Copper(II) salicylideneimine complexes revisited: From a novel derivative and extended characterization of two homologues to interaction with BSA and antiproliferative activity

Article abstract of DOI:10.1016/j.ica.2021.120460

A novel compound bis[N-n-propyl-5-chloro-2-oxy-κO-benzylideneimine-κN-(1–)]copper(II) was prepared, along with two previously reported homologues having N-n-propylsalicylideneimine and N-n-buthyl-5-chlorosalicylideneimine ligands. Single crystal X-ray det

Full text of DOI:10.1016/j.ica.2021.120460

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Research paper
Copper(II) salicylideneimine complexes revisited: From a novel derivative
and extended characterization of two homologues to interaction with BSA and
antiproliferative activity
Mahira Memišević, Adnan Zahirović, Aleksandar Višnjevac, Amar
Osmanović, Dijana Žilić, Marijeta Kralj, Senada Muratović, Irena Martin-
Kleiner, Davorka Završnik, Emira Kahrović
PII:
DOI:
Reference:
S0020-1693(21)00216-4
https://doi.org/10.1016/j.ica.2021.120460
ICA 120460
To appear in:
Inorganica Chimica Acta
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Revised Date:
Accepted Date:
1 April 2021
29 April 2021
16 May 2021
Please cite this article as: M. Memišević, A. Zahirović, A. Višnjevac, A. Osmanović, D. Žilić, M. Kralj, S.
Muratović, I. Martin-Kleiner, D. Završnik, E. Kahrović, Copper(II) salicylideneimine complexes revisited: From
a novel derivative and extended characterization of two homologues to interaction with BSA and antiproliferative
activity, Inorganica Chimica Acta (2021), doi: https://doi.org/10.1016/j.ica.2021.120460
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Copper(II) Salicylideneimine Complexes Revisited: From a Novel Derivative
and Extended Characterization of Two Homologues to Interaction with BSA
and Antiproliferative Activity
1
1
2#
3
2
Mahira Memišević , Adnan Zahirović , Aleksandar Višnjevac , Amar Osmanović , Dijana Žilić ,
2
2
2
3
1
Marijeta Kralj , Senada Muratović , Irena Martin-Kleiner , Davorka Završnik , Emira Kahrović *
1
Laboratory for Inorganic and Bioinorganic Chemistry, Department of Chemistry, Faculty of
Science, University of Sarajevo, Sarajevo, Bosnia and Herzegovina
Ruđer Bošković Institute, Zagreb, Croatia
Faculty of Pharmacy, University of Sarajevo, Sarajevo, Bosnia and Herzegovina
2
3
*
Corresponding author:
Prof. dr. Emira Kahrović
University of Sarajevo Faculty of Science
Department of Chemistry
Zmaja od Bosne 35
7
1000 Sarajevo Bosnia and Herzegovina
E-mail: emira_kahrovic@yahoo.com
Phone: +387 33 279 910
#
Corresponding author for crystallography:
Aleksandar Višnjevac, PhD
Laboratory for Chemical and Biological Crystallography
Ruđer Bošković Institute
Bijenička cesta 54
HR-10000 Zagreb
e-mail: aleksandar.visnjevac@irb.hr
Phone: +385 1 457 1405
Page 1 of 29

Abstract
A novel compound bis[N-n-propyl-5-chloro-2-oxy-κO-benzylideneimine-κN-(1–)]copper(II) was
prepared, along with two previously reported homologues having N-n-propylsalicylideneimine
and N-n-buthyl-5-chlorosalicylideneimine ligands. Single crystal X-ray determination of three
complexes reveals almost ideal CuO
2
2
N trans-square planar geometry. All three compounds
crystalize in P-1 space group. Infrared spectra confirm coordination of iminophenolate via
azomethine nitrogen and deprotonated phenolic oxygen. Electronic spectra showed strong
iminophenolate-to-Cu(II) charge transfer transitions in the region 359-371 nm and d-d transition
9
2
2
centered around 600 nm. The location of an unpaired electron of d configuration Cu(II), in dx −y
orbital is confirmed based on g-tensor values obtained by X-band ESR spectroscopy that also
corresponds to square planar geometry. Cyclic voltammograms in acetonitrile solution showed
one-electron reduction with cathodic peaks in the range –0.598 to –0.618 V as a result of the fast
decomplexation and disproportionation of the Cu(I) complex. The interaction with BSA (Bovine
3
5
-1
serum albumin) showed 1:1 complex-protein binding with K constants values in 10 –10 M
b
range. Docking calculations point out only hydrophobic and π-π interactions between copper(II)
complexes and BSA with average distances between two tryptophan residues and copper
complexes in 20 – 24 Å range. Antiproliferative testing on two tumor cell lines H460 and HCT116
and non-tumor HEK293T showed non-selectivity and close IC50 values for tumor and control lines.
Keywords: Copper(II) Schiff bases, N-alkylsalicyiladimines, BSA, docking study,
antiproliferative activity
Page 2 of 29

