Antimicrobial and antifungal activities of bifunctional cooper(ii) complexes with non-steroidal anti-inflammatory drugs, flufenamic, mefenamic and tolfenamic acids and 1,10-phenanthroline

Article abstract of DOI:10.1515/chem-2020-0180

Cooper(ii) complexes represent a promising group of compounds with antimicrobial and antifungal properties. In the present work, a series of Cu(ii) complexes containing the non-steroidal anti-inflammatory drugs, tolfenamic acid, mefenamic acid and flufenamic acid as their redox-cycling functionalities, and 1,10-phenanthroline as an intercalating component, has been studied. The antibacterial activities of all three complexes, [Cu(tolf-O,O′)2(phen)] (1), [Cu(mef-O,O′)2(phen)] (2) and [Cu(fluf-O,O′)2(phen)] (3), were tested against the prokaryotic model organisms Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) and their antifungal activities were evaluated towards the yeast, Saccharomyces cerevisiae (S. cerevisiae). The antibacterial activity of both strains has been compared with the antibiotic Neomycin. The calculated IC50 values revealed slight differences in the antibacterial activities of the complexes in the order 1 ～3 > 2. The most profound growth inhibition of E. coli was observed, at its highest concentration, for the complex 1, which contains chlorine atoms in the ligand environment. The trend obtained from IC50 values is generally in agreement with the determined MIC values. Similarly, the complex 1 showed the greatest growth inhibition of the yeast S. cerevisiae and the overall antifungal activities of the Cu(ii) complexes were found to follow the order 1 > 3 ? 2. However, for complex 2, even at the highest concentration tested (150 μM), a 50% decrease in yeast growth was not achieved. It appears that the most potent antimicrobial and antifungal Cu(ii) complexes are those containing halogenated NSAIDs. The mechanisms by which Cu(ii) complexes cause antibacterial and antifungal activities can be understood on the basis of redox-cycling reactions between cupric and cuprous species which lead to the formation of free radicals. The higher efficacy of the Cu(ii) complexes against bacterial cells may be due to an absence of membrane-protected nuclear DNA, meaning that on entering a cell, they can interact directly with its DNA. Contrastingly, for the complexes to interact with the DNA in yeast cells, they must first penetrate through the nuclear membrane.

Full text of DOI:10.1515/chem-2020-0180

Open Chemistry 2020; 18: 1444–1451
Research Article
Lenka Hudecova, Klaudia Jomova, Peter Lauro, Miriama Simunkova*, Saleh H. Alwasel,
Ibrahim M. Alhazza, Jan Moncol, Marian Valko
Antimicrobial and antifungal activities of bifunctional
copper(II) complexes with non-steroidal anti-inflammatory
drugs, flufenamic, mefenamic and tolfenamic acids
and 1,10-phenanthroline
https://doi.org/10.1515/chem-2020-0180
growth inhibition of E. coli was observed, at its highest
concentration, for the complex 1, which contains chlorine
atoms in the ligand environment. The trend obtained
from IC50 values is generally in agreement with the
determined MIC values. Similarly, the complex 1 showed
the greatest growth inhibition of the yeast S. cerevisiae
and the overall antifungal activities of the Cu(II) complexes
were found to follow the order 1 > 3 ≫ 2. However, for
complex 2, even at the highest concentration tested
(150 μM), a 50% decrease in yeast growth was not
achieved. It appears that the most potent antimicrobial
and antifungal Cu(II) complexes are those containing
halogenated NSAIDs. The mechanisms by which Cu(II)
complexes cause antibacterial and antifungal activities
can be understood on the basis of redox-cycling reactions
between cupric and cuprous species which lead to the
formation of free radicals. The higher efficacy of the
Cu(II) complexes against bacterial cells may be due to
an absence of membrane-protected nuclear DNA, meaning
that on entering a cell, they can interact directly with its
DNA. Contrastingly, for the complexes to interact with the
DNA in yeast cells, they must first penetrate through the
nuclear membrane.
received September 7, 2020; accepted October 22, 2020
Abstract: Copper(II) complexes represent a promising
group of compounds with antimicrobial and antifungal
properties. In the present work, a series of Cu(II) com-
plexes containing the non-steroidal anti-inflammatory
drugs, tolfenamic acid, mefenamic acid and flufenamic
acid as their redox-cycling functionalities, and 1,10-phe-
nanthroline as an intercalating component, has been stu-
died. The antibacterial activities of all three complexes,
[Cu(tolf-O,O′)2(phen)] (1), [Cu(mef-O,O′)2(phen)] (2) and
[Cu(fluf-O,O′)2(phen)] (3), were tested against the
prokaryotic model organisms Escherichia coli (E. coli)
and Staphylococcus aureus (S. aureus) and their anti-
fungal activities were evaluated towards the yeast,
Saccharomyces cerevisiae (S. cerevisiae). The antibac-
terial activity of both strains has been compared with
the antibiotic Neomycin. The calculated IC50 values
revealed slight differences in the antibacterial activities
of the complexes in the order 1 ∼ 3 > 2. The most profound

