A theobromine derived silver N-heterocyclic carbene: Synthesis, characterization, and antimicrobial efficacy studies on cystic fibrosis relevant pathogens

Article abstract of DOI:10.1039/b907726j

The increasing incidence of multidrug-resistant (MDR) pulmonary infections in the cystic fibrosis (CF) population has prompted the investigation of innovative silver based therapeutics. The functionalization of the naturally occurring xanthine theobromine

Full text of DOI:10.1039/b907726j

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N-Heterocyclic Carbenes
Guest Editor Ekkehardt Hahn
Westfälische Wilhelms-Universität, Münster, Germany
Published in issue 35, 2009 of Dalton Transactions
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www.rsc.org/dalton | Dalton Transactions
A theobromine derived silver N-heterocyclic carbene: synthesis,
characterization, and antimicrobial efficacy studies on cystic fibrosis relevant
pathogens†
Matthew J. Panzner,a Khadijah M. Hindi,b Brian D. Wright,a Jane B. Taylor,b Daniel S. Han,b
Wiley J. Youngs*a and Carolyn L. Cannon*b
Received 17th April 2009, Accepted 24th July 2009
First published as an Advance Article on the web 6th August 2009
DOI: 10.1039/b907726j
The increasing incidence of multidrug-resistant (MDR) pulmonary infections in the cystic fibrosis (CF)
population has prompted the investigation of innovative silver based therapeutics. The
functionalization of the naturally occurring xanthine theobromine at the N1 nitrogen atom with an
ethanol substituent followed by the methylation of the N9 nitrogen atom gives the N-heterocyclic
carbene precursor 1-(2-hydroxyethyl)-3,7,9-trimethylxanthinium iodide. The reaction of this
xanthinium salt with silver acetate produces the highly hydrophilic silver carbene complex SCC8. The
in vitro antimicrobial efficacy of this newly synthesized complex was evaluated with excellent results on
a variety of virulent and MDR pathogens isolated from CF patients. A comparative in vivo study
between the known caffeine derived silver carbene SCC1 and SCC8 demonstrated the ability of both
complexes to improve the survival rates of mice in a pneumonia model utilizing the clinically isolated
infectious strain of Pseudomonas aeruginosa PA M57-15.
expulsion eventually leading to airway obstruction which creates
an excellent environment for bacterial growth.
Pseudomonas aeruginosa, Staphylococcus aureus, and
Burkholderia cepacia complex organisms are amongst the most
Introduction
The investigation of transition metal complexes of N-heterocyclic
carbenes (NHCs) for applications other than catalysis has been
gaining attention in recent years.1,2 Of particular interest is
prevalent pathogens found in the lungs of CF patients and are
the use of silver carbene complexes (SCCs) as a new class of
responsible for the majority of morbidity and mortality associated
with the disease.19 Current treatments for these infections include
the use of large continuous doses of b-lactam antibiotics and
aminoglycosides, in particular nebulized tobramycin. Of major
concern is the development of antibiotic resistance to standard
treatments which is becoming increasingly more prevalent due to
the ability of these pathogens to form biofilms.19 We have recently
published several articles demonstrating the exceptional in vitro
and in vivo antimicrobial efficacy of SCCs against a variety of
relevant CF pathogens.4-15 In continuing our pursuit to address
the need for more effective antimicrobials to treat virulent CF
infections, herein we report the synthesis and characterization of
a highly water soluble theobromine derived SCC (SCC8) along
with an in vitro antimicrobial efficacy studies and in vivo efficacy
study comparing SCC8 to a previously reported caffeine derived
SCC (SCC1).
