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Synlett 2010(7): 1063-1066  
DOI: 10.1055/s-0029-1219781
LETTER
© Georg Thieme Verlag Stuttgart ˙ New York

Isoxazolopyrimidines as Novel ΔF508-CFTR Correctors

Gui Jun Yua, Baoxue Yangb, A. S. Verkman*b, Mark J. Kurth*a
a Department of Chemistry, University of California, One Shields Avenue, Davis, CA 95616, USA
Fax: +1(530)7528995; e-Mail: mjkurth@ucdavis.edu;
b Departments of Medicine and Physiology, University of California, San Francisco, CA 94143, USA
Fax: +1(415)6653847; e-Mail: alan.verkman@ucsf.edu;
Further Information

Publication History

Received 10 December 2009
Publication Date:
17 March 2010 (online)

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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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  • Representative Spectral Data
  • 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]+.

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30

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] .