the actin - bundling 
 protein fascin has been linked to tumor progression , 
 invasion , and metastasis , a fatal development of the disease . fascin has recently emerged as a novel therapeutic target for treatment 
 of cancer metastasis . a cell - based fascin bioassay identified compounds 
 with potential antimetastasis functions , with diverse chemical structural 
 features . however , no follow - up studies 
 of biological activities were reported to confirm that the compounds 
 indeed target fascin to inhibit migration , invasion , and metastasis . from shape - based molecular modeling and 
 subsequent rational design 
 and synthesis , we have recently identified a thiazole lead compound 1 ( figure 1 ) that effectively blocked 
 cell migration and invasion via interactions with the protein fascin . with potent activities ( ic50 in the 
 100 nm range ) in inhibiting migration and invasion of several metastatic 
 human breast cancer cell lines such as mda - mb-231 , hela , and a549 , 
 this compound exhibited no cytotoxicity at concentrations exceeding 
 100 m . the finding of thiazole compounds as antimigration and 
 antiinvasion agents opened up new possibilities of fascin - targeting 
 molecules that can be further optimized for greater potency and bioavailability 
 while maintaining minimal cytotoxicity . to further explore and optimize 
 the structure  activity relationships , we have designed and 
 synthesized 63 additional thiazole derivatives where we sought to 
 ( 1 ) homologize the two lead structures by varying the substitution 
 on the thiazole nitrogen , ( 2 ) change the linker structure between 
 thiazole and phenyl groups , and ( 3 ) modify other substitution groups 
 on both the thiazole ring and the phenyl rings ( figure 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 structure  activity - relationships . for the most 
 potent thiazole derivatives , an in vivo assay utilizing chick embryo 
 chorioallantoic membrane ( cam ) finally we investigated their mode of action by overexpressing the 
 protein fascin in cancer cell lines to determine if the activities 
 of the compounds can abrogate the enhanced migration and invasion 
 of the transfected cell lines . as shown in scheme 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 were prepared 
 following the literature procedure . 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 temperature in dichloromethane , 
 and further methylation of 14 led to the desired analogues 15 and their corresponding isomers 16 . a transwell 
 migration assay was used to determine the effects of the synthesized 
 thiazole derivatives on the migratory capacity of mda - mb-231 , an invasive 
 and metastatic breast cancer cell line . the cancer cells were seeded 
 at a density of 2.5  10 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 containing 5% fbs was measured by counting 
 the total number of cells in the lower chamber after 24 h. the concentration 
 of individual thiazole compounds at which inhibition of migration 
 is observed at 50% is 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 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 , revealing some interesting trends that may 
 help our understanding of the structure  activity - relationships 
 for further optimization of pharmacological index of thiazole derivatives . compounds 5l  r are homologues 
 from the lead structure 1 that were designed and prepared 
 to investigate the effect of thiazole - n substitution on gain or loss 
 of the antimigration activity . for comparison , 1 , the 
 most potent thiazole derivative from our previous study , was also included in the assay . when the thiazole n - methyl is changed to an ethyl group ( 5l ) , 
 there was a marked decrease of activity from 0.218 to 1.945 m . interestingly , an n - propyl substitution at the same 
 position ( 5 m ) confers similar antimigration activity 
 with an ic50 of 0.292 m . this trend continues as 
 the alkyl chain length increased to n - butyl ( 5n , ic50 = 0.196 m ) , n - hexyl 
 ( 5o , ic50 = 0.045 m ) , and n - dodecyl ( 5p , ic50 = 0.024 m ) . in 
 addition , allyl substitution at the thiazole nitrogen ( 5q ) yielded activity comparable to n - propyl ( 5 m ) , but changing the substitution ( 5r ) to propynyl 
 leads to a significant loss of the activity , resulting in an ic50 value of 3.89 m . on another note , putting the propynyl 
 group on the amide nitrogen ( 6 ) appears to restore the 
 activity . if the 
 amide linker is replaced by a sulfamide structure , antimigration activity 
 was largely lost in 12 but not in 11 . thiazole n - methyl substitution 
 confers , whereas the amide n - methyl substitution 
 deprives , antimigration activity . the lead compound 2 was a potent thiazole derivative 
 discovered in a previous study , which also displayed significant cytotoxicity 
 in several cancer cell lines at 10 m . in the current study we further investigated if modifications on n - alkyl substitutions could improve the activity and toxicity 
 profile . thus , when the alkyl substitution at the thiazole nitrogen 
 varied from methyl ( 5j ) , ethyl ( 7a ) , propyl 
 ( 7b ) to allyl ( 7c ) and propynyl ( 7d ) , the cytotoxicity of the compounds remained significant , with relative 
 survival ratio after 5-day treatment at 10 m ranging from 20.4% 
 to 70.9% . however , the cytotoxicity is largely abrogated when the 
 methyl and ethyl substitution is on the amide nitrogen ( 4e , 8a , 8b ) while strong antimigration activity 
 was retained . it was also noted that these compounds ( 7a  d ) exhibited moderate to strong inhibition of 
 cell growth , suggesting that , in addition to blocking fascin - mediated 
 migration , these compounds may have other molecular targets that regulate 
 cell survival and growth . next we examine how modifications 
 on the thiazole ring affect antimigration 
 activity . a methyl substitution on the 5-position of the thiazole 
 ring ( 14h ) results in a near complete loss of activity . 
 adding a fluorine atom at the para - position of the 1-thiazole phenyl 
 brings back cytotoxicity without a significant gain of antimigration 
 activity ( 14i ) . an attempt to move the thiazole phenyl 
 ring to the 5-position results in a more linear analogue that has 
 stronger growth inhibitory effect but also more antimigratory ( 14j ) . interestingly , adding a thiazole n - methyl 
 group to the above three analogues gives a more potent 15h ( compared to 14h ) and 15i ( compared to 15i ) but an inactive 15j ( compared to 14j ) . in comparison of 15j ( thiazole n - methyl ) 
 to 16f ( amide n - methyl ) , it was noted 
 that the analogue with amide n - methyl substitution 
 displayed significantly greater toxicity and antimigration activity 
 than that with a thiazole n - methyl group . modifications 
 on the thiazole phenyl ring by halogen and trifluoromethyl 
 substitutions afforded six analogues 14b , 14c , 14d , 14e , 14f , 14 g , of which only 14e and 14f showed strong 
 antimigration activities with moderate inhibition of cell growth at 
 the same time . it was noted that chlorine substitution at meta position 
 ( 14e ) is far superior to ortho position ( 14b ) . adding a methyl group on the thiazole nitrogen to some of these 
 analogues appears to endow more potent cytotoxicity while increasing 
 the overall inhibitory effect on cell motility . 15c , 15d , 15e , 15f , and 15 g all displayed potent antimigration activities 
 with ic50 values ranging from 0.096 m ( 15f ) to 1.07 m ( 15 g ) . attempts to modify both 
 thiazole phenyl and amide phenyl ring yielded 
 eight analogues ( 14k through 14r ) . these 
 compounds showed almost no antimigration activities except 14o with an ic50 value of 1.03 m . however , if these 
 analogues are modified further by adding a thiazole n - methyl substitution ( 15k through 15 m ) , 
 some modest increases in the activity are obtained . for example , 15l exhibited an ic50 value of 0.758 m . on the other hand , if the methyl substitution is on the amide nitrogen 
 ( 16 g through 16 m ) , the results are mixed . for instance , compared to 14k , ic50 value 
 of 16 g improved from > 25 to 0.312 m , an increase 
 of potency by nearly 100-fold . also , from 15o to 16j , the position change of methyl substitution from thiazole 
 nitrogen to amide nitrogen resulted in an increase of antimigration 
 activity by 4 times . to determine the antiinvasion activities 
 of the thiazole compounds , the most potent antimigration analogues 
 were selected for a matrigel invasion assay . as shown in figure 2 , at 10 m , all tested compounds demonstrate 
 50% or greater inhibition of cell invasion through the matrigel . notably , 
 compound 5o exhibited the greatest potency in blocking 
 the cancer cell invasion . it is also noted that the antiinvasion activities 
 of the selected thiazole derivatives do not always correlate with 
