Synthesis, characterization and biological activity of copper(II) complexes with ligands derived from β-amino acids

Article abstract of DOI:10.1007/s11243-018-0270-0

Two copper(II) complexes with ligands derived from β-amino acids, 2-(1-aminocyclohexyl)acetic acid L1 and 2-(1-amino-4-(tert-butyl)cyclohexyl)acetic acid L2, were synthesized and characterized by microanalysis, infrared, UV–Vis and EPR spectra. The spectr

Full text of DOI:10.1007/s11243-018-0270-0

Transition Metal Chemistry
https://doi.org/10.1007/s11243-018-0270-0
Synthesis, characterization and biological activity of copper(II)
complexes with ligands derived from β‑amino acids
Andriana M. Bukonjić1 · Dušan Lj. Tomović1 · Ana S. Stanković1 · Verica V. Jevtić2 · Zoran R. Ratković2 ·
Jovana V. Bogojeski2 · Jelena Z. Milovanović3 · Dragana B. Đorđević3 · Aleksandar N. Arsenijević3 ·
Marija Z. Milovanović3 · Ivan Potočňák4 · Srećko R. Trifunović2 · Gordana P. Radić1
Received: 12 June 2018 / Accepted: 2 August 2018
© Springer Nature Switzerland AG 2018
Abstract
Two copper(II) complexes with ligands derived from β-amino acids, 2-(1-aminocyclohexyl)acetic acid L1 and 2-(1-amino-
4-(tert-butyl)cyclohexyl)acetic acid L2, were synthesized and characterized by microanalysis, infrared, UV–Vis and EPR
spectra. The spectroscopically predicted structure of the square-planar copper(II) complex with 2-(1-aminocyclohexyl)-acetic
acid C1 was confrmed by single-crystal X-ray analysis. The biological activities (antitumor activities and interaction with
DNA) of the compounds were also investigated. The interactions of both complexes with calf thymus (CT) and herring
testes (HT) DNA were examined by stopped-fow spectroscopy, by absorption (UV–Vis) and by emission spectral studies
(ethidium bromide displacement studies). Both complexes were found to react a bit faster with HT-DNA than with CT-DNA.
The obtained binding constants suggested a moderate intercalative binding mode between the complexes and DNA. In addi-
tion, fuorescence spectrometry of bovine serum albumin with the complexes showed a good fuorescence quenching of the
complexes. The obtained copper(II) complexes have a relatively low cytotoxic efect on murine mammary carcinoma cell
line, 4T1, a moderate efect on murine colon carcinoma cell line, CT26, and a relatively high cytotoxicity toward murine
lung cancer cells, LLC1.
Introduction
Copper is a trace element, but it plays a very important role
in all living organisms. Thus, it participates in redox reac-
Electronic supplementary material The online version of this
tions, in growth and development of organisms, and is a
article (https://doi.org/10.1007/s11243-018-0270-0) contains
component of numerous enzymes [1]. A deviation from
supplementary material, which is available to authorized users.
homeostatic copper concentrations can lead to the develop-
ment of various diseases, including Wilson’s disease and
Menkes disease, and is also thought to be responsible for
malignant angiogenesis, i.e., invasiveness of tumor cells and
the onset of metastases [1–4].
* Jovana V. Bogojeski
jrosic@kg.ac.rs
* Gordana P. Radić
vasic_gordana@yahoo.com
The biological activity of transition metal complexes is
increasingly attracting researchers’ attention with the aim of
fnding potential pharmacotherapeutics [5, 6]. In this con-
text, copper(II) complexes have also been investigated, but
with an insufciently clear mechanism of action [5, 7]. Pre-
vious investigations have shown that the properties of copper
complexes mainly depend on the nature of the ligands and
their donor atoms, but also on the diferences in activities of
the copper(I) and copper(II) complexes [7]. In general, coor-
dination of copper(II) is realized through oxygen, nitrogen,
sulfur and phosphorus donor ligands. Copper(II) complexes
1
Department of Pharmacy, Faculty of Medical Sciences,
University of Kragujevac, Svetozara Markovića 69,
Kragujevac 34000, Republic of Serbia
2
Department of Chemistry, Faculty of Science, University
of Kragujevac, Radoja Domanovića 12, Kragujevac 34000,
Republic of Serbia
3
Department of Microbiology and Immunology, Center
for Molecular Medicine and Stem Cell Research, Faculty
of Medical Sciences, University of Kragujevac, Svetozara
Markovića 69, Kragujevac 34000, Republic of Serbia
4
Institute of Chemistry, P.J. Safárik University, Moyzesova 11,
041 54 Kosice, Slovakia
Vol.:(0123456789)
1 3

