Group 11 metal compounds with tripodal bis(imidazole) thioether ligands. applications as catalysts in the oxidation of alkenes and as antimicrobial agents

Article abstract of DOI:10.3390/molecules16086701

New group 11 metal complexes have been prepared using the previously described tripodal bis(imidazole) thioether ligand (N-methyl-4,5-diphenyl-2- imidazolyl)₂C(OMe)C(CH₃)₂S(tert-Bu) ({BIT ^OMe,StBu}, 2). The pincer ligand offers a N₂S donor atom set that can be used to coordinate the group 11 metals in different oxidation states [Au^I, Au^III, Ag^I, Cu^I and Cu^II]. Thus the new compounds [Au{BIT^OMe,StBu}Cl] [AuCl₄]₂ (3), [Au{BI^TOMe,StBu}Cl] (4), [Ag{BIT^OMe,StBu}X] (X = OSO₂CF₃ - 5, PF6 - 6) and [Cu{BI^TOMe,StBu}Cl₂] (7) have been synthesized from reaction of 2 with the appropriate metal precursors, and characterized in solution. While attempting characterization in the solid state of 3, single crystals of the neutral dinuclear mixed AuIIIAuI species [Au₂{BI ^TOMe,S}Cl₃] (8) were obtained and its crystal structure was determined by X-ray diffraction studies. The structure shows a AuIII center coordinated to the pincer ligand through one N and the S atom. The soft AuI center coordinates to the ligand through the same S atom that has lost the tert-butyl group, thus becoming a thiolate ligand. The short distance between the AuI-AuIII atoms (3.383 A) may indicate a weak metal-metal interaction. Complexes 2-7 and the previously described CuI compound [Cu{BI ^TOMe,StBu}]PF₆ (9) have been evaluated in the oxidation of biphenyl ethylene with tert-butyl hydrogen peroxide (TBHP) as the oxidant. Results have shown that the AuI and AgI complexes 4 and 6 (at 10 mol % loading) are the more active catalysts in this oxidative cleavage. The antimicrobial activity of compounds 2-5, 7 and 9 against Gram-positive and Gram-negative bacteria and yeast has also been evaluated. The new gold and silver compounds display moderate to high antibacterial activity, while the copper derivatives are mostly inactive. The gold and silver complexes were also potent against fungi. Their cytotoxic properties have been analyzed in vitro utilizing HeLa human cervical carcinoma cells. The compounds displayed a very low cytotoxicity on this cell line (5 to 10 times lower than cisplatin) and on normal primary cells derived from C57B6 mouse muscle explants, which may make them promising candidates as potential antimicrobial agents and safer catalysts due to low toxicity in human and other mammalian tissues.

Full text of DOI:10.3390/molecules16086701

Molecules 2011, 16, 6701-6720; doi:10.3390/molecules16086701
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Group 11 Metal Compounds with Tripodal Bis(imidazole)
Thioether Ligands. Applications as Catalysts in the Oxidation of
Alkenes and as Antimicrobial Agents
Fangwei Liu 1, Reema Anis 1, Eunmi Hwang 1, Rafael Ovalle 2, Armando Varela-Ramírez 3,
Renato J. Aguilera 3 and María Contel 1,*
1
Department of Chemistry, Brooklyn College and The Graduate Center, The City University of
New York, Brooklyn, NY 11210, USA
2
Department of Biology, Brooklyn College, The City University of New York, Brooklyn,
NY 11210, USA
3
Department of Biological Sciences, The University of Texas at El Paso, El Paso, TX 79968, USA
* Author to whom correspondence should be addressed; E-Mail: mariacontel@brooklyn.cuny.edu;
Tel.: +1-7189515000 ext. 2833; Fax: +1-7189514607.
Received: 8 July 2011; in revised form: 22 July 2011 / Accepted: 2 August 2011 /
Published: 8 August 2011
Abstract: New group 11 metal complexes have been prepared using the previously
described tripodal bis(imidazole) thioether ligand (N-methyl-4,5-diphenyl-2-
imidazolyl)2C(OMe)C(CH3)2S(tert-Bu) ({BITOMe,StBu}, 2). The pincer ligand offers a N2S
donor atom set that can be used to coordinate the group 11 metals in different
oxidation states [AuI, AuIII, AgI, CuI and CuII]. Thus the new compounds
[Au{BITOMe,StBu}Cl][AuCl4]2 (3), [Au{BITOMe,StBu}Cl] (4), [Ag{BITOMe,StBu}X] (X =
-
-
OSO2CF3 5, PF6 6) and [Cu{BITOMe,StBu}Cl2] (7) have been synthesized from reaction of
2 with the appropriate metal precursors, and characterized in solution. While attempting
characterization in the solid state of 3, single crystals of the neutral dinuclear mixed AuIII-
AuI species [Au2{BITOMe,S}Cl3] (8) were obtained and its crystal structure was determined
by X-ray diffraction studies. The structure shows a AuIII center coordinated to the pincer
ligand through one N and the S atom. The soft AuI center coordinates to the ligand
through the same S atom that has lost the tert-butyl group, thus becoming a thiolate ligand.
The short distance between the AuI-AuIII atoms (3.383 Å) may indicate a weak
metal-metal interaction. Complexes 2-7 and the previously described CuI compound
[Cu{BITOMe,StBu}]PF6 (9) have been evaluated in the oxidation of biphenyl ethylene with

Molecules 2011, 16
tert-butyl hydrogen peroxide (TBHP) as the oxidant. Results have shown that the AuI and
6702
AgI complexes 4 and 6 (at 10 mol % loading) are the more active catalysts in this oxidative
cleavage. The antimicrobial activity of compounds 2-5, 7 and 9 against Gram-positive and
Gram-negative bacteria and yeast has also been evaluated. The new gold and silver
compounds display moderate to high antibacterial activity, while the copper derivatives are
mostly inactive. The gold and silver complexes were also potent against fungi. Their
cytotoxic properties have been analyzed in vitro utilizing HeLa human cervical carcinoma
cells. The compounds displayed a very low cytotoxicity on this cell line (5 to 10 times
lower than cisplatin) and on normal primary cells derived from C57B6 mouse muscle
explants, which may make them promising candidates as potential antimicrobial agents and
safer catalysts due to low toxicity in human and other mammalian tissues.
Keywords: tripodal bis(imidazol) thioether pincer ligands; group 11 metals; oxidation
alkenes; antimicrobial; non-toxic
1. Introduction
In the last few years the use of mixed-donor pincer ligands has increased due to the catalytic
potential of their metal complexes in different organic transformations [1]. They are important in
homogenous catalysis since they provide stabilization and temporary coordinative saturation to the
metal center via chelation until an active site is required. A large number of donor ligands with many
donor type permutations (C, O, S, N, P, etc…), charge and coordination number have been
described [2]. Among these various combinations, mixed-donor ligands of the type N,S are particularly
interesting. They are currently used as modeling active sites for metalloenzymes [3] and they have
found applications in homogeneous catalysis [4] and medicinal chemistry (brain-imaging [5],
antimicrobial properties [6]). They display a hard donor (N) and a soft donor (S) atom that can
coordinate to a wide range of metals. They can bind to the same metal in a particular coordination state
providing a different bond strength and lability for each donor atom. There is also a current interest in
the incorporation of these N,S donor atoms (usually the ligands contain combinations of thiolato or
thioether functional groups and amines, pyrazolyl or imidazole groups) in macrocycles [7] and in
polydentate and tripodal ligands [3-6,8] in order to probe the biologically relevant coordination
chemistry, the supramolecular chemistry (and metal ion recognition), or the catalytic reactions of
different metals.
Recently, biomimetic tripodal bis(imidazole)thioether (BIT) ligands have been reported by
Nicholas et al. incorporating biologically most relevant N-imidazole groups which display a
significantly different basicity and donor-acceptor properties with respect to the other common
nitrogen ligands (such as amines and pyrazolyl ligands) [9-11]. These ligands provide a sterically
encumbered environment that could prevent, in principle, the formation of bimetallic species.
In the search for copper complexes that accurately mimic the electronic and features of the CuM site
of copper hydroxylase enzymes, compounds of CuI have been successfully synthesized with these
tripodal BIT ligands (Scheme 1) [9-11]. Their reactivity towards dioxygen was studied and new

