Novel mixed metal Ag(I)-Sb(III)-metallotherapeutics of the NSAIDs, aspirin and salicylic acid: Enhancement of their solubility and bioactivity by using the surfactant CTAB

Article abstract of DOI:10.1016/j.jinorgbio.2015.04.014

The already known Ag(I)-Sb(III) compound of the formula {Ag(Ph₃Sb)₃(NO₃)} (1) and two novel mixed metal Ag(I)-Sb(III) metallotherapeutics of the formulae {Ag(Ph₃Sb)₃(SalH)}(2) and {Ag(Ph₃Sb)₃(Asp)}(3) (SalH₂ = salicylic acid, AspH = aspirin or 2-acetylsalicylic acid and Ph₃Sb = triphenyl antimony(III)) have been synthesised and characterised by m.p., vibrational spectroscopy (mid-FT-IR), ¹³C-,¹H-NMR, UV-visible (UV-vis) spectroscopic techniques, high resolution mass spectroscopy (HRMS) and X-ray crystallography. Compounds 1,- 3 were treated with the surfactant cetyltrimethylammonium bromide (CTAB) in order to enhance their solubility and as a consequence their bioactivity. The resulting micelles a-c were characterised with X-ray powder diffraction (XRPD) analysis, X-ray fluorescence (XRF) spectroscopy, Energy-dispersive X-ray spectroscopy (EDX), conductivity, Thermal gravimetry-differential thermal analysis (TG-DTA), and atomic absorption. Compounds 1-3 and the relevant micelles a-c were evaluated for their in vitro cytotoxic activity against human cancer cell lines: MCF-7 (breast, estrogen receptor (ER) positive), MDA-MB-231 (breast, ER negative) and MRC-5 (normal human fetal lung fibroblast cells) with sulforhodamine B (SRB) colorimetric assay. The results show significant increase in the activity of micelles compared to that of the initial compounds. Moreover, micelles exhibited lower activity against normal cells than tumor cells. The binding affinity of a-c towards the calf thymus (CT)-DNA, lipoxygenase (LOX) and glutathione (GSH) was studied by the fluorescent emission light and UV-vis spectroscopy.

Full text of DOI:10.1016/j.jinorgbio.2015.04.014

Novel mixed metal Ag(I)-Sb(III)-metallotherapeutics of the NSAIDs, aspirin
and salicylic acid. Enhancement of their solubility and bioactivity by using
the surfactant CTAB
E.I. Gkaniatsou, C.N. Banti, N. Kourkoumelis, S. Skoulika, M. Manoli,
A.J. Tasiopoulos, S.K. Hadjikakou
PII:
DOI:
Reference:
S0162-0134(15)00108-7
doi: 10.1016/j.jinorgbio.2015.04.014
JIB 9714
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
9 February 2015
17 April 2015
17 April 2015
Please cite this article as: E.I. Gkaniatsou, C.N. Banti, N. Kourkoumelis, S. Sk-
oulika, M. Manoli, A.J. Tasiopoulos, S.K. Hadjikakou, Novel mixed metal Ag(I)-Sb(III)-
metallotherapeutics of the NSAIDs, aspirin and salicylic acid. Enhancement of their
solubility and bioactivity by using the surfactant CTAB, Journal of Inorganic Biochemistry
(2015), doi: 10.1016/j.jinorgbio.2015.04.014
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ACCEPTED MANUSCRIPT
1
Novel mixed metal Ag(I)-Sb(III)-metallotherapeutics of the NSAIDs, aspirin and salicylic
acid. Enhancement of their solubility and bioactivity by using the surfactant CTAB.
E.I. Gkaniatsou[a], C.N. Banti[a], N. Kourkoumelis*[b], S. Skoulika[c], M. Manoli[d], A.J.
Tasiopoulos[d] and 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] Section of Physical Chemistry, Department of Chemistry, University of Ioannina, Ioannina,
Greece
[d] Department of Chemistry, University of Cyprus, 1678, Nicosia, Cyprus
*All correspondence should be addressed to:
* Dr N. Kourkoumelis (Assistant Professor): e-mail: nkourkou@uoi.gr; Tel xx30-26510-07594
* Dr S.K. Hadjikakou (Professor); e-mail: shadjika@uoi.gr; tel. xx30-26510-08374
fax xx30-26510-08786
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Abstract
The already known Ag(I)-Sb(III) compound of the formula {Ag(Ph3Sb)3(NO3)} (1) and two
novel mixed metal Ag(I)-Sb(III) metallotherapeutics of the formulae {Ag(Ph3Sb)3(SalH)}(2) and
{Ag(Ph3Sb)3(Asp)}(3) (SalH2= salicylic acid, AspH = aspirin or 2-acetylsalicylic acid and Ph3Sb=
triphenyl antimony(III)) have been synthesised and characterised by m.p., vibrational spectroscopy
13
(mid-FT-IR), C-,1H-NMR, UV-visible (UV-vis) spectroscopic techniques, high resolution mass
spectroscopy (HRMS) and X-ray crystallography. Compounds 1,-3 were treated with the surfactant
cetyltrimethylammonium bromide (CTAB) in order to enhance their solubility and as a
consequence their bioactivity. The resulting micelles a-c were characterised with X-ray powder
diffraction (XRPD) analysis, X-ray fluorescence (XRF) spectroscopy, Energy-dispersive X-ray
spectroscopy (EDX), conductivity, Thermal gravimetry–differential thermal analysis (TG–DTA),
and atomic absorption. Compounds 1-3 and the relevant micelles a-c were evaluated for their in
vitro cytotoxic activity against human cancer cell lines: MCF-7 (breast, estrogen receptor (ER)
positive), MDA-MB-231 (breast, ER negative) and MRC-5 (normal human fetal lung fibroblast
cells) with sulforhodamine B (SRB) colorimetric assay. The results show significant increase in the
activity of micelles compared to that of the initial compounds. Moreover, micelles exhibited lower
activity against normal cells than tumor cells. The binding affinity of a-c towards the calf thymus
(CT)-DNA, lipoxygenase (LOX) and glutathione (GSH) was studied by the fluorescent emission
light and UV-vis spectroscopy.
Keywords:
Bioinorganic Chemistry; silver(I)-antimony(III) compounds; anti-inflammatory drugs; micelles;
cytotoxic activity.
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1. Introduction
Metal-based drugs play a crucial role in medicine [1-2]. Compounds with antimony (anti-
protozoal), bismuth (anti-ulcer), gold (anti-arthritic), iron (anti-malaria), silver (anti-microbial) and
platinum (anti-cancer) are typical examples of metal-based drugs for the treatment of various
diseases [1-7]. Despite the widespread use of these metallotherapeutics, the developed cell
resistance, their side effects and their low solubility prompt the research towards designing new and
safe drugs [8-9]. Moreover, research is focused on limiting the toxicity while enhancing the
reactivity.
