Thrombomodulin induction in cultured human endothelial cells by 9-cis-locked retinoic acid analogues

Article abstract of DOI:10.1016/j.bmc.2006.04.043

9-cis-Retinoic acid (RA) analogues devised to lock the 9-cis double bond by ring formation were synthesized using two stereoselective carbon-carbon bond formation reactions as key steps. The palladium-mediated Suzuki reaction was adopted to construct a 7E

Full text of DOI:10.1016/j.bmc.2006.04.043

Bioorganic & Medicinal Chemistry 14 (2006) 5099–5109
Thrombomodulin induction in cultured human endothelial cells
by 9-cis-locked retinoic acid analogues
Shiro Ikegami,a,* Takamasa Iimori,a Minoru Sudo,a Maroka Kitsukawa,a
Alireza Foroumadi,a Takeshi Yonemura,a Hideyo Takahashi,a
Keiichiro Kizakia and Hidemi Ishiib
aFaculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-0195, Japan
bShowa Pharmaceutical University, Higashi-Tamagawagakuen, Machida, Tokyo 194-8543, Japan
Received 1 March 2006; revised 3 April 2006; accepted 4 April 2006
Available online 19 May 2006
Abstract—9-cis-Retinoic acid (RA) analogues devised to lock the 9-cis double bond by ring formation were synthesized using two
stereoselective carbon–carbon bond formation reactions as key steps. The palladium-mediated Suzuki reaction was adopted to
construct a 7E-double bond (RA numbering) and the Horner–Emmons olefination was employed for stereoselective 11E-double
bond (RA numbering) formation. The synthesized 9-cis-RA analogues that are locked by five-membered ring systems (cyclopentene,
dihydrofuran, and dihydrothiophene) were shown to have comparable thrombomodulin induction activities to that of 9-cis RA.
Conformational analysis of these compounds showed their similarity to 9-cis RA in the spatial orientation of the side chain and
the terminal carboxy group.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
Recently, much attention has been paid to 9-cis-RA,
because it has been identified as a second endogenous
RA that binds both retinoic acid receptors (RARs)
and retinoid X receptors (RXRs) with nanomolar affin-
ity.6–8 This finding prompted us to investigate the struc-
ture–activity relationships of RA analogues to TM
induction. Figure 1 shows our preliminary observations
of TM induction by geometric isomers of RAs (ATRA,
9-cis-RA, 11-cis-RA, and 13-cis-RA). The TM expres-
sion depended on the amount of all of Ras,9 but a
significant increase was observed at 0.1 lM 9-cis-RA,
which was most potent among examined. Thus, our
attention was centered around the structural modifica-
tion which fixes the cis-double bond of 9-cis-RA.
Retinoic acid (RA) is well known to play a critical role
in many aspects of cell proliferation and differentiation,
and has proved useful for the treatment of dermatologic
diseases and certain cancers.1 We have demonstrated
that all-trans-RA (ATRA) causes an increase in throm-
bomodulin (TM) antigen in human umbilical vein endo-
thelial cells (HUVECs) in vitro.2,3 TM is an endothelial
membrane surface glycoprotein and represents one of
the most important antithrombotic factors in the antico-
agulant system. TM forms a 1:1 stoichiometric complex
with thrombin and then thrombin loses its procoagulant
functions such as fibrin formation. Additionally, the
TM–thrombin complex markedly promotes activation
of protein C, which proteolytically degrades coagulation
factors.4,5 Since TM functions as an antithrombotic
agent, RA has the ability to change the coagulant bal-
ance by controlling TM expression.
2. Results and discussion
2.1. Molecular design
Our design of 9-cis-RA analogues presented herein was
based on locking its 9-cis-double bond by introducing
an appropriate ring system.10
Keywords: Retinoic acid; 9-cis-Retinoic acid; Suzuki coupling;
Thrombomodulin.
*
As shown in Figure 2, two modes of ring formation can
be applied to lock the 9-cis-double bond. Mode A forms
Corresponding author. Tel./fax: +81 42 685 3729; e-mail: shi-ike@
pharm.teikyo-u.ac.jp
0968-0896/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.04.043

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S. Ikegami et al. / Bioorg. Med. Chem. 14 (2006) 5099–5109
300
Retinoic Acids
COOH
250
l
)
o
all-trans-retinoic acid
[ ATRA ]
ontr
c
200
150
of
%
(
e
t
ysa
l
COOH
l
l
e
9-cis-RA
11-cis-RA
13-cis-RA
ATRA
9-cis-retinoic acid
[ 9-cis-RA ]
n
c
100
0
n
i
e
antig
COOH
TM
11-cis-retinoic acid
[ 11-cis-RA ]
0
.01
.1
1
10
COOH
13-cis-retinoic acid
[ 13-cis-RA ]
Figure 1. Thrombomodulin induction in human endothelial cells by geometric isomers of RAs.
B
B
A
A
COOH
9-cis-RA
2a
COOH
1a
COOH
Figure 2. Two modes of ring formation of 9-cis-locked RA.
a ring system inside the molecule, and mode B forms one
outside. Some groups used mode A ring formation to
lock the 11-cis-double bond in their studies on the mech-
anism of vision in which cis–trans isomerization of
retinal tissue plays an important role.11–14 Successful
results were obtained in those studies, although mode
A ring formation was considered to alter the three-di-
mensional skeleton of the parent cis-RA. Molecular
mechanics calculations have been widely employed to
investigate stable conformations of biologically interest-
ing molecules, and Dawson’s group has adopted this
technique to discuss the functionally active conforma-
tion of 9-cis-RA based on comparison with RAR- and
RXR-selective retinoids.15,16 Mohamadi et al. conduct-
ed a similar computational analysis17 of 9-cis-locked
RAs 1a and 2a having five-membered ring systems as
representative molecules for our design. Monte Carlo
conformational searches18 of 1a and 2a showed that
they have different conformational properties. The most
stable conformer of 9-cis-RA, which was selected as the
functionally active conformer based on Jong et al.’s
assumption,15 is superimposed on the corresponding
similar conformers of 1a or 2a to visualize their
differences (polytube model in Fig. 3, colored: 9-cis-
RA and black: 9-cis-locked analogue).
Apparently, ring formation in 1a causes distinct loca-
tions of the side chain and the terminal carboxyl group.
Figure 3. Molecular design of 9-cis-locked RA by inducing a ring
system. Left, 1a superimposed on the most stable conformer of
9-cis-RA; right, 2a superimposed on the most stable conformer of
9-cis-RA.

S. Ikegami et al. / Bioorg. Med. Chem. 14 (2006) 5099–5109
5101
On the contrary, mode B ring formation (2a) retains
these locations nearly unchanged. Employment of a
six-membered or seven-membered ring system resulted
in analogous conformational properties. Katsuta et al.
have reported the synthesis of 9-cis-RA analogues
employing mode A ring formation (six-membered ring)
and showed that it was less effective than 9-cis-RA.19
Their result is consistent with our computational consid-
eration because the carboxyl group of their derivative
was also calculated to be differently positioned from that
of 9-cis-RA. Since 2a was found to have a similar
conformational property based on the inspection of this
computational molecular modeling, we focused our
attention on the ‘outer locked’ 9-cis-RAs (mode B ring
formation). Recently, the cyclohexene derivative 2b
(Ro 25-6603), which is one of our target molecules,
has been reported to be an RXR-selective ligand.20,21
Similarly, analogues of 9-cis-RA incorporating an
alicyclic ring, which included our target molecules
2a–c, have been synthesized and their binding affinities
for the RXR receptor were determined.22 Conforma-
tionally defined 6-s-trans-RA analogues employing a
similar ring formation have also been reported.23
In the first stage of this study, we prepared cycloalkane
and benzene derivatives 2a–d. After performing biolog-
ical evaluations showing that the cyclopentene deriva-
tive 2a was most effective (vide infra), we next
examined the incorporation of heteroatoms into five-
membered ring systems (2e–h).
2.2. Chemistry
9-cis-Locked RA analogues 2a–h were synthesized using
two stereoselective carbon–carbon bond formation reac-
tions as key steps22 (Scheme 1).
