Identification of novel 7-amino-5-methyl-1,6-naphthyridin-2(1H)-one derivatives as potent PI3K/mTOR dual inhibitors

Inhibition of the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway is one of the most intensively studied approaches to cancer therapy. Rational design led to the identification of novel 7-amino-5-methyl-1,6-naphthyridin-2(1H)-one derivatives as potent PI3K/mTOR dual inhibitors. Design, synthesis and structure activity relationship are reported.     

Synthesis and structure–activity relationships of PI3K/mTOR dual inhibitors from a series of 2-amino-4-methylpyrido[2,3-d]pyrimidine derivatives

Inhibition of the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway by PI3K/mTOR dual inhibitors provides a promising new approach to the treatment of cancers. In this Letter, we identified structurally novel and potent PI3K/mTOR dual inhibitors from a series of 2-amino-4-methylpyrido[2,3-d]pyrimidine derivatives. Their synthesis and structure–activity relationships are reported.     

Discovery of 2-arylthieno[3,2-d]pyrimidines containing 8-oxa-3-azabi-cyclo[3.2.1]octane in the 4-position as potent inhibitors of mTOR with selectivity over PI3K

2-Aryl-4-morpholinothieno[3,2-d]pyrimidines are known PI3K inhibitors. This class of compounds also potently inhibited the homologous enzyme mTOR. Replacement of the morpholine group in these compounds with an 8-oxa-3-azabicyclo[3.2.1]octane group led to mTOR inhibitors with selectivity over PI3K. Optimization of the 2-aryl substituent led to the discovery of 2-(4-ureidophenyl)-thienopyrimidines as highly potent (IC₅₀ <1 nM) mTOR inhibitors with excellent selectivity (up to >1000-fold) over PI3K and good potency in a cellular proliferation assay (IC₅₀ <50 nM).     

Signaling through the Phosphatidylinositol 3-Kinase (PI3K)/Mammalian Target of Rapamycin (mTOR) Axis Is Responsible for Aerobic Glycolysis mediated by Glucose Transporter in Epidermal Growth Factor Receptor (EGFR)-mutated Lung Adenocarcinoma

Oncogenic epidermal growth factor receptor (EGFR) signaling plays an important role in regulating global metabolic pathways, including aerobic glycolysis, the pentose phosphate pathway (PPP), and pyrimidine biosynthesis. However, the molecular mechanism by which EGFR signaling regulates cancer cell metabolism is still unclear. To elucidate how EGFR signaling is linked to metabolic activity, we investigated the involvement of the RAS/MEK/ERK and PI3K/AKT/mammalian target of rapamycin (mTOR) pathways on metabolic alteration in lung adenocarcinoma (LAD) cell lines with activating EGFR mutations. Although MEK inhibition did not alter lactate production and the extracellular acidification rate, PI3K/mTOR inhibitors significantly suppressed glycolysis in EGFR-mutant LAD cells. Moreover, a comprehensive metabolomics analysis revealed that the levels of glucose 6-phosphate and 6-phosphogluconate as early metabolites in glycolysis and PPP were decreased after inhibition of the PI3K/AKT/mTOR pathway, suggesting a link between PI3K signaling and the proper function of glucose transporters or hexokinases in glycolysis. Indeed, PI3K/mTOR inhibition effectively suppressed membrane localization of facilitative glucose transporter 1 (GLUT1), which, instead, accumulated in the cytoplasm. Finally, aerobic glycolysis and cell proliferation were down-regulated when GLUT1 gene expression was suppressed by RNAi. Taken together, these results suggest that PI3K/AKT/mTOR signaling is indispensable for the regulation of aerobic glycolysis in EGFR-mutated LAD cells.     

Development of novel chromeno[4,3-c]pyrazol-4(2H)-one derivates containing piperazine as inhibitors of PI3Kα

PI3K pathway has been heavily studied and is one of the most potential targets for various cancer treatment. Herein, we designed and synthesized a series of novel chromeno[4,3-c]pyrazol-4(2H)-one derivates contained piperazine based on our previous research. They were evaluated for their PI3Kα wild-type and H1047R mutant inhibitory activities and anticancer effects in vitro. Most of these compounds displayed the potential antiproliferative activities against four cancer cell lines (HCT-116, A549, Huh7 and HL60). Among them, Compound 4p revealed the remarkable antiproliferative activity and was selected for further biological evaluation. Compound 4p displayed the potent activity against both PI3Kα wild-type and H1047R mutant, and a certain degree of selectivity for PI3Kα over PI3Kβ, γ and δ, and meanwhile it can remarkable down-regulate the phosphorylation of Akt. In addition, compound 4p was found to induce cell apoptosis via upregulation of Bax and cleaved-caspase 3/9, and downregulation of Bcl-2. The above results suggested that compound 4p could be considered as a promising PI3Kα inhibitor.     

