Development of a Mitochondriotropic Antioxidant Based on Caffeic

Article pubs.acs.org/jmc

Development of a Mitochondriotropic Antioxidant Based on Caffeic Acid: Proof of Concept on Cellular and Mitochondrial Oxidative Stress Models José Teixeira,†,‡,∇ Fernando Cagide,†,∇ Sofia Benfeito,†,∇ Pedro Soares,† Jorge Garrido,†,§ Inês Baldeiras,∥,⊥ José A. Ribeiro,† Carlos M. Pereira,† António F. Silva,† Paula B. Andrade,# Paulo J. Oliveira,*,‡ and Fernanda Borges*,† †

CIQUP/Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto, Porto 4169-007, Portugal CNCCenter for Neuroscience and Cell Biology, University of Coimbra, UC-Biotech Building, Biocant Park, Cantanhede 3060-197, Portugal § Department of Chemical Engineering, School of Engineering (ISEP), Polytechnic Institute of Porto, Porto 4200-072, Portugal ∥ Faculty of Medicine, University of Coimbra, Coimbra 3004-504, Portugal ⊥ Laboratory of Neurochemistry, Coimbra University Hospital (CHUC), Coimbra 3000-075, Portugal # REQUIMTE/LAQV-Laboratory of Pharmacognosy, Department of Chemistry, Faculty of Pharmacy, University of Porto, Porto 4050-313, Portugal ‡

S Supporting Information *

ABSTRACT: Targeting mitochondrial oxidative stress is an effective therapeutic strategy. In this context, a rational design of mitochondriotropic antioxidants (compounds 22−27) based on a dietary antioxidant (caffeic acid) was performed. Jointly named as AntiOxCINs, these molecules take advantage of the known ability of the triphenylphosphonium cation to target active molecules to mitochondria. The study was guided by structure−activity−toxicity−property relationships, and we demonstrate in this work that the novel AntiOxCINs act as mitochondriotropic antioxidants. In general, AntiOxCINs derivatives prevented lipid peroxidation and acted as inhibitors of the mitochondrial permeability transition pore. AntiOxCINs toxicity profile was found to be dependent on the structural modifications performed on the dietary antioxidant. On the basis of mitochondrial and cytotoxicity/antioxidant cellular data, compound 25 emerged as a potential candidate for the development of a drug candidate with therapeutic application in mitochondrial oxidative stress-related diseases. Compound 25 increased GSH intracellular levels and showed no toxicity on mitochondrial morphology and function.

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INTRODUCTION

targeting that organelle to prevent its disruption is a promising therapeutic strategy that is not always easily achievable.8 Mitochondria-targeted therapies based on bioactive molecules, namely, involving lipophilic cations carriers such as TPP (triphenylphosphonium), that can cross mitochondrial membranes and accumulate within the mitochondrial matrix are being developed.9 The most studied mitochondria-targeted antioxidants are MitoQ10, based on coenzyme Q, and SkQ derivatives, based on plastoquinone, both covalently linked to TPP by a 10-carbon alkyl chain (dTPP). MitoQ10 and SkQ1 are currently in clinical trials for different pathologies, including hepatitis C and dry-eye condition.10,11 Other active molecules have also been used coupled to TPP in order to improve their mitochondrial addressing, such as quercetin, resveratrol, metformin, or vitamin E succinate.12−15

Hydroxycinnamic acids (HCAs), such as caffeic acid, interact with biological systems in multiple ways, including through antioxidant activity mediated by different mechanisms: (i) direct free radical scavenging; (ii) chelation of pro-oxidant transition metals; (iii) modulation of antioxidant-related gene expression; (iv) inhibition of radical generating enzymatic systems. Still, bioavailability and druggability drawbacks limit their use for drug development.1−3 Among other reasons, this gap may be related to pharmacokinetics restraints4 and to the fact that often antioxidants do not reach relevant sites of free radical generation, including mitochondria, a primary target for oxidative damage.5 In fact, mitochondrial function and regulation of redox/oxidative balance are fundamental in controlling cellular life and death.6 Increasing evidence suggests that mitochondrial alterations resulting from pathological oxidative stress play a crucial role in disease.7 While the role of mitochondria in disease pathogenesis is rather consensual, © 2017 American Chemical Society

Received: May 19, 2017 Published: July 26, 2017 7084

DOI: 10.1021/acs.jmedchem.7b00741 J. Med. Chem. 2017, 60, 7084−7098

Journal of Medicinal Chemistry

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Along this framework, a mitochondrial-directed antioxidant based on the natural dietary antioxidant caffeic acid (compound 1, Figure 1) was developed by our group.16 Experimental data

physicochemical properties, antioxidant activity, and biological toxicity in different in vitro models.

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RESULTS Chemistry. AntiOxCINs (compounds 22−27) synthetic strategy is shown in Figure 2. Briefly, di- (2) or trimethoxycinnamic (3) acids were linked by an amidation reaction to suitable bifunctionalized alkyl spacers with a variable length (cinnamic derivatives 4−9). The alcohol functions of the derivatives were further activated with a leaving group (-OSO2CH3) to obtain the cinnamic derivatives 10−15. The terminal group was afterward displaced via a nucleophilic substitution reaction with triphenylphosphine (PPh3) to attain the triphenylphonium cations 16−21 throughout classic or microwave-assisted reactions. The reaction time was 1 h and 30 min, in contrast with 48 h needed in the classic reaction.16 The use of microwave radiation allowed obtaining AntiOxCINs precursors in an accelerated and environmentally friendly process, although no improvement in the reaction yields was observed. Finally, AntiOxCINs (compounds 22−27) were

Figure 1. Compound 1, a lead mitochondriotropic antioxidant inspired on a natural antioxidant (caffeic acid), originally described by Teixeira et al.16

demonstrated that compound 1 is a mitochondriotropic antioxidant operating in its reduced form. Herein, we report the synthesis of new mitochondriotropic antioxidants (compounds 22−27), jointly named as AntiOxCINs. The lead optimization process was guided by the assessment of

Figure 2. Synthetic strategy used in the optimization of the cinnamic mitochondriotropic antioxidant (compound 1). Reagents and conditions were the following: (i) ethyl chloroformate, amino alcohol, rt; (ii) methanesulfonyl chloride, rt; (iii) triphenylphosphine, 130 °C (18 h) or 150 °C (microwave, 1 h 30 min); (iv) BBr3, from −70 °C (10 min) to room temperature (12 h). 7085



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Table 1. Antioxidant, Redox Potential (Ep), and Lipophilic Properties of Mitochondria-Targeted Cinnamic Antioxidants IC50 (μM)

a

compd

MW (g mol−1)

caffeic acid 1 22 23 24 25 26 27

180.16 563.60 619.71 632.52 675.81 635.71 663.76 691.82

DPPH•

ABTS•+

± ± ± ± ± ± ± ±

17.9 ± 1.3 33.3 ± 0.6 30.5 ± 0.7 27.9 ± 0.1 23.5 ± 0.9 12.2 ± 0.1 8.7 ± 0.1 7.5 ± 0.3

18.1 35.4 29.5 28.0 25.9 19.0 14.7 13.7

1.6 1.1 0.6 0.8 0.5 0.4 0.8 0.8

GO•

Ep (V)

Etr (V)

± ± ± ± ± ± ± ±

0.168 0.166a 0.164 0.170 0.174 0.034 0.046 0.057

0.572 0.396 0.345 0.291 0.498 0.423 0.377

3.4 4.5 4.1 2.8 2.7 3.1 2.3 2.5

0.1 0.1 0.1 0.2 0.1 0.1 0.1 0.1

See details by Teixeira et al.16

the order 1 < 22 < 23 < 24 and for pyrogallol based series, 25 < 26 < 27. AntiOxCINs Chelating Properties. Free iron can undergo Fenton reactions with hydrogen peroxide and generate hydroxyl radicals (HO·), which can then quickly react with biomolecules, causing severe oxidative damage with implications in cell survival. In this context, the use of metal chelating agents, or antioxidants that operate by more than one mechanism, can function as an alternative therapeutic approach to prevent metal-induced toxicity.24,25 AntiOxCINs free ironchelating properties were determined by the ferrozine assay using EDTA as reference.26 The iron chelating properties of caffeic acid and MitoQ10 were also evaluated. EDTA was found to chelate all available iron as it completely inhibited the formation of the colored ferrozine−Fe(II) complex. Unlike MitoQ10, AntiOxCINs (both catechol and pyrogallol series) were able to chelate ferrous iron similarly to EDTA (Figure 3). Compounds 22 and 25 displayed the higher iron chelation properties.

obtained by a demethylation reaction using boron tribromide (BBr3) solution. Evaluation of AntiOxCINs Total Antioxidant Activity. Antioxidant ranking activity hierarchy was established by performing total antioxidant capacity (TAC) assays (DPPH, ABTS, and GO).17−19 AntiOxCINs (compounds 22−27) showed effective antioxidant activity, when compared with caffeic acid and compound 1, and the attained IC50 values followed the same ranking hierarchy in the different assays (Table 1). Compounds 25−27 displayed a higher antioxidant activity than compounds 22−24. In fact, compounds 25−27 showed a similar or superior antioxidant activity than caffeic acid and the lead compound (1). AntiOxCINs Redox and Lipophilic Properties. Redox behavior was studied at physiological pH (7.4) by differential pulse and cyclic voltammetry using a glassy carbon working electrode. Caffeic acid and its catechol analogues (compounds 22−24) showed a redox potential (Ep) characteristic of the presence of a catechol group (Ep = 0.164−0.174 V) (Table 1).20 However, a significant decrease in redox potentials was observed for the pyrogallol derivatives 25−27 (Ep = 0.034− 0.057 V) (Table 1). Cyclic voltammetry data obtained for caffeic acid and analogues are characteristic of an electrochemical reversible reaction, as one single anodic peak and one cathodic peak in the reverse scan were observed (Supporting Information Figure S1). Pyrogallol systems appear to suffer an irreversible oxidation reaction as no reduction wave was seen on the cathodic sweep (Table 1, Supporting Information Figure S1). The voltammograms presented a diffusion peak and an adsorption postpeak at a more anodic potential corresponding to the oxidation of the dissolved and adsorbed forms, respectively. The oxidation waves can be related to the oxidation process of the pyrogallol moiety.21 AntiOxCINs lipophilic properties were evaluated at physiological pH by electrochemistry. This technique is often used to mimic transfer of ionic drugs through biological membranes as the process occurs at the interface between two immiscible electrolyte solutions (ITIES).22,23 In the ITIES model, the transfer potential (Etr) becomes less positive with the increasing of the drug lipophilic character. For AntiOxCINs different current charge increments were found (Table 1, Supporting Information Figure S2). In general, an increment of AntiOxCINs lipophilicity, expressed as Etr, was observed as a function of the length of the alkyl spacer (Table 1). For the same increment in spacer length (e.g., 24 vs 27) the introduction of an additional hydroxyl function increased AntiOxCINs hydrophilicity (0.291 and 0.377 V, respectively). For catechol-based series, the relative lipophilicity increased in

Figure 3. Evaluation of iron chelating properties of AntiOxCINs and MitoQ10. EDTA (chelating agent) was used as reference. All test compounds as well as ferrozine were used at a final concentration of 100 μM. Data are the mean ± SEM of three independent experiments and are expressed as % of Fe(II) chelation. Statistically significant compared with control group (EDTA = 100%) using one-way ANOVA. Significance was accepted with (∗) P < 0.05, (∗∗∗∗) P < 0.0001.

AntiOxCINs Mitochondrial Uptake. Mitochondrial AntiOxCINs uptake was assessed in isolated rat liver mitochondria (RLM) in response to the transmembrane electric potential (ΔΨ).27 The addition of complex II substrate succinate resulted in ΔΨ generation and consequent decrease in the extramitochondrial compound concentration. The accumulated AntiOxCINs were then released from mitochondria once ΔΨ was abolished by the K+-ionophore valinomycin (Figure 4A). 7086



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Figure 4. (A) AntiOxCINs uptake by energized rat liver mitochondria, as measured by using a TPP-selective electrode. (B) AntiOxCINs aromatic ring pattern substitution and alkyl carbon side chain effects on lipophilicity (---) and mitochondrial accumulation ratio (−). (C) AntiOxCINs accumulation ratio in rat liver mitochondria.

Figure 5. Effect of AntiOxCINs on mitochondrial lipid peroxidation: (A) TBARS levels and (B) oxidation-derived oxygen consumption under different oxidative conditions. Data are the mean ± SEM from three and six independent experiments and are expressed as % of control (control = 100%) for TBARS and oxidation-derived oxygen consumption assays, respectively. The comparisons between control preparation vs AntiOxCINs (5 μM) preincubations were performed by using one-way ANOVA. (C) Effects of AntiOxCINs on mitochondrial swelling after induction of the mitochondrial permeability transition pore (mPTP). Data are the mean ± SEM of three independent experiments and are expressed as Δabsorbance at 540 nm. The comparisons were performed using one-way ANOVA between control preparations (Ca2+) vs different AntiOxCINs treatment groups. The most relevant concentrations are shown in the figure (for details see Supporting Information Figure S6). Significance was accepted with (∗) P < 0.05, (∗∗) P < 0.01, (∗∗∗) P < 0.0005, (∗∗∗∗) P < 0.0001.

