Structural-activity relationship study of highly-functionalized imidazolines as potent inhibitors of nuclear transcription factor-κB mediated IL-6 production

Article abstract of DOI:10.1016/j.bmc.2009.03.002

We herein describe the synthesis and anti-inflammatory properties of a small library of imidazoline-based NF-κB inhibitors. The structure-activity relationship of various substituents on an imidazoline core structure was evaluated for the ability to inhibit NF-κB mediated IL-6 production. Optimization of the scaffolds was pursued by correlating luciferase-based NF-κB reporter assays with inhibition of IL-6 production in IL-1β stimulated human blood. Several derivatives were found to inhibit NF-κB mediated IL-6 production in the nanomolar range in IL-1β stimulated human blood.

Full text of DOI:10.1016/j.bmc.2009.03.002

Bioorganic & Medicinal Chemistry 17 (2009) 3093–3103
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structural–activity relationship study of highly-functionalized imidazolines
as potent inhibitors of nuclear transcription factor-
jB mediated IL-6 production
Daljinder K. Kahlon a, Theresa A. Lansdell a, Jason S. Fisk a, Jetze J. Tepe a,b,
*
a Department of Chemistry, Michigan State University, East Lansing, MI 48824, USA
b TCH Pharmaceuticals INC, Ann Arbor, MI 48104, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We herein describe the synthesis and anti-inflammatory properties of a small library of imidazoline-
Received 19 January 2009
Revised 2 March 2009
Accepted 3 March 2009
Available online 9 March 2009
based NF-
structure was evaluated for the ability to inhibit NF-
scaffolds was pursued by correlating luciferase-based NF-
j
B inhibitors. The structure–activity relationship of various substituents on an imidazoline core
B mediated IL-6 production. Optimization of the
B reporter assays with inhibition of IL-6 pro-
j
j
duction in IL-1b stimulated human blood. Several derivatives were found to inhibit NF-
jB mediated IL-6
production in the nanomolar range in IL-1b stimulated human blood.
Keywords:
NF-kappaB
IL-6
Ó 2009 Elsevier Ltd. All rights reserved.
Imidazolines
Inflammation
1. Introduction
It is well documented that NF-jB stimulates the expression of
multiple genes responsible for many aspects of inflammatory re-
The mammalian transcription factor NF-kappaB (NF-jB) is an
sponses and the pathogenesis of inflammatory diseases.27,28 For in-
ubiquitous transcription factor responsible for the regulation of
more than 150 genes1,2 impacting virtually every aspect of cellular
adaptation including responses to stress,3 inflammatory stimuli,4
activation of immune cell function,5 cellular proliferation, pro-
grammed cell death (apoptosis),6 and oncogenesis.7–11 In normal
stance, NF-jB increases the production of inflammatory enzymes
such as inducible cylooxygenase-2 (COX-2) and inducible nitric
oxide synthase (iNOS).29 Recruitment of immune cells into in-
flamed tissue is regulated by chemokines (IL-8, MIP-1
a, RANTES)
produced by NF- B transcription. NF- B also expresses cell surface
j
j
non-stimulated cells, NF-
plasm by NF- B’s inhibitory protein, I
an appropriate extracellular signal, such as pro-inflammatory
cytokines TNF- and IL-1b, the NF- B pathway becomes acti-
vated.5,13,14,2,15 During this process I
B undergoes a phosphory-
lated-driven polyubiquination and proteasomal degradation,
resulting in the release of NF- B allows for
B.12 Liberation of NF-
its rapid translocation into the nucleus.16 Following its nuclear
translocation, NF- B binds to DNA and initiates the transcription
of a host of pro-inflammatory signaling genes. Subsequently,
deregulation of the NF- B pathway has been directly implicated
j
B is typically sequestered in the cyto-
adhesion proteins (ICAM-1, VCAM-1 and E-selectin), which are re-
quired for adhesion of immune cells to the blood vessel endothe-
lium and thus, initiation of an inflammatory response.30
j
j
B.12 Upon stimulation by
a
j
j
Furthermore, cytokines (IL-1, IL-2, IL-6 and TNF-
NF- B induce the amplification of inflammatory signals. Of these
cytokines, IL-631–33 and TNF- 34–38 have been identified as key tar-
a) expressed by
j
a
j
j
gets in rheumatoid arthritis (RA) and other inflammatory disor-
ders. Pharmacologic intervention in RA was improved drastically
with the advent of biologicals that specifically target IL-632 or
j
TNF-a.
39–41 Unfortunately, these and alternative treatment options
j
for RA are limited, suffer from high costs and involve undesirable
methods of administration. Furthermore, these therapies lack data
pertaining to their long-term safety, tolerability and sustained effi-
cacy.40 In addition, variability in responses to these anti-inflamma-
tory drugs is found due to the complex network of alternative
cytokine-mediated pathways.42 Inhibition of pro-inflammatory
in the pathogenesis of inflammatory diseases such as rheumatoid
arthritis (RA),17,18 inflammatory bowel disease,19–21 helicobacter
pylori-associated gastritis,22 atheroscelerosis,23 multiple sclero-
sis24 and asthma.25 Given the critical role of NF-
jB-mediated
expression of cytokines, this transcription factor has been actively
pursued as a therapeutic target for these types of inflammatory
disorders.4,26
transcription factors, such as NF-
ter alternative to modulate the complex cytokine network that in-
duces inflammatory response.27 In addition, inhibition of NF-
jB, may therefore represent a bet-
jB
mediated gene transcription by a small molecule would represent
an attractive therapeutic alternative to the current clinical options,
* Corresponding author. Tel.: +1 517 355 9715; fax: +1 517 353 1793.
E-mail address: tepe@chemistry.msu.edu (J.J. Tepe).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.03.002

3094
D. K. Kahlon et al. / Bioorg. Med. Chem. 17 (2009) 3093–3103
Table 1
which still primarily include anti TNF-
tor antibodies.28,43
a
mAbs and anti IL-6 recep-
EC50 values of inhibition of luciferase production in pNF-
jB-luc HeLa cells following
TNF- activation. IC50 values for inhibition of IL-6 production in human whole blood
a
Previously in our lab, we prepared a class of imidazoline scaf-
folds as potent inhibitors of NF- B mediated gene transcription.
The molecular target of these compounds is still elusive but inhibi-
tion of NF- B mediated gene transcription was shown to proceed
via the modulation of I
degradation.44,45 A small series of this
class of imidazolines was investigated for its potential anti-inflam-
matory activity and found to inhibit both TNF- and IL-6 produc-
following IL-1b stimulation
j
Entry
Inhibition of HeLa
NF- B-luc EC50
Inhibition of IL-6 in
human blood IC50 (lM)
j
(
lM)
j
j
Ba
N
CO2Me
R1
N
a
tion in human blood stimulated by IL-1b.46 In our efforts to
improve the potency and develop a better understanding of the
structural requirements for biological activity, we herein describe
the synthesis and anti-inflammatory properties of several imidaz-
oline derivatives functionalized at the R1–R4 positions (Scheme 1).
Biological evaluation of this library of imidazolines led to the dis-
covery of several analogues with the ability to substantially inhibit
R1
1
2
3
4
5
CH3
CH(CH3)2
Ph
>20
ꢀ20
7.5
5.3
13
3
7.1
3
4.1
16.1
Bn
Indoyl-3-methyl
NF-
tion in stimulated human blood. Optimization of the scaffolds was
pursued by correlating luciferase-based NF- B reporter assays
with inhibition of IL-6 production in IL-1b stimulated human blood
to ensure the optimization was through NF- B mediated IL-6 inhi-
jB mediated transcription in cell culture and cytokine produc-
j
Table 2
EC50 values of inhibition of luciferase production in pNF-
jB-luc HeLa cells following
TNF- activation. IC50 values for inhibition of IL-6 production in human whole blood
a
j
following IL-1b stimulation
bition. The results indicated discrete structure–activity relation-
ships for these highly-substituted imidazolines with respect to
NF-jB mediated gene transcription.
Entry
Inhibition of HeLa NF- Inhibition of IL-6 in
jB-luc EC50
(
lM)
human blood IC50 (lM)
N
R2
2. Results and discussion
2.1. Chemistry
N
R2
The imidazoline derivatives were prepared via a 1,3-dipolar
cycloaddition reaction previously developed in our laboratory
(Scheme 1).47–50 Cyclodehydration of N-benzoyl protected amino
acid derivatives with TFAA provided azlactones without the need
for further purification. The azlactones were subsequently treated
with the desired imine in the presence of TMSCl to provide the imi-
dazoline carboxylate, which was isolated by filtration. Diversity at
the R1 position could be easily accessed by varying the starting
amino acid. The resulting imidazoline carboxylates were treated
with TMSCHN2 to yield the methyl esters 1–5 in excellent yield
(Table 1). Next, we examined the variability that could be tolerated
at the R2 position by synthesizing esters 6–8 via the acid chloride
(Table 2). Imidazolines 9 and 10 were synthesized by treating dl-
(4R,5R)-1-benzyl-4,5-dihydro-2,4,5-triphenyl-1H-imidazole-4-car-
boxylic acid48 with EDCI and DMAP in the presence of benzyl alco-
hol and (NH4)2CO3, respectively. To study the effect of the R3 and R4
domains, a number of imines (prepared from their corresponding
aldehydes and amines) underwent the 1,3-dipolar cycloaddition
to afford imidazolines 11–25 (Tables 3 and 4). Imidazoline 12
was reduced to imidazoline 13 utilizing SnCl2 in refluxing ethanol.
3
6
7
8
9
CO2Me
CO2Et
7.5
2.5
2.3
3.5
3.5
>20
3
0.8
1.9
1.9
5.9
>20
CO2nPr
CO2iPr
CO2Bn
CONH2
10
Imidazoline 6 underwent hydrogenolysis using 10% Pd/C in cyclo-
hexene to afford debenzylated imidazoline 26. Imidazoline 26 was
treated with acetic anhydride, benzoyl chloride, or tosyl chloride in
the presence of DMAP and TEA to generate imidazolines 27, 28 and
29, respectively.
Table 3
EC50 values of inhibition of luciferase production in pNF-
TNF- activation. IC50 values for inhibition of IL-6 production in human whole blood
following IL-1b stimulation
jB-luc HeLa cells following
a
Entry
Inhibition of HeLa NF- Inhibition of IL-6 in
jB-luc EC50
(l
M)
human blood IC50 (lM)
R3
N
H
N
Cl
R3
O
CO2Et
R4
Ph
O
N
H
N
O
O
N
O
Bz
TFAA
TMSCl
R4
Ph
OH
R1
N
R1
R1
N
R3
TMS
R3
6
11
12
13
14
15
16
17
18
Ph
4-Pyr
4-Ph–NO2
4-Ph–NH2
4-Ph–OMe
4-Ph–F
4-Ph–Cl
4-Ph–CF3
2-Furan
2.5
>20
3.4
10.4
3.6
5
1.5
1.4
7.6
0.8
9.4
0.5
0.5
1.3
1
0.3
0.8
1.9
R4
Cl
N
R3
O
R4
N
R4
N
R3
R2
R1
O
N
R3
CO2H
H2O
H
Ph
Ph
Ph
R1
N
N
R1
TMS
Scheme 1. Synthesis of imidazoline scaffolds.

D. K. Kahlon et al. / Bioorg. Med. Chem. 17 (2009) 3093–3103
3095
Table 4
bition of NF-
jB mediated luciferase activity in HeLa cells (Table 1).
