Pathogenic Gram-positive bacteria are highly sensitive to triphenylphosphanegold(O-alkylthiocarbamates), Ph3PAu[SC(OR)=N(p- tolyl)] (R = Me, et and iPr)

Article abstract of DOI:10.1007/s13404-013-0091-z

The phosphanegold(I) thiocarbamides, Ph₃PAu{SC(OR)=NC ₆H₄Me-4} for R = Me (1), Et (2) and iPr (3), have been shown to have essentially linear gold atom coordination geometries defined by phosphane-P and thiolate-S atoms, and exhibit minimum inhibitory concentration (MIC) values in the range of 1-37 μg/ml against four Gram-positive bacteria, namely Bacillus cereus, Enterococcus faecalis, Enterococcus faecium and Staphylococcus aureus; compounds 1-3 are less potent against a broad panel of 16 Gram-negative bacteria. As the minimum bactericidal concentration values were quite similar to the MIC values, compounds 1-3 are effective bactericidal agents. The specific action against the four Gram-positive bacteria suggests they function by inhibition of peptidoglycan synthesis.

Full text of DOI:10.1007/s13404-013-0091-z

Gold Bull
DOI 10.1007/s13404-013-0091-z
ORIGINAL PAPER
Pathogenic Gram-positive bacteria are highly sensitive
to triphenylphosphanegold(O-alkylthiocarbamates),
Ph3PAu[SC(OR)=N(p-tolyl)] (R=Me, Et and iPr)
Chien Ing Yeo & Jiun-Horng Sim & Chai-Hoon Khoo &
Zheng-Jie Goh & Kok-Pian Ang & Yoke-Kqueen Cheah &
Zainal Abidin Fairuz & Siti Nadiah Binti Abdul Halim &
Seik Weng Ng & Hoi-Ling Seng & Edward R. T. Tiekink
#
The Author(s) 2013. This article is published with open access at Springerlink.com
. .
Keywords Gold(I) compounds Thiocarbamides
Abstract The phosphanegold(I) thiocarbamides,
Ph3PAu{SC(OR)=NC6H4Me-4} for R=Me (1), Et (2)
and iPr (3), have been shown to have essentially linear
gold atom coordination geometries defined by
phosphane-P and thiolate-S atoms, and exhibit minimum
inhibitory concentration (MIC) values in the range of 1–
37 μg/ml against four Gram-positive bacteria, namely
Bacillus cereus, Enterococcus faecalis, Enterococcus
faecium and Staphylococcus aureus; compounds 1–3 are
less potent against a broad panel of 16 Gram-negative
bacteria. As the minimum bactericidal concentration
values were quite similar to the MIC values, compounds
1–3 are effective bactericidal agents. The specific action
against the four Gram-positive bacteria suggests they
function by inhibition of peptidoglycan synthesis.
.
.
Phosphanegold thiolates Antimicrobial activity
.
Gram-positive bacteria Metal-based drugs
Introduction
It is now widely recognised that the increase in bacterial
resistance, the rapid emergence of new infections, and even
overprescription of antibiotics have significantly decreased
the efficacy of drugs employed in the treatment of patholo-
gies instigated by certain microorganisms [1, 2]. Infections
caused by Gram-positive organisms are of major concern
due to the increased incidence and the high level of
multidrug resistance [3, 4]. Increasing resistance in both
enterococci (lactic acid bacteria) and staphylococci (skin-
colonising bacteria) [5] has accelerated the need for the
development of new antimicrobial agents to treat these
Gram-positive infections [6]. Herein, the specific antimicro-
bial activity against four Gram-positive bacteria exhibited
by a series of gold compounds is described.
Electronic supplementary material The online version of this article
(doi:10.1007/s13404-013-0091-z) contains supplementary material,
which is available to authorized users.
:
:
:
:
C. I. Yeo Z. A. Fairuz S. N. B. A. Halim S. W. Ng
:
H.<L. Seng E. R. T. Tiekink (*)
Disease-modifying antirheumatic drugs based on gold are
well established in the treatment of rheumatoid arthritis [7,
8]. Gold(I) compounds used in this context range from
charged, polymeric and water-soluble gold(I) thiolates
such as sodium gold(I) thiomalate (Myocrisin®) and
the neutral, monomeric and lipophilic phosphanegold(I)
thiolate, triethylphosphanegold(I) tetraacetylthioglucose
(Auranofin®) [9, 10]. While research continues in de-
veloping new antirheumatic agents and in understanding
the mechanism of action [11–14], much recent attention
has focussed upon investigating the anticancer potential
Department of Chemistry, University of Malaya, 50603,
Kuala Lumpur, Malaysia
e-mail: Edward.Tiekink@gmail.com
:
:
:
:
J.<H. Sim C.<H. Khoo Z.<J. Goh K.<P. Ang Y.<K. Cheah (*)
Department of Biomedical Science, Faculty of Medicine
and Health Sciences, University Putra Malaysia, 43400,
Serdang, Selangor Darul Ehsan, Malaysia
e-mail: ykcheah@medic.upm.edu.my
S. W. Ng
Chemistry Department, Faculty of Science, King Abdulaziz
University, PO Box 80203, Jeddah, Saudi Arabia

Gold Bull
of gold(I) and, especially, gold(III) compounds [15–24].
Other ailments/diseases, too, have been investigated
such as anti-viral (HIV AIDS), anti-asthma (acute corti-
costeroid) and even tropical diseases, e.g. malaria and
Chagas disease [13, 14]. Attention has also been direct-
ed towards antimicrobial activity, the focus of the pres-
ent report.
A range of synthetic gold compounds shown strong inhib-
itory and antimicrobial effects. Exploiting the anti-
inflammatory activity of Auranofin® as well as its ability to
inhibit thiol-based redox enzymes renders this an exciting
prospect for the development of novel antimicrobial agents
[24]. The majority of research in this area has in fact focussed
upon analogues of Auranofin®, i.e. phosphanegold(I)
thiolates [24–27], but with attention also being directed to
the study of N-heterocyclic carbene [28], thiolate [29] and
carboxylate [30, 31] derivatives. Relatively less consideration
has been devoted to gold(III) species [32]. In the present study,
phosphanegold(I) thiolates 1–3 (Fig. 1) will be shown to
display marked biological activities. Specifically, 1–3 exhibit
selective and antimicrobial activities against four Gram-
positive bacteria suggesting that these may be potentially
developed as alternative bactericidal agents.