broad spectrum antimicrobials for the treatment of pulmonary
infections.3-14 Much of the research in this area has focused on
the development and testing of SCCs on virulent and multidrug-
resistant (MDR) pathogens associated with cystic fibrosis (CF),
although the in vitro efficacy of SCCs against biosafety level
3 pathogens has also recently been reported.15
CF is a genetic disorder affecting more than 60 000 people
worldwide. The disease arises from a mutation in the genetic
encoding of the cystic fibrosis transmembrane conductance regu-
lator (CFTR), ultimately affecting the lungs and digestive system
of those afflicted.16,17 The CFTR gene is responsible for the
production of the protein that regulates the transport of Cl- and
Na+ across cell membranes. Mutations of the CFTR gene in CF
patients causes problems with the production of this regulatory
protein. The result is a disruption of the normal transfer of Cl-
and Na+ across cell membranes leading to the dehydration and
increased viscosity of cellular secretions.18 In the lungs of CF
patients this increase in mucus viscosity interferes with its natural
Results and discussion
Synthesis of theobromine derived SCC8
aDepartment of Chemistry, The University of Akron, Akron, OH 44325-
3601, USA. E-mail: youngs@uakron.edu; Fax: 330-972-6085; Tel: 330-
972-5362
Based upon previous results obtained from the synthesis of
an electronically stabilized silver NHC complex of methylated
caffeine (SCC1, Fig. 1), it was determined that in general
xanthine derivatives could serve as useful building blocks for
SCC antimicrobials.5 Like caffeine, theobromine is a naturally
occurring molecule which is produced by the cacao tree and is
the chief alkaloid found in chocolate. The synthetic advantage of
bDepartment of Pediatrics and Molecular Microbiology and Microbial
Pathogenesis, Washington University School of Medicine, St. Louis, MO
63110-1002, USA. E-mail: cannon_c@kids.wustl.edu; Fax: 314-286-2895;
Tel: 314-286-2954
† CCDC reference numbers 737693 and 737694 for 1-(2-hydroxyethyl)-
3,7,9-trimethylxanthinium iodide and SCC8. For crystallographic data in
CIF or other electronic format see DOI: 10.1039/b907726j
7308 | Dalton Trans., 2009, 7308–7313
This journal is
The Royal Society of Chemistry 2009
©

loss of the xanthinium hydrogen resonance at 9.33 ppm. The 13
C
NMR further confirmed the structure of SCC8 with an observed
shift of the C8 carbon atom from 139.6 to 186.5 ppm.
Crystals of SCC8 suitable for single crystal X-ray diffraction
studies were grown by slow evaporation from a concentrated
sample in methanol, Fig. 3. The asymmetric unit contains one full
molecule of SCC8. The silver atom is bonded to the carbene car-
Fig. 1 Molecular structure of SCC1.
using theobromine as a NHC building block is the availability of
the N1 position for substitution (Fig. 2). The N1 nitrogen atom
of theobromine is a much better nucleophile when deprotonated
than the N9 nitrogen atom, a feature that aids in the isolation
of singular substitution products. The addition of an alcohol to
the N1 position of theobromine was selected in anticipation that
such a functional group would produce an SCC with greater water
solubility than SCC1 which is a modest 11 mg/mL.
˚
bon atom (C1-Ag1 = 2.072(4) A) and one of the oxygen atoms of
˚
the acetate counter ion (O4-Ag1 = 2.118(3) A). Bond distances and
angles are consistent with those of previously reported for other
xanthine derived carbene silver acetate complexes (Ccarbene-Ag =
5,15
˚
2.067(3)–2.096(4) A). The solid state structure of SCC8 also
shows a short intermolecular Ag-Ag interaction of approximately
˚
3.2 A, which is shorter than the sum of the two van der Waals
˚
˚
radii (3.44 A) of the silver atoms but longer than the 2.89 A bond
distance observed in metallic silver.20,21 NHC silver complexes with
argentophilic interactions of similar distances and shorter have
been previously reported in the literature.3,11,15,22,23
Fig. 2 Numbering scheme for theobromine.
The synthesis of SCC8 was carried out in three steps utilizing
theobromine as the main chemical building block (Scheme 1).
