solution (pH 7.0, 0.1 M, 6.0 mL) was added Burkholderia
cepacia lipase (Amano PS-IM, 615 mg). After vigorous stirring
for 48 h at 30 C, the mixture was filtered through a pad of
Celite to remove insoluble materials, and then extracted with
EtOAc. The organic layer was washed with brine, dried over
workup, the residue was purified by preparative TLC using MeOH
: CHCl (1: 10, v/v) as mobile phase to yield 1a (88.6 mg, 88%) as
3
◦
◦
1b
◦
a white solid. Mp 184 C [lit. mp 185 C]. Its ee and the absolute
configuration were confirmed after derivation to dihexanoate
2
6
5
(2R,3R,4R)-1b. Colorless oil; [a] +114 (c 0.68, CHCl ) [lit.
[a] -126 (c 0.66, CHCl
3
D
2
D
3
1
anhydrous Na
2
SO
4
, and concentrated in vacuo. The residue was
3
) for (2S,3S,4S)-1b]. Its IR and H
purified by silica gel column chromatography (30 g). Elution
with hexane/EtOAc = 4 : 1 afforded (1S,2S,3S)-2,3-epoxy-5-
N-[(2-hydroxybenzoyl)amino]-4,4-dimethoxycyclohex-5-en-1-ol
NMR spectra were identical with those reported previously. HPLC
ꢀ
R
[CHIRALCEL AD-H, 0.46 cm ¥ 25 cm; hexane-2-propanol
-
1
(7 : 1); flow rate 0.5 mL min ; detected at 290 nm]: t (min) =
R
(
2a, 110.0 mg, 40%) and (1R,2R,3R)-2,3-epoxy-5-N-[(2-
28.3 (single peak). There was no peak at 30.9 min ascribable to
hydroxybenzoyl)amino]-4,4-dimethoxycyclohex-5-enyl hexanoate
(2S,3S,4S)-1b.
(
2c, 211.0 mg) as a colorless oil.
2
5
1
(
1S,2S,3S)-2a: [a] -3.2 (c 1.00, CHCl
3
); H NMR d (ppm):
D
Biological assays
3
1
.25 (s, 3H), 3.62 (s, 3H), 3.62 (s, 1H), 3.62 (s, 1H), 4.73 (brs,
H), 6.66 (s, 1H), 6.87 (dd, J = 7.2, 8.0 Hz, 1H), 6.98 (d, J = 8.0
Materials
Hz,1H), 7.31 (d, J = 7.0 Hz, 1H), 7.41 (dd, J = 7.0, 7.2 Hz, 1H),
1
3
8
6
1
1
.21 (s, 1H), 11.9 (s, 1H); C NMR d (ppm): 50.4, 50.9, 51.6, 53.1,
5.1, 95.6, 114.7, 115.6, 118.9, 119.0, 125.2, 127.8, 134.7, 161.7,
68.4; IR nmax 3410, 3298, 2981, 2943, 2835, 1736, 1647, 1527,
6-OHDA was purchased from Sigma (St. Louis, MO). Human
neuroblastoma SK-N-SH cells were obtained from Riken Bio-
Resource Center (Tsukuba, Japan).
-
1
450, 1354, 1234, 1126, 1041, 937, 906, 756 cm . Anal. Calcd for
C
15
H
17NO
6
: C, 58.63; H, 5.58; N, 4.56. Found: C, 58.81; H, 6.19;
Cell culture
ꢀ
R
N, 3.79. HPLC [CHIRALCEL AD-H, 0.46 cm ¥ 25 cm; hexane-
-
1
2
-propanol (3 : 1); flow rate 0.5 mL min ; detected at 290 nm]:
SK-N-SH cells were grown in Minimum Essential Medium Alpha
(MEMa, Invitrogen Carlsbad, CA) supplemented with 10%
heat-inactivated fetal bovine serum (Nichirei Biosciences, Tokyo,
t
R
(min) = 14.6 (single peak). The separation of enantiomers was
confirmed by the analysis of its racemate, t
4.6 (50.2%).
1R,2R,3R)-2c: H NMR d (ppm); 0.88 (t, J = 7.0 Hz, 3H), 1.31
R
(min) = 12.4 (49.8%),
-
1
-1
1
Japan), 200 mg mL kanamycin, 100 U mL penicillin G, 600 mg
1
-1
-1
(
mL L-glutamine, and 2.2 g L NaHCO3.
