Novel Spirocyclic Trichothecanes, Spirotenuipesine A and B, Isolated from Entomopathogenic Fungus, Paecilomyces tenuipes

Article abstract of DOI:10.1021/jo035137x

Entomopathogenic fungi forming fruiting bodies have been employed as tonics and antitussives from ancient times. Paecilomyces tenuipes, which is also called Isaria japonica, is a very popular entomopathogenic fungus and is often considered a health food i

Full text of DOI:10.1021/jo035137x

Novel Sp ir ocyclic Tr ich oth eca n es, Sp ir oten u ip esin e A a n d B,
Isola ted fr om En tom op a th ogen ic F u n gu s, P a ecilom yces ten u ipes
Haruhisa Kikuchi,† Yasuhiro Miyagawa,† Yuko Sahashi,‡ Satoshi Inatomi,§
Asami Haganuma,† Norimichi Nakahata,† and Yoshiteru Oshima*,†
Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-yama,
Aoba-ku, Sendai 980-8578, J apan, Nitto Denko Corporation, 1-1-2, Shimohozumi,
Ibaraki, Osaka 567-8680, J apan, and HokutoCorporation, 800-8,
Shimokomazawa, Nagano 381-0008, J apan
oshima@mail.pharm.tohoku.ac.jp
Received August 1, 2003
Entomopathogenic fungi forming fruiting bodies have been employed as tonics and antitussives
from ancient times. Paecilomyces tenuipes, which is also called Isaria japonica, is a very popular
entomopathogenic fungus and is often considered a health food in northeast Asian countries such
as China, Korea, and J apan. We cultivated the fruiting bodies of Paecilomyces tenuipes. Among
the large-scale cultivations, fruiting body grown in barley grain contained two novel spirocyclic
trichothecane derivatives, spirotenuipesine A (1) and B (2), and known trichothecane mycotoxins.
Compounds 1 and 2 showed potent activity in neurotrophic factor biosynthesis in glial cells. The
isolation of these compounds indicated that P. tenuipes is a promising source for producing various
biologically active substances including trichothecanes. It is noteworthy that trichothecane
mycotoxins are present in Paecilomyces tenuipes, which is typically used in medicinal health food.
In tr od u ction
from Cordyceps militaris, and cordyanhydride A and B,4
maleic anhydride derivatives with unique skeletons from
Cordyceps pseudomilitaris.
A group of entomopathogenic fungi that form fruiting
bodies mainly belong to Cordyceps, Paecilomyces, and
Hirsutella genera and are known as caterpillar fungi. In
J apanese they are called “Toh-Chu-Kasou”, which trans-
lates as winter worm and summer grass. These fungi are
parasites on the pupae of various insects. Mycelia grow
in the intestine and fruiting bodies are formed from the
body. Some of these fungi have been employed as tonics
and antitussives from ancient times.1 Some compounds,
which were isolated from a culture broth or mycelium of
Cordyceps and Paecilomyces species, have been reported
such as ISP-I (myriocin),2 an immunosuppressant from
Isaria sinclairii (which is a synonym of Paecilomyces
cicadae), cordycepin (3′-deoxyadenosine),3 an antibiotic
Paecilomyces tenuipes, also called Isaria japonica, is a
very popular entomopathogenic fungi and is a common
health food in northeast Asian countries such as China,
Korea, and J apan. Although phytochemists have inves-
tigated the chemical components and biological activity,
there are no scientific reports on the fruiting bodies. We
undertook a large-scale cultivation of the fruiting body
from this fungus and investigated the secondary metabo-
lites. This paper describes the isolation of two novel
spirocyclic trichothecanes, spirotenuipesine A (1) and B
(2), and known trichotecane mycotoxins from a methanol
extract. Their potency in neurotrophic factor biosynthesis
in glial cells is also described.
Resu lts a n d Discu ssion
* To whom correspondence should be addressed. Phone: +81-22-
217-6822. Fax: +81-22-217-6821.
Isola tion . Paecilomyces tenuipes was cultivated on a
medium composed of barley grain and brewers’ yeast. The
cultured fruiting bodies (6.8 kg) were extracted three
times with methanol at room temperature to yield the
extract (2.1 kg). The ethyl acetate soluble fraction (159
g) of the extract was separated by repeated column
chromatography over silica gel and ODS to yield a novel
trichothecane 1 (80 mg) and six conventional tricho-
thecenes (3-8). An especially large amount of 7 (22.1 g)
was isolated. In the same manner, a novel compound 2
† Tohoku University.
