Target For Anticancer Drug Discovery Research Articles

Abstract IA19: Targeting the PRC2 complex through EED for anti-cancer therapy

Abstract The Polycomb Repressive Complex-2 (PRC2) plays important roles in regulating gene expression through its histone H3 lysine methyl transferase activity. Dysregulation of PRC2 is observed in multiple cancers. For example, gain of function mutations of the EZH2 catalytic subunit of PRC2 have been reported in B-cell lymphomas, melanoma and parathyroid carcinoma. Components of the complex are also overexpressed in a number of cancers. The PRC2-complex have therefore become a major target for anti-cancer drug discovery. We and others, have reported anti-tumor activity of small molecular weight agents targeting the SAM-binding pocket of EZH2 in preclinical models and a number of EZH2 inhibitors ore showing promising activity in clinical trials. EED and SUZ12 are two additional components of the PRC2 core complex required for regulation of its catalytic function. High resolution structures and other reports have showed that binding of EED to tri-methylated histone H3 Lysine 27 (H3K27Me3) further stimulates the catalytic effect of EZh2. This suggests that small molecular weight compounds binding to and interfering with the EED stimulatory effect on Ezh2 may be able to modulate PRC2 activity. Following this possibility, we launched a comprehensive effort to discover non-catalytic site PRC2 binders. The discovery and characterization of EED226, a highly potent and selective EED-binding inhibitor of PRC2 will be presented. Data will be presented to demonstrate that EED226 inhibits PRC2 function via a distinct allosteric mechanism by binding to the H3K27Me3 binding pocket of EED. Furthermore, EED226 potently inhibits cellular H3K27Me3 levels, modulates target gene expression similar to the SAM competitive Ezh2 inhibitors and inhibits the proliferation of cell lines carrying mutant Ezh2. Oral administration of EED226 to EZH2-mutant tumor bearing mice leads to profound tumor regression. Our data therefore demonstrates that the multi-subunit PRC2 complex can be targeted at non-catalytic binding pockets to achieve anti-tumor activity. Some unique features of the anti-tumor activity achieved through targeting PRC2 will be discussed. Citation Format: Peter W. Atadja. Targeting the PRC2 complex through EED for anti-cancer therapy [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr IA19.

Molecular modeling of cationic porphyrin-anthraquinone hybrids as DNA topoisomerase IIβ inhibitors

Human DNA Topoisomerase II has been regarded as a promising target in anticancer drug discovery. In the present study, we designed six porphyrin-anthraquinone hybrids bearing pyrazole or pyridine group as meso substituents and evaluated their potentials as DNA Topoisomerase IIβ inhibitor. First, we investigated the binding orientation of porphyrin hybrids into DNA topoisomerase IIβ employing AutoDock 4.2 and then performed 20-ns molecular dynamics simulations to see the dynamic stability of each porphyrin-Topo IIβ complex using Amber 14. We found that the binding of porphyrin hybrids occured through intercalation and groove binding mode in addition interaction with the amino acid residues constituting the active cavity of Topo IIβ. Each porphyrin-Topo IIβ complex was stabilized during 20-ns dynamics simulations. The MM-PBSA free energy calculation shows that the binding affinities of porphyrin hybrids were modified with the number of meso substituent. Interestingly, the affinity of all porphyrin hybrids to Topo IIβ was stronger than that of native ligand (EVP), indicating the potential of the designed porphyrin to be considered in experimental research.

Targeting Ras with Macromolecules.

Activating Ras mutations are associated with ∼30% of all human cancers and the four Ras isoforms are highly attractive targets for anticancer drug discovery. However, Ras proteins are challenging targets for conventional drug discovery because they function through intracellular protein-protein interactions and their surfaces lack major pockets for small molecules to bind. Over the past few years, researchers have explored a variety of approaches and modalities, with the aim of specifically targeting oncogenic Ras mutants for anticancer treatment. This perspective will provide an overview of the efforts on developing "macromolecular" inhibitors against Ras proteins, including peptides, macrocycles, antibodies, nonimmunoglobulin proteins, and nucleic acids.