Transition Metal Chemistry
can show a signifcant cytotoxic activity for ligands that con-
tain polydentate Schif bases, fve-membered aromatic het-
erocycles (imidazole, pyrazole, triazole) or six-membered
aromatic heterocycles such as bipyridine (bipy) or tertiary
phosphines [1, 4]. These complexes lead to the inhibition of
Staphylococcus aureus and Escherichia coli [8], while the
introduction of several diferent heterocyclic bases increases
the microbial spectrum of the efect [9, 10].
Vario III CHNOS Elemental Analyzer, Elemental Analysen-
systeme GmbH. For infrared spectra, a PerkinElmer FTIR
31725-X spectrophotometer and KBr pellet technique were
employed.
The 9.8 GHz EPR spectra were recorded at room temper-
ature on a Bruker Elexsys II 540 EPR spectrometer under the
following conditions: microwave power 6.325 mW, modula-
tion amplitude 5 G, modulation frequency 100 kHz, conver-
sion time 240 ms. The samples (30 μL of 0.5 mM aqueous
solution, in deionized 18 MΩ water) were drawn into gas-
permeable Tefon tubes (Zeus industries, Raritan, NJ). The
spectra were recorded and analyzed using the Bruker Xepr
software.
Recent studies are increasingly being focused on the
synthesis of complexes of α-amino acid ligands and their
derivatives. These include the study of the stability of such
complexes, their binding to DNA, and their biological activi-
ties [11–16]. A copper(II) complex of bacitracin showed a
signifcant oxidative activity [16]. The comparative analysis
of three complexes of copper(II) with glycine (gly), alanine
(ala) and a combination of ligands (gly and bipy) showed
that the complex with both amino acid and heterocyclic
ligands showed the strongest interaction with DNA and the
highest cytotoxic activity [12].
Kinetics of the substitution reactions of the copper(II)
complexes with CT and HT-DNA were studied in a stopped-
fow spectrophotometer (PerkinElmer Lambda 35) in 25 mM
Hepes bufer, pH≈7.2 (Acros Organics, Belgium).
Syntheses
Based on previously published results, we can conclude
that, in general, β-amino acids show better biological activi-
ties in comparison with α-amino acids [17–19]. We reasoned
that if such β-amino acids were in the coordination sphere
of copper(II), the biological activities of the resulting com-
plexes might be better still. Because this area has not yet
been fully explored, in this paper our investigations are
focused on the synthesis of copper(II) complexes of deriva-
tives of β-amino acids, namely (2-(1-aminocyclohexyl)acetic
acid L1 and 2-(1-amino-4-(tert-butyl)cyclohexyl)-acetic acid
L2) as well as the biological activities (antitumor activities
and interactions with DNA) of the obtained compounds. The
structures of the complexes were deduced on the basis of
elemental microanalysis and infrared spectra; moreover, the
square-planar structure was confrmed by an X-ray structural
study of the copper(II) complex of L1. The interactions of
the copper(II) complexes with CT-DNA and HT-DNA were
investigated. Since it is considered that some transition metal
complexes manifest their antitumor activity by interactions
with certain proteins [20, 21], we considered it important
to examine the afnity of these new complexes with a pro-
tein, namely bovine serum albumin (BSA). In this study, we
report on the cytotoxicities of the two proligands and their
corresponding copper(II) complexes against murine colon
carcinoma cell line, CT26, murine mammary carcinoma
cells line, 4T1, and murine lung cancer cell line, LLC1.
Synthesis of proligands
The proligands, 2-(1-aminocyclohexyl)acetic acid L1 and
2-(1-amino-4-(tert-butyl)cyclohexyl)acetic acid L2, were
obtained by using the previously described process [22].
A mixture of cyclohexanone or 4-tert-butyl-cyclohexanone
(1 mol), malonic acid (1 mol) and ammonium acetate
(1 mol) in 1-butanol (600 mL) was refuxed for 1.5–3.0 h
until CO2 was no longer evolved. The postreaction mixtures
were transparent solutions. All volatile compounds boiling
to 135 °C were distilled of; a colorless flm of ammonium
malonate was deposited in the Liebig condenser. The reac-
tion mixture was diluted with 1-butanol (300 mL), and the
distillation was repeated. The residue was distilled in a vac-
uum of 10–20 mmHg, until the boiling point of the distillate
reached 128–130 °C. The resulting viscous oil was cooled
to 20 °C and stirred with acetone (700 mL). Afterward it
was allowed to stand for no less than 10 h, and the result-
ing amino acid precipitates were fltered of, washed with
acetone and dried at 100 °C.
2‑(1‑aminocyclohexyl)acetic acid (L1) IR (KBr, cm−1):
3424 (w), 2945 (s), 2166 (w), 1619 (s), 1570 (s), 1492
(m), 1467 (m), 1383 (m), 1266 (w), 1200 (w), 1133 (w),
1060 (w), 946 (w), 899 (w), 764 (w), 717 (w), 603 (w).
1H NMR (200 MHz, CDCl3, δ ppm): 1.25–1.47 (m, 6H,
3CH2), 1.47–1.58 (m, 4H, 2CH2), 2.09 (s, 2H, CH2). 13C
NMR (50 MHz, CDCl3, δ ppm): 42.5 (C–), 48.0 (CH2– from
acetic acid); 41.2; 21.9; 22.6 (CH2– from cyclohexyl group);
177.3 (COOH).
Experimental
Materials and measurements
All reagents were obtained commercially and used without
2‑(1‑amino‑4‑(tert‑butyl)cyclohexyl)acetic acid (L2) IR
further purifcation. Elemental analyses were obtained on a
(KBr, cm−1): 3433 (w), 2954 (s), 2153 (w), 1639 (s), 1583
1 3

