Chem. Rev.

2009, 109, 3859–3884 3859

The Medicinal Applications of Imidazolium Carbene-Metal Complexes

Khadijah M. Hindi,† Matthew J. Panzner,† Claire A. Tessier,† Carolyn L. Cannon,‡ and Wiley J. Youngs*,†
Department of Chemistry, The University of Akron, Akron, Ohio 44325-3601, and Department of Pediatrics and Molecular Microbiology and Microbial
Pathogenesis, Washington University School of Medicine, St. Louis, Missouri 63110

Received October 27, 2008

Contents 5.2. Synthesis and Antimicrobial Properties of 3880

Rhodium- and Ruthenium-NHC Complexes
1. Introduction 3859 6. Palladium 3881
2. Imidazolium Salts as Antimicrobials 3861 6.1. Medicinal Uses of Palladium Compounds 3881
3. Silver 3862 6.2. Synthesis and Antitumor Properties of 3882
3.1. Medical Uses of Silver Compounds 3862 Palladium-NHC Complexes
3.1.1. Mechanism of Silver Activity 3862 6.2.1. Mechanism of Palladium-NHC Complex 3882
3.1.2. Toxicity of Silver 3863 Activity
3.1.3. Resistance to Silver 3863 7. Conclusion 3882
3.2. Antimicrobial Properties of Silver-NHC 3864 8. Acknowledgments 3883
Complexes 9. Note Added after ASAP Publication 3883
3.2.1. Synthesis and Antimicrobial Properties of 3864 10. References 3883
Pyridine-Linked Pincer Silver-NHC
Complexes 1. Introduction
3.2.2. Synthesis and Antimicrobial Properties of 3865
Silver-NHC Complexes Derived from Öfele and Wanzlick reported the synthesis of the first
Xanthines N-heterocyclic carbene (NHC)-metal complexes in 1968.1,2
3.2.3. Synthesis and Antimicrobial Properties of 3867 The isolation of the first free carbene by Arduengo in 1991
Silver-NHC Complexes Derived from set the scene for an ever-growing interest and advancement
4,5-Dichloroimidazole in the field of N-heterocyclic carbene chemistry.3 Shortly
3.2.4. Synthesis and Antimicrobial Properties of 3867 thereafter, the use of these ligands in organometallic chem-
Silver-NHC Complexes Derived from istry, particularly in catalysis, dramatically increased.4,5
1-Benzyl-3-tert-butylimidazole N-heterocyclic carbenes are neutral two-electron donors
3.3. Antitumor Properties of Silver-NHC 3869 with an ability to bond to both hard and soft metals making
Complexes them more versatile ligands than phosphines.6 As an added
3.3.1. Synthesis and Antitumor Properties of 3869 advantage, not only are NHCs easier to synthesize and
Silver-NHC Complexes Derived from functionalize than phosphines but they also form a stronger
4,5-Dichloroimidazole bond to metals and therefore form more stable metal
4. Gold 3869
4.1. Medical Uses of Gold Compounds 3869
4.1.1. Mechanism of Gold Activity 3872
4.1.2. Toxicity of Gold 3873
4.2. Antimicrobial Properties of Gold-NHC 3873
Complexes
4.2.1. Synthesis and Antimicrobial Properties of 3873
Gold-NHC Complexes Derived from
1,3-Diorganylimidazolidin-2-ylidenes
4.2.2. Synthesis and Antimicrobial Properties of 3874
Gold-NHC Complexes Derived from
1-Benzyl-3-tert-butylimidazole
4.3. Synthesis, Antimitochondrial, and Antitumor 3874
Properties of Gold-NHC Complexes
5. Ruthenium and Rhodium 3877
5.1. Medical Uses of Ruthenium and Rhodium 3877 Khadijah Hindi was born in Amman, Jordan. She received a B.S. with
Compounds honors in chemistry from the University of Akron in the fall of 2003. She
5.1.1. Mechanisms of Ruthenium and Rhodium 3878 earned a Ph.D. degree in the summer of 2008 at the University of Akron,
Activity under the direction of Professor Wiley J. Youngs, focusing on the synthesis
and study of silver N-heterocyclic carbene complexes, including their
* To whom correspondence should be addressed. Phone: (330) 972-5362.
application as antitumor agents, as well as antimicrobials active against
E-mail: [email protected]. a wide array of Gram-negative and Gram-positive bacteria. Currently, she
†
The University of Akron. is a Postdoctoral Fellow with Dr. Carolyn Cannon at Washington University
‡
Washington University School of Medicine. School of Medicine in St. Louis, MO.

10.1021/cr800500u CCC: $71.50  2009 American Chemical Society

Published on Web 07/06/2009
3860 Chemical Reviews, 2009, Vol. 109, No. 8 Hindi et al.

Matthew Panzner grew up in Copley Township several miles west of Akron, Carolyn Cannon is a native Texan, who graduated from Texas A&M in
Ohio. He received his B.S. in chemistry from the University of Akron in 1982 with a B.S. in bioengeneering. She received a M.S. in electrical
2001. He subsequently entered graduate school and received his Ph.D. engineering from Worcester Polytechnic Institute in Massachusetts in 1985
under the guidance of Wiley Youngs in 2006. During his time in the Youngs before returning to Texas to pursue a medical deqree and further graduate
laboratory, he studied the synthesis of N-heterocyclic carbene metal training. She obtained her medical degree from the University of Texas
complexes with an emphasis on silver. He currently works as a Project Medical School at Houston and a Ph.D. in Physiology and Cell Biology
Manager for Wiley Youngs focusing on the advancement of silver carbene from the associated Graduate School of Biomedical Sciences, both in
antimicrobials and the synthesis and study of new inorganic backbone 1993. She was a pediatric resident at the Children’s Hospital in Boston
polymer fuel cell membrane materials. and continued training as a pediatric pulmonary fellow, until Dr. Cannon
subsequently joined the faculty a Harvard Medical School in 1999. She
held a joint appointment as a Research Fellow at The Channing Laboratory
where she completed postdoctoral training. In 2003, Dr. Cannon joined
the faculty at Washington University in St. Louis and acts as the Co-
Director of the Cystic Fibrosis Center at St. Louis Children’s Hospital. Dr.
Cannon’s laboratory has focused on the pathogenesis of infection in cystic
fibrosis, as well as the development of the silver-carbene antimicrobials
synthesized in Dr. Youngs’ laboratory to treat these infections. Thus, the
efforts in the laboratory center on the confluence of molecular microbiology,
microbial pathogenesis, drug discovery, pharmocology, toxicology, and
respiratory cell biology.

Claire Tessier began her scientific career as an ACS Project SEED student
in the laboratory of Klaus Wulff at the University of Vermont. She continued
her studies at the University of Vermont and received a B.S. in chemistry
in 1975. In 1982, she earned a Ph.D. degree, under the direction of O. T.
Beachley, Jr., from the State University of New York at Buffalo. In
1981-1982, she completed a postdoctoral position with D. F. Shriver at
Northwestern University. She began her current position as a professor
at the University of Akron in 1990. Her research interests include
phosphorus and silicon-based inorganic-backbone polymers, biomineral-
ization, and metal-NHC chemistries.

complexes than metal-phosphine complexes.7,8 The N- Wiley Youngs is originally from northern New York State. He received a
heterocyclic carbene ligands interact with metal centers B.A. in Psychology from the State University of New York at Albany and
primarily through strong σ-donation and to a lesser degree returned to school to further pursue his undergraduate education in
chemistry at SUNY Potsdam and Clarkson University. He received his
through π-back-donation (Figure 1).9,10 Ph.D. in 1980 under the supervision of Melvyn Churchill at SUNY Buffalo.
Ghosh and co-workers11-16 as well as others17-19 took Following a postdoctoral fellowship with James Ibers at Northwestern
special interest in the exceptional stability of several University, he joined the faculty at Case Western Reserve University in
metal-NHC complexes and conducted in depth analyses in 1983. In 1990, he moved to the University of Akron where he is currently
order to gain better insights into the structure and bonding. a Full Professor of Chemistry. Dr. Youngs’ research interests include
In particular, the metal-ligand donor-acceptor interactions N-heterocyclic carbenes, silver-based antimicrobials, metal-based cancer
drugs, and fuel cell membrane materials.
were inspected using charge decomposition analysis (CDA).
CDA is a tool used to quantitatively estimate the degree of donation was observed in Pd-NHC complexes exhibiting
NHC f metal σ-donation, designated by d, and NHC r lower d/b ratios ranging between 2.59 and 3.9913,14 and
metal π-back-donation, designated by b.20,21 Thus a higher Au-NHC complexes with d/b ratios ranging between 5.23
d/b ratio emphasizes the ability of NHC to function as an and 5.8815,16 compared with the Ag-NHC complexes with
effective σ-donor, whereas a lower d/b ratio highlights the d/b ratios ranging between 7.8 and 12.68.11,12,16 This observa-
greater NHC r metal π-back-donation. Interestingly, in the tion could attest to why silver-NHC complexes are par-
studies conducted by Ghosh, greater NHC r metal π-back- ticularly better transmetalating agents.
Medicinal Imidazolium Carbene-Metal Complexes Chemical Reviews, 2009, Vol. 109, No. 8 3861

Chart 1. Structure of 3-Alkylthiomethyl-1-ethylimidazolium

Chlorides 1a-f22 and 3-Alkoxymethyl-1-methylimidazolium
salts 2a-l, 3a-l, and 4a-l24

Figure 1. Orbital diagram of NHC bonding to metal center.

The newly emerging interest in the medicinal applications

of stable metal-NHCs led us to examine the few accounts
available in the literature dealing with this area of research.
This review will discuss in detail the medicinal applications
of various transition metal-NHC complexes including silver,
gold, rhodium, ruthenium, and palladium. The antimicrobial,
antitumor, and resistance properties, along with proposed values were determined against the bacterial strains Micro-
mechanisms of action to suppress the bacterial growth or coccus luteus, Staph. epidermidis, Staph. aureus, methicillin-
proliferation of tumor cells, will be discussed. resistant Staph. aureus (MRSA), Entercoccus hirae, Escher-
ichia coli, Proteus Vulgaris, K. pneumoniae, and
Pseudomonas aeruginosa, and the fungal strains Candida
2. Imidazolium Salts as Antimicrobials albicans and Rhodotorula rubra (results not shown). The
Before the evaluation of N-heterocyclic carbene-metal terms bactericidal and fungicidal indicate that the test
complexes as metallopharmaceuticals, an overview of the compound kills the tested bacteria and fungi, respectively.
medicinal applications of imidazolium salt precursors is Although it was shown that the salts did possess antibacte-
appropriate. Imidazolium salts are often used as precursors rial and antifungal activities, their efficacy depended greatly
to metal-NHC complexes and are commonly one of the on the length of the alkyl chain. The type of anion did not
byproducts produced from the degradation of NHC-metal exert any notable effect on the activity of the salts. The most
complexes. After a thorough search of the literature, it was active compounds against most bacterial strains were 2-4h,
evident that only a handful of groups have investigated the 2-4i, 2-4j, and 2-4k, bearing 10, 11, 12, and 14 carbon
antimicrobial and antifungal properties of imidazolium salts atoms, respectively, with the dodecyloxymethyl group as the
in relation to various N-substituents. Pernak and co-workers most effective with MIC values ranging between 25 and 395
examined a number of 3-alkylthiomethyl-1-ethylimidazolium µM. This range equates to approximately 7.9-125 µg/mL.
chlorides 1a-f (Chart 1).22 The salts were synthesized by There was a general increase in the activity with increasing
treating 1-ethylimidazole with the appropriate chloromethy- chain length against the fungal strains tested.
lalkyl sulfide in refluxing anhydrous benzene for 4 h. The Additional studies done by Cetinkaya verified the same
products were isolated by extraction with hot hexanes trends as observed earlier.25 They explored the effects of
affording hydroscopic oils with yields of 90%. lipophilic substituents, anions, and backbone substituents on
The antimicrobial activity was reported in terms of the the antimicrobial activity associated with a series of 1,3-
minimum inhibitory concentration (MIC) values, which are diazolidinium salts (5) and the effect of ring size of
defined as the lowest concentration of an antimicrobial that pyrimidinium salts (6) as a comparison (Chart 2). The
visibly inhibits the growth of the bacteria after an overnight complexes were tested against a number of bacterial organ-
incubation.23 The antimicrobial activity was evaluated against isms, including E. coli, Staph. epidermidis, Staph. aureus,
Staphylococcus aureus, Gaffkya tetragena, Sarcina lutea, Enterococcus faecalis, Enterobacter cloacae, and P. aerugi-
Klebsiella pneumoniae, Serratia, marcescens, Rhodothorula nosa, and C. albicans and were compared with ampicillin, a
glutinis, and Bacillus subtilis. The results are summarized β-lactam antibiotic used to treat general bacterial infections.
in Table 1. The most notable feature observed among 30 derivatives
The antimicrobial activity was determined in relation to synthesized is the remarkable enhancement of the antimi-
the hydrophobicity of the imidazolium salts bearing the crobial activity against Gram-negative and Gram-positive
different alkyl substituents. It was found that the activity was bacteria of three derivatives of 5 by the addition of two
related to the length of the alkyl chain with 1d and 1e mesityl or two mesitylmethyl substituents on both nitrogen
showing the best response relative to their analogues. The atoms (bulky and lipophilic groups). Variation of the
MIC values in terms of micrograms per milliliters equate to counterion of the imidazolidinium salt of 5, with R1 and R3
1.13-11.9 µg/mL for 1d and 0.69-46 µg/mL for 1e. It is as mesitylmethyl, greatly affected its antimicrobial activity.
important to point out that the results in Table 1 indicate The mesitylmethyl derivative of 5 showed more effective-
that alkyl chain substituents adversely influence the antimi- ness, especially with X- ) Cl- than its counterparts with
crobial activity of the imidazolium salts if they are too short MIC values of 3.12 µg/mL against Staph. aureus, Ec.
or too long. faecalis, and P. aeruginosa and 6.25 µg/mL against Staph.
Further, Pernak investigated a series of 3-alkoxymethyl- epidermidis. The same derivative completely lost its activity
1-methylimidazolium salts (Chart 1) with the effects of three when X- was changed to PF6- or BF4-. Furthermore,
different anions, Cl-, BF4-, and PF6-.24 Twelve different variations to the backbone such as substitutions from
complexes were made for each anion, 2a-l (Cl-), 3a-l hydrogens to methyl, cyclohexyl, or benzene groups were
(BF4-), and 4a-l (PF6-) (Table 2). The MIC and the determined to decrease the activity of the NHC salts. Lastly,
minimum bactericidal or fungicidal concentration (MBC) the antimicrobial activity of several derivatives of 5 was
3862 Chemical Reviews, 2009, Vol. 109, No. 8 Hindi et al.

Table 1. The Minimum Inhibitory Concentration log(1/MIC) for Complexes 1a-f22

log(1/MIC)a
compound Staph. aureus G. tetragena Sa. lutea K. pneumoniae Se. marcescens R. glutinis B. Subtilis
1a 4.05 3.50 3.52 2.82 2.20 3.85 3.35
1b 4.35 4.35 3.75 3.75 2.84 4.05 4.05
1c 4.69 5.05 4.69 4.39 3.79 5.00 4.39
1d 5.12 5.45 5.12 4.99 5.12 5.12 4.43
1e 5.08 5.70 5.39 5.08 3.87 5.08 4.47
1f 4.83 5.27 5.13 4.75 3.95 4.93 4.49
a
MIC results are based on 24 h incubation @ 37 °C. Values are expressed as mol/L.