In the first step, the Suzuki reaction of alkenyl triflates
(3a–c, e, and g) or aryl iodides (3d and f) with alkenylbo-
rane 4 was employed to construct the 7E-double bond
(RA numbering). This palladium-catalyzed cross-cou-
pling has been known to proceed with complete reten-
tion of stereochemistry of the double bond,24,25 and
completely stereochemically defined coupling products
5a–g were obtained in high yield (90%-quantitative).
The esters 5a–g thus obtained were converted to
alcohols 6a–g using an appropriate reducing reagent.
O
B
Y
Y
O
4
( 1.5 equiv )
X
Pd(PPh3)4 ( 4.0 mol% )
KOH, benzene, reflux
COOR
COOR
5a 97%
5b 98%
5c 96%
5d 96%
5e 93%
5f 98%
5g 97%
3a- c, e, g X = OTf
3d, f, X = I
Y
Y
LiAlH4 ( a - d, f )
DIBAH ( e, g )
MnO2 ( a - d )
CH2OH
CHO
7a 95%
(COCl)2, DMSO, NEt3
-78 ˚C ~ rt ( e - g )
6a 90%
7b 96%
7c 94%
7d 92%
7e 92%
7f 92%
7g 88%
7h 89%
6b 90%
6c 94%
6d 95%
6e 97%
6f 97%
6g 95%
MnO2
Y
O
(EtO)2P
Y
COOMe
( 2.0 equiv )
NaOH / aq. EtOH
LDA ( 2.0 equiv )
THF, rt, 30 min
8a 91%
8b 92%
8c 90%
8d 92%
8e 94%
8f 94%
8g 96%
8h 93%
COOMe
2a 96%
2b 99%
2c 96%
2d 98%
2e 94%
2f 97%
2g 96%
2h 96%
COOH
c
f
b
d
e
a
g
h
S
Y
O
O
S
Scheme 1. Reagents: (a) LiAlH4, THF (for 6a–d, f); DIBAL, CH2Cl2, (for 6e, g); (b) MnO2, pet. ether (for 7a–d); (COCl)2, DMSO, Et3N, CH2Cl2,
(for 7e–g); (COCl)2, DMSO, Et3N, CH2Cl2, then MnO2, pet. ether (for 7h from 6g); (c) NaOH, aq EtOH.

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S. Ikegami et al. / Bioorg. Med. Chem. 14 (2006) 5099–5109
In the case of dihydrofuran and dihydrothiophene deriv-
atives (6e and g), DIBAL was used in place of LiAlH4
because ring cleavage of these heterocycles was ob-
served. After conversion of 6a–g to aldehydes 7a–g in
MnO2 or Swern oxidations, Horner–Emmons olefin-
ation resulted in a stereoselective coupling reaction to
form an 11E-double bond (RA numbering) in 90–98%
yield.26 Thiophene aldehyde 7h was prepared by oxida-
tive aromatization of 7g and was similarly converted
into 8h. Although a small amount (ca. <5% estimated
by 1H NMR) of stereoisomers was formed in the olefin-
ation, they were easily removed by recrystallization at
the final stage. Hydrolysis of esters 8a–h by NaOH in
aqueous EtOH afforded the desired acids 2a–h. In this
straightforward synthesis, RA analogues were prepared
stereoselectively in high overall yield (65–81%).
was comparable to the increase induced by 2a. Aroma-
tization of heterocycles (2f and h) significantly lowered
TM induction potency.
2.4. Discussion
Based on the result that 9-cis-RA induced TM most
effectively, we designed RA analogues that retain the
conformational property of 9-cis-RA. The computation-
al conformational searches of RA analogues showed
that 2a–c could have a low-energy conformation similar
to the ‘active conformation’ of 9-cis-RA. Incorporation
of a five-membered ring system especially modifies the
spatial orientation of the side chain relative to 9-cis-
RA, as shown in Figure 2. On the other hand, a similar
conformation of the aromatic analogue 2d was calculat-
ed to locate ca. +1 kcal/mol above the global minimum,
and the distinct conformation was found to be stable.
This conformational analysis suggested a relation
between the conformational properties and the activity
of TM induction. The five-membered ring system
(in 2a, e, and g), which mimicked the most stable confor-
mation of 9-cis-RA, could be used as an effective lock
with retention of biological activity. The low potency
of TM induction by the aromatic analogues 2d, f, and
h was also consistent with the conformational properties
mentioned above. Although it was difficult to determine
the relation between ring size and activity, it appears
plausible that the lower activity of 2b and c was the
result of changes in steric bulk caused by introduction
of the six- or seven-membered ring system.
2.3. Biological activity
HUVECs were treated with various concentrations of
ATRA, 9-cis-RA, and synthesized RA analogues 2a–d
for 24 h, and TM antigen levels in cell lysates were mea-
sured in an enzyme immunoassay.27
As shown in Figure 4, the effects of 9-cis-RA and cyclo-
pentene derivative 2a were dose-dependent and the TM
antigen levels increased to 150% of the baseline level at
the dose of RAs of 0.001 lM and to 180% at 0.01 lM.
Cyclohexene derivative 2b showed a similar activity to
9-cis-RA at 0.001 lM, but lower activity was observed
at higher concentrations (1–10 lM). The other deriva-
tives 2c and d were less potent in this assay.
Since the analogues described herein are designed to
mimic 9-cis-RA, which activates both RARs and RXRs,
it is conceivable that they are pan-agonists (activating
both RARs and RXRs). However, recent reports on
RAR- and RXR-selective ligands,22,23 in which the
cyclohexene derivative 2b was shown to be highly
The effects of heterocyclic analogues 2e–h were also
measured at the concentrations of 0.001 lM, 0.1 lM,
and 10 lM (Fig. 5). Dihydrofuran and dihydrothioph-
ene derivatives 2e and
g increased TM antigen
levels to 200% of the baseline level at 0.1 lM, which
250
200
9-cis-RA
COOH
COOH
COOH
150
100
n
9-cis-RA
2a
2b
2c
2d
2a ( n = 1 )
2b ( n = 2 )
2c ( n = 3 )
0
10
.0001 .001 .01
.1
1
0
2d
Figure 4. TM induction by 9-cis-locked RA analogues (2a–d).

S. Ikegami et al. / Bioorg. Med. Chem. 14 (2006) 5099–5109
5103
250
ol)
200
2a
COOH
150
100
X
2a
2e
2g
2f
COOH
2e ( X=O )
2g ( X=S )
2h
X
T
M
a
n
t
i
g
e
n
i
n
c
e
l
l
l
y
s
a
t
e
(
%
o
f
c
o
n
t
r
0
0
10
.001
.01
.1
1
2f ( X=O )
2h ( X=S )
COOH
Figure 5. TM induction by 9-cis-locked RA analogues (2e–h).
RXR selective, have suggested that the 9-cis-locked
analogues are also RXR selective. Although there are
many reports on the RXR selectivity of 9-cis-RA,28,29
we have not clarified which receptor(s), RAR and/or
RXR, is involved in the expression of TM induction; a
study concerning the precise role of RAs in the TM
induction is now underway.
3.1.2. Computer modeling. Monte Carlo conformational
searches18 were performed using MacroModel 4.017 with
the MM2* derivative of the MM2 forcefield.
3.1.3. Ethyl 2-[(E)-2-(2,6,6-trimethylcyclohex-1-enyl)eth-
enyl]cyclopent-1-enecarboxylate (5a).22 To a solution of
Pd(PPh3)4 28 mg (0.024 mmol) in benzene (2 ml) was
added enol triflate 3a 172 mg (0.60 mmol) in 3 ml of
benzene. After stirring for 15 min under Ar, 0.9 M ben-
zene solution of alkenylborane 428 1.0 ml (0.90 mmol)
and 4.0 M KOH aqueous solution 0.23 ml (0.90 mmol)
were added at room temperature. The mixture was heat-
ed to reflux for 1 h, and the residual organoborane was
oxidized by 3 M NaOH (0.1 ml) and 30% H2O2 (0.1 ml)
for 1 h at room temperature. The reaction mixture was
filtered through Celite, and the filtrate was extracted
with ether. The organic layer was washed with water
and brine, and dried over MgSO4. The solvent was evap-
orated and the crude residue was purified by silica gel
column chromatography (hexane/ethyl acetate 40:1 as
an eluent) to afford 5a 167 mg (97% yield) as a colorless
3. Conclusion
Molecular design aided by computational chemistry
efficiently creates 9-cis-locked RA analogues that re-
tain the ‘active conformation’ of the parent 9-cis-
RA using appropriate ring formation. The analogues
thus designed were synthesized stereoselectively and,
as expected, induced TM with potency comparable
to that of 9-cis-RA. It is also noteworthy that the
synthesis of the analogues is very efficient (65–88%
overall yield) and thus provides a large quantity of
the compounds for various types of biological
evaluation.