 their antimigration activities . given the association of cell migration 
 with angiogenesis and the possible involvement of fascin in angiogenesis , we next investigated if the thiazole compounds 
 might have any antiangiogenic properties using the chick chorioallantoic 
 membrane ( cam ) assay . in this test the vascular system of a fertilized 
 chicken embryo is used as a model . figure 3 demonstrates the effects of 5o and 5p on 
 the development of embryonal blood vessels compared to a negative 
 control ( pbs ) and a positive control ( 10 ng / plug basic fibroblast 
 growth factor ( bfgf ) and 30 ng / plug vascular endothelial growth factor 
 ( vegf ) ) without the thiazole compounds . the antiangiogenesis activity 
 of the three thiazole compounds was determined by the suppression 
 of angiogenic action of bv ( bfgf + vegf ) when each compound was added 
 to a collagen containing bv and placed on the chorioallantoic membrane 
 of 10-day old chick embryos for 3 days . as shown in figure 3 , the analogues 5o and 5p were seen to potently block the angiogenic action of bv . effect of 5o and 5p on angiogenesis in 
 chick embryo ex ovo chorioallantoic membrane ( cam ) assay . angiogenesis 
 was scored by two scorers in a blinded fashion on a scale from 0 to 
 3 where 0 contained no new vessels and 3 exhibited extreme angiogenesis 
 determined by the presence of new vessels radiating out from the plug 
 in addition to the regular repeating pattern of the normal cam vasculature . dashed lines in representative images ( below ) indicate the top edges 
 of the collagen and drug - containing plugs from which angiogenesis 
 was determined . in these experiments , the thiazole analogues 5o and 5p significantly inhibited the induction 
 of angiogenesis by bfgf and vegf ( bv ) after 3 days . our previous 
 study found that the thiazole derivatives 
 blocked migration and 
 invasion of cancer cells by impairing the cytoskeleton dynamics accompanied 
 by reduced colocalization of the actin - bundling protein fascin . to 
 provide further evidence that fascin might be involved in the antimigratory 
 action of the newly synthesized thiazole analogues , we overexpressed 
 fascin in mda - mb-231 cells to investigate the resulting cell motility 
 and the effect of the thiazole compounds on the cells overexpressing 
 fascin . figure 4 shows the higher level of 
 fascin expression in the transfected cells than the native level of 
 fascin in the control cells ( figure 4a ) . consistent 
 with previous findings , the fascin+ cells exhibited significantly higher ( 2.4-fold increase ) 
 migration than the control cells ( vector ) . treatment of the fascin+ 
 cells with our most potent thiazole analogues , 5o and 5p , nearly completely abrogated the enhanced migration as 
 illustrated in figure 5 . at 10 m 5o and 5p , the control mda - mb-231 cells lost 
 about 80% migratory capacity . the thiazole compounds also inhibited 
 the fascin+ mda - mb-231 cells by about the same percentage ( figure 5 ) . these results provided additional evidence that 
 the thiazole derivatives likely acted through interaction with fascin . enhanced cell migration induced by fascin - overexpression is abrogated 
 by thiazole analogues 5l , 5o , and 5p . to investigate if the thiazole 
 analogues inhibit cell migration and invasion by impairing the f - actin 
 network , actin filaments of hela cells treated with selected thiazole 
 analogues were stained with phalloidin tagged with fluorescent agent 
 ( acti - stain 488 fluorescent phalloidin ) . as shown in figure 6 , the control cells showed a distinct presence of 
 f - actin filaments along the cells and had more evenly distributed 
 f - actin structures in the cytoplasm . in contrast , after treatment 
 with the three thiazole analogues 5o , 5p , and 8b each at 10 m , the cells 
 exhibited various degrees of disorganization of actin filaments and 
 a decrease of f - actin staining intensity near the cell membranes . these results suggest that the antimigration and antiinvasion activities 
 of the thiazole analogues may be mediated by interfering with the 
 actin filament network . effects of thiazole analogues 5o , 5p , 
 and 8b on the f - actin structure of hela cells . cells 
 were plated on glass coverslips and incubated with vehicle ( dmso ) , 5o , 5p , or 8b for 24 h and were 
 fixed and stained with acti - stain 488 phalloidin . cells were observed 
 under a fluorescent microscope , a digital ccd camera , and 40 
 objective . in 
 summary , the 63 new thiazole derivatives designed and prepared 
 by further structural modifications of the previously discovered lead 
 compounds 1 and 2 produced more potent fascin 
 inhibitors . among the active compounds , the 5 series 
 of analogues with longer alkyl chain substitutions on the thiazole 
 nitrogen exhibited greater antimigration activities than those with 
 other structural motifs . the most potent analogue , 5p , inhibited 50% of cell migration at a concentration of 24 nm . as 
 expected , all thiazole derivatives that were selected for matrigel 
 invasion assays were found to have potent antiinvasion activities . in addition to antimigration and antiinvasion properties , two representative 
 thiazole derivatives , 5o and 5p , also showed 
 strong antiangiogenesis activity , blocking new blood vessel formation 
 in a chicken embryo membrane assay . a functional study in which enhanced 
 cell motility due to fascin overexpression was nearly completely abrogated 
 by the most potent analogues 5o and 5p provided 
 additional evidence that the action of the thiazole derivatives involved 
 targeting the f - actin bundling protein fascin . this proposed mechanism 
 of action is further supported by the observation that cellular f - actin 
 structures were significantly impaired by the treatment of the thiazole 
 analogues . all 
 reagents and solvents were purchased 
 from ak scientific , sigma - aldrich chemical co. , fisher scientific , 
 acros , and pharmco - aaper and were used as received . all organic solvents 
 ( pharmco - aaper ) used were of reagent grade quality and were used without 
 further purification . crude synthetic 
 products were purified by the following methods : chromatography on 
 silica gel ( 60100 mesh , fisher scientific ) column . analytical 
 thin layer chromatography ( tlc ) was performed on 250 m fluorescent 
 plates ( agela technologies , de , usa ) and visualized by using uv light . for all products , the purity was ascertained to be greater than 95% 
 by the hplc method using a shimadzu ( columbia , md ) 2010 hplc uv / ms 
 system with a c-18 reverse phase column and by gc  ms analyses 
 using an agilent technologies 5975c inert msd mass spectrometer . to a cooled mixture of nah ( 0.26 g , 60% in oil , 
 6.5 mmol ) in thf ( 20 ml ) was added a solution of compound 3 or 4 ( 5 mmol ) in thf ( 10 ml ) dropwise . after that , 
 the mixture was cooled to 0 c again and mei or rbr ( 6.5 mmol ) 
 was added dropwise . the mixture was then warmed to room temperature 
 and stirred for 2 h. water ( 5 ml ) was added to quench the reaction , 
 and the mixture was further diluted with water ( 50 ml ) . after removal of all the solvent , the residue was purified by silica 
 gel chromatography ( hexane / etoac = 4:1 ) to afford products 5 ( more polar ) and 6 or 7 ( more polar ) and 8 as solids . h nmr ( 300 mhz , cdcl3 ) : 8.37 ( dd , j = 
 1.8 and 8.1 hz , 2h ) , 7.497.42 ( m , 3h ) , 7.187.10 ( m , 
 3h ) , 6.45 ( s , 1h ) , 4.35 ( m , 1h ) , 3.82 ( m , 1h ) , 1.24 ( t , j = 6.9 hz , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174.1 , 
 167.8 , 140.1 , 137.82 , 137.75 , 137.2 , 131.3 , 131.2 , 130.6 , 129.2 , 128.0 , 
 127.2 , 126.8 , 106.8 , 42.1 , 21.3 , 19.6 , 13.8 . hrms ( esi(+ ) ) calcd for c20h21n2os ( m + h ) : 337.1375 . found : 337.1365 . h nmr 
 ( 300 mhz , cdcl3 ) : 8.36 ( m , 2h ) , 7.527.42 
 ( m , 3h ) , 7.177.09 ( m , 3h ) , 6.45 ( s , 1h ) , 4.28 ( m , 1h ) , 3.71 
 ( m , 1h ) , 2.41 ( s , 3h ) , 2.13 ( s , 3h ) , 1.70 ( m , 2h ) , 0.81 ( t , j = 7.5 hz , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174.0 , 168.1 , 140.0 , 138.2 , 137.7 , 137.2 , 131.3 , 131.2 , 130.7 , 
 129.2 , 128.0 , 127.3 , 126.8 , 106.8 , 48.5 , 21.8 , 21.3 , 19.6 , 11.2 . gc  ms : 
 350 ( m ) . hrms ( esi(+ ) ) calcd for c21h23n2os ( m + h ) : 351.1531 . found : 351.1525 . h nmr 
 ( 300 mhz , cdcl3 ) : 8.36 ( m , 2h ) , 7.527.42 
 ( m , 3h ) , 7.177.09 ( m , 3h ) , 6.45 ( s , 1h ) , 4.33 ( m , 1h ) , 3.73 
 ( m , 1h ) , 2.40 ( s , 3h ) , 2.14 ( s , 3h ) , 1.64 ( m , 2h ) , 1.22 ( m , 2h ) , 0.81 
 ( t , j = 7.5 hz , 3h ) . c nmr ( 75 mhz , 
 cdcl3 ) : 174.1 , 168.1 , 140.1 , 138.2 , 137.7 , 137.2 , 131.3 , 
 131.2 , 130.7 , 129.2 , 128.0 , 127.3 , 126.8 , 106.9 , 46.7 , 30.4 , 21.3 , 
 19.9 , 19.6 , 13.6 . gc  ms : 364 ( m ) . hrms ( esi(+ ) ) 
 calcd for c22h25n2os ( m + h ) : 365.1688 . 
