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Synthesis, Characterization, and Cu2+ Coordination Studies of a 3‑Hydroxy-4-pyridinone Aza Scorpiand Derivative Luis M. López-Martínez,†,‡ Javier Pitarch-Jarque,‡ À lvar Martínez-Camarena,‡ Enrique García-España,*,‡ Roberto Tejero,§ Hisila Santacruz-Ortega,† Rosa-Elena Navarro,† Rogerio R. Sotelo-Mundo,†,⊥ Mario Alberto Leyva-Peralta,† Antonio Doménech-Carbó,∥ and Begoña Verdejo*,‡ †

Departamento de Investigación en Polímeros y Materiales, Universidad de Sonora, Calle Rosales y Blvd. Luis Encinas s/n, Col. Centro, Hermosillo, Sonora 83000, México ‡ Instituto de Ciencia Molecular, C/Catedrático José Beltrán 2, 46980 Paterna, Valencia, Spain § Departamento de Química Física, Universidad de Valencia, C/Dr Moliner 50, 46100 Burjassot, Valencia, Spain ⊥ Laboratorio de Estructura Biomolecular. Centro de Investigación en Alimentación y Desarrollo (CIAD), A. C. Carretera a Ejido La Victoria Km 0.6, Hermosillo, Sonora 83304, México ∥ Departamento de Química Analítica, Universidad de Valencia, C/Dr Moliner 50, 46100 Burjassot, Valencia, Spain S Supporting Information *

ABSTRACT: The synthesis, acid−base behavior, and Cu2+ coordination chemistry of a new ligand (L1) consisting of an azamacrocyclic core appended with a lateral chain containing a 3-hydroxy-2-methyl-4(1H)-pyridinone group have been studied by potentiometry, cyclic voltammetry, and NMR and UV− vis spectroscopy. UV−vis and NMR studies showed that phenolate group was protonated at the highest pH values [log K = 9.72(1)]. Potentiometric studies point out the formation of Cu2+ complexes of 1:2, 2:2, 4:3, 1:1, and 2:1 Cu2+/L1 stoichiometries. UV−vis analysis and electrochemical studies evidence the implication of the pyridinone moieties in the metal coordination of the 1:2 Cu2+/L1 complexes. L1 shows a stronger chelating ability than the reference chelating ligand deferiprone. While L1 shows no cytotoxicity in HeLa and ARPE-19 human cell lines (3.1−25.0 μg/mL), it has significant antioxidant activity, as denoted by TEAC assays at physiological pH. The addition of Cu2+ diminishes the antioxidant activity because of its coordination to the pyridinone moiety phenolic group.

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INTRODUCTION Metal ions play pivotal roles in many biological processes. However, imbalances in the biological concentrations or exposure to metal ions from various sources (poisoning, overdose, and/or accumulation) may result in severe toxic effects for human health.1,2 An excess of metal ions can interfere with functions of various organ systems like the central nervous system and liver, among others. Diagnostic testing and subsequently decreasing the body’s burden of these metal ions through chelation therapy should be an integral part of the overall treatment regimen for individuals with a metal poisoning symptomatology or a known exposure to these substances. In fact, chelation therapy is considered as an important intervention for controlling metal levels in the body.3 Although there is a wide spectrum of chelating agents for metal ions, many of them have disadvantages associated with adverse side effects to human health.4 In this sense, it is critical to develop molecules that act as more specific and advanced chelating agents, not only to solve specific poisoning and/or intoxication processes but also to achieve full recovery in © XXXX American Chemical Society

disorders associated with metal-ion overload (Wilson’s disease, neurodegenerative disorders, etc.). Although the brain needs functional metals such as iron, copper, or zinc, alterations in their homeostatic mechanisms coupled with oxidative stress have been implicated in neurological disorders such as Huntington’s, Alzheimer’s, or Parkison’s diseases.5 In order to achieve enhanced metal mobilization from the body, a new perspective on the treatment of these disorders is the use of two structurally different chelating agents so that lower doses of potentially toxic chelating agents are required.6−11 Moreover, supplementation with antioxidants offers good therapeutic results.12−15 A combination of these two properties, ionophoric and antioxidant activity for regulation of reactive oxygen species (ROS), in one single molecule might prove useful in combating these types of disorders. During the last years, some of us have focused our research in the development of different polyazamacrocycles with antioxidant activity.16 On the basis of these types of receptors and Received: April 22, 2016

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DOI: 10.1021/acs.inorgchem.6b01006 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

anticancer agents.5a Moreover, because these types of chelators bind divalent transition metal ions like Cu2+ and Zn2+, the study of the formation of Cu2+ complexes is required because longterm chelation therapies can lead to problems related to essential metal depletion, especially of zinc, copper, and manganese.3,20 In this first report, we have analyzed the synthesis, acid−base behavior, and Cu2+ coordination properties of the new receptor, L1. The antioxidant activity of L1 at physiological pH has been evaluated by Trolox equivalent antioxidant capacity (TEAC) assay.

their antioxidant properties, herein we report on a new receptor of this series functionalized with a 3-hydroxy-2-methyl-4(1H)pyridinone group (L1 in Chart 1). L1 contains two different Chart 1

