The bromodomain and extraterminal (BET) protein BRD4 regulates gene expression via recruitment of transcriptional regulatory complexes to acetylated chromatin. Pharmacological targeting of BRD4 bromodomains by small molecule inhibitors has proven to be an effective means to disrupt aberrant transcriptional programs critical for tumor growth and/or survival. Herein, we report AZD5153, a potent, selective, and orally available BET/BRD4 bromodomain inhibitor possessing a bivalent binding mode. Unlike previously described monovalent inhibitors, AZD5153 ligates two bromodomains in BRD4 simultaneously. The enhanced avidity afforded through bivalent binding translates into increased cellular and antitumor activity in preclinical hematologic tumor models. In vivo administration of AZD5153 led to tumor stasis or regression in multiple xenograft models of acute myeloid leukemia, multiple myeloma, and diffuse large B-cell lymphoma. The relationship between AZD5153 exposure and efficacy suggests that prolonged BRD4 target coverage is a primary efficacy driver. AZD5153 treatment markedly affects transcriptional programs of MYC, E2F, and mTOR. Of note, mTOR pathway modulation is associated with cell line sensitivity to AZD5153. Transcriptional modulation of MYC and HEXIM1 was confirmed in AZD5153-treated human whole blood, thus supporting their use as clinical pharmacodynamic biomarkers. This study establishes AZD5153 as a highly potent, orally available BET/BRD4 inhibitor and provides a rationale for clinical development in hematologic malignancies. Mol Cancer Ther; 15(11); 2563-74. ©2016 AACR.

ABSTRACT:The Wnt pathway is an evolutionarily conserved and tightly regulated signaling network with important roles in embryonic development and adult tissue regeneration. Impaired Wnt pathway regulation, arising from mutations in Wnt signaling components, such as Axin, APC, and β-catenin, results in uncontrolled cell growth and triggers oncogenesis. To explore the reported link between CK2 kinase activity and Wnt pathway signaling, we sought to identify a potent, selective inhibitor of CK2 suitable for proof of concept studies in vivo. Starting from a pyrazolo[1,5-a]pyrimidine lead (2), we identified compound 7h, a potent CK2 inhibitor with picomolar affinity that is highly selectivity against other kinase family enzymes and inhibits Wnt pathway signaling (IC 50 = 50 nM) in DLD-1 cells. In addition, compound 7h has physicochemical properties that are suitable for formulation as an intravenous solution, has demonstrated good pharmacokinetics in preclinical species, and exhibits a high level of activity as a monotherapy in HCT-116 and SW-620 xenografts. KEYWORDS: CK2 kinase, pyrazolo[1,5-a]pyrimidine, Wnt, β-catenin T he serine/threonine protein kinase CK2 is a constitutively active heterotetrameric complex composed of two catalytic (α or α′) and two regulatory (β) subunits, 1 which has emerged as an attractive drug discovery target in oncology. 2 Researchers from Cylene have recently advanced CX-4945, a selective, orally available inhibitor of CK2 into the clinic for treatment of patients with solid tumors and hematological malignancies. 3 Among its diverse functions, CK2 interacts with and regulates multiple components of the Wnt pathway, an evolutionarily conserved signaling network that regulates embryonic development and the regeneration of intestinal epithelial cells. 4 Certain cancers, including colorectal carcinoma (CRC), arise due to gene mutations among constituents of the Wnt pathway, including the CK2 substrates dishevelled (Dvl), APC, and β-catenin. 5 Inhibition of CK2, either by RNA knockdown or with small molecules, decreases β-catenin−Tcfmediated transcription of Wnt target genes such as survivin and leads to cell death and apoptosis in a range of CRC lines. 6,7 In addition, elevated levels of CK2 activity have been reported in CRC tissue samples and expression levels correlate with poor prognosis in CRC patients. 8,9 Taken together, these data illustrate the potential utility of CK2 inhibitors in CRC and other cancers characterized by aberrant Wnt pathway activity.We sought to identify a potent, selective inhibitor of CK2 kinase for hypothesis testing in vivo using preclinical models of CRC. An early probe from our previously described series of ATP-competitive pyrazolo[1,5-a]pyrimidine-derived inhibitors of CK2 (1, 2; Figure 1) was used to assess the link between CK2 inhibition and Wnt signaling. 10 Treatment of DLD-1(APC mutant) cells with 2 inhibits β-catenin phosphorylation and decreases Wnt-mediated gene transcription as shown in a Luciferase reporter assay in APC

In this paper we describe a series of 3-cyano-5-aryl-7-aminopyrazolo[1,5-a]pyrimidine hits identified by kinase-focused subset screening as starting points for the structure-based design of conformationally constrained 6-acetamido-indole inhibitors of CK2. The synthesis, SAR, and effects of this novel series on Akt signaling and cell proliferation in vitro are described.

In this letter, we describe the design, synthesis, and structure−activity relationship of 5-anilinopyrazolo [1,5-a]pyrimidine inhibitors of CK2 kinase. Property-based optimization of early leads using the 7-oxetan-3-yl amino group led to a series of matched molecular pairs with lower lipophilicity, decreased affinity for human plasma proteins, and reduced binding to the hERG ion channel. Agents in this study were shown to modulate pAKT S129 , a direct substrate of CK2, in vitro and in vivo, and exhibited tumor growth inhibition when administered orally in a murine DLD-1 xenograft. KEYWORDS: CK2 kinase, pyrazolo[1,5-a]pyrimidine, matched molecular pair, oxetane T he serine/threonine protein kinase CK2, a tetrameric complex containing two catalytic (α or α′) and two regulatory (β) subunits, controls cell growth, proliferation, and evasion of apoptosis by phosphorylation of a range of substrates in critical cellular signaling pathways including PI3K (phosphatidylinositol 3-kinase)/AKT (protein kinase B), NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells) and Wnt (wingless type MMTV integration site family).1−3 Overexpression of the CK2α subunit correlates with tumor aggressiveness and disease severity in certain cancers, while compensatory increases in CK2α′ levels have been observed in response to RNAi treatment in mice.4,5 Several academic and industrial research groups have been actively engaged in developing small molecule inhibitors of CK2 to provide further pharmacological validation of the compelling in vitro and in vivo data amassed to date. 6 The recent discovery of CX-4945, a selective, orally available inhibitor of CK2 by researchers from Cylene, represents an important first step in evaluating the clinical potential of this novel target in man. 7 We have recently described the design of a series of conformationally constrained inhibitors of CK2 containing the pyrazolo[1,5-a]pyrimidine nucleus.8 Members of this series of compounds exhibited potent inhibition of the enzyme, possessed a high degree of kinase selectivity, and depleted cellular levels of pAKT S129 , a direct substrate of CK2 believed to hyperactivate the AKT pathway.9 Although our attempts to enhance both cellular potency and physical properties in this series were unsuccessful, these studies resulted in an understanding of the structure−property relationships within the scaffold and provided additional insights into ligand−receptor binding. In particular, we found that N-methylation of the acetamide of 1a, to give ring-constrained analogue 1b, preserved enzymatic and cellular activity. In subsequent designs, we proposed to release the constraint in 1b and introduce a new conformational constraint, exemplified by indoline 2, that would enforce the crystallographically observed cisoid conformation of the acetamide in 1 (Figure 1). Compound 2 is a potent inhibitor

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