Synthesis, antimicrobial and antiproliferative activity of novel silver(I) tris(pyrazolyl)methanesulfonate and 1,3,5-triaza-7-phosphadamantane complexes

Article abstract of DOI:10.1021/ic201714c

Five new silver(I) complexes of formulas [Ag(Tpms)] (1), [Ag(Tpms)(PPh ₃)] (2), [Ag(Tpms)(PCy₃)] (3), [Ag(PTA)][BF₄] (4), and [Ag(Tpms)(PTA)] (5) {Tpms = tris(pyrazol-1-yl)methanesulfonate, PPh₃ = triphenylphosp

Full text of DOI:10.1021/ic201714c

ARTICLE
pubs.acs.org/IC
Synthesis, Antimicrobial and Antiproliferative Activity of Novel
Silver(I) Tris(pyrazolyl)methanesulfonate and
1,3,5-Triaza-7-phosphadamantane Complexes
Claudio Pettinari,*,† Fabio Marchetti,‡ Giulio Lupidi,† Luana Quassinti,† Massimo Bramucci,†
Dezemona Petrelli,† Luca A. Vitali,† M. Fꢀatima C. Guedes da Silva,*,§,^ Luísa M. D. R. S. Martins,§,
Piotr Smoleꢀnski,*,X and Armando J. L. Pombeiro*,§
||
†School of Pharmacy and ‡School of Science and Technology, Universitꢁa degli Studi di Camerino, via S Agostino 1,
62032 Camerino MC, Italy
§Centro de Química Estrutural, Complexo I, Instituto Superior Tꢀecnico, Technical University of Lisbon, Av. Rovisco Pais,
1049-001 Lisbon, Portugal
^Universidade Lusꢀofona de Humanidades e Tecnologias, ULHT Lisbon, Campo Grande 376, 1749-024 Lisboa, Portugal
ꢀ
Area Departamental de Engenharia Química, Instituto Superior de Engenharia de Lisboa, Rua Conselheiro Emídio Navarro, 1059-007,
Lisboa, Portugal
XFaculty of Chemistry, University of Wroclaw, ul. F. Joliot-Curie 14,50-383 Wroclaw, Poland
S Supporting Information
b
ABSTRACT: Five new silver(I) complexes of formulas [Ag(Tpms)] (1), [Ag(Tpms)-
(PPh3)] (2), [Ag(Tpms)(PCy3)] (3), [Ag(PTA)][BF4] (4), and [Ag(Tpms)(PTA)]
(5) {Tpms = tris(pyrazol-1-yl)methanesulfonate, PPh3 = triphenylphosphane, PCy3 =
tricyclohexylphosphane, PTA = 1,3,5-triaza-7-phosphaadamantane} have been syn-
1
thesized and fully characterized by elemental analyses, H, 13C, and 31P NMR,
electrospray ionization mass spectrometry (ESI-MS), and IR spectroscopic techni-
ques. The single crystal X-ray diffraction study of 3 shows the Tpms ligand acting in
the N3-facially coordinating mode, while in 2 and 5 a N2O-coordination is found, with
the SO3 group bonded to silver and a pendant free pyrazolyl ring. Features of the tilting in the coordinated pyrazolyl rings in these
cases suggest that this inequivalence is related with the cone angles of the phosphanes. A detailed study of antimycobacterial and
antiproliferative properties of all compounds has been carried out. They were screened for their in vitro antimicrobial activities
against the standard strains Enterococcus faecalis (ATCC 29922), Staphylococcus aureus (ATCC 25923), Streptococcus pneumoniae
(ATCC 49619), Streptococcus pyogenes (SF37), Streptococcus sanguinis (SK36), Streptococcus mutans (UA159), Escherichia
coli (ATCC 25922), and the fungus Candida albicans (ATCC 24443). Complexes 1ꢀ5 have been found to display effective
antimicrobial activity against the series of bacteria and fungi, and some of them are potential candidates for antiseptic or
disinfectant drugs. Interaction of Ag complexes with deoxyribonucleic acid (DNA) has been studied by fluorescence spectro-
scopic techniques, using ethidium bromide (EB) as a fluorescence probe of DNA. The decrease in the fluorescence of DNAꢀEB
system on addition of Ag complexes shows that the fluorescence quenching of DNAꢀEB complex occurs and compound 3 is
particularly active. Complexes 1ꢀ5 exhibit pronounced antiproliferative activity against human malignant melanoma (A375)
with an activity often higher than that of AgNO3, which has been used as a control, following the same order of activity inhibition
on DNA, i.e., 3 > 2 > 1 > 5 > AgNO3. 4.
’ INTRODUCTION
anti-infective coatings in medical devices (e.g., in dental work or
catheters), and healing of burn wounds.5ꢀ12 They were shown to
have a pleiotropic effect on the bacterial cell and most probably
interact with proteins involved in maintaining the proton motive
force through the cytoplasmic membrane or in the respiration of
the bacteria,13,14 although other target sites remain a possibility.15
The interaction of silver ions with thiol groups in enzymes and
Biomedical inorganic chemistry is an important new area of
chemistry that plays a significant role in therapeutic and diag-
nostic medicine for the discovery and development of new
metallodrugs.1 Among the metal complexes used for treatment
of several diseases,2 some silver complexes are currently applied
as antibacterial agents in therapeutic protocols.3,4 In view of their
potent antimicrobial properties and low human toxicity, metallic
silver or silver complexes have been used in a variety of applica-
tions such as water purification, wound management, eye-drops,
Received: August 6, 2011
Published: October 14, 2011
r
2011 American Chemical Society
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Inorganic Chemistry
ARTICLE
hence being suitable ligands for the synthesis of structural models
for N,N,O-binding in metalloenzymes.30a The new silver com-
plexes here reported were obtained from reaction of AgBF4 with
Tpms, also in the presence of some monodentate phosphanes
(triphenylphosphane, PPh3, tricyclohexylphosphane, PCy3, and
the water-soluble 1,3,5-triaza-7-phosphaadamantane, PTA).30
Their antimicrobial activity is also reported. In addition, since
the anticancer activity of silver complexes was recently demo-
nstrated,31ꢀ34 we have also investigated their antiproliferative
activity against human malignant melanoma cells.
Figure 1. Lithium derivative of Tpms ligand.
proteins plays an essential role in its antimicrobial action,
although other cellular contributions, like hydrogen bonding,
may also be involved. In addition to their effects on bacterial
enzymes, silver ions cause marked inhibition of bacterial growth.
They deposit in the vacuole and cell wall as granules, inhibit cell
division and damage the cell envelope and contents of bacteria.16
Bacterial cells increase in size, and the cytoplasmic membrane,
cytoplasmic contents, and outer cell layers exhibit structural
abnormalities. An interaction of silver ions with the bases of
nucleic acids has been also described,17 although the implication
of this in terms of their bactericidal activity is still questioned.
Many silver complexes are sparingly soluble in common
solvents, especially water, which limits their uses in oil treatments
and creams, as for the most used topical antibacterial agent
silversulfadiazine (SSD).18 Moreover, since antimicrobial resis-
tance is growing at a frightening rate (microorganisms such as
methicillin-resistant Staphylococcus aureus, staphylococci with
decreased susceptibility to vancomycin, vancomycin-resistant
enterococci, multidrug resistant Gram-negatives, as well as
Streptococcus pneumoniae with decreased susceptibility to peni-
cillin and other antibacterials are frequently isolated in both
hospital and community settings), there is a growing need for
novel agents or an improvement of the existing ones to combat
resistant organisms.
