Design,synthesis and biological evaluation of 4-aminopyrimidine-5-cabaldehyde oximes as dual inhibitors of c-Met and VEGFR-2

The synergistically collaboration of c-Met/HGF and VEGFR-2/VEGF leads to development of tumor angiogenesis and progression of various human cancers. Therefore, inhibiting both HGF/c-Met and VEGF/VEGFR signaling may provide a novel and effective therapeutic approach for treating patients with abroad spectrum of tumors. Toward this goal, we designed and synthesized a series of derivatives bearing 4-aminopyrimidine-5-cabaldehyde oxime scaffold as potent dual inhibitors of c-Met and VEGFR-2. The cell proliferation assay in vitro demonstrated most target compounds have inhibition potency both on c-Met and VEGFR-2 with IC₅₀ values in nanomolar range, especially compound 14i, 18a and 18b. Based on the further enzyme assay in vitro, compound 18a was considered as the most potent one, the IC₅₀s of which were 210 nM and 170 nM for c-Met and VEGFR-2, respectively. Following that, we docked the compound 10 and 18a with the proteins c-Met and VEGFR-2, and interpreted the SAR of these analogs. All the results indicate that 18a is a dual inhibitors of c-Met and VEGFR-2 that holds promising potential.     

N3-Arylmalonamides: A new series of thieno[3,2-b]pyridine based inhibitors of c-Met and VEGFR2 tyrosine kinases

A family of thieno[3,2-b]pyridine based small molecule inhibitors of c-Met and VEGFR2 were designed based on lead structure 2. These compounds were shown to have IC₅₀ values in the low nanomolar range in vitro and were efficacious in human tumor xenograft models in mice in vivo.     

Discovery of a novel and potent series of thieno[3,2-b]pyridine-based inhibitors of c-Met and VEGFR2 tyrosine kinases

A series of thieno[3,2-b]pyridine-based inhibitors of c-Met and VEGFR2 tyrosine kinases is described. The compounds demonstrated potency with IC₅₀ values in the low nanomolar range in vitro while the lead compound also showed in vivo activity against various human tumor xenograft models in mice. Further exploration of this class of compounds is underway.     

Structure-based design,synthesis, and evaluation of imidazo[1,2-b]pyridazine and imidazo[1,2-a]pyridine derivatives as novel dual c-Met and VEGFR2 kinase inhibitors

To identify compounds with potent antitumor efficacy for various human cancers, we aimed to synthesize compounds that could inhibit c-mesenchymal epithelial transition factor (c-Met) and vascular endothelial growth factor receptor 2 (VEGFR2) kinases. We designed para-substituted inhibitors by using co-crystal structural information from c-Met and VEGFR2 in complex with known inhibitors. This led to the identification of compounds 3a and 3b, which were capable of suppressing both c-Met and VEGFR2 kinase activities. Further optimization resulted in pyrazolone and pyridone derivatives, which could form intramolecular hydrogen bonds to enforce a rigid conformation, thereby producing potent inhibition. One compound of particular note was the imidazo[1,2-a]pyridine derivative (26) bearing a 6-methylpyridone ring, which strongly inhibited both c-Met and VEGFR2 enzyme activities (IC₅₀ = 1.9, 2.2 nM), as well as proliferation of c-Met-addicted MKN45 cells and VEGF-stimulated human umbilical vein endothelial cells (IC₅₀ = 5.0, 1.8 nM). Compound 26 exhibited dose-dependent antitumor efficacy in vivo in MKN45 (treated/control ratio [T/C] = 4%, po, 5 mg/kg, once-daily) and COLO205 (T/C = 13%, po, 15 mg/kg, once-daily) mouse xenograft models.     

Targeted dual inhibition of c-Met/VEGFR2 signalling by foretinib improves antitumour effects of nanoparticle paclitaxel in gastric cancer models

