Antitrypanosomal structure-activity-relationship study of synthetic cynaropicrin derivatives

Article abstract of DOI:10.1016/j.bmcl.2013.12.099

Cynaropicrin is a guaianolide sesquiterpene lactone with a 5-7-5 tricyclic skeleton, four exo-olefins, and two hydroxyl groups. Recently, it was found that the compound is a potent in vitro and in vivo inhibitor of the protozoan parasite Trypanosoma brucei, which causes human African trypanosomiasis (HAT; sleeping sickness). In this Letter, chemical derivatization of cynaropicrin and the structure-activity-relationship (SAR) study against T. brucei is described.

Full text of DOI:10.1016/j.bmcl.2013.12.099

Bioorganic & Medicinal Chemistry Letters 24 (2014) 794–798
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antitrypanosomal structure–activity-relationship study of synthetic
cynaropicrin derivatives
a
a
a
a
Toyonobu Usuki a, , Makiko Sato , Shihori Hara , Yukiko Yoshimoto , Ryosuke Kondo ,
⇑
Stefanie Zimmermann b,c, Marcel Kaiser c, Reto Brun c, Matthias Hamburger b, Michael Adams b
a Department of Materials and Life Sciences, Faculty of Science and Technology, Sophia University, 7-1 Kioicho, Chiyoda-ku, Tokyo 102-8554, Japan
b Division of Pharmaceutical Biology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland
c Department of Medical Parasitology and Infection Biology, Swiss Tropical and Public Health Institute, Socinstrasse 57, CH-4002 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
Cynaropicrin is a guaianolide sesquiterpene lactone with a 5-7-5 tricyclic skeleton, four exo-olefins, and
two hydroxyl groups. Recently, it was found that the compound is a potent in vitro and in vivo inhibitor of
the protozoan parasite Trypanosoma brucei, which causes human African trypanosomiasis (HAT; sleeping
sickness). In this Letter, chemical derivatization of cynaropicrin and the structure–activity-relationship
(SAR) study against T. brucei is described.
Received 9 December 2013
Revised 20 December 2013
Accepted 23 December 2013
Available online 2 January 2014
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Cynaropicrin
Guaianolide
Trypanosoma brucei
SAR
Derivatization
Cynaropicrin (1, Fig. 1) was first isolated by Šorm and co-work-
ers in 1960 from Cynara scolymus L. and is the source of the bitter
taste of artichokes.1 The compound was later also reported from
other Asteraceae such as, for example, Centaurea solstitialis L.,2
Hemisteptia lyrata B.,3 and Saussurea calcicola.4 Classified as a guai-
anolide sesquiterpene lactone, 1 has a 5-7-5 fused tricyclic skele-
ton with six carbon stereocenters, four exo-olefins, and two
hydroxyl groups. Cynaropicrin 1 has been shown to possess vari-
ous biological activities, such as anti-inflammatory properties,5
Figure 1. Structures of cynaropicrin (1) and deacylcynaropicrin (2). The carbon
numbering of 1 was adapted from Ref. 2.
inhibition of NF-j
B,6 and distinct bitterness mediated through
activation of bitter sensory receptors.7
In a screening of 1800 plant and fungal extracts and follow-up
identification of active compounds, guaianolide cynaropicrin 1
was discovered as a compound with potent in vitro activity against
the protozoan parasite Trypanosoma brucei,8 the causative agent of
human African trypanosomiasis (HAT), also known as ‘sleeping
sickness’.9,10 The compound was able to reduce parasitaemia in a
murine model for HAT, although it did not cure the animals. HAT
is transmitted by blood feeding tsetse flies occurring in sub-Saha-
ran Africa and is fatal when left untreated. Approximately 95% of
HAT cases are caused by T. b. gambiense, which occurs in Central
and West Africa. The remaining 5% in East and Southern Africa
are due to T. b. rhodesiense, which evokes a more acute form of
the disease. HAT appears in two stages: an initial hemolymphatic
stage followed by an encephalitic second stage characterized by
the invasion of the parasite into the central nervous system
(CNS), which causes the breakdown of neurological functions.11,12
Presently, only a few drugs are available to treat HAT, all of
which are donated to the World Health Organization (WHO) by
the producers. Melarsoprol, an arsenic, is still the only option for
the treatment of second stage T. b. rhodesiense HAT and is accom-
panied by potentially lethal side effects.13 While the number of
cases has decreased since 2001,14 the complete extermination of
HAT is still considered to be very difficult with the current diagnos-
tic methods and drugs, and the poor economic and unstable polit-
ical conditions in the affected regions.
The antitrypanosomal natural product 1 has been described as a
⇑
promising new drug lead, with potent effects in vivo.8 Its
Corresponding author. Tel.: +81 3 3238 3446; fax: +81 3 3238 3361.
