ABSTRACT
Pyridyl aminothiazoles comprise a novel class of ATP-competitive Chk1 inhibitors with excellent inhibitory potential. Modification of the core with ethylenediamine amides provides compounds with low picomolar potency and very high residence times. Investigation of binding parameters of such compounds using X-ray crystallography and molecular dynamics simulations revealed multiple hydrogen bonds to the enzyme backbone as well as stabilization of the conserved water molecules network in the hydrophobic binding region.
Subject(s)
Antineoplastic Agents/chemical synthesis , Protein Kinase Inhibitors/chemical synthesis , Protein Kinases/chemistry , Pyridines/chemical synthesis , Thiazoles/chemical synthesis , Amides/chemistry , Antineoplastic Agents/pharmacology , Binding Sites , Checkpoint Kinase 1 , Crystallography, X-Ray , Drug Design , Ethylenediamines/chemistry , Humans , Hydrogen Bonding , Kinetics , Molecular Dynamics Simulation , Molecular Structure , Protein Binding , Protein Kinase Inhibitors/pharmacology , Protein Kinases/metabolism , Pyridines/pharmacology , Structure-Activity Relationship , Thiazoles/pharmacology , Water/chemistryABSTRACT
Translation of significant biochemical activity of pyridyl aminothiazole class of Chk1 inhibitors into functional CEA potency required analysis and adjustment of both physical properties and kinase selectivity profile of the series. The steps toward optimization of cellular potency included elimination of CDK7 activity, reduction of molecular weight and polar surface area and increase in lipophilicity of the molecules in the series.
Subject(s)
Antineoplastic Agents/chemical synthesis , Protein Kinase Inhibitors/chemical synthesis , Protein Kinases/chemistry , Pyridines/chemical synthesis , Thiazoles/chemical synthesis , Antineoplastic Agents/pharmacology , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cell Membrane Permeability , Checkpoint Kinase 1 , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/chemistry , Drug Design , Halogenation , Humans , Kinetics , Molecular Structure , Protein Kinase Inhibitors/pharmacology , Protein Kinases/metabolism , Pyridines/pharmacology , Structure-Activity Relationship , Thiazoles/pharmacology , Cyclin-Dependent Kinase-Activating KinaseABSTRACT
We describe herein a novel series of 3-amino-4-hydrazine-cyclobut-3-ene-1,2-diones as potent and selective inhibitors against the CXCR2 chemokine receptor and IL-8-mediated chemotaxis of a CXCR2-expressing cell line. Furthermore, these alkyl-hydrazine series inhibitors such as 5b demonstrated acceptable metabolic stability when incubated in human and rat microsomes.
Subject(s)
Anti-Inflammatory Agents/chemical synthesis , Hydrazines/chemical synthesis , Receptors, Interleukin-8B/antagonists & inhibitors , Animals , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/pharmacology , CHO Cells , Chemotaxis/drug effects , Cricetinae , Cricetulus , Drug Design , Humans , Hydrazines/chemistry , Hydrazines/pharmacology , Interleukin-8/metabolism , Microsomes, Liver/metabolism , Rats , Receptors, Interleukin-8B/metabolism , Structure-Activity RelationshipABSTRACT
Inhibition of kinesin spindle protein (KSP) is a novel mechanism for treatment of cancer with the potential to overcome limitations associated with currently employed cytotoxic agents. Herein, we describe a C2-hydroxymethyl dihydropyrrole KSP inhibitor ( 11) that circumvents hERG channel binding and poor in vivo potency, issues that limited earlier compounds from our program. However, introduction of the C2-hydroxymethyl group caused 11 to be a substrate for cellular efflux by P-glycoprotein (Pgp). Utilizing knowledge garnered from previous KSP inhibitors, we found that beta-fluorination modulated the p K a of the piperidine nitrogen and reduced Pgp efflux, but the resulting compound ( 14) generated a toxic metabolite in vivo. Incorporation of fluorine in a strategic, metabolically benign position by synthesis of an N-methyl-3-fluoro-4-(aminomethyl)piperidine urea led to compound 30 that has an optimal in vitro and metabolic profile. Compound 30 (MK-0731) was recently studied in a phase I clinical trial in patients with taxane-refractory solid tumors.
Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Kinesins/antagonists & inhibitors , Neoplasms/enzymology , Piperidines/pharmacology , Pyrroles/pharmacology , Taxoids/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Antineoplastic Agents/therapeutic use , Enzyme Inhibitors/therapeutic use , Humans , Neoplasms/drug therapy , Taxoids/therapeutic useABSTRACT
Inspired by previous efforts in the pyrazolobenzoxazine class of KSP inhibitors, the design and synthesis of 1,4-diaryl-4,5-dihydropyrazole inhibitors of KSP are described. Crystallographic evidence of binding mode and in vivo potency data is also highlighted.
Subject(s)
Drug Design , Hydrogen/chemistry , Kinesins/antagonists & inhibitors , Kinesins/metabolism , Mitosis/drug effects , Pyrazoles/chemistry , Pyrazoles/pharmacology , Binding Sites , Crystallography, X-Ray , Humans , Models, Molecular , Molecular Structure , Pyrazoles/chemical synthesis , Structure-Activity RelationshipABSTRACT
3,5-diaryl-4,5-dihydropyrazoles were discovered to be potent KSP inhibitors with excellent in vivo potency. These enzyme inhibitors possess desirable physical properties that can be readily modified by incorporation of a weakly basic amine. Careful adjustment of amine basicity was essential for preserving cellular potency in a multidrug resistant cell line while maintaining good aqueous solubility.
Subject(s)
Amides/chemical synthesis , Amides/pharmacology , Antimitotic Agents/chemical synthesis , Antimitotic Agents/pharmacology , Kinesins/antagonists & inhibitors , Mitosis/drug effects , Pyrazoles/chemical synthesis , Pyrazoles/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/antagonists & inhibitors , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Chemical Phenomena , Chemistry, Physical , Drug Design , Drug Resistance, Neoplasm , Genes, MDR/drug effects , Humans , Indicators and Reagents , Solubility , Stereoisomerism , Structure-Activity RelationshipABSTRACT
The development of 2,5-dihydro-4H-pyrazolo[4,3-c]quinolin-4-ones as inhibitors of Chk1 kinase is described. Introduction of a fused ring at the C7/C8 positions of the pyrazoloquinolinone provided an increase in potency while guidance from overlapping inhibitor bound Chk1 X-ray crystal structures contributed to the discovery of a potent and solubilizing propyl amine moiety in compound 52 (Chk1 IC(50)=3.1 nM).
Subject(s)
Protein Kinase Inhibitors/pharmacology , Protein Kinases/drug effects , Quinolones/pharmacology , Checkpoint Kinase 1 , Crystallography, X-Ray , Models, Molecular , Protein Kinase Inhibitors/chemistry , Quinolones/chemistry , Structure-Activity RelationshipABSTRACT
Observations from two structurally related series of KSP inhibitors led to the proposal and discovery of dihydropyrazolobenzoxazines that possess ideal properties for cancer drug development. The synthesis and characterization of this class of inhibitors along with relevant pharmacokinetic and in vivo data are presented. The synthesis is highlighted by a key [3+2] cycloaddition to form the pyrazolobenzoxazine core followed by diastereospecific installation of a quaternary center.
