Is Bismuth Really the "green" Metal? Exploring the Antimicrobial Activity and Cytotoxicity of Organobismuth Thiolate Complexes

Article abstract of DOI:10.1021/acs.inorgchem.9b03550

Antimicrobial resistance is becoming an ever-increasing threat for human health. Metal complexes and, in particular, those that incorporate bismuth offer an attractive alternative to the typically used organic compounds to which bacteria are often able to develop resistance determinants. Herein we report the synthesis, characterization, and biological evaluation of a series of homo- and heteroleptic bismuth(III) thiolates incorporating either one (BiPh₂L), two (BiPhL₂), or three (BiL₃) sulfur-containing azole ligands where LH = tetrazolethiols or triazolethiols (thiones). Despite bismuth typically being considered a nontoxic heavy metal, we demonstrate that the environment surrounding the metal center has a clear influence on the safety of bismuth-containing complexes. In particular, heteroleptic thiolate complexes (BiPh₂L and BiPhL₂) display strong antibacterial activity yet are also nonselectively cytotoxic to mammalian cells. Interestingly, the homoleptic thiolate complexes (BiL₃) were shown to be completely inactive toward both bacterial and mammalian cells. Further biological analysis of the complexes revealed the first insights into the biological mode of action of these particular bismuth thiolates. Scanning electron microscopy images of methicillin-resistant Staphylococcus aureus (MRSA) cells have revealed that the cell membrane is the likely target site of action for bismuth thiolates against bacterial cells. This points toward a nonspecific mode of action that is likely to contribute to the poor selectivity's demonstrated by the bismuth thiolate complexes in vitro. Uptake studies suggest that reduced cellular uptake could explain the marked difference in activity between the homo- and heteroleptic complexes.

Full text of DOI:10.1021/acs.inorgchem.9b03550

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Is Bismuth Really the “Green” Metal? Exploring the Antimicrobial
Activity and Cytotoxicity of Organobismuth Thiolate Complexes
Liam J. Stephens,† Sarmishta Munuganti,† Rebekah N. Duffin, Melissa V. Werrett,
and Philip C. Andrews*
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ABSTRACT: Antimicrobial resistance is becoming an ever-increasing threat for human health. Metal complexes and, in particular,
those that incorporate bismuth offer an attractive alternative to the typically used organic compounds to which bacteria are often
able to develop resistance determinants. Herein we report the synthesis, characterization, and biological evaluation of a series of
homo- and heteroleptic bismuth(III) thiolates incorporating either one (BiPh2L), two (BiPhL2), or three (BiL3) sulfur-containing
azole ligands where LH = tetrazolethiols or triazolethiols (thiones). Despite bismuth typically being considered a nontoxic heavy
metal, we demonstrate that the environment surrounding the metal center has a clear influence on the safety of bismuth-containing
complexes. In particular, heteroleptic thiolate complexes (BiPh2L and BiPhL2) display strong antibacterial activity yet are also
nonselectively cytotoxic to mammalian cells. Interestingly, the homoleptic thiolate complexes (BiL3) were shown to be completely
inactive toward both bacterial and mammalian cells. Further biological analysis of the complexes revealed the first insights into the
biological mode of action of these particular bismuth thiolates. Scanning electron microscopy images of methicillin-resistant
Staphylococcus aureus (MRSA) cells have revealed that the cell membrane is the likely target site of action for bismuth thiolates
against bacterial cells. This points toward a nonspecific mode of action that is likely to contribute to the poor selectivity’s
demonstrated by the bismuth thiolate complexes in vitro. Uptake studies suggest that reduced cellular uptake could explain the
marked difference in activity between the homo- and heteroleptic complexes.
Metal and metal-based compounds have become increasingly
investigated as novel antimicrobials because microbes often
face difficulties in acquiring resistant determinants to these
compounds. This contrasts to organic compounds, for which
bacteria have already proven themselves to be adept at evolving
mechanisms to break down or inactivate.5
Metal Antimicrobials. One of the more commonly known
and widely used metals in antibacterial agents is silver.
However, as the use of silver continues to expand, debates in
INTRODUCTION
■
Antimicrobial Resistance (AMR). The potential impact
that rapidly developing multiresistant bacteria could have on
human health cannot be overstated. AMR is identified by the
World Health Organization as one of the greatest threats we
face globally, now and into the future.1 Infections, which were
once easily treated with a range of antibiotics, are now often
resistant to our last line of defense compounds and in some
cases have become fatal.2,3 Despite efforts to discover new
antibiotics, the rapid rates at which bacteria develop resistance
is alarming. Novel antibiotics can become ineffective within
months of market release, which can translate to a low return
on investment to pharmaceutical companies. This challenging
commercial model (coupled with the fact that antibiotic
treatments are usually short-term) often means reduced
antibiotic research and investment from large companies.4
Special Issue: Bismuth - The Magic Element
Received: December 6, 2019
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Scheme 1. General Synthesis of Tetrazolethiols (a−f) and Triazolethiols (g−l) and the Corresponding Bismuth(III)
Complexes 1a−1l, 2a−2f, and 3a−3l
the literature have arisen that focus on major knowledge gaps
relating to silver (nanoparticle) toxicity, environmental
accumulation, and acquired bacterial resistance.6,7 As such,
we have turned our efforts toward investigating bismuth and its
complexes as novel metal-based antimicrobial agents.
Bismuth. The lead-free movement has resulted in the
replacement of lead with bismuth in a wide range of materials
that encompass medicinal applications such as computed
tomography contrast agents, to impart radiopacity, and a
treatment for infections caused by the Gram-negative
bacterium Helicobacter pylori. Most of these uses stem from
the well-accepted nontoxicity of bismuth, which has been
largely described as a surprisingly harmless element consider-
ing its position in the periodic table.8 Bismuth compounds are
most commonly known for their highly effective antimicrobial
properties coupled with generally low human and environ-
mental toxicity. As such, a number of bismuth compounds are
used globally in combination therapies for the treatment of
infections caused by H. pylori including bismuth subsalicylate
(Pepto-Bismol) and colloidal bismuth subcitrate (CBS or De-
Nol).9,10
A recent breakthrough by Sun and co-workers demonstrated
that CBS and related bismuth(III) compounds can inhibit
metallo-β-lactamases (MBLs), the enzymes responsible for
deactivating the β-lactam class of antibiotics.11 Inhibiting
MBLs is potentially crucial in revitalizing the efficacy of β-
lactam antibiotics, for which many are becoming obsolete
because of AMR.
Despite the vast studies that have investigated the synthesis,
characterization, and antimicrobial efficacy of novel bismuth
complexes, there is still much to be explored regarding their
structure−activity relationships and antimicrobial mode of
action. Studies have demonstrated that the efficacy of
organobismuth complexes against microbes is very dependent
on the number and type of coordinating ligands.12−16 Despite
recent progress in this field, few studies have investigated the
effects of these novel complexes toward human or human-like
cells because bismuth is generally considered to be nontoxic.8
This aspect is critical in understanding the selectivity of novel
compounds for the target microbe, over “normal/healthy”
cells. To elaborately state that new bismuth complexes will be
nontoxic based on the known previous safe history of bismuth
in medicine is often misconstrued. Metal toxicity is typically
misunderstood and can be difficult to measure. It is important
to understand that the toxicity of a metal is not constant and is
affected by the surrounding ligands, oxidation state, solubility,
particle size, and shape, as well as the environment that the
metal/metal complex is exposed to.17 Even for bismuth, it is
known that all of the aforementioned factors can have a
significant influence on their stability (e.g., pH-dependent
ligand exchange and cluster formation), targeted activity, and
toxicity.
There are a few studies that look at the toxic side effects of
bismuth compounds, including but not limited to reduced cell
viability from in vitro studies, to observations of neurotoxicity
in humans. It has been reported that prolonged high level
exposure to certain bismuth compounds can lead to altered
neurofunction. Bismuth-induced neurotoxicity (or encephal-
opathy) is a rare side effect that has been reported in patients
taking high and/or prolonged doses of bismuth subnitrate,
subgallate, or subsalicylate.18−20 However, generally with-
drawal of the bismuth drug leads to complete recovery in
short time periods.20 An in vitro study from 2005 reports the
reduced viability of testicular macrophages upon exposure to
B
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concentrations of bismuth citrate as low as 6.25 μM bismuth
content.21 These examples emphasize the importance of
assessing the toxicity of novel bismuth compounds and how
they can affect the viability of cells: we cannot assume they will
be safe.
conditions (300 W, 50 °C) in ethanol. After removal of the
reaction vessel from the microwave, fine needles developed
rapidly from the reaction solution and were filtered to give
complexes 1a−1f.
The formation of bismuth monotriazolethiolate complexes
1g−1l (BiPh2L, where L-Na is a sodium triazolethiolate
synthesized in situ) was achieved following a salt metathesis
reaction between BiPh2Cl and the sodium salts of triazolethiols
g−l. The desired complexes precipitated from the reaction
mixture and were isolated as pure solids after filtration and
washing with cold water and acetone.
Bistetrazolethiolate complexes (2a−2f; BiPhL2) were
synthesized from the reaction of BiPh3 with tetrazolethiols
(a−f) in a 1:2.2 ratio. After 40 min of reaction under
microwave conditions (300 W, 70 °C), the desired complexes
were isolated as mixtures of mono (1a−1f) and bis complexes
(2a−2f). Purification of the crude mixtures was achieved by
trituration, recrystallization, and/or reverse-phase high-perfo-
mance liquid chromatography (HPLC), which resulted in the
isolation of compounds 2a−2f. Unfortunately, bistriazole
complexes could not be obtained as pure complexes because
the aforementioned reaction conditions with triazolethiols g−l
yielded a mixture of bismuth(III) mono- and bisthiolates that
proved to be inseparable by a number of purification
techniques. Attempts were made to control the formation of
the bistriazoles by using solvent-free conditions, salt metathesis
at reflux, and low temperatures (−75 and 0 °C), varying the
reaction times and different solvent systems [tetrahydrofuran
(THF), toluene, and hexane]. However, all of the afore-
mentioned conditions resulted in the unsuccessful formation of
the desired bistriazoles as single products. The mixtures that
were isolated from the reaction solutions were unable to be
separated via crystallization and a range of chromatography
techniques (flash and HPLC) for the reason that the mono-
and bistriazole species appeared to be in flux in solution. Given
the lack of purity, these mixtures were not considered for use
in biological studies.
Bismuth Heterocyclic Thiols. N-Heterocyclic compounds
play an important role in medicinal chemistry and are the
active components in a range of drug molecules. Their
medicinal properties include but are not limited to antiviral,
antiinflammatory, analgesic, anticonvulsant, antiparasitic, anti-
diabetic, antihistaminic, antineuropathic, antihypertensive, as
well as antifungal and antibacterial.22,23 Resulting from the
increased interest in these organic molecules and improved
synthetic techniques, research has expanded into their use in
bioinorganic chemistry, exploring the coordination of N-
heterocyclic compounds to metals, with subsequent inves-
tigations into their medicinal properties. The motivation
behind the inclusion of a sulfur moiety in these N-heterocyclic
compounds is due to the thiophilic nature of bismuth and the
hydrolytic and thermodynamic stability of bismuth thiolates.
