Bioactive indole alkaloids and phenyl ether derivatives from a marine-derived Aspergillus sp. fungus

Article abstract of DOI:10.1021/np300707x

Two new prenylated indole alkaloids, 17-epi-notoamides Q and M (1 and 2), and two new phenyl ether derivatives, cordyols D and E (9 and 13), together with 10 known compounds (3-8, 10-12, 14) were isolated from a marine-derived Aspergillus sp. fungus. Among them, 1/5 and 2/4 were pairs of epimers. The planar structures and absolute configurations of the new compounds were determined by extensive NMR spectroscopic data as well as CD spectra. The absolute configuration of 3 was confirmed by single-crystal X-ray diffraction analysis for the first time. All isolated metabolites (1-14) and eight synthetic phenyl ether derivatives (12a, 14a-14g) were evaluated for their antibacterial activities in vitro. The polybromide phenyl ether 14g showed pronounced antibacterial activity against Staphylococcus epidermidis with an MIC value of 0.556 μM, stronger than that of the positive control ciprofloxacin (MIC = 3.13 μM).

Full text of DOI:10.1021/np300707x

Article
pubs.acs.org/jnp
Bioactive Indole Alkaloids and Phenyl Ether Derivatives from a
Marine-Derived Aspergillus sp. Fungus
Min Chen,† Chang-Lun Shao,† Xiu-Mei Fu,† Ru-Fang Xu,† Juan-Juan Zheng,† Dong-Lin Zhao,†
Zhi-Gang She,*,‡ and Chang-Yun Wang*,†
†Key Laboratory of Marine Drugs, the Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China,
Qingdao 266003, People’s Republic of China
‡School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou 510275, People’s Republic of China
S
* Supporting Information
ABSTRACT: Two new prenylated indole alkaloids, 17-epi-
notoamides Q and M (1 and 2), and two new phenyl ether
derivatives, cordyols D and E (9 and 13), together with 10
known compounds (3−8, 10−12, 14) were isolated from a
marine-derived Aspergillus sp. fungus. Among them, 1/5 and
2/4 were pairs of epimers. The planar structures and absolute
configurations of the new compounds were determined by
extensive NMR spectroscopic data as well as CD spectra. The absolute configuration of 3 was confirmed by single-crystal X-ray
diffraction analysis for the first time. All isolated metabolites (1−14) and eight synthetic phenyl ether derivatives (12a, 14a−14g)
were evaluated for their antibacterial activities in vitro. The polybromide phenyl ether 14g showed pronounced antibacterial
activity against Staphylococcus epidermidis with an MIC value of 0.556 μM, stronger than that of the positive control ciprofloxacin
(MIC = 3.13 μM).
arine microorganisms, especially marine fungi, have
Mattracted increasing attention from those seeking new
pharmaceutically useful natural products in recent years.1 During
the past decade, a growing number of structurally unique and
biologically active compounds have been isolated from marine
fungi.1,2 For example, the notoamides from Aspergillus spp. are
prenylated indole alkaloids that incorporate complex
bicyclo[2.2.2]diazaoctane or diketopiperazine ring systems and
show a wide range of biological activities.3−6
In our ongoing research on marine fungi from the South China
Sea, we have isolated several new compounds with antibacterial,
cytotoxic, antiviral, and antifouling activities.7−11 Recently, a
chemical investigation of the marine fungal strain Aspergillus sp.
XS-20090066, isolated from the gorgonian Dichotella gemmacea,
RESULTS AND DISCUSSION
■
Compound 1 was isolated as a white powder and has the
molecular formula C27H33N3O5 (13 degrees of unsaturation) as
determined by HRESIMS. The 1H NMR spectrum of 1 (Table
1) showed five singlet methyl signals at δH 3.31, 1.43, 1.40, 1.08,
and 1.01, four doublet olefinic or aromatic signals at δH 6.96 (d, J
= 7.8 Hz), 6.67 (d, J = 9.6 Hz), 6.37 (d, J = 7.8 Hz), and 5.74 (d, J
= 9.6 Hz), three doublet−doublet olefinic signals at δH 6.11 (dd, J
= 17.4, 10.8 Hz), 5.06 (dd, J = 10.8, 1.2 Hz), and 4.99 (dd, J =
17.4, 1.2 Hz), and two exchangeable proton signals at δH 9.65 and
6.22. The 13C NMR and DEPT data revealed that 1 contained
three carbonyl groups (δC 180.3, 167.8, and 165.7), 10 olefinic or
aromatic carbon atoms, and five methyl groups (including a
methoxy carbon signal at δC 52.1). These spectroscopic features
was carried out. The EtOAc extract of the fungal culture showed
antibacterial activity against a panel of pathogenic bacteria. In this
study, we report the isolation, structure elucidation, and
antibacterial activities of two new prenylated indole alkaloids,
17-epi-notoamides Q and M (1 and 2), and two new phenyl ether
derivatives, cordyols D and E (9 and 13), from the bioactive
extract. Ten known compounds, notoamide C (3),6,12
notoamide M (4),13−15 notoamide Q (5),14,15 dehydronotoa-
mide C (6),5 (+)-notoamide B (7),12,16 (−)-stephacidin A
(8),16,17 3,3′-O-dimethylviolaceol-I (10), cordyol C (11),18 4-
methoxycarbonyldiorcinol (12),19 and diorcinol (14),18 are also
described. All isolated metabolites (1−14) and eight synthetic
phenyl ether derivatives (12a and 14a−14g) were evaluated for
their antibacterial activity in vitro. Preliminary structure−activity
relationships (SAR) of the phenyl ether derivatives to the
antibacterial activity are also discussed.
