb
WiDrc
A-549d
MCF-7e
2
5
5.7
6.8
7.8
10
6.4
5.3
4.0
8.5
doxorubicin
0.11
0.12
0.21
0.12
a Concentration that inhibits 50% of the growth of human tumor
cell lines after 72 h exposure.19 b Human liver carcinoma. c Human
colon adenocarcinoma. d Human lung carcinoma. e Human breast ad-
enocarcinoma. f Compounds 1, 3, 4, and 6-12 were inactive (ED50
10 µg/mL).
>
Table 2. Anti-inflammatory Effect of Compounds 1-11 and
Genistein (10 µg/mL) on Elastase Release as Tested on Human
Neutrophilsa
(+)-Ambrein (1R,2R,4aS,8aS,3′E,1′′S)-(+)-1-[6′-(2′′,2′′-Dimethyl-
6′′-methylenecyclohexyl)-4′-methylhex-3′-enyl]-2,5,5,8a-tetrameth-
yldecahydronaphthalen-2-ol (1): colorless, needle-shaped crystals; mp
80-82 °C (lit. 81.5-82.5 °C); [R]D +17.2 (c 0.2, CH2Cl2); 13C NMR
(CDCl3, 75 MHz) δ 39.8 (t, C-1), 24.9 (t, C-2), 42.1 (t, C-3), 33.2 (s,
C-4), 56.2 (d, C-5), 18.5 (t, C-6), 44.6 (t, C-7), 74.3 (s, C-8), 61.5 (d,
C-9), 39.2 (s, C-10), 25.6 (t, C-11), 31.5 (t, C-12), 124.7 (d, C-13),
135.9 (s, C-14), 38.3 (t, C-15), 23.8 (t, C-16), 53.7 (d, C-17), 35.0 (s,
C-18), 36.4 (t, C-19), 20.6 (t, C-20), 32.6 (t, C-21), 149.4 (s, C-22),
15.5 (q, C-23), 33.3 (q, C-24), 21.6 (q, C-25), 23.8 (q, C-26), 16.4 (q,
C-27), 26.3 (q, C-28), 28.5 (q, C-29), 108.9 (t, C-30); EIMS m/z 428
[H]+; FABMS m/z 451 [M + Na]+, 429 [M + H]+, C30H52O.
Reaction of 1 with SeO2. Selenium dioxide (885 mg, 7.90 mmol)
was added to a solution of 1 (3.38 g, 7.90 mmol) in dry 1,4-dioxane
(10 mL), and the reaction mixture was stirred at room temperature for
1 h. Water was then added, and the product was extracted with CH2-
Cl2. The CH2Cl2 layer was washed with saturated NaHCO3 solution
and dried over anhydrous Na2SO4. The solvent was evaporated and
the residue (3.98 g) was chromatographed on a silica gel column (70
g) using a gradient of CH2Cl2-MeOH for elution to afford 1 (665 mg),
a mixture M1, 4 (164 mg, yield 4.9%), 2 (100 mg, total yield 7.5%),
and a mixture M2. M1 (302 mg) was separated on a silica gel column
using n-hexane-EtOAc (100:0 to 1:1) to yield 5 (25 mg, yield 0.7%).
A similar fractionation of M2 (589 mg) yielded 2 (54 mg), a 5:6 mixture
of 2 and 3 (223 mg, 1H NMR integration), and 3 (135 mg, total
yield 7.6%).
compound
% of control
1
2
3
4
5
6
7
8
4.6 ( 1.10b
132.7 ( 2.50
79.3 ( 5.66
74.4 ( 6.68
95.1 ( 4.63
115.5 ( 7.18
85.6 ( 7.08
75.6 ( 5.52
92.2 ( 5.62
82.0 ( 6.99
81.3 ( 4.18
51.6 ( 5.89
9
10
11
genisteinc
a Results are presented as mean ( SEM (n ) 3), p < 0.05. DMSO
was used as negative control (100%). b Concentration necessary for 50%
inhibition. c Positive control.
correlations between H-3 (δH 3.38) and H-26 (δH 1.26) and H-27
(δH 1.11) were consistent with the â-orientation of H-13 and
R-orientation of the OH group at C-14. These data are in accordance
with the structure assigned to 11. Compound 12 was determined
as 4-(2,2-dimethyl-6-methylenecyclohexyl)butan-2-one by spec-
troscopic comparison with literature data.9 Notably, Ambrox (13)
was not obtained under these conditions.
The in vitro cytotoxic activities of 1-12 were investigated against
human liver carcinoma (Hepa59T/VGH), human colon adenocar-
cinoma (WiDr), human lung carcinoma (A-549), and human breast
adenocarcinoma (MCF-7) cell lines, using the MTT assay. The
results are shown in Table 1.16 Among the compounds tested, 2
and 5 exhibited moderate cytotoxic activity against all four tumor
cell lines.
Preparation of (R)- and (S)-MTPA Esters of 2 and 3. To a solution
of 2 or 3 (2 mg in 0.5 mL of pyridine) was added S-(+)- or R-(-)-
MTPA chloride (one drop), and the solution was allowed to stand at
room temperature for 7 h. After purification using preparative TLC,
the ester (1.8 mg, 90% yield) was analyzed by 1H NMR spectroscopic
measurement, and ∆δ ) δS - δR was calculated separately for 2
and 3.
