Synthesis, stability, and antimicrobial studies of electronically tuned silver acetate N-heterocyclic carbenes

Article abstract of DOI:10.1021/jm0708679

A series of methylated imidazolium salts with varying substituents on the 4 and 5 positions of the imidazole ring were synthesized. These salts were reacted with silver acetate to afford their corresponding silver N-heterocyclic carbene (NHC) complexes. These complexes were then evaluated for their stability in water as well as for their antimicrobial efficacy against a variety of bacterial strains associated with cystic fibrosis and chronic lung infections.

Full text of DOI:10.1021/jm0708679

J. Med. Chem. 2008, 51, 1577–1583
1577
Synthesis, Stability, and Antimicrobial Studies of Electronically Tuned Silver Acetate
N-Heterocyclic Carbenes
Khadijah M. Hindi,† Tammy J. Siciliano,† Semih Durmus,† Matthew J. Panzner,† Doug A. Medvetz,† D. Venkat Reddy,†
Lisa A. Hogue,‡ Christine E. Hovis,‡ Julia K. Hilliard,‡ Rebekah J. Mallet,‡ Claire A. Tessier,† Carolyn L. Cannon,*,‡ and
Wiley J. Youngs*,†
Department of Chemistry, UniVersity of Akron, Akron, Ohio 44325-3601, and DiVision of Allergy and Pulmonary Medicine, Department of
Pediatrics, St. Louis Children’s Hospital, Washington UniVersity School of Medicine, St. Louis, Missouri 63110-1002
ReceiVed July 18, 2007
A series of methylated imidazolium salts with varying substituents on the 4 and 5 positions of the imidazole
ring were synthesized. These salts were reacted with silver acetate to afford their corresponding silver
N-heterocyclic carbene (NHC) complexes. These complexes were then evaluated for their stability in water
as well as for their antimicrobial efficacy against a variety of bacterial strains associated with cystic fibrosis
and chronic lung infections.
Scheme 1. Synthesis of 2
Introduction
Our group has a long standing interest in the synthesis of
silver N-heterocyclic carbene (NHC) complexes for their
antimicrobial activity, in particular for the treatment of cystic
fibrosis (CF) and chronic lung infections. Silver has been used
for centuries in a variety of forms for its antimicrobial properties.
Medicinally, silver was introduced in the 1880s and used as a
prophylactic 2% silver nitrate eye solution to prevent ophthalmia
neonatorum in newborns. Silver nitrate was also used to treat a
variety of other problems such as skin ulcers, postsurgical
wounds, and suppurating wounds.1 In the late 1960s, silver
sulfadiazine (SSD) was introduced, and it remains one of the
most effective topical burn treatments.2 The activity of silver
against Gram-positive and Gram-negative bacteria has long been
established. In an inert atmosphere, silver displays no effect
against bacterial organisms. In the presence of oxygen, however,
Ag+ ions are produced. It has been noted that silver cations are
responsible for the bactericidal activity.2–5 However, the mech-
anisms of its antimicrobial action are not completely understood.
Fortunately, despite its wide use in both clinical and nonclinical
applications, only a few accounts of silver resistance have been
reported.6–10As an added advantage, silver has no effect upon the
mammalian cell membrane and has limited toxicity to humans.
Based on the benefits of silver and the pressing interest in
finding silver based antimicrobials active against resistant
organisms, we have synthesized and investigated a variety of
NHC silver complexes. Typically these complexes rapidly
decompose in aqueous solutions.5,11,12 Recently, a newly
synthesized silver complex of caffeine showed promising
antimicrobial properties as well as stability in water for several
days (Scheme 1).13
Scheme 2. Synthesis of 4
In this paper, we report the preparation and characterization
of several imidazolium salts with different substituents at the 4
and 5 positions of the imidazole ring and their silver complexes.
The stability of the complexes in water is compared. Further-
more, the antimicrobial activity of two of the silver complexes
against Pseudomonas aeruginosa and Burkholderia cepacia
complex species as well as control Escherichia coli strains is
reported.
Results and Discussion
1-Methyl-4,5-dichloroimidazole (3) is formed from the depro-
tonation of 4,5-dichloroimidazole with potassium hydroxide and
subsequent methylation with iodomethane in acetonitrile. The
imidazolium salt 1,3-dimethyl-4,5-dichloroimidazolium iodide
(4) is formed by the reaction of 1-methyl-4,5-dichloroimidazole
(3) with excess iodomethane (Scheme 2).
1
The H and 13C NMR spectra of 4 are consistent with its
molecular structure. In the 1H NMR spectrum of 4, the
imidazolium proton appears at 9.42 ppm. This shift is consistent
with the general C2-H acidic proton shift of imidazolium salts
(δ 8–10 ppm).14 The 13C NMR shift of the N-C-N sp2 carbon,
which later becomes the carbene center, appears at 136.6 ppm
for 4.
These results led us to hypothesize that the presence of
electron withdrawing groups on the 4 and 5 positions of the
imidazole ring may lead to more stable silver systems. Our goal
is to synthesize a silver carbene system that is stable enough to
withstand systemic delivery prior to its degradation by salts or
biological molecules present in the body.
Single crystals of 4 suitable for X-ray diffraction analysis
were obtained by slow evaporation from a concentrated solution
of acetonitrile. The molecular structure of the cationic part of 4
is depicted in Figure 1.