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EXPERIMENTAL SECTION

Synthesis of L1. The synthesis of the ligand 6-[2′-(3′-hydroxy-2″methyl-4′-pyridinone)ethyl]-3,6,9-triaza-1(2,6)-pyridinacyclodecaphane (L1) was carried out following a general procedure described in the literature for the preparation of analogous receptors, which consists of the reaction of the corresponding polyamine, in this case 5(2-aminoethyl)-2,5,8-triaza[9]-2,6-pyridinophane (L2),21 with 3(benzyloxy)-2-methyl-4-pyrone in a 1:1 molar ratio using sodium hydroxide in a refluxing ethanol/water mixture.22 Subsequently, treatment with HBr/AcOH/PhOH led to the removal of the benzyl group to give the final product L1 as a hydrobromide salt (Scheme 1). Synthesis of 6-[2′-[3′-(Benzyloxy)-2″-methyl-4′-pyridinone]ethyl]3,6,9-triaza-1(2,6)-pyridinacyclodecaphane Trichlorohydrate. A total of 0.434 g (2.0 mmol) of 3-(benzyloxy)-2-methyl-4-pyrone and 0.5 g (2.0 mmol) of 6-(2′-aminoethyl)-3,6,9-triaza-1(2,6)-pyridinacyclodecaphane (L2) were suspended in an ethanol/water mixture (1/ 0.7 mL) with 2 M NaOH (0.2 mL), and the mixture was left to reflux (70−75 °C) for 24 h with stirring. After cooling, 2 M HCl was added until pH = 1, and the ethanol was evaporated. To the remaining residue was added water (2.5 mL), and this solution was extracted with ether (5 × 2.5 mL). The aqueous phase was alkalinized with 10 M NaOH until pH = 10, and then it was extracted with dichloromethane (5 × 2.5 mL). The organic solution was dried with anhydrous sodium sulfate, and the solvent was evaporated to dryness. The residue obtained was taken into methanol (2 mL) and acidified with HCl until pH = 1. Subsequently, dry methanol/acetonitrile with excess acetone was added to obtain the pure product as the corresponding hydrochloride salt. 1H NMR (D2O, 300 MHz): δH 8.25 (d, J = 7.14 Hz, 1H), 8.00 (t, J = 7.8 Hz, 1H), 7.50−7.47 (s, 7H), 7.18 (d, J = 7.2 Hz, 1H), 5.20 (s, 2H), 4.67 (s, 4H), 4.51 (t, J = 7.9 Hz, 2H), 3.27 (t, J = 5.8 Hz, 4H), 3.16 (t, J = 7.9 Hz, 2H), 3.03 (t, J = 5.8 Hz, 4H), 2.48 (s, 3H). 13C NMR (D2O, 75.43 MHz): δC 161.49 (CO), 140.14, 129.53, 129.06, 122.04, 74.91, 55.02, 52.15, 50.90, 49.30, 46.12, 12.82.

coordination domains, the azamacrocyclic cavity and hydroxypyridinone subunit, offering the possibility of binding metal ions of different kinds, soft metal ions through the nitrogen atoms of the macrocycle, and hard ones through the oxygen atoms of the hydroxypyridinone moiety. An interesting feature of hydroxypyridinones is that they can be easily modified to produce a wide variety of new compounds with different chemical properties without significantly affecting the affinity for the metal ion.17 A well-known example of these characteristics is deferiprone, or 1,2-dimethyl-3-hydroxy-4pyridinone, a classical chelating agent for iron developed as an alternative to the use of deferoxamine for the treatment of iron overload and other related diseases.18 The low molecular weight and high lipophilicity of deferiprone contribute to the ability of this chelator to cross the brain−blood barrier and facilitate the oral administration of this drug.19 Furthermore, hydroxypyridinone derivatives have also been reported as ligands in metallodrugs: complexes of Ga3+ and Gd3+ as nuclear or magnetic resonance imaging diagnostic probes; complexes with Zn2+, VO2+, and MoO22+ as insulin mimetics or organometallic ruthenium and osmium arene complexes as Scheme 1

