Synthesis, characterization, and binding properties towards CT-DNA and lipoxygenase of mixed-ligand silver(I) complexes with 2-mercaptothiazole and its derivatives and triphenylphosphine

Article abstract of DOI:10.1007/s00775-014-1089-6

Mixed-ligand silver(I) complexes of formulae [AgCl(TPP)₂(MTZD)] (1), {[AgCl(TPP)₂(MBZT)]·(MBZT)· 2(toluene)} (2), and [AgCl(TPP)₂(CMBZT)] (3) were obtained by refluxing toluene solutions of silver(I) chloride with tripheny

Full text of DOI:10.1007/s00775-014-1089-6

J Biol Inorg Chem (2014) 19:449–464
DOI 10.1007/s00775-014-1089-6
ORIGINAL PAPER
Synthesis, characterization, and binding properties towards
CT-DNA and lipoxygenase of mixed-ligand silver(I) complexes
with 2-mercaptothiazole and its derivatives and triphenylphosphine
•
L. Kyros C. N. Banti N. Kourkoumelis
•
•
•
M. Kubicki I. Sainis S. K. Hadjikakou
•
Received: 19 August 2013 / Accepted: 29 December 2013 / Published online: 21 January 2014
ꢀ SBIC 2014
1
Abstract Mixed-ligand silver(I) complexes of formulae
[AgCl(TPP)2(MTZD)] (1), {[AgCl(TPP)2(MBZT)]ꢀ(MBZT)ꢀ
2(toluene)} (2), and [AgCl(TPP)2(CMBZT)] (3) were
obtained by refluxing toluene solutions of silver(I) chloride
with triphenylphosphine (TPP) and the appropriate het-
erocyclic thioamides 2-mercaptothiazolidine (MTZD),
2-mercaptobenzothiazole (MBZT), and 5-chloro-2-mer-
captobenzothiazole (CMBZT). The complexes were char-
acterized by the melting point, vibrational spectroscopy
(Fourier transform mid-IR), H-NMR spectroscopy, UV–
vis spectroscopy, and X-ray crystallography. DNA binding
tests indicate the ability of complexes 1–3 to modify the
activity of cells. The binding constants of 1–3 towards calf-
thymus DNA (CT-DNA) [(3.5 8.5) 9 104 M-1 for 1,
(10.0 0.0) 9 104 M-1 for 2, and (46.4 7.0) 9
104 M-1 for 3] indicate strong interaction of 3. Changes in
the fluorescence of ethidium bromide in the presence of
DNA suggest intercalation into or electrostatic interactions
with DNA. The corresponding apparent binding constants
(Kapp) towards CT-DNA calculated through fluorescence
spectra are (3.5 0.7) 9 104 M-1 for 1, (10.0 0.0) 9
104 M-1 for 2, and (46.4 7.0) 9 104 M-1 for 3. Dock-
ing studies on DNA complexes confirm the binding of 1
and 2 in the major groove of CT-DNA and of 3 in the
minor groove. Moreover, the influence of 1–3 on the cat-
alytic peroxidation of linoleic acid to hydroperoxylinoleic
acid by the enzyme lipoxygenase was studied kinetically
and theoretically. The antibacterial effect of 1–3 against the
bacterial species Pseudomonas aeruginosa and Escherichia
coli was evaluated. Complex 1 exhibits the strongest
activity.
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L. Kyros ꢀ C. N. Banti ꢀ S. K. Hadjikakou (&)
Section of Inorganic and Analytical Chemistry,
Department of Chemistry,
University of Ioannina,
45110 Ioannina, Greece
e-mail: shadjika@uoi.gr
N. Kourkoumelis
Medical Physics Laboratory,
Medical School,
University of Ioannina,
45110 Ioannina, Greece
Keywords Bioinorganic chemistry ꢀ Silver(I) chloride
complexes ꢀ Heterocyclic thioamides ꢀ DNA binding ꢀ
Antibacterial activity
M. Kubicki
Department of Chemistry,
Adam Mickiewicz University,
Umultowska 89b,
Introduction
´
61-614 Poznan, Poland
Silver has been recognized as an effective antimicrobial
agent, specifically in the form of silver nitrate, since the
seventeenth and eighteenth centuries [1]. It has also been
used in the treatment of chronic skin ulcers, open wounds,
and suppurating wounds [2]. Nowadays, silver sulfadiazine
I. Sainis
Cancer Biobank Center,
University of Ioannina,
45110 Ioannina, Greece
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J Biol Inorg Chem (2014) 19:449–464
H
N
H
N
H
N
Cl
S
S
S
P
S
S
S
MBZT
MTZD
CMBZT
TPP
Structure 1
remains one of the most effective and widely used topical
burn treatments [3, 4]. The astringent properties of silver
against a wide range of bacteria have been recognized, and
although the cytotoxic effects of silver against Gram-
positive and Gram-negative bacteria have long been
established, the exact mechanisms of action are not com-
pletely understood [1]. It has been reported that silver-
treated bacterial cells exhibited a region in their cytoplasm
with condensed DNA molecules [5]. Condensed DNA
molecules lose their ability to replicate. Another mecha-
nism was proposed, suggesting that the silver moiety in
silver sulfadiazine is dissociated from sulfadiazine and
binds to components within the cell. The subsequent
inhibition of bacterial growth is due to the amount of silver
bound to bacterial DNA [6]. Recently, the antitumor
activity of silver(I) ions has been attributed to their inter-
action with nucleic acids, preferentially with the bases in
DNA rather than with the phosphate groups [7].
apoptosis through the mitochondrial pathway both in vivo
and in vitro [14]. The literature data on bacterial LOXs are
extremely limited, since these enzymes have only recently
been found in prokaryotes (e.g., Pseudomonas aeruginosa)
[15]. No bacterial LOXs have been detected in Escherichia
coli [15]. A possible biological role of LOXs is to facilitate
the dynamic plasticity of membranes in bacteria [15].
