Synthesis, structural characterization and biological studies of novel mixed ligand Ag(I) complexes with triphenylphosphine and aspirin or salicylic acid

Article abstract of DOI:10.1016/j.ica.2011.04.032

Two new mixed ligand silver(I) complexes of formulae {[Ag(tpp) ₃(asp)](dmf)} (1) (aspH = o-acetylsalicylic acid and tpp = triphenylphosphine) and [Ag(tpp)₂(o-Hbza)] (2) (o-HbzaH = o-hydroxy-benzoic acid) were synthesized and characte

Full text of DOI:10.1016/j.ica.2011.04.032

Inorganica Chimica Acta 375 (2011) 114–121
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Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Synthesis, structural characterization and biological studies of novel mixed ligand
Ag(I) complexes with triphenylphosphine and aspirin or salicylic acid
d
f
ˇ ˇ e
ˇ
M. Poyraz a,1, C.N. Banti a,h, N. Kourkoumelis b, V. Dokorou a,c, M.J. Manos , M. Simcic , S. Golic-Grdadolnik ,
T. Mavromoustakos g, A.D. Giannoulis a, I.I. Verginadis h, K. Charalabopoulos h, S.K. Hadjikakou a,
⇑
a Inorganic and Analytical Chemistry, Department of Chemistry, University of Ioannina, 45110 Ioannina, Greece
b Medical Physics Laboratory, Medical School, University of Ioannina, 45110 Ioannina, Greece
c X-Ray Unit, Department of Chemistry, University of Ioannina, 45110 Ioannina, Greece
d Department of Chemistry, University of Cyprus, 1678 Nicosia, Cyprus
e Laboratory of Biomolecular Structure, National Institute of Chemistry, Hajdrihova 19, SI-1001 Ljubljana, Slovenia
f EN-FIST Centre of Excellence, Dunajska 156, SI-1000 Ljubljana, Slovenia
g Organic Chemistry Laboratory, Department of Chemistry, University of Athens, Greece
h Department of Experimental Physiology, Medical School, University of Ioannina, 45110 Ioannina, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
Two new mixed ligand silver(I) complexes of formulae {[Ag(tpp)3(asp)](dmf)} (1) (aspH = o-acetylsali-
cylic acid and tpp = triphenylphosphine) and [Ag(tpp)2(o-Hbza)] (2) (o-HbzaH = o-hydroxy-benzoic acid)
were synthesized and characterized by elemental analyses, spectroscopic techniques and X-ray crystal-
lography at ambient conditions. Three phosphorus and one carboxylic oxygen atoms from a de-proton-
ated aspirin ligand in complex 1 and two phosphorus and two carboxylic oxygen atoms from a
chelating o-Hbza anion in complex 2 form a tetrahedral geometry around Ag(I) ions in both complexes.
Complexes 1 and 2 and the silver(I) nitrate, tpp, aspNa and o-HbzaH were tested for their in vitro cyto-
toxic activity against leiomyosarcoma cells (LMS), human breast adenocarcinoma cells (MCF-7) and nor-
mal human fetal lung fibroblasts (MRC-5) cells with Thiazolyl Blue Tetrazolium Bromide (MTT) assay. For
both cell lines 1 and 2 were found to be more active than cisplatin. Additionally, 1 and 2 exhibit lower
activity on cell growth proliferation of MRC-5 cells. The type of LMS cell death caused by 1 and 2 were
evaluated in vitro by use of flow cytometry assay. The results show that at concentrations of 1.5 and
Received 21 January 2011
Received in revised form 16 April 2011
Accepted 20 April 2011
Available online 5 May 2011
Keywords:
Bioinorganic chemistry
Silver(I) complexes
Aspirin
Crystal structures
Cytotoxic activity
STD 1H NMR
1.9
l
V of complex 1, 44.1% and 69.4%, respectively of LMS cells undergo programmed cell death (apop-
M of 2, LMS cells death was by 29.6% and 81.3%,
tosis). When LMS cells were treated with 1.6 and 2.3
l
respectively apoptotic. Finally, the influence of the complexes 1 and 2, upon the catalytic peroxidation of
linoleic acid to hydroperoxylinoleic acid by the enzyme lipoxygenase (LOX) was kinetically and theoret-
ically studied. The binding of 1 and 2 towards LOX was also investigated by Saturation Transfer Difference
(STD) 1H NMR experiments.
Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction
associations between aspirin/NSAID use and a lower death rate
from cancers of the esophagus, stomach, breast, lung, prostate, uri-
Aspirin (aspH) (o-acetyl-salicylic acid) was the first member of
the class of drugs known as nonsteroidal, anti-inflammatory drugs
(NSAIDs) discovered. Aspirin was introduced as an anti-pyretic,
anti-inflammatory and analgesic drug at the end of nineteenth cen-
tury, while today, is one of the most commonly drug in use, world-
wide [1]. A number of epidemiological studies have indicated that
long term aspirin/NSAID use is associated with 30–50% reduction
in risk of colorectal cancer or adenomatous polyps or death from
colorectal cancer [2a]. Other epidemiologic studies also found
nary bladder and ovary [2b,c]. Salicylic acid (o-HbzaH) (o-hydroxy-
benzoic acid), is a precursor of aspirin, while it has been recently
shown that organotin complexes of o-HbzaH possess strong anti-
proliferative activity [3]. The biomedical applications and uses of
silver(I) complexes, on the other hand, are related to their antibac-
terial action [1b,4] which appears to involve interaction with DNA
[4,5]. Recently, Ag(I) complexes have also been studied for their
antitumor activity [5,6]. Despite the importance of aspirin only
few structures of complexes are available up to now. These include
the complexes of aspirin with calcium(II) [7a], copper(II) [7b–d],
nickel(II) [7e], tin(IV) [7f], cadmium(II) [7g], zinc(II) [7h]. The
silver(I) complex of salicylic acid, however, with formula
{[Ag(o-Hbza)]2} is already known [8].
