Guaianolides as immunomodulators. Synthesis and biological activities of dehydrocostus lactone, mokko lactone, eremanthin, and their derivatives

Article abstract of DOI:10.1021/np980092u

The naturally occurring guaianolides, namely mokko lactone (1), dehydrocostus lactone (2), eremanthin (3), and related guaianolides, 16, 17, 21, 22, 28-31, 33, 36, 37, and 39, have been synthesized starting from l-α- santonin in an effort to examine their structure-activity relationship as inhibitors of the killing function of cytotoxic T lymphocytes (CTL) and the induction of intercellular adhesion molecule-1 (ICAM-1). It was observed during the present study that the guaianolides possessing an α-methylene γ- lactone moiety, i.e., 2, 3, 30, 33, 37, and 39, exhibited significant inhibitory activity toward the killing function of CTL and the induction of ICAM-1.

Full text of DOI:10.1021/np980092u

22
J . Nat. Prod. 1999, 62, 22-30
Gu a ia n olid es a s Im m u n om od u la tor s. Syn th esis a n d Biologica l Activities of
Deh yd r ocostu s La cton e, Mok k o La cton e, Er em a n th in , a n d Th eir Der iva tives
Saori Yuuya,† Hisahiro Hagiwara,† Toshio Suzuki,† Masayoshi Ando,*,‡ Atsushi Yamada,§ Kouji Suda,§
Takao Kataoka,§ and Kazuo Nagai§
Graduate School of Science and Technology and Department of Applied Chemistry, Faculty of Engineering, Niigata University,
Ikarashi 2-8050, Niigata 950-2181, J apan, and Department of Bioengineering, Tokyo Institute of Technology,
4259 Nagatsuda-cho, Midori-ku, Yokohama, 226-8501, J apan
Received March 12, 1998
The naturally occurring guaianolides, namely mokko lactone (1), dehydrocostus lactone (2), eremanthin
(3), and related guaianolides, 16, 17, 21, 22, 28-31, 33, 36, 37, and 39, have been synthesized starting
from l-R-santonin in an effort to examine their structure-activity relationship as inhibitors of the killing
function of cytotoxic T lymphocytes (CTL) and the induction of intercellular adhesion molecule-1 (ICAM-
1). It was observed during the present study that the guaianolides possessing an R-methylene γ-lactone
moiety, i.e., 2, 3, 30, 33, 37, and 39, exhibited significant inhibitory activity toward the killing function
of CTL and the induction of ICAM-1.
The guaianolides represent one of the largest groups of
sesquiterpene lactones covering over 500 known naturally
occurring compounds.1 Some of these compounds have been
reported to possess high antitumor,2-7 antishistosomal,2,8
anthelmintic,9 contraceptive,10 root-growth stimulatory,2,11
root-growth and germination inhibitory activities,12 and
antiulcer activity.13,14 This diverse bioactivity of guaiano-
lides makes them attractive synthetic targets since the
availability of these compounds from natural sources is
very limited. It is important to make these natural products
available in substantial quantities to explore in detail their
bioactivities. Therefore, it is of interest to synthesize these
compounds from easily available starting materials.
Among the guaianolides, Mokko lactone (1) and dehy-
drocostus lactone (2) were initially isolated from mokko
(Sausurea lappa Clarke),15,16 a plant that is used for
medicinal purposes. Subsequent work from various labo-
ratories17,18 led the structures of 1 and 2. The related
compound eremanthin (3) was isolated from the heartwood
oil of Eremanthus elaeagnus and Vanillosposis eryth-
roppa.8,19 It is interesting to note that 3 showed strong
prophylactic action against the human parasite Schisto-
soma mansoni.
As a part of our ongoing research program, we screened
∼100 herbal medicines for inhibitors of the killing function
of cytotoxic T lymphocyte (CTL) and found that only the
extract of S. lappa showed considerable inhibitory activity.
One of the active principles in the extract, namely 2, seems
to bind the cystein residues of tyrosine kinase and conse-
quently inhibit the kinase activity in T-cell.20
Since the above-mentioned activity exhibited by 2 falls
in the category of immunomodulators, it appeared worth-
while to undertake the synthesis of 2 and its congeners to
correlate structure-activity relationships among these
compounds. The present work was initiated with this aim
in mind. It was realized that these compounds could be
derived via a common cationic intermediate I, which in
turn could be generated from mesylate (4) through sol-
volytic rearrangement.21 In fact, I is presumed to be an
intermediate during the biosynthesis of these compounds
in nature (Figure 1).
Resu lts a n d Discu ssion
The starting material used for the synthesis of key
intermediate (11S)-1â-(mesyloxy)eudesm-4(14)-eno-13,6R-
lactone (4) was the R,â-unsaturated ketone 6, which was
prepared from R-santonin (5) in six steps with an overall
yield of 59%3,22 (Scheme 1). Ketalization of 6 and subse-
quent isomerization of the double bond at C-2 by heating
in ethylene glycol in the presence of TsOH at 145 °C gave
a mixture of 7, 8, and 9 (70:1:0.3). Treatment of this
mixture with boiling 50% AcOH-H2O gave the desired â,γ-
unsaturated ketone 10 in 64% overall yield. Dehydroge-
nation of 10 with DDQ in the presence of anhydrous TsOH
at room temperature gave an exo-dienone 11 in 82% yield
accompanied by an endo-dienone 12 in 7% yield. Treatment
of 11 with zinc amalgam in refluxing AcOH gave γ,δ-
unsaturated ketone 13 in 93% yield. Selective reduction
of the C-1 carbonyl group of 13 with LiAl(t-BuO)3H
furnished the desired â-alcohol 14 and the corresponding
R-alcohol 15 in 81% and 13% yields, respectively. The latter
compound could be converted to 14 in 63% yield by Collins
oxidation followed by reduction of the resulting ketone with
LiAl(t-BuO)3H. Mesylation of 14 with MsCl in pyridine at
room temperature gave mesylate 4 in 97% yield. The exo-
dienone 11 was also prepared by an alternative procedure.
Dehydrogenation of 6 with a large excess of DDQ in the
presence of anhydrous TsOH in refluxing toluene gave 11
in 57% yield based on recovered 6.
Solvolytic rearrangement21 of 4 with 0.5 M KOAc in
AcOH provided a mixture (2:1:2.4) of tetra-, tri-, and
disubstituted olefins 16, 17, and 1 in 63% yield. In addition,
the solvolysis reaction also produced a mixture of olefins
18, 19, and 20 (5:4:7) in 16% yield, presumably formed by
the epimerization of the exo-olefinic bond at C-4 to the
thermodynamically more stable olefin during the reaction
conditions employed. Separation of the crude reaction
products by preparative HPLC gave compound 20, along
with a mixture (5:4) of 18 and 19, and the compound 16,
along with a mixture (1:1.6:4) of 16, 17, and 1. Since the
* To whom correspondence should be addressed. Phone: +81-25-262-
7326. Fax: +81-25-262-7326; +81-25-262-7010. E-mail: mando@eng.niigata-
u.ac.jp.
† Graduate School of Science and Technology, Niigata University.
‡ Department of Applied Chemistry, Faculty of Engineering, Niigata
University.
§ Department of Bioengineering, Tokyo Institute of Technology.
10.1021/np980092u CCC: $18.00
© 1999 American Chemical Society and American Society of Pharmacognosy
Published on Web 12/04/1998

Guaianolides as Immunomodulators
J ournal of Natural Products, 1999, Vol. 62, No. 1 23
Phenylselenenylation of 31 followed by treatment of the
resulting phenylseleno lactone 32 with 31% H2O2 gave an
R-methylene γ-lactone (33). Similarly, phenylselenenyla-
tion of â-epoxide 21 gave a mixture of R- and â-phenylse-
leno lactones 34 and 35 in 7% and 78% yields, respectively.
The oxidative syn elimination of 34 and 35 with 31% H2O2
gave endo- and exo-unsaturated γ-lactones 36 and 37 in
86% and 85% yields, respectively. In an analogous manner,
phenylselenenylation of 22 and successive treatment of
phenylselenide 38 with 31% H2O2 gave an R-methylene
γ-lactone 39 in 50% overall yield. Isodehydrocostus lactone
(40) was also obtained from 20 by employing the same
procedure.27
In h ibitor y Activity of Killin g F u n ction of Cytotoxic
T Lym p h ocytes. Cytotoxic T lymphocytes (CTL) are
important in the elimination of virus-infected cells and
tumors and in graft rejection in human body. Recently, we
observed that dehydrocostus lactone (2) showed efficient
inhibitory activity toward the killing function of CTL.20
This result prompted us to examine the structure-activity
relationships of 2 and the 14 related compounds that were
synthesized as per the above-mentioned methods as inhibi-
tors of killing function of CTL. The compounds were
screened for inhibitors of cytotoxic activity of CTL clone
OE4.
F igu r e 1. Retrosynthetic analyses and possible biosynthetic route of
target guaianolides 1-3.
tetrasubstituted olefin 16 was unstable, it was epoxidized
to give a â-epoxide (21) and an R-epoxide (22) in 44% and
41% yields, respectively. Since the complete separation of
16, 17, and 1 by preparative HPLC was not achieved, the
mixture of 16, 17, and 1 (1:1.6:4) was used as such for the
next step.
Phenylselenenylation of the above-mentioned mixture
with LDA and diphenyl diselenide23 afforded a mixture of
phenylseleno lactones, which was separated by HPLC to
give 23, together with a mixture of 24 and 25 (1:6), 26, a
mixture of 17 and 1 (1:4.3), and 27 in 7%, 25%, 12%, 6%,
and 41% yields, respectively. The oxidative syn elimina-
tion23,24 of 23 with 31% H2O2 gave an endo-unsaturated
γ-lactone 28 in 87% yield. The physical constants and
spectral data of 28 were in good agreement with those
reported for 11,13-dihydro-7,11-dehydro-3-desoxyzaluzanin
C.25
Treatment of the mixture (1:6) of 24 and 25 with 31%
H2O2 furnished 3 and the corresponding endo-unsaturated
γ-lactone (29) in 76% and 13% yields, respectively. The
physical constants and spectral data of 3 were in good
accordance with those of natural eremanthin reported in
the literature.8, 19
Similarly, the oxidative syn elimination of 26 with 31%
H2O2 gave an un-natural R-methylene γ-lactone 30 as an
unstable compound. The structure of 30 was established
with the help of spectral data including HREIMS. In a
similar way, treatment of 27 with 31% H2O2 gave 2 in 99%
yield. The physical constants and spectral data of 2 were
in good agreement with those of natural dehydrocostus
lactone reported in the literature.17,18,20,26
Selective epoxidation of the trisubstituted double bond
of 17 using a mixture of 17 and 1 (1:4.3) gave an epoxide
31 and 1 in 13% and 48% yields, respectively. The
compounds 1 and 31 were easily isolated in pure form from
the crude reaction product by HPLC. The physical con-
stants and spectral data of 1 were identical with those of
natural mokko lactone reported in the literature.17,20
In conclusion, the natural products mokko lactone (1),
dehydrocostus lactone (2), and eremanthin (3) have been
synthesized from l-R-santonin in overall yields of 6.9% (13
steps), 4.8% (15 steps), and 2.2% (15 steps), respectively.
