ABSTRACT
In this article we describe the identification of unprecedented ATP-competitive ChoKα inhibitors starting from initial hit NMS-P830 that binds to ChoKα in an ATP concentration-dependent manner. This result is confirmed by the co-crystal structure of NMS-P830 in complex with Δ75-ChoKα. NMS-P830 is able to inhibit ChoKα in cells resulting in the reduction of intracellular phosphocholine formation. A structure-based medicinal chemistry program resulted in the identification of selective compounds that have good biochemical activity, solubility and metabolic stability and are suitable for further optimization. The ChoKα inhibitors disclosed in this article demonstrate for the first time the possibility to inhibit ChoKα with ATP-competitive compounds.
Subject(s)
Adenosine Triphosphate/antagonists & inhibitors , Choline Kinase/antagonists & inhibitors , Cyclohexanes/pharmacology , Protein Kinase Inhibitors/pharmacology , Adenosine Triphosphate/metabolism , Choline Kinase/metabolism , Cyclohexanes/chemical synthesis , Cyclohexanes/chemistry , Dose-Response Relationship, Drug , Humans , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Structure-Activity RelationshipABSTRACT
Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase responsible for the development of different tumor types. Despite the remarkable clinical activity of crizotinib (Xalkori), the first ALK inhibitor approved in 2011, the emergence of resistance mutations and of brain metastases frequently causes relapse in patients. Within our ALK drug discovery program, we identified compound 1, a novel 3-aminoindazole active on ALK in biochemical and in cellular assays. Its optimization led to compound 2 (entrectinib), a potent orally available ALK inhibitor active on ALK-dependent cell lines, efficiently penetrant the blood-brain barrier (BBB) in different animal species and highly efficacious in in vivo xenograft models. Moreover, entrectinib resulted to be strictly potent on the closely related tyrosine kinases ROS1 and TRKs recently found constitutively activated in several tumor types. Entrectinib is currently undergoing phase I/II clinical trial for the treatment of patients affected by ALK-, ROS1-, and TRK-positive tumors.
Subject(s)
Antineoplastic Agents/pharmacology , Benzamides/pharmacology , Drug Discovery , Indazoles/pharmacology , Lung Neoplasms/drug therapy , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Administration, Oral , Anaplastic Lymphoma Kinase , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/chemistry , Benzamides/administration & dosage , Benzamides/chemistry , Blood-Brain Barrier/drug effects , Blotting, Western , Cell Membrane Permeability/drug effects , Cell Proliferation/drug effects , Crystallization , Crystallography, X-Ray , Dogs , Humans , Indazoles/administration & dosage , Indazoles/chemistry , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Mice , Mice, Nude , Mice, SCID , Microsomes, Liver/drug effects , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins/antagonists & inhibitors , Rats , Rats, Wistar , Receptor, trkA/antagonists & inhibitors , Receptor, trkB/antagonists & inhibitors , Receptor, trkC/antagonists & inhibitors , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Compound 1, a hit from the screening of our chemical collection displaying activity against JAK2, was deconstructed for SAR analysis into three regions, which were explored. A series of compounds was synthesized leading to the identification of the potent and orally bioavailable JAK2 inhibitor 16 (NMS-P830), which showed an encouraging tumour growth inhibition in SET-2 xenograft tumour model, with evidence for JAK2 pathway suppression demonstrated by in vivo pharmacodynamic effects.
Subject(s)
Amides/chemical synthesis , Antineoplastic Agents/chemical synthesis , Janus Kinase 2/antagonists & inhibitors , Leukemia, Megakaryoblastic, Acute/drug therapy , Protein Kinase Inhibitors/chemical synthesis , Pyrroles/chemical synthesis , Amides/pharmacology , Animals , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Female , Gene Expression , High-Throughput Screening Assays , Humans , Janus Kinase 2/chemistry , Janus Kinase 2/genetics , Janus Kinase 2/metabolism , Leukemia, Megakaryoblastic, Acute/enzymology , Leukemia, Megakaryoblastic, Acute/genetics , Leukemia, Megakaryoblastic, Acute/pathology , Megakaryocyte Progenitor Cells/drug effects , Megakaryocyte Progenitor Cells/enzymology , Megakaryocyte Progenitor Cells/pathology , Mice , Mice, Nude , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Kinase Inhibitors/pharmacology , Pyrroles/pharmacology , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
We report herein the discovery, structure guided design, synthesis and biological evaluation of a novel class of JAK2 inhibitors. Optimization of the series led to the identification of the potent and orally bioavailable JAK2 inhibitor 28 (NMS-P953). Compound 28 displayed significant tumour growth inhibition in SET-2 xenograft tumour model, with a mechanism of action confirmed in vivo by typical modulation of known biomarkers, and with a favourable pharmacokinetic and safety profile.
Subject(s)
Antineoplastic Agents/pharmacology , Janus Kinase 2/antagonists & inhibitors , Neoplasms, Experimental/drug therapy , Protein Kinase Inhibitors/pharmacology , Pyrimidines/pharmacology , Pyrroles/pharmacology , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Crystallography, X-Ray , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Janus Kinase 2/metabolism , Mice , Mice, SCID , Models, Molecular , Molecular Structure , Neoplasms, Experimental/pathology , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Pyrimidines/chemical synthesis , Pyrimidines/chemistry , Pyrroles/chemical synthesis , Pyrroles/chemistry , Structure-Activity Relationship , Substrate SpecificityABSTRACT
Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase involved in the development of several human cancers and, as a result, is a recognized target for the development of small-molecule inhibitors for the treatment of ALK-positive malignancies. Here, we present the crystal structures of the unphosphorylated human ALK kinase domain in complex with the ATP competitive ligands PHA-E429 and NVP-TAE684. Analysis of these structures provides valuable information concerning the specific characteristics of the ALK active site as well as giving indications about how to obtain selective ALK inhibitors. In addition, the ALK-KD-PHA-E429 structure led to the identification of a potential regulatory mechanism involving a link made between a short helical segment immediately following the DFG motif and an N-terminal two-stranded beta-sheet. Finally, mapping of the activating mutations associated with neuroblastoma onto our structures may explain the roles these residues have in the activation process.