The in situ deprotonation of 4 with silver acetate in a 1:2
molar ratio in dichloromethane afforded the corresponding NHC
* To whom correspondence should be addressed. (W.J.Y.) Phone: 330-
972-5362. Fax: 330-972-7370. E-mail: youngs@uakron.edu. (C.L.C.) Phone:
314-286-2954. Fax: 314-286-2895. E-mail: cannon_c@kids.wustl.edu.
†
University of Akron.
Washington University School of Medicine.
‡
10.1021/jm0708679 CCC: $40.75
2008 American Chemical Society
Published on Web 02/21/2008

1578 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Hindi et al.
Scheme 3. Synthesis of 7
1,3-Dimethylimidazolium iodide (6) was easily formed by
the reaction of methylimidazole and excess iodomethane in
diethylether by using a modified literature procedure.15 The
reaction of the imidazolium salt (6) with 2 molar equiv of silver
acetate in refluxing methylene chloride for 40 h afforded silver
complex 7 in 64% yield (Scheme 3).
The 1H and 13C NMR of 6 are consistent with the structure,
1
with the imidazolium proton observed at 9.06 ppm in the H
NMR, and with the imidazolium carbon observed at 137.0 ppm
in the 13C NMR. The carbene carbon of 7 is observed at 178.8
ppm in the 13C NMR spectrum, which is similar to the cases of
2 and 5. The solid state structure of 7 was determined by single
crystal X-ray diffraction (Figure 3). Crystals were obtained from
slow evaporation of a saturated methylene chloride solution.
The C6-Ag2 bond length is 2.064(6) Å, and the geometry at
the Ag(I) atom is almost linear with a C6-Ag2-O3 bond angle
of 175.97(18)°.
Figure 1. Crystal structure of the cationic portion of 4 with thermal
ellipsoids shown at 50% probability.
Treatment of urocanic acid with sodium bicarbonate and
excess iodomethane in acetonitrile results in the formation of
the iodide salt of dimethylated methyl urocanate (8), eq 2. In
1
the H NMR spectrum of 8, the imidazolium proton (C2-H)
appears at 9.16 ppm, which is consistent with the molecular
structure of 8. The imidazolium carbon (C2) appears at 138.7
ppm in the 13C NMR spectrum. Single crystals of 8 suitable
for X-ray diffraction studies were grown from a concentrated
acetonitrile solution (Figure 4).
Figure 2. Crystal structure of [(4)AgOAc] (5). Thermal ellipsoids are
shown at 50% probability.
silver acetate complex 5 in good yield (79%), eq 1. The most
significant feature of the 13C NMR spectrum of 5 is the signal
at 179.7 ppm corresponding to the carbene carbon atom, which
appears in the typical range of other NHC complexes of Ag(I),
and the loss of the resonance at 136.6 ppm. The 1H NMR
spectrum shows a disappearance of the imidazolium proton
signal at ca. 9 ppm, which further demonstrates the formation
of the expected NHC silver acetate complex. The intensities of
the signals in the 13C NMR of the carbon atoms of the 4 and 5
positions of the imidazole ring are low due to the increase in
the T1 relaxation time upon replacement of hydrogen with
chlorine.
(2)
The silver(I) complex 9 was synthesized by the reaction of 8
with 2.4 molar equiv of silver acetate in dichloromethane. The
reaction mixture was stirred for 1 h at room temperature to
afford the NHC silver acetate complex 9 as a white solid in
88% yield, eq 3. In the 1H and 13C spectra of 9, the loss of the
imidazolium proton (C2-H) and the imidazolium carbon (C2)
signals at 9.16 and 138.7 ppm, respectively, suggests the
formation of the silver(I) NHC complex. In the 13C NMR
(1)
Single crystals of 5 were grown from a concentrated sample
in THF (Figure 2). The asymmetric unit contains three molecules
of water and one molecule of 5. The geometry at the Ag atom
deviates from linearity significantly with a C1-Ag-O1 bond
angle of 168.9(2)°. The C1-Ag bond length is 2.030(8) Å.
To evaluate the stability introduced by the presence of
electron withdrawing groups on the 4 and 5 positions of the
imidazole ring, silver complex 5 was compared to two analogous
silver complexes. Silver complexes 7 and 9 have hydrogen
atoms and a methyl ester group on the alkene carbon atoms,
respectively.
Figure 3. Crystal structure of [(6)AgOAc] (7). Thermal ellipsoids are
shown at 50% probability.

Electronically Tuned SilVer Acetate NHCs
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1579
proximately, 16% of silver complex 5 degrades into the
imidazolium cation within the first hour. This degradation is
accompanied by the appearance of the resonance for the methyl
1
groups of 4 at 3.84 ppm in the H NMR and 34.7 ppm in the
13C NMR. After 2 weeks, approximately 22% of the imidazo-
lium cation 4 is formed, and after 17 weeks, 34% of 5 has
degraded to 4. Complex 7 shows rapid decomposition within
the first 2 h when added to D2O, as approximately 95% degrades
into the starting compound 6. The appearance of the resonance
at 7.47 ppm for the C-H protons on the 4 and 5 positions of
the imidazolium cation and the simultaneous disappearance of
the resonance at 7.29 ppm for the C-H protons on the 4 and 5
1
positions of the imidazole ring of complex 7 in the H NMR
are the basis for the above observation. Furthermore, complex
9 also proves to be quite unstable in D2O, as approximately
94% of the imidazolium cation 8 is formed within 1.5 h.