The data in Table 1 indicate increased hydrophobicity at the R1 po-
EC50 values of inhibition of luciferase production in pNF-jB-luc HeLa cells following
TNF-a activation. IC50 values for inhibition of IL-6 production in human whole blood
following IL-1b stimulation
sition (compounds 1–4) leads to a decrease in NF-jB mediated
luciferase production.
Entry
Inhibition of HeLa NF- Inhibition of IL-6 in
R4
The imidazoline library was further evaluated for it’s ability to in-
hibit NF- B mediated cytokine production in stimulated whole hu-
man blood. Since the cytokine IL-6 is one of the key gene products
resulting from NF- B activation, all compounds were investigated
jB-luc EC50
(l
M)
human blood IC50 (lM)
j
N
CO2Et
j
N
for their ability to inhibit the production of IL-6. Human whole blood
was pretreated with the imidazolines 1–29 for 2 h, followed by IL-1b
thus inducing an NF-j
B mediated cytokine response.52,53 Plasma
R4
was harvested and IL-1b induced IL-6 production was measured
22 h after stimulation using a human IL-6 ELISA (R&D Systems).
The circulating IL-6 levels in IL-1b stimulated samples were signifi-
cantly higher (>1000-fold) than in unstimulated or the vehicle trea-
ted blood (see Supplementary data). Pretreatment of the blood for
2 h with the imidazolines 1–29, followed by IL-1b stimulation re-
sulted in a strong dose-dependent inhibition of IL-6 production, as
compared to the no-drug treated control (see Supplementary data).
Exploration of the R1 domain revealed consistency with the
6
19
20
21
22
23
24
25
26
27
28
29
Bn
2.5
5
0.8
1.4
4.6
1.2
1.6
0.5
1.5
1.3
>20
>20
6.6
6.6
4-Bn–OMe
4-Bn–Me
4-Bn–F
4-Bn–Cl
4-Bn–Br
4-Bn–CF3
2-Methylfuran
H
1.7
5.2
4.2
2.7
6.7
5.5
>20
>20
ꢀ20
>20
Ac
Bz
Tos
HeLa NF-
inhibitors with IC50 values for inhibition of IL-1b stimulated IL-6
production of 3.0 M and 4.1 M, respectively. Interestingly, even
jB-luc assay, compounds 3 and 4 were the most potent
l
l
though compound 1 proved to be inactive in the cell-based assay it
demonstrated good activity in the blood assay. This may be in part
due to its relatively increased hydrophilicity helping to provide im-
proved solubility in the blood assay. Nonetheless, to ensure that
2.2. Biological evaluation and structure–activity relationships
The potential of these highly-functionalized imidazolines to in-
hibit the NF-
first method, the ability of the imidazolines 1–29 to modulate
NF- B mediated gene transcription was evaluated using a lucifer-
ase reporter assay in human cervical epithelial (HeLa) cells con-
taining a stably transfected NF- B-luc gene (Panomics, Fremont,
j
B pathway was assessed using two methods. In the
optimization of the scaffold was for NF-jB mediated inhibition of
IL-6 production, only compounds that indicated a good correlation
between the two assays were pursued for further derivatization.
Since compound 3 proved to be slightly more active than com-
pound 4 while maintaining consistent activity in both assays, a
phenyl group was chosen to be the preferred moiety of R1 position
(R1 = Ph, Scheme 1).
j
j
CA). These cells maintain, through hygromycin selection, a chro-
mosomal integration of a luciferase reporter construct regulated
by multiple copies of the NF-
activated with TNF- (25 ng/mL) in the presence or absence of imi-
dazolines 1–29. Cells were pretreated for 30 min with the imidaz-
j
B response element. The cells were
The next series of analogues focused on evaluating the ester
functional group at the R2 domain. Previously, we reported that
this moiety is critical for stability of the imidazoline scaffold and
the ester functionality was found to be optimal at the R2 position.46
We extended this series to include n-propyl and isopropyl esters (7
and 8). Again, the inhibition of IL-6 production in stimulated hu-
man blood corresponded well with the inhibition of luciferase pro-
duction in the HeLa cells (Table 2). All esters (compounds 3, 6–9)
maintained activity in the low micromolar range, whereas the
amide 10 was devoid of any significant activity in both assays (Ta-
ble 2). An increase in potency was observed when comparing
methyl ester 3 to ethyl ester 6, although no significant improve-
ment in biological activity was observed upon evaluating more ste-
rically demanding ester moieties (compounds 7–9). Even though
most of the esters illustrated similar activity in the luciferase-
based cellular assay, the ethyl ester 6 was found to be the most po-
a
olines followed by TNF-
a stimulation. The proteasome inhibitor
MG-132 (N-Cbz-Leu-Leu-leucinal)51 and DMSO (vehicle) were
used as positive and negative controls, respectively, and luciferase
production was evaluated after 8 h (see Supplementary data data).
All samples were normalized to the TNF-
results are summarized in Tables 1–4. Treatment of HeLa/NF-
luc cells with imidazolines without any TNF- activation did not
induce a significant amount of luciferase activity, indicating that
the imidazolines did not stimulate the NF- B pathway. In order
a
activation control. The
j
B-
a
j
to validate the results from this integrated cell line and demon-
strate the reliability of this high-throughput screen, HeLa cells
were transiently transfected with 6x
jB driver reporter gene
pNF- B-Luc and the internal control plasmid pRL-TK, which pro-
j
vides low levels of Renilla luciferase production (see Supplemen-
tary data).46 Similar results were observed using both luciferase-
based reporter assays, indicating the data obtained from the stable
tent compound in the blood assay yielding an IC50 value of 0.8 lM.
Due to its potency and stability against hydrolysis by esterases,46
the ethyl ester 6 was considered our lead compound and was fur-
ther evaluated for functionalization of the R3 and R4 moieties
(Tables 3 and 4).
HeLa/NF-jB-luc cell line was representative of NF-jB mediated
gene transcription.
In order to determine the key structural requirements for bio-
logical activity of these highly-substituted imidazolines, we first
examined the R1 domain of the scaffold. These derivatives were
readily accessible by starting with the natural amino acid precur-
sors: alanine, valine, phenylglycine, phenylalanine and tryptophan,
to provide compounds 1–5, respectively (Table 1). Pre-treatment of
The R3 position was functionalized by a range of aromatic substit-
uents to study the significance of this region on biological activity
(Table 3). The 4-pyridino-substituted imidazoline 11 was the only
compound in this series to be void of any activity in the luciferase-
based reporter assay and consequently showed weak activity in
our whole blood studies (Table 3, IC50 9.4 lM). Furthermore, the 2-
the HeLa NF-
lowed by TNF-
j
a
B-luc cells with imidazolines 1–5 for 30 min, fol-
stimulation resulted in a dose-dependent decrease
furan derivative 18 was only moderately active in both assays indi-
cating hetero-aromatic substituents provide decreased potency
compared to the phenyl moiety at the R3 position. The para-
chloro-substituted imidazoline 16 proved to be the most potent ana-
in luciferase production for three of the compounds (Table 1). The
imidazoline methyl esters 3 and 4 were the most active com-
pounds with an EC50 value of 7.5 and 5.3
lM, respectively, for inhi-
logue in this series possessing an IC50 value of 0.3 lM in the whole

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D. K. Kahlon et al. / Bioorg. Med. Chem. 17 (2009) 3093–3103
blood assay. As seen previously, the two assays corresponded rela-
tively well in terms of their relative potencies. For example, several
analogues demonstrated low micromolar activity in cell culture
(compounds 12, 16 and 17) and had nanomolar activity in the blood
assay. The only exception was the aniline-substituted imidazoline
cule NF-jB inhibitors as potential anti-inflammatory agents is
currently under investigation in our laboratories.
4. Experimental
13, which provided moderate activity in the HeLa NF-
j
B-luc assay
4.1. General Information
but potent activity (Table 3, IC50 0.5
lM) in our whole blood assay.
Substitutions on the phenyl group of the R3 position resulted in po-
tencyequalto andbetterthanthatof theleadcompound6indicating
thatoptimizationoftheR3 positioncanleadtoimidazolineswithim-
proved activity.
All commercial reagents were purchased from commercial sup-
pliers and used without further purification. MG-132 (N-Cbz-Leu-
Leu-leucinal) was obtained from Calbiochem (San Diego, CA) and
IL-1b was obtained from Roche Applied Sciences (Indianapolis,
IN). All solvents were reagent grade. THF was freshly distilled from
sodium/benzophenone under nitrogen. TEA and TMSCl were
freshly distilled from calcium hydride 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 ElectrothermalÒ capillary melting point
apparatus and are uncorrected. Column chromatography was car-
ried out on silica gel 60 (230–400 mesh) supplied by EM Science.
Yields refer to chromatographically and spectroscopically pure
compounds unless otherwise stated. Infrared spectra were re-
corded on a Nicolet IR/42 spectrometer. Proton and carbon NMR
spectra were recorded on a Varian Unity Plus-500 spectrometer.
High-resolution 3KV EI mass spectra was obtained at the RTSF
Mass Spectrometry Facility of the Michigan State University, using
a Q-ToF Ultima API (Waters Corp., Milford, MA).
We found structural changes to the fourth domain (Table 4, R4
moiety) to be the most sensitive to derivatization. The haloge-
nated-benzyl series (compounds 21–23) demonstrated that biolog-
ical activity increased with decreasing polarity on the benzyl group
with the 4-bromo-benzyl derivative being slightly more potent
than the parent compound 6 in the blood assay. Imidazoline 25 re-
vealed that substitution of the benzyl functional group with a het-
erocycle resulted in moderate biological activity however; potency
was decreased slightly when compared to the compound 6. Dele-
tion of the R4 domain, as illustrated by compound 26, rendered
the scaffold inactive in both assays. Replacement of the benzyl
group with electron-withdrawing substituents, such as acyl, ben-
zoyl or tosyl (compounds 27–29, respectively), resulted in com-
pounds which were unable to restore overall activity to the
potency of the parent compound 6.
2.3. Pharmacological significance of imidazolines
4.1.1. General procedure for imidazoline derivatives 1–5
A flame-dried flask under nitrogen was charged with the corre-
sponding imidazoline carboxylate47–49 (0.50 g, 1.07 mmol) in 9:1
benzene:methanol (50 mL) and cooled to 0 °C. After 5 min, (trimeth-
ylsilyl)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.