Table 2. 3-Alkoxymethyl-1-methylimidazolium Salts 2-424

Cl- BF4- PF6-
no. R no. R no. R
2a C3H7 3a C3H7 4a C3H7 Figure 2. Structure of silver sulfadiazine.
2b C4H9 3b C4H9 4b C4H9
2c C5H11 3c C5H11 4c C5H11 The antimicrobial efficacy of the compounds was deter-
2d C6H13 3d C6H13 4d C6H13 mined against Bac. subtillis, Staph. aureus, resistant Staph.
2e C7H15 3e C7H15 4e C7H15 aureus, E. coli, and Salmonella typhimurium bacterial strains
2f C8H17 3f C8H17 4f C8H17
2g C9H19 3g C9H19 4g C9H19 plus C. albicans fungal strain. The preferred carbon chain
2h C10H21 3h C10H21 4h C10H21 lengths that produced the most efficient antimicrobial activity
2i C11H23 3i C11H23 4i C11H23 are 7c-f with MIC values ranging between 4 and 8 µg/mL
2j C12H25 3j C12H25 4j C12H25 for most of the bacterial strains tested. Generally, as can be
2k C14H29 3k C14H29 4k C14H29
2l C16H33 3l C16H33 4l C16H33
deduced from the various experiments that were described
above, hydrophobic alkyl substituents such as long alkyl
chains or sterically bulky groups have demonstrated their
Chart 2. Structures of 1,3-Diazolidinium Salts (5) and enhancement of the antimicrobial activity of imidazolium
Pyrimidinium Salts (6)25
salts.

3. Silver
3.1. Medical Uses of Silver Compounds
Silver has been recognized as an effective antimicrobial
Chart 3. Structures of 1-Alkyl-2-methylimidazolium agent, specifically in the form of silver nitrate since the 17th
Complexes 7a-7f26 and 18th centuries.27 The popularity of silver nitrate led to
its utilization as a treatment of chronic skin ulcers, open
wounds, and suppurating wounds well into the early 19th
century.28 Soon after the astringent properties of silver against
a wide range of bacteria were recognized, a German
obstetrician, C.F. Crede, introduced a prophylactic 2% silver
nitrate eye solution to prevent ophthalmia neonatorum in
newborns in 1880.27 The emergence of penicillin and other
antibiotics after World War II led to the abandonment of
silver-based antimicrobials.27 Shortly thereafter, resistant
organisms such as P. aeruginosa, Proteus mirabilis, and
Proteus morgani surfaced and led to the revival of silver
directly compared with that of 6 with identical substituents, nitrate by Moyer in 1965.29 As a result, silver sulfadiazine
and it was established that the ring size plays a crucial role. (Figure 2) was introduced by Fox in 1968,30 and it remains
The imidazolidinium salts were much more active than the one of the most effective and widely used topical burn
pyrimidinium salts. The mechanism of activity of these treatments.31-33
complexes has not been studied, but it is thought that
lipophilic side chains can disrupt intermolecular interactions 3.1.1. Mechanism of Silver Activity
and thus cause the dissociation of cellular membrane bilayers
of the bacterial cell, which compromises cellular permeability Although the cytotoxic effects of silver against Gram-
positive and Gram-negative bacteria have long been estab-
and induces leakage of cellular contents.26
lished, the mechanisms of action are not completely under-
More recently Huen Lee and his group synthesized a series stood. Sporadic studies of the cell toxicity mechanisms of
of quaternary imidazolium salts (7a-f) among others and silver suggest that silver ions kill organisms through a variety
tested their antimicrobial activity.26 The general structure is of ways. Two excellent reviews by Lansdown and Hugo
shown in Chart 3. The salts were obtained by the deproto- discuss the antimicrobial properties and the mechanism of
nation of the imidazole starting material with sodium or action of silver.34,35
sodium ethanoate and the subsequent alkylation with the Briefly, studies done by Feng and co-workers suggest that
appropriate alkyl bromide or chloride in refluxing methanol treatment of E. coli and Staph. aureus with Ag+ causes
or acetonitrile. significant morphological changes in the bacterial cells as
Medicinal Imidazolium Carbene-Metal Complexes Chemical Reviews, 2009, Vol. 109, No. 8 3863

Scheme 1. Synthesis of 8a, 8b, 9a, and 9b63

observed by transmission electron microscopy (TEM) and polymer protein solution. Also, the use of antismoking tablets
X-ray microanalysis.36 Silver-treated cells exhibited an and gum, which contain silver acetate, have been reported
electron-light region in their cytoplasm with condensed DNA to cause generalized argyria.43,44 Several reports of localized
molecules. Condensed DNA molecules lose their ability to and generalized argyria surfaced after the use of topical
replicate. Electron-dense granules were detected around the solutions of silver nitrate45 and silver sulfadiazine cream.43
cell wall inside the cytoplasm, and the cytoplasmic membrane It is important to note that argyria is rarely the cause of any
experienced shrinkage and detachment from the cell wall ill effect or death and that there are no effective treatments
thereafter. Furthermore, X-ray microanalysis of the electron- for removing silver deposits from the body.46 This can be
dense granules revealed the presence of silver and sulfur. explained by the fact that silver sulfide is highly insoluble
This was an indication that silver ions interact with thiol with a Ksp of 10-50.
groups leading to the deactivation of enzymatic proteins. Besides argyria, other toxic effects of silver may include
Another proposed mechanism by Fox and Modak sug- upper and lower respiratory tract irritation. Based on some
gested that the silver moiety in silver sulfadiazine dissociated studies, these effects could be a result of the carrier molecule
from sulfadiazine and became bound to components within or anion such as the nitrate in some silver-based compounds
the cell. The subsequent inhibition of bacterial growth was rather than the silver itself.47 Other effects involve the
attributed to the amount of silver bound to bacterial DNA.37 accumulation and binding of silver ions to reduced glu-
The role of each component was determined using radioac- tathione in the liver and directing it to the bile.48 Reduction
tive silver sulfadiazine (110AgSD) and radioactive sulfadiazine in glutathione concentrations could be of concern considering
(35SD). It was established that the amount of SD entering its role in preventing damage to red blood cells by neutral-
the cell was not significant, and it is thus unlikely for the izing the toxic chemicals that enter the body. Although silver
bacterial inhibition to be attributed to the SD moiety of accumulation has been reported in a number of different
AgSD. Furthermore, based on an in ViVo model of Pseudomo- tissues and internal organs throughout the body, only a few
nas infected mice, silver seemed to function best if paired cases of silver toxicity have been presented. Some studies
with sulfadiazine. AgSD had superior activity over other reported that silver was toxic in isolated cells, such as
silver compounds perhaps due to the slow dissociation of lymphocytes,49 keratinocytes,50 hepatocytes,51,52 and fibro-
the complex and thus continual release of Ag+ over time.38 blasts53 by inhibition of proliferation. Similarly, cases involv-
In addition, several groups investigated the effect of Ag+ ing nephrotoxic syndrome54 and leucopenia55,56 have been
ions on the respiratory electron chain of E. coli.39-41 Their reported in patients with severe burns treated with silver
studies lead to the conclusion that Ag+ ions are highly toxic sulfadiazine. Other studies have shown that silver is virtually
to microorganisms due to inhibition of the respiratory chain nontoxic. The issue of silver toxicity is still under debate,
at multiple sites. but ultimately, silver is known to be one of the least toxic
metals.
3.1.2. Toxicity of Silver
3.1.3. Resistance to Silver
Although silver is generally nontoxic to humans, it is
widely known that a prolonged and excessive exposure to Silver is considered a broad-spectrum antibiotic. Silver,
silver causes the development of a rare and irreversible unlike conventional organic-based antibiotics, is active
pigmentation of the skin (argyria), the eyes (argyrosis), or against a wide range of Gram-positive and Gram-negative
both. This is a characteristic blue-black discoloration formed bacteria and targets multiple sites on or within the bacterial
by the interaction of silver ions with melanin and proteins cell. The multifaceted mode of action of Ag+ ions is a major
in the wound exudates to give silver sulfide.42 Argyria occurs contributor to the scarcity of reports of silver resistance
after the topical application of silver compounds to wounds despite its liberal use. It is worth mentioning however that
(localized argyria) or when silver is taken orally, injected silver resistance was reported as early as 1975 when McHugh
directly into the blood stream, inhaled, or applied to mucosal described the emergence of a Salmonella silver-resistant
surfaces (generalized argyria).43 In the case of the latter, silver strain, which caused the deaths of three patients at Mas-
is absorbed and carried to different parts of the body where sachusetts General Hospital burn unit.57 Since then, this area
it is deposited, most commonly, in the eyes, internal organs, has been intensely researched and the underlying molecular
and sun-exposed body parts such as hand, arms, face, nails, basis is presently much more established.58,59
etc.43 Silver and co-workers provided a detailed report of the
Several forms of silver are thought to cause argyria. genetic makeup of a 180 kb pMG101 plasmid isolated from
Extended use of colloidal silver proteins, which have been a silver-resistant Salmonella strain.60 The silver-resistant
used as allergy and cold medications and for a number of region within the plasmid was cloned and sequenced and
other ailments, can cause generalized argyria.43 Colloidal was found to contain nine genes, two of which have unknown
silver proteins are metallic silver particles suspended in a functions. The remaining seven genes are as follows: silE
3864 Chemical Reviews, 2009, Vol. 109, No. 8 Hindi et al.

Scheme 2. Synthesis of 10 and 1164

Figure 3. Molecular structure of 9a.63

Table 3. MIC of 9a, 9b, and AgNO363

E. coli P. aeruginosa Staph. aureus
Test
Ag (mg/mL) day 1 day 2 day 1 day 2 day 1 day 2
compd
9a 1186 - - - - - -
1DF - + - - - +
3.2.1. Synthesis and Antimicrobial Properties of
2DF - + - + + Pyridine-Linked Pincer Silver-NHC Complexes
3DF + + +
4DF + + + The first Ag(I)-NHC complexes possessing antimicrobial
9b 1125 - - - - - - activity against E. coli, Staph. aureus, and P. aeruginosa
1DF - + - + - + were reported by Youngs in 2004.63 The pincer ligands (8a,
2DF - + - + - 8b) were prepared by the reaction of 2,6-bis(imidazolem-
3DF + + + ethyl)pyridine with 2-iodoethanol or 3-bromopropanol, re-
4DF + + +
AgNO3 3176 - + - + + spectively. The corresponding silver complexes 9a and 9b
1DF + + + were readily obtained by the reaction of 8a and 8b with Ag2O
2DF + + + in aqueous methanol or water (Scheme 1).
3DF + + + The formation of (9a, 9b) was confirmed by the disap-
4DF + + + pearance of key resonance peaks at 9.13 ppm and 9.36 ppm
DF ) dilution factor; + ) growth; - ) no growth. associated with the imidazolium protons in the 1H NMR
spectra of 8a and 8b, respectively, and the appearance of a
encodes a 143-amino-acid, periplasmic, Ag+-specific metal- resonance at ca. 181 ppm in the 13C NMR spectra corre-
binding protein (SilE). This protein contains ten histidine sponding to the formation of a C-Ag bond. Furthermore,
residues that bind five Ag+ cations. Upstream of the silE the solid-state structure of 9a illustrates the formation of a
gene is the gene pair, silRS. This pair consists of a membrane one-dimensional polymer with a nearly linear geometry at
kinase sensor (SilS) and a regulatory responder protein the Ag atom with a bond angle of 174.7(4)° (Figure 3).
(SilR). This is followed by the silCBA genes, which The minimum inhibitory concentration (MIC) of 9a and
determine a three-polypeptide cation/proton exchange com- 9b against a panel of pathogens, E. coli, Staph. aureus, and
plex that is potential-driven. The components of this efflux P. aeruginosa, was determined. AgNO3 was used as a
system are SilA, which forms a pathway for Ag+ ions from reference, and the corresponding salts 8a and 8b served as
the cytoplasm to the outer membrane protein, SilC, and control. The results are summarized in Table 3. The silver
finally SilB, the membrane fusion protein whose function is complexes were dissolved in Luria broth (LB) culture
to maintain physical contact between SilA and SilC. Lastly, medium, upon which a precipitate formed, presumably AgCl,
SilP, the last member of the silver-resistant determinant is a and the solution was filtered. Serial dilutions of the silver
member of the heavy-metal resistance efflux P-type ATPase complex-LB broth mixtures were prepared, and a volume
family. The unique silver-resistant determinant described of 20 µL of freshly grown organisms was added daily.
above consists of two metal ion efflux pumps, SilCBA As shown in Table 3, 9a and 9b exhibited better bacte-
(the three-component cation/proton exchange complex) riostatic activity, even at much lower concentrations, com-
and SilP (the P-type ATPase), as well as SilE (the pared with AgNO3. This could be attributed to the fact that
periplasmic metal-binding protein). A more thorough the pincer ligands stabilize their corresponding silver com-
review of the molecular biology of bacterial silver plexes to a certain extent, thereby controlling the release of
resistance is provided by Silver.61 the Ag+ in the culture medium. AgNO3 on the other hand
could precipitate as the insoluble AgCl salt, thus reducing
3.2. Antimicrobial Properties of Silver-NHC the concentration of the biologically active Ag+ in the media.
In turn, the active Ag+ species in AgNO3 is eventually
Complexes consumed by the Cl- adduct, whereas the Ag+ species in
For a review on the recent progress in the synthetic both silver complexes 9a and 9b are released at a much
methods available for silver-NHC complexes, comprehen- slower rate and keep exerting their antimicrobial effects until
sive structural analysis, and a detailed look at the uses of consumed.
silver-NHCs as transfer agents, potential antimicrobials, and Another NHC cyclophane gem-diol salt was prepared by
catalysts, the reader is directed to the recent contribution by the reaction of 2,6-bis(imidazolemethyl)pyridine with 1,3-
Garrison and Youngs.62 dichloroacetone.64 The reaction appeared to proceed via an
Medicinal Imidazolium Carbene-Metal Complexes Chemical Reviews, 2009, Vol. 109, No. 8 3865

Scheme 3. Synthesis of 12 and 1366

Table 4. MIC of 1266

species name MIC (mg/mL)
P. aeruginosa 50
E. coli 50
Staph. aureus 100
Figure 4. Molecular Structure of 11.64 C. albicans 150
A. niger 75
Sac. cereVisiae 150

The electrospun fibers were obtained by creating an

electrically charged jet of solution containing the Techophilic
polymer/complex 11 mixture. This method can produce fiber
mats that range from a few nanometers to several microme-
ters in diameter.65 Furthermore, TEM was used to character-
ize the resulting fibers. It was found that exposing the silver
encapsulated fibers to a humid environment for up to 65 h
caused the slow decomposition of complex 11, which
released silver particles into the polymer matrix (Figure 5).
The antimicrobial properties of the silver encapsulated
polymer mat were evaluated against bacterial pathogens E.
coli, Staph. aureus, and P. aeruginosa. By comparing the
bactericidal activity of complex 11, AgNO3, and the 11/
Figure 5. TEM image of the encapsulated complex 11 in Techophilic fiber mat, it was revealed that after a 48 h
Tecophilic polymer fiber mat showing deposition of silver ions over incubation period, AgNO3 (MIC of 433 µg/mL) showed a
time: (a) electrospun fiber; (b) fiber after exposure to water vapor better antimicrobial activity than the unencapsulated complex
for 30 min; (c) fiber after exposure to water vapor for 65 h.64 11 (MIC of 838 µg/mL). However, the 11/Techophilic fiber
mat with the least amount of silver at 140 µg/mL showed
acid-catalyzed process to yield (10). A subsequent reaction sustained release of silver ions and thus enhanced antimi-
with Ag2O yielded the corresponding silver complex (11) crobial activity over a period of days. Furthermore, the
in good yield (Scheme 2). The formation of 11 was verified encapsulated silver NHC fiber mats exhibited a faster kill
using NMR spectroscopy, which confirmed the loss of the rate with lower silver content ([Ag+] ) 140 µg/mL or 424
imidazolium proton at 9.35 ppm in the 1H NMR and the µg/mL) compared with silver sulfadiazine with [Ag+] ) 3020
appearance of an apparent doublet in the 13C NMR spectrum µg/mL or 0.5% silver nitrate with [Ag+] ) 3176 µg/mL.
at 184-186 ppm. This resonance is presumably due to the This observation could be attributed to the greater surface
coupling between the carbene carbon of 11 and 109Ag and area provided by the polymer mats, through which active
107
Ag nuclei. Furthermore, the X-ray crystal structure shown silver species can be released. Ultimately the study showed
in Figure 4 confirmed the formation of 11. that the bioavailability of active silver species can be
In this report, a new method to ensure the slow release of increased through encapsulation of the silver-NHC into the
silver ions was employed. Complex 11 was encapsulated into polymer mat.
a medical grade polymer, Tecophilic, capable of absorbing
up to 150% of its dry weight in water content. This feature 3.2.2. Synthesis and Antimicrobial Properties of
is important for two reasons, both of which are essential for Silver-NHC Complexes Derived from Xanthines
optimal wound healing. First, in the event of applying this
silver encapsulated hydrophilic polymer to a wound site, the Further investigations of biologically active silver-NHC
water content will allow for the release of silver ions from complexes led to the synthesis of a modified caffeine silver
the polymer matrix. Second, the Techophilic will maintain acetate complex.66 This was of particular interest due to the
a moist environment at the wound site, thereby accelerating use of a xanthine derivative, caffeine, as a carrier molecule
the healing process. Besides its exceptional hydrophilic that has low toxicity and is readily available. Xanthines are
properties, Techophilic polymer is ethanol soluble, which biologically relevant molecules that have been used medici-
made it feasible to electrospin both the polymer and silver nally as diuretics and central nervous system stimulants and
complex 11 together to achieve a homogeneous mixing of to promote smooth muscle relaxation by inhibition of cyclic
the as-spun fiber. adenosine monophosphate (cAMP) phosphodiesterase.67
3866 Chemical Reviews, 2009, Vol. 109, No. 8 Hindi et al.

