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.     

Design and synthesis of 3-(4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridin-2-yl)-1H-quinolin-2-ones as VEGFR-2 kinase inhibitors

A series of 3-(4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridin-2-yl)-1H-quinolin-2-ones have been identified as a new class of VEGFR-2 kinase inhibitors. A variety of (4,5,6,7-tetrahydro-imidazo[5,4-c]pyridin-2-yl)-acetic acid ethyl esters were synthesized, and their VEGFR-2 inhibitory activity was evaluated. Described herein are the preparation of the series and the effects of the compounds on VEGFR-2 kinase activity.     

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.     

The P2Y2 nucleotide receptor mediates vascular cell adhesion molecule-1 expression through interaction with VEGF receptor-2 (KDR/Flk-1)

UTP stimulates the expression of pro-inflammatory vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells through activation of the P2Y(2) nucleotide receptor P2Y(2)R. Here, we demonstrated that activation of the P2Y(2)R induced rapid tyrosine phosphorylation of vascular endothelial growth factor receptor (VEGFR)-2 in human coronary artery endothelial cells (HCAEC). RNA interference targeting VEGFR-2 or inhibition of VEGFR-2 tyrosine kinase activity abolishes P2Y(2)R-mediated VCAM-1 expression. Furthermore, VEGFR-2 and the P2Y(2)R co-localize upon UTP stimulation. Deletion or mutation of two Src homology-3-binding sites in the C-terminal tail of the P2Y(2)R or inhibition of Src kinase activity abolished the P2Y(2)R-mediated transactivation of VEGFR-2 and subsequently inhibited UTP-induced VCAM-1 expression. Moreover, activation of VEGFR-2 by UTP leads to the phosphorylation of Vav2, a guanine nucleotide exchange factor for Rho family GTPases. Using a binding assay to measure the activity of the small GTPases Rho, we found that stimulation of HCAEC by UTP increased the activity of RhoA and Rac1 (but not Cdc42). Significantly, a dominant negative form of RhoA inhibited P2Y(2)R-mediated VCAM-1 expression, whereas expression of dominant negative forms of Cdc42 and Rac1 had no effect. These data indicate a novel mechanism whereby a nucleotide receptor transactivates a receptor tyrosine kinase to generate an inflammatory response associated with atherosclerosis.     

De novo design of VEGFR-2 tyrosine kinase inhibitors based on a linked-fragment approach

Vascular endothelial growth factor receptor-2 (VEGFR-2) tyrosine kinase inhibitors have been demonstrated to possess substantial antitumor activity. VEGFR-2 tyrosine kinase inhibitors are crucial for development of antitumor drugs. Based on the crystal structure of VEGFR-2 tyrosine kinase, a linked-fragment strategy was employed to design novel VEGFR-2 tyrosine kinase inhibitors, and 1000 compounds were generated in this process. Absorption, distribution, metabolism, excretion and toxicity (ADMET) were used to screen the 1000 compounds, and 59 compounds were acceptable. Scaffold hopping was then used for further screening, and only four compounds were obtained in this way. Then, the binding energy of the four molecules to VEGFR-2 tyrosine kinase was calculated using molecular docking, and their values were found to be lower than that of Sorafenib. Finally, molecular dynamics simulations were performed on the complex of the compound with the lowest binding energy with VEGFR-2 tyrosine kinase, and the binding model was analyzed. At the end, four chemical entities with novel structures were obtained, and were suggested for experimental testing in future studies.     

Structure and dual function of vascular endothelial growth factor receptor-1 (Flt-1)

Vascular endothelial growth factor receptor-1 (VEGFR-1/Flt-1) is structurally a typical tyrosine kinase receptor of about 180 kDa, and carries seven Ig-like domains in the extracellular region and a tyrosine kinase domain with a long kinase insert. Recent studies have revealed that the VEGFR-1 gene and its gene product have several unique characteristics structurally and functionally. In addition to the full length receptor, VEGFR-1 gene encodes for a soluble form carrying only six Ig domains via an alternative splicing. Both the full length and soluble form of VEGFR-1 show strong binding affinity for VEGF, but the kinase activity of the full length receptor is one order of magnitude lower than that of VEGFR-2 (KDR/Flk-1). Early in embryogenesis, null mutation of VEGFR-1 gene results in lethality due to a disorganization of blood vessels and an overgrowth of endothelial-like cells, suggesting a regulatory role in vivo. Mice carrying the extracellular domain of VEGFR-1 gene without the tyrosine kinase domain develop an almost normal circular system and survive. Thus, the extracellular region of VEGFR-1 is necessary and sufficient for physiological angiogenesis at the early stage of embryogenesis, possibly acting to trap VEGF and suppress VEGF levels to an appropriate range. The tyrosine kinase domain of VEGFR-1, although much weaker than that of VEGFR-2, transduces signals for endothelial cells. Furthermore, VEGFR-1 is involved in the VEGF-dependent migration and gene expression of monocyte/macrophages. Therefore, VEGFR-1 functions both in a positive and negative manner in different cellular systems and biological conditions.     

