A thiopyranchromenone and other chromone derivatives from an endolichenic fungus, Preussia africana

Article abstract of DOI:10.1021/np2009362

The first example of a naturally occurring thiopyranchromenone, preussochromone A (1), and five other new chromone derivatives, preussochromones B-F (2-6), were isolated from solid cultures of an endolichenic fungus, Preussia africana. The structures of 1-6 were established primarily by NMR experiments, and 2 and 4 were further confirmed by X-ray crystallography. The absolute configurations of 1 and 2 were determined by the application of electronic circular dichroism (ECD), whereas those of C-5 in 3, C-6 in 4, and the 6,7-diol in 5 were deduced via the CD data of the in situ formed [Rh ₂(OCOCF₃)₄] complex, the modified Mosher method, and Snatzke's method, respectively. Compounds 1 and 3 showed significant cytotoxicity against A549 cells.

Full text of DOI:10.1021/np2009362

Article
pubs.acs.org/jnp
A Thiopyranchromenone and Other Chromone Derivatives from an
Endolichenic Fungus, Preussia africana
Fan Zhang,†,‡ Li Li,§ Shubin Niu,† Yikang Si,§ Liangdong Guo,† Xuejun Jiang,† and Yongsheng Che*,†,⊥
†State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100190, People’s Republic of China
‡Graduate School of Chinese Academy of Sciences, Beijing 100039, People’s Republic of China
§Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050,
People’s Republic of China
⊥Beijing Institute of Pharmacology & Toxicology, Beijing 100850, People’s Republic of China
S
* Supporting Information
ABSTRACT: The first example of a naturally occurring thiopyranchromenone,
preussochromone A (1), and five other new chromone derivatives, preussochro-
mones B−F (2−6), were isolated from solid cultures of an endolichenic fungus,
Preussia africana. The structures of 1−6 were established primarily by NMR
experiments, and 2 and 4 were further confirmed by X-ray crystallography. The
absolute configurations of 1 and 2 were determined by the application of electronic
circular dichroism (ECD), whereas those of C-5 in 3, C-6 in 4, and the 6,7-diol in 5
were deduced via the CD data of the in situ formed [Rh2(OCOCF3)4] complex, the
modified Mosher method, and Snatzke’s method, respectively. Compounds 1 and 3
showed significant cytotoxicity against A549 cells.
ichens are combinations of a fungus (the mycobiont) and
an algal partner (the photobiont or phycobiont). In addi-
L
tion to fungal mycobionts, some nonobligate microfungi,
endolichenic fungi, are also found to live asymptomatically in
the bodies (thalli) of lichens.1 Although endolichenic fungi
inhabit the lichen thalli similarly to endophytes living in the
intercellular spaces of healthy plant tissues, the chemistry of
this class of fungi remained largely unexplored. To date, only a
limited number of investigations have been documented
regarding the isolation and identification of bioactive secondary
metabolites from their organisms.1−6 Our prior work on the
endolichenic fungus Coniochaeta sp. grown in a solid-substrate
fermentation culture afforded four relatively rare ring-expanded
(oxepinochromenone) and one ring-contracted (furochro-
menone) xanthones,4 as well as the first examples of naturally
occurring thienols and thiepinols possessing unique 2,3-
dihydrothieno[2,3-b]chromen-4-one and 4,5-dihydro-2H-
thiepino[2,3-b]chromen-6(3H)-one skeletons, respectively.5
During an ongoing search for new cytotoxic natural products
from this unique source, the fungus Preussia africana (Sporor-
miaceae) isolated from the lichen Ramalina calicaris (L.) Fr.
(Ramalinaceae), which was collected from Zixi Mountain,
Yunnan Province, People’s Republic of China, was subjected to
chemical investigation. Fractionation of an EtOAc extract
prepared from a solid-substrate fermentation culture led to the
isolation of a naturally occurring thiopyranchromenone named
preussochromone A (1), five other new chromone derivatives,
preussochromones B−F (2−6), and a known xanthone (7).7
Details of the isolation, structure elucidation, and cytotoxicity
of these metabolites are reported herein.
RESULTS AND DISCUSSION
■
Preussochromone A (1) gave a pseudomolecular ion [M + Na]+
by HRESIMS, consistent with a molecular formula of C15H14O7S
1
(nine degrees of unsaturation). Its H and 13C NMR spectra
showed resonances for three exchangeable protons (δH 5.71,
5.97, and 12.42, respectively), two methyl groups (one methoxy),
two methines including an oxymethine, eight aromatic/olefinic
carbons with three protonated, one oxygenated sp3 quaternary
carbon, one carboxylic carbon (δC 173.5), and one α,β-unsaturated
Received: December 2, 2011
Published: February 10, 2012
© 2012 American Chemical Society and
American Society of Pharmacognosy
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Table 1. NMR Data for 1−3
1
2
3
a
b
a
c
d
c
d
pos.
δC, mult.
δH (J in Hz)
HMBC
δC, mult.
δH (J in Hz)
δC, mult.
