Bioorganic & Medicinal Chemistry Letters 25 (2015) 198202

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Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier.com/locate/bmcl

Curcumolide, a unique sesquiterpenoid with anti-inammatory

properties from Curcuma wenyujin
Jianyong Dong a,, Weiwei Shao a, Pengcheng Yan a, Xiaoqing Cai b, Lianglian Fang a, Xiaowei Zhao c,
Weiwei Lin a, Yuan Cai a
a
b
c

School of Pharmacy, Wenzhou Medical University, Wenzhou 325035, Peoples Republic of China
Wenzhou University, Wenzhou 325035, Peoples Republic of China
The First Afliated Hospital of Wenzhou Medical University, Wenzhou 325000, Peoples Republic of China

a r t i c l e

i n f o

Article history:
Received 23 August 2014
Revised 4 November 2014
Accepted 27 November 2014
Available online 5 December 2014
Keywords:
Curcumolide
Sesquiterpenoid
Curcuma wenyujin
Anti-inammatory effects

a b s t r a c t
Curcumolide, a novel sesquiterpenoid with a unique 5/6/5 tricyclic skeleton, was isolated from Curcuma
wenyujin. The structure and absolute conguration were elucidated by extensive NMR, ECD data analysis,
and a single-crystal X-ray study. This molecule exhibited signicant anti-inammatory effects in
LPS-induced RAW 264.7 macrophages. It suppressed LPS-induced NF-jB activation, including the nuclear
translocation and DNA binding activity of NF-jB, and decreased tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6), interleukin-1b (IL-1b), nitric oxide (NO) and reactive oxygen species (ROS) production.
Therefore, Curcumolide may have therapeutic potential for treating inammatory diseases by inhibiting
NF-jB activation and pro-inammatory mediator production.
2014 Elsevier Ltd. All rights reserved.

Chronic inammation is a hallmark of many human diseases,

such as rheumatic arthritis, arteriosclerosis, obesity, diabetes, neurodegenerative diseases and even cancer.16 Steroids and cyclooxygenase inhibitors have remarkable effects on anti-inammatory, which
have been applied in clinic for more than one century. However, they
often suffer from several side effects in patients, such as atrial
brillation, gastrointestinal erosions, and renal and hepatic insufciency.7,8 Thus, there is an urgent need to nd new and safe
anti-inammatory compounds. The nuclear factor-kappa B (NF-jB)
pathway has been implicated in the pathogenesis of chronic inammatory diseases.9 NF-jB, mainly the p50p65 heterodimer, normally
exists as an inactive state tightly bound to the inhibitory protein of
IjB in the cytoplasm. In response to a variety of stimuli, such as
inammatory cytokines, bacterial lipopolysaccharides (LPS), and
reactive oxygen species (ROS), the proteasome dependent degradation of IjB allows the translocation of NF-jB to the nucleus, interacting with jB elements in the promoter region of a variety of
inammatory response genes, and induces the transcription of proinammatory mediators, including tumor necrosis factor (TNF)-a,
interleukin (IL) 1b, IL-6, inducible nitric-oxide synthase (iNOS), and
cyclooxygenase (COX). These mediators play important roles in
mediating inammatory responses.10 Inhibition of these mediators

Corresponding author. Tel./fax: +86 577 86699572.

E-mail address: jianyd@wmu.edu.cn (J. Dong).
http://dx.doi.org/10.1016/j.bmcl.2014.11.075
0960-894X/ 2014 Elsevier Ltd. All rights reserved.

