Cynaropicrin and inhibition of NF-κB activation: A structure activity relationship study

Article abstract of DOI:10.1016/j.bmcl.2019.04.004

Cynaropicrin is a guaianolide sesquiterpene lactone with a 5-7-5 tricyclic skeleton, four exo-olefins, and two hydroxyl groups. This natural product has various biological activities including anti-inflammatory properties and antitrypanosomal activity. It was also found to suppress photoaging of the skin by inhibiting the transcription activity of nuclear factor-kappa B (NF-κB). In this paper, nine chemical derivatives of cynaropicrin were prepared, and the structure activity relationship (SAR) study for NF-κB inhibition by measuring relative light units (RLU) was conducted.

Full text of DOI:10.1016/j.bmcl.2019.04.004

Accepted Manuscript
Cynaropicrin and inhibition of NF-κB activation: A structure activity relation-
ship study
Jing Li, Yusuke Yoshida, Manami Kurita, Toyonobu Usuki
PII:
S0960-894X(19)30213-6
https://doi.org/10.1016/j.bmcl.2019.04.004
BMCL 26371
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 March 2019
1 April 2019
3 April 2019
Please cite this article as: Li, J., Yoshida, Y., Kurita, M., Usuki, T., Cynaropicrin and inhibition of NF-κB activation:
A structure activity relationship study, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/
10.1016/j.bmcl.2019.04.004
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Cynaropicrin and inhibition of NF-B activation: A structure
activity relationship study
Jing Li a, Yusuke Yoshida b, Manami Kurita a, Toyonobu Usuki a,*
In memory of Professor Koji Nakanishi
a Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia
University, 7-1 Kioicho, Chiyoda-ku, Tokyo 102-8554, Japan
b Adjusted Cell Experiment Laboratory Inc,
5-4-30 Nishi-Hashimoto, Midori-ku, Sagamihara 252-0131, Japan
* Corresponding author. Tel.: +81 3 3238 3446; fax: +81 3 3238 3361; E-mail address:
t-usuki@sophia.ac.jp (T. Usuki)
1

Abstract:
Cynaropicrin is a guaianolide sesquiterpene lactone with a 5-7-5 tricyclic skeleton, four
exo-olefins, and two hydroxyl groups. This natural product has various biological activities
including anti-inflammatory properties and antitrypanosomal activity. It was also found to
suppress photoaging of the skin by inhibiting the transcription activity of nuclear factor-kappa
B (NF-B). In this paper, nine chemical derivatives of cynaropicrin were prepared, and the
structure activity relationship (SAR) study for NF-B inhibition by measuring relative light
units (RLU) was conducted.
Graphical abstract:
Keywords: cynaropicrin; NF-B; SAR; derivatization
2

Main text:
Cynaropicrin (1, Figure 1) is one of the sesquiterpene lactone which was first isolated
by Šorm and co-workers in 1960 from artichokes (Cynara scolymus L).1 The compound 1 was
later also reported to be found in other various Asteraceae, such as Centaurea solstitialis L.,2
Hemisteptia lyrata B.,3 and Saussurea calcicola.4 Cynaropicrin 1, a guaianolide sesquiterpene
lactone, has a 5-7-5 fused tricyclic skeleton with six carbon stereocenters, four exo-olefins,
and two hydroxyl groups. 1 has been shown to exert various biological activities, such as
anti-inflammatory properties,5 distinct bitterness mediated through activation of bitter sensory
receptors,6 and potent in vitro and in vivo inihibitory activity against Trypanosoma brucei, the
protozoan parasite that causes human African trypanosomiasis (HAT or sleeping sickness).7 In
our previous effort, an in vitro structure activity relationship (SAR) study of 1 derivatives
activities against T. brucei was conducted, the results of which suggested that derivatization of
the two hydroxyl groups (OH-3 and OH-19) in 1 does not significantly affect the
antitrypanosomal activity.8 In addition to the antitrypanosomal activity, 1 was also found to
suppress photoaging of skin by inhibiting the transcription activity of nuclear factor-kappa B
(NF-B).9
Figure 1. Structure of cynaropicrin (1). The carbon numbering of 1 was adapted from ref. 2.
3

