Copper(ii) complexes with tridentate halogen-substituted Schiff base ligands: synthesis, crystal structures and investigating the effect of halogenation, leaving groups and ligand flexibility on antiproliferative activities

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Copper(II) complexes with tridentate

halogen-substituted Schiﬀ base ligands:

synthesis, crystal structures and investigating

the eﬀect of halogenation, leaving groups and

ligand ﬂexibility on antiproliferative activities†

b

Cite this: Dalton Trans., 2021, 50,

3990

Nazanin Kordestani,^aHadi Amiri Rudbari, *^aAlexandra R. Fernandes,

Luís R. Raposo, ^bAndré Luz,^bPedro V. Baptista, ^bGiuseppe Bruno,^c

*

Rosario Scopelliti, ^dZohreh Fateminia,^aNicola Micale, ^cNikolay Tumanov,

Johan Wouters, ^eAbolghasem Abbasi Kajani^fand Abdol-Khalegh Bordbar

e

a,g

To investigate the eﬀect of diﬀerent halogen substituents and leaving groups and the ﬂexibility of ligands on

the anticancer activity of copper complexes, sixteen copper(^II) complexes with eight diﬀerent tridentate

Schiﬀ-base ligands containing pyridine and 3,5-halogen-substituted phenol moieties were synthesized and

characterized by spectroscopic methods. Four of these complexes were also characterized by X-ray crystal-

lography. The cytotoxicity of the complexes was determined in three diﬀerent tumor cell lines (i.e. the A2780

ovarian, HCT116 colorectal and MCF7 breast cancer cell line) and in a normal primary ﬁbroblast cell line.

Complexes were demonstrated to induce a higher loss of cell viability in the ovarian carcinoma cell line

(A2780) with respect to the other two tumor cell lines, and therefore the biological mechanisms underlying

this loss of viability were further investigated. Complexes with ligand L₁(containing a 2-pycolylamine-type

motif) were more cytotoxic than complexes with L₂(containing a 2-(2-pyridyl)ethylamine-type motif). The

loss of cell viability in A2780 tumor cells was observed in the order Cu(Cl₂-L₁)NO₃> Cu(Cl₂-L₁)Cl > Cu(Br₂-

L₁)Cl > Cu(BrCl-L₁)Cl. All complexes were able to induce reactive oxygen species (ROS) that could be related

to the loss of cell viability. Complexes Cu(BrCl-L₁)Cl and Cu(Cl₂-L₁)NO₃were able to promote A2780 cell

apoptosis and autophagy and for complex Cu(BrCl-L₁)Cl the increase in apoptosis was due to the intrinsic

pathway. Cu(Cl₂-L₁)Cl and Cu(Br₂-L₁)Cl complexes lead to cellular detachment allowing to correlate with

the results of loss of cell viability. Despite the ability of the Cu(BrCl-L₁)Cl complex to induce programmed

cell death in A2780 cells, its therapeutic window turned out to be low making the Cu(Cl₂-L₁)NO₃complex

the most promising candidate for additional biological applications.

Received 18th November 2020,

Accepted 14th February 2021

DOI: 10.1039/d0dt03962d

rsc.li/dalton

^aDepartment of Chemistry, University of Isfahan, Isfahan 81746-73441, Iran.

E-mail: hamiri1358@gmail.com, h.a.rudbari@sci.ui.ac.ir

^bUCIBIO, Departamento Ciências da Vida, Faculdade de Ciências e Tecnologia,

Universidade NOVA de Lisboa, 2829-516 Caparica, Portugal.

E-mail: ma.fernandes@fct.unl.pt

Introduction

Cancer is the second leading cause of death worldwide and its

incidence is increasing.¹Transition metal complexes with

their versatile structures, redox behavior and physicochemical

properties have been found to be useful as active agents in

chemotherapeutic applications.²The serendipitous discovery

of cisplatin as an anticancer drug has initiated the investi-

gation in the medicinal bioinorganic research field. Cisplatin

[cis-diamminedichlorideplatinum(^II)], as one of the foremost

and widely used metal-based anticancer drugs,^3,4is currently

used for the treatment of a variety of cancers.⁵However, its

application is mostly limited by both its side-eﬀects and

acquired cellular resistance.^6,7Therefore, tremendous eﬀorts

have been made for the development of metal-based anti-

cancer compounds with less toxicity and higher eﬃciency,^8,9

^cDepartment of Chemical, Biological, Pharmaceutical and Environmental Sciences,

University of Messina, Viale Ferdinando Stagno D’Alcontres 31, I-98166 Messina,

Italy

^dInstitut des Sciences et Ingénierie Chimiques, École Polytechnique Fédérale de

Lausanne (EPFL), CH-1015 Lausanne, Switzerland

^eDepartment of Chemistry, Namur Institute of Structured Matter, University of

Namur, 5000 Namur, Belgium

^fApplied Physiology Research Center, Cardiovascular Research Institute, Isfahan

University of Medical Sciences, Isfahan, 81746-73461, Iran

^gCalifornia Institute for Quantitative Biosciences (QB3), University of California,

Berkeley, CA 94720, USA

†Electronic supplementary information (ESI) available. CCDC 1872089–1872092.

For ESI and crystallographic data in CIF or other electronic format see DOI:

10.1039/d0dt03962d

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particularly biocompatible copper(II) complexes that bind to complexes, such as chloride, nitrate and acetate, is necessary,

and cleave DNA under physiological conditions.¹⁰Copper has as reported in previous reports.²⁵

a long history of medical use, and its prospective antitumor

The significant aspect of this work is the investigation of

properties have recently attracted attention because it is the eﬀect of diﬀerent halogen substituents, the flexibility of

thought to be less toxic than nonessential metals such as ligands and leaving groups on the synthesized copper(^II) com-

platinum.^11,12Moreover, its complexes with tunable coordi- plexes with N,N,O-tridentate Schiﬀ-base containing phenolic

nation geometry in a redox active environment could find and pyridine rings and study of their potential applications in

better eﬃcacy at the cellular level.¹³Since 1969, copper has cancer research. For this purpose, 3,5-halogen-substituted

been found to possess high DNA binding aﬃnity.¹⁴This phenol moieties with similar and diﬀerent halogens were used

binding was dependent on the copper complex size, electron to investigate the role of halogen groups in anticancer activity.

aﬃnity, and geometry of the formed adduct, inducing an irre- Two diﬀerent substituted pyridine amines (2-(2-pyridyl)ethyl-

versible modification of the DNA conformational structure. amine and 2-picolylamine) were employed in order to consider

According to these observations, a large number of copper the ligand flexibility, and two kinds of copper(^II) salts, i.e. (Cu

complexes have been considered as DNA-targeting metallo- (NO₃)₂·H₂O and CuCl₂·H₂O), were also used to investigate the

drugs.¹⁵The premise that stoichiometric mixtures of copper eﬀect of the leaving group on anticancer activity.

ions and organic chelators can form a new class of protea-

Hence, we have synthesized and characterized eight

some inhibitors has been recently investigated by Dou’s ligands and sixteen complexes which are depicted in Tables 1

group.^16–18Recent studies have shown that a square-planar Cu and 2, respectively. These compounds were characterized by

(^II) complex chelated by an N,N,O-tridentate Schiﬀ-base con- elemental analysis and FT-IR and NMR spectroscopy.

taining phenolic and pyridine rings as coordinating groups Molecular structures of four complexes were solved by X-ray

has a remarkable capability to cleave DNA.^19,20However, 50% crystallography. In order to fulfill the aim of this research

of the top leading drugs on the market are halogen-substi- work, the antiproliferative potential of the complexes was

tuted.²¹In the drug design process, halogen atoms are often evaluated on three carcinoma cell lines (MCF-7, A2780 and

introduced in order to increase membrane permeability and HCT116) and the cytotoxicity profile on normal human

to prolong the drug’s half-life by delaying the catabolic dermal fibroblast cells.

process, which leads to drug degradation and loss of pharma-

cological activity in the organism.²²In fact, halogen for hydro-

gen substitution on aromatic rings of drugs aﬀords com-

pounds where the carbon–halogen bonds are catabolically

more stable than the corresponding C–H bonds. Usually,

halogen atoms in drugs or drug-like molecules are understood

to be involved in non-directional hydrophobic interactions or

just pointed into relatively empty spaces or cavities which they

tend to occupy without being involved in major stabilizing

contacts. However, since potential electron-rich sites such as

oxygen, nitrogen, and sulfur atoms as well as aromatic

π-electron systems are abundant in proteins, halogen atoms

can also form, stabilizing interactions such as halogen bonds

with the surrounding amino acids. A recent systemic investi-

gation of halogen bonds in protein–ligand complexes by

Hardegger et al. showed that halogen bonds can serve as a

powerful tool in increasing binding selectivity and binding

aﬃnity.²³Similarly, some metal complexes with the asym-

Table 1 All synthesized ligands

R₁

R₂

n

Br

Cl

I

Br

Cl

I

Br

Cl

I

Cl

Br

Cl

I

1

2

Br₂-HL₁

Cl₂-HL₁

I₂-HL₁

BrCl-HL₁

Br₂-HL₂

Cl₂-HL₂

I₂-HL₂

Br

Cl

BrCl-HL₂

Table 2 All synthesized complexes

R₁R₂

X

N

metric

N

_pyridineN′_amineO_phenolate

2,4-di-X-6-((pyridine-2-yl-

Br

Cl

I

Br

Cl

I

Br

Cl

I

Br

Cl

I

Br

Cl

I

Cl

Br

Cl

I

Cl

Br

Cl

I

Cl

Br

Cl

I

Cl

NO₃

Cl

1

2

Cu(Br₂-L₁)Cl

Cu(Cl₂-L₁)Cl

Cu(I₂-L₁)Cl

methylamino)methyl)phenol ligands HLx (where X = H, tert-

butyl, bromo- or iodo-substitued) were synthesized by Verani’s

group.^24–28The results showed that the bromo or iodo substi-

tuted species were eﬀective against cisplatin-resistant neuro-

Cu(BrCl-L₁)Cl

Cu(Br₂-L₁)NO₃

Cu(Cl₂-L₁)NO₃

Cu(I₂-L₁)NO₃

Cu(BrCl-L₁)NO₃

Cu(Br₂-L₂)Cl

Cu(Cl₂-L₂)Cl

Cu(I₂-L₂)Cl

Cu(BrCl-L₂)Cl

Cu(Br₂-L₂)NO₃

Cu(Cl₂-L₂)NO₃

Cu(I₂-L₂)NO₃

Cu(BrCl-L₂)NO₃

blastoma as well as for

a number of other tumors.