Received: October 10, 2012
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Table 1. 1H (600 MHz) and 13C (150 MHz) NMR
Spectroscopic Data for Compounds 1 and 2 (acetone-d6)
(Table 2). It could be deduced that the relative configuration of
C-11 and C-17 in 1 was different from that of 5. In the NOESY
experiments for 1, no correlation was observed between H-11
and 17-OMe, suggesting that these protons are located on
opposite faces of the diketopiperazine ring. This was supported
by the selective NOE experiments for 1, revealing that the
irradiation of 17-OMe resulted in no obvious enhancement of H-
11. These results suggested that one of the two stereogenic
centers in 1 (either C-11 or C-17) was inverted relative to the
corresponding center in 5, indicating two possibilities for the
absolute configurations at C-11 and C-17 of 1, either 11R,17R or
11S,17S. The biogenesis of the notoamides and related
compounds has been proposed on the basis of the genome
sequencing, bioinformatic mining, and biochemical studies.15,20
Therefore, on the basis of biogenetic considerations, the
configuration of C-11 in 1 is proposed as S arising from the L-
Trp moiety. Accordingly, the configuration at C-17 of 1 is
assigned as S.
1
2
δH, mult. (J in
δH, mult. (J in
position
δC, type
Hz)
δC, type
Hz)
1
2
3
4
5
6
7
8
9
10
9.65, brs
9.78, brs
180.3, C
56.9, C
180.1, C
55.8, C
127.1, CH
109.2, CH
153.7, C
106.1, C
139.9, C
122.5, C
6.96, d (7.8)
6.37, d (7.8)
126.3, CH
108.4, CH
152.8, C
105.3, C
139.0, C
121.6, C
6.95, d (7.8)
6.35, d (7.8)
32.6, CH2 2.66, dd (14.4,
4.8); 2.46, dd
31.8, CH2 2.68, dd (13.8,
5.4); 2.57, dd
(14.4, 8.4)
(13.8, 8.4)
11
56.7, CH
167.8, C
3.60, m
56.3, CH
167.3, C
3.56, ddd (8.4,
5.4, 4.8)
The absolute configuration of C-3 in 1 was established
according to its CD spectrum (Figure 1) combined with the X-
12
14
15
16
17
18
19
20
46.2, CH2 3.43−3.49, m
14.3, CH2 1.83−1.89, m
38.1, CH2 2.33, m; 1.58, m
91.8, C
45.1, CH2 3.52, m; 3.38, m
19.3, CH2 2.01, m; 1.87, m
37.1, CH2 2.15, m; 2.09, m
86.6, C
165.7, C
167.0, C
6.22, brs
6.25, d (4.8)
113.8, CH2 5.06, dd (10.8,
1.2); 4.99, dd
113.0,CH2
5.02, dd (10.8,
1.2); 4.95, dd
(17.4, 1.2)
(17.4, 1.2)
21
144.5, CH
43.2, C
6.11, dd (17.4,
10.8)
143.5, CH
42.3, C
6.08, dd (17.4,
10.8)
22
23
24
25
26
27
28
29
23.3, CH3 1.01, s
20.4, CH3 1.08, s
22.5, CH3 0.99, s
21.2, CH3 1.05, s
118.1, CH
131.0, CH
76.5, C
6.67, d (9.6)
5.74, d (9.6)
117.2, CH
130.1, CH
75.7, C
6.66, d (9.6)
5.73, d (9.6)
27.7, CH3 1.40, s
28.0, CH3 1.43, s
52.1, CH3 3.31, s
27.2, CH3 1.39, s
26.9, CH3 1.42, s
Figure 1. CD spectra of compounds 1−5.
17-
OCH3
17-OH
5.99, brs
ray structure (Figure 2) of notoamide C (3). As the CD spectrum
of 1 matched closely with that of 3 and the X-ray structure of 3
unambiguously determined the S configuration at C-3 (see
below), the absolute configuration of 1 could also be assigned as
3S. Thus, the structure of 1 was determined with the absolute
configuration of 3S,11S,17S and named 17-epi-notoamide Q.
Compound 2 was also isolated as a white powder, possessing
the same molecular formula, C26H31N3O5, as notoamide M
suggested that 1 belongs to the family of prenylated indole
alkaloids and is very similar to notoamide Q (5), which was
obtained from the culture of a marine-derived Aspergillus sp. and
was reported to have the 3S,11S,17R configurations.14,15 The
significant differences between these two compounds were the
chemical shifts of H-11 (δH 3.60 in 1 vs δH 4.25 in 5) and 17-
OCH3 (δH 3.31 in 1 vs δH 3.01 in 5) in their 1H NMR spectra
(4).13−15 Careful comparison of the H NMR spectroscopic
1
Table 2. Selected 1H NMR (600 MHz) Spectroscopic Data for Compounds 1−5 (acetone-d6)
1
2
3
4
5
position
10
δH, mult. (J in Hz)
δH, mult. (J in Hz)
δH, mult. (J in Hz)
δH, mult. (J in Hz)
δH, mult. (J in Hz)
2.46, dd (14.4, 8.4); 2.66, dd 2.57, dd (13.8, 8.4); 2.68, dd 2.73, dd (15.0, 4.8); 2.73, dd 2.75, dd (14.4,5.4); 2.65, dd 2.66, dd (14.4, 6.0); 2.90,
(14.4, 4.8)
(13.8, 5.4)
(15.0, 4.8)
(14.4, 3.0)
overlapped
11
14
15
16
17
3.60, m
3.56, ddd (8.4, 5.4, 4.8)
3.52, m; 3.38, m
2.01, m; 1.87, m
2.15, m; 2.09, m
4.03, brt (4.8)
4.15, m
4.25, dd (6.0, 1.8)
3.48, m; 3.23, m
1.76, m; 1.62, m
1.98, m; 1.20, m
3.43−3.49, m
1.83−1.89, m
2.33, m; 1.58, m
3.42, m; 3.20, m
1.69−1.74, m
1.99, m; 1.26, m
3.36−3.41, m
1.71, m; 1.53, m
1.95, m; 1.52, m
3.91, ddd (10.2, 6.6, 1.8)
17-
OCH3
3.31, s
3.01, s
B
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signals (Table 3) were unambiguously accomplished by analysis
of 1D and 2D NMR data. In the HMBC spectrum, correlations
from 2-OCH3 to C-2, 3-OCH3 to C-3, and 3′-OCH3 to C-3′
confirmed the three methoxy groups were attached to C-2, C-3,
and C-3′, respectively. However, because the correlations from
2′-OH to C-1′ and C-2′ were observed simultaneously in the
HMBC spectrum, it was not possible to unambiguously
determine whether these two tetrasubstituted benzene rings
were joined via a C1−O−C1′ linkage or a C1−O−C2′ linkage.
