ABSTRACT
The series of 4-(benzylaminomethylene)isoquinoline-1,3-(2H,4H)-dione and 4-[(pyridylmethyl)aminomethylene]isoquinoline-1,3-(2H,4H)-dione derivatives reported here represents a novel class of potential antitumor agents, which potently and selectively inhibit CDK4 over CDK2 and CDK1. In the benzylamino headpiece, a 3-OH substituent is required on the phenyl ring for CDK4 inhibitory activity, which is further enhanced when an iodo, aryl, heteroaryl, t-butyl, or cyclopentyl substituent is introduced at the C-6 position of the isoquinoline-1,3-dione core. To circumvent the metabolic liability associated with the phenolic OH group on the 4-substituted 3-OH phenyl headpiece, we take two approaches: first, introduce a nitrogen o- or p- to the 3-OH group in the phenyl ring; second, replace the phenyl headpiece with N-substituted 2-pyridones. We present here the synthesis, SAR data, metabolic stability data, and a CDK4 mimic model that explains the binding, potency, and selectivity of our CDK4 selective inhibitors.
Subject(s)
Antineoplastic Agents/chemical synthesis , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Isoquinolines/chemical synthesis , Pyridines/chemical synthesis , Adenosine Triphosphate/metabolism , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Binding Sites , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Screening Assays, Antitumor , Humans , Hydrogen Bonding , In Vitro Techniques , Isoquinolines/chemistry , Isoquinolines/pharmacology , Microsomes, Liver/metabolism , Models, Molecular , Phosphorylation , Pyridines/chemistry , Pyridines/pharmacology , Rats , Retinoblastoma Protein/metabolism , Stereoisomerism , Structure-Activity RelationshipABSTRACT
Insulin-like growth factor receptor (IGF-1R) is a growth factor receptor tyrosine kinase that acts as a critical mediator of cell proliferation and survival. This receptor is over-expressed or activated in tumor cells and is emerging as a novel target in cancer therapy. Efforts in our "Hit to Lead" group have generated a novel series of submicromolar IGF-1R inhibitors based on a isoquinolinedione template originating from a Lance enzyme HTS screen. Chemical triage and parallel synthesis incorporating focused library arrays were instrumental in moving these investigations through the Wyeth exploratory medicinal chemistry process. The strategies, synthesis, and SAR behind this interesting kinase scaffold will be described.
Subject(s)
Antineoplastic Agents/pharmacology , Isoquinolines/pharmacology , Receptor, IGF Type 1/antagonists & inhibitors , Antineoplastic Agents/chemistry , Dose-Response Relationship, Drug , Drug Design , Drug Screening Assays, Antitumor , Isoquinolines/chemistry , Models, Molecular , Molecular Structure , Small Molecule Libraries , Structure-Activity RelationshipABSTRACT
The cyclin-dependent kinases (CDKs), as complexes with their respective partners, the cyclins, are critical regulators of cell cycle progression. Because aberrant regulations of CDK4/cyclin D1 lead to uncontrolled cell proliferation, a hallmark of cancer, small-molecule inhibitors of CDK4/cyclin D1 are attractive as prospective antitumor agents. The series of 4-(phenylaminomethylene)isoquinoline-1,3(2H,4H)-dione derivatives reported here represents a novel class of potent inhibitors that selectively inhibit CDK4 over CDK2 and CDK1 activities. In the headpiece of the 4-(phenylaminomethylene)isoquinoline-1,3(2H,4H)-dione, a basic amine substituent is required on the aniline ring for the CDK4 inhibitory activity. The inhibitory activity is further enhanced when an aryl or heteroaryl substituent is introduced at the C-6 position of the isoquinoline-1,3(2H,4H)-dione core. We present here SAR data and a CDK4 mimic model that explains the binding, potency, and selectivity of our CDK4 selective inhibitors.
Subject(s)
Antineoplastic Agents/chemical synthesis , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Isoquinolines/chemical synthesis , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Screening Assays, Antitumor , Humans , Isoquinolines/chemistry , Isoquinolines/pharmacology , Models, Molecular , Phosphorylation , Retinoblastoma Protein/metabolism , Stereoisomerism , Structure-Activity RelationshipABSTRACT
A series of 4-dimethylamino-but-2-enoic acid [4-(3,6-dioxo-cyclohexa-1,4-dienylamino)-7-ethoxy-quinazolin-6-yl]-amide derivatives were prepared. These compounds have two independent reactive centers and were designed to function as dual irreversible inhibitors of the kinase domains of both Epidermal Growth Factor Receptor (EGFR) and Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) where each reactive center targets a different, non-conserved, cysteine residue located in the ATP binding pocket of these enzymes. The compounds contain a 6-(4-(dimethylamino) crotonamide) Michael acceptor group that targets Cys-773 in EGFR and a 4-(amino-[1,4]benzoquinone) moiety that targets Cys-1045 in VEGFR-2. In vitro studies indicated that most of these compounds are relatively potent inhibitors of each enzyme. These inhibitors were compared with reference compounds that lack one or both of the reactive centers. The relative dependence of the IC(50) values on the concentration of ATP used in the assays suggests that these compounds appear to function as irreversible inhibitors of each kinase.
