Synthetic approach to exo-endo cross-conjugated cyclohexadienones and its application to the syntheses of dehydrobrachylaenolide, isodehydrochamaecynone, and trans-isodehydrochamaecynone

Article abstract of DOI:10.1021/np0205250

Methodology for synthesis of exo-endo cross-conjugated dienones with trans- and cis-decalin systems has been reported. Bromination of the silyl enol ether of α′-methyl α,β-unsaturated ketones with PTAB and successive dehydrobromination of the resulting α′-bromo-α′-methyl α,β-unsaturated ketones under three conditions (DBU/PhH; TBAF/THF; Li₂CO₃, LiBr/DMF) gave the desired exo-endo cross-conjugated dienones in good yield. This method was applied to the syntheses of dehydrobrachylaenolide (1), isodehydro-chamaecynone (5c), and trans-isodehydrochamaecynone (11) starting from tuberiferine (7), chamaecynone (5a), and trans-chamaecynone (9). Eudesmanolides possessing an α-methylene γ-lactone moiety, i.e., 1, 7, and 13, exhibited significant inhibitory activity toward the induction of the intercellular adhesion molecule-1 (ICAM-1). Compound 1 showed greater activity than 7 and 13. All compounds possessing an ethynyl group, 5d, 9, 11, and 14, showed the same degree of termiticidal activity, and the exo-endo cross-conjugated dienone structure in 11 had no influence on the activity.

Full text of DOI:10.1021/np0205250

588
J . Nat. Prod. 2003, 66, 588-594
Syn th etic Ap p r oa ch to Exo-En d o Cr oss-Con ju ga ted Cycloh exa d ien on es a n d Its
Ap p lica tion to th e Syn th eses of Deh yd r obr a ch yla en olid e,
Isod eh yd r och a m a ecyn on e, a n d tr a n s-Isod eh yd r och a m a ecyn on e
Yohsuke Higuchi,† Fumito Shimoma,† Rei Koyanagi,§ Kouji Suda,§ Tomokazu Mitsui, Takao Kataoka,§,
Kazuo Nagai,§ and Masayoshi Ando*,‡
Graduate School of Science and Technology, and Department of Chemistry and Chemical Engineering, Faculty of Engineering,
Niigata University, 2-8050 Ikarashi, Niigata 950-2181, J apan, and Department of Bioengineering, Research Center for
Experimental Biology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, J apan
Received November 6, 2002
Methodology for synthesis of exo-endo cross-conjugated dienones with trans- and cis-decalin systems has
been reported. Bromination of the silyl enol ether of R′-methyl R,â-unsaturated ketones with PTAB and
successive dehydrobromination of the resulting R′-bromo-R′-methyl R,â-unsaturated ketones under three
conditions (DBU/PhH; TBAF/THF; Li2CO3, LiBr/DMF) gave the desired exo-endo cross-conjugated dienones
in good yield. This method was applied to the syntheses of dehydrobrachylaenolide (1), isodehydro-
chamaecynone (5c), and trans-isodehydrochamaecynone (11) starting from tuberiferine (7), chamaecynone
(5a ), and trans-chamaecynone (9). Eudesmanolides possessing an R-methylene γ-lactone moiety, i.e., 1,
7, and 13, exhibited significant inhibitory activity toward the induction of the intercellular adhesion
molecule-1 (ICAM-1). Compound 1 showed greater activity than 7 and 13. All compounds possessing an
ethynyl group, 5d , 9, 11, and 14, showed the same degree of termiticidal activity, and the exo-endo cross-
conjugated dienone structure in 11 had no influence on the activity.
A series of eudesmane type natural products possesses
an exo-endo cross-conjugated cyclohexadienone structure
in the A ring, such as dehydrobrachylaenolide (1),1 gerin,2
Resu lts a n d Discu ssion
As shown in Figure 1, a general synthetic method for
the exo-endo cross-conjugated dienone (structure c) from
the corresponding R′-methyl R,â-unsaturated ketone (struc-
ture a ) in trans- and cis-decalin derivatives has not been
reported in the literature. Because of the interest in the
expected biological activities and synthetic use of the
compounds possessing structure c, we wanted to establish
a general synthetic method for this functional group
starting from structure a . After several unsuccessful at-
tempts,9 we found that enolsilylation10 of compounds 2a ,
3a , and 4a with trimethylsilyl trifluoromethanesulfonate
(TMSOTf) and Et3N in CH2Cl2 and successive treatment
of the resulting silyl enol ethers with phenyltrimethylam-
monium tribromide (PTAB) gave the R′-bromo-R′-methyl
R,â-unsaturated ketones 2b, 3b, and 4b in 86%, 91%, and
67% yields, respectively. Additional functional groups in
encelin,3 virginin,4 and farinosin.5 Although the biological
activities of these compounds have not been reported, they
are expected to show some activities because they have a
Michael acceptor, the active site in the form of an exo-endo
cross-conjugated dienone, in addition to R-methylene γ-lac-
tone, R-methylene ester, and R-methyl γ-lactone moieties.
It is well known that biologically active compounds often
express their activities by binding the nucleophilic portion
of biomolecules such as the cysteine residues of protein.
Exo-endo cross-conjugated dienones are also important as
synthetic intermediates in the preparation of biologically
active natural products. Thus exo-endo cross-conjugated
cyclopentadienones were reported as synthetic intermedi-
ates for hirsutic acid and coriolin.6,7 We have also reported
the effective use of isodehydrochamaecynone (5c),8 which
possesses an exo-endo cross-conjugated cyclohexadienone
moiety, in the synthesis of dehydrochamaecynenol8 and
norsesquibenihiol.8 The crucial step of this conversion was
the efficient preparation of 5c, and various attempts to
improve the yield at that time were unsuccessful.
* To whom correspondence should be addressed. Tel and Fax: +81-25-
262-7326. E-mail: mando@eng.niigata-u.ac.jp.
† Graduate School of Science and Technology, Niigata University.
‡ Department of Chemistry and Chemical Engineering, Niigata Univer-
sity.
§ Department of Bioengineering, Tokyo Institute of Technology.
Research Center for Experimental Biology, Tokyo Institute of Technol-
ogy.
10.1021/np0205250 CCC: $25.00
© 2003 American Chemical Society and American Society of Pharmacognosy
Published on Web 04/12/2003

Exo-Endo Cross-Conjugated Cyclohexadienones
J ournal of Natural Products, 2003, Vol. 66, No. 5 589
Sch em e 1a
F igu r e 1. Conversion of R′-methyl R,â-unsaturated ketones a with
trans- and cis-decalin systems to exo-endo cross-conjugated dienones
c.
a
The yields in parentheses are based on recovered starting material.
the molecule such as a cis-fused γ-lactone, trans-fused
γ-lactone, or isopropenyl groups did not affect the results.