* Corresponding author: Miriama Simunkova, Department of
Keywords: Cu(II) complexes, antimicrobial activity, anti-
fungal activity, NSAID, Escherichia coli, Saccharomyces
cerevisiae
Physical Chemistry, Faculty Chemical and Food Technology,
Slovak University of Technology, 812 37, Bratislava, Slovakia,
e-mail: miriama.simunkova@stuba.sk
Lenka Hudecova: Department of Chemistry, Constantine the
Philosopher University in Nitra, 949 74, Nitra, Slovakia
Klaudia Jomova: Department of Chemistry, Constantine the
Philosopher University in Nitra, 949 74, Nitra, Slovakia
Peter Lauro: Department of Chemistry, Constantine the Philosopher
University in Nitra, 949 74, Nitra, Slovakia
1 Introduction
It has been reported that in the USA alone, more than
50,000 patients die annually from multiresistant bac-
terial infections [1]. Since the antibiotic resistance of
various bacteria is becoming a serious medical threat
globally, there is an urgent requirement to discover new
classes of antibiotics, of which redox metal-based com-
plexes with biologically active ligands are promising
examples [2–7]. Copper is one of the most abundant
Saleh H. Alwasel: Zoology Department, College of Science,
King Saud University, Riyadh, Saudi Arabia
Ibrahim M. Alhazza: Zoology Department, College of Science,
King Saud University, Riyadh, Saudi Arabia
Jan Moncol: Department of Inorganic Chemistry, Faculty Chemical
and Food Technology, Slovak University of Technology, 812 37,
Bratislava, Slovakia
Marian Valko: Faculty Chemical and Food Technology, Slovak
University of Technology, 812 37, Bratislava, Slovakia
Open Access. © 2020 Lenka Hudecova et al., published by De Gruyter.
This work is licensed under the Creative Commons Attribution 4.0
International License.