Based on the above experiment, silver complex 5 bearing
two Cl atoms shows improved stability when compared to those
bearing hydrogens or a methyl ester group. We believe that the
chlorine atoms in complex 5 stabilize the silver complex by
acting as σ-withdrawing and π-donating substituents, thereby
withdrawing electron density from the carbene carbon, making
it less susceptible to attack by protons in an aqueous environ-
ment. The hydrogen atoms of complex 7 do not appear to
contribute any stability. These results compliment the previous
findings of Peris in that the chlorine substituents in the 4 and 5
positions of the imidazole ring lead to a reduction of σ-donor
capability compared to the nonchlorinated equivalents.16 The
methyl ester substituent in complex 9 is both σ-withdrawing
and π-withdrawing. This feature does not appear to add any
water stability to the complex. This is most likely due to the
free rotation of the methyl ester in solution, which serves to
break the conjugation with the electronic framework of the
imidazole ring.
Figure 4. Crystal structure of the cationic portion of 8 with thermal
ellipsoids shown at 50% probability.
Figure 5. Crystal structure of [(8)AgOAc] (9). Thermal ellipsoids are
shown at 50% probability.
Table 1. Stability of Silver Complexes in D2O
silver complex
stability in D2O
MIC and MBC of 5. The minimum inhibitory concentration
(MIC) of 5 against a panel of pathogens, primarily respiratory
isolates of P. aeruginosa and the Burkholderia species, was
determined in two different growth media, the Clinical and
Laboratory Standards Institute (CLSI) standard Mueller-Hinton
(M-H) as well as Luria broth (LB) (Table 2). A 10 mg/mL stock
solution of 5 in water was prepared and diluted 1:1 into M-H
or LB containing 105 colony forming units (CFU) of each
bacterial strain to yield the appropriate test concentration (0, 1,
2, 4, 6, 8, or 10 µg of 5/mL). The MIC50 and MIC90 for 5 against
the panel of 36 respiratory pathogens grown in M-H were 1
and 4 µg/mL, respectively. The MIC50 for 5 against the same
pathogens grown in LB was higher at 2 µg/mL, although the
MIC90 was the same at 4 µg/mL. Although little difference was
seen in the MIC50 and MIC90 of 5 against strains grown in M-H
compared with LB, a few strains were very sensitive to growth
inhibition by 5 in M-H but were not sensitive in LB at any of
the concentrations tested. E. coli strains J53, a silver-sensitive
strain, and J53+pMG101, a silver-resistant strain, were used
as positive and negative controls. Determination of the minimum
bactericidal concentration (MBC) of 5 against a subset of strains
was performed and found to depend markedly on the growth
media in which the tested strain was grown, as well as the strain
itself (Table 2). Compound 5 appears to be bactericidal for
numerous strains, particularly if grown in M-H, but only
bacteriostatic for other strains at the concentrations tested. Thus,
compound 5 appears capable of killing numerous bacterial
strains at concentrations achievable clinically.
2
5
7
9
3 days
17 weeks +
2 h
1.5 h
spectrum of 9, the carbene carbon (C2) signal was observed at
182.0 ppm, which is similar to other silver(I) NHC systems.
Single crystals of complex 9 suitable for X-ray diffraction
were grown from a concentrated solution of acetonitrile. The
solid state structure of 9 is shown in Figure 5. The geometry at
the Ag(I) atom deviates from linearity, with a C1-Ag1-O3
bond angle of 167.28(18)° and Ag(1)-C(1) and Ag(1)-O(3)
bond distances of 2.062(5) and 2.129(4) Å, respectively. The
Ag-C distance of 2.062(5) Å is similar to that of the other
reported silver NHC complexes. In the solid state structure, the
conjugated methyl ester group appears to be in the plane of the
silver acetate complex.
(3)
Water Stability Studies. The stability of each silver complex
in water is monitored by following the changes in the 13C NMR
and 1H NMR spectra in D2O (Table 1). A minor degradation is
observed initially when complex 5 is added to D2O. Ap-
Comparison of the Antimicrobial Activities of 5 and 7.
For a sample panel of four respiratory pathogens, two P.

1580 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Table 2. MIC and MBC of 5
Hindi et al.