Having achieved the synthesis of the target compounds,
it was of interest to prepare some derivatives to examine
structure-activity relationships. Hence, we attempted
syntheses of some R-methylene γ-lactone derivatives such
as 33, 37, and 39 as depicted in Scheme 1.
All compounds possessing an R-methylene γ-lactone
moiety, such as dehydrocostus lactone (2), eremanthin (3),
30, 33, 37, 39, and isodehydrocostus lactone (40),27 showed
significant inhibitory activity on killing function of CTL.
Hence, an R-methylene γ-lactone moiety in the molecule
seems to be essential for the activity of these compounds.
In fact, the activity of endo-unsaturated γ-lactones 28, 29,
and 36 decreased markedly as compared with that of the
corresponding R-methylene γ-lactones 2, 3, and 37. It is
noteworthy that R-methyl γ-lactones (saturated γ-lactones)
such as isocostus lactone (20), mokko lactone (1), and other
related compounds such as 21, 22, and 31 showed no
significant activity (Table 1).
The positions of two double bonds in A and B rings of
the molecule were not related in any way to the expression
of bioactivity. Double-bond isomers such as dehydrocostus
lactone (2), eremanthin (3), compound 30, and isodehydro-
costus lactone (40) showed approximately the same activity.
It was observed that the magnitude of inhibitory activity
was influenced by replacing the double bond in the B ring
with an epoxide ring. Introduction of the epoxide moiety
enhanced the inhibitory activity.
In summary, it has been conclusively established during
the present study that the compounds 33, 37, and 39
showed promising inhibitory activity on the killing function
of cytotoxic T lymphocytes, and the order of the inhibitory
activity in the test samples investigated was 33 ) 37 ) 39
g 3 g 2 g 30 g 40 > 28 > 20, 29 > 31 g 21 > 1, 22, 36.
In h ibitor y Activity on Exp r ession of In ter cellu la r
Ad h esion Molecu le-1 (ICAM-1) In d u ced by In ter leu -
k in -1. Expression of intercellular adhesion molecule-1
(ICAM-1) is induced by interleukin-1 (IL-1) on the surface
of endothelial cells of blood vessels. ICAM-1 on the acti-
vated endothelial cells interacts with lymphocyte function-
associated antigen-1 (LFA-1) on leucocytes in the blood
stream, and the leucocytes begin rolling, adhere to the
surface of endothelium, and finally migrate from the inside
of the blood vessel to the inflammatory portion by chemo-
taxis. The attack of leucocytes causes serious damage to
the inflammatory tissue. Expression of excess amount of
ICAM-1 on the surface of endothelial cells of a blood vessel
plays an important role in the progress of inflammatory

24 J ournal of Natural Products, 1999, Vol. 62, No. 1
Yuuya et al.
Sch em e 1a
a
The yields are based on the recovered starting material.
Ta ble 1. Inhibitory Activity of Killing Function of Cytotoxic T Lymphocytes (CTL)a
R-methylene γ-lactones
(exo-unsaturated γ-lactones)
endo-unsaturated
R-methyl γ-lactones
(saturated γ-lactones)
γ-lactones
compounds
30
3
2
33
37
39
40
28
29
36
20
1
21
22
31
IC50 (µM)
15
11
14
8
7
7
16
50
>100
>1000
>100
>1000
597
>1000
422
a
To examine the dose response of the compounds, OE4 (1 × 104 cells/well) was preincubated with various concentrations of the compounds
for 2 h, mixed with P815 (1 × 103 cells/well) labeled with [3Η]thymidine, and incubated for 4 h. The experiments were carried out in
triplicate cultures. IC50 was deduced from the 50% inhibition of the specific release, which was calculated by using the formula described
in the Materials and Methods for Bioassays.
reaction. These facts suggest that the inhibitors of induc-
tion of ICAM-1 may turn out to yield a new type of
antiinflammatory agent. With this in mind, we began to
examine 2 and the 12 related compounds that were
synthesized by us for determining their inhibitory activity
on the induction of ICAM-1 through bioassay.

Guaianolides as Immunomodulators
J ournal of Natural Products, 1999, Vol. 62, No. 1 25
Ta ble 2. Inhibitory Activity of Induction of Intercellular Adhesion Molecule-1 (ICAM-1)a
R-methylene γ-lactones
(exo-unsaturated γ-lactones)
endo-unsaturated
R-methyl γ-lactones
(saturated γ-lactones)
γ-lactones
compounds
30
3
2
33
37
39
28
442
29
36
1
21
22
31
IC50 (µM)
8
46
10
6
10
7
142
136
251
504
253
117
a
A549 (3 ×104 cells/well) was pretreated with various concentrations of the compounds for 1 h and then incubated with the addition
of IL-1â for 6 h. Absorbance of 415 nm was assayed after treatment of the cells with primary and secondary antibodies and addition of
the enzyme substrate as described in the Materials and Methods for Bioassays. The experiments were carried out in triplicate cultures.
IC50 was calculated by using the formula in Materials and Methods for Bioassays.
°C for 22 min, cooled, poured into a saturated aqueous solution
of NaCl (250 mL), and extracted with EtOAc (3 × 150 mL).
The combined extracts were washed successively with satu-
rated aqueous NaCl (3 × 30 mL), dried (Na2SO4), and
concentrated to give a crude product (2.94 g), which was
chromatographed over silica gel [195 g, 5.5-cm i.d., EtOAc-
hexane (2:8)].
The first elution gave a mixture (70:1:0.3) of compounds 7-9
(1.97 g, 73%), which was employed without further purification
for the next reaction. A part of this mixture was recrystallized
The above-mentioned compounds were screened for
inhibition of induction of ICAM-1 using culture cells A 549
(lung carcinoma, ATCC CCL 185), an in vitro model of
human endothelial cell. All compounds possessing an
R-methylene γ-lactone moiety, such as dehydrocostus lac-
tone (2), eremanthin (3), 30, 33, 37, and 39, showed
significant inhibitory activity on induction of ICAM-1.
Hence, it is inferred that an R-methylene γ-lactone moiety
in the molecule must be essential for activity in the case
of these compounds. In fact, the R-methylene γ-lactone (37)
showed 14-fold stronger activity than the corresponding
endo-unsaturated γ-lactone (36) and 50-fold stronger activ-
ity than the corresponding saturated R-methyl γ-lactone
(21) (Table 2).
The extent of inhibitory activity expressed was influ-
enced by the location of the double bond in the B-ring and
by the introduction of an epoxide ring at the C-9 or C-1(10)
positions. Thus, the epoxide 33 showed 8-fold stronger
activity than that of the corresponding unsaturated com-
pound, eremanthin (3). The most active compound obtained
was the R-methylene γ-lactone derivative 33, and the order
of the inhibitory activity exhibited in the test samples was
33 g 39 g 30 g 37 ) 2 > 3 > 31 > 36 g 29 > 1 ) 22 > 28
> 21.
from ether to give 7 as colorless prisms: mp 124-126 °C; [R]20
D
+105.1° (c 1.25, CHCl3); IR (CHCl3) νmax 1770 cm-1; H NMR
1
δ 1.03 (3H, s, H-15), 1.22 (3H, d, J ) 6.8 Hz, H-12), 1.86 (3H,
br s, Wh/2 ) 5.0 Hz, H-14), 2.69 (1H, br d, J ) 11.5 Hz, H-5),
3.75-4.18 (5H, m, -O(CH2)2O-, H-6), 5.32 (1H, br s, Wh/2
)
10.0 Hz, H-3); 13C NMR δ 12.39 (q, C-12), 15.75 (q, C-15), 22.54,
28.38 (t, C-8 and C-9), 23.50 (q, C-14), 34.49 (t, C-2), 40.57 (d,
C-11), 43.84 (s, C-10), 48.43 (d, C-5), 53.23 (d, C-7), 64.81, 65.49
(t, -OCH2CH2O-), 81.37 (d, C-6), 110.88 (s, C-1), 120.42 (d,
C-3), 133.99 (s, C-4), 179.58 (s, C-13); anal. C 69.70%, H 8.27%,
calcd for C17H24O4, C 69.83%, H 8.27%.
The second elution gave recovered 6 (304 mg, 13%). Al-
though the mixture of compounds 7-9 appeared as a broad
single peak in HPLC [A, EtOAc-hexane (2:8), 3.0 mL/min],
the pure compounds 8 and 9 were isolated by repeated HPLC,
using the mother liquor of 7, by following the above-mentioned
conditions.
In our preliminary results, it appears that these com-
pounds inhibit induction of ICAM-1 by controlling the
nuclear transportation of NF-κB, which is a transcription
factor of ICAM-1 gene. It is interesting to note that the
possible target of compounds in inhibition of the ICAM-1
induction may be different from those in the inhibition of
the killing function of CTL. Details regarding the mode of
action of these compounds will be reported elsewhere.
Compound 8 was obtained as colorless needles (EtOAc): mp
152 °C; [R]20 +75.4° (c 0.68, CHCl3); IR (CHCl3) νmax 1777
D
cm-1; H NMR δ 1.06 (3H, s, H-15), 1.22 (6H, d, J ) 6.8 Hz,
1
H-12, H-14), 2.14-2.34 (2H, m, H-4, 11), 3.42-4.20 (4H, m,
-O(CH2)2O-), 3.88 (1H, dd, J ) 10.7, 10.7 Hz, H-6), 5.50 (1H,
dd, J ) 10.0, 2.1 Hz, H-3), 5.61 (1H, dd, J ) 10.0, 2.2 Hz, H-2);
13C NMR δ 12.40 (q, C-12), 15.97 (q, C-15), 20.55 (q, C14), 23.09
(t), 29.52 (t), 32.80 (d), 40.83 (d), 43.44 (s, C-10), 48.29 (d, C-5),
52.00 (d, C-7), 65.11, 65.16 (t, -OCH2CH2O-), 84.28 (d, C-6),
108.93 (s, C-1), 125.45 (d, C-3), 135.76 (d, C-2), 179.45 (s, C-13);
anal. C 69.74%, H 8.28%, calcd for C17H24O4, C 69.83%, H
8.27%.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. All melting points
1
are uncorrected. H NMR spectra were recorded at 200 MHz
in CDCl3, and 13C NMR spectra were recorded at 50 MHz in
CDCl3 unless otherwise stated. The 1H NMR assignments were
determined by decoupling and H-H COSY experiments. The
13C NMR assignments were determined by DEPT and C-H
COSY experiments or DEPT, HMQC, and HMBC experiments
(at 125.7 MHz). Reactions were run under an atmosphere of
N2 or Ar. THF was distilled from sodium benzophenone ketyl.
