1788 Journal of Natural Products, 2006, Vol. 69, No. 12
Notes
Table 1. 1H NMR Data for Compounds 1-3a
NH2 (CH3)2+, 353.2474], which was further corroborated by the
NMR data. The structure of 2 was established by comparing its
NMR data with those of compound 1. The 1H and 13C NMR spectra
of 2 were very similar to those of 1 except for the presence of one
extra exchangeable proton (δ 8.15, 2H, brs) and two instead of
three methyls attached to nitrogen (1H δ 2.77, 6H, t, J ) 5.5 Hz;
13C δ 35.5), indicating the replacement of NH(CH3)3+ in 1 by NH2-
position
1
2
3
1
2
4.02 (t, 7.3)
2.37 (m)
4.02 (t, 7.1)
2.39 (m)
4.01 (t, 7.1)
2.38 (m)
3
4
5.36 (dt, 15.4, 6.1) 5.34 (dt, 15.4, 6.4) 5.34 (dt, 15.4, 6.5)
5.38 (dd, 15.4, 7.0) 5.40 (dd, 15.4, 7.3) 5.39 (dd, 15.4, 7.0)
5
6
7
8
10
11
12
NH
2.06 (m)
1.27 (m)
1.92 (m)
5.07 (t, 6.5)
1.58 (s)
0.94 (d, 6.6)
1.67 (s)
9.75 (brs)
2.07 (m)
1.27 (m)
1.92 (m)
5.07 (t, 6.5)
1.58 (s)
0.95 (d, 6.9)
1.68 (s)
8.15 (brs)
2.77 (t, 5.5)
2.06 (m)
1.26 (m)
1.92 (m)
5.07 (t, 6.4)
1.58 (s)
0.94 (d, 6.6)
1.67 (s)
7.55 (brs)
+
(CH3)2 in 2. Therefore the structure of 2 is dimethylammonium
5,9-dimethyl-(3E)-3,8-decadienyl-1-sulfate.
Compound 3 exhibited an anion peak at m/z 261.1155 in the
negative HRESIMS (calcd for C12H21O4S- 261.1161) and a
pseudomolecular ion at m/z 493.3322 in the positive HRESIMS
[calcd for C12H21O4S-‚C6H14NO+ + C6H14NO+, 493.3311], which
was further corroborated by the NMR data. On the basis of the 1H
NMR and 13C NMR spectra, 3 contained the same sulfate alkene
core structure as in 1 and 2, with a different ammoniun counterion.
N-CH3 2.96 (d, 3.7)
3′
5′
1′, 6′
2.94 (s)
2.21 (s)
1.46 (s)
1
The H NMR spectrum of 3 revealed three exchangeable protons
a Spectra were recorded in CDCl3 at 600 MHz for 1H using TMS as
internal standard.
at δ 7.55 (3H, brs) and no N-CH3 signals, as well as three
additional methyls and one additional methylene, which were also
observed by 13C NMR (methyls at δ 31.0, 25.7, 25.7 and methylene
at δ 49.9). Additionally, one carbonyl was observed at δ 208.7.
The HMBC data revealed that these carbons belonged to a 2-methyl-
4-oxobutan-2-yl ammonium ion (C6H14NO). Therefore the structure
of compound 3 is 2′-methyl-4′-oxobutan-2′-ylammonium 5,9-
dimethyl-(3E)-3,8-decadienyl-1-sulfate.
Since compounds 2 and 3 possess the same core structure and
nearly identical optical rotations as 1, they were assigned the same
absolute stereochemistry as 1. Compounds 1-3 were evaluated for
their cytotoxicities against the P388 and A-549 cell lines by the
MTT method.9 Only 3 was found to be weakly active against the
P388 cell line (IC50 290 µM).
While sulfated alkenes are common in marine invertebrates, those
that contain complex counterions are rare. To the best of our
knowledge, this sulfated carbon skeleton is reported for the first
time from nature and might represent a biodegradation product of
a sesquiterpene. Alternatively, these metabolites could result from
the addition of an acetate group to the oxidized end of a
monoterpene.
Table 2. 13C NMR Data for Compounds 1-3a
position
1
2
3
1
2
3
4
67.8 CH2
32.6 CH2
123.1 CH
139.1 CH
36.2 CH
68.3 CH2
32.6 CH2
123.0 CH
139.4 CH
36.3 CH
68.2 CH2
32.5 CH2
123.1 CH
139.3 CH
36.3 CH
5
6
7
8
9
36.9 CH2
25.6 CH2
124.5 CH
131.1 qC
17.6 CH3
20.5 CH3
25.5 CH3
45.3 CH3
37.0 CH2
25.7 CH2
124.6 CH
131.2 qC
17.7 CH3
20.5 CH3
25.7 CH3
35.5 CH3
37.0 CH2
25.7 CH2
124.6 CH
131.2 qC
17.7 CH3
20.5 CH3
25.7 CH3
10
11
12
N-CH3
2′
3′
4′
5′
1′, 6′
53.2 qC
49.9 CH2
208.7 qC
31.0 CH3
25.7 CH3
a Spectra were recorded in CDCl3 at 150 MHz for 13C using TMS
as internal standard.
