Preclinical characterization of ABT-348, a kinase inhibitor targeting the aurora, vascular endothelial growth factor receptor/platelet-derived growth factor receptor, and Src kinase families.

ABT-348 [1-(4-(4-amino-7-(1-(2-hydroxyethyl)-1H-pyrazol-4-yl)thieno[3,2-c]pyridin-3-yl)phenyl)-3-(3-fluorophenyl)urea] is a novel ATP-competitive multitargeted kinase inhibitor with nanomolar potency (IC(50)) for inhibiting binding and cellular autophosphorylation of Aurora B (7 and 13 nM), C (1 and 13 nM), and A (120 and 189 nM). Cellular activity against Aurora B is reflected by inhibition of phosphorylation of histone H3, induction of polyploidy, and inhibition of proliferation of a variety of leukemia, lymphoma, and solid tumor cell lines (IC(50) = 0.3-21 nM). In vivo inhibition of Aurora B was confirmed in an engrafted leukemia model by observing a decrease in phosphorylation of histone H3 that persisted in a dose-dependent manner for 8 h and correlated with plasma concentration of ABT-348. Evaluation of ABT-348 across a panel of 128 kinases revealed additional potent binding activity (K(i) < 30 nM) against vascular endothelial growth factor receptor (VEGFR)/platelet-derived growth factor receptor (PDGFR) families and the Src family of cytoplasmic tyrosine kinases. VEGFR/PDGFR binding activity correlated with inhibition of autophosphorylation in cells and inhibition of vascular endothelial growth factor (VEGF)-stimulated endothelial cell proliferation (IC(50) ≤ 0.3 nM). Evidence of on-target activity in vivo was provided by the potency for blocking VEGF-mediated vascular permeability and inducing plasma placental growth factor. Activity against the Src kinase family was evident in antiproliferative activity against BCR-ABL chronic myeloid leukemia cells and cells expressing the gleevec-resistant BCR-ABL T315I mutation. On the basis of its unique spectrum of activity, ABT-348 was evaluated and found effective in representative solid tumor [HT1080 and pancreatic carcinoma (MiaPaCa), tumor stasis] and hematological malignancy (RS4;11, regression) xenografts. These results provide the rationale for clinical assessment of ABT-348 as a therapeutic agent in the treatment of cancer.

Pyrazole diaminopyrimidines as dual inhibitors of KDR and Aurora B kinases.

In an effort to identify kinase inhibitors with dual KDR/Aurora B activity and improved aqueous solubility compared to the Abbott dual inhibitor ABT-348, a series of novel pyrazole pyrimidines structurally related to kinase inhibitor AS703569 were prepared. SAR work provided analogs with significant cellular activity, measureable aqueous solubility and moderate antitumor activity in a mouse tumor model after weekly ip dosing. Unfortunately these compounds were pan-kinase inhibitors that suffered from narrow therapeutic indices which prohibited their use as antitumor agents.

Exploration of diverse hinge-binding scaffolds for selective Aurora kinase inhibitors.

Four hinge-binding scaffolds have been explored for novel selective Aurora kinase inhibitors. The structure activity relationship, selectivity and pharmacokinetic profiles have been evaluated.

Thienopyridine ureas as dual inhibitors of the VEGF and Aurora kinase families.

In an effort to identify multi-targeted kinase inhibitors with a novel spectrum of kinase activity, a screen of Abbott proprietary KDR inhibitors against a broad panel of kinases was conducted and revealed a series of thienopyridine ureas with promising activity against the Aurora kinases. Modification of the diphenyl urea and C7 moiety of these compounds provided potent inhibitors with good pharmacokinetic profiles that were efficacious in mouse tumor models after oral dosing. Compound 2 (ABT-348) of this series is currently undergoing Phase I clinical trials in solid and hematological cancer populations.

Discovery of potent and selective thienopyrimidine inhibitors of Aurora kinases.

In an effort to discover Aurora kinase inhibitors, an HTS hit revealed an amide containing pyrrolopyrimidine compound. Replacement of the pyrrolopyrimidine residue with a thienopyrimidine moiety led to a series of potent and selective Aurora inhibitors.

Synthesis and evaluation of a new generation of orally efficacious benzimidazole-based poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors as anticancer agents.

Small molecule inhibitors of PARP-1 have been pursued by various organizations as potential therapeutic agents either capable of sensitizing cytotoxic treatments or acting as stand-alone agents to combat cancer. As one of the strategies to expand our portfolio of PARP-1 inhibitors, we pursued unsaturated heterocycles to replace the saturated cyclic amine derivatives appended to the benzimidazole core. Not only did a variety of these new generation compounds maintain high enzymatic potency, many of them also displayed robust cellular activity. For example, the enzymatic IC(50) and cellular EC(50) values were as low as 1 nM or below. Compounds 24 (EC(50) = 3.7 nM) and 44 (EC(50) = 7.8 nM), featuring an oxadiazole and a pyridine moiety, respectively, demonstrated balanced potency and PK profiles. In addition, these two molecules exhibited potent oral in vivo efficacy in potentiating the cytotoxic agent temozolomide in a B16F10 murine melanoma model.

