Discovery of novel 2-aryl-4-benzoyl-imidazoles targeting the colchicines binding site in tubulin as potential anticancer agents

Article abstract of DOI:10.1021/jm100884b

A series of 2-aryl-4-benzoyl-imidazoles (ABI) was synthesized as a result of structural modifications based on the previous set of 2-aryl-imidazole-4- carboxylic amide (AICA) derivatives and 4-substituted methoxylbenzoyl-aryl- thiazoles (SMART). The average IC₅₀ of the most active compound (5da) was 15.7 nM. ABI analogues have substantially improved aqueous solubility (48.9 μg/mL for 5ga vs 0.909 μg/mL for SMART-1, 0.137 μg/mL for paclitaxel, and 1.04 μg/mL for combretastatin A4). Mechanism of action studies indicate that the anticancer activity of ABI analogues is through inhibition of tubulin polymerization by interacting with the colchicine binding site. Unlike paclitaxel and colchicine, the ABI compounds were equally potent against multidrug resistant cancer cells and the sensitive parental melanoma cancer cells. In vivo results indicated that 5cb was more effective than DTIC in inhibiting melanoma xenograph tumor growth. Our results suggest that the novel ABI compounds may be developed to effectively treat drug-resistant tumors.

Full text of DOI:10.1021/jm100884b

7414 J. Med. Chem. 2010, 53, 7414–7427
DOI: 10.1021/jm100884b
Discovery of Novel 2-Aryl-4-benzoyl-imidazoles Targeting the Colchicines Binding Site
in Tubulin As Potential Anticancer Agents
Jianjun Chen,† Zhao Wang,† Chien-Ming Li,‡,§ Yan Lu,† Pavan K. Vaddady,† Bernd Meibohm,† James T. Dalton,‡,§
Duane D. Miller,†,§ and Wei Li*,†
†Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, Memphis, Tennessee 38163, United States,
‡Division of Pharmaceutics, College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United States, and
§GTx, Inc., Memphis, Tennessee 38163, United States
Received July 14, 2010
A series of 2-aryl-4-benzoyl-imidazoles (ABI) was synthesized as a result of structural modifications
based on the previous set of 2-aryl-imidazole-4-carboxylic amide (AICA) derivatives and 4-substituted
methoxylbenzoyl-aryl-thiazoles (SMART). The average IC50 of the most active compound (5da)
was 15.7 nM. ABI analogues have substantially improved aqueous solubility (48.9 μg/mL for 5ga vs
0.909 μg/mL for SMART-1, 0.137 μg/mL for paclitaxel, and 1.04 μg/mL for combretastatin A4).
Mechanism of action studies indicate that the anticancer activity of ABI analogues is through inhibition
of tubulin polymerization by interacting with the colchicine binding site. Unlike paclitaxel and
colchicine, the ABI compounds were equally potent against multidrug resistant cancer cells and the
sensitive parental melanoma cancer cells. In vivo results indicated that 5cb was more effective than DTIC
in inhibiting melanoma xenograph tumor growth. Our results suggest that the novel ABI compounds
may be developed to effectively treat drug-resistant tumors.
Introduction
promise in overcoming P-glycoprotein (Pgp) mediated MDR
in vitro.3 Despite the high activity, most SMART compounds
are hydrophobic and have limited water solubility, which requires
extensive useof surfactants to solubilize a sufficient amount of
drug for effective dosing or the use of advanced drug delivery
strategies.3 In addition, it has been reported that the thiazole
ring, which is present in the SMART compounds, may under-
go oxidative cleavage, resulting in a nitroxide and ultimately
in reduced in vivo stability for SMART compounds.4
Cancer is one of the main causes of death, ranked only after
heart disease. While existing therapies are effective in treating
various cancers in their early stages, efficacy against metastatic
cancers is far from satisfactory. With the rapid increasing of
cancers in the U.S. and worldwide, clearly, there is an urgent
need to develop highly effective anticancer drugs, which is the
goal of our research. We previously described 2-aryl-imidazole-
4-carboxylic amide (AICAa) derivatives (Figure 1) as potent
antiproliferative agents for melanoma.1 The most potent
compounds in the AICA series have average IC50 values
ranging from 3.5 to 10μM on melanoma cells.1 More recently,
we reported a series of novel substituted methoxylbenzoyl-
aryl-thiazole (SMART) compounds that possess nanomolar
activity in inhibiting melanoma and prostate cancer cell
growth invitro.2 Preliminarystudiesshowed thatthe SMART
compounds disrupt tubulin polymerization and therefore
effectively prevent the formation of functional microtubules
and block cell mitosis.2 SMART compounds also showed great
We hypothesized that replacing the thiazole ring of the
SMART compounds with an imidazole to give aryl-benzoyl-
imidazoles (ABI) would be highly beneficial for the following
reasons. First, the imidazolemoiety ispresentinmany existing
drugs and has been proven to be very stable.5 Second, compared
with the thiazole ring, the imidazole ring is much more hydro-
philic, is expected to have improved aqueous solubility, and
thus may simplify formulation and in vivo use. Finally, the
imidazoleis slightly acidicunder physiological conditions. Itis
well-known that the microenvironment inside a tumor is slightly
acidic because ofa higher productionoflactic acid fromglyco-
lysis (the Warburg effect).6 The ABI analogues should exist
predominantly in the un-ionized state under these conditions.
As a result, ABI analogues may more easily pass through the
cancer cell membrane and conceivably achieve higher intra-
tumoral drug concentrations and greater in vivo potency.
In this paper, we describe our efforts to synthesize a focused
set of ABI analogues (Figure 1) to understand their structure-
activity relationships, to assess their efficacy in vivo, and to
further elucidate their mechanism of action. The ABI analogues
consist ofthree conjugated aromaticrings(denoted asrings A,
B, and C in Figure 1). An imidazole ring was introduced as the
B ring in the ABI series to replace the thiazole ring in the
SMART series. The linker between the B and C rings was
*To whom correspondence should be addressed. Phone: (901)448-
7532. Fax: (901)448-6828. E-mail: wli@uthsc.edu.
a Abbreviations: ABI, 2-aryl-4-benzoyl-imidazole; AICA, 2-aryl-imida-
zole-4-carboxylic amide; SMART, 4-substituted methoxylbenzoyl-aryl-
thiazole; MDR, multidrug resistant; IC50, 50% inhibition concentration;
nM, nanomolar per liter; μM, micromolar per liter; SPA, scintillation
proximity assay; SAR, structure-activity relationship; DTIC, dacarbazine;
EDG, electron donating group; EWG, electron withdrawing group; Pgp,
P-glycoprotein; DBU, 1,8-diazabicycloundec-7-ene; SRB, sulforhodamine
B assay; DMSO, dimethyl sulfoxide; DMF, dimethylformamide; THF,
tetrahydrofuran; TMS, tetramethylsilane; NMR, nuclear magnetic reso-
nance; SEM, standard error of the mean; rt, room temperature; RP-HPLC,
reverse phase-high performance liquid chromatography; ESI, electrospray
ionization; LC-MS, liquid chromatography-mass spectrometry; PDB,
Protein Data Bank; TLC, thin layer chromatography.
r
pubs.acs.org/jmc
Published on Web 10/04/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7415
modified from an amide in the AICA derivatives to a carbonyl
group in ABIs. For the C ring, different substituted phenyls
were introduced. We show that replacing the thiazole ring
with an imidazole ring retained the antiproliferative activity
with significantly improved water solubility. Mechanism of
action studies indicated that the ABI analogues work by com-
petitively binding to the colchicine binding site in the R/β
tubulin dimer and disrupting tubulin polymerization. How-
ever, unlike existing drugs targeting tubulin such as paclitaxel
and vinblastine, the ABI compounds are equally effective against
multidrug-resistant cancer cells, therefore, they hold great
promise as potential drugs to treat resistant cancer.
Chemistry
The general synthesis of the ABI analogues is outlined in
Schemes 1-3. First, a series of imidazolines (7b-x) was syn-
thesized by reacting the appropriately substituted benzalde-
hyde with ethylene diamine in the presence of iodine and
potassium carbonate (Scheme 1).7 Second, the imidazoline
(7b-x) was oxidized to the corresponding imidazolecatalyzed
by diacetoxyiodobenzene.7 Compounds 2b and 2x were gen-
erated by this method. However, it was not successful when
Figure 1. General structures of AICA, SMART, and ABI.
Scheme 1a
a Reagents and conditions: (a) t-BuOH, I2, ethylenediamine, K2CO3, reflux; (b) PhI (OAc)2, K2CO3, DMSO; (c) DBU, CBrCl3, DMF; (d) NaH,
PhSO2Cl, THF, 0 °C-rt; (e) t-BuLi, substituted benzoyl chloride, THF, -78 °C; (f) Bu4NF, THF, rt.