Despite this stability, Bi−S bonds are labile, which often
translates to interesting interactions in biological systems with
sulfur-containing proteins and peptides. While bismuth(III)
thiolate complexes have been reported previously,24,25 only a
small number have been assessed for their antimicrobial
activity.12,13,26−28
Herein we explore the synthesis, characterization, and
biological evaluation of a series of tetrazole- and triazolethiols
(thiones) and their corresponding bismuth(III) complexes. To
probe for possible structure−activity trends, both homoleptic
(BiL3) and heteroleptic (BiPh2L and BiPhL2) bismuth
thiolates were targeted, where LH is either a series of
substituted 1-(phenyl)-1H-tetrazole-5-thiols (thiones) or
1,2,4-triazole-3-thiols (thiones).
Free tetrazole- and triazolethiols (thiones) and their
corresponding bismuth(III) complexes were tested against
four clinically relevant strains of bacteria; methicillin-resistant
Staphylococcus aureus (MRSA), vancomycin-resistant Enter-
ococcus faecalis (VRE), Escherichia coli (E. coli), and
Pseudomonas aeruginosa (P. aeruginosa). Furthermore, to
probe their selectivity, the complexes were all assessed against
cos-7 monkey kidney cells using the CellTiter-Blue viability
assay and red blood cells using a hemolysis assay.
Tristhiolato complexes (3a−3l; BiL3, where LH is a
tetrazole- or triazolethiol) were prepared following the
overnight reaction of Bi(OtBu) with either tetrazolethiols
(a−f) or triazolethiols (g−l) in a 1:3 ratio under Schlenk
conditions. The synthetic pathways for all bismuth thiolates are
outlined in Scheme 1.
Characterization. The compositions of all of the
triazolethiols were confirmed by analytical methods such as
1H NMR, which matched the previously reported data.30 The
RESULTS AND DISCUSSION
■
Synthesis. Tetrazolethiols (thiones) were synthesized
based on previously published methods,29 via the [3 + 2]
cycloaddition of a substituted aryl isothiocyanate with sodium
azide. After 5 h of heating at reflux in water, the desired
compounds were acquired in good-to-moderate yields.
Triazolethiols (thiones) were synthesized in a two-step
reaction process according to previously published methods.30
First, a hydrazine carbothioamide derivative was formed
following the reaction of an aryl isothiocyanate and isonicotinic
hydrazide. Oxidative cyclization to the corresponding triazole
was achieved after the addition of sodium hydroxide at reflux.
The synthetic pathways for both the tetrazole- and
triazolethiols and their corresponding yields (%) are outlined
in Scheme 1 below.
1
compositions of the tetrazolethiols were confirmed by H and
13C NMR, IR spectroscopy and electrospray ionization mass
spectrometry (ESI-MS). The structures and compositions of
the heteroleptic complexes 1a−1l (BiPh2L) and 2a−2f
(BiPhL2) and the homoleptic complexes 3a−3l (BiL3) were
also confirmed by 1H and 13C NMR, IR spectroscopy, ESI-MS,
and elemental analysis (see the Experimental Section and
Supporting Information for details). In general, the hetero-
leptic bismuth(III) thiolates displayed a downfield shift of the
1
phenyl signals in both H and 13C NMR, consistent with
previously published bismuth complexes.13,15,28 For instance,
in the case of complex 1c, the phenyl protons are shifted to
8.27 (o), 7.57 (m), and 7.38 (p) ppm, all at lower frequencies
relative to the analogous protons in BiPh3 [δ = 7.76 (o), 7.38
(m), and 7.31 (p)]. The chemical shifts and integrals of the
residual phenylbismuth and thiolate ligand signals in 1a−1l
and 2a−2f also support the composition of bismuth(III)
The bismuth monotetrazolethiolate complexes 1a−1f
(BiPh2L, where LH is a tetrazolethiol) were prepared by
treating BiPh3 with one of a series of substituted 1-(phenyl)-
1H-tetrazole-5-thiols (a−f) in a 1.2:1 ratio under microwave
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Figure 1. Extended polymeric structure of 1a showing thermal ellipsoids at 50% probability. Hydrogen atoms have been omitted for clarity.
Selected bond lengths (Å): Bi(1)−S(1), 2.647(2); Bi(1)−N(2), 2.779(7); Bi(1)−C(1), 2.243(8); Bi(1)−N(2), 2.779(7); Bi(1)−C(7), 2.237(8).
Selected bond angles (deg): S(1)−Bi(1)−N(2), 171.07(16); C(1)−Bi(1)−S(1), 85.9(2); C(1)−Bi(1)−N(2), 90.0(3); C(7)−Bi(1)−S(1),
89.2(2).
complexes. For the homoleptic complexes 3a−3l (BiL3), the
disappearance of the stretching frequency corresponding to S−
H at approximately 2550−2600 cm−1 suggests coordination to
the bismuth metal center through the sulfur (thiolate forms of
a−l). High-resolution mass spectrometry also confirmed
formation of the homoleptic complexes 3a−3l. A number of
structures were also confirmed by single-crystal X-ray
diffraction (XRD), which confirmed the data obtained from
other analytical techniques (see below).
X-ray Crystallography. The crystals of 11 heteroleptic
bismuth(III) thiolate complexes were obtained from the slow
evaporation of either toluene, dimethyl sulfoxide (DMSO), or
DMSO/toluene solutions of the isolated complexes. Using
single-crystal XRD, the solid-state structures were elucidated.
The asymmetric units observed for the monotetrazole and
triazolethiolate complexes show structural similarities; how-
Figure 2. Molecular structure of 1l showing thermal ellipsoids at 50%
ever, they do not appear to be analogous. Between the mono-
and bisbismuth(III) structures (complex series 1 and 2,
respectively), the bismuth(III) core adopts five- and six-
coordinate environments. The environments vary from
distorted tetrahedral, distorted disphenoidal/trigonal-bipyra-
midal, and square-pyramidal geometries, with all structures
incorporating an empty void, presumed to be occupied by the
stereochemically active lone pair (SALP). Variations in the
geometry and coordination number of these structures appear
to be influenced by the solvent system used for crystallization
and can be observed in each structure.
Complexes 1a, 1d, 1f, and 1g all appear to be isostructural
and crystallize as a weakly linked 1D polymer, with the
bismuth(III) center adopting a six-coordinate, distorted
octahedral geometry (Figure 1). The length of the bismuth
core interaction for each complex is within the sum of the van
der Waals radii of bismuth and nitrogen, and the Bi−S bond
distances listed are typical of bismuth(III) arylthiolate
complexes.13,28,31,32 These structural motifs have been
observed within previously synthesized bissulfonate com-
plexes.33 Similar polymeric chain-like structures have been
observed where the four-coordinate bismuth(III) atoms are
bridged between a dative nitrogen interaction from either an
adjacent triazole or tetrazole group or pyridyl nitrogen. The
structures present near-linear S−Bi−N bond angles with an
average value of 170.6°.
probability. Hydrogen atoms have been omitted for clarity. Selected
bond lengths (Å): Bi(1)−C(1), 2.263(6); Bi(1)−C(7), 2.238(6);
Bi(1)−O(1), 2.616(4); Bi(1)−S(1), 2.6601(13). Selected bond
angles (deg): S(1)−Bi(1)−O(1), 158.39(9); C(7)−Bi(1)−C(1),
97.6(2); S(1)−Bi(1)−C(1), 92.83(14).
The spatial arrangement of this homometallic monomer unit is
similar to previously synthesized heteroleptic sulfonate
complexes.33 The planes formed by the S−Bi−O and C−
Bi−C interactions display angles of 158.1(5) and 99.0(11)°,
leaving a large void across the S−Bi−O bond and trans to the
two phenyl groups. Again, this void is expected to be occupied
by the SALP.31
Analysis of the bis structures 2b and 2d shows the bismuth
core adopting a five-coordinate geometry, where the fifth
interaction can be attributed to a tetrazolato nitrogen. In the
example 2d, the overall geometry of the structure is a
dimerized distorted square-based pyramid as a result of the
covalent Bi−O and Bi−S bonds and two dative interactions
(Figure 3). The involvement of the SALP from the bismuth
core forming two dative interactions with one DMSO and a
nitrogen atom from a neighboring tetrazolethiolate can be
observed, with bond lengths of 2.447(4) and 2.700(4) Å. The
O−Bi−S and S−Bi−N angles both sit at 167.64(10) and
167.61(10)°; these angles are consistent with previously
reported bismuth thiolate structures.13
Complexes 1h, 1k, and 1l obtained from mixtures of DMSO
and toluene all adopt distorted disphenoidal motifs (Figure 2).
Antibacterial Studies. Disk Diffusion and Minimum
Inhibition Concentrations (MICs). All of the parent tetrazole-
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Figure 3. Molecular structure of 2d showing thermal ellipsoids at 50% probability. Hydrogen atoms have been omitted for clarity. Selected bond
lengths (Å): Bi(1)−S(7), 2.6859(13); Bi(1)−S(3), 2.7421(14); Bi(1)−N(30), 2.706(4); Bi(1)−C(33), 2.248(5); Bi(1)−O(4), 2.444(4); Bi(3)−
S(11), 2.7475(14); Bi(3)−S(1), 2.6876(13); Bi(3)−N(46), 2.700(4); Bi(3)−O(9), 2.447(4); Bi(3)−C(2), 2.252(5). Selected bond angles (deg):
S(7)−Bi(1)−N(30), 167.61(10); O(4)−Bi(1)−S(3), 168.18(9); C(33)−Bi(1)−S(7), 82.60(13); C(33)−Bi(1)−N(30), 87.13(16); N(30)−
Bi(1)−S(3), 88.77(10). One of two complex moieties of the asymmetric unit is shown. Disorder of the secondary residual DMSO solvent molecule
has been modeled over two positions and omitted for clarity (Figure S1).
and triazolethiols and their corresponding bismuth(III)
complexes were screened against four clinically relevant strains
of bacteria: two Gram-positive (MRSA and VRE) and two
Gram-negative (E. coli and P. aeruginosa). The preliminary
activity of the complexes against the bacteria was assessed
using a Kirby−Bauer disk diffusion assay, at a set concentration
of 400 μg/mL.