δH (J in Hz)
1
159.8, qC
111.3, CH
135.9, CH
106.5, CH
155.2, qC
39.5, CH
168.8, qC
71.3, CH
71.3, qC
160.0, qC
110.7, CH
135.4, CH
107.0, CH
156.5, qC
37.3, CH2
165.0, qC
71.3, qC
167.4, qC
101.1, CH
166.7, qC
105.8, qC
146.4, qC
64.9, qC
2
6.80, d (8.4)
7.63, t (8.4)
7.14, d (8.4)
1, 4, 8a
1, 4a
6.77, d (8.0)
7.60, t (8.0)
6.99, d (8.0)
6.62, s
3
4
2, 4a, 8a
4a
5
3.95, q (7.2)
3.64, d (7.2)
9
2.57, d (20); 3.17, d (20)
5a
6
5, 7, 7a, 9
174.7, qC
101.4, CH
7
71.8, CH
4.04, d (3.5)
4.00, d (3.5)
5.73, s
7a
115.2, qC
178.3, qC
109.0, qC
15.7, CH3
8
43.7, CH
113.6, qC
182.2, qC
110.7, qC
25.8, CH3
171.3, qC
51.3, CH3
190.4, qC
106.3, qC
159.1, qC
8a
9
1.37, d (7.2)
5, 5a, 6
10
9a
10
11
12
OH-1
OH-5
OH-6
OH-7
173.5, qC
51.9, CH3
1.49, s
70.3, CH2
56.8, CH3
57.6, CH3
4.35, d (12); 4.69, d (12)
3.60, s
3.95, s
3.95, s
13.20, s
5.80, s
3.69, s
12.42, s
12.50, s
5.71, d (7.2)
5, 7
4.28, s
5.97, s
6, 7a
4.58, s
a
b
c
d
Recorded at 150 MHz in DMSO-d6. Recorded at 600 MHz in DMSO-d6. Recorded at 125 MHz in acetone-d6. Recorded at 500 MHz in
acetone-d6.
ketone carbon (δC 178.3). Analysis of the NMR data of 1
(Table 1) revealed the presence of the same 5-hydroxy-4H-
chromen-4-one moiety as that typically found in xanthones
(e.g., 7),7 but the remaining portion was significantly different,
warranting a detailed 2D NMR analysis (Figure S13). In
calculation. The MMFF94 conformational search was further
optimized using TDDFT at the B3LYP/6-31G(d) basis set
level, affording five lowest energy conformers for the enan-
tiomer pairs 1a and 1b, two for 1c and 1d, two for 1e and 1f,
and three for 1g and 1h, respectively (Figures S17−S20). The
overall calculated ECD spectra of enantiomers 1a and 1b were
then generated by Boltzmann weighting of the five lowest
energy conformers with 86.00, 12.56, 1.35, 0.01, and 0.07%
populations, respectively, by their relative free energies. In a
similar fashion, the overall calculated ECD spectra of 1c−1h
were also generated. The absolute configuration of 1 was then
extrapolated by comparison of experimental and calculated
ECD spectra of 1a−1h after a UV correction of 20 nm (Figure
S21). The overall pattern of the experimental CD spectrum of
1 was comparable only to that of the calculated ECD spectrum
of (5S,6S,7R)-1 (1b), both showing similar negative Cotton
effects (CEs) in the regions of 200−225 and 250−350 nm,
respectively (Figure 1). The specific optical rotations of the two
main conformations of 1b (Con 1 and Con 2) (Figure S17)
have been calculated at the B3LYP/6-31++G(d,p) level using
B3LYP/6-31G(d) geometries, with the results given in Table
S1. The population-averaged specific optical rotations were
compared with the experimental data of 1 in MeOH at 589 nm.
The calculated specific rotation of 1b is consistent with the
experimental data of 1, further supporting the absolute config-
uration deduced from the ECD spectra. Therefore, the absolute
configuration of 1 was deduced to be 5S, 6S, 7R.
1
addition to the above-mentioned fragment, the H−1H COSY
NMR data of 1 showed the isolated spin-system of C-9−C-5−
C-6 (including OH-6). HMBC correlations from the O-methyl
proton signal H3-11 to the carboxylic carbon at 173.5 ppm
located the O-methyl group at C-10, while correlations from
H-6 to C-7 and C-7a and from the exchangeable proton at 4.54
ppm (OH-7) to C-6 and C-7a indicated that C-7 is attached to
both C-6 and C-7a. Considering the chemical shifts of the C-5
methine (δH/δC 3.95/39.5) and by comparison to a similar unit
(δH/δC 3.96/38.2) in an analogous synthetic compound,
2-methylthiopyrano[2,3-b]chromen-5(2H)-one,8 the sulfur atom
present in 1 was shown to be attached to C-5 to complete a
dihydrothiopyran ring; this was supported by a weak, but distinct
four-bond W-type HMBC correlation from H3-9 to C-5a.9 The C-
10 carboxylic carbon is now required to attach to C-7 to satisfy the
unsaturation requirement of 1, even though no additional evidence
for this linkage was provided by the HMBC data. Collectively,
these data permitted assignment of the planar structure of 1.
The absolute configuration of 1 was determined by compar-
ison of experimental and simulated electronic circular dichro-
ism (ECD) spectra, which were generated by time-dependent
density functional theory (TDDFT).10 Due to the presence
of three stereogenic centers, one of the eight stereoisomers,
(5R,6R,7S)-1, (5S,6S,7R)-1, (5S,6S,7S)-1, (5R,6R,7R)-1,
(5S,6R,7S)-1, (5R,6S,7R)-1, (5R,6S,7S)-1, and (5S, 6R,7R)-1
(1a−1h; Figure 1), must represent the actual configuration of
1. A systematic conformational analysis was performed for 1a−
1h via the Molecular Operating Environment (MOE) software
package using the MMFF94 molecular mechanics force field
The elemental composition of preussochromone B (2) was
established as C16H16O7 (nine degrees of unsaturation) by
HRESIMS. Although the 1H and 13C NMR data of 2 (Table 1)
revealed the presence of the same 5-hydroxy-4H-chromen-4-
one moiety as found in 1, the remaining portion was signifi-
cantly different. The C-7−C-8 (including OH-7) fragment was
established on the basis of 1H−1H COSY correlations observed
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Figure 1. Experimental CD spectrum of 1 in MeOH and the calculated ECD spectra of 1a−1h after a UV correction of 20 nm. Structures 1a−1h
represent eight possible stereoisomers of 1.
