DNA binding, antibacterial and antifungal activities of copper(II) complexes with some S-alkenyl derivatives of thiosalicylic acid

Article abstract of DOI:10.1007/s11243-018-0201-0

The biological activities of two binuclear copper(II) complexes containing S-alkenyl derivatives of thiosalicylic acid are reported [alkenyl?=?propenyl (L1), isobutenyl (L2)]. The structure of the complex with the S-isobutenyl derivative (C2) was confirmed by single-crystal X-ray structure analysis, which revealed that the structure consists of centrosymmetric, dinuclear complex molecules [Cu₂(S-i-butenyl-thiosal)₄(DMSO)₂] containing two Cu(II) centers bridged by four S-isobutyl-thiosalicylate ligands in a paddle-wheel type structure. The Cu(II) atom is situated in a distorted square-pyramidal environment formed by carboxylate oxygen atoms in the basal plane and a DMSO ligand in the axial position. The reactivities of the complexes toward guanosine-5′-monophosphate (5′-GMP) were investigated. Complex C2 ([Cu₂(S-i-butenyl-thiosal)₄(H₂O)₂]) reacted more rapidly with 5′-GMP than complex C1. The interactions of complexes C1 and C2 with calf thymus DNA (CT-DNA) were examined by absorption (UV–Vis) and emission spectral studies (ethidium bromide displacement studies), revealing good DNA interaction abilities. The antimicrobial activities of the free ligands and their complexes were tested by microdilution method, and both minimal inhibitory and microbicidal concentrations were determined. All the tested substances demonstrated selective and moderate antibacterial activity on gram-positive bacteria, but low antibacterial activity on gram-negative bacteria. Also, the tested substances demonstrated low antifungal activity.

Full text of DOI:10.1007/s11243-018-0201-0

Transition Metal Chemistry
https://doi.org/10.1007/s11243-018-0201-0
DNA binding, antibacterial and antifungal activities of copper(II)
complexes with some S‑alkenyl derivatives of thiosalicylic acid
Dušan Lj. Tomović1 · Andriana M. Bukonjić1 · Verica V. Jevtić2 · Zoran R. Ratković2 · Jovana V. Bogojeski2 ·
Ana Đeković5 · Ivana D. Radojević3 · Ljiljana R. Čomić3 · Slađana B. Novaković4 · Goran A. Bogdanović4 ·
Srećko R. Trifunović2 · Gordana P. Radić1 · Snežana Cupara1
Received: 25 October 2017 / Accepted: 1 January 2018
© Springer International Publishing AG, part of Springer Nature 2018
Abstract
The biological activities of two binuclear copper(II) complexes containing S-alkenyl derivatives of thiosalicylic acid are
reported [alkenyl = propenyl (L1), isobutenyl (L2)]. The structure of the complex with the S-isobutenyl derivative (C2) was
confrmed by single-crystal X-ray structure analysis, which revealed that the structure consists of centrosymmetric, dinuclear
complex molecules [Cu2(S-i-butenyl-thiosal)4(DMSO)2] containing two Cu(II) centers bridged by four S-isobutyl-thiosal-
icylate ligands in a paddle-wheel type structure. The Cu(II) atom is situated in a distorted square-pyramidal environment
formed by carboxylate oxygen atoms in the basal plane and a DMSO ligand in the axial position. The reactivities of the com-
plexes toward guanosine-5′-monophosphate (5′-GMP) were investigated. Complex C2 ([Cu2(S-i-butenyl-thiosal)4(H2O)2])
reacted more rapidly with 5′-GMP than complex C1. The interactions of complexes C1 and C2 with calf thymus DNA
(CT-DNA) were examined by absorption (UV–Vis) and emission spectral studies (ethidium bromide displacement studies),
revealing good DNA interaction abilities. The antimicrobial activities of the free ligands and their complexes were tested by
microdilution method, and both minimal inhibitory and microbicidal concentrations were determined. All the tested sub-
stances demonstrated selective and moderate antibacterial activity on gram-positive bacteria, but low antibacterial activity
on gram-negative bacteria. Also, the tested substances demonstrated low antifungal activity.
Introduction
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11243-018-0201-0) contains
supplementary material, which is available to authorized users.
Numerous complexes of transition metals are known to be
active biological agents, with a wide range of indications
for medical applications [1–4]. In particular, complexes of
copper(II) can have remarkable potential for the expression
of antimicrobial, antiviral, anti-infammatory and cytotoxic
activity [1–3, 5, 6]. Thiosalicylic acid and its derivatives
have a variety of applications, from the treatment of infam-
matory, allergic and respiratory diseases [7] to potential
applications in the prevention of cirrhosis of the liver [8]
and in investigation of the contact sensitivity of drugs [9].
Derivatives of thiosalicylic acid can also be coordinated with
many transition metal ions [10, 11]. Esters of thiosalicylic
acid may also be used as efective ligands for metal compl-
exation [12–14].
* Gordana P. Radić
vasic_gordana@yahoo.com
* Snežana Cupara
snezanacupara@yahoo.com
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 Biology and Ecology, Faculty of Science,
University of Kragujevac, Radoja Domanovića 12,
Kragujevac 34000, Republic of Serbia
4
Complexes of copper(II) with a fve-membered aromatic
heterocyclic ligand proved to be capable of bacterial growth
inhibition against Staphylococcus aureus and Escherichia
coli [15]. The complexes of modifed ligands with difer-
ent heterocyclic Schif bases showed a broad spectrum of
Laboratory of Theoretical Physics and Condensed
Matter Physics, Vinča Institute of Nuclear Sciences,
University of Belgrade, PO Box 522, Belgrade 11001,
Republic of Serbia
5
State University of Novi Pazar, Vuka Karadzića bb,
Novi Pazar 36300, Republic of Serbia
Vol.:(0123456789)
1 3