(1R,2R,4aS,8aS,3′E,5′S,1′′S)-(+)-1-[6′-(2′′,2′′-Dimethyl-6′′-meth-
ylenecyclohexyl)-4′-methyl-5′-hydroxyhex-3′-enyl]-2,5,5,8a-tetram-
ethyldecahydronaphthalen-2-ol (2): colorless crystals; mp 145 °C;
[R]D +20 (c 0.2, CH2Cl2); IR (CH2Cl2) νmax 3420 (OH), 2928 (CH),
1647 (double bond), 1124 (C-O) cm-1; 1H NMR (CDCl3, 300 MHz)
δ 1.71 (1H, m, H-1a), 1.67 (1H, m, H-1b), 1.54 (2H, m, H-2), 1.39
(1H, m, H-3a), 1.16 (1H, m, H-3b), 0.87 (1H, m, H-5), 1.58 (1H, m,
H-6a), 1.42 (1H, m, H-6b), 1.86 (2H, m, H-7), 1.03 (1H, m, H-9), 1.47
(1H, m, H-11a), 1.33 (1H, m, H-11b), 2.07 (2H, m, H-12), 5.39 (1H,
t, J ) 6.6 Hz, H-13), 3.91 (1H, dd, J ) 9.6, 0.9 Hz, H-15), 2.05 (1H,
m, H-16a), 1.59 (1H, m, H-16b), 2.03 (1H, m, H-17), 1.48 (1H, m,
H-19a), 1.25 (1H, m, H-19b), 1.67 (1H, m, H-20a), 1.24 (1H, m, H-20b),
2.03 (2H, m, H-21), 0.77 (3H, s, H-23), 0.84 (3H, s, H-24), 0.77 (3H,
s, H-25), 1.11 (3H, s, H-26), 1.60 (3H, s, H-27), 0.82 (3H, s, H-28),
0.92 (3H, s, H-29), 4.78 (1H, s, H-30a), 4.60 (1H, s, H-30b); 13C NMR
(CDCl3, 75 MHz) δ 39.7 (t, C-1), 23.6 (t, C-2), 42.0 (t, C-3), 33.2 (s,
C-4), 56.1 (d, C-5), 18.4 (t, C-6), 44.4 (t, C-7), 74.1 (s, C-8), 61.2 (d,
C-9), 39.1 (s, C-10), 25.2 (t, C-11), 30.8 (t, C-12), 125.7 (d, C-13),
138.1 (s, C-14), 75.1 (d, C-15), 32.5 (t, C-16), 49.8 (d, C-17), 34.6 (s,
C-18), 36.2 (t, C-19), 20.5 (t, C-20), 32.5 (t, C-21), 149.5 (s, C-22),
15.3 (q, C-23), 33.3 (q, C-24), 21.4 (q, C-25), 23.8 (q, C-26), 12.2 (q,
C-27), 26.1 (q, C-28), 28.3 (q, C-29), 109.2 (t, C-30); EIMS m/z 444
[M]+; FABMS m/z 467 [M + Na]+, 427 [M - OH]+, 409 [M - H2O
- OH]+; HRESIMS m/z 427.3940 (calcd for C30H51O, 427.3940).
(1R,2R,4aS,8aS,3′E,5′R,1′′S)-(-)-1-[6′-(2′′,2′′-Dimethyl-6′′-meth-
ylenecyclohexyl)-4′-methyl-5′-hydroxyhex-3′-enyl]-2,5,5,8a-tetram-
The in vitro anti-inflammatory activities of 1-11 were also
screened by observation of elastase release as tested on human
neutrophils (Table 2). Genistein was used as a positive control
compound. Compounds 3, 4, and 8 exhibited weak anti-inflamma-
tory activities as compared to compound 1, which showed the most
potent anti-inflammatory activity at 10 µg/mL. In contrast, com-
pounds 2 and 6 possessed inflammatory effect (over 100%) under
the same conditions.
Experimental Section
General Experimental Procedures. Optical rotations were recorded
on a JASCO DIP-1000 polarimeter. IR and UV spectra were measured
on Hitachi T-2001 and Hitachi U-3210 spectrophotometers, respectively.
