Bruton's tyrosine kinase (BTK) is a promising drug target for the treatment of multiple diseases, such as B-cell malignances, asthma, and rheumatoid arthritis. A series of novel aminotriazines were identified as highly selective inhibitors of BTK by a scaffold-hopping approach. Subsequent SAR studies of this series using two conformationally different BTK proteins, an activated form of BTK and an unactivated form of BTK, led to the discovery of a highly selective BTK inhibitor, 4b. With significant efficacy in models in vivo and good ADME and safety profiles, 4b was advanced into preclinical studies.

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A bacterial strain that grew on 4-amino-3-hydroxybenzoic acid was isolated from farm soil. The isolate, strain 10d, was identified as a species of Bordetella. Cell extracts of Bordetella sp. strain 10d grown on 4-amino-3-hydroxybenzoic acid contained an enzyme that cleaved this substrate. The enzyme was purified to homogeneity with a 110-fold increase in specific activity. The purified enzyme was characterized as a meta-cleavage dioxygenase that catalyzed the ring fission between C2 and C3 of 4-amino-3-hydroxybenzoic acid, with the consumption of 1 mol of O 2 per mol of substrate. The enzyme was therefore designated as 4-amino-3-hydroxybenzoate 2,3-dioxygenase. The molecular mass of the native enzyme was 40 kDa based on gel filtration; the enzyme is composed of two identical 21-kDa subunits according to SDS/PAGE. The enzyme showed a high dioxygenase activity only for 4-amino-3-hydroxybenzoic acid. The K m and V max values for this substrate were 35 lM and 12 lmolAEmin, respectively. Of the 2-aminophenols tested, only 4-aminoresorcinol and 6-aminom-cresol inhibited the enzyme. The enzyme reported here differs from previously reported extradiol dioxygenases, including 2-aminophenol 1,6-dioxygenase, in molecular mass, subunit structure and catalytic properties.

Introduction: Bruton's tyrosine kinase (BTK) is a member of the Tec family of cytoplasmic tyrosine kinase. BTK plays a crucial role in the BCR signaling, essential for B-cell development, and BTK has been recognized as a validated therapeutic target for B-cell malignancies. Ibrutinib, the first FDA-approved BTK inhibitor, has been appreciated as a promising targeted therapy for patients with B-cell malignancies. Ibrutinib covalently binds to the Cys481 residue of BTK to inhibit BTK enzymatic activity, but recent studies suggested that the C481S mutation in BTK disrupts the irreversible binding of ibrutinib, and resulted in ibrutinib-resistance in patients. Therefore a non-covalent BTK inhibitor is highly demanded to overcome ibrutinib resistance. We identified a highly selective, orally available non-covalent inhibitor of BTK, CB1763. Here, we report the characterization of CB1763, capable of potently inhibiting BTK [C481S] mutant, both in vitro and in vivo. Material and Methods: We produced ibrutinib-resistant BTK [C481S] mutant recombinant enzyme to assess efficacy of compounds against the drug-resistant enzyme. To evaluate cellular potency, phosphorylations of BTK and PLC- γ were analyzed by Western blotting in C481S BTK mutant transfected HEK293 cells. ABC-type DLBCL cell line, OCI-Ly10 was used to evaluate antitumor efficacy of BTK inhibitors. Kinase selectivity profiling was performed to confirm inhibitor selectivity. Results: In addition to optimization of CB988, previously reported by our group, we identified CB1763 as a highly selective non-covalent BTK inhibitor. CB1763 exhibited strong inhibitory activity for both wild type and C481S mutant BTKs with sub-nanomolar enzyme inhibitory potency in a reversible manner (IC50 = 0.85 and 0.99 nM for wild type and C481S, respectively). In cellular assays, CB1763 significantly reduced Tyr223 autophosphorylation of BTK protein at nano-molar concentration in C481S BTK mutant transfected HEK293 cells. Interestingly, persistent inhibition of autophosphorylation was observed after washout of CB1763, while no prolonged inhibition was observed by ibrutinib treatment. CB1763 demonstrated excellent antitumor activity in the BTK driven OCI-Ly10 xenograft mouse model. Conclusions: BTK [C481S] mutation has been reported in ibrutinib-relapsed CLL and MCL patients. Orally available novel non-covalent BTK inhibitor, CB1763, strongly inhibited C481S mutant BTK in vitro and in vivo, suggesting that CB1763 has the potential to treat patients who are relapsed/refractory to ibrutinib. Citation Format: Tokiko Asami, Wataru Kawahata, Shigeki Kashimoto, Masaaki Sawa. CB1763, a highly selective, novel non-covalent BTK inhibitor, targeting ibrutinib-resistant BTK C481S mutant [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B152.