Diarylheptanoids, new phytoestrogens from the rhizomes of Curcuma comosa: Isolation, chemical modification and estrogenic activity evaluation

Article abstract of DOI:10.1016/j.bmc.2008.05.051

Three new diarylheptanoids, a 1:2 mixture of (3S)- and (3R)-1-(4-methoxyphenyl)-7-phenyl-(6E)-6-hepten-3-ol (13a and 13b) and 1-(4-hydroxyphenyl)-7-phenyl-(6E)-6-hepten-3-one (15), together with two synthetically known diarylheptanoids 1,7-diphenyl-(1E,3E,5E)-1,3,5-triene (9) and 1-(4-hydroxyphenyl)-7-phenyl-(4E,6E)-4,6-heptadien-3-one (16), and nine known diarylheptanoids, 2, 8, 10-12, 14, a 3:1 mixture of 17a and 17b, and 18, were isolated from the rhizomes of Curcuma comosa Roxb. The absolute stereochemistry of the isolated compounds has also been determined using the modified Mosher's method. The isolated compounds and the chemically modified analogues were evaluated for their estrogenic-like transcriptional activity using RT-PCR in HeLa cell line. Some of the isolated diarylheptanoids and their modified analogues exhibited estrogenic activity comparable to or higher than that of the phytoestrogen genistein. Based on the transcriptional activation of both estrogenic targets, Bcl-xL and ERβ gene expression, the structural features for a diarylheptanoid to exhibit high estrogenic activity are the presence of an olefinic function conjugated with the aromatic ring at the 7-position, a keto group at the 3-position, and a phenolic hydroxyl group at the p-position of the aromatic ring attached to the 1-position of the heptyl chain.

Full text of DOI:10.1016/j.bmc.2008.05.051

Bioorganic & Medicinal Chemistry 16 (2008) 6891–6902
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Diarylheptanoids, new phytoestrogens from the rhizomes of Curcuma comosa:
Isolation, chemical modification and estrogenic activity evaluation
b
a
a,
Apichart Suksamrarn a, , Mathurose Ponglikitmongkol , Kanjana Wongkrajang , Anon Chindaduang
Suthadta Kittidanairak b, Aroon Jankam a,à, Boon-ek Yingyongnarongkul a, Narin Kittipanumat a,
Ratchanaporn Chokchaisiri a, Pichit Khetkam a, Pawinee Piyachaturawat c
,
*
a Department of Chemistry, Faculty of Science, Ramkhamhaeng University, Bangkok 10240, Thailand
b Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
c Department of Physiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history:
Three new diarylheptanoids, a 1:2 mixture of (3S)- and (3R)-1-(4-methoxyphenyl)-7-phenyl-(6E)-6-hep-
ten-3-ol (13a and 13b) and 1-(4-hydroxyphenyl)-7-phenyl-(6E)-6-hepten-3-one (15), together with two
synthetically known diarylheptanoids 1,7-diphenyl-(1E,3E,5E)-1,3,5-triene (9) and 1-(4-hydroxyphenyl)-
7-phenyl-(4E,6E)-4,6-heptadien-3-one (16), and nine known diarylheptanoids, 2, 8, 10–12, 14, a 3:1 mix-
ture of 17a and 17b, and 18, were isolated from the rhizomes of Curcuma comosa Roxb. The absolute
stereochemistry of the isolated compounds has also been determined using the modified Mosher’s
method. The isolated compounds and the chemically modified analogues were evaluated for their estro-
genic-like transcriptional activity using RT-PCR in HeLa cell line. Some of the isolated diarylheptanoids
and their modified analogues exhibited estrogenic activity comparable to or higher than that of the phy-
toestrogen genistein. Based on the transcriptional activation of both estrogenic targets, Bcl-xL and ERb
gene expression, the structural features for a diarylheptanoid to exhibit high estrogenic activity are the
presence of an olefinic function conjugated with the aromatic ring at the 7-position, a keto group at
the 3-position, and a phenolic hydroxyl group at the p-position of the aromatic ring attached to the
1-position of the heptyl chain.
Received 24 March 2008
Revised 22 May 2008
Accepted 23 May 2008
Available online 28 May 2008
Keywords:
Diarylheptanoid
Curcuma comosa
Chemical modification
Estrogenic activity
Structure–activity relationship
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
the menopausal symptom is dramatically increased as they have
less adverse effects compared to the classical steroid hormone
Phytoestrogens are naturally occurring compounds from plant
with estrogenic-like biological activity.1,2 They exert actions
mainly by binding to specific estrogen receptors (ER) which exists
estrogen.
The rhizome of Curcuma comosa Roxb. (Zingiberaceae), com-
monly known as Waan Chak Modluk in Thai, has long been used
in indigenous medicine in Thailand as an anti-inflammatory
agent. It has been used widely for the treatment of postpartum
uterine bleeding and as an aromatic stomachic. On the basis of
local use, we investigated the biological effects of the extracts
of the plant rhizomes and found that the hexane extract exhib-
ited estrogenic-like activities by causing an increase in uterine
weight and cornification of vaginal epithelium,12,13 whereas the
polar extracts showed a potent choleretic effect.14 In an early
chemical study, the methanol extract of its rhizomes was shown
to have nematocidal activity and the diarylheptanoids 1–5 were
isolated from the less polar part of the extract.15 We have re-
cently investigated the ethyl acetate and butanol extracts of this
plant species and the known diarylheptanoids 3, 6 and 7, and a
in two forms, ERa and ERb, and activate the transcription of spe-
cific target genes in the nucleus.3,4 Phytoestrogens have several
pharmacological effects in a large number of tissues including
protection against development of cancer,5,6 cardiovascular dis-
ease,7,8 and osteoporosis9 in menopausal women.10 Other biolog-
ical effects independent of the ER such as anti-oxidant and anti-
inflammatory effects of phytoestrogens have also been as-
cribed.10,11 Currently, the interest of using product containing
phytoestrogens as alternatives to the steroid hormone to alleviate
* Corresponding author. Tel.: +662 3190931; fax: +662 3191900.
E-mail addresses: s_apichart@ru.ac.th, asuksamrarn@yahoo.com (A. Suksamrarn).
Present address: National Nanotechnology Center, National Science and Tech-
nology Development Agency, Pathumthani 12120, Thailand.
new phloracetophenone glucoside, 4,6-dihydroxy-2-O-(b-D-gluco-
à
pyranosyl)acetophenone (8), were isolated. Compounds 6, 7, and
Present address: Department of Chemistry, Faculty of Science, Ubon Ratchathani
the glucoside 8 exhibited choleretic activity in rats.16 In the
Rajabhat University, Ubon Ratchathani 34000, Thailand.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.05.051

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A. Suksamrarn et al. / Bioorg. Med. Chem. 16 (2008) 6891–6902
search for compounds with estrogenic activity, a number of
plant species have been investigated for phytoestrogens by our
group. This work deals with the isolation, absolute stereochemis-
try determination, chemical modification, and estrogenic activity
evaluation of diarylheptanoids from the rhizomes of C. comosa.
The diarylheptanoids are a new group of phytoestrogens which
may have health benefit potential particularly for postmeno-
pausal women.
saen district, Nakhon Pathom province were used for this study.
The pulverized, dry rhizome was extracted successively with n-
hexane, CHCl3, and MeOH to yield the hexane, CHCl3, and MeOH
extracts, respectively (see Scheme 1).
The hexane extract of C. comosa rhizomes was subjected to
column chromatography (Scheme 2) to yield five known diaryl-
heptanoids 2, 9, 10, 11, and 12. Compound 2 was identical to
that isolated previously from this plant species.15 Compound 9
has not previously been reported as
a naturally occurring
OR1
R2
Curcuma comosa rhizome
3
(10.4 kg dry wt)
R3
Soxhlet extraction
1, R1 = Ac, R2 = R3 = H
n-Hexane
3, R1 = R2 = R3 = H
6, R1 = R2 = H, R3 = OH
7, R1 = H, R2 = R3 = OH
Hexane extract
(350.0 g)
Marc
O
CHCl3
Chloroform extract
(770.1 g)
Marc
MeOH
R
2, R = H
15, R = OH
20, R = OMe
24, R = OAc
MeOH extract
(322.4 g)
Marc
OH
Scheme 1. Extraction of the pulverized, dry rhizome of Curcuma comosa.
Hexane extract
(100.0 g)
4
O
OH
column chromatography (cc)
n-Hexane → n-Hexane-CHCl3→
CHCl3 → CHCl3-MeOH
R
5, R = H
Fraction H3
Fraction H4
31, R = OH
Fraction H6
Fraction H2
(11.55 g)
COMe
(23.88 g)
CH Cl
O
OH
OH
n-Hexane →
n-Hexane-CH2Cl2
HO
O
→
2
2
→
n-Hexane → n-Hexane-CHCl3
OH
HO
→
CHCl3
CHCl3-MeOH
Compound 9
(20 mg)
OH
Subfraction 6
(87 mg)
Subfraction 7 Subfraction 15
(8.18 g)
8
cc
repeated
cc
2. Results and discussion
2.1. Structural elucidation of diarylheptanoids
Compound 11 Compounds 11+12
(310 mg)
(86 mg)
cc
Preliminary examination of the chemical constituents of
C. comosa rhizomes collected from different locations in Thailand
revealed that some of them contained a relatively high quantity
of the major constituents and constituted additional number of
minor diarylheptanoids which have not previously been reported
in this plant species. The rhizomes of C. comosa from Kampaeng-
Compound 2
Compound 10
(20 mg)
(8 mg)
Compound 12
(60 mg)
Scheme 2. Chromatographic separation of diarylheptanoids from the hexane
extract.