1
Introduction
Copper, playing the important role in many biochemical processes like redox reactions, cell growth
and angiogenesis, is considered to be responsible for various pathological conditions [1-4]. On the
other hand, new copper complexes with potential activity against various diseases are being
intensively designed and tested.
Geometry of copper compounds is less predictable compared to the other first-row transition metal
9
complexes, due to an unusual penchant for distorted geometries. With d electronic configuration,
copper(II) is able to adopt different geometries including square planar, trigonal pyramidal,
octahedral and square pyramidal, with the latter one being often displayed by complexes with
bridging ligands. Outstanding possibilities for adjusting Cu(II)/Cu(I) redox potential arise from
the different hard soft acid-base (HSAB) character of Cu(II) and Cu(I) [5]. The effect of the
geometry on the redox potential depends on the tetragonal distortion tendency, characteristic for
copper Cu(II), towards tetrahedral geometry typical for Cu(I).
Schiff bases have been intensively studied for decades primarily due to their ability to bind
numerous metals into stable five- or six-membered rings, as well as for their biological properties.
The diversity of precursors, carbonyl- and amine compounds, resulted in thousands of new
compounds.
Many copper complexes with salicylaldehyde based Schiff bases were reported to have
antimicrobial, antifungal, antitumor and biomimetic activity [6-10]. To our knowledge, less
attention has been focused on the synthesis and study of copper compounds with Schiff base
derived from derivative of salicylaldehyde and alkylamines. In the early seventies to eighties there
were several reports on syntheses and molecular structures of metal complexes from the end of the
first transition series including copper with Schiff bases derived from alkyl amines or their iso-,
and cyclic derivatives, however they are only modestly described [11-13].
Recent publications mainly refer to copper compounds bearing substituted ethylamine based Schiff
bases with special emphasis on their antiproliferative activity. The general feature of these
complexes is square planar geometry with two nitrogen and two oxygen atoms in trans position.
Copper(II) compounds with iminophenolate ligands derived from 2-methoxyethylamine and
various salicylaldehyde derivatives demonstrated strong antitumor activity [14, 15], while some
Page 3 of 29

were described as precursors for preparation of metal oxide nanoparticles by its thermal
decomposition [16]. Besides to methoxyethylamine derivative, copper(II) compounds with 1-(1-
naphthyl)ethylamine based Schiff base were prepared and tested for antiproliferative activity [17].
In this paper we report the synthesis, characterization and biological properties of three copper
complexes with ligands based on salicylaldehyde or its 5-chloro derivative with low molecular n-
alkylamines. Literature reveals molecular structures of copper(II) complexes with N-n-propyl-
salicylideneimine [18] and N-n-butyl-5-chloro-salicylidenimine [19, 20] for which a complete
physico-chemical characterization is lacking. This inspired us to undertake the present study
including the revisiting of molecular structures of two previously reported compounds, along with
the synthesis and structure of one novel homologue. Furthermore, the biological activity based on
the interaction with bovine serum albumin (BSA) and in vitro antitumor activity for all three
compounds was tested.
2
2
Experimental
.1 Material and methods
All chemicals for synthesis and spectroscopic measurements were reagent grade, used as
purchased without further purification. Lyophilized powder of bovine serum albumin (≥ 96%) was
purchased from Sigma.
Elemental analysis was performed on a Perkin Elmer 2400 Series CHNS/O analyzer. High-
resolution matrix-assisted laser desorption ionization-time-of-flight/time-of-flight mass spectra
were recorded on a 4800 Plus MALDI TOF/TOF analyzer in the positive ion reflector mode in
-1
9
9-1500 Da region using dithranol (10 mg mL in acetonitrile) as a matrix. Infrared spectra were
-1
recorded on Perkin Elmer BX FTIR spectrometer in the range 4000 – 400 cm with KBr pallets.
-5
Electronic spectra of 2 × 10 M copper(II) complexes in methanol solution were recorded in 200
1100 nm range using Perkin Elmer BioLambda 35. In addition, electronic spectra of a more
–
-3
concentrated solutions (2 × 10 M) in the range of 500 – 800 nm were recorded in order to get an
insight into d-d transitions.
The electron spin/paramagnetic resonance (ESR/EPR) measurements were performed from room
down to liquid nitrogen temperature on a Bruker Elexsys 580 FT/CW spectrometer. The spectra
were recorded under the following parameters: microwave frequency around 9.7 GHz, magnetic
Page 4 of 29

field modulation amplitude 0.5 mT and modulation frequency 100 kHz. The measurements were
obtained on powder samples.
Cyclic voltammograms of copper(II) complexes were collected on an Autolab potentiostat /
galvanostat (PGSTAT 12) electrochemical workstation using a three electrode system. A Pt wires
were used as working and counter electrodes and Ag/AgCl as a reference electrode. Cyclic
voltammograms of copper(II) complexes in 2 mM solutions were recorded in dried and
deoxygenated acetonitrile using 0.1 M tetrabutylammonium hexafluorophosphate as a supporting
electrolyte. The start potential was +0.5 V, while -1.5 V was used as first and +2.0 V as second
-1
vertex potential. Cyclic voltammograms were recorded at varying scan rate 0.1 – 0.3 V s .
2
2
.2 Syntheses
.2.1 Preparation of Schiff Bases
1
3
Ligands, hereinafter designated as HL -HL , were prepared by simple condensation of an aldehyde
1
and n-alkyl amine at room temperature. For HL an appropriate volume of n-propylamine was
added to stoichiometric amount of salicylaldehyde followed by the rapid development of an intense
2
3
yellow color of Schiff base. In the case of HL – HL which refers to N-propyl- and N-butyl-5-
chlorosalicylideneimines, appropriate volume of an amine was added to the stoichiometric amount
of aldehyde in ethanol. Ligands were always prepared just before use for the synthesis of copper(II)
compounds.
2
.2.2 Synthesis of copper(II) complexes
The complex compounds of the general formula [Cu(L -N,O)
] abbreviated as 1 – 3, where L1-3
1
-3
2
1
2
3
is the anion of deprotonated corresponding Schiff base (HL for (1), HL for (2) and HL for (3)),
were prepared from solution, as follows.
General procedure
To the solution of 100 mg of Cu(CH
3
COO)
2
·H O (0.5 mmol) in 15 mL of ethanol, an appropriate
2
1
3
volume containing 1 mmol of freshly prepared HL -HL was added. The resulting brown solution
was stirring under reflux for 2.5 hours and kept to evaporate slowly for several days. The green
crystalline solids were filtered out, washed with cold water and dried in vacuum desiccator.
Page 5 of 29

