Bioactive anthraquinone derivatives from the mangrove-derived fungus stemphylium sp. 33231

Article abstract of DOI:10.1021/np500340y

Four new anthraquinone derivatives (1-4) and four new alterporriol-type anthranoid dimers (14-17), along with 17 analogues, were isolated from the solid rice fermentation of the fungus Stemphylium sp. 33231 obtained from the mangrove Bruguiera sexangula v

Full text of DOI:10.1021/np500340y

Article
pubs.acs.org/jnp
Bioactive Anthraquinone Derivatives from the Mangrove-Derived
Fungus Stemphylium sp. 33231
Xue-Ming Zhou,† Cai-Juan Zheng,† Guang-Ying Chen,* Xiao-Ping Song, Chang-Ri Han, Gao-Nan Li,
Yan-Hui Fu, Wen-Hao Chen, and Zhi-Gang Niu
Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, Hainan Normal University, Haikou 571158, People’s
Republic of China
S
* Supporting Information
ABSTRACT: Four new anthraquinone derivatives (1−4) and four new alterporriol-type anthranoid dimers (14−17), along with
17 analogues, were isolated from the solid rice fermentation of the fungus Stemphylium sp. 33231 obtained from the mangrove
Bruguiera sexangula var. rhynchopetala collected from the South China Sea. Their structures were elucidated using comprehensive
spectroscopic methods. The absolute configurations of 1, 3, and 4 were determined by single-crystal X-ray diffraction of their
derivatives (1a, 3b, and 4a). The absolute configurations of the chiral 17−19 were determined by comparing their CD spectra with
21. The inhibitory activities of most of the compounds against seven terrestrial pathogenic bacteria and two cancer cell lines were
evaluated.
ndophytic fungi are well known as rich sources of new
Enatural products with promising biological and pharmaco-
logical activities.1−3 Fungi in the genus Stemphylium produce
various bioactive metabolites. Among them, the anthraquinone
derivatives possess an especially wide range of biological activ-
ities.4,5 For instance, the tetrahydroanthraquinone altersolanol
A has shown cytotoxic activity against the K562 cell line,6 while
anthranoid dimer alterporriol D showed selective inhibition of
bacteria.7 In our investigation of natural antibacterial and cyto-
toxic products from mangrove fungi in the South China Sea,
two new antibacterial α-pyrone derivatives were obtained from
Stemphylium sp. 33231 in potato glucose liquid medium.8 In
order to search for additional bioactive natural products from
Stemphylium sp. 33231, the culture condition was changed to a
solid rice fermentation. The EtOAc extract of the fungal culture
exhibited cytotoxic activity against the A549 cell lines and
antibacterial activity against Staphylococcus albus. Bioassay-
guided fractionation of the EtOAc extract led to the isolation
of four new anthraquinone derivatives, auxarthrol C (1), macro-
sporin 2-O-(6′-acetyl)-a-D-glucopyranoside (2), 2-O-acetylal-
tersolanol B (3), and 2-O-acetylaltersolanol L (4), and four
alterporriol-type anthranoid dimers, alterporriols T−W (14−17).
Also 17 known analogues were isolated: dihydroaltersolanol A
(5),9 macrosporin (6),10 macrosporin-7-O-sulfate (7),11 altersolanols
A−C (8−10)9,10,12−14 and L (11),15 ampelanol (12),11 tetra-
hydroaltersolanol B (13),16 and alterporriols A, B, D, and E
(18−21)14,17−19 and C, N, R, and Q (22−25).9 Herein, we re-
port the isolation, structure elucidation, and biological activities
of these compounds.
RESULTS AND DISCUSSION
■
Auxarthrol C (1) was obtained as a white powder, with the mole-
cular formula C16H16O9 (nine degrees of unsaturation) from
HRESIMS data combined with 1H and 13C NMR spectroscopic
data (Tables 1 and 2). In the 1H NMR spectrum one hydrogen-
bonded hydroxy group at δH 11.14 (s), two meta-coupled
aromatic hydrogens at δH 6.91 (d, J = 2.4 Hz) and 6.84 (d, J =
2.4 Hz), three oxygenated methine signals at δH 4.43 (d, J = 7.2),
4.41 (dd, J = 7.8, 6.0 Hz), and 3.27 (dd, J = 7.8, 6.6 Hz), one
methoxy group at δH 3.88 (s), and one singlet methyl group
at δH 1.12 (s) were observed. In the 13C NMR spectrum,
two carbonyl carbons (δC 193.4 and 191.0), six olefinic carbons
(δC 165.2, 161.8, 134.3, 109.8, 106.8, and 105.8), six O-bearing
carbons (δC 73.8, 71.6, 68.2, 67.4, 67.0, and 66.9), one methoxy
Received: April 16, 2014
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
dx.doi.org/10.1021/np500340y | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Chart 1
carbon (δC 56.2), and one methyl carbon (δC 21.6) were
observed. These spectroscopic features suggested that 1 has a
hydroanthraquinone skeleton. The H NMR data (Table 1)
with a Flack parameter of 0.02(5). Thus, the absolute configu-
ration of 1 was determined as 1R,2R,3R,4R,1aS,4aR (Figure 1).
Compound 2 was obtained as a yellow powder, with the
molecular formula C24H24O11 calculated from HRESIMS data.
The 1H and 13C NMR spectroscopic data (Tables 3 and 4) indi-
cated that 2 was composed of one macrosporin (6)10 subunit,
one glucose subunit, and one acetoxy group. The location of
the glucose at C-2 and the acetoxy group at C-6′ was confirmed
by the HMBC correlation between H-1′ and C-2, and H-6′ and
C-7′. The glycone part of 2, a glucose moiety, was identified and
characterized by the presence of six carbon signals (δC 97.7, 72.9,
71.3, 71.1, 70.0, 63.3) and a signal for one anomeric proton
doublet at δH 5.71 (J = 3.6 Hz; H-6′), respectively. The large
1
were very similar to those of auxarthrol A,20 except for the
absence of a C-1 methylene signal and the presence of a hydroxy
signal δH 5.19 (d, J = 6.0 Hz) and a methine signal δH 4.41 (dd,
J = 7.8, 6.0 Hz) for C-1 in 1. These data indicated that one proton
on the methylene in auxarthrol A was replaced by a hydroxy in 1.