for relevant protons. HMBC cross-peaks from H2-5 to C-5a,
C-6, C-7, and C-8a and from H-7 and H-8 to C-8a established
the cyclohexene moiety fused to the 5-hydroxy-4H-chromen-4-
one unit at C-5a/C-8a. HMBC correlations of H3-10 with C-5,
C-5a,9 and C-7 located the methyl group at C-6, while those
from H-8 and H3-12 to C-11 connected the methyl formate
unit to C-8. The remaining exchangeable proton at 4.28 ppm
was assigned as OH-6 by default. Collectively, the planar
structure of 2 was established, which was further confirmed by
X-ray crystallography (Figure 2). The X-ray data also allowed
assignment of the relative configuration of 2.
(6R,7R,8R)-2 (2b) after a UV correction of 20 nm (Figure S23).
The MMFF94 conformational search followed by B3LYP/6-
31G(d) DFT reoptimization afforded four lowest energy
conformers (Figure S22). The calculated ECD spectra of
enantiomers 2a and 2b were then generated by Boltzmann
weighting of the four conformers (Figure 3). The CD spectrum
Figure 2. Thermal ellipsoid representation of 2. (Note: A different num-
bering system is used for the structural data deposited with the CCDC.)
The absolute configuration of 2 was also determined by
comparison of experimental and calculated ECD spectra,
which were generated for enantiomers (6S,7S,8S)-2 (2a) and
Figure 3. Experimental CD spectrum of 2 in MeOH and calculated
ECD spectra of two enantiomers, (6S,7S,8S)-2 (2a) and (6R,7R,8R)-2
(2b), after a UV correction of 20 nm.
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Table 2. NMR Data for 4−6
4
5
6
a
b
c
b
a
b
pos.
δC, mult.
δH (J in Hz)
δC, mult.
δH (J in Hz)
δC, mult.
δH (J in Hz)
1
162.8, qC
109.0, CH
139.2, CH
108.1, CH
162.1, qC
46.6, CH
86.0, CH
82.3, CH
88.0, qC
162.0, qC
108.8, CH
138.3, CH
107.8, CH
161.9, qC
43.3, CH
83.8, CH
77.7, CH
83.8, qC
162.8, qC
110.4, CH
139.3, CH
108.4, CH
162.0, qC
49.9, CH
79.5, CH
210.5, qC
79.9, qC
2
6.37, d (8.5)
7.38, t (8.5)
6.37, d (8.5)
6.41, d (8.0)
7.39, t (8.0)
6.41, d (8.0)
6.49, d (8.0)
7.43, t (8.0)
6.46, d (8.0)
3
4
4a
5
2.83, m
2.53, m
3.13, m
5a
4.82, dd (4.0, 8.0)
4.23, d (5.5)
4.91, dd (4.0)
4.29, d (7.5)
5.31, dd (4.0)
6
7
7a
52.8, CH
198.0, qC
109.4, qC
11.4, CH3
173.2, qC
52.8, CH3
3.98, d (8.0)
1.20, d (7.5)
56.1, CH
197.2, qC
109.7, qC
8.2, CH3
174.5, qC
52.5, CH3
3.45, d (4.0)
53.7, CH
196.2, qC
109.4, qC
7.8, CH3
171.5, qC
53.8, CH3
3.83, d (4.0)
1.33, d (7.0)
8
8a
9
1.27, d (7.0)
3.78, s
10
11
OH-1
OH-6
OH-7
3.79, s
11.94, s
4.77, s
3.82, s
11.83, s
11.69, s
d
4.53,
4.37,
s
s
d
4.54, br s
5.69, s
a
b
c
d
Recorded at 100 MHz in acetone-d6. Recorded at 500 MHz in acetone-d6. Recorded at 125 MHz in acetone-d6. These assignments are
interchangeable.
recorded for 2 matches the calculated ECD curve of 2a, but is
opposite to that of 2b. Therefore, 2 was deduced to have the
6S, 7S, 8S absolute configuration.
carbon, one carboxylic carbon (δC 173.2), and one α,β-unsatu-
rated ketone carbon (δC 198.0). Analysis of its NMR data
revealed the same 1,2,3-trisubstituted aryl ring with a hydroxy
group attached to C-1, as appeared in 1 and 2. The C-8 ketone
group (δC 198.0) was connected to C-8a on the basis of the
downfield 1H NMR chemical shift for OH-1 (δH 11.94) and the
four-bond W-type HMBC correlations from H-2 and H-4 to
C-8.4 The 1H−1H COSY NMR data showed the second isolated
spin-system of C-7a−C-5a−C-5/C-9−C-6 (including OH-6).
HMBC correlations from H-7a to C-7, C-8, and C-10 and from
H-6 to C-7, C-7a, and C-10 completed a cyclopentane ring that
was connected to C-8 and C-10 at C-7a and C-7, respectively.
An HMBC cross-peak from H3-11 to C-10 located the
O-methyl group at C-10. Considering the chemical shift values
of C-4a (δC 162.1) and C-5a (δC 86.0) and the unsaturation
requirement for 4, they were attached to the remaining oxygen
atom to complete the planar structure of 4 as shown.