Subject(s)
ErbB Receptors/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Quinazolines/chemistry , Quinazolines/pharmacology , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors , Adenosine Triphosphate/chemistry , Adenosine Triphosphate/metabolism , Binding Sites , Biological Assay , Cells, Cultured , ErbB Receptors/chemistry , ErbB Receptors/metabolism , Humans , Inhibitory Concentration 50 , Models, Molecular , Molecular Structure , Protein Conformation/drug effects , Protein Kinase Inhibitors/chemical synthesis , Quinazolines/chemical synthesis , Vascular Endothelial Growth Factor Receptor-2/chemistry , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
The p38-mitogen-activated protein kinases (p38-MAPKs) belong to a family of serine-threonine kinases activated by pro-inflammatory or stressful stimuli that are known to be involved in several diseases. Their biological importance, related to the release of inflammatory pro-cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1), has generated many studies aiming at the development of selective inhibitors for the treatment of inflammatory diseases. In this work, we developed receptor-based three dimensional (3D) quantitative structure-activity relationship (QSAR) models for a series of 33 pyridinyl imidazole compounds [Liverton et al. (1999) 42:2180], using a methodology named free-energy force-field (FEFF) [Tokarski and Hopfinger (1997) 37:792], in which scaled intra- and intermolecular energy terms of the Assisted Model Building Energy Refinement (AMBER) force field combined with a hydration-shell solvation model are the independent variables used in the QSAR studies. Multiple temperature molecular-dynamics simulations (MDS) of ligand-protein complexes and genetic-function approximation (GFA) were employed using partial least squares (PLS) as the fitting functions to develop FEFF-3D-QSAR models for the binding process. The best model obtained in the FEFF-3D-QSAR receptor-dependent (RD) method shows the importance of the van der Waals energy change upon binding and the electrostatic energy in the interaction of ligands with the receptor. The QSAR equations described here show good predictability and may be regarded as representatives of the binding process of ligands to p38-MAPK. Additionally, we have compared the top FEFF-3D-QSAR model with receptor independent (RI) 4D-QSAR models developed in a recent study [Romeiro et al. (2005) 19:385].
Subject(s)
Imidazoles/chemistry , Protein Kinase Inhibitors/chemistry , Quantitative Structure-Activity Relationship , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Hydrophobic and Hydrophilic Interactions , Imidazoles/pharmacology , Ligands , Protein Binding , Protein Conformation , Static Electricity , Temperature , ThermodynamicsABSTRACT
A binding mode of HTI-286, a synthetic analogue of the peptidic antimitotic agent hemiasterlin, to tubulin is proposed. The binding mode was derived from iterative docking experiments directed at regions of the tubulin interdimer interface that are believed to be consistent with all current experimental data regarding the HTI-286/tubulin interaction. These data include (1) competitive inhibition of the tubulin binding of the Vinca alkaloids and other antimitotic agents, (2) proximity to stretches of amino acid residues identified in two separate photoaffinity-labeling experiments, (3) structure-activity relationships for HTI-286 and its analogues, (4) saturation transfer difference nuclear magnetic resonance (NMR) experiments, and (5) NMR transfer nuclear Overhauser effect spectroscopy (NOESY) experiments that potentially identify the bioactive conformation. The predicted binding mode thus affords a means to understand the mode of action of hemiasterlin, HTI-286, and other closely related molecules.
Subject(s)
Binding Sites , Oligopeptides/metabolism , Tubulin/metabolism , Antimitotic Agents/chemistry , Binding, Competitive , Electron Spin Resonance Spectroscopy , Models, Molecular , Molecular Conformation , Oligopeptides/chemistry , Structure-Activity Relationship , Tubulin/chemistryABSTRACT
A series of 2-(quinazolin-4-ylamino)-[1,4] benzoquinone derivatives that function as potent covalent-binding, irreversible inhibitors of the kinase domain of vascular endothelial growth factor receptor-2 (VEGFR-2) has been prepared by ceric ammonium nitrate oxidation of substituted (2,5-dimethoxyphenyl)(6,7-disubstituted-quinazolin-4-yl)amines and by displacement of the chlorine atom of substituted 2-chloro-5-(6,7-disubstituted-quinazolin-4-ylamino)-[1,4]benzoquinones with various amines, anilines, phenols, and alcohols. Enzyme studies were conducted in the absence and presence of glutathione and plasma. Several of the compounds inhibit VEGF-stimulated autophosphorylation in intact cells. Kinetic experiments were performed to study the reactivity of selected inhibitors toward glutathione. Reactivities correlated with LUMO energies calculated as averages of those of individual conformers weighted by the Boltzmann distribution. These results and molecular modeling were used to rationalize the biological observations. The compounds behave as non-ATP-competitive inhibitors. Unequivocal evidence, from mass spectral studies, indicates that these inhibitors form a covalent interaction with Cys-1045. One member of this series displays antitumor activity in an in vivo model.