NF-B was first identified by Barnes as a transcription factor regulating the
expression of K light chains in mouse B lymphocytes in 1986.10 NF-B plays a central role in
regulating of diverse biological processes including immune response, cell development,
growth, and survival.11 NF-B is activated in keratinocytes by stimulation such as by
ultraviolet (UV) rays, which causes photoaging of the skin, characterized by skin wrinkling,
laxity, and pigmentation. In addition, it is activated by inflammatory cytokines such as
interleukin-2 (IL-2), IL-6, IL-8, granulocyte-macrophage colony stimulating factor
(GM-CSF),12 and induces the expression of various genes such as those of basic fibroblast
growth factor (bFGF) and matrix metalloprotease-1 (MMP-1). Moreover, UV irradiation
induces skin cells to secrete bFGF, which is responsible for the proliferation of melanocytes
and keratinocytes9 and promotes the proliferation of skin keratinocytes and melanocytes.13 It
was found that UVB, IL-1, and tumor necrosis factor (TNF) induced NF-B activation, and
produced MMP-1 and bFGF in HaCaT keratinocytes and skin fibroblasts. Thus, inhibition of
the NF-B activation pathway would block the vicious cycle elicited by UV irradiation and
effectively prevent UVB-mediated cutaneous alterations.13 Herein, we screened the effects of
nine derivatizations of cynaropicrin 1 on TNF-mediated NF-B dependent gene expression
by measuring relative light units (RLU).9
As the starting material for preparation of the derivatives, a gram-scale isolation of
cynaropicrin 1 was carried out from Cynara scolymus L. (Asteraceae) according to the
literature procedure.8,14 Derivatization of the obtained natural product 1 was then carried out
as shown in Scheme 1. We envisaged that the side chain of cynaropicrin would be essential
for inhibition of NF-B activation, and aimed to synthesize deacylcynaropicrin 4 at first. For
the synthesis of 4, protection of the two hydroxyl groups (OH-3 and OH-19) was conducted.
4

We have previously reported the synthesis of 4 via introduction of a methoxymethyl (MOM)
group on the two hydroxyl groups.8 However, optical rotation ([]2D0 = +14.5) of 4 via
di-MOM cynaropicrin (2) and deacylcynaropicrin with MOM (3) was not in good agreement
with that of the natural product ([]2D0 = +40.6)3 probably due to the epimerization and/or
racemization of chiral carbon caused by intense conditions during MOM group removal
(pyridinium p-toluenesulfonate (PPTS), tert-buthanol, reflux, 24 h). As an alternative route,
the synthesis of 4 by introduction of a tert-butyl dimethylsilyl (TBS) group on the two
hydroxyl groups of 115 was carried out via compound 3’ followed by removal of the TBS
using tetrabutyl ammonium fluoride (TBAF) in tethrahydrofuran (THF), and thus the optical
rotation ([]2D0 = +37.5) was in agreement with that of the natural one.3
5

H
O
H
O
H
O
H
O
O
O
HO
HO
O
O
OH
O
O
O
ref. 8
ref. 8
O
OH
O
OH
H
H
H
H
O
O
O
O
O
4
9
2
3
ref. 8
a
c
H
O
O
HO
OH
O
H
O
H
H
H
O
O
O
HO
TBSO
TBSO
OH
OTBS
17
O
OH
ref. 15
ref. 15
O
H
H
H
18
11
O
O
13
10
O
O
O
cynaropicrin (1)
2'
3'
ref. 8
b
O
O
H
O
H
H
H
O
O
O
O
HO
O
O
HO
O
O
O
O
O
O
O
O
O
H
H
H
H
O
O
O
O
O
O
O
5
6
7
8
Scheme 1. Reagents and conditions: (a) TBAF, THF, rt, 6 h, 75%; (b) (MeO)2SO2, K2CO3,
acetone, reflux, 6 h, 6% (7), 30% (8); (c) NaBH4, MeOH, rt, 24 h, 45% (9), 6% (10).
6