This was referred by the 13C NMR spectrum (Table 2), where the
C-1 signal moved downfield to δC 67.0 (CH) in 1 compared to δC
35.9 (CH2) in auxarthrol A. The large 3JH‑1, H‑2 = 8.4 Hz showed
that H-1 and H-2 have a trans diaxial relationship. The NOESY
correlation of H-2/3-Me indicated that H-2 and 3-Me should
be placed on the same face. The relative configuration of the
remaining stereocenters of 1 could not be firmly established due
to the lack of evidence. To complete the assignment, an X-ray
crystal structure was desired. However, single crystals of 1 were
not obtained. To improve the crystallinity, many attempts were
made to structurally modify 1. Ultimately, opening of the epoxide
with CH3O−Na+−CH3OH yielded 1a (Supporting Information,
Figure S67).21 Crystallization of 1a from CH3OH−H2O (10:1)
resulted in colorless crystals, which gave an X-ray crystal structure
3
3
3JH‑2′, H‑3′ = 9.2 Hz, JH‑3′, H‑4′ = 9.6 Hz, and JH‑4′, H‑5′ = 9.6 Hz
revealed the axial−axial relationship for these protons. These
data suggested that the glycosyl moiety was α-glucose. Acid
hydrolysis of 2 produced glucose as the sole sugar identified
on the basis of TLC by comparing with an authentic sugar
sample. The glucose isolated from the hydrolysate gave a posi-
tive optical rotation, [α]24 +45 (c 0.2, H2O), and indicated
D
that it is D-glucose. Therefore, the glycosyl moiety was an
B
dx.doi.org/10.1021/np500340y | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1
Table 1. H NMR Data for 1 (600 MHz), 3, 4, and 16 (400
MHz) (δ in ppm, J in Hz)
a
b
a
b
position
1
1
3
4
16
4.41, dd
(7.8, 6.0)
2.79, dd (19.2, 1.59, dd
7.6 H-1ax) (12.4, 12.0 H-1ax)
3.04, dd (19.2, 2.11, ddd
5.6 H-1eq)
(12.4, 4.8, 4.0 H-1eq)
2
4
3.27, dd
(7.8, 6.6)
5.00, dd
(7.6, 5.6)
4.90 dd (12.0, 4.8)
3.84, d (2.4)
4.43, d (7.2) 2.68, d
(19.2 H-4ax)
2.91, d
(19.2 H-4eq)
8.16, br s
5
6.91, d (2.4) 7.14, d (2.4)
6.84, d (2.4) 6.60, d (2.4)
6.76, dd (2.4, 1.2)
6.34, d (2.4)
7.24, d (2.4)
6.64, d (2.4)
7
10
1a
4.66, dd (10.8, 6.8)
2.76, ddd
(12.4, 12.0, 4.0)
Figure 1. X-ray structure of 1a.
4a
2.25, ddd
(12.4, 10.8, 2.4)
was confirmed by the HMBC correlation between H-2 and C-
11. In the NOESY spectrum 3-Me showed a correlation to H-2,
indicating that 3-Me and H-2 should be placed on the same
face. To assign the absolute configuration of 3, a hydrolysis reac-
tion was adopted for 3 to obtain 3a. Furthermore, altersolanol B
2,3-O-acetonide (3b) was prepared from 3a according to a
previously reported method.18 Finally, an X-ray crystal structure of
3b with a Flack parameter of 0.02(11) was obtained (Figure 2).
Ultimately, the absolute configuration of 3 was established
as 2R,3S.
12
2.14, s
2.05, s
1.15, s
3.82, s
3-Me
1.12, s
1.34, s
3.89, s
2.43, s
3.95, s
6-OMe 3.88, s
1-OH
2-OH
3-OH
4-OH
8-OH
10-OH
5.19, d (6.0)
4.66, d (6.6)
4.55, s
4.57, s
5.50, d (7.2)
11.14, s
5.29, d (6.0)
12.86, s
12.21, s
12.63, s
5.67, d (6.8)
a
b
DMSO-d6. Acetone-d6.
2-O-Acetylaltersolanol L (4) was obtained as a white powder.
Its molecular formula of C18H22O8 (eight degrees of unsaturation)
Table 2. 13C NMR Data for 1 (125 MHz), 3, 4, and 16
(100 MHz) (δ in ppm)
1
was determined by HRESIMS. Careful comparison of the H
NMR and 13C NMR spectra of 4 (Tables 1 and 2) with those
of altersolanol L (11)15 showed a close structural relationship
except for the presence of an acetoxy group (δC 170.3, 21.0 and
δH 2.05, s) in 4. The location of the acetoxy group at C-2 was
established by the HMBC correlation between H-2 and C-11. The
a
b
a
b
position
1
3
4
16
1
67.0, CH
71.6, CH
68.2, C
26.6, CH2
73.7, CH
69.5, C
26.0, CH2
73.5, CH
71.3, C
128.5, C
159.9, C
125.5, C
131.1, CH
107.4, CH
167.0, C
106.6, CH
166.2, C
189.2, C
182.1, C
133.0, C
132.1, C
111.8, C
135.9, C
2
3
3
3
4
66.9, CH
106.8, CH
165.2, C
105.8, CH
161.8, C
193.4, C
191.0, C
67.4, C
36.1, CH2
108.0, CH
166.0, C
106.1, CH
164.3, C
187.3, C
183.4, C
141.4, C
142.1, C
109.5, C
133.6, C
170.6, C
21.2, CH3
25.6, CH3
56.1, CH3
72.3, CH
104.3, CH
165.9, C
99.0, CH
164.5, C
203.6, C
66.0, CH
41.2, CH
45.7, CH
108.9, C
152.0, C
170.3, C
21.0, CH3
23.1, CH3
55.7, CH3
large JH‑10, H‑4a = 10.8 Hz and JH‑4a, H‑1a = 12.4 Hz showed that
H-10 and H-4a/H-4a and H-1a have trans diaxial relationships,
which was corroborated by the correlations of H-1a with H-10
and H-2 and H-2 with 3-Me in the NOESY spectrum. To assign
the full relative and absolute configuration of 4, a hydrolysis reac-
tion was adopted for 4 to obtain 4a (Figure S69).9 Crystallization
of 4a from CH3OH−CHCl3 (1:1) resulted in colorless crystals,
which gave an X-ray crystal structure with a Flack parameter of
−0.03(15). Thus, the absolute configuration of 4 was established
as 2R,3S,4R,1aS,4aS,10R (Figure 3).