Thereby, a methylation experiment was carried out for 9. The 1H
and 13C NMR spectrum of the methylated derivative of 9
indicated a symmetrical diphenyl ether structure, suggesting a
C1−O−C1′ linkage for the two benzene rings. The structure of 9
was given the trivial name cordyol D.
Compound 13 was also obtained as a colorless oil with the
molecular formula C15H16O3. The 1D and 2D NMR data
indicated that 13 consisted of a 3-methoxy-5-hydroxytoluene
unit connected to a 3,5-dihydroxytoluene residue via a C1−O−
C1′ linkage. Aromatic carbon signals at δC 158.7, 158.3, and 140.5
were assigned to C-1′, C-3′, and C-5′ in the dihydroxytoluene
residue, respectively. With another methyl group, the 3-methoxy-
5-hydroxytoluene unit was observed at δC 161.0, 158.2, and 140.5
for the aromatic carbons C-3, C-1, and C-5, respectively.
Compound 13 was finally determined by detailed 2D NMR
analysis and was named cordyol E.
Figure 2. Perspective ORTEP drawing for 3.
features of 2 with those of 4 showed significant differences of the
coupling constants of H-11 and H-10 as well as the chemical
shifts of H-16 and H-15 (Table 2), indicating a structural pattern
for 2 vs 4 similar to 1 vs 5. The determination of the
configurations for 2 was just like that for 1. The configuration of
the C-17 stereocenter in 2 was inverted compared to that of 4.
Combined with its CD spectrum (Figure 1), 2 was finally
determined as 17-epi-notoamide M, with the absolute config-
uration of 3S,11S,17S.
The structures of the known compounds (3−8, 10−12, and
1
14) were identified on the basis of their H NMR, 13C NMR,
ESIMS, and specific rotation data and by comparison with data
previously reported in the literature.5,12−19 A structural revision
for notoamide C (3) was proposed recently by Williams and co-
workers, in which the 3R configuration was revised as 3S based
on biosynthetic discussions.6 In the present study, the absolute
configuration of notoamide C (3) was confirmed by single-
crystal X-ray diffraction analysis using Cu Kα radiation and was
unambiguously determined as the 3S configuration (Figure 2).
Consequently, as the CD spectra of the known analogues (4 and
Compound 9 was isolated as a colorless oil. Its molecular
formula of C17H20O5 (eight degrees of unsaturation) was
determined by HRESIMS. The IR spectrum of 9 showed a
broad absorption band at 3460 cm−1 (OH). The H and 13C
1
NMR data were suggestive of a diphenyl ether derivative for 9
very similar to that of cordyol C (11).18 The significant difference
1
was the presence of three methoxy groups in the H NMR
spectrum of 9. Detailed assignments for proton and carbon
Table 3. 1H and 13C NMR Spectroscopic Data for 9, 10, and 13 at 600 (1H) and 150 (13C) MHz
a
a
b
9
10
13
position
δC, type
δH, mult.
δC, type
δH, mult.
δC, type
δH, mult. (J in Hz)
1
150.1, C
143.9, C
134.6, C
147.7, C
108.0, CH
129.1, C
112.4, CH
21.3, CH3
143.9, C
134.6, C
147.7, C
108.0, CH
129.1, C
112.4, CH
21.3, CH3
158.2, C
2
153.4, C
102.1, CH
161.0, C
6.36, t (1.8)
6.53, brs
3
137.9, C
4
107.9, CH
133.7, C
6.50, brs
6.49, brs
109.6, CH
140.5, C
5
6
112.5, CH
21.6, CH3
144.1, C
6.36, brs
2.24, s
6.40, brs
2.23, s
111.6, CH
20.7, CH3
158.7, C
6.39, brs
2.22, s
7
1′
2′
3′
4′
5′
6′
7′
134.8, C
103.2, CH
158.3, C
6.26, t (1.8)
6.30, brs
147.7, C
108.6, CH
128.9, C
6.49, brs
6.49, brs
110.5, CH
140.5, C
112.6, CH
21.3, CH3
61.2, CH3
56.1, CH3
56.2, CH3
6.38, brs
2.23, s
3.86, s
3.85, s
3.89, s
6.40, brs
2.23, s
111.2, CH
20.6, CH3
6.44, brs
2.27, s
2-OCH3
3-OCH3
3′-OCH3
2-OH
2′-OH
3′-OH
56.3, CH3
56.3, CH3
3.88, s
54.8, CH3
3.75, s
3.88, s
5.62, brs
5.62, brs
5.72, brs
8.43, brs
a
b
Recorded in CDCl3. Recorded in acetone-d6.