Transition Metal Chemistry
antibacterial (Escherichia coli, Pseudomonas aeruginosa,
Salmonella typhi and Staphylococcus aureus) and antifun-
gal (Candida albicans, Aspergillus favus, Aspergillus niger
and Cladosporium) activities [16, 17]. Also, copper(II) com-
plexes of some antibiotics (such as ofoxacin and norfoxa-
cin) have been obtained, which is a good approach to the
development of antibiotics with reduced levels of resistance
[18]. A new concept in improving the bactericidal efect of
copper(II) complexes involves selection of ligands based on
the lipophilicity of the resulting complex. Increasing lipo-
philicity enhances the penetration of the complex through
the bacterial membranes [19]. The antimicrobial activity
of dinuclear and mononuclear copper(II) complexes with
substituted carboxylate ligands against bacteria, yeasts and
molds has been demonstrated [13, 19–29].
acid and their corresponding copper(II) complexes [30, 31].
The in vitro antibacterial and antifungal screening of the
two proligands, S-alkenyl derivatives of thiosalicylic acid
[alkenyl = propenyl (L1) and isobutenyl (L2)], and their cor-
responding copper(II) complexes (C1 and C2) is described.
We have also investigated the interactions of the complexes
with guanosine-5′-monophosphate (Scheme 1) and with CT-
DNA. The X-ray crystal structure of complex C2 is also
presented.
Experimental
Materials and measurements
In this paper, we report on the biological activities of
previously synthesized S-alkenyl derivatives of thiosalicylic
All reagents were obtained commercially and used without
further purifcation. Elemental analyses were performed on
Scheme 1 Structures of the Cu(II) complexes and 5′-GMP
1 3

Transition Metal Chemistry
a Vario III C, H, N, S Elemental Analyzer in CHS mode.
Infrared spectra were recorded on a PerkinElmer FTIR
31725-X spectrophotometer using KBr pellets. Conductiv-
ity measurements were made at room temperature on freshly
prepared 10−3 M DMSO solutions using a coronation digital
conductivity meter. Kinetics and mechanism of substitution
reactions of the complexes with selected ligands were stud-
ied in a stopped-fow spectrophotometer in 25 mM Hepes
bufer, pH ≈ 7.2 (Acros Organics, Belgium).
source and CCD detector. Data reduction and empirical
absorption corrections were performed with CrysAlisPro
[32]. The structure was solved by direct methods, using
SIR2002 [33], and refned using the SHELXL program
[34]. The hydrogen atoms attached to C atoms were placed
at geometrically idealized positions with C–H distances
fxed at 0.93 from Csp2 and 0.97 and 0.96 Å from meth-
ylene and methyl Csp3, respectively. The isotropic dis-
placement parameters were set at 1.2Ueq and 1.5Ueq of
the parent C atoms. The DMSO ligand was found to be
disordered over two positions, with site occupation fac-
tors of 0.80 and 0.20. The treatment of the S atom led to a
considerable improvement in the crystal structure model,
while the displacement parameters of the disordered C
atoms were restrained to isotropic parameters due to insta-
bility of the further refnement. All attempts to model the
disorder of the S-isobutenyl substituents of the thiosal-
icylate ligands were unsuccessful. The crystallographic
data are listed in Table 1. The PARST [35] and PLATON
[36] programs were used to perform geometrical calcula-
tions, and the program Mercury [37] was employed for
molecular graphics.
Syntheses
The S-alkenyl derivatives of the thiosalicylic acid [alk-
enyl = propenyl (L1), isobutenyl (L2)] were prepared [30,
31] by reaction of thiosalicylic acid with the corresponding
alkenyl halides in aqueous alkali ethanol solution.
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
the required S-alkenyl thiosalicylate [alkenyl = propenyl
(L1)—0.1607 g, 0.8278 mmol; isobutenyl (L2)—0.1724 g,
0.8278 mmol] was added. The mixture was heated for 3 h,
during which a solution of LiOH (0.0199 g, 0.8278 mmol)
in water (10.0 mL) was added in small portions. The solu-
tion was then fltered and evaporated to small volume. The
blue precipitate was separated by fltration, washed with cold
water and air-dried [30, 31]. In order to obtain single crys-
tals of [Cu2(S-isobutenyl-thiosal)4(H2O)2] (C2) suitable for
X-ray difraction, the complex was recrystallized from H2O/
DMSO mixture.
Table 1 Crystallographic data for C2
Empirical formula
Formula weight
Color, crystal shape
Crystal size (mm)
Temperature (K)
Wavelength (Å)
Crystal system
Space group
Unit cell dimensions
a (Å)
C48H56O10Cu2S6
1112.4
Green, prism
0.322 × 0.270 × 0.120
293(2)
0.71,073
Monoclinic
P21/n
Complex C1 [Cu2(S-propenyl-thiosal)4(H2O)2] =
Cu2C41H43O10S4 (Mr = 951.126): Calcd.: C, 51.77; H,
4.56; S, 13.49. Found: C, 51.54; H, 4.42; S, 13.29%.
μ(294 K) = 1.84 μB. Molar conductivity 5.06 μS/cm (48s).
IR (KBr, cm−1): 3441s, 3076s, 2918m, 1610s, 1549s,
1460m, 1435m, 1400s, 1281w, 1258w, 1229w, 1156w,
1062m, 1044w, 846w, 744m, 695w, 658w, 556w.
10.8516(4)
10.2909(2)
24.7049(8)
100.417(3)
2713.4(2)
2
b (Å)
c (Å)
β (°)
V (Å3)
Complex C2 [Cu2(S-isobutenyl-thiosal)4(H2O)2] =
Cu2C45H51O10S4 (Mr = 1007.232): Calcd.: C, 53.66;
H, 5.10; S, 12.73. Found: C, 53.47; H, 5.18; S, 12.64%.
μ(294 K) = 1.86 μB. Molar conductivity 9.43 μS/cm (48s)
IR (KBr, cm−1): 3446s, 2969s, 2915m, 1613s, 1589s, 1400s,
1281w, 1257w, 1157w, 1062m, 847w, 744m, 719w, 656w,
510w [31].
Z
Dcalc (Mg/m3)
1.366
1.066
μ (mm−1
)
θ range for data collection (°)
Refections collected
Independent refections, Rint
Completeness (%) to θ = 26°
Refnement method
2.59–28.94
12,000
6152, 0.0232
99.9
Full-matrix least squares on F2
4576/308
1.021
Single‑crystal X‑ray crystallography
Data/parameters
Goodness of ft on F2
Single-crystal difraction data for complex C2 were col-
lected at room temperature on an Oxford Diffraction
Gemini S difractometer equipped with a Mo Kα radiation
Final R1/wR2 indices [I > 2σ(I)]
0.0486/0.1257
1 3

