Spiroleptosphol isolated from Leptosphaeria doliolum

Article abstract of DOI:10.1016/j.bmcl.2008.05.087

A novel γ-methylidene-spirobutanolide spirolephtoshol (1) was isolated from ascomycetous fungus Leptosphaeria doliolum as a cytotoxic compound. The relative structure was established by the NMR analysis involving the NOE experiments. Absolute structure of the bicyclic moiety was determined by chemical derivation followed by the CD analysis. The relative and absolute stereochemistry of the side chain was established by comparison of the ¹H NMR spectra and the chiral GC chromatograms of the degradation product with the synthetic samples.

Full text of DOI:10.1016/j.bmcl.2008.05.087

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4228–4231
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Spiroleptosphol isolated from Leptosphaeria doliolum
*
Masaru Hashimoto , Taro Tsushima, Takanori Murakami, Masahiro Nomiya, Noboru Takada,
Kazuaki Tanaka
Faculty of Agriculture and Life Science, Hirosaki University, 3-Bunkyo-cho, Hirosaki 036-8561, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel c-methylidene-spirobutanolide spirolephtoshol (1) was isolated from ascomycetous fungus Lep-
Received 6 March 2008
Revised 11 May 2008
Accepted 15 May 2008
Available online 24 May 2008
tosphaeria doliolum as a cytotoxic compound. The relative structure was established by the NMR analysis
involving the NOE experiments. Absolute structure of the bicyclic moiety was determined by chemical
derivation followed by the CD analysis. The relative and absolute stereochemistry of the side chain
was established by comparison of the 1H NMR spectra and the chiral GC chromatograms of the degrada-
tion product with the synthetic samples.
Keywords:
Isolation
Ó 2008 Elsevier Ltd. All rights reserved.
Structure determination
NMR spectra
CD spectra
Absolute stereochemistry
Degradation
Chiral GC
In the course of studies investigating metabolites from fungi
with unique ecologies, we isolated spiroleptosphol (1), a novel
methylidene-spirobutanolide, as a cytotoxic compound from sap-
rophytic ascomycete Leptosphaeria doliolum isolated from mug-
wort stems.1 Here, we report the structure of 1 involving its
absolute stereochemistry.
ysis of HMBC and HSQC spectra suggested a 3-methylidene-2-oxa-
spiro[4.5]decan-8-en-1-one framework possessing hydroxy groups
at C4, C6, and C7 positions. Strong adsorption at 1785 cmꢀ1 in the
IR spectrum indicated the existence of a butanolide ring in the
molecule, which consisted with the above assumption. These re-
sults led us to propose the planar structure of 1.
c-
Spiroleptosphol (1, Figure 1) was isolated as oil from the fer-
mentation broth of Leptosphareia doliolum by culturing with pota-
to-sucrose medium with shaking (110 rpm) for 14 days and the
following extraction with ethyl acetate and silica gel column chro-
matography.2 Biological assay revealed that 1 exhibited cytotoxic-
The stereochemistries of C4, C6, and C7 alcohol groups were
studied employing tribenzoate 23 prepared by a reaction with
BzCl/DMAP in pyridine (Scheme 1). In the 1H NMR of 2, the C4-H,
C6-H, and C7-H signals were shifted to higher frequency
(Dd = 1.08, 2.30, and 1.73 ppm, respectively), which confirmed
ity against P388 murine leukemia (EC50 = 20
l
g/mL). The EI mass
the alcohol positions.4 The coupling constant between C6-H and
C7-H of 2 was 8.1 Hz, suggesting that these protons took pseudo-
1,2-diaxial relationship. Irradiation of the signal for C6-H induced
the NOEs at C4-H and C10-H to establish the relative stereochem-
istry around the spirobicyclic moiety. Only the 4R*,5R*,6R*,7R*,10R*
isomer satisfies these results.
indicated the molecular ion signal at m/z = 336.1905 suggesting
its molecular formula as C19H28O5.
