Anaplastic lymphoma kinase (ALK) fusion genes promote a variety of human malignancies. Although several ALK inhibitors have significantly improved disease prognosis in patients with ALK positive cancers, the persistent emergence of acquired drug-resistant mutations remain the major problem in clinic treatment. Adoption of new therapeutic strategies such as proteolysis targeting chimera (PROTAC) to overcome drug resistance in BTK/AR-related cancers have shown promising prospect. Herein, we reported the integrate ALK PROTACs through overall optimization of linker, revealed that subtle structural differences can lead to significant activity difference, indicating the key role of conformation of PROTACs in inducing the formation of E3-PROTAC-target protein ternary complexes. A series of rigid ALK PROTACs were developed through conjugation of Ceritinib and thalidomide, orally bioavailable PROTAC 4B (F = 14.22 %) was obtained by overall optimization of molecular properties. 4B effectively induced long lasting degradation of ALK fusion proteins and strong repression of downstream pathway in Karpas 299 cells (DC50 = 119.33 nM, Dmax = 97.1 %) and showed comparable anti-proliferative activity to Ceritinib (IC50 = 3.11 ± 0.08 nM vs IC50 = 1.31 ± 0.43 nM). Furthermore, 4B significantly inhibited the growth of Karpas 299 xenografts in vivo with TGI of 49.5 % and showed superior anti-proliferative activity against G1202R mutation to Ceritinib (IC50 = 52.82 nM vs IC50 = 109.5 nM). Overall, 4B is expected to be a potential treatment for ALK-driven malignancies.Copyright © 2024 Elsevier Masson SAS. All rights reserved.