Design, synthesis, and evaluation of imidazo[1,2-b]pyridazine derivatives having a benzamide unit as novel VEGFR2 kinase inhibitors
Graphical abstract
Introduction
Angiogenesis, which is the formation of new capillary blood vessels from preexisting ones, is a crucial process that promotes tumor growth, survival, and metastasis.1 Since it was hypothesized that the inhibition of angiogenesis could be an effective strategy for cancer therapy in 1971,2 several regulators of angiogenesis, such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and angiopoietin, have been identified.3 Among them, the VEGF signaling pathway that acts through the VEGF receptor 2 (VEGFR2, also known as KDR) has been shown to play a key role in the regulation of tumor angiogenesis, in which the binding of VEGF to VEGFR2 leads to receptor dimerization, which is followed by the autophosphorylation of tyrosine residues in the intracellular kinase domain, resulting in potent mitogenic and chemotactic effects on endothelial cells.4, 5 The expression of VEGF is upregulated by tumor-related changes, such as hypoxia, protooncogene activation, and the aberration of tumor-suppressor genes.6, 7, 8 The overexpression of VEGF correlates with poor prognosis and the clinical stage of patients with solid tumors.9, 10, 11 Therefore, VEGF/VEGFR2 signaling has been thought to be an attractive target for the treatment of cancer. A humanized anti-VEGF monoclonal antibody (bevacizumab)12 and small-molecule VEGFR2 kinase inhibitors (sorafenib,13 sunitinib,14 and pazopanib15) have been approved, and these have demonstrated clinical benefits in the treatment of some tumors with manageable side effects. Many angiogenesis inhibitors are also being evaluated in clinical trials for the treatment of various cancers.16
In our previous study, we reported that the pyrrolo[3,2-d]pyrimidine derivative 1 (Fig. 1) had potent inhibitory activity against VEGFR2 kinase (IC50 = 6.2 nM) and showed antitumor effects in a xenograft nude mouse model with human prostate cancer DU145 cells.17 The crystal structure analysis of compound 1 in complex with VEGFR2 revealed that 1 bound to the inactive form of VEGFR2 (Fig. 2). When 1 bound to VEGFR2, the N1-nitrogen of the pyrrolo[3,2-d]pyrimidine core formed a hydrogen bond with the backbone NH group of Cys919 in the hinge region. The urea NH and carbonyl groups interacted with Glu885 and Asp1046, respectively. The external phenyl group occupied a hydrophobic pocket that was created by the rearrangement of the protein. These observations were similar to those in the crystal structure of the known pyridine- and pyrimidine-based compounds that were bound with VEGFR2.18 Because the VEGFR2 kinase inhibitors possessing a fused or nonfused pyridine/pyrimidine core have been well investigated, our interest moved to new scaffolds for VEGFR2 kinase inhibitors, which are expected to acquire different kinase selectivities and novelties. Our strategy was the replacement of the pyrrolo[3,2-d]pyrimidine by other ring systems that have a hydrogen bond acceptor (HBA) nitrogen, so that the compounds retain the essential hydrogen bonding with the main chain of Cys919. Based on this strategy, we moved the HBA site from the 6-membered ring to the 5-membered ring, which resulted in the discovery of an imidazo[1,2-b]pyridazine core as a novel hinge-binding template (2, Fig. 1). In fact, compound 2 showed moderate inhibitory activity against VEGFR2 kinase with an IC50 value of 5200 nM. Thus, we investigated phenoxy substituents (–Y–R) with the aim of finding more effective interactions with Glu885, Asp1046, and the hydrophobic pocket. In this paper, we describe the synthesis, structure–activity relationships, and characterization of these new inhibitors.
Section snippets
Chemistry
The target compounds were prepared according to the synthetic routes shown in Scheme 1, Scheme 2. The 6-chloroimidazo[1,2-b]pyridazine (3)19 was treated with aminophenols in the presence of K2CO3 in order to obtain the 6-phenoxyimidazo[1,2-b]pyridazines 4a–c, which were converted to the ureas 2 and 5a–d with appropriate phenyl isocyanates (Scheme 1). The benzamides 6a and 6b were obtained by treatment of 4b with benzoyl chloride and 3-(trifluoromethyl)benzoyl chloride, respectively.
For the
Results and discussion
The imidazo[1,2-b]pyridazine derivatives that are depicted in Table 1, Table 2 were evaluated for their inhibitory activities against human VEGFR2 kinase with a non-RI assay using the amplified luminescent proximity homogeneous assay (AlphaScreen) system.20 AlphaScreen is based on the transfer of energy from donor to acceptor microbeads that are brought together by a biomolecular interaction. In this system, an anti-phosphotyrosine antibody is immobilized with the acceptor beads, and the
Conclusion
Utilizing the crystal structure information of the pyrrolo[3,2-d]pyrimidine 1 with VEGFR2, we designed and synthesized novel VEGFR2 inhibitors. Scaffold hopping from 1 resulted in the discovery of the imidazo[1,2-b]pyridazine derivative 2, which showed moderate inhibitory activity against VEGFR2 kinase. Optimization of the phenylureido group of 2 revealed that meta-substitution was optimal, which led to the identification of 5a. As a result of further examination, the benzamide derivative 6a
Experimental section
Commercial reagents and solvents were used without additional purification. Reaction progress was determined by thin layer chromatography (TLC) on Silica Gel 60 F254 plates (Merck) or NH TLC plates (Fuji Silysia). Column chromatography was carried out using a Purif Purification System (Moritex) with silica gel (Purif-Pack SI-60, Fuji Silysia) or basic silica gel (Purif-Pack NH-60, Fuji Silysia) cartridges. Melting points were determined on a Yanagimoto micro melting point, BÜCHI Melting Point
Acknowledgment
The authors thank Ms. T. Yoshida for in vitro assays and Dr. K. Kamiyama for helpful discussion.
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