Scheme 2a
a Reagents and conditions: (a) NH4OH, oxalaldehyde, ethanol, rt; (b) NaH, PhSO2Cl, THF, 0 °C-rt; (c) t-BuLi, substituted benzoyl chloride, THF,
-78 °C; (d) Bu4NF, THF, rt; (e) BBr3, CH2Cl2; (f) c-HCl, AcOH, reflux.

7416 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Chen et al.
Scheme 3a
a Reagents and conditions: (a) NaH, substituted benzoyl chloride, THF.
Table 1. In Vitro Growth Inhibitory Effects of Compounds without A Ring Substitutions
applied to other analogues with different substitutions in the
A ring. Consequently, bromotrichloromethane and DBU were
used as the oxidizing agents to convert imidazoline to the
corresponding imidzaole.8 Although compound 2c was pro-
duced by this means, this method was discontinued because of
poor yield (<5%). Other attempts were also made by using
different oxidizing agents including the activated carbon-O2
system9 and palladium-carbon system,10 but neither of these
methods gave a satisfactory yield (<2%). In our efforts to
find an alternative way to construct the imidazole ring, we
tried starting with iminoether11 but abandoned this approach
because of the multistep synthesis. Finally, a simple, one-step
synthesis of the key intermediate (2a-k) was found (Scheme 2)
by reacting the appropriate benzaldehyde (1a-h) in ethanol
with oxalaldehyde and ammonia hydroxide to construct the
imidazole ring system (2a-k).12 The yield from this method
was not high (approximately 20-40%), but it was acceptable
to conduct the subsequent reactions. 2-Aryl-1H-imidazole
(2a-k) was then converted to the N-phenylsulfonyl protected
2-aryl-imidazoles (3a-k) by treating with phenylsulfonyl
chloride and sodium hydride in THF.13 Compounds 3a-k
were coupled with an appropriately substituted benzoyl
chloride in the presence of tert-butyl lithium to obtain the
aryl (2-aryl-1-(phenylsulfonyl)-1H-imidazol-4-yl) methanone
(4ab-jb).13 Removing the protecting group from 4ab-jb
by tetrabutylammonium fluoride in THF gave the desired
ABI agents (5aa-hb).13 Compounds 5aba and 5aaa were
straightforwardly prepared based on the reaction of 2-phenyl-
1H-imidazole (2a) with an appropriate benzoyl chloride
following a known method (Scheme 3).14 Compounds 6ea,
6fa, and 6ha were afforded by treating 5ea, 5fa, and 5ha,
respectively, with BBr3 to remove the methyl groups. Like-
wise, debenzylation of 5ja and 5jb by concentrated HCl
provided 5ka and 5kb, respectively.
Biological Results and Discussion
We first assessed the in vitro antiproliferative activities of
these compounds using three melanoma cell lines (one murine
melanoma cell line, B16-F1, and two human metastatic mela-
noma cell lines, A375 and WM-164) and four human prostate
cancer cell lines (LNCaP, PC-3, Du 145, and PPC-1). The
results are summarized in Tables 1-4.
Effects of Substitutions on the C Ring (Table 1). A variety
of compounds (5aa-5ai) with an unsubstituted A-ring and
different C-ring substituents generally showed moderate
activity (Table 1), with IC50 values in the μM range (unless
specified, the IC50 value for a specific compound discussed in
the text is referred to as an average of all seven cell lines). The
most potent compound of this series was 5aa, with an average
IC50 value of 160 nM. The removal of one of the methoxy
groups from the 3,4,5-trimethoxy on the C ring (5ad, 5ae) led
to a significant loss of activity (IC50 >10 μM for 5ae and an
IC50 of 3.1 μM for 5ad). This finding is consistent with results

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7417
Table 2. In Vitro Growth Inhibitory Effects of Compounds with Substitutions on A Ringa
a ND: not determined.
from SMART compounds in which the 3,4,5-trimethoxy sub-
stituted compound was most potent. It should be noted that
the compound with 4-fluoro on the C ring (5af) also showed
relatively good activity (IC50 =0.91 μM), a finding that has
an important implication because replacing the trimethoxy
moiety with a 4-fluoro group may provide good activity and
improved metabolic stability. Interestingly, the position of
the fluorine on the C ring was critical for activity because
a shift from 4-fluoro to 3-fluoro resulted in a total loss of
activity (IC50 > 10 μM for 5ag compared with 0.91 μM for
5af). This result suggested that a potential hydrogen bond
donor is present close to the 4-position of this ring. As shown
in the molecular modeling studies below, this hydrogen bond
donor is likely to be the thiol group in Cys-241 in loop 7 of
the β-substituent in R/β-tubulin dimer. Other substituents
such as methoxy and methyl at the 3 or 4 position on the C
ring (5ab, 5ac, 5ah, 5ai) were also evaluated, but none showed
good activity (IC50>10 μM). As clearly indicated in Table 1,
the positions of the A and C rings were critical. A simple shift
of the C-ring moiety from position 4 to position 1 in the imida-
zole ring (B ring) resulted in total loss of activity (IC50>10 μM
for 5aba, 5aaa, 3a, 3x, 3j). This result is consistent with
recent reports in which the position of the aryl group was
found to be important for antiproliferative activity.15,16
Bellina and co-workers reported potent antitumor activity
for a series of 1,5- and 1,2-diaryl-1H-imidazole analogues.
While the 1,5-diaryl-imidazole analogues have nanomolar
activity, a simple shift of the diaryl substitution from the
1,5-position to 1,2-position resulted in significantly lower
activity down to the micromolar range.15 Similarly, Wang
and co-workers reported that 4,5-disubstituted and 1,5-disub-
stituted imidazoles are much more active than the corre-
sponding 1,2-disubstituted imidazoles.16
Effects of Substitutions on the A Ring (Table 2). Because
compounds with 3,4,5-trimethoxy and 4-fluoro substitutions
on the C ring showed good activity, a series of compounds
was synthesized with fixed substitutions on the C ring (4-fluoro
or 3,4,5-trimethoxy) and different substitutions on the A ring
(Table 2). These compounds demonstrated excellent anti-
proliferative activity with IC50 values as low as 8.0 nM on the
WM164 cell line (5da). In general, compounds incorporating
a single substituent on the para-position of the A ring were
more potent as can be seen from the activities of 5ca, 5cb,
5da, 5db, 5fa, 5fb, 5ga, and 5gb (IC50 = 7.9-110 nM). 5db-
HCl salt (IC50=172 nM) showed slightly diminished activity
compared with the corresponding free base 5db (IC50=109nM).
Compound 5fb (IC50=63.7 nM), which has a single halogen
substituent in the para-position of the A and C rings, demon-
strated potency and was devoid of a methoxy moiety. Com-
pounds with 3,4,5-trimethoxy substituents on the A ring lost
activity completely (IC50 > 10 μM for 5ea, 5eb), suggesting
very different binding environments near the A ring and C

7418 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Chen et al.
Table 3. In Vitro Growth Inhibitory Effects of Compounds with Protection on B Ring
ring. Removal of the 5-methoxy substituent from the A-ring
improved activity significantly (IC50=330 nM and >10 μM
for 5ha,5ea, respectively). Demethylation of the 3,4,5-trimethoxy
decreased activity sharply from 43 nM (5fa) to 3.89 μM (6fa).
Similar results were observed for 6ea, 5ka, 5kb, and 6ha due
to the demethylation of subsituents on either the A or C ring.
Electron-donating groups (4-methoxy, 4-dimethylamino,
4-methyl) and electron-withdrawing groups (4-chloro, 4-tri-
fluoromethyl) on the A ring did not show substantial differ-
ences in activity. The introduction of a trifluoromethyl group
at the para-position of the A ring (IC50=193 nM for 5la) led
to a 12-fold decrease of activity compared with compound
5da (IC50=15.7 nM), which has a methyl group at the para-
position. In addition, a shift of the trifluoromethyl group
from para to ortho position in the A ring caused complete
loss of activity (IC50>10 μM for 5ia, 5ib), consistent with the
results with other substituents. The presence of a benzoloxy
group at the para-position of the A ring (IC50 = 75 nM for
5jb) resulted in a 440-fold increase in activity when compared
with the para-hydroxy compound 5kb (IC50 = 33 μM). It is
worthwhile to note that compound 5jb, with the 4-fluoro in
the C ring, has better activity than does its counterpart 5ja,
which has a 3,4,5-trimethoxy group in the C ring (IC50 is
75 nM for 5jb and 7.3 μM for 5ja).
Effects of Additional Substitutions on the B Ring (Table 3).
