Binucleating Hydrazonic Ligands and Their μ-Hydroxodicopper(II) Complexes as Promising Structural Motifs for Enhanced Antitumor Activity

Article

Cite This: Inorg. Chem. XXXX, XXX, XXX −XXX

pubs.acs.org/IC

Binucleating Hydrazonic Ligands and Their μ‑Hydroxodicopper(II)

Complexes as Promising Structural Motifs for Enhanced Antitumor

Activity

†

‡

§

Jesica Paola Rada, Beatriz S. M. Bastos, Luciano Anselmino, Chris H. J. Franco,

Mauricio Lanznaster, Renata Diniz, Claudio O. Fernandez, Mauricio Menacho-Mar

́ ́

quez,^‡

∥

⊥

‡

Ana Maria Percebom,^†and Nicolas

A. Rey*

,†

́

†

‡

Pontiﬁcal Catholic University of Rio de Janeiro, Rio de Janeiro, 22451-900, Brazil

Max Planck Laboratory for Structural Biology, Chemistry and Molecular Biophysics of Rosario (MPLbioR, UNR-MPIbpC) and

macos de Rosario (IIDEFAR, UNR-CONICET), Universidad Nacional

Instituto de Investigaciones para el Descubrimiento de Far

de Rosario, S2002LRK Rosario, Argentina

́

§

Federal University of Juiz de Fora, Juiz de Fora, 36036-900, Brazil

∥

Fluminense Federal University, Niteroi

́

, 24020-150, Brazil

Federal University of Minas Gerais, Belo Horizonte, 31270-901, Brazil

S Supporting Information

⊥

*

ABSTRACT: Very few inorganic antineoplastic drugs have

entered the clinic in the last decades, mainly because of

toxicity issues. Because copper is an essential trace element of

ubiquitous occurrence, decreased side eﬀects could be

expected in comparison with the widely used platinum

anticancer compounds. In the present work, two novel

hydrazonic binucleating ligands and their μ-hydroxo dicopper-

(

II) complexes were prepared and fully characterized. They

diﬀer by the nature of the aromatic group present in their

aroylhydrazone moieties: while H L1 and its complex, 1,

3

possess a thiophene ring, H L2 and 2 contain the more polar furan heterocycle. X-ray diﬀraction indicates that both

3

coordination compounds are very similar in structural terms and generate dimeric arrangements in the solid state. Positive-ion

electrospray ionization mass spectrometry analyses conﬁrmed that the main species present in a 10% dimethyl sulfoxide

+

(

DMSO)/water solution should be [Cu (HL)(OH)] and the DMSO-substituted derivative [Cu (L)(DMSO)] . Scattering

2

techniques [dynamic light scattering (DLS) and small-angle X-ray scattering] suggest that the complexes and their free ligands

interact with bovine serum albumin (BSA) in a reversible manner. The binding constants to BSA were determined for the

complexes through ﬂuorescence spectroscopy. Moreover, to gain insight into the mechanism of action of the compounds, calf

thymus DNA binding studies by UV−visible and DLS measurements using plasmid pBR322 DNA were also performed. For the

complexes, DLS data seem to point to the occurrence of DNA cleavage to Form III (linear). Both ligands and their dicopper(II)

complexes display potent antiproliferative activity in a panel of four cancer cell lines, occasionally even in the submicromolar

range, with the complexes being more potent than the free ligands. Our data on cellular models correlate quite well with the

DNA interaction experiments. The results presented herein show that aroylhydrazone-derived binucleating ligands, as well as

their dinuclear μ-hydroxodicopper(II) complexes, may represent a promising structural starting point for the development of a

new generation of highly active potential antitumor agents.

2

INTRODUCTION

carboplatin, and oxaliplatin. Moreover, because of the

fundamental diﬀerences between the chemistry of copper

complexes and those of platinum, one can also expect diverse

mechanisms of action. In a very recent Perspective published

■

Since the fortuitous discovery of the antitumor activity of

cisplatin by Rosenberg et al. in the second half of the 1960s,

1

the search for new metal-based anticancer agents has increased

dramatically. In spite of this, very few new inorganic

antineoplastic drugs have entered the clinic in the last decades,

mainly because of toxicity issues. In this context and because

copper is an essential trace element of ubiquitous occurrence,

decreased side eﬀects could be expected in comparison with

the widely used platinum anticancer compounds cisplatin,

3

by Wehbe and co-workers, the potentiality of copper

complexes as a novel class of therapeutics for an extensive

range of diseases, including cancer, is discussed. The paper

Received: April 24, 2019

©

XXXX American Chemical Society

A

DOI: 10.1021/acs.inorgchem.9b01195

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

Article

4

,5

41

comments on Casiopeinas, a series of mononuclear mixed-

chelate copper-based antineoplastic compounds that have

entered phase I clinical trials in Mexico. In such a highly active

ﬁeld of research, a large variety of ligands have been used to

synthesize copper complexes with potential antiproliferative

nanomolar range, when tested in HeLa and Caco-2, as well

42 48

as in U937 and MCF7, cells. Some of these works have

reported the induction of apoptosis as a putative mechanism of

43,45

action.

However, regardless of the large number of promising

reports on the antiproliferative eﬀects of copper(II) complexes,

their poor aqueous solubility, as well as an in vivo decreased

activity related to their extensive and nonselective binding to

biomolecules (a process known as “speciation”), creates an

additional barrier for the compounds’ translation into clinical

trials. In order to overcome these diﬃculties, recent studies

have shown the beneﬁt of encapsulating copper(II) complexes

6

action. Santini and co-workers published a comprehensive

3

review about coordination compounds of this metal reported

between 2008 and 2012 as having antitumor activity. Even

more recently, cytotoxic copper complexes comprising a

diversity of ligands have been described, among them

hydrazides and N,N-heterocycles, derivatives of coumarin,

aroylhydrazones, thiosemicarbazones, β-diketones and N-

49

7

8

9

10

1

12

13

3,49

heterocycles, phenanthroline, tripodal pyrazolylamines,

in nanoliposomes, with encouraging results.

1

4

15,16

17

nalidixic acid, benzimidazoles, ₁ﬂuoroquinolones, and a

On the other hand, aroylhydrazones constitute a diverse

family of bidentate nitrogen-and oxygen-donor atoms with a

vast spectrum of biological activities. Our hypothesis is that

8

1

,2,4-triazole-derived Schiﬀ base. In some cases, their ability

7,14,16,17

50

to induce apoptotic cell death was demonstrated.

The

capacity to interact with DNA and the DNA-cleaving

properties of copper compounds have also been con-

modifying previously known compartmental ligands, such as 3-

[(2-hydroxybenzyl)(2-pyridylmethyl)amine]-2-hydroxy-5-

1

4,15,19−23

22

51,52

ﬁrmed.

For example, Patil et al. synthesized a

methylbenzaldehyde (HBPAMFF),

through the addition

series of cobalt, nickel, and copper complexes using the same

organic ligands and showed that the copper(II) derivatives

possess a better cleavage activity than the corresponding cobalt

and nickel compounds.

of an aroylhydrazone moiety represents a simple way to

develop a set of versatile unsymmetrical binucleating ligands.

These ligands allow for the synthesis of interesting novel

dicopper(II) compounds displaying diﬀerent geometries and

even distinct coordination numbers at each cupric center. In

During the past decade, the use of dicopper(II) compounds

has constituted an interesting tendency in this area because

bimetallic copper cores are abundant in nature. A typical

example is that of catechol oxidases (COs), a group of

ubiquitous type 3 dicopper enzymes. The crystal structure of

the sweet-potato CO was already described and possesses, in

53

fact, in a current publication by the research group of Belle,

two new unsymmetrical ligands derived from bis(2-

pyridylmethyl)aminomethyl-2-hydroxy-5-methyl benzaldehyde

and 4-methyl or 4,4-dimethyl 3-thiosemicarbazide, as well as

their dinuclear and tetranuclear phenoxo-bridged copper(II)

complexes, were described and extensively studied by

structural, spectroscopic, electrochemical, and electrospray

ionization mass spectrometry (ESI-MS) methods.

In this stimulating scenario and motivated by the search of

novel structural motifs for the development of potential

antiproliferative agents, the present work describes the ﬁrst two

hydroxo-bridged dicopper(II) complexes containing unsym-

metrical hydrazonic binucleating ligands and evaluates them as

candidates for prospective antitumor drugs.

II

its resting Cu Cu state, two cupric ions bridged by an

24

exogenous hydroxo ligand. This result triggered scientiﬁc

interest in the synthesis of biomimetic models for the active

2

5,26

site of COs.

Some years ago, we demonstrated that, in

addition to the predictable catecholase-like activity, these CO-

inspired complexes display phosphate diester hydrolysis

27

activity, which makes them promiscuous catalysts. The

catalytic mechanism of hydrolysis probably involves terminal

coordination of the phosphate diester to one of the cupric ions

and subsequent intramolecular nucleophilic attack by the

28,29

bridging hydroxide.

The phosphatase-like activity was

EXPERIMENTAL SECTION

■

(

proven in plasmid DNA (as well the nuclease activity),

suggesting that this class of compounds could also perform

properly as potential antineoplastic agents. This was, in fact,

conﬁrmed toward the GLC4 (small-cell lung carcinoma) and

Chemicals. Acetone (DMK), acetonitrile (ACN), diethyl ether

Et O), dimethyl sulfoxide (DMSO), dichloromethane (DCM),

2

ethanol (EtOH), methanol (MeOH), isopropyl alcohol (iPrOH),

triethylamine (Et N), sodium tetrahydroborate (NaBH ), hydro-

3

4

30

K562 (myelogenous leukemia) cell lines. In the past few

years, several other dicopper(II) complexes have been

synthesized and studied regarding their anticancer properties.

chloric acid (HCl), chloroform, anhydrous sodium sulfate, sodium

chloride (NaCl), and potassium hydroxide (KOH) were obtained

from Vetec (Sigma-Aldrich); copper(II) perchlorate hexahydrate

3

1−43

[

Cu(ClO ) ·6H O] and sodium bicarbonate (NaHCO ) were

Some authors demonstrated the DNA binding

DNA cleavage

and/or

4

2

3

3,36,42,44,45

purchased from Sigma-Aldrich; tris(hydroxymethyl)aminomethane

Tris) was acquired from BIO-RAD; furan-2-carbohydrazide (FCH)

abilities of these compounds. Very

(

recently, Cao et al. proved that alteration of the chelating arms

on the ligands allows for regulation of the reactivity of their

and thiophene-2-carbohydrazide (TCH) were purchased from Acros

Organics and Sigma-Aldrich; bovine serum albumin (BSA) was

purchased from Fluka. Plasmid DNA pBR322 was from Thermo

Fisher and calf thymus DNA (ctDNA) sodium salt from Sigma-

Aldrich. All of these chemicals were used without any type of

treatment or further puriﬁcation.