’ EXPERIMENTAL SECTION
Materials and Physical Measurements. All chemicals were
purchased from Aldrich (Milwaukee) and used as received. Lithium
derivative of the scorpionate Tpms ligand was synthesized as previously
reported.29a All of the reactions and manipulations were performed in
the air. Solvent evaporations were always carried out under vacuum
conditions using a rotary evaporator. The samples for microanalyses
were dried in vacuo to constant weight (20 ꢀC, ca. 0.1 Torr). Elemental
analyses (C, H, N, S) were performed in-house with a Fisons Instru-
ments 1108 CHNS-O Elemental Analyzer. IR spectra were recorded
from 4000 to 400 cmꢀ1 with a Perkin-Elmer Spectrum 100 FT-IR
instrument. 1H, 31P{1H}, and 13C{1H} NMR spectra were recorded on
a 400 Mercury Plus Varian instrument operating at room temperature
(400 MHz for 1H, 162.1 MHz for 31P, and 100 MHz for 13C). H and C
chemical shifts (δ) are reported in parts per million (ppm) from SiMe4
(1H and 13C calibration by internal deuterium solvent lock) while P
chemical shifts (δ) are reported in ppm versus 85% H3PO4. Melting
points are uncorrected and were taken on an STMP3 Stuart scientific
instrument and on a capillary apparatus. The electrical conductivity
measurements (ΛM, reported as S cm2 molꢀ1) of dimethylsulfoxide
(DMSO), acetone, and water solutions of the silver derivatives were
taken with a Crison CDTM 522 conductimeter at room temperature
(r.t.). The positive and negative electrospray mass spectra were obtained
with a Series 1100 MSI detector HP spectrometer, using an acetonitrile
mobile phase. Solutions (3 mg/mL) for electrospray ionization mass
spectrometry (ESI-MS) were prepared using reagent-grade acetonitrile.
For the ESI-MS data, mass and intensities were compared to those
calculated using IsoPro Isotopic Abundance Simulator, version 3.1.35
Peaks containing silver(I) ions were identified as the center of an isotopic
cluster.
Silver complexes are also particularly interesting since the
antimicrobial activity and other desirable properties can be tuned
by varying the number and type of ligands. For example, the
silver(I) imidazolate complex has excellent antibacterial and
antifungal properties,19 while its PPh3 adduct essentially does
not show antimicrobial activity.20 Silver(I) sulfadiazine, a poly-
meric complex releasing silver ions slowly, is one of the most
popular silver based antimicrobial agents for treating infections
on severe burn sites.11,12,21ꢀ24
Synthesis of the Silver Compounds 1ꢀ5. [Ag(Tpms)] (1). A
solution of Li(Tpms) (0.077 g, 0.26 mmol) in methanol (10 mL) was
added dropwise to a solution of AgBF4 (0.050 g, 0.26 mmol) in
methanol (10 mL). After 6 h stirring at r.t., the suspension was filtered
off, and the grayish-rose precipitate of 1 was dried under reduced
pressure. 1 is soluble in DMSO and poorly soluble in acetonitrile. Yield
0.083 g, 80%. Mp 310 ꢀC (dec). Anal. Calcd. for C10H9AgN6O3S: C,
29.94; H, 2.26; N, 20.95; S, 7.99. Found: C, 29.65; H, 2.29; N, 20.64; S,
7.71%. ΛM (DMSO, 298 K, 10ꢀ3 mol/L) 1.3 S cm2 molꢀ1. IR
(KBr, cmꢀ1): 3145w, 3133w, 3108w ν(CaromꢀH), 1522 m ν(CdN),
1427 m, 1396 m, 1325 m, 1268vs, 1259s, 1214w, 1199w, 1104 m, 1097
m, 1061 m, 1056 m, 1046s ν(SO3), 1104s, 1096 ν(ring), 866 m, 857s,
Following a recent series of studies25ꢀ28 on complexes formed
upon reactions of silver(I) oxyanions salts with (mostly) uni- and
bidentate ligands based on pyridine and azoles, we now report
the synthesis and bioactivity of new silver(I) derivatives with a
different type of ligand, of the family of anionic scorpionates, that
is, tris(pyrazol-1-yl)methanesulfonate (Tpms). This has recently
been shown to possess interesting structural and electronic
features with respect to classical tris(pyrazol-1-yl)borate (Tp).29,30
Tpms (Figure 1) bears a methanesulfonate group at the place of
the BꢀH moiety in Tp, which confers good stability toward
hydrolysis and increased solubility in polar solvents, such as water
or methanol.
1
847s ν(CꢀN), 635s ν(CꢀS). H NMR (CD3CN, 298 K): δ, 6.40dd
1
(3H, 4-H (pz)), 7.54d (3H, 3,5-H (pz)), 8.20d (3H, 3,5-H (pz)). H
NMR (DMSO-d6, 298 K): δ, 6.36dd (3H, 4-H (pz)), 7.46d (3H, 3,5-H
(pz)), 8.11d (3H, 3,5-H (pz)). 13C NMR (DMSO-d6, 298 K): δ, 95.41s
(CSO3) 106.53s (C4pz), 132.84s (C5pz), 140.34s (C3pz). ESI+-MS
(CH3CN) m/z: 149 [Ag(CH3CN)]+, 190 [Ag(CH3CN)2]+, 550
[Ag2(Tpms)(CH3CN)]+, 591 [Ag2(Tpms)(CH3CN)2]+. ESIꢀ-MS
Tpms and analogous species having different substituents in
the 3-position of the pyrazole rings exhibit a marked coordina-
tion versatility, acting either as a tripodal or a bipodal ligand (i.e.,
with N3-, N2O-, N2-, or NO- coordination modes) with the
possibility of involving the sulfonate moiety in the coordination,
(CH3CN) m/z:
=
293 [Tpms]ꢀ, 694 [Ag(Tpms)2]ꢀ, 1096
[(Ag)2(Tpms)3]ꢀ.
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[Ag(Tpms)(PPh3)] (2). AgBF4 (0.050 g, 0.26 mmol) was dissolved in
methanol(20 mL) and thesolution stirredfor 30 min, then PPh3 (0.067 g,
0.26 mmol) and Li(Tpms) (0.077 g, 0.26 mmol) were added. After 24 h
stirring at r.t. the precipitate was removed by filtration. The solution was
evaporated to dryness, and the solid dissolved in diethyl ether (5 mL).
Slow evaporation afforded a pale-rose powder of 2, which was dried
under reduced pressure. 2 is very soluble in alcohols, chlorinated
solvents, acetonitrile, acetone, and DMSO. Yield 0.103 g, 60%. Mp
185ꢀ186 ꢀC. Anal. Calcd. for C28H24AgN6O3PS: C, 50.69; H, 3.65; N,
12.67; S, 4.83. Found: C, 50.21; H, 3.67; N, 12.33; S, 4.32%. ΛM
(CH3CN, 298 K, 10ꢀ3 mol/L) 4.7 S cm2 molꢀ1. IR (KBr, cmꢀ1):
3156w, 3114w, 3125w, 3056w ν(CaromꢀH), 1522 m ν(CdN), 1480 m,
1435 m, 1414 m, 1381 m, 1330 m, 1313 m, 1272s, 1260vs, 1245vs, 1200
m, 1051vs, 1041s ν(SO3), 1096vs ν(ring), 857 m, 846 m ν(CꢀN), 628s
ν(CꢀS), 524s, 505s ν(PPh3). 1H NMR (CDCl3, 298 K): δ, 6.38dd (3H,
4-H (pz)), 7.25ꢀ7.45 m br (15H, PꢀC6H5), 7.62d (3H, 3,5-H (pz)),
7.98d (3H, 3,5-H (pz)). 1H NMR (CDCl3, 253 K): δ, 6.36dd (3H, 4-H
(pz)), 7.26ꢀ7.52 m br (15H, PꢀC6H5), 7.74d (3H, 3,5-H (pz)), 8.01d
(3H, 3,5-H (pz)). 1H NMR (CDCl3, 223 K): δ, 6.37br (3H, 4-H (pz)),
7.30ꢀ7.55 m br (15H, PꢀC6H5), 7.75br (3H, 3,5-H (pz)), 8.01br (3H,
3,5-H (pz)), 8.01d (3H, 3,5-H (pz)). 13C NMR (acetone-d6, 298 K): δ,
27.0d, 27.8d, 32.0d, 32.5d (Ccy), 107.5s (C4pz), 134.9 (C5pz), 142.6s
(C3pz). 31P{1H} NMR (CDCl3, 298 K): δ, 42.6dd (1J(31P-109Ag): 753
Hz); (1J(31P-107Ag): 653 Hz). 31P{1H} NMR (Acetone-d6, 298 K): δ,
44.2dd (1J(31P-109Ag): 748 Hz); (1J(31P-107Ag): 648 Hz). 19F{1H}
NMR (DMSO-d6, 298 K): δ, ꢀ148.7. 19F{1H} NMR (D2O, 298 K):
δ, ꢀ150.1. ESI+-MS (CH3CN) m/z (%) = 190 [Ag(CH3CN)2]+,
429 [Ag(PCy3)(CH3CN)]+, 591 [(Ag)2(Tpms)(CH3CN)2]+, 687
[Ag(PCy3)2(H2O)]+, 1070 [(Ag)2(Tpms)(PCy3)2]+.
