Cerebrosides of the halotolerant fungus Alternaria raphani isolated from a sea salt field

Article abstract of DOI:10.1021/np9002299

In order to search for structurally novel and bioactive natural compounds from marine-derived fungi, a halotolerant fungal strain (THW-18) identified as Alternaria raphani was isolated from sediment collected in the Hongdao sea salt field. From the ethyl

Full text of DOI:10.1021/np9002299

J. Nat. Prod. 2009, 72, 1695–1698
1695
Cerebrosides of the Halotolerant Fungus Alternaria raphani Isolated from a Sea Salt Field
Wenliang Wang,†,‡ Yi Wang,†,‡ Hongwen Tao,† Xiaoping Peng,† Peipei Liu,† and Weiming Zhu*,†
Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean UniVersity of China,
Qingdao 266003, People’s Republic of China
ReceiVed April 19, 2009
In order to search for structurally novel and bioactive natural compounds from marine-derived fungi, a halotolerant
fungal strain (THW-18) identified as Alternaria raphani was isolated from sediment collected in the Hongdao sea salt
field. From the ethyl acetate extract of Alternaria raphani, three new cerebrosides, alternarosides A-C (1-3), and a
new diketopiperazine alkaloid, alternarosin A (4), together with 15 known compounds were isolated and identified by
spectroscopic and chemical methods, as well as X-ray crystal diffraction analysis. Compounds 1-4 showed weak
antibacterial activity against Escherichia coli, Bacillus subtilis, and Candida albicans with MIC values ranging from 70
to 400 µM.
Halotolerant microorganisms are those microbes that are able to
grow well in variable salinity. The marine environment is the major
source of the halotolerant microorganisms. Some halotolerant
microorganisms living in extreme environments might activate silent
genes and induce unique biosynthetic pathways.1 Therefore,
investigating the secondary metabolites of halotolerant microbes
isolated from hypersaline ecological niches may increase the
chances of finding bioactive compounds with novel structures. In
order to screen halotolerant “talented strains”2 and find structurally
novel and bioactive secondary metabolites, we are continuing to
isolate and identify microbes from hypersaline environments. As a
result, a marine-derived halotolerant fungal strain (THW-18),
authenticated as Alternaria raphani, was isolated from the sediments
collected in the Hongdao sea salt field, Qingdao, China. Only five
metabolites, tetramic acid, alternariol, altenuene, altertoxin I, and
tenuazonic acid, have been previously identified from this fungus.3,4
The EtOAc extract of A. raphani, showing significant cytotoxicity
against the mouse cdc2 mutant cell line (tsFT210), was subjected
to flash column chromatography over silica gel, Sephadex LH-20
chromatography, and HPLC separation to afford three new cere-
broside compounds, alternarosides A-C (1-3), and a new dike-
topiperazine alkaloid, alternarosin A (4), together with 15 known
compounds, cerebroside C (5),5,6 bisdethiobis(methylthio)acetyl-
Analysis of the 1H-1H COSY spectrum led to the identification of
an isolated proton spin-system corresponding to the C-1-C-7