‡ Nitto Denko Corporation.
§ Hokuto Corporation.
(1) (a) Namba, T. Coloured Illustrations of WAKAN-YAKU;
Hoikusha: Osaka, J apan, 1980; Vol. II, pp 233-234. (b) Mizuno, T.
Int. J . Med. Mushrooms 1999, 1 (3), 251-261. (c) Liu, Z.-Y.; Yao, Y.-
J .; Liang, Z. Q.; Liu, A.-Y.; Pegler, D. N.; Chase, M. W. Mycol. Res.
2001, 105 (7), 827-832.
(2) Fujita, T.; Inoue, K.; Yamamoto, S.; Ikumoto, T.; Sasaki, S.;
Toyama, R.; Chiba, K.; Hoshino, Y.; Okumoto, T. J . Antibiot. 1994,
47, 208-215.
(3) (a) Cunningham, K. G.; Manson, Wm.; Spring, F. S.; Hutchinson,
S. A. Nature 1950, 166, 949. (b) Cunningham, K. G.; Hutchinson, S.
A.; Manson, Wm.; Spring, F. S. J . Chem. Soc. 1951, 2299-2300. (c)
Kredich, N. M.; Guarino, A. J . Biochim. Biophys. Acta 1960, 41, 363-
365.
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2000, 41, 1657-1660.
10.1021/jo035137x CCC: $27.50 © 2004 American Chemical Society
Published on Web 12/18/2003
352
J . Org. Chem. 2004, 69, 352-356

Novel Spirocyclic Trichothecanes
TABLE 1. 13C a n d 1H NMR Sp ectr a l Da ta of Sp ir oten u ip esin e A (1) a n d B (2)a
spirotenuipesine A (1)
spirotenuipesine B (2)
13C
1H
13C
1H
2R
â
43.1
2.22 (1H, ddd, J ) 11.6, 3.6, 1.3 Hz)
1.48 (1H, dq, J ) 11.6, 1.3 Hz)
4.44 (1H, quint, J ) 1.3 Hz)
43.1
2.17 (1H, br dd, J ) 11.2, 3.7 Hz)
1.46 (1H, br dd, J ) 11.2, 1.5 Hz)
4.44 (1H, t, J ) 1.5 Hz)
3
76.1
40.8
75.7
41.1
4R
â
5
6
7R
â
8R
â
9
10
11
12
13R
â
14
15
16
2.36 (1H, ddd, J ) 12.9, 3.6, 1.3 Hz)
1.19 (1H, br d, J ) 12.9 Hz)
2.27 (1H, ddd, J ) 12.7, 3.7, 1.5 Hz)
1.16 (1H, br d, J ) 12.7 Hz)
49.1
52.9
24.4
49.5
48.3
23.8
2.10 (1H, dddd, J ) 13.6, 4.5, 3.2, 1.7 Hz)
1.50 (1H, td, J ) 13.6, 3.4 Hz)
1.84 (1H, dtd, J ) 13.8, 3.6, 1.7 Hz)
1.26 (1H, td, J ) 13.8, 3.6 Hz)
1.60 (1H, td, J ) 13.8, 3.6 Hz)
1.48 (1H, dtd, J ) 13.8, 3.6, 1.2 Hz)
37.0
1.68 (1H, td, J ) 13.6, 3.2 Hz)
31.8
1.87 (1H, dddd, J ) 13.6, 4.5, 3.4, 1.7 Hz)
69.2
137.0
130.5
90.0
69.6
60.8
59.4
89.9
63.9
5.70 (1H, dd, J ) 10.5, 1.7 Hz)
5.46 (1H, dd, J ) 10.5, 1.7 Hz)
3.07 (1H, dd, J ) 4.0, 1.2 Hz)
3.47 (1H, dd, J ) 4.0, 1.7 Hz)
64.8
3.82 (1H, d, J ) 12.1 Hz)
3.71 (1H, d, J ) 12.1 Hz)
1.01 (3H, s)
4.99 (1H, s)
1.26 (3H, s)
3.85 (1H, d, J ) 12.7 Hz)
3.78 (1H, d, J ) 12.7 Hz)
1.22 (3H, s)
5.22 (1H, s)
1.30 (3H, s)
15.7
102.9
27.5
15.2
101.2
22.3
a
500 MHz for 1H and 125 MHz for 13C in CDCl3.
(42 mg) and a known trichothecene (9) were afforded from
the n-butanol soluble fraction (365 g) of the extract.
