Eudesmane-type sesquiterpene lactones inhibit multiple steps in the NF-κB signaling pathway induced by inflammatory cytokines

Article abstract of DOI:10.1016/j.bmcl.2011.11.029

Inflammatory cytokines, such as interleukin-1α (IL-1α) and tumor necrosis factor-α (TNF-α), induce the intracellular signaling pathway leading to the activation of nuclear factor κB (NF-κB). A series of eudesmane-type sesquiterpene lactones possessing an

Full text of DOI:10.1016/j.bmcl.2011.11.029

Bioorganic & Medicinal Chemistry Letters 22 (2012) 207–211
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Eudesmane-type sesquiterpene lactones inhibit multiple steps in the NF-
signaling pathway induced by inflammatory cytokines
jB
Ryuichi Tamura a, Yanjie Chen b, Miwako Shinozaki c, Kei Arao d, Liyan Wang d, Wanxia Tang d,
Seiya Hirano a, Hirotsugu Ogura b, Tomokazu Mitsui b, Shigeru Taketani a, Masayoshi Ando c,
,
Takao Kataoka a,b,
⇑
a Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan
b Center for Biological Resources and Informatics, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan
c Department of Chemistry and Chemical Engineering, Niigata University, Ikarashi 2-8050, Nishi-ku, Niigata 950-2181, Japan
d Graduate School of Science and Technology, Niigata University, Ikarashi 2-8050, Nishi-ku, Niigata 950-2181, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Inflammatory cytokines, such as interleukin-1
intracellular signaling pathway leading to the activation of nuclear factor
mane-type sesquiterpene lactones possessing an -methylene
ketone group were synthesized and evaluated for their inhibitory effects on the NF-
a
(IL-1
a
) and tumor necrosis factor-
B (NF-
-lactone group and/or an a
a
j
(TNF-
B). A series of eudes-
-bromo
B-dependent gene
-methylene -lac-
a), induce the
Received 10 August 2011
Revised 7 November 2011
Accepted 9 November 2011
Available online 16 November 2011
j
a
c
j
a
expression and signaling pathway. Our present study reveals that eudesmane-type
c
tones and
and TNF-
a
.
-bromo ketones inhibit multiple steps in the NF-
j
B signaling pathway induced by IL-1
a
Keywords:
a
Inflammatory cytokine
Interleukin-1
Eudesmane
Ó 2011 Elsevier Ltd. All rights reserved.
Nuclear factor jB
Sesquiterpene lactone
Tumor necrosis factor-
a
Inflammatory cytokines, such as interleukin-1 (IL-1) or tumor
necrosis factor- (TNF- ), mainly induce the nuclear factor
(NF- B family of transcription fac-
B) signaling pathway.1 The NF-
to induce the activation of the I
naling molecules.9,10 Immediately after phosphorylated I
ubiquitinated and subsequently hydrolyzed by 26S proteasome,
the NF- B dimers, such as the p65/p50 heterodimer, become free
and translocate to the nucleus.7,8 The NF-
B dimers bind specifi-
cally to their responsive elements and induce the transcriptional
activation of various target genes, such as intercellular adhesion
molecule-1 (ICAM-1).11
j
B kinase complex as common sig-
a
a
jB
jB is
j
j
tors plays a major role in the induction of a variety of genes essen-
j
tial for the regulation of inflammation, innate immunity, and
j
survival.1 NF-
jB has been shown to be responsible for chronic-
inflammation-related diseases, such as autoimmune diseases, can-
cer, and metabolic disorders.2–4 Many types of natural and syn-
thetic small molecules have been reported to block the NF-
signaling pathway induced by inflammatory cytokines.5
NF-jB exists as hetero- or homodimers consisting of five mem-
bers (p65(RelA), RelB, c-Rel, p105/p50, and p100/p52), which share
j
B
Sesquiterpene lactones are a large group of secondary metabo-
lites of many medicinal plants and exhibit a variety of biological
activities. In our previous studies, a series of guaiane-type and
eudesmane-type sesquiterpene lactones were synthesized and
evaluated for their immunomodulatory activities.12–14 It has been
an N-terminal Rel homology domain responsible for DNA binding
