Development of ruthenium(ii) complexes as topical antibiotics against methicillin resistant Staphylococcus aureus

Article abstract of DOI:10.1039/c3dt52879k

A series of ruthenium(ii) bis(2,2′-bipyridyl) complexes containing N-phenyl-substituted diazafluorenes (Ru-C₁, Ru-C₆, Ru-C₇ and Ru-F) was synthesized and their potential antibacterial activity against methicillin resistant Staphylococcus aureus (MRSA) was investigated. The Ru-C₇ complex showed significant improvement in both minimum inhibitory concentration (MIC, 6.25 μg mL^-1) and minimum bactericidal concentration (MBC, 25 μg mL^-1) towards MRSA when compared with those of methicillin (positive control) (MIC = 25 μg mL^-1 and MBC = 100 μg mL^-1). The Ru-C₇ complex possessed much stronger antibacterial effects than the Ru-C₆ complex (MIC, 25 μg mL^-1, MBC, >100 μg mL^-1). Both Ru-C₆ and Ru-C₇ complexes were also demonstrated to be biologically safe when tested on normal human skin keratinocytes.

Full text of DOI:10.1039/c3dt52879k

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Development of ruthenium(II) complexes as topical
antibiotics against methicillin resistant
Staphylococcus aureus†
Cite this: Dalton Trans., 2014, 43,
3949
P.-L. Lam,a G.-L. Lu,b,c K.-M. Hon,b K.-W. Lee,b C.-L. Ho,b X. Wang,b J. C.-O. Tang,a
K.-H. Lam,a R. S.-M. Wong,d S. H.-L. Kok,e Z.-X. Bian,f H. Li,*g K. K.-H. Lee,*e
R. Gambari,*h C.-H. Chui*f and W.-Y. Wong*b,i
A series of ruthenium(II) bis(2,2’-bipyridyl) complexes containing N-phenyl-substituted diazafluorenes
(Ru-C1, Ru-C6, Ru-C7 and Ru-F) was synthesized and their potential antibacterial activity against methicil-
lin resistant Staphylococcus aureus (MRSA) was investigated. The Ru-C7 complex showed significant
improvement in both minimum inhibitory concentration (MIC, 6.25 µg mL−1) and minimum bactericidal
concentration (MBC, 25 µg mL−1) towards MRSA when compared with those of methicillin (positive
control) (MIC = 25 µg mL−1 and MBC = 100 µg mL−1). The Ru-C7 complex possessed much stronger anti-
bacterial effects than the Ru-C6 complex (MIC, 25 µg mL−1, MBC, >100 µg mL−1). Both Ru-C6 and Ru-C7
complexes were also demonstrated to be biologically safe when tested on normal human skin
keratinocytes.
Received 13th October 2013,
Accepted 14th December 2013
DOI: 10.1039/c3dt52879k
www.rsc.org/dalton
previously remediable pathogens, especially methicillin-resist-
ant Staphylococcus aureus (MRSA), are now becoming incurable
Introduction
Owing to the global emergence of drug resistant bacteria in and fatal. MRSA can induce severe skin and soft tissue infec-
response to clinically applied antibiotics, it is urgently needed tions, pneumonia and death.1 The morbidity and mortality
to discover new classes of antimicrobials to overcome the over- rate per 100 000 people is estimated to be 4.6 and 0.5, respecti-
whelming antibiotic resistance problems in our community. Some vely.2,3 In order to overcome the challenge of MRSA infections,
metal complexes with improved biological properties have
been developed and synthesized. Metal ions are essential for
aState Key Laboratory of Chirosciences, Department of Applied Biology and Chemical
Technology, The Hong Kong Polytechnic University, Hong Kong, P.R. China
bInstitute of Molecular Functional Materials, Department of Chemistry and Institute
of Advanced Materials, Hong Kong Baptist University, Hong Kong, P.R. China.
