Nuclear factor-κB mediated inhibition of cytokine production by imidazoline scaffolds

Article abstract of DOI:10.1021/jm8013162

The mammalian nuclear transcription factor NF-κB is responsible for the transcription of multiple cytokines, including the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). Elevated levels of pro-inflammatory cytokines play an important role in the pathogenesis of inflammatory disorders such as rheumatoid arthritis (RA). Inhibition of the pro-inflammatory transcription factor NF-κB has therefore been identified as a possible therapeutic treatment for RA. We describe herein the synthesis and biological activity of a series of imidazoline-based scaffolds as potent inhibitors of NF-κB mediated gene transcription in cell culture as well as inhibitors of TNF-α and IL-6 production in interleukin 1 beta (IL-1β) stimulated human blood.

Full text of DOI:10.1021/jm8013162

1302
J. Med. Chem. 2009, 52, 1302–1309
Nuclear Factor-KB Mediated Inhibition of Cytokine Production by Imidazoline Scaffolds
Daljinder K. Kahlon,† Theresa A. Lansdell,† Jason S. Fisk,† Christopher D. Hupp,† Timothy L. Friebe,‡ Stacy Hovde,†,§
A. Daniel Jones,†,§ Richard D. Dyer,| R. William Henry,§ and Jetze J. Tepe*,†
Department of Chemistry, Michigan State UniVersity, East Lansing, Michigan 48824, Department of Biochemistry & Molecular Biology,
Michigan State UniVersity, East Lansing, Michigan 48824, TCH Pharmaceuticals Inc., Ann Arbor, Michigan 48104
ReceiVed October 17, 2008
The mammalian nuclear transcription factor NF-κB is responsible for the transcription of multiple cytokines,
including the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-R) and interleukin 6 (IL-6).
Elevated levels of pro-inflammatory cytokines play an important role in the pathogenesis of inflammatory
disorders such as rheumatoid arthritis (RA). Inhibition of the pro-inflammatory transcription factor NF-κB
has therefore been identified as a possible therapeutic treatment for RA. We describe herein the synthesis
and biological activity of a series of imidazoline-based scaffolds as potent inhibitors of NF-κB mediated
gene transcription in cell culture as well as inhibitors of TNF-R and IL-6 production in interleukin 1 beta
(IL-1ꢀ) stimulated human blood.
Introduction
erodimer is the most abundant.17 Each NF-κB dimer has unique
transcriptional activity, as transcriptional activators (RelA(p65)/
p50, p50/c-rel, RelA(p65)/RelA(p65), and p65/c-rel) and as
repressors (p50/p50 and p52/p52).18 NF-κB is sequestered in
the cytoplasm by the IκBs in normal nonstimulated cells.21 Upon
cellular stimulation, IκB is phosphorylated and subsequently
polyubiquinated and proteolytically degraded by the 26S
proteasome.21-24 These events unmask the nuclear localization
signal (NLS) of NF-κB, allowing it to translocate into the
nucleus.23 Following its nuclear translocation, NF-κB binds to
DNA and induces gene transcription of a wide variety of genes,
including those responsible for proinflammatory responses. The
levels of NF-κB-DNA binding are consequently much greater
in tissues from RA patients compared to control patients.20,25,26
Hence, regulation of the NF-κB mediated expression of cytok-
ines has been actively pursued as a means of therapeutic
intervention in these types of inflammatory disorders.27,28
Rheumatoid arthritis (RAa) is a common and often debilitating
human autoimmune disease that involves chronic inflammation
of joints, tissues, and organs.1 Compared to tissues in a normal
joint, RA joint tissues have increased cell densities with an
abundance of neutrophils, macrophages, and T lymphocytes.
Analysis of the synovial fluid from patients with RA has
revealed that this tissue is rich in primarily two cytokines: tumor
necrosis factor alpha (TNF-R) and interleukin 1 beta (IL-1ꢀ).2-4
Elevated production of both TNF-R and IL-1ꢀ have been found
in primary fibroblast-like synoviocytes from patients with
rheumatoid arthritis (RA) as well as osteoarthritis (OA).
Furthermore, cultured rheumatoid synovial cells continuously
expressed IL-1 and TNF mRNA, indicating that these cytokines
play important roles in the pathology of rheumatoid arthritis.5
It is well-documented that TNF-R and IL-1ꢀ also act as pivotal
signaling agents for the production of many additional cytokines,
such as IL-6,6-8 via the activation of the mammalian nuclear
transcription factor, NF-κB.9-12 IL-6 is found in abundance in
RA synovium, and due to its role in B cell and hematocytic
cell growth, differentiation, and proliferation of synovial
fibroblasts, IL-6′s overproduction contributes to many RA
symptoms.13-15 The activation of this pro-inflammatory cytokine
signaling cascade induces the release of matrix metalloprotein-
ases (MMPs), which lead to the destruction of bone and
cartilage, resulting in permanent tissue damage.16
Pharmacologic intervention in RA was improved drastically
with the advent of biologicals that target TNF-R, providing a
proof-of-principle for therapeutic intervention in RA by targeting
TNF-R.29-31 Unfortunately, these and alternative treatment
options for RA are limited and suffer from high costs,
undesirable methods of administration, and lack of data on long-
term safety, tolerability, and sustained efficacy. In addition,
variability in responses to these anti-inflammatory drugs is found
due to the complex network of alternative cytokine-mediated
pathways.32 Inhibition of pro-inflammatory transcription factors,
such as NF-κB, may therefore represent a better alternative to
modulate the complex cytokine network that induces inflam-
matory responses.33 Consequently, inhibition of NF-κB by a
small molecule would represent an attractive therapeutic alterna-
tive to the current clinical options.12,34
TNF-R and IL-1ꢀ are considered classical (or canonical)
activators of the NF-κB pathway.17-20 NF-κB is formed from
various members of the Rel family of transcription factors (NF-
κB1(p50), NF-κB2(p52), c-Rel, RelA(p65), and RelB) as either
a homodimer or heterodimer, where the RelA(p65)/p50 het-
We previously described the synthesis and biological activity
of a series of imidazolines as potent small molecule inhibitors
of NF-κB transcription, with imidazoline 1 being the most
effective in cell culture.35,36 The imidazolines are formed via
trimethylsilyl chloride mediated 1,3-dipolar cycloaddition of
oxazol-5-(4H)-ones and imines (Scheme 1).37,38 Even though
the parent molecule 1 displayed excellent activity in cell culture,
additional studies found that the imidazoline scaffold 1 was
fairly unstable upon standing and easily converted to a zwitte-
rionic form 2 (Scheme 2, X-ray crystal structures of 1 and 2
* To whom correspondence should be addressed: Phone: +1-517-355-
9715. Fax: +1-517-353-1793. E-mail: tepe@chemistry.msu.edu.
†
Department of Chemistry, Michigan State University.