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Figure 6. (A) Cytotoxicity profile of compounds 25 (○) and 24 (●) on human hepatocellular carcinoma cells (HepG2) mass. Statistically significant compared with control group using one-way ANOVA. AntiOxCINs prevented the (B) hydrogen peroxide- and (C) iron-induced HepG2 cell mass decrease. The comparisons were performed by using one-way ANOVA between the control (FeSO4 or H2O2) vs preparation where AntiOxCINs were preincubated. Data are the mean ± SEM of four independent experiments, and the results are expressed as percentage of control (control = 100%), which represents the cell density without any treatment in the respective time point. (D) Nontoxic concentrations of compounds 25 and 24 (100 μM and 2.5 μM, respectively) did not alter nuclear morphology and mitochondrial polarization. The images are representative of three independent experiments. (E) Determination of intracellular reduced glutathione (GSH) content in HepG2 cells. Statistically significant compared with control group using one-way ANOVA. Data are the mean ± SEM of four independent experiments, and the results are expressed as ηmol GSH/μg protein. Significance was accepted with (∗) P < 0.05, (∗∗) P < 0.01, (∗∗∗) P < 0.0005, (∗∗∗∗) P < 0.0001.

compound 22, pyrogallol-based AntiOxCINs were more effective in delaying membrane lipid peroxidation. Compound 27 was the most effective compound followed by compound 22. AntiOxCINs Toxicity on Liver Mitochondrial Bioenergetics. AntiOxCINs and MitoQ 10 toxicity on liver mitochondrial bioenergetics, namely, on ΔΨ and respiration parameters, was evaluated.30 AntiOxCINs and MitoQ10 were tested at antioxidant-relevant concentrations (Supporting Information Tables S1−S8). For mitochondrial respiration assays, glutamate/malate and succinate were used as substrates. Succinate is a substrate for mitochondrial complex II, while glutamate/malate transport and further metabolism in mitochondria generate NADH that reduces complex I. The rates for state 2 (basal oxygen consumption under nonphosphorylative conditions), state 3 (excess ADP-stimulated respiration in the presence of inorganic phosphate), state 4 (basal respiration after full ADP phosphorylation to ATP), oligomycin-inhibited and FCCPuncoupled respiration (maximal respiration) are shown in Supporting Information Figure S4. The mitochondrial oxidative phosphorylation coupling index, known as respiratory control ratio (RCR, state 3/state 4 respiration), and ADP/O index (coupling between ATP synthesis and oxygen consumption, which indicates phosphorylation efficiency) were also calculated

Different AntiOxCINs mitochondrial accumulation values were measured, which were dependent on their aromatic pattern substitution and spacer length (Figure 4B). The following ranking order was attained: 1 < 22 < 24 < 23 (catechol series); 25 < 27 < 26 (pyrogallol series) (Figure 4C). Interestingly, despite the structural differences, compounds 22, 24, and 25 displayed approximately the same accumulation ratio. AntiOxCINs Effects on Mitochondrial Lipid Peroxidation. AntiOxCINs activity against lipid peroxidation of RLM membranes was determined. Two different oxidative stressors, FeSO4/H2O2/ascorbate and ADP/FeSO4, and two end-points, thiobarbituric acid reactive species (TBARS) production and oxygen-consumption, have been used. MitoQ10 was used as reference (Figure 5). In the TBARS assay, compounds 22 (catechol series) and 27 (pyrogallol series) were the most effective molecules in preventing mitochondrial lipid peroxidation (Figure 5A). Time-dependent oxygen consumption (Supporting Information Figure S3), resulting from the lipid peroxidation of RLM membranes, was also monitored.28,29 The time lag phase that followed ADP/Fe2+ addition was used to measure AntiOxCINs efficiency (Figure 5B). In agreement with results from TBARS assay, the ability of AntiOxCINs to inhibit lipid peroxidation in RLM decreased in the following ranking MitoQ10 > 27 > 22 ≫ 25 ∼ 26 > 24 ∼ 23 > 1 > caffeic acid. With the exception of 7088



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(Supporting Information Tables S1−S8). The RCR was 6.42 ± 0.57 and 4.90 ± 0.66 for the control, using glutamate−malate or succinate, respectively. For the same substrates, the ADP/O index was 2.64 ± 0.10 and 1.58 ± 0.09, respectively. AntiOxCINs altered mitochondrial respiration in a dosedependent manner. In general, AntiOxCINs increased state 2, state 4, and oligomycin-inhibited respiration at concentrations higher than 2.5 μM in a process that was mainly dependent on their lipophilicity and not relying on their aromatic pattern (catechol vs pyrogallol) (Supporting Information Figure S4). A dual dose-dependent effect on state 3 respiration was observed, with a decrease observed for the less lipophilic compounds (22 and 25) and an increase for the more lipophilic compounds (23, 24, 26, and 27) (Supporting Information Figure S4). Specific respiratory alterations when using complex I substrates resulted in a RCR significant decrease. Data when measuring the ADP/O index (Supporting Information Tables S2−S8) showed that AntiOxCINs also decreased the oxidative phosphorylation efficiency in a dose-dependent manner. Direct effects of AntiOxCINs on ΔΨ were also measured. AntiOxCINs caused a slight dose-dependent ΔΨ depolarization. Moreover, incubation of RLM with compounds 23 and 24 or 26 and 27 at 2.5 μM promoted an initial slight hyperpolarization of 10 or 25 mV, respectively. However, incubations with compounds 24 (5 μM) (Supporting Information Table S5) and 27 (10 μM) (Supporting Information Table S8) resulted in a significant decrease in the ΔΨ of succinate-energized mitochondria. AntiOxCINs ranking toxicity hierarchy on the mitochondrial bioenergetics apparatus was established: 1 < 22 < 23 < 24 for catechol series and 25 < 26 < 27 for pyrogallol series. AntiOxCINs Inhibited the Mitochondrial Permeability Transition Pore. Mitochondrial permeability transition pore (mPTP) opening is involved in the toxicity of different xenobiotics or in the pathophysiology of several diseases.31 AntiOxCINs as well as MitoQ10 had no inducing effect on mPTP opening (data not shown). Interestingly, the more lipophilic compounds (23, 24, 26, 27) caused an inhibition of calcium-dependent mPTP opening. For the catechol based compounds the effect was similar to that of cyclosporin A (1 μM), a classic mPTP desensitizer.32 MitoQ10 had no inhibitory effect in calcium-induced mPTP opening (Figure 5C and Supporting Information Figure S5). Cytotoxicity and Antioxidant Activity of AntiOxCINs on HepG2 Cells. The cytotoxicity of two selected AntiOxCINs (compounds 24 and 25) was assessed using the human cell line HepG2 (Figure 6). From the data obtained, compound 24 (catechol moiety) exhibited higher toxicity than compound 25 (pyrogallol moiety) (Figure 6A). Although compound 24 inhibited cell proliferation at concentrations of >2.5 μM, remarkably compound 25 did not show any effect on cell proliferation for all tested concentrations (0.5−100 μM). The antioxidant cellular profile of compounds 24 and 25 was also assessed under different oxidative stress conditions by treating cells with nontoxic concentrations (2.5 μM and 100 μM, respectively). Both AntiOxCINs significantly prevented iron- and hydrogen peroxide-induced HepG2 cytotoxicity, which was measured as a decrease in cell mass (Figure 6B and Figure 6C). Morphological changes in mitochondrial network and nuclei chromatin condensation were also measured.33 Compounds 24 and 25 did not induce apoptotic-like alterations, including nuclear morphological changes or

mitochondrial depolarization, when incubated with cells for 48 h (Figure 6D). AntiOxCINs Did Not Decrease the Intracellular Reduced Glutathione (GSH) Pools in HepG2 Cells. HepG2 cells were treated with compounds 24 (2.5 μM) and 25 (100 μM) for 48 h, and reduced glutathione (GSH) intracellular content was measured as a cytotoxicity end-point. GSH content in control cells was 17.81 nmol/μg protein. In cells treated with AntiOxCINs compounds (24 and 25), GSH content increased to 20.85 nmol/μg protein and 26.94 nmol/ μg protein, respectively (Figure 6E). Sublethal concentrations of AntiOxCINs did not cause depletion in intracellular GSH content. Remarkably, compound 25 significantly increased intracellular GSH (Figure 6E). In summary, at the assay concentrations, AntiOxCINs 24 and 25 are not cytotoxic as measured by cell mass, GSH content, and fluorescence microscopy.

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DISCUSSION The regulation of mitochondrial redox processes and the development of mitochondriotropic antioxidants capable of specifically accumulating in that organelle are considered to be a promising therapeutic strategy for oxidative-stress related diseases.9,34 Phenolic dietary antioxidants are potent regulators of cellular redox status and have been used as scaffolds in drug discovery programs.21,35−37 Although a general success was obtained in preclinical studies, little benefit on clinical trials was obtained, most likely because the novel molecules were unable to cross biological barriers and reach intracellular target sites.38 As compound 116 showed a lower antioxidant profile than MitoQ10,39 a lead optimization program was started in which two different series of hydroxycinnamic mitochondria-targeted antioxidants were designed and obtained. The antioxidant ranking hierarchy of each series was established. The data from TAC assays indicated that the pyrogallol series displayed a higher antioxidant activity than their catechol counterparts. The decrease observed in antioxidant activity caused by the introduction a TPP cation was attenuated by the increment of the length spacer and/or the introduction of an additional hydroxyl group of the aromatic ring. Redox potentials are correlated with the ability of an antioxidant to donate a hydrogen atom and/or an electron to a free radical.40 Generally, low oxidation potentials (Ep) are associated with a superior antioxidant performance.41 The existence of an additional phenolic group in pyrogallol vs catechol systems seemed to influence the semiquinone intermediate stabilization, expressed by the lower Ep observed, and in turn the oxidative mechanism. The number of hydroxyl substituents on the aromatic ring was found to be directly related to AntiOxCINs antioxidant and electrochemical properties.21,36 AntiOxCINs lipophilicity modulated by the alkyl spacer attached to TPP cation linearly increased with the increment of spacer length, as observed by the potential difference at which the drug transfer across a liquid−liquid interface was measured.23 The data correlated well with AntiOxCINs mitochondrial uptake. Iron and/or copper chelation activity is often ascribed to phenolic acids, which is important in the context of their antioxidant activity, since transition metals can be catalysts of Fenton/Haber Weiss reactions.42,43 Despite the chemical modifications performed, AntiOxCINs still presented a noteworthy capacity to chelate iron, similar to the chelating agent 7089



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concentrations than the ones needed to exert antioxidant effects, independent of their mechanism. Although the mPTP physiology is not fully understood,51 it is consensual that it has an active role in mitochondrial dysfunction. The inhibition of calcium-dependent mPTP opening, which can have important therapeutic interest,52 has already been described for caffeic acid amide and ester derivatives,49,53 although their mechanism of action is still unknown. As caffeic derivatives and AntiOxCINs share antiradical and chelating properties, it is likely that they can interact with the oxidative processes that regulate the mPTP. However, other mechanisms may also be involved, including regulation of mitochondrial calcium movements53 or a direct interaction with a pore component. In summary, the tailored structural modifications on the lead (compound 1) led to a significant improvement of its mitochondriotropic properties. The structure−activity−toxicity relationships studies allowed moving on the drug discovery program by selecting two candidates: compounds 24 (with a catechol core) and 25 (with a pyrogallol core). From the cytotoxic profile on HepG2 cells, it was concluded that compound 24 exhibited a higher toxicity than 25. As mitochondria-targeted antioxidants containing the TPP+ moiety can freely pass through cellular phospholipid bilayers, with the extent of anchoring being mainly dependent upon their hydrophobicity,54 and 24 accumulated approximately at the same extension as 25, it was thought that other toxicity processes including catechol redox chemistry, often linked to deleterious effects, must be foreseen. Additionally, the novel mitochondriotropic antioxidants (compounds 24 and 25) significantly prevented oxidative stress-induced HepG2 cytotoxicity, compound 25 being the most efficient antioxidant, which is in agreement with the data from TAC, redox, and RLM assays. Finally, at concentrations associated with their protective effect, compounds 24 and 25 did not cause noticeable apoptotic-like alterations, suggesting again a safety profile. Phenolic compounds are frequently well-thought-out to be redox-active metabolites that can exhibit multiple actions, such as metal chelation, redox cycling, and protein reactivity and interfere in assay readouts. Particularly, with the presence of catechol- and Michael acceptor-substructures on natural compounds, some drugs, drug candidates, or leads have been described as pan-assay-interference compound (PAINS) as they may contribute to toxicity events.55 However, some authors consider that they can also act as potent regulators of the cellular redox status by inducing cellular defense responses (the so-called hormetic-like effect) promoting health benefits in oxidative stress-induced conditions. As GSH is an important reducing agent, and the main antioxidant within cells responsible for the control of the redox status, involved in detoxification of toxic xenobiotics and reactive oxygen species,56 it was important to evaluate the AntiOxCINs (24 and 25) effects on GSH levels. The data showed that sublethal concentrations of AntiOxCINs did not affect the intracellular reduced glutathione (GSH) content. Remarkably, for compound 25, an increase of the GSH intracellular content was observed, suggesting a putative role on stimulation of GSH redox cycle. The data strengthen compound 25 safety and beneficial properties.

EDTA and caffeic acid. Importantly, the iron chelation property of AntiOxCINs was not shared by MitoQ10. This is an important observation, namely, for the treatment of mitochondrial and metabolic disorders involving iron overload. AntiOxCINs accumulated inside mitochondria driven by ΔΨ, achieving intramitochondrial millimolar concentrations.16 However, a linear increase on AntiOxCINs lipophilicity was not directly translated into an increase in the ratio of mitochondrial matrix accumulation. The most lipophilic compounds (compounds 24 and 27) showed a lower accumulation ratio probably due to the cutoff membrane effect.44 In general, the pyrogallol series was less lipophilic than catechol series, and consequently their accumulation in the mitochondrial matrix was less effective. Even so, all AntiOxCINs presented an accumulation ratio comparable to that of MitoQ10 and higher than the lead compound (compound 1).16,44 Mitochondrial membranes possess high concentration of polyunsaturated fatty acids, which are particularly prone to oxidation as they are located near ROS producing sites.45 The efficacy of pyrogallol vs catechol systems toward preventing lipid peroxidation was already demonstrated for other polyphenols.46 AntiOxCINs presented small reactivity differences on different assays that are most likely related to their iron-chelating properties and the type of oxidative stressor used in the assays. The presence of ascorbate in the FeSO4/H2O2/ ascorbate system maintained iron in its ferrous form, which is prone to be chelated by AntiOxCINs. Within the ADP/Fe2+ system, ROS react with Fe3+ and ADP generating the ADPFe3+-O2•− complex, which is the initiator of radical chain reactions. Although AntiOxCINs displayed strong ironchelating properties, the complex formation is assumed to be kinetically favorable.47 As some AntiOxCINs have a similar lipid peroxidation inhibitory profile to that of MitoQ10, it is likely that their iron-chelating property, as opposed to MitoQ10, can contribute to inhibit iron-mediated ROS generation. Mitochondrial fractions (RLM) are often used in preclinical drug development to detect direct effect of chemicals on the mitochondrial bioenergetics apparatus, thus predicting chemical toxicity.30 The direct effect of AntiOxCINs on mitochondrial bioenergetics apparatus suggested that in general the novel compounds increased proton leakage through the mitochondrial inner membrane, resulting from a membrane permeabilization effect or a proton shuttling activity. This effect may lead to a stimulation of nonphosphorylation respiration and to a small ΔΨ depolarization.48 The data also showed that some of the AntiOxCINs tested interfered with the mitochondrial phosphorylative system. The dual dose-dependent effect on state 3 respiration observed may stem from inhibition/ stimulation of the ADP phosphorylative system or from an inhibitory effect on the respiratory chain, more visible when using complex I substrates. Hereupon, we propose that AntiOxCINs mitochondrial toxicity may be associated with the lipophilicity of the spacer and/or the presence of a TPP moiety and not related to their aromatic substitution pattern (catechol vs pyrogallol).49 Still, the presence of the TPP cation and a lipophilic spacer on mitochondria-targeted antioxidants is required for an efficient mitochondrial accumulation.50 So a suitable lipophilic balance must be attained to circumvent toxicity in the lead optimization process of mitochondriotropic antioxidants. For example, MitoQ10 at 5 μM effectively inhibited lipid peroxidation but at a lower concentration (2.5 μM) caused toxicity on the mitochondrial bioenergetic apparatus. Overall, AntiOxCINs toxicity was detected at higher 7090