B


Article

Inorganic Chemistry Synthesis of 6-]2′-(3′-Hydroxy-2″-methyl-4′-pyridinone)ethyl]3,6,9-triaza-1(2,6)-pyridinacyclodecaphane Tetrabromohydrate (L1·4HBr). A total of 0.5 g (1.12 mmol) of 6-[2′-[3′-(benzyloxy)-2″methyl-4′-pyridinone]ethyl]-3,6,9-triaza-1(2,6)-pyridinacyclodecaphane and 4.01 g (42.64 mmol) of phenol were dissolved in 41.37 mL of 33% HBr/AcOH. The mixture was heated at 90 °C with stirring for 48 h. After cooling, the solution was vacuum-evaporated, and the product obtained was treated with an excess of acetone. After the solution was allowed to stand for at least 3 h, a light-brown precipitate was obtained. The excess solvent was removed and washed with acetone to give the pure product. 1H NMR (D2O, 300 MHz): δH 8.10 (d, J = 7.05 Hz, 1H), 8.00 (t, J = 7.85 Hz, 1H), 7.49 (d, J = 7.85 Hz, 2H), 7.10 (d, J = 7.05 Hz, 1H), 4.67 (s, 4H), 4.58 (t, J = 7.78 Hz, 2H), 3.28 (t, J = 5.88 Hz, 4H), 3.27 (t, J = 7.78 Hz, 2H), 3.09 (t, J = 5.88 Hz, 4H), 2.67 (s, 3H). 13C NMR (D2O, 75.43 MHz): δC 158.48 (C O), 148.65, 142.78, 142.40, 139.63, 138.64, 122.09, 111.21, 55.02, 51.97, 51.00, 50.47, 49.10, 46.17, 12.40. Mp: 232−233 °C. IR (KBr, cm−1): νOH 3600−3200, νCO 1640, νpolysubstituted pyridine 1565, νsubstituted quinone 831. Anal. Calcd for C19H27N5O2·4HBr (681.106 g/ mol): C, 33.50; H, 4.59; N, 10.28. Found: C, 33.83; H, 4.57; N, 9.83. Synthesis of [H2L1](ClO4)2·0.5H2O (1). Under an argon atmosphere, a solution of L1 (10 mg, 0.015 mmol) in water (2 mL) was acidified with perchloric acid until pH = 4. Slow evaporation of the solution afforded the formation of colorless crystals suitable for X-ray diffraction in 95% yield. Electromagnetic Field (EMF) Measurements. The potentiometric titrations were carried out at 298.1 ± 0.1 K using 0.15 M NaClO4 as the supporting electrolyte. The experimental procedure (buret, potentiometer, cell, stirrer, microcomputer, etc.) has been fully described elsewhere.23 The acquisition of the EMF data was performed with the computer program PASAT.24 The reference electrode was an Ag/AgCl electrode in a saturated KCl solution. The glass electrode was calibrated as a hydrogen-ion-concentration probe by the titration of previously standardized amounts of HCl with CO2free NaOH solutions and the equivalent point determined by the Gran’s method,25 which gives the standard potential, E°′, and the ionic product of water [pKw = 13.73(1)]. The computer program HYPERQUAD was used to calculate the protonation and stability constants.26 The pH range investigated was 2.0−11.0, and the concentrations of Cu2+ and the ligand ranged from 1 × 10−3 to 5 × 10−3 mol/dm3, with Cu2+/L1 molar ratios varying from 2:1 to 1:2. The different titration curves for each system (at least two) were treated either as a single set or as separated curves without significant variations in the values of the stability constants. Finally, the sets of data were merged together and treated simultaneously to give the final stability constants. NMR Measurements. The 1H NMR spectra were obtained in D2O solutions at a probe temperature of approximately 23 °C. The internal reference was sodium 2,2-dimethyl-2-silapentane-5-sulfonate. For studies of the pD dependence, a minimum quantity of a dilute KOD or DCl solution was used for to adjust the pD of the sample solutions. The pH value of each sample solution was measured with a long-stem combination electrode inserted into the NMR tube after NMR experiments. The electrode was calibrated with standard aqueous buffers, and the measured pH values were converted to the pD values by the relation pD = pHmeas + 0.4.27 Spectrophotometric Titrations. Absorption spectra were recorded on a Shimadzu UV-2501 PC spectrophotometer. HCl and NaOH were used to adjust the pH values, which were measured with a Metrohm 713 pH meter in both cases. Crystallographic Analysis. Analysis on single crystals of 1 was carried out with an Oxford Diffraction Supernova diffractometer using Mo Kα radiation (λ = 0.71073 Å) at 120 K. The structure was solved by direct methods using SHELXT28 and refined by full-matrix least squares on all F2 using SHELXL28 with the OLEX229 suite. Molecular drawings were produced with MERCURY30 The crystal data, data collection parameters, and results of analysis are listed in Table S1. CCDC 1472832 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from The

Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/ data_request/cif. Electrochemical Measurements. Cyclic voltammetry experiments were performed with 10−3 M aqueous solutions (0.15 M NaClO4 as the supporting electrolyte) of L1 at pH = 7.2. For the study of the electrochemistry of the metal complexes at different molar ratios (1:1 and 1:2 M/L1), equimolar amounts of Cu(ClO4)2·6H2O and the ligand were dissolved in 0.15 M NaClO4, previously degassed, with nitrogen for 10 min, and then the voltammograms were recorded. Electrochemical experiments were performed with BAS CV 50W and Metrohm PGSTAT 101 Autolab in a conventional threecompartment cell with a glassy-carbon working electrode. Prior to the series of experiments, the working electrode was cleaned and activated. Before each run, the electrode was polished with an aqueous suspension of alumina on a soft surface, dried, and cleaned. AgCl (3 M NaCl)/Ag and a platinum-wire auxiliary electrode completed the three-electrode configuration. The cyclic voltammograms were recorded at scan rates of 10−1000 mV/s. The pH was adjusted to the required value by adding appropriate amounts of aqueous HCl and/or NaOH 0.1 M solutions. Density Functional Theory (DFT) Calculations. DFT calculations of the Cu2+ complexes were performed with DFT as a computational method using B3LYP/6-31G** and B3LYP/ LANDL2DZ combinations. Specifically, the 6-31G** basis set was used for the nonmetallic atoms, while the LANDL2DZ was used for the transition metals, in which it has been generated ab initio effective core potentials to replace the Coulomb, core orthogonality, and exchange effects of the chemically inert core electrons in Cu2+. The Gaussian 09 package was used for these DFT calculations and the Molden package for the modeling analysis.31 TEAC Assay. The method used was described by Quirós-Sauceda et al.,32 and it is based on the capacity of a sample to inhibit the 2,2′azinobis(3-ethylbenzotriazoline-6-sulfonic acid radical (ABTS•+) compared with a reference antioxidant standard (Trolox). The ABTS•+ radical was generated by the chemical reaction of 19.2 mg (0.0369 mmol) of ABTS, dissolved in 5 mL of HPLC-grade water and 88 μL of potassium persulfate (K2S2O8; 0.139 mol/L)). It was incubated in the dark at room temperature for 12−16 h (the time required for formation of the radical); then 1 mL of the ABTS activated radical was taken, and 88 mL of pure ethanol was added. The radical solution was adjusted at an absorbance of 0.7 ± 0.02 at 734 nm. The reaction was initiated by adding 245 μL of ABTS•+ and 5 μL of the sample under study. The absorbance was monitored at 734 nm at 1 and 6 min. The percentage of inhibition was calculated, and the results were expressed as μmol of TE/100 g. The measured absorbances at a wavelength of 734 nm were read using an Omega microplate (BMG Labtech Inc.) reader device. Assessment of the Antiproliferative Agents by 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) Assay. Antiproliferative assay was carried out following the methodology reported by Leyva-Peralta et al.33 MTT assay was used to evaluate the antiproliferative activity. It is a colorimetric assay based in the fact that mitochondrial oxidoreductase enzymes are capable of reducing the tetrazolium dye MTT to insoluble formazan, which has a purple color. The cellular oxidoreductase enzymes may, under given conditions, reflect the number of viable cells present. Briefly, cells were seeded in a 96-well plate with Dulbecco’s modified Eagle medium (high glucose, supplemented with 5% fetal bovine serum) at a density of 10000 cells/well. Different concentrations of methanol extract and fractions were added followed by 48 h of incubation. All experiments were conducted in parallel with the controls (0.06−0.5% DMSO). A total of 10 μL of MTT (5 mg/mL; Sigma) was added to each well at the end of the treatment period and incubated at 37 °C for 4 h. Formazan crystals were dissolved with acidic isopropyl alcohol, and the plates were read in an ELISA plate reader, using a test wavelength of 570 nm and a reference wavelength of 630 nm. The plates were read within 10 min after the addition of isopropyl alcohol. The antiproliferative activity of the extracts was reported as IC50 values (IC50 value was defined as the concentration of the extract that inhibits cell proliferation by 50%). C