E-mail address: t-usuki@sophia.ac.jp (T. Usuki).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.12.099

T. Usuki et al. / Bioorg. Med. Chem. Lett. 24 (2014) 794–798
795
mechanism of action was recently described by Zimmermann
et al., and is due to its depleting of intracellular trypanothione as
well as the inhibition of trypanosomal ornithine decarboxylase.15
Herein, we describe the results of a structure–activity-relationship
(SAR) study of 1 derivatives in vitro activities against T. brucei. Pre-
viously, deacylcynaropicrin (2, Fig. 1) prepared via the direct
hydrolysis of 1, was found to exhibit activity against T. brucei less
than ten times that of natural 1, suggesting that the side chain moi-
ety of 1 plays a crucial role in its inhibitory activity.8
As the starting material for preparation of derivatives, a gram-
scale isolation of cynaropicrin 1 was carried out from C. scolymus
L. (Asteraceae) according to the literature procedure.8,16 The iden-
tity was confirmed by complete 1H and 13C NMR assignments via
the 2D-NMR spectrometry.3,4 Derivatization of the obtained natu-
ral product 1 was then carried out as shown in Scheme 1. First,
introduction of a methoxymethyl (MOM) group on the two hydro-
xyl groups (OH-3 and OH-19) was performed by treatment with
MOMCl, iPr2NEt, and tetrabutylammonium iodide (TBAI) in dichlo-
romethane (CH2Cl2) under reflux conditions for 19 h to afford the
3,19-di-MOM derivative 3 in 93% yield. When the reaction was
run at room temperature for 20 h, the desired compound 3 was
obtained in 79% yield.
Cheirolophus species,19 was prepared in 68% yield from 4 by using
PPTS in tBuOH for 28 h. The yield was significantly improved when
compared to that previously reported for the direct hydrolysis of
1.8
Cynaropicrin 1 was acetylated in order to prepare additional
derivatives (Scheme 1). Treatment of 1 with acetic anhydride
(Ac2O) and Et3N in CH2Cl2 at 0 °C for 24 h gave the primary alcohol
product 19-Ac cynaropicrin 7 along with 3,19-di-Ac cynaropicrin 8
in 50% and 8% yield, respectively. Compounds 7 and 8 have also
been isolated from plants, such as Centaurea behen,20 C. omephalo-
tricha,21 and Saussurea katochaete.22 Overall, the organic synthesis
of a total of seven derivatives from natural 1 was achieved.23
All of the compounds prepared in the synthetic study were
tested for in vitro activity against T. b. rhodesiense, and for cytotox-
icity in mammalian cells (rat skeletal myoblasts, L6 cells)
(Table 1).24 Parent compound 1 had half maximum inhibitory con-
centration (IC50) values of 0.28 0.001 and 2.19 0.27 lM
(mean standard error of the mean) against T. b. rhodesiense and
L6 cells, respectively. The selectivity index (SI) value calculated
as IC50 L6 cells/IC50 T. b. rhodesiense cells was 7.8. Deacyl derivative
2 showed weaker T. b. rhodesiense antitrypanosomal activity and
cytotoxicity (IC50 of 4.92 0.34 and 4.94 2.08 lM, respectively),
Hydrolysis of 3 was then conducted in aqueous NaOH at room
temperature for 16 h to give 3-MOM deacylcynaropicrin 4 in 84%
yield. Esterification of alcohol 4 via a Yamaguchi esterification17
using methacrylic acid, 2,4,6-trichlorobenzoyl chloride, triethyl-
amine (Et3N), and N,N-dimethylamino pyridine (DMAP) in toluene
was then carried out at reflux for 7.5 h to afford ester 5. Treatment
of 5 with pyridinium p-toluenesulfonate (PPTS) in tBuOH at reflux
for 26 h afforded cynaropicrin derivative 6 in 89% yield. Compound
6 is also known as the natural product aguerin B18 and has a C-19
methyl group on the side chain, rather than the methyl alcohol
found in 1. In addition, deacylcynaropicrin 2, which has also been
isolated as a natural product from Centaurea canariensis18 and
suggesting that the side chain at OH-8 in 1 was necessary for po-
tent activity against T. b. rhodesiense.8 In compound 3, introduction
of MOM groups at OH-3 and OH-19 positions did not decrease the
antitrypanosomal activity (IC50 of 0.48 0.15
cytotoxicity (IC50 of 0.17 0.07 M). The introduction of a MOM
group at OH-3 position only (compound 4) did not significantly en-
hance the antitrypanosomal activity (IC50 of 2.21 0.43 M), but
did result in higher cytotoxicity (IC50 of 0.86 0.51 M) compared
to that of 2. It was also found that derivatization at OH-19 only did
not affect antitrypanosomal activity (5: IC50 of 0.34 0.01 M; 6:
IC50 of 0.17 0.04 M) when compared to compound 1. However
cytotoxicity in L6 cells was increased (5: IC50 of 0.30 0.16 M;
lM), but increased
l
l
l
l
l
l
Scheme 1. Reagents and conditions: (a) MOMCl, iPr2NEt, TBAI, CH2Cl2, reflux, 19 h, 93%; (b) 2 N NaOH, rt, 16 h, 84%; (c) methacrylic acid, 2,4,6-trichlorobenzoyl chloride,
Et3N, DMAP, toluene, reflux, 7.5 h, 43%; (d) PPTS, tBuOH, reflux, 26 h, 89%; (e) PPTS, tBuOH, reflux, 28 h, 68%; (f) Ac2O, Et3N, CH2Cl2, 0 °C, 24 h, 50% (7), 8% (8).