⇑
Corresponding author. Tel.: +30 26510 08374; fax: +30 26510 08786.
E-mail address: shadjika@uoi.gr (S.K. Hadjikakou).
1
On leave from Chemistry Department, Afyon Kocatepe University, Turkey
0020-1693/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ica.2011.04.032

M. Poyraz et al. / Inorganica Chimica Acta 375 (2011) 114–121
115
O
OH
silver(I) nitrate complex with tetramethylethylenediamine
[Ag((CH3)2NCH2CH2N(CH3)2)]NO3 was 40
ꢀ1 cm2 molꢀ1, in excel-
O
OH
X
lent agreement with values for fully dissociated 1:1 electrolytes,
such as sodium nitrate, in DMSO [11b]. The corresponding molar
conductivity of DMSO solutions, measured for the ionic AgX salts
(X = NO3ꢀ, BF4ꢀ) with (1-benzyl-2-imidazolyl)diphenylphosphine
O
CH3
OH
O
[(Bzlm)Ph2P] are 302 and 320
X
ꢀ1 cm2 molꢀ1 [11c] indicating the
formation of three ionic species in DMSO solution.
Attempts for the preparation of the initial sample by reacting
AgNO3 and aspirin in aqueous NH3 40% solution lead to the forma-
tion of the [Ag(o-Hbza)]2 (3) complex, where a hydrolysis of the es-
ter (aspirin) to its o-HbzaH processor has been occurred. The crystal
structure of 3 was also determined (crystallized in P21/c with
a = 7.3973(2), b = 8.6899(2), c = 10.5388(3) Å, b = 107.2560(10)°)
and is almost identical with those reported earlier [8a] (P21/c,
a = 7.405(1), b = 8.826(2), c = 10.683(2) Å, b = 107.48(4)°).
(A)
(B)
Scheme 1. Molecular formulae of aspH (A) and o-HbzaH (B).
Lipoxygenase (LOX) is an enzyme which catalyzes the oxidation
of arachidonic acid to leukotrienes, in an essential mechanism for
the cell life involving in inflammation mechanism [9a,b]. LOX inhi-
bition is found to induce apoptosis [9c], while the lipid peroxides
derived from fatty acids metabolism by LOX can regulate cellular
proliferation [9d]. Thus, LOX inhibition provides a potential novel
target for the treatment and chemoprevention for a number of dif-
ferent cancers.
In the course of our studies on metallotherapeutics [10], we
have synthesized new silver(I) complexes of formulae {[Ag(tp-
p)3(asp)](dmf)} (1) (aspH = o-acetylsalicylic acid (Scheme 1A) and
tpp = triphenylphosphine) and [Ag(tpp)2(o-Hbza)] (2) (o-Hbza-
H = o-hydroxy-benzoic acid (Scheme 1B). The complexes were
characterized by elemental analyses, spectroscopic techniques
and X-ray crystallography at ambient conditions. The anti-cancer
cell screening results against LMS and MCF-7 showed that both
complexes 1 and 2 are more active than cisplatin and less active
against MRC-5 cells proliferation. These findings are compared
with the results of the influence of 1 and 2, on the catalytic perox-
idation of linoleic acid to hydroperoxylinoleic acid by the enzyme
LOX.
2.2. Crystal and molecular structures of {[Ag(tpp)3(asp)](dmf)} (1) and
[Ag(tpp)2(o-Hbza)] (2)
ORTEP diagrams of 1 and 2 along with their selected bond dis-
tances and angles are shown in Figs. 1 and 2.
In case of 1, three phosphorus atoms from tpp ligands and one
deprotonated carboxylic oxygen atom from aspirin form a tetrahe-
dral geometry around Ag(I) ion. Two phosphorus atoms from tpp
and two oxygen atoms from the deprotonated carboxylic group
which chelates Ag(I) ion form the tetrahedral geometry around sil-
ver(I) in case of 2. The average Ag–P bond distance in 1 is 2.53 Å
(Ag1–P1 = 2.515(2), Ag1–P2 = 2.554(3), Ag1–P3 = 2.528(3)), while
in 2 is 2.43 (Ag(1)–P(1) = 2.4030(6), Ag(1)–P(2) = 2.4589(7)). The
corresponding bond distances observed in silver(I) mixed ligand
complexes of phosphines and carboxylic acids are: 2.6026(8),
2.5441(7) and 2.5432(7) Å in (Ph3P)3AgOC(O)C2F5 [12a], 2.543(2),
2.563(2) and 2.546(3) Å in [Ag(2-sbaH)(PPh3)3] (2-sbaH2 = 2-sulfo-
benzoic acid) [12b], 2.524(7), 2.545(8) and 2.503(8) Å in [Ag(dp-
pe)(tfa)]n (dppe = diphenyl-phosphinethane, tfa = F3CCO2) [12c],
2.365(2) and 2.344(2) Å in [Ag2(CH3CO2)2(dppf)]2 (dppf =
2. Results and discussion
2.1. General aspects
Crystals of the complexes 1 and 2 have been grown as follows:
AgNO3 reacts with the sodium salts of aspirin or o-hydroxy-ben-
zoic and white precipitations are formed. Slow evaporation of
DMF solutions of these precipitations in the presence of triphenyl-
phosphine give crystals of 1 and 2. The formula of the complexes
was firstly deduced from its elemental analysis, m.p. and their
spectroscopic data (see Section 4). The X-ray crystal structures of
complexes were also determined. The crystals of the complexes
are air stable when they store in darkness at room temperature.