Combining properties of different classes of PI3Kα inhibitors to understand the molecular features that confer selectivity

Phosphoinositide 3-kinases (PI3Ks) are major regulators of many cellular functions, and hyperactivation of PI3K cell signalling pathways is a major target for anticancer drug discovery. PI3Kα is the isoform most implicated in cancer, and our aim is to selectively inhibit this isoform, which may be more beneficial than concurrent inhibition of all Class I PI3Ks. We have used structure-guided design to merge high-selectivity and high-affinity characteristics found in existing compounds. Molecular docking, including the prediction of water-mediated interactions, was used to model interactions between the ligands and the PI3Kα affinity pocket. Inhibition was tested using lipid kinase assays, and active compounds were tested for effects on PI3K cell signalling. The first-generation compounds synthesized had IC50 (half maximal inhibitory concentration) values >4 μM for PI3Kα yet were selective for PI3Kα over the other Class I isoforms (β, δ and γ). The second-generation compounds explored were predicted to better engage the affinity pocket through direct and water-mediated interactions with the enzyme, and the IC50 values decreased by ∼30-fold. Cell signalling analysis showed that some of the new PI3Kα inhibitors were more active in the H1047R mutant bearing cell lines SK-OV-3 and T47D, compared with the E545K mutant harbouring MCF-7 cell line. In conclusion, we have used a structure-based design approach to combine features from two different compound classes to create new PI3Kα-selective inhibitors. This provides new insights into the contribution of different chemical units and interactions with different parts of the active site to the selectivity and potency of PI3Kα inhibitors.

Development of a pan-cancer 15 gene expression signature to detect a subgroup driven by EMT/MAPK signalling.

11616 Background: Epithelial–mesenchymal transition (EMT) is the conversion of epithelial cells to mesenchymal cells and involves loss of cell–cell adhesion and cell polarity and increased motility, invasiveness and metastases. The EMT process has therefore been under investigation as a new target for anticancer drug discovery. The aim of this work was to develop an EMT biomarker suitable for formalin-fixed paraffin embedded (FFPE) tissue that could be used for patient treatment selection. Methods: Unsupervised hierarchical clustering of ovarian cancer gene expression data (TCGA. 2011) previously identified an EMT subgroup. We confirmed this EMT subgroup in FFPE tissue using 265 high grade serous ovarian cancer (HGSOC) FFPE samples. The analysis was extended to show existence of an EMT subgroup across a range of solid tumours including colon, lung, melanoma and prostate cancer. Further to this, a common gene list was generated to include only transcripts with high variability and expression across diseases, and used as a starting list for development of a 15 transcript assay which can be used to prospectively identify the EMT subgroup from archived tissue. Results: The 15 gene expression assay was a poor prognostic marker in Colorectal, (Relapse free survival: HR = 1.46 [95% CI:1.07-1.98]); Lung, (Relapse free survival: HR = 2.18 [95% CI:1.33-3.56]); Prostate cancer, (Biochemical recurrence: HR 2.49 CI: 1.43-4.34) and was associated with activated MAPK (phospho-MAPK) in preclinical models and clinical samples (p < 0.05). The assay score was reduced by MEK inhibition (p < 0.05) and elevated by KRAS, NRAS and MEK1 overexpression (p < 0.05). The assay predicted response to the MEK inhibitors Trametinib and Selumetinib in cell line models (p < 0.001). Conclusions: A 15 gene expression assay has been developed from FFPE samples across multiple diseases to detect an EMT molecular subgroup associated with MAPK signalling. The assay predicted sensitivity to MEK inhibitors in pre-clinical model systems. Further work aims to validate the assay as a predictive biomarker in clinical samples from patients treated with EMT or MEK targeted therapies.

Design, synthesis, biological evaluation and molecular docking studies of novel 3-aryl-4-anilino-2H-chromen-2-one derivatives targeting ERα as anti-breast cancer agents

The estrogen receptor (ER) has played an important role in breast cancer development and progression and is a central target for anticancer drug discovery. In order to develop novel selective ERα modulators (SERMs), we designed and synthesized 18 novel 3-aryl-4-anilino-2H-chromen-2-one derivatives based on previously reported lead compounds. The biological results indicated that most of the compounds presented potent ERα binding affinity and possessed better anti-proliferative activities against MCF-7 and Ishikawa cell lines than the positive control tamoxifen. The piperidyl substituted compounds such as 16d and 18d demonstrated strong ERα binding affinities and excellent anti-proliferative activities respectively. Compound 18d displayed the most potent ERα binding affinity with RBA value of 2.83%, while 16d exhibited the best anti-proliferative activity against MCF-7 cells with IC50 value of 4.52±2.47μM. Further molecular docking studies were also carried out to investigate binding pattern of the newly synthesized compounds with ERα. All these results together with the structure–activity relationships (SARs) indicated that these 3-aryl-4-anilino-2H-chromen-2-one derivatives with basic side chain could serve as promising leads for further optimization as novel SERMs.