 found : 365.1682 . h nmr ( 300 mhz , cdcl3 ) : 8.36 ( dd , j = 
 1.5 and 7.8 hz , 2h ) , 7.527.42 ( m , 3h ) , 7.177.09 ( m , 
 3h ) , 6.45 ( s , 1h ) , 4.33 ( m , 1h ) , 3.72 ( m , 1h ) , 2.41 ( s , 3h ) , 2.14 
 ( s , 3h ) , 1.61 ( m , 2h ) , 1.231.17 ( m , 6h ) , 0.81 ( t , j = 7.5 hz , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174.1 , 168.0 , 140.1 , 138.1 , 137.7 , 137.2 , 131.3 , 131.2 , 130.7 , 
 129.2 , 128.0 , 127.3 , 126.8 , 106.9 , 46.9 , 31.1 , 28.2 , 26.2 , 22.4 , 21.3 , 
 19.6 , 13.9 . hrms ( esi(+ ) ) calcd 
 for c24h29n2os ( m + h ) : 393.2001 . 
 found : 393.1986 . h nmr ( 300 mhz , cdcl3 ) : 8.35 ( m , 2h ) , 7.497.26 
 ( m , 3h ) , 7.177.09 ( m , 3h ) , 6.44 ( s , 1h ) , 4.32 ( m , 1h ) , 3.73 
 ( m , 1h ) , 2.40 ( s , 3h ) , 2.13 ( s , 3h ) , 1.591.50 ( m , 4h ) , 1.281.17 
 ( m , 16h ) , 0.88 ( t , j = 7.5 hz , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174.1 , 168.1 , 140.0 , 138.1 , 137.7 , 
 137.3 , 131.3 , 131.2 , 130.7 , 129.2 , 128.0 , 127.3 , 126.8 , 106.9 , 46.9 , 
 32.8 , 31.9 , 29.7 , 29.6 , 29.5 , 29.4 , 29.3 , 28.9 , 28.2 , 26.5 , 25.8 , 
 22.7 , 21.3 , 19.6 , 14.1 . ms ( esi ) : 477 ( m + h ) . hrms ( esi(+ ) ) calcd 
 for c30h41n2os ( m + h ) : 477.2940 . 
 found : 477.2945 . h nmr ( 300 mhz , cdcl3 ) : 8.35 ( m , 2h ) , 7.517.41 
 ( m , 3h ) , 7.147.07 ( m , 3h ) , 6.46 ( s , 1h ) , 5.87 ( m , 1h ) , 5.10 
 ( dd , j = 1.2 and 10.2 hz , 1h ) , 4.98 ( dd , j = 1.2 and 17.1 hz , 2h ) , 4.42 ( m , 1h ) , 2.40 ( s , 3h ) , 2.13 
 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174.2 , 168.1 , 
 140.1 , 137.8 , 137.1 , 131.3 , 131.1 , 130.8 , 129.2 , 128.0 , 127.0 , 126.6 , 
 118.4 , 106.8 , 49.1 , 21.3 , 19.7 . hrms ( esi(+ ) ) calcd for c21h21n2os 
 ( m + h ) : 349.1375 . found : 349.1370 . h nmr ( 300 mhz , cdcl3 ) : 8.40 ( m , 2h ) , 7.527.42 
 ( m , 3h ) , 7.237.11 ( m , 3h ) , 6.47 ( s , 1h ) , 4.80 ( m , 2h ) , 2.40 
 ( s , 3h ) , 2.21 ( m , 1h ) , 2.19 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174.4 , 168.1 , 140.4 , 138.1 , 137.1 , 136.9 , 131.6 , 131.4 , 
 130.8 , 129.4 , 128.1 , 126.9 , 126.4 , 106.9 , 77.3 , 72.4 , 36.0 , 21.4 , 
 19.9 . ms ( esi ) : 347 ( m + h ) . hrms ( esi(+ ) ) calcd for c21h19n2os ( m + h ) : 347.1218 . found : 347.1216 . h nmr ( 300 mhz , cdcl3 ) : 8.36 ( dd , j = 
 1.5 and 8.1 hz , 2h ) , 7.527.43 ( m , 4h ) , 7.20 ( s , 1h ) , 7.18 
 ( s , 2h ) , 4.75 ( m , 2h ) , 2.42 ( s , 3h ) , 2.22(m , 1h ) , 2.21 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174.7 , 167.3 , 141.0 , 138.5 , 
 136.2 , 135.9 , 131.9 , 131.6 , 130.5 , 129.5 , 128.1 , 127.3 , 124.8 , 98.0 , 
 76.97 , 72.7 , 37.1 , 21.5 , 19.6 . ms ( esi ) : 347 ( m + h ) . hrms ( esi(+ ) ) 
 calcd for c21h19n2os ( m + h ) : 347.1218 . 
 found : 347.1210 . h nmr 
 ( 300 mhz , cdcl3 ) : 8.37 ( dd , j = 1.5 and 
 7.5 hz , 2h ) , 7.517.40 ( m , 8h ) , 6.52 ( s , 1h ) , 4.26 ( q , j = 6.9 hz , 2h ) , 1.34 ( t , j = 6.9 hz , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174.2 , 168.1 , 139.1 , 137.1 , 
 130.9 , 129.6 , 129.4 , 129.2 , 128.9 , 128.0 , 107.2 , 42.5 , 14.0 . hrms ( esi(+ ) ) calcd for c18h17n2os ( m + h ) : 309.1062 . h nmr 
 ( 300 mhz , cdcl3 ) : 8.36 ( dd , j = 1.2 and 
 7.5 hz , 2h ) , 7.507.39 ( m , 8h ) , 6.52 ( s , 1h ) , 4.19 ( q , j = 7.5 hz , 2h ) , 1.76 ( m , 2h ) , 0.83 ( t , j = 7.5 hz , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174.1 , 
 168.5 , 139.3 , 137.2 , 131.3 , 130.9 , 129.6 , 129.5 , 129.2 , 128.8 , 128.7 , 
 128.0 , 126.0 , 107.2 , 48.8 , 21.9 , 11.1 . hrms ( esi(+ ) ) calcd for c19h19n2os ( m + h ) : 323.1218 . found : 323.1210 . h nmr 
 ( 300 mhz , cdcl3 ) : 8.35 ( dd , j = 1.8 and 
 6.3 hz , 2h ) , 7.497.43 ( m , 8h ) , 6.55 ( s , 1h ) , 5.98 ( m , 1h ) , 
 5.19 ( d , j = 10.2 hz , 1h ) , 4.98 ( d , j = 17.4 hz , 1h ) , 4.82 ( d , j = 5.1 hz , 2h ) . c nmr ( 75 mhz , cdcl3 ) : 174.3 , 168.5 , 139.3 , 137.0 , 
 131.9 , 131.4 , 130.6 , 129.7 , 129.6 , 129.2 , 128.7 , 128.0 , 118.0 , 107.1 , 
 49.6 . hrms ( esi(+ ) ) calcd for c19h17n2os ( m + h ) : 321.1062 . found : 321.1054 . h nmr ( 300 mhz , cdcl3 ) : 8.40 ( d , j = 
 6.6 hz , 2h ) , 7.577.44 ( m , 8h ) , 6.56 ( s , 1h ) , 4.92 ( d , j = 2.1 hz , 2h ) . c nmr ( 75 mhz , cdcl3 ) : 174.4 , 168.5 , 138.6 , 136.8 , 131.6 , 130.1 , 129.9 , 129.44 , 129.37 , 
 129.0 , 128.1 , 77.8 , 72.8 , 36.9 . . hrms 
 ( esi(+ ) ) calcd for c19h15n2os ( m 
 + h ) : 319.0905 . found : 319.0897 . h nmr ( 300 mhz , cdcl3 ) : 7.92 ( m , 2h ) , 7.557.48 ( m , 5h ) , 7.42 ( m , 2h ) , 7.34 ( m , 
 1h ) , 7.25 ( s , 1h ) , 4.27 ( q , j = 7.5 hz , 2h ) , 1.38 
 ( t , j = 7.5 hz , 3h ) . c nmr ( 75 mhz , 
 cdcl3 ) : 170.6 , 159.0 , 149.5 , 135.2 , 134.8 , 130.4 , 128.68 , 
 128.65 , 127.8 , 126.7 , 126.0 , 109.2 , 45.2 , 14.1 . hrms ( esi(+ ) ) calcd for c18h17n2os ( m + h ) : 309.1062 . found : 309.1053 . h nmr ( 300 
 mhz , cdcl3 ) : 7.91 ( dd , j = 1.5 and 6.9 
 hz , 2h ) , 7.537.32 ( m , 8h ) , 7.24 ( s , 1h ) , 4.18 ( q , j = 7.5 hz , 2h ) , 1.83 ( m , 2h ) , 0.83 ( t , j = 7.5 hz , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 170.8 , 
 159.3 , 149.5 , 135.2 , 134.8 , 130.4 , 128.68 , 128.66 , 128.5 , 127.9 , 127.0 , 
 126.0 , 109.2 , 51.6 , 21.9 , 11.2 . hrms ( esi(+ ) ) calcd for c19h19n2os 
 ( m + h ) : 323.1218 . found : 323.1211 . to a solution of 9 ( 0.61 g , 
 3.0 mmol ) and pyridine ( 0.96 ml , 12 mmol ) in anhydrous dichloromethane 
 was added benzenesulfonic chloride ( 0.76 ml , 6 mmol ) in dichloromethane 
 dropwise at 0 c . after being stirred at room temperature for 
 2 h under nitrogen atmosphere , the reaction mixture was concentrated 
 in vacuo . the saturated na2co3 solution was 
 added to quench the reaction , and the solution was extracted with 
