Antimitotic activity of lobaric acid and a new benzofuran, sakisacaulon A from Stereocaulon sasakii

Article abstract of DOI:10.1016/j.bmcl.2009.03.170

Lobaric acid (1) has been isolated from lichen, Stereocaulon sasakii together with a new benzofuran, sakisacaulon A (2). Lobaric acid (1) inhibited the polymerization of tubulin. Structure-activity relationship of lobaric acid and its derivatives on inhib

Full text of DOI:10.1016/j.bmcl.2009.03.170

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3679–3681
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antimitotic activity of lobaric acid and a new benzofuran, sakisacaulon
A from Stereocaulon sasakii
a
a
a
b
a
Hiroshi Morita a, , Tomoe Tsuchiya , Koji Kishibe , Sayaka Noya , Motoo Shiro , Yusuke Hirasawa
*
a Faculty of Pharmaceutical Sciences, Hoshi University, Ebara 2-4-41 Shinagawa-ku, Tokyo 142-8501, Japan
b X-ray Research Laboratory, Rigaku Corporation, Akishima, Tokyo 196-8666, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Lobaric acid (1) has been isolated from lichen, Stereocaulon sasakii together with a new benzofuran, sakis-
acaulon A (2). Lobaric acid (1) inhibited the polymerization of tubulin. Structure–activity relationship of
lobaric acid and its derivatives on inhibitory activity of tubulin polymerization was discussed.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 23 February 2009
Revised 25 March 2009
Accepted 26 March 2009
Available online 12 May 2009
Keywords:
Stereocaulon sasakii
Lobaric acid
Tubulin
Antimitotic activity
Lichen
Tubulin and microtubule proteins formed by the self-associa-
tion of the ,b-tubulin heterodimers interact with a large number
The whole bodies of S. sasakii were extracted with MeOH, and
the MeOH extract was in turn partitioned with hexane, EtOAc,
a
of structurally diverse natural products from natural sources,
which cause cells to arrest in mitosis.1 These antimitotic agents
are of interest for the insight they can provide potential activity
in the treatment of cancer diseases.2 Among them, paclitaxel is po-
tent inhibitor of cell proliferation and arrest cells in mitosis, but in
contrast to vinblastine, promote the polymerization of tubulin,
causing stabilization and bundling of microtubules.3 Recently
much effort has been directed to the isolation and synthesis of
new antimitotic drugs that target the tubulin/microtubule system
and display efficacy against drug-refractory carcinomas.4 The anti-
mitotic agents have potential applications in drug development.
In our search for bioactive compounds targeting the tubulin/
microtubules from natural sources,5 we found that the extract
from lichen, Stereocaulon sasakii remarkably inhibited the polymer-
ization of tubulin. Lichens are symbiotic associations of fungi and
algae and are known to contain unique substances such as dep-
sides and depsidones.6 Our efforts on identifying new agents that
target tubulin resulted in the isolation of lobaric acid (1)7 from
the whole bodies of S. sasakii together with a new benzofuran,
sakisacaulon A (2), whose structures were established by spectro-
scopic data. This Letter describes effects of a lobaric acid and its
derivatives (4–7) on tubulin assembly as well as structure elucida-
tion of sakisacaulon A (2).
12
11
O
10
7
O
OH
HO
9O
1
10
4'
3'
O
2
2' OR2
5'
O
O
8
O
8
4
12
3'
2'
7
2
9
6
5
4'
5'
11
6
1
1'
3
OR1
7'
7' 6'
8'
5
OH
4
1'
6'
8'
OMe
3
9'
O
MeO
9'
10'
10'
R1 R2
11'
11'
H
H
H
lobaric acid 1:
sakisacaulon A (2)
12'
Me
H
4:
5:
6:
7:
Ac
Me Ac
H
COEt
O
O
OH
H
O
OH
OMe
* Corresponding author. Tel./fax: +81 354985778.
E-mail address: moritah@hoshi.ac.jp (H. Morita).
3
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.170

3680
H. Morita et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3679–3681
Table 1
and n-BuOH. The EtOAc-soluble materials inhibiting the polymer-
1H [dH (J, Hz)] and 13C NMR Data (dC) of Sakisacaulon A (2) and Phenoxybenzofuran
(3)
ization of tubulin were subjected to a silica gel column (CH2Cl2/
MeOH, 1:0?0:1) followed by
a C18 column (CH3OH/H2O,
dH
dC
3:2?1:0) and C18 HPLC (CH3OH/0.1% TFA, 4:1) to afford lobaric
acid (1, 0.2%) and a new benzofuran, sakisacaulon A (2, 0.004%).