5
6
7
8
9
10
1a
4a
73.8, C
9a
109.8, C
134.3, C
10a
11
12
3-Me
6-OMe
Alterporriol T (14) was obtained as an orange, amorphous
powder, with the molecular formula C32H30O13 from HRESIMS
data. The 1H and 13C NMR spectroscopic data (Tables 3 and 4)
indicated that 14 was comprised one altersolanol C (10)9 sub-
21.6, CH3
56.2, CH3
17.3, CH3
56.6, CH3
9
1
unit and one altersolanol B (9) subunit. In the H NMR
spectrum of 14, two aromatic proton signals were assigned to
H-7 in the altersolanol C moiety at δH 6.87 (s) and to H-5′ in
the altersolanol B moiety at δH 7.27 (s). In anthranoid dimer
systems the two meta-coupled aromatic protons H-5 (H-5′) and
H-7 (H-7′) differ considerably with regard to their chemical
shifts, with H-5 (H-5′) consistently resonating more downfield
(∼7.2 ppm) than H-7 (H-7′, ∼6.8 ppm).22 This observation
proved vital to assign the position of the intersubunit linkage in
14. Therefore, 14 was lacking a proton signal at C-5 in the
aromatic altersolanol C moiety and at C-7′ in the altersolanol
B moiety. Furthermore, the HMBC spectrum showed that
H-7 correlated to C-6 and C-8, while H-5′ correlated to C-7′
a
b
DMSO-d6. Acetone-d6.
α-D-glucopyranoside, and the absolute configuration of 2 is as
shown.
2-O-Acetylaltersolanol B (3) was obtained as an orange-
yellow powder. Its molecular formula of C18H18O7 (10 degrees
of unsaturation) was determined by HRESIMS. The mole-
cular formula was also corroborated by H and 13C NMR
1
spectroscopic data (Tables 1 and 2). Its H and 13C NMR
1
spectra closely resembled those of altersolanol B (9)9 except
for the presence of one acetoxy signal (δC 170.6, 21.2 and
δH 2.14, s) in 3. The location of the acetoxy group at C-2
C
dx.doi.org/10.1021/np500340y | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1
Table 3. H NMR Data for 2, 14, 15, and 17 (400 MHz) (δ in ppm)
a
b
b
a
c
position
1
2
14
15
17
17
7.87, s
2.53, dd (19.2, 9.2 H-1ax)
2.98, dd (19.2, 6.0 H-1eq)
3.94, m
2.73−2.90, m
2
4
3.80, dd (5.6, 5.2)
2.68, dd (19.2, 3.2)
2.76, dd (19.2, 2.4)
8.01, br s
4.38, d (6.0)
7.88, br s
8.10, d (0.4)
5
7.18, d (2.8)
6.86, d (2.8)
2.39, br s
3.93, s
7.11, d (2.8)
6.64, d (2.8)
2.10, s
7.26, d (2.8)
6.63, d (2.8)
3.91, s
7
6.87, s
6.86, s
1.22, s
3.84, s
3-Me
6-OMe
4-OH
1′
1.39, s
3.84, s
3.88, s
2.34, d (0.4)
4.43, d (6.0)
2.73−2.90, m
5.71, d (3.6)
3.51 m
2.73−2.90, m
2.34, dd (19.2, 9.6 H-1ax)
2.72, dd (19.2, 6.0 H-1eq)
3.69, dd (9.6, 6.0)
2.51, dd (19.2, 9.6 H-1ax)
3.00, dd (19.2, 6.0 H-1eq)
3.88, dd (9.6, 6.0)
2′
3′
4′
3.83, m
3.76, dd (5.6, 5.6)
3.69, ddd (9.6, 9.2, 5.2)
3.23 m
2.69, d (18.8)
2.78, d (18.8)
7.27, s
2.41, d (19.2)
2.58, d (19.2)
7.29, s
4.10, s
6.80, s
4.29, s
6.88, s
5′
6′
3.61, ddd (9.6, 6.4, 2.4)
4.10, dd (12.0, 6.4 H-6′ ax)
4.16 dd (12.0, 2.4 H-6′ eq)
7′
8′
1.85, s
3′-Me
6′-OMe
8-OH
1′-OH
2′-OH
3′-OH
4′-OH
8′-OH
1.31, s
3.83, s
13.06, s
1.31, s
3.84, s
13.11, s
1.16, s
3.65, s
13.03, s
1.34, s
3.74, s
12.75, s
5.35, d (3.6)
5.21, d (5.2)
5.33, d (6.4)
4.69, br s
4.36, br s
5.56, br s
12.82, s
12.28, s
12.27, s
a
b
c
DMSO-d6. Acetone-d6. CD3OD.
and C-10′. These results indicated that the two monomers
were joined via a C-5−C-7′ linkage. Thus, alterporriol T (14)
was identified as a new anthranoid dimer with a C-5 and C-7′
linkage. Due to the biosynthetic pathway, the configuration
of 14 was tentatively assigned as 2R,3R,4R,2′R,3′S,10 but the
absolute configurations for the axes of chirality in 14 have not
been determined.
of macrosporin (6)10 showed a close structural relationship
except for the absence of the aromatic proton at δH 7.52 (H-1)
in 16 and the fact that the chemical shift of C-1 moved
significantly downfield (δC 128.5, C in 16 vs 111.0, CH in 2).