Interestingly, some of the compounds with a phenylsulfonyl
protection group attached to the nitrogen of the imidazole
ring (4cb, 4db, 4fb, 4ga, 4gb, 4ha, 4jb) were also very active,
with IC50 in the nM range (Table 3). Generally, the activities
of these compounds are comparable to their corresponding
unprotected counterparts as exemplified by comparing the
activities of 4cb (43 nM), 4db (111 nM), 4fb (72 nM), 4ga
(285 nM), 4gb (87 nM), 4ha (268 nM), and 4jb (61 nM) with
their corresponding unprotected counterparts 5cb (36 nM),
5db (109 nM), 5fb (64 nM), 5ga (131 nM), 5gb (72 nM), 5ha
(330 nM), and 5jb (75 nM). Other compounds (4ab-4ag,
4ea, 4eb, 4hb, 4ia, and 4ib, 1-50 μM) were generally much
less active, also in line with their counterparts (5ab-5ag, 5ea,
5eb, 5hb, 5ia, and 5ib, 1-50 μM). The comparable activity of
these N-protected compounds with their unprotected coun-
terparts may suggest that they may have similar binding inter-
actions to their target. It is possible that the N-protection
group does not contribute significantly to the binding for
these compounds, as we will see from the molecular modeling
studies for compounds 4cb and 5cb.
ABI Compounds Are Effective against Multidrug Resistant
Melanoma Cells (Table 4). Pgp-mediated drug efflux repre-
sents a major mechanism for cancer cells to prevent the build
up of effective anticancer intracellular drug concentrations.
We compared the activity of the ABI compounds against multi-
drug resistant melanoma cells (MDA-MB-435/LCCMDR1)
and their parental nonresistant cancer cells (MDA-MB-435).
Although MDA-MB-435 was originally designated as a breast
cancer cell line, it has been shown definitively to originate
from the M14 melanoma cell line.17-19 This pair of cell lines
have been well validated and widely used to assess abilities of
drugs overcoming Pgp-mediated MDR.20-23 Compounds
5cb, 4cb, and 4fb, together with other tubulin-targeting
agents including colchicine, paclitaxel, and vinblastine, were
tested on both the MDR melanoma cell line and its parental
melanoma cell line (Table 4). Compounds 5cb, 4cb, and 4fb
had much better resistance indices (1.3 for 5cb, 0.8 for 4cb,
0.7 for 4fb) than colchicine (65.8), paclitaxel (69.3), and
vinblastine (27.5). Although colchicine, paclitaxel, and vin-
blastine showed excellent activity in nonresistant melanoma
cell lines (0.5-10 nM), these compounds were significantly
less potent in the MDR melanoma cell line (277-658 nM). In
contrast, 5cb, 4cb, and 4fb had essentially equivalent potency
on both MDR (30, 30, and 35 nM for 5cb, 4cb, and 4fb,
respectively) and nonresistant melanoma cellines (24, 38, and
50 nM for 5cb, 4cb, and 4fb, respectively).
ABI Compound 5cb is Highly Active in Vivo. To evaluate
efficacy of ABI analogues in vivo, we tested the antitumor
activity of compound 5cb on mice melanoma B16-F1
xenograph. DTIC, the gold standard in malignant melano-
ma treatment, was used as a positive control.24 Compound

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7419
5cb was selected for the in vivo studies because it is more
(30 mg/kg) treatment group. Each mouse was injected with
0.5 million B16-F1 melanoma cells subcutaneously. Seven
days after tumor inoculation, treatment started with each
compound injected intraperitoneally daily (Figure 2). Tumor
volume was significantly (p < 0.05) reduced 47%, 51%, and
73% for 5cb (10mg/kg), DTIC(60mg/kg), and5cb (30mg/kg),
respectively, after 14 days of treatment. No significant weight
loss was observed in any of the treatment groups during the
experiment.
stable compared with compound 5da; although 5da (IC50
15.7 nM) showed higher in vitro potency than 5cb (IC50
=
=
35 nM), it has been proven to be susceptible to demethylation
due to the presence of the 3,4,5-trimethoxy moiety on the
C-ring.25 Twenty female C57/BL mice were divided into four
groups: a vehicle control group, a DTIC (60 mg/kg) treat-
ment group,24 a 5cb (10 mg/kg) treatment group, and a 5cb
Table 4. In Vitro Growth Inhibitory Effects of the ABI Compounds
in Comparison to Other Anticancer Drugs on Multidrug-Resistant
Melanoma Cell Line (MDR cell) and the Matching Sensitive Parent
Cell Line (Normal Melanoma Cell)
ABI Analogues Have Significantly Higher Water Solubility
than SMART Analogues (Table 5). We compared the HPLC
retention times of ABI compound 5ga (1.5 min) and its corres-
ponding SMART analogue (SMART-1, 2.2 min) using 80/20
methanol/water mobile phase at 1 mL/min flow rate and a
reversed phase column, indicating that the imidazole deri-
vative was more hydrophilic than its corresponding SMART
analogue. The calculated log P values for ABI compound
5ga and the corresponding SMART analogue (SMART-1)
were approximately 2.9 and 4.4, respectively. We also deter-
mined the aqueous solubility of compound 5ga and its
corresponding SMART analogue (SMART-1) using a mini-
tuarized shake-flask method and LC-MS/MS quantification.
The aqueous solubility of compound 5ga was 48.9 and
11.3 μg/mL in buffer pH7.0 and water, respectively, or about
50 and 10 times greater than its SMART counterpart,
SMART-1 (0.909 and 0.83 μg/mL in buffer pH7.0 and water,
respectively). Combretastatin A4 (CA-4) and paclitaxel were
used as positive controls because of their well-established
antitumor activity and extensive studies on these two com-
pounds. It is also very interesting to compare the aqueous
solubility of ABI analogues with CA-4 and paclitaxel be-
cause they all target tubulin polymerization (see below for
mechanism studies for ABI analogues). The aqueous solu-
bility determined in this parallel experiment was 1.04 μg/mL
in buffer pH7.0 and 2.83 μg/mL in water for CA-4, and
0.137 μg/mL in buffer pH 7.0 and 0.021 μg/mL in water for
paclitaxel. Collectively, these results indicate that 5ga and
presumably other ABI analogues, due to the high structural
similarity with 5ga, have much higher aqueous solubility
than their SMART counterparts.
IC50 ( SEM (nM) (n = 3)
MDA-MB-
435
MDA-MB-435/
LCC6MDR1
resistance
indexa
compd ID
5cb
4cb
24 ( 2
38 ( 3
50 ( 6
4 ( 1
30 ( 4
30 ( 2
35 ( 3
277 ( 41
11 ( 1
658 ( 50
1.3
0.8
4fb
paclitaxel
0.7
69.3
27.5
65.8
vinblastine 0.4 ( 0.1
colchicine 10 ( 1
a Resistance indexes were calculated by dividing IC50 values on
multidrug-resistant cell line MDA-MB-435/LCC6MDR1 by IC50 values
on the matching sensitive parental cell line MDA-MB-435.
Mechanism of Action Studies. We hypothesized that the
ABI compounds have a similar mechanism of action to the
SMART compounds that involve binding to the colchicine
site in tubulin R/β-heterodimer. We first performed experi-
ments to confirm the inhibition of tubulin polymerization by
Figure 2. In vivo study of 5cb against B16-F1 melanoma tumors in
C57/BL mice.
Table 5. Aqueous Solubility of ABI and Its SMART Counterpart (SMART-1) As Well As CA-4 and Paclitaxel

7420 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Chen et al.
Figure 3. (A) Effect of ABI compounds on tubulin polymerization in vitro. Tubulin (0.4 mg/assay) was exposed to 10 μM ABI compounds
(vehicle control, 5% DMSO). Absorbance at 340 nm was monitored at 37 °C every minute for 15 min. (B) [3H]-Colchicine competition-binding
scintillation proximity assay showed that ABI compounds competitively bound to tubulin colchicine binding site.
Figure 4. Proposed binding mode of ABI analogues in the colchicine binding site.
ABI compounds. Bovine brain tubulin (>97% pure) was incu-
bated with three potent ABI compounds, 5cb, 5da, and 5db,
at a concentration of 10 μM to determine the effect of these
ABI compounds on tubulin polymerization (Figure 3A).
Tubulin polymerization was completely inhibited by com-
pound 5da, while ∼80% inhibition was observed during incu-
bation with compounds 5cb and 5db.
Three ligand binding sites in tubulin R/β-heterodimer have
been reported: paclitaxel binding site,26 vinblastine binding
site,26,27 and colchicine binding site.26-28 The structural
similarity with the SMART compounds and the effects on
tubulin polymerization prompted us to hypothesize that ABI
compounds bind to the colchicine binding site in tubulin.
