Sensitization of cancer cells to DNA damaging agents by imidazolines

Article abstract of DOI:10.1021/ja060273f

Apoptosis, or programmed cell death, is a cellular mechanism used to regulate cell number and eliminate damaged or mutated cells. Concomitant with the initiation of the apoptotic cell signal, chemotherapeutic agents also induce anti-apoptotic factors, suc

Full text of DOI:10.1021/ja060273f

Published on Web 06/23/2006
Sensitization of Cancer Cells to DNA Damaging Agents by
Imidazolines
Vasudha Sharma, Satyamaheshwar Peddibhotla, and Jetze J. Tepe*
Contribution from the Department of Chemistry, Michigan State UniVersity,
East Lansing, Michigan 48824
Received March 2, 2006; E-mail: tepe@chemistry.msu.edu
Abstract: Apoptosis, or programmed cell death, is a cellular mechanism used to regulate cell number and
eliminate damaged or mutated cells. Concomitant with the initiation of the apoptotic cell signal,
chemotherapeutic agents also induce anti-apoptotic factors, such as NF-κB, which compromise the overall
efficacy of chemotherapeutic anticancer treatment. Here we describe an adjuvant therapy in which a small
molecule is used to sensitize cancer cells toward apoptosis induced by chemotherapeutics. Our results
indicate that the imidazoline 1d modulates the pro-survival NF-κB pathway and selectively sensitizes cancer
cells toward DNA damaging agents, thus enhancing the overall efficacy of the treatment. Pretreatment of
cancer cells with the noncytotoxic imidazoline 1d (10 nM) resulted in a significant increase in apoptosis
and anticancer efficacy of the clinically significant DNA damaging agents camptothecin and cisplatin.
Noncancerous cells remained unaffected during this regimen.
Introduction
is abnormally down-regulated and provides an intrinsic survival
advantage in many types of cancers.9 Additional cellular
Apoptosis (or programmed cell death) is a sequential, stepwise
defensive mechanism to remove infected, mutated, or damaged
cells.1 Conventional anticancer therapy involving chemotherapy
or ionizing radiation is aimed at the induction of apoptosis in
cancer cells.2 Camptothecin (CPT) is a plant alkaloid that
exhibits specificity for DNA topoisomerase I and induces a
stable ternary topoisomerase I-DNA cleavable complex.3,4
Stabilization of this cleaved DNA complex is recognized as
damaged DNA and initiates an apoptotic signaling pathway,
culminating in cell death.4,5 The clinically used DNA damaging
agent, cisplatin (CDDP), exerts its anticancer activity via the
covalent binding to DNA base pairs, which triggers a similar
apoptotic signaling pathway.6 In response to this drug-induced
DNA assault, a range of signaling cascades involved in
apoptosis, cell cycle arrest, and DNA repair are activated. This
is tightly controlled by several regulatory pathways, including
the pro-apoptotic p53 and the pro-survival NF-κB pathways.7,8
Unfortunately, due to mutations that interfere with drug-induced
initiation or execution of apoptosis, the pro-apoptotic response
resistance has been attributed to the activation of anti-apoptotic
signaling pathways such as EGFR and NF-κB, which are
induced after CPT treatment.10,11 Strategies using combinations
of inducers of apoptosis and/or inhibitors of anti-apoptotic
factors with traditional chemotherapeutic drugs may therefore
provide an improved alternative to conventional chemo-
therapy.12-14 Hence, the search for new chemotherapeutic
strategies has therefore shifted to small molecules that can
selectively induce apoptosis in cancer cells or retard the cellular
chemoresistance.15,16
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V.; Melendez-Zajgla, J.; Ortega, A. Mutat. Res. 1997, 381, 67-75. (c)
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2000, 408, 433-439.
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J. AM. CHEM. SOC. 2006, 128, 9137-9143
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Sharma et al.
Scheme 1. Synthesis of the trans-Imidazolinesa
a (a) Acid chloride, NaOH (aq), Et2O, overnight; (b) EDCI, CH2Cl2, 2 h, room temperature, 70-76% overall; (c) TMSCl, imine, CH2Cl2, reflux 4 h, room
temperature overnight, 65-76%, (structures and activities of 1a-j, Supporting Information Figure 1).
Figure 1. (A) Pretreated with the imidazoline 1d. Caspase 3/7 activity in CEM cells treated with no drug control, imidazoline 1d (10 nM), CPT (10 nM),
and the combination of imidazoline 1d (10 nM) followed by CPT (10 nM) after 0.5 h. (B) Post-treated with the imidazoline 1d. Caspase 3/7 activity in CEM
cells treated with no drug control, imidazoline 1d (10 nM), CPT (10 nM), and the combination of CPT (10 nM) followed by imidazoline 1d (10 nM) after
2 h. (C) Top left, CPT (10 nM), 12 h treatment; top right, combination pretreatment with 1d, 12 h; bottom left, CPT (10 nM), 48 h; bottom right, combination
pretreatment with 1d, 48 h. All treatments stained with YoPro-1 and visualized using fluorescence microscopy.
