Trichocladinols A-C, cytotoxic metabolites from a Covdyceps-colomzing ascomycete Trichocladium opacum

Article abstract of DOI:10.1002/ejoc.200900735

Trichocladinols A-C (1-3), three new metabolites, and the known massarigenin A (4), have been isolated from cultures of a Cordyceps-colonizing ascomycete Trichocladium opacum. Their structures were elucidated by NMR spectroscopy and X-ray crystallography.

Full text of DOI:10.1002/ejoc.200900735

FULL PAPER
DOI: 10.1002/ejoc.200900735
Trichocladinols A–C, Cytotoxic Metabolites from a Cordyceps-Colonizing
Ascomycete Trichocladium opacum
Huijuan Guo,[a] Bingda Sun,[a] Hao Gao,[b] Shubin Niu,[a] Xingzhong Liu,[a] Xinsheng Yao,[b]
and Yongsheng Che*[a]
Keywords: Cytotoxicity / Fungal metabolites / Structure elucidation / Natural products / Configuration determination
Trichocladinols A–C (1–3), three new metabolites, and the
known massarigenin A (4), have been isolated from cultures
(S)-MTPA ester. Compounds 1–3 showed modest cytotoxic
effects against the human tumor cell lines HeLa and MCF-7.
of a Cordyceps-colonizing ascomycete Trichocladium opacum. Structurally, compounds 1 and 2 possess a previously unde-
Their structures were elucidated by NMR spectroscopy and
X-ray crystallography. The absolute configuration of 1 was
assigned by using the modified Mosher method and that of
3 was determined by X-ray crystallographic analysis of its
scribed 2,9-dioxatricyclo[5.2.1.03,8]dec-4-ene skeleton.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2009)
Introduction
Cordyceps sinensis (Berk.) Sacc. (Anamorph: Hirsutella
sinensis),[1] is a unique black, blade-shaped fungus found
primarily at high altitude on the Qinghai-Tibetan plateau
parasitizing on dead caterpillars of the moth Hepilus spp.
A combination of the fungus and the dead caterpillars is
known as “Dong Chong Xia Cao” (winter-worm, summer-
grass) and has been widely used as a tonic and/or medicine
for hundreds of years in the Orient.[2] Even though many
medical benefits, including antitumor activity, have been re-
ported for this fungus,[3] whether the active components are
produced by C. sinensis or its colonizing fungi remains to
be investigated. During an ongoing search for new bioactive
natural products from the Cordyceps-colonizing isolates,[4,5]
the fungus Trichocladium opacum (X116), isolated from a
sample of C. sinensis collected in Linzhi, Tibet, P. R. China,
was grown in a solid-substrate fermentation culture. The
extract in organic solvent displayed cytotoxicity against two
human tumor cell lines, HeLa and MCF-7. Fractionation
of the extract afforded three new metabolites, which we have
named trichocladinols A–C (1–3), and the known com-
pound massarigenin A (4; Figure 1).[6] Details of the isola-
tion, structure elucidation, and biological activity of these
metabolites are reported herein.
Figure 1. Metabolites 1–4 from T. opacum.
Results and Discussion
The molecular formula of trichocladinol A (1; atom
numbering shown and used for NMR assignments not in
accord with the systematic IUPAC-consistent name) was as-
signed as C13H18O5 (five degrees of unsaturation) on the
basis of its HRESIMS spectrum (m/z = 277.1046 [M +
Na]+, ∆ = –0.4 mmu). Analysis of the H, 13C, and HMQC
1
NMR spectroscopic data (Table 1) of 1 revealed one ex-
changeable proton (δ = 4.46 ppm), three methyl groups, one
oxygenated methylene unit, four methines (three oxygen-
ated), one disubstituted olefin, two sp3 quaternary carbon
atoms (one oxygenated), and one carboxy carbon atom. In-
[a] Key Laboratory of Systematic Mycology & Lichenology, Insti-
tute of Microbiology, Chinese Academy of Sciences,
Beijing 100190, P. R. China
1
terpretation of the H–1H COSY NMR spectroscopic data
identified two isolated proton spin systems, C-6–C-10 (in-
cluding C-12) and C-4–4-OH. HMBC correlations from 6-
H, 7-H, 10-H, and 12-H to C-5 reveal the connection of C-
5 to C-6 and C-10, completing the cyclohexene ring in 1.
Correlations from 1a-H to C-4, C-5, C-6, and C-10, and
from 4-H to C-1 and C-6 indicate that both C-1 and C-4
Fax: +86-10-82618785
E-mail: cheys@im.ac.cn
[b] Institute of Traditional Chinese Medicine and Natural Prod-
ucts, College of Pharmacy, Jinan University,
Guangzhou 510632, P. R. China
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.200900735.
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1
Table 1. H and 13C NMR spectroscopic data for 1–4 in [D6]acetone and [D6]DMSO.
Pos.