A. Suksamrarn et al. / Bioorg. Med. Chem. 16 (2008) 6891–6902
6893
diarylheptanoid, but it has been synthesized for a conjugated
carbanion study.17 The spectroscopic data of compound 9 were
consistent with those of the reported values.17,18 Compound 10
was identical to the diarylheptanoid isolated from C. xanthorrh-
iza by spectroscopic (IR, 1H NMR and mass spectra)
comparison.19
ester 11x. Compound 11 was also subjected to esterification with
(S)-(+)-MTPA chloride to yield the corresponding (R)-MTPA ester
11y. The
shown in Figure 1. Negative
D
d values (
D
d = dSꢁMTPA-dRꢁMTPA) were determined as
d values were found for H-5, H-
D
6, and H-7, while positive
D d values were obtained for H-1. Fol-
lowing the MPTA rules,21 these data indicated an S configuration
at C-3 (see Fig. 1).
The closely related compound 12 was another optically ac-
tive diarylheptanoid isolated from C. comosa. The 1H NMR data
of 12 were consistent with compound 4 isolated previously
from this plant species15 and other Curcuma species,19 but the
stereochemistry at C-3 was not reported. The absolute configu-
ration of 12 was determined by the same analogy to that of
compound 11 (see Fig. 1). The spatial arrangement of the
groups at the asymmetric C-3 in 12 was the same as that of
11. However, the presence of an additional double bond led
to the assignment of the absolute configuration at C-3 of 12
to be R.
9
O
The diarylheptanoids isolated from the CHCl3 extract included
the already isolated compounds 11 and 12, and compounds 13–
16 (see Scheme 3).
10
Compound 13 was a diarylheptanoid existed as a mixture of
two C-3 enantiomers, 13a and 13b. The HR-TOFMS (ES+)
showed a pseudomolecular ion [M+H]+ at m/z 297.1839, com-
patible with the molecular formula C20H24O2. The IR absorption
band at 3331 cmꢁ1 indicated the presence of a hydroxyl group.
Assignments of the 1H and 13C NMR data were achieved by
COSY, DEPT, HMQC, and HMBC techniques. The presence of
the vinylic function was evident from the double triplet signal
at d 6.20 (J = 15.8, 6.8 Hz, H-6) and the broad doublet signal
at d 6.39 (J = 15.8 Hz, H-7) in the 1H NMR spectrum, which cor-
responded to the 13C NMR at d 130.26 and 130.27. The carbi-
nolic and methoxy protons appeared as a multiplet signal and
a singlet signal at d 3.68 and 3.78, respectively. The 1H NMR
features of the heptyl chain were very similar to those of com-
pound 11. However, the 1H NMR features of the aromatic pro-
tons of 13 were different from those of 11 by the presence of a
para-disubstituted aromatic ring as evident from the two dou-
blets (J = 8.2 Hz) at d 6.81 (2H, H-30 and H-50) and d 7.10 (2H,
H-20 and H-60). Placement of the methoxyl group at the 40-posi-
tion was confirmed by HMBC correlation of the methoxy pro-
tons with C-40 (see Section 4). The attachment of the 4-
methoxyphenyl moiety to the 1-position was confirmed by
the HMBC correlations of H-1 with C-10 and C-20/C-60, H-2 with
C-10, and H-20/H-60 with C-1 (see Section 4). The attachment of
the phenyl moiety to the 7-position was also confirmed by
HMBC correlations of H-6 with C-100, and H-7 with C-100 and
C-200/C-600.
OR1
2"
2'
5
1
7
R2
R3
3
6
4
2
4"
6"
6'
5'
11, R1 = R2 = R3 = H
13a, R1 = R2 = H, R3 = OMe
17a, R1 = R2 = H, R3 = OH
18, R1 = H, R2 = R3 = OH
19, R1 = Me, R2 = R3 = H
22, R1 = Ac, R2 = R3 = H
OR
12, R = H
23, R = Ac
OR1
3
R2
R3
Compound 13 exhibited low optical rotation ð½a D27
ꢀ
þ 2:4ꢂ in
13b, R1 = R2 = H, R3 = OMe
17b, R1 = R2 = H, R3 = OH
EtOH). The absolute stereochemistry at C-3 and the enantio-
meric ratio were determined by the modified Mosher’s meth-
od.21–23 Thus the diarylheptanoid 13a and 13b mixture was
transformed to a 1:2 mixture of the corresponding (S)-MTPA es-
ters 13ax and 13bx, and (R)-MTPA esters 13ay and 13by. Anal-
ysis of the 1H NMR spectra of the two Mosher ester mixtures
(Fig. 1) established the absolute configuration at C-3 of the
diarylheptanoid mixture as S and R in a ratio of 1:2. The diaryl-
heptanoid 13 was thus concluded as a 1:2 mixture of (3S)- and
(3R)-1-(4-methoxyphenyl)-7-phenyl-(6E)-6-hepten-3-ol (13a and
13b).
The diarylheptanoid 3 was isolated previously from a number
of plant species.15,16,20 From the previous isolation of this com-
pound in C. comosa,15 it was reported to exist as a racemic mix-
ture. In this study, it was isolated as the enantiomer 11, which
showed identical 1H NMR data as those reported for compound
319 and exhibited low optical rotation ð½a 2D7
ꢀ
ꢁ 2:6ꢂ in EtOH). Com-
pound 11 was obtained as the major component from this plant
species. Determination of the absolute configuration at C-3 of this
diarylheptanoid using the modified Mosher’s method,21,22 it was
found to be S as indicated in the structure 11. Thus, the diaryl-
heptanoid 11 was subjected to esterification with (R)-(ꢁ)-MTPA
chloride in dry pyridine23 to yield the corresponding (S)-MTPA
Compound 14 was a minor diarylheptanoid isolated from
the CHCl3 extract. The 1H NMR data were consistent with
the structure and were in agreement with those of reported
values.24 In the present work, the C-5 absolute configuration
was determined to be R by the modified Mosher’s method
(see Fig. 1).

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A. Suksamrarn et al. / Bioorg. Med. Chem. 16 (2008) 6891–6902
7-position was also confirmed by HMBC correlations of H-6 with
O
O
OH
5
C-100, and H-7 with C-100 and C-200/C-600. The structure of the diaryl-
heptanoid 15 was concluded as 1-(4-hydroxyphenyl)-7-phenyl-
(6E)-6-hepten-3-one.
Compound 16 was obtained as prisms, mp 129–130 °C. The HR-
TOFMS (ES+) at m/z 279.1367 [M+H]+ was compatible with the
molecular formula C19H18O2. The IR absorption bands at 3380
and 1673 cmꢁ1 revealed the presence of a hydroxyl group and a
conjugated keto group, respectively. The 13C NMR of the conju-
14
gated keto group appeared at d 200.1. The presence of
a,b,c,d-
unsaturated keto system was evident from the 1H NMR signals at
d 6.26 (d, J = 15.6 Hz, H-4), d 6.84 (dd, J = 15.6, 10.1 Hz, H-6), d
6.92 (d, J = 15.6 Hz, H-7), and d 7.33 (partially overlapping signal,
H-5). The saturated H-1 and H-2 resonances appeared as a broad
singlet at d 2.86. The attachment of the phenyl group to the 7-posi-
tion of the heptyl chain was confirmed from the HMBC correlations
between H-6 and C-100, H-7 and C-200/C-600, and H-200/H-600 and C-7,
whereas that of the 4-hydroxyphenyl moiety to the 1-position was
confirmed by the HMBC correlations between H-1/H-2 and C-10,
and H-20/H-60 and C-1 (see Section 4). The spectroscopic data of
16 were consistent with 1-(4-hydroxyphenyl)-7-phenyl-(4E,6E)-
4,6-heptadien-3-one which has been synthesized recently.25
The MeOH extract was chromatographed (Scheme 4) and two
phenolic diarylheptanoids were isolated. The first eluted compo-
nent was shown to be a 3:1 mixture of S and R enantiomers, 17a
and 17b, by the already mentioned modified Mosher’s method
(see Fig. 1). The 1H NMR data of the two enantiomeric mixture
were consistent with the reported data of compound 6.26 The sec-
ond, more polar diarylheptanoid was shown to be 18 by compari-
son of the 1H NMR data with those of reported values of compound
7,26 and determination of the absolute configuration at C-3 was
shown to be S by the modified Mosher’s method (see Fig. 1). The
last and most polar compound was the phloracetophenone gluco-
side 8 isolated previously from this plant species by our group. The
identity of this compound was based on 1H NMR spectral compar-
ison with the reported values.16
OR
16, R = H
21, R = Me
25, R = Ac
OR1
26, R1 = H
28, R1 = Ac
R1
O
R2
27, R1 = R2 = H
29, R1 = H, R2 = OH
32, R1 = R2 = OH
O
OH
OH
It was worth noting that, for diarylheptanoids with one asym-
metric carbon at the 3-position and existed as only one enantiomer
(compounds 11, 12, 14 and 18), the spatial arrangement of func-
tional groups at the asymmetric carbon was the same (S configura-
tion in compounds 11 and 18 and R configuration in compounds 12
and 14). For the existence of two enantiomers (compounds 13a and
13b, and compounds 17a and 17b), especially those with different
ratios of S and R enantiomers (1:2 for 13a and 13b, and 3:1 for 17a
and 17b), the present data did not permit a reasonable explanation
of these observations.
30
O
33
2.2. Chemical modification of diarylheptanoids
Compound 15 was isolated from C. comosa as colorless viscous
oil. The HR-TOFMS (ES+) at m/z 281.1525 [M+H]+ established a
molecular formula C19H20O2. The IR absorption bands indicated
the presence of a hydroxyl group and a saturated keto group at
3360 and 1697 cmꢁ1, respectively. The 13C NMR resonance at d
210.1 confirmed that this compound is a saturated ketone. The
characteristic vinylic function was evident from the double triplet
signal at d 6.14 (J = 15.9, 6.7 Hz, H-6) and the doublet signal at d
6.37 (J = 15.9 Hz, H-7). The placement of the keto group at the 3-
position was deduced from the presence of two triplet signals
(J = 7.3 Hz) of H-1 and H-2 at d 2.84 and 2.72, respectively. The
p-substituted nature of the phenolic hydroxyl group was evident
from the two doublet signals (J = 8.2 Hz) at d 6.76 (H-30 and H-50)
and d 7.01 (H-20 and H-60). The placement of the 4-hydroxyphenyl
moiety at the 1-position was confirmed by the HMBC correlations
of H-1 with C-10 and C-20/C-60, H-2 with C-10, and H-20/H-60 with
C-1 (see Section 4). The attachment of the phenyl moiety to the
A number of diarylheptanoids isolated from C. comosa rhizomes
have been used for chemical modification to diarylheptanoid ana-
logues for estrogenic evaluations (see Schemes 5 and 6). The iden-
tities of the modified compounds were achieved by spectroscopic
(IR, 1H and 13C NMR, and mass spectra) techniques. In order to
see the effects of hydrogen bonding on the estrogenic activity of
the parent diarylheptanoids, the corresponding methyl ethers
and acetates were prepared by standard methods. Starting from
the alcohol 11, and the phenols 15 and 16, the methyl ether ana-
logues 19, 20, and 21 were prepared by reacting the starting com-
pounds with methyl iodide in DMF in the presence of silver oxide
to yield, after column chromatographic purification, the required
methyl ethers 19, 20, and 21 in 58, 86, and 82% yields, respectively
(see Schemes 5 and 6). The spectroscopic (IR, 1H and 13C NMR, and
mass spectra) data of 19, 20 and 21 were in agreement with the
structures (see Section 4). The alcohols 11 and 12, and the phenols
15 and 16 were subjected to acetylation by the standard procedure,