Highly Potent, Selective, and Orally Bioavailable 4-Thiazol-N-(pyridin-2-yl)pyrimidin-2-amine Cyclin-Dependent Kinases 4 and 6 Inhibitors as Anticancer Drug Candidates: Design, Synthesis, and Evaluation.

Cyclin D dependent kinases (CDK4 and CDK6) regulate entry into S phase of the cell cycle and are validated targets for anticancer drug discovery. Herein we detail the discovery of a novel series of 4-thiazol-N-(pyridin-2-yl)pyrimidin-2-amine derivatives as highly potent and selective inhibitors of CDK4 and CDK6. Medicinal chemistry optimization resulted in 83, an orally bioavailable inhibitor molecule with remarkable selectivity. Repeated oral administration of 83 caused marked inhibition of tumor growth in MV4-11 acute myeloid leukemia mouse xenografts without having a negative effect on body weight and showing any sign of clinical toxicity. The data merit 83 as a clinical development candidate.

A novel peptide, 9R-P201, strongly inhibits the viability, proliferation and migration of liver cancer HepG2 cells and induces apoptosis by down-regulation of FoxM1 expression

Overexpression of FoxM1 was closely related to the proliferation, metastasis, chemo-resistance and poor prognosis of various cancers. FoxM1 was regarded as the Achilles’ heel of cancer and a potential target for anti-cancer drug discovery. We previously obtained several high affinity peptides from the phage random library against the DNA binding domain of FoxM1c (FoxM1c-DBD) protein. Here in this paper, we found that 9R-P201, one of the novel peptides, showed stronger inhibition to HepG2 cancer cells than those of DU145, HUVEC and L-02 cells with an IC50 of 43.6µg/ml (13.1µM). The peptide was highly effective to liver cancer cells with an IC50 for L-02 cells of 2855.9µg/ml. We confirmed that 9R-P201 aggregated in the cell nucleus and the expression of FoxM1 was significantly down-regulated at both transcriptional and translational levels in HepG2 cells, leading to the suppression of cell proliferation, migration, angiogenesis, and induction of apoptosis. Whole genomic RNA sequencing analysis revealed that 357 genes were significantly and differentially expressed, most of them were enriched in cancer-associated biological processes. Finally, treatment of HepG2 xenografts with 9R-P201 resulted in growth inhibition and down-regulation of foxM1 expression in tumors. Collectively, our findings suggested that 9R-P201 could strongly inhibit the viability, proliferation and migration of liver cancer HepG2 cells and induce apoptosis by down-regulation of FoxM1 and regulation of related gene expression in signal transduction passways. Thus, 9R-P201 holds great potential as a lead anti-cancer drug targeting FoxM1.

Development of Pyridazinone Chemotypes Targeting the PDEδ Prenyl Binding Site.

The K-Ras GTPase is a major target in anticancer drug discovery. However, direct interference with signaling by K-Ras has not led to clinically useful drugs yet. Correct localization and signaling by farnesylated K-Ras is regulated by the prenyl binding protein PDEδ. Interfering with binding of PDEδ to K-Ras by means of small molecules provides a novel opportunity to suppress oncogenic signaling. Here we describe the identification and structure-guided development of novel K-Ras-PDEδ inhibitor chemotypes based on pyrrolopyridazinones and pyrazolopyridazinones that bind to the farnesyl binding pocket of PDEδ with low nanomolar affinity. We delineate the structure-property relationship and in vivo pharmacokinetic (PK) and toxicokinetic (Tox) studies for pyrazolopyridazinone-based K-Ras-PDEδ inhibitors. These findings may inspire novel drug discovery efforts aimed at the development of drugs targeting oncogenic Ras.

