# Anticancer Activity of Ag(I) N-Heterocyclic Carbene Complexes Derived from 4,5-Dichloro-1H-Imidazole

Authors: Doug A. Medvetz; Khadijah M. Hindi; Matthew J. Panzner; Andrew J. Ditto; Yang H. Yun; Wiley J. Youngs
Journal: Metal-Based Drugs
 (2008)

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## Abstract

A class of Ag(I) N-heterocyclic carbene silver complexes,1–3, derived from 4,5-dichloro-1H-imidazole has been evaluated for their anticancer activity against the human cancer cell lines OVCAR-3 (ovarian), MB157 (breast), and Hela (cervical). Silver complexes 1–3 are active against the ovarian and breast cancer cell lines. A preliminary in vivo study shows 1 to be active against ovarian cancer in mice. The results obtained in these studies warrant further investigation of these compounds in vivo.


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## Body

## 1. Introduction

Research
efforts into the development of new chemotherapeutic anticancer agents play an
important role in the future treatment of cancer.  The divergence from platinum-based agents is
becoming a point of emphasis with the goal being to find drugs that are as
effective as platinum without the severity of side effects.  Metallocene dichlorides and dirhodium
carboxylates have shown anticancer activity, however neither class of compounds
has demonstrated sufficient effectiveness to pursue past phase II clinical
trials [1, 2].Recently,
silver complexes have been reported to have anticancer activity in vitro. Egan has reported that silver complexes of
coumarin derivatives possess anticancer activity against certain types of
cancer [3]. Zhu has reported that silver carboxylate
dimers possess anticancer activity against human carcinoma cells [4]. McKeage has shown phosphine complexes of
silver to be active anticancer agents, even against cisplatin resistant cell
lines [5].Research
into using silver is essential as toxicity is believed to be quite low. In fact, silver has been detected in 29 human
tissues in trace amounts; however, there is no known physiological function for
silver. In vitro studies have shown that silver salts do have an effect
on dermal fibroblasts; however, the effect usually does not lead to cell death [6]. Also, silver is being used in vivo to coat foreign
materials. Artificial heart valves,
along with cardiac and urinary catheters, are being coated with silver in medical applications to reduce or prevent the infection rate of various microbes [7–10]. The problem with ingestion of silver is a permanent
discoloration of the skin known as argyria.
However, argyria is not believed to be harmful to the body physically and
it takes an excessive consumption of silver to develop this condition [11].Based on the
previously discussed reports of the antitumor activity of silver and our
expertise in silver N-Heterocyclic carbene complexes [12–15], along with other reported N-Heterocyclic carbene complexes possessing
anticancer activity [16–18], we have begun to examine the anticancer activity of Ag(I) complexes of N-Heterocyclic
carbenes. Herein, we report the
anticancer activity of three Ag(I)-N-Heterocyclic carbene complexes derived
from 4,5-dichloro-1H-imidazole. These
silver complexes appear to be stable to light and water making them viable candidates
for use as chemotherapeutic agents.