We measured the binding affinity of compound 5cb using
3H-labeled colchicine and a competitive binding scintillation
proximity assay (SPA).29 The results confirmed the strong bind-
ing of 5cb with a binding affinity of 3.4 ( 1.5 μM (Figure 3B).
Colchicine bound tubulin with an IC50 value of 1.8 ( 0.5 μM
under these conditions. These results clearly indicated that
ABI compounds effectively inhibit tubulin polymerization,
similar to the protocol SMART compounds.
which may indicate substantial conformation changes upon
ligand binding. In fact, Dorleans et al. solved the crystal struc-
tures of both the empty tubulin dimer and the ligand-tubulin
complex.30 They found that, without the presence of ligand,
loop 7 (T7, residues 244-251, Figure 4) in the β-monomer
folds in to occupy the binding pocket, while it flips out upon
ligand binding. The associated helix 7 (H7, residues 224-243)
and helix 8 (H8, residues 252-260) were displaced upon
ligand binding. It is conceivable that the extent to which T7 is
displaced depends on the size of individual ligand. This
flexibility presents a significant challenge to understand the
precise binding modes for individual ligands without solving
actual crystal structures. Nevertheless, careful analysis of the
possible binding modes could provide some insights into the
binding of different ligands.
The binding modes of 5cb and 4cb (stick model) are shown
in parts A and B of Figure 4. For comparison, we also displayed
the crystal structure complexes of ABT-751 and DAMA-
colchicine (wire models) along with ABI-5cb/tubulin com-
plex in Figure 4A. For clarity, only the related secondary
structures forming the binding pocket in β-tubulin are shown
in Figure 4A. The overall structures of 5cb, ABT-751, and
DAMA-colchine overlapped very well in the binding pocket.
Several potential hydrogen bonding interactions between
compound 5cb and tubulin were identified. The carbonyl
group in 5cb was in sufficient proximity to form two hydrogen
bond interactions with the backbone NH of Leu-252 in H8
and the side chain of Asp-251 in T7 of the tubulin β-monomer.
Proposed Binding Mode of ABI Binding at the Colchicine
Binding Site. We next investigated with molecular modeling
the possible binding mode for these compounds in tubulin.
Several crystal structures of the ligand-tubulin complex are
available in the Protein Data Bank (PDB),27,28,30 with the most
recent one from Dorleans et al.30 In general, the colchicine
binding pocket tolerates a variety of molecular structures,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7421
Figure 5. SAR relationship of the ABI analogues.
The para-fluorine substituent in the C-ring was close to the
side chain of Cys241 in T7 and Tyr202 in S6, possibly
forming one or two hydrogen bonds. The imidazole proton
is very close and likely to form a hydrogen bond to Thr179 in
T5 loop (residues 173-182) of the tubulin R-monomer
(Figure 4A). Many of these interactions are consistent with
literature reports with closely related ligands. Together with
the hydrophobic interactions provided by the aromatic rings,
the likely formation of these hydrogen bonds would contri-
bute to the high binding affinity to the tubulin dimer, resulting
in high antiproliferative potency.
The binding mode of 4cb will be conceivably less defined
because two of the three aromatic rings may occupy the bind-
ing pocket in the β-monomer while the third ring may extend
toward the interface of the R/β-monomers, similar to how the
side chain of DAMA-colchicine binds. Our modeling indi-
cates that the protecting group likely extends to the tubulin
dimer interface, while the A and C rings of 4cb occupy similar
binding pocket and orientation as 5cb (Figure 4B). This may
explain the similar activity between the two compounds
even though 4cb has an extra ring system. If this is true,
it is possible to design new generations of ligands by properly
incorporating more hydrophilic groups to further improve
aqueous solubility.
work is underway to test these compounds in other types of
cancers to further optimize their efficacy.
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. The solvents for moisture-
sensitive reactions were freshly distilled, and the reactions were
carried out under an argon atmosphere. Routine thin layer chro-
matography (TLC) was performed on aluminum-backed Uni-
plates (Analtech, Newark, DE). Melting points were measured
with Fisher-Johns melting point apparatus (uncorrected). NMR
spectra were obtained on a Varian Inova-500 spectrometer or a
Bruker AX 300 (Billerica, MA) spectrometer. Chemical shifts
are reported as parts per million (ppm) relative to TMS in CDCl3.
Mass spectra were collected on a Bruker ESQUIRE electrospray/
ion trap instrument in positive and negative ion modes. The purity
of the final compounds was tested via RP-HPLC on a Waters
2695 HPLC system installed with a photodiode array detector.
Two RP-HPLC methods were conducted using a Supelco Ascentis
5 μM C-18 column (250 mmꢀ4.6 mm) at ambient temperature
and a flow rate of 0.7 mL/min. HPLC1: gradient, solvent A (water)
and solvent B (methanol), 0-20 min 40-100%B (linear gradient),
20-27 min 100%B. HPLC2: gradient, solvent A (water) and
solvent B (methanol), 0-15 min 40-100%B (linear gradient),
15-25 min 100%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.
Conclusions
We synthesized a set of novel 2-aryl-4-benzoyl-imidazole
(ABI) derivatives that showed potent activity in a number of
cancer cell lines as well as in a xenograft model. The compounds
inhibited tubulin polymerization by binding to the colchicine
binding site. Structure-activity relationships (SAR) (Figure 5)
were investigated by introducing different substituents into
the A and C rings. Compared with the earlier SMART com-
pounds and other well-established anticancer agents such as
paclitaxel and DTIC, ABI analogues showed three improve-
ments. First, aqueous solubility was significantly improved
(5ga, 48.9 μg/mL) compared with that of SMART analogues
(SMART-1, 0.909 μg/mL). Compared with CA-4, which has a
similar mechanism of action, 5ga has at least 15-fold higher
aqueous solubility. This is animportant improvement because
one general problem with drugs targeting tubulin polymeri-
zation suchas paclitaxel is the poor aqueous solubility. In fact,
in the ongoing clinical trial with CA-4, due to its poor aqueous
solubility, its phosphate pro-drug (CA-4P) has to be used.31
Second, compound 5cb showed a much better resistance index
(1.3) compared with colchicine (65.8), paclitaxel (69.3), and
vinblastine (27.5). The fact that 5cb can overcome multidrug
resistance suggests a promising future development of more
drug-like agents. Third, our in vivo study showed that com-
pound 5cb at a dose of 30 mg/kg gave better tumor suppres-
sion than that of DTIC at a dose of 60 mg/kg. Additional
General Procedure for the Preparation of 2-Aryl-1H-imidazole
(2a-l,x). Method A (essential for only 2b, 2x): To a solution of
2-aryl-4, 5-dihydro-1H-imidazole 7 (35 mmol) in DMSO (100 mL)
was added potassium carbonate (38.5 mmol) and diacetoxy-
iodobenzene (38.5 mmol). The reaction mixture was stirred
overnight in darkness. Water was added, followed by extraction
with dichloromethane. The organic layer was dried over mag-
nesium sulfate and concentrated. The residue was subjected to
flash column chromatography (hexane:ethyl acetate 3:2) to give
a white solid. Yield: 30-50%. This method worked for only 2b
and 2x but not for the other compounds for unknown reasons.
Method B (essential for only 2c): To a solution of 2-aryl-4,5-
dihydro-1H-imidazole 7 (50 mmol) in DMF (70 mL) was added
DBU (55 mmol) and CBrCl3 (55 mmol). The reaction mixture
was stirred overnight, and a saturated NaHCO3 (aqueous) solu-
tion was added followed by extraction with dichloromethane.
The organic layer was dried over magnesium sulfate and con-
centrated. The residue was subjected to flash column chroma-
tography (chloroform:methanol 50:1) to yield a white solid.
Yield: 7%.
Method C (essential for 2a, 2d-l): To a solution of appro-
priate 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

7422 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Chen et al.
residue was subjected to flash column chromatography with
dichloromethane as eluent to yield the titled compound as a yellow
powder. Yield: 20-40%.
benzaldehyde1 (60mmol) in t-BuOH (300 mL) was added ethyle-
nediamine (66 mmol) and stirred for 30 min at room tempera-
ture. Potassium carbonate (75 mmol) and iodine (180 mmol)
were added to the reaction mixture sequentially, followed by
stirring at 70 °C for 3 h. Sodium sulfite (Na2SO3) was added, and
the mixture was extracted by chloroform. The organic layer was
dried over magnesium sulfate and concentrated. The residue was
purified by flash column chromatography (chloroform:methanol
20:1) to give a white solid. Yield: 50-60%.
General Procedure for the Preparation of Aryl (2-Aryl-1H-
imidazol-4-yl) Methanone-HCl Salt (5db-HCl). To a solution of
5db (0.5 mmol) in methanol (20 mL) was added 2 M solution of
hydrogen chloride (5 mmol) in ethyl ether and stirred overnight
at room temperature. The reaction mixture was concentrated,
and the residue was washed by CH2Cl2 to yield the titled com-
pound. Yield: 95%.
