Discovery of multi-target receptor tyrosine kinase inhibitors as novel anti-angiogenesis agents

Recently, we have identified a biphenyl-aryl urea incorporated with salicylaldoxime (BPS-7) as an anti-angiogenesis agent. Herein, we disclosed a series of novel anti-angiogenesis agents with BPS-7 as lead compound through combining diarylureas with N-pyridin-2-ylcyclopropane carboxamide. Several title compounds exhibited simultaneous inhibition effects against three pro-angiogenic RTKs (VEGFR-2, TIE-2 and EphB4). Some of them displayed potent anti-proliferative activity against human vascular endothelial cell (EA.hy926). In particular, two potent compounds (CDAU-1 and CDAU-2) could be considered as promising anti-angiogenesis agents with triplet inhibition profile. The biological evaluation and molecular docking results indicate that N-pyridin-2-ylcyclopropane carboxamide could serve as a hinge-binding group (HBG) for the discovery of multi-target anti-angiogenesis agents. CDAU-2 also exhibited promising anti-angiogenic potency in a tissue model for angiogenesis.

Pathological angiogenesis plays a critical role in numerous diseases including cancer, rheumatoid arthritis, and retinopathies 1 . Recently, many efforts have been focused on the discovery of novel anti-angiogenesis agents for the treatment of cancer, ocular, joint or skin disorders 2,3 . Receptor tyrosine kinases (RTKs) display a variety of biological activities including cell proliferation and migration. Multiple RTKs are involved in pathological angiogenesis and have been identified as valid targets for developing anti-angiogenesis agents. Inhibition of angiogenic RTKs has been also considered as a systemic strategy for tumor treatment. Vascular endothelial growth factor (VEGF) is one of the most potent pro-angiogenic factors, and its binding to the receptors (VEGFRs) promotes endothelial cell survival, proliferation, and migration 4 . VEGFR families mainly consists of VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1), and VEGFR-3 (Flt-4) 5,6 . Among all of them, VEGFR-2 plays an essential role in tumor angiogenesis 7 . Likewise, angiogenic growth factors (Ang) and their tyrosine kinase with Ig and EGF homology domain-2 (TIE-2) have been implicated in sprouting, branching, remodeling and maturation of the blood vessels [8][9][10] . Recent research indicated that erythropoietin producing hepatocyte receptor B4 (EphB4) and its transmembrane-type ligand (ephrin B2) were crucial for angiogenesis, vessel maturation, and pericyte recruitment 11 . EphB4 belongs to the Eph family which is the largest subfamily of RTKs 12 . Meaningfully, EphB4-ephrinB2 signaling pathway play critical roles in tumor vessel development and maturation 13,14 . Tumor growth is dependent on multiple factors, including the physiological process of angiogenesis. It has been demonstrated that tumors are capable of secreting multiple angiogenic growth factors. Inhibiting angiogenesis have been identified as a new approach for inhibition of tumor growth, and its clinical efficacy are being evaluated now. Through this approach, some promising small-molecule inhibitors towards various angiogenic RTKs including VEGFR-2, FGFR, PDGFR, TIE-2 and EphB4 are reported. However, it has been also demonstrated that inhibition of a single angiogenic pathway in angiogenesis could induce the acquired resistance. Maximizing the potential of anti-angiogenic therapy is likely to require a broader therapeutic approach using a new generation of multi-target anti-angiogenesis agents. We speculate that simultaneous inhibition of multiple RTKs might be a good strategy to address this issue and achieve maximal clinical efficacy. There are several small molecular multi-target RTK inhibitors in clinical trials as anti-angiogenesis agents. Four drugs with different inhibition profile against angiogenic RTKs have been approved including sorafenib 15 , linifanib 16 , vandetanib 17 , and cabozantinib 18 . Herein, we described here the first discovery of multiple inhibitors of VEGFR-2/EphB4/TIE-2 as novel anti-angiogenesis agents. and ATP-binding site in the active conformation of these RTKs are notably similar. In addition, it is reasonable to assume that all the ATP-competitive RTK inhibitors with anti-angiogenesis potency share the same binding sites. Structural and sequence comparison of three RTKs (VEGFR-2, TIE-2, and EphB4) were depicted in Fig. 1. It is indicated that extensive sequence homology is evident along the kinase domain among VEGFR-2, TIE-2, and EphB4. Moreover, they share several short motifs including glycine-rich loop, conserved glutamate, gatekeeper domain, hinge region, and DFG-motif. All these similar motifs are essential for their catalytic activity and design of novel inhibitors.