MIC (µg/mL)
MBC (µg/mL)
species (genomovar)
strain
PAO1-V
PAM57–15
PA 2192
PA 6294
PA N6
M-H
LB
M-H
LB
note
Pseudomonas aeruginosa
2
4
1
2
1
2
1
1
1
1
1
1
4
2
2
4
2
2
1
1
1
1
1
1
1
1
2
1
4
4
4
4
4
2
1
6
2
2
2
2
2
2
1
4
4
4
2
2
2
>10
6
2
>10
4
4
10
6
>10
2
>10
2
8
>10
>10
8
laboratory strain
mucoid CF clinical isolate
mucoid CF clinical isolate
invasive corneal clinical isolate
nonmucoid CF clinical isolate
nonmucoid CF clinical isolate
nonmucoid CF clinical isolate
nonmucoid CF clinical isolate
nonmucoid CF clinical isolate
mucoid CF clinical isolate
standard Bcc panel strain
standard Bcc panel strain
CF clinical isolate
PAJG3
PA N13
PA 1061
PA N8
PA 324
PC783
4
10
>10
>10
8
>10
nda
nda
nda
nda
2
>10
4
>10
>10
10
Burkholderia cepacia (I)
Burkholderia multiVorans (II)
2
1
HI2229
AU8170
AU5735
AU7484
AU5248
J2315
nda
nda
nda
nda
4
CF clinical isolate
CF clinical isolate
CF clinical isolate
epidemic strain
Burkholderia cenocepacia (III)
Burkholderia stabilis (IV)
1
HI2718
>10
2
1
6
2
4
1
4
4
standard Bcc panel strain
ATTC BAA-245
ATTC BAA-67
HI2210
1
LMG 16656, UK CF patient, 1989
LMG 14294, Belgium CF patient, 1993
standard Bcc panel strain
standard Bcc panel strain
standard Bcc panel strain
LMG 18941, first CF B. dolosa isolate
CF clinical isolate
>10
>10
1
Burkholderia Vietnamiensis (V)
Burkholderia dolosa (VI)
PC259
AU0645
ATTC BAA-246
AU4459
AU5404
AU4298
AU3556
AU4881
AU9248
AU4894
AU3123
AU4750
HI2468
2
2
1
1
2
2
4
2
2
2
2
1
2
1
4
6
6
6
1
nda
nda
nda
nda
nda
nda
nda
nda
>10
4
CF clinical isolate
CF clinical isolate
CF clinical isolate
CF clinical isolate
CF clinical isolate
CF clinical isolate
CF clinical isolate
CF clinical isolate
nda
nda
nda
nda
nda
nda
nda
4
Burkholderia ambifaria (VII)
Burkholderia anthina (VIII)
Burkholderia pyrocinia (IX)
Escherichia coli
2
1
1
2
standard Bcc panel strain
standard Bcc panel strain
standard Bcc panel strain
susceptible strain
AU1293
BC11
J53
1
1
>10
>10
1
>10
>10
J53+pMG101
>10
>10
J53 + plasmid with silver resistance genes
a nd ) not done.
Table 3. MIC Comparison of 5 and 7
MIC (µg/mL)
species (genomovar)
strain
5
7
note
Pseudomonas aeruginosa
PAO1-V
PAM57-15
J2315
AU4459
J53
2
1
1
1
1
1
laboratory strain
mucoid CF clinical isolate
epidemic strain
CF clinical isolate
susceptible strain
4
2
4
2
Burkholderia cenocepacia (III)
Burkholderia dolosa (VI)
Escherichia coli
J53+pMG101
>10
>10
J53 + plasmid with silver resistance genes
1
aeruginosa strains and two strains of the Burkholderia
species, the MIC50 of 5 was 2 µg/mL, and the MIC90 was 4
µg/mL. In contrast, the MIC of 7 was uniformly 1 µg/mL
(Table 3) for all of the strains tested. E. coli strains J53, a
silver-sensitive strain, and J53+pMG101, a silver-resistant
strain, were again used as positive and negative controls. If
compared on a molar basis, the MIC50 values of 5 and 7
against this panel of pathogens are essentially the same,
namely 6.0 × 10-6 mmol/mL (MIC90 for 5 is 12 × 10-6
mmol/mL) and 3.8 × 10-6 mmol/mL, respectively. The result
is somewhat surprising, given the significant difference in
degradation rates between 5 and 7, suggesting either that the
degradation rate of 5 in bacteria-laden media is dramatically
accelerated or that the intact complex has antimicrobial
activity. In order to assess the stability of silver complexes
5 and 7, 10 mg/mL stock solutions of 5 and 7 in D2O were
made and added in a 1:1 ratio to M-H and LB made in D2O.
This was done to mimic the conditions of the MIC determi-
nation experiments. Changes in the H NMR spectrum were
monitored at 0 °C, room temperature, and 37 °C. Complex
5 demonstrated stability in the M-H/D2O broth mixture at 0
°C, room temperature, and 37 °C when monitored over a
maximum of 72 h. It also appeared stable in the LB/D2O
broth mixture at room temperature when monitored over the
same length of time. However, complex 7 demonstrated very
fast degradation when mixed with the M-H/D2O broth
mixture at 0 °C. Based on the 1H NMR spectrum, this
compound degraded within the first minute after the initial
mixing of the components. However, in support of the first
hypothesis is the observation that a precipitate formed upon
the addition of 5 to the M-H broth. Thus, a portion of 5 may
degrade immediately, releasing an active silver cation, and
the stable 1H NMR spectrum may represent that of the
remainder of intact 5. These results strongly support the
possibility that the intact compound has antimicrobial activity

Electronically Tuned SilVer Acetate NHCs
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1581
discarded, and the volatile components were removed in vacuo to
yield a yellow crystalline solid. Crystals suitable for single crystal
X-ray diffraction studies were grown from acetonitrile. Yield: (1.32
g, 8.72 mmol, 97%); mp: 56–58 °C. Anal. Calcd for C4H4Cl2N2:
C, 31.82; H, 2.67; N, 18.55. Found: C, 32.31; H, 2.74; N, 17.51.
given the equivalent MIC50 concentrations of the stable 5
and the unstable 7.