1
2
3
δH [ppm], (mult. J [Hz])[a] δC [ppm][b]
δH [ppm], (mult. J [Hz])[a]
δC [ppm][b]
δH [ppm], (mult. J [Hz])[c]
δC [ppm][d]
1a
1b
3
4.22 (d, 12)
4.32 (d, 12)
62.4, CH2
176.0, Cquat
174.0, Cquat
110.6, Cquat
78.4, CH
110.8, Cquat
80.1, CH
155.6, Cquat
68.1, CH
4
3.78 (d, 6.0)
3.80 (d, 6.5)
5.12 (d, 7.0)
5
6
52.3, Cquat
31.0, CH
136.6, CH
60.9, Cquat
35.0, CH
134.9, CH
56.0, Cquat
28.7, CH
28.0, CH2
2.56 (ddq, 3.0, 2.0, 7.5)
5.67 (dd, 10, 2.0)
2.80 (ddq, 3.0, 2.0, 7.5)
5.67 (dd, 10, 2.0)
2.10 (ddq, 6.5, 5.0, 7.0)
1.20 (ddd, 13, 6.5, 4.0)
2.25 (tt, 13, 5.0)
1.30 (ddd, 13, 10, 4.0)
1.60 (ddd, 13, 6.5, 5.0)
3.50 (td, 10, 5.0)
3.70 (dd, 10, 4.0)
4.53 (d, 2.5)
7a
7b
8a
8b
9
10
11a
11b
12
13
14
5.84 (ddd, 10, 4.5, 3.0)
124.7, CH
5.90 (ddd, 10, 4.5, 3.0)
124.6, CH
28.1, CH2
3.96 (t, 4.5)
4.31 (d, 4.5)
1.41 (s)
69.7, CH
76.4, CH
14.7, CH3
3.99 (t, 4.5)
4.69 (d, 4.5)
1.41 (s)
69.1, CH
78.4, CH
14.6, CH3
70.3, CH
71.4, CH
88.0, CH2
4.72 (d, 2.5)
0.91 (d, 7.0)
1.12 (d, 7.5)
2.04 (s)
16.2, CH3
171.1, Cquat
20.8, CH3
1.20 (d, 7.5)
3.76 (s)
17.6, CH3
52.8, CH3
16.7, CH3
OH-4 4.46 (d, 6.0)
OH-9
OH-10
5.19 (d, 6.5)
5.99 (d, 7.0)
4.63 (d, 5.0)
5.33 (d, 4.0)
[a] Recorded at 500 MHz in [D6]acetone. [b] Recorded at 100 MHz in [D6]acetone. [c] Recorded at 500 MHz in [D6]DMSO. [d] Recorded
at 100 MHz in [D6]DMSO.
are attached to C-5. Those from 4-OH to C-3 and from 11-
H to C-3 and C-4 establish the connections of the doubly
oxygenated sp3 quaternary carbon C-3 (δ = 110.6 ppm) to
C-4 and C-11. Key correlations from the oxymethine pro-
tons 9-H and 10-H to C-3 establish the tetrahydrofuran and
1,3-dioxolane fragments in 1. In addition, NMR resonances
corresponding to an acetyl group (δ = 2.04/20.8, 171.1 ppm)
were observed, which indicates that the C-1 oxygen of 1 was
acylated. This observation was confirmed by an HMBC
cross-peak from 1-H to the carboxy carbon C-13. On the
basis of these data, the gross structure of 1 was established,
as shown in Figure 1.
The relative configuration of 1 was assigned by analysis
of the 1H–1H coupling constants and NOESY data.
NOESY correlation of 6-H with 10-H indicates that both
protons are in pseudoaxial orientations in the cyclohexene
ring. The small coupling constant of 4.5 Hz observed be-
tween 9-H and 10-H suggests a cis relationship, with 9-H
adopting the same orientation as 6-H. NOESY correlation
of 12-H with 4-H indicates that 4-H is opposite 6-H with
respect to the ring system, whereas the NOESY correlation
of 4-OH with 11-H reveals a trans relationship between 4-
H and 11-H. Finally, the structure of 1 was confirmed by
single-crystal X-ray diffraction analysis (Figure 2).
Figure 2. Thermal ellipsoid representation of 1.
The modified Mosher method was applied to assign the
absolute configuration of 1.[7,8] Treatment of 1 with (S)- and
(R)-MTPACl afforded the (R)- and (S)-MTPA esters 1a and
1b, respectively. The differences in the chemical shift values
(∆δ = δS – δR) for the diastereomeric esters 1b and 1a were
calculated to assign the 4R absolute configuration. There-
Figure 3. ∆δ values (∆δ = δS – δR; in ppm) obtained for (R)- and
(S)-MTPA esters 1a and 1b.
Trichocladinol B (2) was assigned the molecular formula
fore, the 3R,4R,5R,6S,9R,10S absolute configuration was C12H16O5 by HRESIMS analysis (m/z = 263.0898 [M +
1
proposed for 1 on the basis of the ∆δ results summarized Na]+, ∆ = –0.8 mmu). Analysis of the H and 13C NMR
in Figure 3.
spectroscopic data (Table 1) of 2 revealed nearly identical
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Cytotoxic Metabolites from a Colonizing Ascomycete
structural features to those found in 1, except that the oxy-
genated methylene carbon atom (C-1) in 1 is replaced by a
carboxy carbon atom (δ = 176.0 ppm), and the acetyl unit
is replaced by an O-methyl group (δ = 3.76/52.7 ppm) in the
spectra of 2. These observations were supported by HMBC
correlations from 10-H and 13-H to C-1. The relative con-
figuration of 2 was deduced to be the same as that of 1 by
1
comparison of its H–1H coupling constants and NOESY
data with those of 1. The absolute configuration of 2 was
presumed to be analogous to that of 1.