 ethyl acetate , dried over mgso4 , and concentrated in vacuo . the residue was purified by flash chromatography to give product 10 ( 0.57 g , 55% yield ) as a solid . h nmr ( 300 
 mhz , cdcl3 ) : 9.85 ( bs , 1h ) , 7.93 ( d , j = 7.8 hz , 2h ) , 7.537.43 ( m , 3h ) , 7.16 ( d , j = 7.8 hz , 1h ) , 7.087.04 ( m , 3h ) , 6.26 ( s , 1h ) , 2.34 and 
 2.32 ( ds , 6h ) . c nmr ( 75 mhz , cdcl3 ) : 168.7 , 
 141.9 , 140.1 , 136.3 , 136.1 , 132.2 , 132.0 , 128.9 , 128.7 , 127.2 , 126.5 , 
 125.7 , 103.6 , 21.2 , 20.4 . hrms 
 ( esi(+ ) ) calcd for c17h17n2o2s2 ( m + h ) : 345.0731 . found : 345.0727 . to a cooled 
 mixture of nah ( 0.07 g , 60% in oil , 1.6 mmol ) in thf ( 5 ml ) was added 
 a solution of compound 10 ( 0.34 g , 1 mmol ) in thf ( 5 ml ) 
 dropwise . after that , the mixture was cooled to 0 c again and 
 mei ( 0.25 ml , 4.0 mmol ) was added dropwise . the mixture was then warmed 
 to room temperature and stirred for 2 h. water ( 3 ml ) was added to 
 quench the reaction , and the mixture was further diluted with water 
 ( 10 ml ) . after removal of all the solvent , the residue was 
 purified by silica gel chromatography ( hexane / etoac = 4:1 ) to afford 
 products 11 ( more polar ) ( 0.27 g , 75% yield ) and 12 ( 0.034 g , 9% yield ) as solids . 11 : h nmr ( 300 mhz , cdcl3 ) : 8.058.02 ( m , 2h ) , 
 7.587.43 ( m , 3h ) , 7.127.01 ( m , 3h ) , 6.27 ( s , 1h ) , 
 3.19 ( s , 3h ) , 2.36 ( s , 3h ) , 2.11 ( s , 3h ) . c nmr ( 75 mhz , 
 cdcl3 ) : 166.8 , 142.3 , 140.6 , 139.1 , 137.6 , 131.9 , 131.4 , 
 130.5 , 128.6 , 127.1 , 126.6 , 126.4 , 103.5 , 33.6 , 21.3 , 19.5 . hrms ( esi(+ ) ) calcd for c18h19n2o2s2 ( m + h ) : 359.0888 . found : 
 359.0879 . 12 : h nmr ( 300 mhz , cdcl3 ) : 
 7.83 ( d , j = 7.2 hz , 2h ) , 7.587.38 ( m , 4h ) , 
 7.026.98 ( m , 2h ) , 6.88 ( s , 1h ) , 3.45(s , 3h ) , 2.32 and 2.30 
 ( ds , 6h ) . c nmr ( 75 mhz , cdcl3 ) : 160.0 , 151.2 , 
 137.9 , 136.8 , 135.9 , 133.8 , 131.8 , 131.4 , 129.4 , 129.3 , 127.4 , 126.6 , 
 112.0 , 36.7 , 21.2 , 21.1 . hrms ( esi(+ ) ) 
 calcd for c18h19n2o2s2 ( m + h ) : 359.0888 . found : 359.0880 . to 
 a solution of 13 ( 0.01 mol ) and 
 dmap ( 1.24 g , 0.01 mol ) in anhydrous dichloromethane was added the 
 acyl chloride in dichloromethane dropwise at 0 c . after being 
 stirred at room temperature for 2 h under nitrogen atmosphere , the saturated na2co3 solution was added to quench the reaction , and the 
 solution was extracted with ethyl acetate , dried over mgso4 , and concentrated in vacuo . the residue was purified by flash chromatography 
 to give product 14 as a solid . h nmr ( 300 mhz , cdcl3 ) : 7.99 ( dd , j = 1.5 and 8.4 hz , 2h ) , 7.527.47 ( m , 3h ) , 6.55 
 ( d , j = 0.9 hz , 1h ) , 2.11 ( s , 3h ) . c 
 nmr ( 75 mhz , cdcl3 ) : 172.0 , 165.3 , 159.3 , 146.8 , 133.1 , 
 132.8 , 132.3 , 130.0 , 128.9 , 128.3 , 127.8 , 108.4 , 16.4 . hrms ( esi(+ ) ) calcd for c11h11n2os ( m + h ) : 219.0592 . found : 219.0589 . h nmr ( 300 mhz , cdcl3 ) : 11.5 ( bs , 1h ) , 7.75 ( dd , j = 1.2 and 8.4 hz , 
 2h ) , 7.63 ( dd , j = 2.1 and 7.2 hz , 1h ) , 7.517.45 
 ( m , 2h ) , 7.387.28 ( m , 3h ) , 7.197.08 ( m , 2h ) . c nmr ( 75 mhz , cdcl3 ) : 165.3 , 158.5 , 146.3 , 132.8 , 
 132.7 , 132.0 , 131.7 , 130.8 , 130.4 , 128.9 , 128.7 , 127.4 , 126.8 , 113.2 . . hrms ( esi(+ ) ) calcd for c16h12cln2os ( m + h ) : 315.0359 . found : 315.0351 . h nmr ( 300 mhz , cdcl3 ) : 11.68 ( bs , 1h ) , 8.177.15 ( m , 10h ) . c nmr 
 ( 75 mhz , cdcl3 ) : 171.3 , 165.3 , 160.1 , 148.3 , 135.9 , 133.5 , 
 133.1 , 133.0 , 131.7 , 131.1 , 130.3 , 130.2 , 130.1 , 129.5 , 129.3 , 128.9 , 
 128.4 , 127.8 , 124.9 , 122.9 , 109.3 . hrms ( esi(+ ) ) calcd for c16h12brn2os ( m + h ) : 358.9854 . found : 358.9847 . h nmr ( 300 mhz , cdcl3 ) : 10.34 ( bs , 1h ) , 7.90 ( m , 2h ) , 7.677.41 ( m , 7h ) , 7.20 ( s , 
 1h ) . c nmr ( 75 mhz , cdcl3 ) : 164.9 , 158.7 , 
 149.1 , 133.1 , 133.0 , 131.83 , 131.79 , 129.0 , 127.6 , 127.4 , 122.1 , 108.5 . gc  ms : 358 , 360 ( m ) . hrms ( esi(+ ) ) calcd for c16h12brn2os ( m + h ) : 358.9854 . found : 
 358.9848 . h nmr ( 300 mhz , cdcl3 ) : 10.19 ( bs , 1h ) , 7.90 ( m , 2h ) , 7.77 ( t , j = 1.8 
 hz , 1h ) , 7.667.56 ( m , 2h ) , 7.497.44 ( m , 1h ) , 7.327.26 
 ( m , 2h ) , 7.20 ( s , 1h ) . c nmr ( 75 mhz , cdcl3 ) : 164.8 , 158.5 , 148.7 , 135.9 , 134.7 , 133.0 , 131.8 , 130.0 , 129.0 , 
 128.0 , 127.4 , 126.2 , 124.1 , 109.1 . hrms ( esi(+ ) ) calcd for c16h12cln2os ( m + h ) : 315.0359 . found : 315.0353 . h nmr ( 300 mhz , cdcl3 ) : 10.08 ( bs , 1h ) , 8.24 ( s , 2h ) , 7.93(m , 2h ) , 7.77 ( s , 1h ) , 7.60 
 ( m , 1h ) , 7.517.45 ( m , 2h ) , 7.39 ( s , 1h ) . c nmr 
 ( 75 mhz , cdcl3 ) : 165.0 , 159.1 , 147.3 , 136.3 , 133.4 , 132.5 , 
 132.0 , 131.7 , 129.3 , 127.6 , 126.2 , 125.3 , 121.7 , 110.9 . hrms ( esi(+ ) ) calcd for c18h11f6n2os ( m + h ) : 417.0496 . found : 417.0486 . h nmr ( 300 
 hz , cdcl3 ) : 10.10 ( 1h , bs ) , 7.93 ( 2h , j = 7.2 hz , d ) , 7.59 ( 1h , m ) , 7.527.47 ( 2h , m ) , 7.13 ( 1h , 
 s ) , 7.04 ( 2h , s ) , 3.92 ( 6h , s ) , 3.87 ( 3h , s ) . c nmr ( 75 
 hz , cdcl3 ) : 164.6 , 158.1 , 153.4 , 150.0 , 138.1 , 133.0 , 131.8 , 
 130.0 , 129.0 , 127.3 , 107.6 , 103.3 , 61.0 , 56.2 . ms - ei : 370 ( m ) . hrms ( esi(+ ) ) calcd for c19h19n2o4s ( m + h ) : 371.1066 . found : 371.1052 . hrms ( esi(+ ) ) 
 calcd for c19h19n2o4s 
 ( m + h ) : 371.1066 . found : 371.1060 . h nmr ( 300 mhz , cdcl3 ) : 8.20 ( d , j = 6.9 hz , 2h ) , 8.09 ( d , j = 7.2 hz , 2h ) , 7.627.43 ( m , 6h ) , 2.51 ( s , 2h ) . c nmr ( 75 mhz , cdcl3 ) : 171.3 , 165.2 , 157.1 , 144.4 , 133.7 , 
 133.3 , 132.8 , 131.9 , 130.3 , 130.1 , 128.8 , 128.4 , 128.3 , 128.1 , 127.9 , 
 122.0 , 12.0 . hrms ( esi(+ ) ) calcd 
 for c17h15n2os ( m + h ) : 295.0905 . 