Disk diffusion assays revealed that only the bismuth(III)
monothiolate (1a−1l; BiPh2L) and bisthiolate (2a−2f;
BiPhL2) complexes inhibited the growth of bacteria at 400
μg/mL, with clear zones of inhibition observed for all of these
particular complexes against all four strains of bacteria. The
parent tetrazole- and triazolethiols (thiones), as well as the
bismuth(III) tristhiolate complexes (3a−3l; BiL3), did not
show any antibacterial activity, with a confluent lawn of
bacteria observed against all strains of bacteria. This marked
difference in activity between homo- and heteroleptic bismuth-
(III) complexes has been observed previously.13,16
The in vitro activity of the active heteroleptic complexes
1a−1l and 2a−2f was next evaluated by calculating the MIC
against the aforementioned strains of bacteria using a broth
microdilution method (see the Experimental Section for
details). The MIC results demonstrated the significant
antibiotic activity of all of the heteroleptic (mono and bis)
bismuth(III) complexes from both classes of azole compounds,
against all four bacterial strains tested (Table 1). The
complexes (1a−1l and 2a−2f) proved to be a highly active
species, with MIC values ranging from 0.39 to 21.50 μM
(0.67−25 μg/mL; Table 1). In general, higher activities were
seen for the tetrazole-containing complexes (1a−1f and 2a−
2f) relative to the triazole complexes (1g−1l). In order to
confirm the lack of activity of the homoleptic bismuth
complexes 3a−3l (as shown in disk diffusion assays), MIC
assays were conducted for these compounds against the four
strains of bacteria. Unsurprisingly, all homoleptic complexes
Table 1. Antibacterial Activities of Complexes 1a−1l, 2a−2f,
a
and BiPh3 against MRSA, VRE, E. coli, and P. aeruginosa
μM (μg/mL)
MRSA
VRE
E. coli
P. aeruginosa
1a
1b
1c
1d
1e
1f
2a
2b
2c
2d
2e
2f
1g
1h
1i
1j
1k
1l
2.82 (1.56)
2.68 (1.56)
2.68 (1.56)
2.72 (1.56)
2.74 (1.56)
5.33 (3.13)
4.67 (3.13)
4.30 (3.13)
4.30 (3.13)
4.41 (3.13)
4.46 (3.13)
4.27 (3.13)
12.5 (9.90)
12.5 (10.25)
12.5 (10.65)
12.5 (21.50)
12.5 (10.35)
12.5 (10.60)
28.40 (12.5)
5.63 (3.13)
5.36 (3.13)
5.36 (3.13)
5.44 (3.13)
2.74 (1.56)
2.67 (1.56)
2.33 (1.56)
2.15 (1.56)
2.15 (1.56)
2.20 (1.56)
2.23 (1.56)
2.14 (1.56)
3.13 (2.47)
3.13 (2.56)
6.25 (5.33)
12.5 (10.75)
6.25 (5.18)
6.25 (5.30)
14.20 (6.25)
1.41 (0.78)
1.34 (0.78)
0.67 (0.39)
0.68 (0.39)
1.37 (0.78)
2.67 (1.56)
2.33 (1.56)
2.15 (1.56)
2.15 (1.56)
2.20 (1.56)
2.23(1.56)
2.14 (1.56)
12.5 (9.90)
12.5 (10.25)
12.5 (10.65)
12.5 (21.50)
12.5 (10.35)
6.25 (5.30)
(>50)
2.82 (1.56)
2.68 (1.56)
2.68 (1.56)
2.72 (1.56)
2.74 (1.56)
5.33 (3.13)
4.67 (3.13)
4.30 (3.13)
4.30 (3.13)
4.41 (3.13)
4.46 (3.13)
4.27 (3.13)
25 (19.80)
25 (20.50)
25 (21.30)
25 (21.50)
25 (10.35)
25 (21.20)
(>50)
BiPh3
a
The MICs of complexes 3a−3f were not included in the table
because these complexes were shown to possess no antibacterial
activities at 400 μg/mL in disk diffusion assays.
(3a−3l) showed no activity at the highest concentration tested
(400 μg/mL).
Interestingly, for the tetrazole complexes (1a−1f and 2a−
2f), the lowest MICs were observed against the Gram-negative
strain, E. coli. This result was surprising because it has been
previously shown that bismuth(III) complexes tend to inhibit
Gram-positive bacteria at much lower concentrations13,28,34 (as
E
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Figure 4. Cellular uptake of bismuth(III) tetrazolethiolate complexes 1f, 2f, and 3f (a and b) and bismuth(III) triazolethiolate complexes 1i, 3i, and
BiPh3 (c and d), as measured by ICP-MS.
was the case with triazole complexes 1g−1l), believed to be a
result of the inability of such compounds to cross the
additional outer membrane present in Gram-negative
bacteria.35 The potent activity of the tetrazole compounds
(1a−1f and 2a−2f) relative to the triazole complexes (1g−1l)
against the Gram-negative pathogens E. coli and P. aeruginosa is
not a consequence of their respective lipophilicities. C log P
values (predicted using ChemDraw 18.1; Table S2) suggest
that there is no significant difference in their projected
lipophilicities that may have contributed to one set of
complexes being more active than the other against these
particular strains of Gram-negative bacteria.
The small range of MICs observed across the spectrum of
complexes suggests that subtle changes in the thiolate ligand
have little impact on the antibacterial activity. For instance,
against MRSA, both the active tetrazole- (1a−1f and 2a−2f)
and triazole-containing complexes (1g−1l) have only a 2-fold
difference in their MICs between the most active complexes in
their series (e.g., 12.5 μM: 1g−1h and 1k−1l) and the least
active complex (e.g., 25 μM: 1j).
Despite there being a clear difference in the activity between
the homo- and heteroleptic bismuth thiolate complexes, the
MIC results demonstrated that the degree of thiolate
substitution (i.e., mono- vs bisthiolate) also has little impact
on the biological activity of the complexes. When the MICs of
the mono complexes 1a−1f (BiPh2L) are compared with the
analogous bis complexes 2a−2f (BiPhL2), there are no
substantial differences in their respective MICs against all
four strains of bacteria (Table 1).
thiolates and the heteroleptic analogues, the cellular uptake of
Bi209 in MRSA and E. coli was evaluated and quantified using
inductively coupled plasma mass spectrometry (ICP-MS). The
assay was conducted in order to further understand whether
the increased biological activity of the heteroleptic bismuth-
(III) complexes could be a direct result of an increase in the
cellular uptake of these particular complexes relative to their
homoleptic counterparts. Since previous studies had indicated
that, within each class of compound, there was little difference
in the antimicrobial activity (Table 1), only one ligand for each
class (tetrazole and triazole) was chosen for this study (f and
i). A control, with no bismuth complex added, and BiPh3 were
included in this study. In order to directly compare the uptake
of different complexes at the same concentration, complex
concentrations of 5.3 μM (for complexes 1f, 2f, and 3f) and
12.5 μM (for complexes 1i, 3i, and BiPh3) were added to
MRSA and E. coli bacteria.
After the complexes were exposed to bacterial cells for 24 h,
the cells were first washed and digested, before Bi209 content
was analyzed via ICP-MS (see the Experimental Section for
details of the procedure). The results obtained from ICP-MS
revealed that there is a significant increase in the cellular
uptake of Bi209 for the most biologically active (heteroleptic)
complexes (as shown by MICs), 1f, 1i, and 2f, relative to their
homoleptic bismuth(III) tristhiolate counterparts, 3f and 3i, as
well as BiPh3. This trend is clearly evident from the uptake
graphs shown in Figure 4. The values obtained were also
consistent between bacterial strains (MRSA and E. coli), with
the Bi209 uptake significantly lower for homoleptic bismuth-
(III) complexes (3f and 3i), further suggesting a direct
correlation between the complex cell uptake and biological
Bacterial Cell Uptake. To investigate the differences in
the antibacterial activity between the homoleptic bismuth
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activity. It is clearly evident that complexes of the type BiPh2L
have the greatest uptake in bacterial cells, followed by the bis
complexes (BiPhL2), with the tristetrazole/triazole complexes
(BiL3) the lowest (mono > bis > tris). This reinforces the
observation that higher uptake is correlated with higher
antibacterial activity and that the activity is clearly linked to the
lepticity of the complex. In general, it can be concluded from
this assay that there is a direct correlation between the
antibacterial activity and complex cellular uptake, with the
most active complexes giving the largest readings of Bi209/106
bacterial cells.
Time-Kill Assays. Continuing to probe the mode of action
(bacteriostatic vs bactericidal) for the bismuth thiolates, we
performed time-kill assays on example complexes 1b and 2b
using MRSA and E. coli. Aliquots of brain heart infusion (BHI)
broth containing overnight cultured bacteria with various
concentrations of complexes 1b and 2b were serially diluted in
phosphate-buffered solution (PBS) and plated on freshly
prepared nutrient agar or Luria−Bertani (LB) plates at 0, 1, 3,
6, and 24 h. Incubation of the inoculated plates at 37 °C for 24
h revealed the bacterial survivors as colony-forming units
(CFU) per milliliter.
Colony counting of bacterial survivors revealed the
concentration-dependent killing profiles of complexes 1b and
2b, with the antimicrobial action observed as being
bacteriostatic at MIC and MIC × 2 concentrations (Figure
5). The number of viable cells remained consistent with the
starting inoculum (approximately 5 × 105 CFU/mL) at MIC
and MIC × 2 concentrations at all time points analyzed in the
24 h assay. However, at MIC × 4 concentrations, complexes
1b and 2b both displayed rapid bactericidal activity against
both strains of bacteria (Figure 5). After just 1 h, no viable cells
were observed when E. coli was exposed to complexes 1b and
2b at MIC × 4 concentrations. For MRSA, slower killing
kinetics were shown as no viable cells were observed at the 6 h
time point for complex 1b, while no viable cells were present
after 24 h of exposure of cells to complex 2b. It is, however,
important to note that no time points were analyzed between 6
and 24 h, and it is likely that complex 2b (at MIC × 4) is
sufficient enough to completely eradicate all remaining
bacterial survivors within these two particular time frames.
Bacterial Membrane Integrity (Scanning Electron
Microscopy, SEM). On the basis of the results of the uptake
and time-kill assays, we were encouraged to further probe the
mechanism by which the bismuth thiolates were killing
bacterial cells, using SEM imaging. Complex 1b was used as
a representative example, and SEM images were captured of
MRSA cells exposed to this complex at a range of different
concentrations (MIC × 1/2, MIC, MIC × 2, and MIC × 4) for
20 h. Untreated MRSA cells, used as a no additive control,
remained morphologically intact, retaining their typical grape-
like structures with smooth surfaces and no visible cell damage
(Figure 6a). In contrast, cells exposed to complex 1b (at all
concentrations), looked visually unhealthy with distorted cell
membranes (Figure 6b−e). As expected, at MIC × 4
concentrations, there was a significant decrease in the number
of bacterial cells (viable and lysed) present on the surface,
while the limited cells that did remain displayed a high degree
of distortion in their cell shape. Surprisingly, and in contrast to
time-kill assays, at MIC × 2, there was a significant reduction
in the number of cells that remained after overnight exposure,
and those that did remain visually looked impaired. Even at
lower concentrations (sub-MIC and MIC), cells appeared
morphologically unhealthy with bleb-like structures.36 The
increased abundance of extracellular debris, present on the
surface of films exposed to 1b, further suggests cell membrane
destruction, and hence these SEM images are the first
definitive evidence that bismuth(III) thiolate complexes
interfere with bacterial membrane integrity.
