Smardaesidins -G, isopimarane and 20 nor-isopimarane diterpenoids from Smardaea sp., a fungal endophyte of the moss Ceratodon purpureus (1)

Article abstract of DOI:10.1021/np2000864

Five new isopimarane diterpenes, smardaesidins -E (1- 5) and two new 20-nor-isopimarane diterpenes, smardaesidins F (6) and G (7), together with sphaeropsidins A (8) and C-F (10-13) were isolated from an endophytic fungal strain, Smardaea sp. AZ0432, occurring in living photosynthetic tissue of the moss Ceratodon purpureus. Of these, smardaesidins B (2) and C (3) were obtained as an inseparable mixture of isomers. Chemical reduction of sphaeropsidin A (8) afforded sphaeropsidin B (9), whereas catalytic hydrogenation of 8 yielded 7-O-15,16-tetrahydrosphaeropsidin A (14) and its new derivative, 7-hydroxy-6-oxoisopimara-7-en-20-oic acid (15). The acetylation and diazomethane reaction of sphaeropsidin A (8) afforded two of its known derivatives, 6-O-acetylsphaeropsidin A (16) and 8,14-methylenesphaeropsidin A methyl ester (17), respectively. Methylation of 10 yielded sphaeropsidin C methyl ester (18). The planar structures and relative configurations of the new compounds 1-7 and 15 were elucidated using MS and 1D and 2D NMR experiments, while the absolute configurations of the stereocenters of 4 and 6-8 were assigned using a modified Mosher's ester method, CD spectra, and comparison of specific rotation data with literature values. Compounds 1-18 were evaluated for their potential anticancer activity using several cancer cell lines and cells derived from normal human primary fibroblasts. Of these, compounds 8, 11, and 16 showed significant cytotoxic activity. More importantly, sphaeropsidin A (8) showed cell-type selectivity in the cytotoxicity assay and inhibited migration of metastatic breast adenocarcinoma (MDA-MB-231) cells at subcytotoxic concentrations.

Full text of DOI:10.1021/np2000864

Article
pubs.acs.org/jnp
Smardaesidins A−G, Isopimarane and 20-nor-Isopimarane
Diterpenoids from Smardaea sp., a Fungal Endophyte of the Moss
Ceratodon purpureus1
Xiao-Ning Wang,†,‡ Bharat P. Bashyal,† E. M. Kithsiri Wijeratne,† Jana M. U’Ren,§ Manping X. Liu,†
Malkanthi K. Gunatilaka,§ A. Elizabeth Arnold,§ and A. A. Leslie Gunatilaka*,†
†SW Center for Natural Products Research and Commercialization, School of Natural Resources and the Environment, College of
Agriculture and Life Sciences, University of Arizona, 250 E. Valencia Road, Tucson, Arizona 85706-6800, United States
‡School of Pharmaceutical Sciences, Shandong University, 44 West Wenhua Road, Jinan 250012, P.R. China
§School of Plant Sciences, College of Agriculture and Life Sciences, University of Arizona, Tucson, Arizona 85721-0036,
United States
S
* Supporting Information
ABSTRACT: Five new isopimarane diterpenes, smardaesidins A−E (1− 5) and two new 20-nor-isopimarane diterpenes,
smardaesidins F (6) and G (7), together with sphaeropsidins A (8) and C−F (10−13) were isolated from an endophytic fungal
strain, Smardaea sp. AZ0432, occurring in living photosynthetic tissue of the moss Ceratodon purpureus. Of these, smardaesidins
B (2) and C (3) were obtained as an inseparable mixture of isomers. Chemical reduction of sphaeropsidin A (8) afforded
sphaeropsidin B (9), whereas catalytic hydrogenation of 8 yielded 7-O-15,16-tetrahydrosphaeropsidin A (14) and its new
derivative, 7-hydroxy-6-oxoisopimara-7-en-20-oic acid (15). The acetylation and diazomethane reaction of sphaeropsidin A (8)
afforded two of its known derivatives, 6-O-acetylsphaeropsidin A (16) and 8,14-methylenesphaeropsidin A methyl ester (17),
respectively. Methylation of 10 yielded sphaeropsidin C methyl ester (18). The planar structures and relative configurations of
the new compounds 1−7 and 15 were elucidated using MS and 1D and 2D NMR experiments, while the absolute configurations
of the stereocenters of 4 and 6−8 were assigned using a modified Mosher’s ester method, CD spectra, and comparison of specific
rotation data with literature values. Compounds 1−18 were evaluated for their potential anticancer activity using several cancer
cell lines and cells derived from normal human primary fibroblasts. Of these, compounds 8, 11, and 16 showed significant
cytotoxic activity. More importantly, sphaeropsidin A (8) showed cell-type selectivity in the cytotoxicity assay and inhibited
migration of metastatic breast adenocarcinoma (MDA-MB-231) cells at subcytotoxic concentrations.
Understanding their mechanisms is one of the main challenges
ell-based anticancer drug discovery efforts rely frequently
Con potency rather than selectivity. In our search for for exploratory and applied cancer research. A number of
molecular pathways and cellular mechanisms that underlie the
multistage process of cancer metastasis have been recently
identified; these include tumor invasion, tumor cell dissem-
ination through the bloodstream or the lymphatic system,
colonization of distant organs, and finally, fatal outgrowth.4 As
all these processes involve cell migration, compounds that
effectively inhibit cell migration could prove to be very useful
anticancer therapeutics.
potential anticancer agents from endosymbiotic fungi of the
Sonoran desert bioregion, we have continued to address this
deficiency by employing cell-based target-oriented assays,2
including an assay for cancer cell migration inhibition,2b in
addition to an assay for inhibition of the proliferation of tumor
cell lines with the goal of discovering cytotoxic agents with
selective activity.2a Approximately 90% of all cancer deaths are
caused by metastatic spread of primary tumors.3 Of all the
processes involved in carcinogenesis, local invasion and the
formation of metastases are the most relevant clinically, but
they are the least understood at the molecular level.
Received: January 27, 2011
Published: October 14, 2011
© 2011 American Chemical Society and
American Society of Pharmacognosy
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Endophytic fungi, consisting mostly of Ascomycota,
represent one of the largest (conservatively ca. 106 species)
and one of the least-explored resources of biologically active
small-molecule natural products.5 These fungi colonize internal
tissues of plants6 and are a fundamental feature of plant biology
in biomes ranging from Arctic tundra to hot deserts and
tropical rain forests.7 Although in many cases the specific
interactions between endophytes and their hosts have not been
characterized, many endophytes produce small-molecule
natural products8 that may protect hosts from herbivores,
plant pathogens, and abiotic stressors such as drought and
thermal stress.8b Although endophytes were first observed with
certainty well over a century ago, this group of microorganisms
did not receive significant attention until the recent realization
of their ecological relevance7 and the potential of yielding
metabolites with diverse structures and biological functions.8
Almost all endophytic fungi investigated thus far for their
secondary metabolites have been isolated from vascular plants,
and endophytes associated with bryophytes, especially mosses,
have received relatively little attention. We and others have
recently encountered endophytic fungi in photosynthetic
tissues of mosses and found them to be diverse and frequently
abundant, even in relatively arid ecosystems.9
first report of secondary metabolites from a moss-inhabiting
endophyte and a fungal strain of the genus Smardaea.