Complexes 1 and 2 are soluble in MeCN, CHCl3, CH2Cl2 DMSO,
DMF and CH3OH. Since a dissociation of the Ag–P bonds have been
observed for silver(I) complexes with phosphines and carboxylic
acids in solution [11], the stability of the complexes 1 and 2 in
DMSO solutions were tested by UV–Vis spectra and conductivity
measurements. No any changes were observed between the initial
UV spectrum and the corresponding one measured after 48 h for
both complexes (Fig. S1). The period of 48 h for the stability testing
of the complexes was chosen since biological experiments require
48 h of incubation with complexes. In order to assure that no any
ionic species are also formed in DMSO or DMSO/water solutions,
the molar conductance (Km) values of the complexes 1 and 2 in
DMSO solution (10ꢀ3 M) were determined (1: 15.0 (0 h), 15.2
(48 h), 15.0 (72 h) and 2: 17.8 (0 h), 18.1 (48 h), 14.4
Fig. 1. ORTEP diagram together with labeling scheme of 1. Thermal ellipsoids drawn
at the 50% probability level. Selected bond lengths (Å) and angles [°]: Ag1–
P1 = 2.515(2), Ag1–P2 = 2.554(3), Ag1–P3 = 2.528(3), Ag1–O1 = 2.395(10), O1–
C55 = 1.144(18), O2–C55 = 1.287(17), O3–C62 = 1.16(2), O4–C61 = 1.388(17),
O4–C62 = 1.303(18), P1–Ag1–P2 = 109.32(9), P1–Ag1–P3 = 115.37(8), P1–Ag1–O1 =
110.4(2), P2–Ag1–P3 = 117.09(8), P2–Ag1–O1 = 101.4(2),P3–Ag1–O1 = 101.9(3);
solvated DMF: O5–C64 = 1.07(3), N1–C64 = 1.21(3).
(72 h)
X
ꢀ1 cm2 molꢀ1). These values showed that the solutions of
the complexes are not conducting, confirming their stability in
DMSO or DMSO/water media. The molar conductance of the

116
M. Poyraz et al. / Inorganica Chimica Acta 375 (2011) 114–121
Fig. 2. ORTEP diagram together with labeling scheme of 2. Thermal ellipsoids drawn at the 50% probability level. Selected bond lengths (Å) and angles [°]: Ag(1)–
P(1) = 2.4030(6), Ag(1)–P(2) = 2.4589(7), Ag(1)–O(1) = 2.4045(12), Ag(1)–O(2) = 2.5230(12), O(1)–C(37) = 1.2535(17), O(2)–C(37) = 1.2752(18), O(3)–C(43) = 1.3539(19),
O(3)–HA = 0.7768, P(1)–Ag(1)–O(1) = 125.49(3), O(1)–Ag(1)–P(2) = 99.22(3), P(1)–Ag(1)–P(2) = 133.358(16), , P(1)–Ag(1)–O(2) = 112.55(3), P(2)–Ag(1)–O(2) = 104.02(3),
O(1)–Ag(1)–O(2) = 53.57(4).
1,l0-bis(dipheny1-phosphino) ferrocene) [12d], 2.340(4) and
2.364(5) Å in [Ag2(C6H5CO2)2(dppf)] [12d], 2.523(5), 2.544(5),
2.496(5) Å in [Ag2(HCO2)2(dppf)3] [12d], 2.5199(7), 2.5237(6),
2.4953(7) Å in [Ag(PPh3)3O2CH]HCO2H [12e], 2.527(2), 2.533(2),
2.510(3) Å in [Ag(PPh3)3O2CH] [12e], 2.482, 2.508, 2.439, Å in
[Ag(dppe)3(CF3COO)]2 (dpph = 1,6-bis(diphenyl-phosphino) hex-
ane) [12f]. The Ag–O bond distance in 1 is 2.395(10) Å and in 2
are 2.4045(12) and 2.5230(12) Å, while the corresponding bond
distances measured are: 2.433(2) Å in (Ph3P)3AgOC(O)C2F5 [12a],
2.559(11) Å in [Ag(2-sbaH)(PPh3)3] (2-sbaH2 = 2-sulfobenzoic acid)
[12b], 2.499(1) Å in [Ag(dppe)(tfa)]n (dppe = diphenyl-phosphine-
thane, tfa = F3CCO2) [12c], 2.268(2) Å in [Ag2(CH3CO2)2(dppf)]2
(dppf = 1,l0-bis(dipheny1-phosphino) ferrocene) [12d], 2.219(9) Å
in [Ag2(C6H5CO2)2(dppf)] [12d], 2.65(2) Å in [Ag2(HCO2)2(dppf)3]
[12d], 2.5199(7), 2.509(2) Å in [Ag(PPh3)3O2CH]HCO2H [12e],
2.507(9) Å in [Ag(PPh3)3O2CH] [12e].