1
oil. IR (neat) 2930, 1701, 1227, 1107 cmÀ1. H NMR
(400 MHz, CDCl3) d 1.07 (6H, s, C(CH3)2), 1.30 (3H,
t, J = 7.0 Hz, COOCH2CH3), 1.47 (2H, m, CH2), 1.62
(2H, m, CH2), 1.77 (3H, s, CH3), 1.88 (2H, m, CH2),
2.04 (2H, t, J = 6.3 Hz, CH2), 2.73 (4H, t, J = 7.6 Hz,
2(CH2)), 4.21 (2H, q, J = 7.0 Hz, COOCH2CH3), 6.21
(1H, d, J = 16.5 Hz, C@CH), 7.34 (1H, d, J = 16.5 Hz,
C@CH). MS (EI) m/z 288 (M+).
3.1. Experimental
3.1.1. General. Melting points were determined on a
Yanagimoto micro melting point apparatus and
are uncorrected. IR spectra were recorded on a
JASCO FT/IR-8000 spectrometer. 1H NMR spectra
were recorded on a JEOL GSX-400 spectrometer
(400 MHz). High and low mass spectra were conducted
on an JEOL SX-102A spectrometer. Elemental analysis
was performed by Perkin-Elmer Series II CHNS/O ana-
lyzer 2400. Tetrahydrofuran and benzene were distilled
from sodium metal prior to use. Methylene chloride
was distilled from CaH2. Column chromatography was
performed by using BW-820 (Fuji Silysia) or Wakogel
C-200, and thin-layer chromatography was carried out
on 0.25-mm E. Merck precoated silica-gel glass plate
(Art. 5715).
Similar reactions of 3b, 3c, 3e, and 3g gave the following
compounds.
3.1.4. Ethyl 2-[(E)-2-(2,6,6-trimethylcyclohex-1-enyl)eth-
enyl]cyclohex-1-enecarboxylate (5b).22 Yield: 98%. IR
(neat) 2930, 1709, 1208 cmÀ1 1H NMR (400 MHz,
.
CDCl3)
d 1.02 (6H, s, C(CH3)2), 1.30 (3H, t,
J = 7.0 Hz, COOCH2CH3), 1.46 (2H, m, CH2), 1.61
(2H, m, CH2), 1.67 (4H, m, 2(CH2)), 1.72 (3H, d,
J = 0.6 Hz, CH3), 2.01 (2H, t, J = 6.2 Hz, CH2), 2.39

5104
S. Ikegami et al. / Bioorg. Med. Chem. 14 (2006) 5099–5109
(4H, m, 2(CH2)), 4.21 (2H, q, J = 7.0 Hz,
COOCH2CH3), 6.32 (1H, d, J = 16.3 Hz, C@CH), 6.83
(1H, d, J = 16.3 Hz, C@CH). MS (EI) m/z 302 (M+).
Similar reactions of 3f gave the following compound.
3.1.9.
enyl)ethenyl]furan-3-carboxylate (5f). Yield: 98%. IR
(neat) 2928, 1728, 1148, 1094 cmÀ1 1H NMR
Methyl
4-[(E)-2-(2,6,6-trimethylcyclohex-1-
3.1.5. Ethyl 2-[(E)-2-(2,6,6-trimethylcyclohex-1-enyl)eth-
enyl]cyclohept-1-enecarboxylate (5c).22 Yield: 95%. IR
.
(400 MHz, CDCl3) d 1.06 (6H, s, C(CH3)2), 1.48 (2H,
m, CH2), 1.63 (2H, m, CH2), 1.77 (3H, s, CH3), 2.03
(2H, t, J = 6.3 Hz, CH2), 3.83 (3H, s, COOCH3), 6.49
(1H, d, J = 16.6, C@CH), 6,55 (1H, d, J = 16.6 Hz,
C@CH), 7.55 (1H, d, J = 1.7 Hz, Ar), 7.97 (1H, d,
J = 1.7 Hz, Ar). MS (EI) m/z 274 (M+).
(neat) 2924, 1703, 1200 cmÀ1 1H NMR (400 MHz,
.
CDCl3)
d 1.02 (6H, s, C(CH3)2), 1.29 (3H, t,
J = 7.1 Hz, COOCH2CH3), 1.46 (2H, m, CH2), 1.51–
1.64 (6H, m, 3(CH2)), 1.71 (3H, d, J = 0.6 Hz, CH3),
1.76 (2H, m, CH2), 2.01 (2H, t, J = 6.3 Hz, CH2), 2.53
(4H, m, 2(CH2)), 4.20 (2H, q, J = 7.1 Hz,
COOCH2CH3), 6.33 (1H, d, J = 16.2 Hz, C@CH), 6.74
(1H, d, J = 16.2 Hz, C@CH). MS (EI) m/z 316 (M+).
3.1.10.
2-[(E)-2-(2,6,6-trimethylcyclohex-1-enyl)ethen-
yl]cyclopent-1-enemethanol (6a).22 To a suspension of
LiAlH4 65 mg (1.7 mmol) in THF (5 ml) was added
dropwise ester 5a 119 mg (0.41 mmol) in THF (3 ml)
at 0 °C and the reaction mixture was stirred for
30 min. Saturated aqueous Na2SO4 solution (1 ml) was
slowly added to this mixture, and precipitate was re-
moved by filtration through Celite. The filtrate was
dried over MgSO4 and concentrated in vacuo. Alcohol
6a 92 mg (90% yield) was obtained as a colorless oil after
purification by silica gel column chromatography (hex-
ane/ethyl acetate 8:1 as an eluent) IR (neat) 3312,
3.1.6.
enyl)ethenyl]-2,5-dihydrofuran-3-carboxylate (5e). Yield:
93%. IR (neat) 2930, 1715, 1237 cmÀ1 1H NMR
Methyl
4-[(E)-2-(2,6,6-trimethylcyclohex-1-
.
(400 MHz, CDCl3) d 1.05 (6H, s, C(CH3)2), 1.47 (2H,
m, CH2), 1.62 (2H, m, CH2), 1.77 (3H, s, CH3), 2.05
(2H, t, J = 6.2 Hz, CH2), 3.77 (3H, s, COOCH3), 4.89
(2H, t, J = 4.2 Hz, OCH2), 4.99 (2H, t, J = 4.2 Hz,
OCH2), 6.26 (1H, d, J = 16.6 Hz, C@CH), 7.19 (1H, d,
J = 16.6 Hz, C@CH). MS (EI) m/z 276 (M+).
1
2926, 1456, 1030 cmÀ1. H NMR (400 MHz, CDCl3) d
3.1.7.
Methyl
4-[(E)-2-(2,6,6-trimethylcyclohex-1-
(5g).