Molecules 2011, 16
6703
dimeric hydroxo-copper(II) derivatives were obtained. In order to incorporate the ligand set of the
CuM site of the copper hydroxylase enzymes, the BIT ligands were modified according to the
imidazole C(Ph or H) and N(H or Me) substituents as well as the positions (2- or 4-) of the tripodal
attachment [11]. The authors concluded that N-Me substitution and 4-tethering on the imidazole unit
increased oxidation and oxygenation reactivity on the derived CuI complexes while Ph-substitution and
2-tethering decreased reactivity [11].
Scheme 1. Preparation of new gold, silver and copper complexes (3-7) and previously
described copper(I) 9 [10] incorporating the pincer BITOMe,StBu ligand 2 [10].
We thought that some of these tripodal ligands (specifically BITOR,SR’) could be useful in the
synthesis of group 11 metal complexes with the metals in different oxidation states. We wanted a
chelating (pincer) system able to stabilize group 11 metal derivatives, but with basicity and electronic
properties different from those of amines. We were especially interested in the preparation of gold
complexes in different oxidation states (I and III) with the same set of ligands (or same set of mixed-
donor atoms within the ligand) to study their catalytic (oxidation reactions) and biological (cytotoxic,
antimicrobial) behavior as a function of the oxidation state of the gold center. We also wanted to study

Molecules 2011, 16
6704
the activity of different metals from the same triad with the same ligand. We report here on
the preparation and characterization of group 11 metal complexes with previously described
potentially pincer tripodal bis(imidazole) thioether ligand (N-methyl-4,5-diphenyl-2-
imidazolyl)2C(OMe)C(CH3)2S(tert-Bu)
{BITOMe,StBu
}
2.
Thus,
new
compounds
-
-
[Au{BITOMe,StBu}Cl][AuCl4]2 (3), [Au{BITOMe,StBu}Cl] (4), [Ag{BITOMe,StBu}X] (X = OSO2CF3 5, PF6
6) and [Cu{BITOMe,StBu}Cl2] (7) have been synthesized from reaction of 2 (Scheme 1) with the
appropriate metal precursors, and characterized in solution. Their catalytic activity in the oxidation of
biphenyl ethylene with tert-butyl hydrogen peroxide (TBHP) as the oxidant has been studied. AuI and
especially AgI complexes displayed moderate catalytic activity. The antimicrobial activity of
compounds 2-5 and 7 and ligand 2 [10] and [Cu{BITOMe,StBu}(NCCH3)]PF6 (9) [10] were tested
against Gram-positive and Gram-negative bacteria and yeast. The cytotoxic properties of these novel
compounds were analyzed in vitro utilizing HeLa human cervical carcinoma cells. AuI, AuIII and AgI
derivatives displayed high to moderate antibacterial/antifungal activity, while all the compounds were
less cytotoxic against the HeLa tumor cells than cisplatin and even less toxic to normal primary cells
derived from C57B6 mouse muscle explants.
2. Results and Discussion
2.1. Chemistry
BIT ligands with bulkier S-alkyl groups (tert-Bu or CPh3) such as BITOMe,StBu (2) had been
employed by Nicholas and co-workers in an attempt to inhibit dinuclear Cu complex formation to
potentially kinetically stabilize mono-Cu-O2 species. However, reactions of the CuI derivatives afford
mainly dinuclear Cu alkoxo and hydroxo complexes and the oxygenation reactivity of the CuI
complexes depends markedly on the carbinol and thioether susbtituents [10].
The oxygenation of CuI complex with ligand BITOH,SMe affords a dinuclear CuII compound featuring
bridging BIT-alkoxo ligands and terminal –SOMe2. The incorporation of methyl in BITOMe,SR’ ligands
prevents the oxidation of the sulfur atom. The different R’ substituent affects the reactivity of the CuI
towards oxygenation and thus sterically hindered ligand BITOMe,StBu (2) affords a slower
oxygenation [10]. We choose ligand 2 as a candidate for the preparation of different complexes of Au,
Ag and Cu to be applied in homogeneous oxidation reactions as well as to study their
biological activity.
Ligand 2 was prepared as previously reported [10] but we isolated and fully characterized precursor
ligand BITOH,StBu (1) and prepared 2 from isolated 1 (see the Experimental section). The addition of
ligand 2 to solutions of different salts of group 11 metals (see Experimental for details) affords
compounds [Au{BITOMe,StBu}Cl][AuCl4]2 (3), [Au{BITOMe,StBu}Cl] (4), [Ag{BITOMe,StBu}X] (X =
-
-
OSO2CF3 5, PF6 6) and [Cu{BITOMe,StBu}Cl2] (7) (Scheme 1). Depending on the metals, 2 acts as a
pincer ligand (see Scheme 1). All complexes were isolated in moderate yields as air-stable solids. The
stoichiometry of the compounds is proposed on the basis of their spectroscopic characterization,
microanalysis, mass spectrometry and conductivity studies (see experimental). The geometries for the
metal in these derivatives (square-planar for AuIII, linear for AuI and AgI and, based on the X-ray
structure of related mononuclear [(BITOMe,SMe)CuII(DMF)2]2+ [10] square-pyramidal geometry for the