and dimerization.6 In unstimulated cells, NF-
j
B dimers are associ-
B) family proteins and seques-
tered as an inactive complex in the cytosol.6 Upon stimulation with
IL-1 and TNF- , IL-1 receptor and TNF receptor 1 induce the
shown that 3-oxoeudesm-11(13)-eno-12,6
oeudesma-1,11(13)-dieno-12,6 -lactone (7) (the latter also known
as (+)-tuberiferin), but not (11S)-3-oxoeudesmano-12,6 -lactone
(2), inhibit cell-surface ICAM-1 expression in response to IL-1
stimulation.13
Moreover, (11S)-2 -bromo-3-oxoeudesmano-
12,6 -lactone (1), initially designated as santonin-related
compound 2, was found to inhibit ICAM-1 expression and I
degradation induced by IL-1
or IL-1b.14 Those studies proposed
that eudesmane derivatives possessing either an -methylene
-lactone group or an -bromo ketone group inhibit ICAM-1
a-lactone (4) and 3-ox-
ated with the inhibitor of NF-
j
B (Ij
a
a
a
a
recruitment of different sets of adaptor proteins and initiate dis-
a
tinct signaling processes.7,8 These bifurcated processes converge
a
jBa
a
⇑
Corresponding author. Tel./fax: +81 75 724 7752.
E-mail address: takao.kataoka@kit.ac.jp (T. Kataoka).
a
c
a
Present address: 22-20 Keiwa-machi, Taihaku-ku, Sendai 982-0823, Japan.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.029

208
R. Tamura et al. / Bioorg. Med. Chem. Lett. 22 (2012) 207–211
1,11(13)-dieno-12,6a
-lactone (8)18 were synthesized from com-
pound 1 (Scheme 1 and Supplementary data).
Human lung carcinoma A549 cells were pretreated with com-
pounds 1–8 for 1 h and then incubated with IL-1
6 h. Compounds 1, 3, 4, 5, 7, and 8 inhibited cell-surface ICAM-1
expression induced by IL-1 or TNF- in a dose-dependent man-
a or TNF-a for
a
a
ner, whereas compounds 2 and 6 were inactive (Fig. 2). The IC50
values of compounds 1, 3, 4, 7, and 8 for the cell-surface ICAM-1
expression were mostly in the range of 3–10 lM (Table 1). The
IC50 values of compound 5 were calculated to be several times
higher than those of compounds 1, 3, 4, 7, and 8 (Table 1). The
amount of total ICAM-1 protein as translation product was then
analyzed by Western blotting. Compounds 1, 3, 4, 5, 7, and 8
diminished total ICAM-1 protein expression in a manner similar
to cell-surface ICAM-1 expression (Supplementary Fig. 1). Thus,
these data indicate that
-bromo ketones exhibit strong inhibitory activities on ICAM-1
expression at the protein level. In addition, it seems likely that
an ,b-unsaturated carbonyl group in the A ring (present in com-
pounds 5 and 7) is necessary for moderate inhibitory activities
on ICAM-1 expression induced by IL-1 and TNF-
a-methylene c-lactones as well as
a
a
a
a.
To evaluate cytotoxicity, A549 cells were incubated for 24 h in
the presence of compounds 1–8. The IC50 values of compounds 3,
4, 7, and 8 were calculated to be approximately 100 lM (Table 1
and Supplementary Fig. 2). Although those values were at least
10-fold higher than the values required to inhibit cell-surface
ICAM-1 expression, it seems that
a-methylene c-lactones exhibit
cytotoxicity particularly upon prolonged exposure. By contrast,
compounds 1 and 5 only partially decreased cell viability at 100–
Figure 1. Structures of eudesmane derivatives 1–8.
320
compound 1 at 100
viability up to 7 h, as judged by MTT assay and crystal violet stain-
ing (Supplementary Fig. 3). These data suggest that -bromo ke-
l
M during 24 h incubation (Supplementary Fig. 2). In fact,
expression by targeting the NF-
explore the structure–activity relationship, eight eudesmane
derivatives 1–8 (Fig. 1) were synthesized and evaluated for their
j
B signaling pathway. To further
lM did not exert any inhibitory effects on cell
a
inhibitory activities on ICAM-1 expression as well as the NF-
naling pathway induced by inflammatory cytokines.
jB sig-
tone 1 is the most selective compound that inhibits ICAM-1
expression with high potency and low cytotoxicity.