Figure 8. TEM of B. dolosa strain AU4459 of (a) normal cell

and (b) cell treated with complex 13.66

Scheme 4. Synthesis of 14 and 1571

Figure 6. Molecular structure of the cationic portion of 12.66

which the imidazolium proton appeared at 9.30 ppm, and

by 13C NMR, in which the imidazolium carbon appeared at
139.6 ppm. The crystal structure of 12 is shown in Figure 6.
The subsequent formation of the silver acetate complex
(13) was achieved via the in situ deprotonation of 12 with 2
Figure 7. Molecular structure of 13.66 mol equiv of silver acetate in methanol (Scheme 3). The
disappearance of the resonance at 139.6 ppm corresponding
Table 5. MIC of 1366 to the imidazolium carbon of 12 in the 13C NMR and the
MIC (µg/mL) appearance of a characteristic resonance at 186.2 ppm
species (genomovar) strain M-H LB corresponding to the carbene carbon atom further demon-
strated the formation of 13. The crystal structure of 13 is
P. aeruginosa 27853-ATCC 4 10
PAO1-V 4 4 shown in Figure 7.
PA M57-15 2 4 The antimicrobial activity of 12 against bacterial and
PA 2192 4 2 fungal strains was studied (Table 4). It is not surprising that
PA 6294 6 6 12 exhibited some antimicrobial properties because caffeine
PA N6 1 10 has been known to induce mutations in bacteria and fungi
PA JG3 1 8
PA N13 1 6 by binding to DNA and interfering with normal cell cycle
PA 1061 1 8 checkpoint functions.68-70 Additionally, preliminary toxicity
PA N8 1 6 studies on a small number of Sprague Dawley rats showed
PA 324 1 6 that 12 exhibited very low toxicity (1.068 g/kg) when
PA FRD1 1 10
B. cepacia (I) PC783 6 6
administered intravenously.
B. multiVorans (II) HI2229 6 10 The in Vitro antimicrobial activity of silver complex 13
AU8170 2 4 was evaluated in detail against a panel of highly resistant
AU5735 8 2 pathogens recovered from the respiratory tract of cystic
AU7484 6 2 fibrosis (CF) patients (Table 5). In addition, E. coli J53 strains
AU5248 2 2
B. cenocepacia (III) J2315 1 4 with and without the silver-resistant plasmid pMG101 were
HI2718 6 4 used as positive and negative controls in the MIC evaluation
ATTC BAA-245 1 4 of 13. The results demonstrated the efficacy of 13 against
B. stabilis (IV) ATTC BAA-67 1 1 E. coli J53 lacking the pMG101 plasmid and the resistance
HI2210 2 2 of J53+pMG101 to 13 with MICs of <1 µg/mL and >5 µg/
B. Vietnamiensis (V) PC259 4 4
B. dolosa (VI) AU0645 4 8 mL, respectively. The silver was the responsible moiety for
ATTC BAA-246 1 4 the antimicrobial activity of complex 13. Not only was
AU4459 6 4 complex 13 active against a number of bacterial strains, but
AU5404 6 6 it also showed a fungicidal effect on Aspergillus niger and
AU4881 1 4 Saccharomyces cereVisiae, with MIC values of 13 and 4 µg/
AU9248 1 2
AU4894 1 4 mL, respectively, and fungistatic activity against C. albicans
B. ambifaria (VII) HI2468 6 6 with a MIC value of 4 µg/mL.
B. anthina (VIII) AU1293 4 4 In an attempt to understand the antimicrobial mechanism,
B. pyrocinia (IX) BC11 4 4 a Burkholderia dolosa strain was treated with a 5 µg/mL
E. coli J53 1 4
J53 + pMG101 >5000 >5000
dose of complex 13 for 1 h at 37 °C. TEM was used to
evaluate the surface morphology of both the normal bacterial
cell (Figure 8a) and the treated cell (Figure 8b) in LB
The methylated caffeine derivative (12) was synthesized medium. The treated cell showed major damage, which was
by the reaction of caffeine with an excess of methyl iodide characterized by cell “ghosts” completely lacking cytoplasm.
in DMF (Scheme 3). The formation of the water-soluble and The small dark clusters observed in the image (Figure 8b)
air-stable imidazolium salt was confirmed by 1H NMR, in were thought to be silver salts incorporated into the
Medicinal Imidazolium Carbene-Metal Complexes Chemical Reviews, 2009, Vol. 109, No. 8 3867

and it was within the typical range of other NHC-Ag(I)

complexes. The solid-state molecular structure is shown in
Figure 9.
To conduct a thorough investigation of the effect of NHC
substituents on Ag-NHC stability, two similar Ag(I)-N-
heterocyclic carbene complexes, one lacking any substituents
on the 4 and 5 positions of the imidazole ring (16) and the
second bearing a methyl ester group on the 4(5) position
(17), were synthesized (Chart 4). Both complexes were
synthesized by the general procedure of making the imida-
zolium iodide salt using iodomethane and the subsequent
synthesis of the corresponding silver complex by adding 2
mol equiv of silver acetate in dichloromethane.
Figure 9. Molecular structure of 15.71
A stability analysis of all three silver complexes, 15, 16,
Chart 4. Structures of 16 and 1771 and 17, was performed by monitoring the degradation of the
complexes in D2O over time by 1H and 13C NMR spectros-
copy. The results revealed that the presence of Cl atoms
improved the stability of the silver acetate complex 15 over
its analogues (Table 6). It was postulated that the Cl atoms,
acting as σ-withdrawers and π-donators, led to a reduction
of σ-donor capability72 thereby leaving the carbene carbon
Table 6. Stability of Ag(I) Complexes 13, 15, 16, and 1771 center with less electron density and, therefore, less suscep-
silver complexes stability in D2O tible to attack by protons present in an aqueous environment.
13 3d The MIC of silver complex 15 was evaluated against a
15 17 weeks + panel of highly resistant respiratory pathogens, mainly strains
16 2h of P. aeruginosa and Burkholderia species in two different
17 1.5 h growth media, Mueller-Hinton (M-H) and Luria broth (LB)
(Table 7). In addition, E. coli J53 strain (silver-sensitive)
and J53 + pMG101 strain (silver-resistant) were used as
membrane of the treated cells. Those silver salts could be
positive and negative controls in the MIC evaluation of 15.
the cause of the apparent damage to the structural integrity
The MBC was also evaluated for 15 and was found to depend
of the cellular membrane.
on two factors, the growth media in which the tested strain
was grown and the strain itself. Complex 15 seemed to have
3.2.3. Synthesis and Antimicrobial Properties of a better bactericidal activity in M-H broth against some of
Silver-NHC Complexes Derived from the tested strains, but only showed a bacteriostatic activity
4,5-Dichloroimidazole for other strains at the concentrations examined. As defined
The Youngs group noted that complex 13 had stability in previously, the term bactericidal indicates that the compound
water for up to three days. It was hypothesized that the kills the tested bacteria, while bacteriostatic indicates that
presence of electron-withdrawing substituents on the 4 and the compound inhibits the growth of the bacteria but does
5 positions of the imidazole ring may have helped to stabilize not necessarily kill it. It has been demonstrated that 15 is
the silver complex from rapid degradation when exposed to bactericidal for numerous strains at clinically achievable
concentrations (Table 7).
water. Stable silver N-heterocyclic carbene complexes could
play a very vital role in the systemic delivery of silver A comparative antimicrobial study with complexes 15 and
antimicrobials that could potentially reach and thereby treat 16 on representative strains of P. aeruginosa and Burkhold-
infections prior to degradation by salts/biological molecules eria species found that the MIC50 values were basically the
present in the blood stream. Keeping this in mind, a novel same when compared on a molar basis. This result was
N-heterocyclic carbene derived from 4,5-dichloroimidazole irrespective of the difference in the rate of degradation
was synthesized.71 between the two silver complexes, which was a further
indication that the Ag+ ions are the active moiety within
The imidazolium salt 1,3-dimethyl-4,5-dichloroimidazo- any Ag(I)-NHC.
lium iodide (14) was formed by the deprotonation of 4,5-
dichloroimidazole with potassium hydroxide (KOH) and the 3.2.4. Synthesis and Antimicrobial Properties of
subsequent methylation with iodomethane in acetonitrile Silver-NHC Complexes Derived from
(Scheme 4). Characterization using 1H NMR spectroscopy 1-Benzyl-3-tert-butylimidazole
revealed the presence of the imidazolium proton resonance
at 9.42 ppm. In the 13C NMR spectrum, the chemical shift Another important contribution by the Ghosh research
at 136.6 ppm was consistent with the imidazolium carbon group led to the synthesis and antimicrobial evaluation of
atom. an (NHC)AgCl complex (18), which exhibited significant
antimicrobial activity.73 Complex 18 was synthesized ac-
The formation of the corresponding silver complex (15) cording to Scheme 5 where the 1-benzyl-3-tert-butylimida-
was achieved via the in situ deprotonation of 14 with 2 mol zolium chloride salt precursor was treated with half an
equiv of silver acetate in dichloromethane (Scheme 4). The equivalent of Ag2O in dichloromethane. The formation of
most notable chemical shift at 179.7 ppm in the 13C NMR 18 was further demonstrated by the disappearance of the
spectrum denoted the carbene carbon atom of complex 15, imidazolium proton resonance in the 10 ppm region in the
3868 Chemical Reviews, 2009, Vol. 109, No. 8 Hindi et al.

Table 7. MIC and MBC of 1571

MIC (µg/mL) MBC (µg/mL)
species (genomovar) strain M-H LB M-H LB
P. aeruginosa PA O1-V 2 2 >10 >10
PA M57-15 4 1 6 4
PA 2192 1 2 4 >10
PA 6294 2 2 >10 >10
PA N6 1 2 4 >10
PA JG3 2 2 4 8
PA N13 1 2 10 4
PA 1061 1 2 6 10
PA N8 1 2 >10 >10
PA 324 1 1 2 >10
B. cepacia (I) PC783 1 4 2 8
B. multiVorans (II) HI2229 1 4 1 >10
AU8170 4 4 a a
AU5735 2 2 a a
AU7484 2 2 a a
AU5248 4 2 a a
B. cenocepacia (III) J2315 2 1 4 >10
HI2718 2 >10 2 >10
ATTC BAA-245 2 2 a a
B. stabilis (IV) ATTC BAA-67 1 >10 6 >10
HI2210 1 >10 2 >10
B. Vietnamiensis (V) PC259 1 1 6 >10
B. dolosa (VI) AU0645 1 2 1 6
ATTC BAA-246 1 2 4 6
AU4459 1 1 4 6
AU5404 1 2 a a
AU4298 2 2 a a
AU3556 1 2 a a
AU4881 4 4 a a
AU9248 4 2 a a
AU4894 4 2 a a
AU3123 4 2 a a
AU4750 4 2 a a
B. ambifaria (VII) HI2468 2 1 4 >10
B. anthina (VIII) AU1293 1 2 1 4
B. pyrocinia (IX) BC11 1 1 1 1
E. coli J53 2 4 >10 >10
J53 + pMG101 >10 >10 >10 >10
a
Not determined.

Scheme 6. Synthesis of 19a, 19b, 20a, and 20b74

The antimicrobial activity of 18 against Bac. subtilis and

E. coli was evaluated using different concentrations of 18
Figure 10. Percent inhibition of Bac. subtilis 168 cell proliferation and measuring the bacterial growth at different time inter-
by complex 18.73 vals.73 The growth of the Gram-positive Bac. subtilis was
Scheme 5. Synthesis of 1873
inhibited as demonstrated by Figure 10, whereas no effect
on the growth of the Gram-negative E. coli was observed.
The antimicrobial activity of the 1-benzyl-3-tert-butyl-
imidazolium chloride salt precursor was also evaluated,
but no effect on the growth of the bacterial strains was
noted, further demonstrating that the silver moiety of
complex 18 was responsible for the reported activity.
Furthermore, for the wild-type Bac. subtilis 168, the half-
1
H NMR spectrum and the appearance of the NCN-Ag maximal inhibitory concentration (IC50) and MIC for
characteristic resonance at 177.7 ppm in the 13C NMR complex 18 were determined to be 9 ( 1.5 µM and 25 (
spectrum. 3.2 µM, respectively.
Medicinal Imidazolium Carbene-Metal Complexes Chemical Reviews, 2009, Vol. 109, No. 8 3869