46

dinuclear copper(II) complexes toward DNA. The in vitro

antiproliferative activity of novel dicopper(II) compounds has

also been tested. As an example, the complexes synthesized by

Godlewska and co-workers showed a moderate antineoplastic

47

activity in U937 cells. However, in most cases, those

compounds presented a better antiproliferative proﬁle than

cisplatin itself. In this sense, the authors reported new

Syntheses of Ligands and Their Dicopper(II) Complexes.

Precursors. The synthesis of the pendant arm (2-hydroxybenzyl)(2-

pyridylmethyl)amine (HBPA) from 2-(aminomethyl)pyridine and 2-

5

4,55

hydroxybenzaldehyde has been previously reported.

The con-

complexes with IC₅₃₀ values in the micromolar range for

ditions of the reaction were slightly modiﬁed or optimized in the

present study: 2-(aminomethyl)pyridine (7.9 g, 73 mmol) and 2-

hydroxybenzaldehyde (7.7 mL, 73 mmol) were stirred in 50 mL of

MeOH for 1 h, with the subsequent gentle, portionwise addition of

5−39

HepG2 cancer cells.

Additionally, remarkable activities

35 39

have been described in the HeLa and A549, SMMC-7721,

and MGC-803 and BEL-7404 cell lines. In other studies, the

compounds have shown even lower IC₅₀values, in the

43

solid NaBH (2.8 g, 73 mmol) at 0 °C. The mixture was stirred

4

B

DOI: 10.1021/acs.inorgchem.9b01195

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

Article

Scheme 1. Synthesis of the Chemical Precursors HBPA and HBPAMFF, the Binucleating Ligands H L1 and H L2, and Their

3

Respective μ-Hydroxodicopper(II) Complexes 1 and 2

overnight, and then the pH was adjusted to 5−6 with a 4 mol L⁻¹HCl

aqueous solution. The solvent was removed under reduced pressure

on a rotary evaporator at 45 °C and the product redissolved in 60 mL

of chloroform and then washed with 5 × 40 mL portions of a

to 40 mL of a CMFF (2.0 g, 10.4 mmol) solution in DCM. The

mixture was stirred for 3 h at room temperature and then washed and

extracted, as described above for HBPA. The product was allowed to

stand in iPrOH at −18 °C (freezer conditions) for 12 h, and a white

crystalline solid was separated.

saturated NaHCO solution and once with water, waiting 15 min

3

between the separations. The product was extracted with chloroform

Mp = 103−104 °C. Yield = 2.9 g (8 mmol, 77%).

(3 × 30 mL) and the organic phase dried over anhydrous sodium

Ligands. H L1. This ligand was prepared by dropwise adding a

3

sulfate. Chloroform was evaporated and the product allowed to stand

methanolic solution (10 mL) of TCH (586.3 mg, 4 mmol) to 30 mL

of a slightly heated solution containing the precursor HBPAMFF

in Et O in a freezer for 3 days. A colorless crystalline solid was

2

separated.

(1.45 g, 4 mmol) in MeOH/Et O (1:1). The mixture was left under

2

Mp = 63 °C. Yield = 9.0 g (42 mmol, 58%).

reﬂux for 2 h, and after 30 min of cooling, the product was ﬁltered oﬀ

51

Precursor HBPAMFF was synthesized as described previously

from HBPA and (2-chloromethyl-4-methyl-6-formyl)phenol

and washed with ice-cold Et O.

2

Mp = 187 °C. Yield = 1.8 g (3.7 mmol, 92%). Recrystallization in

MeOH aﬀorded light-yellow crystals with hexagonal geometry.

56

(

CMFF), according to the following modiﬁed conditions: HBPA

(

2.3 g, 10.4 mmol) and Et N (1.5 mL, 10.4 mmol) were both

Optical photographs of H L1 crystals can be found in Figure S1.

3

dissolved together in 40 mL of DCM and then slowly added at 0 °C

The single-crystal size was very small for X-ray diﬀraction (XRD)

C

DOI: 10.1021/acs.inorgchem.9b01195

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Inorganic Chemistry

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−

1

analysis. Elem anal. Calcd for C H O N S (486.63 g mol ): C,

6

Raman spectra were collected in a dark room with an exposure time of

2 s and 20 accumulations. An objective lens with 10× magniﬁcation

was used to acquire the optical photographs of the compounds. The

spectra were processed by using the LabSpec 6 software, provided by

the manufacturer.

2

7

26

3

4

6.6; H, 5.4; N, 11.5; S, 6.6. Found: C, 66.1; H, 5.5; N, 11.8; S, 6.7.

H L2. This ligand was prepared from FCH (514.7 mg, 4 mmol) in

mL of MeOH and HBPAMFF (1.45 g, 4 mmol) following the same

3

5

procedure as that used to synthesize H L1.

3

Mp = 185 °C. Yield = 1.6 g (3.4 mmol, 85%). Light-yellow crystals

X-ray Crystallography. Single-crystal data of complexes 1 and 2

were collected using a Rigaku Agilent SuperNova diﬀractometer with

Mo Kα radiation (λ = 0.71073 Å) at room temperature (293−294 K).

Data collection, cell reﬁnement, and data reduction were performed

with hexagonal geometry were obtained by recrystallization in MeOH

(

Figure S1). Single crystals were analyzed by XRD, but peaks

presented low intensity. Elem anal. Calcd for C H O N (470.57 g

2

7

26

4

−

1

58

mol ): C, 68.9; H, 5.6; N, 11.9. Found: C, 68.4; H, 5.6; N, 12.3.

using the CrysAlisPro software. The structures were solved and

59

Copper Complexes. [Cu (μ-OH)(C H N O S)]ClO ·2H O (1).

reﬁned using the SHELXS and SHELXL packages. The method

2

27 24

4

3

4

2

60

Cu(ClO ) ·6H O (378.1 mg, 1.0 mmol) was dissolved in 3 mL of

described by Larson was used to reﬁne an empirical isotropic

extinction parameter, x, by applying a multiscan absorption

4

2

MeOH and dropwise added to a solution of the H L1 ligand (243.32

3

61

mg, 0.5 mmol) in 35 mL of a MeOH/ACN (6:1) mixture. The

reactants were heated for 40 min, and then 1 mL of a methanolic

KOH solution (1 M, 1 mmol) was added. Heating and stirring were

maintained for another 20 min. After 12 h, the green solid was ﬁltered

oﬀ and washed with ice-cold MeOH. The dry solid was recrystallized

in EtOH, aﬀording dark-green crystals (Figure S2 ) suitable for XRD

analysis.

correction. The structures of the compounds were drawn with the

62

Mercury program. All atoms were reﬁned with anisotropic

parameters, except hydrogen atoms, which were located from Fourier

diﬀerence maps, set in calculated positions and reﬁned as riding

atoms. For both compounds, disordered solvent molecules were

63

observed in the crystal lattice. Because of this fact, the SQUEEZE

methodology was applied to obtain a better description of the

complexes. In this method, the electronic density of the solvent was

removed from the crystal data, which allowed for a description of the

compound based on an improved model. Solvent-accessible volumes

Yield = 220 mg (0.29 mmol, 58%). Elem anal. Calcd for 1 (764.21

−

1

g mol ): C, 42.4; H, 3.8; N, 7.3; S, 4.2. Found: C, 42.9; H, 3.9; N,

.5; S, 4.0. ICP-OES. Calcd: Cu, 16.6. Found: Cu, 15.8. TGA: for

7

3

complex 1, the weight loss was approximately 5.3% (calcd 4.7%)

between 25 and 278 °C, corresponding to the removal of two

per unit cell equal to 469 and 282 Å were found for 1 and 2,

respectively. These results agree with elemental analyses of the

compounds, which indicate 2 mol of crystallization water molecules in

complex 1 but only 1 mol in complex 2 because the voids in the

former are almost twice the size of those observed in the latter.

CHNS and Copper Content. Elemental analysis was performed to

determine the CHNS content in ligands and complexes on a

ThermoElectron analyzer (model Flash EA 1112). All of the

measurements were carried out in triplicate with 1% as the maximum

standard deviation. The copper content in the complexes was

estimated using inductively coupled plasma optical emission

spectrometry (ICP-OES; PerkinElmer Optima 7300 DV). Samples

were treated with nitric acid and diluted in water. Measurements were

performed with a dye laser pulsed in the 324.7 nm line.

−

1

2

hydration water molecules. Molar conductivity in ACN: 180 Ω cm

−

1

57

mol (1:1 electrolyte system), consistent with the presence of a

single perchlorate as the counterion.

[

Cu (μ-OH)(C H N O )]ClO ·H O (2). This complex was prepared

2 27 24 4 4 4 2

according to the same procedure as that used to synthesize 1.

Complex 2 was recrystallized in DCM/DMK/EtOH (1:1:1),

obtaining dark-green crystals (Figure S2 ) suitable for XRD analysis.

Yield = 266 mg (0.36 mmol, 72%). Elem anal. Calcd for 2 (730.12

−1

g mol ): C, 44.4; H, 3.7; N, 7.7. Found: C, 44.0; H, 3.7; N, 7.4. ICP-

OES. Calcd: Cu, 17.4. Found: Cu, 17.3. TGA: in the case of 2, the

weight loss between 25 and 221 °C was in accordance to the

elimination of only one hydration water molecule. Molar conductivity

−

1

2

−1

57

in ACN: 144 Ω cm mol (1:1 electrolyte system), consistent

with the presence of a single perchlorate as the counterion.

Caution! Perchlorate salts of metal complexes containing organic

ligands are potentially explosive and should be handled with care. Only

small amounts should be prepared.

