Discovery of 4-aryl-2-benzoyl-imidazoles as tubulin polymerization inhibitor with potent antiproliferative properties

Article abstract of DOI:10.1021/jm4001117

A series of 4-aryl-2-benzoyl-imidazoles were designed and synthesized based on our previously reported 2-aryl-4-benzoyl-imidazole (ABI) derivatives. The new structures reversed the aryl group and the benzoyl group of previous ABI structures and were named as reverse ABI (RABI) analogues. RABIs were evaluated for biological activity against eight cancer cell lines including multidrug-resistant cancer cell lines. In vitro assays indicated that several RABI compounds had excellent antiproliferative properties, with IC₅₀ values in the low nanomolar range. The average IC₅₀ of the most active compound 12a is 14 nM. In addition, the mechanism of action of these new analogues was investigated by cell cycle analysis, tubulin polymerization assay, competitive mass spectrometry binding assay, and molecular docking studies. These studies confirmed that these new RABI analogues maintain their mechanisms of action by disrupting tubulin polymerization, similar to their parental ABI analogues.

Full text of DOI:10.1021/jm4001117

Article
pubs.acs.org/jmc
Discovery of 4‑Aryl-2-benzoyl-imidazoles as Tubulin Polymerization
Inhibitor with Potent Antiproliferative Properties
Min Xiao,†,§ Sunjoo Ahn,‡,§ Jin Wang,† Jianjun Chen,† Duane. D. Miller,†,‡ James T Dalton,‡
and Wei Li*,†
†Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, Room 327B, 847 Monroe Avenue,
Memphis, Tennessee 38163, United States
‡GTx, Inc., Memphis, Tennessee 38163, United States
S
* Supporting Information
ABSTRACT: A series of 4-aryl-2-benzoyl-imidazoles were designed and
synthesized based on our previously reported 2-aryl-4-benzoyl-imidazole (ABI)
derivatives. The new structures reversed the aryl group and the benzoyl group of
previous ABI structures and were named as reverse ABI (RABI) analogues. RABIs
were evaluated for biological activity against eight cancer cell lines including
multidrug-resistant cancer cell lines. In vitro assays indicated that several RABI
compounds had excellent antiproliferative properties, with IC50 values in the low
nanomolar range. The average IC50 of the most active compound 12a is 14 nM.
In addition, the mechanism of action of these new analogues was investigated by
cell cycle analysis, tubulin polymerization assay, competitive mass spectrometry
binding assay, and molecular docking studies. These studies confirmed that these
new RABI analogues maintain their mechanisms of action by disrupting tubulin
polymerization, similar to their parental ABI analogues.
reversed the two major substitutions on the B ring to produce
the 4-aryl-2-benzoyl-imidazoles (reverse ABI, or RABI)
compounds. We developed a one-pot synthetic strategy to
synthesize RABI analogues in good yields based on the
literature for synthesizing similar scaffold.9 Finally, we system-
atically incorporated additional substitutions in the B ring of the
RABI analogues to determine molecular shape/conformational
requirements for their anticancer potency. Biological testing of
those RABI compounds revealed their excellent antiprolifer-
ative activity against several cancer cell lines including
multidrug-resistant cancer cell lines. Mechanism of action of
RABIs was investigated using cell cycle analysis, tubulin
polymerization assay, competitive mass spectrometry binding
assay, and molecular modeling. These studies showed that their
antitumor activity was achieved through the antimitotic effect
by the inhibition of tubulin polymerization, similar to their
parental ABI analogues.
INTRODUCTION
■
Cancer remains one of the leading causes of mortality
worldwide.1,2 While current therapies are effective in treating
early stage cancers, the efficacy against advanced cancers,
especially multidrug-resistant cancers, is limited. Thus,
developing novel anticancer agents that can effectively
overcome multidrug resistance will provide significant improve-
ment of quality of life in cancer patients.
We previously reported the discovery of ABI analogues
targeting the colchicine binding site in tubulin as potent
antiproliferative agents.3−8 Compared with existing tubulin-
targeting agents such as paclitaxel, colchicine, or vinblastine,
ABI compounds have comparable in vitro and in vivo potency
but can effectively circumvent several clinically relevant
multidrug resistant mechanisms, including drug resistance
mediated by P-glycoprotein (Pgp), multidrug resistance-
associated proteins (MRPs), and breast cancer resistant
proteins (BCRP).5,6 ABI compounds have also shown excellent
oral bioavailability,5 a potential advantage over existing tubulin
inhibitors which can only be administrated by intravenous
injection. To further optimize the potency of ABI analogues
and to gain further insight in their structure−activity relation-
ships (SARs), we designed and synthesized several new series
of ABI analogues (summarized in Figure 1) by introducing
three major changes to the parental ABI scaffold as described
below.
CHEMISTRY
■
The general synthesis of the A ring modified analogues (5a−c)
of ABI compounds is outlined in Scheme 1 using the same
protocol as the method reported previously.3,4,7 The general
synthesis of the substituted imidazoles (8a−e) follows in
Scheme 2. A series of diketones (7a−e)10 in ethanol reacted
with 3,4,5-trimethoxy benzeneacetaldehyde 6 and ammonium
hydroxide to generate a series of substituted imidazoles.11 RABI
First, we varied the substitutions at the para-position on the
A ring of ABI analogues. This was accomplished by using
previously established synthetic strategies.3,4,7 Second, we
Received: January 18, 2013
Published: April 2, 2013
© 2013 American Chemical Society
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Figure 1. Design protocol for synthesis of RABI analogues.
a
Scheme 1
a
Reagents and conditions: (a) oxalaldehyde, NH4OH, EtOH, 0 °C−rt; (b) NaH, PhSO2CI, THF, 0 °C−rt; (c) t-BuLi, substituted benzoyl chloride,
THF, −78 °C; (d) Bu4NF, THF, rt.
Scheme 2
Scheme 3
compounds (11−14) were synthesized utilizing a one-pot, one-
step reaction, which is outlined in Scheme 3.9 The arylglyoxal12
reacts with 3,4,5-trimethoxyphenyl glyoxal in the presence of
ammonium acetate in ethanol to give four products with similar
yields around 20% in one pot. The ratio of compounds 12a−i
to 13a−i is approximately 1:1. Two dimensional 1H−13C
heteronuclear multiple bond correction spectroscopy (HMBC)
NMR experiments were used to distinguish the structures
between 12a−i and 13a−i (Figure S1, Supporting Informa-
tion). Strategies to incorporate additional substitutions on the
B ring of the RABI compounds are shown in Scheme 4. In
Scheme 4, there are three conditions to introduce substitution
to the N1-position. In condition a, compound 12a react with
methyl iodide, ethyl bromide, and benzyl bromide in the
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a
modification did not generate a more potent compound than
5da, we decided to design and synthesize analogues by
modifying the B ring.
Scheme 4
The Ketone Linker Remains Critical for RABI Ana-
logues. We previous reported that a ketone linker is essential
for the activity of the parental ABI and its related analogues;16
to test whether this is still a critical requirement, we synthesized
five RABI analogues containing a methylene linker instead of a
carbonyl linker (Scheme 2). The biological activity of these five
compounds is shown in Table 2. All of them were basically
inactive, consistent with results reported previously which
indicated the essential role of the carbonyl linker.
a
Reagents and conditions: (a) NaH, CH3I for 15a, CH3CH2Br for
Effect of Substitutions on the A Ring of RABIs. After
confirming the essential role of the carbonyl linker in the RABI
analogues, we converted the methylene linker to carbonyl linker
using a slightly modified approach as shown in Scheme 3 and
produced RABI analogues 11−13. All RABI compounds were
evaluated for their antiproliferative activity, and the results were
shown in Table 3. Compound 12a was most potent with IC50
values ranging from 6 to 24 nM. Introducing an electron
withdrawing group with increasing sizes to the para position on
the A ring (4-fluoro, 4-chloro, 4-bromo, and 4-trifluoromethyl)
generally decreased activity (compare 12b−e with 12a).
Among the three halogen substituted RABI compounds, the
trend of activity was F < Cl < Br. Introduction of an electron
donating group such as methyl to the compound maintained
the activity (12f IC50, 10−27 nM), while the introduction of
methoxy and dimethylamino group caused loss of activity to
some extent (12g IC50, 30−210 nM; 12h IC50, 96−263 nM).
Compound 12i with phenol group as A ring was the least
potent one among compounds 12a−i. These results are
consistent with the structure−activity relationships identified
in the parental ABI analogues (Table 1 and earlier studies3,7).