‡
Current address: Chemistry Department, Eastern Michigan University.
Department of Biochemistry & Molecular Biology, Michigan State
§
University.
|
TCH Pharmaceuticals Inc.
a Abbreviations: RA, rheumatoid arthritis; TNF-R, tumor necrosis factor
R; IL-1ꢀ, interleukin-1ꢀ; OA, osteoarthritis, IL-6, interleukin-6; NLS,
nuclear localization signal; TFAA, trifluoroacetic anhydride; TMS, trim-
ethylsilyl; EDCI, 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide; DMAP,
4-dimethylaminopyridine.
10.1021/jm8013162 CCC: $40.75
2009 American Chemical Society
Published on Web 02/16/2009

NF-κB Mediated Inhibition of Cytokine Production
Scheme 1. Synthesis of the trans-Imidazolinesa
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1303
used as positive control and DMSO (vehicle) served as the
negative control. Luciferase production was evaluated after 8 h,
and all samples were normalized to the TNF-R activation
control. Treatment of HeLa/NF-κB-luc cells with imidazolines
without any TNF-R activation did not induce any significant
amount of luciferase activity at 20 µM, indicating that the
imidazolines alone did not stimulate the NF-κB pathway.
Pretreatment of the cells with the imidazolines followed by
TNF-R stimulation resulted in a dose-dependent decrease in
luciferase production for several of the analogues of 1, 7-9,
whereas the zwitterion 2 was devoid of activity (Table 1). While
the decarboxylated products 4 and 5 displayed significantly
reduced activity, the imidazoline ester 8 was the most active
alternative to compound 1 with an EC50 value in HeLa cells of
2.5 µM (Figure 1A). The imidazole 6, amide 10, and alcohol
11 were significantly less active, with EC50 values at or greater
than 20 µM. To verify that the results for luciferase production
in this integrated cell line were due to inhibition of NF-κB and
not to cell death, HeLa cells were transiently transfected with
6× κB driven reporter gene pNF-κB-luc (Stratagene) and the
internal control plasmid pRL-TK (Stratagene), which provides
low levels of Renilla luciferase expression. After activation with
TNF-R in the presence or absence of imidazoline 8, luciferase
production was assayed and the data was normalized to the
Renilla luminescence (see Supporting Information). The two
luciferase assays corresponded well with one another, confirming
the results from the luciferase assay using the stable HeLa/NF-
κB-luc cell line. These results indicate that the ester moiety
represented a comparable alternative to imidazoline 1 for NF-
κB inhibition.
a (a) Benzoyl chloride, 1 N NaOH (aq), overnight; (b) TFAA, CH2Cl2,
2 h, room temperature, 77% overall; (c) TMSCl, imine, CH2Cl2, reflux,
overnight, 68%.
are provided in the Supporting Information). Zwitterion 2 is
readily decarboxylated to form the ylide 3,39 which provided a
mixture of three products: the cis-dihydroimidazoline 4, the
trans-dihydroimidazoline 5, and the fully aromatized imidazole
6 (Scheme 2). Furthermore, the cis- and trans-dihydroimida-
zolines (4 and 5) isomerized upon standing and dehydrogenated
to the imidazole 6 at room temperature when exposed to polar
solvents such as DMSO. This cis/trans isomerization and
oxidation of the decarboxylated form of diaryl-substituted
imidazolines is well-documented,40-42 and thus this zwitterion
induced decarboxylation needed to be addressed to pursue its
further clinical potential. The instability of imidazoline 1 results
in undesirable pharmacological properties and could be the
reason for the inconsistent activity seen when imidazoline 1
was tested in cell culture and whole blood (Table 1). Fortunately,
chemical manipulation and derivatization of the carboxyl moiety
readily restores stability and potency. In this paper, we describe
the synthesis of derivatives of imidazoline 1 aimed to prevent
the aforementioned degradation and the new compounds were
evaluated for their stability and inhibition of NF-κB mediated
TNF-R and IL-6 production. Biological evaluation indicated that
three analogues substantially inhibited NF-κB mediated tran-
scription in cell culture and inhibited TNF-R and IL-6 production
inIL-1ꢀstimulatedhumanbloodatlowmicromolarconcentrations.
Inhibition of TNF-r and IL-6 Production by Imidazo-
lines in Human Whole Blood. The results from cell culture
prompted the analysis of these imidazolines for their anti-
inflammatory potential in whole blood. The imidazolines were
subsequently evaluated for their ability to inhibit a NF-κB
mediated cytokine response in IL-1ꢀ stimulated human blood.
Human blood samples were pretreated with imidazolines 4-11
for 2 h followed by IL-1ꢀ stimulation. Plasma was harvested
22 h after stimulation and IL-1ꢀ induced TNF-R production
was measured using a human TNF-R ELISA (R&D Systems)
assay. The circulating TNF-R levels in IL-1ꢀ stimulated blood
samples were significantly higher than in unstimulated or the
vehicle treated blood. Pretreatment of the blood for 2 h with
the imidazolines 4-11, followed by IL-1ꢀ stimulation, resulted
in a strong dose-dependent inhibition of TNF-R production, as
compared to the vehicle treated control (Figure 1C). The amide
10 and the alcohol 11 were inactive (Table 1). Therefore,
imidazolines 7-9 were effective in decreasing TNF-R levels,
presumably via inhibition of NF-κB mediated gene transcription.
Synthesis and Biological Evaluation
Synthesis of Imidazoline Derivatives. We envisioned that
the replacement of the carboxylic acid in 1 with esters or a less
acidic alcohol or amide functionalities would avoid the forma-
tion of the zwitterion. The imidazoline scaffold was prepared
via our previously reported protocol using a silicon mediated
1,3-dipolar cycloaddition (Scheme 1).37,38 The resulting
imidazoline·HCl salt 1 was treated with TMSCHN2 to yield
the methyl ester 7 in excellent yield (Scheme 2). The ethyl ester
8 was prepared via the acid chloride. To form the benzyl ester
9, the imidazoline 1 was coupled with benzyl alcohol, EDCI,
and DMAP. To determine the importance of the ester moiety,
imidazoline 1 was converted to the amide 10 using similar
coupling conditions and (NH4)2CO3. Finally, the ester was
reduced to the alcohol 11 with lithium aluminum hydride to
study the significance of the carbonyl moiety for activity
(Scheme 2).
To evaluate the effect of inhibiting TNF-R production on
other downstream pro-inflammatory cytokines, such as IL-6,
the same plasma samples were subjected to an IL-6 ELISA
(R&D Systems). As seen with the TNF-R response, IL-1ꢀ
stimulation caused a robust increase in IL-6 production when
compared to the unstimulated control or the vehicle-only treated
blood samples. Pretreatment of the blood for 2 h with the
imidazolines 4, 5, 7-11, followed by IL-1ꢀ stimulation, resulted
in a strong dose-dependent inhibition of IL-6 production, as
compared to the vehicle treated control (Figure 1D). Consistent
with the TNF-R data as well as the NF-κB mediated luciferase
activity, compounds 10 and 11 were significantly less active.
