Multidrug resistance (MDR) reduces the sensitivity of tumor cells to various chemotherapeutic drugs with unrelated structures, thereby considerably reducing their efficacy and even resulting in the failure of cancer chemotherapy [1,2]. P-glycoprotein (Pgp/ABCB1/MDR1) is widely overexpressed in MDR cancer cells and is considered to be the main cause of the onset of MDR [[3], [4], [5]]. Pgp is the first of the ATP-Binding Cassette (ABC) transporters to be identified. Its efflux activity decreases the intracellular accumulation of anticancer drugs (e.g., imatinib, paclitaxel, and vinca alkaloids), thus causing MDR and resulting in poor clinical outcomes [4]. Inhibiting the efflux function of Pgp can restore the intracellular accumulation of anticancer drugs, so as to efficiently kill cancer cells. Therefore, Pgp is a key drug target in overcoming Pgp-mediated MDR in cancer treatment [[6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18]].

Over the past few decades, three generations of Pgp modulators have been developed, which include verapamil (Vrp), dexverapamil, tariquidar (Tqd), and zosuquidar. Unfortunately, owing to toxicity, poor therapeutic efficacy, and other shortcomings, none of the Pgp inhibitors have been approved for clinical use [19]. Recently, multiple groups have developed highly active modulators based on the structural evolutionary trends of the three generations of Pgp modulators [[7], [8], [9],13,14,17,18]. One such example is the compound d7, whose reversal fold (RF) is up to 6011, approximately 20 times higher than that of Tqd (third-generation Pgp modulator) [9]. Nonetheless, the cytotoxic effects of these Pgp modulators are yet to be alleviated. Several groups have developed novel potential Pgp modulators with low cytotoxicity from various natural products [20]. The development of hypotoxic, effective, and structurally novel molecules for combatting Pgp-mediated MDR is an important alternative approach. However, such Pgp modulators are mostly less active than Tqd and should be improved further.

Pyxinol (see Fig. 1) is a human liver metabolite of 20S-protopanaxadiol (20S-PPD), which is the pharmacophore of ginsenoside, the main active ingredient of Panax ginseng [21,22]. Owing to its established safety profile with a strip-shaped scaffold structure that is similar to most Pgp modulators in appearance, an attempt was made to develop pyxinol derivatives to treat Pgp-mediated MDR [[23], [24], [25], [26], [27]]. Y30, an amide derivative of pyxinol, was identified to effectively suppress Pgp-mediated MDR and be orally available [25]. G3 is another Pgp modulator that has been developed from pyxinol, and its activity is superior to that of Vrp (first-generation Pgp modulator) [26]. Both Y30 and G3 have been confirmed to be substrate inhibitors of Pgp that can stimulate basal Pgp-ATPase activity as well as Vrp, Tqd, and zosuquidar [25,26]. Recently, a prominent pyxinol derivative, S54, modified with l-proline residue was developed, which can effectively inhibit Pgp efflux function and suppress both basal and stimulated Pgp-ATPase activity [27]. S54 could not be pumped out by Pgp and bound preferentially to its putative allosteric site (PAS) in the outward-facing conformation but not the general drug-binding domain (DBD) [27]. S54 is quite different from other common Pgp modulators and is termed as a nonsubstrate allosteric inhibitor of Pgp. Nevertheless, the MDR reversal activity of these pyxinol derivatives is lower than that of Tqd.

In this study, based on the structural optimization of the derivative S54, 29 novel pyxinol amide derivatives bearing an aliphatic heterocycle were designed and synthesized. Their MDR reversal activity was investigated in KBV cells, and the effect of Pgp on the intracellular accumulation of potent pyxinol derivatives and Vrp was analyzed using liquid chromatography–tandem mass spectrometry (LC–MS/MS). The most potent derivative 4c, which exhibited better MDR reversal activity than Tqd with less cytotoxicity, was further examined. Its selectivity and mechanism of reversing Pgp-mediated MDR were elucidated. In addition, the binding property of 4c toward Pgp and its effect on KBV cell migration and colony formation were investigated.