[Ag(PTA)(MeOH)][BF4] (4). AgBF4 (0.050 g, 0.26 mmol) was
dissolved in methanol (20 mL), and the solution stirred for 30 min,
then PTA (0.041 g, 0.26 mmol) was added. The resulting suspension
was stirred at r.t. for 1 h, then the solid was filtered off, washed with
methanol and dried under vacuum giving a grayish powder of 4. It is
soluble in water, acetonitrile, acetone, and DMSO. Yield 0.089 g, 90%.
Mp 208 ꢀC (dec.). Anal. Calcd. for C7H16AgBF4N3OP: C, 21.90; H,
4.20; N, 10.95. Found: C, 21.61; H, 4.23; N, 11.26%. ΛM (H2O, 298 K,
10ꢀ3 mol/L) 98.7 S cm2 molꢀ1. IR (KBr, cmꢀ1): 3553 m ν(OꢀH),
3157w ν(CꢀH), 1674 m δ(OꢀH), 1288 m, 1234s, 950vs ν(PTA),
1052vs, 1035vs, 1024vs, 1004s ν(BF4). 1H NMR (D2O, 298 K): δ, 4.27s
(6H, PCH2N, PTA), 4.65d and 4.77d (6H, JAB = 13 Hz, NCHAHBN,
PTA). 31P{1H} NMR (D2O, 298 K): δ, ꢀ77.9vbr. 31P{1H} NMR
(D2O, 278 K): δ, ꢀ78.6s br. ESI+-MS (H2O) m/z: 283
[Ag(PTA)(H2O)]+, 422 [Ag(PTA)2]+. ESI+-MS (CH3CN) m/z: 306
[Ag(PTA)(CH3CN)]+ 422 [Ag(PTA)2]+.
1
3,5-H (pz)). H NMR (CD3OD, 298 K): δ, 6.47dd (3H, 4-H (pz)),
7.44ꢀ7.52 m br (15H, PꢀC6H5), 7.66d (3H, 3,5-H (pz)), 8.08d (3H,
1
3,5-H (pz)). H NMR (acetone-d6, 298 K): δ, 6.46t (3H, 4-H (pz)),
7.50ꢀ7.65 m br (15H, PꢀC6H5), 7.75d (3H, 3,5-H (pz)), 8.24d (3H,
3,5-H (pz)). 1H NMR (acetone-d6, 203 K): δ, 6.46br (3H, 4-H (pz)),
7.40ꢀ7.50 m br, 7.60ꢀ7.65 m br (15H, PꢀC6H5), 7.95br (3H, 3,5-H
(pz)), 8.22br (3H, 3,5-H (pz)). 1H NMR (acetone-d6, 183 K): δ, 6.45br,
6.60br (3H, 4-H (pz)), 7.40ꢀ7.50 m br, 7.60ꢀ7.65 m br (15H,
PꢀC6H5), 8.00br, 8.22br (3H, 3,5-H (pz)), 8.50br, 8.59br (3H, 3,5-H
[Ag(Tpms)(PTA)] (5). To a solution of AgBF4 (0.050 g, 0.26 mmol) in
methanol (20 mL) was added Li(Tpms) (0.077 g, 0.26 mmol) and after
30 min, PTA (0.036 g, 0.25 mmol). The resulting suspension was stirred
at r.t. for 3 h. The solid was filtered off, washed with several small
portions of methanol and dried under vacuum giving a grayish-brown
powder of 5. It can also be obtained by using water as solvent. It is soluble
in DMSO and water, slightly soluble in acetonitrile and acetone. Yield
(pz)). 13C NMR (acetone-d6, 298 K): δ, 107.6s (C4pz), 130.3d (Carom
,
11.0 Hz), 132.0d (Carom, 1.6 Hz), 132.2d br (Cipso) 134.7d (Carom, 17.0
Hz) 135.2 (C5pz), 143.1s (C3pz). 31P{1H} NMR (CDCl3, 298 K): δ,
16.5dbr (1J(31P-109/107Ag): 696 Hz). 31P{1H}NMR (acetone-d6, 298
K): δ, 16.9dbr (1J(31P-109/107Ag): 688 Hz). 31P{1H} NMR (acetone-d6,
0.074 g, 50%. Mp 216ꢀ218 ꢀC. Anal. Calcd. for C16H21AgN9O3PS
3
1
263 K): δ, 16.4dd (1J(31P-109Ag): 775 Hz; J(31P-107Ag): 674 Hz).
MeOH: C, 34.59; H, 4.27; N, 21.35; S, 5.43. Found: C, 34.33; H, 4.12; N,
21.08; S, 5.72%. ΛM (DMSO, 298 K, 10ꢀ3 mol/L) 1.1 S cm2 molꢀ1. IR
(KBr, cmꢀ1): 3610w, 3545w ν(OꢀH), 3146w ν(CaromꢀH), 1640w
δ(OꢀH), 1522 m ν(CdN), 1426 m, 1392 m, 1324 m, 1283 m, 1241sh,
1227vs, 1052vs ν(SO3), 1095s ν(ring), 1285 m, 1270 m, 957s, 949s
ν(PTA), 861s, 848 m ν(CꢀN). 1H NMR (DMSO-d6, 298 K): δ, 4.23s
(6H, PCH2N, PTA), 4.42d and 4.59d (6H, JAB = 15 Hz, NCHAHBN,
PTA), 6.46s br (3H, 4-H (pz)), 7.64s br (3H, 3,5-H (pz)), 8.08s br (3H,
3,5-H (pz)). 1H NMR (CD3CN, 298 K): δ, 4.24d (6H, PCH2N, PTA),
4.50d and 4.60d (6H, NCHAHBN,PTA), 6.49dd (3H, 4-H (pz)), 7.71d
(3H, 3,5-H (pz)), 8.12d (3H, 3,5-H (pz)). 13C NMR (CD3CN, 298 K):
δ, 51.46d (CPTA), 73.66 (CPTA), 107.96 (C4pz), 134.96s (C5pz), 143.16
(C3pz).31P{1H} NMR (DMSO-d6, 298 K): δ, ꢀ84.4s br. ESI+-MS
(CH3CN) m/z: 190 [Ag(CH3CN)2]+, 591 [(Ag)2(Tpms)(CH3CN)2]+,
707 [(Ag)2(Tpms)(CH3CN)(PTA)]+, 823 [(Ag)2(Tpms)(PTA)2]+.
X-ray Crystallography of Compounds 2, 3, and 5. X-ray
Crystal Structure Determinations. The X-ray diffraction data of 2, 3, and
5 were collected using a Bruker AXS-KAPPA APEX II diffractometer
with graphite monochromated MoꢀKα radiation. Data were collected
using ω scans of 0.5ꢀ per frame, and a full sphere of data was obtained.
Cell parameters were retrieved using Bruker SMART software and
refined using Bruker SAINT36 on all the observed reflections. Absorp-
tion corrections were applied using SADABS.36 Structures were solved
by direct methods using the SHELXSꢀ97 package37a and refined with
SHELXL-97.37b Calculations were performed with the WinGX System-
Version 1.80.03.38 All hydrogens were inserted in calculated positions.
Least square refinements with anisotropic thermal motion parameters
for all the nonꢀhydrogen atoms and isotropic for the remaining atoms
were employed. There are disordered solvent molecules in the structure
of complex 5; PLATON/SQUEEZE39 was used to correct the data.
31P{1H} NMR (acetone-d6, 233 K): δ, 15.9dd (1J(31P-109Ag): 776 Hz;
1J(31P-107Ag): 672 Hz). 31P{1H} NMR (acetone-d6, 203 K): δ, 15.4ddbr
(1J(31P-109Ag): 737 Hz; 1J(31P-107Ag): 638 Hz). 31P{1H} NMR
(acetone-d6, 183 K): δ, 15.8ddbr (1J(31P-109Ag): 745 Hz; 1J(31P-107Ag):
650 Hz), 15.1ddbr (1J(31P-109Ag): 786 Hz; 1J(31P-107Ag): 686 Hz),
13.1ddbr (1J(31P-109Ag): 713 Hz; 1J(31P-107Ag): 616 Hz). ESI+-MS
(CH3OH) m/z: 632 [Ag(PPh3)2]+, 686 [Ag(Tpms)(PPh3)Na]+, 1034
[(Ag)2(Tpms)(PPh3)2]+.