There are three choices for dehydrobromination of R′-
bromo-R′-methyl R,â-unsaturated ketone (structure b) to
the desired exo-endo cross-conjugated dienone (structure
c): (A) DBU in PhH at room temperature; (B) tetrabuty-
lammonium fluoride (TBAF) in THF at room temperature;
(C) Li2CO3, LiBr in DMF at 120 °C. The reaction must be
controlled by E2 type elimination in order to obtain the
desired structure c. Since the structure c is a good Michael
acceptor, the bases employed in the dehydrobromination
reactions must be a hard base such as fluoride anion (F-)
or a bulky base such as DBU to avoid 1,4-addition of the
base to the product. Since reaction conditions C needed
high reaction temperature (120-150 °C) and long reaction
time (3-4 h), a part of the desired exo-structure c seemed
to isomerize gradually to thermodynamically more stable
endo-dienone (structure d ). Dehydrobromination of 2b by
method A gave the desired 2c in 75% yield and by method
B gave a 5:2 mixture of 2c and 2d . Dehydrobromination of
3b and 4b gave the desired 3c and 4c by method B in 56%
and 88% yields, respectively.
Then we planned to prepare isodehydrochamaecynone
(5c) from chamaecynone (5a ) and isochamaecynone (6a )
by an analogous method. Enolsilylation of 5a with TMSOTf
and Et3N in CH2Cl2 and successive treatment of the
resulting silyl enol ether with PTAB gave R′-bromo-R′-
methyl R,â-unsaturated ketone (5b)11 in 60% yield ac-
companied by dehydrochamaecynone (5d ) in 35% yield.
Analogously, 6a gave 5b and 5d in 57% and 28% yields,
respectively. Dehydrobromination of 5b by method B
(TBAF in THF at room temperature) gave the desired 5c
in 30% yield. Dehydrobromination of 5b by method C (Li2-
CO3, LiBr in DMF at 120 °C) gave the desired 5c in 40%
yield and 5d in 37% yield. Isodehydrochamaecynone (5c)
is a synthetic intermediate for dehydrochamaecynenol and
norsesquibenihiol, and their overall yields were much
improved by the improvement of the yield of 5c.8
Sch em e 2a
a
The yields in parentheses are based on recovered starting material.
wanted to synthesize dehydrobrachylaenolide (1) from
tuberiferine (7)12 (Scheme 1). Enolsilylation of 7 with
TMSOTf and Et3N in CH2Cl2 and successive treatment of
the resulting silyl enol ether with PTAB gave bromide 8
in 74% yield. Dehydrobromination of 8 with DBU (method
A) gave 1 in only 26% yield. On the contrary, treatment of
8 with TBAF in THF gave 1 in 63% yield. The physical
constants and spectral data of 1 were in good agreement
with those of natural dehydrobrachylaenolide reported in
the literature.1
Finally, we wanted to introduce the synthesis of trans-
isodehydrochamaecynone (11) (Scheme 2). Enolsilylation
of trans-chamaecynone (9)13 with TMSOTf and Et3N in
CH2Cl2 and successive treatment of the resulting silyl enol
ether with PTAB gave bromide 10 in 92% yield. Treatment
of 10 with DBU gave a complex mixture probably because
the acetylide anion was formed under the reaction condi-
tions. On the other hand, treatment of 10 with TBAF in
THF gave 11 in 65% yield accompanied by 5d in 8% yield.
The yield of 11 was improved to 77% by dehydrobromina-
tion of 10 with Li2CO3 and LiBr in DMF at 120 °C.
In conclusion, dehydrobrachylaenolide (1), isodehydro-
chamaecynone (5c), and trans-isodehydrochamaecynone
(11) have been synthesized by bromination of the silyl enol
ether of each substrate with PTAB and successive dehy-
drobromination with TBAF or Li2CO3 and LiBr.
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 th e In -
fla m m a tor y Cytok in e In ter leu k in -1 (IL-1). Expression
of ICAM-1 is induced by IL-1 on the surface of endothelial
cells of blood vessels. ICAM-1 on the activated endothelial
cells interacts with lymphocyte function-associated anti-
gen-1 (LFA-1) on leukocytes in the blood stream, and the
From an interest in the influence of the exo-endo dienone
moiety on the biological activities of the compound, we

590 J ournal of Natural Products, 2003, Vol. 66, No. 5
Higuchi et al.
12
Ta ble 1.
Inhibitory Activity on Induction of ICAM-1a
12
13
7
1
IC50 (µM)
>1000
17
7.4
3.0
a
A549 cells (3 × 104 cells/well) were pretreated with various
concentrations of the compounds for 1 h and then incubated in
the presence 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 Experi-
mental Section. The experiments were carried out in triplicate
cultures. IC50 was calculated by using the formula in the Experi-
mental Section.
leukocytes begin rolling, adhere to the surface of endothe-
lium, and finally migrate from the inside of the blood vessel
to the inflammatory portion by chemotaxis. The attack of
leukocytes occasionally causes serious damage to the
inflammatory tissue. Expression of excess ICAM-1 on the
surface of endothelial cells of a blood vessel plays an
important role in the progress of inflammatory reaction.
These facts suggest that inhibitors of induction of ICAM-1
may yield a new type of antiinflammatory agent. With this
in mind, we examined 1 and the three related compounds
7, 12,12 and 1312 for their inhibitory activity on the
F igu r e 2. Dehydrobrachylaenolide (1) inhibits the induction of
ICAM-1 in response to IL-1. A549 cells were pretreated with various
concentrations of compound 1 for 1 h and then incubated in the
presence of IL-1â for 6 h. Open circles represent the ICAM-1 expression
(mean ( SD of triplicate cultures). A549 cells were incubated with
various concentrations of compound 1 for 24 h, and cell viability was
measured by MTT assay (filled circles). Data points represent mean
(SD of triplicate cultures.
Ta ble 2. Termiticidal Activities of Chamaecynone (5a ) and
trans-Chamaecynone (9) against Coptotermes formosanus by
Filter Paper Contact Method
mortality (%) (days)
compound
concentration (ppm)
1
2
5
7
5a
100
1000
100
0
27
0
0
97
0
27
100
43
87
100
100
100
9
1000
20
80
100
induction of ICAM-1 through bioassay. The compounds
were screened for inhibition of induction of ICAM-1 using
the human cultured A549 cell line (lung carcinoma, ATCC
CCL 185), an in vitro model of human endothelial cells.
All compounds possessing an R-methylene γ-lactone
moiety, such as 1, 7, and 13, 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, no inhibitory activity on ICAM-1 expression was
observed in compound 12, which contains no R,â-unsatur-
ated carbonyl residues. However, its structural analogues
that contain unsaturated carbonyl residues prevented
ICAM-1 expression, greater with increasing number of R,â-
unsaturated carbonyl residues (compound 1 > compound
7 > compound 13 > compound 12) (Table 1). Compound 1
inhibited ICAM-1 expression in a dose-dependent manner
(IC50 ) 3 µM) under concentrations that did not decrease
cell viability. Toxic activity of compound 1 (IC50 ) 55 µM)
required about 20-fold higher concentrations than those
required for the inhibition of ICAM-1 expression (Figure
2).
The transcription of the ICAM-1 gene induced by IL-1
is largely dependent on the transcription factor NF-κB.
Upon IL-1 stimulation, NF-κB translocates from the cyto-
plasm into the nucleus and activates a variety of target
genes. As reported previously,14 (11S)-2R-bromo-3-oxoeudes-
mano-12,6R-lactone, devoid of an R-methylene γ-lactone
moiety, inhibits the signaling pathway that triggers the
nuclear translocation of NF-κB. In our preliminary re-
sults,15 it appears that these compounds possessing an
R-methylene γ-lactone moiety control the signaling path-
way upstream of the nuclear translocation of NF-κB.