These data confirmed that compound 16 was a symmetrical
dimer of 2 with a C-1 and C-1′ linkage. Thus, alterporriol V
(16) was identified as a new symmetrical dimer with a C-1 and
C-1′ linkage, but the absolute configurations for the axes of
chirality in 16 have not been determined.
Alterporriol U (15) was also obtained as an orange,
amorphous powder. Its molecular formula, C32H30O12
(18 degrees of unsaturation), was obtained from HRESIMS
Alterporriol W (17) was obtained as a red, amorphous
powder with the molecular formula C32H26O12 from HRESIMS
1
data. The H and 13C NMR (Tables 3 and 4) spectroscopic
data indicated that 15 comprised two altersolanol B (9)9 units,
data. The H and 13C NMR spectroscopic data (Tables 3
1
1
but it is not a symmetrical dimer. In the H NMR spectrum,
and 4) indicated that 17 was composed of one altersolanol C
(10)9 subunit and one macrosporin (6)10 subunit. In the H
1
two aromatic protons at δH 6.86 (s) and 7.29 (s) were observed.
These results indicated that the two monomers were also joined
via a C-5−C-7′ linkage as in 14. This was corroborated by the
HMBC spectrum (H-7 correlated with C-6 and C-8, and
H-5′ correlated with C-7′ and C-10′). Thus, 15 was identified
as a new anthranoid dimer with a C-5 and C-7′ linkage. Due
to the biosynthetic pathway, the configuration of 15 was also
tentatively assigned as 2R,3S,2′R,3′S.10 However, the absolute
configurations for the axes of chirality in 15 have not been
determined.
NMR spectrum, four aromatic proton signals were observed,
among which three were due to C-4, C-5, and C-7 in the
macrosporin moiety at δH 7.88 (br s), 7.11 (d, J = 2.8 Hz), and
6.64 (d, J = 2.8 Hz) and one due to C-7′ in the altersolanol C
moiety at δH 6.80 (s). Therefore, 17 lacked proton signals
corresponding to C-1 in 6 and to C-5′ in 10. These results
indicated that the two monomers were joined via a C-1−C-5′
linkage. The configuration of 17 also has been tentatively
assigned as 2′R,3′R,4′R.10
Alterporriol V (16) was obtained as a yellow, amorphous
powder, with the molecular formula C32H22O10 from
HRESIMS data. However, there were only 16 signals present
in the 13C NMR spectrum. This result indicated that compound
An interesting feature of the isolated anthranoid dimers is
their axial chirality. CD spectra of 18/19 and 20/21 recorded in
MeOH showed a near-quasi-mirror images pattern (Figure 4).
This is due to the chiral axis occurring within the chromophore,
and it is expected to dominate the observed CD spectrum.22,23
So, these CD spectra indicated that they (18 and 19, 20 and 21)
1
16 was a symmetrical dimer. Careful comparison of the H
NMR and 13C NMR spectra of 16 (Tables 1 and 2) with those
D
dx.doi.org/10.1021/np500340y | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 4. 13C NMR Data for 3, 14, 15, and 17 (100 MHz)
(δ in ppm)
a
b
b
a
position
2
14
15
17
1
110.8, CH
159.8, C
135.0, C
129.6, CH
107.3, CH
166.0, C
106.0, CH
164.6, C
186.2, C
180.8, C
133.0, C
127.0, C
110.2, C
134.8, C
16.3, CH3
56.4, CH3
97.7, CH
71.3, CH
72.9, CH
70.0, CH
71.1, CH
63.3, CH2
170.1, C
20.4, CH3
29.6, CH2
68.7, CH
73.4, C
30.3, CH2
71.9, CH
70.6, C
131.0, C
164.2, C
131.0, C
129.3, CH
105.5, CH
165.3, C
104.8, CH
163.2, C
189.3, C
178.5, C
130.6, C
125.6, C
110.5, C
136.0, C
56.4, CH3
17.6, CH3
28.8, CH2
67.0, CH
72.0, C
2
3
4
70.9, CH
117.5, C
165.8, C
104.5, CH
165.5, C
189.8, C
184.8, C
142.6, C
144.6, C
110.4, C
133.5, C
22.3, CH3
57.1, CH3
30.3, CH2
71.9, CH
70.6, C
36.3, CH2
117.6, C
165.8, C
104.5, CH
165.2, C
189.6, C
184.5, C
142.5, C
143.9, C
110.2, C
131.8, C
25.4, CH3
57.1, CH3
30.6, CH2
71.8, CH
70.5, C
5
6
7
8
9
10
1a
4a
Figure 3. X-ray structure of 4a.
9a
10a
3-Me
6-OMe
1′
2′
3′
4′
5′
6′
7′
overall absolute configuration of 17 has been tentatively assigned
as aR,2′R,3′R,4′S.
Another interesting phenomenon of 18 and 19 is their
crystallization. When they were in a 1:1 mixture, it was easy to
crystallize. However, it was difficult to obtain a single crystal
when they were separated. Ultimately, the absolute config-
urations of 18 and 19 were confirmed by mixed crystal X-ray
crystallography (Figure S66). Thus, the absolute configurations
of 18 and 19 have been assigned as aS,1′S,2′R,3′S,4′R and
aR,1′S,2′R,3′S,4′R, respectively.
