Abstract Mcl-1 is a member of the Bcl/Mcl family of proteins that promotes cell survival by preventing induction of apoptosis in a broad range of cancers. High expression of Mcl-1 has been linked to tumor development and resistance to anticancer therapies, underscoring the potential of Mcl-1 inhibitors as anticancer drugs. We have previously shown that AZD5991, a rationally designed macrocycle with sub-nanomolar affinity for Mcl-1 and high selectivity, induces rapid and irreversible commitment to apoptosis in Mcl-1-dependent cancer cells in a manner dependent on proapoptotic BAK. Here, we demonstrate that AZD5991 exhibits cytotoxic activity (GI50<100nM) in various MM and AML cell lines in vitro with an activity profile distinct from the selective Bcl2 inhibitor venetoclax. In vivo, AZD5991 shows potent antitumor activity with complete (100%) tumor regression in several mouse MM and AML xenograft models after a single tolerated dose. AZD5991 shows enhanced efficacy in vivo when combined with standard-of-care agents. Pharmacodynamic studies confirmed that AZD5991 kills cancer cells by activation of the mitochondrial apoptotic pathway. Ex vivo analysis indicates that AZD5991 has single agent activity in primary AML patient samples with LC50 values in the low nanomolar range. Consistent with our findings in AML cell lines, the activity profile for AZD5991 in AML primary samples was distinct from venetoclax, highlighting the unique therapeutic opportunity for AZD5991. Based on these data a phase I clinical trial has been launched for evaluation of AZD5991 in patients with MM and other hematologic malignancies (NCT03218683). Citation Format: Adriana E. Tron, Matthew A. Belmonte, Steven Criscione, Edwin A. Clark, Eric Gangl, Francis D. Gibbons, Jeffrey W. Tyner, Stephen E. Kurtz, Qing Ye, Alexander W. Hird, Alwin Schuller, J. Paul Secrist. Selective Mcl-1 inhibition by AZD5991 induces on-target cell death and achieves antitumor activity in multiple myeloma and acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 302.

Mcl-1 is a member of the Bcl-2 family of proteins that promotes cell survival by preventing induction of apoptosis in many cancers. High expression of Mcl-1 causes tumorigenesis and resistance to anticancer therapies highlighting the potential of Mcl-1 inhibitors as anticancer drugs. Here, we describe AZD5991, a rationally designed macrocyclic molecule with high selectivity and affinity for Mcl-1 currently in clinical development. Our studies demonstrate that AZD5991 binds directly to Mcl-1 and induces rapid apoptosis in cancer cells, most notably myeloma and acute myeloid leukemia, by activating the Bak-dependent mitochondrial apoptotic pathway. AZD5991 shows potent antitumor activity in vivo with complete tumor regression in several models of multiple myeloma and acute myeloid leukemia after a single tolerated dose as monotherapy or in combination with bortezomib or venetoclax. Based on these promising data, a Phase I clinical trial has been launched for evaluation of AZD5991 in patients with hematological malignancies (NCT03218683).

Mcl-1, a member of the Bcl/Mcl family, is a key protein involved in evasion of apoptosis in a wide variety of tumors. Its amplification and overexpression have also been implicated in innate and acquired resistance to anticancer drugs. Mcl-1 is capable of preventing induction of apoptosis, both by binding and inactivating the pro-apoptotic executioner Bcl-2 protein, Bak, as well as by sequestering other pro-apoptotic BH3-only proteins such as Bim and Noxa. AZD5991 is a rationally designed macrocycle with sub-nanomolar affinity for Mcl-1. It demonstrates all the hallmarks of a true Mcl-1 inhibitor: 1. potent, selective, and rapid apoptosis in Mcl-1-dependent cell lines (e.g., GI 50 as low as 10 nM in multiple myeloma cell lines); 2. loss of activity upon overexpression of Bcl-xL or siRNA-mediated knockout of Bak; 3. Mcl-1:Bak complex disruption as demonstrated by co-immunoprecipitation. AZD5991 is active in vivo, with complete (100%) tumor regression demonstrated in several mouse xenograft models after a single tolerated dose. We have also demonstrated synergistic in vivo efficacy in combination with standard-of-care agents. Analysis of ex vivo activity in primary samples from leukemia patients indicates that a high percentage of leukemia patients should respond to drug treatment, which supports our plan for a phase I trial of AZD5991 in patients with hematologic cancers. Citation Format: Alexander W. Hird, J. Paul Secrist, Ammar Adam, Matthew A. Belmonte, Eric Gangl, Frank Gibbons, David Hargreaves, Jeffrey W. Johannes, Stephen L. Kazmirski, Jason G. Kettle, Stephen E. Kurtz, Michelle L. Lamb, Martin J. Packer, Bo Peng, Craig R. Stewart, Jeffrey W. Tyner, Wenzhan Yang, Qing Ye, XiaoLan Zheng, Edwin A. Clark. AZD5991: A potent and selective macrocyclic inhibitor of Mcl-1 for treatment of hematologic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr DDT01-02. doi:10.1158/1538-7445.AM2017-DDT01-02