Alignment of the crystal structures of three RTKs is depicted in Fig. 2. Structural alignment and comparison of independence and full sets of inter-residue distances indicated that there were no significant difference in root mean square distance (RMSD). The difference in RMSD for them are 1.178 Å, 0.949 Å, and 0.981 Å, respectively. As they displayed a similar two-lobed architecture, we believe that the large size of the RTK family and the similarity of kinase domains make the multiplex inhibition strategy a feasible drug discovery approach.
Numbers of biphenyl-aryl ureas incorporated with salicylaldoxime have been developed as potent and selective VEGFR-2 inhibitors. We have identified salicylaldoxime as a novel HBG of multi-target RTK inhibitors. A novel biphenyl-aryl urea bearing salicylaldoxime (BPS-7) has been developed with natural alkaloid taspine as lead compound (Fig. 3). Fortunately, BPS-7 also displayed selective inhibition against angiogenic EphB4 and TIE-2. Moreover, it significantly inhibited the proliferation and migration of human umbilical vein endothelial cells 19 . Based on our previous findings, we propose that multiplex inhibition of VEGFR-2, TIE-2 and EphB4 would enhance anti-angiogenesis effect and reduce the occurrence of drug resistance 20 .
In order to continue our study in discovery of novel anti-angiogenesis agents, we explored the structural diversity of hinge-binding group (HBG) of BPS-7. N-pyridin-2-ylcyclopropane carboxamide was incorporated as HBG of VEGFR-2/TIE-2/EphB4 multiple inhibitors. We supposed that it might simultaneously form hydrogen  bonds with hinge of three RTKs and therefore provide an opportunity to improve affinity. Moreover, the two methoxyl groups on biphenyl have been removed in order to reduce the steric hindrance of inhibitors when binding with receptors. In summary, we designed and synthesized a series of diaryl ureas incorporation of N-pyridin-2-ylcyclopropane carboxamide as novel multi-target anti-angiogenesis agents (Fig. 4). Biological evaluation and molecular modeling of these multiple VEGFR-2/TIE-2/EphB4 inhibitors were also carried out.
The synthetic steps of ureas (8a-8n) were similar to the synthesis of intermediates (3a-3k). The key intermediates (9a-9n) were prepared from corresponding ureas (8a-8n) and 2-amino-6-bromopyridine (6) by Pd-catalyzed Suzuki coupling reaction. Finally, cyclopropanecarbonyl chloride coupling of (9a-9n) through the acylation reaction afford the title compounds (CDAU-12~CDAU-25). All the title compounds characterized by 1 H-NMR, 13 C-NMR, high resolution mass spectrum (HRMS), and melting point analysis and their purity were above 95% determined by LC-MS (Supplementary Information).  RTK inhibitory activity. All the title compounds were evaluated for their inhibitory potency against VEGFR-2, TIE-2 and EphB4 with sorafenib as positive control. Tyrosine kinase inhibition were tested by luminescent ADP-Glo TM assay. As observed in Table 1, several compounds exhibited simultaneous inhibition against the three angiogenic RTKs. In particular, compound CDAU-1 and CDAU-2 displayed the most potent activity against VEGFR-2, TIE-2 and EphB4 with IC 50 values of 1.11 nM, 7.20 nM, 5.34 nM (CDAU-1) and 1.01 nM, 8.32 nM, 5.11 nM (CDAU-2), respectively. In addition, another two compounds (CDAU-3 and CDAU-5) bearing trifluoromethyl group exhibited potent inhibitory activity against VEGFR-2 and TIE-2. The results indicated that dual-halogen substituent and trifluoromethyl were the most favorable for their enzymatic inhibitory activity. However, the compounds incorporated with aliphatic amine at terminal (CDAU-9, CDAU-10 and CDAU-11) showed the poorest activity. It might be due to the deficiency of interaction with allosteric site of RTKs.   For title compounds with cyclopropancarboxamid at meta-position of biphenyl (Table 2), the majority of them exhibited poor activity except for compound bearing 3-trifluoromethyl substituent on terminal aniline (CDAU-21). It displayed moderate RTKs (VEGFR-2, TIE-2 and EphB4) inhibitory activities with IC 50 values of 6.27 nM, 42.76 nM, and 161.74 nM, respectively. These results indicated that the position of the cyclopropancarboxamid plays critical role in tyrosine kinase inhibition. Para-position was more favorable than meta-position for their potency. We speculate that para-position was beneficial for interaction with the hinge region. In addition, terminal anilines incorporated with trifluoromethyl were beneficial for VEGFR-2/TIE-2/EphB4 inhibitory activities. The inhibition against three angiogenic RTKs indicated that N-pyridin-2-ylcyclopropane carboxamide could serve as novel hinge-binding group for VEGFR-2/TIE-2/EphB4 multi-kinase inhibitors.
Kinase Selectivity assays. We investigated the kinase selectivity of the most potent compounds (CDAU-1, CDAU-2) against other four kinases including B-Raf, FGFR-1, EGFR, and Src for its selective profile. The results were summarized in Table 3. The results revealed that they showed less potency against FGFR-1, EGFR, and Src compared with VEGFR-2, TIE-2, and EphB4. Moreover, they also exhibited potent B-Raf inhibitory activity. It was indicated that they exhibited moderate selectivity for VEGFR-2/TIE-2/EphB4 relative to other RTKs including FGFR-1, EGFR, and Src.