subunit of 1. HMBC correlations from H-6, H-7, and H-19 to the
carbonyl suggested that the carbonyl linked C-7 and C-9 (Figure
1). Methanolysis of 1 afforded methyl 2-hydroxyoctadeca-3-enoate,
which was identified by GC-MS (tR 18.61 min, m/z 312 [M]+,
Figure S13), and methyl glucopyranosides ([R]25D +75, ESIMS m/z
195 [M + H]+),20 indicating that 1 contained a C18-sphingosine
and a glucose moiety. ESIMS/MS showed key fragments at m/z
588.6 [M + H - 180]+, 570.6 [588.6 - H2O]+, 290.3 [570.6 -
280.3]+, and 280.3 [570.6 - 290.3]+ (Figures S10 and S16). The
loss of m/z 280.3 (C18H32O2) and the fragments at m/z 280.3
(C18H34NO) and 290.3 (C19H32NO) confirmed that the fatty acid
unit and sphingosine unit were 2-hydroxyoctadec-3-enoic acid and
2-amino-1,3-dihydroxy-9-methyleneoctadec-4-en-8-one, respec-
tively. Thus the constitution of 1 was established. Acidic hydrolysis
of the methyl glucopyranosides afforded D-glucose, which was
identified by directly comparing the specific rotation ([R]25D +49)
and Rf value (0.30 and 0.35/CHCl3-MeOH-H2O, 7:3:0.5) with
those of standard D/L-glucose ([R]25D +54/-56).21 The NMR signals
of the anomeric proton and carbon at δH/C 4.12 (d, J ) 7.8)/103.5
(CH) suggested the ꢀ-configuration of the glucoside. The coupling
constants of J3′,4′ (15.1 Hz) and J4,5 (15.6 Hz) suggested the
E-configuration of the 3′,4′- and 4,5-double bonds. The specific
rotation of the corresponding methyl 2R-hydroxyoctadec-3-enoate
aranotin (6),7 cerebroside D,5 acetylaranotin,8 N-acetyltyramine,9
cyclo-(Tyr-Pro),10 (22E,24R)-3ꢀ,5R-dihydroxy-23-methylergosta-
7,22-dien-6-one,11,12 (22E,24R)-3ꢀ,5R,9R-trihydroxyergosta-7,22-
dien-6-one,12 (22E,24R)-23-methylergosta-7,22-diene-3ꢀ,5R,6ꢀ-
triol,13 cerevisterol,14 6ꢀ-methoxyergosta-7,22-diene-3ꢀ,5R-diol,14
ergosterol peroxide,15 ergosterol,16 alterperylenol,17 and altertoxin
I.18,19 The new compounds exhibited weak antibacterial activity,
but did not show cytotoxicity against P388, A-549, BEL-7402, and
HL-60 cells (IC50 > 50 µM) and 1,1-diphenyl-2-picrylhydrazyl
(DPPH) radical scavenging activity (IC50 > 500 µM).
Alternaroside A (1) was obtained as a colorless, amorphous
powder. Its molecular formula was suggested as C43H77NO10
according to its HRESIMS at m/z 768.5662 [M + H]+. The 1D
NMR spectra of 1 indicated the presence of aliphatic chain(s), an
amide linkage, and a sugar residue, which strongly suggested a
cerebroside structure for 1 (Table 1). Except for terminal double-
bond signals (δH/C 6.09 and 5.79/124.5, CH2) and a carbonyl signal
(δC 200.7, qC) instead of an allylic methyl signal (δH/C 1.54/15.7,
CH3) and a methine signal (δH/C 5.09/123.5, CH), respectively, the
1D NMR spectra of 1 were similar to those of cerebroside C (5).5,6
* To whom correspondence should be addressed. Tel.: 0086-532-
82031268. Fax: 0086-532-82031268. E-mail: weimingzhu@ouc.edu.cn.
† Ocean University of China.
‡ These authors made equal contribution to this paper.
10.1021/np9002299 CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/17/2009

1696 Journal of Natural Products, 2009, Vol. 72, No. 9
Notes
Table 1. 1H and 13C NMR Data for 1-3 (600, 150 MHz, DMSO-d6, TMS, δ ppm)
1
2
3
no.