General Procedure for the Preparation of 2-Aryl-1-(phenyl-
sulfonyl)-1H-imidazole (3a-l,x). To a solution of 2-aryl-1H-
imidazole 2 (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 solu-
tion (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 column
chromatography (hexane:ethyl acetate 2:1) to give a pale solid.
Yield: 40-50%.
General Procedure for the Preparation of Aryl (2-Aryl-1-(phenyl-
sulfonyl)-1H-imidazol-4-yl) Methanone (4aa-ai, ba, ca, cb, da,
db, ea, eb, fa, fb, ga, gb, ha, hb, ia, ib, ja, jb, la). To a solution of
2-aryl-1-(phenylsulfonyl)-1H-imidazole (6.0 mmol) 3in 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 chromatography (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: 4aa, 4ad, 4ae, 4ai, 4ba, 4ca, 4da, 4fa, 4ja).
Yield: 15%-40%.
General Procedure for the Preparation of Aryl (2-Aryl-1H-
imidazol-4-yl) Methanone (5aa-ai, ba, ca, cb, da, db, ea, eb, fa,
fb, ga, gb, ha, hb, ia, ib, ja, jb, la). 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 ammonium
fluoride (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 chromatography (hexane:ethyl
acetate 3:1) or recrystallized from water and methanol to give
a white solid. Yield: 80-95%.
General Procedure for the Preparation of (2-(4-Hydroxyphenyl)-
1H-imidazol-4-yl) (Aryl) Methanone (5ka, 5kb). To a solution of
(2-(4-(benzyloxy) phenyl)-1H-imidazol-4-yl)(aryl) methanone
5 (5ja or 5jb, 1 mmol) in AcOH(20 mL) was added concentrated
HCl (2 mL) and refluxed overnight. After removing the solvent,
the residue was recrystallized from dichloromethane to give the
titled compound as a yellow solid. Yield: 70-85%.
2-Phenyl-1H-imidazole (2a). Yield: 36.8%. 1H NMR(500 MHz,
DMSO) δ 12.52 (br, 1H), 7.95 (d, J = 7.0 Hz, 2H), 7.44 (t, J =
7.5 Hz, 2H), 7.34 (t, J=7.0 Hz, 1H), 7.25-7.27 (m, 1H), 7.04-7.07
(m, 1H). MS (ESI): calculated for C9H8N2, 144.1; found, 167.1
[M þ Na]þ.
2-(4-Fluorophenyl)-1H-imidazole (2b). Yield: 56.5%. 1H NMR
(300 MHz, DMSO) δ 12.46 (br, 1 H), 7.94-7.99 (m, 2 H), 7.24-
7.30 (m, 2 H), 7.00-7.03 (m, 2 H). MS (ESI): calculated for
C9H7FN2, 162.1; found, 163.0 [M þ H]þ, 160.6 [M - H]-.
2-(4-Methoxyphenyl)-1H-imidazole (2c). Yield: 22.2%. 1H NMR
(500 MHz, CDCl3) δ 7.80 (d, J=10.0 Hz, 2 H), 7.15 (s, 2 H), 3.86
(s, 3 H). MS (ESI): calculated for C10H10N2O, 174.1; found, 175.0
[M þ H]þ, 172.8 [M - H]-.
1
2-(p-Tolyl)-1H-imidazole (2d). Yield: 36.1%. H NMR (500
MHz, CDCl3) δ 7.64 (d, J = 7.5 Hz, 2 H), 7.16 (d, J = 7.5 Hz,
2 H), 7.12 (s, 1 H), 7.02 (s, 1 H). MS (ESI): calculated for C10H10N2,
158.1; found 159.0 [M þ H]þ, 156.8 [M - H]-.
2-(3,4,5-Trimethoxyphenyl)-1H-imidazole (2e). Yield: 26.0%.
1H NMR (500 MHz, CDCl3) δ 7.26 (s, 2 H), 7.08 (d, J=1.5 Hz,
2 H), 3.86 (s, 3 H), 3.82 (s, 6 H). MS (ESI): calculated for C12H14-
N2O3, 234.1; found, 234.9 [M þ H]þ.
2-(4-Chlorophenyl)-1H-imidazole (2f). Yield: 19.8%. 1H NMR
(500 MHz, DMSO) δ13.60 (br, 1 H), 7.94 (d, J=8.5 Hz, 2 H), 7.51
(d, J=8.0 Hz, 2 H), 7.27 (s, 1 H), 7.03 (s, 1 H). MS (ESI): calcu-
lated for C9H7ClN2, 178.0; found, 178.9 [M þ H]þ.
4-(1H-Imidazol-2-yl)-N,N-dimethylaniline (2g). Yield: 16.5%.
1H NMR (300 MHz, CDCl3) δ 7.70 (dd, J=7.0 Hz, 2.0 Hz, 2 H),
7.10 (s, 2 H), 6.75 (dd, J=9.0 Hz, 2.0 Hz, 2 H), 3.02 (s, 6 H). MS
(ESI): calculated for C11H13N3, 187.1; found, 187.9 [M þ H]þ,
185.8 [M - H]-.
2-(3,4-Dimethoxyphenyl)-1H-imidazole (2h). Yield: 22.0%.
1H NMR (500 MHz, CDCl3) δ 7.52 (d, J=1.5 Hz, 1 H), 7.27-
7.28 (m, 1 H), 7.14 (s, 2 H), 6.88 (d, J=8.0 Hz, 1 H), 3.91 (s, 3 H),
3.87 (s, 3 H). MS (ESI): calculated for C11H12N2O2, 204.1;
found, 205.1 [M þ H]þ, 202.8 [M - H]-.
General Procedure for the Preparation of Aryl (2-Phenyl-1H-
imidazol-1-yl) Methanone (5aba, 5aaa). To a solution of 2-phenyl-
1H-imidazole 2a (10 mmol) in THF (20 mL) was added NaH
(15 mmol) and substituted benzoyl chloride (12 mmol) at 0 °C. The
reaction mixture was stirred overnight and diluted by saturated
NaHCO3 solution followed by extraction with ethyl acetate. The
organic layer was dried over magnesium sulfate and concentrated.
The residue was purified by flash column chromatography
(chloroform) to give a white solid. Yield: 12-16%.
2-(2-(Trifluoromethyl)phenyl)-1H-imidazole (2i). Yield: 25.5%.
1H NMR (500 MHz, DMSO) δ 12.31 (br, 1 H), 7.84 (d, J=8.0 Hz,
1 H), 7.76 (t, J=8.0 Hz, 1 H), 7.65 (t, J=7.5 Hz, 1 H), 7.16 (br,
2 H). MS (ESI): calculated for C10H7F3N2, 212.1; found, 212.9
[M þ H]þ, 210.7 [M - H]-.
2-(4-(Benzyloxy)phenyl)-1H-imidazole (2j). Yield: 12.1%. 1H
NMR (500 MHz, CDCl3) δ 7.77 (d, J=8.5 Hz, 2 H), 7.36-7.47
(m, 5 H), 7.10-7.18 (m, 2 H), 7.06 (d, J=9.0 Hz, 2 H), 5.13 (s,
2 H). MS (ESI): calculated for C16H14N2O, 250.1; found, 251.1
[M þ H]þ, 248.8 [M - H]-.
General Procedure for the Preparation of (2-Aryl-1H-imidazol-
4-yl) (3,4,5-Trihydroxyphenyl) Methanone 6 (6ea, 6fa, 6ha). To a
solution of aryl (2-aryl-1H-imidazol-4-yl) methanone 5 (5ea,
5fa, 5ha) (0.5 mmol) in CH2Cl2 (6.0 mL) was added 1.0 M of
BBr3 (2 mmol) in CH2Cl2 and stirred for 1 h at room tempera-
ture. Water was added to destroy excess BBr3. The precipitated
solid was filtered and recrystallized from MeOH to afford a
yellow solid. Yield: 60-80%.
2-(4-(Trifluoromethyl)phenyl)-1H-imidazole (2l). Yield: 26.2%.
1H NMR (CDCl3, 500 MHz) δ 8.03 (d, J=8.0 Hz, 2 H), 7.66 (d,
J=8.0 Hz, 2 H), 7.25 (s, 2 H). MS (ESI) calculated for C10H7F3N2,
212.1; found, 213.1 [M þ H]þ.
2-(4-Nitrophenyl)-1H-imidazole (2x). Yield: 53.7%. 1H NMR
(500 MHz, DMSO) δ 12.97 (br, 1 H), 8.32 (d, J=9.0 Hz, 2 H),
8.17 (d, J = 9.0 Hz, 2 H), 7.42 (s, 1 H), 7.17 (s, 1H). MS (ESI):
calculated for C9H7N3O2, 189.1; found, 189.9 [M þ H]þ, 187.8
[M - H]-.