Effect of Substitutions on the C Ring of RABIs. Several
RABI compounds (11a−i) which did not have the 3,4,5-
trimethoxy moiety on C ring showed poor activity as shown in
Table 3. The RABI compounds with unsubstituted phenyl ring
as A ring and C ring caused complete loss of activity (11a IC50
> 50 μM). Compounds with a single halogen substituent in the
para position of A and C rings lost activity completely (IC50 > 5
μM for 11b−d). When methyl, methoxy, trifluoromethyl,
dimethylamino, or hydroxyl was introduced to the para position
of the A and C rings, the activity was also lost. All these results
suggested the essential role of 3,4,5-trimethoxy substituents on
the C ring. Interestingly, when a 3,4,5-trimethoxy group was
put on the A ring, the activity of two compounds 13a and 13d
returned to some extent (13a IC50, 195−5770 nM; 13d IC50,
131−429 nM) even though there is no 3,4,5-trimethoxy
substituents on the C ring, perhaps suggesting that the
15b, and BnBr for 15c; (b) K2CO3, ACN, CH3CH2CH2I for 15d,
(CH3)2CHI for 15e, and cyclopentyl bromide for 15f; (c) Cul,
Cs2CO3, DMF, ligand, 2-bromo-pyridine for 15g, 2-bromothiophene
for 15h.
presence of sodium hydride in anhydrous THF to generate
compounds 15a−c.7 In condition b, compound 12a reacts with
n-propyl iodide, i-propyl iodide, and cyclopentyl bromide in the
presence of potassium carbonate in acetonitrile to generate
compounds 15d−f.13,14 In condition c, copper iodide, cesium
carbonate, and a ligand are used to introduce a pyridine ring or
a thiophene ring to N1-position of compound 12a to make
compound 15g−h.15 A similar method to that in Scheme 3 was
employed to synthesize a series of 5-substituted RABI
compounds (17a−c), as shown in Scheme 5.
BIOLOGICAL RESULTS AND DISCUSSION
■
All the compounds were evaluated for their cytotoxicity in
human melanoma cell lines and prostate cancer cell lines.
Colchicine, vinblastine and docetaxel, the well-known anti-
mitotic agents were included in the assays, serving as positive
controls and as basis for comparison. The results are
summarized in Tables 1−4.
Effect of Substitutions on the A Ring of ABI
Analogues. As shown in Table 1, compound 5a (average
IC50 = 18 nM) maintained potent activity in most cancer cells
compared with compound 5da3,6 (average IC50 = 10nM),
which has a methyl substitution on the A ring para position.
Introducing a larger isopropyl group (5b, average IC50 = 254
nM) into this position on the A ring caused a 20-fold decrease
of potency compared with compound 5da. Further increasing
the size of this substitution using a tert-butyl group to this
position resulted in a 100-fold loss of activity compared with
5da. The activity trend in terms of para substitution in this A
ring is Me > Et > H > i-Pr > t-Bu, clearly suggesting a relatively
small binding pocket to the receptor around the A ring, with a
methyl group being the optimal size. Because A ring
Scheme 5
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a
Table 1. In Vitro Growth Inhibitory Effects of ABI Compounds with A Ring Substitution
a
ND: not determined.
a
Table 2. In Vitro Growth Inhibitory Effects of RABI Compounds with Methylene Linker
a
ND: not determined.
a
Table 3. In Vitro Growth Inhibitory Effects of RABI Compounds without B Ring Substitution
a
ND: not determined.
orientation of the compound changed when binding to tubulin.
One results worth noting is that when 3,4,5-trimethoxy group
was introduced to both the A ring and the C ring, the activity
was lost completely. This is consistent with results in previous
sections that a bulky A ring containing a 3,4,5-trimethoxy
moiety cannot be tolerated at the receptor.
Effect of Substitutions on the B Ring of RABIs.
Modifications on the B ring in two different sites were
investigated: the N1- and 5-position of the imidazole ring.
Introducing methyl, ethyl, or propyl at the 5-position of the
imidazole ring resulted in inactive compounds (17a−c) as
shown in Table 4. A trend in activity for the substituted alkyl
groups, Me > Et > Pr, was also observed, which suggests big
bulky groups at this position are detrimental to activity. In
contrast, when different substitution groups were introduced to
the N1-position of the imidazole ring, the activity was
maintained with only minimal loss compared with the parent
compound, 12a. First, some alkyl groups were tried on the N1-
position including methyl, ethyl, n-propyl, i-propyl, and
cyclopentyl groups. Introduction of methyl did not affect the
activity compared with 12a (15a IC50, 9−26 nM), while the
activity began to lose as the size of the alkyl groups increased,
suggesting that a bulky alkyl group at this position was
unfavorable. Benzyl group, which was also a relatively big group
at the N1-position of the imidazole ring, decreased the activity
(15c IC50, 34−160 nM). Surprisingly, when the substituents
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a
Table 4. In Vitro Growth Inhibitory Effects of RABI Compounds with B Ring Substitution
a
ND: not determined.
were changed from alkyl groups to heterocyclic rings, the
“effect of big size” disappeared. The introduction of a pyridine
ring to the N1-position generated a very potent compound,
with IC50 ranging from 6 to 37 nM. The introduction of a
thiophene ring also produced a potent compound, with IC50
ranging from 8 to 20 nM. The excellent activity of 15g and 15h
promises the future optimization at this position using other
ring systems.
Effect of RABI Compounds against Multidrug-Resist-
ant Melanoma Cells. Pgp-mediated drug efflux represents a
major mechanism for cancer cells to prevent the buildup of
effective intracellular drug concentrations. We have previously
shown that the parent ABI analogues can effectively overcome a
variety of clinically relevant multidrug resistant mechanisms
including Pgp-mediated drug resistance.5,6 To determine
whether the new RABI analogues maintain this ability, we
compared the activity of the RABI compounds against
multidrug-resistant melanoma cells (MDA-MB-435/
LCCMDR1) and their parental nonresistant cancer cells
(MDA-MB-435). This pair of cell lines have been well validated
and widely used to assess abilities of drugs overcoming Pgp-
mediated MDR.17−20 Compound 12a, 12d, and 12e, together
with colchicine, vinblastine, and paclitaxel, were tested on both
the MDR melanoma cells and their parental melanoma cell
lines (Table 5). Compounds 12a, 12d, and 12e showed much
even better activity against Pgp-overexpressed melanoma cells
than their parental, nonresistant melanoma cells.
Mechanism of Action Studies on RABIs. Because the
parental ABI analogues kills cancer cells by inhibiting mitotic
process, we hypothesized that RABIs maintain their mechanism
of action. To test this hypothesis, we first performed the cell
cycle analysis after the treatment of RABIs on PC3 cells. Cell
cycle distribution was determined by propidium iodide (PI)
staining. Treated cells were fixed with 70% ice-cold ethanol, and
the fixed cells were stained with PI in the presence of RNase A.
Cell cycle distribution was analyzed by fluorescence-activated
cell sorting (FACS) analysis. Compound 5a, 12a, 12d, 12f, 15a,
and 15b were treated on PC3 cells for 24 h. Four different
concentrations, 1, 10, 50, and 100 nM, of each compound were
chosen to examine the dose effect. Results indicated that RABIs
arrest cells in the G2/M phase (Figure. 2A). In the vehicle
treated group, about 18% of PC3 cells were distributed in the
G2/M phase. RABIs increased the proportion of cells in G2/M
phase up to 70% approximately in a concentration-dependent
manner (Figure. 2B). The potency of the different concen-
trations in arresting cells in the G2/M phase positively
correlated with in vitro cell growth inhibitory activity.
On the basis of their effect on cell cycle distribution, we next
investigated the effect on tubulin polymerization of RABI
analogues. To determine the effect of drug on tubulin
polymerization, a fluorescence-enhanced tubulin polymeriza-
tion assay kit was used. The control drug, vinblastine, inhibited
tubulin polymerization and destabilized microtubule, while
paclitaxel promoted microtubule stability (Figure S2, Support-
ing Information). RABI compounds 12a and 15a inhibited
tubulin polymerization as the tubulin destabilizer, vinblastine in
a concentration-dependent manner (Figure S2, Supporting
Information), and 15a showed more potent inhibitory effect
than 12a. In the competitive mass spectrometry binding assay,
the amount of unbound colchicine in the presence or absence
of any competitor would explain whether there is the
competition of compounds and colchicine to bind in tubulin.
12a and 15g competed effectively with colchicine for tubulin
binding (Figure S3, Supporting Information) with potency
similar to podophyllotoxin. Vinblastine, the negative control,
did not inhibit colchicine binding to tubulin. Collectively, these
data confirmed that new RABI analogues maintain their
antimitotic mechanisms of action, most likely binding to the
colchicine binding site in tubulin, as is the case for the parental
ABI analogues.6,7
Table 5. In Vitro Growth Inhibitory Effects of RABI
Compounds Comparison to Other Anticancer Drugs on
Multidrug-Resistant Melanoma Cells and Parent Cell Line
IC50 SEM (nM)
MDA-MB-
435
MDA-MB-435/
LCC6MDR1
resistance
index
compd
12a
24
3
11
2
0.5
0.7
12d
58 14
27 11
43 11
12e
21
643
11
5
9
1
0.8
colchicine
vinblastine
paclitaxel
11
2
58.5
27.5
69.3
0.4 0.1
4
1
277 41
better resistance index (0.5 for 12a, 0.7 for 12d, 0.8 for 12e)
than colchicine (58.5), vinblastine (27.5), and paclitaxel (69.3).
Although colchicine, vinblastine, and paclitaxel showed
excellent activity in nonresistant melanoma cell lines, they
were much less potent in resistant melanoma cell lines. In
contrast, compounds 12a, 12d, and 12e showed comparable or
Molecular Modeling Studies. One surprising results from
the analysis of the structure−activity relationships for the RABI
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Figure 2. Effect of compounds 5a, 12a, 12d, 12e, 15a, and 15b on cell cycle.
analogues is that incorporating a 5- or 6-membered heterocyclic
ring substitution at the N1-position in the B ring produced
highly active compounds, while both similar-sized alkyl
substitutions or a larger benzyl substitution resulted in
significantly reduced activity (Table 4). To better understand
how the N1-substituted RABI analogues interact with tubulin,
the potential binding modes for two of the most potent
compounds, 12a and 15g, were investigated at the colchicine
binding site in tubulin dimer using the Schrodinger 2011
molecular modeling suite (Schrodinger, Inc., New York, NY).