ESI-MS Analyses. ESI-MS analyses were carried out on a

PerkinElmer SQ-300 mass spectrometer. Stock solutions containing

the samples were prepared by dissolving 1.0 mg of the compounds in

100 μL of DMSO (MS grade) and diluting the initial solutions by the

addition of 900 μL of deionized water. Both H L1 and H L2 ligands

3

The whole synthetic route described in this section is summarized

in Scheme 1.

precipitated after the dilution step. Thus, 900 μL of MeOH (MS

grade) was used in the preparation of the ligands’ stock solutions

instead of water. Aliquots of 50 μL of the stock solutions of all

samples were diluted in 950 μL of MeOH and analyzed by direct

infusion. Analyses of the stock solutions of the complexes were also

performed after 24 h of aging. Standard conﬁguration parameters for

the positive mode were used, with the capillary output voltage

(CAPEX) set at 50 V. Isotopic patterns were simulated using the

Measurements. Spectroscopic Studies. UV−visible spectra over

the wavelength range 900−250 nm were recorded in a PerkinElmer

Lamba 35 spectrophotometer and an Agilent Cary 100 spectropho-

tometer, after dissolving the compounds in DMSO or 10% DMSO in

water. The binucleating ligands were characterized by 1D and 2D

NMR spectroscopy. The spectra were recorded on a Bruker Avance

III HD-400 spectrometer and calibrated with reference to the residual

64

Isotope Distribution Calculator and Mass Spec Plotter tool.

1

13

peaks for DMSO-d : 2.50 ( H) and 39.52 ( C) ppm. J coupling

Thermogravimetric Analysis (TGA) and Electrical Conductivity.

TGA curves were acquired in a PerkinElmer Pyris 1 thermogravi-

metric analyzer. TGA scans were performed from 25 to 900 °C at 10

6

constants are given in hertz. Chemical shifts are reported in parts per

million. Spectra were processed by using the TopSpin 3.5 software.

Mid-infrared vibrational spectra were acquired on a PerkinElmer

Spectrum 400 Fourier transform infrared (FTIR) spectrophotometer,

−

1

°C min under a ﬂowing dry-air atmosphere. Curve optimization and

calculations were processed in the Pyris v 8.0.0.0172 software. The

conductivity measurements were performed at room temperature in a

650MA electrical conductivity meter. In order to obtain a ﬁnal

−1

at room temperature, in KBr pellets, with a resolution of 4 cm in the

−

1

4

1

000−450 cm range. Each measurement corresponds to a total of

−

3

−1

6 scans. The spectra were processed by using the PerkinElmer

concentration of 1 × 10 mol L , the copper(II) complexes were

Spectrum 10.03.09 software. On the other hand, Raman spectra were

collected at room temperature by using a micro-Raman confocal

Horiba Xplora ONE Jobin Yvon spectrometer with a spectral

dissolved in ACN.

BSA Binding Studies. The interaction between BSA and the

synthesized compounds was conﬁrmed by UV−visible, while binding

constants were determined only for the complexes using ﬂuorescence

spectroscopy. For the latter measurements, a PerkinElmer LS 55

spectroﬂuorimeter with an optical light ﬁlter corresponding to 50% of

light attenuation was used. The excitation wavelength was ﬁxed at 280

nm, and the emission was recorded in the range from 290 to 500 nm.

The excitation and emission slits were 6 nm. All measurements were

performed at 25 °C. Samples were prepared with a ﬁxed

concentration of BSA (1 μM) and by variation of the concentrations

resolution of 10 cm⁻in the 3500−700 cm range. The Raman

microscope was equipped with a charge-coupled-device (CCD)

detector and with an integrated camera and a Kohler illumination for

transmission and reﬂection illumination. The laser beam was focused

onto the samples through a microscope objective of 50×. Diﬀerent

lasers were used as the excitation source: 532 nm (for the ligands) and

1

−1

7

85 nm (for the complexes). The samples were placed on quartz

sample slides. In order to achieve a suﬃcient signal-to-noise ratio,

D

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1

of the complexes from 0 to 8 μM. For that, a BSA solution was

prepared in a buﬀer (10 mM Tris-HCl containing 10 mM NaCl, pH

solution (25 μg mL ) was prepared in 10 mM Tris-HCl, at pH 7.40.

Samples containing DNA with diﬀerent compounds were set to

contain 10% DMSO. For that, the DNA concentration was ﬁxed (2.5

7

.40) and stored at 4 °C. The concentration of BSA was determined

−1

using the molar absorptivity of 43824 M cm at 280 nm. Stock

solutions of the complexes (1 mM) were prepared in DMSO.

Mixtures of BSA and complex 1 or 2 solutions in the appropriate

proportions were analyzed after 3 min of incubation at 25 °C. This

time point was chosen on the basis of previous preliminary tests

performed on the system, which indicated that the equilibrium

condition was fully reached upon such a reaction time. For UV−

visible measurements, the samples were prepared with a BSA/

compound molar ratio of 1:5, and blanks with the same concentration

of compounds were subtracted.

μg mL ) and the concentrations of the complexes were varied from 0

to 50 μM. The inﬂuence of the ligands and Cu(ClO ) ·6H O (50

μM) was also analyzed. Blanks of DNA (in 10% DMSO) and of the

tested compounds in the absence of DNA were measured.

4

2

For small-angle X-ray scattering (SAXS) measurements, BSA

samples were analyzed in the same conditions as those previously

described for the DLS technique, excluding sample ﬁltration. All

SAXS measurements were performed on the beamline at the Brazilian

Synchrotron Light Laboratory (LNLS, Campinas, Brazil). The

temperature was set at 25.0 ± 0.5 °C and controlled using a water

bath. The sample holder was a mica window cell, and the data frames

were collected during exposure intervals of 120 s. The beam

wavelength was 1.548 Å. SAXS data were acquired using a 2D X-

ray detector (CCD-MAR165; MarResearch USA), and the sample-to-

detector distance was 902 mm, covering a scattering vector range of 5

DNA Binding Studies. In order to study the interaction of the

synthesized compounds with DNA, the corresponding compound

DNA binding constant (K ) was determined by UV−visible

b

spectroscopy. For that, an Agilent Cary 100 spectrophotometer, in

the range 600−250 nm, was used. Samples were placed in a quartz

cuvette with a 1 cm path length. The absorbance was measured for

samples with ﬁxed concentrations of the synthesized compounds (25

μM, 3 mL) and increasing concentrations (0−50 μM) of DNA. The

absorbance corresponding to free DNA was subtracted by adding an

equal amount of DNA to the samples and reference solution. The

stock solution was prepared by dissolving sodium salt ctDNA (2 mg

−

1

< q < 0.1 nm . Experimental curves are represented as the X-ray

scattered intensity, I(q), as a function of the scattering vector, q. SAXS

experimental data were ﬁtted to the Generalized Gaussian Coil

67,68

69

model

using the Sasfit program to calculate the gyration radius,

R_g, of BSA in the absence and presence of diﬀerent concentrations of

the complexes and a single concentration of the ligands and

Cu(ClO ) ·6H O.

−1

mL ) in a buﬀer solution (50 mM Tris-HCl containing 50 mM

NaCl, pH 7.40) and keeping the solution cold overnight. The DNA

concentration per nucleotide was determined by diluting 20 times an

aliquot of the stock solution and using the molar absorptivity value of

4

2

Cytotoxicity Assays. Cell Culture. In the present study, we used a

panel of cell lines to evaluate the antitumoral potential of the

described compounds. This panel includes two human colorectal

cancer cell lines (HCT116 and HT29), a human triple-negative breast

cancer cell line (MDA-MB-231), and a melanoma cell line derived

from mice (B16F10). As a nontumoral control, we used Madin-Darby

canine kidney cells. All cells were incubated at 37 °C with 5% CO₂

and cultured in a Dulbecco’s modiﬁed Eagle’s medium (DMEM)

1

−1

65,66

6

−

600 M⁻cm at 260 nm.

A ctDNA stock solution was stored at

20 °C until use. All samples were analyzed in a proportion of 10%

DMSO and 90% Tris-HCl (50 mM, 50 mM NaCl, pH 7.40) at 25 °C.

The 260/280 nm absorbance ratio of a ctDNA-diluted (20×) stock

solution was 1.8−1.9, indicating that DNA was suﬃciently free of

protein. Analyses of the ctDNA solution in pure buﬀer and in

mixtures containing 10% DMSO and 90% buﬀer presented identical

spectra.

−

1

supplemented with 10% fetal calf serum, penicillin (10 μg mL ), and

streptomycin (100 μg mL ).

Proliferation. For in vitro studies of cell proliferation inhibition by

−

1

3

−1

Scattering Studies. For dynamic light scattering (DLS) experi-

ments, 2 mL of each sample was transferred to dispensable cells,

which were incubated for 5 min (BSA samples) or 15 min (DNA

samples) at 25.0 ± 0.1 °C inside the instrument, to achieve thermal

stabilization. BSA samples were ﬁltered through a poly-

the diﬀerent compounds on cells, 5 × 10 cells well were plated on

96-well culture plates. After attachment, diﬀerent concentrations of

drugs were added and cells were allowed to grow for 36 h. The

number of living cells was estimated by the tetrazolium salt reduction

method (MTT, Sigma-Aldrich) as described before. Proliferation

was expressed as the percentage of control untreated samples. The

concentrations of drugs decreasing the cell proliferation by 50%

(IC ) were estimated from the absorbance curves as a function of the

concentration of the analyzed compound by means of the GraphPad

Prism 7 program. The ﬁnal concentration of DMSO in the culture

media was never higher than 1%, and we have carefully checked that

the solvent has no eﬀect on the cell growth at such a low level.

70

(tetraﬂuoroethylene) syringe ﬁlter (0.45 μm pore size and 0.25 μm

diameter, LCR). As a control, some nonﬁltered samples were also

tested in order to verify the possibility of the formation of particles

with higher hydrodynamic radii. Measurements were carried out using

a compact Horiba Scientiﬁc SZ-100 nanoparticle analyzer. The

instrument was equipped with a semiconductor excitation solid laser

5

0

(532 nm, 10 mW) light source and photomultiplier-tube detector.