Elevated expression of multiple growth factors and receptors including c-Met and VEGFR has been reported in gastric adenocarcinoma (GAC) and thus provides a potentially useful therapeutic target. The therapeutic efficacy of foretinib, a c-Met/VEGFR2 inhibitor, was determined in combination with nanoparticle paclitaxel (NPT) in GAC. Animal studies were conducted in NOD/SCID mice in subcutaneous and peritoneal dissemination xenografts. The mechanism of action was assessed by Immunohistochemical and Immunoblot analyses. In c-Met overexpressing MKN-45 cell-derived xenografts, NPT and foretinib demonstrated inhibition in tumour growth, while NPT plus foretinib showed additive effects. In c-Met low-expressing SNU-1 or patient-derived xenografts, the foretinib effect was smaller, while NPT had a similar effect compared with MKN-45, as NPT plus foretinib still exhibited an additive response. Median mice survival was markedly improved by NPT (83%), foretinib (100%) and NPT plus foretinib (230%) in peritoneal dissemination xenografts. Subcutaneous tumour analyses exhibited that foretinib increased cancer cell death and decreased cancer cell proliferation and tumour vasculature. NPT and foretinib suppressed the proliferation of GAC cells in vitro and had additive effects in combination. Further, foretinib caused a dramatic decrease in phosphorylated forms of c-Met, ERK, AKT and p38. Foretinib led to a decrease in Bcl-2, and an increase in p27, Bax, Bim, cleaved PARP-1 and cleaved caspase-3. Thus, these findings highlight the antitumour impact of simultaneous suppression of c-Met and VEGFR2 signalling in GAC and its potential to enhance nanoparticle paclitaxel response. This therapeutic approach might lead to a clinically beneficial combination to increase GAC patients’ survival.     

Synthesis and biological evaluation of benzamides and benzamidines as selective inhibitors of VEGFR tyrosine kinases

A series of benzamidines and benzamides was synthesized as selective inhibitors of vascular endothelial growth factor receptor (VEGFR) tyrosine kinases, and tested for inhibitory activity toward autophosphorylation by the enzyme assay. Selective inhibition of VEGFR-2 tyrosine kinase was observed in the salicylic amide 4e and the anthranilic amidine 5a, and their percent inhibitions of VEGFR-2 tyrosine kinase were 44-60% at a 10 microM concentration of compounds. The salicylic amide 4a showed inhibition of both VEGFR-1 and VEGFR-2 tyrosine kinases at a 10 microM concentration.     

Discovery of novel 2-aminopyridine-3-carboxamides as c-Met kinase inhibitors

A series of 2-aminopyridine-3-carboxamide derivatives against c-Met were designed and synthesized by employing bioisosteric replacement of heterocyclic moieties with the amide bond. The structure-activity relationship (SAR) at various positions of the scaffold was explored. In this study, a promising compound (S)-24o with a c-Met IC(50) of 0.022μM was identified. The compound exhibited dose-dependent inhibition of the phosphorylation of c-Met as well as downstream signaling in EBC-1 cells. Furthermore, the interactive binding model of (S)-24o with c-Met was elucidated by virtue of a molecular modeling study.     

Identification of a novel series of potent RON receptor tyrosine kinase inhibitors

A novel series of N-(3-fluoro-4-(2-substituted-thieno[3,2-b]pyridin-7-yloxy)phenyl)-1-phenyl-5-(trifluoromethyl)-1H-pyrazole-4-carboxamides targeting RON receptor tyrosine kinase was designed and synthesized. SAR study of the series allowed us to identify compounds possessing either inhibitory activity of RON kinase enzyme in the low nanomolar range with low residual activity against the closely related c-Met or potent dual inhibitory activity against RON and c-Met, ? with no significant activity against VEGFR2 in both cases.     

Licochalcone A induces apoptosis through endoplasmic reticulum stress via a phospholipase Cγ1-, Ca2+-, and reactive oxygen species-dependent pathway in HepG2 human hepatocellular carcinoma cells

Licochalcone A (LicA), an estrogenic flavonoid, induces apoptosis in multiple types of cancer cells. In this study, the molecular mechanisms underlying the anti-cancer effects of LicA were investigated in HepG2 human hepatocellular carcinoma cells. LicA induced apoptotic cell death, activation of caspase-4, -9, and -3, and expression of endoplasmic reticulum (ER) stress-associated proteins, including C/EBP homologous protein (CHOP). Inhibition of ER stress by CHOP knockdown or treatment with the ER stress inhibitors, salubrinal and 4-phenylbutyric acid, reduced LicA-induced cell death. LicA also induced reactive oxygen species (ROS) accumulation and the anti-oxidant N-acetylcysteine reduced LicA-induced cell death and CHOP expression. In addition, LicA increased the levels of cytosolic Ca²⁺, which was blocked by 2-aminoethoxydiphenyl borate (an antagonist of inositol 1,4,5-trisphosphate receptor) and BAPTA-AM (an intracellular Ca²⁺ chelator). 2-Aminoethoxydiphenyl borate and BAPTA-AM inhibited LicA-induced cell death. Interestingly, LicA induced phosphorylation of phospholipase Cγ1 (PLCγ1) and inhibition of PLCγ1 reduced cell death and ER stress. Moreover, the multi-targeted receptor tyrosine kinase inhibitors, sorafenib and sunitinib, reduced LicA-induced cell death, ER stress, and cytosolic Ca²⁺ and ROS accumulation. Finally, LicA induced phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) and c-Met receptor and inhibition of both receptors by co-transfection with VEGFR2 and c-Met siRNAs reversed LicA-induced cell death, Ca²⁺ increase, and CHOP expression. Taken together, these findings suggest that induction of ER stress via a PLCγ1-, Ca²⁺-, and ROS-dependent pathway may be an important mechanism by which LicA induces apoptosis in HepG2 hepatocellular carcinoma cells.     