Transition Metal Chemistry
Kinetic measurements
Ethidium bromide displacement studies
The substitution reactions of complexes C1 and C2 with
the nucleophile 5′-GMP were studied spectrophotometri-
cally. Spectral changes resulting from the mixing of the
complex and nucleophile solutions were recorded over the
wavelength range from 200 to 400 nm in order to establish
a suitable wavelength at which kinetic measurements could
be performed.
The relative binding of the complexes to CT-DNA was deter-
mined by calculating the quenching constant (Ksv) from the
slopes of straight lines obtained from Stern–Volmer Eq. (2).
I0∕I = 1 + Ksv[Q]
(2)
where I0 and I are the emission intensities in the absence and
the presence of the quencher (complex C1 or C2), respec-
tively, [Q] is the total concentration of quencher and Ksv is
the Stern–Volmer quenching constant which can be obtained
from the slope of the plot of I0/I versus [Q].
Substitution reactions were initiated by mixing equal
volumes of complex and ligand solutions directly in the
stopped-fow instrument and followed for at least eight half-
lives. The substitution reaction was monitored by the change
in absorbance with time under pseudo-frst-order conditions.
The observed pseudo-frst-order rate constants, kobs, were
calculated as the average values from four to six independent
kinetic runs using the program OriginPro 8. Experimental
data are presented in Tables S1 and S2.
Viscosity measurements
The viscosities of DNA solutions were measured in the
presence of increasing amounts of complexes C1 and C2.
The fow time was measured with a digital stopwatch. Each
sample was measured three times, and the average fow time
was calculated. The data were analyzed as (η/η0)1/3 against
r, where η is the viscosity of DNA in the presence of the
complex and η0 is the viscosity of DNA alone in the bufer
solution. The viscosities 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.
UV–Vis DNA interactions
A stock solution of CT-DNA was prepared in PBS bufer,
giving a ratio of UV absorbances at 260 and 280 nm
(A260/A280) of ca. 1.8–1.9, indicating that the DNA was suf-
fciently free of protein. The concentration was determined
from the UV absorbance at 260 nm (ε = 6600 M−1 cm−1)
[38]. 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 meas-
urements were made on an RF-1501 PC spectrofuorometer
(Shimadzu, Japan). The fuorescence spectra were recorded
in the range 550–750 nm upon excitation at 527 nm in all
cases. The excitation and emission bandwidths were both
10 nm.
In vitro antimicrobial assays
The test compounds were dissolved in DMSO and then
diluted with nutrient liquid medium to achieve a concen-
tration of 10%. DMSO was purchased from Acros Organ-
ics. Resazurin was obtained from Alfa Aesar. Doxycycline
(Galenika A.D., Belgrade) was dissolved in Mueller–Hinton
broth (Torlak, Belgrade), while antimycotics, fuconazole
(Pfzer Inc.) and ketoconazole (Sigma-Aldrich) were dis-
solved in Sabouraud dextrose broth (Torlak).
The antimicrobial activities were assessed against 18
microorganisms. These included six strains of pathogenic
bacteria, including four standard strains (Escherichia coli
ATCC 25922, Pseudomonas aeruginosa ATCC 27853,
Proteus mirabilis ATCC 12453 and Staphylococcus aureus
ATCC 25923), plus two clinical isolates (Bacillus cereus
and Salmonella enterica). Three species of probiotic bac-
teria (Bifdobacterium animalis subsp. lactis PMFKGP33,
Lactobacillus plantarum PMFKG-P31 and Bacillus subti-
lis IP 5832 PMFKG-P32), seven mold species (Aspergil-
lus niger ATCC 16404, Aspergillus favus ATCC 9170,
Aspergillus fumigatus ATTC 204305, Trichoderma viridae
ATCC 13233, Penicillium chrysogenum, Penicillium itali-
cum and Penicillium expansum) and two yeast species (Can-
dida albicans ATCC 10231 and Saccharomyces boulardii
PMFKGP34) were also tested. The clinical isolates were
UV–Vis absorption studies
In order to quantitatively compare the binding strength
of the complexes, the intrinsic binding constants Kb were
determined by monitoring the changes in absorption of the
MLCT band with increasing concentration of CT-DNA,
using 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], [DNA] is the
concentration of DNA in base pairs, εA = Aobs/[complex],
εf is the extinction coefcient for the unbound complex and
εb is the extinction coefcient for the complex in the fully
bound form.
1 3

