Copper(I)âPhosphine Polypyridyl Complexes: Synthesis

Article pubs.acs.org/IC

Copper(I)−Phosphine Polypyridyl Complexes: Synthesis, Characterization, DNA/HSA Binding Study, and Antiproliferative Activity Wilmer Villarreal,† Legna Colina-Vegas,† Gonzalo Visbal,‡,§ Oscar Corona,∥ Rodrigo S. Corrêa,†,⊥ Javier Ellena,# Marcia Regina Cominetti,∇ Alzir Azevedo Batista,† and Maribel Navarro*,‡,⊗ †

Departamento de Química, Universidade Federal de São Carlos, CEP 13565-905 São Carlos, SP, Brazil Diretoria de Metrologia Aplicada às Ciências da Vida, Instituto Nacional de Metrologia, Qualidade e Tecnologia, CEP 25250-020 Xerém, RJ, Brazil § Centro de Desenvolvimento Tecnológico em Saúde (CDTS), Fundaçaõ Oswaldo Cruz - Fiocruz, CEP 21040-361 Rio de Janeiro, RJ, Brazil ∥ Centro de Química, Instituto Venezolano de Investigaciones Científicas (IVIC), Carretera Panamericana Km. 11, Apartado 20632, Altos de Pipe, 1020A Estado Miranda, Venezuela ⊥ ICEB, Departamento de Química, Universidade Federal de Ouro Preto, CEP 35400-000 Ouro Preto, MG, Brazil # Instituto de Física de São Carlos, Universidade de São Paulo, CEP 13560-970 São Carlos, SP, Brazil ∇ Departamento de Gerontología, Universidade Federal de São Carlos, CEP 13565-905 São Carlos, SP, Brazil ‡

S Supporting Information *

ABSTRACT: A series of copper(I)−phosphine polypyridyl complexes have been investigated as potential antitumor agents. The complexes [Cu(PPh3)2dpq]NO3 (2), [Cu(PPh3)2dppz]NO3 (3), [Cu(PPh3)2dppa]NO3 (4), and [Cu(PPh3)2dppme]NO3 (5) were synthesized by the reaction of [Cu(PPh3)2NO3] with the respective planar ligand under mild conditions. These copper complexes were fully characterized by elemental analysis, molar conductivity, FAB-MS, and NMR, UV−vis, and IR spectroscopies. Interactions between these copper(I)−phosphine polypyridyl complexes and DNA have been investigated using various spectroscopic techniques and analytical methods, such as UV−vis titrations, thermal denaturation, circular dichroism, viscosity measurements, gel electrophoresis, and competitive fluorescent intercalator displacement assays. The results of our studies suggest that these copper(I) complexes interact with DNA in an intercalative way. Furthermore, their high protein binding affinities toward human serum albumin were determined by fluorescence studies. Additionally, cytotoxicity analyses of all complexes against several tumor cell lines (human breast, MCF-7; human lung, A549; and human prostate, DU-145) and nontumor cell lines (Chinese hamster lung, V79-4; and human lung, MRC-5) were performed. The results revealed that copper(I)− phosphine polypyridyl complexes are more cytotoxic than the corresponding planar ligand and also showed to be more active than cisplatin. A good correlation was observed between the cytostatic activity and lipophilicity of the copper(I) complexes studied here.

■

INTRODUCTION According to the World Health Organization (WHO), cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. Furthermore, there are over 100 different known cancers that affect humans. This disease has caused approximately 8.2 million deaths and an estimated 13% of all deaths worldwide. In 2012, there were approximately 14.1 million new cancer cases in the world (not including skin cancer other than melanoma).1 Clearly, the current situation regarding cancer shows an urgent need to develop effective and affordable pharmaceutical agents to treat these diseases. Transition metals, particularly platinum © 2017 American Chemical Society

derivatives, have been tested in clinical trials against several cancers. Cisplatin is one of the most powerful chemotherapy drugs, which has worldwide approval for clinical practice; however, its use is limited due to its severe side effects. The other two FDA-approved agents are carboplatin and oxaliplatin, while nedaplatin, lobaplatin, and heptaplatin have received restricted approval for clinical use.2 Various strategies have been designed to synthesize anticancer metallotherapeutic drugs Received: October 10, 2016 Published: March 14, 2017 3781

DOI: 10.1021/acs.inorgchem.6b02419 Inorg. Chem. 2017, 56, 3781−3793

Article

Inorganic Chemistry

Figure 1. Synthesis procedures: 1:1 molar ratio copper complex:polypyridine ligand, dichloromethane, room temperature, and 1 h of reaction. Complex 1 was previously reported;15 however, the synthetic method used here was different from that one.

Table 1. Data Obtained from Different Spectroscopical and Analytical Methods for Copper−Phosphine Polypyridyl Complexes Studied elemental analysis complex

calcd

found

FAB-MS [M − NO3]+ (m/z)

2

%C 65.6 %H 4.3 %N 7.6 %C 67.6 %H 4.3 %N 7.3 %C 64.7 %H 4.0 %N 6.8

65.8 4.5 7.8 67.3 4.7 7.1 64.3 4.2 6.7

820

%C 63.7 %H 4.1 %N 6.5

64.0 4.3 5.6

3

4

5

a

ν ν ν ν ν ν ν ν ν ν ν ν ν ν

869

913

a

IR (cm−1)

665

CC: 1623 CN: 1580 NO3: 1384 CC: 1629 CN: 1583 NO3: 1384 CC: 1627 CN: 1578 NO3: 1385 HO−CO: 1709 CC: 1624 CN: 1579 NO3: 1385 Me−O−CO: 1721

ΛM(DMSO) (Ω−1 cm2 mol−1) 66.3

66.2

70.3

68.9

Correspond to [M − PPh3 − NO3]+

mentioned above and including dpq (dipyrido[3,2-d:2′,3′f ]quinoxaline/2,3-di-2-pyridylquinoxaline) was tested against antitumor cells. They showed strong partial intercalation and cleaved DNA in the presence of the reducing agent.10 Recently, various copper(II) polypyridyl complexes, DNA intercalators, with significant anticancer properties have been reported.11 Regarding copper(I) complexes as antitumor drugs, which is the focus of this paper, their utility is due to the fact that [Cu(dppz)2]BF4 showed high activity against leishmania parasites associated with the interaction between the copper− dppz complexes and the DNA by intercalation.12 In fact, compounds that bind non-covalently, such as by intercalation, have attracted particular interest in cancer therapy. Our interest in the compounds studied here is also due to the cytotoxic activity reported for copper(I)−phosphine compounds.13,14 Bearing these considerations in mind, our strategy in searching for a new copper(I)-based drug with high activity and low toxicity is to coordinate polypyridyl ligands (well-

using different metals beside platinum, such as ruthenium, titanium, gold, and copper.3 Coordination compounds of copper(I) and copper(II) have also been tested as promising candidates for cancer treatment, in the search for less toxic compounds with better activities (based on the premise that copper is an essential trace element for plants and animals including human beings) than platinum metallodrugs.4−6 A wide range of copper(II) complexes have been investigated as cytotoxic agents, for example, casiopeinas, copper-based drugs with general formula Cu(N−N)(A− A)]NO3, where N−N represents a dimine donor-like phen or bipy and A−A stands for amino acidate or acetyl acetonate, which have shown antineoplasic activities both in vitro and in vivo.7 This biological activity could be related to damage caused in the potential targets, such as mitochondria and DNA.8 Recently, structural changes to this promising drug class have been made as a strategy to obtain new active compounds.9 Another series of Cu(II) complexes with the same N−N ligand 3782


Article

Inorganic Chemistry

Figure 2. ORTEP view of complex 3 [Cu(PPh3)2(dppz)]NO3, showing the atom labels and 50% of the probability of ellipsoids.

molecular ion [M − NO3]+, but it displayed the peak corresponding to the molecular ion [M − PPh3 − NO3]+. The absorption spectrum of each copper polypyridyl ligand showed the same pattern of signals observed for the corresponding ligand spectrum. Likewise, the IR spectrum displayed peaks associated with ligand coordinated to copper and the bands assigned to ν(N−O) modes of nitrate groups. All 1H NMR resonances could be assigned on the basis of the 1 H chemical shift variation of each signal with respect to those of the free ligand (Δδ) as a parameter to deduce the coordination mode of the polypyridyl ligand to the copper. As a general trend, only one set of signals was observed, where proton Ha signals of polypyridyl ligand were shifted to downfield, while proton Hc peaks were shifted to a higher field after the coordination to copper ion, indicating that the coordination of the polypyridyl ligand to copper is through the iminic nitrogen as a bidentate ligand. As an example, the aromatic region of the 1H NMR spectrum of complex 3 is shown in Figure S1. Additionally, the relative integrals correspond to two phosphine ligands for one polypyridyl ligand on each copper complex. The 31P{1H} NMR spectra of the complexes 2−5 showed a signal corresponding to coordinated PPh3 in the range of δ 3.02−4.07 ppm (see Experimental Section for details). Oxidation state of copper(I) was also confirmed by the silent EPR spectrum for each complex 2−5. The structure of complex 3 was confirmed using the X-ray diffraction technique and its ORTEP structure is shown in Figure 2. This complex crystallized in the triclinic system, with space group P1̅, contains one molecule of the complex, one nitrate as the counterion and one methanol as the solvent in the asymmetric unit. The metal is tetra-coordinated by the binding of two P-atoms of the PPh3 ligand and two N-atoms of the dppz ligand. The complex presents a slightly distorted tetrahedral geometry, as can be seen by the bond angles around the Cu(I) metal center (see Table 2), in which only the N1−Cu1− P2 bond angle is close to 109.5°. The distances for the Cu−P [2.269(1)−2.274(1) Å], Cu−N [2.093(3)−2.098(3) Å] bond

known DNA intercalator agents) and triphenylphosphine as the auxiliary ligand into the coordination sphere of copper(I). The main aim of our study is to increase the activity of copper(I) complexes against cancer cells based on their interactions with DNA, such as intercalation, electrostatic, and/or cleavage. Therefore, in this paper, we show the easy preparation of four copper(I)−bis-triphenylphosphine polypyridyl complexes (Figure 1), which are able to target the DNA mainly through an intercalation binding mode. The interaction properties of the compounds with DNA were studied using spectroscopic titration with UV−visible spectroscopy, thermal denaturation, viscosity measurements, gel electrophoretical assays, circular dichroism studies, and fluorescent intercalator displacement assay. In this article, their interactions with HSA by fluorescence quenching and their growth inhibitory effect on three tumor and two non-tumor cell lines were also evaluated.