To verify the imidazolines are non-cytotoxic and represent
potentially viable anti-inflammatory agents, all compounds were
evaluated for cell death by MTS assay. The results indicated that
none of the compounds inhibited proliferation as tested at 10 lM
for 24 h in 184B5 cells (transformed healthy breast cells) (see Sup-
plementary data). Furthermore, the lead compound, imidazoline 6,
was analyzed by FACS analysis in HeLa-NF-jB-luc cells. It was
found that after 8 h no change in cell cycle was observed, indicat-
ing that upon treatment with imidazoline 6 the cells remained
healthy (see Supplementary data). To confirm the cell viability data
in the whole blood experiments, human white blood cell counts
4.1.1.1. dl-(4R,5R)-Methyl 1-benzyl-4-methyl-2,5-diphenyl-4,5-
dihydro-1H-imidazole-4-carboxylate (1). The reaction mixture
was concentrated to minimal residue and purified via flash column
chromatography on silica gel (9:1 ethyl acetate:hexane as eluant)
to yield a clear oil (43 mg, 83% yield). 1H NMR (500 MHz, CDCl3):
d 1.53 (s, 3H), 3.11 (s, 3H), 3.83 (d, 1H, J = 16.0 Hz), 4.28 (s, 1H),
4.72 (d, 1H, J = 16.0 Hz), 6.93–6.94 (m, 2H), 7.22–7.29 (m, 6H),
7.30–7.33 (m, 2H), 7.46–7.47 (m, 3H), 7.74–7.76 (m, 2H); 13C
NMR + DEPT (125 MHz, CDCl3): d 27.05 (–CH3), 48.96 (–CH2),
51.54 (–CH3), 73.14 (–CH), 77.97 (quaternary –C), 127.51 (aro-
matic –CH), 127.70 (aromatic –CH), 127.79 (aromatic –CH),
128.14 (aromatic –CH), 128.28 (aromatic –CH), 128.64 (aromatic
–CH), 128.80 (aromatic –CH), 130.32 (aromatic –CH), 130.68 (aro-
matic quaternary –C), 136.53 (aromatic quaternary –C), 137.11
also indicated no significant cellular toxicity at 10 lM or less imi-
dazoline 6 (see Supplementary data). Therefore, the inhibition of
luciferase production and inhibition of IL-6 production by imidaz-
oline 6 was not due to a decrease in cell number but rather due to
an inhibition of NF-jB transcription. To demonstrate that imidaz-
oline 6 was pharmacologically relevant in the treatment of inflam-
mation, human blood samples were treated with imidazoline 6 2 h
after initiation of an inflammatory response induced by IL-1b. Imi-
dazoline 6 was still capable of reducing IL-6 levels after its induc-
(aromatic quaternary –C), 165.90, 172.49; IR (neat): 3031 cmÀ1
,
tion resulting in an IC50 value of 2.5 l
M after 22 h.46
2967 cmÀ1, 2949 cmÀ1, 1734 cmÀ1, 1595 cmÀ1; HRMS (ESI): m/z
calcd for C25H25N2O2 [M+H], 385.1916; found, 385.1899.
3. Conclusion
4.1.1.2. dl-(4R,5R)-Methyl 1-benzyl-4-isopropyl-2,5-diphenyl-4,5-
dihydro-1H-imidazole-4-carboxylate (2). The reaction mixture
was concentrated to minimal residue and purified via flash column
chromatography on silica gel (1:1 ethyl acetate:hexane as eluant)
to yield a white solid (197 mg, 76% yield). Mp 128–131 °C; 1H
NMR (500 MHz, CDCl3): d 0.70 (d, 3H, J = 6.5 Hz), 0.98 (d, 3H,
J = 6.5 Hz), 2.17 (septet, 1H, J = 6.75 Hz), 2.92 (s, 3H), 3.74 (d, 1H,
J = 15.0 Hz), 4.37 (s, 1H), 4.59 (d, 1H, J = 15.0 Hz), 6.86–6.88 (m,
2H), 7.20–7.22 (m, 4H), 7.26–7.29 (m, 1H), 7.31–7.34 (m, 2H),
7.47–7.49 (m, 3H), 7.72–7.74 (m, 2H); 13C NMR + DEPT
(125 MHz, CDCl3): d 16.22 (–CH3), 18.69 (–CH3), (–CH2), 36.64
(–CH), 48.95 (–CH2), 51.07 (–CH3), 69.52 (–CH), 85.19 (quaternary
–C), 127.72 (aromatic –CH), 127.95 (aromatic –CH), 128.24
Herein we describe the structure–activity relationships of an
imidazoline scaffold capable of inhibiting NF- B mediated gene
j
transcription. Evaluation of the biological activity of this library
indicates there is a delicate balance between hydrophobicity and
hydrophilicity in the imidazoline scaffold to achieve potent activity
in cell culture and human blood. Six compounds from this library
were found to be capable of reducing IL-6 production in stimulated
human blood at nanomolar IC50 values. Furthermore, the activity of
the imidazolines in cellular NF-jB reporter assays correlated well
with the relative activity of the compounds in stimulated whole
human blood assays. The further clinical potential and determina-
tion of biological mechanism of these non-cytotoxic small mole-

D. K. Kahlon et al. / Bioorg. Med. Chem. 17 (2009) 3093–3103
3097
(aromatic –CH), 128.39 (aromatic –CH), 128.59 (aromatic –CH),
128.66 (aromatic –CH), 128.84 (aromatic –CH), 130.08 (aromatic –
CH), 135.78 (aromatic quaternary –C), 138.19 (aromatic quaternary
2855 cmÀ1, 1728 cmÀ1, 1593 cmÀ1; HRMS (ESI): m/z calcd for
C33H30N3O2 [M+H], 500.2338; found, 500.2345.
–C), 165.37, 172.41; IR (neat): 3031 cmÀ1, 2967 cmÀ1, 1732 cmÀ1
,
4.1.2. General procedure for imidazoline derivatives 6–8
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
acid48 (20.0 g, 46.3 mmol,) and dichloromethane (300 mL). After
cooling the reaction mixture to 0 °C, oxalyl chloride (11.7 mL,
1597 cmÀ1
413.2229; found, 413.2229.
; HRMS (ESI): m/z calcd for C27H29N2O2 [M+H],
4.1.1.3. dl-(4R,5R)-Methyl 1-benzyl-4,5-dihydro-2,4,5-triphenyl-
1H-imidazole-4-carboxylate (3). The reaction mixture was con-
centrated to minimal residue and purified by running through a
small silica plug to give the product as a white solid (240 mg,
50% yield). Mp 114–116 °C; 1H NMR (500 MHz, CDCl3) d 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) d 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,
138.8 mmol) was added over 5 min followed by DMF (10 lL/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 etha-
nol (6), n-propanol (7) or isopropanol (8) (equal volume to CH2Cl2)
was added. After stirring for an addition 2.5 h the alcohol was re-
moved on the rotary evaporator and dichloromethane (300 mL)
was added. The organic solution was washed with saturated
NaHCO3 solution (1 Â 100 mL) and H2O (1 Â 100 mL), dried over
MgSO4, filtered and the solvent was removed under reduced
pressure.
130.5, 136.6, 137.9, 144.0, 165.6, 171.4; IR (neat): 3061 cmÀ1
,
3031 cmÀ1, 2948 cmÀ1, 1734 cmÀ1, 1595 cmÀ1; HRMS (ESI): m/z
calculated for [C30H26N2O2+H]+ 447.2070; found: 447.2070.
4.1.2.1. dl-(4R,5R)-Ethyl 1-benzyl-4,5-dihydro-2,4,5-triphenyl-
1H-imidazole-4-carboxylate (6). The product was purified using
flash column chromatography on silica gel (1:1 hexane:ethyl ace-
tate as eluant) to give a white solid (16.1 g, 76% yield). Mp 86–
88 °C; 1H NMR (500 MHz, CDCl3) d 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) d 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,
4.1.1.4. dl-(4R,5R)-Methyl 1,4-dibenzyl-2,5-diphenyl-4,5-dihydro-
1H-imidazole-4-carboxylate (4). The reaction mixture was
concentrated to minimal residue and purified via flash column
chromatography on silica gel (1:1 ethyl acetate:hexane as eluant)
to yield a white solid (74 mg, 78% yield). Mp 97–99 °C; 1H NMR
(500 MHz, CDCl3): d 3.16 (d, 1H, J = 14.0 Hz), 3.17 (s, 3H), 3.40 (d,
1H, J = 16.0 Hz), 3.71 (d, 1H, J = 16.0 Hz), 4.27 (d, 1H, J = 16.0 Hz),
4.54 (s, 1H), 4.47 (d, 2H, J = 7.5 Hz), 7.04–7.07 (m, 2H), 7.10–7.13
(m, 1H), 7.20 (d, 2H, J = 7.0 Hz), 7.26–7.28 (m, 4H), 7.30–7.36 (m,
4H), 7.43 (d, 1H, J = 1.5 Hz), 7.44 (d, 1H, J = 2.0 Hz), 7.64–7.66 (m,
2H); 13C NMR + DEPT (125 MHz, CDCl3): d 44.49 (–CH2), 48.88
(–CH2), 51.50 (–CH3), 70.11 (–CH), 81.92 (quaternary –C), 126.66
(aromatic –CH), 127.11 (aromatic –CH), 127.44 (aromatic –CH),
127.73 (aromatic –CH), 128.03 (aromatic –CH), 128.20 (aromatic
–CH), 128.37 (aromatic –CH), 128.49 (aromatic –CH), 128.50 (aro-
matic –CH), 130.10 (aromatic –CH), 130.83 (aromatic quaternary –
C), 131.41 (aromatic –CH), 135.86 (aromatic quaternary –C), 136.12
(aromatic quaternary –C), 137.17 (aromatic quaternary –C),
144.1, 165.3, 170.7; IR (neat): 3173 cmÀ1, 3061 cmÀ1, 3031 cmÀ1
,
2981 cmÀ1, 1732 cmÀ1, 1597 cmÀ1; HRMS (ESI): m/z calculated
for [C31H28N2O2+H]+ 461.2229; found: 461.2225.
4.1.2.2. dl-(4R,5R)-Propyl 1-benzyl-2,4,5-triphenyl-4,5-dihydro-
1H-imidazole-4-carboxylate (7). The product was purified using
flash column chromatography on silica gel (1:1 hexane:ethyl ace-
tate as eluant) to give a white solid (170 mg, 67% yield). Mp 88–
90 °C; 1H NMR (500 MHz, CDCl3): d 0.63 (t, 3H, J = 7.25 Hz), 1.20
(sextet, 2H, J = 7.0 Hz), 3.40 (dt, 1H, J = 10.5, 6.5 Hz), 3.89 (dt, 1H,
J = 10.5, 6.5 Hz), 3.81 (d, 1H, J = 16.0 Hz), 4.60 (d, 1H, J = 16.0 Hz),
4.91 (s, 1H), 6.74 (d, 2H, J = 7.0 Hz), 7.05–7.08 (m, 2H), 7.10–7.13
(m, 1H), 7.24–7.27 (m, 1H), 7.30–7.33 (m, 3H), 7.34–7.39 (m,
4H), 7.46–7.47 (m, 3H), 7.73–7.76 (m, 4H); 13C NMR + DEPT
(125 MHz, CDCl3): d 10.23 (–CH3), 21.30 (–CH2), 48.63 (–CH2),
66.54 (–CH2), 73.79 (–CH), 82.92 (quaternary –C), 126.81 (aro-
matic –CH), 127.17 (aromatic –CH), 127.35 (aromatic –CH),
127.37 (aromatic –CH), 127.82 (aromatic quaternary –C), 127.98
(aromatic –CH), 128.14 (aromatic –CH), 128.31 (aromatic –CH),
128.41 (aromatic –CH), 128.49 (aromatic –CH), 128.58 (aromatic
–CH), 128.82 (aromatic –CH), 130.33 (aromatic –CH), 136.58 (aro-
matic quaternary –C), 137.85 (aromatic quaternary –C), 144.02
166.20, 172.08; IR (neat): 3063 cmÀ1, 3029 cmÀ1, 2948 cmÀ1
,
2926 cmÀ1, 1732 cmÀ1, 1595 cmÀ1; HRMS (ESI): m/z calcd for
C31H29N2O2 [M+H], 461.2229; found, 461.2245.