E-mail: rwywong@hkbu.edu.hk; Fax: +852-3411 7348; Tel: +852-3411 7074
cAuckland Cancer Society Research Centre, Faculty of Medical and Health Sciences,
The University of Auckland, New Zealand
human beings. The capability of identifying and realizing the
functions of metal ions for disease treatments remains impor-
tant in bioinorganic and medicinal inorganic chemistry. A
series of cobalt, copper, nickel, zinc and silver complexes have
been reported to have antimicrobial activities.4,5
Among various metal-containing compounds, ruthenium
compounds are considered stable under both highly acidic
and alkaline conditions.6 Applications of ruthenium com-
plexes or compounds have a long history. This includes
antimicrobial activity,7–18 anti-cancer activity,18–20 DNA
binding,18,21–23 and chemiluminescence.24 The antibacterial
activity of mononuclear tris(bidentate)-ruthenium(II) and
-iron(II) complexes containing polypyridyl ligands has been
reported by Dwyer and co-workers more than fifty years ago.7,8
The potential effects on microbial growth inhibition of ruthe-
nium complexes have been re-studied recently. Keene, Collins
and co-workers have shown that inert dinuclear polypyridyl-
ruthenium(II) complexes in which the metal centres are linked
by a flexible alkane chain, [{Ru(phen)2}2{μ-bbn}]4+ {phen =
1,10-phenanthroline and bbn = bis[4(4′-methyl-2,2′-bipyridyl)]-
1,n-alkane (n = 2, 5, 7, 10, 12 or 16)}, exhibited excellent
dDepartment of Medicine and Therapeutics, Prince of Wales Hospital,
The Chinese University of Hong Kong, Hong Kong, P.R. China
eSchool of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong,
P.R. China. E-mail: kaholee@cuhk.edu.hk; Fax: +852-2603 5139;
Tel: +852-3943 6785
fClinical Division, School of Chinese Medicine, Hong Kong Baptist University,
Hong Kong, P.R. China. E-mail: chchui@hkbu.edu.hk; Fax: +852-2637 5396;
Tel: +852-2632 1500
gCollege of Life and Environmental Sciences, Minzu University of China,
Beijing 100081, P.R. China. E-mail: lihuamuc@gmail.com
hCentre of Biotechnology, Department of Life Sciences and Biotechnology, University
of Ferrara, Ferrara, Italy. E-mail: gam@unife.it; Fax: +39-532-202723;
Tel: +39-532-974443
iInstitute of Polymer Optoelectronic Materials and Devices, State Key Laboratory of
Luminescent Materials and Devices, South China University of Technology,
Guangzhou 510640, P.R. China
†CCDC 956920. For crystallographic data in CIF or other electronic format see
DOI: 10.1039/c3dt52879k
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growth inhibition (1 to 2 μg mL−1) against both Gram positive
Staphylococcus aureus (S. aureus) and methicillin-resistant
S. aureus (MRSA), and Gram negative Escherichia coli (E. coli)
and Pseudomonas aeruginosa (P. aeruginosa).9,10 The prelimi-
nary cytotoxicity study revealed that Ru(bbn)-type complexes
with good antibacterial activity did not possess significant
cytotoxic effects towards human red blood cells and a human
monocytic leukemia cell line (THP-1).9 This research group
further demonstrated that the antibacterial activity of chloride-
containing polypyridyl-ruthenium(II) complexes (1 to 2 μg mL−1
)
was generally stronger than that of iridium(III) chloro complexes
(2 to 64 μg mL−1) against a range of Gram positive and Gram
negative bacteria.11,12 Higher cellular uptake as well as quicker
membrane permeability and depolarization have been demon-
strated to be crucial to the antimicrobial activity of ruthenium
complexes.10,13 Krishnamoorthy et al.14 reported the binuclear
ruthenium(II) carbonyl complexes which possessed better
growth inhibition against the Gram positive S. aureus, as well as
the Gram negative E. coli and Klebsiella pneumoniae (K. pneumo-
niae) than the free ligand because of Tweedy’s chelation
theory.15 Gabra et al. also reported that the Ru(II) complex
[Ru(dmb)2IIP]2+, where dmb is 4,4′-dimethyl-2,2′-bipyridine and
IIP is 2-(1H-indol-3-yl)-1H-imidazo[4,5-f] [1,10]phenanthroline,
showed antibacterial activities against E. coli and S. aureus at
20 µg mL−1 due to Tweedy’s chelation theory.16 The chelation
reduces the polarity of the metal atom due to partial sharing of
its positive charge with the donor groups and possible electron
delocalization over the entire ring. This results in an increased
lipophilicity of the metal chelates. The increased lipophilicity
facilitates the penetration of metal complexes into the lipid
layers of the bacterial membrane which blocks the metal
binding sites on the enzymes of microorganisms. Additionally,
these complexes interfere with the respiration process of the
bacterial cells, and therefore avoid the protein synthesis and
inhibit further growth of the microorganisms.15–17
Fig. 1 Chemical structures of new ruthenium(II) complexes containing
various N-phenyl-substituted 4,5-diazafluorenes.
Experimental
General
All reactions were carried out under a nitrogen atmosphere
with the use of standard Schlenk techniques, but no special pre-
cautions were taken to exclude oxygen during work-up. Solvents
were predried and distilled from appropriate drying agents. All
chemicals, unless otherwise stated, were obtained from com-
mercial sources and used as received. 1H NMR spectra were
measured in appropriate solvents on a JEOL EX270 or a Varian
INOVA 400 MHz FT-NMR spectrometer, with the chemical shifts
quoted relative to SiMe4. Fast atom bombardment (FAB) mass
spectra were recorded on a Finnigan MAT SSQ710 mass spectro-
meter. Electronic absorption spectra were obtained with a
Hewlett Packard 8453 UV-visible spectrometer.
