Synthesis and antimicrobial studies of silver N-heterocyclic carbene complexes bearing a methyl benzoate substituent

Article abstract of DOI:10.1016/j.ica.2010.08.008

Due to the properties of silver as an antimicrobial, our research group has synthesized many different silver carbene complexes. Two new silver N-heterocyclic carbene complexes derived from 4,5-dichloroimidazole and theobromine bearing methyl benzoate sub

Full text of DOI:10.1016/j.ica.2010.08.008

Inorganica Chimica Acta 364 (2010) 125–131
Contents lists available at ScienceDirect
Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Synthesis and antimicrobial studies of silver N-heterocyclic carbene complexes
bearing a methyl benzoate substituent
⇑
Amanda R. Knapp, Matthew J. Panzner, Doug A. Medvetz, Brian D. Wright, Claire A. Tessier, Wiley J. Youngs
Department of Chemistry, The University of Akron, Akron, OH 44325-3601, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 10 August 2010
Due to the properties of silver as an antimicrobial, our research group has synthesized many different sil-
ver carbene complexes. Two new silver N-heterocyclic carbene complexes derived from 4,5-dichloroim-
idazole and theobromine bearing methyl benzoate substituents were synthesized by in situ carbene
formation using silver acetate as the base in the reaction. The new compounds were fully characterized
by several methods including NMR spectroscopy and X-ray crystallography. Preliminary antimicrobial
efficacy studies against Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli were con-
ducted. The results of this study demonstrated antimicrobial efficacy of the two complexes comparable
to silver nitrate, showing their potential for use in the treatment of bacterial infections.
Ó 2010 Elsevier B.V. All rights reserved.
Dedicated to Arnold L. Rheingold.
Keywords:
Silver
N-heterocyclic carbene
Antimicrobial
Methyl benzoate
Silver carbene complex
1. Introduction
[8–10]. Due to this complication Wang and Lin developed a meth-
od of creating metal carbenes in situ using silver oxide [11]. The
Silver has a long standing use as an antimicrobial agent, partic-
ularly in modern medicine for the prevention and treatment of
bacterial infections associated with severe burn wounds [1]. This
is evidenced by the use of silver sulfadiazine, trademarked as Sil-
vadeneÒ, in burn wards worldwide since 1968 [1–3]. Silver has
many properties that make it an attractive candidate for use as a
medicinal agent. It is present in the human body in single digit
synthesis began with an imidazolium cation and deprotonation
was done using the silver salt as the base. These milder conditions
lead to a more versatile method for making silver carbene com-
plexes that remain stable under aerobic conditions.
Due to its promise as an antimicrobial, our research group has
focused delivering silver by coupling silver (I) to NHCs to increase
its stability in the body. The complexes developed by our research
group have been synthesized by in situ carbene formation using
silver acetate as a basic silver salt. The silver carbene complexes
(SCCs) have utilized different backbones and substituents to allow
for changes in water stability, water or organic solubility, and low
toxicity of the degradation products. Most of these SCCs are stable
to aerobic conditions, as well as in solution. They have been
tested against a variety of bacterial species and have all shown
effectiveness in vitro while some have also shown in vivo efficacy
[12–20].
Herein we report the synthesis of two new SCCs utilizing the
methyl-3-methylbenzoate group as a substituent on theobromine
and 4,5-dichloroimidazole. Theobromine is a biocompatible natu-
ral product found in chocolate and 4,5-dichloroimidazole has been
shown to give added stability to the silver complexes due to the
electron withdrawing nature of the chloride groups of the back-
bone [15]. The benzoate group as a substituent serves as a site to
complex targeting groups that can aid in the delivery of the
compound to bacteria cells in the body. These two SCCs have been
successfully synthesized, characterized, and tested for their stabil-
ity and antimicrobial activity.
lg/L concentrations despite no known biological function [4]. This
fact indicates that the body can tolerate the presence of silver in
low doses without toxic effect. In fact, its low toxicity is one of
the greatest advantages of silver over other medicinally relevant
metals. A cosmetic condition known as argyria, which is a blue-
gray discoloration of the skin caused by extreme excess exposure
of silver salts, is one of the only known side effects [5,6]. In general
silver does not accumulate in the body, but rather is readily ex-
creted, an important aspect for its use as a potential internalized
therapeutic agent.
The first silver carbene was isolated by Arduengo et al. in 1993
[7]. The synthesis involved first isolating the free carbene by using
a strong base such as KH or KOtBu, followed by the reaction of the
free carbene with silver triflate. Although it is possible to generate
a stable free carbene in this manner, the conditions required
resulted in decomposition when applied to complexes with meth-
ylene groups
a to the nitrogen of the N-heterocyclic carbene (NHC)
⇑
Corresponding author. Tel.: +1 330 972 5362; fax: +1 330 972 7370.