Yield: 97%. IR (neat) 2928, 1715, 1599, 1435, 1219,
1.01 (6H, s, C(CH3)2), 1.46 (2H, m, CH2), 1.61 (2H,
m, CH2), 1.71 (3H, d, J = 0.9 Hz, CH3), 1.90 (2H, m,
CH2), 2.01 (2H, t, J = 6.1 Hz, CH2), 2.58 (4H, t,
J = 7.5 Hz, 2(CH2)), 4.33 (2H, s, CH2OH), 6.05 (1H,
d, J = 15.8 Hz, C@CH), 6.35 (1H, d, J = 158 Hz,
C@CH). MS (EI) m/z 246 (M+).
enyl)ethenyl]-2,5-dihydrothiophen-3-carboxylate
1103 cmÀ1. H NMR (400 MHz, CDCl3) d 1.05 (6H, s,
1
C(CH3)2), 1.47 (2H, m, CH2), 1.62 (2H, m, CH2), 1.76
(3H, d, J = 0.6 Hz, CH3), 2.05 (2H, t, J = 6.1 Hz,
CH2), 3.77 (3H, s, COOCH3), 4.06 (2H, m, SCH2),
4.13 (2H, m, SCH2), 6.41 (1H, dd, J = 16.5, 0.6 Hz,
C@CH), 7.43 (1H, d, J = 16.5 Hz, C@CH). MS (EI)
m/z 292 (M+).
Similar reactions of 5b–5d and 5f gave the following
compounds.
3.1.11. 2-[(E)-2-(2,6,6-Trimethylcyclohex-1-enyl)ethen-
yl]cyclohex-1-enemethanol (6b).22 Yield: 90%. IR (neat)
3.1.8.
Methyl
2-[(E)-2-(2,6,6-trimethylcyclohex-1-
1
enyl)ethenyl]benzoate (5d). To a solution of Pd(PPh3)4
17 mg (0.015 mmol) in benzene (1 ml) was added
methyl o-iodobenzoate 3d 78 mg (0.30 mmol) in 1 ml
of benzene. After stirring for 15 min under Ar,
0.9 M benzene solution of alkenylborane 424 0.50 ml
(0.45 mmol) and 4.0 M KOH aqueous solution
0.34 ml (1.4 mmol) were added at room temperature.
The mixture was heated to reflux for 2 h, and the
residual organoborane was oxidized by 3 M NaOH
(0.1 ml) and 30% H2O2 (0.1 ml) for 1 h at room tem-
perature. The reaction mixture was filtered through
Celite, and the filtrate was extracted with ether. The
organic layer was washed with water and brine, and
dried over MgSO4. The solvent was evaporated and
the crude residue was purified by silica gel column
chromatography (hexane/ethyl acetate 30:1 as an elu-
ent) to afford 5d 82 mg (96% yield) as a colorless
3227, 2924, 1458 cmÀ1. H NMR (400 MHz, CDCl3) d
1.01 (6H, s, C(CH3)2), 1.46 (2H, m, CH2), 1.61 (2H,
m, CH2), 1.66 (4H, m, 2(CH2)), 1.70 (3H, d,
J = 0.6 Hz, CH3), 2.00 (2H, t, J = 6.1 Hz, CH2), 2.26
(4H, m, 2(CH2)), 4.23 (2H, s, CH2OH), 6.11 (1H, dd,
J = 16.2, 0.6 Hz, C@CH), 6.48 (1H, d, J = 16.2 Hz,
C@CH). MS (EI) m/z 260 (M+).
3.1.12. 2-[(E)-2-(2,6,6-Trimethylcyclohex-1-enyl)ethen-
yl]cyclohept-1-enemethanol (6c).22 Yield: 94%. IR (neat)
1
3252, 2923, 1451 cmÀ1. H NMR (400 MHz, CDCl3) d
1.01 (6H, s, C(CH3)2), 1.45–1.55 (6H, m, 3(CH2)), 1.62
(2H, m, CH2), 1.70 (3H, d, J = 0.6 Hz, CH3), 1.78
(2H, m, CH2), 2.02 (2H, t, J = 6.1 Hz, CH2), 2.42 (4H,
m, 2(CH2)), 4.26 (2H, s, CH2OH), 6.11 (1H, d,
J = 15.9 Hz, C@CH), 6.38 (1H, d, J = 15.9 Hz, C@CH).
MS (EI) m/z 256 (M+ÀH2O).
oil. IR (neat) 2924, 1723, 1244, 1076 cmÀ1. H NMR
1
(400 MHz, CDCl3) d 1.10 (6H, s, C(CH3)2), 1.50
(2H, m, CH2), 1.65 (2H, m, CH2), 1.80 (3H, d,
J = 0.9 Hz, CH3), 2.04 (2H, t, J = 6.3 Hz, CH2), 3.89
(3H, s, COOCH3), 6.57 (1H, dd, J = 16.2, 0.9 Hz,
C@CH), 7.06 (1H, d, J = 16.2 Hz, C@CH), 7.27 (1H,
dt, J = 1.1, 7.6 Hz, Ar), 7.47 (1H, dt, J = 1.1, 7.6 Hz,
Ar), 7.61 (1H, dd, J = 1.1, 7.6 Hz, Ar), 7.86 (1H,
dd, J = 1.1, 7.6 Hz, Ar). MS (EI) m/z 284 (M+).
3.1.13. 2-[(E)-2-(2,6,6-Trimethylcyclohex-1-enyl)ethen-
yl]benzyl alcohol (6d). Yield: 95%. IR (neat) 3330,
1
2926, 1455, 1099 cmÀ1. H NMR (400 MHz, CDCl3) d
1.07 (6H, s, C(CH3)2), 1.50 (2H, m, CH2), 1.65 (2H,
m, CH2), 1.78 (3H, d, J = 0.9 Hz, CH3), 2.04 (2H, t,
J = 6.3 Hz, CH2), 4.76 (2H, s, CH2OH), 6.58 (1H, dd,
J = 16.2, 0.9 Hz, C@CH), 6.65 (1H, d, J = 16.2 Hz,
C@CH), 7.24 (1H, dt, J = 1.4, 7.4 Hz, Ar), 7.30 (1H,

S. Ikegami et al. / Bioorg. Med. Chem. 14 (2006) 5099–5109
5105
dt, J = 1.4, 7.4 Hz, Ar), 7.37 (1H, dd, J = 1.4, 7.4 Hz,
Ar), 7.54 (1H, dd, J = 1.4, 7.4 Hz, Ar). MS (EI) m/z
256 (M+).
Similar reactions of 6b–d gave the following compounds.
3.1.18. 2-[(E)-2-(2,6,6-Trimethylcyclohex-1-enyl)ethenyl]-
cyclohex-1-enecarbaldehyde (7b). Yield: 96%. IR (neat)
1
3.1.14. 4-[(E)-2-(2,6,6-Trimethylcyclohex-1-enyl)ethen-
yl]furan-3-methanol (6f). Yield: 92%. IR (neat) 3330,
2932, 1661, 1154 cmÀ1. H NMR (400 MHz, CDCl3) d
1.03 (6H, s, C(CH3)2), 1.48 (2H, m, CH2), 1.66–1.72
(6H, m, 3(CH2)), 1.73 (3H, d, J = 0.9 Hz, CH3), 2.04
(2H, t, J = 6.3 Hz, CH2), 2.32 (2H, t, J = 6.1 Hz,
CH2), 2.49 (2H, m, CH2), 6.46 (1H, dd, J = 15.9,
0.9 Hz, C@CH), 6.96 (1H, d, J = 15.9 Hz, C@CH),
10.31 (1H, s, CHO). MS (EI) m/z 258 (M+).
1
2928, 1458, 1055 cmÀ1. H NMR (400 MHz, CDCl3) d
1.05 (6H, s, C(CH3)2), 1.48 (2H, m, CH2), 1.63 (2H,
m, CH2), 1.75 (3H, d, J = 0.9 Hz, CH3), 2.02 (2H, t,
J = 6.1 Hz, CH2), 4.64 (2H, s, CH2OH), 6.17 (1H, d,
J = 16.4, C@CH), 6.48 (1H, d, J = 16.4 Hz, C@CH),
7.39 (1H, d, J = 1.5 Hz, Ar), 7.45 (1H, d, J = 1.5 Hz,
Ar), MS (EI) m/z 246 (M+).