Details regarding the mode of action of these compounds
will be reported elsewhere.
Ta ble 3. Termiticidal Activities of Norsesquiterpenoids with
Trans Ring Fusion against Coptotermes formosanus by Filter
Paper Contact Method
mortality (%) (days)
compound
concentration (ppm)
1
2
5
14
100
1000
100
1000
100
0
0
0
47
0
33
0
0
93
3
80
3
93
0
0
100
100
100
17
100
7
9
11
5d
1000
100
1000
13
90
100
at C-7 showed significant termiticidal activity. The differ-
ence in the stereochemistry of ring fusion did not influence
the potency of the termiticidal activity. We then examined
the termiticidal activity of 9 and three related compounds
5d , 11, and 1413 (Table 3). All compounds possessing an
ethynyl group at C-7 showed significant termiticidal activ-
ity. The termiticidal activity was increased by introduction
of a double bond at the R,â-position of 14. However, further
introduction of an exo- or endo-double bond at the R′,â′-
position had no effect.
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
are uncorrected. 1H NMR and 13C NMR spectra were recorded
at 200 (500) MHz and at 50 (125) MHz, respectively, in CDCl3.
1H NMR assignments were determined by decoupling and
H-H COSY experiments. 13C NMR assignments were deter-
mined by DEPT, C-H COSY, HMQC, and HMBC experi-
ments. All reactions were run under an atmosphere of N2.
Benzene, CH2Cl2, DMF, diisopropylamine, and triethylamine
were distilled from CaH2. MeOH was distilled from Mg(OMe)2.
THF was distilled from sodium benzophenone ketyl. The
Ter m iticid a l Activity. The termiticidal activity of 5a
and the corresponding trans-fused enone 9 was examined
(Table 2). These compounds possessing an ethynyl group

Exo-Endo Cross-Conjugated Cyclohexadienones
J ournal of Natural Products, 2003, Vol. 66, No. 5 591
column codes are as follows: A, 25 × 0.46 cm i.d. stainless
steel column packed with 10 µm silica gel; B, 15 × 0.46 cm
i.d. stainless steel column packed with 5 µm silica gel. Silica
gel (230-400 mesh) was employed for flash chromatography,
and 70-230 mesh silica gel was employed for column chro-
matography.
(3:7)] to give 3b (67.9 mg, 91%) as colorless prisms (CHCl3-
EtOAc): mp (dec) 140 °C; [R]20 -59.8° (c 0.89, CHCl3); IR
D
1
(CHCl3) νmax 1784, 1692 cm-1; H NMR (CDCl3, 200 MHz) δ
6.81 (1H, d, J ) 9.9 Hz, H-1), 6.11 (1H, d, J ) 9.9 Hz, H-2),
4.14 (1H, dd, J ) 11.5, 10.0 Hz, H-6), 3.09 (1H, d, J ) 11.5
Hz, H-5), 2.37 (1H, dq, J ) 12.2, 6.9 Hz, H-11), 2.08 (3H, s,
H-15), 1.28 (3H, d, J ) 6.9 Hz, H-13), 1.12 (3H, s, H-14); 13C
NMR (CDCl3, 50 MHz) δ 193.2 (s, C-3), 178.3 (s, C-12), 157.6
(d, C-1), 125.8 (d, C-2), 78.6 (d, C-6), 60.7 (s, C-4), 57.2 (d, C-5),
52.6 (d, C-7), 40.7 (d, C-11), 39.7 (s, C-10), 38.8 (t), 24.2 (q,
C-15), 22.9 (t), 22.3 (q, C-14), 12.5 (q, C-13); anal. C 54.98%,
H 5.85%, calcd for C15H19O3Br, C 55.06%, H 5.85%.
(11S)-4r-Br om o-3-oxoeu d esm -1-en o-12,6â-la cton e (2b).
To a stirred solution of 2a (19.6 mg, 0.0789 mmol) and Et3N
(65.6 µL, 0.473 mmol) in CH2Cl2 (0.9 mL) was added TMSOTf
(45.8 µL, 0.237 mmol) at 0 °C. After 2.5 h, the mixture was
treated with PTAB (89.1 mg, 0.237 mmol) in CH2Cl2 (0.5 mL)
and stirred at this temperature for 2 h. The reaction was
quenched with a mixture of 10% aqueous solution of Na2S2O3
and a saturated aqueous solution of NaHCO3, and the mixture
was extracted with CH2Cl2. The combined extracts were dried
(Na2SO4), concentrated, and purified by flash chromatography
[2.5 g; 1.2 cm i.d.; EtOAc-hexane (3:7)] to give 2b (22.1 mg,
86%) as colorless needles (CHCl3-EtOAc): mp (dec) 134 °C;
(11S)-3-Oxoeu d esm a -1,4(15)-d ien o-12,6r-la cton e (3c):
Deh yd r obr om in a tion of 3b by Meth od B. A solution of 3b
(52.6 mg, 0.161 mmol) and TBAF (1 M in THF, 322 µL) in
THF (3 mL) was stirred at room temperature for 16 h. Then
the additional TBAF (1 M in THF, 161 µL) was added, and
stirring was continued for 7.5 h. Since the starting material
3b was detected by TLC, TBAF (1 M in THF, 161 µL) was
further added. The reaction mixture was stirred again for 2
h, poured into a saturated aqueous solution of NH4Cl, and
extracted with CH2Cl2. The dried (Na2SO4) and concentrated
product was separated by flash chromatography [8 g; 1.6 cm
i.d.; EtOAc-hexane (3:7)]. The faster running gave 3c (22.2
mg, 56%) as colorless prisms (EtOAc-hexane): mp 123-125
[R]20D -225.6° (c 1.68, CHCl3); IR (CHCl3) νmax 1776, 1684 cm-1
;
1H NMR (CDCl3, 200 MHz) δ 6.73 (1H, d, J ) 10.0 Hz, H-1),
6.00 (1H, d, J ) 10.0 Hz, H-2), 5.31 (1H, dd, J ) 4.3, 2.2 Hz,
H-6), 2.59 (1H, d, J ) 2.2 Hz, H-5), 2.42 (1H, q, J ) 7.7 Hz,
H-11), 2.14 (1H, m, H-7), 2.10 (3H, s, H-15), 1.86 (1H, m, H-8),
1.38 (3H, d, J ) 7.7 Hz, H-13), 1.32 (3H, s, H-14); 13C NMR
(CDCl3, 50 MHz) δ 194.5 (s, C-3), 179.4 (s, C-12), 159.3 (d, C-1),
124.0 (d, C-2), 77.8 (d, C-6), 70.3 (s, C-4), 55.1 (d, C-5), 43.6 (d,
C-11), 42.4 (d, C-7), 38.4 (t, C-9), 37.7 (s, C-10), 27.4 (q, C-15),
23.6 (t, C-8), 21.3 (q, C-14), 14.1 (q, C-13); anal. C 54.85%, H
5.82%, calcd for C15H19O3Br, C 55.06%, H 5.85%.