RESULTS AND DISCUSSION
■
Smardaesidin A (1) was obtained as a white amorphous solid. A
molecular formula of C20H28O6 was determined for 1 on the
basis of its HRESIMS, accounting for seven degrees of
unsaturation. Its IR spectrum had absorption bands at 3396,
1755, and 1726 cm−1 indicating the presence of OH and CO
1
groups. The H NMR spectrum (Table 1) of 1 displayed
signals for three tertiary methyl protons at δ 1.11, 1.16, and
1.20, one oxygenated methine proton at δ 4.56, a pair of
oxygenated methylene protons at δ 3.70 and 3.45, two aliphatic
methine protons at δ 2.96 and 0.96, and a group of aliphatic
methylene and methine protons at δ 1.10−2.03. The 13C NMR
data (Table 2) analyzed with the help of HSQC data indicated
that smardaesidin A contained 20 carbons, which included
three methyls, six methylenes, one of which is oxygenated
(δ 63.2), four methines of which one is oxygenated (δ 77.9),
seven quaternary carbons of which two are oxygenated (δ 77.4
and 87.4), and two carbonyl carbons (δ 179.4 and 208.1),
1
suggesting that 1 was a polyoxygenated diterpene. The H−1H
COSY spectrum of 1 indicated the presence of three
spin systems, CH2(1)−CH2(2)−CH2(3), CH2(11)−CH2(12),
and CH(14)−CH(15)−CH2(16) (Figure 1). These partial
structures, methyls, and methines are joined together through
nonprotonated carbons on the basis of HMBC correlations to
form a tetracyclic diterpene framework (Figure 1). Specifically,
HMBC correlations of CH3-17 (δ 1.20) to C-14 (δ 27.4) and
C-15 (δ 32.6), H2-16 (δ 3.70 and 3.45) to C-13 (δ 23.2) and
C-14, and H-14 (δ 0.96) and H-15 (δ 1.26) to C-8 (δ 87.4)
suggested the presence of a cyclopropane ring bearing CH3 and
CH2OH substituents at C-13 and C-15, respectively. HMBC
correlations of H-5 and H-7 to C-6 (δ C 208.1) and those of
H-1α and H-5 to C-20 (δC 179.4) also helped to locate the
ketone (δ 208.1) and lactone (δ 179.4) carbonyl carbons at C-6
and C-20, respectively. The molecular formula, the remaining
one double bond equivalent, and the considerably deshielded
nature of C-8 (δC 87.4)17 all suggested the presence of a
lactone ring between C-8 and C-10. The deshielded nature of
In the course of our ongoing efforts to discover potential
anticancer agents,1,2 an EtOAc extract derived from a solid
(potato dextrose agar, PDA) culture of an endophytic fungal
strain, Smardaea sp. AZ0432 (Pyronemataceae, Ascomycota),
isolated from the fire moss (Ceratodon purpureus, Ditrichaceae)
was found to be active in the resazurin (alamarBlue) cell
viability assay10 for cancer cell proliferation/survival. Fire moss
is a globally distributed species currently used as a model
system in plant development and physiology.11 It is known for
its capacity to colonize relatively extreme environments,
including nutrient-poor or polluted soils and sites previously
consumed by fire.12 Bioactivity-guided fractionation of the
bioactive extract of Smardaea sp. AZ0432 provided five new
isopimarane diterpenes, smardaesidins A−E (1−5), two new
20-nor-isopimarane diterpenes, smardaesidins F (6) and G (7),
and five known diterpenes, sphaeropsidins A (8),13 C (10),14
D
(11),15 E (12),15 and F (13)16 (Chart 1). This constitutes the
Chart 1
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a
1
Table 1. H NMR Spectroscopic Data of Compounds 1−4
position
1
2
3
4
1a
1.31, m (α-H)
1.85, m (β-H)
1.52, m (α-H)
2.03, m (β-H)
1.14, m (α-H)
1.39, m (β-H)
2.96, s
4.09, dd (12.0, 4.8)
3.76, dd (10.8, 5.6)
4.10, dd (11.7, 5.0)
1b
2a
1.75, m (α-H)
1.99, m (β-H)
1.74, m
1.95, m
1.29, m
1.47, m
2.38, m
1.97, m
1.71, m
1.90, m (α-H)
2b
3a
1.80, q (12.0) (β-H)
3.25, dd (12.1, 4.0)
1.39, ddd (13.8, 13.8, 3.6) (α-H)
1.52, m (β-H)
3b
5
2.31, dd (12.8, 6.0)
2.41, m (2H)
2.27, dd (12.5, 5.7)
6a
2.51, dd (18.4, 5.7) (α-H)
2.41, dd (18.4, 12.5) (β-H)
6b
7
4.56, d (0.8)
11a
11b
12a
12b
14
1.56, ddd (14.4, 4.4, 2.4) (α-H)
1.34, m (β-H)
2.00, m (α-H)
2.41, m (β-H)
1.98, m (α-H)
1.51, m (β-H)
6.70, d (2.0)
1.79, m
2.02, dt (14.8, 3.5) (α-H)
2.14, td (14.8, 3.5) (β-H)
1.90, m (α-H)
2.19, m
1.89, ddd (14.8, 14.4, 4.4) (α-H)
1.77, ddd (14.8, 6.0, 2.4) (β-H)
0.96, d (5.6)
2.18, m
1.54, m
1.48, dddd (13.6, 3.5, 3.4, 1.8) (β-H)
6.69, d (1.8)
5.81, d (1.6)
5.91, dd (17.6, 10.8)
5.04, dd (17.6, 1.2)
4.93, dd (10.8, 1.2)
1.10, s (3H)
0.87, s (3H)
1.11, s (3H)
3.78, d (12.0)
4.08, d (12.0)
15
1.26, m
5.90, dd (17.6, 10.4)
5.09, dd (17.6, 0.8)
5.02, dd (10.4, 0.8)
1.15, s (3H)
5.88, dd (17.5, 10.7)
5.09, dd (17.5, 1.0)
5.02, dd (10.7, 1.0)
1.10, s (3H)
16a
16b
17
3.70, dd (11.6, 6.0)
3.45, dd (11.6, 8.4)
1.20, s (3H)
18
1.16, s (3H)
0.90, s (3H)
0.96, s (3H)
19
1.11, s (3H)
1.05, s (3H)
0.87, s (3H)
20a
20b
4.28, d (12.0)
3.88, d (12.0)
0.95, s (3H)
a
Data were measured in CD3OD at 400 MHz. Chemical shifts (δ) are expressed in ppm, and J values are presented in Hz.
Table 2. 13C NMR Spectroscopic Data of Compounds 1−7 and 15
a
a
a
a
b
b
b
b
position
1
2
3
4
5
6
7
15
1
26.1
18.7
43.5
34.1
56.2
208.1
77.9
87.4
77.4
56.0
27.5
28.9
23.2
27.4
32.6
63.2
21.4
32.8
20.1
179.4
74.0
31.3
40.9
34.2
42.5
38.5
203.4
139.0
75.2
49.5
31.2
31.4
39.8
147.9
147.9
112.5
24.2
33.2
21.5
70.3
30.6
40.1
34.6
42.1
34.1
95.8
140.9
73.6
46.3
29.8
32.3
39.7
131.9
149.2
111.2
26.0
32.5
20.5
65.8
69.5
38.7
76.2
39.7
40.7
37.5
203.7
138.1
75.6
47.3
30.1
31.3
40.0
148.4
147.6
112.7
24.1
28.1
15.1
11.3
22.0
18.6
38.4
31.1
44.1
78.1
73.6
136.5
72.0
53.3
30.9
71.2
44.5
136.1
142.4
118.5
25.2
31.8
22.7
178.1
27.6
18.6
40.6
33.8
140.0
197.9
76.3
76.2
74.5
155.3
28.7
33.5
35.1
40.3
146.5
112.6
31.8
26.4
29.7
63.6
28.5
33.7
34.2
141.6
199.6
76.9
76.5
74.5
151.4
29.9
33.8
35.2
40.2
146.7
112.9
31.8
25.1
30.3
35.1
19.4
42.7
33.0
57.8
193.2
143.2
121.8
47.8
51.3
23.7
35.9
33.9
37.2
37.3
7.8
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21.4
32.3
20.2
179.4
62.9
a
b
Recorded at 100 MHz in CD3OD. Recorded at 100 MHz in CDCl3. Chemical shifts (δ) are expressed in ppm.