Identification of new pyrrolo[2,3-d]pyrimidines as potent VEGFR-2 tyrosine kinase inhibitors: Design,synthesis, biological evaluation and molecular modeling

Vascular endothelial growth factor receptor-2 (VEGFR-2) plays a crucial role in cancer angiogenesis. In the current study, a series of novel pyrrolo[2,3-d]pyrimidine based-compounds was designed and synthesized as VEGFR-2 inhibitors, in accordance to the structure activity relationship (SAR) studies of known type II VEGFR-2 inhibitors. The newly synthesized compounds were evaluated for their ability to inhibit VEGFR-2 kinase enzyme in vitro. All the tested compounds demonstrated highly potent dose-related VEGFR-2 inhibition with IC₅₀ values in nanomolar range. Among these compounds, pyrrolo[2,3-d]pyrimidine derivatives carrying biaryl urea moieties (12d and 15c) exhibited IC₅₀ values of 11.9 and 13.6 nM respectively. Additionally, most of the newly synthesized final compounds were tested on 60 human cancer cell lines. Docking of these compounds into the inactive conformation of VEGFR-2 was performed which showed comparable binding modes to that of the FDA approved VEGFR-2 kinase inhibitors. These newly discovered potent kinase inhibitors could be considered as potential candidates for the development of new targeted anticancer agent.     

Receptor chimeras indicate that the vascular endothelial growth factor receptor-1 (VEGFR-1) modulates mitogenic activity of VEGFR-2 in endothelial cells

Vascular endothelial growth factor (VEGF) provokes angiogenesis in vivo and stimulates growth and differentiation of endothelial cells in vitro. Although VEGF receptor-1 (VEGFR-1) and VEGFR-2 are known to be high affinity receptors for VEGF, it is not clear which of the VEGFRs are responsible for the transmission of the diverse biological responses of VEGF. For this purpose we have constructed a chimeric receptor for VEGFR-1 (CTR) and VEGFR-2 (CKR) in which the extracellular domain of each receptor was replaced with the extracellular domain of human colony-stimulating factor-1 receptor (CSF-1R), and these receptors were expressed in pig aortic endothelial (PAE) cells. We show that CKR individually expressed in PAE cells is readily tyrosine-phosphorylated in vivo, autophosphorylated in vitro, and stimulates cell proliferation in a CSF-1-dependent manner. In contrast, CTR individually expressed in PAE cells showed no significant in vivo, in vitro tyrosine phosphorylation and cell growth in response to CSF-1 stimulation. The kinase activity of CKR was essential for its biological activity, since mutation of lysine 866 to arginine abolished its in vivo, in vitro tyrosine phosphorylation and mitogenic signals. Remarkably, activation of CTR repressed CKR-mediated mitogen-activate protein kinase activation and cell proliferation. Similar effects were observed for VEGFR-2 co-expressed with VEGFR-1. Collectively, these findings demonstrate that VEGFR-2 activation plays a positive role in angiogenesis by promoting endothelial cell proliferation. In contrast, activation of VEGFR-1 plays a stationary role in angiogenesis by antagonizing VEGFR-2 responses.     

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.     