Molecules 2011, 16
6705
Cu(II)) are the most plausible ones considering all these data. It is clear that the two silver compounds
-
5 with the triflate ligand and 6 with the PF6 anion are different. Compound 5 is a neutral compound
with the triflate coordinated as a covalent (instead of ionic) group [12], as confirmed by its IR and
conductivity measurements (see Experimental). However, polynuclear structures cannot be excluded
for the silver derivatives. The monomeric nature of the CuII derivative [Cu{BITOMe,StBu}Cl2] (7) was
confirmed by EPR studies of solutions of 7 in CH3CN at 77 K. The X-band spectrum shows signals
that can be interpreted as being due to a paramagnetic entity (S ) 1/2) with nearly axial symmetry,
whose gyromagnetic tensor was determined by g || = 2.35 and g ⊥ = 2.073, and is typical for
monomeric or isolated CuII complexes [13]. Unfortunately we could not get crystals of enough quality
for single crystal X-ray diffraction studies of 4-7.
We obtained high quality red crystals from CH2Cl2/Et2O solutions of the Au(III) compound
[Au{BITOMe,StBu}Cl][AuCl4]2 (3). The structure corresponds to a new neutral dinuclear Au(III)-Au(I)
compound (8 in Scheme 1). The structure is shown in Figure 1, while selected bond and angles are
presented in Table 1. The tripodal ligand is coordinated to both a AuIII center and a AuI center via the
sulfur atom which has lost the tert-Bu group becoming a thiolato functional group instead of the
original thioether. The AuIII ion is four-coordinated in a square-planar geometry as expected with
angles close to 90. The AuI is two-coordinated, but the coordination geometry around the gold atom is
slightly distorted from linearity, with a S(1)-Au(2)-Cl(3) angle of 174.81(8). The AuIII-Cl bond lengths
are 2.3066(18) and 2.31119(19) Å, similar to those found for the only two crystal structures of mixed
Au(III)-Au(I) species with a S atom as a bridge and containing chlorides, [Na(15-crown-
5)]4[AuCl2]2[Au4Cl6S2] (2.34(1)-2.31(1)) and [Na(15-crown-5)] [Au4Cl6S2] (2.332(3)-2.328(3)) [14].
The AuIII-S bond length (2.327(2) Å) compares well with the value found for one of the few Au(III)
thiolate complexes described [Au2Cl4(μ-SPh)2] (2.332(5)-2.339(5) Å) [15] and is shorter than those
found for mixed AuIII-AuI thiolate complexes with ligands coordinated to the AuIII other than
chlorides (e.g., [(C6F5)3Au(μ2-2-SC6H4NH2)(AudppmAu)(μ2-2-SC6H4NH2)Au(C6F5)] (2.372(3)-
2.375(5) Å) [16], and 2.380(2) [(C6F5)3Au(μ2-SC6F5)(AudppfAu)( μ2-SC6F5)Au(C6F5)3]) [17]. The
AuI-S and AuI-Cl distances of 2.248(2) Å and 2.258(2) Å are very close to those found in the above
mentioned crystal structures of [Na(15-crown-5)]4[AuCl2]2[Au4Cl6S2] (2.24(2) and 2.24(1) Å) and
[Na(15-crown-5)] [Au4Cl6S2] (2.247(3) and 2.282(2) Å) [14].
Figure 1. Molecular drawing of neutral dinuclear mixed AuIII-AuI species
[Au2{BITOMe,S}Cl3] 8 (obtained while crystallizing compound[Au{BITOMe,StBu}Cl][AuCl4]2
3) with the atomic numbering scheme.

Molecules 2011, 16
Table 1. Selected bond distances (Å) and angles (°) in species 8.
6706
Au1 N1 2.038(5) Å
Au1 Cl1 2.3066(18) Å
N1 Au1 Cl1 177.4(2)°
Cl2 Au1 S1 174.35(8)°
N1 Au1 Cl2 91.01(17)°
Cl1 Au1 Cl2 90.66(7)°
N1 Au1 S1 89.42(17)
Au1 Cl2 2.3119(19) Å
Au1 S1 2.327(2) Å
Au2 S1 2.248(2) Å
Au2 Cl3 2.258(2) Å
Au1 Au2 3.383 Å
Cl1 Au1 S1 89.11(7)°
S1 Au2 Cl3 174.81(8)°
Au2 S1 Au1 95.37(6)°
The AuI-AuIII distance is 3.383 Å and it is comparable to the shortest distances obtained in two
other bridging thiolate and sulfide mixed AuI-AIII complexes. Thus the AuI-AuIII distances obtained for
tetranuclear [(C6F5)3Au(μ2-SC6F5)(AudppfAu)(μ2-SC6F5)Au(C6F5)3] are 3.2812(7) and 3.3822(7) Å
[3.2923(7) and 3.4052(7) Å] [17] and, in the case of [{S(Au2dppf)}2{Au(C6F5)2}][CF3SO3], 3.2923(7)
and 3.4052(7) Å [18]. The above mentioned distances (including that in 8) are shorter than
those reported in other sulfide, thiolate, or selenide mixed derivatives [3.404(1)-4.011(1) Å] [17-20].
This distance may be indicative of the presence of a weak interaction between the AuI and AuIII
centers [17] and is similar to that for AuI-AuI contacts in many molecules.
Although the loss of the tert-Bu group in the [Cu(BITOMe,StBu)] complexes was not described,
Nicholas et al. [10] reported the loss of the trityl group (CPh3) from [Cu(BITOMe,STr)] derivatives in
reactions of ligand ((BITOMe,STr) and [Cu(CH3CN)4]PF6 with O2 or under Ar in the presence of
tert-BuNC. The compounds obtained were [(BITOMe,S)2Cu2(μ-OH)2](PF6)2 in the first case or
[(BITOMe,S)2Cu2(tBuNC)2] and [(BITOMe,S)Cu2 (tBuNC)2]PF6 in the second [10]. The authors reported
that the loss of trityl group occured before oxygenation. In our case this cleavage occurs with
concomitant reduction of one counter ion [AuCl4]- to AuCl, which then coordinates to the S atom.
Some thioether ligands can afford Au(I) derivatives by reduction of HAuCl4 [21] and the reduction of
AuIII complexes with calix[4]arenes modified by thioether groups on the upper rim to AuI has been
reported as well [22]. Carbon-sulfide bond cleavage in spontaneously adsorbed thioether-based
monolayers at gold was described and gold-bound thiolate species were detected [23]. The nature of
-
the N2S chelating ligand 2 may allow for a coordination of a counter ion AuIIICl4 and reduction to
AuICl with a concomitant C-S bond cleavage and loss of tert-butyl alcohol.
2.2. Catalysis
Oxidation is a very important method for the synthesis of chemical intermediates in the manufacture
of high-tonnage commodities, high-value fine chemicals, agrochemicals and pharmaceuticals.
Oxidation of olefins provides a way to turn mineral oil into chemicals of commercial value. Gold has
been reported to be a catalyst in homogeneous and heterogeneous [24] oxidation reactions. The
number of reports on truly homogeneous catalytic oxidation reactions with gold compounds is more
limited. AuCl in combination with nitrogen or phosphorous containing ligands has been used as a
catalyst in the aerobic oxidation of alcohols [25] and the oxidative cleavage of double [26], C-H, [27]
and single and triple bonds [27]. Very importantly, AuIII cations have been used in the oxidation of
methane to methanol (in the presence of strong acids) [28,29] while AuIII-chlorides and