During our studies on the development of new metal-
lotherapeutics [8–10, 16–30] which would be able to
overcome the cell’s resistance while still interacting with
intracellular components and leading to cell death, we have
synthesized novel silver(I) chloride complexes with TPP
and the heterocyclic thioamides 2-mercaptothiazolidine
(MTZD), MBZT, and 5-chloro-2-mercaptobenzothiazole
(CMBZT) (Structure 1) of formulae [AgCl(TPP)2(MTZD)]
(1), {[AgCl(TPP)2(MBZT)]ꢀ(MBZT)ꢀ2(toluene)} (2), and
[AgCl(TPP)2(CMBZT)] (3). The complexes were charac-
terized by their melting point, vibrational spectroscopy
1
We have recently shown that silver(I) complexes of the
anti-inflammatory drugs salicylic acid (salH2), aspirin
(aspH), and naproxen (naprH) of formulae [Ag(TPP)2
(salH)], {[Ag(TPP)3(asp)](DMF)}, {[Ag(TPP)3(napr)](H2O)},
and [Ag(TPTP)2(napr)] [where TPP is triphenylphosphine,
DMF is dimethylformamide, and TPTP is tri(p-tolyl)phos-
phine] interfere strongly with both calf-thymus DNA (CT-
DNA) and lipoxygenase (LOX), leading to cell death
through apoptosis [8, 9]. The binding affinity of the mixed-
ligand silver(I) complex [AgI(TPP)2(MBZT)] (MBZT
is 2-mercaptobenzothiazole) towards CT-DNA is minimal
and towards LOX is negligible, causing mild cytotoxicity
[10]. However, this complex does not interact with gluta-
thione, which is responsible for the deactivation of cisplatin
and for the development of the cells’s resistance during
chemotherapy, which makes the study of complexes of this
type very interesting.
(Fourier transform mid-IR), H-NMR spectroscopy, UV–
vis spectroscopy, and X-ray crystallography. Although the
crystal structure of 1 is identical with the structure previ-
ously reported [31], we proceeded with its refinement once
again for comparison with the structures of 2 and 3.
Moreover, the use of toluene instead of acetonitrile affor-
ded 1 in 120 min and thus reduced the reaction time from
24 h. The binding affinity of 1–3 towards the intracellular
molecules CT-DNA and LOX was studied for the evalua-
tion of the mechanism of cell death. The ability to bind CT-
DNA was tested by UV–vis, fluorescence, and docking
studies. Kinetics and theoretical studies were used to
investigate the influence of complexes 1–3 on the catalytic
peroxidation of linoleic acid by the enzyme LOX. The
antibacterial effect of 1–3 against the bacterial species
P. aeruginosa and E. coli was also evaluated.
LOX is an enzyme that takes part in the metabolism of
arachidonic acid. LOX catalyzes the oxidation of arachi-
donic acid to leukotrienes, in an essential mechanism for
cell life [11, 12]. Prostaglandins, the final products formed
from the metabolism of arachidonic acid, contribute to
tumorigenesis as angiogenic factors [13]. Studies have
shown that LOX inhibitors induce the release of cyto-
chrome c from mitochondria into the cytosol, causing
Results and discussion
General aspects
Complexes 1–3 were synthesized by refluxing for 2 h tol-
uene solutions of silver(I) chloride, TPP, and the appro-
priate thioamide in a 1:2:1 molar ratio.
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J Biol Inorg Chem (2014) 19:449–464
451
UV light was also investigated. Irradiation of chloroform
solutions of 1–3 with UV radiation at room temperature
causes the gradual decomposition of the complex. Figure
S5 shows the UV spectral changes in the region from 240
to 400 nm of a 2.5 9 10-5 M CHCl3 solution of com-
plexes 1–3 during irradiation (0–600 s) with UV light
(kmax = 254 nm) at room temperature using a low-pressure
(15-W) mercury lamp (germicidal). A monotonic reduction
of the absorbance is observed, with no appearance of new
bands indicating photodecomposition of the complex. For
the characterization of the photoproducts, a solution of
60 mg of complexes 1–3 in 10 ml chloroform was irradi-
ated for 1 h in a quartz conical flask under aerobic condi-
tions. The solutions were filtered and high-resolution mass
spectra of the filtrates were recorded. The main species in
these solutions are the [Ph3P=O]? ion (1, 2) and the
[Ph3P=O•Na]? ion (3), indicating the decomposition of the
initial complexes with formation of phosphine oxide, on
irradiation of compounds 1–3 (Figs. S6–S8).
Fig. 1 Anisotropic ellipsoid diagram of [AgCl(TPP)2(MTZD)] (1),
where TPP is triphenylphosphine and MTZD is 2-mercaptothiazoli-
dine, together with the partial numbering scheme. The ellipsoids are
drawn at the 33 % probability level. The intramolecular hydrogen
bond is shown as a dashed line
Vibrational spectroscopy
Thioamide vibrational bands
I and II appear at
1,303–1,478 cm-1 (1) and 1,313–1,478 cm-1 (2, 3) in the
IR spectra of the complexes 1–3 (Figs. S7–S9), which are
attributed to the m(C=N) and m(C–N) vibrations mainly
undergoing a shift as compared with the corresponding
vibrational bands of the free forms of the ligands, which
are observed at 1,296–1,433 cm-1 (MTZD), 1,319–
1,496 cm-1 (MBZT), and 1,312–1,453 cm-1 (CMBZT).
Thioamide bands III and IV, attributed to the m(C=S) and
m(C–S) vibrations, were observed at 697–1,050 cm-1
(MTZD), 751–1,035 cm-1 (MBZT), and 739–1,038 cm-1
(CMBZT) in the spectra of the free forms of the ligands
and at 747–1,047 cm-1 (1), 744–1,025 cm-1 (2), and
745–1,022 cm-1 (3) in the spectra of complexes 1–3. The
band at 1,090 cm-1 in the IR spectra of 1–3 is assigned to
the antisymmetric m(C–P) vibrations and the bands at
507 cm-1 (1), 513 cm-1 (2), and 516 cm-1 (3) are
assigned to the symmetric m(C–P) vibrations of 1–3. The
corresponding m(C–P) bands of the free form of the TPP
ligand are observed at 1,088 cm-1 for the antisymmetric
vibration and at 509 cm-1 for the symmetric vibration.
toluene
AgCl þ 2 TPP þ LS ꢁꢁꢁꢁꢁꢁ! ½AgCl(TPP)2ðLSÞꢂ
reflux; 2 hrs
LS is MTZD ð1Þ; MBZT ð2Þ and CMBZT ð3Þ:
Complex 1 was previously synthesized by heating, under
reflux, the suspension which had been formed by sil-
ver(I) chloride and TPP in 1:2 ratio in acetonitrile for 24 h
[31]. The white solid formed was then filtered off and was
suspended in chloroform with MTZD in 1:1 silver-to-thi-
one ratio. The suspension was stirred until a clear solution
was obtained. Complex 1 was then recrystallized from
dichloromethane from the solid that precipitated after
evaporation of the solvent. However, here 1 is synthesized
in high yield in one step. The full re-refinement of its
structure was done for comparison with the structures of 2
and 3, and the geometrical data are also required for the
docking studies (see computational part). Crystals of
complexes 1–3 are stable in air but were kept in darkness.