Pioneering studies by Baldwin et al. demonstrated the control
of inducible chemoresistance through inhibition of NF-κB via
the incorporation of a mutated form of I-κBR, a natural inhibitor
of NF-κB.11,17 Piette et al. showed that the overexpression of
mutated IκB-R regulated the cytotoxicity caused by camptoth-
ecin.18 These studies illustrated the clinical potential of inhibition
of NF-κB in combination chemotherapy. The most successful
clinical example is the proteasome inhibitor bortezomib (PS-
341), which may be used as a single agent or in combination
regimens with classical anticancer agents providing a more than
additive apoptotic response.12,15,19,20
This library was evaluated for its ability to enhance CPT-
induced apoptosis in CEM cells using a caspase-based Apo-
One assay (Supporting Information Figure 1). Caspase activation
plays a central role in the execution of apoptosis via the
proteolytic cleavage of multiple protein substrates by caspase-
3, -6, and -7.23 NF-κB regulates programmed cell death via cross
talk with the caspases.8 Figure 1A illustrates the effect of the
imidazoline 1d on CEM cells when incubated with CPT over a
48-h time period. Enhancement of CPT-induced apoptosis in
CEM cells was investigated at concentrations e10 nM CPT,
which causes DNA aberrations but no significant apoptosis in
leukemia cells.17 Treatment of the cells with the imidazolines
had no effect on the level of apoptosis (Figure 1A and
Supporting Information Figure 3A), indicating that imidazolines
do not exhibit significant cell cytotoxicity. Treatment with 10
nM CPT resulted in some cell death starting after 12 h.
Combination treatment of the noncytotoxic imidazoline 1d (10
nM) followed by CPT (10 nM) after 0.5 h resulted in complete
apoptotic cell death after 48 h (Figure 1A, pretreatment).
Interestingly, treatment of the cells with CPT (10 nM) followed
by the addition of the imidazoline 1d after 2 h did not show
any enhancement of efficacy (Figure 1B, post-treatment).
Consistent with the Apo-One assay, an increased cell perme-
ability characterized by the increase in the fluorescence was
observed when the Yo-Pro1 stained cell population was
subjected to fluorescence microscopy (Figure 1C).
Results and Discussion
Recently, we reported the imidazoline scaffold as a potent
inhibitor of the NF-κB signaling pathway via the inhibition of
the degradation of its inhibitory protein IκB.21 Here, we report
the ability of imidazoline 1d to selectively sensitize cancer
cells toward CPT and CDDP in CEM (acute leukemia), HT1080
(fibrosarcoma), and HeLa (cervical carcinoma) cells but not in
the noncancerous SL89 (fibroblast) cell line. The imidazoline
1d was identified as the most effective compound from a
small library prepared via our previously reported TMSCl-
mediated 1,3-dipolar cycloaddition of oxazolones and imines
(Scheme 1).22
(17) Cusack, J. C., Jr.; Liu, R.; Baldwin, A. S., Jr. Cancer Res. 2000, 60, 2323-
2330.
(18) Habraken, Y.; Piret, B.; Piette, J. Biochem. Pharmacol. 2001, 62, 603-
Post-Treatment with Imidazoline Does Not Potentiate
CPT-Induced Apoptosis. Previously, we had shown that
pretreatment of CEM cells with imidazoline 1d inhibited the
DNA binding activity of NF-κB, degradation of I-κBR, and
616.
(19) An, J.; Sun, Y.; Fisher, M.; Rettig, M. B. Leukemia 2004, 18, 1699-1704.
(20) (a) Teicher, B. A.; Ara, G.; Herbst, R.; Palombella, V. J.; Adams, J. Clin.
Cancer Res. 1999, 5, 2638-2645. (b) Orlowski, R. Z.; et al. J. Clin. Oncol.
2002, 20, 4420-4427. (c) Adams, J. Curr. Opin. Chem. Biol. 2002, 6,
493-500.
(21) Sharma, V.; Lansdell, T. A.; Peddibhotla, S.; Tepe, J. J. Chem. Biol. 2004,
11, 1689-1699.
(22) (a) Peddibhotla, S.; Tepe, J. J. Synthesis 2003, 9, 1433-1440. (b)
Peddibhotla, S.; Jayakumar, S.; Tepe, J. J. Org. Lett. 2002, 4, 3533-3535.