CHCl3 was distilled from CaH2. Silica gel (70-200 mesh) was
employed for column chromatography and silica gel (230-400
mesh) for flash column chromatography. To describe HPLC
conditions, we designate column, solvent, flow rate (mL/min),
and retention time (tR (min)) as per following order. The
column codes are as follows: A, 250- × 4.6-mm i.d. stainless
column packed with 10-mm silica gel; B, 250- × 8-mm i.d.
stainless column packed with 10-mm silica gel; C, 300- × 10-
mm i.d. glass column packed with 10-mm silica gel; D, 500- ×
30-mm i.d. stainless column packed with 25-40-mm silica gel.
(11S )-1,1-(E t h y le n e d io x y )e u d e s m -3-e n o -13,6R-la c -
ton e (7). A mixture of 6 (2.30 g, 9.26 mmol), ethylene glycol
(137 mL), and TsOH‚H2O (305 mg, 1.60 mmol) in dry benzene
(500 mL) was refluxed in a flask equipped with a Dean-Stark
column packed with molecular sieves for 24 h, and then
benzene was removed. The residue was heated at 145-150
Compound 9 was obtained as colorless needles (EtOAc): mp
129 °C; [R]20 +135.4° (c 1.09, CHCl3); IR (CHCl3) νmax 1772,
D
1652 cm-1; 1H NMR δ 0.95 (3H, s, H-15), 1.19 (3H, d, J ) 6.8
Hz, H-12), 2.59 (1H, J ) 11.0 Hz, H-5), 3.80-4.06 (5H, m,
-O(CH2)2O-, H-6), 4.80 (1H, s, H-14a), 4.94 (1H, s, H-14b);
13C NMR δ 12.42 (q, C-12), 15.91 (q, C-15), 22.73 (t), 28.94 (t),
31.27 (t), 32.43 (t), 41.05 (d, C-11), 45.94 (s, C-10), 49.96 (d,
C-5), 51.74 (d, C-7), 65.12, 65.22 (t, -OCH2CH2O-), 79.63 (d,
C-6), 110.00 (t, C-14), 111.52 (s, C-1), 143.37 (s, C-4), 179.40
(s, C-13); anal. C 70.03%, H 8.34%, calcd for C17H24O4, C
69.83%, H 8.27%.
(11S)-1-Oxoeu d esm -3-en o-13,6R-la cton e (10). A solution
of the mixture (70:1:0.3) of ketals 7-9 (3.65 g, 12.5 mmol) in
an aqueous solution of acetic acid (260 mL) was refluxed for
22 min. The mixture was cooled, poured into a saturated
aqueous solution of NaCl (300 mL), and extracted with EtOAc
(3 × 100 mL). The combined extracts were worked up to give
a crude product, which was purified by the combination of
column chromatography [silica gel 130 g, 5.5-cm i.d., EtOAc-
hexane (2:8, 3:7, and 1:1)] and HPLC [B, EtOAc-hexane (2:
8), 9.9 mL/min] affording a mixture (38:1) of desired com-
pounds 10 and 13 (2.79 g, 87%), which was employed as such
for the next reaction. Slow elution of column chromatography
gave the R,â-unsaturated ketone 6 (105 mg, 3%).

26 J ournal of Natural Products, 1999, Vol. 62, No. 1
Yuuya et al.
The mixture of 10 and 13 was recrystallized from EtOAc to
(EtOAc): mp 154-155 °C; [R]20 +154.7° (c 0.60, CHCl3); IR
D
give 10 as colorless needles: mp 138-139 °C; [R]20 +61.1° (c
(CHCl3) νmax 1782, 1710 cm-1; H NMR δ 1.13 (3H, s, H-15),
1
D
1.74, CHCl3); IR (CHCl3) νmax 1774, 1714 cm-1; 1H NMR δ 1.13
(3H, s, H-15), 1.24 (3H, d, J ) 6.9 Hz, H-12), 1.96 (3H, br s,
Wh/2 ) 5.0 Hz, H-14), 2.32 (1H, dq, J ) 12.3, 6.7 Hz, H-11),
2.60 (1H, br d, J ) 11.2 Hz, H-5), 2.84 (1H, ddd, J ) 22.0, 5.2,
2.6 Hz, H-2ax), 3.01 (1H, ddd, J ) 22.0, 4.8, 1.6 Hz, H-2eq),
4.09 (1H, dd, J ) 11.2, 9.7 Hz, H-6), 5.52-5.60 (1H, m, H-3);
13C NMR δ 12.40 (q, C-12), 16.85 (q, C-15), 22.13 (q, C-14),
22.13 (t), 31.64 (t), 38.33 (t, C-2), 40.51 (d, C-11), 48.10 (s, C-10),
48.25 (d, C-5), 52.74 (d, C-7), 80.25 (d, C-6), 119.18 (d, C-3),
135.52 (s, C-4), 179.04 (s, C-13), 212.24 (s, C-1); anal. C 72.47%,
H 8.04%, calcd for C15H20O3, C 72.55%, H 8.12%.
1.24 (3H, d, J ) 6.9 Hz, H-12), 4.12 (1H, dd, J ) 10.4, 10.4
Hz, H-6), 5.08 (1H, s, H-14a), 5.21 (1H, s, H-14b); 13C NMR δ
12.45 (q, C-12), 18.12 (q, C-15), 22.49 (t), 31.47 (t), 33.98 (t,
C-2), 37.60 (t, C-3), 41.02 (d, C-11), 50.23 (s, C-10), 51.78 (d,
C-7), 52.22 (d, C-5), 78.58 (d, C-6), 112.63 (t, C-14), 140.75 (s,
C-4), 178.87 (s, C-13), 211.99 (s, C-1); anal. C 72.37%, H 8.25%,
calcd for C15H20O3, C 72.55%, H 8.25%.
(11S)-1â-Hyd r oxyeu d esm -4(14)-en o-13,6R-la cton e (14)
an d (11S)-1R-Hydr oxyeu desm -4(14)-en o-13,6R-lacton e (15).
To a stirred solution of 13 (1.14 g, 4.58 mmol) in THF (20 mL)
at 0 °C was added LiAl(t-BuO)3H (4.67 g, 18.32 mmol). The
mixture was stirred for 1 h at 0 °C, 2 M HCl (20 mL) was
added, and stirring was continued for 10 min at 0 °C. The
mixture was then filtered through Celite under reduced
pressure. The filtrate was poured into a saturated aqueous
solution of NaCl (200 mL) and extracted with EtOAc (3 × 60
mL). The combined extracts were worked up as usual to give
a pale yellow oil (1.21 g), which was purified by preparative
HPLC [D, EtOAc-hexane (3:7), 9.0 mL/min] to give 14 (tR 7.8
min, 843 mg, 73%) and 15 (tR 5.4 min, 98 mg, 9%). The mixture
of 14 and 15 (181 mg) obtained as the middle fraction in the
above-mentioned preparative HPLC was further purified by
another HPLC [B, EtOAc-hexane (3:7), 10 mL/min] to give
14 (tR 6.4 min, 86.7 mg, 8%) and 15 (tR 4.0 min, 51 mg, 4%).
The total yields of 14 and 15 were 930 mg (81%) and 149 mg
(13%), respectively.
(11S)-1-Oxoeu d esm -2,4(14)-d ien o-13,6R-la cton e (11). A
mixture of TsOH‚H2O (1.97 g, 10.4 mmol) and benzene (70 mL)
was refluxed in a flask equipped with a Dean-Stark column
packed with molecular sieves to eliminate H2O for 1.5 h, and
then the mixture (38:1) of 10 and 13, together with DDQ (1.94
g, 8.29 mmol), and benzene (40 mL) was added at 25 °C. After
being stirred for 20 min, the mixture was filtered through
Celite, and the filtrate was poured into a saturated aqueous
solution of NaCl (150 mL) and extracted with EtOAc (3 × 50
mL). The combined extracts were worked up to give spectro-
scopically pure 11 (935 mg, 55%) as a crystalline solid. The
mother liquor was purified by column chromatography [silica
gel, 56 g; 3.3-cm i.d., EtOAc-hexane (2:8)] and HPLC [B,
EtOAc-hexane (2:8), 9 mL/min; tR: 11, 5.6 min; 12, 7.6 min]
to give spectroscopically pure 11 (465 mg, 27%) and 12 (125
mg, 7%). The combined yield of compound 11 was 82%.
Compound 11 was obtained as colorless needles (EtOAc):
mp 110-113 °C; [R]20D +389.9° (c 0.70, CHCl3); IR (CHCl3) νmax
1782, 1678 cm-1; 1H NMR δ 1.08 (3H, s, H-15), 1.26 (3H, d, J
) 6.9 Hz, H-12), 2.35 (1H, dq, J ) 12.1, 6.9 Hz, H-11), 2.83
(1H, br d, J ) 10.4 Hz, H-5), 4.17 (1H, dd, J ) 10.4, 10.4 Hz,
H-6), 5.54 (1H, d, J ) 1.8 Hz, H-14a), 5.82 (1H, d, J ) 1.8 Hz,
H-14b), 5.87 (1H, d, J ) 9.9 Hz, H-2), 7.05 (1H, d, J ) 9.9 Hz,
H-3); 13C NMR δ 12.36 (q, C-12), 17.74 (q, C-15), 22.35 (t), 31.47
(t), 40.58 (d, C-11), 47.17 (s, C-10), 49.49 (d, C-5), 51.91 (d,
C-7), 79.35 (d, C-6), 122.06 (t, C-14), 125.13 (d, C-2), 139.71
(s, C-4), 146.51 (d, C-3), 178.82 (s, C-13), 202.34 (s, C-1); anal.
C 73.08%, H 7.57%, calcd for C15H18O2, C 73.14%, H 7.37%.
Compound 12 was obtained as colorless needles (EtOAc):
mp 142-145 °C; [R]20D -102.3° (c 0.87, CHCl3); IR (CHCl3) νmax
Compounds 14 was obtained as colorless needles (EtOAc):
mp 145-146 °C; [R]20 +130.45° (c 0.892, CHCl3), IR (CHCl3)
D
νmax 3520, 1774 cm-1; 1H NMR δ 0.83 (3H, s, H-15), 1.23 (3H,
d, J ) 6.9 Hz, H-12), 2.11 (1H, br d, J ) 10.4 Hz, H-5), 3.50
(1H, dd, J ) 11.5, 4.7 Hz, H-1), 4.05 (1H, dd, J ) 10.4, 10.4
Hz, H-6), 4.83 (1H, d, J ) 1.4 Hz, H-14a), 4.97 (1H, d, J ) 1.4
Hz, H-14b); 13C NMR δ 11.59 (q, C-12), 12.45 (q, C-15), 22.95
(t), 31.18 (t), 33.46 (t, C-3), 35.89 (t, C-2), 41.13 (d, C-11), 42.79
(s, C-10), 52.25 (d, C-7), 52.40 (d, C-5), 78.15 (d, C-1), 79.29
(d, C-6), 110.23 (t, C-14), 142.75 (s, C-4), 179.36 (s, C-13); anal.
C 71.87%, H 8.87%, calcd for C15H22O3, C 71.97%, H 8.86%.