This molecular formula was supported by observing 19 reso-
nances in the 13C NMR spectrum as shown in Table 1. The 1H
NMR spectrum provided 28 proton signals including three
exchangeable protons. The COSY spectral analysis disclosed an
(E)-3,5-dimethyl-1-heptenyl side chain attached to a methine car-
bon of which 1H resonance was 3.45 ppm. The E-geometry of the
double bond in the side chain was established based on a large
coupling constants (J = 15.5 Hz). The triplet signals observed at
4.68 and 4.80 ppm were coupled each other with 2.3 Hz, suggest-
ing an exo-methylene group. These signals were further coupled
with a proton appeared at 5.16 ppm both in 2.3 Hz. Detailed anal-
The absolute configuration of 1 was investigated by the CD
spectral analysis. Since tribenzoate 2 gave only broad positive Cot-
ton curve, as shown in Figure 2, but not distinct exciton coupling
probably due to cancellation by existence of more than three chro-
mophores,5 1 was converted into dibenzoate 4 in order to simplify
the CD spectrum.6
Hydrogenation using PtO2 catalyst provided 3 as a single dia-
stereomer.7 The stereochemistry at C3 position was assigned by
an NOE between C3-H and C4-H. Heating 3 with BzCl/DMAP in
pyridine gave the desired 4.8 Sterically hindered C4-OH retained
under this condition. The coupling constant between C6-H and
* Corresponding author. Tel./fax: +81 172 39 3782.
E-mail address: hmasaru@cc.hirosaki-u.ac.jp (M. Hashimoto).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.05.087

M. Hashimoto et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4228–4231
4229
11
CH2
3
5
O
4
1
OH
O
HO
19
6
12
10
9
14
20
13
16
HO
7
8
15
17
18
Figure 1. Structure of spiroleptosphol (1).
Figure 2. The CD spetra of tribenzoate 2 and dibenzoate 4.
Table 1
NMR spectral data of 1 in CDCl3
1) O3
Position
13C
1H
HMBC
HO
spiroleptosphol (1)
(R)
(R)
1
3
4
174.63
157.11
68.69
—
58.74
72.22
—
70.00
—
127.73
129.78
38.95
89.08
—
—
6, 10
4, 11
11
—
6, 9, 10
4, 8
—
6, 9
2) NaBH4
(65% 2 steps)
nat-5
5.16 (dt, 6.5, 2.3)
3.36 (brd, 6.5)
—
3.84 (brd, 6.8)
4.40 (br)
4.71 (br)
4.39 (br)
5.75 (dt, 10.2, 2.8)
5.58 (ddd, 1.6, 2.8, 10.2)
3.45 (dq, 8.4, 2.8)
4.68 (t, 2.3)
4-OH
5
6
6-OH
7
7-OH
8
9
10
11
HO
HO
(S)-6
rac-6
1) Tf2O
2) Evans's alkylation
(91%, 2 steps)
ref. 10
Bn
—
10
3) HPLC separation
Bn
10, 12
4, 8, 9, 12, 13
4
O
N
O
N
(S)
(S)
(R)
(R)
4.80 (t, 2.3)
O
O
O
O
12
13
14
15
124.32
141.67
34.28
5.33 (dd, 8.4, 15.5)
5.43 (dd, 7.6, 15.5)
2.16 (m)
0.98 (ddd, 5.3, 8.0, 13.4)
1.24 (ddd, 5.1, 8.6, 13.4)
1.32 (m)
9, 10, 13, 14
10, 14
12, 13, 15
(2S,4S)-7
LiBH4
(78%)
(2S,4S)-5
(2R,4R)-7
LiBH4
(the yield not optimized)
see ref. 13
43.86
13, 14, 17, 19, 20
(2R,4R)-5
chiral GC analysis
16
17
18
19
20
31.57
29.77
11.89
20.86
19.04
17
16, 18, 20
16, 17
1.11 (m) 1.28 (m)
0.85 (t, 7.2)
rac-5
nat-5
0.91 (d, 6.7)
0.80 (d, 6.3)
13, 14, 15
15, 16, 17
Scheme 2. Stereochemical determination of the side chain.
