GCCMS: 308 (M+). thiazole analogues showed strong antiangiogenesis activity, obstructing new blood vessel formation inside a chicken embryo membrane assay. Finally, a functional study was carried out to investigate the mechanism of action via interaction with the F-actin bundling protein fascin. Intro The actin-bundling protein fascin has been linked to tumor progression, invasion, and metastasis, a fatal development of the disease.1?5 Fascin has recently emerged like a novel therapeutic target for treatment of cancer metastasis.6?8 A cell-based fascin bioassay identified compounds with potential antimetastasis functions, with diverse chemical structural features.9 However, no follow-up studies of biological activities were reported to confirm the compounds indeed target fascin to inhibit migration, invasion, and metastasis.9 From shape-based molecular modeling and subsequent rational design and synthesis, we have recently identified a thiazole lead compound 1 (Number ?(Number1)1) that effectively blocked cell migration and invasion via interactions with the protein fascin.10 With potent activities (IC50 in the 100 nM array) in inhibiting migration and invasion of several metastatic human breast cancer cell lines such as MDA-MB-231, HeLa, and A549, ZPK this compound exhibited no cytotoxicity at concentrations exceeding 100 M. The getting of thiazole compounds as antimigration and antiinvasion providers opened up fresh possibilities of fascin-targeting molecules that can be further optimized for higher potency and bioavailability while keeping minimal cytotoxicity. To further explore and enhance the structureCactivity human relationships, we have designed and synthesized 63 additional thiazole derivatives where we wanted to (1) homologize the two lead constructions by varying the substitution within the thiazole nitrogen, (2) modify the linker structure between thiazole and phenyl organizations, and (3) change other substitution groups on both the thiazole ring and the phenyl rings (Physique ?(Figure1).1). These thiazole analogues were then biologically evaluated to determine their cytotoxicity, antimigration, and antiinvasion activities. Further, molecular modeling was performed to assist in the elucidation of observed structureCactivity-relationships. For the most potent thiazole derivatives, an in vivo assay utilizing chick embryo chorioallantoic membrane (CAM) was Indirubin performed to assess their in vivo antimetastasis potential by inhibiting angiogenesis. Finally we investigated their mode of Indirubin action by overexpressing the protein fascin in malignancy cell lines to determine if the activities of the compounds can abrogate the enhanced migration and invasion of the transfected cell lines. Open in a separate window Physique 1 Structures of lead compounds 1 and 2 and their analogues from lead modifications. Results and Conversation Chemistry As shown in Plan 1, compounds 5 and 7 and their isomers 6 and 8 were respectively obtained from the N-alkylation of the amides 3 and 4 which Indirubin were prepared following the literature process.10 4-(2,4-Dimethylphenyl)thiazol-2-amine (9) was treated with benzenesulfonic chloride to give 10 which was transformed to the analogue 11 and its isomer 12 by the N-methylation reaction in THF. The acylation of the 2-aminothiazoles 13 by acyl chloride provided the amides 14 at room heat in dichloromethane, and further methylation of 14 led to the desired analogues 15 and their corresponding isomers 16. Open in a separate window Plan 1 Synthesis of Analogues of Lead Compounds Antimigration Activity and Cytotoxicity A Transwell migration assay was used to determine the effects of the synthesized thiazole derivatives around the migratory capacity of MDA-MB-231, an invasive and metastatic breast malignancy cell collection. The malignancy cells were seeded at a density of 2.5 104 per well in the upper chamber in serum-free media. In the presence of varying concentrations of the thiazole derivatives, the cells ability to migrate to the lower chamber with media made up of 5% FBS was measured by counting the total quantity of cells in the lower chamber after 24 h. The concentration of individual thiazole compounds at which inhibition of migration is usually observed at 50% is usually defined as the IC50 value listed in Table 1. To evaluate possible contributions of cell viability loss to reduced migration, the thiazole derivatives were also subjected to cell survival assays to determine their cytotoxicities. These were conducted by treating the MDA-MB-231 cells with individual thiazole compounds for 5 days and counting the surviving cells. The 63 newly synthesized thiazole derivatives displayed a wide range of antimigration activities as reflected in the IC50 values going from a Indirubin low of 24 nM to greater than 50 M or no apparent activity. Such variations in activity appears to be dependent on minor structural modifications, exposing some interesting styles that may help our understanding of the structureCactivity-relationships for further optimization of pharmacological index of thiazole derivatives. Compounds 5lCr are homologues from your lead structure 1 that were designed and prepared to investigate the effect of thiazole-N substitution on gain or loss of Indirubin the antimigration activity. For comparison, 1, the most potent thiazole derivative.