is benecial for the treatment of inammatory diseases and has

become an important strategy.11
Curcuma wenyujin Y.H. Chen et C. Ling (Zingiberaceae) is cultivated
in the southeast of China, its rhizomes have been used as a Traditional
Chinese Medicine for the treatment of arthralgia, jaundice, thoracicabdominal pain, and dysmenorrhea.12 Phytochemical studies on this
plant have led to the isolation of various sesquiterpenoids including
guaiane, cadinane, germacrane and eudesmane with different
oxygenations and cleavage patterns,1320 which shows antiinammatory,21 antitumor,2224 antioxidant,17 and inhibition of
platelet aggregation.25 In order to determine the anti-inammatory
constituents, a novel sesquiterpenoid with a rare 5/6/5 tricyclic skeleton, namely curcumolide (1), was discovered from this plant. In this
study, the isolation, structural elucidation, and anti-inammatory
activities of the compound are reported.
Dried rhizomes of C. wenyujin (20 kg) were powdered and
extracted with 95% EtOH (10 L  3) at room temperature. The
Ethanol extract was evaporated to dryness under reduced pressure
and the residue (1700 g) was suspended in water and then partitioned successively with petroleum ether (bp 6090 C), EtOAc,
and n-BuOH to give three corresponding portions. The EtOAc
extract (600 g) was subjected to silica gel column chromatography
and eluted with a petroleum etherMe2CO (50:1 to 1:1) gradient
solvent system to afford eight major fractions (18). Fraction 4
(35 g) was subjected to silica gel column chromatography by
elution with petroleum etherEtOAc (20:1 to 1:1) to afford four

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J. Dong et al. / Bioorg. Med. Chem. Lett. 25 (2015) 198202

subfractions (4a4d). Subfraction 4d (8 g) was separated on silica

gel column chromatography eluting with petroleum etherMe2CO
(20:1), and further puried by an ODS column (C-18, eluted with
MeOHH2O 70:30) to obtain compound 1 (46 mg, >99% purity, as
determined by HPLC).

1
5

3
4
15

14
10
9
7

11
13

12

OH

Curcumolide (1)

Curcumolide (1),26 obtained as colorless crystals, had a molecular

formula of C15H22O3 as determined by HRESIMS (m/z 273.1469
[M+Na]+, calcd 273.1461) and NMR data, indicating ve degrees of
unsaturation. The 1H NMR spectrum of 1 showed four methyl group
signals, including an olenic methyl singlet at dH 1.71 (s, H3-14), a
methyl doublet at dH 1.06 (d, J = 6.2 Hz, H3-15)], and two methyl
singlets at dH1.21 (s, H3-12) and dH 1.33 (s, H3-13), in addition to
an olenic proton signal at dH 5.94 (s, H-9). The 13C NMR spectrum
presented a total of fteen carbon resonances, including a carbonyl
carbon (dC 179.4), two olenic carbons [dC 139.4 (qC) and 121.7
(CH)], two oxygenated carbons [dC 92.1 (qC) and 70.7 (qC)]
(Table 1).
The HMQC spectrum assigned all protons and corresponding
carbons in the molecule. Two degrees of unsaturation, accounted
for by the functional groups (a carbonyl and an olenic double
bond) from ve in the molecule, suggested a tricyclic structure in
1. Detailed analysis of 2D NMR spectroscopic data established a
rare structure of the 5/6/5-membered tricyclic backbone. The
1
H1H COSY correlations allowed to establish the partial structure
of consecutive proton system extending from H-1 [dH 2.49 (1H, t,
J = 11.4 Hz)] to H3-15 via H2-2 [dH 1.91 (1H, m), 1.59 (1H, m)],
H2-3 [dH 2.07 (1H, m), 1.54 (1H, m)], and H-4 (dH 2.08 (1H, m)).
The HMBC correlations observed from H3-15 to C-3 (dC 29.9), C-4
(dC 38.4), and C-5 (dC 92.1), from H3-14 to C-1 (dC 51.2), C-10
(dC 139.4), and C-9 (dC 121.7), from H-1 to C-4, C-5, and C-6
(dC 37.1), and from H2-6 [dH 2.34 (1H, d, J = 10.8 Hz), 1.77 (1H, d,