Discovery of 4,6-Disubstituted Pyrimidine Derivatives as Novel Dual VEGFR2/FGFR1 Inhibitors

Abnormalities in the FGFRs signaling pathway and VEGFR2 amplification often occur in a variety of tumors, and they synergistically promote tumor angiogenesis. Studies have shown that the up-regulation of FGF-2 is closely related to the resistance of VEGFR2 inhibitors. Activation of the FGFRs signal is a signal of compensatory angiogenesis after VEGFR2 resistance. Dual VEGFR2/FGFR1 inhibitors contribute to overcoming the resistance of VEGFR2 inhibitors and inhibit tumor growth significantly. Based on this, we designed and synthesized a series of 4,6-disubstituted pyrimidine derivatives as dual VEGFR2/FGFR1 inhibitors by the molecular hybridization strategy. 3-(2,6-Dichloro-3,5-dimethoxyphenyl)-1-{6-[(4-methoxyphenyl)amino]pyrimidin-4-yl}-1-methylurea ( 8b ) had the best inhibitory activities against VEGFR2 and FGFR1 at 10 μM (82.2 % and 101.0 %, respectively), it showed moderate antiproliferative activities against A549 and KG-1 cell lines as well. Besides, molecular docking was also carried out to study the binding mode of 3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-{6-[(4-methoxyphenyl)-amino]-pyrimidin-4-yl}-1-methylurea ( 8b ) with VEGFR2 and FGFR1. These studies reveal that this series of compounds deserve further optimization.     

N-(4-(6,7-Disubstituted-quinolin-4-yloxy)-3-fluorophenyl)-2-oxo-3-phenylimidazolidine-1-carboxamides: A novel series of dual c-Met/VEGFR2 receptor tyrosine kinase inhibitors

A series of N-(4-(6,7-disubstituted-quinolin-4-yloxy)-3-fluorophenyl)-2-oxo-3-phenylimidazolidine-1-carboxamides targeting c-Met and VEGFR2 tyrosine kinases was designed and synthesized. The compounds were potent against these two enzymes with IC₅₀ values in the low nanomolar range in vitro, possessed favorable pharmacokinetic profiles and showed high efficacy in vivo in several human tumor xenograft models in mice.     

N-(3-fluoro-4-(2-arylthieno[3,2-b]pyridin-7-yloxy)phenyl)-2-oxo-3-phenylimidazolidine-1-carboxamides: A novel series of dual c-Met/VEGFR2 receptor tyrosine kinase inhibitors

A series of N-(3-fluoro-4-(2-arylthieno[3,2-b]pyridin-7-yloxy)phenyl)-2-oxo-3-phenylimidazolidine-1-carboxamides targeting c-Met and VEGFR2 tyrosine kinases was designed and synthesized. The compounds were potent against these two enzymes with IC₅₀ values in the low nanomolar range in vitro, possessed favorable pharmacokinetic profiles and showed high efficacy in vivo in several human tumor xenograft models in mice.     

Discovery of aminopyridines substituted with benzoxazole as orally active c-Met kinase inhibitors

We report the synthesis and biological evaluation of aminopyridines substituted with benzoxazole. The SAR of the aminopyridines was explored to improve the inhibitory activity against c-Met and to decrease hERG affinity. These studies led to the discovery of amide 24 which showed good c-Met inhibitory potency, low affinity to hERG and favorable pharmacokinetic properties in rats.     

