Structural Requirements for Activity of Phenoxazines fro Reversal of Drug Resistance in the Cancer Cells

Abstract

In the course of a chemical program aimed at identifying chemically
useful modulators of MDR in cancer therapy, a series of twentyone 2-trifluoromethyl-
N¹⁰-substituted phenoxazines (1-21) has been synthesised.
The novel 2-trifluoromethylphenoxazine (1) was prepared by the pyrolytic
condensation of 2-bromophenol and 4-chloro-3-nitrobenzotrifluoride as
outlined in Scheme-I. This compound undergoes N-alkylation in the
presence of phase transfer catalyst (PTC). Stirring of 2-trifluoromethylphenoxazine
with 1-bromo-3-chloropropane or 1-bromo-4-chlorobutane in
a two phase system consisting of an organic solvent (benzene) and 6N
potassium hydroxide in the presence of tetrabutylammonium bromide leads
to the formation of compounds 2 and 9 in good yield. N-(ω-chloroalkyl)and
N-(chloroacetyl) analogues were found to undergo iodide catalysed
nucleophilic substitution reaction with various secondary amines including
N,N-diethylamine, N,N-diethanol-amine, morpholine, piperidine, pyrrolidine
and (β-hydroxyethyl)-piperazine. Purified products were characterized
by UV, IR, ¹H and ¹³C-NMR and mass-spectral data. The
lipophilicity expressed in log₁₀ P and pKa of compounds were determined.
The effect of 1-21 at 100 μM on the steady-state accumulation of vinblastine
(VLB) was studied in KBCh^R-8-5 cells and the data revealed that the
compounds (3-8, 10, 12-15) exhibited a significant VLB uptake enhancing
effect (8.3-58.5-fold relative to control) compared to a standard modulator,
verapamil (VRP) (7.5-fold). These eleven compounds caused a 1.10-7.82-
fold greater uptake of VLB than did a similar concentration of VRP.
Comparison of the derivatives for their ability to potentiate the uptake of
VLB revealed that they largely follow the order: N¹⁰-propyl > N¹⁰-butyl
> N¹⁰-acetyl compounds. To determine whether the increase in VLB uptake
upon coincubation with 1-21 modulators was due to a slowing of P-gp
mediated efflux, KBCh^R-8-5 cells were loaded with [³H] VLB in the
absence of modulator and efflux examined in the absence or presence of
100 μM of 8 or VRP. Less than 10% in the absence or about 40% of cell
associated VLB in the presence of 100 μM 8 remained at the end of a 2 h
efflux period, suggesting that modulator 8, like VRP, is able to inhibit
p-glycoprotein (P-gp) mediated efflux. Cytotoxicity was determined and
the IC₁₀ and IC₅₀ values lie respectively in the range 0.1-30.9 μM and
2.1-70.9 μM for KBCh^R-8-5 cells. Substitution of hydrogen by CF₃ in

C-2 of phenoxazine ring caused a greater enhancement in the antiproliferative
protency by 1.1-3.3-fold for KBCH^R-8-5 cells than their counterparts,
presumably due to increased hydrophobicity. Compounds at IC₁₀ were
evaluated for their efficacy to modulate the cytotoxicity of VLB in KBCh^R-
8-5 cells and compounds 3, 5, 11 and 13, like VRP, were able to completely
reverse the 24-fold resistance of KBCh^R-8-5 cells to VLB. The structural
features for reversal of MDR seem to include a hydrophobic phenoxazine
ring with a —CF₃ in 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 anti-MDR
activity showed no correlation suggesting that lipophilicity is not the sole
determinant of potency for biological activity.