Typically, highly lipophilic cell membrane targeting
compounds act in a nonspecific detergent-like manner, which
often excludes them for use as potential novel antibiotics.37
Consequently, we wanted to address these potential selectivity
concerns and assess the effect of bismuth thiolate complexes
on the viability of mammalian cells.
Mammalian Cell Cytotoxicity. In attempting to develop
novel antibacterial compounds, it is crucial that the inhibition
of bacterial growth occurs at low compound concentrations,
with high degrees of selectivity for prokaryotic cells over
eukaryotic (mammalian) cells. All bismuth complexes were
assessed against cos-7 cells using the CellTiter-Blue viability
assay. The homoleptic bismuth(III) complexes, 3a−3l (BiL3),
did not affect the cell viability up to concentrations of 50 μM.
However, when cos-7 cells were exposed to the heteroleptic
bismuth(III) thiolate complexes for 24 h (1a−1l and 2a−2g),
a significant reduction in the cellular viability was observed,
with the half-maximal inhibition concentrations (IC50) in the
low micromolar range (Figure 7). Similar to the trends
observed in the antibacterial assays, the monothiolate
complexes 1a−1l (BiPh2L) showed generally lower IC50 values
(i.e., more significant reduction in the cellular viability)
compared to the bisthiolate complexes (Figures 7 and 8). It
is clear that the degree of thiolate substitution on the bismuth
metal center has a marked influence on not only the
antibacterial activity but also the mammalian cell toxicity.
Figure 5. Time-kill assays of complexes 1b and 2b (at various
concentrations) against E. coli (top) and MRSA (bottom).
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1
Figure 6. SEM images of (a) untreated MRSA cells and MRSA cells exposed to complex 1b at (b) MIC × /2, (c) MIC, (d) MIC × 2, and (e)
MIC × 4. Scale bars: 1 μM (a−c and e) and 100 nm (d).
Figure 7. Comparison of percent cell viabilities of both tetrazole- and triazolethiolate complexes 1a−1l (BiPh2L) against cos-7 cells. Dose response
curves were generated over a range of concentrations (24 nM to 50 μM) in the appropriate culture media from 10 mM DMSO stock solutions. All
readings were compared spectroscopically to that of a nontreated control, and the percentage growth inhibition was calculated. A DMSO control
was also included at the same range of concentrations.
On the basis of the cell viability studies, the trends show that
there is little variation between the bismuth(III) triazole- and
tetrazolethiolates. Small chemical changes in the thiolates show
some structure−activity relationships. Interestingly, complex 1l
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Figure 8. Comparison of percent cell viability of the tetrazolethiolate complexes 2a−2f (BiPhL2) against cos-7 cells. Dose response curves were
generated over a range of concentrations (24 nM to 50 μM) in the appropriate culture media from 10 mM DMSO stock solutions. All readings
were compared spectroscopically to that of a nontreated control, and the percentage growth inhibition was calculated. A DMSO control was also
included at the same range of concentrations.
(IC50 = 0.44 μM), which contains two methyl groups, has an
IC50 value that is an order of magnitude more potent than any
other complex reported. This could be a consequence of its
apparent increase in lipophilicity (Table S2).
destruction of MRSA cells by bismuth(III) thiolates, could
explain this poor selectivity. This is due to similarities in the
membrane of bacteria and mammalian cells, which share many
common structural and biochemical features.
After assessment of both the antimicrobial activity and effect
on the cell viability against cos-7 cells, it is vital to evaluate the
ability of the complexes to selectivity inhibit pathogenic
bacteria in the presence of mammalian cells. Consequently, the
selectivity index (SI) values of all biologically active complexes
have been calculated. SI is defined as the ratio between the
antibacterial activity (MIC) and IC50 against cos-7 cells, i.e., SI
= IC50/MIC. High SIs are favorable because they show a drug’s
potential to have a safe in vitro profile.38 The SI values of
complexes 1a−1l and 2a−2f using MRSA as the reference
bacterial strain are shown in Table 2. The calculated SIs
Determination of the Hemolytic Activity. Due in large
part to the rapid bactericidal activity displayed by the
complexes in time-kill assays as well as the apparent
cytotoxicity effects against cos-7 cells, complexes 1b, 2b, and
3b as well as BiPh3 were evaluated for their hemolytic activity
against red blood cells at concentrations exceeding MIC × 4
for 1b and 2b (25 μg/mL). Trixton X-100 at 2% served as a
positive control, while DMSO at 2% served as a negative
control. The release of hemoglobin, measured at 540 nm, was
used to quantify the membrane-damaging properties of the
bismuth tetrazolethiolates. At this particular concentration (25
μg/mL), all three of the bismuth tetrazolethiolates (1b, 2b,
and 3b) as well as BiPh3 displayed no significant hemolytic
activity39 relative to the positive control (Figure 9).
a
Table 2. SI Values of Complexes 1a−1l and 2a−2f
complex
SI
complex
SI
1a
1b
1c
1d
1e
1f
2a
2b
2c
1.63
1.19
0.83
2.03
1.81
0.78
1.22
1.71
1.50
1g
1h
1i
1j
1k
1l
2d
2e
2f
0.46
0.37
0.31
0.39
0.81
0.03
1.58
1.03
1.22
a
The SI is calculated using the MICs against MRSA and the IC50
values against cos-7 cells.
highlight the poor selectivity for bacterial cells that the
bismuth(III) thiolate complexes (1a−1l and 2a−2f) possess.
In general, the SIs of bismuth tetrazole complexes 1a−1f and
2a−2f are just over 1, showing a small and nonsignificant
affinity for bacterial cells over mammalian cells. Conversely,
the bismuth triazole complexes 1g−1l seem to display a greater
affinity toward mammalian cos-7 cells, with complex 1l being
the most selectively cytotoxic. The aforementioned complex
(1l) has an IC50 value over 10 times lower than its respective
MIC against MRSA. Accordingly, it can be concluded that the
these bismuth(III) thiolate complexes possess such poor SIs
that to consider them as novel antimicrobials without extensive
structural modifications is highly implausible. The previously
obtained SEM images, which point toward the cell membrane
Figure 9. Percentage of hemolysis induced by complexes 1b, 2b, and
3b and BiPh3.
Interestingly, despite showing considerable cytotoxicity against
cos-7 cells, the hemolytic activity of 1b and 2b suggests a much
greater selectivity toward bacterial cells over mammalian cells.
In comparison to the positive control (100% hemolysis), at 25
μg/mL complexes 1b and 2b caused only 11 and 14%
hemolysis, respectively. The stark difference between the
membrane-destructive effects of compounds against cos-7 cells
and red blood cells is not uncommonly observed in the
literature and has been attributed to the differences in the
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frozen (−80 °C) glycerol stocks (15% v/v) on either freshly prepared
nutrient agar (Gram-positive) or LB agar (Gram-negative) at 37 °C
for 24 h. Single colonies were then placed in either 3 mL of BHI
media or LB media and incubated at 37 °C and 180 rpm for 18 h.
Disk Diffusion. The antibiotic activity of the synthesized complexes
was determined using a Kirby−Bauer disk diffusion method. Briefly,
200 μL of a 1:100 dilution of overnight-cultured bacteria was applied
to either nutrient agar or LB agar plates in order to form the confluent
bacterial lawn. Sterile disks, inoculated with 60 μL of synthesized
complexes, were then placed on the streaked agar plates before being
incubated for 24 h at 37 °C. After incubation, the zone of inhibition
(if it existed) was recorded and measured in millimeters.
composition of the aforementioned membranes and their
respective glycocalyx.40 It can therefore be hypothesized that
the biologically active bismuth(III) thiolates may have a higher
affinity for bacterial and fibroblast-like cell membranes relative
to the membrane of red blood cells. Conversely, the reduced
time frame at which the red blood cells are exposed to the
complexes (2 h) is much shorter than that in cytotoxicity (24
h) and bacterial (20 h) studies, and it may also be apparent
that this has a significant effect on the reduced toxicity toward
erythrocytes.
MIC. The MIC of the synthesized complexes was determined using
a broth microdilution method according to Clinical Standard
Laboratory Institute (CSLI) guidelines. Briefly, 25 μL of a 1:100
dilution of an overnight culture of bacteria (to give approximately 5 ×
105 CFU/mL) was added to falcon tubes containing 1 mL of serially
diluted complexes in either BHI (Gram-positive) or LB (Gram-
negative) media. The MIC was determined as the lowest
concentration of complex where visible growth of the bacteria was
inhibited following overnight incubation (37 °C, 180 rpm) for 20 h.
Time-Kill Assay. The mode of inhibitory activity of the synthesized
complexes was determined by measuring the decrease in the CFU
with time. First, an overnight culture of bacteria was centrifuged for
15 min at 4000 rpm and washed three times with 1 mL of PBS. The
bacterial pellets were resuspended in either BHI or LB and diluted to
approximately 5 × 105 CFU/mL before varying concentrations of
complexes were added and incubated at 37 °C and 180 rpm. Bacterial
survivors were determined by plating serial dilutions onto agar plates
at 0, 1, 3, 6, and 24 h after incubation.
SEM. The cell morphology of MRSA cells exposed to bismuth
complexes was analyzed by SEM. A single colony of MRSA was added
to a falcon tube containing 3 mL of BHI broth and incubated
overnight at 37 °C and 180 rpm. The overnight-cultured bacteria was
then diluted to approximately 5 × 105 CFU/mL before complexes
were added and incubated overnight at 37 °C and 180 rpm for a
further 20 h. Following overnight incubation, the bacterial solutions
were centrifuged at 4000 rpm for 15 min, with the supernatant
discarded for use. Pelleted cells were first washed three times with 1
mL of PBS before being fixed in 2.5% glutaraldehyde and 2%
paraformaldehyde (in 0.1 M sodium cacodylate buffer) for 1 h at
room temperature. The fixed cells were then washed three times in a
fresh sodium cacodylate buffer before being post-fixed in 1% osmium
tetroxide in a cacodylate buffer at room temperature. Post-fixed cells
were washed three times with Milli-Q water, and 100 μL aliquots were
incubated on round coverslips coated with 0.1% polyethylenimine for
45 min. Following incubation, excess cells were washed off by
immersing the coverslips in water, and the coverslips with adhered
cells were then dehydrated in increasing concentrations of ethanol,
consisting of 30, 50, 70, 90, and 2 × 100% ethanol for 10 min each.
Dehydrated cells on the coverslips were dried with a Bal-Tec CPD
030 critical point drier. The coverslips were then mounted, using
carbon adhesive tape, onto SEM specimen stubs before being coated
with iridium. Analyses were carried out on a JEOL JSM-7001F field-
emission-gun scanning electron microscope.