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Inorganic Chemistry Statistical Analysis. All data were expressed as mean ± standard deviation (SD). Data were subjected to statistical analysis of variance (ANOVA) by comparing the means with a Tukey test (p < 0.05). The IBM SPSS 20 statistical program was used for all statistical analyses.

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RESULTS AND DISCUSSION Acid−Base Behavior. Table 1 collects the stepwise protonation constants of L1 determined in 0.15 M NaClO4 Table 1. Stepwise Protonation Constants for L1 Obtained by EMF Measurements and UV−Vis and NMR Spectroscopy L1 reaction

a

H + H−1L ⇆ L H + L ⇆ HL H + HL ⇆ H2L H + H2L ⇆ H3L log β

EMF

b

NMR

EMFd

9.789(2)

10.07(3)

9.86 3.70

2.910(6)

3.19(5)

UV e

9.72 (1) 9.07(1) 7.70(3) 2.61(3) 29.10(4)

L3 b

c

13.56

a

Charges omitted. bDetermined in 0.15 M NaClO4. cDetermined in D2O. dDetermined in 0.15 M NaCl.34 eValues in parentheses are standard deviations in the last significant figure.

at 298.1 K along with those previously reported for the parent compound L3.34 The distribution diagram of L1 is collected in Figure S1. The receptor L1 presents in the pH range of study (2.5− 11.0) four stepwise protonation constants. The first three values obtained for L1 can be assigned to protonation of the hydroxyl oxygen and the secondary amino groups of the macrocyclic cavity. The fourth one, with a much lower value, corresponds to protonation of the pyridyl nitrogen of the pyridinone moiety. Furthermore, analysis of the variation with the pH of the pyridinone band in UV−vis spectra with the HypSpec program,35 allows one to calculate two pK values of 9.789(2) and 2.910(6) (see Figure 1 and Table 1). These values, which are in agreement with those previously reported for analogous compounds containing hydroxypyridinone moieties (see Table 1, L3) and with the results obtained by the potentiometric measurements for the first and fourth protonation constants, can therefore be assigned to protonation of the hydroxyl oxygen and the nitrogen atom of the pyridinone moiety. On the other hand, 1H NMR experiments also gave hints about the protonation sequence, followed by polyamine ligands because it is well-known that, upon deprotonation, the hydrogen nuclei bound to the α-carbon atom with respect to the nitrogen atoms bearing the deprotonation processes are those experimenting the largest upfield shifts.36 The upfield shift of all of the 1H signals associated with the pyridinone moiety (a−c; see Figure 2 for the labeling) upon going from pH = 2 to pH = 4 supports that first deprotonation ([H3L1]3+ to [H2L1]2+) occurs at the pyridinium nitrogen. In fact, the triplet signal attributable to the hydrogen atoms of the methylene group (h) nearest to this pyridinium nitrogen also shows an upfield shift in the same pH range. The remaining signals associated with the pyridinic aromatic spacer (m and n), the macrocyclic core (l, k, and j), and the pendant arm (i) do not undergo significant changes from pH = 2 to pH 8. However, an upfield shift of these signals occurs at pH = 8−9, in correspondence with the second and third deprotonation steps ([H2L1]2+ to [HL1]+ and [HL1]+ to [L1]), suggesting that these two steps occur at a larger extent at

Figure 1. pH dependence of the absorption spectrum of L1: (A) from pH = 2 to pH = 4; (B) from pH = 8 to pH = 11. [L1] = 10−4 M in 0.15 M NaClO4.