[Ag(Tpms)(PCy3)] (3). AgBF4 (0.050 g, 0.26 mmol) was dissolved
in methanol (20 mL) and stirred for 30 min, then PCy3 (0.072 g,
0.26 mmol) and Li(Tpms) (0.077 g, 0.26 mmol) were added. After 24 h
stirring at r.t. the precipitate (LiBF4) was removed by filtration. Slow
evaporation of the solution afforded a brown crystalline solid of 3 which
was dried under reduced pressure. Compound 3 is very soluble in
alcohols, chlorinated solvents, acetonitrile, acetone, and DMSO. Yield
0.141 g, 80%. Mp 315 ꢀC (dec). Anal. Calcd. for C28H42AgN6O3PS: C,
49.34; H, 6.21; N, 12.33; S, 4.70. Found: C, 49.37; H, 6.62; N, 12.20; S,
5.16%. ΛM (CH3CN, 298 K, 10ꢀ3 mol/L) 2.9 S cm2 molꢀ1. IR
(KBr, cmꢀ1): 3162w, 3124w, 3109w ν(CaromꢀH), 2930 m, 2854 m,
1522s ν(CdN), 1445 m, 1430 m, 1398 m, 1320 m, 1267vs, 1248vs, 1200
m, 1045vs ν(SO3), 1096s ν(ring), 857s, 843s ν(CꢀN), 625s, 612
ν(CꢀS), 515s, 473 m, 459 m, 407 m ν(PCy3). 1H NMR (CDCl3, 298
K): δ, 1.25ꢀ1.84 m br (33H, P-(C6H5)3) 6.42dd (3H, 4-H (pz)), 7.64d
(3H, 3,5-H (pz)), 8.01d (3H, 3,5-H (pz)). 1H NMR (CDCl3, 273 K): δ,
1.25ꢀ1.84 m br (33H, P-(C6H5)3) 6.42dd (3H, 4-H (pz)), 7.64d (3H,
1
3,5-H (pz)), 8.01d (3H, 3,5-H (pz)). H NMR (CDCl3, 253 K): δ,
1.25ꢀ1.84 m br (33H, P-(C6H5)3) 6.42dd (3H, 4-H (pz)), 7.64d (3H,
1
3,5-H (pz)), 8.01d (3H, 3,5-H (pz)). H NMR (CDCl3, 233 K): δ,
1.25ꢀ1.84 m br (33H, P-(C6H5)3) 6.42dd (3H, 4-H (pz)), 7.64d (3H,
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Crystallographic data have been deposited at the CCDC and
allocated the deposition numbers CCDC 837589ꢀ837591.
Scheme 1
Antimicrobial Activity. Bacterial Strains. Silver derivatives were
tested against a panel of microorganisms including Staphylococcus aureus
ATCC 25923, Escherichia coli ATCC 25922, Pseudomonas aeruginosa
ATCC 27853, Streptococcus pneumoniae ATCC 49619, Enterococcus
faecalis ATCC 29212, Streptococcus pyogenes SF370, Streptococcus sangui-
nis SK36, Streptococcus mutans UA159, Candida albicans ATCC 24433.
Bacterial strains were cultured overnight at 37 ꢀC (Streptococcus spp in
5% CO2 atmosphere) in blood agar plates with the exception of Candida
that was grown in RPMI1640.
Disc Diffusion Method. Antibiotic susceptibility testing was per-
formed by the paper disk diffusion method.40 Growth media used for
the antimicrobial assay were as indicated by the international guidelines
of the CLSI.41 Briefly, a suspension of 108 cells per mL prepared in saline
(106 per mL for Candida) was spread on the solid media plates using a
sterile cotton swab. Sterile filter paper discs (6 mm in diameter) were
placed on the surface of inoculated plates and spotted with 20 μL of each
silver derivative with a concentration of 1 mg/mL (0.1%). Each silver
derivative was dissolved in DMSO. The plates were incubated for 18 h at
35 ꢀC (Streptococcus spp in 5% CO2 atmosphere). The diameters of zone
inhibition (including the 6 mm disk) were measured with callipers. A
reading of more than 6 mm indicated growth inhibition. No zone
inhibition was observed using DMSO alone. Silver nitrate was used for
comparison.
DNA Binding Study by Fluorescence Spectroscopy. UVꢀvis absor-
bance values were measured on a Varian cary-1 spectrophotometer in
10 mM phosphate buffer (pH 7.0) containing 50 mM NaCl at r.t.
(25 ꢀC). A stock solution of ct-DNA was prepared by dissolving the solid
material in the same phosphate buffer. The concentration of DNA was
determined by UV absorbance at 260 nm using the molar absorption
coefficient ε = 260 (6600 Mꢀ1 cmꢀ1).42 The competitive binding
experiment was carried out by maintaining the EB and ct-DNA
concentration at 5 μM and 55.7 μM, respectively, while increasing the
concentration of the Ag complex (Ag-X). The fluorescence spectra of a
series of solutions with various concentrations of the silver derivative and
a constant EB-c-DNA complex were measured. All the fluorescence data
are corrected for absorption of exciting and emitted light according to
the relationship (eq 1):17
derivatives 1ꢀ5 ranging from 0.01 μM to 100 μM and incubated for
72 h. The cell viability was assessed through an MTT (3-(4,5-dimethyl-
thiozol-2-yl)-2,5-diphenyl-tetrazolium bromide; Sigma, St. Louis, MO)
conversion assay as described.13,44 The optical density of each sample
was measured with a microplate spectrophotometer reader Titertek
Multiscan microElisa (Labsystems, Helsinki, Finland) at 540 nm. Cyto-
toxicity is expressed as the concentration of compound inhibiting cell
growth by 50% (IC50). The IC50 values were determined using the
GraphPad Prism 4 computer program (GraphPad Software, San Diego,
CA, U.S.A.). AgNO3 was used as comparison compound.
’ RESULTS AND DISCUSSION
Synthesis and Spectral Characterization of the Silver
Complexes 1ꢀ5. The complex [Ag(Tpms)] (1) has been
synthesized by reaction of the lithium salt of tris(pyrazol-1-yl)-
methanesulfonate, Li(Tpms), with AgBF4 in methanol, at r.t.. 1 is
a grayish-rose solid, stable in air, poorly soluble in DMSO and
acetonitrile, suggesting an oligomeric or a polymeric structure,
where SO3 and pyrazolyl rings of Tpms could be involved in
coordination to silver atoms, a feature previously observed in
binary M(Tpms) derivatives.30a,d,45 1 is a nonelectrolyte in
DMSO, and the existence of silverꢀTpms interactions in this
solvent is also confirmed by proton NMR spectroscopy. The
presence of a unique set of resonances for the H atoms of Tpms
in the 1H NMR of 1 in DMSO-d6 suggests a N3-coordination to
silver, but we cannot exclude a dissociation of the polymeric solid
1 upon dissolution in polar DMSO, which can cause breaking of
Fc ¼ Fm ꢁ eðA1 þ A2Þ=2
ð1Þ
where Fc and Fm are the corrected and measured fluorescence, respec-
tively. A1 and A2 are the values of absorbance of silver derivatives at the
exciting and emission wavelengths. Fluorescence quenching spectra
were recorded using an ISS-Greg 200 spectrofluorophotometer with
an excitation wavelength of 500 nm and emission spectrum at 520ꢀ
700 nm. For fluorescence quenching experiments, the SternꢀVolmer’s
eq 2 was used:43
SO3 Ag interactions eventually present in the solid com-
3 3 3
pound. The IR spectrum of 1 displays the expected ν(CdN +
CdC) of the pyrazolyl rings at 1522 cmꢀ1, together with several
bands likely due to the SO3 group vibration modes at 1268,
F0=F ¼ 1 þ kQ τ0½Ag-Xꢂ ¼ 1 þ KSV½Ag-Xꢂ
ð2Þ
1251, 1199, 1061, 1056, and 1046 cmꢀ1 45
Additionally, three
.
where F0 and F represent the fluorescence intensity in the absence and in
the presence of drug. [Ag-X] is the concentration of the silver derivative
and KSV is the SternꢀVolmer constant which is equal to kQ ꢁ τ0, where
kQ is the bimolecular quenching rate constant and τ0 is the average
fluorescence lifetime of the fluorophore in the absence of drug.