## 2. Results and Discussion

The imidazolium
salts and their silver acetate complexes1, 2, and 3 were synthesized similarly
to procedures in a previously published procedure from our group [19]. These silver complexes have been shown to be very stable and can be synthesized efficiently [19].Silver complexes of
type1, 2, and 3 have been chosen over the use of silver salts because of their
stability. The use of silver salts in vivo is not practical because the
free silver ions will form complexes with salts in the bloodstream.The
MTT assay was run against the cancer cell lines OVCAR-3 (ovarian), MB157
(breast), and Hela (cervical) to determine the in vitro anticancer efficacy of1–3, the imidazolium salt precursors, silver nitrate, and silver
acetate relative to that of Cisplatin.
Cancer cells were plated at cell densities of 5000 cells per well in
96-well plates and allowed to incubate overnight. The following day, the test compounds were
dissolved in DMSO [20] and diluted into the respective cell culture media to the
desired micromolar concentrations. The
media in the wells was replaced with fresh media containing test compound or
DMSO for control, and the cells were incubated for 72 hours. Following this test, period MTT protocol was
followed by adding 10 μL of MTT in PBS to each well, and the plates were
incubated for four hours. In viable,
metabolically active cells, MTT is reduced in the mitochondria by the enzyme
succinate dehydrogenase forming insoluble bluish purple formazan crystals. These formazan crystals are then resolublized
by addition of 100 μL of SDS in dilute HCl.
The optical density of each well is then read at 570 nm. The imidazolium salts showed no activity
against the cancer cell lines, while the silver complexes 1–3 were very efficacious against the ovarian and breast cancer
cell lines, while showing very minimal effect on the cervical cancer cell line. The IC50 concentrations for active
compounds, where IC50 stands for the concentration that causes a 50%
reduction in cell viability, are reported in Table 1.Table 1
IC50 concentrations of silver drugs.
OVCAR-3(a)MB157(a)Hela(a)Cisplatin122525AgNO335550AgOAc2012NA1358>20023020>20032010>200IC(a)50 concentrations are reported in micromolar. NA = not achievable due to solubility of AgOAc.To
determine the effect of silver complexes1–3 and Cisplatin on the morphology of the OVCAR-3 and MB157 cells in culture, phase
contrast pictures were taken of tested cell culture. The cells were dyed with Hoesch for clarity
purposes only. Cells were plated in 24-well
plates and allowed to grow to confluency. Test compounds were dissolved in DMSO
and diluted into the respective cell culture media to the desired micromolar
concentrations. The media in the wells
was replaced with fresh media containing test compound or DMSO for
control. Cells were incubated with test
compounds at 50 μM for 36 hours. Selected
results are shown as pictures in Figures 2–7.Figure 1
Imidazolium salts precursors of (a)1, (b) 2, (c) 3.Scheme 1Figure 2
OVCAR-3 control.Figure 3
OVCAR-3 incubated with Cisplatin.Figure 4
OVCAR-3 incubated with1.Figure 5
MB157 control.Figure 6
MB157 incubated with Cisplatin.Figure 7
MB157 incubated with1.The above described pictures show that these
silver compounds have significant effect on cell viability. These results qualitatively show that both
the OVCAR-3 and MB157 cell lines did not survive the exposure to1–3 at 50 μM. All of the silver complexes demonstrated
superior effectiveness as compared to Cisplatin at the same concentration.To
quantify the efficacy of the silver complexes, a live/dead cell assay was performed.
Selected fluorescence images are shown in Figures8–13. Cells were plated in 24-well plates and
allowed to grow to confluency. Test
compounds were dissolved in DMSO and diluted into the respective cell culture
media to the desired micromolar concentrations.
The media in the wells was replaced with fresh media containing test
compound or DMSO for control. Cells were
incubated with test compounds at 50 μM for 36 hours. Live cells fluoresced red due to their
metabolic activity that converted the stain C12-resazurin into
fluorescent C12-resorufin.
Dead cells accumulated the Sytox green stain since their cell membranes
had been compromised, resulting in green fluorescence. The silver complexes 1–3 produced significantly higher death among the OVCAR-3 cell
compared to Cisplatin and control OVCAR-3 cells (P<.0001). The viabilities of OVCAR-3 cells exposed to 1–3 were 11%, 0%, and 0%,
respectively. Meanwhile, OVCAR-3 cells
exposed to Cisplatin resulted in 78% viability, which was not significantly
different than the control cells 93% viability (P=.5579). All silver complexes and Cisplatin produced
significant death among MB157 breast cancer cells. The live/dead assay revealed 10% cell
viability for MB157 cells exposed to Cisplatin, which was significantly
different than the 92% viability for the MB157 control cells (P<.0001). Based on the live/dead assay, 1–3, showed significantly superior cell
lysing capabilities with ovarian cancer compared to Cisplatin. These silver complexes also were shown to
completely kill the breast cancer cell line MB157. Our findings suggest silver
complexes of this type may be excellent candidates for therapeutic drugs for
certain types of cancer.Figure 8
OVCAR-3 control.Figure 9
OVCAR-3 incubated with Cisplatin.Figure 10
OVCAR-3 incubated with1.Figure 11
MB157 control.Figure 12
MB157 incubated with Cisplatin.Figure 13
MB157 incubated with1.The cell viability percentages were
tallied from the live/dead assay data and shown in Figure14. The data from
microscopy and live/dead assay coincides with the MTT assay except for the Cisplatin
data. This discrepancy can be attributed
to the fact that Cisplatin takes longer incubation periods to be fully
effective against the cells lines. In
the MTT, assay cells had to be incubated for 72 hours to see maximum efficacy,
here the incubation time was only 36 hours.Figure 14
Percent viability of OVCAR-3 and MB157 after testing with cisplatin (Cis), silver acetate, (AgOAc), and silver nitrate (AgNO3), 1, 2, and 3. The percentages are based on cell counts from the live/dead assay data after cells were tested for 36 hours. Cells were incubated with test compounds at 50 μM.Due to the
activity of these silver complexes, in
vitro, a preliminary in vivo study was run to determine if1 was active against an ovarian cancer xenograft model. Ten million OVCAR-3
cells were injected subcutaneously into the back of female athymic nude
mice. Upon visible tumor growth,
approximately six weeks, silver complex 1 was injected subcutaneously at the tumor site every third day for ten
days. Each dose consisted of 333 mg/kg
of 1 for a total of 1000 mg/kg over
the ten day period. The high dose was
chosen because a parallel study was being run, where IP injections were given at
a dose of 100 mg/kg per day and it seemed relevant to give the same total dose
over this ten-day period.  Injections
were only given every third day because the compound absorbed more slowly in
the subcutaneous study. The mice were then necropsied to determine what effect 1 had on the tumors, as well as, on the
internal organs. Selected pictures of
the tumors, along with the internal organs, are shown in Figures 15–17. According to pathological results, 1 caused major cell death of the
tumors, however, showed no ill-effects to the major organs of the mice.Figure 15
Necrotic tumor mass.Figure 16
Normal tumor mass.Figure 17
View of the internal organs.