2.3. Biological tests
2.3.1. Cytotoxicity
Complexes 1 and 2, were tested for their in vitro cytotoxic activ-
ity against leiomyosarcoma cancer cells (LMS) (mesenchymal tis-
sue) from the Wistar rat, polycyclic aromatic hydrocarbons (PAH,
benzo[a]pyrene) carcinogenesis, human breast adenocarcinoma
cells (MCF-7) and normal human fetal lung fibroblasts (MRC-5)
cells. The cell growth proliferation activities were evaluated with
Thiazolyl Blue Tetrazolium Bromide (MTT) assay. The IC50 values
of the complexes and their ligands against the cell lines tested
are summarized in Table 1. The final IC50 values for cell growth
proliferation after 48 h incubation with 1 and 2 against LMS cells
are: 1.5 0.1 and 1.6 0.3
cells are: 1.6 0.2 and 2.5 0.5
proliferation activity of cisplatin against LMS cells, evaluated by
l
V, respectively, while against MCF-7
l
V, respectively. The cell growth
The AgꢁꢁꢁOcarboxylic in 1 distance is 3.248 Å which is shorter than
the sum of van der Waals radius of silver and oxygen (3.65–4.08 Å)
[13]. However, this distance could not be considered as bonding
interaction since the geometry around the metal center is tetrahe-
dron as the bond angles varied between 101.4(2)° and 117.09(8)°
(Fig. 1), whereas bonding interaction should lead to square pyrami-
dal arraignment with bond angle around 90°. The close AgꢁꢁꢁOcarboxylic
distance is explained to the steric effect caused to the sp2 hybridism
of the carboxylic carbon which is also bonded to the carboxylic oxy-
gen donor atom. The corresponding carbonylic oxygen-silver dis-
tances observed are: 3.413 Å in (Ph3P)3AgOC(O)C2F5 [12a], 3.425 Å
in [Ag(2-sbaH)(PPh3)3] (2-sbaH2 = 2-sulfobenzoic acid) [12b],
3.094 Å in [Ag(dppe)(tfa)]n (dppe = diphenyl-phosphinethane,
tfa = F3CCO2) [12c], 3.116 Å in [Ag2(HCO2)2(dppf)3] [12d] and they
are all ascribed as non bonding interactions [12]. The Ag–O bond dis-
tances in 2 are Ag1–O978 = 2.5230, Ag1–O987 = 2.4043 indicating
the chelation of carboxylic group to silver(I) ions.
Table 1
IC50 values of the complexes and their ligands against the cell lines tested.
Compounds
IC50
(
l
V)
References
LMS
MCF-7
MRC-5
*
*
*
*
*
*
1
2
1.5 0.1
1.6 0.3
3.7 0.3
>350
>400
67.4 13.9 56.5 10.6 >160
0.8 0.08
1.5 0.06
1.6 0.2
2.5 0.5
3.3 0.2
>350
2.9 0.1
3.1 0.3
27.7 3.3
>350
AgNO3
AspNa
o-HbzaH
tpp
[AgCl(tptp)3] (0.5 H2O)
[AgI(tptp)3]
[AgCl(cmbzt)(tptp)2]
Cisplatin
>400
>400
[5]
[5]
[5]
[10a,14a]
8
0.39
25
20
–
*
This work.

M. Poyraz et al. / Inorganica Chimica Acta 375 (2011) 114–121
117
MTT assay, was found to be 25
uated with the same assay, is given equal to 20
l
V [10a], while against MCF-7 eval-
V [14a]. Thus,
dependent cytotoxic response in LMS cells through apoptosis.
Although, no direct comparison can be made due to the different
l
both complexes 1 and 2 were found to exhibit significant higher
activity (10-fold) than that of cisplatin against LMS and MCF-7
cells. Also, complexes 1 and 2 also show significantly stronger
activity than their ligands (tpp, aspNa and o-HbzaH) (Table 1)
and stronger activity (2-fold) than that of silver(I) nitrate (a known
antimicrobial agent [1b]). Additionally, 1 and 2 was found to exhi-
bit lower activity on cell growth proliferation of MRC-5 cells (nor-
mal human fetal lung fibroblast) with IC50 values 2.9 0.1 and
cell lines used, the apoptosis of HeLa cells induced by 33.75 lV
solution of organogold(III) complexes containing the ‘‘pincer’’
iminophosphorane ligand (2-C6H4-PPh2 = NPh) of formula
[Au{j
2-C,N-C6H4(PPh2 = N(C6H5)-2}Cl2] is 23.3% [14b].
2.4. Study of the peroxidation of linoleic acid by the enzyme
lipoxygenase in the presence of complexes 1 and 2 and their ligands
3.1 0.3 lV, respectively.
Since LOX inhibition is found to induce apoptosis [9c] the influ-
ence of complexes 1 and 2 on the oxidation of linoleic acid by the
enzyme LOX were studied in a wide concentration range. The de-
gree of LOX activity (A, %) in the presence of these complexes
was calculated according to the method described previously
[15]. Fig. 4 compares the inhibitory effect of 1, 2 and cisplatin in
various concentrations The IC50 values found for complexes are
2.3.2. Flow cytometry
A flow cytometry assay was used to quantify apoptotic or necro-
tic cells, treated with compounds 1 and 2. Treated and untreated
LMS cells were stained with Annexin V-FITC and Propidium Iodide
(PI). Fig. 3, shows the dose-dependent cytotoxic response in LMS
cells through apoptosis when treated with 1. Compared to un-
treated LMS cells which showed a total of 15.2% of background cells
death (10.4% apoptosis and 4.8% necrosis), the cells treated with
7.2 (1), 2.3 (2) and 65.9 (cisplatin) lM, respectively. Thus, both
complexes exhibit significantly higher inhibitory activity of LOX
than cisplatin. LOX inhibition activity is found to be related to
the cytotoxic activity against LMS cells [10]. Therefore, the high
cytostatic activity caused by 1 and 2 (see above) might be attrib-
uted to their LOX inhibitory activity.
