Synthesis and analysis of structural features of phenoxazine analogues needed to reverse vinblastine resistance in multidrug resistant (MDR) cancer cells

In an attempt to find clinically useful modulators of multidrug resistance (MDR), a series of twentyone 2-chloro-N-10-substituted phenoxazines has bee n synthesized. The novel 2-chlorophenoxazine is prepared by the pyrolytic c ondensation of 2-bromophenol and 2,5-dichloronitrobenzene. This compound un dergoes N-alkylation in the presence of phase transfer catalyst (PTC). Stir ring of 2-chlorophenoxazine with 1-bromo-3-chloropropane or 1-bromo-4-chlor obutane in a two phase system consisting of an organic solvent (benzene) an d 6N potassium hydroxide in the presence of tetrabutylammonium bromide lead s to the formation of compounds 2 and 9 in good yield. N-(omega -chloroalky l) and N-(chloroacetyl) analogues have been found to undergo iodide-catalyz ed nucleophilic substitution on reaction with various secondary amines. Pro ducts have been characterized by UV, IR, H-1 and C-13 NMR, mass-spectral da ta and elemental analyses. The lipophilicity expressed in log(10) P, and pK (a) of compounds have been determined. All the compounds have been examined for their ability to increase the uptake of vinblastine (VLB) in MDR KBCh( R)-8-5 cells and the results show that compounds 3, 4, 7, 8 and 10-15 at 10 0 muM concentration exhibit enhanced accumulation of VLB by 2.0-5.8-fold gr eater than a similar concentration of verapamil. However, the effects on VL B uptake are specific because these derivatives have little activity in the parental drug-sensitive line KB 3-1. The effect of these compounds on the cellular accumulation of VLB in low P-glycoprotein containing MDR rhabdomyo sarcoma cell line (Rh30) has also been examined. Most of the chlorophenoxaz ines at 100 muM concentration except 2, 9, 16 and 18 enhance significantly the accumulation of VLB in Rh30 cells by 10.9-53-fold with respect to contr ol. Substitution of hydrogen by chlorine in position C-2 of the phenoxazine ring increases the ability to enhance the uptake of VLB in KBCh(R)-8-5 cel ls by 1.15-19.7-fold. The effect of compounds 3, 5, 6, 12 and 17-21 on the efflux of VLB from KBCh(R)-8-5 cells has been examined and the results show that these compounds except 21 significantly inhibit the efflux of VLB con sistent with being competitors for P-glycoprotein. Efflux of VLB from Rh30 cells in the presence of 100 muM of 1, 5, 12, 17, 20 and 21 result in 43-65 % of the accumulated VLB being retained at 2 hr, suggesting that the phenox azines have relatively little effect on VLB efflux from Rh30 cells. The pre vious work using DiOC(3) (3) has shown that the dye is a part of P-glycopro tein-mediated MDR phenotype. The KBCh(R)-8-5 cells are loaded with 360 nM o f DiOC(3) (3) and efflux experiments are done in the absence or presence of 6, 12, or 21 by monitoring the fluorescence signal with time and the resul ts in almost no efflux of the dye from the cells. These efflux data in KBCh (R)-8-5 and Rh30 cells suggest that 2-chlorophenoxazines may act through bo th P-glycoprotein mediated and independent mechanisms. Cytotoxicity has bee n determined and the IC50 values lie in the range 3.2-42.1 muM for N-10-chl oropropyl, 2.7-16.7 muM for N-10-chlorobutyl and 51.6-68.6 muM for N-10-chl oroacetyl derivatives against KBCh(R)-8-5 cells suggesting that the antipro liferative activity decreases in the order: - butyl > - propyl > - acetyl a nalogues. Further, substitution of hydrogen by chlorine in C-2 of phenoxazi ne ring causes a greater enhancement in the antiproliferative potency by 1. 1-2.6-fold for KBCh(R)-8-5 cells than their respective counterparts, presum ably due to increased hydrophobicity. Compounds at IC,, have been evaluated for their efficacy to modulate the cy totoxicity of VLB in KBCh(R)-8-5 cells and compound 6 exhibits the greatest MDR reversal effect (136-fold) followed by compounds 12, 10, 13, 11 and so on. The structural features for reversal of MDR seem to include a hydropho bic phenoxazine ring with a -Cl group in the C-2 position and a tertiary am ino group at a distance of three or four carbon chain from the tricyclic ri ng. Examination of the relationship between partition coefficient and cytot oxicity or anti-MDR activity shows no correlation suggesting that lipophili city is not the sole determinant of potency for biological activity.