E-mail address: youngs@uakron.edu (W.J. Youngs).
0020-1693/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.ica.2010.08.008

126
A.R. Knapp et al. / Inorganica Chimica Acta 364 (2010) 125–131
1H, CHbenzyl), 7.92 (s, 1H, CHbenzyl), 8.04 (s, 1H, NCHN). 13C NMR
2. Experimental
(500 MHz, d6-DMSO): d 30.0 (NCH3), 33.7 (NCH3), 43.9 (NCH2),
52.2 (OCH3), 107.1 (COCN), 128.4 (Cbenzyl), 129.0 (Cbenzyl), 129.3
(Cbenzyl), 130.1 (Cbenzyl), 133.2 (Cbenzyl), 138.8 (Cbenzyl), 142.8
(NCHN), 143.3 (NCN), 151.4 (NCON), 154.8 (NCON), and 166.6
(COOCH3). Crystal data for 1: C16H16N4O4, M = 328.33, triclinic,
2.1. Materials and general methods
All reactions were carried out under aerobic conditions. 4,5-
Dichloroimidazole, theobromine, iodomethane, methyl-3-bromo-
methyl benzoate, silver acetate, and silver nitrate were purchased
from Alfa Aesar. All solvents were purchased from Fisher Scien-
tific. All products and solvents were used as received without fur-
ther purification. LB Brother Miller (DIFCO) and Bacto-agar
(DIFCO) were prepared according to manufacturer’s instructions
and sterilized before use. 1H and 13C NMR were collected on a
Varian 500 MHz instrument. All spectra were referenced to deu-
terated solvents. Elemental analyses were performed by the Uni-
versity of Illinois microanalysis laboratory. Mass spectrometry
was performed by the Ohio State University mass spectrometry
and proteomics facility (compounds 1, 3, and 4) where exact mass
was determined, as well as by the University of Akron mass spec-
trometry lab (compounds 2, 5, and 6) where nominal mass was
determined. Crystals of the compounds were coated in paratone
oil, mounted on a CryoLoop, and placed on a goniometer under
a stream of nitrogen. Crystal structure data sets were collected
on a Bruker SMART Apex CCD diffractometer with graphite-
a = 8.434(4) Å, b = 8.897(4) Å, c = 10.825(5) Å,
b = 104.970(7)°,
= 108.245(7)°, V = 736.9(6) Å3, T = 100(2) K, space
(Mo K
) = 0.109 mmÀ1, 6442 reflections mea-
sured, 3339 independent reflections (Rint = 0.0207). The final R1 val-
ues were 0.0459 (I > 2
(I)). The final wR(F2) values were 0.1164
(I > 2
a = 93.242(8)°,
c
ꢀ
group P1, Z = 2,
l
a
r
r
(I)). The final R1 values were 0.0554 (all data). The final
wR(F2) values were 0.1244 (all data). The goodness of fit on F2
was 1.034.
Synthesis of 2. Compound 1 (8.00 mmol, 2.75 g) was placed in a
pressure tube with 10 mL of DMF. Methyl iodide (15 mL) was added
and the pressure tube was heated at 85 °C for 24 h. The volatiles
were removed and methylene chloride was added. A small amount
of white precipitate was filtered and the solution was concentrated
to 10 mL. Ether was added and the sticky orange solid that re-
mained on the flask and filter paper was dissolved in methanol.
The volatiles were removed by rotary evaporation to yield the prod-
uct as a shiny orange solid. Crystals were grown from acetonitrile/
ethylacetate. Yield: 3.16 g, 6.51 mmol, 81.4%. mp: 146–147 °C. Anal.
Calc. for C17H19N4O4I: C, 43.42; H, 4.07; N, 11.91. Found: C, 42.63; H,
4.04; N, 11.46%. ES-MS (m/z): calc., 343.1406 [MÀIÀ]+; found, 343.2.
1H NMR (500 MHz, d6-DMSO): d 3.76 (s, 3H, NCH3), 3.86 (s, 3H,
OCH3), 4.09 (s, 3H, NCH3), 4.16 (s, 3H, NCH3), 5.16 (s, 2H, NCH2C),
7.51 (t, J = 7.70 Hz, 1H, CHbenzyl), 7.63 (d, J = 7.83 Hz, 1H, CHbenzyl),
7.88 (d, J = 7.83 Hz, 1H, CHbenzyl), 7.98 (s, 1H, CHbenzyl), 9.34 (s, 1H,
monochromated Mo K
a radiation (k = 0.71073 Å). The data was
integrated using SAINT and a Bruker SHELXTL package was used for
structure solution, refinement, and modeling of the crystals
[21–23]. This data is represented in Table 1.