4.1.1.5. dl-(4R,5R)-Methyl 4-((1H-indol-2-yl)methyl)-1-benzyl-
2,5-diphenyl-4,5-dihydro-1H-imidazole-4-carboxylate (5). The
reaction mixture was concentrated to minimal residue and purified
via flash column chromatography on silica gel (9:1 ethyl ace-
tate:hexane as eluant) to yield a yellow solid (11 mg, 14% yield).
Mp 175–176 °C; 1H NMR (500 MHz, CDCl3):
d 2.79 (d, 1H,
J = 14.5 Hz), 2.87 (d, 1H, J = 15.0 Hz), 3.45 (s, 3H), 3.89 (d, 1H,
J = 16.0 Hz), 4.70 (d, 1H, J = 16.0 Hz), 4.98 (s, 1H), 6.83–6.84 (m
1H), 6.87–6.90 (m, 1H), 6.94–6.98 (m, 3H), 7.07 (d, 1H,
J = 8.0 Hz), 7.20–7.26 (m, 5H), 7.28–7.35 (m, 4H), 7.44–7.46 (m,
3H), 7.40–7.76 (m, 2H), 8.16 (br s, 1H); 13C NMR + DEPT
(125 MHz, CDCl3): d 31.91 (–CH2), 48.76 (–CH2), 52.24 (–CH3),
69.69 (–CH), 79.41 (quaternary –C), 110.51 (aromatic quaternary
–C), 110.60 (aromatic –CH), 118.58 (aromatic –CH), 118.84 (aro-
matic –CH), 121.08 (aromatic –CH), 123.65 (aromatic –CH),
127.46 (aromatic –CH), 128.01 (aromatic quaternary –C), 128.09
(aromatic –CH), 128.44 (aromatic –CH), 128.56 (aromatic –CH),
128.59 (aromatic –CH), 128.72 (aromatic –CH), 130.16 (aromatic
–CH), 130.97 (aromatic quaternary –C), 135.56 (aromatic quater-
nary –C), 136.05 (aromatic quaternary –C), 136.70 (aromatic qua-
(aromatic quaternary –C), 165.47, 170.68; IR (neat): 3064 cmÀ1
,
3030 cmÀ1, 2967 cmÀ1, 1732 cmÀ1, 1595 cmÀ1; HRMS (ESI): m/z
calcd for C32H31N2O2 [M+H], 475.2386; found, 475.2383.
4.1.2.3. dl-(4R,5R)-Isopropyl 1-benzyl-2,4,5-triphenyl-4,5-dihy-
dro-1H-imidazole-4-carboxylate (8). The product was purified
using flash column chromatography on silica gel (1:1 hexane:ethyl
acetate as eluant) to give a white solid (118 mg, 47% yield). Mp
109–111 °C; 1H NMR (500 MHz, CDCl3): d 0.63 (d, 3H, J = 6.5 Hz),
0.85 (d, 3H, J = 6.5 Hz), 3.77 (d, 1H, J = 15.5 Hz), 4.48 (septet, 1H,
J = 6.25 Hz), 4.58 (d, 1H, J = 15.5 Hz), 4.90 (s, 1H), 6.73 (d, 2H,
ternary –C), 166.01, 175.58; IR (neat): 3059 cmÀ1, 2924 cmÀ1
,

3098
D. K. Kahlon et al. / Bioorg. Med. Chem. 17 (2009) 3093–3103
J = 7.5 Hz), 7.06–7.09 (m, 2H), 7.11–7.14 (m, 1H), 7.24–7.28 (m,
1H), 7.30–7.34 (m, 3H), 7.35–7.37 (m, 4H), 7.46–7.47 (m, 3H),
7.72–7.74 (m, 4H); 13C NMR + DEPT (125 MHz, CDCl3): d 20.75
(–CH3), 21.21 (–CH3), 48.52 (–CH2), 68.58 (–CH), 73.58 (–CH),
82.72 (quaternary –C), 126.76 (aromatic –CH), 127.17 (aromatic
–CH), 127.28 (aromatic –CH), (aromatic –CH), 127.29 (aromatic –
CH), 127.92 (aromatic –CH), 128.26 (aromatic –CH), 128.34
(aromatic –CH), 128.39 (aromatic –CH), 128.52 (aromatic –CH),
128.77 (aromatic –CH), 130.17 (aromatic –CH), 130.77 (aromatic
quaternary –C), 136.80 (aromatic quaternary –C), 138.17 (aromatic
quaternary –C), 144.26 (aromatic quaternary –C), 165.20, 170.09;
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 cmÀ1
,
3063 cmÀ1, 3031 cmÀ1, 2926 cmÀ1, 1688 cmÀ1, 1591 cmÀ1; HRMS
(FAB): m/z calculated for [C29H25N3O+H]+ 432.2076; found:
432.2073.
4.1.3. General procedure for compounds 11, 12, 14–18
A solution of benzyl amine (0.2 g, 2.11 mmol) and appropriate
aldehyde (0.2 g, 2.11 mmol) in 50 mL of anhydrous benzene was
refluxed under nitrogen for 3 h and then concentrated in vacuo.
The resulting residue was redissolved into 50 mL of anhydrous
dichloromethane. Then 2,4-diphenyl-5(4H)-oxazolone (0.5 g, 2.11
mmol) and chlorotrimethylsilane (0.3 g, 2.74 mmol) were added
and the mixture was refluxed under nitrogen for 22 h. The solution
was concentrated in vacuo and the resulting residue was resus-
pended in ethyl acetate producing a white solid precipitate
(0.5 g) which was isolated via filtration. The crude solid was then
dissolved into 15 mL of ethanol, treated with 1 mL of concentrated
H2SO4, and refluxed for 21 h. The solution was concentrated in va-
cuo and then dissolved into dichloromethane before being washed
with saturated NaHCO3 solution (3 Â 20 mL) and brine solution
(1 Â 20 mL). The organic fraction was dried over sodium sulfate
and then concentrated in vacuo.
IR (neat): 3063 cmÀ1
,
3030 cmÀ1
,
2980 cmÀ1
,
2936 cmÀ1
,
1728 cmÀ1, 1595 cmÀ1; HRMS (ESI): m/z calcd for C32H31N2O2
[M+H], 475.2386; found, 475.2388.
4.1.2.4. 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,5R)-(1-benzyl-4,5-dihydro-2,4,5-triphenyl-1H-imidazole-4-car-
boxylic acid48 (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 Â 5 mL), saturated NaHCO3 solu-
tion (1 Â 5 mL), H2O (1 Â 5 mL) and brine solution (1 Â 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 (120 mg, 54% yield). 1H NMR (500 MHz, CDCl3) d 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) d 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
4.1.3.1. dl-(4R,5R)-Ethyl 1-benzyl-2,4-diphenyl-5-(pyridin-4-yl)-
4,5-dihydro-1H-imidazole-4-carboxylate (11). The resulting crude
residue was purified via column chromatography using silica gel
(100% ethyl acetate) to afford the product as a clear oil (220 mg,
1
23% yield). H NMR (500 MHz, CDCl3): d 0.83 (t, 3H, J = 7.5 Hz), 3.66
(dq, 1H, J = 11, 7 Hz), 3.75 (dq, 1H, J = 11, 7.5 Hz), 3.83 (d, 1H,
J = 15.5 Hz), 4.62 (d, 1H, J = 15.5 Hz), 4.86 (s, 1H), 6.74 (d, 2H,
J = 7.5 Hz), 7.05–7.08 (m, 2H), 7.12–7.14 (m, 1H), 7.28–7.37 (m, 5H),
7.49–7.52 (m, 3H), 7.71–7.72 (m, 2H), 7.79–7.81 (m, 2H), 8.63 (d,
2H, J = 6.0 Hz); 13C NMR + DEPT (125 MHz, CDCl3): d 13.39 (–CH3),
49.24 (–CH2), 61.20 (–CH2), 72.62 (–CH), 83.06 (quaternary –C),
122.95 (aromatic –CH), 126.49 (aromatic –CH), 127.14 (aromatic
–CH), 127.56 (aromatic –CH), 127.61 (aromatic –CH), 128.11 (aro-
matic –CH), 128.43 (aromatic –CH), 128.65 (aromatic –CH), 128.77
(aromatic –CH), 129.93 (aromatic quaternary –C), 130.60 (aromatic
–CH), 135.86 (aromatic quaternary –C), 143.34 (aromatic quaternary
–C), 147.28 (aromatic quaternary –C), 149.93 (aromatic –CH), 165.71,
(neat): 3063 cmÀ1, 3032 cmÀ1, 2924 cmÀ1, 1740 cmÀ1, 1593 cmÀ1
;
HRMS (ESI): m/z calculated for [C36H30N2O2+H]+ 523.2386; found:
523.2390.
170.23; IR (neat): 3063 cmÀ1, 2982 cmÀ1, 1734 cmÀ1, 1597 cmÀ1
;
HRMS (ESI): m/z calcd for C30H27N3O2 [M+H], 462.2182; found,
4.1.2.5. dl-(4R,5R)-1-Benzyl-4,5-dihydro-2,4,5-triphenyl-1H-imid-
azole-4-carboxamide (10). To a stirred solution of dl-(4R,5R)-(1-
benzyl-4,5-dihydro-2,4,5-triphenyl-1H-imidazole-4-carboxylic acid48
(100 mg, 0.21 mmol,), N-hydroxybenzotriazole (32 mg, 0.23
mmol), 1-ethyl-(3-dimethylaminopropyl) carbodiimide (45 mg,
0.23 mmol) in anhydrous THF (2 mL) was added N,N-diisopropyl-
ethylamine (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 re-
duced pressure. The product was purified using column chroma-
tography on silica gel (3:7 hexane:ethyl acetate as eluant) to give
a light yellow solid (65 mg, 71% yield). Mp 65–68 °C; 1H NMR
462.2177.
4.1.3.2. dl-(4R,5R)-Ethyl 1-benzyl-5-(4-nitrophenyl)-2,4-diphe-
nyl-4,5-dihydro-1H-imidazole-4-carboxylate (12). The resulting
crude solid was purified via column chromatography (1:1 ethyl
acetate:hexane as eluant) affording the title compounds as a white
solid (540 mg, 51% yield). Mp 173–174 °C; 1H NMR (500 MHz,
CDCl3): d 0.83 (t, 3H, J = 7.5 Hz), 3.64 (dq, 1H, J = 11, 7 Hz), 3.74
(dq, 1H, J = 11, 7 Hz), 3.81 (d, 1H, J = 16 Hz), 4.60 (d, 1H,
J = 16 Hz), 4.93 (s, 1H), 6.71 (d, 2H, J = 7.5 Hz), 7.03–7.10 (m, 2H),
7.10–7.13 (m, 1H), 7.27–7.35 (m, 3H), 7.48–7.51 (m, 3H), 7.54 (d,
2H, J = 8.5 Hz), 7.67–7.69 (m, 2H), 7.77–7.80 (m, 2H), 8.21 (d, 2H,
J = 9.5 Hz); 13C NMR + DEPT (125 MHz, CDCl3): d 15.59 (–CH3),
49.46 (–CH2), 61.30 (–CH2), 73.16 (–CH), 83.32 (quaternary –C),
123.61 (aromatic –CH), 126.49 (aromatic –CH), 127.25 (aromatic
–CH), 127.69 (aromatic –CH), 127.75 (aromatic –CH), 128.24 (aro-
matic –CH), 128.53 (aromatic –CH), 128.76 (aromatic –CH), 128.87
(aromatic –CH), 128.96 (aromatic –CH), 129.98 (aromatic –CH),
130.74 (aromatic –CH), 135.83 (aromatic quaternary –C), 143.45
(aromatic quaternary –C), 146.09 (aromatic quaternary –C),
147.76 (aromatic quaternary –C), 165.78, 170.42; IR (neat):
3063 cmÀ1, 2982 cmÀ1, 1732 cmÀ1, 1597 cmÀ1, 1350 cmÀ1; HRMS
(ESI): m/z calcd for C31H27N3O4 [M+H], 506.2080; found, 506.2076.