The reduction of IL-6 levels clearly indicates that inhibition of
NF-κB activation causes a decline in the production of additional
pro-inflammatory cytokines. Even though the decarboxylated
Inhibition of NF-KB-Mediated Gene Transcription. The
ability of the imidazolines 1, 2 and 4-11 to modulate NF-κB-
mediated gene transcription was evaluated using a luciferase
reporter assay in human cervical epithelial (HeLa) cells with a
stably transfected NF-κB-luc gene. These cells maintain, through
hygromycin selection, a chromosomal integration of a luciferase
reporter construct regulated by multiple copies of the NF-κB
response element. The cells were activated with TNF-R (25 ng/
mL) in the presence or absence of imidazolines 1, 2, 4-11.
Because activation of the NF-κB pathway requires proteasomal
degradation of IκB, a classic proteasome inhibitor 12 (N-CBz-
Leu-Leu-leucinal, also known as MG-132, Figure 1B)43 was

1304 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Scheme 2. Synthesis of Imidazolines Derivativesa
Kahlon et al.
a (a) NaHCO3 (aq). (b) DMSO, rt or THF, reflux. (c) TMSCHN2, benzene:MeOH (9:1), 0 °C. (d) (1) (COCl)2, CH2Cl2, 0 °C; (2) EtOH; (e) EDCI, DMAP,
benzyl alcohol, CH2Cl2. (f) EDCI, HOBt, DIPEA, (NH4)2CO3, THF. (g) LiAlH4, THF.
Table 1
zoline mediated cell death, both the HeLa cells and white blood
cells were evaluated for cell death at various concentrations of
imidazoline 8. An LDH release assay showed that incubation
of HeLa/NF-κB-luc cells with up to 10 µM imidazoline 8 for
8 h did not induce any significant amount of cell death (see
Supporting Information). FACS analysis of these samples
confirmed the cells were healthy (see Supporting Information).
To confirm the cell viability data in the whole blood experi-
ments, human white blood cell counts also indicated no
significant cell cytotoxicity at 10 µM or less imidazoline 8 (see
Supporting Information). Therefore, the inhibition of luciferase
production and inhibition of TNF-R and IL-6 production by
imidazoline 8 was not due to a decrease in cell number but
rather due to inhibition of NF-κB transcription.
HeLa NF-κB-luc whole blood TNF-R whole blood IL-6
compd
EC50a, µM
IC50b, µM
IC50c, µM
1
4
5
6
7
8
0.95d
4.6
11.0
>20
7.5
>20
NTe
NTe
NTe
2.8
>20
2.5
2.4
NTe
3.0
2.5
1.2
0.8
8a (R,R)
8b (S,S)
9
10
11
1.6
0.6
0.2
2.9
0.7
0.6
3.5
3.2
5.9
∼20
>20
11.9
>20
∼20
>20
a EC50 values for inhibition of luciferase production in pNF-κB-luc HeLa
cells following TNF-R activation. b IC50 values for inhibition of TNF-R
production in human whole blood following IL-1ꢀ stimulation. c IC50 values
for inhibition of IL-6 production in human whole blood following IL-1ꢀ
stimulation. EC50 and IC50 values were calculated from logarithmic curves
(see Supporting Information for complete list of log values and standard
errors). d Reference 35. e NT: Not tested.
Stability of Imidazoline 8. Sterically accessible carboxylic
esters are often susceptible to hydrolysis by blood and cellular
esterases, a conversion that could present a potential problem
due to the reoccurrence of the zwitterionic form 2 and hence
lead to instability of the imidazoline scaffold. The ethyl ester
derivative (compound 8) was therefore incubated in whole
human blood for 24 h and evaluated for hydrolysis. Compound
8 was found to be stable to blood esterolytic activity for up to
24 h at 37 °C as determined by LC/MS analysis of compound-
spiked human blood. LC/MS analysis indicated no detection of
cleavage products over this 24 h incubation period (see
Supporting Information). These results indicated that imidazoline
8 is the active compound in the assays and not a pro-drug for
imidazoline 1.
compounds (4 and 5) inhibited IL-6 production in the low
micromolar range, their ability to isomerize and aromatize to
the imidazole 6 made them unattractive as potential drug
candidates.
From this data, compound 8 was determined to be the superior
compound of this series based on the activities in all three
assays. Considering the difference between the types of assays,
the activity of the compounds in cell culture corresponded very
well with the activity in IL-1ꢀ stimulated whole blood for both
TNF-R and IL-6 inhibition (Table 1). This data indicates that
imidazoline 8 is a very effective inhibitor of NF-κB mediated
gene transcription, resulting in a decrease in the levels of the
pro-inflammatory cytokines TNF-R and IL-6 in IL-1ꢀ stimulated
human blood. To demonstrate the pharmacological relevance
of these imidazolines, human blood samples were first stimulated
with IL-1ꢀ and then treated with imidazoline 8 at various doses
(see Supporting Information). The IC50 value for post-treatment
was comparable to that for pretreatment, suggesting the imi-
dazolines inhibit inflammatory response after it has been
initiated.
Evaluation of Enantiomers. To optimize the anti-inflam-
matory efficacy of our scaffold, each enantiomer (8a-(R,R) and
8b-(S,S)) of the racemic compound 8 was evaluated for its
biological activity. Previously, we found that both enantiomers
of compound 1 were active in cell culture, however the (R,R)
enantiomer of imidazoline 1 was approximately 5-fold more
potent than the (S,S) enantiomer.36 To evaluate the two
enantiomers of compound 8, the enantiomers were readily
resolved by chiral HPLC (see Supporting Information). This
procedure gives excellent resolution (∼5 min between enanti-
omers), and both enantiomers were isolated and evaluated for
their activities. Similar to the imidazoline 1, both enantiomers
showed strong inhibition of NF-κB mediated gene transcription,
Cytotoxicity of Imidazoline 8. To exclude the possibility
of decreased cytokine production due to an increase of imida-

NF-κB Mediated Inhibition of Cytokine Production
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1305
Figure 1. Dose-response activity of imidazoline 8 in (A) inhibition of luciferase production in HeLa-NF-κB-luc cells, (C) inhibition of TNF-R
production in IL-1ꢀ stimulated human blood, and (D) inhibition of IL-6 production in IL-1ꢀ stimulated human blood. (A) Cells were unstimulated/
stimulated with 25 ng/mL TNF-R in the absence or presence of imidazoline 8 in 1% DMSO. Fold-induction (%) of the luciferase activity, normalized
for all samples to TNF-R stimulation, is shown. (B) Structure of peptide aldehyde 12. (C) Human blood (1:10 RPMI) were unstimulated/stimulated
with 200 U/mL IL-1ꢀ in the absence or presence of imidazoline 8 in 1% DMSO. Fold-induction of the TNF-R levels of stimulated (calculated from
unstimulated) cells was determined. (D) Human blood (1:10 RPMI) were unstimulated/stimulated with 200 U/mL IL-1ꢀ in the absence or presence
of imidazoline 8 in 1% DMSO. Fold-induction of the IL-6 levels of stimulated (calculated from unstimulated) cells was determined. All experiments
were run in duplicate.
