Design, Synthesis and Characterization of ABCG2 Inhibitors with a Focus on Water Solubility and Stability in Plasma

ATP-binding cassette (ABC) transporters are gatekeepers at all major tissue barriers, e.g. the intestinal barrier or the blood-brain barrier (BBB). On one hand, these proteins extrude endogenous substrates and on the other hand, they protect cells against the entry of numerous xenobiotics, including a variety of drugs such as anticancer agents or neuropharmaceuticals. The three main drug transporters are ABCB1, ABCC1 and ABCG2, with ABCG2 being predominant at the human BBB. They constitute a severe impediment for the oral bioavailability and brain penetration of drugs, and confer multidrug resistance (MDR) to cancer cells. Thus, inhibitors of ABCG2 are required as molecular tools for examinations on the patho(physiological) role of this transporter and as potential drugs to overcome tissue barriers (especially the BBB) and MDR in cancer. So far, the discovery of ABCG2 inhibitors had largely been driven by potency, while drug-like properties – imperative for application in vivo – had been overlooked. Moreover, the mechanism of transport inhibition had been enigmatic. Therefore, the present thesis aimed at creating novel ABCG2 inhibitors with improved drug-like properties, especially stability in blood plasma and water solubility, and at elucidating the underlying transport inhibition mechanism. Tariquidar analogs had been described as potent and selective ABCG2 inhibitors. Yet, their susceptibility to hydrolysis limited their applicability in vivo. Chapter 2 of this thesis comprises the synthesis (performed by Prof. König’s group) and characterization (performed by our group, including the author of this thesis) of new tariquidar-related inhibitors, obtained by bioisosteric replacement of the labile moieties in the previous tariquidar analog UR-ME22-1. CuAAC (“click” reaction) gave access to a triazole core as a substitute for the labile amide group and the unstable ester moiety was replaced by acyl groups. An HPLC assay proved enhanced stability in blood plasma. Compounds UR-MB108 and UR-MB136 inhibited ABCG2 in a Hoechst 33342 transport assay with an IC50 value of about 80 nM, thus belong to the most potent ABCG2 inhibitors described so far. Compound UR-MB108 was highly selective for ABCG2, while its PEGylated analog UR-MB136 showed some potency at ABCB1. Both UR-MB108 and UR-MB136 produced an ABCG2 ATPase-depressing effect, which is in agreement with a precedent cryo-EM study identifying UR MB136 as an ATPase inhibitor that exerts its effect via locking the inward-facing conformation of ABCG2. As reference substances, compounds UR-MB108 and UR-MB136 allow additional mechanistic studies on ABCG2 inhibition. Stable in blood plasma, they are also applicable in vivo. Chapter 3 of this thesis is focused on water solubility, which is an indispensable prerequisite for all bioactive compounds – seemingly trivial, however often underestimated. Many drug development failures have been attributed to poor aqueous solubility. ABCG2 inhibitors are especially prone to be insoluble since they have to address the extremely large and hydrophobic multidrug binding site in ABCG2. A case in point is UR-MB108, which showed high potency (79 nM), but very low aqueous solubility (78 nM). To discover novel potent ABCG2 inhibitors with improved solubility, a fragment-based approach was pursued. Substructures of UR-MB108 were optimized and the fragments ‘enlarged’ to obtain inhibitors, in part supported by molecular docking studies. Syntheses were achieved, i.a., via Sonogashira coupling, click chemistry and amide coupling. A kinetic solubility assay revealed that UR-MB108 and most of the new inhibitors did not precipitate during the short time period of the applied biological assays. The solubility of the compounds in aqueous media at equilibrium was investigated in a thermodynamic solubility assay, where UR-Ant116, UR-Ant121, UR-Ant131 and UR-Ant132 excelled with solubilities between 1 µM and 1.5 µM – an up to 19-fold improvement compared to UR-MB108. Moreover, these novel N-phenyl-chromone-2-carboxamides inhibited ABCG2 in a Hoechst 33342 transport assay with potencies in the low three-digit nanomolar range, reversed MDR in ABCG2-overexpressing cancer cells, were non-toxic and proved stable in blood plasma. Altogether, the aforementioned compounds are attractive candidates for in vivo studies and in vitro assays requiring long-term incubation, both needing sufficient solubility at equilibrium. UR-Ant121 and UR-Ant132 were highly ABCG2-selective, a precondition for developing PET tracers. The triple ABCB1/C1/G2 inhibitor UR-Ant116 qualifies for potential therapeutic applications, given the concerted role of the three transporter subtypes at many tissue barriers, e.g. the BBB. Taken together, this thesis provides ABCG2 inhibitors that are potent and effective as well as water-soluble, stable in blood plasma and nontoxic, which may be of interest as molecular biological tools and as drugs for patients with cancer or brain disorders