36.1, CH2
103.4, CH
163.6, C
120.1, C
161.7, C
190.1, C
184.3, C
143.8, C
144.6, C
110.8, C
133.5, C
25.5, CH3
56.8, CH3
36.1, CH2
103.3, CH
163.8, C
120.1, C
161.4, C
189.9, C
184.3, C
141.9, C
144.5, C
110.8, C
133.5, C
25.5, CH3
56.8, CH3
69.1, CH
125.5, C
164.6, C
102.8, CH
163.2, C
188.5, C
183.6, C
141.5, C
144.5, C
108.8, C
131.8, C
21.9, CH3
56.0, CH3
8′
9′
The structures of known compounds 5−13 and 18−25 were
10′
1a′
4a′
9a′
10a′
3′-Me
6′-OMe
1
identified by comparison of their H/13C NMR spectra and
[α]24 with those in the literature.
D
Compounds 1−9, 11, 15−22, and 25 were evaluated for
cytotoxic activities against the mouse melanoma cell line
(B16F10) and the human lung adenocarcinoma cell line
(A549). However, in the assay all IC50 values obtained were
higher than 10 μM and were defined as inactive.
a
b
DMSO-d6. Acetone-d6.
The antibacterial activities of all compounds were
determined against seven terrestrial pathogenic bacteria:
Micrococcus tetragenus, Escherichia coli, Staphylococcus albus,
Bacillus cereus, S. aureus, Kocuria rhizophila, and Bacillus subtilis
(Table 5). Compounds 3, 8, and 9 exhibited only weak broad-
spectrum antibacterial activities against E. coli, S. aureus, and B.
subtilis, while 8 exhibited weak antibacterial activity against S.
aureus with an MIC value 2.07 μM. These results indicated
that anthraquinone derivatives showed better antibacterial activ-
ities than anthraquinone dimers in these assays. All compounds
were tested for brine shrimp lethality using Artemia salina (brine
shrimp).24 Only compound 2 displayed a moderate lethality
effect, with an LD50 value of 10 μM.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were mea-
sured on a JASCO P-1020 digital polarimeter. UV spectra were recorded
on a Beckman DU 640 spectrophotometer. CD spectra were recorded
on a MOS-450 spectrometer. IR spectra were recorded on a Nicolet
6700 spectrophotometer. NMR spectra were recorded on a Bruker AV
Figure 2. X-ray structure of 3b.
have different axial chiralities. However, the axial chiralities of
18 and 19 have not yet been assigned. In order to assign axial
chiralities of 17−19, their CD spectra were compared with
known compound 21. Regarding their biaryl chromophoric
system, 17 and 19 showed the same spectral feature in the 205−
340 nm range as 21 (Figure 5) and their CD are primarily
determined by their axial chirality, so they must have the same aR
axial chirality, whereas 18 has an aS configuration.7,18 Thus, the
1
spectrometer (400 MHz for H and 100 MHz for 13C) and a JEOL
JEM-ECP NMR spectrometer (600 MHz for 1H and 150 MHz for 13C).
TMS was used as an internal standard. HRESIMS spectra were mea-
sured on a Q-TOF Ultima Global GAA076 LC mass spectrometer.
Silica gel (Qing Dao Hai Yang Chemical Group Co.; 200−300 mesh),
octadecylsilyl silica gel (YMC; 12 nm−50 μm), and Sephadex LH-20
(GE) were used for column chromatography (CC). Precoated silica gel
plates (Yan Tai Zi Fu Chemical Group Co.; G60, F-254) were used for
E
dx.doi.org/10.1021/np500340y | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Figure 4. CD spectra of 18, 19, 21, and 22.
Fungal Materials. The fungal strain Stemphylium sp. 33231 was iso-
lated from the mangrove Burguiera sexangula var. rhynchopetala
collected in the South China Sea in August 2012.8 The strain was
deposited in the Key Laboratory of Tropical Medicinal Plant Chemistry
of Ministry of Education, College of Chemistry and Chemical
Engineering, Hainan Normal University, P. R. China, with a GenBank
accession number KF479349.
Fermentation, Extraction, and Isolation. The fungal strain
was grown on solid rice cultures in 1 L Erlenmeyer flasks (276 flasks;
100 mL of natural seawater from the South China Sea was added
to 100 g of commercially available rice, autoclave sterilization) at
25 °C without shaking for 4 weeks. The fermentation was extracted
three times with an equal volume of EtOAc. The combined EtOAc
layers were evaporated to dryness under reduced pressure to give an
EtOAc extract (225.4 g), which was subjected to silica gel CC
(petroleum ether, EtOAc, MeOH v/v, gradient) to generate nine
fractions (Frs. 1−9). Fr. 2 was isolated by CC on silica gel eluting with
petroleum ether−EtOAc (5:1) to obtain 6 (5.8 g). Fr. 4 was isolated
by CC on silica gel eluting with petroleum ether−EtOAc (1:1) and
then subjected to repeated Sephadex LH-20 CC eluting with mix-
tures of CHCl3−MeOH (1:1) to obtain 9 (25 mg), 10 (15.2 mg), 16
(8.8 mg), 22 (5.5 mg), and 25 (7.5 mg). Fr. 5 was isolated by CC
Figure 5. CD spectra of 17, 19, and 22.
thin-layer chromatography (TLC). X-ray diffraction data were collected
on an Aglient Technologies Gemini A Ultra system.
a
Table 5. Antimicrobial Activities of Isolated Compounds
MIC (μM)
compound
M. tetragenus
E. coli
S. albus
B. cereus
S. aureus
K. rhizophila
B. subtilis
1
3
6
8
9
>10
7.8
9.8
3.9
>10
3.9
>10
>10
>10
>10
>10
>10
>10
8.3
>10
3.9
>10
>10
>10
>10
7.8
>10
3.9
4.6
4.6
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
8.9
9.2
>10
4.1
8.2
4.1
2.07
7.8
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.3
7.8
7.8
10
>10
7.3
>10
>10
>10
>10
>10
5.0
>10
>10
>10
>10
>10
>10
>10
>10
0.3
8.8
13
>10
>10
>10
>10
>10
>10
>10
0.6
15
>10
>10
>10
7.5
16
8.1
19
7.9
20
10.0
2.50
>10
0.6
21
5.0
>10
>10
0.16
22
>10
0.3
b
ciprofloxacin
0.6
a
b
Compounds 2, 4, 5, 7, 11, 12, 14, 17, 18, and 23−25 are inactive for all terrestrial pathogenic bacteria used (MIC > 10 μM/L). Ciprofloxacin was
used as a positive control.