Transition Metal Chemistry
(s), 1506 (m), 1466 (m), 1395 (m), 1364 (m), 1336 (m),
1262 (w), 1173 (w), 1055 (w), 950 (w), 901 (w), 764 (w),
714 (w), 623 (w). 1H NMR (200 MHz, CDCl3, δ ppm): 2.41
(s, 2H, CH2), 1.52–1.75 (m, 8H, 4CH2), 1.41 (s, H, CH),
0.49 (m, 9H, 3CH3). 13C NMR (50 MHz, CDCl3, δ ppm):
42.8 (C-from cyclohexyl group), 48.0 (CH2– from acetic
acid); 20.7; 39.6 (CH2– from cyclohexyl group); 32.5 (CH–
from cyclohexyl group); 27.6 (CH3– from t-butyl group);
177.3 (COOH).
water molecules were found in the diference Fourier map
and refned using a riding model; all other hydrogen atoms
were placed in calculated positions and refned riding on
their parent C or N atoms. An analysis of bond distances
and angles was performed using SHELXL2013. DIAMOND
[27] was used for molecular graphics.
Kinetic measurements
The interactions of complexes C1 and C2 with CT- and
HT-DNA were studied by stopped-fow spectroscopy. In
a typical experiment, one syringe contained various con-
centrations of CT- or HT-DNA in PBS bufer (pH= 7.2),
and the other contained 1.0 mM of complex C1 or C2. For
each run, equal volumes of both solutions from separate
syringes were rapidly mixed in the mixing chamber and the
changes in absorbance were monitored for a chosen period
of time. The temperature of both drive syringes, the cell
and the mixing chamber was maintained at 37.0 0.2 °C
using a circulating water bath. The absorbance change was
measured at λ=260 nm after the addition of either complex.
The observed pseudo-frst-order rate constants, kobsd, were
calculated as the average values from four to six independent
kinetic runs using the program OriginPro 8. Experimental
data are presented in Tables S2–S5.
Synthesis of the complexes
Copper(II) nitrate trihydrate (0.1000 g, 0.4139 mmol) was
dissolved in water (10.0 mL) on a steam bath, and 2-(1-ami-
nocyclohexyl)acetic acid L1 (0.1301 g, 0.8278 mmol)
or 2-(1-amino-4-(tert-butyl)cyclohexyl)acetic acid L2
(0.1766 g, 0.8278 mmol) was added. The reaction mixture
was heated for 3 h, and during this period 10.0 mL of an
aqueous lithium hydroxide solution (0.0199 g, 0.8278 mmol)
was added in small portions. The solutions were the fltered
and evaporated to small volume.
Single crystals of the copper(II) complex with 2-(1-ami-
nocyclohexyl)acetic acid (C1) suitable for single-crystal
X-ray difraction analysis were obtained by slow evaporation
of a water solution. Yield: 0.1256 g (80.75%). Anal. Calc.
for C1=CuC16H28N2O4 (Mr =375.95): C, 51.12; H, 7.51; N,
7.45%. Found: C, 51.27; H, 7.42; N, 7.42%. IR (KBr, cm−1):
3375 (s), 3293 (s), 3243 (s), 2933 (m), 2865 (m), 1576 (s),
1408 (s), 1375 (s), 1198 (m), 1139 (w), 1065 (w), 957 (w),
804 (w), 730 (w), 678 (w).
DNA interactions
Stock solutions of CT-DNA and HT-DNA were prepared in
PBS bufer. The ratio of UV absorbances at 260 and 280 nm
(A260/A280) of ca. 1.8–1.9 indicated that the DNA was suf-
fciently free from protein. The concentration was deter-
mined by the UV absorbance at 260 nm (ε=6600 M−1 cm−1)
[28]. The UV–Vis spectra were obtained on a PerkinElmer
Lambda 35 or 25 double beam spectrophotometer, using
1.0 cm path length quartz cuvettes (3.0 mL). Fluorescence
measurements were taken on an RF-1501 PC spectrofuor-
ometer (Shimadzu, Japan). The fuorescence spectra were
recorded in the range of 550–750 nm upon excitation at
527 nm in all cases. The excitation and emission bandwidths
were both 10 nm.
Complex (C2) was obtained as a blue powder from a water
solution by slow evaporation. Yield: 0.1581 g (78.24%).
Anal. Calc. for C2=CuC24H44N2O4 (Mr =488.16): C, 59.05;
H, 9.08; N, 5.74%. Found: C, 58.98; H, 9.01; N, 5.69%. IR
(KBr, cm−1): 3369 (s), 3297 (s), 3239 (s), 2949 (m), 2866
(m), 1577 (s), 1448 (s), 1429 (s), 1408 (s), 1326 (s), 1209
(m), 1176 (m), 1121 (w), 1109 (w), 993 (w), 973 (w), 959
(w), 900 (w), 733 (w), 680 (w).
Single‑crystal X‑ray crystallography
A summary of the X-ray difraction experiment and structure
refnement for C1 is given in Table S1. The crystal struc-
ture of C1 was determined using an Oxford Difraction
Xcalibur2 difractometer equipped with a Sapphire2 CCD
detector using graphite monochromated Mo Kα radiation
(λ=0.71073 Å). Crysalis CCD [23] was used for data collec-
tion, while Crysalis RED [23] was used for cell refnement,
data reduction and absorption correction. The structure was
solved by SUPERFLIP [24] and subsequent Fourier synthe-
ses using SHELXL2013 [25], as implemented in the WinGX
program suite [26]. Anisotropic displacement parameters
were refned for all non-H atoms. Hydrogen atoms from the
UV–Vis absorption studies
In order to compare the binding strengths of the complexes
with DNA, the intrinsic binding constants Kb were deter-
mined by monitoring the changes in absorption at the MLCT
band with increasing concentrations of CT-DNA, using the
following Eq. 1;
(
)
(
)
[
(
)]
[DNA]∕ ꢀA−ꢀf = [DNA]∕ ꢀb−ꢀf + 1∕ Kb ꢀb−ꢀf
(1)
where Kb is given by the ratio of slope to the y intercept
in plots of [DNA]/(εA − εf) versus [DNA], where [DNA]
1 3