The molecular formula of preussochromone C (3) was
determined to be C14H12O7 (nine degrees of unsaturation) on
1
the basis of its HRESIMS data. Analysis of the H and 13C
NMR data of 3 (Table 2) revealed significantly different
structural features from 1 and 2. On the basis of HMBC cross-
peaks from H-2 to C-1, C-4, C-8a, and C-94,11 and from the
intramolecularly hydrogen-bonded phenolic proton (δH 13.2)
to C-1, C-2, and C-8a, plus consideration of the chemical shift
value of C-4a (δC 146.4), an aryl ring was established with a
hydroxy group and an upfield carboxylic carbon (δC 159.1)
attached to C-1 and C-4, respectively. HMBC correlations from
the exchangeable proton at 5.80 ppm to C-4a, C-5, and C-10
located OH-5 at C-5 and enabled connection of C-5 to C-4a.
Further correlations from H-10a to C-4a and C-5 and from H-
10b to C-9 established a δ-lactone moiety fused to the aryl ring
at C-4/C-4a. In turn, correlations from H-10b to C-6 and from
H-7 to C-5, C-6, C-8, and C-8a indicated that the C-6/C-7
olefin is linked to C-5 and C-8, and C-8 is connected to C-8a,
completing the 3,3a-dihydrobenzo[de]isochromene-1,6-dione
skeleton, as found in corymbiferone.12 Finally, the remaining
two O-methyls were attached to C-3 and C-6, respectively, on
the basis of relevant HMBC cross-peaks.
The relative configuration of 4 was assigned by X-ray crystal-
lography (Figure 4), while the absolute configuration was deduced
The absolute configuration of the C-5 secondary alcohol was
deduced via the CD data of the in situ formed [Rh2(OCOCF3)4]
complex,13 with the inherent contribution subtracted. The Rh
complex of 3 showed a positive E band at ca. 350 nm (Figure
S14), correlating to the 5S absolute configuration by applying
the bulkiness rule.13,14
Preussochromone D (4) gave a pseudomolecular ion [M + H]+
peak by HRESIMS, indicating the molecular formula C15H16O7
(eight degrees of unsaturation). Its NMR data (Table 2)
showed three exchangeable protons (δH 4.54, 4.77, and 11.94,
respectively), two methyl groups including one O-methyl,
four methines with two oxygenated, six aromatic carbons (three
of which were protonated), one oxygenated sp3 quaternary
Figure 4. Thermal ellipsoid representation of 4. (Note: A different
numbering system is used for the structural data deposited with the
CCDC.)
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using the modified Mosher method.15 Treatment of 4 with
(S)- and (R)-MTPA Cl afforded the (R)- (4a) and (S)-MTPA
(4b) monoesters, respectively. The difference in chemical shift
values (Δδ = δS − δR) for 4b and 4a was calculated to assign the
6R configuration (Figure S16). Therefore, the 5S, 5aR, 6R, 7R,
7aR absolute configuration is proposed for 4.
cancer cells), and HCT116 (colon cancer cells) (Table 3).
Compounds 1 and 3 showed significant cytotoxic effects against
a
Table 3. Cytotoxicity of Compounds 1 and 3
IC50 (μM)
Preussochromone E (5) was assigned the same molecular
formula, C15H16O7 (eight degrees of unsaturation), as 4 by
HRESIMS. Interpretation of its 1H and 13C NMR spectroscopic
data (Table 2) revealed the same planar structure as 4, which
compound
A549
MCF-7
HeLa
HCT116
1
8.34 0.41
5.75 0.69
5.51 0.23
25.52 0.73
25.87 2.62
15.1 1.11
11.3 0.41
3
41.17 1.33 29.96 3.03
11.62 0.18 9.3 0.46
cisplatin
1
was supported by relevant H−1H COSY and HMBC data,
a
2 and 4−6 were inactive at 20 μg/mL.
suggesting that 5 is a stereoisomer of 4. The relative configu-
1
ration of 5 was deduced by analysis of H−1H coupling con-
A549 cells, with IC50 values of 8.34 and 5.75 μM, respectively,
while the positive control cisplatin showed an IC50 value of
5.51 μM. Other compounds did not show detectable cyto-
toxicity against the four cell lines at 20 μg/mL.
stants and NOESY data. The same coupling constant of 4.0 Hz
between H-5 and H-5a in 5 and 4, together with a NOESY
correlation of H-5a with H3-9, revealed a cis relationship for H-
5a and H3-9. A coupling constant of 4.0 Hz observed for H-7a
in 5, compared to 8.0 Hz for the same proton in 4, suggested a
trans relationship for H-5a and H-7a, which was also supported
by a NOESY correlation of H-5 with H-7a. A coupling constant
of 7.5 Hz observed for H-6 (5.5 Hz in 4) and the NOESY
correlations of H-6 with H-5a and H3-9 established a trans
relationship for H-6 and H3-9.16 A NOESY correlation of H-6
with H3-11 placed these protons on the same face of the
cyclopentane ring, thereby allowing determination of the
relative configuration of 5.