Molecules 2011, 16
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[AuCl2(NO3)(thioether)] complexes have been employed in sulfoxide [30,31] and thioether oxidations
[32,33]. Common complexes such as Na[AuCl4] or [AuCl(PPh3)] have been used in the oxidation of
alkanes (with very poor conversions) [34]. AuCl3 and AuCl2 with NO ligands have been used in the
epoxidation of alkenes [35] and [Au(OAc)3] in the aerobic oxidation of dibenzylamine [36]. Very
recently, oxidative transformations of Au-C(sp2) bonds [37] have been reported. In these processes
there was strong evidence for the existence of AuI/AuIII catalytic cycles. This has opened up a new area
of gold catalysis where in situ-generated Au-C(sp2) bonds can be oxidatively transformed in the
presence of external oxidants [37].
We wanted to study the catalytic activity of the new AuIII and AuI complexes with the same set of
ligands BITOMe,StBu (2) [10] and chlorides. We chose the reaction depicted in Table 2: the oxidative
cleavage of diphenyl ethylene in the presence of catalyst and tert-butyl hydroperoxide to produce
benzophenone.
Table 2. Oxidative Cleavage of Diphenyl Ethylene.
Run
Catalyst
Solvent
Temp.
(°C)
90
Cat. mol Conversion % a
%
1
2
3
4
5
6
7
8
9
10
11
12
13
3
3
4
4
4
4
5
5
6
6
7
7
9
Toluene / Acetonitrile
Toluene / Acetonitrile
Toluene
5
10
5
26
31
0
90
RT
RT
90
90
90
90
90
90
90
Toluene
Toluene
Toluene
10
5
10
5
10
5
10
5
0
43
60
42
48
42
80
0
Toluene / Acetonitrile
Toluene / Acetonitrile
Toluene / Acetonitrile
Toluene / Acetonitrile
Toluene / Acetone
Toluene / Acetone
Toluene
90
10
10
7
10
90
a By 1H-NMR.
AuCl in the presence of neocuproine (5 mol %) in different solvents catalyzed the reaction at 90 °C
in 10 hours. The reaction could be run in water and the amount of catalyst and ligand could be
decreased to 1 mol %. The replacement for AuCl3 salts afforded a very low conversion to
benzophenone [26]. In our case, we tested the AuIII and AuI compounds 3 and 4 (entries 1-6) in either
toluene or mixtures of toluene/acetonitrile for the cationic AuIII compound 3. The compounds were
active although the conversions were lower than those described for AuCl and neocuproine. The AuI
complex was a better catalyst than the AuIII derivative as previously described [26]. The increase of the
amount of catalyst improved the conversion but did not double it. Our reaction time was higher (24 h
instead of 10 h) and the reactions were run at 90 °C. We also tested the silver derivatives 5 and 6 and
found a similar activity to the AuI for the one with covalent triflate (5) whereas the cationic silver

Molecules 2011, 16
6708
-
derivative with PF6 anion (6) was the most active of all studied with a conversion of 80% (10 mol %
cat). Homogeneous oxidation of organic products with silver derivatives is limited to a couple of not
very detailed patents with inorganic silver salts (like AgNO3) [38,39]. To our knowledge, these are the
first examples of well-defined organic soluble silver catalysts in oxidation reactions. In contrast,
neither the CuI or CuII derivatives showed activity in this oxidative cleavage with TBHP. CuI
complexes of 2 had been described as having a lower reactivity towards oxygen in comparison to other
BIT ligands [11].
2.3. Biological Activity
2.3.1. Antimicrobial Activity
The antimicrobial activity of the new compounds 3-5, 7 as well as that of compound 9 and the
ligand 2 were evaluated against yeast (Saccharomyces cerevisiae) Gram-negative (Escherichia coli
and Salmonella tiphymurium) and Gram-positive (Bacillus cereus and Staphyloccocus aureus) bacteria
(Table 3).
The ligand {BITOMe,StBu} 2 was totally inactive against S. cerevisiae and the representative
Gram-positive and Gram-negative bacteria. S. cerevisiae showed clearing at the highest concentration
tested for gold and silver compounds, but was unresponsive towards the copper compounds; these
compounds should be tested against fungi and other microbes to determine general toxicity ranges for
eukaryotes. AuIII complex 3 showed moderate toxicity toward both Gram-positive bacteria, but had
low toxicity towards the Gram-negative bacteria. Though AuI complex 4 showed low toxicity towards
most bacteria, Escherichia coli was highly vulnerable to this compound, which may be a precursor for
an narrow-range antibacterial. AgI complex 5 was highly toxic to all bacteria tested and is a promising
candidate for a broad-range antibacterial. CuII complex 7 was highly toxic to Bacillus cereus, but
completely innocuous to other bacteria, implying that a targeted antibacterial can be developed from
compound 7. CuI complex 9 was not toxic to the bacteria tested in this study.
Table 3. Toxicity assesment of compounds 3-5, 7, 9 and ligand 2 against microbial organisms.a
Yeast
Gram-negative
Gram-positive
Compound
S. Cerevisiae
(X2180-1A)
E. Coli S. Tiphymurium B. Cereus
S. Aureus
2 b
---
100
100
100
---
---
100
0.1
10
---
100
100
10
---
10
---
10
10
---
---
10
100
10
---
---
3 (AuIII) c
4 (AuI) c
5 (AgI) c
7 (CuII) c
9 (CuI) c
---
---
---
---
---
a
Minimun number of μg required to create a zone of clearing of 0.5 cm diameter on a lawn of
fungal or bacterial cells: 1, 10, 100 or --- no toxicity. b Ligand 2 was dissolved in CH2Cl2. c Metallic
compounds dissolved in CH3CN.
We found that the silver derivative 5 is the most potent against Gram-negative bacteria with the
exception of a lower toxicity for the AuI species towards the Gram-negative Escherichia coli bacteria.
AuIII and AgI derivatives are more toxic towards Gram-positive bacteria than the AuI analogue. This in