The 1H, 13C NMR, COSY, HMQC, HMBC, and NOESY spectra were
recorded on a Bruker FT-300 or a Varian Unity INOVA FT-500 NMR
1
spectrometer at 500 MHz for H and 125 MHz for 13C, respectively,
using TMS as internal standard. Chemical shifts are given in δ (ppm)
and coupling constants in Hz. Low-resolution EIMS and FABMS were
recorded on a VG Quattro 5022 mass spectrometer. HRMS were
recorded on a Finnigan MAT-95XL mass spectrometer. An UVGL-25
mineral light lamp (Upland, CA) operating at 254 nm was used for
photoreaction. Silica gel 60 (Merck) was used for column chromatog-
raphy, and precoated silica gel plates (Merck, Kieselgel 60 F-254, 1
mm) were used for preparative TLC. Sephadex LH-20 (Amersham

Chemical Transformation and Biological ActiVities of Ambrein
Journal of Natural Products, 2007, Vol. 70, No. 2 151
ethyldecahydronaphthalen-2-ol (3): amorphous solid; [R]D -21 (c
0.2, CH2Cl2); IR (CH2Cl2) νmax 3420 (OH), 2938 (CH), 1647 (double
(1R,2R,4aS,8aS,3′E,5′S,1′′S)-(+)-1-[6′-(2′′,2′′-Dimethyl-6′′-meth-
ylenecyclohexyl)-4′-methyl-5′-benzoyloxyhex-3′-enyl]-2,5,5,8a-tet-
ramethyldecahydronaphthalen-2-ol (6): amorphous solid; [R]D +60.4
(c 0.2, CH2Cl2); UV λmax (MeOH) (log ꢀ) 238 (4.53), 272 (3.39) nm;
IR (CH2Cl2) νmax 3502 (OH), 2933, 2866 (CH), 1718 (CdO, ester),
1
bond), 1124 (C-O) cm-1; H NMR (CDCl3, 300 MHz) δ 1.65 (2H,
m, H-1), 1.54 (2H, m, H-2), 1.41 (2H, m, H-3), 0.95 (1H, m, H-5),
1.61 (1H, m, H-6a), 1.43 (1H, m, H-6b), 1.88 (2H, m, H-7), 1.04 (1H,
m, H-9), 1.52 (1H, m, H-11a), 1.34 (1H, m, H-11b), 2.09 (2H, m, H-12),
5.23 (1H, t, J ) 7.0 Hz, H-13), 4.48 (1H, br d, J ) 8.9 Hz, H-15),
2.20 (1H, m, H-16a), 2.06 (1H, m, H-16b), 2.08 (1H, m, H-17), 1.72
(2H, m, H-19), 1.54 (1H, m, H-20a), 1.30 (1H, m, H-20b), 2.10 (2H,
m, H-21), 0.77 (3H, s, H-23), 0.87 (3H, s, H-24), 0.78 (3H, s, H-25),
1.13 (3H, s, H-26), 1.72 (3H, s, H-27), 0.85 (3H, s, H-28), 0.95 (3H,
s, H-29), 4.83 (1H, s, H-30a), 4.64 (1H, s, H-30b); 13C NMR (CDCl3,
75 MHz) δ 39.9 (t, C-1), 23.8 (t, C-2), 42.0 (t, C-3), 33.5 (s, C-4),
56.1 (d, C-5), 18.6 (t, C-6), 44.6 (t, C-7), 74.3 (s, C-8), 60.9 (d, C-9),
39.1 (s, C-10), 25.8 (t, C-11), 29.8 (t, C-12), 127.2 (d, C-13), 138.0 (s,
C-14), 67.7 (d, C-15), 30.3 (t, C-16), 49.9 (d, C-17), 34.7 (s, C-18),
36.2 (t, C-19), 20.6 (t, C-20), 32.2 (t, C-21), 149.8 (s, C-22), 15.4 (q,
C-23), 33.3 (q, C-24), 21.6 (q, C-25), 24.0 (q, C-26), 18.2 (q, C-27),
26.4 (q, C-28), 28.4 (q, C-29), 109.3 (t, C-30); EIMS m/z 426 [M -
H2O]+; FABMS m/z 409 [M - H2O - OH]+; HRESIMS m/z 427.3937
(calcd for C30H51O, 427.3940).
1
1646 (double bond), 1276 (C-O, ester), 1118 (C-O), 711 cm-1; H
NMR (CDCl3, 300 MHz) δ 1.75 (2H, m, H-1), 1.60 (2H, m, H-2),
1.35 (1H, m, H-3a), 1.16 (1H, m, H-3b), 0.84 (1H, m, H-5), 1.60 (1H,
m, H-6a), 1.42 (1H, m, H-6b), 1.88 (2H, m, H-7), 1.08 (1H, m, H-9),
1.42 (2H, m, H-11), 2.56 (1H, m, H-12a), 2.13 (1H, m, H-12b), 5.26
(1H, t, J ) 6.2 Hz, H-13), 5.73 (1H, dd, J ) 9.0, 1.5 Hz, H-15), 2.10
(1H, m, H-16a), 1.60 (1H, m, H-16b), 1.96 (1H, m, H-17), 1.51 (1H,
m, H-19a), 1.26 (1H, m, H-19b), 1.63 (1H, m, H-20a), 1.30 (1H, m,
H-20b), 2.10 (2H, m, H-21), 0.77 (3H, s, H-23), 0.80 (3H, s, H-24),
0.77 (3H, s, H-25), 1.16 (3H, s, H-26), 1.72 (3H, s, H-27), 0.90 (3H,
s, H-28), 0.94 (3H, s, H-29), 4.82 (1H, s, H-30a), 4.50 (1H, s, H-30b),
8.07 (2H, d, J ) 7.5 Hz, H-3′,7′), 7.46 (2H, t, J ) 7.5 Hz, H-4′,6′),
7.57 (1H, t, J ) 7.5 Hz, H-5′); 13C NMR (CDCl3, 75 MHz) δ 39.9 (t,
C-1), 23.6 (t, C-2), 42.1 (t, C-3), 33.4 (s, C-4), 56.0 (d, C-5), 18.6 (t,
C-6), 44.6 (t, C-7), 74.2 (s, C-8), 60.0 (d, C-9), 39.0 (s, C-10), 25.0 (t,
C-11), 30.1 (t, C-12), 129.7 (d, C-13), 133.6 (s, C-14), 72.6 (d, C-15),
30.5 (t, C-16), 50.0 (d, C-17), 34.7 (s, C-18), 36.2 (t, C-19), 20.7 (t,
C-20), 32.5 (t, C-21), 148.4 (s, C-22), 15.5 (q, C-23), 33.4 (q, C-24),
21.6 (q, C-25), 23.6 (q, C-26), 18.4 (q, C-27), 26.3 (q, C-28), 28.4 (q,
C-29), 110.1 (t, C-30), 165.9 (s, C-1′), 130.8 (s, C-2′), 129.7 (d, C-3′,7′),
128.4 (d, C-4′,6′), 132.8 (d, C-5′); EIMS m/z 426; FABMS m/z 531
[M - OH]+, 409 [M - PhCOOH - OH]+; HRESIMS m/z 531.4201
(calcd for C37H55O2, 531.4202).
