Tetrahedron 61 (2005) 883–887
New glycolipid inhibitors of Myt1 kinase
Bing-Nan Zhou,a Shoubin Tang,a Randall K. Johnson,b Michael P. Mattern,b John S. Lazo,c
Elizabeth R. Sharlow,c Kim Harichd and David G. I. Kingstona,*
a
Department of Chemistry, M/C 0212, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA
b
GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, King of Prussia, PA 19406-0939, USA
c
Department of Pharmacology, University of Pittsburgh, Pittsburgh, PA, USA
d
Department of Biochemistry, M/C 0459, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA
Received 5 May 2004; revised 9 November 2004; accepted 9 November 2004
Available online 10 December 2004
Abstract—A crude extract of a marine alga showed activity against the enzyme Myt1 kinase. Bioassay-directed fractionation led to the
isolation of two bioactive glycoglycerolipids. Lipid 1 was identified as sn-1,2-dipalmityl-3-(N-palmityl-6-deoxy-6-amino-a-D-glucosyl)
glycerol and lipid 2 as sn-1-palmityl-2-myristyl-3-(N-stearyl-6-deoxy-6-aminoglucosyl)glycerol. Compounds 1 and 2 had IC50 values of 0.12
and 0.43 mg/mL, respectively, in the Myt1 kinase inhibitory bioassay, and were inactive against Akt and Chk1 kinases.
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
The enzyme Myt1 kinase, which is a Thr-14 and Tyr-15
specific cdc2 kinase, has been shown to be an important
regulator of cdc2/cyclin B kinase activity. It has been
reported that the inhibitory phosphorylation of cdc2 is
important for the timing of entry into mitosis, and studies
have shown that premature activation of cdc2 leads to
mitotic catastrophe and cell death.1–3 Inhibition of Myt1
kinase is predicted to cause premature activation of cdc2,
and inhibitors of this enzyme would thus be expected to kill
rapidly proliferating cells and abrogate normal cell cycle
checkpoints. Such inhibitors would thus be attractive for the
treatment of cancer, because they could be used in
conjunction with conventional chemotherapies to overcome
drug resistance and enhance their cytotoxicity by abrogating
cell cycle checkpoints.
As a part of our systematic search for potential anti-cancer
agents from plants and marine organisms,4–6 the methanol
extract of an alga designated UM 2972M was found to show
activity in a bioassay for inhibitors of Myt1 kinase, with an
IC50 of 4 mg/mL. It was thus selected for fractionation for
isolation of its bioactive constituents.
The algae comprise a large group of marine organisms, and
have been subjected to intensive chemical studies. Many
Keywords: Marine; Myt1 kinase; Glycolipid.
* Corresponding author. Tel.: C1 540 231 6570; fax: C1 540 231 3255;
e-mail: dkingston@vt.edu
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.11.013
bioactive compounds, including the neurotoxic amino acids
kainic acid and domoic acid7 and the hypocholesterolemic
betain lipids8 have been isolated from them, as well as
carotenoids,9 steroids,10 halogenated compounds,11,12 fatty
acids,13–17 phenolic compounds,18,19 and terpenoids.20–22
2. Results and discussion
The methanol extract (UM 2972 M) from an unknown algal
species was subjected to partition between aqueous MeOH
and organic solvents, and the aqueous MeOH fraction was
then stripped of its MeOH and extracted with n-butanol to
give a bioactive n-butanol fraction (IC50Z1 mg/mL).
Column chromatography of the n-butanol fraction on
Sephadex LH 20 with elution with CH2Cl2–MeOH (3:1),
followed by repeated chromatography on RP-18 reversed
phase silica gel with MeOH–H2O (9:1) gave the two
bioactive compounds 1 and 2. Compounds 1 (1.05 mg,
0.025%) and 2 (1.46 mg, 0.033%) had activities of 0.12 and
0.43 mg/mL, respectively, in the Myt1 kinase bioassay.