C
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a
Table 4. Antibacterial Activities of Compounds 8−14, 12a, and 14a−14g
MIC (μM)
compound
S. epidermidis
S. aureus
V. anguillarum
V. parahemolyticus
P. putida
8
9
14.5
>100
>100
100
>100
>100
100
>100
>100
100
>100
>100
>100
100
>100
>100
>100
100
10
11
100
100
12
2.71
100
10.9
100
21.7
9.23
25.6
100
21.7
>100
51.2
100
21.7
>100
51.2
12a
13
25.6
27.2
100
51.2
54.3
>100
>100
20.8
>100
46.0
>100
1.11
0.780
14
100
100
14a
14b
14c
14d
14e
14f
46.0
>100
10.4
>100
23.0
>100
1.11
0.0975
100
>100
20.8
>100
46.0
18.7
0.556
3.13
>100
10.4
100
>100
10.4
>100
100
2.87
18.7
1.11
0.195
>100
14g
ciprofloxacin
1.11
0.195
b
a
b
Data are expressed in MIC values (μM). Ciprofloxacin was used as a positive control.
5) matched closely with that of 3, the absolute configurations of
these two compounds could be assigned as 3S. The co-isolated
dehydronotoamide C (6) was also deduced as 3S on the basis of
biogenetic considerations. In addition, it seems that 6 may arise
from 2 and 4 via dehydration during the extraction or purification
processes. However, when 4 was dissolved in a mixed solvent
with freshly prepared silica gel and stirred at 40 °C for 1 week, 6
was not detected in the solution. Therefore, 6 is proposed as a
new natural product possessing a double bond between C-16 and
C-17, although a synthetic sample has been reported.5 It should
be noted that compound 10, whose structure was listed in
SciFinder Scholar, had no associated reference. Thus we report
its spectroscopic and MS data in the Experimental Section.
The antibacterial activities of the isolated compounds 1−14
were evaluated with four Gram-positive bacteria (Bacillus cereus,
Micrococcus tetragenus, Staphylococcus epidermidis, S. aureus) and
four Gram-negative bacteria (Escherichia coli, Vibrio anguillarum,
V. parahemolyticus, and Pseudomonas putida), using a standard
screening protocol (Table 4).21 The results demonstrated that
most of prenylated indole alkaloids (1−8) showed weak or no
activity against these pathogenic bacteria except for stephacidin A
(8), which revealed selectivity toward S. epidermidis (MIC = 14.5
μM).
Most of the phenyl ether derivatives (11−14) exhibited
moderate to strong inhibitory activity against five of the tested
pathogenic bacteria (S. epidermidis, S. aureus, V. anguillarum, V.
parahemolyticus, and P. putida). Especially, 12 showed
pronounced antibacterial activity against S. epidermidis with an
MIC value of 2.71 μM, which was close to the positive control
ciprofloxacin (MIC = 3.13 μM). It is noteworthy that the activity
of 13 was stronger than that of 14, and the only difference
between their structures is the methylation of the 3-OH group in
13. To elucidate the contribution of the two hydroxy groups (3-
OH and 3′-OH) to antibacterial activity and also to further study
the SAR of these compounds, a series of derivatives (12a, 14a−
14g) was designed and synthesized. As expected, different
chemically modified analogues exhibited different inhibitory
activities. It was found that the alkylation of a single hydroxy
group (14c and 14e) was helpful for the activity. The
corresponding benzoylation (14f) resulted in stronger activity
against S. epidermidis and V. parahemolyticus than observed for
the parent compound 14. The dialkylation or diacylation of two
hydroxy groups (14b and 14d) reduced activity. These results
suggested that one free hydroxy plays a critical role in
antibacterial activity.
Interestingly, compound 12, with a methoxycarbonyl group at
C-4, was more active than 14, while the antibacterial intensities of
the polybromide derivatives of 12 and 14 (12a and 14g) were
reversed. 2,4,6,2′,4′,6′-Hexabromodiorcinol (14g) exhibited
promising antibacterial activity against five tested pathogenic
bacteria (S. epidermidis, S. aureus, V. anguillarum, V. para-
hemolyticus, and P. putida) with MIC values ranging from 0.556
to 1.11 μM (Table 4), 1 order of magnitude more active than that
of the parent compound 14. In particular, 14g showed the
highest activity toward S. epidermidis, with an MIC value of 0.556
μM, approximately 5-fold more potent than that of ciprofloxacin
(MIC = 3.13 μM). However, comparison of the antibacterial
activity of 2,6,2′,4′,6′-pentabromo-4-methoxycarbonyldiorcinol
(12a) with that of its parent compound (12) indicated that
bromination of the benzene ring decreased the activity. The
above results revealed that an ester functionality or bromination
could increase activity, but the simultaneous presence of both an
ester and a brominated benzene ring caused loss of activity.
Halogenated biphenols are well known as antibacterial agents.
Triclosan, with chlorine substitutions, is a topical antibacterial
ingredient added to many consumer products such as clothing,
kitchenware, furniture, soaps, and body washes to reduce or
prevent bacterial contamination.22 The polybrominated phenyl
ether derivative 14g displayed potent antibacterial activity against
a panel of pathogenic bacteria and has potential for antibacterial
agent development.
Compounds 1−14 were also assessed for their cytotoxic
activity against human erythroleukemia K562 and human
promyelocytic leukemia HL-60 cell lines. Compound 12 showed
cytotoxic activity against the K562 cell line with an IC50 value of
5.94 μM. Other compounds were found to be inactive (IC50 > 10
μM) against these tumor cell lines.
