XMD-17-51 Inhibits DCLK1 Kinase and Prevents Lung Cancer Progression.

Doublecortin-like kinase 1 (DCLK1) is a cancer stem cell marker that is highly expressed in various types of human cancer, and a protein kinase target for cancer therapy that is attracting increasing interest. However, no drug candidates targeting DCLK1 kinase have been developed in clinical trials to date. XMD-17-51 was found herein to possess DCLK1 kinase inhibitory activities by cell-free enzymatic assay. In non-small cell lung carcinoma (NSCLC) cells, XMD-17-51 inhibited DCLK1 and cell proliferation, while DCLK1 overexpression impaired the anti-proliferative activity of XMD-17-51 in A549 cell lines. Consequently, XMD-17-51 decreased Snail-1 and zinc-finger-enhancer binding protein 1 protein levels, but increased those of E-cadherin, indicating that XMD-17-51 reduces epithelial-mesenchymal transition (EMT). Furthermore, sphere formation efficiency was significantly decreased upon XMD-17-51 treatment, and XMD-17-51 reduced the expression of stemness markers such as ß-catenin, and pluripotency factors such as SOX2, NANOG and OCT4. However, the percentage of ALDH+ cells was increased significantly following treatment with XMD-17-51 in A549 cells, possibly due to EMT inhibition. In combination, the present data indicated that XMD-17-51 inhibited DCLK1 kinase activity in a cell-free assay with an IC50 of 14.64 nM, and decreased DCLK1 protein levels, cell proliferation, EMT and stemness in NSCLC cell lines. XMD-17-51 has the potential to be a candidate drug for lung cancer therapy.

Imaging Mutant Huntingtin Aggregates: Development of a Potential PET Ligand.

Mutant huntingtin (mHTT) protein carrying the elongated N-terminal polyglutamine (polyQ) tract misfolds and forms protein aggregates characteristic of Huntington's disease (HD) pathology. A high-affinity ligand specific for mHTT aggregates could serve as a positron emission tomography (PET) imaging biomarker for HD therapeutic development and disease progression. To identify such compounds with binding affinity for polyQ aggregates, we embarked on systematic structural activity studies; lead optimization of aggregate-binding affinity, unbound fractions in brain, permeability, and low efflux culminated in the discovery of compound 1, which exhibited target engagement in autoradiography (ARG) studies in brain slices from HD mouse models and postmortem human HD samples. PET imaging studies with 11C-labeled 1 in both HD mice and WT nonhuman primates (NHPs) demonstrated that the right-hand-side labeled ligand [11C]-1R (CHDI-180R) is a suitable PET tracer for imaging of mHTT aggregates. [11C]-1R is now being advanced to human trials as a first-in-class HD PET radiotracer.

3,4-Disubstituted isothiazoles: novel potent inhibitors of VEGF receptors 1 and 2.

Novel derivatives of isothiazoles are described as potent ATP-competitive inhibitors of vascular endothelial growth factor receptors I and II (VEGFR-1/2). A number of compounds exhibited VEGFR-2 inhibitory activity comparable to that of Vatalanib in both HTRF enzymatic and cellular assays. Several derivatives featuring bulky meta-substituents in the amide portion of the molecule displayed 4- to 8-fold specificity for VEGFR-2 versus VEGFR-1. Active molecules also showed high intrinsic permeability (> 30 x 10(-5) cm/min) across Caco-2 cell monolayer.

(1,2,3-Triazol-4-yl)benzenamines: synthesis and activity against VEGF receptors 1 and 2.

Derivatives of (1,2,3-triazol-4-yl)benzenamines are described as potent and ATP-competitive inhibitors of vascular endothelial growth factor receptors I and II (VEGFR-1/2). A number of compounds exhibited VEGFR-2 and VEGFR-1 inhibitory activity comparable to that of Vatalanib in both HTRF enzymatic and cellular assays.

Arylphthalazines as potent, and orally bioavailable inhibitors of VEGFR-2.

A series of arylphthalazine derivatives were synthesized and evaluated as antagonists of VEGF receptor II (VEGFR-2). IM-094482 57, which was prepared in two steps from commercially available starting materials, was found to be a potent inhibitor of VEGFR-2 in enzymatic, cellular and mitogenic assays (comparable activity to ZD-6474). Additionally, 57 inhibited the related receptor, VEGF receptor I (VEGFR-1), and showed excellent exposure when dosed orally to female CD-1 mice.

Synthesis and evaluation of heteroaryl-ketone derivatives as a novel class of VEGFR-2 inhibitors.

