Homopetasinic acid isolated from Diaporthe sp. strain 1308-05

Article abstract of DOI:10.1016/j.tetlet.2016.01.095

Homopetasinic acid (1) was isolated from fungi of the Diaporthe sp. strain 1308-05. NMR spectroscopic structural analysis revealed a petasol (3) substructure and a (4E,6E)-7-carboxy-3-hydroxy-2-methylhepta-4,6-dienoate side chain. The absolute configuration of the petasol moiety was established by the specific rotation value after basic hydrolysis. The (2′S,3′S)-configuration of the side chain was determined by NMR empirical methods as well as comparison of the spectral data with related model compounds. The absolute structure of the side chain moiety was established on the basis of ECD spectral analyses involving theoretical calculations. The biological activities of 1 are also discussed.

Full text of DOI:10.1016/j.tetlet.2016.01.095

Tetrahedron Letters 57 (2016) 1117–1119
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Tetrahedron Letters
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Homopetasinic acid isolated from Diaporthe sp. strain 1308-05
Atsushi Ito a, Ikuya Kumagai b, Miku Maruyama c, Hayato Maeda b, Akio Tonouchi b, Tatsuo Nehira d,
Ken-ichi Kimura a,c, Masaru Hashimoto a,b,
⇑
a The United Graduate School of Agricultural Science, Iwate University, 3-18-8 Ueda, Morioka 020-8550, Japan
b Faculty of Agriculture and Bioscience, Hirosaki University, 3-Bunkyo-cho, Hirosaki 036-8561, Japan
c Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka 020-8550, Japan
d Graduate School of Integrated Arts and Sciences, Hiroshima University, 1-7-1, Kagamiyama, Higashi-Hiroshima 739-8521, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Homopetasinic acid (1) was isolated from fungi of the Diaporthe sp. strain 1308-05. NMR spectroscopic
structural analysis revealed a petasol (3) substructure and a (4E,6E)-7-carboxy-3-hydroxy-2-methyl-
hepta-4,6-dienoate side chain. The absolute configuration of the petasol moiety was established by the
specific rotation value after basic hydrolysis. The (20S,30S)-configuration of the side chain was determined
by NMR empirical methods as well as comparison of the spectral data with related model compounds.
The absolute structure of the side chain moiety was established on the basis of ECD spectral analyses
involving theoretical calculations. The biological activities of 1 are also discussed.
Received 16 December 2015
Revised 21 January 2016
Accepted 27 January 2016
Available online 28 January 2016
Keywords:
Homopetasinic acid
Chemical correlation
Electron circular dichroism analysis
Cytotoxicity
Ó 2016 Elsevier Ltd. All rights reserved.
Introduction
1308-05. Homopetasinic acid (1) afforded a protonated molecular
ion at m/z = 417.2296. The mass spectrum also showed the sodium
Petasin, an eremophilane-type sesquiterpenoid, was first iso-
lated from roots of Petasites hybridus (L.) in 1955.1 S-Petasin is
the (Z)-3-(methylthio)acrylate derivative found in P. officinalis2 as
well as P. formosanus Kitamura.3 The latter has been used as a folk
medicine in Taiwan.4 Since S-petasin exhibits remarkable biologi-
cal properties, such as inhibition of cumulative histamine,3 spas-
molytic activity,2 and anti-inflammatory activity,5 it is
anticipated to be a novel drug. Although these were isolated from
plant resources, we herein report homopetasinic acid (1), a 3-O-
[(4E,6E,2S,3S)-3-hydroxy-2-methylocta-4,6-dienedioate] analogue
of petasol (3), from the filamentous fungus Diaporthe sp. strain
1308-05. The structure was elucidated by ESIMS, 1H and 13C
NMR, chemical derivatizations, and ECD analyses.
adduct ion at m/z = 439.2115, which was helpful to establish the
molecular formula of 1 as C24H32O6 ([M+H]+: 417.2277, [M+Na]+:
439.2097). This molecule also gave a dehydrated ion at m/z =
399.2187, suggesting a hydroxy group ([MꢀOH]+: 399.2171).
The NMR spectral data of 1 in CDCl3 are summarized in Table 1.