Complexes 1–3 were soluble in toluene, acetonitrile, ace-
tone, DMF, and dimethyl sulfoxide (DMSO).
Crystal and molecular structures of 1–3
Since dissociation of the Ag–P bonds might occur in
solution [32], the stability of complexes 1–3 in DMSO
solutions was tested by UV–vis spectra and 1H-NMR
spectra for 2 h as this is the duration of the biological
experiments (interaction with DNA and LOX). No changes
were observed between the initial UV and 1H-NMR spectra
and the corresponding spectra when measured after 2 h
(Figs. S1, S2–S4). The stability of the complexes under
Crystals of compounds 1–3 suitable for single-crystal
X-ray analysis were grown by slow evaporation of the
liquid remaining after filtration of the initial crops of solid
material from the reactions of silver(I) chloride with the
heterocyclic thioamides MTZD, MBZT, or CMBZT and
TPP in a molar ratio of 1:1:2, respectively, in toluene.
Although the crystal and molecular structures of compound
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J Biol Inorg Chem (2014) 19:449–464
Fig. 2 Anisotropic ellipsoid diagram of {[AgCl(TPP)2(MBZT)]ꢀ
(MBZT)ꢀ2(toluene)} (2), where MBZT is 2-mercaptobenzothiazole,
together with the partial numbering scheme. The ellipsoids are drawn
at the 33 % probability level. The intramolecular hydrogen bond is
shown as a dashed line. For clarity, other components of the crystal
structure (free form of the ligand and solvent molecules) are not
shown
1 were previously reported [31], we have reevaluated its
structure here for comparison with the structures of 2 and
3; its geometrical parameters were also used for the
docking studies.
Figures 1, 2, and 3 show the displacement ellipsoid
representations of complexes 1–3.
Fig. 3 a Anisotropic ellipsoid diagram of [AgCl(TPP)2(CMBZT)]
(3), where , and CMBZT is 5-chloro-2-mercaptobenzothiazole,
together with the partial numbering scheme. The ellipsoids are drawn
at the 33 % probability level. The intramolecular hydrogen bond is
shown as a dashed line. b Packing of complex 2 after removal of the
solvent molecules: the voids in the crystal structure are easily visible
In all three cases, the silver centers are four-coordi-
nated—by two phosphorous atoms from TPP ligands, one
sulfur atom from MBZT or CMBZT, and one chlorine
atom, in a slightly distorted tetrahedral fashion; the dis-
tortion is mainly caused by the disposition of bulky TPP
ligands. Table 1 lists the relevant geometrical parameters.
In general, the Ag–P and Ag–Cl bond distances agree well
with those found in similar structures (e.g., in
[AgCl(TPP)2(MTZD)] [31], [AgCl(TPP)2(pmtH)] (pmtH
is pyrimidine-2-thione) [33], {[AgCl(CMBZT)(TPTP)2]ꢀ
(MeOH)} (1) [17], [AgBr(TPP)(pmtH)]2 [34] and [AgI
(TPP)2(MBZT)] [10]). In all complexes the intramolecular
N–HꢀꢀꢀCl hydrogen bonds additionally strengthen the tet-
rahedral coordination (Table 1 lists also the details of these
interactions). The Ag–S bond distances in 2 and 3 are
slightly longer than the corresponding bond distances
reported for the terminal Ag–S bonds found in [AgCl
ones found in [AgI(TPP)2(MBZT)], where the Ag-S
˚
bond distances are 2.7825 (11) A (molecule A) and
˚
2.7159 (11) A (molecule B) [10]. The variations in Ag–S
bond distances observed among [AgCl(PR3)(LS)] (PR3 is
triarylphospine, LS is thioamide) complexes might be
attributed to the different electronic effects caused by the
ligands.
In the case of 2 a neutral thioamide ligand MBZT is also
cocrystallized in the unit cell, and it is also hydrogen-
bonded with the chlorine atom. In this crystal structure the
solvent (toluene) molecules occupy the empty spaces
(Fig. 3b shows a fragment of the crystal packing after
removal of the toluene molecules).
´
˚
(TPP)2(MTZD)] [2.6629 (8) A] [31], [AgCl(TPP)2(pmtH)]
˚
[2.6151 (4) A] [33], {[AgCl(CMBZT)(TPTP)2]ꢀ(MeOH)}
The calculated volumes of the Hirshfeld surfaces
´
3
(Fig. 4) are 903.13 A (1), 1,391.51 A (2), and
3
˚
(1) [2.6486 (16) A] [17], and [AgBr(TPP)(pmtH)]2
˚
[2.5548 (9) A] [34] but they are similar in length to the
˚
˚
3
998.18 A (3).
˚
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453
˚
Table 1 Selected bond distances (A), angles (ꢁ) and hydrogen bond
data of [AgCl(TPP)2(MTZD)] (1), {[AgCl(TPP)2(MBZT)]ꢀ(MBZT)ꢀ
2(toluene)} (2), and [AgCl(TPP)2(CMBZT)] (3), where TPP is
triphenylphosphine, MTZD is 2-mercaptothiazolidine, MBZT
is 2-mercaptobenzothiazole, and CMBZT is 5-chloro-2-
mercaptobenzothiazole
1
2
3
˚
Bond distances (A)
Ag1–Cl1
2.5596 (9)
2.4735 (8)
2.4832 (7)
2.6861 (10)
1.674 (4)
2.6819 (11)
2.5106 (10)
2.4677 (9)
2.7037 (13)
1.672 (5)
2.5585 (7)
2.4715 (7)
2.4704 (7)
2.7777 (8)
1.665 (3)
Ag1–P2
Ag1–P3
Ag1–S42
C42–S42
Bond angles (ꢁ)
Cl1–Ag1–P2
Cl1–Ag1–P3
Cl1–Ag1–S42
P2–Ag1–P3
P2–Ag1–S42
P3–Ag1–S42
Hydrogen bonds
H43ꢀꢀꢀCl1
112.53 (3)
102.77 (3)
102.68 (3)
123.05 (3)
110.25 (3)
110.25 (3)
105.61 (4)
109.98 (3)
104.91 (4)
123.12 (3)
99.90 (4)
113.14 (3)
109.49 (3)
101.65 (2)
126.69 (2)
93.63 (3)
111.59 (4)
107.69 (3)
2.17
2.24
2.19
N43ꢀꢀꢀCl1
3.082 (4)
173
3.102 (3)
174
3.046 (2)
178
N43–H43ꢀꢀꢀCl1
DNA binding studies
UV–vis spectroscopy
Absorption spectroscopy was used for the study of the
binding properties of metal complexes with DNA [35].