General Procedure for the Preparation of 2-Aryl-4,5-dihy-
dro-1H-imidazole 7 (7b, 7c, 7x). To a solution of appropriate

Article
2-Phenyl-1-(phenylsulfonyl)-1H-imidazole (3a). Yield: 50.3%.
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7423
(d, J=8.5 Hz, 2 H), 3.88-3.95 (m, 4 H). MS (ESI): calculated for
C9H9N3O2, 191.1; found, 191.9 [M þ H]þ, 189.7 [M - H]-.
2-(4-Fluorophenyl)-4,5-dihydro-1H-imidazole (7b). Yield: 60.2%.
1H NMR (500 MHz, CDCl3) δ7.80(q, J=7.0 Hz, 2 H), 7.11 (d, J=
10.0 Hz, 2 H), 3.82 (br, 4 H). MS (ESI): calculated for C9H9FN2,
164.1; found, 165 [M þ H]þ.
1H NMR (500 MHz, CDCl3) δ 7.64-7.67 (m, 1H), 7.56 (t, J=
9.0 Hz, 1H), 7.32-7.48 (m, 9H), 7.12-7.16 (m, 1H). MS (ESI):
calculated for C15H12N2O2S, 284.1; found, 307.1 [M þ Na]þ.
2-(4-Fluorophenyl)-1-(phenylsulfonyl)-1H-imidazole (3b). Yield:
56.9%. 1H NMR (500 MHz, CDCl3) δ 7.66 (d, J=2.0 Hz, 1 H),
7.58 (t, J=10.0 Hz, 1 H), 7.36-7.42 (m, 6 H), 7.12 (d, J=2.0 Hz,
1 H), 7.06 (t, J = 10.0 Hz, 2 H). MS (ESI): calculated for
C15H11FN2O2S, 302.1; found, 300.8 [M - H]-.
2-(4-Methoxyphenyl)-4,5-dihydro-1H-imidazole (7c). Yield:
1
56.9%. H NMR (500 MHz, CDCl3) δ 7.84 (d, J = 8.5 Hz,
2 H), 6.94 (d, J=9.0 Hz, 2 H), 3.87 (s, 3 H), 3.85 (br, 4 H). MS
(ESI): calculated for C10H12N2O, 176.1; found, 177.0 [M þ H]þ.
(4-Methoxyphenyl)(2-phenyl-1-(phenylsulfonyl)-1H-imidazol-
4-yl)methanone (4ab). Yield: 26.3%; mp 118-120 °C. 1H NMR
(500 MHz, DMSO) δ 8.37 (d, J = 1.0 Hz, 1 H), 8.15-8.18 (m,
2 H), 8.12 (d, J = 9.0 Hz, 2 H), 7.56-7.64 (m, 5 H), 7.46-7.50
(m, 3 H), 7.16 (d, J = 8.0 Hz, 2 H), 3.90 (s, 3 H). MS (ESI):
calculated for C23H18N2O4S, 418.1; found, 419.1 [M þ H]þ.
HPLC2: tR 17.72 min, purity 95.7%.
2-(4-Methoxyphenyl)-1-(phenylsulfonyl)-1H-imidazole (3c).
Yield: 40.9%. 1H NMR (500 MHz, CDCl3) δ 7.62 (d, J=5.0 Hz,
1 H), 7.56 (tt, J = 15.0 Hz, 5.0 Hz, 1 H), 7.32-7.43 (m, 6 H),
7.10 (d, J = 5.0 Hz, 1 H), 6.88 (dt, J = 16.0 Hz, 6.0 Hz, 2 H),
3.87 (s, 3 H). MS (ESI): calculated for C16H14N2O3S, 314.1;
found, 337.1 [M þ Na]þ, 312.9 [M - H]-.
1-(Phenylsulfonyl)-2-(p-tolyl)-1H-imidazole (3d). Yield: 46.6%.
1H NMR (500 MHz, CDCl3) δ 7.63 (d, J=1.0 Hz, 1 H), 7.55 (t,
J=8.0 Hz, 1 H), 7.42 (d, J=8.0 Hz, 2 H), 7.35 (t, J=7.5 Hz, 2 H),
7.27-7.29 (m, 2 H), 7.16 (d, J=7.5 Hz, 2 H), 7.10 (s, 1 H), 2.41 (s,
3 H). MS (ESI): calculated for C16H14N2O2S, 298.1; found, 321.1
[M þ Na]þ.
(3-Methoxyphenyl)(2-phenyl-1-(phenylsulfonyl)-1H-imidazol-
4-yl)methanone (4ac). Yield: 31.2%; mp 136-138 °C. 1H NMR
(500 MHz, CDCl3) δ 8.35 (s, 1 H), 7.86 (d, J=8.0 Hz, 1 H),7.72
(s, 1 H), 7.60 (t, J = 7.5 Hz, 1 H), 7.51 (t, J = 7.5 Hz, 1 H),
7.35-7.42 (m, 9H), 7.14 (dd, J = 8.0 Hz, 2.0 Hz, 1 H), 3.88 (s,
3 H). MS (ESI): calculated for C23H18N2O4S, 418.1; found,
419.1 [M þ H]þ. HPLC2: tR 17.56 min, purity 97.4%.
1-(Phenylsulfonyl)-2-(3,4,5-trimethoxyphenyl)-1H-imidazole (3e).
Yield: 55.7%. 1H NMR (500 MHz, CDCl3) δ 7.68 (d, J=1.5 Hz,
1 H), 7.55 (t, J=7.0 Hz, 1 H), 7.42 (d, J=7.5 Hz, 2 H), 7.35 (t,
J=8.5 Hz, 2 H), 7.11 (d, J=1.5 Hz, 2 H), 6.60 (s, 1 H), 3.90 (s,
3 H), 3.79 (s, 6 H). MS (ESI): calculated for C18H18N2O5S,
374.1; found, 397.1 [M þ Na]þ.
(2-Phenyl-1-(phenylsulfonyl)-1H-imidazol-4-yl)(p-tolyl)methanone
(4ah). Yield: 28.9%; mp 108-110 °C. 1H NMR (500 MHz, CDCl3)
δ 8.00 (d, J=7.5 Hz, 2 H), 7.98 (q, J=8.0 Hz, 1.5 Hz, 2 H), 7.91
(d, J=8.0 Hz, 1 H), 7.81 (s, 1 H), 7.44-7.48 (m, 3 H), 7.35-7.40
(m, 2 H), 7.30 (t, J=8.0 Hz, 2 H), 7.20 (s, 2 H), 2.42 (s, 3 H). MS
(ESI): calculated for C23H18N2O3S, 402.1; found, 403.1 [M þ H]þ.
HPLC2: tR 16.06 min, purity 96.2%.
2-(4-Chlorophenyl)-1-(phenylsulfonyl)-1H-imidazole (3f). Yield:
54.9%. 1H NMR (500 MHz, CDCl3) δ 7.65 (d, J=2.0 Hz, 1 H),
7.58 (t, J=7.5 Hz, 1 H), 7.43 (d, J=8.5 Hz, 2 H), 7.38 (t, J=8.0
Hz, 2 H), 7.34-7.36 (m, 4 H), 7.12 (d, J=1.5 Hz, 1 H). MS (ESI):
calculated for C15H11ClN2O2S, 318.0; found, 341.0 [M þ Na]þ.
N,N-Dimethyl-4-(1-(phenylsulfonyl)-1H-imidazol-2-yl) aniline
(3g). Yield: 48.3%. 1H NMR (300 MHz, CDCl3) δ 7.59 (d, J=
2.0 Hz, 1 H), 7.55 (t, J=8.0 Hz, 1 H), 7.45 (d, J=7.5 Hz, 2 H),
7.28-7.38 (m, 4 H), 7.07 (d, J=2.0 Hz, 1 H), 6.68 (d, J=8.5 Hz,
2 H), 3.04 (s, 3 H). MS (ESI): calculated for C17H17N3O2S,
327.1; found, 350.0 [M þ Na]þ, 325.9 [M - H]-.
(4-Fluorophenyl)(2-phenyl-1-(phenylsulfonyl)-1H-imidazol-4-yl)
Methanone (4af). Yield: 25.4%; mp 114-116 °C. 1H NMR (500
MHz, CDCl3) δ 8.10 (q, J=3.5 Hz, 5.5 Hz, 2 H), 7.88 (d, J=7.5
Hz, 2 H), 7.67 (t, J=7.5 Hz, 1 H), 7.48-7.54 (m, 3 H), 7.38-7.41
(m, 5 H), 7.24 (t, J = 8.5 Hz, 2 H). MS (ESI): calculated
for C22H15FN2O3S, 406.1; found, 429.1 [M þ Na]þ. HPLC2:
t
R 15.43 min, purity 96.1%.