The binding of 1 and 2 towards LOX was also investigated by
STD 1H NMR experiment. On the top of Fig. 5A and B the aromatic
region of the off-resonance NMR spectra are shown and on the bot-
tom the on-resonance STD 1H NMR spectra of the complexes 1 and
2. Peaks attributed to the aspirin or o-hydroxy-benzoic acid mole-
cules ca 6.9, 7.4–7.5 ppm and 7.8 ppm and peaks attributed to tri-
phenylphosphine ca 7.6–7.7 ppm of the complexes 1 and 2 are
complex 1 (1.5
(Ann+/PIꢀ) and late apoptotic cells (Ann+/PI+)) and 8.8% necrosis.
When LMS cells were treated with 1.9 M of 1, 69.4% of the cells
were early and late apoptotic and 8.9% necrotic. LMS cells treated
with the complex 2 (1.6 V), show 29.6% apoptosis (early and late
apoptotic cells) and 5.0% necrosis, while when LMS cells were trea-
ted with 2.3 M of 2, 81.3% of the cells were early and late apoptotic
lM) showed 44.1% apoptosis (early apoptotic cells
l
l
l
and 1.6% of the cells undergo necrosis. In this case the untreated
LMS cells show a total of 10.5% of background cells death (7.9%
apoptosis and 2.6% necrosis). Thus, complex 1 and 2 cause a dose
Fig. 3. Flow cytometry assay summary results for LMS cells, treated with various concentrations of 1 (1.5 and 1.9 lV) for 48 h of incubation, in comparison with the untreated
cells (control).

118
M. Poyraz et al. / Inorganica Chimica Acta 375 (2011) 114–121
100
75
1
2
Cis platin
50
25
0
0
10
20
30
40
50
60
70
80
Concentration (
)
Fig. 4. The inhibitory effect against LOX caused by 1, 2 and cisplatin in various concentrations.
7.80
7.70
7.60
7.50
7.40
7.30
7.20
7.10
7.00
ppm (t1)
(A)
7.80
7.70
7.60
7.50
7.40
7.30
7.20
7.10
7.00
ppm (f1)
(B)
Fig. 5. The off-resonance reference NMR spectrum (top) and the on-resonance STD NMR spectrum (bottom) for the 1-protein (A) and 2-protein (B) complexes. The on-
resonance STD NMR spectrum has approximately 40-fold in case of 1 and 200-fold lower in case of 2, lower intensity compared to the reference spectrum.
present in the binding mode of the protein. Thus, both components
of the complexes, aspirin or o-hydroxy-benzoic acid and triphenyl-
phosphine have binding affinity 10ꢀ3–10ꢀ8 M at LOX receptor [16].
This experiment provides direct evidence for the binding affinity of
the complexes with LOX and supports experimental results re-
ported above.
Complexes are docked into different pockets indicating different
interaction relationships towards the enzyme inhibition.
The complex 1 binds into the space defined by the larger hydro-
phobic pocket of LOX of approximate volume of 573 Å3 and a smal-
ler one of 160 Å3. Relevant amino acids are: Ala76, Arg767, Asn128,
Asn769, Asp760, Asp768, Gln766, Glu78, Glu761, Gly75, Gly765,
His771, Leu74, Lys110, Met15, Phe782, Pro770, Thr73, Trp772
and Val762. Hydrogen bonding interaction appears between the
acetyl-O and the N atom of Arg767. The lowest binding energy
upon EI (E = enzyme, I = inhibitor) complex formation, was found
to be -55.7 kJ/mol. On the contrary, 2 resides into a pocket of
2.5. Computational studies – molecular docking
In order to investigate further the binding of complexes 1 and 2
towards LOX activity, theoretical docking studies were performed.

M. Poyraz et al. / Inorganica Chimica Acta 375 (2011) 114–121
119
eration activity with IC50 values 1.5 and 1.6
2.5
l
V (1) and 1.6 and
l
M (2) against LMS and MCF-7 cells, respectively. These values
indicate stronger activity than the corresponding of cisplatin
[10a,14a]. Silver(I) complexes of formula {[AgCl(tptp)3] (0.5
H2O)}, [AgI(tptp)3] (tptp = tris(p-tolyl)phosphine) or [AgCl(cmbzt)
(tptp)2] (cmbzt = 5 chloro-2-mercapto-benzimidazole) with tetra-
hedral geometry around the metal center, on the other hand, also
exhibit strong cytostatic activity against LMS cells with IC50 values
0.8 0.08, 1.5 0.06 and 8 0.39
Flow cytometry assay (in vitro) show that when LMS cells are trea-
ted with 1 at concentration of 1.5 V, 44.1% of cells undergo pro-
grammed cell death (apoptosis), while when they were treated
with 2 at 1.6 V 29.6% were apoptotic. However, 62.4% of LMS cell
were apoptotic when they treated with [AgCl(cmbzt)(tptp)2] com-
plex at 15 V [5]. This indicates that 1 and 2 likely acts though
lV, respectively (Table 1) [5].
l
l
l
apoptosis. This is further supported by the high LOX inhibitory
activity caused by these complexes with IC50 values 7.2 (1) and
2.3 (2) lV, respectively, since LOX inhibition, is found to induce
Fig. 6. The binding sites for EI complexes (and ESI in the case of 1). The sphere
indicates the active site as this was resulted from our previous work [10f].
apoptosis [9c]. STD 1H NMR studies also show that 1 has high bind-
ing affinity to LOX.