Transition Metal Chemistry
is the concentration of DNA in the base pairs, εA = Aobsd
/
serum-free PBS. The number of viable cells was determined
by trypan blue exclusion.
[complex], εf is the extinction coefcient for the unbound
complex, and εb is the extinction coefcient for the complex
in the fully bound form.
The efects of the test compounds on cell viability were
determined using the MTT colorimetric technique [29]. Cells
were diluted in growth medium to (5×104 cells/mL), and ali-
quots (5×103 cells/100 mL) were placed in individual wells
in 96 multiplates. The next day, the medium was exchanged
with 100 μL solution of the test compounds, which had been
serially diluted twofold in the medium such that concentra-
tions ranged from 1000 to 7.8 μM in growth medium. Each
compound was tested in triplicate. The cells were incubated
at 37 °C in 5% CO2 for 72 h. After the incubation, the super-
natant was removed and 15% MTT solution (5 mg/mL in
PBS, 10 μL) in DMEM medium without FBS was added to
each well. After an additional 4 h of incubation at 37 °C in a
5% CO2, the medium with MTT was removed and DMSO
(150 μL) with glycine bufer (20 μL) was added to dissolve the
crystals. The plates were shaken for 10 min. The optical den-
sity of each well was determined at 595 nm using a microplate
Zenyth 3100 Multimode detector. The percentage of cytotoxic-
ity was calculated as a percentage using the formula:
Ethidium bromide (EB) displacement studies
The binding strengths of complexes to CT-DNA and HT-
DNA were determined by calculating the quenching con-
stant (Ksv) from the slopes of straight lines obtained from
the Stern–Volmer equation (Eq. 2).
I0∕I = 1 + Ksv[Q]
(2)
where I0 and I represent the emission intensities in the
absence and the presence of the quencher (complex C1 or
C2), respectively; [Q] is a total concentration of quencher;
Ksv is the Stern–Volmer quenching constant which can be
obtained from the slope of the plot of I0/I versus [Q].
Viscosity measurements
The viscosity of DNA solutions was measured in the pres-
ence of increasing amounts of complexes C1 and C2. The
fow time was measured with a digital stopwatch. Each sam-
ple was measured three times, and the average fow time
was calculated. The data are presented as (η/η0)1/3 against r,
where η is the viscosity of DNA in the presence of the com-
plex and η0 is the viscosity of DNA alone in the bufer solu-
tion. The viscosity values were calculated from the observed
fow time of the DNA-containing solutions (t), corrected for
the fow time of the bufer alone (t0), η=(t−t0)/t0.
% cytotoxicity = 100 − ((E − B)∕(S − B) × 100)
where B is for the background medium alone, S is for total
viability/spontaneous death of untreated target cells, and E
is for the experimental well. All experiments were carried
out in triplicate.
For the apoptosis assays, the cells were plated in T25 cul-
ture fasks and allowed to grow overnight. After the cells
reached subconfuency, the medium was replaced with the
complex (15 μM). The exposed cells were kept at 37 °C in a
5% CO2 incubator for 24 h. The cultured cells were washed
twice with PBS and resuspended in 1× binding bufer (10×
binding bufer: 0.1 M Hepes/NaOH (pH 7.4), 1.4 M NaCl,
25 mM CaCl2) at a concentration 1×106/mL. Annexin FITC
and propidium iodide (PI) were added to 100 mL of cell sus-
pension and incubated for 15 min at room temperature (25 °C)
in the dark. After the incubation, 400 mL of 1× binding bufer
was added to each tube and the stained cells were analyzed
within 1 h by using FACS Calibur (BD, San Jose, USA) and
Flow Jo software (Tri Star). Since Annexin V FITC staining
precedes the loss of membrane integrity that accompanies the
later stage identifed by PI, Annexin FITC positive, PI negative
indicates early apoptosis, while the viable cells are Annexin V
FITC negative, PI negative. Cells that are in late apoptosis or
already dead are both Annexin V FITC and PI positive.
In vitro cytotoxicity studies
The complexes were dissolved in 10% DMSO in distilled
water at a concentration of 10 mM and fltered through
a 0.22-mm Millipore filter. These stock solutions were
diluted in culture medium immediately before use. MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro-
mide) was dissolved (5 mg/mL) in a phosphate bufer saline
having a pH of 7.2 and fltered through a 0.22-mm Millipore
flter before use. All reagents were purchased from Sigma
Chemicals.
CT26, 4T1 and LLC1 cells were purchased from the Amer-
ican Type Culture Collection (ATCC, Manassas, USA). The
cells were maintained in DMEM (Sigma-Aldrich, Munich,
Germany) supplemented with 10% fetal bovine serum (FBS,
Sigma-Aldrich, Munich, Germany), penicillin (100 IU/mL)
and streptomycin (100 μg/mL) in a humidifed atmosphere of
95% air/5% CO2 at 37 °C. Subconfuent monolayers, in log
growth phase, were harvested by brief treatment with 0.25%
trypsin and 0.02% EDTA in phosphate bufered saline (PBS,
Sigma-Aldrich, Munich, Germany) and washed three times in
1 3