Table 1
H (600 MHz) and

J = 10.8 Hz)] to C-7 (dC 56.8) and C-9, led to the assignment of a
cyclopentane moiety (ring A) fused to a six-membered ring B at
C-1 and C-5, and ring B contained a methyl group and double bond
that were located at C-10 and C-9/C-10, respectively. Additional
HMBC correlations from both H3-12 and H3-13 to a hydroxylated
quaternary carbon C-11 at dC 70.7 (qC), two methyl carbon at dC
24.9 (CH3) and 26.1 (CH3), and a quaternary carbon C-7 indicated
the attachment of a 2-hydroxyisopropane group to C-7. Moreover,
the presence of a c-lactone moiety (ring C) fused to ring B at C-5
and C-7 was revealed by the HMBC correlations from H2-6 to an
oxygenated quaternary carbon C-5, C-7, and carbonyl carbon C-8
(dC 179.4), and from olenic proton H-9 to C-6, C-7, and C-8, in
association with the IR absorption at 1739 cm 1. Consequently,
the gross structure of 1 was elucidated as shown in Figure 1.
The relative stereochemistry of 1 was established by NOESY
spectroscopic analysis (Fig. 1). The key NOE cross peaks were
observed between H-1/H-4, H-1/H-6a, H-6a/H3-12, and H-6a/
H3-13, suggesting that H-1, H-4, H-6a, and 2-hydroxyisopropane
group were oriented toward the same side, and requiring rings
A/B and B/C were trans- and cis-fused, respectively. An X-ray
crystallography analysis was performed. A perspective of single
molecule of 1 was given in Figure 2,27 which conrmed the structure deduced by NMR studies.
The absolute conguration of 1 was established by measurement
of the ECD spectrum and comparison with calculated ECD data.28
According to the established relative conguration, curcumolide
(1) should be one of the two enantiomers (1R,4R,5R,7R)- or
(1S,4S,5S,7S)-. As shown in Figure 3, the experimental ECD spectra
agreed well with the calculated ECD curves of (1S,4S,5S,7S)enantiomer. Thus, the absolute conguration of 1 was established
as 1S,4S,5S,7S.
In order to identify the anti-inammatory effect of 1, the
pro-inammatory cytokines (TNF-a, IL-1b, IL-6), nitric oxide
(NO), ROS, and NF-jB were measured in LPS-induced RAW 264.7
macrophages treated with 1, and the viability of cultured RAW

OC

OH
O

H-1H COSY H H

HMBC H C

ROESY H H

Figure 1. Key 2D NMR correlations of 1.

13

C (150 MHz) NMR Data of 1 in CDCl3 (d in ppm, J in Hz)

No.

dH

1
2a
2b
3a
3b
4
5
6a
6b
7
8
9
10
11
12
13
14
15

2.49
1.91
1.59
2.07
1.54
2.08

dC
(1H,
(1H,
(1H,
(1H,
(1H,
(1H,

t, 11.4)
m)
m)
m)
m)
m)

2.34 (1H, d, 10.8)

1.77 (1H, d, 10.8)

5.94 (1H, s)

1.21
1.33
1.71
1.06

(3H,
(3H,
(3H,
(3H,

s)
s)
s)
d, 6.2)

51.2 CH
23.3 CH2
29.9 CH2
38.4 CH
92.1 qC
37.1 CH2
56.8 qC
179.4 qC
121.6 CH
139.4 qC
70.7 qC
24.9 CH3
26.1 CH3
20.5 CH3
12.4 CH3

Data were assigned by HSQC, HMBC, 1H1H COSY, and ROESY spectra.

Figure 2. X-ray structure of 1.

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J. Dong et al. / Bioorg. Med. Chem. Lett. 25 (2015) 198202

Figure 3. Comparison of the experimental ECD spectrum of 1 in MeOH with

calculated ECD spectra for 1R,4R,5R,7R and 1S,4S,5S,7S.

Figure 4. Curcumolide (1) inhibits LPS-induced expression of TNF-a, IL-1b and IL-6
in RAW 264.7 macrophages. Cells were pretreated with 1 (020 lM) or hydrocortisone (10 lM) for 2 h followed by a further 22 h treatment with LPS. The (A) TNF-a,
(B) IL-1b and (C) IL-6 contents in the culture medium were determined by ELISA.
Data are expressed as means SEM from four independent experiments.

indicates a signicant difference compared to control group at p <0.01;

# ##
,
indicates a signicant difference compared to LPS alone group at p <0.05 and
0.01, respectively.

Figure 5. Curcumolide (1) inhibits LPS-induced NO production in RAW 264.7

macrophages. Cells were treated with 1 (020 lM) or hydrocortisone (10 lM) for
2 h followed by a further 22 h treatment with LPS. The nitrite production in the
culture medium was measured by the Griess reaction. Data are expressed as
means SEM from four independent experiments. indicates a signicant difference compared to control group at p <0.01, ## indicates a signicant difference
compared to LPS alone group at p <0.01.

