Pipersentan: A De Novo Synthetic Endothelin Receptor Antagonist that Inhibits Monocrotaline- and Hypoxia-Induced Pulmonary Hypertension

Figures

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  FIGURE 1. Chemical Synthesis of Pipersentan. Pipersentan was synthesized in three steps. First, the commercially available 1-(5-bromo-6-chloropyrimidin-4-yl)-N-(2-methoxyethyl)azanesulfonamide (M1) underwent nucleophilic substitution with ethylene glycol (M2) to give the intermediate 1-{5-bromo-6-[(2-hydroxyethyl)oxy]pyrimidin-4-yl}-N-(2-methoxyethyl)azanesulfonamide (M3). Then, the intermediate 1-[5-(benzo[d][1,3]dioxol-5-yl)-6-[(2-hydroxyethyl)oxy]pyrimidin-4-yl]-N-(2-methoxyethyl)azanesulfonamide (M5) was obtained by the Suzuki coupling reaction from M3 and 2-(benzo[d][1,3]dioxol-5-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (M4). Finally, pipersentan was obtained through the nucleophilic substitution reaction from M5 and 5-bromo-2-chloropyrimidine (M6).

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  FIGURE 2. Effects of Pipersentan on hPASMC Proliferation, Migration and Calcium Mobilization (A–D) hPASMCs were treated with DMSO, Pipersentan (0.01–100 μM), Macitentan (0.01–100 μM), SSP (0.2 μM) or Triton and exposed to DMSO or ET-1 (1 μM) for 24 h. (A,B) Proliferation was determined by detecting DNA synthesis in adherent cells (n = 5). (C,D) Cytotoxicity was determined by detecting LDH released into the cell culture medium (n = 5) (E,F) hPASMCs were treated with DMSO, Macitentan (10 μM) or Pipersentan (0.1–10 μM) and exposed to DMSO or ET-1 (1 μM) for 24 h. (E) Representative images of migrated cells (scale bars, 500 μm, Original magnification: 200). (F) Quantification of the crystal violet levels (n = 3) (G) The antagonistic activity against hPASMCs was determined by detecting the intracellular calcium mobilization (n = 3). The IC50 of Macitentan and Pipersentan was 0.51 ± 0.56 nM and 1.66 ± 0.51 nM. The data are presented as the mean ± standard deviation, *** p < 0.001 vs. ET-1 (-); ### p < 0.001, ## p < 0.01, # p < 0.05 vs. ET-1 (+), n. s., not significant, ANOVA with Tukey’s post hoc in (A–F); n. s., not significant, unpaired Student’s t-tests in (G); SSP, staurosporine; Pip,…

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  FIGURE 3. Pipersentan Improved Cardiopulmonary Function in Rats with MCT-induced PH (n = 12, 10, 13, 12, 13 and 13 for each group) (A) Diagrammatic sketch of the animal protocol. Rats were subcutaneously injected with MCT at a dose of 60 mg/kg or with vehicle. Treatments by daily oral gavage started on day 14 and were continued for 2 weeks. Measurement of PH parameters was performed on day 28 (B–D) Hemodynamic measurements. (B) Representative images. (C) RVSP. (D) mPAP (E–G) UCG analyses. (E) Representative images. (F) TAPSE. (G) RVWT. The data are presented as the mean ± standard deviation, *** p < 0.001 vs. veh, ### p < 0.001 vs. MCT + veh, ## p < 0.01 vs. MCT + veh, # p < 0.05 vs. MCT + veh, ANOVA with Tukey’s post hoc test. Veh, vehicle; Mac, macitentan; Pip, Pipersentan; RVSP, right ventricular systolic pressure; mPAP, mean pulmonary artery pressure; UCG, ultrasonic cardiogram; TAPSE, tricuspid annular plane systolic excursion; RVWT, right ventricular wall thickness.

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  FIGURE 4. Pipersentan Improved Cardiopulmonary Remodeling in Rats with MCT-induced PH (n = 12, 10, 13, 12, 13 and 13 for each group) (A,B) Histopathological analysis. (A) Representative images of pulmonary arterioles (scale bars, 50 μm, Original magnification: 200). (B) MWT (C) RVHI. The data are presented as the mean ± standard deviation, *** p < 0.001 vs. veh, ### p < 0.001 vs. MCT + veh, ## p < 0.01 vs. MCT + veh, # p < 0.05 vs. MCT + veh, ANOVA with Tukey’s post hoc test. Veh, vehicle; Mac, macitentan; Pip, Pipersentan; MWT, medial wall thickness; RVHI, right ventricular hypertrophy index.

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  FIGURE 5. Pipersentan Improved Cardiopulmonary Function in Rats with Hypoxia-induced PH (n = 12) (A) Diagrammatic sketch of the animal protocol. Rats were housed in a hypobaric hypoxia chamber (with a barometric pressure of approximately 380 mmHg) or in normal air (with a barometric pressure of approximately 760 mmHg) for 28 days. Treatments by daily oral gavage started on day 14 and were continued for 2 weeks. Measurement of PH parameters was performed on day 28 (B–D) Hemodynamic measurements. (B) Representative images. (C) RVSP. (D) mPAP (E–G) UCG analyses. (E) Representative images. (F) TAPSE. (G) RVWT. The data are presented as the mean ± standard deviation, *** p < 0.001 vs. veh, ### p < 0.001 vs. Hypoxia + veh, ## p < 0.01 vs. Hypoxia + veh, # p < 0.05 vs. Hypoxia + veh, ANOVA with Tukey’s post hoc test. Veh, vehicle; Mac, macitentan; Pip, Pipersentan; RVSP, right ventricular systolic pressure; mPAP, mean pulmonary artery pressure; UCG, ultrasonic cardiogram; TAPSE, tricuspid annular plane systolic excursion; RVWT, right ventricular wall thickness.

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  FIGURE 6. Pipersentan improved Cardiopulmonary Remodeling in Rats with Hypoxia-induced PH (n = 12). (A,B) Histopathological analysis. A Representative images of pulmonary arterioles (scale bars, 50m, Original magnification: 200). B MWT. (C) RVHI. The data are presented as the mean standard deviation, ***P < 0.001 vs veh, ###P < 0.001 vs MCT+veh, ##P < 0.01 vs MCT+veh, #P < 0.05 vs MCT+veh, ANOVA with Tukeys post hoc test. Veh, vehicle; Mac, macitentan; Pip, Pipersentan; MWT, medial wall thickness; RVHI, right ventricular hypertrophy index.