Discovery and optimization of 5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one derivatives as mTORC1/mTORC2 dual inhibitors

New potent mTORC1/mTORC2 dual inhibitors, 5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one derivatives, were obtained by optimizing functional groups on our previously reported PI3Kα inhibitor. All the target compounds were synthesized and structural optimization on the structure of the lead compound based on cytotoxic activity. The results showed that some of the target compounds exhibited moderate to high cytotoxic activity against cell line U87MG and PC-3. The activities against mTOR kinase were investigated and the compound 12q showed excellent activity with an IC₅₀ value of 54 nM in the same level of the positive control BEZ235 with IC₅₀ value of 55 nM under the same test conditions. The western blot and cell cycle results demonstrate that compound 12q is a candidate as an mTORC1/mTORC2 dual-target inhibitor. The theoretical calculations were also performed to better understanding the binding modes of the compound 12q in the mTOR active site.     

Potent antitumour of the mTORC1/2 dual inhibitor AZD2014 in docetaxel-sensitive and docetaxel-resistant castration-resistant prostate cancer cells

Recent studies indicate mammalian target of rapamycin (mTOR) may play an important role in PCa progression and drug resistance. Here, we investigated the effects of a novel mTORC1/C2 dual inhibitor, AZD2014, on naive and docetaxel (Doc)-pre-treated castration-resistant PCa (CRPC) cells and explored its therapeutic potential in CRPCs. In the current study, AZD2014 has a greater inhibitory effect against 4EBP1 and AKT phosphorylation than rapamycin in CRPC cells and prevented the feedback activation of AKT signalling. Importantly, AZD2014 suppressed CRPC cell growth in vitro by suppressing proliferation, apoptosis, cell cycle arrest at G1 phase and autophagy to a greater extent than rapamycin. Moreover, AZD2014 was more efficacious than rapamycin in inhibiting migration, invasion and EMT progression in Doc-sensitive and Doc-resistant CRPC cells. Overall, AZD2014 showed significant antitumour effects. Thereby, the current study highlights a reliable theoretical basis for the clinical application of AZD2014 in both Doc-sensitive and Doc-resistant CRPCs.     

Discovery of AZD3514, a small-molecule androgen receptor downregulator for treatment of advanced prostate cancer

Removal of the basic piperazine nitrogen atom, introduction of a solubilising end group and partial reduction of the triazolopyridazine moiety in the previously-described lead androgen receptor downregulator 6-[4-(4-cyanobenzyl)piperazin-1-yl]-3-(trifluoromethyl)[1,2,4]triazolo[4,3-b]pyridazine (1) addressed hERG and physical property issues, and led to clinical candidate 6-(4-{4-[2-(4-acetylpiperazin-1-yl)ethoxy]phenyl}piperidin-1-yl)-3-(trifluoromethyl)-7,8-dihydro[1,2,4]triazolo[4,3-b]pyridazine (12), designated AZD3514, that is being evaluated in a Phase I clinical trial in patients with castrate-resistant prostate cancer.     

Synthesis and evaluation of an AZD2461 [18F]PET probe in non-human primates reveals the PARP-1 inhibitor to be non-blood-brain barrier penetrant

Poly(ADP-ribose)polymerase-1 inhibitor (PARPi) AZD2461 was designed to be a weak P-glycoprotein (P-gp) analogue of FDA approved olaparib. With this chemical property in mind, we utilized the AZD2461 ligand architecture to develop a CNS penetrant and PARP-1 selective imaging probe, in order to investigate PARP-1 mediated neuroinflammation and neurodegenerative diseases, such as Alzheimer’s and Parkinson’s. Our work led to the identification of several high-affinity PARPi, including AZD2461 congener 9e (PARP-1 IC₅₀ = 3.9 ± 1.2 nM), which was further evaluated as a potential ¹⁸F-PET brain imaging probe. However, despite the similar molecular scaffolds of 9e and AZD2461, our studies revealed non-appreciable brain-uptake of [¹⁸F]9e in non-human primates, suggesting AZD2461 to be non-CNS penetrant.     

AZD2014 Radiosensitizes Oral Squamous Cell Carcinoma by Inhibiting AKT/mTOR Axis and Inducing G1/G2/M Cell Cycle Arrest

Background

Oral squamous cell carcinoma (OSCC) is one of the most common malignant neoplasms in Taiwan. Activation of the mTOR signaling pathway has been linked to decreased radiation responsiveness in human oral cancer, thus it limits efficacy of radiotherapy. To address this question, we investigated the effect of AZD2014, a novel small molecular ATP-competitive inhibitor of mTORC1 and mTORC2 kinase, as a radiosensitizer in primary OSCC and OSCC-derived cell line models.