■

RESULTS AND DISCUSSION Synthesis and Characterization of Copper Polypyridyl Complexes. A series of copper polypyridyl complexes were synthesized by the reaction between [Cu(PPh3)2NO3] with the respective polypyridyl ligands under mild conditions, as summarized in Figure 1. This enabled us to evaluate the effect of the length of the polypyridyl ligands coordinated to copper ion on the biological properties of these complexes. Complexes 2−5 were fully characterized by a combination of different analytical and spectroscopy techniques including elemental analysis, molar conductivity, UV−vis, infrared (IR), FAB-MS, ESI-MS, and NMR (1H and 31P{1H}) spectroscopy, as described in the Experimental Section and shown in Table 1. Elemental analyses of the copper complexes are in agreement with the molecular formula proposed. The molar conductivity values obtained for these copper−phosphine polypyridyl complexes 2−5 are in the range for electrolyte 1:1 dissolved in DMSO.16 The FAB-MS or ESI-MS spectra of compounds 2−4 showed a moderate intensity peak related to the molecular ion [M − NO3]+ and the fragment ion [M − PPh3 − NO3]+, except complex 5 which did not show the corresponding 3783


Article

Inorganic Chemistry

some electronic perturbation in complexes 1−5 (see Figure S2). For each of these complexes, hypochromicity and small red shifts were observed, and the data are given in Table 3. The

Table 2. Selected Interatomic Distances (Å) and Angles (deg) for Complex 3a Cu1−N1 Cu1−N2 Cu1−P1 Cu1−P2

2.093(3) 2.098(3) 2.269(1) 2.274(1)

N1−Cu1−N2 N1−Cu1−P1 N2−Cu1−P1 N1−Cu1−P2 N2−Cu1−P2 P1−Cu1−P2

79.88(10) 108.83(8) 112.29(8) 109.54(8) 110.73(8) 125.95(3)

Table 3. Summary of Data Obtained from Titrations: Binding Constants (Kb) for Interaction of Complexes 1−5 with DNA and DNA Melting Temperatures complex 1 2 3 4 5

a

Atoms are labeled as shown in Figure 2. The numbers between parentheses are the estimated standard deviations of the last significant figures.

Kb (104mol L−1) 2.24 ± 0.42 4.28 ± 0.86 1.80 ± 0.17 0.33 ± 0.04 18.50 ± 3.22

−1

λ (nm) 370 380 370 380 370

hypochromism (%)

ΔTm

± ± ± ± ±

21 18 19 21 17

25.45 33.12 18.64 24.76 24.40

2.74 2.57 0.74 2.06 4.83

values of the binding constant, Kb, were determined for these complexes as described in the Experimental Section, and all values are shown in Table 3. As can be seen, the Kb values show the following trends 5 > 2 > 1 > 3 > 4. In comparison, these values are similar to those ones found by other copper complexes with planar ligands.10,22,23 This observation provides suitable evidence of the reversible interaction between these copper(I) complexes and DNA,24 commonly associated with the strength of intercalative interaction. Nevertheless, based on these results found by electronic absorption studies, the possibility of groove binding or electrostatic interaction cannot be completely excluded. In fact, other experiments using spectroscopic techniques are needed to complement these data in order to prove the binding modes and draw decisive conclusions. As was mentioned before, spectroscopic titration data provided essential information about the occurrence of the interaction of these copper complexes to DNA. However, additional evidence is needed to support an intercalative binding mode. Therefore, it was important to carry out hydrodynamic assays. These experiments have often been used to evaluate structural changes in the DNA helix and to determine intercalation interaction of the metal complexes to DNA. Measurements of DNA viscosity in the presence of different concentrations of complexes 1−5 are shown in Figure 4. The relative viscosity of CT-DNA increased in the presence of complexes 1−5, as a result of the double helix lengthening due to the intercalation of the complexes by the biomolecule base pairs. Similar behavior was also reported for other metal complexes with planar ligands such as Ru(II),25 Au(III),26 Ir(II),27 Cu(I)10 and Cu(II)19,28 and for the ethidium bromide, which is a classical intercalator.29 These results indicate that complexes 1−5 are able to intercalate between the DNA base pairs, which are consistent with the findings obtained from electronic absorption spectroscopy. For further elucidation of their interactions with the DNA, the melting temperature (Tm) was determined from the thermal denaturation curves of DNA obtained, as described in the Experimental Section, and data are shown in Table 3. The thermal denaturation technique is also an important tool to determine the nature of the metal complex−DNA interactions. It has been reported that covalent binding to DNA induces a marked destabilization of the double helix. This destabilizing interaction tends to decrease the thermal denaturation temperature of DNA in the presence of metal complexes, whereas a stabilizing interaction will increase it due to intercalation binding.30,31 The observed DNA melting temperature in the absence of the copper complexes was 65

lengths found for compound 3 are within the normal range found for similar Cu(I) complexes.17−19 In the crystal structure, the methanol molecule is hydrogenbonded to the NO3− anion. The O1sH1s···O2N5 hydrogen bond contact is separated by 2.039 Å. In addition, considering crystal packing stabilization, the π−π stacking contacts involving planar rings of the dppz ligand can also be observed (Figure 3). Passing a plane on all atoms of the dppz

Figure 3. Representation of π−π interactions occurring in the dppz ligand, contributing to crystal packing stabilization of complex 3.

rings, an interplanar distance of 3.407 Å is observed. This intermolecular π−π interaction, in the solid state, shows the possibility of this complex binding to DNA by intercalation stabilized by π-stacking interactions involving the dppz ligand. Based on these results, we propose that all the Cu(I)− phosphine polypyridyl complexes studied here are diamagnetic. This is characteristic considering the presence of copper(I) (d10) and should present a tetracoordinated configuration with 18-electrons, as is shown by the X-ray structure of complex 3, which by analogy with the reported crystalline structures informed for complexes [Cu(PPh3)2biqui)]NO3 and [Cu(PPh3)2bipy)]NO3,20,21 should adopt a distorted tetrahedral geometry. DNA Interaction Studies. The interaction of complexes 1−5 with DNA causes electronic perturbations in these complexes. These perturbations can be observed from spectroscopic and emission studies. In fact, in the spectroscopic titration, the increase in the CT-DNA concentration showed 3784


Article

Inorganic Chemistry

Figure 4. Effect of increasing concentration of the copper(I) complexes 1−5 on the relative viscosity of CT-DNA at 25 °C.

Figure 5. Circular dichroism (CD) spectra of CT DNA incubated 18 h with complexes 1−5 at different [complex]/[DNA] ratios at 37 °C.

°C, while the Tm of CT-DNA in the presence of complexes 1− 5 increased (Table 3). These studies show that the interaction of CT-DNA with complexes 1−5 lead to the stabilization of the double helix of DNA, indicating intercalation of the extended planar ligand moiety between the DNA bases. The results also suggest that these studied compounds have non-covalent interaction with the CT-DNA.23,32 Interestingly, a druginduced increase in the melting temperature (Tm) of DNA is a common feature of various anticancer drugs such as anthracyclines.27 Circular dichroism (CD) was also recorded, because this technique is very sensitive to diagnosing changes in the DNA structure. The CD spectrum of CT-DNA consists of a negative

band at a positive band near 248 nm and at 275 nm, attributed to the right-handed helicity and base stacking characteristic of B-DNA, respectively. Simple groove binding and electrostatic interaction of the complexes with DNA showed little or no perturbation of base stacking and helicity bands, while intercalation enhanced the intensities of both bands.33 Figure 5 shows the CD of CT-DNA alone and in the presence of complexes 1−5 at different [complex]/[DNA] ratios. It can be observed that all compounds increase the ellipticity of both bands for B-DNA and wavelength shifts were observed in the region of DNA absorption. This kind of behavior has already been reported for several metal complexes with planar ligands as evidence of intercalative interaction with DNA.20,25,34,35 3785