Transition Metal Chemistry
The infrared spectra of both complexes confrm a biden-
tate mode by means of the amino nitrogen atoms. The
isolated copper(II) complexes show asymmetric stretching
frequencies of the primary amino group at 3375 cm−1 for
C1 and 3369 cm−1 for C2. Also, the complexes have no
absorption bands from protonic amino groups, whereas
in the spectra-free amino acids the signals of the protonic
amino group appear at about 3300–2300 cm−1 (specifcally
2944 cm−1 L1 and 2954 cm−1 L2).
Results and discussion
Synthesis and chemical characterization
Two β-amino acids derivatives 2-(1-aminocyclohexyl)
acetic acid L1 and 2-(1-amino-4-(tert-butyl)cyclohexyl)
acetic acid L2) were prepared [22] by direct reaction of
cyclohexanone, malonic acid and ammonium acetate.
The corresponding copper(II) complexes C1 and C2 were
obtained by direct reaction of copper(II) nitrate with L1
and L2, respectively, in a molar ratio of 1:2, followed addi-
tion of an equimolar amount of aqueous lithium hydroxide,
with satisfactory yields of about 80% (Scheme 1).
Based on the composition of the complexes and their IR
spectra alone, it cannot be concluded with certainty how
the β-amino acid derivatives are coordinated to copper(II).
However, a single-crystal X-ray difraction study of com-
plex C1 revealed that the metal is coordinated to L1 in a
square-planar copper(II) complex with trans(O)-trans(N)-
geometry. Since ligand L2 is a structural analog of L1,
a similar geometry can be expected for the structure of
corresponding copper(II) complex C2.
Infrared spectra of the isolated complexes were recorded
in order to identify the coordination mode of β-amino
acids ligands. In particular, the asymmetric stretching
frequencies of the carboxylic groups were used to deter-
mine whether these groups were uncoordinated (absorp-
tion bands expected in the range from 1700 to 1750 cm−1)
or coordinated (absorption bands are located in the range
from 1600 to 1650 cm−1) to the metal center [30–32].
Thus, the isolated copper(II) complexes both show dou-
ble sharp and strong asymmetric stretching frequencies
of the carboxylic groups at about 1650–1570 cm−1. The
observed clear double bands for the complexes suggest
small diferences in coordination of the ligand carboxylic
groups to the metal.
The electronic spectra of both complexes were recorded
in water, with a concentration of 1 × 10−4 mol/dm3 at
room temperature. The spectra show an absorption band
at 264 nm for C1 and at 256 nm for C2.
Figure 1 shows room temperature EPR spectra of the
two copper(II) complexes in 0.5 mM aqueous solution.
Both spectra are anisotropic with axial symmetry and
clearly show diferent Cu signals, most likely due to the
diferent ligands. The spectrum of complex C1 is char-
acterized by g‖ = 2.16, and g⊥ = 2.07. The spectrum of
complex C2 shows a poorly resolved Cu signal (S = 1/2,
I = 3/2) hyperfne coupling, with g‖ = 2.23, and g⊥ = 2.09.
Scheme 1 Synthesis of L1
(R=H) and L2 (R=tert-butyl)
and the corresponding com-
plexes C1 and C2
1 3

Transition Metal Chemistry
Fig. 1 Room temperature
X-band EPR spectra of com-
plexes a C1 and b C2 in water
(0.5 mM)
Table 1 Selected bond lengths (Å) and angles [°] for C1
Cu1–O1
Cu1–N1
O1–C1
O2–C1
N1–C3
C1–C2
C3–C4
C3–C2
C3–C8
C4–C5
C8–C7
C6–C7
C6–C5
1.9238(15)
1.9852(17)
1.280(3)
1.245(3)
1.500(3)
1.520(3)
1.532(3)
1.536(3)
1.538(3)
1.530(3)
1.533(3)
1.522(3)
1.524(3)
O1–Cu1–N1i
O1i–Cu1–N1
O1–Cu1–N1
C1–O1–Cu1
C3–N1–Cu1
O2–C1–O1
O2–C1–C2
O1–C1–C2
N1–C3–C4
N1–C3–C2
C4–C3–C2
N1–C3–C8
C4–C3–C8
C2–C3–C8
C1–C2–C3
C5–C4–C3
C7–C8–C3
C7–C6–C5
C6–C7–C8
C6–C5–C4
87.45(7)
87.45(7)
92.55(7)
130.35(14)
113.79(13)
121.47(19)
118.47(19)
120.01(19)
107.70(16)
107.31(17)
112.82(17)
109.70(17)
109.48(17)
109.75(17)
117.62(17)
112.32(17)
113.06(17)
111.95(18)
111.31(18)
111.11(19)
Fig. 2 Molecular structure of C1 with thermal ellipsoids shown at
40% probability. Dashed lines represent hydrogen bonds (i: −x+1,
−y, −z+1)
Crystal structure of complex C1
The X-ray crystal structure of C1, shown in Fig. 2, reveals
a neutral complex molecule consisting of a Cu(II) atom and
two bidentate L1 ligands. These are coordinated to Cu1 by
their O1 atoms after deprotonation of the carboxylic groups,
and by their amino N1 atoms in trans-positions forming a
square-planar coordination sphere around Cu1. As the Cu1
atom is localized at the center of symmetry, the complex unit
contains only one independent L1 ligand.
Symmetry transformations used to generate equivalent atoms: i:
−x+1, −y, −z+1
[33]. However, only one of these Cu(II) complexes is
square planar [34] as the coordination number of the
Cu(II) atom in all the others is increased, mostly due to
coordinated solvent molecule(s).
The bonds in the carboxylic group of ligand L1 are clearly
delocalized (see Table S2), while all other bond lengths are
typical for single C–C and C–N bonds. The cyclohexyl ring
is in a chair conformation. All three molecules of crystal
water are involved in hydrogen bonds (Table S6), stabiliz-
ing the crystal structure. Each water molecule is in a typical
tetrahedral environment provided by other water molecules,
amine or carbonyl groups. Complex units in the structure
form layers in the bc plane which are linked by hydrogen
bonds from layers of crystal water in order to form an infnite
3D system (Fig. 3).
The selected bond lengths and angles for C1 are sum-
marized in Table 1. A deformed square-planar geometry
around the central atom is confrmed by the N1–Cu1–O1
and N1–Cu1–O1i bite angles (i = 1 − x, − y, 1 − z) which
difer a little from 90° as expected for atoms coordinated
in cis-positions, and also by the Cu1–O1 bond which is
slightly shorter than the Cu1–N1 bond due to the smaller
covalent radius of oxygen. Nevertheless, the bond dis-
tances around the Cu1 atom are similar to those observed
in twelve similar Cu(II) complexes with diferent β-amino
acids as reported in the Cambridge Structure Database
1 3

