Practical total synthesis of fuligocandins A and B

Article abstract of DOI:10.1055/s-0030-1258547

Fuligocandins A and B, cycloanthranilylproline derivatives isolated from the myxomycete Fuligo candida, were synthesized efficiently by a Meyer-Schuster rearrangement, which gave Z-isomer with high selectivity. The absolute stereochemistry at C-11a of the

Full text of DOI:10.1055/s-0030-1258547

2498
LETTER
Practical Total Synthesis of Fuligocandins A and B
P
ractical Tota
l
Synthes
i
is of
F
d
o
Aand
B
ri A. Arai,* Junya Seto, Firoj Ahmed, Kento Uchiyama, Masami Ishibashi*
Graduate School of Pharmaceutical Sciences, Chiba University, Inage, Chiba 263-8522, Japan
E-mail: mish@p.chiba-u.ac.jp; E-mail: marai@p.chiba-u.ac.jp
Received 15 July 2010
O
Abstract: Fuligocandins A and B, cycloanthranilylproline deriva-
tives isolated from the myxomycete Fuligo candida, were synthe-
sized efficiently by a Meyer–Schuster rearrangement, which gave
Z-isomer with high selectivity. The absolute stereochemistry at C-
11a of the natural products was determined to be S by comparison
N
11a
N
H
H
of the optical rotation of the natural products with synthetic coun-
terparts derived from L-proline. This report is the first total synthe-
sis of optically active fuligocandin B.
O
O
3
6
14
15
5
4
N
5a
7
2
8
2'
11a
3'
3a'
1
Key words: total synthesis, fuligocandin, Meyer–Schuster rear-
rangement, myxomycete, TRAIL
10
9a
9
11
N
H
1'NH
4'
H
12
13
7a'
O
5'
7'
6'
14
1
2
Fuligocandins A (1) and B (2), novel natural compounds,
were isolated from field-collected fruit-bodies of myxo-
mycete Fuligo candida and structurally characterized
with spectroscopic methods by our group.1 Among more
than 200 natural compounds in our library, we previously
reported tumor necrosis factor related apoptosis-inducing
ligand (TRAIL)-resistance-overcoming activity of 2.2
However, the absolute stereochemistry at the C-11a posi-
tion of 1 and 2 remained undetermined. Here, we describe
the determination of the absolute stereochemistry of C-
11a by total synthesis of optically active fuligocandins A
and B (Figure 1).
Figure 1 The structures of fuligocandins A (1) and B (2)
and 2 by Eschenmoser coupling reaction of the thione
compound.4 This basic condition resulted in racemization
at C-11a. Our retrosynthetic analysis of 1 and 2 is an ap-
proach which includes the Meyer–Schuster rearrange-
ment to make a,b-unsaturated ketone (Scheme 1).
As shown in Scheme 2, coupling of N-Boc-anthranilic
acid 3 and L-proline methyl ester (4) gave compound 6 in
96% yield. After hydrolysis of the methyl ester with
LiOH, the intramolecular cyclization gave N-Boc-cyclo-
anthranilylproline 8 in 79% yield (two steps). To obtain 1
containing a,b-unsaturated ketone, a Meyer–Schuster re-
arrangement was selected as a key step.5
More et al. reported the first total synthesis of 1 in six
steps from azide derivatives,3 while the optical purity of
synthetic 1 was not reported. Bergman et al. synthesized 1
O
N
O
O
N
N
N
H
H
N
H
N
H
OH
H
H
O
O
Meyer–Schuster
rearrangement
aldol condensation
1
+
CHO
NH
O
N
2
H
N
N
H
H
O
Scheme 1 Retrosynthesis of fuligocandins A (1) and B (2)
SYNLETT 2010, No. 16, pp 2498–2502
x
x
.x
x
.2
0
1
0
Advanced online publication: 26.08.2010
DOI: 10.1055/s-0030-1258547; Art ID: U06610ST
© Georg Thieme Verlag Stuttgart · New York

LETTER
Practical Total Synthesis of Fuligocandins A and B
2499
estingly, the rearrangement gave only the Z-isomer of 1 in
90% yield. The Z-geometry was established by NOE ex-
periment.6 As shown in the proposed mechanism
(Scheme 3), removal of the Boc group would occur first.