Article

Inorganic Chemistry

observed, when the copper complex/DNA ratio increases, the mobility of the plasmid changes. It can also be observed that changes occur from the SC to the L forms of the DNA when the complex/DNA ratio increases. In the highest concentrations of the complexes (Ri 0.25−1.0), the pBR322 bands cannot be observed for all complexes (except for complex 4), due to the DNA fragmentation or the complete displacement of ethidium bromide used as a fluorescence intercalator.37 Similar patterns of mobility have been described for some copper and platinum complexes reported by us and other researchers.20,24,38,39 Thus, these complexes are also able to perform a DNA cleavage based on the results from the electrophoresis experiment. These results were compatible with the aforementioned concerning the stabilizing effects of DNA, which is a consequence of the interaction of these copper complexes with the biomolecule, observed by the thermal denaturation techniques and CD spectroscopy. In order to evaluate the intercalate capacity of copper complexes 1−5, a competitive fluorescent intercalator displacement assay was performed as described in the Experimental Section. CT-DNA was incubated with thiazole orange (TO), and then copper complexes 1−5 were added at different concentration ratios. TO is known as an effective intercalator and it is used in fluorescent intercalator displacement assays with DNA due to the capacity of increasing the fluorescence of DNA by ca. 3000-fold.40 Figure 7a−d shows that all the copper complexes displaced the TO due to the loss fluorescence. It can also be observed in this figure that the DNA-TO adduct emission was clearly quenched by all the complexes at different

In addition, gel electrophoresis mobility assays have also been examined. A plasmid DNA is characterized by presenting the supercoiled (SC), the open circular (OC), and the linear (L) forms. Changes in electrophoretic mobility of any of the mentioned forms are commonly taken as evidence of direct metal−DNA interactions.36 In Figure 6, the gel electrophoretic

Figure 6. Effects of the concentration of complexes 1−5 on the conformation of pBR322 plasmid DNA. Molecular weight marker (MW) and DNA in DMSO (10%).

separation of pBR322 DNA alone is shown and after incubation with different concentrations of copper complexes 1−5. As

Figure 7. Effects of the concentrations of complexes 1−5 on the fluorescence of the DNA-TO adduct. 3786


Article

Inorganic Chemistry

Table 4. Stern−Volmer Quenching Constant (KSV), Biomolecular Quenching Rate Constant (Kq), Biomolecular Binding Constant (Kb), Number of Binding Sites (n), ΔG0, ΔH0, and ΔS0 Values for Complex−HSA Systems at Different Temperatures complex

T (K)

1

300 310 300 310 300 310 300 310 300 310

2 3 4 5

KSV (×105) 0.93 0.92 0.75 0.76 2.27 2.77 1.18 1.01 1.43 1.30

± ± ± ± ± ± ± ± ± ±

0.02 0.01 0.08 0.06 0.03 0.04 0.05 0.02 0.01 0.01

Kq (×1015) 0.46 0.50 0.37 0.38 1.13 1.40 0.60 0.50 0.71 0.64

± ± ± ± ± ± ± ± ± ±

0.03 0.04 0.03 0.03 0.01 0.02 0.09 0.02 0.02 0.03

n 1.13 1.38 0.90 0.91 1.38 1.41 1.15 1.22 1.40 1.37

± ± ± ± ± ± ± ± ± ±

Kb 0.01 0.02 0.01 0.08 0.01 0.01 0.03 0.02 0.04 0.03

(2.07 (3.13 (2.90 (3.15 (1.12 (1.54 (5.00 (8.53 (5.27 (5.52

± ± ± ± ± ± ± ± ± ±

0.10)× 0.12)× 0.17)× 0.22)× 0.12)× 0.12)× 0.35)× 0.77)× 0.07)× 0.18)×

106 106 104 104 107 107 105 105 106 106

ΔG

ΔH

ΔS

−36.26 −38.55 −25.83 −26.70 −41.27 −42.64 −34.06 −35.20 −38.60 −40.00

−32.20

−13.60 −20.55 −64.56 −65.26 −55.93 −58.56 23.90 19.45 −116.42 −117.20

−6.46 −24.50 −41.22 −3.68

Table 5. IC50 and Log D Values for Copper Complexes toward Tumor Cells (A549, MCF-7, DU-145) and Non-tumor Cells (V79-4 and MRC-5) after 48 h of Incubationa IC50 (μM) complex

A549

MCF-7

DU-145

V79-4

MRC-5

IS

Log D

[Cu(PPh3)2(phen)]NO3 (1) [Cu(PPh3)2(dpq)]NO3 (2) [Cu(PPh3)2(dppz)]NO3 (3) [Cu(PPh3)2(dppa)]NO3 (4) [Cu(PPh3)2(dppme)]NO3 (5) phen dpq dppz cisplatin [CuI[P(CH2OH)3]2L]b [CuI(PCN)(X)]c [CuI(N∩N) (PCN)(X)]c [CuI−pris(pyrazolyl)borate]d [CuII−polypididyl]e

0.47 ± 0.04 0.38 ± 0.02 0.36 ± 0.01 0.92 ± 0.08 0.32 ± 0.01 >50 >50 >100 14.42 ± 1.45 2.10−25.13 2.74−3.37 0.22−7.52 0.84−22.51 0.60−38.20

1.75 ± 0.35 3.50 ± 0.50 2.63 ± 0.25 5.25 ± 0.25 3.50 ± 0.40 >50 >50 >100 13.98 ± 2.02 1.55−16.70 4.51−6.98 0.25−6.98 0.73−19.25

1.34 ± 0.04 1.24 ± 0.03 0.78 ± 0.04 2.20 ± 0.15 0.80 ± 0.01 >50 >50 >100 2.33 ± 0.40

0.40 ± 0.06 0.47 ± 0.02 0.34 ± 0.04 1.05 ± 0.05 0.31 ± 0.06 >50 >50 >100 21.60 ± 1.28

0.70 ± 0.03 3.57 ± 0.90 0.52 ± 0.02 1.83 ± 0.13 0.71 ± 0.06 >50 >50 >100 29.09 ± 0.78

1.48 9.40 1.44 1.98 2.20

0.41 ± 0.01 0.41 ± 0.01 0.96 ± 0.03 −0.33 ± 0.01 0.78 ± 0.03

2.01

a Selectivity index IC50 MRC-5/IC50 A549. The dppa and dppme ligands were not evaluated due to their low solubility. bL= acetonitrile or bis(1,2,4triazol-1-yl)acetate ligands.49 cX = Cl or Br; N∩N = 2,2′-bipyridine, 1,10-phenanthroline, 5,6-dimethyl-1,10-phenanthroline, or dipyrido-[3,2-d:2′,3′f ]-quinoxaline; and PCN = tris(2-cyanoethyl)phosphine; 72 h of incubation.50 d72 h of incubation.51 eRef 11.

values are not changed for the complexes 1 and 2, increasing for 3, and decreasing for 4 and 5. Moreover, the biomolecular quenching rate constant (Kq) values for all these copper complexes were in the range of (0.37−1.40) × 1015 mol L−1 s−1, much greater than 2.0 × 1010 mol L−1 s−1, maximum value for dynamic quenching,43 indicated that the fluorescence quenching was triggered via the static quenching mechanism. The numbers of binding sites (n) were approximately 1 (0.90− 1.41), indicating a unique binding site between the HSA and the complexes with binding constants (Kb) between 105 and 107 M−1. These values or behavior are similar to the ones reported for [Cu(flumequine)2(α-diimines)] α-diimines, i.e., 2,2′-bipyridine, 1,1′-phenanthroline, or 2,2′-bipyridylamine,44 and Cu(II) complexes with 2-benzoylpyridine-4-methylthiosemicarbazone (Bp4mt) or 2-benzoylpyridine thiosemicarbazone (HBpt) ligands.45 The thermodynamic parameters, i.e., enthalpy change (ΔH), entropy change (ΔS), and free energy change (ΔG), are the main evidence to determine the binding mode between these copper complexes and the HSA protein.46 The thermodynamic values (Table 4) show that this family of Cu(I) compounds have two different interactions with the protein: the first one showed negative ΔH and ΔS values for complexes 1, 2, 3, and 5, which have structures analogous with those of the extended planar N−N ligands, showing van der Waals or hydrogen bond formation. The second case

extents, following trend 5 > 3 > 2 > 1 (Figure 7f). This effect seems to be directly related to the extended length of the intercalating ligand, and the minor changes in the dppz ligand structure (complex 5) that could lead to a significant influence on the competitive displacement of TO from DNA. In contrast, complex 4 did not show such an effect (Figure 7e), probably due to deprotonation of the carboxylic acid group under the conditions used in this experiment (pH 7.4). This generates repulsion between the carboxylate and phosphate groups of DNA and affects the efficiency of intercalation of this kind of ligand. HSA Interaction Studies. The interaction of small molecules with serum proteins is an important aspect of metal-based drug metabolism, affecting the distribution and biotransformation and ultimately the mechanism of action and the bioavailability of the medicinal agent.41 HSA is an important protein, involved in transporting hydrophobic metabolites as fatty acids and metal ions, including copper.42 For this reason, the interaction between the copper complexes 1−5 and HSA were performed using fluorescence quenching experiments at different temperatures (300 and 310 K), upon increasing the concentration of the complexes, a regular reduction of the fluorescence intensity was caused (as shown in Figure S3), in Table 4 is shown that with an increase of temperature, the Stern−Volmer quenching constant (KSV) 3787