Transition Metal Chemistry
Table 2 Rate constants for the reactions of C1 and C2 with CT- and
HT-DNA at pH=7.2 (PBS bufer) and 310 K
CT-DNA
HT-DNA
k2 (M−1 s−1
)
k2(M−1 s−1
)
C1
C2
(3.8 0.1)×103
(2.9 0.1)×103
(6.3 0.2)×103
(3.4 0.1)×103
The results presented in Table 1 clearly show that the
rate constants depend on the nature of the ligand in the
complexes such that k2 value for the reaction of C1 has a
greater value than that for C2. This can be attributed to the
presence of the tert-butyl group on the ligand, which causes
an increased steric efect for complex C2. Also, it can be
seen that the reaction goes faster with HT-DNA than with
CT-DNA.
Fig. 3 Hydrogen bonds (dashed lines) forming a 3D supramolecular
structure in C1. Hydrogen atoms not involved in hydrogen bonds are
omitted for clarity
DNA interaction studies
Interactions of the complexes with DNA
Electronic absorption spectroscopy is widely used to deter-
mine the DNA binding afnity of metal complexes. Hence,
the potential binding abilities of C1 and C2 for CT- and
HT-DNA were studied by UV spectroscopy. Typical titra-
tion curves for C1 and C2 in the presence of either DNA
at diferent concentrations are given in Figs. 5 and S2; the
other curves are similar and hence not given. Upon increas-
ing the concentration of either DNA, a signifcant hyperchro-
mic efect with the appearance of a new band at 258, 262
or 266 nm, but with only insignifcant absorption changes
in the region of 300–500 nm was observed, as shown in
Fig. 5 (see also Figures S2, S3 and S4 in the Supporting
Information). A signifcant hyperchromic shift with the
appearance of a new signal in the UV–Vis spectra clearly
suggests a strong interaction between the complexes and
DNA [35, 36]. Moreover, the intrinsic binding constants Kb
(Table 3) obtained for the two complexes with CT- and HT-
DNA follow the order C1>C2. Once again, for complex C2
the steric hindrance of the two tert-butyl groups results in a
reduced interaction with DNA.
The kinetics of the interactions both complexes with calf
thymus (CT) and herring testes (HT) DNA was investi-
gated spectrophotometrically by following the change in
absorbance at 260 nm as a function of time using stopped-
fow spectroscopy. All measurements were taken at 7.2 and
a temperature of 310 K. To keep a constant pH of 7.2, a
PBS bufer was used for all measurements (PBS = phos-
phate buffer solution = 0.01 M, c(NaCl) = 0.137,
c(KCl) = 0.0027 M, pH 7.4).
The substitution reactions of C1 and C2 with CT- and
HT-DNA can be represented by Eq. (3):
k2
DNA + C ⇌ DNA-C
(3)
k1
where C=C1 or C2; DNA=CT- or HT-DNA.
Here, k2 is the second-order rate constant for the for-
ward reaction, characterizing formation of the product, and
k1 is the rate constant for the reverse reaction. The rate of
the reaction is then described by Eq. (4). All kinetic runs
could be ftted by a single exponential function. Plots of
kobsd versus DNA concentration revealed a linear depend-
ence with negligible intercept for both complexes. The
values of the constants k2 are listed in Table 2.
In order to further characterize of the binding of these
complexes with DNA, competitive binding experiments
were carried out on both EB-CT-DNA and EB-HT-DNA
by varying the concentration of the complexes. EB is a pla-
nar cationic molecule emitting intense fuorescence at about
600 nm in the presence of DNA, due to its strong intercala-
tion between adjacent DNA base pairs [37]. The enhanced
fuorescence can be quenched upon the addition of a second
molecule which can displace the EB and/or bound or break
the secondary structure of the DNA [38]. The interactions
of C1 and C2 with CT-DNA and HT-DNA were studied by
monitoring the fuorescence intensity of EB in PBS bufer
kobsd = k2[P] + k1[Cl−] ≈ k2[P]
(4)
Each pseudo-frst-order rate constant, kobsd, was calcu-
lated as the average of six or seven independent runs, and the
values are given in Tables S2–S5. The experimental results
for the substitution reactions of C1 and C2 with CT- and
HT-DNA are shown in Fig. 4 (see also Figure S1 of the
Supporting Information).
1 3