Silver ions have been extensively used for a variety of medical purposes [10-16]. Silver
sulphadiazine is an example of silver containing drug [10]. The anti-bacterial activity of silver
compounds has been attributed to its ionized form Ag(I), which interact with thiol-containing
proteins and DNA causing cell damage [10,14,17]. This interaction of silver compounds with
proteins and DNA combined with their very low toxicity to humans, create a new era for the
development of anti-cancer metallotherapeutics [10]. Recent studies have shown selectivity of silver
compounds with carboxylic acids towards adenocarcinoma cells [10].
Antimonials (such as potassium antimony tartrate or the less toxic pentavalent, sodium
stibogluconate (pentostam) and melglumine antimoniate (glucantime)) have been used for the
treatment of leismaniasis (tropical disease) [18-23]. The potential anti-cancer activity of antimony
compounds is also under consideration [18-23]. Antimony(III) complexes are more active, mainly,
against carcinoma, but less active against lymphoma [18]. It is also notable, that antimony(III)
complexes exhibit strong anti-proliferative activity against human cancer cells, which is not
observed against murine cells [18]. Specifically, the antimony(III) complexes exhibit selectivity
against sarcoma and carcinoma cells [18].
Aspirin (AspH) (Figure 1) and its active metabolite, salicylic acid (SalH2) (Figure 1) belong
to the class of non-steroidal anti-inflammatory drugs (NSAIDs). A number of studies relate the use
of NSAIDs with a reduction in the incidence of colorectal, bladder, prostate, lung cancer and breast
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cancer, identifying them as selective anticancer agents [24-28]. NSAIDs also target lipoxygenase
(LOX). LOX is a class of enzymes responsible for the conversion of arachidonic, linoleic and other
polyunsaturated fatty acids into biologically active metabolites [29]. LOX is mainly distributed to
the mitochondrion [15]. Studies on the relationship between NSAID-induced apoptosis and the
inhibition of lipoxygenase, concluded that LOX is an additional molecular target [11-16,30-32].
Micelles, on the other hand, can be used as drug carriers with the added benefit of increasing
the bioavailability of the drug allowing them to accumulate in areas with tumours [33-35]. The
solubility can be improved using surfactants like the cationic cetyltrimethylammonium bromide
(CTAB) (Figure 1) [36]. CTAB has been widely used in the preparation of gold nanoparticles [37-
38] and in several studies concerning the encapsulation of poorly soluble drugs in water [39]. Its
low toxicity in clinical trials [40] and its potential anticancer activity [41] have been the key factors
in our study.
Since DNA is targeted, by current chemotherapeutics of cancers [11-16] the development of
new DNA binders that intercalate into either minor of major grooves is a critical aspect for the
development of new drugs. Moreover, glutathione (GSH), plays an important role in multi-drug
resistance since GSH readily interacts with chemotherapeutics preventing substantial quantities of
them from reaching DNA in the nucleus and ultimately limit their efficacy [12]. High levels of GSH
ave been found in many tumours including breast cancer that show different degrees of multidrug
resistance, including breast cancer [12].
Breast cancer is hormone dependent malignancy [12]. Since estrogen receptors (ERs) are
expressed in human breast cancer, they probably act as both a growth and a survival factors for
breast cancer [12]. MCF-7 (breast, ER positive) and MDA-MB-231 (breast, ER negative) are two
human breast cancer cell lines where the ERs are devoid (MDA-MB-231), or present (MCF-7) [12].
Our studies aim at the development of new metallotherapeutics [11-16,18-23] which
overcome the cell resistance while they can interact with ERs causing a selective inhibition of
breast cancer cells. In the course of these studies we have synthesize silver(I) complex
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{Ag(Ph3Sb)3(NO3)} (1) (Ph3Sb= triphenyl antimony) (Figure 1)) [42], was used as a precursor for
the synthesis of {Ag(Ph3Sb)3(SalH)}(2). The complex {Ag(Ph3Sb)3(Asp)}(3) is also prepared. The
compounds 1-3 were characterised by m.p., FT-IR, 1H-,13C-NMR, UV-vis spectroscopic
techniques, HRMS and single crystal X-ray diffraction analysis. The stability of the complexes in
1
DMSO solution has been verified by means of H- NMR, UV-vis and conductivity measurements.
The micelles a-c was characterized by atomic absorption, conductivity, Thermal gravimetry–
differential thermal analysis (TG–DTA) (TG–DTA), X-ray powder diffraction (XRPD) analysis, X-
ray fluorescence (XRF) spectroscopy and Energy-dispersive X-ray spectroscopy (EDX). The in
vitro cytotoxic activity of the compounds 1-3 and their micelles a-c against MCF-7 (breast, ER
positive), MDA-MB-231 (breast, ER negative) and MRC-5 (normal human fetal lung fibroblast
cells) was evaluated and the results are discussed in comparison. The binding affinities of micelles
a-c towards the intracellular molecules of calf thymus (CT)-DNA, glutathione (GSH) and
lipoxygenase (LOX) were studied by fluorescence and UV-vis spectroscopies.
Figure 1
2. Results and discussion
2.1. General aspects: Compound 1 was synthesised through the reaction of AgNO3 with Ph3Sb
(Diagram 1). Crystals of 1 (Figure S1) (1st preparation: P21/n, a= 17.815(2), b= 14.488(2), c=
19.648(4) Å, β= 97.130(10)o; 2nd preparation: P21/n, a= 17.3575(7), b= 14.4695(5), c= 19.4437(9)
Å, β= 96.051(4)ο) are similar to the known one (P21/n: a= 19.602(5), b= 14.455(1), c= 17.727(2),
β= 97.19(2)ο) [42]. However, the small variations in the unit cell parameters (β angle) led us to
refine the structure again. Complex 1 was then used as a precursor for the formation of compound
2.
The reaction of 1 with salicylic acid in the presence of an excess of (CH2CH3)3N in
CH3OH/CH3CN solution yielded the compound 2 (Diagram 1). X-Ray Powder Diffraction (XRPD)
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analysis was performed in order to compare the two products. Compound 2 was also prepared by
the reaction of SalH2 with AgNO3, followed by the addition of Ph3Sb in DMSO solution (Diagram
1). The diffraction patterns in XRPD indicate that the two products of 2 obtained from these two
reactions are identical. (Figure S2). Moreover the intermediate complex silver salicylate, [Ag(Sal)]
(Figure S3) was isolated during this procedure as an intermediate: space group P21/c; monoclinic;
a= 7.4100(10), b= 8.8350(10) and c= 10.687(2) Å; β= 107.630(10)o;V= 666.79(17) Å3 Z= 4, T=
293(2) K, D(calc)= 2.4404(7) g cm-3; μ= 2.964 mm-1 which is identical to the known one (P21/c, a=
7.405(1), b= 8.826(2), c= 10.Å, β = 107.48(4)o) [16,43].