Recrystallization was performed from diethyl ether giving green crystals suitable for single crystal
diffraction studies. Yields: 60–80%.
Complex 1. Bis[N-n-propyl-2-oxy-κO-benzylidenimine-κN-(1–)]copper(II). Anal. Calcd. mass
w/% for C20
H
24
N
2
2
O Cu (Mr = 387.95): C 61.91, H 6.24, N 7.22; found: C 61.50, H 5.97, N 7.08.
+
-1
−1
MALDI-TOF MS [M+Na] m/z: 410.1029. UV-Vis (MeOH) LMCT λmax/nm: 359 (log ε / M cm
–
1
3
.86). IR (KBr), νmax/cm 1624 (C=N), 1330 (C–O), 545 (Cu–O), 425 (Cu–N).
Complex 2. Bis[N-n-propyl-5-chloro-2-oxy-κO-benzylidenimine-κN-(1–)]copper(II). Anal.
Calcd. mass w/%, for C20
H22Cl
2
N
2
O
2
Cu (Mr = 456.85): C 52.58, H 4.85, N 6.13; found: C 52.31,
+
H 5.05, N 6.32. MALDI-TOF MS [M+Na] m/z: 478.0246. UV-Vis (MeOH) LMCT λmax/nm: 371
-1
−1
–1
(
log ε / M cm 3.96). IR (KBr), νmax/cm 1624 (C=N), 1327 (C–O), 560 (Cu–O), 424 (Cu–N).
Complex 3. Bis[N-n-butyl-5-chloro-2-oxy-κO-benzylidenimine-κN-(1–)]copper(II). Anal. Calcd.
mass w/%, for C22
H26Cl N
2 2
O
2
Cu (Mr = 484.90): C 54.49, H 5.40, N 5.78; found: C 54.13, H 5.60,
+
N 6.09. MALDI-TOF MS [M+Na] m/z: 506.0560. UV-Vis (MeOH) LMCT λmax/nm: 371 (log ε /
-1
−1
–1
M cm 3.99). IR (KBr), νmax/cm 1625 (C=N), 1329 (C–O), 570 (Cu–O), 424 (Cu–N).
2
.3 X-ray crystallography
Crystal data collections were conducted at the room temperature [298 (2) K], on an Oxford
diffraction Xcalibur Nova Ruby device and Cu radiation (=1.54184 Å) was employed for all
samples. Data reduction and cell refinement were carried out using the CRYSALIS PRO software
[
21]. Structures were solved by direct methods with SIR2014 [22] and refined by a full matrix
least-squares refinement based on F2, with SHELXL [23]. Molecular illustrations were prepared
with MERCURY [24]. Calculations of molecular geometries parameters were performed with
PLATON [25]. All software used is included into the WinGX package [26]. Hydrogen atoms were
either included in their geometrically calculated positions, and refined according to the riding
model, or (where sustainable throughout the refinement) located from the difference map and
refined freely. Multi-scan absorption correction procedure [27] was applied for all data collections.
Page 6 of 29

2
2
.4 Biological evaluation
.4.1 Interaction with BSA
Spectrofluorimetric titration
Spectrofluorimetric titration of bovine serum albumin with copper(II) complexes was carried out
-6
by adding 10 µL aliquots of 1 – 3 (0.2 mM) to solution of BSA (2.000 mL, 3.68 × 10 M) in 10
mM Tris-HCl buffer (pH = 7.42) and recording the emission spectra in 290 – 430 nm range with
2
79 nm as excitation wavelength using a PerkinElmer LS55 Luminescence. The synchronous
spectra were recorded under the similar conditions as described above while recording the
emission spectra in 250 – 310 nm range with Δλ = 15 nm and Δλ = 60 nm.
Molecular docking study
The docking study was set up in YASARA 19.12.14 [28, 29] and performed using AutoDock 4.2
[
30]. The crystal structure of BSA (PDB ID: 3V03) from Protein Data Bank was used as the target
molecule. The protein structure was prepared by removing water molecules, adding polar
hydrogen atoms and optimizing in the AMBER03 force field [31]. The geometries of the copper(II)
complex molecules were exported from crystallographic data and optimized by the density
functional theory (DFT) (B3LYP/LanL2DZ basis set) using Gaussian 09 [32]. First, a blind
docking approach was used, searching the whole protein for potential binding sites. The lowest
energy docked structures were obtained this way and those positions were used as a center for
further analyses using grid maps with 58×56×42 (for complex 1), 58×44×48 (for complex 2) and
5
8×54×46 (for complex 3) points, and grid point spacing of 0.375 Å for all three complexes. The
Lamarckian genetic algorithm was employed with the following parameters: 150 docking runs
with a maximum of 17,500,000 energy evaluations and 27,000 generations for each run.
2
.4.2 Antiproliferative activity in vitro
Cell culturing
The experiments were carried out on 3 human cell lines, HEK293T (embryonic kidney), H460
(lung carcinoma) and HCT116 (colon carcinoma). The cells were cultured as monolayers and
maintained in Dulbecco's modified Eagle medium (DMEM), supplemented with 10% fetal bovine
Page 7 of 29