Transition Metal Chemistry
kobsd /s-1
HT -DNA
3
2
1
0
CT -DNA
10 4[CT- or HT-DNA]/M
0
1
2
3
4
5
0.53
0.51
0.49
0.47
0.45
0.41
0.4
A
A
II
I
0.39
0.38
0.37
0.36
HT- DNA
CT-DNA
t(s)
t(s)
0
1
2
3
4
5
6
0
2
4
Fig. 4 Pseudo-frst-order rate constants plotted as a function of CT-
or HT-DNA concentration for the reactions between the complexes
and DNA at pH=7.2 and 310 K in PBS bufer, λ=260 nm. Graph
I—absorbance-time traces for the reaction between C1 and HT-DNA
(2×10−4 M), pH=7.2, 310 K, PBS bufer; Graph II—absorbance-
time traces for the reaction between C1 and CT-DNA (2×10−4 M),
pH=7.2, 310 K, PBS bufer
0.5
(pH=7.2). In all cases, there was signifcant quenching of
fuorescence intensity due to the displacement of EB from
the EB-DNA complex. The quenching parameters for the C1
and C2 were calculated using the Stern–Volmer equation.
Increasing the concentration of C1 or C2 (0–10 μM) resulted
in a signifcant decrease in EB fuorescence intensity, with
a noticeable redshift (see Figs. 6 and S3, the other depend-
ences are similar and are not given). These results indicate
loss of EB from the EB-DNA complex due to displacement
by complexes C1 and C2 [37, 39, 40].
A
5
Complex 2
4
0.4
3
2
0.3
1
0.2
0
4 10-6[DNA]
0
2
6
8
The fuorescence intensity at 606, 609, 611 or 612 nm
(depending on the complex-DNA combination) was used
to estimate Ksv (Table 3). The fuorescence quenching data
were also used to determine the number of binding sites
(n) and the equilibrium binding constant Kbin by using the
Scatchard equation [41–43];
0.1
0
200
250
300
350
400
450
500
(nm)
(
)
log I0 − I ∕I = log Kbin + n log [Q]
Fig. 5 UV–Vis titration spectra for complex C2 (10 μM) in PBS
bufer (phosphate bufer solution=0.01 M, c(NaCl)=0.137,
c(KCl)=0.0027 M, pH 7.4) with increasing concentration of HT-
DNA (0–10 μM). The arrow shows hyperchromism in the spectral
band, λmax =258 nm. Inset graph: plots of [DNA]/(εA −εf) versus
[DNA]
The obtained values for Kbin and the number of binding
sites (n) are given in Table S7. These values were calculated
from plots of log(I0 − I)/I versus log[Q] (see also Figure S4
in the Supporting Information; the other dependences are
1 3

Transition Metal Chemistry
Table 3 Binding constants for
interactions of C1 and C2 with
CT- and HT-DNA and BSA in
PBS bufer
Complex C1
Kb (M−1
Complex C2
Kb [M−1
)
Ksv (M−1
)
]
Ksv (M−1
)
CT-DNA
HT-DNA
BSA
(5.9 0.1)×105
(9.8 0.1)×103
(2.4 0.1)×104
(1.75 0.2)×105
(3.8 0.1)×105
(9.6 0.1)×103
(2.3 0.1)×104
(1.0 0.1)×105
(6.2 0.1)×105
/
(4.0 0.1)×105
/
Kb =intrinsic binding constant; Ksv =Stern–Volmer constant; BSA=bovine serum albumin
2.9
2.4
1.9
1.4
0.9
0.4
I (a.u.)
160
120
80
40
0
0
1
2
3
4
5
6
550
570
590
610
λ (nm)
630
650
670
690
710
10-5 [Q] M
Fig. 6 Fluorescence titration spectra of EB-CT-DNA and of EtBr
(10 μM) bound to CT-DNA (10 μM) in the presence of varying
amounts of complex C2, λmax =609 nm. [The arrow shows changes
in fuorescence intensity upon increasing the concentration of C2
(0–10 μM).] Inset graph: Stern–Volmer plots for EB-DNA fuores-
cence titration with C2
similar and they are not given). The calculated number of
binding sites is in a range from 0.8 to 1.5, hence approxi-
mately equal to 1, suggesting a single DNA binding site for
both complexes.
Protein binding studies
Qualitative analysis of the binding of metal complexes to
BSA can be performed by means of fuorescence spectros-
copy, which can also provide useful information about the
structure, dynamics and folding of proteins [46–48]. The
changes in the fuorescence spectrum of BSA upon addition
of diferent concentrations of complex C1 or C2 (0–10 µM)
in the range of 300–500 nm (λex =295 nm) are presented in
Figs. 7 and S5 (The other dependences are similar and so
not given.) The observed decrease in fuorescence intensity
at 365 nm is indicative of an interaction between the com-
plexes and BSA protein. Fluorescence quenching data were
analyzed using the Stern–Volmer equation, and the quench-
ing constant (Ksv) was calculated by using from a plot of I0/I
versus [Q], as shown in Fig. 7.
According to the values of the constants presented in
Table 3, a strong interaction of both complexes with CT-
or HT-DNA is evident. Complex C1 has a greater afnity
for CT-DNA than complex C2, again due to greater steric
efects for C2. To some extent, both complexes interact more
strongly with HT-DNA than with CT-DNA, which can be
attributed to the higher proportion of guanine bases in the
former.
Viscosity measurements
Viscosity measurements were taken to further characterize
the interactions of complexes C1 and C2 with CT-DNA.
Classical intercalation results in lengthening and stifening
of the double helix of DNA, leading to an increase in vis-
cosity [44, 45]. The addition of increasing amounts (up to
r = 1.0) of C1 and C2 to a CT-DNA solution (0.01 mM)
resulted in an increase in the relative viscosity of CT-DNA
(Figure S6), which was more pronounced upon the addition
of C1. Therefore, the observed results suggest that the com-
plexes bind to CT-DNA by intercalation.
The equilibrium binding constant Kbin and n (see also
Table S7) as the number of binding sites can be analyzed by
the Scatchard equation (see also Figure S7 of the Support-
ing Information, the other dependences are similar and so
are not given). The values of n for the studied complex are
0.9 or 1.1 which strongly suggested the existence of a single
binding site in BSA for these complexes.
According to the obtained constants, both complexes
interact strongly with the BSA protein (Table 3). By
1 3