## 3. Conclusion

In
conclusion, we have shown a class of Ag(I) N-Heterocyclic carbenes to possess
anticancer activity, in vitro,
against ovarian (OVCAR-3) and breast (MB157) cancer; however, these silver
complexes have little effect on cervical (Hela) cancer. Each compound,1–3, seems to be more active against the breast cancer cell line
MB157 with IC50’s of around 10 μM.Preliminary in vivo studies show that1 is active against an ovarian cancer
xenograft model by subcutaneous injection.
Further in vivo studies
are underway to determine the effect of 1 on the ovarian cancer xenograft model, where the mode of delivery is
through intraperitoneal injection. These
findings indicate that N-Heterocyclic carbene silver complexes may be useful in
cancer chemotherapy depending on the type of cancer.


## 4. Experimental

### 4.1. General

4,5-dichloro-1H-imidazole was purchased from  TCI (Wellesly Hills, Ma, USA).
1-bromohexane was purchased from Aldrich (Louis Street, Mo ,USA). 2-bromomethylnaphthalene was purchased from Alfa (Ward Hill, Ma, USA). All chemicals were used without further
purification.  All mass spectrometry was performed at CCIC at The Ohio State
University.H1 and C13
NMR spectra were obtained on a Varian 300 MHz or Varian 500 MHz instrument. All elemental analyses were performed at the University of Illinois
at Urbana-Champaign. Pathology reports from the mouse study were
performed at BioReliance.