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20 mL), 10% aqueous sodium bicarbonate (NaHCO3) (2 × 20 mL) and dried over anhydrous sodium sulfate (Na2SO4). After filtration, the solvent was evaporated and a pale yellow compound was obtained. (E)-3-(3,4-Dimethoxyphenyl)-N-(6-hydroxyhexyl)prop-2-enamide (4). Yield: 81%. 1H (400 MHz, CDCl3): δ = 1.31 (4H, m, H3′, H4′), 1.49 (4H, m, H2′, H5′), 3.30 (2H, m, H1′), 3.55 (2H, t, J = 6.5 Hz, H6′), 3.80 (3H, s, OCH3), 3.81 (3H, s, OCH3), 6.03 (1H, t, J = 5.6 Hz, CONH), 6.25 (1H, d, J = 15.5 Hz, Hα), 6.75 (1H, d, J = 8.3 Hz, H5), 6.94 (1H, d, J = 1.9 Hz, H2), 6.99 (1H, dd, J = 1.7, 8.4 Hz, H6), 7.48 (1H, d, J = 15.5 Hz, Hβ). 13C (100 MHz, CDCl3): δ = 25.4 (C3′), 26.6 (C4′), 30.0 (C2′), 32.7 (C5′), 39.7 (C1′), 56.0 (2 × (OCH3), 62.7 (C6′), 109.9 (C2), 111.2 (C5), 118.9 (Cα), 121.9 (C6), 128.0 (C1), 140.8 (Cβ), 149.2 (C4), 150.6 (C3), 166.5 (CONH). EI/ MS m/z (%): 307 (M+, 17), 206 (62), 192 (27), 191 (100), 189 (29). (E)-3-(3,4,5-Trimethoxyphenyl)-N-(6-hydroxyhexyl)prop-2-enamide (5). Yield: 88%. 1H (400 MHz, CDCl3): δ = 1.37 (4H, m, H3′, H4′), 1.56 (4H, m, H2′, H5′), 3.36 (2H, m, H1′), 3.62 (2H, t, J = 6.6 Hz, H6′), 3.85 (6H, s, 2 × OCH3), 3.86 (3H, s, OCH3), 6.35 (1H, t, J = 5.6 Hz, CONH), 6.40 (1H, d, J = 15.5 Hz, Hα), 6.72 (2H, s, H2, H6), 7.52 (1H, d, J = 15.5 Hz, Hβ). 13C (100 MHz, CDCl3): δ = 25.1 (C3′), 26.2 (C4′) 29.8 (C2′), 32.3 (C5′), 39.3 (C1′), 55.9 (2 × OCH3), 60.7 (OCH3), 62.3 (C6′), 104.7 (C2, C6), 120.2 (Cα), 130.3 (C1), 139.2 (C4), 140.4 (Cβ), 153.1 (C3, C5), 166.0 (CONH). EI/ MS m/z (%): 337 (M+, 64), 336 (41), 236 (27), 222 (58), 221 (100). (E)-3-(3,4-Dimethoxyphenyl)-N-(8-hydroxyoctyl)prop-2-enamide (6). Yield: 83%. 1H (400 MHz, CDCl3): δ = 1.26−1.40 (6H, m, H3′, H4′, H5′), 1.51−1.62 (4H, m, H2′, H6′), 1.70−1.81 (2H, m, H7′), 3.37 (2H, dd, J = 7.0, 13.0 Hz, H1′), 3.63 (2H, t, J = 6.6 Hz, H8′), 3.89 (3H, s, OCH3), 3.89 (3H, s, OCH3), 5.82 (1H, bs, CONH), 6.29 (1H, d, J = 15.5 Hz, Hα), 6.84 (1H, d, J = 8.3 Hz, H5), 7.02 (1H, d, J = 1.9 Hz, H2), 7.07 (1H, dd, J = 8.3, 1.9 Hz, H6), 7.55 (1H, d, J = 15.5 Hz, Hβ). 13C (100 MHz, CDCl3): δ = 25.6 (C6′), 26.8 (C3′), 29.2 (C4′), 29.3 (C5′), 29.7 (C2′), 32.7 (C7′), 39.7 (C1′), 55.9 (OCH3), 56.0 (OCH3), 62.9 (C8′), 109.8 (C2), 111.1 (C5), 118.8 (C6), 121.9 (Cα), 127.9 (C1), 140.6 (Cβ), 149.1 (C4), 150.5 (C3), 166.2 (CONH). EI/ MS m/z (%): 336 (M + 1, 40), 335 (M+, 71), 206 (53), 192 (75), 191 (100), 151 (63). (E)-3-(3,4,5-Trimethoxyphenyl)-N-(8-hydroxyoctyl)prop-2-enamide (7). Yield: 89%. 1H (400 MHz, CDCl3): δ = 1.28−1.41 (6H, m, H3′, H4′, H5′) 1.44−1.70 (6H, m, H2′, H6′, H7′), 3.38 (2H, dd, J = 7.0, 13.0 Hz, H1′), 3.64 (2H, t, J = 6.6 Hz, H8′), 3.87 (3H, s, OCH3), 3.88 (6H, s, 2 × OCH3), 5.67 (1H, t, J = 7.0 Hz, CONH), 6.30 (1H, d, J = 15.5 Hz, Hα) 6.73 (2H, s, J = 6.7 Hz, H2, H6), 7.53 (1H, d, J = 15.5 Hz, Hβ). 13C (100 MHz, CDCl3): δ = 25.6 (C6′), 26.8 (C3′), 29.2 (C4′), 29.3 (C5′), 29.7 (C2′), 32.7 (C7′), 39.8 (C1′), 56.2 (2 × OCH3), 61.0 (OCH3), 63.0 (C8′), 105.0 (C2, C6), 120.2 (Cα), 130.5 (C1), 139.6 (C4), 140.8 (Cβ), 153.4 (C3, C5), 165.8 (CONH). EI/ MS m/z (%): 366 (M + 1, 39), 365 (M+, 98), 236. (45), 221 (100) 181 (37). (E)-3-(3,4-Dimethoxyphenyl)-N-(10-hydroxydecyl)prop-2-enamide (8). Yield: 78%. 1H (400 MHz, CDCl3): δ = 1.21−1.41 (10H, m, H3′, H4′, H5′,H6′, H7′), 1.49−1.62 (4H, m, H2′, H8′), 1.75−2.00 (2H, m, H9′), 3.32−3.42 (2H, m, H1′), 3.64 (2H, t, J = 6.6 Hz, H10′), 3.90 (6H, s, 2 × OCH3), 5.79 (1H, bs, CONH), 6.29 (1H, d, J = 15.5 Hz, Hα), 6.84 (1H, d, J = 8.3 Hz, H5), 7.02 (1H, d, J = 1.7 Hz, H2), 7.08 (1H, dd, J = 8.3, 1.7 Hz, H6), 7.56 (1H, d, J = 15.5 Hz, Hβ). 13C (100 MHz, CDCl3): δ = 25.8 (C8′), 27.0 (C3′), 29.3 (C4′), 29.45 (C5′), 29.49 (C6′), 29.6 (C7′), 29.8 (C2′), 32.9 (C9′), 39.9 (C1′), 56.0 (OCH3), 56.1 (OCH3), 63.2 (C10′), 109.9 (C2), 111.3 (C5), 118.8 (C6), 122.0 (Cα), 128.0 (C1), 140.9 (Cβ), 149.3 (C4), 150.7 (C3), 166.3 (CONH). EI/MS m/z (%): 364 (M + 1, 433), 363 (M+, 89), 206 (54), 192 (72), 191 (100), 151 (46). (E)-3-(3,4,5-Trimethoxyphenyl)-N-(10-hydroxydecyl)prop-2-enamide (9). Yield: 69%. 1H (400 MHz, CDCl3): δ = 1.23−1.42 (10H, m, H3′, H4′, H5′,H6′, H7′), 1.51−1.61 (4H, m, H2′, H8′), 1.89−2.06 (2H, m, H9′), 3.33−3.43 (2H, m, H1′), 3.64 (2H, t, J = 6.6 Hz, H10′), 3.87 (3H, s, OCH3), 3.88 (6H, s, 2 × OCH3)), 5.82 (1H, bs, CONH), 6.33 (1H, d, J = 15.6 Hz, Hα), 6.73 (2H, s, H2, H6), 7.55 (1H, d, J = 15.5 Hz, Hβ). 13C (100 MHz, CDCl3): δ = 25.8 (C8′), 27.0 (C3′), 29.3 (C4′), 29.45 (C5′), 29.50 (C6′), 29.6 (C7′), 29.8 (C2′), 32.9

CONCLUSION The tailored structural modifications performed on the lead compound (1) led to a significant improvement of its mitochondriotropic antioxidant properties. From the structure−activity−toxicity relationships study, compound 25 emerge as a potential candidate for the development of a first class drug with therapeutic application in mitochondrial oxidative stress-related diseases and to mitigate the effects of mitochondrial and metabolic disorders involving iron overload. Compound 25 did not disturb mitochondrial morphology and polarization and showed remarkable antioxidant and ironchelation properties, not shared by MitoQ10, preventing ironand hydrogen peroxide-induced damage on cells. Additionally, it was found that compound 25 can play a role on the maintenance of intracellular GSH homeostasis by increasing its supply. Overall, physicochemical, pharmacological, and toxicological properties of compound 25 are very dissimilar from the natural scaffold, and so it cannot be considered PAINS. The present study showed that taming natural scaffolds by chemical modulation of its structural architecture is a valid strategy to attain innovative antioxidants without toxicity liabilities. The present study is part of a drug discovery project to perform a lead optimization process, carried out by tailored structural modification on a lead compound in order to improve mitochondrial delivery and reduce toxicity. Accordingly, we used preclinical biological models such as isolated rat liver mitochondria and cell lines. This initial stage allowed us to select the more promising molecule to be used and validated in more robust animal models of disease.

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EXPERIMENTAL SECTION

Chemistry. Reagents, General Methods, and Apparatus. All reagents were purchased from Sigma-Aldrich (Barcelona, Spain) and used without additional purification. The solvents were pro analysis grade and were acquired from Panreac and Sigma-Aldrich. Reaction progress was assessed by thin layer chromatography (TLC) analyses on aluminum silica gel sheets 60 F254 plates (Merck, Darmstadt, Germany) in dichloromethane, ethyl acetate, and dichloromethane/ methanol, in several proportions. The spots were detected using UV detection (254 and 366 nm). Flash column chromatography was performed using silica gel 60 (0.040−0.063 mm) (Carlo Erba Reactifs, SDS, France). The workup solvents were then evaporated under reduced pressure in a Buchi rotavapor. 1H and 13C NMR spectra were acquired at room temperature and recorded on a Bruker Avance III operating at 400 and 100 MHz, respectively. Chemical shifts are expressed in δ (ppm) values relative to tetramethylsilane (TMS) as internal reference, and coupling constants (J) are given in Hz. Assignments were also made from DEPT (distortionless enhancement by polarization transfer) (underlined values). Mass spectra (MS) were recorded on a Bruker Microtof (ESI) or Varian 320-MS (EI) apparatus and referred in m/z (% relative) of important fragments. The purity of AntiOxCINs was evaluated by high-performance liquid chromatography (HPLC) using the conditions previously described in Teixeira et al.16 The purity of the compounds was verified to be >97%. Synthesis of AntiOxCINs. General Synthetic Procedure for Obtention of Cinnamic Acid Amides (4−9). 3,4-Dimethoxycinnamic acid (2) or 3,4,5-trimethoxycinnamic acid (3) (1 mmol) was dissolved in dichlomethane (10 mL) and triethylamine (2 mmol). To the stirred solution kept in an ice bath, ethyl chloroformate (2 mmol) was added dropwise. After stirring 2 h at room temperature, the mixture was cooled again in an ice bath and the intended amino alcohol (2 mmol) was added dropwise. The reaction was stirred during 10 h at room temperature. After extraction with dichloromethane (20 mL), the combined organic phases were washed with HCl 1 M (3 × 7091