C
H
CH3OH/CH3CN
O
H
3
Ag(Ph3Sb)3NO3
AgNO3 + 3Ph3Sb
AgNO3 + SalH2
/
C
H
C
N
3
S
a
l
H
,
(
C
H
(1)
2
C
H
3
)
3N
2
Ag(Ph3Sb)3(SalH)
)
b
S
h
P
3
(2)
(
g
A
(CH3CH2)3N
DMSO
[Ag(SalH)]
O
S
M
D
Diagram 1
Compound 3 was synthesised with an alternative method to overcome the hydrolysis of
aspH which occurs when 1 reacts with aspH directly (Diagram 2, procedure I). The esterification
reaction of the salicylic acid of 2 was attempted (Diagram 2, procedure II). Compound 2 was
treated with acetic anhydride in the presence of small amounts of the catalyst, phosphoric acid.
However, the new product obtained was the diacetic-triphenyl antimony, [Ph3Sb(Ac)2]. The product
was characterised by single crystal X-ray diffraction (Figure 2). Compound 3 was finally obtained
by the reaction of aspirin with AgNO3 in the presence of excess of (CH2CH3)3N [44], followed by
the addition of Ph3Sb in DMSO solution (Diagram 2, procedure III).
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Hydrolysis of the acetyl ester of AspH
Final product (2)
N
C
3
H
C
/
H
N
O
)
3
3
H
2
H
C
C
3
H
C
(
CH3OH/CH3CN
NaOH
Hydrolysis of the acetyl ester of AspH
Final product (2)
(I)
Ag(Ph3Sb)3NO3 + AspH
a
c
e
(1)
t
o
n
e
/
H
N
a
2 O
2 C
O
3
No reaction. Final roduct (1)
Ag(Ph3Sb)3(SalH) + (CH3CO)2O
Ph3Sb(CH3COO)2
(II)
(CH3CH2)3N
3Ph3Sb
DMSO
Ag(Ph3Sb)3(Asp)
[Ag(Asp)]
AgNO3 + AspH
(III)
DMSO
(3)
Diagram 2
Figure 2
Crystals of the compounds 1 and 2 have been formed through slow evaporation of
CH3OH/CH3CN solutions, whereas crystals of the complex 3 have been grown in CH2Cl2/toluene
solution. The formulae of the compounds were initially examined with spectroscopic methods and
their structures were confirmed by single crystal X-ray diffraction analysis. Compound 1 and 2
were soluble in CH3OH, CH3CN CHCl3, CH2Cl2, (CH3)2CO, DMSO and DMF, while compound 3
was poorly soluble in the above solvents. Micelles a-c were formed by adding drop wise aqueous
solution of CTAB to a methanolic-aqueous solution (1:3) of the corresponding compound 1-3
(Scheme 1)
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N+
N+
N+
N+
N+
N+
N+
N+
Br-
N+
[Ag(Ph3Sb)3L]
N+
[Ag(Ph3Sb)3L] + n
N+
N+
N+
N+
N+
N+
N+
Scheme 1
2.2 Characterisation Studies of compounds 1, 2 and 3:
2.2.1. Solid state studies:
Crystal and molecular structure of {Ag(Ph3Sb)3(NO3)} (1), {Ag(Ph3Sb)3(SalH)}(2) and
{Ag(Ph3Sb)3(Asp)}(3): The structure of compound 1 (Figure S1) is known [42], (1: crystallized in
P21/n, a= 17.815(2), b= 14.488(2), c= 19.648(4) Å, β= 97.130(10)o; NOPQUE: P21/n, a=
19.602(5), b= 14.455(1), c= 17.727(2) Å, β= 97.19(2) o, [42]) (Figure S1). ORTEP diagram of 2-3
are shown in Figures 3-4.
Figures 3-4
In the case of 2 and 3, three antimony atoms from Ph3Sb and one oxygen atom from the
deprotonated carboxylic group of salicylic acid and aspirin respectively, form a tetrahedral
geometry around Ag(I) ion. The average Ag-Sb bond distance in 2 is 2.737 Å (Ag-Sb1= 2.716, Ag-
Sb2= 2.695 and Ag-Sb3= 2.799) and in 3 is 2.727 Å (Ag-Sb1= 2.7233(8), Ag-Sb2= 2.7393(7) and
Ag-Sb3= 2.7179(7)). The Ag–O bond distance in 2 is 2.297 Å and in 3 is 2.391(6) Å, while the
corresponding bond distances observed in silver(I) mixed ligand complexes of triphenylphospine
with salicylic acid or aspirin were 2.4045(12) Å and 2.5230(12) Å, respectively [16].
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Vibrational spectroscopy: The spectrum of compound 1 shows vibrational bands at (i) 1406 cm-1
which is assigned to νas (NO2), (ii) 1384 cm-1 of the νs (NO2) and (iii) 1036 cm-1 for the ν(NO)
vibration. The Ph-Sb frequency is near 450 cm-1, but it is hidden by the strong phenyl band at 454
cm-1 (Figure S4). The IR spectrum of the complex 2 shows a vibrational band at 3443 cm−1, which
is assigned to v(phenolic OH) [45]. The corresponding vibrational band v(O-H) of the free ligand
appears at 3340 cm−1. The vas(COO−) vibration of the free ligand appears at 1659 cm−1 and the
vs(COO−) at 1325 cm−1. The corresponding vas(COO−) vibration on 2 is observed at 1589 cm−1 and
the vs(COO−) at 1254cm−1 (Figure S5). For compound 3, the vibrational band at 1769 cm-1
corresponds to ν(C=O) of the ester group whereas the v(C=O) of the carboxylic group appears at
1743 cm-1 [16]. The respective vibrational bands in the free ligand are found at 1753 cm-1 and 1694
cm-1. The ν(C-O) vibration can been seen at 1221 cm-1 and 1217 cm-1 in the free ligand and 3
respectively [16] (Figure S6).
2.2.2. Solution studies:
1H-NMR studies: Compound 1 has three different types of protons 2-ortho (Ha), 2-meta (Hb) and 1-
para (Hc) in each phenyl group of Ph3Sb (Figure S7, Figure 1). The resonance signals of these
protons appear as a multiple peak in the 1H-NMR spectrum at 7.51-7.42 ppm in DMSO-d6 solution.