Molecules 2011, 16
6709
contrast to results found for AuI complexes containing phosphanes, including our work with AuI water-
soluble phosphanes [40]. The fact that gold compounds are more toxic for Gram-positive bacteria than
Gram-negative ones or fungi has been noted previously with auranofin [41] and some other AuI
phosphane derivatives [40,42] and N-heterocyclic carbene-AuI derivatives [43]. Compound 3 is one of
the very few examples of AuIII derivatives exhibiting antimicrobial properties [44]. Dinuclear
AgI-oxygen bonding complexes derived from camphanic acic ligands have displayed a wide spectrum
of effective antimicrobial activity [45]. Some N-heterocyclic carbene AgI complexes inhibit more
efficiently the growth of Gram-positive bateria including B. subtilis [46-48], Gram-negative E. coli
[47-50], and P. aeruginosa [47,49,50] and even antibiotic resistant strains of S. aureus. [50,51]
Binuclear and polymeric AgI complexes with a tridentate heterocyclic N- and S- ligand 8-(9pyridin-3-
yl)methylthio) quinoline were good inhibitors of Gram-positive bacteria and some Gram-negative
bacteria (P. aeruginosa) but they were very poor against E. coli [52]. The behaviour of the silver
compound 5 is similar to that of silver-carbene derivatives, [46-51] silver fluorinated tris(pyrazolyl)-
borate complexes [53] and AgNO3 and silver(I) sulfadiazine [52] inhibiting grow of both Gram-
negative and Gram-positive bacteria. However in the case of the tris(pyrazolyl)borate complexes it was
demonstrated that the effect on Gram-positive species was due to the ligand whereas the activity for
Gram-negative species was truly due to the silver ion [53]. In our case it seems that the activity
displayed is due to the presence of the metal. Thus 5 has potential applications in the broad spectrum
of antimicrobial field.
2.3.2. Cytotoxicity
The cytotoxic properties of the AuIII, AuI, AgI, CuII and CuI compounds were analyzed in vitro as
previously described utilizing human HeLa cervical carcinoma cell line that expresses the Green
Fluorescence Protein (GFP) in the nucleus [54,55]. Before use, all test compounds were dissolved in
DMSO and used at a final solvent concentration of 0.1% that has no discernible effect on cell viability.
The well known anticancer cytotoxic agent, cisplatin, was used as a positive control for cell death
induction. It is apparent that the complexes displayed low to very low cytotoxicity towards HeLa cells.
Gold compounds 3, 4 were 1/10 as effective as cisplatin in causing death of HeLa cells, whereas AgI
(5) was 1/5 as effective. The cytotoxicity of 6 could not be evaluated due to the formation of crystals in
the culture media. Due to the low solubility of the copper compounds 7, 9 in DMSO-H2O, their
toxicity towards HeLa cells was very low (less than 10%) and could not be fully evaluated. If the
values obtained are an indication of their cytotoxic potential at higher concentrations they would still
fall far below the toxicity values of the other compounds and cisplatin.
Moreover, we studied the cytotoxicity of compounds 3-5,7 and 9 against normal primary cells
(Figure 2). Cytotoxicity was monitored by using live-cell imaging of a normal primary culture of
adherent cells obtained from C57B6 mouse muscle explants after 22 hrs of incubation with the
compounds. The cytotoxicity of these compounds against the normal cells was very low although there
were some differences since complexes of AuI 4 and CuI (9) were slightly more toxic than the AuIII (3),
AgI (5) and CuII (7) derivatives.

Molecules 2011, 16
Figure 2. In vitro cytotoxicity of compounds 3-5, 7 and 9 against normal cells (conc. μM).
6710
Cytotoxicity was monitored by using live-cell imaging of a normal primary culture of
adherent cells obtained from C57B6 mouse muscle explants after 22 hrs of incubation with
the compounds. Each bar represents average value of three measurements and error bars
represent standard deviations. Results for compound 3 at 200 µM are not shown since there
was no cytotoxicity observed. Three controls were included; untreated cells (Unt), treated
with 0.5% v/v diluent of the chemical compounds (DMSO) and treated with 1 mM H2O2.
This lack of toxicity in cancerous and normal mammalian cell lines makes them promising
candidates as potential antimicrobial agents (low toxicity in human tissues) and as safer and
greener catalysts.
Table 4. IC50 values for HeLa-GPF cells of compounds 3-7 and ligand 2 [10] and complex
9 [10], compared to cisplatin.
IC50(μM) a
Cell line
Cisplatin
2 b
3
4
5
6 b
7c
9 c
HeLa-GPF
14.9
----
155
155
73.6
---
---
---
a
IC50 is defined as the concentration of drug required to disrupt the plasma membrane of 50% of cell
population, compared to untreated cells, after 22 hours of incubation. Cells with compromised plasma
membrane were monitored using propidium iodide (PI) and flow cytometry. Cisplatin was used as reference
compound. b Compound 6 was found to be soluble in DMSO but when 1 µL was added to culture media with
cells the compound came out of solution forming crystals. Since it was difficult to evaluate how much of the
compound was actually in solution, an accurate IC50 value could not be determined. In the case of compound
2, it was possible to examine its cytotoxicity against HeLa-GFP cells at 250 µM, but its cytotoxicity was very
c
poor (5.4 ± 2.1%). Due to the low solubility of the copper compounds in the DMSO-H2O mixture, the
maximum concentration possible tested for 7 and 9 was 50 and 25µM, and their percent of cytotoxicity was
7.4 and 9, respectively.

Molecules 2011, 16
6711
3. Experimental
3.1. General
Solvents were purified by use of a PureSolv purification unit from Innovative Technology, Inc.
(Newburyport, MA, USA); all other chemicals were used as received. Elemental analyses were carried
out by Atlantic Microlab, Inc. (Norcross, GA, USA). Infrared spectra (4000-400 cm−1) were recorded
1
on a Nicolet 380 FT-IR infrared spectrophotometer on KBr pellets. The H-, and 13C{1H}-NMR
spectra were recorded in CDCl3 solutions at 25 °C on a Bruker 400 and spectrometer (δ, ppm; J, Hz);
1H and 13C{1H} were referenced using the solvent signal as internal standard. EPR studies were
conducted on a Bruker E500 EPR spectrometer. The mass spectra (ESI) were recorded from CH2Cl2 or
CH3CN solutions by the mass spectrometry facility of the University of California Riverside
(Riverside, CA, USA). X-ray data were collected with a Nonius Kappa CCD (Hunter College, The
City University of New York, New York, NY, USA). Conductivity was measured in an Oakton
pH/conductivity meter. Ligand 2 was prepared as previously reported [10], but we isolated and
characterized precursor ligand BITOH,StBu (1) and prepared 2 from isolated 1. All other chemicals and
solvents were purchased from Sigma-Aldrich and Strem Chemicals.
1-Methoxy-2-methyl-1,1-bis(N-methyl-4,5-diphenyl-2-imidazolyl)2-tert-butylthiopropanol (1, BITOH,StBu).
To a solution of N-methyl-4,5-diphenylimidazole (1.40 g, 6.0 mmol) in 30 mL of dry THF under N2
cooled to −78 °C was added n-BuLi dropwise (2.5 M, 2.40 mL, 6.0 mmol). The mixture was stirred at
−78 °C for 3 h to give a red-brown solution. After the addition of ethyl-2-methyl-tert-butylthio-
propanonate (0.41 g, 3.0 mmol), the mixture was allow to warm to room temperature overnight to give
a yellow solution. The reaction was quenched with 80 mL of water and extracted with diethyl ether (20
mL) three times. The organic phase was dried over MgSO4, filtered through Celite, and vacuum dried
to give 1 as a pure white compound. Yield: 1.40 g, 75%. Anal. Calc. for C40H42N4OS (626.92): C,
75.71; H, 6.88; N, 8.80; S, 4.78; found: C, 75.74; H, 6.68; N, 8.76; S, 4.54. MS (ESI+) [m/z]: 627.3
[M+1]+. IR (cm−1): ν (C=N, C3N2) = 2958, ν (C-N, C3N2) = 1501, ν (C-OCH3) = 1074, ν (S-C (CH3)2)
1
= 970. H-NMR: δ = 7.18−7.68 (m, 20H, Hp+Ho+Hm, C6H5 Bz); 5.70 (s, 1H, OH); 3.08 (s, 6H,
13
N-CH3); 2.28 (s, 6H, SC (CH3)2); 1.49 (s, 9H, C (CH3)3). C{1H}-NMR: δ = 15.24 [C(CH3)3], 27.59
(N-CH3), 32.05 [SC(CH3)2], 33.46 [C(CH3)3], 47.28 [SC(CH3)2], 59.73 (O-CH3), 126.17 (C3, C3N2),
126.50 (C2, C3N2), 128.04 (Co, Ph), 128.08 (Cp, Ph) 131.07 (Cm, Ph), 134.67 (C1, Ph), 146.25
(C1, C3N2).
1-Methoxy-2-methyl-1,1-bis(N-methyl-4,5-diphenyl-2-imidazolyl)2-tert-butylthiopropane (2, BITOH,StBu
)
[10] can be prepared from isolated 1 in 80% yield as follows: to a suspension of sodium hydride
(0.048 g, 1.92 mmol) in 10 mL of dry THF under N2 was added the solution of 1 (1.003 g, 1.6 mmol)
in 10 mL of dry THF, and the resulting mixture was stirred for 1 h at room temperature. Methyl iodide
(0.12 mL, 1.92 mmol) was added dropwise, and then the reaction mixture was stirred at reflux under
N2 for 22 h. The reaction mixture was quenched with 80 mL of water and extracted with
dichloromethane (20 mL) three times. The organic phase was dried over MgSO4, filtered through
celite, and vacuum dried to give 2 as a yellow powder. Yield: 0.870 g, 80%.