1
δC
δ
H (J in Hz)
δC
δ
H (J in Hz)
δC
δH (J in Hz)
68.5, CH2
3.94, m;
68.6, CH2
3.94, m;
68.6, CH2
3.95, m;
3.51, dd (10.2, 4.1)
3.78, m
3.98, m
5.40, dd (15.6, 6.9)
5.58, dt (15.6, 6.4)
2.16, m
3.51, dd (10.6, 4.1)
3.78, m
3.97, m
5.37, dd (15.6, 6.9)
5.57, dt (15.6, 6.2)
1.94, m
3.51, dd (10.1, 4.1)
3.78, m
3.97, m
5.36, dd (15.6, 6.9)
5.56, dt (14.2, 6.2)
1.97, m
2
3
4
5
6
7
8
9
52.9, CH
70.5, CH
131.3, CH
130.0, CH
26.6, CH2
36.7, CH2
200.7, qC
147.9, qC
30.3, CH2
28.0, CH2
29.1, CH2
29.1, CH2
31.3, CH2
22.1, CH2
13.9, CH3
124.5, CH2
172.0, qC
71.9, CH
129.0, CH
131.0, CH
31.7, CH2
29.1, CH2
31.3, CH2
22.1, CH2
13.9, CH3
103.5, CH
73.4, CH
76.6, CH
70.0, CH
76.9, CH
61.1, CH2
52.9, CH
70.5, CH
130.9, CH
130.9, CH
27.4, CH2
32.1, CH2
123.5, CH
134.9, qC
39.1, CH2
27.3, CH2
29.1, CH2
28.7, CH2
22.1, CH2
13.9, CH3
15.7, CH3
52.9, CH
70.5, CH
131.0, CH
130.7, CH
32.0, CH2
28.6, CH2
129.5, CH
130.2, CH
28.7, CH2
31.9, CH2
29.0, CH2
28.9, CH2
31.3, CH2
22.1, CH2
13.9, CH3
2.73, t (7.6)
1.94, m
5.09, br t (6.2)
1.97, m
5.38, “t”-like (3.3)
5.38, “t”-like (3.3)
1.97, m
1.23, m
1.23, m
1.23, m
1.23, m
1.23, m
0.85, t (6.8)
10
11
12-14
15
16
17
18
19
1′
2.15, m
1.23, m
1.23, m
1.23, m
1.23, m
1.23, m
0.85, t (6.9)
6.09, br s; 5.79, br s
1.94, m
1.23, m
1.23, m
1.23, m
1.23, m
0.85, t (6.9)
1.54, br s
172.0, qC
71.9, CH
129.0, CH
130.9, CH
31.6, CH2
29.0, CH2
31.3, CH2
22.1, CH2
13.9, CH3
103.5, CH
73.4, CH
76.5, CH
70.0, CH
76.9, CH
61.0, CH2
172.0, qC
71.9, CH
129.0, CH
130.9, CH
31.7, CH2
29.0, CH2
31.3, CH2
22.1, CH2
13.9, CH3
103.5, CH
73.4, CH
76.5, CH
70.0, CH
76.9, CH
61.0, CH2
2′
4.30, br t (5.1)
5.44, dd (15.1, 5.1)
5.67, dt (15.1, 6.9)
1.93, dt (6.9, 6.8)
1.23, m
1.23, m
1.23, m
0.85, t (6.6)
4.12, d (7.8)
2.96, m
3.14, m
3.05, m
3.09, m
3.67, br dd (11.0, 6.0)
3.46, ddd (11.0, 6.0, 5.0)
7.39, d (9.1)
4.97, m
5.78, d (5.0)
4.99, m
4.30, br t (4.6)
5.44, dd (15.4, 5.3)
5.68, dt (15.4, 6.4)
1.94, m
1.23, m
1.23, m
4.30, br t (5.1)
5.43, dd (15.1, 5.5)
5.68, dt (15.1, 6.9)
1.97, m
1.23, m
1.23, m
3′
4′
5′
6′-15′
16′
17′
18′
1′′
2′′
3′′
4′′
5′′
6′′
1.23, m
1.23, m
0.85, t (6.9)
4.11, d (7.8)
2.96, m
3.14, m
3.04, m
0.85, t (6.8)
4.12, d (7.8)
2.96, m
3.14, m
3.04, m
3.08, m
3.09, m
3.66, br dd (9.7, 6.0)
3.43, ddd (9.7, 6.0, 5.5)
7.39, d (9.7)
4.95, d (5.5)
5.78, d (5.0)
4.98, d (4.6)
4.92, d (5.0)
4.94, d (4.6)
4.52, t (6.0)
3.67, ddd (11.3, 5.9, 1.9)
3.43, ddd (11.3, 5.9, 5.0)
7.39, d (9.6)
4.95, d (5.5)
5.78, d (4.6)
4.99, d (4.6)
4.92, d (5.0)
4.93, d (5.0)
4.52, t (5.9)
NH
OH-3
OH-2′
OH-2′′
OH-3′′
OH-4′′
OH-6′′
4.93, m
4.94, m
4.53, t (6.0)
([R]20D -42) indicated the R-configuration at C-2′ ([R]25D -45.3).22
at m/z 280.3 (C18H34NO) and 262.3 (C18H32N) corresponded to a
sphingosine unit with a methylated odd-carbon straight chain and
a hydroxyloctadecenoyl moiety. The coincidence of NMR data from
C-1 to C-10 and the similar specific rotation to that of cerebroside
C (5)6 supported the configuration of the sphingosine unit as
(2S,3R,4E,8E). Methanolysis of 2 also afforded methyl 2R-
hydroxyoctadec-3-enoate and methyl D-glucopyranosides. Accord-
ingly, the structure of 2 was established as nor-cerebroside C, i.e.,
(2R,3E)-2-hydroxy-N-[(2S,3R,4E,8E)-l-ꢀ-D-glucopyranosyloxy-3-
hydroxy-9-methylheptadec-4,8-dien-2-yl]octadec-3-enamide.