RX1 FTIR spectrophotometer. Elemental analyses were
performed on a Perkin Elmer PE 2400 CHN Elemental
Analyser. Melting points were determined on a Krüss
KSP1N melting point meter.
All chemicals and solvents were sourced commercially and
used as received. All reactions were carried out under ambient
conditions. The thiocarbamides, ROC(=S)N(H)C6H4Me-4 for
R=Me, Et and iPr, were prepared in quantitative yields as
described in the literature [33]. For example,
MeOC(=S)N(H)C6H4Me-4 was prepared from the stoichio-
metric reaction of S=C=NC6H4Me-4 and MeOH in the pres-
ence of equimolar NaOH with MeOH serving as the solvent.
T h e p h o s p h a n e g o l d ( I ) t h i o c a r b a m i d e s ,
Ph3PAu{SC(OR)=NC6H4Me-4} for R=Me, Et and iPr, were
obtained from the reaction of Ph3PAuCl precursor
(synthesised by the reduction of KAuCl4 by sodium sulfite
followed by addition of the stoichiometric amount of
triphenylphosphane) with one mole equivalent of
ROC(=S)N(H)C6H4Me-4 in the presence of NaOH, following
standard procedures [34]. Details for the synthesis of 3 are
given here; 1 and 2 were prepared in an analogous fashion.
NaOH (0.5 mmol) in MeOH (5 mL) was added to a suspen-
sion of Ph3PAuCl (0.5 mmol) in MeOH (20 mL), followed by
addition of iPrOC(=S)N(H)C6H4Me-4 in MeOH (20 mL).
The resulting mixture was stirred for 3 h at 50 °C. An
equivolume of dichloromethane was added and the solution
was left for slow evaporation at room temperature, yielding
colourless crystals after 2 weeks.
Experimental
General
1H and 13C{1H} NMR spectra were recorded in CDCl3
solution on a Bruker Avance 400 MHz NMR spectrometer
with chemical shifts relative to tetramethylsilane as internal
reference. 31P{1H} NMR spectra were recorded in CDCl3
solution on the same instrument but with the chemical shifts
recorded relative to 85 % aqueous H3PO4 as external refer-
ence; abbreviations for NMR assignments: s, singlet; d,
doublet; t, triplet; q, quartet; sept, septet; and m, multiplet.
IR spectra were obtained as KBr pellets on a Perkin Elmer
Characterisation
MeOC(=S)N(H)C6H4Me-4
1H NMR (400 MHz, CDCl3, 25 °C): δ 8.70 (br s, 1H, NH),
7.13 (br s, 4H, aryl-H), 4.11 (s, 3H, OCH3), 2.32 (s, 3H,
aryl-CH3) ppm. 13C{1H} NMR (400 MHz, CDCl3, 25 °C):
δ 189.6 (Cq), 135.5 (C1), 134.4 (C4), 129.6 (C3), 122.0
(C2), 58.8 (OCH3), 21.0 (C5) ppm. Anal. Calc. for
C8H9NOS: C, 59.64; H, 6.17; N, 7.73 %. Found: C,
59.26; H, 6.29; N, 7.74 %. IR (KBr disc, per centimeter):
3,234(br) ν(N–H), 1,451(s) ν(C–N), 1,204(s) ν(C=S),
1,061(s) ν(C–O). M.pt: 78–80 °C.
EtOC(=S)N(H)C6H4Me-4
1H NMR (400 MHz, CDCl3, 25 °C): δ 8.68 (br s, 1H, NH),
7.13 (br s, br, 4H, aryl-H), 4.62 (s, br, 2H, OCH2), 2.32 (s, 3H,
aryl-CH3), 1.39 (t, 3H, CH3, J=7.10 Hz) ppm. 13C{1H} NMR
(400 MHz, CDCl3, 25 °C): δ 188.6 (Cq), 135.2 (C1), 134.6
(C4), 129.6 (C3), 121.6 (C2), 68.7 (OCH2), 21.0 (C5), 14.1
(CH3) ppm. Anal. Calc. for C9H11NOS: C, 61.50; H, 6.71; N,
7.17 %. Found: C, 61.30; H, 6.82; N, 7.23 %. IR (KBr disc,
Fig. 1 Chemical structures for 1–3. The red arrow indicates flexibility
in the orientation of the thiolate anion

Gold Bull
per centimter): 3,236(br) ν(N–H), 1,450(s) ν(C–N), 1,202(s)
ν(C=S), 1,042(s) ν(C–O). M.pt: 64–67 °C.
1,431(s) ν(C=N), 1,118(s) ν(C–O), 1,101(s) ν(C–S). M.pt:
131–133 °C.
iPrOC(=S)N(H)C6H4Me-4
Ph3PAu{SC(O-iPr)=NC6H4Me-4} (3)
1H NMR (400 MHz, CDCl3, 25 °C): δ 8.65 (br s, 1H, NH),
7.13 (br s, 4H, aryl-H), 5.65 (sept, 1H, OCH, J=6.22 Hz),
2.32 (s, 3H, aryl-CH3), 1.39 (d, 6H, CH3, J=6.20 Hz) ppm.
13C{1H} NMR (400 MHz, CDCl3, 25 °C): δ 187.7 (Cq),
135.0 (C1), 134.7 (C4), 129.5 (C3), 121.5 (C2), 73.7
(OCH), 21.7 (CH3), 20.9 (C5) ppm. Anal. Calc. for
C10H13NOS: C, 63.12; H, 7.22; N, 6.69 %. Found: C,
63.35; H, 7.33; N, 6.70 %. IR (KBr disc, per centimeter):
3,221(br) ν(N–H), 1,462(s) ν(C–N), 1,205(s) ν(C=S),
1,087(s) ν(C–O). M.pt: 91–92 °C.