The N1 nitrogen atom of theobromine was first deprotonated
with potassium carbonate followed by condensation with 2-iodo-
ethanol to give 1-(2-hydroxyethyl)-3,7-dimethylxanthine 1. Ini-
tially it was noticed that the use of stronger bases such as potassium
hydroxide produced multiple products in this step. Subsequent
methylation afforded the carbene precursor 1-(2-hydroxyethyl)-
3,7,9-trimethylxanthinium iodide 2 which was reacted with two
equivalents of silver acetate to give SCC8 in 74% yield. The
addition of the ethanol functionality off the N1 nitrogen atom
served to enhance the solubility of SCC8 ten fold (123 mg/mL)
over that of SCC1.
Fig. 3 Thermal ellipsoid plot of silver complex SCC8 with thermal
ellipsoids shown at 50% probability.
MIC and MBC of SCC8
The minimum inhibitory concentration (MIC) of SCC8
against strains of P. aeruginosa, Alcaligenes xylosoxidans,
Stenotrophomonas maltophilia, S. aureus, and Burkholderia species
was determined by broth microdilution (Table 1).
A 10 mg/mL stock solution of SCC8 in sterile water was
prepared and diluted 1:1 into M-H containing 105 colony form-
ing units (CFU) of each bacterial strain to yield the appropriate
test concentration (1, 2, 4, 6, 8, or 10 mg of SCC8/mL). The
E. coli strain J53 is known to be sensitive to killing by silver
cations and serves as a positive control. In contrast, the J53 +
pGM101 is a J53 derivative that harbors the pGM101 plasmid
originally conferring silver-resistance to a burn ward isolate of
Salmonella and serves as a negative control.24 The MIC90 of SCC8
was 1 mg/mL for non-silver resistant organisms tested (Table 1).
The MIC for J53 lacking the silver resistant plasmid was 2 mg/mL,
whereas the MIC of SCC8 for J53 containing pMG101 was greater
than 10 mg/mL demonstrating that the antimicrobial activity of
SCC8 is primarily due to the silver moiety. Determination of the
minimum bactericidal concentration (MBC) of SCC8 against the
tested strains was performed. With the exception of the silver
resistant E. coli strain, SCC8 appeared to be bactericidal for all of
the strains tested, which indicates that SCC8 is capable of killing
numerous bacterial strains at clinically achievable concentrations.
Scheme 1 Synthesis of silver carbene SCC8.
In vivo efficacy of SCC8 and SCC1
1H and 13C NMR were used to confirm the formation of the
silver complex. The most notable change in the H NMR is the
To compare the in vivo efficacy of SCC1 and SCC8 in a mouse
pneumonia model, four 50 mg doses of aerosolize SCC1 and
1
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The Royal Society of Chemistry 2009
Dalton Trans., 2009, 7308–7313 | 7309
©

Table 1 MIC and MBC values for SCC8
Species
strain
MIC
(mg/mL)
MBC
(mg/mL)
Pseudomonas aeruginosa
PA O1-Va
1
1
1
1
2
6
1
2
PA M57-15b
PA HP3c
PA SB05c
Alcaligenes xylosoxidans
AX RE05c
1
1
2
1
AX OT05c
Stenotrophomonas maltophilia
SM BE06c
1
1
2
8
SM AH06c
Staphylococcus aureus
SA EH06d
1
1
6
6
SA LL06d
Burkholderia multivorans
BM DP06c
1
2
2
4
BM CS06c
Burkholderia cenocpeacia
J2315e
1
1
1
1
8
6
1
2
Burkholderia dolosa
AU4459e
Burkholderia thailandensis
E264f
Burkholderia gladioli
BG CM06c
Escherichia coli
J53g
2
>10
2
>10
J53+pMG101h
a Common laboratory strain. b Mucoid strain from a CF patient. c MDR
strain from a CF patient. d MRSA strain from a CF patient. e Epidemic
clinical strain. f Wild type strain. g Silver sensitive strain. h Strain harboring
silver resistance plasmid.