### 4.2. Cell Lines

The human
cancer cell lines OVCAR-3 and MB157 were purchased from ATCC (Manassas, Va, USA). The OVCAR-3 cell line was grown in RPMI 1640
media with 2 mM L-glutamine and modified to contain 10 mM HEPES, 1 mM sodium
pyruvate, 4.5 g/L glucose, and 1.5 g/L sodium bicarbonate and was supplemented
with .01 mg/mL bovine insulin and 20% fetal bovine serum. The cells were grown at37°C with
5%  CO2 and passed every 2-3 days. The MB157 cell line was grown in DMEM with 4 mM
L-glutamine modified to contain 4.5 g/L glucose and 1.5 g/L sodium bicarbonate
and supplemented with 10% fetal bovine serum.
The cells were grown at 37°C with 5%  CO2 and
passed every 2-3 days. The human cancer cell line Hela S3 was donated by Dr. Yang Yun of the Biomedical Engineering department at the University of Akron. The Hela cell line was grown in
DMEM/F-12K media supplemented with 1% antibiotic/antimicotic and 10% fetal
bovine serum. The cells were grown at 37°C with 5%  CO2 and passed once a week.


### 4.3. MTT Assay

The MTT
assay was purchased from Molecular Probes (Part of Invitrogen). The respective cell lines
were grown to confluency and plated in 96-well plates at 5000 cells per well in
triplicate and allowed to incubate overnight. Compounds1, 2, 3, the imidazolium salts precursors, Cisplatin,
silver acetate, and silver nitrate were dissolved in DMSO to a concentration
of 0.1 M and diluted into cell culture
media to the desired testing concentrations.
Media in each well was removed and replaced with the fresh media
containing test compounds. The test
compounds were allowed to incubate for 24, 48, or 72 hours after which the MTT
protocol was followed. A stock solution
of MTT was prepared by adding 1 mL of PBS to the preweighed vial containing
MTT. 10 μL of this stock solution were
added to each well and allowed to incubate for 4 hours. A stock solution of sodium dodecyl sulfate
was prepared by adding 10 mL of 0.01 M HCl to the preweighed SDS vial. Following the 4 hour incubation period, 100 μL
of the SDS : HCl solution were added and incubated overnight. The optical density was read at 570 nm on Molecular
Devices Spectramax M2 plate reader.


### 4.4. Microscopy

Cells were
plated at 50 000 cells per well in 24-well plates and the culture was allowed
to grow to confluency. Compounds1, 2, 3, and Cisplatin were dissolved in
DMSO to a concentration of 0.1 M and
diluted into cell culture media to the desired testing concentrations. Media in each well was removed and replaced
with the fresh media containing test compounds.
The test compounds were incubated for 36 hours. Afterwards, cells were rinsed with PBS and
fixed for 10 minutes with freshly prepared 0.5% formaldehyde solution. After fixation, cells were rinsed and
permeabilized with 0.2% Triton X solution for 10 minutes. Hoechst nuclear dye was prepared according to
the manufacturer’s recommendations and carefully applied to the cells. After 30 minutes of incubation, cells were
rinsed three times with PBS and visualized with fluorescence microscopy. All images were captured using AxioVision 200
by Zeiss with a 10x Plan Neofluor Zeiss objective and a high-resolution CCD HRm
camera. The flourescent and phase
contrast images were colocalized using the AxioVision software version 4.6.


### 4.5. Live/Dead Assay

The live/dead assay was purchased from Invitrogen (Carlsbad, Ca, USA). Cells were plated in 24-well plates. Compounds1, 2, 3, and Cisplatin were dissolved in DMSO
to a concentration of 0.1 M and diluted into cell culture media to the desired
testing concentrations. Media in each
well was removed and replaced with the fresh media containing test
compounds. The test compounds were
allowed to incubate for 36 hours. The live/dead
stains were then prepared by dissolving 40 μg of C12-Resazurin in
100 μL of DMSO as a 1 mM stock solution.
A 50 μM C12-Resazurin solution was then made up by diluting
2.5 μL of the stock solution into 47.5 μL of DMSO. The Sytox green solution was prepared by
diluting 5 μL of a 10 μM stock solution in 45 μL of DMSO. The staining solution was prepared by
diluting 48 μL of 50 μM C12-Resazurin and 48 μL of Sytox green stain
into 4.704 mL of 1X PBS for final concentrations of 500 nM. The cell media was then removed from culture,
and the cells were washed with 1X PBS.
Cells were then covered with 200 μL of live/dead staining solution and
incubated for 15 minutes at room temperature protected from light. Lastly, the cells were visualized using a
fluorescent microscope. Three random
images were taken per well, and the channels were combined, and the images were
processed using Axiovision software as previously described. Cell viabilities were found to be significant
using Tukey’s multiple comparison among means (α=0.05).


