Diarylheptanoids from Curcuma kwangsiensis and their inhibitory activity on nitric oxide production in lipopolysaccharide-activated macrophages

Article abstract of DOI:10.1016/j.bmcl.2011.07.012

Eleven diarylheptanoids (1-11) were isolated from rhizomes of Curcuma kwangsiensis, together with seven known compounds. Their structures were elucidated by 1D and 2D NMR, circular dichroism (CD), and accurate mass measurements. Inhibitory effects of the

Full text of DOI:10.1016/j.bmcl.2011.07.012

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5363–5369
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Diarylheptanoids from Curcuma kwangsiensis and their inhibitory activity
on nitric oxide production in lipopolysaccharide-activated macrophages
Jun Li a,b, Chun-Ru Liao a,b, Jun-Qi Wei a,b, Li-Xia Chen a,b, Feng Zhao c, , Feng Qiu a,b,
⇑
⇑
a Department of Natural Products Chemistry, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China
b Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China
c School of Pharmacy, Yantai University, No. 32 Road QingQuan, Laishan District, Yantai 264005, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Eleven diarylheptanoids (1–11) were isolated from rhizomes of Curcuma kwangsiensis, together with
seven known compounds. Their structures were elucidated by 1D and 2D NMR, circular dichroism
(CD), and accurate mass measurements. Inhibitory effects of the isolated compounds on nitric oxide pro-
duction in lipopolysaccaride-activated macrophages were evaluated. Compounds 1, 2, and 3 showed
Received 1 April 2011
Revised 22 June 2011
Accepted 6 July 2011
Available online 18 July 2011
strong inhibitory activity on NO production with IC50 values of 3.13, 2.81 and 2.41 lM, respectively.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Curcuma kwangsiensis
Diarylheptanoid
Inhibition of nitric oxide production
Macrophages
Curcuma kwangsiensis S.G. Lee et C.F. Ling, a perennial herba-
ceous plant of Zingiberaceae, spreads widely in the southwest of
China, including Guangxi, Sichuan, Guangdong and Yunnan Prov-
inces. The dried rhizome of C. kwangsiensis is an important crude
drug, frequently listed in prescriptions of traditional Chinese med-
icine for the treatment of stomach trouble and ‘Oketsu’,1 various
syndromes caused by the obstruction of blood circulation, such
as arthralgia, psychataxia, and dysmenorhea. Major secondary
metabolites of genus of Curcuma have been reported to be rich in
diarylheptanoids and sesquiterpenoids.2–5 These substances
showed various bioactivities, such as estrogenic,6 vasorelaxant,7
heptoprotective,8 and antifungal activities.9
Our previous paper reported the isolation and characterization
of eighteen diarylheptanoids from EtOH extracts of C. kwangsien-
sis.10 In a continued search for bioactive constituents from this
plant, eleven new diarylheptanoids (1–11), together with seven
known ones (7a, 9a, 12–16) were obtained (Fig. 1). This Letter
describes the isolation and structural elucidation of these new
compounds, and reports the inhibitory effect of the compounds
on NO production in LPS-activated macrophages.
and spectroscopic methods. Interestingly, all isolated diarylhepta-
noids possessed the 3, 5-dihydroxyheptane moiety that is different
from our previous studies on this plant.10
Compound 1,12
½
a 2D5
= +0.20 (c 0.1, MeOH), was isolated as a yel-
ꢀ
lowish oil. The HR-ESI-MS spectrum exhibited a unique [M+Na]+ ion
peak at m/z 355.1514 corresponding to the molecular formula of
C
19H24O5 (calcd. for [M+Na]+: 355.1516). The 1H NMR spectrum of
1 revealed a 1,3,4-trisubstituted aromatic ring [dH 6.67 (1H, d,
J = 8.4 Hz), 6.63 (1H, d, J = 2.0 Hz), and 6.50 (1H, dd, J = 8.4, 2.0 Hz)]
and a 1,4-disubstituted aromatic ring [dH 6.69 (2H, d, J = 8.2 Hz),
7.00 (2H, d, J = 8.2 Hz)]. The deduction was supported by its 13C
NMR data, the two oxygenated aromatic rings were evidenced from
the resonances at dC 156.5, 146.3, 144.3, 135.4, 134.6, 130.4 (ꢁ2),
120.8, 116.7, 116.4, and 116.3 (ꢁ2). 1H NMR, 13C NMR and IR spectra
of 1 were similar to those of the known compound 9a,13 which had
been obtained previously from Tacca chantrieri. However, distinctive
differences in the 13C NMR spectrum were observed between these
molecules. The chemical shifts of C-2, C-4, and C-6 of 1 were upfield
0.3, 0.6, and 0.5 ppm, respectively, while those of C-3 and C-5 were
downfield 2.4 ppm in the 13C NMR spectrum of 1 (Fig. 1). These facts
suggested that 1 is a stereoisomer of 9a. Since 9a was optically active
Dried rhizomes of C. kwangsiensis were extracted with EtOH–
H2O (7:3, v/v) and fractionated with cyclohexane, EtOAc, and
n-BuOH. From these extracts and by using combined chromato-
graphic separations, eleven new and seven known compounds were
isolated.11 Their strctures were elucidated using physicochemical
(½a 2D5
¼ þ1:7, c 0.12, MeOH) while 1 was optically inactive, which
ꢀ
suggested that the two hydroxyl groups in C-3 and C-5 were of the
syn type.14,15 Furthermore, the observed COSY and HMBC correla-
tions of 1 are in agreement with the elucidated structure of 9a. On
the basis of these data, compound 1 was established as rel-(3R,5S)-
3,5-dihydroxy-1-(3,4-dihydroxyphenyl)-7-(4-hydroxyphenyl) heptane.
⇑
Corresponding authors. Tel.: +86 24 23986463; fax: +86 24 23993994 (F.Q.).