A 1,3-shift of the hydroxy group would generate the cor-
responding hydroxyallene 14. After tautomerization be-
tween 15 and 16, the Z-isomer 1 would be obtained. This
noteworthy high prediction for formation of the Z-isomer
would be due to the hydrogen bond of NH in intermediate
15.7 However, it was found that the enantiomeric excess
of 1 was decreased to 40% (Table 1, entry 1).8 We con-
firmed that no racemization occurred in compounds 8 and
10 by chiral HPLC analysis.9 As shown in Scheme 4, a
propargylic cation 17 would be generated by dehydration
from 12, followed by an imine (18)–enamine (19) tau-
tomerization. To prevent this unfavorable reaction, the re-
arrangement was performed at –20 °C, which improved
the ee of 1 to 70% (Table 1, entry 2).
O
CO2H
N
a
b, c
CO2Me
2Me
NHBoc
NHBoc
CO
3
6 (S):
7 (R):
CO2Me
O
O
N
N
d
H
N
H
N
OH
Boc
O
Boc
8 (S):
9 (R):
H
10 (S):
11 (R):
H
H
H
Scheme 2 Reagents and conditions: (a) L-proline methyl ester hy-
drochloride (4) or D-proline methyl ester hydrochloride (5), PyBOP,
DIPEA, CH2Cl2, r.t., 2 h, 96% [6 (S)], 87% [7 (R)]; (b) LiOH, THF–
MeOH–H2O, r.t., 5 h; (c) PyBOP, DIPEA, CH2Cl2, r.t., 72 h, 79% [8
(S), 2 steps from 6], 79% [9 (R), 2 steps from 7]; (d) propynyllithium,
THF, –78 °C, 40 min, 50% [10 (S)], 41% [11 (3R)].
Table 1 Meyer–Schuster Rearrangement of 10
O
N
Meyer–Schuster
rearrangement
O
O
1
H
OH
N
TFA–CH2Cl2 (1:1)
N
N
H+
– H+
10
Boc
H
H
O
N
N
OH2
H
H
+
O+
H
10
12
13
H
Entry Temp (°C) Time (h) Ratio E/Z Yield (%) ee (%)
O
O
N
N
N
1
2
0
4
4
0:1
0:1
90
90
40
70
H
H
H
H+
–20
N
H
N
N
H
OH
HO
O
1
The H NMR and 13C NMR spectra of synthetic 1 were
compared with those of the natural product, showing that
they were completely identical. Synthetic 1 (70% ee)
14
15
16
showed dextrorotation {[a]14 +403 (c = 1.4, MeOH)}.
Z-isomer (1)
D
We reported that natural product 1 also shows dextrorota-
tion {[a]26D +657 (c = 1.4, MeOH)}.1 To ensure the abso-
lute configuration of the natural product, we also
synthesized (–)-fuligocandin A [(ent)-1] starting from D-
proline methyl ester (5). Compound 9 was obtained in
79% yield (two steps from 7). Each reaction proceeded
smoothly and gave comparable yields to those with L-pro-
line. A Meyer–Schuster rearrangement at 0 °C gave (ent)-
1 in 99% yield as a single Z-isomer, which showed levoro-
Scheme 3 Proposed mechanism
Using in situ prepared propynyllithium, propargylic alco-
hol 10 was obtained as a single stereoisomer. Moderate
chemoselective addition of propynyllithium might be due
to the electron deficiency of N-Boc amide in two amide
units of 8. Next, we examined the Meyer–Schuster rear-
rangement of 10 under acidic conditions (Table 1). Inter-
O
O
O
N
N
N
– H2O
12
H
H
+
N
N
N
H
H
18
H2O
rac-14
17
19
Scheme 4 Proposed racemization mechanism
Synlett 2010, No. 16, 2498–2502 © Thieme Stuttgart · New York

2500
M. A. Arai et al.
LETTER
tation.10 Therefore, the absolute stereochemistry at C-11a
in 1 was determined as S.