Yield: 0.297 g (89 %) colourless crystals from the slow
evaporation from a dichloromethane/methanol mixture
(1:1v/v). H NMR (400 MHz, CDCl3, 25 °C): δ 7.53–7.42
(br m, 15H, Ph3P), 6.83 (d, 2H, o-aryl-H, JCP=8.04 Hz), 6.72
(d, 2H, m-aryl-H, JCP=8.16 Hz), 5.28 (sept, 1H, OCH,
J=6.19 Hz), 2.05 (s, 3H, aryl-CH3), 1.32 (d, 6H, CH3,
J=6.20 Hz) ppm. 13C{1H} NMR (400 MHz, CDCl3,
25 °C): δ 162.9 (Cq), 148.9 (Ph, C1), 134.2 (d, o-PC6H5,
1
JCP=55.2 Hz), 131.6 (d, p-PC6H5, JCP=7.8 Hz), 131.2 (Ph,
C4), 129.5 (Ph, C3), 129.5 (d, i-PC6H5, JCP=226.8 Hz), 129.1
(d, m-PC6H5, JCP=45.8 Hz), 121.7 (Ph, C2), 70.3 (OCH),
22.1 (CH3), 20.8 (aryl-CH3) ppm. 31P{1H}NMR (400 MHz,
CDCl3, 25 °C): δ 37.8 ppm. Anal. Calc. for C28H27AuNOPS:
C, 52.18; H, 4.38; N, 2.10 %. Found: C, 52.15; H, 4.25; N,
2.15 %. IR (KBr disc, per centimeter): 1,437(s) ν(C=N),
1,132(s) ν(C–O), 1,093(s) ν(C–S). M.pt: 148–151 °C.
Ph3PAu{SC(OMe)=NC6H4Me-4} (1)
Yield: 0.297 g (93 %) colourless crystals from the slow
evaporation from a dichloromethane/methanol mixture
1
(1:1v/v). H NMR (400 MHz, CDCl3, 25 °C): δ 7.53–7.40
(m, br, 15H, Ph3P), 6.82 (d, 2H, o-aryl-H, JCP=8.04 Hz),
6.73 (d, 2H, m-aryl-H, JCP=8.20 Hz), 3.90 (s, 3H, OCH3),
2.03 (s, 3H, aryl-Me) ppm. 13C{1H} NMR (400 MHz,
CDCl3, 25 °C): δ 164.4 (Cq), 148.6 (Ph, C1), 134.3 (d,
o-PC6H5, JCP=55.3 Hz), 131.6 (d, p-PC6H5, JCP=8.8 Hz),
131.5 (Ph, C4), 129.6 (Ph, C3), 129.5 (d, i-PC6H5,
X-ray crystallography
Intensity measurements for a colourless block (0.06×0.06×
0.11 mm) of 3 were made at 100 K on an Agilent Supernova
dual diffractometer with an Atlas (Mo) detector (ω scan
technique) using graphite monochromatized Mo Kα radiation
[35]. The structure was solved by direct methods (SHELXS97
[36] through the WinGX Interface [37]) and refined (aniso-
tropic displacement parameters, H atoms in the riding model
approximation and a final weighting scheme of the form
w=1/[σ2(F 2)+0.018P2] where P=(F 2 +2F2)/3) with
J
CP=226.9 Hz), 129.1 (d, m-PC6H5, JCP=45.8 Hz), 121.8
(Ph, C2), 55.3 (OCH3), 20.8 (aryl-Me) ppm. 31P{1H} NMR
(400 MHz, CDCl3, 25 °C): δ 38.0 ppm. Anal. Calc. for
C26H23AuNOPS: C, 50.71; H, 3.94; N, 2.19 %. Found: C,
50.93; H, 3.64; N, 2.18 %. IR (KBr disc, per centimeter):
1,436(s) ν(C=N), 1,146(s) ν(C–O), 1,100(s) ν(C–S). M.pt:
143–145 °C.
o
o
c
SHELXL97 on F2 [36]. Crystal data for C29H29AuNOPS:
M=667.53, monoclinic, P21/c, a=9.7098(3), b=12.5573(4),
c=21.9413(6) Å, β=100.931(3)º, V=2,626.74(14) Å3, Z=4,
Dx=1.688 g cm−3, F(000)=1,320, μ=5.763 mm−1, no. meas.
data=10,940, no. unique data=6,043, Rint=0.023, no. of pa-
rameters=310, R (5,249 data with I≥2σ(I))=0.024, wR (all
data)=0.050. Maximum and minimum residual electron den-
sity peaks=0.81 and −0.77 e Å−3. A view of the molecular
structure is shown in Fig. 2, drawn at the 50 % probability
level with ORTEP-3 for Windows [37], and the overlay dia-
gram (Fig. 3) was drawn with QMol [38]. The crystal packing
analysis was conducted with the aid of PLATON [39] and
Fig. 4 was drawn with DIAMOND [40].