 found : 295.0897 . h nmr ( 300 mhz , cdcl3 ) : 10.6 ( bs , 1h ) , 7.82 ( dd , j = 1.2 and 8.1 hz , 
 2h ) , 7.537.41 ( m , 5h ) , 7.01 ( m , 2h ) . c nmr ( 75 
 mhz , cdcl3 ) : 164.7 , 155.1 , 144.2 , 132.7 , 132.0 , 130.7 , 
 130.0 , 129.9 , 128.8 , 127.4 , 122.2 , 115.4 , 115.2 , 12.1 . hrms ( esi(+ ) ) calcd for c17h14fn2os ( m + h ) : 313.0811 . found : 313.0802 . h nmr ( 300 mhz , cdcl3 ) : 10.01 ( bs , 1h ) , 7.92 ( d , j = 7.5 hz , 2h ) , 7.69 
 ( d , j = 8.1 hz , 2h ) , 7.577.45 ( m , 3h ) , 7.19 
 ( d , j = 8.1 hz , 2h ) , 7.14 ( s , 1h ) , 2.37 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 165.2 , 158.9 , 150.3 , 137.9 , 
 132.7 , 131.9 , 131.4 , 129.4 , 128.7 , 127.4 , 126.0 , 107.3 , 21.2 . hrms ( esi(+ ) ) calcd for c17h15n2os ( m + h ) : 295.0905 . found : 295.0899 . h nmr ( 300 mhz , cdcl3 ) : 10.92 ( bs , 1h ) , 7.80 ( d , j = 8.7 hz , 2h ) , 7.10 
 ( s , 1h ) , 6.94 ( s , 2h ) , 6.84 ( d , j = 8.7 hz , 2h ) , 
 3.86 ( s , 6h ) , 3.84(s , 3h ) , 3.83 ( s , 3h ) . c nmr ( 75 mhz , 
 cdcl3 ) : 164.6 , 163.1 , 159.1 , 153.2 , 149.9 , 137.9 , 130.0 , 
 129.4 , 124.0 , 114.0 , 107.5 , 103.3 , 60.8 , 56.0 , 55.4 . hrms ( esi(+ ) ) 
 calcd for c20h21n2o5s 
 ( m + h ) : 401.1171 . found : 401.1165 . h nmr ( 300 mhz , cdcl3 ) : 11.43 ( bs , 1h ) , 7.73 ( m , 2h ) , 7.30 ( m , 2h ) , 7.12 ( s , 1h ) , 6.88 
 ( s , 2h ) , 3.85 ( s , 6h ) , 3.83(s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 164.4 , 159.0 , 153.3 , 150.1 , 139.1 , 138.0 , 130.3 , 129.7 , 
 129.0 , 128.8 , 107.9 , 103.3 , 60.8 , 56.0 . hrms ( esi(+ ) ) calcd for c19h18cln2o4s ( m + h ) : 405.0676 . found : 405.0664 . h nmr ( 300 mhz , cdcl3 ) : 11.07 ( bs , 1h ) , 7.44 ( dd , j = 1.8 and 8.1 hz , 
 1h ) , 7.39 ( s , 1h ) , 7.11 ( s , 1h ) , 6.92 ( s , 2h ) , 6.78 ( d , j = 8.4 hz , 1h ) , 3.92 ( s , 3h ) , 3.86 ( s , 6h ) , 3.83(s , 3h ) , 3.82 ( s , 
 3h ) . c nmr ( 75 mhz , cdcl3 ) : 164.8 , 159.2 , 
 153.3 , 152.8 , 150.1 , 148.9 , 138.0 , 129.9 , 124.2 , 120.8 , 110.4 , 110.3 , 
 107.6 , 103.3 , 60.9 , 56.0 , 55.8 . hrms ( esi(+ ) ) calcd for c21h23n2o6s ( m + h ) : 431.1277 . found : 431.1265 . h nmr ( 300 mhz , cdcl3 ) : 11.03 ( bs , 1h ) , 8.28 ( d , j = 9.0 hz , 1h ) , 7.10 
 ( s , 2h ) , 7.09 ( s , 1h ) , 6.68 ( dd , j = 2.1 and 9.0 
 hz , 1h ) , 6.56 ( d , j = 2.1 hz , 1h ) , 4.11 ( s , 3h ) , 
 3.96(s , 6h ) , 3.90 ( s , 3h ) , 3.89 ( s , 3h ) . c nmr ( 75 mhz , 
 cdcl3 ) : 164.8 , 162.6 , 159.2 , 158.1 , 153.4 , 149.9 , 138.0 , 
 134.5 , 130.4 , 112.0 , 107.5 , 106.1 , 103.4 , 98.7 , 60.9 , 56.4 , 56.2 , 
 55.7 . hrms ( esi(+ ) ) calcd for c21h23n2o6s ( m + h ) : 431.1277 . found : 431.1270 . h nmr ( 300 mhz , cdcl3 ) : 11.50 ( bs , 1h ) , 7.13 ( d , j = 0.3 hz , 1h ) , 7.06 
 ( s , 2h ) , 6.90 ( s , 2h ) , 3.87 ( s , 3h ) , 3.84(s , 6h ) , 3.82 ( s , 3h ) , 3.78 
 ( s , 6h ) . c nmr ( 75 mhz , cdcl3 ) : 165.1 , 159.3 , 
 153.3 , 153.1 , 150.2 , 141.9 , 138.1 , 129.6 , 126.8 , 107.8 , 104.8 , 103.2 , 
 60.9 , 60.8 , 56.1 , 56.0 . hrms ( esi(+ ) ) calcd for c22h25n2o7s ( m + h ) : 461.1382 . found : 461.1374 . h nmr ( 300 mhz , cdcl3 ) : 11.38 ( bs , 1h ) , 7.84 ( dd , j = 1.5 and 7.8 hz , 
 1h ) , 7.247.15 ( m , 3h ) , 7.12 ( s , 2h ) , 4.11 ( s , 3h ) , 3.96(s , 
 6h ) , 3.92 ( s , 3h ) , 3.89 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 162.6 , 157.5 , 153.4 , 152.6 , 150.1 , 148.0 , 138.1 , 130.3 , 
 124.8 , 124.0 , 123.0 , 117.0 , 107.6 , 103.4 , 61.9 , 60.9 , 56.2 . hrms ( esi(+ ) ) 
 calcd for c21h23n2o6s 
 ( m + h ) : 431.1277 . found : 431.1268 . h nmr ( 300 mhz , cdcl3 ) : 11.19 ( bs , 1h ) , 8.33 ( dd , j = 1.5 and 7.8 hz , 
 1h ) , 7.57 ( dt , j = 1.5 and hz , 1h ) , 7.197.07 
 ( m , 5h ) , 4.14 ( s , 3h ) , 3.96 ( s , 6h ) , 3.89 ( s , 3h ) . c 
 nmr ( 75 mhz , cdcl3 ) : 162.7 , 157.8 , 157.7 , 153.4 , 150.0 , 
 138.0 , 134.5 , 132.6 , 130.3 , 121.7 , 119.0 , 111.6 , 107.7 , 103.4 , 60.9 , 
 56.4 , 56.2 . hrms ( esi(+ ) ) calcd for c20h21n2o5s ( m + h ) : 401.1171 . h nmr ( 300 mhz , cdcl3 ) : 10.75 ( bs , 1h ) , 7.417.39 ( m , 2h ) , 7.31 ( m , 1h ) , 7.12 ( s , 
 1h ) , 7.05 ( m , 1h ) , 6.96 ( s , 2h ) , 3.88 ( s , 6h ) , 3.84 ( s , 3h ) , 3.77 
 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 164.8 , 159.8 , 
 158.5 , 153.3 , 150.1 , 138.0 , 133.1 , 129.9 , 129.8 , 119.4 , 119.2 , 112.2 , 
 107.6 , 103.3 , 60.9 , 56.0 , 55.3 . hrms ( esi(+ ) ) calcd for c20h21n2o5s ( m + h ) : 401.1171 . found : 
 401.1164 . to a cooled mixture of nah ( 0.26 g , 60% in oil , 
 6.5 mmol ) in thf 
 ( 20 ml ) was added a solution of compound 14 ( 5 mmol ) 
 in thf ( 10 ml ) dropwise . after that , the mixture was cooled to 0 c 
 again and mei ( 6.5 mmol ) was added dropwise . the mixture was then 
 warmed to room temperature and stirred for 2 h. water ( 5 ml ) was added 
 to quench the reaction , and the mixture was further diluted with water 
 ( 50 ml ) . after removal of all the solvent , the residue was 
 purified by silica gel chromatography ( hexane / etoac = 4:1 ) to afford 
 products 15 ( more polar ) and 16 as solids . h nmr 
 ( 300 mhz , cdcl3 ) : 7.83 ( dd , j = 1.8 and 
 7.8 hz , 2h ) , 7.477.43 ( m , 3h ) , 6.29 ( d , j = 1.2 hz , 1h ) , 3.79 ( s , 3h ) , 2.31 ( d , j = 1.2 hz , 
 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174.0 , 169.0 , 
 137.1 , 134.4 , 131.3 , 129.2 , 128.0 , 104.3 , 32.9 , 14.4 . hrms ( esi(+ ) ) calcd for c12h13n2os ( m + h ) : 233.0749 . found : 233.0741 . h nmr 
 ( 300 mhz , cdcl3 ) : 8.38 ( dd , j = 
 1.8 and 7.8 hz , 2h ) , 7.547.39 ( m , 7h ) , 6.60 ( s , 1h ) , 3.63 
 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174.3 , 168.4 , 
 136.9 , 136.3 , 134.8 , 132.3 , 131.53 , 131.49 , 130.0 , 129.9 , 129.2 , 128.1 , 
 127.3 , 108.1 , 34.0 . hrms ( esi(+ ) ) 
 calcd for c17h14cln2os ( m + h ) : 329.0515 . 