Compound 15 was obtained as colorless needles (EtOAc):
mp 146-148 °C; [R]20D +166.5° (c 0.92, CHCl3), IR (CHCl3) νmax
3520, 1774 cm-1; 1H NMR δ 0.86 (3H, s, H-15), 1.22 (3H, d, J
) 6.9 Hz, H-12), 2.70 (1H, d, J ) 11.4 Hz, H-5), 3.46 (1H, dd,
J ) 2.6, 2.6 Hz, H-1), 4.03 (1H, dd, J ) 11.4, 10.1 Hz, H-6),
4.80 (1H, d, J ) 1.5 Hz, H-14a), 4.95 (1H, d, J ) 1.5 Hz, H-14b);
13C NMR δ 12.42 (q, C-15), 18.18 (q, C-12), 22.90 (t), 29.90 (t),
30.07 (t), 33.20 (t), 41.08 (d, C-11), 42.71 (s, C-10), 47.03 (d,
C-5), 51.95 (d, C-7), 74.18 (d, C-1), 80.06 (d, C-6), 108.91 (t,
C-14), 144.58 (s, C-4), 179.61 (s, C-13); anal. C 71.85%, H
8.74%, calcd for C15H22O3, C 71.97%, H 8.86%.
Oxid a tion of 15 w ith Cr O3‚2P y. F or m a tion of 13 fr om
15. CrO3 (720 mg, 7.2 mmol) was added to a mixture of CH2-
Cl2 (6 mL) and pyridine (1.16 mL, 14.4 mmol) at 0 °C and
stirred for 10 min. Then, 15 (90 mg, 0.36 mmol) dissolved in
CH2Cl2 (4 mL) was added over 5 min. The mixture was stirred
at 0 °C for 3 h and then at room temperature for 2 h and
filtered through Celite under reduced pressure. The filtrate
was worked up to give a crystalline crude product of 13 (79
mg), which was subsequently purified by HPLC [C, EtOAc-
hexane (3:7), 3.0 mL/min] to give 13 (69 mg, 78%) as colorless
needles (EtOAc): mp 155 °C.
(11S)-1â-(Mesyloxy)eu d esm -4(14)-en o-13,6R-la cton e (4).
To a stirred solution of 14 (1.20 g, 4.80 mmol) in pyridine (50
mL) was added MsCl (533 µL, 7.20 mmol). The mixture was
stirred for 3.5 h at 25 °C and worked up to give spectroscopi-
cally pure 4 (1.53 g, 97%). Compound 4 was obtained as
colorless prisms (ether): mp 122-124 °C; [R]20D +98.9° (c 0.27,
CHCl3); IR (CHCl3) νmax 1774, 1338, 1174 cm-1; 1H NMR δ 0.92
(3H, s, H-15), 1.23 (3H, d, J ) 6.8 Hz, H-12), 3.03 (3H, s,
-OMs), 4.02 (1H, dd, J ) 10.4, 10.4 Hz, H-6), 4.57 (1H, dd, J
)11.5, 4.7 Hz, H-1), 4.89 (1H, d, J ) 1.3 Hz, H-14a), 5.04 (1H,
d, J ) 1.3 Hz, H-14b); 13C NMR δ 12.35 (q, C-15), 12.44 (q,
C-12), 22.60 (t), 28.98 (t), 32.88 (t), 35.83 (t), 38.90 (q, -OMs),
40.90 (d, C-11), 42.08 (s, C-10), 51.91 (d, C-7), 52.29 (d, C-5),
1788, 1670, 1638 cm-1 1H NMR δ 1.26 (3H, d, J ) 6.9 Hz,
;
H-12), 1.34 (3H, s, H-15), 2.17 (3H, d, J ) 1.8 Hz, H-14), 2.37
(1H, dq, J ) 11.9 Hz, 6.9, H-11), 4.72 (1H, br d, J ) 10.4 Hz,
H-6), 6.04 (1H, d, J ) 9.8 Hz, H-2), 6.84 (1H, d, J ) 9.8 Hz,
H-3); 13C NMR δ 12.31 (q, C-12), 18.95 (q, C-15), 23.23 (t), 26.10
(q, C-14), 35.33 (t), 40.91 (d, C-11), 50.41 (s, C-10), 53.05 (d,
C-7), 81.62 (d, C-6), 121.29 (s, C-5), 123.38 (d, C-2), 143.04 (s,
C-4), 148.93 (d, C-3), 177.92 (s, C-13), 204.40 (s, C-1); anal. C
73.14%, H 7.47%, calcd for C15H18O3, C 73.14%, H 7.37%.
Alter n a te Rou te for th e P r ep a r a tion of 11. Deh yd r o-
gen a tion of 6 w ith DDQ. A solution of anhydrous TsOH (139
mg), 6 (100 mg, 0.40 mmol), and DDQ (187 mg, 0.80 mmol) in
toluene (3 mL) was refluxed for 7 h. During this period, more
DDQ (94.3 mg, 0.40 mmol) was added into the reaction mixture
in two lots. After cooling, the reaction mixture was filtered
through Celite under reduced pressure. The filtrate was
worked up to give a crude oily product (112 mg), which was
purified by the combination of column chromatography [silica
gel, 8 g; 1.8-cm i.d., EtOAc-hexane (2:8)] and HPLC [A,
EtOAc-hexane (2:8), 3 mL/min] to give 11(tR 6.0 min, 42 mg,
41%) and recovered 6 (tR 7.2 min, 29 mg, 28%).
(11S)-1-Oxoeu d esm -4(14)-en o-13,6R-la cton e (13). A mix-
ture of 11 (495 mg, 2.01 mmol) and Zn-Hg28 in AcOH (13 mL)
was refluxed for 1 h under Ar and filtered. The filtrate was
poured into a saturated aqueous solution of NaCl (30 mL) and
extracted with EtOAc (4 × 10 mL). The combined extracts were
worked up to give spectroscopically pure 13 (409 mg, 82%) as
colorless needles. The mother liquor was subjected to the
combination of column chromatography [silica gel, 3.6 g; 1.3-
cm i.d., EtOAc-hexane (2:8)] and HPLC [A; EtOAc-hexane
(2:8), 3.0 mL/min] to give 13 (55 mg, 11%). The combined yield
of 13 was 93%. Compound 13 was obtained as colorless needles

Guaianolides as Immunomodulators
J ournal of Natural Products, 1999, Vol. 62, No. 1 27
78.48 (d, C-6), 87.66 (d, C-1), 111.43 (t, C-14), 140.98 (s, C-4),
178.89 (s, C-13); HREIMS m/z 328.1340 (calcd for C16H24O5S
328.1345).
matography [silica gel, 12 g; 2.0-cm i.d., EtOAc-hexane (2:8)]
and HPLC [A, EtOAc-hexane (2:8), 3.0 mL/min]. The first
peak (tR 3.2 min) gave spectroscopically pure 21 (62.6 mg, 44%)
Solvolysis of 4. F or m a tion of (11S)-Gu a ia -1(10),4(14)-
d ien o-13,6R-la cton e (16), (11S)-Gu a ia -4(14),9-d ien o-12,6R-
la cton e (17), a n d (11S)-Gu a ia -4(14),10(15)-d ien o-13,6R-
la cton e (1). A mixture of 4 (1.49 g, 4.55 mmol) and 0.5 M
KOAc in AcOH (120 mL) was refluxed under stirring for 22 h,
cooled, poured into a saturated aqueous solution of NaCl (150
mL), and extracted with EtOAc (3 × 50 mL). The combined
extracts were worked up to give a crude oily material (1.06 g)
which was passed through short column of silica gel [40 g; 3-cm
i.d., EtOAc-hexane (1:9)]. The eluent was further purified by
HPLC [C, EtOAc-hexane (5:95), 9.0 mL/min].
as colorless needles (EtOAc): mp 76-78 °C; [R]20 +20.8° (c
D
0.46, CHCl3); IR (CHCl3) νmax 1772 cm-1; 1H NMR δ 1.19 (3H,
d, J ) 6.9 Hz, H-12), 1.36 (3H, s, H-15), 2.82 (1H, dd, J )
10.0, 2.5 Hz, H-5), 3.99 (1H, dd, J ) 10.0, 9.8 Hz, H-6), 5.12
(1H, d, J ) 1.9 Hz, H-14a), 5.41 (1H, d, J ) 2.1 Hz, H-14b);
13C NMR δ 12.35 (q, C-12), 22.94 (q, C-15), 22.94 (t), 31.08 (t),
32.41 (t), 34.19 (t), 41.08 (d, C-11), 49.03 (d, C-5), 53.40 (d,
C-7), 63.15, 73.10 (s, C-1, C-10), 82.19 (d, C-6), 110.67 (t, C-14),
148.28 (s, C-4), 178.65 (s, C-13); HREIMS m/z 248.1414 (calcd
for C15H20O3 248.1413).
The second peak (tR 5.2 min) gave 22 (58.8 mg, 41%) as
The first peak (tR 4.0 min) gave a mixture (5:4) of (11S)-
guaia-1(10),3-dieno-13,6R-lactone (18) and (11S)-guaia-3,9-
dieno-13,6R-lactone (19) (73.1 mg, 7%). A part of this mixture
was purified by repeated HPLC to give 18 as colorless needles
colorless needles (EtOAc): mp 63-64 °C; [R]20D -21.2° (c 0.53,
1
CHCl3); IR (CHCl3) νmax 1774 cm-1; H NMR δ 1.23 (3H, d, J
) 6.7 Hz, H-12), 1.36 (3H, s, H-15), 2.29 (1H, dq, J ) 12.6, 6.7
Hz, H-11), 2.61 (1H, d, J ) 11.3 Hz, H-5), 3.74 (1H, dd, J )
11.3, 9.8 Hz, H-6), 5.15 (2H, br s, H-14); 13C NMR δ 12.42 (q,
C-12), 20.49 (q, C-15), 25.00 (t), 30.04 (t), 30.44 (t), 37.42 (t),
42.25 (d, C-11), 53.96 (d, C-5), 55.52 (d, C-7), 62.82, 72.51 (s,
C-1, C-10), 81.28 (d, C-6), 112.06 (t, C-14), 147.04 (s, C-4),
177.74 (s, C-13); anal. C 72.68, H 8.08, calcd for C15H20O3, C
72.55, H 8.12.
(EtOAc): mp 80 °C; [R]25 +8.9° (c 0.41, CHCl3); IR (CHCl3)
D
ν
max 1762 cm-1; 1H NMR δ 1.22 (3H, d, J ) 6.9 Hz, H-12), 1.72
(3H, br s, H-15), 1.91 (3H, br s, H-14), 2.96 (2H, br s, H-2),
3.30 (1H, br d, J ) 9.9 Hz, H-5), 3.66 (1H, dd, J ) 9.9, 9.9 Hz,
H-6), 5.52 (1H, br s, H-3); anal. C 77.03%, H 8.65%, calcd for
C
15H20O2, C 77.55%, H 8.68%. 19: 1H NMR (based on the
mixture of 18 and 19) δ 1.23 (3H, d, J ) 7.0 Hz, H-12), 1.79
(3H, br s, H-15), 1.91 (3H, br s, H-14), 4.03 (1H, dd, J ) 9.3,
9.3 Hz, H-6), 5.46 (1H, br d, J ) 7.7 Hz, H-9), 5.52 (1H, br s,
H-3).