H-2β (δ 2.03) as a result of the magnetic anisotropic effect
from the C-20 carbonyl provided additional evidence for the
presence of this lactone moiety (Figure 2). The 13C NMR
chemical shift of C-9 (δ 77.4) was in agreement with those
reported for related diterpenes with an α-OH at this
position.13−16,18 The relative stereochemistry of 1 was
determined with the help of NOEDIFF data (Figure 2).
Irradiation of H-5 (δ 2.96) led to significant enhancements of
H-1α, H-3α, H-7, and CH3-18, suggesting that they were
α-cofacial and that OH-7 had a β-configuration. Strong NOE
between H-7 and H-15 confirmed α-orientation of the latter
proton. Consequently, the structure of smardaesidin A was
identified as 7β,9α,16-trihydroxy-8β,20-epoxy-14α,15β-cyclo-
isopimara-6,20-dione (1).
Smardaesidins B (2) and C (3) were obtained as a white
amorphous solid of an inseparable mixture of two isomers in a
ratio of approximately 4:1, as determined by 1H NMR
spectroscopy. All our attempts to separate 2 and 3 by
chromatographic methods (normal and reversed-phase TLC
and HPLC) failed. Therefore, the structures of these two
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of 2 and 3 would lead to isolable product(s) from any of the
individual isomers. This mixture when acetylated with Ac2O/
pyridine afforded 20-O-acetylsmardaesidin B (2a) and 1,20-di-
O-acetylsmardaesidin B (2b) as the only isolable products, both
of which were derived from the major component of the
mixture.
Smardaesidin D (4) was obtained as a white amorphous
powder, the molecular formula of which was determined as
C20H30O4 by a combination of HRESIMS and NMR data. The
1H and 13C NMR data (Tables 1 and 2) showed signals that
closely resembled those of 9α-hydroxyisopimara-8(14),15-dien-
7-one.20 The only difference was the presence of two
oxygenated methines (δH/δC 4.10/69.5 and 3.25/76.2) in 4,
instead of two methylene groups in 9α-hydroxyisopimara-
8(14),15-dien-7-one. These were placed at C-1 and C-3,
respectively, by the CH(1)−CH2(2)−CH(3) spin system
Figure 1. Selected 1H−1H COSY (
(H→C) of 1.
) and HMBC correlations
1
identified from the H−1H COSY spectrum and also by the
HMBC correlations of CH3-20/C-1, CH3-18/C-3, and CH3-
19/C-3 (Supporting Information). In the NOEDIFF experi-
ment, irradiation of any one of the protons H-5, H-3, and H-1
gave rise to enhancement of the other two, indicating that they
are all α-cofacial and hence both 1-OH and 3-OH are
equatorially β-oriented. Stereochemistry at other positions
were determined to be the same as that of 9α-hydroxyisopi-
mara-8(14),15-dien-7-one20 by NOEDIFF experiments (Sup-
porting Information). Therefore, the structure of smardaesidin
D was identified as 1β,3β,9α-trihydroxyisopimara-8(14),15-
dien-7-one (4).
Figure 2. Selected NOEs observed in the NOEDIFF experiments of 1.
compounds were elucidated as a mixture. The HRESIMS
displayed the same quasi molecular ion [M + H]+ peak at m/z
335.2217 that allowed establishment of the common molecular
The HRESIMS of smardaesidin E (5) obtained as a white
amorphous solid displayed a pseudo molecular ion consistent
1
formula C20H30O4 for both 2 and 3. The H and 13C NMR
spectra (Tables 1 and 2) of the major component (2) displayed
signals due to three tertiary methyls at δH/δC 1.15/24.2
(CH3-17), 0.90/33.2 (CH3-18), and 1.05/21.5 (CH3-19), an
oxygenated methylene group (δH 4.28 and 3.88; δC 62.9), an
oxygenated methine group (δH 4.09; δC 74.0), a vinyl group
(δH 5.90, 5.09, and 5.02), and an enone moiety (δ H 6.70; δC
203.4, 139.0, and 147.9). These data indicated that the
structure of 2 closely resembled that of sphaeropsidin C
(10).14 The major differences were the presence of a −CH2OH
group in 2 in place of a −CO2H group in 10 and an additional
OH group in 2. The oxymethylene protons at δ 4.28 and 3.88
showed HMBC correlations with C-1 (δ 74.0), C-5 (δ 42.5),
C-9 (δ 75.2), and C-10 (δ 49.5), confirming the above
observation and locating the −CH2OH and OH groups at C-10
and C-1, respectively. Irradiation of both H-5 and H-3α caused
enhancement of H-1, suggesting that H-1 is axially α-oriented
(Supporting Information). The stereochemistry at other
positions was determined to be the same as that of 10 by
NOEDIFF experiments (Supporting Information). Thus, the
structure of smardaesidin B was determined as 1β,9α,20-
trihydroxyisopimara-8(14),15-dien-7-one (2). The minor com-
ponent, smardaesidin C (3), was identified as the C-7−C-20
hemiketal derived from 2. This was confirmed by the high-field
shift of H-14 in 3 (δ 5.81) in comparison with that of 2
(δ 6.70), the presence of a dioxygenated carbon of a hemiketal
(δ 95.8), and the absence of a ketone carbonyl in 3. The
NOEDIFF experiments (Supporting Information) were used to
determine the stereochemical disposition of groups on the
diterpene carbon skeleton. Thus, the structure of smardaesidin
C was determined as 1β,7α,9α-trihydroxy-7β,20-epoxyisopi-
mara-8(14),15-diene (3). Although inseparable isomeric
mixtures of natural products are known,19 it was of interest
to determine whether derivatization of the equilibrium mixture
1
with the molecular formula C20H28O5. The H and 13C NMR
spectra (Tables 3 and 2, respectively) of 5 revealed three
tertiary methyls (δH/δC 1.00/25.2 (CH3-17), 1.03/31.8 (CH3-
18), and 0.85/22.7 (CH3-19)), a vinyl group (δH 5.77, 5.21,
5.36; δC 142.4 and 118.5 for C-15 and C-16, respectively), a
trisubstituted double bond (δH 5.71; δC 136.5 and 136.1 for
C-8 and C-14, respectively), three oxygenated methine protons
(δH 4.52, 4.29, and 3.79; δC 78.1, 73.6, and 71.2 for C-6, C-7
and C-12, respectively), an oxygenated quaternary carbon (δ C
72.0, C-9), and a lactone carbonyl carbon (δ C 178.1, C-20).