The scattering angle (θ) was set at 90°, and each sample was

measured 20 times for 120 s at 25.0 ± 0.1 °C. The SZ-100 Horiba for

Windows software was used to record and ﬁt the correlation functions,

obtaining the size distributions and mean hydrodynamic diameters

through the CONTIN algorithm. The samples were prepared from a

stock solution of BSA (essentially fatty acid free), with a

concentration of 5 × 10⁻M, 100 mM NaCl, and 50 mM Tris

buﬀer, pH 7.40. The BSA concentration in the stock solution was

determined by its absorbance at 280 nm (ε = 43824 M cm ).

Stock solutions of the dicopper(II) complexes 1 and 2 were prepared

in DMSO. Diﬀerent aliquots of the complexes were added to 2800 μL

of a BSA stock solution in order to obtain samples with BSA/complex

molar ratios of 1:1, 1:7, 1:14, 1:21, and 1:28. Moreover, an additional

volume of DMSO was used to obtain complex/BSA solutions with

RESULTS AND DISCUSSION

■

Syntheses. Both ligands, as well as their respective

dicopper(II) complexes, were obtained in good yield and in

crystalline form. Because of the tetrahedral amine nitrogen

atom displaying three diﬀerent substituents, all of the ligands

and complexes were isolated as a pair of optical isomers.

Characterization of the Ligands and Complexes.

Solution NMR of the Ligands. Ligands were fully charac-

terized using multinuclear NMR. The experimental data set

5

−

1

−1

1

13

includes, along with 1D H and C spectra, COSY, HSQC,

and HBMC contour maps. Some representative NMR plots

can be found in Figures S3 −S8. It is well-known that

aroylhydrazone derivatives can present geometric isomerism

with respect to the azomethine group as well as amide-related

10% DMSO. For the sake of comparison, samples containing only

BSA in 10% DMSO or only the complexes (in the absence of BSA) in

Tris-HCl/NaCl buﬀer were also analyzed. Ligands/BSA and starting

salt/BSA measurements were carried out in a unique BSA/compound

molar ratio of 1:28, in order to compare their eﬀect with that caused

by the copper complexes on the protein. To evaluate the stability of

the copper(II) complex/BSA systems over time, measurements were

taken every 3 h for at least 24 h. On the other hand, the DNA stock

71−73

tautomerization processes.

In both ligands, the E isomer is

virtually the only one existing. However, duplicated sets of

1

13

signals in the H and C NMR spectra of H L1 indicate the

3

presence, in a DMSO-d solution, of both iminol and amido

6

E

DOI: 10.1021/acs.inorgchem.9b01195

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

Article

Scheme 2. Amide-Related Tautomerization Process in H L1 (with the Tautomers’ Percentages at Equilibrium) and the

3

Labeling System Employed for the Assignment of the NMR Data According to the XRD Numbering Scheme

tautomers (Scheme 2). Because in the ¹³C NMR spectrum of

this ligand the carbonyl signal at 161.04 ppm is not part of the

main set, we suggest that the iminol form constitutes the

predominating species. This is expected because the tautomer

is stabilized by intramolecular hydrogen bonding between

and 963 for H L2. These peaks were assigned, respectively, to

3

the sodium-associated monomeric and dimeric species [H L +

3

+

+ +

Na ] and [(H L) + Na ] .

3

2

Crystal Structures of 1 and 2. Figure 1 shows the structures

of the cationic complexes present in 1 and 2. Selected bond

distances and angles are listed in Table 1. Detailed crystal data,

collection, and reﬁnement parameters can be found in Tables

S2 and S4 .

O1−H and N2. In contrast, H L2 does not show any

3

duplicated signal and, thus, exists in just one tautomeric form.

The absence of the carbonyl resonance above 160 ppm

represents supporting evidence that, for both ligands, the same

species prevail at equilibrium. Because iminol [−(HO)C

NNC−] seems to be much more stable in solution for these

systems, the present NMR analysis focuses only on this

tautomer.

The crystal structures indicate that both complexes generate

dimeric arrangements containing two partially deprotonated

2−

(HL1 or HL2 ) ligands and four divalent copper centers. In

fact, compounds 1 and 2 are quite similar, exhibiting two

crystallographically independent metal sites (Cu1 and Cu2)

with intermetallic distances of 2.944 and 2.923 Å, respectively.

Cu1, which is coordinated by the hydrazonic moiety of the

ligand, comprising the donor atoms N2 and O1 (correspond-

ing to the iminolate form), shows a square-planar geometry

with a slightly distorted plane [the average distances among

the atoms and the best plane are 0.012(4) and 0.03(1) Å,

correspondingly, for complexes 1 and 2]. Endogenous (O3)

phenoxo and exogenous (O2) hydroxo bridges complete the

coordination sphere. On the other hand, Cu2 exhibits an

N O -type octahedral environment distorted by a pronounced

A complete assignment of the hydrogen and carbon nuclei

for the new ligands H L1 and H L2 is reported (Table S1).

3

The chemical shifts previously published by our research group

for the precursor HBPAMFF were employed as a starting point

for the assignment of the nonhydrazonic moiety of the

5

2

ligands. 2D COSY maps helped us to conﬁrm the identity of

aromatic hydrogen nuclei because some signals appear to be

overlapped and, in the case of H L1, duplicated because of the

3

1

13

iminol/amide tautomerism. As expected, the H and C NMR

spectra of H L2 are very similar to those of H L1 except for

3

2

4

the absence of tautomeric equilibrium in the former under the

conditions of the present study.

Jahn−Teller eﬀect. The equatorial plane contains the tertiary

amine N3 and the pyridine N2 atoms, in addition to the O3

phenoxo and O2 hydroxo bridges. The apical positions are

occupied by the protonated phenol oxygen O4 and by the

ESI-MS Analysis of the Ligands. Two prominent peaks

+

were observed at m/z 509 and 995 for H L1 and at m/z 493

3

F

DOI: 10.1021/acs.inorgchem.9b01195

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

Article

Figure 1. (Left) Crystal structure representations of 1 (A) and 2 (B). The ellipsoids were drawn with 30% probability. Only one of the enantiomers

is shown. Perchlorate counterions, disordered water molecules, and most of the hydrogen atoms were omitted for the sake of clarity. Symmetry

code: (i) −x + 1, −y + 1, −z + 1. (Right) Corresponding dimers, formed by a pair of enantiomers interacting through trifurcated hydroxo bridges

i

(

O2- and O2 -donor atoms), with the copper(II) coordination spheres highlighted as colored ellipsoids.

i

1555 cm⁻¹is assigned to the amide N−H bending vibration. In

−1

74

bridging hydroxo O2 atom of another dinuclear unit,

constituting a nice example of a trifurcated bridge, with the

structural function of maintaining the dimer’s integrity. When

the compounds are in solution, those interactions are probably

disrupted, and Cu2 should assume a square-pyramidal

geometry. The furan and thiophene rings are not involved in

coordination.

the TCH precursor, this mode appears at 1542 cm . The

complete absence of these bands in the FTIR spectrum of 1, as

well as the pronounced shift of the ν

band to lower

CO

wavenumbers, indicates that the ligand loses the amide

hydrogen atom and is coordinated in its iminolate form,

which is in agreement with the acidic nature of this group and

the use of stoichiometric sodium hydroxide during the last step

of the complexes’ syntheses.

Two perchlorate counterions, located in voids, were also

observed in the crystal lattice for each complex dimer. Dimers

present moderate O···O hydrogen bonds between both the

terminal endogenous (O4) and bridging exogenous (O2)

hydroxides and the perchlorate counterions. The crystal

packing is also stabilized by weak CH···O interactions

involving perchlorate and methylene groups. Moreover, for

complex 1, CH···S interactions between the ligands could be

identiﬁed also. A complete list of the hydrogen-bonding

geometries present in complexes 1 and 2 can be found in

Tables S3 and S5, respectively.

As expected, speciﬁc bands for the perchlorate counterions

are also present in the vibrational spectra of complexes 1 and 2.

The main vibrational frequencies of H L1, H L2, and their

3

coordination compounds, along with the proposed assign-

ments, are summarized in Table S6.

UV−Visible Spectroscopy Studies of the Compounds.

Both H L1 and H L2 display ﬁve foremost absorptions in the

3

250−500 nm spectral range, as shown in Figure 2 (top), in

which the spectra of the precursor HBPAMFF and the

respective hydrazides are also shown for the sake of

comparison. Three of those absorptions are also present in

the precursors and are probably related to the thiophene/furan

and HBPAMFF transitions.

Vibrational Properties of the Compounds. Both H L1 and

H L2 and their corresponding dicopper(II) complexes 1 and 2

3

were studied under the perspective of vibrational (IR and

Raman) spectroscopy. All of the spectra are available in Figures

S10 −S15 , where the IR spectrum of the HBPAMFF precursor

The most intense UV bands occur at 312 nm (H L1) and

3

(

Figure S9 ) is also included. When the vibrational bands of the

310 nm (H L2), followed by strong absorptions centered at

3

free ligands are compared to those of their metal compounds

Figures S10 and S11 ), clear signs of complexation are

evidenced. Probably, the most patented among them are

related to deprotonation of the hydrazonic group upon

300 nm (H L1) and 298 nm (H L2), corresponding to the

3

(

aroylhydrazone transitions, because they are completely absent

in the spectra of the precursors. The upper half of Table S7

displays the absorption bands of both ligands along with their

respective molar absorptivity coeﬃcients and suggested

assignments.

coordination. For example, in the IR spectrum of H L1, the

3

−

1

NH stretching mode is observed at 3151 cm , and the band at

G

DOI: 10.1021/acs.inorgchem.9b01195

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

Article

Table 1. Selected Geometric Parameters for Complexes 1

and 2

references. Notice that, in this medium, the ligand and

complex absorptions are slightly shifted.

a

Stability of the Ligands. Over the ﬁrst 12 h of assay, the

absorptions of the ligands’ solutions decrease by around 90%

1

2

Bond Distances (Å)

1.910(1)

(

Figure S16). With time, the bands associated with the

Cu1−O1

Cu1−O2

Cu1−O3

Cu1−N2

Cu2−O2

Cu2−O3

Cu2−O4

Cu2−N3

Cu2−N4

Cu2−O2

1.895(2)

1.924(2)

1.908(2)

1.905(2)

1.949(2)

1.956(2)

2.570(2)

2.003(2)

1.976(2)

2.590(2)

hydrazone group remain in the same position. However, the

wavelenghts from the HBPAMFF-related absorptions are

slightly blue-shifted for both ligands, approaching that of the

free HBPAMFF precursor. This absorption band of

HBPAMFF, at about 340 nm, has a molar absorptivity that

is around one-third of those related to the associated bands in

H L1 and H L2. Therefore, our results suggest that, in

1.933(1)

1.914(1)

1.911(2)

1.952(1)

1.960(1)

2.574(2)

2.005(2)

1.984(2)

3

aqueous solution, the ligands are hydrolyzed in a relatively fast

way. The ligands were also assayed in pure (100%) DMSO and

in 10% DMSO/90% phosphate-buﬀered saline (PBS; pH 7.4).

