In vitro activity of novel dual action MDR anthranilamide modulators with inhibitory activity on CYP-450 (Part 2)

Abstract

Synthesis and in vitro cytotoxicity assays of new anthranilamide MDR modulators have been performed to assess their inhibition potency on the P-glycoprotein (P-gp) transporter. Previous studies showed that the replacement of the aromatic spacer group between nitrogen atoms (N¹ and N²) in the P-gp inhibitor XR9576 with ethyl or propyl chain is optimal for P-gp inhibition potency. To confirm that observation, the ethyl or the propyl linker arm was replaced with a pyrrolidine or an alicyclic group such as cyclohexyl. In addition, an arylpiperazinyl group and two methoxyl groups onto the anthranilic part were introduced to assess their effect on the anti P-gp activity. Five molecules were prepared and evaluated on CEM/VLB500. All new anthranilamides were more potent than verapamil, most of them exhibited a lower cytotoxicity than XR9576. Compound 5 was the most potent and its inhibition activity was similar to XR9576. Interestingly, in vitro biotransformation studies of compounds 4 and 5 using human CYP-450 isoforms revealed, that conversely to XR9576, compounds 4 and 5 inhibited CYP3A4, an enzyme that colocalizes with P-gp in the intestine and contributes to tumor cell chemoresistance by enhancing the biodisposition of numerous drugs, notably paclitaxel. In that context, 5 might be suitable for further drug development.

Introduction

Multidrug resistance (MDR) in tumor cells is a major impediment to successful cancer therapy.¹ Multidrug resistance is a phenomenon related to tumor cells having been exposed to a cytotoxic agent and subsequently developed cross-resistance to a wide range of structurally and functionally unrelated compounds such as anthracyclines, vinca alkaloids, podophyllotoxins, and taxus alkaloids.² The chemoresistance that tumor cells develop often results from the overexpression of particular proteins such as multidrug-resistance-associated protein (MRP) and P-glycoprotein (P-gp).³ P-gp is an ATP-binding cassette transporter encoded by the MDR1 gene.⁴ P-gp contributes to drug resistance via ATP-dependent drug efflux of the cytotoxic drugs out of the cancer cells, thus lowering the intracellular concentrations at innocuous levels.5, 6 In addition, MDR genes are expressed intrinsically in some cancer cells without previous exposure to chemotherapy agents.⁷ Therefore, molecules able to antagonize P-gp activity are of utmost importance to restore the efficacy of clinically used antineoplastics. To that end, several studies have demonstrated that P-gp activity can be lowered both in vitro and in vivo by a wide variety of structurally heterogeneous molecules such as verapamil,⁸ cyclosporin A,⁹ tacrolimus,¹⁰ quinidine,¹¹ dihydropyridines,¹² and other drugs.13, 14 Consequently, numerous clinical trials using clinically relevant P-glycoprotein modulators such as verapamil,15, 16 tamoxiphen,¹⁷ progesterone,¹⁸ and cyclosporin A¹⁹ have been conducted with limited successes mainly because of their intrinsic toxicity or the unfavorable pharmacokinetics of the accompanying anticancer drugs. A third generation of P-gp modulators including cyclosporin D analogues,²⁰ anthranilamide derivatives (XR9576, tariquidar™),21, 22, 23 acridonecarboxamide derivatives (elacridar™),²⁴ and cyclopropyldibenzosuberane derivatives (zosuquidar™)²⁵ have displayed improved P-gp selectivity and pharmacological properties.²⁶

The anthranilamide derivative XR9576 (Fig. 1) was shown to reverse primary doxorubicin, vinblastine, and paclitaxel resistance in advanced breast cancer. Unfortunately, a recent phase II clinical trial on breast carcinoma and a phase III on lung carcinoma assessing the efficacy of tariquidar™ in combination with vinorelbine versus vinorelbine alone were discontinued due to a significant proportion of undisclosed adverse events in the tariquidar™ cohort.²⁷ In addition, a third clinical trial assessing the efficacy of paclitaxel/carboplatin/tariquidar™ in combination was also terminated due to poor response rates and toxicity.²⁸ Therefore, there are unmet needs for newer P-gp inhibitors with reduced intrinsic toxicity.

We report here the synthesis of novel P-gp inhibitors based on the anthranilamide derivative XR9576 (Fig. 1). As aforementioned, it is known that anthranilamides abrogate cell chemoresistance at low nanomolar levels. We have already published a study of ten anthranilamide derivatives (Fig. 2) that were designed to assess the importance of the aromatic spacer and the distance between N¹ and N² by replacing the aromatic spacer with alkyl chains of various lengths.²⁹

In that paper, we have shown that an ethyl or a propyl carbon spacer between N¹ and N² is optimal for P-gp inhibition. The most potent P-gp modulator synthesized was P03 (Fig. 2) and it is fourfold more active than verapamil, but fourfold less active than XR9576. To optimize the anti P-gp activity of compound P03, we have rigidified the spacer group between N¹ and N² found in portion C of the molecule (Fig. 2). In that context, we have evaluated the effect of modifying the ethyl group of P03 to the inhibitory activity on P-gp by choosing a group with the same length and more rigidity such as R or S pyrrolidinyl group and cyclohexyl group (Fig. 3). We have decided also to modify the linker by an arylpiperazinyl group instead of ethyl linker because the distance between N¹ and N² is similar to the distance between N¹ and N² of XR9576. We prepared arylpiperazinyl derivatives bearing two methoxy-groups on the anthralinidyl moiety (Fig. 3) to be similar to XR9576. All compounds were compared to verapamil (VRP) and XR9576. Herein we describe our efforts to identify new anthranilamide-based P-gp inhibitors.