In compound 2, the aromatic protons of the Ph3Sb groups appear as a multiple at 7.51-7.42 ppm
(Figure S8, Figure 1). The hydroxyl proton, Hh of compound 2 does not appear in the range of 9-10
ppm, where it is expected [45], suggesting fast exchange with DMSO-d6 (Figure 1). The doublet
signal at 7.77-7.74 ppm is attributed to the proton Hd of the phenyl group, as it is closer to the
carboxyl group (i.e. less shielded). The triplet signal at 7.23-7.17 ppm is assigned to the Hf proton,
whereas Ηe, Hg appears as a multiplet at 6.71-6.64 ppm. The respective signals of the free ligand
(SalH2) are observed at 11.5 ppm (s, Ηh), 7.81-7.78 ppm (d, Hd), 7.48-7.45 ppm (t, Ηf), 6.94-6.25
ppm (d, Ηg) and 6.91-6.68 ppm (t, He) [15-16]. Compound 3, shows a singlet at 2.58 ppm that
corresponds to the methyl protons Hh of the ligand Asp [15-16] (Figure S9a). The doublet signal at
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7.93-7.90ppm is attributed to the less shielded proton Ηd. The triplet at 7.28-7.22 ppm corresponds
to the proton He and the doublet at 7.05-7.02 ppm to the proton Hg. The signal of the proton Hf
which is expected to be observed at ~7.4 ppm and it is overlapped with the multiplet signal of the
the Ph3Sb protons (~7.48 ppm) (Figure S9b). The respective signals of the free ligand (AspH) are
observed at 2.30 ppm (s, Ηh), 8.05 ppm(d, Ηd), 7.36 ppm (t, He), 7.15 ppm (d, Hg), whereas the
triplet of Hf, appears at 7.62 ppm [15-16].
Stability studies: The stability of 1-3 was tested by UV-vis, and 1H-NMR spectroscopy for a period
of 1 week in DMSO-d6 solutions. No changes were observed between the initial UV spectrum and
the corresponding spectra of 1-3 measured after 24, 48 hrs and 1 week for all three compounds
indicating no disintegration. (Figure S10). There was no change observed in the 1H-NMR spectra of
compound 1-3 (Figure S11-S13).
2.3. Characterisation Studies of micelles a, b and c:
2.3.1. Solid state studies:
X-ray fluorescence spectroscopy: Semi-quantitative XRF analysis has been used in order to assert
the encapsulation of compounds 1-3 in the micelles' structures a-c respectively (Figure 5). The
results testify the presence of the compounds in the final structures. The ratios of Sb:Ag were
determined from the integration of the peaks, Ag Kα (22.1 keV) and Sb Kα (26.3 keV) and the
sensitivity curve of the device, in the region of 11-40 keV. The peaks of indium derive from the
shielding material of the source. The presence of Ag and Sb was initially confirmed by EDX
measurements. However, reliable elemental quantification was not achieved due to the low
concentration of each element and the inhomogeneous nature of the samples [46]. A typical electron
microscopy (SEM) image (micelle b) accompanied by qualitative EDX analysis for complex 2, are
shown in Figure S14.
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Figure 5
Thermal analysis: The weight loss of a-c was monitored while the temperature increased at a rate of
10°C min-1 from ambient up to 600°C, under nitrogen-air flow (Figure S15). TG/DTA analysis
o
shows an endothermic peak between 200-280 (a) 210-290 (b) and 200-300 (c) C, which is
attributed to the loss of all ligands and surfactant molecules. The 10% (a), 4% (b) and 5% (c) TG
residues consist of Ag0/Sb2O3 mixture. While silver oxide forms as the compounds start to
decompose, the oxide melts at 280oC and finally elemental silver is formed [47]. On the other hand,
Sb2O3 that is produced from the Ph3Sb groups remains stable up to 653oC [48].
2.3.2. Solution studies:
Atomic Absorption: The mass of the silver contained in each micelle was measured via graphite
furnace atomic absorption (GFAAS). The percentage (%) of the compound engulfed in the micellar
structure is concluded by the amount of silver detected (Table 1). The percentage of the compounds
1-3 in the micelle a-c are 0.66(1), 0.60(2) and 1.76(3) %.
Table 1
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Conductivity measurements for critical micelle concentration (CMC) determination: In order to
assert the formation of micelles the determination of the CMC via the conventional conductivity
method [49] at 303 K was performed. The results were compared with the corresponding CMC of
pure CTAB and those found in the presence of the co-solvents (CH3OH/CH3CN). Below the CMC
the solubility of hydrophobic substances is enhanced by providing regions of interaction with the
solvent (vesicular cavities). Above the CMC, surfactant molecules self-aggregate to form micelles.
A break-point is observed in the values of conductivity of the micellar solution with the
formation of the micelles (Figure 6). The changes on the CMC in the presence of the compounds 1-
3 were studied -in the same volumes of the co-solvents- and found to be 1.40 x 10-3, 2.0 x 10-3 and
2.0 x 10-3 mol dm-3 respectively (Figure 6). By taking into account the concentration of the
complexes used (2.5 x 10-5 mol dm-3) the CTAB complex ratio is 56 (1), 80 (2-3). The formation of
the micelles has been confirmed by the concentrations that had been used for their preparation
which were much greater than the found CMCs. The CMC of pure CTAB in water was determined
and it agrees with the values reported (~ 1.25 x 10-3 mol dm-3) [49]. The effect of the co-solvents
used for the dissolution of the compounds was investigated (CH3OH and CH3CN 3 mL, ratio 1:1)
on CMC. A twofold increase of the CMC (~ 2.50 x 10-3 mol dm-3) was observed.
Figure 6
2.4. Biological Tests – Cytotoxicity: Complexes 1-3 and micelles a-c were tested for their in vitro
cytotoxic activity against MCF-7 (breast, ER positive), MDA-MB-231 (breast, ER negative) and
MRC-5 (normal human fetal lung fibroblast cells). MCF-7 and MDA-MB-231 are two human
breast cancer cell lines where the ERs are absent (MDA-MB-231), or present (MCF-7) [12] MRC-5
cells (normal human fetal lung fibroblast cells) are used to evaluate the toxicity of the compounds.
The IC50 values (50 % of cell viability) are summarized in Table 2. Cell survival was estimated by
means of SRB assay after 48 hrs of incubation. The results reveal that MCF-7 cells are more
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sensitive to compounds 1-3 with IC50 values 3.05 (1), 3.19 (2) and 5.88 (3) μΜ, than MDA-MB-231
cells IC50 values 5.67 (1), 7.26 (2) and 14.86 (3) μΜ) indicating involvement of estrogen receptors
in their mechanism of action. The compounds (1-3) were toxic on normal cells (4.36 (1), 5.03 (2)
and 4.75 (3) μΜ). The activities of 2 and 3 against MCF-7 and MRC-5 are lower than the
corresponding ones
[Ag(tpp)2(SalH)],
{[Ag(tpp)3(asp)](dmf)},
[Ag(tptp)2(salH)]
and
[Ag(tmtp)2(salH)] [11,16] (Table 2) where instead of triphenylstibine, the ligand is
triphenylphospine, tri(p-tolyl)phosphine (tptp) or tri(m-tolyl)phosphine (tmtp).