Ph3PAu{SC(OEt)=NC6H4Me-4} (2)
Yield: 0.278 g (85 %) colourless crystals from the slow
evaporation from a dichloromethane/ethanol mixture (1:1
1
v/v). H NMR (400 MHz, CDCl3, 25 °C): δ 7.54–7.40 (m,
br, 15H, Ph3P), 6.83 (d, 2H, o-aryl-H, JCP=8.04 Hz), 6.73
(d, 2H, m-aryl-H, JCP=8.20 Hz), 4.34 (q, 2H, OCH2,
J=7.09 Hz), 2.05 (s, 3H, aryl-CH3), 1.33 (t, 3H, CH3,
J=7.10 Hz) ppm. 13C{1H} NMR (400 MHz, CDCl3,
25 °C): δ 163.8 (Cq), 148.7 (Ph, C1), 134.3 (d, o-PC6H5,
J
CP=55.3 Hz), 131.6 (d, p-PC6H5, JCP=9.0 Hz), 131.4 (Ph,
C4), 129.5 (Ph, C3), 129.5 (d, i-PC6H5, JCP=226.8 Hz),
129.1 (d, m-PC6H5, JCP=45.6 Hz), 121.8 (Ph, C2), 63.8
(OCH2), 20.8 (aryl-CH3), 14.7 (CH3) ppm. 31P{1H}NMR
(400 MHz, CDCl3, 25 °C): δ 38.0 ppm. Anal. Calc. for
C27H25AuNOPS: C, 51.46; H, 4.16; N, 2.14 %. Found: C,
51.68; H, 3.91; N, 2.22 %. IR (KBr disc, per centimeter):
Screening for antibacterial activity
Antibacterial screening was performed using the disc diffu-
sion method in accordance with the National Committee for
Clinical Laboratory Standards (NCCLS) guideline. A total

Gold Bull
Fig. 4 A view in projection down the a-axis of the unit cell contents
for 3. The edge-to-face C–H…π interactions are represented as purple
dashed lines
Fig. 2 Molecular structure of 3 show atom labelling scheme
of 20 bacterial strains were used in this study: Staphylococcus
aureus American Type Culture Collection (ATCC) 25923,
Bacillus cereus ATCC 10876, Enterococcus faecalis ATCC
29212, Enterococcus faecium ATCC 19434, Pseudomonas
aeruginosa ATCC 27853, Vibrio parahaemolyticus ATCC
17802, Aeromonas hydrophilla ATCC 35654, Escherichia coli
ATCC 25922, Proteus mirabilis ATCC 25933, Proteus
vulgaris ATCC 13315, Enterobacter cloacae ATCC 35030,
Enterobacter aerogenes ATCC 13048, Shigella flexneri ATCC
12022, Shigella sonnei ATCC 9290, Salmonella typhimurium
ATCC 14028, Salmonella paratyphi A ATCC 9150, Klebsiella
pneumoniae ATCC 700603, Acinetobacter baumannii ATCC
19606, Stenotrophomonas maltophilia ATCC 13637 and
Citrobacter freundii ATCC 8090. All bacterial cultures were
obtained from ATCC. The inoculum suspension of each bac-
terial strain was adjusted to 0.5 McFarland standard turbidity
(corresponding to approximately 108 CFU/ml) by adding
Mueller–Hinton broth. This suspension was then swabbed on
the surface of Mueller–Hinton agar plates using a sterile cotton
swab. The tested compounds were dissolved in DMSO to the
test concentration of 1 mg/ml. Sterile 6 mm filter paper discs
were aseptically placed on Mueller–Hinton agar surfaces and
10 μl of the dissolved compounds were immediately added to
discs. Each plate contained one standard antibiotic paper disc
served as positive control, one disc served as negative control
(10 μl broth) and one disc served as solvent control (10 μl
DMSO). The plates were incubated at 37 °C for 24 h.
Antibacterial activity was evaluated by measuring the diameter
of inhibition zone against the test bacterial strains. Each
experiment was performed in duplicate.
Determination of minimum inhibitory concentration
and minimum bactericidal concentration
The minimum inhibitory concentration (MIC) was determined
by the broth microdilution method according to the NCCLS.
The tested compounds were serially threefold diluted in
DMSO to the test concentrations of 1,000, 330, 110, 37, 12,
4, 1 and 0.5 μg/ml and then placed into each well of a 96-well
microplate. An inoculum suspension with density of
105 CFU/ml of exponentially growing bacterial cells was
added into each well. The 96-well microplates were incubated
at 37 °C for 24 h. All tests were performed in triplicate. Four
controls comprising medium with standard antibiotic (positive
control), medium with DMSO (solvent control), medium with
inoculum bacterial cells (negative control), and medium with
broth only (negative growth control) were included in each
test. Bacterial growth was detected by adding 50 μl of a
0.2 mg/ml p-iodonitrotetrazolium violet (INT) indicator solu-
tion into each of the microplate wells and incubated at 37 °C
for 30 min under aerobic agitation. Where bacterial growth
was inhibited, the suspension in the well remained clear after
incubation with INT. By contrast, the INT changed from clear
to red in the presence of bacterial activity. The lowest concen-
tration of the tested compound which completely inhibited
Fig. 3 Overlay diagram for 1 (red image), 2 (green) and 3 (blue)
whereby the P1, Au and S1 atoms have been superimposed. The
diagram highlights the different orientations of the thiolate ligand
which can place either the oxygen atom or arene ring in close proxim-
ity to gold

Gold Bull
bacterial growth was taken as the MIC. After MIC determi-
nation of each tested compound, an aliquot of 100 μl from
each well which showed no visible growth was spread onto
MHA at 37 °C for 30 min. The minimum bactericidal con-
centration (MBC) was defined as the lowest concentration of
the tested compound producing a 99.9 % reduction in bacterial
viable count on the MHA.
mode of complexation of the anions indicated in Fig. 1. This
is confirmed by a crystal structure determination of 3.
The molecular structure of 3 is illustrated in Fig. 2. The gold
atom is linearly coordinated by the phosphane-P1 and thiolate-S1
atoms with bond lengths of 2.2561(7) and 2.3171(7) Å, respec-
tively. The C1–S1 and C1–N1 bond lengths of 1.776(3) and
1.269(4) Å, respectively, have significantly elongated and short-
ened compared to related “free” thiocarbamide molecules [33],
and are consistent with the notion that the molecule was
deprotonated during the synthesis and coordinates as a thiolate.
The P1–Au–S1 angle of 172.88(3)° deviates significantly from
the ideal 180° and this feature is attributed to the close approach
of the O1 atom to gold, i.e. Au…O1 is 2.887(2) Å.