Antimicrobial and antifungal activities of bifunctional copper(II) complexes

1445
elements in the human body and is also a redox active 2 Experimental
metal, whose positive effects on human health were first
noted as early as the late 19th century, when it was
reported by Doctor Luton that a mixture of copper chloride
and sodium salicylate was effective in the treatment of
2.1 Materials
rheumatoid arthritis, rheumatic fever and other disorders Copper acetate, methanol, 1,10-phenanthroline, flufe-
[8]. This observation was later augmented during the first namic acid, tolfenamic acid and mefenamic acid were
half of the 20th century in Finland, where miners who all obtained from Merck (Germany). To evaluate antimi-
worked in copper mines were found to suffer a consider- crobial activity of Cu(II) complexes, E. coli DH5α and
ably lower incidence of arthritis than the majority of S. aureus ATCC25923 were used, as obtained from the
population.
Institute of Plant Genetics of the Slovak Academy of
Copper occurs most commonly in biological systems Sciences in Nitra. S. cerevisiae CCY27-22-6 was used in
in either +2 or +1 oxidation states (referred to as cupric or order to determine the antifungal activities of the Cu(II)
cuprous species, respectively) [9]. To date, there is no complexes. The particular strain was obtained from the
direct proof that Cu(III) species exist in biological systems, Institute of Chemistry of the Slovak Academy of Sciences,
which would require the presence of a redox active co- Bratislava.
factor. Most common are those complexes containing
copper in the +2 oxidation state and these show inhibi-
tory effects toward the growth of a variety of bacteria,
fungi and viruses [10–13]. Copper(II) complexes con- 2.2 Preparation of Cu(II) complexes with
taining coordinated, structurally flat ligands, such as
1,10-phenanthroline, exhibit cytotoxic effects which have
been attributed predominantly to their ability to cleave
phenanthroline and non-steroidal anti-
inflammatory drugs
DNA, as a result of either complete or partial intercalation
of the phenanthroline moiety in between the DNA base
pairs [14–16]. In addition to this intercalation mechanism,
a process of redox cycling, with the formation of DNA
[Cu(tolf-O,O′)2(phen)] (1). To 50 mL of Cu(II) acetate
(0.200 g) methanol solution (0.02 M) 0.198 g of phenan-
throline monohydrate was added. The solution was
stirred for 10 min and then 0.523 g of solid tolfenamic
acid was added to achieve the 1:2 ratio of phenanthroline:
tolfenamic acid. The resulting olive-coloured mixture
was stirred for a further 24 h until a precipitate was
formed, which was filtered off and air-dried for one
damaging ROS, may occur.
In the present work, we report the antibacterial and
antifungal activities of Cu(II) complexes bearing 1,10-
phenanthroline as an intercalating ligand and the non-
steroidal anti-inflammatory drugs: tolfenamic acid,
month. Suitable monocrystals for X-ray analysis were
mefenamic acid and flufenamic acid, to provide redox-
obtained by slow evaporation of the solvent.
cycling functionalities (Figure 1). The antibacterial activ-
[Cu(mef-O,O′)2(phen)] (2). To 50 mL of Cu(II) acetate
ities of these Cu(II) complexes were gauged against the
(0.200 g) dissolved in methanol (0.02 M) 0.198 g of phe-
frequently studied prokaryotic model organisms, E. coli
nanthroline monohydrate was added. The solution was
stirred for 10 min and then 0.483 g of solid mefenamic
acid was added to achieve the 1:2 ratio of phenanthroline:
mefenamic acid. The resulting mixture was stirred for
and S. aureus, and their antifungal activities were deter-
mined against S. cerevisiae, a yeast that is widely employed
in the baking and brewing industries.
Figure 1: The structures of ligands used for preparation of studied Cu(II) complexes.