Conclusion
In conclusion, we have shown that imidazolium salts 4, 6,
and 8 did not act as antibacterial agents in low concentrations,
but when combined with silver acetate the systems exhibited
excellent bacteriostatic activity. Most of the silver complexes
dissociated completely in the presence of water within a few
hours or days with rapid removal of all silver. However,
complex 5 showed a moderate initial dissociation, with continual
release of silver over the time observed. Thus, we have presented
evidence of the electronic tuning of an NHC ligand by
introducing chlorine substituents on the 4 and 5 positions of
the imidazole ring. The chlorine substituents act in a σ-with-
drawing and π-donating fashion, thereby significantly stabilizing
the NHC silver complex 5 compared to its nonchlorinated
analogue 7 and its σ-withdrawing and π-withdrawing methyl
ester analogue 9.
We are currently exploring other systems bearing a variety
of electron withdrawing groups as well as other halogens. Our
goal is to explore if other halogens will act similarly to the
chlorines in further stabilizing the NHC silver complexes and
if other electron withdrawing groups that act strictly as
σ-withdrawing and π-withdrawing groups will have a similar
affect. This study will ultimately help us find the ideal silver
NHC that may survive systemic delivery.
1
ESI-MS (m/z): calcd, 157.0, [M + Li]+; found, 156.9. H NMR
(300 MHz, DMSO-d6): δ 3.60 (s, 3H, CH3), 7.76 (s, 1H, NCHN).
13C {1H} NMR (75 MHz, DMSO-d6): δ 32.3 (NCH3), 136.2
(NCHN), 124.0 (C-Cl), 112.6 (C-Cl).
X-ray crystal structure analysis of 3: formula C4H4Cl2N2, MW
) 150.99, colorless crystal 0.27 × 0.16 × 0.07 mm3, a ) 7.128(3)
Å, b ) 7.195(3) Å, c ) 11.938(4) Å, R ) 84.108(6)°, ꢀ )
78.185(5)°, γ ) 88.354(6)°. T ) 100(2) K, Z ) 4, triclinic, space
3
group P1, V ) 596.1(4) Å , Dcalc ) 1.682 Mg ·m-3, λ ) 0.71073
j
Å, µ ) 0.969 mm-1, 4634 reflections collected, 2383 independent
(Rint ) 0.0334), 147 refined parameters, R1/wR2 (I > 2σ(I)) )
0.0415/0.1120 and R1/wR2 (all data) ) 0.0445/0.1139, maximum
(minimum) residual electron density 0.739(-0.358) e ·Å-3
.
Synthesis of 1,3-Dimethyl-4,5-dichloroimidazolium Iodide
(4). Compound 3 (1.32 g, 8.72 mmol) was dissolved in acetonitrile
(30 mL), and excess iodomethane (2 mL, 32.1 mmol) was added.
The mixture was refluxed at 85 °C for 2 days. The volatile
components were removed in vacuo to yield a green crystalline
solid. Crystals suitable for single crystal X-ray diffraction were
grown from acetonitrile. Yield: (2.45 g, 8.37 mmol, 96%); mp:
179–182 °C. Anal. Calcd for C5H7Cl2N2I: C, 20.50; H, 2.41; N,
9.56. Found: C, 20.31; H, 2.27; N, 9.22. ESI-MS (m/z): calcd, 165.0,
1
[C5H7Cl2N2+]; found, 164.9. H NMR (300 MHz, DMSO-d6): δ
3.83 (s, 6H, CH3), 9.42 (s, 1H, NCHN). 13C {1H} NMR (75 MHz,
DMSO-d6): δ 35.0 (NCH3), 136.6 (NCHN), 118.9 (C-Cl).
X-ray crystal structure analysis of 4: formula C5H7Cl2N2I, MW
) 292.93, colorless crystal 0.50 × 0.10 × 0.05 mm3, a )
7.1338(12) Å, b ) 7.4652(13) Å, c ) 8.7890(15) Å, R ) 90°, ꢀ )
92.125(3)°, γ ) 90°. T ) 100(2) K, Z ) 2, monoclinic, space group
P2(1), V ) 467.74(14) Å3, Dcalc ) 2.080 Mg ·m-3, λ ) 0.71073
Å, µ ) 3.928 mm-1, 3971 reflections collected, 2104 independent
(Rint ) 0.0295), 93 refined parameters, R1/wR2 (I > 2σ(I)) )
0.0368/0.0909 and R1/wR2 (all data) ) 0.0374/0.0913, maximum
Experimental Section
General Procedures. All manipulations were carried out under
aerobic conditions. 4,5-Dichloroimidazole and iodomethane were
purchased from Alfa Aesar. Silver(I) oxide, silver acetate, and
methylimidazole were purchased from Acros Organics. Silver nitrate
was purchased from Fisher Scientific, and urocanic acid was
purchased from TCI. LB Broth Miller (DIFCO) and Bacto-agar
(DIFCO) were prepared according to manufacturer’s instruction and
sterilized before use. All solvents were obtained from a PureSolv
solvent purification system. 1H and 13C NMR data were collected
on a Varian Gemini 300 MHz instrument. 1H NMR stability studies
for complexes 5 and 7 in M-H/D2O and LB/D2O broth mixtures
were done on a Varian INOVA 400 MHz instrument. The 1H and
13C NMR spectra obtained were referenced to the residual protons
of the deuterated solvents and the solvent resonances, respectively.
