Discovery of a novel aminopyrazine series as selective PI3Kα inhibitors.

We report the discovery of a novel aminopyrazine series of PI3Kα inhibitors, designed by hybridizing two known scaffolds of PI3K inhibitors. We describe the progress achieved from the first compounds plagued with poor general kinase selectivity to compounds showing high selectivity for PI3Kα over PI3Kß and excellent general kinase selectivity. This effort culminated with the identification of compound 5 displaying high potency and selectivity, and suitable physiochemical and pharmacokinetic properties for oral administration. In vivo, compound 5 showed good inhibition of tumour growth (86% tumour growth inhibition at 50mg/kg twice daily orally) in the MCF7 xenograft model in mice.

Discovery of a series of 8-(1-phenylpyrrolidin-2-yl)-6-carboxamide-2-morpholino-4H-chromen-4-one as PI3Kß/δ inhibitors for the treatment of PTEN-deficient tumours.

Attempts to lock the active conformation of compound 4, a PI3Kß/δ inhibitor (PI3Kß cell IC50 0.015µM), led to the discovery of a series of 8-(1-phenylpyrrolidin-2-yl)-6-carboxamide-2-morpholino-4H-chromen-4-ones, which showed high levels of potency and selectivity as PI3Kß/δ inhibitors. Compound 10 proved exquisitely potent and selective: PI3Kß cell IC50 0.0011µM in PTEN null MDA-MB-468 cell and PI3Kδ cell IC50 0.014µM in Jeko-1 B-cell, and exhibited suitable physical properties for oral administration. In vivo, compound 10 showed profound pharmacodynamic modulation of AKT phosphorylation in a mouse PTEN-null PC3 prostate tumour xenograft after a single oral dose and gave excellent tumour growth inhibition in the same model after chronic oral dosing. Based on these results, compound 10 was selected as one of our PI3Kß/δ preclinical candidates.

Discovery of a series of 8-(2,3-dihydro-1,4-benzoxazin-4-ylmethyl)-2-morpholino-4-oxo-chromene-6-carboxamides as PI3Kß/δ inhibitors for the treatment of PTEN-deficient tumours.

We report the discovery and optimisation of a series of 8-(2,3-dihydro-1,4-benzoxazin-4-ylmethyl)-2-morpholino-4-oxo-chromene-6-carboxamides, leading to compound 16 as a potent and selective PI3Kß/δ inhibitor: PI3Kß cell IC50 0.012 µM (in PTEN null MDA-MB-468 cell) and PI3Kδ cell IC50 0.047 µM (in Jeko-1 B-cell), with good pharmacokinetics and physical properties. In vivo, 16 showed profound pharmacodynamic modulation of AKT phosphorylation in a mouse PTEN-deficient PC3 prostate tumour xenograft after a single oral dose and gave excellent tumour growth inhibition in the same model after chronic oral dosing. Compound 16 was selected as a preclinical candidate for the treatment of PTEN-deficient tumours.

Discovery of 1-(4-(5-(5-amino-6-(5-tert-butyl-1,3,4-oxadiazol-2-yl)pyrazin-2-yl)-1-ethyl-1,2,4-triazol-3-yl)piperidin-1-yl)-3-hydroxypropan-1-one (AZD8835): A potent and selective inhibitor of PI3Kα and PI3Kδ for the treatment of cancers.

Starting from potent inhibitors of PI3Kα having poor general kinase selectivity (e.g., 1 and 2), optimisation of this series led to the identification of 25, a potent inhibitor of PI3Kα (wild type, E545K and H1047R mutations) and PI3Kδ, selective versus PI3Kß and PI3Kγ, with excellent general kinase selectivity. Compound 25 displayed low metabolic turnover and suitable physical properties for oral administration. In vivo, compound 25 showed pharmacodynamic modulation of AKT phosphorylation and near complete inhibition of tumour growth (93% tumour growth inhibition) in a murine H1047R PI3Kα mutated SKOV-3 xenograft tumour model after chronic oral administration at 25mg/kg b.i.d. Compound 25, also known as AZD8835, is currently in phase I clinical trials.

Discovery of 9-(1-anilinoethyl)-2-morpholino-4-oxo-pyrido[1,2-a]pyrimidine-7-carboxamides as PI3Kß/δ inhibitors for the treatment of PTEN-deficient tumours.