with the (R,R) enantiomer 8a (EC50 ) 1.6 µM) somewhat more
potent than the (S,S) enantiomer 8b (EC50 ) 2.9 µM) (Table
1). The enantiomers were also tested for their ability to inhibit
cytokine production in whole human blood. Once more, the
(R,R) enantiomer 8a was found to be slightly more potent than
the (S,S) enantiomer 8b. The IC50 values for TNF-R and IL-6
inhibition for the (R,R) enantiomer 8a were 0.6 and 0.2 µM,
respectively. The IC50 values for the (S,S) enantiomer were in
the same range, albeit slightly less potent. These results indicate
that both enantiomers of 8 are able to inhibit cytokine production
at the low micromolar level, with the (R,R) enantiomer to be
slightly superior.
derivative 8 was found to be a potent inhibitor of NF-κB
mediated gene transcription in HeLa cells with low micromolar
EC50 values and stable against degradation and hydrolysis by
esterases. The cellular activity of the imidazolines translated
well in whole human blood assays, resulting in nanomolar IC50
values for TNF-R and IL-6 production after IL-1ꢀ mediated
NF-κB stimulation. Furthermore, evaluation of isolated enan-
tiomers revealed the (R,R) enantiomer of imidazoline 8 to be
slightly more potent than the (S,S) enantiomer at inhibiting NF-
κB mediated gene transcription and cytokine production. The
further clinical potential of these stable noncytotoxic small
molecule NF-κB inhibitors as potential anti-inflammatory agents
is currently under investigation in our laboratories.
Conclusion
The NF-κB inhibitor imidazoline 1 was previously found to
be a potent inhibitor of NF-κB mediated gene transcription in
cell culture. The carboxylic acid functionality of this class of
compounds was found to be responsible for subsequent decom-
position (Scheme 1). Herein, we described the synthesis and
biological activity of structurally modified imidazolines, inca-
pable of forming a zwitterionic species. The ethyl ester
Experimental Section
General Information. All commercial reagents were purchased
from commercial suppliers and used without further purification.
All solvents were reagent grade. Peptide aldehyde 12 was obtained
from Calbiochem (San Diego, CA), TNF-R was purchased from
Invitrogen (Carlsbad, CA), and IL-1ꢀ was obtained from Roche
Applied Sciences (Indianapolis, IN). THF was freshly distilled from

1306 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Kahlon et al.
sodium/benzophenone under nitrogen. CH2Cl2 was dispensed from
a delivery system, which passes the solvents through a column
packed with dry neutral alumina. Melting points were obtained using
an electrothermal capillary melting point apparatus and are uncor-
rected. Column chromatography was carried out on silica gel 60
(230-400 mesh) supplied by EM Science. Yields refer to chro-
matographically and spectroscopically pure compounds unless
otherwise stated. Infrared spectra were recorded on a Nicolet IR/
42 spectrometer. Proton and carbon NMR spectra were recorded
on a Varian Unity Plus-500 spectrometer. High-resolution 70 eV
EI mass spectra and FAB mass spectra were obtained at the RTSF
Mass Spectrometry Facility of the Michigan State University, using
a JEOL AX-505H and a JEOL HX-110 double focusing mass
spectrometer (JEOL USA, Peabody, MA), respectively. Combustion
analysis was preformed on a PerkinElmer 2400 Series II CHNS/O
analyzer. All final compounds possess purity >95% as determined
by combustion analysis, high-resolution mass spectrometry, or
HPLC.
DL-(4R,5R)-1-Benzyl-2,4,5-triphenyl-4,5-dihydro-1H-imidazole-
4-carboxylic Acid Hydrochloride (1). Melting point 152-155 °C.
1H NMR (500 MHz, DMSO-d) δ 4.18 (d, 1H, J ) 16.0 Hz), 4.86
(d, 1H, J ) 16.0 Hz), 5.59 (s, 1H), 6.71 (d, 2H, J ) 7.5 Hz), 7.10
(t, 2H, J ) 7.7 Hz), 7.19 (t, 2H, J ) 7.5 Hz), 7.45-7.59 (m, 7H),
7.72-7.77 (m, 4H), 7.84 (t, 1H, J ) 7.2 Hz), 7.97 (d, 2H, J ) 7.5
Hz), 12.86 (br s, 1H), 13.99 (br s, 1H). 13C NMR (125 MHz,
DMSO-d) δ 48.7, 73.8, 75.9, 121.4, 127.2, 128.3, 128.7, 128.9,
129.0, 129.1, 129.4, 129.5, 129.9, 132.8, 133.2, 134.3, 139.2, 165.7,
167.6.
then washed with saturated NaHCO3 solution (2 × 20 mL) and
brine solution (1 × 20 mL). The solution was then dried over
Na2SO4 and concentrated in vacuo. The resulting crude residue was
purified via flash column chromatography using silica gel (1:9
methanol:dichloromethane as eluant), affording the product as a
1
yellow oil (77 mg, 20%). H NMR (500 MHz, CDCl3) δ 3.85 (d,
1H, J ) 15.5 Hz), 4.77 (d, 1H, J ) 16 Hz), 4.92 (d, 1H, J ) 11.5
Hz), 5.55 (d, 1H, J ) 11 Hz), 6.90-6.96 (m, 5H), 6.97-7.00 (m,
4H), 7.02-7.09 (m, 3H), 7.25-7.29 (m, 3H), 7.50-7.53 (m, 3H),
7.81-7.83 (m, 2H). 13C NMR (125 MHz, CDCl3) δ 49.0, 68.4,
72.9, 126.2, 127.1, 127.3, 127.5, 127.8, 127.9, 127.9, 128.1, 128.5,
128.6, 128.7, 130.2, 131.2, 136.6, 136.8, 139.3, 167.1. IR (neat):
3030 cm-1, 2924 cm-1, 1595 cm-1. HRMS (FAB): m/z calculated
for [C28H24N2 + H]+, 389.2018; found, 389.2017.