Table 8. Half-Maximal Inhibitory Concentrations (IC50) the number of live cells as well as the number of dead cells.
Measured Using the MTT Assay74 Live cells produced a red fluorescence due to their ability to
IC50 (µM)a metabolize C12-resazurin, and dead cells produced a green
test compound OVCAR-3 MB157 HeLa fluorescence due to their compromised cell membranes
15 35 8 >200 allowing the accumulation of the Sytox Green stain. The
20a 30 20 >200 images shown in Figure 11a-f represent fluorescence images
20b 20 10 >200 from OVCAR-3 and MB157 control, incubation with cis-
AgNO3 35 5 50 platin, and incubation with complex 15.
AgOAc 20 12 b Based on the quantitative measurements of the assay
cisplatin 12 25 25
(Figure 12), the viability of OVCAR-3 cells exposed to silver
a
IC50 results are based on 72 h incubation period. b Not determined complexes 15, 20a, and 20b was 11%, 0%, and 0%,
due to solubility limitations of AgOAc. respectively. These results were superior to those for
OVCAR-3 cells treated with cisplatin, which resulted in 78%
3.3. Antitumor Properties of Silver-NHC viability. The OVCAR-3 control cells were 93% viable.
Complexes Furthermore, all three silver complexes and cisplatin were
3.3.1. Synthesis and Antitumor Properties of Silver-NHC equally active against MB157 cancer cell lines with 10%
Complexes Derived from 4,5-Dichloroimidazole cell viability compared with the control cells, which exhibited
92% viability. The cell viability percent values were based
Several factors led the Youngs research group to test and on cell counts after incubation of the cells with the test
report the efficacy of Ag(I)-NHC complexes 15, 20a, and compounds for 36 h at 50 µM. Cell viabilities were found
20b against the human cancer cell lines OVCAR-3 (ovarian), to be significant using Tukey’s multiple comparison among
MB157 (breast), and HeLa (cervical).74 One of those factors means (R ) 0.05).
was the urgent need to find new chemotherapeutic agents The effect that silver imposed on the morphology of the
effective against cisplatin-resistant tumor cell lines with OVCAR-3 and MB157 cells can be seen in Figure 13a-f in
milder toxic effects. Another important factor was the current which the cells were incubated with complex 15 at 50 µM
interest in other metal-N-heterocyclic carbene complexes for 36 h. The cells were stained blue with Hoesch stain. The
demonstrating notable tumor cytotoxicity.73,75,76 Furthermore, results were compared with the morphology of cisplatin-
Ag(I)-phosphine complexes with antitumor activity were treated cells. Silver complex 15 had a considerable effect
first reported by Sadler in 1988.77 Since the start of this work, on the viability of the treated cells as compared with the
other reports of silver-based complexes possessing anticancer control.
activity have appeared in the literature.78-80 The in Vitro results with the silver complexes 15, 20a,
Complexes (20a, 20b) were synthesized according to and 20b showed excellent activity specifically against
Scheme 6. The 4,5-dichloroimidazolium iodide salt precur- OVCAR-3 ovarian cancer cells and MB157 breast cancer
sors (19a, 19b) were synthesized by the deprotonation of cells. This sparked interest in the activity of these silver
4,5-dichloroimidazole with KOH followed by the substitution complexes in ViVo. A preliminary study was conducted in
with 1 equiv of the appropriate alkyl or aryl bromide in which a small number of athymic nude mice were inoculated
acetonitrile and subsequent methylation with an excess with 107 OVCAR-3 cells subcutaneously in their backs. The
amount of iodomethane. Subsequent in situ deprotonation tumors were allowed to grow for approximately 6 weeks after
of 19a and 19b with silver acetate in a 1:2 molar ratio in which subcutaneous (SubQ) injections of silver complex 15
dichloromethane afforded the corresponding NHC silver were performed along with control injections (diluent alone).
acetate complexes. One complex 15 dose group (333 mg/kg per injection) was
The in Vitro efficacy of complexes 15, 20a, 20b, the SubQ injected every third day for 10 days. The total SubQ
imidazolium cation precursors 14, 19a, and 19b, silver doses (∼1000 mg/kg) were within the range of another
nitrate, silver acetate, and cisplatin was determined against parallel study where intraperitoneal (IP) injections (100 mg/
the human cancer cell lines OVCAR-3 (ovarian), MB157 kg per injection) were administered for 10 consecutive days.
(breast), and HeLa (cervical) using the MTT assay (Table After day 10, all mice were humanely sacrificed. All dead
8). In the MTT assay, the enzyme succinate dehydrogenase mice underwent necropsy to evaluate the spleen size,
present in the mitrochondria of living cells cleaves the evidence of gastrointestinal toxic effects, and the condition
tetrazolium rings of the yellow MTT to form insoluble purple of the liver and kidneys along with the effect of 15 on the
formazan crystals. Sodium dodecyl sulfate (SDS) is a tumors. Figure 14 gives a view of the internal organs of one
solubilizing solution added to dissolve the formazan. The of the treated mice, in which all organs looked grossly
absorbance of the resulting colored solution was measured normal. This result was further confirmed by a histopatho-
at a wavelength of 570 nm. The number of surviving cells logical assessment of the brain, liver, lungs, heart, kidneys,
is directly proportional to the amount of formazan present. and spleen. The tumors treated with silver, however, were
The imidazolium salts 14, 19a, and 19b showed much determined to be necrotic, whereas the control tumors were
higher IC50 values and were therefore not active against the still viable (Figure 15).
tumor cell lines tested (results not shown). As shown in Table
8, complexes 15, 20a, and 20b were comparable in activity 4. Gold
to cisplatin against OVCAR-3 and MB157 cell lines.
However, the silver complexes showed minimal activity 4.1. Medical Uses of Gold Compounds
against HeLa cells. This was an indication that the reported
silver complexes are selective in their preference against The discovery of the bacteriostatic properties of gold
certain cancers. cyanide, K[Au(CN)2], by Robert Koch in 1890 against
The live/dead assay was used to measure cell viability. It tubercle bacillus marked the start of its use in modern
is a two-color flourescent assay that simultanously determines medicine.85,86 By 1920, the wide use of various gold salts as
3870 Chemical Reviews, 2009, Vol. 109, No. 8 Hindi et al.

Figure 11. Live/dead assay images of OVCAR-3 and MB157 cells:74 (a) OVCAR-3 control; (b) OVCAR-3 incubated with cisplatin; (c)
OVCAR-3 incubated with 15; (d) MB157 control; (e) MB157 incubated with cisplatin; (f) MB157 incubated with 15.

Figure 12. Percent viability of OVCAR-3 and MB157 after treatment with cisplatin (Cis), silver acetate, (AgOAc), silver nitrate (AgNO3),
15, 20a, and 20b. The percentages are based on cell counts from the live/dead assay data after incubation with test compounds at 50 µM
for 36 h.74

a treatment for tuberculosis was underway. The belief that Besides the antiarthritic applications of gold compounds,
rheumatoid arthritis was an atypical form of tuberculosis led ophthalmologists use metallic gold to treat a condition called
to the use of gold(I) salts for the treatment of this disease. lagophthalmos, which is the inability to close the eyelids
By the early 1930s, gold therapy (chrysotherapy) was completely. This is done by surgically implanting metallic
discontinued as a treatment of tuberculosis based on its gold “weights” in the upper eyelid to help it close fully.86
ineffectiveness; however it is still considered the most The antimicrobial activity of gold compounds was inves-
effective available therapy for the management of rheumatoid tigated in light of the early evidence of their activity against
arthritis.87 No major therapeutic advancements have taken tubercle bacillus presented by Robert Koch. Recent studies
place in this field except for the antiarthritic gold compound looked at the effect of a gold(I) thiocyanate complex,
auranofin (Figure 16), which was introduced in the early Au(SCN)(PMe3), against a number of Gram-positive bacterial
1980s. Auranofin or triethylphosphine(2,3,4,6-tetra-O-acetyl- strains including MRSA, methicillin-sensitive Staph. aureus,
β-1-D-(thiopyranosato-S)gold(I) was introduced as an orally Ec. faecalis, coagulase-negative staphylococci, and strepto-
bioavailable drug designed in hopes of improving the cocci.81 The compound was mostly active against Ec. faecalis
pharmacokinetic profile, as well as reducing the cytotoxic and Staph. aureus with MIC50 values of 0.77 and 0.33 µg/
effects (discussed in a later section), encountered with other mL, respectively.81 Furthermore, a comparative in Vitro
gold compounds.85-87 toxicity study against CHO mammalian cell lines demon-
Medicinal Imidazolium Carbene-Metal Complexes Chemical Reviews, 2009, Vol. 109, No. 8 3871

Figure 13. Morphology of OVCAR-3 and MB157 cells:74 (a) OVCAR-3 control; (b) OVCAR-3 incubated with cisplatin; (c) OVCAR-3
incubated with 15; (d) MB157 control; (e) MB157 incubated with cisplatin; (f) MB157 incubated with 15.

gold(I) complexes were evaluated and have shown marked

antitumor activity. For a comprehensive table, the reader is
referred to a previous review in Chemical ReViews by
Shaw.87 The screening efforts of the different complexes led
to the evaluation of a cationic tetrahedral gold(I) phosphine
complex, [Au(dppe)2]Cl (Figure 17).82 It is important to note
that the bis(diphenylphosphino) ethane (dppe) ligand exhibits
antitumor activity in itself, and it was suggested that the gold
serves to protect it against degradation.77,82 However, ex-
tensive studies done comparing the antitumor activity of dppe
alone to [Au(dppe)2]Cl and other Au(I), Ag(I), and Cu(I)
phenyl-substituted diphosphine complexes demonstrated that
the antitumor activity was comparable, but the metal
complexes were 20-fold more potent.77,82 This result suggests
Figure 14. View of the internal organs of a mouse treated with that the effect of such metals expands beyond protection
three SubQ injections of complex 15 of 333 mg/kg dose over a 10
day period.74 against degradation. Although the gold complex
[Au(dppe)2]Cl exhibited excellent antitumor activity, it never
made it to clinical trials due to the extensive cardiovascular
strated the selectivity of the Au(I) thiocyanate complex for
bacteria over mammalian cells with a MIC50 at least ten times toxicity unveiled during the preclinical toxicology studies.83
greater than the bacterial MIC values.81 A number of gold(III) Similar cationic gold(I) phosphine complexes in which
compounds, namely, AuCl2(damp) and Au(OAc)2(damp), pyridyl groups replaced the phenyl substituents in
were also tested (Chart 5). Although both complexes were [Au(dppe)2]Cl were synthesized for the purpose of evaluating
adequately active against Gram-positive bacteria and E. coli, the effects of decreased lipophilicity (Chart 6).84 The presence
the more water-soluble complex Au(OAc)2(damp) showed of the pyridyl ligands and the position of the N atom
enhanced selectivity against Ec. faecalis and Staph. aureus.81 introduce significant changes within the interactions between
Another major and current area of study involves the use the compounds and the solvent. As evidence, the 4-pyridyl
of gold in the treatment of cancer. Auranofin was found to complex exists as a simple monomeric cation in the solid
have a limited antitumor activity against HeLa cells (cervical state and in solution. In contrast, the 2-pyridyl complex exists
cancer) in Vitro and P388 leukemia cells in ViVo but was as a dimer in the solid state, and in solution, it exists in a
inactive in solid tumors, which prompted the evaluation of constant equilibrium state between monomeric, dimeric, and
other gold-based complexes. A number of triphenylphosphine tetrameric clusters. This further provides an explanation as
3872 Chemical Reviews, 2009, Vol. 109, No. 8 Hindi et al.

Figure 15. Image of the solid OVCAR-3 tumors allowed to grow in athymic nude mice: (a) healthy tumor before SubQ injection of
complex 15; (b) necrotic tumor after three SubQ injections of 333 mg/kg dose of 15 over a 10 day period.74

A dependence on lipophilicity was observed when the

Au(I) complexes were tested in ViVo against the murine colon
38 adenocarcinoma. The drugs were administered daily for
10 days. The compound with the intermediate lipophilicity,
namely, the 2-pyridyl complex, showed the most antitumor
activity, significant tumor growth delay of 9 days, decreased
dose-limiting toxicity, and higher gold concentrations in the
tumor. The most lipophilic, namely, [Au(dppe)2]Cl, and the
Figure 16. Structure of auranofin. most hydrophilic, namely, the 4-pyridyl complex, had no
marked effect on tumor growth delay. The lipophilicity factor
was also used to explain the toxicity observed between the
compounds mentioned. It is thought that very lipophilic
aromatic cations such as [Au(dppe)2]Cl exhibit high and
nonspecific binding to proteins, which might explain the high
host toxicity. In contrast, highly hydrophilic molecules such
as the 4-pyridyl complex demonstrate low protein binding
Figure 17. Structure of antitumor Au(dppe)2 chloride complex. and high rates of excretion, which might explain its inactivity.
Chart 5. Structures of Antimicrobial Gold(III) Complexes For more comprehensive reviews that address the early
developments in the medicinal chemistry of gold complexes,
the reader is directed to reviews by Fricker,85 Merchant,86
and Shaw.87

4.1.1. Mechanism of Gold Activity

Although gold has been used in the clinical setting for
almost 80 years, its cytotoxic mechanisms are not completely
Chart 6. Structures of Antitumor understood. It has been established that gold complexes with
Bis[1,2-bis(di-n-pyridylphosphino)ethane] Gold(I) Chloride different geometries exert their effect through different modes
Complexes Where n ) 2 or 4 of action. For instance, the monomeric linear complex,
auranofin, which is well-known for its antiarthritic properties,
has the ability to undergo ligand exchange reactions where
it displaces both of its ligands and forms different metabo-
lites, one of which is [Au(CN)2]-, thought to target and
inhibit certain immune cell functions involved in the inflam-
matory response of rheumatoid arthritis.87 Auranofin, which
was also found to exhibit antitumor activity,88,89 was found
to inhibit the growth of tumor cells via an antimitochondrial
to why the 4-pyridyl has much more water solubility than mechanism,90,91 which involves the inhibition of mammalian
its analogue, the 2-pyridyl complex. It was found that the selenoenzyme mitochondrial thioredoxin reductase (TrxR).92,93
2-pyridyl complex had a similar activity against P388 This is thought to occur through the binding of Au(I) to the
leukemia compared with [Au(dppe)2]Cl, and it was also C-terminal redox active -Cys-Sec- center.94
active against B16 melanoma in ViVo. On the other hand, On the other hand, the cationic tetrahedral gold(I) phos-
the 4-pyridyl complex was inactive against all tumor cell phine complexes do not undergo ligand dissociation reactions
lines tested and was toxic to mice. due to the added stability by two chelating diphosphine
Medicinal Imidazolium Carbene-Metal Complexes Chemical Reviews, 2009, Vol. 109, No. 8 3873

ligands. The stability of such complexes in the presence of Scheme 7. Synthesis of 21-2698
thiols, disulfides, and serum proteins was assessed by
monitoring 31P NMR spectroscopy.82 The 31P NMR reso-
nance was unaffected by the addition of glutathione (GSH
and GSSH, thiol and disulfide sources) and after incubation
with bovine serum. As judged by the NMR spectra, the gold
phosphine complexes do not readily undergo ligand displace-
ment reactions in the presence of thiols in aqueous media.
This is perhaps a result of the high stability of the
gold-phosphorus bonds.
Furthermore, it was recognized that the mitochondria might
be a critical intracellular target involved in the antitumor
activity of the drugs. Studies using isolated rat hepatocyte
mitochondria were carried out in order to assess the effects
exerted by [Au(dppe)2]Cl complex.84 The cationic and
lipophilic nature of this particular complex could have aided
its uptake into the mitochondria. Once there, [Au(dppe)2]Cl
is thought to cause a number of crucial modifications to the
mitochondria including loss of the inner membrane potential many synthetic studies, the reader is directed to the recent
difference, efflux of Ca2+, increased mitochondrial respira- contribution by Cronje and Raubenheimer.95
tion, mitochondrial swelling, and finally increased perme-
ability of the inner mitochondrial membrane and uncoupling 4.2.1. Synthesis and Antimicrobial Properties of
of oxidative phosphorylation.90 These effects are thought to Gold-NHC Complexes Derived from
be particular to the lipophilic nature of [Au(dppe)2]+ and 1,3-Diorganylimidazolidin-2-ylidenes
other tetrahedral bis(dipyridylphosphino)gold(I) complexes.
This property could play an important role in improving the As discussed previously, two-coordinate gold(I) phosphine
selectivity of the mitochondrial-targeted drugs by fine-tuning complexes were reported to have potent antimicrobial
their degree of liphophilicity. activity.85,96,97 However, Cetinkaya was the first to report the
For a more comprehensive review of this topic, the reader antimicrobial activity of six gold(I)-NHC complexes
is directed to two excellent reviews by Berners-Price that (21-26).98 The gold-carbene complexes were synthesized
address the possible role of mitochondria in the mechanisms following a general and simple procedure. The synthesis was
of cytotoxicity and antitumor activity of gold complexes,90 carried out under argon or nitrogen atmosphere by reacting
as well as the recent strategies on specifically targeting the 1 equiv of AuCl(PPh)3 with either 2 equiv of 1,3-dimesi-
mitochondrial cell death pathway with gold compounds.93 tylimidazolidin-2-ylidene (21) or equal equivalents of
the corresponding bis(1,3-dialkylimidazolidin-2-ylidene)
(22-26) (Scheme 7). The reaction mixtures were refluxed
4.1.2. Toxicity of Gold in toluene for 2 h and then cooled to room temperature where
Although the [Au(dppe)2]Cl complex has shown in ViVo hexanes were added to obtain a creamy solid, which was
activity against P388 leukemia, M5078 reticulum cell recrystallized from a CH2Cl2/Et2O mixture. The resulting
sarcoma, B16 melanoma, mammary adenocarcinoma 16/C, gold complexes (21-26) were analyzed and confirmed
and intraperitoneal and subcutaneous transplanted tumors, through a variety of analytical techniques including melting
it was determined that this complex was severely hepatotoxic point, elemental analysis, IR, 1H NMR, and 13C NMR
upon the in ViVo evaluation in male beagle dogs.90 As spectroscopies.98
mentioned above, this [Au(dppe)2]Cl -induced cytotoxicity The cationic gold(I)-NHC complexes (21-26) were
to liver cells could be a direct effect of its ability to cause evaluated for their in Vitro antimicrobial activity against a
the uncoupling of oxidative phosphorylation, which is related variety of Gram-positive and Gram-negative bacteria and
to the increased permeability of the inner mitochondrial fungal species. The results were compared with those of
membrane.90 standard drugs ampicillin and flucytosine (Table 9).
Normal cells rely on oxygen consumption and oxidative Based on the results from Table 9, gold complexes 21-23
phosphorylation for ATP production, whereas solid tumors showed good and selective activity against both Gram-
have been shown to rely primarily on glucose uptake and positive and Gram-negative bacteria with complex 22 show-
glycolysis for ATP production. Therefore, antitumor agents ing the most activity against the strains tested, as well as
that target the oxidative phosphorylation pathways (i.e., some activity against the fungus C. albicans. The 1,3-
[Au(dppe)2]Cl) are likely to cause toxicity in normal cells.90 dimesitylmethylimidazolinium chloride (Mmi) salt exhibited
The pronounced difference between normal tissues and solid effective and selective antibacterial activity against three
tumors related to their reliance on oxidative phosphorylation Gram-positive (Staph. epidermidis, Staph. aureus, and Ec.
vs glycolysis for ATP production can be utilized in order to faecalis) and one Gram-negative (P. aeruginosa) bacterial
design selective and therefore effective antitumor agents. strains. However, the derived gold(I) complex 24 did not
show enhanced activity against any of the strains tested. It
4.2. Antimicrobial Properties of Gold-NHC is apparent that the functionalization of the nitrogen atoms
Complexes of the NHC ligands and the complexation with Au(I) at the
C2 site influence the antimicrobial activity. Some of the Au(I)
For a review on the recent progress in the synthetic complexes presented became completely inactive and some
methods available for gold-NHC complexes, their medicinal with marked activity. In light of this evidence, it can be
applications, and theoretical calculations that accompanied deduced that the gold atoms do not necessarily influence the
3874 Chemical Reviews, 2009, Vol. 109, No. 8 Hindi et al.