1H NMR spectrum was measured at 40 °C.
On the other hand, (2R,4R)-2,4-dimethylhexan-1-ol (nat-5) was
obtained by ozonolysis of 1 followed by reductive workup as
shown in Scheme 2. The relative stereochemistry of nat-5 was esti-
mated to be 2R*,4R* by 1H NMR spectral comparison with those of
the corresponding epimeric isomers in the literature.10 Unfortu-
nately, the reported optical rotation value of 5 was quite small
NOE
H
O
H
H
OBz
CH2
6
BzCl
BzO
BzO
spiroleptosphol (1)
7
R
Py
H
O
(½a 2D4
ꢁ
þ 3:7 for 2R,4R-isomer).11 To make matters worse, vaporiza-
H2, PtO2
2
3JHC6C7H = 8.1 Hz
tion of nat-5 during purification process was not disregardable in
such small scale to provide only approximately 6 mg of nat-5 from
H3C
NOE
H
OH
O
O
H
H
3
H
H
H
BzCl
BzO
6
CH3
DMAP
4
BzO
BzO
O
OH
7
HO
7
BzO
Py, Δ
R
H
H
R
O
HO
4
3JHC6C7H = 10.3 Hz
3
Scheme 1. Stereochemical determination of the bicyclic moiety.
C7-H in the 1H NMR spectrum was 10.3 Hz, which is a typical
value for 1,2-diaxial protons in cyclohexanes taking chair confor-
mation. As expected, 4 gave a pair of exciton-split curve with
negative Cotton effect at 238 nm (
D
e238ꢀ18.9) and positive one
at 222 nm ( 222+10.9) in the CD spectrum in MeOH to indicate
D
e
a negative chirality for the C6–C7 glycol function based on the
‘dibenzoate rule’.9 These results revealed the absolute stereo-
chemistry around the bicyclic moiety to be 4R,5R,6R,7R,10R as
depicted (see Figure 1).
Figure 3. Total ion chromatograms of nat-5, (2S,4S)-5, (2R,4R)-5, and rac-5 by GC-
EIMS using chiral capillary column (RESTEC Rt-bDEXmTM, 30 m, 0.25 mm ID) at 75°
(constant temperature).

4230
M. Hashimoto et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4228–4231
Table 2
Stereochemistries of 3-methylidene-2-oxaspiro[4.5]decan-1-ones in the literature
Compound
C4
C5 C6 C7
C10
R
Producers
Spiroleptosphol (this report)
Oxaspirol15
R
—
S
R
R
—
S
R
—
S
R
—
S
(3R,5R)-(E)-3,5-dimethylhept-1-en-1-yl Leptosphaeria doliolum
a
a
a
a
a
—
(E,E)-3,5-dimethylhepta-1,4-dien-1-yl
Rhodotorula glutinis T-110
Arthropsis truncata
Chaetomium subspirale
Paecilomyces sp.
CH2
Arthropsolide Ac16
Oxaspirodion17
Rb
(E)-but-2-en-1-yl
(E)-but-2-en-1-yl
(E)-but-2-en-1-yl
methyl
3
O
S* keto form S*
d
d
—
—
4
Paecilospirone18
Massarigenin A19
Mycosporulone20
S*
S*
R* S* keto form S*
S* R* S* R*
OH
O
R
10
HO
6
Massarina tunicata
5
keto form S* R* keto form S*
methyl
methyl
methyl
Coniothyrium sporulosum
Microsphareopsis sp. FO-5050IV
Mycospharella rosigena
6-epi-50-hydroxy- mycosporulone21 S*
R* R* keto form
R* R* keto form S*
R
7
Rosigenine22
S*
HO
a
b
c
Not described.