109Ag NMR data were collected on a Varian INOVA 750 MHz
instrument. Mass spectrometry data were collected on a Bruker
Daltons (Billerica, MA) Esquire-LC mass spectrometer equipped
with ESI. Elemental analyses were performed at the Microanalyses
Laboratory at The University of Illinois and Galbraith Laboratories.
X-ray Crystallographic Structure Determination Details.
Crystals of 3, 4, 5, 7, 8, and 9 were coated in paratone oil, mounted
on a CryoLoop, and placed on a goniometer under a stream of
nitrogen. X-ray data were collected using a Bruker Apex CCD
diffractometer with graphite-monochromated Mo KR radiation (λ
) 0.71073 Å). The data was integrated using SAINT. An empirical
absorption correction and other corrections were applied by using
multiscan SADABS. A Bruker SHELXTL package was used for
the structure solution, refinement, and modeling of the crystals. The
structures were determined by full-matrix least-squares refinement
of F2 and the selection of appropriate atoms from the generated
difference map.
(minimum) residual electron density 2.252(-2.467) e ·Å-3
.
Synthesis of (1,3-Dimethyl-4,5-dichloroimidazole-2-ylidene)-
silver(I)acetate (5). Compound 4 (1.51 g, 5.15 mmol) was dissolved
in dichloromethane (50 mL), and silver acetate (1.78 g, 10.7 mmol)
was added. The mixture was stirred at room temperature for 2.5 h.
The yellow precipitate, presumably AgI, was filtered and discarded.
The volume of the reaction mixture was reduced under pressure to
5 mL. Hexane (1 L) was added, and the fine white precipitate was
filtered and washed with 20 mL of hexane. Crystals suitable for
single crystal X-ray diffraction studies were grown from THF.
Yield: (1.35 g, 4.07 mmol, 79%); mp: 187–188 °C. Anal. Calcd
for C7H9AgCl2N2O2: C, 25.33; H, 2.73; N, 8.44. Found: C, 25.37;
H, 2.69; N, 8.01. ESI-MS (m/z): calcd, 165.0, [C5H7Cl2N2+], 272.8
[C5H6AgCl2N2+], 436.8 [C10H12AgCl4N4]; found, 164.9, 272.7, and
1
436.7. H NMR (300 MHz, DMSO-d6): δ 1.79 (s, 3H, COCH3),
3.77 (s, 6H, N-CH3). 13C {1H} NMR (75 MHz, DMSO-d6): δ
179.7 (C-Ag), 175.7 (C-O), 116.9 (C-Cl), 37.5 (N-CH3), 23.4
(COCH3). 109Ag (35 MHz, DMSO-d6): δ 352.55 (s, C-Ag-O).
X-ray crystal structure analysis of 5: formula C7H15AgCl2N2O5,
MW ) 385.98, colorless crystals 0.48 × 0.16 × 0.10 mm3, a )
6.4992(10) Å, b ) 13.491(2) Å, c ) 15.744(3) Å, R ) 90°, ꢀ )
90°, γ ) 90°. T ) 100(2) K, Z ) 4, orthorhombic, space group
P2(1)2(1)2(1), V ) 1380.4(4) Å3, Dcalc ) 1.828 Mg ·m-3, λ )
0.71073 Å, µ ) 1.856 mm-1, 12260 reflections collected, 3298
independent (Rint ) 0.0594), 157 refined parameters, R1/wR2 (I >
2σ(I)) ) 0.0673/0.1767 and R1/wR2 (all data) ) 0.0825/0.1876,
maximum (minimum) residual electron density 4.484(-1.219)
Synthesis of 1-Methyl-4,5-dichloroimidazole (3). 4,5-Dichlor-
oimidazole (1.23 g, 9.00 mmol) and potassium hydroxide (2.24 g,
40.0 mmol) were stirred in acetonitrile (50 mL) for 2 h at room
temperature. The excess KOH was filtered from the solution, and
iodomethane (0.562 mL, 9.00 mmol) was added. The reaction
mixture was stirred at room temperature for 24 h. The volatile
components were removed, and the crude product was redissolved
in dichloromethane. The solid, presumably KI, was filtered and
e·Å-3
.
Synthesis of (1,3-Dimethylimidazole-2-ylidene)silver(I)acetate
(7). Compound 6 (0.91 g, 4.0 mmol) was dissolved in dichlo-
romethane (50 mL), AgOAc (1.37 g, 8.20 mmol) was added, and
the mixture was refluxed for 40 h. The precipitate, presumably AgI,
was filtered, and a clear solution was obtained. The volatile

1582 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6
Hindi et al.
components were removed in vacuo to produce a white sticky solid.
The solid was washed with diethyl ether (3 × 10 mL) and dried
under reduced pressure for 7 h to yield 7. Crystals suitable for single
crystal X-ray diffraction studies were grown from methylene
chloride. Yield: (0.67 g, 2.6 mmol, 64%); mp: 109–111 °C. Anal.
Calcd for C7H11N2O2Ag: C, 31.96; H, 4.22; N, 10.65. Found: C,
32.39; H, 4.57; N, 10.05. ESI-MS (m/z): Calcd for C5H8N2Ag
[MsOAc]+: 202.97 and 204.97, found 203.00 and 205.00. 1H NMR
(300 MHz, DMSO-d6): δ 7.41 (s, 2H, CH), 3.75 (s, 6H, CH3), 1.77
(s, 3H, CH3). 13C {1H} NMR (75 MHz, DMSO-d6): δ 178.6 (s,
CsAg), 174.8 (s, CdO), 122.9 (s, CdC), 37.9 (s, CH3), 23.2 (s,
CH3). 109Ag (35 MHz, DMSO-d6): δ 343.62 (s, CsAgsO).