Antiproliferative Activity. MTT Cytotoxicity Assay. A375 cells
(human malignant melanoma) were cultured in Dulbecco’s Modified
Eagle’s Medium (DMEM) with 2 mM L-glutamine, 100 IU/mL
penicillin, 100 μg/mL streptomycin, and supplemented with 10% heat
inactivated fetal bovine serum (HI-FBS). Cells were cultured in a
humidified atmosphere at 37 ꢀC in presence of 5% CO2. Briefly, cells
were seeded at the initial density of 2 ꢁ 104 cells/mL in 96-well
microtiter plates (Iwaki, Tokyo, Japan). After incubation for 24 h at
37 ꢀC, cells were treated with different concentrations of silver
absorptions at 866, 857, and 847 cmꢀ1 are assigned to ν(CꢀN),
the former at 866 cmꢀ1 having about half the intensity of the
latter ones, likely indicating the presence of both coordinated and
uncoordinated pyrazolyl rings, as recently proposed by Kl€aui
et al.:45 their “IR criterion”45 for the assignment of the coordina-
tion mode of a similar TpmstBu ligand (TpmstBu = tris(3-tert-
butylpyrazol-1-yl)methanesulfonate) is in fact based on the
presence of a ν(CꢀN) band of coordinated pyrazolyl rings at
about 855 cmꢀ1 for N3-coordinated TpmsR ligands, whereas a
band at 865 cmꢀ1 should also be observed in the presence of
uncoordinated pyrazolyl ring, as it is the case of N2O-coordi-
nation. The positive ESI-MS spectrum of 1 in acetonitrile
shows isotopic clusters centered at m/z 149 and 190 due to
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Scheme 2
16.9 ppm (1J(31P-Ag) = 688 Hz) is detected, while on cooling at
ꢀ10 ꢀC a well resolved double doublet at 16.4 ppm arises
(1J(31P-109Ag): = 775 Hz; 1J(31P-107Ag) = 674 Hz). At
ꢀ70 ꢀC it shifts to 15.4 ppm (with a lowering of the silverꢀ
Scheme 3
phosphorus coupling constants: J(31P-109Ag) = 737 Hz; J-
(31P-107Ag) = 638 Hz) and broadens. Finally, at ꢀ90 ꢀC three
broad double doublets are observed at δ 15.8 (1J(31P-109Ag) =
745 Hz; 1J(31P-107Ag) = 650 Hz), 15.1 (1J(31P-109Ag) = 786 Hz;
1J(31P-107Ag) = 686 Hz) and 13.1 (1J(31P-109Ag) = 713 Hz;
1J(31P-107Ag) = 616 Hz), the latter with half the intensity of the
former ones. This behavior supports the existence of at least three
silverꢀphosphorus species in solution such as those depicted in
the Scheme 2, the Tpms likely acting as N3-, N2O-, or N2-donor.
The positive ESI-MS spectrum of 2 in acetonitrile is char-
acterized by clusters centered at m/z 632, 686, and 1034 due to
[Ag(PPh3)2]+, [Ag(Tpms)(PPh3)Na]+, and [(Ag)2(Tpms)
(PPh3)2]+ species, respectively. The positive ESI-MS of 3 in
acetonitrile shows peaks at 190 ([Ag(CH3CN)2]+), 429
([Ag(PCy3)(CH3CN)]+), 591 ([(Ag)2(Tpms)(CH3CN)2]+),
687 ([Ag(PCy3)2(H2O)]+), and 1070 [(Ag)2(Tpms)(PCy3)2]+.
By treatment of a MeOH solution of AgBF4 with PTA at r.t.,
[Ag(PTA)(MeOH)][BF4] (4) is obtained, as a high melting
brown powdered solid, soluble in water, acetonitrile, acetone,
and DMSO. The conductivity measurement of a water solution
of 4 exhibits a ΛM value of 98.7 S cm2 molꢀ1, close to those
reported for 1:1 electrolytes in water (118ꢀ131 S cm2 molꢀ1)47,
in accordance with an extensive dissociation. The IR spectrum of
4 confirms the presence of both the coordinated PTA ligand30b
1
1
[Ag(MeCN)]+ and [Ag(MeCN)2]+, respectively, together with
peaks at m/z 550 and 591, due to [(Ag)2(Tpms)(MeCN)]+ and
[(Ag)2(Tpms)(MeCN)2]+, while in the negative ESI-MS spec-
trum the peaks at m/z 694 and 1096 are assigned to
[Ag(Tpms)2]ꢀ and [(Ag)2(Tpms)3]ꢀ, in accordance with the
presence in solution of mononuclear silver species but also of
aggregates containing two silver centers.
Complexes [Ag(Tpms)(PPh3)] (2) and [Ag(Tpms)(PCy3)]
(3) have been prepared in methanol at r.t. by reaction of
Li(Tpms) with AgBF4 in the presence of an equivalent amount
of the appropriate phosphane (Scheme 1).
They are air-stable solids, well-soluble in water, acetonitrile,
acetone, DMSO, alcohols, and chlorinated solvents. In the IR
spectra of 2 and 3, ν(CdC) and ν(CdN) of the pyrazolyl rings
are observed in the range 1624ꢀ1521 cmꢀ1, whereas ν(SO),
ν(pz), ν(CꢀN), and ν(CꢀS) are detected in the range
1300ꢀ600 cmꢀ1 45
.
Strong bands between 550 and 400 cmꢀ1
confirm the presence of coordinated PPh3 and PCy3.46
The r.t. 1H NMR spectra of 2 and 3 show the pyrazolyl and
phosphane proton resonances always deshielded with respect to
those in the free ligands. The presence of a unique set of
resonances for each group could be in favor of a N3-coordination
of Tpms, but, although this is in accordance with the solid state
molecular structure of 3, it contrasts with the structure of 2
containing instead a N2O-bonded Tpms ligand (see below X-ray
diffraction studies). On the basis of this evidence, we have
decided to run 1H NMR spectra in deutero-acetone and CD2Cl2
at low temperatures, and we have observed for 2 a progressive
broadening of the H resonances of Tmps and splitting into two
sets with integral ratios 2:1 below ꢀ70 ꢀC, in accordance with a
fluxionality operating in solution between silver species contain-
ing N3- and N2O-bonded Tpms at r.t.. This behavior has been
recently observed also for similar copper(I) Tpms phosphane-
compounds.30b However, for our silver complexes the fluxion-
ality persists well below the temperature of ꢀ70 ꢀC for which two
quite well-resolved sets of resonances were observed in analo-
gous copper(I) derivatives. The r.t. 31P{1H} NMR spectrum in
CDCl3 of 2 exhibits a doublet at δ 16.5, with 1J(31PꢀAg) = 696
Hz, while for 3 a well resolved double doublet at δ 42.6, with
1J(31P-109Ag) = 753 Hz and 1J(31P-107Ag) = 653 Hz, is observed.
The variable temperature 31P{1H} NMR study of our acetone-d6
solution of 2 was also carried out: at r.t. a broad doublet at
and the [BF4]ꢀ group.48a In detail, the band at about 1050 cmꢀ1
,
because of this counterion, displays a fine splitting, indicative of
its involvement in interactions with Ag through F atoms. We can
hypothesize a polymeric structure for 4 where both [BF4]ꢀ and
PTA could play the role of bridging ligands.30k,l,49 The 1H NMR
spectrum of 4 in D2O shows two types of methylene protons,
typical of the PTA ligand. One of them, assigned to the
PꢀCH2ꢀN moiety, occurs as a singlet at δ 4.27 whereas the
other one, corresponding to the NꢀCH2ꢀN group, displays an
AB spin system centered at δ 4.73 (JAB = 13 Hz), attributed to the
NꢀCHaxꢀN and NꢀCHeqꢀN protons.30b The 31P{1H} NMR
spectrum exhibits a broad singlet at δ ꢀ77.9, the 107Agꢀ P and
31
109Agꢀ P spinꢀspin coupling not being observed because of
31
the fast ligand exchange.30k,50 The ESI-MS spectra of 4 carried
out in water and acetonitrile show peaks due to silver species
always containing the PTA ligand. The ionic dissociation of 4 in
ionizing solvents is further confirmed by the 19F NMR spectrum
of 4 in D2O and DMSO, typical of a ionic BF4 group.48b
Finally, by reaction of a methanol solution of AgBF4 with
Li(Tpms) and PTA at r.t., the derivative [Ag(PTA)(Tpms)] (5)
has been isolated (Scheme 3). Because of stronger coordination
ability of PTA to Ag(I) in comparison with Tpms, to prevent
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Figure 2. Ortep diagram of [Ag(Tpms)(PPh3)] (2), drawn at 50% probability. Hydrogen atoms and chloroform molecule were ommitted for clarity.