Figure 4. Key NOESY correlations for 3.
Trichocladinol C (3) gives a pseudomolecular ion [M +
Na]+ peak at m/z = 251.0892 (∆ = –0.2 mmu) in the HRES-
IMS analysis, which corresponds to an elemental composi-
1
tion of C11H16O5 (four degrees of unsaturation). The H
the three esters 3a, 3b, and 3c were obtained by reversed-
phase HPLC purification. The calculations of all of the rel-
evant signals of 3b and 3a showed anomalous values for 6-
H, 7-H, and 8-H (Figure 5), possibly due to interference
between the two MTPA units. Ultimately, the absolute con-
figuration 3 was established as 4R,5R,6R,9S,10S by single-
crystal X-ray crystallographic analysis of its (S)-MTPA es-
ter 3c (Figure 6).
and 13C NMR spectra of 3 show signals for three exchange-
able protons, one methyl group, two methylene units, four
methines (three oxygenated), one terminal olefin, one sp3
quaternary carbon atom, and one carboxy carbon atom.
The proton spin system corresponding to the C-6–C-10 (in-
cluding C-12) subunit of 3 was established on the basis of
relevant 1H–1H COSY correlations. HMBC cross-peaks
from 6-H, 10-H, 10-OH, and 12-H to C-5 completed the
cyclohexane ring in 3. The exocyclic sp2 methylene protons
(δ = 4.53 and 4.72 ppm) are correlated to C-3 and C-4 in
the HMBC spectrum of 3, which indicates that C-4 is allylic
to the C-3/C-11 terminal olefin. Correlations from 6-H and
10-H to C-4 shows the connection of C-4 to C-5, which is
further supported by HMBC cross-peaks from the ex-
changeable proton at 4-OH (δ = 5.99 ppm) to C-3, C-4, and
C-5. An HMBC correlation from 10-H to C-1 indicates that
C-1 is directly attached to C-5. The remaining two ex-
changeable protons (δ = 4.63 and 5.33 ppm, respectively)
were assigned as C-9-OH and C-10-OH on the basis of
HMBC correlations from 9-OH to C-8, C-9, and C-10, and
Figure 5. Structures of 3a–c and ∆δ values (∆δ = δS – δR; ppm)
obtained for (R)- and (S)-MTPA esters 3a and 3b.
from 10-OH to C-5, C-9, and C-10. On the basis of the 13
C
NMR chemical shifts of the C-3/C-11 exocyclic olefin (δ
= 88.0 and 155.6 ppm, respectively) and the unsaturation
requirement for 3, C-3 and the carboxy carbon C-1 must
be attached to the only remaining oxygen atom to form an
ester linkage, thereby completing the highly functionalized
3-methylidene-2-oxaspiro[4.5]decan-1-one structure of 3.
The relative configuration of 3 was determined by analy-
1
sis of its H–1H coupling constants and NOESY data (Fig-
ure 4). The large trans diaxial-type coupling constant of
10 Hz observed between 9-H and 10-H indicates that they
are pseudoaxially oriented, whereas the NOESY correlation
of 10-H with 12-H indicates that 6-H is pseudoequatorially
oriented. The NOESY correlation of 4-H with 9-H reveals
their proximity in space, which suggests that the two rings
of the spirocycle are nearly perpendicular to each other.
Therefore, the relative configuration of trichocladinol C
shown in Figure 4 was proposed.
Figure 6. Thermal ellipsoid representation of 3c.
The absolute configuration of 3 was initially assigned by
application of the modified Mosher method (Figure 5).
Treatment of 3 with (S)- and (R)-MTPACl afforded the (R)-
The known compound massarigenin A (4) was also iso-
MTPA ester 3a and the (S)-MTPA esters 3b and 3c, respec- lated from the crude extract, and its structure was identified
tively. However, selectivity for the acylation of one of the by comparison of its NMR and MS data with those re-
C-4, C-9, and C-10 hydroxy groups was not achieved, and ported previously.[6] Massarigenin A is a metabolite pos-
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H. Guo, B. Sun, H. Gao, S. Niu, X. Liu, X. Yao, Y. Che
FULL PAPER
sessing the 3-methylidene-2-oxaspiro[4.5]decan-1-one skel-
eton and was initially isolated from the aquatic fungus Mas-
sarina tunicata as an antimicrobial agent.
Trichocladinols A–C (1–3) were evaluated for their cyto-
toxicity against the human tumor cell lines HeLa and
MCF-7 (Table 2). Trichocladinol B (2) was the most active
metabolite against the two cell lines, showing IC50 values of
13.5 and 8.3 µ, respectively.
Table 2. Cytotoxicity of compounds 1–3.
Compd.