°C; [R]20 -49.2° (c 1.65, CHCl3); IR (CHCl3) νmax 1780, 1678,
D
1
1624 cm-1; H NMR (CDCl3, 200 MHz) δ 6.80 (1H, d, J ) 9.9
Hz, H-1), 6.27 (1H, dd, J ) 2.3, 1.0 Hz, H-15), 6.03 (1H, dd, J
) 9.9, 0.8 Hz, H-2), 5.67 (1H, ddd, J ) 2.3, 1.0, 0.8 Hz, H-15),
4.13 (1H, dd, J ) 10.8, 9.8 Hz, H-6), 2.89 (1H, ddd, J ) 10.8,
2.3, 2.3 Hz, H-5), 2.38 (1H, dq, J ) 12.1, 6.8 Hz, H-11), 1.26
(3H, d, J ) 6.8 Hz, H-13), 1.07 (3H, s, H-14); 13C NMR (CDCl3,
50 MHz) δ 187.8 (s, C-3), 178.7 (s, C-12), 158.6 (d, C-1), 140.7
(s, C-4), 127.4 (d, C-2), 122.0 (t, C-15), 78.9 (d, C-6), 52.2 (d),
52.1 (d), 40.7 (d, C-11), 39.6 (s, C-10), 36.3 (t), 22.7 (t), 19.6 (q,
C-14), 12.5 (q, C-13); anal. C 72.90%, H 7.40%, calcd for
(11S)-3-Oxoeu d esm a -1,4(15)-d ien o-12,6â-la cton e (2c):
Deh yd r obr om in a tion of 2b by Meth od A. A solution of 2b
(38.2 mg, 0.117 mmol) and DBU (52.5 µL, 0.351 mmol) in PhH
(1 mL) was stirred at room temperature for 47 h, poured into
1 M HCl (5 mL), and extracted with EtOAc. The combined
extracts were washed with a saturated aqueous solution of
NaHCO3 and a saturated aqueous solution of NaCl, dried (Na2-
SO4), concentrated (35 mg), and purified by flash chromatog-
raphy [2.5 g; 1.2 cm i.d.; EtOAc-hexane (3:7)] to give 2c (21.6
C
15H18O3, C 73.15%, H 7.37%. The slower running gave
R-santonin (3d ) (2.2 mg, 6%) as colorless crystals.
4r-Br om o-7âH-eu desm a-1,11-dien -3-on e (4b). To a stirred
solution of 4a (19.0 mg, 0.0870 mmol) and Et3N (36.2 µL, 0.261
mmol) in CH2Cl2 (0.6 mL) was added TMSOTf (25.3 µL, 0.131
mmol) at 0 °C. After 1 h, the mixture was treated with PTAB
(36.0 mg, 0.0957 mmol) in CH2Cl2 (0.3 mL) and stirred at this
temperature for 10 min. The reaction mixture was poured into
10% aqueous solution of Na2S2O3 and extracted with CH2Cl2.
The combined extracts were dried and separated by flash
chromatography [2.5 g; 1.2 cm i.d.; EtOAc-hexane (5:95)]. The
faster running gave 7âH-eudesma-1,4(15),11-trien-3-one (4c)
mg, 75%) as colorless plates (EtOAc): mp 134-137 °C; [R]20
D
-345.8° (c 1.65, CHCl3); IR (CHCl3) νmax 3004, 1774, 1676, 1624
1
cm-1; H NMR (CDCl3, 200 MHz) δ 6.75 (1H, d, J ) 9.9 Hz,
H-1), 6.32 (1H, dd, J ) 2.1, 0.8 Hz, H-15), 6.01 (1H, dd, J )
9.9, 0.8 Hz, H-2), 5.74 (1H, ddd, J ) 2.1, 0.8, 0.8 Hz, H-15),
4.91 (1H, dd, J ) 4.3, 2.6 Hz, H-6), 2.73 (1H, ddd, J ) 2.6, 2.1,
2.1 Hz, H-5), 2.42 (1H, q, J ) 7.6 Hz, H-11), 2.13 (1H, ddd, J
) 11.4, 6.8, 4.3 Hz, H-7), 1.35 (3H, d, J ) 7.6 Hz, H-13), 1.09
(3H, s, H-14); 13C NMR (CDCl3, 50 MHz) δ 188.6 (s, C-3), 179.6
(s, C-12), 159.9 (d, C-1), 141.3 (s, C-4), 126.7 (d, C-2), 121.7 (t,
C-15), 76.4 (d, C-6), 49.0 (d), 43.7 (d), 41.8 (d), 36.9 (s, C-10),
34.9 (t), 23.6 (t), 19.7 (q), 14.1 (q); HREIMS m/z 246.1256 (calcd
for C15H18O3, 246.1256).
Deh yd r obr om in a tion of 2b by Meth od B. A solution of
2b (45.3 mg, 0.138 mmol) and TBAF (1 M in THF, 276 µL) in
THF (2.5 mL) was stirred at room temperature for 9 h. Then
additional TBAF (1 M in THF, 276 µL) was added, and stirring
was continued for 3.3 h. Since 2b was still detected in the
reaction mixture, TBAF (1 M in THF, 69.0 µL) was further
added and stirring was continued for 1.8 h. The reaction
mixture was poured into a saturated aqueous solution of NH4-
Cl and extracted with CH2Cl2. The combined extracts were
dried (Na2SO4), concentrated, and purified by flash chroma-
tography [3 g; 1.2 cm i.d.; EtOAc-hexane (3:7)] to give a
regioisomeric mixture of 2c and 6â-santonin 2d (18.9 mg, 55%).
The ratio (2c:2d ) 5:2) was determined by 1H NMR integration
values.
(1.0 mg, 5%) as a colorless oil: [R]20 -34.5° (c 0.67, CHCl3);
D
1
IR (CHCl3) νmax 3096, 1672, 1624 cm-1; H NMR (CDCl3, 500
MHz) δ 6.77 (1H, d, J ) 9.8 Hz, H-1), 6.07 (1H, dd, J ) 2.2,
1.5 Hz, H-15), 5.96 (1H, d, J ) 9.8 Hz, H-2), 5.19 (1H, br s,
Wh/ 2 ) 3 Hz, H-15), 4.93 (1H, ddd, J ) 1.5, 1.5, 1.5 Hz, H-12),
4.80 (1H, br s, Wh/ 2 ) 4 Hz, H-12), 2.69 (1H, dddd, J ) 12.5,
2.2, 2.2, 2.2 Hz, H-5), 2.49 (1H, br s, Wh/ 2 ) 12 Hz, H-7), 2.04
(1H, dddd, J ) 13.9, 2.2, 2.2, 2.2 Hz, H-6), 1.98 (1H, m, H-8),
1.83 (1H, dddd, J ) 14.1, 14.1, 5.7, 4.0 Hz, H-8), 1.75-1.67
(2H, H-6 and -9), 1.73 (3H, d, J ) 0.5 Hz, H-13), 1.43 (1H,
ddd, J ) 12.7, 4.0, 3.2 Hz, H-9), 0.98 (3H, s, H-14); 13C NMR
(CDCl3, 125 MHz) δ 189.8 (s, C-3), 162.1 (d, C-1), 146.4 (s),
145.9 (s), 126.6 (d, C-2), 117.8 (t, C-15), 111.3 (t, C-12), 42.8
(d, C-5), 38.2 (s, C-10), 37.8 (d, C-7), 32.6 (t, C-9), 25.1 (t, C-6),
23.1 (t, C-8), 22.7 (q, C-13), 17.4 (q, C-14); HREIMS m/z
216.1527 (calcd for C15H20O, 216.1514). The slower running
gave 4b (17.3 mg, 67%) as colorless crystals: mp 108-111 °C;
[R]20D -56.5° (c 0.74, CHCl3); IR (CHCl3) νmax 3100, 1680 cm-1
;
(11S)-4r-Br om o-3-oxoeu d esm -1-en o-12,6r-la cton e (3b).