Quinazolines with intra-molecular hydrogen bonding scaffold (iMHBS) as PI3K/mTOR dual inhibitors

Intra-molecular hydrogen bonding was introduced to the quinazoline motif to form a pseudo ring (intra-molecular H-bond scaffold, iMHBS) to mimic our previous published core structures, pyrido[2.3-D]pyrimidin-7-one and pteridinone, as PI3K/mTOR dual inhibitors. This design results in potent PI3K/mTOR dual inhibitors and the purposed intra-molecular hydrogen bonding structure is well supported by co-crystal structure in PI3Kγ enzyme. In addition, a novel synthetic route was developed for these analogs.     

N-Phenyl-4-hydroxy-2-quinolone-3-carboxamides as selective inhibitors of mutant H1047R phosphoinositide-3-kinase (PI3Kα)

This work describes our efforts to optimize the lead PI3Kα inhibitor N-benzyl 4-hydroxy-2-quinolone-3-carboxamide using structure-based design and molecular docking. We identified a series of N-phenyl 4-hydroxy-2-quinolone-3-carboxamides as selective inhibitors of mutant H1047R versus wild-type PI3Kα and we also showed that the cell growth inhibition by these compounds likely occurs by inhibiting the formation of pAKT and induction of apoptosis.     

Triazines incorporating (R)-3-methylmorpholine are potent inhibitors of the mammalian target of rapamycin (mTOR) with selectivity over PI3Kα

Potent inhibitors of the mammalian target of rapamycin (mTOR) which contain the triazine scaffold and the (R)-3-methyl morpholine moiety have been identified. Such compounds also demonstrated good selectivity over the related lipid kinase PI3Kα. Incorporation of additional functionality at the 4-position of the arylureidophenyl ring resulted in compounds with enhanced cellular activity.     

Helicobacter pylori promotes eukaryotic protein translation by activating phosphatidylinositol 3 kinase/mTOR

The innate immune response elicited by Helicobacter pylori in the human gastric mucosa involves a range of cellular signalling pathways, including those implicated in metabolism regulation. In this study, we analysed H. pylori-induced PI3K/Akt/mTOR signalling, which regulates glycolysis and protein synthesis and associates thereby with cellular energy- and nutrients-consuming processes such as growth and proliferation. The immunohistochemical analysis demonstrated that Akt kinase phosphorylation is abundant in gastric biopsies obtained from gastritis, gastric adenoma and adenocarcinoma patients. Infection with H. pylori led to the phosphorylation of Akt effectors mTOR and S6 in a type 4 secretion system (T4SS)-independent manner in AGS cells. We observed that the activation of these molecules was dependent on PI3K and the Src family tyrosine kinases. Furthermore, H. pylori induced the phosphorylation of 4E-BP1 and eIF4E and suppressed the phosphorylation of eEF2, which are important regulators of protein synthesis. Inhibition of PI3K and Akt kinase prevented the phosphorylation of 4E-BP1, suggesting that PI3K signalling is involved in the regulation of translation initiation during H. pylori infection. Metabolic labelling showed that infected cells had higher rates of [³⁵S]methionine/cysteine incorporation, and this effect could be prevented using LY294002, an PI3K inhibitor. Thus, H. pylori activates PI3K/Akt signalling, mTOR, eIFs and protein translation, which might impact H. pylori-related gastric pathophysiology.     

Activation of the PI3K/mTOR/AKT Pathway and Survival in Solid Tumors: Systematic Review and Meta-Analysis

Background

Aberrations in the phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR)/AKT pathway are common in solid tumors. Numerous drugs have been developed to target different components of this pathway. However the prognostic value of these aberrations is unclear.

Methods

PubMed was searched for studies evaluating the association between activation of the PI3K/mTOR/AKT pathway (defined as PI3K mutation [PIK3CA], lack of phosphatase and tensin homolog [PTEN] expression by immunohistochemistry or western-blot or increased expression/activation of downstream components of the pathway by immunohistochemistry) with overall survival (OS) in solid tumors. Published data were extracted and computed into odds ratios (OR) for death at 5 years. Data were pooled using the Mantel-Haenszel random-effect model.

