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

Abstract

In an attempt to find clinically useful modulators of multidrug resistance (MDR), a series of twentyone 2-chloro-<i>N</i>¹⁰-substituted phenoxazines has been synthesized. The novel 2-chlorophenoxazine is prepared by the pyrolytic condensation of 2-bromophenol and 2,5-dichloronitrobenzene. This compound undergoes <i>N</i>-alkylation in the presence of phase transfer catalyst (PTC). Stirring of 2-chlorophenoxazine with 1-bromo-3-chloropropane or l-bromo-4-chlorobutane in a two phase system consisting of an organic solvent (benzene) and 6<i>N </i>potassium hydroxide in the presence of tetrabutylammonium bromide leads to the formation of compounds <b>2</b> and <b>9</b> in good yield. <i>N</i>-(ω-chloroalkyl) and <i>N</i>-(chloroacetyl) analogues have been found to undergo iodide-catalyzed nucleophilic substitution on reaction with various secondary amines. Products have been characterized by UV, IR, ¹H and ¹³C NMR, mass-spectral data and elemental analyses. The lipophilicity expressed in log₁₀ <i>P, </i>and <i>pK_a </i>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 <b>3, 4, 7, 8</b> and <b>10-15</b> at 100 μ<i>M </i>concentration exhibit enhanced accumulation of VLB by 2.0-5.8-fold greater than a similar concentration of verapamil. However, the effects on VLB 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 rhabdomyosarcoma cell line (Rh30) has also been examined. Most of the chlorophenoxazines at 100 μ<i>M</i> concentration except <b>2, 9, 16</b> and <b>18</b> enhance significantly the accumulation of VLB in Rh30 cells by 10.9-53-fold with respect to control. 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 cells by 1.15-19.7-fold. The effect of compounds <b>3, 5, 6, 12</b> and <b>17-21</b> on the efflux of VLB from KBCh^R-8-5 cells has been examined and the results show that these compounds except <b>21</b> significantly inhibit the efflux of VLB consistent with being competitors for <i>P</i>-glycoprotein. Efflux of VLB from Rh30 cells in the presence of 100 μ<i>M </i>of <b>1, 5, 12, 17, 20</b> and <b>21</b> result in 43-65% of the accumulated VLB being retained at 2 hr, suggesting that the phenoxazines have relatively little effect on VLB efflux from Rh30 cells. The previous work using DiOC₃ (3) has shown that the dye is a part of P-glycoprotein-mediated MDR phenotype. The KBCh^R-8-5 cells are loaded with 360 n<i>M</i> of DiOC₃ (3) and efflux experiments are done in the absence or presence of <b>6, 12</b>, or <b>21</b> by monitoring the fluorescence signal with time and the results 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 both <i>P</i>-glycoprotein mediated and independent mechanisms. Cytotoxicity has been determined and the <i>IC</i>₅₀values lie in the range 3.2-42.1 μ<i>M </i>for <i>N</i>¹⁰-chloropropyl, 2.7-16.7 μ<i>M </i>for <i>N</i>¹⁰<i>-</i>chlorobutyl and 51.6-68.6 μ<i>M </i>for <i>N</i>¹⁰-chloroacetyl derivatives against KBCh^R-8-5 cells suggesting that the antiproliferative activity decreases in the order: - butyl > - propyl > - acetyl analogues. Further, substitution of hydrogen by chlorine in C-2 of phenoxazine 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, presumably due to increased hydrophobicity. Compounds at <i>IC</i>₁₀<i> </i>have been evaluated for their efficacy to modulate the cytotoxicity of VLB in KBCh^R-8-5 cells and compound <b>6</b> exhibits the greatest MDR reversal effect (136-fold) followed by compounds <b>12</b>, <b>10, 13, 11</b> and so on. The structural features for reversal of MDR seem to include a hydrophobic phenoxazine ring with a -Cl group in the C-2 position and a tertiary amino group at a distance of three or four carbon chain from the tricyclic ring. Examination of the relationship between partition coefficient and cytotoxicity or antiMDR activity shows no correlation suggesting that lipophilicity is not the sole determinant of potency for biological activity.