P h en ylselen en yla tion of a m ixtu r e (1:1.6:4) of 16, 17,
a n d 1. F or m a tion of 11R-(P h en ylselen o)gu a ia -4(14),10-
(15)-d ien o-13,6R-la cton e (23), 11R-(P h en ylselen o)gu a ia -
4(14),9-dien o-13,6R-lacton e (24), 11â-(P h en ylselen o)gu aia-
4(14),9-dien o-13,6R-lacton e (25), 11â-(P h en ylselen o)gu aia-
1(10),4(14)-d ien o-13,6R-la ct on e (26), a n d 11â-(P h en yl-
selen o)gu a ia -4(14),10(15)-d ien o-13,6R-la cton e (27). A solu-
tion of the mixture of 16, 17, and 1 (400 mg, 1.72 mmol) in
THF (6 mL) was slowly added to a cooled (-76 °C) solution of
lithium diisopropylamide [prepared from diisopropylamine
(746 µL, 5.31 mmol), 1.6 M BuLi in hexane (3.3 µL, 5.31 mmol)]
in THF (6 mL). After 1 h, a solution of diphenyl diselenide
(1.66 g, 5.31 mmol) in THF (5 mL) and HMPA (926 µL, 5.31
mmol) was added at -76 °C. The reaction mixture was stirred
at -76 °C for 30 min and then warmed to -40 °C over period
of 30 min, and stirring was continued for an additional 1.5 h.
The reaction was quenched by addition of 0.2 M aqueous
solution of HCl (40 mL) at -10 °C. The mixture was extracted
with EtOAc (3 × 20 mL). The combined extracts were worked
up to give a crude oily product (2.3 g), which was passed
through a short column of silica gel. The eluent was then
separated by HPLC [B, EtOAc-hexane (5:95), 3.0 mL/min].
The first peak (tR 2.8 min) gave 23 (47.7 mg, 7%) as a pale
The second peak (tR 5.6 min) gave (11S)-guaia-3,10(15)-
dieno-12,6R-lactone (20) (95.1 mg, 9%) as a pale yellow oil:
[R]25 +71.4° (c 0.64, CHCl3); IR (CHCl3) νmax 1760, 1640, 902
D
1
cm-1; H NMR δ 1.24 (3H, d, J ) 6.8 Hz, H-12), 1.83 (3H, br
s, H-14), 2.78 (1H, br dd, J ) 9.0, 9.7 Hz, H-5), 3.10 (1H, q, J
) 7.0 Hz, H-1), 3.98 (1H, dd, J ) 9.7, 9.7 Hz, H-6), 4.84 (1H,
d, J ) 1.7 Hz, H-15), 4.87 (1H, d, J ) 1.7 Hz, H-15), 5.53 (1H,
br s, H-3); HREIMS m/z 232.1450 (calcd for C15H20O2 232.1463).
The third peak gave 16 (169.1 mg, 16%) as a highly unstable
1
viscous oil: IR (CHCl3) νmax 1770 cm-1; H NMR δ 1.23 (3H,
d, J ) 6.9 Hz, H-12), 1.75 (3H, s, H-15), 3.24 (1H, br dd, J )
10.1, 1.2 Hz, H-5), 3.65 (1H, dd, J ) 9.9, 9.9 Hz, H-6), 5.08
(1H, d, J ) 1.2 Hz, H-14a), 5.13 (1H, d, J ) 1.2 Hz, H-14b).
The fourth peak gave a mixture (1:1.6:4) of 16, 17, and 1
(496.8 mg, 47%), which was employed for the next reaction
without further purification. 17: 1H NMR (scanned on the total
mixture of 16,17, and 1) δ 1.23 (3H, d, J ) 7.0 Hz, H-12), 1.82
(3H, s, H-15), 3.95 (1H, dd, J ) 9.8, 9.8 Hz, H-6), 5.52 (1H, br
d, J ) 7.7 Hz, H-9). Repeated HPLC of the mixture gave pure
1
Mokko Lactone (1) as a colorless oil: [R]20 +26.9° (c 0.31,
yellow oil: IR (CHCl3) νmax 1770, 1644 cm-1; H NMR δ 1.42
D
CHCl3); IR (CHCl3) νmax 1744, 1676 cm-1; 1H NMR (500 MHz)
δ 1.24 (3H, d, J ) 7.0 Hz, H-12), 1.30 (1H, dddd, J ) 12.1,
12.1, 11.7, 5.1 Hz, H-8), 1.86 (1H, dddd, J ) 17.2, 10.3, 4.5,
1.5 Hz, H-2), 1.95 (2H, m, H-2, H-7), 2.05 (1H, ddd, J ) 12.1,
12.1, 5.1 Hz, H-9), 2.11 (1H, dddd, J ) 12.5, 10.3, 5.0, 1.7 Hz,
H-3), 2.21 (1H, dq, J ) 11.7, 7.0 Hz, H-11), 2.52 (3H, m, H-3,
H-8, H-9), 2.80 (1H, dd, J ) 9.4, 8.0 Hz, H-5), 2.88 (1H, ddd,
J ) 8.0, 8.0, 4.5 Hz, H-1), 3.92 (1H, dd, J ) 9.4, 9.4 Hz, H-6),
4.78 (1H, br s, Wh/2 ) 3.0 Hz, H-15a), 4.88 (1H, br s, Wh/2 ) 3.0
Hz, H-15b), 5.05 (1H, d, J ) 2.2 Hz, H-14a), 5.20 (1H, d, J )
2.0 Hz, H-14b); 13C NMR (125 MHz) δ 13.23 (q, C-12), 30.18
(t, C-2), 32.49 (t, C-3, C-8), 37.64 (t, C-9), 42.05 (d, C-11), 47.07
(d, C-1), 49.88 (d, C-7), 51.95 (d, C-5), 85.25 (d, C-6), 109.17 (t,
C-14), 111.82 (t, C-15), 149.94 (s, C-10), 151.68 (s, C-4), 178.66
(s, C-13); HREIMS m/z 232.1456 (calcd for C15H20O2 232.1463).
Ep oxid a tion of 16 w ith m -CP BA. F or m a tion of (11S)-
1â,10â-Ep oxygu a i-4(14)-en o-13,6R-la cton e (21) a n d (11S)-
1R,10R-Ep oxygu a i-4(14)-en o-13,6R-la cton e (22). A solution
of 16 (134.0 mg, 0.577 mmol) in CHCl3 (12 mL) was stirred
with 85% m-CPBA (117.1 mg, 0.577 mmol) for 1 h at 0 °C,
poured into a mixture of a 0.1 M aqueous solution of KI (15
mL) and a saturated aqueous solution of NaCl (10 mL), and
extracted with CHCl3 (4 × 10 mL). The combined extracts were
washed with a 0.1 M aqueous solution of Na2S2O3 (3 × 8 mL)
and a saturated aqueous solution of NaCl (3 × 8 mL), dried
(Na2SO4), and concentrated to give a crude oily product (175
mg), which was purified by the combination of column chro-
(3H, s, H-12), 3.91 (1H, dd, J ) 9.6, 9.6, H-6), 4.74 (1H, br s,
H-15a), 4.84 (1H, br s, H-15b), 5.00 (1H, br s, H-14a), 5.09 (1H,
br s, H-14b), 7.27-7.42 (3H, m, Ph), 7.67-7.73 (2H, m, Ph).
The second peak (tR 3.6 min) gave a mixture (1:6) of 24 and
25 (168.2 mg, 25%). Repeated purification of this mixture by
HPLC employing the same conditions gave spectroscopically
pure 25 as colorless crystals: mp 104-106 °C; [R]20 +95.4°
D
(c 0.31, CHCl3); IR (CHCl3) νmax 1758, 1662 cm-1; 1H NMR (500
MHz) δ 1.53 (3H, s, H-12), 1.82 (3H, br s, H-15), 1.96 (1H, ddd,
J ) 11.5, 10.1, 3.2 Hz, H-7), 4.17 (1H, dd, J ) 10.1, 10.1 Hz,
H-6), 4.99 (1H, br s, H-14a), 5.18 (1H, br s, H-14b), 5.57 (1H,
ddd, J ) 8.1, 2.7, 1.5 Hz, H-9), 7.27-7.45 (3H, m, Ph), 7.58-
7.68 (2H, m, Ph); 13C NMR δ 22.10 (q, C-12), 26.82 (t, C-8),
27.84 (q, C-15), 29.14 (t), 29.80 (t), 47.58 (d, C-5), 51.28 (s,
C-11), 51.56 (d, C-1), 52.69 (d, C-7), 81.42 (d, C-6), 110.56 (t,
C-14), 121.25 (d, C-9), 124.55 (s, Ph), 128.94 (d, m-Ph), 129.72
(d, p-Ph), 138.33 (d, o-Ph), 138.44 (s, C-10), 150.28 (s, C-4),
1
175.76 (s, C-13). H NMR of 24 (scanned on the mixture of 24
and 25) δ 1.41 (3H, s, H-12), 1.77 (3H, br s, H-15), 3.99 (1H,
dd, J ) 9.6, 9.6 Hz, H-6), 4.95 (1H, br s, H-14a), 5.08 (1H, br
s, H-14b), 5.45-5.55 (1H, m, H-9), 7.27-7.45 (3H, m, Ph),
7.68-7.72 (2H, m, Ph).
The third peak (tR 4.4 min) gave 26 (78.5 mg, 12%) as a
pale yellow oil: [R]20 +69.9° (c 0.39, CHCl3); IR (CHCl3) νmax
D
1768 cm-1; 1H NMR δ 1.53 (3H, s, H-12), 1.77 (3H, br s, H-15),
2.06 (1H, ddd, J ) 10.3, 10.3, 2.9 Hz, H-7), 3.20 (1H, br d, J )
10.3 Hz, H-5), 4.08 (1H, dd, J ) 10.3, 10.3 Hz, H-6), 5.09 (1H,

28 J ournal of Natural Products, 1999, Vol. 62, No. 1
Yuuya et al.
br s, H-14a), 5.14 (1H, br s, H-14b), 7.28-7.44 (3H, m, Ph),
7.58-7.64 (2H, m, Ph); 13C NMR δ 21.84 (q, C-12), 23.04 (q,
C-15), 25.46 (t), 31.57 (t), 32.74 (t), 34.71(t), 50.77 (s, C-11),
51.99 (d, C-5), 60.51 (d, C-7), 79.48 (d, C-6), 110.61 (t, C-14),
124.21 (s, Ph), 128.86 (d, m-Ph), 129.58 (d, p-Ph), 131.79 (s,
C-1), 135.91 (s, C-10), 138.29 (d, o-Ph), 149.68 (s, C-4), 175.43
(s, C-13); HREIMS m/z 388.0950 (calcd for C21H24O2Se
388.0942).