Hyperchromism or hypochromism observed in the UV
spectrum of the CT-DNA complex with respect to the
corresponding spectrum of free CT-DNA is related to the
configuration of the double helix structure of DNA [8].
The intercalative mode of binding usually results in hyp-
ochromism along with or without a small redshift or
blueshift due to the strong stacking interaction between an
aromatic chromophore and the base pairs of DNA [35].
The absorption spectra of 1–3 in the absence and pre-
sence of CT-DNA are shown in Fig. 5. The bands of 1–3
undergo hypochromism of about 22.3 % (1), 6.1 % (2), and
9.4 % (3). Moreover, in the case of 1, a redshift of 5 nm is
also observed, indicating that 1 binds to DNA through the
conjugation of the aromatic rings with the stacking base
pairs of the DNA helix [35]. In the case of complexes 2 and
3, on the other hand, the simultaneous blueshifts of 2 and
1 nm, respectively, suggest binding through intercalation
and electrostatic interaction of 2 and 3 with DNA bases
[36].
Fig. 4 Volumes of the Hirshfeld surfaces of 1 (a), 2 (b), and 3 (c)
determined [8] (Fig. 6, Table 2). The calculated Kb values
for 1–3 are (3.5 8.5) 9 104, (10.0 0) 9 104, and
(46.4 7.0) 9 104 M-1 respectively, confirming the
interaction of 1–3 with CT-DNA (Table 2). Complexes 1–
3 bind to CT-DNA in a manner similar to cisplatin
To compare quantitatively the binding affinity of 1–3
towards CT-DNA, their binding constants (Kb) were
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J Biol Inorg Chem (2014) 19:449–464
Fig. 5 UV spectra of calf-
thymus DNA (CT-DNA) in
buffer solution in the absence
and presence of 1–3 at r values
of 0, 0.05, 0.07, 0.09, 0.11, and
0.13 (r = [complex]/[DNA],
5 9 10-5 M DNA). The insets
show A/Ao versus the
concentration of the complex at
kmax
(Kb = (5.73 0.45) 9 104 M-1 [8]). The binding affinity
of 3 towards CT-DNA is highest among the mixed-ligand
silver(I) complexes of triarylphosphine and anti-
inflammatory drugs or thioamides tested by our group
(Table 3). The Kb value of 3 is 1.5-fold higher than the
corresponding value of {[Ag(TPP)3(napr)](H2O)} (naprH
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J Biol Inorg Chem (2014) 19:449–464
455
Fig. 6 UV spectra of
complexes 1–3 in the absence
and presence of CT-DNA at
r values of 1, 0.5, 0.25, 0.17,
0.125, and 0.1 (r = [complex]/
[DNA], 10 lM complex,
10–100 lM CT-DNA). The
insets show [DNA]/(ea - ef)
versus DNA concentration
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J Biol Inorg Chem (2014) 19:449–464
610 nm of EtBr (2.3 lL) solutions containing CT-DNA
(26 lL) in the absence or presence of various concentra-
tions of complexes 1–3 (0–250 lL). The fluorescence
quenching spectra illustrate that on increasing the con-
centration of the complexes 1–3, the emission band at
610 nm exhibited hypochromism of 9.0, 16.4, and 19.1 %,
respectively.
Table 2 The DNA binding constants (Kb, Kapp) of complexes 1–3
Complex Hypochromicity Dk
Kb (9104)
(M-1
Kapp (9104)
(M-1
(%)
(nm)
)
)
1
2
3
22.3
6.1
5
-2
-1
3.5 0.7
1.1 0.02
1.8 0.44
2.2 0.24
10.0
0
9.4
46.4 7.0
The apparent binding constant (Kapp) was calculated
using the equation [41]
is naproxen, an anti-inflammatory drug) and is fourfold
higher than that of {[Ag(TPP)3(asp)](DMF)} (aspH
is aspirin, an anti-inflammatory drug) [8, 9]. It is also
twofold higher than the corresponding affinity of
[Ag(TPP)2(p-Hbza)], which contains p-hydroxybenzoic
acid (p-HbzaH), a well-known antioxidant agent. The Kb
KEtBr½EtBrꢂ ¼ Kapp½drugꢂ50;
where [drug]50 is the concentration of the complex causing
a 50 % reduction of the fluorescence, KEtBr = 107 M-1
,
and [EtBr] = 2.3 lL [41]. The concentration of the drug
causing a 50 % reduction of the fluorescence was
calculated from the plot of I0/I versus the concentration
of the complex [Q] (Fig. 7), where I0 and I are the
fluorescence intensities of the CT-DNA in the absence and
presence of complexes 1–3, respectively. The linear Stern–
Volmer equation is as follows:
value of [AgI(TPP)2(MBZT)] [(10.7 2.6) 9 104 M-1
]
(Table 3) [10] is in accordance with the corresponding
value determined for 2, which contains the same ligand.
Thus, the kind of halogen which is coordinated to the silver
ion (chlorine instead of iodine) exerts a minor influence on
the binding properties of the complexes towards CT-DNA.
Compound 2 also binds to DNA with Kb similar to that of
[Ag(TPTP)2(napr)] [(11 2.8) 9 104 M-1] [9].
I0=I ¼ 1 þ KSV ꢃ ½Q ꢂ;
where KSV is the Stern–Volmer dynamic quenching con-
stant and [Q] is the total concentration of the quencher
(complexes 1–3) [37].
Fluorescence spectroscopy studies
The Kapp values calculated for complexes 1–3 are
(3.5 0.7) 9 104 M-1 (1) (10.0 0) 9 104 M-1 (2),
and (46.4 7.0) 9 104 M-1 (3) (Table 3), suggesting an
intercalative or minor groove binding of the complexes.