Transition Metal Chemistry
a generous gift from the Institute of Public Health, Kragu-
jevac. The other microorganisms were provided from the
collection held by the Laboratory of Microbiology Faculty
of Science, University of Kragujevac.
Results and discussion
Synthesis and chemical characterization
The bacterial suspensions were prepared by the direct
colony method. The turbidity of the initial suspension was
adjusted using a densitometer (DEN-1, BioSan, Latvia).
When adjusted to the turbidity of the 0.5 McFarland’s
standard [39], the bacterial suspensions contained about 108
colony-forming units (CFU)/mL and the yeast suspensions
contained 106 CFU/mL. Tenfold dilutions of the initial sus-
pension were additionally prepared in sterile 0.85% saline.
The suspensions of fungal spores were prepared by gentle
stripping of spores from slopes with growing aspergilli. The
resulting suspensions were diluted 1:1000 in sterile 0.85%
saline.
S-alkenyl (R = propenyl (L1), isobutenyl (L2)) derivatives
of thiosalicylic acid were prepared [30, 31] by alkylation
of thiosalicylic acid with the corresponding alkenyl halo-
genides in alkaline water–ethanol solution (Scheme 2). The
corresponding complexes were obtained by direct reaction of
copper(II) nitrate with the S-alkenyl derivatives (molar ratio
1:2) in aqueous solution with satisfactory yields (more than
80%) (Scheme 3). The structure of complex C2 was deter-
mined by X-ray crystallography. In order to obtain single
crystals of [Cu2(S-isobutenyl-thiosal)4(H2O)2] (C2) suitable
for X-ray difraction analysis, the complex was recrystallized
from H2O/DMSO solvent mixture. It can be assumed that
S-propenyl complex C1 has the same overall structure as the
S-isobutenyl complex C2. The molar conductance values of
both complexes in DMSO measured at 10−3 M concentra-
tion are in the range of 5–10 μS/cm, indicating that both
complexes behave as non-electrolytes.
Antimicrobial activities were tested by determining the
minimum inhibitory concentration (MIC) and minimum
microbicidal concentration (MMC) using the microdilution
plate method with resazurin [40]. The 96-well plates were
prepared by dispensing 100 µL of nutrient broth, Muel-
ler–Hinton broth for bacteria and Sabouraud dextrose broth
for fungi, into each well. A 100-µL aliquot from the stock
solution of the test compound (concentration 2000 µg/mL)
was added into the frst row of the plate. Then, twofold serial
dilutions were performed with a multichannel pipette. The
obtained concentration range was from 1000 to 7.8 µg/mL.
The method has been described in detail in a previous paper
[30].
The X-ray crystal structure of complex C2 con-
sists of centrosymmetric, dinuclear complex molecules
[Cu2(S-isobutenyl-thiosal)4(DMSO)2] in which two Cu(II)
centers are bridged by four S-isobutenyl-thiosalicylate
ligands in a paddle-wheel type structure (Fig. 1). Each
Cu(II) center is situated in a distorted square-pyramidal
environment formed by carboxylate oxygen atoms in the
basal plane and a DMSO ligand in the axial position.
Selected geometrical parameters for the crystal struc-
ture are given in Table 2. The Cu atom is displaced from
the square base toward the axial O atom by 0.205(1) Å.
The Cu…Cu distance of 2.6430(7) Å is slightly longer in
comparison with the value of 2.614 Å found in dimeric
copper(II) acetate [41], but closely comparable to those
found in polymorphic [Cu2(S-propyl-thiosal)4(DMSO)2],
which has values of 2.6407(4) and 2.6398(9) Å in poly-
morphs I and II, respectively [29].
Doxycycline, fuconazole and ketoconazole were used as
positive controls. 10% DMSO (as a solvent control test) was
found not to inhibit the growth of the microorganisms. Each
test included growth and sterility controls.
All the tests were performed in duplicate, and the MICs
were constant. Minimum bactericidal and fungicidal con-
centrations were determined by plating 10 µL of samples
from wells where no indicator color change or no mycelia
growth was recorded, onto nutrient agar medium. At the end
of the incubation period, the lowest concentration with no
growth (no colony) was defned as the minimum microbi-
cidal concentration.
The coordination bond lengths of the S-isobutenyl-
thiosalicylate ligand (Table 2) are within the values
Scheme 2 Synthesis method of S-alkenyl derivates of thiosalicylic acid
1 3

Transition Metal Chemistry
Scheme 3 Synthesis of copper(II)-complex with S-alkenyl derivates of thiosalicylic acid
Table 2 Selected bond lengths (Å) and angles (°) of C2
Cu1…Cu1i
Cu1–O1a
Cu1–O2a
Cu1–O1b
Cu1–O2b
Cu1–O3
O1a–C1a
O2a–C1a
O1b–C1b
O2b–C1b
C3a–S1a
C8a–S1a
C3b–S1b
C8b–S1b
2.6430(7)
1.974(2)
1.968(2)
1.952(2)
1.967(2)
2.148(2)
1.257(4)
1.254(4)
1.256(4)
1.256(4)
1.762(3)
1.817(5)
1.761(4)
1.799(7)
O1a–Cu1–O1b
O1a–Cu1–O2bi
O1b–Cu1–O2ai
O2ai–Cu1–O2bi
O1a–Cu1–O3
O1b–Cu1–O3
O2ai–Cu1–O3
O2bi–Cu1–O3
O1a–Cu1–O2ai
O1b–Cu1–O2bi
C1a–C2a–C3a
O1a–C1a–O2a
O1b–C1b–O2b
Cu1i–Cu1–O3
88.4(1)
89.8(1)
90.0(1)
89.4(1)
98.1(1)
98.1(1)
93.9(1)
93.9(1)
168.1(1)
167.98(9)
127.8(3)
125.7(3)
125.6(3)
175.17(8)
Symmetry codes: (i) − x, − y, − z + 1
Fig. 1 Crystal structure of [Cu2(S-isobutenyl-thiosal)4(DMSO)2]
with the atom-labeling scheme. For the sake of clarity, only the major
component of the DMSO ligand is presented
angles between the mean planes of phenyl C2/C7 and car-
boxyl groups are 28.0(3) and 52.1(2)° in ligands A and
B, respectively (Fig. S1). The maximal dihedral angle of
57.3° for complexes of this type is observed in the S-pro-
pyl derivative [29], while in the complex with benzoate
ligands [42] the corresponding dihedral angles range from
7.9° to 22.4°. Another conformational diference between
the two diferent ligands of C2 concerns the S-isobutenyl
substituent, which in ligand A prefers an extended form
with a C3a–S1a–C8a–C9a torsion angle of −174.4(3)°
(Fig. S1). In ligand B, the larger twisting observed for the
reported for other complexes of S-propyl derivatives
[1.957(4)–1.982(4) Å], as well as those reported for the
equivalent Cu(II) complex with the unsubstituted benzo-
ate ligand (1.948–1.985 Å) [42]. In complex C2 (Fig. 1),
ligands A and B show signifcant conformational difer-
ences. The phenyl rings display diferent rotation about
the formally single C1–C2 bonds; hence, the dihedral
1 3