Experimental Section
General Experimental Procedures. Melting points were measured
using a Yanaco MP-500D micro-melting point apparatus and were
uncorrected. Optical rotations were obtained on a JASCO P-1020 digital
polarimeter. UV spectra were recorded on a Beckmen DU 640
spectrophotometer. IR spectra were taken on a Nicolet Nexus 470
spectrophotometer in KBr disks. All NMR data were recorded on a
JEOL Eclips-600 spectrometer using TMS as internal standard, and
the chemical shifts were recorded as δ values. ESIMS was measured
on a Q-TOF Ultima Global GAA076 LC mass spectrometer. Semi-
prepartive HPLC was performed using a RP-18 column (YMC-Pack
ODS-A, 10 × 250 mm, 5 µm, 4 mL/min).
atmosphere of 5% CO2 and 95% air at 37 °C (tsFT210 cell line at 32
°C). A 200 µL portion of this cell suspension at a density of 5 × 104
cell mL-1 was plated in 96-well microtiter plates and incubated for 24
h at the above conditions. Then 2 µL of the test compound solutions
(in DMSO) at different concentrations was added to each well and
further incubated for 72 h in the same conditions. A 20 µL volume of
the MTT solution (5 mg/mL in IPMI-1640 medium) was added to each
well and incubated for 4 h. The medium (150 µL) containing MTT
was then replaced with DMSO and gently mixed to dissolve any
formazan crystals formed. UV absorbance was then measured on a
Spectra Max Plus plate reader at 540 nm.
Animal Material. T. hardwickii (5 kg) was collected in Jiaozhou
Bay, Qingdao (-15 m), and was frozen at -18 °C until extracted. The
animals were identified by Professor Xiaoqi Zeng, and a voucher
specimen of the organism is preserved at Ocean University of China.
Extraction and Isolation. The frozen animals were homogenized
in EtOH (15 L) and extracted for 12 h. After centrifugation of the extract
the solvent was removed and the concentrated aqueous residue was
partitioned against EtOAc and subsequently against n-BuOH. The
n-BuOH extract was subjected to Si gel CC using a step gradient of
CHCl3/MeOH, and the 85:15 CHCl3/MeOH fraction was collected.
Further chromatography on Sephadex LH-20 (MeOH) followed by
repeated C18 RP-HPLC (CH3CN/H2O, 1:3) gave compounds 1 (80 mg),
2 (10 mg), and 3 (15 mg).
Oxidation of Compound 1. To a solution of 424 mg (1.98 mmol)
NaIO4 in 5 mL of water was added 7.0 mg (0.044 mmol) of KMnO4.6
After stirring for 30 min at 25 °C, 30.4 mg (0.22 mmol) of anhydrous
K2CO3, 1.25 mL of t-BuOH, and 35 mg (0.11 mmol) of compound 1
in 1.25 mL of t-BuOH were added while maintaining the solution
temperature at 20-30 °C. The resulting reddish-purple suspension was
stirred for 2 h at 25 °C. After the addition of 0.1 mL (1.5 mmol) of
ethylene glycol and further stirring for 2 h, the reaction was acidified
to pH 4 with 1 M aqueous HCl and then extracted with Et2O (20 mL
× 3). The organic extract was washed with brine and dried over
anhydrous Na2SO4. The solvent was removed in vacuo, and the residue
was purified on RP-18 HPLC using CH3OH/H2O (4:6) as the mobile
phase to afford (2R)-2-methylpentanedioic acid (1a, 4.9 mg): [R]25
D
1
-16.8 (c 0.067, ethanol); H NMR (CDCl3, 600 MHz) δ 11.4 (2H,
Biological Assays. The fractions were assayed using the MTT
method9 with the mouse temperature-sensitive p34cdc2 mutant cell line
tsFT210. Cytotoxic activity of the three compounds was evaluated by
the MTT method using P388 and A-549 cell lines. The cell lines were
grown in RPMI-1640 supplemented with 10% FBS under a humidified
brs, COOH), 2.55 (1H, m, H-2), 2.47 (2H, t, J ) 7.1 Hz, H-4), 2.02
(1H, m, H-3a), 1.84 (1H, m, H-3b), 1.22 (3H, d, J ) 7.0 Hz, CH3); 13
C
NMR (CDCl3, 150 MHz) δ 182.6, 179.7, 38.6, 31.7, 28.1, 16.8; negative
ESIMS m/z 145 [M - H]-.