In the organic medium, both H L1 and H L2 demonstrate a

i

2.599(2)

Bond Angles (deg)

98.49(6)

3

Cu1−O2−Cu2

Cu1−O3−Cu2

O1−Cu1−O2

O1−Cu1−O3

O1−Cu1−N2

O2−Cu1−O3

O2−Cu1−N2

O3−Cu1−N2

O2−Cu2−O3

O2−Cu2−N3

O2−Cu2−N4

97.99(9)

98.32(9)

102.05(9)

175.67(10)

83.06(10)

81.54(8)

174.72(10)

93.42(10)

79.69(8)

171.60(9)

102.72(9)

82.11(9)

97.48(9)

92.20(9)

177.43(9)

86.42(9)

83.39(9)

85.43(10)

remarkable stability, keeping more than 90% of the initial

absorbance values during the 24 h of monitoring at room

temperature. Although not as stable as in pure DMSO, the

ligands are more resistant to hydrolysis in the “buﬀered”

medium than in 10% DMSO/90% water. The initial

98.90(6)

103.49(6)

175.23(6)

82.46(7)

80.89(5)

174.02(6)

93.14(7)

absorbance value decrease for H L1 was only 31% over 24

3

h, while that for H L2 was 63% under the same experimental

3

conditions, as can be observed in Figure S17A,B.

79.25(6)

Stability of the Complexes. Over 24 h, both complexes’

solutions in 10% DMSO contain 85% (1) and 91% (2) of their

initial absorbances (Figure S18) and, in this sense, are quite

resistant to dissociation. Because hydrolysis of the hydrazones

begins with protonation of the azomethine nitrogen atom,

coordination through this donor atom should prevent that

reaction. Upon dissolution in a 10% DMSO/90% PBS (pH

171.01(6)

103.26(6)

85.79(6)

97.38(8)

92.15(6)

177.48(6)

86.59(7)

81.36(7)

i

O2−Cu2−O2

O3−Cu2−O4

O3−Cu2−N3

O3−Cu2−N4

O4−Cu2−N3

O4−Cu2−N4

N3−Cu2−N4

7.4) medium, the stabilities of complexes 1 and 2 remain

relatively unchanged (Figure S17C,D ). The structural integrity

of the complexes in aqueous solution, up to 24 h at room

temperature (23 ± 2 °C), was conﬁrmed by ESI-MS analyses

85.37(7)

a

Symmetry code: (i) −x + 1, −y + 1, −z + 1.

(

Figure 3). DMSO/water (1:9) stock solution aliquots were

analyzed immediately and after 24 h, by direct infusion, after a

previous dilution in MeOH (5:95). The MS spectra of both

The absorption proﬁles of the complexes are very similar to

+

those of their respective ligands. Nevertheless, the bands

related to the hydrazone group and to the HBPAMFF-derived

central phenol system are bathochromically shifted in the

spectra of 1 and 2 (Figure 2, bottom). The presence of the

iminolate tautomeric form in the complexes, as well as

deprotonation of the central (bridging) phenol group upon

coordination, increases electron delocalization all over this

moiety of the ligand. In both complexes, a broad low-intensity

complexes are similar, with three main peaks at m/z 611, 625,

+

and 671 for 1 and m/z 627, 641, and 687 for 2, which were

+

assigned respectively to the species [Cu

(HL)(OH)] ,

2

+

[Cu (HL)(CH O)] , and [Cu (L)(DMSO)] , the latter

2 3 2

without any exogenous bridging group between the copper(II)

+

ions. In the speciﬁc case of complex 1, additional peaks at m/z

663 and 741 were also observed in the spectrum after 24 h of

+

aging and attributed to the species [Cu (HL)(OH)(OH ) ]

2 2 2

+

(

Table S7 , lower half) absorption is also observed from 550 to

and [Cu (HL)(OH)(OH ) (DMSO)] . All of the discussed

2 2 2

8

00 nm. These bands, centered at 667 nm for both 1 and 2, are

assignments were supported by comparisons between the

experimental and simulated isotopic patterns (Figure S19). No

evidence for the dissociation or hydrolysis of coordinated

attributed to d−d transitions between the electronic states of

the copper centers. The presence of charge-transfer bands can

certainly not be ruled out, although their observation may be

impaired because of overlap with the intraligand absorptions.

Hydrolysis Studies. Substances containing the hydrazone

group are known to be prone to dissociate into the respective

carbonyl compound and hydrazide in water. However, an

aqueous medium is necessary for most biological tests and,

even more signiﬁcant, water constitutes the universal

physiological solvent. For this reason, the stabilities of the

ligands and complexes were monitored by UV−visible, over 24

h, in water solutions containing 10% DMSO to ensure for

complete dissolution of the compounds. The hydrazone-

related bands at 312/299 nm (H L1), 308/299 nm (H L2),

ligands was found. In this sense and considering that the

+

species [Cu (HL)(CH

2

O)] constitutes an “artefact” because

3

of the presence of MeOH used in the dilutions for ESI-MS

measurements, we can conclude that, under the experimental

conditions employed in the biological assays described below,

the main metal-containing species present should be the

dinuclear cations themselves characterized by XRD,

+

[Cu (HL)(OH)] , and the DMSO-substituted derivative

2

+

[Cu (L)(DMSO)] , in which the μ-hydroxo bridge is replaced

2

by a terminal solvent molecule.

Evaluation of BSA Binding through UV−Visible and

Fluorescence Spectroscopies. Because of the importance of

serum albumin in the transportation of drugs in vertebrates,

3

43/330 nm (1), and 333/320 nm (2) were used as

H

DOI: 10.1021/acs.inorgchem.9b01195

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Inorganic Chemistry

Article

−

5

−5

Figure 2. (Top) UV−visible spectra of the ligands H L1 (left, 4.6 × 10 M) and H L2 (right, 4.8 × 10 M), as well as their respective precursors

3

HBPAMFF (10.6 × 10⁻M), TCH (7.0 × 10 M), and FCH (4.0 × 10 M), in DMSO at room temperature. (Bottom) UV−visible spectra of

5

−5

−

5

−5

complexes 1 (left, 6.2 × 10 M) and 2 (right, 7.0 × 10 M) recorded in DMSO at room temperature. The spectra of H L1 (3.4 × 10 M) and

3

−5

−3

H L2 (2.9 × 10 M) are included for the sake of comparison. Insets: Visible absorbance of 1 (5.0 × 10 M) and 2 (5.0 × 10 M) in the 500−

3

900 nm region.

this class of proteins has been extensively studied, as well as

their interaction with potential pharmacological agents,

including organic ligands and coordination complexes. The

bovine-variant BSA presents chemical properties similar to

ligands (Figure 4B). The band at 280 nm, on the other hand, is

associated with the aromatic amino acids in the protein.

91

Although, upon addition of the copper(II) complexes, its

intensity seems to remain unchanged (Figure 4A, inset), this

absorption band increases slightly in the presence of the

ligands, indicating that the interaction of H L1 and H L2 with

75

those of human serum albumin (HSA), with only a few

7

6

diﬀerences. Thus, BSA has been largely used as a model in

3

order to study the interactions with many compounds,

BSA modiﬁes the microenvironments of the residues of their

aromatic amino acids.

18,77−80

92

including copper complexes.

These complexes are

well-known by their BSA binding propensity, showing a strong

On the other hand, ﬂuorescence spectroscopy has been

widely used as an eﬃcient technique for analysis of the BSA

conformational changes and binding with diﬀerent com-

8

1−84

aﬃnity for the protein.

In recent studies, authors

evaluated the interactions of both HSA and BSA with

copper(II) complexes. The values found for the binding

constants, as well as for the number of binding sites, are in

15

pounds. Parts C and D of Figure 4 present the eﬀect of

copper(II) complexes in the ﬂuorescence emission of BSA.

Following the addition of 1 or 2 to a BSA solution, the protein

ﬂuorescence intensity at 353 nm gradually decreases when the

concentrations of the complexes increase, suggesting that the

quenching mechanism is primarily of the static (or contact)

type, i.e., through the generation of a protein/complex adduct

in the ground state (before excitation). Because of signiﬁcant

absorption of the complex/BSA solutions at the emission

wavelength, the ﬂuorescence measurement was treated to

8

5−88

agreement,

although exceptions, such as the work of

89

Manna and co-workers, were identiﬁed.

UV−visible spectroscopy is a common and useful technique

15,90

to study the structural changes of albumin proteins.

In the

presence of complexes 1 and 2, the absorption band at 205 nm,

91

related to the peptide bonds of the protein, is red-shifted and

an intense absorption decrease is observed (Figure 4A). This

hypochromic eﬀect is probably due to the induced

perturbation of BSA α helices by the complexes, while the

shift toward longer wavelengths is produced by the changes in

93

correct the “inner ﬁlter” eﬀect (eq 1). This equation is

limited to solutions with absorbances lower than 0.3.