F
dx.doi.org/10.1021/np500340y | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
on silica gel eluting with CHCl3−MeOH (35:1) and then subjected to
Sephadex LH-20 CC eluting with CHCl3−MeOH (1:1) and further
purified by using octadecylsilyl silica gel eluting with 40% MeOH−
H2O to obtain 8 (28.5 mg), 4 (12.5 mg) 13 (43.5 mg), 14 (18.2 mg),
15 (8.7 mg), and 17 (6.3 mg). Fr. 6 was isolated by CC on silica gel
eluting with CHCl3−MeOH (25:1) and then subjected to Sephadex
LH-20 CC eluting with CHCl3−MeOH (1:1) and further purified by
using octadecylsilyl silica gel eluting with 45% MeOH−H2O to obtain
1 (13.9 mg), 3 (48.8 mg), 5 (9.2 mg), 8 (5.7 mg), 11 (10.6 mg),
and 23 (12.5 mg). Fr. 7 was isolated by CC on silica gel eluting
with CHCl3−MeOH (15:1) and then subjected to Sephadex LH-20
CC eluting with CHCl3−MeOH (1:1) and further purified by using
octadecylsilyl silica gel eluting with 50% MeOH−H2O to obtain 2
(3.8 mg), 18 (11.2 mg), and 19 (8.7 mg). Fr. 8 was isolated by CC
on silica gel eluting with CHCl3−MeOH (9:1) and then subjected
to Sephadex LH-20 CC eluting with CHCl3−MeOH (1:1) and further
purified by using octadecylsilyl silica gel eluting with 50% MeOH−
H2O to obtain 7 (88.7 mg), 24 (6.1 mg), 21 (5.4 mg), and 20
(10.5 mg) respectively.
direct methods (SHELXS-97) and refined using full-matrix least-
squares difference Fourier techniques. Carbon and oxygen atoms were
refined anisotropically. Hydrogen atoms were either refined freely with
isotropic displacement parameters or positioned with an idealized
geometry and refined riding on their parent C atoms. Crystals suitable
for X-ray diffraction (1a and 3b) were obtained by slow evaporation of
a solution in MeOH−H2O, and 4a was obtained by slow evaporation
of a solution in MeOH−CHCl3. The mixed crystal of 18 and 19 was
obtained by slow evaporation of a solution in MeOH−DMSO.
Crystallographic data (excluding structure factors) for 1a, 3b, and 4a
and the mixed crystal of 18 and 19 have been deposited with the
Cambridge Crystallographic Data Centre: CCDC reference numbers
972307, 979866, 972306, and 979867. These data can be obtained,
free of charge, from the Cambridge Crystallographic Data Centre via
http://www.ccdc.cam.ac.uk/data_request/cif.
Crystal data for 1a: C17H20O10, M = 384.33, space group P212121
with a = 6.1647(7) Å, b = 11.40078(15) Å, c = 23.0795(3) Å, α = β =
γ = 90°, V = 1622.09(4) Å3, Z = 4, T = 293(2) K, Dc = 1.574 g/cm3,
μ(Cu Kα) = 1.130 mm−1, F(000) = 808, 8631 reflections measured,
2807 independent reflections (Rint = 0.0219). The final R1 values were
0.0265 [I > 2σ(I)]. The final wR2 (F2) values were 0.0653 [I > 2σ(I)].
The final R1 values were 0.0283 (all data). The final wR2 (F2) values
were 0.0672 (all data). Flack parameter = 0.02(5).
Crystal data for 3b: C19H20O6, M = 344.35, space group P212121
with a = 7.97730(10) Å, b = 20.0228(3) Å, c = 20.8545(3) Å, α =
β = γ = 90°, V = 3331.05(8) Å3, Z = 8, T = 119.99(18) K, Dc =
1.373 g/cm3, μ(Cu Kα) = 0.851 mm−1, F(000) = 1456, 20 601
reflections measured, 5564 independent reflections (Rint = 0.0362). The
final R1 values were 0.0303 [I > 2σ(I)]. The final wR2 (F2) values were
0.0704 [I > 2σ(I)]. The final R1 values were 0.0335 (all data). The final
wR2 (F2) values were 0.0725 (all data). Flack parameter = 0.02(11).
Crystal data for 4a: C16H20O7, M = 324.32, space group P212121
with a = 5.66941(19) Å, b = 10.8760(4) Å, c = 23.9077(7) Å, α =
β = γ = 90°, V = 1474.16(8) Å3, Z = 4, T = 110.02(10) K, Dc =
1.461 g/cm3, μ(Cu Kα) = 0.971 mm−1, F(000) = 688, 6945 reflections
measured, 2514 independent reflections (Rint = 0.0286). The final R1
values were 0.0313 [I > 2σ(I)]. The final wR2 (F2) values were 0.0765
[I > 2σ(I)]. The final R1 values were 0.0330 (all data). The final wR2
(F2) values were 0.0779 (all data). Flack parameter = −0.03(15).
Crystal data for mixed crystal of 18 and 19: 3(C2H6OS)·
2(C32H26O13)·6(O), M = 1567.44, space group P1 with a = 9.18695(15) Å,
b = 9.48126(17) Å, c = 21.1713(3) Å, α = 82.3800(14)°, β =
83.5029(13)°, γ = 79.8305(14)°, V = 1791.47(5) Å3, Z = 1, T =
120.0(2) K, Dc = 1.453 g/cm3, μ(Cu Kα) = 1.780 mm−1, F(000) = 818,
Auxarthrol C (1): white powder; [α]24 −160 (c 0.4, MeOH); UV
D
(MeOH) λmax (log ε) 358 (0.85), 310 (0.98), and 249 (2.29) nm; IR
(KBr) νmax 3421, 3011, and 1722 cm−1; 1H and 13C NMR, see Tables
1 and 2; HRESIMS m/z 351.0711 [M − H]− (calcd for C16H15O9,
351.0716).