Acknowledgment
We thank Prof. Toshiaki Sunazuka (Kitasato Institute for Life Sci-
ences & Graduate School of Infection Control Sciences) for kind
discussion about synthesis of 2. This study was supported by a
Grants-in-Aid for Scientific Research from the Japan Society for the
Promotion of Science (JSPS) and by a Grants-in-Aid to M.A.A.
from the Takeda Science Foundation and from the Naito Fundation.
Fuligocandin B (2) was synthesized by aldol condensation
with indole aldehyde 20 and 1 (Scheme 5). The 1H NMR
and 13C NMR spectra of synthetic 2 were completely iden-
tical to those of the natural product (see Supporting Infor-
mation). Synthetic 2 (61% ee)11 showed dextrorotation
{[a]18D +248 (c = 0.6, MeOH)} and natural product 2 also
showed dextrorotation {[a]26D +149 (c = 0.6, MeOH)}.1,12
This result strongly supports the S configuration at the C-
11a position.
References and Notes
(1) Nakatani, S.; Yamamoto, Y.; Hayashi, M.; Komiyama, K.;
Ishibashi, M. Chem. Pharm. Bull. 2004, 52, 368.
(2) Hasegawa, H.; Yamada, Y.; Komiyama, K.; Hayashi, M.;
Ishibashi, M.; Sunazuka, T.; Izuhara, T.; Sugahara, K.;
Tsuruda, K.; Masuda, M.; Takasu, N.; Tsukasaki, K.;
Tomonaga, M.; Kamihira, S. Blood 2007, 110, 1664.
(3) For a reported synthesis of fuligocandin A, see: More, S. S.;
Shanmughapriya, D.; Lingam, Y.; Patel, N. B. Synth.
Commun. 2009, 39, 2058.
CHO
N
TIPS
20
(4) For the synthesis of racemic fuligocandins A and B, see:
Pettersson, B.; Hasimbegovic, V.; Bergman, J. Tetrahedron
Lett. 2010, 51, 238.
1) LDA, THF, –78 °C to 0 °C
1
2
2) 1 M HCl, 63% (2 steps)
(5) For a reported Meyer–Schuster rearrangement of N-Boc
lactam, see: Sun, C.; Lin, X.; Weinreb, S. J. Org. Chem.
2006, 71, 3159.
(6) NOE (5.0%) was observed between a-proton of a,b-
unsaturated ketone and a proton at C-1 position in five-
membered ring.
Scheme 5 Synthesis of fuligocandin B (2)
TRAIL, a member of the tumor necrosis factor superfam-
ily, selectively induces apoptosis in various cancer cells
with little toxicity toward normal cells. This property of
TRAIL has prompted attempts to develop drugs for cancer
treatment. However, several types of cancer have shown
resistance against TRAIL. Compounds that induce
TRAIL sensitivity would be important candidates as
drugs for TRAIL-resistant cancer cells. We previously
found fuligocandin B (2) has activity against adult T-cell
leukemia/lymphoma with no toxicity against normal pe-
ripheral blood mononuclear cells.2 In this study, we fo-
cused on human gastric adenocarcinoma (AGS) cells.
Combined treatment with TRAIL and synthesized com-
pounds, synthetic fuligocandin B (2) showed TRAIL-
resistance-overcoming activity in a dose-dependent man-
ner, although the optical purity of synthetic 2 is moderate
(see Supporting Information). Interestingly, fuligocandin
A (1) and (ent)-1 did not show any activity. This result in-
dicates that the indole moiety is important to the activity.
(7) A Meyer–Schuster rearrangement of N-alkylated
succinimide gave E-isomer as a main product: Mamouni, A.;
Daïch, A.; Decroix, B. Synth. Commun. 1998, 28, 1839.