A. Suksamrarn et al. / Bioorg. Med. Chem. 16 (2008) 6891–6902
6895
OR
OR
-0.054 -0.095
-0.063 -0.115
-0.051
+0.115
+0.123
S
R
-0.039
+0.067
+0.052
-0.097
11, R = H
11x, R = (S)-MTPA
11y, R = (R)-MTPA
12, R = H
12x, R = (S)-MTPA
12y, R = (R)-MTPA
OR
OR
+0.054 +0.114
-0.061 -0.113
-0.052
-0.114
-0.065
+0.116 +0.066
+0.022
+0.008
-0.022
-0.007
OMe
S
R
+0.053
OMe
13a, R = H
13ax, R = (S)-MTPA
13ay, R = (R)-MTPA
13b, R = H
13bx, R = (S)-MTPA
13by, R = (R)-MTPA
1 : 2
OR
OR
O
-0.131
-0.129
-0.066 -0.133
+0.072
+0.019
+0.005
+0.113
OR
OR
R
S
+0.081 +0.073
-0.055
+0.075
14, R = H
14x, R = (S)-MTPA
14y, R = (R)-MTPA
18, R = H
18x, R = (S)-MTPA
18y, R = (R)-MTPA
OR
OR
+0.061 +0.129
-0.061 -0.110
-0.041
-0.124
+0.124 +0.072
-0.072
+0.013
OR
-0.021
OR
S
R
+0.051
17a, R = H
17ax, R = (S)-MTPA
17ay, R = (R)-MTPA
17b, R = H
17bx, R = (S)-MTPA
17by, R = (R)-MTPA
3 : 1
Figure 1.
D
d = (D
dS
ꢁ
DdR) values obtained from the MTPA esters of compounds 11, 12, 13a and 13b mixture, 14, 17a and 17b mixture, and 18 in CDCl3.
using acetic anhydride and dry pyridine, to afford the correspond-
ing acetates 22, 23, 24 and 25 in 76, 79, 87 and 78% yields, respec-
tively (see Schemes 5 and 6). The 1H NMR data of 22 and 23 were
consistent with the reported values,15,19 whereas the spectroscopic
(IR, 1H and 13C NMR and mass spectra) data of 24 and 25 were in
agreement with the structures (see Section 4).
Chloroform extract
(300.0 g)
cc
→
n-Hexane → n-Hexane-CH2Cl2
→
CH2Cl2
CH2Cl2-MeOH
The contribution of the olefinic function at the 6-position of the
alkyl chain of the diarylheptanoids was evaluated by comparison
of the estrogenic activity of the parent compound 11 and its ace-
tate 22, and the parent compounds 2 and 15 with those of the cor-
responding dihydro analogues 26, 28, 27, and 29, which were
prepared by hydrogenation reaction using palladium on charcoal
as a catalyst (Schemes 5 and 6). The spectroscopic data of 26 and
28 were in agreement with the structures, whereas those of 27
and 29 were consistent with those reported previously.27
The natural diarylheptanoid 15 was chemically modified by
placing the olefinic function at the 1-position of the molecule
(see Scheme 6). Thus, the dihydro analogue 29 was subjected to
dehydrogenation with DDQ in benzene at reflux to give the enone
30 in 47% yield. The presence of two doublets, J = 16.1 Hz, at d 6.60
(H-2) and d 7.70 (H-1) was compatible with the structure of 30. The
analogue with an olefinic moiety at the 4-position was also pre-
pared. Thus. compound 15 was subjected to allylic hydroxylation
Fractions C2+C3 Fraction C5
Fraction C7
CH2Cl2
Fraction C11
CH2Cl2-MeOH
Compounds 11+12
→
(132 mg)
Compound 16
(72 mg)
Subfraction 3 Subfraction 7
cc
Compounds 13a+13b
Compound 14
Compound 15
(150 mg)
(6 mg)
(16 mg)
Scheme 3. Chromatographic separation of diarylheptanoids from the chloroform
extract.

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A. Suksamrarn et al. / Bioorg. Med. Chem. 16 (2008) 6891–6902
2.3. Structures and estrogenic-activity relationships of
diarylheptanoids
Methanol extract
(210.0 g)
cc
CH2Cl2
MeOH
Among various in vitro determinations of estrogenic activity of
plant compounds, the analysis of the transcriptional regulation of
endogenous estrogen responsive genes in cell lines is crucial for
the assessment of classical estrogenic-like action. There are a num-
ber of estrogen target genes whose estrogen responsive elements
(ERE) have already been identified.28–30 The estrogen receptors
mediate the biological responses to estrogens by binding directly
to ERE after forming hormone-receptor complex capable of activat-
ing gene transcription. Among these, anti-apoptotic Bcl-xL and
estrogen receptor b (ERb) genes were shown to respond to estro-
gens at both the transcriptional and the translational levels.31–34
Although a wide range of concentrations (10ꢁ6 M to 10ꢁ10 M) of
17b-estradiol (E2), the most effective form of estrogens, could ex-
ert physiological effects, it was shown that 10ꢁ8 M gave the maxi-
mal result.35 In this study, we measured the alteration of
endogenous Bcl-xL and ERb gene expression in human cervical
cancer HeLa cell line by RT-PCR technique after exposure to various
diarylheptanoids at the final concentrations of 10ꢁ8 M as compared
to 10ꢁ8 M E2 which was used as a positive control. The diluent
DMSO at the same final concentration of 0.01% was used as a neg-
ative control. Cytotoxicity test has been performed to ensure that
the selected concentration of the pure compound (10ꢁ8 M) had
no effect on cell viability (data not shown). Our data showed that
most of diarylheptanoids investigated exhibited estrogenic activity
by inducing gene expression although with different levels. The
estrogenic activity of diarylheptanoids is expressed in percentage
relative to the reference compound, E2, as summarized in Table
1. The explanation for the estrogenic activity of diarylheptanoids
has not yet been established. However, we believed that the estro-
genic activity could possibly be due to the ability of this class of
compounds to adjust the alignment of the molecule to bind to
the estrogen receptor.
→
CH2Cl2-MeOH
→
Fraction M2
Fraction M6
(22.0 g)
Compounds 15+16
(4 mg)
(11.0 g)
Fraction M1
Fraction M5
(90.0 g)
CH2Cl2-MeOH
Compounds 11+12
(8 mg)
Compound 8
(18 mg)
(45.0 g)
CH2Cl2-MeOH
Compounds 17a+17b
(14 mg)
Compound 18
(21 mg)
Scheme 4. Chromatographic separation of diarylheptanoids and phloracetophe-
none glucoside from the methanol extract.
using selenium dioxide in dioxane at reflux to yield the hydroxyl
analogue 31, which was hydrogenated to the corresponding dihy-
dro analogue 32, which in turn was dehydrated using p-toluenesul-
fonic acid as a catalyst to afford the enone 33 in 20% overall yield
from compound 15. The presence of an
a,b-unsaturated double
bond in 33 was evident from the 1H NMR signals at d 6.08 (d,
J = 15.9 Hz, H-4) and d 6.81 (dt, J = 15.9, 7.1 Hz, H-5). The modified
compounds were evaluated for their estrogenic activity.
Compound 9 is a polyconjugated olefin and is the only diaryl-
heptanoid without oxygen function on the heptyl chain. It exhib-
ited moderately high estrogenic activity (78% for Bcl-xL and 76%
for ERb) comparing to that of E2 (100%). The unconjugated ketone
2 also showed moderately high activity (74% for Bcl-xL and 85% for
ERb). The corresponding conjugated ketone 10, however, was
much less active (44% for Bcl-xL and 34% for ERb). The result indi-
cated that, in the case of the keto analogue, addition of one more
extra double bond led to significant decrease in activity.
Addition of a phenolic hydroxyl group to the p-position of the
phenyl group at the 1-position of the heptyl chain of compound
2 gave rise to the ketone 15. Compound 15 exhibited the highest
estrogenic activity (79% for Bcl-xL and 91% for ERb) among the
diarylheptanoids. Interestingly, addition of a phenolic hydroxyl
group to the p-position of the phenyl group attached to the 1-posi-
tion of the heptyl chain of compound 10 to the conjugated ketone
It should be noted that the magnitude of the optical rotations of
the diarylheptanoids with two similar, saturated substituent
groups is small (for example, ꢁ2.6 and ꢁ5.2° for compounds 11
and 18). Without the preparation of the Mosher’s salts, they might
be mistaken for the existence of racemic mixtures. The magnitude
of the optical rotations of the corresponding methyl ethers and
acetate derivatives is only slightly higher than the parent alcohols
(for example, ꢁ5.3 and ꢁ7.3° for the methyl ether 19 and the ace-
tate 22 of the parent compound 11). Larger ester group increased
the magnitude of the optical rotation of the parent alcohol, for
example, ꢁ19.8° and ꢁ 47.4° for the benzoate and naphthalene-
2-carboxylate esters, respectively (experimental data for the prep-
aration of these latter two esters are not shown). The presence of
different substituent groups, for example, the unsaturated substi-
tuent in compound 12, led to a significant increase in the optical
rotation (ꢁ64.2° for compound 12).
OR
5
OR
c
4
11, R = H
26, R = H
28, R = Ac
b
a
19, R = Me
22, R = Ac
12, R = H,
23, R = Ac,
b
b
Scheme 5. Reagents: (a) MeI, Ag2O, DMF; (b) Ac2O, pyridine; (c) H2, Pd–C, EtOH.