The stereochemistry in the text was inconsistent with that in the figure in the literature.
The C3 is sp3, but its stereochemistry was not assigned.
Not assigned.
d
e
The C3 is sp3, and its stereochemistry was assigned to be S*.
318 (21, [MꢀH2O]+), 300 (7.6, [Mꢀ2H2O]+), 121(88), 69 (100), FDMS (rel. int.)
m/z 359 (43, [M+Na]+), 337 (100, MH+), 336 (43, M+), EIHRMS found m/z
336.1905. calcd for C19H28O5; M+: 336.1936.
of 1 (24 mg). This amount was insufficient to judge the absolute
chemistry confidently based on its optical rotation.
3. Physical data of 2: IR 2960, 1808, 1731, 1675, 1260, 1115, 1090, 710 cmꢀ1 1H
.
We dissolved this subject by direct comparison of the chiral GC
chromatograms. Prior to the analysis, we prepared both enantio-
mers (2S,4S)-5 and (2R,4R)-5 as well as racemate rac-5. The
(2S,4S)-5 was synthesized by the Organ’s protocol via (2S,4S)-7.10
The enantiomer (2R,4R)-5 was obtained by the similar methodol-
ogy but employing racemic alcohol rac-612 and enantiomeric
Evans’s auxiliary, (S)-4-benzyl-3-propionyloxazolidin-2-one.13
Although the alkylation gave a 1:1 mixture of (2R,4R)-7 and the
corresponding (4S)-isomer, these were successfully separated by
HPLC (Develosil 60, AcOEt:hexane = 7:93).14 The racemate rac-5
was readily prepared by combining (2R,4R)-5 and (2S,4S)-5.
As the authentic samples in hand, these were analyzed by
GCMS. It was found that a chiral capillary column (RESTEC Rt-
bDEXmTM, 30 m, 0.25 mm ID) was effective to distinguish these
enantiomers with sufficient reproducibility (Figure 3). Both GC
peaks provided signals at m/z = 112 [M-H2O]+, 101 [M-(CH2CH3)]+,
and 57 [CH(CH3)CH2CH3]+ with the same intensities to confirm the
peak assignment. These results clearly proved that the sample de-
rived from the natural product has 2R,4R configuration, establish-
ing the (3R,5R)-(E)-3,5-dimethyl-1-heptenyl side chain attached
to the C10 position.
As described, we achieved in disclosing the structure of spirolep-
tosphol (1) including its absolute configuration as depicted in Figure
1. Several 3-methylidene-2-oxaspiro[4.5]decan-1-one derivatives,
oxaspirol,15 arthropsolide A,16 oxaspirodion,17 paecilospirone,18
massarigenin A,19 mycosporulone,20 6-epi-50-hydroxymycosporu-
lone,21 and rosigenin22 have been isolated from basidomycetous
and ascomycetous fungi. These exhibited various biological activi-
ties. Interestingly, the stereochemistries of this bicyclic unit are di-
verse in these compounds as shown in Table 2. Although two types
of biogeneses for this moiety have been proposed, neither has
reached to the final conclusion.22,23 Further biological assays and
biosynthetic studies of 1 are now under investigation in our labora-
tories. Taking high productivity (120 mg from 5.0 L of culture med-
ium) into account, Leptosphaeria doliolum would produce sufficient
amount of samples for these investigations.