IC50 [µ]
HeLa
MCF-7
1
2
3
118.1
13.5
43.9
59.1
8.3
65.8
Conclusions
Scheme 1. Proposed biogenesis of 1–3.
Trichocladinols A (1) and B (2) are unique metabolites
featuring an unprecedented 2,9-dioxatricyclo[5.2.1.03,8]dec-
4-ene skeleton, the presence of the 1,3-dioxolane moiety
also being relatively rare in natural products; to the best of
our knowledge, only a few natural products incorporating
the 1,3-dioxolane unit have been reported, for example, 6α-
hydroxy-1α,4α,4,5-diepoxyxanth-10(14)-ene from the leaves
of Pluchea dioscoridis,[9] isagarin from the roots of Pentas
longiflora,[10] and exo-7-ethyl-5-methyl-6,8-dioxabicyclo-
[3.2.1]oct-3-ene from the urine of adult male mice as a
chemical signal of the male state.[11] Although the known
compound spiroleptosphol B (5) possesses the same hexa-
hydrobenzofuran substructure and substitution pattern as 1
and 2, it possesses a different 7,9-dioxatricyclo[6.2.1.01,6]-
undec-3-en-10-one skeleton.[12] These metabolites may
share the same biosynthetic processors on the basis that
the 2,9-dioxatricyclo[5.2.1.03,8]dec-4-ene skeleton in 1 and 2
could be formed from the 7,9-dioxatricyclo[6.2.1.01,6]undec-
3-en-10-one skeleton or vice versa through rearrangement
reactions. Trichocladinol C (3) is closely related to massari-
genin A (4),[6] both belonging to the γ-methylidene-spirobu-
tanolide class of metabolites, which have mainly been iso-
lated from the basidiomycetous and ascomycetous
fungi.[12–21] Biogenetically, by comparison of the biogenesis
of arthropsolide A,[20] the γ-methylidene-spirobutanolides
could be derived from the condensation of a tetraketide pre-
cursor and a malic acid unit followed by a series of reac-
tions including reduction, elimination, and epoxidation.
The carbon skeleton could also be derived from a polyke-
tide chain through some unusual rearrangement reac-
tions.[21] The possible biosynthetic pathways of trichocladi-
nols A–C (1–3) are proposed in Scheme 1. Compounds 1–
4 are the first natural products to be reported from the
Cordyceps-colonizing ascomycete Trichocladium opacum,
and the discovery of these metabolites implies that Cordy-
ceps-colonizing fungi could be a useful source of diverse
bioactive natural products.
Experimental Section
General: Optical rotations were measured with a Perkin–Elmer 241
polarimeter and UV data were recorded with a Shimadzu Biospec-
1601 spectrophotometer. The CD spectra were recorded with a
JASCO J-815 spectropolarimeter using MeOH as the solvent. IR
data were recorded using a Nicolet Magna-IR 750 spectrometer.
1H and 13C NMR spectroscopic data were acquired with Varian
Mercury-400 and Inova-500 spectrometers using the solvent signals
as references ([D6]acetone: δ = 2.05/29.8, 206.0 ppm; [D6]DMSO: δ
= 2.50/39.5 ppm). The HMQC and HMBC experiments were opti-
mized for 145.0 and 8.0 Hz, respectively. ESIMS and HRESIMS
data were recorded with a Mariner ESI-TOF mass spectrometer.
Fungal Material: The culture of Trichocladium opacum was isolated
by Dr. Mu Wang from a sample of C. sinensis (Berk.) Sacc. col-
lected in Linzhi, Tibet, on March 1, 2004. The isolate was identified
based on sequence (Genbank accession number GQ179993) analy-
sis of the ITS region of the rDNA and assigned the accession
number X116 in the culture collection at the Institute of Microbiol-
ogy, Chinese Academy of Sciences, Beijing. The isolate was subcul-
tured on slants of potato dextrose agar (PDA) at 25 °C for 5 d.
Fermentation was carried out in six 500-mL Erlenmeyer flasks,
each containing 80 g of rice. Distilled H2O (120 mL) was added to
each flask and the contents were soaked overnight before autoclav-
ing at 15 psi for 30 min. The flasks were cooled to room tempera-
ture and inoculated with 3.0 mL of a hyphal cell suspension and
incubated at 25 °C for 40 d.
Extraction and Isolation: The fermented rice substrate was ex-
tracted repeatedly with EtOAc (3ϫ1.0 L) and the organic solvent
was evaporated to dryness under a vacuum to afford a crude extract
(6.0 g) that was fractionated by silica gel vacuum column
chromatography (CC; 5ϫ8 cm) using petroleum ether/EtOAc gra-
dient elution. The fraction eluted with 25% EtOAc (200 mg) was
further separated by Sephadex LH-20 CC eluting with MeOH. One
subfraction (50 mg) was further separated by semipreparative
RPHPLC (Agilent Zorbax SB-C18 column, 5 µm, 9.4ϫ250 mm,
2 mLmin–1) to afford trichocladinol A (1; 15.0 mg, tR = 14.25 min;
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Cytotoxic Metabolites from a Colonizing Ascomycete
45% MeOH in H2O for 5 min followed by 45–60% for 25 min).