the secondary amino groups of the macrocyclic core. Above pH = 9, a new upfield on the 1H signals associated with the pyridinone moiety is observed. This behavior can be attributed to the last deprotonation ([L1] to [H−1L1]−). Using the NMR chemical shifts of the proton nuclei in the pyridinone ring, two protonation constants could be calculated by the HypNMR program (see Table 1), which are in reasonable agreement with the values previously determined by pH-metric and UV (see Scheme 2). X-ray Diffraction Studies. The crystal structure of [H2L1](ClO4)2·H2O consists of [H2L1]2+ cations, ClO4− anions, and water molecules (Figure 3). These divalent cations adopt an extended conformation in which the pendant arm lies far apart from the charged macrocyclic core, facilitating the formation of intermolecular hydrogen bonds between the carbonyl and hydroxyl groups (O2−H···O1 = 2.679 Å) belonging to two different pyridinone moieties, giving rise to dimeric units. At the difference with other aza scorpiand-like ligands previously reported,21 in this case intramolecular π−π stacking or hydrogen bonding does not seem to occur. D


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Inorganic Chemistry

Figure 2. Labeling and titration curve for observed chemical shifts as a function of pD in the aliphatic (A) and aromatic (B) regions. [L1] = 5 × 10−3 M in D2O.

Scheme 2

Figure 3. Ball and stick representation of the cation [H2L1]2+.

Table 2. Logarithms of the Formation Constants for the Cu2+ Complexes of L1 Determined in 0.15 M NaClO4 at 298.1 K

Furthermore, the different dimeric units are interconnected between them through a network of hydrogen bonds, which involve both protonated secondary amino groups of the macrocyclic core, the carbonyl group of the pyridinone moiety, and one of the perchlorate anions (see Figure S2 in the Supporting Information). Cu2+ Complexation Studies. The formation of Cu2+ complexes with L1 was studied in solution by potentiometric measurements and UV−vis spectroscopy. The pH-metric titrations carried out at 298.1 K in 0.15 M NaClO4 provided stability constants and model species that are collected in Tables 2 and S2. The results previously reported for L3 are also included in the table for comparison.37 Potentiometric titrations show that L1 is able to form species with 1:2, 1:1, 2:2, 4.3, and 2:1 Cu2+/L1 stoichiometries, whose percentage of formation is highly dependent on the molar ratio and initial concentrations (see Figure 4).

entry

reaction

1 2 3

Cu + H−1L ⇆ Cu(H−1L) Cu(H−1L) + H2O ⇆ Cu(H−1L)(OH) + H Cu(H−1L)(OH) + H2O ⇆ Cu(H−1L) (OH)2 + H Cu2L(H−1L) + H ⇆ Cu2L2 Cu2(H−1L)2 + H ⇆ Cu2L(H−1L) 2Cu + 2H−1L ⇆ Cu2(H−1L)2 Cu(HL)(L) + H ⇆ Cu(HL)2 CuL2 + H ⇆ Cu(HL)(L) Cu(L)(H−1L) + H ⇆ CuL2 Cu(H−1L)2 + H ⇆ Cu(L)(H−1L) Cu + 2(H−1L) ⇆ Cu(H−1L)2 2Cu + H−1L + 2H2O ⇆ Cu2(H−1L)(OH)2 + 2H 4Cu + 3(H−1L) ⇆ Cu4(H−1L)3

4 5 6 7 8 9 10 11 12 13

L3c

L1 a

16.39(3) −9.05(4) −11.01(6) 7.76(7) 8.68(1) 37.14(7) 6.88(5) 8.69(5) 9.14(5) 9.89(9) 21.96(6) 4.90(6)

b

10.42

2.89 19.09

64.42(5)

a

Charges omitted. bValues in parentheses are standard deviations in the last significant figure. cDetermined in KCl 0.1 M.37

E


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Inorganic Chemistry

Figure 4. Distribution diagrams of the L1/Cu2+ system as a function of the pH in aqueous solution in 0.15 M at 298.1 K: (A) [L1] = [Cu2+] = 10−3 M; (B) [L1] = 10−3 M, and [Cu2+] = 5 × 10−4 M; (C) [L1] = 10−3 M, and [Cu2+] = 1.33 × 10−3 M.

The 1:2 Cu2+/L1 complexes have formulas varying from [Cu(HL)2]4+ to [Cu(H−1L)2]. A joint analysis of the stability constants and UV−vis spectral data is illustrative to find out whether the Cu2+ binding involves the macrocyclic core, the hydroxypyridinone moiety, or both of them. First, the stability constant obtained for the [Cu(H−1L1)2] species (Table 2) is slightly higher than that previously reported for the corresponding complex of L3. Second, the values of the constants for the four protonation steps of [Cu(H−1L1)2] are high and comparable to the protonation constants of the free ligands (entries 7−10 in Table 2). Both of these facts suggest that coordination of the metal ion occurs through the two deprotonated hydroxypyridinone moieties, with the secondary amino groups of the macrocyclic cores remaining available to be protonated. A similar coordination mode has been reported for complexes of several hydroxypyridinone derivatives.38 The UV−vis spectra of solutions containing Cu2+ and L1 in a 1:2 molar ratio and recorded at variable pH, in agreement with previously reported studies for L3,37 are characterized by low absorptivities of the d−d band in the visible region. However, a detailed analysis of the UV region of the spectra can provide information about the role of the pyridinone moiety as an anchorage point for the metal ion (see Figure 5). Variation as a function of the pH of the intensity of the band at 310 nm (attributable to the pyridinone ring in its phenolate form) shows that deprotonation of the hydroxy group occurs as soon as the 1:2 complexes start to be formed. Indeed, an increase in the intensity of the band is observed as the [Cu(HL1)2]4+ species appears, reaching a plateau when its maximum percentage in solution is achieved. A further increase in the