Gu a ia -1(10),4(14),11(12)-tr ien o-13,6R-la cton e (30). The
oxidative syn elimination of 26 by the method mentioned above
gave 30 (61%) as a colorless oil: IR (CHCl3) νmax 1768 cm-1
;
1H NMR δ 1.76 (3H, br s, H-15), 3.33 (1H, br d, J ) 10.2 Hz,
H-5), 3.64 (1H, dd, J ) 10.2, 10.2 Hz, H-6), 5.10 (1H, br s,
H-14a), 5.17 (1H, br s, H-14b), 5.38 (1H, d, J ) 3.1 Hz, H-12a),
6.11 (1H, J ) 3.3 Hz, H-12b); 13C NMR δ 23.34 (q, C-15), 25.87
(t, C-8), 31.78 (t), 32.82 (t), 34.45 (t, C-9), 52.40 (d, C-5), 53.14
(d, C-7), 81.97 (d, C-6), 110.61 (t, C-14), 117.52 (t, C-12), 131.43
(s), 136.00 (s), 140.42 (s, C-11), 149.52 (s, C-4), 169.81 (s, C-13);
HREIMS m/z 230.1307 (calcd for C15H18O2 230.1307).
The fourth peak (tR 6.4 min) gave a mixture (1:4.3) of 17
and 1 (23.22 mg, 6%).
The fifth peak (tR 7.2 min) gave 27 (269.6 mg, 41%) as
colorless prisms (EtOAc): mp 127-128 °C; [R]20 +19.3° (c
Deh yd r ocostu s La cton e (2). The oxidative syn elimina-
tion of 27 by the method mentioned above gave dehydrocostus
D
0.92, CHCl3); IR (CHCl3) νmax 1754, 1644 cm-1; 1H NMR δ 1.55
(3H, s, H-12), 2.14 (1H, dd, J ) 9.5, 3.5 Hz, H-5), 4.08 (1H,
dd, J ) 9.5, 9.5 Hz, H-6), 4.81 (1H, s, H-15a), 4.92 (1H, s,
H-15b), 5.05 (1H, d, J ) 2.1 Hz, H-14a), 5.15 (1H, d, J ) 1.8
lactone 2 (99%) as colorless needles (EtOAc): mp 49 °C; [R]20
D
-14.8° (c 0.42, CHCl3); IR (CHCl3) νmax 1762, 1644 cm-1; H
1
NMR (500 MHz) δ 1.42 (1H, dddd, J ) 13.2, 11.5, 10.2, 5.6
Hz, H-8), 1.87 (1H, ddd, J ) 13.2, 4.7, 2.7 Hz, H-2), 1.95 (1H,
ddd, J ) 13.2, 7.6, 1.7 Hz, H-2), 2.16 (1H, dddd, J ) 12.2, 10.4,
5.6, 5.6 Hz, H-9), 2.24 (1H, dddd, J ) 13.2, 8.2, 5.8, 4.4 Hz,
H-8), 2.87 (1H, dd, J ) 9.3, 3.0 Hz, H-5), 2.88-2.95 (2H, m,
H-1, H-7), 3.97 (1H, dd, J ) 9.3, 9.3 Hz, H-6), 4.82 (1H, s,
H-15a), 4.90 (1H, s, H-15b), 5.07 (1H, br d, J ) 2.0 Hz, H-14a),
5.27 (1H, br d, J ) 2.0 Hz, H-14b), 5.49 (1H, d, J ) 3.2 Hz,
H-12a), 6.22 (1H, d, J ) 3.3 Hz, H-12b); 13C NMR (125 MHz)
δ 30.28 (t, C-2), 30.91 (t, C-8), 32.57 (t, C-3), 36.24 (t, C-9),
45.11 (d, C-7), 47.60 (d, C-1), 52.01 (d, C-5), 85.22 (d, C-6),
109.59 (t, C-14), 112.60 (t, C-15), 120.16 (t, C-12), 139.73 (s,
C-11), 149.20 (s, C-10), 151.21 (s, C-4), 170.24 (s, C-13); anal.
C 77.67%, H 7.97%, calcd for C15H18O2, C 78.23%, H 7.88%.
Selective Ep oxid a tion of 17 in a Mixtu r e (1:4.3) of 17
a n d Mok k o La cton e (1) To Give 9R,10R-Ep oxygu a i-4(14)-
en o-13,6R-la cton e (31). A solution of a mixture (1:4.3) of 17
and 1 (42 mg, 0.064 mmol) in CHCl3 (0.8 mL) was stirred with
78% m-CPBA (2.94 mg, 0.013 mmol) for 1 h at 0 °C. The
reaction mixture was worked up to give a colorless oil (15 mg),
which was purified by the combination of column chromatog-
raphy [silica gel, 2 g; 1.2-cm i.d., EtOAc-hexane (1:9 and 2:8)]
and HPLC [A, EtOAc-hexane (5:95), 3.0 mL/min]. The peak
(tR 6.8 min) gave Mokko Lactone (1) (4.0 mg, 48%) on
evaporation of the solvent. Slow elution gave 31 (2.1 mg, 13%)
Hz, H-14b), 7.28-7.44 (3H, m, Ph), 7.58-7.64 (2H, m, Ph); 13
C
NMR δ 22.28 (q, C-12), 29.76 (t), 29.91 (t), 32.14 (t), 37.01 (t),
47.10 (d, C-1), 51.40 (s, C-11), 51.78 (d, C-7), 54.60 (d, C-5),
82.49 (d, C-6), 109.73 (t, C-15), 112.13 (t, C-14), 124.75 (s, Ph),
128.92 (d, m-Ph), 129.72 (d, p-Ph), 138.35 (d, o-Ph), 149.85 (s,
C-10), 151.08 (s, C-4), 175.82 (s, C-13); HREIMS m/z 388.0949
(calcd for C21H24O2Se 388.0942).
Gu a ia -4(14),7(11),10(15)-tr ien o-13,6R-la cton e (28). A so-
lution of 23 (4.2 mg, 0.01 mmol) in THF (200 µL) containing
AcOH (2.0 µL, 0.03 mmol) was treated at 0 °C with 31% H2O2
(6.0 µL, 0.06 mmol) for 1 h. The reaction mixture was poured
into a saturated aqueous solution of NaHCO3 (5 mL) and
extracted with EtOAc (3 × 4 mL). The combined extracts were
worked up to give a crude oily product (3.5 mg), which was
purified by HPLC [A, EtOAc-hexane (2:8), 2.4 min] to give
28 (2.2 mg, 87%) as colorless plates (EtOAc): mp 82-84 °C;
[R]20D +28.1° (c 0.17, CHCl3); IR (CHCl3) νmax 1744, 1670 cm-1
;
1H NMR δ 1.80 (H, br s, H-12), 2.49 (1H, m, H-5), 2.88 (1H,
m, H-1), 4.63 (1H, br d, J ) 12.1 Hz, H-6), 4.94 (1H, d, J ) 1.7
Hz, H-15a), 4.96 (1H, br s, H-15b), 5.11 (1H, br s, H-14a), 5.19
(1H, br s, H-14b); 13C NMR δ 8.58 (q, C-12), 28.91 (t), 29.74
(t), 30.41 (t), 30.68 (t), 48.86 (d, C-1), 51.57 (d, C-5), 80.63 (d,
C-6), 111.21 (s), 112.21 (t, C-14), 113.12 (t, C-15), 122.97 (s),
149.28 (s, 2C), 162.73 (s, C-13); HREIMS m/z 230.1308 (calcd
for C15H18O2 230.1307).
as colorless plates (EtOAc): mp 114-116 °C; [R]20 -12.0° (c
D
1
Er em a n th in (3) a n d Gu a ia -4(14),7(11),9-tr ien o-13,6R-
la cton e (29). The oxidative syn elimination of a mixture (1:
6) of 24 and 25 (152.8 mg, 0.39 mmol) by the method
mentioned above gave a mixture of eremanthin (3) and the
endo-unsaturated γ-lactone (29), which were separated by
HPLC [A, EtOAc-hexane (5:95), 3.0 mL/min]. The first peak
(tR 2.8 min) gave 3 (69.3 mg, 76%) as colorless needles
(EtOAc): mp 63 °C; [R]20D -106.6° (c 0.55, CHCl3); IR (CHCl3)
1.0, CHCl3); IR (CHCl3) νmax 1766, 1660 cm-1; H NMR (500
MHz) δ 1.23 (3H, d, J ) 6.8 Hz, H-12), 1.38 (3H, s, H-15), 1.66
(1H, m, H-3), 1.84 (1H, dd, J ) 15.1, 12.0 Hz, H-8ax), 2.00
(1H, dddd, J ) 12.3, 12.0, 10.1, 2.7 Hz, H-7), 2.06 (1H, m, H-2),
2.27 (1H, dq, J ) 12.3, 6.8 Hz, H-11), 2.40 (1H, ddddd, J )
17.6, 9.3, 9.3, 2.2, 2.2 Hz, H-3), 2.49 (1H, ddd, J ) 15.1, 5.4,
2.7 Hz, H-8eq), 2.52-2.62 (3H, m, H-1, H-2, H-5), 3.03 (1H, d,
J ) 5.4 Hz, H-9), 3.65 (1H, dd, J ) 10.1, 10.1 Hz, H-6), 4.95
(1H, br s, Wh/2 ) 5.0 Hz, H-14a), 5.10 (1H, br s, Wh/2 ) 5.0 Hz,
H-14b); HREIMS m/z 248.1409 (calcd for C15H20O3 248.1416).
9R,10R-Epoxy-11â-(ph en ylselen o)gu aia-4(14)-en o-13,6R-
la cton e (32). Phenylselenenylation of 31 gave 32 and unre-
acted 31, which were separated by flash chromatography. The
first elution gave 32 (44%) as colorless needles (EtOAc): mp
152-154 °C; IR (CHCl3) νmax 1760, 1660 cm-1; 1H NMR δ 1.38
(3H, s, H-15), 1.53 (3H, s, H-12), 3.10 (1H, d, J ) 5.5 Hz, H-9),
3.82 (1H, dd, J ) 9.8, 9.8 Hz, H-6), 4.94 (1H, br s, Wh/2 ) 5.0
Hz, H-14a), 5.09 (1H, br s, Wh/2 ) 5.0 Hz, H-14b), 7.26-7.47
(3H, m, Ph), 7.55-7.65 (2H, m, Ph).