(23) (a) Nicholson, D. W.; Thornberry, N. A. Science 2003, 299, 214-215. (b)
Thornberry, N. A.; Lazebnik, Y. Science 1998, 281, 1312-1316. (c)
Thornberry, N. A. Chem. Biol. 1998, 5, R97-103.
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Sensitizing Cancer Cells to DNA Damaging Agents
A R T I C L E S
Figure 2. (A) Treatment of CEM cells with tubulin inhibitor Taxol. (B) EMSA of NF-κB activation in CEM cells by Taxol. DNA ) oligonucleotide with
NF-κB binding domain; TNF-R ) endogenous activator of NF-κB; R-p65 ) specific antibody for p65 unit of NF-κB; Taxol ) chemotherapeutic agent.
transcriptional activity of NF-κB induced by CPT.21 To further
evaluate the relationship between the NF-κB signaling events
and apoptosis induction by CPT, we determined the effect of
pre- and post-treatment with imidazoline on CPT-induced
apoptosis in CEM cells. Figure 1A (and Supporting Information
Figure 3B) demonstrates that pretreatment of the cells with
imidazoline 1d significantly enhances apoptosis, whereas ad-
dition of the imidazoline 2 h after CPT treatment (Figure 1B)
does not affect the induction of apoptosis (for both washed and
continuous exposure, see Supporting Information Figure 3C).
This differential effect of pre- or post-treatment with 1d indicates
that the pro-survival activation of NF-κB by CPT is likely to
precede the apoptotic signaling in CEM cells.
Potentiation by the Imidazoline Scaffold Requires DNA
Damage. We investigated whether imidazoline 1d could
potentiate drug-mediated cell death by various anticancer agents
including taxol, vincristine, camptothecin, and cisplatin. In CEM
cells, agents that induced DNA damage (cisplatin and camp-
tothecin) consistently showed a strong enhancement of apoptosis
on pretreatment with imidazoline. However, the imidazoline 1d
failed to significantly enhance tubulin inhibitors such as taxol
(Figure 2A) and vincristine (Supporting Information Figure 4A)
in CEM cells. Further investigation revealed that while taxol
did not activate NF-κB in CEM cells, CPT activated NF-κB in
a dose-dependent manner (Figures 2B and 4A). Activation of
the NF-κB signaling cascade requires an intact nucleus and
topoisomerase-1-mediated nuclear damage in CEM cells.24 This
suggests that the drug-induced apoptotic and anti-apoptotic
signaling pathways related to DNA damage are crucial to the
enhancement of efficacy by noncytotoxic imidazoline 1d. This
is in contrast to proteasome inhibitor bortezomib, which inhibits
the NF-κB pathway and potentiates the effect of tubulin inhibitor
taxol as well as DNA damaging agents, doxorubicin and
cisplatin.13,19
Selective Potentiation of Drug-Mediated Apoptosis In-
duced by Imidazolines in Cancer Cells. The effect of
imidazoline 1d on cancer cells CEM, HT1080, HeLa, and
noncancerous fibroblast cells (SL89) was quantified by calculat-
ing the concentration at which CPT and CDDP induced 95%
apoptosis (CC95) with and without pretreatment of the cells with
imidazoline 1d (100 nM for 0.5 h, CC95 combination). Table 1
clearly indicates that only the cancerous cell lines exhibited
strong enhancement of apoptosis.
The CC95 values of both CPT and CDDP dropped drastically
(up to 74-fold) upon pretreatment of cancerous cells with the
imidazoline (Table 1 and Figure 3A). No increase in apoptosis
was seen in the noncancerous fibroblasts (SL89) upon pretreat-
ment with the noncytotoxic imidazoline 1d (Table 1 and Figure
3B).
Correlation between Potentiation and NF-κB Activation.
Our data indicate that sensitization of cells to chemotherapeutic
agents using imidazoline 1d exhibits a strong correlation with
NF-κB activation. For example, CEM cells showed only
moderate activation of NF-κB by CDDP (g3 µM, Figure 4B)
resulting in moderate fold enhancement (14-fold) compared to
(24) Huang, T. T.; Wuerzberger-Davis, S. M.; Seufzer, B. J.; Shumway, S. D.;
Kurama, T.; Boothman, D. A.; Miyamoto, S. J. Biol. Chem. 2000, 275,
9501-9509.