NMR (400 MHz in CDCl3) d 0.84 (3H, d, J = 6.5), 0.85 (3H, t, J = 7.5), 0.97 (3H, d,
J = 6.5), 1.06 (1H, ddd, J = 5.5, 9.3, 13.7), 1.14 (1H, dq, J = 13.4, 7.5), 1.31 (2H, m),
1.42 (1H, m), 2.27 (1H, m), 3.50 (1H, dq, J = 7.7, 2.6), 4.51 (1H, dd, J = 2.2, 3.2),
4.88 (1H, dd, J = 2.2, 3.2), 5.50 (1H, dd, J = 7.7, 15.5), 5.59 (1H, dt, J = 10.6, 2.6),
5.60 (1H, dd, J = 7.7, 15.5), 5.91 (1H, dt, J = 10.6, 2.6), 6.14 (1H, d, J = 8.1), 6.24
(1H, t, J = 2.2), 6.44 (1H, dt, J = 8.1, 2.6) 7.35–7.7 (12H, m), 7.99 (4H, m), 8.14
(2H, m), 13C NMR (100 MHz, in CDCl3) d 11.22, 18.92, 21.12 (each CH3), 29.99
(CH2), 31.87, 34.50, 41.28 (each CH), 43.97 (CH2), 56.42 (C), 71.82, 72.70, 70.43
(each CH), 90.60 (CH2), 123.22, 125.51, 125.51, 128.26, 128.33. 128.50, 128.79,
128.84, 129.42. 129.77, 129.78, 129.85, 130.07, 132.97, 133.52, 134.14 (each
CH), 152.69, 165.03, 165.22, 169.9, 165.97 (each C), ESIMS (rel. int.) m/z 1319
(7.5, [2M+Na]+), 671 (100, [M+Na]+), ESIHRMS found m/z 671.2606. calcd for
C
40H40O8Na; [M+Na]+.
4. Addition of D2O led to serious spectral broadening in the 1H NMR spectrum of
1.
5. Ayer et al. investigated the stereochemistry of tri-O-benzoate of arthropsolide
A in their structural studies. They judged its absolute stereochemistry based on
a possitive cotten effect in the CD spectrum. Interestingly, they concluded the
enantiomeric configuration for the C6 and the C7. In their report, the
stereochemistry of arthropsolide A in the text did not accord with that in the
figure (see Ref. 16).
6. We have not succeeded in preparing the 6,7-O-dibenzoate of 1 so far we
examined. Benzoylation with BzCl in pyridine gave
a
mixture of
monobenzoates, 4,6-O-dibenzoate, and tribenzoate 2.
7. Physical data of 3: IR (film) 3460, 2930, 1735, 1055 cmꢀ1. 1H NMR (400 MHz,
acetone-d6) d 0.82 (3H, d, J = 7.4), 0.83 (3H, d, J = 6.5), 0.84 (3H, t, J = 6.7), 0.89–
1.20 (5H, m), 1.22 (1H, dt, J = 13.4, 6.6) 1.29 (1H, m) 1.29 (3H, dd, J = 1.5, 6.3),
1.31–1.43 (3H, m), 1.63 (1H, ddt, J = 1.5, 7.5, 9.5), 1.74 (2H, m), 1.81 (1H, m),
1.88 (1H, dt, J = 12.7, 4.4), 3.32 (1H, dd, J = 2.9, 9.2), 3.83 (1H, d, 4.9), 3.91 (1H,
dddd, J = 4.4, 4.9, 9.2, 11.3), 4.21 (1H, d, J = 2.9), 4.64 (1H, dd, J = 1.5, 5.4), 4.73
(2H, m), 13C NMR (100 MHz, acetone-d6) d 11.51, 14.99, 19.97, 20.77 (each
CH3), 26.70 30.05 (each CH2), 30.93 (CH), 31.37 (CH2), 32.46 (CH), 32.87, 35.86
(each CH2), 39.12 (CH), 45.46 (CH2), 58.18 (C), 70.65, 77.19, 79.29, 81.59 (each
CH), 177.46 (C), ESIMS (rel. int.) m/z 343 (100, [M+H]+), 325 (24, [MꢀH2O+H]+),
260 (9), ESIHRMS found m/z 343.2503. Calcd for C 19H35O5; [M+H]+: 343.2484.