serum (FBS), 2mM L-glutamine, 100 U ml-1 penicillin and 100 μg ml streptomycin in a
-1
humidified atmosphere with 5% CO at 37˚C.
2
Proliferation assays
The panel cell lines were inoculated onto a series of standard 96-well microtiter plates on day 0,
4
-1
at 1.6×10 cells mL , depending on the doubling times of specific cell line. Test agents were then
-8
-4
added in five 10-fold dilutions (10 to 10 M) and incubated for a further 72 hours. Working
dilutions were freshly prepared on the day of testing. After 72 hours of incubation the cell growth
rate was evaluated by performing the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) assay [33, 34].
3
3
Results and discussion
.1 Synthesis and characterization
Three copper(II) complexes with Schiff bases derived from salicylaldehyde or 5-chloro-derivative
and low molecular n-alkyl amines with discrete alteration on the CuO center were prepared
2
N
2
1
-3
and characterized. The preparation of ligands (HL ) was performed by a simple condensation of
salicylaldehyde or 5-chloro derivative with n-propyl- and n-butylamine in absolute ethanol without
heating. The traces of finely dispersed water were not removed from the ligand prior to use. The
synthesis of copper(II) complexes 1 – 3 was conducted in ethanol solution using Cu(OAc)
2
·H O
2
as the starting compound and ligands HL1-3 in molar ratio 1:2 under the reflux for several hours.
The solid crystalline complex compounds 1 – 3 are fairly stable for at least a few months under the
ambient conditions.
To our knowledge, syntheses of compounds 1 and 3 with the same molecular formula as reported
here are published. However, the crystal structure of compound 1 deserves redetermination and
re-refinement which are reported here. On the other hand, crystal structure of compound 3 has
been reported previously twice [19, 20]. These two reported structures have different
crystallographic data of molecular structure and are different from the one reported in this paper
(Table 1). Also these syntheses were performed under different experimental conditions, including
temperature and starting material.
Page 8 of 29

Mentioned Schiff bases with two donor atoms, azomethine (-CH=N-) nitrogen and deprotonated
1
-3
phenolic group (C-O-) were found to bind copper(II) in neutral complex species [Cu(L -N,O) ],
2
adopting the square planar geometry (Scheme 1.)
Scheme 1. Syntheses of copper(II) complexes 1 – 3.
The purity of the complexes was checked by elemental analysis (Table S2). The mass spectra
+
recorded as [M + Na] where M denotes neutral complex species, showed the isotopic distribution
characteristic for copper(II) species, thus confirming molecular formulation 1 – 3 (Table S3, Figure
S1-S6).
Infrared spectra 1 – 3 are very similar due to discrete changes on salicylaldehyde and alkyl moieties
of ligand. Coordination of anionic L1-3 through phenolic oxygen and azomethine nitrogen was
-1
confirmed based on the shift of -HC=N- toward lower wavenumbers for 9 – 11 cm and phenolic
-1
C-O for 49 – 51 cm to the higher region compared to the free ligands (Table S4, Figure S7). A
considerable shift of phenolic C-O(H) frequencies corresponds to weak binding of deprotonated
oxygen to copper(II) which reinforced C-O bond. New low-frequency bands in spectra 1 – 3 were
assigned to M-L bonds. Copper(II)-oxygen frequencies were found in the region 545 – 570 cm-1
-1
while Cu-N bonds appeared at 419 – 425 cm [35]. Asymmetric and symmetric stretching
-1
frequencies of CH
3
group appear in the region 2963 – 2923 and 2865 – 2853 cm , respectively.
Strong bands attributed to symmetric deformation of CH
2
and CH groups were found at 1469 –
3
-1
-1
1
465 cm and 1392 – 1388 cm , respectively.
The electronic spectra of ligands and copper(II) complexes were recorded in methanol (Figure 1,
1
-3
Table S5). Ligands HL have showed three bands (I-III) arising from intraligand transitions π→
π* and n→ π*. The spectra of 1 – 3 have additional bands as a result of band I splitting arising
Page 9 of 29

from extended aromatic ring conjugation [36, 37], strong iminophenolate-to-Cu(II) charge transfer
transition (LMCT) and d-d transition (Figure S8). In a more concentrated methanol solution, a
2
2
2
weak band for 1 and shoulder for 2 and 3, centered about 600 nm, were assigned to dz → dx -y
transition of copper(II) in square planar geometry [38, 39] (Figure 1 inset).
-5
Figure 1. Electronic spectra of copper(II) complexes 1 – 3 in methanol solution (2 × 10 M). Inset:
-3
The region of d-d transition (2 × 10 M).
3
.2 Molecular structures
The molecular structures of the three mononuclear copper(II) complexes with Schiff bases 1 – 3
were determined by single-crystal X-ray diffraction analysis. Experimental data collection,
structure solution and refinement are given in Table S1.
The structure of 1 was previously reported and stored in the CSD under the refcode PSCACP, but
largely ill-refined. Refinement of our structure 1 went down to the final R value of 0.064, as
1
compared to 0.098 for the PSCACP. Precision of the unit cell and geometrical parameters is
improved in the structure of 1 for an order of magnitude with respect to the analogous values in
PSCACP. The average of shift/e.s.d, in the final least-squares cycle was 0.00 as compared to 0.275
for PSCACP. These crystallographic improvements which were achieved with the new
determination, along with the context of this report impose the re-publication with high quality
diffraction data and a more reliable refinement as highly beneficial [18]. The structure of
compound 3 was, relatively recently, deposited two times in CCDC, both times with a unit cell
Page 10 of 29