A. Suksamrarn et al. / Bioorg. Med. Chem. 16 (2008) 6891–6902
6897
O
O
5
c
4
R
R
2, R = H
27, R = H
29, R = OH
15, R = OH
a
20, R = OMe
24, R = OAc
16, R = OH,
21, R = OMe,
25, R = OAc,
b
a
d
O
b
R = OH,
OH
OH
e
30
OH
O
OH
O
c
OH
31
32
f
O
OH
33
Scheme 6. Reagents and conditions: (a) MeI, Ag2O, DMF; (b) Ac2O, pyridine; (c) H2, Pd–C, EtOH; (d) DDQ, benzene, reflux; (e) SeO2, dioxane, reflux; (f) p-TsOH, benzene, 50 °C.
Table 1
16, clearly was due to the influence of the extra phenolic hydroxyl
group in 15 and 16. Comparison of the structures of 15 and 16 with
Relative transcriptional activation activity of diarylheptanoids and genistein to 17b-
estradiol (which was set as 100%) at the same concentration (10ꢁ8 M) on Bcl-xL and
that of E2, the additional phenolic hydroxyl group could possibly
ERb genes in HeLa cell line
act as either the 3- or 17-hydroxyl group of E2, and it might exert
Compound
Bcl-xL
ERb
its binding ability to the receptor in similar fashion to that of E2.
Compound 11, the hydroxyl analogue of the ketone 2, on the
other hand, was much less active than compound 2 (53% for Bcl-
xL and 42% for ERb). The finding implied that the polar hydroxyl
group on the alkyl chain caused the decrease in estrogenic activity.
This was further confirmed by the relatively less active alcohol 17
(58% for Bcl-xL and 53% for ERb) comparing with the highly active
corresponding ketone 15. However, the same trend could not be
applied to the alcohol 12, which was more active than the ketone
10 (63% for Bcl-xL and 75% for ERb). It should be noted that the
presence of an extra hydroxyl group on the same aromatic ring
as in compound 18 did not improve its activity (see Table 1).
To see whether it was the steric factor or electronic factor that
made the alcohol 11 less active than the corresponding ketone 2,
the methyl ether 19 and the acetate 22 were prepared from the
parent compound 11. It was found that significant increase in
activity was observed in both the methyl ether 19 and the acetate
22 (73% for Bcl-xL and 75% for ERb, and 71% for Bcl-xL and 76% for
ERb, respectively). The results indicated that it was the electronic
factor that caused decrease in estrogenic activity of the alcohol
11. The hydrogen-bond forming ability of the hydroxyl group to
the in-appropriate position of the receptor might lead to decrease
in activity. The higher estrogenic activity of the ketone 2 than the
corresponding alcohol 11 could possibly be due to the fact that the
former functional group is located at a suitable position for the
receptor to form a strong hydrogen bonding with the keto function
of the diarylheptanoid. However, the same explanation could not
17b-Estradiol (E2)
Genistein
2
100
100
72.85 6.84
74.30 0.54
77.50 3.54
43.97 8.23
52.85 2.91
62.51 9.19
78.51 0.37
74.54 0.62
57.73 7.58
45.98 7.36
72.50 0.71
71.40 0.57
76.77 3.64
71.06 9.70
47.96 3.19
75.30 4.96
49.82 9.79
40.00 9.90
42.18 1.20
55.00 7.07
74.41 8.36
47.08 10.71
75.51 2.37
85.45 3.54
76.46 2.12
33.72 5.66
42.48 3.54
75.42 7.07
90.56 2.83
87.75 8.48
52.50 9.19
51.50 7.78
74.97 9.90
58.51 3.55
72.00 7.06
75.50 9.19
58.36 3.33
84.00 8.48
68.45 1.95
37.03 3.58
57.50 10.61
58.97 2.83
70.33 9.43
50.86 3.08
9
10
11
12
15
16
17
18
19
20
21
22
23
24
25
26
27
28
30
33
Means of intensities SD after normalization with those of internal standard are
shown.
16 resulted in 2- to 3-fold increase in activity. The increment of
estrogenic activity in going from the non-phenolic compound 2
to the phenolic compound 15, especially from compound 10 to

6898
A. Suksamrarn et al. / Bioorg. Med. Chem. 16 (2008) 6891–6902
be applied to the dienol 12 and its acetate 23, where the latter
exhibited lower activity. The conflicting results on the low activity
of the alcohol 11 and the high activity of the alcohol 12 need addi-
tional postulation. The lower activity of compound 11 than that of
compound 12 could result from the too flexible heptyl chain of the
former, while the additional conjugated double bond in 12 lowered
the free rotation of the chain, so that the alignment of diarylhepta-
noid molecule 12 in the receptor was optimized. The lower activity
of the conjugated dienone 10 than that of the non-conjugated en-
one 2 could possibly be due to the extended conjugation of the for-
mer resulted in a too rigid molecule to accommodate itself easily to
the receptor. The explanation was further supported by the fact
that, instead of a significant increase in activity in similar fashion
to that in going from 11 to 12, the activity of the conjugated die-
none 16 was only comparable to the non-conjugated enone 15.
In order to see whether the high estrogenic activity of the
diarylheptanoids 15 and 16 was due to the presence of free hydro-
xyl group, compounds 15 and 16 were subjected to methylation
and acetylation to the respective methyl ethers 20 and 21, and
the acetates 24 and 25. As expected, the methyl ethers and the ace-
tates were not more active than the parent compounds; some of
them were even less active than their parent compounds (see Table
1). The results have demonstrated that the free phenolic hydroxyl
group in the diarylheptanoids 15 and 16 contributed to high estro-
genic activity in similar manner to that occurred in E2.
analogues exhibited lower estrogenic activity than the natural hor-
mone 17b-estradiol, some of them showed high activity. Induction
of gene transcription by diarylheptanoids of both Bcl-xL and ERb
genes confirmed the novel activity of these compounds. The study
on structure-activity relationship has led to a conclusion that,
based on the transcriptional activation of both estrogenic targets,
Bcl-xL and ERb gene expression, the structural features for a diaryl-
heptanoid from C. comosa to exhibit high estrogenic activity are the
presence of an olefinic function conjugated with the aromatic ring
at the 7-position, a keto group at the 3-position, and a phenolic hy-
droxyl group at the p-position of the aromatic ring attached to the
1-position of the heptyl chain to exhibit high estrogenic activity.
The saturated heptyl-chain analogues, the 3-hydroxy analogues,
and the analogues with an olefinic function at the 1- and 4-posi-
tions were relatively less active. Evaluations of these diarylhepta-
noids in vivo system are undergoing to establish their biological
activities.
4. Experimental
4.1. General
Melting points were determined using an Electrothermalmelting
point apparatus and were uncorrected. IR spectra were recorded in
KBr or as neat on a Perkin-Elmer FT-IR Spectrum BX spectrophotom-
eter. 1H and 13C NMR spectra were recorded on a BrukerAVANCE 400
spectrometer operating at 400 and 100 MHz, respectively. Mass
spectra were obtained using a Finnigan LC-Q mass spectrometer.
High resolution mass spectra were obtained using a Bruker micrO-
TOF mass spectrometer. Unless indicated otherwise, column chro-
matography and TLC were carried out using Merck silica gel 60
(finerthan 0.063 mm)and precoated silica gel 60 F254 plates, respec-
tively. Spots on TLC were detected under UV light and by spraying
with anisaldehyde-H2SO4 reagent followed by heating.
Next, we would also like to see whether the olefinic function
adjacent to the aromatic ring attached to the 7-position contrib-
uted to estrogenic activity of the diarylheptanoids. Compounds
11, 2 and 22 were hydrogenated to the corresponding dihydro ana-
logues 26, 27, and 28 and it was found that all the saturated ana-
logues were significantly less active than their parent compounds
(see Table 1). The assay results indicated that the olefinic function
conjugated with the 7-phenyl group contributed to high estrogenic
activity.
We would also like to further investigate whether placement of
the double bond function at the 1-position, instead of the 6-posi-
tion, would affect estrogenic activity. The enone 30 was therefore
synthesized and it was found that this analogue exhibited lower
activity (74% for Bcl-xL and 70% for ERb) than the parent compound
15 (79% for Bcl-xL and 91% for ERb). Moreover, we have also syn-
thesized the isomeric ketone 33 and this conjugated ketone was
even much less active than compound 30 (47% for Bcl-xL and
51% for ERb).
4.2. Plant material
The rhizomes of C. comosa were collected from Kampaengsaen
district, Nakhon Pathom province, Thailand, in January, 2005. A
voucher specimen (Apichart Suksamrarn, No. 052) is deposited at
the Faculty of Science, Ramkhamhaeng University.
4.3. Extraction and isolation
The results indicated that the diarylheptanoids 2, 9, 15, 16, 19,
21, 22 and 24 exhibited estrogenic activity comparable to the phy-
toestrogen genistein. Some of these diarylheptanoids (compounds
2, 15, and 16) showed even higher activity than genistein. Com-
pound 15 has been selected as a structure lead for further struc-
tural modification.
The fresh rhizomes (80 kg) were sliced and oven-dried at 40 °C.
The pulverized rhizome (10.4 kg) was extracted successively with
n-hexane, CHCl3, and MeOH in a Soxhlet extraction apparatus to
yield the hexane (350.0 g), CHCl3 (770.1 g), and MeOH (322.4 g) ex-
tracts, respectively (see Scheme 1). The hexane extract (100.0 g)
was chromatographed (Merck silica gel, 0.063–0.200 mm, see
Scheme 2), using a gradient of hexane, hexane–CHCl3, CHCl3, and
CHCl3–MeOH, to give 6 fractions (H1–H6). Fraction H2 (11.55 g)
was chromatographed, using hexane, hexane–CH2Cl2, and CH2Cl2as
eluent, to yield compound (9)17,18 (20 mg) as an off-white solid.
Fractions H3 and H4 (23.88 g) were combined and chromato-
graphed, eluting with hexane–CHCl3, CHCl3, and CHCl3–MeOH to
afford 15 subfractions.
3. Conclusion
From the rhizomes of Curcuma comosa Roxb., three new
diarylheptanoids, a 1:2 mixture of (3S)- and (3R)-1-(4-methoxy-
phenyl)-7-phenyl-(6E)-6-hepten-3-ol (13a and 13b) and 1-(4-hydro-
xyphenyl)-7-phenyl-(6E)-6-hepten-3-one (15), together with two
synthetically known diarylheptanoids 1,7-diphenyl-(1E,3E,5E)-
1,3,5-triene (9) and 1-(4-hydroxyphenyl)-7-phenyl-(4E,6E)-4,6-
heptadien-3-one (16), and nine known diarylheptanoids, 2, 8,
10–12, 14, a 3:1 mixture of 17a and 17b, and 18, were isolated.
The absolute stereochemistry of the isolated alcohols has also been
determined using the modified Mosher’s method. Compounds 2,
11, 12, 15 and 16 were chemically modified for biological evalua-
tion. The isolated diarylheptanoids and the modified analogues
were subjected to estrogenic activity evaluation. Although all nat-
urally occurring diarylheptanoids and their chemically modified
Subfraction 6 (87 mg) was chromatographed to give two im-
pure compounds, which upon repeated column chromatography,
yielded compound (2)15 (20 mg) as colorless oil, and 1019,20
(8 mg) as pale yellow needles from MeOH, mp 62–63 °C. Subfrac-
tion 7 (8.18 g) was chromatographed, using similar eluting solvent
system, to give compound 11 (310 mg), together with compound
12 contaminated with compound 11 (86 mg). Compound 11 was
obtained as colorless needles from CH2Cl2–hexane, mp 46–48 °C
(lit.19 47–49 °C), ½a 2D7
ꢀ
ꢁ 2:6ꢂ (EtOH,c 0.30). Repeated column chro-