The 1- and 2-D NMR data of 5 closely resembled those of
sphaeropsidin B (9)14 except that dioxygenated carbon (C-6)
of the hemiketal (δ 111.0) was replaced with an oxygenated
methine carbon (δH 4.52; δC 78.1). In the HMBC spectrum
(Figure 3), the correlations of this methine proton (δ H 4.52) to
C-4 (δC 31.1), C-5 (δC 44.1), C-7 (δC 73.6), C-8 (δC 136.5),
and C-10 (δC 53.3) further supported its position as C-6. The
oxygenated methine at δH 4.29 coupled with H-6 was shown to
be CH-7, and this was verified by HMBC correlations of H-7 to
C-5, C-8, C-9, and C-14. The HMBC correlation of H-6/C-20
indicated that a γ-lactone ring was present between C-6 and
C-20. Additionally, the third oxygenated methine at δH/δC
3.79/71.2 was positioned at C-12 by HMBC correlations of
CH3-17/C-12, H-14/C-12, and H-12/C-9. The stereochemical
assignments of 5 were made by NOEDIFF experiments and
proton coupling constants. Unlike sphaeropsidin B (9),
irradiation of either H-5 or H-7 gave no enhancement of
the other one of the pair, while enhancement of H-14 was
observed on irradiation of H-7 (Figure 4); therefore, H-7 was
determined to be β-oriented in 5. The coupling constants of
H-12 (δ 3.79) with both protons at C-11 and a W-type long-
range coupling with H-14 indicated that H-12 had an equatorial
orientation, and this was further verified by NOEDIFF data of
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1
Table 3. H NMR Spectroscopic Data of Compounds 5−7 and 15
position
5
6
7
15
1a
1.67, ddd (13.2, 13.2, 5.6) (α-H)
1.99, m (β-H)
1.55, m (α-H)
1.43, m (β-H)
1.19, m (α-H)
1.38, m (β-H)
2.79, s
2.51, dddd (19.2, 4.4, 4.4, 1.6) (α-H)
2.16, ddd (19.2, 9.6, 6.0) (β-H)
1.58, m (α-H)
4.63, br s
2.56, d (12.9)
1.12, m
1b
2a
1.72, m (α-H)
1.69, m (β-H)
1.45, m (α-H)
1.66, m
2b
1.71, m (β-H)
1.49, m
3a
1.55, m (α-H)
1.36, m
3b
1.37, m (β-H)
1.89, ddd (14.2, 14.2, 2.4) (β-H)
1.20, m
5
2.33, m
6
4.52, d (4.8)
7
4.29, d (4.8)
4.48, br s
4.47, br s
9
2.33, m
11a
11b
12a
12b
14a
14b
15
1.98, dd (14.8, 4.0) (α-H)
2.13, dd (14.8, 2.0) (β-H)
1.87, td (14.0, 2.8) (α-H)
1.98, dt (14.0, 3.2) (β-H)
1.75, m (α-H)
1.88, td (14.0, 2.8) (α-H)
2.32, dt (14.0, 3.2) (β-H)
1.76, m (α-H)
1.86, m
1.12, m
1.48, m
3.79, ddd (4.0, 2.0, 1.6)
5.71, d (1.6)
0.96, td (14.8, 2.4) (β-H)
2.01, dd (14.8, 2.8) (α-H)
0.99, d (14.8) (β-H)
6.06, dd (17.6, 10.8)
5.11, d (17.6)
1.36, m (β-H)
1.21, m
2.05, dd (14.8, 3.2) (α-H)
0.96, d (14.8) (β-H)
6.05, dd (17.6, 10.8)
5.15, dd (17.6, 0.8)
5.12, dd (10.8, 0.8)
0.88, s (3H)
2.68, d (14.8)
1.52, m
5.77, dd (17.6, 10.8)
5.21, dd (17.6. 0.8)
5.36, dd (10.8, 0.8)
1.00, s (3H)
1.27, q (7.3, 2H)
0.84, t (7.3, 3H)
16a
16b
17
5.10, d (10.8)
0.87, s (3H)
0.69, s (3H)
1.22, s (3H)
0.96, s (3H)
18
1.03, s (3H)
1.13, s (3H)
1.06, s (3H)
19
0.85, s (3H)
1.33, s (3H)
1.44, s (3H)
7-OH
8-OH
9-OH
3.61, br s
3.56, br s
2.78, br s
2.87, br s
2.89, br s
2.96, br s
a
Data were measured in CDCl3 at 400 MHz. Chemical shifts (δ) are expressed in ppm, and J values are presented in Hz.
H-12/CH3-17. Stereochemistry at other positions was
determined to be the same as that of sphaeropsidin B (9) by
NOEDIFF data (Figure 4). On the basis of these data the
structure of smardaesidin E was identified as 7α,9α,12α-
trihydroxy-6β,20-epoxyisopimara-8(14),15-dien-20-one (5).
Smardaesidin F (6), a white amorphous solid, showed a
HRESIMS pseudo molecular ion corresponding to the
1
molecular formula C19H28O4. The H NMR data (Table 3)
showed three tertiary methyls, a vinyl group, three D2O
exchangeable signals for hydroxy groups, and an oxygenated
methine proton at δ 4.48 (br s, H-7). The 13C NMR data
(Table 2) indicated that compound 6 contained 19 carbons,
which included three methyls, seven methylenes, one of which
is olefinic (δ 112.6), two methines, one of which is oxygenated
(δ 76.3) and one is olefinic (δ 146.5), and seven quaternary
carbons, two of which are oxygenated (δ 74.5 and 76.2) and
two are olefinic (δ 140.0 and 155.3), and one carbonyl
(δ 197.9), as discerned from its HSQC spectrum. Analysis of
1
the H−1H COSY, HSQC, and HMBC spectra of 6 furnished
the planar structure of this compound. Two spin systems
1
(Figure 5) were determined from the H−1H COSY spectrum
of 6. A methyl and a vinyl linked to C-13, as present in
compounds 2−5, were verified by HMBC correlations of
Figure 3. Selected 1H−1H COSY (
) and HMBC correlations
(H→C) of 5.
Figure 5. Selected 1H−1H COSY (
) and HMBC correlations
Figure 4. Selected NOEs observed in the NOEDIFF experiments of 5.
(H→C) of 6.
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CH3-17 to C-12, C-13, C-14, and C-15, H-15 to C-14, and
CH2-16 to C-13. The presence of an α,β-unsaturated ketone
moiety in 6 was discerned from the carbon resonances at δC
140.0 (C-5), 155.3 (C-10), and 197.9 (C-6), and this was
placed at the junction of rings A and B by the HMBC
correlations of CH3-18 and CH3-19 to C-3, C-4, and C-5,
CH2-1 to C-5 and C-10, H-1α to C-9, and CH2-11 to C-10
(Figure 5). The HMBC correlations of H-7 to C-6, C-8, C-9,
and C-14, OH-8 (δ 2.78, br s) to C-7, C-8, C-9, and C-14,
CH2-11 to C-9, and H-14β to C-7, C-8, and C-9 indicated that
the quaternary carbons, C-8 and C-9, joining the rings B and C
of 6 were oxygenated and that the OH was linked to C-7.
These data suggested that compound 6 was a 20-nor-
isopimarane-type diterpenoid. The relative stereochemistry of
6 was determined by the application of NOEDIFF data
(Supporting Information). Irradiation of CH3-17 caused
enhancement of H-15, H-16a, H-12β, H-12α, H-14β, and
H-14α, indicating that CH3-17 was equatorially oriented in ring
C. Key NOEDIFF data of H-1α/H-11β, H-1β/H-11β, and
H-1β/H-12β indicated that rings B and C were cis-fused.
Therefore, the OH groups at C-8 and C-9 should be
α-oriented. It is also interesting to note that as B/C rings
were cis-fused in 6, both H-12β and H-14β were just above the
plane of the α,β-unsaturated ketone moiety (see the Supporting
Information for a 3D structure), and the shielding magnetic
anisotropic effect of this moiety rendered both protons to
resonate at considerably higher fields (δ 0.96 and 0.99) than
normal aliphatic methylene protons. The irradiation of H-7
caused enhancements of OH-7, OH-8, and OH-9, while no
enhancement of CH2-14 was observed, suggesting that
the OH-7 had a β orientation. Consequently, the structure of
smardaesidin F was identified as 7β,8α,9α-trihydroxy-20-
norisopimara-5(10),15-dien-6-one (6).
Figure 6. ΔδH values (Δδ (in ppm) = δS − δR) obtained for (S)- and
(R)-MTPA esters (4a,b, respectively) of smardaesidin D (4) in
pyridine-d5.
1R,3S,5S,9S,10R,13R (4). The absolute configuration at C-9 of
smardaesidin F (6) was established by the analysis of its CD
spectrum (Supporting Information). For a series of enones with
doubly endocyclic CC bonds as in 6, it has been reported that
an additional ring fused to this bond has very little influence on
their CD spectra.23 Cotton effects displayed by 6 (Supporting
Information) are similar to those reported for β-rotunol,23 with a
positive Cotton effect at 250 nm (π → π* of enone) and a
negative Cotton effect at 333 nm (n → π* of enone), suggesting
the same absolute configuration (S) at C-9 of 6 as for β-rotunol.