Alternaroside C (3) was obtained as a colorless, amorphous
powder, and HRESIMS at m/z 740.5656 [M + H]+ suggested the
same molecular formula as for 2. The NMR spectra of 3 were quite
similar to those of 5 except for the substitution of an olefinic
methine resonance (δH/C 5.38/130.2) for the vinyl methyl signal
(δH/C 1.54/15.7) and the olefinic quaternary carbon signal (δC 134.8).
The values of δC-1-C-5 and δC-1′-C-4′ and the specific rotation ([R]20
D
-11) of 1 were close to those of cerebroside C (5) ([R]20 -8),
D
suggesting that 1 shared the same 2S,2′R,3R-configuration with 5.
Thus, the structure of 1 is proposed as (2R,3E)-2-hydroxy-N-
[(2S,3R,4E)-l-ꢀ-D-glucopyranosyloxy-3-hydroxy-9-methylene-8-
oxooctadec-4-en-2-yl]octadec-3-enamide.
The molecular formula of alternaroside B (2) was determined
to be C42H77NO9 by HRESIMS at m/z 740.5666 [M + H]+. The
1H and 13C NMR data of 2 were almost the same as those of
cerebroside C (5),5,6 except for subtle differences in the chain length
of the sphingosine unit. The ESIMS/MS showed key fragments at
m/z 560.6 [M + H - 180]+, 542.6 [560.6 - H2O]+, 524.6 [542.6
- H2O]+, 281.3 [560.6 - 279.3]+, 280.3 [560.6 - 280.3 or 524.6
- 244.3]+, and 262.3 [280.3 - H2O or 524.6 - 262.3]+ (Figures
S11 and S17). The loss of m/z 279.3 (C18H33NO) and the fragments
1
Figure 1. Key H-1H COSY and HMBC correlations of 1 and 3.

Notes
Journal of Natural Products, 2009, Vol. 72, No. 9 1697
Table 2. 1H and 13C NMR Data for 4 (600, 150 MHz, DMSO-d6, TMS, δ ppm)
no.
δC
δH (J in Hz)
no.