Table 2
The encapsulation of the compounds 1-3 in the micelles a-c, results in a boost of their
cytotoxic activity (Table 2). Micelles show 67.8-fold (a) 66.5-fold (b) and 57.1-fold (c), increased
activity against MCF-7 and 135-fold (a) 165-fold (b) and 57.1-fold (c), against MDA-MB-231.
Moreover, MRC-5 cells are more resistant to the micelles' action, when compared in the cancer cells
lines. The therapeutic potency index (TPI) [12] of the tested compounds, which is defined as the
IC50 value against normal cell lines divided by the IC50 value against cancer cell lines of the same or
similar tissue are also determined. The higher the TPI value is, the better the potency of the
metallotherapeutic agent. Although, the complexes exhibit high toxicity, their micelles are not toxic
indicating better therapeutic index (Table 2). Finally, the encapsulated complexes 1-3, into micelles
a-c, exhibit higher selectivity to tumor cells than normal.
The mechanisms of action of a-c were further investigated by the DNA binding studies,
LOX inhibitory activity and their interaction with GSH.
2.5. DNA binding studies:
2.5.1. UV-absorption spectral studies:
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The interaction of the micelles with CT-DNA has been investigated by UV absorption
spectroscopy. There are three modes of DNA binding (a) Electrostatic interaction with the
negatively charged nucleic sugar-phosphate structure, which are along the external DNA double
helix and do not possess selectivity (b) binding interaction with two grooves of DNA double helix
and (iii) intercalation between the stacked base pairs of native DNA [50]. Especially, the
intercalation can be clarified by the hypochromism as a result of the disconfirmation of DNA in the
helix axis. A slight decrease in the absorption intensity (at λmax= 261 nm) is observed upon the
presence and absence of a-c at various r values (r= [complex]/[DNA], [DNA]= 5x10-5 M) which is
attributed to the intercalation binding between DNA and micelles a-c (Figure S16).
The binding constants (Kb) of micelles a-c towards CT-DNA were evaluated by monitoring
the changes in the absorbance of the UV spectra ([a]= 3.9 mM, [b]= 3.0 mM, [c]= 5.0 mM), at 300-
310 nm, with increasing concentration of CT-DNA (Figure S17). Kb is obtained as previously
reported in the Ref’s [11-16]. The calculated Kb values for a-c are: (3.3±0.1)× 104 (a), (10.0±0.1)
×104 (b) and (2.5±0.1) ×104 (c) M-1 respectively, suggesting interaction with CT-DNA (Table 3)
Table 3
2.5.2. Fluorescence spectroscopic studies: Fluorescence spectroscopy is an effective method to
study metal complexes DNA interaction [11]. Ethidium bromide (EB) emits in the presence of
DNA due to its strong intercalation between the adjacent DNA base pairs [11]. The displacement of
EB (quantified by fluorescence) by the titration of a compound is suggestive of an intercalative
binding [11]. The emission spectra at 610 nm of the EB (2.3 μΜ) solutions which contain CT-DNA
(26 μΜ) in the absence or presence of various concentrations of micelles a-c (0-5.4 μΜ) were
recorded upon their excitation at 527 nm (Figure 7). The apparent binding constant (Kapp) was
calculated as described earlier [11]. Their Kapp values are 2239±178×104 (a), 2262±134×104 (b) and
1129±251×104 (c) M-1 suggesting an intercalative or minor groove binding of both complexes
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(Table 3). The high cytotoxic activity of the micelles might be attributed to the significant high
Kapp values which is due to their intercalating interaction with DNA.
Figure 7
2.6. Study of the peroxidation of linoleic acid by the enzyme lipoxygenase in the presence of
micelles a-c: LOX inhibition is known to induce apoptosis [11-16] so the influence of micelles a-c
on the oxidation of linoleic acid by the enzyme LOX was studied in a wide concentration range
(Figure S18). The degree of LOX activity (A, %) in the presence of these compounds was
calculated according to the method described previously [11-16]. The a shows high inhibitory
activity with IC50 value of 8.4 μΜ in contract to the lower activity exhibited by b (IC50= 29.4 μΜ)
(Table 3). However, no IC50 value was determined for c, at the concentrations tested (IC50 >30 μΜ)
(Table 3).
2.7. Reaction of micelles a-c with GSH as Monitored by UV Absorption: As the deactivation of
cisplatin, caused by glutathione seems to be an important determinant of its cytotoxic effects, the
reaction of micelles a-c with GSH was investigated. The initial UV spectra of the solutions (final
volume of 2.0 mL) containing different concentrations of a-c (68 (A), 140 (B) and 250 (C) μM (a);
150(A), 310 (B) and 580 (C) μΜ (b); 360 (A), 730 (B) and 1360 (C) μM (c)) in 100 mM Tris-HCl,
pH= 7.4 with a high excess of GSH (16.5 mM) at 37° C and after 1, 2, 3, 4 and 5 hrs are shown in
Figures S19-21. The increasing absorption intensity of the solution with time is partially due to the
reaction of metal complex with GSH (formation of metal-sulphur bonds) and partially due to the
oxidation of GSH (formation of the disulfide GSSG), as evidence by the increased absorption with
time for GSH alone [12]. However, as the rate of disulfide formation is low, the concentration of
GSH was assumed not to change much over the time course due to the GSH-complex reaction.
Because the GSH concentration is higher than that of a-c, the reaction of GSH with the complexes
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does not deplete GSH [12]. The absorbance associated with GSH-complex formation is obtained,
by subtracting the absorbance due to the disulfide from the observed absorbance (Figures S22-S24).
The absorbance at 280 nm is mainly due to the formation of Ag-GSH products [12].
The initial rate of the reaction between GSH and a metallotherapeutic agent is calculated as
described previously [12]. The difference curve is fitted to the equation: Id= C + A1 exp(-b1t) + A2
exp(-b2t) and the initial reaction slope (So) is then derived from the equation So= -(A1b1 + A2b2)
[12]. Table 4 summarizes the results for b-c whereas micelle a does not interact with GSH. The
order of the reaction towards a metallotherapeutic agent is calculated by the equation Soi= k
[complex]ix and alternatively from the ln(So) vs. ln([complex]) graph (Table 4). The x value shows
that the reaction is of first order towards complex b-c.