(500 MHz, CDCl3)
d 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) d 48.4, 74.3, 81.6,

D. K. Kahlon et al. / Bioorg. Med. Chem. 17 (2009) 3093–3103
3099
4.1.3.3. dl-(4R,5R)-Ethyl 1-benzyl-5-(4-methoxyphenyl)-2,4-
diphenyl-4,5-dihydro-1H-imidazole-4-carboxylate (14). The
resulting crude solid was purified via column chromatography
using silica gel (1:1 ethyl acetate:hexane as eluant) to afford the
product as an off-white solid (336 mg, 36% yield). Mp 143–
144 °C; 1H NMR (500 MHz, CDCl3): d 0.85 (t, 3H, J = 7 Hz), 3.64
(dq, 1H, J = 10.5, 7.5 Hz), 3.74 (dq, 1H, J = 10.5, 7 Hz), 3.80 (d, 1H,
J = 16.0 Hz), 3.81 (s, 3H), 4.57 (d, 1H, J = 15.5 Hz), 4.86 (s, 1H),
6.74 (d, 2H, J = 7.5 Hz), 6.89 (d, 2H, J = 8.5 Hz), 7.04–709 (m, 2H),
7.09–7.20 (m, 1H), 7.25–7.33 (m, 5H), 7.44–7.47 (m, 3H), 7.72–
7.74 (m, 4H); 13C NMR + DEPT (125 MHz, CDCl3): d 13.58 (–CH3),
48.45 (–CH2), 55.25 (–CH3), 60.94 (–CH2), 73.33 (–CH), 82.66
(quaternary –C), 113.82 (aromatic –CH), 126.79 (aromatic –CH),
127.12 (aromatic –CH), 127.27 (aromatic –CH), 127.30 (aromatic
–CH), 127.94 (aromatic –CH), 128.37 (aromatic –CH), 128.53 (aro-
matic –CH), 128.77 (aromatic –CH), 129.34 (aromatic –CH),
129.76 (aromatic –CH), 130.20 (aromatic –CH), 130.76 (aromatic
quaternary –C), 136.76 (aromatic quaternary –C), 144.17 (aro-
matic quaternary –C), 159.59 (aromatic quaternary –C), 165.30,
4.1.3.6. dl-(4R,5R)-Ethyl 1-benzyl-2,4-diphenyl-5-(4-(trifluoro-
methyl)phenyl)-4,5-dihydro-1H-imidazole-4-carboxylate (17).
The resulting crude solid was purified via column chromatogra-
phy using silica gel (4:6 ethyl acetate:hexane as eluant) to afford
the product as a white crystalline solid (159 mg, 14% yield). Mp
155–156 °C; 1H NMR (500 MHz, CDCl3) (TMS): d 0.78 (t, 3H,
J = 7.0 Hz), 3.63 (dq, 1H, J = 11, 7 Hz), 3.73 (dq, 1H, J = 10.5,
7 Hz), 3.82 (d, 1H, J = 16.0 Hz), 4.62 (d, 1H, J = 15.5 Hz), 4.94 (s,
1H), 6.73 (d, 2H, J = 7.5 Hz), 7.04–7.10 (m, 2H), 7.11–7.13 (m,
1H), 7.27–7.32 (m, 1H), 7.32–7.36 (m, 2H), 7.48–7.50 (m, 3H),
7.52 (d, 2H, J = 7.5 Hz), 7.64 (d, 2H, J = 8.0 Hz), 7.71–7.73 (m,
2H), 7.78–7.80 (m, 2H); 13C NMR + DEPT (125 MHz, CDCl3)
(TMS):
d 13.36 (–CH3), 49.05 (–CH2), 61.13 (–CH2), 73.21
(–CH), 83.07 (quaternary –C), 125.35 (q, J = 3.6 Hz, aromatic
–CH), 123.97 (q, J = 270 Hz, quaternary –C), 126.62 (aromatic –
CH), 127.16 (aromatic –CH), 127.51 (aromatic –CH), 127.56
(aromatic –CH), 128.10 (aromatic –CH), 128.44 (aromatic –CH),
128.65 (aromatic –CH), 128.83 (aromatic –CH), 130.46 (q,
J = 32 Hz, quaternary –C), 130.53 (aromatic –CH), 136.13 (aro-
matic quaternary –C), 142.44 (aromatic quaternary –C), 143.68
170.90; IR (neat): 3032 cmÀ1, 2934 cmÀ1, 1732 cmÀ1, 1595 cmÀ1
;
HRMS (ESI): m/z calcd for C32H30N2O3 [M+H], 491.2335; found,
491.2332.
(aromatic quaternary –C), 165.59, 170.49; IR (neat): 3065 cmÀ1
,
2982 cmÀ1, 1734 cmÀ1, 1597 cmÀ1; HRMS (ESI): m/z calcd for
C32H27N2O2F3 [M+H], 529.2103; found, 529.2110.
4.1.3.4. dl-(4R,5R)-Ethyl 1-benzyl-5-(4-fluorophenyl)-2,4-diphe-
nyl-4,5-dihydro-1H-imidazole-4-carboxylate (15). The resulting
crude solid was purified via column chromatography using silica
gel (1:1 ethyl acetate:hexane) to afford the product as a white solid
(124 mg, 12% yield). Mp 96–97 °C; 1H NMR (500 MHz, CDCl3): d
0.84 (t, 3H, J = 7.5 Hz), 3.67 (dq, 1H, J = 10.5, 7.5 Hz), 3.76 (dq, 1H,
J = 10.5, 7 Hz), 3.84 (d, 1H, J = 16.0 Hz), 4.63 (d, 1H, J = 15.5 Hz),
4.92 (s, 1H), 6.75–6.78 (m, 2H), 7.06–7.12 (m, 4H), 7.12–7.16 (m,
1H), 7.28–7.42 (m, 5H), 7.48–7.54 (m, 3H), 7.72–7.80 (m, 4H);
13C NMR + DEPT (125 MHz, CDCl3): d 13.53 (–CH3), 48.73 (–CH2),
61.04 (–CH2), 73.01 (–CH), 82.78 (quaternary –C), 115.35 (d,
J = 21.6 Hz, aromatic –CH), 126.68 (aromatic –CH), 127.11 (aro-
matic –CH), 127.39 (aromatic –CH), 127.43 (aromatic –CH),
128.01 (aromatic –CH), 128.40 (aromatic –CH), 128.59 (aromatic
–CH), 128.77 (aromatic –CH), 129.73 (d, J = 8.0 Hz, aromatic –CH),
130.37 (aromatic –CH), 130.43 (aromatic –CH), 133.77 (d,
J = 3.0 Hz, aromatic quaternary –C), 136.40 (aromatic quaternary
–C), 143.90 (aromatic quaternary –C), 162.61 (d, J = 245 Hz, aro-
4.1.3.7. dl-(4R,5R)-Ethyl 1-benzyl-5-(furan-2-yl)-2,4-diphenyl-
4,5-dihydro-1H-imidazole-4-carboxylate (18). The resulting
crude residue was purified via column chromatography using sil-
ica gel (4:6 ethyl acetate:hexane) to afford the product as a yellow
oil (238 mg, 25% yield). 1H NMR (500 MHz) (CDCl3): d 1.01 (t, 3H,
J = 7.0 Hz), 3.86 (dq, 1H, J = 10.5, 7 Hz), 3.94 (dq, 1H, J = 10.5,
7 Hz), 3.88 (d, 1H, J = 15.5 Hz), 4.54 (d, 1H, J = 15.5 Hz), 5.01 (s,
1H), 6.36–6.39 (m, 2H), 6.81–6.83 (m, 2H), 7.08–7.13 (m, 2H),
7.25–7.28 (m, 1H), 7.31–7.34 (m, 2H), 7.43–7.46 (m, 4H), 7.71–
7.75 (m, 4H); 13C NMR + DEPT (125 MHz) (CDCl3): d 13.72 (–
CH3), 49.01 (–CH2), 61.27 (–CH2), 67.97 (–CH), 81.21 (quaternary
–C), 109.30, (aromatic –CH), 110.50 (aromatic –CH), 126.55 (aro-
matic –CH), 127.08 (aromatic –CH), 127.30 (aromatic –CH),
127.43 (aromatic –CH), 128.03 (aromatic –CH), 128.39 (aromatic
–CH), 128.46 (aromatic –CH), 128.68 (aromatic –CH), 130.17 (aro-
matic –CH), 130.65 (aromatic quaternary –C), 136.51 (aromatic
quaternary –C), 142.51 (aromatic –CH), 143.34 (aromatic quater-
nary –C), 151.41 (aromatic quaternary –C), 165.47, 170.75; IR
(neat): 3063 cmÀ1, 2980 cmÀ1, 1734 cmÀ1, 1597 cmÀ1; HRMS
(ESI): m/z calcd for C29H26N2O3 [M+H], 451.2022; found, 451.2005.
matic quaternary –C), 165.39, 170.70; IR (neat): 3063 cmÀ1
,
2982 cmÀ1, 1732 cmÀ1, 1597 cmÀ1; HRMS (ESI): m/z calcd for
C31H27N2O2F [M+H], 479.2135; found, 479.2130.
4.1.3.5. dl-(4R,5R)-Ethyl 1-benzyl-5-(4-chlorophenyl)-2,4-diphe-
nyl-4,5-dihydro-1H-imidazole-4-carboxylate (16). The resulting
crude solid was recrystallized using ethyl acetate/hexane to afford
the product as a white crystalline solid (6.23 g, 50% yield). Mp 165–
166 °C; 1H NMR (500 MHz, CDCl3) (TMS): d 0.86 (t, 3H, J = 7.0 Hz),
3.66 (dq, 1H, J = 11, 7.5 Hz), 3.75 (dq, 1H, J = 11, 7.5 Hz), 3.80 (d, 1H,
J = 15.5 Hz), 4.61 (d, 1H, J = 15.5 Hz), 4.87 (s, 1H), 6.74 (d, 2H,
J = 7.0 Hz), 7.05–7.08 (m, 2H), 7.11–7.12 (m, 1H), 7.26–7.29 (m,
1H), 7.31–7.36 (m, 6H), 7.47–7.49 (m, 3H), 7.70–7.72 (m, 2H),
7.75–7.77 (m, 2H); 13C NMR + DEPT (125 MHz, CDCl3) (TMS): d
13.55 (–CH3), 48.83 (–CH2), 61.12 (–CH2), 73.10 (–CH), 82.87 (qua-
ternary –C), 126.68 (aromatic –CH), 127.17 (aromatic –CH), 127.46
(aromatic –CH), 127.49 (aromatic –CH), 128.06 (aromatic –CH),
128.45 (aromatic –CH), 128.63 (aromatic –CH), 128.65 (aromatic
–CH), 128.81 (aromatic –CH), 129.49 (aromatic –CH), 130.41 (aro-
matic quaternary –C), 130.43 (aromatic –CH), 134.08 (aromatic
quaternary –C), 136.35 (aromatic quaternary –C), 136.63 (aromatic
quaternary –C), 143.84 (aromatic quaternary –C), 165.48, 170.65;
IR (KBr): 3063 cmÀ1, 2980 cmÀ1, 1732 cmÀ1, 1595 cmÀ1; HRMS
(ESI): m/z calcd for C31H27N2O2Cl [M+H], 495.1833; found,
495.1834.