δH (J in Hz)
δC
1
2
3
163.9, qC
71.0, qC
39.2, CH2
1′
2′
3′
164.3, qC
69.8, qC
38.9, CH2
3.25, dt (15.1, 2.3)
2.96, br d (15.1)
3.13, dt (15.1, 2.3)
2.94, br d (15.1)
4
5
7
8
9
10
111.0, qC
137.0, CH
139.9, CH
105.3, CH
71.1, CH
59.6, CH
14.0, CH3
20.6, CH3
169.4, qC
4′
5′
7′
8′
9′
111.1, qC
136.5, CH
137.8, CH
110.4, CH
71.1, CH
63.0, CH
14.1, CH3
6.78, t (2.3)
6.68, t (2.3)
6.45, dd (8.2, 2.3)
4.72, dd (8.2, 2.3)
5.62, dt (8.3, 2.3)
4.96, br d (8.3)
2.21, s
6.29, dd (8.3, 2.3)
4.80, dd (8.3, 2.3)
4.38, tt (7.3, 2.3)
4.75, br d (7.3)
2.20, s
10′
2-SCH3
9-O COCH3
9-O COCH3
2′-SCH3
9′-OH
1.98, s
5.29, d (7.3)
Experimental Section
Table 3. MIC Values of Antibacterial Activities against
Escherichia coli, Bacillus subtilis, and Candida albicans for
Compounds 1-4 (µM)
General Experimental Procedures. Specific rotations were obtained
on a JASCO P-1020 digital polarimeter. UV spectra were recorded on
a Beckman DU 640 spectrophotometer. NMR data were recorded on a
JEOL JNM-ECP 600 spectrometer using TMS as internal standard,
and chemical shifts were recorded as δ values. ESIMS was measured
on a Q-TOF ULTIMA GLOBAL GAA076 LC mass spectrometer.
Vacuum-liquid chromatography (VLC) utilized silica gel H (Qingdao
Marine Chemical Factory, Qingdao, China). Semipreparative HPLC
was performed using an ODS column [YMC-pack ODS-A, 10 × 250
mm, 5 µm, 4 mL/min]. The GC-MS system consisted of an Agilent
6890 gas chromatograph and an Agilent 5973 mass selective detector
in the electron-ionization mode.
Fungal Material. The fungus THW-18 was isolated from sediment
collected in the Hongdao sea salt field, Qingdao, China. According to
its morphological characteristics and 18S rRNA sequences (Supporting
Information; GenBank GQ354822), THW-18 was identified as Alter-
naria raphani by Prof. Chengxiang Fang, China Center for Type Culture
Collection. The voucher specimen was deposited in our laboratory at
-80 °C. The producing strain was prepared on potato dextrose agar
slants and stored at 4 °C.
Fermentation and Extraction. A. raphani was grown under static
conditions at 20 °C for 45 days in 250 1 L conical flasks containing
the liquid medium (300 mL/flask, pH 7.0) composed of mannitol (20
g/L), maltose (20 g/L), glucose (10 g/L), monosodium glutamate (10
g/L), KH2PO4 (0.5 g/L), MgSO4 ·7H2O (0.3 g/L), yeast extract (3 g/L),
corn steep liquor (1 g/L), and sea salt (100 g/L). The fermented whole
broth (75 L) was filtered through cheese cloth to be separated into filtrate
and mycelia. The filtrate was concentrated under reduced pressure to
about 25% the original volume and then extracted three times with
equal volumes of EtOAc to give an EtOAc solution, while the mycelia
were extracted three times with acetone. The acetone solution was
concentrated under reduced pressure to afford an aqueous solution. The
aqueous solution was extracted three times with equal volumes of
EtOAc to give another EtOAc solution. The EtOAc extracts were
combined and concentrated under reduced pressure to give a crude
extract (42 g).