3.1.19. 2-[(E)-2-(2,6,6-Trimethylcyclohex-1-enyl)ethenyl]-
cyclohept-1-enecarbaldehyde (7c). Yield: 86%. IR (neat)
1
3.1.15. 4-[(E)-2-(2,6,6-Trimethylcyclohex-1-enyl)ethenyl]-
2,5-dihydrofuran-3-methanol (6e). To a solution of ester
5e 104 mg (0.015 mmol) in 3 ml CH2Cl2 was added
1.76 M DIBAL in hexane 0.64 ml (1.13 mmol) at
À78 °C and the reaction mixture was stirred for
30 min at the same temperature. Methanol 0.3 ml and
saturated aqueous Rochelle salt 4 ml were sequentially
added to this mixture. After being stirred for 90 min at
room temperature, the two-phase mixture was extracted
with CH2Cl2. The organic layer was washed with brine
and dried over MgSO4. The solvent was evaporated
and the crude residue was purified by silica gel column
chromatography (hexane/ethyl acetate 2:1 as an eluent)
to afford 5e 90 mg (97% yield) as a colorless oil. IR
2924, 1659, 1165 cmÀ1. H NMR (400 MHz, CDCl3) d
1.04 (6H, s, C(CH3)2), 1.43–1.50 (4H, m, 2(CH2)),
1.57–1.66 (4H, m, CH2 and CH2), 1.74 (3H, d,
J = 0.9 Hz, CH3), 1.80 (2H, m, CH2), 2.04 (2H, t,
J = 6.4 Hz, CH2), 2.58 (4H, m, 2(CH2)), 6.40 (1H, dd,
J = 15.9, 0.9 Hz, C@CH), 6.77 (1H, d, J = 15.9 Hz,
C@CH), 10.13 (1H, s, CHO). MS (EI) m/z 272 (M+).
3.1.20. 2-[(E)-2-(2,6,6-Trimethylcyclohex-1-enyl)ethenyl]-
benzaldehyde (7d). Yield: 92%. IR (neat) 2928, 1698,
1
1595, 1188 cmÀ1. H NMR (400 MHz, CDCl3) d 1.09
(6H, s, C(CH3)2), 1.51 (2H, m, CH2), 1.66 (2H, m,
CH2), 1.81 (3H, d, J = 0.9 Hz, CH3), 2.06 (2H, t,
J = 6.1 Hz, CH2), 6.60 (1H, dd, J = 16.1, 0.9 Hz,
C@CH), 7.17 (1H, d, J = 16.1 Hz, C@CH), 7.38 (1H,
dt, J = 7.6, 1.3 Hz, Ar), 7.55 (1H, dt, J = 7.6, 1.3 Hz,
Ar), 7.59 (1H, dd, J = 7.6, 1.3 Hz, Ar), 7.84 (1H, dd,
J = 7.6, 1.3 Hz, Ar), 10.33 (1H, s, CHO). MS (EI) m/z
254 (M+).
(neat) 3407, 2928, 1456, 1362, 1071 cmÀ1 1H NMR
.
(400 MHz, CDCl3) d 1.00 (6H, s, C(CH3)2), 1.46 (2H,
m, CH2), 1.61 (2H, m, CH2), 1.71 (3H, s, CH3), 2.01
(2H, t, J = 6.3 Hz, CH2), 4.40 (2H, s, CH2OH), 4.84
(2H, d, J = 3.4 Hz, OCH2), 4.86 (2H, d, J = 3.4 Hz,
OCH2), 5.91 (1H, d, J = 16.3 Hz, C@CH), 6.24 (1H, d,
J = 16.3 Hz, C@CH). MS (EI) m/z 248 (M+).
3.1.21. 4-[(E)-2-(2,6,6-Trimethylcyclohex-1-enyl)ethenyl]-
2,5-dihydrofuran-3-carbaldehyde (7e). To a solution of
oxalyl chloride 0.11 ml (1.3 mmol) in CH2Cl2 (4 ml)
was added DMSO 0.18 ml (2.5 mmol) at À78 °C under
Ar and the mixture was stirred for 3 min. To this solu-
tion alcohol 6e 240 mg (0.97 mmol) in CH2Cl2 (3 ml)
was added and stirring was continued for an additional
15 min. Triethylamine 0.68 ml (4.9 mmol) was added,
and the reaction mixture was stirred for 5 min and
allowed to warm to room temperature. Water 10 ml
was added and the aqueous layer was reextracted with
CH2Cl2. The combined organic layer was washed with
brine and dried (MgSO4). Purification on silica gel
column chromatography (hexane/ethyl acetate 10:1 as
an eluent) afforded 7e 218 mg (92%) as a colorless oil.
Similar reactions of 5g gave the following compound.
3.1.16. 4-[(E)-2-(2,6,6-Trimethylcyclohex-1-enyl)ethenyl]-
2,5-dihydrothiophen-3-methanol (6g). Yield: 97%. IR
(neat) 3395, 2930, 1456, 1109 cmÀ1
.
1H NMR
(400 MHz, CDCl3) d 1.00 (6H, s, C(CH3)2), 1.46 (2H,
m, CH2), 1.62 (2H, m, CH2), 1.70 (3H, s, CH3), 2.01
(2H, t, J = 6.1 Hz, CH2), 3.99 (4H, m, CH2SCH2),
4.37 (2H, s, CH2OH), 6.07 (1H, d, J = 16.2 Hz, C@CH),
6.33 (1H, d, J = 16.2 Hz, C@CH). MS (EI) m/z 264
(M+).
3.1.17. 2-[(E)-2-(2,6,6-Trimethylcyclohex-1-enyl)ethenyl]-
cyclopent-1-enecarbaldehyde (7a). To a solution of alco-
hol 6a 126 mg (0.51 mmol) in 20 ml of petroleum ether
(boiling range 30–60 °C) was added 898 mg of MnO2.
After being stirred for 3.5 h at room temperature, the
reaction mixture was filtered through Celite and the fil-
trate was concentrated in vacuo to afford aldehyde 7a
119 mg (95%) as a yellow oil. IR (neat) 2930, 1659,
IR (neat) 2932, 1659, 1613, 1225 cmÀ1 1H NMR
.
(400 MHz, CDCl3) d 1.06 (6H, s, C(CH3)2), 1.49 (2H,
m, CH2), 1.64 (2H, m, CH2), 1.77 (3H, s, CH3), 2.07
(2H, t, J = 6.3 Hz, CH2), 4.93 (2H, t, J = 3.9 Hz,
OCH2), 5.05 (2H, t, J = 3.9 Hz, OCH2), 6.38 (1H, d,
J = 16.2 Hz, C@CH), 6.85 (1H, d, J = 16.2 Hz, C@CH),
10.11 (1H, s, CHO). MS (EI) m/z 246 (M+).
1
1611, 1208 cmÀ1. H NMR (400 MHz, CDCl3) d 1.05
(6H, s, C(CH3)2), 1.49 (2H, m, CH2), 1.64 (2H, m,
CH2), 1.75 (3H, d, J = 0.6 Hz, CH3), 1.92 (2H, m,
CH2), 2.06 (2H, t, J = 6.1 Hz, CH2), 2.68 (2H, t,
J = 7.5 Hz, CH2), 2.82 (2H, m, CH2), 6.52 (1H, dd,
J = 15.9, 0.9 Hz, C@CH), 6.98 (1H, d, J = 15.9 Hz,
C@CH), 10.19 (1H, s, CHO). MS (EI) m/z 244 (M+).
Similar reactions of 6a and 6g gave the following
compounds.
3.1.22. 4-[(E)-2-(2,6,6-Trimethylcyclohex-1-enyl)ethen-
yl]furan-3-carbaldehyde (7f). Yield: 92%. IR (neat)
2928, 1694, 1530, 1146 cmÀ1 1H NMR (400 MHz,
.

5106
S. Ikegami et al. / Bioorg. Med. Chem. 14 (2006) 5099–5109
CDCl3) d 1.06 (6H, s, C(CH3)2), 1.48 (2H, m, CH2), 1.63
(2H, m, CH2), 1.77 (3H, s, CH3), 2.03 (2H, t, J = 6.1 Hz,
CH2), 6.48 (1H, d, J = 16.6 Hz, C@CH), 6.70 (1H, d,
J = 16.6 Hz, C@CH), 7.57 (1H, s, Ar), 8.01 (1H, d,
J = 1.6 Hz, Ar), 10.01 (1H, s, CHO). MS (EI) m/z 244
(M+).
Similar reactions of 7b–7h gave the following
compounds.