1446

Lenka Hudecova et al.
24 h until a precipitate was formed, which was filtered off was maintained to equal exactly 1% of the total volume
and air-dried for one month. Suitable monocrystals for of the test culture. The cultures were incubated at 37°C,
X-ray analysis were obtained by slow evaporation of the with continuous stirring at 150 rpm.
solvent.
The effect of the studied substance on the bacterial
[Cu(fluf-O,O′)2(phen)] (3). The Cu(II) complex with cell growth was monitored from an absorbance mea-
flufenamic acid was prepared analogously to above surement made every two hour, over a period of 24 h.
described complexes. To 50 mL of Cu(II) acetate (0.200 g) The growth profiles of bacterial cultures, determined
dissolved in methanol (0.02 M) 0.198 g of phenanthroline as a function of multiple concentrations of each test
monohydrate was added. The solution containing copper substance, were compared with those for a control
acetate and phenanthroline in methanol was stirred for culture where no test substances were present. Data
10 min. Into this solution, 0.483 g of solid flufenamic acid were obtained in triplicate and used for the calcula-
was added to achieve the 1:2 ratio of phenanthroline: flufe- tion of the IC50 values (i.e. the concentration of the
namic acid. The reaction mixture was stirred for 24 h until a test substance that causes 50% inhibition of bacterial
precipitate was formed, which was filtered off and air-dried growth).
for one month. Suitable monocrystals for X-ray analysis
were obtained by slow evaporation of the solvent.
Since its fraction in the medium did not exceed 1%,
the effect of the DMSO solvent on the growth curve could
be excluded in the consideration of the antibacterial
activities of the Cu(II) complexes 1–3 [17].
In order to determine minimum inhibitory concentra-
tion (MIC) values of the Cu(II) complexes against E. coli
and S. aureus, the rapid p-iodonitrotetrazolium chloride
(INT) colorimetric assay was used [18]. Briefly, both
E. coli and S. aureus were diluted with sterile broth
(Müller and Hinton) to reach final inocula of cca. 106
CFU mL−1. Stock solutions of copper(II) complexes were
dissolved in DMSO to reach the concentration of 25 mg
mL−1. 100 µL solution of each Cu(II) complex was diluted
with redistilled and deionized water (Merck Millipore) in
a 96 well microtiter microplates (Thermo Fischer scientific)
to reach final concentrations ranging from 3 to 300 µL
mL−1. As a positive control against bacterial strains,
1 mg mL−1 solution of the antibiotic Neomycin (Sigma-
Aldrich, FRG) was applied with final concentrations in
the range 2.5–270 µL mL−1. Individual bacteria (100 µL)
were added to wells and incubated at 36.8°C for 24 h.
p-Iodonitrotetrazolium chloride (45 µL of 0.2 mg mL−1
solution) from Sigma Aldrich (FRG) was used to indicate
the bacterial growth at 36.8°C for 24 h. p-Iodonitrotetra-
zolium undergoes colour change from colourless to red
upon its reduction by active organisms. The values of
MIC were obtained visually as the lowest concentrations
resulting in growth inhibition.
2.3 Determination of the antibacterial
activity for Cu(II) complexes
To investigate the antibacterial activity of the complexes,
the prokaryotic model organism E. coli DH5α and S.
aureus were used, as obtained from the Institute of
Plant Genetics of the Slovak Academy of Sciences in
Nitra. DH5-strain alpha was used because it appears to
be suitable for high efficiency transformation in a wide
variety of routine applications such as plasmid isolation
and other procedures.
Bacterial cells were incubated in a liquid LB (Lysogen
Broth, Luria-Bertani) medium containing 0.5% yeast
extract, 1% tryptone and 1% NaCl. For solid LB media,
the composition was enriched with 2% agar. The media
were sterilized at 125°C for 30 min. Prior to inoculation of
the bacterial cells, an ampicillin selection agent (50 mg/mL)
was added to the medium.
The bacterial culture was seeded under sterile condi-
tions into 20 mL of prepared LB medium, and the cultiva-
tion was performed in a 250 mL Erlenmeyer flask at 37°C
over 16 h, with continuous stirring at 150 rpm (Heidolf
Unimax 1010, Germany). Once grown, the culture was
diluted with pure LB medium to achieve an optical den-
sity (OD600) of 0.1, which represents a concentration of
1–2 × 108 cells/mL. The optical density was determined
spectrophotometrically (Varioskan Flash, Thermo Scientific).
Thus prepared, the inoculum was pipetted into 25 mL
Erlenmeyer flasks.
2.4 Determination of antifungal activity of
Cu(II) complexes
To each flask was added different concentrations of A model organism S. cerevisiae CCY 27-22-6 was used in
the substance to be tested, but this additional volume order to determine the antifungal activities of the Cu(II)

