Despite recent advances in the inhibition of EGFR (epidermal growth factor receptor), there remains a clinical need for new EGFR Exon20 insertion (Ex20Ins) inhibitors that spare EGFR WT. Herein, we report the discovery and optimization of two chemical series leading to ether 23 and biaryl 36 as potent, selective, and brain-penetrant inhibitors of Ex20Ins mutants. Building on our earlier discovery of alkyne 5 which allowed access to CNS property space for an Ex20Ins inhibitor, we utilized structure-based design to move to lower lipophilicity and lower CLint compounds while maintaining a WT selectivity margin. During optimization, aldehyde oxidase (AO) metabolism was identified as a human clearance risk, and through SAR exploration, lower AO metabolism was achieved. Potency and WT margin were optimized across a range of Ex20Ins mutants including the potential acquired resistance T790M mutant and efficacy demonstrated in an LXF2478 Ex20Ins ASV model with margin to EGFR WT in vivo.

Herein, we report the identification and optimization of a series of potent inhibitors of EGFR Exon20 insertions with significant selectivity over wild-type EGFR. A strategically designed HTS campaign, multiple iterations of structure-based drug design (SBDD), and tactical linker replacement led to a potent and wild-type selective series of molecules and ultimately the discovery of 36. Compound 36 is a potent and selective inhibitor of EGFR Exon20 insertions and has demonstrated encouraging efficacy in NSCLC EGFR CRISPR-engineered H2073 xenografts that carry an SVD Exon20 insertion and reduced efficacy in a H2073 wild-type EGFR xenograft model compared to CLN-081 (5), indicating that 36 may have lower EGFR wild-type associated toxicity.

Legends for Supplementary Figures 1 - 7.

Supplementary Figure 1 contains a Western blot showing the amount of estrogen receptor remaining after treatment of MCF-7 cells with AZD9496, fulvestrant and estradiol alongside GAPDH protein levels as an internal gel loading control; Supplementary Figure 2 shows the rate of decreased detection by mass spectrometry of an estrogen receptor heavy isotype labelled peptide over time after treatment of cells with AZD9496, fulvestrant and tamoxifen and the calculated half life; Supplementary Figure 3 shows the degradation of estrogen receptor α by AZD9496, fulvestrant and estradiol in the presence of +/- 10 μM proteosomal inhibitor MG132; Supplementary Figure 4 contains a Western blot showing the re-emergence over 48 hours of estrogen receptor α following removal of AZD9496, fulvestrant and estradiol compounds from MCF-7 cells; Supplementary Figure 5 shows a bar chart of the measured estradiol levels in the blood of mice over 42 days after implantation of the mice with estrogen pellets to support growth of the MCF-7 cells as a tumour in mice; Supplementary Figure 6 shows the effects of AZD9496, fulvestrant and tamoxifen on mRNA levels of estrogen receptor regulated genes in vitro and in vivo; Supplementary Figure 7 shows the levels of estrogen receptor α protein remaining in tumours from the HCC1428 LTED model after treatment with AZD9496 and fulvestrant.

Supplementary Table 1 contains crystallographic data collection and refinements statistics for AZD9496 binding to the human estrogen receptor α ligand binding domain, Supplementary Table 2 shows the rate constants for association, dissociation and equilibrium dissociation constant for AZD9496 binding to the human estrogen receptor α ligand binding domain, Supplementary Table 3 displays IC50 values for AZD9496 binding to androgen, glucocorticoid, progesterone and estrogen receptor β ligand binding domains, Supplementary Table 4 displays IC50s values for cell growth inhibition of a panel of estrogen receptor positive and negative cell lines using AZD9496.

In this article, we report the discovery of a series of pyrimidopyridones as inhibitors of IRAK4 kinase. From a previously disclosed 5-azaquinazoline series, we found that switching the pyridine ring for an N-substituted pyridone gave a novel hinge binding scaffold which retained potency against IRAK4. Importantly, introduction of the carbonyl established an internal hydrogen bond with the 4-NH, establishing a conformational lock and allowing truncation of the large basic substituent to a 1-methylcyclopyl group. Subsequent optimisation, facilitated by X-ray crystal structures, allowed identification of preferred substituents at both the pyridone core and pyrazole. Subsequent combinations of optimal groups allowed control of lipophilicity and identification of potent and selective inhibitors of IRAK4 with better in vitro permeability and lower clearance.

In this article, we report our efforts towards improving in vitro human clearance in a series of 5-azaquinazolines through a series of C4 truncations and C2 expansions. Extensive DMPK studies enabled us to tackle high Aldehyde Oxidase (AO) metabolism and unexpected discrepancies in human hepatocyte and liver microsomal intrinsic clearance. Our efforts culminated with the discovery of 5-azaquinazoline 35, which also displayed exquisite selectivity for IRAK4, and showed synergistic in vitro activity against MyD88/CD79 double mutant ABC-DLBCL in combination with the covalent BTK inhibitor acalabrutinib.

