Mitochondrial-Targeted Triphenylphosphonium-Conjugated Ionophores with Enhanced Cytotoxicity in Cancer Cells

Abstract

Salinomycin and monensin represent a class of natural ionophore antibiotics with strong anticancer properties. In this paper we report on chemical modification of these compounds by conjugation with phosphonium cations for targeting conjugates to the mitochondria of cancer cells. Our findings indicate that this approach yields conjugates with enhanced anticancer activity and selectivity, outperforming not only the parent compounds but also the widely used chemotherapeutic agent, doxorubicin. Comprehensive biological and biophysical analyses proved that the conjugates target the mitochondria in cancer cells, with some of the derivatives additionally promoting generation of mitochondrial reactive oxygen species (mtROS). This targeted strategy holds significant promise for the development of effective mitochondrial-targeted novel anticancer agent.

Keywords: antiproliferative activity; ionophore antibiotics; mitochondria-targeting conjugates; monensin; salinomycin; triphenylphosphonium cation.

Figure 1

Chemical structures of the two main representatives of ionophore antibiotics—salinomycin and monensin.

Scheme 1

Synthesis of propargyl phosphonium salts as precursors for click reaction.

Scheme 2

Synthesis of phosphonium salts of SAL. Reagents and conditions: (a) TMS(CH₂)₂OH, DMAP, TCFH, CH₂Cl₂, 0 °C to rt, 24 h; (b) PPh₃, DIAD, DPPA, THF, 0 °C to rt, 24 h; (c) TBAF, THF, rt, 24 h; (d) PPh₃, H₂O, THF, rt, 24 h; (e) (3-carboxypropyl)triphenylphosphonium bromide, TEA, DMF, 0 °C to rt, 24 h; (f) triphenylpropargylphosphonium bromide, 3, 4 or 5, DIPEA, CuI, CH₃CN, rt, 24 h; (g) succinic anhydride, pyridine, 60 °C, 24 h; (h) DBU, 1,6-dibromohexane, toluene, reflux, 24 h; (i) PPh₃, CH₃CN, reflux, 72 h.

Scheme 3

Synthesis of phosphonium salts of MON. Reagents and conditions: (a) TsCl, Na₂CO_3(aq), toluene, rt, 72 h; (b) NaN₃, DMSO, rt, 72 h; (c) H₂, Pd/C, MeOH, rt, 72 h; (d) (3-carboxypropyl)triphenylphosphonium bromide, ethyl chloroformate, TEA, DMF, −5 °C to rt, 24 h; (e) triphenylpropargylphosphonium bromide or 4, DIPEA, CuI, CH₃CN, rt, 24 h; (f) 3 or 5, sodium ascorbate, CuSO₄, H₂O/MeOH, rt, 24 h; (g) DBU, (3-carboxypropyl)triphenylphosphonium bromide, toluene, reflux, 24 h; (h) succinic anhydride, pyridine, 60 °C, 24 h; (i) DBU, (3-carboxypropyl)triphenylphosphonium bromide, toluene, reflux, 24 h.

Figure 2

Flow cytometry analysis of MitoTracker RedCMXRos and MitoTracker GreenFM mitochondrial staining in A549 and MDA-MB-231 cells after treatment with SAL, MON and their derivatives 1a, 1f, 2a and 2f for 48 h at their IC₅₀ concentrations. Untreated cells were used as the control. The bars % of MitoTrackers positive cells as compared to the control. The results are presented by means ± SD, n = 4. Statistical significance was determined at p < 0.05 (*); p < 0.01 (**); p < 0.001 (***) and p < 0.0001 (****) by 1-way ANOVA.

Figure 3

Results of a representative experiment by MitoTracker Red CMXRos and MitoTracker Green FM fluorescence assays of A549 (a) and MDA-MB-231 (b) cells after treatment with SAL, MON and their derivatives 1a, 1f, 2a, 2f, analyzed by flow cytometry. Ctrl means control.

Figure 3

Results of a representative experiment by MitoTracker Red CMXRos and MitoTracker Green FM fluorescence assays of A549 (a) and MDA-MB-231 (b) cells after treatment with SAL, MON and their derivatives 1a, 1f, 2a, 2f, analyzed by flow cytometry. Ctrl means control.

Figure 4

The effect of SAL, MON and their derivatives 1a, 1f, 2a and 2f on mitochondrial ROS production in A549 and MDA-MB-231 cells. The bars show the fold change in the mean fluorescent intensity as compared to the control. The cells were incubated for 6 h and 12 h with the compounds studied at their IC₅₀ concentrations, labeled with MitoROS 580 dye and analyzed by microplate-based fluorimetry. Untreated cells were used as the control. The results are presented by means ± SD, n = 4. Statistical significance was determined at p < 0.05 (*); p < 0.01 (**) and p < 0.001 (***), by 1-way ANOVA.

Figure 5

Impact of SAL derivatives on the oxygen consumption rate in whole non-perforated A549 cells cultured in starving DMEM (Dulbecco’s Minimal Essential Medium). Bars illustrate the effects of the tested compounds at different concentrations (0.1, 0.3 and 1 μM) on the oxygen consumption rate of A549 cells, measured using high-resolution respirometry. The compounds analyzed include SAL analogs: (A) 1a, (B) 1d, (C) 1e and (D) 1f; their effects are normalized to the oxygen consumption rate recorded after stabilization, prior to any compound addition. Complete mitochondrial uncoupling was induced with 2 μM FCCP. Data are presented as mean ± SD, with n = 4. Statistical significance was assessed by one-way ANOVA, with p-values of <0.01 (**), <0.001 (***), and p < 0.0001 (****) considered significant.

Figure 6

Impact of MON derivatives on the oxygen consumption rate in whole non-perforated A549 cells cultured in starving DMEM (Dulbecco’s Minimal Essential Medium). Bars illustrate the effects of the tested compounds at different concentrations (0.1, 0.3 and 1 μM) on the oxygen consumption rate of A549 cells, measured using high-resolution respirometry. The compounds analyzed include MON analogs: (A) 2a, (B) 2d, (C) 2e and (D) 2f; their effects are normalized to the oxygen consumption rate recorded after stabilization, prior to any compound addition. Complete mitochondrial uncoupling was induced with 2 μM FCCP. Data are presented as mean ± SD, with n = 4. Statistical significance was assessed by one-way ANOVA, with p-values of <0.05 (*), <0.01 (**) and <0.001 (***), and p < 0.0001 (****) considered significant.

Figure 7

Current–time recordings and current–voltage (I–V) relationships for ionic currents across lipid bilayer membranes under a 50/150 mM KCl (cis/trans) gradient. The left panel displays representative current traces recorded at −60 mV in control conditions and in the presence of 3 or 10 μM of (A) compound 1d, (B) compound 1f. The right panel shows the corresponding current–voltage (I–V) relationships for these compounds at concentrations of 0.1, 0.3, 1, 3, and 10 μM, across a voltage range from −60 mV to +60 mV. Average current values (n = 4) and their standard deviations (SD) are plotted, with dashed lines representing baseline (control) current levels in the absence of compounds. The ↕ symbol denotes representative amplitudes of current observed in the presence of each compound. All experiments were repeated four times. Statistical significance was evaluated using one-way ANOVA, with values of p < 0.001 (***) considered highly significant.