Riccardin D-N induces lysosomal membrane permeabilization by inhibiting acid sphingomyelinase and interfering with sphingomyelin metabolism in vivo

Lysosomes are important targets for anticancer drug discovery. Our previous study showed that Riccardin D-N (RD-N), a natural macrocylic bisbibenzyl derivative produced by Mannich reaction, induced cell death by accumulating in lysosomes. Experiments were performed on human lung squamous cell carcinoma tissue from left inferior lobar bronchus of patient xenografts and H460 cells. RD-N was administrated for 25days. The specimens of xenografts in Balb/c athymic (nu+/nu+) male mice were removed for immunohistochemistry, subcellular fractionation, enzyme activities and Western blotting analysis. mRFP-GFP-LC3 reporter was used to examine autophagy in H460 cells. Sphingomyelin assay was evaluated by thin-layer chromatography and assay kit. Lysosomal membrane permeabilization (LMP) caused by acid sphingomyelinase (ASM) inhibition and subsequent changes of sphingomyelin (SM) metabolism selectively destabilized the cancer cell lysosomes in RD-N-treated H460 cells in vitro and tumor xenograft model in vivo. The destabilized lysosomes induced the release of cathepsins from the lysosomes into the cytosol and further triggered cell death. These results explain the underlying mechanism of RD-N induced LMP. It can be concluded that a more lysosomotropic derivative was synthesized by introduction of an amine group, which could have more potential applications in cancer therapy.

4-Oxo-1,4-dihydroquinoline-3-carboxamide Derivatives as New Axl Kinase Inhibitors.

Axl is a new potential target for anticancer drug discovery. A series of 4-oxo-1,4-dihydroquinoline-3-carboxamides were designed and synthesized as highly potent Axl kinase inhibitors. One of the most promising compounds, 9im, tightly bound with Axl protein and potently inhibited its kinase function with a Kd value of 2.7 nM and an IC50 value of 4.0 nM, respectively, while was obviously less potent against most of the 403 wild-type kinases evaluated at a relatively high concentration. The compound dose-dependently inhibited the TGF-β1-induced epithelial-mesenchymal transition (EMT) and suppressed the migration and invasion of MDA-MB-231 breast cancer cells. In addition, 9im also demonstrated reasonable pharmacokinetics properties in rats and exhibited in vivo therapeutic effect on hepatic metastasis in a xenograft model of highly metastatic 4T1 murine breast cancer cells. Compound 9im may serve as a lead compound for new anticancer drug discovery and a valuable research probe for further biological investigation on Axl.

Discovery and Optimization of 1-(4-chloro-3-(trifluoromethyl)- phenyl)-3-(2-(amino)pyridin-3-yl)ureas as Novel KDR Kinase Inhibitors.

Kinase insert Domain-containing Receptor (KDR) is one of the currently validated targets for anticancer drug discovery and development. Herein, a series of o-amino-arylurea derivatives have been synthesized and evaluated for their kinase inhibitory activity. The optimization on the basis of biological screening and molecular modeling resulted in obvious increase in KDR kinase inhibitory activity compared with the hit compound. Eventually, we identified a potent inhibitor 5a of 1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(2-((quinolin-4-ylmethyl) amino)pyridin-3-yl)urea scaffold against KDR (IC50 = 0.0689 µM), which can serve as good starting point for further KDR inhibitor optimization and development.

Structure-Based Drug Design of Small Molecule Peptide Deformylase Inhibitors to Treat Cancer.

Human peptide deformylase (HsPDF) is an important target for anticancer drug discovery. In view of the limited HsPDF, inhibitors were reported, and high-throughput virtual screening (HTVS) studies based on HsPDF for developing new PDF inhibitors remain to be reported. We reported here on diverse small molecule inhibitors with excellent anticancer activities designed based on HTVS and molecular docking studies using the crystal structure of HsPDF. The compound M7594_0037 exhibited potent anticancer activities against HeLa, A549 and MCF-7 cell lines with IC50s of 35.26, 29.63 and 24.63 μM, respectively. Molecular docking studies suggested that M7594_0037 and its three derivatives could interact with HsPDF by several conserved hydrogen bonds. Moreover, the pharmacokinetic and toxicity properties of M7594_0037 and its derivatives were predicted using the OSIRIS property explorer. Thus, M7594_0037 and its derivatives might represent a promising scaffold for the further development of novel anticancer drugs.