2.2. Synthesis
NCHN). 13C NMR (500 MHz, d6-DMSO):
d 32.0 (NCH3), 34.9
Synthesis of 1. Theobromine (15.0 mmol, 2.70 g) was added to a
250 mL round bottom flask and 100 mL of dry DMF was added.
Potassium carbonate (16.5 mmol, 2.28 g) was added and the mix-
ture was stirred 30 min. Methyl-3-(bromomethyl)benzoate
(16.0 mmol, 3.66 g) was added and the mixture was stirred at
120 °C for 18 h. The white KBr precipitate was filtered and the vol-
atiles were removed. The resulting oil was dissolved in 20 mL of
methylene chloride and 200 mL of ether was added to yield the
crude product as a cream colored solid. The solid was filtered and
purified by stirring with 200 mL methanol for 30 min followed by
vacuum filtration to isolate the desired product as soft white pow-
der. Crystals were grown from methylene chloride. Yield: 2.35 g,
9.00 mmol, 60.0%. mp: >260 °C. Anal. Calc. for C16H16N4O4: C,
58.53; H, 4.91; N, 17.06. Found: C, 58.14; H, 4.83; N, 16.41%. ES-
MS (m/z): calc., 351.1069 [M+Na]+; found, 351.1058. 1H NMR
(500 MHz, d6-DMSO): d ppm 3.42 (s, 3H, NCH3), 3.84 (s, 3H,
OCH3), 3.89 (s, 3H, NCH3), 5.09 (s, 2H, NCH2N), 7.45 (t, J = 7.76 Hz,
1H, CHbenzyl), 7.58 (d, J = 1.51 Hz, 1H, CHbenzyl), 7.83 (d, J = 7.57 Hz,
(NCH3), 36.2 (NCH3), 44.9 (NCH2), 52.7 (OCH3), 108.3 (COCN),
128.8 (Cbenzyl), 129.3(Cbenzyl), 129.4(Cbenzyl), 130.3 (Cbenzyl), 133.4
(Cbenzyl), 137.5(Cbenzyl), 140.2 (NCHN), 140.4 (NCN), 150.6 (NCON),
153.7 (NCON), and 166.5 (COOCH3) ppm. Crystal data for 2:
2(C17H19N4O4)Á2(I)ÁO, M = 956.52, triclinic, a = 8.9713(15) Å,
b = 11.6793(19) Å, c = 19.215(3) Å,
a = 106.366(3)°, b = 92.729(3)°,
3
ꢀ
c
= 93.658(3)°, V = 1923.3(6) Å , T = 100(2) K, space group P1, Z
= 2, (Mo K
) = 1.698 mmÀ1, 13814 reflections measured, 6729
independent reflections (Rint = 0.0363). The final R1 values were
0.0824 (I > 2 (I)).
(I)). The final wR(F2) values were 0.1899 (I > 2
l
a
r
r
The final R1 values were 0.0975 (all data). The final wR(F2) values
were 0.1961 (all data). The goodness of fit on F2 was 1.233.
Synthesis of 3. Compound 2 (1.82 mmol, 0.814 g) was dissolved
in 30 mL methylene chloride. Silver acetate (3.64 mmol, 0.608 g)
was added and the mixture was stirred at room temperature for
3 h. AgI precipitate was filtered and the solution was concentrated
to 5 mL. Hexanes (100 ml) were added and the desired product
Table 1
Crystallographic experimental data for compounds 1–6.