Five eudesmane derivatives, 1, 2, 2
11(13)-eno-12,6 -lactone (3), 4, and 7, were synthesized from
the starting material (ꢀ)-
-santonin as described previously.14,15
(11S)-3-Oxoeudesm-1-eno-12,6
-lactone (5),16 (11S)-3b-hydrox-
yeudesm-1-eno-12,6
-lactone (6),17 and 3b-hydroxyeudesma-
a
-bromo-3-oxoeudesm-
We further investigated the inhibitory effects of eudesmane
a
derivatives on the nuclear translocation of NF-
by IL-1 or TNF- . A549 cells were pretreated with compounds 1–
8 for 1 h and then stimulated with IL-1 or TNF- for 30 min. In
unstimulated A549 cells, NF- B subunits p65 and p50 were detect-
jB subunits induced
a
a
a
a
a
a
a
j
Scheme 1. Synthesis of compounds 5, 6, and 8.

R. Tamura et al. / Bioorg. Med. Chem. Lett. 22 (2012) 207–211
209
Table 1
Biological activities of eudesmane derivatives
Compound
ICAM-1 expressiona
Cell viabilityb
IL-1a
TNF-a
1
2
3
4
5
6
7
8
10c
4.6c
>320
>320
88d
>320
3.4c
6.2c
52
>320
2.8c
4.3c
34
>320
4.3c
6.1c
110d
>320
>320
120d
140d
>320
10c
23c
a
A549 cells were preincubated with serial dilutions of compounds for 1 h and
then incubated with IL-1 (0.25 ng/mL) or TNF- (2.5 ng/mL) for 6 h in the presence
or absence of compounds. ICAM-1 expression (%) was determined by the Cell ELISA
a
a
assay using triplicate cultures. The means of IC50 values (lM) were calculated from
at least three independent experiments, including Fig. 2.
b
A549 cells were incubated with serial dilutions of compounds for 24 h. Cell
viability (%) was determined by MTT assay using triplicate cultures. The means of
IC50 values (lM) were calculated from four independent experiments, including
Supplementary Fig. 2.
c
Statistical significance was assessed by one-way analysis of variance, followed
by the Tuker test for multiple comparisons. The IC50 values of compounds 1, 3, 4, 7,
and 8 for ICAM-1 expression induced by IL-1
a and TNF-a were not statistically
significant (P >0.05).
d
Statistical significance was assessed by one-way analysis of variance, followed
by the Tuker test for multiple comparisons. The IC50 values of compounds 3, 4, 7,
and 8 for cell viability were not statistically significant (P >0.05).
tion of p65 and p50 induced by IL-1
affected that induced by TNF- (Fig. 3B).
IL-1 and TNF- rapidly induced the phosphorylation of I
within 5 min and phosphorylated I was subsequently hydro-
lyzed by the ubiquitin–proteasome system within 10–15 min in
A549 cells.19 Consistent with our previous study,14
degrada-
tion induced by IL-1 was inhibited by compound 1 at 100
(Fig. 4A). However, surprisingly, we found that compound 1 did
not inhibit or only weakly inhibited the TNF- -induced I deg-
radation at 100 M (Fig. 4B). Similarly, compound 1 strongly inhib-
ited the IL-1 -induced I phosphorylation but only weakly
affected the TNF- -induced I phosphorylation (Fig. 5A and B).