 found : 329.0505 . h nmr ( 300 mhz , cdcl3 ) : 7.83 ( dd , j = 
 1.8 and 8.1 hz , 2h ) , 7.64 ( m , 1h ) , 7.59 ( m , 1h ) , 7.517.36 
 ( m , 6h ) , 6.60 ( s , 1h ) , 3.75 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174.4 , 168.9 , 137.8 , 136.8 , 132.8 , 132.5 , 132.2 , 131.6 , 
 130.5 , 129.3 , 128.1 , 127.8 , 123.0 , 107.8 , 34.9 . hrms ( esi(+ ) ) calcd for c17h14brn2os ( m + h ) : 373.0010 . found : 373.0006 . h nmr 
 ( 300 mhz , cdcl3 ) : 8.37 ( dd , j = 
 1.5 and 8.1 hz , 2h ) , 7.65 ( d , j = 6.6 hz , 2h ) , 7.517.43 
 ( m , 4h ) , 7.28 ( d , j = 7.2 hz , 1h ) , 6.58 ( d , j = 0.9 hz , 1h ) , 3.74 ( d , j = 2.1 hz , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174.3 , 168.9 , 138.2 , 136.9 , 
 132.3 , 131.9 , 131.6 , 130.8 , 129.5 , 128.1 , 124.2 , 107.4 , 34.9 . hrms ( esi(+ ) ) calcd for c17h14brn2os ( m + h ) : 373.0010 . found : 373.0000 . h nmr 
 ( 300 mhz , cdcl3 ) : 8.37 ( d , j = 
 7.8 hz , 2h ) , 7.517.43 ( m , 6h ) , 7.32 ( m , 1h ) , 6.59(s , 1h ) , 
 3.75 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174.4 , 
 168.9 , 137.9 , 136.8 , 135.0 , 132.3 , 131.6 , 130.3 , 129.9 , 129.4 , 129.3 , 
 128.1 , 127.4 , 107.8 , 34.9 . hrms 
 ( esi(+ ) ) calcd for c17h14cln2os ( m 
 + h ) : 329.0515 . found : 329.0508 . h nmr ( 300 mhz , cdcl3 ) : 8.37 ( dd , j = 
 1.5 and 8.1 hz , 2h ) , 8.03 ( s , 1h ) , 7.91 ( s , 2h ) , 7.507.46 
 ( m , 3h ) , 6.73 ( s , 1h ) , 3.77 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174.4 , 168.9 , 136.4 , 136.0 , 132.8 , 132.7 , 132.4 , 131.7 , 
 129.2 , 128.0 , 123.3 , 120.9 , 109.4 , 34.8 . hrms ( esi(+ ) ) calcd for c19h13f6n2os ( m + h ) : 431.0653 . found : 431.0646 . h nmr ( 300 hz , cdcl3 ) : 8.37 ( 2h , j = 7.8 hz , d ) , 7.507.43 ( 3h , m ) , 6.60 ( 2h , s ) , 
 6.56 ( 1h , s ) , 3.92 ( 3h , s ) , 3.90 ( 6h , s ) , 3.76 ( 3h , s ) . c nmr ( 75 hz , cdcl3 ) : 174.3 , 168.7 , 153.5 , 139.4 , 139.1 , 
 137.0 , 131.5 , 129.2 , 128.1 , 125.9 , 106.7 , 106.6 , 61.0 , 56.4 , 35.0 . hrms ( esi(+ ) ) calcd for c20h21n2o4s ( m + h ) : 385.1222 . found : 
 385.1215 . h nmr ( 300 mhz , cdcl3 ) : 8.37 ( dd , j = 
 1.8 and 8.1 hz , 2h ) , 7.537.42 ( m , 6h ) , 7.337.30 ( m , 
 2h ) , 3.61 ( s , 3h ) , 2.17 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174.0 , 167.0 , 137.2 , 134.1 , 131.3 , 130.4 , 129.8 , 129.5 , 
 129.2 , 129.1 , 128.0 , 117.8 , 35.0 . hrms ( esi(+ ) ) calcd for c18h17n2os ( m + h ) : 309.1062 . found : 309.1055 . h nmr ( 300 mhz , cdcl3 ) : 8.36 ( dd , j = 
 0.9 and 7.8 hz , 2h ) , 7.477.44 ( m , 3h ) , 7.327.22 ( m , 
 4h ) , 3.60 ( s , 3h ) , 2.15 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174 . 0 , 167.0 , 164.9 , 161.6 , 137.1 , 133.0 , 132.4 , 132.2 , 
 131.4 , 129.2 , 128.0 , 125.74 , 125.70 , 118.2 , 116.5 , 116.2 , 34.9 , 12.2 . . hrms ( esi(+ ) ) calcd for c18h16fn2os ( m + h ) : 327.0967 . found : 327.0957 . h nmr ( 300 mhz , cdcl3 ) : 8.38 ( dd , j = 
 1.5 and 7.8 hz , 2h ) , 7.487.44 ( m , 3h ) , 7.30 ( s , 4h ) , 6.53 
 ( s , 1h ) , 3.74 ( s , 3h ) , 2.44 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174.2 , 168.9 , 139.8 , 139.6 , 137.1 , 131.4 , 129.6 , 129.23 , 
 129.17 , 128.0 , 127.7 , 106.6 , 34.9 , 21.4 . hrms ( esi(+ ) ) calcd for c18h17n2os 
 ( m + h ) : 309.1062 . found : 309.1054 . h nmr 
 ( 300 mhz , cdcl3 ) : 8.33 ( d , j = 
 9.0 hz , 2h ) , 6.95 ( d , j = 9.0 hz , 2h ) , 6.60 ( s , 2h ) , 
 6.53 ( s , 1h ) , 3.92 ( s , 3h ) , 3.90(s , 6h ) , 3.88 ( s , 3h ) , 3.74 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 173.9 , 168.4 , 162.4 , 153.5 , 
 139.2 , 139.1 , 131.1 , 129.7 , 126.0 , 113.2 , 106.6 , 106.4 , 61.0 , 56.3 , 
 55.3 , 34.9 , 29.7 . hrms ( esi(+ ) ) calcd for c21h23n2o5s ( m + h ) : 415.1328 . found : 415.1321 . h nmr ( 300 mhz , cdcl3 ) : 8.30 ( d , j = 
 8.7 hz , 2h ) , 7.42 ( d , j = 8.7 hz , 2h ) , 6.60 ( s , 2h ) , 
 6.58 ( s , 1h ) , 3.93 ( s , 3h ) , 3.90 ( s , 6h ) , 3.75 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 173.2 , 168.8 , 153.6 , 139.5 , 139.3 , 
 137.6 , 135.5 , 130.7 , 128.3 , 125.7 , 106.8 , 106.7 , 61.0 , 56.4 , 35.0 . 