Molecules 2011, 16
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[Au{BITOMe,StBu}Cl][AuCl4]2 (3): To a solution of K[AuCl4] (0.166 g, 0.44 mmol) in acetonitrile
(2 mL) a solution of BITOMe,StBu (2, 0.140 g, 0.22 mmol) in CH2Cl2 (20 mL) was added dropwise at
0 °C. The resulting solution was allowed to stir for over 40 min at RT and the solvent was removed
under vacuum to ca. 1 mL. Addition of Et2O/n-hexane (1:1,15 mL) and cooling to –5 °C afforded 3 as
a pale orange solid that was filtered and dried under vacuum. Yield: 0.213 g, 62%. Anal. Calc. for
C41H44N4OSAu3Cl9 (1550.85): C, 31.75; H, 3.61; N, 2.07; found: C, 32.04; H, 3.17; N, 3.69; MS
(ESI+) [m/z]: 641.3 [Ligand -H]+. IR (cm−1): ν(C = N, C3N2) = 2958, ν(C-N, C3N2) = 1505, ν(C-OCH3)
= 1090, ν[S-C(CH3)2] = 973. 1H-NMR: 7.18-7.54 (m, 20H, Hp+Ho+Hm, C6H5 Ph); 3.78 (s, 3H, O-CH3)
13
3.47(s, 3H, N-CH3); 2.76 (s, 3H, N-CH3); 1.65 [s, 6H, SC (CH3)2]; 1.28 [s, 9H, C (CH3)3]. C{1H}-
NMR: δ = 24.37 [C(CH3)3], 28.12 (N-CH3), 29.53 [SC(CH3)2], 33.51 [C(CH3)3], 36.44 [SC(CH3)2],
45.80 (COCH3), 93.27 (COCH3), 126.70 (C3, C3N2), 127.99 (C2, C3N2), 128.94 (Cp, Ph), 129.45 (d,
Cm, Ph) 129.82 (Co, Ph), 130.53 (C- C3N2, Ph), 173.73 (C1, C3N2). Λ(CH3CN) = 321 Ω–1cm2mol–1, 3 ions
(1+/2-).
[Au{BITOMe,StBu}Cl] (4): To a solution of [AuCl(tht)] (0.032 g, 0.10 mmol) in CH2Cl2 (10 mL) a
solution of BITOMe,StBu (2, 0.064 g, 0.10 mmol) in CH2Cl2 (10 mL) was added dropwise at 0 °C. The
resulting solution was allowed to stir for over 40 min at RT and the solvent was removed under
vacuum to ca. 1 mL. Addition of Et2O/n-hexane (1:1, 15 mL) and cooling to –5 °C afforded 4 as a
white solid that was filtered and dried under vacuum. Yield: 0.045 g, 69%. Anal. Calc. for
C41H44N4OSAuCl (873.30): C, 56.39; H, 5.08; N, 6.42; found: C, 56.78; H, 5.16; N, 6.50; MS (ESI+)
[m/z]: 873 [M + 1]. IR (cm–1): ν(C=N, C3N2) = 2954, ν(C-N, C3N2) = 1501, ν(C-OCH3) = 1087, ν [S-
C(CH3)2] = 960. 1H-NMR: δ =7.18-7.54 (m, 20H); 3.75 (s, 3H); 3.75 (s, 3H); 3.41 (s, 6H); 1.65 (s, 6H)
1.34 (s. 9H). 13C{1H}-NMR: δ = 23.69 [C(CH3)3], 27.62 (N-CH3), 28.02 [SC(CH3)2], 33.46 [C(CH3)3],
47.41 [SC(CH3)2], 55.05 (COCH3), 88.37 (COCH3), 125.73 (C3, C3N2), 126.56 (C2, C3N2), 127.94
(Co, Ph), 128.96 (Cm, Ph), 130.53 (Cp, Ph), 138.27 (C1, C3N2). Λ(CH3CN) = 17 Ω–1cm2mol–1, neutral.
[Ag{BITOMe,StBu}X] (X = OSO2CF3 5, PF6 6): To a solution of AgOSO2CF3 (0.026 g, 0.1 mmol, 5) or
AgPF6 (0.075 g, 0.3 mmol, 6) in Et2O or CH3CN (5 mL) a solution of BITOMe,StBu (2) (0.064 g,
0.10 mmol, 5 or 0.192 g, 0.3 mmol, 6) in CH2Cl2 (10 mL) was added dropwise at 0 °C. The resulting
mixtures were allowed to stir for over 40 min at 0 °C protected from light exposure. Subsequent
removal of the solvent under vacuum to ca. 1 mL, addition of Et2O/n-hexane (1:1, 15 mL) and cooling
to –5 °C afforded 5 and 6 as a white solids that were filtered and dried under vacuum. Compound 5:
yield: 0.050 g, 56%. Anal. Calc. for C42H44N4OS2AgF3 (897.81): C, 56.57; H, 4.29; N, 6.28; S, 7.19
found: C, 56.13; H, 4.67; N, 5.93; S, 7.29. MS (ESI+) [m/z]: 748.22 [M]+. IR (cm–1): ν(C = N, C3N2) =
2953, ν(C-N, C3N2) = 1505, ν(SO) = 1248 (br, s), 1157 (s), 1023 (m), ν(S-CF3) = 640 (signals of
-
-
1
C-OCH3 and S-C(CH3)2 have been overlapped by the signals of covalent OSO2CF3). H-NMR: δ =
7.23−7.74 (m, 20H, Hp+Ho+Hm, C6H5 Ph); 4.24 (t, 6H, N-CH3); 1.36 [s, 6H, SC(CH3)2]; 0.99 [s, 9H,
C(CH3)3]. 13C{1H}-NMR: δ =14.06 [C(CH3)3], 25.36 (N-CH3), 28.18 [SC(CH3)2], 33.48 [C(CH3)3],
35.79 [SC(CH3)2], 57.05 (COCH3), 61.14 (COCH3), 126.59 (C3, C3N2), 127.92 (C2, C3N2), 128.93 (Co,
Ph), 129.76 (Cm, Ph), 131.12 (Cp, Ph), 132.81(C1, C3N2). Λ(CH3CN) = 93 Ω–1cm2mol–1, neutral.
Compound 6: yield: 0.153 g, 60%. Anal. Calc. for C43H47N5SOAgPF6 (934.77): C, 55.25; H, 5.07; N,
7.49; found: C, 55.01; H, 4.90; N, 7.04. MS(ESI+) [m/z]: 747.22 [M -1]+. IR (cm-1):