These results suggest that -bromo ketone 1 selectively blocks
the early NF- B signaling pathway induced by IL-1 but not
TNF- . In addition, compound 1 at 100 M only weakly inhibited
the TNF- -induced phosphorylation and degradation
(Figs. 4B and 5B), but almost completely inhibited the TNF-
induced NF- B translocation (Fig. 3B), suggesting that compound
1 does not affect I B kinase activation but selectively inhibits the
translocation process of NF- B subunits in the TNF- -dependent
NF- B signaling pathway. Compounds 3, 4, and 7 at 100
strongly inhibited I phosphorylation and degradation induced
by IL-1 (Figs. 4A and 5A) and TNF- (Figs. 4B and 5B). By contrast,
compound 8 at 100 M did not inhibit I phosphorylation and
degradation induced by IL-1 (Figs. 4A and 5A) and TNF-
(Figs. 4B and 5B). Since compound 8 at 100 M inhibited the IL-
- and TNF- -induced NF- B translocation (Fig. 3A and B), it
seems that compound 8 does not affect I B kinase activation but
inhibits NF- B translocation in the TNF- and IL-1-dependent
NF- B signaling pathways. Taken together, it is very likely that
-methylene -lactones are necessary to inhibit I phosphory-
lation and degradation in the common NF- B signaling pathway,
whereas a hydroxyl group in the A-ring (as found in compound
8) may attenuate the inhibitory activity of -methylene -lactones.
Sesquiterpene lactones that contain ,b-unsaturated carbonyl
moieties, such as -methylene -lactones, are reactive to cysteine
a (Fig. 3A) and partly
a
a
a
jBa
jBa
IjBa
a
lM
a
jBa
l
a
jBa
a
jBa
a
j
a
a
l
a
IjBa
a-
j
Figure 2. Effect of eudesmane derivatives on cell-surface ICAM-1 expression
induced by IL-1 and TNF- . A549 cells were preincubated with various concen-
trations of compounds 1–8 for 1 h and then incubated with IL-1 (0.25 ng/mL; filled
circles) or TNF- (2.5 ng/mL; open circles) for 6 h in the presence of compounds 1–
8. ICAM-1 expression (%) was determined by the Cell ELISA assay. Results are shown
as means SD of triplicate cultures and representative of at least three independent
experiments.
j
a
a
j
a
a
j
lM
a
jBa
a
a
l
jBa
a
a
l
1
a
a
j
able only in the cytoplasm, whereas a portion of p65 and p50 sub-
j
units translocated to the nucleus upon treatment with IL-1
(Fig. 3A) or TNF- (Fig. 3B). Compounds 1, 3, 4, 7, and 8 at 30–
100 M inhibited the nuclear translocation of p65 and p50 induced
by IL-1 or TNF- (Fig. 3A and B), indicating that -methylene
-lactones as well as -bromo ketones inhibit the NF- B transloca-
tion induced by IL-1 and TNF- . As their inhibitory concentra-
a
j
a
a
j
l
a
c
jBa
a
a
a
j
c
a
j
a
a
a
c
tions were higher than those required for the inhibition of
ICAM-1 expression at the protein level, it seems that compounds
1, 3, 4, 7, and 8 complementarily affect late processes downstream
a
a
c
thiol groups in the Michael-type addition. Sesquiterpene lactones
of the NF-
DNA binding, transcriptional activation and mRNA/protein synthe-
sis. Compound 5 at 100 M barely affected the nuclear transloca-
jB translocation leading to ICAM-1 expression, such as
and related compounds containing those functional moieties have
been identified to inhibit the NF-j jB kinase
B signaling pathway.5 I
l

210
R. Tamura et al. / Bioorg. Med. Chem. Lett. 22 (2012) 207–211
Figure 4. Effect of eudesmane derivatives on
inflammatory cytokines. A549 cells were preincubated with indicated concentra-
tions of compounds 1–8 for 1 h and then incubated with (+) IL-1 (0.25 ng/mL; A)
or TNF-
IjBa degradation induced by
a
a
(2.5 ng/mL; B) or without cytokines (ꢀ) for 15 min in the presence of
compounds 1–8. Cell lysates were analyzed by Western blotting using anti-I
and anti-b-actin antibodies.