CONCLUSION
■
Despite bismuth generally being considered to be nontoxic, we
have demonstrated that its complexes should not be regarded
as inherently safe just because of their metal center. The
bismuth(III) thiolates detailed in this study demonstrate that
the particular ligand class and number surrounding the metal
center play significant roles in the cytotoxicity of these bismuth
complexes. A total of 30 new bismuth(III) thiolates were
synthesized from the reaction of tetrazole- and triazolethiols
(thiones) with either BiPh3, BiPh2Cl, or Bi(OtBu). Preliminary
antibacterial assays conducted on these compounds revealed
that the degree of substitution around the bismuth metal
center is key, with potent activity only seen for heteroleptic
complexes (BiPh2L or BiPhL2) containing either one or two
azole ligands. Homoleptic complexes (BiL3) with three
tetrazole or triazole ligands coordinated to the metal center
were shown to be completely inactive, and the results obtained
from ICP-MS analysis suggest that this is a direct result of a
decrease in their cellular uptake relative to their heteroleptic
counterparts. Further mode of action studies conducted on a
number of complexes point toward the cell membrane being
the target site of action for bismuth(III) thiolates. The cell
membranes of bacterial and mammalian cells share many
common structural and biochemical characteristics. Hence, at
this point in the drug discovery program, we became
concerned that the bismuth(III) thiolates could have a
nonspecific mode of action, resulting in cytotoxic effects and
poor SIs. There are a plethora of compounds, both naturally
derived and chemically synthesized, that can kill pathogenic
bacteria, but many of these candidates often also perturb the
membrane of mammalian cells. Subsequently, in vitro cell
viability assays were conducted on bismuth(III) thiolates using
cos-7 cells, which revealed their potent cytotoxic nature. The
heteroleptic complexes that were shown to be active against
bacteria were also shown to be cytotoxic, with no significant
degree of selectivity for bacterial cells. However, in contrast,
hemolytic assays were performed on a number of these
complexes, and no significant hemolysis was observed.
Consequently, this study has highlighted the importance of
using a number of different in vitro cytotoxicity assays in the
development of potentially novel pharmaceuticals. As was the
case with bismuth(III) thiolates, new complexes can appear to
be nontoxic (hemolysis assay) in some circumstances and
considerably cytotoxic in others (CellTiter-Blue, cos-7). These
findings highlight the complexities involved in determining the
metal toxicity and raises the question of whether bismuth really
is the green metal.
Cell Uptake Assay. Overnight cultures of MRSA and E. coli were
first diluted to approximately 1 × 106 CFU/mL and added to falcon
tubes containing the complexes diluted in BHI so that the overall
concentration of the active components was 5.3 μM (for complexes
1f, 2f, and 3f) and 12.5 μM (for complexes 1i and 3i) in solution.
After incubation for 20 h (37 °C, 180 rpm), the falcon tubes were
centrifuged for 15 min at 4000 rpm, with the resulting supernatant
discarded. The leftover pelleted cells were then washed six times with
1 mL of PBS before being resuspended in 3 mL of BHI in order to
determine the CFU/mL of bacteria present (serial dilution and
overnight incubation of nutrient agar plates). The falcon tubes were
then recentrifuged (15 min, 4000 rpm) and the pelleted bacteria
washed a further six times with 1 mL of PBS before 200 μL of
concentrated HNO3 was added and left to sit at 60 °C overnight. The
EXPERIMENTAL SECTION
■
Microbiology. Bacterial Growth. MRSA (M118797, methicillin-
resistant S. aureus), VRE (M846910, vancomycin-resistant E. faecalis),
P. aeruginosa (ATCC27853), and E. coli (G102) were recovered from
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solutions were then diluted with 4.8 mL of 2% HNO3 before the Bi209
composition was measured by ICP-MS.
late ligand (1 equiv) was added. The resulting solution was reacted at
70 °C for 20 min before being left to cool to room temperature. Upon
cooling, the desired compound crashed out of the solution as pure
crystalline solids, which were filtered and collected without the need
for further purification.
GP2: Heteroleptic Monotriazolebisphenylbismuth(III) Complex
Synthesis. All of the following manipulations were carried out under a
nitrogen atmosphere using Schlenk techniques. BiPh2Cl (1 equiv) was
added to a solution of the previously synthesized triazole (1 equiv)
with NaOH (1 equiv) in dry methanol. The resulting solution was
stirred at room temperature for 4 h. After the elapsed time, the
precipitate that formed was filtered and washed with cold water and
acetone to yield the desired complex.
GP3: Heteroleptic Bistetrazolemonophenylbismuth(III) Complex
Synthesis. All of the following manipulations were carried out using a
CEM discover LabMate microwave as follows: BiPh3 (1 equiv) was
first dissolved in 5 mL of methanol before the previously synthesized
tetrazolethiolate ligand (2.2 equiv) was added. The resulting solution
was reacted at 50 °C for 40 min before being left to cool to room
temperature. After subsequent cooling for 2 h, impurities that crashed
out of the solution were removed by filtration, with the resulting
filtrate concentrated in vacuo. The remaining crude mixture was then
washed with cold diethyl ether to remove any unreacted BiPh3, with
the leftover solid being the desired complex.
GP4: Homoleptic Tetrazole/triazolebismuth(III) Complex Syn-
thesis. All of the following manipulations were carried out under a
nitrogen atmosphere using Schlenk techniques. Bi(OtBu)3 (1 equiv)
was added to a solution of the tetrazole/triazolethiolate (3 equiv) in
dry THF at room temperature. The resulting solution was allowed to
stir overnight before being concentrated in vacuo. The crude solid
obtained was washed with cold diethyl ether and filtered to yield the
desired compound.
ICP-MS. Determination of the bismuth content following uptake
experiments was conducted using a PerkinElmer NexION 350
inductively coupled plasma mass spectrometer, measuring bismuth
at 208.98 amu. Raw count rates from the analyses were externally
standardized by means of a calibration curve constructed using a
commercially available bismuth stock solution. An internal standard of
80 ppb lead was used for bismuth analysis. The precision is on the
order of <5% relative standard deviation from the single analyses. The
final bismuth content was averaged from three independent bacterial
experiments.
Cell Viability Assay (Cytotoxicity). All media and supplements
were purchased from Gibco and maintained as directed. Cos-7 cells
were cultured in Dulbecco’s modified Eagle’s medium previously
supplemented with 1% penicillin/streptomycin, 1% glutamax, and
10% fetal bovine serum. The cells were maintained at 37 °C in a 5%
CO2 incubator. A CellTiter-Blue Cell Viability Assay kit was
purchased from Promega Corp. and used to determine the effect
that bismuth complexes had on the viability of cos-7 cells.
Falcon 96-well flat-bottom plates were initially seeded at 105 cells/
mL with adherent cos-7 cells for 24 h. Compounds 1a−1l, 2a−2h,
and 3a−3l were then dissolved in DMSO to make up a 10 mM
working stock and sequentially diluted out in the appropriate culture
media (50 μM to 24 nM). Following the addition of compounds to
the adherent cells, plates were incubated for an additional 24 h.
CellTiter-Blue was added to the plates at 15% total well volume
according to the manufacturer’s protocol. Assays were then measured
spectroscopically using fluorescence excitation at 544 nm and
emission at 590 nm to determine the cell viability. The compounds
were compared to a no-drug negative control as well as a positive
control of untreated cells to determine the percent inhibition and
percent viability calculated. Fluorescence measurements were
conducted on a BMG-Labtech ClarioStar microplate reader. Three
independent assays were conducted for each compound. Standard
deviations and IC50 values were modeled through a nonlinear
regression (curve fit) on the GraphPad Prism 8 program.
[Bi(Ph)2(C8H7N4S)] (1a). Following GP1, 1-(p-tolyl)-1H-tetrazole-
5-thiol (90 mg, 0.47 mmol) and BiPh3 (247 mg, 0.56 mmol) were
stirred in 7 mL of methanol. The title compound was isolated as light-
1
orange crystals (190 mg, 73%). H NMR (400 MHz, DMSO-d6): δ
8.30−8.23 (m, 4H), 7.59 (t, J = 7.4 Hz, 4H), 7.55−7.49 (m, 2H),
7.39−7.33 (m, 4H), 2.37 (s, 3H). 13C NMR (101 MHz, DMSO-d6):
δ 157.6, 139.2, 137.8, 137.3, 132.0, 131.3, 130.3, 129.8, 127.7, 124.3,
20.7. IR (film, cm−1): ν 3047 (C−H), 1370 (C−N), 821 (C−S). Mp:
184−186 °C. MS (ESI). Calcd for C20H17BiN4S: m/z 555.1059 [(M
+ H)+]. Elem anal. Calcd C, 43.31; H, 3.09; N, 10.11. Found: C,
43.58; H, 2.96; N, 10.00. CCDC 1968625.
Hemolysis Assay. Hemolysis assays were performed as detailed by
Ciornei et al.40 Blood was first centrifuged at 800g for 10 min before
the supernatant and buffy coat layer were discarded before for use.
This step was repeated a further two times before the resulting
erythrocytes were resuspended in PBS (5% v/v). Next, complexes
(overall concentration = 25 μg/mL) were added to individual PCR
tubes containing 1.5 mL of the erythrocytes and incubated for 2 h at
37 °C with gentle end-overend rotation. 2% Trixton X-100 served as a
positive control, while 2% DMSO served as a negative control. After
incubation, the samples were centrifuged at 800g for 10 min. The
percent hemolysis was determined by measuring the absorbance of
the supernatant (release of hemoglobin) at 540 nm and expressed as a
percentage of the positive control’s induced hemolysis.
[Bi(Ph)2(C9H10N5S)] (1b). Following GP1, 1-(4-dimethylamino)-
phenyl)-1H-tetrazole-5-thiol (100 mg, 0.45 mmol) and BiPh3 (237
mg, 0.54 mmol) were stirred in 7 mL of methanol. The title
1
compound was isolated as light-orange crystals (150 mg, 57%). H
NMR (400 MHz, DMSO-d6): δ 8.28−8.23 (m, 4H), 7.58 (dd, J = 8.2
and 7.0 Hz, 4H), 7.40−7.31 (m, 4H), 6.82−6.76 (m, 2H), 2.95 (s,
6H). 13C NMR (101 MHz, DMSO-d6): δ 157.3, 150.6, 137.8, 137.3,
131.3, 130.3, 127.7, 125.4, 122.8, 111.8. IR (film, cm−1): ν 3048 (C−
H), 1371 (C−N), 822 (C−S). Mp: 159 °C. MS (ESI). Calcd for
C21H20BiN5S: m/z 584.1423 [(M + H)+]. Elem anal. Calcd: C, 43.23;
H, 3.40; N, 12.00. Found: C, 42.9; H, 3.13; N, 11.95. CCDC
1968624.