(
(
m, 2H), 1.31 (m, 2H), 1.66 (m, 2H), 2.39 (t, J = 7.6 Hz, 2H), 3.26
s, 3H), 3.58, (d, J = 4.5 Hz, 1H), 3.62 (s, 3H), 3.63 (ddd, J = 2.3,
Plasmid construction and reporter luciferase assays
2
2
8
8
.4, 4.5 Hz, 1H), 5.87 (dd, J = 2.2, 2.4 Hz, 1H), 6.56 (dd, J = 2.2,
.3 Hz, 1H), 6.87 (ddd, J = 1.0, 7.2, 8.2 Hz, 1H), 6.97 (dd, J = 1.0,
.2 Hz, 1H), 7.30 (dd, J = 1.5, 7.2 Hz, 1H), 7.40 (ddd, J = 1.5,
.2, 8.2 Hz, 1H), 8.30 (s, 1H), 11.8 (s, 1H). Its ee was confirmed
The human wild-type (5¢-GTGACTCAGCA-3¢) and mutant (5¢-
GCGACTCAGCA-3¢) ARE sequences of human NQO1 were
cloned upstream of the luciferase reporter gene by using sense
and antisense oligonucleotides synthesized to have BglII and
XhoI sites, respectively. The oligonucleotides were annealed,
phosphorylated using T4 polynucleotide kinase, and cloned into
the respective sites in the pGL4.17 firefly luciferase reporter vector
by derivation to the corresponding dihexanoate (1R,2R,3R)-2b.
HPLC analysis was performed in the same manner as above: t
min) = 23.5 (83.1%), 26.7 (16.9%).
R
(
(
2R,3S)-2,3-Epoxy-5-N -[(2-hydroxybenzoyl)amino]-4,4-di-
methoxycyclohex-5-en-1-one (8). To a solution of 2a (128.0 mg,
.42 mmol) in CH Cl was added activated MnO (Eastman
(
Promega, Madison, WI). Then, SK-N-SH cells were transfected
with wild-type or mutant ARE sequence vector, or empty vectors,
and phRL TK vectors (Promega) using lipofectamine LTX
Reagent (Invitrogen). After a 24 h incubation at 37 C, the cells
were treated with chemicals, and then 18 h later the luciferase
activity was measured. The results were normalized with the
Renilla luciferase activity.
0
2
2
2
TM
Kodak Co., 50199, 185 mg). After having been stirred for 24 h
at room temperature, the mixture was filtered through a pad of
Celite to remove insoluble materials. The residue was washed with
EtOAc. The combined filtrate and washings were concentrated in
◦
2
4
vacuo to afford 8 (124.8 mg, 98%) as a yellow oil; [a] +200 (c
D
1
1.00, CHCl
3
). Its IR and H NMR spectra were identical with
3
those reported previously. This compound was employed for the
next step without further purification.
Reverse transcription (RT)-PCR
SK-N-SH cells were grown to confluence in 60 mm dishes and
treated with (2R,3R,4R)-1a. Total RNA was prepared from the
cells by using a High-Capacity cDNA Reverse Transcription Kit
(Applied Biosystems, Carlsbad, CA). The following primers were
used for PCR: HO-1 Forward, 5¢-ATGACACCAAGGACCAGA-
GC-3¢; HO-1 Reverse, 5¢-GTGTAAGGACCCATCGGAGA-
3¢; NQO1 Forward, 5¢-CACTGATCGTACTGGCTCACTC-
3¢; NQO1 Reverse, 5¢- AAGGGTCCTTTGTCATACATGG-3¢;
GCLC Forward, 5¢-CCCATGGAGGTGCAATTAAC-3¢; GCLC
Reverse, 5¢-TGCGATAAACTCCCTCATCC-3¢; b-actin For-
ward, 5¢-TTCTACAATGAGCTGCGTGT-3¢; b-actin Reverse, 5¢-
GTCAGGTCCCGGCCAGCCAG-3¢.
(
2R,3R,4R)-2,3-Epoxy-4-hydroxy-5-N -[(2-hydroxybenzyl)-
amino]cyclohex-5-en-1-one (1a). A solution of 8 (118.0 mg, 0.39
mmol) in TFA (0.78 mL) was stirred for 1.5 h at 40 C. The
mixture was concentrated in vacuo to afford (2R,3S)-2,3-epoxy-5-
N-[(2-hydroxybenzoyl)amino]cyclohex-5-ene-1,4-dione (99.9 mg,
9%) as a yellow solid; [a]D +51.3 (c 1.00, MeOH). Its IR and H
NMR spectra were identical with those reported previously for
the racemic form. This compound was employed for the next step
without further purification.
According to the reported procedure, this diketone (99.9 mg,
.39 mmol) was reduced with NaBH(OAc) in MeOH. After
◦
2
4
1
9
1,3
0
3
4
640 | Org. Biomol. Chem., 2011, 9, 4635–4641
This journal is © The Royal Society of Chemistry 2011