jBa
ing activity.24,25 Epoxyquinoids contain an epoxide structure that is
known to react with nucleophiles, such as cysteine thiol groups. It
has been reported that IjB kinase b or p65, or both, are directly
inactivated by the modification of their cysteine groups with
epoxyquinone A monomer, Jesterone dimer or (ꢀ)-dehydroxyme-
thylepoxyquinomicin.26–28 In addition to those proteins, TGF-b
activating kinase 1 (TAK1) has been identified to be a molecular
target of epoxyquinol B.29 Collectively, those studies propose that
plural signaling proteins essential for the NF-jB signaling pathway
are highly sensitive to thiol-reactive sesquiterpene lactones and
epoxyquinoids. This notion is consistent with our present findings
that eudesmane derivatives inhibit multiple steps in the NF-jB sig-
Figure 3. Effect of eudesmane derivatives on nuclear translocation of NF-jB
subunits p65 and p50 induced by inflammatory cytokines. A549 cells were
preincubated with indicated concentrations of compounds 1–8 for 1 h and then
incubated with (+) IL-1
a (0.25 ng/mL; A) or TNF-a (2.5 ng/mL; B) or without
cytokines (ꢀ) for 30 min in the presence of compounds 1–8. Cytoplasmic and
nuclear lysates were analyzed by Western blotting using anti-p65 and anti-p50
antibodies.
naling pathway. However, it remains unclear how thiol-reactive
small molecules bind specific cysteine residues of target proteins
and selectively inactivate only limited steps in the NF-
jB signaling
b contains a critical cysteine residue (Cys-179) in the activation
pathway.
loop within the catalytic domain.20,21 Parthenolide and artemiso-
In conclusion, our present study revealed for the first time that
eudesmane-type sesquiterpene lactones possessing an -methy-
lene -lactone group or an -bromo ketone group inhibit multiple
steps in the NF- B signaling pathway (Supplementary Fig. 4). A
comparison of the inhibitory profiles of compounds 1–8 revealed
lide have been reported to inhibit the catalytic activity of I
j
B ki-
a
nase b via the modification of Cys-179.22,23 The NF-
jB subunit
c
a
p65 bears a critical cysteine residue (Cys-38) in the Rel homology
domain essential for dimerization and DNA binding. It has been
shown that helenalin and parthenolide directly target Cys-38 of
p65 and thereby prevent either nuclear translocation or DNA bind-
j
that
a
-methylene
c-lactones 4 and 7 inhibit I
jBa
phosphorylation
(step 3) and compound 8, also an
a
-methylene-c-lactone, inhibits

R. Tamura et al. / Bioorg. Med. Chem. Lett. 22 (2012) 207–211
211
Acknowledgment
This work was supported in part by a Grant-in-Aid for Scientific
Research (KAKENHI) from Japan Society for the Promotion of Sci-
ence (JSPS).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.11.029.
References and notes
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16. Compound
5
as colorless needles: mp 140.5–141.5 °C; IR (CHCl3) 1782,
1676 cmꢀ1
;
1H NMR (200 MHz)
d
1.18 (3 H, s, C10-CH3), 1.23 (3H, d,
J = 6.9 Hz, C11–CH3), 1.36 (3H, d, J = 6.9 Hz, C4-CH3), 2.30 (1H, dq, J = 12.9,
6.9 Hz, C11-H), 2.58 (1 H, dq, J = 13.0, 6.9 Hz, C4-H), 4.00 (1H, dd, J = 10.7, 9.9 Hz,
C6-H), 5.89 (1H, d, J = 9.9 Hz, C2-H), 6.71 (1H, d, J = 9.9 Hz, C1-H); 13C NMR
(50 MHz, in CDCl3) d 12.4 (q, C13), 14.5 (q, C15), 19.2 (q, C14), 22.7(t, C8), 37.4 (t,
C9), 38.4 (s, C10), 40.5 (d, C11), 42.2 (d, C4), 51.7 (d, C5), 52.8 (d, C7), 81.8 (d, C6),
126.6 (d, C2), 158.1 (d, C1), 178.8 (s, C12), 200.6 (s, C3); ½a D20
ꢁ
+4.0 (c 1.67, CHCl3).