X-ray crystal structure analysis of 9: formula C11H15AgN2O5,
MW ) 363.11, colorless crystals 0.26 × 0.05 × 0.02 mm3, a )
3.9393(11) Å, b ) 13.676(4) Å, c ) 14.281(4) Å, R ) 63.854(4)°,
ꢀ ) 84.961(4)°, γ ) 81.923(4)°. T ) 100(2) K, Z ) 2, triclinic,
3
space group P1, V ) 683.5(3) Å , Dcalc ) 1.764 Mg ·m-3, λ )
j
0.71073 Å, µ ) 1.492 mm-1, 5856 reflections collected, 3152
independent (Rint ) 0.0669), 176 refined parameters, R1/wR2 (I >
2σ(I)) ) 0.0563/0.1250 and R1/wR2 (all data) ) 0.0704/0.1277,
maximum (minimum) residual electron density 1.428(-0.671)
e·Å-3
.
Crystallographic data for compounds 3, 4, 5, 7, 8, and 9 have
been deposited with the Cambridge Crystallographic Data Center
(666021, 655235, 655236, 655237, 655238, 655239).
X-ray crystal structure analysis of 7: formula C7H11AgN2O2, MW
) 263.05, colorless crystal 0.11 × 0.05 × 0.04 mm3, a ) 10.508(2)
Å, b ) 10.521(2) Å, c ) 12.881(3) Å, R ) 95.984(4)o, ꢀ )
96.090(4)o, γ ) 93.974(4)o, V ) 1403.7(5) Å3, Dcalc ) 1.867
Bacteria. All bacterial strains were maintained as glycerol stocks
at -80 °C. For experiments, the bacteria were streaked onto either
tryptic soy agar (TSA) or blood agar plates and incubated overnight
at 37 °C. Dr. Simon Silver of Chicago, IL, kindly provided the
silver sensitive and silver resistant E. coli strains. Dr. Thomas Ferkol
of St. Louis, MO, provided PAM57-15. Dr. Gerald Pier of Boston,
MA, provided all other strains of P. aeruginosa. B. cepacia complex
species either were obtained from the Clinical Microbiology
Laboratory at St. Louis Children’s Hospital (CF clinical isolates
of B. multiVorans) or were provided by Dr. John LiPuma of Ann
Arbor, MI, Dr. Johannes Huebner of Boston, MA (CF clinical
isolates of B. dolosa), or the American Type Culture Collection.
Stock Solutions of 5 and 7. Compound 5 was diluted in sterile,
<20 MΩ water at a concentration of 10 mg/mL and stored in small
aliquots at -80 °C. Individual aliquots of stock solution 5 were
thawed and stored at 4 °C for periods no longer than 2 weeks.
Compound 7 was dissolved in dimethylsulphoxide (DMSO) to give
a final concentration of 30 mg/mL. Stock solutions of 7 were not
stored for longer than the day of the experiment.
In Vitro Antimicrobial Activity. Minimal inhibitory concentra-
tions (MICs) were determined by broth microdilution as previously
described by using standard CLSI (formerly the National Committee
on Clinical Laboratory Standards, NCCLS) protocols.13,17 Briefly,
bacteria from fresh overnight plates were suspended in standard
Mueller-Hinton broth (M-H), or for some experiments in Luria broth
(LB), to an optical density at 650 nm (OD650) of 0.2 and were grown
in a shaking incubator to an OD650 of 0.4 (∼2 × 108 CFU/mL).
The bacteria were diluted in the broth to a concentration of 105 in
100 µL, which was added to triplicate wells, containing 100 µL of
either 5 or 7 diluted in water to various concentrations from 10
mg/mL stocks. The final concentrations tested were 1, 2, 4, 6, 8,
and 10 µg/mL. The MIC was the lowest of these concentrations,
at which each of the triplicate wells was clear after incubation of
the plate for 18–20 h at 37 °C. The MIC50 is, by definition, the
concentration at which growth of 50% of the tested strains is
inhibited, and similarly the MIC90 is the concentration at which
90% of the tested strains fail to grow. Minimal bactericidal
concentrations (MBCs) of 5 were determined by plating all of the
clear wells of the MIC experiments on TSA plates, incubating the
plates overnight at 37 °C, and noting the concentration at which
there were no colonies.
Mg·m-3, µ ) 2.117 mm-1, Z ) 6, triclinic, space group P1, λ )
j
0.71073 Å, T ) 100 K, ω and ꢁ scans, 12369 reflections collected,
6448 independent (Rint ) 0.0337), 344 refined parameters, R1/wR2
(I g 2σ(I)) ) 0.0534/0.1101 and R1/wR2 (all data) ) 0.0801/
0.1166, maximum (minimum) residual electron density 1.058
(-0.915) e ·Å-3
.