Article

Inorganic Chemistry

Figure 8. Morphological study under an inverted microscope (100×) of A549 lung tumor cell line control cells and cells treated with the IC50 concentrations of complexes 1−5 after 24 and 48 h. In all panels, the images are representative of many pictures taken in n = 3 experiments.

showed negative ΔH and positive ΔS values, which corresponds to complex 4, where the difference is the presence of one free carboxylic group in the planar N−N ligand. This group seems to have a significant effect on the interaction with the protein, which could be attributed to the deprotonation of the carboxylic acid present in the dppa ligand (complex 4), favoring electrostatic interactions. All complexes showed negative values of ΔG, indicating the spontaneous interaction with this protein. Distribution Coefficient (Log D). Lipophilicity is the most important property that governs the pharmacokinetics and the pharmacodynamics of drugs.47 It is directly related to the ability of a compound to permeate through biological membranes. The most widely used method to measure lipophilic properties of chemical compounds is the distribution coefficient between equal volumes of water or buffer and n-octanol. The value distribution coefficient (Log D) for complexes 1−5 are shown in Table 5. These values are within the range of −0.33−0.96. These results show a good correlation with the extent of the planar aromatic rings of the binders, which increases the lipophilicity of the complexes (Figure S4). In the case of complex 4, a negative value of Log D was obtained, probably due to the possibility of deprotonation of carboxylic acid present in the binder (the experiment was conducted at pH 7.4), leading to a better affinity of the complex for the aqueous phase. Cytotoxicity Assay and Cell Morphology Analysis. The cytotoxicity profile of complexes 1−5 was investigated in different types of tumor cells such as A549 (human lung), MCF-7 (human breast), DU-145 (human prostate), and the non-tumor cells lines V79-4 (Chinese hamster lung) and MRC5 (human lung). The cell viability was determined by mitochondrial-dependent reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to formazan.48 Before performing the biological screening, the stability of the complexes was tested using the 1H and 31P{1H} NMR and UV−vis techniques in DMSO solutions (Figures S5−S9). After 48 h, the spectra of the complexes were the same when compared with those recorded using fresh solutions. While the percent cell viability was calculated by dividing the average absorbance of cells treated with the copper(I)−phosphine polypyridyl complexes by that of the control, the percentage of cell viability versus drug concentration (logarithmic scale) was plotted to determine the IC50 (drug concentration at which 50% of the cells are viable relative to the control), with its

estimated error derived from the average of 3 trials. The results obtained using this assay for complexes 1−5, their respective organic ligand and cisplatin used as a positive control are listed in Table 5. It has been found that all the complexes, especially complexes 3 and 5, exhibit potent cytotoxicity. The IC50 of all copper complexes 1−5 are much lower than their respective polypyridyl ligand against all tumor cell lines evaluated, demonstrating that copper atom has a very important effect against cancer cells. Furthermore, the studied complexes 1−5 also showed to be more active than cisplatin. Likewise, complexes 1, 2, 3, and 5 displayed in vitro antitumor activity against cancers cell lines of lung (A549) and breast (MCF-7) similar to the ones reported for other Cu(I)−tris(hydroxymethyl)phosphine,49 Cu(I)−phosphine polypyridyl,50 and Cu(I)−phosphine tris(pyrazolyl)borate complexes.51 The IC50 values found for complex 1−5 can be compared with those recently reported for Cu(II) polypyridyl complexes in the same tumor cell A549,11 and another human lung cancer cell line (H460),52 finding that all these copper(I) complexes studied in this work, except complex 4, showed better cytotoxic effects than the best Cu(II) compound reported in those studies. The cytotoxicity toward tumor cells lines, exhibited by complexes 1−5, indicated that the coordination of both phosphine and polypyridyl chelating ligands in one single molecule made it possible to create copper(I) complexes with an increased antitumor activity. Two other interesting observations that can be made when analyzing all these results: (i) A good correlation can be seen for complexes 3 and 5 that showed greater lipophilic character and also displayed better cytotoxicity activity. (ii) A general linear relationship can be established between their level of DNA interaction (Figure 6) and their biological activity, indicating that DNA intercalation is an important recognition of their anticancer response. Therefore, the significant cytotoxicity shown for these copper(I) complexes can be associated with the combination of several desirable biological features, such as intercalation with DNA, lipophilic character and HSA affinity. Most importantly, complexes 2 and 5, particularly complex 2, exhibited much higher cytotoxicity in the A549 tumor cell line than in the MRC-5 non-tumor cells. On the other hand, cisplatin exhibited similar cytotoxicity in the tumor cells and non-tumor cells. These results demonstrated that, in general, 2 and 5 had a higher activity than cisplatin in tumor cells and less 3788


Article

Inorganic Chemistry

Yellow crystals of the [Cu(PPh3)2(dppz)]NO3 complex were grown by slow evaporation of a mixture of methanol/dichloromethane/ether (1:1:1 v/v) solution at room temperature. To perform the X-ray diffraction experiment, crystal was mounted on glass fiber and positioned on the goniometer head. Intensity data were measured with the crystal at 298 K on an Enraf-Nonius Kappa-CCD diffractometer (95 mm CCD camera on φ-goniostat) using graphite monochromated Mo Kα radiation (λ = 0.71073 Å). The final unit cell parameters were based on all reflections. Data was collected using the COLLECT program;59 integration and scaling of the reflections were performed with the HKL Denzo-Scalepack system of programs.60 Gaussian absorption correction was applied.61 The structure was solved by direct methods using SHELXS-97.62 The model was refined by full-matrix least-squares on F2 with SHELXL-97.62 Hydrogen atoms of the aromatic rings of the PPh3 and dppz ligands were set isotropic with a thermal parameter 20% greater than the equivalent isotropic displacement parameter of the atom to which each one was bonded, in addition, for the methanol solvent the O−H hydrogen and CH3 hydrogen atoms, this percentage was set to 50%. The WinGX63 program was used to prepare material for publication. The structural analysis and figures were made using the MERCURY64 and ORTEP3.65 Crystallographic data and experimental details of the structural analysis are summarized in Table S1. Synthesis and Characterization. 11-Carboxy-dipyrido(3,2a:2,3-c)phenazine (dppa). 1,10-Phenanthroline-5,6-dione (500 mg, 2.36 mmol) was stirred in ethanol at reflux until total dissolution, then 3,4-diaminobenzoic acid (364 mg, 2.4 mmol) was added, the reaction was stopped when a gray solid was precipitated. The gray solid was filtrated and washed with diethyl ether. Yield: 91%. 1H NMR (300 MHz; CDCl3/TFA-d) 11.58 ppm (s, 1H, O−H), 10.26 ppm (dd, 2H, Hc,c′), 9.41 ppm (dd, 2H, Ha,a′), 9.39 ppm (s, 1H, Hf), 8.78 ppm (dd, 2H, He), 8.69 ppm (d, 2H, Hd), 8.45 ppm (dd, 2H, Hb), 8.42 ppm (dd, 2H, Hb′). Dipyrido[3,2-a:2′,3′-c]phenazine-11-carboxylic Acid Methyl Ester (dppme). The dppa (100 mg; 0.31 mmol) was added to a solution of methanol and sulfuric acid (5%), observing that the solution turned to light red. The dissolution was left to stir and under reflux for 2 h. The solution turned to amber again. Addition of 15 mL of distilled water obtained a beige solid. This solid was purified using a Soxhlet extractor in chloroform. The final solution was evaporated and a yellow ochre solid was obtained (87 mg; Yield: 83%). 1H NMR (300 MHz; CDCl3) 9.61 ppm (dd, 1H, Hc), 9.59 ppm (s, 1H, Hc′), 9.30 ppm (dd, 1H, Ha), 9.29 ppm (dd, 1H, Ha′), 9.05 ppm (d, 1H, Hf), 8.49 ppm (dd, 1H, Hd), 8.36 ppm (d, 1H, He), 7.82 ppm (dd, 1H, Hb), 7.80 ppm (dd, 1H, Hb′), 4.10 ppm (s, 3H, CH3−O−CO). General Description of the Synthesis of Copper Complexes. A solution of [Cu(PPh3)2NO3] in CH2Cl2 (15 mL) was added to the specific polypyridyl ligand at 1:1 molar ratio. The mixture was stirred and kept under argon, at room temperature for a period of 1 h. Afterward, it was transferred by cannula to the hexane solvent and the respective solid precipitate. Finally, it was filtered and the yellow solid was dried under vacuum. It is worth mentioning that complex [Cu(PPh3)2(phen)]NO3 (1) has been previously reported using another methodology15 that we present here. [Cu(PPh3)2(phen)]NO3 (1). Yellow solid. Yield (123 mg; 95%). Elemental analysis C48H38N3P2O3Cu·1/9CH2Cl2: % calcd C (68.8), H (4.6), N (5.0); % Found C (68.8), H (4.5), N (5.1). FAB-MS (DCM/ NBA) [M − NO3]+: 767 m/z, [M − PPh3 − NO3]+: 505 m/z. 1H NMR CDCl3 δ ppm: 8.69 (dd, 2H, Ha) 8.63 (dd, 2H, Hc), 8.09 (s, 2H, Hd), 7.82 (dd, 2H, Hb), 7.34 (m, 6H, PPh3), 7.18 (m, 12H, PPh3), 7.09 (m, 12H, PPh3). 31P{1H} NMR CDCl3 δ ppm: 3.88 (s, PPh3). IR (cm−1) ν(C−H) 3435; ν(CC, CN)1624, 1598; ν(PPh3) 1434; ν(NO3) 1384. ΛM(DMSO) = 63.2 Ω−1 cm2 mol−1. [Cu(PPh3)2(dpq)]NO3 (2). Light yellow solid. Yield (133 mg; 97%). Elemental analysis C50H38N5P2O3Cu·1/2CH2Cl2: % calcd C (65.6), H (4.3), N (7.6); % Found C (65.8), H (4.5), N (7.8). FAB-MS (DCM/ NBA) [M − NO3]+: 820 m/z, [M − PPh3 − NO3]+: 557 m/z. 1H NMR CDCl3 δ ppm: 9.62 (d, 2H, Hc), 9.15 (s, 2H, Hd), 8.88 (dd, 2H, Ha), 8.00 (dd, 2H, Hb), 7.32 (m, 6H, PPh3), 7.18 (m, 24H, PPh3).