Compound 1 had a molecular weight of 967, as indicated by
a pseudomolecular ion (MCNa)C at m/zZ990 in its
MALDI TOF mass spectrum, and a composition of
C57H109NO10, as indicated by FAB-HRMS. Three spin–
spin systems were identified from its 1H NMR and COSY
spectra (Table 1). The first spin system indicated the
presence of a 1,2 diacylated glyceryl moiety [dH 4.51 ppm
(1H, dd, JZ11.9, 3.1 Hz, Hsn-1a), 4.18 ppm (1H, dd, JZ
12.1, 7.0 Hz, Hsn1a), 5.30 ppm (1H, m, Hsn-2), 4.11 ppm
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B.-N. Zhou et al. / Tetrahedron 61 (2005) 883–887
Table 1. Partial NMR data of lipids 1 and 2a,b
Position
sn-1
sn-2
sn-3
G-1
G-2
G-3
G-4
G-5
G-6
CH3–
(–CH2–)n
a-CH2–
b-CH2–
–COO–
–COO–
–COO–
a
b
1
2
dH and J
dC
dH and J
dC
4.51 (dd, JZ12.0, 3.0)
4.18 (dd, JZ12.0, 6.9)
5.31 (m)
4.10 (dd, JZ10.8, 5.2)
3.57 (dd, JZ10.8, 6.4)
4.76 (d, JZ3.6)
3.40 (dd, JZ3.6, 9.5)
3.63 (dd, JZ9.5, 9.6)
3.08 (dd, JZ9.6, 9.2)
4.07 (ddd, JZ9.2, 9.4, 2.0)
3.34 (dd, JZ14.4, 2.0)
2.91 (dd, JZ14.4, 9.4)
0.89 (3!CH3–, t, JZ6.8)
1.0–1.5
2.33 (3!–CH2–, m)
1.59 (3!–CH2–, m)
64.3
4.50 (dd, JZ12.0, 3.0)
4.18 (dd, JZ12.0, 6.9)
5.30 (m)
4.10 (dd, JZ10.7, 5.3)
3.56 (dd, JZ10.7, 6.3)
4.75 (d, JZ3.7)
3.39 (dd, JZ3.6, 9.6)
3.62 (dd, JZ9.6, 9.7)
3.07 (dd, JZ9.7, 9.3)
4.06 (ddd, JZ9.3, 8.4, 2.0)
3.34 (dd, JZ14.3, 2.0)
2.91 (dd, JZ14.3, 8.4)
0.89 (3!CH3–, t, JZ7.0)
1.0–1.5
2.33 (3!–CH2–, m)
1.60 (3!–CH2–, m)
64.3
71.7
67.1
101.0
73.5
75.0
74.9
69.9
54.2
14.5
35.0, 35.2
26.0
174.4
175.2
175.2
71.7
67.1
100.0
73.5
75.0
74.9
68.9
54.2
14.5
35.0; 35.2
26.0
174.4
175.2
175.1
d values are in ppm and J values are in Hz.
Chemical shifts were assigned by DQ COSY, HMQC, and HMBC spectra.
(1H, dd, JZ11.8, 5.2 Hz, Hsn-3a), and 3.57 ppm (1H, dd, JZ
11.8, 6.4 Hz, Hsn-3b)]. The cross-peaks in the HMBC
spectrum [dH/dC: 5.31 (Hsn-2)/174.4 (COO); 2.33 (a-CH2)/
174.4 (COO); 4.51 and 4.18 (Hsn-1)/175.2 (COO); 2.33
(a-CH2)/175.2 (COO)] also indicated the presence of acyl
groups on the 1 and 2 positions of a glycerol moiety. The
second group of spin systems contained signals for three
long chain fatty acids [dH 0.89 ppm (9H, t, JZ6.83 Hz, 3!