The 1H-decoupling 13C NMR and HMQC spectra reveal 24 carbon
atoms comprising four methyl, four methylene (involving an exo-
methylene), ten methine, and six quaternary carbons (involving
three carbonyls). These analyses also reveal 30 hydrogen atoms
directly linked with carbons. The 13C NMR signal at 198.46 ppm
is assigned as a ketone, while signals appearing at 169.76 and
174.79 ppm suggest carboxy groups. These were supported by
broad but strong absorption found at 1650–1720 cmꢀ1 in the IR
spectrum. Treatment of 1 with trimethylsilyldiazomethane in
methanol gave the monomethyl ester 2 (m/z = 431.2434), revealing
a carboxylic acid functionality in the molecule. Detailed NMR anal-
Results and discussion
yses disclosed that
1 comprises a (4E,6E,2S,3S)-3-hydroxy-2-
Isolation and structural elucidation
methylocta-4,6-dienedioate side chain unit and a petasol substruc-
ture. Relative configuration of the petasol moiety was determined
by NOEs found at Hb-1/H-3, Hb-1/H3-14, H-7/H3-14, and H3-14/H3-
15. The (E,E)-configuration for the C40,C60-diene component was
In the course of exploring novel and bioactive secondary
metabolites from fungi in the Shirakami mountainous area Japan,
homopetasinic acid (1) was isolated along with phomolide C6
and UCS1025A7 from the culture broth of Diaporthe sp. strain
2
established by observing large spin couplings at JH-4/H-5 and
2JH–6/H-7 (15.5 Hz for both).
As shown in Figure 1, alkaline hydrolysis of 1 gave an approxi-
mate 1:1 mixture of petasol (3) and isopetasol (4) as well as
⇑
Corresponding author. Tel./fax: +81 172 39 3782.
E-mail address: hmasaru@hirosaki-u.ac.jp (M. Hashimoto).
http://dx.doi.org/10.1016/j.tetlet.2016.01.095
0040-4039/Ó 2016 Elsevier Ltd. All rights reserved.

1118
A. Ito et al. / Tetrahedron Letters 57 (2016) 1117–1119
Table 1
NMR spectral data of homopetasinic acid (1) in CDCl3⁄
Position
1
d
13C, type
d1H (J in Hz)
: 2.36 ddd (2.5, 4.4, 15.1)
b: 2.50 ddt (1.4, 4.9, 15.1)
: 1.48 dddd (4.4, 11.2, 15.1, 16.3)
b: 2.14 dddd (2.5, 4.5, 4.9, 16.3)
4.91 dt (4.5, 11.2)
HMBC
NOESY
30.49, CH2
31.47, CH2
a
Hb-1, Ha-2, H-9
2, 9, 10
H
H
H
a-1, H-3, H3-14
a-1, Hb-2
a-2,
2
a
3
4
5
6
74.08, CH
47.06, CH
40.00, C
4, 15, 10
3, 5, 6, 14, 15
H3-14, H3-15
Ha-6, H3-15
1.63 dq (11.2, 6.7)
41.61, CH2
a: 1.88 t (14.0)
4, 5, 7, 8, 14,
5, 7, 8, 10, 14
6, 8, 11, 12, 13
H-4, Hb-6
-6, H-7, H3-14, H3-15,
Hb-6, H-12, H3-13, H3-14
b: 2.03 dd (4.3, 14.0)
3.12 dd (4.3, 14.0)
Ha
7
8
9
10
11
12
50.30, CH
198.46, C
124.79, CH
166.24, C
143.21, C
114.54, CH2
5.8 d (1.4)
1, 6, 7,
Ha-1
4.83 brs
5.00 brs
1.74 s
1.23. s
0.96 d (6.7)
7, 13,
7, 13
7, 11, 12
4, 5, 6, 10
3, 4, 5,
H-7
H3-13
13
14
15
10
20.05, CH3
17.17, CH3
10.39, CH3
174.79, C
45.35, CH
73.47, CH
142.62, CH
129.10, CH
145.56, CH
120.88, CH
169.76, C
H-3, Hb-1, Hb-6, H-7, H3-15
H-3, H-4, Hb-6, H3-14
20
2.64 quint (6.7)
4.37 dt (0.5, 6.7)
6.15 dd (6.7, 15.5)
6.48 dd (11.3, 15.5)
7.32 dd (11.3, 15.5)
5.93 d (15.5)
10, 30, 40, 90
10, 20, 40, 50, 90
30, 60
30, 60, 70
40, 50, 80
50, 80
H-30, H-40, H3-90,
H3-90, H-20, H-40, H-50, H-60
H-20, H-30, H-60, H3-90
H-40, H-70
H-40, H-50
H-50
30
40
50
60
70
80
90
14.13, CH3
1.25 d (6.7)
10, 20, 30
H-20, H-30
O
O
side chain unit
petasol unit
+
1
9
10
8
O
HO
HO
OH
O
1) NaOH
aq. THF
13
3
8'
5
6'
4'
11
RO
2'
4
7
3
(ca. 1 : 1)
4
3'
1'
7'
5'
O
1
14
2) TMSCHN2,
MeOH
O
15
12
9'
OR
O
1: R = H
2: R = Me
OH
O
H3CO
2
3
2
OCH3
3
Ph
OMe
O
Figure 1. Structures of homopetasinic acid (1) and its methyl ester 2.