To a stirred solution of 3a (56.5 mg, 0.228 mmol) and Et3N
(126 µL, 0.909 mmol) in CH2Cl2 (1.8 mL) was added TMSOTf
(88.1 µL, 0.456 mmol) at 0 °C. After 3 h, the mixture was
treated with PTAB (94.4 mg, 0.251 mmol) in CH2Cl2 (0.7 mL)
and stirred at this temperature for 15 min. The reaction
mixture was poured into 10% aqueous solution of Na2S2O3 and
extracted with CHCl3. The dried product (180 mg) was purified
by flash chromatography [8 g; 1.6 cm i.d.; EtOAc-hexane
1H NMR (CDCl3, 200 MHz) δ 6.77 (1H, d, J ) 9.9 Hz, H-1),
5.96 (1H, d, J ) 9.9 Hz, H-2), 4.95 (2H, br s, Wh/ 2 ) 6 Hz, Hs-
12), 2.67 (1H, dd, J ) 12.8, 2.2 Hz, H-5), 2.53 (1H, br s, Wh/ 2
)
11 Hz, H-7), 2.33 (1H, dddd, J ) 14.0, 2.2, 2.2, 2.2 Hz, H-6),
1.81 (6H, H-13 and -15), 1.19 (3H, s, H-14); 13C NMR (CDCl3,
50 MHz) δ 195.4 (s, C-3), 161.1 (d, C-1), 145.3 (s, C-11), 124.4
(d, C-2), 111.7 (t, C-12), 70.7 (s, C-4), 49.3 (d), 39.1 (s, C-10),
38.4 (d), 35.9 (q), 25.3 (q), 25.1 (t), 23.3 (q), 22.7 (t), 20.4 (t);
HREIMS m/z 296.0745 (calcd for C15H21OBr, 296.0776).

592 J ournal of Natural Products, 2003, Vol. 66, No. 5
Higuchi et al.
Deh yd r obr om in a tion of 4b by Meth od B. A solution of
4b (15.7 mg, 0.0528 mmol) and TBAF (1 M in THF, 106 µL)
in THF (1 mL) was stirred at room temperature for 16.3 h.
Then the mixture was treated with additional TBAF (1 M in
THF, 52.8 µL) and stirred for a further 10.3 h. Since 4b was
still detected in the reaction mixture, TBAF (1 M in THF, 52.8
µL) was further added. After 1.3 h, the reaction mixture was
poured into a saturated aqueous solution of NH4Cl and
extracted with CH2Cl2. The combined extracts were dried and
purified by flash chromatography [2.5 g; 1.2 cm i.d.; EtOAc-
hexane (3:97)] to give 4c (10.1 mg, 88%) as a colorless oil.
4â-Br om o-5âH -13-n or eu d esm -1-en -11-yn -3-on e (5b ):
Br om in a tion of Ch a m a ecyn on e (5a ). To a stirred solution
of 5a (16.7 mg, 0.0826 mmol) and Et3N (34.4 µL, 0.248 mmol)
in CH2Cl2 (0.6 mL) was added TMSOTf (24.0 µL, 0.124 mmol)
at 0 °C. After 1.5 h, the mixture was treated with PTAB (34.2
mg, 0.0909 mmol) in CH2Cl2 (0.3 mL) and stirred at this
temperature for 20 min. The reaction mixture was poured into
10% aqueous Na2S2O3 and extracted with CH2Cl2. The com-
bined extracts were dried (Na2SO4) and separated by flash
chromatography [2.5 g; 1.2 cm i.d.; EtOAc-hexane (5:95)]. The
faster running gave 5b (13.9 mg, 60%) as a colorless oil: IR
(CHCl3) νmax 3316, 2120, 1682 cm-1; 1H NMR (CDCl3, 200 MHz)
δ 6.54 (1H, d, J ) 10.3, 1.5 Hz, H-1), 6.01 (1H, d, J ) 10.3 Hz,
H-2), 2.84 (1H, br s, Wh/ 2 ) 13 Hz, H-7), 2.68 (1H, ddd, J )
10.4, 5.2, 1.5 Hz, H-5), 2.15 (1H, d, J ) 2.3 Hz, H-12), 1.93
(3H, s, H-15), 1.59 (3H, s, H-14); 13C NMR (CDCl3, 50 MHz) δ
193.2 (s, C-3), 157.5 (d, C-1), 125.0 (d, C-2), 86.0 (s, C-11), 70.4
(d, C-12), 59.8 (s, C-4), 49.1 (d, C-5), 38.2 (s, C-10), 36.8 (t),
31.0 (t), 29.6 (q, C-14), 28.7 (q, C-15), 27.0 (t), 26.6 (d, C-7).
The slower running gave dehydrochamaecynone (5d ) (5.7 mg,
i.d.; EtOAc-hexane (5:95)]. The faster running gave 5c (6.1
mg, 40%) as colorless crystals. The slower running gave 5d
(5.7 mg, 37%) as colorless crystals.
4â-Br om o-5âH -13-n or eu d esm -1-en -11-yn -3-on e (5b ):
Br om in a tion of Isoch a m a ecyn on e (6a ). To a stirred solu-
tion of 6a (16.4 mg, 0.0811 mmol) and Et3N (33.7 µL, 0.243
mmol) in CH2Cl2 (0.6 mL) was added TMSOTf (23.6 µL, 0.122
mmol) at 0 °C. After 1 h, the mixture was treated with PTAB
(42.6 mg, 0.113 mmol) in CH2Cl2 (0.3 mL) and stirred for 20
min. The reaction mixture was poured into 10% aqueous
Na2S2O3 and extracted with CHCl3. The combined extracts
were washed, dried, and separated by flash chromatography
[2.5 g; 1.2 cm i.d.; EtOAc-hexane (5:95)]. The faster running
gave 5b (12.9 mg, 57%) as colorless crystals. The slower
running gave 5d (4.5 mg, 28%) as colorless crystals.
4r-Br om otu ber ifer in e (8). To a stirred solution of 7 (37.1
mg, 0.151 mmol) and Et3N (104.7 µL, 0.755 mmol) in CH2Cl2
(1.1 mL) was added TMSOTf (73.1 µL, 0.378 mmol) at 0 °C.