Essential Role for Endocytosis in the Growth Factor-stimulated Activation of ERK1/2 in Endothelial Cells

Vascular endothelial growth factor (VEGF) stimulates angiogenesis by binding to VEGF receptor 2 (VEGFR2) on endothelial cells (ECs). Downstream activation of the extracellular related kinases 1/2 (ERK1/2) is important for angiogenesis to proceed. Receptor internalization has been implicated in VEGFR2 signaling, but its role in the activation of ERK1/2 is unclear. To explore this question we utilized pitstop and dynasore, two small molecule inhibitors of endocytosis. First, we confirmed that both inhibitors block the internalization of VEGFR2 in ECs. We then stimulated ECs with VEGF in the presence and absence of the inhibitors and examined VEGFR2 signaling to ERK1/2. Activation of VEGFR2 and C-Raf still occurred in the presence of the inhibitors, whereas the activation of MEK1/2 and ERK1/2 was abrogated. Therefore, although internalization is not required for activation of either VEGFR2 or C-Raf in ECs stimulated with VEGF, internalization is necessary to activate the more distal kinases in the cascade. Importantly, inhibition of internalization also prevented activation of ERK1/2 when ECs were stimulated with other pro-angiogenic growth factors, namely fibroblast growth factor 2 and hepatocyte growth factor. In contrast, the same inhibitors did not block ERK1/2 activation in fibroblasts or cancer cells stimulated with growth factors. Finally, we show that these small molecule inhibitors of endocytosis block angiogenesis in vitro and in vivo. Therefore, receptor internalization may be a generic requirement for pro-angiogenic growth factors to activate ERK1/2 signaling in human ECs, and targeting receptor trafficking may present a therapeutic opportunity to block tumor angiogenesis.     

Pharmacophore modeling and virtual screening studies to identify new c-Met inhibitors

Mesenchymal epithelial transition factor (c-Met) is an attractive target for cancer therapy. Three-dimensional pharmacophore hypotheses were built based on a set of known structurally diverse c-Met inhibitors. The best pharmacophore model, which identified inhibitors with an associated correlation coefficient of 0.983 between their experimental and estimated IC(50) values, consisted of two hydrogen-bond acceptors, one hydrophobic, and one ring aromatic feature. The highly predictive power of the model was rigorously validated by test set prediction and Fischer's randomization method. The high values of enrichment factor and receiver operating characteristic (ROC) score indicated the model performed fairly well at distinguishing active from inactive compounds. The model was then applied to screen compound database for potential c-Met inhibitors. A filtering protocol, including druggability and molecular docking, were also applied in hits selection. The final 38 molecules, which exhibited good estimated activities, desired binding mode and favorable drug likeness were identified as potential c-Met inhibitors. Their novel backbone structures could be served as scaffolds for further study, which may facilitate the discovery and rational design of potent c-Met kinase inhibitors.     

c-Met inhibitors with novel binding mode show activity against several hereditary papillary renal cell carcinoma-related mutations

c-Met is a receptor tyrosine kinase often deregulated in human cancers, thus making it an attractive drug target. One mechanism by which c-Met deregulation leads to cancer is through gain-of-function mutations. Therefore, small molecules capable of targeting these mutations could offer therapeutic benefits for affected patients. SU11274 was recently described and reported to inhibit the activity of the wild-type and some mutant forms of c-Met, whereas other mutants are resistant to inhibition. We identified a novel series of c-Met small molecule inhibitors that are active against multiple mutants previously identified in hereditary papillary renal cell carcinoma patients. AM7 is active against wild-type c-Met as well as several mutants, inhibits c-Met-mediated signaling in MKN-45 and U-87 MG cells, and inhibits tumor growth in these two models grown as xenografts. The crystal structures of AM7 and SU11274 bound to unphosphorylated c-Met have been determined. The AM7 structure reveals a novel binding mode compared with other published c-Met inhibitors and SU11274. The molecule binds the kinase linker and then extends into a new hydrophobic binding site. This binding site is created by a significant movement of the C-helix and so represents an inactive conformation of the c-Met kinase. Thus, our results demonstrate that it is possible to identify and design inhibitors that will likely be active against mutants found in different cancers.     

Synthesis and anti-tumor activity of [1,4] dioxino [2,3-f] quinazoline derivatives as dual inhibitors of c-Met and VEGFR-2

Both c-Met and VEGFR-2 were important targets for cancer therapies. In order to develop reversible and non-covalent c-Met and VEGFR-2 dual inhibitors, a series of [1,4]dioxino[2,3-f]quinazoline derivatives were designed and synthesized. The enzyme assay demonstrated that most target compounds had inhibition potency on both c-Met and VEGFR-2 with IC₅₀ values in nanomolar range especially compounds 7m and 7k. Based on further cell proliferation assay in vitro, compound 7k showed significantly anti-tumor activity in vivo on a hepatocellular carcinoma (MHCC97H cells) xenograft mouse model. We docked the compound 7m with c-Met and VEGFR-2 kinases, and interpreted the SAR of these analogues. All results indicated that the target compounds were dual inhibitors of c-Met and VEGFR-2 kinases that held promising potential in cancer therapy.     