3.1.26. Methyl (E,E)-3-methyl-5-[2-[(E)-2-(2,6,6-trimeth-
ylcyclohex-1-enyl)ethenyl]cyclohex-1-enyl]penta-2,4-dieno-
ate (8b). Yield: 91%. A yellow oil. IR (nujol) 2926, 1715,
1
1605, 1154 cmÀ1. H NMR (400 MHz, CDCl3) d 1.04
3.1.23. 4-[(E)-2-(2,6,6-Trimethylcyclohex-1-enyl)ethenyl]-
2,5-dihydrothiophen-3-carbaldehyde (7g). Yield: 88%. A
yellow crystalline solid, mp 56–58 °C IR (nujol) 2932,
(6H, s, C(CH3)2), 1.48 (2H, m, CH2), 1.64 (2H, m,
CH2), 1.69 (4H, m, 2(CH2)), 1.75 (3H, d, J = 0.6 Hz,
CH3), 2.04 (2H, t, J = 6.3 Hz, CH2), 2.32 (2H, m, CH2),
2.36 (3H, d, J = 1.2 Hz, CH3), 2.40 (2H, m, CH2), 3.71
(3H, s, COOCH3), 5.81 (1H, s, C@CH), 6.23 (1H, d,
J = 15.8 Hz, C@CH), 6.28 (1H, d, J = 15.8 Hz, C@CH),
6.40 (1H, d, J = 15.8 Hz, C@CH), 7.34 (1H, d,
J = 15.8 Hz, C@CH). MS (EI) m/z 354 (M+).
1
1665, 1456, 1173 cmÀ1. H NMR (400 MHz, CDCl3) d
1.05 (6H, s, C(CH3)2), 1.49 (2H, m, CH2), 1.64 (2H,
m, CH2), 1.76 (3H, s, CH3), 2.07 (2H, t, J = 6.4 Hz,
CH2), 4.02 (2H, t, J = 3.7 Hz, SCH2), 4.22 (2H, t,
J = 3.7 Hz, SCH2), 6.52 (1H, d, J = 16.0 Hz, C@CH),
6.99 (1H, d, J = 16.0 Hz, C@CH), 10.17 (1H, s, CHO).
MS (EI) m/z 262 (M+).
3.1.27. Methyl (E,E)-3-methyl-5-[2-[(E)-2-(2,6,6-trimeth-
ylcyclohex-1-enyl)ethenyl]cyclohept-1-enyl]penta-2,4-dieno-
ate (8c). Yield: 92%. A yellow oil. IR (nujol) 2924, 1715,
3.1.24. 4-[(E)-2-(2,6,6-Trimethylcyclohex-1-enyl)ethen-
yl]thiophen-3-carbaldehyde (7h). To a stirred solution
of aldehyde 7g 102 mg (0.39 mmol) in 15 ml of petro-
leum ether (boiling range 30–60 °C) was added 998 mg
MnO2. After being stirred for 6 h at room temperature,
the reaction mixture was filtered though Celite and the
filtrate was concentrated in vacuo. Purification on silica
gel column chromatography (hexane/ethyl acetate 10:1
as an eluent) afforded 7h 89 mg (89%) as a yellow oil.
1
1605, 1154 cmÀ1. H NMR (400 MHz, CDCl3) d 1.04
(6H, s, C(CH3)2), 1.47–1.54 (6H, m, 3(CH2)), 1.64 (2H,
m, CH2), 1.76 (3H, s, CH3), 1.77 (2H, m, CH2), 2.05
(2H, t, J = 6.1 Hz, CH2), 2.36 (3H, d, J = 0.9 Hz, CH3),
2.53 (4H, m, 2(CH2)), 3.71 (3H, s, COOCH3), 5.81 (1H,
s, C@CH), 6.23 (1H, d, J = 15.9 Hz, C@CH), 6.30 (1H,
d, J = 15.9 Hz, C@CH), 6.62 (1H, d, J = 15.9 Hz,
C@CH), 7.27 (1H, d, J = 15.9 Hz, C@CH). MS (EI) m/
z 360 (M+).
IR (neat) 2928, 1686, 1435, 911 cmÀ1
.
1H NMR
(400 MHz, CDCl3) d 1.08 (6H, s, C(CH3)2), 1.49 (2H,
m, CH2), 1.64 (2H, m, CH2), 1.79 (3H, s, CH3), 2.04
(2H, t, J = 6.3 Hz, CH2), 6.59 (1H, d, J = 16.4 Hz,
C@CH), 6.93 (1H, d, J = 16.4 Hz, C@CH), 7.33 (1H,
dd, J = 32, 0.8 Hz, Ar), 8.09 (1H, d, J = 3.2 Hz, Ar),
10.03 (1H, s, CHO). MS (EI) m/z 260 (M+).
3.1.28. Methyl (E,E)-3-methyl-5-[2-[(E)-2-(2,6,6-trimeth-
ylcyclohex-1-enyl)ethenyl]phenyl]penta-2,4-dienoate (8d).
Yield: 92%. A yellow-green oil. IR (nujol) 2926, 1719,
1
1456, 1156 cmÀ1. H NMR (400 MHz, CDCl3) d 1.08
(6H, s, C(CH3)2), 1.51 (2H, m, CH2), 1.65 (2H, m,
CH2), 1.80 (3H, d, J = 0.9 Hz, CH3), 2.05 (2H, t,
J = 6.3 Hz, CH2), 2.40 (3H, d, J = 0.9 Hz, CH3), 3.73
(3H, s, COOCH3), 5.90 (1H, s, C@CH), 6.49 (1H, dd,
J = 16.2, 0.9 Hz, C@CH), 6.62 (1H, d, J = 16.2 Hz,
C@CH), 6.70 (1H, d, J = 15.9 Hz, C@CH), 7.22–7.30
(3H, m, C@CH, Ar), 7.46 (1H, dd, J = 1.5, 7.6 Hz,
Ar), 7.50 (1H, d, J = 7.6 Hz, Ar). MS (EI) m/z 350 (M+).
3.1.25. Methyl (E,E)-3-methyl-5-[2-[(E)-2-(2,6,6-trimeth-
ylcyclohex-1-enyl)ethenyl]cyclopent-1-enyl]penta-2,4-dieno-
ate (8a). To LDA in THF (3 ml) prepared from 0.11 ml
(0.75 mmol) of diisopropylamine and 0.51 ml of 1.5 M
BuLi solution in hexane (0.75 mmol) was added the
phosphate26 188 mg (0.75 mmol) at À78 °C under Ar
and the reaction mixture was stirred for 15 min. A
solution of aldehyde 7a 119 mg (0.38 mmol) in THF
(2 ml) was then added at the same temperature, and
the reaction mixture was allowed to warm to room
temperature and stirred for further 15 min in the dark.
Saturated aqueous NH4Cl solution was added and the
mixture was extracted with ether, washed with brine,
and dried (MgSO4). The solvent was removed, and
chromatography of the residue on silica gel (hexane/
ethyl acetate 30:1 as an eluent) afforded methyl ester
8a 150 mg (91%) as a yellow crystalline solid, mp
3.1.29. Methyl (E,E)-3-methyl-5-[2-[(E)-2-(2,6,6-trimeth-
ylcyclohex-1-enyl)ethenyl]2,5-dihydrofuran-3-yl]penta-
2,4-dienoate (8e). Yield: 94%. A yellow crystalline solid,
mp 78–81 °C. IR (nujol) 2926, 1717, 1595, 1154 cmÀ1
.
1H NMR (400 MHz, CDCl3) d 1.03 (6H, s, C(CH3)2),
1.48 (2H, m, CH2), 1.63 (2H, m, CH2), 1.76 (3H, s,
CH3), 2.05 (2H, t, J = 6.1 Hz, CH2), 2.36 (3H, d,
J = 0.9 Hz, CH3), 3.72 (3H, s, COOCH3), 4.89 (2H, d,
J = 3.5 Hz, OCH2), 4.93 (2H, d, J = 3.5 Hz, OCH2),
5.82 (1H, s, C@CH), 6.02 (1H, d, J = 16.2 Hz, C@CH),
6.03 (1H, d, J = 16.2 Hz, C@CH), 6.44 (1H, d,
J = 16.2 Hz, C@CH), 6.94 (1H, d, J = 16.2 Hz, C@CH).