Starting from TGX-221, we designed a series of 9-(1-anilinoethyl)-2-morpholino-4-oxo-pyrido[1,2-a]pyrimidine-7-carboxamides as potent and selective PI3Kß/δ inhibitors. Structure-activity relationships and structure-property relationships around the aniline and the amide substituents are discussed. We identified compounds 17 and 18, which showed profound pharmacodynamic modulation of phosphorylated Akt in the PC3 prostate tumour xenograft, after a single oral dose. Compound 17 also gave significant inhibition of tumour growth in the PC3 prostate tumour xenograft model after chronic oral dosing.

Small-molecule androgen receptor downregulators as an approach to treatment of advanced prostate cancer.

Chemical starting points were investigated for downregulation of the androgen receptor as an approach to treatment of advanced prostate cancer. Although prototypic steroidal downregulators such as 6a designed for intramuscular administration showed insufficient cellular potency, a medicinal chemistry program derived from a novel androgen receptor ligand 8a led to 6-[4-(4-cyanobenzyl)piperazin-1-yl]-3-(trifluoromethyl)[1,2,4]triazolo[4,3-b]pyridazine (10b), for which high plasma levels following oral administration in a preclinical model compensate for moderate cellular potency.

Synthesis and structure-activity relationships of thieno[2,3-b]pyrroles as antagonists of the GnRH receptor.

A new class of small-molecule GnRH antagonists, the thieno[2,3-b]pyrroles, was designed. Herein, the synthesis and structure-activity relationships are described. Substitution at the C4 position was investigated; during this study, it was observed that introducing piperazines and piperidines improved the physical properties of the compounds while retaining good in vitro potency. This exploration led to the discovery of amidopiperidines with improved pharmacokinetic properties.

The use of 18F-Fluoro-deoxy-glucose positron emission tomography (18F-FDG PET) as a non-invasive pharmacodynamic biomarker to determine the minimally pharmacologically active dose of AZD8835, a novel PI3Kα inhibitor.

BACKGROUND: The phosphatidyl inositol 3 kinase (PI3K), AKT and mammalian target of rapamycin (mTOR) signal transduction pathway is frequently de-regulated and activated in human cancer and is an important therapeutic target. AZD8835 is a PI3K inhibitor, with selectivity against PI3K α and δ isoforms, which is currently in Phase 1 clinical trials. 18F-Fluoro-deoxy-glucose positron emission tomography (18F-FDG PET) is a non-invasive pharmacodynamic imaging biomarker that has become an integral part of drug development. It has been used widely with PI3K inhibitors both clinically and pre-clinically because of the role of the PI3K pathway in glucose metabolism. In this study we investigated the potential of 18F-FDG PET as a non-invasive pharmacodynamic biomarker for AZD8835. We sought to understand if 18F-FDG PET could determine the minimally effective dose of AZD8835 and correlate with other pharmacodynamic biomarkers for validation of its use in clinical development. 18F-FDG PET scans were performed in nude mice in the BT474C breast xenograft model. Mice were fasted prior to imaging and static 18F-FDG PET was performed. Treatment groups received AZD8835 by oral gavage at a dose volume of 10ml/kg. Treatment groups received either 3, 6, 12.5, 25 or 50mg/kg AZD8835. Tumour growth was monitored throughout the study, and at the end of the imaging procedure, tumours were taken and a full pharmacodynamic analysis was performed. RESULTS: Results showed that AZD8835 reduced 18F-FDG uptake at a dose of 12.5, 25 and 50mg/kg with no significant reduction at doses of 3 and 6mg/kg. These results were consistent with other pharmacodynamics biomarkers measured and show 18F-FDG PET as a sensitive biomarker with the ability to determine the minimal effective dose of AZD8835. CONCLUSIONS: Our pre-clinical studies support the use of 18F-FDG PET imaging as a sensitive and non- invasive pharmacodynamic biomarker (understanding the role of PI3K signalling in glucose uptake) for AZD8835 with a decrease in 18F-FDG uptake observed at only two hours post treatment. The decrease in 18F-FDG uptake was dose dependent and data showed excellent PK/PD correlation. This data supports and parallels observations obtained with this class of compounds in patients.

Inhibiting PI3Kß with AZD8186 Regulates Key Metabolic Pathways in PTEN-Null Tumors.