E-mail addresses: zhaofeng74@yahoo.com.cn (F. Zhao), fengqiu2000@tom.com
Compound 2,16
½
a 2D5
ꢀ
¼ þ0:4 (c 0.1, MeOH), was isolated as a yel-
lowish oil. The HR-ESI-MS spectrum exhibited a unique [M+Na]+
(F. Qiu).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.07.012

5364
J. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5363–5369
OH OH
OR1 OR2
R1
4'
HO
R3
4'
1'
1''
4
1'
1''
3'
3''
R
3'
3R' 5S'
3R 5R
4''
OH
4''
HO
OH
5'
R2
R1
Ac
H
Ac
H
R2
H
H
R3
H
H
R4
H
H
OH
OH
H
R1
OH
OCH3
OCH3
R2
H
H
7
7a
8
8a
9
9a
11
12
13
1
2
3
H
H
Ac
H
H
H
Ac
OH
OH
OH
OH
OH
OCH3
OH
OH
O
3
OH
Ac
R1
R
3
1'
1'' 3''
3'
H
H
5S
Ac
H
Ac
H
4''
R4
4'
R2
OH
OH
R1
H
H
H
R2
R3
H
R4
H
OH
OR1 OR2
5S
4
5
5a
6
15
16
OH
OH
R3
1''
R
4
OH
3'
3''
1'
OCH3 OCH3 OCH3
OH
3S
4''
OH
4'
HO
OCH3
H
H
OH
OH
OH
H
OH
OCH3 OH
OH OH
OH
R1
Ac
H
R2
H
H
R3
OH
OH
R4
H
H
10
10a
1'
1''
3R 5S
4''
OH
4'
HO
14
Figure 1. Structures of compounds 1–16, 5a, 7a, 8a, 9a, and 10a.
ion peak at m/z 369.1671 corresponding to the molecular formula
of C20H26O5 (calcd. for [M+Na]+: 369.1672). The 1H NMR spectrum
of 2 revealed a 1,3,4-trisubstituted aromatic ring [dH 6.75 (1H, br s),
6.68 (1H, d, J = 7.7 Hz), and 6.61 (1H, br d, J = 7.7 Hz)] and a 1,4-
disubstituted aromatic ring [dH 6.69 (2H, d, J = 8.2 Hz), 6.99 (2H,
d, J = 8.2 Hz)]. The deduction was supported by its 13C NMR data,
the two oxygenated aromatic rings were evidenced from the reso-
nances at dC 156.5, 149.0, 145.6, 135.4, 134.6, 130.4 (ꢁ2), 121.9,
116.3 (ꢁ3), and 113.3. 1H NMR, 13C NMR and IR spectra of 2 were
similar to those of the known compound (3R,5R)-3,5-dihydroxy-1-
(4-hydroxy-3-methoxyphenyl)-7-(4-hydroxyphenyl)heptane.13
However, distinctive differences in the 13C NMR spectrum were
observed between these molecules. The chemical shifts of C-2,
C-4, and C-6 of 2 were upfield 0.4, 0.6, and 0.2 ppm, respec-
tively, while those of C-3 and C-5 were downfield 2.3 ppm in
the 13C NMR spectrum of 2 (Fig. 1). These facts suggested that
2 is a stereoisomer of the known compound. In addition, 2
was optically inactive, which suggested that the two hydroxyl
groups in C-3 and C-5 were also of the syn type. On the basis
of the 1D and 2D (COSY, HSQC, and HMBC) NMR data (Table
1), the structure of 2 was determined as rel-(3R,5S)-3,5-dihy-
droxy-1-(4-hydroxy-3-methoxyphenyl)-7-(4-hydroxyphenyl)-
heptane.
was supported by HSQC and HMBC spectra. The HMBC correlation
of H-1 (dH 2.52, 2.62) with C-10 (dC 134.7), C-20 (dC 104.9), and C-60
(dC 110.0), and H-7 (dH 2.57, 2.67) with C-100 (dC 134.6), C-200 and C-
600 (dC 130.4 ꢁ 2) confirmed that the 1,3,4,5-tetrasubstituted phe-
nyl moiety was connected to C-1 and the 1,4-disubstituted phenyl
moiety was connected to C-7. Additionally, 3 was optically inac-
tive, suggesting that the relative configuration at C-3 and C-5
was also of the syn type. This deduction was supported by its 1H
and 13C NMR data in the non-aromatic region (Table 1). On the
basis of the 1D and 2D (COSY, HSQC, and HMBC) NMR data, the
structure of 3 was determined as rel-(3R,5S)-3,5-dihydroxy-1-(3-
methoxy-4,5-dihydroxyphenyl)-7-(4-hydroxyphenyl)heptane.
Compound 4,16 was obtained as a yellowish oil. The molecular
formula of 4 was determined to be C19H22O3 on the basis of HR-
ESI-MS (m/z 316.1904 [M+NH4]+) and NMR data (Table 2). Analysis
of the 1D and 2D (HMQC, HMBC) NMR spectroscopic data, the pla-
nar structure of 4 was assigned as 5-hydroxy-1-(4-hydroxy-
phenyl)-7-phenyl-3-heptanone.2 The absolute configuration of
the hydroxyl group at C-5 was determined by application of the
circular dichroism (CD) spectrum. The absolute configuration of
hydroxyl group in analogous compounds showing negative Cotton
effect associated with the carbonyl n ? p⁄ transition in the region
of about 300 nm was determined to be 5S, and those showing po-
sitive Cotton effect was determined as 5R using CD spectra in
Compound 3,16 a yellowish oil, showed the molecular formula
C
20H26O6, as determined by HR-ESI-MS m/z 363.1802 [M+H]+
CHCl3.17 Thus, the negative Cotton effect at 297.0 nm (
D
e
ꢂ0.050,
(calcd. for C20H27O6, 363.1802) and NMR data. The 1H NMR spec-
trum of 3 revealed a 1,3,4,5-tetrasubstituted benzene ring [dH
6.35 (2H, d, J = 2.2 Hz, H-20 and H-60)] and a 1,4-disubstituted aro-
matic protons [dH 6.71 (2H, d, J = 8.5 Hz), 7.03 (2H, d, J = 8.5 Hz)].
The 13C NMR spectrum displayed 20 signals consistent with seven
methylene [dC 32.0, 32.8, 40.8, 41.0, 45.0, and 71.0 (ꢁ2)], one meth-
oxyl (dC 56.7), and 12 aromatic ring carbons, and suggesting a
diarylheptanoid structure. The 1H NMR spectrum exhibited some
similarities between 3 and 2, except that the H-50 signal of aro-
matic ring was absent in 3, being replaced by an OH group. Fur-
thermore, the 13C NMR spectrum showed a significant downfield
shift for C-50 (dC 146.6) compared to that of 2. This deduction
in CHCl3) in the CD spectrum suggested the S configuration at
C-5 in compound 4, and the structure of 4 was determined (5S)-
5-hydroxy-1-(4-hydroxyphenyl)-7-phenyl-3-heptanone.
Compound 5,18 was obtained as a yellowish oil. The molecular
formula of 5 was determined to be C19H22O5 on the basis of HR-
ESI-MS (m/z 353.1358 [M+Na]+) and NMR data (Table 2). Analysis
of the 1D and 2D (HMQC, HMBC) NMR spectroscopic data identi-
fied the planar structure of 5 as 5-hydroxy-1-(4-hydroxyphenyl)-
7-(3,4-dihydroxyphenyl)-3-heptanone.19 Due to being less soluble
in CHCl3, 5 was methylated to give the corresponding trimethyl
derivative 5a (Fig. 1), with trimethylsilyldiazomethane in MeOH.20
The CD spectrum of 5a in CHCl3 exhibited a negative Cotton effect

J. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5363–5369
5365
Table 1
1H and 13C NMR spectroscopic data for compounds 1–3 in CD3OD
No.