Antimicrobial and antifungal activities of bifunctional copper(II) complexes

1447
complexes. The particular strain was obtained from the 3 Results and discussion
Institute of Chemistry of the Slovak Academy of Sciences,
Bratislava. Yeast cells were cultured in a liquid YPD The aim of the present work was to evaluate both anti-
(Yeast Extract–Peptone–Dextrose) medium containing: bacterial and antifungal properties of selected bifunc-
2% peptone, 2% glucose and 1% yeast extract. In the tional Cu(II) complexes, bearing NSAID ligands to provide
case of solid YPD media, the composition was enriched redox-cycling functionalities and with 1,10-phenanthro-
by adding 2% agar. The media were sterilized for 30 min line as an intercalating agent. The antimicrobial activities
at 125°C and the yeast culture was inoculated under of the Cu(II) complexes were determined using the pro-
sterile conditions into 20 mL of the prepared liquid YPD karyotic organisms, E. coli and S. aureus, and the eukar-
medium. The cultivation was made over 16 h in a 250 mL yotic organism, S. cerevisiae, was used for the determina-
sterile Erlenmeyer flask maintained at 30°C, with contin- tion of antifungal activity.
uous stirring at 150 rpm (Heidolf Unimax 1010, Germany).
Once grown, the culture was diluted with pure YPD
medium, so that the optical density OD600 reached value
of 0.1, which corresponded to 1–2 × 108 cells/mL. The 3.1 Structure of Cu(II) complexes with
optical density was determined spectrophotometrically
(Varioskan Flash, Thermo Scientific). Thus prepared,
the yeast culture was used as a vegetative inoculum for
all yeast cell assays, inoculums, and was pipetted into
25 mL Erlenmeyer flasks. To each Erlenmeyer flask was
(fluf-O,O′)2(phen)] (3) are shown in Figure 2. All three
NSAIDs and 1,10-phenanthroline
The molecular structures of the copper(II) complexes [Cu
(tolf-O,O′)2(phen)] (1), [Cu(mef-O,O′)2(phen)] (2) and [Cu
then added the complex to be studied at different con-
centrations so that the volume of the solution occupied
1% of the total volume. The cultures were incubated at
30°C and stirred continuously at 150 rpm. The change
in yeast culture growth caused by the tested complex
was monitored from spectrophotometric measurements,
made every two hour, over a 24 h period. The growth of
bacterial cultures in the presence of the tested complexes
was compared with a control culture in which these com-
plexes were absent. Each measurement was performed in
triplicate and the data obtained were used to calculate
IC50 values (i.e. the concentration of the test substance
required to cause a 50% inhibition in the growth of the
yeast culture).
complexes are monomeric and crystallize in the mono-
clinic space group, and in each case, the Cu(II) ion is
chelated in the equatorial plane by the two 1,10-phenan-
throline nitrogen atoms and a total of four carboxylate
oxygen atoms from two molecules of the particular
NSAID [14]. The equatorial plane of all three complexes
(2N2O) has a regular planar geometry. Conversely, the
axial positions of the Cu(II) complexes are distorted,
due to the limited bonding capacity of the bidentate,
tolfenamte, mefanamate and flufenamate anions. In the
solid state, the molecular structures of the Cu(II) com-
plexes are stabilized by intermolecular hydrogen bonds,
which are, however, disrupted upon their introduction
into the fluid environment.
Figure 2: Molecular structures of the Cu(II) complexes. (a) Molecular structure of complex [Cu(tolf-O,O′)2(phen)] (1). (b) Molecular structure
of complex [Cu(mef-O,O′)2(phen)] (2). (c) Molecular structure of complex [Cu(fluf-O,O′)2(phen)] (3).

