4-[2-[1-(2-Cyano-3-hydroxybut-2-enonylamino)ethyl]-4-ethyl-
sulfamoylbenzo[b]furan-7-yloxy]butyric acid (26e)
Acknowledgements
We express our appreciation to the staff of the Instrumental
Analytical Center of Mukogawa Women’s University for the
NMR and MS measurements and elemental analyses.
To a solution of 26d (1.1 g, 2.2 mmol) in ethanol (60 ml) was
added 10% NaOH aqueous solution (7.0 ml). The mixture was
heated at 60 ЊC for 2.5 h, and then the solvent was evaporated.
The residue was treated according the usual manner to give a
yellow powder. The powder was recrystallized from ethanol to
give 26e (0.46 g) as a colorless powder.
Notes and References
1 Part of this work has been published as
a preliminary
communication: E. Tsuji, K. Ando, J. Kunikomo, M. Yamashita,
S. Ohta, S. Kohno and Y. Ohishi, Org. Biomol. Chem., 2003, 1, 3139–
3141.
2-[1-(But-2-enonyl)aminoethyl]-7-methoxybenzo[b]furan (27a)
Procedure for 27b from 23f
2 (a) M. Bäck, Life Sciences, 2002, 71, 611–622; (b) J. F. Evans,
Prostaglandins & Other Lipid Mediators, 2002, 68–69, 587–597;
(c) C. E. Heise, B. F. O’Dowd, D. J. Figueroa, N. Sawyer, T. Nguyen,
D.-S. Im, R. Stocco, J. N. Bellefeuille, M. Abramovitz, R. Cheng,
D. V. Williams Jr., Z. Zeng, Q. Liu, L. Ma, M. K. Clements,
N. Coulombe, Y. Liu, C. P. Austin, S. R. George, G. P. O’Neill, K. M.
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30531–30536; (d ) H.-P. Nothacker, Z. Wang, Y. Zhu, R. K.
Reinscheid, S. H. S. Lin and O. Civelli, Mol. Pharmacol., 2000, 58,
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Catalytic reduction of 23e (1.5 g, 7.3 mmol) in a similar manner
as for 23a gave amine (24e, 0.61 g) as a yellow oil. Crotonyl
chloride (0.5 ml, 4.8 mmol) was added to a solution of the oil in
anhydrous THF (30 ml). After stirring at 25 ЊC for 6 h, the
mixture was worked up in the usual way to afford a yellow
powder which was recrystallized from ethyl acetate to give 27a
(0.41 g) as pale yellow prisms.
3 W. Laurence, N. Xavier, B. Magnus, G. Lean Pierre, D. Sven-Erik
and B. Charies, Br. J. Pharm., 2002, 137, 1339–1345.
7-Methoxy-2-[1-[(3-phenylprop-2-enonyl)]aminoethyl]benzo[b]-
furan (27c) Procedure for 27d from 23f
4 H. Galczenski, J. Hutchinson, D. J. Figueroa, J. Scheigetz, R. Young
and J. F. Evans, American Thoracic Society – 98th International
Conference (May 2002, Atlanta) [D38] [Poster F4] Characterization
of DUO-LT : A Novel Dual Cysteinyl Receptor Antagonist.
5 I. Fleming, Frontier Orbitals and Organic Chemical Reactions, Wiley,
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6 Unstable 2-ethyl-3,5-diaminobenzo[b]furan obtained by reduction
of 2-ethyl-3,5-dinitrobenzo[b]furan with H2 over Pd/C in ethanol
easily gave 5-amino-2-ethyl-3(2H )-benzo[b]furanone by treatment
with HCl. On the other hand, catalytic reduction of 2-ethyl-3,5-
diaminobenzo[b]furan in acetic anhydride gave stable 2-ethyl-3,5-
diacetylaminobenzo[b]furan (unpublished data obtained in our
laboratory).
Catalytic reduction of 23e (0.8 g, 3.9 mmol) in a similar manner
as for 23a gave the crude amine (24e) as a yellow oil. trans-
Cinnamic acid chloride [prepared by treatment of trans-
cinnamic acid (0.76 g, 4.64 mmol) with SOCl2 (5 ml, 4.8 mmol)]
was added to a solution of the oil in anhydrous THF (30 ml).
After stirring at 65 ЊC for 3 h, work up in the usual way gave a
yellow oil which was purified on silica gel column chromato-
graphy [hexane–ethyl acetate (10 : 1)] to give 27c (0.73 g) as
colorless prisms.
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Calcium release experiments
Calcium mobilization assays were carried out using stably
transfected HEK 293T-cysLT2R or CHO-cysLT1R cells loaded
with Fura 2-AM fluorescent indicator dye (Molecular Probes,
Eugene, OR) in the fluorescent imaging plate reader system
(Hamamatsu Photonics, Japan). Briefly, the cells were seeded
in microtiter plate at 1.0 × 105 cells/well (HEK 293T cell) or
0.4 × 105 cells/well (CHO cell) and incubated at 37 ЊC for 24 h
(5% CO2). The cells were loaded with 7.5 µM Fura 2-AM
in Dulbecco’s modified Eagle’s medium (HEK 293T cell) or
F-12 medium (CHO cell) containing 10% fetal bovine serum,
20 mM HEPES (pH 7.4) and 2.5 mM probenecid at 37 ЊC
for 30 min. The cells were washed with Hanks’ balanced salt
solution containing 20 mM HEPES (pH 7.4). LTD4 (100 nM)
were added the cells, and fluorescence values were taken
at 3-second intervals for 90 seconds. Test compounds were
added 1 min before LTD4 (100 nM) stimulation, and the
LTD4 response was obtained by calculating peak fluorescence
values.
11 S. S. Sangapure and S. Y. Agasimundin, Indian J. Chem., 1976, 14B,
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12 G. Viti, D. Giannotti, R. Nannicini, R. Ricci and V. Pestellini,
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13 G. S. Harwalkar, S. S. Sangapure and Y. S. Agasimundin, Indian
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Radioligand binding studies
Stably transfected HEK 293T-cysLT2R cells were grown and
harvested, and the membranes were prepared according to the
procedure reported by Obata et al.22 For competition binding
studies, the membranes (125 µg of total membrane protein)
were incubated with 1.0 nM [3H]LTD4 (Perking Elmer, Boston,
MA) and various concentrations of test compounds in assay
buffer (pH 7.4) containing 10 mM HEPES, 20 mM CaCl2 and
20 mM -penicillamine at room temperature for 1 h. The reac-
tions were stopped by ice-cold wash buffer (pH 7.4) containing
10 mM HEPES and 0.01% bovine serum albumin, and the
bound ligand was captured on Whatman GF/B filters. Radio-
activity was quantitated by liquid scintillation counter. Non-
specific binding was determined in the presence of 1 µM
unlabelled LTD4.
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acetoacetate in our laboratory [A. Graul and J. Castañer, Drugs of
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18 E. Kuo, P. Hambleton, D. Kay, P. Evans, S. Matharu, E. Little,
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O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 6 2 5 – 6 3 5
634