(8) The ee was determined by a chiral phase HPLC;
CHIRALPAK AD-RH, MeOH–H2O = 75:25, flow rate: 1
mL/min, l = 254 nm detection, tR = 11.5 min [(ent)-1)], tR =
14.3 min (1) {or CHIRALPAK IA, hexane–i-PrOH = 4:1,
flow rate: 0.5 mL/min, l = 254 nm detection, tR = 18.6 min
[(ent)-1)], tR = 29.7 min (1)}. Natural product 1: >99% ee.
(9) Racemization was checked by comparing retention times of
8 with 9, 10 with 11 by CHIRALPAK AD-RH.
(10) Optical rotation of (ent)-1 (52% ee): [a]18D –224 (c = 1.4,
MeOH).
(11) Compound 1 which was synthesized at 0 °C was used for the
synthesis of 2. Therefore, the ee of 2 was not decreased by
this reaction. The ee was determined by a chiral phase
HPLC; CHIRALPAK AD-RH, MeOH–H2O = 1:1, flow
rate: 1 mL/min, l = 370 nm detection, tR = 56.3 min [(ent)-
2], tR = 68.1 min (2) {or CHIRALPAK IA, hexane–
i-PrOH = 4:1, flow rate: 0.5 mL/min, l = 370 nm detection,
tR = 58.7 min [(ent)-2)], tR =79.1 min (2)}. Natural product
2: >99% ee.
In conclusion, a practical total synthesis of optically
active fuligocandins A (1), B (2) was achieved using
Meyer–Schuster rearrangement as a key step.13 Com-
pound 1 was obtained as a Z-isomer with high selectivity.
The absolute stereochemistry at C-11a of the natural prod-
uct 1 and 2 was determined to be S by comparison of the
optical rotation of the natural product with synthetic 1
(70% ee), and synthetic 2 (61% ee) derived from L-pro-
line. This report is the first total synthesis of optically ac-
tive fuligocandin B (2). Moreover, as a preliminaly result,
we found the TRAIL-resistance-overcoming activity of 2
against AGS cells.
(12) Optical rotation of (ent)-2 (57% ee): [a]18D –181 (c = 0.6,
MeOH).
(13) (S)-1-(2-tert-Butoxycarbonylaminobenzoyl)pyrrolidine-
2-carboxylic Acid Methyl Ester (6): A solution of 3 (167
mg, 0.70 mmol), PyBOP (474 mg, 0.91 mmol), and DIPEA
(0.48 mL, 2.80 mmol) in CH2Cl2 (7.0 mL) was cooled to 0
°C under an argon atmosphere. The mixture was stirred for
10 min, and then L-proline methyl ester hydrochloride (4,
151 mg, 0.91 mmol) was added. The reaction mixture was
allowed to warm to r.t. and stirred for 1 h. The reaction
mixture was quenched with 5% aq KHSO4 solution and
extracted with EtOAc. The combined organic layers were
washed with sat. aq NaHCO3 solution, brine, dried over
Na2SO4, filtered and concentrated in vacuo. The residue was
chromatographed on silica gel (hexane–EtOAc, 1:1) to
afford 6 (210 mg, 0.60 mmol, 86%) as a white amorphous
solid. Data for 6: 1H NMR (400 MHz, CDCl3): d = 8.37 (br
Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synlett. Included are
general information and experimental procedures for preparation
and compound characterization data, copies of NMR spectra.