Table 4
3. Conclusions
The new silver(I)-antimony compounds of the anti-inflammatory drugs salicylic acid and
aspirin 2-3 have been synthesized and fully characterized. Compounds 1-3 were poorly soluble in
most solvents (including H2O) and therefore were treated with the surfactant CTAB to enhance their
properties (solubility and bioavailability).
The compounds and their micelles were tested for their in vitro cytotoxicity against the
cancer cell line MCF-7 (positive to ER) and MDA-MB-231 (negative to ER) and the normal cell
line MRC-5. MCF-7 (positive to ER) cells are more sensitive to 1-3 than MDA-MB-231 (negative
to ER) indicating involvement of estrogen receptors in their mechanism of action. Compounds, 1
and 2 showed the better cytotoxic activity against cancerous cells. The activities of 2 and 3 against
cells are lower than the corresponding silver(I) compounds where the ligand is tpp, tptp or tmtp
instead of Ph3Sb (Table 2) demonstrating the role of the ligand type. Micelles a-c, exhibit improved
cytotoxicity ranging from 67.8 to 135 times higher than the initial compounds against MCF-7 and
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MDA-MB-231 which is attributed to the better solubility of the compounds. The encapsulated
complexes 1-3, into micelles a-c, exhibit higher selectivity to tumor cells than normal which is also
observed in the initial compounds 1-3 (Table 2). Thus, the encapsulated compounds 1-3 retained
their selectivity against tumor cells.
The mechanism of action of the micelles a-c was investigated by mean of their DNA
binding affinity, their LOX inhibitory activity and their interaction with GSH. The compounds a-c
strongly intercalates DNA (Kapp values are in the magnitude of 1000 x 104 M-1). The positive
charged micelles (due to the CTAB) (Scheme 2) might be attracted by the negative phosphoric
groups of DNA backbone, increasing the binding affinity of the compounds towards DNA. Micelles
a and b which contain the nitrate and salycilate compounds 1 and 2 respectively, interact stronger
with DNA than micelle of c (which contain aspirinate compound 3). The micelles a-c also inhibit
LOX (an enzyme which mainly distributed in the mitochondrion [15]) with the same manner as
they interact with DNA. Thus micelles a and b inhibit LOX activity. The higher binding affinity of
1-2 against DNA and their stronger inhibitory activity against LOX towards the corresponding ones
of 3, might be attributed to the ionic nature of compound 1 and to the hydrogen bonding affinity of
2 through the –OH group of SalH2 towards AspH. Recently, cisplatin was found to interact with a
number of other possible targets such as RNA, sulfur-containing enzymes (metallothionein and
glutathione) and mitochondrion as well [51]. Similarly, to cisplatin compounds a and b were found
to interact with nuclear DNA and maybe with mitochondrion [51].
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 salicylic acid, acetylsalicylic acid,
triphenylantimony(III), silver(I) nitrate and cetyltrimethylammonium bromide (Sigma-Aldrich,
Merck) were used without further purification. MDA-MB-231 cells were purchase from the
American Type Culture Collection (ATCC, Rockville, MD, USA) and the Imperial Cancer
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Research Fund (ICRF), London. MCF-7 cells are of the most cited breast cell lines, while MRC-5 is
the most common control cell line. Melting points were measured in open tubes with a Stuart
scientific apparatus and are uncorrected. X-ray powder diffraction patterns were obtained using a
Bruker AXS D8 Advance diffractometer in Bragg–Brentano geometry equipped with a Cu sealed-
tube radiation source (λ=1.54178 Å) and a secondary beam graphite monochromator. The whole 2θ
range was measured in steps of 0.02 with a counting time of 5 s per step. Infra-red spectra in the
region of 4000–370 cm-1 were obtained in KBr discs, with a Perkin–Elmer Spectrum GX FT-IR
1
spectrophotometer. The H- NMR spectra were recorded on a Bruker AC250 MHz FT-NMR
1
instrument in DMSO-d6 solution. Chemical shifts δ are reported in ppm using H TMS as an
internal reference. Thermal studies were carried out on a Shimadzu DTG-60 simultaneous DTA-TG
apparatus, under air-N2 flow (50 cm3 min-1) with a heating rate of 10 ˚C min-1. A Jasco UV/Vis/NIR
V570 series spectrophotometer was used to obtain electronic absorption spectra. XRF measurement
was carried out using an Am-241 radioisotopic source (exciting radiation 59.5 keV). For the
detection of X-ray fluorescence a Si (Li) detector was used. The measuring time was chosen so as to
collect ~ 2000 data on the weaker Kα peak. Graphite furnace atomic absorption (GFAAS) was
measured in a Shimadzu AA6300 instrument. Conductivity measurements were carried out at 293 K
in H2O-MeOH/MeCN solutions with a WTF LF-91 conductivity meter.
4.2. Synthesis and crystallisation: (1): A solution of 0.5 mmol AgNO3 (0.085 g) in CH3OH/CH3CN
(3 mL, 1:1) was mixed with to a stirred solution of 1.5 mmol Ph3Sb (0.530 g) in CH3OH/CH3CN (7
mL, 1:1). The mixture was stirred for 30min at room temperature and the resulting clear solution
was kept in darkness. Colourless crystals of 1 suitable for X-ray analysis were grown from slow
evaporation of the solution after 2 days. (2): A solution of 0.08 mmol 1 (0.100 g) in
CH3OH/CH3CN (3 mL, 1:1) was mixed with to a solution of 0.08 mmol SalH2 (0.011 g) in
CH3OH/CH3CN (3 mL, 1:1). The mixture was treated with 3.5 mmol (CH3CH2)3N (0.5 mL), added
dropwise. The resulting solution was stirred for 30 min at room temperature. Colourless crystals of
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2 were grown after 24 hrs. (3): Compound 3 was prepared with a different method from 2. A
solution of 0.33 mmol AgNO3 (0.056 g) in DMSO (2 mL) was mixed with to a solution of 0.33
mmol AspH (0.059 g) in DMSO (2 mL), followed by the treatment of 3.5 mmol (CH3CH2)3N (0.5
mL), added dropwise. The mixture was left stirring for 30 min at room temperature, before the
addition of 1 mmol Ph3Sb (0.353 g) in DMSO (4 mL). The final solution was left stirring in
darkness for 24 hrs. The light purple precipitate was filtered off and left in the dryer for another
24hrs. Finally, the powder was dissolved in 20 mL CH2Cl2/toluene (1:1) and left in the fridge for 1
week, when colourless crystals of the product were collected. The powder was compared with the
crystal via XRPD and their diffraction peaks indicate that are identical (Figure S25).