796
T. Usuki et al. / Bioorg. Med. Chem. Lett. 24 (2014) 794–798
Table 1
In vitro antitrypanosomal activity and L6 cytotoxicity of cynaropicrin (1) and derivatives 2–8
a
Values are expressed in
Selectivity index (SI): calculated as IC50 L6 cells/IC50 T. b. rhodesiense cells.
l
M. Each value corresponds to the mean standard error of the mean from at least three independent bioassays.
b
6: IC50 of 0.33 0.18
1.9). Furthermore, acetylation at OH-19 and/or OH-3 positions de-
creased antitrypanosomal activity (7: IC50 of 1.86 0.31 M; 8:
IC50 of 2.13 0.34 M) and cytotoxicity (7: IC50 of 6.39 2.67
M; 8: IC50 of 4.94 2.08 M), leading to a similar decrease in
l
M), resulting in lower SI values (5: 0.9; 6:
versity) for their preliminary experiments. This work was sup-
ported, in part, by a Grant-in-Aid for Young Scientists (B) from
the Ministry of Education, Culture, Sports, Science and Technology
of Japan (to T.U.).
l
l
l
l
the SI values (7: 3.4; 8: 2.3) as compared to that of 1.
Supplementary data
Based on these results of the SAR study, the following conclu-
sions could be drawn: (1) an acyl side chain at OH-8 is essential
for antitrypanosomal activity and the 2-hydroxylmethyl-2-prope-
noic acid side chain affects SI value; (2) changing the hydrophilicity
of the OH-3 substituent affects neither antitrypanosomal activity
nor cytotoxicity; (3) changing the hydrophilicity of OH-19 group
does not affect the antitrypanosomal activity, but does influence
the cytotoxicity; (4) formation of an ester at the OH-19 position
decreases both the antitrypanosomal activity and cytotoxicity;
and (5) introduction of Ac groups at OH-19 and OH-3 positions
leads to a decrease in both the antitrypanosomal activity and
cytotoxicity.
In summary, synthesis of derivatives of cynaropicrin 1 and
determination of their in vitro activities against T. b. rhodesiense
and L6 cells were carried out as part of a SAR study. The results
suggest that derivatization of the two hydroxyl groups (OH-3 and
OH-19) in 1 does not significantly affect the antitrypanosomal
activity. This fact will be useful for the design of labeled deriva-
tives, such as photo-affinity, spin-probe labels, etc, for further
biological studies of sesquiterpene lactones.
Supplementary data (1H and 13C NMR spectra of new com-
pounds 3, 4, and 5) associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
12.099.
References and notes
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Akao, Y. Bioorg. Med. Chem. Lett. 2013, 23, 518.
7. Cravotto, G.; Nano, G. M.; Binello, A.; Spagliardi, P.; Seu, G. J. Sci. Food Agric.
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8. Zimmermann, S.; Kaiser, M.; Brun, R.; Hamburger, M.; Adams, M. Planta Med.
2012, 78, 553.
9. http://www.who.int/mediacentre/factsheets/fs259/en/.
10. Brun, R.; Blum, J.; Chappuis, F.; Burri, C. Lancet 2010, 375, 148.
11. Buguet, A.; Bert, J.; Tapie, P.; Tabaraud, F.; Doua, F.; Lonsdorfer, J.; Bogue, P.;
Dumas, M. J. Clin.Neurophysiol 1993, 10, 190.
Acknowledgments
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Infect. Dis 2008, 8, 16.
13. Blum, J.; Nkunku, S.; Burri, C. Trop. Med. Int. Health 2001, 6, 390.
We thank Professor Motoo Tori (Tokushima Bunri University)
for his kind support, Mr. Takuya Sato and Ms. Jing Li (Sophia Uni-

T. Usuki et al. / Bioorg. Med. Chem. Lett. 24 (2014) 794–798
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14. Simarro, P. P.; Franco, J. R.; Diarra, A.; Ruiz Postigo, J. A.; Jannin, J. Res. Rep. Trop.
Med. 2013, 4, 1.
15. Zimmermann, S.; Oufir, M.; Leroux, A.; Krauth-Siegel, R. L.; Becker, K.; Kaiser,
M.; Brun, R.; Hamburger, M.; Adams, M. Bioorg. Med. Chem. 2013, 21, 7202.
16. Adams, M.; Zimmermann, S.; Kaiser, M.; Brun, R.; Hamburger, M. Nat. Prod.
Commun. 2009, 4, 1377.