5a
5b
: R = H
: R = Bz
6
BzCl/pyridine
(2E,4E)-6-hydroxy-7-methylocta-2,4-dienedioic acid. The latter
was isolated in the form of dimethyl ester 5a after treatment with
trimethylsilyldiazomethane in MeOH. It is known that alkaline
hydrolytic conditions accompany the isomerization8 at the C11-
5a
threo-6 erythro-6
2JH-2/H-3 (Hz)
7.3
7.2
4.2
2-CH3 (ppm)
14.07 14.22
11.45
olefin. Although 3 and 4 were not separated, the mixture showed
25
Scheme 1. Degradation of 1, chemical transformation of the degradant 5a, and
some characteristic spectral data of 5a and model compound 6.
[
a
]
+99 (c 0.52, CHCl3), which is the mean value of pure 3 and
D
4 from the literature (+130 and +70, respectively).4,9 This allowed
us to conclude the (5R)-configuration of 1.
The configuration of the side chain moiety was determined as
follows. The C2, C3 methine carbons, and methyl signal attached
to C2 of 5a appear at 73.53, 45.15, and 14.07 ppm, respectively,
in the 13C NMR spectrum in CDCl3. Application of Heathcock’s
empirical rule suggested a threo-configuration between the C2-
CH3 and C3-OH in 5a.10 This was further investigated with syn-
thetic model compounds threo-6 and erythro-6, which were selec-
tively prepared by applying Crimmins’s titanium ion mediated
Evans’s aldol reactions.11–13 The 2JH-2/H-3 spin coupling of naturally
degraded 5a is 7.3 Hz in CDCl3 (see Scheme 1), which shows good
accordance with the corresponding value of threo-6 (7.2 Hz). In
contrast, that of erythro-6 gives an obviously smaller coupling con-
stant (4.2 Hz). The 2-CH3 of 5a resembles that of threo-6
(14.22 ppm) rather than erythro-6 (11.45 ppm). These results
establish the (20S⁄,30S⁄)-configuration for 1.
of a benzoyl group (generally, kmax = 230 nm, e = 15,300) at C3-
OH as another chromophore would induce an ECD exciton cou-
pling.14 Benzoylation of 5a smoothly proceeded to give 5b under
conventional conditions. As expected, 5b affords a distinct nega-
tively split ECD curve comprising a negative Cotton effect at
251 nm (
D
e
ꢀ13) and a positive one at 226 nm (
De +15) (Fig. 2).
Wavelengths of these Cotton effects agree with the kmax of 5b.
Molecular modeling calculations suggest that stable conformers
of the (2S,3S)-5b isomer should show a negative chiral relationship
between the chromophores. Theoretical ECD and UV spectra of
(2S,3S)-5b show good accordance with the experimental spectra
calculated with B3LYP/6-31G⁄.6,15–17 These studies establish the
entire structure of homopetasinic acid (1) shown in Figure 1.
Biological properties
Next, we investigated the absolute configuration of the side
Homopetasinic acid (1) inhibited the growth of Cochliobolus
chain. The fragment 5a possesses
a dienoate chromophore
miyabeanus at 24 lM (IC50). This molecule inhibits the growth of
(kmax = 256 nm, = 20,000). Thus, we expected that introduction
e

A. Ito et al. / Tetrahedron Letters 57 (2016) 1117–1119
1119
Figure 2. UV and ECD spectra of experimental 5b (solid line) and theoretical (2S,3S)-5b (dashed line) as well as the 3D structure of its most stable conformation.
3. Ko, W.-C.; Lei, C.-B.; Lin, Y.-L.; Chen, C.-F. Planta Med. 2001, 68, 224–229.
human colon adenocarcinoma (COLO 201) cells (IC50 ca. 10 lM)
4. Sugama, K.; Hayashi, K.; Nakagawa, T.; Mitsuhashi, H.; Yoshida, N.
Phytochemistry 1983, 22, 1619–1622.