Furthermore, by comparing the results of some previous

reports, it can be concluded that the size of the ring that was

formed during the formation of a complex, or, in other words,

the ligand flexibility can be eﬀective on anticancer properties

of the compounds.^25–29Finally, to enrich the delivery

eﬃciency and enhance the anticancer activity and selectivity

of anticancer metal agents, having good leaving group(s) on

Cl

NO₃

Br

Cl

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As shown in Fig. 1–3, two mononuclear complexes con-

nected to each other by an oxygen atom of one nitrate group

(O(6)). The Cu(1)–O(6)–Cu(2) angles in Cu(Cl₂-L₂)NO₃, Cu(Br₂-

Results and discussion

Synthesis and characterization

The ligands were synthesized by Schiﬀ condensation of L₂)NO₃and Cu(BrCl-L₂)NO₃are 96.88(1), 96.11(1) and 96.22

2-hydroxy-3,5 halogen-substituted salicylaldehyde with 2-(2- (1)°, respectively, and are very near to each other (Table 4). The

pyridyl)ethylamine and 2-picolylamine in distilled water at O(6)–Cu(1) bond distance is 2.5750(1) Å in Cu(Cl₂-L₂)NO₃,

room temperature separately, according to our published work 2.5499(1) Å in Cu(Br₂-L₂)NO₃and 2.5604(5) Å in Cu(BrCl-L₂)

(Table 1).³⁰

NO₃(Table 4). These bond distances in all three structures are

The synthesis of the Cu(II) complexes was performed using a in the range of upper values for a long coordination distance

common procedure, by reaction of a stoichiometric amount of in Cu(^II) compounds. These distances clearly indicate a rela-

CuCl₂·H₂O and Cu(NO₃)₂·H₂O with the corresponding ligands, tively weak coordination of O(6) to Cu(1) atom. Without this

in methanol. These reactions yielded distorted square planar oxygen–copper interaction, we can assume these binuclear

mononuclear species in which the metal ion was surrounded complexes as two mononuclear complexes. Surely, this inter-

by a tridentate ligand (N, N, O) and one leaving group (Cl or action exists only in solid state and in the solution we cannot

NO₃) (Table 2). The sixteen resulting complexes, as green color see this oxygen–copper weak coordination. Perhaps, one of the

production, were analyzed by elemental analysis and FT-IR reasons for this lack of dimerization is some steric hindrance

spectroscopy and four of them were studied by X-ray diﬀraction for the coordination of any of the O-atoms due to the non-pla-

analysis. The shift of the imine band by complexation suggests narity of the chelate ring formed by the py–CH₂–CH₂–Nv arm

coordination via the imine nitrogen atom.³⁰Also the stretching of ligands.

vibration of the phenolic OH group of ligands disappeared in

the FT-IR spectra of the complexes.

Also, the distances between the Cu(1) atom and O-atom

(O3) of another nitrate group are 2.5920(1), 2.6823(1) and

2.6673(5) Å in Cu(Cl₂-L₂)NO₃, Cu(Br₂-L₂)NO₃and Cu(BrCl-L₂)

NO₃complexes, respectively (Table 4). These distances also

clearly indicate a relatively weak coordination of O(3) to Cu(1)

atom. The strong coordination of O(2) and weak coordination

of O(3) or, in other words, the asymmetric bidentate coordi-

nation of the nitrate (NO₃⁻) suggest the localization of the

negative charge predominantly on O(2). This is also reflected

in the longer N(3)–O(2) bond lengths (1.2848(1) Å in Cu(Cl₂-L₂)

NO₃, 1.2908(1) Å in Cu(Br₂-L₂)NO₃and 1.295(11) Å in Cu(BrCl-

L₂)NO₃than the N(3)–O(3) bond lengths (1.2828(1) Å in Cu

(Cl₂-L₂)NO₃, 1.2418(1) Å in Cu(Br₂-L₂)NO₃and 1.231(10) Å in

Cu(BrCl-L₂)NO₃. Without consideration of these two weak

X-ray crystal structures

Description of the crystal structures Cu(Cl₂-L₂)NO₃, Cu(Br₂-

L₂)NO₃and Cu(BrCl-L₂)NO₃. Views of the molecular structures

of Cu(Cl₂-L₂)NO₃, Cu(Br₂-L₂)NO₃and Cu(BrCl-L₂)NO₃complexes

with the common atom numbering scheme are shown in

Fig. 1–3, respectively. The crystallographic data and selected

bond lengths and angles are collected in Tables 3 and 4, respect-

ively. Single crystal X-ray analysis reveals that all complexes crys-

tallize in monoclinic P2₁/n space groups. All three complexes

have identical structures. Hence, the bond lengths and angles in

the three complexes are comparable to each other.

Fig. 1 ORTEP representation of Cu(Cl₂-L₂)NO₃. Displacement ellipsoids are drawn at the 50% probability level and H atoms are shown as small

−

spheres of arbitrary radii. Cu–O short contact is shown as open dashed lines. One NO₃groups are disordered over two sites and reﬁned with site

occupancy factors 0.73 : 0.27. Only the major component of the disordered NO₃⁻group is shown.

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Fig. 2 ORTEP representation of Cu(Br₂-L₂)NO₃. Displacement ellipsoids are drawn at the 50% probability level and H atoms are shown as small

spheres of arbitrary radii. Cu–O short contact is shown as open dashed lines.

Fig. 3 ORTEP representation of Cu(BrCl-L₂)NO₃. Displacement ellipsoids are drawn at the 50% probability level and H atoms are shown as small

spheres of arbitrary radii. Cu–O short contact is shown as open dashed lines.

oxygen–copper interactions, Cu(1)–O(3) and Cu(1)–O(6), the angles for the Cu(1) center is 699.7, 703.4 and 703.4° for Cu

two copper centers (Cu(1) and Cu(2)) exhibit distorted square (Cl₂-L₂)NO₃, Cu(Br₂-L₂)NO₃and Cu(BrCl-L₂)NO₃complexes,

planar geometry and consist of one tridentate Schiﬀ-base respectively, while the sum (∑) of the six inter-bond angles for

ligand for each copper center. The coordinating sites of copper the Cu(2) center is 689.6, 689.5 and 689.1° for Cu(Cl₂-L₂)NO₃,

atoms are occupied by one phenolate oxygen, one imine nitro- Cu(Br₂-L₂)NO₃and Cu(BrCl-L₂)NO₃complexes, respectively

gen and one pyridine-N atom of the mono-negative tridentate (Table 4). It deviates from the ideal angle of 720° in ideal

Schiﬀ-base ligand. The remaining site is occupied by one square planar geometry, suggesting that the copper(^II) centers

oxygen atom of the nitrate group.

have distorted square planar geometry, and that the deviation

The sum (∑) of the six inter-bond angles in four co- of square planar geometry in the Cu(2) center is greater than

ordinated complexes is a criterion for the deviation amount in that in the Cu(1) center. The reason for this greater deviation

square planar geometry. The sum (∑) of the six inter-bond of square planar geometry around Cu(2) is the existence of

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Table 3 Crystal data and structure reﬁnements

Cu(Cl₂-L₂)NO₃

C₂₈H₂₂Cl₄Cu₂N₆O₈

839.39

140.00(10)

1.54184

Monoclinic

P2₁/n

Cu(BrCl-L₂)NO₃

C₂₈H₂₂Br₂Cl₂Cu₂N₆O₈

928.31

120(2)

0.71073

Monoclinic

P2₁/n

Cu(Br₂-L₂)NO₃

C₂₈H₂₂Br₄Cu₂N₆O₈

1017.23

140.00(10)

1.54184

Monoclinic

P2₁/n

Cu(I₂-L₂)Cl

C₁₄H₁₁ClCuI₂N₂O

576.04

100.01(10)

0.71073

Triclinic

ˉ

P1

Empirical formula

Formula weight

Temperature (K)

Wavelength (Å)

Crystal system

Space group

Unit cell dimensions

a (Å)

b (Å)

c (Å)

α (°)

β (°)

14.1249(3)

10.0756(3)

21.5993(6)

90

90.8729(18)

90

14.3113(6)

10.1603(12)

21.679(4)

90

91.547(7)

90

14.25361(14)

10.15411(9)

21.8598(2)

90

90.3364(9)

90

7.6342(4)

8.3051(6)

13.3598(10)

74.533(6)

88.995(5)

79.331(5)

801.80(9)

2

γ (°)

Volume (Å³)

Z

3073.59(14)

4

3151.1(7)

4

3163.77(5)

4

Density (calculated) (Mg

1.814

1.957

2.136

2.386

m⁻³

)

Absorption coeﬃcient

5.453

4.117

8.110

5.377

(mm⁻¹

)

F(000)

Theta range for data

collection (°)

1688

3.713 to 75.978

1832

1.423 to 30.000

1976

3.692 to 75.303

538

3.379 to 29.597

Index ranges

−17 ≤ h ≤ 17

−12 ≤ k ≤ 12

−26 ≤ l ≤ 26

8565

98.5

−20 ≤ h ≤ 19

−14 ≤ k ≤ 14

−30 ≤ l ≤ 30

42 539

9213

99.5

Semi-empirical from

equivalents

Full-matrix least-squares on

F²

−11 ≤ h ≤ 17

−12 ≤ k ≤ 12

−27 ≤ l ≤ 27

22 226

6440

100.0

−10 ≤ h ≤ 10

−10 ≤ k ≤ 10

−8 ≤ l ≤ 18

3771

99.1

Reflections collected

Independent reflections

Data completeness (%)

Absorption correction

Gaussian

Analytical

Gaussian

Refinement method

Full-matrix least-squares

on F²

Full-matrix least-squares

on F²

Data/restraints/parameters 8565/87/463

9213/0/428

1.034

6440/0/435

1.101

3771/0/191

1.142

Goodness-of-fit on F²

1.055

Final R indices [I > 2σ(I)]

R₁= 0.0452

wR₂= 0.1350

R₁= 0.0483

wR₂= 0.1378

R₁= 0.0786

wR₂= 0.2150

R₁= 0.0953

wR₂= 0.2335

3.305 and −0.930

R₁= 0.0272

wR₂= 0.0724

R₁= 0.0281

wR₂= 0.0730

0.852 and −0.517

R₁= 0.0418

wR₂= 0.0751

R₁= 0.0568

wR₂= 0.0816

1.595 and −0.949

R indices (all data)

Largest diﬀ. peak and hole 0.444 and 0.1222

(e Å⁻³

CCDC number

)

1872092

1872091

1872090

1872089

O(6) of the N(4)N(5)O(5) plane with a deviation value of 0.915, (1)–C(8)–C(9)–C(10)–N(2) and C(23) in Cu(2)–N(4)–C(22)–C

0.910 and 0.925 Å for Cu(Cl₂-L₂)NO₃, Cu(Br₂-L₂)NO₃and Cu (23)–C(24)–N(5)) is displaced by 0.685 and 0.754 Å in Cu(Cl₂-

(BrCl-L₂)NO₃complexes, respectively.