SCC8 were delivered 12 hours apart in a nose-only fashion to
P. aeruginosa-infected mice in a multi-dosing chamber (Fig. 4A).
Treatment with SCC1 resulted in 100% survival for an 83%
survival advantage compared with water-treated animals (Fig. 4B:
6/6, 100% SCC1 survival versus 1/6, 17% water; p = 0.0043)
and treatment with SCC8 resulted in 83% survival for a 66%
survival advantage (Fig. 4B: 5/6, 83% SCC8 survival versus
1/6, 17% water; p = 0.0043). The small difference in survival
between SCC1 and SCC8 may be explained by the slightly
decreased molar dose of Ag(I) delivered to the SCC8 group based
on the differences in molecular weights of the two compounds
(MW SCC1: 375.13, SCC8: 405.16. Thus, 14.4 mg Ag(I) delivered
as SCC1 and 13.3 mg Ag(I) as SCC8 an 8% difference). The
differences in survival between SCC1 and SCC8 were not evident
in the clinical appearance of the animals, as both treatments
resulted in significant and identical improvements in clinical scores
by 24 hours after inoculation compared to the water-treated group
(SCC1 versus water and SCC8 versus water both p < 0.001 by
ANOVA). The weight loss was identical at 24 hours in all of the
treatment groups, but by 48 hours, the slope of the weight loss
curve in the SCC1 and SCC8-treated animals had decreased and
the mean weight loss in those groups differed significantly from
that of the single surviving water-treated mouse (SCC1 versus
water, p = 0.0395 and SCC8 versus water, p = 0.0212 by 1 sample
t-test). By 72 hours, the SCC1 and 5 surviving SCC8-treated mice
were gaining weight (SCC1 versus water, p = 0.0037, SCC8 versus
water, p = 0.0356 by 1 sample t-test).
Fig. 4 Effect of nebulized SCC1 and SCC8 on survival, clinical scores,
and weight loss in a mouse model of Pseudomonas pneumonia. A)
Treatment protocol. Mice received treatment with nebulized SCC1, SCC8
or water one hour after infection with P. aeruginosa PA M57-15, and
again every 12 h for a total of 4 doses. Mice were evaluated on day 3
(72 h). B) Kaplan-Meier survival curves. The survival of mice treated with
SCC1, SCC8 and water were 100%, 83% and 17%, respectively (SCC1
vs. water p = 0.0043, SCC8 vs. water p = 0.0096). C) Clinical scores.
Clinical scores for each of the three treatment groups are displayed as
mean SD. Numbers in parentheses indicate the number of surviving
animals available for analysis, if less than the initial group size of 6. At the
24 hour time point, SCC1 vs. water and SCC8 vs. water both p < 0.001
by ANOVA. D) Weight loss. Displayed are the mean % weight changes
from inoculation weight SD for SCC1, SCC8 and water-treated mice.
The SCC1 and SCC8 treatment groups were compared at 48 and 72 hours
to the single living water-treated animal by 1 sample t-test (48 h: SCC1
vs. water, p = 0.0395, SCC8 vs. water, p = 0.0212; 72 h: SCC1 vs. water,
p = 0.0037, SCC8 vs. water, p = 0.0356).
The gross pathology of the lungs mirrored the outcome of the
SCC1, SCC8 and water-treated animals. The lungs of the SCC1
and surviving SCC8-treated animals were pink and collapsed upon
opening the chest cavity, whereas the lungs of 4 water-treated
animals that were opened at the time of death (3 of those that
7310 | Dalton Trans., 2009, 7308–7313
This journal is
The Royal Society of Chemistry 2009
©

died before 48 hours that were observed at the precise moment
of death and the sole survivor) were inflamed and stiff. Spleens
were harvested from the 4 water-treated animals and the SCC1-
and all SCC8-treated animals to assay for bacteria as a marker
of dissemination from the lung. All of the animals that received
only nebulized water had bacteria in the spleen (Fig. 5). The only
animal that had bacteria in the spleen among the SCC1- and
SCC8-treated animals was the single SCC8 animal that died. Thus,
the SCC1 and SCC8 animals had a significantly less dissemination
of bacteria from the lungs to the spleen (SCC1 versus water, p =
0.0067; SCC8 versus water, p = 0.0357 by Fisher’s exact test of
the contingency table). Thus, as we have seen in previous studies,
survival appears related to bacteremia, as all of the animals that
died had bacteria recovered from their spleens.13,14 These findings
indicate that treatment with SCC1 and SCC8 can effectively
decrease the likelihood of bacteremia and death in a P. aeruginosa
pneumonia model.