Subject(s)
Benzoxazines/chemistry , Benzoxazines/pharmacology , Drug Design , Kinesins/antagonists & inhibitors , Kinesins/metabolism , Mitosis/drug effects , Pyrazoles/chemistry , Animals , Benzoxazines/chemical synthesis , Benzoxazines/pharmacokinetics , Cell Line , Dogs , Humans , Hydrogen/chemistry , Molecular Structure , Structure-Activity RelationshipABSTRACT
Installation of a C2-aminopropyl side chain to the 2,4-diaryl-2,5-dihydropyrrole series of kinesin spindle protein (KSP) inhibitors results in potent, water soluble compounds, but the aminopropyl group induces susceptibility to cellular efflux by P-glycoprotein (Pgp). We show that by carefully modulating the basicity of the amino group by beta-fluorination, this series of inhibitors maintains potency against KSP and has greatly improved efficacy in a Pgp-overexpressing cell line. The discovery that cellular efflux by Pgp can be overcome by carefully modulating the basicity of an amine may be of general use to medicinal chemists attempting to transform leading compounds into cancer cell- or CNS-penetrant drugs.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/physiology , Fluorine/metabolism , Kinesins/antagonists & inhibitors , Propylamines/pharmacology , Pyrroles/pharmacology , Biological Transport , Cytoskeleton , Hydrogen-Ion Concentration , Kinesins/metabolism , Solubility , WaterABSTRACT
The kinesin spindle protein (KSP), a microtubule motor protein, is essential for the formation of bipolar spindles during mitosis. Inhibition of KSP activates the spindle checkpoint and causes apoptosis. It was shown that prolonged inhibition of KSP activates Bax and caspase-3, which requires a competent spindle checkpoint and couples with mitotic slippage. Here we investigated how Bax is activated by KSP inhibition and the roles of Bax and p53 in KSP inhibitor-induced apoptosis. We demonstrate that small interfering RNA-mediated knockdown of Bax greatly attenuates KSP inhibitor-induced apoptosis and that Bax activation is upstream of caspase activation. This indicates that Bax mediates the lethality of KSP inhibitors and that KSP inhibition provokes apoptosis via the intrinsic apoptotic pathway where Bax activation is prior to caspase activation. Although the BH3-only protein Puma is induced after mitotic slippage, suppression of de novo protein synthesis that abrogates Puma induction does not block activation of Bax or caspase-3, indicating that Bax activation is triggered by a posttranslational event. Comparison of KSP inhibitor-induced apoptosis between matched cell lines containing either functional or deficient p53 reveals that inhibition of KSP induces apoptosis independently of p53 and that p53 is dispensable for spindle checkpoint function. Thus, KSP inhibitors should be active in p53-deficient tumors.
Subject(s)
Apoptosis Regulatory Proteins/biosynthesis , Apoptosis/physiology , Caspase 3/metabolism , Kinesins/physiology , Proto-Oncogene Proteins/biosynthesis , Tumor Suppressor Protein p53/physiology , bcl-2-Associated X Protein/biosynthesis , Antibiotics, Antineoplastic/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/drug effects , Cell Line, Tumor , Doxorubicin/pharmacology , Humans , Kinesins/antagonists & inhibitors , Paclitaxel/pharmacology , Protein Processing, Post-Translational , Signal Transduction , Spindle ApparatusABSTRACT
The development of 3-(indol-2-yl)indazoles as inhibitors of Chek1 kinase is described. Introduction of amides and heteroaryl groups at the C6 position of the indazole ring system provided sufficient Chek1 potency and selectivity over Cdk7 to permit escape from DNA damage-induced arrest in a cellular assay. Enzyme potency against Chek1 was optimized by the incorporation of a hydroxymethyl triazole moiety in compound 21 (Chek1 IC(50)=0.30nM) that was shown by X-ray crystallography to displace one of three highly conserved water molecules in the HI region of the ATP-binding cleft.
Subject(s)
Indazoles/chemistry , Indazoles/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Protein Kinases/chemistry , Protein Kinases/metabolism , Cell Cycle/drug effects , Cell Line, Tumor , Checkpoint Kinase 1 , Crystallography, X-Ray , Humans , Indazoles/metabolism , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/metabolism , Structure-Activity RelationshipABSTRACT
Molecular modeling in combination with X-ray crystallographic information was employed to identify a region of the kinesin spindle protein (KSP) binding site not fully utilized by our first generation inhibitors. We discovered that by appending a propylamine substituent at the C5 carbon of a dihydropyrazole core, we could effectively fill this unoccupied region of space and engage in a hydrogen-bonding interaction with the enzyme backbone. This change led to a second generation compound with increased potency, a 400-fold enhancement in aqueous solubility at pH 4, and improved dog pharmacokinetics relative to the first generation compound.
Subject(s)
Drug Design , Kinesins/antagonists & inhibitors , Pyrazoles/chemistry , Pyrazoles/pharmacology , Alkylation , Allosteric Site , Amination , Animals , Crystallography, X-Ray , Dogs , Hydroxylation , Kinesins/chemistry , Kinesins/metabolism , Mitosis , Models, Molecular , Molecular Structure , Pyrazoles/chemical synthesis , Pyrazoles/pharmacokinetics , Solubility , Structure-Activity Relationship , WaterABSTRACT
2,4-Diaryl-2,5-dihydropyrroles have been discovered to be novel, potent and water-soluble inhibitors of KSP, an emerging therapeutic target for the treatment of cancer. A potential concern for these basic KSP inhibitors (1 and 2) was hERG binding that can be minimized by incorporation of a potency-enhancing C2 phenol combined with neutral N1 side chains. Aqueous solubility was restored to these, and other, non-basic inhibitors, through a phosphate prodrug strategy.
