6652
J.-H. Choi et al. / Tetrahedron 67 (2011) 6649e6653
ESIMS m/z 285 [2MþNa]þ; HRESIMS m/z 285.1071 [2MþNa]þ
added to the solution. The resulting mixture was stirred for 24 h at
room temperature. The products were purified by silica gel flash
column chromatography (CH2Cl2; CH2Cl2/acetone 9:1, 5:5; acetone;
acetone/MeOH 5:5) and then reverse-phase HPLC (Capcell pak C18
AQ, 50% MeOH) to give 1 (7.7 mg, 33%); 1H and 13C NMR, see Table 1.
(calcd for C10H18N2NaO6, 285.1063).18
3.4.4. Ethyl 2-(phenethylamino)acetate (6). 2-Phenethylamine
(6 mmol, 755
ture of ethyl bromoacetate (6 mmol, 660
(0.6 mmol, 60 L) was added to the solution. The resulting mixture
mL) was dissolved in pyridine (500
mL) and a mix-
mL) and diethylamine
m
3.5. Bioassay
was stirred for 24 h at room temperature. The products were pu-
rified by silica gel flash column chromatography (acetone) and then
reversed-phase HPLC (Capcell pak C18 AQ, 50% MeOH) to obtain 6
(45.4 mg, 3.6% yield). Compound 6: 1H NMR (CDCl3, 270 MHz)
Neuro2a cells were obtained from the Health Science Research
Resources Bank, Japan, and maintained in the Dulbecco‘s modified
Eagles medium (D-MEM) (SIGMA, USA) supplemented with 10%
(v/v) fetal bovine serum (FBS), unless particularly noted. Cell via-
bility analysis was performed by 3-(4,5-dimethyl-2-thiazolyl)2,5-
diphenyl-2H-tetrazolium bromide (MTT) assay. MTT assay was
performed as follows; Neuro2a cells were cultured in 96-well
plates at cell density 5000 cells/well and after one-day cultiva-
tion, the cells were cultured in D-MEM without FBS, and with
d
7.12e7.29 (5H, m), d 4.14 (2H, q, 7.0), d 3.31 (2H, s), d 2.87 (2H, t,
2.7),
d
2.77 (2H, t, 2.7),
d
1.23 (3H, t, 7.0); ESIMS m/z 208 [MþH]þ;
HRESIMS m/z 208.1338 [MþH]þ (calcd for C12H19N1O2, 208.1359).19
3.4.5. Ethyl 2-(N-phenethylacetamido)acetate (7). Compound
(100 mmol, 20 mg) was dissolved in pyridine (500 L) and
a mixture of acetyl chloride (0.4 mmol, 30 L) and DIPCD
(0.4 mmol, 60 L) was added to the solution. The resulting mixture
6
m
m
0.5
mg/mL of tunicamycin (or 20 nM thapsigargin) and varying
m
concentrations of test compounds were applied to the medium.
The cells were incubated for 24 h, and then the viability was
measured by MTT assay, as described previously.22 Briefly,
0.25 mg/mL of MTT in D-MEM without FBS were added onto the
cells and incubated for 2 h. The incubation was terminated by
addition of 20% SDS (v/w) and 50% DMSO (v/v) in water. The
absorbance at 570 nm of the reaction mixture was measured by
a microplate reader (Molecular Devices, USA).
was stirred for 24 h at room temperature. The products were
purified by silica gel flash column chromatography (CH2Cl2;
CH2Cl2/acetone 9:1, 5:5; acetone; acetone/MeOH 5:5) and then
reversed-phase HPLC (Capcell pak C18 AQ, 50% MeOH) to give 7
(4.7 mg, 19% yield). Compound 7 was obtained a set of rotational
isomers (7a and 7b). The molar ratio of 7a to 7b was 2.5 to 1.
Compound 7a: 1H NMR (CD3OD, 270 MHz)
200, H-300, H-400, H-500, H-600), 4.18 (2H, q, 7.0, H-1000),
H-2),
3.63 (2H, t, 7.2, H-10), 2.88 (2H, t, 7.1, H-20),
NCOeMe),
1.26 (3H, t, 7.0, H-2000). 7b:
H-300, H-400, H-500, H-600), 4.18 (2H, q, 7.0, H-1000),
d 7.19e7.32 (5H, m, H-
d
d
4.04 (2H, s,
1.86 (3H, s,
d
d
d
3.6. Statistical analysis
d
d
7.19e7.32 (5H, m, H-200,
d
d
4.09 (2H, s, H-2),
Data collected were analyzed statistically using TukeyeKramer
multiple comparisons tests to determine significant difference in
the data among the groups. P values less than 0.01 were considered
significant. The values are expressed as meanꢀSEM.
d
3.55 (2H, t, 7.7, H-10),
d
2.80 (2H, t, 7.6, H-20),
d
2.01 (3H, þs,
NCOeMe),
d
1.26 (3H, t, 7.0, H-2000); ESIMS m/z 272 [MþNa] ;
HRESIMS m/z 272.1254 [MþNa]þ (calcd for C14H19N1NaO3,
272.1262).