Both compounds were docked into two different tubulin crystal
structure (PDB ID code: 1SA0 or 3HKD), representing two
potential binding geometries for colchicine site ligands.
Interestingly, nonsubstituted RABI analogue 12a demonstrated
best Glide docking score of −9.2 in 3HKD compared with a
score of −6.4 in 1SA0, while substituted RABI analogue 15g
showed best Glide score of −9.0 in 1SA0 and could not fit into
the binding pocket of 3HKD.
The overview of the binding site of 12a and TN-16 (native
ligand of 3HKD) is shown in Figure 3A. This binding pocket is
located on the interface between the α- and β-subunits of the
tubulin dimer and extended slightly out of the β-subunit.21,22
Figure 3B illustrated the close view of the potential binding
pose. Generally, 12a (green stick) overlapped well with TN-16
(blue stick). The A ring of 12a went deep into the pocket and
overlapped very well with the first phenyl ring of TN-16. There
is very little space between the A ring and its surrounding
amino acids in the β-subunit (Figure 3B), and this explains why
little tolerance is allowed for larger substitution in the A ring for
both ABI and RABI compounds. The imidazole ring over-
lapped well with the pyrrolidinedione ring of TN-16. A
potential hydrogen bond is formed between the imidazole NH
of 12a and VAL238 in β-H7 (Figure 3B), similar to the one
formed between the native ligand TN-16 and VAL238 in β-H7.
This hydrogen bond stabilized the interaction of 12a with the
binding pocket. The 3,4,5-trimethoxybenzoyl group (C ring) of
12a extends toward the α/β-tubulin interface, similar to the
mode of the active parental ABI analogues.4,7
Unlike 12a, which does not possess a large N1-substitution,
the much “fatter” RABI 15g cannot fit into the cylinder shaped
binding pocket in 3HKD but docks reasonably well into the
shallower pocket in 1SA0. The potential binding mode of 15g
is shown in Figure 3C,D. Figure 3C shows the general view of
the binding site of 15g and the native ligand colchicine in 1SA0.
Figure 3D illustrates the closed view of the potential binding
pose. Interestingly, in this binding mode, part of 15g (green
stick) overlapped well with the native ligand colchicine (blue
stick), while the original A ring of 15g extended out of the
colchicine binding pocket into the α/β-tubulin interface. The
pyridine ring substitute on the N1- position occupied the site
where the 7-membered ring in colchicine binds, while the 3,4,5-
trimethoxybenzoyl group (C ring) of 15g overlapped very well
with the 3,4,5-trimethoxyphenyl ring in colchicine. A hydrogen
bond between the oxygen of 4-OMe in 15g and SH group of β-
CYS241 stabilized the interaction. A similar hydrogen bond was
also observed between the oxygen of one methoxy group in
colchicine and SH of β-CYS241. This binding mode is well
consistent with the structure−activity relationships observed for
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Figure 3. (A) The overview of the binding modes of 12a and the native ligand TN-16 in tubulin crystal structure 3HKD. (B) The close view of the
potential binding pose of 12a and TN-16 in 3HKD. (C) The overview of the binding modes of 15g and the native ligand colchicine in tubulin crystal
structure 1SA0. (D) The close view of the potential binding pose of 15g and colchicine in 1SA0.
Figure 4. SAR of RABI analogues.
substituted RABI compounds: a smaller, alkyl substitution
could not fill this region of the binding pocket and lacks the
planar geometry required for binding, while too large a benzyl
substitution could not fit into the pocket and also lacks the
needed geometry. A 5- or 6-membered heterocyclic ring has the
desirable shape and size to fit into the pocket well. These data
also suggest that while the original “A ring” may not be critical
for binding when a suitable N1-substitution is present, an
optimized substitution replacing this original A ring moiety may
take advantage of the added interactions between the ligand
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column chromatography (hexane:ethyl acetate 2:1) to give a pale solid.
Yield: 40−50%.
and receptor and provide even better ligand than 15g. Work
toward this direction is current ongoing and will be reported in
the future.
General Procedure for the Preparation of Aryl (2-Aryl-1-
(phenylsulfonyl)-1H-imidazol-4-yl)methanone (4a−c). To a solution
of 2-aryl-1-(phenylsulfonyl)-1H-imidazole (6.0 mmol) 3 in anhydrous
THF (30 mL) at −78 °C was added 1.7 M tert-butyl lithium in
pentane (5.3 mL, 9.0 mmol) and stirred for 10 min. Appropriate
substituted benzoyl chloride (7.2 mmol) was added at −78 °C and
stirred for overnight. The reaction mixture was diluted with 100 mL of
saturated NaHCO3 solution (aqueous) and extracted by ethyl acetate
(200 mL). The organic layer was dried over magnesium sulfate and
concentrated. The residue was purified by flash column chromatog-
raphy (hexane:ethyl acetate 4:1) to give a white solid. (Note: Due to
the limited amount of starting material or the difficulty of separation,
the following products formed in this step were used without further
purification as a mixture for the next step.) Yield: 15−40%.
CONCLUSIONS
■
In summary, novel RABI analogues were designed and
synthesized based on rational structural modification of
previous ABI analogues. Structure−activity relationships
(Figure 4) were investigated by introducing different
substituents into the A, B, and C rings. Several RABIs showed
excellent antiproliferative activity which was comparable to
existing tubulin-targeting agents such as paclitaxel, colchicine,
or vinblastine but could overcome Pgp-mediated multiple drug
resistance effectively. Among them compound 12a was the
most potent one, with IC50 in the low nanomolar range, while
compound 15g provided very interesting insights in future
optimization of these analogues. Mechanism of action studies
confirmed that RABI analogues maintain their ability to inhibit
tubulin polymerization at colchicine binding site and arresting
cells in G2/M phase. These results strongly suggest that novel
RABI analogues can be further developed as a promising
antitumor agent for the more efficacious treatment of advanced
cancers.
General Procedure for the Preparation of Aryl (2-Aryl-1H-
imidazol-4-yl)methanone (5a−c). To a solution of aryl (2-aryl-1-
(phenylsulfonyl)-1H-imidazol-4-yl) methanone (2.0 mmol) 4 in THF
(20.0 mL) was added 1.0 M tetrabutyl ammoniumfluoride (4.0 mmol)
and stirred overnight. The reaction mixture was diluted by 50 mL of
saturated NaHCO3 solution (aqueous) and extracted by ethyl acetate
(100 mL). The organic layer was dried over magnesium sulfate and
concentrated. The residue was purified by flash column chromatog-
raphy (hexane:ethyl acetate 3:1) or recrystallized from water and
methanol to give a white solid. Yield: 80−95%.
(2-(4-Ethylphenyl)-1H-imidazol-4-yl)(3,4,5-trimethoxyphenyl)-
methanone (5a). 1H NMR (500 MHz, CDCl3) δ 10.40 (br, 1H), 7.92
(d, J = 8.0 Hz, 2H), 7.85 (s, 1H), 7.36 (d, J = 8.0 Hz, 2H), 7.27 (s,
2H), 3.99 (s, 3H), 3.97 (s, 6H), 2.75 (q, d = 7.5 Hz, 2H), 1.313 (t, d =
7.5 Hz,3H). Exact mass for C21H22N2O4: 366.158. HRMS: [M + H]+:
367.1764. HPLC1: tR 4.97 min, purity 97.8%.
EXPERIMENTAL SECTION
■
General. All reagents were purchased from Sigma-Aldrich
Chemical Co., Fisher Scientific (Pittsburgh, PA), Alfa Aesar (Ward
Hill, MA), and AK Scientific (Mountain View, CA) and were used
without further purification. Routine thin layer chromatography
(TLC) was performed on aluminum-backed Uniplates (Analtech,
Newark, DE). NMR spectra were obtained on a Varian Inova-500
spectrometer (Agilent Technologies, Santa Clara, CA) or a Bruker
Ascend 400 (Billerica, MA) spectrometer. Chemical shifts are reported
as parts per million (ppm) relative to TMS in CDCl3. High resolution
mass spectra were collected on a Waters Xevo G2-S Tof instrument.
The purity of the final compounds was tested via Agilent series HPLC
system (Agilent 1100 Series, Agilent 1100 Chemstation, Agilent
Technology Co, Ltd.) installed with a photodiode array detector. Four
RP-HPLC methods were conducted using a Phenomenex 5 μm C-18
column (250 mm × 4.6 mm) at ambient temperature and a flow rate
of 1.0 mL/min. HPLC1: solvent A (water) and solvent B (methanol),
0−30 min 90% B. HPLC2: solvent A (water) and solvent B
(methanol), 0−30 min 85%. HPLC3: solvent A (water) and solvent
B (methanol), 0−30 min 80% B. HPLC4: solvent A (water) and
solvent B (methanol), 0−15 min 50−100% B (linear gradient), 15−25
min 100% B, 25−28 min 100−50% B, 28−33 min 50% B. UV
detection at 254 nm. Purities of the compounds were established by
careful integration of areas for all peaks detected and are reported for
each compound in the following section.