Results

Analysis included 17 studies. Activation of the PI3K/mTOR/AKT pathway was associated with significantly worse 5-year survival (OR:2.12, 95% confidence intervals 1.42–3.16, p<0.001). Loss of PTEN expression and increased expression/activation of downstream components were associated with worse survival. No association between PIK3CA mutations and survival was observed. Differences between methods for assessing activation of the PI3K/mTOR/AKT pathway were statistically significant (p = 0.04). There was no difference in the effect of up-regulation of the pathway on survival between different cancer sites (p = 0.13).

Conclusion

Activation of the PI3K/AKT/mTOR pathway, especially if measured by loss of PTEN expression or increased expression/activation of downstream components is associated with poor survival. PIK3CA mutational status is not associated with adverse outcome, challenging its value as a biomarker of patient outcome or as a stratification factor for patients treated with agents acting on the PI3K/AKT/mTOR pathway.     

Design,synthesis and SAR of new-di-substituted pyridopyrimidines as ATP-competitive dual PI3Kα/mTOR inhibitors

PI3Kα/mTOR ATP-competitive inhibitors are considered as one of the promising molecularly targeted cancer therapeutics. Based on lead compound A from the literature, two similar series of 2-substituted-4-morpholino-pyrido[3,2-d]pyrimidine and pyrido[2,3-d]pyrimidine analogs were designed and synthesized as PI3Kα/mTOR dual inhibitors. Interestingly, most of the series gave excellent inhibition for both enzymes with IC₅₀ values ranging from single to double digit nM. Unlike many PI3Kα/mTOR dual inhibitors, our compounds displayed selectivity for PI3Kα. Based on its potent enzyme inhibitory activity, selectivity for PI3Kα and good therapeutic index in 2D cell culture viability assays, compound 4h was chosen to be evaluated in 3D culture for its IC₅₀ against MCF7 breast cancer cells as well as for docking studies with both enzymes.     

Combination of 2-methoxy-3-phenylsulfonylaminobenzamide and 2-aminobenzothiazole to discover novel anticancer agents

The fragment of 2-substituted-3-sulfonylaminobenzamide has been proposed to replace the fragment of 2-substituted-3-sulfonylaminopyridine in PI3K and mTOR dual inhibitors to design novel anticancer agents based on bioisostere. The combination of the fragment of 2-substituted-3-sulfonylaminobenzamide with the fragment of 2-aminobenzothiazole or 2-aminothiazolo[5,4-b]pyridine, or 2-amino[1,2,4]triazolo[1,5-a]pyridine produced the novel structures of anticancer agents. As a result, nineteen target compounds were synthesized and characterized. Their antiproliferative activities in vitro were evaluated via MTT assay against four human cancer cell lines including HCT-116, A549, MCF-7 and U-87 MG. The SAR of target compounds was preliminarily discussed. Compound 1g with potent antiproliferative activity was examined for its effect on the AKT and p-AKT⁴⁷³. The anticancer effect of 1g was evaluated in established nude mice HCT-116 xenograft model. The results suggested that compound 1g can block PI3K/AKT/mTOR pathway and significantly inhibit tumor growth. These findings strongly support our assumption that the fragment of benzamide can replace the pyridine ring in some PI3K and mTOR dual inhibitor to design novel anticancer agents.     

18F-FDG Is a Surrogate Marker of Therapy Response and Tumor Recovery after Drug Withdrawal during Treatment with a Dual PI3K/mTOR Inhibitor in a Preclinical Model of Cisplatin-Resistant Ovarian Cancer

AIM: Targeting the phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway is a potential means of overcoming chemoresistance in ovarian cancer. We investigated the capability of ¹⁸F-fluororodeoxyglucose (¹⁸F-FDG) small-animal positron emission tomography (SA-PET) to predict the effects of a dual PI3K/mTOR inhibitor (BEZ-235) in a cisplatin-resistant ovarian cancer model. METHODS: In a first experiment, nude rats bearing subcutaneous SKOV3 tumors received BEZ-235 for 3 days given alone or after paclitaxel and were compared to controls (either untreated or that were given the excipients of paclitaxel and BEZ-235). SA-PET was performed at baseline, on day 3, and day 7. In a second experiment aiming at further exploring the kinetics of ¹⁸F-FDG tumor uptake during the first 48 hours following drug cessation, untreated controls were compared to rats receiving BEZ-235, which were imaged at baseline, on day 3, on day 4, and on day 5. SA-PET results were compared to cell proliferation assessment (Ki-67), PI3K/mTOR downstream target expression studies (pAKT and phospho-eukaryotic translation initiation factor 4E-binding protein 1), and apoptosis evaluation (cleaved caspase-3). RESULTS: In the first experiment, BEZ-235, compared to untreated controls, induced a marked decrease in ¹⁸F-FDG uptake on day 3, which was correlated to a significant decrease in cell proliferation and to a significant PI3K/mTOR pathway inhibition. No tumor necrosis or apoptosis occurred. Four days following treatment cessation, tumor recovery (in terms of PI3K/mTOR inhibition and cell proliferation) occurred and was identified by ¹⁸F-FDG SA-PET. Paclitaxel plus BEZ-235 showed results similar to BEZ-235 alone. In the second experiment, PI3K/mTOR pathways exhibited partial recovery as early as 24 hours following treatment cessation, but both ¹⁸F-FDG SA-PET and cell proliferation remained unchanged. CONCLUSIONS:¹⁸F-FDG SA-PET is a surrogate marker of target inhibition during treatment with BEZ-235 and predicts tumor recovery 4 days after drug withdrawal, but not during the first 48 hours following drug cessation, when a lag between PI3K/mTOR pathway recovery and metabolic recovery is observed. ¹⁸F-FDG SA-PET could be used for therapy monitoring of PI3K/mTOR inhibitors, but our results also raise questions regarding the potential impact of the delay between PET imaging and the last drug intake on the accuracy of FDG imaging.     