17. Inanaga, J.; Hirata, K.; Saeki, H.; Katsuki, T.; Yamaguchi, M. Bull. Chem. Soc. Jpn.
1979, 52, 1989.
18. González, A. G.; Bermejo, J.; Cabrera, I.; Massanet, G. M.; Mansilla, H.; Galindo,
A. Phytochemistry 1978, 17, 955.
The combined organic layers were washed with brine, dried over Na2SO4, and
then concentrated in vacuo. Purification on silica gel column chromatography
(hexane/Et2O = 1:1) afforded 4 as a colorless oil (41.9 mg, 0.137 mmol, 84%); Rf
0.45 (hexane/EtOAc = 1:2); ½a D20
ꢀ
+40.2 (c 0.45, CHCl3); IR (neat, cmꢁ1) 3460,
2936, 1762, 1640, 1452, 1273, 1149, 1040, 960, 910, 818; 1H NMR (300 MHz,
CDCl3) d 6.23 (1H, d, J = 3.7 Hz, H13), 6.12 (1H, d, J = 3.3 Hz, H13), 5.45 (1H, s,
H15), 5.30 (1H, s, H15), 5.11 (1H, s, H14), 4.95 (1H, s, H14), 4.75 (1H, d,
J = 7.1 Hz, CH2) 4.64 (1H, d, J = 7.1 Hz, CH2), 4.41 (1H, t, J = 6.6 Hz, H3), 4.14 (1H,
dd, J = 9.1, 1.4 Hz, H6), 3.97–3.91 (1H, m, H8), 3.37 (3H, s, Me), 3.01–2.92 (1H,
m, H8), 2.80–2.64 (3H, m, H5/7/9), 2.31–2.21 (2H, m, H2/9), 1.83–1.73 (1H, m,
H2); 13C NMR (75 MHz, CDCl3) d 169.8, 148.7, 142.9, 138.3, 122.9, 117.0, 114.7,
94.8, 78.5, 77.7, 71.9, 55.4, 52.0, 50.8, 45.8, 41.1, 37.3; ESI-HRMS (m/z) calcd for
19. Marco, J. A.; Sanz-Cervera, J. F.; Garcia-Lliso, V.; Suzanna, A.; Garcia-Jacas, N.
Phytochemistry 1994, 37, 1101.
20. Rustaiyan, A.; Niknejad, A.; Zdero, C.; Bohlmann, F. Phytochemistry 1981, 20,
2427.
C
17H21NaO5 [M+Na]+ 329.1365, found 329.1349.
(1R,3S,5R,6R,7R,8S)-3-(Methoxymethoxy)-13,14,15-trimethylene-12-oxododecahy-
21. Kolli, E. H.; León, F.; Benayache, F.; Estévez, S.; Quintana, J.; Estévez, F.; Brouard,
I.; Bermejo, J.; Benayache, S. J. Braz. Chem. Soc. 2012, 23, 977.
22. Saito, Y.; Iwamoto, Y.; Okamoto, Y.; Gong, X.; Kuroda, C.; Tori, M. Nat. Prod.
Commun. 2012, 7, 447.
droazuleno[6,7-ß]furan-8-yl methacrylate (5): To a solution of methacrylic acid
(55.4
trichlorobenzoyl chloride (0.10 mL, 0.66 mmol, 10.9 equiv) and Et3N (91.5
0.66 mmol, 10.9 equiv). After stirring for 30 min at room temperature,
lL, 0.66 mmol, 10.9 equiv) in toluene (1 mL) was added 2,4,6-
l
L,
a
23. General: All non-aqueous reactions were conducted under an atmosphere of
nitrogen with magnetic stirring. CH2Cl2, iPr2NEt, and toluene were dried using
activated molecular sieves. PPTS was prepared following literature published
procedure (Miyashita, M.; Yoshikoshi, A.; Grieco, P. A. J. Org. Chem. 1977, 42,
3772.), and DMAP was recrystallized from toluene. All reagents were obtained
from commercial suppliers and used without further purification unless
otherwise stated. Analytical thin layer chromatography (TLC) was performed
on silica gel 60 F254 plates (Merck). Column chromatography was performed
solution of 4 (18.5 mg, 0.060 mmol, 1.0 equiv) and DMAP (14.6 mg, 0.12 mmol,
2.0 equiv) in toluene (1.0 mL) was added to the reaction mixture. After stirring
for 7.5 h under reflux condition, the mixture was diluted with Et2O, filtered,
and then concentrated in vacuo. Purification on silica gel column
chromatography (hexane/EtOAc = 3:1) afforded 5 as a yellow oil (10.0 mg,
0.027 mmol, 43%); Rf 0.52 (hexane/EtOAc = 2:1); ½a D25
ꢀ
+65.8 (c 0.15 in CHCl3);
IR (neat, cmꢁ1) 3523, 2929, 1770, 1717, 1637, 1453, 1294, 1268, 1147, 1022,