CH3–), 1–1.5 ppm (multi –CH2–) 1.59 ppm (6H, m, 3!bCH2–), 2.33 ppm (6H, m, 3!a-CH2–). The third spin
system indicated the presence of a glycosyl moiety [dH
4.76 ppm (1H, d, JZ3.8 Hz, HG-1), 3.40 ppm (1H, dd, JZ
9.5, 3.8 Hz, HG-2), 3.63 ppm (1H, d, JZ9.5, 9.1 Hz, HG-3),
3.08 ppm (1H, dd, JZ9.1, 9.7 Hz, HG-4), 4.07 ppm (1H, m,
HG-5), and 2.91 (1H, dd, JZ14.3, 9.3 Hz, HG-6a), and
3.34 ppm (1H, d, JZ14.1, 2.1 Hz, HG-6b)]. The HMBC
cross-peak dH 4.76 (HG-1)/dC 67.1 (Csn-3) showed that
glycosylation was on the 3 position of the glycerol moiety,
and that 1 was a glycoglycerolipid. Its 13C NMR spectrum
supported this conclusion. The chemical shifts and the
coupling constants of the G-1 to G-4 protons of the glycosyl
moiety were very close to those of methyl a-D-glucoside,
indicating that these hydroxyl groups were unacylated. The
chemical shifts of HG-6 in the glycosyl moiety suggested
the presence of an aminoacyl group at this position. The
coupling constant of the anomeric proton (JZ3.8 Hz)
indicated the a configuration, and since glucose has the
D-configuration in most cases in nature compound 1 could
be assigned as an a-D-6-desoxy-6-aminoglucoside.
When 1 was hydrolyzed under various conditions (1%
NaOMe in MeOH, with lipase from Mucor javanicus,23,24
or 0.5% HCl) the only ester that could be detected was
methyl palmitate. The polar fraction remaining after acidic
hydrolysis was reduced by reaction with NaBH4 and the
resulting products acetylated and analyzed by GC–MS.
Analysis of the retention times and fragmentation patterns
of the two products indicated them to be glycerol triacetate
and 6-acetylamino-1,2,3,4,5-penta-acetoxyhexane. This
result confirmed that 1 was a glycoglycero lipid. A negative
Cotton effect between lmax 200–250 nm indicated that 1
was an sn-1, sn-2 diacylglycosylglycero-lipid.25 The
difference between the 13C NMR signals for the two
carbonyl carbons of the two glyceryl esters (0.6 ppm)
indicated that 1 was an sn-1, sn-2 diacyl ester; had it been an
sn-1, sn-3 diacyl ester these signals would have been closer
or even overlapped.26,27 Thus, compound 1 was assigned the
structure sn-1,2-di-palmityl-3-(N-palmityl-6 0 -desoxy-6 0 amino-a-D-glucosyl)-glycerol.
Compound 2 also had a molecular weight 967 and a
composition of C57H109NO10, as indicated by its MALDI
TOF mass spectrum and FAB-HRMS. Its 1H NMR and
COSY spectra (Table 1) were very similar to those of 1, and
showed the presence of a diacylglyceryl moiety [dH
4.50 ppm (1H, dd, JZ12.0, 3.0 Hz, Hsn-1a), 4.18 ppm (1H,
dd, JZ12.0, 6.9 Hz, Hsn-1a), 5.30 ppm (1H, m, Hsn-2),
4.10 ppm (1H, dd, JZ10.7, 5.3 Hz, Hsn-3a), and 3.56 ppm
(1H, dd, JZ10.7, 6.3 Hz, Hsn-3b)].