5. Thomet, O. A. R.; Wiesmann, U. N.; Schapowal, A.; Bizer, C.; Simon, H.-U.
Biochem. Pharmacol. 2001, 61, 1041–1047.
6. Ito, A.; Maeda, H.; Tonouchi, A.; Hashimoto, M. Biosci., Biotechnol., Biochem.
2015, 79, 1067–1069.
7. Nakai, R.; Ogawa, H.; Asai, A.; Ando, K.; Agatsuma, T.; Matsumiya, S.; Akinaga,
S.; Yamashita, Y.; Mizukami, T. J. Antibiot. 2000, 53, 294–296.
8. Herbst, D.; Djerassi, C. J. Am. Chem. Soc. 1960, 82, 4337–4341.
9. Wang, C.; Li, J.; Yang, R.; Zhao, Y.; Cheng, Y.; Zhang, Z.; He, L. Rec. Nat. Prod.
2014, 8, 156–164.
10. Heathcock, C. H.; Pirrung, M. C.; Sohn, J. E. J. Org. Chem. 1979, 44, 4294–4299.
11. Crimmins, M. T.; King, B. W.; Tabet, E. A. J. Am. Chem. Soc. 1997, 119, 7883–
7884.
based on WST-1 assay.18 However it was not annihilated at higher
concentrations (for example 25 and 100
M).19 Further, it exhibits
weak inhibition against human promyelocytic leukemia cells HL60
(IC50 105
M) based on MTT assay.20 Although the cytotoxicity was
comparable to other petasin related penicilleremophilane A [IC50
56.95 (KB cells) or 39.55 M (MCF-7 cells)], penicilleremophilane
l
l
l
B, an analogue without the arylacetate group, has no activities.21
Additionally, S-petasin is known to potently inhibit degranulation
of RBL-2H3 cells induced by DNP-BSA (IC50 1.0 nM),22 1 showed
no activity even in 1 ꢁ 103 higher concentration. (IC50 > 1000 nM).
These results suggest that the structure of the petasin side chains
play an important role in the biological activities.
12. Crimmins, M. T.; McDougall, P. J. Org. Lett. 2003, 5, 591–594.
13. Crimmins, M. T.; King, B. W.; Tabet, E. A.; Chaudhary, K. J. Org. Chem. 2001, 66,
894–902.
14. Harada, N.; Nakanishi, K.; Berova, N. In Comprehensive Chiroptical Spectroscopy:
Applications in Stereochemical Analysis of Synthetic Compounds, Natural Products,
and Biomolecules; Berova, N., Polavarapu, P. L., Nakanishi, K., Woody, R. W.,
Eds.; Wiley: NJ, USA, 2012; pp 115–166.
15. Honmura, Y.; Takekawa, H.; Tanaka, K.; Maeda, H.; Nehira, T.; Hehre, W.;
Hashimoto, M. J. Nat. Prod. 2015, 78, 1505–1510.
16. Arayama, M.; Nehira, T.; Maeda, H.; Tanaka, K.; Miyagawa, H.; Ueno, T.;
Hosokawa, S.; Hashimoto, M. Tetrahedron 2015, 71, 4788–4794.
17. Yasumura, R.; Ashtekar, K. D.; Tonouchi, A.; Nehira, T.; Borhan, B.; Hashimoto,
M. Tetrahedron 2013, 69, 9469–9474.
Acknowledgments
Part of this work was supported by a JSPS Grant-in-Aid for
Exploratory Research (26660093, 15H04491). The authors would
like to thank Enago (www.enago.jp) for the English language
review.
Supplementary data
18. Shirozu, T.; Harada, Y. Mycoscience 2004, 45, 17–23.
19. That was reproducible. At this moment, we do not have the idea to explain
that. Some molecules with cytostatic activity against cancer cells show the
similar dose-independent activity.
20. Kimura, K.; Sakamoto, Y.; Fujisawa, N.; Uesugi, S.; Aburai, N.; Kawada, M.;
Ohba, S.; Yamori, T.; Tsuchiya, E.; Koshino, H. Bioorg. Med. Chem. 2012, 20,
3887–3897.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2016.01.
095.
21. Daengrot, C.; Rukachaisirikul, V.; Tansakul, C.; Thongpanchang, T.;
Phongpaichit, S.; Bowornwiriyapan, K.; Sakayaroj, J. J. Nat. Prod. 2015, 78,
615–622.
References and notes
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Young-Geun, L.; Young-Whan, C.; Dong-Soon, I. Biomol. Ther. (Seoul) 2015, 23,
45–52.
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