After 2 h, the mixture was treated with PTAB (90.8 mg, 0.242
mmol) in CH2Cl2 (0.4 mL) and stirred for 25 min. The reaction
mixture was poured into 10% aqueous Na2S2O3 and extracted
with CHCl3. The combined extracts were washed, dried, and
purified by flash chromatography [2.5 g; 1.2 cm i.d.; EtOAc-
hexane (3:7)] to give 8 (36.2 mg, 74%) as colorless prisms
(EtOAc): mp (dec) 150 °C; [R]20 -63.6° (c 0.50, CHCl3); IR
D
(CHCl3) νmax 1776, 1682, 1624 cm-1; 1H NMR (CDCl3, 200 MHz)
δ 6.84 (1H, d, J ) 9.9 Hz, H-1), 6.17 (1H, d, J ) 3.2 Hz, H-13),
6.12 (1H, d, J ) 9.9 Hz, H-2), 5.50 (1H, d, J ) 3.2 Hz, H-13),
4.14 (1H, dd, J ) 11.5, 11.5 Hz, H-6), 3.21 (1H, d, J ) 11.5
Hz, H-5), 2.67 (1H, m, H-7), 2.09 (3H, s, H-15), 1.12 (3H, s,
H-14); 13C NMR (CDCl3, 50 MHz) δ 193.1 (s, C-3), 169.7 (s,
C-12), 157.4 (d, C-1), 137.9 (s, C-11), 125.8 (d, C-2), 118.1 (t,
C-13), 78.8 (d, C-6), 60.4 (s, C-4), 57.6 (d, C-5), 49.8 (d, C-7),
39.8 (s, C-10), 38.5 (t), 24.3 (q, C-15), 22.3 (q, C-14), 21.2 (t);
anal. C 55.50%, H 5.27%, calcd for C15H17O3Br, C 55.40%, H
5.27%.
35%) as colorless crystals: mp 84 °C; [R]20 -181.9° (c 0.57,
D
CHCl3); IR (CHCl3) νmax 3316, 2120, 1662, 1630, 1612 cm-1
;
1H NMR (CDCl3, 500 MHz) δ 6.71 (1H, d, J ) 9.8 Hz, H-1),
6.21 (1H, d, J ) 9.8 Hz, H-2), 3.05 (1H, ddd, J ) 12.8, 2.1, 2.1
Hz, H-6), 2.32 (1H, dd, J ) 12.8, 12.7 Hz, H-6), 2.26 (1H, m,
H-7), 2.16 (1H, d, J ) 2.2 Hz, H-12), 1.96 (1H, m, H-8), 1.90
(3H, s, H-15), 1.86 (1H, m, H-8), 1.78 (1H, ddd, J ) 13.4, 3.9,
2.4 Hz, H-9), 1.29 (1H, ddd, J ) 13.4, 13.4, 4.4 Hz, H-9), 1.24
(3H, s, H-14); 13C NMR (CDCl3, 125 MHz) δ 186.1 (s, C-3),
156.6 (s, C-5), 156.2 (d, C-1), 130.1 (s, C-4), 126.2 (d, C-2), 86.6
(s, C-11), 69.2 (d, C-12), 39.7 (s, C-10), 36.8 (t, C-9), 33.7 (t,
C-6), 30.5 (d, C-7), 27.5 (t, C-8), 23.4 (q, C-14), 10.6 (q, C-15);
HREIMS m/z 200.1203 (calcd for C14H16O, 200.1201).
Deh yd r obr a ch yla en olid e (1): Deh yd r obr om in a tion of
8 by Meth od A. A solution of 8 (25.2 mg, 0.0775 mmol) and
DBU (23.2 µL, 0.155 mmol) in PhH (0.5 mL) was stirred at
room temperature for 43 h, poured into 1 M HCl, and extracted
with EtOAc. The combined extracts were washed, dried (Na2-
SO4), and passed through a short column of silica gel. The
eluate was concentrated and separated by HPLC [column B;
EtOAc-hexane (3:7); 3.0 mL/min]. The peak (tR 2.8 min) gave
1 (4.9 mg, 26%) as colorless prisms (CHCl3-EtOAc): mp 222-
Isod eh yd r och a m a ecyn on e (5c): Deh yd r obr om in a tion
of 5b by Meth od B. A solution of 5b (6.5 mg, 0.0231 mmol)
and TBAF (1 M in THF, 46.0 µL) in THF (0.5 mL) was stirred
at room temperature for 4.2 h. Since 5b was still detected in
the reaction mixture, TBAF (1 M in THF, 34.2 µL) was further
added. The reaction mixture was stirred for a further 26.2 h,
poured into a saturated aqueous solution of NH4Cl, and
extracted with CH2Cl2. The combined extracts were dried and
separated by flash chromatography [2 g; 1.2 cm i.d.; EtOAc-
hexane (5:95)]. 5c was separated as a colorless oil (1.4 mg,
30%): [R]20D -101° (c 0.09, CHCl3); IR (CHCl3) νmax 3316, 2116,
1670, 1624, 1456 cm-1; 1H NMR (CDCl3, 500 MHz) δ 6.65 (1H,
dd, J ) 10.1, 1.9 Hz, H-1), 6.07 (1H, br s, Wh/ 2 ) 3 Hz, H-15),
6.06 (1H, d, J ) 10.1 Hz, H-2), 5.36 (1H, br s, Wh/ 2 ) 3 Hz,
H-15), 2.92 (1H, ddd, J ) 12.1, 4.5, 1.9 Hz, H-5), 2.83 (1H, br
s, Wh/ 2 ) 11 Hz, H-7), 2.13 (1H, d, J ) 2.4 Hz, H-12), 1.88 (1H,
ddd, J ) 13.5, 13.5, 3.4 Hz, H-9), 1.80-1.72 (2H, H-6 and -8),
1.65 (1H, m, H-6), 1.61 (1H, m, H-9), 1.50 (1H, dddd, J ) 13.5,
13.5, 3.9, 3.7 Hz, H-8), 1.17 (3H, s, H-14); 13C NMR (CDCl3,
125 MHz) δ 187.9 (s, C-3), 158.5 (d, C-1), 145.6 (s, C-4), 128.8
(d, C-2), 122.0 (t, C-15), 86.5 (s, C-11), 70.1 (d, C-12), 46.1 (d,
C-5), 38.2 (s, C-10), 34.4 (t, C-9), 34.0 (t, C-6), 29.2 (q, C-14),
27.6 (t, C-8), 26.0 (d, C-7); HREIMS m/z 200.1196 (calcd for
224 °C; [R]24 +67.9° (c 0.16, CHCl3); IR (CHCl3) νmax 1778,
D
1
1678, 1624 cm-1; H NMR (CDCl3, 500 MHz) δ 6.82 (1H, d, J
) 9.8 Hz, H-1), 6.28 (1H, m, H-15), 6.15 (1H, d, J ) 3.2 Hz,
H-13), 6.04 (1H, d, J ) 9.8 Hz, H-2), 5.72 (1H, m, H-15), 5.48
(1H, d, J ) 3.2 Hz, H-13), 4.13 (1H, dd, J ) 10.7, 10.7 Hz,
H-6), 3.03 (1H, ddd, J ) 10.7, 2.3, 2.3 Hz, H-5), 2.64 (1H, m,
H-7), 2.16 (1H, m, H-8), 1.87 (1H, m, H-9), 1.07 (3H, s, H-14);
13C NMR (CDCl3, 125 MHz) δ 187.7 (s, C-3), 170.0 (s, C-12),
158.4 (d, C-1), 140.4 (s, C-4), 138.1 (s, C-11), 127.5 (d, C-2),
122.3 (t, C-15), 117.7 (t, C-13), 79.1 (d, C-6), 52.6 (d, C-5), 49.4
(d, C-7), 39.7 (s, C-10), 36.0 (t, C-9), 21.1 (t, C-8), 19.6 (q, C-14);
HREIMS m/z 244.1096 (calcd for C15H16O3, 244.1100). The
peak (tR 3.7 min) gave starting material 8 (3.3 mg, 13%).