### 4.6. Ovarian Cancer Xenograft Model

Three-week-old female
athymic nude mice were purchased from Harlan (Indianapolis, In, USA) and housed for one week prior to the
experiment.  Ten million cells were
injected subcutaneously into the back of three animals, right below the
ear. The animals were monitored and the
weights were recorded 2-3 times per week
until the tumors grew to be visible (macroscopic tumors grew in about six
weeks). The next day, and every third
day following for ten days, the animals were injected with a dose of 333 mg/kg
of compound1 subcutaneously at the
tumor site for a total of 1000 mg/kg. After
ten days, the animals were sacrificed, and a full necropsy was performed. The brain, liver, lungs, heart, kidneys,
spleen, and tumors were sent for pathology report. Synthetic, NMR, and crystallographic data for the imidazolium salts and their silver acetate structures are available as Supplementary Material at doi:10.1155/2008/384010.


## 4.1. General

4,5-dichloro-1H-imidazole was purchased from  TCI (Wellesly Hills, Ma, USA).
1-bromohexane was purchased from Aldrich (Louis Street, Mo ,USA). 2-bromomethylnaphthalene was purchased from Alfa (Ward Hill, Ma, USA). All chemicals were used without further
purification.  All mass spectrometry was performed at CCIC at The Ohio State
University.H1 and C13
NMR spectra were obtained on a Varian 300 MHz or Varian 500 MHz instrument. All elemental analyses were performed at the University of Illinois
at Urbana-Champaign. Pathology reports from the mouse study were
performed at BioReliance.


## 4.2. Cell Lines

The human
cancer cell lines OVCAR-3 and MB157 were purchased from ATCC (Manassas, Va, USA). The OVCAR-3 cell line was grown in RPMI 1640
media with 2 mM L-glutamine and modified to contain 10 mM HEPES, 1 mM sodium
pyruvate, 4.5 g/L glucose, and 1.5 g/L sodium bicarbonate and was supplemented
with .01 mg/mL bovine insulin and 20% fetal bovine serum. The cells were grown at37°C with
5%  CO2 and passed every 2-3 days. The MB157 cell line was grown in DMEM with 4 mM
L-glutamine modified to contain 4.5 g/L glucose and 1.5 g/L sodium bicarbonate
and supplemented with 10% fetal bovine serum.
The cells were grown at 37°C with 5%  CO2 and
passed every 2-3 days. The human cancer cell line Hela S3 was donated by Dr. Yang Yun of the Biomedical Engineering department at the University of Akron. The Hela cell line was grown in
DMEM/F-12K media supplemented with 1% antibiotic/antimicotic and 10% fetal
bovine serum. The cells were grown at 37°C with 5%  CO2 and passed once a week.


## 4.3. MTT Assay

The MTT
assay was purchased from Molecular Probes (Part of Invitrogen). The respective cell lines
were grown to confluency and plated in 96-well plates at 5000 cells per well in
triplicate and allowed to incubate overnight. Compounds1, 2, 3, the imidazolium salts precursors, Cisplatin,
silver acetate, and silver nitrate were dissolved in DMSO to a concentration
of 0.1 M and diluted into cell culture
media to the desired testing concentrations.
Media in each well was removed and replaced with the fresh media
containing test compounds. The test
compounds were allowed to incubate for 24, 48, or 72 hours after which the MTT
protocol was followed. A stock solution
of MTT was prepared by adding 1 mL of PBS to the preweighed vial containing
MTT. 10 μL of this stock solution were
added to each well and allowed to incubate for 4 hours. A stock solution of sodium dodecyl sulfate
was prepared by adding 10 mL of 0.01 M HCl to the preweighed SDS vial. Following the 4 hour incubation period, 100 μL
of the SDS : HCl solution were added and incubated overnight. The optical density was read at 570 nm on Molecular
Devices Spectramax M2 plate reader.