1
2
3
1Ha
13Cb
32.2
1Ha
13Cb
32.5
1Ha
13Cb
1a
1b
2
3
4
2.61 (1H, m)
2.51 (1H, m)
1.68 (2H, m)c
3.79 (1H, m)
1.60 (2H, m)
3.79 (1H, m)
1.68 (2H, m)c
2.64 (1H, m)
2.54 (1H, m)
2.65 (1H, m)c
2.56 (1H, m,)c
1.70 (2H, m)c
3.75 (1H, m)
1.61 (2H, m)
3.79 (1H, m)
1.70 (2H, m)c
2.64 (1H, m)c
2.55 (1H, m)c
2.62 (1H, m)
2.52 (1H, m)
1.74 (2H, m)c
3.78 (1H, m)
1.63 (2H, m)
3.78 (1H, m)
1.67 (2H, m)c
2.67 (1H, m)
2.57 (1H, m)
32.8
41.0e
71.1
45.0
71.1
40.9e
32.0
40.9e
71.0
45.0
71.0
41.0e
32.0
41.0e
71.0
45.0
71.0
40.8e
32.0
5
6
7a
7b
10
135.4
116.7
146.3
144.3
116.4
120.8
134.6
130.4
116.3
156.5
116.3
130.4
135.4
113.3
149.0
145.6
116.3
121.9
134.6
130.4
116.3
156.5
116.3
130.4
56.5
134.7
104.9
149.7
133.2
146.6
110.0
134.6
130.4
116.2
156.5
116.2
130.4
56.7
20
6.63 (1H, d, 2.0)
6.75 (1H, br s)
6.35 (1H, d, 2.2)
30
40
50
6.67 (1H, d, 8.4)
6.50 (1H, dd, 8.4, 2.0)
6.68 (1H, d, 7.7)
6.61 (1H, br d, 7.7)
60
6.35 (1H, d, 2.2)
100
200
300
400
500
600
30-OCH3
7.00 (1H, d, 8.2)d
6.69 (1H, d, 8.2)d
6.99 (1H, d, 8.2)d
6.69 (1H, d, 8.2)d
7.03 (1H, d, 8.5)d
6.71 (1H, d, 8.5)d
6.69 (1H, d, 8.2)d
7.00 (1H, d, 8.2)d
6.69 (1H, d, 8.2)d
6.99 (1H, d, 8.2)d
3.81 (3H, s)
6.71 (1H, d, 8.5)d
7.03 (1H, d, 8.5)d
3.82 (3H, s)
a
b
c
600 MHz for 1H NMR; the coupling constants (J) are in parentheses and reported in Hz; chemical shifts are given in ppm.
150 MHz for 13C NMR.
Overlapped signal.
Pseudo-doublet.
Assignments may be interchangeable within the same column.
d
e
Table 2
1H and 13C NMR spectroscopic data for compounds 4–6 in CD3OD
No.
4
5
6
1Ha
13Cb
1Ha
13Cc
1Ha
13Cc
1
2
3
4a
4b
5
2.74 (2H, m)d
2.72 (2H, m)
29.9
46.6
211.9
51.4
2.74 (2H, m)d
2.73 (2H, m)d
29.9
46.5
212.2
51.4
2.72 (2H, m)d
2.71 (2H, m)d
30.4
46.5
212.3
51.4
2.52 (1H, dd,4.3,15.9)
2.57 (1H, m)
4.02 (1H, m)
2.53 (1H, m)
2.57 (1H, m)
4.01 (1H, m)
1.66 (2H, m)
2.51 (1H, m)
2.61 (1H, m)
2.48 (1H, m)
2.59 (1H, m)
3.97 (1H, m)
1.62 (2H, m)
2.56 (1H, m)
2.60 (1H, m)
68.4
40.4
33.0
68.5
40.6
32.3
68.4
40.6
32.0
6
1.70 (2H, m)
7a
7b
10
2.60 (1H, m)
2.73 (1H, m)d
133.4
130.4
116.3
156.9
116.3
130.4
143.5
129.6
129.5
126.9
129.5
129.6
133.4
130.4
116.3
156.7
116.3
130.4
135.1
116.7
146.3
144.4
116.5
120.8
134.3
113.2
149.0
145.8
116.3
121.8
134.2
130.5
116.3
156.4
116.3
130.5
56.5
20
6.98 (1H, d, 8.2)e
6.67 (1H, d, 8.2)e
7.00 (1H, d, 8.4)
6.68 (1H, d, 8.4)
6.62 (1H, d, 1.9)
30
40
50
6.67 (1H, d, 8.2)e
6.98 (1H, d, 8.2)e
6.68 (1H, d, 8.4)
7.00 (1H, d, 8.4)
6.66 (1H, d, 8.1)
60
6.50 (1H, dd, 1.9,8.1)
6.95 (1H, d, 8.4)e
100
200
300
400
500
600
30-OCH3
7.17 (1H, d, 7.4)e
7.24 (1H, t, 7.4)
7.13 (1H, t, 7.4)
7.24 (1H, t, 7.4)
7.17 (1H, d, 7.4)e
6.71 (1H, d, 1.9)
e
6.66 (1H, d, 8.4)
6.66 (1H, d, 8.0)
6.57 (1H, dd, 1.9,8.0)
6.66 (1H, d, 8.4)e
6.95 (1H, d, 8.4)e
3.77 (3H, s)
a
b
c
600 MHz for 1H NMR; the coupling constants (J) are in parentheses and reported in Hz; chemical shifts are given in ppm.
75 MHz for 13C NMR.
150 MHz for 13C NMR.
Overlapped signal.
Pseudo-doublet.
d
e
(292.6 nm), similar to 4. The structure of
5
were therefore
droxy-1-(4-hydroxy-3-methoxyphenyl)-7-(4-hydroxyphenyl)-3-
formulated as (5S)-5-hydroxy-1-(4-hydroxyphenyl)-7-(3,4-dihy-
droxyphenyl)-3-heptanone.
heptanone.22 The CD spectrum of 6 in CHCl3 exhibited a negative
Cotton effect (296.8 nm). The structure of
6 were therefore
Compound 6,21 was obtained as a yellowish oil. The molecular
formula of 6 was determined to be C20H24O5 on the basis of
HR-ESI-MS (m/z 367.1523 [M+Na]+) and NMR data. Analysis of
the NMR spectra, the planar structure of 6 was assigned as 5-hy-
formulated as (5S)-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-
7-(4-hydroxyphenyl)-3-heptanone.