L₂)NO₃, 0.687 and 0.768 Å in Cu(Br₂-L₂)NO₃, and 0.665 and

In all complexes, the six-membered chelate ring formed by 0.767 Å in Cu(BrCl-L₂)NO₃from the mean plane constituted

the salicylaldimine fragment of the ligand is planar (rms devi- by Cu(1)–N(1)–C(8)–C(9)–C(10)–N(2) and Cu(2)–N(4)–C(22)–C

ation: 0.078 and 0.038 Å for Cu(Cl₂-L₂)NO₃, 0.058 and 0.029 Å (23)–C(24)–N(5) rings. The CvN bond distances are in the

for Cu(Br₂-L₂)NO₃, 0.059 and 0.038 Å for Cu(BrCl-L₂)NO₃. range of 1.277(3)–1.297(10) Å for reported structures, which is

However the metal ion is displaced by 0.305 and 0.152 Å in consistent with the CvN bond when coordinated to a metal

Cu(Cl₂-L₂)NO₃, 0.220 and 0.116 Å in Cu(Br₂-L₂)NO₃, and 0.220 center (Table 4).³¹With considering two weak oxygen–copper

and 0.153 Å in Cu(BrCl-L₂)NO₃from the plane constituted by interactions, Cu(1)–O(3) and Cu(1)–O(6), the Cu(1) atom is in

the remaining five atoms. Thus in these complexes, the a distorted octahedral environment, where three donor atoms

chelate ring is folded along the O(1), N(1) line in the O(1)–C of the mono-negative tridentate Schiﬀ-base ligand (N(1), N(2)

(1)–C(6)–C(7)–N(1)–Cu(1) ring and along the O(5), N(4) line in and O(1)) and one oxygen atom of the nitrate group (O(2))

the O(5)–C(15)–C(20)–C(21)–N(4)–Cu(2) ring and they have a form a square planar arrangement around the Cu(1) atom

half-chair like conformation. As expected, the second six- and the two oxygen atoms of two diﬀerent nitrate groups

membered chelate ring formed by the py–CH₂–CH₂–Nv arm weakly coordinate to the Cu(1) at the axial positions.

of the ligand is not planar due to the two methylene groups As shown in Fig. 2, the O(3) atom has excessive deviation

in all complexes. Interestingly, this chelate ring has a half- from axial positions. Surely, this excessive deviation is due to

chair conformation in all complexes (Fig. 4). In the half-chair the required angles for the nitrate group (O–N–O angle in

−

conformation, one of the methylene C-atoms (C(9) in Cu(1)–N NO₃= 60°).

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Table 4 Selected bond lengths (Å) and angles (°) for Cu(Cl₂-L₂)NO₃, Cu(Br₂-L₂)NO₃, Cu(BrCl-L₂)NO₃and Cu(I₂-L₂)Cl

Cu(Cl₂-L₂)NO₃

Cu(BrCl-L₂)NO₃

Cu(Br₂-L₂)NO₃

Cu(I₂-L₂)Cl

Bond lengths (Å)

Cu(1)–O(1)

Cu(1)–O(2)

Cu(1)–O(3)

Cu(1)–O(6)

Cu(1)–N(1)

Cu(1)–N(2)

Cu(2)–O(5)

Cu(2)–O(6)

Cu(2)–N(4)

Cu(2)–N(5)

N(1)–C(7)

N(4)–C(21)

1.915(2)

2.071(9)

2.5920(1)

2.5750(1)

1.952(3)

2.023(3)

1.882(3)

1.998(2)

1.939(3)

1.984(3)

1.281(5)

1.284(5)

—

1.931(6)

2.042(6)

2.6673(5)

2.5604(5)

1.963(6)

2.048(7)

1.993(7)

2.010(5)

1.945(7)

1.993(7)

1.285(10)

1.297(10)

—

1.9223(16)

2.2.0267(17)

2.6823(1)

2.5499(1)

1.9583(18)

2.0407(19)

1.8813(17)

1.9997(16)

1.9445(19)

1.989(2)

1.903(4)

—

1.973(5)

1.987(5)

—

1.286(8)

—

1.277(3)

1.285(3)

—

Cu(1)–Cl(1)

2.2460(17)

Bond angles (°)

O(1)–Cu(1)–N(1)

O(1)–Cu(1)–O(2)

N(1)–Cu(1)–O(2)

O(1)–Cu(1)–N(2)

N(1)–Cu(1)–N(2)

O(2)–Cu(1)–N(2)

O(5)–Cu(2)–N(4)

O(5)–Cu(2)–N(5)

N(4)–Cu(2)–N(5)

O(5)–Cu(2)–O(6)

N(4)–Cu(2)–O(6)

N(5)–Cu(2)–O(6)

Cu(1)–O(6)–Cu(2)

91.72(11)

80.54(15)

166.61(15)

172.77(12)

95.46(12)

92.57(16)

93.77(12)

165.11(12)

95.92(12)

84.65(11)

160.16(11)

89.98(11)

96.88(1)

91.5(2)

81.5(2)

170.3(3)

172.7(3)

94.8(3)

92.6(3)

93.9(3)

165.1(3)

96.2(3)

84.3(2)

159.6(3)

90.0(3)

96.22(1)

91.68(7)

81.33(7)

170.52(7)

172.47(7)

94.96(8)

92.45(7)

93.83(8)

165.79(8)

95.98(8)

84.49(7)

159.61(7)

89.81(7)

96.11(1)

91.14(19)

—

158.7(2)

95.3(2)

—

One of the interesting interactions in crystal packing of all

The Cu(^II) ion is four-coordinate forming a distorted

the complexes is the existence of intermolecular halogen square planar coordination sphere, in which three positions

bonds (X-bonds). Halogen bonds (X-bonds) are interactions are occupied by two N atoms and one O atom from the

that occur between an organic halide (–Cl, –Br, –I) and a mono-negative tridentate Schiﬀ-base ligand, forming five-

bound electronegative atom (Lewis base), such as oxygen, and six-membered chelate rings, and the other one coming

nitrogen, or sulfur; the former of these is referred to as the from a coordinated chloride ion. The CuN₂O unit is located

X-bond donor while the latter is called the X-bond in a well plane with a mean deviation of 0.133 Å, while the

acceptor.^32,33Most of the halogen bonds in reported com- chloride ion is obvious out of the above plane with a devi-

plexes involved halogens bound to aromatic groups. Halogen ation value of 1.413 Å. Similar to Cu(Cl₂-L₂)NO₃, Cu(Br₂-L₂)

bonds involving aromatic rings are generally stronger than NO₃and Cu(BrCl-L₂)NO₃complexes, the five- and six-mem-

those involving aliphatic chains because aromatic moieties bered chelate rings in Cu(I₂-L₂)Cl have half-chair confor-

have electron-withdrawing properties that lead to larger σ- mation. The bond distances of Cu–O, Cu–N and Cu–Cl are in

holes.

the normal range compared to the reported complexes con-

As we can see for example for Cu(BrCl-L₂)NO₃in Fig. 5, only taining the analogous unsymmetrical tridentate Schiﬀ-base

halogens in the para position can involve in intramolecular ligands.^19,31

halogen bonds with one oxygen of the nitrate group in the

The sum (∑) of the six inter-bond angles for Cu(I₂-L₂)Cl

nearest neighbor molecule (O3). These X⋯O contacts are is 682.4°, which deviates from the ideal angle of 720° in

smaller than the sum of Bondi’s van der Waals radii of halo- ideal square planar geometry, suggesting that the geometry

gens (3.180 Å (Cl(4)⋯O(3)) for Cu(Cl₂-L₂)NO₃, 3.209 Å (Br(4) around copper(^II) is distorted square planar. Also, the cis

⋯O(3)) for Cu(Br₂-L₂)NO₃and 3.185 Å (Cl(1)⋯O(3)) for Cu angles in the basal plane involving the phenolate oxygen and

(BrCl-L₂)NO₃. The vdW radii for Br, Cl, and O atoms are 1.85, imine nitrogen (O(1)–Cu(1)–N(1), 91.14(19)°) and that invol-

1.75, and 1.52 Å, respectively, and the corresponding sum of ving imine nitrogen and pyridine nitrogen (N(1)–Cu(1)–N(2),

the vdW radii is Br + O = 3.37 and Cl + O = 3.27 Å.

95.3(2)°) deviate from the ideal value of 90°, suggesting that

Description of the crystal structure Cu(I₂-L₂)Cl. Complex Cu the square planar coordination geometry in Cu(I₂-L₂)Cl is

ˉ

(I₂-L₂)Cl crystallizes in the triclinic P1 space group such that slightly distorted due to the chelate eﬀect. The CvN bond

the asymmetric unit contains one complex molecule. A repre- distance is 1.286(8) Å (N1vC7), which is consistent with a

sentative molecular structure along with a selected atom num- slight elongation of the CvN double bond when coordinated

bering scheme is depicted in Fig. 6, while the crystal structure to a late metal center.³⁴

refinement data and important bond distances and angles are

given in Tables 3 and 4, respectively.

As we can see in Fig. 7, there is intermolecular halogen–

halogen type I interaction in the Cu(I₂-L₂)Cl complex.³⁵The

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Fig. 4 (a) Coordination environments around atoms Cu(1) with considering weak oxygen–copper interaction; (b) coordination environments

around atoms Cu(1) and Cu(2) without considering weak oxygen–copper interaction; (c) half-chair like conformation of the chelate rings formed by

the salicylaldimine fragment of ligands; (d) the half-chair conformation of the chelate rings formed by the py–CH₂–CH₂–Nv arm of ligands.

Fig. 5 Halogen bonds (X-bonds) in Cu(BrCl-L₂)NO₃.

halogen atoms in the ortho and para positions are involved in sum of Bondi’s van der Waals radii of halogens. A statistical

the halogen–halogen interaction with ortho and para halogens study proved that at distances inferior to the sum of van der

of another molecule, respectively, with an I⋯I distance of Waals radii type I interactions are dominant, while at distances

3.754 Å for I_ortho⋯I_orthointeraction and 3.862 Å for I_para⋯I_paraclose to the value of that sum halogen bonds prevail. However,

interaction. This should be noted that the vdW radius for I when the contact distance is superior to the van der Waals

atoms is 1.98 Å and the corresponding sum of the vdW radii is radii sum, type I becomes more frequent, particularly for I⋯I

I + I = 3.96 Å. Then, these I⋯I contacts are smaller than the contacts.³⁶

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Fig. 6 ORTEP representation of Cu(I₂-L₂)Cl. Displacement ellipsoids are drawn at the 50% probability level and H atoms are shown as small spheres

of arbitrary radii.