256 Å3 defined by Ala569, Arg360, Arg588, Asn355, Asn502,
Asn573, Asp408, Asp411, Asp578, Asp584, Cys357, Gln579,
Ile359, Ile412, Leu407, Leu501, Lys587, Met406, Met497, Ser498,
Trp574, Tyr409, Tyr493, Tyr571, Val358, Val570 and Val575. Pro-
tein–ligand hydrogen bonds are formed between the coordinated
O atoms and N atoms of Arg360 and O atom of Tyr493. The binding
energy for the stable EI complex formation was found to be
ꢀ76.7 kJ/mol. To this value, hydrogen bonds contribute by 7%
while significant contribution stems from the ligand steric interac-
tions. The binding sites for EI complexes are shown in Fig. 6, while
the sphere indicates the active site of reversible inhibitors as this
was resulted from our previous work [10f]. It is shown that 1 binds
into LOX in the same pocket that all reversible inhibitors docked,
while 2, in different site away.
The crystal structure of LOX (E) does not include the substrate
(linoleic acid, S) as the co-crystallized cofactor and therefore to test
the binding affinity we have evaluated the binding energy when
the ES complex is formed to be ꢀ100.2 kJ/mol. Linoleic acid docks
exactly at the larger binding cavity of the protein which is the pre-
ferred activity site as shown previously by our group [10b,e,f]. In
the case of 1, when the ESI (enzyme:substrate:inhibitor) complex
is formed, the results are ambiguous concerning the exact binding
pocket. The calculation outcome showed that the variation of the
scoring energies among different sites is minimal. The stability,
as this is reflected by steric and hydrogen bonding factors is com-
parable with the EI complex and constantly lower than that of ES
4. Experimental
4.1. Materials and instruments
All reagents were purchased from commercial sources and used
as received. Solvents used were of reagent grade, while acetylsali-
cylic acid and triphenylphosphine (Sigma–Aldrich, Merck) were
used without further purification. Melting points were measured
in open tubes with a Stuart scientific apparatus and are uncor-
rected. Infra-red spectra in the region of 4000–370 cmꢀ1 were ob-
tained in KBr disks, with a Perkin–Elmer Spectrum GX FT-IR
spectrophotometer. The 1H, 13C NMR spectra were recorded on a
Bruker AC250 MHz FT-NMR instrument in DMSO-d6 solution.
Chemical shifts d are reported in ppm using 1H TMS as an internal
reference. Thermal studies were carried out on a Shimadzu DTG-60
simultaneous DTA-TG apparatus, under N2 flow (50 cm3 minꢀ1
)
with a heating rate of 10 °C minꢀ1. A Jasco UV/Vis/NIR V570 series
spectrophotometer was used to obtain electronic absorption spec-
tra. Conductivity measurements were carried out at 293 K in DMSO
solutions with a WTF LF-91 conductivity meter.
4.2. Synthesis and crystallization of 1 and 2
A solution of 0.5 mmol ligand (o-acetyl-salicylic acid (aspH)
(0.090 g) and o-hydroxy-benzoic acid (o-HbzaH) (0.069 g)) in
by almost 40%. The complex
2
resides into the same
pocket albeit having a different orientation which adds 32 kJ/mol
to the stability of the ESI complex compared to the EI case due to
increased steric interactions. As a result, this compound acts as
inhibitor which always binds at a site away from the substrate
binding site, causing a reduction in the catalytic rate which may
eventually lead to cell death.
water (5 cm3) was added dropwise to
a stirred solution of
0.5 mmol silver(I) nitrate (0.085 g) in water (5 cm3) at room tem-
perature and a clear solution obtained. The resulted solution was
treated by 0.5 cm3 NaOH 1 N and a white powder precipitated
immediately which was filtered off. 0.057 g (1) or 0.040 g (2) of
the resulting powder was dissolved in DMF (10 ml) and 0.132 g tri-
phenylphospine (0.5 mmol) was added. The mixture was stirred
for 30 min at 50 °C and the clear solution was kept in darkness.
White crystals of 1 and 2 suitable for X-ray analysis were grown
from slow evaporation of the solution after 2 days.
3. Conclusions
Since the relationship between inflammation and carcinogene-
sis has been examined in numerous biochemical studies [1a,17],
the synthesis of new metal complexes with anti-inflamatory drugs,
such as aspirin, is one of the strategies employed for the develop-
ment of new metallotherapeutics. Thus, two new mixed ligand
silver(I) complex of formula {[Ag(tpp)3(asp)](dmf)} (1) and
[Ag(tpp)2(o-Hbza)] (2) were synthesized and characterized. The
geometry around the silver(I) ion is tetrahedral in both complexes.