Transition Metal Chemistry
phenyl ring is accompanied by pronounced folding of the
S-isobutenyl substituent, resulting in a C3b–S1b–C8b–C9b
torsion angle of −54.3(5)°. The folding of the S-isobutenyl
moiety in ligand B seems to have an important role in
arranging the bulky S-isobutenyl substituent next to the
S-dimethyl moieties of the axial ligands.
and (4), where Nu = 5′-GMP; the slope of the line represents
k1 or k2. Plots of kobs1,2 versus nucleophile concentration
showed a linear dependence with no meaningful intercept
for all complexes and substitution steps. The results are sum-
marized in Table 3.
kobs1 = k1[Nu]
(3)
In the crystal packing, weak C–H…π interactions
between the neighboring aromatic ligands link the com-
plex molecules into chains extending along the a crystal-
lographic axis [C4b–H4b…πii: C… Cg1 = 3.742(5); H…
Cg1 = 2.89 Å; C–H…Cg1 154°; Cg1: centroid of C2a/C7a
ring; (ii) = −x + 1,− y,− z + 1], as shown in Fig. 2. Tak-
ing into account the major orientation of the axial DMSO
ligand, one can observe short and directional C–H…O con-
tacts between the axial ligands [C12a–H12c…O3iii: H…O
2.47Å, C–H…O 163°; (iii) − x,− y − 1, − z + 1] which
link the molecular chains along the b crystallographic axis
(Fig. S2).
kobs2 = k2[Nu]
(4)
Figure 3 shows the dependence of kobs on nucleophile con-
centration for complex C1 (see also Fig. S3).
As expected, the substitution reactions of these com-
plexes were very rapid, as shown in Table 3. Complex C2
is more reactive toward 5′-GMP than complex C1. Since
these complexes difer only in the S-alkenyl group, the dif-
ference can be attributed to the stronger inductive efect of
the isobutenyl group compared to the propenyl group. For
both complexes, the second substitution step is signifcantly
slower than the frst.
Substitution of the complexes with 5′‑GMP
Interactions of the complexes with DNA
To get an idea of the robustness of these complexes in
living tissue, the substitution reactions of C1 and C2
with a selected nucleophile 5′-GMP, a fragment of DNA
(Scheme 1), were investigated. The change in absorbance
was followed at suitable wavelengths, as a function of time at
310 K and pH ≈ 7.2. The proposed reaction pathways for all
observed substitution processes are presented in Scheme 4.
The substitution reactions of both complexes proceeded in
two successive reaction steps, both of which were dependent
on the nucleophile concentration.
The interactions of complexes C1 and C2 with CT-DNA
were investigated by UV–Vis titrations. The intrinsic
equilibrium binding constants (Kb) were evaluated. Com-
plex absorption titration studies were carried out at room
temperature using a fxed concentration of the complexes
(10 μM) in PBS bufer and varying the amount of CT-DNA
(0–15 μM) [43, 44]. Addition of CT-DNA to a solution
of either complex resulted in a signifcant hyperchromic
efect with the appearance of a new band at 258 nm, but
with only insignifcant absorption changes in the region of
300–500 nm; see Figs. 4 and S4. These observations suggest
a strong interaction between the complexes and CT-DNA
The rate constants for substitution could be determined,
under pseudo-frst-order conditions, from a plot of kobs ver-
sus the total nucleophile concentration, according to Eqs. (3)
Fig. 2 Crystal packing of com-
plex C2. Dashed lines indicate
the C–H…π interactions con-
necting the complex molecules
along the a axis. For the sake
of clarity, only the major com-
ponent of the DMSO ligand is
presented
1 3