Fig. 7 A side view representation of Cu(I₂-L₂)Cl, showing the association of the adjacent molecules through halogen⋯halogen interactions (I(1)⋯I

(1) or I_ortho⋯I_orthodistance: 3.754 Å; I(2)⋯I(2) or I_para⋯I_paradistance: 3.862 Å.

Biological assays. Antiproliferative potential and biological

mechanisms triggered by the complexes

(Cl₂-L₁)NO₃complexes induce a higher antiproliferative activity

in A2780 cells compared to HCT116 cells (Fig. 8 and ESI

Fig. S1 and Table S1†). In contrast, in MCF7 cells only the Cu

The antiproliferative eﬀects of Cu(^II) complexes were evaluated (Cl₂-L₁)NO₃complex, the one with the strongest antiprolifera-

in three human cancer cell lines, namely ovarian carcinoma tive activity in all tumor cell lines, shows a significant antipro-

(A2780), colorectal carcinoma (HCT116) and breast adeno- liferative activity (ESI Fig. S1†). It is interesting to note that

carcinoma (MCF7) using the MTS assay. Amongst all sixteen complexes with L₁appear to be more active than complexes

complexes, Cu(Cl₂-L₁)Cl, Cu(Br₂-L₁)Cl, Cu(BrCl-L₁)Cl and Cu with L₂(Fig. 8, Table 5 and ESI Fig. S1 and Table S1†). The six-

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Fig. 8 Antiproliferative eﬀect of complexes Cu(Cl₂-L₁)Cl, Cu(Br₂-L₁)Cl, Cu(BrCl-L₁)Cl and Cu(Cl₂-L₁)NO₃in A2780 (light grey) and HCT116 (dark

grey) cancer cell lines exposed to increasing concentrations of each complex for 48 h evaluated by the MTS method. The vehicle control condition

was DMSO 0.1% (v/v) (negative control). The results shown are expressed as the mean SD from three independent assays. The symbol * indicates

that p-value <0.05.

Table 5 Values of relative IC₅₀(µM) in HCT116, A2780 and MCF7 cancer lines and in human normal primary dermal ﬁbroblasts. n.d. – not

determined

Cell lines

Cu(Cl₂-L₁)Cl (µM)

Cu(Br₂-L₁)Cl (µM)

Cu(BrCl-L₁)Cl (µM)

Cu(Cl₂-L₁)NO₃(µM)

Cisplatin (µM)

HCT116

A2780

MCF7

25.5 5.52

10.1 2.77

43.4 8.73

31.6 5.19

25.2 7.67

11.0 1.85

>50

29.1 7.35

13.5 3.31

44.2 4.06

25.17 9.50

18.1 1.78

4.2 2.2

29.9 6.86

34.0 6.26

15.6 0.6 (ref. 41)

3.4 0.2 (ref. 42)

>50

Fibroblasts

14.8 8.44

8.8 0.9

membered chelate ring formed by the py–CH₂–CH₂–Nv arm

To evaluate the eﬀect of these complexes in normal human

of ligand L₂, unlike five-membered chelate ring in ligand cells, human primary dermal fibroblasts were also exposed to

L₁,^37–39is not planar due to the two methylene groups in all increasing concentrations of the complexes (Fig. 9). As

complexes and might make it diﬃcult for complexes to exhibit observed in Fig. 9 and Table 5, complex Cu(Br₂-L₁)Cl induces a

cytotoxic activity within cells (Fig. 1–3 and 6). The loss of cell higher antiproliferative activity compared to the other three

viability in A2780 tumor cells was observed in the order Cu complexes Cu(BrCl-L₁)Cl, Cu(Cl₂-L₁)Cl and Cu(BrCl-L₁)NO₃

(Cl₂-L₁)NO₃> Cu(Cl₂-L₁)Cl > Cu(Br₂-L₁)Cl > Cu(BrCl-L₁)Cl (with Cu(BrCl-L₁)Cl

>

Cu(Cl₂-L₁)Cl

>

Cu(Cl₂-L₁)NO₃).

(Fig. 8 and Table 5). Interestingly, independently of the ligand Considering that the IC₅₀of the complex Cu(Br₂-L₁)Cl in

(L₁or L₂), the order of cytotoxicity is Cu(Cl₂-L_n)X > Cu(Br₂-L_n)X dermal fibroblasts is very similar to the IC₅₀in A2780

> Cu(BrCl-L_n)X ∼ Cu(I₂-L_n)X (where L_n: L₁or L₂and X: Cl or (Table 5), no therapeutic window exists for its application

NO₃) and it seems that the cytotoxic activity might be corre- without inducing normal cells cytotoxicity.

lated with the electronegativity of the R₁/R₂substitutions (Cl₂>

If we calculate the in vitro selectivity of complexes in

Br₂> I₂). Compared with cisplatin, Cu(Cl₂-L₁)NO₃displayed a A2780 tumor cells compared to normal cells it is clear that

similar IC₅₀value, and Cu(Cl₂-L₁)Cl and Cu(Br₂-L₁)Cl showed complex Cu(Cl₂-L₁)NO₃is the most promising with a higher

IC₅₀values 3-fold higher (Table 5). When we compare the therapeutic window followed by Cu(Cl₂-L₁)Cl (higher SI;

eﬀect of the leaving group (X = NO₃or Cl) in complexes Cu Table 6).

(Cl₂-L₁)X biological activity, a decreased cytotoxic activity is

observed for X = Cl that might be correlated with NO₃being an followed by Cu(Cl₂-L₁)Cl seems to be highly dependent on the

easier leaving group compared to Cl⁴⁰and/or due to a diﬀerent higher cytotoxic activity of both complexes (Cu(Cl₂-L₁)NO₃

It is interesting to note that this higher SI for Cu(Cl₂-L₁)NO₃

>

internalization within cells (see the below discussion).

Cu(Cl₂-L₁)Cl) in A2780 cells (since their IC₅₀values in normal

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Fig. 9 Cytotoxicity of complexes Cu(Cl₂-L₁)Cl, Cu(Br₂-L₁)Cl, Cu(BrCl-L₁)Cl and Cu(Cl₂-L₁)NO₃in normal human dermal ﬁbroblasts exposed to

increasing concentrations of each complex for 48 h evaluated by the MTS method. The vehicle control condition was DMSO 0.1% (v/v) (negative

control). The results shown are expressed as the mean SD from three independent assays. The symbol * indicates that p-value <0.05.

Table 6 Selectivity index (SI) of Cu(II) complexes and cisplatin in A2780

cells compared to normal primary dermal ﬁbroblasts

Cu(Cl₂-L₁)NO₃, cells were exposed to the IC₅₀concentrations

(Table 5) of each complex for 48 h and analyzed for the pres-

ence of cells in apoptosis (early or late) and necrosis (Fig. 10).

Cu(Cl₂-L₁)Cl Cu(Br₂-L₁)Cl Cu(BrCl-L₁)Cl Cu(Cl₂-L₁)NO₃Cisplatin

Our results indicate that the exposure of A2780 cells to the

SI 3.1

1.3

1.5

8.1

2.6

IC₅₀concentrations of Cu(Br₂-L₁)Cl, Cu(BrCl-L₁)Cl and Cu(Cl₂-

L₁)NO₃led to a statistically significant increase of cells in late

apoptosis when compared to the DMSO control (0.6% in cells

exposed to Cu(Cl₂-L₁)Cl, 0.7% in cells exposed to Cu(Br₂-L₁)Cl,

38.2% in cells exposed to Cu(BrCl-L₁)Cl, 2.3% in cells exposed

to Cu(Cl₂-L₁)NO₃compared to 0.4% in DMSO control)

(Fig. 10). Interestingly, exposure of A2780 cells to IC₅₀concen-

tration of Cu(BrCl-L₁)Cl also displayed a very high increase in

cells in early apoptosis when compared to the DMSO control

(15.2% versus 2.2%) (Fig. 10). Interestingly, in the presence of

complex Cu(BrCl-L₁)Cl the number of cells in apoptosis is even

higher than that for cisplatin (38.9% versus 14.2%, respect-

ively). None of the complexes were able to induce necrosis

(Fig. 10). Complex Cu(Cl₂-L₁)Cl induced the lowest levels of

apoptosis, 2.7% of early and 0.6% of late apoptosis when com-

fibroblasts are similar). In contrast, the lower SI for Cu(Br₂-L₁)

Cl and Cu(BrCl-L₁)Cl is due to their higher levels of cytotoxicity

in normal fibroblasts. Moreover, Cu(Cl₂-L₁)NO₃and Cu(Cl₂-L₁)

Cl show a higher SI compared to cisplatin that might be attrib-

uted to the higher cytotoxicity (lower IC₅₀) of cisplatin in

normal fibroblasts (Tables 5 and 6 and ESI Fig. S2†).

Considering the higher sensitivity of the complexes in the

ovarian cancer cell line, A2780, this cell line was selected for

further studies (Fig. 8 and Table 5).

To further characterize the cell death mechanisms respon-

sible for the decrease of A2780 cell viability in the presence of

the complexes, Cu(Cl₂-L₁)Cl, Cu(Br₂-L₁)Cl, Cu(BrCl-L₁)Cl and

Fig. 10 Cell death mechanism induced in A2780 cells after 48 h exposure to the IC₅₀concentrations of Cu(Cl₂-L₁)Cl, Cu(Br₂-L₁)Cl, Cu(BrCl-L₁)Cl

and Cu(Cl₂-L₁)NO₃evaluated by ﬂow cytometry. DMSO 0.1% (v/v) was used as solvent control and cisplatin (3.4 µM; IC₅₀) was used as a positive

control. The results are expressed as the mean SD from three independent assays. The symbol * indicates a p-value <0.05.

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pared to the DMSO control, 2.2% and 0.4%, respectively,

despite with statistical significance (Fig. 10) indicating that

other types of cell death might be involved in the loss of cell

viability.

To further confirm the higher induction of apoptosis in

A2780 cells exposed to Cu(BrCl-L₁)Cl, the mitochondrial mem-

brane potential was analysed using the JC-1 monomer/aggre-

gate ratio (Fig. 11). JC-1 is a monomeric fluorescent dye that

aggregates when in contact with the normal mitochondrial

membrane potential.⁴³The aggregation of the dye causes a

redshift in the emission spectrum.⁴³By measuring the green

and red fluorescence it is possible to obtain a monomer/aggre-

gate ratio. Normalizing the ratio for the vehicle control, DMSO

0.1% (v/v) it is possible to observe a hyperpolarization of the

mitochondrial membrane when the monomer/aggregate ratio

is <1 and a depolarization of the mitochondrial membrane

when the monomer/aggregate ratio is >1 (Fig. 11).