264.7 cells was examined by the MTT assay. No signicant change

in cell viability was observed in RAW 264.7 cells after exposure to
1 for 24 h with different concentrations (0.01100 lM). Then, the
nontoxic concentrations of 1 were used for subsequent experiments. The dosage selection was based on the IC50 values arising
from the inhibition of NO production by 1 in preliminary study.
Pro-inammatory cytokines play an important role in activating
the inammatory response.29 In order to investigate whether 1
acts as an anti-inammatory agent we measured the inhibition
of 1 to the production of pro-inammatory cytokines, TNF-a,
IL-1b, and IL-6 in the culture medium of RAW 264.7 cells by ELISA.
As shown in Figure 4AC, the production of TNF-a, IL-6 and IL-1b
were signicantly elevated with LPS (0.5 lg/mL) treatment.
Inversely, RAW 264.7 cells exposed to 1 (10 and 20 lM) showed
a dose-dependent inhibitory effect on cytokines production after
LPS treatment, including TNF-a (37.62 0.98%, and 55.40 3.65%,
respectively), IL-1b (27.93 2.96%, and 68.02 3.97%, respectively), and IL-6 production (37.30 1.20%, and 78.27 2.12%,
respectively). Meanwhile, we found the similar inhibitory effect
with 1 in the positive control group (10 lM hydrocortisone).
NO is involved in various biological processes including inammation. The inhibition of NO production by iNOS may have potential therapeutic effect to inammation associated diseases.30 NO
production was determined by measuring nitrite content released
into the culture medium using the Griess reagent. As shown in
Figure 5, six-folds of NO production were observed after exposure
to LPS for 22 h compared to the vehicle control. However, the production of NO was signicantly blocked by 1 treatment in a dosedependent manner. The blocked ratio was shown as followed:
25.15 0.36% at 2 lM, 53.88 1.26% at 10 lM, and 71.02 0.53%
at 20 lM. As a positive control, hydrocortisone (10 lM) also
showed a signicant inhibitory effect on NO production after LPS
treatment.
ROS are thought to be involved in activation of the transcription
factor NF-jB, central for mediating production of pro-inammatory cytokines in response to inammatory stimuli.31 To determine
the effect of 1 on intracellular ROS, LPS-induced ROS production
was detected in RAW264.7 cells with or without the presence of
1. The production of ROS induced by LPS was signicantly
increased (2.5-folds) compared to the vehicle control (Fig. 6).
However, the production of ROS was dose-dependently inhibited

J. Dong et al. / Bioorg. Med. Chem. Lett. 25 (2015) 198202

Figure 6. Curcumolide (1) inhibits LPS-induced ROS production in RAW 264.7

macrophages. Cells were treated with 1 (020 lM) for 2 h followed by a further
22 h treatment with LPS. The cells were harvested and incubated with 10 lM DCFHDA for 20 min, and washed twice with cold PBS. The formation of ROS in the cells
was evaluated by the arbitrary uorescence unit. Data are expressed as means
SEM from four independent experiments. indicates a signicant difference
compared to control group at p <0.01; #, ## indicates a signicant difference
compared to LPS alone group at p <0.05 and 0.01, respectively.

201

Figure 8. Curcumolide (1) inhibits LPS-induced DNA binding of NF-jB p65.

RAW264.7 cells were preincubated with different concentrations of 1 (020 lM)
for 1 h and then stimulated with LPS for 30 min. DNA-binding activity of NF-jB p65
was quantied by ELISA using the Trans AM NF-jB p65 transcription factor assay
kit. Data are expressed as means SEM from three independent experiments.

indicates a signicant difference compared to control group at p <0.01;