toxicity in normal cells MRC-5, suggesting that complexes 2 had high selectivity between tumor cells and non-tumor cells. In Figure 8, the A549 lung tumor cell shape can be clearly observed, as well as changes before and after the treatment with complexes 1−5 followed at different times, using an inverted microscope. After 24 h of incubation, only complexes 5 ≫ 3 showed modifications on A549 cell morphology, when compared with control cells. However, the morphology was severely altered after 48 h of incubation in the presence of all copper complexes, causing changes to the spindle-shaped form, reduced cell numbers and loss of adhesion.

■

CONCLUSIONS The new complexes 2−5 were synthesized by the reaction of [Cu(PPh3)2NO3] with each polypyridyl ligand under mild conditions. These complexes were characterized by elemental analysis, molar conductivity, FAB-MS, and UV−vis, IR, and NMR spectroscopies. The crystal structure of complex 3 was determined by single-crystal X-ray diffraction. Results from these spectroscopic and analytical techniques indicated that copper complexes 2, 4, and 5 may have structures similar to that of complex 3. Furthermore, DNA−copper complex interactions were examined by several spectroscopic and analytical techniques, revealing that these copper(I) complexes interact with the DNA by intercalation. The fluorescence quenching experiments further reveal the high binding interaction of the Cu(I) complexes with HSA protein via the static quenching mechanism. Thermodynamic parameters suggest van der Waals interaction or hydrogen bond formation for complexes 1, 2, 3, and 5, while showing an electrostatic interaction with HSA for complex 4, which could be attributed to the deprotonation of the carboxylic acid present in the dppa ligand. It was also demonstrated that all these copper(I) complexes were cytotoxic toward three tumor cell lines, and their IC50 values were lower than that found for cisplatin in the present study. The significant in vitro antitumor activity observed for these new copper(I) complexes shows promising findings for future in vivo cytotoxicity evaluation, particularly complexes 2, 3, and 5.

■

EXPERIMENTAL SECTION

All manipulations were routinely carried out under argon using common Schlenk techniques. Analytical grade solvents were distilled from appropriate drying agents prior to use. All other commercial reagents were used without further purification. The compounds [Cu(PPh3 ) 2]NO3 ,53 1,10-phenantroline-5,6-dione (phendione), dipyrido[3,2-a:2′,3′-h]quinoxoline (dpq), dipyrido[3,2-a:2′,3′-c]phenazine (dppz), 11-carboxy-dipyrido(3,2-a:2,3-c)phenazine (dppa), and dipyrido[3,2-a:2′,3′-c]phenazine-11-carboxylic acid methyl ester (dppme) were synthesized according to reported methods.54−58 C, H and N analyses were performed using a Carlo Erba Model EA1108 elemental analyzer. NMR spectra were obtained from CDCl3, trifluoroacetic acid-d, and DMSO-d6 solutions with a Bruker AVANCE 300 spectrometer. IR spectra were obtained from a Nicolet 5DCX FT spectrometer, Nicolet Magna IR 560 instrument, while UV−vis spectra were recorded on a HP 8453 diode array instrument. FAB mass spectra were obtained from matrices of DMSO and nitrobenzyl alcohol (NBA) at the Analytical Services of the University of California Riverside Mass Spectrometry Facility. ESI-MS spectra were performed on Thermo Finnigan TSQ Quantum Ultra AM. EPR spectra were measured at room temperature in a Bruker EMX spectrometer and conductivity measurements were taken using a LaMotte CDS 5000 conductivity meter. Calf thymus DNA (CT-DNA) from SIGMA type I, No. D-1501 was used for corresponding DNA studies. Emission spectra were recorded on a Synergy H1Multi-Mode Reader (BioTek). 3789


Article

Inorganic Chemistry P{1H} NMR CDCl3 δ ppm: 4.05 (s, PPh3). IR (cm−1) ν(C−H) 3439; ν(CC, CN) 1623, 1580; ν(PPh3) 1434; ν(NO3) 1385. ΛM (DMSO) = 66.3 Ω−1cm2 mol−1. [Cu(PPh3)2(dppz)]NO3 (3). Yellow solid. Yield (135 mg; 95%). Elemental analysis C54H40CuN5P2O3·2/5CH2Cl2: % calcd C (67.6), H (4.3), N (7.3); % found C (67.3), H(4.7), N (7.1). FAB-MS (DCM/ NBA) [M − NO3]+: 869 m/z, [M − PPh3 − NO3]+: 607 m/z. 1H NMR CDCl3 δ ppm: 9.76 (dd, 2H, Hc), 8.79 (dd, 2H, Ha), 8.46 (dd, 2H, Hd), 8.07 (dd, 2H, He), 7.90 (dd, 2H, Hb), 7.36−7.30 (m, 6H, PPh3), 7.22−7.15 (m, 24H, PPh3). 31P{1H} NMR CDCl3 δ ppm: 4.07 (s, PPh3). IR (cm−1): ν(CC, CN) 1629, 1583; ν(C−H) 3404; ν(PPh3) 1435; ν(NO3) 1384. ΛM (DMSO) = 66.2 Ω−1 cm2 mol−1. [Cu(PPh3)2(dppa)]NO3 (4). Yellow ochre solid. Yield (144 mg; 96%). Elemental analysis C55H40CuN5O5P2·2/3CH2Cl2: % calcd C (64.7), H (4.0), N (6.8); % found C (64.3), H (4.2), N (6.7). FAB-MS (DCM/NBA) [M − NO3]+: 913 m/z [M − PPh3 − NO3]+: 651 m/z. 1 H NMR CDCl3 δ ppm: 9.57 (d, 2H, Hc,c′), 9.20 (d, 2H, Ha,a′), 8.97 (d, 1H, Hd), 8.94 (d, 1H, He), 8.49 (s, 1H, Hf), 8.16 (dd, 1H, Hb), 7.98 (dd, 1H, Hb′), 7.74−7.66 (m, 6H, PPh3), 7.57−7.35 (m, 24H, PPh3), 7.67 (m, O−H). 31P{1H} NMR CDCl3 δ ppm: 3.44 (s, PPh3). IR (cm−1) ν(C−H) 3434; ν(HO−CO) 1709; ν(CC, CN) 1627, 1578; ν(PPh3) 1435; ν(NO3) 1384. ΛM (DMSO) = 70.3 Ω−1cm2 mol−1. [Cu(PPh3)2(dppme)]NO3 (5). Yellow ochre solid (143 mg; 95%). C56H42CuN5P2O5·CH2Cl2: % calcd C (63.7), H (4.1), N (6.5); % found C (64.0), H (4.3), N (5.6). ESI-MS (MeOH) [M − PPh3 − NO3]+: 665 m/z. 1H NMR CDCl3 δ ppm: 9.77 (dd, 2H, Hc,c′), 9.16 (d, 1H, Hf), 8.93 (dd, 2H, Ha,a′), 8.61 (dd, 1H, He), 8.51 (d, 1H, Hd), 8.07 (dd, 2H, Hb,b′), 7.37 (m, 30H, PPh3), 4.12 (s, 3H, CH3− O−CO). 31P{1H} NMR CDCl3 δ ppm: 3.02 (s, PPh3). IR (cm−1) ν(C−H) 3421; ν(Me−O−CO) 1721; ν(CC, CN) 1624, 1579; ν(PPh3) 1434; ν(NO3) 1385. ΛM (DMSO) = 68.9 Ω−1cm2 mol−1. DNA Interaction Studies. All the measurements using CT-DNA were carried out in buffer Tris-HCl 5 mM (pH 7.2), 50 mM NaCl. The UV absorbance ratio 260/280 was 1.8−1.9 indicating that the DNA was sufficiently free of protein.66 The CT-DNA concentration per nucleotide was determined by absorption spectrophotometric analysis using the molar absorption coefficient 6600 mol−1 dm3 cm−1 at 260 nm.67 The spectroscopic titrations were carried out by adding increasing amounts of CT-DNA to a solution of the complex at a fixed concentration contained in a quartz cell and recording the UV−vis spectra after each addition. The absorption of CT-DNA was subtracted by adding the same amounts of CT-DNA to the blank. The intrinsic binding constant Kb was determined from the plot of [DNA]/(εa − εf) vs [DNA], where [DNA] is the concentration of DNA in base pairs and the apparent absorption coefficients, εa, εf, and εb, correspond to Aobs/[Cu], the extinction coefficient for the free copper complex, and the extinction coefficient of the copper complex in the totally bound form, respectively. The data were fitted to eq 1, with a slope equal to 1/(εb − εf) and the intercept equal to 1/[Kb(εb − εf)], and Kb was obtained from the ratio of the slope to the intercept.68