This, along with NOEDIFF data for smardaesidin F (see above
and Supporting Information), established its absolute config-
uration as 7R,8R,9S,13S. The CD spectrum of smardaesidin G
(7) was almost superimposable with that of 6 (Supporting
Information), suggesting that they bear the same absolute
configuration at C-9. CD data together with stereochemical
information presented above for 7 established the absolute
configuration of smardaesidin F as 1R,7R,8R,9S,13S. Application
of a Mosher’s ester method for the determination of absolute
configuration of smardaesidin A (1) failed, as it gave rise to its
7,16-bis-MTPA ester, leading to interactions in ΔδS−R effects
among the modification sites, probably due to their close
proximity.24
Smardaesidin G (7) was also a white amorphous solid. The
HRESIMS displayed a pseudo molecular ion consistent with
the molecular formula C19H28O5. The 1H and 13C NMR
spectra (Tables 3 and 2, respectively) of 7 closely resembled
those of 6. The only difference was the absence of one of the
methylenes and the presence of an oxygenated methine (δ H
4.63 (br s); δC 63.6) in 7, suggesting that it was a hydroxylated
derivative of 6. On the basis of 1H−1H COSY and HMBC data
(Supporting Information), this OH group was placed at C-1.
In the NOEDIFF experiment with 7, irradiation of H-1 caused
enhancement of only H-11β, suggesting that H-1 was
1
Six isopimarane analogues, including sphaeropsidin B (9),
were prepared for the purpose of investigating the antiprolifer-
ative activity of this class of diterpenoids. Sphaeropsidin B (9)
was obtained by the chemical reduction of sphaeropsidin A (8)
with NaBH4/MeOH. Catalytic hydrogenation (H2/Pt−C/
EtOH) of 8 afforded 7-O-15,16-tetrahydrosphaeropsidin A
(14) and a new isopimarane analogue which was identified as
7-hydroxy-6-oxo-isopimara-7-en-20-oic acid (15) (see below).
Sphaeropsidin A (8) on acetylation with Ac2O/pyridine yielded
6-O-acetylsphaeropsidin A (16), whereas on methylation with
diazomethane, it afforded 8,14-methylenesphaeropsidin A methyl
ester (17) (for NMR data see the Supporting Information).
Treatment of sphaeropsidin C (10) with MeI/K2CO3 provided
the corresponding methyl ester (18). The molecular formula
C20H30O4 of the new isopimarane analogue 15 obtained by
catalytic hydrogenation of sphaeropsidin A (8) (C20H26O5)
suggested that this reaction led to the addition of four hydrogen
α-oriented (Supporting Information). In addition, in the H
NMR spectrum, H-1 resonated as a broad singlet at δ 4.63. As it
is only coupled to both protons at C-2, this indicated that the
vicinal coupling constants for both protons were very small,
which was consistent with the relevant dihedral angles shown in
the favorable half-chair conformation adopted by ring A with a
β-OH at C-1 (Supporting Information). Thus, the structure of
smardaesidin G was identified as 1β,7β,8α,9α-tetrahydroxy-20-
norisopimara-5(10),15-dien-6-one (7).
The absolute configurations of the stereocenters of 4 and 6−8
were assigned using a modified Mosher’s ester method, CD
spectra, and comparison of specific rotation data with those
reported. The optical rotation of 8 was identical with that
reported, suggesting that it bears the same absolute structure
established previously for sphaeropsidin A.13,21 The configuration
at C-3 of smardaesidin D (4) was determined to be S by the
application of a modified Mosher’s ester method22 (Figure 6).
This, together with the aforementioned NMR data, led to the
establishment of the absolute configurations of smardaesidin D as
1
atoms and removal of one oxygen atom. The H and 13C NMR
spectra of 15 (Tables 3 and 2, respectively) indicated the absence
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Table 4. Cytotoxicities of Compounds 8, 11, and 16 Against a Panel of Seven Tumor Cell Lines and Normal Human Primary
a
Fibroblast Cells
b
cell line
compd
NCI-H460
SF-268
MCF-7
MDA-MB-231
PC-3
PC-3M
MIAPaCa-2
WI-38
8
1.9 ± 0.5
>10
2.1 ± 0.2
7.5 ± 0.5
4.9 ± 0.7
0.6 ± 0.0
3.0 ± 0.7
>10
1.4 ± 0.1
3.7 ± 0.9
2.5 ± 0.1
0.5 ± 0.1
2.5 ± 0.4
9.6 ± 0.4
nt
2.4 ± 0.3
>10
2.0 ± 0.0
9.0 ± 0.5
nt
3.7 ± 0.0
>10
11
16
2.8 ± 0.2
0.3 ± 0.1
2.0 ± 0.7
0.4 ± 0.0
2.7 ± 0.6
0.3 ± 0.0
nt
doxorubicin
0.3 ± 0.0
0.3 ± 0.1
0.8 ± 0.1
b
a
Results are expressed as IC50 values ± standard deviation in μM. Doxorubicin and DMSO were used as positive and negative controls. Key: NCI-
H460, human nonsmall cell lung cancer; SF-268, human CNS cancer (glioma); MCF-7, human breast cancer; MDA-MB-231, human metastatic
breast adenocarcinoma; PC-3, human prostate adenocarcinoma; PC-3M, human metastatic prostate adenocarcinoma; MIAPaCa-2, human pancreatic
cancer; WI-38, normal human primary fibroblast cells; nt, not tested.
Figure 7. Cell migration inhibition assay of compounds 8 and 11 in metastatic cancer cell line MDA-MB-231: (A) DMSO control (negative);
(B) LY294002 (positive control) at 7.5 μM; (C) compound 8 at 2.0 μM; (D) compound 11 at 5.0 μM.
of the oxygenated carbon at C-9 and its replacement with a CH
(δH 2.33 (m); δC 47.8), suggesting that the OH substituent at
C-9 in 8 has undergone hydrogenolysis during the reaction. In its
NMR spectra, 15 had signals due to CH3 (δH 0.84; δC 7.8) and
CH2 (δH 1.27; δC 37.3) of an ethyl moiety, suggesting that the
vinyl group of sphaeropsidin A underwent hydrogenation. The
NMR data also indicated that 1,4-hydrogenation of the enone
moiety of 8 had occurred, resulting in a CH2 at C-14 (δH 2.68
and 1.52; δC 37.2) and opening of the lactone ring with the
generation of a new 7-hydroxyenone moiety in ring B (δ C 193.2
(C-6), 143.2 (C-7), and 121.8 (C-8)) and a carboxylic acid
moiety at C-10 (δC 179.4). The above NMR assignments of 15
concentrations of 1.5 and 4.8 μM, respectively (Supporting
Information). The IC50 and IC25 values for 8 against MDA-MB-
231 cell proliferation/survival were 3.00 ± 0.05 and 1.50 ±
0.24 μM, respectively, and those for 11 were above 5.00 μM.
These results suggested that sphaeropsidin A (8) is capable
of inhibiting migration of this metastatic breast cancer cell line
at a sublethal concentration. Further studies to evaluate its
mechanism of cell migration inhibition are currently in
progress. Previously, sphaeropsidins and some of their
analogues have been shown to have host-selective phytotoxicity
and selective antimycotic activity against several plant
pathogenic fungi.18 This constitutes the first report of the
cancer cell proliferation inhibitory activity of sphaeropsidins
A (8) and D (11) and cancer cell migration inhibitory activity
of 8.
Among the new compounds encountered in this study,
smardaesidin A (1) possesses a rare cyclopropane ring fused
to ring C of the isopimarane skeleton. To the best of our
knowledge, this constitutes the second report of a related
diterpene with cyclopropane ring fused to ring C. The first of
these, mimosol D, containing a cyclopimarane and not a
cycloisopimarane skeleton, has recently been isolated from
Caesalpinia mimosoides.26 Smardaesidins B and C (2 and 3)
coexist in a hemiketal/keto equilibrium, with the keto form 2 as
the major constituent, since the conjugation with the Δ8,14
double bond makes it more stable. Smardaesidins F and G
(6 and 7) are two polyhydroxylated 20-nor-isopimaranes, which
belong to a rare group of diterpenoids.
