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DOI: 10.1055/s-0029-1219781
Isoxazolopyrimidines as Novel ΔF508-CFTR Correctors
Publication History
Publication Date:
17 March 2010 (online)
Abstract
Using a cell-based high-throughput screen, we identified isoxazolo[5,4-d]pyrimidines as novel small-molecule correctors of the cystic fibrosis mutant protein ΔF508-CFTR. 22 Isoxazolo[5,4-d]pyrimidine analogues were synthesized and tested. Synthesis of the key intermediate, 5-amino-3-arylisoxazole-4-carboxamide, was accomplished by nitrile oxide cycloaddition to (2-amino-1-cyano-2-oxoethyl)sodium. Formation of 3-arylisoxazolo-[5,4-d]pyrimidin-4(5H)-one and chlorination gave 4-chloro-3-arylisoxazolo[5,4-d]pyrimidine. Finally, functionalization at C-4 of the pyrimidine ring by nucleophilic substitution gave the targeted isoxazolo[5,4-d]pyrimidines. Six of the reported analogues had low micromolar potency for increasing halide transport in ΔF508-CFTR cells.
Key words
cystic fibrosis - ΔF508-CFTR - corrector - isoxazolopyrimidine
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29a
Compound 4a: ¹H NMR (600 MHz, DMSO): δ = 7.65 (br s, 2 H), 7.63-7.58 (m, 2 H), 7.38-7.32 (m, 2 H). ¹³C NMR (15 MHz, DMSO): δ = 171.70, 164.11, 163.85, 162.22, 159.78, 131.09, 131.04, 125.46, 125.44, 115.93, 115.79, 86.79. ESI-MS: m/z = 222.08 [M + H]+.
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29b
Compound 5a: ¹H NMR (300 MHz, DMSO): δ = 13.17 (br s, 1 H), 8.46 (s, 1 H), 8.38 (m, 2 H), 7.42 (m, 2 H). ESI-MS: m/z = 232.06 [M + H]+.
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29c
Compound 6a: ¹H NMR (600 MHz, CDCl3): δ = 9.02 (s, 1 H), 7.92-7.77 (m, 2 H), 7.28 (m, 2 H). ¹³C NMR (150 MHz, CDCl3): δ = 175.19, 165.60, 164.12, 158.07, 157.03, 156.72, 132.17, 132.11, 122.57, 116.39, 116.24, 110.62, 77.37, 77.16, 76.95.
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29d
Compound 7: ¹H NMR (600 MHz, CDCl3): δ = 8.59 (s, 1 H), 7.85-7.69 (m, 2 H), 7.45 (d, J = 7.9 Hz, 1 H), 7.38-7.29 (m, 2 H), 7.16 (t, J = 7.7 Hz, 1 H), 7.10 (d, J = 7.5 Hz, 1 H), 6.81 (br s, 1 H), 2.31 (s, 3 H), 2.03 (s, 3 H). ¹³C NMR (150 MHz, CDCl3): δ = 176.12, 165.31, 163.63, 159.45, 157.18, 156.18, 138.28, 134.79, 131.06, 130.70, 130.64, 128.85, 126.29, 124.84, 123.31, 117.45, 117.30, 95.73, 20.71, 14.21. ESI-MS: m/z = 335.10 [M + H]+.
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29e
Compound 28: ¹H NMR (600 MHz, CDCl3): δ = 8.67 (s, 1 H), 6.79 (m, 7 H), 6.56 (br s, 1 H), 3.35 (s, 3 H), 2.00 (s, 2 H), 1.96 (s, 3 H). ¹³C NMR (150 MHz, CDCl3): δ = 176.07, 163.97, 162.25, 160.25, 157.52, 144.82, 139.36, 133.03, 130.43, 129.88, 127.08, 123.56, 114.55, 96.77, 40.97, 20.28, 15.68. ESI-MS: m/z = 349.12 [M + H]+.
References and Notes
General Procedure
for Bioassays - Δ508-CFTR Corrector
Activity Assay
Assays were performed by utilizing
FRT epithelial cells stably coexpressing human ΔF508-CFTR
and the high-sensitivity halide-sensing fluorescent protein YFP-H148Q/I152L
used as described previously.¹¹ Cells
were grown at 37 ˚C (95% air/5% CO2)
for 24 h and then incubated for 16-20 h with 50 µL
of medium containing the test compound. At the time of the assay,
cells were washed with PBS and then incubated with PBS containing
forskolin (20 µM) and genistein (50 µM) for 20
min. Measurements were carried out using FLUOstar fluorescence plate
readers (Optima; BMG LABTECH Gmbh), each equipped with 500 ± 10
nm excitation and 535 ± 15 nm emission filters (Chroma Technology
Corp.). Each well was assayed individually for I- influx
by recording fluorescence continuously (200 ms per point) for 2
s (baseline) and then for 12 s after rapid (<1 s) addition
of 165 µL PBS in which 137 mM Cl- was
replaced by I-. I- influx
was computed by fitting the final 11.5 s of the data to an exponential
for extrapolation of initial slope All experiments contained negative
control (DMSO vehicle) and positive control corr-4a¹¹ ({N-[2-(5-chloro-2-meth-oxyphenylamino)-4′-methyl-4,5′-bithiazol-2′-yl]benz-
amide}).
Background I- influx (from DMSO control) was subtracted
to report the increase in I- influx in Table
[¹]
.