Discovery and synthesis of 2-amino-1-methyl-1H-imidazol-4(5H)-ones as GPCR ligands; an approach to develop breast cancer drugs via GPCR associated PAR1 and PI3Kinase inhibition mechanism

Efforts were taken to synthesis and characterize 2-amino-1-methyl-1H-imidazole-4(5H)-one derivatives (4a-u) through a four-step reaction. The achieved compounds in remarkable yield have characterized through standard analytical techniques such as FTIR, LC-MS, NMR, HRMS, and elemental analysis. Present study mainly aimed to evaluate 4a-u as G protein-coupled receptors (GPCR). In the mechanism, stimulation of phosphoinositide 3-kinase (PI3K) and Akt (protein kinase B) is a general reaction activated by a series of membrane-bound receptors such as GPCR. Protease-activated receptor-1 (PAR1) is a subfamily of related GPCR, which triggered by the division of fragment of its extracellular domain. Therefore, molecular docking is done to ensure the inhibition of PAR1 and PI3Kinase. PI3Kinase is a chief enzyme in the development of breast cancer via the Akt/mTOR pathway. Thus, in vitro PI3Kinase inhibition and anti-breast cancer studies has also done to screen medicinally important compounds among (4a-u). Based on the best binding affinity, in vitro relative % activity and IC₅₀ values, compounds 4a, 4g, 4i, 4n, and 4u were screened for further preclinical studies in animal model evaluations.     

Inhibition of chemokine (CXC motif) ligand 12/chemokine (CXC motif) receptor 4 axis (CXCL12/CXCR4)-mediated cell migration by targeting mammalian target of rapamycin (mTOR) pathway in human gastric carcinoma cells

CXCL12/CXCR4 plays an important role in metastasis of gastric carcinoma. Rapamycin has been reported to inhibit migration of gastric cancer cells. However, the role of mTOR pathway in CXCL12/CXCR4-mediated cell migration and the potential of drugs targeting PI3K/mTOR pathway remains unelucidated. We found that CXCL12 activated PI3K/Akt/mTOR pathway in MKN-45 cells. Stimulating CHO-K1 cells expressing pEGFP-C1-Grp1-PH fusion protein with CXCL12 resulted in generation of phosphatidylinositol (3,4,5)-triphosphate, which provided direct evidence of activating PI3K by CXCL12. Down-regulation of p110β by siRNA but not p110α blocked phosphorylation of Akt and S6K1 induced by CXCL12. Consistently, p110β-specific inhibitor blocked the CXCL12-activated PI3K/Akt/mTOR pathway. Moreover, CXCR4 immunoprecipitated by anti-p110β antibody increased after CXCL12 stimulation and G(i) protein inhibitor pertussis toxin abrogated CXCL12-induced activation of PI3K. Further studies demonstrated that inhibitors targeting the PI3K/mTOR pathway significantly blocked the chemotactic responses of MKN-45 cells triggered by CXCL12, which might be attributed primarily to inhibition of mTORC1 and related to prevention of F-actin reorganization as well as down-regulation of active RhoA, Rac1, and Cdc42. Furthermore, rapamycin inhibited the secretion of CXCL12 and the expression of CXCR4, which might form a positive feedback loop to further abolish upstream signaling leading to cell migration. Finally, we found cells expressing high levels of cxcl12 were sensitive to rapamycin in its activity inhibiting migration as well as proliferation. In summary, we found that the mTOR pathway played an important role in CXCL12/CXCR4-mediated cell migration and proposed that drugs targeting the mTOR pathway may be used for the therapy of metastatic gastric cancer expressing high levels of cxcl12.     