Subject(s)
Angiogenesis Inhibitors/chemical synthesis , Benzoquinones/chemical synthesis , Quinazolines/chemical synthesis , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors , Adenosine Triphosphate/metabolism , Angiogenesis Inhibitors/chemistry , Angiogenesis Inhibitors/pharmacology , Animals , Benzoquinones/chemistry , Benzoquinones/pharmacology , Binding Sites , Cell Line , Female , Glutathione/chemistry , Humans , Kinetics , Mice , Mice, Nude , Models, Molecular , Molecular Conformation , Phosphorylation , Protein Binding , Protein Structure, Tertiary , Quantum Theory , Quinazolines/chemistry , Quinazolines/pharmacology , Spectrometry, Mass, Electrospray Ionization , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Structure-Activity Relationship , Vascular Endothelial Growth Factor Receptor-2/chemistry , Vascular Endothelial Growth Factor Receptor-2/metabolism , Xenograft Model Antitumor AssaysABSTRACT
The p38-mitogen-activated protein kinase (p38-MAPK) plays a key role in lipopolysaccharide-induced tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) release during the inflammatory process, emerging as an attractive target for new anti-inflammatory agents. Four-dimensional quantitative structure-activity relationship (4D-QSAR) analysis [Hopfinger et al., J. Am. Chem. Soc., 119 (1997) 10509] was applied to a series of 33 (a training set of 28 and a test set of 5) pyridinyl-imidazole and pyrimidinyl-imidazole inhibitors of p38-MAPK, with IC50 ranging from 0.11 to 2100 nM [Liverton et al., J. Med. Chem., 42 (1999) 2180]. Five thousand conformations of each analogue were sampled from a molecular dynamics simulation (MDS) during 50 ps at a constant temperature of 303 K. Each conformation was placed in a 2 angstroms grid cell lattice for each of three trial alignments. 4D-QSAR models were constructed by genetic algorithm (GA) optimization and partial least squares (PLS) fitting, and evaluated by leave-one-out cross-validation technique. In the best models, with three to six terms, the adjusted cross-validated squared correlation coefficients, Q2adj, ranged from 0.67 to 0.85. Model D (Q2adj = 0.84) was identified as the most robust model from alignment 1, and it is representative of the other best models. This model encompasses new molecular regions as containing pharmacophore sites, such as the amino-benzyl moiety of pyrimidine analogs and the N1-substituent in the imidazole ring. These regions of the ligands should be further explored to identify better anti-inflammatory inhibitors of p38-MAPK.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/chemical synthesis , Drug Design , Models, Molecular , Quantitative Structure-Activity Relationship , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/chemistry , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Catalytic Domain , Imidazoles/chemical synthesis , Imidazoles/pharmacology , Molecular Conformation , Pyridines/chemical synthesis , Pyridines/pharmacologyABSTRACT
It has been previously reported that appropriately substituted 4-anilinoquinoline-3-carbonitriles are potent inhibitors of Src kinase, with biological activity in vitro and in vivo. Structural modifications to these compounds have been explored, providing the 4-anilinobenzo[g]quinoline-3-carbonitriles as a series with enhanced Src inhibitory properties. The synthesis and structure-activity relationships of these 4-anilino-7,8-dialkoxybenzo[g]quinoline-3-carbonitriles are presented here. Analogues with cyclic basic amine groups attached via ethoxy linkages at the C-8 position were the most active in vitro, with subnanomolar IC50 values against Src kinase observed for a majority of the compounds synthesized. Compound 17d was more potent in vitro than the analogously substituted 4-anilinoquinoline-3-carbonitrile SKI-606, which is undergoing evaluation in clinical trials. The most potent analogue synthesized was 17a, with an IC50 of 0.15 nM against Src kinase and with an IC50 of 10 nM against Src-transformed fibroblasts. Molecular modeling studies provided a rationale for the exceptional activity observed for these compounds, with favorable van der Waals interactions playing the major role. Compound 17c was found to be highly selective for Src kinase when tested against a panel of other kinases, with modest selectivity versus the Src family kinases Lyn and Fyn. Following ip dosing at 50 mg/kg, analogues 17c and 17d were shown to have plasma levels that significantly exceeded the cellular IC50 values against Src-transformed fibroblasts. In an Src-transformed fibroblast xenograft model, both compounds exhibited a significant inhibition of tumor growth.