13
41
Chemistry. BiPh3 and Bi(OtBu)3 were synthesized following
literature procedures. Starting materials for the synthesis of thiols
were purchased from the commercial supplies Sigma-Aldrich and
Oakwood Chemicals. BHI broth was obtained from Oxoid Australia
1
Pty. H and 13C NMR spectra were recorded on a Bruker AV600 or
[Bi(Ph)2(C10H11N4S)] (1c). Following GP1, 1-mesityl-1H-tetrazole-
5-thiol (100 mg, 0.45 mmol) and BiPh3 (237 mg, 0.54 mmol) were
stirred in 7 mL of methanol. The title compound was isolated as pale-
Bruker AV400 spectrometer at 600 and 125 MHz or 400 and 100
MHz, respectively. Chemical shifts are quoted in parts per million
(ppm) and are referenced to the residual solvent peak (DMSO). IR
spectroscopy measurements were carried out with a Cary 630 Fourier
transform infrared spectrometer in the range of 4000−500 cm−1. Mass
spectrometry was performed on a Micromass Platform QMS
spectrometer with an electrospray source and a cone voltage of 35
eV. Melting points were obtained using a Stuart Scientific SMP 3
melting point machine. Elemental analysis was carried out by Dr.
Biplop Saha at Monash University.
General Procedures (GP). GP1: Heteroleptic
Monotetrazolebisphenylbismuth(III) Complex Synthesis. All of the
following manipulations were carried out using a CEM discover
LabMate microwave as follows: BiPh3 (1.2 equiv) was first dissolved
in 5 mL of methanol before the previously synthesized tetrazolethio-
1
yellow crystals (163 mg, 63%). H NMR (400 MHz, DMSO-d6): δ
8.27−8.20 (m, 4H), 7.57 (dd, J = 8.3 and 6.9 Hz, 4H), 7.38−7.31 (m,
2H), 7.08−7.03 (m, 2H), 2.31 (s, 3H), 1.79 (s, 6H). 13C NMR (101
MHz, DMSO-d6): δ 140.0, 137.6, 137.3, 135.1, 131.2, 130.3, 130.1,
129.0, 127.7, 20.6, 16.9. IR (film, cm−1): ν 3062 (C−H), 1368 (C−
N), 856 (C−S). Mp: 173 °C. MS (ESI). Calcd for C22H21BiN4S: m/z
505.0855 (PhBiL)+. Elem anal. Calcd C, 45.37; H, 3.63; N, 9.62.
Found: C, 45.39; H, 3.45; N, 9.63.
[Bi(Ph)2(C7H4ClN4S)] (1d). Following GP1, 1-(4-chlorophenyl)-1H-
tetrazole-5-thiol (50 mg, 0.23 mmol) and BiPh3 (125 mg, 0.28 mmol)
were stirred in 5 mL of methanol. The title compound was isolated as
1
light-orange crystals (100 mg, 79%). H NMR (400 MHz, DMSO-
K
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d6): δ 8.31−8.24 (m, 4H), 7.75−7.70 (m, 2H), 7.66−7.56 (m, 6H),
7.36 (td, J = 7.4 and 1.4 Hz, 2H). 13C NMR (151 MHz, DMSO-d6): δ
158.0, 137.8, 133.9, 133.3, 131.4, 130.3 129.5, 127.8, 126.1. IR (film,
cm−1): ν 3047 (C−H), 1368 (C−N), 833 (C−S). Mp: 171−173 °C.
MS (ESI). Calcd for C19H14BiClN4S: m/z 597.0326 [(M + Na)+].
Elem anal. Calcd: C, 39.70; H, 2.45; N, 9.75. Found: C, 39.41; H,
2.49; N, 9.75. CCDC 1968616.
(CC), 824 (C−S), 735 (C−H). Mp: 119−123 °C (dec). MS
(ESI). Calcd for C25H19BiClN4S: m/z 650.0735 [(M + Na)+]. Elem
anal. Calcd: C, 46.13; H, 2.79; N, 8.61. Found: C, 44.95; H, 2.64; N,
8.47.
[Bi(Ph)2(C13H8N4FS)] (1k). Following GP2, BiPh2Cl (200 mg, 0.5
mmol) was reacted with triazolethiol k (148 mg, 0.5 mmol) in
methanol. The title complex was isolated as an off-white solid (121
1
[Bi(Ph)2(C8H7ON4S)] (1e). Following GP1, 1-(4-methoxyphenyl)-
1H-tetrazole-5-thiol (50 mg, 0.22 mmol) and BiPh3 (115 mg, 0.26
mmol) were stirred in 5 mL of methanol. The crude solid present
after the reaction was filtered and then washed with cold diethyl ether
to yield the desired compound as a pale-yellow solid (100 mg, 80%).
1H NMR (400 MHz, DMSO-d6): δ 8.30−8.24 (m, 4H), 7.62−7.52
(m, 6H), 7.35 (ddt, J = 8.8, 7.3, and 1.4 Hz, 2H), 7.12−7.07 (m, 2H),
3.82 (s, 3H). 13C NMR (101 MHz, DMSO-d6): δ 159.7, 137.8, 131.3,
127.7, 127.2, 126.1, 114.5, 55.6. IR (film, cm−1): ν 3008 (C−H),
1367 (C−N), 833 (C−S). Mp: 151 °C. MS (ESI). Calcd for
C20H17BiN4OS: m/z 593.0826 [(M + Na)+]. Elem anal. Calcd: C,
42.11; H, 3.00; N, 9.82. Found: C, 41.83; H, 2.97; N, 10.04.
[Bi(Ph)2(C7H4O2N5S)] (1f). Following GP1, 1-(4-nitrophenyl)-1H-
tetrazole-5-thiol (60 mg, 0.27 mmol) and BiPh3 (142 mg, 0.32 mmol)
were stirred in 5 mL of methanol. The crude solid present after the
reaction was filtered and then washed with cold diethyl ether to yield
the desired compound as a pale-yellow solid (78 mg, 49%). 1H NMR
(400 MHz, DMSO-d6): δ 8.42−8.38 (m, 2H), 8.29 (dt, J = 7.7 and
1.3 Hz, 4H), 8.14 (d, J = 8.7 Hz, 2H), 7.62 (t, J = 7.5 Hz, 4H), 7.37
(tt, J = 7.3 and 1.3 Hz, 2H). 13C NMR (151 MHz, DMSO-d6): δ
158.7, 147.0, 139.4, 137.8, 137.3, 131.4, 130.3, 127.8, 124.9, 124.8. IR
(film, cm−1): ν 3046 (C−H), 1346 (C−N), 852 (C−S). Mp: 175 °C
(dec). MS (ESI). Calcd for C19H14BiN5O2S: m/z 586.0750 [(M +
H)+]. Elem anal. Calcd C, 38.98; H, 2.41; N, 11.96. Found: C, 38.75;
H, 2.05; N, 12.04. CCDC 1968617.
mg, 38%). H NMR (600 MHz, DMSO-d6): δ 8.56−8.46 (m, 1H),
8.25 (dd, J = 7.8 and 1.4 Hz, 2H), 7.56 (t, J = 7.6 Hz, 2H), 7.42−7.30
(m, 2H), 7.25−7.15 (m, 1H). 13C NMR (151 MHz, DMSO-d6): δ
150.0, 137.8, 131.2, 127.6, 121.2, 116.7, 116.6. IR (film, cm−1): ν
1575 (CC), 1460 (C−N), 824 (C−S), 735 (C−H). Mp: 121 °C
(dec). MS (ESI). Calcd for C25H19BiFN4S: m/z 635.10782 [(M + H)
+]. CCDC 1968620.
[Bi(Ph)2(C15H13N4S)] (1l). Following GP2, BiPh2Cl (200 mg, 0.5
mmol) was reacted with triazolethiol l (152 mg, 0.5 mmol) in
methanol. The title complex was isolated as an off-white solid (242 g,
1
75%). H NMR (600 MHz, DMSO-d6): δ 8.49 (d, J = 6.2 Hz, 1H),
8.35−8.18 (m, 3H), 7.52 (t, J = 7.5 Hz, 3H), 7.36−7.26 (m, 2H),
7.21 (dd, J = 4.4 and 1.8 Hz, 2H), 7.19−7.13 (m, 1H), 7.10 (d, J = 7.9
Hz, 1H), 2.34 (s, 2H), 1.83 (s, 2H). 13C NMR (151 MHz, DMSO-
d6): δ 150.1, 137.9, 134.8 (d, J = 40.2 Hz), 131.9, 128.2 (d, J = 38.6
Hz), 127.5, 120.0. IR (film, cm−1): ν 1575 (CC), 1460 (C−N),
824 (C−S), 735 (C−H). Mp: 161−162 °C. MS (ESI). Calcd for
C27H23BiN4S: m/z 645.1507 [(M + H)+]. CCDC 1968623.
[Bi(Ph)(C8H7N4S)2] (2a). Following GP3, 1-(p-tolyl)-1H-tetrazole-
5-thiol (75 mg, 0.39 mmol) and BiPh3 (68 mg, 0.16 mmol) were
stirred in 4 mL of methanol. After the crashed-out solids were filtered
off, the crude mixture was purified by HPLC and then concentrated in
vacuo to yield the desired complex as a pale-yellow solid (35 mg,
1
33%). H NMR (600 MHz, DMSO-d6): δ 8.90 (d, J = 7.1 Hz, 2H),
7.85 (t, J = 7.5 Hz, 2H), 7.58 (d, J = 7.9 Hz, 4H), 7.43 (dd, J = 8.0
and 6.6 Hz, 1H), 7.37 (d, J = 8.1 Hz, 4H), 2.39 (s, 6H). 13C NMR
(151 MHz, DMSO): δ 139.4, 139.1, 133.0, 132.0, 131.3, 129.7, 127.6,
124.3, 20.7. IR (film, cm−1): ν 2922 (C−H), 1375 (C−N), 853 (C−
S). Mp: 147−150 °C (dec). MS (ESI). Calcd for C22H19BiN8S2: m/z
667.0849 [(M − H)−].
[Bi(Ph)2(C13H9N4S)] (1g). Following GP2, BiPh2Cl (200 mg, 0.5
mmol) was reacted with triazolethiol g (138 mg, 0.5 mmol) in
methanol. The title complex was isolated as an off-white solid (302
1
mg, 98%). H NMR (400 MHz, DMSO-d6): δ 8.52 (d, J = 6.2 Hz,
2H), 8.24 (d, J = 7.3 Hz, 4H), 7.54 (t, J = 7.6 Hz, 4H), 7.35−7.28 (m,
1H), 7.28−7.18 (m, 2H), 7.03 (d, J = 8.9 Hz, 1H). 13C NMR (151
MHz, DMSO-d6): δ 149.9, 137.9, 135.2, 134.7, 131.1, 129.7, 129.5,
127.9, 127.5, 121.0. IR (film, cm −1): ν 1599 (CC), 1428 (C−N),
1342 (C−N), 832 (C−S), 722 (C−C). Mp: 214−219 °C. MS (ESI).
Calcd for C25H19BiN4S: m/z 645.1505 [(M + H) +]. CCDC 1968621.