evaporator and the crude material dissolved methanol and◦cooled
1
to give 1 as colorless crystals (7.6 g, 68%). Mp: 201–203 C. H
NMR (500 MHz, DMSO-d6) d 3.39 (s, 3H, CH3), 3.50 (q, 2H,
CH2), 3.87 (s, 3H, CH3), 3.94 (t, 2H, CH2), 4.70 (t, 1H, OH),
1
7.98 (s, 1H, CH). 13C{ H} NMR (125 MHz, DMSO-d6) d 29.3
(CH3), 33.1 (CH3), 42.4 (CH2), 57.7 (CH2), 106.6 (C), 142.8 (CH),
=
=
148.1 (C), 150.9 (C O), 154.4 (C O). Found: C, 48.00; H, 5.33;
N, 24.25. Calc. for C9H12N4O3: C, 48.19; H, 5.40; N, 25.00%.
TOF-MS-EA+: m/z 247 (M+ of C9H12N4O3Na).
1-(2-Hydroxyethyl)-3,7,9-trimethylxanthinium
iodide
(2).
Compound 1 (2.0 g, 8.9 mmol) was added to iodomethane
(30 mL, 92.7 mmol) in a pressure tube and DMF was added
dropwise until the solution appeared clear. The tube was sealed
and heated at 80 ◦C for 2 d with stirring. Volatiles were removed
by rotary evaporator and the resulting solid was washed with
acetone to give 2 as a pale yellow solid (2.4 g, 66%). Mp:
175-176 ◦C. 1H NMR (500 MHz, DMSO-d6) d 3.54 (t, 2H, CH2),
3.73 (s, 3H, CH3), 3.98 (t, 2H, CH2), 4.05 (s, 3H, CH3), 4.15 (s,
1
3H, CH3), 4.47 (bs, 1H, OH), 9.33 (s, 1H, CH). 13C{ H} NMR
(125 MHz, DMSO-d6) d 31.3 (CH3), 35.6 (CH3), 36.9 (CH3),
43.4 (CH2), 57.2 (CH2), 107.7 (C), 139.3 (C), 139.6 (CH) 150.0
=
=
(C O), 153.2 (C O). Found: C, 32.47; H, 4.00; N, 14.53. Calc.
for C10H15N4O3I: C, 32.79; H, 4.13; N, 15.30%. TOF-MS-EA+:
m/z 239 (M+ of C10H15N4O3).
X-Ray crystal structure analysis: formula C10H15IN4O3, Mw
=
˚
366.16, colorless crystal 0.37 ¥ 0.29 ¥ 0.19 mm, a = 15.0967(18) A,
◦
◦
˚
˚
b = 10.4071(13) A, c = 8.708(10) A, a = 90 , b = 104.119(2) ,
◦
g = 90 , V = 1326.8(3) A , Dcalc = 1.833 Mg cm-3, m =
3
˚
Fig. 5 Effects of nebulized SCC1 and SCC8 on the likelihood of bacterial
dissemination to the spleen in a mouse model of Pseudomonas pneumonia.
Bacteremia in all of the mice plotted as number of spleens in each treatment
group (SCC1 vs. water, p = 0.0067; SCC8 vs. water, p = 0.0357).