Preussochromone (1) represents the first example of the
naturally occurring thiopyranchromenone class of compounds
possessing the 3,4-dihydrothiopyrano[2,3-b]chromen-
5(2H)-one skeleton, which was previously found only in syn-
thetic products.8,20 Initially, the prenylated coumarins from
the plant Ferula communis21 showed significant activity against
Mycobacterium tuberculosis, promoting synthesis of their sulfur
isosters (e.g., 8 and 9)8,20 for evaluations. Due to assay limita-
tions, 1 was not tested for its antituberculosis activity. Com-
pound 2 is a new member of the xanthone class, which has
been encountered frequently as the bioactive principles of
plants and fungi.22 However, 2 differs from the known ana-
logues by having different substituents on the aryl and cyclo-
hexene rings. Compound 3 is closely related to corymbiferone,
an antioxidant isolated from Penicillium hordei,12 simonyellin, a
phenylbenzoisochromenone isolated from the lichen Simonyella
variegata,23 and a few other natural products,24 but differs from
the precedents by having different substituents at C-1, C-3, C-5,
C-6, C-7, and C-10. Compounds 4−6 belong to a relatively rare
type of fungal metabolite incorporating the cyclopentabenzopyran-
9-one skeleton. Other examples of this class of natural products
include wrightiadione,25 coniochaetones A and B,26,27 pseudo-
bruceol I,28 remisporine A,29 and diaportheones A and B,30 but
4−6 differ from the above-mentioned compounds by having
either different substitution patterns or substituents on the aryl
and cyclopentane rings.
The absolute configuration of the tert/sec 6,7-diol moiety in
5 was assigned using the in situ dimolybdenum CD method,
developed by Snatzke and Frelek.17,18 Upon addition of di-
molybdenum tetraacteate [Mo2(OAc)4] to 5 in DMSO solu-
tion, a metal complex was generated as an auxiliary chromo-
phore. Since the contribution from the inherent CD resulting
from the C-8 and C-10 carbonyls was subtracted to give the
induced CD of the complex, the observed sign of the Cotton
effect in the induced spectrum originates solely from the
chirality of the vic-diol moiety expressed by the sign of the O−
C−C−O torsion angle. The positive Cotton effect observed at
around 310 and 400 nm, respectively, in the induced CD spec-
trum (Figure S15) permitted deduction of the 6S and 7R abso-
lute configuration on the basis of the empirical rule proposed
by Snatzke. Combining the relative configuration established, 5
was assigned the 5S, 5aR, 6S, 7R, 7aS absolute configuration.
Preussochromone F (6) was isolated as a yellow powder with
a molecular formula of C15H14O7 (nine degrees of unsatura-
tion), established by HRESIMS. The 1H and 13C NMR spectra
of 6 showed resonances similar to those of 4 and 5, except
that the C-6 oxymethine (δH/δC 4.23/82.3) was replaced by a
ketone carbon (δC 210.5), which was confirmed by HMBC
correlations from H-5a, H-7, and H3-9 to C-6. The relative
configurations at C-5, C-5a, C-7, and C-7a in 6 were established
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured on a Perkin-Elmer 241 polarimeter, and UV data were
obtained on a Shimadzu Biospec-1601 spectrophotometer. CD spectra
were recorded on a JASCO J-815 spectropolarimeter. IR data were
1
recorded using a Nicolet Magna-IR 750 spectrophotometer. H and
1
to be the same as those in 5 by analysis of H−1H coupling
13C NMR data were acquired with Varian Mercury-400, Inova-500,
and NMR system-600 spectrometers using solvent signals (acetone-d6:
δH 2.05/δC 29.8, 206.1; DMSO-d6: δH 2.50/δC 39.5) as references.
The HMQC and HMBC experiments were optimized for 145.0 and
8.0 Hz, respectively. ESIMS and HRESIMS data were obtained using
an Agilent Accurate-Mass-Q-TOF LC/MS 6520 instrument equipped
with an electrospray ionization (ESI) source. The fragmentor and
capillary voltages were kept at 125 and 3500 V, respectively. Nitrogen
was supplied as the nebulizing and drying gas. The temperature of
the drying gas was set at 300 °C. The flow rate of the drying gas and
the pressure of the nebulizer were 10 L/min and 10 psi,
respectively. All MS experiments were performed in positive ion
mode. Full-scan spectra were acquired over a scan range of m/z
100−1000 at 1.03 spectra/s.
constants and NOESY data for relevant protons, whereas the
absolute configuration of 6 was deduced by a semisynthetic
method.19 Specifically, treatment of 5 with manganese dioxide
1
afforded an oxidation product of OH-6, with its H NMR data
and specific rotation value identical to those of 6, suggesting the
5R, 5aR, 7R, 7aS absolute configuration for 6.
The remaining known compound 7 was readily identified as
2,8-dihydroxy-3-methyl-9-oxoxanthene-1-carboxylic acid methyl
ester by comparison of its NMR and MS data with those reported.7
Compounds 1−6 were tested for cytotoxicity against the
following four human tumor cell lines, HeLa (cervical epithelial
cells), A549 (lung carcinoma epithelial cells), MCF-7 (breast
234
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Journal of Natural Products
Article
Fungal Material. The culture of Preussia africana (Sporormiaceae)
was isolated from the lichen Ramalina calicaris (L.) Fr. (Ramalinaceae)
collected from Zixi Mountain, Yunnan Province, People’s Republic of
China, in November 2006. The isolate was identified by one of the
authors (L.G.) based on morphology and sequence (Genbank
Accession No. JQ031265) analysis of the ITS region of the rDNA
and assigned the accession number 90-1-6-2 in L.G.’s culture
collection at the Institute of Microbiology, Chinese Academy of
Sciences, Beijing. The fungal strain was cultured on slants of potato
dextrose agar at 25 °C for 10 days. Agar plugs were cut into small
pieces (about 0.5 × 0.5 × 0.5 cm3) under aseptic conditions, and 15
pieces were used to inoculate three Erlenmeyer flasks (250 mL), each
containing 50 mL of media (0.4% glucose, 1% malt extract, and 0.4%
yeast extract); the final pH of the media was adjusted to 6.5 and
sterilized by autoclave. Three flasks of the inoculated media were
incubated at 25 °C on a rotary shaker at 170 rpm for five days to
prepare the seed culture. Spore inoculum was prepared by suspen-
sion in sterile, distilled H2O to give a final spore/cell suspension of
1 × 106/mL. Fermentation was carried out in eight Fernbach flasks
(500 mL), each containing 80 g of rice. Distilled H2O (120 mL) was
added to each flask, and the contents were soaked overnight before
autoclaving at 15 psi for 30 min. After cooling to room temperature,
each flask was inoculated with 5.0 mL of the spore inoculum and
incubated at 25 °C for 40 days.
