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Fig. 3 Stability of quininib and analogues at different pH values and in the presence of esterase. Stability of quininib (A), ace-quininib-HCl (3) (B) and ace-quininib (2) (C) at different pH values at room temperature (20 °C). Stability study (n = 2) was carried out in phosphate buffers at pH 4 (blue circles), 7.4 (red squares) or 9 (green triangles). Compound (1 mg/mL, 10 μL) was added to the phosphate buffer at room temperature. Samples were removed, vortexed and assayed at t = 0, 15, 30, 60, 120, 180, 240 and 1440 min. Compound stability was determined based on the percentage remaining at each timepoint compared to amount at t = 0 min. (D,E) Ace-quininib (2) is converted to quininib (1) in the presence of esterase enzymes. The conversion of ace-quininib (2) to quininib (1) was measured using a porcine esterase enzyme in PBS at pH 7.4. The reaction mixture was incubated in a water bath at 37 °C for 5 min. At t = 5 s, 30 s, 1 min, 2 min and 5 min, aliquots of 500 μL were removed and quenched with ice cold methanol: acetonitrile before analysis by HPLC. N = 3. (E) Retention time of quininib (1) is 4.9 min and ace-quininib (2) is 6.1 min.