Scaffold oriented synthesis. Part 2: Design, synthesis and biological evaluation of pyrimido-diazepines as receptor tyrosine kinase inhibitors.

We report the discovery of the pyrimido-diazepine scaffolds as novel adenine mimics. Structure-based design led to the discovery of analogs with potent inhibitory activity against receptor tyrosine kinases, such as KDR, Flt3 and c-Kit. Compound 14 exhibited low nanomolar KDR enzymatic and cellular potencies (IC(50)=9 and 52 nM, respectively).

3-amino-benzo[d]isoxazoles as novel multitargeted inhibitors of receptor tyrosine kinases.

A series of benzoisoxazoles and benzoisothiazoles have been synthesized and evaluated as inhibitors of receptor tyrosine kinases (RTKs). Structure-activity relationship studies led to the identification of 3-amino benzo[ d]isoxazoles, incorporating a N, N'-diphenyl urea moiety at the 4-position that potently inhibited both the vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor families of RTKs. Within this series, orally bioavailable compounds possessing promising pharmacokinetic profiles were identified, and a number of compounds demonstrated in vivo efficacy in models of VEGF-stimulated vascular permeability and tumor growth. In particular, compound 50 exhibited an ED 50 of 2.0 mg/kg in the VEGF-stimulated uterine edema model and 81% inhibition in the human fibrosarcoma (HT1080) tumor growth model when given orally at a dose of 10 mg/kg/day.

Identification of aminopyrazolopyridine ureas as potent VEGFR/PDGFR multitargeted kinase inhibitors.

Tumor angiogenesis is mediated by KDR and other VEGFR and PDGFR kinases. Their inhibition presents an attractive approach for developing anticancer therapeutics. Here, we report a series of aminopyrazolopyridine ureas as potent VEGFR/PDGFR multitargeted kinase inhibitors. A number of compounds have been identified to be orally bioavailable and efficacious in the mouse edema model.

1,4-Dihydroindeno[1,2-c]pyrazoles with acetylenic side chains as novel and potent multitargeted receptor tyrosine kinase inhibitors with low affinity for the hERG ion channel.

The synthesis of a novel series of 1,4-dihydroindeno[1,2-c]pyrazoles with acetylene-type side chains is described. Optimization of those compounds as KDR kinase inhibitors identified 8, which displayed an oral activity in an estradiol-induced murine uterine edema model (ED50 = 3 mg/kg) superior to Sutent (ED50 = 9 mg/kg) and showed potent antitumor efficacy in an MX-1 human breast carcinoma xenograft tumor growth model (tumor growth inhibition = 90% at 25 mg/kg.day po). The compound was docked into a homology model of the homo-tetrameric pore domain of the hERG potassium channel to identify strategies to improve its cardiac safety profile. Systematic interruption of key binding interactions between 8 and Phe656, Tyr652, and Ser624 yielded 90, which only showed an IC50 of 11.6 microM in the hERG patch clamp assay. The selectivity profile for 8 and 90 revealed that both compounds are multitargeted receptor tyrosine kinase inhibitors with low nanomolar potencies against the members of the VEGFR and PDGFR kinase subfamilies.

Discovery of N-(4-(3-amino-1H-indazol-4-yl)phenyl)-N'-(2-fluoro-5-methylphenyl)urea (ABT-869), a 3-aminoindazole-based orally active multitargeted receptor tyrosine kinase inhibitor.

In our continued efforts to search for potent and novel receptor tyrosine kinase (RTK) inhibitors as potential anticancer agents, we discovered, through a structure-based design, that 3-aminoindazole could serve as an efficient hinge-binding template for kinase inhibitors. By incorporating an N,N'-diaryl urea moiety at the C4-position of 3-aminodazole, a series of RTK inhibitors were generated, which potently inhibited the tyrosine kinase activity of the vascular endothelial growth factor receptor and the platelet-derived growth factor receptor families. A number of compounds with potent oral activity were identified by utilizing an estradiol-induced mouse uterine edema model and an HT1080 human fibrosarcoma xenograft tumor model. In particular, compound 17p (ABT-869) was found to possess favorable pharmacokinetic profiles across different species and display significant tumor growth inhibition in multiple preclinical animal models.

ABT-869, a multitargeted receptor tyrosine kinase inhibitor: inhibition of FLT3 phosphorylation and signaling in acute myeloid leukemia.