Anacardic Acids from Knema hookeriana as Modulators of Bcl-xL/Bak and Mcl-1/Bid Interactions.

Proteins of the Bcl-2 family are key targets in anticancer drug discovery. Disrupting the interaction between anti- and pro-apoptotic members of this protein family was the approach chosen in this study to restore apoptosis. Thus, a biological screening on the modulation of the Bcl-xL/Bak and Mcl-1/Bid interactions permitted the selection of Knema hookeriana for further phytochemical investigations. The ethyl acetate extract from the stem bark led to the isolation of six new compounds, three acetophenone derivatives (1-3) and three anacardic acid derivatives (4-6), along with four known anacardic acids (7-10) and two cardanols (11, 12). Their structures were elucidated by 1D and 2D NMR analysis in combination with HRMS experiments. The ability of these compounds to antagonize Bcl-xL/Bak and Mcl-1/Bid association was determined, using a protein-protein interaction assay, but only anacardic acid derivatives (4-10) exhibited significant binding properties, with Ki values ranging from 0.2 to 18 μM. Protein-ligand NMR experiments further revealed that anacardic acid 9, the most active compound, does not interact with the anti-apoptotic proteins Bcl-xL and Mcl-1 but instead interacts with pro-apoptotic protein Bid.

Direct Inhibitors of Ras-Effector Protein Interactions.

Activating Ras mutations are associated with ~30% of all human cancers, which often respond poorly to standard therapies. The four Ras isoforms are therefore highly attractive targets for anticancer drug discovery. However, Ras proteins function through protein-protein interactions and their surfaces lack any major pockets for small molecules to bind; as a result they have been declared "undruggable" for the past 30 years. Several breakthroughs during the past few years may finally remove Ras from the list of undruggable proteins. This mini-review discusses the current approaches to developing inhibitors especially cyclic peptides that physically block the interaction between Ras and its downstream effector proteins, which is potentially the most effective approach for treating Ras mutant cancers.

Novel 3-substituted-2-oxoindoline-based N-hydroxypropenamides as histone deacetylase inhibitors and antitumor agents.

Histone deacetylases (HDAC) are currently a group of validated targets for anticancer drug discovery and development. In our research program to find novel small molecules targeting these enzymes, we designed and synthesized two series of 3-hydroxyimino-2-oxoindoline- and 3- methoxyimino-2-oxoindoline-based N-hydroxypropenamides (3a-g, 6a-g). The results show that these propenamides potently inhibited HDAC2 with IC50 values in sub-micromolar range, approximately 10-fold lower than that of SAHA (also known as suberoylanilohydroxamic acid). Evaluation of cytotoxicity of these compounds in three human cancer cell lines revealed that most of the synthesized compounds were up to 5-fold more cytotoxic than SAHA. Docking studies showed that the compounds bound to HDAC2 at the binding site with higher binding affinities compared to SAHA. Our present results demonstrate that these novel 3-substituted-2-oxoindoline-based N-hydroxypropenamides are potential for further development as anticancer agents.

Ligand-based chemoinformatic discovery of a novel small molecule inhibitor targeting CDC25 dual specificity phosphatases and displayingin vitroefficacy against melanoma cells

CDC25 phosphatases are important regulators of the cell cycle and represent promising targets for anticancer drug discovery. We recently identified NSC 119915 as a new quinonoid CDC25 inhibitor with potent anticancer activity. In order to discover more active analogs of NSC 119915, we performed a range of ligand-based chemoinformatic methods against the full ZINC drug-like subset and the NCI lead-like set. Nine compounds (3, 5-9, 21, 24, and 25) were identified with Ki values for CDC25A, -B and -C ranging from 0.01 to 4.4 μM. One of these analogs, 7, showed a high antiproliferative effect on human melanoma cell lines, A2058 and SAN. Compound 7 arrested melanoma cells in G2/M, causing a reduction of the protein levels of CDC25A and, more consistently, of CDC25C. Furthermore, an intrinsic apoptotic pathway was induced, which was mediated by ROS, because it was reverted in the presence of antioxidant N-acetyl-cysteine (NAC). Finally, 7 decreased the protein levels of phosphorylated Akt and increased those of p53, thus contributing to the regulation of chemosensitivity through the control of downstream Akt pathways in melanoma cells. Taken together, our data emphasize that CDC25 could be considered as a possible oncotarget in melanoma cells and that compound 7 is a small molecule CDC25 inhibitor that merits to be further evaluated as a chemotherapeutic agent for melanoma, likely in combination with other therapeutic compounds.