( 2 - ( 4 - I s o p r o p y l p h e n y l ) - 1 H - i m i d a z o l - 4 - y l ) ( 3 , 4 , 5 -
1
trimethoxyphenyl)methanone (5b). H NMR (500 MHz, CDCl3) δ
10.38 (br, 1H), 7.82 (d, J = 8.0 Hz, 2H), 7.75 (s, 1H), 7.38 (d, J = 8.0
Hz, 2H), 7.15 (s, 2 H), 3.88 (s, 3H), 3.86 (s, 6H), 2.90 (m, 1H),
1.331(d, d = 6.5 Hz, 6H); Exact Mass for C22H24N2O4: 380.1700;
HRMS: [M + H]+: 381.1818; HPLC1: tR 5.26 min, purity 97.5%.
( 2 - ( 4 - t e r t - B u t y l p h e n y l ) - 1 H - i m i d a zo l - 4 - y l ) ( 3 , 4 , 5 -
1
trimethoxyphenyl)methanone (5c). H NMR (500 MHz, CDCl3) δ
10.42 (br, 1 H), 7.94 (d, J = 8.0 Hz, 2H), 7.84 (s, 1H), 7.54 (d, J = 8.0
Hz, 2H), 7.25 (s, 2H), 3.98 (s, 3H), 3.96 (s, 6H), 1.385(s, 9H). Exact
Mass for C23H26N2O4: 394.1900. HRMS: [M + H]+: 395.2054.
HPLC2: tR 7.75 min, purity >99%.
General Procedure for the Synthesis of 5-(Alkyl or Aryl)-4-phenyl-
2-(3,4,5-trimethoxybenzyl)-1H-imidazole and 4-(Alkyl or Aryl)-5-
phenyl-2-(3,4,5-trimethoxybenzyl)-1H-imidazole (8a−e). To a sol-
ution of the aldehyde 6 (5 mmol) in ethanol (20 mL) at 0 °C was
added the phenyl alkyl dione 7 (5.5 mmol) and a solution of 29%
ammonium hydroxide in water (50 mmol, 7 mL). After stirring for 2−
3 days at room temperature, the reaction mixture was concentrated
and the residue was subjected to flash column chromatography with
dichloromethane as eluent to yield the titled compound as a yellow
powder. Yield: 20−30%.
General Procedure for the Preparation of 2-Aryl-1H-imidazole
(2a−c). To a solution of appropriate benzaldehyde 1 (100 mmol) in
ethanol (350 mL) at 0 °C was added a solution of 40% oxalaldehyde in
water (12.8 mL,110 mmol) and a solution of 29% ammonium
hydroxide in water (1000 mmol, 140 mL). After stirring for 2−3 days
at room temperature, the reaction mixture was concentrated and the
residue was subjected to flash column chromatography with
dichloromethane as eluent to yield the titled compound as a yellow
powder. Yield: 20−40%.
5-Phenyl-2-(3,4,5-trimethoxybenzyl)-1H-imidazole and 4-Phenyl-
1
2-(3,4,5-trimethoxybenzyl)-1H-imidazole (8a). H NMR (500 MHz,
chloroform-d) δ 7.91−7.62 (m, 2H), 7.43−7.34 (m, 2H), 7.27−7.21
(m, 2H), 6.51 (s, 2H), 4.12 (s, 2H), 3.86 (s, 3H), 3.84 (s, 6H). Exact
mass for C19H20N2O3: 324.1500. HRMS: [M + H]+: 325.1684.
5-Methyl-4-phenyl-2-(3,4,5-trimethoxybenzyl)-1H-imidazole and
4-Methyl-5-phenyl-2-(3,4,5-trimethoxybenzyl)-1H-imidazole (8b).
1H NMR (500 MHz, chloroform-d) δ 7.55 (d, J = 7.6 Hz, 2H),
7.43−7.37 (m, 2H), 7.25 (d, J = 6.7 Hz, 1H), 6.49 (s, 2H), 4.03 (s,
2H), 3.84 (d, J = 1.3 Hz, 3H), 3.81 (d, J = 1.2 Hz, 6H), 2.42 (s, 3H).
Exact mass for C20H22N2O3: 338.1600. HRMS: [M + H]+: 339.1799.
5-Ethyl-4-phenyl-2-(3,4,5-trimethoxybenzyl)-1H-imidazole and
4-Ethyl-5-phenyl-2-(3,4,5-trimethoxybenzyl)-1H-imidazole (8c). 1H
NMR (500 MHz, chloroform-d) δ 8.47 (s, 0.42H), 8.39 (s, 0.58H),
7.86−7.62 (m, 2H), 7.41 (m, 2H), 7.35−7.31 (m, 1H), 6.54 (m, 2H),
4.10 (m, 2H), 3.90−3.83 (m, 9H), 2.79 (m, 2H), 1.38−1.17 (m, 3H).
Exact mass for C21H24N2O3: 352.1800. HRMS: [M + H]+: 353.1912.
General Procedure for the Preparation of 2-Aryl-1-(phenyl-
sulfonyl)-1H-imidazole (3a−c). To a solution of 2-aryl-1H-imidazole2
(20 mmol) in anhydrous THF (200 mL) at 0 °C was added sodium
hydride (60% dispersion in mineral oil, 1.2 g, 30 mmol) and stirred for
30 min. Benzenesulfonyl chloride (2.82 mL, 22 mmol) was added, and
the reaction mixture was stirred overnight. After dilution by 100 mL of
saturated NaHCO3 solution (aqueous), the reaction mixture was
extracted by ethyl acetate (500 mL). The organic layer was dried over
magnesium sulfate and concentrated. The residue was purified by flash
3325
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Journal of Medicinal Chemistry
Article
4-Phenyl-5-propyl-2-(3,4,5-trimethoxybenzyl)-1H-imidazole and
5-Phenyl-4-propyl-2-(3,4,5-trimethoxybenzyl)-1H-imidazole (8d).
1H NMR (400 MHz, chloroform-d) δ 8.63 (s, 0.48H), 8.50 (s,
7.26 (s, 1H), 2.49 (d, J = 4.52 Hz, 6H), 2.42 (d, J = 4.95 Hz, 6H);.
Exact mass for C18H16N2O: 276.1263. HRMS: [M + H]+: 277.1385.
HPLC3: tR 10.27 min, purity 98.7%.
0.52H), 7.70−7.64 (m, 1H), 7.41 (m, 3H), 7.37−7.32 (m, 1H), 6.52
(d, J = 1.3 Hz, 2H), 4.08 (d, J = 5.1 Hz, 2H), 3.87 (m, 3H), 3.86−3.84
(m, 6H), 2.73 (m, 2H), 1.88−1.75 (m, 1H), 1.64 (m, 1H), 0.98 (m,
3H). Exact mass for C22H26N2O3: 366.1900. HRMS: [M + H]+:
367.2011.
(4-Methoxyphenyl)(4-(4-methoxyphenyl)-1H-imidazol-2-yl)-
methanone and (4-Methoxyphenyl)(5-(4-methoxyphenyl)-1H-imi-
1
dazol-2-yl)methanone (11g). H NMR (400 MHz, chloroform-d) δ
10.50 (s, 1H), 10.38 (s, 1H), 8.77 (d, J = 8.90 Hz, 2H), 8.60 (d, J =
8.89 Hz, 2H), 7.77 (s, 1H), 7.75 (s, 1H), 7.48 (d, J = 8.75 Hz, 1H),
7.45−7.36 (m, 2H), 7.03−6.87 (m, 5H), 3.86 (s, 2H), 3.85 (s, 2H),
3.84 (s, 2H), 3.80 (s, 2H), 3.79 (s, 2H). Exact mass for C18H10F6N2O:
384.0697;. HRMS: [M + H]+: 385.0790. HPLC2: tR 6.05 min, purity
97.2%.
4,5-Diphenyl-2-(3,4,5-trimethoxybenzyl)-1H-imidazole (8e). 1H
NMR (400 MHz, chloroform-d) δ 7.44−7.36 (m, 4H), 7.28−7.20
(m, 6H), 6.50 (s, 2H), 4.08 (s, 2H), 3.78 (m, 9H). Exact mass for
C25H24N2O3: 400.1800. HRMS: [M + H]+: 401.1977.
(4-(Dimethylamino)phenyl)(4-(4-(dimethylamino)phenyl)-1H-
imidazol-2-yl)methanone and (4-(Dimethylamino)phenyl)(5-(4-
(dimethylamino)phenyl)-1H-imidazol-2-yl)methanone(11h). 1H
NMR (500 MHz, DMSO-d6) δ 13.17 (s, 0.35H), 13.12 (s, 1H),
8.64 (d, J = 8.95 Hz, 2H), 8.50 (d, J = 8.95 Hz, 1H), 7.80−7.69 (m,
4H), 7.50 (s, 1H), 6.82 (d, J = 8.99 Hz, 2H), 6.76 (t, J = 7.70, 7.70 Hz,
5H), 3.07 (s, 6H), 3.05 (s, 4H), 2.95 (s, 4H), 2.93 (s, 6H)). Exact
mass for C18H10F6N2O: 384.0697. HRMS: [M + H]+: 385.0790.
HPLC1: tR 5.43 min, purity 95.3%.