 hrms ( esi(+ ) ) calcd for c20h20cln2o4s ( m + h ) : 419.0832 . found : 419.0824 . h nmr ( 300 mhz , cdcl3 ) : 8.00 ( m , 1h ) , 7.91 ( m , 1h ) , 7.37 
 ( t , j = 7.8 hz , 1h ) , 7.05 ( m , 1h ) , 6.60 ( s , 2h ) , 
 6.57 ( s , 1h ) , 3.93 ( s , 3h ) , 3.90 ( s , 6h ) , 3.89 ( s , 3h ) , 3.76 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 174.1 , 168.8 , 159.5 , 153.5 , 
 139.4 , 139.2 , 138.5 , 129.0 , 125.8 , 121.8 , 118.0 , 113.6 , 106.7 , 106.6 , 
 61.0 , 56.4 , 55.4 , 35.0 . hrms ( esi(+ ) ) calcd for c21h23n2o5s ( m + h ) : 415.1328 . found : 415.1320 . h nmr ( 300 mhz , cdcl3 ) : 8.35 ( dd , j = 
 1.8 and 7.8 hz , 2h ) , 7.497.41 ( m , 3h ) , 6.23 ( d , j = 1.2 hz , 1h ) , 3.95 ( s , 3h ) , 1.75 ( m , 1h ) , 1.02 ( m , 2h ) , 0.73 ( m , 
 2h ) . c nmr ( 75 mhz , cdcl3 ) : 174.0 , 169.1 , 
 140.6 , 137.1 , 131.3 , 129.2 , 128.0 , 103.9 , 33.2 , 8.7 , 5.9 . hrms ( esi(+ ) ) calcd for c14h15n2os ( m + h ) : 259.0509 . found : 259.0519 . h nmr ( 300 mhz , cdcl3 ) : 7.68 ( s , 2h ) , 6.61 ( s , 2h ) , 6.57 
 ( s , 1h ) , 3.96 ( s , 6h ) , 3.93 ( s , 3h ) , 3.92(s , 3h ) , 3.91 ( s , 6h ) , 3.77 
 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 173.7 , 168.8 , 
 153.5 , 152.7 , 141.1 , 139.4 , 139.2 , 132.3 , 125.8 , 106.7 , 106.6 , 106.4 , 
 61.0 , 60.9 , 56.3 , 56.1 , 34.9 . hrms ( esi(+ ) ) calcd for c23h27n2o7s ( m + h ) : 475.1539 . found : 
 475.1526 . h nmr ( 300 mhz , cdcl3 ) : 7.97 ( dd , j = 
 1.8 and 7.8 hz , 1h ) , 7.607.46 ( m , 7h ) , 7.377.23 ( m , 
 2h ) , 3.74 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 
 170.4 , 159.0 , 146.0 , 134.5 , 133.5 , 132.1 , 131.3 , 130.9 , 130.6 , 128.73 , 
 128.66 , 127.6 , 126.9 , 114.6 , 38.5 . hrms ( esi(+ ) ) calcd for c17h14cln2os ( m + h ) : 329.0515 . found : 329.0515 . h nmr ( 300 
 mhz , cdcl3 ) : 8.1 ( s , 1h ) , 7.83 ( d , j = 
 7.8 hz , 1h ) , 7.597.44 ( m , 6h ) , 7.317.28 ( m , 2h ) , 3.76 
 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 170.5 , 160.2 , 
 147.9 , 136.7 , 134.4 , 131.0 , 130.7 , 130.2 , 129.1 , 128.7 , 127.6 , 124.5 , 
 122.9 , 110.2 , 38.5 . hrms ( esi(+ ) ) 
 calcd for c17h14brn2os ( m + h ) : 373.0010 . 
 found : 373.0001 . h nmr ( 300 
 mhz , cdcl3 ) : 7.80 ( d , j = 8.7 hz , 2h ) , 
 7.597.50 ( m , 7h ) , 3.75 ( s , 3h ) . c nmr ( 75 mhz , 
 cdcl3 ) : 170.4 , 160.2 , 148.3 , 134.5 , 133.7 , 131.8 , 131.0 , 
 128.7 , 127.6 , 121.8 , 109.6 , 38.5 . hrms ( esi(+ ) ) calcd for c17h14brn2os ( m + h ) : 373.0010 . found : 373.0005 . h nmr 
 ( cdcl3 ) : 7.617.57 ( 2h , m ) , 7.557.50 ( 3h , 
 m ) , 7.19 ( 1h , s ) , 7.15 ( 2h , s ) , 3.95 ( 6h , s ) , 3.89 ( 3h , s ) , 3.77 ( 3h , 
 s ) . c nmr ( cdcl3 ) : 170.4 , 160.0 , 153.5 , 149.5 , 
 138.3 , 134.5 , 131.0 , 130.5 , 128.7 , 127.6 , 108.9 , 103.4 , 61.0 , 56.2 , 
 38.5 . ms - ei : 384 ( m ) . hrms ( esi(+ ) ) calcd for c20h21n2o4s ( m + h ) : 385.1222 . found : 
 385.1216 . h nmr ( 300 mhz , cdcl3 ) : 7.697.64 ( m , 2h ) , 7.577.48 
 ( m , 5h ) , 7.167.10 ( m , 2h ) , 3.67 ( s , 3h ) , 2.52 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 170.2 , 163.7 , 160.5 , 156.2 , 
 143.8 , 134.6 , 131.6 , 130.8 , 130.1 , 130.0 , 128.6 , 127.6 , 123.3 , 115.4 , 
 115.1 , 37.9 , 12.1 . hrms ( esi(+ ) ) 
 calcd for c18h16fn2os ( m + h ) : 327.0967 . 
 found : 327.0959 . h nmr ( 300 mhz , cdcl3 ) : 7.82 ( d , j = 
 8.1 hz , 2h ) , 7.597.49 ( m , 5h ) , 7.247.19 ( m , 3h ) , 3.76 
 ( s , 3h ) , 2.39 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 170.4 , 159.9 , 149.5 , 137.7 , 134.7 , 132.0 , 130.8 , 129.4 , 128.6 , 
 127.6 , 126.0 , 108.4 , 38.4 , 21.3 . hrms ( esi(+ ) ) calcd for c18h17n2os 
 ( m + h ) : 309.1062 . found : 309.1055 . h nmr ( 300 mhz , cdcl3 ) : 7.58 ( d , j = 8.4 hz , 2h ) , 7.16 ( s , 1h ) , 7.16 
 ( s , 2h ) , 7.00 ( d , j = 8.7 hz , 2h ) , 3.95 ( s , 6h ) , 
 3.89(s , 6h ) , 3.81 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 170.2 , 161.8 , 160.4 , 153.4 , 149.2 , 138.0 , 130.6 , 130.0 , 126.4 , 
 113.9 , 108.7 , 103.4 , 60.9 , 56.2 , 55.4 , 38.8 , 29.7 . hrms ( esi(+ ) ) calcd 
 for c21h23n2o5s ( m + h ) : 
 415.1328 . found : 415.1317 . h nmr ( 300 mhz , cdcl3 ) : 7.237.17 ( m , 3h ) , 7.16 ( s , 2h ) , 6.95 ( d , j = 8.4 hz , 1h ) , 3.96 ( s , 3h ) , 3.95(s , 6h ) , 3.93 ( s , 3h ) , 3.88 ( s , 
 3h ) , 3.82 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 
 170.1 , 160.4 , 153.4 , 151.4 , 149.3 , 149.0 , 138.1 , 130.5 , 126.5 , 121.3 , 
 111.3 , 110.4 , 108.7 , 103.4 , 61.0 , 56.2 , 56.0 , 38.8 , 29.7 . hrms ( esi(+ ) ) calcd for c22h25n2o6s ( m + h ) : 445.1433 . found : 445.1426 . h nmr ( 300 mhz , cdcl3 ) : 7.35 ( d , j = 8.4 hz , 1h ) , 7.16 ( s , 3h ) , 6.60 
 ( dd , j = 2.4 and 8.4 hz , 1h ) , 6.52 ( d , j = 2.1 hz , 1h ) , 3.94(s , 6h ) , 3.88 ( s , 3h ) , 3.87 ( s , 3h ) , 3.84 ( s , 
 3h ) , 3.64 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 
 168.9 , 162.8 , 159.9 , 157.1 , 153.4 , 149.0 , 138.0 , 130.7 , 129.9 , 117.2 , 
 108.5 , 105.0 , 103.4 , 98.5 , 60.9 , 56.2 , 55.6 , 55.5 , 36.7 . hrms ( esi(+ ) ) 
 calcd for c22h25n2o6s 
 ( m + h ) : 445.1433 . found : 445.1424 . h nmr ( 300 mhz , cdcl3 ) : 7.19 ( s , 
 1h ) , 7.16 ( s , 2h ) , 6.82 ( s , 2h ) , 3.95 ( s , 6h ) , 3.92 ( s , 3h ) , 3.90 
 ( s , 6h ) , 3.89(s , 3h ) , 3.80 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 170.1 , 160.2 , 153.5 , 153.3 , 149.4 , 140.3 , 138.2 , 130.5 , 
 129.5 , 108.9 , 105.1 , 103.4 , 61.01 , 60.98 , 56.3 , 56.2 , 38.7 . hrms ( esi(+ ) ) 
 calcd for c23h27n2o7s 
 ( m + h ) : 475.1539 . found : 475.1530 . h nmr ( 300 mhz , cdcl3 ) : 7.207.18 ( m , 2h ) , 7.15 ( s , 2h ) , 7.05 ( dd , j = 1.5 and 8.1 hz , 1h ) , 6.93 ( dd , j = 1.5 and 7.8 
 hz , 1h ) , 3.95(s , 6h ) , 3.93 ( s , 3h ) , 3.88 ( s , 3h ) , 3.86 ( s , 3h ) , 3.63 
 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 168.4 , 159.4 , 
 153.4 , 152.7 , 149.1 , 145.2 , 138.0 , 130.5 , 130.0 , 124.9 , 119.0 , 114.0 , 
 108.7 , 103.4 , 61.7 , 60.9 , 56.2 , 55.9 , 36.7 . hrms ( esi(+ ) ) calcd for 
 c22h25n2o6s ( m + h ) : 445.1433 . 