twice as large as the one reported hereby, and with a refined model featuring the entire complex
molecule within the asymmetric unit, unlike in our complex 3, where expectedly, only one half
centrosymmetric complex molecule is present in the asymmetric unit, and with a copper(II) ion
lying on the inversion center. Table 1 summarizes these structure reports, including our structure
solution for quick comparison. The structure of novel compound 2 having Schiff base derived from
5
-chloro-salicylaldehyde and n-propyl amine is presented in Figure 2. Molecular structures of
compounds 1 and 3 are given in Figures S9 – S10.
Table 1. Parameters and data of the previously reported structures of compound 3, and the
analogous data from our structure solution.
CSD refcode
Publ. year
Space group
a (Å)
b (Å)
c (Å)
MOCGAN [19]
2008
P-1
MOCGAN01 [20]
2012
P-1
3
P-1
10.061 (3)
10.098 (3)
11.569 (3)
97.09 (3)
90.79 (3)
105.75 (3)
1121.21
2
10.090 (1)
10.100 (1)
11.527 (1)
96.86 (1)
91.11 (1)
105.66 (1)
1121.35
2
7.4389 (9)
8.4528 (7)
9.1008 (9)
74.401 (8)
88.640 (9)
83.104 (8)
547.17 (10)
1
α (°)
β (°)
γ (°) 3
V (Å )
Z
R
1
[I>2σ(I)]
0.0603
0.0487
0.0382
All three compounds crystallize in the P-1 space group with half a complex molecule in the
asymmetric unit. In all structures, copper(II) ion lies on an inversion center and reveals an almost
perfect trans square planar coordination configuration, being bound by an azomethine nitrogen
and a deprotonated hydroxyl group from two bidentate ligand molecules. The geometrical
parameters of the coordination spheres are summarized in Table 2, and lie well within the expected
value range for the related complexes, with no notable differences among the hereby reported
structures. Search of the current version of the CSD [40] for trans square planar N
2
2
O copper(II)
complexes, with an exclusion of disordered, ionic and structures with a final R factor above 5%,
resulted with 484 structures with a mean value of the Cu-N coordination contact of 1.98 (4) Å, and
of the Cu – O coordination contact of 1.91 (4) Å, which corresponds very well with the analogous
values reported in the Table 2. With an expected exception of terminal n-propyl (in 1 and 2) and
n-butyl (in 3) groups, complex molecules of all three hereby reported complexes are virtually
planar. The accordingly calculated ls planes reveal the maximal atomic distance from such a plane
Page 11 of 29

of 0.072 Å (for Cu1) in 1, 0.036 Å (for Cu1) in 2, and 0.157 Å (for Cu1) in 3. No hydrogen bonds
nor significant stacking interactions are present in the structures of 1, 2 and 3.
Table 2. Coordination spheres geometries for copper(II) complexes 1 – 3.
1
2
3
Cu1 – O1 (Å)
Cu1 – N1 (Å)
1.888 (3)
2.023 (3)
180.
91.77 (12)
88.23 (12)
180.
1.8919 (16)
2.0191 (17)
180.
91.33 (7)
88.67 (7)
180.
1.8938 (14)
2.0199 (17)
180.
91.11 (6)
88.98 (6)
180.
O1 – Cu1 – O1i (°)
O1 – Cu1 – N1 (°)
O1 – Cu1 – N1i (°)
N1 – Cu1 – N1i (°)
N1 – Cu1 – O1i (°)
O1i – Cu1 – N1i (°)
88.23 (12)
91.77 (12)
88.67 (7)
91.33 (7)
88.98 (6)
91.11 (6)
Figure 2. Molecular structure od compound 2 with atom labelling. Thermal ellipsoids are scaled
at the 50% probability level.
Page 12 of 29

3
.3 Magnetic properties
The magnetic properties of three mononuclear copper(II) complexes were studied by X-band ESR
spectroscopy. The representative ESR spectra of the investigated Cu(II) complexes, obtained at a
few selected temperatures, are shown in Figure 3. The simulations were performed using EasySpin
software [41] using only the Zeeman term in spin-Hamiltonian:
푯 = 휇 푩품푺.
(1)
퐵
In Eq. (1) 푩 is external magnetic field, 품 is 품-tensor, 푺 is electron spin operator for copper(II) ions
(
S = 1/2) and the constant μ
B
is Bohr magneton. Due to large distances between copper(II) ions
(the closest Cu-Cu distance is around 6.7, 7.5 and 7.4 Å in complex 1, 2 and 3, respectively)
electron-electron interaction could be neglected. The simulated spectra are also shown in Figure
, while the parameters used for the simulations are given in supplementary Table S6. The spectra
3
were simulated using the same spin-Hamiltonian parameters in all temperature range, taking into
account only temperature change of the width of Lorentzian lines. All simulations were performed
by using also 품-strain parameters, that includes Gaussian distribution of g-values [41]. Low-
intensity lines that could be noticed in the recorded spectra (in a range 320-340 mT) do not hail
from hyperfine interaction between electron and nuclear spins but are due to static-electricity-
effect between powder samples and quartz tubes i.e. the samples were scattered across the tubes.
Figure 3. The experimental (black lines) and simulated (red lines) ESR spectra of the Cu(II)
complexes (1 – 3) at the indicated temperatures.
Page 13 of 29

