Superior Pyrimidine Derivatives as Selective ABCG2 Inhibitors and Broad-Spectrum ABCB1, ABCC1, and ABCG2 Antagonists

Abstract

In the search for highly effective modulators addressing ABCG2mediated MDR, 23 pyrimidines were synthesized and biologically assessed. Seven derivatives with (a) nitrogen-and/or halogen-containing residue(s) had extraordinary potencies against ABCG2 (IC 50 < 150 nM). The compounds competitively inhibited ABCG2-mediated Hoechst 33342 transport but were not substrates of ABCG2. The most potent MDR reverser, compound 19, concentration-dependently increased SN-38-mediated cancer cell death at 11 nM (EC 50 ), time-dependently doubled SN-38 toxicity in a period of 7 days at 10 nM, and half-maximally accelerated cell death combined with SN-38 at 17 nM. No induction of ABCG2 was observed. Furthermore, 11 pyrimidines were revealed as triple ABCB1/ABCC1/ABCG2 inhibitors. Five possessed IC 50 values below 10 μM against each transporter, classifying them as some of the 50 most potent multitarget ABC transporter inhibitors. The most promising representative, compound 37, reversed ABCB1-, ABCC1-, and ABCG2-mediated MDR, making it one of the three most potent ABC transporter inhibitors and reversers of ABC transporters-mediated MDR.

Scaffold fragmentation and substructure hopping reveal potential, robustness, and limits of computer-aided pattern analysis (C@PA)

“…3 Visualization of the classification of modulators of ABCB1, ABCC1, and ABCG2 as proposed earlier [15] : ‘class 7 compounds’ are defined as triple ABCB1, ABCC1, and ABCG2 inhibitors that exert their half-maximal effect against these transporters below 10 µM. This has up to date been reported for 56 compounds [15] , [43] , [45] , [62] , [67] , [75] , [93] , [94] , [95] , [98] , [99] , [101] , [103] , [107] , [109] , [110] , [111] , [112] , [113] , [118] , [119] , [120] , [121] , [122] , [123] , [126] . Amongst these molecules are the compounds revealed by C@PA, 8–9 and 11 .…”

“…Compounds 16 – 25 were screened at 10 µM in calcein AM (ABCB1 and ABCC1) as well as pheophorbide A (ABCG2) fluorescence accumulation assays using either ABCB1-overexpressing A2780/ADR, ABCC1-overexpressing H69AR, or ABCG2-overexpressing MDCK II BCRP cells, respectively, as reported earlier [15] , [43] , [45] , [67] , [101] . Calcein AM and pheophorbide A are substrates of ABCB1 and ABCC1 as well as ABCG2, respectively, which passively diffuse into the used cells and become extruded by the corresponding ABC transporter.…”

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Fig. 7 Preliminary screening of compounds 16 – 25 against ABCB1 (A), ABCC1 (B), and ABCG2 (C) in calcein AM (A and B) and pheophorbide A (C) assays, respectively, using ABCB1-overexpressing A2780/ADR (A), ABCC1-overexpressing H69AR (B), and ABCG2-overexpressing MDCK II BCRP (C) cells as described earlier [15] , [43] , [45] , [67] , [101] . The data were normalized by defining 100% inhibition by the effect value of 10 µM of the reference inhibitors 2 (ABCB1; A), 26 (ABCC1, B), and 27 (ABCG2, C) as reported earlier [67] , [101] .

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“…The vast majority of reports specified structure-based retrospective computational approaches, in which observed biological effects of compounds were underpinned mostly through molecular docking experiments with cryo-EM structures or homology models. Most pronounced are, again, ABCB1 [37] , [38] , [39] , [40] , [41] , [42] and ABCG2 [43] , [44] , [45] , [46] , [47] , [48] , [49] , [50] , [51] , [52] , [53] . Less- or under-studied ABC transporters are barely reflected in the literature.…”

“…As indicated above, ABCB1, ABCC1, and ABCG2 are the most investigated and understood ABC transporters, and hence, represent model targets for the generation of pan-ABC transporter inhibitors [15] . However, even for these well-studied ABC transporters, only 133 broad-spectrum ABCB1, ABCC1, and ABCG2 inhibitors were described to date [15] , [43] , [45] , [62] , [67] , [75] , [93] , [94] , [95] , [96] , [97] , [98] , [99] , [100] , [101] , [102] , [103] , [104] , [105] , [106] , [107] , [108] , [109] , [110] , [111] , [112] , [113] , [114] , [115] , [116] , [117] , [118] , [119] , [120] , [121] , [122] , [123] , [124] , [125] , [126] , amongst which only 56 exerted their effects below 10 µM [15] , [43] , [45] , [62] , [67] , [75] , [93] , [94] , [95] , [98] , [99] , [101] , [103] , [107] , [109] , [110] , [111] , [112] , [113] , [118] , [119] , [120] , [121] ,…”