Figure 5. Distribution diagram for the system Cu2+/L1 where [L] = 10−4 M [Cu2+] = 5 × 10−5 M (2:1 molar ratio). The UV−vis spectroscopic parameters at 310 nm are overlaid: (blue ●) Cu2+/L1; (red ●) free L1. (a) Cu(HL)2; (b) Cu2L2; (c) Cu(HL)(L); (d) CuL2; (e) Cu(L)(H−1L); (f) Cu(H−1L)2; (g) Cu(H−1L)(OH); (h) Cu(H−1L)(OH)2; (i) Cu2L(H−1L); (j) Cu(H−1L); (k) Cu2(H−1L)2.

absorbance is observed with the formation of the remaining protonated 1:2 Cu2+/L complexes. Similar changes were observed for the free ligand only above pH = 9 in F


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Inorganic Chemistry

Figure 6. DFT-optimized minimum-energy structure of [Cu(HL1)2]4+.

dimer species of [Cu 2(H xL1) 2 ] (4+x)+ (x = −1, 0, 1) stoichiometries. The distribution calculated for a 1:1 molar ratio and [Cu2+] = 10−3 M shows that such species prevail in a broad pH = 2−10 range. The monomeric species would predominate above pH = 10 at this concentration. This distribution also holds when the initial concentration is decreased by 1 order of magnitude ([Cu2+] = 10−4 M) even though the extent of formation of the monomer species at basic pH values is greater. The formation of dimeric species is supported by DFT calculations on the [Cu2(H−1L)2]2+ species. Each Cu2+ would be coordinated by the macrocyclic core of one of the ligands and the O1 atom of the other ones (see Figure 7C) in a distorted square-pyramidal geometry (τ = 0.125). The axial position would be occupied in this case by the tertiary amine. Two hydrogen bonds formed between O2 of one ligand and the proton of the N3 atom of the other ligand (O2···H−N3,

correspondence with deprotonation of the hydroxyl group of the pyridinone moiety (vide supra). DFT calculations performed for the [Cu(HL1)2]4+ complex support the proposed binding mode. The calculation shows (Figure 6) that the complex has a distorted square-pyramidal coordination geometry (τ = 0.178), in which the equatorial positions are occupied by the oxygen atoms of the two pyridinone moieties and the axial one is occupied by a water molecule. Table S3 collects representative distances and angles of the coordination sphere. Analysis of the species occurring in solution for 1:1 Cu2+/L1 molar ratio is, however, more complex. First, the stability constant of the [Cu(H−1L1)]+ complex is clearly higher than that reported for the analogous complex of pyridinone (L3), while it is comparable to that for the macrocycle L4 (log K = 17.78),39 which constitutes the macrocyclic core of L1. This might suggest that coordination of the metal ion should occur at the macrocyclic cavity. However, the only variation observed in the UV−vis band attributable to the hydroxyl group of the pyridinone ring occurs around pH = 4, while no further changes are observed for higher pH values (see Figure S3). This last point might, however, suggest a certain degree of participation of the pyridinone moiety in the complexation event. The initial 1H NMR spectrum of a solution containing L1 and Cu(ClO4)2 at pD = 4.9 shows as signals a and b attributable to the CH protons as well as signal c of the methyl group of the pyridinone experiment significant broadening due to the vicinity of the paramagnetic center. The spectrum collected after a while still shows some broadening of the aromatic pyridinone signals, but in this case, a low field is also observed for all of the signals of the macrocyclic ring (see Figure S4). These results might be suggesting an initial coordination of the metal through the pyridinone ring, followed by a reorganization to finally also involve the macrocycle in the binding. All of these experimental facts can be conciliated by assuming the formation of dimeric 2:2 Cu2+/L1 complexes in which the metal ions are coordinated by the pyridinone moiety of one of the ligands and the macrocyclic part of the other one. Such species have been evidenced, for instance, in a Ni2+ complex of a related ligand having a carboxylate group in the pendant arm.40 The potentiometric titrations indicate the formation of

Figure 7. DFT-optimized minimum-energy structures of (A and B) [Cu(H−1L1)]+ and (C) [Cu2(H−1L1)2]2+. G


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Inorganic Chemistry

Figure 8. DFT-optimized minimum-energy structures of [Cu4(H−1L1)3]5+.

[L]total = 10 μM at pH = 7.4. L1 has a pCu value of 2 orders of magnitude higher than the one obtained for L3. Another illustrative way to compare both systems is to calculate the distribution diagram for a ternary system with [Cu2+] = [L1] = [L3] = 10−3 M and compare the percentage of Cu2+ complexation by the two ligands, assuming that there is no formation of mixed complexes. As is evident in Figure 9, the percentage of Cu2+ complexation of L1 is much higher than that of L3 throughout all of the studied pH range.