Cell growth inhibition.
On the basis of RTK inhibition assays, thirteen title compounds were selected and further evaluated for their anti-proliferative activity against human vascular endothelial cell (EA.hy926) with sorafenib as positive control. As shown in Table 4, the majority of them displayed moderate to high anti-proliferative activities with IC 50 values ranging from 16     respectively. These two compounds represent the first class of multiplex inhibitors with a "triplet" inhibition profile as well as anti-angiogenesis potency. They might not only inhibit the process of angiogenesis, but also prevent the occurrence of resistance. Anti-angiogenesis potency on the tissue model for angiogenesis. A novel tissue model for angiogenesis that imitated new blood vessels formation in vivo had been established in the previous study 23 . Here, it was used to evaluate the anti-angiogenesis potency of the most potent CDAU-2. The results indicated that CDAU-2 could effectively inhibit the blood vessels formation (Fig. 8). CDAU-2 inhibited vessels outgrew from the periphery of the lung tissues evidently at 15.6 μ M, and exhibited good reproducibility. Furthermore, CDAU-2 could potently inhibit vessels growth at the tested concentrations.

Discussion
Herein, we disclosed the first class of multi-target inhibitors with a "triplet" inhibition profile. Extensive investigations linked drug resistance with compensatory activation of angiogenic RTKs, especially for VEGFR-2, TIE-2, and EphB4. Moreover, complexity and heterogeneity of angiogenesis make it difficult to be treated with single target agents. Accordingly, we proposed that multiple inhibition of RTKs could enhance the efficacy and overcome the resistance on the basis of vascular normalization concept. Meanwhile, it is feasible to develop multiple inhibitors against VEGFR-2/TIE-2/EphB4 because of their highly conserved DFG-out conformation. These novel strategies have yielded promising results in the discovery of anti-angiogenesis agents. We have developed the first class of multiple inhibitors of VEGFR-2/TIE-2/EphB4. Simultaneous blockade of VEGFR-2/TIE-2/EphB4 signaling pathways leads to inhibition of endothelial cell survival, vascular permeability, migration, and proliferation within angiogenesis (Fig. 9). These novel inhibitors might contribute to the discovery of novel anti-angiogenesis agents with VEGFR-2/TIE-2/EphB4 as multiple targets.

Conclusion
In conclusion, we described the discovery of multi-target inhibitors as novel anti-angiogenesis agents. N-pyridin-2-ylcyclopropane carboxamide was firstly introduced to diaryl urea core as hinge-binding group. A series of diarylureas incorporated of N-pyridin-2-ylcyclopropane carboxamide were designed, synthesized and evaluated as multi-target inhibitors of VEGFR-2/TIE-2/EphB4. The biological evaluation revealed that compounds CDAU-1 and CDAU-2 exhibited the most potent inhibitory activity against VEGFR-2, TIE-2 and EphB4. Meanwhile, they displayed potent anti-proliferative activity against human vascular endothelial cell (EA. hy926). These two compounds represented the first class of multiple VEGFR-2/EphB4/TIE-2 inhibitors with a "triplet" inhibition profile.  The most potent CDAU-2 also exhibited promising anti-angiogenic potency in a tissue model for angiogenesis. Moreover, biological evaluation and molecular docking indicated that N-pyridin-2-ylcyclopropane carboxamide is beneficial for potency of these multi-target inhibitors. It could be considered as a novel hinge-binding group for further discovery of multi-target anti-angiogenesis agents. Our results may contribute to the discovery of novel anti-angiogenesis agents for the intervention of pathological angiogenesis-related diseases.   (2) (1.50 g, 6.85 mmol) in anhydrous CH 2 Cl 2 (10 mL) was added and continued stirring for 20 min. Subsequently, the ice bath was removed, and the mixture was reacted at room temperature overnight. After completion of the action, the reaction was quenched with dilute NaHCO 3 . The organic layer was washed with water and brine, and dried over Na 2 SO 4 . After filtration and concentration in vacuo, the residues was purified by silica gel flash chromatography (PE/AcOEt = 7:1) affording (3a) as white solid (1.77 g, 63.67%).