F IGURE 1. Planar structure of spirotenuipesine A (1).
St r u ct u r e E lu cid a t ion . HREI-MS (m/z 266.1528
[M]+), 1H, and 13C NMR spectra indicated that the
molecular formula of 1 was C15H22O4. The 13C NMR
spectrum of 1 showed the presence of two olefinic, an
acetal, two oxygenated quaternary, an oxymethine, an
oxymethylene, two quaternary, four methylene, and two
methyl carbons (Table 1). 1H-1H COSY revealed that
C-2-C-3-C-4, C-7-C-8, and C-10-C-11 were connected.
The methyl protons at C-16 were correlated to three
carbons (C-8, -9, and -10) and the five protons at C-7, -8,
-10, -11, and -15 were correlated with the quaternary
carbon (C-6) in the HMBC spectrum, which suggests a
substituted cyclohexene ring (Figure 1A). The methyl
proton at C-14 coupled with three carbons (C-4, -5, and
-12) and the two protons at C-2 and -13 were correlated
to the quaternary carbon (C-5), which implied a substi-
tuted cyclopentane ring (Figure 1B). The correlations
between peaks H-7-C-5, H-15-C-5, and H-14-C-6 in-
dicated that the partial structures A and B were con-
nected through the C-5-C-6 bond. The intramolecular
acetal moiety was deduced by the correlational peaks of
methine proton at C-15 with the two carbons at C-3 and
C-12 in the HMBC spectrum. Therefore, Figure 1C
depicts the planar structure of 1 for a trichothecane
sesquiterpenoid.
F IGURE 2. Relative structure of spirotenuipesine A (1).
The cross-peak between H-7â and H-16 in the NOESY
spectrum indicated that the configuration of the methyl
group at position 16 was axial in the cyclohexene ring.
The methyl group at position 14 faced the same direction
as C-16 because of the NOEs between H-7â and H-14.
The correlational peak between H-4R and H-7R deter-
mined the relative configuration of C-5 and C-6, and the
configuration of the other stereocenters at C-3, C-12, and
C-15 was fixed by steric constraints of the caged structure
(Figure 2). Structural rigidity and distortion of the spiro
and tricyclo rings in 1 allowed for the observation of
W-couplings between H-2R-H-4R (J ) 1.3 Hz), H-7R-
H-11 (J ) 1.7 Hz), and H-8â-H-10 (J ) 1.3 Hz).
The HREIMS of 2 (m/z 282.1470 [M]+) gave a molec-
ular formula, C15H22O5, that differs from that of 1 by one
oxygen atom. The 1H NMR spectrum of 2 was nearly
identical with that of 1, though 2 lacked the olefinic
proton signals (δH 5.70 and 5.46) which were replaced
by two other signals (δH 3.47 and 3.07). The correspond-
J . Org. Chem, Vol. 69, No. 2, 2004 353

Kikuchi et al.
SCHEME 1a
a
Reagents and conditions: (a) Oxone, CH2Cl2-MeOH-phosphate buffer (pH 9.2)-acetone (1:4:2:0.3), 0 °C; (a′) Oxone, CH2Cl2-MeOH-
phosphate buffer (pH 9.2)-acetone (1:4:2:0.3), rt; (b) benzyl bromide, sodium hydride, THF, 0 °C; (c) 10% HCl-MeOH, rt; (d) (R)- or
(S)-R-methoxyphenylacetic acid, EDCI‚HCl, DMAP, CH2Cl2, 0 °C.
ing signals in the 13C NMR spectrum of 2 were at δC 59.4
and 60.8. In view of the above findings, we concluded that
2 has an epoxy ring between positions 10 and 11 (Table
1). This is supported by the fact that the epoxidation of
1 with dimethyldioxirane, which was prepared from
acetone and Oxone in situ at 0 °C, gives a high yield of
2 (synthetic [R]25 +4.6 (c 0.252, CHCl3), natural [R]25
D
D
+5.5 (c 0.832, CHCl3)) (Scheme 1), although the reaction
at room temperature yields the overoxidized product (10).