1
2
3
4
5
6
C
16H16N4O4
2(C17H19N4O4)Á2(I)ÁO
C19H21AgN4O6ÁC2H4O2
monoclinic
P2(1)/c
C12H10Cl2N2O2
triclinic
P1
C13H13Cl2N2O2ÁI
C30H30Ag2Cl4N4O8
monoclinic
C2/c
Lattice system
Space group
a (Å)
b (Å)
c (Å)
triclinic
P1
triclinic
triclinic
ꢀ
ꢀ
ꢀ
ꢀ
P1
P1
8.434(4)
8.9713(15)
11.6793(19)
19.215(3)
106.366(3)
92.729(3)
93.658(3)
0.0824
0.1899
0.0975
0.1961
6.718(4)
36.73(2)
9.112(6)
90.00
104.689(10)
90.00
0.0756
0.1673
0.1238
0.1822
7.836(4)
9.003(5)
9.383(5)
68.826(8)
84.052(9)
81.241(8)
0.0367
0.0989
0.0414
0.1036
5.980(3)
7.203(3)
19.686(9)
86.077(7)
83.139(7)
66.480(7)
0.0879
.02260
0.0925
0.2280
47.430(20)
12.106(5)
29.818(12)
90.00
123.715(7)
90.00
0.0770
0.1553
0.1501
0.1862
8.897(4)
10.825(5)
93.242(8)
104.970(7)
108.245(7)
0.0459
0.1164
0.0554
0.1244
a
(°)
b (°)
(°)
c
R1 (I > 2
r
(I))
r
wR(F2) (I > 2
R1 (all data)
(I))
wR(F2) (all data)
Goodness-of-fit (GOF)
1.034
1.233
0.925
1.054
1.267
1.013

A.R. Knapp et al. / Inorganica Chimica Acta 364 (2010) 125–131
127
was filtered as a white precipitate. The solid was stirred in 300 mL
ether for 30 min to remove the acetic acid and the product was col-
lected by vacuum filtration. Crystals were grown from methylene
chloride. Yield: 0.829 g, 1.70 mmol, 93.4%. mp: 159–161 °C. Anal.
Calc. for AgC19H21N4O6: C, 44.72; H, 4.35; N, 10.98. Found: C,
44.39; H, 4.40; N, 9.65%. ES-MS (m/z): calc., 343.1406 [MÀAgOAc]+
and 531.0410 [M+Na]; found, 343.1272 and 531.0554. 1H NMR
(500 MHz, d6-DMSO): d 1.85 (s, 3H, OCCH3), 3.73 (s, 3H, NCH3),
3.84 (s, 3H, OCH3), 4.02 (s, 3H, NCH3), 4.15 (s, 3H, NCH3), 5.11 (s,
2H, NCH2C), 7.47 (t, J = 7.80 Hz, 1H, CHbenzyl), 7.59 (d, J = 6.65 Hz,
1H, CHbenzyl), 7.85 (d, J = 7.76 Hz, 1H, CHbenzyl), 7.94 (s, 1H, CHbenzyl).
13C NMR (500 MHz, d6-DMSO): d 22.8 (COCH3), 32.1 (NCH3), 34.3
(NCH3), 38.8 (NCH3), 44.6 (NCH2), 52.7 (OCH3), 109.4 (COCN),
128.7 (Cbenzyl), 129.1 (Cbenzyl), 129.3 (Cbenzyl), 130.2 (Cbenzyl), 133.2
(Cbenzyl), 138.0 (Cbenzyl), 141.4 (NCN), 150.9 (NCON), 153.5 (NCON),
166.5 (COOCH3), 174.3 (NCN), and 186.5 (COCH3) ppm. Crystal data
for 3: C19H21AgN4O6ÁC2H4O2, M = 569.32, monoclinic, a = 6.718(4) Å,
(NCH2), 52.9 (OCH3), 118.7 (CCl), 120.5 (CCl), 129.9 (Cbenzyl),
130.1 (Cbenzyl), 130.2 (Cbenzyl), 130.9 (Cbenzyl), 133.8 (Cbenzyl), 137.3
(Cbenzyl), 139.7 (NCHN), and 166.2 (COOCH3) ppm. Crystal data
for 5:
C
13H13Cl2N2O2ÁI, M = 427.05, triclinic, a = 5.980(3) Å,
b = 7.203(3) Å, c = 19.686(9) Å,
a
= 86.077(7)°, b = 83.139(7)°,
3
ꢀ
c
= 66.480(7)°, V = 771.7(6) Å , T = 100(2) K, space group P1, Z = 2,
l(Mo K
a
) = 2.423 mmÀ1, 6621 reflections measured, 3477 inde-
pendent reflections (Rint = 0.0377). The final R1 values were
0.0879 (I > 2r r(I)).
(I)). The final wR(F2) values were 0.2260 (I > 2
The final R1 values were 0.0925 (all data). The final wR(F2) values
were 0.2280 (all data). The goodness of fit on F2 was 1.264.
Synthesis of 6. Compound 5 (1.00 mmol, 0.427 g) was dissolved
in 25 mL of methylene chloride in a 100 mL round bottom flask. Sil-
ver acetate (2.00 mmol, 0.338 g) was added and the mixture was
stirred for 3 h at room temperature. The precipitate presumably
AgI was filtered and the solution was concentrated to 2 mL. Ether
was added and the product was crystallized in the freezer over-
night. The resulting white fluffy solid was filtered and dried on a
vacuum line to yield the clean, dry product. Crystals were grown
from methylene chloride. Yield: 0.218 g, 0.470 mmol, 47.0%. mp
92 °C. Anal. Calc. for AgC15H15N2O2Cl2: C, 38.57; H, 3.45; N, 6.00.