A. Suksamrarn et al. / Bioorg. Med. Chem. 16 (2008) 6891–6902
6899
matography of compounds 11 and 12 mixture afforded pure 1215
(60 mg) which was recrystallized from CH2Cl2–hexane as white
(C-30, C-50), 126.5 (C-6), 127.2 (C-200, C-600), 128.8 (C-300, C-500),
129.2, 129.3, 129.4 (C-4, C-20, C-60, C-400), 133.0 (C-10), 135.9 (C-
100), 141.6 (C-7), 143.0 (C-5), 154.0 (C-40), 200.1 (C-3); HMBC corre-
lations: H-1/H-2 (C-3, C-10), H-4 (C-2, C-3, C-6), H-5 (C-3, C-7), H-6
(C-5, C-100), H-7 (C-5, C-200/C-600), H-20/H-60 (C-1, C-40), H-30/H-50 (C-
10, C-40), H-200/H-600 (C-7); ESMS (+ve): m/z 279 [M+H]+, 301 [M-
Na]+, 579 [2M+Na]+; HR-TOFMS (ES+): m/z 279.1367 [M+H]+; calcd
for C19H18O2+H, 279.1380.
powder, mp 79–80 °C (lit.19 78–79 °C), ½a D27
ꢀ
ꢁ 64:2ꢂ (EtOH, c 0.30).
The CHCl3 extract (300.0 g) was chromatographed (Merck silica
gel, 0.063–0.200 mm, see Scheme 3), using a gradient of hexane,
hexane–CH2Cl2, CH2Cl2, and CH2Cl2–MeOH (5% increment in the
polar solvent for 250 mL of each proportion) to give 11 combined
fractions (C1–C11).
Fractions C2 and C3 contained additional mixture of 11 and 12
(132 mg). Fraction C5 was chromatographed using CH2Cl2–MeOH
under gradient condition to give seven combined subfractions.
Subfraction 3, eluted by pure CH2Cl2, was further subjected to re-
peated column chromatography to yield a mixture of two enantio-
The MeOH extract (210.0 g) was chromatographed (Merck silica
gel, 0.063–0.200 mm, see Scheme 4), using a gradient CH2Cl2,
CH2Cl2–MeOH, and MeOH, to give six combined fractions (M1–
M6). Fraction M1 contained a small quantity (8 mg) of compounds
11 and 12, whereas fraction M2 constituted a small quantity
(4 mg) of compounds 15 and 16 mixture.
A half portion (45.0 g) of fraction M5 was chromatographed,
using ingredient elution with CH2Cl2-MeOH, to yield a mixture of
two enantiomers 17a and 17b (14 mg), mp 94–95 °C (from
mers 13a and 13b (6 mg), compound 14 (16 mg, ½a D27
ꢀ
þ 3:4ꢂ (EtOH,
c 0.25), and compound 15 (150 mg). Fraction C7, upon repeated
column chromatography using gradient elution with CH2Cl2 and
CH2Cl2–MeOH, afforded compound 16 (72 mg).
CH2Cl2–hexane) (lit.26 94–95 °C), ½a D26
ꢀ
þ 1:73ꢂ (MeOH, c 0.17), and
4.3.1. Compounds 13a and 13b mixture
the 1H NMR data of which were identical to the reported values
of compound 6.26 Further elution with increasing proportion of
MeOH afforded compound 18 (21 mg), mp 100–101 °C (from
Colorless viscous oil; ½a D27
ꢀ
þ 2:4ꢂ (EtOH,c 0.23); IR (neat) mmax
:
3331, 3026, 2945, 2915, 2831, 1610, 1512, 1453, 1439, 1336,
1302, 1243, 1180, 1081, 1035, 971 cmꢁ1
;
1H NMR (CDCl3,
CH2Cl2–hexane) (lit.16 99–100 °C), ½a D28
ꢀ
ꢁ 5:2ꢂ (EtOH,c 0.41), and
400 MHz): d 1.64 (m, 2H, H-4), 1.74 (m, 2H, H-2), 2.31 (m, 2H, H-
5), 2.61 (m, 1H, H-1a), 2.73 (m, 1H, H-1b), 3.68 (m, 1H, H-3),
3.78 (s, 3H, OMe), 6.20 (dt, J = 15.8, 6.8 Hz, 1H, H-6), 6.39 (br d,
J = 15.8 Hz, 1H, H-7), 6.81 (d, J = 8.2 Hz, 2H, H-30, H-50), 7.10 (d,
J = 8.2 Hz, 2H, H-20, H-60), 7.16–7.32 (m, 5H, H-200-H-600); 13C NMR
(CDCl3, 100 MHz): d 29.2 (C-5), 31.0 (C-1), 37.0 (C-4), 39.3 (C-2),
55.2 (OMe), 70.8 (C-3), 113.8 (C-30, C-50), 125.9 (C-200, C-600), 126.9
(C-400), 128.2, 128.4 (C-300, C-500), 129.2 (C-20, C-60), 130.26, 130.27
(C-6, C-7), 134.0 (C-10), 137.5 (C-100), 157.7 (C-40); HMBC correla-
tions: H-1 (C-2, C-3, C-10, C-20/C-60), H-2 (C-1, C-4, C-10), H-3 (C-
1, C-5), H-4 (C-2, C-3, C-5, C-6), H-5 (C-3, C-4, C-6, C-7), H-6 (C-4,
C-5, C-7, C-100), H-7 (C-5, C-6, C-100, C-200/C-600), OMe (C-40), H-20/
H-60 (C-1, C-30/C-50, C-40), H-30/H-50 (C-10, C-40); ESMS (+ve): m/z
297 [M+H]+, 615 [2M+Na]+; HR-TOFMS (ES+): m/z 297.1839
[M+H]+; calcd for C20H24O2+H, 297.1849.
the 1H NMR data of this compound were identical to those re-
ported for compound 7.26 A half portion (11.0 g) of fraction M6
was similarly subjected to column chromatography and, after re-
peated column chromatography, the glucoside 816 (18 mg), mp
222–224 °C, was resulted.
4.4. Determination of C-3 absolute configuration of
diarylheptanoids
To a solution of the diarylheptanoid 11 (2.1 mg, 0.0078 mmol)
in dry pyridine (100
l
L) was added (R)-(ꢁ)-MTPA chloride
(15 L) at 10 °C and the mixture was stirred for 5 min. Stirring con-
l
tinued at ambient temperature and the completion of reaction was
monitored by TLC. Two milliliters of n-hexane was added to the
reaction mixture and the hexane-soluble part was subjected to
flash column chromatography using hexane and 3% EtOAc/hexane
as eluting solvent to give the (S)-MTPA ester 11x (3.1 mg). The pro-
cedure was repeated, but using (S)-(+)-MTPA chloride in place of
(R)-(ꢁ)-MTPA chloride, to yield the (R)-MTPA ester 11y (4.5 mg).
The 1H NMR spectra of 11x and 11y were recorded in CDCl3; the
chemical shift differences of the proton resonances between the
(S)-MTPA ester 11x and the (R)-MTPA ester 11y were calculated
and the results are summarized in Figure 1.
4.3.2. Compound 15
Colorless viscous oil; IR (neat)
1697, 1611, 1595, 1514, 1445, 1365, 1263, 1224, 1173, 967,
832 cmꢁ1 1H NMR (CDCl3, 400 MHz): d 2.46 (dt, J = 7.0, 6.8 Hz,
mmax: 3360, 3025, 2919, 2860,
;
2H, H-5), 2.56 (t, J = 6.8 Hz, 2H, H-4), 2.72 (t, J = 7.3 Hz, 2H, H-2),
2.84 (t, J = 7.3 Hz, 2H, H-1), 6.14 (dt, J = 15.9, 6.7 Hz, 1H, H-6),
6.37 (d, J = 15.9 Hz, 1H, H-7), 6.76 (br d, J = 8.2 Hz, 2H, H-30, H-50),
7.01 (br d, J = 8.2 Hz, 2H, H-20, H-60), 7.16–7.33 (m, 5H, H-200-H-
600); 13C NMR (CDCl3, 100 MHz): d 27.0 (C-5), 28.8 (C-1), 42.5 (C-
4), 44.6 (C-2), 115.3 (C-30, C-50), 125.9 (C-200, C-600), 127.0 (C-400),
128.4 (C-300, C-500), 128.7 (C-6), 129.3 (C-20, C-60), 130.7 (C-7),
132.7 (C-10), 137.3 (C-100), 154.0 (C-40), 210.1 (C-3); HMBC correla-
tions: H-1 (C-2, C-3, C-10, C-20/C-60), H-2 (C-1, C-3, C-4, C-10), H-4
(C-2, C-3, C-5, C-6), H-5 (C-3, C-4, C-6, C-7), H-6 (C-4, C-5, C-7, C-
100), H-7 (C-5, C-100, C-200/C-600), H-20/H-60 (C-1, C-30/C-50), H-30/H-
50 (C-10); ESMS (-ve): m/z 279 [MꢁH]ꢁ; 559 [2MꢁH]ꢁ; HR-TOFMS
(ES+): m/z 281.1525 [M+H]+; calcd for C19H20O2+H, 281.1536.
Following the above procedure, the absolute configurations of
compounds 12, 13a and 13b, 14, 17a and 17b, and 18 were deter-
mined and the results are summarized in Figure 1.
4.5. Chemical modifications of diarylheptanoids
The procedures for chemical modifications of the diarylhepta-
noids 11, 12, 15 and 16 are summarized in Schemes 5 and 6.
4.5.1. (3S)-1,7-Diphenyl-3-methoxy-(6E)-6-heptene (19)
To a solution of compound 11 (100 mg, 0.376 mmol) in DMF
(5 mL) were added methyl iodide (0.7 mL, d = 2.27 g/mL,
11.28 mmol) and silver oxide (784 mg, 3.38 mmol), and the mix-
ture was stirred at ambient temperature for 24 h. Water (30 mL)
was added and the mixture was extracted with EtOAc
(3 ꢃ 40 mL). The combined organic layer was washed with water,
dried over anhydrous Na2SO4, and the solvent was evaporated in
vacuo. The residue was purified by column chromatography under
isocratic condition using hexane/EtOAc (10:1) to afford compound
4.3.3. Compound 16
Prisms from EtOAc–hexane, mp 129–130 °C (lit.25 129.3–
130.0 °C). IR (KBr)
mmax: 3380, 3026, 2928, 1673, 1638, 1618,
1595, 1513, 1445, 1368, 1219, 1175, 1100, 1009, 829, 752 cmꢁ1
;
1H NMR (CDCl3, 400 MHz): d 2.86 (br s, 4H, H-1, H-2), 6.26 (d,
J = 15.6 Hz, 1H, H-4), 6.72 (d, J = 8.4 Hz, 2H, H-30, H-50), 6.84 (dd,
J = 15.6, 10.1 Hz, 1H, H-6), 6.92 (d, J = 15.6 Hz, 1H, H-7), 7.06 (d,
J = 8.4 Hz, 2H, H-20, H-60), 7.25–7.35 (m, 3H, H-300-H-500), 7.33 (par-
tially overlapping signal, 1H, H-5), 7.45 (br d, J = 7.1 Hz, 2H, H-200,
H-600); 13C NMR (CDCl3, 100 MHz): d 29.4 (C-1), 42.5 (C-2), 115.3
19 (61 mg, 58%) as colorless oil; ½a D27
ꢀ
ꢁ 5:3ꢂ (EtOH, c 0.29); IR (neat)
m
max: 3025, 2929, 2856, 1600, 1494, 1452, 1182, 1096, 965, 742,