Purification. The extract (42 g), showing a cytotoxic effect on K562
cells at 100 µg/mL, was separated into five fractions on a silica gel
VLC column using step gradient elution with CHCl3-petroleum ether
(0-100%) and then with MeOH-CHCl3 (0-50%). Fraction 2 (1.5 g)
was further separated into seven subfractions by Sephadex LH-20,
eluting with MeOH-CHCl3 (1:1). Ergosterol (175 mg) was obtained
from fraction 2-1 after recrystallization from MeOH-CHCl3. Fraction
3 (1.8 g) was subjected to Sephadex LH-20, eluting with MeOH-CHCl3
(1:1), to afford six subfractions. Subfraction 3-2 was (121 mg) further
purified by semipreparative HPLC (70% MeOH-H2O, 4.0 mL/min)
to yield 4 (6.2 mg), 6 (42 mg), and acetylaranotin (26 mg). Ergosterol
peroxide (48 mg) was obtained from subfraction 3-3 after recrystalli-
zation from MeOH-CHCl3. The mother liquor of fraction 3-3 was
purified by semipreparative HPLC (gradient elution 70-100% MeOH,
4.0 mL/min) to afford 6ꢀ-methoxyergosta-7,22-diene-3ꢀ,5R-diol (7.3
mg) and (22E,24R)-23-methylergosta-7,22-diene-3ꢀ,5R,6ꢀ-triol (8.9
mg). Fraction 4 (8.5 g) was separated into 17 subfractions by Sephadex
LH-20, eluting with MeOH-CHCl3 (1:1). Subfractions 4-3 and 4-4
were combined and purified by semipreparative HPLC (gradient elution
92-100% MeOH, 4.0 mL/min) to afford 1 (22.8 mg), 2 (16.2 mg), 3
1
2
3
4
Escherichia coli
Bacillus subtilis
Candida albicans
130
130
260
135
68
270
135
135
270
203
203
406
These data combined with the same ESIMS/MS pattern as that of
2 (Figures S12 and S18) indicated a hydroxyoctadecenoyl moiety
1
and a 2-amino-1,3-dihydroxyoctadec-4,8-diene moiety. A H-1H
COSY experiment showed the connections from C-1 to C-6 and
from C-2′ to C-4′. HMBC correlations from H-5 (δ 5.56) and H-8
(δ 5.38) to C-6 (δ 32.0), C-7 (δ 28.6), and C-10 (δ 28.7) located
the second double bond at C-8/C-9 in 3 (Figure 1). Methanolysis
of 3 gave methyl 2R-hydroxyoctadec-3-enoate and methyl D-
glucopyranosides. The “t”-like multiplet of two olefinic protons (δ
5.38) and the chemical shifts of C7 and C10 (δ 28.7 and 28.9,
respectively) indicated the Z-configuration of the 8,9-double bond.23
Thus, the structure of 3 was established as (2R,3E)-2-hydroxy-N-
[(2S,3R,4E,8Z)-l-ꢀ-D-glucopyranosyloxy-3-hydroxyoctadec-4,8-
dien-2-yl]octadec-3-enamide.
Alternarosin A (4) was obtained as a colorless, amorphous
powder. Its molecular formula was determined as C22H24N2O7S2
according to its HRESIMS peak at m/z 515.0917 [M + Na]+.
Diagnostic IR absorption peaks were observed for hydroxy and
amide carbonyl groups at 3418 and 1648 cm-1, respectively. Its
1D NMR spectra revealed three carbonyls, four quaternary carbons
(two sp2), 10 methines (six sp2), two methylenes, and three methyl
groups (Table 2). Except for the absence of an acetyl group, the
1D NMR spectra of 4 were quite similar to those of bisdethiodi(m-
ethylthio)acetylaranotin (6),7 implying that 4 was the 9- or
9′-deacetyl derivative of 6. This conclusion was further confirmed
by the HMBC correlations between 9′-OH (δH 5.29, s) and C-8′
(δC 110.4, CH), C-9′ (δC 71.1, CH), and C-10′ (δC 63.0, CH).
Acetylation of 4 with acetic anhydride in pyridine afforded
compound 6, and the absolute configuration of 6 was determined
by single-crystal X-ray diffraction analysis.24 Thus, the structure
of
4 was elucidated as 9′-O-deacetylbisdethiobis(methylth-
io)acetylaranotin.
The new isolates 1-4 were evaluated for cytotoxicity against
P388 and HL-60 cancer cells with the SRB method,25 and the A549
and BEL-7402 cancer cells with the MTT methods.26 Their
antimicrobial activities against Escherichia coli, Bacillus subtilis,
and Candida albicans were also evaluated by an agar dilution
method.27 Compounds 1-4 were also evaluated for their DPPH
radical scavenging activity.28 Compounds 1-4 showed very weak
antimicrobial activities against Escherichia coli, Bacillus subtilis,
and Candida albicans with MIC values from 70 to 400 µM (Table
3), while they showed no cytotoxic effect on the four cancer cell
lines (IC50 >100 µM) and no DPPH radical scavenging activity (IC50
> 500 µM).