We have discovered novel inhibitors of VEGFR-2 kinase with low nanomolar potency in both enzymatic and cell-based assays. Active series are heteroaryl-ketone compounds containing a central aromatic ring with either an indazolyl or indolyl keto group in the ortho orientation to the benzylic amine group (Fig. 1). The best compounds were demonstrated to be inactive against a small select panel of tyrosine and serine/threonine kinases with the exception of VEGFR-1 kinase, a close family member. In addition, the lead candidate 8 displayed acceptable exposure levels when administered orally to mice.

Sea urchin embryo as a model organism for the rapid functional screening of tubulin modulators.

Identification of antimitotic molecules that affect tubulin dynamics is a multistep procedure. It includes in vitro tubulin polymerization assay, studies of a cell cycle effect, and general cytotoxicity assessment. To simplify this lengthy screening protocol, we have introduced and validated an assay system based on the sea urchin embryos. The proposed two-step procedure involves the fertilized egg test for mitotic arrest and the behavioral assessment of a free-swimming blastula. In order to validate the assay, we have analyzed the effect of a panel of known antiproliferative agents on the sea urchin embryo. For all tubulin destabilizing drugs, we observed rapid spinning and lack of forward movement of an embryo. Both effects are likely to result from the in vivo microtubule disassembly caused by test molecules. Notably, the described assay yields rapid information on antiproliferative, antimitotic, cytotoxic, and tubulin destabilizing activities of the molecules along with their solubility and permeability potential. Moreover, measured potencies of the test articles correlated well with the reported values in both in vitro and cell based assays.

Synthesis of polysubstituted 4-fluoroquinolinones.

[reaction: see text] A convenient one-pot synthesis of 4-fluoroquinolinones that are active against KDR kinase is described. The mechanism of the reaction is believed to involve the formation of a quinone methide intermediate.

Discovery of Dual VEGFR-2 and Tubulin Inhibitors with in Vivo Efficacy.

In an effort to develop potent, orally bioavailable compounds for the treatment of neoplastic diseases, we developed a class of dual VEGFR-2 kinase and tubulin inhibitors. Targeting the VEGFR receptor kinase and tubulin structure allows for inhibition of both tumor cells and tumor vasculature. Previously, a combination of two compounds, a VEGF receptor tyrosine kinase inhibitor and tubulin agent, was demonstrated to produce an enhanced antitumor response in animal studies. We have reaffirmed their results, with the added benefit that both activities are found in one compound.

Novel inhibitors of VEGF receptors-1 and -2 based on azole-5-carboxamide templates.

We have developed a series of novel potent 1-(2-(pyridin-4-yl)ethyl)-1H-azole-5-carboxamides active against kinases VEGFR-2 and -1. Both specific and dual ATP-competitive inhibitors of VEGFR-2 were identified. Kinase selectivity could be controlled by varying the 5-carboxamide substituent at the azole ring. The most specific molecules displayed >10-fold selectivity for VEGFR-2 over VEGFR-1. Compound activities in vitro and in cell-based assays (IC(50)<100 nM) were similar to those of reported clinical and development candidates, including PTK787 (Vatalanib(trade)) and ZD6474 (Vandetanib(trade mark)). High permeability of active compounds across the Caco-2 cell monolayer (>40 x 10(-5)cm/min) is indicative of their potential for intestinal absorption upon oral administration.

1H-1,2,4-triazol-3-yl-anilines: novel potent inhibitors of vascular endothelial growth factor receptors 1 and 2.

Novel derivatives of 1,2,4-triazoles are described as potent ATP-competitive inhibitors of vascular endothelial growth factor receptors I and II (VEGFR-1/2). A number of compounds display VEGFR-2 inhibitory activity comparable to that of Vatalanib and Vandetanib in both homogenous time-resolved fluorescence enzymatic and cellular assays. Several active molecules feature high intrinsic permeability (>30 x 10(-5) cm/min) across Caco-2 cell monolayer.

ortho-Substituted azoles as selective and dual inhibitors of VEGF receptors 1 and 2.

We have developed a series of novel potent ortho-substituted azole derivatives active against kinases VEGFR-1 and VEGFR-2. Both specific and dual ATP-competitive inhibitors of VEGFR-2 were identified. Kinase activity and selectivity could be controlled by varying the arylamido substituents at the azole ring. The most specific molecule (17) displayed > 10-fold selectivity for VEGFR-2 over VEGFR-1. Compound activities in enzymatic and cell-based assays were in the range of activities for reported clinical and development candidates (IC50 < 100 nM), including Novartis' PTK787 (Vatalanib). High permeability of active compounds across the Caco-2 cell monolayer (> 30x10(-5) cm/min) is indicative of their potential for intestinal absorption upon oral administration.

2-((1H-Azol-1-yl)methyl)-N-arylbenzamides: novel dual inhibitors of VEGFR-1/2 kinases.