The cross-peak between H-11 and H-15 in the NOESY
spectrum revealed that the stereochemistry of the ep-
oxide was â. Approach of dimethyldioxirane on 1 thus
occurs from the less hindered â face of the ring. This fact
was supported by transacetalization at C-15 under acidic
conditions described later.
F IGURE 3. ∆δRS values for (R)-MPA ester (13a ) and (S)-MPA
ester (13b).
rotrophic factors synthesized in 1321N1 cells, which
promote the differentiation of PC-12 cells.6 The culture
medium conditioned with 1 µM 1 or 2 and 100 nM
phorbol 12-myristate 13-acetate (PMA), which is an
activator of neurotrophic factor biosynthesis, enhanced
the extension of neurite outgrowth of PC-12 cells (Figure
4). 1 and 2 caused a concentration-dependent differentia-
tion of PC-12 cells through the biosynthesis of nerotrophic
factors from 1321N1 cells (Figure 5). These results
indicate that 1 and 2 biosynthesize and release neu-
rotrophic factors from 1321N1 cells and the released
neurotrophic factors promote neuronal differentiation of
PC-12 cells. Therefore, 1 and 2 are active in neurotrophic
factor biosynthesis, suggesting that both may be lead
compounds in drug synthesis for serious neuronal dis-
orders such as Alzheimer’s disease.
Deter m in a tion of Absolu te Con figu r a tion . To de-
termine the absolute configurations of 1 and 2, the
following transformations were conducted (Scheme 1). 13-
O-Benzyl ether (11) was prepared by treating 2 with
benzyl bromide. Under acidic conditions, epoxide opening
and transacetalization yielded a sole product 12. The
â-configuration of the epoxide between C-10 and C-11 was
substantiated by this reaction via hemiacetal intermedi-
ate a in addition to the NOE data mentioned above.
Esterification of 12 with (R)-R-methoxyphenylacetic acid5
in the presence of EDCI and DMAP yielded only 3-O-
(R)-R-methoxyphenylacetate (13a ). In a similar manner,
3-O-(S)-R-methoxyphenylacetate (13b) was afforded. The
∆δRS value of each proton was calculated from the
difference in chemical shifts between 13a and 13b
(Figure 3). Then, the structure of 12 was fitted into the
proposed model of R-methoxyphenylacetate5 in accor-
dance with the sign of ∆δRS to give the absolute config-
uration of 12 as 3R, 5R, 6S, 9S, 10R, 11S, 12R, and 15S.
Therefore, the absolute configurations of 1 and 2 were
based on 12 and determined to be (3R, 5R, 6R, 9S, 12R,
15R) and (3R, 5R, 6S, 9S, 12R, 15R), respectively.
Biologica l Act ivit ies. To investigate the biological
effects of 1 and 2, 1321N1 human astrocytoma cells (glial
cell line) were incubated for 2 days with each compound.
Then rat pheochromocytoma (PC-12) cells were cultivated
for 2 days in the conditioned 1321N1 culture medium.
The culture medium has been shown to contain neu-
Con clu sion
Although a number of trichothecane-type sesquiterpen-
oids have been isolated as fungal metabolites produced
by various species of Fusarium, Myrothecium, Tricho-
derma, Cephalosporium, Verticimonosporium, and Stachy-
botrys,8 1 and 2 are uniquely cyclized trichothecanes
containing spirocyclic and tricyclo ring systems. It is
speculated that an analogue of trichotriol (17) and FS-2
(18) biosynthesized 1 and 2.9 Numerous biological studies
(6) Obara, Y.; Nakahata, N.; Ohizumi, Y. Brain Res. 1998, 806, 79-
88.
(7) Obara, Y.; Kobayashi, H.; Ohta, T.; Ohizumi Y.; Nakahata, N.
Mol. Pharmacol. 2001, 59, 1287-1297.
(8) (a) Grove, J . F. Nat. Prod. Rep. 1988, 187-209. (b) Grove, J . F.
Prog. Chem. Org. Nat. Prod. 1996, 69, 1-70.
(9) Corley, D. G.; Rottinghaus, G. E.; Tempesta, M. S. J . Org. Chem.
1987, 52, 4405-4408.