(1R,2R,4aS,8aS,3′E,5′R,1′′S)-(+)-1-[6′-(2′′,2′′-Dimethyl-6′′-meth-
ylenecyclohexyl)-4′-methyl-5′-benzoyloxyhex-3′-enyl]-2,5,5,8a-tet-
ramethyldecahydronaphthalen-2-ol (7): amorphous solid; [R]D -20.3
(c 0.2, CH2Cl2); UV λmax (MeOH) (log ꢀ) 238 (4.51), 273 (3.13) nm;
IR (CH2Cl2) νmax 3502 (OH), 2932 (CH), 1715 (CdO, ester), 1646
(double bond), 1601 (aromatic), 1276 (C-O, ester), 1113 (C-O), 711
cm-1; 1H NMR (CDCl3, 300 MHz) δ 1.72 (2H, m, H-1), 1.56 (2H, m,
H-2), 1.38 (1H, m, H-3a), 1.14 (1H, m, H-3b), 0.89 (1H, m, H-5), 1.46
(2H, m, H-6), 1.88 (2H, m, H-7), 1.07 (1H, m, H-9), 1.44 (2H, m,
H-11), 2.20 (2H, m, H-12), 5.55 (1H, t, J ) 7.3 Hz, H-13), 5.38 (1H,
t, J ) 7.2 Hz, H-15), 1.93 (2H, m, H-16), 1.75 (1H, m, H-17), 1.54
(1H, m, H-19a), 1.33 (1H, m, H-19b), 1.71 (2H, m, H-20), 2.25 (1H,
m, H-21a), 2.02 (1H, m, H-21b), 0.78 (3H, s, H-23), 0.85 (3H, s, H-24),
0.78 (3H, s, H-25), 1.14 (3H, s, H-26), 1.70 (3H, s, H-27), 0.85 (3H,
s, H-28), 0.93 (3H, s, H-29), 4.82 (1H, s, H-30a), 4.72 (1H, s, H-30b),
8.04 (2H, d, J ) 7.2 Hz, H-3′,7′), 7.43 (2H, t, J ) 7.2 Hz, H-4′,6′),
7.54 (1H, t, J ) 7.2 Hz, H-5′); 13C NMR (CDCl3, 75 MHz) δ 39.8 (t,
C-1), 23.8 (t, C-2), 41.9 (t, C-3), 33.2 (s, C-4), 56.1 (d, C-5), 18.4 (t,
C-6), 44.7 (t, C-7), 74.1 (s, C-8), 61.3 (d, C-9), 39.1 (s, C-10), 25.0 (t,
C-11), 31.0 (t, C-12), 131.4 (d, C-13), 131.5 (s, C-14), 80.4 (d, C-15),
28.7 (t, C-16), 50.1 (d, C-17), 35.0 (s, C-18), 37.2 (t, C-19), 20.6 (t,
C-20), 33.2 (t, C-21), 148.4 (s, C-22), 15.4 (q, C-23), 33.4 (q, C-24),
21.5 (q, C-25), 23.8 (q, C-26), 11.3 (q, C-27), 25.3 (q, C-28), 28.5 (q,
C-29), 109.4 (t, C-30), 165.7 (s, C-1′), 131.1 (s, C-2′), 129.5 (d, C-3′,7′),
128.2 (d, C-4′,6′), 132.6 (d, C-5′); EIMS m/z 426, 408; FABMS m/z
531 [M - OH]+; HRESIMS m/z 531.4200 (calcd for C37H55O2,
531.4202).