In 15% to 30% of patients with acute myeloid leukemia (AML), aberrant proliferation is a consequence of a juxtamembrane mutation in the FLT3 gene (FMS-like tyrosine kinase 3-internal tandem duplication [FLT3-ITD]), causing constitutive kinase activity. ABT-869 (a multitargeted receptor tyrosine kinase inhibitor) inhibited the phosphorylation of FLT3, STAT5, and ERK, as well as Pim-1 expression in MV-4-11 and MOLM-13 cells (IC(50) approximately 1-10 nM) harboring the FLT3-ITD. ABT-869 inhibited the proliferation of these cells (IC(50) = 4 and 6 nM, respectively) through the induction of apoptosis (increased sub-G(0)/G(1) phase, caspase activation, and PARP cleavage), whereas cells harboring wild-type (wt)-FLT3 were less sensitive. In normal human blood spiked with AML cells, ABT-869 inhibited phosphorylation of FLT3 (IC(50) approximately 100 nM), STAT5, and ERK, and decreased Pim-1 expression. In methylcellulose-based colony-forming assays, ABT-869 had no significant effect up to 1000 nM on normal hematopoietic progenitor cells, whereas in AML patient samples harboring both FLT3-ITD and wt-FLT3, ABT-869 inhibited colony formation (IC(50) = 100 and 1000 nM, respectively). ABT-869 dose-dependently inhibited MV-4-11 and MOLM-13 flank tumor growth, prevented tumor formation, regressed established MV-4-11 xenografts, and increased survival by 20 weeks in an MV-4-11 engraftment model. In tumors, ABT-869 inhibited FLT3 phosphorylation, induced apoptosis (transferase-mediated dUTP nick-end labeling [TUNEL]) and decreased proliferation (Ki67). ABT-869 is under clinical development for AML.

Thienopyridine urea inhibitors of KDR kinase.

A series of substituted thienopyridine ureas was prepared and evaluated for enzymatic and cellular inhibition of KDR kinase activity. Several of these analogs, such as 2, are potent inhibitors of KDR (<10 nM) in both enzymatic and cellular assays. Further characterization of inhibitor 2 indicated that this analog possessed excellent in vivo potency (ED50 2.1 mg/kg) as measured in an estradiol-induced mouse uterine edema model.

Isothiazolopyrimidines and isoxazolopyrimidines as novel multi-targeted inhibitors of receptor tyrosine kinases.

A series of isothiazolopyrimidines and isoxazolopyrimidines were synthesized and identified as potent KDR inhibitors. SAR studies led to isothiazolopyrimidine urea analogs that potently inhibit VEGFR tyrosine kinases (KDR enzymatic and cellular IC(50) values below 10 nM) as well as cKIT and TIE2. The selected compounds 8 and 13 display 56% and 48% oral bioavailability in mice, respectively.

Preclinical activity of ABT-869, a multitargeted receptor tyrosine kinase inhibitor.

ABT-869 is a structurally novel, receptor tyrosine kinase (RTK) inhibitor that is a potent inhibitor of members of the vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) receptor families (e.g., KDR IC50 = 4 nmol/L) but has much less activity (IC50s > 1 micromol/L) against unrelated RTKs, soluble tyrosine kinases, or serine/threonine kinases. The inhibition profile of ABT-869 is evident in cellular assays of RTK phosphorylation (IC50 = 2, 4, and 7 nmol/L for PDGFR-beta, KDR, and CSF-1R, respectively) and VEGF-stimulated proliferation (IC50 = 0.2 nmol/L for human endothelial cells). ABT-869 is not a general antiproliferative agent because, in most cancer cells, >1,000-fold higher concentrations of ABT-869 are required for inhibition of proliferation. However, ABT-869 exhibits potent antiproliferative and apoptotic effects on cancer cells whose proliferation is dependent on mutant kinases, such as FLT3. In vivo ABT-869 is effective orally in the mechanism-based murine models of VEGF-induced uterine edema (ED50 = 0.5 mg/kg) and corneal angiogenesis (>50% inhibition, 15 mg/kg). In tumor growth studies, ABT-869 exhibits efficacy in human fibrosarcoma and breast, colon, and small cell lung carcinoma xenograft models (ED50 = 1.5-5 mg/kg, twice daily) and is also effective (>50% inhibition) in orthotopic breast and glioma models. Reduction in tumor size and tumor regression was observed in epidermoid carcinoma and leukemia xenograft models, respectively. In combination, ABT-869 produced at least additive effects when given with cytotoxic therapies. Based on pharmacokinetic analysis from tumor growth studies, efficacy correlated more strongly with time over a threshold value (cellular KDR IC50 corrected for plasma protein binding = 0.08 microg/mL, >or=7 hours) than with plasma area under the curve or Cmax. These results support clinical assessment of ABT-869 as a therapeutic agent for cancer.

Inhibition of phosphorylation of the colony-stimulating factor-1 receptor (c-Fms) tyrosine kinase in transfected cells by ABT-869 and other tyrosine kinase inhibitors.