[Bi(Ph)2(C14H11N4S)] (1h). Following GP2, BiPh2Cl (200 mg, 0.5
mmol) was reacted with triazolethiol h (145 mg, 0.5 mmol) in
methanol. The title complex was isolated as an off-white solid (274
[Bi(Ph)(C9H10N5S)2] (2b). Following GP3, 1-(4-dimethylamino)-
phenyl)-1H-tetrazole-5-thiol (50 mg, 0.22 mmol) and BiPh3 (45 mg,
0.10 mmol) were stirred in 4 mL of methanol. After the crashed-out
solids were filtered off, the crude leftover mixture was triturated with
methanol, with the remaining solids being the desired complex,
1
isolated as a dark-yellow solid (29 mg, 40%). H NMR (400 MHz,
DMSO-d6): δ 8.91−8.84 (m, 2H), 7.83 (t, J = 7.7 Hz, 2H), 7.42 (tt, J
= 7.4 and 1.3 Hz, 1H), 7.33−7.26 (m, 4H), 6.78−6.73 (m, 4H), 2.95
(s, 12H). 13C NMR (151 MHz, DMSO-d6): δ 151.1, 139.8, 133.4,
128.1, 125.9, 123.1, 112.1. IR (film, cm−1): ν 2890 (C−H), 1357 (C−
N), 843 (C−S). Mp: 180−182 °C. MS (ESI). Calcd for
C24H25BiN10S2: m/z 761.1170 [(M + Cl)−].
[Bi(Ph)(C10H11N4S)2] (2c). Following GP3, 1-mesityl-1H-tetrazole-
5-thiol (100 mg, 0.45 mmol) and BiPh3 (90 mg, 0.21 mmol) were
stirred in 5 mL of methanol. After the crashed-out solids were filtered
off, the crude mixture was purified by HPLC and then concentrated in
vacuo to yield the desired complex as a yellow solid (52 mg, 34%). 1H
NMR (600 MHz, DMSO-d6): δ 8.84 (dd, J = 7.8 and 1.5 Hz, 2H),
7.77 (t, J = 7.7 Hz, 2H), 7.38 (ddd, J = 8.9, 6.4, and 2.7 Hz, 1H), 7.05
(s, 4H), 2.30 (s, 6H), 1.77 (s, 12H). 13C NMR (101 MHz, DMSO-
d6): δ 140.5, 139.9, 135.5, 133.3, 130.6, 129.4, 128.0, 21.1, 17.4. IR
(film, cm−1): ν 2919 (C−H), 1374 (C−N), 851 (C−S). Mp: 134 °C.
MS (ESI). Calcd for C26H27BiN8S2: m/z 759.1292 [(M + Cl)−]. Elem
anal. Calcd: C, 43.09; H, 3.76; N, 15.46. Found: C, 42.93; H, 3.45; N,
15.72. CCDC 1968618.
[Bi(Ph)(C7H4ClN4S)2] (2d). Following GP3, 1-(4-chlorophenyl)-1H-
tetrazole-5-thiol (80 mg, 0.36 mmol) and BiPh3 (74 mg, 0.17 mmol)
were stirred in 5 mL of methanol. After the crashed-out solids were
filtered off, the crude leftover mixture was triturated with methanol,
with the remaining solids being the desired complex, isolated as a
yellow solid (42 mg, 35%). 1H NMR (600 MHz, DMSO-d6): δ 8.92−
8.89 (m, 2H), 7.86 (t, J = 7.6 Hz, 2H), 7.70−7.66 (m, 4H), 7.62−
7.59 (m, 4H), 7.44 (tt, J = 7.3 and 1.3 Hz, 1H). 13C NMR (151 MHz,
1
mg, 87%). H NMR (400 MHz, DMSO-d6): δ 8.52 (d, J = 6.2 Hz,
2H), 8.24 (d, J = 7.3 Hz, 4H), 7.54 (t, J = 7.6 Hz, 4H), 7.35−7.28 (m,
1H), 7.28−7.18 (m, 2H), 7.03 (d, J = 8.9 Hz, 1H). 13C NMR (151
MHz, DMSO-d6): δ 150.0, 137.8, 137.3, 131.1, 130.3, 130.2, 127.6,
127.6, 121.0. IR (film, cm−1): ν 1575 (CC), 1460 (C−N), 1390
(C−N), 824 (C−S), 735 (C−H). Mp: 161−162 °C. MS (ESI). Calcd
for C25H19BiN4S: m/z 645.1507 [(M + H)+]. CCDC 1968619.
[Bi(Ph)2(C14H11N4OS)] (1i). Following GP2, BiPh2Cl (200 mg, 0.5
mmol) was reacted with triazolethiol i (153 mg, 0.5 mmol) in
methanol. The title complex was isolated as an off-white solid (273
1
mg, 85%). H NMR (400 MHz, DMSO-d6): δ 8.59−8.45 (m, 2H),
8.24 (d, J = 7.3 Hz, 4H), 7.54 (t, J = 7.6 Hz, 4H), 7.41−7.28 (m, 2H),
7.29−7.17 (m, 4H), 7.03 (d, J = 8.9 Hz, 2H), 3.80 (s, 3H). 13C NMR
(151 MHz, DMSO-d6): δ 150.0, 137.8, 131.1, 128.3 (d, J = 236.8
Hz), 121.0, 114.8, 55.5. IR (film, cm −1): ν 1575 (CC), 1460 (C−
N), 1390 (C−N), 824 (C−S), 735 (C−H). Mp: 119−123 °C. Elem
anal. Calcd C, 48.30; H, 3.27; N, 8.67. Found: C, 48.95; H, 3.33; N,
8.69. MS (ESI). Calcd for C25H19BiN4S: m/z 647.1302 [(M + H) +].
[Bi(Ph)2(C13H8N4ClS)] (1j). Following GP2, BiPh2Cl (200 mg, 0.5
mmol) was reacted with triazolethiol j (155 mg, 0.5 mmol) in
methanol. The title complex was isolated as an off-white solid (173
1
mg, 53%). H NMR (400 MHz, DMSO-d6): δ 8.52 (d, J = 6.2 Hz,
4H), 8.26 (d, J = 7.3 Hz, 2H), 7.55 (dd, J = 8.2 and 6.1 Hz, 5H), 7.33
(dd, J = 8.1 and 6.0 Hz, 3H), 7.29−7.15 (m, 2H). 13C NMR (151
MHz, DMSO-d6): δ 131.7, 130.3, 114.8. IR (film, cm−1): ν 1575
L
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DMSO-d6): δ 139.8, 134.2, 133.8, 133.6, 129.8, 128.8, 128.2, 126.4.
IR (film, cm−1): ν 3056 (C−H), 1371 (C−N), 836 (C−S). Mp:
174−176 °C. MS (ESI). Calcd for C20H13BiCl2N8S2: m/z 742.9615
[(M + Cl)−]. Elem anal. Calcd: C, 33.86; H, 1.85; N, 15.80. Found:
C, 34.09; H, 1.67; N, 15.52. CCDC 1968622.
110−112 °C. MS (ESI). Calcd for C24H21BiN12O3S3: m/z 865.0500
[(M + Cl)−].
[Bi(C7H4O2N5S)3] (3f). Following GP4, 1-(4-nitrophenyl)-1H-
tetrazole-5-thiol (100 mg, 0.45 mmol) and Bi(OtBu)3 (64 mg, 0.15
mmol) were stirred in 6 mL of THF. The crude mixture obtained was
purified by recrystallization in ethyl acetate and hexane to yield the
[Bi(Ph)(C8H7ON4S)2] (2e). Following GP3, 1-(4-methoxyphenyl)-
1H-tetrazole-5-thiol (80 mg, 0.34 mmol) and BiPh3 (70 mg, 0.16
mmol) were stirred in 5 mL of methanol. After the crashed-out solids
were filtered off, the crude mixture was purified by HPLC and then
concentrated in vacuo to yield the desired complex as a yellow solid
1
desired complex as a yellow solid (116 mg, 90%). H NMR (400
MHz, DMSO-d6): δ 8.49 (d, J = 8.8 Hz, 6H), 8.37 (d, J = 8.9 Hz,
6H). 13C NMR (151 MHz, DMSO-d6): δ 124.3, 123.0. IR (film,
cm−1): ν 2924 (C−H), 1364 (C−N), 854 (C−S). Mp: 195−198 °C
(dec). MS (ESI). Calcd for C21H12BiN15O6S3: m/z 909.9750 [(M +
Cl)−].
[Bi(C13H9N4S)3] (3g). Following GP4, triazolethiol g (380 mg, 1.5
mmol) and Bi(OtBu)3 (215 mg, 0.5 mmol) were stirred in 10 mL of
THF. The title complex was isolated as a yellow solid (280 g, 58%).
1H NMR (400 MHz, DMSO-d6): δ 8.53 (d, J = 5.2 Hz, 2H), 7.51 (s,
3H), 7.37 (d, J = 6.2 Hz, 2H), 7.21 (d, J = 5.1 Hz, 2H). 13C NMR
(151 MHz, DMSO-d6): δ 150.0, 134.7, 133.6, 129.4, 128.58, 121.6. IR
(film, cm−1): ν 1495 (C−N), 1298 (C−C−N), 1285 (C−N), 830
(C−S). Mp: 197 °C (dec). MS (ESI). Calcd for C29H36BiCl2N8O6S3:
m/z 935.4728 ([L2Bi·CH2Cl2·6H2O]+).
1
(47 mg, 42%). H NMR (400 MHz, DMSO-d6): δ 8.89 (dd, J = 7.8
and 1.4 Hz, 2H), 7.84 (t, J = 7.6 Hz, 2H), 7.50−7.45 (m, 4H), 7.42
(tt, J = 7.4 and 1.3 Hz, 1H), 7.08−7.02 (m, 4H), 3.81 (s, 6H). IR
(film, cm−1): ν 2998 (C−H), 1368 (C−N), 848 (C−S). Mp: 170 °C
(dec). MS (ESI). Calcd for C22H19BiN8O2S2: m/z 737.0593 [(M +
Cl)−]. Elem anal. Calcd: C, 37.72; H, 2.73; N, 16.00. Found: C,
37.80; H, 2.69; N, 15.64.
[Bi(Ph)(C7H4O2N5S)2] (2f). Following GP3, 1-(4-nitrophenyl)-1H-
tetrazole-5-thiol (95 mg, 0.42 mmol) and BiPh3 (75 mg, 0.17 mmol)
were stirred in 5 mL of methanol. After the crashed-out solids were
filtered off, the crude leftover mixture was washed with cold diethyl
ether, with the remaining solids being the desired complex, isolated as
a yellow solid (72 mg, 36%). 1H NMR (600 MHz, DMSO-d6): δ 8.92
(d, J = 7.4 Hz, 2H), 8.39−8.34 (m, 4H), 8.12 (d, J = 8.6 Hz, 4H),
7.91 (t, J = 7.5 Hz, 2H), 7.45 (t, J = 7.4 Hz, 1H). 13C NMR (151
MHz, DMSO-d6): δ 147.0, 139.5, 139.4, 133.4, 127.9, 125.7, 124.9,
124.7, 118.2. IR (film, cm−1): ν 3078 (C−H), 1358 (C−N), 853 (C−
S). Mp: 170 °C (dec). MS (ESI). Calcd for C20H13BiN10O4S2: m/z
765.0046 [(M + Cl)−].