6900
A. Suksamrarn et al. / Bioorg. Med. Chem. 16 (2008) 6891–6902
696 cmꢁ1
;
1H NMR (CDCl3, 400 MHz): d 1.60–1.76 (m, 2H, H-4),
4.5.5. (3R)-3-Acetoxy-1,7-diphenyl-(4E,6E)-4,6-heptadiene (23)
Compound 12 (25 mg, 0.0.094 mmol) was acetylated in the
same manner described for the preparation of 22 from 11 to afford
1.76–1.92 (m, 2H, H-2), 2.22–2.33 (m, 2H, H-5), 2.60–2.80 (m,
2H, H-1), 3.27 (m, 1H, H-3), 3.38 (s, 3H, OMe), 6.23 (dt, J = 15.8,
6.8 Hz, 1H, H-6), 6.41 (d, J = 15.8 Hz, 1H, H-7), 7.15–7.25 (m, 4H,
H-20, H-40, H-60, H-400), 7.25–7.38 (m, 6H, H-30, H-50, H-200, H-300,
H-500, H-600); 13C NMR (CDCl3, 100 MHz): d 28.7 (C-5), 31.5 (C-1),
33.0 (C-4), 35.2 (C-2), 56.4 (OMe), 79.5 (C-3), 125.7 (C-400), 125.9
(C-200, C-600), 126.8 (C-40), 128.3 and 128.4 (C-20, C-30, C-50, C-60, C-
300, C-500), 130.0 (C-7), 130.5 (C-6), 137.7 (C-100), 142.3 (C-10); ESMS
(+ve): m/z 281 [M+H]+; HR-TOFMS (ES+): m/z 281.1886 [M+H]+;
calcd for C20H24O+H, 281.1900.
compound 23 (23 mg, 79%) as colorless oil; ½a D27
ꢀ
þ 12:3ꢂ (EtOH, c
0.30). The spectroscopic (IR, 1H NMR, and MS) data were consistent
with the reported data of the acetate derivative of compound 4 iso-
lated from other plant species.19
4.5.6. 1-(4-Acetoxyphenyl)-7-phenyl-(6E)-6-hepten-3-one (24)
Compound 15 (20 mg, 0.071 mmol) was acetylated in the same
manner described for the preparation of 22 from 11 to afford com-
pound 24 (20 mg, 87%) as colorless oil; IR (neat)
mmax: 3407, 3029,
4.5.2. 1-(4-Methoxyphenyl)-7-phenyl-(6E)-6-hepten-3-one (20)
Compound 15 (20 mg, 0.071 mmol) was subjected to methyla-
tion in the same manner to the preparation of 19 from 11, except
that the reaction time was only 2 h, to give, after usual column
chromatographic purification, the product 20 (18 mg, 86%) as col-
2924, 2855, 1760, 1713, 1639, 1505, 1447, 1370, 1198, 1015 cmꢁ1
;
1H NMR (CDCl3, 400 MHz): d 2.27 (s, 3H, OAc), 2.46 (dt, J = 7.2,
6.9 Hz, 2H, H-5), 2.55 (t, J = 7.2 Hz, 2H, H-4), 2.73 (t, J = 7.6 Hz,
2H, H-2), 2.88 (t, J = 7.6 Hz, 2H, H-1), 6.15 (dt, J = 15.7, 6.8 Hz, 1H,
H-6), 6.37 (d, J = 15.8 Hz, 1H, H-7), 6.95 (d, J = 8.4 Hz, 2H, H-30, H-
50), 7.16 (br d, J = 8.4 Hz, 2H, H-20, H-60), 7.16–7.32 (m, 5H, H-200-
H-600); 13C NMR (CDCl3, 100 MHz): d 21.0 (OCOMe), 27.0 (C-5),
29.0 (C-1), 42.4 (C-4), 44.3 (C-2), 121.4 (C-30, C-50), 126.0 (C-200,
C-600), 127.0 (C-400), 128.4 (C-300, C-500), 128.7 (C-6), 129.2 (C-20, C-
60), 130.8 (C-7), 137.3 (C-100), 138.6 (C-10), 148.9 (C-40), 169.5
(OCOMe), 208.8 (C-3); ESMS (+ve): m/z 345 [M+Na]+, 667
[2M+Na]+; HR-TOFMS (ES+): m/z 323.1644 [M+H]+; calcd for
orless needles (from CHCl3–hexane), mp 69 °C; IR (KBr) mmax
3033, 2947, 2929, 2832, 1704, 1610, 1512, 1510, 1460, 1438,
1413, 1373, 1301, 1278, 1242, 1180, 1092, 1036, 969, 750 cmꢁ1
:
;
1H NMR (CDCl3, 400 MHz): d 2.45 (dt, J = 7.1, 6.9 Hz, 2H, H-5),
2.54 (t, J = 6.9 Hz, 2H, H-4), 2.70 (t, J = 7.5 Hz, 2H, H-2), 2.83 (t,
J = 7.5 Hz, 2H, H-1), 3.75 (s, 3H, OMe), 6.14 (dt, J = 15.8, 6.8 Hz,
1H, H-6), 6.36 (d, J = 15.8 Hz, 1H, H-7), 6.79 (br d, J = 8.5 Hz, 2H,
H-30, H-50), 7.07 (br d, J = 8.5 Hz, 2H, H-20, H-60), 7.19–7.32 (m,
5H, H-200-H-600); 13C NMR (CDCl3, 100 MHz): d 26.9 (C-5), 28.8
(C-1), 42.3 (C-4), 44.5 (C-2), 55.1 (OMe), 113.8 (C-30, C-50), 125.9
(C-200, C-600), 126.9 (C-400), 128.3 (C-300, C-500), 128.7 (C-6), 129.1
(C-20, C-60), 130.6 (C-7), 132.9 (C-10), 137.3 (C-100), 157.8 (C-40),
209.1 (C-3); ESMS (+ve): m/z 317 [M+Na]+, 611 [2M+Na]+; HR-
TOFMS (ES+): m/z 295.1691 [M+H]+; calcd for C20H22O2+H,
295.1693.
C21H22O3+H, 323.1642.
4.5.7. 1-(4-Acetoxyphenyl)-7-phenyl-(4E,6E)-4,6-heptadien-3-
one (25)
Compound 16 (15 mg, 0.054 mmol) was acetylated in the same
manner described for the preparation of 22 from 11 to afford com-
pound 25 (14 mg, 78%) as colorless oil; IR (KBr)
3035, 2925, 2855, 1759, ca. 1700 (shoulder), 1636, 1610, 1508,
1450, 1370, 1218, 1197, 1167, 1017, 913 cmꢁ1 1H NMR (CDCl3,
mmax: 3433, 3063,
;
4.5.3. 1-(4-Methoxyphenyl)-7-phenyl-(4E,6E)-4,6-heptadien-3-
one (21)
Compound 16 (25 mg, 0.09 mmol) was subjected to methyla-
tion in the same manner to the preparation of 20 from 15 to give,
after usual column chromatographic purification, the product 21
400 MHz): d 2.27 (s, 3H, OAc), 2.86–2.98 (m, 4H, H-1, H-2), 6.26
(d, J = 15.4 Hz, 1H, H-4), 6.85 (dd, J = 15.5, 10.3 Hz, 1H, H-6), 6.93
(d, J = 15.6 Hz, 1H, H-7), 6.98 (d, J = 8.4 Hz, 2H, H-30, H-50), 7.20
(d, J = 8.4 Hz, 2H, H-20, H-60), 7.26–7.37 (m, 3H, H-300-H-500), 7.33
(obscured signal, 1H, H-5), 7.45 (br d, J = 7.1 Hz, 2H, H-200, H-600);
13C NMR (CDCl3, 100 MHz): d 21.1 (COMe), 29.4 (C-1), 42.2 (C-2),
121.4 (C-30, C-50), 126.6 (C-6), 127.2 (C-200, C-600), 128.8 (C-300, C-
500), 129.2 (C-400), 129.3 (C-20, C-60), 129.4 (C-4), 135.9 (C-100),
138.8 (C-10), 141.4 (C-7), 142.7 (C-5), 148.9 (C-40), 169.6 (COMe),
199.1 (C-3); ESMS (+ve): m/z 663 [2M+Na]+; HR-TOFMS (ES+):
m/z 321.1493 [M+H]+; calcd for C21H20O3+H, 321.1485.
(22 mg, 82%) as colorless oil; IR (neat)
mmax: 2928, 1707, 1612,
1512, 1450, 1247, 1178, 1034, 827, 752, 699 cmꢁ1; 1H NMR (CDCl3,
400 MHz): d 2.84–2.94 (m, 4H, H-1, H-2), 3.77 (s, 3H, OMe), 6.26 (d,
J = 15.4 Hz, 1H, H-4), 6.82 (d, J = 8.5 Hz, 2H, H-30, H-50), 6.84 (dd,
J = 15.5, 10.0 Hz, 1H, H-6), 6.92 (d, J = 15.5 Hz, 1H, H-7), 7.12 (d,
J = 8.5 Hz, 2H, H-20, H-60), 7.28 (dd, J = 15.4, 10.1 Hz,, 1H, H-5),
7.28–7.38 (m, 3H, H-300-H-500), 7.45 (br d, J = 7.1 Hz, 2H, H-200, H-
600); 13C NMR (CDCl3, 100 MHz): d 29.3 (C-1), 42.6 (C-2), 55.2
(OMe), 113.9 (C-30, C-50),126.6 (C-6), 127.2 (C-200, C-600), 128.8 (C-
300, C-500), 129.1 (C-400), 129.2 (C-20, C-60), 129.5 (C-4), 133.3 (C-10),
136.0 (C-100), 141.3 (C-7), 142.6 (C-5), 157.9 (C-40), 199.5 (C-3);
ESMS (+ve): m/z 293 [M+H]+, 607 [2M+Na]+; HR-TOFMS (ES+):
m/z 293.1531 [M+H]+; calcd for C20H20O2+H, 293.1536.
4.5.8. (3S)-1,7-Diphenylheptan-3-ol (26)
Compound 11 (50 mg, 0.187 mmol) in EtOH (5 mL) was hydro-
genated at atmospheric pressure for 20 min, with 10% Pd-C (20 mg,
0.018 mmol) as a catalyst. The mixture was filtered through Celite;
the solid was washed with EtOAc and the filtrate was concentrated
in vacuo. The residue was purified by column chromatography un-
der isocratic condition using hexane/EtOAc (80:20) to afford com-
pound 26 (49 mg, 97%) as colorless needles (from EtOAc–hexane),
4.5.4. (3S)-3-Acetoxy-1,7-diphenyl-(6E)-6-heptene (22)
To a solution of compound 11 (50 mg, 0.187 mmol) in pyri-
dine (4 mL) was added acetic anhydride (0.3 mL, d = 1.081 g/mL,
3.15 mmol), and the mixture was stirred at ambient tempera-
ture for 3 h. Water (30 mL) was added and the mixture was ex-
tracted with EtOAc (3ꢃ 40 mL). The combined organic layer was
washed with water, dried over anhydrous Na2SO4, and the sol-
vent was evaporated in vacuo. The residue was purified by col-
umn chromatography (silica gel, 0.063–0.20 mm) under isocratic
condition using hexane/EtOAc (10:1) to yield compound 22
mp 59–60 °C; ½a 2D7
ꢀ
þ 8:0ꢂ (EtOH, c 0.27); IR (KBr)
mmax: 3416, 2927,
2856, 1627, 1550, 1497, 1452, 1382, 1020, 749, 698 cmꢁ1; 1H NMR
(CDCl3, 400 MHz): d 1.30–1.54 (m, 4H, H-5, H-6), 1.61 (m, 2H, H-4),
1.74 (m, 2H, H-2), 2.62 (t, J = 7.7 Hz, 2H, H-7), 2.65 (m, 1H, H-1a),
2.77 (m, 1H, H-1b), 3.60 (m, 1H, H-3), 7.10–7.20 (m, 6H,
H-20, H-40, H-60, H-200, H-400, H-600), 7.22–7.30 (m, 4H, H-30, H-50,
H-300, H-500); 13C NMR (CDCl3, 100 MHz): d 25.2 (C-5), 31.4 (C-4),
32.0 (C-1), 35.8 (C-7), 37.4 (C-6), 39.0 (C-2), 71.2 (C-3), 125.6 and
125.7 (C-40, C-400), 128.2 and 128.3 (C-20, C-30, C-50, C-60, C-200, C-
300, C-500, C-600), 142.1 (C-10), 142.5 (C-100); ESMS: m/z 291
[M+Na]+; HR-TOFMS (ES+): m/z 291.1717 [M+Na]+; calcd for
(44 mg, 76%) as colorless oil; ½a D28
ꢀ
ꢁ 7:3ꢂ (EtOH, c 0.26); The
spectroscopic (IR, 1H NMR and MS) data were consistent with
the reported data of compound 1 isolated earlier from this
plant species.15
C19H24O+Na, 291.1719.