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Table 1. Cancer Cell Specific Enhancement of Efficacya
cell line
drug
CC95
CC95 combination
fold enhancement
CEM
CPT
CDDP
CPT
CDDP
CPT
CDDP
CPT
CDDP
44 nM
15 µM
86 µM
1.48 µM
53 µM
0.3 µM
15 µM
0.5 µM
0.6 nM
1.1 µM
6.7 µM
0.06 µM
5.2 µM
0.06 µM
16 µM
74
14
13
24
10
5
CEM
HT1080
HT1080
HeLa
HeLa
SL89
1
SL89
0.6 µM
1
a Data reported as an average of two independent experiments after 48
h of drug exposure. The imidazoline itself induced no significant levels of
apoptosis (tested up to 10 µM). CPT ) camptothecin, CDDP ) cisplatin.
Figure 4. (A) EMSA for NF-κB-DNA binding in CEM, HT1080, and
HeLa cells activated with various concentrations of CPT (lanes 5, 7-11:
10 µM, 1 µM, 0.1 µM, 0.01 µM, 1 nM, 0.1 nM). TNF-R was used as a
positive control (lane 2), and CPT was used as a positive control (lane 5).
A p65-specific antibody (R-p65) was used in lane 3 (TNF-R activation)
and lane 6 (10 µM CPT activation) as a positive control. (B) EMSA for
NF-κB-DNA binding in CEM, HT1080, and HeLa cells activated with
various concentrations of CDDP (lanes 5, 7-11: 10 µM, 3 µM, 1 µM, 0.3
µM, 0.1 µM, and 0.03 µM). TNF-R was used as a positive control (lane 2),
and CDDP was used as a positive control (lane 5). A p65-specific antibody
(R-p65) was used in lane 3 (TNF-R activation) and lane 6 (10 µM CDDP
activation) as a positive control.
Figure 3. (A) CC95 comparison of CPT with and without imidazoline 1d
in CEM leukemia cells. (B) CC95 of CPT with and without imidazoline 1d
in noncancerous fibroblast cells. Data reported as an average of two
independent experiments after 48 h of drug exposure.
the strong activation (g0.01 µM, Figure 4A) and enhancement
(74-fold) with CPT. This observation was noticeable across the
panel of cell lines (Figure 4 and Table 1), in particular SL89
cells, which showed no enhancement with the imidazoline
treatment (Figure 3B, Table 1) and were completely insensitive
to NF-κB activation when treated with varying concentrations
of either CPT, CDDP, or TNF-R (Figure 5).
This observation was further supported by the NF-κB driven
transcription studied via a luciferase-based reporter assay in
HeLa and SL89 cells. Both TNF-R as well as CPT activated
NF-κB and enhanced NF-κB-based transcription in HeLa cells
(Figure 6). SL89 cells were devoid of any significant NF-κB
transcriptional activity (Figure 6), correlating well with the
inability to show potentiation of DNA damage in the presence
of imidazoline 1d. However, it should be noted that even though
the imidazoline is a potent modulator of the NF-κB signaling
pathway via the inhibition of I-κB degradation, it is not yet
known if NF-κB is the primary target of the imidazoline.
Figure 5. EMSA for NF-κB-DNA binding in SL89 cells. Cells were
activated with TNF-R and CPT (10 µM, 1 µM, 0.1 µM, 0.01 µM, 1 nM)
and CDDP (10 µM, 3 µM, 1 µM, 0.3 µM, and 0.1 µM). Hela cells activated
with TNF-R were used as a positive control. The supershift with antibody
specific for the p65 unit is shown in lane 12.
Chemical Resolution of 1d and Biological Activity of
Enantiomers 2 and 3. Racemic 1d was chemically resolved
using an EDCI-mediated esterification with R-(+)-1-phenyle-
thanol yielding two diastereomeric esters, which on hydrolysis
afforded the (S,S) enantiomer 2 and (R,R) enantiomer 3 in overall
44% yield (Scheme 2). The enantiomers were crystallized, and
their absolute configuration was determined by X-ray crystal-
lography (Scheme 2).
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Sensitizing Cancer Cells to DNA Damaging Agents
A R T I C L E S
Scheme 2. Chemical Resolution of 1d and X-ray Crystal
Structures of 2 (S,S) and 3 (R,R)a
Figure 6. SL89 cells do not show NF-κB-mediated transcriptional activity
with either TNF-R or CPT. pFCMEKK causes constitutive activation of
NF-κB. pCIS-CK does not contain the enhancer element for NF-κB and
serves as a negative control. TNF-R and CPT are known to activate NF-κB
in a variety of cell lines.
a (a) (R)-Phenylethanol, EDCI, DMAP, CH2Cl2; (b) 2 N NaOH/EtOH,
reflux, 6 h, then HCl, 44% overall.