General Procedure for the Preparation of (4 or 5)-Aryl-2-aryloyl-
(1H)-Imidazole derivatives (11−14). To ammonium acetate (10
mmol) in ethanol (5 mL) and water (0.3 mL) was added arylglyoxal
hydrate 9 (1 mmol) in ethanol (5 mL) and 3,4,5-trimethoxyphenyl
glyoxal hydrate 10 (1 mmol) in ethanol (10 mL). The mixture was
stirred at room temperature for 30−45 min. The reaction was stopped
after the consumption of the starting material monitored by TLC. The
mixture was then extracted with ethyl acetate. The organic layer was
washed with brine, dried over anhydrous sodium sulfate, and
concentrated to get the crude product. The crude was purified by
flash chromatography (dichloromethane:methanol 50:1).
Phenyl-(4-phenyl-1H-imidazol-2-yl)methanone and phenyl-(5-
phenyl-1H-imidazol-2-yl)methanone (11a). 1H NMR (500 MHz,
DMSO-d6): δ 13.80 (s, 0.25H), 13.63 (s, 1H), 8.60 (d, J = 7.76 Hz,
2H), 8.47 (d, J = 7.7 Hz, 0.5H), 8.08 (s, 1H), 7.97 (d, J = 7.95 Hz,
0.5H), 7.94 (d, J = 7.64 Hz, 2H), 7.79 (s, 0.25H), 7.69 (t, J = 7.1 Hz,
1H), 7.66 (t, J = 7.6 Hz, 0.25H), 7.60 (t, J = 7.6 Hz, 2H), 7.57 (t, J =
8.1 Hz, 0.5H), 7.47 (t, J = 7.55 Hz, 0.5H), 7.42 (t, J = 7.7 Hz, 2H),
7.37 (t, J = 7.1 Hz, 0.25H), 7.28 (t, J = 7.3 Hz, 1H). Exact mass for
C16H12N2O: 248.095. HRMS: [M + H]+: 249.1058. HPLC4: tR 10.83
min, purity 97.6%.
(4-Hydroxyphenyl)(4-(4-hydroxyphenyl)-1H-imidazol-2-yl)-
methanone and (4-Hydroxyphenyl)(5-(4-hydroxyphenyl)-1H-imida-
1
zol-2-yl)methanone (11i). H NMR (500 MHz, DMSO-d6) δ 13.37
(s, 0.37H), 13.29 (s, 1H), 10.39 (s, 1H), 9.46 (s, 1H), 8.60 (d, J = 7.88
Hz, 3H), 8.47 (s, 1H), 7.79 (s, 2H), 7.73 (d, J = 7.48 Hz, 4H), 7.55 (s,
1H), 6.92 (d, J = 8.02 Hz, 4H), 6.81 (d, J = 8.03 Hz, 4H). Exact mass
for C16H12N2O3: 280.0800. HRMS: [M + H]+: 281.0967. HPLC2: tR
3.54 min, purity 99.8%.
(4-Phenyl-1H-imidazol-2-yl)(3,4,5-trimethoxyphenyl)methanone
and (5-Phenyl-1H-imidazol-2-yl)(3,4,5-trimethoxyphenyl)-
1
methanone (12a). H NMR (400 MHz, chloroform-d) δ 10.63 (s,
0.48H), 10.47 (s, 1H), 8.19 (s, 2H), 7.98 (s, 1H), 7.82 (t, J = 1.67,
1.67 Hz, 1H), 7.81 (t, J = 1.11, 1.11 Hz, 1H), 7.60−7.53 (m, 1H), 7.51
(d, J = 1.97 Hz, 1H), 7.46−7.30 (m, 3H), 7.29−7.22 (m, 1H), 3.95 (s,
5H), 3.91 (s, 3H), 3.91 (s, 3H), 3.89 (s, 1H). Exact mass for
C19H18N2O4: 338.1267. HRMS: [M + H]+: 339.1423. HPLC2: tR 6.40
min, purity 95.0%.
(4-Fluorophenyl)(4-(4-fluorophenyl)-1H-imidazol-2-yl)-
methanone and (4-Fluorophenyl)(5-(4-fluorophenyl)-1H-imidazol-
1
2-yl)methanone (11b). H NMR (400 MHz, chloroform-d) δ 10.68
(s, 1H), 10.52 (s, 1H), 8.93−8.82 (dd, J = 5.89, 8.64 Hz, 2H), 8.72
(dd, J = 5.60, 8.70 Hz, 0.39H), 7.89 (dd, J = 5.39, 8.72 Hz, 2H), 7.63
(dd, J = 5.05, 8.25 Hz, 0.46H), 7.59 (d, J = 2.2 Hz, 0.29 H), 7.55 (d, J
= 2.8 Hz, 1H) 7.25−7.20 (m, 2H), 7.20−7.13 (m, 2H. Exact mass for
C16H10F2N2O: 284.0761. HRMS: [M + H]+: 285.0830. HPLC4: tR
13.97 min, purity 98.0%.
(4-(4-Fluorophenyl)-1H-imidazol-2-yl)(3,4,5-trimethoxyphenyl)-
methanone and (5-(4-Fluorophenyl)-1H-imidazol-2-yl)(3,4,5-
1
trimethoxyphenyl)methanone (12b). H NMR (400 MHz, chloro-
form-d) δ 10.60 (s, 0.30H), 10.46 (s, 1H), 8.15 (s, 2H), 7.97 (s, 1H),
7.77 (dd, J = 5.35, 8.90 Hz, 2H), 7.58−7.50 (dd, J = 5.10, 8.20 Hz,
0.47 H), 7.48 (s, 0.46 H), 7.46 (s, 1H), 7.45 (s, 1H), 7.10−7.02 (m,
2H), 3.94 (s, 6H), 3.92 (s, 3H), 3.91 (s, 2H), 3.89 (s, 1H). Exact mass
for C19H17FN2O4: 356.1172. HRMS: [M + H]+: 357.1245. HPLC2: tR
6.90 min, purity 99.3%.
(4-Chlorophenyl)(4-(4-chlorophenyl)-1H-imidazol-2-yl)-
methanone and (4-Chlorophenyl)(5-(4-chlorophenyl)-1H-imidazol-
1
2-yl)methanone (11c). H NMR (400 MHz, chloroform-d) δ 10.70
(s, 0.65 H), 10.55 (s, 1H), 8.78 (d, J = 8.65 Hz, 2H), 8.63 (s, 1H), 7.87
(s, 2H), 7.66−7.52 (m, 5H), 7.50−7.39 (m, 2H). Exact mass for
C16H10Cl2N2O: 316.017. HRMS: [M + H]+: 317.0292. HPLC4: tR
16.30 min, purity 98.8%.
4-Bromophenyl-(4-(4-bromophenyl)-1H-imidazol-2-yl)ketone
and 4-Bromophenyl-(5-(4-bromophenyl)-1H-imidazol-2-yl)-
methanone (11d). 1H NMR (500 MHz, DMSO-d6) δ 13.91 (s,
0.16H), 13.73 (s, 1H), 8.51 (d, J = 8.4 Hz, 2H), 8.42 (d, J = 8.3 Hz,
0.32H), 8.16 (s, 1H), 7.93 (d, J = 8.15 Hz, 0.32H), 7.89 (d, J = 8.35
Hz, 2H),7.83 (d, J = 8.45 Hz,2H), 7.80 (d, J = 8.4 Hz, 0.32H), 7.79
(s,0.16H), 7.67 (d, J = 8.05 Hz, 0.32H), 7.62 (d, J = 8.35 Hz, 2H).
Exact mass for C16H10Br2N2O: 403.916. HRMS: [M + H]+: 404.9241.
HPLC4: tR 16.53 min, purity 95.5%.
(4-(4-Chlorophenyl)-1H-imidazol-2-yl)(3,4,5-trimethoxyphenyl)-
methanone and (5-(4-Chlorophenyl)-1H-imidazol-2-yl)(3,4,5-
1
trimethoxyphenyl)methanone (12c). H NMR (400 MHz, chloro-
form-d) δ 10.57 (s, 0.33H), 10.45 (s, 1H), 8.15 (s, 2H), 7.97 (s,
0.49H), 7.81−7.68 (m, 2H), 7.51−7.47 (m, 1H), 7.36−7.30 (m, 2H),
3.94 (s, 6H), 3.91 (s, 3H), 3.89 (s, 0.75H). Exact mass for
C19H17ClN2O4: 372.0877. HRMS: [M + H]+: 373.0992. HPLC4: tR
15.76 min, purity 95.6%.
(4-(4-Bromophenyl)-1H-imidazol-2-yl)(3,4,5-trimethoxyphenyl)-
methanone and (5-(4-Bromophenyl)-1H-imidazol-2-yl)(3,4,5-
1
trimethoxyphenyl)methanone (12d). H NMR (400 MHz, chloro-
form-d) δ 10.77 (s, 0.36H), 10.59 (s, 1H), 8.24 (s, 2H), 8.06 (s, 1H),
7.78 (d, J = 1.86 Hz, 1H), 7.76 (d, J = 1.98 Hz, 1H), 7.69−7.47 (m,
4H), 4.03 (s, 6H), 4.01 (s, 3H), 4.00 (s, 2H), 3.99 (s, 1H). Exact mass
for C19H17BrN2O4: 416.0372. HRMS: [M + H]+: 417.0496. HPLC1:
tR 6.37 min, purity 96.7%.