A. Suksamrarn et al. / Bioorg. Med. Chem. 16 (2008) 6891–6902
6901
4.5.9. 1,7-Diphenylheptan-3-one (27)
Compound 2 (10 mg, 0.039 mmol) was hydrogenated in the
same manner to that of compound 11 to afford compound 27
202.3 (C-3); ESMS (ꢁve): m/z 279 [MꢁH]ꢁ, 559 [2MꢁH]ꢁ; HR-TOF-
MS (ES+): m/z 281.1536 [M+H]+; calcd for C19H20O2+H, 281.1536.
(9 mg, 95%) as colorless oil; IR (neat)
m
max: 3027, 2930, 2858,
4.5.12. 1-(4-Hydroxyphenyl)-7-phenyl-(4E)-4-hepten-3-one
(33)
1712, 1603, 1495, 1453, 1408, 1371, 1099, 1026, 747, 700 cmꢁ1
;
1H NMR (CDCl3, 400 MHz): d 1.58 (m, 4H, H-5, H-6), 2.37 (t,
J = 6.5 Hz, 2H, H-4), 2.58 (t, J = 6.9 Hz, 2H, H-7), 2.68 (t, J = 7.8 Hz,
2H, H-2), 2.87 (t, J = 7.8 Hz, 2H, H-1), 7.10–7.20 (m, 6H, H-20, H-
40, H-60, H-200, H-400, H-600), 7.22–7.30 (m, 4H, H-30, H-50, H-300, H-
500); 13C NMR (CDCl3, 100 MHz): d 23.3 (C-5), 29.7 (C-1), 30.8 (C-
6), 35.6 (C-7), 42.7 (C-4), 44.2 (C-2), 125.7 and 126.0 (C-40, C-400),
128.2, 128.3 and 128.4 (C-20, C-30, C-50, C-60, C-200, C-300, C-500, C-
600), 141.0 (C-10), 142.1 (C-100), 209.9 (C-3); ESMS (+ve): m/z 267
[M+H]+, 533 [2M+H]+; HR-TOFMS (ES+): m/z 267.1748 [M+H]+;
calcd for C19H22O+H, 267.1743.
Compound 15 (60 mg, 0.143 mmol) and SeO2 (60 mg,
0.541 mmol) in dioxane (3 mL) were refluxed for 4 h. The evapo-
rated mixture was chromatographed (isocratic elution with hex-
ane/EtOAc, 3:1) to give 1-(4-hydroxyphenyl)-7-phenyl-(4E)-4-
hepten-5-ol-3-one (31) (27 mg), which was subsequently hydroge-
nated as described for the preparation of compound 26 from 11 to
afford compound 32, ESMS (+ve): m/z 299 [M+H]+, 619 [2M+Na]+;
HR-TOFMS (ES+): m/z 299.1638 [M+H]+; calcd for C19H22O3+H,
299.1642, which in turn was subjected to dehydration using p-
TsOH (10 mg) in benzene (3 mL) at 50 °C for 1 h to give, after col-
umn chromatography under isocratic condition using hexane/
EtOAc (3:1), compound 33 (12 mg) as colorless viscous oil. The
overall yield of 33 from compound 15 was 20%.
4.5.10. (3S)-3-Acetoxy-1,7-diphenylheptane (28)
Compound 26 (20 mg, 0.075 mmol) was acetylated in the same
manner described for the preparation of 23 from 12 to afford com-
pound 28 (20 mg, 86%) as colorless oil; ½a D27
ꢀ
ꢁ 4:2ꢂ (EtOH, c 0.48);
4.5.12.1. 1-(4-Hydroxyphenyl)-7-phenyl-(4E)-4-hepten-5-ol-3-
1H NMR (CDCl3, 400 MHz): d 1.30 (m, 2H) and 1.57 (m, 4H) (H-4,
H-5, H-6), 1.80 (m, 2H, H-2), 2.00 (s, 3H, OAc), 2.60 (m, 4H, H-1,
H-7), 4.90 (m, 1H, H-3), 7.12–7.19 (m, 6H, H-20, H-40, H-60, H-200,
H-400, H-600), 7.23–7.29 (m, 4H, H-30, H-50, H-300, H-500); 13C NMR
(CDCl3, 100 MHz): d 21.1 (OCOMe), 24.8 (C-5), 31.2 (C-4), 31.7 (C-
7), 33.9 (C-6), 35.7 (C-1, C-2), 73.8 (C-3), 125.6 and 125.8 (C-40,
C-400), 128.2 and 128.3 (C-20, C-30, C-50, C-60, C-200, C-300, C-500, C-
600), 141.6 (C-10), 142.4 (C-100), 170.8 (OCOMe); ESMS (+ve): m/z
333 [M+Na]+; HR-TOFMS (ES+): m/z 333.1825 [M+Na]+; calcd for
one (31). Amorphous solid; IR (KBr)
1514, 1412, 1310, 1233, 1105, 1018, 816, 751 cmꢁ1
(CDCl3, 400 MHz): 2.68 (d, J = 5.9 Hz, 2H, H-4), 2.75 (t,
m
max: 3381, 2917, 1698, 1597,
;
1H NMR
d
J = 6.8 Hz, 2H, H-2), 2.84 (t, J = 6.8 Hz, 2H, H-1), 4.72 (dt, J = 6.1,
5.9 Hz, 1H, H-5), 6.15 (dd, J = 15.9, 6.1 Hz, 1H, H-6), 6.59 (d,
J = 15.9 Hz, 1H, H-7), 6.72 (d, J = 8.3 Hz, 2H, H-30, H-50), 7.02 (d,
J = 8.3 Hz, 2H, H-20, H-60), 7.20–7.35 (m, 5H, H-200-H-600); 13C NMR
(CDCl3, 100 MHz): d 28.6 (C-1), 45.4 (C-2), 49.3 (C-4), 68.5 (C-5),
115.3 (C-30, C-50), 126.4 (C-200, C-600), 127.7 (C-400), 128.5 (C-300, C-
500), 129.4 (C-20, C-60), 130.0 (C-6), 130.4 (C-7), 132.7 (C-10), 136.1
(C-100), 153.9 (C-40), 210.3 (C-3); ESMS (+ve): m/z 615 [2M+Na]+;
HR-TOFMS (ES+): m/z 319.1297 [M+Na]+; calcd for C19H20O3+Na,
319.1305.
C21H26O2+Na, 333.1825.
4.5.11. 1-(4-Hydroxyphenyl)-7-phenyl-(1E)-1-hepten-3-one
(30)
Compound 15 (40 mg, 0.143 mmol) was hydrogenated as de-
scribed for the preparation of compound 19 from 11 to afford com-
4.5.12.2. 1-(4-Hydroxyphenyl)-7-phenyl-(4E)-4-hepten-3-one
(33). Amorphous solid; IR (KBr)
mmax: 3375, 3026, 2927, 2860,
pound 29 (38 mg, 94%).
A portion (30 mg, 0.106 mmol) of
1658, 1620, 1515, 1451, 1367, 1227, 1109, 975, 829, 700 cmꢁ1
;
compound 29 was heated with DDQ (120 mg, 0.528 mmol) in ben-
zene (2 mL) at reflux for 3 h to give, after column chromatography
(hexane/EtOAc, 3:1), compound 30 (14 mg, 47%).
1H NMR (CDCl3, 400 MHz): d 2.50 (dt, J = 7.3, 7.1 Hz, 2H, H-6),
2.74 (t, J = 7.3 Hz, 2H, H-7), 2.78 (d, J = 6.1 Hz, 2H, H-2), 2.82 (d,
J = 6.1 Hz, 2H, H-1), 4.58 (br s, 1H, OH), 6.08 (d, J = 15.9 Hz, 1H,
H-4), 6.72 (d, J = 8.2 Hz, 2H, H-30, H-50), 6.81 (dt, J = 15.9, 7.1 Hz,
1H, H-5), 7.02 (d, J = 8.2 Hz, 2H, H-20, H-60), 7.15 (d, J = 7.2 Hz, 2H,
H-200, H-600), 7.19 (br t, J = 7.2 Hz, 1H, H-400), 7.27 (br t, J = 7.2 Hz,
2H, H-300, H-500); 13C NMR (CDCl3, 100 MHz): d 29.2 (C-1), 34.0 (C-
6), 34.3 (C-7), 41.9 (C-2), 115.2 (C-30, C-50), 126.1 (C-400), 128.2
(C-200, C-600), 128.4 (C-300, C-500), 129.4 (C-20, C-60), 130.6 (C-4),
133.3 (C-10), 140.6 (C-100), 146.2 (C-5), 153.8 (C-40), 199.6 (C-3);
ESMS (+ve): m/z 615 [2M+Na]+; HR-TOFMS (ES+): m/z 281.1530
[M+H]+; calcd for C19H20O2+H, 281.1536.
4.5.11.1. Compound 29. Colorless viscous oil; IR (neat) mmax
3390, 3025, 2932, 2859, 1702, 1614, 1515, 1452, 1372, 1226,
1172, 1099, 1030, 828, 748, 700 cmꢁ1 1H NMR (CDCl3,
:
;
400 MHz): d 1.58 (m, 4H, H-5, H-6), 2.39 (t, J = 6.8 Hz, 2H, H-4),
2.58 (t, J = 7.0 Hz, 2H, H-7), 2.67 (t, J = 7.5 Hz, 2H, H-2), 2.81 (t,
J = 7.5 Hz, 2H, H-1), 6.74 (d, J = 8.4 Hz, 2H, H-30, H-50), 7.01 (d,
J = 8.4 Hz, 2H, H-20, H-60), 7.10–7.19 (m, 3H, H-300, H-400, H-500),
7.20–7.27 (m, 2H, H-200, H-600); 13C NMR (CDCl3, 100 MHz): d 23.3
(C-5), 28.9 (C-1), 30.9 (C-6), 35.6 (C-7), 42.8 (C-4), 44.4 (C-2),
115.3 (C-30, C-50), 125.7 (C-400), 128.2 ( C-300, C-500), 128.3 (C-200, C-
600), 129.3 (C-20, C-60,), 133.0 (C-10), 142.1 (C-100), 153.9 (C-40),
210.6 (C-3); ESMS (ꢁve): m/z 281 [MꢁH]ꢁ, 562 [2MꢁH]ꢁ; HR-TOF-
MS (ES+): m/z 283.1686 [M+H]+; calcd for C19H22O2+H, 283.1693.
4.6. Assay for estrogenic activity in human cervical cancer HeLa
cell line
The estrogenic activity of diarylheptanoids was investigated in a
cervical carcinoma HeLa (ATCC) cell line using RT-PCR assay. HeLa
cells were grown in Dulbecco minimal essential medium (DMEM)
(GIBCO Invitrogen Corporation, USA) supplemented with 5% FBS,
4.5.11.2. Compound 30. Pale yellow amorphous solid; IR (KBr)
m
max: 3379, 3058, 3024, 2938, 2927, 2864, 1676, 1585, 1516,
1438, 1371, 1268, 1170, 1104, 1076 cmꢁ1 1H NMR (CDCl3,
;
100 U/mL of penicillin G, 100 lg/mL of streptomycin, and 2.5 lg/
400 MHz): d 1.53–1.75 (m, 4H, H-5, H-6), 2.63 (t, J = 7.0 Hz, 2H,
H-7), 2.68 (t, J = 6.8 Hz, 2H, H-4), 6.60 (d, J = 16.1 Hz, 1H, H-2),
6.88 (d, J = 8.6 Hz, 2H, H-30, H-50), 7.13–7.19 (m, 3H, H-300-H-500),
7.22–7.30 (m, 2H, H-200, H-600), 7.42 (d, J = 8.6 Hz, 2H, H-20, H-60),
7.70 (d, J = 16.1 Hz, 1H, H-1); 13C NMR (CDCl3, 100 MHz): d 24.3
(C-5), 31.0 (C-6), 35.7 (C-7), 40.5 (C-4), 116.2 (C-30, C-50), 123.3
(C-2), 125.8 (C-400), 126.5 (C-10), 128.3 and 128.4 (C-200, C-300, C-500,
C-600), 130.5 (C-20, C-60), 142.1 (C-100), 143.9 (C-1), 159.1 (C-40),
mL of amphotericin B in 5% CO2 incubator at 37 °C for 24 h, and
then subcultured (at 7.5 ꢃ 105 cells/plate) onto phenol red-free
DMEM supplemented with 5% charcoal stripped FBS (GIBCO Invit-
rogen) for another 24 h to remove serum steroids. Cells were
washed with PBS once and exposed to non-toxic final concentra-
tion of 10ꢁ8 M of pure compounds in the same phenol red-free
medium for 24 h. Cultures were also treated with freshly prepared
17b-estradiol (solubilized with DMSO and diluted with the med-