was separated from the sample and mounted on a glass fiber, and data
were collected using a Rigaku RAXIS RAPID IP diffractometer with
graphite-monochromated Mo Kα radiation, λ = 0.71073 Å at 293(2) K.
́
Crystal data: C16H16O7, M = 320.29, space group orthorhombic,
́
́
P2(1)2(1)2(1); unit cell dimensions a = 8.5972(17) Å, b = 10.312(2) Å,
c = 16.512(3) Å, V = 1463.9(5) Å , Z = 4, Dcalcd = 1.453 mg/m3, μ =
0.115 mm−1, F(000) = 672. The structure was solved by direct
methods using SHELXL-9732 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 positions and refined as riding atoms
with the relative isotropic parameters. Absorption corrections were
performed using the Siemens Area Detector Absorption Program
(SADABS).33 The 1933 measurements yielded 1933 independent
reflections after equivalent data were averaged, and Lorentz and
polarization corrections were applied. The final refinement gave R1 =
0.0392 and wR2 = 0.0605 [I > 2σ(I)].
3
́
́
Preussochromone C (3): yellow, amorphous solid; [α]25D +174.5 (c
0.11, MeOH); UV (MeOH) λmax (log ε) 239 (3.32), 341 (3.19), 376
(3.25) nm; CD (c 3.4 × 10−4 M, CH2Cl2) λmax (Δε) 329 (+1.1) nm,
293 (−1.0) nm, 255.5 (+4.6) nm; IR (neat) νmax 3434 (br), 2924,
1709, 1646, 1587, 1460, 1371, 1252, 1210, 1068 cm−1; H and 13C
1
NMR data see Table 1; HMBC data (acetone-d6, 500 MHz) H-2 →
C-1, 4, 8a, 9; H-7 → C-5, 6, 8, 8a; H-10a → C-4a, 5; H-10b → C-4a, 5,
6, 8a, 9; H3-11 → C-3; H3-12 → C-6; OH-1 → C-1, 2, 8a; OH-5 →
C-4a, 5, 10; HRESIMS m/z 293.0652 (calcd for C14H13O7, 293.0656).
Absolute Configuration of the Tertiary Alcohol in 3 (refs 13, 14).
A sample of 3 (0.5 mg) was dissolved in a dry solution of [Rh2-
(OCOCF3)4] complex (1.5 mg) in CH2Cl2 (200 μL). The first CD
spectrum was recorded immediately after mixing, and its time evolu-
tion was monitored until stationary (ca. 10 min after mixing). The
inherent CD was subtracted. The observed sign of the E band at ca.
350 nm in the induced CD spectrum was correlated to the absolute
configuration of the C-5 tertiary moiety.
Extraction and Isolation. The fermented material was extracted
repeatedly with EtOAc (4 × 1.0 L), and the organic solvent was evap-
orated to dryness under vacuum to afford the crude extract (3.0 g),
which was fractionated by silica gel vacuum liquid chromatography
using petroleum ether−EtOAc gradient elution. The fraction (100
mg) eluted with 10% EtOAc was separated again by Sephadex LH-20
column chromatography (CC) using 1:1 CH2Cl2−MeOH as eluents,
and the resulting subfractions were further purified by RP HPLC
(Agilent Zorbax SB-C18 column; 5 μm; 9.4 × 250 mm; 50−75%
MeOH in H2O over 40 min; 2 mL/min) to afford 7 (2.0 mg, tR 35.9
min). Fractions (350 mg) eluted with 12, 15, and 18% EtOAc were
fractionated again by Sephadex LH-20 CC using 1:1 CH2Cl2−MeOH
as eluent. The resulting subfractions were further purified by semi-
preparative RP HPLC (Agilent Zorbax SB-C18 column; 5 μm; 9.4 ×
250 mm; 43% MeOH in H2O for 40 min; 2 mL/min) to afford 6
(8.0 mg, tR 34.0 min). Fractions eluted with 20 (97 mg), 25 (160 mg),
35 (250 mg), and 40 and 45% (300 mg) EtOAc were individually
separated by Sephadex LH-20 CC, eluting with 1:1 CH2Cl2−MeOH.