Quinoxalinone (Part II). Discovery of (Z)-3-(2-(pyridin-4-yl)vinyl)quinoxalinone derivates as potent VEGFR-2 kinase inhibitors

Inhibition of VEGFR-2 kinase has been highlighted as one of the well-defined strategies to suppress tumor growth via blockade of angiogenesis. Guided by the principles of bioisosteric replacement and pharmacophoric fragment migration, a series of novel quinoxalinone derivates were designed, synthesized and evaluated for their VEGFR-2 inhibitory potencies. Among them, compounds 7c, 8b, 8c, 8e and 10b displayed antiangiogenic abilities via the in vitro tube formation assay (cellular level) and ex vivo rat aortic ring assay (tissue level) at a low concentration (0.1 μM). By means of in vivo zebrafish embryo model, two (Z)-3-(2-(pyridin-4-yl)vinyl)quinoxalinone derivates 8c and 8e showed significant antiangiogenesis effects, suggesting they have potentials to be developed into antiangiogenesis agents via further structural optimization. Moreover, these two compounds also demonstrated potent inhibition toward VEGFR-2 and B-raf kinases in a low concentration (1 μM). A possible interpretation of our evaluation result has been presented by a molecular docking study by docking representative compound 8c with VEGFR-2.     

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.     

Discovery and molecular docking of quinolyl-thienyl chalcones as anti-angiogenic agents targeting VEGFR-2 tyrosine kinase

Vascular endothelial growth factor Receptor-2 (VEGFR-2) kinase inhibition is one of the well established strategies to promptly tackle tumor growth by suppression of angiogenesis. In the current study, structure-based virtual screening methodology of a series of quinolyl-thienyl chalcones indicated their strong potential as VEGFR-2 kinase inhibitors. In vitro VEGFR-2 kinase inhibitory activity was found to be significant (compound 19, IC(50): 73.41nM). All compounds showed significant inhibition of human umbilical vein endothelial cells (HUVEC) proliferation (compound 19, IC(50): 21.78nM). Molecular interactions of the compounds were studied using molecular docking studies.     

(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.     

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.     

Differential regulation of vascular endothelial growth factor receptors (VEGFR) revealed by RNA interference: interactions of VEGFR-1 and VEGFR-2 in endothelial cell signaling

Vascular endothelial growth factor (VEGF) plays a central role in vascular homeostasis. VEGF receptors (VEGFRs) include several subtypes that may have a differential role in endothelial signal transduction, but interactions among these receptors are incompletely understood. In these studies, we designed small interfering RNA (siRNA) duplexes that targeted specific VEGFR subtypes in bovine aortic endothelial cells (BAEC). siRNA-mediated downregulation of VEGFR-2 by its cognate siRNA resulted in a significant attenuation of VEGF-mediated signaling. Compared to control siRNA-treated cells, VEGFR-2 siRNA markedly inhibited VEGF-mediated activation of PI3K/Akt/GSK3-beta as well as MAP kinase and PKC pathways. VEGFR-2 siRNA also blocked VEGF-stimulated phosphorylation and dephosphorylation of endothelial nitric oxide synthase (eNOS) at Ser(1179) and Ser(116), respectively. VEGFR-2-specific siRNA had no effect on the abundance of VEGFR-1 protein. By contrast, VEGFR-1-specific siRNA markedly not only downregulated the abundance of VEGFR-1 but also significantly reduced VEGFR-2 protein and mRNA abundance. VEGFR-1 siRNA had no effect on the stability of VEGFR-2 protein or mRNA. However, VEGFR-1 siRNA significantly inhibited VEGFR-2 promoter activity, as determined in luciferase assays using VEGFR-2 promoter fusion constructs in transfected BAEC. Deletion of either the 5' E box or the 3' E box and the GATA element in the VEGFR-2 promoter completely abolished the inhibition of VEGFR-2 promoter activity elicited by VEGFR-1 siRNA. Taken together, our data suggest that VEGFR-1 receptor is a critical determinant of VEGFR-2 abundance, while VEGFR-2 is the key receptor directly responsible for endothelial cell signaling stimulated by VEGF.     

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.     

Selective inhibition of vascular endothelial growth factor receptor-2 (VEGFR-2) identifies a central role for VEGFR-2 in human aortic endothelial cell responses to VEGF