Oncogenic and RASopathy-associated K-RAS mutations relieve membrane-dependent occlusion of the effector-binding site

K-RAS4B (Kirsten rat sarcoma viral oncogene homolog 4B) is a prenylated, membrane-associated GTPase protein that is a critical switch for the propagation of growth factor signaling pathways to diverse effector proteins, including rapidly accelerated fibrosarcoma (RAF) kinases and RAS-related protein guanine nucleotide dissociation stimulator (RALGDS) proteins. Gain-of-function KRAS mutations occur frequently in human cancers and predict poor clinical outcome, whereas germ-line mutations are associated with developmental syndromes. However, it is not known how these mutations affect K-RAS association with biological membranes or whether this impacts signal transduction. Here, we used solution NMR studies of K-RAS4B tethered to nanodiscs to investigate lipid bilayer-anchored K-RAS4B and its interactions with effector protein RAS-binding domains (RBDs). Unexpectedly, we found that the effector-binding region of activated K-RAS4B is occluded by interaction with the membrane in one of the NMR-observable, and thus highly populated, conformational states. Binding of the RAF isoform ARAF and RALGDS RBDs induced marked reorientation of K-RAS4B from the occluded state to RBD-specific effector-bound states. Importantly, we found that two Noonan syndrome-associated mutations, K5N and D153V, which do not affect the GTPase cycle, relieve the occluded orientation by directly altering the electrostatics of two membrane interaction surfaces. Similarly, the most frequent KRAS oncogenic mutation G12D also drives K-RAS4B toward an exposed configuration. Further, the D153V and G12D mutations increase the rate of association of ARAF-RBD with lipid bilayer-tethered K-RAS4B. We revealed a mechanism of K-RAS4B autoinhibition by membrane sequestration of its effector-binding site, which can be disrupted by disease-associated mutations. Stabilizing the autoinhibitory interactions between K-RAS4B and the membrane could be an attractive target for anticancer drug discovery.

Investigating the Role of 3‐Phosphoinositide‐Dependent Protein Kinase 1 in the Spatiotemporal Regulation of the Purinosome

The enzymes involved in human de novo purine biosynthesis form a transient multienzyme complex, the “purinosome,” under purine starvation. To determine cellular regulatory mechanisms of purinosome assembly/disassembly, fluorescence live‐cell imaging has been used to monitor purinosome dynamics following small molecular inhibition/activation of protein kinases involved in cell signaling. To date, we observe that pharmacological inhibition of 3‐phosphoinositide‐dependent protein kinase 1 (PDK1) resulted in purinosome formation, but not purinosome dissociation, in HeLa and Hs578T cells. Subsequent inhibition of protein kinase C, one of downstream kinases of PDK1, resulted in purinosome formation as well. However,small‐molecule inhibition of other downstream targets of PDK1, including Akt and mammalian Target of Rapamycin (mTOR), exhibited no effect on purinosome assembly. We hypothesize that PDK1‐associated signaling pathways negatively regulate purinosome formation in an Akt‐independent manner. Collectively, our work has the potential to demonstrate the functional role of PDK1 on de novo purine biosynthesis, thus providing a novel target for anti‐cancer drug discovery.

Discovery of biphenyl-based VEGFR-2 inhibitors. Part 3: Design, synthesis and 3D-QSAR studies

VEGFR-2 plays an essential role in angiogenesis and is a central target for anticancer drug discovery. In order to develop novel VEGFR-2 inhibitors, we designed and synthesized 33 biphenyl amides based on our previously reported lead compound. The biological results indicated that four compounds (18b, 20e, 20h and 20j) are potent VEGFR-2 inhibitors which are comparable to positive control. Compound 18b displayed the most potent VEGFR-2 inhibition with IC50 value of 2.02nM. Moreover, it exhibited promising antiproliferative activity against MCF-7 and SMMC-7721 cells with IC50 values of 1.47μM and 5.98μM, respectively. Molecular docking and 3D-QSAR studies were also carried out. The results indicated that these biphenyl amides could serve as promising leads for further optimization as novel VEGFR-2 inhibitors.