Complex 3 exhibits a high Kapp value (Table 3). This might
be due to the presence of the chlorine atoms which
are involved in hydrogen-bonding interactions with the
DNA bases. The mixed-ligand complex with naprH
{[Ag(TPP)3(napr)](H2O)}, on the other hand, exhibits a
higher Kapp value than 3, indicating its greater intercalative
interaction with DNA or its better binding at the minor
Ethidium bromide (EtBr) is one of the most sensitive
fluorescent probes that bind to DNA through intercalation
[35]. The displacement of EtBr (quantified by fluorescence)
by the titration of a compound is indicative of an interca-
lative or minor groove binding [37–42]. Competitive
binding of drugs to DNA with EtBr could provide infor-
mation with regard to the DNA binding affinity [35]. Two
mechanisms have been proposed for the reduction in the
emission intensity: the replacement of EtBr, and/or elec-
tron transfer [40]. Figure 7 shows the emission spectra at
Table 3 DNA binding constants (Kb, Kapp) and half-maximal inhibitory concentrations (IC50) towards lipoxygenase (LOX) caused by 1–3 and
other related Ag(I) complexes
Complex
Jb (9104) (M-1
)
Kapp (9104) (M-1
)
IC50 (lL) LOX
Reference
1
3.5 0.7
1.1 0.02
1.8 0.44
2.2 0.24
1.5 0.1
2.9 0.3
1.6 0.4
–
[30
[30
[30
[30
5.1
This work
This work
This work
[10]
2
10.0
0
3
46.4 7.0
10.7 2.6
32.8 8.5
4.7 1.8
11 2.8
[AgI(TPP)2(MBZT)]
{[Ag(TPP)3(napr)](H2O)}
[Ag(TPTP)2(napr)]
{[Ag(TPP)3(asp)](DMF)}
[Ag(TPP)2(salH)]
[Ag(TPP)2(p-Hbza)]
[9]
[30
7.2
[9]
[8]
13.3 6.5
27.7 7.9
–
2.3
[8]
–
7.6
[8]
aspH is aspirin, naprH is naproxen, and salH2 is salicylic acid.
MBZT 2-mercaptobenzotiazole, p-Hbza p-hydroxybenzoic acid, TPP triphenylphosphine, TPTP tri(p-tolyl)phosphine
123

J Biol Inorg Chem (2014) 19:449–464
457
Fig. 7 Emission spectra of
ethidium bromide bound to
DNA in the presence of 1–3
(2.3 lL ethidium bromide,
26 lL DNA, 0–250 lL
complex, kex = 527 nm). The
arrow shows the intensity
changing on increasing the
concentration of complexes 1–3.
The insets shows I0/I versus the
concentration of the complex
Inhibitory activity of 1–3 towards the peroxidation
of linoleic acid by the enzyme LOX
groove. It is known that compounds which inhibit malig-
nant cells interact with DNA [42], and small DNA binders
interact with DNA either by intercalating in between the
base pairs or by docking in the minor groove, or both.
Therefore, 3 may be an efficient new small DNA binder.
LOX inhibition is known to induce apoptosis [8, 43, 44].
The influence of complexes 1–3 on the oxidation of linoleic
123

458
J Biol Inorg Chem (2014) 19:449–464
acid by the enzyme LOX was studied over a wide con-
centration range. The degree of LOX activity in the pre-
sence of 1–3 was calculated according to the method
described previously [8]. Figure 8 shows LOX activity
versus various concentrations of complexes 1–3. All three
complexes show no inhibitory activity towards LOX,
and we were unable to determined the half-maximal
inhibitory concentrations (IC50) within the concentra-
tions used (0–30 lM) (IC50 [ 30 lM) (Fig. 8, Table 3).
This is in agreement with the results obtained for
[AgI(TPP)2(MBZT)], where also no inhibitory activity was
detected [10]. Thus, 1–3 may act through interaction with
DNA rather than interfering with LOX. Moreover,
although mixed-ligand silver(I) complexes of anti-inflam-
matory drugs as ligands (aspH, naprH, or salH2) and tria-
rylphosphines inhibit LOX activity in very low concentrations
([Ag(TPP)2(salH)], IC50 = 2.3 lL;, {[Ag(TPP)3(napr)]
(H2O)}, IC50 = 5.1 lL); {[Ag(TPP)3(asp)](DMF)}, IC50
=
7.2 lL) (Table 3), the mixed-ligand silver(I) complexes of
thioamides and triarylphosphines exhibit no such activity.
Therefore, the anti-inflammatory drugs affect the LOX
inhibitory activity.
Fig. 8 Lipoxygenase activity
versus various concentrations of
complexes 1–3
125
100
75
50
25
0
0
5
10
15
20
25
30
35
Concentration of Complex (µM)
1
2
3
Fig. 9 Binding sites and hydrogen-bonding interactions of 1–3 towards CT-DNA
123

J Biol Inorg Chem (2014) 19:449–464
Computational studies
DNA docking
459
binding affinity of complexes 1–3 was explored by
molecular docking methods to evaluate their potential
inhibitory effectiveness.
All complexes dock away from the central iron atom,
which was identified as the active site of the enzyme [47],
and owing to their large dimensions they prefer surface
cavities to exert their binding activity. Furthermore, they
cannot reach the largest pocket (green sphere in Fig. 10),
which has been suggested as a possible site associated with
an enhanced inhibitory effect [22, 23] in agreement with
the experimental results where no inhibitory activity
towards LOX was found for the complexes studied.
The results showed that 1 resides in the cavity/surface
formed by Ala540, Arg242, Asn245, Asn537, Gln544,
Glu106, Glu179, Glu236, Glu256, Ile257, Leu541, Lys17,
Lys260, Met536, Phe238, Pro241, Tyr132, Tyr239,
Knowledge of how cisplatin inhibits DNA replication and
transcription has led to a dramatic increase in the number of
new metal complexes as promising metallotherapeutics
[45]. DNA docking studies were performed for all three
complexes with regard to CT-DNA. The lowest-energy
conformations show that 1 and 2 bind to the major groove
near the domain of guanines G19 and G4 (Fig. 9). Com-
plex 1 forms two hydrogen bonds with O2 and O5 of G19 at
˚
2.52 and 2.69 A with the protonated nitrogen atom, and 2
˚
form a hydrogen bond with the O6 atom of G4 at 2.15 A.