Selected bond distances (Å) and angles (deg): P1ꢀAg1 2.3286(10), P2ꢀAg2 2.3259(12), O11ꢀAg1 2.551(3), O21ꢀAg2 2.513(3), N11ꢀAg1
2.259(4), N21ꢀAg1 2.307(4), N41ꢀAg2 2.307(4), N51ꢀAg2 2.282(4), N11ꢀN12 1.367(4), N21ꢀN22 1.360(4), N31ꢀN32 1.356(5), N51ꢀN52
1.350(5); N11ꢀAg1ꢀN21 79.41(13), N11ꢀAg1ꢀP1 147.14(10), N21ꢀAg1ꢀP1 130.53(10), N11ꢀAg1ꢀO11 78.31(12), N21ꢀAg1ꢀO11
74.68(12), P1ꢀAg1ꢀO11 118.52(7), N51ꢀAg2ꢀN41 78.14(14), N51ꢀAg2ꢀP2 141.36(10), N41ꢀAg2ꢀP2 137.60(10), N51ꢀAg2ꢀO21
78.56(12), N41ꢀAg2ꢀO21 77.10(12), P2ꢀAg2ꢀO21 117.41(7), N31ꢀC10ꢀN12 107.8(4), N31ꢀC10ꢀN22 107.1(4), N12ꢀC10ꢀN22
112.7(4), N61ꢀC20ꢀN52 107.7(3), N61ꢀC20ꢀN42 107.2(3), N52ꢀC20ꢀN42 112.3(3).
cleavage AgꢀN(Tpms) bond, addition of PTA to the reaction
mixture should be slow, after reaction of Tpms with Ag(I) center
and in lower amount than stoichiometric, to yield 5 in high purity.
We have tried to apply the “IR criterion” of Kl€aui et al.45 also to
our Tpms derivatives with phosphanes, but it appears not to be
valid for our compounds 2, 3, and 5. In fact, in their IR spectra we
have always detected two ν(CꢀN) absorptions, in spite of the
different solid state N2O- (in 2 and 5) and N3-coordination
modes of Tpms (in 3), as shown (see below) by X-ray diffraction
studies. On the other hand, the criterion based on the number of
IR active sulfonate vibrations51 is not straightforward. Capwell
states that a “free” sulfonate (C3v symmetry) should exhibit
only two IR active vibration modes, whereas a coordinated one
(Cs symmetry) should instead exhibit at least three bands.51a In
the IR of 2, 3, and 5 at least three bands are always observed in the
The positive ESI-MS spectrum in acetonitrile of 5 shows
peaks at m/z 190 ([Ag(CH3CN)2]+), 591 ([(Ag)2(Tpms)
(CH3CN)2]+), 707 ([(Ag)2(Tpms)(CH3CN)(PTA)]+), and
823 ([(Ag)2(Tpms)(PTA)2]+), in accordance with possible
aggregation in solution and formation of dinuclear species.
X-ray Diffraction Studies of Silver Compounds 2, 3, and 5.
X-ray quality crystals of 2, 3, and 5 were obtained from chloro-
form (2, 3) or methanol (5) solutions. Ortep diagrams are shown
in Figures 2ꢀ4 with selected bond distances and angles listed in
the respective legends. Crystallographic data are presented in
Table 1. In the structure of 2 there are two molecules of the silver
complex apart from a CHCl3 solvent molecule, probably because
of conformational differences as depicted in the Supporting
Information, Figure S1. In the structures of 3 and 5 the asym-
metric unit comprises only one of the entire complex entities.
All these complexes have four-coordinate, highly distorted
tetrahedral silver atoms. The Tpms ligand coordinates to silver in
k3-N2O (2 and 5) or k3-N3 (3) fashion; the fourth coordination
positions are occupied by the P atoms of the phosphanes. Houser
et al.52 introduced a parameter (τ4) to describe the geometry
of a four-coordinate metal system which is determined by the
equation
ꢀ
ranges expected for the vibration modes of the SO3 group,
indifferently to its free or silver-coordinated nature.
The r.t. 1H NMR spectrum of 5 in DMSO-d6 similarly to 4,
shows two types of methylene protons for the coordinated PTA.
One of them, occurs as a singlet at 4.23 ppm while the second one
displays an AB spin system centered at 4.50 ppm (JAB = 15
1
Hz).30b Similarly to the other silver Tpms derivatives, the H
NMR spectrum of 5 at r.t. reveals the presence of three
equivalent pyrazolyl rings (three broad singlets at δ 8.08, 7.64,
and 6.46 corresponding to the pyrazolyl protons in positions 5, 3
and 4). The r.t. 31P{1H} NMR spectrum, equally to 4, exhibits a
τ4 ¼ ½360ꢀ ꢀ ðβ þ αÞꢂ=141ꢀ
where β and α are the largest angles involving the metal. By
means of this simple criterium, perfect square planar or tetra-
hedral geometries should have τ4 values of 0 or 1, respectively.
For our complexes such a parameter assumes values of 0.58 and
0.57 (2), 0.62 (3) or 0.53 (5), thus showing that the structures
are distorted tetrahedral. The distortions result from the
31
31
broad singlet at δ ꢀ84.4, and no 107Agꢀ P or 109Agꢀ P spinꢀ
spin coupling was observed, likely because of the fast ligand
exchange.50 This effect was previously reported for other PTA-
Ag(I) complexes.30k
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Figure 4. Ortep diagram of [Ag(Tpms)(PTA)] (5), drawn at 50%
probability. Hydrogen atoms were ommitted for clarity. Selected bond
distances (Å) and angles (deg): P1ꢀAg1 2.3431(14), O1ꢀAg1
2.607(4), N4ꢀAg1 2.477(5), N7ꢀAg1 2.256(5) N4ꢀN5 1.360(6),
N6ꢀN7 1.379(6), N8ꢀN9 1.374(6); P1ꢀAg1ꢀN4 139.49(11),
P1ꢀAg1ꢀN7 145.98(12), N4ꢀAg1ꢀN7 73.46(15), N6ꢀC10ꢀN8
108.9(4), N5ꢀC10ꢀN8 109.2(4), N56ꢀC10ꢀN6 110.7(4).
Figure 3. Ortep diagram of [Ag(Tpms)(PCy3)] (3), drawn at 50%
probability. Hydrogen atoms were ommitted for clarity. Selected bond
distances (Å) and angles (deg): P1ꢀAg1 2.3555(4), N11ꢀAg1
2.4058(14), N21ꢀAg1 2.4137(13), N31ꢀAg1 2.3848(14), N11ꢀ
N12 1.3627(19), N21ꢀN22 1.3571(18), N31ꢀN32 1.3614(18);
N11ꢀAg1ꢀN21 77.44(5), N31ꢀAg1ꢀN11 76.86(5), N31 Ag1ꢀ
N21ꢀ75.74(5), P1ꢀAg1ꢀN11 138.98(3), P1ꢀAg1ꢀN21 130.03(3),
P1ꢀAg1ꢀN31 133.28(4), N22ꢀC10ꢀN12 109.39(13), N32ꢀC10ꢀ
N12 109.80(12), N32ꢀC10 N22ꢀ109.99(12).
respectively. (Table 2). These angles would be 180ꢀ in the
absence of tilting. The consideration of the AgꢀNꢀNꢀCmethine
torsion angles, as proposed by Templeton et al.,60 is an alter-
native for measuring such distortions, and would equal 0ꢀ for an
undistorted geometry. For 3 and 5 such angles (Table 2) are
opposite in sign and considerably greater than those for 2,
therefore reinforcing the fact that the metal atom is considerably
away from the planes of the pyrazole rings in the former cases.