1-Benzyl-2,4,5-triphenyl-1H-imidazole (6). To a flame-dried
100 mL round-bottom flask was added 2,4,5-triphenylimidazole (2
g, 6.76 mmol) and anhydrous THF (60 mL). Sodium hydride (432
mg, 10.81 mmol) was then added and the mixture stirred for 5
min. Then benzyl chloride (1.24 mL, 10.81 mmol) was added
dropwise and the mixture refluxed overnight under a nitrogen
atmosphere. The solid precipitate was filtered off and the THF was
concentrated down. The crude residue was put into solution using
ethyl acetate (50 mL) and was washed with brine solution (1 × 30
mL). The organics were dried using anhydrous sodium sulfate and
concentrated. The crude residue was purified by flash column
chromatography using silica gel (8:2 hexane:ethyl acetate as eluant)
affording the product as a white powder (1.33 g, 51%); mp
159-161 °C. 1H NMR (500 MHz, CDCl3): δ 5.1 (s, 2H), 6.78-6.81
(m, 2H), 7.1-7.23 (m, 8H), 7.28-7.4 (m, 6H), 7.57-7.6 (m, 2H),
7.64-7.67 (m, 2H). 13C NMR (125 MHz, CDCl3): δ 48.2, 125.9,
126.2, 126.7, 127.2, 128.0, 128.51, 128.52, 128.55, 128.72, 128.81,
129.0, 130.0, 130.9, 131.0, 134.4, 137.5, 138.0, 148.0. IR (NaCl)
3065 cm-1, 3032 cm-1, 1956 cm-1, 1889 cm-1, 1812 cm-1, 1762
cm-1. HRMS (ESI): m/z calculated for [C28H22N2 + H]+, 387.1861;
found, 387.1873.
DL-(4R,5R)-1-Benzyl-2,4,5-triphenyl-4,5-dihydro-1H-imidazol-
3-ium-4-carboxylate (2). DL-(4R,5R)-1-benzyl-2,4,5-triphenyl-4,5-
dihydro-1H-imidazole-4-carboxylic acid hydrochloride (1) was
dissolved in dichloromethane. Then it was washed 2× saturated
NaHCO3 solution. The organic layer was separated, dried over
MgSO4, and concentrated in vacuo to yield the zwitterion 2 as an
1
off-white solid; mp 119-121 °C. H NMR (500 MHz, CDCl3) δ
3.78 (d, 1H, J ) 16.0 Hz), 4.60 (d, 1H, J ) 16.0 Hz), 4.91 (s, 1H),
6.60 (d, 2H, J ) 7.5 Hz), 7.06 (t, 2H, J ) 7.5 Hz), 7.15 (t, 1H, J
) 7.5 Hz), 7.26-7.33 (m, 5H), 7.38-7.41 (m, 5H), 7.49 (t, 1H, J
DL-(4R,5R)-Methyl 1-Benzyl-4,5-dihydro-2,4,5-triphenyl-1H-
imidazole-4-carboxylate (7). A flame-dried flask under nitrogen
was charged with DL-(4R,5S)-1-benzyl-4,5-dihydro-2,4,5-triphenyl-
1H-imidazole-4-carboxylic acid (1) (0.50 g, 1.07 mmol) in 9:1
benzene:methanol (50 mL) and cooled to 0 °C. After 5 min,
(trimethylsilyl)diazomethane (1.1 mL, 2.13 mmol) was added
dropwise and then the reaction mixture was allowed to stir at 0 °C
for 3 h. The reaction mixture was concentrated to minimal residue
and purified by running through a small silica plug to give the
product as a cream-colored solid (0.24 g, 50%); mp 114-116 °C.
1H NMR (500 MHz, CDCl3) δ 3.18 (s, 3H), 3.84 (d, 1H, J ) 16.0
Hz), 4.63 (d, 1H, J ) 16.0 Hz), 4.91 (s, 1H), 6.73 (d, 2H, J ) 7.5
Hz), 7.05 (t, 2H, J ) 7.5 Hz), 7.10 (t, 1H, J ) 7.5 Hz), 7.25-7.28
(m, 1H), 7.31-7.34 (m, 3H), 7.37 (d, 4H, J ) 4.5 Hz), 7.47 (d,
1H, J ) 1.5 Hz), 7.48(d, 1H, J ) 2.0 Hz), 7.73 (m, 2H), 7.77 (d,
1H, J ) 3.5 Hz), 7.78 (d, 1H, J ) 1.5 Hz). 13C NMR (125 MHz,
CDCl3) δ 48.7, 51.8, 73.9, 83.1, 126.8, 127.2, 127.4, 127.5, 128.0,
128.1, 128.3, 128.4, 128.5, 128.6, 128.9, 130.4, 130.5, 136.6, 137.9,
) 7.5 Hz), 7.54 (d, 2H, J ) 7.5 Hz), 7.82 (d, 2H, J ) 7.0 Hz). 13
C
NMR (125 MHz, DMSO-d) δ 48.7, 75.8, 79.3, 123.3, 125.9, 127.0,
127.7, 128.2, 128.5 (2 peaks), 129.1, 129.2, 129.3, 129.3, 129.6,
133.2, 134.0, 136.2, 143.3, 165.1, 168.4.
DL-(4R,5R)-1-Benzyl-2,4,5-triphenyl-4,5-dihydro-1H-imida-
zole (4). The synthesis of compound 4 has been previously
reported.44 The title compound was isolated in small amounts via
a decarboxylation of imidazoline 1. A solution of imidazoline 1
(0.7 g, 1.62 mmol) in 100 mL of THF was refluxed for 5 h, resulting
in complete decomposition of starting material. The solution was
then concentrated in vacuo, resulting in a crude mixture of
imidazolines 4 and 5 and imidazole 6 in a 2:1:1 ratio, respectively.
The crude residue was then purified via flash column chromatog-
raphy using silica gel (1:9 methanol:dichloromethane as eluant),
isolating the product as a white solid (112 mg, 18%-unoptimized);
mp 78-80 °C. 1H NMR (500 MHz, CDCl3) δ 3.99 (d, 1H, J ) 16
Hz), 4.41 (d, 1H, J ) 8.5 Hz), 4.77 (d, 1H, J ) 15.5 Hz), 5.07 (d,
1H, J ) 8.5 Hz), 7.00-7.01 (m, 2H), 7.18-7.23 (m, 2H),
7.24-7.30 (m, 4H), 7.31-7.33 (m, 2H), 7.34-7.39 (m, 3H),
7.42-7.45 (m, 2H), 7.53-7.55 (m, 3H), 7.88-7.90 (m, 2H). 13C
NMR (125 MHz, CDCl3) δ 49.6, 72.6, 77.9, 126.7, 127.0, 127.1,
127.4, 127.7, 127.9, 128.3, 128.4, 128.6, 128.6, 128.8, 130.1, 131.3,
136.4, 141.8, 143.9, 165.9. IR (neat): 3063 cm-1, 3030 cm-1, 1595
cm-1. HRMS (FAB): m/z calculated for [C28H24N2 + H]+,
389.2018; found, 389.2020.
144.0, 165.6, 171.4. IR (NaCl): 3061 cm-1, 3031 cm-1, 2948 cm-1
,
1734 cm-1, 1595 cm-1. HRMS (ESI): m/z calculated for
[C30H26N2O2 + H]+, 447.2070; found, 447.2070.