From the obtained g-values given in Table S6, it could be seen that g
complexes which points out that unpaired copper(II) electron is located predominantly in dx2−y2
orbital [42]. The same conclusion could be obtained from parameter R = (g -g ) / (g -g ) < 1, given
x
≈g
y
<g
z
for all investigated
y
x
z
y
in Table S6. These results are ni agreement with square planar geometry of copper(II) ions,
obtained from single-crystal X-ray diffraction analysis [43].
3
.4 Electrochemical studies
Electrochemical behavior of Cu(II) complexes depends on geometry. In the case of square planar
Cu(II) the reduction to Cu(I) species is related to the flexibility of the structural reorganization to
tetrahedral geometry, electronic states and crystal packing forces [44]. In the case of rigid planarity
of Cu(II) coordination sphere, a reversible transfer of an electron to corresponding Cu(III) complex
also may occur. In addition to geometry, the potential of Cu(II)/Cu(I) strongly depends on the
HSAB character of the donor atoms. Soft ligands in the coordination sphere increase the redox i.e.
in Cu-proteins the values vary from + 183 mV to + 785 mV [45]. In the case of oxygen-donor
ligands, the stabilization of Cu(II) and decrease of potential could be expected. Redox behavior of
copper(II) complexes 1 – 3 was investigated by cyclic voltammetry in acetonitrile solution with
tetrabutylammonium hexafluorophosphate as a supporting electrolyte in the range -1.5 to +2.0 V
(Figure 4).
All complexes were found to have similar pattern of electrochemical response. The cathodic peak
퐼퐼
퐼
−
(
A) corresponding to one-electron reduction of complex species [퐶푢 퐿 ] + 푒 ↔ [퐶푢 퐿 ] was
2 2
found at –0.608, –0.598, and –0.618 V for 1 – 3, respectively. Chemical reversibility is omitted by
퐼
−
ꢀ
−
the fast decomposition of electrogenerated Cu(I) complex species (B) [퐶푢 퐿 ] → 퐶푢 + ꢁ퐿
2
followed by the process 퐶푢ꢀ → 퐶푢2ꢀ + 푒 (C) and the corresponding reduction peak (A’). In the case of
complex 1, the appearance of another peak (D) in the back scan reveals the process known as anodic
stripping [46]. This peak refers to the reoxidation of metallic copper that has been deposited on
the electrode surface during Cu(I)/Cu(0) process. Unlike to complexes 2 and 3, the peak (D) occurs
-1
at all scan rate values (100 – 300 mV s ) in cyclic voltammograms of complex 1.
Page 14 of 29

-3
Figure 4. Cyclic voltammogram of 1 – 3 (2 × 10 M) in MeCN, supporting electrolyte 0.1 M
-1
Bu
4
NPF , scan rate 0.1 V s . Inset: Enlarged region 0 – 0.6 V.
6
3
3
.5 Biological evaluation
.5.1 Interaction with BSA
Proteins, especially the most abundant albumins, are generally considered as potential targets of
many biologically active copper(II) complexes [47-49]. The interaction of BSA with copper(II)
complexes 1 – 3 was investigated by spectrofluorimetry. Typical titration is shown in Figure 5a.
Tryptophan, Trp-213, in the hydrophobic pocket of subdomain IIA and Trp-134 at the surface of
protein, along with tyrosine residues, show intrinsic fluorescence in aqueous solution with
emission maximum near 340 nm when the excitation is carried our near 280 nm [50]. The presence
of copper(II) complexes 1 – 3 in increasing amounts caused the decrease of the BSA emission band
intensity indicating the alteration in the secondary structure of the BSA (Figure S11).
Page 15 of 29

1
000
1.8
a)
b)
1
1
1
1
.6
.4
.2
.0
8
6
4
2
00
00
00
00
0
4
y = 9.88 × 10 x + 0.98
2
r
= 0.9956
0.8
0
4x10-6 8x10-6
[
complex] / M
0
.0
c)
-
-
-
-
-
0.2
0.4
0.6
0.8
1.0
y = 1.11 x + 5.52
2
r
= 0.9992
-1.2
-
6.2 -6.0 -5.8 -5.6 -5.4 -5.2 -5.0
log[complex]
2
80 300 320 340 360 380 400 420 440

/ nm
Figure 5. Interaction of copper(II) complex 1 with BSA: a) spectrofluorimetric titration of BSA
with 1; b) graphical determination of Stern-Volmer constant; c) graphical determination of binding
constant and number of binding sites.
The fluorescence linearly decreases with the increase of copper(II) complex concentration and the
change obeys to classical Stern-Volmer relation (Eq. (2))
ꢂ0
=
1 + 퐾 [푐표푚푝푙푒푥]
(ꢁ)
푆푉
ꢂ
where I and I are emission intensities in the absence and the presence of complex, respectively
0
and KSV is Stern-Volmer quenching constant and [complex] is concentration of the complex.
Stern-Volmer quenching constants were determined graphically as the slopes of the [complex] vs
0
I /I plot (Figure 5b) and the corresponding values are summarized in Table 3. The KSV values are
5
-1
in (0.89 – 2.13) × 10 M range confirming that copper(II) complexes 1 – 3 induce significant
fluorescence quenching. The KSV values decrease in order 2 > 3 > 1. The quenching rate constants
1
3
-1 -1
(k
q
) are in (0.89 – 2.13) × 10 M s range and indicate that copper(II) complexes cause static
quenching of BSA fluorescence. In the case of dynamic quenching maximum value of k is 2 ×
q
1
0
-1 -1
1
0 M s [51].
Page 16 of 29