Further purification of the resulting subfractions by RP HPLC (Agilent
Zorbax SB-C18 column; 5 μm; 9.4 × 250 mm) afforded 1 (2.0 mg, tR
36.0 min; 29% CH3CN in H2O for 40 min; 2 mL/min) and 5 (1.8 mg,
tR 39.9 min; the same gradient as in purification of 1), 4 (10.0 mg, tR
32.4 min; 27% CH3CN in H2O for 2 min, followed by 27−30% over
33 min; 2 mL/min), 2 (3.0 mg, tR 22.9 min; 44% MeOH in H2O for
30 min; 2 mL/min), and 3 (10.5 mg, tR 20.7 min; 35% MeOH in H2O
for 2 min, followed by 35−50% over 28 min; 2 mL/min), respectively.
Preussochromone D (4): white needles (acetone); mp 162−163 °C;
[α]25 −28.2 (c 0.17, MeOH); UV (MeOH) λmax (log ε) 300
D
(3.24), 338 (3.24), 375 (3.32) nm; IR (neat) νmax 3494 (br), 2972,
1
1707, 1644, 1627, 1460, 1375, 1279, 1211, 1079 cm−1; H and 13C
NMR data see Table 2; HMBC data (acetone-d6, 500 MHz) H-2 →
C-1, 4, 8, 8a; H-3 → C-1, 2, 4; H-4 → C-2, 8; H-5 → C-5a, 6, 7, 9;
H-5a → C-6, 7, 9; H-6 → C-5a, 7, 7a, 9, 10; H-7a → C-5a, 7, 8, 10;
H3-9 → C-5, 5a, 6; H3-11 → C-10; OH-1 → C-1, 2, 3; HRESIMS m/z
309.0968 (calcd for C15H17O7, 309.0969).
X-ray Crystallographic Analysis of 4 (ref 34). Upon
crystallization from acetone−H2O (20:1) using the vapor diffusion
method, colorless crystals were obtained for 4. A crystal (0.56 × 0.14 ×
0.14 mm) was separated from the sample and mounted on a glass
fiber, and data were collected using a Rigaku RAPID IP diffractometer
́
with graphite-monochromated Mo Kα radiation, λ = 0.71073 Å at
173(2) K. Crystal data: C15H16O7, M = 308.28, space group mono-
Preussochromone A (1): white powder; [α]25 −232.0 (c 0.10,
clinic, P2(1); unit cell dimensions a = 9.2581(19) Å, b = 5.2295(10) Å,
́
́
D
3
c = 14.650(3) Å, V = 698.9(2) Å , Z = 2, Dcalcd = 1.465 mg/m3, μ =
0.117 mm−1, F(000) = 324. The structure was solved by direct
methods using SHELXL-9732 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 positions and refined as riding atoms
with the relative isotropic parameters. Absorption corrections were
performed using SADABS.33 The 5776 measurements yielded 1597
independent reflections after equivalent data were averaged, and Lorentz
and polarization corrections were applied. The final refinement gave R1 =
0.0483 and wR2 = 0.1497 [I > 2σ(I)].
́
́
MeOH); UV (MeOH) λmax (log ε) 240 (3.49), 347 (3.36), 368 (3.26)
nm; CD (c 1.5 × 10−3 M, MeOH) λmax (Δε) 207.5 (−2.81), 241
(+1.90), 262 (−6.61), 289.5 (−4.14) nm ; IR (neat) νmax 3466 (br),
1
2954, 1735, 1646, 1601, 1471, 1413, 1280, 1093 cm−1; H, 13C NMR
and HMBC data see Table 1; HRESIMS m/z 361.0357 (calcd for
C15H14O7SNa, 361.0352).
Preussochromone B (2): white needles (MeOH); mp 166−167 °C;
[α]25 −71.9 (c 0.16, MeOH); UV (MeOH) λmax (log ε) 239 (3.25),
D
341 (3.12), 350 (3.15) nm; CD (c 1.6 × 10−3 M, MeOH) λmax (Δε)
212.5 (−7.02), 252.5 (−1.79), 266.5 (−2.22), 326.5 (−0.83) nm; IR
(neat) νmax 3507 (br), 2956, 2916, 1748, 1651, 1622, 1596, 1475,
1
1349, 1233, 1083 cm−1; H and 13C NMR data see Table 1; HMBC
Preparation of (R)- (4a) and (S)-MTPA (4b) Esters. A sample of
4 (1.3 mg, 0.004 mmol), (S)-MPTA Cl (2.5 μL, 0.013 mmol), and
pyridine-d5 (0.5 mL) were allowed to react in an NMR tube at
ambient temperature for 12 h. The mixture was purified by RP HPLC
(Agilent Zorbax SB-C18 column; 5 μm; 9.4 × 250 mm; 4.6 × 250 mm;
55% MeOH in H2O for 2 min, followed by 55−100% for 33 min) to
correlations (acetone-d6, 500 MHz) H-2 → C-1, 4, 9a; H-3 → C-1, 4a;
H-4 → C-2, 4a; H2-5 → C-5a, 6, 7, 8a, 10; H-7 → C-5, 6, 8a, 10;
H-8 → C-5a, 7, 8a, 11; H3-10 → C-5, 5a, 7; H3-12 → C-11; OH-1 →
C-1; HRESIMS m/z 321.0964 (calcd for C16H17O7, 321.0969).