4.1.3.8. dl-(4R,5R)-Ethyl 5-(4-aminophenyl)-1-benzyl-2,4-diphe-
nyl-4,5-dihydro-1H-imidazole-4-carboxylate (13). A solution of
dl-(4R,5R)-ethyl-1-benzyl-5-(4-nitrophenyl)-2,4-diphenyl-4,5-dihy-
dro-1H-imidazole-4-carboxylate (12) (0.1 g, 0.2 mmol) H2O (36 mg,
.
2.0 mmol) in 10 mL of ethanol was treated with SnCl2 2H2O (0.3 g,
1.2 mmol). The solution was heated to reflux for 2 h and cooled to
room temperature before being poured over ice (ꢀ50 g). The pH of
the resulting aqueous solution was adjusted (pH 8) using NaHCO3
powder. The solution was then washed with EtOAc (3 Â 50 mL).
The combined EtOAc washes weredried over sodium sulfate and con-
centrated in vacuo. The resulting residue was purified via column
chromatography using silica gel (100% ethyl acetate as eluant) to af-
ford the product as a white solid (57 mg, 60% yield). Mp 60–62 °C; 1H
NMR (500 MHz, CDCl3): d 0.91 (t, 3H, J = 7.5 Hz), 3.68–3.82 (m, 4H)
3.86 (d, 1H, J = 16 Hz), 4.60 (d, 1H, J = 15.5 Hz), 4.86 (s, 1H), 6.68 (d,
2H, J = 8.5 Hz), 6.78 (d, 2H, J = 7.5 Hz), 7.08–7.16 (m, 3H), 7.18 (d,
2H, J = 8 Hz), 7.27–7.30 (m, 1H), 7.35–7.36 (m, 2H), 7.47–7.50 (m,
3H), 7.75–7.78 (m, 4H); 13C NMR + DEPT (125 MHz, CDCl3): d13.57
(–CH3), 48.24 (–CH2), 60.86 (–CH2), 73.48 (–CH), 82.51 (quaternary
–C), 114.84 (aromatic –CH), 126.78 (aromatic –CH), 127.07 (aromatic
–CH), 127.16 (aromatic –CH), 127.19 (aromatic –CH), 127.86

3100
D. K. Kahlon et al. / Bioorg. Med. Chem. 17 (2009) 3093–3103
(aromatic –CH), 128.29 (aromatic –CH), 128.46 (aromatic –CH),
128.70 (aromatic –CH), 129.18 (aromatic –CH), 130.09 (aromatic –
CH), 130.84 (quaternary aromatic –C), 136.88 (quaternary aromatic
–C), 144.24 (quaternary aromatic –C), 146.51 (quaternary aromatic
–CH), 128.18 (aromatic –CH), 128.35 (aromatic –CH), 128.46 (aro-
matic –CH), 128.74 (aromatic –CH), 128.98 (aromatic –CH), 130.18
(aromatic –CH), 130.57 (aromatic quaternary –C), 133.39 (aromatic
quaternary –C), 136.87 (aromatic quaternary –C), 137.88 (aromatic
quaternary –C), 144.03 (aromatic quaternary –C), 165.39, 170.71; IR
–C), 165.19, 170.96; IR (KBr): 3460 cmÀ1, 3373 cmÀ1, 3063 cmÀ1
,
1732 cmÀ1, 1614 cmÀ1; HRMS (FAB): m/z calcd for C31H29N3O2
(neat): 3058 cmÀ1, 3031 cmÀ1, 2978 cmÀ1, 2925 cmÀ1, 1732 cmÀ1
,
[M+H], 476.2338; found, 476.2332.
1597 cmÀ1; HRMS (ESI): m/z calcd for C32H31N2O2 [M+H], 475.2386;
found, 475.2414.
4.1.4. General procedure for imidazoline derivatives 19–25
A solution of appropriate amine (0.2 g, 2.11 mmol) and appro-
priate benzylaldehyde (0.2 g, 2.11 mmol) in 50 mL of anhydrous
benzene was refluxed under nitrogen for 12 h and then concen-
trated in vacuo. The resulting residue was redissolved into 50 mL
of anhydrous dichloromethane. Then 2,4-diphenyl-5(4H)-oxazo-
lone (0.5 g, 2.11 mmol) and chlorotrimethylsilane (0.3 g, 2.74
mmol) were added and the mixture was refluxed under nitrogen
for an additional 12 h. The solution was concentrated in vacuo
and the resulting residue was resuspended in ethyl acetate produc-
ing a white solid precipitate (0.4 g) which was isolated via filtra-
tion. The white solid was then dissolved into 50 mL of
dichloromethane, cooled to 0 °C, and treated with oxalyl chloride
4.1.4.3. dl-(4R,5R)-Ethyl 1-(4-fluorobenzyl)-2,4,5-triphenyl-4,5-
dihydro-1H-imidazole-4-carboxylate (21). The resulting crude
residue was purified via column chromatography using silica gel
(1:1 ethyl acetate:hexane) to afford the product as a white solid
(144 mg, 74% yield). Mp 67–69 °C; 1H NMR (500 MHz, CDCl3): d
0.78 (t, 3H, J = 7.0 Hz), 3.68 (dq, 1H, J = 10.6, 7.0 Hz), 3.80 (d, 1H,
J = 15.5 Hz), 4.54 (d, 1H, J = 16.0 Hz), 4.85 (s, 1H), 6.65–6.69 (m,
2H), 6.70–6.74 (m, 2H), 7.24–7.29 (m, 1H), 7.31–7.35 (m, 4H),
7.36–7.37 (m, 3H), 7.46–7.49 (m,3H), 7.73–7.76 (m, 4H); 13C
NMR + DEPT (125 MHz, CDCl3): d 13.44 (–CH3), 48.11 (–CH2),
61.01 (–CH2), 73.89 (–CH), 82.76 (quaternary –C), 115.24 (d,
J = 21.5 Hz, aromatic –CH,), 126.74 (aromatic –CH), 127.46 (aro-
matic –CH), 128.02 (aromatic –CH), 128.08 (aromatic –CH),
128.37 (aromatic –CH), 128.50 (aromatic –CH), 128.63 (aromatic
–CH), 128.83 (aromatic –CH), 128.84 (d, J = 8.2 Hz, aromatic –CH),
130.45 (aromatic –CH), 132.24 (d, J = 3.7 Hz, aromatic quaternary
–C), 137.75 (aromatic quaternary –C), 144.02 (aromatic quaternary
–C), 161.98 (d, J = 244 Hz, aromatic quaternary –C), 165.35, 170.50;
(0.3 g, 2.68 mmol) and DMF (30 lL). The solution was stirred for
2 h, concentrated in vacuo, and the residue was cooled to 0 °C.
The residue was redissolved into 20 mL of ethanol before being left
to stir overnight. The solution was concentrated in vacuo and then
dissolved into dichloromethane before being washed with
saturated NaHCO3 solution (3 Â 50 mL) and brine solution
(1 Â 50 mL). The organic fraction was dried over sodium sulfate
and then concentrated in vacuo.
IR (neat): 3063 cmÀ1
,
3033 cmÀ1
,
2982 cmÀ1
,
2928 cmÀ1
,
1732 cmÀ1, 1597 cmÀ1; HRMS (ESI): m/z calcd for C31H28N2O2F
[M+H], 479.2135; found, 479.2126.
4.1.4.1. dl-(4R,5R)-Ethyl 1-(4-methoxybenzyl)-2,4,5-triphenyl-4,5-
dihydro-1H-imidazole-4-carboxylate (19). The resulting crude
residue was purified via column chromatography using silica gel
(1:1 ethyl acetate:hexane) to afford the product as clear oil
(88 mg, 51% yield). 1H NMR (500 MHz, CDCl3): d 0.77 (t, 3H,
J = 7.25 Hz), 3.56 (dq, 1H, J = 10.9, 7.0 Hz), 3.66 (dq, 1H, J = 10.9,
7.0 Hz), 3.67 (s, 3H), 3.74 (d, 1H, J = 15.5 Hz), 4.53 (d, 1H,
J = 15.5 Hz), 4.88 (s, 1H), 6.62 (d, 2H, J = 8.5 Hz), 6.75 (d, 2H,
J = 8.5 Hz), 7.23–7.25 (m, 1H), 7.29–7.32 (m, 3H), 7.34–7.39 (m,
4H), 7.45–7.46 (m, 3H), 7.71–7.72 (m, 2H), 7.74–7.76 (m, 2H);
13C NMR + DEPT (125 MHz, CDCl3): d 13.38 (–CH3), 48.10 (–CH2),
55.05 (–CH3), 60.87 (–CH2), 73.46 (–CH), 82.71 (quaternary –C),
113.69 (aromatic –CH), 126.73 (aromatic –CH), 127.22 (aromatic
–CH), 127.88 (aromatic –CH), 128.10 (aromatic –CH), 128.19
(aromatic –CH), 128.36 (aromatic –CH), 128.37 (aromatic quater-
nary –C), 128.43 (aromatic –CH), 128.49 (aromatic –CH), 128.80
(aromatic –CH), 130.24 (aromatic –CH), 130.54 (aromatic quater-
nary –C), 137.88 (aromatic quaternary –C), 144.04 (aromatic qua-
ternary –C), 158.76 (aromatic quaternary –C), 165.38, 170.67; IR
4.1.4.4. dl-(4R,5R)-Ethyl 1-(4-chlorobenzyl)-2,4,5-triphenyl-4,5-
dihydro-1H-imidazole-4-carboxylate (22). The resulting crude
residue was purified via column chromatography using silica gel
(1:1 ethyl acetate:hexane) to afford the product as a white solid
(110 mg, 52% yield). Mp 112–115 °C; 1H NMR (500 MHz, CDCl3):
d 0.78 (t, 3H, J = 7.25 Hz), 3.59 (dq, 1H, J = 10.6, 7.0 Hz), 3.68 (dq,
1H, J = 10.6, 7.0 Hz), 3.79 (d, 1H, J = 16.0 Hz), 4.53 (d, 1H,
J = 16.0 Hz), 4.85 (s, 1H), 6.64 (d, 2H, J = 8.5 Hz), 6.99–7.02 (m,
2H), 7.27–7.30 (m, 1H), 7.32–7.35 (m, 4H), 7.36–7.37 (m, 3H),
7.46–7.48 (m, 3H), 7.73–7.76 (m, 4H); 13C NMR + DEPT
(125 MHz, CDCl3): d 13.44 (–CH3), 48.11 (–CH2), 61.03 (–CH2),
74.02 (–CH2), 82.77 (quaternary –C), 126.73 (aromatic –CH),
127.50 (aromatic –CH), 128.05 (aromatic –CH), 128.41 (aromatic
–CH), 128.45 (aromatic –CH), 128.52 (aromatic –CH), 128.54 (aro-
matic –CH), 128.64 (aromatic –CH), 128.78 (aromatic –CH), 130.46
(aromatic –CH), 133.15 (aromatic quaternary –C), 135.11 (aromatic
quaternary –C), 137.67 (aromatic quaternary –C), 143.96 (aromatic
quaternary –C), 165.30, 170.52; IR (neat): 3061 cmÀ1, 3030 cmÀ1
,
(neat): 3063 cmÀ1, 3031 cmÀ1, 2980 cmÀ1, 2936 cmÀ1, 1732 cmÀ1
1597 cmÀ1
HRMS (ESI): m/z calcd for C32H31N2O3 [M+H],
491.2335; found, 491.2328.