DL-(4R,5R)-Ethyl 1-Benzyl-4,5-dihydro-2,4,5-triphenyl-1H-
imidazole-4-carboxylate (8). Into a flame-dried flask under nitrogen
was placed DL-(4R,5S)-(1-benzyl-4,5-dihydro-2,4,5-triphenyl-1H-
imidazole-4-carboxylic acid (1) (20.0 g, 46.3 mmol) and dichlo-
romethane (300 mL). After cooling the reaction mixture to 0 °C,
oxalyl chloride (11.7 mL, 138.8 mmol) was added over 5 min,
followed by DMF (10 µL/1 mL of dichloromethane). The reaction
mixture was allowed to stir at 0 °C for 2 h, after which the solvent
was removed on the rotary evaporator. The residue was placed on
a vacuum line for 1 h. The flask was then placed under nitrogen
and cooled to 0 °C and ethanol (400 mL) was added. After stirring
for an addition 2.5 h, the ethanol was removed on the rotary
evaporator and dichloromethane (300 mL) was added. The organic
solution was washed with saturated NaHCO3 solution (1 × 100
mL) and H2O (1 × 100 mL), dried over MgSO4, filtered, and the
Because of the difficulty in separation and to verify the structures
of compounds 5 and 6, each compound was prepared individually
via an alternative route.
DL-(4R,5S)-1-Benzyl-2,4,5-triphenyl-4,5-dihydro-1H-imida-
zole (5). A solution of DL-(4R,5S)-2,4,5-triphenyl-4,5-dihydro-1H-
imidazole45 (0.3 g, 1.01 mmol) and benzyl bromide (0.2 g, 1.06
mmol) in 20 mL of anhydrous benzene was treated with triethyl
amine (0.2 g, 2.02 mmol). The solution was refluxed for 15 h and

NF-κB Mediated Inhibition of Cytokine Production
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1307
solvent was removed under reduced pressure to give a yellow oil.
The product was purified using flash column chromatography on
silica gel (1:1 hexane:ethyl acetate as eluant) to give a white solid
(16.1 g, 76%); mp 86-88 °C. 1H NMR (500 MHz, CDCl3) δ 0.83
(t, 3H, J ) 7.2 Hz), 3.63 (qq, 1H, J ) 7.2 Hz), 3.73 (qq, 1H, J )
7.2 Hz), 3.86 (d, 1H, J ) 16.0 Hz), 4.64 (d, 1H, J ) 15.5 Hz),
4.96 (s, 1H), 6.78 (d, 2H, J ) 7.0 Hz), 7.09 (t, 2H, J ) 7.2 Hz),
7.14 (t, 1H, J ) 7.2 Hz), 7.28-7.32 (m, 1H), 7.34-7.38 (m, 3H),
7.39-7.45 (m, 4H), 7.49-7.51 (m, 3H), 7.79-7.81(m, 4H). 13C
NMR (125 MHz, CDCl3) δ 13.4, 48.6, 60.9, 73.7, 82.8, 126.7,
127.1, 127.2, 127.3, 127.9, 128.1, 128.2, 128.3, 128.4, 128.5, 128.8,
130.2, 130.6, 136.6, 137.9, 144.1, 165.3, 170.7. IR (NaCl): 3061
cm-1, 3031 cm-1, 2981 cm-1, 1732 cm-1, 1597 cm-1. HRMS (ESI):
m/z calculated for [C31H28N2O2 + H]+, 461.2229; found, 461.2225.
DL-(4R,5R)-Benzyl 1-Benzyl-4,5-dihydro-2,4,5-triphenyl-1H-
imidazole-4-carboxylate (9). To a stirred solution of DL-(4R,5S)-
1-benzyl-4,5-dihydro-2,4,5-triphenyl-1H-imidazole-4-carboxylic acid
(1) (200 mg, 0.43 mmol) in dichloromethane was added 1-ethyl-
(3-dimethylaminopropyl) carbodiimide (120 mg, 0.64 mmol). After
5 min was added 4-(N,N-dimethylamino)pyridine (52 mg, 0.43
mmol). After stirring for an addition 10 min, benzyl alcohol (92
mg, 0.85 mmol) was added. The reaction mixture was stirred at
room temperature overnight. The reaction mixture was washed with
10% HCl solution (1 × 5 mL), saturated NaHCO3 solution (1 × 5
mL), H2O (1 × 5 mL), and brine solution (1 × 5 mL). The product
was extracted using dichloromethane, dried over MgSO4, filtered,
and the solvent was removed under reduced pressure. The product
was purified using column chromatography on silica gel (9:1
dichloromethane:ethyl acetate as eluant) to give a light-yellow oil
4,5-dihydro-2,4,5-triphenyl-1H-imidazole-4-carboxylate (7) (50 mg,
0.11 mmol) in THF (3 mL) was added and the reaction mixture
was stirred under nitrogen and warmed to room temperature. The
reaction mixture was quenched using saturated NH4Cl solution (3
mL). The mixture was allowed to stir for 10 min and then placed
in a separatory funnel. The product was extracted using ethyl acetate
(20 mL). The aqueous layer was washed with fresh ethyl acetate
(5 mL). The combined organic layers were washed with brine
solution, dried over MgSO4, filtered, and the solvent was removed
under reduced pressure. The crude material was purified via column
chromatography on silica gel (100% ethyl acetate as eluant) to yield
a white solid (29 mg, 62%); mp 138-141 °C. 1H NMR (500 MHz,
CDCl3) δ 2.78 (br s, 1H), 3.47-3.50 (m, 1H), 3.53-3.56 (m, 1H),
3.87 (d, 1H, J ) 15.5 Hz), 4.59 (s, 1H), 4.72 (d, 1H, J ) 15.5 Hz),
6.72 (d, 2H, J ) 6.5 Hz), 7.05-7.10 (m, 3H), 7.21-7.22 (m, 1H),
7.32 (t, 2H, J ) 7.5 Hz), 7.35-7.41 (m, 6H), 7.54 (t, 3H, J ) 3.5
Hz), 7.84-7.85 (m, 2H). 13C NMR (125 MHz, CDCl3) δ 49.0, 67.0,
73.8, 76.9, 125.8, 126.8, 127.5, 127.6, 128.1, 128.4, 128.4, 128.5,
128.8, 128.8, 130.5, 130.7, 135.7, 135.9, 146.3, 166.5. IR (NaCl):
3183 cm-1 (broad), 3061 cm-1, 3029 cm-1, 2924 cm-1, 1597 cm-1
.
HRMS (ESI): m/z calculated for [C29H26N2O + H]+, 419.2123;
found 419.2125.