The diastereomeric esters and the enantiomers 2 and 3 were
tested for enhancement of apoptosis for a period of 48 h with
CPT (Figure 7A). None of the compounds induced any
significant cell death over 48 h; however, the (R,R) enantiomer
3 potentiated the apoptotic effect of CPT (CC95 combination )
0.53 nM, 83-fold enhancement) nearly 5 times more than the
(S,S) enantiomer 2 (CC95 combination ) 2.4 nM, 18-fold
enhancement). The esters did not show a strong enhancement
of apoptosis as determined by the caspase-based Apo-One assay
(data not shown).
FACS Analysis. Apoptosis is a complex cellular mechanism
involving characteristic changes within the nucleus, loss of mito-
chondrial function, membrane blebbing, etc.1 CEM cells were
treated with 1d, 2, and 3 for 30 min, followed by treatment
with 0.01 µM CPT for 12 h and 48 h. At the desired time points,
cells were harvested, stained, and sorted. As confirmed by the
flow cytometry experiments, compounds 1d, 2, and 3 did not
induce apoptosis themselves (Figure 7B, top panel), but
enhanced apoptosis in combination with CPT (Figure 7B, bottom
panel).
penicillin (614 ng/mL), streptomycin (10 µg/mL), and HEPES buffer,
pH 7.2 at 37 °C, 5% CO2. HT1080 and Hela cells were cultured in
DMEM supplemented with 10% fetal bovine serum, penicillin (614
ng/mL), streptomycin (10 µg/mL), and HEPES buffer, pH 7.2 at 37
°C, 5% CO2. SL89 cells were cultured in MEM supplemented with
10% fetal bovine serum, penicillin (614 ng/mL), and streptomycin (10
µg/mL) at 37 °C, 5% CO2.
Optimization of Concentrations of Chemotherapeutic Agents for
Apoptosis. Cells were cultured as described above. DMSO was used
as the vector for all drugs and added in the control experiments. Cell
cultures were treated with various concentrations of the camptothecin
or cisplatin and allowed to incubate at 37 °C, 5% CO2. At time points
0, 6, 12, 24, and 48 h, an aliquot of the cell culture was transferred to
a 96-well plate and mixed with an equal volume of Apo-ONE
Homogeneous Caspase-3/7 assay reagent (Promega Corporation). The
contents of the plate were gently mixed and allowed to incubate for 30
min at room temperature. The fluorescence of each well was then
measured on a Molecular Imager FXPro at 532 nm. All reported data
is the average of two independent experiments unless otherwise
indicated.
Conclusion
Enhancement of Apoptosis. CEM cells (CCRF-CEM) were cultured
as described above. Cell cultures were treated with 0.1 µM, 0.01 µM,
or 1 nM of the imidazoline 1d and allowed to incubate at 37 °C, 5%
CO2 atmosphere for 30 min. Camptothecin was added at concentrations
50 µM, 1 µM, 0.3 µM, 0.1 µM, 0.03 µM, 0.01 µM, 3 nM, 1 nM, or
0.3 nM and further incubated. An aliquot was transferred to a 96-well
plate at various time points 0, 6, 12, 24, and 48 h and mixed with an
equal volume of Apo-ONE Homogeneous Caspase-3/7 assay (Promega
Corporation) reagent. The contents of the plate were gently mixed and
allowed to incubate for 1 h. The fluorescence of each well was then
measured on a Molecular Imager FX Pro at 532 nm. All reported data
is the average of two independent experiments unless otherwise
indicated. The data were then analyzed using GraphPad prism 4.0, and
CC95 values were calculated. Data were normalized between maximum
and minimum cell death relative to the respective control sets.
EMSA Assay for NF-κB-DNA Binding. CEM cells were cultured
as described above. Cells (1.6 × 106 cells/mL) were treated with TNF-R
(0.4 pg) or camptothecin (Sigma-Aldrich) (10 µM, 1 µM, 0.1 µM, 0.01
µM, 1 nM, 0.1 nM) or CDDP (100 µM, 30 µM, 10 µM, 3 µM, 1 µM,
0.3 µM, 0.1 µM) at 37 °C and 5% CO2 for 25 min or 2 h, respectively.
SL89 cells were cultured as described above. Cells at 70% confluence
were treated with TNF-R (0.4 pg) or camptothecin (Sigma-Aldrich)
(10 µM, 1 µM, 0.1 µM, 0.01 µM, 1 nM) or CDDP (10 µM, 3 µM, 1
µM, 0.3 µM, 0.1 µM) at 37 °C and 5% CO2 for 25 min or 2 h,
Conventional chemotherapy suffers a compromised clinical
efficacy due to the induction of anti-apoptotic factors, concurrent
with the desired induction of programmed cell death. The
imidazoline 1d is the lead compound from a novel class of
nontoxic small molecules that enhance the efficacy of the DNA
damaging agents. This is postulated to proceed via the inhibition
of the pro-survival signaling pathways, such as NF-κB, in a
highly cell-specific manner. In particular, imidazoline 1d
selectively sensitized cancer cells to genotoxic stress at low
nanomolar concentrations. No such effect was seen in noncan-
cerous fibroblasts. These studies indicate the potential of small-
molecule NF-κB inhibitors to sensitize tumor cells toward
classical anticancer agents in order to enhance the overall
efficacy of anticancer treatment. Work toward identifying the
target, detailed mechanism of action, and clinical potential of
these agents is currently under investigation in our laboratories.