The HMBC cross-peaks [dH/dC: 5.30 (Hsn-2)/174.4 (–COO–);
2.33(a-CH2–)/174.4 (–COOH); 4.50 and 4.18 (Hsn-1)/175.2
(–COO–); 2.33 (a-CH2–)/175.2 (–COO–)] supported this
result. Overlapped signals for three long chain fatty acids
[dH 0.89 ppm (9H, t, JZ6.83 Hz, 3!CH3–), 1–1.5 ppm
(multi –CH2–), 1.59 ppm (6H, m, 3!b-CH2–), 2.33 ppm
(6H, m, 3!a-CH2–) could also be found in its NMR
spectrum. NMR signals for a similar glycosyl moiety to that
of 1 [dH 4.75 ppm (1H, d, JZ3.7 Hz, HG-1), 3.39 ppm (1H,
dd, JZ9.6, 3.7 Hz, HG-2), 3.62 ppm (1H, d, JZ9.6, 9.7 Hz,
HG-3), 3.07 ppm (1H, dd, JZ9.3, 9.7 Hz, HG-4), 4.06 ppm
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B.-N. Zhou et al. / Tetrahedron 61 (2005) 883–887
(1H, m, HG-5), and 2.91 (1H, dd, JZ14.3, 8.4 Hz, HG-6a),
and 3.34 ppm (1H, d, JZ14.3, 2.0 Hz, HG-6b) were also
observed. All the spectroscopic data for 2 were
consistent with its assignment as an a-D-6-desoxy-6amino-glucoside.
Hydrolysis of 2 with 0.5% HCl followed by 1% NaOMe in
methanol and then by treatment with diazomethane yielded
approximately equimolar amounts of methyl myristate,
methyl palmitate and methyl stearate as determined by GC–
MS. The polar fraction remaining after hydrolysis was
reduced with NaBH4 and acetylated, and the resulting
product analyzed by GC–MS. Analysis of the retention
times and fragmentation patterns of the two products
indicated than to be glycerol triacetate and 6-acetylamino1,2,3,4,5-penta-acetoxyhexane. These results indicated
that 2 was a glycoglycero lipid with three different acyl
groups, so the locations of these groups needed to be
determined.
When 2 was hydrolyzed with 1% NaOMe in absolute
methanol, only methyl palmitate and methyl myristate were
detected by GC–MS. Since amides are hydrolyzed more
slowly than esters under alkaline conditions, this result
indicated that the stearyl moiety acylated the 6-amino
position of glucose, and that the myristyl and palmityl
groups both acylated the glyceryl moiety. When 2 was
hydrolyzed with lipase from M. javanicus23 and the
products treated with diazomethane, methyl stearate and
methyl palmitate were both isolated. This indicates that
the myristyl group is located at the more hindered sn-2
position. Compound 2 was thus assigned as sn-1-palmityl-2myristyl-3-(N-stearyl-6 0 -desoxy-6 0 -amino-a-D-glucosyl)glycerol.
Three 6 0 -desoxy-6 0 -amino-glucosylglycerolipids have
previously been reported in the literature,28 but the acyl
groups in these compounds were different from ours.
Compounds 1 and 2 are thus new natural products.
As noted earlier, compounds 1 and 2 had IC50 values of
0.12G0.07 and 0.43G0.01 mg/mL, respectively, in the
Myt1 kinase bioassay. The bioactivity of compounds of this
type as inhibitors of Myt1 kinase is new, although
diacylglycerol is well known as an activator of protein
kinase C.29 The inhibition against Myt1 kinase appeared to
be selective as we observed no inhibition of two other
protein kinases, Chk1 and Akt, with concentrations of
compounds 1 and 2 as high as 100 mg/mL.
Although simple acylglycerols are not normally thought of
as ‘druggable’ entities, they can serve as prototypes for
compounds with inproved pharmacological properties.
Thus, conformationally constrained diacylglycerol-bislactones have been investigated as diacylglycerol analogs,30
and the conversion of an O-glycoside to a C-glycoside
boosted the potency of a ceramide O-glycoside by two to
three orders of magnitude.31 In addition, it is worth
noting that a sea alga has yielded a sulfoquinovosyldiacylglycerol which acts as a potent inhibitor of
eukaryotic DNA polymerases and HIV reverse transcriptase
type 1.32
3. Experimental
3.1. General experimental procedures
CD and NMR spectra were obtained as previously
described;33 a 9 Hz optimization was employed for the
long-range coupling pathway in HMBC determinations.