Complexes 1 and 2 were tested for their in vitro cell growth prolif-
Compound 1: White crystal, Yield: 0.135 g; melting point: 162–
165 °C. Elemental analysis, Anal. Calc. for C66H55AgN1O5P3: C:
69.36; H: 4.85; N, 1.23. Found: C: 69.23; H: 4.80; N: 1.33%. IR
(cmꢀ1), (KBr): 3052 m, 1747s, 1665s, 1594vs, 1479vs,1435vs,
1092vs,743vs, 694vs, 512vs, 494vs; Far-IR (cmꢀ1), (polyethylene):
277vs, 254s, 242m, 225m, 202s; 1H-NMR (ppm) in DMSO-d6:
7.47–7.23 ppm (m, H(Caromatic), 2.87 and 2.71 ppm (s, H(CH3–
of DMF)), 2.07 ppm (s, H(CH3– of acetyl group)); MS m/z: 632.9

120
M. Poyraz et al. / Inorganica Chimica Acta 375 (2011) 114–121
[Ag(tpp)2]+, 368.8 [Ag(tpp)]+, 261.9 [tpp]+, UV–Vis (DMSO):
kmax = 259 nm (log = 4.6).
ment. Cells were washed with PBS, treated with media containing
various concentrations of 1 and 2 (1.5 and 1.9 V (1) and 1.6 and
2.3 V (2)) in DMSO (0.008–0.020% v/v) and incubated for 48 h.
e
l
Compound 2: White crystal, Yield: 0.174 g; melting point: 189–
191 °C. Elemental analysis, Anal. Calc. for C43H35AgO3P2: C: 67.12;
H: 4.58. Found: C: 67.43; H: 4.71%. IR (cmꢀ1), (KBr): 3052m,
1619vs, 1591vs, 1562vs, 1457vs, 1437vs, 1352vs, 1305vs, 511vs,
434m; Far-IR (cmꢀ1), (polyethylene): 279vs, 253s, 247s; 1H NMR
(ppm) in DMSO-d6: 15.88 ppm (br, H(HO), 7.68–6.55 ppm ((m,
H(Caromatic); MS m/z: 632.9 [Ag(tpp)2]+, 368.8 [Ag(tpp)]+, 261.9
l
Supernatants and cells collected were centrifuged and cell pellets
were suspended in calcium buffer 1ꢂ at a rate 105 cells/100
ll.
Cells were stained with Annexin (BD 556420) and Propidium
Iodide (Sigma P4864) in a dark room for 15 min. DNA content
was determined on a FACScan flow cytometer (Partec ML, Partec
GmbH, Germany). Percentage of apoptotic, necrotic and decom-
pensate cells were calculated over all viable cells (100%). Two
replicates were performed for each complex.
[tpp]+, UV–Vis (DMSO): kmax = 261 nm (log
e = 4.6).
4.3. X-ray structure determination
4.5. Study of lipoxygenase inhibition mechanism
Data for 1 studied were collected by the h–2h scan technique in
the h range 5.11–13.96° (1) on a Bruker P4 diffractometer, while
intensity data for the crystals of 2 and 3 were collected by the h–
2h scan technique in the h range 2.82–28.93° (2), 2.88–32.54° (3),
on an Oxford Diffraction CCD instrument [18a], using graphite-
These studies were performed as previously reported [10f,15].
4.6. Saturation Transfer Difference 1H NMR experiments
monochromated Mo Ka (k = 0.71073 Å) radiation. Cell parameters
were determined by least-squares fit from 32 reflections in the
range. The structures were solved with direct methods with SHEL-
XS-97 [18b] and refined by full-matrix least-squares procedures
on F2 with SHELXL-97 [18c]. All non-hydrogen atoms were refined
anisotropically, hydrogen atoms were located at calculated posi-
tions (C–H = 0.93–0.97 Å) and refined as a ‘riding model’.
NMR samples for the STD experiments were prepared in 99.9%
D2O buffer containing 20 mM TRIS (98% D11), 7 mM (ND4)2SO4
(98% D8), 3.5 mM MgCl2 and 0.3 mM DTT (98% D10), pD 9. Ligand
concentration was 0.4 mM and the protein concentration was
0.004 mM resulting in protein–ligand ratio of 1:100.
The STD NMR experiments [16] were recorded on Varian Direct-
Drive 800 MHz spectrometer equipped with a Cryoprobe probe
with spectral width of 8117 Hz, 8192 complex data points, and
5000 scans at 30 °C. Relaxation delay was set to 9.6 s according
to the longest 1H T1 relaxation time of the ligands. Selective on-res-
onance irradiation frequency was set to 0.32 ppm with saturation
time of 0.4 s. Selective pulse consisted of a train of 50 ms Gauss-
shaped pulses separated by a 1 ms delay. Off-resonance irradiation
frequency for the reference spectrum was applied at 30 ppm.
Water suppression was achieved with excitation sculpting [19].
Spectra were zero filled twice and line broadening function of
1 Hz was applied.
Compound 1: C66H59AgN1O5P3: MW = 1146.9, triclinic, space
ꢀ
group P1, a = 12.724(2) Å, b = 13.662(3) Å, c = 19.746(5) Å,
a
= 95.98(2)°, b = 102.66(2)°,
Z = 2, T = 293(2),
(calc) = 1.309 g cmꢀ3
1174. The refinement converged to final R [for 6304 reflections with
I > 2 (I)] = 0.0945. wR = 0.3023 (all 9931).
c
= 117.490(10)°, V = 2886.1(11) Å3,
q
, l
= 0.481 mmꢀ1, F(0 0 0) =
r
Compound 2: C43H35AgO3P2: MW = 769.5, monoclinic, space
group P21/n, a = 8.909(5) Å, b = 19.527(5) Å, c = 20.754(5) Å, b =
97.229(5)°, V = 3582(2) Å3, Z = 4, T = 293(2),
l
q ,
(calc) = 1.427 g cmꢀ3
= 0.692 mmꢀ1, F(0 0 0) = 1576. The refinement converged to final
R [for 8935 reflections with I > 2r(I)] = 0.0226. wR = 0.0571 (all
54953).