2.421 mm-1, Z = 4, monoclinic, space group P21/c (No. 14),
˚
l = 0.71073 A, T = 100 K, w and j scans, 10406 reflections
collected, 2684 independent (Rint) 0.0498, 167 refined parameters,
R1/wR2 (I ≥ 2s(I)) = 0.0410/0.1014 and R1/wR2 (all data) =
0.0489/0.1060, maximum (minimum) residual electron density
-3
˚
1.963 (-1.374) e A .
Experimental
1-(2-Hydroxyethyl)-3,7,9-trimethylxanthin-8-ylidene silver ac-
etate (SCC8). Xanthinium salt 2 (1.8 g, 4.9 mmol) was dissolved
in methanol (200 mL) and silver acetate (1.6 g, 9.8 mmol) was
added with stirring. The reaction was stirred 1.5 h during which
time a yellow precipitate of AgI was formed. The reaction mixture
was filtered and the volatiles removed by rotary evaporation. The
resulting solid was washed with acetone a◦nd dried to afford SCC8
as a white solid (1.5 g, 74%). Mp: 212-214 C. 1H NMR (500 MHz,
DMSO-d6) d 1.74 (s, 3H, CH3 of OAc), 3.54 (t, 2H, CH2), 3.75
(s, 3H, CH3), 3.98 (t, 2H, CH2), 4.07 (s, 3H, CH3), 4.20 (s, 3H,
General considerations
2-Iodoethanol, iodomethane, theobromine, and silver acetate
were purchased from Alfa Aesar and used as received. All
solvents were purchased from Fisher Scientific and used without
further purification. All reactions were carried out under aerobic
1
conditions. H and 13C NMR data were obtained on a Varian
500 MHz instrument. The spectra were referenced to deuterated
solvents. Elemental analyses were preformed by the University
of Illinois microanalysis laboratory. Mass spectrometry analyses
were preformed by the Ohio State University mass spectrometry
and proteomics facility. Crystal structure data sets were collected
on a Bruker SMART Apex CCD diffractometer with graphite-
1
CH3). 13C{ H} NMR (125 MHz, DMSO-d6) d 23.4 (CH3 of
OAc), 31.5 (CH3), 37.8 (CH3), 39.1 (CH3), 43.2 (CH2), 57.4 (CH2),
=
=
=
108.9 (C), 140.5 (C), 150.4 (C O), 153.2 (C O), 173.6 (C O of
OAc), 186.5 (C-Ag). Found: C, 35.48; H, 4.14; N, 13.40. Calc. for
C12H17N4O5Ag: C, 35.64; H, 4.24; N, 13.86%. TOF-MS-EA+: m/z
345/347 (M+ of C10H14N4O3Ag).
˚
monochromated Mo Ka radiation (l = 0.71073 A). Unit cell
determination was achieved by using reflections from three
different orientations.
X-Ray crystal structure analysis: formula C12H17AgN4O5, Mw =
˚
1-Hydroxyethyl-3,7-dimethylxanthine
(1). Theobromine
405.17, colorless crystal 0.43 ¥ 0.08 ¥ 0.07 mm, a = 23.896(3) A,
◦
◦
˚
˚
(9.0 g, 50 mmol) and potassium carbonate (7.6 g, 55 mmol)
were dissolved in DMF (150 mL). The solution was brought to
reflux at which time 2-iodoethanol (3.9 mL, 50 mmol) was added.
The solution was stirred for 1 h and an additional equivalent of
2-iodoethanol (3.9 mL, 50 mmol) was added. The reaction was
then stirred at reflux for 17 h. Volatiles were removed by rotary
b = 8.2797(11) A, c = 14.8598(19) A, a = 90 , b = 94.104(2) , g =
◦
90 , V = 2932.5(6) A , Dcalc = 1.835 Mg cm-3, m = 1.405 mm-1,
3
˚
˚
Z = 8, monoclinic, space group C2/c (No. 15), l = 0.71073 A,
T = 100 K, w and j scans, 11365 reflections collected, 2985
independent (Rint) 0.0476, 204 refined parameters, R1/wR2 (I ≥
2s(I)) = 0.0395/0.0931 and R1/wR2 (all data) = 0.0535/0.1001,
This journal is
The Royal Society of Chemistry 2009
Dalton Trans., 2009, 7308–7313 | 7311
©

maximum (minimum) residual electron density 1.160
Delivery
-3
˚
(-0.658) e A .