1
were supported by the analysis of H−1H COSY, HSQC, and
HMBC spectra (see the Supporting Information). Thus, the
hydrogenation product of sphaeropsidin A (8) was characterized
as 7-hydroxy-6-oxo-isopimara-7-en-20-oic acid (15).
Compounds 1−18 were first evaluated for in vitro inhibition
of cell proliferation using a panel of five cancer cell lines: NCI-
H460, SF-268, MCF-7, PC-3M, and MDA-MB-231. The cells
were exposed to 10 μM concentration of test compounds for
72 h in RPMI-1640 media supplemented with 10% fetal bovine
serum, and cell viability was evaluated by the alamarBlue
assay.10 Only sphaeropsidins A (8) and D (11) and 6-O-
acetylsphaeropsidin A (16) were active at this concentration
(Supporting Information). IC50 values of these compounds
were then determined against a panel of seven cancer cell lines,
NCI-H460, SF-268, MCF-7, MDA-MB-231, PC-3, PC-3M, and
MIAPaCa-2, and normal human fibroblast (WI-38) cells by this
assay (Table 4). It is noteworthy that sphaeropsidins A (8) and
D (11) exhibited marginal selectivity in inhibiting proliferation
of the metastatic human breast adenocarcinoma cell line MDA-
MB-231 used in this study. Therefore, it was of interest to
evaluate these for inhibition of migration of this metastatic cell
line.2b Compounds 8 and 11 inhibited migration of MDA-MB-
231 cells at concentrations of 2.0 and 5.0 μM, respectively
(Figure 7). The extent of cell migration when determined
using NIH ImageJ software25 suggested that 8 and 11 caused
ca. 50% inhibition of the migration of MDA-MB-231 cells at
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured with a JASCO Dip-370 digital polarimeter. UV spectra
were recorded on a Shimadzu UV-1601 UV−vis spectrophotometer.
IR spectra were recorded on a Shimadzu FTIR-8300 spectrometer
using KBr disks. NMR spectra were recorded in CDCl3 or CD3OD
1
with a Bruker Avance III instrument at 400 MHz for H NMR and
100 MHz for 13C NMR, respectively, using residual CHCl3 or CH3OH
as internal reference. Low-resolution and high-resolution MS were
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recorded on Shimadzu LCMS-QP8000α and Bruker Apex-ultra FT-
ICR spectrometers, respectively. CD spectra were recorded in MeOH
on a Jasco 810 spectrometer at 25 °C, using a quartz cell with 1 mm
optical path length. Column chromatography was performed on silica
gel type 60 (Baker) or Sephadex LH-20 (25−100 μm; GE
Healthcare). Thin-layer chromatography (TLC) and preparative
TLC were performed on silica gel 60 GF254 plates (Merck). LRP-2
Whatman (Catalog No. 4776-001) was used for reversed-phase (RP)
column chromatography. RP-TLC separations were performed on
Merck RP-18 F254S precoated aluminum sheets. Preparative HPLC
was performed on a Waters Delta Prep 4000 system equipped with
a Waters 996 photodiode array detector and a Waters Prep LC
controller utilizing Empower Pro software and using a RP column
(Phenomenex Luna 5 μm, C18, 100 Å, 250 × 10 mm); chromato-
grams were acquired at 254 and 205 nm.
Fungal Isolation and Identification. In June 2007, a small mat
(ca. 5 cm2) of Ceratodon purpureus was collected from sandy soil in a
mixed coniferous forest ca. 9.5 km northwest of the Southwestern
Research Station in the eastern Chiricahua Mountains of southeastern
Arizona, USA (2100 m.a.s.l.; 31°53′00″ N, 109°12′18″ W).9b Healthy
photosynthetic tissue was cut into 2 × 2 mm segments, washed in tap
water, surface-sterilized by agitating sequentially in 95% EtOH for 30 s,
0.5% NaOCl for 2 min, and 70% EtOH for 2 min following previous
procedures,27 and then surface-dried under sterile conditions before
plating on 2% malt extract agar (MEA) in a Petri plate. The plate was
sealed with Parafilm and incubated under ambient light/dark
conditions at room temperature (ca. 21.5 °C). One of the emergent
fungi was isolated into pure culture on 2% MEA, vouchered in sterile
water, and deposited as a living voucher at the Robert L. Gilbertson
Mycological Herbarium at the University of Arizona under accession
No. ARIZ0432 (AZ0432). The fungal strain lacked the reproductive
structures necessary for taxonomic identification; thus, the nuclear
ribosomal internal transcribed spacers and 5.8s gene (ITS rDNA; ca.
600 bp) and an adjacent portion of the nuclear ribosomal large subunit
(LSU rDNA; ca. 500 bp) were amplified by PCR and sequenced
bidirectionally as described previously.9b
C (62.1 mg), D (22.7 mg), E (364.1 mg), F (51.6 mg), G (91.0 mg),
H (114.1 mg), I (40.6 mg), J (11.0 mg), and K (28.0)). Fraction C was
chromatographed over a silica gel (2.0 g) column with hexanes/
2-propanol as eluent (50/1 and 30/1, 150 mL each) to afford six
subfractions (C1−C6). C3 (7.8 mg) was purified by preparative
HPLC (MeOH/H2O, 75:25, 2.6 mL/min) to give 6 (3.1 mg, tR
28.80 min). The fraction C5 (10.7 mg) was purified by preparative
HPLC (MeCN/H2O, 55/45, 2.6 mL/min) to give 5 (1.8 mg, tR
=
=
12.21 min). Fraction E was separated over a column of silica gel
(10.0 g) and eluted with hexane/2-propanol (100/1, 300 mL) to
afford a major compound, which on treatment with hexanes/acetone
(10/1) afforded 8 as a white amorphous solid (280.0 mg). Fraction F
was divided into five fractions (F1−F5) by silica gel (2.0 g) column
chromatography with hexanes/2-propanol as eluent (50/1 and 30/1,
150 mL each). Fraction F3 (7.7 mg) was further purified by
preparative HPLC (MeCN/H2O, 40/60, 2.8 mL/min) to give 7 (1.7
mg, tR = 10.90 min). Fraction G was separated by silica gel (3.5 g)
column chromatography with hexane/2-propanol as eluent (50/1,
30/1, and 20/1, 200 mL each) to afford eight fractions (G1−G8).
Fraction G2 (8.7 mg) was purified by preparative RP-TLC developed
with MeOH/H2O (50/50) to give 11 (2.1 mg, Rf = 0.35). G4
(13.0 mg) was separated by preparative HPLC (MeOH/H2O, 60/40,
2.8 mL/min) to yield 2 and 3 as a mixture (4.1 mg, tR = 14.10 min)
and 12 (2.1 mg, tR = 47.90 min). Fraction H was further fractionated
by silica gel column chromatography using hexanes/2-propanol
mixtures (50/1, 30/1, 20/1, and 15/1, 150 mL each) to afford 10
subfractions (H1−H10). From fractions H1 and H5, respectively, 10
(24.0 mg) and 13 (3.5 mg) were obtained as crystals. H7 (12.5 mg)
was separated by a small column packed with reversed-phase silica gel
using MeOH/H2O (50/50) as the eluent to afford 1 (4.0 mg). H9
(11.2 mg) was purified by preparative HPLC (MeOH/H2O, 60/40,
2.6 mL/min) to yield 4 (3.5 mg, tR = 23.62 min).
25
Smardaesidin A (1): white amorphous powder; [α]D = +1.3
(c 0.163, MeOH); UV (EtOH) λmax (log ε) 221 (3.03) nm; IR (KBr)
1
νmax 3396, 3298, 2930, 1755, 1726, 1462, 1070, 945 cm−1; for H
and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 387.1778
[M+Na]+ (calcd for C20H28O6Na, 387.1778).