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(C9′), 39.9 (C1′), 56.0 (2 × OCH3), 61.1 (OCH3), 63.2 (C10′), 105.1 (C2, C6), 120.3 (Cα), 130.6 (C1), 139.7 (C4), 141.0 (Cβ), 153.5 (C3, C5), 165.9 (CONH). EI/MS m/z (%): 394 (M + 1, 40), 393 (M+, 100), 236 (37) 222 (86), 221 (93). General Synthetic Procedure for Obtention of Methanesulfonates Derivatives (10−15). Cinnamic acid amide (4−9) (1 mmol) was dissolved in a mixture of tetrahydrofuran (10 mL) and triethylamine (2 mmol) and stirred at room temperature over a period of 10 min. A solution of methanesulfonyl chloride (1.3 mmol) in tetrahydrofuran (5 mL) was then added dropwise. After stirring at room temperature for 12 h, the mixture was neutralized and the solvent partially evaporated. The resulting reaction mixture was extracted with dichloromethane (3 × 20 mL), and the combined organic phases were washed with water (3 × 20 mL), 10% aqueous NaHCO3 (2 × 20 mL), dried with anhydrous sodium sulfate (Na2SO4), filtered, and evaporated. The crude product was used without further purification in the next step. (E)-(6-(3-(3,4-Dimethoxyphenyl)prop-2-enamide)hexyl)methanesulfonate (10). Yield: 87%. 1H (400 MHz, CDCl3): δ = 1.42 (4H, m, H3′, H4′), 1.66 (4H, m, H2′, H5′), 3.00 (3H, s, OSO2CH3), 3.38 (2H, m, H1′), 3.88 (3H, s, OCH3), 3.89 (3H, s, OCH3), 4.22 (2H, t, J = 6.4 Hz, H6′), 5.97 (1H, t, J = 5.6 Hz, CONH), 6.33 (1H, d, J = 15.5 Hz, Hα), 6.84 (1H, d, J = 8.3 Hz, H5), 7.03 (1H, d, J = 1.9 Hz, H2), 7.07 (1H, dd, J = 1.9, 8.3 Hz, H6), 7.55 (1H, d, J = 15.5 Hz, Hβ). 13C (100 MHz, CDCl3): δ = 24.9 (C3′), 26.0 (C4′), 28.8 (C2′), 29.3 (C5′), 37.2 (OSO2CH3), 39.2 (C1′), 55.7 (2 × OCH3), 69.8 (C6′), 109.5 (C2), 110.9 (C5), 118.6 (Cα), 121.7 (C6), 127.7 (C1), 140.4 (Cβ), 148.9 (C4), 150.3 (C3), 166.1 (CONH). (E)-(6-(3-(3,4,5-Trimethoxyphenyl)prop-2-enamide)hexyl)methanesulfonate (11). Yield: 95%. 1H (400 MHz, CDCl3): δ = 1.36 (4H, m, H3′, H4′), 1.60 (4H, m, H2′, H5′), 2.95 (3H, s, OSO2CH3), 3.32 (2H, m, H1′), 3.80 (6H, s, 2 × OCH3), 3.81 (3H, s, OCH3), 4.16 (2H, t, J = 6.4 Hz, H6′), 6.07 (1H, t, J = 5.7 Hz, CONH), 6.34 (1H, d, J = 15.5 Hz, Hα), 6.68 (2H, s, H2, H6), 7.46 (1H, d, J = 15.6 Hz, Hβ). 13 C (100 MHz, CDCl3): δ = 25.5 (C3′), 26.6 (C4′), 29.4 (C2′), 29.8 (C5′), 37.8 (OSO2CH3), 39.9 (C1′), 56.5 (2 × OCH3), 61.4 (OCH3), 70.5 (C6′), 105.4 (C2, C6), 120.8 (Cα), 131.0 (C1), 139.8 (C4), 141.0 (Cβ), 154.8 (C3, C5), 166.4 (CONH). (E)-(8-(3-(3,4-Dimethoxyphenyl)prop-2-enamide)octyl)methanesulfonate (12). Yield: 95%. 1H (400 MHz, CDCl3): δ = 1.27−1.47 (8H, m, H3′, H4′, H5′, H6′), 1.48−1.64 (2H, m, H2′), 1.66−1.79 (2H, m, H7′), 3.00 (3H, s, OSO2CH3), 3.37 (2H, dd, J = 13.1, 6.7 Hz, H1′), 3.88 (3H, s, OCH3), 3.89 (3H, s, OCH3), 4.21 (2H, t, J = 6.5 Hz, H8′), 5.97 (1H, bs, CONH), 6.33 (1H, d, J = 15.5 Hz, Hα), 6.83 (1H, d, J = 8.3 Hz, H5), 7.03 (1H, s, H2), 7.07 (1H, d, J = 8.1 Hz, H6), 7.55 (1H, d, J = 15.5 Hz, Hβ). 13C (100 MHz, CDCl3): δ = 25.3 (C6′), 26.7 (C3′), 28.8 (C4′), 29.00 (C5′), 29.05 (C2′), 29.6 (C7′), 37.4 (OSO2CH3), 39.7 (C1′), 55.87 (OCH3), 55.95 (OCH3), 70.2 (C8′), 109.7 (C2), 111.1 (C5), 118.9 (C6), 121.9 (Cα), 128.0 (C1), 140.5 (Cβ), 149.1 (C4), 150.5 (C3), 166.2 (CONH). (E)-(8-(3-(3,4,5-Trimethoxyphenyl)prop-2-enamide)octyl)methanesulfonate (13). Yield: 96%. 1H (400 MHz, CDCl3): δ = 1.29−1.45 (6H, m, H3′, H4′, H5′), 1.52−1.63 (4H, m, H2′, H6′), 1.65−1.80 (2H, m, H7′), 3.00 (3H, s, OSO2CH3), 3.38 (2H, td, J = 13.1, 7.0 Hz, H1′), 3.87 (3H, s, OCH3), 3.88 (6H, s, 2 × OCH3), 4.23 (2H, t, J = 6.5 Hz, H8′), 5.64 (1H, t, J = 7.0 Hz, NH), 6.30 (1H, d, J = 15.5 Hz, Hα), 6.73 (2H, s, H2, H6), 7.53 (1H, d, J = 15.5 Hz, Hβ). 13 C (100 MHz, CDCl3): δ = 25.3 (C6′), 26.7 (C3′), 28.8 (C7′), 29.0 (C4′), 29.1 (C5′), 29.6 (C2′), 37.4 (OSO2CH3), 39.7 (C1′), 56.2 (2 × OCH3), 61.0 (OCH3), 70.1 (C8′), 105.0 (C2, C6), 120.1 (Cα), 130.5 (C1), 139.6 (C4), 140.8 (Cβ), 153.4 (C3, C5), 165.8 (CONH). (E)-(10-(3-(3,4-Dimethoxyphenyl)prop-2-enamide)decyl)methanesulfonate (14). Yield: 98%. 1H (400 MHz, CDCl3): δ = 1.20−1.45 (12H, m, H3′, H4′, H5′, H6′, H7′, H8′), 1.49−1.64 (2H, m, H2′), 1.67−1.83 (2H, m, H9′), 3.01 (3H, s, OSO2CH3), 3.38 (2H, dd, J = 10.9, 6.3 Hz, H1′), 3.90 (6H, s, 2 × OCH3), 4.23 (2H, t, J = 6.6 Hz, H10′), 5.82−5.95 (1H, m, CONH), 6.32 (1H, d, J = 15.5 Hz, Hα), 6.85 (1H, d, J = 8.2 Hz, H5), 7.03 (1H, s, H2), 7.08 (1H, d, J = 8.2 Hz, H6), 7.57 (1H, d, J = 15.5 Hz, Hβ). 13C (100 MHz, CDCl3): δ = 25.4 (C8′), 27.0 (C3′), 29.0 (C5′), 29.2 (C4′), 29.28 (C6′), 29.34

(C7′), 29.4 (C2′), 29.8 (C9′), 37.5 (CH3SO3), 39.9 (C1′), 55.97 (OCH3), 56.05 (OCH3), 70.3 (C10′), 109.8 (C2), 111.2 (C5), 118.8 (C6), 122.0 (Cα), 128.0 (C1), 140.8 (Cβ), 149.2 (C4), 150.6 (C3), 166.3 (CONH). (E)-(10-(3-(3,4,5-Trimethoxyphenyl)prop-2-enamide)decyl)methanesulfonate (15). Yield: 96%. 1H (400 MHz, CDCl3): δ = 1.18−1.47 (12H, m, H3′, H4′, H5′, H6′, H7′, H8′), 1.51−1.64 (2H, m, H2′), 1.68−1.82 (2H, m, H9′), 3.00 (3H, s, OSO2CH3), 3.32−3.45 (2H, m, H1′), 3.87 (3H, s, OCH3)), 3.88 (6H, s, 2 × OCH3), 4.22 (2H, t, J = 6.6 Hz, H10′), 5.84 (1H, bs, CONH), 6.34 (1H, d, J = 15.5 Hz, Hα), 6.74 (2H, s, H2, H6), 7.54 (1H, d, J = 15.5 Hz, Hβ). 13C (100 MHz, CDCl3): δ = 25.5 (C8′), 27.0 (C3′), 29.0 (C5′), 29.0(C4′), 29.2 (C6′), 29.3 (C7′), 29.35 (C2′), 29.40 (C9′), 37.5 (OSO2CH3), 40.0 (C1′), 56.3 (2 × OCH3), 60.1 (OCH3),70.3 (C10′), 105.2(C2, C6), 105.2(C6), 120.1 (Cα), 130.6 (C1), 139.8 (C4), 141.8 (Cβ), 153.6 (C3, C5), 166.1 (CONH). General Synthetic Procedure for Obtention of Triphenylphosphonium salts (16−21). Obtention of Triphenylphosphonium Salts 16 and 17. Methanesulfonate (10 or 11) (1 mmol) was thoroughly mixed with triphenylphosphine (1 mmol) in a microwave vial and sealed under argon. The reaction mixture was placed under microwave irradiation at 150 °C for 1 h and 30 min with magnetic stirring. Upon completion, the reaction mixture was cooled at room temperature and the crude product was purified by flash chromatography, using dichloromethane/methanol [9:1 ratio (v/v)] as elution system. The fractions containing the intended compound were combined, and the solvent was evaporated. The resulting residue was then dissolved with a minimum amount of dichloromethane and triturated with excess ethyl ether. The solvent was decanted and the final solid residue was dried under vacuum to give triphenylphosphonium methanesulfonate salt. (E)-(6-(3-(3,4-Dimethoxyphenyl)prop-2-enamide)hexyl)triphenylphosphonium Methanesulfonate (16). Yield: 73%. 1H (400 MHz, CDCl3): δ = 1.38 (4H, m, H3′, H4′), 1.47 (4H, m, H2′, H5′), 3.17 (2H, d, J = 5.1 Hz, H1′), 3.29 (2H, m, H6′), 3.69 (3H, s, OCH3), 3.71 (3H, s, OCH3), 6.62 (1H, d, J = 8.3 Hz, H5), 6.82 (1H, d, J = 15.7 Hz, Hα), 6.85 (1H, dd, J = 1.9, 8.3 Hz, H6), 7.04 (1H, s, H2), 7.29 (1H, d, J = 15.7 Hz, Hβ), 7.54−7.63 (15H, m, PPh3), 8.30 (1H, t, J = 5.3 Hz, CONH). 13C (100 MHz, CDCl3): δ = 21.2 (d, JCP = 51.8 Hz, C6′), 25.0 (C5′) 28.1 (C4′), 28.9 (C3′), 38.2 (C2′), 39.0 (C1′), 55.4 (2 × OCH3), 109.2 (C2), 110.3 (C5), 117.5 (d, JCP = 85.9 Hz, C1″), 120.3 (Cα), 121.3 (C6), 128.1 (C1), 130.0 (d, JCP = 12.5 Hz, C3″, C5″), 132.8 (d, JCP = 9.9 Hz, C2″, C6″), 134.5 (d, JCP = 2.8 Hz, C4″), 138.0 (Cβ), 148.4 (C4), 149.3 (C3), 166.4 (CONH).EI/MS m/z (%): 277 (25), 195 (33), 85 (85), 83 (100). (E)-(6-(3-(3,4,5-Trimethoxyphenyl)prop-2-enamido)hexyl)triphenylphosphonium Methanesulfonate (17). Yield: 65%. 1H (400 MHz, DMSO): δ = 1.33 (4H, m, H3′, H4′), 1.52 (4H, m, H2′, H5′), 3.15 (2H, m, H1′), 3.59 (2H, m, H6′), 3.69 (3H, s, OCH3), 3.82 (6H, s, 2 × OCH3), 6.68 (1H, d, J = 15.7 Hz, Hα), 6.90 (2H, s, H2, H6), 7.34 (1H, d, J = 15.7 Hz, Hβ), 7.76−7.84 (15H, m, PPh3), 8.18 (1H, t, J = 5.6 Hz, CONH). 13C (100 MHz, DMSO): δ = 20.2 (d, JCP = 49.7 Hz C6′), 21.8 (C5′), 25.6 (C4′), 28.8 (C3′), 29.6 (C2′), 38.5 (C1′), 55.9 (2 × OCH3), 60.2 (OCH3), 104.9 (C2, C6), 118.6 (d, JCP = 85.7 Hz, C1″), 121.9 (Cα), 130.3 (d, JCP = 12.4 Hz, C3″, C5″), 130.7 (C1), 133.6 (d, JCP = 10.1 Hz, C2″, C6″), 134.9 (d, JCP = 2.4 Hz, C4″), 138.5 (Cβ), 153.1 (C3, C5), 156.3 (C4), 165.0 (CONH). EI/MS m/z (%): 278 (24), 277 (48), 263 (34), 262 (100), 261 (22), 184 (22), 183 (75), 108 (38). Obtention of Triphenylphosphonium Salts 18−21. Methanesulfonate (12−15) (1 mmol) was heated with triphenylphosphine (1 mmol) under argon atmosphere at 130 °C for 48 h. The crude product was purified by flash chromatography, using dichloromethane/methanol [9:1 ratio (v/v)] as elution system. The fractions containing the intended compound were combined and the solvent was evaporated. The resulting residue was then dissolved with a minimum amount of dichloromethane and triturated with excess ethyl ether. The solvent was decanted and the final solid residue was dried under vacuum to give triphenylphosphonium methanesulfonate salt. 7092



