Purpose: Non-small cell lung cancers (NSCLCs) harboring ALK gene rearrangements (ALK þ ) typically become resistant to the first-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor (TKI) crizotinib through development of secondary resistance mutations in ALK or disease progression in the brain. Mutations that confer resistance to second-generation ALK TKIs ceritinib and alectinib have also been identified. Here, we report the structure and first comprehensive preclinical evaluation of the next-generation ALK TKI brigatinib. Experimental Design: A kinase screen was performed to evaluate the selectivity profile of brigatinib. The cellular and in vivo activities of ALK TKIs were compared using engineered and cancer-derived cell lines. The brigatinib-ALK co-structure was determined.Results: Brigatinib potently inhibits ALK and ROS1, with a high degree of selectivity over more than 250 kinases. Across a panel of ALK þ cell lines, brigatinib inhibited native ALK (IC 50 , 10 nmol/L) with 12-fold greater potency than crizotinib. Superior efficacy of brigatinib was also observed in mice with ALK þ tumors implanted subcutaneously or intracranially. Brigatinib maintained substantial activity against all 17 secondary ALK mutants tested in cellular assays and exhibited a superior inhibitory profile compared with crizotinib, ceritinib, and alectinib at clinically achievable concentrations. Brigatinib was the only TKI to maintain substantial activity against the most recalcitrant ALK resistance mutation, G1202R. The unique, potent, and pan-ALK mutant activity of brigatinib could be rationalized by structural analyses. Conclusions: Brigatinib is a highly potent and selective ALK inhibitor. These findings provide the molecular basis for the promising activity being observed in ALK þ , crizotinib-resistant patients with NSCLC being treated with brigatinib in clinical trials.

In the treatment of echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase positive (ALK+) non-small-cell lung cancer (NSCLC), secondary mutations within the ALK kinase domain have emerged as a major resistance mechanism to both first- and second-generation ALK inhibitors. This report describes the design and synthesis of a series of 2,4-diarylaminopyrimidine-based potent and selective ALK inhibitors culminating in identification of the investigational clinical candidate brigatinib. A unique structural feature of brigatinib is a phosphine oxide, an overlooked but novel hydrogen-bond acceptor that drives potency and selectivity in addition to favorable ADME properties. Brigatinib displayed low nanomolar IC50s against native ALK and all tested clinically relevant ALK mutants in both enzyme-based biochemical and cell-based viability assays and demonstrated efficacy in multiple ALK+ xenografts in mice, including Karpas-299 (anaplastic large-cell lymphomas [ALCL]) and H3122 (NSCLC). Brigatinib represents the most clinically advanced phosphine oxide-containing drug candidate to date and is currently being evaluated in a global phase 2 registration trial.

In the treatment of chronic myeloid leukemia (CML) with BCR-ABL kinase inhibitors, the T315I gatekeeper mutant has emerged as resistant to all currently approved agents. This report describes the structure-guided design of a novel series of potent pan-inhibitors of BCR-ABL, including the T315I mutation. A key structural feature is the carbon-carbon triple bond linker which skirts the increased bulk of Ile315 side chain. Extensive SAR studies led to the discovery of development candidate 20g (AP24534), which inhibited the kinase activity of both native BCR-ABL and the T315I mutant with low nM IC(50)s, and potently inhibited proliferation of corresponding Ba/F3-derived cell lines. Daily oral administration of 20g significantly prolonged survival of mice injected intravenously with BCR-ABL(T315I) expressing Ba/F3 cells. These data, coupled with a favorable ADME profile, support the potential of 20g to be an effective treatment for CML, including patients refractory to all currently approved therapies.

The BCR-ABL inhibitor imatinib has revolutionized the treatment of chronic myeloid leukemia. However, drug resistance caused by kinase domain mutations has necessitated the development of new mutation-resistant inhibitors, most recently against the T315I gatekeeper residue mutation. Ponatinib (AP24534) inhibits both native and mutant BCR-ABL, including T315I, acting as a pan-BCR-ABL inhibitor. Here, we undertook a combined crystallographic and structure-activity relationship analysis on ponatinib to understand this unique profile. While the ethynyl linker is a key inhibitor functionality that interacts with the gatekeeper, virtually all other components of ponatinib play an essential role in its T315I inhibitory activity. The extensive network of optimized molecular contacts found in the DFG-out binding mode leads to high potency and renders binding less susceptible to disruption by single point mutations. The inhibitory mechanism exemplified by ponatinib may have broad relevance to designing inhibitors against other kinases with mutated gatekeeper residues.

Huang is an employee and shareholder of Theseus Pharmaceuticals and a former employee of ARIAD. Y. Hu is an employee of Takeda. F. Li is a former employee of ARIAD. M.T. Greenfield is a former employee of ARIAD. S.G. Zech is an employee and shareholder of Amgen Inc. and a former employee of ARIAD. B. Das is a former employee of ARIAD. N.I. Narasimhan is a former employee of ARIAD. T. Clackson is an employee and shareholder of Xilio Therapeutics and is a former employee of ARIAD. D. Dalgarno is an employee and shareholder of Theseus Pharmaceuticals and a former employee of ARIAD. W.C. Shakespeare is an employee and shareholder of Theseus Pharmaceuticals and a former employee of ARIAD. M. Fitzgerald is a former employee Research.