Results and discussion
Synthesis and characterisation
Crystallography, NMR and theoretical studies confirm that
the ROC(=S)N(H)C6H4Me-4 molecules exist as amides
rather than thiols [33]. The Ph3PAu{SC(OR)=NC6H4Me-
4}, for R=Me (1), Et (2) and iPr (3), compounds were
prepared by the facile reaction of Ph3PAuCl with the respec-
tive ROC(=S)N(H)C6H4Me-4 in the presence of NaOH in
good yields (≥85 %), each as colourless crystals. The com-
pounds are light and air-stable. They are soluble in chlori-
nated solvents, partially soluble in acetonitrile and DMSO,
sparingly soluble in methanol, ethanol and acetone, and
insoluble in water.
Crystal structure determinations have been reported previ-
ously for each of 1 [41] and 2 [42]. As seen from the overlay
diagram in Fig. 3, a very similar mode of coordination is
found for 1 and 3, which conforms to the norm for structures
of this type [43]. However, a variation is noted in 2, where the
arene ring is oriented towards the gold atom rather than the O1
atom; intra- and intermolecular Au…π(arene) interactions
have been reviewed recently [44]. Such variations have been
discussed in some detail in the literature and arise owing to the
combination of subtle electronic and steric effects of the P-, O-
and N-bound substituents [41].
NMR spectroscopy (1H, 13C{1H} and 31P{1H}) show the
expected resonances and integration. Notable in the 1H NMR
spectra of 1–3 was the absence of the resonance due to N-H
found in ROC(=S)N(H)C6H4Me-4, and notable upfield shifts
for the OCH and tolyl-Me-H protons. In the 13C{1H} spectra,
significant upfield shifts were observed for the quaternary
(25 ppm) and O-bound (3–5 ppm) carbon nuclei compared
with their positions in the spectra of ROC(=S)N(H)C6H4Me-
4. These results confirm coordination of anionic forms of the
ligands and a significant reduction in delocalisation of π-
electron density over the central OC(S)N chromophore com-
pared with that in the ROC(=S)N(H)C6H4Me-4 molecules [33].
It is noteworthy that the systematic variation in
the OCH (1H) resonances and in the quaternary and O-bound
(13C{1H}) resonances that are correlated with the electron-
donating ability of R observed in the spectra of
ROC(=S)N(H)C6H4Me-4, i.e. downfield for R=Me compared
with upfield for iPr, persists in the spectra of 1–3. In the
31P{1H} NMR, a single resonance was observed for each of
1–3 at approximately 38.0 ppm, i.e. deshielded with respect to
uncoordinated Ph3P (−5.2 ppm). As an indication of the stabil-
Figure 4 illustrates the crystal packing in 3. Molecules
associate into a three-dimensional architecture by a combina-
tion of edge-to-face C–H…π interactions, there being no other
significant intermolecular interactions of note; see [45] for
geometric parameters describing the intermolecular interac-
tions. The interactions pivotally involve the p-tolyl ring in that
it forms a donor interaction to a P-bound phenyl ring, and
accepts two interactions from two P-bound phenyl rings.
Antimicrobial activity
The antibacterial activity of the gold compounds were de-
termined against a wide variety of pathogens comprising
four strains of Gram-positive bacteria and 16 strains of
Gram-negative bacteria. The tested bacterial species vary
with respect to their disease modality and virulence. Of the
20 tested bacterial species, only four bacterial species that
belong to Gram-positive (B. cereus, E. faecalis, E. faecium
and S. aureus) showed consistent inhibition zones towards
1–3 in disc diffusion assay; see data in Table 1.
Briefly, no inhibitory activity was detected from 1 to 3
against any of the Gram-negative bacteria. The results re-
vealed the specific effectiveness of the tested compound 1–3
on Gram-positive bacteria (which have thick peptidoglycan
as the outermost layer in the cell wall), and not against
Gram-negative bacteria (which have an outer membrane
composed of phospholipids and lipopolysaccharide). As
Gram-positive bacteria are dependent on peptidoglycan for
their cell wall protection and to maintain osmotic pressure
1
ity of 1–3 in solution, time-dependent H NMR spectra were
measured. These studies showed that in DMSO solution no
change in the resonances occurred over a period of 2 weeks.
Infrared spectroscopy confirmed the absence of ν(N–H)
in the spectra of 1–3. Compared to that observed for
ROC(=S)N(H)C6H4Me-4, systematic shifts to lower, lower,
and higher wavenumber are noted for absorptions due to
ν(C–N), ν(C–O) and ν(C–S) in 1–3 consistent with the

Gold Bull
Table 1 Antibacterial activity
measured by zone of inhibition
of 1–3 and standard antibiotics
Microorganism
1
2
3
Standard anti-biotics (30 μg/disc)
Gram-Positive bacteria
B. cereus ATCC 10876
8.0
10.5
8.0
8.0
10.5
8.0
8.0
10.5
8.0
15.0 (Tetracycline)
E. faecalis ATCC 29212
E. faecium ATCC 19434
S. aureus ATCC 25923
10.5 (Tetracycline)
20.0 (Chloramphenicol)
20.0 (Chloramphenicol)
9.0
9.0
9.5
Gram-negative bacteria
A. baumannii ATCC 19606
A. hydrophilla ATCC 35654
C. freundii ATCC 8090
E. cloacae ATCC 35030
E. aerogenes ATCC 13048
E. coli ATCC 25922
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
20.0 (Tetracycline)
25.0 (Tetracycline)
25.0 (Tetracycline)
20.0 (Tetracycline)
22.0 (Tetracycline)
25.0 (Tetracycline)
15.0 (Tetracycline)
12.0 (Tetracycline)
25.0 (Tetracycline)
25.0 (Tetracycline)
24.0 (Tetracycline)
22.0 (Tetracycline)
26.0 (Chloramphenicol)
19.0 (Chloramphenicol)
19.0 (Chloramphenicol)
24.0 (Tetracycline)
K. pneumoniae ATCC 700603
P. aeruginosa ATCC 27853
S. typhimurium ATCC 14028
S. paratyphi A ATCC 9150
S. flexneri ATCC 12022
S. sonnei ATCC 9290
S. maltophilia ATCC 13637
P. mirabilis ATCC 25933
P. vulgaris ATCC 13315
V. parahaemolyticus ATCC 17802
The diameter of inhibition zones
in millimetres (mm) were mea-
sured around the disc after 24 h
incubation; –, no zone of
inhibition
[46], the mechanism for the antibacterial action of 1–3
might be related to the inhibition of peptidoglycan synthesis
in the cell walls of Gram-positive bacteria leading to the
death of the bacteria.