1.713 Å) contribute also to stabilization of the complex. Table S3 collects representative distances and angles of the coordination sphere. DFT calculations on [Cu(H−1L1)]+ confirm the impossibility of the simultaneous participation of the pyridinone moiety and the macrocyclic core of a single crystal in the binding of the metal ion. While in one of the minimum-energy structures the coordination sphere would be completed by the four nitrogen atoms of the macrocycle core and one water molecule, in the other one, the water molecule would be removed, giving rise to a sort of cation−π interaction between the metal ion and pyridinone ring (see Figure 7A,B). The speciation studies show also the formation of a species of 4:3 Cu2+/L stoichiometry, which should imply coordination of three Cu2+ by the three macrocyclic cavities and of another one by the pyridinone fragments of the three macrocycles (see Figure 8). This species, which is necessary for obtaining a good fitting of the data, prevails in solution from pH = 7 to 9 for 4:3 Cu2+/L molar ratios (Figure 4C). In order to compare the relative affinities of L1 and the reference ligand deferiprone (L3) for Cu2+, we had calculated pCu values from the equilibrium constants obtained at 298.1 K.41,42 Table 3 shows the pCu values obtained for L1 and L3, where pCu refers to −log [Cu2+] when [Cu2+]total = 1 μM and Table 3. Equilibrium Free Metal-Ion Concentrations Expressed as pCu = −log [Cu2+], when [Cu2+]total = 1 μM and [L]total = 10 μM at pH = 7.4 ligand

pCu

L1 L3

13.16 11.25

Figure 9. Representation of Cu2+ complexation as a function of the pH for a system containing equivalent amounts of L1, L3, and Cu2+ (1:1:1 molar ratio; 10−3 M concentration). H


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Inorganic Chemistry

The corresponding voltammograms for Cu2+/L1 solutions at pH = 7.2 and different molar ratios of M/L1 (1:2 and 1:1) are also depicted in Figure 10. The voltammetric response of the 1:1 complex is similar to that of L1, but both the initial anodic wave and its coupled cathodic peak are significantly enhanced, thus suggesting that the complexation lowers the rate of the secondary post-electron-transfer reaction. The same, although weaker, effect was observed in the voltammogram of the 1:2 complex. Figure S5 shows repetitive cyclic voltammetry on a 1:1 Cu2+/ L1 solution at pH = 7.2. One can see that an almost reversible couple appears at potentials of +0.10 (cathodic) and +0.20 V (anodic), accompanied by a cathodic signal at ca. −0.45 V. These signals can, in principle, be attributable to the oneelectron reduction processes of Cu2+ species but, remarkably, are considerably weaker than those corresponding to the L1centered signals in Figure 10. A similar weak response was obtained in solutions of the 1:2 complex. These features suggest that such signals correspond to a minor fraction of Cu2+ from dissociated complexes, while the metal center in the Cu2+/ L1 complexes remains electrochemically silent. This idea was reinforced by the fact that the ratio between the L1-centered and Cu2+-centered signals was essentially the same at the entire range of tested scan rates, 10−1000 mV/s, a situation as expected for relatively fast dissociation equilibrium. It is pertinent to note that the redox properties of the copper complexes depend on the capacity of electron transfer, and such an ability, in turn, depends on several factors: the type of coordination geometry, rigidity, cavity size, shape, and chemical environment of the ligand.45 Then, the significant blocking of the metal-centered electrochemistry observed under our experimental conditions suggests that the metal center becomes occluded for electron-transfer processes, although it contributes to facilitate electron transfer from/to the pyridinone moiety. Evaluation of the Antioxidant Capacity by TEAC Assay. Evaluation of the antioxidant capacity of synthetic compounds today is of great importance because of its many applications in different areas confined to health (food industry and pharmaceutical, biological, and medical areas).46 L1 in its free form (0.0746 g of ETa/mol) has a higher antioxidant capacity compared to its copper complexes. The antioxidant capacity of the copper complexes was in the range of 0.0399− 0.0017 g of ETa/mol). The ability to neutralize free radicals in both the ligand and its copper complexes was compared with ascorbic acid; L1 has an antioxidant activity of 56%, while the copper complexes exhibit activity below 30%. Ascorbic acid at the same molar concentration has an activity of 0.1337 g of ETa/mol (Figure 11). The antioxidant capacity observed for L1 could be due to the hydroxyl group of the pyridinone moiety. Puglisi et al. mentioned that hydroxypyridinone derivatives are able to donate hydrogen atoms in a manner similar to that of αtocopherol (vitamin E) to quench free radicals, and this ability was quantified by ABTS radical assay.47 On the other hand, it was mentioned that the presence of phenolic groups in the structure of many natural and synthetic compounds shows improved antioxidant capacity over other compounds lacking such groups. Phenolic acids and flavonoids are compounds containing phenolic groups and are often reported in neutralizing ROS.48 However, in accordance with the electrochemical measurements, the copper complexes of L1 present poor antioxidant capacity. This behavior can be attributed to the implication of the hydroxyl group in coordination to the metal ion, losing its

Electrochemical Response. Figure 10 shows the cyclic voltammogram at the platinum electrode of L1 at pH = 7.2.

Figure 10. Cyclic voltammograms at the platinum electrode recorded for L1 solutions in 0.15 M NaClO4 at pH = 7.2: (black) L1; (red) 1:1 M:L1; (blue) 1:2 M/L1. Potential scan rate = 100 mV/s.