The fractions (1.0 g) eluted with 30–45% EtOAc were combined
and fractionated by Sephadex LH-20 CC using CH2Cl2/MeOH
(1:1) as eluent, and one of the subfractions (800 mg) was separated
again by silica gel CC (2ϫ13 cm) eluting with CH2Cl2/MeOH gra-
dients. One resulting subfraction (50 mg) eluted with 1% MeOH
In a similar fashion, a sample of 1 (2.0 mg, 0.008 mmol), CH2Cl2
(3.0 mL), DMAP (10.0 mg), and (R)-MTPACl (10.0 µL,
0.052 mmol) were allowed to react in a 10-mL round-bottomed
flask at room temperature for 18 h and the reaction mixture was
processed as described above for 1a to afford 1b (1.3 mg) as a white
powder. 1H NMR (500 MHz, CDCl3, 25 °C): δ = 5.93 (ddd, J =
was purified by RPHPLC to afford trichocladinol C (3; 10.0 mg, 10, 4.0, 3.0 Hz, 1 H, 8-H), 5.71 (dd, J = 10, 2.0 Hz, 1 H, 7-H), 5.18
tR = 18.70 min; 35% MeOH in H2O for 5 min followed by 35–50%
for 25 min) and another subfraction (20 mg) also eluted with 1%
MeOH was purified by RPHPLC using a different gradient (40%
(s, 1 H, 4-H), 4.44 (d, J = 4.0 Hz, 1 H, 10-H), 4.13 (t, J = 4.0 Hz,
1 H, 9-H), 4.08 (d, J = 12 Hz, 1 H, 1b-H), 3.96 (d, J = 12 Hz, 1
H, 1a-H), 2.50 (ddq, J = 7.5, 3.0, 2.0 Hz, 1 H, 6-H), 2.04 (s, 3 H,
MeOH in H2O for 5 min followed by 40–60% for 25 min) to afford 14-H), 1.37 (s, 3 H, 11-H), 0.98 (d, J = 7.5 Hz, 3 H, 12-H) ppm.
trichocladinol B (2; 4.0 mg, tR = 14.30 min) and massarigenin A
Trichocladinol B (2): Colorless oil. [α]2D2 = +141 (c = 0.06, CH3OH).
(4; 8.0 mg, tR = 16.00 min).
UV (CH3OH): λmax [log(ε/–1 cm–1)] = 203 [4.24] nm. IR (neat):
ν
= 3426 (br), 2942, 1708, 1439, 1393 cm–1. For 1H and 13C
˜
max
Trichocladinol
[5.2.1.03,8]dec-4-en-7-yl)methyl Acetate] (1): Colorless oil. [α]2D2
+78 (c = 0.03, CH3OH). UV (CH3OH): λmax [log(ε/–1 cm–1)] =
200 [3.62] nm. IR (neat): ν = 3465 (br), 2974, 1739, 1453, 1382,
A
[(10-Hydroxy-1,6-dimethyl-2,9-dioxatricyclo-
NMR spectroscopic data, see Table 1. HMBC data (400 MHz,
[D6]acetone, 25 °C): 4-H Ǟ C-6; 6-H Ǟ C-4, C-7, C-8, C-12; 7-H
Ǟ C-5, C-6, C-8, C-9, C-12; 9-H Ǟ C-5, C-7, C-10; 10-H Ǟ C-1,
C-3, C-4, C-9; 11-H Ǟ C-3, C-4; 12-H Ǟ C-5, C-6, C-7; 13-H Ǟ
C-1. NOESY correlations (400 MHz, [D6]acetone, 25 °C): 4-H ↔
12-H; 9-H ↔ 11-H. HRMS (ESI): calcd. for C12H16O5Na [M +
Na]+ 263.0890; found 263.0898.
=
˜
max
1242 cm–1. For H and 13C NMR spectroscopic data, see Table 1.
HMBC data (400 MHz, [D6]acetone, 25 °C): 1a-H Ǟ C-4, C-5, C-
6, C-10, C-13; 1b-H Ǟ C-13; 4-H Ǟ C-1, C-6; 6-H Ǟ C-4, C-5,
C-7, C-8, C-12; 7-H Ǟ C-5, C-6, C-8, C-9, C-12; 8-H Ǟ C-6, C-7,
C-9, C-10; 9-H Ǟ C-3, C-5, C-7, C-10; 10-H Ǟ C-3, C-4, C-5, C-
6, C-9; 11-H Ǟ C-3, C-4; 12-H Ǟ C-5, C-6, C-7; 14-H Ǟ C-13; 4-
OH Ǟ C-3, C-4, C-5. NOESY correlations (400 MHz, [D6]acetone,
25 °C): 4-H ↔ 12-H; 6-H ↔ 10-H; 4-OH ↔ 11-H. HRMS (ESI):
calcd. for C13H18O5Na [M + Na]+ 277.1046; found 277.1050.
1
Trichocladinol C (3): Colorless oil. [α]2D2 = +60 (c = 0.07, CH3OH).