Moreover, in both molecules of 2 the methine carbon is away
(though very slightly) from such planes, in contrast to structures
3 and 5. The somewhat unexpected (because of the way the
ligand coordinates to the metal) higher degree of tilting of
complex 3 is shown in the Supporting Information, Figure S2.
It is in 3, however, that the PꢀAgꢀCsp3 angle is higher
(174.85ꢀ), averaging 166.12ꢀ in complex 2 and equal to
160.21ꢀ in 5. Despite those discrepancies, the NꢀCsp3ꢀN
angles, which should reflect the way the ligand binds to the
metal, are similar in all the structures and adopt values from
109.73 (in 3) to 112.47ꢀ (in 2).
The degree of tilting of the pyrazolyl rings in several metal
complexes has previously been related61 to unfavorable non-
bonding interactions resulting from either bulky substituents on
the pyrazolyl rings or from the size of the metal atoms. In our
cases, we have tentatively related several structural parameters
with the cone angles of the phosphanes61 and summarized them
in Table 2. Taking into account that the mode of coordination
of the scorpionate ligand in 2 and 5 differs from that in 3, it
appears that for the former cases (k3-NNO coordination type) a
smaller cone angle of the phosphane gives rise to smaller
chelation of the Tpms ligand, which shows bite angles in the
ranges of 74.7ꢀ79.4ꢀ (2), 75.7ꢀ77.4ꢀ(3), and 73.4ꢀ79.4ꢀ (5).
The AgꢀN bond distances adopt values of 2.259(4)ꢀ2.307(4)
Å (2), 2.3848(14)ꢀ2.4137(13) (3), and 2.256(5)ꢀ2.477(5) Å
(5). These distances compare well with those found in other
pseudo tetrahedral silver complexes, for example, with bis-
(pyrazolyl)alkane ligands such as in [Ag{RR0C(pz)2}2]X
(R = R0 = Ph; R = H, R0 = Ph; R = H, R0 = CH2Ph; X = PF6.53
R = R0 = Me, X = ClO454), with the bitopic bis(pyrazolyl)-
methane ligands as in [Ag2(μ-L)2]X2 (L = CH(pz)2(CH2)2CH-
(pz)2 or CH(pz)2(CH2)3CH(pz)2; X = BF4 or CF3SO3)55 or
even with the multitopic 1,2,4,5-C6H2[CH2OCH2CH(pz)2]4
ligand;56 in all cases, the AgꢀN bond distances are in the
2.234(2)ꢀ2.5092(19) Å range.
The PꢀAg bond distances, in the 2.3259(12)ꢀ2.3555(4) Å
range, are considerably shorter than those usually found in both
neutral57 or cationic58 silver phosphane complexes and even
shorter than that in the T-shaped three-coordinate complex
[Ag{P(C6H4CH2NMe2-2)3}(OCOCF3)].59 Moreover, the
OꢀAg bond distances, ranging from 2.513(3) to 2.607(4) Å in
our complexes, are much longer than the normal covalent
silver(I)ꢀoxygen bond length of about 2.3 Å indicating that
the sulfonate arms in 2 and 5 are only weakly bonded to
the metal.
Although the pyrazolyl rings in our structures are all nearly
planar, the silver atoms lie out of these planes [average values of
1.810 (5), 1.352 (3), and 0.417 and 0.199 Å (2), and considering
only the coordinated rings] with the AgꢀNꢀNꢀCpyr torsion
angles averaging 167.5, 141.1, and 140.2ꢀ for 2, 3, and 5,
AgNNCpz torsion angles, shorter intraligand Ncoord Ncoord
but longer metal-N distances. Therefore, the pyrazolyl rings of
,
3 3 3
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Inorganic Chemistry
Table 1. Crystallographic Data for Compounds [Ag(Tpms)(PPh3)] (2), [Ag(Tpms)(PCy3)] (3), and [Ag(Tpms)(PTA)] (5)
ARTICLE
2 CHCl3
3
5
3
empirical formula
2(C28H24AgN6O3PS), CHCl3
C28H42AgN6O3PS
C16H21AgN9O3PS
formula weight
1446.23
681.58
558.32
crystal system
orthorhombic
monoclinic
orthorhombic
space group
P212121
P21/n
Pbca
a (Å)
13.0005(5)
12.6027(3)
7.1753(7)
b (Å)
21.2408(10)
15.1631(5)
15.0485(12)
c (Å)
21.7244(9)
16.2546(5)
41.898(4)
β (deg)
90
104.472(2)
90
V (Å3)
5999.0(4)
3007.63(15)
4524.1(7)
Z
4
4
4
density (calculated) (Mg/m3)
1.601
1.505
1.639
absorption coefficient (mmꢀ1
)
0.970
0.833
1.092
F(000)
2920
1416
2256
reflections collected/unique
goodness-of-fit on F2
final R indices [I > 2σ(I)]
59812/13212 [R(int) = 0.0905]
1.000
45299/12082 [R(int) = 0.0408]
0.964
35133/4123 [R(int) = 0.0845]
1.004
R1 = 0.0451, wR2 = 0.0700
R1 = 0.0341, wR2 = 0.0745
R1 = 0.0519, wR2 = 0.1439
Table 2. Some Structural Parameters of 2, 3, and 5 and the Cone Angles of the Phosphanes
2
3
5
intraligand Ncoord
N
coord distance (Å)
av. 2.965
av. 2.979
av. 141.1
2.835
3 3 3
AgꢀNꢀNꢀCpz torsion angle (deg)
166.6, ꢀ170.1 (Ag1)
161.1, ꢀ172.1 (Ag2)
1.7, ꢀ3.6 (Ag1)
ꢀ5.3, ꢀ8.1 (Ag2)
av. 2.288
av. 140.2
AgꢀNꢀNꢀCmethine torsion angle (deg)
av. 35.97
ꢀ28.4, ꢀ44.9
AgꢀN (Å)
phosphane cone angle (deg)
av. 2.401
av. 2.367
145
170
118
Table 3. Disc Diffusion Test of Compounds 1ꢀ5 against a Panel of Reference Bacterial Strainsa
compound
S. aureus
S. pyogenes
S. pneumonae
S. sanguins
S. mutans
E. faecalis
P. aeruginosa
E. coli
C. albicans
1
11.0
6.0
12.0
6.0
11.0
6.0
12.0
6.0
11.0
6.0
10.0
6.0
15.0
6.0
11.0
6.0
12.0
6.0
2
3
12.0
14.0
15.0
11.0
14.0
15.0
16.0
13.0
13.0
15.0
16.0
13.0
13.0
14.0
16.0
11.0
14.0
14.0
16.0
12.0
9.0
15.0
17.0
17.0
14.0
11.0
13.0
15.0
11.0
15.0
17.0
20.0
14.0
4
9.0
5
10.0
11.0
AgNO3
a Diameters of zone inhibition are expressed in millimeters.
the scorpionate ligand tilted to overcome unfavorable interac-
tions from the bulky phosphane, which is further evidence of the
versatility of those ligands.
two Gram-negative species tested, our results demonstrate that
compound 4 is less active against E. coli but with a diameter of the
inhibition zone still wider than that of the comparison test
compound. The active compounds 4 and 5 significantly inhibit
the growth of the P. aeruginosa strain. The recorded diameters
were considerably wide (17 mm), even if a general higher
susceptibility toward silver is clear from the diameter of the silver
nitrate (14 mm against a mean value of 12). It is possible that
Pseudomonas is basically more susceptible to the action of the
silver ions. At last, compound 5 shows a very good activity against
C. albicans, with a mean diameter size of 20 mm.
Antibacterial Activity. The antimicrobial activities of the
compounds 1ꢀ5 against bacteria and fungi were examined
qualitatively by agar diffusion tests (Table 3). Whereas the
lithium derivative of Tpms shows no activity, the silver derivatives
possess a wide spectrum of effective antibacterial and antifungal
activities. In general, compounds bearing PTA ligand (4 and 5)
show a significantly higher antibacterial activity than silver nitrate
(P < 0.01 by the Fisher’s least significant difference procedure).