Cancer cells harboring MET gene amplification activate alternative signaling pathways to escape MET inhibition but remain sensitive to Hsp90 inhibitors

Hepatocyte growth factor/scatter factor (HGF/SF) receptor c-Met is implicated in growth, invasion and metastasis of many tumors. Tumor cells harboring MET gene amplification are initially sensitive to c-Met tyrosine kinase inhibitors (TKI), but escape from long-term treatment has not been investigated. C-Met is a client of heat shock protein 90 (Hsp90) and is destabilized by Hsp90 inhibitors, suggesting that these drugs may inhibit tumors driven by MET amplification, although tumor escape under these conditions also has not been explored. Here, we evaluated the initial inhibitory effects of, and the likelihood of escape from, the Hsp90 inhibitor 17-allylamino-17-demethoxy-geldanamycin (17-AAG) and the c-Met TKI SU11274, using two cell lines harboring MET gene amplification. 17-AAG inhibited cell growth in both cell lines and induced substantial apoptosis, whereas SU11274 was only growth inhibitory in one cell line. In both cell lines, c-Met-dependent Akt, Erk and/or STAT3 signaling, as well as activation of the EGFR family, resumed shortly after treatment with c-Met TKI despite sustained c-Met inhibition. PKC δ upregulation may participate in reactivation of c-Met downstream signaling in both cell lines. In contrast to c-Met TKI, 17-AAG destabilized c-Met protein and durably blocked reactivation of downstream signaling pathways and EGFR family members. Our data demonstrate that downstream signaling in tumor cells over-expressing c-Met is not stably suppressed by c-Met TKI, even though c-Met remains fully inhibited. In contrast, Hsp90 inhibitors provide long-lasting suppression of c-Met-dependent signaling, and these drugs should be further evaluated in tumors driven by MET gene amplification.     

Discovery of novel substituted benzo-anellated 4-benzylamino pyrrolopyrimidines as dual EGFR and VEGFR2 inhibitors

The quinazoline scaffold is the main part of many marketed EGFR inhibitors. Resistance developments against those inhibitors enforced the search for novel structural lead compounds. We developed novel benzo-anellated 4-benzylamine pyrrolopyrimidines with varied substitution patterns at both the molecular scaffold and the attached residue in the 4-position. The structure-dependent affinities towards EGFR are discussed and first nanomolar derivatives have been identified. Docking studies were carried out for EGFR in order to explore the potential binding mode of the novel inhibitors. As the receptor tyrosine kinase VEGFR2 recently gained an increasing interest as an upregulated signaling kinase in many solid tumors and in tumor metastasis we determined the affinity of our compounds to inhibit VEGFR2. So we identified novel dually acting EGFR and VEGFR2 inhibitors for which first anticancer screening data are reported. Those data indicate a stronger antiproliferative effect of a VEGFR2 inhibition compared to the EGFR inhibition.     

Etk/Bmx transactivates vascular endothelial growth factor 2 and recruits phosphatidylinositol 3-kinase to mediate the tumor necrosis factor-induced angiogenic pathway

Tumor necrosis factor (TNF), via its receptor 2 (TNFR2), induces Etk (or Bmx) activation and Etk-dependent endothelial cell (EC) migration and tube formation. Because TNF receptor 2 lacks an intrinsic kinase activity, we examined the kinase(s) mediating TNF-induced Etk activation. TNF induces a coordinated phosphorylation of vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) and Etk, which is blocked by VEGFR2-specific inhibitors. In response to TNF, Etk and VEGFR2 form a complex resulting in a reciprocal activation between the two kinases. Subsequently, the downstream phosphatidylinositol 3-kinase (PI3K)-Akt signaling (but not signaling through phospholipase C-gamma) was initiated and directly led to TNF-induced EC migration, which was significantly inhibited by VEGFR2-, PI3K-, or Akt-specific inhibitors. Phosphorylation of VEGFR2 at Tyr-801 and Tyr-1175, the critical sites for VEGF-induced PI3K-Akt signaling, was not involved in TNF-mediated Akt activation. However, TNF induces phosphorylation of Etk at Tyr-566, directly mediating the recruitment of the p85 subunit of PI3K. Furthermore, TNF- but not VEGF-induced activation of VEGFR2, Akt, and EC migration are blunted in EC genetically deficient with Etk. Taken together, our data demonstrated that TNF induces transactivation between Etk and VEGFR2, and Etk directly activates PI3K-Akt angiogenic signaling independent of VEGF-induced VEGFR2-PI3K-Akt signaling pathway.