Subject(s)
Models, Molecular , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/chemistry , Pyrimidines/chemistry , Anaplastic Lymphoma Kinase , Animals , Cell Line , Enzyme Inhibitors/chemistry , Humans , Protein Structure, Tertiary , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Receptor Protein-Tyrosine Kinases , SpodopteraABSTRACT
We have recently reported CDK inhibitors based on the 6-substituted pyrrolo[3,4-c]pyrazole core structure. Improvement of inhibitory potency against multiple CDKs, antiproliferative activity against cancer cell lines and optimization of the physico-chemical properties led to the identification of highly potent compounds. Compound 31 (PHA-793887) showed good efficacy in the human ovarian A2780, colon HCT-116 and pancreatic BX-PC3 carcinoma xenograft models and was well tolerated upon daily treatments by iv administration. It was identified as a drug candidate for clinical evaluation in patients with solid tumors.
Subject(s)
Antineoplastic Agents/chemistry , Cyclin-Dependent Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Pyrazoles/chemistry , Pyrroles/chemistry , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacokinetics , Binding Sites , Cell Line, Tumor , Crystallography, X-Ray , Cyclin-Dependent Kinases/metabolism , HCT116 Cells , Humans , Injections, Intravenous , Mice , Mice, Nude , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacokinetics , Pyrazoles/chemical synthesis , Pyrazoles/pharmacokinetics , Pyrroles/chemical synthesis , Pyrroles/pharmacokinetics , Xenograft Model Antitumor AssaysABSTRACT
We have recently reported a new class of CDK2/cyclin A inhibitors based on a bicyclic tetrahydropyrrolo[3,4-c]pyrazole scaffold. The introduction of small alkyl or cycloalkyl groups in position 6 of this scaffold allowed variation at the other two diversity points. Conventional and polymer-assisted solution phase chemistry provided a way of generating compounds with improved biochemical and cellular activity. Optimization of the physical properties and pharmacokinetic profile led to a compound which exhibited good efficacy in vivo on A2780 human ovarian carcinoma.
Subject(s)
Cyclin-Dependent Kinase 2/antagonists & inhibitors , Drug Design , Protein Kinase Inhibitors/classification , Protein Kinase Inhibitors/chemical synthesis , Pyrazoles/chemistry , Pyrroles/chemistry , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/classification , Antineoplastic Agents/metabolism , Bridged Bicyclo Compounds, Heterocyclic/administration & dosage , Bridged Bicyclo Compounds, Heterocyclic/chemical synthesis , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Bridged Bicyclo Compounds, Heterocyclic/metabolism , Caco-2 Cells , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin-Dependent Kinase 2/metabolism , Growth Inhibitors/administration & dosage , Growth Inhibitors/chemical synthesis , Growth Inhibitors/classification , Growth Inhibitors/metabolism , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism , Mice , Mice, Nude , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/metabolismABSTRACT
The synthesis of the major metabolite of a potent 3-aminopyrazole CDK2/cyclin A inhibitor is presented. A stereoconservative approach starting from malic acid was employed to construct the hydroxy-substituted pyrrolidinone moiety. In the key step of the synthesis the use of cyanoborohydride immobilized on Amberlyst 26 in trifluoroethanol represented a valid alternative to conventional solution-phase reducing agents.
Subject(s)
Cyclin A/antagonists & inhibitors , Cyclin-Dependent Kinase 2/antagonists & inhibitors , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Pyrrolidinones/chemistry , Pyrrolidinones/pharmacology , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin A/metabolism , Cyclin-Dependent Kinase 2/metabolism , Humans , Mass Spectrometry , Molecular Structure , Protein Kinase Inhibitors/chemistry , Pyrrolidinones/metabolismABSTRACT
Inhibitors of cyclin-dependent kinases (CDK) such as CDK2/cyclin A-E are currently undergoing clinical trials to verify their potential as new anticancer agents. In a previous article we described the lead discovery process of a 3-aminopyrazole class of CDK2/cyclin A-E inhibitors. The endpoint of this process was PNU-292137, a compound endowed with in vivo antitumor activity in a mouse tumor xenograft model. We optimized this lead compound to improve some physicochemical properties, notably solubility and plasma protein binding. This lead optimization process brought us to the discovery of (2S)-N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-[4-(2-oxo-1-pyrrolidinyl)phenyl]propanamide (PHA-533533, 13), a compound with a balanced activity vs druglike profile. Compound 13 inhibited CDK2/cyclin A with a K(i) of 31 nM, counteracting tumor cell proliferation of different cell lines with an IC(50) in the submicromolar range. Solubility was improved more than 10 times over the starting lead, while plasma protein binding was decreased from 99% to 74%. With exploitation of this globally enhanced in vitro profile, 13 was more active than PNU-292137 in vivo in the A2780 xenograft model showing a tumor growth inhibition of 70%. Proof of mechanism of action was obtained in vivo by immunohistochemical analysis of tumor slices of 13-treated vs untreated animals.