While the treatment of gastrointestinal stromal tumors (GISTs) has been revolutionized by the application of targeted tyrosine kinase inhibitors capable of inhibiting KIT-driven proliferation, diverse mutations to this kinase drive resistance to established therapies. Here we describe the identification of potent pan-KIT mutant kinase inhibitors that can be dosed without being limited by the tolerability issues seen with multitargeted agents. This effort focused on identification and optimization of an existing kinase scaffold through the use of structure-based design. Starting from a series of previously reported phenoxyquinazoline and quinoline based inhibitors of the tyrosine kinase PDGFRα, potency against a diverse panel of mutant KIT driven Ba/F3 cell lines was optimized, with a particular focus on reducing activity against a KDR driven cell model in order to limit the potential for hypertension commonly seen in second and third line GIST therapies. AZD3229 demonstrates potent single digit nM growth inhibition across a broad cell panel, with good margin to KDR-driven effects. Selectivity over KDR can be rationalized predominantly by the interaction of water molecules with the protein and ligand in the active site, and its kinome selectivity is similar to the best of the approved GIST agents. This compound demonstrates excellent cross-species pharmacokinetics, shows strong pharmacodynamic inhibition of target, and is active in several in vivo models of GIST.

Isoquinolines activated with sulfamoyl chlorides were reacted with indoles in a 3-component reaction to generate a library of dihydroisoquinoline derivatives. Using a differential protecting group strategy, products could be further derivatised. Synthesis of isoquinoline starting materials using several different methods is also described.

We report the discovery of a novel series of 3-cinnoline carboxamides as highly potent and selective ataxia telangiectasia mutated (ATM) kinase inhibitors. Optimization of this series focusing on potency and physicochemical properties (especially permeability) led to the identification of compound 21, a highly potent ATM inhibitor (ATM cell IC50 0.0028 μM) with excellent kinase selectivity and favorable physicochemical and pharmacokinetics properties. In vivo, 21 in combination with irinotecan showed tumor regression in the SW620 colorectal tumor xenograft model, superior inhibition to irinotecan alone. Compound 21 was selected for preclinical evaluation alongside AZD0156.

Title: AZD9496: An oral estrogen receptor inhibitor that blocks the growth of ER1 positive and ESR1 mutant breast tumours in preclinical models. 2 3 Running title: Pharmacology of SERD inhibitor in breast cancer models 4 5 Hazel M. Weir, Robert H. Bradbury, Mandy Lawson, Alfred A. Rabow, David Buttar, 6 Rowena J. Callis, Jon O. Curwen, Camila de Almeida, Peter Ballard, a Michael Hulse, 7 Craig S. Donald, Lyman J. L. Feron, Galith Karoutchi, Philip MacFaul, Thomas Moss, 8 Richard A. Norman, Stuart E. Pearson, Michael Tonge, Gareth Davies, Graeme E. 9 Walker, Zena Wilson, Rachel Rowlinson, Steve Powell, Claire Sadler, Graham 10 Richmond, Brendon Ladd, d Ermira Pazolli, Anne Marie Mazzola, Celina D’Cruz, Chris 11 De Savi. 12 13 Authors’ Affiliations: 14 Oncology iMed, AstraZeneca, Mereside, Alderley Park, Macclesfield SK10 4TG, UK; 15 Discovery Sciences, AstraZeneca, Mereside, Alderley Park, Macclesfield SK10 4TG UK; c 16 H3 Biomedicine, Technology Sq, Cambridge, MA 02139,Oncology iMed, AstraZeneca R&D 17 Boston, Gatehouse Drive, Waltham, MA 02451, U.S. 18 19

A probe compound is described to inhibit ALK1, a target of interest for oncology due to its connection to angiogenesis.

Abstract Fulvestrant is an estrogen receptor (ER) antagonist administered to breast cancer patients by monthly intramuscular injection. Given its present limitations of dosing and route of administration, a more flexible orally available compound has been sought to pursue the potential benefits of this drug in patients with advanced metastatic disease. Here we report the identification and characterization of AZD9496, a nonsteroidal small-molecule inhibitor of ERα, which is a potent and selective antagonist and downregulator of ERα in vitro and in vivo in ER-positive models of breast cancer. Significant tumor growth inhibition was observed as low as 0.5 mg/kg dose in the estrogen-dependent MCF-7 xenograft model, where this effect was accompanied by a dose-dependent decrease in PR protein levels, demonstrating potent antagonist activity. Combining AZD9496 with PI3K pathway and CDK4/6 inhibitors led to further growth-inhibitory effects compared with monotherapy alone. Tumor regressions were also seen in a long-term estrogen-deprived breast model, where significant downregulation of ERα protein was observed. AZD9496 bound and downregulated clinically relevant ESR1 mutants in vitro and inhibited tumor growth in an ESR1-mutant patient-derived xenograft model that included a D538G mutation. Collectively, the pharmacologic evidence showed that AZD9496 is an oral, nonsteroidal, selective estrogen receptor antagonist and downregulator in ER+ breast cells that could provide meaningful benefit to ER+ breast cancer patients. AZD9496 is currently being evaluated in a phase I clinical trial. Cancer Res; 76(11); 3307–18. ©2016 AACR.