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cooled at a temperature below −70 °C. To this solution, boron tribromide (3 mmol, 1 M solution in dichloromethane) was added. Once the addition was completed, the reaction was kept at −70 °C for 10 min and then allowed to warm to room temperature with continuous stirring for 12 h. After BBr3 destruction with water, the purification process was carried out straightforwardly. After water removal the resulting product was dissolved in methanol and dried over anhydrous Na2SO4, filtered and the solvent evaporated. (E)-(6-(3-(3,4-Dihydroxyphenyl)prop-2-enamido)hexyl)triphenylphosphonium Methanesulfonate (22). Yield: 30%. 1H (400 MHz, DMSO): δ = 1.35 (4H, m, H3′, H4′), 1.50 (4H, m, H2′, H5′), 3.17 (2H, d, J = 2.8 Hz, H1′), 3.58 (2H, m, H6′), 6.34 (1H, d, J = 15.7 Hz, Hα), 6.75 (1H, d, J = 8.0 Hz, H5), 6.82 (1H, dd, J = 1.9, 8.0 Hz, H6), 6.94 (1H, d, J = 1.9 Hz, H2), 7.20 (1H, d, J = 15.7 Hz, Hβ), 7.74−7.92 (15H, m, PPh3), 7.99 (1H, t, J = 5.6 Hz, CONH), 9.14 (1H, s, OH), 9.39 (1H, s, OH). 13C (100 MHz, DMSO): δ = 20.2 (d, JCP = 50.2 Hz, C6′), 21.8 (C5′) 25.6 (C4′), 28.9 (C3′), 29.6 (C2′), 38.4 (C1′), 113.8 (C2), 115.8 (C5), 118.4 (d, JCP = 85.6 Hz, C1″), 119.0 (Cα), 120.3 (C6), 126.4 (C1), 130.3 (d, JCP = 12.4 Hz, C3″, C5″), 133.6 (d, JCP = 10.1 Hz, C2″, C6″), 134.9 (d, JCP = 2.4 Hz, C4″), 138.8 (Cβ), 145.5 (C4), 147.2 (C3), 165.3 (CONH). ESI/MS m/z (%): 525 (M+ + H − CH3SO3, 53), 524 (M − CH3SO3, 100), 434 (10). (E)-(8-(3-(3,4-Dihydroxyphenyl)acrylamido)octyl)triphenylphosphonium Methanesulfonate (23). Yield: 55%. 1H (400 MHz, MeOD): δ = 1.23−1.41 (6H, m, H3′, H4′, H5′), 1.47−1.59 (4H, m, H2′, H6′), 1.60−1.73 (2H, m, H7′), 3.25 (2H, t, J = 7.0 Hz, H1′), 3.33−3.44 (2H, m, H8′), 6.36 (1H, d, J = 15.7 Hz, Hα), 6.75 (1H, d, J = 8.2 Hz, H5), 6.87 (1H, dd, J = 8.2, 2.0 Hz, H6), 6.99 (1H, d, J = 2.0 Hz, H2), 7.36 (1H, d, J = 15.7 Hz, Hβ), 7.68−7.94 (16H, m, PPh3, CONH). 13C (100 MHz, MeOD): δ = 22.7 (d, JCP = 51.0 Hz, C8′), 22.5 (d, JCP = 4.4 Hz, C6′), 27.7 (C3′), 29.7 (C4′), 29.9 (C5′), 30.4 (C2′), 31.4 (d, JCP = 16.0 Hz, C7′), 40.4 (C1′), 115.1 (C2), 116.5 (C5), 118.6 (C6), 120.0 (d, JCP = 86.3 Hz, C1″), 122.1 (Cα), 128.3 (C1), 131.6 (d, JCP = 12.6 Hz, C3″, C5″), 134.8 (d, JCP = 9.9 Hz, C2″, C6″), 136.3 (d, JCP = 3.0 Hz, C4″), 142.1 (Cβ), 146.8 (C4), 148.8 (C3), 169.2 (CONH). ESI/MS m/z (%): 553 (M+ + H − CH3SO3, 73), 552 (M − CH3SO3, 100), 462 (10). (E)-(10-(3-(3,4-Dihydroxyphenyl)acrylamido)decyl)triphenylphosphonium Methanesulfonate (24). Yield: 80%. 1H (400 MHz, MeOD): δ = 1.20−1.40 (10H, m, H3′, H4′, H5′, H6′, H7′), 1.47− 1.57 (4H,m, H2′, H8′), 1.59−1.71 (2H, m, H9′), 3.26 (2H, t, J = 7.1 Hz, H1′), 3.33−3.41 (2H, m, H10′), 6.36 (1H, d, J = 15.7 Hz, Hα), 6.75 (1H, d, J = 8.2 Hz, H5), 6.88 (1H, dd, J = 8.4, 2.1 Hz, H6), 6.99 (1H, d, J = 2.1 Hz, H2), 7.36 (1H, d, J = 15.7 Hz, Hβ), 7.68−7.98 (16H, m, PPh3, CONH). 13C (100 MHz, MeOD): δ = 22.7 (d, JCP = 51.0 Hz, C10′), 23.5 (d, JCP = 4.4 Hz, C8′), 27.9 (C3′), 29.8 (C4′), 30.2 (C5′, C6′), 30.3 (C7′), 30.4 (C2′), 31.5 (d, JCP = 16.1 Hz, C9′), 40.5 (C1′), 115.0 (C2), 116.5 (C5), 119.8 (C6), 120.0 (d, JCP = 86.3 Hz, C1″), 122.0 (Cα), 128.3 (C1), 131.5 (d, JCP = 12.6 Hz, C3″, C5″), 134.8 (d, JCP = 10.0 Hz, C2″, C6″), 136.3 (d, JCP = 3.0 Hz, C4″), 142.0 (Cβ), 146.8 (C4), 148.7 (C3), 169.2 (CONH). ESI/MS m/z (%): 581 (M+ + H − CH3SO3, 85) 580 (M+ − CH3SO3, 100), 490 (20). (E)-(6-(3-(3,4,5-Trihydroxyphenyl)prop-2-enamido)hexyl)triphenylphosphonium Methanesulfonate (25). Yield: 50%. 1H (400 MHz, DMSO): δ = 1.35 (4H, m, H3′, H4′), 1.50 (4H, m, H2′, H5′), 2.72 (2H, m, H1′), 3.58 (2H, m, H6′) 6.28 (1H, d, J = 15.6 Hz, Hα), 6.47 (2H, s, H2, H6), 7.10 (1H, d, J = 15.6 Hz, Hβ), 7.75−7.79 (15H, m, PPh3), 8.00 (1H, t, J = 5.6 Hz, CONH). 13C (100 MHz, DMSO): δ = 19.8 (d, JCP = 49.5 Hz, C6′), 21.3 (C5′), 25.2 (C4′), 26.2 (C3′), 28.5 (C2′), 38.2 (C1′), 106.3 (C2, C6), 118.2 (d, JCP = 85.1 Hz, C1″), 118.6 (Cα), 124.9 (C1), 129.8 (d, JCP = 12.4 Hz, C3″, C5″), 133.2 (d, JCP = 10.1 Hz, C2″, C6″), 134.5 (d, JCP = 2.8 Hz, C4″), 134.7 (C4), 138.8 (Cβ), 145.7 (C3, C5), 164.9 (CONH). ESI/MS m/z (%): 541 (M+ + H − CH3SO3, 53), 540 (M+ − CH3SO3, 100). (E)-(8-(3-(3,4,5-Trihydroxyphenyl)acrylamido)octyl)triphenylphosphonium Methanesulfonate (26). Yield: 88%. 1H (400 MHz, MeOD): δ = 1.25−1.42 (6H, m, H3′, H4′, H5′), 1.46−1.61 (4H, m, H2′, H6′), 1.59−1.74 (2H, m, H7′), 3.24 (2H, t, J = 7.0 Hz, H1′), 3.35 (3H, s, OSO2CH3), 3.43−3.32 (2H, m, H8′), 6.33 (1H, d, J = 15.6 Hz, Hα), 6.56 (2H, s, H2, H6), 7.28 (1H, d, J = 15.6 Hz, Hβ), 7.70−7.92

(E)-(8-(3-(3,4-Dimethoxyphenyl)acrylamido)octyl)triphenylphosphonium Methanesulfonate (18). Yield: 53%. 1H (400 MHz, MeOD): δ = 1.25−1.40 (6H, m, H3′, H4′, H5′), 1.49−1.60 (4H, m, H2′, H6′), 1.61−1.73 (2H, m, H7′), 2.68 (3H, s, OSO2CH3), 3.26 (2H, t, J = 7.1 Hz, H1′), 3.43−3.33 (2H, m, H8′), 3.85 (3H, s, OCH3), 3.86 (3H, s, OCH3), 6.48 (1H, d, J = 15.7 Hz, Hα), 6.96 (1H, d, J = 8.3 Hz, H5), 7.11 (1H, dd, J = 2.0, 8.3 Hz, H6), 7.15 (1H, d, J = 2.0 Hz, H2), 7.44 (1H, d, J = 15.7 Hz, Hβ), 7.70−7.94 (16H, m, PPh3, CONH). 13C (100 MHz, MeOD): δ = 22.6 (d, JCP = 51.2 Hz, C8′), 23.5 (d, JCP = 4.4 Hz, C6′), 27.7 (C3′), 29.7 (C4′), 29.9 (C5′), 30.4 (C2′), 31.4 (d, JCP = 16.0 Hz, C7′), 39.5 (OSO2CH3), 40.4 (C1′), 56.4 (OCH3), 56.5 (OCH3), 111.4 (C2), 112.8 (C5), 119.6 (C6), 120.0 (d, JCP = 85.8 Hz, C1″), 123.2 (Cα), 129.4 (C1), 131.5 (d, JCP = 12.6 Hz, C3″, C5″), 134.8 (d, JCP = 9.9 Hz, C2″, C6″), 136.3 (d, JCP = 3.0 Hz, C4″), 141.5 (Cβ), 150.7 (C4), 152.2 (C3), 168.9 (CONH). ESI/MS m/z (%): 581 (M+ + H − CH3SO3, 45), 580 (M+ − CH3SO3, 54), 462 (100). (E)-(8-(3-(3,4,5-Trimethoxyphenyl)acrylamido)octyl)triphenylphosphonium Methanesulfonate (19). Yield: 96%. 1H (400 MHz, CDCl3): δ = 1.18−1.46 (6H, m, H3′, H4′, H5′), 1.51−1.66 (6H, m, H2′, H6′, H7′), 2.68 (3H, s, OSO2CH3), 3.33 (2H, dd, J = 12.3, 6.3 Hz, H1′), 3.58−3.43 (2H, m, H8′), 3.83 (3H, s, OCH3), 3.85 (6H, s, 2 × OCH3), 6.85 (2H, s, H2, H6), 6.92 (1H, d, J = 15.7 Hz, Hα), 7.46 (1H, d, J = 15.6 Hz, Hβ), 7.62−7.85 (15H, m, PPh3), 7.99 (1H, t, J = 5.2 Hz, CONH). 13C (100 MHz, CDCl3): δ = 21.9 (d, JCP = 50.2 Hz, C8′), 22.3 (d, JCP = 4.5 Hz, C6′), 25.9 (C4′), 27.8 (C3′), 28.0 (C5′), 28.8 (C2′), 29.5 (d, JCP = 16.1 Hz, C7′), 39.3 (OSO2CH3), 39.7 (C1′), 56.3 (2 × OCH3), 60.9 (OCH3), 105.1 (C2, C6), 118.5 (d, JCP = 85.8 Hz, C1″), 122.4 (Cα), 130.5 (d, JCP = 12.5 Hz, C3″, C5″), 131.5 (C1), 133.5 (d, JCP = 9.9 Hz, C2″, C6″), 135.1 (d, JCP = 2.9 Hz, C4″), 138.8 (C4), 139.0 (Cβ), 153.2 (C3, C5), 166.7 (CONH). ESI/ MS m/z (%): 611 (M+ + H − CH3SO3, 46) 610 (M+ − CH3SO3, 100). (E)-(10-(3-(3,4-Dimethoxyphenyl)acrylamido)decyl)triphenylphosphonium Methanesulfonate (20). Yield: 61%. 1H (400 MHz, MeOD): δ = 1.18−1.41 (10H, m, H3′, H4′, H5′,H6′, H7′), 1.46−1.59 (4H, m, H2′, H8′), 1.60−1.72 (2H, m, H9′), 2.68 (3H, s, OSO2CH3), 3.27 (2H, t, J = 7.1 Hz, H1′), 3.32−3.42 (2H, m, H10′), 3.85 (3H, s, OCH3), 3.86 (3H, s, OCH3), 6.48 (1H, d, J = 15.7 Hz, Hα), 6.95 (1H, d, J = 8.2 Hz, H5), 7.11 (1H, dd, J = 8.2, 1.9 Hz, H6), 7.14 (1H, d, J = 1.9 Hz, H2), 7.44 (1H, d, J = 15.7 Hz, Hβ), 7.95−7.68 (16H, m,PPh3, CONH). 13C (100 MHz, MeOD): δ = 22.6 (d, JCP = 51.1 Hz, C10′), 23.5 (d, JCP = 4.5 Hz, C8′), 27.9 (C3′), 29.8 (C4′), 30.2 (C5′, C6′), 30.35 (C7′), 30.42 (C2′), 31.5 (d, JCP = 16.1 Hz, C9′), 39.5 (OSO2CH3), 40.5 (C1′), 56.4 (OCH3), 56.5 (OCH3), 111.4 (C2), 112.8 (C5), 119.8 (C6), 120.0 (d, JCP = 85.8 Hz, C1″), 123.2 (Cα), 129.4 (C1), 131.5 (d, JCP = 12.5 Hz, C3″, C5″), 134.8 (d, JCP = 9.9 Hz, C2″, C6″), 136.3 (d, JCP = 3.0 Hz, C4″), 141.5 (Cβ), 150.7 (C4), 152.2 (C3), 168.9 (CONH). ESI/MS m/z (%): 610 (M+ + H − CH3SO3, 73) 609 (M+ − CH3SO3, 100), 491 (33), 490 (67). (E)-(10-(3-(3,4,5-Trimethoxyphenyl)acrylamido)decyl)triphenylphosphonium Methanesulfonate (21). Yield: 69%. 1H (400 MHz, MeOD): δ = 1.20−1.41 (10H, m, H3′, H4′, H5′, H6′, H7′), 1.48− 1.59 (4H, m, H2′, H8′), 1.60−1.72 (2H, m, H9′), 2.68 (3H, s, OSO2CH3), 3.28 (2H, t, J = 7.1 Hz, H1′), 3.35−3.45 (2H, m, H10′), 3.78 (3H, s, OCH3), 3.86 (6H, s, 2 × OCH3), 6.55 (1H, d, J = 15.7 Hz, Hα), 6.86 (2H, s, H2, H6), 7.43 (1H, d, J = 15.7 Hz, Hβ), 7.70− 7.96 (16H, m, PPh3, CONH). 13C (100 MHz, MeOD): δ = 22.6 (d, JCP = 51.0 Hz, C10′), 23.5 (d, JCP = 4.4 Hz, C8′), 27.9 (C3′), 29.8 (C4′), 30.2 (C5′, C6′), 30.37 (C7′), 30.42 (C2′), 31.5 (d, JCP = 16.0 Hz, C9′), 39.5 (OSO2CH3), 40.5 (C1′), 56.7 (2 × OCH3), 61.2 (OCH3), 106.3 (C2, C6), 120.0 (d, JCP = 86.3 Hz, C1″), 121.5 (Cα), 132.2 (C1), 131.5 (d, JCP = 12.5 Hz, C3″, C5″), 134.8 (d, JCP = 10.0 Hz, C2″, C6″), 136.3 (d, JCP = 3.0 Hz, C4″), 140.7 (C4), 141.5 (Cβ), 154.8 (C3, C5), 168.6 (CONH). ESI/MS m/z (%): 640 (M+ + 2 − CH3SO3, 100), 639 (M+ + H − CH3SO3, 100), 418 (33). General Synthetic Procedure for Obtention of Mitochondriotropic Antioxidants (22−27). The triphenylphosphonium compound (16−21) (1 mmol) was dissolved in anhydrous dichloromethane (15 mL). The reaction mixture was stirred under argon and 7093