92

the polarities of their surroundings. BSA presents similar

spectral changes for this absorption in the presence of free

F

⁼^Fobs ^aⁿ^tⁱ^l^o^g^[⁽^Aex + A )/2]

(1)

corr

em

I

DOI: 10.1021/acs.inorgchem.9b01195

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Inorganic Chemistry

Article

Figure 3. ESI-MS(+) spectra measured from fresh DMSO/MeOH solutions of H L1 (A) and H L2 (B). (C) Complex 1 in a fresh DMSO/water

3

solution. (D) 1 in a DMSO/water solution after 24 h at 23 ± 2 °C. (E) Complex 2 in a fresh DMSO/water solution. (F) 2 in a DMSO/water

solution after 24 h at 23 ± 2 °C. The capillary output voltage was set at 50 V. All compounds were diluted in MeOH (5:95) before injection.

where F_c_o_r_randF_o_b_sare respectively the corrected and measured

ﬂuorescence intensities. A_e_xcorresponds to the absorbance

value at the excitation wavelength used to acquire the

^ﬂ^u^o^r^e^s^c^eⁿ^c^e^s^p^e^c^t^r^a^aⁿ^d^Aem to that at the emission

wavelength in a 1.0-cm-path-length cuvette. The Stern−

quenching agent. [Q] is the quenching agent concentration, K_q

is the biomolecular quenching rate constant, and τ is the

0

average lifetime of the protein in the absence of a quencher,

−

8

95

equal to 10 s. The Stern−Volmer constant for the adduct

formation, K , can be determined directly from the slope of

SV

94

Volmer equation (eq2) was used to infer in a more accurate

way the main quenching mechanism and to determine the

respective constants

the straight line obtained when I /I versus [Q] is plotted. The

0

K_S_Vand K constants for complexes 1 and 2 can be found in

q

Table 2.

The maximum value of the K parameter for a mechanism to

I /I = 1 + K [Q] = 1 + K τ [Q]

q

0

SV

q 0

(2)

1

0

be considered of a pure dynamic quenching type is 2.0 × 10

−

1

−1

where I₀and I are, correspondingly, the steady-state

ﬂuorescence intensities in the absence and presence of the

M

s . Complexes 1 and 2 presented K values of about 3

q

order of magnitude higher than that expected for a diﬀusion-

J

DOI: 10.1021/acs.inorgchem.9b01195

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Inorganic Chemistry

Article

Figure 4. UV−visible spectra of the BSA (1 μM) in a buﬀer solution (10 mM Tris-HCl and 10 mM NaCl at pH 7.40) in the absence and presence

of the complexes (A) and ligands (B) at a 5 μM concentration. Insets: Details of the 250−320 nm region. Fluorescence spectra, at 25 °C, of BSA (1

μM; λ = 280 nm; λ = 353 nm) in the absence (red curve) and presence (black curves) of increasing concentrations (1−8 μM) of the

ex

em

dicopper(II) complexes 1 (C) and 2(D). Insets: Corresponding Stern−Volmer and Scatchard plots.

Table 2. Quenching and Binding Parameters for the

Interaction of Complexes 1 and 2 with BSA at 25 °C

Inﬂuence of the Compounds in BSA Conformation by

Scattering Techniques. Scattering techniques have recently

been used as an alternative tool to other spectroscopic

methods for characterizing the structures formed due to the

5

−1

K_q(×10¹³M⁻¹s⁻¹

K (×10 M⁻¹)

4

compound K_S_V(×10 M

)

n

b

1

2

2.08 ± 0.04

1.90 ± 0.04

2.08

1.90

9.23

3.29

0.93

0.85

97−102

interaction between diﬀerent compounds and BSA.

Figure 5 presents the size distributions of BSA before and

after the addition of diﬀerent concentrations of 1 and 2. The

corresponding autocorrelation functions are presented in

Figure S20. Native BSA has only one population with a

hydrodynamic diameter of 8.6 ± 0.1 nm. Both the ligands and

complexes are too small to be detected by DLS and do not

exhibit signiﬁcant scattering intensity. Upon the addition of 1,

the diameter gradually increases by up to 18 nm and a new

population of larger particles (from 133 to 225 nm) are

observed, which are probably related to the formation of

aggregates. Nonﬁltered compound/BSA samples, i.e., preserv-

ing the whole spectrum of the particles’ sizes, were also

analyzed and indicate that the addition of 1 to BSA solutions

leads to the formation of another population of aggregates

larger than 1 μm (which are not adequate to be precisely

characterized by DLS because they present a high scattering

intensity, making the samples more turbid). Similar trends

9

2

controlled ﬂuorescence quenching, indicating that the

systems under study are dominated by a static quenching

mechanism, caused by the interaction between BSA and the

complexes.

To determine the binding constants, K , and the number of

complexes bound to each BSA unit, n, the Scatchard equation

b

96

was employed (eq 3).

log[(I − I)/I] = log K + n log [Q]

In the linear ﬁt plot of eq 3, the intercept corresponds to log K_b

and the slope to n.

The calculated protein binding constants (Table 2) are 9.23

10 M and 3.29 × 10 M for complexes 1 and 2,

respectively. Therefore, the aﬃnity of 1 for BSA is 2.8 times

(3)

0

b

4

−1

4

−1

×

higher than that of 2. The K values found are perfectly

were observed for 2 and ligands H L1 and H L2, as is also

3 3

b

comparable to those obtained for the interaction of BSA with

shown in Figures 5 and S20 . However, ligands caused a minor

change in the hydrodynamic size of BSA compared with their

respective complexes under the same conditions (BSA/

compound molar ratio of 1:28). The obtained results indicate

77

previously reported copper(II) complexes. The calculated n

parameter indicates a single binding site in BSA for the

complexes.

K

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Inorganic Chemistry

Article

compound (pterodontic acid) leads to an increase of the

hydrodynamic diameter of BSA from 7.3 to 22.8 nm and to the

arising of a second population of larger particles.

Over time, the frequency of the ﬁrst BSA population

nonaggregated) increases, while the frequency of the

(

population corresponding to aggregated BSA decreases. This

observation indicates that aggregation is reversible and could

be related to a trend of release of the copper complexes with

time and return of the protein to its native form. A comparison

between the frequencies of BSA populations in the presence of

1

and 2 after an incubation period of 24 h shows that the

protein disaggregates faster for complex 2 than for complex 1.

This behavior could be expected based on the value for the

binding constant obtained by ﬂuorescence spectroscopy, which

is lower for complex 2 than for complex 1.

To better evaluate the conformational changes of BSA,

SAXS can complement DLS because X-rays have a shorter

wavelength than the visible light and are more adequate for

evaluating smaller dimensions. Figure 6 presents the

Figure 5. Hydrodynamic diameter (nm) distribution, at 25 °C, of

BSA samples (5 × 10⁻M) in the absence and presence of complexes

5

1

(A) and 2 (B) at BSA/complex molar ratios of 1:1, 1:7, 1:14, 1:21,

and 1:28. (C) BSA in the absence and presence of Cu(ClO ) ·6H O

4

2

and free ligands H L1 and H L2 at a BSA/compound molar ratio

3

equal to 1:28. Scattering angle: 90°. Samples (2 mL, in 100 mM NaCl

and 50 mM Tris buﬀer at pH 7.40) were ﬁltered (0.25 μm) before the

scattering measurements were performed.

Figure 6. SAXS curves, at 25 °C, of free BSA (5 × 10⁻M, in 100

mM NaCl and 50 mM Tris buﬀer at pH 7.40) in the absence and

presence of complexes 1 (A) and 2 (B) at BSA/complex molar ratios

of 1:1, 1:7, 1:14, 1:21, and 1:28. All samples were ﬁltered (0.45 μm)

before the scattering measurements were performed. The curve

ﬁttings, using a Generalized Gaussian Coil model, for free BSA and

BSA/complexes at 1:28 are included.

5

that interactions of the ligands and especially the complexes

with BSA cause its aggregation. Although the increment in the

size of the ﬁrst population is not signiﬁcant to evidence the

occurrence of conformational changes in the secondary

structure of BSA, the obtained values agree with the results

from the literature. By DLS measurements, Adel and co-

1

03

workers reported a hydrodynamic radius for BSA of 3.8 ±

experimental curves of the scattering intensity, I, as a function

of the scattering vector, q, obtained for samples containing

BSA and diﬀerent concentrations of 1 and 2. Samples

containing only the complexes do not present signiﬁcant

scattering intensity. The ﬁtted curve corresponding to the

101

0

.2 nm, whereas Yu and collaborators obtained a 7.13 nm

hydrodynamic diameter for this protein. In the same direction,

Zocchi reported a value of 8 nm for the native BSA

hydrodynamic diameter, using micromechanical measure-

1

04

ments.

colleagues

Also in accordance with our data, Li and

solution of free BSA provided a gyration radius, R , of 3.5 nm.

g

1

00

observed that the addition of an organic

Upon the addition of complexes, the intensity is increased in

L

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Inorganic Chemistry

Article

Figure 7. Absorption spectra and 1/(A − A) versus 1/[DNA] plots (insets) of the copper(II) complexes 1 (A) and 2 (B) and the ligands H L1

0

3

(

C) and H L2(D) (25 μM, 3 mL, in 10% DMSO and 90% 50 mM Tris-HCl, pH 7.40) in the absence (red curve) and with increasing

3

concentrations (black curves) of ctDNA (5−50 μM) at 25 °C. Spectra were registered after an incubation time of 3 min from each ctDNA

addition.

2

2,85,106

the low-q region but is not aﬀected in the high-q region. This

indicates that the form factor of BSA is not signiﬁcantly

aﬀected by the presence of the complexes, although there is an

aggregation generating larger particles. The samples with

higher concentrations of complexes were also ﬁtted consider-

ing the existence of two populations. The best ﬁtting resulted

ability to bind to and cleave DNA.

In the present study,

UV−visible spectroscopy was used to investigate the

interactions of the synthesized compounds with ctDNA

through titration experiments. Upon the addition of increasing

amounts ctDNA to solutions containing 1 and 2, a clear

hypochromic eﬀect is observed in the complexes’ absorption

bands, which suggests an interaction with the biopolymer

Figure 7A,B). Hypochromism is commonly associated with

intercalative binding. The free ligands H L1 and H L2

present a similar behavior in the presence of ctDNA (Figure

in R values of 3.8 and 68 nm for 1 and 3.9 and 52 nm for 2.

g

(

However, the size of the second population is too large for the

available q region, and one can only aﬃrm that there are

98

3

aggregates with R ≥ 68 nm (1/BSA system) and 52 nm (2/

g

7

C,D).