Upon scanning of the potential in the positive direction, an anodic peak appears at 1.04 V vs Ag/AgCl, followed, in the subsequent cathodic scan, by two small reduction peaks at 0.78 and 0.17 V. The first of such peaks can be attributed to the cathodic counterpart of the initial oxidation process, resulting, similar to the electrochemistry of L3 described by Yadegari et al., in terms of the oxidation of the pyridinone moiety to the dione.43,44 The electron-transfer process would possibly be quasi-reversible and influenced by the rate of protonation/ deprotonation. The overall electrochemical oxidation process, however, would be accompanied by a competing follow-up reaction, giving rise to a secondary product that is electrochemically reduced at a potential clearly less positive than the potentials at which the main redox couple occurs (see Scheme 3). Scheme 3. Tentative Scheme for L1-Centered Electrochemical Processes

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Inorganic Chemistry

studied by potentiometry, UV−vis spectroscopy, and electrochemistry, showing high affinity for Cu2+. The formation of Cu2+/L1 of 2:1, 4:3, 2:2, 1:1 and 1:2 stoichiometries has been detected in aqueous solution. The stoichiometry of the complexes formed is highly dependent on the pH, concentration, and metal-to-ligand ratio. The solution studies and DFT calculations support the implication of the pyridinone moiety for most of the stoichiometries. L1 shows higher affinity for Cu2+ than the related ligand L3. The antioxidant capacity for L1 and its copper complexes in different molar ratios has been measured by TEAC assays at physiological pH. The receptor L1 shows an interesting antioxidant capacity comparable to phenolic acids. However, the presence of hydroxyl groups in the pyridinone moiety involved in coordination to the metal ion can be related with the poor antioxidant capacity observed for the metal complexes. Preliminary studies with two cell lines, HeLa and ARPE-19, do not reveal toxicity and show normal growth. Future studies will devoted to addressing the Fe 3+ coordination behavior of L1 as well as the possibility of binding Fe3+ and Cu2+ in a concerted manner.

Figure 11. Antioxidant capacity of L1 and its copper complexes (at different 2:1, 1:1, 1:2, and 3:4 M/L1 molar ratios) by TEAC assay. Ascorbic acid and a solution of Cu(ClO4)2 were used as controls. Bars represent the standard error of the mean (±SEM) of triplicate experiments. ETa (Trolox equivalents). Reaction time = 10 min.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.6b01006. Distribution diagrams, crystallographic data, distances and angles, cyclic voltammograms, and observation in an inverted microscope of the antiproliferative activity (% cell viability) of L1 and its copper complexes on cancer cell line HeLa and normal cell line ARPE-19 (PDF)

antioxidant capacity. The antioxidant capacity of copper [Cu(ClO4)2] was undetectable. On the basis of our results, L1 has an antioxidant capacity comparable to those of phenolic acids but lower than those of flavonoids and vitamins C and E. Determination of the Antiproliferative Activity. The antiproliferative activities of L1 and of their copper complexes were evaluated using MTT colorimetric assay on two cell lines; a cancer cell line (HeLa, human cervix carcinoma) and normal cell line (ARPE-19, human retinal pigmented epithelium, normal cell line). The concentrations used for each compound vary from 3.1 to 25.0 μg/mL. Each assay was performed in triplicate, and all experiments were carried out in parallel with a control of normal growth of each cell line. Tables S4 and S5 show the results obtained for in vitro antiproliferative activity assays, expressed as percentage values of cellular viability ± SD after 48 h of continuous exposure to different concentrations of each test compound. The results were compared with those established by the United States National Cancer Institute, which in the case of pure compounds can be considered active if the IC50 values are lower than 4 μg/mL.49 However, this study suggests that copper complexes do not show antiproliferative activity in the two cell lines used, with values above 60% cell viability at the highest concentration used of 25 μg/mL and viability close to 100% at a concentration of 3.125 μg/mL after 48 h of exposure and worked conditions. In accordance with these results, microscope observations show the integrity of the cell morphology and growth upon exposure to each of the compounds under study; this fact was observed in both cell lines without significant changes (see Figures S6−S9). IC50 values were not calculated because they were not achieved at the highest concentration tested.

ACKNOWLEDGMENTS Financial support by the Spanish Ministerio de Economiá y Competitividad (Projects Consolider Ingenio CSD-201000065, CTQ2013-48917-C3-1-P, and Unidad de Excelencia MDM 2015-0038) and Generalitat Valenciana (Project PROMETEOII2015-002) is gratefully acknowledged. L.M.L.M. is thankful for a CONACYT graduate scholarship, travel support from Universidad de Sonora, and a travel scholarship ́ (beca mixta) from CONACYT and “Red Temática de Quimica Supramolecular, convenio 271884”. We also acknowledge Q. B.Mónica Alejandra Villegas Ochoa from Centro de Investigación en Alimentación y Desarrollo (CIAD), A. C., and Dr. Juan Carlos Gálvez Ruiź from the Departamento de Ciencias ́ Qui mico Biológ icas, Universidad de Sonora, for their collaboration. The NMR spectrometer is operated under the support of the Secretariá de Educación Pública, México (Program P/PIFI 2014).

CONCLUSIONS A new polyazamacrocyclic ligand containing an 3-hydroxy-2methyl-4(1H)-pyridinone group in its pendant arm has been synthesized. Coordination of Cu2+ by this new ligand was

(1) Agency for Toxic Substances and Disease Registry, Atlanta, GA, http://www.atsdr.cdc.gov/csem/csem.asp?csem=7&po=10. (2) Scott, L. E.; Orvig, C. Chem. Rev. 2009, 109, 4885−4910.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: (+34)963544879. * E-mail: [email protected]. Phone: (+34)963544401. Notes

The authors declare no competing financial interest.

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