Novel potent derivatives of (azol-1-yl)methyl-N-arylbenzamides with improved solubility (>3mM) are described as ATP-competitive inhibitors of vascular endothelial growth factor receptor 2 (VEGFR-2). Many compounds display VEGFR-2 inhibitory activity reaching IC(50)<100 nM in the enzymatic assay. The compounds also inhibit the related tyrosine kinase, VEGFR-1, with similar potencies. Several compounds containing bulky lipophilic substituents at the benzamide pharmacophore yielded 10- to 17-fold selectivity for the VEGFR-2 versus VEGFR-1 kinase.

N-(Aryl)-4-(azolylethyl)thiazole-5-carboxamides: novel potent inhibitors of VEGF receptors I and II.

Novel potent derivatives of N-(aryl)-4-(azolylethyl)thiazole-5-carboxamides are described as inhibitors of vascular endothelial growth factor receptor II (VEGFR-2). Several compounds display VEGFR-2 inhibitory activity reaching IC(50)<100 nM in both enzymatic and cellular assays. The compounds also inhibit the related tyrosine kinase, VEGFR-1. By controlling the substitution pattern on the 5-carboxamido pharmacophore, both dual and specific VEGFR-2 thiazoles were identified.

4-(Azolylphenyl)-phthalazin-1-amines: Novel inhibitors of VEGF receptors I and II.

Novel potent derivatives of phthalazine are described as ATP-competitive inhibitors of vascular endothelial growth factor receptors I and II (VEGFR-1/2). A number of compounds display VEGFR-2 inhibitory activity reaching that of Vatalanib 3 (IC50 < 100 nm) in an HTRF enzymatic assay. Several derivatives also show good potential for the development as VEGFR-2 specific inhibitors showing 15-20-fold selectivity over VEGFR-1.

Inhibitors of VEGF receptors-1 and -2 based on the 2-((pyridin-4-yl)ethyl)pyridine template.

We have developed a series of novel potent ((pyridin-4-yl)ethyl)pyridine derivatives active against kinases VEGFR-1 and -2. Both specific and dual ATP-competitive inhibitors of VEGFR-2 were identified. Kinase selectivity could be controlled by varying the arylamino substituent at the 1,3,4-oxadiazole ring. The most specific molecules displayed >10-fold selectivity for VEGFR-2 over VEGFR-1. Compound activities in vitro and in cell-based assays (IC(50)<100nM) were similar to those of reported clinical and development candidates, including PTK787 (Vatalanibtrade mark). High permeability of active compounds across the Caco-2 cell monolayer (>30x10(-5)cm/min) is indicative of their potential for intestinal absorption upon oral administration.

Hetaryl imidazoles: a novel dual inhibitors of VEGF receptors I and II.

A novel potent derivatives of hetaryl imidazoles were described as inhibitors of vascular endothelial growth factor receptor II (VEGFR-2). Several compounds display VEGFR-2 inhibitory activity reaching IC(50)<100 nM in both enzymatic and cellular assays. The compounds also inhibit the related tyrosine kinase, VEGFR-1. By controlling the substitution pattern on the 5-carboxamido functionality, both dual and specific VEGFR-2 thiazoles were identified.

1-(Azolyl)-4-(aryl)-phthalazines: novel potent inhibitors of VEGF receptors I and II.

Novel potent derivatives of phthalazine are described as an adenosine triphosphate-competitive inhibitors of vascular endothelial growth factor receptors I and II. A number of compounds display vascular endothelial growth factor receptor II inhibitory activity reaching that of Vatalanib A (IC(50): < 50 nm) in an homogenous time-resolved fluorescence enzymatic assay.

1-[4-(1H-Benzoimidazol-2-yl)-phenyl]-3-[4-(1H-benzoimidazol-2-yl)-phenyl]-urea derivatives as small molecule heparanase inhibitors.

A novel class of 1-[4-(1H-benzoimidazol-2-yl)-phenyl]-3-[4-(1H-benzoimidazol-2-yl)-phenyl]-ureas are described as potent inhibitors of heparanase. Among them are 1,3-bis-[4-(1H-benzoimidazol-2-yl)-phenyl]-urea (7a) and 1,3-bis-[4-(5,6-dimethyl-1H-benzoimidazol-2-yl)-phenyl]-urea (7d), which displayed good heparanase inhibitory activity (IC(50) 0.075-0.27 microM). Compound 7a showed good efficacy in a B16 metastasis model.

Arylphthalazines. Part 2: 1-(Isoquinolin-5-yl)-4-arylamino phthalazines as potent inhibitors of VEGF receptors I and II.

A novel class of 1-(isoquinolin-5-yl)-4-arylamino-phthalazines is described as inhibitors of vascular endothelial growth factor receptor II (VEGFR-2). Many compounds display VEGFR-2 inhibitory activity with an IC(50) as low as 0.017 microM in an HTRF enzymatic assay. The compounds also inhibit VEGFR-1, a related tyrosine kinase.