Purpose: PTEN-null tumors become dependent on the PI3Kß isoform and can be targeted by molecules such as the selective PI3Kß inhibitor AZD8186. However, beyond the modulation of the canonical PI3K pathway, the consequences of inhibiting PI3Kß are poorly defined.Experimental Design: To determine the broader impact of AZD8186 in PTEN-null tumors, we performed a genome-wide RNA-seq analysis of PTEN-null triple-negative breast tumor xenografts treated with AZD8186. Mechanistic consequences of AZD8186 treatment were examined across a number of PTEN-null cell lines and tumor models.Results: AZD8186 treatment resulted in modification of transcript and protein biomarkers associated with cell metabolism. We observed downregulation of cholesterol biosynthesis genes and upregulation of markers associated with metabolic stress. Downregulation of cholesterol biosynthesis proteins, such as HMGCS1, occurred in PTEN-null cell lines and tumor xenografts sensitive to AZD8186. Therapeutic inhibition of PI3Kß also upregulated PDHK4 and increased PDH phosphorylation, indicative of reduced carbon flux into the TCA cycle. Consistent with this, metabolomic analysis revealed a number of changes in key carbon pathways, nucleotide, and amino acid biosynthesis.Conclusions: This study identifies novel mechanistic biomarkers of PI3Kß inhibition in PTEN-null tumors supporting the concept that targeting PI3Kß may exploit a metabolic dependency that contributes to therapeutic benefit in inducing cell stress. Considering these additional pathways will guide biomarker and combination strategies for this class of agents. Clin Cancer Res; 23(24); 7584-95. ©2017 AACR.

The use of (18)F-fluorodeoxyglucose positron emission tomography ((18)F-FDG PET) as a pathway-specific biomarker with AZD8186, a PI3Kß/δ inhibitor.

BACKGROUND: The phosphatidylinositol 3 kinase (PI3K) signalling pathway is frequently altered in human cancer and a promising therapeutic target. AZD8186 (AstraZeneca) is a PI3Kß/δ inhibitor, currently in phase 1 clinical trials. (18)F-fluorodeoxyglucose positron emission tomography ((18)F-FDG PET) is often used as a biomarker for inhibitors targeting the PI3K axis because of the association of this pathway with glucose metabolism. In this study, we assessed if (18)F-FDG PET could be used as a pharmacodynamic marker to monitor PI3Kß inhibition by AZD8186, and hence have potential as a clinical biomarker of PI3Kß pathway activation, and for patient selection. (18)F-FDG PET scans were performed in nude mice bearing 786-0 renal, U87-MG glioma, and BT474C breast xenograft models. Mice were fasted prior to imaging and static (18)F-FDG PET imaging was performed. Tumour growth was monitored throughout each study, and at the end of the imaging procedure, tumours were taken and a full pharmacodynamic analysis performed. RESULTS: Results showed that in PTEN null tumour xenograft models, 786-0 and U87-MG, the PI3Kß inhibitor AZD8186 reduces (18)F-FDG uptake at a dose of 50 mg/kg, the same dose which causes tumour inhibition, while it has no impact in a PI3Kα mutant tumour xenograft BT474C. Consistent with the change in (18)F-FDG uptake, AZD8186 also modulated AKT and associated glucose pathway biomarkers in the PTEN null tumour xenografts but not in PTEN wild-type tumours. CONCLUSIONS: Our pre-clinical studies support the use of (18)F-FDG PET imaging as a sensitive and non-invasive pharmacodynamic biomarker for use in clinical studies with AZD8186.

Intermittent High-Dose Scheduling of AZD8835, a Novel Selective Inhibitor of PI3Kα and PI3Kδ, Demonstrates Treatment Strategies for PIK3CA-Dependent Breast Cancers.

The PIK3CA gene, encoding the p110α catalytic unit of PI3Kα, is one of the most frequently mutated oncogenes in human cancer. Hence, PI3Kα is a target subject to intensive efforts in identifying inhibitors and evaluating their therapeutic potential. Here, we report studies with a novel PI3K inhibitor, AZD8835, currently in phase I clinical evaluation. AZD8835 is a potent inhibitor of PI3Kα and PI3Kδ with selectivity versus PI3Kß, PI3Kγ, and other kinases that preferentially inhibited growth in cells with mutant PIK3CA status, such as in estrogen receptor-positive (ER(+)) breast cancer cell lines BT474, MCF7, and T47D (sub-µmol/L GI50s). Consistent with this, AZD8835 demonstrated antitumor efficacy in corresponding breast cancer xenograft models when dosed continuously. In addition, an alternative approach of intermittent high-dose scheduling (IHDS) was explored given our observations that higher exposures achieved greater pathway inhibition and induced apoptosis. Indeed, using IHDS, monotherapy AZD8835 was able to induce tumor xenograft regression. Furthermore, AZD8835 IHDS in combination with other targeted therapeutic agents further enhanced antitumor activity (up to 92% regression). Combination partners were prioritized on the basis of our mechanistic insights demonstrating signaling pathway cross-talk, with a focus on targeting interdependent ER and/or CDK4/6 pathways or alternatively a node (mTOR) in the PI3K-pathway, approaches with demonstrated clinical benefit in ER(+) breast cancer patients. In summary, AZD8835 IHDS delivers strong antitumor efficacy in a range of combination settings and provides a promising alternative to continuous dosing to optimize the therapeutic index in patients. Such schedules merit clinical evaluation. Mol Cancer Ther; 15(5); 877-89. ©2016 AACR.