914, 814; 1H NMR (300 MHz, CDCl3) d 6.21 (1H, d, J = 3.8 Hz, H13), 6.18 (1H, s,
H18), 5.67 (1H, s, H18), 5.59 (1H, d, J = 3.1 Hz, H13), 5.48 (1H, s, H15), 5.34 (1H,
s, H15), 5.14–5.06 (2H, m, H8/14), 4.80 (1H, s, H14), 4.78 (1H, d, J = 6.9 Hz, CH2),
4.65 (1H, d, J = 6.9 Hz, CH2), 4.34 (1H, t, J = 6.3 Hz, H3), 4.25 (1H, dd, J = 10.7,
9.3 Hz, H6), 3.39 (3H, s, Me), 3.21–3.13 (1H, m, H7), 3.00 (1H, dd, J = 18.2,
8.6 Hz, H1), 2.79 (1H, t, J = 10.7 Hz, H5), 2.70 (1H, dd, J = 14.8, 5.2 Hz, H9), 2.39
(1H, dd, J = 14.8, 3.4 Hz, H9), 2.34–2.24 (1H, m, H2), 1.99 (3H, s, H19), 1.80 (1H,
ddd, J = 13.8, 10.3, 6.2 Hz, H2); 13C NMR (75 MHz, CDCl3) d 169.2, 166.4, 148.5,
142.0, 137.8, 136.0, 126.5, 122.4, 118.0, 115.1, 94.8, 78.3, 77.6, 74.0, 55.5, 52.1,
47.4, 45.9, 37.1, 36.9, 18.2; ESI-HRMS (m/z) calcd for C21H26NaO6 [M+Na]+
397.1627, found 397.1628.
with acidic silica gel 60 (spherical, 40–50 lm; Kanto Chemicals). Optical
rotations were measured on a JASCO P-2200 digital polarimeter at the sodium
lamp (k = 589 nm) D line and are reported as follows: ½a DT
ꢀ
(c g/100 mL, solvent).
Infrared (IR) spectra were recorded on a JASCO FT-IR 4100 spectrometer and
are reported in wave numbers (cmꢁ1). 1H and 13C NMR spectra were recorded
on a JEOL JNM-EXC 300 spectrometer (300 MHz) or a JEOL JNM-ECA 500
spectrometer (500 MHz). 1H NMR data are reported as follows: chemical shift
(d, 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 (d, ppm). EI-MS spectra were recorded
on a JEOL JMS-700 instrument. ESI-MS spectra were recorded on a JEOL JMS-
T100LC instrument.
(1R,3S,5R,6R,7R,8S)–3-Hydroxy-13,14,15-trimethylene-12-oxododecahydroazuleno
[6,7-ß]furan-8-yl methacrylate (6): Compound
5
(8.7 mg, 0.023 mmol,
Isolation of cynaropicrin (1): Extraction and isolation of cynaropicrin from
artichoke leaves was achieved by following procedure published protocol.8,16
Dried artichoke leaves (100 g) purchased from Dixa (Switzerland) were
exhaustively extracted three times with ethyl acetate (900 mL) yielding 5.4 g
1.0 equiv) and PPTS (20.7 mg, 0.084 mmol, 3.6 equiv) were dissolved in
tBuOH (3 mL) with stirring under nitrogen atmosphere. After stirring for 26 h
under reflux condition, the mixture was diluted with EtOAc, and then
quenched with
a saturated NaHCO3 solution. The aqueous layer was
of crude extract. The extract was separated on
a
silica gel column
extracted with EtOAc. The combined organic layers were washed with brine,
dried over Na2SO4, and then concentrated in vacuo. Purification on silica gel
chromatography with ethyl acetate as mobile phase. Fractions containing 1
were concentrated to give 1.7 g of impure cynaropicrin. Purification via silica
gel column chromatography (hexane/EtOAc = 1:2) afforded 1 as a yellow solid
column chromatography (hexane/EtOAc = 1:1) afforded
6
ꢀ
as
a yellow oil
(6.6 mg, 0.020 mmol, 89%); Rf 0.15 (hexane/EtOAc = 2:1); ½a D25
+114.5 (c 0.14 in
(1.4 g); Rf 0.62 (hexane/EtOAc = 1:3; ½a D25
ꢀ
+130.4 (c 0.18, CHCl3); IR (neat,
CHCl3); IR (neat, cmꢁ1) 3481, 2927, 1769, 1636, 1454, 1269, 1159, 1015, 908,
815; 1H NMR (300 MHz, CDCl3) d 6.22 (1H, d, J = 3.4 Hz, H13), 6.19 (1H, s, H18),