(1R,2R,4aS,8aS,3′E,1′′S)-(+)-1-[6′-(2′′,2′′-Dimethyl-6′′-methyle-
necyclohexyl)-4′-hydroxymethylhex-3′-enyl]-2,5,5,8a-tetramethyl-
decahydronaphthalen-2-ol (4): amorphous solid; [R]D +20.4 (c 0.2,
CH2Cl2); IR (CH2Cl2) νmax 3420 (OH), 2928 (CH), 1646 (double bond),
1134 (C-O) cm-1; 1H NMR (CDCl3, 300 MHz) δ 1.63 (2H, m, H-1),
1.54 (2H, m, H-2), 1.43 (2H, m, H-3), 0.95 (1H, m, H-5), 1.45 (2H,
m, H-6), 1.87 (2H, m, H-7), 1.05 (1H, m, H-9), 1.30 (2H, m, H-11),
1.88 (2H, m, H-12), 5.45 (1H, t, J ) 7.1 Hz, H-13), 2.17 (2H, m,
H-15), 1.52 (2H, m, H-16), 1.68 (1H, m, H-17), 1.47 (1H, m, H-19a),
1.26 (1H, m, H-19b), 1.72 (2H, m, H-20), 2.07 (2H, m, H-21), 0.79
(3H, s, H-23), 0.87 (3H, s, H-24), 0.79 (3H, s, H-25), 1.12 (3H, s,
H-26), 4.03 (2H, s, H-27), 0.83 (3H, s, H-28), 0.91 (3H, s, H-29), 4.78
(1H, s, H-30a), 4.59 (1H, s, H-30b); 13C NMR (CDCl3, 75 MHz) δ
39.8 (t, C-1), 23.7 (t, C-2), 42.0 (t, C-3), 33.4 (s, C-4), 56.0 (d, C-5),
18.4 (t, C-6), 44.3 (t, C-7), 74.0 (s, C-8), 61.2 (d, C-9), 39.1 (s, C-10),
25.6 (t, C-11), 27.1 (t, C-12), 126.8 (d, C-13), 139.5 (s, C-14), 30.7 (t,
C-15), 25.5 (t, C-16), 54.4 (d, C-17), 34.9 (s, C-18), 36.2 (t, C-19),
20.5 (t, C-20), 32.4 (t, C-21), 149.1 (s, C-22), 15.4 (q, C-23), 33.2 (q,
C-24), 21.5 (q, C-25), 23.8 (q, C-26), 66.7 (t, C-27), 26.3 (q, C-28),
28.3 (q, C-29), 109.0 (t, C-30); EIMS m/z 426 [M - H2O]+; FABMS
m/z 427 [M - OH]+, 409 [M - H2O - OH]+; HRESIMS m/z 427.3943
(calcd for C30H51O, 427.3940).
(1R,2R,4aS,8aS,3′E,1′′S)-(+)-1-[6′-(2′′,2′′-Dimethyl-6′′-methyle-
necyclohexyl)-4′-methyl-5′-oxohex-3′-enyl]-2,5,5,8a-tetramethyl-
decahydronaphthalen-2-ol (5): amorphous solid; [R]D +22.4 (c 0.2,
CH2Cl2); UV λmax (MeOH) (log ꢀ) 238 (3.9), 272 (0.9) nm; IR (CH2-
Cl2) νmax 3490 (OH), 2930 (CH), 1718 (CdO, ester), 1645 (double
1
bond), 1083 (C-O), 738 cm-1; H NMR (CDCl3, 300 MHz) δ 1.65
(2H, m, H-1), 1.50 (2H, m, H-2), 1.46 (1H, m, H-3a), 1.37 (1H, m,
H-3b), 0.91 (1H, m, H-5), 1.46 (2H, m, H-6), 2.00 (2H, m, H-7), 1.11
(1H, m, H-9), 1.27 (2H, m, H-11), 2.33 (2H, m, H-12), 6.69 (1H, t, J
) 7.0 Hz, H-13), 2.85 (2H, m, H-16), 2.65 (1H, m, H-17), 1.55 (2H,
m, H-19), 1.68 (2H, m, H-20), 2.05 (2H, m, H-21), 0.81 (3H, s, H-23),
0.88 (3H, s, H-24), 0.81 (3H, s, H-25), 1.18 (3H, s, H-26), 1.76 (3H,
s, H-27), 0. 88 (3H, s, H-28), 0.95 (3H, s, H-29), 4.68 (1H, s, H-30a),
4.38 (1H, s, H-30b); 13C NMR (CDCl3, 75 MHz,) δ 39.9 (t, C-1), 24.1
(t, C-2), 42.0 (t, C-3), 33.3 (s, C-4), 56.2 (d, C-5), 18.4 (t, C-6), 44.8
(t, C-7), 74.3 (s, C-8), 61.4 (d, C-9), 39.1 (s, C-10), 23.9 (t, C-11),
35.3 (t, C-12), 142.2 (d, C-13), 137.4 (s, C-14), 201.8 (s, C-15), 34.5
(t, C-16), 49.2 (d, C-17), 35.0 (s, C-18), 38.7 (t, C-19), 20.6 (t, C-20),
32.4 (t, C-21), 149.5 (s, C-22), 15.4 (q, C-23), 33.4 (q, C-24), 21.6 (q,
C-25), 23.7 (q, C-26), 12.0 (q, C-27), 24.5 (q, C-28), 28.9 (q, C-29),
108.2 (t, C-30); FABMS m/z 465 [M + Na]+, 443 [M + H]+;
HRESIMS m/z 443.3886 (calcd for C30H51O2, 443.3889).
Benzoylation of 4. Compound 4 (60 mg, 0.14 mmol) was subjected
to benzoylation, and chromatographic purification of the product using
the same methods mentioned under benzoylation of 2 and 3, to yield
8 (9 mg, yield 12.2%).