We next turned our attention to the derivatization of the two hydroxyl groups (OH-3
and OH-19). 3,19-Di-Ac cynaropicrin 5 and 19-Ac cynaropicrin 6 were prepared as reported
in our previous study.8 In addition to these derivatives, methylation of 1 was conducted with
(MeO)2SO2 in acetone at reflux for 31 h16 to give 3,19-di-Me cynaropicrin 7 and 19-Me
cyaropicrin 8 in 6% and 30% yield, respectively. Attempt of reduction of exo-olefines of 1
with NaBH4 in MeOH at room temperature for 24 h afforded 9 and 10 in 45% and 6% yield,
respectively. Exo-olefines of both C17-C18 and C11-C13 were much more reactive than other
exo-olefines, probably due to the chelation between borane and -carbonyl group.
Moreover, tetrapropylammonium perruthenate (TPAP) oxidation,17 oxidation by
Dess-Martin periodinane,18 Trost-Masuyama oxidation,19 and palladium-catalyzed
hydrogenation of 1 were carried out to acquire more derivatives for SAR study. However, no
reaction occurred at all or complex mixtures were obtained. Preparation of the nine
derivatives from natural 1 was achieved through high performance liquid chromatography
(HPLC) purification.20
7

Nine compounds prepared in the synthetic study and natural 1 were tested with
luciferase reporter gene (luc2) for in vitro antiphotoaging activity via inhibition of NF-B
activation (Table 1).21 The DNA element specifically binds to NF-upstream and introduce
the expression vector having a luciferase report gene downstream into HEK293 cell
(transfection). NF-activated by TNF-stimulation then binds to upstream of an
expression to guide onset of gene. The expression of the luciferase gene produces luciferase
protein, which will emit light in the presence of luciferin. Herein the emission of light was
detected for NF-activity. We examined three other concentration of samples (0.75 μM, 1.5
μM, and 3.0 μM), however, no compound showed effective or acceptable activity for 0.75 μM.
Parent compound 1 had strong antiphotoaging activity of 72.85 ± 4.02 and 5.67 ± 1.16 (mean
± standard error of the mean) for 1.5 μM and 3.0 μM, respectively. Di-MOM compound 2
showed moderate activity of 98.35 ± 3.52 for 1.5 μM and 8.97 ± 1.85 for 3.0 μM. Meanwhile,
deacyl derivative 3 showed weak inhibition of NF-activation (125.06 ± 1.91 for 1.5 μM
and 100.42 ± 4.42 for 3.0 μM, respectively), suggesting that the side chain at OH-8 of 1 was
necessary for potent inhibition of NF-B activation.8 Accordingly, deacylcynaropicrin 4 did
not show inhibitory of NF-B activation (118.54 ± 1.80 for 1.5 μM and 96.38 ± 2.06 for 3.0
μM, respectively).
8

Table 1. Relative light units (RLU) of cynaropicrin (1) and derivatives 1-10.
Compd R1
R2
R3-R4
0.75 μM
1.5 μM
3.0 μM
1
H
C=CH2 134.36 ± 8.06 72.85 ± 4.02
C=CH2 154.19 ± 6.19 98.35 ± 3.52
5.67 ± 1.16
8.97 ± 1.85
2
3
4
H
H
C=CH2 126.80 ± 3.59 125.06 ± 1.91 100.42 ± 4.42
C=CH2 126.83 ± 1.90 118.54 ± 1.80 96.38 ± 2.06
H
5
C=CH2 170.79 ± 2.49 141.92 ± 6.60 93.49 ± 1.68
C=CH2 130.03 ± 5.33 109.99 ± 2.31 33.84 ± 2.47
C=CH2 160.54 ± 7.34 151.32 ± 8.69 137.26 ± 4.02
C=CH2 173.60 ± 3.39 158.28 ± 4.89 133.55 ± 6.38
6
H
7
CH3
H
8
9
H
CH-CH3 122.95± 4.74
138.86 ± 3.88 136.65 ± 4.02
10
H
C=CH2 122.95 ± 4.74 135.03 ± 2.50 136.88 ±7.26
a Each value corresponds to the mean ± standard derivation and error of the mean from at
9