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A. Suksamrarn et al. / Bioorg. Med. Chem. 16 (2008) 6891–6902
ium) at the final concentration of 10ꢁ8 M or 0.01%DMSO alone
which were then used as positive and negative controls, respec-
tively. After 24 h treatment, cells were collected and total RNA
was purified using TRIzol Reagent (Pacific Science Co., Ltd). cDNA
was synthesized by SuperscriptTM II RNase Hꢁ Reverse-Transcrip-
tase kit (Invitrogen). The primers used included primers for Bcl-
xL (sense 50-CTGGTGGTTGACTTTCTCTC-30 and anti-sense 50-GAG-
TTCATTCACTACCTGTTC-30), ERb (sense 50-CGCTAGAACA CAC-
CTTACCTG-30 and anti-sense 50-TTCACCATTCCCACTTCGTA-30) and
their internal standard, HPRT (sense 50-TGTGATGAAGGAGATGG-
GAGG-30 and anti-sense 50-AAGCTTGCGACCTTGACCATCT-30). PCR
amplification was performed using an automated PTC-200Ò Peltier
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This work was supported by the research team strengthening
grant of the National Center for Genetic Engineering and Biotech-
nology. Support from the Center for Innovation in Chemistry: Post-
graduate Education and Research Program in Chemistry is
gratefully acknowledged. We are grateful to Dr. Thaya Jenjittikul,
Department of Biology, Mahidol University, Bangkok, for identify-
ing the Curcuma plant species from different parts of Thailand.
We are indebted to Suttiporn Pikulthong, Department of Chemis-
try, Faculty of Science, Mahidol University, Bangkok, for recording
the high resolution mass spectra.
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