High throughput screening followed by a lead generation campaign uncovered a novel series of urea containing morpholinopyrimidine compounds which act as potent and selective dual inhibitors of mTORC1 and mTORC2. We describe the continued compound optimization campaign for this series, in particular focused on identifying compounds with improved cellular potency, improved aqueous solubility, and good stability in human hepatocyte incubations. Knowledge from empirical SAR investigations was combined with an understanding of the molecular interactions in the crystal lattice to improve both cellular potency and solubility, and the composite parameters of LLE and pIC(50)-pSolubility were used to assess compound quality and progress. Predictive models were employed to efficiently mine the attractive chemical space identified resulting in the discovery of 42 (AZD3147), an extremely potent and selective dual inhibitor of mTORC1 and mTORC2 with physicochemical and pharmacokinetic properties suitable for development as a potential clinical candidate.

The discovery of an orally bioavailable selective estrogen receptor downregulator (SERD) with equivalent potency and preclinical pharmacology to the intramuscular SERD fulvestrant is described. A directed screen identified the 1-aryl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole motif as a novel, druglike ER ligand. Aided by crystal structures of novel ligands bound to an ER construct, medicinal chemistry iterations led to (E)-3-(3,5-difluoro-4-((1R,3R)-2-(2-fluoro-2-methylpropyl)-3-methyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl)acrylic acid (30b, AZD9496), a clinical candidate with high oral bioavailability across preclinical species that is currently being evaluated in phase I clinical trials for the treatment of advanced estrogen receptor (ER) positive breast cancer.

Wide-ranging exploration of analogues of an ATP-competitive pyrrolopyrimidine inhibitor of Akt led to the discovery of clinical candidate AZD5363, which showed increased potency, reduced hERG affinity, and higher selectivity against the closely related AGC kinase ROCK. This compound demonstrated good preclinical drug metabolism and pharmacokinetics (DMPK) properties and, after oral dosing, showed pharmacodynamic knockdown of phosphorylation of Akt and downstream biomarkers in vivo, and inhibition of tumor growth in a breast cancer xenograft model.

Lactate dehydrogenase A (LDHA) catalyzes the conversion of pyruvate to lactate, utilizing NADH as a cofactor. It has been identified as a potential therapeutic target in the area of cancer metabolism. In this manuscript we report our progress using fragment-based lead generation (FBLG), assisted by X-ray crystallography to develop small molecule LDHA inhibitors. Fragment hits were identified through NMR and SPR screening and optimized into lead compounds with nanomolar binding affinities via fragment linking. Also reported is their modification into cellular active compounds suitable for target validation work.

Abstract AKT is a key node in the most frequently de-regulated signaling pathway in human cancer and has been shown to mediate resistance to a range of cytotoxic, anti-hormonal and targeted therapies. We decided to explore inhibitors of AKT as potential new anti-cancer therapeutics. Here we disclose for the first time the discovery and structure of AZD5363, an orally bioavailable, potent ATP-competitive inhibitor of AKT. We evaluated a range of chemical starting points arising from our previous collaboration with the Institute of Cancer Research and Astex Therapeutics Ltd. Ultimately AZD5363 was discovered following a long journey that started from a pyrrolopyrimidine series of compounds. Our first challenge was to improve potency and a second challenge was to improve ROCK selectivity. ROCK is an AGC kinase like AKT but is involved in regulation of vascular tone and thus blood pressure. Extensive SAR studies exploring the series revealed that achieving selectivity over ROCK while retaining AKT potency was quite challenging. Eventually we discovered ways which could improve both selectivity and potency. However, these compounds had significant activity against the hERG ion channel which is implicated in the development of Torsades de Pointes and cardiac death. The next phase of work therefore had to focus on reducing hERG activity, while at the same time not adversely impacting either AKT potency or ROCK selectivity. Finally we discovered that introduction of a key substituent group provided a compound that achieved reduced hERG potency and, surprisingly, also achieved a further small improvement in both AKT potency and ROCK selectivity. This compound was AZD5363. A crystal structure of AZD5363 bound to AKT has revealed some of the key interactions that may contribute to its potency. For example, the pyrrolopyrimidine appears to form hydrogen bonds to the hinge region of the kinase. AZD5363 inhibits all known AKT isoforms with a potency of <10 nM and inhibits phosphorylation of the AKT substrate, PRAS40 in BT474c cells with a potency of 0.31 μM. Activity in in vivo pharmacodynamic and xenograft models has also been demonstrated. A synthetic route suitable for scale-up has been developed. In conclusion, AZD5363 is a potent inhibitor of AKT in vitro and in cells. It has good hERG and ROCK selectivity. It has pharmacodynamic and xenograft activity in vivo. AZD5363 has potential in cancer therapy and is currently in phase 1 clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4478. doi:10.1158/1538-7445.AM2011-4478