Design, synthesis, and evaluation of 5-methyl-4-phenoxy-5H-pyrrolo[3,2-d]pyrimidine derivatives: Novel VEGFR2 kinase inhibitors binding to inactive kinase conformation
Introduction
Angiogenesis, the formation of new capillary blood vessels from preexisting vasculature, is the process by which solid tumors are supplied with oxygen and nutrients.1 Angiogenesis is increased in various types of cancers, and high microvessel density in the tumor correlates with poor prognosis in patients.2, 3 Therefore, the inhibition of tumor angiogenesis is considered to be a promising approach for the treatment of many human malignancies.4
The vascular endothelial growth factor (VEGF) family is a large family of angiogenic and lymphangiogenic growth factors, and VEGF plays an important role in tumor angiogenesis.5 In solid tumors, VEGF expression is upregulated by cancer-related changes, such as proto-oncogene activation,6 loss of tumor suppressor function,7, 8 growth factor stimuli,9 and hypoxic status.10, 11 Angiogenesis is triggered by the binding of VEGF to vascular endothelial growth factor receptor (VEGFR); the different subtypes of VEGFR include VEGFR1 (also known as Flt1), VEGFR2 (also known as KDR), and VEGFR3 (also known as Flt4).5 Binding of VEGF to VEGFR induces conformational changes in VEGFR followed by receptor dimerization, autophosphorylation of tyrosine residues in the intracellular kinase domain and exerts potent mitogenic and chemotactic effects on endothelial cells.12 VEGF overexpression correlates with poor prognosis and clinical staging in the majority of solid tumor patients.13, 14, 15 VEGF/VEGFR signaling is, therefore, regarded as an attractive therapeutic target for inhibition of tumor angiogenesis. Bevacizumab, a recombinant humanized monoclonal antibody against VEGF, has been approved as first-line therapy for various conditions such as metastatic colorectal cancer.16, 17, 18
The majority of known kinase inhibitors such as gefitinib are classified as type-I kinase inhibitors, which bind in and around the region occupied by the adenine ring of ATP.19 On the other hand, type-II kinase inhibitors (e.g., imatinib) induce the DFG-out conformation of the activation loop, enabling them to occupy the adenine binding site and an adjacent hydrophobic pocket created by this rearrangement.20 The X-ray co-crystal structures of kinase-bound type-II kinase inhibitors also generally reveal two conserved hydrogen-bond interactions between the ligand and protein: one with the side chain of a conserved glutamic acid in the αC-helix (Glu885 in VEGFR2), and the other with the backbone amide of aspartic acid in the DFG motif (Asp1046 in VEGFR2). Type-II kinase inhibitors have several advantages over type-I kinase inhibitors, including improved kinase selectivity and slower off-rates.21, 22 Therefore, we focused on the identification of novel type-II VEGFR kinase inhibitors. Studies to identify novel kinase inhibitors showed that the pyrrolo[3,2-d]pyrimidine scaffold could serve as a novel kinase hinge binding template.23 This finding encouraged us to generate novel type-II kinase inhibitors by incorporating a substituted benzene ring at the C4-position of the pyrrolo[3,2-d]pyrimidine core via an appropriate linker (X) (Fig. 1). To rationalize the designed compound as a good starting point for type-II inhibitors, the binding model of VEGFR2 with the pyrrolo[3,2-d]pyrimidine derivative (A) (X = O, R′ = H, Y-R = H in Fig. 1) was generated. The binding model suggested the N1-nitrogen of the pyrrolo[3,2-d]pyrimidine interacts with the main chain NH group of Cys919 in the hinge region and the phenoxy group extended toward the back hydrophobic pocket. Thus, we designed the compounds possessing a hydrogen bond donor–acceptor pair (Y) and a lipophilic group (R) on the benzene ring, aiming interaction with Glu885, Asp1046, and the hydrophobic pocket. In this paper, we report the synthesis, structure–activity relationships (SAR), and characterization of these new inhibitors.
Section snippets
Chemistry
A general synthesis of 4-phenoxy-pyrrolo[3,2-d]pyrimidine derivatives possessing urea, amide, thiourea, and benzimidazole functionalities is shown in Scheme 1, Scheme 2. Synthesis was initiated by treating pyrrolo[3,2-d]pyrimidine 124, 25 with methyl methanesulfonate to afford 2, which was coupled with 3- or 4-aminophenol in the presence of potassium carbonate to provide aniline 3 or 6, respectively. Aniline 3 was converted to the corresponding urea 4 or amide 5 using phenyl isocyanate or
Results and discussion
The pyrrolo[3,2-d]pyrimidine derivatives depicted in Table 1, Table 2, Table 3, Table 4 were evaluated for their inhibitory activity against human VEGFR2 kinase by a non-RI assay using the amplified luminescent proximity homogeneous assay (AlphaScreen®) system. AlphaScreen® is based on the transfer of energy from donor to acceptor microbeads brought together by a biomolecular interaction.26 In this system, an anti-phosphotyrosine antibody is immobilized with acceptor beads, and the biotinylated
Conclusion
In order to generate novel type-II inhibitors of VEGFR kinase, we designed and synthesized a series of pyrrolo[3,2-d]pyrimidine derivatives. Incorporation of a diphenylurea moiety at the C4-position of the pyrrolo[3,2-d]pyrimidine core via an oxygen linker afforded potent VEGFR2 kinase inhibitors. Moreover, meta-substitution of the urea terminal benzene ring and substitution with a small lipophilic group at the 2-position of the central benzene ring enhanced HUVEC inhibitory activity. In
Experimental
Melting points were determined on a BÜCHI Melting Point B-545, and were not corrected. Proton nuclear magnetic resonance (1H NMR) spectra were recorded on Varian Mercury 300 (300 MHz) or Bruker DPX300 (300 MHz) instruments. Chemical shifts are reported as δ values (ppm) downfield from internal tetramethylsilane of the indicated organic solution. Peak multiplicities are expressed as follows: s, singlet; d, doublet; t, triplet; q, quartet; dd, doublet of doublet; ddd, doublet of doublet of
Acknowledgments
The authors thank Mr. T. Hirayama, Dr. T. Ichikawa, and Dr. Y. Nishikimi for synthetic support; Ms. T. Yoshida, Dr. A. Mizutani, and Ms. Y. Nagase for in vitro and in vivo assays. The authors thank Mr. K. Iwamoto and Ms. A. Hirokawa for molecular biology and protein expression. The authors thank Mr. S. Yamasaki and Ms. Y. Watanabe for PK evaluation.
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