Discovery of (R)-8-(1-(3,5-difluorophenylamino)ethyl)-N,N-dimethyl-2-morpholino-4-oxo-4H-chromene-6-carboxamide (AZD8186): a potent and selective inhibitor of PI3Kß and PI3Kδ for the treatment of PTEN-deficient cancers.

Several studies have highlighted the dependency of PTEN deficient tumors to PI3Kß activity and specific inhibition of PI3Kδ has been shown activity against human B-cell cancers. We describe the discovery and optimization of a series of 8-(1-anilino)ethyl)-2-morpholino-4-oxo-4H-chromene-6-carboxamides as PI3Kß/δ inhibitors, which led to the discovery of the clinical candidate 13, also known as AZD8186. On the basis of the lower lipophilicity of the chromen-4-one core compared to the previously utilized pyrido[1,2-a]pyrimid-4-one core, this series of compounds displayed high metabolic stability and suitable physical properties for oral administration. Compound 13 showed profound pharmacodynamic modulation of p-Akt in PTEN-deficient PC3 prostate tumor bearing mice after oral administration and showed complete inhibition of tumor growth in the mouse PTEN-deficient PC3 prostate tumor xenograft model. 13 was selected as a clinical candidate for treatment of PTEN-deficient cancers and has recently entered phase I clinical trials.

Feedback suppression of PI3Kα signaling in PTEN-mutated tumors is relieved by selective inhibition of PI3Kß.

In PTEN-mutated tumors, we show that PI3Kα activity is suppressed and PI3K signaling is driven by PI3Kß. A selective inhibitor of PI3Kß inhibits the Akt/mTOR pathway in these tumors but not in those driven by receptor tyrosine kinases. However, inhibition of PI3Kß only transiently inhibits Akt/mTOR signaling because it relieves feedback inhibition of IGF1R and other receptors and thus causes activation of PI3Kα and a rebound in downstream signaling. This rebound is suppressed and tumor growth inhibition enhanced with combined inhibition of PI3Kα and PI3Kß. In PTEN-deficient models of prostate cancer, this effective inhibition of PI3K causes marked activation of androgen receptor activity. Combined inhibition of both PI3K isoforms and androgen receptor results in major tumor regressions.

Inhibition of PI3Kß signaling with AZD8186 inhibits growth of PTEN-deficient breast and prostate tumors alone and in combination with docetaxel.

Loss of PTEN protein results in upregulation of the PI3K/AKT pathway, which appears dependent on the PI3Kß isoform. Inhibitors of PI3Kß have potential to reduce growth of tumors in which loss of PTEN drives tumor progression. We have developed a small-molecule inhibitor of PI3Kß and PI3Kδ (AZD8186) and assessed its antitumor activity across a panel of cell lines. We have then explored the antitumor effects as single agent and in combination with docetaxel in triple-negative breast (TNBC) and prostate cancer models. In vitro, AZD8186 inhibited growth of a range of cell lines. Sensitivity was associated with inhibition of the AKT pathway. Cells sensitive to AZD8186 (GI50 < 1 µmol/L) are enriched for, but not exclusively associated with, PTEN deficiency. In vivo, AZD8186 inhibits PI3K pathway biomarkers in prostate and TNBC tumors. Scheduling treatment with AZD8186 shows antitumor activity required only intermittent exposure, and that increased tumor control is achieved when AZD8186 is used in combination with docetaxel. AZD8186 is a potent inhibitor of PI3Kß with activity against PI3Kδ signaling, and has potential to reduce growth of tumors dependent on dysregulated PTEN for growth. Moreover, AZD8186 can be combined with docetaxel, a chemotherapy commonly used to treat advanced TBNC and prostate tumors. The ability to schedule AZD8186 and maintain efficacy offers opportunity to combine AZD8186 more effectively with other drugs.