X-ray Crystallographic Analysis of 2 (ref 31). Upon
crystallization from MeOH−H2O (30:1) using the vapor diffusion method,
colorless crystals were obtained for 2. A crystal (0.40 × 0.30 × 0.20 mm)
1
afford 4a (0.8 mg, tR 27.6 min): white powder; H NMR (acetone-d6,
500 MHz) δ 11.77 (1H, s, OH-1), 7.45 (1H, t, J = 8.5 Hz, H-3), 6.46
235
dx.doi.org/10.1021/np2009362 | J. Nat. Prod. 2012, 75, 230−237

Journal of Natural Products
Article
(2H, d, J = 8.5 Hz, H-2/H-4), 5.54 (1H, d, J = 3.0 Hz, H-6), 4.94 (1H,
dd, J = 3.0, 7.0 Hz, H-5a), 4.00 (1H, d, J = 7.0 Hz, H-7a), 3.74 (3H, s,
H3-11), 3.19 (1H, m, H-5), 0.97 (3H, d, J = 7.5 Hz, H3-9).
Similarly, a sample of 4 (0.7 mg, 0.002 mmol), (R)-MPTA Cl
(2.5 μL, 0.013 mmol), and pyridine-d5 (0.5 mL) were allowed to react
in an NMR tube at ambient temperature for 5 h, and the reaction
mixture was processed as described above for 4a to afford 4b (0.5 mg):
white powder; 1H NMR (acetone-d6, 500 MHz) δ 11.78 (1H, s,
OH-1), 7.45 (1H, t, J = 8.5 Hz, H-3), 6.46 (2H, d, J = 8.5 Hz, H-2/
H-4), 5.61 (1H, d, J = 3.0 Hz, H-6), 4.98 (1H, dd, J = 3.0, 7.0 Hz,
H-5a), 4.00 (1H, d, J = 7.0 Hz, H-7a), 3.65 (3H, s, H3-11), 3.24 (1H,
m, H-5), 1.23 (3H, d, J = 7.5 Hz, H3-9).
solution of a compound in DMSO and serial dilutions; the test
compounds showed good solubility in DMSO and did not precipitate
when added to the cells). The plate was incubated for 48 h at 37 °C in
a humidified, 5% CO2 atmosphere. Proliferation was assessed by
adding 20 μL of MTS (Promega) to each well in the dark, followed by
a 90 min incubation at 37 °C. The assay plate was read at 490 nm
using a microplate reader.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra of 1−6, CD spectrum of Rh complex
of 3, CD spectrum of 5 in DMSO containing Mo2(OAc)4, and
UV and CD calculations for 1 and 2. This material is available
free of charge via the Internet at http://pubs.acs.org.
Preussochromone E (5): white powder; [α]25 +30.0 (c 0.10,
D
MeOH); UV (MeOH) λmax (log ε) 298 (3.28), 334 (3.28), 371
(3.45); CD (c 3.2 × 10−4 M, DMSO) λmax (Δε) 272 (−3.34), 344
(+0.04) nm; IR (neat) νmax 3380 (br), 2955, 1737, 1639, 1626, 1463,
1
1463, 1365, 1222, 1061 cm−1; H and 13C NMR data see Table 2;
AUTHOR INFORMATION
■
HMBC data (acetone-d6, 500 MHz) H-2 → C-1, 4; H-3 →C-1, 2, 4;
H-4 →C-2, 8; H-5 →5a, 6, 7, 9; H-5a → C-6, 7; H-6 → C-7, 10; H-7a
→ C-7, 8, 10; H3-9 → C-5, 5a, 6; H3-11 → C-10; NOESY correlations
(acetone-d6, 500 MHz) H-5 ↔ H-7a; H-5a ↔ H3-9; H-6 ↔ H-5a, H3-
9, H3-11; HRESIMS m/z 309.0966 (calcd for C15H17O7, 309.0969).
Absolute Configuration of the 6,7-Diol Moiety in 5 (refs 17, 18).
HPLC grade DMSO was dried with 4 Å molecular sieves. According to
the published procedure,35 a mixture of 1:1.3 diol/Mo2(OAc)4 for 5
was subjected to CD measurements at a concentration of 0.5 mg/mL.
The first CD spectrum was recorded immediately after mixing, and its
time evolution was monitored until stationary (about 10 min after
mixing). The inherent CD was subtracted. The observed signs of the
diagnostic bands at around 310 and 400 nm in the induced CD
spectrum were correlated to the absolute configuration of the 6,7-diol
moiety.
Corresponding Author
*Tel/Fax: +86 10 82618785. E-mail: cheys@im.ac.cn.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge financial support from the National
Natural Science Foundation of China (30925039), the Beijing
Natural Science Foundation (5111003), the Ministry of
Science and Technology of China (2010ZX09401-403 and
2012ZX09301-003), and the Chinese Academy of Sciences
(KSCX2-EW-G-6).
Preussochromone F (6): yellow powder; [α]25 +7.0 (c 0.10,
D
MeOH); UV (MeOH) λmax (log ε) 298 (3.11), 319 (3.21), 340 (3.19)
nm; IR (neat) νmax 3433 (br), 2941, 1755, 1656, 1625, 1583, 1471,
DEDICATION
■
1
1356, 1215, 1139, 1059 cm−1; H and 13C NMR data see Table 2;
Dedicated to Professor Jiangchun Wei on the occasion of his
80th birthday.
HMBC data (acetone-d6, 400 MHz) H-2 → C-1, 4, 8; H-3 → C-1; H-
4 → C-2; H-5 → C-5a, 6, 9; H-5a → C-6, 7; H-7a → C-5a, 8; H3-9 →
C-5, 5a, 6; H3-11 → C-10; OH-1 → C-1, 2; OH-7 → C-6, 7, 7a, 10;
NOESY correlation (acetone-d6, 500 MHz) H-5a ↔ H3-9, H3-11;
HRESIMS m/z 307.0808 (calcd for C15H15O7, 307.0812).
REFERENCES
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