least three independent bioassays.
Surprisingly, the activity of di-Ac compound 5 was weaker (RLU 141.92 ± 6.60 for
1.5 μM and 93.49 ± 1.68 for 3.0 μM, respectively) than that of compound 2 which has
di-MOM groups at the same positions. As well as compound 5, compound 7 having di-Me
groups at the same positions showed weaker activity (RLU 151.32 ± 8.69 for 1.5 μM and
137.26 ± 4.02 for 3.0 μM, respectively) than compound 2, whereas compound 6 which has a
Me group at OH-19 showed slightly weaker activity (RLU 109.99 ± 2.31 for 1.5 μM and
33.84 ± 2.47 for 3.0 μM, respectively) than 2. It was thus found that protecting groups at
OH-3 and OH-19 positions affect NF-B activity when compared to compound 1.
We also screened derivatives 9 and 10, and found that neither of them inhibited
NF-B activation (9: RLU 138.86 ± 3.88 for 1.5 μM and 136.65 ± 4.02 for 3.0 μM; 10: RLU
135.03 ± 2.50 for 1.5 μM and 136.88 ±7.26 for 3.0 μM, respectively). This was thus
suggested that exo-olefin at C-17 affect NF-B activity while exo-olefin at C-11 did not.
Based on these results of the SAR study, the following conclusions could be drawn:
1) an acyl side chain at OH-8 is essential for inhibition of NF-B activation; 2) di-MOM
cynaropicrin showed similar activity with natural cynaropicrin 1; 3) an exo-olefine at C-17
affects inhibition of NF-B activation; 4) an exo-olefine at C-11 does not affect NF-B
activity.
10

In summary, synthesis of derivatives of cynaropicrin 1 and determination of their
antiphotoaging activities via inhibitory NF-B activity were carried out as part of a SAR
study. The results suggest that side chain of 1, the hydrophilicity of the OH-3 and OH-19
substituent, and the exo-olefin (C17-C18) significantly affect inhibitory activity against
NF-B. These results will be useful for the design of labeled derivatives, such as
photo-affinity, spin-probe labels, etc, for further biological studies of sesquiterpene lactones.
Acknowledgements
This paper is dedicated to Professor Koji Nakanishi (Columbia University). We thank
Dr. Michael Adams (University of Basel, currently Bacoba AG, Switzerland) for his supports
and suggestions.
Supplementary data
Supplementary data (1H and 13C NMR spectra of compounds 7, 8, 9 and 10, and
summary of bioassay) associated with this article can be found in the online version, at http://
11