The serial dilution assay suggested that the inhibitory
effects of 1–3 towards different pathogens in the disc diffu-
sion assay were dose dependent. Table 2 collects the MIC
and MBC values for the four Gram-positive pathogens in
response to treatment with 1–3 and standard antibiotics. The
MIC values were in the range of 1–37 μg/ml, which may be
considered as high antibacterial activity in comparison with
the standard antibiotic tetracycline for which the range in
MIC values is 4–37 μg/ml. The most susceptible strains
were B. cereus (1–4 μg/ml) and E. faecalis (4 μg/ml), while
E. faecium (37 μg/ml) and S. aureus (37 μg/ml) showed the
lowest sensitivity towards 1–3.
The MBC, time kill curve and serum bactericidal titre are the
in vitro microbiological techniques uses to determine whether an
antibacterial agent is bacteriostatic or bactericidal [47]. MBC was
the method used in this study to determine the bactericidal
activity of the tested compounds towards the inhibited pathogens.
According to Levison [48], antibacterial agents are regarded as
bactericidal if the MBC is no more than fourfold higher than the
MIC. By contrast, the MBC of bacteriostatic agents are many
fold higher than their MIC. This is because the bacteriostatic
agents only prevent the growth of bacteria, whereas bactericidal
Table 2 MIC (in microgram per milliliter) and MBC (in microgram per milliliter) of 1–3 and standard anti-biotics against selected Gram-positive
bacteria
Microorganism
1
2
3
Tetracycline
Chloramphenicol
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
B. cereus ATCC 10876
E. faecalis ATCC 29212
E. faecium ATCC 19434
S. aureus ATCC 25923
1.0
1.0
4.0
4.0
4.0
4.0
37.0
ND
ND
4.0
ND
ND
ND
ND
ND
ND
ND
ND
ND
4.0
4.0
4.0
4.0
4.0
4.0
0.05
0.05
ND
37.0
37.0
37.0
37.0
37.0
37.0
37.0
37.0
37.0
37.0
37.0
37.0
MIC minimum inhibitory concentration, MBC minimum bactericidal concentration, –bacteria grown across all tested dilution which showed
bacteriostatic properties, ND not determined

Gold Bull
6. Jones RN (2003) Global epidemiology of antimicrobial resistance
among community-acquired and nosocomial pathogens: a five-
year summary from the SENTRY Antimicrobial Surveillance
Program (1997–2001). Semin Respir Crit Care Med 24:121–134.
doi:10.1055/s-2003-37923
agents kill the bacteria [49]. The MBC values of 1–3 were same
as their MIC, indicating good bactericidal effects against B.
cereus, E. faecalis, E. faecium and S. aureus.
Data from this study suggest that 1–3 could be potent against
Bacillus spp., Staphylococcus spp. and Enterococcus spp., an
observation in agreement with the recent report of Sordo et al.
[31], who found high antibacterial activity of certain gold
sulfanylcarboxylates against Bacillus spp. and Staphylococcus
spp. Previous reports suggested the potential importance of
bactericidal drugs for treating bacterial infections in severely
neutropenic patients, who exhibit abnormally low level of neu-
trophils in the blood and a lack of host defences to clear the
infecting bacteria if bacteriostatic drugs are introduced [47, 48,
50]. In addition, it is noted that the Gram-positive bacteria are an
important cause of infection in neutropenic patients [47, 51, 52].
In summary, the easily synthesised, stable and crystalline
gold compounds 1–3 may be potentially developed as alter-
native bactericidal agents for use in the treatment of micro-
bial disease especially for recently emerging multidrug-
resistant strains of methicillin-resistant S. aureus (MRSA)
[49] and Enterococcus spp. [53] which are known to cause
life-threatening infections in humans. Efforts are underway
to explore the antibacterial activities of related derivatives
with variations in P-, O-, and N-substituents and into possi-
ble mechanisms of action.
7. Eisler E (2003) Chrysotherapy: a synoptic review. Inflamm Res
52:487–501. doi:10.1007/s00011-002-1208-2
8. Kean WF, Kean IRL (2008) Clinical pharmacology of gold.
Inflammopharmacology 16:112–125. doi:10.1007/s10787-007-0021-x
9. Pacheco EA, Tiekink ERT, Whitehouse MW (2009) Gold com-
pounds and their applications in medicine. In: Mohr F (ed) Gold
chemistry. Applications and future directions in the life sciences.
Wiley, Germany, pp 283–319
10. Berners-Price SJ (2011) Gold-based therapeutic agents: a new
perspective. In: Alessio E (ed) Bioinorganic medicinal chemistry.
Wiley, Weinheim, pp 197–222
11. Bhabak K, Bhuyan BJ, Mugesh G (2011) Bioinorganic and me-
dicinal chemistry: aspects of gold(I)-protein complexes. Dalton
Trans 40:2099–2111. doi:10.1039/C0DT01057J
12. Trávníček Z, Štarha P, Vančo J, Šilha T, Hošek J, Suchý P Jr,
Prazanova G (2012) Anti-inflammatory active gold(I) complexes
involving 6-substituted-purine derivatives. J Med Chem 55:4568–
4579. doi:10.1021/jm201416p
13. Pacheco EA, Tiekink ERT, Whitehouse MW (2009) Biomedical ap-
plications of gold and gold compounds. In: Corti C, Holliday R (eds)
Gold: science and applications. CRC Press, Boca Raton, pp 217–230
14. Berners-Price SJ, Filipovska A (2011) Gold compounds as thera-
peutic agents for human diseases. Metallomics 3:863–873.
doi:10.1039/c1mt00062d
15. Ott I (2009) On the medicinal chemistry of gold complexes as
anticancer drugs. Coord Chem Rev 253:1670–1681. doi:10.1016/
j.ccr.2009.02.019
16. Fregona D, Ronconi L, Aldinucci D (2009) Groundbreaking
gold(III) anticancer agents. Drug Discov Today 14:1075–1076
17. Tiekink ERT (2002) Gold derivatives for the treatment of cancer.
Crit Rev Hematol Oncol 42:225–248. doi:10.1016/S1040-
8428(01)00216-5
Acknowledgments The Ministry of Higher Education (Malaysia) is
thanked for funding medicinal chemistry studies through the High-
Impact Research scheme (UM.C/HIR-MOHE/SC/12). Support from
the University of Malaya is also gratefully acknowledged (UMRG:
RG125).