1448

Lenka Hudecova et al.
3.2 Antibacterial activity
2.5
2.0
1.5
1.0
0.5
0.0
Antibacterial activity of all three Cu(II) complexes was
studied against E. coli and S. aureus. The effect of the
Cu(II) complexes on the growth of prokaryotic E. coli
was investigated over a 24 h period. During the initial
12 h, data were recorded at two hourly intervals. The con-
centration range employed for all three Cu(II) complexes
was 10–150 μmol. At the lowest concentration tested
(10 μmol), a slight decrease in absorbance for complexes
1 and 3 was observed, but practically no effect for the
complex 2, as compared with the control (Figures 3–5).
A gradual decrease in the absorbances, with increasing
concentrations of the Cu(II) complexes, indicates a sensi-
tivity of the bacterial culture to their presence, which is
manifested by an inhibition of cell culture growth.
Control
10µmol
30µmol
80µmol
100µmol
150µmol
0
5
10
15
20
25
Time [h]
Figure 4: Growth curves of E. coli during the course of a 24 h culti-
vation in the presence of 10; 30; 80; 100; and 150 μmol of the
complex 2.
From the measured absorbance values determined
following 24 h of cell culture growth, the IC50 values
were calculated for each complex (Table 1). The IC50
value corresponds to the concentration of the complex
that causes 50% inhibition of bacterial growth. Although,
at first sight, the graphs (Figures 3–5) indicate the effects
of the Cu(II) complexes to be similar, the calculated IC50
values reflect slight activity differences in the order of 1 ∼
3 > 2. The most profound growth inhibition was observed
at the highest concentration of the complex 1, which con-
tains coordinated NSAIDs, each bearing a chlorine atom.
A slightly lower inhibitory activity was observed for com-
plex 3, which contains NSAIDs, each with three fluorine
atoms. The lowest antibacterial activity was observed
for the complex 2, with its methyl-substituted NSAID
ligands. From the growth curves of all three Cu(II) com-
plexes, and at all concentrations tested, the stationary
phase of bacterial culture growth of E. coli is apparent
after 12 h. For the complex 3, at concentrations of 80 μmol
and higher, decreases in the absorbance are measured
after 12 h (Figure 5), which indicates bacterial cell death.
Minimum inhibitory concentrations for both Gram-
negative E. coli and Gram-positive S. aureus bacteria
were determined. The standard antibiotic Neomycin
was used as a control for antibacterial activity of both
strains. The results are summarized in Table 2. From
the obtained results, we can see a relatively good correla-
tion with above-presented IC50 values for E. coli. The best
inhibitory activity against both E. coli as well as S. aureus
has been obtained for complex 1, followed by complex 3.
Complex 2 shows the least activity against both strains.
From the obtained results, it can be concluded that all
three Cu(II) complexes exhibit better inhibitory activity
2.5
2.0
1.5
2,5
2,0
1,5
1,0
1.0
Control
Control
10µmol
10µmol
0.5
0.0
30µmol
80µmol
100µmol
150µmol
0,5
30µmol
80µmol
100µmol
0,0
150µmol
0
5
10
15
20
25
0
5
10
15
20
25
time [h]
Time [h]
Figure 3: Growth curves of E. coli during the course of a 24 h culti-
vation in the presence of 10; 30; 80; 100; and 150 μmol of the
complex 1.
Figure 5: Growth curves of E. coli during the course of a 24 h culti-
vation in the presence of 10; 30; 80; 100; and 150 μmol of the
complex 3.