Compound 7,23 was obtained as a colorless oil and gave the
molecular formula C21H26O5, on the basis of the HR-ESI-MS m/z

5366
J. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5363–5369
381.1675 [M+Na]+ (calcd for C21H26O5Na, 381.1672). The 1H and
13C NMR data of 7 in the aromatic regions were similar with those
of the known compound 7a (Fig. 1),13 suggesting that 7 also pos-
sessed two 1,4-disubstituted benzene rings. However, distinctive
differences in the 13C NMR spectrum were observed between these
molecules. The chemical shifts of C-2, and C-4 of 7 were upfield 3.3
and 2.6 ppm, respectively, while that of C-3 was downfield
4.3 ppm; meanwhile, an acetyl carbon signal at dC 21.3 and 173.3
was present in the 13C NMR spectrum of 7. Notably, the HMBC cor-
relations of methyl protons dH 2.05 with the ester carbonyl carbon
(dC 173.3) and C-3 (dC 73.0), and H-3 (dH 5.13) with the ester car-
bonyl carbon (dC 173.3) were observed, respectively. These facts
suggested that 7 was the C-3 acetylated derivative of 7a.
Compounds 10 and 11,25 were obtained as a mixture, in a yel-
lowish oil form, and could not be further separated in achiral sta-
tionary phase. The HR-ESI-MS spectrum exhibited
a unique
[M+Na]+ ion peak at m/z 397.1618 corresponding to the molecular
formula C21H26O6 (calcd. for C21H26O6Na, 397.1622). The 1D and
2D (HMQC, HMBC) NMR spectra of the mixture demonstrated
two sets of similar resonances due to diarylheptanoid moieties,
but with not identical intensities in the 1H NMR spectrum (a ratio
of 10 and 11, about 1:2). Detailed analysis of the 1H NMR spectrum
in the aromatic region revealed that two sets of 1,3,4-trisubstituted
aromatic protons [dH 6.65 (1H, d, J = 8.2 Hz), 6.48 (1H, dd, J = 8.2,
2.0 Hz), and 6.60 (1H, d, J = 2.0 Hz) weakly, and dH 6.64 (1H, d,
J = 8.2 Hz), 6.46 (1H, dd, J = 8.2, 2.0 Hz), and 6.58 (1H, d,
J = 2.0 Hz) intensely, respectively] and two sets of 1,4-disubstituted
aromatic protons [dH 6.66 (2H, d, J = 8.4 Hz) and 6.96 (2H, d,
J = 8.4 Hz) weakly, and at dH 6.67 (2H, d, J = 8.4 Hz) and 6.98 (2H,
d, J = 8.4 Hz) intensely, respectively]. Careful investigation of the
13C NMR data of 10 and 11 in the nonaromatic region revealed that
10 and 11 possessed also the same monoacetylated 3,5-dihydroxy-
heptane moiety as 7 and 8 in comparison with those of 7 and 8.
In the HMBC spectrum, long range correlations were observed
between the proton and carbon pairs of 10 and 11, example, HMBC
correlations from H-1 to C-2, C-3, C-10, C-20, and C-60, from H-7 to
C-5, C-6, C-100, C-200, and C-600, and from H-3 to C-1, C-2, C-4, C-5,
and ester carbonyl carbon, respectively, indicating 1,3,4-trisubsti-
tuted phenyl moiety was connected to C-1, 1,4-disubstituted phe-
nyl moiety was connected to C-7, and the acetoxyl group was
connected to C-3, respectively. Therefore, all the spectroscopic data
disclosed that 10 and 11 are a pair of stereoisomers, possessing the
same planar structure 3-acetoxy-5-hydroxy-1-(3,4-dihydroxy-
phenyl)-7-(4-hydroxyphenyl)heptane. The mixture was hydro-
lyzed with HCl/MeOH to give deacetyl mixture (10a and 11a),
however, whose 1H and 13C NMR spectra displayed only a set of
resonances that were identical to those of 9a, especially, at C-3
and C-5 (dC 68.2 in 9a and deacetyl mixture, unlike dC 71.1 in 1)
in the 13C NMR spectrum. These facts suggested that the two hy-
droxyl groups at C-3 and C-5 in 10 and 11 were of the anti type.13,14
In additon, the deacetyl hydrolyzate prepared from mixture of 10
and 11,24 exhibited positive optical rotation in MeOH, and 11a de-
rived from 11 should predominate in the enantiomeric mixture of
10a and 11a, and consequently, 11a rather than 10a is the same as
In order to determine the absolute configuration at C-3 and C-5,
compound 7 was deacetylated with HCl/MeOH gave the corre-
sponding 3-deacetyl derivative 7a,24 by comparing their 1H and
13C NMR spectroscopic data and optical rotation with the reported
in the literature.13 Therefore, compound 7 was assigned as (3R,5R)-
3-acetoxy-5-hydroxy-1,7-bis(4-hydroxyphenyl)heptane.
Compound 8,25 was obtained as a yellowish oil. The HR-ESI-MS
of 8 showed a [M+Na]+ ion peak at m/z 413.1578 (calcd for
C21H26O7Na, 413.1571), consistent with the molecular formula
C21H26O7. The 1H NMR spectrum of 8 showed the presence of
two 1,3,4-trisubstituted benzene rings [dH 6.67 (1H, d, J = 8.0 Hz),
6.49 (1H, dd, J = 8.0, 3.1 Hz), and 6.59 (1H, d, J = 3.1 Hz); dH 6.66
(1H, d, J = 8.0 Hz), 6.46 (1H, dd, J = 8.0, 3.1 Hz), and 6.61 (1H, d,
J = 3.1 Hz)]. The 13C NMR spectrum displayed 21 signals consistent
with seven methylene (dC 32.1, 32.4, 38.1, 41.1, 43.1, 68.2 and
73.0), one acetoxy (dC 21.2 and 173.2), and 12 aromatic ring car-
bons, and suggesting an acetylated diarylheptanoid structure. This
deduction was supported by COSY, HSQC and HMBC spectra (Table
3). The attachment of 3,4-dihydroxyphenyl moieties at C-1 and
C-7, respectively, were confirmed by the HMBC correlations of
H-1 with C-10, C-20, and C-60, H-2 with C-10, H-20/H-60 with C-1,
H-7 with C-100, C-200, and C-600, H-6 with C-100, H-200/H-600 with
C-7. Meanwhile, 8 was hydrolyzed with HCl/MeOH to give 8a
(Fig. 1),24 by comparing their 1H and 13C NMR spectroscopic
data and optical rotation with the reported in the literature.13
Therefore, 8 was assigned as (3R,5R)-3-acetoxy-5-hydroxy-1,7-
bis(3,4-dihydroxyphenyl)heptane.