Transition Metal Chemistry
Scheme 4 Substitution reactions of copper(II)-complexes with S-alkenyl derivates of thiosalicylic acid
Table 3 Rate constants for the frst and second reaction step of the
substitution reactions of the Cu(II)-complexes with 5′-GMP at
pH = 7.2 (25 mM Hepes bufer) at 310 K
it strongly emits at ~ 600 nm due to intercalation of EB
between DNA base pairs. The addition of either complex
C1 or C2 to the CT-DNA-EB complex led to signifcant
quenching in fuorescence intensity, due to the displace-
ment of EB. The quenching parameters for the complexes
C1 and C2 were calculated using the Stern–Volmer equa-
tion. Quantitative EB displacement studies were per-
formed by changing the concentrations of the complex
and monitoring the emission intensity of the EB-DNA
[43, 44]. Increasing concentration of C1 or C2 (0–30 μM)
resulted in a signifcant decrease in fuorescence intensity,
with a noticeable redshift. Hence, both complexes C1 and
C2 are capable of displacing EB from the EB-DNA com-
plex and can therefore strongly interact with DNA binding
sites (Fig. 5) [47–49].
5′-GMP
First step k2 [M−1 s−1
]
Second step k2 [M−1 s−1
]
Complex 1
Complex 2
(6.7 0.2) 106
(3.1 0.1) 106
(5.2 0.1) 105
(2.3 0.1) 105
through external contacts, presumably hydrogen bonding
and electrostatic interactions [45, 46].
The interactions of complexes C1 and C2 with CT-
DNA were also investigated by ethidium bromide (EB)
displacement studies. EB fuorescences weakly in free
solution [45, 46]; however, in the presence of CT-DNA
1 3

Transition Metal Chemistry
1200
kobs/s-1
kobs/s-1
80
40
0
first step
second step
800
400
0
[5'-GMP]/M
[5'-GMP]/M
0
0.0001
0.0002
0.0003
0
0.0001
0.0002
0.0003
Fig. 3 Pseudo-frst-order rate constants plotted as a function of nucleophile concentration for the frst and second steps of the substitution reac-
tions of complex C1 with 5′-GMP at pH = 7.2 and 310 K in 25 mM Hepes bufer
18
12
6
8
6
4
2
0
1.2
0.9
0.6
0.3
0
I0/I
25
20
15
10
5
0
1
2
3
4
10-4 [Q]
0
0
10
20
10-11[DNA]
0
550
600
650
Wavelength (nm)
700
230
280
330
380
Wavelength(nm)
Fig. 5 Fluorescence titration spectra of EB (10 μM) bound to DNA
(10 μM) in the presence of varying amounts of complex C1. [Arrow
shows changes in fuorescence intensity upon increasing concentra-
tion of C1 (4–30 μM)]. Inset: Stern–Volmer plots for EB-DNA fuo-
rescence titration with C1
Fig. 4 UV–Vis titration spectra of complex C1 (10 μM) in PBS
bufer (phosphate bufer solution 0.01 M, CNaCl 0.137,
=
=
C
KCl = 0.0027 M, pH 7.4) with increasing concentration of CT-DNA
(0–15 μM). Arrow shows hyperchromism in the spectral band. Inset:
plot of [DNA]/(εA − εf) versus [DNA]
Table 4 Obtained constants for interaction between complexes C1
and C2 with CT-DNA
According to the constants presented in Table 4, these
complexes interact strongly with CT-DNA, in good agree-
ment with the UV–Vis spectroscopic studies.
CT-DNA
Kb [M−1
]
Ksv [M−1
]
In order to further characterize the binding of these
complexes to DNA, viscosity measurements of DNA solu-
tions were performed in the presence and absence of the
complexes. The viscosity of DNA is sensitive to changes in
length, and in the absence of X-ray crystal structure data,
this is regarded as the most critical evidence in identifying
the DNA binding mode in solution [50, 51]. The addition
Complex 1
Complex 2
(1.3 0.1) × 106
(1.1 0.1) × 106
(1.7 0.1) × 105
(1.6 0.1) × 105
1 3

Transition Metal Chemistry
of increasing amounts (up to r = 1.0) of complexes C1 and
C2 to a DNA solution (0.01 mM) resulted in an increase in
the relative viscosity of DNA (Fig. S5), which was more
pronounced upon the addition of complex C1. In the case
of classic intercalation, the DNA base pairs are separated to
host the bound compound, resulting in an increase in DNA
viscosity, the magnitude of which is usually proportional to
the strength of the interaction. Hence, the observed viscos-
ity increase for these complexes is another indication of an
intercalative interaction with DNA.
higher sensitivities than the gram-negative strains. Among
gram-positive bacteria, Staphylococcus aureus ATCC
25923 demonstrated the highest resistance (MICs/MMCs
are 1000/> 1000 µg/mL). The probiotic bacteria showed the
highest sensitivities. The most sensitive was Bifdobacterium
animalis subsp. lactis, with an MIC value of 31.25 µg/mL
for complex C1. Overall the copper(II) complexes showed
low antifungal activities. The test compounds showed little
or no efect on the growth of yeasts and other fungi, with
MIC and MMC values which were from 250 to > 1000 µg/
mL.
Microbiological studies
In previous work, the Cu(II) complexes of some other
S-alkyl derivatives of thiosalicylic acid (alkyl = benzyl;
methyl; ethyl; propyl; butyl) were tested against gram-pos-
itive and gram-negative bacteria as well as fungi [13, 29].
In general, the activities of the complexes were higher or
similar than those of the corresponding free ligands. All the
tested complexes demonstrated moderate or selective anti-
bacterial activity and low antifungal activity, which is in
accordance with the present results.
The results of in vitro testing of antimicrobial activities for
both the free ligands and their copper(II) complexes are
shown in Table 5. MIC and MMC values, along with those
for doxycycline, fuconazole and ketoconazole, are listed in
Table 6. All of the test compounds showed moderate and
selective antimicrobial activities. MIC and MMC values for
the complexes were in range 31.25 to > 1000 µg/mL. The
best results were however still lower than the positive control
values.
Previous research into the antimicrobial activities of
dinuclear and mononuclear copper(II) complexes of car-
boxylate ligands has provided diverse conclusions. Some
binuclear Cu(II) carboxylate complexes show no antibacte-
rial activity [20]. The lack of specifcity suggests that the
sensitivity of various microorganisms to the complexes is
not related to the diferent cell wall structures [20]. There
In general, the activities of the complexes were higher
than, or similar to the corresponding free ligands. The excep-
tion, where the free ligands gave higher activities, are bac-
teria Bacillus cereus (for L1 and L2) and Salmonella enter-
ica (for L2). Gram-positive bacteria demonstrated slightly
Table 5 In vitro antimicrobial activity of the ligands L1 and L2 and the copper(II)-complexes C1 and C2 with positive controls
Species
ML06—L1
MICa
MN06—C1
MIC
ML07—L2
MIC
MN07—C2
MMCb
MMC
MMC
MIC
MMC
Bifdobacterium animalis subsp. lactis
Lactobacillus plantarum
Bacillus subtilis IP 5832
250
500
500
500
1000
1000
31.25
500
500
500
500
1000
1000
1000
500
1000
1000
1000
500
500
500
500
500
500
Bacillus cereus
62.5
1000
1000
> 1000
1000
500
500
1000
250
1000
1000
1000
1000
1000
> 1000
250
1000
1000
1000
1000
1000
1000
500
1000
500
250
> 1000
1000
500
1000
500
1000
1000
> 1000
> 1000
> 1000
1000
1000
1000
> 1000
> 1000
> 1000
1000
62.5
250
1000
1000
1000
500
1000
1000
500
500
250
250
1000
500
1000
1000
> 1000
500
> 1000
> 1000
1000
500
1000
> 1000
> 1000
1000
1000
1000
1000
Staphylococcus aureus ATCC 25923
Pseudomonas aeruginosa ATCC 27853
Proteus mirabilis ATCC 12453
Escherichia coli ATCC 25922
Salmonella enterica
Saccharomyces boulardii
Candida albicans ATCC 10231
Penicillium chrysogenum
Penicillium italicum
Penicillium expansum
Trichoderma viridae ATCC 13233
Aspergillus favus ATCC 9170
Aspergillus fumigatus ATTC 204305
Aspergillus niger ATCC 16404
> 1000
> 1000
> 1000
> 1000
1000
1000
> 1000
1000
1000
1000
1000
1000
> 1000
> 1000
> 1000
1000
250
1000
1000
500
> 1000
> 1000
> 1000
> 1000
1000
> 1000
> 1000
> 1000
> 1000
> 1000
1000
500
> 1000
1000
1000
1000
> 1000
1000
1000
1000
1000
1000
> 1000
500
1000
500
1000
> 1000
aMIC values (µg/mL)—inhibitory activity
bMMC values (µg/mL)—microbicidal activity; “/”—not tested
1 3