The results indicate that exposure of A2780 cells to Cu(BrCl-

L₁)Cl promotes a depolarization of the mitochondrial mem-

brane potential, at levels similar to cisplatin (Fig. 11).

Depolarization of mitochondrial membrane potential is an

early event in the induction of apoptosis through intracellular

signalling, the intrinsic pathway.⁴⁴These results indicate that

Cu(BrCl-L₁)Cl cytotoxicity may be due to induction of intrinsic

apoptosis in the A2780 ovarian cancer cell line (Fig. 11).

Considering that for Cu(Cl₂-L₁)Cl and Cu(Br₂-L₁)Cl com-

plexes a lower level of apoptosis was triggered (Fig. 10);

another type of cell death mechanism – autophagy – was

studied for all complexes (Fig. 12).

Fig. 12 Induction of autophagy in the A2780 cell line by ﬂow cytome-

try. Cells were exposed to IC₅₀concentrations of Cu(Cl₂-L₁)Cl, Cu(Br₂-

L₁)Cl, Cu(BrCl-L₁)Cl and Cu(Cl₂-L₁)NO₃for 48 h. Controls were per-

formed with cells treated with DMSO 0.1% (v/v), vehicle control, and

Rapamycin 0.02 μM as autophagy positive control. Cisplatin 3.4 µM, was

also used as a positive control. Results were normalized to the DMSO

0.1% control and expressed as the mean SD from three independent

assays. The symbol * indicates a p-value <0.05.

(Fig. 12). However, this eﬀect was much more pronounced for

When compared to A2780 cells exposed to the DMSO 0.1% Cu(BrCl-L₁)Cl, Cu(Cl₂-L₁)NO₃and cisplatin, respectively, with

(v/v) control, all complexes were able to trigger autophagy 2.2×, 4.2× and 5.5× more autophagic cells in A2780 exposed for

48 h compared to the control (Fig. 12). This result indicates

that for all complexes but particularly, for Cu(BrCl-L₁)Cl and

Cu(Cl₂-L₁)NO₃the loss of cell viability is due to the induction

of both apoptosis and autophagy. What is more, results of cell

viability (IC₅₀) correlate with the autophagic potential particu-

larly for Cu(Cl₂-L₁)NO₃and cisplatin in the order cisplatin >

Cu(Cl₂-L₁)NO₃≫ Cu(Cl₂-L₁)Cl, Cu(Br₂-L₁)Cl and Cu(BrCl-L₁)Cl.

Mitochondria are the main site for reactive oxygen species

(ROS) formation. An increase in the levels of ROS has been

described and correlated with the induction of programmed

cell death in eukaryotic cells.^44,45Thus, an evaluation of intra-

cellular ROS production was performed in A2780 cells exposed

to IC₅₀concentrations of the complexes Cu(Cl₂-L₁)Cl, Cu(Br₂-

L₁)Cl, Cu(BrCl-L₁)Cl and Cu(Cl₂-L₁)NO₃(Fig. 13).

The exposure of A2780 cells to the IC₅₀concentrations of

Cu(Cl₂-L₁)Cl, Cu(Br₂-L₁)Cl, Cu(BrCl-L₁)Cl and Cu(Cl₂-L₁)NO₃

revealed that intracellular ROS were, respectively, 1.3×, 1.2×,

1.5× and 2.4× higher, with statistical significance, compared to

A2780 cells exposed to DMSO 0.1% (v/v), (Fig. 13). Our results

Fig. 11 Normalized mitochondrial membrane potential ratios of A2780

cells incubated for 48 h with cisplatin 3.4 µM (positive control) and with seem to point that the increase in intracellular ROS plays an

16.5 µM (IC₅₀concentration) of Cu(BrCl-L₁)Cl determined by ﬂow cyto-

metry using JC-1 dye. Ratios were normalized with the vehicle control

DMSO 0.1% (v/v). The ﬂuorescence was measured by ﬂow cytometry in

BL1 (530/30 nm) and BL2 (574/26 nm) channels. The results are

important role in the cytotoxicity of Cu(Cl₂-L₁)Cl, Cu(Br₂-L₁)Cl,

Cu(BrCl-L₁)Cl and Cu(Cl₂-L₁)NO₃in the A2780 cell line

(Fig. 13) and might be correlated with the loss of cell viability

(Fig. 8) and induction of cell death via apoptosis and auto-

phagy (Fig. 10–12). Once again the higher loss of cell viability

expressed as the mean SD from three independent assays. The symbol

* indicates a p-value <0.05.

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Fig. 14 Accumulation of complexes in the cellular fraction as evaluated

by ICP-AES determination of Cu in the cellular fractions of A2780

ovarian cancer cultures after by 3 h exposure to the respective IC₅₀of

the complexes. The results presented are average (normalized to

Fig. 13 Reactive oxygen species (ROS) induced by 48 h exposure of

A2780 cells to IC₅₀concentrations of Cu(Cl₂-L₁)Cl, Cu(Br₂-L₁)Cl, Cu

(BrCl-L₁)Cl and Cu(Cl₂-L₁)NO₃evaluated by ﬂow cytometry. DMSO 0.1%

(v/v) was used as the solvent control and H₂O₂25 µM and cisplatin

3.4 µM were used as positive controls. The results are expressed as the

control)

SD of three independent assays. The symbol * indicates a

p-value <0.05.

mean

SD from three independent assays. The symbol * indicates a

p-value <0.05.

Cu(Cl₂-L₁)NO₃and high intracellular ROS and cell death

(Fig. 8 and 12–14). Higher levels of intracellular ROS have been

described as either a consequence of or a trigger of diﬀerent

(lower IC₅₀) (Table 5) is observed for cisplatin and Cu(Cl₂-L₁) types of cell death.^46–49However, in A2780 cells exposed to Cu

NO₃in agreement with the higher ROS induction (Fig. 13) trig- (Cl₂-L₁)Cl, only a small increase in autophagy was observed

gering a higher cell death (autophagy) (Fig. 12).

(1.4× higher compared to DMSO control) (Fig. 12). Similarly,

To better explain these results, we have assessed the intern- A2780 cell line exposure to Cu(Br₂-L₁)Cl led to a statistically

alization of complexes by measuring intracellular Cu by significant but small increase of late apoptosis, 0.7% when

ICP-AES in A2780 cells exposed to the IC₅₀of each complex compared to 0.4% of late apoptotic cells observed in the

(Fig. 14). Since copper is an essential metal present within our DMSO control (Fig. 10).

cells, results were normalized by the amount of Cu within cells

To try to add more insights into the diﬀerences in apoptosis

not exposed to the Cu(^II) complexes. Interestingly, when we and autophagy observed for Cu(Cl₂-L₁)Cl and Cu(Br₂-L₁)Cl

compare the % of intracellular Cu after exposure of cells to Cu complexes, A2780 cells were exposed for 48 h to 0.1× IC₅₀, IC₅₀

(Cl₂-L₁)NO₃or Cu(Cl₂-L₂)NO₃it is clear that there is an and 10× IC₅₀concentrations of complexes Cu(Cl₂-L₁)Cl and Cu

increased internalization of complex with the L₁ligand com- (Br₂-L₁)Cl and cell death was evaluated with the trypan blue

pared to complex with the L₂ligand (Fig. 14) that might corre- exclusion method (Fig. 15 and Fig. S3†). Trypan blue is a vital

late with its higher cytotoxicity towards A2780 cells (Table 5 dye impermeable to viable cells and stains cells with a compro-

and Fig. 8). Moreover, when we compare the four complexes mised plasma membrane (dead cells will become blue).⁵⁰

with the L₁ligand, Cu(Cl₂-L₁)NO₃, Cu(BrCl-L₁)Cl, Cu(Cl₂-L₁)Cl Using the trypan blue method, we are able to stain not only

and Cu(Br₂-L₁)Cl, the % of intracellular Cu follows the intra- the adherent cells but also cells in the supernatant (detached

cellular ROS levels and autophagic cell death, with Cu(Cl₂-L₁) from the bottom of the culture wells) and scored them as live

NO₃> Cu(BrCl-L₁)Cl > Cu(Cl₂-L₁)Cl > Cu(Br₂-L₁)Cl, (Fig. 13 and or dead (Fig. 15 and S3†). Results presented in Fig. 15 are rep-

12, respectively).

resented as percentage of cells and in ESI Fig. S3† as the total

Taken together, our results show that 48 h of exposure to number of cells.

the IC₅₀concentration of Cu(BrCl-L₁)Cl in the A2780 cell line

It is possible to observe that a concentration of 0.1 × IC₅₀of

leads to the induction of intracellular ROS which trigger the complexes Cu(Cl₂-L₁)Cl and Cu(Br₂-L₁)Cl induces an increase

induction of apoptosis, via the depolarization of the mitochon- of adherent dead cells (Fig. 15A and Fig. S3A†). When the con-

drial membrane and autophagy (Fig. 10–13). For complex Cu centrations of complexes Cu(Cl₂-L₁)Cl and Cu(Br₂-L₁)Cl raise

(Cl₂-L₁)NO₃the same eﬀect is observed but autophagy seems to the IC₅₀, there is an increase of both living and dead cells in

to be the preferable mechanism of cell death triggered by the culture media supernatant, which indicates a detachment

complex exposure (Fig. 10–13). Also the % of intracellular Cu of cells from the surface of the culture wells (Fig. 15B and

correlates with the induction of higher cytotoxicity levels for Fig. S3B†).

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Fig. 15 Evaluation by the trypan blue exclusion method of the percentage of live and dead cells in the population of adherent and in the culture

media supernatant (non-adherent) A2480 cells. Cells were incubated for 48 h with DMSO 0.01% (v/v) (A), DMSO 0.1% (v/v) (B) and DMSO 1% (C) as

solvent controls and with 0.1 × IC₅₀(A), 1 × IC₅₀(B) and 10 × IC₅₀(C) concentrations of complexes Cu(Cl₂-L₁)Cl and Cu(Br₂-L₁)Cl. The results shown

are expressed as the mean SD from three independent assays.