MS (EI) m/z 342 (M+). HRMS Calcd for C22H30O3:
342.2195. Found: 342.2196.
1
77–79 °C. IR (nujol) 2924, 1719, 1154 cmÀ1. H NMR
(400 MHz, CDCl3) d 1.04 (6H, s, C(CH3)2), 1.48 (2H,
m, CH2), 1.64 (2H, m, CH2), 1.75 (3H, d, J = 0.9 Hz,
CH3), 1.93 (2H, m, CH2), 2.05 (2H, t, J = 6.3 Hz,
CH2), 2.37 (3H, d, J = 0.9 Hz, CH3), 2.66 (4H, m,
2(CH2)), 3.71 (3H, s, COOCH3), 5.81 (1H, s, C@CH),
6.19 (1H, d, J = 15.8 Hz, C@CH), 6.20 (1H, d,
J = 15.8 Hz, C@CH), 6.58 (1H, d, J = 15.8 Hz,
C@CH), 7.12 (1H, d, J = 15.8 Hz, C@CH). MS (EI)
m/z 340 (M+). HRMS Calcd for C23H32O2: 340.2403.
Found: 340.2409.
3.1.30. Methyl (E,E)-3-methyl-5-[2-[(E)-2-(2,6,6-trimeth-
ylcyclohex-1-enyl)ethenyl]furan-3-yl]penta-2,4-dienoate (8f).
Yield: 96%. A yellow oil. IR (neat) 2928, 1717, 1613, 1237,
1
1157 cmÀ1. H NMR (400 MHz, CDCl3) d 1.06 (6H, s,
C(CH3)2), 1.49 (2H, m, CH2), 1.64 (2H, m, CH2), 1.77
(3H, s, CH3), 2.04 (2H, t, J = 6.1 Hz, CH2), 2.37 (3H, s,

S. Ikegami et al. / Bioorg. Med. Chem. 14 (2006) 5099–5109
5107
1
CH3), 3.72 (3H, s, COOCH3), 5.80 (1H, s, C@CH), 6.17
(1H, d, J = 16.5 Hz, C@CH), 6.38 (1H, d, J = 16.5 Hz,
C@CH), 6.61 (1H, d, J = 16.2 Hz, C@CH), 6.84 (1H, d,
J = 16.2 Hz, C@CH), 7.45 (1H, s, Ar), 7.55 (1H, s, Ar).
MS (EI) m/z 340 (M+).
1651 cmÀ1. H NMR (400 MHz, CDCl3) d 1.04 (6H, s,
C(CH3)2), 1.48 (2H, m, CH2), 1.64 (2H, m, CH2), 1.70
(4H, m, 2(CH2)), 1.75 (3H, s, CH3), 2.05 (2H, t,
J = 6.1 Hz, CH2), 2.33 (2H, m, CH2), 2.37 (3H, d,
J = 0.9 Hz, CH3), 2.40 (2H, m, CH2), 5.83 (1H, s,
C@CH), 6.24 (1H, d, J = 15.8 Hz, C@CH), 6.31 (1H,
d, J = 15.8 Hz, C@CH), 6.71 (1H, d, J = 15.8 Hz,
C@CH), 7.38 (1H, d, J = 15.8 Hz, C@CH). MS (EI)
m/z 340 (M+). Anal. Calcd for C23H32O2/0.75H2O: C,
78.03; H, 9.54. Found: C, 77.65; H, 9.21.
3.1.31. Methyl (E,E)-3-methyl-5-[2-[(E)-2-(2,6,6-trimeth-
ylcyclohex-1-enyl)ethenyl]-2,5-dihydrothiophen-3-yl]penta-
2,4-dienoate (8g). Yield: 94%. A yellow crystalline solid,
mp 145–146 °C. 1H NMR (400 MHz, CDCl3) d 1.03
(6H, s, C(CH3)2), 1.49 (2H, m, CH2), 1.64 (2H, m,
CH2), 1.75 (3H, s, CH2), 2.05 (2H, t, J = 6.0 Hz, CH2),
2.37 (3H, d, J = 0.9 Hz, CH2), 3.72 (3H, s, COOCH3),
4.01 (2H, m, SCH2), 4.06 (2H, m, SCH2), 5.84 (1H, s,
C@CH), 6.20 (2H, d, J = 16.0 Hz, C@CH), 6.56 (1H,
d, J = 16.0 Hz, C@CH), 7.08 (1H, d, J = 16.0 Hz,
C@CH). MS (EI) m/z 358 (M+). HRMS Calcd for
C22H30O2S: 358.1997. Found: 358.1996.
3.1.35. (E,E)-3-Methyl-5-[2-[(E)-2-(2,6,6-trimethylcyclo-
hex-1-enyl)ethenyl]cyclohept-1-enyl]penta-2,4-dienoic acid
(2c)22. Yield: 96%. Yellow needles, mp 143–145 °C
(CH2Cl2/hexane). IR (nujol) 3445, 2924, 1684 cmÀ1. H
1
NMR (400 MHz, CDCl3) d 1.04 (6H, s, C(CH3)2),
1.47–1.55 (6H, m, 3C(CH3)2), 1.64 (2H, m, CH2), 1.76
(3H, s, CH3), 1.78 (2H, m, CH2), 2.05 (2H, t,
J = 6.1 Hz, CH2), 2.37 (3H, s, CH3), 2.53 (4H, m,
2(CH2)), 5.83 (1H, s, C@CH) 6.24 (1H, d, J = 15.8 Hz,
C@CH), 6.33 (1H, d, J = 15.8 Hz, C@CH), 6.62 (1H, d,
J = 15.8 Hz, C@CH), 7.30 (1H, d, J = 15.8 Hz, C@CH).
MS (EI) m/z 354 (M+). Anal. Calcd for C24H34O2/
0.5H2O: C, 79.29; H, 9.70. Found: C, 78.90; H, 9.65.
3.1.32. Methyl (E,E)-3-methyl-5-[2-[(E)-2-(2,6,6-trimeth-
ylcyclohex-1-enyl)ethenyl]-thiophen-3-yl]penta-2,4-dieno-
ate (8h). Yield: 93%. A yellow oil. IR (neat) 2928, 1717,
1
1613, 1237, 1156 cmÀ1. H NMR (400 MHz, CDCl3) d
1.07 (6H, s, C(CH3)2), 1.50 (2H, m, CH2), 1.65 (2H,
m, CH2), 1.78 (3H, s, CH3), 2.05 (2H, t, J = 6.1 Hz,
CH2), 2.39 (3H, d, J = 0.9 Hz, CH3), 3.72 (3H, s,
COOCH3), 5.86 (1H, s, H-14), 6.36 (1H, d,
J = 16.2 Hz, C@CH), 6.48 (1H, d, J = 16.2 Hz, C@CH),
6.68 (1H, d, J = 16.0 Hz, C@CH), 6.98 (1H, d,
J = 16.0 Hz, C@CH), 7.24 (1H, d, J = 3.2 Hz, Ar),
7.39 (1H, d, J = 3.2 Hz, Ar). MS (EI) m/z 356 (M+).
3.1.36. (E,E)-3-Methyl-5-[2-[(E)-2-(2,6,6-trimethylcyclo-
hex-1-enyl)ethenyl]phenyl]penta-2,4-dienoic acid (2d).