Biological Methods. Cell Culture. The human cell line HeLa-
NF-κB-luc was purchased from Panomics Inc. (Fremont, CA). The
cells were maintained in Dulbecco’s modified Eagle’s medium
(DMEM) containing 4.5 g/L glucose, 3.7 g/L bicarbonate, and
supplemented with 5% fetal bovine serum, 100 U/mL penicillin,
100 µg/mL streptomycin, 1 mM sodium pyruvate, 0.2 mM
L-glutamine, and 100 µg/mL of hygromycin B. The human cell
line HeLa was purchased from ATCC. The cells were maintained
in DMEM media containing 4.5 g/L glucose, 3.7 g/L bicarbonate,
and supplemented with 10% fetal bovine serum, 100 U/mL
penicillin, 100 mg/mL streptomycin, 1 mM sodium pyruvate, and
0.2 µM L-glutamine. Cells were cultured at 37 °C, 5% CO2
atmosphere, 97% relative humidity and were routinely passaged
by trypsin-EDTA treatment to maintain a cell density between 2
× 105 to 5 × 105.
1
(120 mg, 54%). H NMR (500 MHz, CDCl3) δ 3.79 (d, 1H, J )
15.5 Hz), 4.50 (d, 1H, J ) 12.5 Hz), 4.61 (d, 1H, J ) 15.5 Hz),
4.76 (d, 1H, J ) 12.5 Hz), 4.92 (s, 1H), 6.74 (d, 2H, J ) 7.5 Hz),
6.95 (dd, 2H, J ) 7.5, 2.5 Hz), 7.04-7.08 (m, 2H), 7.10-7.13 (m,
1H), 7.17-7.21 (m, 3H), 7.27-7.29 (m, 4H), 7.30-7.34 (m, 4H),
7.47-7.49 (m, 3H), 7.74-7.77 (m, 4H). 13C NMR (125 MHz,
CDCl3) δ 48.6, 66.6, 73.7, 82.7, 126.8, 127.1, 127.4, 127.5, 127.7,
127.9.0, 128.0 (2 peaks), 128.4 (2 peaks), 128.5, 128.6, 128.8, 130.4,
135.4, 136.3, 137.3, 143.5, 165.6, 170.3. IR (NaCl): 3063 cm-1
,
Viability Assays. Human white blood cells were counted
manually using a BD Unopette reservoir and a hemacytometer.
NF-KB-luc Reporter Assay. HeLa NF-κB-luc cells (5.0 × 105
cells/mL) were seeded into a 96-well white opaque plate using
DMEM medium supplemented with 5% fetal bovine serum, 100
U/mL penicillin, 100 µg/mL streptomycin, 1 mM sodium pyruvate,
0.2 mM L-glutamine, and 100 µg /mL hygromycin B. After 24 h,
the cell culture medium was replaced with DMEM medium
supplemented with 100 U/mL penicillin and 100 µg/mL strepto-
mycin. Cell cultures were pretreated with vehicle (1% DMSO), 50
µM peptide aldehyde 12 (positive control), or imidazoline (final
concentrations were 20, 10, 5, 1, 0.5, 0.1, 0.05 µM) for 30 min at
37 °C in 5% CO2. TNF-R was added to a final concentration of 25
ng/mL, and the samples were further incubated for 8 h at 37 °C in
5% CO2. Cells were assayed for firefly luciferase production using
the Steady-Glo luciferase reporter assay (Promega, Madison, WI)
according to manufacturer’s protocol. The luminescence of each
well was measured using a Veritas microplate luminometer. All
reported data are the average of two independent experiments unless
otherwise indicated. The data was analyzed using GraphPad Prism
4.0. The data was normalized to TNF-R activation, and the EC50
values were calculated using the equation for the sigmodial curve
for variable slope.
NF-KB-luc/Renilla-luc Dual Reporter Assay. HeLa cells (3.5
× 105 cells/mL) were seeded into a 6-well plate using using DMEM
media supplemented with 10% fetal bovine serum and no antibiot-
ics. Transient transfections were performed using lipofectamine
2000. Briefly, 0.8 µg of plasmid DNA was combined with 1.5 µL
lipofectamine 2000. HeLa cells were transiently cotransfection with
internal control pRL-TK. A constitutively activated plasmid pFC-
MEKK served as positive control and pCIS-CK was the negative
control. The mixture was incubated at room temperature for 20
min and mixed with cells in Opti-MEM I medium. Cells were
transfected for 5-6 h at 37 °C in 5% CO2. Cells were allowed to
3032 cm-1, 2924 cm-1, 1740 cm-1, 1593 cm-1. HRMS (ESI): m/z
calculated for [C36H30N2O2 + H]+, 523.2386; found, 523.2390.
DL-(4R,5R)-1-Benzyl-4,5-dihydro-2,4,5-triphenyl-1H-imidazole-
4-carboxamide (10). To a stirred solution of DL-(4R,5S)-1-benzyl-
4,5-dihydro-2,4,5-triphenyl-1H-imidazole-4-carboxylic acid (1) (100
mg, 0.21 mmol,), N-hydroxybenzotriazole (32 mg, 0.23 mmol), and
1-ethyl-(3-dimethylaminopropyl) carbodiimide (45 mg, 0.23 mmol)
in anhydrous THF (2 mL) was added N,N-diisopropylethylamine
(30 mg, 0.23 mmol). The reaction mixture was stirred at ambient
temperature for 10 min. Then ammonium carbonate (50 mg, 0.64
mmol) was added in one portion and the resulting suspension was
stirred at ambient temperature overnight. The reaction mixture was
concentrated to minimal residue. The residue was treated with 1:1
saturated NaHCO3 solution:H2O (2 mL) and stirring was continued
for 2 h. The mixture was placed into a separatory funnel, and the
product was extracted using ethyl acetate, dried over MgSO4, and
the solvent was removed under reduced pressure. The product was
purified using column chromatography on silica gel (3:7 hexane:
ethyl acetate as eluant) to give a light-yellow solid (65 mg, 71%);
1
mp 65-68 °C. H NMR (500 MHz, CDCl3) δ 3.80 (d, 1H, J )
16.5 Hz), 4.56 (d, 1H, J ) 16.5 Hz), 4.86 (s, 1H), 5.15 (br s, 1H),
6.71 (d, 2H, J ) 7.5 Hz), 7.05 (t, 2H, J ) 7.5 Hz), 7.11 (t, 1H, J
) 7.2 Hz), 7.26-7.28 (m, 1H), 7.30-7.36 (m, 7H), 7.50-7.54
(m, 3H), 7.71-7.73 (m, 2H), 7.79 (d, 2H, J ) 7.5 Hz). 13C NMR
(125 MHz, CDCl3) δ 48.4, 74.3, 81.6, 126.3, 126.9, 127.3, 127.4,
128.1, 128.3, 128.4, 128.5, 128.8, 130.6, 136.7, 137.8, 144.8, 165.4,
173.4. IR (NaCl): 3442 cm-1, 3063 cm-1, 3031 cm-1, 2926 cm-1
,
1688 cm-1, 1591 cm-1. HRMS (FAB): m/z calculated for
[C29H25N3O + H]+, 432.2076; found 432.2073.