The properties of several multitargeted receptor tyrosine kinase inhibitors have been studied for their inhibition of colony-stimulating factor-1 receptor (CSF-1R) signaling. A structurally novel, multitargeted tyrosine kinase inhibitor (ABT-869), imatinib (STI571), and four compounds currently in clinical development (AG013736, BAY 43-9006, CHIR258, and SU11248) were tested for inhibition of CSF-1R signaling in both the enzymatic and cellular assays. ABT-869 showed potent CSF-1R inhibition in both the enzyme and cell-based assays (IC50s < 20 nmol/L). In contrast to a previous report, we have found that imatinib has activity against human CSF-1R in both assays at submicromolar concentrations. In enzyme assays, we have found that the inhibition of CSF-1R by both ABT-869 and imatinib are competitive with ATP, with Ki values of 3 and 120 nmol/L, respectively. SU11248 is a potent inhibitor of CSF-1R in the enzyme assay (IC50 = 7 nmol/L) and inhibits receptor phosphorylation in the cellular assay (IC50 = 61 nmol/L). AG013736 was also a potent inhibitor of CSF-1R in both assays (enzyme, IC50 = 16 nmol/L; cellular, IC50 = 21 nmol/L), whereas BAY 43-9006 is less potent in the enzyme assay (IC50 = 107 nmol/L) than in the cellular system (IC50 = 20 nmol/L). In contrast, we found that CHIR258 had less activity in the cellular assay (IC50 = 535 nmol/L) relative to its enzymatic potency (IC50 = 26 nmol/L). These results show the use of a cell-based assay to confirm the inhibitory activity of lead compounds and drug candidates, such as ABT-869, against the CSF-1R protein in situ.

High-level bacterial expression and purification of human SirT2 protein for NMR studies.

Silent information regulator 2 (Sir2) proteins are a class of protein deacetylase enzymes that play key roles in transcriptional gene silencing, DNA repair, and aging. Here, we describe the high-level bacterial expression and purification of a human SirT2 construct that yields high resolution NMR spectra. By removing the N-terminal helix alpha0 and using Thioredoxin as a fusion partner, greater than 10 mg/L of purified protein can be obtained from minimal media. The protein is fully functional and enables NMR-based screening and structural studies of this important protein.

Thienopyrimidine ureas as novel and potent multitargeted receptor tyrosine kinase inhibitors.

A series of novel thienopyrimidine-based receptor tyrosine kinase inhibitors has been discovered. Investigation of structure-activity relationships at the 5- and 6-positions of the thienopyrimidine nucleus led to a series of N,N'-diaryl ureas that potently inhibit all of the vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) receptor tyrosine kinases. A kinase insert domain-containing receptor (KDR) homology model suggests that these compounds bind to the "inactive conformation" of the enzyme with the urea portion extending into the back hydrophobic pocket adjacent to the adenosine 5'-triphosphate (ATP) binding site. A number of compounds have been identified as displaying excellent in vivo potency. In particular, compounds 28 and 76 possess favorable pharmacokinetic (PK) profiles and demonstrate potent antitumor efficacy against the HT1080 human fibrosarcoma xenograft tumor growth model (tumor growth inhibition (TGI) = 75% at 25 mg/kg.day, per os (po)).

Fluorescence assay of SIRT protein deacetylases using an acetylated peptide substrate and a secondary trypsin reaction.

A novel fluorescent substrate was devised for the sirtuin (SIRT) class of human protein deacetylases comprised of a peptide sequence containing a single acetyl-lysine residue, with a fluorescent group (tetramethylrhodamine-6-carboxylic acid, 6-TAMRA) near the carboxyl terminus and a nonfluorescent quenching group (QSY-7) near the amino terminus. The peptide sequence is modeled after the p53 acetylation site but is unreactive toward trypsin because all other lysine and arginine residues have been replaced by serine. However, the SIRT-deacetylated peptide is readily cleaved by trypsin, resulting in a maximal 30-fold enhancement of the 6-TAMRA fluorescence. Nicotinamide at millimolar concentrations stops the deacetylation but does not inhibit trypsin, and a microtiter plate assay of the SIRTs has been devised using the fluorescent substrate and these reagents. Using this method, the kinetics of the reaction of the cosubstrate nicotinamide adenine dinucleotide and the competitive inhibitor nicotinamide with SIRT1 and SIRT2 has been analyzed. Several nicotinamide analogs have also been tested as inhibitors and found to have much lower affinity for these enzymes than does the parent compound.

Heterocyclic ketones as inhibitors of histone deacetylase.

Several heterocyclic ketones were investigated as potential inhibitors of histone deacetylase. Nanomolar inhibitors such as 22 and 25 were obtained, the anti-proliferative activity of which were shown to be mediated by HDAC inhibition.