Synthesis of N-phenyl-substituted diazafluorenes (Scheme 1)
4,5-Diazafluoren-9-one (0.15 g, 0.824 mmol), 97% of 4-substi-
tuted aniline (0.16 g, 0.828 mmol) and 4-toluenesulfonic acid
(0.0072 g, 0.038 mmol) were added to toluene (20 mL) and the
mixture was brought to reflux under 100 °C using a Dean and
Stark apparatus in order to drive the reaction to the product
side. At the beginning, the reactants were not soluble in the
solvent. After a while, all reactants dissolved to form a yellow
solution. After 24 h of reflux, a pale yellow solution was
obtained and it was then concentrated to about 2 mL and
5 mL ethanol was also added. The first crop of product was
obtained by filtration followed by washing with ethanol (3 mL)
twice to obtain a yellow powder. The filtrate was evaporated to
dryness using a rotary evaporator and the residue was then
As a topically used remedy for skin bacterial infections, the
developed antimicrobial agents should exhibit satisfactory
antibacterial effects, and more importantly, they should have
low cytotoxicity towards human beings. Ruthenium complexes
have been reported to display the cytotoxic effects towards
cancer cells. The high cytotoxicity of [Ru(bpy)2(4,4′-bbtb)]Cl2
(bpy
=
2,2′-bipyridyl); [Ru(bpy)2(4,5′-bbtb)]Cl2 and [Ru-
(bpy)2(5,5′-bbtb)]Cl2, where bbtb is bis(benzothiazol-2-yl)-2,2′-
bipyridine (1–10 µM) against a range of cancer cells including
MCF-7 and MDA-MB-468 (human breast carcinoma), HT29
and RKO (human colon carcinoma) and SkMel (human mela-
noma) has been demonstrated.19 The mixed ligand ruthenium(II)
complexes [Ru(bbdo)(dppz)](ClO4)2 (bbdo = 1,8-bis(benzimidazol-
2-yl)-3,6-dithiooctane; dppz = dipyrido[3,2-a:2′,3′-c]-phenazine)
exhibited a much stronger cytotoxicity against the melanoma
cancer cell line (A375) than that of cisplatin with low cytotoxicity
to normal cells.20 In this study, four new ruthenium(II) bis(2,2′-
bipyridyl) complexes containing N-phenyl-substituted diaza-
fluorenes were synthesized (Fig. 1) and their antibacterial acti-
vities towards MRSA and cytotoxic effects on HaCaT human
skin keratinocytes were investigated.
Scheme 1
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recrystallized from ethanol and water. Another batch of 8.18–8.28 (m, Ar), 8.35–8.40 (m, Ar), 8.74–8.80 (m, Ar). FAB-MS
product was thus obtained. All components of the series were (m/z): 928.9 ([M − PF6]+). Elemental analysis calcd (%) for
satisfactorily characterized by IR, 1H-NMR and FAB-MS C44H41N7ORuP2F12: C 49.17, H 3.84, N 9.12; found: C 49.30,
techniques.
L-C7: Yield: 0.16 g (52%). 1H NMR δ (ppm): 0.91 (t, J =
H 4.02, N 9.33.
Synthesis of Ru-C6. cis-Dichlorobis(2,2′-bipyridine)ruthe-
6.5 Hz, CH3), 1.32–1.36 (m, CH2), 1.46–1.55 (m, CH2), nium (0.05 g, 0.103 mmol) and ligand L-C6 (0.03 g,
1.76–1.84 (m, CH2), 4.02 (t, J = 6.6 Hz, CH2), 6.96–7.12 (m, Ar), 0.084 mmol) were refluxed in 25 mL of ethanol for 4 h. The
7.37–7.40 (m, Ar), 8.25 (m, Ar), 8.65 (m, Ar), 8.80 (m, Ar). solution was filtered hot under vacuum, and ammonium hexa-
FAB-MS (m/z): 370.8.
fluorophosphate (0.27 g) was added to the solution mixture.
L-C6: Yield: 0.19 g (61%). 1H NMR δ (ppm): 0.94 (t, J = After that, the solution was allowed to stand overnight while
6.7 Hz, CH3), 1.33–1.40 (m, CH2), 1.44–1.55 (m, CH2), red crystals of Ru-C6 were formed. Then, the crystals were
1.77–1.87 (m, CH2), 4.03 (t, J = 6.7 Hz, CH2), 6.95–7.12 (m, Ar), repeatedly recrystallized from acetone–water and ethanol–
7.35–7.40 (m, Ar), 8.25 (m, Ar), 8.65 (m, Ar), 8.79 (m, Ar). water. Orange-red precipitates were obtained in 67% yield
1
FAB-MS (m/z): 356.9.
(0.06 g). H NMR δ (ppm): 0.94 (t, J = 7.0 Hz, CH3), 1.35–1.53
L-C1: Yield: 0.18 g (63%). 1H NMR δ (ppm): 3.89 (s, CH3), (m, CH2), 1.76–1.86 (m, CH2), 4.09 (t, J = 6.5 Hz, CH2),
6.94–7.10 (m, Ar), 7.36–7.41 (m, Ar), 8.26 (d, J = 7.6 Hz, Ar), 7.07–7.18 (m, Ar-H), 7.42–7.74 (m, Ar), 7.90 (d, J = 5.4 Hz, Ar),
8.66 (d, J = 4.8 Hz, Ar), 8.81 (d, J = 4.86 Hz, Ar). FAB-MS (m/z): 8.02 (d, J = 4.6 Hz, Ar), 8.17–8.27 (m, Ar), 8.41–8.47 (m, Ar),
286.8.
8.78–8.83 (m, Ar). FAB-MS (m/z): 916.5 ([M − PF6]+). Elemental
L-F: Yield: 0.15 g (66%). 1H NMR δ (ppm): 7.01–7.30 (m, analysis calcd (%) for C43H39N7ORuP2F12: C 48.69, H 3.71,
Ar), 7.54 (m, Ar), 8.28 (m, Ar), 8.67 (m, Ar), 8.80 (m, Ar). N 9.24; found: C 48.87, H 3.89, N 9.13.