Synlett 2010, No. 16, 2498–2502 © Thieme Stuttgart · New York

LETTER
s, 1 H), 8.18 (d, 1 H, J = 8.3 Hz), 7.36–7.40 (m, 2 H), 7.01
Practical Total Synthesis of Fuligocandins A and B
2501
Fuligocandin A (1): To a solution of 10 (10 mg, 33.0 mmol)
in CH2Cl2 (0.8 mL) was added TFA (0.8 mL) at –20 °C. The
reaction mixture was stirred for 4 h at –20 °C. After addition
of toluene, TFA was carefully evaporated in vacuo at r.t. To
this mixture was added sat. aq NaHCO3 solution, then the
mixture was stirred at r.t. The mixture was extracted with
CH2Cl2 and the combined organic layers were dried over
Na2SO4, filtered and concentrated in vacuo. The residue was
chromatographed on silica gel (hexane–EtOAc = 1:1) to
afford 1 (7.6 mg, 29.7 mmol, 90%, 70% ee). The ee value was
determined by CHIRALPAK AD-RH (46 × 150 mm);
eluent: 75% MeOH; flow rate: 1.0 mL/min; tR: (ent)-1 (14.4
min), 1 (22.6 min). Data for 1: [a]18D +225 (c = 1.4, MeOH).
1H NMR (400 MHz, CDCl3): d = 12.6 (br s, 1 H), 7.97 (dd,
1 H, J = 1.5, 7.7 Hz), 7.45 (dt, 1 H, J = 1.5, 7.9 Hz), 7.21 (dt,
1 H, J = 1.5, 7.9 Hz), 7.02 (d, 1 H, J = 7.9 Hz), 5.29 (s, 1 H),
4.30 (d, 1 H, J = 7.9 Hz), 3.78–3.85 (m, 1 H), 3.61–3.69 (m,
1 H), 2.39–2.41 (m, 1 H), 2.20 (s, 3 H), 2.06–2.16 (m, 3 H).
13C NMR (100 MHz, CDCl3): d = 198.4, 165.7, 158.8, 137.1,
132.4, 131.1, 127.2, 124.4, 122.1, 91.1, 55.3, 46.9, 30.0,
27.0, 23.4. IR (ATR): 2987, 2877, 1629, 1591, 1557, 1406,
1262, 1250, 754, 733, 696, 683 cm–1. EIMS: m/z = 256 [M+].
HRMS (EI): m/z [M+] calcd for C15H16N2O2: 256.1212;
found: 256.1211.
Fuligocandin B (2): To a solution of prepared LDA (0.79
mmol) in THF (4.0 mL) was added 1 (51 mg, 0.20 mmol) in
THF via cannula at –78 °C under an argon atmosphere. The
solution was stirred for 30 min at this temperature. The
resulting solution was then transferred to a THF solution of
20 (298 mg, 1.00 mmol) via cannula at –78 °C. The solution
was stirred for 20 min at this temperature. The reaction
mixture was allowed to warm to 0 °C. The reaction mixture
was stirred for 1 h at 0 °C and H2O was then added. The
mixture was extracted with EtOAc. The organic layer was
washed with brine, dried over Na2SO4, filtered and
(t, 1 H, J = 7.6 Hz), 4.67–4.71 (m, 1 H), 3.79 (s, 3 H), 3.58–
3.64 (m, 1 H), 3.48–3.54 (m, 1 H), 2.32–2.41 (m, 1 H), 1.95–
2.08 (m, 2 H), 1.85–1.92 (m, 1 H), 1.50 (s, 9 H). 13C NMR
(100 MHz, CDCl3): d = 172.6, 168.8, 153.0, 137.3, 131.0,
127.2, 123.5, 121.6, 120.2, 80.3, 59.1, 52.5, 50.0, 29.4, 28.3,
25.3. IR (ATR): 3336, 2978, 1730, 1625, 1588, 1522, 1456,
1416, 1242, 1199, 1158 cm–1. EIMS: m/z = 348 [M+]. HRMS
(EI): m/z [M+] calcd for C17H24N2O5: 348.1685; found:
348.1689.