(5) Trost, B. M.; Balletire, J . L.; Godleski, S.; McDougal, P. G.;
Balkovec J . M. J . Org. Chem. 1986, 51, 2370-2374.
354 J . Org. Chem., Vol. 69, No. 2, 2004

Novel Spirocyclic Trichothecanes
is a promising source for producing various biologically
active compounds including trichothecanes.
Recently, two groups also reported the isolation of 4â-
acetoxyscirpene-3,15-diol (7) from the fruiting body of P.
tenuipes and its apoptosis-inducing activities.10,11 This
study also demonstrated that the fruiting body of P.
tenuipes cultured on barley grain produced a large
amount of 7. This is noteworthy since P. tenuipes
cultivated on media that contain grains were typically
used in medcinal health foods.
Exp er im en ta l Section
Or ga n ism a n d Cu ltu r e Con d ition s. The strain MH19912
of P. tenuipes (synonym of Isaria japonica) was isolated at
Yamanashi Prefecture, J apan, and preserved at Hokuto
Corporation (Nagano, J apan). This strain was cultured on a
medium composed of barley grain, brewers’ yeast, and distilled
water (weight ratio 57:9:120). Millet grain instead of barley
grain or only living silkworm was also used in the medium.
This medium was poured into a plastic bag, autoclaved at 120
°C for 40 min, and inoculated with a mycelial agar disk (5 mm
in diameter) of this strain precultured on the potato dextrose
agar medium. The culture was incubated at 25 °C for 21 days.
After mycelia grew sufficiently, the surface of the culture was
scratched and the culture was transferred to the condition for
formation of fruiting bodies (synnemata), at 20 °C, 90% relative
humidity, with a lighting cycle of 15 min light and 45 min dark,
for 21 days. After cultivation, the fruiting bodies were har-
vested and dried.
F IGURE 4. Glial cell-mediated morphological change of PC-
12 cells by spirotenuipesine A (1) and B (2). PC-12 cells were
incubated for 2 days in conditioned medium of 1321N1 human
astrocytoma cells (glial cells) with or without 1 µM 1 or 2, or
with 100 nM PMA for 2 days. Phase contrast microscopies of
PC-12 cells are shown.
Isola tion of 1 a n d 2. The cultured fruiting bodies (dry
weight 6.79 kg) of P. tenuipes were extracted three times with
methanol (90 L) at room temperature to give the extract (2.06
kg). This extract was partitioned with ethyl acetate and water
to yield ethyl acetate solubles (159 g) and a water layer, which
was extracted with n-butanol three times to afford n-butanol
solubles (365 g). The ethyl acetate solubles were chromato-
graphed over SiO2, and the column eluted with n-hexanes-
ethyl acetate mixtures with increasing polarity. The ethyl
acetate eluent (4.00 g) was further chromatographed over SiO2
with chloroform-methanol (39:1) and then ODS with water-
methanol (1:99) to give spirotenuipesine A (1) (80 mg). The
n-butanol solubles were chromatographed over SiO2, and the
column was eluted with ethyl acetate-methanol mixtures with
increasing polarity. Ethyl acetate eluent (5.36 g) was further
chromatographed over ODS with water-methanol (1:99) and
then SiO2 with chloroform-methanol (19:1) to give spiro-
tenuipesine B (2) (42 mg). 1 ((3R,5R,6R,9S,12R,15R)-7′-hy-
droxymethyl-3′,4-dimethylspiro[cyclohex-2-ene-1,2′-[8,9]-di-
oxatricyclo[3.3.1.03,7]nonan]-4-ol): colorless oil; [R]25D +32.8 (c
0.417, CHCl3); 1H NMR and 13C NMR data are shown in Table
1; EI-MS m/z 266 [M]+, 248, 220, 189, 172, 145, 43 (base);
HREI-MS m/z 266.1528 (266.1518 calcd for C15H22O4). 2
((3R,5R,6S,9S,12R,15R)-2,3-epoxy-7′-hydroxymethyl-3′,4-di-
methylspiro[cyclohexane-1,2′-[8,9]dioxatricyclo[3.3.1.03,7]nonan]-4-ol):
F IGURE 5. Effects of spirotenuipesine A (1) and B (2) on glial
cell-mediated morphological changes of PC-12 cells. PC-12 cells
were incubated for 2 days in a conditioned medium of 1321N1
human astrocytoma cells (glial cells) with varying concentra-
tions of 1 or 2, or PMA for 2 days. The differentiation of PC-
12 cells was evaluated as previously described.7 Each column
represents the mean with SE of three or four experiments.