2-(3, 4-Dimethoxyphenyl)-1-(phenylsulfonyl)-1H-imidazole (3h).
Yield: 60.3%. 1H NMR (500 MHz, CDCl3) δ 7.64 (d, J=7.0 Hz,
1 H), 7.55 (t, J=7.5 Hz, 1 H), 7.40 (dd, J=8.5 Hz, 1.5 Hz, 2 H),
7.35 (t, J=8.0 Hz, 2H), 7.09 (d, J=2.0 Hz, 1 H), 7.02 (dd, J=
8.0 Hz, 2.0 Hz, 1 H), 6.89 (d, J=1.5 Hz, 1 H), 6.86 (d, J=8.0 Hz,
1 H), 3.95 (s, 3 H), 3.81 (s, 3 H). MS (ESI): calculated for
C17H16N2O4S, 344.1; found, 367.0 [M þ Na]þ.
(3-Fluorophenyl)(2-phenyl-1-(phenylsulfonyl)-1H-imidazol-4-yl)
Methanone (4ag). Yield: 18.3%; mp 102-104 °C. 1H NMR (500
MHz, CDCl3) δ 8.14 (d, J=7.5 Hz, 1 H), 7.76-7.87 (m, 3 H), 7.74
(d, J=9.0 Hz, 1 H), 7.37-7.57 (m, 10 H), 7.38-7.41 (m, 5 H), 7.24
(t, J = 8.5 Hz, 2 H). MS (ESI): calculated for C22H15FN2O3S,
406.1; found, 429.1 [M þ Na]þ. HPLC2: tR 15.75 min, purity
96.5%.
1-(Phenylsulfonyl)-2-(2-(trifluoromethyl) phenyl)-1H-imidazole
(3i). Yield: 58.6%. 1H NMR (500 MHz, CDCl3) δ 7.64-7.67 (m,
2 H), 7.61-7.63 (m, 3 H), 7.40-7.46 (m, 5 H), 7.16 (d, J=1.5 Hz,
1 H). MS (ESI): calculated for C16H11F3N2O2S, 352.1; found,
353.1 [M þ H]þ.
(4-Fluorophenyl)(2-(4-methoxyphenyl)-1-(phenylsulfonyl)-1H-
imidazol-4-yl) Methanone (4cb). Yield: 23.5%; mp 135-137 °C.
1H NMR (500 MHz, CDCl3) δ 8.00 (d, J = 5.5 Hz, 2 H),
7.74-7.76 (m, 2 H), 7.54-7.58 (m, 1 H), 7.40 (d, J = 7.0 Hz,
2 H), 7.28-7.30 (m, 3H), 7.14-7.16 (m, 2 H), 6.80-6.82 (m, 2 H),
3.80 (s, 3 H). MS (ESI): calculated for C23H17FN2O4S, 436.1;
found, 459.0 [M þ Na]þ, 434.9 [M - H]-. HPLC2: tR 16.53 min,
Purity 96.1%.
(4-Fluorophenyl)(1-(phenylsulfonyl)-2-(p-tolyl)-1H-imidazol-
4-yl) Methanone (4db). Yield: 18.6%; mp 142-144 °C. 1H NMR
(500 MHz, CDCl3) δ 8.07 (q, J=8.5 Hz, 5.5 Hz, 2 H), 7.88 (d,
J =7.5 Hz, 2 H), 7.64 (t, J=8.0 Hz, 1 H), 7.49 (d, J =8.0 Hz,
2 H), 7.38 (s, 1H), 7.30 (d, J=8.0 Hz, 2 H), 7.18-7.24 (m, 4 H),
2.43 (s, 3 H). MS (ESI): calculated for C23H17FN2O3S, 420.1;
found, 443.0 [M þ Na]þ, 418.9 [M - H]-. HPLC2: tR 17.28 min,
purity 97.3%.
2-(4-(Benzyloxy)phenyl)-1-(phenylsulfonyl)-1H-imidazole (3j).
1
Yield: 62.0%; mp 102-104 °C. H NMR (500 MHz, CDCl3)
δ 7.56 (d, J=1.0 Hz, 1 H), 7.46 (t, J=8.0 Hz, 1 H), 7.20-7.40
(m, 11 H), 7.03 (d, J=1.0 Hz, 1H), 6.89 (t, J=8.0 Hz, 2 H), 5.08
(s,2 H). MS (ESI): calculated for C22H18N2O3S, 390.1; found,
413.1 [M þ Na]þ. HPLC2: tR 18.22 min, purity 95.9%.
1-(Phenylsulfonyl)-2-(4-(trifluoromethyl)phenyl)-1H-imidazole
(3l). Yield: 36.7%. 1H NMR (CDCl3, 500 MHz) δ 7.75 (d, J =
2.0 Hz, 1 H), 7.69 (d, J=8.0 Hz, 2 H), 7.65 (t, J=8.0 Hz, 1 H),
7.60 (d, J=8.0 Hz, 2 H),, 7.48 (d, J=7.5 Hz, 2 H), 7.43 (t, J=8.0
Hz, 2 H), 7.22 (d, J=2.0 Hz, 1 H). MS (ESI) calcd for C16H11F3-
N2O2S, 352.1; found, 553.1 [M þ H]þ..
(1-(Phenylsulfonyl)-2-(3,4,5-trimethoxyphenyl)-1H-imidazol-
4-yl)(3,4,5-trimethoxyphenyl) Methanone (4ea). Yield: 21.1%;
mp 135-137 °C. 1H NMR (500 MHz, CDCl3) δ7.91(d, J=8.0Hz,
2 H), 7.65 (t, J=7.5 Hz, 1 H), 7.51 (t, J=8.0 Hz, 2 H), 7.44 (s,
1 H), 7.34 (s, 2 H), 6.60 (s, 2 H), 3.98 (s, 3 H), 3.96 (s, 6 H), 3.91 (s,
3 H), 3.73 (s, 6 H). MS (ESI): calculated for C28H28N2O9S, 568.2;
found, 569.2 [M þ H]þ. HPLC1: tR 17.86 min, purity 98.9%.
(4-Fluorophenyl)(1-(phenylsulfonyl)-2-(3,4,5-trimethoxyphenyl)-
1H-imidazol-4-yl) Methanone (4eb). Yield: 18.8%; mp 135-137 °C.
2-(4-Nitrophenyl)-1-(phenylsulfonyl)-1H-imidazole (3x). Yield:
50%; mp 145-147 °C. 1H NMR (500 MHz, DMSO) δ 8.28 (d,
J=8.5 Hz, 2 H), 8.03 (d, J=1.5 Hz, 1 H), 7.78 (t, J=7.5 Hz, 1 H),
7.64-7.68 (m, 4H), 7.60 (t, J=8.0 Hz, 2 H), 7.30 (d, J=1.5 Hz,
1 H). MS (ESI): calculated for C15H11N3O4S, 329.1; found, 352.0
[M þ Na]þ, 327.9 [M - H]-. HPLC2: tR 14.87 min, purity 98.8%.
2-(4-Nitrophenyl)-4,5-dihydro-1H-imidazole (7x). Yield: 70.3%.
1H NMR (500 MHz, CDCl3) δ 8.30 (d, J = 9.0 Hz, 2 H), 7.98

7424 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Chen et al.
1H NMR (500 MHz, CDCl3) δ 8.11 (q, J= 5.5 Hz, 3.0 Hz, 1 H),
8.00-8.03 (m, 1 H), 7.82 (d, J=7.5 Hz, 1 H), 7.78 (s, 1 H), 7.64 (t,
J=7.0 Hz, 1 H), 7.48 (t, J=8.0 Hz, 1 H), 7.42 (s, 1 H), 7.21-7.26
(m, 4 H), 6.62 (s, 1 H), 3.98 (s, 3 H), 3.96 (s, 6 H), 3.93 (s, 3 H). MS
(ESI):calculatedforC25H21FN2O6S, 496.1; found, 497.1 [M þ H]þ.
HPLC2: tR 15.26 min, purity 98.5%.
(1-(Phenylsulfonyl)-2-(4-(trifluoromethyl)phenyl)-1H-imidazol-
4-yl)(3,4,5-trimethoxyphenyl) Methanone (4la). Yield: 36.7%.
1H NMR (CDCl3, 500 MHz) δ 8.06 (d, J = 7.5 Hz, 2 H), 7.78
(t, J=8.0 Hz, 1 H), 7.72 (d, J=8.0 Hz, 2 H), 7.62 (d, J=8.0 Hz,
2 H),, 7.59 (d, J=8.0 Hz, 2 H), 7.50 (s, 1 H), 7.37 (s, 2 H), 4.04
(s, 3 H), 4.02 (s, 6 H). MS (ESI) calculated for C26H21F3N2O6S,
546.1; found, 547.1 [M þ H]þ.