Table 3. Data on interaction of copper(II) complexes 1 – 3 with BSA.
Compound
K
sv /M-1
[Cu]50% /M
1.04 × 10-5
4.47 × 10-6
4.66 × 10-6
k
q
/ M-1 s-1
K
b
/ M-1
n
Δλ / nm
9.88 × 104
2.13 × 105
1.96 × 105
9.88 × 1012
2.13 × 1013
1.96 × 1013
3.32 × 105 1.11
5.34 × 103 0.71
2.33 × 103 0.64
5
4
7
1
2
3
The change in the position of the emission maximum of BSA correlates with the change in the
polarity around fluorophore. All three copper(II) complexes 1 – 3 cause hypsochromic shift of 4
to 7 nm indicating that the polarity around tryptophan is decreased i.e. the hydrophobicity has
increased.
To investigate whether the changes in the conformation of BSA are more pronounced around
tryptophan or tyrosine synchronous spectra were recorded. When the difference between the
emission and the excitation wavelength is kept fixed (Δλ = λem – λex) the emission maximum is in
correlation with tyrosine for Δλ = 15 nm and with tryptophan for Δλ = 60 nm. The synchronous
spectra of BSA in the presence of an increasing concentrations of copper(II) complex 1 are shown
in Figure 5. The spectra clearly show that the fluorescence quenching arising from tryptophan
intrinsic fluorescence (Figure 5b) is more affected by the presence of the copper(II) complex 1
compared to the tyrosine intrinsic fluorescence (Figure 5a). Moreover, small hypsochromic shift
(Δλ = 1 nm) of the peak maximum was observed for tryptophan fluorescence.
2
1
1
00
50
00
800
600
a)
b)
4
2
00
00
0
5
0
0
2
40
260
280
300
320
240
260
280
300
320

/ nm
/ nm
Figure 5. Synchronous emission spectra of BSA in presence of increasing concentrations of
copper(II) complex (1) at: a) Δλ = 15 nm; b) Δλ = 60 nm.
Page 17 of 29

The values of the binding constant (K ) and the number of the binding sites (n) are determined
b
using equation (4):
ꢂ0 ꢃ ꢂ
푙표푔
= 푙표푔퐾 + 푛 log[푐표푚푝푙푒푥]
(3)
푏
ꢂ
by plotting log[(I – I)/I] vs [complex] (Figure 5c). The slope of the diagram is the number of the
0
binding sites while the intercept is the logarithmic value of the binding constant. The calculated
values are summarized in Table 3. All complexes have number of the biding sites close to 1
indicating that the complexes prefer 1:1 binding with albumin. The binding constant values are in
3
5
-1
2
.33 × 10 – 3.32 × 10 M range. The largest value of the binding constant was observed for
5
-1
complex 1 (3.32 × 10 M ) having Schiff base ligand derived from propylamine and
salicylaldehyde. The values are generally higher from complexes having propyl structural moiety
and decrease in order 1 ≫ 2 > 3.
Literature reveals that the interaction of many copper(II) complexes with BSA were studied since
this interaction is closely related to drug efficiency in the treatment of many diseases and strongly
depends on their binding properties with the carrier protein. Molecules with a K binding constant
b
4
6
-1
in order 10 –10 M are thought to be acceptable for drug carriers in the blood [52], accordingly,
complex 1 can be considered in this light. For homoleptic or heteroleptic Cu(II) complexes with
ligands derived from salicylaldehydes, naftaldehyde, phenantroline and phenol derivatives we
2
6
-1
found reports on binding properties that differ according to K in order 10 – 10 M [17, 53, 54],
b
5
-1
6
-1
6
9
-1
1
0 M [55], 10 M [56], 10 – 10 M [57, 58], respectively.
Page 18 of 29

3
.5.2 Molecular docking study
A molecular docking technique was employed to analyse the interaction and binding modes
between copper(II) complexes and BSA. The docking results are shown in Table 4 and Figures 6
and 8.
Table 4. Contacting receptor residues involved in copper(II) complexes-BSA formation and free
binding energy for the best-calculated docking positions.
Binding
Copper(II)
complex
Contacting receptor residues (amino acids)
energy
-
1
(
kcal mol )
Asp-108, Arg-144, His-145, Pro-146, Tyr-147, Phe-148, Leu-
189, Ser-192, Ala-193, Arg-196, Leu-197, Glu-424, Val-425,
Ser-428, Leu-454, Ile-455, Asn-457, Arg-458, Val-461
Asp-107, Asp-108, Ser-109, Pro-110, Asp-111, Leu-112, Arg-
144, His-145, Pro-146, Tyr-147, Leu-189, Thr-190, Ser-192,
Ala-193, Arg-196, Glu-424, Ser-428, Arg-458
Ser-109, Pro-110, Asp-111, Leu-112, Pro-113, Lys-114, Leu-
115, Ile-141, Arg-144, Arg-185, Pro-420, Glu-424, Arg-427,
Arg-458, Thr-518, Glu-519, Ile-522
1
-9.99
2
3
-8.67
-8.02
As shown in Figure 6, all copper(II) complexes occupied the same domain of BSA (Pro-110, His-
45, Ala-193, Glu-424, Arg-458, etc.) and bound to BSA with binding energies of similar values
Table 4).
1
(
Figure 6. Binding poses of copper(II) complexes 1 – 3 (a – c) with BSA (PDB ID: 3V03) as
assessed by molecular docking study.
Page 19 of 29

The trend in binding energies (absolute values) for copper(II) complexes is 1 > 2 > 3, indicating
that complex 1 has the highest affinity toward BSA, and complex 3 the lowest. No hydrogen bonds
were observed in the formations, only hydrophobic and π-π interactions.
Figure 7. Copper(II) complex 2 close-up projection of the binding mode within the BSA.
The distances between copper(II) complexes with tryptophan residues in BSA were also calculated
from the docking study (Figure 8).
Figure 8. Distances between tryptophan residues in BSA and copper(II) complexes 1 – 3 (a – c)
as assessed by molecular docking study.
Obtained results showed that distances between two tryptophan residues and complexes were as
follows: 16.62 Å (Trp-213) and 22.65 Å (Trp-134) with an average of 19.63 Å for complex 1;
2
0.43 Å (Trp-213) and 21.45 Å (Trp-134) with an average of 20.94 Å for complex 2; 23.00 Å
(Trp-213) and 25.67 Å (Trp-134) with an average of 24.33 Å for complex 3.
Page 20 of 29