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(16H, m, PPh3, CONH). 13C (100 MHz, MeOD): δ = 22.7 (d, JCP = 51.1 Hz, C8′), 23.5 (d, JCP = 4.4 Hz, C6′), 27.7 (C3′), 29.7 (C4′), 29.9 (C5′), 30.3 (C2′), 31.4 (d, JCP = 16.1 Hz, C7′), 40.4 (C1′, CH3SO3), 108.3 (C2, C6), 118.7 (Cα), 120.0 (d, JCP = 86.3 Hz, C1″), 127.4 (C1), 131.6 (d, JCP = 12.5 Hz, C3″, C5″), 134.8 (d, JCP = 9.9 Hz, C2″, C6″), 136.3 (d, JCP = 3.0 Hz, C4″), 126.8 (C4), 142.4 (Cβ), 147.2 (C3, C5), 169.2 (CONH). ESI/MS m/z (%): 569 (M+ + H − CH3SO3, 43), 568 (M+ − CH3SO3, 100). (E)-(10-(3-(3,4,5-Trihydroxyphenyl)acrylamido)decyl)triphenylphosphonium Methanesulfonate (27). Yield: 53%. 1H (400 MHz, MeOD): δ = 1.19−1.43 (10H, m, H3′, H4′, H5′, H6′, H7′), 1.49− 1.60 (4H, m, H2′, H8′), 1.60−1.73 (2H, m, H9′), 3.28 (2H, t, J = 7.0 Hz, H1′), 3.34−3.43 (2H, m, H10′), 6.34 (1H, d, J = 15.6 Hz, Hα), 6.58 (2H, s, H2, H6), 7.31 (1H, d, J = 15.6 Hz, Hβ), 7.71−7.95 (16H, m, PPh3, CONH). 13C (100 MHz, MeOD): δ = 22.7 (d, JCP = 51.0 Hz, C10′), 23.5 (d, JCP = 4.3 Hz, C8′), 27.9 (C3′), 29.8 (C4′), 30.2 (C5′, C6′), 30.3 (C7′), 30.4 (C2′), 31.5 (d, JCP = 15.9 Hz, C9′), 40.5 (C1′), 108.2 (C2, C6), 118.7 (Cα), 120.0 (d, JCP = 86.3 Hz, C1″), 127.3 (C1), 131.5 (d, JCP = 12.5 Hz, C3″, C5″), 134.8 (d, JCP = 9.9 Hz, C2″, C6″), 136.3 (d, JCP = 3.0 Hz, C4″), 136.7 (C4), 142.4 (Cβ), 147.1 (C3, C5), 169.2 (CONH). ESI/MS m/z (%): 597 (M+ + H − CH3SO3, 67), 596 (M+ − CH3SO3, 100) 418 (14). Evaluation of AntiOxCINs Total Antioxidant Activity. The radical scavenging activity of AntiOxCINs was evaluated by means of total antioxidant capacity assays based on the spectrophotometric DPPH•, ABTS•+, and GO•.57 DPPH• Radical Assay. DPPH• radical scavenging activity was performed as previously described.19,58 Briefly, solutions of the test compounds with increasing concentrations (range between 50 μM and 500 μM) were prepared in ethanol. A DPPH• ethanolic solution (6.85 mM) was also prepared and then diluted to reach the absorbance of 0.72 ± 0.02 at 515 nm. Each compound solution (20 μL) was added to 180 μL of DPPH• solution in triplicate, and the absorbance at 515 nm was recorded every minute over 45 min. The percent inhibition of the radical was based in the comparison between the blank (20 μL of ethanol and 180 μL of DPPH• solution), which corresponded to 100% of radical, and test compounds solutions. The dose−response curves allowed the determination of IC50 values. ABTS•+ Radical Cation Assay. ABTS•+ scavenging activity was evaluated as previously described.17 Briefly, ethanolic solutions of the test compounds with increasing concentrations (range between 10 μM and 500 μM) were prepared. ABTS•+ radical cation solution was obtained by addition of 150 mM aqueous potassium persulfate solution (163 μL) to 10 mL of 7 mM aqueous ABTS solution followed by storage in the dark at room temperature for 16 h (2.45 mM final concentration). The solution was then diluted in ethanol to reach the absorbance of 0.72 ± 0.02. After addition, in triplicate, of the compound (20 μL) to ABTS•+ solution (180 μL) the spectrophotometric measurement was carried out each minute over a total of 15 min. The percent inhibition of radical was based on comparison between the blank (20 μL of ethanol and 180 μL of ABTS•+ solution), which corresponds to 100% of radical, and test compounds solutions. The dose−response curves allowed the determination of IC50 values. GO• Radical Assay. GO• radical scavenging protocol was adapted from the literature.59 Solutions of test compounds with concentrations from 5 μM to 75 μM were prepared in ethanol. An ethanolic solution of 5 mM GO• was prepared and diluted to reach the absorbance of 1.00 ± 0.02 at 428 nm. The addition (20 μL) in triplicate of compound solution to GO• solution (180 μL) was followed by absorbance measurement at 428 nm over 30 min, in the dark, at room temperature. The percent inhibition of radical was based on comparison between the blank (20 μL of ethanol and 180 μL of GO• solution), which corresponds to 100% of radical, and test compounds solutions. The dose−response curves allowed the determination of IC50 values. Evaluation of AntiOxCINs Redox and Lipophilic Properties. Electrochemical data were obtained using a computer-controlled potentiostat Autolab PGSTAT302N (Metrohm Autolab, Utrecht, The Netherlands). Cyclic voltammetry (CV) was performed at a scan rate of 50 mV s−1. The experimental conditions for differential pulse

voltammetry (DPV) were the following: step potential of 4 mV, pulse amplitude of 50 mV, and scan rate of 8 mV s−1. The electrochemical data were monitored by the General Purpose Electrochemical System (GPES), version 4.9, software package. All electrochemical experiments were performed in an electrochemical cell at room temperature, which was placed in a Faraday cage in order to minimize the contribution of background noise to the analytical signal. Evaluation of Redox Properties. Voltammetric curves were recorded using a three-electrode system. A glassy carbon electrode (GCE, d = 2 mm) was used as working electrode. The counter electrode was a platinum wire, with a saturated Ag/AgCl reference electrode completing the circuit. Stock solutions of each compound (10 mM) were prepared by dissolving the appropriate amount in ethanol. The voltammetric working solutions were prepared in the electrochemical cell, at a final concentration of 0.1 mM. The supporting electrolyte at pH 7.4 was prepared by diluting 6.2 mL of 0.2 M dipotassium hydrogen phosphate and 43.8 mL of 0.2 M potassium dihydrogen phosphate to 100 mL. Representative voltammograms are shown in Figure S1. Evaluation of Lipophilicity. The experimental electrochemical cell used in the evaluation of AntiOxCINs lipophilicity was a fourelectrode system with arrays of micro liquid−liquid interfaces (μITIES).60 The system contained two Ag/AgCl reference electrodes, prepared by electrochemical oxidation of an Ag wire in NaCl 1 M solution, and two counter electrodes of Pt, one in each phase (Figure S2A). The used organic electrolyte salt bis(triphenylphosphoranylidene)ammonium tetrakis(4-chlorophenyl)borate (BTPPATPBCl) was prepared by the metathesis of BTPPACl (97%) and KTPBCl (98%), and 1,6-dichlorohexane (98%) was purified according to a procedure described elsewhere.61 In this system, a microporous membrane consisting of a 12 μm thick PET membrane with 66 holes, 10 μm hole diameter, and 100 μm separation between the holes centers was used. The microhole arrays were kindly supplied by Prof. Hubert Girault, Institute of Chemical Sciences and Engineering, ISIC Laboratory of Physical and Analytical Electrochemistry, Switzerland. The electrochemical cell used had a geometrical water/organic solvent interface of 5.2 × 10−5 cm2. The microporous membrane was sealed with a fluorosilicone sealant (Dow Corning 730) onto a glass cylinder which was filled with 4.0 mL of the aqueous phase, where the aliquots from concentrated AntiOxCINs solution were added in order to change the concentration of the species in the aqueous phase. The membrane was then immersed in the organic phase contained in the cell. The organic phase reference solution (a 2 mM BTPPACl + 2 mM NaCl aqueous solution) was mechanically stabilized by a gel.60 The aqueous supporting electrolyte solution used in the studies was a TrisHCl buffer 10 mM, pH 7.0. An example of representative data is depicted in Figure S2B. Evaluation of AntiOxCINs Iron Chelating Properties. The iron chelation capacity of the novel mitochondria-targeted antioxidants was evaluated by the spectrophotometric ferrozine method using a BioTek Synergy HT plate reader, which measured the absorbance of the [Fe(ferrozine)3]2+ complex at 562 nm.26 The assay was performed in ammonium acetate buffer (pH 6.7) using a solution of ammonium iron(II) sulfate in ammonium acetate as the source of ferrous ions. In each well, a solution of the test compound (100 μM) and ammonium iron(II) sulfate in ammonium acetate (20 μM) were added, incubated for 10 min, and the absorbance was read at 562 nm. An aqueous 5 mM solution of ferrozine was freshly prepared and then added to each well (96 μM final concentration). After a new incubation at 37 °C for a 10 min period, the absorbance of [Fe(ferrozine)3]2+ complex was measured at 562 nm. Blank wells were run using DMSO instead of the test compounds. All compounds (caffeic acid, cinnamic derivatives, EDTA, and MitoQ10) as well as ferrozine were used at a final concentration of 100 μM. The absorbance of the first reading was subtracted from the final values to discard any absorbance due to the test compounds. Data are the mean ± SEM of three independent experiments and are expressed as % of Fe(II) chelation (EDTA = 100%). EDTA, used as reference, was found to chelate all available iron as it completely inhibited the formation of the colored ferrozine−Fe(II) complex. 7094



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Evaluation of AntiOxCINs Functional Mitochondrial Toxicity Profile. Isolation of Rat Liver Mitochondria. Rat liver mitochondria (RLM) were prepared by tissue homogenization followed by differential centrifugations in ice-cold buffer containing 250 mM sucrose, 10 mM HEPES (pH 7.4), 1 mM EGTA, and 0.1% fat-free bovine serum albumin. After obtaining a crude mitochondrial preparation, pellets were washed twice and resuspended in washing buffer (250 mM sucrose and 10 mM HEPES, pH 7.4).49 The protein concentration was determined by the biuret assay using bovine serum albumin (BSA) as a standard.62 Measurement of AntiOxCINs Mitochondria Uptake. The uptake of AntiOxCINs by energized RLM was evaluated by using an ionselective electrode, according to previously established methods, which measures the distribution of tetraphenylphosphonium (TPP+).27 An Ag/AgCl electrode was used as reference. To measure AntiOxCINs uptake, RLM (0.5 mg protein/mL) were incubated with constant stirring, at 37 °C, in 1 mL of KCl medium (120 mM KCl, 10 mM HEPES, pH 7.2, and 1 mM EGTA). Five sequential 1 μM additions of AntiOxCINs were performed to calibrate the electrode response in the presence of rotenone (1.5 μM). Succinate (SUC, 10 mM) was then added to generate ΔΨ, and valinomycin (VAL, 0.2 μg/mL) was added at the end of the assay to dissipate ΔΨ. The mitochondrial accumulation ratio was calculated by the disappearance of AntiOxCINs from extra- to intramitochondrial medium assuming an intramitochondrial volume of ∼0.5 μL/mg protein and a binding correction expected for the mitochondrial uptake of TPP compounds. Evaluation of AntiOxCINs Effect on RLM Lipid Peroxidation. The effects of AntiOxCINs on RLM lipid peroxidation were evaluated by two methods. (A) RLM lipid peroxidation was measured by oxygen consumption as described by Sassa et al.28 The oxygen consumption of 2 mg of RLM in a total volume of 1 mL of a reaction medium consisting of 100 mM KCl, 10 mM Tris-HCl, pH 7.6, using glutamate/malate (5 mM/ 2.5 mM) as respiratory substrate, was monitored at 37 °C with a Clark-type oxygen electrode. RLM were incubated for 5 min period with the different compounds (5 μM), and the lipid peroxidation process was started by adding 10 mM ADP and 0.1 mM FeSO4 (final concentrations). The saturated concentration of O2 in the incubation medium was assumed to be 217 μM at 37 °C. Time-dependent changes on oxygen consumption resulting from peroxidation of RLM membranes by a pro-oxidant pair (1 mM ADP/0.1 mM FeSO4) were recorded. The traces are the mean ± SEM recorded from six independent experiments. The time lag phase associated with the slower oxygen consumption that followed the addition of ADP/Fe2+ was used to measure the effectiveness of the tested compounds to prevent lipid peroxidation. Data are the mean ± SEM from six independent experiments and are expressed as % of control (control = 100%). (B) Lipid peroxidation was also measured by thiobarbituric acid reactive species (TBARS) assay.16 RLM (2 mg of protein/mL) were incubated in 0.8 mL of medium containing 100 mM KCl, 10 mM TrisHCl, pH 7.6, at 37 °C, supplemented with 5 mM glutamate/2.5 mM malate as substrate. RLM were incubated for 5 min period with the different tested compounds (5 μM), and then mitochondria were exposed to oxidative stress condition by the addition of 100 μM FeSO4/500 μM H2O2/5 mM ascorbate for 15 min at 37 °C. After exposure to oxidative stress, 60 μL of 2% (v/v) butylated hydroxytoluene in DMSO was added, followed by 200 μL of 35% (v/v) perchloric acid and 200 μL of 1% (w/v) thiobarbituric acid. Samples were then incubated for 15 min at 100 °C, allowed to cool down and the supernatant was transferred to a glass tube. After addition of 2 mL of MiliQ water and 2 mL of butan-1-ol, samples were vigorously vortexed for few seconds. The two phases were allowed to separate. The fluorescence of aliquots (250 μL) of the organic layer was analyzed in a plate reader (λEx = 515 nm; λEm = 553 nm) for TBARS. The TBARS background production in RLM energized with glutamate/malate was found to be negligible. Data are the mean ± SEM of three independent experiments and are expressed as % of control (control = 100%).