Experimental Section
Cell Culture. CEM cells ((CCRF-CEM); American Type Culture
Collection, Rockville, MD) were grown in RPMI-1640 media (Gibco-
BRL, Rockville, MD) supplemented with 10% fetal bovine serum,
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Sharma et al.
Figure 7. (A) Caspase 3/7 activity measured in CEM cells. CC95 comparison for CPT in CEM leukemia cells in the presence and absence of racemic 1d
and the enantiomers 2 and 3. Data reported as an average of two separate experiments. (B) FACS analysis (top) cells alone, racemic 1d (0.1 µM), 3 (0.1
µM), 2 (0.1 µM); (bottom) CPT (0.01 µM), 1d f CPT (0.01 µM), 3 f CPT (0.01 µM), 2 f CPT (0.01 µM). The lower right quadrant indicates the %
apoptosis ((standard deviation) for the sample.
respectively. The cells were harvested by centrifugation and washed
in ice-cold PBS, and the nuclear extracts were prepared as previously
described.21 The protein concentration of the extracts was determined
according to the Bradford method (1976) with BioRad reagents. Nuclear
extracts (20 µg total protein) were incubated for 20 min at room
temperature with a double-stranded Cy3-labeled NF-κB consensus
oligonucleotide, 5′-AGTTGAGGGGACTTTC CCAGGC-3′ (0.16 pmol).
The binding mixture (25 µL) contained 10 mM HEPES-NaOH, pH
7.9, 4 mM tris-HCl, pH 7.9, 6.0 mM KCl, 1 mM EDTA, 1 mM DTT,
10% glycerol, 0.3 mg/mL bovine serum albumin, and 1 µg of poly
(dI.dC). The mixture was loaded on a 4% polyacrylamide gel prepared
in 1X trisborate/EDTA buffer and was electrophoresed at 200 V for
20 min. After electrophoresis, the gel was analyzed using a phospho-
rimager (Biorad FX plus) for detection of the NF-κB-DNA binding.
then harvested, stained with Yo-Pro-1, and visualized under a fluores-
cence microscope Olympus CK2 using a BP480-550C filter.
Compound 1d. dl-(3S,4S)-1-Benzyl-2,4,5-triphenyl-4,5-dihydro-
1H-imidazole-4-carboxylic Acid. A solution of benzaldehyde (0.6 g,
5.7 mmol) and benzylamine (0.61 g, 5.7 mmol) in dry dichloromethane
(120 mL) was refluxed under nitrogen for 2 h. 2,4-Diphenyl-4H-
oxazolin-5-one (1.35 g, 5.7 mmol) and chlorotrimethylsilane (0.8 g,
7.4 mmol) were added, and the mixture was refluxed under nitrogen
for 6 h and then stirred overnight at room temperature. The product
was purified by silica gel column chromatography with 1:5 ethanol/
ethyl acetate to afford 2.1 g of the product in 65% yield as an off-
1
white solid. Mp 153-155 °C dec. H NMR (300 MHz) (CDCl3): δ
3.8 (1H, d, J ) 15.6 Hz), 4.62 (1H, d, J ) 15.6 Hz), 4.98 (1H, s), 6.58
(2H, d, J ) 8.1 Hz), 7.05-7.65 (16H, m), 7.9 (2H, d, J ) 7.2 Hz); 13
C
NF-κB-Mediated Transcription in HeLa (Cervical Carcinoma)
and SL89 (Noncancerous Fibroblast) Cells. Transient transfections
were performed using lipofectamine 2000. Briefly, 0.8 µg of plasmid
DNA was combined with lipofectamine 2000 (Invitrogen) (1.5 µL).
The mixture was incubated at room temperature for 20 min and mixed
with the cells without serum. Cells were transfected for 5 h at 37 °C
in 5% CO2. Cells were allowed to grow in complete medium for 15 h
in 5% CO2. Cells were then treated with TNF-R (10 ng/µL) or
camptothecin (3 µM) for 5 h or 10.5 h, respectively. Cells were washed
with 1 × PBS. Washed cells were assayed for luciferase production
using the manufacturer’s protocol for both luciferase as well as renilla
luciferase (co-reporter) using the Dual-Glo reporter assay (Promega).