In conclusion, two new prenylated indole alkaloids (1 and 2)
and two new phenyl ether derivatives (9 and 13), together with
10 known compounds (3−8, 10−12, and 14), were isolated from
the marine-derived fungus Aspergillus sp. XS-20090066. Eight
chemically modified phenyl ether derivatives (12a, 14a−14g)
D
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Journal of Natural Products
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cm−1; 1H NMR (acetone-d6, 600 MHz) and 13C NMR (acetone-d6, 150
MHz), see Table 1; ESIMS m/z 502.4 [M + Na]+; HRESIMS m/z
502.2319 (calcd for C27H33N3O5Na, 502.2312).
were also prepared. The absolute configuration of 3 was
confirmed by single-crystal X-ray diffraction for the first time,
and the absolute configurations of 1−5 were unambiguously
determined as 3S based on their CD spectra. The antibacterial
activities of the isolated metabolites and eight synthesized
derivatives were evaluated in vitro, and the preliminary SAR of
phenyl ether derivatives was also examined. Particularly, the
polybrominated phenyl ether derivative 14g showed pronounced
antibacterial activity against S. epidermidis, approximately 5-fold
more potent than that of ciprofloxacin.
17-epi-Notoamide M (2): white powder; mp 194−196 °C; [α]25
D
−8.6 (c 0.20, MeOH); UV (MeOH) λmax (log ε) 252 (2.52), 282 (1.65),
294 (1.14) nm; CD (0.83 mM, MeOH) λmax (Δε) 231 (11.1), 250
(−4.9), 278 (5.2) nm; IR (KBr) νmax 3465, 2940, 1653, 1457, 1118
cm−1; 1H NMR (acetone-d6, 600 MHz) and 13C NMR (acetone-d6, 150
MHz), see Table 1; ESIMS m/z 488.2 [M + Na]+; HRESIMS m/z
466.2343 (calcd for C26H32N3O5, 466.2336).
Cordyol D (9): colorless oil; UV (MeOH) λmax (log ε) 234 (1.87), 276
(0.46) nm; IR (KBr) νmax 3460, 2926, 1637, 1452, 1103, 821 cm−1; 1H
NMR (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz), see Table
3; ESIMS m/z 327.1 [M + Na]+, 631.3 [2 M + Na]+; HRESIMS m/z
327.1197 (calcd for C17H20O5Na, 327.1203).
EXPERIMENTAL SECTION
■
General Experimental Procedures. Melting points were
determined on an X-6 micromelting point apparatus and are
uncorrected. Optical rotations were measured on a JASCO P-1020
digital polarimeter. UV spectra were obtained on a Beckman DU 640
spectrophotometer. CD spectra were recorded on a JASCO J-810
circular dichroism spectrometer. IR spectra were recorded on a Nicolet-
Nexus-470 spectrometer using KBr pellets. NMR spectra were acquired
using a JEOL JEM-ECP NMR spectrometer (600 MHz for 1H and 150
MHz for 13C), using TMS as internal standard. ESIMS spectra were
obtained from a Micromass Q-TOF spectrometer. Single-crystal data
were measured on an Agilent Gemini Ultra diffractometer (Cu Kα
radiation). Semipreparative HPLC was performed on a Waters 1525
system using a semipreparative C18 (Kromasil, 5 μm, 10 × 250 mm)
column coupled with a Waters 2996 photodiode array detector. Silica gel
(Qing Dao Hai Yang Chemical Group Co.; 200−300 mesh), Sephadex
LH-20 (Amersham Biosciences), and octadecylsilyl silica gel (Unicorn;
45−60 μm) were used for column chromatography (CC). Precoated
silica gel plates (Yan Tai Zi Fu Chemical Group Co.; G60, F-254) were
used for thin layer chromatography (TLC).
3,3′-O-Dimethylviolaceol-I (10): colorless oil; UV (MeOH) λmax
(log ε) 238 (2.15), 280 (0.93) nm; IR (KBr) νmax 3440, 2962, 1658,
1
1457, 1098, 795 cm−1; H NMR (CDCl3, 600 MHz) and 13C NMR
(CDCl3, 150 MHz), see Table 3; ESIMS m/z 313.1 [M + Na]+, 603.3 [2
M + Na]+; HRESIMS m/z 313.1041 (calcd for C16H18O5Na, 313.1046).
Cordyol E (13): colorless oil; UV (MeOH) λmax (log ε) 232 (1.84),
274 (0.72) nm; IR (KBr) νmax 3416, 2921, 1606, 1452, 1149, 810 cm−1;
1H NMR (acetone-d6, 600 MHz) and 13C NMR (acetone-d6, 150 MHz),
see Table 3; ESIMS m/z 245.1 [M + H]+; HRESIMS m/z 245.1172
(calcd for C15H17O3, 245.1172).
