Design, Synthesis, and Cardioprotective Effects of N-Mercapto-Based Hydrogen Sulfide Donors

Abstract

Hydrogen sulfide (H2S) is a signaling molecule which plays regulatory roles in many physiological and/or pathological processes. Therefore, regulation of H2S levels could have great potential therapeutic value. In this work, we report the design, synthesis, and evaluation of a class of N-mercapto (N-SH)-based H2S donors. Thirty-three donors were synthesized and tested. Our results indicated that controllable H2S release from these donors could be achieved upon structural modifications. Selected donors (NSHD-1, NSHD-2, and NSHD-6) were tested in cellular models of oxidative damage and showed significant cytoprotective effects. Moreover, NSHD-1 and NSHD-2 were also found to exhibit potent protective effects in a murine model of myocardial ischemia reperfusion (MI/R) injury.

Scheme 1. Design of NSHDs

Scheme 2. Synthesis of NSHDs

Figure 1

Cysteine and GSH-mediated H₂S release from NSHD-1.

Scheme 3. Chemical Synthesis of 1-Mercapto-3-phenylurea-Based Donors

Scheme 4. Proposed Mechanism of H₂S Release from NSHDs

Figure 2

Cytotoxicity of NSHDs in the absence or presence of cysteine. H9c2 cells were incubated with NSHD-1, NSHD-2, and NSHD-6 at varied concentrations (20–160 μM) for 1 h in the absence or presence of cysteine (480 μM). Excess NSHDs were then washed away with PBS. The CCK-8 assay was applied to measure cell viability. Data were expressed as the mean ± SEM. *P < 0.05, **P < 0.01 vs the control group.

Figure 3

Effects of H₂O₂ on cell viability. H9c2 cells were treated with varied concentrations of H₂O₂ for 5 h. The CCK-8 assay was performed to detect cell viability. Data were expressed as the mean ± SEM. **P < 0.01 vs the control group.

Figure 4

Effects of H₂S donors (A–E) and NSHD byproducts (F) on H₂O₂-induced cellular damage. Prior to the 5-h H₂O₂ (400 μM) treatment, H9c2 cells were preconditioned with NSHD-1 (A), NSHD-2 (B), and NSHD-6 (C) in the presence of cysteine (480 μM) at the indicated concentrations for 1 h. GYY4137 (D) and NaHS (E) were used as positive controls under similar conditions. Effects of H₂S releasing byproducts, such as NBC and BZM (160 μM), and cysteine alone (480 μM) were also investigated (F). The CCK-8 assay was performed to detect cell viability. Data were expressed as the mean ± SEM. **P < 0.01 vs the control group. ^†P < 0.05, ^‡P < 0.01 vs the H₂O₂ treatment group.

Figure 5

Effects of NSHDs on H₂O₂-induced LDH release. H9c2 cells were preconditioned with NSHD-1, NSHD-2, and NSHD-6 (160 μM) in the presence of cysteine (480 μM) for 1 h followed by treatment with H₂O₂ (400 μM) for 5 h. LDH in cells and medium was measured with a commercial kit. Data were expressed as the mean ± SEM. **P < 0.01 vs the control group. ^‡P < 0.01 vs the H₂O₂ treatment group.

Figure 6

Effects of H₂S donors on H₂O₂-induced MMP loss in H9c2 cells. (A–E) Rh123 staining followed by photofluorography to observe MMP in H9c2 cells. (A) Control group. (B) Cells were pretreated with cysteine (480 μM) for 1 h, then exposed to H₂O₂ (400 μM) for 5 h. Cells were preconditioned with 160 μM NSHD-1 (C), NSHD-2 (D), and NSHD-6 (E) in the presence of cysteine (480 μM) for 1 h prior to H₂O₂ treatment. (F) Quantitative analysis for Rh123 in panels A–E. Mean fluorescence intensities (MFI) were measured using IMAGEJ software. Data were expressed as the mean ± SEM. **P < 0.01 vs the control group. ^‡P < 0.01 vs the H₂O₂ treated group.

Figure 7

Cardioprotective effects of NSHD-1 and NSHD-2 in myocardial ischemia-reperfusion injury. Doses of NSHD-1, NSHD-2, or vehicle were injected at the time of reperfusion. (Left) Myocardial area-at-risk (AAR) per left ventricle (AAR/LV) and infarct per area-at-risk (INF/AAR) were assessed in vehicle (n = 12), NSHD-1 (50 μg/kg) treated animals (n = 6), and NSHD-1 (100 μg/kg) treated animals (n = 12). AAR/LV was similar among all groups. INF/AAR was significantly smaller in the 100 μg/kg dose treated animals. (Right) Myocardial area-at-risk (AAR) per left ventricle (AAR/LV) and infarct per area-at-risk (INF/AAR) were assessed in the vehicle (n = 12), NSHD-2 (50 μg/kg) treated (n = 12), and NSHD-2 (100 μg/kg) treated animals (n = 17). AAR/LV was similar among all groups. INF/AAR was significantly smaller in animals treated with either dose.

Figure 8

Blood was collected at 4 h of reperfusion, and circulating cardiac troponin I levels were measured. Troponin I level was significantly reduced with either 50 μg/kg or 100 μg/kg of NSHD-2 treatment.