Reference and notes
1. Suchý, M.; Herout, V.; Šorm, F. Collect. Czech. Chem. Commun. 1960, 25, 2777.
2. Wang, Y.; Hamburger, M.; Cheng, C. H. K.; Costall, B.; Naylor, R. J.; Jenner, P.;
Hostettmann, K. Helv. Chim. Acta 1991, 74, 117.
3. Ha, T. J.; Jang, D. S.; Lee, J. R.; Lee, K. D.; Lee, J.; Hwang, S. W.; Jung, H. J.; Nam, S.
H.; Park, K. H.; Yang, M. S. Arch. Pharm. Res. 2003, 26, 925.
4. Choi, S. Z.; Choi, S. U.; Lee, K. R. Arch. Pharm. Res. 2005, 28, 1142.
5. Cho, J. Y.; Baik, K. U.; Jung, J. H.; Park, M. H. Eur. J. Pharm. 2000, 398, 399.
6. Cravotto, G.; Nano, G. M.; Binello, A.; Spagliardi, P.; Seu, G. J. Sci. Food Agric. 2005, 85,
1757.
7. Zimmermann, S.; Kaiser, M.; Brun, R.; Hamburger, M.; Adams, M. Planta Med. 2012, 78,
533.
8. Usuki, T.; Sato, M.; Hara, S.; Yoshimoto, Y.; Kondo, R.; Zimmermenn, A.; Kaiser, M.;
Brun R.; Hamburger, M.; Adams, M. Bioorg. Med. Chem. Lett. 2014, 24, 794.
9. Tsuda-Tanaka, Y.; Tanaka, K.; Kojima, H.; Hamada, T.; Masutani, T.; Tsuboi, M.; Akao, Y.
Bioorg. Med. Chem. Lett. 2013, 23, 518.
10. Barnes, J. Int. J. Biochem. Cell Biol. 1997, 29, 867.
11. Xiao, G.; Rabosn, B, A.; Young, W.; Qing, G.; Qu, Z. Cytokine & Growth Factor Reviews
2006, 17, 281.
12. Okamoto, T.; Sakurada, S.; Yang, J, P. Merin, J, P. Curr. Top. Cell. Regul. 1997, 35, 149.
13. Tanaka, K.; Hasegawa, J.; Asamitsu, K.; Okamoto, T. J. Pharmacol. Exp. Ther. 2005, 315,
624.
14. Adams, M.; Zimmrmann, S.; Kaiser, M.; Brun, R.; Hamburger, M. Nat. Prod. Commun.
2009, 4, 1377.
15. Sato, T.; Hara, S.; Sato, M.; Ogawa, K.; Adams, M.; Usuki, T. Bioorg. Med. Chem. Lett.
2015, 25, 5504.
16. Tanaka, A.; Arai, Y.; Kim, S. N.; Ham, J.; Usuki, T. J. Asian Nat. Prod. Res. 2011, 13,
290.
17. Griffith, W. P.; Ley, S. V.; Whitcombe, G. P.; White, A. D. J. Chem. Soc., Chem. Commun.
1987, 1625.
18. Dess, D. B.; Martin, J. C. J. Org. Chem. 1983, 48, 4155.
19. (a) Trost, B. M.; Masuyama, Y. Tetrahedron Lett. 1984, 25, 173. (b) Yoshimura, F.;
Miyashita, M. Chem. Eur. J. 2009, 15, 6626.
20. General: All non-aqueous reactions were conducted under an atmosphere of nitrogen
with magnetic stirring. CH2Cl2 was dried using activated molecular sieves. All reagents
were obtained from commercial suppliers and used without further purification unless
otherwise stated. Analytical thin layer chromatography (TLC) was performed on silica
12