Specific rotations for known compounds 3−8: notoamide C (3):
[α]25 +97 (c 0.40, MeOH) [lit. [α]27 +23 (c 0.255, MeOH)];12
D
D
notoamide M (4): [α]25D +34 (c 0.10, MeOH) [lit. [α]23D +51 (c 1.7,
MeOH)];13 notoamide Q (5): [α]25D +78 (c 0.10, MeOH) [lit. [α]20
D
+82 (c 2.2, MeOH)];13 dehydronotoamide C (6): [α]25D +30 (c 0.10,
CH2Cl2) [lit. [α]25D +81.3 (c 0.07, CHCl3)];5 notoamide B (7): [α]25
D
+118 (c 0.20, MeOH) [lit. [α]D +102 (c 0.05, MeOH); [α]27D −118 (c
0.064, MeOH)];16,12 stephacidin A (8): [α]25 −67 (c 0.06, MeOH−
D
Extraction and Isolation. The fungus Aspergillus sp. XS-20090066
was isolated from the inner part of the fresh gorgonian Dichotella
gemmacea, which was collected from the Xisha Islands coral reef in the
South China Sea in December 2009. The strain was deposited at the Key
Laboratory of Marine Drugs, the Ministry of Education of China, School
of Medicine and Pharmacy, Ocean University of China, Qingdao, PR
China, with the GenBank (NCBI) accession number HM535361. The
fermentation was carried out statically in a rice medium (100 mL
seawater, 100 g rice, 0.6 g peptone) in 1 L Erlenmeyer flasks for 28 days
at room temperature (rt). The fermented rice substrate (30 flasks) was
extracted repeatedly with EtOAc (3 × 300 mL for each flask), and the
solvent was combined and concentrated in vacuo to afford a residue
(21.5 g), which was subjected to silica gel column chromatography using
a step gradient elution with EtOAc−petroleum ether (0−100%) and
then with MeOH−CHCl3 (0−100%) to provide seven fractions (Fr.1−
Fr.7). Fr.2 was subjected to Sephadex LH-20 CC eluting with a mixture
of CHCl3−MeOH (v/v, 1:1) and then was further purified by using
semipreparative HPLC (Kromasil, 5 μm, 10 × 250 mm, 2 mL/min)
eluting with 90% MeOH−H2O to give compounds 13 (4.5 mg) and 12
(11.9 mg). Fr.3 was isolated on silica gel CC by using repeated gradient
elution of petroleum ether−EtOAc to yield 14 (190 mg) and subfraction
Fr.3-1, which was further purified by HPLC (80% MeOH−H2O) to give
9 (6.2 mg). Fr.4 was subjected to silica gel CC using gradient elution
with petroleum ether−EtOAc and then was subjected to an ODS
column eluting with 95% MeOH−H2O to provide 10 (4.9 mg) and 11
(5.1 mg). Fr.5 was separated on silica gel CC by using gradient elution of
petroleum ether−EtOAc and then further purified on an ODS column
eluting with 85% MeOH−H2O to produce 7 (24 mg) and 8 (65 mg).
Fr.6 was isolated by silica gel CC eluting with CHCl3−MeOH (v/v,
30:1) to obtain 3 (16 mg) and two subfractions, Fr.6-1 and Fr.6-2. Fr.6-1
was further purified by HPLC (62% MeOH−H2O) to afford 1 (3.1 mg)
and 5 (6 mg). Fr.6-2 was further purified on HPLC (55% MeOH−H2O)
to afford 2 (2.7 mg), 4 (4.9 mg), and 6 (3.7 mg).
CH2Cl2, 1:1) [lit. [α]D −32 (c 0.05, MeOH−CH2Cl2, 1:1); [α]D +61.5
(c 0.26, MeOH−CH2Cl2, 1:1)].16
X-ray Crystallographic Analysis of 3. Colorless crystals of 3 were
obtained from MeOH. Single-crystal X-ray diffraction data were
collected at 150 K on an Agilent Gemini Ultra diffractometer with Cu
Kα radiation (λ = 1.54178 Å). The structure was solved by direct
methods (SHELXS-97) and refined using full-matrix least-squares
difference Fourier techniques. All non-hydrogen atoms were refined
anisotropically, and all hydrogen atoms were placed in idealized
positions and refined relatively isotropically with a riding model.
Crystallographic data for 3 have been deposited in the Cambridge
Crystallographic Data Centre with the deposition number 886124.
Copies of the data can be obtained, free of charge, on application to the
Director, CCDC, 12 Union Road, Cambridge CB21EZ, UK [fax: t44-
(0)1223-336033, or e-mail: deposit@ccdc.cam.ac.uk].
Crystal data for 3: C26H3N31O4, Mr = 449.54, monoclinic, a =
14.9294(2) Å, b = 10.36530(10) Å, c = 15.2344(2) Å, α = 90°, β =
92.6220(10)°, γ = 90°, V = 2355.02(5) Å3, space group P21, Z = 4, Dx =
1.268 mg/m3, μ(Cu Kα) = 0.695 mm−1, and F(000) = 960. Crystal
dimensions: 0.43 × 0.39 × 0.34 mm3. Independent reflections: 8378
(Rint = 0.0293). The final R1 values were 0.0370, wR2 = 0.0970 (I >
2σ(I)). Flack parameter = 0.01(12).
Methylation Experiment for 9. To a solution of 9 (3 mg) in dry
acetone (1.0 mL) were added (CH3)2SO4 (5 μL) and K2CO3 (10 mg) at
rt, and the reaction mixture was stirred for 5 h. The solvent was
evaporated in vacuo, and the residue was purified by HPLC (95%
MeOH−H2O) to give 1.9 mg of the methylated derivative of 9 as a
colorless oil: 1H NMR (600 MHz, CDCl3, δ, ppm) 6.49 (2H, brs), 6.30
(2H, brs), 3.87 (6H, s), 3.85 (6H, s), 2.23 (6H, s); 13C NMR (150 MHz,
CDCl3, δ, ppm) 153.4 (C × 2), 150.1 (C × 2), 137.8 (C × 2), 133.6 (C ×
2), 112.4 (CH × 2), 108.2 (CH × 2), 61.1 (CH3 × 2), 56.1 (CH3 × 2),
21.6 (CH3 × 2); ESIMS m/z 319.1 [M + H]+.
Preparation of Derivatives of 12 and 14. Brominated
Derivatives 12a and 14g. To a stirred solution of 12 or 14 (10 mg,
respectively) in acetone (1.0 mL) was added slowly bromine (2.0 mL) at
rt, and the reaction mixture was stirred for 30 min. After the starting
material was consumed, the mixture was concentrated in vacuo to give a
17-epi-Notoamide Q (1): white powder; mp 179−180 °C; [α]25
D
−9.3 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 245 (2.30), 278 (1.10),
296 (0.67) nm; CD (0.64 mM, MeOH) λmax (Δε) 230 (9.3), 248
(−7.5), 272 (2.7) nm; IR (KBr) νmax 3445, 2921, 1647, 1447, 1154
E
dx.doi.org/10.1021/np300707x | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
residue, which was purified by HPLC (70% MeOH−H2O) to give
compound 12a or 14g (9 or 11 mg, 30.9% or 36.4%) as a white solid.