FAB-MS (m/z): 276.3.
Synthesis of Ru-C1. cis-Dichlorobis(2,2′-bipyridine)ruthe-
nium (0.194 g, 0.401 mmol) and ligand L-C1 (0.11 g,
0.382 mmol) were refluxed in 25 mL of ethanol for 4 h. The
solution was filtered hot under vacuum. Then, ammonium
hexafluorophosphate (0.33 g) was added to the solution
mixture. After that, the solution was left to stand overnight,
and crystals of Ru-C1 were formed. After repeated recrystalliza-
tion from acetone–water and ethanol–water, orange-red pre-
cipitates were obtained in 53% yield (0.20 g). 1H NMR δ (ppm):
3.86 (s, OCH3), 7.06–7.17 (m, Ar), 7.43–7.61 (m, Ar), 7.68–7.73
(m, Ar), 7.88 (d, J = 5.4 Hz, Ar), 8.00 (d, J = 5.4 Hz, Ar),
8.15–8.25 (m, Ar), 8.40–8.46 (m, Ar), 8.79–8.81 (m, Ar). FAB-MS
(m/z): 845.5 ([M − PF6]+). Elemental analysis calcd (%) for
C38H29N7ORuP2F12: C 46.07, H 2.95, N 9.90; found: C 45.92, H
3.10, N 10.20.
Synthesis of new ruthenium(II) complexes (Scheme 2)
A suspension of RuCl3·nH2O, 2,2′-bipyridine and LiCl in DMF
was heated under reflux for 12 h. The reaction mixture
was allowed to cool to 20 °C and acetone was added. Finally,
the mixture was stored in
a refrigerator overnight to
yield crude dark-green crystals. Filtration and washing
with water proceeded until the filtrate became colourless.
Repeated washing with aliquots of diethyl ether gave the
complex of pure dark-green crystals. Then, the crystals were
dried under reduced pressure to give cis-RuCl2(bpy)2·2H2O in
good yield.
Synthesis of Ru-C7. cis-Dichlorobis(2,2′-bipyridine)ruthe-
nium (0.05 g, 0.103 mmol) and ligand L-C7 (0.03 g,
0.084 mmol) were refluxed in 25 mL of ethanol for 4 h. The
solution was filtered hot under vacuum, and ammonium hexa-
fluorophosphate (0.27 g) was added to the solution mixture.
After that, the solution was allowed to stand overnight while
red crystals of Ru-C7 were formed. Then, the crystals were
repeatedly recrystallized from acetone–water and ethanol–
water. Orange-red precipitates were obtained in 64% yield
Synthesis of Ru-F. cis-Dichlorobis(2,2′-bipyridine)ruthenium
(0.09 g, 0.186 mmol) and ligand L-F (0.05 g, 0.182 mmol) were
refluxed in 25 mL of ethanol for 4 h. The solution was filtered
hot under vacuum. Then, ammonium hexafluorophosphate
(0.35 g) was added to the solution mixture. Crystals of Ru-F
were formed upon standing and they were repeatedly recrystal-
lized from acetone–water and ethanol–water. Orange-red pre-
1
cipitates were obtained in good yield (0.12 g, 68%). H NMR δ
1
(0.07 g). H NMR δ (ppm): 0.93 (t, J = 7.0 Hz, CH3), 1.35–1.49
(ppm): 7.16–7.21 (m, Ar), 7.26–7.33 (m, Ar), 7.40–7.45 (m, Ar),
7.56–7.64 (m, Ar), 7.74 (q, Ar), 7.90 (dd, J = 5.4 Hz, Ar), 8.03
(dd, J = 5.4 Hz, Ar), 8.15–8.26 (m, Ar), 8.40–8.46 (m, Ar),
8.77–8.83 (m, Ar). FAB-MS (m/z): 834.0 ([M − PF6]+). Elemental
analysis calcd (%) for C37H26N7RuP2F13: C 45.41, H 2.68, N
10.02; found: C 45.64, H 2.75, N 9.93.
(m, CH2), 1.79–1.88 (m, CH2), 4.04 (t, J = 6.4 Hz, CH2),
7.01–7.16 (m, Ar), 7.42–7.68 (m, Ar), 7.90–8.08 (m, Ar),
X-Ray crystallography
X-ray diffraction data were collected at 293 K using graphite-
monochromated Mo-Kα radiation (λ = 0.71073 Å) on a Bruker
Axs SMART 1000 CCD diffractometer. The collected frames
were processed with the software SAINT+25 and an absorption
correction (SADABS)26 was applied to the collected reflections.