gel 60 F254 plates produced by Merck KGaA (Darmstadt, Germany). Column
chromatography was performed with acidic silica gel 60 (spherical, 40-50 m; Kanto
Chemicals Co. Ltd, Tokyo, Japan). Optical rotations were measured on a JASCO P-2200
digital polarimeter at the sodium lamp (= 589 nm) D line and are reported as follows:
[]DT (c g/100 mL, solvent). Infrared (IR) spectra were recorded on a JASCO FT-IR 4100
spectrometer and are reported in wavenumbers (cm-1). 1H and 13C nuclear magnetic
resonance (NMR) spectra were recorded on a JEOL JNM-EXC 300 spectrometer (300
MHz). 1H NMR data are reported as follows: chemical shift (, ppm), integration,
multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad), coupling
constants (J) in Hz, assignments. 13C NMR data are reported in terms of chemical shift (,
ppm). Fast atom bombardment (FAB) MS spectra were recorded on a JEOL JMS-700
instrument. Electrospray ionization (ESI) MS spectra were recorded on a JEOL
JMS-T100LC instrument.
Isolation of cynaropicrin (1): Extraction and isolation of cynaropicrin from artichoke
leaves were achieved by the following published protocol.8,14 Dried artichoke leaves (100
g) obtained from Dixa AG (Gallen, Switzerland) through Dr. Michael Adams were
exhaustively extracted three times with ethyl acetate (900 mL) yielding 5.4 g of crude
extract. The extract was separated on a silica gel column chromatography with ethyl
acetate as mobile phase. Fractions containing 1 were concentrated to give 1.7 g of impure
cynaropicrin 1. Purification via silica gel column chromatography (hexane/EtOAc = 1:2)
afforded 1 as a yellow solid (1.4 g).
(1R,3S,5R,6R,7R,8S)-3-Hydroxy-13,14,15-trimethylene-12-oxododecahydroazuleno[
6,7-]furan-8-yl methacrylate (4): To a solution of 3’ (24 mg, 0.064 mmol, 1.0 eq)
under dry nitrogen atmosphere in THF (1.0 mL) was added TBAF (46.0 μL, 0.92 mmol,
2.5 eq) at 0 °C. After stirring 6 h under room temperature, the mixture was diluted with
EtOAc and quenched with saturated NH4Cl solution. The aqueous layer was then
extracted with EtOAc. The combined organic layers were washed with brine, dried over
Na2SO4, and concentrated in vacuo. Purification on silica gel column chromatography
(hexane/EtOAc = 1:1) afforded 4 as a colorless oil (12.7 mg, 0.048 mmol, 75%); Rf 0.20
1
20
(hexane/EtOAc = 1:1); [] +37.5 (c 1.2, CHCl3); H NMR (300 MHz, CDCl3) 
D
6.27-6.26 (1H, d, J = 3.0 Hz, H13), 6.15-6.14 (1H, d, J = 3.0 Hz, H13), 5.48 (1H, s, H15),
5.34 (1H, s, H15), 5.13 (1H, s, H14), 4.98 (1H, s, H14), 4.55-4.54 (1H, m, H3), 4.18-4.08
(1H, m, H6), 3.99-3.93 (1H, m, H8), 3.01 (1H, m, H1), 2.92-2.67 (3H, m, H5/7/9),
2.32-2.18 (2H, m, H2/9), 1.78-1.67 (1H, m, H2); FAB-MS (m/z) calcd for C15H19O4
[M+H]+ 263.12, found 263.12.
(1R,3S,5R,6R,7R,8S)-3-Methoxy-13,14,15-trimethylene-12-oxododecahydroazuleno[
6,7-]furan-8-yl 17-(methoxy)acrylate (7) and
13