The latter procedure was also used for the preparation of 2 with the respective ligand
(SalH2) and the yielded powder product found to be identical with the one synthesised before
(Figure S2). When the reaction was stopped at the first step, before the addition of the Ph3Sb
solution, we were able to isolate crystals suitable for X-ray of the already know compound of silver
salicylate [43]. The same try was also made in the case of 3, but no crystals were formed from the
already know [AgAsp] compound [44].
1: Colourless crystal, Yield: 70%; melting point: 209-212 °C; Elemental analysis found: C: 52.30;
H: 3.61, N: 1.48 %; calculated for C54H45AgNO3Sb3: C: 52.77; H: 3.69; N: 1.14 %. IR (cm-1),
(KBr): 3065m, 1576s,1431s, 1406vs, 1384vs, 1259s, 1066s, 996s, 743s 733s, 694vs, 454s; 448s;
1H-NMR (ppm) in DMSO-d6: 7.51-7.42 ppm (m, H(Caromatic of Ph3Sb)); UV-vis (DMSO): λmax
263 nm (logε = 4.64) (Figures S4, S7, S10).
=
2: Colourless crystal, Yield: 50%; melting point: 167-170 °C; Elemental analysis found: C: 55.96;
H: 3.65 %; calculated for C61H50AgO3Sb3: C: 56.18; H: 3.86 %. IR (cm-1), (KBr): 3443m,
1
1624s,1589 vs, 1577s, 1478s, 1382s, 1254vs, 1066s, 997s, 731s, 695s 453s; H-NMR (ppm) in
DMSO-d6: 7.53- 7.40 ppm (m, H(Caromatic of Ph3Sb)), 7.72-7.72 ppm (d, (Hd, HSal), 7.69-7.68
ppm (d, (Hg, HSal), 7.21-7.12 ppm (t, (He, HSal), 6.68-6.58 ppm (d, (Hf, HSal); UV-vis (DMSO):
λmax= 263 nm (logε= 4.59) (Figures S5, S8, S10).
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3: Colourless crystal, Yield: 75%; melting point: 184-187 °C; Elemental analysis found: C: 56.44;
H: 3.91 %; calculated for C63H52AgO4Sb3: C: 56.21; H: 3.89 %. IR (cm-1), (KBr): 3045m,
1769vs,1743vs, 1589s, 1558s, 1364s, 1221vs, 1067s, 731s, 695s, 454vs; 1H-NMR (ppm) in DMSO-
d6: 7.53- 7.37 ppm (m, H(Caromatic of Ph3Sb)), 7.94-7.92 ppm (d, (Hd, HAsp), 7.90-7.89 ppm (d,
(Hg, Hasp), 7.28-7.21 ppm (t, (He, HAsp), 7.05-7.01 ppm (d, (Hf, HAsp), 2.58 ppm (s, H(-COOCH3,
HAsp)),; UV-vis (DMSO): λmax= 263 nm (logε= 4.54) (Figures S6, S9, S10).
4.3. X-Ray Crystal-Structure Determination of compounds 1, 2, 3, [Ag(Sal)]n and [Ph3Sb(Ac)2]:
The structures 1, 2, 3 and [Ag(Sal)] were solved by direct methods and refined by full-matrix least-
squares techniques using the SHELXL-97 package. All non-hydrogen atoms have been located by
difference Fourier maps and refined anisotropically. All hydrogen atoms except the hydroxyl one
(2, [Ag(Sal)]), have been placed on calculated positions and refined isotropically by using the riding
model. The hydroxyl proton has been located by difference Fourier maps and refined isotropically
with restrained O-H distance and COH angle. In case of compound 1 the two oxygen atoms of the
-
NO3 group were refined over two positions with occupancy factors 0.5.
Intensity data for the crystals of 1 (second time) and [Ph3Sb(Ac)2] were collected on an
Oxford Diffraction CCD instrument, using graphite monochromated Mo radiation (k = 0.71073 Å).
Cell parameters were determined by least-squares refinement of the diffraction data from 25
reflections. All data were corrected for Lorentz-polarization effects and absorption [52-53]. The
structures were solved with direct methods with SHELXS 97 [54] and refined by full-matrix least-
squares procedures on F 2 with SHELXL 97 [55]. All non-hydrogen atoms were refined
anisotropically, hydrogen atoms were located at calculated positions and refined via the ’’riding
model’’ with isotropic thermal parameters fixed at 1.2 (1.3 for CH3 groups) times the Ueq value of
the appropriate carrier atom.
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1 (1st preparation): formula, C54H45AgNO3Sb3: Mr 1229.03; Monoclinic, space group P21/n, a =
17.815(2), b = 14.488(2), c = 19.648(4) Å, β = 97.13(1)°; Z = 4, V = 5032.0(14) Å3, Dcalc. = 1.622
Mg/m3, MoKa radiation (0.71073 Å), T = 293(2) K; 9780 reflections collected on a Bruker P4
diffractometer, 8713 unique (Rint=0.0398), 4949 unique reflections with I>2σ(I); For I>2σ(I) R1 =
0.0660, wR2 = 0.1771, GOF= 1.036.
1(2nd preparation): formula, C54H45AgNO3Sb3: Mr 1229.03; Monoclinic, space group P21/n, a =
17.3575(7), b = 14.4695(5), c = 19.4437(9) Å, β = 96.051(4)°; Z = 4, V = 4856.2(3) Å3, Dcalc. =
1.700 Mg/m3, MoKa radiation (0.71073 Å), T = 100(2) K; 23480 reflections collected, 8542 unique
(Rint= 0.043), 6986 unique reflections with I>2σ(I); For I>2σ(I) R1 = 0.0459, wR2 = 0.1699, GOF=
1.07.
2: formula, C61H50AgO3Sb3: Mr 1304.13; Triclinic, space group P-1, a = 13.288(1), b = 13.920(1), c
= 14.943(2) Å, α = 91.69(1)°, β = 90.75(1)°, γ = 102.96(1)°; Z = 2, V = 2691.9(5) Å3, Dcalc. = 1.609
Mg/m3, MoKa radiation (0.71073 Å), T = 293(2) K; 10716 reflections collected on a Bruker P4
diffractometer, 9395 unique (Rint=0.0238), 7250 unique reflections with I>2σ(I); For I>2σ(I) R1 =
0.0462, wR2 = 0.1201, GOF=1.041.
3: formula, C63H52AgO4Sb3: Mr 1346.17; Triclinic, space group P-1, a = 13.211(2), b = 13.527(1), c
= 16.372(2) Å, α = 86.64(1)°, β = 75.82(1)°, γ = 80.95(1)°; Z = 2, V = 2800.7(6) Å3, Dcalc. = 1.596
Mg/m3, MoKa radiation (0.71073 Å), T = 293(2) K; 11169 reflections collected on a Bruker P4
diffractometer, 9790 unique (Rint=0.0327), 6927 unique reflections with I>2σ(I); For I>2σ(I) R1 =
0.0426, wR2 = 0.0940, GOF=1.010.