Abstract Mcl-1, a member of the Bcl/Mcl family, is a key protein involved in evasion of apoptosis in a wide variety of tumors. Its amplification and overexpression have also been implicated in innate and acquired resistance to anticancer drugs. Mcl-1 is capable of preventing induction of apoptosis, both by binding and inactivating the pro-apoptotic executioner Bcl-2 protein, Bak, as well as by sequestering other pro-apoptotic BH3-only proteins such as Bim and Noxa. AZD5991 is a rationally designed macrocycle with sub-nanomolar affinity for Mcl-1. It demonstrates all the hallmarks of a true Mcl-1 inhibitor: 1. potent, selective, and rapid apoptosis in Mcl-1-dependent cell lines (e.g., GI50 as low as 10 nM in multiple myeloma cell lines); 2. loss of activity upon overexpression of Bcl-xL or siRNA-mediated knockout of Bak; 3. Mcl-1:Bak complex disruption as demonstrated by co-immunoprecipitation. AZD5991 is active in vivo, with complete (100%) tumor regression demonstrated in several mouse xenograft models after a single tolerated dose. We have also demonstrated synergistic in vivo efficacy in combination with standard-of-care agents. Analysis of ex vivo activity in primary samples from leukemia patients indicates that a high percentage of leukemia patients should respond to drug treatment, which supports our plan for a phase I trial of AZD5991 in patients with hematologic cancers. Citation Format: Alexander W. Hird, J. Paul Secrist, Ammar Adam, Matthew A. Belmonte, Eric Gangl, Frank Gibbons, David Hargreaves, Jeffrey W. Johannes, Stephen L. Kazmirski, Jason G. Kettle, Stephen E. Kurtz, Michelle L. Lamb, Martin J. Packer, Bo Peng, Craig R. Stewart, Jeffrey W. Tyner, Wenzhan Yang, Qing Ye, XiaoLan Zheng, Edwin A. Clark. AZD5991: A potent and selective macrocyclic inhibitor of Mcl-1 for treatment of hematologic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr DDT01-02. doi:10.1158/1538-7445.AM2017-DDT01-02

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 (IC50 = 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.

During the lead generation and optimization of PARP inhibitors blocking centrosome clustering, it was discovered that increasing hydrogen bond acceptor (HBA) strength improved cellular potency but led to elevated Caco2 and MDR1 efflux and thus poor oral bioavailability. Conversely, compounds with lower efflux had reduced potency. The project team was able to improve the bioavailability by reducing efflux through systematic modifications to the strength of the HBA by changing the electronic properties of neighboring groups, whilst maintaining sufficient acceptor strength for potency. Additionally, it was observed that enantiomers with different potency showed similar efflux, which is consistent with the promiscuity of efflux transporters. Eventually, a balance between potency and low efflux was achieved for a set of lead compounds with good bioavailability which allowed the project to progress towards establishing in vivo pharmacokinetic/pharmacodynamic relationships.

The propensity for cancer cells to accumulate additional centrosomes relative to normal cells could be exploited for therapeutic benefit in oncology. Following literature reports that suggested TNKS1 (tankyrase 1) and PARP16 may be involved with spindle structure and function and may play a role in suppressing multi-polar spindle formation in cells with supernumerary centrosomes, we initiated a phenotypic screen to look for small molecule poly (ADP-ribose) polymerase (PARP) enzyme family inhibitors that could produce a multi-polar spindle phenotype via declustering of centrosomes. Screening of AstraZeneca’s collection of phthalazinone PARP inhibitors in HeLa cells using high-content screening techniques identified several compounds that produced a multi-polar spindle phenotype at low nanomolar concentrations. Characterization of these compounds across a broad panel of PARP family enzyme assays indicated that they had activity against several PARP family enzymes, including PARP1, 2, 3, 5a, 5b, and 6. Further optimization of these initial hits for improved declustering potency, solubility, permeability, and oral bioavailability resulted in AZ0108, a PARP1, 2, 6 inhibitor that potently inhibits centrosome clustering and is suitable for in vivo efficacy and tolerability studies.

The potent and selective 3-amido-4-anilinoquinoline CSF-1R inhibitor AZ683 suffered from cardiovascular liabilities, which were linked to the off-target activities of the compound and ion channel activity in particular. Less basic and less lipophilic examples from both the quinoline and cinnoline series demonstrated cleaner secondary pharmacology profiles. Cinnoline 31 retained the required potency and oral PK profile, and was progressed through the safety screening cascade to be nominated into development as AZD7507.

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.

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.

Abstract Casein Kinase 2 (CK2) is a highly conserved and ubiquitously expressed serine/threonine kinase that regulates several key oncogenic signaling pathways, including PI3K/AKT, and NFkB and Wnt, and whose targeted overexpression in transgenic animal models results in neoplastic growth. Herein we describe the identification of AZ285, a potent pyrazolo[1,5-a]pyrimidine-derived inhibitor of CK2 with a high degree of kinase selectivity. AZ285 depletes levels of the CK2 substrates pAKTS129 and PTENS370 and induces apoptosis in cells in a concentration-dependent manner. Optimization of early leads was aided in part by cell wash-out experiments and X-ray cocrystallography with huCK2a and resulted in an agent with physical properties and DMPK characteristics suitable for evaluation in disease model studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3907. doi:1538-7445.AM2012-3907

3-Amido-4-anilinocinnolines have been identified as potent and highly selective inhibitors of CSF-1R. The synthesis and SAR of these compounds is reported, along with some physical property, pharmacokinetic and kinase selectivity data.

A series of amidoheteroaryl compounds were designed and synthesized as inhibitors of B-Raf kinase. Several compounds from the series show excellent potency in biochemical, phenotypic and mode of action cellular assays. Potent examples from the series have also demonstrated good plasma exposure following an oral dose in rodents and activity against the Ras-Raf pathway in tumor bearing mice.