Fig. 1. Synthesis and characterization of the novel TRAM-34 derivatives mitoTRAM-34 and rev-mitoTRAM.

a Structure of TRAM-34 (left) and synthesis of mitoTRAM-34 (right). Reagents and conditions: (i) LiAlH₄, THF, Et₂O, room temperature for 3.5 h, 94% yield; (ii) (1) NaH, DMF, 0 °C for 1 h; (2) benzyl bromide, room temperature for 3.5 h, 94% yield; (iii) (1) n-butyllithium, THF, −70 °C for 1 h; (2) 2-chlorobenzophenone, from −70 °C to room temperature overnight, 80% yield; (iv) acetyl chloride, toluene, reflux for 45 min; (v) pyrazole, CH₃CN, reflux overnight, 83% yield; (vi) Pd/C, H₂, EtOAc, room temperature for 75 min, 90% yield; (vii) LiCl, 2,4,6-trimethylpyridine, methanesulfonyl chloride, DMF, room temperature for 15 h, 89% yield; (viii) NaI, PPh₃, CH₃CN, 95 °C for 6 h, 81% yield. b Synthesis of rev-mitoTRAM. Reagents and conditions: (i) DMAP, 3-chloropropylamine hydrochloride, DCM, THF, room temperature for 3.5 h, 88% yield; (ii) K₂CO₃, benzyl bromide, acetone, 70 °C for 15 h, 92% yield; (iii) (1) n-butyllithium, THF, −70 °C for 1 h; (2) 2-chlorobenzophenone, from −70 °C to room temperature overnight, 50% yield; (iv) acetyl chloride, toluene, 100 °C for 1.5 h; (v) pyrazole, CH₃CN, 100 °C for 17 h, 68% yield; (vi) Pd/C, H₂, EtOAc, room temperature for 1.5 h, 87% yield; (vii) DMAP, 8, CH₃CN, 50 °C for 20 h, 82% yield; (viii) NaI, PPh₃, CH₃CN, 95 °C for 6 h, 72% yield. c Levels of rev-mitoTRAM and its hydrolysis product (TRAM-34-OH) in the medium and in B16F10 cells after 2, 4, 6, 8, or 24 h of incubation with 5 µM rev-mitoTRAM. Mean ± SEM, N = 3. d MitoTRAM-34 and TRAM-34-OH block plasma membrane K_Ca3.1 currents in GL-15 cells. Left: Time-course of the current at 0 mV measured from the current ramps obtained by applying linear gradients of potential from −100 to 140 mV (Vh of 0 mV) repeated every 5 s. 0 pA current level is indicated by the dashed line. The K_Ca3.1 current was activated by DC-EBIO (1 mM) and ionomycin (500 nM) (EBIO/ionomycin), and the chamber was then perfused sequentially (in the presence of the activators) with mitoTRAM-34, TRAM-34-OH and finally with TRAM-34 (all 2 µM). Individual data points in the trace represent the current conducted by K_Ca3.1 channels following activation by EBIO/ionomycin and inhibition by the indicated drugs. Center: Representative current ramps recorded at the time points indicated on the left panel (same color code). Right: Mean residual K_Ca3.1 current recorded at 0 mV after mitoTRAM-34 and TRAM-34-OH application, expressed as % of the difference between the current recorded after full activation and after complete block of K_Ca3.1 with 3 µM TRAM-34 (n = 5). Leak current was not subtracted.