(1R,3S,5R,6R,7R,8S)-3-hydroxy-13,14,15-trimethylene-12-oxododecahydroazuleno[6
,7-]furan-8-yl 17-(methoxy)acrylate (8): To a solution of cynaropicrin 1 (50.0 mg,
0.144 mmol, 1.0 eq) and K2CO3 (80.0 mg, 0.576 mmol) in acetone (2.0 mL) was added
(MeO)2SO2 (55.0 µL, 0.576 mmol). After stirring at reflux for 6 h, the reaction mixture
was cooled to room temperature and filtered. After concentration to remove acetone,
EtOAc and saturated NaHCO3 solution were added and stirred for 5 min, then extracted
with EtOAc. The combined organic layers were washed with 2 M HCl, saturated
NaHCO3 solution, brine, dried over Na2SO4, and concentrated in vacuo. Purification by
silica gel column chromatography (hexane/EtOAc = 1:1) afforded 7 as a colorless oil (3.0
1
20
mg, 0.008 mmol, 6%); [] +108.1 (c 0.5, CHCl3); H NMR (300 MHz, CDCl3) 6.37
D
(1H, s, H19), 6.21-6.20 (1H, d, J = 6.0 Hz, H13), 5.93 (1H, s, H19), 5.62-5.61 (1H, d, J =
3.0 Hz, H13), 5.42 (1H, s, H15), 5.27 (1H, s, H15), 5.16-5.10 (2H, m, H8, H14),
4.90-4.89 (1H, m, H14), 4.32-4.26 (1H, m, H3), 4.17-4.03 (2H, m, H18), 4.01-4.00 (1H, t,
J = 4.5 Hz, H6), 3.40 (1H, s, Me), 3.31 (1H, s, Me), 3.17-3.10 (1H, m, H7), 3.04-2.98
(1H, m, H1), 2.78-2.66 (1H, m, H5), 2.43-2.24 (1H, m, H9), 1.81-1.72 (1H, m, H2), 1.59
(1H, m, H2); 13C NMR (75 MHz, CDCl3) 167.6, 165.3, 150.3, 148.6, 144.1, 137.6,
126.1, 122.6, 117.6, 115.6, 81.4, 60.1, 56.9, 52.1, 46.9, 44.9, 38.2, 37.3, 34.7, 14.5;
ESI-HRMS (m/z) calcd for C20H24NaO6 [M+Na]+ 383.1471, found 383.1451. Purification
on silica gel column chromatography (hexane/EtOAc = 1:2) afforded 8 as a colorless oil
(15.4 mg, 0.043 mmol, 30%); Rf 0.21 (hexane/EtOAc = 1:2); 1H NMR (300 MHz,
CDCl3) 6.37-6.36 (1H, m, H19), 6.22-6.21 (1H, d, J = 3.0 Hz, H13), 5.94 (1H, m, H19),
5.65-5.64 (1H, d, J = 3.0 Hz, H13), 5.49 (1H, m, H15), 5.35 (1H, m, H15), 5.14-4.94 (2H,
m, H8/H14), 4.58-4.53 (1H, m, H14), 4.27-4.16 (2H, m, H18), 3.39 (3H, s, Me),
3.23-3.15 (1H, m, H7), 3.02-2.93 (1H, m, H1), 2.87-2.81 (1H, m, H5), 2.73-2.62 (1H, m,
H9), 2.42-2.36 (1H, m, H9), 2.27-2.18 (1H, m, H2), 1.78-1.67 (1H, m, H2); ESI-HRMS
(m/z) calcd for C21H26NaO6 [M+Na]+ 397.1627, found 397.1630.
(1R,3S,5R,6R,7R,8S)-3-Hydroxy-14,15-trimethylene-13-methyl-12-oxododecahydroa
zuleno[6,7-]furan-8-yl 17-methyl-methacrylate (9) and
(1R,3S,5R,6R,7R,8S)-3-hydroxy-13,14,15-trimethylene-12-oxododecahydroazuleno[6
,7-]furan-8-yl 17-methyl-methacrylate (10): To a solution of 1 (48.0 mg, 0.139 mmol,
1.0 eq) under dry nitrogen atmosphere, were dissolved in 1.0 ml of MeOH. NaBH4 (10.5
mg, 0.278 mmol, 2.0 eq) was added and the mixture was magnetically stirred for 24 h at
room temperature. The reaction was monitored by TLC, elunted with hexane/EtOAc =
1:3 (Rf 9 = 0.40, Rf 10 = 0.30). For work-up, the reacted mixture was diluted with H2O
and acidified with 2 M H2SO4. The organic phase was washed with H2O and saturated
brine, dried over anhydrous Na2SO4, and concentrated in vacuo. Purification by silica gel
column chromatography (hexane/EtOAc = 1:3) afforded 9 as a colorless oil (22.0 mg,
14