,
2980 cmÀ1, 2930 cmÀ1, 1732 cmÀ1, 1595 cmÀ1; HRMS (ESI): m/z
;
calcd for C31H28N2O2Cl [M+H], 495.1839; found, 495.1862.
4.1.4.5. dl-(4R,5R)-Ethyl 1-(4-bromobenzyl)-2,4,5-triphenyl-4,5-
dihydro-1H-imidazole-4-carboxylate (23). The resulting crude
residue was purified via column chromatography using silica gel
(1:1 ethyl acetate:hexane) to afford the product as a white solid
(490 mg, 48% yield). Mp 111–113 °C; 1H NMR (500 MHz, CDCl3): d
0.78 (t, 3H, J = 7.25 Hz), 3.58 (dq, 1H, J = 10.6, 7.2 Hz), 3.68 (dq,
1H, J = 10.6, 7.2 Hz), 3.77 (d, 1H, J = 16.0 Hz), 4.51 (d, 1H,
J = 15.5 Hz), 4.84 (s, 1H), 6.58 (d, 2H, J = 8.5 Hz), 7.15–7.17 (m,
2H), 7.24–7.31 (m, 1H), 7.32–7.35 (m, 3H), 7.35–7.37 (m, 4H),
7.46–7.48 (m, 3H), 7.72–7.76 (m, 4H); 13C NMR + DEPT (125 MHz,
CDCl3): d 13.46 (–CH3), 48.17 (–CH2), 61.02 (–CH2), 74.05 (–CH),
82.88 (quaternary –C), 121.24 (aromatic quaternary –C), 126.76
(aromatic –CH), 127.511 (aromatic –CH), 128.06 (aromatic –CH),
128.40 (aromatic –CH), 128.52 (aromatic –CH),128.65 (aromatic –
4.1.4.2. dl-(4R,5R)-Ethyl 1-(4-methylbenzyl)-2,4,5-triphenyl-4,5-
dihydro-1H-imidazole-4-carboxylate (20). The resulting crude
residue was purified via column chromatography using silica gel
(1:1 ethyl acetate:hexane) to afford the product as a clear oil
(85 mg, 28% yield). 1H NMR (500 MHz, CDCl3): d 0.80 (t, 3H,
J = 7.0 Hz), 2.23 (s, 3H), 3.58 (dq, 1H, J = 10.5, 7.0 Hz), 3.68 (dq, 1H,
J = 10.5, 7.0 Hz), 3.77 (d, 1H, J = 16.0 Hz), 4.57 (d, 1H, J = 15.5 Hz),
4.92 (s, 1H), 6.62 (d, 2H, J = 8.0 Hz), 6.86 (d, 2H, J = 7.5 Hz), 7.25–
7.29 (m, 1H), 7.31–7.35 (m, 4H), 7.36–7.38 (m, 3H), 7.46–7.47 (m,
3H), 7.73–7.77 (m, 4H); 13C NMR + DEPT (125 MHz, CDCl3): d 13.38
(–CH3), 20.86 (–CH3), 48.27 (–CH2), 60.85 (–CH2), 73.56 (–CH),
82.75 (quaternary –C), 126.74 (aromatic –CH), 127.01 (aromatic –
CH), 127.21 (aromatic –CH), 127.88 (aromatic –CH), 128.09 (aromatic

D. K. Kahlon et al. / Bioorg. Med. Chem. 17 (2009) 3093–3103
3101
CH), 128.77 (aromatic –CH), 128.79 (aromatic –CH), 130.42
(aromatic –CH), 130.45 (aromatic quaternary –C), 131.49 (aromatic
–CH), 135.74 (aromatic quaternary –C), 137.77 (aromatic quater-
nary –C), 144.05 (aromatic quaternary –C), 165.29, 170.61; IR
128.47, 129.81, 131.16, 140.04, 143.25, 162.83, 171.33; IR (neat):
3368 cmÀ1, 3063 cmÀ1, 2982 cmÀ1, 1730 cmÀ1, 1599 cmÀ1; HRMS
(ESI): m/z calcd for C24H22N2O2 [M+H], 371.1760; found, 371.1761.
(neat): 3061 cmÀ1, 3034 cmÀ1, 2984 cmÀ1, 2930 cmÀ1, 1732 cmÀ1
,
4.1.4.9. dl-(4R,5R)-Ethyl 1-acetyl-2,4,5-triphenyl-4,5-dihydro-
1H-imidazole-4-carboxylate (27). A solution of dl-(4R,5R)-ethyl-
2,4,5-triphenyl-4,5-dihydro-1H-imidazole-4-carboxylate (26) (100
mg, 0.3 mmol), acetic anhydride (32.7 mg, 0.32 mmol), triethyl-
amine (30.1 mg, 0.3 mmol) and 20 mL of anhydrous dichlorometh-
ane was treated with DMAP (ꢀ10 mg). The solution was stirred for
48 h and then washed with 2 M HCl solution (2 Â 20 mL) and brine
solution (1 Â 20 mL). The organic fraction was dried over sodium
sulfate and concentrated in vacuo . The resulting crude residue
was purified via column chromatography using silica gel (3:7 ethyl
acetate:hexane) to afford the product as a white solid (93 mg, 84%
yield). Mp 59–61 °C 1H NMR (500 MHz, CDCl3): d 0.75 (t, 3H,
J = 7.0 Hz), 1.72 (s, 3H), 3.62 (dq, 1H, J = 10.5, 7.5 Hz), 3.70 (dq,
1H, J = 10.5, 7 Hz), 5.88 (s, 1H), 7.30–7.38 (m, 4H), 7.40–7.49 (m,
4H), 7.50–7.53 (m, 1H), 7.73 (d, 2H, J = 7.0 Hz), 7.78–7.80
(m, 2H); 13C NMR + DEPT (125 MHz, CDCl3): d 13.35 (–CH3),
24.84 (–CH3), 61.41 (–CH2), 72.77 (–CH), 82.42 (quaternary –C),
126.47 (aromatic –CH), 127.36 (aromatic –CH), 127.71 (aromatic
–CH), 128.24 (aromatic –CH), 128.51 (aromatic –CH), 128.57 (aro-
matic –CH), 128.64 (aromatic –CH), 131.13 (aromatic –CH), 131.36
(quaternary aromatic –C), 138.07 (quaternary aromatic –C), 141.04
(quaternary aromatic –C), 160.01, 167.43, 169.31; IR (neat):
3065 cmÀ1, 2982 cmÀ1, 1736 cmÀ1, 1684 cmÀ1, 1624 cmÀ1; HRMS
(ESI): m/z calcd for C26H24N2O3 [M+H], 413.1865; found, 413.1906.
1595 cmÀ1
539.1334; found, 539.1374.
; HRMS (ESI): m/z calcd for C31H28N2O2Br [M+H],
4.1.4.6. dl-(4R,5R)-Ethyl 2,4,5-triphenyl-1-(4-(trifluoromethyl)-
benzyl)-4,5-dihydro-1H-imidazole-4-carboxylate (24). The result-
ing crude residue was purified via column chromatography using
silica gel (1:1 ethylacetate:hexane) to afford the product as a white so-
lid (135 mg, 64% yield). Mp 83–85 °C; 1H NMR (500 MHz, CDCl3): d
0.78 (t, 3H, J = 7.0 Hz), 3.59 (dq, 1H, J = 10.7, 7.1 Hz), 3.69 (dq, 1H,
J = 10.6, 7.1 Hz), 3.89 (d, 1H, J = 16.5 Hz), 4.61 (d, 1H, J = 16.0 Hz),
4.85 (s, 1H), 6.82 (d, 2H, J = 8.0 Hz), 7.28–7.30 (m, 3H), 7.32–7.35 (m,
3H), 7.36–7.37 (m, 4H), 7.41–7.48 (m, 3H), 7.74–7.77 (m, 4H); 13C
NMR + DEPT (125 MHz, CDCl3): d 13.41 (–CH3), 48.35 (–CH2), 61.08
(–CH2), 74.25 (–CH), 82.75 (quaternary –C), 123.86 (q, J = 270 Hz, aro-
matic quaternary –C), 125.32 (q, J = 3.8 Hz, aromatic –CH), 126.69
(aromatic –CH), 127.29 (aromatic –CH), 127.63 (aromatic –CH),
128.03 (aromatic –CH), 128.08 (aromatic –CH), 128.51 (aromatic –
CH), 128.57 (aromatic –CH), 128.71 (aromatic –CH), 128.76 (aromatic
–CH), 129.63 (q, J = 32 Hz, aromatic quaternary –C), 130.62 (aromatic
–CH), 137.53 (aromatic quaternary –C), 140.82 (aromatic quaternary –
C), 143.87 (aromatic quaternary –C), 165.33, 170.38;; IR (neat):
3063 cmÀ1, 3036 cmÀ1, 2983 cmÀ1, 1732 cmÀ1, 1597 cmÀ1; HRMS
(ESI): m/z calcd for C32H28N2O2F3 [M+H], 529.2103; found, 529.2104.