(1R,2R,4aS,8aS,3′E,1′′S)-(+)-1-[6′-(2′′,2′′-Dimethyl-6′′-methyle-
necyclohexyl)-4′-benzoyloxymethylhex-3′-enyl]-2,5,5,8a-tetrameth-
yldecahydronaphthalen-2-ol (8): amorphous solid; [R]D +23 (c 0.2,
CH2Cl2); UV λmax (MeOH) (log ꢀ) 229 nm (4.36); IR (CH2Cl2) νmax
3450 (OH), 2932 (CH), 1716 (CdO, ester), 1646 (double bond), 1605
(aromatic), 1270 (C-O, ester), 1111 (C-O), 711 cm-1 1H NMR
;
(CDCl3, 300 MHz) δ 1.64 (2H, m, H-1), 1.58 (2H, m, H-2), 1.40 (1H,
m, H-3a), 1.14 (1H, m, H-3b), 0.97 (1H, m, H-5), 1.62 (1H, m, H-6a),
1.42 (1H, m, H-6b), 1.40 (1H, m, H-7a), 1.18 (1H, m, H-7b), 1.08
(1H, m, H-9), 1.58 (2H, m, H-11), 2.01 (2H, m, H-12), 5.60 (1H, t, J
) 5.3 Hz, H-13), 2.13 (2H, m, H-15), 1.35 (2H, m, H-16), 1.73 (1H,
m, H-17), 1.52 (1H, m, H-19a), 1.26 (1H, m, H-19b), 1.73 (2H, m,
H-20), 2.05 (2H, m, H-21), 0.79 (3H, s, H-23), 0.86 (3H, s, H-24),
0.79 (3H, s, H-25), 1.14 (3H, s, H-26), 4.75 (2H, s, H-27), 0.82 (3H,
s, H-28), 0.90 (3H, s, H-29), 4.75 (1H, s, H-30a), 4.58 (1H, s, H-30b),
8.07 (2H, d, J ) 7.6 Hz, H-3′,7′), 7.44 (2H, t, J ) 7.6 Hz, H-4′,6′),
Benzoylation of 2 and 3. A mixture of 2 and 3 (200 mg, 0.45 mmol)
was treated with benzoyl chloride (0.1 mL) and pyridine (2 mL) at 50
°C for 15 h. The crude residue (287 mg) was separated on Sephadex
LH-20 using CH2Cl2-MeOH (1:1) to give four fractions (F1-F4).
Fraction F2 (127 mg) was chromatographed using normal-phase HPLC
and n-hexane-CH2Cl2-MeOH (8:8:1) to yield three fractions (A-
C). Fraction B (55 mg) was further separated using normal-phase HPLC
and n-hexane-EtOAc (12:1) to yield 6 (24 mg, yield 9.7%) and 7 (12
mg, yield 4.9%).

152 Journal of Natural Products, 2007, Vol. 70, No. 2
Shen et al.
7.54 (1H, t, J ) 7.6 Hz, H-5′); 13C NMR (CDCl3, 75 MHz) δ 39.8 (t,
C-1), 23.6 (t, C-2), 42.0 (t, C-3), 33.2 (s, C-4), 56.1 (d, C-5), 18.5 (t,
C-6), 44.6 (t, C-7), 74.0 (s, C-8), 61.5 (d, C-9), 39.1 (s, C-10), 25.5 (t,
C-11), 27.5 (t, C-12), 128.3 (d, C-13), 134.5 (s, C-14), 31.0 (t, C-15),
25.3 (t, C-16), 54.4 (d, C-17), 34.8 (s, C-18), 36.1 (t, C-19), 20.6 (t,
C-20), 32.3 (t, C-21), 149.1 (s, C-22), 15.4 (q, C-23), 33.4 (q, C-24),
21.5 (q, C-25), 23.8 (q, C-26), 69.5 (t, C-27), 26.3 (q, C-28), 28.3 (q,
C-29), 109.1 (t, C-30), 166.5 (s, C-1′), 130.9 (s, C-2′), 129.6 (d, C-3′,7′),
128.3 (d, C-4′,6′), 132.8 (d, C-5′); EIMS m/z 309 [M - PhCOOH -
OH]+; FABMS m/z 571 [M + Na]+; HRESIMS m/z 571.4130 (calcd
for C37H56O3Na, 571.4127).
Reaction with p-Toluenesulfonyl Chloride. A mixture of 3 (100
mg, 0.45 mmol), p-toluenesulfonyl chloride (0.1 mL), and pyridine (1.5
mL) was stirred at 50 °C for 12 h. Ether was added, and the mixture
was washed with saturated aqueous NaHCO3 solution and brine, dried,
and evaporated. The resulting crude residue (127 mg) was chromato-
graphed on a silica gel column using n-hexane-EtOAc (100:0 to 10:
1) to afford two fractions (A and B). Fraction A (89 mg) was separated
by silica gel preparative TLC (GF254) using n-hexane-CH2Cl2 (3:1) to
yield 9 (20 mg, 20.8%) and 10 (42 mg, 43.8%).