Sequence data for AZ0432, as well as 95 additional plant- and
lichen-associated fungi isolated from the same site, were aligned with
data from 56 closely related endophytic, endolichenic, and
saprotrophic fungi from the same site9b and eight described species
of Pyronemataceae (Ascomycota) chosen on the basis of highest-
affinity BLASTn matches to the GenBank nonredundant (nr) database
in NCBI28 and recent phylogenetic analyses within the family.29−31
Subsequent phylogenetic analyses were conducted in GARLI32 using
the GTR+I+G model of evolution as determined by ModelTest,33
followed by 1000 bootstrap replicates to assess support for the
resulting topology. The analysis placed AZ0432 with strong statistical
support in Smardaea (Supporting Information).
Smardaesidins B and C (2 and 3): white amorphous powder;
25
[α]D −20.8 (c 0.115, MeOH); UV (EtOH) λmax (log ε) 245 (3.80)
nm; IR (KBr) νmax 3396, 2930, 1666, 1601, 1462, 1265, 1068, 1018
cm−1; for 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z
335.2217 [M + H]+ (calcd for C20H31O4, 335.2217).
25
Smardaesidin D (4): white amorphous powder; [α]D = −5.8
(c 0.148, MeOH); UV (EtOH) λmax (log ε) 244 (3.79) nm; IR (KBr)
νmax 3425, 2963, 1672, 1601, 1460, 1369, 1279, 1001 cm−1; for 1H and
13C NMR data, see Tables 1 and 2; HRESIMS m/z 335.2215 [M +
H]+ (calcd for C20H31O4, 335.2217).
25
Smardaesidin E (5): white amorphous powder; [α]D = +30.6
(c 0.120, MeOH); UV (EtOH) λmax (log ε) 218 (3.10) nm; IR (KBr)
Culturing, Extraction, and Isolation of Metabolites. For
secondary metabolite isolation, the fungus was cultured in 20 T-flasks
(800 mL), each containing 135 mL of PDA coated on four sides of the
flasks, maximizing the surface area for fungal growth (total surface
area/flask ca. 400 cm2). After incubation for 16 days at 28 °C, MeOH
(200 mL/T-flask) was added and the flasks were shaken (at 200 rpm)
for 1 h followed by sonication for 1 h at 25 °C, and the resulting
extract was filtered through Whatman No. 1 filter paper and a layer of
Celite 545. The filtrate was concentrated to ca. 25% of its original
volume by evaporation under reduced pressure and extracted with
EtOAc (1 L × 4). Combined EtOAc extract was evaporated under
reduced pressure to afford a dark brown semisolid (1.40 g), which was
partitioned between hexane and 80% aqueous MeOH. The cytotoxic
80% aqueous MeOH fraction was diluted to 50% aqueous MeOH by
addition of water and then extracted with CHCl3. Evaporation of
CHCl3 under reduced pressure yielded a dark brown semisolid
(1.05 g). A large portion (1.00 g) of this was subjected to gel permea-
tion chromatography over a column of Sephadex LH-20 (40.0 g) made
up in hexanes/CH2Cl2 (1/4) and eluted with hexanes/CH2Cl2 (1/4,
200 mL) followed by CH2Cl2/acetone (3/2, 150 mL). A total of 35
fractions (10 mL each) were collected and combined on the basis of
their TLC patterns to yield 12 fractions (A (31.4 mg), B (108.7 mg),
1
νmax 3435, 2928, 1771, 1636, 1462, 1113, 1067, 1001 cm−1; for H
and 13C NMR data, see Tables 3 and 2; HRESIMS m/z 371.1835
[M + Na]+ (calcd for C20H28O5Na, 371.1829).
Smardaesidin F (6): white amorphous powder; [α]D = +77.5
25
(c 0.150, MeOH); UV (EtOH) λmax (log ε) 241 (3.87) nm; IR (KBr)
νmax 3425, 2930, 1670, 1636, 1597, 1458, 1113, 1053, 947 cm−1; for
1H and 13C NMR data, see Tables 3 and 2; HRESIMS m/z 343.1879
[M + Na]+ (calcd for C19H28O4Na, 343.1880).
25
Smardaesidin G (7): white amorphous powder; [α]D = +62.1
(c 0.14, MeOH); UV (EtOH) λmax (log ε) 235 (3.76) nm; IR (KBr)
1
νmax 3427, 2932, 1678, 1636, 1458, 1140, 1043, 947 cm−1; for H
and 13C NMR data, see Tables 3 and 2; HRESIMS m/z 359.1825
[M + Na]+ (calcd for C19H28O5Na, 359.1829).
25
Sphaeropsidin A (8): white amorphous powder; [α]D = +111.4
(c 1.20, MeOH) (lit.21 +112.4); H and 13C NMR data were fully
1
consistent with those reported.13,21
Sphaeropsidin B (9): A solution of sphaeropsidin A (8, 1.0 mg) in
MeOH (0.5 mL) was treated with NaBH4 (1.0 mg) at 0 °C and stirred
for 45 min. The solution was treated with a few drops of water and
evaporated in vacuo. The residue was treated with CH2Cl2, filtered
through plug of silica gel, and eluted with 10% MeOH to give
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Article
sphaeropsidin B (9, 1.0 mg) as a white solid. 1H and 13C NMR
chemical shifts were identical with the reported data.14
normal human primary fibroblast cells (WI-38). The cancer cells
were all cultured under standard culture conditions. The test
compounds or vehicle control (DMSO) were added to appropriate
wells, and the cells were incubated for 72 h. Then 20 μL/well
alamarBlue solution was added into the assay plates for a final assay
volume of 200 μL/well, yielding a final concentration of 10%
alamarBlue. After they were shaken for 10 s, plates were returned to
the incubator and kept for 4 h. The plates were then exposed to
an excitation wavelength of 560 nm, and the fluorescence emitted at
590 nm was read. The percent viability was expressed as fluorescence
counts in the presence of test compound as a percentage of that in the
vehicle control. Doxorubicin and DMSO were used as positive and
negative controls, respectively.
Preparation of 7-O-15,16-Tetrahydrosphaeropsidin A (14)
and 7-Hydroxy-6-oxo-isopimara-7-en-20-oic acid (15). A sol-
ution of sphaeropsidin A (8, 10.0 mg) in EtOH (2 mL) was added to a
suspension of 5% Pt−C (3.0 mg) in EtOH (1 mL) and 1 N HCl
(15 μL). Hydrogenation was carried out at room temperature and under
atmospheric pressure with continuous stirring. After 45 min, the
reaction mixture was filtered through a short bed of silica gel and
evaporated under reduced pressure. The residue thus obtained was
purified by HPLC (70% MeCN/H2O containing 0.025% ammonium
formate for 11 min and then 100% MeCN at 4 mL/min) to give 14
(4.8 mg, 47.4%, tR = 7.01 min) and 15 (1.1 mg, 11.4%, tR = 16.01 min).
7-O-15,16-Tetrahydrosphaeropsidin A (14): white solid; H and
Cell Migration Inhibition Assay (CMIA).2b For CMIA (also
known as wound healing assay) the metastatic breast cancer (MDA-
MB-231) cells were cultured in DMEM/Ham’s F-12 medium
containing 10% fetal bovine serum and gentamicin (50 μg/mL). Cells
were grown under a 5% CO2 atmosphere at 37 °C, and the cells were
harvested at or above 80% confluence. The cells were plated onto sterile
24-well plates at a density of 150 000 cells per well and were allowed to
recover for 24 h until a confluent cell monolayer formed in each well
(>90% confluence). Wounds were then inflicted to each cell monolayer
using a sterile toothpick, media were removed, the cell monolayers were
washed once with PBS, and fresh media were added to each well. Test
samples of compounds 8 and 11 were prepared in DMSO at different
concentrations and added to the plates, each in duplicate along with
the two controls: phosphotidylinositol (PtdIns) 3-kinase inhibitor
LY29400235 at 7.5 μM (positive control) and DMSO (negative
control). The plates were incubated for 40 h, during which the wells
treated with DMSO had healed entirely and the wells treated with
LY294002 and samples containing cell motility inhibitors had wounds
present. All treatments, including the controls, were documented
photographically. A treatment was considered active if there was a
wound present in duplicate wells at the completion of the assay.