This detachment of cells is dependent on the concentration of them by X-ray crystallography. In vitro cytotoxicity assay

of complexes, since both the percentage and number of cells revealed an antiproliferative activity of Cu(Cl₂-L₁)Cl, Cu(Br₂-L₁)

in the supernatant increase when the concentration of com- Cl, Cu(BrCl-L₁)Cl and Cu(Cl₂-L₁)NO₃towards the A2780

plexes is raised to 10 × IC₅₀(Fig. 15C and Fig. S3C†). ovarian carcinoma cell line compared to the other tumor cell

Therefore, incubation with complexes Cu(Cl₂-L₁)Cl and Cu lines studied (HCT116 and MCF7). Complex Cu(Cl₂-L₁)NO₃

(Br₂-L₁)Cl leads to A2780 cellular detachment from culture demonstrated a higher selectivity index towards A2780 cells

plates (Fig. 15 and Fig. S3†). These results may shed some compared to normal fibroblasts. Interestingly, when we com-

light into the reason why a low level of apoptosis or even auto- pared internalization of Cu(Cl₂-L₁)NO₃and Cu(Cl₂-L₂)NO₃

phagy was detected in A2780 exposed to the IC₅₀concen- clearly the amount of intracellular Cu correlates with cyto-

trations of complexes Cu(Cl₂-L₁)Cl and Cu(Br₂-L₁)Cl (Fig. 10 toxicity (Cu(Cl₂-L₁)NO₃≫ Cu(Cl₂-L₂)NO₃). Moreover, the % of

and 13). The methods used for the detection of both types of internalized Cu was higher for Cu(Cl₂-L₁)NO₃compared with

cell death rely on protocols designed to test adherent cells and the other Cu(^II) complexes with the L₁ligand also agreeing

not cells in the culture medium (supernatant), meaning that with the cytotoxicity and higher selectivity towards A2780 cells.

an underestimation of apoptosis and autophagy through ana-

lysis by flow cytometry might occur.

Exposure of A2780 cells to the four complexes, Cu(Cl₂-L₁)Cl,

Cu(Br₂-L₁)Cl, Cu(BrCl-L₁)Cl and Cu(Cl₂-L₁)NO₃leads to an

Comparing our cytotoxicity results with similar complexes increase in the intracellular ROS that could be related to the

described the literature is not an easy task since diﬀerent induced cell death and loss of cell viability. Indeed, the

methodologies (e.g. diﬀerent cell lines used and time points) amount of Cu internalized due to the incubation of cells with

are used. However, a comparison might be made with results the four complexes could be correlated with the level of intra-

of Maheswari et al., 2006 where the authors used Cu(^II) com- cellular ROS and trigger of cell death.

plexes with Schiﬀ-base ligands that include substituted salicy-

The Cu(BrCl-L₁)Cl complex was able to trigger apoptosis via

laldehyde, Hpyrimol, salicylaldehyde semicarbazone and sali- the intrinsic pathway, and also autophagy. Although the Cu

cylaldehyde hydrazone ligands. In this work the authors report (Cl₂-L₁)NO₃complex also triggered both cell death mecha-

an IC₅₀value of 3.4 µM in the A2780 cancer cell line for a Cu nisms, a higher level of autophagy was induced. Incubation of

(^II) complex with a Hpyrimol ligand, 3× lower than the com- A2780 cells with Cu(Cl₂-L₁)Cl seemed to trigger cell death via

plexes here described.¹⁹Other studies report IC₅₀values autophagy and Cu(Br₂-L₁)Cl triggered apoptosis. Nevertheless,

ranging from 1 to 55 µM in diﬀerent cell lines that include Cu(Cl₂-L₁)Cl and Cu(Br₂-L₁)Cl complexes lead to cellular

A2780, MCF7, MOLT-4, A549, SK-II, HCT8, ME180 and detachment, underestimating the number of cells measured

L1210.^{19,20,51–55}These Cu(II) complexes have also been by flow cytometry and allowing to correlate with the results of

described to trigger apoptosis which is in line with the results loss of cell viability. Cu(^II) complexes with Schiﬀ-base ligands

indicated in the present work.^{19,20,52–56}

have been previously reported to trigger apoptosis in vitro

which supports our findings.

Conclusion

Experimental section

Chemicals and instrumentation

To investigate the eﬀect of diﬀerent halogen substituents,

leaving groups and flexibility of ligands on the anticancer

activity of copper complexes, a series of copper(^II) complexes Copper(^II) chloride, copper(II) nitrate, 3,5-dibromosalicylalde-

containing tridentate Schiﬀ-base ligands have been prepared hyde, 3,5-diiodosalicylaldehyde, 3,5-dichlorosalicylaldehyde,

and characterized by several spectroscopic methods, and some 3-bromo-5-chlorosalicylaldehyde, salicylaldehyde, 2-(2-pyridyl)

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ethylamine and 2-picolylamine were purchased from Sigma- to the instructions of the manufacturer, cells were detached

Aldrich and used without further purification. The commercial with trypsin and washed with PBS 1× before being incubated

solvents were distilled and then used for preparation of at room temperature for 15 minutes with Annexin V-Alexafluor

ligands and complexes. The FT-IR spectrum was recorded on a 488 assay solution and 10 μg mL⁻¹propidium iodide.^43,57Cells

JASCO, FT/IR-6300 spectrometer (4000–400 cm⁻¹) in KBr were analyzed in an Attune acoustic focusing cytometer

pellets. The elemental analysis was performed on Leco, (ThermoFisher Scientific) and results were analyzed with the

CHNS-932 and PerkinElmer 7300 DV elemental analyzers.

respective software (Attune Cytometric Software, vs. 2.1).

Biological assays

Alterations of mitochondrial membrane potential

Cell culture. A2780 and HCT116 cancer derived cell lines JC-1 dye, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolo-

(from human ovary and colorectal carcinomas, respectively) carbocyanine iodide, (Abnova, Taipei, Taiwan) was used to

and normal human dermal fibroblasts were cultivated in RPMI evaluate alterations in the mitochondrial membrane

1640 medium (A2780) or in Dulbecco’s modified Eagle’s potential.^43,57JC-1 is a green fluorescent dye in the monomeric

medium (DMEM) (HCT116 and fibroblasts). All culture media form. However, when the dye is in contact with the intact

were supplemented with 1% (v/v) PenStrep solution and 10% electrochemical gradient of the mitochondrial membrane, it

(v/v) fetal bovine serum (FBS) (all from Thermo Fischer aggregates, which results in a redshift of the emission

Scientific, Waltham, MA, USA). Cells were grown at 37 °C, spectrum.^43,57As described above, 6-well plates with 2 × 10⁵

under an atmosphere with high humidity and 5% (v/v) CO₂, as A2780 cells per well were seeded and incubated for 24 h before

described elsewhere. The A2780 cancer cell line was purchased being incubated with 0.1% (v/v) DMSO, cisplatin 3.4 µM and

from Merck (Darmstadt, Germany) and HCT116 cancer cell with IC₅₀concentration of the Cu(BrCl-L₁)Cl complex. After

line and normal epidermal human fibroblasts from American 48 h incubation, JC-1 dye was added to the cells according to

Type Culture Collection (ATCC, Manassas, VA, USA).^57–59

the manufacturer’s instructions. An Attune focusing flow cyt-

ometer (ThermoFisher Scientific) was used to quantify the

fluorescence of the monomer (BL-1 filter) and the aggregate

(BL-2 filter) with the respective software (Attune Cytometric

Viability assays in A2780 and HCT116 cancer cell lines and in

normal human dermal fibroblasts

Cells were seeded in 96 well-plates (7500 cells/100 µL well). Software, vs. 2.1). The values obtained for DMSO treated cells

After 24 h incubation, cells were incubated with DMSO were used to normalize the JC-1 ratios obtained for cisplatin

(vehicle control), cisplatin (positive control) or increasing con- 3.4 µM and Cu(BrCl-L₁)Cl.

centrations of the Cu(Cl₂-L₁)Cl, Cu(Br₂-L₁)Cl, Cu(BrCl-L₁)Cl

and Cu(Cl₂-L₁)NO₃complexes. Cell cultures were then incu-

bated for 48 h and cell viability was determined with the

Intracellular reactive oxygen species (ROS) production in the

A2780 cell line

CellTiter 96® aqueous non-radioactive proliferation assay The method uses 2′,7′-dichlorodihydrofluorescein diacetate

(MTS

Dehydrogenases present in metabolically active cells reduce 3- lar ROS. Intracellular esterases remove acetate groups from

(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sul- this dye allowing to be reduced, which increases its fluo-

assay)

(Promega,

Madison,

WI,

USA).^57,58dye (H2DCF-DA) (ThermoFisher Scientific) to detect intracellu-

fophenyl)-2H-tetrazolium, inner salt (MTS), producing forma- rescence. In 6-well plates, 2 × 10⁵A2780 cells per well are incu-

zan which can be measured at 490 nm in a Tecan microplate bated for 24h. Cells were then incubated with DMSO 0.1% (v/v)

reader, Infinite M200 (Tecan, Männedorf, Switzerland). The (vehicle control), 25 µM H₂O₂and 3.4 µM cisplatin (positive

amount of formazan is directly proportional to viable cells. controls) and IC₅₀concentrations of Cu(Cl₂-L₁)Cl, Cu(Br₂-L₁)

The IC₅₀values are the concentrations that inhibit cellular pro- Cl, Cu(BrCl-L₁)Cl and Cu(Cl₂-L₁)NO₃complexes for 48 h. Then

liferation by 50% compared to the vehicle control (DMSO 0.1% an incubation of A2780 cancer cells with 10 μM H2DCF-DA

v/v). The IC₅₀values are used to compare the biological activity (ThermoFisher Scientific) was performed for 30 min at 37 °C.

of the diﬀerent complexes. IC₅₀values were determined with Cells were then analyzed in an Attune acoustic focusing cyt-

GraphPad Prism 8.2.1 software (GraphPad Software, La Jolla, ometer (ThermoFisher Scientific) and the experimental data

CA, USA) through analysis of the dose response curves.

were analyzed with the respective software (Attune Cytometric

Software, vs. 2.1).^43,57

Apoptosis induction in the A2780 cell line

Autophagy induction in the A2780 cell line

Annexin V-Alexafluor 488/PI dead cell apoptosis assay

(Thermofisher Scientific) was used to quantify apoptosis in An autophagy assay kit (Abcam) was used to detect A2780 cells

A2780 cells by flow cytometry. Six well plates with A2780 cells in autophagy. A2780 cells were seeded in 6-well plates at a

(2 × 10⁵cells/1 mL well) were seeded and incubated for 24 h, density of 2 × 10⁵cells per well and incubated for 24 h. Then,

and then incubated for 48 h with DMSO 0.1% (v/v), as the A2780 cells were incubated with DMSO 0.1% (v/v) and with

vehicle control, cisplatin 3.4 µM (positive control) and with rapamycin 0.2 μM (vehicle and positive control, respectively)

IC₅₀concentrations of Cu(Cl₂-L₁)Cl, Cu(Br₂-L₁)Cl, Cu(BrCl-L₁) and with IC₅₀concentrations of Cu(Cl₂-L₁)Cl, Cu(Br₂-L₁)Cl, Cu

Cl and Cu(Cl₂-L₁)NO₃complexes. As the positive control, (BrCl-L₁)Cl and Cu(Cl₂-L₁)NO₃complexes for 48 h. According

A2780 cells were also treated with cisplatin 3.4 μM. According to the manufacturer’s instructions, cells were detached with

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trypsin and washed with reaction buﬀer 1×. Then an incu- GraphPad Prism v8.2.1 program was used to perform statistical

bation of A 2780 cells with green stain solution was performed analysis (GraphPad Software, La Jolla, CA, USA).

for 30 min at room temperature. Before analysis, cells were

Synthesis of the complexes

washed and then resuspended with reaction buﬀer 1×. The

Attune acoustic focusing cytometer (ThermoFisher Scientific) A general synthetic route was used for all complexes in which a

and the respective software (Attune Cytometric Software, vs. solid sample of CuCl₂or Cu(NO₃)₂(1 mmol) was dissolved in

2.1) were used to analyze cells.^43,57

2 mL of MeOH and then 30 mL of MeOH solution containing

the appropriate ligand (1 mmol) was added. The resulting

solution was refluxed for 2 h.