8. Physical data of 4: IR (film) 3460, 2930, 1730, 1230, 710 cmꢀ1 1H NMR
.
(400 MHz, CDCl3) d 0.83 (3H, d, J = 6.6), 0.85 (3H, d, J = 6.7), 0.86 (3H, t,
J = 7.3 Hz), 0.92, 1.06 (each 1H, m), 1.35 (3H, d, J = 6.5), 1.68 (1H, dt, J = 5.0,
11.3), 1.69 (1H, dt, J = 4.3, 10.0), 1.9–2.2 (3H, m), 2.42 (1H, dq, J = 12.7, 3.4), 4.53
(1H, quint, J = 6.5), 4.60 (1H, brd, J = 6.5), 5.60 (1H, d, J = 10.3), 5.73 (1H, dt,
J = 4.7, 10.3 Hz), 7.31 (2H, t, J = 7.9), 7.36 (2H, t, J = 7.6), 7.44 (1H, brt, J = 7.9),
7.50 (1H, brt, J = 7.6), 7.85 (2H, brd, J = 7.6), 7.94 (2H, brd, J = 7.9 Hz), 13C NMR
(100 MHz, CDCl3) d 11.27, 14.45, 19.70, 20.27, 24.85, 28.99, 29.28, 30.20, 30.30,
31.69, 35.18, 38.85, 44.59, 56.53, 72.50, 75.08, 78.04, 78.51, 128.20, 128.60,
128.64, 129.52, 129.80, 129.93, 132.77, 133.73, 165.52, 166.33, 175.13, ESIMS
(rel. int.) m/z 573 (100, [M+Na]+), 551 (15, [M+H]+), ESIHRMS found m/z
551.3010. calcd for C33H43O7; [M+H]+: 551.3009.
9. Harada, N.; Nakanishi, K. J. Am. Chem. Soc. 1969, 91, 3989.
10. Organ, M. G.; Bilokin, Y. V.; Bratovanov, S. J. Org. Chem. 2002, 67, 5176.
11. White, J. D.; Johnson, A. T. J. Org. Chem. 1994, 59, 3347.
Acknowledgments
We thank Mr. Junsuke Miyanishi and Ms. Yuki Yagihashi for
their experimental assistance. Cyitotoxicity of 1 was determined
by Dr. Kaoru Yamada of Nagoya University.
12. (R)-2-Methylbutanol was not available commercially. Organ et al. synthesized
this compound from methyl (S)-(+)-3-hydroxy-2-methylpropionate through
six steps. Since microgram scale was enough in our case, we adopted the
described scheme providing desired (2R,4R)-7 directly.
13. Evans, D. A.; Ennis, M. D.; Mathre, D. J. J. Am. Chem. Soc. 1982, 104, 1737.
14. The reduction of (2R,4R)-7 was performed using 1.0 mg of the HPLC purified
sample. The NMR spectrum of the product (2S,4S)-5 was identical to that of
(2S,4S)-5. Although the accurate amount of the product was not available due
to the small scale, the amount was still enough for GC analysis.
References and notes
1. Otani, Y.; Mikawa, T. Mem. Natn. Sci. Mus. Tokyo 1971, 30, 71.
2. Physical property of 1: IR (film) 3360, 2960, 1785, 1680, 1200, 1100,
15. Doi, J.; Hirota, A.; Nakagawa, M.; Sakai, H.; Isogai, A. Agric. Biol. Chem. 1985, 49, 2247.
1065 cmꢀ1. ½a 2D5
ꢁ
ꢀ 230ꢂ (c = 1.10, CHCl3). EIMS (rel. int.) m/z 336 (5.2, M+),

M. Hashimoto et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4228–4231
4231
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17. Rether, J.; Erkel, G.; Anke, T.; Sterner, O. J. Antibiot. 2004, 57, 493.
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M.; Komiyama, K.; Omura, S. J. Antibiot. 1999, 52, 501.
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