5.68 (1H, s, H18), 5.61 (1H, d, J = 3.1 Hz, H13), 5.50 (1H, s, H15), 5.37 (1H, s,
H15), 5.14–5.06 (2H, m, H8/14), 4.94 (1H, s, H14), 4.57 (1H, t, J = 7.6 Hz, H3),
4.25 (1H, t, J = 9.3 Hz, H6), 3.23–3.15 (1H, m, H7), 3.03–2.94 (1H, m, H1), 2.85
(1H, J = 10.3 Hz, H5), 2.71 (1H, dd, J = 14.5, 5.2 Hz, H9), 2.38 (1H, dd, J = 14.5,
3.8 Hz, H9), 2.29–2.19 (1H, m, H2), 1.99 (3H, s, H19), 1.74 (1H, ddd, J = 13.1,
10.7, 7.6 Hz, H2); 13C NMR (75 MHz, CDCl3) d 169.3, 166.5, 152.5, 142.0, 137.6,
136.2, 126.7, 122.8, 118.2, 113.7, 78.7, 74.2, 74.0, 51.5, 47.9, 45.5, 39.3, 37.3,
cmꢁ1) 3419, 3080, 2931, 1765, 1712, 1643, 1456, 1403, 1270, 1152, 1054, 958,
910, 815, 642; 1H NMR (500 MHz, CDCl3) d 6.29 (1H, s, H18), 6.17 (d, J = 2.3 Hz,
H13), 5.94 (1H, s, H18), 5.59 (1H, 1.7 Hz, H13), 5.44 (1H, s, H15), 5.33 (1H, s,
H15), 5.13–5.09 (2H, m, H8/14), 4.91 (1H, s, H14), 4.53 (1H, t, J = 6.9 Hz, H3),
4.35 (2H, s, H19), 4.25 (1H, t, J = 9.7 Hz, H6), 3.20–3.14 (1H, m, H7), 2.94 (1H,
dd, J = 19.3, 8.4, H1), 2.81 (1H, t, J = 10.3 Hz, H5), 2.69 (1H, dd, J = 14.9, 4.6 Hz,
H9), 2.36 (1H, dd, J = 14.9, 3.4 Hz, H9), 2.23–2.13 (1H, m, H2), 1.71 (1H, m, H2);
13C NMR (125 MHz, CDCl3) d 169.4 (C12), 165.4 (C16), 152.2 (C4), 141.8 (C10),
139.4 (C17), 137.4 (C11), 126.8 (C18), 122.9 (C13), 118.3 (C14), 113.6 (C15),
78.6 (C6), 74.4 (C8), 73.7 (C3), 62.1 (C19), 51.4 (C5), 47.6 (C7), 45.4 (C1), 39.1
(C2), 37.0 (C9); EI-MS (m/z) calcd for C19H22O6 [M]+ 346.1, found 346.0.
(1R,3S,5R,6R,7R,8S)-3-(Methoxymethoxy)-13,14,15-trimethylene-12-
18.4; ESI-HRMS (m/z) calcd for
C
19H22NaO5 [M+Na]+ 353.1365, found
353.1351.
(1R,3S,5R,6R,7R,8S)-3-Hydroxy-13,14,15-trimethylene-12-oxododecahydroazuleno
[6,7-ß]furan-8-yl methacrylate (2): 5 (8.6 mg, 0.028 mmol, 1.0 equiv) and PPTS
(16.8 mg, 0.070 mmol, 2.4 equiv) were dissolved in tBuOH (3 mL) with stirring
under nitrogen atmosphere. After stirring for 28 h under reflux condition, the
mixture was diluted with EtOAc, and quenched with a saturated NaHCO3
solution. The aqueous layer was then extracted with EtOAc. The combined
organic layers were washed with brine, dried over Na2SO4, and then
concentrated in vacuo. Purification on silica gel column chromatography
(hexane/EtOAc = 1:2) afforded 2 as a yellow oil (5.0 mg, 0.019 mmol, 68%); Rf
0.29 (hexane/EtOAc = 2:1); 1H NMR (300 MHz, CDCl3) d 6.27 (1H, dd, J = 3.5,
0.7 Hz, H13), 6.15 (1H, dd, J = 3.4, 0.7 Hz, H13), 5.50 (1H, m, H15), 5.35 (1H, m,
H15), 5.14 (1H, s, H14), 4.99 (1H, s, H14), 4.56 (1H, m, H3), 4.15 (1H, m, H6),
3.97 (1H, m, H8), 2.98 (1H, m, H1), 2.85–2.68 (3H, m, H5/7/9), 2.33–2.19 (2H,
m, H2/9), 1.74 (1H, m, H2).
oxododecahydroazuleno[6,7-ß]furan-4-yl 17-((methoxymethoxy)methyl)acrylate
(3): MOMCl (41.4 lL, 0.55 mmol, 6.0 equiv) was added to a flask containing
cynaropicrin 1 (31.6 mg, 0.091 mmol, 1.0 equiv), iPr2NEt (0.19 mL, 1.09 mmol,
12.0 equiv), and TBAI (16.8 mg, 0.046 mmol, 0.5 equiv)) in CH2Cl2 (3 mL) at
0 °C. After stirring for 19 h under reflux condition, the mixture was diluted
with CH2Cl2, and quenched with a saturated NaHCO3 solution. The aqueous
layer was then extracted with CH2Cl2. The combined organic layers were
washed with brine, dried over Na2SO4, and then concentrated in vacuo.