ν
max 1762, 1660 cm-1; 1H NMR (500 MHz) δ 1.54 (1H, m, H-2),
1.84 (3H, br s, H-15), 2.01 (1H, m, H-8), 2.17 (1H, m, H-2),
2.37 (1H, m, H-3), 2.50-2.73 (5H, m, H-1, H-3, H-5, H-7, H-8),
3.94 (1H, dd, J ) 10.3, 9.4 Hz, H-6), 5.04 (1H, br s, H-14a),
5.21 (1H, br s, H-14b), 5.47 (1H, d, J ) 3.2 Hz, H-12a), 5.53
(1H, br d, J ) 7.7 Hz, H-9), 6.20 (1H, d, J ) 3.4 Hz, H-12b);
13C NMR (125 MHz) δ 27.95 (q, C-15), 29.10 (t, C-8), 29.52 (t,
C-3), 30.51 (t, C-2), 45.27 (d, C-7), 47.03 (d, C-1), 52.49 (d, C-5),
83.21 (d, C-6), 110.92 (t, C-14), 119.39 (t, C-12), 120.68 (d, C-9),
138.09 (s, C-10), 139.99 (s, C-11), 149.92 (s, C-4), 170.00 (s,
C-13); anal. C 77.94%, H 8.07%, calcd for C15H18O2, C 78.23%,
H 7.88%.
Further flash chromatography gave recovered 31 (36%).
9R, 10R-E p oxygu a ia -4(14),11(12)-d ien o-13,6R-la ct on e
(33). The oxidative syn elimination of 32 gave 33 (95%) as
The second peak (tR 4.6 min) gave 29 (11.9 mg, 13%) as a
colorless oil: [R]20 +91.2° (c 0.07, CHCl3); IR (CHCl3) νmax
D
1
1744, 1674 cm-1; H NMR (500 MHz) δ 1.78 (3H, br s, H-15),
colorless needles (EtOAc): mp 136-138 °C; [R]20 -36.6° (c
D
1
1.81 (3H, dd, J ) 3.2, 1.5 Hz, H-12), 1.94 (2H, m, H-2), 2.39
(1H, dd, J ) 9.2, 7.7 Hz, H-5), 2.51 (1H, m, H-3), 2.89 (1H, dd,
J ) 13.2, 7.7 Hz, H-1), 3.13 (1H, dd, J ) 18.6, 6.7 Hz, H-8),
3.29 (1H, d, J ) 18.6 Hz, H-8), 4.67 (1H, br d, J ) 9.2 Hz,
H-6), 5.05 (1H, dd, J ) 4.4, 2.2 Hz, H-14a), 5.29 (1H, dd, J )
4.4, 2.3 Hz, H-14b), 5.49 (1H, m, H-9); 13C NMR (125 MHz) δ
8.41 (q, C-15), 22.94 (q, C-12), 25.91 (t, C-8), 27.26 (t, C-2),
30.82 (t, C-3), 43.74 (d, C-1), 51.17 (d, C-5), 82.85 (d, C-6),
108.95 (t, C-14), 118.23 (d, C-9), 139.63 (s, C-10), 151.12 (s,
2C), 160.57 (s, C-7), 174.58 (s, C-13); HREIMS m/z 230.1320
(calcd for C15H18O2 230.1307).
0.40, CHCl3); IR (CHCl3) νmax 1770, 1664 cm-1; H NMR (500
MHz) δ 1.39 (3H, s, H-15), 1.66 (1H, dddd, J ) 12.9, 12.9, 10.7,
8.8 Hz, H-2ax), 1.91 (1H, dd, J ) 15.4, 12.0 Hz, H-8ax), 2.07
(1H, m, H-2eq), 2.40 (1H, ddddd, J ) 17.6, 8.8, 8.8, 2.0, 2.0
Hz, H-3), 2.55 (1H, m, H-3), 2.63 (1H, dd, J ) 12.9, 6.2 Hz,
H-1), 2.68 (1H, dd, J ) 10.7, 6.2 Hz, H-5), 2.63 (1H, ddd, J )
15.4, 9.3, 4.9 Hz, H-8eq), 2.90 (1H, dddd, J ) 12.0, 9.5, 3.4,
3.4 Hz, H-7), 3.09 (1H, d, J ) 4.9 Hz, H-9), 3.65 (1H, dd, J )
10.7, 9.5 Hz, H-6), 4.99 (1H, br s, Wh/2 ) 4.0 Hz, H-14a), 5.12
(1H, br s, Wh/2 ) 4.0 Hz, H-14b), 5.48 (1H, d, J ) 3.4 Hz,
H-12a), 6.19 (1H, d, J ) 3.4 Hz, H-12b); 13C NMR (125 MHz)

Guaianolides as Immunomodulators
J ournal of Natural Products, 1999, Vol. 62, No. 1 29
δ 26.23 (q, C-15), 27.63 (t), 28.29 (t), 28.37 (t), 40.90 (d, C-7),
46.61 (d), 52.07 (d), 61.64 (d, C-9), 63.40 (s, C-10), 81.58 (d,
C-6), 111.16 (t, C-14), 119.11 (t, C-12), 139.59 (s, C-11), 148.50
(s, C-4), 169.66 (s, C-13); HREIMS m/z 246.1257 (calcd for
H-2, H-7), 2.73 (1H, br d, J ) 11.0 Hz, H-5), 3.75 (1H, dd, J )
11.0, 10.0 Hz, H-6), 5.18 (2H, d, J ) 1.2 Hz, H-14), 5.44 (1 H,
d, J ) 3.0 Hz, H-12a), 6.16 (1H, d, J ) 3.5 Hz, H-12b); 13C
NMR (125 MHz) δ 20.69 (q, C-15), 23.52 (t, C-8), 30.02 (t, C-2),
30.45 (t, C-3), 36.89 (t, C-9), 51.90 (d, C-7), 54.19 (d, C-5), 67.72
(s, C-10), 72.61 (s, C-1), 81.35 (d, C-6), 112.18 (t, C-14), 118.48
(t, C-12), 139.72 (s, C-11), 146.79 (s, C-4), 169.32 (s, C-13); anal.
C 72.69%, H 7.33%, calcd for C15H18O3, C 73.14%, H 7.37%.
C
15H18O3 246.1256).
P h en ylselen en yla t ion of 21. F or m a t ion of 1â,10â-
E p oxy-11R-(p h e n ylse le n o)gu a ia -4(14)-e n o-13,6R-la c-
t on e (34) a n d 1â,10â-E p oxy-11â-(p h en ylselen o)gu a ia -
4(14)-en o-13,6R-la ct on e (35). Phenylselenenylation of 21
gave 34 and 35, which were separated by flash chromatogra-
phy. The first elution gave 34 (7%) as a pale yellow oil: 1H
NMR δ 1.33 (3H, s, H-15), 1.40 (3H, s, H-12), 2.49 (1H, br d,
J ) 9.8 Hz, H-5), 4.10 (1H, dd, J ) 9.8, 9.8 Hz, H-6), 5.08 (1H,
d, J ) 2.1 Hz, H-14a), 5.33 (1H, d, J ) 2.2 Hz, H-14b), 7.27-
7.45 (3H, m, Ph), 7.60-7.70 (2H, m, Ph).
Ma ter ia ls a n d Meth od s for Bioa ssa ys
Cells. A mouse H-2d-specific CD8+ CTL clone, OE4, and
a human lung adenocarcinoma clone, A549, were main-
tained in RPMI 1640 medium supplemented with 10% (v/
v) fetal calf serum, 5% (v/v) rat-conditioned medium
(culture supernatant of rat splenocytes stimulated with 5
mg/mL of concanavalin A for 24 h), 50 mM 2-mercapto-
ethanol, 50 mg/mL of kanamycin, and 8 mg/mL of tylosin
tartrate. OE4 was stimulated with mitomycin C-treated
splenocytes from BALB/c mice (female, 6-8 weeks old,
Charles River Co., Ltd., Yokohama, J apan) every other
week and used for experiments at least 5 days after the
stimulation. P815 mastocytoma was maintained in the
medium described above, but without rat-conditioned
medium.
An tibod ies a n d Cytok in e. C167 (mouse anti human
ICAM-1 antibody) was purchased from Leinco Technologies
Inc., Ballwin, MO, and sheep peroxidase-linked anti-mouse
Ig antibody was from Amersham International, Bucking-
hamshire, U.K. Recombinant human IL-1â was a com-
mercial product of genzyme diagnostics, Cambridge, MA.
Cyt ot oxicit y Assa y. The target cell (TC) P815(H-2d)
was incubated with [3H]thymidine (1 mci/mL) for 18 h and
washed three times with the medium before use. OE4 (1
× 104 cells/well in 100 mL) was incubated with or without
test compounds for 2 h in U-bottom microtiter plates and
mixed with 100 mL of TC P815 (1 × 104 cells/mL). The
plates were centrifuged (300g) for 3 min and incubated for
4 h. To assess DNA degradation, 10 mL of 2% (v/v) Triton
X-100 was added to each well at the end of the incubation,
and the cells were solubilized by pipetting and centrifuged
(600g, 5 min). One hundred microliters of supernatant was
removed and measured for radioactivity. The percentage
of specific lysis was calculated by using the following
formula as described in a previous paper:20
The second elution gave 35 (78%) as a pale yellow oil: IR
1
(CHCl3) νmax 1764, 1662 cm-1; H NMR δ 1.37 (3H, s, H-15),
1.49 (3H, s, H-12), 4.35 (1H, dd, J ) 10.1, 9.2 Hz, H-6), 5.12
(1H, d, J ) 1.9 Hz, H-14a), 5.41 (1H, d, J ) 2.1 Hz, H-14b),
7.27-7.44 (3H, m, Ph), 7.58-7.65 (2H, m, Ph); 13C NMR δ
21.06 (t), 21.84 (q, C-15), 22.86 (q, C-12), 31.09 (t), 32.41 (t),
34.25 (t), 49.06 (d, C-5), 50.01 (s, C-11), 57.37 (d, C-7), 62.84
(s), 72.88 (s), 80.22 (d, C-6), 110.71 (t, C-14), 124.33 (s, Ph),
128.88 (d, m-Ph), 129.63 (d, p-Ph), 138.29 (d, o-Ph), 148.15 (s,
C-4), 175.96 (s, C-13).
1â,10â-Epoxygu a ia -4(14),7(11)-d ien o-13,6R-la cton e (36).
The oxidative syn elimination of 34 gave spectroscopically pure
36 (86%) as a colorless oil: IR (CHCl3) νmax 1764, 1646 cm-1
;
1H NMR δ 1.39 (3H, s, H-15), 1.80 (3H, s, H-12), 4.74 (1H, d,
J ) 10.3 Hz, H-6), 5.19 (1H, br s, Wh/2 ) 4.0 Hz, H-14a), 5.43
(1H, br s, H-14b); HREIMS m/z 246.1256 (calcd for C15H18O3
246.1256).
1â,10â-Epoxygu aia-4(14),11(12)-dien o-13,6R-lacton e (37).