MALDI spectra were determined on a Kratos Kompact SEQ
(Kratos Analytical, Manchester, UK) time of flight mass
spectrometer, and FAB mass spectra were obtained on a
JEOL JMS-HX-110 instrument. The conditions for GC–MS
conditions for fatty acid methyl ester: Hewlett Packard
HP5710A Gas Chromatograph with HP-5 column (30 M!
0.32 mM i.d.), oven temperature programmed from 80 to
280 8C at 8 8C/min for 1 and at 4 8C/min for 2, with He
carrier gas at 12 psi. A VG7070E-HF mass spectrometer,
scanned from 50 to 500 amu at 1.5 s/scan, was used for
detection. Sephadex LH-20 (Sigma) was employed for gel
permeation chromatography. Column chromatography was
carried out on LRP-2 (RP-18) and reversed-phase TLC on
MKC18F silica gel 60 A (RP-18) from Whatman.
3.2. Bioassay methods
The crude extract, fractions, and pure compounds were
assayed for inhibitory activity against Myt1 kinase by a
modification of a previously described microtiter-based
fluorescence polarization assay.34 Briefly, 10 mg recombinant glutathione-S-transferase (GST) tagged-Myt1 protein
was incubated in kinase buffer (50 mM Tris–HCl, pH 7.5,
20 mM MgCl 2 and 1 mM DTT) containing 2.5 nM
fluorescein-labeled Cdc2-derived peptide (containing
Myt1 phosphorylation target sites Thr-14 and Tyr-15)
(Molecular Devices, Sunnyvale, CA), 100 mM ATP, and
compounds solubilized in dimethyl sulfoxide. The kinase
reaction was incubated for 1 h at room temperature after
which an anti-phospho-Cdc2 antibody (1 mg/mL) (Cell
Signaling Technology, Beverly, MA) was added and
incubated for an additional hour. Fluorescence polarization
measurements were taken in black 96 well microtiter plates
(Costar, Acton, MA) with a Wallac Victor 1420 multilabel
microtiter plate reader (Perkin Elmer, Boston, MA).
Duplicate samples were subjected to 25 flashes and
experiments were performed 2–3 times. Dimethyl sulfoxide
was used as the vehicle control; staurosporine (Sigma, St.
Louis, MO) was used as the positive control and it had an
IC50 value of 1.9 mM in this assay.
The effects of 1 and 2 on Akt and Chk1 activity were also
studied with 384 well microtiter-based IMAP (Molecular
Devices, Sunnyvale, CA) fluorescence polarization assays.
Briefly, compounds 1 or 2 (0.1 to 100 mg/mL) were
incubated for 30–60 min at room temperature in the kinase
buffer (10 mM Tris–HCl, pH 7.2, 10 mM MgCl2, 0.1%
bovine serum albumin, and 0.05% NaN3) with 0.2 U/mL
Akt or 0.4 U/mL Chk1, 100 nM fluorescently labeled Akt
substrate or Chk1 crosstide substrate peptide, 5 mM ATP in
a total volume of 20 mL. IMAP binding solution (15–60 mL)
was added to each well and incubated at room temperature
for 30 min. Data were collected on an Analyst GT
(Molecular Devices) and analyzed using SoftMaxPro software. Triplicate experiments were performed 2–3 times.
Staurosporine (Sigma-Aldrich, St. Louis, MO) was used as a
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B.-N. Zhou et al. / Tetrahedron 61 (2005) 883–887
positive control for Akt (IC50Zw60 nM) and UCN-01
(National Cancer Institute, Bethesda, MD) was the positive
control for Chk1 (IC50Zw2 mM).