Table 9. MIC of 21-2698

MIC (µg/mL)a
test compound Staph. epidermidis Staph. aureus Ec. faecalis Eb. cloacae P. aeruginosa E. coli C. albicans
21 800 3.12 800 3.12 1600 800 800
22 6.25 3.12 3.12 1600 3.12 1600 200
23 >1600 >1600 >1600 >1600 >1600 3.12 >1600
24 >1600 200 >1600 100 >1600 200 >1600
25 >1600 50 800 12.5 >1600 1600 1600
26 >1600 50 >1600 800 >1600 400 >1600
Mmib 6.25 3.12 3.12 1600 3.12 400 200
ampicillin <3.12 >3.12 6.25 <3.12 25 <3.12
flucytosine 6.25
a
MIC results are based on 24 h incubation at 37 °C for bacteria and 48 h incubation at 30 °C for fungus. Mmi ) 1,3-dimesitylmethylimidazolinium
b

chloride25

previously of the silver complex 18 with (SMe2)AuCl

(Scheme 8). According to the 13C NMR spectrum, the
diagnostic NCN-Au resonance appeared at 169.2 ppm,
which is observed for analogous Au-NHC complexes.
The antimicrobial efficacy of the gold complex 27 was
evaluated against Bac. subtilis and E. coli. Similarly to the
Ag-NHC complex 18, the Au-NHC complex 27 exhibited
antimicrobial activity against Bac. subtilis (Figure 18) but
had no effect against E. coli. Furthermore, the IC50 and the
MIC values of 27 obtained for the growth of the wild-type
Bac. subtilis 168 were calculated to be 4 ( 0.8 and 15 (
2.3 µM, respectively.
In an effort to understand the antimicrobial mechanism
Figure 18. Percent inhibition of Bac. subtilis 168 cell proliferation of complex 27, the morphology of the Bac. subtilis cells was
by complex 27.73 evaluated after their incubation with 4 µM of 27 for 4 h
(Figure 19). The elongation of the Bac. subtilis cells by 3.5-
fold from 2.2 ( 0.5 µm to 7.3 ( 3.1 µm was observed
compared with the control cells grown in the absence of 27.
This elongation suggested that 27 inhibited bacterial prolif-
eration by blocking the cytokinesis step of cell division.73

4.3. Synthesis, Antimitochondrial, and Antitumor

Properties of Gold-NHC Complexes
Baker and co-workers explored the potential of five
compounds of the general formula [(R2Im)2Au]+, where Im
represents an imidazole, and a series of dinuclear Au(I)
Figure 19. Effects of complex 27 on the morphology of Bac. complexes as possible antimitochondrial antitumor agents.99,76
subtilis. Cells were incubated for 4 h in the absence and in the Considering the fact that mitochondrial regulation of apop-
presence of 27, and the cell morphology was visualized by DIC
microscopy. Scale bar represents 10 µm.73
tosis in tumor cells could be manipulated for therapeutic gain,
complexes (28-39) of different lipophilicities were evaluated
Scheme 8. Synthesis of 2773 for their efficacy in inducing mitochondrial membrane
permeabilization (MMP) in isolated rat liver mitochondria.
MMP is regulated by the mitochondrial permeability transi-
tion pore (MPT), which is considered vital to the mitochon-
drially induced apoptosis process.
The imidazolium salt precursors were deprotonated in situ
with lithium hexamethyldisilazide (LiHMDS) in DMF at
room temperature followed by the reaction with half the
equivalent amounts of (Me2S)AuCl to yield the correspond-
antimicrobial activity but rather it is the type of functional
ing [(R2Im)2Au]+ complexes 28-32 (Scheme 9).99 The
groups bound to the carrier ligands that do.
lipophilicity of the resultant gold-NHC complexes 28-32
varied according to the alkyl substituents on the imidazolium
4.2.2. Synthesis and Antimicrobial Properties of salt precursors.
Gold-NHC Complexes Derived from
The gold complexes of bridging bidentate NHCs 33-39
1-Benzyl-3-tert-butylimidazole
were synthesized by reacting an equimolar mixture of the
Ghosh and co-workers reported the antimicrobial activity desired imidazolium salt with (Me2S)AuCl in DMF at 100
of a gold(I)-NHC complex, [1-benzyl-3-tert-butylimidazole- °C after which a carboxylate salt (sodium acetate, lithium
2-ylidene]AuCl (27).73 The complex was synthesized fol- acetate, or lithium butyrate) was added to the solution and
lowing the carbene-transfer route from the reaction discussed allowed to react for an additional 30 min at 120 °C.100
Medicinal Imidazolium Carbene-Metal Complexes Chemical Reviews, 2009, Vol. 109, No. 8 3875

Chart 7. Structures of Antimitochondrial Gold(I) Complexes 33-3976

Scheme 9. Synthesis of 28-3299 These results clearly demonstrate the direct dependence of
the antimitochondrial activity on lipophilicity.
On the other hand, the dinuclear Au(I)-carbene complexes
(33-39) were found to induce significant mitochondrial
swelling at a concentration of 10 µM. Complex 34 induced
marked swelling at a submicromolar concentration of 0.5
µM compared with its analogues. A time-dependent assay
of Au(I) uptake into the mitochondria of complexes 33-39
was performed to establish whether the degree of Au(I)
uptake was associated with increased swelling of the mito-
chondria. However there was no apparent correlation between
Complexes 33-39 shown in Chart 7 precipitated from the
the two activities indicating that the ability of those
reaction mixture.
complexes to induce MMP does not correlate with their
The 1H and 13C NMR spectral data were in good
ability to enter the mitochondria. A possible mechanism by
agreement with the proposed structures. Notably, the 1H
which complexes 22-39 function could be through the
NMR spectrum was missing the acidic imidazolium C2-H
disruption of a certain enzyme or the interaction with
proton signal and the 13C NMR spectrum showed the
components of the MPT.
appearance of the C2 carbene signal downfield of the C2
carbon signal of the imidazolium salt confirming the forma- Baker and co-workers also reported the synthesis of linear
tion of the Au-NHC complex. The resulting Au-NHC Au(I)-NHC complexes as the first NHC analogues for the
complexes were further characterized crystallographically. Au(I) phosphine drug auranofin.101 The neutral 2,3,4,6-tetra-
The complexes were synthesized as the halide salt but O-acetyl-β-D-glucopyranosyl-1-thiolato complexes
converted to the hexafluorophosphate salt using KPF6 in [(R2Im)Au(SR′)] (40-44) were synthesized by the treatment
order to obtain crystals suitable for single X-ray diffraction of [(R2Im)AuCl] complexes with tetra-O-acetyl-β-D-glu-
analysis. The structures of complexes 28 · PF6- and 30 · PF6- copyranose (HSR′) under basic conditions (Scheme 10). The
are shown in Figure 20. goal was to take advantage of the ease by which N-
As mentioned previously, the lipophilicity of complexes heterocyclic carbenes can be chemically manipulated to
28-32 was varied based on the alkyl substituents of the NHC synthesize a range of structurally similar complexes with
ligands. The results were as expected with complex 28 · Br- varying degrees of lipophilicity for biological evaluation. It
(R ) Me) as the least lipophilic and complex 32 · Cl- (R ) was stated that according to preliminary biological studies,
Cy) as the most lipophilic. The effect of complexes 28-32 the antimitochondrial activity of these complexes correlated
on inducing MMP was monitored by measuring the degree with their lipophilicity but the results were not shown.
of mitochondrial swelling based on the absorbance at 540 Several recent papers came out addressing the antitumor
nm as a function of time at varying drug concentrations of activity and the underlying cytotoxic mechanisms of
1 or 10 µM. At a drug concentration of 1 µM, the time taken Au(I)-NHC complexes.102-104 In addition, one recent review
to induce mitrochondrial swelling decreased as the degree by Berners-Price addressed recent strategies on targeting the
of lipophilicity increased. Therefore, the least lipophilic mitochondrial cell death pathway with Au(I)-NHC com-
complex 28 · Br- was virtually inactive, whereas the rest of plexes.93 Raubenheimer reported the cytotoxicity of a bis-
the complexes showed significant activity. At 10 µM (NHC)Au(I) complex (45) carrying an electrophoric, cyto-
however, complexes 29-32 · Cl- showed rapid induction of toxic ferrocene moiety.102 Complex 45 was synthesized
mitochondrial swelling, and interestingly enough, complex through a multistep synthetic procedure (Scheme 11) involv-
28 · Br- showed modest activity at the higher concentration. ing the diazotation and subsquent coupling of ferrocene to
3876 Chemical Reviews, 2009, Vol. 109, No. 8 Hindi et al.

Figure 20. The cationic portion of complexes (a) 28 · PF6- and (b) 30 · PF6-. Reproduced by permission of The Royal Chemical Society.99

Scheme 10. Synthesis of 40-44101 against HeLa and Jurkat cell lines with considerably lower
IC50 values. Jurkat cells were the most sensitive with an IC50
value of 0.253 ( 0.031 µM compared with that of cisplatin
(0.783 ( 0.054 µM), while the CoLo cells were the least
sensitive with an IC50 value of 1.007 ( 0.081 µM compared
with that of cisplatin (0.407 ( 0.043 µM). This was an
indication that complex 45 is selective in its performance
against certain types of cancer. The effect of complex 45 on
normal human lymphocytes was also investigated. It was
determined that normal cells were less sensitive to complex
45, which further highlights its selectivity to cancer cells.102
Filipovska and co-workers investigated the antitumor
activity and cytotoxic mechanism of a previously discussed
Au(I)-NHC complex 29 against two liver progenitor cell
(LPC) lines, one nontumorigenic [p53-immortalized liver
(PIL) 4] and the other tumorigenic (PIL2).103 The goal was
Scheme 11. Synthesis of 45102 to exploit the increase in mitochondrial membrane potential
(∆Ψm) in cancer cells for the development of mitochondria-
targeted chemotherapeutics that selectively target tumor cells.
This can be achieved by the use of delocalized lipophilic
cations (DLCs), which can pass easily through the lipid
bilayer as a consequence of their positive charge and
preferentially accumulate in the mitochondria of tumorigenic
cells because of the large ∆Ψm thereby directly exerting their
destructive effect.93 The lipophilic, cationic Au(I)-NHC
complex 29 exhibited notable selective toxicity for the
tumorigenic PIL2, with elevated ∆Ψm compared with
nontumorigenic PIL4 cells.103
Complex 29 was shown to accumulate in the mitochondria
of PIL2 cells by measuring the mitochondrial gold concen-
tration using ICP-MS. The mitochondrial concentration of
gold increased with increasing concentrations of complex
29 given. When PIL2 cells were treated with 4 µM of
complex 29, >50% cell growth inhibition was observed and
>80% of the gold content was found accumulated within the
mitochondria. The distribution of the gold was found shifted
to the cytoplasm with the dissipation of ∆Ψm with the
uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhy-
4-(1H-imidazole-1-yl)aniline, followed by alkylation with drazone. This further proves that the cationic gold(I) complex
1-bromo-2-butene in CH2Cl2 and anion exchange in acetone. accumulates in the mitochondria of cells driven by the ∆Ψm.
The final bis(NHC)Au(I) ferrocenyl complex was obtained Furthermore, to demonstrate the selective inhibition of PIL
after the reaction with Ag2O and the subsequent carbene cell growth by complex 29, PIL2 and PIL4 cells were treated
ligand transfer to (CH3)2SAuCl in the presence of tetraethy- with Au(I) complex over 72 h, and it was shown that the
lammonium chloride. growth of PIL2 cells was inhibited by 50% after a 48 h
The in Vitro efficacy of complex 45 was determined against incubation and >80% of total ATP was lost after 72 h. In
human cancer cell lines HeLa (cervical), CoLo 320 DM contrast, the growth and total ATP in the PIL4 cells were
(colon), Jurkat (leukemia), and MCF-7 (breast) using the not affected.103 These data substantiate the selective toxicity
MTT assay. Complex 45 was more effective than cisplatin of complex 29 toward the tumorigenic PIL2 cells compared
Medicinal Imidazolium Carbene-Metal Complexes Chemical Reviews, 2009, Vol. 109, No. 8 3877

Scheme 12. Synthetic Routes to Ruthenium(II)-Carbene intermediate lipophilicity (log P ) -0.83) and thus superior
Complexes120,121 cytotoxic potency and selectivity.99,104 Complex 29 exhibited
the same selective mitochondrial targeting characteristics on
MDA-MB-231, MDA-MB-468, and HMEC as those ob-
served against tumorigenic PIL2 and nontumorigenic PIL4
cells. Additionally, MDA-MB-231 cells were treated with
increasing concentrations of complex 29 for 6 h. TrxR
activity was inhibited by nearly 50% with a 5 µM concentra-
tion of complex 29. These data underscore the successful
attempt at the design of mitochondria-targeted chemothera-
peutics that are selectively toxic to cancer cells as well as
allow targeting and thus selective inhibition of mitochondrial
selenoproteins, such as TrxR.104