Found: C, 38.07; H, 3.36; N, 5.81%. ES-MS (m/z): calc., 487.0
[M+Na]+ and 299.0 [MÀAgOAc]+; found, 486.9 and 299.0. 1H NMR
(500 MHz, d6-DMSO): d ppm 1.79 (s, 3H, OCCH3), 3.84 (s, 3H,
OCH3), 3.86 (s, 3H, NCH3), 5.55 (s, 2H, NCH2C), 7.54 (t, J = 8.30 Hz,
1H, CHbenzyl), 7.59 (d, J = 7.83 Hz, 1H, CHbenzyl), 7.92 (d, J = 7.58,
1H, CHbenzyl), 7.95 (s, 1H, CHbenzyl). 13C NMR (500 MHz, d6-DMSO):
d 23.8 (COCH3, 38.3 (NCH3), 52.8 (COOCH3), 53.5 (NCH2), 116.9
(CCl), 118.4 (CCl), 128.5 (Cbenzyl), 129.4 (Cbenzyl), 129.9 (Cbenzyl),
130.6 (Cbenzyl), 132.6 (Cbenzyl), 136.7 (Cbenzyl), 166.3 (COOCH3),
176.2 (NCN), and 181.2 (COCH3) ppm. Crystal data for 6:
b = 36.73(2) Å,
c = 9.112(6) Å,
= 90.00°, V = 2175(2) Å3, T = 100(2) K, space group P2(1)/c, Z = 4,
(Mo K
) = 0.985 mmÀ1, 18708 reflections measured, 5240 inde-
a
= 90.00°,
b = 104.689(10)°,
c
l
a
pendent reflections (Rint = 0.1250). The final R1 values were 0.0756
(I > 2r r(I)). The final
(I)). The final wR(F2) values were 0.1673 (I > 2
R1 values were 0.1238 (all data). The final wR(F2) values were
0.1822 (all data). The goodness of fit on F2 was 0.925.
Synthesis of 4. 4,5-Dichloroimidazole (5.00 mmol, 0.685 g) was
dissolved in 30 mL DMF in a 100 mL round bottom flask. K2CO3
(5.50 mmol, 0.760 g) was added and the mixture was stirred until
dissolved. Methyl-3-(bromomethyl)benzoate (5.00 mmol, 1.14 g)
was added and the mixture was stirred at reflux (120 °C) overnight.
The solid that was formed, presumably KBr, was filtered from the
solution and the volatiles were removed from the filtrate by rotary
evaporation. Dichloromethane was added and stirred resulting in a
yellow solid that was filtered. The solid was dried under vacuum to
yield clean product. Crystals were grown from acetonitrile. Yield:
1.25 g, 4.39 mmol, 87.7%. mp: 85–86 °C. Anal. Calc. for
C
30H30Ag2Cl4N4O8, M = 932.12, monoclinic, a = 47.430(20) Å,
b = 12.106(5) Å, c = 29.818(12) Å, = 90.00°, b = 123.715(7)°,
= 90.00°, V = 14242(10) Å3, T = 100(2) K, space group C2/c, Z = 16,
(Mo K
) = 1.453 mmÀ1, 60807 reflections measured, 16796 inde-
a
c
l
a
C
12H10N2O2Cl2: C, 50.55; H, 3.54; N, 9.82. Found: C, 50.50; H,
pendent reflections (Rint = 0.1181). The final R1 values were
3.53; N, 9.72%. ES-MS (m/z): calc., 307.0017 [M+Na]+; found,
306.9994. 1H NMR (500 MHz, d6-DMSO): d 3.85 (s, 3H, OCH3),
5.34 (s, 2H, NCH2C), 7.51 (d, J = 9.00 Hz, 1H, CHbenzyl), 7.55 (t,
J = 7.30 Hz, 1H, CHbenzyl), 7.83 (s, 1H, CHbenzyl), 7.90 (d, J = 7.58 Hz,
1H, CHbenzyl), 8.03 (s, 1H, NCHN). 13C NMR (500 MHz, d6-DMSO): d
49.0 (NCH2), 52.7 (OCH3), 112.8 (CCl), 125.3 (NCHN), 128.3 (Cbenzyl),
129.3 (Cbenzyl), 129.9 (Cbenzyl), 131.0 (Cbenzyl), 132.6 (Cbenzyl), 137.0
(Cbenzyl), and 166.3 (COOCH3) ppm. Crystal data for 4:
0.0770 (I > 2r r(I)).