[Bi(C14H11N4S)3](3h). Following GP4, triazolethiol h (400 mg, 1.5
mmol) and Bi(OtBu)3 (215 mg, 0.5 mmol) were stirred in 10 mL of
THF. The title complex was isolated as a yellow solid (347 mg, 69%).
1H NMR (400 MHz, DMSO-d6): δ 8.52 (d, J = 6.2 Hz, 2H), 8.24 (d,
J = 7.3 Hz, 4H), 7.54 (t, J = 7.6 Hz, 4H), 7.35−7.28 (m, 1H), 7.28−
7.18 (m, 2H), 7.03 (d, J = 8.9 Hz, 1H). 13C NMR (151 MHz, DMSO-
d6): δ 149.9, 138.8, 133.9, 129.8, 128.3, 121.4, 20.8. IR (film, cm−1): ν
1423 (C−N), 1259 (C−C−N), 821 (C−S). Mp: 190 °C (dec). MS
(ESI). Calcd for C42H33BiN12S3: m/z 1033.9881 [(M − H)−].
[Bi(C14H11N4OS)3] (3i). Following GP4, triazolethiol i (425 mg, 1.5
mmol) and Bi(OtBu)3 (215 mg, 0.5 mmol) were stirred in 10 mL of
THF. The title complex was isolated as an orange solid (280 mg,
[Bi(C8H7N4S)3] (3a). Following GP4, 1-(p-tolyl)-1H-tetrazole-5-
thiol (75 mg, 0.39 mmol) and Bi(OtBu)3 (55 mg, 0.13 mmol) were
stirred in 5 mL of THF. The title compound was isolated as a bright-
1
yellow solid (95 mg, 94%). H NMR (400 MHz, DMSO-d6): δ 7.79
(d, J = 8.3 Hz, 6H), 7.29 (d, J = 8.1 Hz, 6H), 2.36 (s, 8H). 13C NMR
(101 MHz, DMSO-d6): δ 137.5, 134.4, 129.4, 124.1, 21.2. IR (film,
cm−1): ν 2858 (C−H), 1376 (C−N), 837 (C−S). Mp: 161 °C (dec).
MS (ESI). Calcd for C24H21BiN12S3: m/z 817.0677 [(M + Cl)−].
[Bi(C9H10N5S)3] (3b). Following GP4, 1-(4-dimethylamino)phenyl)-
1H-tetrazole-5-thiol (100 mg, 0.45 mmol) and Bi(OtBu)3 (65 mg,
0.15 mmol) were stirred in 5 mL of THF. The title compound was
1
53%). H NMR (400 MHz, DMSO-d6): δ 8.62−8.37 (m, 2H), 7.26
(dd, J = 20.5 and 6.7 Hz, 4H), 7.04 (d, J = 8.4 Hz, 2H), 3.81 (s, 3H).
13C NMR (151 MHz, DMSO-d6): δ 159.6, 129.8, 121.6, 114.6, 55.4.
IR (film, cm−1): ν 1251 (C−C−N), 826 (C−S). Mp: 191 °C (dec).
MS (ESI). Calcd for C30H42BiN8O10S2: m/z 947.4937 ([L2Bi·
2CH3OH·6H2O]+).
1
isolated as a dark-orange solid (95 mg, 94%). H NMR (400 MHz,
DMSO-d6): δ 7.70−7.59 (m, 6H), 6.82−6.71 (m, 6H), 2.93 (s, 18H).
13C NMR (151 MHz, DMSO-d6): δ 149.8, 125.0, 111.6, 40.1. IR
(film, cm−1): ν 2854 (C−H), 1357 (C−N), 814 (C−S). Mp: 150 °C
(dec). MS (ESI). Calcd for C27H25BiN15S3: m/z 904.1466 [(M +
Cl)−].
[Bi(C13H8N4ClS)3] (3j). Following GP4, triazolethiol j (408 mg, 1.5
mmol) and Bi(OtBu)3 (215 mg, 0.5 mmol) were stirred in 10 mL of
THF. The title complex was isolated as a bright-orange solid (303 mg,
1
57%). H NMR (400 MHz, DMSO-d6): δ 8.53 (d, J = 5.1 Hz, 2H),
7.56 (d, J = 8.3 Hz, 2H), 7.39 (d, J = 8.2 Hz, 2H), 7.21 (d, J = 5.1 Hz,
2H). 13C NMR (151 MHz, DMSO-d6): δ 113.0, 100.4, 96.07, 93.4,
92.3, 91.2, 84.4. IR (film, cm−1): ν 1492 (C−H), 1424 (C−N), 1091
(C−C−N), 826 (C−S). Mp: 192 °C (dec). MS (ESI). Calcd for
C28H20BiCl6N8S2: m/z 951.4693 ([L2Bi·2CH2Cl2]+).
[Bi(C13H8N4FS)3] (3k). Following GP4, triazolethiol k (432 mg, 1.5
mmol) and Bi(OtBu)3 (215 mg, 0.5 mmol) were stirred in 10 mL of
THF. The title complex was isolated as a yellow solid (336 mg, 66%).
1H NMR (400 MHz, DMSO-d6): δ 8.62−8.43 (m, 2H), 7.30−7.10
(m, 5H). 13C NMR (151 MHz, DMSO-d6): δ 162.9, 161.2, 150.0,
148.5, 133.6, 131.1, 131.0, 130.9, 121.6, 116.4, 116.3. IR (film, cm−1):
ν 1602 (C−C), 1427 (C−N), 1216 (C−C−N), 839 (C−S), 827 (C−
S). Mp: 237 °C (dec). MS (ESI). Calcd for C39H24BiF3N12S3: m/z
1087.2122 [(M + MeOH)−].
[Bi(C15H13N4S)3] (3l). Following GP4, triazolethiol l (423 mg, 1.5
mmol) and Bi(OtBu)3 (215 mg, 0.5 mmol) were stirred in 10 mL of
THF. The title complex was isolated as a yellow solid (463 mg, 88%).
1H NMR (400 MHz, DMSO-d6): δ 8.54 (d, J = 5.2 Hz, 2H), 7.49−
6.90 (m, 5H), 2.35 (s, 3H), 1.97 (s, 3H). 13C NMR (151 MHz,
DMSO-d6): δ 150.2, 139.6, 135.5, 133.5, 131.7, 131.2, 128.8, 127.9,
120.6, 20.8, 17.3. IR (film, cm−1): ν 1298 (C−N), 825 (C−S). Mp:
171 °C (dec). MS (ESI). Calcd for C45H39BiN12S3: m/z 1129.2619
[(M + DMSO)−].
[Bi(C10H11N4S)3] (3c). Following GP4, 1-mesityl-1H-tetrazole-5-
thiol (75 mg, 0.35 mmol) and Bi(OtBu)3 (50 mg, 0.12 mmol) were
stirred in 6 mL of THF. The title compound was isolated as a bright-
1
yellow solid (80 mg, 85%). H NMR (400 MHz, DMSO-d6): δ 7.06
(s, 6H), 2.31 (s, 9H), 1.85 (s, 18H). 13C NMR (101 MHz, DMSO-
d6): δ 135.3, 129.0, 20.7, 17.1. IR (film, cm−1): ν 2917 (C−H), 1375
(C−N), 851 (C−S). Mp: 136−137 °C. MS (ESI). Calcd for
C30H33BiN12S3: m/z 901.1693 [(M + Cl)−].
[Bi(C7H4ClN4S)3] (3d). Following GP4, 1-(4-chlorophenyl)-1H-
tetrazole-5-thiol (75 mg, 0.34 mmol) and Bi(OtBu)3 (50 mg, 0.12
mmol) were stirred in 6 mL of THF. The title compound was isolated
1
as a bright-yellow solid (82 mg, 82%). H NMR (600 MHz, DMSO-
d6): δ 8.15−8.12 (m, 6H), 7.55−7.52 (m, 6H). 13C NMR (151 MHz,
DMSO-d6): δ 162.2, 136.4, 131.6, 128.8, 125.2, 40.5. IR (film, cm−1):
ν 2969 (C−H), 1374 (C−N), 839 (C−S). Mp: 147−149 °C. MS
(ESI). Calcd for C21H12BiCl3N12S3: m/z 876.9181 [(M + Cl)−].
[Bi(C8H7ON4S)3] (3e). Following GP4, 1-(4-methoxyphenyl)-1H-
tetrazole-5-thiol (100 mg, 0.45 mmol) and Bi(OtBu)3 (64 mg, 0.15
mmol) were stirred in 6 mL of THF. The crude mixture obtained was
purified by recrystallization in ethyl acetate and hexane to yield the
1
desired complex as a yellow solid (108 mg, 87%). H NMR (400
MHz, DMSO-d6): δ 7.89−7.78 (m, 6H), 7.05−6.95 (m, 6H), 3.79 (s,
9H). 13C NMR (151 MHz, DMSO-d6): δ 158.0, 130.1, 125.1, 113.5,
55.4. IR (film, cm−1): ν 2934 (C−H), 1374 (C−N), 830 (C−S). Mp:
M
https://dx.doi.org/10.1021/acs.inorgchem.9b03550
Inorg. Chem. XXXX, XXX, XXX−XXX

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Forum Article
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.inorgchem.9b03550.
■
sı
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Experimental data (preparation of tetrazole and triazole
thiolates), crystallography details, and NMR (1H and
13C) spectra (PDF)
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Accession Codes
CCDC 1968616−1968625 contain the supplementary crys-
tallographic data for this paper. These data can be obtained
free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by
emailing data_request@ccdc.cam.ac.uk, or by contacting The
Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
AUTHOR INFORMATION
Corresponding Author
Philip C. Andrews − Monash University School of Chemistry,
Clayton, Victoria 3800, Australia; orcid.org/0000-0002-
3971-7311; Email: phil.andrews@monash.edu
■
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Authors
Liam J. Stephens − Monash University School of Chemistry,
Clayton, Victoria 3800, Australia
Sarmishta Munuganti − Monash University School of
Chemistry, Clayton, Victoria 3800, Australia
Rebekah N. Duffin − Monash University School of Chemistry,
Clayton, Victoria 3800, Australia
Melissa V. Werrett − Monash University School of Chemistry,
Clayton, Victoria 3800, Australia; orcid.org/0000-0001-
6811-4504
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.inorgchem.9b03550
Author Contributions
†The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript. These authors contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors acknowledge the use of facilities within the
Monash X-ray Platform and the assistance of Dr. Craig Forsyth
for assistance with single-crystal X-ray crystallography. The
authors also acknowledge the use of facilities within the
Monash Centre for Electron Microscopy and the Ramaciotti
Centre for Cryo Electron Microscopy and are thankful to the
assistance provided by Dr. Xiya Fang and Dr. Simon Crawford,
respectively. The authors also acknowledge the use of facilities
within the Monash Analytical Platform.
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