UV (CH3OH) λmax [log(ε/–1 cm–1)] = 204 [3.95] nm. IR (neat):
ν
= 3489, 3320 (br), 2970, 1789, 1692, 1463, 1392 cm–1. For
˜
max
1H and 13C NMR spectroscopic data, see Table 1. HMBC data
(400 MHz, [D6]DMSO, 25 °C): 4-H Ǟ C-3, C-5, C-6, C-10, C-11;
6-H Ǟ C-4, C-5, C-8, C-10, C-12; 7a-H Ǟ C-8; 7b-H Ǟ C-5, C-
8, C-12; 8a-H Ǟ C-7, C-9; 8b-H Ǟ C-7; 9-H Ǟ C-10; 10-H Ǟ C-
1, C-4, C-5, C-9; 11-H Ǟ C-3, C-4; 12-H Ǟ C-5, C-6, C-7; 4-OH
Ǟ C-3, C-4, C-5; 9-OH Ǟ C-8, C-9, C-10; 10-OH Ǟ C-5, C-10.
NOESY correlations (400 MHz, [D6]DMSO, 25 °C): 4-H ↔ 10-
OH; 8a-H ↔ 12-H; 9-H ↔ 4-H, 7b-H; 10-H ↔ 8a-H, 12-H.
HRMS (ESI): calcd. for C11H16O5Na [M + Na]+ 251.0890; found
251.0892.
X-ray Crystallographic Analysis of 1: Upon crystallization from
acetone/H2O (10:1) using the vapor diffusion method, colorless
crystals were obtained for 1 and a crystal (0.58ϫ0.51ϫ0.45 mm)
was separated from the sample and mounted on a glass fiber, and
data were collected using a Rigaku R-AXIS RAPID IP dif-
fractometer with graphite-monochromated Mo-Kα radiation(λ =
0.71073 Å) at 173(2) K. Crystal data: C13H18O5, M = 254.27, space
group monoclinic, P21, unit cell dimensions: a = 7.3075(15), b =
9.2713(19), c = 10.017(2) Å, V = 638.1(2) Å3, Z = 2, Dcalcd.
=
Preparation of the (R)-MTPA Ester 3a and the (S)-MTPA Esters
3b and 3c: A sample of 3 (2.0 mg, 0.009 mmol) was dissolved in
CH2Cl2 (4.0 mL) in a 10-mL round-bottomed flask. DMAP
(10.0 mg) and (S)-MTPACl (10.0 µL, 0.052 mmol) were quickly
added and the flask was sealed and the contents stirred at room
temperature for 24 h. The mixture was evaporated to dryness and
purified by semipreparative RPHPLC (Agilent Zorbax SB-C18 col-
umn, 5 µm, 9.4ϫ250 mm, 80% CH3OH in H2O for 5 min followed
1.323 mgm–3, µ = 0.101 mm–1, F(000) = 272. The structure was
solved by direct methods using SHELXL-97[22] and refined by
using full-matrix least-squares difference Fourier techniques. All
non-hydrogen atoms were refined with anisotropic displacement
parameters and all hydrogen atoms were placed in idealized posi-
tions and refined as riding atoms with the relative isotropic param-
eters. Absorption corrections were performed by using the Siemens
Area Detector Absorption Program (SADABS).[23] The 5238 mea-
surements yielded 1555 independent reflections after equivalent
data had been averaged and Lorentz and polarization corrections
applied. The final refinement gave R1 = 0.0476 and wR2 = 0.1132
[IϾ2σ(I)].[24]
by 80–100% for 25 min, 2 mLmin–1) to afford 3a (1.2 mg, tR
=
22.50 min) as a white powder. 1H NMR (500 MHz, CDCl3, 25 °C):
δ = 6.47 (s,1 H, 4-H), 4.97 (d, J = 1.5 Hz, 1 H, 11b-H), 4.71 (td, J
= 10, 4.0 Hz, 1 H, 9-H), 4.68 (d, J = 1.5 Hz, 1 H, 11a-H), 4.14 (d,
J = 10 Hz, 1 H, 10-H), 2.25 (tt, J = 13, 5.0 Hz, 1 H, 7b-H), 2.07
(ddd, J = 7.0, 6.5, 5.0 Hz, 1 H, 6-H), 1.68 (ddd, J = 13, 6.5, 5.0 Hz,
1 H, 8b-H), 1.30 (ddd, J = 13, 10, 4.0 Hz, 1 H, 8a-H), 1.16 (ddd,
J = 13, 6.5, 4.0 Hz, 1 H, 7a-H), 1.08 (d, J = 7.0 Hz, 3 H, 12-
H) ppm.