Scheme 2
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The structure was solved by the direct method (SHELXTL)27 in suspended at various treatment concentrations (for Ru-C7 and
conjunction with standard difference Fourier techniques and methicillin) in phosphate buffer saline (PBS) containing 30 μM
subsequently refined by full-matrix least-squares analyses on of 2′-7′-dichlorodihydrofluorescein diacetate (DCFH-DA) dye at
F2. Hydrogen atoms were generated in their idealized positions 37 °C for 60 min in a shaker. Afterwards, the treated bacterial
and all non-hydrogen atoms were refined anisotropically. culture was washed twice with PBS, pelleted after centrifu-
Crystal data: C38H29N7F12O2P2Ru, Mw = 990.69, tetragonal, gation and finally re-suspended in a total volume of 1 mL of
space group P43, a = 17.6845(7), b = 17.6845(7), c = 12.6644(9) PBS. Fluorescence values were then measured at an excitation
Å, V = 3960.7(4) Å3, Z = 4, ρcalcd = 1.661 Mg m−3, μ(MoKα) = wavelength of 485 nm and emission wavelength of 515 nm in a
0.576 mm−1, F(000) = 1984, T = 293 K. 19 999 reflections 96-well black plate (Perkin Elmer) using a Perkin Elmer Victor
measured, of which 5879 were unique (Rint = 0.0975). Final V multiwell plate reader.29
R1 = 0.0581 and wR2 = 0.1314 for 3559 observed reflections
with I > 2σ(I) (CCDC-956920).
Cytotoxicity assay
Human cells including HaCaT skin keratinocytes were
Determination of minimum inhibitory concentrations (MICs)
removed from 75 mL sterile cell culture flasks with trypsin and
neutralized with fetal bovine serum. After washing with phos-
and minimum bactericidal concentrations (MBCs)
The concentration of methicillin-resistant Staphylococcus
aureus (MRSA) was adjusted to 1 × 106 cell mL−1. The
minimum inhibitory concentrations (MICs) of our synthesized
compounds were determined by the Luria Broth (LB) dilution
method. Various concentrations of synthesized compounds
were added from a starting concentration of 100 μg mL−1 and
they were diluted serially in the 96 wells microtitre plate.
Methicillin was used as a positive control while 0.2% DMSO
was used as a negative control. They were then incubated at
37 °C for 24 h. The minimum concentrations of compounds
that induced a complete growth inhibition will be recorded as
MICs.28 Ten microliters of samples obtained from the wells
having no visible growth will be plated on the agar plates and
the ones which did not have any colony formed with a
minimum concentration will be the MBCs.
phate buffered saline and centrifugation, skin cells were re-
suspended in complete cell culture medium at a concentration
of approximately 1 × 105 cells mL−1 and counted manually
using a haematocytometer under an inverted microscope.
Human cells seeded in the 96 wells microtitre plates for 24 h
were prepared for the screening of our compounds. The com-
plexes Ru-C6 and Ru-C7 with the concentration of 50 μg mL−1
,
0.1% DMSO (as negative control) and 2 µg mL−1 doxorubicin
(as positive control) were incubated with cells for a further
period of 24 h. The cytotoxicity assessment was conducted by
the sulforhodamine B protein staining method. Skin cells were
fixed with trichloroacetic acid, washed with deionized water
and stained with sulforhodamine B. Afterwards, cells were
washed again with acetic acid. Photomicrographs of skin
keratinocytes were captured and optical absorptions were then
measured at 575 nm after dissolving in Tris-base buffer using
a Perkin Elmer Victor V multiwell plate reader.28,30,31
Zone of clearance study
MRSA was used to study the effectiveness of the complexes
Ru-C6 and Ru-C7 against its growth in culture. MRSA was
diluted with broth medium and plated on the nutrient agar
plate and the holes were created on the agar using a sterile
transfer pipette. The compounds with two concentrations (5 μg
Results
Synthesis of ruthenium(II) complexes
and 10 μg), 0.1% DMSO (as negative control) and 5 μg methicil- Synthesis of the N-(p-alkoxyphenyl)-substituted 4,5-diazafluor-
lin (as positive control) were placed in the holes of agar. The enes as well as the fluoro-substituted analogue and their ruthe-
plates were then incubated at 37 °C for 24 h and the inhibition nium(II) bis(2,2′-bipyridyl) complexes (Ru-C1, Ru-C6, Ru-C7 and
zones (mm) of MRSA on the agar plates were recorded.28
Ru-F) is shown in Scheme 2. The p-alkoxyphenyl-N-(4,5-diaza-
fluorenylene)aniline precursors were prepared by condensation
of 4,5-diazafluoren-9-one with an appropriate substituted
aniline in toluene in the presence of 4-toluenesulfonic acid as
a catalyst. The Ru(II) complexes were synthesized through the
reaction of cis-[Ru(2,2′-bipyridyl)2Cl2] with each of the above
ligands followed by metathesis with NH4PF6 and recrystalliza-
tion. The geometry of Ru-C1 was confirmed by X-ray crystallo-
graphy (Fig. 2). These Ru(II) complexes display a visible
absorption band at around 441–450 nm at 298 K presumably
due to a mixture of the π to π* electronic transitions of the N-
Time-kill kinetics assay
MRSA bacterial culture (1 × 106 cell mL−1, 1 mL) was treated
with different concentrations of Ru-C7 complex at 37 °C in an
orbital shaker. Time-kill assays were analyzed by determining
the reduction in viable count at 2 and 4 h and comparing
those with that at 0 h.
