title: Design and synthesis of novel 7-aminosubstituted pyrido[2,3-b]pyrazines exhibiting anti-breast cancer activity.
abstract: Breast cancer (BrCa) remains an unmet medical need despite the revolutionary development of antibody treatments and protein kinase inhibitors. In the current study, a series of novel substituted pyridopyrazine derivatives have been rationally designed and evaluated as multi-kinase inhibitors in the PI3K pathway. The target compounds were prepared from 6-amino-2-picoline, which upon nitration and selective reduction was converted to suitably substituted 6-methyl-7-aminopyrido[2,3-b]pyrazines. Suitable manipulation of the former amines provided the designed analogues, which were then assessed in vitro against several BrCa cell lines using the MTT cytotoxicity assay. The most potent compounds underwent evaluation in a broad spectrum of protein kinases, while their pharmacokinetic parameters were measured by LC-MS/MS. In vivo evaluation of a hit compound (14a) was performed in a HER2 amplified BrCa xenograft model (HCC1954) and efficacy was determined using Western blot based phosphokinase assays and immunohistochemistry. This derivative showed low micromolar cytotoxic potency in all BrCa cell lines, a mild inhibition of the PI3Kα wild type and H1047R mutated enzyme and excellent pharmacokinetic parameters following oral and intraperitoneal administration at the designed dose of 10 mg/kg, with absence of in vivo phenotypic toxicity. Interestingly, compound 14a inhibited the growth of xenografted tumors. Analysis of excised tumors from the treated animals showed a significantly reduced population of Ki-67 positive cells, as well as downregulated levels of phosphorylated AKT, ERK1/2 and SRC compared to vehicle treated animals. Finally, the specificity of 14a was assessed in a panel of 31 kinases where a mild, but direct, inhibition of the MET receptor tyrosine kinase was observed.
authors.affiliation: Division of Pharmacology-Pharmacotechnology, Biomedical Research Foundation Academy of Athens (BRFAA), Athens, 11527, Greece. Electronic address: ore_argyros@yahoo.com.
authors.forename: Orestis
authors.initials: O
authors.lastname: Argyros
authors.affiliation: School of Health Sciences, Department of Pharmacy, Division of Pharmaceutical Chemistry, National and Kapodistrian University of Athens, Panepistimiopolis, Zografou, Athens, 15771, Greece. Electronic address: nlougiak@pharm.uoa.gr.
authors.forename: Nikolaos
authors.initials: N
authors.lastname: Lougiakis
authors.affiliation: Division of Pharmacology-Pharmacotechnology, Biomedical Research Foundation Academy of Athens (BRFAA), Athens, 11527, Greece. Electronic address: eva_kouvari@yahoo.gr.
authors.forename: Eva
authors.initials: E
authors.lastname: Kouvari
authors.affiliation: Institute of Molecular Biology and Biotechnology, Department of Biomedical Research, Foundation for Research and Technology Hellas, 45110, Ioannina, Greece; Laboratory of Biological Chemistry, Department of Medicine, School of Health Sciences, University of Ioannina, 45110, Ioannina, Greece. Electronic address: apapafot@cc.uoi.gr.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Papafotika
authors.affiliation: Institute of Nanoscience and Nanotechnology, N.C.S.R "Demokritos", Athens, 15310, Greece. Electronic address: c.raptopoulou@inn.demokritos.gr.
authors.forename: Catherine P
authors.initials: CP
authors.lastname: Raptopoulou
authors.affiliation: Institute of Nanoscience and Nanotechnology, N.C.S.R "Demokritos", Athens, 15310, Greece. Electronic address: v.psycharis@inn.demokritos.gr.
authors.forename: Vassilis
authors.initials: V
authors.lastname: Psycharis
authors.affiliation: Institute of Molecular Biology and Biotechnology, Department of Biomedical Research, Foundation for Research and Technology Hellas, 45110, Ioannina, Greece; Laboratory of Biological Chemistry, Department of Medicine, School of Health Sciences, University of Ioannina, 45110, Ioannina, Greece. Electronic address: schristo@uoi.gr.
authors.forename: Savvas
authors.initials: S
authors.lastname: Christoforidis
authors.affiliation: School of Health Sciences, Department of Pharmacy, Division of Pharmaceutical Chemistry, National and Kapodistrian University of Athens, Panepistimiopolis, Zografou, Athens, 15771, Greece. Electronic address: pouli@pharm.uoa.gr.
authors.forename: Nicole
authors.initials: N
authors.lastname: Pouli
authors.affiliation: School of Health Sciences, Department of Pharmacy, Division of Pharmaceutical Chemistry, National and Kapodistrian University of Athens, Panepistimiopolis, Zografou, Athens, 15771, Greece. Electronic address: marakos@pharm.uoa.gr.
authors.forename: Panagiotis
authors.initials: P
authors.lastname: Marakos
authors.affiliation: Division of Pharmacology-Pharmacotechnology, Biomedical Research Foundation Academy of Athens (BRFAA), Athens, 11527, Greece. Electronic address: ctamvakop@bioacademy.gr.
authors.forename: Constantin
authors.initials: C
authors.lastname: Tamvakopoulos
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000138:chemical synthesis* / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000473:pathology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D060326:Chemistry Techniques, Synthetic; D015195:Drug Design; D005260:Female; D006801:Humans; D051379:Mice; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011719:Pyrazines / Q000138:chemical synthesis* / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D023041:Xenograft Model Antitumor Assays
chemical_list: D000970:Antineoplastic Agents; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011719:Pyrazines
keywords: Breast cancer; In vivo activity; Met translational medicine; PI3K; Pharmacokinetic parameters; Pyridopyrazine