The results were read on a Veritas microplate luminometer (Turner
Biosystems, CA) as relative light units. Values from the luciferase
activity were normalized to those with renilla luciferase.
NMR (75 MHz) (CDCl3): δ 48.3, 75.6, 79.1, 123.1, 125.7, 126.7, 127.3,
127.4, 127.9, 128.1, 128.2, 128.8, 128.9, 129, 129.3, 132.9, 133.8, 136,
143.1, 164.8, 168.1; IR (neat): 3400 cm-1 (very broad), 1738 cm-1
;
HRMS (EI): calcd for C29H24N2O2 [(M - H) - CO2]+ 387.1526 and
obsd [(M - H) - CO2]+ 387.1539.
Compounds 2 and 3. In a flame-dried flask, under nitrogen, a well-
stirred suspension of 1-benzyl-2,4,5-triphenyl-4,5-dihydro-1H-imida-
zole-4-carboxylic acid (0.1 g, 0.18 mmol) in dry methylene chloride
(20 mL) was maintained at 0 °C. To this mixture was added EDCI‚
HCl (36 mg, 0.18 mmol), followed by DMAP (22 mg, 0.18 mmol),
and stirred for 20 min. (R)-(+)-1-phenylethanol (43.5 mg, 2 equiv, 0.36
mmol) was added, and the mixture was stirred overnight at room
temperature. The reaction mixture was washed with 2 N HCl (2 × 20
mL), with saturated sodium bicarbonate (2 × 20 mL), and then with
brine (20 mL). The organic layer was dried over sodium sulfate and
evaporated under reduced pressure. The crude product was purified by
column silica gel chromatography using 40% ether/hexane mixture.
These were characterized and subjected to hydrolysis under reflux with
2 N NaOH/EtOH separately. The reaction mixtures were cooled and
acidified to pH ) 3 and extracted with CHCl3. The organic layer was
dried and evaporated in vacuo to obtain the two enantiomers in 44%
yield.
FACS Analysis. CEM cells were cultured as described above. Cell
cultures were treated with 0.1 µM imidazoline 1d, 2, or 3 and allowed
to incubate at 37 °C, 5% CO2 atmosphere for 30 min. Camptothecin
was added at 0.01 µM and further incubated for 12 and 48 h to mark
early and late apoptotic events. Cells were then harvested and washed
in 1 × PBS. Cells were stained with Yo-Pro1 and propidium iodide to
mark the apoptotic cells. These were then analyzed on a BD Biosciences
Vantage SE (San Jose, CA) flow cytometer using a 530/30 band-pass
filter for Yo-Pro1 and 660/20 (band-pass filter) for PI, respectively.
S,S,R-1-Benzyl-2,4,5-triphenyl-4,5-dihydro-1H-imidazole-4-car-
boxylic Acid 1-Phenylethyl Ester: [R]D +19.45°, c 1, CHCl3; 1H
NMR (300 MHz), CDCl3: δ 0.873 (d, J ) 6.6, 3H), 3.73 (d, J ) 15
Hz, 1H), 4.56 (d, J ) 15 Hz, 1H), 4.93 (s, 1H), 5.23 (q, J ) 6.5, 1H),
6.73-7.72 (m, 25H); 13C NMR (74.4 MHz) CDCl3: 21.80, 48.53,
72.77, 73.26, 82.32, 125.73, 126.75, 127.23, 127.30, 127.38, 127.94,
128.26, 128.37, 128.54, 128.58, 128.74, 130.29, 130.34, 136.47, 137.69,
Fluorescence Microscopy. CEM cells (CCRF-CEM) were cultured
as described above. Cell cultures were treated with 0.1 µM imidazoline
1d, 2, or 3 and allowed to incubate at 37 °C, 5% CO2 atmosphere for
30 min. Camptothecin was added at 0.01 µM and further incubated
for 12 and 48 h to mark early and late apoptotic events. Cells were
9
9142 J. AM. CHEM. SOC. VOL. 128, NO. 28, 2006

Sensitizing Cancer Cells to DNA Damaging Agents
A R T I C L E S
141.37, 143.58, 165.38, 169.79; IR(CHCl3): 3584.2, 3063.4, 3032.5,
2926.4, 2855.0, 1736.1, 1595.3, 1495.0, 1412.1, 1064.8 cm-1; EIMS:
m/z ) 537.2 (M + H), 387.3 (M - COOCH(Me)Ph); HRMS (FAB+)
(C37H33O2N2) (M + H) 537.2542; found, 537.2542.
