Irreversible inhibition of disease-associated proteins with small molecules is a powerful approach for achieving increased and sustained pharmacological potency. Here, we introduce α-chlorofluoroacetamide (CFA) as a novel warhead of targeted covalent inhibitor (TCI). Despite weak intrinsic reactivity, CFA-appended quinazoline showed high reactivity toward Cys797 of epidermal growth factor receptor (EGFR). In cells, CFA-quinazoline showed higher target specificity for EGFR than the corresponding Michael acceptors in a wide concentration range (0.1–10 μM). The cysteine adduct of the CFA derivative was susceptible to hydrolysis and reversibly yielded intact thiol but was stable in solvent-sequestered ATP-binding pocket of EGFR. This environment-dependent hydrolysis can potentially reduce off-target protein modification by CFA-based drugs. Oral administration of CFA quinazoline NS-062 significantly suppressed tumor growth in a mouse xenograft model. Further, CFA-appended pyrazolopyrimidine irreversibly inhibited Bruton’s tyrosine kinase with higher target specificity. These results demonstrate the utility of CFA as a new class warheads for TCI.

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We thank Y. Ichinose, National Hospital Organization, Kyushu Cancer Center (Fukuoka, Japan) for kindly providing PC-9 cells. We also thank M. Fujita, Kyushu University (Fukuoka, Japan) for kindly providing HEK293 cells. This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas “Chemistry for Multimolecular Crowding Biosystems” (JSPS KAKENHI Grant No. JP17H06349) and Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under Grant Number JP18am0101091. A.O. acknowledges Takeda Science Foundation for its financial support. N.S. acknowledges Grant-in-Aid for Young Scientists B (JSPS KAKENHI Grant No. JP17K15483) for its financial support. H.F. acknowledges JSPS Research Fellowships for Young Scientists. I.T.b.M. is supported by the World Premier International Research Center Initiative, Japan. K.K. acknowledges Grant-in-Aid for Scientific Research on Innovative Areas (JSPS KAKENHI Grant No. JP15H05955).

Author information

Author notes

  1. These authors contributed equally: Naoya Shindo, Hirokazu Fuchida and Mami Sato.


  1. Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan

    • Naoya Shindo
    • , Hirokazu Fuchida
    • , Mami Sato
    • , Kosuke Watari
    • , Tomohiro Shibata
    • , Chizuru Miura
    • , Kei Okamoto
    • , Yuji Hatsuyama
    • , Keisuke Tokunaga
    • , Seiichi Sakamoto
    • , Satoshi Morimoto
    • , Yoshito Abe
    • , Mitsunori Shiroishi
    • , Jose M. M. Caaveiro
    • , Tadashi Ueda
    • , Naoya Matsunaga
    • , Takaharu Nakao
    • , Satoru Koyanagi
    • , Shigehiro Ohdo
    • , Mayumi Ono
    •  & Akio Ojida
  2. Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Nagoya, Japan

    • Keiko Kuwata
  3. Graduate School of Engineering, Kyoto University, Kyoto, Japan

    • Tomonori Tamura
    •  & Itaru Hamachi
  4. Faculty of Pharmaceutical Sciences, Nagasaki International University, Sasebo, Japan

    • Yasuchika Yamaguchi


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A.O. conceived and directed the study. N.S., H.F., M. Sato. C.M., K.O., Y.H., and K.T. synthesized compounds, designed and executed chemical, biochemical and cellular experiments and analyzed data. K.W., T.S., and M.O. performed in vivo animal studies. S.S., S.M., Y.A., M. Shiroishi., J.M.M.C., and T.U. performed protein expression and X-ray crystallography experiments. K.K., T.T., and I.H. performed proteome analysis by mass spectrometry. A.O., N.M., T.N., S.K., and S.O. performed the pharmacokinetics studies. Y.Y. assisted the data analysis of the structure–activity relationship. A.O., N.S., and H.F. wrote the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Akio Ojida.

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