Transition Metal Chemistry
Table 6 In vitro antimicrobial
activity of the positive controls
(doxycycline for bacteria;
fuconazole and ketoconazole
for fungi)
Species
Doxycycline
Fluconazole
Ketoconazole
MICa
MMCb
MIC
MMC
MIC
MMC
Bifdobacterium animalis subsp. lactis
Lactobacillus plantarum
Bacillus subtilis IP 5832
31.25
0.45
62.50
7.81
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
1.95
0.98
15.63
7.81
/
/
/
/
Bacillus cereus
Staphylococcus aureus ATCC 25923
Pseudomonas aeruginosa ATCC 27853
Proteus mirabilis ATCC 12453
Escherichia coli ATCC 25922
Salmonella enterica
Saccharomyces boulardii
Candida albicans ATCC 10231
Penicillium chrysogenum
Penicillium italicum
Penicillium expansum
Trichoderma viridae ATCC 13233
Aspergillus favus ATCC 9170
Aspergillus fumigatus ATTC 204305
Aspergillus niger ATCC 16404
0.22
62.5
3.75
125
/
/
/
/
15.63
15.63
62.5
31.25
/
/
/
/
15.63
31.25
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
31.25
31.25
62.5
1000
/
1000
1000
500
1000
/
31.25
62.5
125
62.5
62.5
125
125
62.5
/
/
62.50
/
125
/
62.5
62.5
aMIC values (µg/mL)—inhibitory activity
bMMC values (µg/mL)—microbicidal activity; “/”—not tested
are studies that show good antimicrobial activities of
Cu(II) complexes, including activities better than standard
drugs [19, 25–27]. Others Cu(II) carboxylate complexes
exhibit moderate antibacterial activity [21–28], which is
in accordance with our previous [13] and current research.
In most studies, as in the present case, weak antifungal
activity is observed [22, 23, 25].
activities against gram-negative bacteria. Also, the tested
substances showed low antifungal activities.
Supplementary data
CCDC 1535567 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/retr
ieving.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.
Conclusion
In the crystal structure of the dinuclear complex
[Cu2(S-isobutenyl-thiosal)4(DMSO)2], the two Cu(II) cent-
ers are bridged by four S-isobutenyl-thiosalicylate ligands
in a paddle-wheel type structure. The metal atoms are sit-
uated in a distorted square-pyramidal environment, pro-
vided by oxygen atoms of carboxylate ligands in the basal
plane, and a DMSO ligand in the axial position. The order
of reactivity of the studied complexes toward 5′-GMP
showed that the S-isobutenyl derivative reacted more rap-
idly than the S-isobutenyl derivative as free ligand. Both
of these complexes exhibited strong interactions with
DNA. Both the free ligands and their copper(II) complexes
demonstrated selective and moderate antibacterial activi-
ties against gram-positive bacteria and low antibacterial
Acknowledgements This work was financially supported by the
Ministry of Education, Science and Technological Development of
the Republic of Serbia (Projects 172016, 173032, 172034, 172035,
172011).
References
1. Tisato F, Marzano C, Porchia M, Pellei M, Santini C (2010)
Med Res Rev 30:708–749
2. Marzano C, Pellei M, Tisato F, Santini C (2009) Anticancer
Agents Med Chem 9:185–211
3. Prudhomme M (2013) Advances in anticancer agents in medici-
nal chemistry. Bentham Science Publishers, Clermont-Ferrand
1 3