4.1.4.7. dl-(4R,5R)-Ethyl 1-(furan-2-ylmethyl)-2,4,5-triphenyl-4,5-
dihydro-1H-imidazole-4-carboxylate (25). The resulting crude
residue was purified via column chromatography using silica gel
(1:1 ethyl acetate:hexane) to afford the product as a yellow oil
(88 mg, 41% yield). 1H NMR (500 MHz, CDCl3): d 0.81 (t, 3H,
J = 7.25 Hz), 3.57 (dq, 1H, J = 10.5, 7.2 Hz), 3.67 (dq, 1H, J = 10.5,
7.2 Hz), 3.90 (d, 1H, J = 16.5 Hz), 4.41 (d, 1H, J = 16.0 Hz), 4.98 (s,
1H), 5.72 (d, 1H, J = 3.0 Hz), 6.06 (dd, 1H, J= 4.0, 2.0 Hz), 7.09 (dd,
1H, J = 1.5, 1.0 Hz), 7.09 (dd, 1H, J = 1.5, 1.0 Hz), 7.24–7.27 (m,
1H), 7.28–7.37 (m, 5H), 7.41–7.42 (m, 2H), 7.46–7.49 (m, 3H),
7.70–7.72 (m, 2H), 7.78–7.80 (m, 2H); 13C NMR (125 MHz, CDCl3):
d 13.44 (–CH3), 42.63 (–CH2), 61.02 (–CH2), 74.55 (–CH), 82.95
(quaternary –C), 108.15 (aromatic –CH), 109.93 (aromatic –CH),
126.75 (aromatic –CH), 127.26 (aromatic –CH),128.00 (aromatic –
CH), 128.08 (aromatic –CH), 128.26 (aromatic –CH), 128.37 (aro-
matic –CH), 128.50 (aromatic –CH), 128.87 (aromatic –CH),
130.38 (aromatic –CH), 137.86 (aromatic quaternary –C), 142.26
(aromatic –CH), 143.98 (aromatic quaternary –C), 149.92 (aromatic
4.1.4.10. dl-(4R,5R)-Ethyl 2,4,5-triphenyl-1-benzoyl-4,5-dihydro-
1H-imidazole-4-carboxylate (28). A solution of dl-(4R,5R)-ethyl-
2,4,5-triphenyl-4,5-dihydro-1H-imidazole-4-carboxylate (26) (100
mg, 0.27 mmol) and triethylamine (30.4 mg, 0.3 mmol) in 20 mL of
anhydrous dichloromethane was treated with benzoyl chloride
(45.0 mg, 0.32 mmol) and DMAP (ꢀ20 mg). The solution was stirred
at room temperature for 24 h and then washed with 2 M HCl solution
(2 Â 20 mL), saturated NaHCO3 solution (2 Â 20 mL), and brine solu-
tion (1 Â 20 mL). The solution was then dried oversodium sulfate and
concentrated invacuo. The resulting residue was purified viasilica gel
column chromatography using silica gel (3:7 ethyl acetate:hexane as
eluant) to afford the product as a white solid (94 mg, 73% yield). Mp
61–63 °C; 1H NMR (500 MHz, CDCl3): d 0.82 (t, 3H, J = 6.5 Hz), 3.71
(dq, 1H, J = 11, 7.5 Hz), 3.79 (dq, 1H, J = 10.5, 7 Hz), 5.91 (s, 1H),
7.02–7.06 (m, 4H), 7.18–7.24 (m, 3H), 7.28–7.31 (m, 1H), 7.36–7.40
(m, 4H), 7.45–7.49 (m, 4H), 7.65–7.67 (m, 2H), 7.85–7.87 (m, 2H);
13C NMR (125 MHz, CDCl3 + DEPT): d 131.41 (–CH3), 61.44 (–CH2),
74.49 (–CH), 82.86 (quaternary –C), 126.51 (aromatic –CH), 127.54
(aromatic –CH), 127.77 (aromatic –CH), 128.04 (aromatic –CH),
128.32 (aromatic –CH), 128.35 (aromatic –CH), 128.57 (aromatic –
CH), 128.59 (aromatic –CH), 128.61 (aromatic –CH), 128.90 (aromatic
–CH), 130.62 (aromatic quaternary –C), 130.80 (aromatic –CH),
131.27 (aromatic –CH), 134.62(aromatic quaternary–C), 137.95(aro-
matic quaternary –C), 140.59 (aromatic quaternary –C), 161.12,
quaternary –C), 165.76, 170.52; IR (neat): 3061 cmÀ1, 2982 cmÀ1
,
2934 cmÀ1, 1734 cmÀ1, 1597 cmÀ1; HRMS (ESI): m/z calcd for
C29H27N2O3 [M+H], 451.2022; found, 451.2013.
4.1.4.8. dl-(4R,5R)-Ethyl 2,4,5-triphenyl-4,5-dihydro-1H-imidaz-
ole-4-carboxylate (26). A solution of dl-(4R,5R)-ethyl-1-benzyl-
2,4,5-triphenyl-4,5-dihydro-1H-imidazole-4-carboxylate (6) (790
mg, 1.72 mmol), 10 mL of cyclohexene and 50 mL of anhydrous
THF was treated with 300 mg of 10% Pd/C. The solution was stirred
under reflux for 24 h and then filtered through Celite. The resulting
solution was concentrated in vacuo resulting in a yellowish crude
solid. The solid was recrystallized (ethyl acetate:hexane) to afford
the product as a crystalline white solid (553 mg, 87% yield). Mp
140–142 °C; 1H NMR (500 MHz, CDCl3): d 0.75 (t, 3H, J = 7.0 Hz),
3.40–3.53 (m, 1H), 3.67 (dq, 1H, J = 11, 7.5 Hz), 5.53 (br s, 1H),
6.13 (br s, 1H), 7.25–7.34 (m, 6H), 7.37–7.40 (m, 2H), 7.42–7.45
(m, 2H), 7.48–7.51 (m, 1H), 7.78 (d, 2H, J = 8.0 Hz), 8.00 (d, 2H,
J = 7.5 Hz); 13C NMR (125 MHz, CDCl3): d 13.23, 29.80, 61.61,
83.95, 126.43, 127.69, 127.72, 127.81, 127.93, 128.19, 128.30,
168.76, 169.41; IR (neat): 3061 cmÀ1, 2982 cmÀ1, 1749 cmÀ1
,
1721 cmÀ1, 1599 cmÀ1; HRMS (ESI): m/z calcd for C31H27N2O3
[M+H], 475.2022; found, 475.2028.
4.1.4.11. dl-(4R,5R)-Ethyl 2,4,5-triphenyl-1-tosyl-4,5-dihydro-
1H-imidazole-4-carboxylate (29). A solution of dl-(4R,5R)-ethyl-
2,4,5-triphenyl-4,5-dihydro-1H-imidazole-4-carboxylate (26) (155
mg, 0.42 mmol) and triethylamine (42.5 mg, 0.42 mmol) in 20 mL
of anhydrous dichloromethane was treated with tosyl chloride
(87.4 mg, 0.46 mmol) and DMAP (ꢀ10 mg). The solution was
stirred at room temperature for 22 h and then washed with 2 M
HCl solution (2 Â 20 mL), saturated NaHCO3 solution (2 Â 20 mL),
and brine solution (1 Â 20 mL). The solution was then dried over

3102
D. K. Kahlon et al. / Bioorg. Med. Chem. 17 (2009) 3093–3103
sodium sulfate and concentrated in vacuo. The resulting residue
was purified via precipitation (dichloromethane:hexane) to afford
the product as a white solid (160 mg, 73% yield). Mp 150–151 °C;
1H NMR (500 MHz, CDCl3): d 0.71 (t, 3H, J = 7.0 Hz), 2.24 (s, 3H),
3.49 (dq, 1H, J = 10.5, 7 Hz), 3.57 (dq, 1H, J = 10.5, 7 Hz), 5.78 (s,
1H), 6.79 (d, 2H, J = 7.5 Hz), 6.96 (d, 2H, J = 8.5 Hz), 7.23–7.36 (m,
3H), 7.37–7.44 (m, 3H), 7.49–7.56 (m, 4H), 7.57–7.60 (m, 2H),
7.89–7.91 (m, 2H); 13C NMR (125 MHz, CDCl3): d 13.29, 21.43,
61.59, 75.16, 83.26, 126.67, 127.05, 127.34, 127.47, 127.79,
128.11, 128.54, 128.61, 129.20, 129.81, 130.25, 131.58, 134.74,
50
(final concentrations were 20, 10, 5, 1, 0.5, 0.1, 0.05
30 min at 37 °C in 5% CO2. TNF- (Invitrogen Cytokines & Signaling,
l
M MG-132 (EMD Biosciences, San Diego, CA) or imidazoline
l
M) for
a
Camarillo, CA) 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. The
plate and Steady-Glo luciferase assay reagent (Promega Corpora-
tion) were equilibrated to room temperature. An equal volume of
Steady-Glo assay reagent was added to each well. The contents
of the plate were gently mixed for 5 min and the luminescence
of each well was measured using a Veritas microplate luminometer
(Turner Biosystems, CA). All reported data are the average of two
independent experiments unless otherwise indicated. The data
were analyzed using GraphPad Prism 4.0. The data was normalized
138.53, 142.20, 144.08, 160.07, 169.04; IR (neat): 3065 cmÀ1
,
3034 cmÀ1, 1736 cmÀ1, 1599 cmÀ1; HRMS (ESI): m/z calcd for
C31H28N2O4S [M+H], 525.1848; found, 525.1859.
to TNF-a activation and the EC50 values were calculated using the
4.2. Biological methods
4.2.1. Cell culture
equation for the sigmoidal curve for variable slope.
4.2.1.4. Human whole blood IL-1b challenge. After obtaining
the appropriate approval for de-identified human cell lines, human
whole blood was obtained through the Jasper Research Clinic, Kal-
amazoo, MI, from a single healthy, fasted human volunteer and
was collected in glass citrated tubes by venipuncture. Only sam-
ples with a white blood count falling within the normal range
(4800–10,800 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,
The human cell line HeLa-NF-jB-luc was purchased from
Panomics Inc (Fremont, CA). The cells were maintained in Dul-
becco’s Modified Eagle’s medium (DMEM, Gibco Invitrogen, Fred-
rick, MD) containing 4.5 g/L glucose, 3.7 g/L bicarbonate, and
supplemented with 5% fetal bovine serum, 100 U/mL penicillin,
100
lg/mL streptomycin, 1 mM sodium pyruvate, 0.2 mM
L-gluta-
mine and 100
lg/mL of hygromycin B (Roche). The human cell line
184B5 was purchased from ATCC (Rockville, MD). 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 pyru-
100 U/mL penicillin, and 100 lg/mL streptomycin. Aliquots of di-
luted blood (1 mL) were preincubated with vehicle (0.1% DMSO, fi-
nal concentration) or imidazoline (final concentrations were 10, 3,
vate, and 0.2
lM
L-glutamine. Cells were cultured at 37 °C, 5% CO2
1, 0.3 and 0.1 lM) for 2 h at 37 °C, 5% CO2. IL-1b (Roche) was added
atmosphere, 97% relative humidity and were routinely passaged by
trypsin–EDTA (Life technologies, Gran Island NY) treatment
according to ATCC protocol.
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
À80 °C. IL-6 levels were determined by ELISA (R & D Systems).
4.2.1.1. Viability assays. Human white blood cells were counted
manually using a BD Unopette Reservoir (Becton Dickenson,
Franklin Lakes, NJ) and a hemacytometer. The inhibitory effect of
imidazolines on healthy cells 184B5 was assessed by MTS ([3-(4,5-
dimethyl thiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophe-
nyl)-2H-tetrazolium], inner salt) assay (Promega, Madison, WI).
184B5 cells were seeded in 96 well plates at 5000 cells/well 24 h
prior to treatment. The cells were treated with vehicle or imidazo-
line and incubated at 37 °C, 5% CO2. Twenty-four hours after treat-
Acknowledgments
The authors gratefully thank Dr. James Hamby and Dr. Alex
Bridges for their helpful discussions. The authors gratefully
acknowledge funds from the American Cancer Society (RSG CDD-
106972), Michigan Universities Commercialization Initiative
(MUCI), Michigan State University and funding from the TCH Phar-
maceuticals Inc. The authors also thank Rahman Saleem for assis-
tance in acquiring HRMS data.
ment 20 lL MTS reagent was added to each well and the plate was
further incubated for 4 h at 37 °C, 5% CO2. Absorbance was mea-
sured at 490 nm using a spectrophotometer (Molecular Devices,
Sunnyvale, CA) (see Supplementary data).
Supplementary data
Experimental data for reporter assays, ELISA IL-6, FACS, MTS,
cell counting experiments for all compounds are provided. Supple-
mentary data associated with this article can be found, in the on-
line version, at doi:10.1016/j.bmc.2009.03.002.
4.2.1.2. FACS analysis. For the analysis of cell cycle parameters
the adherent cell cultures were trypsinized and resuspended in
1 mL of 1 Â 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 1Â PBS con-
taining 10% FBS. Then the cell pellet was resuspended in staining
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