The oxidative syn elimination of 35 gave 37 (85%) as colorless
plates (EtOAc): mp 125-126 °C; [R]20D +34.6° (c 0.10, CHCl3);
1
IR (CHCl3) νmax 1766, 1660 cm-1; H NMR (500 MHz) δ 1.38
(3H, s, H-15), 1.49 (1H, dddd, J ) 13.9, 12.9, 11.2, 2.7 Hz, H-8),
1.68 (1H, ddd, J ) 13.9, 8.8, 3.7 Hz, H-2), 1.86 (1H, dddd, J )
13.9, 4.7, 3.3, 1.7 Hz, H-8), 2.05 (1H, ddd, J ) 15.6, 12.9, 3.3
Hz, H-9), 2.15 (1H, ddd, J ) 13.9, 9.8, 9.8 Hz, H-2), 2.24 (1H,
ddd, J ) 15.6, 4.7, 2.7 Hz, H-9), 2.30 (1H, ddd, J ) 11.2, 10.3,
1.7 Hz, H-7), 2.48 (1H, m, H-3), 2.61 (1H, m, H-3), 2.94 (1H,
br dd, J ) 10.3, 2.3 Hz, H-5), 3.98 (1H, dd, J ) 10.3, 10.3 Hz,
H-6), 5.15 (1H, dd, J ) 4.6, 2.0 Hz, H-14a), 5.44 (1H, d, J )
2.9 Hz, H-12a), 5.47 (1H, dd, J ) 4.4, 2.2 Hz, H-14b), 6.18 (1H,
d, J ) 3.4 Hz, H-12b); 13C NMR (125 MHz) δ 21.70 (t, C-8),
23.01 (q, C-15), 31.02 (t, C-3), 32.44 (t, C-2), 34.04 (t, C-9), 49.39
(d, C-5), 49.70 (d, C-7), 63.25 (s, C-10), 73.10 (s, C-1), 82.38 (d,
C-6), 110.90 (t, C-14), 118.65 (t, C-12), 138.89 (s, C-11), 147.93
(s, C-4), 170.11 (s, C-13); anal. C 72.99%, H 7.43%, calcd for
% of specific lysis ) (experimental release -
spontaneous release)/(maximum release -
spontaneous release) × 100
C
15H18O3, C 73.14%, H 7.37%.
1R,10R-Epoxy-11â-(P h en ylselen o)gu aia-4(14)-en o-13,6R-
la cton e (38). Phenylselenenylation of 22 gave 38 and recov-
ered 22, which were separated by flash chromatography. The
first elution gave 38 (60%) as colorless needles (EtOAc): mp
Assa y of ICAM-1 Exp r ession . After 1 h of A549 cells
with or without test compounds in a microtiter plate at 3
× 104 cells/well in 150 mL, 50 mL of IL-1â was added to
the culture at a final concentration of 280 U/mL, and the
cells were further incubated for 6 h. The cells were washed
once with phospate-buffered saline (PBS) and fixed by 15
min of incubation with 1% paraformaldehyde-PBS for 15
min and then washed once with PBS. After blocking with
1% bovine serum albumin-PBS for 60 min at 37 °C, the
cells were treated with mouse anti-human ICAM-1 anti-
body for 60 min at 37 °C. After being washed with 0.02%
Tween-PBS for three times, the cells were treated with
peroxidase-linked anti-mouse Ig antibody for 60 min at 37
°C.
218-220 °C; [R]20 -33.0° (c 0.09, CHCl3); IR (CHCl3) νmax
D
1
1772, 1660 cm-1; H NMR δ 1.39 (3H, s, H-15), 1.55 (3H, s,
H-12), 2.55 (1H, d, J ) 11.2, Hz, H-5),4.03 (1H, dd, J ) 11.2,
9.5 Hz, H-6), 5.15 (2H, br s, H-14), 7.27-7.46 (3H, m, Ph),
7.60-7.68 (2H, m, Ph); 13C NMR δ 20.41 (q, C-15), 21.97 (q,
C-12), 22.99 (t), 29.89 (t), 30.29 (t), 37.28 (t), 50.94 (s, C-11),
54.06 (d, C-5), 59.55 (d, C-7), 62.76 (s), 72.33 (s), 79.14 (d, C-6),
112.16 (t, C-14), 124.18 (s, Ph), 129.03 (d, m-Ph), 129.79 (d,
p-Ph), 138.16 (d, o-Ph), 146.92 (s, C-4), 175.13 (s, C-13);
HREIMS m/z 404.0918 (calcd for C21H24O3Se 404.0891). The
second elution gave recovered 22 (14%).
1R,10R-Epoxygu aia-4(14),11(12)-dien o-13,6R-lacton e (39).
The oxidative syn elimination of 38 gave 39 (84%) as colorless
needles (EtOAc): mp 130-131 °C; [R]20 -54.7° (c 0.17,
D
CHCl3); IR (CHCl3) νmax 1774, 1662 cm-1; 1H NMR (500 MHz)
δ 1.36 (3H, s, H-15), 1.46 (1H, dddd, J ) 13.7, 12.9, 10.7, 1.2
Hz, H-8), 1.57 (1H, m, H-9), 1.74 (1H, dddd, J ) 14.7, 9.3, 5.4,
1.0 Hz, H-3), 2.16 (1H, dddd, J ) 13.7, 6.7, 3.2, 1.5 Hz, H-8),
2.21 (1H, ddd, J ) 14.7, 10.1, 8.1 Hz, H-2), 2.34 (1H, ddd, J )
14.3, 6.7, 1.2 Hz, H-9), 2.52 (1H, m, H-3), 2.58-2.69 (2H, m,
The cells were washed three times with 0.02% Tween-
PBS. The cells were incubated with the substrate (0.1%
o-phenylenedianine dihydrochloride and 0.02% H2O2 in 0.2
M sodium citrate, pH 5.3) for 15 min in the dark and
assayed for the absorbance at 415 nm by using a microplate

30 J ournal of Natural Products, 1999, Vol. 62, No. 1
Yuuya et al.
(13) Giordano, O. S.; Guerreiro, E.; Pestchanker, M. J .; Guzman, J .; Pastor,
D.; Guardin, T. J . Nat. Prod. 1990, 53, 803-809.
reader. Expression of ICAM-1 was calculated as follows.
(14) Guardia, T.; Guzman, J . A.; Pestchanker, M. J .; Guerreiro, E.;
Giordano, O. S. J . Nat. Prod. 1994, 57, 507-509.
Expression of ICAM-1 (% of control) )
(absorbance of sample -
(15) Semmler, F. W.; Feldstein, J . Ber. 1914, 47, 2433-2437.
(16) Ukita, T. Yakugaku Zasshi 1939, 59, 80-82.
(17) Hikino, H.; Meguro, K.; Kusano, G.; Takemoto, T. Chem. Pharm. Bull.
1964, 12, 632-634.
absorbance without IL-1â treatment)/
(absorbance with IL-1â treatment -
absorbance without IL-1â treatment) × 100
(18) Mathur, S. B.; Hiremath, S. V.; Kulkarni, G. H.; Kelkar, G. R.;
Bhattacharyya, S. C.; Simonovic, D.; Rao, A. S. Tetrahedron 1965,
21, 3575-3590.
(19) Corbel, A.; Gariboldi, P.; J ommi, G.; Orsini, F.; Ferrari, G. Phy-
tochemistry 1974, 13, 459-465.
(20) Taniguchi, M.; Kataoka, T.; Suzuki, H.; Uramoto, M.; Ando, M.; Arao,
K.; Magae, J .; Nishimura, T.; Otake, N.; Nagai, K. Biosci. Biotech.
Biochem. 1995, 59, 2064-2067.
Ack n ow led gm en t. We thank Mr. T. Sato and Mrs. H.
Ando of the Instrument Analysis Center for Chemistry,
Tohoku University, for HREIMS and microanalyses.
(21) Solvolytic rearrangements of trans-decalin derivatives to guaianes.
(a) Heathcock, C. H.; Ratcliffe, R. J . Am. Chem. Soc. 1971, 93, 1746-
1757. (b) Ando, M.; Akahane, A.; Yamaoka, H.; Takase, K. J . Org.
Chem. 1982, 47, 3909-3916. (c) Ando, M.; Kusaka, H.; Ohara, H.;
Takase, K.; Yamaoka, H.; Yanagi, Y. J . Org. Chem. 1989, 54, 1959-
1960. (d) Ando, M.; Yoshimura, H. J . Org. Chem. 1993, 58, 4127-
4131.
Refer en ces a n d Notes
(1) Connolly, J . D.; Hill, R. A. Dictionary of Terpenoids; Chapman and
Hall: London, 1991; Vol. 1, pp 476-541.
(2) Rodriguez, E.; Towers, G. H. N.; Mitchell, J . C. Phytochemistry 1976,
15, 1573-1580.
(22) Ando, M.; Akahane, A.; Takase, K. Bull. Chem. Soc. J pn. 1978, 51,
283-289.
(23) Grieco, P. A.; Miyashita, M. J . Org. Chem. 1974, 39, 120-122.
(24) Sharpless, K. B.; Young, M. W.; Lauer, R. F. Tetrahedron Lett. 1973,
1979-1982.
(25) Bohlmann, F.; Le Van, N. Phytochemistry 1977, 16, 1304-1306.
(26) Yoshioka, H.; Babry, T. J .; Timmermann, B. N. Sesquiterpene
Lactones; University Tokyo Press: Tokyo, 1973; p 333.
(27) Ando, M.; Ibayashi, K.; Minami, N.; Nakamura, T.; Isogai, K.;
Yoshimura, H. J . Nat. Prod. 1994, 57, 433-445.
(28) Zn-Hg was prepared by the following procedure: Zn powder (2.5 g,
38.2 mmol) and 2 M HCl (5 mL) were stirred for 6 min, and the
aqueous solution was then decanted. HgCl2 (437 mg, 1.61 mmol) and
0.5 M HCl (4 mL) were added, and after being stirred for 5 min, the
aqueous solution was decanted. The residue in the flask was used
for the reaction immediately.
(3) Ando, M. J . Synth. Org. Chem., J pn 1992, 50, 858-874.
(4) Kupchan, S. M.; Eakin, M. A.; Thomas, A. M. J . Med. Chem. 1971,
14, 4, 1147-1152.
(5) Lee, K.-H.; Hung, E.-S.; Piantadosi, C. P.; Pagano, J . S.; Geissmas,
T. A. Cancer Res. 1971, 31, 1649-1654.
(6) Ogura, M.; Cordell, G. A.; Farnsworth, N. R. Phytochemistry 1978,
17, 957-961.
(7) Ando, M.; Kusaka, H.; Ohara, H.; Takase, K.; Yamaoka, H.; Yanagi,
Y. J . Org. Chem. 1989, 54, 1952-1960.
(8) Vichnewski, W.; Gilbert, B. Phytochemistry 1972, 11, 2563-2566.
(9) Sa´nchez-Viesca, F.; Romo, J . Tetrahedron 1963, 19, 1285-1291.
(10) Bates, R. B.; Cekan, Z.; Procha´zka, V.; Herout, V. Tetrahedron Lett.
1963, 1127-1130.
(11) Osawa, T., Taylar, D.; Suzuki, A.; Tamura, S. Tetrahedron Lett. 1977,
1169-1172.
(12) Asakawa, Y.; Takemoto, T. Phytochemistry 1979, 18, 285-288.
NP980092U