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6-Ethynylthieno[3,2-d]- and 6-ethynylthieno[2,3-d]pyrimidin-4-anilines as tunable covalent modifiers of ErbB kinases

  1. Edgar R. Wood a ,
  2. Lisa M. Shewchuk b ,
  3. Byron Ellis c ,
  4. Perry Brignola c ,
  5. Ronald L. Brashear d ,
  6. Thomas R. Caferro e ,
  7. Scott H. Dickerson b ,
  8. Hamilton D. Dickson e ,
  9. Kelly H. Donaldson e ,
  10. Michael Gaul e ,
  11. Robert J. Griffin f ,
  12. Anne M. Hassell b ,
  13. Barry Keith g ,
  14. Robert Mullin g ,
  15. Kimberly G. Petrov e ,
  16. Michael J. Reno e ,
  17. David W. Rusnak g ,
  18. Sarva M. Tadepalli f ,
  19. John C. Ulrich f ,
  20. Craig D. Wagner a ,
  21. Dana E. Vanderwall b ,
  22. Alex G. Waterson e ,
  23. Jon D. Williams b ,
  24. Wendy L. White b , and
  25. David E. Uehling e , h
  1. Departments of aAssay Development,
  2. bComputational and Structural Sciences,
  3. cBiological Reagents and Assay Development,
  4. fMetabolic DMPK,
  5. eOncology Chemistry,
  6. dPhysical Properties and Developability, and
  7. gOncology Biology, GlaxoSmithKline, Research Triangle Park, NC 27709

  1. Edited by John Kuriyan, University of California, Berkeley, CA, and approved December 12, 2007 (received for review September 12, 2007)

Abstract

Analysis of the x-ray crystal structure of mono-substituted acetylenic thienopyrimidine 6 complexed with the ErbB family enzyme ErbB-4 revealed a covalent bond between the terminal carbon of the acetylene moiety and the sulfhydryl group of Cys-803 at the solvent interface. The identification of this covalent adduct suggested that acetylenic thienopyrimidine 6 and related analogs might also be capable of forming an analogous covalent adduct with EGFR, which has a conserved cysteine (797) near the ATP binding pocket. To test this hypothesis, we treated a truncated, catalytically competent form of EGFR (678–1020) with a structurally related propargylic amine (8). An investigation of the resulting complex by mass spectrometry revealed the formation of a covalent complex of thienopyrimidine 8 with Cys-797 of EGFR. This finding enabled us to readily assess the irreversibility of various inhibitors and also facilitated a structure–activity relationship understanding of the covalent modifying potential and biological activity of a series of acetylenic thienopyrimidine compounds with potent antitumor activity. Several ErbB family enzyme and cell potent 6-ethynyl thienopyrimidine kinase inhibitors were found to form covalent adducts with EGFR.

Footnotes

  • hTo whom correspondence should be addressed. E-mail: david.e.uehling{at}gsk.com

  • Author contributions: E.R.W., L.M.S., T.R.C., S.H.D., K.H.D., M.G., R.J.G., B.K., S.M.T., J.C.U., C.D.W., D.E.V., and D.E.U. designed research; E.R.W., L.M.S., B.E., P.B., R.L.B., T.R.C., S.H.D., H.D.D., K.H.D., M.G., R.J.G., A.M.H., R.M., K.G.P., M.J.R., D.W.R., S.M.T., C.D.W., A.G.W., J.D.W., W.L.W., and D.E.U. performed research; B.E., P.B., T.R.C., H.D.D., K.H.D., M.G., R.M., K.G.P., S.M.T., and A.G.W. contributed new reagents/analytic tools; E.R.W., L.M.S., R.L.B., T.R.C., H.D.D., K.H.D., M.G., R.J.G., A.M.H., B.K., R.M., K.G.P., M.J.R., D.W.R., S.M.T., J.C.U., C.D.W., A.G.W., J.D.W., W.L.W., and D.E.U. analyzed data; and D.E.U. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • Data deposition: The structure of the ErbB-4 kinase domain complexed with thienopyrimidine inhibitor 6 has been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 2R4B).

  • This article contains supporting information online at www.pnas.org/cgi/content/full/0708281105/DC1.

  • ↵ i We use full-length numbering for EGFR, ErbB-2, and Erb-4 (EGFR: UniProtKB/Swiss-Prot entry P00533, ErbB-2 UniProtKB/Swiss-Prot entry P04626, ErbB-4 UniProtKB/Swiss-Prot entry Q15303, and ref. 3 uses the numbering for mature protein.