[Ag(Sal)]: formula, C7H5AgO3: Mr 244.98; Monoclinic, space group P21/c, a = 7.410(1), b =
8.835(1), c = 10.687(2) Å, β = 107.63(1)°; Z = 4, V = 666.79(17) Å3, Dcalc. = 2.440 Mg/m3, MoKa
radiation (0.71073 Å), T = 293(2) K; 1444 reflections collected on a Bruker P4 diffractometer, 1177
unique (Rint=0.0184), 1031 unique reflections with I>2σ(I); For I>2σ(I) R1 = 0.0268, wR2 =
0.0670, GOF=1.069.
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[Ph3Sb(Ac)2]: formula, C22H21O4Sb: Mr 471.15; Monoclinic, space group C2/c, a = 12.7257(9), b =
9.8981(7), c = 15.6343(12) Å, β = 106.972(8)°; Z = 4, V = 1883.5(3) Å3, Dcalc. = 1.6615(3) Mg/m3,
MoKa radiation (0.71073 Å), T =100 K; 11169 reflections collected on a Bruker P4 diffractometer,
9790 unique (Rint=0.0327), 6927 unique reflections with I>2σ(I); For I>2σ(I) R1 = 0.0426, wR2 =
0.0940, GOF=1.010.
The crystallographic data of the compounds have been deposited with the Cambridge
Crystallographic Data Centre as supplementary publication number CCDC-1011503 (1; 1st
preparation), 1011697(1; 2nd preparation), 1011504 (2), 1011505 (3), 1011506 ([Ag(Sal)]),
1011696 ([Ph3Sb(Ac)2]). Copies of the data can be obtained, free of charge, via the internet
(http://www.ccdc.cam.ac.uk/data_request/cif), e-mail (data_request@ccdc.cam.ac.uk), or fax (+44-
1223-336033).
4.4. Micellar Synthesis: 0.038 mmol (0.047 g (1), 0.049 g (2) and 0.051 g (3)), were dissolved in
CH3OH/CH3CN (3 cm3, 1:1) and stirred at 303 K. 10 cm3 of distilled H2O were added to the
solution forming a suspension; we would use the low solubility of the compounds in water to
identify later the point at which they had been fully engulfed in the micelles. A separate solution of
8 mmol CTAB (3 g) in distilled H2O (30 cm3) was prepared, stirred at 303 K and added stepwise (1
cm3 steps) to the first solution of the respective compound until it became clear, which meant that
the majority of the suspended particles were inside the micellar structures. The clear mixture was
left stirring for 30 min at 303 K; then it was filtered off and left to evaporate slowly overnight. The
micelles were collected as colourless aggregates.
4.5. Cytotoxic activity, DNA binding and LOX inhibitory activity studies: This study was performed
as was previously reported [11-16].
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5. Table of Abbreviations
AspH
CMC
CTAB
CT-DNA
EB
Aspirin
Crticical Micelle Concentration
Cetyltrimethylammonium Bromide
Calf Thymus DNA
Ethidium Bromide
Er
Estrogen Receptors
GFAAS
GSH
Graphite Furnace Atomic Absorption
Glutathione
GST
Kapp
Glutathione S-Transferases
Apparent Binding Constant
Lipoxygenase
LOX
MRC-5 cells
NSAIDs
SalH2
So
Normal Human Fetal Lung Fibroblast Cells
Non-Steroidal Anti-Inflammatory Drugs
Salicylic Acid
Initial Reaction Slope
SRB assay
tmtp
Sulforhodamine B Assay
Tri(m-tolyl)phosphine
TPI
Therapeutic Potency Index
Tri(p-tolyl)phosphine
tptp
XRPD
X-Ray Powder Diffraction
Acknowledgements
This work was carried out in partial fulfillment of the requirements for the graduate studies (Master)
MSc thesis of Mrs. E.I.G. under the supervision of S.K.H. 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 fluorimetry instrument to the Department of
Chemistry of the University of Ioannina. Dr. C. Papachristodoulou is acknowledged for the XRF
measurements. The authors would like to thank the Unit of bioactivity testing of xenobiotics, the
University of Ioannina, for providing access to the facilities. CNB and SKH acknowledge the Josef
and Esther Gani Foundation for the financial support. CNB and SKH also acknowledge the COST
Action CM1105 “Functional metal complexes that bind to biomolecules” for the stimulating
discussions. CNB and SKH acknowledge the National Scholarships Foundation of Greece (IKY) for
the post doctoral research fellowship of excellence program (IKY)-Siemens.
Appendix A. Supplementary Data
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Table 1: Atomic absorption analysis results
Micelle
mtot (μg)
11600
9360
mAg (μg)
6.77
Percentage (%)
0.66
0.60
1.76
a
b
c
4.65
54170
76.3
Where, mtot, the total mass of the micelle that was analysed,
mAg, the detected mass of silver in the sample and
Percentage (%), the percentage of the compound in the respective micelle.
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Table 2: IC50 values for cell viability found for complexes 1–3 and their micelles a–c against MCF-7 (breast, ER positive), MDA-MB-231 (breast, ER
negative) and the normal human lung cell line MRC-5 (normal human fetal lung fibroblast cells).
IC50 (μΜ)
Compound
MCF-7
MDA-MB-231
MRC-5
TPI MCF-7
TPI MDA-MB-231
Ref.
Αg(Ph3Sb)3(NO3) (1)
Micelle a
3.05±0.11
0.045±0.004
3.19±0.07
5.67±0.17
0.042±0.002
7.26±0.18
4.36±0.44
0.070±0.008
5.03±0.12
1.42
1.56
1.58
1.29
0.81
0.77
1.66
0.69
1.41
0.32
Αg(Ph3Sb)3(SalH) (2)
Micelle b
0.048±0.006
5.88±0.39
0.044±0.004
14.86±0.86
0.062±0.005
4.75±0.82
Αg(Ph3Sb)3(Asp) (3)
Micelle c
0.103±0.011
2.3±0.3
0.157±0.013
0.263±0.050
2.55
1.35
1.26
1.67
[Ag(tpp)2(salH)]
-
-
-
-
3.1±0.3
-
-
-
-
[16]
[16]
[11]
[11]
{[Ag(tpp)3(asp)](dmf)}
[Ag(tptp)2(salH)]
[Ag(tmtp)2(salH)]
2.3±0.3
2.9±0.1
1.7±0.3
-
-
5.9±0.8
29