(4-(4-(Trifluoromethyl)phenyl)-1H-imidazol-2-yl)(3,4,5-
trimethoxyphenyl)methanone and (5-(4-(Trifluoromethyl)phenyl)-
1H-imidazol-2-yl)(3,4,5trimethoxyphenyl)methanone (12e). 1H
NMR (400 MHz, chloroform-d) δ 10.90 (s, 0.16H), 10.67 (s, 1H),
8.26 (s, 2H), 8.08 (s, 0.36H), 8.01 (d, J = 7.30 Hz, 2H), 7.80−7.88 (m,
0.79H), 7.76−7.62 (m, 3H), 4.08−3.95 (m, 11H). Exact mass for
C20H17F3N2O4: 406.114. HRMS: [M + H]+: 407.1319. HPLC2: tR
9.60 min, purity 95.1%.
(4-(Ttrifluoromethyl)phenyl)(4-(4-(trifluoromethyl)phenyl)-1H-
imidazol-2-yl)methanone and (4-(Trifluoromethyl)phenyl)(5-(4-
(trifluoromethyl)phenyl)-1H-imidazol-2-yl)methanone (11e). 1H
NMR (400 MHz, chloroform-d) δ 10.83 (s, 0.38H), 10.60 (s, 1H),
8.78 (d, J = 8.19 Hz, 2H), 8.64 (d, J = 8.13 Hz, 0.48H), 7.93 (d, J =
8.34 Hz, 2H), 7.76 (d, J = 8.56 Hz, 2H), 7.66−7.59 (m, 3H). Exact
mass for C18H10F6N2O: 384.0697. HRMS: [M + H]+: 385.0790.
HPLC1: tR 7.76 min, purity 98.3%.
p-Tolyl(4-p-tolyl-1H-imidazol-2-yl)methanone and p-Tolyl(5-p-
tolyl-1H-imidazol-2-yl)methanone (11f). 1H NMR (400 MHz,
chloroform-d) δ 10.96 (s, 1H), 10.73 (s, 1H), 8.71 (d, J = 8.26 Hz,
2H), 8.54 (d, J = 8.23 Hz, 2H), 7.82 (d, J = 8.11 Hz, 2H), 7.67−7.49
(m, 4H), 7.37 (t, J = 7.62, 7.62 Hz, 4H), 7.30 (s, 1H), 7.28 (s, 2H),
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Journal of Medicinal Chemistry
Article
(4-p-Tolyl-1H-imidazol-2-yl)(3,4,5-trimethoxyphenyl)methanone
and (5-p-Tolyl-1H-imidazol-2-yl)(3,4,5-trimethoxyphenyl)-
2H), 7.53 (d, J = 31.95 Hz, 2H), 7.07 (s, 2H), 6.82 (dd, J = 12.16,
30.47 Hz, 6H), 3.90 (s, 5H), 3.88 (s, 8H), 3.84 (s, 6H). Exact mass for
C19H18N2O5: 354.1216. HRMS: [M + H]+: 355.1339. HPLC1: tR 4.00
min, purity 97.4%.
3,4,5-Trimethoxyphenyl-(4-(3,4,5-trimethoxyphenyl)-1H-imida-
zol-2-yl)methanone and 3,4,5-Trimethoxyphenyl-(5-(3,4,5-trime-
thoxyphenyl)-1H-imidazol-2-yl)methanone(14). 1H NMR (400
MHz, chloroform-d) δ 10.76 (s, 0.40H), 10.61 (s, 1H), 8.20 (s,
2H), 7.95 (s, 1H), 7.48 (d, J = 1.90 Hz, 0.40H), 7.47 (d, J = 2.34 Hz,
1H), 7.06 (s, 2H), 6.75 (s, 1H), 3.93 (s, 5H), 3.91 (d, J = 0.84 Hz,
4H), 3.89 (s, 1H), 3.87 (s, 2H), 3.86 (s, 5H) 3.83 (s, 1H), 3.82 (s,
2H). Exact mass for C22H24N2O7: 428.1584. HRMS: [M + H]+:
429.1677. HPLC3: tR 9.60 min, purity 96.4%.
General Procedure for the Preparation of (4 or 5)-Aryl-2-aryloyl-
(1H)-imidazole Derivatives (15a−c). To a solution of 12a (135 mg,
0.4 mmol) in THF (10 mL) in ice-bath was added sodium hydride
(60% dispersion in mineral oil, 28 mg, 0.60 mmol) followed by adding
methyl iodide (85 mg, 0.60 mmol) (for 15a) or ethyl iodide (93 mg,
0.60 mmol) (for 15b) or benzyl bromide (102 mg, 0.60 mmol) (for
15c). The resulting reaction mixture was stirred overnight under reflux
condition. After dilution by 50 mL of saturated NaHCO3 solution
(aqueous), the reaction mixture was extracted by ethyl acetate (100
mL). The organic layer was dried over magnesium sulfate and
concentrated. The residue was purified by flash column chromatog-
raphy.
(1-Methyl-4-phenyl-1H-imidazol-2-yl)(3,4,5-trimethoxyphenyl)-
methanone (15a). 1H NMR (500 MHz, chloroform-d) δ 7.97 (d, J =
2.38 Hz, 2H), 7.85 (d, J = 6.01 Hz, 2H), 7.46−7.39 (m, 3H), 7.28 (d, J
= 2.39 Hz, 1H), 4.16−4.10 (m, 3H), 3.99 (d, J = 2.82 Hz, 9H). Exact
mass for C20H20N2O4: 352.1423. HRMS: [M + H]+: 353.1527.
HPLC1: tR 6.03 min, purity 96.4%.
1
methanone (12f). H NMR (400 MHz, chloroform-d) δ 10.57 (s,
0.77H), 10.44 (s, 1H), 8.18 (s, 2H), 7.96 (s, 1H), 7.71 (d, J = 1.87 Hz,
1H), 7.69 (d, J = 1.88 Hz, 1H), 7.47 (d, J = 2.44 Hz, 2H), 7.22 (s, 1H),
7.16 (s, 1H), 3.94 (s, 6H), 3.92 (s, 3H), 3.90 (s, 3H), 3.89 (s, 2H).
Exact mass for C20H20N2O4: 352.1423. HRMS: [M + H]+: 353.1527.
HPLC1: tR 5.63 min, purity 95.9%.
( 4 - ( 4 - M e t h o x y p h e n y l ) - 1 H - i m i d a z o l - 2 - y l ) ( 3 , 4 , 5 -
trimethoxyphenyl)methanone and (5-(4-Methoxyphenyl)-1H-imi-
dazol-2-yl)(3,4,5-trimethoxyphenyl)methanone (12g). 1H NMR
(400 MHz, chloroform-d) δ 10.60 (s, 1H), 10.50 (s, 1H), 8.27 (s,
2H), 8.05 (s, 1H), 7.84 (s, 1H), 7.82 (s, 1H), 7.59 (d, J = 8.87 Hz,
1H), 7.54 (d, J = 1.90 Hz, 1H), 7.51 (d, J = 2.31 Hz, 1H), 7.05−6.97
(m, 4H), 4.04 (s, 5H), 4.01 (s, 3H), 4.00 (s, 3H), 3.98 (s, 2H), 3.89 (s,
2H), 3.88 (s, 3H). Exact mass for C20H20N2O5: 368.1372. HRMS: [M
+ H]+: 369.1572. HPLC4: tR 13.78 min, purity 96.5%.
(4-(4-(Dimethylamino)phenyl)-1H-imidazol-2-yl)(3,4,5-
trimethoxyphenyl)methanone and (5-(4-(Dimethylamino)phenyl)-
1H-imidazol-2-yl)(3,4,5-trimethoxyphenyl)methanone (12h). 1H
NMR (500 MHz, chloroform-d) δ 10.67 (s, 1H), 10.54 (s, 0.49H),
8.28 (s, 1H), 8.03 (s, 2H), 7.78 (s, 1H), 7.76 (s, 1H), 7.53 (d, J = 8.79
Hz, 2H), 7.50 (d, J = 1.56 Hz, 1H), 6.78 (m, 4H), 4.03 (s, 4H), 3.99
(s, 8H), 3.97 (s, 3H), 3.04 (s, 6H), 3.02 (s, 4H). Exact mass for
C21H23N3O4: 381.1689. HRMS: [M + H]+: 382.1842. HPLC1: tR 5.53
min, purity 96.0%.
(4-(4-Hydroxyphenyl)-1H-imidazol-2-yl)(3,4,5-trimethoxyphenyl)-
methanone and (5-(4-Hydroxyphenyl)-1H-imidazol-2-yl)(3,4,5-
1
trimethoxyphenyl)methanone) (12i). H NMR (500 MHz, chloro-
form-d) δ 11.19 (s, 1H), 10.75 (s, 1H), 8.23 (s, 2H), 7.98 (s, 2H), 7.77
(s, 1H), 7.75 (s, 1H), 7.54 (d, J = 5.13 Hz, 3H), 7.49 (s, 1H), 6.90 (t, J
= 9.24, 9.24 Hz, 4H), 4.01 (s, 6H), 3.99 (s, 3H), 3.97 (s, 5H), 3.96 (s,
3H). Exact mass for C19H18N2O5: 354.1216. HRMS: [M + H]+:
355.1378. HPLC2: tR 4.46 min, purity 98.1%.
(1-Ethyl-4-phenyl-1H-imidazol-2-yl)(3,4,5-trimethoxyphenyl)-
1
methanone (15b). H NMR (400 MHz, chloroform-d) δ 7.95 (s,
2H), 7.89−7.83 (m, 2H), 7.51 (s, 1H), 7.43 (t, J = 7.61, 7.61 Hz, 2H),
7.36−7.31 (m, 1H), 4.56 (q, J = 7.19, 7.19, 7.19 Hz, 2H), 3.99 (s, 6H),
3.98 (s, 3H), 1.30 (t, J = 7.19, 7,19 Hz, 3H). Exact mass for
C21H22N2O4: 366.158. HRMS: [M + H]+: 367.1725. HPLC1: tR 6.47
min, purity 97.1%.