Internalization of complexes in the A2780 cell line

ICP-AES, inductively coupled plasma atomic emission spec-

Cu(Br₂-L₁)Cl: After addition of CuCl₂to a solution of

troscopy, was used to determine the amount of Cu in A2780 ligand, a green precipitate was formed. This green precipitate

cells exposed to complexes Cu(Cl₂-L₁)Cl, Cu(Br₂-L₁)Cl, Cu was collected by filtration, washed with cold methanol and

(BrCl-L₁)Cl, Cu(Cl₂-L₁)NO₃and Cu(Cl₂-L₂)NO₃. In 25 cm²

T

dried in air. Yield: 86%. Anal. Calcd for C₁₃H₉Br₂ClCuN₂O: C,

flasks, 5 × 10⁶cells were seeded and incubated for 24 h before 33.36; H, 1.94; N, 5.99%. Found: C, 33.38; H, 1.92; N, 5.95%.

culture media were removed and A2780 cells were incubated Selected IR data (KBr, cm⁻¹): 3433 (m), 2921 (w), 1623 (s,

with the IC₅₀concentrations of the above described complexes CvN), 1440 (m), 1413 (m), 1316 (w), 1280 (w), 1155 (m), 1051

for 48 h, before the culture media were removed and washed (w), 859 (w), 716 (m).

twice with PBS. Vehicle control used was with DMSO 1%. The

Cu(Cl₂-L₁)Cl: After addition of CuCl₂to a solution of ligand,

media and PBS were combined and treated with freshly made a green precipitate was formed. This green precipitate was col-

aqua regia (3 : 1 HCl : HNO₃). Cells were detached from the lected by filtration, washed with cold methanol and dried in

flasks with triple express (Thermo Fischer Scientific) and air. Yield: 90%. Anal. Calcd for C₁₃H₉Cl₃CuN₂O: C, 41.19; H,

treated with fresh aqua regia. Pelleted cells formed the cellular 2.39; N, 7.39%. Found: C, 41.25; H, 2.36; N, 7.42%. Selected IR

fraction of the sample and culture media, and PBS from the data (KBr, cm⁻¹): 3433 (m), 3071 (w), 2924 (w), 1636 (s, CvN),

wash, formed the extracellular fraction of the samples. 1519 (w), 1449 (m), 1285 (w), 1214 (w), 1168 (m), 1023 (w), 765

Untreated A2780 cells were also prepared in the same way to (m), 580 (w).

provide the same biological matrix for Cu standards. Samples

Cu(I₂-L₁)Cl: After addition of CuCl₂to a solution of ligand,

were incubated for 24 h in a hood fume at room temperature a green precipitate was formed. This green precipitate was col-

and delivered to Laboratório de Análises/LAQV were ICP-AES lected by filtration, washed with cold methanol and dried in

was performed. Results were shown as % of intracellular Cu air. Yield: 93%. Anal. Calcd for C₁₃H₉ClCuI₂N₂O: C, 27.78; H,

(normalized to control cells) SD.

1.61; N, 4.98%. Found: C, 27.73; H, 1.65; N, 5.02%. Selected IR

data (KBr, cm⁻¹): 3432 (w), 3053 (m), 1619 (s, CvN), 1570 (w),

1485 (w), 1432 (w), 1409 (m), 1312 (w), 1218 (w), 1149 (m),

1052 (w), 995 (w), 824 (w), 759 (w), 464 (w).

Cytotoxicity of the Cu(Cl₂-L₁)Cl and Cu(Br₂-L₁)Cl complexes in

the A2780 cell line using the trypan blue exclusion method

Strober et al. described the trypan blue exclusion method.⁵⁰

Cu(BrCl-L₁)Cl: After addition of CuCl₂to a solution of

The method relies on an exclusion dye, trypan blue, that ligand, a green precipitate was formed. This green precipitate

cannot stain viable cells. Only dead cells will become blue due was collected by filtration, washed with cold methanol and

to the lack of a functional plasmatic membrane. A2780 cells dried in air. Yield: 89%. Anal. Calcd for C₁₃H₉BrCl₂CuN₂O: C,

were seed in 6-well plates (2 × 10⁵cells per well) and after an 36.86; H, 2.14; N, 6.61%. Found: C, 36.84; H, 2.16; N, 6.64%.

initial 24 h incubation, cells were incubated with 3 diﬀerent Selected IR data (KBr, cm⁻¹): 3434 (m), 3059 (w), 2916 (w),

concentrations of the vehicle control, DMSO (0.01%, 0.1% and 1624 (s, CvN), 1509 (w), 1441 (m), 1416 (m), 1211 (w), 1159

1% (v/v)) and of Cu(Cl₂-L₁)Cl and Cu(Br₂-L₁)Cl complexes (0.1 (m), 1051 (w), 860 (w), 750 (m), 584 (w), 470 (w).

× IC₅₀, 1 × IC₅₀, and 10 × IC₅₀). After an incubation of 48 h,

Cu(Br₂-L₁)NO₃: After addition of Cu(NO₃)₂to a solution of

A2780 cells attached to the wells and cells in the culture ligand, a green precipitate was quickly formed. The precipitate

medium supernatant were counted and the live and dead cells was collected by filtration, washed with cold methanol and

were scored. Cellular suspensions were mixed with trypan blue dried in air. Yield: 92%. Anal. Calcd for C₁₃H₉Br₂CuN₃O₄: C,

0.4% (w/v) (ThermoFisher Scientific) in a 4 : 1 ratio (cells: 31.57; H, 1.83; N, 8.50%. Found: C, 31.62; H, 1.53; N, 8.45%.

trypan blue) as previously described.^50,60The cell suspensions Selected IR data (KBr, cm⁻¹): 3433 (m), 3060 (w), 2920 (w),

were counted on a hemocytometer (Hirschmann, Eberstadt, 1629 (s, CvN), 1507 (w), 1471 (s), 1422 (s), 1289 (s), 1215 (w),

Germany) with an inverted microscope Olympus CXX41 1153 (m), 1013 (m), 762 (w), 713 (w), 586 (w), 475 (w).

(Tokyo, Japan).

Cu(Cl₂-L₁)NO₃: After addition of Cu(NO₃)₂to a methanolic

solution of ligand, the color immediately changes from yellow-

ish to green without any precipitation. The resulting solution

Statistical analysis

Results are presented throughout this manuscript as the mean was refluxed for about 2 h. The solution was allowed to stand

SD of at least three independent experiments. The Student at room temperature. The dark green precipitate was obtained

t-test was used to determine statistical significance. Results at room temperature by slow evaporation of the solvent. Yield:

were considered statistically significant if p < 0.05. The 87%. Anal. Calcd for C₁₃H₉Cl₂CuN₃O₄: C, 38.49; H, 2.24; N,

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10.36%. Found: C, 38.51; H, 2.20; N, 10.38%. Selected IR data solution was refluxed for about 2 h. The solution was allowed

(KBr, cm⁻¹): 3432 (m), 3068 (w), 2928 (w), 1631 (s, CvN), 1518 to stand at room temperature. The dark green precipitate was

(w), 1473 (s), 1444 (s), 1290 (s), 1164 (m), 1051 (w), 1014 (m), obtained at room temperature by slow evaporation of the

866 (w), 762 (m), 591 (w).

solvent. Yield: 81%. Anal. Calcd for C₁₄H₁₁BrCl₂CuN₂O: C,

Cu(I₂-L₁)NO₃: After addition of Cu(NO₃)₂to a solution of 38.43; H, 2.53; N, 6.40%. Found: C, 38.51; H, 2.49; N, 6.45%.

ligand, a green precipitate was quickly formed. The precipitate Selected IR data (KBr, cm⁻¹): 3433 (s), 2914 (w), 1620 (s, CvN),

was collected by filtration, washed with cold methanol and 1479 (w), 1429 (s), 1411 (m), 1319 (w), 1207 (m), 869 (w), 737

dried in air. Yield: 91%. Anal. Calcd for C₁₃H₉I₂CuN₃O₄: C, (m).

26.53; H, 1.54; N, 7.14%. Found: C, 26.49; H, 1.68; N, 7.09%.

Cu(Br₂-L₂)NO₃: After addition of Cu(NO₃)₂to a methanolic

Selected IR data (KBr, cm⁻¹): 3433 (m), 3046 (w), 1629 (s, solution of ligand, the color of the solution changed from

CvN), 1570 (m), 1471 (s), 1319 (w), 1285 (s), 1149 (m), 1009 yellow to dark green without any precipitation. The resulting

(m), 871 (w), 696 (w), 582 (w).

solution was refluxed for about 2 h. The solution was allowed

Cu(BrCl-L₁)NO₃: After addition of Cu(NO₃)₂to a methanolic to stand at room temperature. The dark green crystals were

solution of ligand, the color immediately changes from yellow- obtained at room temperature by slow evaporation of the

ish to green without any precipitation. The resulting solution solvent. Yield: 87%. Anal. Calcd for C₁₄H₁₁Br₂CuN₃O₄: C,

was refluxed about 2 h. The solution was allowed to stand at 33.06; H, 2.18; N, 8.26%. Found: C, 33.17; H, 2.21; N, 8.34%.

room temperature. The dark green precipitate was obtained at Selected IR data (KBr, cm⁻¹): 3434 (m), 3054 (w), 2957 (w),

room temperature by slow evaporation of the solvent. Yield: 1623 (m, CvN), 1381 (s), 1280 (w), 1155 (w), 959 (w), 866 (w),

87%. Anal. Calcd for C₁₃H₉BrClCuN₃O₄: C, 34.69; H, 2.02; N, 758 (m), 584 (w).