Compound 3: C7H5AgO3: MW = 245.0, monoclinic, space group
P21/c, a = 7.4030 Å, b = 8.7010 Å, c = 10.5450 Å, b = 107.2660°,
5. Computational details
V = 646.96(3) Å3, Z = 4, T = 293(2),
q
(calc) = 2.505 g cmꢀ3
,
l =
3.055 mmꢀ1, F(0 0 0) = 468. The refinement converged to final R
Molecular docking was performed with the grid based version
of the MOLDOCK scoring function [20] as this is implemented in MOLE-
GRO VIRTUAL DOCKER software (www.molegro.com). Flexible torsions
were assigned automatically and no constrains were applied;
geometry optimizations based on the X-ray structures of the com-
plexes were carried out using the Forcite module (www.accelrys.
com) to confirm that no significant divergence in the conforma-
tions of the complexes exist due to crystal packing effects. The uni-
versal forcefield, which is parameterized for the full periodic table,
was adopted for the minimizations. The three dimensional coordi-
nates of LOX (pdb ID: 1F8N) were obtained from the Protein Data
Bank. (www.rcsb.org/pdb) and solvent molecules within the pro-
tein structure were removed prior to the docking procedure. The
docking space was adequate to cover an extensive domain and
was defined as a sphere of 34 Å in diameter with its origin at the
center of the enzyme. Due to the stochastic nature of the ligand–
protein docking search algorithm, 15 runs were conducted and
ten poses were retained for each ligand. The docked derivatives
were scored and reranked using the ‘‘reranking score’’ scheme
which is a weighted linear combination of the intermolecular
interactions (steric, Van der Waals, hydrogen bonding, electro-
static) between the ligand and the protein, and intramolecular
interactions (torsional, sp2–sp2, steric, Van der Waals, hydrogen
bonding, electrostatic) of the ligand. Steric interactions between
the ligands and the protein or the cofactor were assigned and
calculated using the piecewise linear potential (PLP) Energy
[for 2338 reflections with I > 2
r(I)] = 0.0164. wR = 0.0409 (all 9931).
4.4. Biological tests
4.4.1. MTT assay
Cell growth inhibition was analyzed using the 3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. LMS,
MCF-7 and MRC-5 cells cultured on 96-well plates and maintained
in DMEM with 10% fetal calf serum (FCS), incubated at 37 °C, 5%
CO2. A DMSO solution (0.008–0.020% v/v DMSO in DMEM for the
complexes and 0.01–0.1% v/v DMSO in DMEM for the ligands) con-
taining different concentrations of the compounds tested (0.5–
20
lV for the complexes and 10–400
lV for the ligands) was then
added. After incubation for 48 h, 50
well from a stock solution (3 mg/ml), and incubated for an addi-
tional 3 h. Blue formazans were eluted from cells by adding
200 ll of DMSO under gentle shaking and absorbance was deter-
mined at 550 nm (subtract background absorbance measured at
690 nm) using a microplate spectrophotometer (Diagnostics Pas-
teur LP 400). Three to five independent experiments were per-
formed for each compound.
ll of MTT was added in each
4.4.2. Flow cytometry
LMS cells were seeded onto six-well plates at a density of
6 ꢂ 104 cells per well and incubated for 24 h before the experi-

M. Poyraz et al. / Inorganica Chimica Acta 375 (2011) 114–121
121
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orientation was taken into account for the final ranking.
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Acknowledgments
(b) M.J. Knapp, J.P. Klinman, Biochemistry 42 (2003) 11466;
(c) X.-Z. Ding, C.A. Kuszynski, T.H. El-Metwally, T.E. Adrian, Biochem. Biophys.
Res. Commun. 266 (1999) 392;
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K.V. Honn, Cancer Metastasis Rev. 26 (2007) 503.
M.P. acknowledges the Ministry of Education of Greece for the
financial support (decision number 94975/I/04.08.2009). The
NMR studies were supported by EN-FIST Centre of Excellence
(Dunajska 156, SI-1000 Ljubljana, Slovenia) and Ministry of Higher
Education, Science and Technology of Slovenia. T.M. is a recipient
of a grant by East-NMR program.
[10] (a) K. Lazarou, B. Bednarz, M. Kubicki, I.I. Verginadis, K. Charalabopoulos, N.
Kourkoumelis, S.K. Hadjikakou, Inorg. Chim. Acta 363 (2010) 763;
(b) I. Ozturk, S. Filimonova, S.K. Hadjikakou, N. Kourkoumelis, V. Dokorou, M.J.
Manos, A.J. Tasiopoulos, M.M. Barsan, I.S. Butler, E.R. Milaeva, J. Balzarini, N.
Hadjiliadis, Inorg. Chem. 49 (2010) 488;
(c) K.N. Kouroulis, S.K. Hadjikakou, N. Kourkoumelis, M. Kubicki, L. Male, M.
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doi:10.1155/2008/654137 (Article ID 654137).;
(f) M.N. Xanthopoulou, S.K. Hadjikakou, N. Hadjiliadis, E.R. Milaeva, J.A.
Gracheva, V.Yu. Tyurin, N. Kourkoumelis, K.C. Christoforidis, A.K. Metsios, S.
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(g) M.N. Xanthopoulou, S.K. Hadjikakou, N. Hadjiliadis, M. Kubicki, S. Skoulika,
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Charalabopoulos, J. Inorg. Biochem. 96 (2003) 425.
Appendix A. Supplementary material
CCDC 776894, 791214 and 791213 contain the supplementary
crystallographic data for compounds 1, 2 and 3, respectively. These
data can be obtained free of charge from The Cambridge Crystallo-
graphic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ica.2011.04.032.
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