3.3. Isolation of bioactive compounds
The crude algal extract UM 2972M35 (4.04 g) was
partitioned between 50% aq MeOH and n-hexane, and the
aq MeOH layer was then partitioned with CH2Cl2. The 50%
aq MeOH fraction was then evaporated in vacuo to remove
MeOH, and the residual H2O fraction was extracted with
BuOH. The BuOH fraction (1.56 g) was active in the Myt1
kinase bioassay (IC50Z1.0 mg/mL) and was subjected to
column chromatography on Sephadex LH 20 (20 g, eluted
with CH2Cl2–MeOH (3:1). A total of 45 fractions (5 mL/
each fraction) was collected and fractions 8–12 (119.0 mg),
which showed the characteristic 1H NMR spectra of a
glycoglycerolipid, were the most active (IC50Z0.4–1.0 mg/
mL). The active fractions 9–10 (IC50Z0.4 mg/mL) were
repeatedly purified by chromatography on an RP-18 column
with MeOH–H2O (9:1) as eluant. Compound 1 (1.05 mg,
0.026% based on crude extract) was isolated from fractions
13–28, and compound 2 (1.35 mg, 0.033% based on crude
extract) from fraction 11. The inhibitory activities of the
isolated compounds were IC50Z0.12 mg/mL for 1 and
IC50Z0.43 mg/mL for 2.
3.3.1. Compound 1. MALDI TOF mass spectrum: m/z 990
(MCNa)C. HRFABMS m/z 968.8059 [MCH]C; (calcd for
C57H110NO10 968.8130). [a]23
D ZC41.8 (c 0.12, MeOH).
1
H NMR and 13C NMR spectra: see Table 1.
3.3.2. Alkaline hydrolysis of 1. Compound 1 (0.1 mg) in
MeOH (0.2 mL) was treated with 1% NaOMe–MeOH
solution (0.8 mL) and the solution was stirred at room
temperature for 5 h. After partition between n-hexane and
MeOH, the n-hexane extract was subjected on GC–MS.
Methyl palmitate was detected with retention time 15 0 14 00
and EIMS m/z 270 (M)C, 227, 143, 87, 74, 57, 55. Its
retention time and fragmentation peaks were same as the
data from a standard sample in the data base.
3.3.3. Enzymatic hydrolysis of 1. Compound 1 (0.1 mg)
was mixed with lipase (1 mg) from M. javanicus (Fluka
Chemie AG, CH-9471, Switzerland, EEC No. 2326199) in
dioxane–H2O (1:1, 1 mL) and the reaction mixture was
incubated at 38 8C for 4 h. After removal of the dioxane and
H2O, the residue was dissolved in 2 mL MeOH and
partitioned with n-hexane. The n-hexane extract was treated
with diazomethane and analyzed by GC–MS. Methyl
palmitate was detected as described above.
3.3.4. Complete hydrolysis of 1. Compound 1 (0.12 mg)
was treated with 0.5% HCl at 65 8C overnight. The reaction
mixture was evaporated to dryness in vacuo and the residue
mixed with 1% NaOMe in MeOH and incubated at room
temperature for 6 h. After partitioning with n-hexane, the
n-hexane fraction was evaporated to dryness and treated
with diazomethane and analyzed by GC–MS as described
above. Methyl palmitate was the only ester detected. The
aqueous fraction was evaporated to dryness and treated with
NaBH4 in MeOH at room temperature for 4 h with stirring.
After evaporation of the solvent, the reaction mixture was
acetylated with Ac2O (0.5 mL) and dry pyridine (20 mL)
with stirring overnight. After removal of the reagents with
N2, the reaction mixture was extracted with CH2Cl2. The
CH2Cl2 extract was subjected to GC–MS and glycerol
triacetate (retention time: 7 0 20 00 , EIMS m/z 158 (MK60)C,
145, 116, 115, 183, 74, 73, and 61) and 6-acetylamino1,2,3,4,5-penta-acetoxyhexane (retention time: 17 0 47 00 , and
EIMS m/z 433 (M)C, 374, 331, 304, 289, 259, 207, and 87)
were detected.
3.3.5. Compound 2. MALDI TOF mass spectrum: m/zZ
990 (MCNa)C. HRFABMS m/z 968.8076 [MCH]C;
(calcd for C57H110NO10 968.8130). [a]23
D ZC31.1 (c 0.10,
MeOH). 1H NMR and 13C NMR spectrum data: see Table 1.