(1R,2R,4aS,8aS,3′R,4′E,1′′S)-(+)-1-[6′-(2′′,2′′-Dimethyl-6′′-meth-
ylenecyclohexyl)-4′-methylhex-4′-enyl]-2,3′-epoxy-2,5,5,8a-tetram-
ethyldecahydronaphthalen-2-ol (9): colorless, needle-shaped crystals;
mp 112 °C; [R]D +25.2 (c 0.2, CH2Cl2); IR (CH2Cl2) νmax 3400 (OH),
2928 (CH), 1646 (double bond), 1074 (C-O) cm-1; 1H NMR (CDCl3,
300 MHz) δ 1.54 (2H, m, H-1), 1.53 (2H, m, H-2), 1.35 (2H, m, H-3),
0.96 (1H, m, H-5), 1.42 (2H, m, H-6), 1.83 (2H, m, H-7), 1.60 (1H,
m, H-9), 1.79 (1H, m, H-11a), 1.55 (1H, m, H-11b), 1.61 (2H, m, H-12),
4.12 (1H, dd, J ) 9.9, 1.2 Hz, H-13), 5.34 (1H, t, J ) 6.6 Hz, H-15),
2.22 (1H, m, H-16a), 2.08 (1H, m, H-16b), 1.79 (1H, m, H-17), 1.46
(1H, m, H-19a), 1.26 (1H, m, H-19b), 1.60 (2H, m, H-20), 2.06 (1H,
m, H-21a), 2.00 (1H, m, H-21b), 0.80 (3H, s, H-23), 0.86 (3H, s, H-24),
0.80 (3H, s, H-25), 1.21 (3H, s, H-26), 1.60 (3H, s, H-27), 0.83 (3H,
s, H-28), 0.93 (3H, s, H-29), 4.74 (1H, s, H-30a), 4.50 (1H, s, H-30b);
13C NMR (CDCl3, 75 MHz) δ 39.1 (t, C-1), 23.7 (t, C-2), 42.1 (t, C-3),
33.3 (s, C-4), 56.4 (d, C-5), 18.5 (t, C-6), 43.5 (t, C-7), 74.9 (s, C-8),
51.9 (d, C-9), 37.1 (s, C-10), 26.4 (t, C-11), 15.9 (t, C-12), 75.6 (d,
C-13), 136.3 (s, C-14), 125.7 (d, C-15), 24.9 (t, C-16), 54.0 (d, C-17),
35.0 (s, C-18), 37.2 (t, C-19), 20.2 (t, C-20), 33.2 (t, C-21), 149.4 (s,
C-22), 15.0 (q, C-23), 33.4 (q, C-24), 21.5 (q, C-25), 25.9 (q, C-26),
12.4 (q, C-27), 25.3 (q, C-28), 28.6 (q, C-29), 108.8 (t, C-30); EIMS
m/z 409 [M - OH]+; FABMS m/z 449 [M + Na]+, 427 [M + H]+;
HRESIMS m/z 427.3941 (calcd for C30H51O, 427.3940).
C-27), 24.7 (q, C-28), 28.8 (q, C-29), 108.7 (t, C-30); EIMS m/z 409
[M - OH]+; FABMS m/z 449 [M + Na]+, 427 [M + H]+; HRESIMS
m/z 427.3942 (calcd for C30H51O, 427.3940).
Photoreaction of Ambrein (1). Ambrein (1, 300 mg) was dissolved
in acetone (5 mL) containing a few drops of H2O and irradiated with
shortwave UV light (254 nm, UV lamp) for 72 h at room temperature.
The mixture was stirred continuously, and the evaporated solvent was
replaced. The solution was then evaporated, and the residue (285 mg)
was separated on a silica gel column using n-hexane-EtOAc (100:1
to 2:1) to afford 12 (6 mg) and 11 (25 mg, 8.3%).
(1R,2R,4aS,8aS,3′S,4′S,1′′S)-(+)-1-[6′-(2′′,2′′-Dimethyl-6′′-meth-
ylenecyclohexyl)-4′-hydroxy-4′-methylhexyl]-2,3′-epoxy-2,5,5,8a-tet-
ramethyldecahydronaphthalen-2-ol (11): amorphous solid; [R]D
-19.8 (c 0.2, CH2Cl2); IR (CH2Cl2) νmax 3446 (OH), 2926 (CH), 1645
(double bond), 1088 (C-O) cm-1; 1H NMR (CDCl3, 300 MHz) δ 1.57
(2H, m, H-1), 1.54 (2H, m, H-2), 1.36 (2H, m, H-3), 1.00 (1H, m,
H-5), 1.50 (2H, m, H-6), 1.79 (2H, m, H-7), 1.15 (1H, m, H-9), 1.65
(2H, m, H-11), 1.65 (2H, m, H-12), 3.38 (1H, dd, J ) 9.9, 2.0 Hz,
H-13), 2.06 (2H, m, H-15), 1.48 (2H, m, H-16), 1.65 (1H, m, H-17),
1.53 (2H, m, H-19), 1.21 (2H, m, H-20), 2.00 (2H, m, H-21), 0.74
(3H, s, H-23), 0.87 (3H, s, H-24), 0.80 (3H, s, H-25), 1.26 (3H, s,
H-26), 1.11 (3H, s, H-27), 0.92 (3H, s, H-28), 0.85 (3H, s, H-29), 4.75
(1H, brs, H-30a), 4.56 (1H, brs, H-30b); 13C NMR (CDCl3, 75 MHz)
δ 39.1 (t, C-1), 23.9 (t, C-2), 42.1 (t, C-3), 33.3 (s, C-4), 57.6 (d, C-5),
18.6 (t, C-6), 41.9 (t, C-7), 73.3 (s, C-8), 56.4 (d, C-9), 36.7 (s, C-10),
26.6 (t, C-11), 19.9 (t, C-12), 75.2 (d, C-13), 74.9 (s, C-14), 36.5 (t,
C-15), 18.2 (t, C-16), 54.7 (d, C-17), 35.1 (s, C-18), 35.4 (t, C-19),
20.7 (t, C-20), 32.7 (t, C-21), 149.4 (s, C-22), 15.6 (q, C-23), 33.3 (q,
C-24), 21.3 (q, C-25), 29.7 (q, C-26), 23.7 (q, C-27), 26.1 (q, C-28),
28.5 (q, C-29), 108.9 (t, C-30); FABMS m/z 467 [M + Na]+; HRESIMS
m/z 467.3865 (calcd for C30H52O2Na, 467.3865).