SCC1, SCC8, and water were delivered via an Aeroneb Lab
apparatus (Aerogen Inc., Galway, Ireland) connected to a multi-
dosing animal chamber. The Aerogen nebulizer is based on
micropump technology that produces 4 to 6 mm particles in a low
velocity aerosol. The multi-dosing chamber is a square plexiglass
box with inner dimensions of 8 ¥ 8 ¥ 4.5 inches height. The
nebulizer is mounted in the center of the lid.
Bacteria
The laboratory strain PAO1-V was provided by Dr Maynard
Olson (University of Washington, Seattle). The mucoid clinical
isolate of P. aeruginosa PA M57-15 was provided by Dr Thomas
Ferkol (Washington University, St. Louis, MO). The PA HP3, PA
SB05, AX RE05, AX OT05, SM BE06, SM AH06, SA EH06,
SA LL06, BM DP06, BM CS06, and BG CM06 strains were
cultured from the sputum of cystic fibrosis patients at St. Louis
Children’s Hospital. Burkholderia cepacia complex organisms
J2315, and AU4459 were provided by Dr John Lipuma (University
of Michigan, Ann Arbor, MI). Burkholderia thailandensis strain
E264 was provided by Dr Josie Gray (University of Washington,
Seattle). The silver sensitive and silver resistant E. coli strains J53
and J53+pMG101, were provided by Dr Simon Silver (University
of Chicago, Chicago, IL).24 All bacterial strains were maintained
as glycerol stocks at -80 ◦C.
Pulmonary infection model
Mice (16–23 g) were anesthetized using a cocktail composed
of a 1:1 ratio of ketamine HCL (100 mg/mL, Fort Dodge
Animal Health, Fort Dodge, IA) and xylazine sterile solution
(20 mg/mL, Bedford, OH). Each mouse received an intramuscular
injection of approximately 1 mL/gm body weight of the cocktail
mix. A 100 mL aliquot of PA M57-15 in LB (1.9 ¥ 106 CFU)
was delivered to each mouse intranasally. Beginning 1 hour
after inoculation, the treatment groups of 6 mice each received
5 mL of either SCC1 (50 mg total dose), SCC8 (50 mg total
dose), or water via 15-minute nebulization exposure periods every
12 hours for a total of four doses. To ensure nose-only delivery
of the drugs, mice were individually placed in CH-247 tubes (CH
Technologies, Westwood, NJ) prior to placement into a multi-
dosing chamber. Mice were weighed and scored based on their
clinical appearance daily. Three days after infection (72 hour
after the initial inoculation), mice were euthanized with carbon
dioxide and their lungs and spleens were visualized for signs of
inflammation. Spleens were harvested, homogenized in 1 mL of
LB (PowerGen 125 homogenizer, Fischer Scientific) and 10 mL
aliquots of the homogenate were plated in duplicate on TSA plates
and grown overnight at 37 ◦C. Presence or absence of bacterial
colonies was noted to determine bacteremia.
Drugs
Stocks of SCC1 (Ksp of 11 mg/mL) and SCC8 (Ksp of
125 mg/mL) were reconstituted in sterile water (Milli-Q Synthesis
System, Millipore Corp., Billerica, MA) to a concentration of
10 mg/mL and aliquots of 1 mL were stored at -80 ◦C until use.