Compound 9,25 was obtained as a yellowish oil. The molecular
formula C23H28O7 ([M+Na]+ 439.1731, calcd for 439.1727), was
determined by HR-ESI-MS. The 1H NMR spectrum of 9 displayed
a 1,3,4-trisubstituted benzene ring [dH 6.68 (1H, d, J = 8.0 Hz),
6.61 (1H, d, J = 2.0 Hz), and 6.48 (1H, dd, J = 8.0, 2.0 Hz)] and a
1,4-disubstituted benzene ring [dH 6.70 (2H, d, J = 8.5 Hz) and
6.99 (2H, d, J = 8.5 Hz)]. The 13C NMR spectrum showed the pres-
ence of eleven non-aromatic carbons in 9; five methylenes (dC
31.9, 32.1, 37.8, 37.9, and 39.6), two acetoxy [dC 21.2 (ꢁ2),
172.88, and 172.90], suggesting a diarylheptanoid structure. The
13C NMR data of 9 were similar to that of the known compound
9a (Fig. 1), except that two acetyl units was present in 9. Mean-
while, the chemical shifts of C-2, C-4, and C-6 of 9 were upfield
3.5, 6.0, and 3.5 ppm, respectively, while that of C-3 and C-5 were
downfield 2.9 ppm in the 13C NMR data of 9. These facts suggested
that 9 is a diacetylated derivative of 9a. Deacetylation of 9 with
HCl/MeOH gave the corresponding 3, 5-dihydroxyl derivative 9a
which was identified as (3R,5R)-dihydroxy-1-(3,4-dihydroxy-
phenyl)-7-(4-hydroxyphenyl)heptane,24 by comparing their 1H
and 13C NMR spectroscopic data and optical rotation with the re-
ported in the literature.13
9a according to the optical rotation (½a D25
ꢀ
¼ þ1:7, c 0.12, MeOH).13
Therefore, the pair of diarylheptanoid enantiomers were identified
as (3S,5S)-3-acetoxy-5-hydroxy-1-(3,4-dihydroxyphenyl)-7-(4-
hydroxyphenyl)heptane (10) and (3R,5R)-3-acetoxy-5-hydroxy-1-
(3,4-dihydroxyphenyl)-7-(4-hydroxyphenyl)heptane
(11),
respectively.(HPLC chromatogram of the enantioseparation
of mixtures of 10 and 11 see Supplementary data.)
In addition to eleven new diarylheptanoids (1–11), seven
known ones, (3R,5R)-3,5-dihydroxy-1,7-bis(4-hydroxyphenyl
)heptane (7a),13 (3R,5R)-3,5-dihydroxy-1-(3,4-dihydroxyphen
yl)-7-(4-hydroxyphenyl)heptane (9a),13 (3R,5R)-3,5-dihy-
droxy-1-(4-hydroxy-3-methoxyphenyl)-7-(3,4-dihydroxyphenyl)-
heptane (12),13 (3 R,5R)-3,5-diacetoxy-1,7-bis(3,4-dihydroxyphenyl)-
heptane (13),22 (3R,5S)-3,5-dihydroxy-1,7-bis(4-hydroxyphenyl)-
heptane (14),26 (5S)-5-hydroxy-1,7-bis(4-hydroxyphenyl)heptan-
3-one (15),27 and (5S)-5-hydroxy-1-(4-hydroxy-3-methoxy-
phenyl)-7-(3,4-dihydroxyphenyl)heptan-3-one (16),15 were
isolated and identified by comparison of their spectroscopic data
with those reported in the literature.
All isolated compounds were evaluated for their inhibitory ef-
fects on NO production induced by LPS in macrophages (Table 4).
Cell viability was determined by the MTT method to find whether
inhibition of NO production was due to cytotoxicity of the test
compounds.28 As shown in Table 4, indomethacin (IC50 12.96
Finally, the assignment of all protons and carbons were
made unambiguously by COSY, HMQC and HMBC NMR exper-
iments (Table 3). On the basis of the above evidence, 9 was
determined as (3R,5R)-3,5-diacetoxy-1-(3,4-dihydroxyphenyl)
-7-(4-hydroxyphenyl)heptane.
1.16 lM) and hydrocortisone (IC50 40.64 3.22 lM) were used as

Table 3
1H and 13C NMR spectroscopic data for compounds 7–11 in CD3OD
No.
7
8
9
10
11
1Ha
13Cb
1Ha
13Cb
1Ha
13Cb
1Ha
13Cb
1Ha
13Cb
1
2
3
4a
4b
5
2.59 (2H, m)g
1.88 (2H, m)
5.13 (1H, m)
1.78 (1H, ddd, 13.1, 10.3, 3.0)
1.68 (1H, ddd, 13.1, 9.2, 3.0)
3.58 (1H, m)
32.2
38.1
73.0
43.1
2.48 (2H, m)g
1.81 (2H, m)
5.08 (1H, m)
1.71 (1H, m)
1.59 (1H, m)
3.34 (1H, m)
1.64 (2H, m)
32.1
38.1
73.0
43.1
2.46 (2H, t, 7.9)
1.78 (2H, m)g
4.93 (1H, m)
1.82 (2H, m)
32.1
37.8c
71.6d
39.6
2.45 (2H, m)g
1.80 (2H, m)
5.06 (1H, m)
1.71 (1H, m)
1.58 (1H, m)
3.51 (1H, m)
1.64 (2H, m)
2.62 (1H, m)
2.49 (1H, m)g
31.9
38.2
73.0
43.1
2.45 (2H, m)g
1.80 (2H, m)
5.06 (1H, m)
1.71 (1H, m)
1.58 (1H, m)
3.51 (1H, m)
1.64 (2H, m)
2.62 (1H, m)
2.49 (1H, m)g
32.1c
38.1
73.1
43.1
68.2
41.2
31.9
68.2
41.1
32.4
4.93 (1H, m)
71.7d
37.9c
31.9
68.2
41.1
32.4
68.2
41.2
32.2c
6
1.71 (2H, m)
1.78 (2H, m)g
2.51 (2H, t, 8.0)
7a
7b
10