(1-Benzyl-4-phenyl-1H-imidazol-2-yl)(3,4,5-trimethoxyphenyl)-
methanone (15c). 1H NMR (500 MHz, chloroform-d) δ 7.94 (d, J =
1.70 Hz, 2H), 7.84 (d, J = 7.73 Hz, 2H), 7.47−7.28 (m, 9H), 5.74 (s,
2H), 3.98 (s, 9H). Exact mass for C26H24N2O4: 428.1736. HRMS: [M
+ H]+: 429.1931. HPLC1: tR 6.70 min, purity 95.4%.
General Procedure for the Preparation of (4 or 5)-Aryl-2-aryloyl-
(1H)-Imidazole Derivatives (15d−f). To a solution of 12a (135 mg,
0.4 mmol) in ACN (10 mL) was added potassium carbonate (82 mg,
0.60 mmol) followed by n-propyl iodide (82 mg, 0.48 mmol) (for
15d) or i-propyl iodide (82 mg, 0.48 mmol) (for 15e) or cyclopentyl
bromide (72 mg, 0.48 mmol) (for 15f). The resulting reaction mixture
was stirred overnight under reflux condition. After dilution by 50 mL
of saturated NaHCO3 solution (aqueous), the reaction mixture was
extracted by ethyl acetate (100 mL). The organic layer was dried over
magnesium sulfate and concentrated. The residue was purified by flash
column chromatography.
Phenyl(4-(3,4,5-trimethoxyphenyl)-1H-imidazol-2-yl)methanone
and Phenyl(5-(3,4,5-trimethoxyphenyl)-1H-imidazol-2-yl)-
1
methanone(13a). H NMR (400 MHz, chloroform-d) δ 10.67 (s,
1H), 10.51 (s, 1H), 8.68−8.63 (m, 2H), 8.53−8.49 (m, 1H), 7.62−
7.53 (m, 2H), 7.53−7.44 (m, 5H), 7.04 (s, 2H), 6.75 (s, 1H), 3.89 (s,
6H), 3.89 (s, 3H), 3.83 (s, 2H), 3.82 (s, 3H). Exact mass for
C19H18N2O4: 338.1267. HRMS: [M + H]+: 339.1348. HPLC2: tR 5.23
min, purity 97.4%.
(4-Bromophenyl)(4-(3,4,5-trimethoxyphenyl)-1H-imidazol-2-yl)-
methanone and (4-Bromophenyl)(5-(3,4,5-trimethoxyphenyl)-1H-
1
imidazol-2-yl)methanone(13d). H NMR (400 MHz, chloroform-d)
δ 10.66 (s, 1H), 10.57 (s, 1H), 8.72−8.62 (m, 2H), 8.56−8.50 (m,
1H), 7.80−7.75 (m, 1H), 7.75−7.67 (m, 4H), 7.57 (q, J = 2.04 Hz,
2H), 7.12 (s, 2H), 6.82 (s, 1H), 3.99 (s, 6H), 3.98 (s, 4H), 3.93 (s,
2H), 3.92 (s, 3H). Exact mass for C19H17BrN2O4: 416.0372. HRMS:
[M + H]+: 417.0454. HPLC2: tR 7.28 min, purity 97.9%.
(4-Methoxyphenyl)(4-(3,4,5-trimethoxyphenyl)-1H-imidazol-2-
yl)methanone and (4-Methoxyphenyl)(5-(3,4,5-trimethoxyphenyl)-
1H-imidazol-2-yl)methanone (13g). 1H NMR (400 MHz, chloro-
form-d) δ 10.80 (s, 1H), 10.66 (s, 1H), 8.87−8.79 (m, 2H), 8.73−8.67
(m, 1H), 7.56 (d, J = 1.83 Hz, 1H), 7.53 (d, J = 2.32 Hz, 1H), 7.14 (s,
2H), 7.05 (dd, J = 3.59, 8.90 Hz, 3H), 6.83 (s, 1H), 3.99 (s, 6H), 3.96
(s, 3H), 3.95 (s, 3H), 3.94 (s, 2H), 3.92 (s, 2H), 3.91 (s, 3H). Exact
mass for C20H20N2O5: 368.1372. HRMS: [M + H]+: 369.1494.
HPLC2: tR 5.94 min, purity 97.4%.
(4-(Dimethylamino)phenyl)(4-(3,4,5-trimethoxyphenyl)-1H-imi-
dazol-2-yl)methanone and (4-(Dimethylamino)phenyl)(5-(3,4,5-tri-
methoxyphenyl)-1H-imidazol-2-yl)methanone (13h). 1H NMR (400
MHz, chloroform-d) δ 11.20 (s, 1H), 10.94 (s, 1H), 8.94−8.75 (m,
2H), 8.74−8.61 (m, 1H), 7.54 (s, 1H), 7.50 (s, 1H), 7.15 (d, J = 2.41
Hz, 2H), 6.86 (s, 1H), 6.76 (dd, J = 5.30, 9.05 Hz, 3H), 3.99 (s, 7H),
3.93 (s, 4H), 3.91 (s, 6H), 3.14 (s, 6H), 3.13 (s, 4H). Exact mass for
C21H23N3O4: 381.1689. HRMS: [M + H]+: 382.1842. HPLC1: tR 4.82
min, purity 97.8%.
(4-Phenyl-1-propyl-1H-imidazol-2-yl)(3,4,5-trimethoxyphenyl)-
1
methanone (15d). H NMR (500 MHz, chloroform-d) δ 7.94 (s,
2H), 7.90−7.82 (m, 2H), 7.47 (s, 1H), 7.42 (t, J = 7.67 Hz, 2H),
7.34−7.28 (m, 1H), 4.51−4.40 (m, 2H), 3.98 (d, J = 1.49 Hz, 9H),
1.96 (h, J = 7.40 Hz, 2H), 1.03 (t, J = 7.42 Hz, 3H). Exact mass for
C22H24N2O4: 380.1700. HRMS: [M + H]+: 381.1897. HPLC1: tR 6.88
min, purity 98.0%.
( 1 - I s o p r o p y l - 4 - p h e n y l - 1 H - i m i d a z o l - 2 - y l ) ( 3 , 4 , 5 -
1
trimethoxyphenyl)methanone (15e). H NMR (400 MHz, chloro-
form-d) δ 7.79 (s, 3H), 7.78−7.73 (m, 1H), 7.57 (s, 1H), 7.34 (dd, J =
6.87, 8.44 Hz, 2H), 7.25−7.21 (m, 1H), 5.66−5.21 (m, 1H), 3.89 (s,
9H), 1.52 (s, 6H). Exact mass for C22H24N2O4: 380.1700. HRMS: [M
+
+ H] : 381.1937. HPLC1: tR 6.90 min, purity 98.3%.
(1-Cyclopentyl-4-phenyl-1H-imidazol-2-yl)(3, 4, 5-
(4-Hydroxyphenyl)(4-(3,4,5-trimethoxyphenyl)-1H-imidazol-2-yl)-
1
trimethoxyphenyl)methanone (15f). H NMR (500 MHz, chloro-
methanone and (4-Hydroxyphenyl)(5-(3,4,5-trimethoxyphenyl)-1H-
1
imidazol-2-yl)methanone (13i). H NMR (500 MHz, chloroform-d)
form-d) δ 7.90−7.82 (m, 4H), 7.61 (d, J = 1.7 Hz, 1H), 7.42 (td, J =
7.7, 1.7 Hz, 2H), 7.33−7.28 (m, 1H), 5.88−5.32 (m, 1H), 3.98 (t, J =
δ 11.81 (s, 1H), 11.45 (s, 1H), 8.62−8.44 (m, 2H), 8.39−8.19 (m,
3327
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Journal of Medicinal Chemistry
Article
1.9 Hz, 9H), 2.64−2.20 (m, 2H), 2.00−1.77 (m, 6H). Exact mass for
C24H26N2O4: 406.1893. HRMS: [M + H]+: 407.2103. HPLC1: tR 8.07
min, purity 98.3%.
Cell Culture and Cytotoxicity Assay. We examined the
antiproliferative activity of the RABI compounds in four human
melanoma cell lines (A375 and WM-164, MDA-MB-435, and MDA-
MB-435/LCC6MDR1) and four human prostate cancer cell lines
(LNCaP, DU 145, PC-3, and PPC-1). All these cell lines were
purchased from ATCC (American Type Culture Collection, Manassas,
VA). Melanoma cells were cultured in DMEM (Cellgro Mediatech
Inc., Herndon, VA), and prostate cancer cells were cultured in RPMI
1640 (Cellgro Mediatech, Inc., Herndon, VA) supplemented with 10%
fetal bovine serum (Cellgro Mediatech). Cultures were maintained at
37 °C in a humidified atmosphere containing 5% CO2. Then 1000−
5000 cells were plated into each well of 96-well plates depending on
growth rate and exposed to different concentrations of a test
compound for 48 h (fast growing melanoma cells) or 96 h (slow
growing prostate cancer cells) in three−five replicates. Cell numbers at
the end of the drug treatment were measured by the sulforhodamine B
(SRB) assay. Briefly, the cells were fixed with 10% trichloroacetic acid
and stained with 0.4% SRB, and the absorbances at 540 nm were
measured using a plate reader (DYNEX Technologies, Chantilly VA).