9.34%. Found: C, 34.81; H, 1.93; N, 10.03%. Selected IR data

Cu(Cl₂-L₂)NO₃: After addition of Cu(NO₃)₂to a methanolic

(KBr, cm⁻¹): 3433 (m), 2925 (w), 1631 (s, CvN), 1511 (w), 1473 solution of ligand, the color of the solution changed from

(s), 1442 (m), 1291 (s), 1212 (w), 1157 (m), 1015 (w), 866 (w), yellow to dark green without any precipitation. The resulting

749 (m), 470 (w).

solution was refluxed for about 2 h. The solution was allowed

Cu(Br₂-L₂)Cl: After addition of Cu(NO₃)₂to a methanolic to stand at room temperature. The dark green crystals were

solution of ligand, the color of the solution changed from obtained at room temperature by slow evaporation of the

yellow to dark green without any precipitation. The resulting solvent. Yield: 82%. Anal. Calcd for C₁₄H₁₁Cl₂CuN₃O₄: C,

solution was refluxed for about 2 h. The solution was allowed 40.06; H, 2.64; N, 10.01%. Found: C, 40.19; H, 2.73; N, 9.1%.

to stand at room temperature. The dark green precipitate was Selected IR data (KBr, cm⁻¹): 3434 (s), 2921 (w), 1631 (s, CvN),

obtained at room temperature by slow evaporation of the 1445 (m), 1380 (m), 1210 (w), 1164 (m), 1014 (w), 763 (m), 591

solvent. Yield: 87%. Anal. Calcd for C₁₄H₁₁Br₂ClCuN₂O: C, (w).

34.88; H, 2.30; N, 5.81%. Found: C, 34.81; H, 2.35; N, 5.78%.

Selected IR data (KBr, cm⁻¹): 3433 (m), 2913 (w), 1625 (s, solution of ligand, the color of the solution changed from

CvN), 1448 (s), 1214 (w), 1153 (m), 871 (w), 775 (w). yellow to dark green without any precipitation. The resulting

Cu(I₂-L₂)NO₃: After addition of Cu(NO₃)₂to a methanolic

Cu(Cl₂-L₂)Cl: After addition of Cu(NO₃)₂to a methanolic solution was refluxed for about 2 h. The solution was allowed

solution of ligand, the color of the solution changed from to stand at room temperature. The dark green precipitate was

yellow to dark green without any precipitation. The resulting obtained at room temperature by slow evaporation of the

solution was refluxed for about 2 h. The solution was allowed solvent. Yield: 91%. Anal. Calcd for C₁₄H₁₁CuI₂N₃O₄: C, 27.90;

to stand at room temperature. The dark green precipitate was H, 1.84; N, 6.97%. Found: C, 27.90; H, 1.84; N, 7.11%. Selected

obtained at room temperature by slow evaporation of the IR data (KBr, cm⁻¹): 3409 (m), 1627 (s, CvN), 1383 (s), 1282

solvent. Yield: 88%. Anal. Calcd for C₁₄H₁₁Cl₃CuN₂O: C, 42.77; (w), 1166 (w), 761 (m).

H, 2.82; N, 7.13%. Found: C, 42.71; H, 2.97; N, 7.08%. Selected

Cu(BrCl-L₂)NO₃: After addition of Cu(NO₃)₂to a methanolic

IR data (KBr, cm⁻¹): 3433 (m), 3056 (w), 2912 (w), 1620 (s, solution of ligand, the color of the solution changed from red

CvN), 1516 (w), 1435 (s), 1323 (w), 1166 (m), 961 (w), 867 (w), to dark green without any precipitation. The resulting solution

759 (s), 584 (w), 538 (w).

was refluxed for about 2 h. The solution was allowed to stand

Cu(I₂-L₂)Cl: After addition of CuCl₂to a solution of ligand, at room temperature. The dark green crystals were obtained at

a green precipitate was formed. The precipitate was collected room temperature by slow evaporation of the solvent. Yield:

by filtration, washed with cold methanol and dried in air. 89%. Anal. Calcd for C₁₄H₁₁BrClCuN₃O₄: C, 36.23; H, 2.39; N,

Green crystals were formed from a methanolic solution of the 9.05%. Found: C, 36.36; H, 2.32; N, 8.84%. Selected IR data

complex by slow evaporation of the solvent after standing for (KBr, cm⁻¹): 3432 (m), 3067 (w), 2922 (w), 1625 (s, CvN), 1447

one week in air. Yield: 79%. Anal. Calcd for C₁₄H₁₁ClCuI₂N₂O: (s), 1383 (w), 1283 (s), 1210 (w), 1160 (m), 1007 (w), 866 (w),

C, 29.19; H, 1.92; N, 4.86%. Found: C, 29.28; H, 1.83; N, 748 (m).

4.91%. Selected IR data (KBr, cm⁻¹): 3433 (s), 2920 (w), 1610

(s, CvN), 1484 (w), 1430 (s), 1146 (s), 857 (w), 763 (m), 585 (w).

X-ray crystal structure determination

Cu(BrCl-L₂)Cl: After addition of Cu(NO₃)₂to a methanolic The diﬀraction data (except compound Cu(BrCl-L₂)NO₃) were

solution of ligand, the color of the solution changed from measured at low temperature using Cu or Mo K_αradiation on

yellow to dark green without any precipitation. The resulting a Rigaku SuperNova dual system in combination with an Atlas

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type CCD detector. The data reduction was carried out using

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CrysAlis^{Pro 61}

The data for crystal structure Cu(BrCl-L₂)NO₃

.

were collected at 120 K using Mo Kα radiation on a Bruker

APEX II CCD diﬀractometer equipped with a kappa geometry

goniometer. The dataset was reduced using EvalCCD⁶²and

then corrected for absorption.⁶³

The solution analysis and refinements were performed 10 Y. An, S.-D. Liu, S.-Y. Deng, L.-N. Ji and Z.-W. Mao, J. Inorg.

using SHELXT⁶⁴and SHELXL,⁶⁵respectively. The crystal struc-

Biochem., 2006, 10, 1586–1593.

tures were refined using full-matrix least-squares based on F²11 S. Zhang, C. Tu, X. Wang, Z. Yang, J. Zhang, L. Lin, J. Ding

with all non-hydrogen atoms anisotropically defined. and Z. Guo, Eur. J. Inorg. Chem., 2004, 20, 4028–4035.

Hydrogen atoms were placed in calculated positions by means 12 R. S. Kumar, K. Sasikala and S. Arunachalam, J. Inorg.

of the “riding” model. Pseudo merohedral twinning was found Biochem., 2008, 2, 234–241.

for all crystal structures and treated directly with CrysAlis^{Pro 61}13 S. Dhar and A. R. Chakravarty, Inorg. Chem., 2003, 8, 2483–

or with the TWINROTMAT routine of PLATON.⁶⁶Constraints

2485.

and rigid bond restraints (EADP and RIGU cards) were 14 G. L. Eichhorn and Y. A. Shin, J. Am. Chem. Soc., 1968, 26,

employed during the last stages of refinement of Cu(BrCl-L₂) 7323–7328.

NO₃in order to obtain acceptable anisotropic displacement 15 C. Santini, M. Pellei, V. Gandin, M. Porchia, F. Tisato and

parameters. More details concerning the crystal structures and C. Marzano, Chem. Rev., 2013, 1, 815–862.

their refinement can be found in Table 3 or in the corres- 16 K. G. Daniel, D. Chen, S. Orlu, Q. C. Cui, F. R. Miller and

ponding CIFs. Q. P. Dou, Breast Cancer Res., 2005, 6, R897.

In particular, for compound Cu(BrCl-L₁)NO₃, the obtained 17 K. G. Daniel, P. Gupta, R. H. Harbach, W. C. Guida and

twin law is −1 0 0 0 −1 0 0.082 0 1 and the BASF parameter is Q. P. Dou, Biochem. Pharmacol., 2004, 6, 1139–1151.

0.123(1). Applying the twin improved the refinement by about 18 D. Chen and Q. P. Dou, Expert Opin. Ther. Targets, 2008, 6,

4.5% and reduced the residual electron densities by about 2. 739–748.

Nevertheless, some relatively high peaks still reside close to 19 P. U. Maheswari, S. Roy, H. den Dulk, S. Barends, G. van

heavy atoms (Br, Cu) and this could be likely due to absorption

eﬀects.

Wezel, B. Kozlevcar, P. Gamez and J. Reedijk, J. Am. Chem.

Soc., 2006, 3, 710–711.

20 V. Rajendiran, R. Karthik, M. Palaniandavar, H. Stoeckli-

Evans, V. S. Periasamy, M. A. Akbarsha, B. S. Srinag and

H. Krishnamurthy, Inorg. Chem., 2007, 20, 8208–8221.

21 Y. Lu, Y. Wang and W. Zhu, Phys. Chem., 2010, 18, 4543–4551.

22 E. Parisini, P. Metrangolo, T. Pilati, G. Resnati and

G. Terraneo, Chem. Soc. Rev., 2011, 5, 2267–2278.

Conﬂicts of interest

The authors declare no competing financial interest.

23 L. A. Hardegger, B. Kuhn, B. Spinnler, L. Anselm,

R. Ecabert, M. Stihle, B. Gsell, R. Thoma, J. Diez and

J. Benz, Angew. Chem., Int. Ed., 2011, 1, 314–318.

24 R. Shakya, F. Peng, J. Liu, M. J. Heeg and C. N. Verani,

Inorg. Chem., 2006, 16, 6263–6268.

25 S. S. Hindo, M. Frezza, D. Tomco, M. J. Heeg,

L. Hryhorczuk, B. R. McGarvey, Q. P. Dou and C. N. Verani,

Eur. J. Med. Chem., 2009, 11, 4353–4361.

Acknowledgements

The authors are grateful to the Research Council of the

University of Isfahan for financial support of this work. This

work was supported by the Applied Molecular Biosciences

Unit-UCIBIO which is financed by national funds from FCT/

MCTES (UIDB/04378/2020).

26 R. Shakya, C. Imbert, H. P. Hratchian, M. Lanznaster,

M. J. Heeg, B. R. McGarvey, M. Allard, H. B. Schlegel and

C. N. Verani, Dalton Trans., 2006, 21, 2517–2525.

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Dalton Trans., 2021, 50, 3990–4007 | 4007

Article Doi

Copper(ii) complexes with tridentate halogen-substituted Schiff base ligands: synthesis, crystal structures and investigating the effect of halogenation, leaving groups and ligand flexibility on antiproliferative activities

DOI: 10.1039/d0dt03962d

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Full text of DOI:10.1039/d0dt03962d

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