Subject(s)
Ether-A-Go-Go Potassium Channels/metabolism , Kinesins/antagonists & inhibitors , Prodrugs , Pyrroles/chemical synthesis , Pyrroles/pharmacology , Animals , Area Under Curve , Dogs , Protein Binding , Pyrroles/metabolism , Pyrroles/pharmacokinetics , Rats , Solubility , Spindle Apparatus/chemistry , WaterABSTRACT
The evolution of 2,4-diaryl-2,5-dihydropyrroles as inhibitors of KSP is described. Introduction of basic amide and urea moieties to the dihydropyrrole nucleus enhanced potency and aqueous solubility, simultaneously, and provided compounds that caused mitotic arrest of A2780 human ovarian carcinoma cells with EC(50)s<10nM. Ancillary hERG activity was evaluated for this series of inhibitors.
Subject(s)
Kinesins/antagonists & inhibitors , Pyrroles/chemistry , Pyrroles/pharmacology , Cell Line, Tumor , Female , Humans , Models, Molecular , Ovarian Neoplasms/pathology , Pyrroles/chemical synthesis , Spindle Apparatus/chemistryABSTRACT
The inhibition of KSP causes mitotic arrest by activating the spindle assembly checkpoint. While transient inhibition of KSP leads to reversible mitotic arrest, prolonged exposure to a KSP inhibitor induces apoptosis. Induction of apoptosis by the KSP inhibitor couples with mitotic slippage. Slippage-refractory cells show resistance to KSP inhibitor-mediated lethality, whereas promotion of slippage after mitotic arrest enhances apoptosis. However, attenuation of the spindle checkpoint confers resistance to KSP inhibitor-induced apoptosis. Furthermore, sustained KSP inhibition activates the proapoptotic protein, Bax, and both activation of the spindle checkpoint and subsequent mitotic slippage are required for Bax activation. These studies indicate that in response to KSP inhibition, activation of the spindle checkpoint followed by mitotic slippage initiates apoptosis by activating Bax.
Subject(s)
Apoptosis , Genes, cdc/physiology , Kinesins/antagonists & inhibitors , Mitosis/drug effects , Proto-Oncogene Proteins c-bcl-2/metabolism , Spindle Apparatus/physiology , Caspase 3 , Caspases/metabolism , Cell Cycle Proteins/metabolism , Drug Resistance , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , HeLa Cells , Humans , Kinesins/genetics , Kinesins/metabolism , Molecular Structure , Protein Kinases/metabolism , Protein Serine-Threonine Kinases , Pyrroles/pharmacology , bcl-2-Associated X ProteinABSTRACT
A series of macrocyclic piperazinone compounds with dual farnesyltransferase/geranylgeranyltransferase-I inhibitory activity was prepared. These compounds were found to be potent inhibitors of protein prenylation in cell culture. A hypothesis for the binding mode of compound 3o in FPTase is proposed.
Subject(s)
Alkyl and Aryl Transferases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Heterocyclic Compounds/pharmacology , Piperazines/pharmacology , Tumor Cells, Cultured/drug effects , Enzyme Inhibitors/chemistry , Heterocyclic Compounds/chemistry , Humans , Inhibitory Concentration 50 , Molecular Structure , Piperazines/chemistry , Protein Prenylation , Structure-Activity Relationship , Tumor Cells, Cultured/enzymologyABSTRACT
Farnesyl:protein transferase (FPTase) catalyzes the covalent addition of the isoprenyl moiety of farnesylpyrophosphate to the C-terminus of the Ras oncoprotein and other cellular proteins. Inhibitors of FPTase (FTIs) have been developed as potential anticancer agents, and several compounds have been evaluated in clinical trials. To facilitate the identification of cell-active FTIs with high potency, the authors developed a method that uses a radiolabeled FTI that serves as a ligand in competitive displacement assays. Using high-affinity [(3)H]-labeled or [(125)I]-labeled FTI radioligands, they show that specific binding to FPTase can be detected in intact cells. Binding of these labeled FTI radioligands can be competed with a variety of structurally diverse FTIs, and the authors show that inhibition of FTI radioligand binding correlates well with inhibition of FPTase substrate prenylation in cells. This method provides a rapid and quantitative means of assessing FTI potency in cells and is useful for guiding the discovery of potent, novel inhibitors of FPTase. Similar methods could be employed in the optimization of inhibitors for other intracellular drug targets.