The structure of 1 was identical with lobaric acid by 2D NMR
analysis.7 Lobaric acid (1) was crystallized from methanol-water
as colorless needles, mp 196–198 °C and was analyzed by X-ray
crystallography.8 The asymmetric unit contains one molecule of
1, giving a calculated density of 1.387 g cmꢀ3. The ORTEP drawing
of 1 was shown in Figure 1. The conformation of the two aromatic
rings (C-1–C-6 and C-10–C-60) is essentially twisted at 125.28° with
extended antiparallel side chains at C-6 and C-60. This conforma-
tion in 1 obtained from X-ray analysis corresponded well to those
in the case of the other depsidones such as excelsione and garcid-
epsidone A.9
2
3
2
3
1
2
3
4
5
6
7
8
108.0
159.9
102.9
168.9
101.2
149.7
167.7
102.6
39.7
105.9
159.0
100.6
167.1
98.8
155.2
169.5
80.8
34.0
26.4
22.1
13.0
114.5
153.3
101.1
147.1
132.2
135.3
26.9
28.8
31.8
21.9
12.9
6.08 (1H, d, 1.4)
6.69 (1H, d, 1.4)
6.05 (1H, d, 1.4)
6.66 (1H, d, 1.4)
5.47 (1H, d, 1.4)
1.76 (2H, m)
1.43 (2H, m)
1.41 (2H, m)
0.95 (3H, t, 7.1)
9
2.17 (2H, m)
2.06 (2H, m)
1.34 (2H, m)
0.90 (3H, t, 7.1)
6.23 (1H, d, 2.8)
10
11
12
10
26.8
23.6
14.4
108.7
156.7
102.9
151.4
133.6
138.2
31.3
20
Compound 2 showed the pseudomolecular ion peak at m/z 453
(M+Na)+ in the ESIMS, and the molecular formula C24H30O7 was
established by HRESIMS [m/z 453.1890 (M+Na)+].10 IR absorptions
30
6.30 (1H, d, 2.8)
6.39 (1H, d, 2.8)
40
50
implied the presence of hydroxyl (3360 cmꢀ1
) and carbonyl
60
(1740 cmꢀ1) functionalities. The 1H NMR data (Table 1) showed
the presence of four aromatic protons, a pentyl and a butyl side
chains, and a methoxy group. The 13C NMR data (Table 1) revealed
twenty-four carbon signals due to eight sp2 quaternary carbons,
four sp2 methines, one ester carbonyl, one sp3 quaternary carbon,
seven sp3 methylenes, one methoxy, and two methyl groups.
Partial structures C-9 to C-12 (unit a) and C-70 to C-110 (unit b)
were deduced from detailed analysis of the 1H–1H COSY spectrum
of 2 (Fig. 2). The HMBC cross-peaks of H-5 to C-1 and C-4, H-3 to C-
1, C-2, C-4, and C-5, H-10 to C-20 and C-50, and H-30 to C-20, C-40, and
C-50 indicated the presence of two aromatic rings. Connection be-
tween the unit a and the aromatic ring through C-8 were eluci-
dated by an HMBC correlation for H-5 to C-8 and a NOESY
correlation between H-5 and H2-9. The HMBC correlation for H-10
70
2.37 (2H, m)
1.51 (2H, m)
1.24 (2H, m)
1.20 (2H, m)
0.82 (3H, t, 7.1)
3.77 (3H, s)
2.46 (2H, m)
1.39 (2H, m)
1.22 (2H, m)
1.21 (2H, m)
0.80 (3H, t, 7.1)
3.77 (3H, s)
80
30.9
32.8
23.4
14.3
90
100
110
4-OMe
10-Me
56.7
55.1
2.09 (3H, s)
10.0
O
O
OH
a
HO
9
7
8
10
1
4'
12
O
2
3'
6
5'
11
2'
3
5
6'
7'
OH
4
1'
8'
9'
OMe
1H- 1H COSY
HMBC
NOESY
10'
b
11'
Figure 2. Selected 2D NMR correlations for Sakisacaulon A (2).
to C-70 placed the unit b at C-60. Connection between C-2 and C-
50 through an ether linkage was supported by the NOESY correla-
tion between H-3 and H2-70. Thus, 2 was a new phenoxybenzofu-
ran derivative and was named sakisacaulon A. The
stereochemistry at C-8 may be racemic due to a weak optical rota-
tion. The presence of a benzofuran moiety was also supported by
comparing of the spectroscopic data to those of a phenoxybenzofu-
ran
3 derived from 1 by sodium borohydride/ chloroethyl
acetate.11
In this study, it was found that lobaric acid (1) remarkably
inhibited the polymerization of tubulin. Microtubule polymeriza-
tion and depolymerization were monitored by the increase and
the decrease in turbidity. Inhibitory effects of 1 to tubulin polymer-
ization are shown in Figure 3, in which tubulin polymerization was
inhibited in a concentration-dependent manner (IC50 100 lM). On
the other hand, sakisacaulon A (2) and phenoxybenzofuran (3)
with bond cleavage of ester linkage of depsidone did not show
inhibition of polymerization of tubulin. On the other hand, some
derivatives of lobaric acid (1) such as methyl ester (4), acetate
(5), methyl ester acetate (6), and propionate (7), which were pre-
pared by treatment of 1 with trimethylsilyl diazomethane and/or
acetic anhydride or propionyl chloride in pyridine, were found to
be less potent than lobaric acid itself, indicating that the presence
of the carboxylic acid and hydroxy at C-10 and C-20, respectively, is
important for the activity.12
Figure 1. ORTEP drawing for lobaric acid (1).