17. Compound 6 as colorless plates: mp 163–165 °C; IR (CHCl3) 3605, 3524,
1772 cmꢀ1;1H NMR (200 MHz, in CDCl3) d 1.06 (3 H, s, C10-CH3), 1.20 (3H, d,
J = 6.9 Hz, C11-CH3), 1.24 (3H, d, J = 6.5 Hz, C4-CH3), 2.26 (1H, dq, J = 12.5,
6.9 Hz, C11-H), 3.79 (1H, d, J = 8.0 Hz, C3-H), 3.92 (1H, dd, J = 11.0, 9.6 Hz, C6-H),
5.52 (2H, s, C1-H, C2-H); 13C NMR (50 MHz, in CDCl3) d 12.3 (q, C13), 17.1 (q,
C15), 21.3 (q, C14), 22.9 (t, C8), 37.5 (d, C4), 38.0 (t, C9), 38.6 (s, C10), 40.6 (d, C11),
50.6 (d, C5), 53.8 (d, C7), 75.0 (d, C3), 82.0 (d, C6), 127.8 (d, C2), 139.3 (d, C1),
179.5 (s, C12); ½a D20
ꢁ
+63.6 (c 2.25, CHCl3); Anal. Calcd for C15H22O3: C, 71.97; H,
8.86. Found: C, 71.49; H, 9.26.
18. Compound 8 as colorless prisms: mp 124–126 °C; IR (CHCl3) 3620, 3472,
1768 cmꢀ1 1H NMR (200 MHz) d 1.05 (3H, s, C10-CH3), 1.29 (3H, d, J = 6.3 Hz,
;
C4-CH3), 2.51 (1H, ddddd, J = 10.6, 10.6, 3.2, 3.2, 3.2 Hz, C7-H), 3.81 (1H, d,
J = 8.4 Hz, C3-H), 3.91 (1H, dd, J = 10.6, 10.6 Hz, C6-H), 5.39 (1H, d, J = 3.2 Hz,
C
13-Hb), 5.54 (2H, s, C1-H, C2-H), 6.06 (1H, d, J = 3.2 Hz, C13-Ha); 13C NMR
(50 MHz, in CDCl3) d 17.1 (q, C15), 21.2 (t, C8), 21.3 (q, C14), 37.5 (d, C4), 37.7 (t,
C9), 38.7 (s, C10), 51.1 (d, C7), 51.2 (d, C5), 74.9 (d, C3), 82.3 (d, C6), 116.6 (t, C13),
127.9 (d, C2), 138.9 (s, C11), 139.1 (d. C1), 170.7 (s, C12); Anal. Calcd for
C
15H20O3: C, 72.55; H, 8.12. Found: C, 71.82; H, 7.89.
Figure 5. Effect of eudesmane derivatives on I
inflammatory cytokines. A549 cells were preincubated with (+) or without (ꢀ) the
proteasome inhibitor MG-132 (20 M) or various concentrations of compounds 1–8
for 1 h and then incubated with (+) IL-1 (0.25 ng/mL; A) or TNF- (2.5 ng/mL; B) or
without cytokines (ꢀ) for 15 min in the presence of compounds 1–8. MG-132 was
included to stabilize phosphorylated I by preventing its proteasomal degrada-
tion. Cell lysates were analyzed by Western blotting using antibodies reactive to
phospho-I , I and b-actin.
jBa phosphorylation induced by
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28. Yamamoto, M.; Horie, R.; Takeiri, M.; Kozawa, I.; Umezawa, K. J. Med. Chem.
2008, 51, 5780.
l
a
a
jBa
jBa jBa
NF-
In addition, we found that
pound 1, are essential to inhibit the IL-1-dependent I
phorylation (step 1) and the TNF- -dependent NF-
translocation (step 4) at the earliest points of the pathway. It seems
that compound 3 mainly targets I phosphorylation (step 3) in
B signaling pathway, as a combined phenotype
-methylene- -lactone and -bromo ketone
j
B translocation (step 4) at the earliest points of the pathway.
-bromo ketones, specifically com-
phos-
a
jBa
a
jB
jBa
the common NF-
j
29. Kamiyama, H.; Usui, T.; Sakurai, H.; Shoji, M.; Hayashi, Y.; Kakeya, H.; Osada, H.
Biosci., Biotechnol., Biochem. 2008, 72, 1894.
caused by both
a
c
a
groups.
the early NF-
1 stimulation.
a
-Bromo ketone 1 may be the most useful tool to clarify
jB signaling pathway particularly in response to IL-