presented as (η/η0)1/3 versus the ratio [complex]/[DNA], where η and η0 are the specific viscosity of CT-DNA in the presence and absence of the complex, respectively. The values of η and η0 were calculated from the formula (t − tb)/tb, where t is the observed flow time and tb is the flow time for the buffer alone. Relative viscosities of CT-DNA were calculated from (η/η0).25 CD spectra were recorded on a spectropolarimeter JASCO J720 between 400 and 200 nm in continuous scanning mode (200 nm/ min). The final data are expressed in molar ellipticity (millidegrees). All of the CD spectra were generated and represented the averages from three scans. Stock solutions (1.5 mM) of each complex were freshly prepared in DMSO prior to use. An appropriate volume of each solution was added to samples of a freshly prepared solution of CTDNA (100 μM) in Tris-HCl buffer to achieve molar ratios ranging from 0.03 to 0.12 drug/DNA. The samples were incubated at 37 °C for 18 h. Next, 10 μL portions of pBR322 plasmid DNA in Tris-HCl buffer were incubated at 37 °C for 18 h with molar ratios of the Cu(I) compounds between 0.03 and 1.0. After incubation, 5 μL of each sample were separated by electrophoresis in a 1% agarose gel for 45 min at 100 V using Tris−borate−EDTA buffer (TBE) and stained with ethidium bromide (2 μL ethidium bromide per 40 mL agarose gel mixture). Samples of free DNA and DNA + DMSO were used as controls. The DNA bands were visualized by imaging with UV light transilluminator (ChemiDoc MP, Bio-Rad). Competitive displacement assay was carried out by adding TO to CT-DNA solution. The TO-CT-DNA solutions, incubated for 24 h, containing 1 μM of TO and 10 μM of CT-DNA was excited at 480 nm, and fluorescence emission spectra were recorded between 505 and 700 nm. The Cu(I) polypyridyl complexes were incubated from 0 to 1 μM with the TO-CT-DNA solutions and were excited at 480 nm. The fluorescence emission spectra were recorded between 505 and 700 nm Albumin Binding Studies. The protein binding study was performed by tryptophan fluorescence quenching experiments using human serum albumin (HSA, 3 μM) in Tris-HCl buffer. The quenching of the emission intensity of tryptophan residues of HSA at 305 nm was monitored using complexes 1−5 as a quencher with increasing concentration at different temperatures (300 and 310 K). The fluorescence spectra were recorded from 300 to 500 nm at an excitation wavelength of 270 nm. The fluorescence spectra of complexes 1−5 in buffer solutions were recorded under the same experimental conditions and not exhibited emission spectra. The Stern−Volmer and Scatchard equations and graphs were used in order to study the interaction of each quencher with human serum albumin and calculate the Stern−Volmer quenching constant (KSV, M−1), biomolecular quenching rate constant (Kq, M−1 s−1), binding constant (Kb, M−1), and the number of binding sites (n). The thermodynamic parameters were calculated from the van’t Hoff equation, ΔH0 (kJ mol−1), ΔS0 (J mol−1 K), and ΔG0 (kJ mol−1). Furthermore, the change in free energy (ΔG) was calculated from the following equation: ΔG = −RT ln K = ΔH − TΔS. Distribution Coefficient (Log D). Buffer−n-octanol distribution coefficients were determined using the shake flask method.69 A UV− vis calibration curve was prepared in the range of 10−100 μM in noctanol. The determination was carried out at pH 7.4 in a mixture of equal volumes of buffer:n-octanol and continuously shaking for 18 h at room temperature. The concentration of complex in n-octanol was measured spectrophotometrically in order to determine values of D = [compound] (in n-octanol)/[compounds] (in buffer). Biological Tests: Cell Culture, Antiproliferative Assays, and Morphological Observations in Tumor Cells. In vitro cytotoxicity assays on cultured human tumor cell lines still represent the standard method for initial screening of antitumor agents. Thus, as a first step to assess their pharmacological properties, these copper(I) complexes were assayed against the human lung tumor cell line A549 (ATCC CCL-185), human breast tumor cell line MCF-7 (ATCC HTB-22), human prostate tumor cell line DU-145 (ATCC HTB-81) and the normal cell line V79-4 (ATCC CCL-93) and MRC-5 (ATCC CCL171). The cells were routinely maintained with Dulbecco’s Modified Eagle’s medium (DMEM for A549, V79-4, and MRC-5) or Roswell

31

[DNA]/(εa − εf ) = [DNA]/(εb − εf ) + 1/[Kb(εb − εf )]

(1)

The interaction of copper complexes 1−5 with CT-DNA was measured in thermal denaturation experiments. Melting curves were recorded in media containing 50 mM NaClO4 and 5 mM Tris-HCl buffer (pH = 7.29). The absorbance at 260 nm was monitored for solutions of CT-DNA (70 μM) before and after incubation with a solution of the copper complexes under study (15 μM in Tris-HCl buffer) for 1 h at room temperature. The temperature was increased by 1 °C/min between 55 and 70 °C and by 3 °C/min between 40 and 55 °C and between 70 and 95 °C.25 Viscosity measurements were carried out using an Oswald viscometer, immersed in a water bath at 25 °C. The CT-DNA concentration was kept constant in all samples, but the copper complex concentration was increased from 0 to 120 μM. The flow time was measured with a digital stopwatch. The sample flow times were measured at least six times and the mean value was used. Data are 3790


Article

Inorganic Chemistry Park Memorial Institute medium (RPMI 1640 for MCF-7 and DU145) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified 5% CO2 atmosphere. To evaluate the growth inhibition, cells were seeded in 96-well plates (Corning Costar) at a concentration of 1.5 × 104 cells/well and grown for 24 h in complete medium. Solutions of the compounds were prepared by diluting a freshly prepared stock solution (20 mM in DMSO) and 0.75 μL of each complex sample was added to 150 μL of medium. The percentage of DMSO in the culture medium never exceeded 0.5%: at this concentration, DMSO has no effect on the cell viability. Cells were exposed to the complex for a 48 h period. Following 48 h drug exposure, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) was added 30 μL (1 mg/mL) to the cells and incubated for 3−4 h, then the culture medium was removed and the violet formazan dissolved in isopropanol. The optical density of each well (96-well plates) was quantified at 540 nm using a multiwell plate reader and the percentage of surviving cells was calculated from the ratio of absorbance between treated and untreated cells. The IC50 value was calculated as the concentration reducing the proliferation of the cells by 50% and is presented as a mean (±SE) of at least three independent experiments. For the morphological study, A549 lung tumor cells were seeded at a density of 0.3 × 105 cells/well into 12-well plates. After allowing 48 h to adhere, images of cells treated with or without complexes 1−5 were taken at 0, 24, and 48 h.

■

assistance at IVIC and INMETRO. This work was partially supported by CNPq, FAPERJ and CAPES in Brazil.