J ) 15 Hz, 1H), 4.84 (d, J ) 15 Hz, 1H), 5.09 (s, 1H), 6.61 (2H, d, J
) 8.1 Hz), 7.12-7.77 (16H, m), 7.97 (2H, d, J ) 7.2 Hz); 13C NMR
(74.4 MHz) CDCl3: 48.76, 74.96, 77.03, 121.45, 125.90, 126.57,
127.15, 128.29, 128.48, 128.57, 128.81, 129.12, 129.39, 129.56, 129.65,
130.09, 132.47, 132.84, 133.01, 134.14, 139.96, 165.84, 168.34; IR-
R,R,R-1-Benzyl-2,4,5-triphenyl-4,5-dihydro-1H-imidazole-4-car-
boxylic Acid 1-Phenylethyl Ester. [R]D -7.77°, c 1, CHCl3; 1H NMR
(300 MHz), CDCl3: δ 1.205 (d, J ) 6.6, 3H), 3.73 (d, J ) 15 Hz,
1H), 4.56 (d, J ) 15 Hz, 1H), 4.95 (s, 1H), 5.23 (q, J ) 6.5, 1H),
6.80-7.81 (m, 25H); 13C NMR (74.4 MHz) CDCl3: 21.75, 48.50,
73.42, 73.49, 82.87, 126.01, 126.87, 127.14, 127.29, 127.35, 127.95,
127.99, 128.13, 128.20, 128.40, 128.57, 128.75, 130.23, 36.72, 137.28,
141.00, 143.95, 165.50, 169.94; IR(CHCl3): 3584.2, 3063.4, 3032.5,
2924.5, 2855.0, 1736.2, 1595.3, 1495.0, 1412.1, 1064.8 cm-1; EIMS:
m/z ) 537.3(M + H), 387.3 (M - COOCH (Me) Ph) HRMS (FAB+)
(C37H33O2N2) (M + H) 537.2542; found, 537.2542.
(4S,5S)-1-Benzyl-4, 5-dihydro-2,4,5-triphenyl-1H-imidazole-4-
carboxylic Acid (2): Mp 148-149 °C dec. [R]D -47.58°, c 1, CHCl3;
1H NMR (300 MHz), CDCl3: δ 3.96 (d, J ) 15 Hz, 1H), 4.81 (d, J )
15 Hz,1H), 5.05 (s, 1H), 6.61 (2H, d, J ) 8.1 Hz), 7.09-7.92 (16H,
m), 7.90 (2H, d, J ) 7.2 Hz), 11.42 (s, 1H); 13C NMR (74.4 MHz)
CDCl3: 49.37, 75.33, 76.06, 121.29, 125.93, 127.65, 129.24, 129.40,
129.42, 129.86, 130.02, 130.14, 130.76, 131.69, 131.82, 132.18, 134.62,
138.54, 166.69, 167.71; IR(CHCl3): 3584.2, 3063.4, 2924.5, 1743.9,
1558.9, 1456.4, 1253.9 cm-1; EIMS: m/z ) 387.3 (M - COOH)
HRMS (FAB+) (C29H25O2N2) (M + H) 433.1916; found, 433.1914.
(4R,5R)-1-Benzyl-4,5-dihydro-2,4,5-triphenyl-1H-imidazole-4-
carboxylic Acid (3‚HCl): Mp 147-148 °C dec. [R]D +47.87°, c 1,
CHCl3; mp 147-149 °C dec. 1H NMR (300 MHz), CDCl3: δ 4.02 (d,
(CHCl3): 3584.2, 3063.4, 2924.5, 1741.9, 1556.7, 1456.4, 1221.1 cm-1
;
EIMS: m/z ) 386.3 (M - COOH - H) HRMS (FAB+) (C29H25O2N2)
(M + H) 433.1916; found, 433.1915. LRMS (FAB-) 35, 37.
Acknowledgment. We gratefully acknowledge the American
Cancer Society for their financial support (Research Scholar
Award, RSG-04-018-01-CDD). The authors also thank the MSU
Foundation for financial support. We greatly acknowledge Dr.
Rui Huang for carrying out the X-ray crystallography. V.S.
gratefully acknowledges the College of Natural Science, MSU
for The Graduate School Dissertation Fellowship during the
course of preparation of this manuscript.
Supporting Information Available: Additional cell toxicity
data, initial caspase-based screen, potentiation with CPT and
CDDP in various cell lines, toxicity data with tubulin inhibitors,
post-treatment with imidazoline, X-ray data tables and CIF files,
1H and 13C for all new compounds, and complete ref 20b. This
material is available free of charge via the Internet at
http://pubs.acs.org.
JA060273F
9
J. AM. CHEM. SOC. VOL. 128, NO. 28, 2006 9143