(11aS)-10-tert-Butoxycarbonyl-2,3-dihydro-1H-pyrrolo-
[2,1-c][1,4]benzodiazepine-5,11 (10H,11aH)dione (8):
To a stirred solution of 6 (210 mg, 0.60 mmol) in solvent
(THF–MeOH–H2O = 3:1:1, 2.0 mL) was added LiOH⋅H2O
(50 mg, 1.2 mmol) at 0 °C. The solution was stirred for 5 h
at r.t. The reaction mixture was acidified with 1 M aq HCl to
pH 2, then extracted with EtOAc. The organic layer was
washed with brine, dried over Na2SO4, filtered and
concentrated in vacuo to afford (S)-1-(2-tert-butoxycarbonyl-
aminobenzoyl)pyrrolidine-2-carboxylic acid (195 mg) as a
white powder. To a solution of the carboxylic acid and
PyBOP (406 mg, 0.75 mmol) in CH2Cl2 (20 mL) was added
DIPEA (0.42 mL, 2.32 mmol) at 0 °C under an argon
atmosphere. The reaction mixture was allowed to warm to
r.t. and stirred for 72 h. The reaction mixture was quenched
with 5% aq KHSO4 and extracted with EtOAc. The organic
layer was washed with sat. aq NaHCO3 solution, brine, dried
over Na2SO4, filtered and concentrated in vacuo. The residue
was chromatographed on silica gel (hexane–EtOAc = 1:5) to
afford 8 (148 mg, 0.47 mmol, 79% in 2 steps) as a white
amorphous solid. Data for 8: [a]14D +157 (c = 1.0, MeOH).
1H NMR (400 MHz, CDCl3): d = 7.91 (dd, 1 H, J = 1.5, 7.8
Hz), 7.51 (dt, 1 H, J = 1.5, 7.8 Hz), 7.41 (dt, 1 H, J = 1.2, 7.8
Hz), 7.23 (dd, 1 H, J = 1.2, 7.8 Hz), 4.06–4.15 (m, 1 H),
3.82–3.87 (m, 1 H), 3.52–3.59 (m, 1 H), 2.69–2.73 (m, 1 H),
2.09–2.16 (m, 1 H), 1.94–2.07 (m, 2 H), 1.46 (s, 9 H). 13
C
concentrated in vacuo. The residue was chromatographed on
silica gel (hexane–EtOAc = 10:1 → 1:1) to afford a white
solid. The solution of white solid in solvent (THF–1 N aq
HCl = 1:1, 5.0 mL) was stirred for 17 h at r.t. and sat. aq
NaHCO3 was then added to it and the mixture was then
extracted with EtOAc. The organic layer was washed with
H2O, brine, dried over Na2SO4, filtered and concentrated
in vacuo. The residue was chromatographed on silica gel
(hexane–EtOAc = 1:2 → 0:1) to afford 2 (48 mg, 0.12
mmol, 63%, 61% ee). The ee value was determined by
CHIRALPAK AD-RH (46 × 150 mm); eluent: 50% MeCN;
flow rate: 1.0 mL/min; tR: (ent)-2 (56.3 min), 2 (68.1 min).
Data for 2: [a]18D +248 (c = 0.6, MeOH). 1H NMR (400
MHz, acetone-d6): d = 13.4 (br s, 1 H), 10.9 (br s, 1 H), 8.02
(d, 1 H, J = 8.3 Hz), 7.91 (d, 1 H, J = 15.8 Hz), 7.89 (dd, 1
H, J = 1.7, 7.8 Hz), 7.83 (d, 1 H, J = 2.7 Hz), 7.49–7.54 (m,
2 H), 7.17–7.24 (m, 3 H), 7.13 (d, 1 H, J = 8.1 Hz), 7.02 (d,
1 H, J = 15.8 Hz), 5.81 (s, 1 H), 4.44 (d, 1 H, J = 7.6 Hz),
3.67–3.72 (m, 1 H), 3.55–3.62 (m, 1 H), 2.56–2.63 (m, 1 H),
2.22–2.32 (m, 1 H), 2.05–2.16 (m, 2 H). 13C NMR (100
MHz, acetone-d6): d = 190.2, 165.8, 160.4, 138.7, 138.6,
134.9, 133.1, 131.9, 128.2, 126.4, 124.3, 123.8, 123.4,
122.6, 121.6, 121.2, 114.4, 113.0, 93.4, 56.1, 47.4, 27.6,
24.1. IR (ATR): 3250, 1590, 1567, 1270, 1247, 1137, 1106,
754, 738 cm–1. HRMS (ESI): m/z [M + Na]+ calcd for
C24H21O2N3Na: 406.1514; found: 406.1526.