1698 Journal of Natural Products, 2009, Vol. 72, No. 9
Notes
(14.3 mg), 5 (104 mg), and cerebroside D (59 mg). Subfractions
4-6-4-9 were combined and crystallized from MeOH-CHCl3 (1:1)
to yield cerevisterol (255 mg). The mother liquor was further purified
by semipreparative HPLC (gradient elution 85-100% MeOH, 4.0 mL/
min) to afford (22E,24R)-3ꢀ,5R-dihydroxy-23- methylergosta-7,22-dien-
6-one (9.1 mg) and (22E,24R)-3ꢀ,5R,9R-trihydroxyergosta-7,22-dien-
6-one (7.1 mg). Subfractions 4-11-4-13 were combined and purified
by semipreparative HPLC (gradient elution 45-60% MeOH, 4.0 mL/
min) to yield alterperylenol (4.6 mg) and altertoxin I (7.7 mg).
N-Acetyltyramine (21 mg) and cyclo-(Tyr-Pro) (18 mg) were isolated
from subfraction 4-16 by semipreparative HPLC (gradient elution
15-40% MeOH, 4.0 mL/min).
and 13C NMR data, Table 2; HRESIMS m/z 515.0917 [M + Na]+ (calcd
for C22H24N2O7S2Na, 515.0923).
Cerebroside C (5): colorless, amorphous powder; [R]20D -8 (c 0.1,
MeOH); 1H NMR and 13C NMR data were consistent with those
reported in the literature;6 ESIMS m/z 754.8 [M + H]+, 736.8 [M +
H - H2O]+.
Acknowledgment. This work was supported by the Chinese National
Natural Science Fund (Nos. 30470196 and 30670219) and the Chinese
National High Technology Research and Development Program
(2007AA091502). The cytotoxicity assay was performed at the
Shanghai Institute of Materia Medica, Chinese Academy of Sciences.
The working strain THW-18 was identified by Prof. Chengxiang Fang,
China Center for Type Culture Collection.
Methanolysis of 1. Compound 1 (10 mg) was refluxed with 5%
HCl-MeOH (4 mL) for 10 h. The reaction mixture was immediately
cooled and extracted with n-hexane. The n-hexane layer was concen-
trated to afford methyl 2R-hydroxyoctadec-3-enoate (3.2 mg): [R]20
Supporting Information Available: 1D NMR spectra of 1-4, X-ray
data for 6, ESIMS/MS of 1-3, GC-MS of methanolysis products of
1-3, and bioassay protocols used. This material is available free of
charge via the Internet at http://pubs.acs.org.
D
-42 (c 0.1, CHCl3); GC-MS (30 m × 0.32 mm × 0.25 µm HP-5 MS
column: He, 1 mL/min; 40 °C, 2 min, 40-250 °C, ∆ 15 °C/min, 250
°C, 10 min); tR 18.61 min; m/z (rel int) 312 (M+, 0.6), 253 (M+ - 59,
98), 151 (5), 137 (9), 123 (21), 109 (46), 95 (75), 83 (56), 81 (55), 57
(100), 43 (38); 1H NMR (CDCl3, 600 MHz) δ 0.88 (3H, t, J ) 6.8 Hz,
H-18), 1.25 (24H, m, H-6-H-17), 2.06 (2H, q, J ) 6.8 Hz, H-5), 2.88
(1H, d, J ) 6.0 Hz, 2-OH), 3.80 (3H, 1-OCH3), 4.61 (1H, dd, J ) 6.0,
6.8 Hz, H-2), 4.98 (1H, dd, J ) 16.5, 6.8 Hz, H-3), 5.88 (1H, dt, J )
16.5, 6.8 Hz, H-4). After adding 5 mL of H2O, the aqueous MeOH
layer was concentrated under reduced pressure to remove residual HCl.