Purification on silica gel column chromatography (hexane/EtOAc = 1:1)
afforded
3
as
a
ꢀ
yellow oil (36.7 mg, 0.084 mmol, 93%); Rf 0.84 (hexane/
EtOAc = 1:2); ½a 2D0
+72.4 (c 0.45, CHCl3); IR (neat, cmꢁ1) 2948, 1770, 1717,
1640, 1454, 1400, 1267, 1148, 1047, 915, 816; 1H NMR (CDCl3, 300 MHz) d 6.38
(1H, s, H18), 6.21 (1H, d, J = 3.4 Hz, H13), 5.99 (1H, s, H18), 5.62 (1H, d,
J = 3.1 Hz, H13), 5.48 (1H, s, H15), 5.34 (1H, s, H15), 5.16–5.10 (2H, m, H8/14),
4.91 (1H, s, H14), 4.77 (1H, d, J = 6.9 Hz, CH2), 4.69 (2H, s, CH2), 4.65 (1H, d,
J = 6.9 Hz, CH2), 4.44 (1H, t, J = 6.2 Hz, H3), 4.30 (2H, s, H19), 4.25 (1H, dd,
J = 10.7, 9.3 Hz, H6), 3.39 (6H, s, Me ꢂ 2), 3.21–3.12 (1H, m, H7), 3.02–2.99 (1H,
m, H1), 2.79 (1H, t, J = 9.3 Hz, H5), 2.70 (1H, dd, J = 14.8, 5.2 Hz, H9), 2.40 (1H,
dd, J = 14.8, 3.4 Hz, H9), 2.33–2.24 (1H, m, H2), 1.85–1.75 (1H, m, H2); 13C NMR
(75 MHz, CDCl3) d 169.2, 164.9, 148.6, 142.1, 137.6, 137.1, 127.6, 122.6, 118.2,
115.2, 96.2, 94.9, 78.3, 77.7, 74.3, 65.8, 55.6, 52.2, 47.5, 46.0, 37.2, 36.9; ESI-
HRMS (m/z) calcd for C23H30NaO8 [M+Na]+ 457.1838, found 457.1838.
(1R,3S,5R,6R,7R,8S)-8-Hydroxy-3-(methoxymethoxy)-13,14,15-trimethylene-
decahydroazuleno[6,7-ß]furan-12-one (4): 2 N NaOH solution (1.5 mL) was
(1R,3S,5R,6R,7R,8S)-3-Hydroxy-13,14,15-trimethylene-12-oxododecahydroazuleno
[6,7-ß]furan-8-yl 17-(acetoxymethyl)acrylate (7) and (1R,3S,5R,6R,7R,8S)-3-
acetoxy-13,14,15-trimethylene-12-oxododecahydroazuleno[6,7-ß]furan-8-yl 17-
(acetoxymethyl)acrylate (8): To a solution of 1 (48.4 mg, 0.14 mmol, 1.0 equiv)
in CH2Cl2 (0.4 mL) was added Et3N (28
lL, 0.20 mmol, 1.4 equiv) and acetic
anhydride (14.2 L, 0.15 mmol, 1.05 equiv). After stirring at 0 °C for 24 h, the
l
mixture was diluted with CH2Cl2, and quenched with 1 M HCl solution. The
aqueous layer was then extracted with CH2Cl2. The combined organic layers
were washed with brine, dried over Na2SO4, and then concentrated in vacuo.
Purification on silica gel column chromatography (hexane/EtOAc = 1:2)
afforded 7 as a yellow oil (26.9 mg, 0.070 mmol, 50%) and 8 as a yellow oil
(4.7 mg, 0.011 mmol, 8%), respectively. Compound 7: Rf 0.15 (hexane/
added to
3
(70.5 mg, 0.162 mmol, 1.0 equiv). After stirring at room
EtOAc = 2:1); ½a 2D5
ꢀ
+68.7 (c 0.15 in CHCl3); IR (neat, cmꢁ1) 3499, 2939, 2359,
temperature for 28 h, the mixture was diluted with EtOAc, and quenched
with 2 N H2SO4 solution. The aqueous layer was then extracted with EtOAc.
