1,4-Benzothiazepines with Cyclopropanol Groups and Their Structural Analogues Exhibit Both RyR2-Stabilizing and SERCA2a-Stimulating Activities

Figures

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  Figure 1. (a) 7-Methoxy-2,3,4,5-tetrahydrobenzo [1,4-f]thiazepine derivatives that prevent Ca2+ leak from RyR channels. (b) ARM210 in the pocket of human disease-related RyR2-R2474S channel (PDB: 7UA1).23

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  Scheme 1. Synthesis of Dual-Acting Compounds with Cyclopropanol Residues Reagents and conditions: (a) Jones reagent, aceton; (b) H2SO4, EtOH; (c) EtMgBr, Ti(OiPr)4,31 Et2O; (d) t-BuMe2SiOSO2CF3, 2,6-lutidine, DCM; (e) H2, Pd/C, THF; (f) Dess–Martin reagent, DCM; (g) Na(CH3COO)3BH, 1,2-dichloroethane; (h) NaH, DMF; (i) aq. HF (55%), MeCN (compounds 11a, 12a); 1 M n-Bu4NF in THF (compounds 11b,c and 12b,c).

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  Figure 2. (a) Confocal and STED images (Abberior STED 775 QUAD scan) of live HEK-293 RyR2 R-CEPIA1er cells. Scale bar: overview −5 μm; inserts −2 and 1 μm. (b) R-CEPIA1er assay fluorescence measurements with HEK-293 cells expressing WT RyR2. F/F0 is the ratio between the average fluorescence before (first 90 s) and after (last 100 s) injection of the test compound. The effect of compound addition (25 μM, 1 mM S107) on [Ca2+]ER was normalized to the control (Cnt, 0.1 v/v% DMSO). Dant—dantrolene. Values represent the mean ± S.D., ***P < 0.0005, ****P < 0.0001 vs DMSO by unpaired t test; n = 6–28.

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  Figure 3. Caffeine-induced Ca2+ release assay. HL-1 cells were incubated with the FLIPR Calcium 6 Ca2+ indicator and then treated with various concentrations of compound 12c. Ca2+ influx was initiated by the addition of 10 mM caffeine. The differences between the caffeine-induced peak minus basal fluorescence, ΔFCaff, were taken for the analysis. The inset shows the dose–response curve of 12c. The response on caffeine addition was normalized to the control (Cnt, 0.1 v/v% DMSO). Data represent as mean ± S.D., n = 8 independent measurements.

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  Figure 4. Dose–response curves of compounds 11 and 12a,b. The data were normalized to the 0.1 v/v% DMSO. Values represent the mean ± S.D., n = 8–16.

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  Figure 5. Increase of caffeine-induced Ca2+ release by test compounds. Effects of 10 μM solutions of CDN1163, ARM210, S107, 11, and 12 (25 μM for 11d) on caffeine-induced Ca2+ release. Values represent the mean ± S.D., **P < 0.005, ***P < 0.001, ****P < 0.0001 vs control (Cnt, 0.1 v/v% DMSO) by unpaired t test, n = 8–24.

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  Figure 6. NADH-coupled ATPase assay. (a) Mouse ventricular microsomes were incubated in the assay buffer with and without 100 μM 12a. After ATP injection, the intrinsic fluorescence of NADH decreases due to ATP consumption. The reaction rate of NADH oxidation is greater in the mouse SR sample containing 12a. (b) Compound 12a evokes an enhancement in the kinetic rate of NADH–NAD+ conversion in a dose-dependent manner. The effects of compound 12a are expressed as a percentage of the control effect. Data represented as mean ± S.D., n = 3–15 independent measurements. (c) Effect of 10 μM CDN1163, 11, 12 (3 μM for 12b), and ARM210 on SERCA2a activity in mouse SR. The data were normalized to the control (Cnt, 0.1 v/v% DMSO). Values represent the mean ± S.D., *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001 vs control by unpaired t test; n = 9–24/3–5 mice.

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  Figure 7. Schematic representation of the modulating effect of cyclopropanol compounds 11 and 12 (modified from Niggli et al.56). LTCC = L-type calcium channel; NCX = sodium–calcium exchanger that removes Ca2+ from cells. Phosphorylated RyR2 is depicted by added beige stars with a “P” sign. The binding of Calstabin2 (FKBP12.6) to RyR2 is shown by brown circles.