The conformation energies were calculated to be -87.89
and -77.95 kcal mol-1 for 1 and 2, respectively. In con-
trast, 3 prefers the minor groove, where, in general, the
electronegative AT sequences are closer and exhibit shorter
van der Waals contacts compared with the GC regions. This
result is in agreement with the experimentally found high
Kapp value (Table 2), where significant steric interactions
and better binding affinity were found. Complex 3 exhibits
hydrogen bonding between the N? atom of the ligand and
˚
Val237, and Val240 at an Ag–Fe distance of 18.8 A.
Complexes 2 and 3 share similar behavior and a similar
docking site with the common residues Ala569, Arg360,
Arg588, Asn370, Asn573, Asp408, Asp411, Cys357,
Ile412, Leu501, Lys587, Trp574, Tyr493, Tyr571, Val358,
Val570, and Val575. Significant steric interactions as
revealed by pairwise linear potential calculations are evi-
dent with Arg360, Asn573, Ile412, Tyr493, and Val570.
˚
the O2 atom of thymine T8 at 2.39 A. No hydrogen-bond-
ing interactions were revealed for the chlorine atoms of the
ligand, although they cannot be excluded for conformations
with similar orientation and energy. The latter was calcu-
Effects of complexes 1–3 on the growth of microbial
strains
lated to be -99.95 kcal mol-1
.
The noteworthy high binding affinity of 1–3 towards CT-
DNA led us to evaluate their antibacterial activity. It is well
known that the antibacterial action of silver compounds
appears to involve interaction with DNA [1, 2]. To test the
possible effects of silver complexes on the growth of
LOX docking
Blocking LOX is strongly related to tumor cell prolifera-
tion and apoptosis in several tumor cell types [46]. The
Fig. 10 Binding sites for
complexes 1–3 with regard to
lipoxygenase
123

460
J Biol Inorg Chem (2014) 19:449–464
microbial cells, two microbial species were used:
P. aeruginosa and E. coli. The minimum inhibitory con-
centration (MIC) of 1 that prevents growth overnight of
P. aeruginosa bacteria was found to be 150 lM, whereas
the corresponding MIC of silver nitrate was 60 lM. The
MICs of 2 and 3 with regard to P. aeruginosa are higher
than 400 lM. The MICs of 2 and 3 could not be deter-
mined since the effect of DMSO, which was used as a
solvent for the stock solutions of the complexes (final
DMSO concentration 4 % v/v), is detrimental to cell
growth for high concentrations of the complexes. The
MICs of 1–3 with regard to E. coli cells show higher
activity of 1 [40 lM for 1, more than 400 lM for 2, and
300 lM for 3); the MIC of silver nitrate is 25 lM. The
effect of DMSO on cell growth was visible again above
4 % v/v. The significantly high activity of 1 against both
microbial strains is in agreement with its ability to bind
DNA (see earlier). The negligible activity, however,
observed in the case of 2 and 3 might be due to either
variable resistance of the bacterial strains to these com-
plexes compared with complex 1 [48] or differences in
basal uptake rates and efflux rates for complexes 2 and 3 in
comparison with complex 1. The higher MICs of silver
complexes with regard to P. aeruginosa compared with
E. coli could be assigned to the lower outer membrane
permeability. This hypothesis is strengthened by the fact
that although the multidrug and toxic compound extrusion
gene families are similar in both E. coli and P. aeruginosa,
the P. aeruginosa genome contains many more predicted
multidrug efflux systems [49]. Moreover, P. aeruginosa is
well known for its intrinsic resistance to many front-line
antibiotics, due mainly to its low outer membrane perme-
ability and to active efflux [50].
photodecomposition of the initial compounds on radiation
with UV-C light.
The binding constants (Kb) of 1–3 towards CT-DNA
calculated from the UV–vis spectra (Table 3) indicate that
2 and 3 bind strongly to DNA, whereas 1 exhibits lower
affinity for DNA. Thus, the kind of the thioamide ligand
affects the binding properties. Among 2 and 3, the latter
exhibits 4.5-fold higher activity (Table 3), which might be
attributed to the chlorine substituent of the CMBZT ligand
and its interaction with DNA bases. The Kb value of
[AgI(TPP)2(MBZT)] (Table 3) [10] is similar to the cor-
responding value determined for 2, which contains the
same ligand. Thus, the kind of halogen which is coordi-
nated to the silver ion (chlorine instead of iodine) exerts a
minor influence on the binding properties of the complexes
towards CT-DNA. The corresponding apparent binding
constants (Kapp) towards CT-DNA were calculated from
the changes in fluorescence of EtBr. Complex 3 exhibits a
high Kapp value (Table 3). The mixed-ligand complex with
naprH {[Ag(tpp)3(napr)](H2O)}, on the other hand,
exhibits a higher Kapp value than 3, indicating its greater
intercalative interaction with DNA or its better binding at
the minor groove. The low theoretical binding energy of 1–
3 with regard to CT-DNA reveals that 1 and 2 biud to the
major groove, and 3 binds to the minor groove. This is in
agreement with the higher Kapp value (Table 3) measured
experimentally for 3.
The inhibitory activity of 1–3 towards LOX is negligible
at concentrations of 1–30 lM. This is in accordance with
the inhibitory activity found for [AgI(TPP)2(MBZT)],
which also exhibits no inhibitory activity at the same
concentrations. In contrast, mixed-ligand silver(I) com-
plexes of anti-inflammatory drugs as ligands (aspH, naprH,
or salH2) and triarylphosphines inhibit LOX activity at very
low concentrations (Table 3). Therefore, the presence of
halogen atoms and thioamides in the coordination sphere of
the silver(I) ion eliminates the LOX inhibitory activity of
the complexes, and the anti-inflammatory drugs in the
complexes enhance their LOX inhibitory activity. Molec-
ular docking studies have shown that 1–3 dock far away
from the iron atom, which was identified as the active site
of the enzyme [27], and even further from the larger
pocket, which possibly acts as an allosteric site [22]
(Fig. 10). This orientation of 1–3, far away from the active
or allosteric sites of LOX, might explain their negligible
inhibitory activity found experimentally (see earlier).
Complex 1 was found to inhibit both the P. aeruginosa
and E. coli, whereas 2 and 3 exhibit negligible activity.