Preparation of (R)- and (S)-MTPA Esters 1a and 1b: A sample of
1 (2.0 mg, 0.008 mmol) was dissolved in CH2Cl2 (3.0 mL) in a 10-
mL round-bottomed flask. DMAP (10.0 mg) and (S)-MTPACl
(10.0 µL, 0.052 mmol) were quickly added and the flask was sealed
and the contents stirred at room temperature for 24 h. The mixture
was evaporated to dryness and purified by semipreparative
RPHPLC (Agilent Zorbax SB-C18 column, 5 µm, 9.4ϫ250 mm;
In a similar fashion, a sample of 3 (2.0 mg, 0.009 mmol), CH2Cl2
(4.0 mL), DMAP (10.0 mg), and (R)-MTPACl (10.0 µL,
0.052 mmol) were allowed to react in a 10-mL round-bottomed
80% CH3OH in H2O for 5 min followed by 80–100% for 25 min, flask at room temperature for 18 h and the reaction mixture was
2 mLmin–1) to afford 1a (1.2 mg, tR = 16.30 min) as a white pow-
processed as described above for 3a to afford 3b (1.3 mg) and 3c
der. 1H NMR (500 MHz, CDCl3, 25 °C): δ = 5.94 (ddd, J = 10, (0.9 mg) as white powders. 3b: 1H NMR (500 MHz, CDCl3, 25 °C):
4.0, 3.0 Hz, 1 H, 8-H), 5.71 (dd, J = 10, 2.0 Hz, 1 H, 7-H), 5.19 δ = 6.58 (s, 1 H, 4-H), 4.92 (d, J = 1.5 Hz, 1 H, 11b-H), 4.70 (td,
(s,1 H, 4-H), 4.45 (d, J = 4.0 Hz, 1 H, 10-H), 4.13 (t, J = 4.0 Hz,
1 H, 9-H), 4.07 (d, J = 12 Hz, 1 H, 1b-H), 3.99 (d, J = 12 Hz, 1
H, 1a-H), 2.49 (ddq, J = 7.5, 3.0, 2.0 Hz, 1 H, 6-H), 2.05 (s, 3 H,
14-H), 1.32 (s, 3 H, 11-H), 1.00 (d, J = 7.5 Hz, 3 H, 12-H) ppm.
J = 10, 4.0 Hz, 1 H, 9-H), 4.55 (d, J = 1.5 Hz, 1 H, 11a-H), 4.22
(d, J = 10 Hz, 1 H, 10-H), 2.29 (tt, J = 13, 5.0 Hz, 1 H, 7b-H),
2.20 (ddd, J = 7.0, 6.5, 5.0 Hz, 1 H, 6-H), 1.67 (ddd, J = 13, 6.5,
5.0 Hz, 1 H, 8b-H), 1.44 (ddd, J = 13, 10, 4.0 Hz, 1 H, 8a-H), 1.32
Eur. J. Org. Chem. 2009, 5525–5530
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5529

H. Guo, B. Sun, H. Gao, S. Niu, X. Liu, X. Yao, Y. Che
FULL PAPER
(ddd, J = 13, 6.5, 4.0 Hz, 1 H, 7a-H), 1.02 (d, J = 7.0 Hz, 3 H, 12-
National Natural Science Foundation of China (30870057 and
1
H) ppm. 3c: H NMR (500 MHz, CDCl3, 25 °C): δ = 6.30 (s, 1 H, U0633008).
4-H), 5.82 (d, J = 10 Hz, 1 H, 10-H), 4.82 (td, J = 10, 4.0 Hz, 1 H,
9-H), 4.71 (d, J = 1.5 Hz, 1 H, 11b-H), 4.38 (d, J = 1.5 Hz, 1 H,
11a-H), 2.20 (tt, J = 13, 5.0 Hz, 1 H, 7b-H), 1.60 (ddd, J = 7.0,
6.5, 5.0 Hz, 1 H, 6-H), 1.42 (ddd, J = 13, 6.5, 5.0 Hz, 1 H, 8b-H),
1.20 (ddd, J = 13, 10, 4.0 Hz, 1 H, 8a-H), 1.08 (ddd, J = 13, 6.5,
4.0 Hz, 1 H, 7a-H), 0.99 (d, J = 7.0 Hz, 3 H, 12-H) ppm.
[1]
Y. Q. Chen, N. Wang, L. H. Qu, T. H. Li, W. M. Zhang, Bio-
chem. Syst. Ecol. 2001, 29, 597–607.
R. R. M. Paterson, Phytochemistry 2008, 69, 1469–1495.
J. S. Zhu, G. M. Halpen, K. Jones, J. Altern. Complement. Med.
1998, 4, 289–303.
[2]
[3]
X-ray Crystallographic Analysis of 3c: Upon crystallization from
MeOH using the vapor diffusion method, colorless crystals were
obtained for 3c and a crystal (0.26ϫ0.17ϫ0.03 mm) was separated
from the sample and mounted on a glass fiber. The data were col-
lected by using a Rigaku MicroMax-007HF Saturn 724+ CCD dif-
fractometer with Mo-Kα radiation (λ = 0.71073 Å) at 173(2) K.
Crystal data: C41H37F9O11, M = 876.71, space group triclinic, P1,
unit cell dimensions a = 8.3882(17), b = 11.056(2), c = 21.830(4) Å,
V = 2007.0(7) Å3, Z = 2, Dcalcd. = 1.451 mgm–3, µ = 0.131 mm–1,
F(000) = 904. The structure was solved by direct methods using
SHELXL-97[22] and refined by using full-matrix least-squares dif-
ference Fourier techniques. All non-hydrogen atoms were refined
with anisotropic displacement parameters and all hydrogen atoms
were placed in idealized positions and refined as riding atoms with
the relative isotropic parameters. Absorption corrections were per-
formed by using the Siemens Area Detector Absorption Program
(SADABS).[23] The 33313 measurements yielded 9164 independent
reflections after equivalent data had been averaged and Lorentz
and polarization corrections applied. The final refinement gave R1
= 0.0547 and wR2 = 0.1203 [IϾ2σ(I)].[24]
[4]
[5]
[6]
H. Guo, H. Hu, S. Liu, X. Liu, Y. Zhou, Y. Che, J. Nat. Prod.
2007, 70, 1519–1521.