5. Ruthenium and Rhodium

5.1. Medical Uses of Ruthenium and Rhodium
Compounds
Ruthenium is mostly known for its use in the development
of new antitumor agents. The first Ru(III) complexes
explored for their antitumor activity were chloro-ammino-
Ru(III) complexes, such as cis-[RuCl2(NH3)4]Cl, trans-
[RuCl4(Im)2](HIm), and fac-[RuCl3(NH3)3]. While this class
of Ru(III) complexes was very active against primary tumors,
fac-[RuCl3(NH3)3] was determined unsuitable as an antitumor
agent due to its poor water solubility.105,106 trans-
[RuCl4(Im)2](HIm), known as the Keppler-type complex,
exhibited better tumor inhibition than cyclophosphamide,
Chart 8. Structures of Ru(II) Complexes 48a and 48b121 cisplatin, and 5-fluorouracil when examined against P388
leukemia and B16 melanoma, as well as against platinum-
resistant colorectal tumors. Results show that a single dose
of 72 mg/kg improved the life-span of animals inoculated
with P388 leukemia and multiple doses of 8 mg/kg reduced
tumor weights in animals inoculated with B16 melanoma in
comparison with the control groups.107
A class of chloro-dimethylsulfoxide-Ru(II) complexes
with the nontumorigenic PIL4 cells. Complex 29 was also with significantly lowered toxicity, namely, cis- and trans-
shown to selectively induce cell death in the tumorigenic [RuCl2(dmso)4], was also investigated. The coordinating
PIL2 cells through a mitochondrial apoptotic pathway.103 This dimethylsulfoxide ligands enhanced the selectivity of the
was demonstrated by following a two-step process. The ruthenium complexes for solid tumor metastases but lowered
amount of caspase-3 activation, which is used as a marker their activity compared with cisplatin. The cis-
for apoptosis, was measured. A marked increase in caspase-3 [RuCl2(dmso)4] isomer exhibited less activity against Ehrlich
activation was observed in PIL2 cells after 12 h of incubation ascites carcinoma and L1210 leukemia than cisplatin but was
with complex 29, whereas no caspase-3 activation was significantly less toxic.108
observed for PIL4. This result confirms the induction of cell Among the most promising Ru-based antitumor agents is
death by activating the apoptotic pathway. The next step was the complex (HIm) trans-[RuCl4(dmso-S)(Im)] (Im ) imi-
to confirm that complex 29 induced apoptosis via the dazole, dmso ) dimethylsulfoxide), known as NAMI-A,
mitochondria, which was demonstrated by the incubation of which demonstrated excellent antimetastatic activity against
the PIL2 cells with complex 29 and noting a 3-fold increase solid tumors. It interfered with the growth of malignant lung
in caspase-9 activity.103 metastatic tumors such as Lewis lung carcinoma (LLC), MCa
Next, Filipovska and co-workers aimed to rationally design mammary carcinoma, and TS/A adenocarcinoma. NAMI-A
an antitumor agent that combined selective mitochondrial was the first ruthenium complex to enter and complete phase
targeting along with selective thioredoxin reductase (TrxR) I clinical trials.105,106 A number of other ruthenium complexes
inhibition.104 Increased activity of TrxR was found to bearing various ligands such as arenes,109 2-phenylazopyri-
correlate with the acceleration of tumor growth, and therefore dine (azypy),110 and 2,2′:6′,2′′-terpyridine (terpy)111 among
the inhibition of this system has become an important drug others have been developed and evaluated for their antitumor
target. Au(I) complexes are known to be potent inhibitors activity.
of mammalian TrxR, where their activity is exerted through Rhodium complexes were also investigated for their
Au(I) binding to the C-terminal redox active -Cys-Sec- center antitumor activity. The majority of these tested complexes
of TrxR.93,94 The group investigated the effects of complex were either active but very toxic or not as effective as
29 against cell growth of two highly tumorigenic breast cell cisplatin. In particular, a class of dirhodium complexes
lines, MDA-MB-231 and MDA-MB-468, as well as normal [µ-(RCO2)4Rh2(H2O)2] (R ) Me, Et, Pr), exhibited good in
human mammary epithelial cells (HMEC). Complex 29 was ViVo antitumor activity against several tumor types such as
chosen over two other Au(I)-NHC complexes due to its Ehrlich ascites, sarcoma 180, and P388 lymphocytic leuke-
3878 Chemical Reviews, 2009, Vol. 109, No. 8 Hindi et al.

Table 10. MIC of Ru(II) Complexes 46-48120,121

MIC (µg/mL)a
compound Ec. faecalis Staph. aureus E. coli P. aeruginosa C. albicans C. tropicalis
46a 100 100 >1000 >1000
46b 800 1000 800 1000
46c 200 50 >1000 >1000
46d 200 200 1000 1000
46e 50 25 1000 1000
46f 100 100 1000 1000
47a >800 >800 >800 >800 >800 >800
47b 800 >800 >800 >800 >800 >800
47c 100 100 >800 >800 800 200
47d 100 100 >800 >800 100 100
47e 100 25 >800 >800 100 100
47f >800 >800 >800 >800 400 >800
47g 200 >800 >800 >800 >800 >800
47h 50 50 >800 >800 200 100
47i 50 50 >800 >800 200 200
47j 100 50 >800 >800 200 200
48a 6.25 6.25 1000 1000
48b 25 12.5 800 800
ampicillin 0.78 0.39 3.12 <75
a
MIC results are based on 16-20 h incubation at 35 °C for bacteria and 48 h incubation at 35 °C for fungi.

Scheme 13. Synthetic Routes to Rhodium(I)-Carbene Chart 9. Structures of Rh(I) Complexes 51a, 51b, and 52
Complexes

Table 11. MIC of Rh(I) Complexes 49-52121

MIC (µg/mL)a
compound Ec. faecalis Staph. aureus E. coli P. aeruginosa
49a 5 5 >1000 >1000
49b 25 25 200 400
49c 5 5 1000 1000
50a 400 50 800 1000
50b 50 50 >1000 >1000
50c 200 200 200 200
50d 50 25 1000 >1000
50e 25 25 >1000 >1000
51a 25 25 >1000 >1000
51b 100 25 1000 1000
52 400 400 400 400
ampicillin 0.78 0.39 3.12 <75
a
MIC results are based on 16-20 h incubation at 35 °C.

mia; however severe toxic side effects prevented their

5.1.1. Mechanisms of Ruthenium and Rhodium Activity
advancement.106,112 Fine-tuning of characteristic features,
lipophilicity, charge, solubility, lability of bridging groups, Ruthenium-based complexes are thought to act through
etc., has led to the design of additional dirhodium complexes three major mechanisms, which potentially explain their
in order to reach a compromising median between antitumor selectivity to solid and metastatic tumors. Experiments with
activity and toxic side effects.112 RuCl3 and calf-thymus DNA conducted by Sideris and co-
Other monomeric, square-planar Rh(I) complexes such as workers found that there was little interaction between the
[(COD)(PMI)Rh]Cl (PMI ) 2-pyridinalmethylimine, COD two reagents perhaps through cross-linking.117 To further
) cyclooctadiene) seemed active against metastatic tumors verify this theory, Reedijk and co-workers showed that mer-
including MCa mammary carcinoma and Lewis lung carci- [Ru(terpy)Cl3] bound to guanine derivatives in a trans
noma. Furthermore, Rh(III) complexes analogous to those configuration and formed DNA interstrand cross-links.111
of ruthenium(III) complexes described above were either Therefore, multichloro ruthenium(III) complexes appear to
inactive or possessed modest levels of activity when tested favor interstrand cross-links with guanine N7 sites. This
against primary or metastatic MCa mammary tumors in the mechanism differs from cisplatin, which forms intrastrand
lung.106,113 cross-links.
For more comprehensive and recent reviews on the Moreover, ruthenium has the ability to mimic iron due to
medicinal developments of rhodium and ruthenium metal their chemical similarity and bind to transferrin.106,118 Rapidly
complexes, the reader is directed to the recent contributions dividing tumor cells have a higher requirement for iron in
by Liu,114 Sadler,115 and Reedijk.116 comparison to normal cells and therefore have an increased
Medicinal Imidazolium Carbene-Metal Complexes Chemical Reviews, 2009, Vol. 109, No. 8 3879

Scheme 14. Synthetic Routes to Palladium(II)-Carbene

Complexes73

Figure 21. Molecular structure of 56.73 Table 12. Half-Maximal Inhibitory Concentration (IC50) of
53-55123
IC50 Calu-6 IC50 MCF7
compound pH 6.8 pH 7.4 pH 6.8 pH 7.4
cisplatin 26.04 27.21 122.50 207.66
53 0.32 1.50 1.17 5.31
54 0.10 0.46 0.45 2.17
55 0.47 1.02 0.63 2.43

Table 13. Percent Inhibition of HeLa Cell Proliferation by 56

and 5773
inhibition, %
concentration (µM) 56 57
1 7(3 7(3
5 23 ( 4 57 ( 3
10 35 ( 3 85 ( 2

Table 14. Half-Maximal Inhibitory Concentrations (IC50) of 56c

Compared with Cisplatin Using the SRB Assay73
IC50 (µM)
compound HeLa HCT 116 MCF-7
cisplatin 8(1 16 ( 3 15 ( 2
Figure 22. Molecular structure of 57.73 57 4 ( 0.2 0.8 ( 0.05 1(3

Chart 10. Structures of Pd Complexes 53-55

more active in ViVo than Ru(III). Therefore, Ru(III) complexes
are thought to serve as prodrugs and readily undergo reduction
to Ru(II) by glutathione and other redox proteins present in
ViVo.106,118 This suggested mechanism is known as “activation
by reduction”. Work done by Clarke demonstrating the in-
creased inhibition of growth in HeLa cells with added transferrin
and lower partial pressures of oxygen (PO2) further confirmed
the above suggested mechanisms.119
The in ViVo mechanism of rhodium compounds has not
been investigated thoroughly. However, structural studies
number of transferrin receptors on their cell surface. Once with dirhodium complexes suggested their analogy to cis-
ruthenium reaches the cell surface, apotransferrin can then platin in binding to adjacent guanines on DNA and forming
act as a selective carrier of Ru drugs into the tumor cells. intrastrand cross-links.112 Additionally, rhodium(III) com-
This characteristic is thought to contribute to ruthenium’s plexes are structurally similar to ruthenium(III) complexes
low toxicity. and yet their activation by reduction to rhodium(II) is not
Rapidly growing tumors are also known to possess low likely. This is perhaps due to their enhanced inertness in
oxygen content and lower pH than normal cells. This type of comparison to their ruthenium analogues, which explains
environment favors the binding of Ru(II) complexes, which are their reduced antitumor activity.113
3880 Chemical Reviews, 2009, Vol. 109, No. 8 Hindi et al.

Figure 23. Inhibition of HeLa, HCT 116, and MCF-7 cell proliferation by 57 and cisplatin, measured using the SRB assay.73

Figure 24. Immunofluorescence analysis of HeLa cells arrested at the G2/M phase of the cell cycle after treatment with 57 at 10 and 20
µM. The cells were stained with cyclin B1 antibody. The figure demonstrates the overexpression of cyclin B1 in the treated cells compared
with the control.73 Scale bar represents 10 µM.

5.2. Synthesis and Antimicrobial Properties of Having said that, this section will focus on the role of the
Rhodium- and Ruthenium-NHC Complexes two metal NHCs as antimicrobial agents.
Although the antitumor activity and mechanism of action Cetinkaya and co-workers were the first to investigate the
for both ruthenium and rhodium metals has been discussed in Vitro antimicrobial activity of Rh(I)- and Ru(II)-carbene
above, no accounts of antitumor activity of ruthenium and complexes. The ruthenium complexes (46, 47) were synthe-
rhodium NHCs were found in the literature. Based on their sized by treating the electron-rich olefins (I or II) with half
antitumor efficacy, especially that of Ru, the exploration of the equivalents of the appropriate [(arene)RuCl2]2 in refluxing
the antitumor potential of Ru NHCs is highly recommended. toluene (Scheme 12).120,121 Other similar ruthenium com-
Medicinal Imidazolium Carbene-Metal Complexes Chemical Reviews, 2009, Vol. 109, No. 8 3881

where the electron rich olefin II and its benzimidazole

derivative I were reacted with the chloro-bridged dimer
[RhCl(COD)]2 in refluxing toluene to yield complexes
(49-50) (Scheme 13).121 Complex (51) however was made
Figure 25. Western blot analysis of the cell lysate treated with by utilizing a different Rh(I) compound with two triph-
(a) vehicle, (b) 10 µM 57, and (c) 20 µM 57. Phospho-cdc2 antibody enylphosphine ligands and complex (52) was synthesized by
(p-cdc2), which is specific to the G2 phase, was used. Phospho- the treatment of 1-benzyl-2-imidazoline with [RhCl(COD)]2
rylation was particularly increased in 20 µM 57-treated cells. The (Chart 9).121 All of the reactions described were carried out
figure indicates the inactivation of cdc2 by phosphorylation, which
arrested the cells at the G2 phase. The top band is the phospho-
under oxygen-free conditions. The purity of the complexes
rylated form and the botton band is the unphosphorylated form of was analyzed with the aid of 1H NMR and FT-IR spec-
cdc2.73 troscopies and melting point techniques.
The antimicrobial efficacy of the rhodium(I) complexes
plexes, 48a and 48b, were synthesized following the same 49-52 was investigated against the bacterial strains Ec.
general procedure (Chart 8).121 All of the complexes were faecalis, Staph. aureus, E. coli, and P. aeruginosa.121
analyzed using 1H NMR and FT-IR spectroscopies and Ampicillin was used as a reference compound, and the MIC
melting points. values of the complexes along with the control compound
The antimicrobial activity of the ruthenium complexes are reported in Table 11.
(46-48) was evaluated against Ec. faecalis, Staph. aureus, Based on the results in Table 11, more Rh(I) complexes
E. coli, and P. aeruginosa.120,121 In addition, the antifungal have shown greater antimicrobial activity than structurally
activity of complexes 47a-j against C. albicans and C. similar Ru(II) complexes. Particularly, complexes 49a-c,
tropicalis was investigated.120 Ampicillin was used as a 50b, 50d, 50e, and 51a were mostly effective against the
reference compound. The results are summarized in Table 10. Gram-positive bacteria Ec. faecalis and Staph. aureus with
As observed from Table 10, none of the ruthenium MIC values ranging between 5-50 µg/mL. None of the
complexes tested is as effective at killing bacteria as the complexes shown in Table 11 seemed active against the
reference compound ampicillin. Complex 48a however came Gram-negative bacteria E. coli and P. aeruginosa. The
closest with MIC values of 6.25 µg/mL againt both Ec. enhanced activity of the Ru(II) and Rh(I) complexes against
faecalis and Staph. aureus, followed by 48b with MIC values the Gram-positive bacteria and their diminished activity
of 25 and 12.5 µg/mL againt these bacteria, respectively. against the Gram-negative bacteria could be related to the
Complexes 46a and 46c-f showed some activity with MIC difference in their bacterial cell wall structure. Gram-negative
values ranging between 25 to 100 µg/mL, whereas 46b bacteria have an additional outer membrane protecting their
exhibited no activity against any of the bacterial organisms thin peptidoglycan layer, which could contribute to the
tested. Complexes 47a-j also exhibited limited to no activity. reduced activity against them due to limited penetration of
In particular, complexes 47a, 47b, and 47f were completely the metal complexes through the membrane.
inactive with antibacterial MIC values of 800 or more. More
significant activity against Gram-positive bacteria and fungi 6. Palladium
was observed with complexes 47c, 47d, 47e, 47g, 47h, 47i,
and 47j. None of the Ru(II) complexes mentioned exhibited 6.1. Medicinal Uses of Palladium Compounds
any activity against the Gram-negative bacteria E. coli.
It is worth noting that the investigators looked into the Palladium, being structurally similar to platinum, has been
antimicrobial efficacy of other ruthenium(II) complexes explored as a potential alternative to treat several types of
bearing other nitrogen-donating ligands; however the carbene cancers unresponsive to current chemotherapeutic treatments.
derivatives showed more pronounced activity. Interestingly, Several different palladium complexes were investigated for
more potent activity within this group of compounds was their antiproliferative activity against tumor cells.122-125
observed with those bearing more lipophilic substituents Amtmann and co-workers investigated the antitumor
compared with their analogues. Lipophilic side chains activity of Pd-xanthate complexes on two human cancer
evidently enhance the capability of antimicrobial agents to cell lines, Calu-6 (lung adenocarcinoma) and MCF-7 (mamma
cross the bacterial cellular membrane, thus providing an carcinoma).125 The palladium complexes 53-55, shown in
important insight for the future design of efficacious anti- Chart 10, exhibited superior activity compared with other
microbials that is consistent with previous studies.26 metal-xanthate complexes tested.
As mentioned previously, the antimicrobial efficacy of The IC50 values ranged between 0.1 and 0.47 µM in Calu-6
Rh(I) carbene complexes was also evaluated. The synthetic and between 0.45 and 1.17 µM in MCF-7 cells (Table 12).
procedure followed that of the Ru(II) carbene complexes Further studies on the differential activity of complexes

Figure 26. HeLa cells incubated with 5 and 10 µM of 57 for 24 h and then visualized with propidium iodide staining. Dead or dying cells
are stained red due to the penetration of propidium iodide through the cell membrane, whereas the control cells lack the stain indicating
their viability.73 Scale bar represents 10 µM.
3882 Chemical Reviews, 2009, Vol. 109, No. 8 Hindi et al.