In vitro antimicrobial activity
Minimal inhibitory concentrations (MICs) were determined
by broth microdilution method as previously described by a
standard Clinical and Laboratory Standards Institute (CLSI)
protocol.13 Briefly, bacteria were streaked from frozen glycerol
stocks onto TSA or blood agar plates and incubated overnight
at 37 ◦C. Colonies from the fresh plates were suspended in the
CLSI standard M-H broth to an optical density at 650 nm
(OD650) of 0.2 and grown at 37 ◦C in a shaking incubator at
200 rpm to an OD650 of 0.4, which corresponds to ~5 ¥ 108 colony
forming units (CFU)/mL. The bacteria were diluted in the broth
to a concentration of 105 in 100 mL, which was added to triplicate
wells of a 96-well plate, containing 100 mL of SCC8 diluted in
sterile water to various concentrations from 10 mg/mL stock. The
final concentrations tested were 1, 2, 4, 6, 8, and 10 mg/mL. The
plates were incubated overnight at 37 ◦C. The MIC was the lowest
of these concentrations, at which each of the triplicate wells in
each 96-well plate was clear. Each triplicate measurement was
performed at least in duplicate for a minimum of 6 separate
measurements. The MBC of SCC8 was determined by plating the
wells with growth inhibition (clear) on TSA or blood agar plates
and noting the lowest concentration that resulted in no growth
after an overnight incubation at 37 ◦C.
Clinical score
Mice were examined for features of activity, fur and posture. The
activity scores were as follows: 0 for normal activity, 1 for slow,
but spontaneous walking, 2 for movement only with stimulus and
3 for lack of movement with stimulus. The fur was noted to be
either normal (score of 0) or ruffled (score of 1). The mice were
noted to have normal posture (score of 0) or to be hunched (score
of 1). Thus, clinical scores ranged from 0 for a well animal to 5 for
a moribund animal.
Statistical analysis
All analyses were performed using Prism 4. The in vivo survival
curves in the infection model were compared using a log-rank
test. Changes in animal weights at 24 hours after inoculation were
compared by ANOVA. The average clinical scores for the three
groups at 48 and 72 hours after inoculation were compared by 1
sample t-test using the single living water-treated mouse as the test
mean. Data are given as mean standard deviation. The risk of
bacteremia between the three treatment groups was compared by
Fisher’s exact test of a contingency table (number of animals with
bacteria in the spleen versus number of animals cleared).
Mice
Male C57BL/6J mice (Jackson Laboratories, Bar Harbor, Maine)
at 5 weeks of age were used for these studies that were approved by
the Washington University School of Medicine Animal Studies
Committee. Animals were housed in a barrier facility under
pathogen-free conditions until they were inoculated with bacteria.
7312 | Dalton Trans., 2009, 7308–7313
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The Royal Society of Chemistry 2009
©

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Conclusion
The studies presented demonstrate the potential to take advantage
of naturally occurring chemical moieties for the synthesis of new
silver carbene complexes. In particular, theobromine serves as
an excellent chemical building block for such products, in part
due to its ease of functionalization at the N1 nitrogen atom upon
deprotonation. The addition of alcohols such as a hydroxyethyl
group at this position appears to greatly enhance the water
solubility of the resulting silver carbene complex. In fact SCC8
demonstrated a ten fold enhancement in water solubility over
an analogous caffeine derivative. Like previously reported SCCs,
SCC8 showed single digit mg/mL concentration MICs in vitro on
all relevant CF pathogens tested including MDR strains. In vivo
efficacy studies conducted have demonstrated that nebulized silver
carbene complexes such as SCC1 and SCC8 have great potential
in aiding in the future treatment of virulent and MDR pulmonary
infections associated with CF.
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Acknowledgements
This work was supported by the Washington University School
of Medicine, The University of Akron, The University of Akron
Center for Silver Therapeutics Research, The Goodyear Tire &
Rubber Company, the National Institute of Allergies and Infec-
tious Diseases (1 R01 A106785601) and The National Institute of
General Medical Sciences (1 R01 GM86895-01). We wish to thank
the Ohio Board of Regents and the National Science Foundation
for funds used to purchase the 500 MHz NMR instrument (CHE-
0341701 and DMR-0414599) and the Bruker-Nonius Apex CCD
X-ray diffractometer (CHE-0116041) used in this research.
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Dalton Trans., 2009, 7308–7313 | 7313
©