Y. Zhang, S. Liu, Y. Che, X. Liu, J. Nat. Prod. 2007, 70, 1522–
1525.
H. Oh, D. C. Swenson, J. B. Gloer, C. A. Shearer, J. Nat. Prod.
2003, 66, 73–79.
J. A. Dale, H. S. Mosher, J. Am. Chem. Soc. 1973, 95, 512–519.
I. Ohtani, T. Kusumi, Y. Kashman, H. Kakisawa, J. Am. Chem.
Soc. 1991, 113, 4092–4096.
A. A. Mahmoud, Phytochemistry 1997, 45, 1633–1638.
[7]
[8]
[9]
[10]
L. V. Puyvelde, S. E. Hady, N. D. Kimpe, J. F. Dupont, J. P. De-
clercq, J. Nat. Prod. 1998, 61, 1020–1021.
[11]
[12]
D. P. Wiesler, F. J. Schwende, M. Carmack, M. Novotny, J. Org.
Chem. 1984, 49, 882–884.
T. Murakami, T. Tsushima, N. Takada, K. Tanaka, K. Niher,
T. Miura, M. Hashimoto, Bioorg. Med. Chem. 2009, 17, 492–
495.
[13] J. Doi, A. Hirota, M. Nakagawa, H. Sakai, A. Isogai, Agric.
Biol. Chem. 1985, 49, 2247–2248.
[14] W. A. Ayer, P. A. Craw, J. Neary, Can. J. Chem. 1992, 70, 1338–
1347.
Massarigenin A (4): The 1H NMR, 13C NMR, and ESIMS data
were fully consistent with literature values.[6]
[15] J. Rether, G. Erkel, T. Anke, O. Sterner, J. Antibiot. 2004, 57,
MTT Assay:[4] In 96-well plates, 104 cells were introduced into each
well. After cell attachment overnight, the medium was removed and
each well was treated with 50 µL of medium containing 0.2%
DMSO or the appropriate concentration of test compounds
(10 mgmL–1 as the stock solution of the compound in DMSO and
serial dilutions). Cells were first treated at 37 °C for 4 h in a humid-
ified incubator at 5% CO2 and then grown for another 48 h after
the medium had been changed to fresh Dulbecco’s Modified Eagle
Medium (DMEM). MTT (Sigma) was dissolved in a serum-free
medium or PBS at 0.5 mgmL–1 and sonicated briefly. In the dark,
50 µL of MTT/medium was added to each well after the medium
had been removed from the wells and incubated at 37 °C for 3 h.
Upon removal of the MTT/medium, 100 µL of DMSO was added
to each well and agitated at 60 rpm for 5 min to dissolve the pre-
cipitate. The assay plate was read at 540 nm by using a microplate
reader.
493–495.
[16] A. Hirota, M. Nakagawa, H. Hirota, Agric. Biol. Chem. 1991,
55, 1187–1188.
[17] M. Kaouadji, N. B. D. Gusmao, R. Steiman, F. Seigle-Mur-
andi, J. Nat. Prod. 1993, 56, 2189–2192.
[18] A. Fukami, Y. Taniguchi, T. Nakamura, M.-C. Rho, K. Kawa-
guchi, M. Hayashi, K. Komiyama, S. Omura, J. Antibiot. 1999,
52, 501–504.
[19] M. Hashimoto, T. Tsushima, T. Murakami, M. Nomiya, N.
Takada, K. Tanaka, Bioorg. Med. Chem. Lett. 2008, 18, 4228–
4231.
[20] W. A. Ayer, P. A. Craw, Can. J. Chem. 1992, 70,1348–1355.
[21] A. Albinati, S. Brückner, L. Camarda, N. Gianluca, Tetrahe-
dron 1980, 36, 117–121.
[22] G. M. Sheldrick, SHELXL-97, Program for X-ray Crystal
Structure Solution and Refinement, University of Göttingen,
Göttingen, Germany, 1997.
[23] G. M. Sheldrick, SADABS, Program for Empirical Absorption
Correction of Area Detector Data, University of Göttingen,
Göttingen, Germany, 1999.
[24] CCDC-734059 (for 1) and -734060 (for 3c) contain the supple-
mentary crystallographic data for this paper. These data can
be obtained free of charge from The Cambridge Crystallo-
graphic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
Received: July 3, 2009
Supporting Information (see also the footnote on the first page of
1
this article): H and 13C NMR spectra of trichocladinols A–C (1–
3).
Acknowledgments
We gratefully acknowledge financial support from the Ministry of
Science and Technology of China (2009CB522302) and the
Published Online: September 24, 2009
5530
www.eurjoc.org
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 5525–5530