  • ↵ j Determination of the covalent binding site on EGFR for compound 8: covalently modified EGFR protein bands from a Coomassie-stained SDS/PAGE gel were excised (1-mm cubes), destained, reduced (4 mM DTT), and alkylated (4-vinylpyridine) according to a standard procedure. The gel pieces were then washed, crushed, dried, and digested with thermolysin (10 mM Tris, pH 8.0, 37°C, 18 h). After enzymatic digestion, nonpassively eluting peptides were further extracted from the crushed gel pieces by addition of 50% CH3CN. Analysis of the thermolysin cleavage products by nano-ESI LC/MS/MS definitively confirmed Cys-797 as the site of covalent modification from the EGFR peptide observed at m/z 402.6 corresponding to H2N-FGC-(8)-OH. Additional information concerning the identification of Cys-797 as the covalent binding site on EGFR is detailed in SI Fig. 5.

  • ↵ k Among the handful of kinases with a conserved Cys in the “lip” region of the kinase active site, those that also have an Asp in the same position as ErbB-2 and EGFR include JAK3, ITK, and BLK. Compound 8 was found to be inactive (IC50 > 10 μM) against ITK, whereas no data are available for JAK3 or BLK. In ErbB-4 this residue is glutamate, and because it is also acidic and in a sterically similar orientation, it should function similarly in the proposed mechanism. It should be noted that compound 8 has been shown to have >10-fold selectivity against 31/31 non-ErB family kinases in which it has been tested, and >100-fold selectivity against 30/31 such kinases. The limited number of kinases containing this CysXXAsp sequence is expected to also promote kinase selectivity for HKI-272, which contains a basic amine adjacent to a Michael acceptor.

  • ↵ l The drug exposures in the efficacy studies described in Table 3 with 3 and 8 were not determined. Nevertheless, the dramatic difference in AUCs in a separate but similar PK experiment strongly suggest that exposure of 3 in the efficacy study itself was significantly higher than that of 8, as suggested in the text. In the PK experiment, female CD-1 mice were given a single oral dose of 7 in pH 3 methanesulfonic acid at 113 mg/kg or a single oral dose of 3 as HPMC/Tween suspension at 100 mg/kg versus b.i.d. dosing for 14 days in the efficacy study. Moreover, in another PK experiment, the exposure of 3 at 10 mg/kg in HPMC/0.1% HCl was also found to be much higher than 8 dosed in aqueous methanesulfonic acid at pH3 (1.6 vs. 0.67 h*μg/ml).

  • ↵ m Studies to assess the relative reactivity of agents described herein have demonstrated high selectivity for compounds in the thienopyrimidine series to Cys-797 of EGFR vs. thiols such as glutathionen (unpublished data).

  • ↵ n Representative Sonagashira coupling reaction procedure (compound 13): A mixture of 6-bromo-N-(3-chloro-4-{[(3-fluorophenyl)methyl]oxy}phenyl)thieno[2,3-d]pyrimidin-4-amine hydrochloride (3.31 g, 6.6 mmol) (preparation described in WO 2003053446), Cu(I) I (44 mg, 0.23 mmol), 5-ethynyl-2-pyrazinamine (825 mg, 6.93 mmol, WO 2003053446), and bis (triphenylphosphine)dichloropalladium (II) (324.3 mg, 0.46 mmol) under a nitrogen atmosphere was covered with anhydrous tetrahydrofuran (68 ml) and triethylamine (3.3 ml, 23.8 mmol). The mixture was heated under a nitrogen atmosphere at 40°C for 1.5 h, allowed to cool to room temperature and partitioned between saturated aqueous NaHCO3 and 5:1 CHCl3/i-PrOH. The organic layer was dried over Na2SO4, filtered, and concentrated. Purification of the residue by silica gel chromatography eluting with a hexane/ethyl acetate gradient supplied product 13 (340 mg) along with recovered starting bromide (2.16 g). lH NMR (400 MHz, DMSO-d6) δ 5.25 (s, 2H), 7.17–7.21 (m, 1H), 7.27–7.34 (m, 3H), 7.45–7.50 (m, 1H), 7.67 (dd, 1H, J = 9.0, 2.4 Hz), 7.87 (s, 1H), 8.03 (d, 1H, J = 2.2 Hz), 8.09 (s, 1H), 8.23 (s, 1H), 8.54 (s, 1H), 9.73 (s, 1H). HRMS (M+H)+ 503.0869, Calcd 503.0857.

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