Discovery of 3,6-dihydro-2H-pyran as a morpholine replacement in 6-aryl-1H-pyrazolo[3,4-d]pyrimidines and 2-arylthieno[3,2-d]pyrimidines: ATP-competitive inhibitors of the mammalian target of rapamycin (mTOR)

The morpholine hinge-region binding group on a series of pyrazolopyrimidine and thienopyrimidine mammalian target of rapamycin (mTOR) inhibitors was replaced with 3,6-dihydro-2H-pyran (DHP), giving compounds of equivalent potency and selectivity versus PI3K. These results establish the DHP group as a hinge-region binding motif for the preparation of highly potent and selective mTOR inhibitors.     

Calmodulin and IQGAP1 activation of PI3Kα and Akt in KRAS,HRAS and NRAS-driven cancers

Calmodulin (CaM) binds only oncogenic KRas, but not HRas or NRas, and thus contributes only to KRAS-driven cancers. How CaM interacts with KRas and how it boosts KRAS cancers are among the most coveted aims in cancer biology. Here we address this question, and further ask: Are there proteins that can substitute for CaM in HRAS- and NRAS-driven cancers? Can scaffolding protein IQGAP1 be one? Data suggest that formation of a CaM–KRas–PI3Kα ternary complex promotes full PI3Kα activation, and thereby potent PI3Kα/Akt/mTOR proliferative signaling. CaM binds PI3Kα at the cSH2 and nSH2 domains of its regulatory p85 subunit; the WW domain of IQGAP1 binds cSH2. This raises the question whether IQGAP1, together with an oncogenic Ras isoform, can partially activate PI3Kα. Activated, membrane-bound PI3Kα generates PIP₃. CaM shuttles Akt to the plasma membrane; CaM's release and concomitant phosphoinositide binding stimulates Akt activation. Notably, IQGAP1 directly interacts with, and helps juxtapose, PI3Kα and Akt as well as mTOR. Our mechanistic review aims to illuminate CaM's actions, and help decipher how oncogenic Ras isoforms – not only KRas4B – can activate the PI3Kα/Akt/mTOR pathway at the membrane and innovate drug discovery, including blocking the PI3Kα–IQGAP1 interaction in HRAS- and NRAS-driven cancers.     

Deciphering Combinations of PI3K/AKT/mTOR Pathway Drugs Augmenting Anti-Angiogenic Efficacy In Vivo

Ocular neovascularization is a common pathology associated with human eye diseases e.g. age-related macular degeneration and proliferative diabetic retinopathy. Blindness represents one of the most feared disabilities and remains a major burden to health-care systems. Current approaches to treat ocular neovascularisation include laser photocoagulation, photodynamic therapy and anti-VEGF therapies: Ranibizumab (Lucentis) and Aflibercept (Eylea). However, high clinical costs, frequent intraocular injections, and increased risk of infections are challenges related with these standards of care. Thus, there is a clinical need to develop more effective drugs that overcome these challenges. Here, we focus on an alternative approach by quantifying the in vivo anti-angiogenic efficacy of combinations of phosphatidylinositol-3-kinase (PI3K) pathway inhibitors. The PI3K/AKT/mTOR pathway is a complex signalling pathway involved in crucial cellular functions such as cell proliferation, migration and angiogenesis. RT-PCR confirms the expression of PI3K target genes (pik3ca, pik3r1, mtor and akt1) in zebrafish trunks from 6 hours post fertilisation (hpf) and in eyes from 2 days post fertilisation (dpf). Using both the zebrafish intersegmental vessel and hyaloid vessel assays to measure the in vivo anti-angiogenic efficacy of PI3K/Akt/mTOR pathway inhibitors, we identified 5 µM combinations of i) NVP-BEZ235 (dual PI3K-mTOR inhibitor) + PI-103 (dual PI3K-mTOR inhibitor); or ii) LY-294002 (pan-PI3K inhibitor) + NVP-BEZ235; or iii) NVP-BEZ235 + rapamycin (mTOR inhibitor); or iv) LY-294002 + rapamycin as the most anti-angiogenic. Treatment of developing larvae from 2–5 dpf with 5 µM NVP-BEZ235 plus PI-103 resulted in an essentially intact ocular morphology and visual behaviour, whereas other combinations severely disrupted the developing retinal morphology and visual function. In human ARPE19 retinal pigment epithelium cells, however, no significant difference in cell number was observed following treatment with the inhibitor combinations. Collectively, these results highlight the potential of combinations of PI3K/AKT/mTOR pathway inhibitors to safely and effectively treat ocular neovascularization.