Molecules 2011, 16
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1
ν(C=N, C3N2) =2958, ν(C-N, C3N2) = 1501, ν(C-OCH3) = 1074, ν(S-C(CH3)2) = 979. H-NMR: δ =
7.58-7.27 (m, 20H, Hp+Ho+Hm, C6H5 Ph); 3.95 (s, 3H, N-CH3); 3.05 (br, 3H, O-CH3); 2.62 (s, 3H, N-
CH3); 1.64 [s, 6H, SC (CH3)2]. 13C{1H}-NMR: δ =14.07 [C(CH3)3], 22.62 (N-CH3), 31.21 [SC(CH3)2],
33.51 [C(CH3)3], 35.67 [SC(CH3)2], 51.69 (COCH3), 82.58 (COCH3), 126.41 (C3, C3N2), 128.09 (C2,
31
C3N2), 128.70 (Co, Ph), 129.40 (Cm, Ph), 131.39 (Cp, Ph), 144.16 (C1, C3N2). P{1H}-NMR (CDCl3):
–144.32 (sept). Λ(CH3CN) = 220 Ω–1cm2mol–1, 2 ions (1+/1-).
[Cu{BITOMe,StBu}Cl2] (7): To a solution of CuCl2 (0.0134 g, 0.10 mmol) in CH3CN (2 mL) a solution of
BITOMe,StBu (2) (0.064 g, 0.10 mmol) in CH2Cl2 (10 mL) was added dropwise. The resulting yellow-
green solution was allowed to stir for over 1.5 h at RT and the solvent was removed under vacuum to
ca. 1 mL. Addition of Et2O/n-hexane (1:1, 15 mL) afforded 7 as a green solid that was filtered and
dried under vacuum. Yield: 0.049 g, 63%. Anal. Calc. for C41H44N4OSCuCl2.2H2O (811.38): C, 60.69;
H, 5.96; N, 6.90; found: C, 60.67; H, 5.46; N, 6.92; MS(ESI+) [m/z]: 738 [M-Cl - 1]+. IR (cm−1):
ν(C=N, C3N2) =2958, ν(C-N, C3N2) = 1501, ν(C-OCH3) = 1087, ν[S-C(CH3)2] = 976. Λ(CH3CN)
71.5 Ω–1cm2mol–1, neutral.
=
3.2. Catalytic Experiments
Typical procedure: To a solution of metallic complexes 3-7, 9 (amounts as specified in Table 2) in
toluene (5 mL) or in toluene (3 mL) - acetonitrile (2 mL) or in toluene (3 mL) – acetone (2 m), a
solution of biphenyl ethylene (88 μL, 0.5 mmol) and TBHP (0.171 mL, 70% solution in t-BuOH,
1.2 mmol) were added at room temperature. The reaction mixture was stirred at 90 °C for 24 h. The
conversion of benzophenone was determined by 1H-NMR after work-up of the mixture and isolation of
the crude product.
3.3. Single-Crystal X-ray Diffraction Studies
Crystals of 8 (red prisms) where obtained when a solution of 3 in CH2Cl2 was crystallized by slow
diffusion of Et2O at 0 °C. The intensity data for 8 were measured with a Bruker–Nonius Kappa CCD
diffractometer (graphite monochromated Mo-Kα radiation, λ = 0.71073 Å, φ-ω scans) at 100(2) K
(Table 1). The data were not corrected for absorption. Details of the solution and refinements for this
compound are presented below: the crystal of 8, with approximate dimensions 0.36_0.06_0.02 mm,
was orthorhombic with space group P212121. The final unit-cell constants of 8 were a = 11.032(2) Å,
b = 18.178(4) Å, c = 18.202(4) Å, V = 3650.2(13) Å3, Z = 4, ρ = 1.973 gcm–1, μ = 8.342 mm–1. The
structure of 8 was solved with SHELXS-97 and refined by full-matrix least-squares on F2 with
SHELXL-97. The hydrogen atoms were included in the structure-factor calculations, but their
parameters were not refined. The final discrepancy indices for the 8373 reflections (θ _ 27.52°) were
R = 0.0464 (calculated on F) and Rw = 0.0929 (calculated on F2) with 438 parameters varied. Tables
of thermal parameters and observed and calculated structure factors (crystal structure of compound 8)
have been deposited at the Cambridge Crystallographic Data Center. Any request for this material
should quote a full literature citation and the reference number CCDC 784217 and may be obtained
from The Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (Fax: +441233336033; E-Mail:
deposit@ccdc.cam.ac.uk or www: http://ccdc.cam.ac.uk).