2.70 (1H, m)
2.58 (1H, ddd, 14.1, 8.8, 5.8)
2.60 (1H, m)g
2.48 (1H, m)g
133.9
134.7
116.6c
146.2
144.3d
116.40e
120.7
135.2
116.7c
146.2
144.4d
116.44e
120.8
21.2
134.5
116.6
146.3
144.5
116.5
120.7
133.7
130.4
116.3
156.7
116.3
130.4
21.2
135.2
116.4
146.3
144.3
116.7
120.8
133.9
130.4
116.3
156.6
116.3
130.4
21.3
134.7
116.5
146.3
144.5
116.6
120.7
134.4
130.5
116.2
156.5
116.2
130.5
21.3
20
7.06 (1H, d, 8.4)f
6.76 (1H, d, 8.4)f
130.4c
116.2d
156.5e
116.2d
6.59 (1H, d, 3.1)
6.61 (1H, d, 2.0)
6.60 (1H, d, 2.0)
6.58 (1H, d, 2.0)
30
40
50
6.76 (1H, d, 8.4)f
7.06 (1H, d, 8.4)
6.67 (1H, d, 8.0)
6.49 (1H, dd, 8.0, 3.1)
6.68 (1H, d, 8.0)
6.48 (1H, dd, 8.0, 2.0)
6.65 (1H, d, 8.2)
6.48 (1H, dd, 8.2, 2.0)
6.64 (1H, d, 8.2)
6.46 (1H, dd, 8.2, 2.0)
f
c
60
130.4
134.4
130.5c
116.3d
156.6e
116.3d
130.5c
21.3
100
200
300
400
500
600
3-OAc
7.04 (1H, d, 8.5)f
6.75 (1H, d, 8.5)f
6.61 (1H, d, 3.1)
6.99 (1H, d, 8.5)f
6.70 (1H, d, 8.5)f
6.96 (1H, d, 8.4)f
6.66 (1H, d, 8.4)f
6.98 (1H, d, 8.4)f
6.67 (1H, d, 8.4)f
6.75 (1H, d, 8.5)f
7.04 (1H, d, 8.5)f
2.05 (3H, s)
6.66 (1H, d, 8.0)
6.46 (1H, dd, 8.0, 3.1)
1.99 (3H, s)
6.70 (1H, d, 8.5)f
6.99 (1H, d, 8.5)f
1.978 (3H, s)c
6.66 (1H, d, 8.4)f
6.96 (1H, d, 8.4)f
1.972 (3H, s)
6.67 (1H, d, 8.4)f
6.98 (1H, d, 8.4)f
1.970 (3H, s)
173.3
173.2
172.88e
21.2
173.3
173.3
5-OAc
1.981 (3H, S)c
172.90e
a
600 MHz for 1H NMR; the coupling constants (J) are in parentheses and reported in Hz; chemical shifts are given in ppm.
b
150 MHz for 13C NMR.
c,d,e
Assignments may be interchangeable within the same column.
Pseudo-doublet.
Overlapped signal.
f
g

5368
J. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5363–5369
9:1)], and subfaction E52 was separated by RP-HPLC with MeOH–H2O (1:1) to
Table 4
afford compound 5 (tR 19.8 min, 21.5 mg), compound 15 (tR 27.1 min, 16.5 mg),
and compound 4 (tR 27.2 min, 10.6 mg). Fraction E8 (5.2 g) was subjected to a
Sephadex LH-20 column [CHCl3–MeOH (1:1)] to give four subfractions (E81–
E84). Fraction E82 was purified by RP-HPLC [MeOH–H2O (1:1)] to obtain
compound 16 (tR 23.3 min, 22.5 mg) and compound 6 (tR 28.2 min, 19.7 mg).
Fraction E83 was purified by RP-HPLC [MeOH–H2O (4:6)] to obtain compound
1 (tR 31.3 min, 36.5 mg), compound 12 (tR 35.6 min, 26.1 mg) and compound 2
(tR 40.2 min, 12.7 mg). The n-BuOH extract (82 g) was subjected to silica gel CC
with CHCl3–MeOH (100:2 to 0:100) to obtain eight fractions (B1–B8). Fraction
B3 (15.8 g) was then subjected to Sephadex LH-20 (CHCl3–MeOH, 1:1) and
Inhibitory effect of compounds isolated from Curcuma kwangsiensis on NO production
induced by LPS in macrophagesa
Compound
IC50 SD (
l
M)
Compound
IC50 SD (
>100
11.49 1.03
25.79 2.15
7.98 0.66
>100
46.37 4.22
9.37 0.89
12.96 1.16
40.64 3.22
lM)
1c
3.13 0.33
2.81 0.26
2.41 0.18
75.71 6.46
82.84 6.83
37.51 2.78
32.00 3.28
35.50 3.41
10.67 1.02
9.52 0.87
7a
9a
2
3c
12
4
5
6
13c
14
15
separated by RP-HPLC with MeOH–H2O (7:13) to afford compound
7 (tR
7
16c
35.1 min, 11.8 mg), compound 8 (tR 38.6 min, 13.4 mg), compound 7a (tR
41.5 min, 28.1 mg), and compound 14 (tR 45.3 min, 33.2 mg). Fraction B4
(12.4 g) was purified by ODS open CC [MeOH–H2O (1:4 to 9:1)], and fraction
B43 by RP-HPLC with MeOH–H2O (3:7) to afford the mixture of 10 and 11 (tR
34.2 min, 40.6 mg), compound 13 (tR 39.1 min, 14.2 mg) and compound 9 (tR
42.5 min, 35.2 mg).
8c
Indomethacinb
Hydrocortisoneb
9
10,11c
a
NO concentration of control group: 3.24 0.21
lM. NO concentration of LPS-
12. rel-(3R,5S)-3,5-Dihydroxy-1-(3,4-dihydroxyphenyl)-7-(4-hydroxyphenyl)-
treated group: 33.46 2.13
l
M.
heptane (1): yellowish oil; ½a D25
ꢀ
: +0.2 (c 0.1, MeOH); UV kmax (MeOH) nm (log
b
Positive control.
): 280.6 (3.65); IR (KBr) m
max cm-1: 3319, 2941, 1610, 1514, 1449, 1367, 1239,
c
e
Cytotoxicity (100 lM). Other compounds show no cytotoxicity.
1112, 826; for 1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz),
spectroscopic data, see Table 1; HR-ESI-MS m/z 355.1514 (calcd. for
C
19H24O5Na, 355.1516).