Antimicrobial and antifungal activities of bifunctional copper(II) complexes

1449
Table 1: Inhibitory concentrations of complexes 1–3 required to
cause a 50% decrease in the growth of E. coli bacterial culture (IC50
at 24 h
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
)
Control
10µmol
30µmol
80µmol
100µmol
150µmol
Complex
IC50 (%)
[Cu(tol-O,O′)2(phen)] (1)
[Cu(mef-O,O′)2(phen)] (2)
[Cu(flu-O,O′)2(phen)] (3)
112.4
148.2
120.1
Table 2: Minimum inhibitory concentrations (µg mL−1) of Cu(II)
complexes against S. aureus and E. coli
0
5
10
15
20
time [h]
Complex
Gram (+)
S. aureus
Gram (−) E. coli
Figure 6: Growth curves of S. cerevisiae during a 24 h cultivation in
the presence of 10; 30; 80; 100; and 150 μM of complex 1.
[Cu(tol-O,O′)2(phen)] (1)
[Cu(mef-O,O′)2
(phen)] (2)
65.72
95.62
74.25
105.56
[Cu(flu-O,O′)2(phen)] (3)
Neomycin
77.29
9.35
80.66
3.25
complexes accords with their antibacterial activities
against E. coli.
Based on our in vitro studies, which unequivocally
confirmed that interaction of Cu(II) complexes with DNA
takes place, we assumed that oxidative damage had
occurred at the DNA level with subsequent inhibition of
cell culture growth. The mechanism by which Cu(II) com-
plexes cause toxicity can be understood on the basis of
redox-cycling reactions between cupric and cuprous spe-
cies which result in the formation of free radicals [14,19].
Under aerobic conditions, this redox cycling leads to the
occurrence of oxidative damage by the production of
superoxide radical anions and highly reactive hydroxyl
radicals. The present results demonstrated that the anti-
bacterial activities of the Cu(II) complexes were generally
higher than their antifungal activities, other than the
single exception of the rather high activity of complex 1
against S. aureus. This can be due to the absence of outer
membrane of Gram-positive bacteria S. aureus, which
makes them more susceptible to certain antibiotics or
molecules with antibiotic affects such as Cu(II) com-
plexes. Further modifications in ligand sphere (both
redox-cycling and intercalating functionalities) are
necessary to achieve better antibacterial activity of Cu(II)
complexes.
3.3 Antifungal activity
The effect of the Cu(II) complexes on the growth of the
eukaryotic model microorganism S. cerevisiae was stu-
died over the period of 20 h. During the initial 12 h, as
with the E. coli measurements, we recorded data at two
hourly intervals. The complexes 1–3 were all measured in
the same concentration range, 10–150 μM (Figures 6–8).
The growth curves shown in Figures 6–8 exhibit a
sigmoidal character. In contrast with the growth of the
bacterial cells, in each experiment performed, the yeast
cultures of S. cerevisiae all demonstrated exponential
growth phases up to a period of 12 h. Based on these
growth curves, the Cu(II) complexes under investigation
can be ordered according to their antifungal activity as: 1
> 3 ≫ 2. Similarly, due to its low antibacterial activity, the
results showed that complex 2 exhibits minimal anti-
fungal activity (Table 3). Hence, we may conclude that,
overall, the order of antifungal activities for all three Cu(II)
3.5
3.0
2.5
2.0
Control
1.5
10µmol
30µmol
1.0
80µmol
100µmol
0.5
150µmol
0.0
0
5
10
15
20
time [h]
Figure 7: Growth curves of S. cerevisiae during a 24 h cultivation in
the presence of 10; 30; 80; 100; and 150 μM of complex 2.