 found : 445.1428 . h nmr ( 300 mhz , cdcl3 ) : 7.47 ( m , 1h ) , 7.38 ( dd , j = 1.8 and 7.5 hz , 1h ) , 
 7.18 ( s , 1h ) , 7.16 ( s , 2h ) , 7.08 ( dt , j = 0.9 and 
 7.5 hz , 1h ) , 7.00 ( d , j = 8.4 hz , 1h ) , 3.95 ( s , 6h ) , 
 3.88 ( s , 3h ) , 3.86 ( s , 3h ) , 3.63 ( s , 3h ) . c nmr ( 75 mhz , 
 cdcl3 ) : 168.9 , 155.5 , 153.4 , 149.1 , 138.0 , 131.7 , 130.6 , 
 128.3 , 124.5 , 121.1 , 111.0 , 108.7 , 103.4 , 60.9 , 56.2 , 55.6 , 36.5 . 
 hrms ( esi(+ ) ) calcd for c21h23n2o5s ( m + h ) : 415.1328 . found : 415.1319 . h nmr ( 300 mhz , cdcl3 ) : 7.41 ( m , 1h ) , 7.19 ( s , 1h ) , 7.15 ( s , 2h ) , 7.157.05 ( m , 
 3h ) , 3.95 ( s , 6h ) , 3.89 ( s , 3h ) , 3.86 ( s , 3h ) , 3.76 ( s , 3h ) . c nmr ( 75 mhz , cdcl3 ) : 170.2 , 159.7 , 153.4 , 149.3 , 
 138.1 , 135.7 , 130.5 , 129.8 , 119.5 , 116.8 , 112.8 , 108.9 , 103.4 , 60.9 , 
 56.2 , 55.4 , 38.4 . hrms ( esi(+ ) ) calcd for c21h23n2o5s ( m + h ) : 415.1328 . found : 415.1322 . these cancer cell lines were routinely 
 cultured in dmem medium supplemented with 10% fbs , 4 mm glutamine , 
 1 mm sodium pyruvate , 100 iu / ml penicillin , 100 g / ml streptomycin , 
 and 0.25 g / ml amphotericin . briefly , 
 mda - mb-231 , a549 , or hela cells were seeded at a density of 2.5  
 10 in 500 l of serum - free and phenol red - free media 
 in the upper chamber of a 24-well transwell system . dmem supplemented 
 with fbs ( 5% ) was used as a chemoattractant in the lower wells . after 
 24 h , membranes were scrubbed , fixed with 10% phospho - buffered formalin , 
 permeabilized with 100% ice - cold methanol , and stained with 0.1% crystal 
 violet in 20% methanol . membranes were removed and mounted on glass 
 slides for visualization by light microscopy . for dose dependent migration 
 assays , mda - mb-231 cells were seeded in the transwell migration chambers 
 as described above and treated with 10 , 10 , 10 , and 10 m concentrations of each thiazole compound for measurement of resulting 
 cell migration ; 0.1% dmso was used as vehicle control . data are represented 
 as % of migrated cells treated with vehicle per 100 field of 
 view ( 100 ) . for growth 
 assay in the 
 presence of 10 m individual thiazole compound , 
 mda - mb-231 cells were plated in six - well plates at a density of 50  000 
 per well in dmem medium supplemented with 10% fbs . the cells were 
 then cultured for 5 days , while equal treatment volumes of dmso were 
 used as vehicle control . the ratio of thiazole compound treated 
 cell numbers to vehicle treated cell numbers was defined as survival 
 ratio . matrigel - coated 
 invasion chambers ( bd 
 biosciences , nj ) were used to determine the inhibitory effect of the 
 thiazole derivatives on mda - mb-231 cells . briefly , cells in exponential 
 phase of growth were serum - starved for 24 h prior to seeding , detached 
 by brief trypsinization , and resuspended in medium containing dmso 
 or thiazole derivatives . mda - mb-231 cells 
 ( 2.5  10 cells / well ) were seeded in the upper chamber , 
 and medium supplemented with 10% fbs as chemoattractant containing 
 dmso or 10 m thiazole derivatives was added to the bottom well . after incubation for 24 h , the noninvasive cells were removed from 
 the upper surface of the membrane by  scrubbing  , and 
 the invasive cells on the under surface of the membrane were fixed 
 with 4% formaldehyde for 10 min at room temperature and stained with 
 0.04% crystal violet , counted microscopically at 100 magnification . 
 five fields per membrane the relative invasion of cells was calculated as the percentage 
 invasion through the matrigel membrane relative to that of dmso treated 
 cells . the fascin total rna samples were extracted 
 from hela cells using a purelink total rna purification system ( invitrogen ) 
 and quantitatively analyzed using a nanodrop spectrophotometer ( thermo 
 scientific ) . the fascin cdna was generated using a superscript iii 
 one - step rt - pcr system ( invitrogen ) with the following primers : fascin1-f 
 ( sense ) 5-gaa ttc atg acc gcc aac ggc aca gc-3 and 
 fascin1-r ( antisense ) 5-aag ctt cta gta ctc cca gag cga ggc-3. 
 the rt - pcr reaction was carried out as follows : step 1 , 45 c 
 for 30 min and 94 c for 2 min ; step 2 , for 35 cycles 94 c 
 for 15 s , 51 c for 30 s , and 72 c for 1 min and 30 s ; 
 step 3 , 72 c for 5 min and hold at 4 c . human fascin cdna 
 ( 1.5 kilobases [ kb ] ) samples were then cloned into a pcdna 3.1 vector 
 ( invitrogen ) . for fascin overepression , the mda - mb-231 cells were seeded at a density of 5 
  10 cells par well in six - well plates . at 7080% confluence , the cells were transiently tranfected with 4 g 
 of the fascin - expressing plasmid pcdna - fascin . the transfection was 
 achieved by incubating the cells overnight at 37 c in a co2 incubator with lipofectamine 2000 reagent ( invitrogen ) . the 
 next day , cell medium was replaced with 1000 g / m g418 followed 
 by medium replacement every 23 days for a total 1012 
 days . stable clones were isolated after selection with 500 g / ml 
 g418 for 23 weeks , and fascin expression levels were determined 
 by western blot . fertilized 
 embryos ( charles river laboratories , charleston , sc ) were incubated 
 at 37.5 c for 3 days , removed from their shell using a dremel 
 tool , and placed into a covered weighing boat for 7 further days of 
 incubation . solidified 30 l onplants containing 2.1 mg / ml rat 
 tail collagen ( bd biosciences , bedford , ma ) and 10 ng of bfgf and 
 30 ng of vegf in the presence or absence of thiazole analogues 5o and 5p were placed on the cam over two pieces 
 of nylon mesh approximately 0.5 cm . after 3 additional 
 days of incubation , images were taken of each plug on surviving embryos 
 using a mini - vid camera ( lw scientific ; lawrenceville , ga ) and quantified 
 in a masked fashion on a scale from 0 to 3 with 0 representing no 
 angiogenesis and 3 representing extreme angiogenesis . data from one 
 scorer ( confirmed by a second masked scorer ) are presented as the 
 mean  standard error of the mean . statistical significance was 
 determined by one - way analysis of variance ( anova ) followed by dunnett s 
 multiple comparison test ( graphpad prism 6 , la jolla , ca ) . vehicle or thiazole analogues 
 treated hela cells were fixed for 15 min with 3.7% paraformaldehyde 
 in pbs . cells were washed with pbs and permeabilized in pemeabilization 
 buffer ( 0.5% triton x-100 ( w / v ) in pbs ) for 10 min and washed again 
 with pbs . the cells were then treated for 30 min with 200 l 
 of 100 nm acti - stain 488 phalloidin in the dark . the coverslips were 
 rinsed with pbs and inverted on a drop of antifade mounting media 
 on a glass slide . cells were observed under a fluorescence microscope 
 equipped with a 480ex/535em filter set , a digital ccd camera , and 
 40 objective .
fascin 
 has recently emerged as a potential therapeutic target , 
 as its expression in cancer cells is closely associated with tumor 
 progression and metastasis . following the initial discovery of a series 
 of thiazole derivatives that demonstrated potent antimigration and 
 antiinvasion activities via possible inhibition of fascin function , 
 we report here the design and synthesis of 63 new thiazole derivatives 
 by further structural modifications in search of more potent fascin 
 inhibitors . 
 the 5 series of analogues with longer alkyl 
 chain substitutions on the thiazole nitrogen exhibited greater antimigration 
 activities than those with other structural motifs . 
 the most potent 
 analogue , 5p , inhibited 50% of cell migration at 24 nm . 
 moreover , the thiazole analogues showed strong antiangiogenesis activity , 
 blocking new blood vessel formation in a chicken embryo membrane assay . 
 finally , a functional study was conducted to investigate the mechanism 
 of action via interaction with the f - actin bundling protein fascin .