Determination of reactive oxygen species (ROS)
Intracellular ROS in the MRSA were measured using dichloro- phenyl-substituted 4,5-diazafluorenes and metal-to-ligand
fluorescein diacetate (DCFH-DA) with minor modifications. charge-transfer (MLCT). As compared to the free ligands
MRSA bacterial culture (1 × 106 cell mL−1, 1 mL) was pelleted (420–443 nm), it was shown that the absorption peaks
by centrifugation at 5000g for 10 min. The pellet was re- occurred at longer wavelengths upon metal coordination
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their concentration used was up to 50 µg mL−1. The results
revealed that Ru-C7 could be considered as bactericidal against
MRSA since its MBC was no more than four times its MIC32,33
whereas Ru-C6 could be regarded as bacteriostatic because its
MBC was more than fourfold its MIC.32,33 Additionally, Ru-C7
exhibited much stronger inhibitory and microbicidal effects
against MRSA than methicillin, suggesting that Ru-C7 could be
more effective in killing the bacteria.
Zone of clearance study
It was noticed that Ru-C7 possessed better anti-MRSA activity
relative to that of methicillin (positive control) from the zone
of inhibition assay (Table 2 and Fig. 3). A dose response of
Ru-C6 and Ru-C7 in antibacterial activities is shown. A higher
dose of ruthenium(II) complexes (10 μg) displayed a stronger
inhibitory effect than that with a lower dose (5 μg). Ru-C7 at
doses of 5 μg and 10 μg showed to have a larger inhibition
zone towards MRSA when compared with methicillin.
Although methicillin exhibited inhibition zones towards MRSA
on the agar plates, its inhibition zones did show certain
degree of blurred effects. However, the new ruthenium(II) com-
pounds have clear inhibition zones towards MRSA, revealing
that it could provide a prolonged antibacterial activity against
MRSA. This might be associated with the fact that methicillin
only inhibits an initial growth of bacterial cells and its bac-
terial growth inhibition ability decreases with a subsequent
growth of MRSA. The results showed that Ru-C7 could promote
better growth inhibition of the bacteria as compared to methi-
cillin. DMSO as a negative control did not give any anti-MRSA
activity.
Fig. 2 X-ray crystal structure of the cation of Ru-C1.
Table 1 MICs and MBCs of the synthesized ruthenium(II) complexes.
Results are shown as mean SD from three independent experiments
MRSA
MIC (μg mL−1
)
MBC (μg mL−1
)
Ru-C7
Ru-C6
6.25
25
25
>100
N.D.
N.D.
100
Ru-C1
Ru-F
>100
>100
25
Methicillin (positive control)
N.D., not determined.
Table 2 Zone of clearance (mm) towards MRSA. Results are shown as
accompanied by an increase of the molar absorptivity for the
compounds.
mean SD from three independent experiments
Zone of clearance (mm)
MRSA
Determination of minimum inhibitory concentrations (MICs)
and minimum bactericidal concentrations (MBCs)
5 μg
4.60 0.40
1.70 0.26
10 μg
Table 1 shows the minimum inhibitory concentrations (MICs)
and minimum bactericidal concentrations (MBCs) of four syn-
thesized ruthenium complexes against MRSA. Among the four
complexes, Ru-C6 and Ru-C7 exhibited growth inhibition
towards MRSA. Ru-C7 complex had much stronger antibacter-
ial activities than Ru-C6. This could be shown in the lower MIC
(6.25 µg mL−1) and MBC (25 µg mL−1) of Ru-C7 against MRSA
when compared with those of Ru-C6, with the MIC of 25 µg
mL−1 and MBC of more than 100 µg mL−1. The results indi-
cated that a lower concentration of Ru-C7 was required to
inhibit the MRSA growth (MIC) and kill the MRSA strain
(MBC), leading to a higher efficiency in antibacterial activities.
Furthermore, Ru-C7 did show significant improvement in both
MIC and MBC in contrast to those of methicillin (positive
control) (MIC = 25 µg mL−1 and MBC = 100 µg mL−1) after an
incubation period of 24 h. Both the free ligands L-C6 and L-C7
Ru-C7
Ru-C6
5.67 0.40
3.97 0.45
5 μg Methicillin (positive control)
DMSO (negative control)
3.77 0.25
Nil
did not exhibit any growth inhibition towards MRSA when Fig. 3 Growth inhibition results towards MRSA.
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Fig. 5 Generation of fluorescence signal from MRSA after various treat-
ment with Ru-C7 and methicillin and incubation with 30 μM of
dichlorofluorescein diacetate (DCFH-DA). The presence of ROS would
cleave DCFH-DA to DCFH which would generate a green fluorescence
signal. Results are shown as mean SD from three independent exper-
iments. *P value < 0.05 when compared with control.
Fig. 4 Time-kill kinetics assay for the study of the antibacterial activity
of Ru-C7 at both 25 μg mL−1 (4 MIC) and 50 μg mL−1 (8 MIC) after 2 h
and 4 h of incubation with MRSA.
Time-kill kinetics assay
A time-kill kinetics assay was also conducted to provide the
information on bacterial killing effects at timed intervals.
MRSA was incubated with Ru-C7 at both 25 μg mL−1 (4 MIC)
and 50 μg mL−1 (8 MIC), respectively. The time-kill kinetics of
4 MIC was similar to that of 8 MIC with a slightly less potency.
After 2 h of incubation, about 70% of the bacterial cells could
survive from 4 MIC and about 10% from that of 8 MIC treat-
ments as compared with those from time 0. After 4 h of incu-
bation, the viable bacterial cells further decreased to about 1%
from 4 MIC and about 0.1% from that of 8 MIC treatments
(Fig. 4) relative to those from time 0. The results showed that 8
MIC of Ru-C7 could be characterized as bactericidal as more
than 99.9% reduction in bacterial colonies from the initial
inoculum32,33 was observed after 4 h. However, 4 MIC of Ru-C7
was noticed as bacteriostatic because of less than 99.9% kill of
bacteria32,33 after 4 h. Ru-C7 at 8 MIC could be more effective
to kill the MRSA strain after the 4 h exposure when compared
with that at 4 MIC.