(I)). The final wR(F2) values were 0.1553 (I > 2
The final R1 values were 0.1501 (all data). The final wR(F2) values
were 0.1862 (all data). The goodness of fit on F2 was 1.013.
2.3. MIC Studies
Single colonies of Pseudomonas aeruginosa, Staphylococcus aur-
eus, and Escherichia coli were isolated from LB Agar plates and were
diluted in 5.0 mL LB broth. The solutions were incubated at 37 °C
for 24 h to yield bacteria solutions. Compounds 2, 3, 5, 6, and silver
nitrate (0.01 mmol) were each dissolved separately in 4 mL of
DMSO and diluted to 10 mL with LB broth. LB broth (2 mL) was
added to each of six tubes. Serial dilutions were done for each com-
pound by adding 1 mL of the dissolved compound to the first tube,
taking 1 mL from tube 1 and adding to tube 2 and so on for all 6
tubes. Prepared bacteria solution (0.150 mL) was added to each
tube and tubes were incubated at 37 °C for 24 h in a shaking incu-
bator at 140 rpm. Bacteria growth was noted by turbidity of the
solution in the tubes and MIC was determined by the lowest con-
centration lacking turbidity.
C
12H10Cl2N2O2, M = 285.12, triclinic, a = 7.836(4) Å, b = 9.003(5) Å,
c = 9.383(5) Å, = 68.826(8)°, b = 84.052(9)°, = 81.241(8)°,
V = 609.2(6) Å3, T = 100(2) K, space group P1, Z = 2,
(Mo
) = 0.527 mmÀ1, 4879 reflections measured, 2438 independent
reflections (Rint = 0.0238). The final R1 values were 0.0367
a
c
ꢀ
l
K
a
(I > 2r r(I)). The final
(I)). The final wR(F2) values were 0.0989 (I > 2
R1 values were 0.0414 (all data). The final wR(F2) values were
0.1036 (all data). The goodness of fit on F2 was 1.054.
Synthesis of 5. Compound 4 (1.20 mmol, 0.342 g) was placed in
a 100 mL round bottom flask and dissolved in 25 mL acetonitrile.
Methyl iodide (1.0 mL) was added and the mixture was stirred
for 24 h at reflux. The volatiles were removed by rotary evapora-
tion to yield a yellow solid. The yellow solid was washed with ace-
tone and the off-white precipitate was filtered and collected.
Crystals were grown from acetonitrile. Yield: 0.326 g, 0.750 mmol,
62.5%. mp: 159–160 °C. Anal. Calc. for C13H13N2O2Cl2I: C, 36.56; H,
3.07; N, 6.56. Found: C, 36.45; H, 3.11; N, 6.41%. ES-MS (m/z): calc.,
299.0 [MÀI]+; found, 299.0. 1H NMR (500 MHz, d6-DMSO): d 3.84
(s, 3H, OCH3), 3.87 (s, 3H, NCH3), 5.60 (s, 2H, NCH2C), 7.61 (t,
J = 7.83 Hz, 1H, CHbenzyl), 7.69 (d, J = 7.83 Hz, 1H, CHbenzyl), 8.00
(d, J = 7.58 Hz, 1H, CHbenzyl), 8.08 (s, 1H, CHbenzyl), 9.45 (s, 1H,
3. Results and discussion
3.1. Synthesis and characterization of 3
The synthesis of the theobromine derivative 3 was developed
by adapting synthetic procedures previously established in our
group based on the electronics of the theobromine building block
[16]. The N1 nitrogen in theobromine (Fig. 1a) is a good nucleophile
NCHN). 13C NMR (500 MHz, d6-DMSO):
d 35.4 (NCH3), 51.4

128
A.R. Knapp et al. / Inorganica Chimica Acta 364 (2010) 125–131
by filtration. The product 1 was isolated and N9 was methylated
with excess CH3I. This imidazolium cation served as the carbene
precursor for the synthesis of 3, which was accomplished by the
addition of two equivalents of silver acetate. The first equivalent
of silver acetate produces silver iodide and serves as the base to
deprotonate C8 producing acetic acid and forming the carbene
in situ. The second equivalent of silver acetate then reacts with
the carbene to form 3.
Fig. 1. Atom labeling of (a) theobromine and (b) 4,5-dichloroimidazole.