(53-55) on cell lines Calu-6 and MCF-7 at pH 6.8 and 7.4 6.2.1. Mechanism of Palladium-NHC Complex Activity
(pH of normal cells) proved the enhanced cytotoxicity of
the Pd complexes under slightly acidic conditions, namely, The properties of the Pd(II) complex 57 are of interest
pH 6.8. This is of interest since this is a common occurrence due to its enhanced in Vitro activity against the tumor cell
in solid tumors due to the production of lactic acid. lines tested compared with cisplatin, the most widely used
chemotherapeutic agent. To further understand the mecha-
Gonzalez conducted DNA-binding studies of two Pd(II) nistic pathway employed by 57, HeLa cells were incubated
complexes vs cisplatin. They concluded that their Pd(II) with different concentrations of 57 for 24 h. The goal was
complexes interact with DNA at a faster rate than cisplatin to study the effects of 57 on the cell cycle because cisplatin
and produce the same amount of DNA destabilization at and other analogous Pt(II) complexes are known to induce
lower concentrations.126 Furthermore, a comparative study DNA damage by arresting the cells in the G2/M phase of
evaluating the biological activity of a trans-Pd(II) complex the cell cycle and thus hinder their growth.139,140 Two
bearing a pyrazole containing ligand confirmed its enhanced proteins, cdc2 (also known as cdk1) and cyclin B1, are
cytotoxicity against two human leukemia cell lines, HL-60 known to regulate the progression of cells from the G2 to
and NALM-6, compared with its cis-Pt(II) counterpart with the M phase. Cdc2 is a member of a family of kinases called
the same ligand system.127 Several other palladium(II) cyclin-dependent protein kinases (CDKs), whose role is to
complexes with nitrogen-containing ligands, such as mediate stages of mitosis. Cdc regulates entry into mitosis.
Pd-pyridine126-129 and Pd-amine130-134 complexes have It is phosphorylated on Thr14 and Tyr15 residues prior to
been shown to possess antitumor activity.
its association with the protein cyclin B1 to form an inactive
For more comprehensive and recent reviews on the complex. This complex is inactive because the phosphory-
medicinal developments of palladium metal complexes and lation of the 14 and 15 residues blocks the ATP binding site
their use as chemotherapeutic agents, the reader is directed of the kinase. During transition from the G2 to the M phase,
to the recent contributions by Abdalla135 and Caires.136 the kinase is activated by dephosphorylating Thr14 and Tyr15
residues.
6.2. Synthesis and Antitumor Properties of The HeLa cells were stained with antibodies against cyclin
Palladium-NHC Complexes B1 and phospho-cdc2 in order to evaluate the ability of
Although the biological activity of Pd-based drugs bearing complex 57 to regulate these two proteins and in turn induce
a variety of ancillary ligands has been under intense cell cycle arrest. The results of the study revealed that 57
investigation, only one report of Pd-NHC complexes with caused overexpression of cyclin B1 in the cells (Figure 24),
anticancer activity is found in recent literature. Ghosh took which suggests G2/M arrest and induction of apoptosis.141
the initiative of exploring the cytotoxic capacity of two Pd This only reveals that 57 caused the arrest of the HeLa cells
complexes, (NHC)Pd(pyridine)Cl2 (56) and (NHC)2PdCl2 in the G2/M phase because cyclin B1 is specific to the G2/M
(57) against three human tumor cell lines in Vitro.73 phase in general. The phospho-cdc2 antibody is particularly
specific to the G2 phase, and it revealed the increase in cdc2
Complex 56 was synthesized by the direct reaction of
phophorylation, thus inactivation of cdc2, after treatment with
1-benzyl-3-tert-butylimidazolium chloride with PdCl2 in
pyridine. Complex 57 was obtained by a transmetalation 20 µM 57, subsequently proving that 57-treated cells are
route, employing the previously discussed silver complex arrested in the G2 phase (Figure 25).
18 and (COD)PdCl2 (Scheme 14). Both complexes were Additional studies revealed the ensuing apoptotic cell death
evaluated using 13C NMR, which confirmed the presence of following G2 arrest. Live and dead cells treated with 57 at
the NCN-Pd metal bond by the appearance of resonances at different concentrations (0, 5, and 10 µM) for 24 h were
151.4 ppm and 166.9 ppm for complexes 56 and 57, visualized using propidium iodide staining (Figure 26).
respectively. These shifts fall within the range (δ 175-145 Propidium iodide is excluded by viable cells but can penetrate
ppm) of other Pd-NHC metal complexes.137 the cell membrane of dying or already dead cells. Cells
The formation of the two metal complexes was further treated with 57 stained positive for propidium iodide after
proved by X-ray diffraction studies (Figures 21 and 22). The 24 h, which indicates that the cells were either in late
Pd-metal centers of both complexes were shown to exist apoptosis or had undergone necrosis. The control cells lacked
in square-planer geometries and the substituents on the N-1 propidium iodide staining, indicating that they were still
and N-3 positions of both NHC ligands are oriented trans viable.
with respect to each other.
The Pd-NHC complexes 56 and 57 were evaluated for 7. Conclusion
their antitumor activity in terms of the percent inhibition of It is evident from the chemistry highlighted in this section
HeLa cell proliferation, and the results are summarized in that metal-based pharmaceuticals are highly sought-after for
Table 13. Complex 57, the more cytotoxic of the two Pd their antitumor or antimicrobial properties. There is relatively
complexes, was compared with cisplatin (Table 14). little information known about how metal-based drugs
The results revealed that 57 inhibited the growth of HeLa function and therefore many studies have been aimed at
(cervical cancer), MCF-7 (breast cancer), and HCT 116 exploring the mechanistic pathways employed by these drugs.
(colon adenocarcinoma) cells rather potently in a concentra- One interesting trend observed among some metals such as
tion-dependent manner (Table 14, Figure 23). The cell ruthenium and silver is that each one utilizes multiple
proliferation was measured using the sulforhodamine B biological mechanisms and can work by a variety of different
(SRB) assay, in which tumor cells are incubated with routes. This multifaceted approach could perhaps contribute
different concentrations of the test compound for one cell to their enhanced activity compared with other metals such
cycle.138 Figure 23 shows that 57 has a stronger inhibition as rhodium and low occurrence of resistance toward them
effect compared with cisplatin under similar conditions. compared with platinum.
Medicinal Imidazolium Carbene-Metal Complexes Chemical Reviews, 2009, Vol. 109, No. 8 3883

The pharmaceutical application of NHCs and their metal (22) Pernak, J.; Skrzypczak, A. Eur. J. Med. Chem. 1996, 31, 901–903.
(23) Andrews, J. M. J. Antimicrob. Chemother. 2001, 48, 5–16.
complexes is a relatively new area that has been gaining (24) Pernak, J.; Sobaszkiewicz, K.; Mirska, I. Green Chem. 2003, 5, 52–
interest and has been explored by only a handful of 56.
researchers. NHCs are a versatile class of ligands that can (25) Cetinkaya, E.; Denizci, A.; Ozdemir, I.; Ozturk, H. T.; Karaboz, I.;
be manipulated easily. They possess the ability to bind to Cetinkaya, B. J. Chemother. 2002, 14, 241–245.
(26) Demberelnyamba, D.; Kim, K.-S.; Choi, S.; Park, S.-Y.; Lee, H.;
both hard and soft metals and can be readily functionalized, Kim, C.-J.; Yoo, I.-D. Bioorg. Med. Chem. 2004, 12, 853–857.
which is a promising aspect in terms of designing suitably (27) Klasen, H. J. Burns 2000, 26, 117–130.
targeted pharmaceuticals. The lipophilicity of NHCs and (28) Silver, S.; Phung, L.; Silver, G. J. Ind. Microbiol. Biotechnol. 2006,
most of their metal complexes seems to be important in 33, 627–634.
(29) Moyer, C. A.; Brentano, L.; Gravens, D. L.; Margraf, H. W.; Monafo,
contributing to both their antimicrobial and antitumor effects W. W. Arch. Surg. 1965, 90, 812–867.
as in the case of some imidazolium salts, gold(I)-NHCs, (30) Fox, C. L. Arch. Surg. 1968, 96, 184–188.
and ruthenium(II)-NHCs. Silver NHCs seem to be the most (31) Melaiye, A.; Youngs, W. Expert Opin. Ther. Pat. 2005, 15, 125–
efficacious in terms of their antimicrobial activity and low 130.
(32) Klasen, H. J. Burns 2000, 26, 131–138.
toxicity compared with other antimicrobial metal-NHCs (33) Fakhry, S. M.; Alexander, J.; Smith, D.; Meyer, A. A.; Petterson,
including Ru(II) and Rh(I). H. D. J. Burn Care Rehabil. 1995, 16, 86–90.
(34) Russell, A. D.; Hugo, W. B. Prog. Med. Chem. 1994, 31, 351–370.
(35) Lansdown, A. B. J. Wound Care 2002, 11, 125–130.
8. Acknowledgments (36) Feng, Q. L.; Wu, J.; Chen, G. Q.; Cui, F. Z.; Kim, T. N.; Kim, J. O.
J. Biomed. Mater. Res. 2000, 52, 662–668.
Author K.M.H. would like to thank Dr. Douglas A. (37) Modak, S. M.; Fox, C. L., Jr. Biochem. Pharmacol. 1973, 22, 2391–
Medvetz for helpful discussions in the preparations of this 2404.
manuscript. Also, the authors would like to thank Dr. Michael (38) Fox, C.; Modak, S. Antimicrob. Agents Chemother. 1974, 5, 582–
588.
J. Taschner, Dr. Daniel Ely, and Dr. Peter L. Rinaldi for (39) Holt, K.; Bard, A. Biochemistry 2005, 44, 13214–13223.
useful discussions in the preparation of this review. The (40) Bragg, P. D.; Rainnie, D. J. Can. J. Microbiol. 1974, 20, 883–889.
authors would like to thank the National Institute of Allergies (41) Schreurs, W. J.; Rosenberg, H. J. Bacteriol. 1982, 152, 7–13.
and Infectious Diseases (Grant 1 R01 A106785601) for (42) Lansdown, A. B. J. Wound Care 2002, 11, 173–177.
(43) Drake, P. L.; Hazelwood, K. Ann. Occup. Hyg. 2005, 49, 575–585.
support during the preparation of this manuscript. (44) East, B. W.; Boddy, K.; Williams, E. D.; Macintyre, D.; Mclay, A. L.
Clin. Exp. Dermatol. 1980, 5, 305–311.
9. Note Added after ASAP Publication (45) Lee, S. M.; Lee, S. H. J. Dermatol. 1994, 21, 50–53.
(46) Greene, R. M.; Su, W. P. Am. Fam. Physician 1987, 36, 151–154.
Co-author Claire A. Tessier was added to the manuscript (47) Demling, R. H.; Disanti, L. Wounds 2001, 13 (Suppl A), 5–15.
(48) Baldi, C.; Minoia, C.; Di Nucci, A.; Capodaglio, E.; Manzo, L.
and her name was removed from the Acknowledgments Toxicol. Lett. 1988, 41, 261–268.
section. This paper originally posted to the web on July 6, (49) Hussain, S.; Anner, R. M.; Anner, B. M. Biochem. Biophys. Res.
2009, and reposted on July 24, 2009. Commun. 1992, 189, 1444–1449.
(50) Fraser, J. F.; et al. ANZ J. Surg. 2004, 74, 139–142.
(51) Liu, J.; Kershaw, W. C.; Klaassen, C. D. Toxicol. Appl. Pharmacol.
10. References 1991, 107, 27–34.
(52) Baldi, C.; Minoia, A. C.; DiNucci, A.; Capodaglio, E.; Manzo, L.
(1) Öfele, K. J. Organomet. Chem. 1968, 12, P42–P43. Toxicol. Lett. 1988, 41, 261–268.
(2) Wanzlick, H.-W.; Schönberr, H.-J. Angew. Chem., Int. Ed. Engl. 1968, (53) Hidalgo, E.; Dominguez, C. Toxicol. Lett. 1998, 98, 169–79.
7, 141–142. (54) Owens, C. J.; Yarbrough, D. R.; Brackett, N. C. Arch. Intern. Med.
(3) Arduengo, A. J., III; Harlow, R. L.; Kline, M. J. Am. Chem. Soc. 1974, 134, 332–335.
1991, 113, 361–363. (55) Fullar, F. W.; Engler, P. E. J. Burn Care Rehabil. 1988, 9, 606–609.
(4) Herrmann, W. A. Angew. Chem., Int. Ed. 2002, 41, 1290–1309. (56) Jerrett, F.; Ellerbe, S.; Demling, R. Am. J. Surg. 1978, 135, 818–
(5) Bourissou, D.; Guerret, O.; Gabbai, F. P.; Bertrand, G. Chem. ReV. 819.
2000, 100, 39–91. (57) McHugh, S. L.; Moellering, R. C.; Hopkins, C. C.; Swartz, M. N.
(6) Herrmann, W. A.; Kocher, C. Angew. Chem., Int. Ed. Engl. 1997, Lancet 1975, 1, 235–240.
36, 2162–2187.
(58) Gupta, A.; Silver, S. Nat. Biotechnol. 1998, 16, 888.
(7) Herrmann, W. A.; Goossen, L. J.; Spiegler, M. Organometallics 1998,
(59) Silver, S.; Lo, J-F.; Gupta, A. APUA News 1999, 17, 1–3.
17, 2162–2168.
(8) McGuinness, D. S.; Cavell, K. J.; Skelton, B. W.; White, A. H. (60) Gupta, A.; Matsui, K.; Lo, J-F.; Silver, S. Nat. Med. 1999, 5, 183–
Organometallics 1999, 18, 1596–1605. 188.
(9) Hu, X.; Castro-Rodriguez, I.; Olsen, K.; Meyer, K. Organometallics (61) Silver, S. FEMS Microbiol. ReV. 2003, 27, 341–353.
2004, 23, 755–764. (62) Garrison, J. C.; Youngs, W. J. Chem. ReV. 2005, 105, 3978–4008.
(10) Nemcsok, D.; Wichmann, K.; Frenking, G. Organometallics 2004, (63) Melaiye, A.; Simons, R. S.; Milsted, A.; Pingitore, F.; Wesdemiotis,
23, 3640–3646. C.; Tessier, C. A.; Youngs, W. J. J. Med. Chem. 2004, 47, 973–977.
(11) Samantaray, M. K.; Roy, D.; Patra, A.; Stephen, R.; Saikh, M.; Sunoj, (64) Melaiye, A.; Sun, Z.; Hindi, K.; Milsted, A.; Ely, D.; Reneker, D. H.;
R. B.; Ghosh, P. J. Organomet. Chem. 2006, 691, 3797–3805. Tessier, C. A.; Youngs, W. J. J. Am. Chem. Soc. 2005, 127, 2285–
(12) Samantaray, M. K.; Katiyar, V.; Roy, D.; Pang, K.; Nanavati, H.; 2291.
Stephen, R.; Sunoj, R. B.; Ghosh, P. Eur. J. Inorg. Chem. 2006, (65) Reneker, D. H.; Yarin, A. L.; Fong, H.; Koombhongse, S. J. Appl.
2975–2984. Phys. 2000, 87, 4531–4574.
(13) Ray, L.; Shaikh, M. M.; Ghosh, P. Dalton Trans. 2007, 4546–4555. (66) Kascatan-Nebioglu, A.; Melaiye, A.; Hindi, K.; Durmus, S.; Panzner,
(14) Ray, L.; Barman, S.; Shaikh, M. M.; Ghosh, P. Chem.sEur. J. 2008, M.; Hogue, L.; Mallett, R.; Hovis, C.; Coughenour, M.; Crosby, S.;
6646–6655. Milsted, A.; Ely, D.; Tessier, C.; Cannon, C.; Youngs, W. J. Med.
(15) Ray, L.; Shaikh, M. M.; Ghosh, P. Inorg. Chem. 2008, 47, 230–240. Chem. 2006, 49, 6811–6818.
(16) Samantaray, M. K.; Pang, K.; Shaikh, M. M.; Ghosh, P. Inorg. Chem. (67) Cropp, G. J. Am. J. Med. 1996, 100 (1A), 19S–29S.
2008, 47, 4153–4165. (68) Osman, F.; McCready, S. Mol. Gen. Genet. 1998, 260, 319–334.
(17) Baba, E.; Cundari, T. R.; Firkin, I. Inorg. Chim. Acta 2005, 358, (69) Abratt, V. R.; Peak, M. J.; Peak, J. G.; Santangelo, J. D.; Woods,
2867–2875. D. R. Can. J. Microbiol. 1990, 36, 490–494.
(18) Lee, M.-T.; Hu, C.-H. Organometallics 2004, 23, 976–983. (70) Selby, C. P.; Sancar, A. Prog. Clin. Biol. Res. 1990, 340A, 179–
(19) Nemcsok, D.; Wichmann, K.; Frenking, G. Organometallics 2004, 193.
23, 3640–3646. (71) Hindi, K.; Siciliano, T.; Durmus, S.; Panzner, M.; Medvetz, D.;
(20) Vyboishchikov, S. F.; Frenking, G. Chem.sEur. J. 1998, 4, 1439– Reddy, V.; Hogue, L.; Hovis, C.; Hilliard, J.; Mallett, R.; Tessier,
1448. C.; Cannon, C.; Youngs, W. J. Med. Chem. 2008, 51, 1577–1583.
(21) Frenking, G.; Pidun, U. J. Chem. Soc., Dalton Trans. 1997, 1653– (72) Viciano, M.; Mas-Marza, E.; Sanau, M.; Peris, E. Organometallics
1662. 2006, 25, 3063–3069.
3884 Chemical Reviews, 2009, Vol. 109, No. 8 Hindi et al.