13. Yokosuka, A.; Mimaki, Y.; Sakagami, H.; Sashida, Y. J. Nat. Prod. 2002, 65, 283.
14. Kikuzaki, H.; Usuguchi, J.; Nakatani, N. Chem. Pharm. Bull. 1991, 39, 120.
15. Ma, J.; Jin, X.; Yang, L.; Liu, Z.-L. Phytochemistry 2004, 65, 1137.
positive controls. Compounds 1, 2, and 3 showed strong inhibitory
activity on NO production with IC50 values of 3.13, 2.81 and
2.41 lM, respectively. From the structural features of the diaryl-
heptanoid skeleton, it was found that the stereochemistry of
C-3 and C-5 could influence the inhibitory effects on NO produc-
tion, and these compounds with two syn type of hydroxyl
groups in C-3 and C-5 exhibited conspicuously inhibitory activity
(e.g., 1, 2, 3).
16. rel-(3R,5S)-3,5-Dihydroxy-1-(4-hydroxy-3-methoxyphenyl)-7-(4-hydroxy-
phenyl)heptane (2): yellowish oil; ½a D25
ꢀ
: +0.4 (c 0.1, MeOH); UV kmax (MeOH)
nm (log e
): 282.4 (3.85); IR (KBr) mmax cm-1: 3331, 2940, 1604, 1517, 1451,
1369, 1278, 1152, 1059, 1032, 816; for 1H NMR (CD3OD, 600 MHz) and 13C
NMR (CD3OD, 150 MHz), spectroscopic data, see Table 1; HR-ESI-MS m/z
369.1671 (calcd. for C20H26O5Na, 369.1672). rel-(3R,5S)-3,5-dihydroxy-1-(3-
methoxy-4,5-dihydroxyphenyl)-7-(4-hydroxyphenyl)heptane (3): yellowish
oil; ½a 2D5
ꢀ
: 0 (c 0.1, MeOH); UV kmax (MeOH) nm (log e): 279.2 (3.71); IR (KBr)
mmax cmꢂ1: 3234, 2938, 1677, 1602, 1516, 1452, 1272, 1202, 1128, 1033, 800;
for 1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz), spectroscopic
data, see Table 1; HR-ESI-MS m/z 363.1802 (calcd. for C20H27O6, 363.1802).
(5S)-5-hydroxy-1-(4-hydroxyphenyl)-7-phenyl-3-heptanone (4): yellowish
Acknowledgements
oil; ½a 2D5
ꢀ
: +15.3 (c 0.1, MeOH); UV kmax (MeOH) nm (log e): 278.0 (3.03); IR
The research was supported by National Key Science and
Technology Special Project (2009ZX09301-012) and Program for
Liaoning Excellent Talents in University (LR201037).
(KBr) m
max cmꢂ1: 3389, 2925, 1712, 1614, 1513, 1449, 1403, 1386, 1211, 1116,
1040, 974, 753; CD (CHCl3) kmax
(
D
e
): 297.0 (ꢂ0.050); for 1H NMR (CD3OD,
600 MHz) and 13C NMR (CD3OD, 75 MHz), spectroscopic data, see Table 2; HR-
ESI-MS m/z 316.1904 (calcd. for C19H26O3N, 316.1907).
17. Itokawa, H.; Morita, H.; Midorikawa, I.; Aiyama, R.; Morita, M. Chem. Pharm.
Bull. 1985, 33, 4889.
Supplementary data
18. (5S)-5-Hydroxy-1-(4-hydroxyphenyl)-7-(3,4-dihydroxyphenyl)-3-heptanone
(5): yellowish oil; ½a D25
ꢀ
: +11.2 (c 0.1, MeOH); UV kmax (MeOH) nm (log e): 282.0
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.07.012.
(3.65); IR (KBr) mmax cmꢂ1: 3439, 2943, 1697, 1614, 1516, 1447, 1400, 1370,
1230, 1173, 1106, 974, 823; CD (CHCl3) kmax
(D
e
): 292.6 (ꢂ0.048); for 1H NMR
(CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz), spectroscopic data, see
Table 2; HR-ESI-MS m/z 353.1358 (calcd. for C19H22O5Na, 353.1359).
19. Kato, N.; Hamada, Y.; Shioiri, T. Chem. Pharm. Bull. 1984, 32, 3323.
References and notes
1. Sasaki, Y.; Goto, H.; Tohda, C.; Hatanaka, F.; Shibahara, N.; Shimada, Y.;
Terasawa, K.; Komatsu, K. Biol. Pharm. Bull. 2003, 26, 1135.
2. Suksamrarn, A.; Ponglikitmongkol, M.; Wongkrajang, K.; Chindaduang, A.;
Kittidanairak, S.; Jankam, A.; Yingyongnarongkul, B.-e.; Kittipanumat, N.;
Chokchaisiri, R.; Khetkam, P.; Piyachaturawat, P. Bioorg. Med. Chem. 2008, 16,
6891.
3. Kaewamatawong, R.; Boonchoong, P.; Teerawatanasuk, N. Phytochem. Lett.
2009, 2, 19.
4. Matsuda, H.; Morikawa, T.; Ninomiya, K.; Yoshikawa, M. Bioorg. Med. Chem.
2001, 9, 909.
5. Ohshiro, M.; Kuroyanagi, M.; Ueno, A. Phytochemistry 1990, 29, 2201.
6. Winuthayanon, W.; Suksen, K.; Boonchird, C.; Chuncharunee, A.;
Ponglikitmongkol, M.; Suksamrarn, A.; Piyachaturawat, P. J. Agric. Food Chem.
2009, 57, 840.
7. Matsuda, H.; Morikawa, T.; Ninomiya, K.; Yoshikawa, M. Tetrahedron 2001, 57,
8443.
20. Methylation of Compound 5: The mixture of 5 (4 mg) and trimethyl-
silyldiazomethane (2.0 M solution in n-hexane) (0.8 mL) in MeOH (1.0 mL)
were stirred at room temperature overnight. After the excess of
trimethylsilyldiazomethane was decomposed with AcOH, the reaction
mixture was evaporated to dryness. The resulting residue was purified by
preparative TLC (cyclohexane–acetone, 4:1, Rf = 0.42) and yielded 5a (3.4 mg).
21. (5S)-5-Hydroxy-1-(4-hydroxy-3-methoxyphenyl)-7-(4-hydroxyphenyl)-3-
heptanone (6): yellowish oil; ½a D25
ꢀ
: +12.6 (c 0.1, MeOH); UV kmax (MeOH) nm
(log e
): 282.3 (3.76); IR (KBr) mmax cmꢂ1: 3414, 2938, 1694, 1605, 1515, 1451,
1371, 1273, 1152, 1117, 1033, 958, 815; CD (CHCl3) kmax (De): 296.8 (ꢂ0.047);
for 1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz), spectroscopic
data, see Table 2; HR-ESI-MS m/z 367.1523 (calcd. for C20H24O5Na, 367.1516).