Evaluation of AntiOxCINs Effect on Mitochondrial Respiration. RLM respiration was evaluated polarographically with a Clarktype oxygen electrode connected to a suitable recorder in a 1 mL thermostated water-jacketed chamber with magnetic stirring at 37 °C.49,63 The standard respiratory medium consisted of 130 mM sucrose, 50 mM KCl, 5 mM KH2PO4, 5 mM HEPES (pH 7.3), and 10 μM EGTA. Increasing concentrations of AntiOxCINs (2.5−10 μM) were added to the reaction medium containing respiratory substrates glutamate/malate (10 mM and 5 mM, respectively) or succinate (5 mM) and RLM (1 mg) and allowed to incubate for a 5 min period prior to the assay. State 2 was considered as the respiration during the 5 min incubation time with AntiOxCINs. To induce state 3 respiration, 125 nmol of ADP (using glutamate/malate) or 75 nmol of ADP (using succinate) was added. State 4 was determined after ADP phosphorylation finished. Subsequent addition of oligomycin (2 μg/mL) inhibited ATP-synthase and originated the oligomycininhibition respiration state. Finally, 1 μM FCCP was added to induce uncoupled respiration. The results are the mean ± SEM of seven independent experiments. Evaluation of AntiOxCINs Effect on Mitochondrial Transmembrane Electric Potential (ΔΨ). Mitochondrial transmembrane electric potential (ΔΨ) was estimated through the evaluation of fluorescence changes of safranine (5 μM) and was recorded on a spectrofluorometer operating at excitation and emission wavelengths of 495 and 586 nm, with a slit width of 5 nm.64 Increasing concentrations of AntiOxCINs (2.5−10 μM) were added to the reaction medium (200 mM sucrose, 1 mM KH2PO4, 10 mM Tris (pH 7.4), and 10 μM EGTA) containing respiratory substrates glutamate/ malate (5 mM and 2.5 mM, respectively) or succinate (5 mM) and RLM (0.5 mg in 2 mL final volume) and allowed to incubate for a 5 min period prior to initiate the assay at 25 °C. In this assay, safranine (5 μM) and ADP (25 nmol) were used to initiate the assay and to induce depolarization, respectively. Moreover, 1 μM FCCP was added at the end of all experiments to depolarize mitochondria. ΔΨ was calculated using a calibration curve obtained when RLM were incubated in a K+-free reaction medium containing 200 mM sucrose, 1 mM NaH2PO4, 10 mM Tris (pH 7.4), and 10 μM EGTA, supplemented with 0.4 μg of valinomicin. The extension of fluorescence changes of safranine induced by ΔΨ was found to be similar in the standard and K+-free medium. “Repolarization” corresponded to the recovery of membrane potential after the complete phosphorylation of ADP added. Lag phase reflected the time required to phosphorylate the added ADP. Values are the mean ± SEM of five independent experiments. Isolated RLM developed a ΔΨ ≈ 226 mV and ΔΨ ≈ 202 mV (negative inside) when glutamate/ malate or succinate were used respectively as substrates (Supporting Information Tables S1−S8). Evaluation of AntiOxCINs Effect on Mitochondrial Permeability Transition Pore Opening. The induction of mPTP was measured by following mitochondrial swelling, estimated by alterations of light scattered from mitochondrial suspensions, as monitored spectrophotometrically at 540 nm.65 Increasing concentrations of AntiOxCINs (2.5−10 μM) were added to the reaction medium (200 mM sucrose, 1 mM KH2PO4, 10 mM Tris (pH 7.4), 5 mM succinate, and 10 μM EGTA supplemented with 1.5 μM rotenone) in the presence of RLM (1 mg) and allowed to incubate for a 5 min period before the assay. The experiments were initiated by the addition of a suitable concentration of Ca2+ (15−50 μM), titrated every day. Cyclosporin A (CsA), a PTP desensitizer,32 was added to demonstrate mPTP opening. The reaction was stirred continuously and the temperature maintained at 37 °C. Data are the mean ± SEM of three independent experiments and are expressed as Δabsorbance at 540 nm. Evaluation of Cytotoxicity/Antioxidant Outline in Human Hepatocellular Carcinoma Cells. Cell Culture. Human hepatocellular carcinoma HepG2 cells (ECACC, U.K.) were cultured in highglucose medium composed of Dulbecco’s modified Eagle’s medium (DMEM; D5648) supplemented with sodium pyruvate (0.11g/L), sodium bicarbonate (1.8 g/L) and 10% fetal bovine serum (FBS), and 1% of antibiotic penicillin−streptomycin 100× solution. Cells were 7095



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maintained at 37 °C in a humidified incubator with 5% CO2. HepG2 cells were seeded at density of 4 × 104 cells/mL and grown for 24 h before treatment. Cytotoxicity Screening. Cells were placed on 48-well plate (2 × 104 cells/500 μL) and then were incubated during 48 h with compounds 24 and 25 concentrations ranging from 0.5 μM to 100 μM and from 0.5 μM to 50 μM, respectively. After incubation, the sulforhodamine B (SRB) assay was used for cell density determination based on the measurement of cellular protein content.66 Briefly, after incubation, the medium was removed and wells were rinsed with PBS (1×). Cells were fixed by adding 1% acetic acid in 100% methanol for at least 2 h at −20 °C. Later, the fixation solution was discarded and the plates were dried in an oven at 37 °C. An amount of 250 μL of 0.5% SRB in 1% acetic acid solution was added and incubated at 37 °C for 1 h. The wells were then washed with 1% acetic acid in water and dried. Then, 500 μL of Tris (pH 10) was added and the plates were stirred for 15 min. Finally, 200 μL of each supernatant was transferred in 96-well plates and optical density was measured at 540 nm. Data are the mean ± SEM of four independent experiments, and the results were expressed as percentage of control (control = 100%), which represents the cell density without any treatment in the respective time point. Cellular Antioxidant Screening. Cells were placed in 48-well plate (2 × 104 cells/500 μL) and then were preincubated with nontoxic concentrations of compounds 24 (2.5 μM) or 25 (100 μM) for 1 h. After incubation time, cells were treated with oxidative stress-inducing agents by the addition of 250 μM FeSO4 or 250 μM H2O2 for 48 h. At the end of incubation time, SRB assay was used for cell mass determination as previously described. Data are the mean ± SEM of four independent experiments, and the results are expressed as percentage of control (control = 100%), which represents the cell density without any treatment in the respective time point. Vital Epifluorescence Microscopy. For detection of morphological alterations, including chromatin condensation and mitochondrial polarization and distribution, cells were placed in 6-well plates with a glass coverslip per well (8 × 104 cells/2 mL)33 and then treated with nontoxic concentrations of mitochondriotropic cinnamic derivatives (compounds 24 and 25) for 48 h. Thirty minutes prior the end of the incubation time, the polarized mitochondrial network was stained with TMRM (100 nM) while nuclei were stained with Hoechst 33342 (1 μg/mL), to detect apoptotic chromatin condensation, in HBSS (NaCl 137 mM, KCl 5.4 mM, NaHCO3 4.2 mM, Na2HPO4 0.3 mM, KH2PO4 0.4 mM, CaCl2 1.3 mM, MgCl2 0.5 mM, MgSO4 0.6 mM, and D-glucose 5.6 mM, pH 7.4) at 37 °C under dark conditions. Glass coverslips were removed from the wells and placed on glass slides with a drop of mounting medium. The cell images were acquired using a Zeiss LSM 510Meta microscope and analyzed with ImageJ software, version 1.49. The dyes were maintained with cells during the imaging procedure, and four image fields were randomly collected from each well. The images are representative of three independent experiments. Quantification of Reduced Glutathione (GSH) Content by HPLC. Cells were placed in a 100 mm cell culture dish (4 × 105 cells/10 mL) and then were incubated with nontoxic concentrations of 24 (2.5 μM) or 25 (100 μM) for 48 h. After incubation, cells were collected and the pellet was resuspended in PBS (1×), with samples stored at −80 °C until use. Reduced glutathione content in HepG2 cells was evaluated by HPLC67,68 using a reverse phase column (RP18 Spherisorb, S5 OD2) and MOPHAS as mobile phase. The flow rate was 1 mL/min, and the detection was performed at 515 nm. Statistics. Data were analyzed in GraphPad Prism 5.0 software (GraphPad Software, Inc.), with all results being expressed as the mean ± SEM for the number of experiments indicated. In data analysis, Student’s t test was used for comparison of two mean values, and oneway ANOVA with Dunnet multiple comparison post-test was used to compare more than two groups with one independent variable. Significance was accepted with (∗) P < 0.05, (∗∗) P < 0.01, (∗∗∗) P < 0.0005, (∗∗∗∗) P < 0.0001.

Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00741. Data of the toxicity studies of AntiOxCINs and MitoQ10; set of figures and tables related to their properties (PDF) Molecular formula strings (CSV)

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AUTHOR INFORMATION

Corresponding Authors

*P.J.O.: e-mail, [email protected]. *F.B.: e-mail, [email protected]. ORCID

Fernanda Borges: 0000-0003-1050-2402 Author Contributions ∇

J.T., F.C., and S.B. contributed equally to the work.

Notes

All the compounds, processes, and applications are under patent (NPAT20161000075435). P.J.O. and F.B. are cofounders of CNC spin-off company MitoDIETS. The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by FEDER funds through the Operational Programme Competitiveness FactorsCOMPETE and national funds by FCTFoundation for Science and Technology under Research Grants QUI/UI0081/2013, NORTE-01-0145-FEDER-000028, FTO/2433/2014, POCI01-0145-FEDER-016659, POCI-01-0145-FEDER-007440 and PTDC/DTP-PTDC/DTP-FTO/2433/2014. J.T. (Grants SFRH/BD/79658/2011 and PTDC/DTP-FTO/2433/2014), F.C. (Grant SFRH/BPD/74491/2010), S.B. (Grant SFRH/ BD/99189/2013), J.A.R. (Grant SFRH/BPD/105395/2014) grants are supported by FCT, POPH, and QREN. The authors thank Dr. Mike Murphy (MRC, Cambridge, U.K.) for providing us with MitoQ10.

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ABBREVIATIONS USED CsA, cyclosporin A; CV, cyclic voltammetry; DPV, differential pulse voltammetry; GO, galvinoxyl radical; GSH, glutathione; HCA, hydroxycinnamic acid; ITIES, immiscible electrolyte solutions; mPTP, mitochondrial permeability transition pore; PAINS, pan-assay interference compounds; RCR, respiratory control ratio; RLM, rat liver mitochondria; ROS, reactive oxygen species; SAR, structure−activity relationship; SRB, sulforhodamine B; TAC, total antioxidant capacity; TBARS, thiobarbituric acid reactive species; TMRM, tetramethylrhodamine methyl ester; TPP, triphenylphosphonium; ΔΨ, mitochondrial transmembrane electric potential

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REFERENCES

(1) El-Seedi, H. R.; El-Said, A. M.; Khalifa, S. A.; Goransson, U.; Bohlin, L.; Borg-Karlson, A. K.; Verpoorte, R. Biosynthesis, natural sources, dietary intake, pharmacokinetic properties, and biological activities of hydroxycinnamic acids. J. Agric. Food Chem. 2012, 60, 10877−10895. (2) Jacob, J. K.; Tiwari, K.; Correa-Betanzo, J.; Misran, A.; Chandrasekaran, R.; Paliyath, G. Biochemical basis for functional ingredient design from fruits. Annu. Rev. Food Sci. Technol. 2012, 3, 79−104. 7096



Article

(3) Benfeito, S.; Oliveira, C.; Soares, P.; Fernandes, C.; Silva, T.; Teixeira, J.; Borges, F. Antioxidant therapy: still in search of the “magic bullet”. Mitochondrion 2013, 13, 427−435. (4) Saso, L.; Firuzi, O. Pharmacological applications of antioxidants: lights and shadows. Curr. Drug Targets 2014, 15, 1177−1199. (5) Murphy, M. P. Antioxidants as therapies: can we improve on nature? Free Radical Biol. Med. 2014, 66, 20−23. (6) Wallace, D. C.; Fan, W.; Procaccio, V. Mitochondrial energetics and therapeutics. Annu. Rev. Pathol.: Mech. Dis. 2010, 5, 297−348. (7) Pagano, G.; Talamanca, A. A.; Castello, G.; Cordero, M. D.; d’Ischia, M.; Gadaleta, M. N.; Pallardo, F. V.; Petrovic, S.; Tiano, L.; Zatterale, A. Oxidative stress and mitochondrial dysfunction across broad-ranging pathologies: toward mitochondria-targeted clinical strategies. Oxid. Med. Cell. Longevity 2014, 2014, 541230. (8) Armstrong, J. S. Mitochondrial medicine: pharmacological targeting of mitochondria in disease. Br. J. Pharmacol. 2007, 151, 1154−1165. (9) Smith, R. A.; Hartley, R. C.; Cocheme, H. M.; Murphy, M. P. Mitochondrial pharmacology. Trends Pharmacol. Sci. 2012, 33, 341− 352. (10) Gane, E. J.; Weilert, F.; Orr, D. W.; Keogh, G. F.; Gibson, M.; Lockhart, M. M.; Frampton, C. M.; Taylor, K. M.; Smith, R. A.; Murphy, M. P. The mitochondria-targeted anti-oxidant mitoquinone decreases liver damage in a phase II study of hepatitis C patients. Liver Int. 2010, 30, 1019−1026. (11) Antonenko, Y. N.; Avetisyan, A. V.; Bakeeva, L. E.; Chernyak, B. V.; Chertkov, V. A.; Domnina, L. V.; Ivanova, O. Y.; Izyumov, D. S.; Khailova, L. S.; Klishin, S. S.; Korshunova, G. A.; Lyamzaev, K. G.; Muntyan, M. S.; Nepryakhina, O. K.; Pashkovskaya, A. A.; Pletjushkina, O. Y.; Pustovidko, A. V.; Roginsky, V. A.; Rokitskaya, T. I.; Ruuge, E. K.; Saprunova, V. B.; Severina, II; Simonyan, R. A.; Skulachev, I. V.; Skulachev, M. V.; Sumbatyan, N. V.; Sviryaeva, I. V.; Tashlitsky, V. N.; Vassiliev, J. M.; Vyssokikh, M. Y.; Yaguzhinsky, L. S.; Zamyatnin, A. A., Jr.; Skulachev, V. P. Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 1. Cationic plastoquinone derivatives: synthesis and in vitro studies. Biochemistry (Moscow) 2008, 73, 1273−1287. (12) Mattarei, A.; Biasutto, L.; Marotta, E.; De Marchi, U.; Sassi, N.; Garbisa, S.; Zoratti, M.; Paradisi, C. A mitochondriotropic derivative of quercetin: a strategy to increase the effectiveness of polyphenols. ChemBioChem 2008, 9, 2633−2642. (13) Biasutto, L.; Mattarei, A.; Marotta, E.; Bradaschia, A.; Sassi, N.; Garbisa, S.; Zoratti, M.; Paradisi, C. Development of mitochondriatargeted derivatives of resveratrol. Bioorg. Med. Chem. Lett. 2008, 18, 5594−5597. (14) Boukalova, S.; Stursa, J.; Werner, L.; Ezrova, Z.; Cerny, J.; Bezawork-Geleta, A.; Pecinova, A.; Dong, L.; Drahota, Z.; Neuzil, J. Mitochondrial targeting of metformin enhances its activity against pancreatic cancer. Mol. Cancer Ther. 2016, 15, 2875−2886. (15) Yan, B.; Stantic, M.; Zobalova, R.; Bezawork-Geleta, A.; Stapelberg, M.; Stursa, J.; Prokopova, K.; Dong, L.; Neuzil, J. Mitochondrially targeted vitamin E succinate efficiently kills breast tumour-initiating cells in a complex II-dependent manner. BMC Cancer 2015, 15, 401−412. (16) Teixeira, J.; Soares, P.; Benfeito, S.; Gaspar, A.; Garrido, J.; Murphy, M. P.; Borges, F. Rational discovery and development of a mitochondria-targeted antioxidant based on cinnamic acid scaffold. Free Radical Res. 2012, 46, 600−611. (17) Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; Rice-Evans, C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biol. Med. 1999, 26, 1231− 1237. (18) Munoz-Munoz, J. L.; Garcia-Molina, F.; Varon, R.; Tudela, J.; Garcia-Canovas, F.; Rodriguez-Lopez, J. N. Quantification of the antioxidant capacity of different molecules and their kinetic antioxidant efficiencies. J. Agric. Food Chem. 2010, 58, 2062−2070. (19) Son, S.; Lewis, B. A. Free radical scavenging and antioxidative activity of caffeic acid amide and ester analogues: structure-activity relationship. J. Agric. Food Chem. 2002, 50, 468−472.

