Column chromatography was performed on a 230400 mesh silica gel (Fisher, Somerville, NJ) column. receptor tyrosine kinases (RTKs) have been associated with several cancers where they play a pivotal role in tumor angiogenesis.1,2Angiogenesis requires the transduction of signals from the extracellular domain D149 Dye of endothelial cells to the nucleus which is mediated by RTKs.3Solid tumors require angiogenesis to grow beyond 12 mm in size and metastasis requires the presence of blood vessels to allow transport of tumor cells to sites distal to the primary tumor.3,4Inhibition of tumor angiogenesis prevents the growth and metastasis of several types of solid tumors. Thus, inhibition of angiogenesis via RTK inhibition D149 Dye provides an attractive target for the treatment of cancer.1,5Among the RTKs implicated in tumor progression and angiogenesis are members of the VEGFR family namely VEGFR-1 and VEGFR-2, members of the EGFR family and members of the PDGFR family, D149 Dye namely PDGFR- and PDGFR-.5,6 Small molecule RTK inhibitors targeting the ATP binding site of tyrosine kinases are currently in clinical use while others are in clinical trials as antitumor agents.5,7,8Initial strategies for RTK inhibition focused on single RTK inhibitors such as erlotinib,1and gefitinib,2that were approved for non small cell lung cancer (Figure 1).7,8However, tumors have redundant signaling pathways for angiogenesis and often develop resistance to agents that target one specific pathway.9,10A multitargeted approach that inhibits multiple signaling pathways has shown to be more effective than the inhibition of a single target.1011Sorafenib,3an inhibitor of VEGFR, PDGFR and Raf-1 kinase and sunitinib,4an inhibitor of VEGFR-1, VEGFR-2, fms-like tyrosine kinase-3 (Flt-3), PDGFR, stem D149 Dye cell factor receptor (c-Kit) and colony stimulating factor (CSF-1) have been approved for renal cell carcinoma, and sunitinib most recently for pancreatic cancer.12,13 == Figure 1. == Reported RTK Inhibitors Gangjeeet. al.14previously reported compounds57(Figure 2) as multiple RTK inhibitors in a series ofN4-(3-bromophenyl)-6-substitutedphenylmethyl-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines. It was demonstrated that variation of the phenyl substituents in the 6-benzyl moiety determined both the potency and specificity of inhibitory activity against various RTKs. To further develop the structure-activity relationship, it was of interest to determine if variation in the anilino moiety could similarly influence potency and specificity for RTK inhibition. Thus, compounds813(Figure 2) were synthesized as analogs of57with two different 4-anilino moieties in combination with the 6-benzyl sidechains of57. We elected the 4-chloro anilino and 4-chloro-2-fluoroanilino substitutions in compounds813on the basis of the potent multiple RTK inhibition seen for these anilines in 66 fused systems such as quinazolines, pthalazines and pyrido[2,3-d]pyrimidines.1519Vatalinib bearing the 4-chloro anilino substitution, has shown potent VEGFR-1 and VEGFR-2 inhibition,15,16while quinazolines bearing the 4-chloro-2-fluoroanilino substitution have shown potent, dual VEGFR-2 and EGFR inhibition.17,18The 2-NH2moiety in813was maintained to provide additional hydrogen bonding in the Hinge region of RTKs compared to other known RTK inhibitors that lack this 2-NH2moiety. The flexible 6-benzyl substitutions were incorporated to allow for multiple conformations of this side chain and to perhaps afford interactions with multiple RTKs. == Figure 2. == Target compounds813as analogs of57 == 2. Chemistry == The synthesis of compounds813is shown inScheme 1. The 4-chloro-6-substituted pyrrolo[2,3-d]pyrimidines,141514were synthesized in five steps from the corresponding phenylacetic acids. Treatment with the appropriate aniline,17in isopropanol and a few drops of conc HCl at reflux afforded compounds811. Reaction of1614(synthesized in six steps from 2,5-dimethoxyphenylacetic acid) with the appropriate aniline17in ZNF538 isopropanol and a few drops of conc HCl, followed by depivaloylation with base, at reflux afforded compounds1213. == Scheme 1. == aReagents and Conditions: (a)17,iPrOH, 23 drops conc HCI, 4 h, reflux; (b) KOH, 1,4-dioxane, reflux == 3. Results and Discussions == The RTK inhibitory activities of compounds813were evaluated in human tumor cells known to express high levels of EGFR, VEGFR-2, VEGFR-1 and PDFGR- using a phosphotyrosine ELISA assay.19The effect of compounds813on cell proliferation was measured using A431 cancer cells, known to overexpress EGFR. EGFR is known to play a role in the overall survival of A431 cells.19Cellular evaluations of RTK inhibitory activities afford more meaningful results for translation toin vivostudies than direct enzymatic.
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