Vascular endothelial growth factor receptors (VEGFR) are considered essential for angiogenesis. The VEGFR-family proteins consist of VEGFR-1/Flt-1, VEGFR-2/KDR/Flk-1, and VEGFR-3/Flt-4. Among these, VEGFR-2 is thought to be principally responsible for angiogenesis. However, the precise role of VEGFRs1-3 in endothelial cell biology and angiogenesis remains unclear due in part to the lack of VEGFR-specific inhibitors. We used the newly described, highly selective anilinoquinazoline inhibitor of VEGFR-2 tyrosine kinase, ZM323881 (5-[[7-(benzyloxy) quinazolin-4-yl]amino]-4-fluoro-2-methylphenol), to explore the role of VEGFR-2 in endothelial cell function. Consistent with its reported effects on VEGFR-2 [IC(50) < 2 nM], ZM323881 inhibited activation of VEGFR-2, but not of VEGFR-1, epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), or hepatocyte growth factor (HGF) receptor. We studied the effects of VEGF on human aortic endothelial cells (HAECs), which express VEGFR-1 and VEGFR-2, but not VEGFR-3, in the absence or presence of ZM323881. Inhibition of VEGFR-2 blocked activation of extracellular regulated-kinase, p38, Akt, and endothelial nitric oxide synthetase (eNOS) by VEGF, but did not inhibit p38 activation by the VEGFR-1-specific ligand, placental growth factor (PIGF). Inhibition of VEGFR-2 also perturbed VEGF-induced membrane extension, cell migration, and tube formation by HAECs. Vascular endothelial growth factor receptor-2 inhibition also reversed VEGF-stimulated phosphorylation of CrkII and its Src homology 2 (SH2)-binding protein p130Cas, which are known to play a pivotal role in regulating endothelial cell migration. Inhibition of VEGFR-2 thus blocked all VEGF-induced endothelial cellular responses tested, supporting that the catalytic activity of VEGFR-2 is critical for VEGF signaling and/or that VEGFR-2 may function in a heterodimer with VEGFR-1 in human vascular endothelial cells.     

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.     

The presence of a single tyrosine residue at the carboxyl domain of vascular endothelial growth factor receptor-2/FLK-1 regulates its autophosphorylation and activation of signaling molecules

Vascular endothelial growth factor receptor (VEGFR)-2 plays a critical role in vasculogenesis during embryonic development and pathological angiogenesis, but little is known about the molecular mechanisms governing its functions. Here we investigated the role of tyrosine 1212 on mouse VEGFR-2 autophosphorylation and its signal transduction relay in endothelial cells. Mutation of tyrosine 1212 on VEGFR-2 to phenylalanine severely impaired the ligand-dependent autophosphorylation of VEGFR-2 and its ability to associate with and activate Src. This mutation also reduced the VEGFR-2 ability to phosphorylate phospholipase Cgamma1 and mitogen-activated protein kinase (MAPK). Unlike mutation of tyrosine 1212 to phenylalanine, replacement of tyrosine 1212 with glutamic acid preserved the ligand-dependent activation of VEGFR-2 and activation of VEGFR-2-associated signaling proteins including Src, phospholipase Cgamma1, and MAPK. Further analysis showed that Src activation is not required for activation of VEGFR-2, since cells co-expressing wild type receptor with kinase dead Src or wild type Src displayed no apparent effect in the ligand-dependent autophosphorylation of VEGFR-2. Similarly, expression of wild type VEGFR-2 in fibroblast (SYF) cells obtained from the triple knockout Src family kinases showed normal ligand-dependent autophosphorylation. Collectively, these results suggest that phosphorylation of tyrosine 1212 of VEGFR-2 plays a crucial role in the activation of VEGFR-2 and subsequently VEGFR-2-mediated angiogenesis.     

Expression and purification of the catalytic domain of human vascular endothelial growth factor receptor 2 for inhibitor screening

Vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen, can act in tumor-induced angiogenesis by binding to specific receptors on the surface of endothelial cells. One such receptor, VEGFR-2/KDR, plays a key role in VEGF-induced angiogenesis. Here, we expressed the catalytic domain of VEGFR-2 as a soluble active kinase using Bac-to-Bac expression system, and investigated correlations between VEGFR-2 activity and enzyme concentration, ATP concentration, substrate concentration and divalent cation type. We used these data to establish a convenient, effective and non-radioactive ELISA screening technique for the identification and evaluation of potential inhibitors for VEGFR-2 kinase. We screened 200 RTK target-based compounds and identified one (TKI-31) that potently inhibited VEGFR-2 kinase activity (IC50=0.596 microM). Treatment of NIH3T3/KDR cells with TKI-31 blocked VEGF-induced phosphorylation of KDR in a dose-dependent manner. Moreover, TKI-31 dose-dependently suppressed HUVEC tube formation. Thus, we herein report a novel, efficient method for identifying VEGFR-2 kinase inhibitors and introduce one, TKI-31, that may prove to be a useful new angiogenesis inhibitor.