Determination of reactive oxygen species (ROS)
The possible underlying mechanism on how Ru-C7 showed
anti-MRSA activity was further investigated. As shown in Fig. 5,
it was noted that Ru-C7 could significantly induce the reactive
oxygen species (ROS) generation after incubation with MRSA
for 1 h when compared with methicillin.
Fig. 6 Cytotoxicity evaluation of the ruthenium(II) compounds on
human skin keratinocytes: (A) untreated control; (B) vehicle control
(0.1% DMSO); (C) 50 µg mL−1 Ru-C6 complex; (D) 50 µg mL−1 Ru-C7
complex; (E) 2 µg mL−1 doxorubicin and (F) cell viability of ruthenium(II)
compounds towards human skin keratinocytes. Each experiment was
conducted in triplicate and a mean value was obtained. Three indepen-
Cytotoxicity assay
dent experiments were done. Results are shown as mean
three independent experiments. *P value <0.05 when compared with
untreated control.
SD from
Fig. 6 shows the cell viability of our ruthenium(II) compounds
towards human skin keratinocytes. It was observed that both
the ruthenium compounds at the concentration of 50 µg mL−1
and vehicle control (0.1% DMSO) exhibited satisfactory cell via-
bility and more than 90% of skin cells still survived. However,
the positive control, 2 µg mL−1 doxorubicin, had only about
40% of cell viability towards skin keratinocytes. From Fig. 6, it
shows that ruthenium compounds at 50 µg mL−1 possessed
similar cellular morphology to the untreated control and the
vehicle control (0.1% DMSO) whereas the positive control,
2 µg mL−1 doxorubicin, exhibited a high degree of cellular
shrinkage and condensation towards skin keratinocytes.
3954 | Dalton Trans., 2014, 43, 3949–3957
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since only 99% reduction of bacteria was shown.32,33 The bac-
teria exposed to Ru-C7 at 8 MIC might no longer divide, result-
Discussion
In this work, the antibacterial activities of synthesized ing in stopping their growth.
ruthenium(II) complexes with the ligands of bis(2,2′-bipyridyl)
Many antibiotics, such as ciprofloxacin, execute the antibac-
against MRSA strain were investigated. Collins, Keene and co- terial activity by inducing oxidative stress in bacterial cells.37
workers reported the mononuclear complexes [Ru- Recently, engineered zinc oxide and titanium(IV) oxide nano-
(phen)2(bb7)]2+ and [Ru(phen)2(bb16)]2+, in which the poten- particles were shown to induce oxidative stress after penetrat-
tially bridging bbn ligands are coordinated through only one of ing the E. coli cell membrane, causing bacterial DNA damages
the two Me2bpy (4,4′-dimethyl-2,2′-dipyridyl) chelating groups, and cell death.29 One of the possible antibacterial mechanisms
while the other Me-bpy group, which is separated from the co- of Ru-C7 was further demonstrated by the intracellular gene-
ordinated one by a chain of either 7 or 16 methylenes, was not ration of ROS. Bacterial cells might uptake the Ru-C7 and this
coordinated. Both [Ru(phen)2(bb7)]2+ and [Ru(phen)2(bb16)]2+ compound might further induce the ROS generation. The
had
a significantly stronger cytotoxic effect (MIC = 16 excessive generation of ROS would cause lethal DNA damage
µg mL−1) against the MRSA strain when compared with the and bacterial cell death.29 This may be one of the pathways for
mononuclear complex [Ru(phen)2(Me2bpy)]2+ (without the Ru-C7 to execute its inhibitory and bactericidal effects towards
methylene bridge) (MIC > 128 µg mL−1).9 Accordingly, our MRSA. The next challenge is the development of the topical
results showed that the mononuclear complexes Ru-C7 and antibiotics with the therapeutic selectivity between bacteria
Ru-C6 exhibited MICs of 6.25 µg mL−1 and 25 µg mL−1 against cells and normal human cells. The desired antibacterial
MRSA, respectively, and they were more cytotoxic against agents should have the promising antibacterial effects, but
MRSA than Ru-C1. Similar observation could also be identified with the weak cytotoxic activity towards human cells. The anti-
from their corresponding MBC values. Structurally, we biotics, either bacteriostatic or bactericidal agents, should
observed that the antibacterial activity of our ruthenium com- exhibit no significant cytotoxicity during topical adminis-
plexes may at least in part be dictated by the electron-donating tration. The selectivity of antibacterial compounds between
ability of the substituents. An improved anti-MRSA activity bacterial cells and normal human cells has been demonstrated
could be observed when there was an increase in the electron- previously. Collins, Keene and co-workers demonstrated that
donating effect from fluorine to OC7H15.