18. Tiekink ERT (2003) Gold compounds in medicine: potential anti-
tumour agents. Gold Bull 36:117–124. doi:10.1007/BF03215502
19. Fricker SP (1996) Medical uses of gold compounds: past, present
and future. Gold Bull 29:53–60. doi:10.1007/BF03215464
20. Che C-M, Sun RW-Y (2011) Therapeutic applications of gold
complexes: lipophilic gold(III) cations and gold(I) complexes for
anti-cancer treatment. Chem Commun 47:9554–9560.
doi:10.1039/C1CC10860C
Open Access This article is distributed under the terms of the Creative
Commons Attribution License which permits any use, distribution, and
reproduction in any medium, provided the original author(s) and the
source are credited.
21. Milacic V, Dou QP (2009) The tumor proteasome as a novel
target for gold(III) complexes: implications for breast cancer
therapy. Coord Chem Rev 253:1649–1660. doi:10.1016/
j.ccr.2009.01.032
22. Bindoli A, Rigobello MP, Scutari G, Gabbiani C, Casini A,
Messori L (2009) Thioredoxin reductase: a target for gold com-
pounds acting as potential anticancer drugs. Coord Chem Rev
253:1692–1707. doi:10.1016/j.ccr.2009.02.026
References
1. Hemaiswarya S, Kruthiventi AK, Doble M (2008) Synergism be-
tween natural products and antibiotics against infectious diseases.
Phytomedicine 15:639–652. doi:10.1016/j.phymed.2008.06.008
2. Editor (2013) Editorial: the antibiotic alarm. Nature. doi:10.1038/
495141a
3. Jones RN, Pfaller MA (1998) Bacterial resistance: a worldwide
problem. Diagn Microbiol Infect Dis 31:379–388. doi:10.1016/
S0732-8893(98)00037-6
23. Gabbiani C, Cinellu MA, Maiore L, Massai L, Scaletti F (2012)
Chemistry and biology of three representative gold(III) compounds
as prospective anticancer agents. Inorg Chim Acta 393:115–124.
doi:10.1016/j.ica.2012.07.016
24. Madeira JM, Gibson DL, Kean WF, Klegeris A (2012) The bio-
logical activity of auranofin: implications for novel treatment of
diseases. Inflammopharmacology 20:297–306. doi:10.1007/
s10787-012-0149-1
25. Novelli F, Recine M, Sparatore F, Juliano C (1999) Gold(I) com-
plexes as antimicrobial agents. Il Farmaco 54:232–236. doi:10.1016/
S0014-827X(99)00019-1
26. Noguchi R, Hara A, Sugie A, Nomiya K (2006) Synthesis of novel
gold(I) complexes derived by AgCl-elimination between
4. Pfaller MA, Jones RN, Doern GV, Sader HS, Kugler KC, Beach
ML (1999) Survey of blood stream infections attributable to Gram-
positive cocci: frequency of occurrence and antimicrobial suscep-
tibility of isolates collected in 1997 in the United States, Canada,
and Latin America from the SENTRY Antimicrobial Surveillance
Program. SENTRY Participants Group. Diagn Microbiol Infect
Dis 33:283–297. doi:10.1016/S0732-8893(98)00149-7
5. Otto M (2010) Staphylococcus colonization of the skin and anti-
microbial peptides. Expert Rev Dermatol 5:183–195. doi:10.1586/
edm.10.6

Gold Bull
[AuCl(PPh3)] and silver(I) heterocyclic carboxylates, and their anti-
microbial activities. Molecular structure of [Au(R, S-
Hpyrrld)(PPh3)] (H2pyrrld = 2-pyrrolidone-5-carboxylic acid).
Inorg Chem Commun 9:355–359. doi:10.1016/j.inoche.2006.01.001
27. Marques LL, Oliveira GMD, Lang ES, Campos MMAD, Gris LRS
(2007) New gold(I) and silver(I) complexes of sulfamethoxazole:
synthesis, X-ray structural characterization and microbiological
activities of triphenylphosphine(sulfamethoxazolato-N2)gold(I)
and (sulfamethoxazolato)silver(I). Inorg Chem Commun
10:1083–1087. doi:10.1016/j.inoche.2007.06.005
28. Ray S, Mohan R, Singh JK, Samantaray MK, Shaikh MM, Panda D,
Ghosh P (2007) Anticancer and antimicrobial metallopharmaceutical
agents based on palladium, gold, and silver N-heterocyclic carbene
complexes. J Am Chem Soc 129:15042–15053. doi:10.1021/ja075889z
29. Nomiya K, Takahashi S, Noguchi R (2000) Synthesis and crystal
structure of a hexanuclear silver(I) cluster [Ag(Hmna)]6.4H2O
(H2mna=2-mercaptonicotinic acid) and a supramolecular gold(I) com-
plex H[Au(Hmna)2] in the solid state, and their antimicrobial activities. J
Chem Soc Dalton Trans 2000:2091–2097. doi:10.1039/B001664K
30. Corbi PP, Quintão FA, Ferraresi DKD, Lustri WR, Amaral AC,
Massabni AC (2010) Chemical, spectroscopic characterization,
and in vitro antibacterial studies of a new gold(I) complex with
N-acetyl-L-cysteine. J Coord Chem 63:1390–1397. doi:10.1080/
00958971003782608
31. Barreiro E, Casas JS, Couce MD, Sánchez A, Seoane R, Perez-
Estévez A, Sordo J (2012) Synthesis and antimicrobial activities of
gold(I) sulfanylcarboxylates. Gold Bull 45:23–34. doi:10.1007/
s13404-011-0040-7
32. Dinger MB, Henderson W (1998) Organogold(III) metallacyclic
chemistry. Part 41. Synthesis, characterisation, and biological activity
of gold(III)-thiosalicylate and -salicylate complexes. J Organomet
Chem 560:233–243. doi:10.1016/S0022-328X(98)00493-8
33. Ho SY, Bettens RPA, Dakternieks D, Duthie A, Tiekink ERT (2005)
Prevalence of the thioamide {…H-N-C=S}2 synthon—solid-state (X-
ray crystallography), solution (NMR) and gas-phase (theoretical)
structures of O-methyl-N-aryl-thiocarbamides. CrystEngComm
7:682–689. doi:10.1039/B514254G
40. Brandenburg K (2006) DIAMOND. Crystal Impact GbR, Bonn
41. Kuan FS, Ho SY, Tadbuppa PP, Tiekink ERT (2008) Electronic and
steric control over Au…Au, C–H…O and C–H…π interactions in
the crystal structures of mononuclear triarylphosphinegold(I)
carbonimidothioates: R3PAu[SC(OMe)=NR′] for R′ = Ph, o-tol, m-
tol or p-tol, and R′ = Ph, o-tol, m-tol, p-tol or C6H4NO2-4.