1450

Lenka Hudecova et al.
Cu,Fe
⋅
H2O2 ⟶ OH + OH−
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
When generated in a living cell, hydroxyl radicals
may disrupt the integrity of the bacterial membrane
and cause damage to important cellular components
such as bacterial DNA, RNA and proteins, resulting in
a range of deleterious effects, and even cell death.
Nonetheless, we note that the cytotoxic effects of the added
complexes toward the studied microorganisms may not
necessarily correspond with their SOD mimetic activities,
because in actual living cells, hydroxyl radicals may also
damage other structures and so the spectrum of possible
action mechanisms is accordingly broadened [22,23].
Control
10µmol
30µmol
80µmol
100µmol
150µmol
0
5
10
15
20
time [h]
Figure 8: Growth curves of S. cerevisiae during a 24 h cultivation in
the presence of 10; 30; 80; 100; and 150 μM of complex 3.
4 Conclusions
Table 3: Inhibitory concentrations of complexes 1–3 required to
cause a 50% decrease in the growth of S. cerevisiae (IC50) esti-
mated after a 24 h period
In this paper, the antimicrobial and antifungal properties
of copper(II) complexes containing biologically active
non-steroidal anti-inflammatory drugs, tolfenamic acid,
mefenamic acid and flufenamic acid, and 1,10-phenan-
throline were studied. Based on the IC50 and MIC values,
it can be concluded that Cu(II) complexes exhibit pro-
mising antibacterial activity against E. coli and S. aureus,
of which the most powerful effect is observed for complex
1, which contains a chlorine atom on each tolfenamic
acid moiety. Interestingly, the most potent anticancer
activities, against several cancer cell lines, have been
observed for Cu(II) complexes containing halogenated
NSAIDs. We suggest that, in addition to these complexes
causing ROS-induced damage to bacteria, their NSAID
halogen atoms are involved in hydrogen bond formation
with particular hydrogen atoms at the DNA surface.
With respect to their antifungal activities, the results
indicate that the Cu(II) complexes can be arranged in the
order 1 > 3 ≫ 2. The lower activity observed for Cu(II)
complexes against S. cerevisiae can be accounted for by
the presence of a nuclear membrane in yeast cells, which
protects their DNA from attack by external agents such as
Cu(II) complexes.
Complex
IC50 (%)
[Cu(tol-O,O′)2(phen)] (1)
[Cu(mef-O,O′)2(phen)] (2)
[Cu(flu-O,O′)2(phen)] (3)
95.5
—
147.2
against S. cerevisiae. Indeed, complex 1, with its Cu(II)-
coordinated chlorinated NSAID ligand, appears to be the
most effective agent against both bacteria and fungi. The
overall greater activity of the Cu(II) complexes against
bacteria than fungi may be due to the fact that these
agents can interact directly with the DNA upon entry
into a bacterial cell, which, unlike a fungal cell, does
not have membrane-protected nuclear DNA [20]. In con-
trast, for the complexes to interact with DNA in yeast
cells, they must first penetrate through the nuclear
membrane.
The results obtained demonstrate a correlation between
the cytotoxic effects of the complexes toward the studied
microorganisms and their observed SOD mimetic activ-
ities, as recently reported by our group (1 ∼ 3 > 2) [14].
To recapitulate, briefly, the SOD mimetic activity of the
studied complexes rests upon their ability to dismutate
the superoxide radical anion and produce hydrogen
peroxide, according to the reaction [9].
Acknowledgements: This work was supported by
Scientific Grant Agency (VEGA Project 1/0482/20) and
Research and Development Support Agency (APVV-15-
0079). S. Alwasel would like to extend his sincere appre-
ciation to the Researchers Supporting Project (RSP-2020/
59), King Saud University, Riyadh, Saudi Arabia, for sup-
port. M. S. would like to thank STU Grant scheme Support
of Excellent Teams of Young Researches CuSPK for finan-
cial support.
SOD
⋅
−
2O2 + 2H+ ⟶ H2O2 + O2
Thus formed, hydrogen peroxide can give rise to for-
mation of a highly reactive hydroxyl radical according to
the Fenton reaction [14,21].

Antimicrobial and antifungal activities of bifunctional copper(II) complexes

1451
Conflict of interest: The authors declare no conflicts of [13] Turel I. The interactions of metal ions with quinolone anti-
bacterial agents. Coord Chem Rev. 2002;232:27–47.
interest.
doi: 10.1016/S0010-8545(02)00027-9.
[14] Simunkova M, Lauro P, Jomova K, Hudecova L, Danko M,
Ethical approval: The conducted research is not related to
either human or animal use.
Alwasel S, et al. Redox-cycling and Intercalating Properties of
novel mixed copper(II) complexes with non-steroidal anti-
inflammatory drugs tolfenamic, mefenamic and flufenamic
acids and phenanthroline functionality: structure, SOD-
mimetic activity, interaction with albumin, DNA damage study
and anticancer activity. J Inorg Biochem. 2019;194:97–113.
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