N-(6-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)phenyl)pyridin-2-yl)cyclopropanecarboxamide
Angiogenic RTK inhibition evaluation. The in vitro kinase inhibition assays against VEGFR-2, TIE-2, and EphB4 of all the title compounds were detected using the ADP-Glo ™ kinase assay kit (Promega, Madison) with sorafenib as positive control 24 . The kinase assay was performed in duplicate in a reaction mixture of final volume of 10 μ L. General procedures are as the following: for VEGFR-2 assays, the tyrosine kinase (0.6 ng/mL) were incubated with substrates (0.2 mg/mL), tested title compounds (1.2 × 10 −4~1 2 μ M) and ATP (50 μ M) in a final buffer of Tris 40 mM, MgCl 2 10 mM, BSA 0.1 mg/mL, DTT 1 mM in 384-well plate with the total volume of 5 μ L. The assay plate was incubated at 30 °C for 1 h. After the plate was cooled at room temperature for 5 min, 5 μ L of ADP-Glo reagent was added into each well to stop the reaction and consume the remaining ADP within 40 min. At the end, 10 μ L of kinase detection reagent was added into the well and incubated for 30 min to produce a luminescence signal. As for TIE-2 and EphB4 assays, the tyrosine kinase (2.4 ng/mL) were incubated with substrates (0.2 mg/mL), tested title compounds (1.2 × 10 −4~1 2 μ M) and ATP (50 μ M) in a final buffer of Tris 40 mM, MgCl 2 10 mM, BSA 0.1 mg/mL, DTT 1 mM in 384-well plate with the total volume of 5 μ L. The assay plate was incubated at 30 °C for 4 h. After the plate was cooled at room temperature for 5 min, 5 μ L of ADP-Glo reagent was added into each well to stop the reaction and consume the remaining ADP within 1 h. At the end, 10 μ L of kinase detection reagent was added into the well and incubated for 30 min to produce a luminescence signal. The luminescence was read by VICTOR-X multi-label plate reader. The signal was correlated with the amount of ATP present in the reaction and was inversely correlated with the kinase activity.
Cell growth inhibitory activity in cancer cell lines. Growth inhibitory activities were evaluated against human vascular endothelial cell (EA.hy926) 25 . Thirteen selected title compounds were tested using MTT assay to assess cell proliferation. Exponentially growing cells were harvested and plated in 96-well plates at a concentration of 1 × 1 4 cells/well, and then incubated for 24 h at 37 °C. The cells were treated with title compounds respectively at various concentrations for 48 h. Then, 22 mL fresh MTT (5 mg/mL) was added to each well and incubated for 4 h at 37 °C. Supernatant was discarded, and 150 mL DMSO was added to each well. Absorbance values were determined by a microplate reader (Bio-Rad Instruments) at 490 nm. The IC 50 values were calculated according to inhibition ratios.

Molecular docking modeling.
In order to understand the binding mode of inhibitors with VEGFR-2/ TIE-2/EphB4, molecule docking was performed using Sybyl-X/Surflex-dock module based on the crystal structures of VEGFR-2 (PDB ID: 4ASD), TIE-2 (PDB ID: 2P4I) and EphB4 (PDB ID: 4BB4) 26 . Hydrogen was added and minimized using the Tripos force field and Pullman charges. The most potent compound (CDAU-A2) was depicted with the Sybyl-X/Sketch module (Tripos Inc.) and optimized applying Powell's method with the Tripos force field with convergence criterion set at 0.05 kcal/(Åmol), and assigned with the Gasteiger-Hückel charge. The docking studied was carried out using Surflex-dock module. The residues in a radius 5.0 Å around the ligand of VEGFR-2/TIE-2/ EphB4 in the crystal complex were selected as the active site. Other docking parameters were kept at default. Screening of CDAU-2 on the tissue model for angiogenesis. In brief, the mouse lung tissue was separated and cut into pieces (0.5-1.0 mm 3 ) 27 . Then pieces of lung tissue were placed onto 48-well plate coated with polymerized fibrinogen with thrombin. After consolidation, each group was incubated with 200 μ L/well 1640 medium containing different concentrations of CDAU-2. Control groups were incubated with 2.5% DMSO or 1640 medium alone. The sprouting vessels were observed at the fifth day post treatment.