BSA system). The dimensions of the protein after the addition

of the complexes are only slightly bigger (3.8 and 3.9 nm for 1

and 2, respectively) than those of free BSA. Once more, the

results indicate that there is no signiﬁcant change in size, which

could evidence conformational changes on the secondary

structure. Yet, the interaction between the complexes and BSA

also induces a process of aggregation, conﬁrming the results

obtained by DLS. For the sake of comparison, the copper salt

Cu(ClO ) ·6H O itself does not induce protein aggregation

The binding constants were determined by monitoring the

absorbance bands centered at 322 and 317 nm for complexes 1

and 2, respectively, as well as those at 314 and 309 nm for the

ligands H L1 and H L2, correspondingly. The treatment of the

experimental data was performed by using a modiﬁed form of

the Benesi−Hildebrand equation (eq 4):

3

107,108

1

=

4

2

A − A

0

ε − ε [compound]

b

f

0

(

Figure S21 ).

1

+

Evaluation of the ctDNA Binding Constant through

K (ε − ε ) [compound] [DNA]

(4)

b

f

0

UV−Visible Spectroscopy. To evaluate the properties of the

new compounds as potential anticancer drugs, the study of

their interaction with DNA constitutes a signiﬁcant step in

order to better understand the possible mechanism of action

where A and A are the absorbance values for the compounds

in the absence and presence of DNA, respectively, ε and ε_f

correspond to the molar absorptivities of the fully bound DNA

and of the free compound, correspondingly, [compound] is

0

b

1

05

involved. Copper(II) complexes have demonstrated a great

0

M

DOI: 10.1021/acs.inorgchem.9b01195

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

Article

the initial concentration of the compound, [DNA] is the

concentration of added DNA, and K is the binding constant.

b

A plot of 1/(A − A) versus 1/[DNA] gives an intercept equal

0

to 1/(ε − ε ) × 1/[compound] and a slope equivalent to 1/

b

f

0

K (ε − ε ) × 1/[compound] . Thus, K is determined by the

b

f

0

b

ratio of the intercept to the slope (Figure 7A−D, insets). The

calculated binding constants for complexes 1 and 2 are (3.50 ±

3

4

−1

0

.08) × 10 and (4.38 ± 0.08) × 10 M , respectively. These

values are comparable to those obtained for other reported

copper(II) complexes. However, they are lower than the

values usually found for representative DNA intercalators, such

as ethidium bromide (which is around 10 −10 M ). This

outcome could indicate that intercalation between the base

pairs is not the main mode of interaction of the synthesized

dicopper(II) complexes with DNA. On the other hand, a

98

5

6

−1 109

3

−1

binding constant of (1.64 ± 0.08) × 10 M was calculated

for H L1 and that of (7.91 ± 0.08) × 10 M for H L2.

3

−1

3

According to the binding results, complex 2 presents a

higher aﬃnity than complex 1 toward ctDNA, a trend also

observed with H L2 in comparison to that with H L1. The

3

free ligands showed a minor intercalator ability compared to

that of their respective dinuclear complexes, which is in

agreement with the results previously reported for other

9

aroylhydrazones and their copper(II) complexes. The lower

intercalative aﬃnity of 1 could be related to its higher reactivity

toward DMSO, as shown by ESI-MS(+) measurements,

because the DMSO-substituted species should present an

impaired planarity regarding the intercalative moiety con-

stituted by the central phenol ring and the aroylhydrazone-

derived pendant arm. As mentioned above, the experimental

data indicate that coordination to copper(II) increases the

binding aﬃnity for DNA.

Inﬂuence of the Compounds in a pBR322 Plasmid

DNA Conformation by DLS. In many previous reports, both

mononuclear and dinuclear copper(II) complexes have shown

6

,28,110−112

cleavage activity on plasmid DNA.

The eﬀects of

cleavage agents over plasmids, leading to their transition from

supercoiled (form I) to circular (form II) to linear (form III)

conformations of DNA, have been largely studied by gel and

capillary electrophoresis. Recently, however, scattering techni-

Figure 8. Hydrodynamic radius (nm) distribution of pBR322 plasmid

−1

DNA samples (2.5 μg mL , 2 mL, in 10 mM Tris buﬀer, pH 7.40) in

the presence of (A) complex 1 (10−50 μM) and (B) complex 2 (15−

60 μM). (C) pBR322 plasmid DNA in the presence of H L1, H L2,

3

113

ques have their importance increased in this context.

and the starting salt Cu(ClO ) ·6H O, all of them at a concentration

4 2 2

of 50 μM. Scattering angle: 90°. Incubation: 15 min at 25.0 ± 0.1 °C.

In this work, DLS measurements were also used to evaluate

the inﬂuence of the synthesized compounds in a pBR322

plasmid DNA conformation. To carry out these studies, it was

not possible to use SAXS because the samples do not present

signiﬁcant X-ray scattering in this range of concentrations.

Solutions of pure native plasmid DNA were not detected by

DLS as well, probably because of the absence of suﬃcient

contrast.

Figure 8 presents the size distributions of DNA macro-

molecules in aqueous solutions with di ﬀ erent concentrations of

complexes 1 (A) and 2 (B). Figure S22 presents the

corresponding autocorrelation functions. When complexes

are added to the pBR322 DNA solutions, a population is

observed with mean hydrodynamic radii of 58 ± 4 and 73 ± 6

nm for 1 (10 μM) and 2 (15 μM), respectively. We attribute

this population to the plasmid form I. In previous static light

concentrations of the tested complexes (up to 10 μM for 1 and

15 μM for 2), the interaction is not strong enough to alter

dramatically the conformational structure of pBR322 plasmids.

Nevertheless, taking into account that the free plasmid had not

been detected because of the low contrast of its solution, it is

deduced that the complexes interact with plasmids, causing a

signiﬁcant increase in their contrast, which allowed the

autocorrelation functions to be obtained. The observed

diﬀerences between the hydrodynamic radius of plasmid

form I in the presence of the complexes and the free plasmid

gyration radius estimated by extrapolation is possibly due to an

electrostatic interaction between the positively charged

dicopper(II) complexes and the negatively charged phosphate

groups of pBR322, which can be responsible for DNA

115

scattering studies, the gyration radii (R ) of diﬀerent plasmid

compaction in the solutions containing 1 and 2.

g

1

14

DNAs have been measured.

From these results, the

The further addition of 1 (25 μM) or 2 (30 μM) caused a

signiﬁcant increase in the plasmid hydrodynamic radius to 130

± 50 or 150 ± 70 nm, respectively. The obtained values are

similar to those estimated for the circular plasmid form II

(∼125 nm). However, the generation of a second population

expected radii of pBR322 plasmids, used in the present

work, can be estimated by extrapolation (Figure S23 ) as 89 nm

for form I, 124 nm for form II, and 171 nm for form III. Upon

analysis of the observed radii, it is possible to infer that, at low

N

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Table 3. Growth Inhibition, Expressed as IC ± SD, of Several Cancer Cell Lines [HCT116 (Colon, Human), HT29 (Colon,

50

Human), MDA-MB-231 (Breast, Human), and B16F10 (Melanoma, Mouse)] after Incubation for 36 h in the Presence of the

a

Synthesized Compounds

H L1

3

1

H L2

3

2

cell line

HCT116

HT29

MDA-MB-231

B16F10

MDCK (control)

IC₅₀(μM)

SI

IC₅₀(μM)

SI

IC₅₀(μM)

SI

IC₅₀(μM)

SI

2.97 ± 0.54

2.09 ± 0.36

1.02 ± 0.24

0.88 ± 0.37

8.62 ± 0.34

2.90

4.12

8.45

9.79

0.56 ± 0.55

0.78 ± 0.47

0.90 ± 0.28

0.32 ± 0.46

0.65 ± 0.39

1.16

0.83

0.72

2.03

5.17 ± 0.12

4.20 ± 0.42

0.65 ± 0.26

0.59 ± 0.72

14.7 ± 0.31

2.84

3.5

22.6

24.9

1.42 ± 0.53

1.89 ± 0.48

1.21 ± 0.28

1.23 ± 0.45

2.46 ± 0.38

1.73

1.30

2.03

2.0

a

The selectivity index (SI) was estimated based on the IC₅₀values obtained for the nontumoral control MDCK cells, also included in the table. The

values in bold correspond to those in the submicromolar range.

of larger scattering particles (800 ± 300 and 800 ± 200 nm) is

observed as well. These particles could be related to the

formation of plasmid aggregates, making it diﬃcult to

guarantee the biopolymer form at this point. When plasmid

pBR322 is in the presence of higher concentrations of

complexes 1 (50 μM) or 2 (60 μM), only one population is

observed in each case, with a hydrodynamic radius of 167 ± 7

or 190 ± 10 nm, respectively. A single population indicates

that plasmid DNA disaggregates, and the size that observed

might indicate the occurrence of cleavage to form III, whose R_g

was estimated as ∼170 nm. On the other hand, the free ligands

H L1 and H L2 have not shown this property (Figure 8C). It

evaluated the their eﬀect and the eﬀect of their ligands on cell

proliferation, using a panel of diﬀerent cancer cell lines. By

performing tetrazolium salt reduction assays, we observed that

all of the compounds analyzed eﬀectively decreased the cancer

cell proliferation, in vitro, in a dose-dependent manner (data

not shown). Indeed, IC₅₀values obtained for all of the tested

cell lines ﬁt into the micromolar range, with even some

submicromolar values (Table 3). Interestingly, compounds 1

and 2 seemed to be more eﬀective than their respective

ligands, aﬀecting the cell proliferation at lower doses.

Nevertheless, these complexes showed a decreased selectivity

against cancer cells after analysis of their eﬀect on nontumoral

cells Madin−Darby canine kidney (MDCK).

The higher antiproliferative eﬀects among the compounds

tested are undoubtedly seen for 1, which is also the most

reactive complex regarding plasmid DNA cleavage (as shown

by the DLS studies). However, complex 1 is not the one with

the highest intercalation aﬃnity because 2 presents a binding

constant toward ctDNA 10 times greater than that determined

for 1. Interactions of complexes 1 and 2 with double-stranded

DNA seem to involve electrostatic (because both complexes

are cationic), intercalative, and coordinative components.