Methods

We isolated primary tumor cells from OSCC tissues and cell lines. AZD2014 was administered with and without ionizing radiation. The radiosensitizing effect of AZD2014 were then assessed using cell viability assays, clonogenic survival assays, and cell cycle analyses. Western blotting was used to detect protein expression.

Results

Combination treatment with AZD2014 and irradiation resulted in significant reduction in OSCC cell line and primary OSCC cell colony formation due to the enhanced inhibition of AKT and both mTORC1 and mTORC2 activity. Pre-treatment with AZD2014 in irradiated oral cancer cells induced tumor cell cycle arrest at the G1 and G2/M phases, which led to disruption of cyclin D1-CDK4 and cyclin B1-CDC2 complexes. Moreover, AZD2014 synergized with radiation to promote both apoptosis and autophagy by increasing caspase-3 and LC3 in primary OSCC cells.

Conclusions

These findings suggest that in irradiated OSCC cells, co-treatment with AZD2014, which targets mTORC1 and mTORC2 blockade, is an effective radiosensitizing strategy for oral squamous cell carcinoma.     

Hydantoin based inhibitors of MMP13—Discovery of AZD6605

Piperidine ether and aryl piperazine hydantoins are reported as potent inhibitors of MMP13. A medicinal chemistry campaign focused on replacing the reverse hydroxamate zinc binding group associated with historical inhibitors with a hydantoin zinc binding group then optimising MMP13 potency, solubility and DMPK properties whilst maintaining good selectivity over MMP14. A number of high quality candidates were progressed and following rat and dog safety evaluation, AZD6605 (3m) was identified as a candidate drug.     

mTORC2 regulates PGE2-mediated endothelial cell survival and migration

Prostaglandin E₂ (PGE₂) promotes angiogenesis by in part inducing endothelial cell survival and migration. The present study examined the role of mTOR and its two complexes, mTORC1 and mTORC2, in PGE₂-mediated endothelial cell responses. We used small interfering RNA (siRNA) to raptor or rictor to block mTORC1 or mTORC2, respectively. We observed that down-regulation of mTORC2 but not mTORC1 reduced baseline and PGE₂-induced endothelial cell survival and migration. At the molecular level, we found that knockdown of mTORC2 inhibited PGE₂-mediated Rac and Akt activation two important signaling intermediaries in endothelial cell migration and survival, respectively. In addition, inhibition of mTORC2 by prolonged exposure of endothelial cells to rapamycin also prevented PGE₂-mediated endothelial cell survival and migration confirming the results obtained with the siRNA approach. Taken together these results show that mTORC2 but not mTORC1 is an important signaling intermediary in PGE₂-mediated endothelial cell responses.     

Discovery of AAT-008, a novel,potent, and selective prostaglandin EP4 receptor antagonist

Starting from acylsufonamide HTS hit 2, a novel series of para-N-acylaminomethylbenzoic acids was identified and developed as selective prostaglandin EP4 receptor antagonists. Structural modifications on lead compound 4a were explored with the aim of improving potency, physicochemical properties, and animal PK predictive of QD (once a day) dosing regimen in human. These efforts led to the discovery of the clinical candidate AAT-008 (4j), which exhibited significantly improved pharmacological profiles over grapiprant (1).     

AZD2184: a radioligand for sensitive detection of β-amyloid deposits

The presence of β‐amyloid plaques in brain is a hallmark of Alzheimer’s disease (AD) and serves as a biomarker for confirmation of diagnosis postmortem. Positron emission tomography (PET) radioligands such as Pittsburgh compound B ([¹¹C]‐2‐(3‐fluoro‐4‐methylamino‐phenyl)‐benzothiazol‐6‐ol) (PIB) binds selectively to β‐amyloid and are promising new tools supporting the clinical diagnoses of AD. In addition, such methodology may be useful for evaluation of new drugs aiming at reduction of amyloid plaque load. The objective of this study is to develop a new amyloid selective PET radioligand with higher signal‐to‐background ratio when compared with existing amyloid PET ligands. The lead compound, AZD2184, (2‐[6‐(methylamino)pyridin‐3‐yl]‐1,3‐benzothiazol‐6‐ol) was found to have high affinity for amyloid fibrils in vitro (K_d: 8.4 ± 1.0 nM). Two minutes after i.v. administration in rats, about 1% of the dose was in brain. In vitro autoradiography on cortical brain sections from amyloid‐beta precursor protein/presenilin 1 (APP/PS1) mice and AD patients showed that while [³H]AZD2184 and [³H]PIB are mutually displaceable, [³H]AZD2184 displays a higher signal‐to‐background ratio primarily by virtue of lower background binding levels. The ratio of binding ability in prefrontal cortex (high plaque load) to subcortical white matter (background) was 4.5 for [³H]AZD2184 and 0.8 for [³H]PIB at 1 nM. In adjacent cortical sections from APP/PS1 mouse as well as from AD cortical tissue, [³H]AZD2184 and antibodies to human β‐amyloid labeled identical structures. In vivo administration of [³H]AZD2184 to APP/PS1 mice further showed that [³H]AZD2184 labels amyloid deposits with low non‐specific background binding. Taken together, the pre‐clinical profile of AZD2184 in relation to the reference ligand PIB, suggests that ¹¹C‐labeled AZD2184 is a potential radioligand for PET‐visualization of β‐amyloid deposits in the living human brain.     