Percentages of cell survival versus drug concentrations were plotted,
and the IC50 (concentration that inhibited cell growth by 50% of
untreated control) values were obtained by nonlinear regression
analysis using GraphPad Prism (GraphPad Software, San Diego, CA).
Cell Cycle Analysis. Cell cycle distribution was determined by
propidium iodide (PI) staining. Treated cells were washed with PBS
and fixed with 70% ice-cold ethanol overnight. Fixed cells were then
stained with 20 μg/mL of PI in the presence of RNase A (300 μg/mL)
at 37 °C. for 30 min. Cell cycle distribution was analyzed by
fluorescence-activated cell sorting (FACS) analysis core services at the
University of Tennessee Health Science Center, TN.
General Procedure for the Preparation of (4 or 5)-Aryl-2-aryloyl-
(1H)-imidazole Derivatives (15g−h). Under inert atmosphere, a
Schlenk flask was charged with Cs2CO3 (260 mg, 0.8 mmol), CuI (76
mg, 0.4 mmol), ligand (0.4 mmol), compound 12a (135 mg, 0.4
mmol), 2-pyrimidyl bromide (124 mg, 0.8 mmol) (for 15g), or 2-
bromothiophene (130 mg, 0.8 mmol) (for 15h) and DMF (5 mL).
The reaction mixture was stirred for 30 min at room temperature and
then heated to 110 °C for 2 days. The reaction mixture was monitored
by TLC. After the starting material was completely consumed, the
reaction was stopped and the mixture was cooled to room
temperature. The reaction mixture was directly passed through a
plug of silca gel. After being rinsed with ethyl acetate, the combined
filtrate was washed with saturated brine. After the organic layer was
dried by sodium sulfate, it was concentrated. The residue was purified
by column chromatography on silica gel to provide the desired
product.
(4-Phenyl-1-(pyridin-2-yl)-1H-imidazol-2-yl)(3,4,5-
1
trimethoxyphenyl)methanone (15g). H NMR (500 MHz, chloro-
form-d) δ 8.59 (dt, J = 1.70, 4.33 Hz, 1H), 7.91 (ddt, J = 1.67, 3.99,
7.42 Hz, 3H), 7.86 (d, J = 1.52 Hz, 2H), 7.82 (d, J = 1.57 Hz, 1H),
7.48−7.40 (m, 4H), 7.34 (td, J = 1.38, 7.27 Hz, 1H), 3.98 (d, J = 1.62
Hz, 3H), 3.96 (d, J = 1.41 Hz, 6H). Exact mass for C24H21N3O4:
415.1500. HRMS: [M + H]+: 416.1692. HPLC1: tR 4.85 min purity,
97.9%.
(4-Phenyl-1-(thiophen-2-yl)-1H-imidazol-2-yl)(3,4,5-
1
trimethoxyphenyl)methanone (15h). H NMR (500 MHz, chloro-
form-d) δ 7.87−7.76 (m, 4H), 7.52 (d, J = 1.84 Hz, 1H), 7.37 (td, J =
1.83, 7.63, 8.06 Hz, 2H), 7.30−7.23 (m, 2H), 7.06 (dd, J = 1.95, 3.59
Hz, 1H), 6.97 (dp, J = 1.70, 5.60 Hz, 1H), 3.89 (d, J = 1.94 Hz, 3H),
3.88 (d, J = 1.95 Hz, 6H). Exact mass for C23H20N2O4S: 420.1100.
HRMS: [M + H]+: 421.1298. HPLC1: tR 6.33 min, purity 98.2%.
General Procedure for the Preparation of (4 or 5)-Alkyl-(5 or 4)-
aryl-2-aryloyl-(1H)-imidazole Derivatives (18a−c). To ammonium
acetate (10 mmol) in ethanol (5 mL) and water (0.3 mL) was added
phenyl alkyl diones 17(a-c) (1 mmol) in ethanol (5 mL) and 3,4,5-
trimethoxyphenyl glyoxal hydrate 10 (1 mmol) in ethanol (10 mL).
The mixture was stirred at room temperature for 30−45 min. The
reaction was stopped after the consumption of the starting material
monitored by TLC. The mixture was then extracted with ethyl acetate.
The organic layer was washed with brine, dried over anhydrous
sodium sulfate, and concentrated to get the crude product. The crude
was purified by flash chromatography.
Molecular Modeling. All molecular modeling studies were
performed with Schrodinger Molecular Modeling Suite 2011
(Schrodinger LLC, New York, NY) running on a Dell Linux
workstation. We selected tubulin complex with TN16 (PDB code:
3HKD) and tubulin complex with colchicine (PDB code: 1SA0) as our
modeling system. RABIs were built and prepared using the Ligprep
module, and they were docked into the TN16 site and colchicine site
by the Glide module in the Schrodinger Suite. The best docking
complexes were subject to restricted molecular dynamics to release any
strains by using the Macromodel module with OPLS-2005 force field.
The ligand and its surrounding residues within 15 Å were allowed to
move freely, while the outer atoms are frozen.
ASSOCIATED CONTENT
■
S
* Supporting Information
(5-Methyl-4-phenyl-1H-imidazol-2-yl)(3,4,5-trimethoxyphenyl)-
Structure elucidation of 12a; in vitro tubulin polymerization
assay; competitive binding at the colchicine site in tubulin for
compounds 12a and 15g. This material is available free of
charge via the Internet at http://pubs.acs.org.
methanone and (4-Methyl-5-phenyl-1H-imidazol-2-yl)(3,4,5-
1
trimethoxyphenyl)methanone (18a). H NMR (400 MHz, chloro-
form-d) δ 10.43 (s, 0.59H), 10.32 (s, 1H), 8.24 (s, 2H), 8.05 (s, 1H),
7.87−7.71 (m, 3H), 7.62−7.38 (m, 6H), 4.02 (s, 5H), 4.01 (s, 3H),
3.99 (s, 2H), 3.98 (s, 2H), 3.95 (s, 2H), 2.64 (s,3H), 2.56 (s, 1H).
Exact mass for C19H17ClN2O4: 372.0877. HRMS: [M + H]+:
373.0992. HPLC2: tR 6.42 min, purity 95.4%.
AUTHOR INFORMATION
■
Corresponding Author
(5-Ethyl-4-phenyl-1H-imidazol-2-yl)(3,4,5-trimethoxyphenyl)-
methanone and (4-Ethyl-5-phenyl-1H-imidazol-2-yl)(3,4,5-
*Phone: (901)448-7532. Fax: (901)448-6828. E-mail: wli@
uthsc.edu.
1
trimethoxyphenyl)methanone (18b). H NMR (400 MHz, chloro-
form-d) δ 10.85 (s, 1H), 8.28 (s, 2H), 8.15 (s, 1H), 7.75 (m, 1H),
7.45−7.30 (m, 5H), 4.00 (m, 9H), 3.05 (m, 2H), 1.40 (m, 3H). Exact
Mass for C19H17ClN2O4: 372.0877. HRMS: [M + H]+: 373.0992.
HPLC1: tR 5.37 min, purity 96.3%.
(4-Phenyl-5-propyl-1H-imidazol-2-yl)(3,4,5-trimethoxyphenyl)-
methanone and (5-Phenyl-4-propyl-1H-imidazol-2-yl)(3,4,5-
trimethoxyphenyl)methanone(18c). 1H NMR (400 MHz, chloro-
form-d) δ 10.52 (s, 0.52 H), 10.47 (s, 1H), 8.24 (s, 2H), 8.14 (s, 1H),
7.78 (d, J = 7.39 Hz, 2H), 7.60−7.43 (m, 5H), 7.36 (t, J = 7.40, 7.40
Hz, 1H), 4.01 (s, 5H), 4.00 (s, 3H), 3.99 (s, 4H), 3.00−2.93 (t, J =
7.40, 7.40 Hz, 2H), 2.84 (t, J = 7.50, 7.50 Hz, 1H), 1.94−1.87 (m,
1H), 1.87−1.78 (m, 2H), 1.07 (t, J = 7.33, 7.33 Hz, 3H), 0.98−0.92 (t,
J = 7.10, 7.10 Hz, 1H). Exact mass for C19H17ClN2O4: 372.0877.
HRMS: [M + H]+: 373.0992. HPLC: tR 12.46 min, purity 96.1%.
Author Contributions
§These authors contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the NIH grants R01CA148706,
1S10RR026377-01, and 1S10OD010678-01 and funds from
GTx, Inc. The content is solely the responsibility of the authors
and does not necessarily represent the official views of the
National Institutes of Health.
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Journal of Medicinal Chemistry
Article
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ABBREVIATIONS USED
■
ABI, 2-aryl-4-benzoyl-imidazoles; RABI, reverse ABI; HMBC,
heteronuclear multiple-bond correlation spectroscopy; Pgp, P-
glycoprotein; SAR, structure−activity relationship; ND, not
determined; PI, propidium iodide; FACS, fluorescence-
activated cell sorting; SRB, sulforhodamine B
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