Therefore, in such a multifaceted panorama, pointing out the

most signiﬁcant factor for an improved antiproliferative activity

is not straightforward. On the other hand, our results suggest

that the DNA-damaging capacity is directly related to the

cytotoxicity, while a higher binding aﬃnity toward ctDNA

seems to be associated with an enhanced selectivity index.

Concerning the ligands, hydrolysis constitutes an aspect to

3

is noteworthy that the starting copper salt Cu(ClO ) ·6H O

4

2

has been previously reported as being nonactive toward the

112

cleavage of plasmid pBR322 DNA.

DLS measurements were repeated over 1 day, in order to

test the particles’ stability in the 1/DNA and 2/DNA samples

containing respectively ﬁnal concentrations of 50 and 60 μM.

After 24 h, no signiﬁcant changes were observed in the

hydrodynamic radii of 167 nm (1/DNA) and 190 nm (2/

DNA), which indicates that the process is, overall, irreversible.

The diﬀerence between the reversibility of the interactions

with BSA and those with DNA is mainly related to the

nuclease activity exhibited by the complexes. Although the

interactions of 1 and 2 with DNA certainly possess

electrostatic and intercalative components, covalent binding

involving an oxygen-donor atom from the biopolymer’s

phosphate diester backbone to one coordinatively unsaturated

copper center in the complexes should also be important. This

constitutes the ﬁrst step in the general catalytic mechanism of

hydrolysis proposed for artiﬁcial bioinspired phosphatases/

nucleases displaying bimetallic cores at their active sites. The

metal centers in such a class of catalysts usually work

cooperatively, and, in this sense, dissimilarities in terms of

their coordination spheres could increase the complementarity

needed in order to potentialize cooperativity between the

metal centers.

A smaller amount of complex 1, compared to complex 2, is

needed to cause an increase in the hydrodynamic radius of

pBR322 plasmid DNA, which is, in fact, the opposite trend

observed for their intercalation ability. In general, DLS results

indicate that complexes interact more strongly with DNA than

their free ligands, as expected based on the binding constants

determined by UV−visible spectroscopy. Additionally, the

complexes presented the capacity to cleave DNA, which

constitutes an assumed mode of action for potential anticancer

agents.

be taken into account, particularly in the case of H L2.

3

Nevertheless, they also present an interesting antiproliferative

proﬁle, with IC₅₀values in the low micromolar range. A

possible mechanism for the cytotoxicity exhibited by the

ligands could be related to the formation of metal complexes in

the culture medium or inside the cells. The compounds thus

generated could either be cytotoxic per se, or, alternatively,

their formation could deplete the available amount of some

physiologically relevant metal ions, such as iron(III). However,

additional work would be necessary in order to conﬁrm this

hypothesis. What is certain is that the free ligands are much

less toxic to the MDCK control cells than the dicopper(II)

complexes 1 and 2.

So, from a medicinal chemistry point of view, ligands H L1

3

and, especially, H L2 seem to be more promising than their

3

respective metal compounds because of their higher selectivity

indexes, particularly in the case of the human breast cell line

MDA-MB-231. However, their apparent susceptibility to

hydrolysis should impair this performance, although their

reversible binding to serum albumin could partially protect

Cytotoxic Activity. In order to determine the putative

antineoplastic activity of the synthesized compounds, we

O

DOI: 10.1021/acs.inorgchem.9b01195

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Inorganic Chemistry

Article

them from this undesirable side reaction. On the other hand,

the dicopper(II) complexes are much more stable and are also

able to bind serum albumin in a reversible manner. Despite

their higher intrinsic activity, as mentioned above, they are less

selective toward cancer cells (i.e., more toxic for the MDCK

control cells). Yet, binding to serum albumin could reduce

their toxicity in living systems. A better panorama regarding

the real signiﬁcance of these complexes as anticancer agents

can only be obtained after in vivo tests, which are already

underway and will be the subject of future reports.

can involve the formation of anchoring hydrogen bonds

between 2 and the biopolymer), the thiophene ring can

somehow increase the reactivity of 1 toward DNA. Probably

both complexes would be acting at the DNA level, promoting

replicative stress or (most likely) cleavage, leading to

checkpoint responses and the onset of apoptosis. However,

further studies should be addressed to conﬁrm this hypothesis.

Finally, the present work shows that aroylhydrazone-derived

binucleating ligands and their dinuclear μ-hydroxo dicopper-

(II) complexes may represent a promising structural starting

point for the development of highly active potential antitumor

agents.

CONCLUSIONS

■

Two novel C -symmetric binucleating ligands comprising

1

ASSOCIATED CONTENT

Supporting Information

hydrazonic moieties and their μ-hydroxo dicopper(II)

complexes, 1 and 2, were synthesized and fully characterized.

XRD analyses indicate that both coordination compounds are

very similar in structural terms and generate dimeric

arrangements containing two partially deprotonated ligands

and four divalent copper centers. Although UV−visible

measurements indicate that the free ligands are quite

susceptible to hydrolysis in a 90% water-containing medium,

complexation to copper(II) ions seems to prevent this

reaction. The dinuclear metal compounds reported in this

work are, indeed, very stable in aqueous solution, concerning

both ligand hydrolysis and complex dissociation. ESI-MS(+)

analyses conﬁrmed that the main metal-containing species

■

*

S

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.inorg-

chem.9b01195.

Optical photographs of ligands’ and metal complexes’

crystals (Figures S1 and S2), 1D and 2D NMR spectra

of the ligands (Figures S3−S8), NMR assignments of

the ligands (Table S1), additional crystallographic tables

(

Tables S2−S5), vibrational spectra of the compounds

Figures S9−S15), selected vibrational and UV−visible

absorption properties (Tables S6 and S7), UV−visible

stability proﬁles (Figures S16−S18), ESI-MS(+) exper-

imental and simulated isotopic patterns for the

complexes (Figure S19), DLS autocorrelation functions

and SAXS curves (Figures S20−S22), and estimation of

the diﬀerent plasmid pBR322 form radii by extrapolation

present in a 10% DMSO/water solution should be the

+

dinuclear cations characterized by XRD, [Cu (HL)(OH)] ,

2

as well as the DMSO-substituted derivative [Cu (L)-

2

+

(

DMSO)] , in which the μ-hydroxo bridge is replaced by a

terminal solvent molecule.

(

Figure S23) (PDF)

Both complexes and ligands show a high aﬃnity for BSA,

indicated by the observed static quenching of the protein

ﬂuorescence due to interaction with the compounds. Addi-

tionally, scattering techniques (DLS and SAXS) suggest that

the complexes and their free ligands interact with BSA in a

reversible manner. Because BSA is an important protein

involved in the transportation of drugs in the biological system,

possessing properties similar to those of HSA, the observed

results indicate that the new compounds could be targeted

through blood transport. The biological activity of the

compounds also includes interaction with ctDNA, with the

complexes showing a higher aﬃnity than their respective

ligands. Moreover, the plasmid pBR322 DNA cleavage results

from DLS indicate that the complexes interact electrostatically

with plasmid DNA at low concentrations. At the higher

concentrations of 50 μM (1) or 60 μM (2), in contrast, data

seem to point to the occurrence of DNA cleavage to form III

Accession Codes

CCDC 1851259 and 1851260 contain the supplementary

crystallographic data for this paper. These data can be obtained

free of charge via www.ccdc.cam.ac.uk/data_request/cif , or by

emailing data_request@ccdc.cam.ac.uk , or by contacting The

Cambridge Crystallographic Data Centre, 12 Union Road,

Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

AUTHOR INFORMATION

Corresponding Author

*E-mail: nicoarey@puc-rio.br.

■

ORCID

Ana Maria Percebom: 0000-0002-1511-8656

́

Nicolas A. Rey: 0000-0002-0624-7560

Notes

The authors declare no competing ﬁnancial interest.

(

linear). Over time, the value of the hydrodynamic radius for

plasmid DNA form III remained stable and, therefore, the

process is not reversible. Concerning the ability to cause

damage to plasmid pBR322 DNA, complex 1 is more active

than 2. Both ligands and their dicopper(II) complexes display

potent antiproliferative activity in the cancer cell lines

evaluated, occasionally even in the submicromolar range.

Once the eﬀect of the metal complexes on tumor cell

proliferation was analyzed, it was clear that the addition of

copper to the ligands H L1 and H L2 increased their activity.

Our data on the cellular models correlate quite well with the

DNA interaction experiments because IC₅₀values are smaller

for 1, which showed the higher activity on DNA by DLS

studies. Thus, although the presence of the furan ring seems to

be important for an improved DNA intercalation ability (which

ACKNOWLEDGMENTS

■

N.A.R. thanks Fundaca

Pesquisa do Estado do Rio de Janeiro (FAPERJ, Brazil) and

̧

o

̃

Carlos Chagas Filho de Amparo a

̀

́

Conselho Nacional de Desenvolvimento Cientiﬁco e Tecno-

logico (CNPq, Brazil) for the research fellowships awarded.

́

This work was ﬁnancially supported by FAPERJ, FINEP, and

FAPEMIG. J.P.R. is grateful to LABSO-Bio Laboratory and to

CAPES and CNPq for doctoral fellowships. The authors also

acknowledge the Brazilian National Synchrotron Laboratory

(LNLS-CNPEM) for SAXS beamtime (SAXS1-20160298),

3

́

io (Departamento de Quimica,

Prof. Ricardo Queiroz Aucel

PUC-Rio, Brazil) for the ﬂuorescence spectroscopy facilities,

and Dr. Michel

̀

e Salmain (Institut Parisien de Chimie

P

DOI: 10.1021/acs.inorgchem.9b01195

Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

Moleculaire, Universite

providing the ctDNA sample for the binding studies. The MS

measurement time available at Laboratorio Multiusuario de

Espectrometria de Massas da Universidade Federal Fluminense

is also highly appreciated by the authors.

Article

́

Pierre et Marie Curie, France) for

cleavage and cytotoxicity of mixed ligand copper(II) complexes of the

antibacterial drug nalidixic acid. J. Inorg. Biochem. 2017, 174, 1−13.

15) Hu, J.; Liao, C.; Guo, Y.; Yang, F.; Sang, W.; Zhao, J. A.

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Binucleating Hydrazonic Ligands and Their μ-Hydroxodicopper(II) Complexes as Promising Structural Motifs for Enhanced Antitumor Activity

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