3
.5.3 Antitumor activity in vitro
There is a continuing interest in developing novel strategies and novel compounds in the fight
against cancer. Due to the role of free copper(II) in angiogenesis and progression of malignancy
[
59, 60] the focus of antiangiogenic therapy has been based on copper-chelating agents. On the
other hand, numerous copper(II) complexes are being continuously designed and their
antiproliferative activity has been studied. The most numerous among the copper(II) complexes
are based on thiosemicarbazone ligands, some of which such as MarboranTM or TriapineTM are in
clinical use [61]. The complexes 1 – 3 were tested on two cancer cell lines and control non-tumor
cells. The most pronounced antiproliferative activity was demonstrated by compound 3, while 2
was the least active. Compound 1 was cytotoxic only at the highest tested concentration. No
selectivity between (H460 and HCT116) and non-tumor (HEK293T) cell lines was observed with
an insignificant IC50 ratio (non-tumor/tumor line) which is still the best for compound 1 having
Schiff base derived from non-substituted salicylaldehyde and n-propylamine (Table 5).
Concentration-response profiles for compounds 1 – 3 are presented in supplementary, Figures S12-
S14.
Table 5. IC50 values (µM) for 1 – 3.
Cell Lines
Compound
HEK293T
31±28
33±5
H460
18±6
>100
20±9
HCT116
22±5
58±20
16±3
1
2
3
13±1
Cytotoxic activity of compound 3 has been previously reported against chronic myeloid leukaemia
cells (K562), a human nasopharyngeal epidermoid tumour cell line KB and a human normal cell
line (L02) with IC50 values 34.8, 35.6 and 75.4 μM, respectively [19]. Copper complexes 1 – 3 can
be regarded as moderately active antiproliferative agents if compared to other structurally similar
copper(II) Schiff base complexes featuring CuN
2
2
O coordination core. They are more active
compared to copper(II) complexes with Schiff base ligands derived from substituted
salicylaldehyde and furfurylamine [62], 2-amino-4,6-dimethylpyrimidine [63] or
sulfamethoxazole [64]. Similar or comparable antiproliferative activity was found for Cu(II)
complexes of quinolin-2(1H)-one derived Schiff bases [65] and 2‐[(4‐chlorobenzylidene)amino]
phenol [66]. However, copper(II) Schiff base complexes 1 – 3 are less active compared to Cu(II)-
Page 21 of 29

salicylideneimine complexes of 2-methoxyethylamine [15, 16], 4-morpholinoaniline [67], 1-
naphtylamine [17] or Cu(II)-naphtaldimines complex of 2-methoxyethylamine [14]. Better activity
was also observed for some Cu(II) Schiff base complexes of higher nuclearities [58] or with
tetradentate ligands [68].
4
. Conclusion
Herein, we revisited copper(II) complexes of N-n-alkylsalicylidenimine ligands including
synthesis, physicochemical characterization and the molecular structure of a novel complex, bis[N-
n-propyl-5-chloro-2-oxy-κO-benzylideneimine-κN-(1–)]copper(II) (2), along with the molecular
structures and an extended characterization of its two previously reported homologues (1, 3).
Additional characterization of 1 and 3 in particular relates to the examination of its magnetic
properties by X-band ESR spectroscopy and the determination of the redox potentials by cyclic
voltammetry. All three complexes were tested for its biological properties, starting from in vitro
BSA binding affinity using fluorescence technique to the docking study. Compounds were also
tested for in vitro cytotoxicity against two tumor cell lines H460 and HCT116 and non-tumor
HEK293T. Although the first results for 1 – 3 show the moderate binding affinity for BSA and
rather non-selectivity for the two tumor and control cell lines, there is a potential to pursue further
biological research. This story also implies the design of new copper(II) complexes with Schiff
bases having a similar motif and study of structure activity relationships.
Funding
This work was supported by the Federal Ministry of Education and Science of Bosnia and
Herzegovina (grant no. 05-39-2619-1/18) and by the Croatian Science Foundation under the
project HrZZ IP-2018-01-3168.
Page 22 of 29

Supporting information
CCDC 2073263, 2073264 and 2073265 contain the supplementary crystallographic data. These
data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from
the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax:
(+44) 1223-336-033; or e-mail: deposit@ccdc.cam.ac.uk. Additional data on physicochemical
characterization and biological activity associated with this article are available online in
Electronic Supporting Information (ESI).
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Page 26 of 29

Page 27 of 29

Highlights




Copper(II) compounds with N-n-alkyl-salicylideneimine ligands were revisited and one
novel homologue was prepared
Three compounds were fully described by X-ray diffraction and spectroscopic studies in
solid state and solution
Biological activity was investigated in the light of interaction with BSA and antitumor
activity
Interaction with BSA is the first study of Cu(II) complexes with Schiff bases derived
from n-alkyl amines
Page 28 of 29

CRediT author statement
EK: Conceptualization, Methodology, Supervision, Writing - Original Draft, Review & Editing;
MM: Investigation; AZ: BSA interaction, Visualization, Writing - Original Draft, Review &
Editing; AV: X-ray diffraction, Writing - Original Draft, Review & Editing; AO and DZ: Docking
studies; DŽ and SM: ESR measurements; MK and IMK: antiproliferative testing
Page 29 of 29