1767, 1371, 1267, 1143, 1018, 576, 462; 1H NMR (300 MHz, CDCl3) d 6.45 (1H,
s, H18), 6.22 (1H, d, J = 3.4 Hz, H13), 5.96 (1H, s, H18), 5.61 (1H, d, J = 2.8 Hz,

798
T. Usuki et al. / Bioorg. Med. Chem. Lett. 24 (2014) 794–798
H13), 5.49 (1H, s, H15), 5.36 (1H, s, H15), 5.18–5.12 (2H, m, H8/14), 4.93 (1H, s, Sanofi-Aventis) was used as reference drug. After 70 h of incubation, 10
l
L of
H14), 4.83 (2H, s, H19), 4.56 (1H, t, J = 6.9 Hz, H3), 4.28–4.22 (1H, m, H6), 3.21–
3.15 (1H, m, H7), 2.98 (1H, dd, J = 18.9, 8.3 Hz, H1), 2.85 (1H, t, J = 9.6 Hz, H5),
2.71 (1H, dd, J = 14.5, 4.8 Hz, H9), 2.40 (1H, dd, J = 14.5, 3.4 Hz, H9), 2.31–2.19
(1H, m, H2), 2.10 (3H, s, Me), 1.78–1.68 (1H, m, H2); 13C NMR (125 MHz,
(CD3)2CO) d 170.5, 165.1, 154.6, 144.0, 139.6, 136.9, 128.6, 121.3, 117.7, 111.7,
79.2, 75.5, 73.8, 62.8, 51.6, 47.9, 45.8, 40.1, 37.5, 20.7; ESI-HRMS (m/z) calcd for
Alamar blue marker (12.5 mg resazurin (Sigma–Aldrich) dissolved in 100 mL of
distilled water) was added, and the color change was allowed to develop for 2–
6 h.
A Spectramax Gemini XS micro plate fluorescence reader (Molecular
Devices Cooperation) with an excitation wavelength of 536 nm and an
emission wavelength of 588 nm was used to read the plates. The assay was
carried out at least three independent repetitions, and the IC50s with standard
errors were calculated using sigmoidal growth inhibition curves in the Softmax
Pro software (Molecular Devices) and MS Excel (Microsoft).
C
21H24NaO7 [M+Na]+ 411.1420, found 411.1433. 8: Rf 0.82 (hexane/
EtOAc = 1:2); 1H NMR (CDCl3, 300 MHz) d 6.45 (1H, s, H18), 6.24 (1H, d,
J = 3.4 Hz, H13), 5.97 (1H, s, H18), 5.63 (1H, d, J = 3.1 Hz, H13), 5.59–5.54 (2H,
m, H3/15), 5.37 (1H, s, H15), 5.19–5.13 (1H, m, H8/14), 4.96 (1H, s, H14), 4.84
(2H, s, H19), 4.18 (1H, dd, J = 10.3, 8.9 Hz, H6), 3.24–3.16 (1H, m, H7), 3.03 (1H,
dd, J = 18.6, 7.9 Hz, H1), 2.85 (1H, t, J = 10.0 Hz, H5), 2.69 (1H, dd, J = 14.8,
5.2 Hz, H9), 2.49–2.32 (2H, m, H2/9), 2.10 (3H, s, Me), 2.09 (3H, s, Me), 1.79
(1H, ddd, J = 13.4, 11.0, 7.2 H, H2); ESI-HRMS (m/z) calcd for C23H26NaO8
[M+Na]+ 453.1525, found 453.1533.
Cytotoxicity: Rat skeletal myoblast cells (L6 cells) were seeded in RPMI 1640
medium supplemented with 2 lM L-glutamine, 5.95 g/L HEPES, 2 g/L NaHCO3,
and 10% fetal bovine serum as previously reported.16 The cultures were
maintained at 37 °C under a humidified 5% CO2 atmosphere. The IC50 values
were determined using the Alamar Blue assay as described above against L6-
cells seeded in 100
After 24 h, the medium was removed and replaced with 100
l
L RPMI 1640 in 96-well microtiter plates (4000 cells/well).
l
L of fresh RPMI
24. Antitrypanosomal assay: Trypanosoma brucei rhodesiense (STIB 900) was grown
in minimum essential medium (MEM) with Earle’s salts supplemented with
0.2 mM 2-mercaptoethanol (Baltz, T.; Baltz, D.; Giroud, C.; Crockett, J. EMBO J.
1985, 4, 1273.) and the following modifications: 1 mM sodium pyruvate,
0.5 mM hypoxanthine, and 15% heat-inactivated horse serum. Cultures were
maintained in a humidified 5% CO2 atmosphere at 37 °C. The IC50 values of the
samples were determined using Alamar Blue assay as previously described
(Räz, B.; Iten, M.; Grether-Bühler, Y.; Kaminsky, R.; Brun, R. Acta Trop. 1997, 68,
139.). Serial threefold dilutions were prepared in 96-well microtiter plates, and
1640 with or without a serial threefold drug dilution. Podophyllotoxin (purity:
>95%, Sigma–Aldrich) was used as the reference drug. After 70 h of incubation
under a humidified 5% CO2 atmosphere, 10
above) was added to all of the wells. The plates were incubated for an
additional 2 h. Spectramax Gemini XS micro plate fluorescence reader
lL of the Alamar Blue marker (see
A
(Molecular Devices) was used to read the plates at an excitation wavelength of
536 nm and an emission wavelength of 588 nm. The assay was carried out for
at least three independent repetitions and the IC50s and respective standard
errors were calculated using sigmoidal growth inhibition curves in the Softmax
Pro software (Molecular Devices) and MS Excel (Microsoft).
2000 T. b. rhodesiense STIB 900 bloodstream forms in 50
lL were added to each
well, except for the negative controls. Melarsoprol (ArsobalÒ, purity: >95%;