NMR (100 MHz, CDCl3): d = 169.1, 164.9, 150.6, 135.3,
131.2, 130.9, 129.7, 127.4, 126.1, 84.6, 58.8, 46.6, 27.5,
26.3, 23.5. IR (ATR): 2979, 1773, 1748, 1716, 1646, 1457,
1416, 1238, 1144 cm–1. EIMS: m/z = 316 [M+]. HRMS: (EI):
m/z [M+] calcd for C17H20N2O4: 316.1423; found: 316.1431.
(11aS)-10-tert-Butoxycarbonyl-2,3-dihydro-11-hydroxy-
11-prop-1-ynyl-1H-pyrrolo[2,1-c][1,4]benzodiazepine-
5,11 (10H,11aH)dione (10): To a solution of prepared LDA
(9.0 mmol) in THF (90 mL) was added 1,2-dibromopropane
(0.31 mL, 3.0 mmol) at –78 °C under an argon atmosphere.
The solution was stirred for 10 min at this temperature, and
another 20 min at 0 °C. The resulting lithium acetylide
solution was then transferred to a THF solution of 8 (624 mg,
2.0 mmol) via cannula at –78 °C. The mixture was then
stirred at –78 °C for 40 min and sat. aq NH4Cl solution was
added slowly. The mixture was extracted with EtOAc and
the organic extracts were dried over Na2SO4, filtered and
concentrated in vacuo. The residue was chromatographed on
silica gel (hexane–EtOAc, 2:1) to afford a single isomer 10
(329 mg, 0.92 mmol, 46%) as a white amorphous solid. Data
for 10: 1H NMR (400 MHz, CDCl3): d = 8.44 (br s, 1 H), 8.21
(d, 1 H, J = 7.6 Hz), 7.38 (t, 1 H, J = 7.6 Hz), 7.02 (t, 1 H,
J = 7.6 Hz), 4.80–4.84 (m, 1 H), 3.52–3.66 (m, 2 H), 2.30–
2.40 (m, 1 H), 2.06 (s, 3 H), 1.83–2.11 (m, 3 H), 1.51 (s, 9
H). 13C NMR (100 MHz, CDCl3): d = 185.6, 168.9, 153.0,
137.6, 131.2, 127.3, 123.1, 121.5, 120.2, 92.9, 80.4, 66.5,
29.7, 28.5, 28.3, 25.2, 4.3. IR (ATR): 3336, 2926, 2220,
1727, 1521, 1409, 1340, 1157, 756 cm–1. EIMS: m/z = 356
[M+]. HRMS (EI): m/z [M+] calcd for C20H24N2O4:
TRAIL-Resistance-Overcoming Activity: To assess the
sensitivity to TRAIL, cell viability was evaluated using a
fluorometric microculture cytotoxicity assay (FMCA).
Briefly, AGS cells were seeded in a 96-well plate (6 × 103
cells per well) in 200 mL of RPMI medium containing 10%
356.1736; found: 356.1730.
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FBS. Cells were cultured in 24 h and then treated with test
samples in the absence or presence of TRAIL (100 ng/mL),
and control cells were treated with culture medium
containing 0.1% DMSO. After 24 h incubation, the cells
were washed with PBS, and 200 mL of PBS containing
fluorescein diacetate (10 mg/mL) was added to each well.
After 1 h incubation at 37 °C, fluorescence was measured in
a 96-well scanning spectrofluorometer (Thermo Electron
Corporation) at 538 nm, following excitation at 485 nm.
Synlett 2010, No. 16, 2498–2502 © Thieme Stuttgart · New York