Quantification of Cell Migration Inhibition. ImageJ software
available from the NIH Web site (http://rsb.info.nih.gov/rj/) was
used to quantify CMIA data.25 Three random pictures were taken for
each wound using an inverted microscope at 10× magnification;
photos were taken immediately after a wound was inflicted to the cell
monolayer and uploaded into the ImageJ software, and the area of the
wound was measured by using the rectangle area selection tool and the
three areas per well averaged. After the DMSO wells healed (usually
40 h after infliction of the wound), three random pictures were taken
for each treated well and the areas of the wounds measured as above.
The percentage of wound healed was then calculated using the formula
100 − [(final area)/(initial area) × 100%].25
1
13C NMR and LRMS data were consistent with those reported.18
7-Hydroxy-6-oxo-isopimara-7-en-20-oic acid (15): white solid;
25
[α]D = +108.1 (c 0.28, MeOH); UV (MeOH) λmax (log ε) 280.0
(5.65), 202.0 (5.45) nm; IR (KBr) νmax 3429, 1693, 1662, 1461, 1380,
1
1236, 1207 cm−1; for H and 13C NMR data, see Tables 3 and 2;
HRESIMS m/z 333.2072 [M − H]− (calcd for C20H29O4, 333.2071).
6-O-Acetylsphaeropsidin A (16) and 8,14-Methylenesphaero-
psidin A Methyl Ester (17). These were prepared following the same
procedures described in the literature18 and were characterized by
comparison of NMR data with those reported.18
Sphaeropsidin C Methyl Ester (18). Sphaeropsidin C (10,
5.2 mg) was dissolved in acetone (1.0 mL), mixed with 10.0 mg of
K2CO3 and 0.5 mL of MeI, and stirred at room temperature for 1 h.
The mixture was filtered, evaporated in vacuo, and subjected to a
preparative silica gel TLC (eluent: CH2Cl2/MeOH (96/4)) to give
1
sphaeropsidin C methyl ester (18; 5.0 mg). The H and 13C NMR
data were identical with those reported previously.14
Preparation of the (R)- and (S)-MTPA Ester Derivatives of
Smardaesidin D (4) by a Convenient Mosher’s Ester
Procedure.22 Compound 4 (0.75 mg) was dissolved in pyridine-d5
(0.4 mL) and transferred into an NMR tube. S-(+)-α-Methoxy-α-
(trifluoromethyl)phenylacetyl (MTPA) chloride (10 μL) was added
into the NMR tube and was shaken carefully to mix the sample and the
MTPA chloride evenly. The reaction in the NMR tube was monitored
1
immediately by H NMR. The reaction was found to be complete
within 15 min, to give the mono (R)-MTPA ester derivative (4b) of 4.
1H NMR data of 4b (400 MHz, pyridine-d5; data were assigned on the
1
basis of the correlations of the H−1H COSY, HSQC, and HMBC
spectra): δ 7.014 (1H, d, J = 1.7 Hz, H-14), 5.794 (1H, dd, J = 10.6,
17.5 Hz, H-15), 5.187 (1H, dd, J = 3.9, 12.4 Hz, H-3), 5.023 (1H,
assigned on the basis of HSQC, H-1), 5.017 (1H, assigned based on
HSQC, H-16a), 4.887 (1H, dd, J = 0.9, 10.6 Hz, H-16b), 3.016 (1H, dd,
J = 5.1, 13.3 Hz, H-5), 2.766 (1H, overlapped, H-11a), 2.749 (1H,
overlapped, H-6a), 2.668 (1H, overlapped, H-2a), 2.656 (1H,
overlapped, H-6b), 2.572 (1H, overlapped, H-11b), 2.402 (1H, q, J =
12.1 Hz, H-2b), 2.263 (1H, dist. dt, J = 3.2, 13.3 Hz, H-12a), 1.559 (1H,
m, H-12b), 1.328 (3H, s, H-20), 1.120 (3H, s, H-17), 1.027 (3H, s, H-
19), and 0.852 (3H, s, H-18). In the manner described for 4b, another
portion of compound 4 (0.75 mg) in pyridine-d5 (0.4 mL) was reacted
in a second NMR tube with (R)-(−)-α-MTPA chloride (10 μL) at
room temperature for 15 min, to afford the mono (S)-MTPA ester (4a)
of 4. 1H NMR data of 4a (400 MHz, pyridine-d5): δ 7.014 (1H, d, J =
1.7 Hz, H-14), 5.793 (1H, dd, J = 10.6, 17.5 Hz, H-15), 5.180 (1H, dd,
J = 3.9, 12.4 Hz, H-3), 5.010 (1H, overlapped, assigned on the basis of
HSQC, H-1), 5.018 (1H, overlapped, assigned on the basis of HSQC,
H-16a), 4.886 (1H, dd, J = 0.9, 10.6 Hz, H-16b), 3.029 (1H, dd, J = 5.1,
13.3 Hz, H-5), 2.757 (1H, H-11a), 2.788 (1H, H-6a), 2.612 (1H, H-2a),
2.651 (1H, H-6b), 2.567 (1H, H-11b), 2.238 (1H, H-2b), 2.273 (1H,
H-12a), 1.550 (1H, m, H-12b), 1.2905 (3H, s, H-20), 1.112 (3H, s,
H-17), 1.032 (3H, s, H-19), and 0.974 (3H, s, H-18).
ASSOCIATED CONTENT
■
S
* Supporting Information
Figures and tables giving selected H−1H COSY and HMBC
1
correlations and selected NOEs of compounds 2−4, 6, and 7,
1
1H and 13C NMR, H−1H COSY, HSQC, and HMBC spectra
1
of compounds 1−7, 15, and 17, H NMR of 2a,b, CD spectra
of 6 and 7, cytotoxicity (alamarBlue assay) data of compounds
1−18, and quantitative cell migration assay data for compounds
8 and 11. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: (520) 621-9932. Fax: (520) 621-8378. E-mail: leslieg@
ag.arizona.edu.
■
Cytotoxicity Assay. The resazurin-based colorometric (alamarBl-
ue) assay10,34 was used for the in vitro assay of cytotoxicity to human
nonsmall cell lung cancer (NCI-H460), CNS glioma (SF-268), breast
cancer (MCF-7), human metastatic breast adenocarcinoma (MDA-
MB-231), prostate adenocarcinoma (PC-3), metastatic prostate
adenocarcinoma (PC-3M), pancreatic cancer (MIAPaCa-2), and
ACKNOWLEDGMENTS
■
Financial support for this work from the National Cancer
Institute (Grant R01 CA90265) and the National Institute of
General Medical Sciences (Grant P41 GM094060) are
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Journal of Natural Products
Article
Prod. 2002, 65, 1278−1282. (b) Seco, J. M.; Quinoa,
́
E.; Riguera, R.
̃
gratefully acknowledged. We also thank the National Science
Foundation for supporting collection and identification of
fungal strains (Grant DEB-064099 to A.E.A.), the NSF IGERT
Program in Genomics at The University of Arizona for graduate
fellowship (to J.M.U.), the College of Agriculture and Life
Sciences at The University of Arizona for institutional support
of E.M.K.W. and M.K.G., the China Scholarship Council for a
postdoctoral fellowship (to X.-N.W.), Mr. Timothy Bolton for
his assistance in preparing large-scale cultures, and Dr. Kamal B.
Gunaherath for critically reading the manuscript and suggesting
some changes.
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