Macrosporin 2-O-(6′-acetyl)-a-D-glucopyranoside (2): yellow
powder; [α]24 +120 (c 0.3, MeOH); UV (MeOH) λmax (log ε)
D
310 (1.41), 281 (4.21), and 219 (2.50) nm; IR (KBr) νmax 3498, 1692,
1633, and 1573 cm−1; 1H and 13C NMR, see Tables 3 and 4;
HRESIMS m/z 487.1238 [M − H]− (calcd for C24H23O11, 487.1240).
2-O-Acetylaltersolanol B (3): orange-yellow powder; [α]24 −85.7
D
(c 0.25, MeOH); UV (MeOH) λmax (log ε) 427 (0.89) and 258 (3.11)
1
nm; IR (KBr) νmax 3440, 1643, and 1019 cm−1; H and 13C NMR,
see Tables 1 and 2; HRESIMS m/z 345.0971 [M − H]− (calcd for
C18H17O7, 345.0974).
2-O-Acetylaltersolanol L (4): white powder; [α]24 −48.8 (c 0.35,
D
MeOH); UV (MeOH) λmax (log ε) 316 (1.98), 261 (3.29), and
1
242 (2.02) nm; IR (KBr) νmax 3438, 1638, and 1391 cm−1; H and
13C NMR, see Tables 1 and 2; HRESIMS m/z 367.1391 [M + H]+
(calcd for C18H23O8, 367.1393).
Alterporriol T (14): orange, amorphous powder; [α]24 +64
D
(c 0.2, MeOH); UV (MeOH) λmax (log ε) 438 (0.80), 271 (2.63),
and 239 (1.47) nm; CD (c 1.61 × 10−1 mol/L, MeOH) λmax (Δε) 337
(5.08), 271 (−1.49), and 214 (1.35); IR (KBr) νmax 3441, 1637, and
1
1398 cm−1; H and 13C NMR, see Tables 3 and 4; HRESIMS m/z
621.1603 [M − H]− (calcd for C32H29O13, 621.1608).
12 348 reflections measured, 11 844 independent reflections (Rint
=
Alterporriol U (15): orange, amorphous powder; [α]24 −200
0.0377). The final R1 values were 0.0496 [I > 2σ(I)]. The final wR2
(F2) values were 0.1394 [I > 2σ(I)]. The final R1 values were 0.0531
(all data). The final wR2 (F2) values were 0.1451 (all data). Flack
parameter = 0.010(9).
D
(c 0.3, MeOH); UV (MeOH) λmax (log ε) 438 (1.46) and 255 (3.93)
nm; CD (c 1.65 × 10−1 mol/L, MeOH) λmax (Δε) 327 (−6.78), 265
(0.09), and 212 (−1.52); IR (KBr) νmax 3435, 2971, 1644, and 1397
cm−1; 1H and 13C NMR, see Tables 3 and 4; HRESIMS m/z 605.1653
[M − H]− (calcd for C32H29O12, 605.1659).
Biological Assays. Cytotoxic activity was evaluated by the MTT
method as described previously.25 Two cancer cell lines, the mouse
melanoma cell line B16F10 and the human lung adenocarcinoma cell
line A549, were used. Epirubicin was used as a positive control. Anti-
bacterial activity was determined by the conventional broth dilution
assay.26 Seven terrestrial pathogenic bacteria, M. tetragenus (ATCC
13623), E. coli (ATCC 25922), S. albus (ATCC 8799), B. cereus
(ATCC 14579), S. aureus (ATCC 6538), K. rhizophila (ATCC 9341),
and B. subtilis (ATCC 6633), were used, and ciprofloxacin was used as
a positive control. Brine shrimp toxicity of the isolated compounds was
determined as described previously. Colchicine was used as a positive
control.24
Alterporriol V (16): yellow, amorphous powder; [α]24 −186
D
(c 0.15, MeOH); UV (MeOH) λmax (log ε) 401 (0.49), 287 (1.81),
and 237 (0.95) nm; IR (KBr) νmax 3424, 2923, 1630, 1572, and 1304
cm−1; 1H and 13C NMR, see Tables 1 and 2; HRESIMS m/z 565.1133
[M − H]− (calcd for C32H21O10, 565.1135).
Alterporriol W (17): red, amorphous power; [α]24 +198 (c 0.15,
D
MeOH); UV (MeOH) λmax (log ε) 338 (1.50), 229 (4.29), and 207
(1.89) nm; CD (c 1.66 × 10−1 mol/L, MeOH) λmax (Δε) 289 (−1.93),
258 (2.90), and 232 (2.65); IR (KBr) νmax 3440 and 1610 cm−1;
1H and 13C NMR, see Tables 3 and 4; HRESIMS m/z 601.1342
[M − H]− (calcd for C32H25O12, 601.1346).
Alterporriol A (18): CD (c 1.62 × 10−1 mol/L, MeOH) λmax (Δε)
316 (1.42), 284 (5.21), 267 (−6.96), and 225 (2.19).
ASSOCIATED CONTENT
* Supporting Information
■
S
Alterporriol B (19): CD (c 1.58 × 10−1 mol/L, MeOH) λmax (Δε)
314 (−0.81), 285 (−4.50), 266 (4.40), and 220 (−1.02).
1H, 13C, DEPT, HMQC, HMBC, COSY, NOESY, and HRESIMS
spectra of the new compounds (1−4, 14−17); CIF files and X-ray
crystallographic data for 1a, 3b, 4a, and mixed crystal of 18 and
19. This material is available free of charge via the Internet at
http://pubs.acs.org.