Among Gram-positive bacteria, only E. faecalis was not signifi-
cantly responding to any of the compounds. This result is not
surprising in view of the well-known general tolerance toward
many antibacterial agents that characterizes this species. For the
Fluorescence Studies. Fluorescence quenching may result
from a variety of processes such as excited state reactions, energy
transfer, ground state complex formation, and collisional pro-
cesses. Collisional or dynamic quenching refers to a process
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Inorganic Chemistry
ARTICLE
Table 5. Cytotoxic Activity (IC50) of Compounds 1ꢀ5 on
A375 Human Malignant Melanoma Cell Linea
compounds
IC50 /μM
1 (95% CI)
1.62 (0.87ꢀ2.65)
0.97 (0.69ꢀ1.37)
0.42 (0.24ꢀ0.75)
12.35 (10.32ꢀ15.24)
1.90 (0.75ꢀ3.26)
2.03 (1.22ꢀ3.04)
2 (95% CI)
3 (95% CI)
4 (95% CI)
5 (95% CI)
AgNO3 (95% CI)
a The IC50 value is the concentration of compound that affords a 50%
reduction in cell growth (after 72 h of incubation). CI = confidence
interval.
Figure 5. Fluorescence spectra of EB-DNA in the presence of com-
pound 1 at 293 K. The total concentrations of 1 are 0, 5, 10, 15, 20, 25,
30, 40, 50 μM/L for spectra (aꢀi), respectively.
the linear range (inset of Figure 6). Data for the quenching
constants for the interaction on all the studied Ag+ complexes are
reported in Table 4.
Generally, the linearity of the SternꢀVolmer plot may have
two meanings: the existence of one binding site for the ligand
in the proximity of the fluorophore, or more than one binding
site equally accessible to the ligand. In the case of fluorescence
caused only by EB-ct-DNA and assuming that the silver deri-
vative (1:1 stoichiometry) is responsible for the fluorescence
quenching of the protein, the observed change in fluorescence of
EB-ct-DNA, after binding with increasing concentrations of Ag
complexes, can be related to the following eq 3:62
ꢀ
logðF0 FÞ=F ¼ n log KA ꢀ n log f1=ð½Ag-Xꢂ
Figure 6. Plot of experimental quenching data obtained from titration
plotted following eq 3. Inset: SternꢀVolmer plot.
ꢀ ½EB-ct-DNAꢂðF0 ꢀ FÞ=F0ÞÞg
ð3Þ
where [EB-ct-DNA] and [Ag-X] are the total concentration of
EB-ct-DNA and different Ag complexes tested, respectively. On
the basis of the assumption that n is equal to 1 and where any
assumed conditions concerning free EB-ct-DNA or free/bound
drug concentrations are not required, the plot of log(F0 ꢀ F)/F
versus log(1/([Ag-X] ꢀ n[EB-ct-DNA](F0 ꢀ F)/F0))) is drawn
and fitted linearly, and then the slope n can be obtained.62
Figure 6 shows the plot obtained for the compound 1, and all
values of association constants obtained for the different com-
plexes are reported in Table 4. From the values of the KSV and KA
constants, related to the binding of the different Ag+ complexes
to DNA, we can see that compounds 2 and 3 show the higher
binding activity on DNA. In particular, the interaction of 3 results
in about a 450 fold increase of the binding constant in compar-
ison with that of AgNO3 used as control. The binding activity on
DNA follows the sequence 3 > 2 > 1 > 5 > AgNO3> 4.
Table 4. KSV and KA Values for Binding of Compounds 1ꢀ5
with ct-DNA
compound
KSV103 Mꢀ1
KA 104 Mꢀ1
n
1
7.4((0.38)
14.9((0.48)
3.4((0.15)
2.71((0.19)
3.45((0.5)
3.03((.0.1)
0.63((.06)
5.3((0.7)
0.82
0.93
0.91
0.91
0.62
0.77
2
3
70.7((1.7)
0.05((.01)
0.154((.06)
0.148((.02)
4
5
AgNO3
where the fluorophore and the quencher come into contact
during the lifetime of the excited state, whereas static quenching
concerns the formation of a fluorophoreꢀquencher complex.
The fluorescence spectra for the interaction of the silver com-
pound 1 with EB-ct-DNA at 298 K are shown in Figure 5. EB-ct-
DNA exhibits a strong fluorescence emission at 597 nm on
excitation at 500 nm. All the tested silver complexes have
negligible fluorescence under these experimental conditions.
Even so, to avoid any contribution from ligands to the fluores-
cence of EB-ct-DNA, control sets were subtracted. Addition of
silver compounds to EB-ct-DNA leads to a significant quenching
of the fluorescence intensity of EB-ct-DNA. According to the
SternꢀVolmer eq 2,43 for fluorescence quenching of EB-ct-DNA
(in Figure 5 we report the quenching because of the binding of ct-
DNA by compound 1), F0/F is plotted versus the quencher
concentration [Ag-X] (Figure 6). The SternꢀVolmer quenching
constant KSV was obtained by the slope of the regression curve in
Antineoplastic Activity. Biomedical applications of Ag+ com-
plexes are related mostly to their antibacterial action, which
appears to involve interaction with DNA. A similar interaction of
the metal with DNA may also be the cause of the antitumor
action of some of those complexes. The cytotoxic activities of the
Ag+ compounds 1ꢀ5 as 50% inhibitory concentration (IC50)
values against human malignant melanoma cells (A375) are
shown in Table 5 and compared with AgNO3. Complexes 2
and 3 tested for in vitro cytotoxic activity present pronounced
antiproliferative effects with mean IC50 values of 0.97 and
0.42 μM, respectively, being 2 and 4 times more active than
silver nitrate. The cytotoxic activities of the other complexes are
smaller than those of 2 and 3, following the sequence 3 > 2 > 1 >
5 > AgNO3. 4, in line with the binding activity of these
complexes on DNA.
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Inorganic Chemistry
ARTICLE
’ CONCLUSIONS
(4) (a) Klasen, H. J. Burns 2000, 26, 117–130. (b) Klasen, H. J. Burns
2000, 26, 131–138.
Novel silver(I) complexes have been prepared, bearing the
scorpionate ligand Tpms and also the monodentate phosphanes
PPh3, PCy3, and PTA. In the presence of both Tpms and
phosphane, compounds 2, 3, and 5 are obtained, existing as
mononuclear species in the solid state with tetrahedral silver
atoms coordinated by a phosphane and N3-bonded (in 3) or
N2O-ligated (in 2 and 5) Tpms. The coordination versatility of
the scorpionate ligands is also expressed by the tilting of the
coordinated pyrazolyl rings which appears to be related to the
cone angles of the phosphanes: the higher this parameter, the
higher the AgNNCpz torsion angles, which can be envisaged as a
way of the ligand to avoid unfavorable interactions with other
bulky ligands.
Derivatives 1 and 4, likely polynuclear in the solid state,
dissociate in solution into mononuclear and dinuclear species.
These silver(I) complexes display antibacterial activity which is
compared with that of AgNO3 used as control. Although 2 shows
a lower activity, compounds bearing PTA ligand (4 and 5) are
more active. These findings demonstrate a possible relationship
between structure/activity and selectivity for silver(I) complexes,
which can be further explored toward a more selective antibac-
terial activity.
Our silver compounds also have an antiproliferative activity,
which, in the case of 2 and 3, is two and five times, respectively,
that of AgNO3 used as the control (Table 3). These results are in
line with the values of the binding constant (KA) for the
interaction of silver compounds with DNA (Table 4). From
these data we observe that the affinities of 2 and 3 are higher than
those of the other derivatives and the control (AgNO3). In
particular, for the interaction of the Ag complexes with DNA, we
observe a very high binding activity of complex 3 (450 fold that of
AgNO3), the interaction of which with DNA was the object of
further characterization.
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S
Supporting Information. X-ray crystallographic files
b
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3
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’ AUTHOR INFORMATION
Corresponding Author
*E-mail: claudio.pettinari@unicam.it (C.P.), fatima.guedes@
ist.utl.pt (M.F.G.d.S.), pombeiro@ist.utl.pt (A.J.L.P.), piotr.
smolenski@chem.uni.wroc.pl (P.S.).
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’ ACKNOWLEDGMENT
We thank the University of Camerino Financial Support, the
Fundac-~ao para a Ci^encia e a Tecnologia (FCT), Portugal, and its
Strategy Programme PEst-OE/QUI/UI0100/2011, and the
KBN program (Grant N204 280438), Poland.
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