■

(1) World Health Organization. http://www.who.int/cancer/en/. (2) Farrell, N. P. Multi-platinum anti-cancer agents. Substitutioninert compounds for tumor selectivity and new targets. Chem. Soc. Rev. 2015, 44, 8773−8785. (3) Medici, S.; Peana, M.; Nurchi, V. M.; Lachowicz, J. I.; Crisponi, G.; Zoroddu, M. A. Noble metals in medicine: Latest advances. Coord. Chem. Rev. 2015, 284, 329−350. (4) Marzano, C.; Pellei, M.; Tisato, F.; Santini, C. Copper complexes as anticancer agents. Anti-Cancer Agents Med. Chem. 2009, 9, 185−211. (5) Iakovidis, I.; Delimaris, I.; Piperakis, S. M. Copper and Its Complexes in Medicine: A Biochemical Approach. Mol. Biol. Int. 2011, 2011, 594529. (6) Santini, C.; Pellei, M.; Gandin, V.; Porchia, M.; Tisato, F.; Marzano, C. Advances in Copper Complexes as Anticancer Agents. Chem. Rev. 2014, 114, 815−862. (7) Ruiz- Azuara, L.; Bravo, M. E. Copper Compounds in Cancer Chemotherapy. Curr. Med. Chem. 2010, 17, 3606−3615. (8) Rivero-Müller, A.; De Vizcaya-Ruiz, A.; Plant, N.; Ruiz, L.; Dobrota, M. Mixed chelate copper complex, Casiopeina IIgly, binds and degrades nucleic acids: A mechanism of cytotoxicity. Chem.-Biol. Interact. 2007, 165, 189−199. (9) Křikavová, R.; Vančo, J.; Trávníček, Z.; Buchtík, R.; Dvořaḱ , Z. Copper(II) quinolinonato-7-carboxamido complexes as potent antitumor agents with broad spectra and selective effects. RSC Adv. 2016, 6, 3899−3909. (10) Rajendiran, V.; Karthik, R.; Palaniandavar, M.; Stoeckli-Evans, H.; Periasamy, V. S.; Akbarsha, M. A.; Srinag, B. S.; Krishnamurthy, H. Mixed-Ligand Copper(II)-phenolate Complexes: Effect of Coligand on Enhanced DNA and Protein Binding, DNA Cleavage, and Anticancer Activity. Inorg. Chem. 2007, 46, 8208−8221. (11) Nagababu, P.; Barui, A. K.; Thulasiram, B.; Devi, C. S.; Satyanarayana, S.; Patra, C. R.; Sreedhar, B. Antiangiogenic Activity of Mononuclear Copper(II) Polypyridyl Complexes for the Treatment of Cancers. J. Med. Chem. 2015, 58, 5226−5241. (12) Navarro, M.; Cisneros-Fajardo, E.; Fernandez-Mestre, M.; Arriechi, D.; Marchan, E. Synthesis, characterization, DNA binding study and biological activity against Leishmania mexicana of [Cu(dppz)2]BF4. J. Inorg. Biochem. 2003, 97, 364−369. (13) Berners-Price, S. J.; Johnson, R. K.; Mirabelli, C. K.; Faucette, L. F.; McCabe, F. L.; Sadler, P. J. Copper(I) complexes with bidentate tertiary phosphine ligands: solution chemistry and antitumor activity. Inorg. Chem. 1987, 26, 3383−3387. (14) Bowen, R. J.; Navarro, M.; Shearwood, A. J.; Healy, P. C.; Skelton, B. W.; Filipovska, A.; Berners-Price, S. J. 1:2 Adducts of copper(I) halides with 1,2-bis(di-2-pyridylphosphino)ethane: solid state and solution structural studies and antitumour activity. Dalton Trans. 2009, 10861−10870. (15) Jardine, F. H.; Vohra, A. G.; Young, F. J. Copper(I) nitrato and nitrate complexes. J. Inorg. Nucl. Chem. 1971, 33, 2941−2945. (16) Geary, W. J. The use of conductivity measurements in organic solvents for the characterisation of coordination compounds. Coord. Chem. Rev. 1971, 7, 81−122. (17) Zhang, L.; Yue, S.; Li, B.; Fan, D. A series of [Cu(N−N)(P− P)]BF4 complexes: Luminescence quenching caused by electronconfiguration transformation in excited state. Inorg. Chim. Acta 2012, 384, 225−232. (18) Safin, D. A.; Mitoraj, M. P.; Robeyns, K.; Filinchuk, Y.; Vande Velde, C. M. L. Luminescent mononuclear mixed ligand complexes of copper(I) with 5-phenyl-2,2′-bipyridine and triphenylphosphine. Dalton Trans. 2015, 44, 16824−16832. (19) Makal, A.; Benedict, J.; Trzop, E.; Sokolow, J.; Fournier, B.; Chen, Y.; Kalinowski, J. A.; Graber, T.; Henning, R.; Coppens, P. Restricted Photochemistry in the Molecular Solid State: Structural Changes on Photoexcitation of Cu(I) Phenanthroline Metal-to-Ligand

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.6b02419. CCDC 1488188 contains the supplementary crystallographic data for complex 3. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. Table S1 and Figures S1−S9, including the NMR and spectrophotometric titration spectra for complexes 1−5 and X-ray crystallographic data for complex 3 (PDF) X-ray crystallographic data for complex 3 (CIF)

■

REFERENCES

AUTHOR INFORMATION

Corresponding Author

* E-mail: [email protected] or [email protected]. br. Phone: +55-32-21023310. ORCID

Wilmer Villarreal: 0000-0002-7781-4083 Present Address

́ M.N.: Departamento de Quimica, Instituto de Ciências Exatas, Universidade Federal de Juiz de Fora, CEP 36036900, Juiz de Fora-MG, Brazil ⊗

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This article is dedicated to the memory of Prof. Dr. Roberto Sánchez Delgado, a pioneer in the search of a transition metalbased chemotherapy against parasitic diseases such as malaria, Chagas diseases, and leishmaniasis. He was my mentor and our excellent and beloved friend, who taught some of us that we must always strive for excellence in our research and teaching. The authors are grateful to Ibis Colmenares, Sorenlis González, Nieves Alvarez, Darlin Duarte and Alberto Fuentes for the technical assistance in the preliminary studies at IVIC. We would also like to thank MS and the elemental analysis team 3791


Article

Inorganic Chemistry Charge Transfer (MLCT) Complexes by Time-Resolved Diffraction. J. Phys. Chem. A 2012, 116, 3359−3365. (20) Navarro, M.; Corona, O. A.; González, T. (2,2′-Biquinolineκ2N,N′)bis(triphenyl phosphine-κP)copper(I) nitrate 2,2′-biquinoline hemisolvate hemihydrate. Acta Crystallogr., Sect. E: Struct. Rep. Online 2007, 63 (4), m1153−m1155. (21) Navarro, M.; Corona, O. A.; González, T.; Capparelli, M. V. (2,2′-Bipyridine) bis(triphenylphosphine)copper(I) nitrate chloroform solvate hemihydrate. Acta Crystallogr., Sect. E: Struct. Rep. Online 2008, 64, m533−m534. (22) Navarro, M.; Cisneros-Fajardo, E. J.; Sierralta, A.; FernándezMestre, M.; Silva, P.; Arrieche, D.; Marchán, E. Design of copper DNA intercalators with leishmanicidal activity. J. Biol. Inorg. Chem. 2003, 8, 401−408. (23) Gupta, T.; Dhar, S.; Nethaji, M.; Chakravarty, A. R. Bis(dipyridophenazine)copper(II) complex as major groove directing synthetic hydrolase. Dalton Trans. 2004, 1896−1900. (24) Zou, X.-H.; Ye, B.-H.; Li, H.; Zhang, Q.-L.; Chao, H.; Liu, J.-G.; Ji, L.-N.; Li, X.-Y. The design of new molecular “light switches” for DNA. JBIC, J. Biol. Inorg. Chem. 2001, 6, 143−150. (25) Haq, I.; Lincoln, P.; Suh, D.; Norden, B.; Chowdhry, B. Z.; Chaires, J. B. Interaction of Δ- and Λ-[Ru(phen)2DPPZ]2+ with DNA: A Calorimetric and Equilibrium Binding Study. J. Am. Chem. Soc. 1995, 117, 4788−4796. (26) Navarro, M.; Hernández, C.; Colmenares, I.; Hernández, P.; Fernández, M.; Sierraalta, A.; Marchán, E. Synthesis and characterization of [Au(dppz)2]Cl3. DNA interaction studies and biological activity against Leishmania (L) mexicana. J. Inorg. Biochem. 2007, 101, 111−116. (27) Schäfer, S.; Sheldrick, W. S. Coligand tuning of the DNA binding properties of half-sandwich organometallic intercalators: Influence of polypyridyl (pp) and monodentate ligands (L = Cl, (NH2)2CS, (NMe2)2CS) on the intercalation of (η5-pentamethylcyclopentadienyl)-iridium(III)- dipyridoquinoxaline and -dipyridophenazine complexes. J. Organomet. Chem. 2007, 692, 1300−1309. (28) Jia, L.; Xu, J.; Zhao, X.; Shen, S.; Zhou, T.; Xu, Z.; Zhu, T.; Chen, R.; Ma, T.; Xie, J.; Dong, K.; Huang, J. Synthesis, characterization, and antitumor activity of three ternary dinuclear copper (II) complexes with a reduced Schiff base ligand and diimine coligands in vitro and in vivo. J. Inorg. Biochem. 2016, 159, 107−119. (29) Satyanarayana, S.; Dabrowiak, J. C.; Chaires, J. B. Neither Δnor Λ-tris(phenanthroline)ruthenium(II) binds to DNA by classical intercalation. Biochemistry 1992, 31, 9319−9324. (30) David-Cordonnier, M. H.; Laine, W.; Lansiaux, A.; Rosu, F.; Colson, P.; de Pauw, E.; Michel, S.; Tillequin, F.; Koch, M.; Hickman, J. A.; Pierré, A.; Bailly, C. Covalent binding of antitumor benzoacronycines to double-stranded DNA induces helix opening and the formation of single-stranded DNA: unique consequences of a novel DNA-bonding mechanism. Mol. Cancer Ther. 2005, 4, 71−80. (31) Novakova, O.; Chen, H.; Vrana, O.; Rodger, A.; Sadler, P. J.; Brabec, V. DNA Interactions of Monofunctional Organometallic Ruthenium(II) Antitumor Complexes in Cell-free Media. Biochemistry 2003, 42, 11544−11554. (32) Xiong, Y.; Ji, L. N. Synthesis, DNA-binding and DNA-mediated luminescence quenching of Ru(II) polypyridine complexes. Coord. Chem. Rev. 1999, 185, 711−733. (33) Nordén, B.; Tjerneld, F. Structure of methylene blue−DNA complexes studied by linear and circular dichroism spectroscopy. Biopolymers 1982, 21, 1713−1734. (34) Rajendiran, V.; Murali, M.; Suresh, E.; Palaniandavar, M.; Periasamy, V. S.; Akbarsha, M. A. Non-covalent DNA binding and cytotoxicity of certain mixed-ligand ruthenium(II) complexes of 2,2′dipyridylamine and diimines. Dalton Trans. 2008, 2157−2170. (35) Gençaslan, S.; Sheldrick, W. Bifunctional Bioorganometallic Iridium(III)−Platinum(II) Complexes Incorporating Both Intercalative and Covalent DNA Binding Capabilities. Eur. J. Inorg. Chem. 2005, 2005, 3840−3849. (36) Cutts, S. M.; Masta, A.; Panousis, C.; Parsons, P. G.; Sturm, R. A.; Phillips, D. R. A Gel Mobility Shift Assay for Probing the Effect of