Transition Metal Chemistry
27. Siddiqi ZA, Sharma PK, Shahid M, Khalid M, Siddique A, Kumar
S (2012) Eur J Med Chem 57:102–111
4. Gielen M, Tiekink ER (2005) Metallotherapeutic drugs and
metal-based diagnostic agents: the use of metals in medicine.
Wiley, Chichester
28. Chalkidou E, Perdih F, Turer I, Kessissoglou PD, Psomas G
(2012) J Inorg Biochem 113:55–65
5. Goodman VL, Brewer GJ, Merajver SD (2004) Endocr Relat
Cancer 11:255–263
29. Bukonjić AM, Tomović DL, Nikolić MV et al (2017) J Mol Struct
1128:330–337
6. Iakovidis I, Delimaris I, Piperakis SM (2011) Mol Biol Int
2011:1–13
30. Radić GP, Glođović VV, Radojević ID et al (2012) Polyhedron
31:69–76
7. H. Jacobelli, US Patent 20050267095
31. Tomović DL, Bukonjić AM, Kočović A et al (2017) Serb J Exp
Clin Res. 18:13–18
8. Reif S, Weis B, Aeed H et al (1999) J Hepatol 31:1053–1061
9. Serrano G, Bonillo J, Aliaga A et al (1990) J Am Acad Dermatol
23:479–483
32. Agilent, CrysAlis PRO (2014) PRO. England, Agilent Technolo-
gies, Yarnton, Oxfordshire
10. Ferrer EG, Williams PA (1997) Polyhedron 16:3323–3325
11. Wehr-Candler T, Henderson W (2016) Coord Chem Rev
313:111–155
33. Burla MC, Camalli M, Carrozzini B et al (2003) J Appl Crystal-
logr 36:1103
34. Sheldrick GM (2008) Acta Crystallogr A 64:112–122
35. Nardelli M (1995) J Appl Crystallogr 28:659
36. Spek AL (2003) J Appl Crystallogr 36:7–13
37. Macrae CF, Edgington PR, McCabe P et al (2006) J Appl Crystal-
logr 39:453–457
12. Gilbert JG, Addison AW, Nazarenko AY, Butcher RJ (2001) Inorg
Chim Acta 324:123–130
13. Nikolić MV, Mijajlović MŽ, Jevtić VV et al (2014) Polyhedron
79:80–87
14. Nikolić MV, Mijajlović MŽ, Jevtić VV et al (2016) J Mol Struct
1116:264–271
38. Meadows KA, Liu F, Sou J, Hudson BP, McMillin DR (1993)
Inorg Chem 32:2919–2923
15. Chohan ZH, Shad HA, Youssouf MH, Hadda TB (2010) Eur J
Med Chem 45:2893–2901
39. Andrews JM (2005) J Antimicrob Chemoth 56:60–76
40. Sarker SD, Nahar L, Kumarasamy Y (2007) Methods 42:321–324
41. Van Niekerk JN, Schoening FR (1953) Acta Crystallogr
6:227–232
16. Patil SA, Naik VH, Kulkarni AD, Badami PS (2010) Spectrochim
Acta A Mol Biomol Spectrosc 75:347–354
17. Shebl M, Khalil SM, Ahmed SA, Medien HA (2010) J Mol Struct
980:39
42. Reyes-Ortega Y, Alcantara-Flores JL, Hernandez-Galindo MC
(2005) J Am Chem Soc 127:16312–16317
18. Katsarou ME, Efthimiadou EK, Psomas G, Karaliota A, Vour-
loumis D (2008) J Med Chem 51:470–478
43. Long EC, Barton JK (1990) Acc Chem Res 23:271–273
44. Pasternack RF, Gibbs EJ, Villafranca JJ (1983) J Biochem 22:251
45. Koumousi ES, Zampakou M, Raptopoulou CP et al (2012) Inorg
Chem 51:7699–7710
19. Siddiqi ZA, Khalid M, Kumar S, Shahid M, Noor S (2010) Eur J
Med Chem 45:264–269
20. Betanzos-Lara S, Gracia-Mora I, Granada-Macyas P, Flores-
Alamo M, Barba-Behrens N (2013) Inorg Chim Acta 397:94–100
21. Abou-Hussein AA, Linert W (2012) Spectrochim Acta Part A Mol
Biomol Spectrosc 95:596–609
46. Rizvi MA, Zaki M, Afzal M et al (2015) Eur J Med Chem
90:876–888
47. Meyer-Almes FJ, Porschke D (1993) Biochemistry 32:4246–4253
48. Liu ZC, Wang BD, Yang ZY, Li Y, Qin DD, Li TR (2009) Eur J
Med Chem 44:4477–4484
22. Amer S, El-Wakiel N, El-Ghamry H (2013) J Mol Struct
1049:326–335
49. Howe GM, Wu KC, Bauer WR (1976) Biochemistry
19:4339–4346
23. Creaven BS, Egan DA, Karcz D (2007) J Inorg Biochem
101:1108–1119
50. Li DD, Tian JL, Gu W, Liu X, Yan SP (2010) J Inorg Biochem
104:171–179
24. Efthimiadou EK, Katsarou ME, Karaliota A, Psomas G (2008) J
Inorg Biochem 102:910–920
51. Jiang M, Li Y, Wu Z, Liu Z, Yan C (2009) J Inorg Biochem
103:833–844
25. El-Gamel NE, Zayed MA (2011) Spectrochim Acta Part A
82:414–423
26. Geeta B, Shravankumar K, Muralidhar Reddy P (2010) Spectro-
chim Acta Part A 77:911–915
1 3