Multi-mechanisms are involved in reactive oxygen species regulation of mTORC1 signaling

The mammalian target of rapamycin complex 1(mTORC1) integrates diverse signals to control cell growth, proliferation, survival, and metabolism. Role of reactive oxygen species (ROS) on mTORC1 signaling remains obscure and mechanisms through which ROS modulate mTORC1 are not known. We demonstrate that low doses ROS exposure stimulate mTORC1 while high concentrations or long-term ROS treatment decrease mTORC1 activity in vivo and in a variety of cell lines. The dose/time needed for inhibition or activation are cell type-dependent. In HEK293 cells hydrogen peroxide (H₂O₂) stimulates phosphorylation of AMP-activated kinase (AMPK) (T172) and Raptor (S792), enhances association of activated AMPK with Raptor. Furthermore, AMPK inhibitor compound c inhibits H₂O₂-induced Raptor (S792) phosphorylation and reverses H₂O₂-induced de-phosphorylation of mTORC1 downstream targets p70-S6K1 (T389), S6 (S235/236) and 4E-BP1 (T37/46). H₂O₂ also stimulates association of endogenous protein phosphatase 2A catalytic subunit (PP2Ac) with p70-S6K1. Like compound c, inhibitor of PP2A, okadaic acid partially reverses inactivation of mTORC1 substrates induced by H₂O₂. Moreover, inhibition of PP2A and AMPK partially rescued cells from H₂O₂-induced cell death. High doses of H₂O₂ inhibit while low doses of H₂O₂ activate mTORC1 both in TSC2^?/? P53^?/? and TSC2^+/+ P53^?/? MEFs. These data suggest that PP2A and AMPK-mediated phosphorylation of Raptor mediate H₂O₂-induced inhibition of mTORC1 signaling.     

PGE2-induced colon cancer growth is mediated by mTORC1

The inflammatory prostaglandin E₂ (PGE₂) cytokine plays a key role in the development of colon cancer. Several studies have shown that PGE₂ directly induces the growth of colon cancer cells and furthermore promotes tumor angiogenesis by increasing the production of the vascular endothelial growth factor (VEGF). The signaling intermediaries implicated in these processes have however not been fully characterized. In this report, we show that the mechanistic target of rapamycin complex 1 (mTORC1) plays an important role in PGE₂-induced colon cancer cell responses. Indeed, stimulation of LS174T cells with PGE₂ increased mTORC1 activity as observed by the augmentation of S6 ribosomal protein phosphorylation, a downstream effector of mTORC1. The PGE₂ EP₄ receptor was responsible for transducing the signal to mTORC1. Moreover, PGE₂ increased colon cancer cell proliferation as well as the growth of colon cancer cell colonies grown in matrigel and blocking mTORC1 by rapamycin or ATP-competitive inhibitors of mTOR abrogated these effects. Similarly, the inhibition of mTORC1 by downregulation of its component raptor using RNA interference blocked PGE₂-induced LS174T cell growth. Finally, stimulation of LS174T cells with PGE₂ increased VEGF production which was also prevented by mTORC1 inhibition. Taken together, these results show that mTORC1 is an important signaling intermediary in PGE₂ mediated colon cancer cell growth and VEGF production. They further support a role for mTORC1 in inflammation induced tumor growth.     

Identification of osimertinib (AZD9291) as a lysine specific demethylase 1 inhibitor

Osimertinib (AZD9291) is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) that has been approved for the treatment of EGFR-mutated non-small cell lung cancer (NSCLC). In this study, osimertinib was characterized as a LSD1 inhibitor for the first time with an IC₅₀ of 3.98 ± 0.3 μM and showed LSD1 inhibitory effect at cellular level. These findings provide new molecular skeleton for dual inhibitor for LSD1 and EGFR. Osimertinib could serve as a lead compound for further development for anti-NSCLC drug discovery with dual targeting.     