H. Morita et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3679–3681
3681
Figure 3. Inhibitory effects of lobaric acid (1) and vinblastine to the polymerization of tubulin protein. Various concentrations of 1 were mixed with tubulin protein (1.5 mg/
mL) at 0 °C and incubated at 37 °C. The absorbance at 400 nm was measured.
Hirasawa, Y.; Izawa, E.; Matsuno, Y.; Kawahara, N.; Goda, Y.; Morita, H. Bioorg.
Cytotoxicity of depsidone related compounds has been stud-
Med. Chem. Lett. 2007, 17, 5868; Suzuki, H.; Morita, H.; Shiro, M.; Kobayashi, J.
ied.13 Lichen metabolites such as sphaerophorin, pannarin, induced
Tetrahedron 2004, 60, 2489; Suzuki, H.; Morita, H.; Iwasaki, S.; Kobayashi, J.
apoptosis in human melanoma cells14 and usnic acid was also
Tetrahedron 2003, 59, 5307; Kobayashi, J.; Suzuki, H.; Shimbo, K.; Takeya, K.;
Morita, H. J. Org. Chem. 2001, 66, 6626; Morita, H.; Shimbo, T.; Shigemori, H.;
Kobayashi, J. Bioorg. Med. Chem. Lett. 2000, 10, 469.
shown to induce apoptosis of murine leukemia L1210 cells.15 Com-
mon structural feature among these compounds is the presence of
6. Dobson, F. S. Lichens An Illustrated Guide to British and Irish Species; Richmond
two aromatic rings which can be connected through one or two
atoms bridge spacer. Orientation of the two aromatic rings is re-
quired to be ca. 125°. In addition, the appropriate torsion between
two aromatic planes and the appropriate functions such as carbox-
ylic acid and hydroxy may be important to show activity. In this
work, we found that lobaric acid from lichen, S. sasakii showed
antimitotic activity. Efforts are currently underway to determine
the structure–activity relationship for antimitotic activity of a ser-
ies of depsidones as antimitotic agents.
Publishing Co., Ltd: Slough, UK, 2000. p. 431; Fahselt, D. Symbiosis 1994, 16,
117.
7. Elix, J. A.; Wardlaw, J. H.; Yoshimura, I. Aust. J. Chem. 1997, 50, 763; Sundholm,
E. G.; Huneck, S. Chem. Scr. 1980, 16, 197.
8. All measurements were made on a Rigaku RAXIS RAPID imaging plate area
detector with graphite monochromated Cu-K
colorless platelet crystal, C25H28O8, 0.20 ꢁ 0.08 ꢁ 0.03 mm, triclinic,
a = 4.73139(13) Å, b = 11.3895(4) Å, c = 11.4300(3) Å, = 63.8883(19)°, b =
82.2266(17)°,
= 82.561(2)°, V = 546.30(3) Å3, z = 1, the calculated density is
1.387 g/cm3. Of the 9935 reflections that were collected, 1980 were unique.
The structure was solved by direct methods. R1 = 0.0477 (I > 2.00 (I)). All
calculations were performed using the CrystalStructure crystallographic
a radiation. Crystal data of 1: A
a
c
r
software package except for refinement, which was performed using SHELXL
97.
-
Acknowledgment
9. Lang, G.; Cole, A. L. J.; Blunt, J. W.; Robinson, W. T.; Munro, M. H. G. J. Nat. Prod.
2007, 70, 310; Xu, Y.-J.; Chiang, P.-Y.; Lai, Y.-H.; Vittal, J. J.; Wu, X.-H.; Tan, B. K.
H.; Imiyabir, Z.; Goh, S.-H. J. Nat. Prod. 2000, 63, 1361.
The authors thank Dr. Isao Yoshimura, Hattori Botanical Labora-
tory, for the botanical identification of S. sasakii. This work was
partly supported by a Grant-in-Aid for Scientific Research from
the Ministry of Education, Culture, Sports, Science, and Technology
of Japan, and a grant from the Open Research Center Project.