Cytotoxicity Assay. Cytotoxicity was determined against Hepa59T/
VGH (human liver carcinoma), WiDr (human colon adenocarcinoma),
A-549 (human lung carcinoma), and MCF-7 (human breast adenocar-
cinoma) tumor cells was based on a MTT assay method. The assay
procedure was carried out as previously described.17 ED50 values were
defined by a comparison with the untreated cells as the concentration
of test sample resulting in 50% reduction of absorbance.
Anti-inflammatory Assay (Measurement of Elastase Release).
Degranulation of azurophilic granules was determined by elastase
release as described previously,18 with some modifications. Experiments
were performed using MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide as the
elastase substrate. In brief, after supplementation with MeO-Suc-Ala-
Ala-Pro-Val-p-nitroanilide (100 µM), neutrophils (6 × 105/mL) were
equilibrated at 37 °C for 2 min and incubated with each test compound
for 5 min. Cells were activated by FMLP (100 nM)/CB (0.5 µg/mL),
and changes in absorbance at 405 nm were monitored continuously
for elastase release. The results are expressed as the percentage of the
initial rate of elastase release in the FMLP/CB-activated, test compound-
free (DMSO) control system.
Crystal data for 9: C30H50O1, M 426.72, orthorhombic, P212121 (No.
19), a 6.298(2) Å, b 11.916(2) Å, c 35.057(5) Å, V 2631.1(7) Å3, Z 4,
D
calcd 1.077 g cm-3, F(000) 952, λ(Mo KR) 0.71073 Å, T 298 K, 2232
unique reflections collected. Final GooF 1.35,
R 0.0476, wR
0.0705, 280 parameters, µ 0.062 mm-1, R indices based on 1546
reflections with I > 2σ(I), absorption corrections applied. Copies
of the deposited crystal data can be obtained, free of charge,
on application to CCDC (file number 626138), 12 Union Road,
Cambridge CB2 1EZ, UK [fax: +44(0) 1223 336033 or e-mail:
deposit@ccdc.cam.ac.uk].
Acknowledgment. The authors are grateful to the National Science
Council, Taipei, Taiwan, for financial support (grant #NSC 93-2811-
B-110-004). We thank Ms. S.-C. Yu, NSC Southern MS Instrument
Center at the National Sun Yat-sen University, for measurement of
mass spectra.
(1R,2R,4aS,8aS,5′S,3′E,1′′S)-(+)-1-[6′-(2′′,2′′-Dimethyl-6′′-meth-
ylenecyclohexyl)-4′-methylhex-3′-enyl]-2,5′-epoxy-2,5,5,8a-tetram-
ethyldecahydronaphthalen-2-ol (10): colorless, needle-shaped crystals;
mp 98 °C; [R]D -24.3 (c 0.2, CH2Cl2); IR (CH2Cl2) νmax 3400 (OH),
2928 (CH), 1647 (double bond), 1078 (C-O) cm-1; 1H NMR (CDCl3,
300 MHz) δ 1.65 (1H, m, H-1a), 1.60 (1H, m, H-1b), 1.54 (2H, m,
H-2), 1.44 (1H, m, H-3a), 1.40 (1H, m, H-3b), 0.99 (1H, m, H-5), 1.44
(2H, m, H-6), 1.80 (2H, m, H-7), 1.20 (1H, m, H-9), 2.27 (1H, m,
H-11a), 2.10 (1H, m, H-11b), 1.60 (1H, m, H-12a), 1.49 (1H, m, H-12b),
5.28 (1H, br t, J ) 6.2 Hz, H-13), 3.96 (1H, dd, J ) 9.2, 0.9 Hz,
H-15), 1.62 (2H, m, H-16), 1.84 (1H, m, H-17), 1.50 (1H, m, H-19a),
1.31 (1H, m, H-19b), 1.76 (2H, m, H-20), 2.15 (1H, m, H-21a), 2.00
(1H, m, H-21b), 0.74 (3H, s, H-23), 0.86 (3H, s, H-24), 0.80 (3H, s,
H-25), 1.25 (3H, s, H-26), 1.60 (3H, s, H-27), 0.81 (3H, s, H-28), 0.93
(3H, s, H-29), 4.74 (1H, s, H-30a), 4.47 (1H, s, H-30b); 13C NMR
(CDCl3, 75 MHz) δ 39.1 (t, C-1), 23.8 (t, C-2), 42.0 (t, C-3), 33.3 (s,
C-4), 56.4 (d, C-5), 18.6 (t, C-6), 42.2 (t, C-7), 74.6 (s, C-8), 57.4 (d,
C-9), 36.8 (s, C-10), 24.7 (t, C-11), 31.8 (t, C-12), 126.4 (d, C-13),
135.9 (s, C-14), 75.5 (d, C-15), 18.4 (t, C-16), 53.7 (d, C-17), 35.1 (s,
C-18), 37.7 (t, C-19), 19.9 (t, C-20), 33.8 (t, C-21), 149.3 (s, C-22),
15.6 (q, C-23), 33.4 (q, C-24), 21.3 (q, C-25), 20.7 (q, C-26), 12.0 (q,
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NP068029P