This might be due to either the variable resistance of the
bacterial species against these complexes compared with
complex 1 [48] or differences in basal uptake rates and
efflux rates for 2 and 3 in comparison with 1. This is further
supported by the lower volumes of the Hirshfeld surface
Despite the high activity found for silver nitrate against
both microbial strains, the main problem with the use of
this salt in medicine is its high solubility in water, with its
simultaneous very low lipophilicity. Thus, the silver ions
immediately released after silver nitrate application are
chemically consumed and rapidly inactivated through the
formation of chemical complexes by chloride within a few
hours [4]. Complex 1 overcomes this problem since it has
been designed to be stable and to be soluble in organic
media.
Conclusions
Three silver(I) chloride complexes—1–3—were synthe-
sized and fully characterized. The geometry around the
metal center in 1–3 is tetrahedral. Complexes 1–3 exhibit
photosensitivity on UV radiation, whereas they are stable
during biological experiments. Triphenylphosphine oxide
was detected in the irradiated solution, suggesting
123

J Biol Inorg Chem (2014) 19:449–464
461
calculated for 1 in contrast to 2 and 3. It is also concluded
that the antibacterial activity of 1–3 against E. coli is in the
same order as the volumes of the Hirshfeld surfaces.
Complex 3. Yield 69 %, molar mass 869 g/mol, melting
point 189–194 ꢁC. IR (cm-1) (KBr): 3,449 (m), 3,064 (m),
2,622 (m), 1,655 (m), 1,586 (m), 1,478 (s), 1,434 (vs),
1,313 (s), 1,080 (vs), 744 (vs), 689 (vs), 502 (vs). Solu-
bility: acetone, acetonitrile, toluene, chloroform, DMF,
DMSO.
Materials and methods
Materials and instruments
X-ray structure determination
All solvents used were of reagent grade. Silver(I) chloride
was prepared by mixing aqueous solutions of AgNO3
(Degussa) with an appropriate amount of NaCl (Aldrich).
The precipitates were filtered off and dried in darkness.
TPP and the thioamides MTZD, MBZT, and CMBZT
(Merck) were used with no further purification. DMSO and
Diffraction data for the complexes were collected at room
temperature by x scan with a SuperNova diffractometer
(Atlas detector) with mirror-monochromatized Cu Ka
radiation (k = 1.54178 A) for 1 and 2 and with an Xcali-
bur diffractometer (Eos detector) with graphite-mono-
˚
˚
chromatized Mo Ka radiation (k = 0.71073 A) for 3. The
¨
boric acid were from Riedel-de Haen. Melting points were
data were corrected for Lorentz-polarization and absorp-
tion effects [51]. Accurate unit-cell parameters were
determined by a least-squares fit of the reflections of the
highest intensity, chosen from the whole experiment. The
structures were solved with SIR92 [52] and were refined
with the full-matrix least-squares procedure on F2 by
SHELXL-2013 [53]; most of the calculations were per-
formed within the WinGX program suite [54]. Scattering
factors incorporated in SHELXL-2013 were used. All non-
hydrogen atoms were refined anisotropically; hydrogen
atoms were placed in the calculated positions, and were
refined as a ‘‘riding model’’ with the isotropic displacement
parameters set at 1.2 times (1.5 times for methyl groups)
the Ueq value for appropriate non-hydrogen atoms. In the
crystal structure of 2, the voids are occupied by partially
disordered solvent (toluene) molecules; the geometry and
displacement parameters for the solvent molecules were
restrained. The Alert A in the checkcif of 2 is connected
with the heavily disordered part of the structure; this short
contact is the result of this disorder. Relevant crystal data
are listed in Table 4, together with refinement details.
The volumes of the Hirshfeld surface [55] were calcu-
lated with the program CrystalExplorer [56].
measured in open tubes with a Stuart Scientific apparatus
and are uncorrected. IR spectra in the region from 370 to
4,000 cm-1 were obtained from KBr discs, with a Perk-
inElmer Spectrum GX Fourier transform IR spectropho-
tometer. The 1H-NMR spectra were recorded with a Bruker
AC 250-MHz Fourier transform NMR instrument in
DMSO-d6 solution. A JASCO UV–vis/near IR V570 series
spectrophotometer was used to obtain electronic absorption
spectra. Fluorescence spectra were recorded with a Perk-
inElmer LS55 fluorimeter Conductivity measurements
were conducted at 20 ꢁC in DMSO solutions with a WTF
LF-91 conductivity meter.
Synthesis and crystallization of 1–3
Complexes 1–3 were synthesized as follows.
A toluene suspension (20 ml) containing 0.5 mmol
AgCl (0.072 g), 1 mmol TPP (0.262 g), and 0.5 mmol of
the appropriate thioamide—MTZD (0.059 g) (1), MBZT
(0.167 g) (2), or CMBZT (0.101 g) (3)—was heated under
reflux for 2 h. The clear solutions were filtered and the
filtrates were kept in darkness. After a few days, white
crystals (1), yellow crystals (2), and pale-yellow crystals
(3) suitable for X-ray crystallographic analysis were
precipitated.
Crystallographic data (excluding structure factors) for
the structural analysis have been deposited with the Cam-
bridge Crystallographic Data Centre (CCDC), nos. CCDC-
956172 (1), CCDC-956173 (2), and CCDC-956174 (3).
Copies of this information may be obtained free of charge
from the CCDC (The Director, CCDC, 12 Union Road,
Cambridge, CB2 1EZ, UK; Fax: ?44-1223-336033;
e-mail: deposit@ccdc.cam.ac.uk; website: http://www.
ccdc.cam.ac.uk).
Complex 1. Yield 62%, molar mass 787 g/mol, melting
point 202–209 ꢁC. IR (cm-1) (KBr): 3,450 (m), 3,137 (m),
2,932 (m), 1,709 (w), 1,518 (w), 1,478 (s), 1,433 (s),
1,093 (m), 748 (s), 696 (vs), 508 (m), 486 (m). Solubility:
acetone, acetonitrile, toluene, DMF, DMSO, methanol,
ethanol.
Complex 2. Yield 56 %, molar mass 1,172 g/mol,
melting point 134–138 ꢁC. IR (cm-1) (KBr): 3,449 (m),
3,001 (m), 2,944 (m), 1,897 (w), 1,585 (m), 1,493 (m),
1,431 (s), 1,094 (s), 744 (s), 695 (s), 513 (s), 439 (s).
Solubility: acetone, acetonitrile, toluene, DMF, DMSO,
methanol, ethanol.