Significant difference without drug (*P < 0.05).
colorless oil; [R]25 +5.5 (c 0.832, CHCl3); 1H NMR and 13C
D
NMR data are shown in Table 1; EI-MS m/z 282 [M]+, 264,
206, 187, 227, 43 (base); HREI-MS m/z 282.1470 (282.1467
calcd for C15H22O5).
Con ver sion of 1 in to 2. To a solution of 1 (2.7 mg, 10.1
µmol) in dichloromethane-methanol-phosphate buffer (pH
9.2) (1:4:2) (1.5 mL) was added acetone (30 µL) and 0.25 M
Oxone solution in water (0.5 mL) at 0 °C. After being stirred
for 6 h, this mixture was poured into water (5 mL) and
extracted with 1-butanol (10 mL) three times. The organic
on trichothecanes have focused on their antibacterial,
antifungal, insecticidal, and cytostatic properties, espe-
cially phytotoxicity, but our study demonstrated that 1
and 2 show activity in neurotrophic factor biosynthesis.
This biological activity of trichothecanes and P. tenuipes
(10) Nam, K.-S.; J o, Y.-S.; Kim, Y.-H.; Hyun, J .-W.; Kim, H.-W. Life
Sci. 2001, 69, 229-237.
(11) Oh, G.-S.; Hong, K.-H.; Oh, H.; Pae, H.-O.; Kim, I.-K.; Kim, N.-
Y.; Kwon, T.-O.; Shin, M.-K.; Chung, H.-T. Biol. Pharm. Bull. 2001,
24, 785-789.
J . Org. Chem, Vol. 69, No. 2, 2004 355

Kikuchi et al.
layer was evaporated. The residue was chromatographed over
to afford 12 (0.7 mg, 2.0 µmol, 37%). 12: colorless oil; [R]25
D
silica gel eluted by chloroform-methanol (9:1) to afford 2 (2.6
+312 (c 0.120, chloroform); 1H NMR (400 MHz, CDCl3) δ 7.20-
7.30 (5H, m), 5.59 (1H, s), 5.24 (1H, dd, J ) 5.5, 1.7 Hz), 4.62
(1H, d, J ) 11.8 Hz), 4.50 (1H, d, J ) 11.8 Hz), 4.15 (1H, br s),
4.00 (1H, d, J ) 5.5 Hz), 3.57 (1H, d, J ) 10.7 Hz), 3.42 (1H,
d, J ) 10.7 Hz), 2.15 (1H, ddd, J ) 14.0, 6.6, 1.9 Hz), 1.91-
2.03 (2H, m), 1.90 (1H, dd, J ) 12.5, 8.6 Hz), 1.79 (1H, dd, J
) 14.0, 8.5 Hz), 1.71 (1H, ddd, J ) 12.5, 5.4, 1.9 Hz), 1.55-
1.65 (2H, m), 1.37 (3H, s), 1.20 (3H, s); EI-MS m/z 373 [M -
OH]+, 355, 217, 91 (base), 43; HREI-MS m/z 373.1998 (373.2016
calcd for C22H29O5).
mg, 9.2 µmol, 91%). Synthetic 2: [R]25 +4.6 (c 0.252, CHCl3);
D
other spectral data were identical with those of the natural 2.
