Tubulin and warmth shock protein 27 (Hsp27) are well-characterized molecular targets for anti-cancer drug development. compounds for tubulin. The results revealed several structural moieties of the lead compounds that are critical for Hsp27 inhibition. The modification of these structural fragments eliminated Hsp27 inhibition but did not harm tubulin-targeting effects of the compounds. This result further defined the structure-activity relationship between the tubulin and Hsp27 effects of these compounds. chaperone function of Hsp27 were evaluated. By monitoring dithiothreitol (DTT)-induced insulin aggregation in the presence of Hsp27 with or without the compounds their Hsp27 inhibition can be examined. In this chaperone activity assays Hsp27 exhibited potent inhibition of DTT-induced insulin aggregation. Previous study showed that this corresponding N-methylmethanesulfonamide 5 at 10 μM inhibited Hsp27 functions by 27 % [22]. However both compounds 10 and 12 did not show inhibitory activity against Hsp27 chaperone activity at 10 μM suggesting Hsp27 targeting effect decreased TRICKB in the new compounds. Substitution of the methanesulfonamide at the C moiety of compound 5 Pseudoginsenoside-RT5 with ethanesulfonamide or benzylsulfonamide is usually detrimental for its Hsp27 targeting effect. However this modification did not impact tubulin targeting effects. The results suggest that smaller sulfonamide moiety is preferred for Hsp27 inhibition. 3 Conclusion We synthesized numerous sulfonamide derivatives and acetamide derivatives based on the previously reported compounds 2-5 [22]. The 2 2 5 group which had been demonstrated to be important for the anti-proliferative activity of these compounds was maintained for all the new compounds. The methanesulfonamide group at the C moiety was changed to an acetamide group or a diversity of alkyl/aryl sulfonamide groups. The SAR study revealed that most ethyl- propyl- phenyl- benzyl-sulfonamides showed weaker cell growth inhibition compared to the corresponding methanesulfonamides. Only N-methylethanesulfonamide 10 and N-methylbenzylsulfonamide 12 managed similar potency. Further mechanism investigation indicated that compounds 10 and 12 are potent inhibitors of tubulin polymerization. Their tubulin inhibitory activities are comparable to the corresponding lead compound N-methylmethanesulfonamide 5. However both compounds did not show Hsp27 inhibition. The substitution of methanesulfonamide with ethanesulfonamide or benzylsulfonamide significantly impaired the Hsp27 inhibitory effects. The molecular docking simulation suggested that compounds 10 and 12 may adopt different binding modes to be accommodated in the colchicine binding site of tubulin. Future study will focus on discerning the structural fragments that are important for Hsp27 inhibition and develop new anti-cancer brokers with better potency to Pseudoginsenoside-RT5 target both tubulin and Hsp27. 4 Experimental section 4.1 Chemistry Chemicals were commercially available and used as received without further purification. Moisture sensitive reactions were carried out under a dry argon atmosphere in flame-dried glassware. Thin-layer chromatography was performed on silica gel TLC plates with fluorescence indication 254 nm (Fluka). Flash column chromatography was performed using silica gel 60? (BDH 40 μM). Mass spectra were obtained around the ABI QStar Electrospray mass spectrometer at Cleveland State University MS facility Center. All the NMR spectra were recorded on a Bruker 400 MHz (13C NMR at 100 MHz) using DMSO-= 8.8 Hz) 7.677 (1H d J= 8.4 Hz) 7.623 (1H s) 7.355 (1H d Pseudoginsenoside-RT5 J= 8.8 Hz) 7.141 (1H d J= 2.8 Pseudoginsenoside-RT5 Hz) 7.058 (2H d J= 8.8 Hz) 6.981 (1H d J= 9.2 Hz) 6.859 (1H dd J= 2.8 8.8 Hz) 5.097 (2H s) 3.838 (3H s) 3.805 (3H s) 3.718 (3H s) 2.07 (3H s); 13C NMR δ 168.63 165.15 162.36 153.68 150.9 149.91 136.92 129.98 127.4 126.16 123.99 123.61 114.5 114.05 113.66 112.74 112.24 105.88 65.44 56.39 55.9 55.82 24.06 ESI-MS calculated for C25H27N2O6 [M+H]+ 451.19 found: 450.99 N-[3-(2 5 4 (31): 1H NMR δ 10.056 (1H s) 9.172 (1H s) 7.69 (1H d J= 8.8 Hz) 7.606 (2H m) 7.522 (1H d J= 1.6 Hz) 7.327 (1H d J= 9.2 Hz) 7.142 (1H d J= 3.2 Hz).