(73) Ray, S.; Mohan, R.; Singh, J. K.; Samantaray, M. K.; Shaikh, M. M.; (108) Yasbin, R. E.; Matthews, C. R.; Clarke, M. J. Chem.-Biol. Interactions
Panda, D.; Ghosh, P. J. Am. Chem. Soc. 2007, 129, 15042–15053. 1980, 31, 355–365.
(74) Medvetz, D. A.; Hindi, K. M.; Panzner, M. J.; Ditto, A. J.; Yun, (109) Morris, R. E.; Aird, R. E.; Murdoch, P. S.; Chen, H.; Cummings, J.;
Y. H.; Youngs, W. J. Met.-Based Drugs 2008, 2008, 384010. Hughes, N. D.; Parsons, S.; Parkin, A.; Boyd, G.; Jodrell, D. I.;
(75) Barnard, P.; Wedlock, L.; Baker, M.; Berners-Price, S.; Joyce, D.; Sadler, P. J. J. Med. Chem. 2001, 44, 3616–3621.
Skelton, B.; Steer, J. Angew. Chem., Int. Ed. 2006, 45, 5966–5970. (110) Hotze, A. C. G.; Bacac, M.; Velders, A. H.; Jansen, B. A. J.;
(76) Barnard, P.; Baker, M.; Berners-Price, S.; Day, D. J. Inorg. Biochem. Kooijman, H.; Spek, A. L.; Haasnoot, J. G.; Reedijk, J. J. Med. Chem.
2004, 98, 1642–1647. 2003, 46, 1743–1750.
(77) Berners-Price, S. J.; Johnson, R. K.; Giovenella, A. J.; Faucette, L. F.; (111) Vliet, P. M.; Toekimin, S. M. S.; Haasnoot, J. G.; Reedijk, J.;
Mirabelli, C. K.; Sadler, P. J. J. Inorg. Biochem. 1988, 33, 285–295. Novakova, O.; Vrana, O.; Brabec, V. Inorg. Chim. Acta 1995, 231,
(78) Thati, B.; Noble, A.; Creaven, B.; Walsh, M.; McCann, M.; 57–64.
Kavanagh, K.; Devereux, M.; Egan, D. Cancer Lett. 2007, 248, 321– (112) Chifotides, H. T.; Dunbar, K. R. Acc. Chem. Res. 2005, 38, 146–
331. 156.
(79) Zhu, H.; Zhang, X.; Liu, X.; Wang, X.; Liu, G.; Usman, A.; Fun, H. (113) Mestroni, G.; Alessio, E.; Santi, A. S.; Geremia, S.; Bergamo, A.;
Inorg. Chem. Commun. 2003, 6, 1113–1116. Sava, G.; Boccarelli, A.; Schettino, A.; Coluccia, M. Inorg. Chim.
(80) Liu, J.; Galettis, P.; Farr, A.; Maharaj, L.; Samarasinha, H.; Acta 1998, 273, 62–71.
McGechan, A.; Baguley, B.; Bowen, R.; Berners-Price, S.; McKeage, (114) Qu, P.; He, H.; Liu, X. Huaxue Jinzhan 2006, 18, 1646–1651.
M. J. Inorg. Biochem. 2008, 102, 303–310. (115) Bruijnincx, P. C. A.; Sadler, P. J. Curr. Opin. Chem. Biol. 2008, 12,
(81) Elsome, A. M.; Hamilton-Miller, J. M. T.; Brumfitt, W.; Noble, W. C. 197–206.
J. Antimicrob. Chemother. 1996, 37, 911–918. (116) Reedijk, J. Platinum Met. ReV. 2008, 52, 2–11.
(82) Berners-Price, S. J.; Mirabelli, C. K.; Johnson, R. K.; Mattern, M. R.; (117) Tselepi-Kalouli, E.; Katsaros, N.; Sideris, E. Inorg. Chem. Acta 1986,
McCabe, F. L.; Faucette, L. F.; Sung, C-M.; Mong, S-M.; Sadler, 124, 181–186.
P. J.; Crooke, S. T. Cancer Res. 1986, 46, 5486–5493. (118) Kapitza, S.; Pongratz, M.; Jakupec, M. A.; Heffeter, P.; Berger, W.;
(83) Hoke, G. D.; Macia, R. A.; Meunier, P. C.; Bugelski, P. J.; Mirabelli, Lackinger, L.; Keppler, B. K.; Marian, B. J. Cancer Res. Clin. Oncol.
C. K.; Rush, G. F.; Matthews, W. D. Toxicol. Appl. Pharmacol. 1989, 2005, 131, 101–110.
100, 293–306. (119) Frasca, D.; Ciampa, J.; Emerson, J.; Umans, R. S.; Clarke, M. J.
Met.-Based Drugs 1996, 3, 197–209.
(84) McKeage, M. J.; Berners-Price, S. J.; Galettis, P.; Bowen, R. J.;
(120) Cetinkaya, B.; Ozemir, I.; Binbasioglu, B.; Durmaz, R.; Gunal, S.
Brouwer, W.; Ding, L.; Zhuang, L.; Baguley, B. C. Cancer
Arzneim.-Forsch./Drug Res. 1999, 49, 538–540.
Chemother. Pharmacol. 2000, 46, 343–350.
(121) Cetinkaya, B.; Cetinkaya, E.; Kucukbay, H.; Durmaz, R. Arzneim.-
(85) Fricker, S. P. Gold Bull. 1996, 29, 53–60.
Forsch./Drug Res. 1996, 46, 821–823.
(86) Merchant, B. Biologicals 1998, 26, 49–59. (122) Navarro, M.; Penã, N. P.; Colmenares, I.; González, T.; Arsenak,
(87) Shaw, C. F., III Chem. ReV. 1999, 99, 2589–2600. M.; Taylor, P. J. Inorg. Biochem. 2006, 100, 152–157.
(88) Simon, T. M.; Kunishima, D. H.; Vibert, G. J.; Lorber, A. Cancer (123) Friebolin, W.; Schilling, G.; Zoller, M.; Amtmann, E. J. Med. Chem.
Res. 1981, 41, 94–97. 2005, 48, 7925–7931.
(89) Mirabelli, C. K.; Johnson, R. K.; Sung, C. M.; Faucette, L. F.; (124) Gonzalez, M. L.; Tercero, J. M.; Matilla, A.; Niclos-Gutierrez, J.;
Muirhead, K.; Crooke, S. T. Cancer Res. 1985, 45, 32–39. Fernandez, M. T.; Lopez, M. C.; Alonso, C.; Gonzalez, S. Inorg.
(90) McKeage, M. J.; Maharaj, L.; Berners-Price, S. J. Coord. Chem. ReV. Chem. 1997, 36, 1806–1812.
2002, 232, 127–135. (125) Budzisz, E.; Krajewska, U.; Rozalski, M.; Szulawska, A.; Czyz, M.;
(91) McKeage, M. J. Br. J. Pharmacol. ReV. 2002, 136, 1081–1082. Nawrot, B. Eur. J. Pharmacol. 2004, 502, 59–65.
(92) Rigobello, M. P.; Scutari, G.; Folda, A.; Bindoli, A. Biochem. (126) Kovala-Demertzi, D.; Demertzis, M. A.; Filiou, E.; Pantazaki, A. A.;
Pharmacol. 2004, 67, 689–696. Yadav, P. N.; Miller, J. R.; Zheng, Y.; Kyriakidis, D. A. BioMetals
(93) Barnard, P. J.; Berners-Price, S. J. Coord. Chem. ReV. 2007, 251, 2003, 16, 411–418.
1889–1902. (127) Kuduk-Jaworska, J.; Puszko, A.; Kubiak, M.; Pełczynska, M. J. Inorg.
(94) Urig, S.; Fritz-Wolf, K.; Reau, R.; Herold-Mende, C.; Toth, K.; Biochem. 2004, 98, 1447–1456.
Davioud-Charvet, E.; Becker, K. Angew. Chem., Int. Ed. 2006, 45, (128) Zhao, G.; Lin, H.; Yu, P.; Sun, H.; Zhu, S.; Su, X.; Chen, Y. J. Inorg.
1881–1886. Biochem. 1999, 73, 145–149.
(95) Cronje, S.; Raubenheimer, H. G. Chem. Soc. ReV. 2008, 37, 1998– (129) Kovala-Demertzi, D.; Boccarelli, A.; Demertzis, M. A.; Coluccia,
2011. M. Chemotherapy 2007, 53, 148–152.
(96) Elsome, A. M.; Hamilton-Miller, J. M. T.; Brumfitt, W.; Nobble, (130) Friaza, G. G.; Fernández-Botello, A.; Pérez, J. M.; Prieto, M. J.;
W. C. J. Antimicrob. Chemother. 1996, 37, 911–918. Moreno, V. J. Inorg. Biochem. 2006, 100, 1368–1377.
(97) Nomiya, K.; Noguchi, R.; Ohsawa, K.; Tsuda, K.; Oda, M. J. Inorg. (131) Ruiz, J.; Cutillas, N.; Vicente, C.; Villa, M. D.; López, G. Inorg.
Biochem. 2000, 78, 363–370. Chem. 2005, 44, 7365–7376.
(98) Özdemir, I.; Denizci, A.; Öztürk, T. H.; Çetinkaya, B. Appl. (132) Abu-Surrah, A. S.; Al-Allaf, T. A. K.; Rashan, L. J.; Klinga, M.;
Organometal. Chem. 2004, 18, 318–322. Leskelä, M. Eur. J. Med. Chem. 2002, 37, 919–922.
(99) Baker, M. V.; Barnard, P. J.; Berners-Price, S. J.; Brayshaw, S. K.; (133) Faraglia, G.; Fregona, D.; Sitranb, S.; Giovagninia, l.; Marzanoc,
Hickey, J. L.; Skelton, B. W.; White, A. H. Dalton Trans. 2006, C.; Baccichetti, F.; Casellato, U.; Graziani, R. J. Inorg. Biochem.
3708–3715. http://dx.doi.org/10.1039/b602560a. 2001, 83, 31–40.
(100) Barnard, P. J.; Baker, M. V.; Berners-Price, S. J.; Skelton, B. W.; (134) Suvachittanont, S.; Hohmann, H.; van Eldik, R.; Reedijk, J. Inorg.
White, A. H. Dalton Trans. 2004, 1038–1047. Chem. 1993, 32, 4544–4548.
(101) Baker, M. V.; Barnard, P. J.; Berners-Price, S. J.; Brayshaw, S. K.; (135) Abu-Surrah, A. S.; Al-Sa’doni, H. H.; Abdalla, M. Y. Cancer Ther.
Hickey, J. L.; Skelton, B. W.; White, A. H. J. Organomet. Chem. 2008, 6, 1–10.
2005, 690, 5625–5635. (136) Caires, A. C. F. Anti Canc. Agents Med. Chem. 2007, 7, 484–491.
(102) Horvath, U. E. I.; Bentivoglio, G.; Hummel, M.; Schottenberger, H.; (137) Hermann, W. A.; Bohn, V. P. W.; Gstottmayr, C. W. K.; Grosche,
Wurst, K.; Nell, M. J.; van Rensburg, C. E. J.; Cronje, S.; M.; Reisinger, C.-P.; Weskamp, T. J. Organomet. Chem. 2001, 617-
Raubenheimer, H. G. New J. Chem. 2008, 32, 533–539. 618, 616–628.
(103) Jellicoe, M. M.; Nichols, S. J.; Callus, B. A.; Baker, M. V.; Barnard, (138) Gupta, K.; Bishop, J.; Peck, A.; Brown, J.; Wilson, L.; Panda, D.
P. J.; Berners-Price, S. J.; Whelan, J.; Yeoh, G. C.; Filipovska, A. Biochemistry 2004, 43, 6645–6655.
Carcinogenesis 2008, 29, 1124–1133. (139) Mueller, S.; Schittenhelm, M.; Honecker, F.; Malenke, E.; Lauber,
(104) Hickey, J. L.; Ruhayel, R. A.; Barnard, P. J.; Baker, M. V.; Berners- K; Wesselborg, S.; Hartmann, J. T.; Bokemeyer, C.; Mayer, F. Int.
Price, S. J.; Filipovska, A. J. Am. Chem. Soc. 2008, 130, 12570– J. Oncol. 2006, 29, 471–479.
12571. (140) Billecke, C.; Finniss, S.; Tahash, L.; Miller, C.; Mikkelsen, T.; Farell,
(105) Alessio, E.; Mestroni, G.; Bergamo, A.; Sava, G. Curr. Top. Med. N. P.; Bogler, O. Neuro Oncol. 2006, 8, 215–226.
Chem. 2004, 4, 1525–1535. (141) Hagting, A.; Karlsson, C.; Clute, P.; Jackman, M.; Pines, J. EMBO
(106) Clarke, M.; Zhu, F.; Frasca, D. Chem. ReV. 1999, 99, 2511–2534. J. 1998, 17, 4127–4138.
(107) Keppler, B. K.; Rupp, W. J. Cancer Res. Clin. Oncol. 1986, 111,
166–168. CR800500U