The residue was partitioned between H2O and EtOAc. The H2O layer
was evaporated in vacuo, and the residue was purified by a Sephadex
LH-20 column with MeOH to yield methyl D-glucopyranosides (2.1
mg): [R]25D +75 (c 0.05, MeOH); ESIMS m/z 195 [M + H]+; Rf 0.50/
0.56 (CHCl3-MeOH-H2O, 7:3:0.5). The obtained methyl D-glucopy-
ranosides (2.1 mg) were refluxed with 2 N HCl in H2O (1 mL) for 2 h,
and the reaction mixture was neutralized with NaHCO3 and concentrated
in vacuo. The reaction solid was dissolved in MeOH (10 mL), and the
solution was concentrated in vacuo to give D-glucose (1.6 mg) as a
colorless syrup, which was directly compared with the standard sample
References and Notes
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by Rf value (0.30 and 0.35/CHCl3-MeOH-H2O, 7:3:0.5) and [R]25
D
(+49 vs standard D-glucose +54/L-glucose -56) (c 0.1, H2O). The
same results were also obtained from the methanolysis of 2 and 3.
Chemical Transformation of 4 to 6. Compound 4 (3 mg) was
dissolved in 3 mL of dry pyridine, and Ac2O (10 µL) was added under
an Ar atmosphere at rt. The mixture was stirred at 40 °C for 1 h, and
then 10 mL of H2O was added to quench the reaction. The mixture
was extracted three times with EtOAc (5 mL each). The EtOAc was
evaporated, and the residue was purified by HPLC to afford 6 (1.8
mg): [R]20 -465 (c 0.66, CHCl3), lit. [R]26 -315.1 (c 1, CHCl3).29
D
D
Alternaroside A (1): colorless, amorphous powder; [R]20 -11 (c
D
0.6, MeOH); UV (MeOH) λmax (log ε) 201 (3.4) nm; IR (KBr) νmax
1
3429, 3155, 2836, 1633, 1567, 1056, 1011, 941 cm-1; H NMR and
13C NMR data, Table 1; HRESIMS m/z 768.5662 [M + H]+ (calcd for
C43H78NO10, 768.5625); ESIMS/MS m/z 768.7 [M + H]+, 750.7 [M
+ H - H2O]+, 588.6 [M + H - 180]+, 570.6 [588.6 - H2O]+, 290.3,
and 280.3.
Alternaroside B (2): colorless, amorphous powder; [R]20 -9 (c
D
0.1, MeOH); UV (MeOH) λmax (log ε) 202 (3.2) nm; IR (KBr) νmax
1
3418, 3133, 2819, 1619, 1551, 1048, 1008, 937 cm-1; H NMR and
13C NMR data, Table 1; HRESIMS m/z 740.5666 [M + H]+ (calcd for
C42H78NO9, 740.5677); ESIMS/MS m/z 740.6 [M + H]+, 722.7 [M +
H - H2O]+, 560.6 [M + H - 180]+, 542.6 [560.6 - H2O]+, 524.6
[542.6 - H2O]+, 281.3, 280.3, and 262.3.
Alternaroside C (3): colorless, amorphous powder; [R]20 -4 (c
D
0.1, MeOH); UV (MeOH) λmax (log ε) 201 (3.2) nm; IR (KBr) νmax
1
3430, 3126, 2822, 1648, 1571, 1061, 1021, 962 cm-1; H NMR and
13C NMR data, Table 1; HRESIMS m/z 740.5656 [M + H]+ (calcd for
C42H78NO9, 740.5677); ESIMS/MS m/z 740.6 [M + H]+, 722.7 [M +
H - H2O]+, 560.6 [M + H - 180]+, 542.6 [560.6 - H2O]+, 524.6
[542.6 - H2O]+, 280.3, and 262.3.
(28) Chen, Y.; Wong, M.; Rosen, R. T.; Ho, C.-T. J. Agric. Food Chem.
1999, 47, 2226–2228.
(29) DeLong, D. C.; Lively, D. H.; Neuss, N. U.S. Patent 3,907,988, 1975.
Alternarosin A (4): colorless, amorphous powder; [R]20 -85 (c
D
1.0, CHCl3); UV (MeOH) λmax (log ε) 223 (4.1) nm, 285 (3.6) nm; IR
(KBr) νmax 3418, 3126, 1740, 1648, 1230, 1059, 1031 cm-1; 1H NMR
NP9002299