Fig. 5: C-benzylation prevents redox-cycling of β-lapachone; cathepsin-B-cleavable β-lapachone prodrugs.
a, In vitro redox-cycling capability of 1, 24 and 25 measured by phenol red/horseradish peroxidase reporter assay. Relative increase in redox-cycling ability calculated by (A – A0)/A0, where A is absorbance at 610 nm for the test compound and A0 is absorbance at 610 nm for a sample with PBS added only. The redox activity of 1 saturates the assay above 10 μM, but 24 and 25 show no measurable redox activity up to 25 μM. b, Generalized ROS production by compounds 1, 24 and 25 in HL-60 cells measured with the dye 2′,7′-dichlorodihydrofluorescein diacetate. c, Toxicity to AML cell line HL-60 with and without the ROS quencher NAC (600 mM). IC50 of 24 decreased slightly from 13.6 ± 1.3 µM to 10.5 ± 0.9 µM due to NAC. IC50 of 1 doubled from 0.47 ± 0.26 µM to 1.18 ± 0.18 µM with NAC. d, In vitro 5-LO enzyme target inhibition with and without DTT (1 mM). IC50 of 1 without DTT > 30 µM, with DTT 0.24 ± 0.13 µM. IC50 of 24 without DTT 8.0 ± 2.5 µM, with DTT 10.4 ± 4.1 µM. IC50 of 25 without DTT 11.3 ± 1.1 µM, with DTT 11.5 ± 2.2 µM. e, In vitro methaemoglobin generation measured by absorbance at 630 nm after 1 h incubation with test compounds. f, Cathepsin-B-activatable prodrugs release β-lapachone by linker elimination following enzymatic amide-bond cleavage. g, Release of 1 from 26 at 254 nm after in vitro dipeptide cleavage by protease cathepsin B (MES 20 mM buffer, pH 5). Peaks for PAB-BL 10 and β-lapachone 1 overlap. h, Concentration-dependent methaemoglobin generation by dipeptide prodrugs after 4 h incubation in ovine blood. Methaemoglobin was measured by absorbance at 630 nm following treatment with compounds relative to DMSO control. For a–e and h, data show mean ± s.e.m. from one representative experiment (n = 3).