Deh yd r obr om in a tion of 8 by Meth od B. A solution of 8
(24.0 mg, 0.0738 mmol) and TBAF (1 M in THF, 148 µL) in
THF (2.2 mL) was stirred at room temperature for 5 h, poured
into saturated aqueous NH4Cl (5 mL), and extracted with CH2-
Cl2 (5 × 10 mL). The extracts were washed, dried, and
separated by flash chromatography [3.4 g; 1.2 cm i.d.; EtOAc-
hexane (3:7)]. The eluate was concentrated and further
separated by HPLC [column A; EtOAc-hexane (4:6); 3.0 mL/
min]. The first peak gave 1 (11.3 mg, 63%) as colorless crystals.
The second peak gave starting material 8 (4.1 mg, 17%).
4r-Br om o-13-n or eu d esm -1-en -11-yn -3-on e (10). To a
stirred solution of 9 (23.5 mg, 0.116 mmol) and Et3N (48.2 µL,
0.348 mmol) in CH2Cl2 (0.8 mL) was added TMSOTf (33.6 µL,
0.174 mmol) at 0 °C. After 1.6 h, the mixture was treated with
PTAB (48.1 mg, 0.128 mmol) in CH2Cl2 (0.4 mL) and stirred
at this temperature for 20 min. The reaction mixture was
poured into 10% aqueous Na2S2O3 and extracted with CH2-
C
14H16O, 200.1201).
Deh yd r obr om in a tion of 5b by Meth od C. A mixture of
5b (21.9 mg, 0.0779 mg), Li2CO3 (23.1 mg, 0.312 mmol), and
LiBr (23.1 mg) in DMF (1.6 mL) was stirred at 120 °C for 2 h,
cooled to room temperature, poured into saturated aqueous
NaCl, and extracted with EtOAc. The combined extracts were
washed with 2 M HCl and saturated aqueous NaCl, dried (Na2-
SO4), and separated by flash chromatography [2.5 g; 1.2 cm

Exo-Endo Cross-Conjugated Cyclohexadienones
J ournal of Natural Products, 2003, Vol. 66, No. 5 593
Cl2. The extracts were dried and purified by flash chromatog-
raphy [2.5 g; 1.2 cm i.d.; EtOAc-hexane (5:95)] to give 10 (29.9
mg, 92%) as colorless crystals: mp 115-119 °C; [R]20D -184.1°
After 1 h of A549 cells with or without test compounds in a
microtiter plate at 3 × 104 cells/well in 150 µL, 50 µL of IL-1â
was added to the culture at a final concentration of 0.25 ng/
mL, and the cells were further incubated for 6 h. The cells
were washed once with phosphate-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 overnight, the fixed cells
were treated with mouse anti-human ICAM-1 antibody for 60
min at 37 °C. After being washed with 0.02% Tween-PBS, the
cells were treated with peroxidase-linked anti-mouse IgG
antibody for 60 min at 37 °C. The cells were washed three
times with 0.02% Tween-PBS. The cells were incubated with
the substrate (0.1% o-phenylenediamine dihydrochloride and
0.02% H2O2 in 0.2 M sodium citrate buffer, pH 5.3) for 20 min
in the dark and assayed for the absorbance at 415 nm by using
a microplate reader. Expression of ICAM-1 was calculated as
follows.
(c 1.32, CHCl3); IR (CHCl3) νmax 3316, 2120, 1682 cm-1 1H
;
NMR (CDCl3, 500 MHz) δ 6.79 (1H, d, J ) 9.9 Hz, H-1), 5.99
(1H, dd, J ) 9.9, 0.7 Hz, H-2), 2.49 (1H, dd, J ) 12.7, 2.2 Hz,
H-5), 2.39 (2H, H-6 and -7), 2.15 (1H, dd, J ) 2.2, 0.5 Hz, H-12),
1.95 (1H, m, H-8), 1.80 (3H, d, J ) 0.7 Hz, H-15), 1.58 (1H,
ddd, J ) 13.3, 3.3, 3.3 Hz, H-9), 1.45 (1H, ddd, J ) 13.3, 13.3,
3.9 Hz, H-9), 1.17 (3H, s, H-14); 13C NMR (CDCl3, 125 MHz)
δ 194.8 (s, C-3), 160.0 (d, C-1), 124.7 (d, C-2), 87.2 (s, C-11),
69.2 (s, C-4), 68.7 (d, C-12), 54.3 (d, C-5), 39.4 (t, C-9), 38.0 (s,
C-10), 29.8 (t, C-6), 29.4 (d, C-7), 28.0 (t, C-8), 25.1 (q, C-15),
20.4 (q, C-14); HREIMS m/z 280.0462 (calcd for C14H17OBr,
280.0463).
Deh yd r obr om in a tion of 10 by Meth od A. A solution of
10 (10.4 mg, 0.0370 mmol) and DBU (11.1 µL, 0.0740 mmol)
in PhH (0.3 mL) was stirred at room temperature for 17.5 h.
Additional DBU (5.6 µL, 0.0370 mmol) was added. The mixture
was stirred for 20.5 h, poured into 1 M HCl, and extracted
with EtOAc. The combined extracts were washed, dried, and
separated by flash chromatography [2.5 g; 1.2 cm i.d.; EtOAc-
hexane (5:95)], yielding a mixture of 10, 11, and unknown
product (4.9 mg) (1:1:1). The slower running gave 5d (0.8 mg,
11%) as colorless crystals.
Expression of ICAM-1 (% of control) )
(absorbance with sample treatment -
absorbance without IL-1â treatment)/
(absorbance with IL-1â treatment -
absorbance without IL-1â treatment) × 100
13-Nor eu d esm a -1,4(15)-d ien -11-yn -3-on e (11): Deh y-
d r obr om in a tion of 10 by Meth od B. A solution of 10 (23.4
mg, 0.0832 mmol) and TBAF (1 M in THF, 166 µL) in THF
(1.8 mL) was stirred at room temperature for 14.8 h. The
reaction mixture was treated with additional TBAF (1 M in
THF, 166 µL) and stirred for 5 h, poured into saturated
aqueous NH4Cl, and extracted with CH2Cl2. The combined
extracts were dried and separated by flash chromatography
[3 g; 1.2 cm i.d.; EtOAc-hexane (1:9)]. The eluate was further
separated by HPLC [column A; EtOAc-hexane (1:9); 3.0 mL/
min]. The first peak (tR 2.9 min) gave 11 (10.9 mg, 65%) as
A549 cells were incubated in the presence or absence of test
compounds for 24 h. At the last 4 h of incubation, the cells
were pulsed with 500 µg/mL of 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl tetrazolium bromide (MTT) for 4 h. MTT forma-
zan was solubilized with 5% sodium dodecyl sulfate (SDS)
overnight. Absorbance at 595 nm was measured. Cell viability
(%) was calculated as (experimental absorbance - background
absorbance)/(control absorbance - background absorbance) ×
100.