Hydrogen peroxide impairs insulin-stimulated assembly of mTORC1

Oxidants are well recognized for their capacity to reduce the phosphorylation of the mammalian target of rapamycin (mTOR) substrates, eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and p70 S6 kinase 1 (S6K1), thereby hindering mRNA translation at the level of initiation. mTOR functions to regulate mRNA translation by forming the signaling complex mTORC1 (mTOR, raptor, GβL). Insulin signaling to mTORC1 is dependent upon phosphorylation of Akt/PKB and the inhibition of the tuberous sclerosis complex (TSC1/2), thereby enhancing the phosphorylation of 4E-BP1 and S6K1. In this study we report the effect of H₂O₂ on insulin-stimulated mTORC1 activity and assembly using A549 and bovine aortic smooth muscle cells. We show that insulin stimulated the phosphorylation of TSC2 leading to a reduction in raptor–mTOR binding and in the quantity of proline-rich Akt substrate 40 (PRAS40) precipitating with mTOR. Insulin also increased 4E-BP1 coprecipitating with mTOR and the phosphorylation of the mTORC1 substrates 4E-BP1 and S6K1. H₂O₂, on the other hand, opposed the effects of insulin by increasing raptor–mTOR binding and the ratio of PRAS40/raptor derived from the mTOR immunoprecipitates in both cell types. These effects occurred in conjunction with a reduction in 4E-BP1 phosphorylation and the 4E-BP1/raptor ratio. siRNA-mediated knockdown of PRAS40 in A549 cells partially reversed the effect of H₂O₂ on 4E-BP1 phosphorylation but not on S6K1. These findings are consistent with PRAS40 functioning as a negative regulator of insulin-stimulated mTORC1 activity during oxidant stress.     

Inhibition of mTOR kinase by AZD8055 can antagonize chemotherapy-induced cell death through autophagy induction and down-regulation of p62/sequestosome 1

AZD8055 is an ATP-competitive inhibitor of mammalian target of rapamycin (mTOR) that forms two multiprotein complexes, mTORC1 and mTORC2, and negatively regulates autophagy. We demonstrate that AZD8055 stimulates and potentiates chemotherapy-mediated autophagy, as shown by LC3I-II conversion and down-regulation of the ubiquitin-binding protein p62/sequestosome 1. AZD8055-induced autophagy was pro-survival as shown by its ability to attenuate cell death and DNA damage (p-H2AX), and to enhance clonogenic survival by cytotoxic chemotherapy. Autophagy inhibition by siRNA against Beclin 1 or LC3B, or by chloroquine, partially reversed the cytoprotective effect of AZD8055 that was independent of cell cycle inhibition. The pro-survival role of autophagy was confirmed using ectopic expression of Beclin 1 that conferred cytoprotection. To determine whether autophagy-mediated down-regulation of p62/sequestosome 1 contributes to its pro-survival role, we generated p62 knockdown cells using shRNA that showed protection from chemotherapy-induced cell death and DNA damage. We also overexpressed wild-type (wt) p62 that promoted chemotherapy-induced cell death, whereas mutated p62 at functional domains (PB1, UBA) failed to do so. The ability of ectopic wt p62 to promote cell death was blocked by AZD8055. AZD8055 was shown to inhibit phosphorylation of the autophagy-initiating kinase ULK1 at Ser(757) and inhibited known targets of mTORC1 (p-mTOR Ser(2448), p70S6K, p-S6, p4EBP1) and mTORC2 (p-mTOR Ser(2481), p-AKT Ser(473)). Knockdown of mTOR, but not Raptor or Rictor, reduced p-ULK1 at Ser(757) and enhanced chemotherapy-induced autophagy that resulted in a similar cytoprotective effect as shown for AZD8055. In conclusion, AZD8055 inhibits mTOR kinase and ULK1 phosphorylation to induce autophagy whose pro-survival effect is due, in part, to down-regulation of p62.     

Efficacy and safety of AZD3199 vs formoterol in COPD: a randomized,double-blind study

Background

We investigated the efficacy and safety of AZD3199, a novel inhaled ultra-LABA, with the main aim of establishing a dose that would maintain 24-hour bronchodilation in patients with COPD.

Methods

Patients (n = 329) were randomized to AZD3199 (200, 400 or 800 μg o.d.), formoterol (9 μg b.i.d.) or placebo via Turbuhaler® in a parallel group study. The primary objective of the study was to compare the clinical efficacy of three doses of AZD3199 inhaled once daily with 9 μg formoterol twice daily and placebo, over a 4-week treatment period in adults with moderate-to-severe COPD. After 4 weeks, peak (0–4 h) and trough (24–26 h) forced expiratory volume in 1 second (FEV₁) were assessed as the primary efficacy outcome variables.