DL-(4R,5R)-1-Benzyl-4,5-dihydro-2,4,5-triphenyl-1H-imidazol-
4-yl)methanol (11). In a flame-dried flask a suspension of lithium
aluminum hydride (7 mg, 0.18 mmol) in anhydrous THF (8 mL)
was cooled to 0 °C. Then a solution of DL-(4R,5S)-methyl 1-benzyl-

1308 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Kahlon et al.
grow in complete growth medium for 24 h in 5% CO2. Cell cultures
were pretreated with vehicle (1% DMSO), 50 µM peptide aldehyde
12 (positive control), or imidazoline 8 (final concentrations were
20, 10, 5, 2.5, 1.25, 0.625, 0.312 µM) for 30 min at 37 °C in 5%
CO2. TNF-R was added to a final concentration of 25 ng/mL, and
the samples were further incubated for 8 h at 37 °C in 5% CO2.
Cells were assayed for both firefly luciferase production as well as
Renilla luciferase (coreporter) using the Dual-Glo reporter assay
according to the manufacturer’s protocol. The results were read on
Veritas microplate luminometer as relative light units. Values for
the luciferase activity were normalized to the Renilla luciferase and
analyzed using GraphPad Prism 4.0 (see Supporting Information).
Human Whole Blood IL-1ꢀ Challenge. After obtaining the
appropriate approval for deidentified human cell lines, human whole
blood was obtained through the Jasper Research Clinic, Kalamazoo,
MI, from a single healthy, fasted human volunteer and was collected
in glass citrated endotoxin-free tubes by venipuncture. Only samples
with a white blood count falling within the normal range
(4800-10800 white blood cells per liter) were used. To support
the viability of white blood cells, blood was diluted 1:10 in RPMI-
1640 media supplemented with 10% fetal bovine serum, 100 U/mL
penicillin, and 100 µg/mL streptomycin. Aliquots of diluted blood
(1 mL) were preincubated with vehicle (0.1% DMSO, final
concentration) or imidazoline (final concentrations were 10, 3, 1,
0.3, and 0.1 µM) for 2 h at 37 °C, 5% CO2. IL-1ꢀ was added to a
final concentration of 200 U/mL, and the samples were further
incubated for 18 h at 37 °C, 5% CO2. At the end of the incubation
period, the blood samples were centrifuged at 3000 RPM for 5
min. The plasma was removed, snap frozen, and stored at -80 °C.
TNF-R and IL-6 levels were determined by ELISA (R&D Systems,
Inc.).
After centrifugation (5000g, 15 min; 4 °C), 100 µL of each
supernatant was transferred to an autosampler vial containing 100
µL of 0.15% aqueous formic acid. These solutions were analyzed
by LC/MS on a Waters LCT Premier mass spectrometer equipped
with Shimadzu SIL-5000 autosampler and LC-20AD pumps.
Analyses were performed using a Waters Atlantis dC18 column
(2.1 mm × 50 mm, 3 µm particles) using a flow rate of 0.3 mL/
min. The gradient consisted of 10 mM aqueous ammonium acetate
(solvent A) and acetonitrile (solvent B), starting at 90% A with a
linear gradient to 100% B at 20 min. Volumes of 10 µL were
injected and analyzed using positive mode electrospray ionization.
Extracted ion chromatograms were generated for the protonated
compound 8 (m/z 461.2) and the hydrolyzed product (compound
1, m/z 433.2). Retention times and response factors were obtained
through analysis of authentic standards of compounds 1 and 8.
Acknowledgment. We gratefully acknowledge funds from
the American Cancer Society (RSG CDD-106972), Michigan
Universities Commercialization Initiative (MUCI), Michigan
State University, and funding from the TCH Pharmaceuticals
Inc. We also thank Rui H. Huang of Michigan State University
for helpful discussions and X-ray crystallography, the Mass
Spectrometry Facility at Michigan State University for assistance
in acquiring HRMS data, and Dr. Louis King for assistance in
acquiring FACS data.
Supporting Information Available: Experimental data for
reporter assays, ELISA IL-6, ELISA TNF-R, FACS, and cell lysis
experiments. This material is available free of charge via the Internet
at http://pubs.acs.org.
CytoTox-ONE Homogeneous Membrane Integrity Assay.
HeLa NF-κB-luc cells (3.8 × 105 cells/mL) were seeded into a
96-well black opaque plate using DMEM media supplemented with
2% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL strepto-
mycin, and 100 µg/mL hygromycin B. Cells were treated with either
vehicle (1% DMSO) or imidazoline 8 (final concentrations were
40, 20, 10, 5, 2, 0.5, 0.2 µM) for 8 h at 37 °C, 5% CO2. Media-
only wells served as negative control to determine background
fluorescence. Untreated cells with 1% DMSO served as vehicle
control. The plate and CytoTox-ONE assay reagent (Promega,
Madison, WI) were equilibrated to room temperature. To determine
the maximum LDH release, 2 µL of lysis solution (9% w/v Triton
X-100 in water) was added to high control wells. Background LDH
release from tissue culture cells was considered as low (media)
control. An equal volume of CytoTox-ONE assay reagent was
subsequently added to each well. The contents of the plate were
gently mixed for 30 s and allowed to incubate at room temperature
for 10 min. The fluorescence of each well was recorded on
SpectraMax M5e with an excitation wavelength of 560 nm and an
emission wavelength of 590 nm. All reported data are the average
of four independent experiments unless otherwise indicated. The
data was normalized to maximum LDH release (see Supporting
Information).
FACS Analysis. Cells were treated with either vehicle (1%
DMSO) or imidazoline 8 (final concentrations were 40, 20, 10, 5,
2, 0.5, 0.2 µM) for 8 h at 37 °C, 5% CO2. For the analysis of cell
cycle parameters, the adherent cell cultures were trypsinized and
resuspended in 1 mL of 1× PBS with 50% FBS. Then the cells
were fixed with 70% (v/v) ice-cold ethanol overnight. After
centrifugation of the cells, the pellet was washed two times with 5
mL of ice cold 1× PBS containing 10% FBS. Then the cell pellet
was resuspended in staining solution (PBS containing 50 µg
propidium iodide, 10 µL 0.1 M EDTA, 14 units RNaseA). The
DNA content was analyzed by flow cytometry using a FACS
Vantage SE analyzer with ModFit LT software (see Supporting
Information).
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LC/MS Assay for Stability of Compound 8 in Whole Blood.
Plasma aliquots (50 µL) from blood incubated with imidazoline 7
for different incubation times up to 24 h were added to 200 µL of
ice-cold HPLC-grade acetonitrile in a 1.5 mL microcentrifuge tube.

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