##
indicates a signicant difference compared to LPS alone group at p <0.01.

were treated with 0.5 lg/mL of LPS without or with 2, 10, 20 lM of

1 for 24 h. Then, DNA binding activity of NF-jB p65 was determined
by ELISA (Fig. 8). Stimulation of RAW264.7 cells with LPS strongly
induced DNA binding activity of NF-jB p65. However, treatment of
1 signicantly suppressed LPS-induced DNA binding activity of
NF-jB p65 in a dose-dependent manner, corresponding to
36.49 2.98% at 2 lM, 51.31 3.57% at 10 lM, and 68.00 5.08% at
20 lM Compound 1.
In conclusion, the present work afforded an unprecedented
sesquiterpene skeleton with a 5/6/5 tricyclic ring system from
C. wenyujin. Our bioassay showed that 1 exhibits an inhibitory
effect on LPS-stimulated inammatory effects in RAW 264.7
macrophages. Compound 1 suppressed LPS-induced NO and ROS
production, nuclear translocation, and DNA binding activity of
NF-jB, as well as the expression of TNF-a, IL-6, IL-1b. Our study
suggests that 1 can be a potential anti-inammatory compound.
Acknowledgments

Figure 7. Curcumolide (1) inhibits LPS-induced NF-jB p65 translocation into the
nucleus. RAW 264.7 cells were pretreated with 20 lM Curcumolide for 1 h followed
by treatment with LPS for 30 min. The image visualized using uorescence
microscopy. p65 was detected by Alexa Fluor 488-labeled immunostaining (green);
nuclei were stained by Hoechst33342 (blue). The results shown are from one
experiment, which is representative of two others performed.

Financial support from Natural Science Foundation of China

(81373933) and the Zhejiang Provincial Natural Science Foundation of China (LY13H280003), and the Key Project of Traditional
Chinese Medicine Science and Technology of Zhejiang Province
(2013ZA087) is gratefully acknowledged.
Supplementary data

by 57.90 2.75% and 82.26 1.41% in 1 preincubated cells at concentrations of 10 and 20 lM, respectively.
To further conrm the inhibitory activity of 1 on NF-jB, we
analyzed the effect of 1 on LPS-induced NF-jB p65 nuclear translocation by an immunouorescence assay. As shown in Figure 7, the
p65 subunit mainly localized on the cytoplasm of the non-induced
macrophages, and stimulation of cells with LPS leaded to p65 accumulation in cell nucleus. However, the LPS-induced NF-jB p65
nuclear translocation was markedly impaired after 1 (20 lM)
treatment.
After nuclear translocation, NF-jB subunits bind promoter
elements of various pro-inammatory genes that contain the NFjB consensus sequence.32 Next, we analyzed the effect of 1 on
LPS-induced DNA binding activity of NF-jB p65. RAW264.7 cells

Supplementary data (experimental procedures, NMR, MS, and

IR spectra of 1) associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.bmcl.2014.11.075.
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Curcumolide (1): colorless crystals (CHCl3/MeOH); mp 8687 C; [a]20
7.1 (c
D
0.14, MeOH); UV (MeOH) kmax (log e) 223 (1.55), 206 (2.58) nm; IR (KBr) mmax:
3502, 2975, 2874, 1739, 1157, 933 cm 1; 1H (600 MHz) and 13C NMR (150 MHz)

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273.1461.
Crystal data for curcumolide (1): C15H22O3, M = 250.33, orthorhombic, P 21/n,
a = 6.192 (7) , b = 8.362 (9) , c = 14.187(15) , a = c = 90, b = 95.54 V = 731.2
(14) 3, Z = 4, Dcalcd = 1.137 g/cm 3, l = 0.078 mm 1, 2530 reections measured,
1758 reections independent (Rint = 0.0396), R = 0.0969, wR2 = 0.2401, goodness
of t = 1.030, and T = 298 K. X-ray crystallographic analysis was recorded
on a Bruker Smart-Apex CCD diffractometer equipped with a graphitemonochromatized Mo Ka (k = 0.071073 nm) radiation by using an x-scan
mode. Determination of the crystal class, orientation matrix, and cell dimensions
was performed according to the established procedures. Corrections for Lorentz
polarization and empirical absorption were performed. Most of the nonhydrogen atoms were located by direct methods, and the rests were derived
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methanol solvent. The calculated ECD spectra were compared with the
experimental data after a UV correction. All calculations have been
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