3.3.6. Alkaline hydrolysis of 2. Compound 2 (0.11 mg) in
MeOH (0.2 mL) was treated with 1% NaOMe in MeOH
(1.0 mL) and the solution was stirred at room temperature
for 4 h. After extraction with n-hexane, the n-hexane extract
was analyzed by GC–MS. Methyl myristate was detected
with retention time 17 0 46 00 and EIMS m/z 242 (MC), 199,
143, 87, 74, and 57 and methyl palmitate with retention time
22 00 58 00 and EIMS m/z 270 (MC), 227, 185, 143, 87, 74, and
57. Their retention times and fragmentation patterns were
the same as those of standard samples in the database.
3.3.7. Enzymatic hydrolysis of 2. Compound 2 (0.12 mg)
was hydrolyzed with lipase from M. javanicus and the
n-hexane extract treated with diazomethane and analyzed by
GC–MS as previously described. Two peaks were detected.
One of them was identified as methyl palmitate by its
retention time (22 0 53 00 ) and EIMS (m/z 270 (MC), 239, 227,
185, 171, 143, 129, 115, 99, 87, 74, 57). The second was
identified as methyl stearate by its retention time (27 0 42 00 )
and EIMS (m/z 298 (MC), 267, 255, 213, 199, 185, 157,
143, 129, 97, 87, 74, and 57). The retention times and
fragmentation patterns of these compounds were the same
as those of standard samples in the database.
The MeOH fraction after partition was evaporated to
dryness in vacuo. The residue was treated with 1%
NaOMe in MeOH at room temperature for 4 h. After
extraction with n-hexane, the n-hexane fraction was
concentrated, treated with diazomethane, and analyzed by
GC–MS. The major peak was identified as methyl myristate
by its retention time of 17 0 37 00 and EIMS m/z 242 (MC),
199, 143, 87, 74, 57, and 55.
3.3.8. Complete hydrolysis of 2. Compound 2 (0.10 mg)
was treated with 0.5% HCl solution (1 mL) at 65 8C
overnight, the reaction mixture evaporated to dryness, and
the residue incubated with 1% NaOMe in MeOH (1 mL) at
room temperature for 6 h. After partition with n-hexane, the
n-hexane fraction was evaporated to dryness and treated
with diazomethane and analyzed by GC–MS. The first peak
was identified as methyl myristate by its retention time of
17 0 20 00 and EIMS m/z 242 (MC), 199, 143, 87, 74, 57, and
55. The second peak was identified as methyl palmitate by
its retention time of 22 00 55 00 and EIMS m/z 270 (MC), 239,
227, 185, 143, 87, 74, 57. The third peak was detected as
methyl stearate by its retention time of 27 0 46 00 and EIMS m/z
298 (MC), 267, 255, 213, 199, 143, 87, 74, 57. The aqueous
fraction was evaporated to a small volume and reduced with
B.-N. Zhou et al. / Tetrahedron 61 (2005) 883–887
NaBH4 at room temperature for 4 h with stirring. After
evaporation of the solvent and drying under vacuum
overnight, the reaction mixture was acetylated with Ac2O
(0.5 mL) and dry pyridine (20 mL) with stirring overnight.
After removal of the reagents with N2, the reaction mixture
was extracted with CH2Cl2. The CH2Cl2 extract was
subjected to GC–MS and showed two intense peaks,
which were identified as glycerol triacetate and 6-acetylamino-1,2,3,4,5-penta-acetoxyhexane as previously
described.
Acknowledgements
This work was supported by a National Cooperative Drug
Discovery Group award to the University of Virginia (U19
CA 50771, Dr. S. M. Hecht, Principal Investigator), and this
support is gratefully acknowledged. We also thank Mr. Tom
Glass and Mr. William R. Bebout, Sr., (Virginia Polytechnic
Institute and State University) for spectroscopic support
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