Synthesis of 1,3-Dimethyl-4-acrylic Acid Methyl Ester Imi-
dazolium Iodide (8). A solution of urocanic acid (0.497 g, 3.60
mmol) and sodium bicarbonate (1.2 g, 14 mmol) was refluxed in
acetonitrile (150 mL) for 1 h. After the addition of excess
iodomethane (12.77 g, 90.00 mmol), stirring was continued for 3
days under reflux. The excess sodium bicarbonate was filtered off,
and the volatile components were removed in vacuo. The crude
product was redissolved in 250 mL of dichloromethane and stirred
for 2 h. The solid, presumably NaI, was filtered, and the volatile
components were removed in vacuo to yield a white solid. Crystals
suitable for single crystal X-ray diffraction were grown from
acetonitrile. Yield: (0.9 g, 3 mmol, 81%); mp: 195–197 °C; Anal.
Calcd for C9H13N2O2I: C, 35.08; H, 4.25; N, 9.09. Found: C, 35.08;
H, 3.90; N, 8.91. ESI-MS (m/z): calcd for C9N2O2H13I [M-I]+
1
181.1. Found 180.9. H NMR (300 MHz, DMSO-d6): δ 9.16 (s,
1H, NCHN), 8.32 (s, 1H, NCHC), 7.50 (d, 1H, CCH), 6.70 (d,
1H, CHCdO), 3.92 (s, 3H, CH3), 3.85 (s, 3H, CH3), 3.75 (s, 3H,
CH3). 13C {1H} NMR (75 MHz, DMSO-d6): δ 165.6 (CdO), 138.7
(NCHC), 129.8 (CdC), 127.5 (CdC), 123.6 (CdC), 122.1 (CdC),
52.0 (OCH3), 36.2 (NsCH3), 34.0 (NsCH3).
X-ray crystal structure analysis of 8: formula C9H13N2O2I, MW
) 308.11, colorless crystals 0.25 × 0.21 × 0.09 mm3, a )
9.6726(16) Å, b ) 9.4366(15) Å, c ) 12.868(2) Å, R ) 90°, ꢀ )
99.595(3)°, γ ) 90°. T ) 100(2) K, Z ) 4, monoclinic, space group
P2(1)/n, V ) 1158.1(3) Å3, Dcalc ) 1.767 Mg ·m-3, λ ) 0.71073
Å, µ ) 2.744 mm-1, 9576 reflections collected, 2759 independent
(Rint ) 0.0638), 179 refined parameters, R1/wR2 (I > 2σ(I)) )
0.0375/0.0840 and R1/wR2 (all data) ) 0.0460/0.0875, maximum
(minimum) residual electron density 1.688(-0.812) e ·Å-3
.
Synthesis of (1,3-Dimethyl-4-acrylic Acid Methyl Ester Imi-
dazole-2-ylidene)silver(I)acetate (9). A mixture of 8 (0.20 g, 0.65
mmol) and silver acetate (0.26 g, 1.6 mmol) in dichloromethane
(50 mL) was stirred at room temperature for 1 h. The reaction
mixture was filtered through Celite to remove a yellow precipitate,
presumably AgI, and the volatile components were removed in
vacuo to yield a white solid. Crystals suitable for single crystal
X-ray diffraction were grown from acetonitrile. Yield: (0.20 g, 0.57
mmol, 88%); mp: 122–123 °C. Anal. Calcd for C11H15N2O4Ag: C,
38.06; H, 4.36; N, 8.07. Found: C, 38.85; H, 4.68; N, 7.87. ESI-
MS (m/z): calcd for C11N2O4H15Ag [M-OAc]+ 287.0, 289.0. Found
286.8, 288.8. Calcd for C18N4O4H24Ag [M]+ 467.1, 469.1. Found
467.0, 469.0. 1H NMR (300 MHz, DMSO-d6): δ 8.11 (s, 1H,
NCHC), 6.50 (d, 1H, CCH), 7.51 (d, 1H, CHCdO), 3.88 (s, 3H,
CH3), 3.79 (s, 3H, CH3), 3.73 (s, 3H, CH3), 1.83 (s, 3H, CH3CdO).
13C {1H} NMR (75 MHz, DMSO-d6): δ 182.0 (CsAg), 174.0
(CdO), 166.2 (CdO), 130.0 (CdC), 129.4 (CdC), 124.4 (CdC),
118.7 (CdC), 51.7 (OCH3), 38.5 (NsCH3), 36.6 (NsCH3), 22.4
(COCH3).
Acknowledgment. This work was supported by the Uni-
versity of Akron, Washington University School of Medicine,
and the National Institute of Allergies and Infectious Diseases
(1 R01 A106785601). We wish to thank the National Science
Foundation (CHE-8808587 and CHE-9977144) for funds used
to purchase the Varian Gemini 300 MHz and the Varian INOVA
400 MHz NMZ instruments used in this work. We also wish to
thank the Kresge Foundation and donors to the Kresge Challenge
Program at The University of Akron for funds used to purchase
the Varian INOVA 750 MHz NMR instrument used in this
work. We would like to acknowledge the National Science
Foundation (CHE-0116041) and the Ohio Board of Regents for
funds used to purchase the Bruker-Nonius Apex CCD X-ray
diffractometer used in this research. We thank Dr. Martha Kory

Electronically Tuned SilVer Acetate NHCs
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 6 1583
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Supporting Information Available: Melting point, elemental
1
analysis, H NMR, and 13C NMR data for compounds 3, 4, 5, 7,
8, and 9. 109Ag NMR data for compounds 5 and 7. This material
is available free of charge via the Internet at http://pubs.acs.org.
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