(20) Gaspar, A.; Garrido, E. M.; Esteves, M.; Quezada, E.; Milhazes, N.; Garrido, J.; Borges, F. New insights into the antioxidant activity of hydroxycinnamic acids: Synthesis and physicochemical characterization of novel halogenated derivatives. Eur. J. Med. Chem. 2009, 44, 2092−2099. (21) Teixeira, J.; Silva, T.; Benfeito, S.; Gaspar, A.; Garrido, E. M.; Garrido, J.; Borges, F. Exploring nature profits: development of novel and potent lipophilic antioxidants based on galloyl-cinnamic hybrids. Eur. J. Med. Chem. 2013, 62, 289−296. (22) Ribeiro, J. A.; Silva, F.; Pereira, C. M. Electrochemical study of the anticancer drug daunorubicin at a water/oil interface: drug lipophilicity and quantification. Anal. Chem. 2013, 85, 1582−1590. (23) Alemu, H. Voltammetry of drugs at the interface between two immiscible electrolyte solutions. Pure Appl. Chem. 2004, 76, 697−705. (24) Gozzelino, R.; Arosio, P. Iron homeostasis in health and disease. Int. J. Mol. Sci. 2016, 17, 130−143. (25) Gammella, E.; Recalcati, S.; Cairo, G. Dual role of ROS as signal and stress agents: iron tips the balance in favor of toxic effects. Oxid. Med. Cell. Longevity 2016, 2016, 1−9. (26) Stookey, L. L. Ferrozine—a new spectrophotometric reagent for iron. Anal. Chem. 1970, 42, 779−781. (27) Kamo, N.; Muratsugu, M.; Hongoh, R.; Kobatake, Y. Membrane potential of mitochondria measured with an electrode sensitive to tetraphenyl phosphonium and relationship between proton electrochemical potential and phosphorylation potential in steady state. J. Membr. Biol. 1979, 49, 105−121. (28) Sassa, H.; Takaishi, Y.; Terada, H. The triterpene celastrol as a very potent inhibitor of lipid peroxidation in mitochondria. Biochem. Biophys. Res. Commun. 1990, 172, 890−897. (29) Santos, D. J.; Moreno, A. J. Inhibition of heart mitochondrial lipid peroxidation by non-toxic concentrations of carvedilol and its analog BM-910228. Biochem. Pharmacol. 2001, 61, 155−164. (30) Pereira, C. V.; Moreira, A. C.; Pereira, S. P.; Machado, N. G.; Carvalho, F. S.; Sardao, V. A.; Oliveira, P. J. Investigating drug-induced mitochondrial toxicity: a biosensor to increase drug safety? Curr. Drug Saf. 2009, 4, 34−54. (31) Zamzami, N.; Marchetti, P.; Castedo, M.; Hirsch, T.; Susin, S. A.; Masse, B.; Kroemer, G. Inhibitors of permeability transition interfere with the disruption of the mitochondrial transmembrane potential during apoptosis. FEBS Lett. 1996, 384, 53−57. (32) Soriano, M. E.; Nicolosi, L.; Bernardi, P. Desensitization of the permeability transition pore by cyclosporin a prevents activation of the mitochondrial apoptotic pathway and liver damage by tumor necrosis factor-alpha. J. Biol. Chem. 2004, 279, 36803−36808. (33) Serafim, T. L.; Carvalho, F. S.; Bernardo, T. C.; Pereira, G. C.; Perkins, E.; Holy, J.; Krasutsky, D. A.; Kolomitsyna, O. N.; Krasutsky, P. A.; Oliveira, P. J. New derivatives of lupane triterpenoids disturb breast cancer mitochondria and induce cell death. Bioorg. Med. Chem. 2014, 22, 6270−6287. (34) Brookes, P. S.; Yoon, Y.; Robotham, J. L.; Anders, M. W.; Sheu, S. S. Calcium, ATP, and ROS: a mitochondrial love-hate triangle. Am. J. Physiol.: Cell Physiol. 2004, 287, C817−C833. (35) Hung, C. C.; Tsai, W. J.; Kuo, L. M.; Kuo, Y. H. Evaluation of caffeic acid amide analogues as anti-platelet aggregation and antioxidative agents. Bioorg. Med. Chem. 2005, 13, 1791−1797. (36) Menezes, J. C.; Kamat, S. P.; Cavaleiro, J. A.; Gaspar, A.; Garrido, J.; Borges, F. Synthesis and antioxidant activity of long chain alkyl hydroxycinnamates. Eur. J. Med. Chem. 2011, 46, 773−777. (37) Garrido, J.; Gaspar, A.; Garrido, E. M.; Miri, R.; Tavakkoli, M.; Pourali, S.; Saso, L.; Borges, F.; Firuzi, O. Alkyl esters of hydroxycinnamic acids with improved antioxidant activity and lipophilicity protect PC12 cells against oxidative stress. Biochimie 2012, 94, 961−967. (38) Crozier, A.; Jaganath, I. B.; Clifford, M. N. Dietary phenolics: chemistry, bioavailability and effects on health. Nat. Prod. Rep. 2009, 26, 1001−1043. (39) Tauskela, J. S. MitoQ–a mitochondria-targeted antioxidant. IDrugs 2007, 10, 399−412. 7097



Article

(40) Yang, B.; Kotani, A.; Arai, K.; Kusu, F. Relationship of electrochemical oxidation of catechins on their antioxidant activity in microsomal lipid peroxidation. Chem. Pharm. Bull. 2001, 49, 747−751. (41) Collins, C. A.; Fry, F. H.; Holme, A. L.; Yiakouvaki, A.; AlQenaei, A.; Pourzand, C.; Jacob, C. Towards multifunctional antioxidants: synthesis, electrochemistry, in vitro and cell culture evaluation of compounds with ligand/catalytic properties. Org. Biomol. Chem. 2005, 3, 1541−1546. (42) Hynes, M. J.; O’Coinceanainn, M. n. The kinetics and mechanisms of reactions of iron(III) with caffeic acid, chlorogenic acid, sinapic acid, ferulic acid and naringin. J. Inorg. Biochem. 2004, 98, 1457−1464. (43) Xu, J.; Marzetti, E.; Seo, A. Y.; Kim, J. S.; Prolla, T. A.; Leeuwenburgh, C. The emerging role of iron dyshomeostasis in the mitochondrial decay of aging. Mech. Ageing Dev. 2010, 131, 487−493. (44) Asin-Cayuela, J.; Manas, A. R.; James, A. M.; Smith, R. A.; Murphy, M. P. Fine-tuning the hydrophobicity of a mitochondriatargeted antioxidant. FEBS Lett. 2004, 571, 9−16. (45) Kowaltowski, A. J.; Vercesi, A. E. Mitochondrial damage induced by conditions of oxidative stress. Free Radical Biol. Med. 1999, 26, 463−471. (46) Kappus, H.; Kieczka, H.; Scheulen, M.; Remmer, H. Molecular aspects of catechol and pyrogallol inhibition of liver microsomal lipid peroxidation stimulated by ferrous ion-ADP-complexes or by carbon tetrachloride. Naunyn-Schmiedeberg's Arch. Pharmacol. 1977, 300, 179− 187. (47) Gutteridge, J. M.; Zs.-Nagy, I.; Maidt, L.; Floyd, R. A. ADP-iron as a Fenton reactant: radical reactions detected by spin trapping, hydrogen abstraction, and aromatic hydroxylation. Arch. Biochem. Biophys. 1990, 277, 422−428. (48) Trnka, J.; Elkalaf, M.; Andel, M. Lipophilic triphenylphosphonium cations inhibit mitochondrial electron transport chain and induce mitochondrial proton leak. PLoS One 2015, 10, e0121837. (49) Serafim, T. L.; Carvalho, F. S.; Marques, M. P.; Calheiros, R.; Silva, T.; Garrido, J.; Milhazes, N.; Borges, F.; Roleira, F.; Silva, E. T.; Holy, J.; Oliveira, P. J. Lipophilic caffeic and ferulic acid derivatives presenting cytotoxicity against human breast cancer cells. Chem. Res. Toxicol. 2011, 24, 763−774. (50) Reily, C.; Mitchell, T.; Chacko, B. K.; Benavides, G.; Murphy, M. P.; Darley-Usmar, V. Mitochondrially targeted compounds and their impact on cellular bioenergetics. Redox Biol. 2013, 1, 86−93. (51) Siemen, D.; Ziemer, M. What is the nature of the mitochondrial permeability transition pore and what is it not? IUBMB Life 2013, 65, 255−62. (52) Rao, V. K.; Carlson, E. A.; Yan, S. S. Mitochondrial permeability transition pore is a potential drug target for neurodegeneration. Biochim. Biophys. Acta, Mol. Basis Dis. 2014, 1842, 1267−1272. (53) Wei, X.; Ma, Z.; Fontanilla, C. V.; Zhao, L.; Xu, Z. C.; Taggliabraci, V.; Johnstone, B. H.; Dodel, R. C.; Farlow, M. R.; Du, Y. Caffeic acid phenethyl ester prevents cerebellar granule neurons (CGNs) against glutamate-induced neurotoxicity. Neuroscience 2008, 155, 1098−1105. (54) Ross, M. F.; Prime, T. A.; Abakumova, I.; James, A. M.; Porteous, C. M.; Smith, R. A.; Murphy, M. P. Rapid and extensive uptake and activation of hydrophobic triphenylphosphonium cations within cells. Biochem. J. 2008, 411, 633−645. (55) Bisson, J.; McAlpine, J. B.; Friesen, J. B.; Chen, S.-N.; Graham, J.; Pauli, G. F. Can invalid bioactives undermine natural product-based drug discovery? J. Med. Chem. 2016, 59, 1671−1690. (56) Shi, M. M.; Kugelman, A.; Iwamoto, T.; Tian, L.; Forman, H. J. Quinone-induced oxidative stress elevates glutathione and induces gamma-glutamylcysteine synthetase activity in rat lung epithelial L2 cells. J. Biol. Chem. 1994, 269, 26512−26517. (57) Liu, Z. Q. Chemical methods to evaluate antioxidant ability. Chem. Rev. 2010, 110, 5675−5691. (58) Brand-Williams, W.; Cuvelier, M. E.; Berset, C. Use of a free radical method to evaluate antioxidant activity. LWT - Food Sci. Technol. 1995, 28, 25−30.

(59) Wu, W.-M.; Lu, L.; Long, Y.; Wang, T.; Liu, L.; Chen, Q.; Wang, R. Free radical scavenging and antioxidative activities of caffeic acid phenethyl ester (CAPE) and its related compounds in solution and membranes: A structure−activity insight. Food Chem. 2007, 105, 107− 115. (60) Ribeiro, J. A.; Miranda, I. M.; Silva, F.; Pereira, C. M. Electrochemical study of dopamine and noradrenaline at the water/ 1,6-dichlorohexane interface. Phys. Chem. Chem. Phys. 2010, 12, 15190−15194. (61) Katano, H.; Tatsumi, H.; Senda, M. Ion-transfer voltammetry at 1,6-dichlorohexane|water and 1,4-dichlorobutane|water interfaces. Talanta 2004, 63, 185−193. (62) Gornall, A. G.; Bardawill, C. J.; David, M. M. Determination of serum proteins by means of the biuret reaction. J. Biol. Chem. 1949, 177, 751−766. (63) Estabrook, R. W. Mitochondrial respiratory control and the polarographic measurement of ADP:O ratios. Methods Enzymol. 1967, 10, 41−47. (64) Kowaltowski, A. J.; Castilho, R. F. Ca2+ acting at the external side of the inner mitochondrial membrane can stimulate mitochondrial permeability transition induced by phenylarsine oxide. Biochim. Biophys. Acta, Bioenerg. 1997, 1322, 221−229. (65) Palmeira, C. M.; Wallace, K. B. Benzoquinone inhibits the voltage-dependent induction of the mitochondrial permeability transition caused by redox-cycling naphthoquinones. Toxicol. Appl. Pharmacol. 1997, 143, 338−347. (66) Papazisis, K. T.; Geromichalos, G. D.; Dimitriadis, K. A.; Kortsaris, A. H. Optimization of the sulforhodamine B colorimetric assay. J. Immunol. Methods 1997, 208, 151−158. (67) Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248−254. (68) Pastore, A.; Federici, G.; Bertini, E.; Piemonte, F. Analysis of glutathione: implication in redox and detoxification. Clin. Chim. Acta 2003, 333, 19−39.

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Development of a Mitochondriotropic Antioxidant Based on Caffeic

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