X-ray Crystal Structure Analysis of Compounds 1a, 3b, and
4a and Mixed Crystal of 18 and 19. Crystal X-ray diffraction data
were collected on a Bruker APEX DUO diffractometer with Cu Kα
radiation (λ = 1.5418 and 1.541 84 Å). The structure was solved by
G
dx.doi.org/10.1021/np500340y | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(25) Scudiero, D. A.; Shoemaker, R. H.; Paull, K. D.; Monks, A.;
Tierney, S.; Nofziger, T. H.; Currens, M. J.; Seniff, D.; Boyd, M. R.
Cancer Res. 1988, 48, 4827−4833.
(26) Appendio, G.; Gibbons, S.; Giana, A.; Pagani, A.; Grassi, G.;
Stavri, M.; Smith, E.; Rahman, M. M. J. Nat. Prod. 2008, 71, 1427−
1430.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: 86-89865889422. Fax: 86-89865889422. E-mail:
chgying123@163.com.
Author Contributions
†X.-M. Zhou and C.-J. Zheng are co-first authors.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the National Natural Science
Foundation of China (21162009, 31360069) and The Major
Technology Project of Hainan (ZDZX2013008-4).
REFERENCES
■
(1) Aly, A. H.; Debbab, A.; Kjer, J.; Proksch, P. Fungal Diversity 2010,
41, 1−16.
(2) Aly, A. H.; Debbab, A.; Proksch, P. Appl. Microbiol. Biotechnol.
2011, 90, 1829−1845.
(3) Debbab, A.; Aly, A. H.; Lin, W. H.; Proksch, P. Microbiol.
Biotechnol. 2010, 3, 544−563.
(4) Haraguchi, H.; Abo, T.; Fukuda, A.; Okamura, N.; Yagi, A.
Phytochemistry 1996, 43, 989−992.
(5) Phuwapraisirisan, P.; Rangsan, J.; Siripong, P.; Tip-Pyang, S. Nat.
Prod. Rep. 2009, 23, 1063−1071.
(6) Teiten, M. H.; Mack, F.; Debbab, A.; Aly, A. H.; Dicato, M.;
Proksch, P.; Diederich, M. Bioorg. Med. Chem. 2013, 21, 3850−3858.
(7) Debbab, A.; Aly, A. H.; Edrada-Ebel, R.; Wray, V.; Pretsch, A.;
Pescitelli, G.; Kurtan, T.; Proksch, P. Eur. J. Org. Chem. 2012, 7, 1351−
1359.
(8) Zhou, X. M.; Zheng, C. J.; Song, X. P.; Han, C. R.; Chen, W. H.;
Chen, G. Y. J. Antibiot. 2014, 67, 401−403.
(9) Zheng, C. J.; Shao, C. L.; Guo, Z. Y.; Chen, J. F.; Deng, D. S.;
Yang, K. L.; Chen, Y. Y.; Fu, X. M.; She, Z. G.; Lin, Y. C.; Wang, C. Y.
J. Nat. Prod. 2012, 75, 189−197.
(10) Becker, A. M.; Rickards, R. W.; Schmalzl, K. J.; Yick, H. C. J.
Antibiot. 1978, 31, 324−329.
(11) Aly, A. H.; Edrada-Ebel, R.; Wray, V.; Muller, W. E.; Kozytska,
̈
S.; Hentschel, U.; Proksch, P.; Ebel, R. Phytochemistry 2008, 69, 1716−
1725.
(12) Stoessl, A. Can. J. Chem. 1969, 47, 767−776.
(13) Stoessl, A.; Unwin, C. H.; Stothers, J. B. Can. J. Chem. 1983, 61,
372−377.
(14) Stoessl, A. Can. J. Chem. 1969, 47, 777−784.
(15) Debbab, A.; Aly, A. H.; Edrada-Ebel, R.; Wray, V.; Muller, W. E.
̈
G.; Totzke, F.; Zirrgiebel, U.; Schachtele, C.; Kubbutat, M. H. G.; Lin,
̈
W. H.; et al. J. Nat. Prod. 2009, 72, 626−631.
(16) Stoessl, A.; Stothers, J. B. Can. J. Chem. 1983, 61, 378−382.
(17) Suemitsu, R.; Sakurai, Y.; Nakachi, K.; Miyoshi, I.; Kubota, M.
Agric. Biol. Chem. 1989, 53, 1301−1304.
(18) Kanamaru, S.; Honma, M.; Murakami, T.; Tsushima, T.; Kudo,
S.; Tanaka, K.; Nihei, K.; Nehira, T.; Hashimoto, M. Chirality 2012,
24, 137−146.
(19) Suemitsu, R.; Yamamoto, T.; Miyai, T.; Ueshima, T.
Phytochemistry 1987, 26, 3221−3224.
(20) Alvi, K. A.; Rabenstein, J. J. Ind. Microbiol. Biotechnol. 2004, 31,
11−15.
(21) Kuniaki, T.; Yoshikazu, S.; Tatsuya, T.; Yoshiaki, F.; Seijiro, H.
Tetrahedron Lett. 2011, 22, 138−148.
(22) Bringmann, G.; Menche, D.; Kraus, J.; Muhlbacher, J.; Peters,
̈
K.; Peters, E. M.; Brun, R.; Bezabih, M.; Abegaz, B. M. J. Org. Chem.
2002, 67, 5595−5610.
(23) Bringmann, G.; Mortimer, J. P. A.; Keller, P. A.; Gresser, M. J.;
Garner, J.; Breuning, M. Angew. Chem., Int. Ed. 2005, 44, 5384−5427.
(24) Solis, P. N.; Wright, C. W.; Anderson, M. M. Planta Med. 1993,
59, 250−252.
H
dx.doi.org/10.1021/np500340y | J. Nat. Prod. XXXX, XXX, XXX−XXX