22. Kikuzaki, H.; Kobayashi, M.; Nakatani, N. Phytochemistry 1991, 30, 3647.
23. (3R,5R)-3-Acetoxy-5-hydroxy-1,7-bis(4-hydroxyphenyl)heptane (7):
yellowish oil; ½a D25
ꢀ
: +14.3 (c 0.1, MeOH); UV kmax (MeOH) nm (log e): 279.0
(3.77); IR (KBr) mmax cmꢂ1: 3337, 2941, 1708, 1613, 1513, 1450, 1376, 1262,
1030, 827; for 1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz),
spectroscopic data, see Table 3; HR-ESI-MS m/z 381.1675 (calcd. for
8. Song, E.-K.; Cho, H.; Kim, J.-S.; Kim, N.-Y.; An, N.-H.; Kim, J.-A.; Lee, S.-H.; Kim,
Y.-C. Planta Med. 2001, 67, 876.
9. Cho, J.-Y.; Choi, G. J.; Lee, S.-W.; Jang, K. S.; Lim, H. K.; Lim, C. H.; Lee, S. O.; Cho,
K. Y.; Kim, J.-C. J. Microbiol. Biotechnol. 2006, 16, 280.
C21H26O5Na, 381.1672).
24. Hydrolysis of 7, 8, 9, and mixtures of 10 and 11: To a solution of 7 (6.0 mg) in
MeOH (1.5 mL), a drop of analytical grade HCl was added. The resulting
mixture was stirred at room temperature overnight and dried under vacuum.
The residue was purified by preparative TLC (CHCl3–MeOH, 5:1, Rf = 0.42) and
yielded 7a (4.3 mg). Following the above procedure, the corresponding
deacetyl derivatives (8a, 9a, and mixtures of 10a and 11a) were prepared
from 8, 9, and mixtures of 10 and 11, respectively.
10. Li, J.; Zhao, F.; Li, M. Z.; Chen, L. X.; Qiu, F. J. Nat. Prod. 2010, 73, 1667.
11. The rhizomes of C. kwangsiensis (10 kg) were cut into approximately 2 cm
pieces and repeatedly (ꢁ3) extracted with EtOH–H2O (7:3, v/v, 100 L) for 2 h.
The combined extracts were concentrated in vacuo, suspended in H2O, and
partitioned successively with cyclohexane, EtOAc, and n-BuOH. The EtOAc
extract (65 g) was subjected to silica gel CC with CHCl3–MeOH (100:1 to 0:100)
to obtain nine fractions (E1–E9), which were combined according to TLC
analysis. Fraction E3 (6 g) was chromatographed on a Sephadex LH-20 column
with CHCl3–MeOH (1:1) to give three subfractions (E31–E33). Fraction E32
(2.1 g) was purified by ODS open column chromatography [MeOH–H2O (1:9 to
9:1)], and resolution of fraction E323 by RP-HPLC with MeOH–H2O (3:2) to
afford compound 9a (tR 26.8 min, 16.5 mg) and compound 3 (tR 34.2 min,
40.6 mg). Fraction E5 (8.1 g) was subjected to ODS CC [MeOH–H2O (2:8 to
25. (3R,5R)-3-Acetoxy-5-hydroxy-1,7-bis(3,4-dihydroxyphenyl)heptane
(8):
): 283.2
yellowish oil; ½a D25
ꢀ
: +18.1 (c 0.1, MeOH); UV kmax (MeOH) nm (log
e
(3.64); IR (KBr) mmax cm-1: 3336, 2943, 1708, 1605, 1524, 1445, 1376, 1281,
1114, 1023, 958, 813; for 1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD,
150 MHz), spectroscopic data, see Table 3; HR-ESI-MS m/z 413.1578 (calcd. for
C
21H26O7Na, 413.1571). (3R,5R)-3,5-diacetoxy-1-(3,4-dihydroxyphenyl)-7-(4-
hydroxyphenyl)heptane (9): yellowish oil; ½a D25
ꢀ
: +17.2 (c 0.1, MeOH); UV kmax

J. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5363–5369
5369
(MeOH) nm (log
e
): 280.6 (3.48); IR (KBr) mmax cmꢂ1: 3372, 2936, 1708, 1611,
the Griess reaction. Briefly, RAW264.7 cells were seeded into 96-well tissue
1515, 1444, 1376, 1265, 1114, 1026, 957, 827; for 1H NMR (CD3OD, 600 MHz)
and 13C NMR (CD3OD, 150 MHz), spectroscopic data, see Table 3; HR-ESI-MS m/
z 439.1731 (calcd. for C23H28O7Na, 439.1727). (3S,5S)- and (3R,5R)-3-acetoxy-
5-hydroxy-1-(3,4-dihydroxyphenyl)-7-(4-hydroxyphenyl)heptane (10) and
culture plates at a density of 1 ꢁ 105 cells/well, and stimulated with 1
lg/mL of
LPS in the presence or absence of compounds. After incubation at 37 °C for
24 h, 100 lL of cell-free supernatant was mixed with 100 lL of Griess
containing equal volumes of 2% (w/v) sulfanilamide in 5% (w/v) phosphoric
acid and 0.2% (w/v) of N-(1-naphthyl)ethylenediamine solution to determine
nitrite production. Absorbance was measured in a microplate reader at 550 nm
against a calibration curve with sodium nitrite standards. Experiments were
performed in triplicate, and data are expressed as the mean SD of three
independent experiments.10,29,30
(11): yellowish oil; UV kmax (MeOH) nm (log e): 280.8 (3.55); IR (KBr) mmax
cmꢂ1: 3320, 2942, 1708, 1610, 1514, 1444, 1376, 1261, 1113, 1024, 957,
819; for 1H NMR (CD3OD, 600 MHz) and 13C NMR (CD3OD, 150 MHz),
spectroscopic data, see Table 3; HR-ESI-MS m/z 397.1618 (calcd. for
C
21H26O6Na, 397.1622).
26. Martin, T.; Kikuzaki, H.; Hisamoto, M.; Nakatani, N. J. Am. Oil Chem. Soc. 2000,
77, 667.
27. Shinji, O.; Koyama, M.; Aoki, T.; Takayuki, S. Bull. Chem. Soc. Jpn. 1985, 1985,
2423.
29. Dirsch, V. M.; Stuppner, H.; Vollmar, A. M. Planta Med. 1998, 64, 423.
30. Hong, S. S.; Lee, S. A.; Han, X. H.; Hwang, J. S.; Lee, C.; Lee, D.; Hong, J. T.; Kim, Y.;
Lee, H.; Hwang, B. Y. J. Nat. Prod. 2008, 71, 1055.
28. Determination of NO production and cell viability assay: The nitrite concentration
in the medium was measured as an indicator of NO production according to