CrystEngComm 10:548–564. doi:10.1039/b717198f
42. Tadbuppa PP, Tiekink ERT (2009) [(Z)-O-Ethyl-N-
(p-tolyl)thiocarbamato-κS](triphenylphosphine)-κP]gold(I). Acta
Crystallogr E 65:m1587. doi:10.1107/S1600536809047618
43. Ho SY, Cheng EC-C, Tiekink ERT, Yam VW-W (2006) Luminescent
phosphine gold(I) thiolates: correlation between crystal structure and
photoluminescent properties in [R3PAu{SC(OMe)=NC6H4NO2-4}]
(R=Et, Cy, Ph) and [(Ph2P-R-PPh2){AuSC(OMe)=NC6H4NO2-4}2]
(R=CH2, (CH2)2, (CH2)3, (CH2)4, Fc). Inorg Chem 45:8165–8174.
doi:10.1021/ic0608243
44. Caracelli I, Zukerman-Schpector J, Tiekink ERT (2013)
Supramolecular synthons based on gold…π(arene) interactions.
Gold Bull. doi:10.1007/s13404-013-0088-7
45. Geometric parameters characterising the intermolecular
C–H…π interactions in the crystal structure of 3: C4–H4…Cg(C24-
C29)i = 2.69 Å, C4…Cg(C24-C29)i = 3.473(4) Å, angle at H=140°;
C19–H19…Cg(C2-C7)ii = 2.79 Å, C19…Cg(C2-C7)ii = 3.662(3)Å,
angle at H=153°; and C27–H27…Cg(C2-C7)iii = 2.82 Å, C27…
Cg(C2-C7)iii = 3.515(3)Å, angle at H=131°. Symmetry operations: i,
2-x, ½+y, ½-z; ii, 2-x, -½+y, ½-z; and iii, x, 1½-y, ½+z
46. Schneewind O, Missiakas DM (2012) Protein secretion and sur-
face display in Gram positive bacteria. Phil Trans R Soc B
367:1123–1139. doi:10.1098/rstb.2011.0210
47. Pankey GA, Sabath LD (2004) Clinical relevance of bacteriostatic
versus bactericidal mechanisms of action in the treatment of Gram-
positive bacterial infections. Clin Infect Dis 38:864–870.
doi:10.1086/381972
48. Levison ME (2004) Pharmacodynamics of antimicrobial
drugs. Infect Dis Clin N Am 18:451–465. doi:10.1016/
j.idc.2004.04.012
49. French GL (2006) Bactericidal agents in the treatment of MRSA
infections—the potential role of daptomycin. J Antimicrob
Chemother 58:1107–1117. doi:10.1093/jac/dkl393
34. Hall VJ, Siasios G, Tiekink ERT (1993) Triorganophosphinegold(I)
carbonimidothioates. Aust J Chem 46:561–570. doi:10.1071/
CH9930561
35. CrysAlisPro (2010) Agilent Technologies, Yarnton, Oxfordshire,
England
50. Klastersky J (1986) Concept of empiric therapy with antibiotic
combinations. Am J Med 80:2–12
51. Brown AE (1984) Neutropenia, fever, and infection. Am J Med
76:421–428. doi:10.1016/0002-9343(84)90661-2
52. Pizzo PA, Hathorn JW, Hiemenz J, Browne M, Commers J, Cotton
D, Gress J, Longo D, Marshall D, McKnight J, Rubin M, Skelton
J, Thaler M, Wesley R (1986) A randomized trial comparing
ceftazidime alone with combination antibiotic therapy in cancer
patients with fever and neutropenia. N Engl J Med 315:552–558.
doi:10.1056/NEJM198608283150905
36. Sheldrick GM (2008) A short history of SHELX. Acta Crystallogr
A64:112–122. doi:10.1107/S0108767307043930
37. Farrugia LJ (2012) WinGX and ORTEP for Windows: an update. J
Appl Crystallogr 45:849–854. doi:10.1107/S0021889812029111
38. Gans J, Shalloway D (2001) Qmol: a program for molecular
visualization on Windows based PCs. J Mol Graph Model
19:557–559. doi:10.1016/S1093-3263(01)00090-0
39. Spek AL (2003) Single-crystal structure validation with the pro-
gram PLATON. J Appl Crystallogr 36:7–13. doi:10.1107/
S0021889802022112
53. Arias CA, Contreras GA, Murray BE (2010) Management of
multidrug-resistant enterococcal infections. Clin Microbiol Infect
16:555–562. doi:10.1111/j.1469-0691.2010.03214.x