10. Sakisacaulon A (2): colorless solid, ½a D22
+4 (c 1.0, MeOH); IR (KBr) mmax 3360,
ꢂ
2960, 2940, 1740, and 1620 cmꢀ1 1H and 13C NMR (Table 1); ESIMS m/z 453
;
(M+Na)+; HRESITOFMS m/z 453.1890 (M+Na)+, calcd for C24H30O7Na
453.1889.
11. To a solution of lobaric acid (55 mg) and Et3N (44
lL) in THF (2 mL) at 0 °C was
added ClCO2Et (31 L). After the mixture was stirred for 1.5 h at 0 °C, a solution
l
References and notes
of NaBH4 (39 mg) in H2O (2 mL) was added and stirred for 1.5 h at 0 °C, and
then temperature was allowed to rise to rt. and it was stirred for an additional
2 h. The mixture was neutralized with 1 M HCl and the aqueous layer was
extracted with EtOAc. Purification or the EtOAc extracts by ODS HPLC (85%
MeOH) gave 3 (10 mg): colorless solid, IR (KBr) mmax 3383, 2958, 1736, and
1. Iwasaki, S. Med. Res. Rev. 1993, 13, 183; Kavallaris, M.; Verrills, N. M.; Hill, B. T.
Drug Res. Updates 2001, 4, 392.
2. Shigemori, H.; Kobayashi, J. J. Nat. Prod. 2004, 67, 245.
1612 cmꢀ1 1H and 13C NMR (Table 1); ESIMS m/z 429 (M+H)+; HRESITOFMS m/
;
3. Some examples: Tinley, T. L.; Hlubek, D. A. R.; Leal, R. M.; Jackson, E. M.; Cessac,
J. W.; Quada, J. C., Jr.; Hemscheidt, T. K.; Mooberry, S. L. Cancer Res. 2003, 63,
3211; Verrills, N. M.; Flemming, C. L.; Liu, M.; Ivery, M. T.; Cobon, G. S.; Norris,
M. D.; Haber, M.; Kavallaris, M. Chem. Biol. 2003, 10, 597; Hood, K. A.; West, L.
M.; Rouwé, B.; Northcote, P. T.; Berridge, M. V.; Wakefield, J., St.; Miller, J. H.
Cancer Res. 2002, 62, 3356; Hardt, I. H.; Steinmetz, H.; Gerth, K.; Sasse, F.;
Reichenbach, H.; Höfle, G. J. Nat. Prod. 2001, 64, 847; Mooberry, S. L.; Tien, G.;
Hernandez, A. H.; Plubrukarn, A.; Davidson, B. S. Cancer Res. 1999, 59, 653.
4. Imai, Y.; Tsukahara, S.; Asada, S.; Sugimoto, Y. Cancer Res. 2004, 64, 4346;
Yanase, K.; Tsukahara, S.; Asada, S.; Ishikawa, E.; Imai, Y.; Sugimoto, Y. Mol.
Cancer Ther. 2004, 1129.
z 429.2669 (M+H)+, calcd for C25H33O6 429.2272.
12. Compounds 2–7 did not show inhibition of tubulin polymerization at 200 lM.
13. Li, G.-Y.; Li, B.-G.; Yang, T.; Liu, G.-Y.; Zhang, G.-L. Helv. Chim. Acta 2008, 91,
124; Millot, M.; Tomasi, S.; Articus, K.; Rouaud, I.; Bernard, A.; Boustie, J. J. Nat.
Prod. 2007, 70, 316; Pittayakhajonwut, P.; Dramae, A.; Madla, S.;
Lartpornmatulee, N.; Boonyuen, N.; Tanticharoen, M. J. Nat. Prod. 2006, 69,
1361; Bucara, F.; Schneidera, I.; Ögmundsdottirb, H.; Ingolfsdottir, K.
Phytomedicine 2004, 11, 602; Permana, D.; Lajis, N. H.; Mackeen, M. M.; Ali,
A. M.; Aimi, N.; Kitajima, M.; Takayama, H. J. Nat. Prod. 2001, 64, 976.
14. Russo, A.; Piovano, M.; Lombardo, L.; Garbarino, J.; Cardile, V. Life Sci. 2008, 83,
468.
5. Morita, H.; Tomizawa, Y.; Tsuchiya, T.; Hirasawa, Y.; Hashimoto, T.; Asakawa, Y.
Bioorg. Med. Chem. Lett. 2009, 19, 493; Morita, H.; Hirasawa, Y.; Muto, A.;
Yoshida, T.; Sekita, S.; Shirota, O. Bioorg. Med. Chem. Lett. 2008, 18, 1050;
15. Bezivin, C.; Tomasi, S.; Rouaud, I.; Delcros, J.-G.; Boustie, J. Planta Med. 2004, 70,
874.