(3R,5R,6S,9S,12R)-2,3-Ep oxy-5-h yd r oxym eth yl-1′,4-d i-
methyl-3′-oxospiro[cyclohexane-1,2′-[4]oxa-bicyclo[3.3.0]oc-
ta n e]-4,7′-d iol (10). To a solution of 1 (12.6 mg, 44.7 µmol)
in dichloromethane-methanol-phosphate buffer (pH 9.2) (1:
4:2) (1.5 mL) was added acetone (30 µL) and a 0.25 M Oxone
solution in water (0.6 mL) at room temperature. After being
stirred for 3 h, this mixture was poured into water (5 mL),
then extracted with 1-butanol (10 mL) three times. The organic
layer was evaporated. The residue was chromatographed over
silica gel eluted by chloroform-methanol (9:1) to afford 10 (4.8
(R)- a n d (S)-r-Meth oxyp h en yla ceta te of 12 (13a a n d
13b). To a solution of 12 (0.7 mg, 2.0 µmol) in dichloromethane
(1 mL) was added triethylamine (20 µL), 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide hydrochloride (1.1 mg), (R)-R-
methoxyphenylacetic acid (0.9 mg), and DMAP (0.4 mg) at 0
°C. After being stirred for 6 h, the mixture was poured into
0.25 M hydrochloric acid (5 mL), then extracted with ethyl
acetate three times. The organic layer was collected and
evaporated. The residue was chromatographed over silica gel
eluted by chloroform to afford (R)-MPA ester 13a (0.5 mg, 1.0
µmol, 50%). In the same manner, (S)-MPA ester (13b) (0.6 mg,
1.2 µmol, 29%) was synthesized from 12 (1.5 mg, 4.0 µmol).
13a : colorless oil; 1H NMR (600 MHz, CDCl3) δ 7.25-7.39
(10H, m), 5.35 (1H, s), 5.22 (1H, dd, J ) 5.5, 1.5 Hz), 4.95-
5.01 (1H, m), 4.70 (1H, s), 4.59 (1H, d, J ) 11.9 Hz), 4.46 (1H,
d, J ) 11.9 Hz), 3.95 (1H, d, J ) 5.5 Hz), 3.51 (1H, d, J ) 10.8
Hz), 3.38 (1H, d, J ) 10.8 Hz), 3.37 (3H, s), 2.14 (1H, ddd, J )
14.6, 6.8, 1.2 Hz), 2.02 (1H, br dd, J ) 13.6, 7.5 Hz), 1.90-
1.99 (2H, m), 1.78 (1H, dd, J ) 14.6, 6.9 Hz), 1.75-1.79 (1H,
m), 1.48-1.53 (2H, m), 1.35 (3H, s), 1.19 (3H, s); EI-MS m/z
521 [M - OH]+, 199, 121 (base), 91; HREI-MS m/z 521.2540
(521.2542 calcd for C31H37O7). 13b: colorless oil; 1H NMR (600
MHz, CDCl3) δ 7.26-7.43 (10H, m), 5.36 (1H, s), 5.24 (1H, dd,
J ) 5.5, 1.7 Hz), 4.99-5.04 (1H, m), 4.70 (1H, s), 4.60 (1H, d,
J ) 11.9 Hz), 4.49 (1H, d, J ) 11.9 Hz), 3.95 (1H, d, J ) 5.5
Hz), 3.54 (1H, d, J ) 10.7 Hz), 3.36 (1H, d, J ) 10.7 Hz), 3.36
(3H, s), 2.19 (1H, ddd, J ) 14.3, 6.8, 1.2 Hz), 1.93 (1H, dd, J
) 13.7, 7.1 Hz), 1.86-1.90 (1H, m), 1.83-1.91 (2H, m), 1.70
(1H, br dd, J ) 13.7, 6.0 Hz), 1.32-1.43 (2H, m), 1.33 (3H, s),
1.16 (3H, s); EI-MS m/z 521 [M - OH]+, 199, 121 (base), 91;
HREI-MS m/z 521.2543 (521.2542 calcd for C31H37O7).
mg, 16.0 µmol, 36%). 10: colorless oil; [R]25 +19.9 (c 0.483,
D
MeOH); 1H NMR (400 MHz, CD3OD) δ 4.28 (1H, m), 4.02 (1H,
d, J ) 3.8 Hz), 3.79 (1H, d, J ) 12.7 Hz), 3.70 (1H, d, J ) 12.7
Hz), 3.03 (1H, d, J ) 3.8 Hz), 2.11 (1H, ddd, J ) 14.8, 6.4, 1.2
Hz), 2.06-2.12 (1H, m), 1.98 (1H, ddd, J ) 14.4, 9.4, 3.3 Hz),
1.93 (1H, ddd, J ) 14.8, 5.2, 1.4 Hz), 1.87 (1H, ddd, J ) 13.8,
9.1, 3.3 Hz), 1.78 (1H, ddd, J ) 13.4, 5.1, 1.2 Hz), 1.53 (1H,
ddd, J ) 14.4, 9.1, 3.4 Hz), 1.39 (1H, ddd, J ) 13.8, 9.4, 3.4
Hz), 1.31 (3H, s), 1.30 (3H, s); 13C NMR (100 MHz, CD3OD) δ
179.5, 96.7, 70.9, 68.3, 64.4, 60.2, 58.2, 53.3, 49.5, 48.2, 45.1,
32.2, 26.0, 24.4, 19.1; EI-MS m/z 298 [M]+, 267, 249, 228, 43
(base); HREI-MS m/z 298.1415 (298.1416 calcd for C15H22O6).