Yield: 98%. Yellow plates, mp 176–178 °C (CH2Cl2/hex-
ane). IR (neat) 3445, 2924, 1682, 1458 cmÀ1. H NMR
1
(400 MHz, CDCl3) d 1.09 (6H, s, C(CH3)2), 1.51 (2H,
m, CH2), 1.66 (2H, m, CH2), 1.81 (3H, s, CH3), 2.06
(2H, t, J = 6.3 Hz, CH2), 2.42 (3H, s, CH3), 5.93 (1H,
s, C@CH), 6.50 (1H, d, J = 16.2 Hz, C@CH), 6.62
(1H, d, J = 16.2 Hz, C@CH) 6.74 (1H, d, J = 15.9 Hz,
C@CH), 7.23–7.32 (2H, m, Ar), 7.32 (1H, d,
J = 15.9 Hz, C@CH), 7.47 (1H, dd, J = 7.6, 1.0 Hz,
Ar), 7.52 (1H, dd, J = 7.6, 1.0 Hz, Ar). MS (EI) m/z
336 (M+). Anal. Calcd for C23H28O2: C, 82.10; H,
8.39. Found: C, 81.84; H, 8.41.
3.1.33. (E,E)-3-Methyl-5-[2-[(E)-2-(2,6,6-trimethylcyclo-
hex-1-enyl)ethenyl]cyclopent-1-enyl]penta-2,4-dienoic acid
(2a)22. To a stirred solution of ester 8a 162 mg
(0.48 mmol) in 3 ml of ethanol was added 3.0 ml of
6.0 M KOH in the dark under Ar, and the reaction mix-
ture was heated to 60 °C for 1 h. After hydrolysis was
complete (by TLC), ethanol was evaporated in vacuo,
and the mixture was cooled to 0 °C and acidified with
10% aqueous HCl. The organics were extracted with
ether, washed with brine, dried over MgSO4, and concen-
trated in vacuo. The product was purified by column
chromatography on silica gel (CH2Cl2/ether 5:1) to give
149 mg of desired acid 2a (89%) as a yellow solid. Recrys-
tallization from CH2Cl2/hexane afforded yellow needles,
3.1.37. (E,E)-3-Methyl-5-[2-[(E)-2-(2,6,6-trimethylcyclo-
hex-1-enyl)ethenyl]-2,5-dihydrofuran-3-yl]penta-2,4-dienoic
acid (2e). Yield: 94%. Yellow needles, mp 224–226 °C
(MeOH). IR (neat) 2926, 1694, 1591, 1458, 1181 cmÀ1
.
1H NMR (400 MHz, CDCl3) d 1.03 (6H, s, C(CH3)2),
1.48 (2H, m, CH2), 1.63 (2H, m, CH2), 1.76 (3H, s,
CH3), 2.05 (2H, t, J = 6.0 Hz, CH2), 2.37 (3H, s, CH3),
4.91 (2H, d, J = 3.5 Hz, OCH2), 4.94 (2H, d, J = 3.5 Hz,
OCH2), 5.85 (1H, s, C@CH) 6.02 (2H, d, J = 16.0 Hz,
2(C@CH)), 6.44 (1H, d, J = 16.0 Hz, C@CH), 6.98 (1H,
d, J = 16.0 Hz, C@CH). MS (EI) m/z 328 (M+). Anal.
Calcd for C21H28O3: C, 82.10; H, 8.39. Found: C,
76.47; H, 8.73.
mp 193–194 °C. IR (nujol) 3445, 2924, 1684, 1156 cmÀ1
.
1H NMR (400 MHz, CDCl3) d 1.04 (6H, s, C(CH3)2),
1.48 (2H, m, CH2), 1.64 (2H, m, CH3), 1.76 (3H, s,
CH3), 1.94 (2H, m, CH2), 2.05 (2H, t, J = 6.0 Hz, CH2),
2.38 (3H, s, CH2), 2.67 (4H, m, 2(CH2)), 5.84 (1H, s,
C@CH), 6.20 (1H, d, J = 15.8 Hz, C@CH) 6.23 (1H, d,
J = 15.8 Hz, C@CH), 6.59 (1H, d, J = 15.8 Hz, C@CH),
7.16 (1H, d, J = 15.8 Hz, C@CH). Anal. Calcd for
C22H30O2/0.75H2O: C, 77.72; H, 9.34. Found: C, 77.75;
H, 9.34.
3.1.38. (E,E)-3-Methyl-5-[2-[(E)-2-(2,6,6-trimethylcyclo-
hex-1-enyl)ethenyl]furan-3-yl]penta-2,4-dienoic acid (2f).
Yield: 97%. Colorless needles, mp 175–177 °C (MeOH).
1
Similar reactions of 8b–h gave the following compounds.
IR (nujol) 2928, 1692, 1599, 1574, 1456, 1061 cmÀ1. H
NMR (400 MHz, CDCl3) d 1.06 (6H, s, C(CH3)2), 1.49
(2H, m, CH2), 1.64 (2H, m, CH2), 1.78 (3H, s, CH3),
2.04 (2H, t, J = 6.0 Hz, CH2), 2.38 (3H, d, J = 0.6 Hz,
CH3), 5.83 (1H, s, C@CH), 6.17 (1H, d, J = 16.2 Hz,
C@CH), 6.39 (1H, d, J = 16.2 Hz, C@CH) 6.40 (1H,
3.1.34. (E,E)-3-Methyl-5-[2-[(E)-2-(2,6,6-trimethylcyclo-
hex-1-enyl)ethenyl]cyclohex-1-enyl]penta-2,4-dienoic acid
(2b).22 Yield: 99%. Yellow needles, mp 165–167 °C
(CH2Cl2/hexane). IR (nujol) 3445, 2924, 1682,

5108
S. Ikegami et al. / Bioorg. Med. Chem. 14 (2006) 5099–5109
d, J = 16.0 Hz, C@CH), 6.88 (1H, d, J = 16.0 Hz,
C@CH), 7.46 (1H, s, Ar), 7.57 (1H, d, J = 1.2 Hz, Ar).
MS (EI) m/z 326 (M+). Anal. Calcd for C21H26O3: C,
77.27; H, 8.03. Found: C, 77.11; H, 7.96.
with 50 mM Tris–HCl (pH 7.4) containing 0.15 M
NaCl, 0.5% Triton X-100, and 1 mM benzamidine
hydrochloride. Triton X-100-insoluble material was
removed by centrifugation (18,000g for 15 min at 4 °C),
and TM antigen levels in the cell lysates were measured
by an enzyme immunoassay using mouse monoclonal
anti-human TM antibodies as previously described.
Purified human placental TM was used as a standard.
3.1.39. (E,E)-3-Methyl-5-[2-[(E)-2-(2,6,6-trimethylcyclo-
hex-1-enyl)ethenyl]-2,5-dihydrothiophen-3-yl]penta-2,4-die-
noic acid (2g). Yield: 96%. Yellow needles, mp 231–234 °C
(AcOEt). IR (nujol) 3422, 2924, 1688, 1590, 1458,
1
1197 cmÀ1. H NMR (400 MHz, CDCl3) d 1.04 (6H, s,
C(CH3)2), 1.49 (2H, m, CH2), 1.64 (2H, m, CH2), 1.75
(3H, s, CH3), 2.07 (2H, t, J = 6.3 Hz, CH2), 2.38 (3H,
d, J = 0.6 Hz, CH3), 4.01 (2H, d, J = 3.4 Hz, SCH2),
4.07 (2H, d, J = 3.4 Hz, SCH2), 5.87 (1H, s, C@CH)
6.21 (1H, d, J = 16.0 Hz, C@CH), 6.23 (1H, d,
J = 16.0 Hz, C@CH), 6.56 (1H, d, J = 16.0 Hz, C@CH),
7.12 (1H, d, J = 16.0 Hz, C@CH). MS (EI) m/z 344
(M+). Anal. Calcd for C21H28O2S: C, 73.21; H, 8.19; S,
9.31. Found: C, 72.96; H, 8.38; S, 9.32.
Acknowledgments
We are grateful to Misses K. Ichikawa, J. Shimode, and
J. Nonobe for spectroscopic measurements. This study
was performed through Special Coordination Funds of
the Science and Technology Agency of the Japanese
Government. Partial financial support from the Minis-
try of Education, Science, Sports, and Culture of Japan
is also acknowledged.
3.1.40. (E,E)-3-Methyl-5-[2-[(E)-2-(2,6,6-trimethylcyclo-
hex-1-enyl)ethenyl]thiophen-3-yl]penta-2,4-dienoic acid
(2h). Yield: 97%. Colorless needles, mp 172–174 °C
(AcOEt/hexane). IR (neat) 2926, 1684, 1595, 1256,
References and notes
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