the synthetic inert polypyridylruthenium(II) complexes exhibi-
Ru-C7 could be regarded as a bactericidal agent against ted the greater values of 50% inhibitory concentration (IC50)
MRSA as its MBC (25 µg mL−1) was no more than four times towards human fresh red blood cells and human monocytic
its MIC (6.25 µg mL−1).32,33 Theoretically, MRSA in exposure to THP-1 cells when compared with their MICs against suscep-
Ru-C7 could no longer grow even after the elimination of the tible S. aureus and MRSA, suggesting that the complexes pos-
compound.34 However, Ru-C6 could be characterized as a bac- sessed significantly less cytotoxicity towards two human cell
teriostatic agent because its MBC (>100 µg mL−1) was more lines.9 The synthesized quinoline derivatives 5,7-dibromo-
than fourfold its MIC (25 µg mL−1).32,33 Theoretically, bacterial 2-methylquinolin-8-ol showed the anti-S. aureus activity (MIC =
cells exposed to Ru-C6 at the MIC or higher could grow again 6.25 µg mL−1) and a mild cytotoxicity towards both human
after the removal of the compound.34 Bacteriostatic agents skin keratinocytes HaCaT and human esophageal NE3 cells
generally inhibit the bacterial growth without killing them, with an average IC50 between 18 and 22 µg mL .
−1 38 Our in vitro
and possess their activity by inhibiting protein biosynthesis cytotoxicity study showed that both bacteriostatic Ru-C6 and
and/or blocking DNA replication of bacteria.35 The better anti- bactericidal Ru-C7 at a dose of 50 µg mL−1 did not induce sig-
bacterial effect of Ru-C7 might be due to the increment in cel- nificant cytotoxicity towards human skin keratinocytes. The
lular uptake and lipophilicity with the increased number of results revealed that both Ru-C6 and Ru-C7 could execute their
carbon atoms present in the linking chain of ruthenium(II) anti-MRSA activities at their MICs (Ru-C6, MIC = 25 µg mL−1
complexes.10,36 Both Ru-C6 and Ru-C7 demonstrated a dose and Ru-C7, MIC = 6.25 µg mL−1) without causing noticeable
dependent antibacterial activity in the inhibition zone assay. cytotoxicity towards human skin cells. Both of them would be
The much clear inhibition zone was observed in both ruthe- potential candidates for the formulation of topically applied
nium compounds whereas the blurred zone was shown in antibiotics.
methicillin. These might be associated with the fact that
methicillin at a dose of 5 µg might be bacteriostatic and the
initially inhibited bacteria could recover from the inhibition
zone after the specific time of period. The time-kill kinetics
Conclusions
demonstrated that Ru-C7 possessed a concentration-dependent In summary, we have synthesized a panel of new bioactive
bacterial killing activity that the extent of killing increased ruthenium(II) complexes. Their potential antibacterial activity
with a higher concentration of the compound.33 MRSA strain towards MRSA was studied. We identified that Ru-C7 showed
was liable to killing by Ru-C7 at 8 MIC during the short term much stronger inhibitory and microbicidal effects towards
exposure of 4 h and it was considered bactericidal at this con- MRSA when compared to those of methicillin. When consider-
centration (more than 99.9% bacterial reduction).32,33 In con- ing the possible structural–activity relationship, some
trast to Ru-C7 at 4 MIC, it was determined to be bacteriostatic researchers have speculated that a straightforward correlation
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between affinity and antimicrobial activity was difficult to
9 F. Li, Y. Mulyana, M. Feterl, J. M. Warner, J. G. Collins and
make from the three ruthenium complexes tested on antibac-
F. R. Keene, Dalton Trans., 2011, 40, 5032.
terial activity. They suggested that the ruthenium compounds 10 F. Li, M. Feterl, Y. Mulyana, J. M. Warner, J. G. Collins and
being taken up by bacteria or binding to other targets such as F. R. Keene, J. Antimicrob. Chemother., 2012, 67, 2686.
proteins or ribosomes may also play certain roles in addition 11 Y. Mulyana, J. G. Collins and F. R. Keene, J. Inclusion
to DNA binding activity.6,10,39 Interestingly, Ru-C7 was demon-
Phenom. Macrocyclic Chem., 2011, 71, 371.
strated to be non-toxic towards the human skin keratinocytes 12 M. Pandrala, F. Li, L. Wallace, P. J. Steel, B. Moore II,
HaCaT with at least 8 MIC to that of MRSA. Our results would
encourage further investigation of these ruthenium(II) com-
plexes as antibiotics for topical application.
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Acknowledgements
We acknowledge the financial support from the National Basic
Research Program of China (973 Project 2013CB834702), a
joint research grant under Innovation Technology Commission
and Research Grants Council from PolyU, the research grants:
38-40-116 (Dr C. H. Chui) and 03-16-176 (Prof. Z. X. Bian), the
Areas of Excellence Scheme from University Grants Committee
of HKSAR (AoE/P-03/08), Hong Kong Research Grants Council
(HKBU203011) and Hong Kong Baptist University (FRG2/11-12/
156). H. Li also thanks the 985 Program and 111 Project from
Minzu University of China (CUN985-07-08 and 111 Project
B08044) for financial support. Prof. R. Gambari is supported
by AIRC.
21 H. Song, J. T. Kaiser and J. K. Barton, Nat. Chem., 2012, 4,
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