Biological tests
DNA binding studies
This study was conducted as reported in [8].
123

462
J Biol Inorg Chem (2014) 19:449–464
Table 4 Crystal data, data collection, and structure refinement
1
2
3
Formula
C39H35AgClNP2S2
787.06
C43H35 AgClNP2S2ꢀC7H5NS2ꢀ1.5(C7H8)
1,140.54
C43H34 AgCl2NP2S2
869.54
Formula weight
Crystal system
Space group
Monoclinic
P21/c
Triclinic
Monoclinic
P21/c
P-1
˚
a (A)
14.1445 (8)
10.3249 (6)
25.1116 (17)
90
13.0484 (5)
13.5500 (5)
17.4727 (10)
70.489 (4)
76.483 (4)
78.754 (3)
2,808.1 (2)
2
14.8878 (3)
10.2774 (3)
26.4302 (8)
90
˚
b (A)
˚
c (A)
a (ꢁ)
b (ꢁ)
c (ꢁ)
94.643 (6)
90
92.702 (3)
90
3
˚
V (A )
3,655.3 (4)
4
4,039.5 (2)
4
Z
dx (g cm-3
F(000)
)
1.43
1.35
1.43
1,608
1,174
1,768
l (mm-1
)
7.21
5.55
0.85
H range (ꢁ)
hkl range
3.14–75.61
-17 B h B 17
-12 B k B 12
-31 B l B 26
3.49–70.00
-15 B h B 15
-16 B h B 15
-20 B h B 21
1.37–26.52
-18 B h B 18
-12 B h B 10
-31 B h B 32
Reflections
Collected
21,054
7,395 (0.036)
6,791
23,837
10,616 (0.038)
7,763
17,729
8,166 (0.026)
7,236
Unique (Rint
)
With I [ 2r (I)
Number of parameters
R (F) [I [ 2r (I)]
wR (F2) [I [ 2r (I)]
R (F) (all data)
419
622
460
0.046
0.053
0.038
0.125
0.147
0.108
0.050
0.071
0.043
wR (F2) (all data)
0.129
0.158
0.113
Goodness of fit
Max/min Dq (e-
1.05
1.00
0.80
-3
˚
A
)
1.31/-1.06
0.84/-0.65
0.87/-0.67
Fluorescence spectroscopy studies
Computational details
The fluorescence spectroscopy method using EtBr was
used to determine the relative DNA binding properties of
complexes 1–3 with regard to CT-DNA. The emission
spectra at 610 nm of EtBr (2.3 lL) solutions containing
CT-DNA (26 lL) in the absence or presence of various
concentrations of complexes 1–3 (0, 25, 50, 100, 150, 200,
and 250 lL) were recorded on their excitation at 527 nm
[9, 10, 57].
Molecular docking studies were performed for LOX
(Protein Data Bank ID 1F8N) similarly to the procedure
described in [23]. DNA docking was conducted according
to [8].
Bacterial cultures and estimation of MIC
P. aeruginosa PAO1 and E. coli Mach1 (Invitrogen) bac-
terial strains were grown overnight in Luria–Bertani (LB)
broth with vigorous shaking at 37 ꢁC. After 1 day, LB agar
plates supplemented with serial concentrations of 1–3,
silver nitrate, and DMSO were poured and immediately
inoculated with the appropriate inoculum of either
Study of the peroxidation of linoleic acid by the enzyme
LOX in the presence of complexes
This study was performed as reported in [8].
123

J Biol Inorg Chem (2014) 19:449–464
463
21. Ozturk II, Urgut OS, Banti CN, Kourkoumelis N, Owczarzak
AM, Kubicki M, Charalabopoulos K, Hadjikakou SK (2013)
Polyhedron 52:1403–1410
22. Ozturk I, Filimonova S, Hadjikakou SK, Kourkoumelis N, Dokorou
V, Manos MJ, Tasiopoulos AJ, Barsan MM, Butler IS, Milaeva ER,
Balzarini J, Hadjiliadis N (2010) Inorg Chem 49:488–501
23. Balas VI, Banti CN, Kourkoumelis N, Hadjikakou SK, Geromi-
chalos GD, Sahpazidou D, Male L, Hursthouse MB, Bednarz B,
Kubicki M, Charalabopoulos K, Hadjiliadis N (2012) Aust J
Chem 65:1625–1637
P. aeruginosa PAO1 or E. coli overnight cultures. Plates
were incubated for 24 h at 37 ꢁC. The MIC was defined as
the lowest concentration of the supplements that inhibits
visible bacterial growth after overnight incubation. In all
cases, LB plates without any supplement were also inoc-
ulated (controls). The experiment was performed with three
biological replicates.
Acknowledgments This work was conducted in partial fulfillment of
the requirements for the graduate studies of L.K. (PhD) and C.N.B.
(master) under the supervision of S.K.H within the framework of the
graduate program in bioinorganic chemistry. The National Strategic
Reference Framework, Regional Operational Program of Epirus
2007-2013 (2011/S 29-048013), is acknowledged for supporting the
purchase of an LS55 fluorimeter by the Department of Chemistry of the
UniversityofIoannina. A. Koukou(Department ofChemistry, University
ofIoannina)isacknowledgedforthesupplyofP. aeruginosaPAO1cells.
24. Balas VI, Verginadis II, Geromichalos GD, Kourkoumelis N,
Male L, Hursthouse MB, Repana KH, Yiannaki E, Charalabo-
poulos K, Bakas T, Hadjikakou SK (2011) Eur J Med Chem
46:2835–2844
25. Xanthopoulou MN, Hadjikakou SK, Hadjiliadis N, Schurmann
M, Jurkschat K, Michaelides A, Skoulika S, Bakas T, Binolis J
(2003) J Inorg Biochem 96:425–434
26. Abdellah MA, Hadjikakou SK, Hadjiliadis N, Kubicki M, Bakas
T, Kourkoumelis N, Simos YV, Karkabounas S, Barsan M, Butler
IS (2009) Bioinorg Chem Appl. doi:10.1155/2009/542979
27. Xanthopoulou MN, Hadjikakou SK, Hadjiliadis N, Milaeva ER,
Gracheva JA, Tyurin VY, Kourkoumelis N, Christoforidis KC,
Metsios AK, Karkabounas S, Charalabopoulos K (2008) Eur J
Med Chem 43:327–335
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