The formation of each product was monitored by 1H and 13C
NMR. The hydrogen on the N1 of theobromine has a 1H NMR signal
at 11.12 ppm. After the reaction with methyl-3-bro-
momethylbenzoate, the signal is no longer present indicating that
the substituent was attached. The signal for the C8 proton can also
be monitored to assure that the substituent was not attached to N9.
The theobromine proton signal for C8 is at 7.98 ppm. After the reac-
tion with methyl-3-bromomethylbenzoate the signal only shifts
slightly to 8.04 ppm, indicating that the reaction to form the imi-
dazolium cation at N9 has not occurred. Once the methylation of
1 has taken place, we see a shift in the signal for the proton on
C8 from 8.04 to 9.34 ppm. This downfield shift is indicative of the
imidazolium cation formation. Finally, the reaction of 2 with
AgOAc causes the dissipation of the signal at 9.34 ppm and the
appearance of a signal for the acetate methyl group at 1.85 ppm.
The formation of 3 can further be confirmed by 13C NMR. The signal
for C8 of 2 can be found at 140.4 ppm. After reaction with AgOAc
the signal at 140.4 disappears and one appears further downfield
at 186.5 ppm, a shift indicative of the formation of the carbene. A
signal corresponding to the carbonyl carbon of the silver acetate
portion can also be observed at 174.3 ppm. Further analysis of
the products by elemental analysis and mass spectrometry con-
firmed the presence of compounds 1, 2, and 3. Crystals suitable
for X-ray crystallography were grown of all the compounds and
their X-ray structure was determined as another method of confir-
mation (Figs. 3–5). The asymmetric units of compounds 2 and 3
show a molecule of water present in the crystal. This could be
due to exposure to water in the atmosphere when growing the
crystals or synthesizing the compounds. The Ag–C bond length in
compound 3 is 2.023(6) Å and the Ag–O bond length was found
to be 2.064(4) Å. The C–Ag–O bond angle of compound 3 only devi-
ates slightly from linearity at 178.2(2) Å. Both the bond lengths and
bond angles are consistent with xanthene based silver carbene
complexes [16].
Fig. 2. Xanthene derivatives (a) 7 and (b) 8.
when it is deprotonated making it a possible site for attachment of
different substituents. The nucleophilicity of the N1 nitrogen is
much greater than that of the N9 making it more likely to obtain
single substituted products. Previous studies have taken advantage
of this property where iodoethanol was reacted with theobromine
to yield a hydrophilic silver complex 7 (Fig. 2a). This complex has
more water solubility than the methylated caffeine system 8 and
has been shown to have antimicrobial properties against biosafety
level 3 bacteria (Fig. 2b) [16].
The synthesis of 3 was completed as shown in Scheme 1. The
first step of the synthesis is the deprotonation of the N1 nitrogen
with potassium carbonate in DMF followed by the addition of
methyl-3-bromomethylbenzoate. The deprotonation produces
KBr as a byproduct that falls out of solution and can be removed
Scheme 1. Synthesis of 3 from theobromine.

A.R. Knapp et al. / Inorganica Chimica Acta 364 (2010) 125–131
129
(Fig. 1b). The electronic properties create a more stable silver com-
plex by removing electron density from the carbene carbon,
strengthening the silver–carbon bond in the complex. Due to the
proven stability of these complexes, the methyl-3-methylbenzoate
derivative was also synthesized. Synthesis of 6 was done by adapt-
ing previously reported procedures (Scheme 2) [12,15]. 4,5-Dichlo-
roimidazole was deprotonated with KOH and methyl-3-
bromomethylbenzoate was added. The KBr byproduct was re-
moved by filtration and compound 4 was isolated. This product
was methylated with CH3I to obtain the imidazolium cation 5
and two equivalents of AgOAc were added to afford 6 in 87.7%
yield.
Fig. 3. Crystal structure of 1 with thermal ellipsoids drawn at 50%.
3.2. Synthesis and characterization of 6
One of the objectives in making different silver carbene com-
plexes is finding ones that have enhanced stability in biological
conditions. In an effort to accomplish this purpose, 4,5-dichloroim-
idazole was used as a starting material for several SCCs. The advan-
tage of this precursor compared to others is the stability of its
complexes in water and NaCl solutions. It has been demonstrated
that compared to SCCs synthesized from 1-methylimidazole, those
synthesized from 4,5-dichloroimidazole show increased stability
due to the electron withdrawing chlorides in the 4 and 5 positions
Scheme 2. Synthesis of 6 from 4,5-dichloroimidazole.
Fig. 4. Crystal structure of the cation of 2 with thermal ellipsoids drawn at 50%.
Fig. 5. Crystal structure of 3 with thermal ellipsoids drawn at 50%.