Molecules 2011, 16
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3.4. EPR studies
The electron paramagnetic resonance (EPR) spectra was measured on a Bruker E500 EPR
spectrometer operating at X-band. In the experiment, an ER4122SHQE cavity was used. A solution of
7 (1 mM) in CH3CN at 77 K was introduced in a standard EPR quartz tube (707-SQ from Wilmad) and
was measured at 77 K using an immersion quartz Dewar. The microwave frequency was determined.
The X-band conditions were as follows: 3320 G, 1.0 mW microwave power, 9.51 GHz microwave
frecuency, 23dB attenuation, 77 K, 4.0 G modulation amplitude, 100 kHz modulation frequency,
60 dB gain; 163 ms conversion time, 167 s sweep time. EPR data acquisition and manipulation were
performed using XeprView software (Bruker). CuSO4 in 50% ethylene glycol was used as a standard
for spin quantification by double integration of EPR signal intensities. The signal-to-noise ratio in EPR
spectra was improved by signal averaging when necessary.
3.5. Antimicrobial Assays
The gold compounds were tested for microbial toxicity in a Kirby-Bauer disk diffusion assay. Metal
compounds 3-7 and 9 were dissolved in acetonitrile and ligand 2 (insoluble in CH3CN) in CH2Cl2 to a
concentration of 10 mg/mL and serially diluted in methanol by factors of 10 to create solutions ranging
down to 0.1 mg/mL. A solution (10 μL) was aliquoted onto a paper filter disk (5 mm diameter × 1 mm
thickness) that were then vacuum dried and stored at –20 °C prior to the experiment. For the assay, a
cell suspension (100 μL) containing 3 × 107 cells/mL were spread uniformly on a Mueller-Hinton (MH
for bacteria) or Yeast Extract (YPD for fungi) agar plate (100 mm × 15 mm). After spreading four
paper disks impregnated with either 100, 10, 1, or 0.1 μg of a compound were placed on the agar
surface with a solvent control in the center of the plate. The plates were incubated at either 30 °C
(fungi) or 37 °C (bacteria) for 48 h and resulting zones of growth suppression were measured.
3.6. Cytotoxicity Assays
Adherent HeLa-GFP human cervical carcinoma cells (Kanda et al.) [56] were seeded, in a 24 well
plate format, 100,000 cells/well using DMEM media (1 mL, HyClone, Logan, UT) supplemented with
antibiotics and 10% heat-inactivated newborn calf serum (HyClone). After overnight incubation, to
allow cell attachment, they were exposed for 22 h to several concentrations of chemical compounds.
Floating cells were collected in a ice-cold tube and placed on ice, while attached cells were treated for
15 min with 0.25% of trypsin solution (Invitrogen, Carlsbad, CA, USA), diluted in serum free
DMEM, and incubated at 37 °C. Cells from each individual well, included both those harvested by
trypsinization and those floating, were centrifuged at 1,400 rpm for 5 min at 4 °C The media was then
removed and cells resuspended in staining solution (500 µL) containing propidium iodide (2 µg/mL)
dissolved in FACS buffer (PBS, 0.5 mM EDTA, 2% heat inactivated fetal bovine serum, and 0.1%
sodium azide), incubated in the dark at room temperature for 15 min and analyzed by flow cytometry,
using a Cytomic FC 500 cytometer (Beckman-Coulter, Miami, FL, USA). The data were analyzed
using CXP software (Beckman-Coulter).

Molecules 2011, 16
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3.7. Cytotoxicity against normal primary culture cells
Mouse muscle cultures were initiated using tissue explants [57] from healthy 8 to 10 weeks old
C57B6 mice utilizing DMEM media supplemented with 10% fetal bovine serum and antibiotics (also
referred as complete media). After approximately two weeks of culture, when abundant growth of cells
had appeared from the explants, extensive washes with fresh media were performed to eliminate
floating cells to selectively keep the adherent cells. The morphology of the adherent cells was mostly
of fibroblastic nature (Figure 3). Adherent cells were collected by trypsinization, counted and seeded at
a density of 10,000 cells per well in 200 µL of complete media using a 96 well plate format, incubated
overnight to promote cell adherence, and incubated for 22 h in the presence of compounds. A mixture
of two fluorescent dyes, Propidium iodide (PI; MP Biomedicals, Solon, OH, USA) and Hoechst 33342
(Invitrogen, Eugene, OR, USA) at a final concentration of 1 µg/mL each was added to treated cells 1 h
prior to image capture from each individual well utilizing a BD Pathway 855 Bioimager system (BD
Biosciences Rockville, MD, USA) [58]. While Hoechst dye has the ability to easily cross cell
membranes of healthy and dead cells and stains nuclear DNA, PI is only able to stain dead or dying
cells with compromised plasma membranes. The fluorescence signal emitted from each individual
fluorophore was captured in two separate channels, according with the dye emission requirements. To
acquire adequate cell numbers of cells for statistical analyses, images from nine contiguous image
fields (3X3 montages) were captured per well utilizing a 20x objective. Data analysis determining the
percentage of death cells from each individual well was performed by using BD AttoVision™ v1.6.2
software [58].
4. Conclusions
In conclusion, we have prepared new stable complexes of group 11 metals with the previously
described tripodal bis(imidazole) thioether ligand (N-methyl-4,5-diphenyl-2-imidazolyl)2C(OMe)
C(CH3)2S(tert-Bu) ({BITOMe,StBu}, 2) [10] which offers a pincer N2S donor atom set. The gold and,
especially silver derivatives are active in the oxidative cleavage of diphenylethylene with TBHP at
90 °C. The use of modified BIT ligands (for which it is known that oxygenation reactivity is much
higher for CuI derivatives) [11] may provide more efficient catalysts for this and other homogeneous
oxidation processes. The antimicrobial activity of the ligand 2 and the metal complexes has been
evaluated against Gram-positive and Gram-negative bacteria and yeast. The new gold and silver
compounds display moderate to high antibacterial activity which is not due to the free ligand. Gold and
silver complexes are also potent against fungi. Moreover, silver compound 5 seems to hold potential
applications in the broad spectrum of antimicrobial field. The fact that the compounds display very low
or no cytotoxicity on HeLa Human cervical carcinoma cells and normal mammalian cells makes them
promising candidates as potential antimicrobial agents (low toxicity in human tissues) and as safer and
greener catalysts.

Molecules 2011, 16
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Acknowledgments
We thank the financial support of Brooklyn College (start-up budgets of M.C. and R.O.), PSC-
CUNY research award PSCREG-39-68 (M.C.) and two grants from the National Institute of General
Medical Sciences, SC2GM082307 (M.C.) and 3S06GM076168-02S1 (R.O.) The help of graduate
student Abdelahad Khajo and Prof. Richard Magliozzo (Brooklyn College) to measure the EPR of
compound 7 is gratefully acknowledged. We thank the staff of the Cell Culture and High Throughput
Screening (HTS) Core Facility for services and facilities provided. This core facility is supported by
grant 5G12RR008124 to the Border Biomedical Research Center (BBRC), granted to the University of
Texas at El Paso from the National Center for Research Resources (NCRR) of the NIH. The X-ray
measurement (L. Todaro) was supported by a "Research Centers in Minority Institutions" award,
RR-03037, from the National Center for Research Resources, National Institutes of Health. We thank
undergraduate student Chaya Levine and graduate student Jose González-Pantoja for their assistance
with some of the experiments.
Conflict of Interest
The authors declare no conflict of interest.
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Molecules 2011, 16
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8. For example: Lee, Y.; Lee, D.-H.; Narducci-Sarjeant, A.A.; Zakharov, L.N.; Rheingold, A.L.;
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Sample Availability: Samples of ligand 2 (250 mg) and silver derivatives 5 and 6 (20-30 mg) are
available from the authors.
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