Tetrahedron Lett. To examine further the roles of the amide/thioamide switch and the lipophilicity/hydrophobicity of one amino substituent on P-gp ATPase stimulation, inhibition of P-gp-mediated efflux in whole cells, and transport of the chalcogenorhodamines in whole cells, the series of amide- and thioamide-containing thiorhodamines 1-12 (Chart 1) were prepared. The thiorhodamine core was selected 1) to avoid any ambiguities associated with the chalcogen atom and 2) because a thiorhodamine was the best inhibitor in earlier work.15 The thiorhodamines were evaluated for their ability to stimulate ATPase activity in isolated, wild-type human P-gp, their ability to be transported in multidrug-resistant, P-gp over-expressing cells, and their ability to inhibit P-gp drug transport in Epertinib hydrochloride multidrug-resistant cells. Open in a separate window Chart 1 Thiorhodamine analogues 1-12 examined in this study. 2. Results and Discussion 2.1. Epertinib hydrochloride Synthesis of thiorhodamine analogues 1-12 Thiorhodamines 1-12 were prepared from thioxanthones 13-15 (Chart 2), which were prepared by reported procedures.19,20 Compound 14 replaces a dimethylamino substituent with an azadecalin substituent. The fused aniline equivalent of this substitution is known as julolidine (Chart 2) and compounds derived from 14 are referred to as julolidyl rhodamines in the remainder of the manuscript. Thioxanthone 15 incorporates a trimethyltetrahydroquinoline group (Chart Epertinib hydrochloride 2) and compounds derived from 15 are referred to as half-julolidyl rhodamines in the remainder of the manuscript. The thioxanthone 15 has the same number of carbon atoms as 14, but the are compiled in Table 1 and covered a range from log = 1.2 for dyes 1 and 2 to log ~ 2.6-2.7 for dyes 8, 10 and 12. Based on these values of log 0.05, Student t-test) for dimethylamino analogues 1-4 (6.6-12.1-fold stimulation) relative to their corresponding julolidyl analogues 5-8 (3.5-5.6-fold stimulation) and half-julolidyl analogues 9-12 (4.7-fold stimulation). The latter two groups did not show significant differences. There were no statistically significant differences in values of 0.03 for Epertinib hydrochloride amides, 0.01 for thioamides) for the dimethylamino analogues 1-4 [(5.5-6.4) 10?6 M] relative to either their corresponding julolidyl analogues 5-8 [(1.9-3.6) 10?6 M] or the corresponding half-julolidyl analogues 9 and 10 (2.2 10?6 M and 3.1 10?6 M], respectively). For 11 and 12 where values of 0.03, Student t-test) relative to the corresponding thiorhodamine compounds 1-4 bearing dimethylamino fragments (Table 1). Furthermore, the increased affinity for compounds 5-12 bearing the julolidyl and half-julolidyl fragments gave decreased P-gp ATPase stimulation that was statistically significant ( 0.05, Student t-test) relative to the corresponding thiorhodamine compounds 1-4 bearing dimethylamino fragments. The presence of a thioamide or an amide group in thiorhodamines 1-12 gave no predictable trend in either inhibition Epertinib hydrochloride of P-gp efflux of CAM) and P-gp-mediated VIN efflux in whole cells, values for the whole-cell data are 3- to 30-fold higher Nog (thioamide functionality on a 2-thienyl substituent at the 9-position of the thiorhodamine core and varying in an amine fragment (dimethylamino, julolidyl, half-julolidyl) at the 3-position of the thiorhodamine core. While overall differences are small within this series, certain trends emerge. The net effect of all the compounds 1-12 is to function as an inhibitor of P-gp in the native environment of whole cells as measured by IC50 values for the uptake of CAM. Compounds 9-12 also have the net effect of functioning as inhibitors of P-gp as measured for IC50 values for inhibition of P-gp-mediated efflux of tritiated VIN. Derivatives with julolidyl and half-julolidyl fragments have lower values of IC50 for CAM uptake in whole cells than derivatives with a dimethylamino fragment suggesting that these derivatives have higher affinity for P-gp. Values of = 10 Hz), 7.25 (d, 1 H, = 3.5 Hz), 7.11 (d, 1 H, = 3.5 Hz), 7.09 (d, 2 H, = 2.5 Hz), 7.05 (dd, 2 H, = 2.5, 10 Hz), 4.12 (br s, 2 H), 3.87 (br s,.