Alkylated Derivatives 14c, 14d, and 14e. A mixture of 14 (10
mg), 1-bromopentane (10 μL) or 1-bromopropane (4 μL), and K2CO3
(10 mg) in dry acetone (2.0 mL) was stirred at 30 °C overnight. The
solvent was evaporated in vacuo to give a residue, which was purified by
silica gel column chromatography (petroleum ether−EtOAc, v/v, 9:1 to
5:1) to give 14c (3.7 mg, 28.7%), 14d (8.6 mg, 54.1%), or 14e (4.9 mg,
42.2%).
Acylated Derivatives 14a, 14b, and 14f. To a solution of 14 (10
mg) in dry acetone (2.0 mL) were added Ac2O (9 μL) or BzCl (5 μL)
and K2CO3 (10 mg) at rt, and the reaction mixture was stirred for 2 h.
The solvent was evaporated in vacuo, and the residue was purified by
silica gel column chromatography (petroleum ether−EtOAc, v/v, 9:1 to
5:1) to give 14a (4.1 mg, 35.1%), 14b (6.9 mg, 51.1%), or 14f (4.9 mg,
34.2%).
1.8 Hz), 7.49 (2H, t, J = 7.2 Hz), 6.78 (1H, brs), 6.75 (1H, brs), 6.67
(1H, t, J = 1.2 Hz), 6.44 (1H, brs), 6.41 (1H, brs), 6.32, (1H, t, J = 1.2
Hz), 5.36 (1H, brs), 2.35 (3H, s), 2.26 (3H, s); 13C NMR (150 MHz,
CDCl3, δ, ppm) 165.3 (C), 157.8 (C), 156.7 (C), 151.6 (C), 141.1 (C),
140.8 (C), 133.8 (C), 133.7 (C), 130.3 (CH), 130.2 (CH), 129.4 (CH),
128.7 (CH), 128.6 (C), 117.3 (CH), 117.2 (CH), 112.2 (CH), 111.6
(CH), 109.8 (CH), 103.6 (CH), 21.5 (CH3), 21.5 (CH3); ESIMS m/z
335.1 [M + H]+, 357.0 [M + Na]+; HRESIMS m/z 357.1103 (calcd for
C21H18O4Na, 357.1097).
2,4,6,2′,4′,6′-Hexabromodiorcinol (14g): white powder; mp
204−205 °C; 1H NMR (600 MHz, CD3OD, δ, ppm) 2.58 (s, 6H); 13
C
NMR (150 MHz, CD3OD, δ, ppm) 154.4 (C × 2), 152.6 (C × 2), 139.4
(C × 2), 107.5 (C × 2), 105.5 (C × 2), 104.0 (C × 2), 23.0 (CH3 × 2);
ESIMS m/z 702.3 [M − H]−; HRESIMS m/z 702.5440 (calcd for
C14H779Br381Br3O3, 702.5429).
Antibacterial Assays. Eight bacterial strains, including Gram-
positive Bacillus cereus (ACCC 11077), Micrococcus tetragenus (ATCC
13623), Staphylococcus epidermidis (ATCC 12228), and S. aureus
(ATCC 27154) and Gram-negative Escherichia coli (ATCC 25922),
Vibrio anguillarum (ATCC 19019), V. parahemolyticus (ATCC 17802),
and Pseudomonas putida (ATCC 17848), were used for antibacterial
assay. The specific antibacterial assay was carried out as described
previously.21
2,6,2′,4′,6′-Pentabromo-4-methoxycarbonyldiorcinol (12a):
white powder; mp 180−181 °C; 1H NMR (600 MHz, CDCl3 δ, ppm)
11.77 (1H, s), 6.10 (1H, brs), 4.01 (3H, s), 2.68 (3H, s), 2.61 (3H, s);
13C NMR (150 MHz, CDCl3, δ, ppm) 170.4 (C), 158.3 (C), 152.8 (C),
148.8 (C), 148.6 (C), 140.1 (C), 137.5 (C), 110.3 (C), 110.3 (C), 108.3
(C), 107.1 (C), 100.6 (C), 100.0 (C), 52.4 (CH3), 24.1 (CH3), 22.8
(CH3); ESIMS m/z 682.6 [M − H]−; HRESIMS m/z 682.6375 (calcd
for C16H1079Br281Br3O5, 682.6378).
Cytotoxicity Assays. The cytotoxic activities against the K562 and
HL-60 cell lines were determined by the MTT method.23 Adriamycin
was used as a positive control.
1
3-O-Acetyldiorcinol (14a): colorless oil; H NMR (600 MHz,
CDCl3, δ, ppm, J/Hz) 6.70 (1H, brs), 6.64 (1H, brs), 6.54 (1H, t, J = 2.4
Hz), 6.42 (2H, brs), 6.30 (1H, t, J = 2.4 Hz), 5.00 (1H, brs), 2.32, (3H,
s), 2.27, (3H, s), 2.26, (3H, s); 13C NMR (150 MHz, CDCl3, δ, ppm)
168.7 (C), 156.8 (C), 155.8 (C), 150.3 (C), 140.2 (C), 139.8 (C), 116.2
(CH), 116.2 (CH), 111.3 (CH), 110.6 (CH), 108.6 (CH), 102.6 (CH),
20.5 (CH3), 20.5 (CH3), 20.2 (CH3); ESIMS m/z 295.0 [M + Na]+,
567.1 [2 M + Na]+; HRESIMS m/z 295.0941 (calcd for C16H16O4Na,
295.0941).