Transition Metal Chemistry
600
500
400
300
200
100
0
2.5
2
I (a.u.)
1.5
1
0.5
0
0
1
2
3
4
5
6
7
8
305
355
405
455
λ (nm)
10-6[Q] M
Fig. 7 Emission spectrum of BSA (10 µM; λex, 295 nm; λem 365 nm)
in the presence of increasing amounts of C1 (0–10 µM), λmax =365
nm. The arrow shows the direction of emission intensity changes
upon increasing the concentration of the complex. Inset graph: plots
of I0/I versus [Q]
comparing the obtained quenching constants (Ksv) for
the interactions of complexes C1 and C2 with DNAs and
BSA, it can be seen that studied Cu(II) complexes interact
better with BSA than with DNA (Table 3). Furthermore,
complex C1 interacts more strongly with BSA than com-
plex C2. The tert-butyl groups of C2 most likely lead to
steric hindrance, resulting in a weakened interaction with
BSA. Similar results have been reported for other amino
acid complexes [49, 50].
Anticancer activity of the complexes
The results of the MTT assays indicate that the free proli-
gands L1 and L2 have no cytotoxic efect against the three
tested carcinoma cell lines (CT26, 4T1, LLC1), even at a
concentration of 1000 µM (Fig. 8). The cytotoxic efect
of both copper complexes on all three cell lines proved
to be dose dependent. The complexes showed almost no
cytotoxicity on murine mammary carcinoma cells, 4T1,
up to concentration of 125 µM (Fig. 8). Complex C2 had
better cytotoxic activity against murine lung cancer cell
Fig. 8 Representative graphs
of 4T1, CT26 and LLC1 cell
survival after 72 h cell growth
in the presence of the copper
complexes and free proligands
1 3

Transition Metal Chemistry
line, LLC1 and murine colon carcinoma cell line, CT26
cells than C1 (Fig. 8). The cytotoxic efect of C2 on LLC1
was lower compared to cisplatin (Fig. 8). However, lower
percentages of viable CT26 cells were detected after expo-
sure to C2 at lower concentrations (7.8 and 15.625 µM)
compared to the same concentrations of cisplatin (Fig. 8).
The analysis of IC50 values also indicates that com-
plex C1 has a lower cytotoxic efect against CT26 and
LLC1 cell lines compared with the efect of C2 (Table 4).
Although the cytotoxic activity of C2 toward murine lung
carcinoma cells LLC1 is lower compared with cisplatin,
the IC50 values for C2 (15.61 6.86 µM) on LLC1 are
relatively low.
Conclusion
Two new copper(II) complexes with β-amino acid ligands
were synthesized and characterized. The ligand precur-
sors are coordinated via nitrogen and oxygen atoms to
the copper(II) center, giving square-planar structures as
confrmed by X-ray analysis for complex C1. The interac-
tions with calf thymus and herring testes DNA were stud-
ied by stopped-fow spectroscopy. Based on the obtained
rate constants, it can be concluded that the reaction goes
faster with HT-DNA than with CT-DNA, probably due to
the higher percentage of guanine in the former. The interac-
tions between the complexes and CT-DNA, HT-DNA and
bovine serum albumin were also examined. Overall, both
complexes have good afnities for the both CT- and HT-
DNA. Complex C1 has a greater afnity for DNA than C2,
which can be explained by the steric efect of tert-butyl
groups in C2. Viscosity measurements suggest that the
complexes bind to CT-DNA by intercalation. According to
the obtained quenching constants, it can be concluded that
both copper(II) complexes interact more strongly with BSA
than the DNA. We have also studied the antitumor activi-
ties of these complexes. In conclusion, it can be stated that
copper(II) complex C2 has a relatively low cytotoxic efect
on murine mammary carcinoma cells, a moderate efect on
murine carcinoma cells and a relatively high cytotoxicity
toward murine lung cancer cells.
In order to determine the potential for induction of
apoptotic death by the complexes, fow cytometry analy-
sis of CT26 cells stained with Annexin V and PI after
24 h exposure to the complexes (concentration 15 μM)
was carried out. The results indicate that after 24 h, both
complexes induce apoptotic death of CT26 cells (Fig. 9).
Moreover, both complexes had higher cytotoxic efects on
CT26 compared to cisplatin (concentration 15 mM). These
observations correlate with the MTT assays, which indi-
cate better cytotoxic efects for the copper(II) complexes at
lower concentrations on CT26 cells than cisplatin (15 μM).
Supplementary data
Table 4 IC50 values (μM) for 72 h of action of the copper(II) com-
plexes, free proligands and cisplatin on 4T1, CT26, and LLC1 cells,
as determined by MTT assays
CCDC 1810786 contains the supplementary crystallo-
graphic data for this paper. 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.
Compound
4T1
CT26
LLC1
C1
221.87 16.25
390.58 56.48
>1000
>1000
<7.8
655.42 118.67
39.86 7.09
>1000
>1000
29.11 5.87
195.90 33.05
15.61 6.86
>1000
>1000
<7.8
C2
L1
L2
Cisplatin
Acknowledgements The authors are grateful to the Ministry of Science
and Technological Development of the Republic of Serbia for fnancial
support (Grants Nos. 172016, 172034, 175069, 175103, 172011) and
Faculty of Medical Sciences for grant MP 2014/02. This work was
Data are presented as mean values SD (standard deviation) from
three experiments
Fig. 9 Representative fow plots
showing the percentages of
early apoptotic, late apoptotic
and viable CT26 cells after 24 h
treatment the copper complexes
(concentration: 15 μM)
1 3

Transition Metal Chemistry
23. (2007) Oxford difraction, CrysAlis CCD, CCD data collection
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cal Chemistry, University of Belgrade) for acquiring and analyzing
the EPR spectra.
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27. Brandenburg K (2009) DIAMOND (version 3.2i) Crystal Impact
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1 3