130
A.R. Knapp et al. / Inorganica Chimica Acta 364 (2010) 125–131
Fig. 6. Crystal structure of 4 with thermal ellipsoids drawn at 50%.
Table 2
MIC data (lg/mL and nmol/mL) for 3 and 6 and their corresponding imidazolium
cations against bacteria strains.
Just as with 3, the formation of the products was monitored by
1H and 13C NMR. The hydrogen on N1 of 4,5-dichloroimidazole
gives a signal at 13.33 ppm. After the addition of methyl-3-bro-
momethylbenzoate, that signal is no longer present indicating
the complete transformation to compound 4. The signal for the
proton on C2 shifts slightly from 7.70 to 8.03 ppm. After methyla-
tion, the proton on C2 shifts downfield from 8.03 to 9.45 ppm,
indicative of imidazolium cation formation. The reaction of the cat-
ion with silver acetate causes the disappearance of the C2 proton
peak and the resonance at 1.79 ppm appears representative of
the acetate methyl group. The formation of 6 was also confirmed
by elemental analysis, mass spectrometry, and X-ray crystallogra-
phy (Figs. 6–8). Crystals suitable for X-ray crystallography were
MIC for bacterial strains
Compound
P. Aerugionosa
S. aureus
g/mL
E. coli
g/mL
l
g/mL
nmol/mL
l
nmol/mL
l
nmol/mL
2
3
5
6
>235
64.0
>215
59.0
42.5
>500
126
>503
136
>235
64.0
>215
59.0
42.5
>500
126
>503
136
>235
64.0
>215
59.0
42.5
>500
126
>503
136
AgNO3
250
250
250
grown from concentrated solvents. Just as with compounds 2 and
3, compounds 5 and 6 both have water in the asymmetric unit of
the structure. This could be due to water absorbed from the
atmosphere when growing the crystals or doing the reaction. The
asymmetric unit of compound 6 also contained four molecules that
are interacting through the silver on the silver acetate. It is possible
that this could be due to the desire of silver to be 4 coordinate and
the interaction with another soft silver atom makes it more stable
in crystalline form. Similar to compound 3, the bond length of Ag–C
in 6 was 2.060(8) Å. However, the Ag–O length of 6 was signifi-
cantly larger at 2.127(7) Å than the Ag–O bond length of compound
3 (2.064(4) Å). The C–Ag–O bond angle in 6 is slightly less than lin-
ear at 177.9(3) Å.
Fig. 7. Crystal structure of 5 with thermal ellipsoids drawn at 50%.
3.3. Antimicrobial studies
An important characteristic of our SCCs is their antimicrobial
properties. In order to determine the efficacy of the complexes,
minimum inhibitory concentrations (MICs) are determined. The
MIC is the minimum concentration of compound necessary to inhi-
bit the growth of a bacterial strain. A preliminary study of silver
complexes 3 and 6, and of the corresponding imidazolium cation,
was done to determine their MIC. The results of these compounds
were compared to that of silver nitrate which was used as a posi-
tive control based on its established antimicrobial properties [24].
The compounds were incubated for 24 h with the non-virulent
strains of P. aeruginosa, S. aureus, and E. coli in growth media.
The imidazolium cations 2 and 5 showed no antimicrobial activity
at the concentrations tested (Table 2). Although there is evidence
of imidazolium cations that have shown antimicrobial activity
[25,26], the results from the study of 2 and 5 is consistent with
studies on other SCCs synthesized in our research group in which
no antimicrobial activity is observed [12,15,16]. The silver com-
plexes, however, showed inhibition of bacterial growth with MICs
of 64.0
and 6, respectively. These values were comparable to the MIC for
silver nitrate (42.5 g/mL, 250 nmol/mL), indicating that these
lg/mL (126 nmol/mL) and 59.0 lg/mL (136 nmol/mL) for 3
l
new silver complexes are just as effective. Further studies are nec-
Fig. 8. Crystal structure of 6 with thermal ellipsoids drawn at 50%.
essary to determine the efficacy of these compounds compared to

A.R. Knapp et al. / Inorganica Chimica Acta 364 (2010) 125–131
131
other SCCs and their effectiveness against more virulent strains,
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Acknowledgments
This work was supported by The University of Akron, The
University of Akron Center for Silver Therapeutics Research, the
National Institute of Allergies and Infectious Diseases (1 R01
A106785601) and The National Institute of General Medical Sci-
ences (1 R01 GM86895-01). We wish to thank the Ohio Board of
Regents and the National Science Foundation for funds used to pur-
chase 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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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ica.2010.08.008.