Acknowledgements
3.4.6. 2-(Phenethylamino)acetic
(1 mmol,139 mg) was dissolved in pyridine (500
of 2-phenethylamine (1 mmol, 125
(0.1 mmol, 10 L) was added to the solution. The resulting mixture
acid
(8). Bromoacetic
acid
m
L) and a mixture
We thank V. K. Deo (Shizuoka University) for valuable
discussion.
m
L) and diethylamine
m
was stirred for 48 h at room temperature. The products were
purified by silica gel flash column chromatography (CH2Cl2;
CH2Cl2/acetone 9:1, 5:5; acetone; acetone/MeOH 5:5) and then
reversed-phase HPLC (Capcell pak C18 AQ, 33% MeOH) to obtain 8
(23.4 mg, 13% yield). Compound 8: 1H NMR (CD3OD, 270 MHz)
References and notes
1. Katayama, T.; Imaizumi, K.; Sato, N.; Miyoshi, K.; Kudo, T.; Hitomi, J.; Morihara,
T.; Yoneda, T.; Gomi, F.; Mori, Y.; Nakano, Y.; Takeda, J.; Tsuda, T.; Itoyama, Y.;
Murayama, O.; Takashima, A.; St George-Hyslop, P.; Takeda, M.; Tohyama, M.
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2. Imai, Y.; Soda, M.; Takahashi, R. J. Biol. Chem. 2000, 275, 35661e35664.
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2002, 11, 1505e1515.
d
7.21e7.35 (5H, m), d 3.50 (2H, s), d 3.25 (2H, q, 7.0), d 2.97 (2H, t,
7.0); ESIMS m/z 381 [2MþNa]þ; HRESIMS m/z 381.1782 [2MþNa]þ
(calcd for C20H26N2NaO4, 381.1790).20
4. Nakagawa, T.; Zhu, H.; Morishima, N.; Li, E.; Xu, J.; Yankner, B. A.; Yuan, J. Nature
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3.4.7. 2-(N-Phenethylformamido)acetic acid (9). Glyoxylic acid
monohydrate (10 mmol, 1.84 g) was dissolved in distilled water
(5 mL) and 2-phenethylamine (5 mmol, 630 mL) was added to the
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solution. The resulting mixture was stirred for 24 h at room tem-
perature. The products were purified by silica gel flash column
chromatography (acetone; acetone/MeOH 5:5) and then reversed-
phase HPLC (Capcell pak C18 AQ, 28% MeOH) to afford 9 (113 mg,
11% yield). Compound 9 was obtained a set of rotational isomers (9a
and 9b). The molar ratio of 9a to 9b was 2.8 to 1. Compound 9a: 1H
7. Ueda, K.; Tsujimori, M.; Kodani, S.; Chiba, A.; Kubo, M.; Masuno, K.; Sekiya, A.;
Nagai, K.; Kawagishi, H. Bioorg. Med. Chem. 2008, 16, 9467e9470.
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Koshino, H.; Kawagishi, H. Tetrahedron 2009, 65, 221e224.
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NMR (CD3OD, 270 MHz)
d
7.83 (1H, s),
2.86 (2H, t, 7.0). Compound 9b:
3.99 (2H, s), 3.56 (2H, t, 8.1),
d
7.20e7.28 (5H, m),
d
d
d
4.06
8.02
2.86
(2H, s),
(1H, s),
d
3.61 (2H, t, 7.0),
7.20e7.28 (5H, m),
d
12. Yang, Y. P.; Cheng, M. J.; Teng, C. M.; Chang, Y. L.; Tsai, I. L.; Chen, I. S. Phyto-
d
d
d
(2H, t, 7.0); ESIMS m/z 230 [MþNa]þ; HRESIMS m/z 230.0792
chemistry 2002, 61, 567e572.
13. Mahoney, W. C.; Duksin, D. J. Biol. Chem. 1979, 254, 6572e6576.
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[MþNa]þ (calcd for C11H13N1NaO3, 230.0794).21
15. Hitomi, J.; Katayama, T.; Taniguchi, M.; Honda, A.; Imaizumi, K.; Tohyama, M.
3.4.8. Ethyl 2-(N-phenethylformamido)acetate (1). Compound
(100 mmol, 20 mg) was dissolved in pyridine (500 L) and a mixture
of formic acid (0.4 mmol, 15 L) and DIPCD (0.4 mmol, 60 L) was
6
Neurosci. Lett. 2004, 357, 127e130.
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m
m
m