colorless prisms (EtOAc-hexane): mp 112-115 °C; [R]20
D
-183.6° (c 0.81, CHCl3); IR (CHCl3) νmax 3316, 2120, 1674, 1624
cm-1; 1H NMR (CDCl3, 500 MHz) δ 6.80 (1H, dd, J ) 10.0, 0.5
Hz, H-1), 6.10 (1H, dd, J ) 2.2, 1.0 Hz, H-15), 6.00 (1H, dd, J
) 10.0, 0.7 Hz, H-2), 5.21 (1H, ddd, J ) 2.4, 1.0, 0.7 Hz, H-15),
2.51 (1H, dddd, J ) 12.2, 2.4, 2.2, 2.2 Hz, H-5), 2.38 (1H, m,
H-7), 2.13 (1H, d, J ) 2.4 Hz, H-12), 2.07 (1H, m, H-6), 1.96
(1H, m, H-8), 1.74 (1H, m, H-8), 1.52 (1H, ddd, J ) 13.3, 13.3,
3.9 Hz, H-9), 0.97 (3H, d, J ) 0.5 Hz, H-14); 13C NMR (CDCl3,
125 MHz) δ 189.0 (s, C-3), 160.9 (d, C-1), 145.1 (s, C-4), 127.0
(d, C-2), 118.5 (t, C-15), 87.6 (s, C-11), 68.4 (d, C-12), 47.4 (d,
C-5), 37.2 (s, C-10), 36.3 (t, C-9), 29.9 (t, C-6), 28.9 (d, C-7),
27.9 (t, C-8), 17.7 (q, C-14); anal. C 83.93%, H 8.02%, calcd
for C14H16O, C 83.96%, H 8.05%. The second peak (tR 4.5 min)
gave 5d (1.3 mg, 8%) as colorless crystals.
Ter m iticid a l Activities. The termites used in this test
were the active, healthy workers of Coptotermes formosanus
Shiraki, a subterranean termite commonly found in J apan.
A 1 mL quantity of an acetone solution of each test
compound (1 mg for 1000 ppm and 0.1 mg for 100 ppm test
solutions) was applied to a filter paper disk (9 cm diameter)
placed in a Petri dish. After air-drying, the filter paper was
moistened with 1 mL of distilled H2O and 10 active worker
termites were introduced. Each dish was covered with a lid
and kept at 25 ( 1 °C for 5 or 7 days. The number of dead
termites was counted to calculate the percent mortality at 1,
2, and 5 days after treatment.
Deh yd r obr om in a tion of 10 by Meth od C. A mixture of
10 (20.4 mg, 0.0726 mg), Li2CO3 (21.6 mg, 0.290 mmol), and
LiBr (21.6 mg) in DMF (1.5 mL) was stirred at 120 °C for 30
min and cooled to room temperature. The mixture was
extracted with EtOAc, washed with 2 M HCl and aqueous
NaCl, dried, and separated by flash chromatography [2.5 g;
1.2 cm i.d.; EtOAc-hexane (5:95)]. After the slower running
5d (0.9 mg, 6%) was removed, the faster running was further
separated by HPLC [column A; EtOAc-hexane (5:95); 3.0 mL/
min]. The first peak gave 11 (11.2 mg, 77%) as colorless
crystals. The second peak (tR 5.6 min) gave starting material
10 (1.8 mg, 9%).
In h ibitor y Activity on In d u ction of ICAM-1. Human
lung carcinoma cell line A549 cells were maintained in RPMI
1640 medium (Invitrogen, Carlsbad, CA) supplemented with
10% (v/v) fetal calf serum (J RH Bioscience, Lenexa, KS), 50
µM 2-mercaptoethanol, and penicillin-streptomycin-neomy-
cin antibiotic mixture (Invitrogen).
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. We also
express our gratitude to Ms. H. Yamazaki of Sumitomo
Chemical Co., Ltd., for bioassay of compounds in termiticidal
activity.
Su p p or tin g In for m a tion Ava ila ble: Synthetic outlines of dehy-
drochamaecynenol and norsesquibenihiol are shown in Scheme 1.8
Details of the NMR experiments performed on compounds 1, 9, and
11 are summarized in Tables 1-3. The leukocyte-endothelial cell
interactions are illustrated graphically in Figure 1, and a possible
target of sample is shown in Figure 2. The experimental data of
inhibitory activities of 1 on expression of ICAM-1 and cell viability
are summarized in Table 4. This material is available free of charge
via the Internet at http://pubs.acs.org.
Mouse anti-human ICAM-1 antibody C167 was purchased
from Leinco Technologies, Inc. (Ballwin, MO), and peroxidase-
conjugated goat anti-mouse IgG antibody was obtained from
J ackson ImmunoResearch Laboratories, Inc. (West Grove, PA).
Recombinant human IL-1â was a commercial product of
Genzyme Diagnostic (Cambridge, MA).
Refer en ces a n d Notes
(1) Bohlmann, F.; Zdero, C. Phytochemistry 1982, 21, 647-651.
(2) Rodriguez, E.; Sanchez, B.; Grieco, P. A.; Majetich, G.; Oguri, T.
Phytochemistry 1979, 18, 1741-1742.
(3) Geissman, T. A.; Mukherjee, R. J . Org. Chem. 1968, 33, 656-660.
(4) Sims, J . J .; Berryman, K. A. Phytochemistry 1972, 11, 444-445.

594 J ournal of Natural Products, 2003, Vol. 66, No. 5
Higuchi et al.
(5) Herz, W.; Subramaniam, P. S.; Geissman, T. A. J . Org. Chem. 1968,
33, 3743-3749.
(11) We attempted the bromination of lithium enolate (LDA) of 6a with
CBr4 in THF at -78 °C. The desired 5b was obtained in only 4%
yield, accompanied by undesired monobromide 17 and dibromide 18
(12% and 7%, respectively). Compounds 17 and 18 were produced
via lithium acetylide.
(6) Hashimoto, H.; Tsuzuki, K.; Sakan, F.; Shirahama, H.; Matsumoto,
T. Tetrahedron Lett. 1974, 3745-3748.
(7) Tatsuta, K.; Akimoto, K.; Kinoshita, M. J . Am. Chem. Soc. 1979, 101,
6116-6118.
(8) Ando, M.; Kikuchi, K.; Isogai, K.; Ibe, S.; Asao, T. J . Nat. Prod. 1995,
58, 177-183, and references therein.
(9) Bromination of 2a with PTAB in THF at 0 °C was attempted. The
purified products were a mixture of undesired monobromide 15 and
additional product 16 (97%, 15:16 ) 78:19), which are probably
produced by the following successive reaction: (i) the Michael addition
of Br- of PTAB to the â-position of R,â-unsaturated ketone; (ii)
addition of Br+ to the resulting anion at R-position (formation of 16);
(iii) dehydrobromination of 16 in the reaction conditions.
(12) Compounds 7, 12, and 13, which were used in the bioassay (Table
3), were synthesized by our published method: Ando, M.; Wada, T.;
Kusaka, H.; Takase, K.; Hirata, N.; Yanagi, Y. J . Org. Chem. 1987,
52, 4792-4796.
(13) Unpublished results from our laboratory.
(14) Kawai, S.; Kataoka, T.; Sugimoto, H.; Nakamura, A.; Kobayashi, T.;
Arao K.; Higuchi, Y.; Ando, M.; Nagai, K. Immunopharmacology 2000,
48, 129-135.
(15) Yuuya, S.; Hagiwara, H.; Suzuki, T.; Ando, M.; Yamada, A.; Suda,
K.; Kataoka, T.; Nagai, K. J . Nat. Prod. 1999, 62, 22-30.
(10) Reuss, R. H.; Hassner, A. J . Org. Chem. 1974, 39, 1785-1787.
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