## 4.4. Microscopy

Cells were
plated at 50 000 cells per well in 24-well plates and the culture was allowed
to grow to confluency. Compounds1, 2, 3, and Cisplatin were dissolved in
DMSO to a concentration of 0.1 M and
diluted into cell culture media to the desired testing concentrations. Media in each well was removed and replaced
with the fresh media containing test compounds.
The test compounds were incubated for 36 hours. Afterwards, cells were rinsed with PBS and
fixed for 10 minutes with freshly prepared 0.5% formaldehyde solution. After fixation, cells were rinsed and
permeabilized with 0.2% Triton X solution for 10 minutes. Hoechst nuclear dye was prepared according to
the manufacturer’s recommendations and carefully applied to the cells. After 30 minutes of incubation, cells were
rinsed three times with PBS and visualized with fluorescence microscopy. All images were captured using AxioVision 200
by Zeiss with a 10x Plan Neofluor Zeiss objective and a high-resolution CCD HRm
camera. The flourescent and phase
contrast images were colocalized using the AxioVision software version 4.6.


## 4.5. Live/Dead Assay

The live/dead assay was purchased from Invitrogen (Carlsbad, Ca, USA). Cells were plated in 24-well plates. Compounds1, 2, 3, and Cisplatin were dissolved in DMSO
to a concentration of 0.1 M and diluted into cell culture media to the desired
testing concentrations. Media in each
well was removed and replaced with the fresh media containing test
compounds. The test compounds were
allowed to incubate for 36 hours. The live/dead
stains were then prepared by dissolving 40 μg of C12-Resazurin in
100 μL of DMSO as a 1 mM stock solution.
A 50 μM C12-Resazurin solution was then made up by diluting
2.5 μL of the stock solution into 47.5 μL of DMSO. The Sytox green solution was prepared by
diluting 5 μL of a 10 μM stock solution in 45 μL of DMSO. The staining solution was prepared by
diluting 48 μL of 50 μM C12-Resazurin and 48 μL of Sytox green stain
into 4.704 mL of 1X PBS for final concentrations of 500 nM. The cell media was then removed from culture,
and the cells were washed with 1X PBS.
Cells were then covered with 200 μL of live/dead staining solution and
incubated for 15 minutes at room temperature protected from light. Lastly, the cells were visualized using a
fluorescent microscope. Three random
images were taken per well, and the channels were combined, and the images were
processed using Axiovision software as previously described. Cell viabilities were found to be significant
using Tukey’s multiple comparison among means (α=0.05).


## 4.6. Ovarian Cancer Xenograft Model

Three-week-old female
athymic nude mice were purchased from Harlan (Indianapolis, In, USA) and housed for one week prior to the
experiment.  Ten million cells were
injected subcutaneously into the back of three animals, right below the
ear. The animals were monitored and the
weights were recorded 2-3 times per week
until the tumors grew to be visible (macroscopic tumors grew in about six
weeks). The next day, and every third
day following for ten days, the animals were injected with a dose of 333 mg/kg
of compound1 subcutaneously at the
tumor site for a total of 1000 mg/kg. After
ten days, the animals were sacrificed, and a full necropsy was performed. The brain, liver, lungs, heart, kidneys,
spleen, and tumors were sent for pathology report. Synthetic, NMR, and crystallographic data for the imidazolium salts and their silver acetate structures are available as Supplementary Material at doi:10.1155/2008/384010.



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*Source: 384010-2008-07-01.xml*