Drug−DNA Adducts on DNA-Binding Proteins. Methods Mol. Biol. 1997, 90, 95−106. (37) Ghosh, A.; Mandoli, A.; Kumar, D. K.; Yadav, N. S.; Ghosh, T.; Jha, B.; Thomas, J. A.; Das, A. DNA binding and cleavage properties of a newly synthesised Ru(II)-polypyridyl complex. Dalton Trans. 2009, 9312−9321. (38) Navarro, M.; Castro, W.; Higuera-Padilla, A. R.; Sierraalta, A.; Abad, M. J.; Taylor, P.; Sánchez-Delgado, R. A. Synthesis, characterization and biological activity of trans-platinum(II) complexes with chloroquine. J. Inorg. Biochem. 2011, 105, 1684−1691. (39) Carreira, M.; Calvo-Sanjuán, R.; Sanaú, M.; Zhao, X.; Magliozzo, R. S.; Marzo, I.; Contel, M. Cytotoxic hydrophilic iminophosphorane coordination compounds of d8 metals. Studies of their interactions with DNA and HSA. J. Inorg. Biochem. 2012, 116, 204−214. (40) Tse, W. C.; Boger, D. L. A Fluorescent Intercalator Displacement Assay for Establishing DNA Binding Selectivity and Affinity. Acc. Chem. Res. 2004, 37, 61−69. (41) Thompson, K. H.; Orvig, C. Metal complexes in medicinal chemistry: new vistas and challenges in drug design. Dalton Trans. 2006, 761−764. (42) Sarkar, B. Metal-protein interactions in transport, accumulation, and excretion of metals. Biol. Trace Elem. Res. 1989, 21, 137−144. (43) Zhao, X.; Liu, R.; Chi, Z.; Teng, Y.; Qin, P. New Insights into the Behavior of Bovine Serum Albumin Adsorbed onto Carbon Nanotubes: Comprehensive Spectroscopic Studies. J. Phys. Chem. B 2010, 114, 5625−5631. (44) Tsitsa, I.; Tarushi, A.; Doukoume, P.; Perdih, F.; de Almeida, A.; Papadopoulos, A.; Kalogiannis, S.; Casini, A.; Turel, I.; Psomas, G. Structure and biological activities of metal complexes of flumequine. RSC Adv. 2016, 6, 19555−19570. (45) Liu, Y.; Li, A.; Shao, J.; Xie, C.; Song, X.; Bao, W.; Xu, J. Four Cu(II) complexes based on antitumor chelators: synthesis, structure, DNA binding/damage, HSA interaction and enhanced cytotoxicity. Dalton Trans. 2016, 45, 8036−8049. (46) Ross, P.; Subramanian, S. Thermodynamics of protein association reactions: forces contributing to stability. Biochemistry 1981, 20, 3096−3102. (47) Colina-Vegas, L.; Villarreal, W.; Navarro, M.; de Oliveira, C. R.; Graminha, A. E.; Maia, P. I.; Deflon, V. M.; Ferreira, A. G.; Cominetti, M. R.; Batista, A. A. Cytotoxicity of Ru(II) piano−stool complexes with chloroquine and chelating ligands against breast and lung tumor cells: Interactions with DNA and BSA. J. Inorg. Biochem. 2015, 153, 150−161. (48) Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods 1983, 65, 55−63. (49) Marzano, C.; Pellei, M.; Colavito, D.; Alidori, S.; Lobbia, G. G.; Gandin, V.; Tisato, F.; Santini, C. Synthesis, Characterization, and in Vitro Antitumor Properties of Tris(hydroxymethyl)phosphine Copper(I) Complexes Containing the New Bis(1,2,4-triazol-1-yl)acetate Ligand. J. Med. Chem. 2006, 49, 7317−7324. (50) Gandin, V.; Porchia, M.; Tisato, F.; Zanella, A.; Severin, E.; Dolmella, A.; Marzano, C. Novel Mixed-Ligand Copper(I) Complexes: Role of Diimine Ligands on Cytotoxicity and Genotoxicity. J. Med. Chem. 2013, 56, 7416−7430. (51) Gandin, V.; Tisato, F.; Dolmella, A.; Pellei, M.; Santini, C.; Giorgetti, M.; Marzano, C.; Porchia, M. In Vitro and in Vivo Anticancer Activity of Copper(I) Complexes with Homoscorpionate Tridentate Tris(pyrazolyl)borate and Auxiliary Monodentade Phosphine Ligands. J. Med. Chem. 2014, 57, 4745−4760. (52) Ramakrishnan, S.; Rajendiran, V.; Palaniandavar, M.; Periasamy, V. S.; Srinag, B. S.; Krishnamurthy, H.; Akbarsha, M. A. Induction of Cell Death by Ternary Copper(II) Complexes of L-Tyrosine and Diimines: Role of Coligands on DNA Binding and Cleavage and Anticancer Activity. Inorg. Chem. 2009, 48, 1309−1322. (53) Cotton, F. A .; G oodgame, D. I. L. T etra kis(triphenylphosphine)- silver(I) and -copper(I) Complexes. J. Chem. Soc. 1960, 5267−5269. 3792


Article

Inorganic Chemistry (54) Calderazzo, F.; Marchetti, F.; Pampaloni, G.; Passarelli, V. Coordination properties of 1,10-phenanthroline-5,6-dione towards group 4 and 5 metals in low and high oxidation states. J. Chem. Soc., Dalton Trans. 1999, 24, 4389−4396. (55) Bodige, S.; MacDonnell, F. Synthesis of free and ruthenium coordinated 5,6-diamino-1,10-phenanthroline. Tetrahedron Lett. 1997, 38, 8159−8160. (56) Collins, J. G.; Sleeman, A. D.; Aldrich-Wright, J. R.; Greguric, I.; Hambley, T. W. A 1H NMR Study of the DNA Binding of Ruthenium(II) Polypyridyl Complexes. Inorg. Chem. 1998, 37, 3133−3141. (57) Dickeson, J. E.; Summers, L. A. Derivatives of 1,10Phenanthroline-5,6-quinone. Aust. J. Chem. 1970, 23, 1023−1027. (58) Ossipov, D.; Zamaratski, E.; Chattopadhyaya, J. Dipyrido[3,2a:2′,3′-c]phenazine-Tethered Oligo-DNA: Synthesis and Thermal Stability of Their DNA·DNA and DNA·RNA Duplexes and DNA· DNA·DNA Triplexes. Helv. Chim. Acta 1999, 82, 2186−2200. (59) Enraf-Nonius COLLECT; Nonius BV: Delft, The Netherlands, 1997. (60) Otwinowski, Z.; Minor, W. In Methods in Enzymology, Vol. 276; Carter, C. W., Jr., Sweet, R. M., Eds.; Academic Press: New York, 1997; p 307. (61) Coppens, P.; Leiserowitz, L.; Rabinovich, D. Calculation of absorption corrections for camera and diffractometer data. Acta Crystallogr. 1965, 18, 1035−1038. (62) Sheldrick, G. M. A short history of SHELX. Acta Crystallogr., Sect. A: Found. Crystallogr. 2008, 64, 112−122. (63) Farrugia, L. J. WinGX suite for small-molecule single-crystal crystallography. J. Appl. Crystallogr. 1999, 32, 837−838. (64) Macrae, C. F.; Edgington, P. R.; McCabe, P.; Pidcock, E.; Shields, G. P.; Taylor, R.; Towler, M.; van de Streek, J. Mercury: visualization and analysis of crystal structures. J. Appl. Crystallogr. 2006, 39, 453−457. (65) Farrugia, L. J. ORTEP-3 for Windows - a version of ORTEP-III with a Graphical User Interface (GUI). J. Appl. Crystallogr. 1997, 30, 565−565. (66) Hay, P. J.; Wadt, W. R. Ab initio effective core potentials for molecular calculations. Potentials for K to Au including the outermost core orbitals. J. Chem. Phys. 1985, 82, 299−310. (67) Marmur, J. A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J. Mol. Biol. 1961, 3, 208−218. (68) Reichmann, M. E.; Rice, S. A.; Thomas, C. A.; Doty, P. A Further Examination of the Molecular Weight and Size of Desoxypentose Nucleic Acid. J. Am. Chem. Soc. 1954, 76, 3047−3053. (69) Baka, E.; Comer, J. E.A.; Takács-Novák, K. Study of equilibrium solubility measurement by saturation shake-flask method using hydrochlorothiazide as model compound. J. Pharm. Biomed. Anal. 2008, 46, 335−341.

3793


Copper(I)âPhosphine Polypyridyl Complexes: Synthesis

Recommend Documents

Copper(I)âPhosphine Polypyridyl Complexes: Synthesis