ASSOCIATED CONTENT
* Supporting Information
■
S
1H, 13C, HMQC, HMBC, and MS spectra of compounds 1, 2, 9,
10, and 13; NOESY spectra of 1 and 2; 1D NOE spectrum of 1;
1H, 13C, and MS spectra of compounds 12a and 14a−14g; CIF
file and X-ray crystallographic data for 3; specific rotations for
known compounds 3−8. These materials are available free of
charge via the Internet at http://pubs.acs.org.
3,3′-O-Diacetyldiorcinol (14b): colorless oil; 1H NMR (600 MHz,
CDCl3, δ, ppm, J/Hz) 6.71 (2H, brs), 6.67 (2H, brs), 6.55 (2H, t, J = 2.4
Hz), 2.32 (6H, s), 2.26 (6H, s); 13C NMR (150 MHz, CDCl3, δ, ppm)
168.7 (C × 2), 156.8 (C × 2), 150.8 (C × 2), 140.2 (C × 2), 116.8 (CH
× 2), 116.5 (CH × 2), 109.1 (CH × 2), 20.8 (CH3 × 2), 20.5 (CH3 × 2);
ESIMS m/z 337.1 [M + Na]+, 651.0 [2 M + Na]+; HRESIMS m/z
337.1051 (calcd for C18H18O5Na, 337.1046).
AUTHOR INFORMATION
Corresponding Author
■
1
3-O-Pentyldiorcinol (14c): colorless oil; H NMR (600 MHz,
*Tel/Fax: 86-532-82031536 (C.-Y.W.). Tel/Fax: 86-20-
84034096 (Z.-G.S.). E-mail: changyun@ouc.edu.cn (C.-Y.W.);
cesshzhg@mail.sysu.edu.cn (Z.-G.S.).
CDCl3, δ, ppm, J/Hz) 6.48 (1H, brs), 6.40 (2H, brs), 6.38 (2H, brs),
6.29 (1H, d, J = 2.4 Hz), 4.87 (1H, brs), 3.89 (2H, t, J = 6.6 Hz), 2.28
(3H, s), 2.26 (3H, s), 1.76 (2H, m), 1.34−1.42 (4H, m), 0.92 (3H, t, J =
7.2 Hz); 13C NMR (150 MHz, CDCl3, δ, ppm) 159.7 (C), 157.9 (C),
157.2 (C), 155.9 (C), 140.3 (C), 139.9 (C), 111.5 (CH), 111.4 (CH),
110.3 (CH), 110.1 (CH), 102.6 (CH), 102.3 (CH), 67.5 (CH2), 28.4
(CH2), 27.6 (CH2), 21.9 (CH3), 21.1 (CH3), 20.8 (CH3), 13.4 (CH2);
ESIMS m/z 299.5 [M − H]−, 599.2 [2 M − H]−; HRESIMS m/z
299.1649 (calcd for C19H23O3, 299.1642).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the Key Program of National
Natural Science Foundation of China (No. 41130858), the
National Natural Science Foundation of China (Nos. 40976077;
30901879; 81172977; 41176121), the Natural Science Founda-
tion of Shandong Province (ZR2011DQ019), the Program for
New Century Excellent Talents in University, Ministry of
Education of China (No. NCET-11-0472), and the Research
Fund for the Doctoral Program of Higher Education, Ministry of
Education of China (No. 20090132110002).
3,3′-O-Dipentyldiorcinol (14d): colorless oil; 1H NMR (600
MHz, CDCl3, δ, ppm, J/Hz) 6.46 (2H, brs), 6.39 (2H, brs), 6.36 (2H,
brs), 3.88 (4H, t, J = 6.6 Hz), 2.27 (6H, s), 1.74 (4H, dt, J = 13.8, 6.6 Hz),
1.34−1.43 (8H, m), 0.91 (6H, t, J = 6.6 Hz); 13C NMR (150 MHz,
CDCl3, δ, ppm) 159.6 (C × 2), 157.5 (C × 2), 139.8 (C × 2), 111.2 (CH
× 2), 109.8 (CH × 2), 101.9 (CH × 2), 67.4 (CH2 × 2), 28.3 (CH2 × 2),
27.6 (CH2 × 2), 21.8 (CH3 × 2), 21.0 (CH3 × 2), 13.4 (CH2 × 2);
ESIMS m/z 371.2 [M + H]+; HRESIMS m/z 371.2583 (calcd for
C24H35O3, 371.2581).
1
3-O-Propyldiorcinol (14e): colorless oil; H NMR (600 MHz,
REFERENCES
CDCl3, δ, ppm, J/Hz) 6.48 (1H, brs), 6.40 (2H, brs), 6.38 (2H, brs),
6.29 (1H, brs), 4.85 (1H, brs), 3.86 (2H, t, J = 6.6 Hz), 2.28 (3H, s), 2.26
(3H, s), 1.78 (2H, m), 1.01 (3H, t, J = 7.2 Hz); 13C NMR (150 MHz,
CDCl3, δ, ppm) 159.5 (C), 157.7 (C), 157.1 (C), 155.8 (C), 140.2 (C),
139.8 (C), 111.4 (CH), 111.2 (CH), 110.2 (CH), 109.9 (CH), 102.5
(CH), 102.1 (CH), 68.9 (CH2), 29.0 (CH2), 21.9 (CH3), 20.9 (CH3),
9.8(CH2); ESIMS m/z 273.1 [M + H]+; HRESIMS m/z 273.1486
(calcd for C17H21O3, 273.1485).
■
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