(3R,5R,6S,9S,12R,15R)-2,3-Ep oxy-7′-ben zyloxym eth yl-
3′,4-d im eth ylsp ir o[cycloh exa n e-1,2′-[8,9]d ioxa -tr icyclo-
[3.3.1.03,7]n on a n ]-4-ol (11). To a solution of 2 (10.0 mg, 35.4
µmol) in THF (1 mL) was added sodium hydride (3.6 mg) and
benzyl bromide (100 µL) at 0 °C. After being stirred for 5 h,
the mixture was poured into water (5 mL) and extracted with
ethyl acetate three times. The organic layer was dried over
anhydrous sodium sulfate and evaporated. The residue was
chromatographed over silica gel eluted by n-hexane-chloro-
form (1:9) to afford 11 (8.7 mg, 23.4 µmol, 66%). 11: colorless
oil; [R]25 +6.9 (c 0.434, chloroform); 1H NMR (400 MHz,
D
CDCl3) δ 7.25-7.36 (5H, m), 5.21 (1H, s), 4.63 (1H, d, J ) 12.0
Hz), 4.56 (1H, d, J ) 12.0 Hz), 4.42 (1H, br s), 3.73 (1H, d, J
) 10.9 Hz), 3.65 (1H, d, J ) 10.9 Hz), 3.50 (1H, dd, J ) 3.9,
1.6 Hz), 3.01 (1H, dd, J ) 3.9, 1.1 Hz), 2.24 (1H, br d, J ) 12.6
Hz), 2.18 (1H, br dd, J ) 11.4, 2.8 Hz), 1.83 (1H, dtd, J ) 13.7,
3.7, 1.6 Hz), 1.77 (1H, br s), 1.60 (1H, td, J ) 13.7, 3.7 Hz),
1.54 (1H, br d, J ) 11.4 Hz), 1.47 (1H, dtd, J ) 13.7, 3.7, 1.1
Hz), 1.29 (3H, s), 1.24 (1H, td, J ) 13.7, 3.7 Hz), 1.20 (3H, s),
1.15 (1H, br d, J ) 12.6 Hz); 13C NMR (100 MHz, CDCl3) δ
137.9, 128.3 (2C), 127.6 (2C), 127.6, 101.2, 89.1, 75.7, 73.9, 71.2,
69.8, 60.5, 59.6, 49.8, 48.5, 43.4, 41.2, 32.1, 23.8, 22.4, 15.5;
EI-MS m/z 372 [M]+, 354, 91 (base), 43; HREI-MS m/z
372.1942 (372.1947 calcd for C22H28O5).
Ack n ow led gm en t. This work was supported in part
by a Grant-in-Aid for Scientific Research (No. 13024208)
from the Ministry of Education, Science, Sports and
Culture of J apan, Shorai Foundation for Science and
Technology, Hokuto Foundation for Biological Science,
and Takeda Science Foundation.
(3R,5R,6S,9S,10R,11S,12R,15S)-5-Ben zyloxym eth yl-9,-
13-dim eth yl-2,4-dioxa-tetr acyclo[8.3.1.03,10.05,9]tetr adecan e-
7,13,14-tr iol (12). Benzyl ether 11 (2.0 mg, 5.4 µmol) was
dissolved in 10% hydrogen chloride containing methanol (0.5
mL). This solution was stirred for 7 h at room temperature,
refluxed for 2.5 h, and evaporated. The residue was chromato-
graphed over silica gel eluted by chloroform-methanol (49:1)
Su p p or tin g In for m a tion Ava ila ble: NMR spectra of new
compounds such as spirotenuipesine A (1) and B (2). This
material is available free of charge via the Internet at
http://pubs.acs.org.
J O035137X
356 J . Org. Chem., Vol. 69, No. 2, 2004