Results

All AZD3199 doses significantly increased mean peak and trough FEV₁ versus placebo (106–171 ml and 97–110 ml increases, respectively), but with no clear dose–response; the level of bronchodilation was comparable to or greater than that achieved with formoterol. Forced vital capacity (FVC) at peak bronchodilation also significantly increased with AZD3199 versus placebo (153–204 ml). COPD symptom scores and reliever use were reduced with AZD3199, while FEV₁ reversibility was unaltered. Adverse events were mild-to-moderate, with no safety concerns identified. Drug exposure was dose-proportional, but lower than predicted from healthy volunteers.

Conclusions

All three doses of AZD3199 produced 24-hour bronchodilation, but with no clear dose–response, suggesting that doses of 200 μg or less may be sufficient to maintain bronchodilation over 24 hours in patients with COPD. No safety concerns were identified. Further studies are required to determine the once-daily AZD3199 dose for COPD.

Trial registration

Clinicaltrials.gov, {"type":"clinical-trial","attrs":{"text":"NCT00929708","term_id":"NCT00929708"}}NCT00929708     

The first radiosynthesis of [11C]AZD8931 as a new potential PET agent for imaging of EGFR,HER2 and HER3 signaling

The reference standard AZD8931{2-(4-((4-((3-chloro-2-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)piperidin-1-yl)-N-methylacetamide} (11a) was synthesized from methyl 4,5-dimethoxy-2-nitrobenzoate or ethyl 4,5-dimethoxy-2-nitrobenzoate and 2-chloro-N-methylacetamide in 11 steps with 2–5% overall chemical yield. The precursor N-desmethyl-AZD8931{2-(4-((4-((3-chloro-2-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)piperidin-1-yl)acetamide} (11b) was synthesized from methyl 4,5-dimethoxy-2-nitrobenzoate or ethyl 4,5-dimethoxy-2-nitrobenzoate and 2-bromoacetamide in 11 steps with 2–4% overall chemical yield. The target tracer [¹¹C]AZD8931 {2-(4-((4-((3-chloro-2-fluorophenyl)amino)-7-methoxyquinazolin-6-yl)oxy)piperidin-1-yl)-N-[¹¹C]methylacetamide} ([¹¹C]11a) was prepared from N-desmethyl-AZD8931 (11b) with [¹¹C]CH₃OTf under basic condition (NaH) through N-[¹¹C]methylation and isolated by HPLC combined with solid-phase extraction (SPE) in 40–50% radiochemical yield based on [¹¹C]CO₂ and decay corrected to end of bombardment (EOB) with 370–1110 GBq/μmol specific activity at EOB.     

Imidazo[1,5-a]pyrazines: orally efficacious inhibitors of mTORC1 and mTORC2

The discovery and optimization of a series of imidazo[1,5-a]pyrazine inhibitors of mTOR is described. HTS hits were optimized for potency, selectivity and metabolic stability to provide the orally bioavailable proof of concept compound 4c that demonstrated target inhibition in vivo and concomitant inhibition of tumor growth in an MDA-MB-231 xenograft model.     

AKAP13 couples GPCR signaling to mTORC1 inhibition

The mammalian target of rapamycin complex 1 (mTORC1) senses multiple stimuli to regulate anabolic and catabolic processes. mTORC1 is typically hyperactivated in multiple human diseases such as cancer and type 2 diabetes. Extensive research has focused on signaling pathways that can activate mTORC1 such as growth factors and amino acids. However, less is known about signaling cues that can directly inhibit mTORC1 activity. Here, we identify A-kinase anchoring protein 13 (AKAP13) as an mTORC1 binding protein, and a crucial regulator of mTORC1 inhibition by G-protein coupled receptor (GPCR) signaling. GPCRs paired to Gα_s proteins increase cyclic adenosine 3’5’ monophosphate (cAMP) to activate protein kinase A (PKA). Mechanistically, AKAP13 acts as a scaffold for PKA and mTORC1, where PKA inhibits mTORC1 through the phosphorylation of Raptor on Ser 791. Importantly, AKAP13 mediates mTORC1-induced cell proliferation, cell size, and colony formation. AKAP13 expression correlates with mTORC1 activation and overall lung adenocarcinoma patient survival, as well as lung cancer tumor growth in vivo. Our study identifies AKAP13 as an important player in mTORC1 inhibition by GPCRs, and targeting this pathway may be beneficial for human diseases with hyperactivated mTORC1.