Subject(s)
Alkyl and Aryl Transferases/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Drug Screening Assays, Antitumor/methods , Enzyme Inhibitors/pharmacology , Radioligand Assay , Alkyl and Aryl Transferases/metabolism , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Cell Line, Transformed , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Farnesyltranstransferase , Humans , KB Cells , Rats , Sensitivity and SpecificityABSTRACT
A series of novel diaryl ether lactams have been identified as very potent dual inhibitors of protein farnesyltransferase (FTase) and protein geranylgeranyltransferase I (GGTase-I), enzymes involved in the prenylation of Ras. The structure of the complex formed between one of these compounds and FTase has been determined by X-ray crystallography. These compounds are the first reported to inhibit the prenylation of the important oncogene Ki-Ras4B in vivo. Unfortunately, doses sufficient to achieve this endpoint were rapidly lethal.
Subject(s)
Alkyl and Aryl Transferases/antagonists & inhibitors , Antineoplastic Agents/chemical synthesis , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Carrier Proteins/metabolism , Crystallography, X-Ray , Drug Screening Assays, Antitumor , HSP40 Heat-Shock Proteins , Heat-Shock Proteins/metabolism , Humans , Mice , Mice, Nude , Models, Molecular , Neoplasm Transplantation , Neoplasms, Experimental/drug therapy , Protein Prenylation , Structure-Activity Relationship , Transplantation, Heterologous , Tumor Cells, Cultured , rap1 GTP-Binding Proteins/metabolism , ras Proteins/metabolismABSTRACT
Farnesyl:protein transferase (FPTase) inhibitors were developed as anti-Ras drugs, but they fail to inhibit Ki-Ras activity because Ki-Ras can be modified by geranylgeranyl:protein transferase type-I (GGPTase-I). L-778,123, an inhibitor of FPTase and GGPTase-I, was developed in part because it can completely inhibit Ki-Ras prenylation. To support the clinical development of L-778,123, we developed pharmacodynamic assays using peripheral blood mononuclear cells (PBMCs) to measure the inhibition of prenylation of HDJ2 and Rap1A, proteins that are FPTase- and GGPTase-I substrates, respectively. We validated these assays in animal models and show that inhibition of HDJ2 prenylation in mouse PBMCs correlates with the concentration of FPTase inhibitors in blood. In dogs, continuous infusion of L-778,123 inhibited both HDJ2 and Rap1A prenylation in PBMCs, but we did not detect inhibition of Ki-Ras prenylation. We reported previously results from the first L-778,123 Phase I trial that showed a dose-dependent inhibition of HDJ2 farnesylation in PBMCs. In this report, we present additional analysis of patient samples from this trial and a second Phase I trial of L-778,123, and demonstrate the inhibition of both HDJ2 and Rap1A prenylation in PBMC samples. This study represents the first demonstration of GGPTase-I inhibition in humans. However, no inhibition of Ki-Ras prenylation by L-778,123 was detected in patient samples. These results confirm the pharmacologic profile of L-778,123 in humans as a dual inhibitor of FPTase and GGPTase-I, but indicate that the intended target of the drug, Ki-Ras, was not inhibited.
Subject(s)
Alkyl and Aryl Transferases/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Imidazoles/pharmacology , Animals , Dogs , Dose-Response Relationship, Drug , Humans , Immunoblotting , Leukocytes/drug effects , Leukocytes, Mononuclear/drug effects , Mice , Models, Chemical , Proto-Oncogene Proteins p21(ras)/metabolism , Time Factors , rap1 GTP-Binding Proteins/metabolismABSTRACT
We have prepared a series of potent, dual inhibitors of the prenyl transferases farnesyl protein transferase (FPTase) and geranyl-geranyl protein transferase I (GGPTase). The compounds were shown to possess potent activity against both enzymes in cell culture. Mechanistic analysis has shown that the compounds are CAAX competitive for FPTase inhibition but geranyl-geranyl pyrophosphate (GGPP) competitive for GGPTase inhibiton.