0.063 mmol, 45%); 1H NMR (300 MHz, CDCl3): 5.40 (1H, m, H15), 5.32-5.29 (1H, m,
H15), 5.09-4.98 (3H, m, H8/14), 4.56-4.51 (1H, m, H3), 4.05-4.02 (1H, m, H6),
3.77-3.73 (2H, m, H19), 2.93-2.87 (1H, m, H1), 2.83-2.80 (1H, m, H17), 2.74-2.68 (2H,
m, H7/11), 2.58/2.52 (1H, m, H5), 2.31-2.18 (3H, m, H2/9), 1.79 (1H, m, H2), 1.42-1.39
(3H, t, J = 9.0 Hz, H13), 1.23 (3H, m, H18); 13C NMR (75 MHz, CDCl3):  177.5, 165.1,
152.5, 141.9, 139.1, 126.3, 117.3, 112.3, 78.7, 76.2, 73.4, 62.1, 53.2, 50.4, 44.1, 41.1,
40.3, 38.6, 15.3; ESI-MS (m/z) calcd for C19H26O6 [M]+ 350.17, found 350.17.
Purification by silica gel column chromatography (hexane/EtOAc = 1:3) afforded 10 as a
colorless oil (2.8 mg, 0.008 mmol, 6%); 1H NMR (300 MHz, CDCl3): 6.14 (1H, m,
H13), 5.65-5.64 (1H, m, H13), 5.42 (1H, m, H15), 5.34-5.32 (1H, m, H15), 5.14 (1H, m,
H8), 5.04-5.02 (2H, m, H14), 4.55 (1H, m, H3), 4.08 (1H, m, H6), 2.95-2.87 (2H, m,
H19), 2.84-2.77 (1H, m, H1), 2.74-2.72 (1H, m, H17), 2.55-2.49 (1H, m, H5), 2.34-2.31
(1H, m, H7), 2.27-2.25 (1H, m, H9), 2.23-2.20 (1H, m, H2), 1.97 (1H, m, H2), 1.23 (3H,
m, H18).
21. NF-kB assay: HEK 293 cells (RIKEN RBC, Japan) were dispensed into the wells of a
multi-well plate (1.5  104 cells/well in 96-well plate) in minimum essential media
(MEM) supplemented with 10% fetal bovine serum (FBS), 1% non-essential amino acids
(NEAA) and incubated at 37 °C for 24 h. Each well of 96-well plate was added with
10μL of transfection reagent containing 0.01 µg pGL4.32 [luc2P/NF-κB-RE/Hygro]
(Promega, USA), 10 μL of MEM and 0.1μL of X-tremeGENE HP reagent (Roche,
Switzerland) and incubated at 37 °C for 24 h. After the supernatants were exchanged for
MEM including 10% FBS, 1% NEAA and compounds, and then incubated at 37 °C for 2
h, 5 μL of 200 ng/mL TNF-α (Sigma-Aldrich, USA) was added to each well of 96-well
plate. After 5 h of culture, 30 μL of supernatant was removed from each well, and 70 μL
of Bright-Glo Luciferase Assay Reagent (Promega, USA) was added. After 2 min of
mixing, the relative light units (RLU) as a NF-κB activity were measured using a
microplate reader Varioskan (Thermo Fisher Scientific, USA).
Cytotoxicity: HEK 293 cells (1.5 X 104 cells/well in 96-well plate) were plated in MEM
supplemented with 10% FBS, 1% NEAA and incubated at 37 °C for 24 h. The
supernatant of each well was changed for MEM including 10% FBS, 1% NEAA and
compounds, and then incubated at 37 °C for 7 h. The viability of the cultures was
determined colorimetrically using WST-8 reagent (Nacalai Tesque, Japan) in a modified
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) reduction assay. In
brief, culture supernatants were replaced by MEM medium including 10% WST-8
reagent and incubated at 37 °C for 1 h. The absorbance was measured at 450 nm using a
microplate reader. Cell viabilities are presented as absorbance changes per hour.
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Graphical abstract
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