(2-(4-Chlorophenyl)-1-(phenylsulfonyl)-1H-imidazol-4-yl)-
(4-fluorophenyl) Methanone (4fb). Yield: 36.8%; mp 153-
(2-Phenyl-1H-imidazol-4-yl)(3,4,5-trimethoxyphenyl) Methanone
(5aa). Yield: 10.1%; mp 227-229 °C. 1H NMR (500 MHz,
CDCl3) δ 8.0-8.03 (m, 2 H), 7.83 (s, 1 H), 7.34-7.38 (m, 3 H),
7.21 (s, 2 H), 3.90 (s, 3 H), 3.84 (s, 6 H). MS (ESI): calculated for
C19H18N2O, 338.1; found 337.1 [M - H]-. HPLC2: tR14.19 min,
purity 96.3%.
1
155 °C. H NMR (500 MHz, CDCl3) δ 8.06 (q, J = 5.5 Hz,
3.0 Hz, 2 H), 7.89 (d, J=7.5 Hz, 2 H), 7.68 (t, J=8.0 Hz, 1 H),
7.52 (t, J=8.0 Hz, 2 H), 7.34-7.38 (m, 5H), 7.23 (t, J=8.5 Hz,
2 H). MS (ESI): calculated for C22H14ClFN2O3S, 440.0; found,
463.0 [M þ Na]þ. HPLC2: tR 17.72 min, purity 97.4%.
(2-(4-(Dimethylamino)phenyl)-1-(phenylsulfonyl)-1H-imidazol-
4-yl)(3,4,5-trimethoxyphenyl) Methanone (4ga). Yield: 32.2%;
mp 157-159 °C. 1H NMR (500 MHz, CDCl3) δ 7.89 (d, J=8.0
Hz, 2 H), 7.62 (t, J=7.5 Hz, 1 H), 7.48 (t, J=8.0 Hz, 2 H), 7.43
(s, 1 H), 7.32 (d, J=8.5 Hz, 2 H), 7.30 (s, 2H), 6.62 (d, J=9.0 Hz,
2 H), 3.97 (s, 3 H), 3.95 (s, 6 H), 3.05 (s, 6 H). MS (ESI):
calculated for C27H27N3O6S, 521.2; found, 544.1 [M þ Na]þ,
519.8 [M - H]-. HPLC2: tR 16.00 min, purity 97.9%.
(4-Methoxyphenyl)(2-phenyl-1H-imidazol-4-yl) Methanone (5ab).
1
Yield: 16.6%; mp 179-181 °C. H NMR (500 MHz, CDCl3)
δ 11.1 (br, 1 H), 8.07-8.10 (m, 2 H), 8.04 (d, J = 8.5 Hz, 2 H),
7.84 (d, J=1.0 Hz, 1 H), 7.49-7.51 (m, 3 H), 7.07 (d, J=9.0 Hz,
2 H), 3.95 (s, 3 H). MS (ESI): calculated for C17H14N2O2, 278.1;
found, 279.0 [M þ H]þ. HPLC1: tR 15.14 min, purity >99%.
(3-Methoxyphenyl)(2-phenyl-1H-imidazol-4-yl) Methanone (5ac).
1
Yield: 22.5%; mp 160-162 °C. H NMR (500 MHz, CDCl3)
(2-(4-(Dimethylamino)phenyl)-1-(phenylsulfonyl)-1H-imidazol-
4-yl)(4-fluorophenyl) Methanone (4gb). Yield: 38.5%; mp 125-
127 °C. 1H NMR (500 MHz, CDCl3) δ 8.04 (q, J=5.5 Hz, 3.5
Hz, 2 H), 7.80 (d, J=7.5 Hz, 2 H), 7.61 (t, J=8.0 Hz, 1 H), 7.45
(t, J=8.0 Hz, 2 H), 7.39 (s, 1 H), 7.35 (d, J=9.0 Hz, 2 H), 7.21
(t, J=8.5 Hz, 2 H), 6.62 (d, J=9.0 Hz, 2 H), 3.05 (s, 6 H). MS
(ESI): calculated for C24H20FN3O3S, 449.1; found, 472.1 [M þ
Na]þ, 447.9 [M - H]-. HPLC2: tR 16.85 min, purity 96.5%.
(2-(3,4-Dimethoxyphenyl)-1-(phenylsulfonyl)-1H-imidazol-4-yl)-
(3,4,5-trimethoxyphenyl) Methanone (4ha). Yield: 28.6%; mp
136-138 °C. 1H NMR (300 MHz, CDCl3) δ 7.92 (dd, J=8.5 Hz,
1.5 Hz, 2 H), 7.66 (t, J=7.5 Hz, 2 H), 7.51 (t, J=7.5 Hz, 2 H),
7.43 (s, 1 H), 7.33 (s, 2 H), 7.02 (dd, J=8.0 Hz, 2.0 Hz, 1 H), 6.91
(d, J=2.0 Hz, 1 H), 6.86 (d, J=8.5 Hz, 1 H), 3.98 (s, 3 H), 3.96
(s, 9 H), 3.77 (s, 3 H). MS (ESI): calculated for C27H26N2O8S,
538.1; found, 561.1 [M þ Na]þ, 536.8 [M - H]-. HPLC2:
δ 11.2 (br, 1 H), 8.10-8.12 (m, 2 H), 7.87 (d, J=1.0 Hz, 1 H), 7.61
(d, J=7.5 Hz, 1 H), 7.48-7.52 (m, 5 H), 7.21 (dd, J=2.5 Hz, 8.5
Hz, 1 H), 3.91 (s, 3 H). MS (ESI): calculated for C17H14N2O2,
278.1; found, 279.0 [M þ H]þ. HPLC2:tR 15.07 min, purity >99%.
(3,5-Dimethoxyphenyl)(2-phenyl-1H-imidazol-4-yl) Methanone
(5ad). Yield: 26.2%; mp 168-170 °C. 1H NMR (500 MHz, CDCl3)
δ 8.04-8.06 (m, 2 H), 7.88 (s, 1 H), 7.50-7.52 (m, 3 H), 7.15 (d,
J=2.0 Hz, 2 H), 6.75 (t, J=1.0 Hz, 1 H), 3.89 (s, 6 H). MS (ESI):
calculated for C18H16N2O3, 308.1; found, 331.1 [M þ Na]þ,
306.9 [M - H]-. HPLC2: tR 15.59 min, purity >99%.
(3,4-Dimethoxyphenyl)(2-phenyl-1H-imidazol-4-yl) Methanone
(5ae). Yield: 18.6%; mp 162-164 °C. 1H NMR (500 MHz,
CDCl3) δ 10.9 (br, 1 H), 8.05 (dd, J=1.5 Hz, 8.0 Hz, 2 H), 7.86
(d, J=1.5 Hz, 1 H), 7.74 (dd, J=2.0 Hz, 8.5 Hz, 1 H), 7.56 (d,
J=2.0 Hz, 1 H), 7.50-7.52 (m, 3 H), 7.04 (d, J=8.5 Hz, 1 H),
4.03 (s, 3 H), 3.99 (s, 3 H). MS (ESI): calculated for C18H16N2O3,
308.1; found, 331.1 [M þ Na]þ, 306.9 [M - H]-. HPLC2:
t
R 14.67 min, purity 98.2%.
t
R 13.54 min, purity >99%.
(2-(3,4-Dimethoxyphenyl)-1-(phenylsulfonyl)-1H-imidazol-4-yl)-
(4-Fluorophenyl)(2-phenyl-1H-imidazol-4-yl) Methanone (5af).
(4-fluorophenyl) Methanone (4hb). Yield: 31.9%; mp 144-145 °C.
1H NMR (300 MHz, CDCl3) δ 8.09 (q, J=5.5 Hz, 3.5 Hz, 2 H),
7.81 (d, J=8.0 Hz, 2 H), 7.62 (t, J=7.5 Hz, 2 H), 7.48 (t, J=7.5
Hz, 2 H), 7.40 (s, 1 H), 7.21-7.25 (m, 2 H), 7.04 (dd, J=8.0 Hz, 2.0
Hz, 1 H), 6.92 (d, J=2.0 Hz, 1 H), 6.86 (d, J=8.5 Hz, 1 H), 3.96
(s, 3 H), 3.79 (s, 6 H). MS (ESI): calculated for C24H19FN2O5S,
466.1; found, 489.1 [M þ Na]þ, 464.8 [M - H]-. HPLC2: tR 15.52
min, purity 97.4%.
1
Yield: 30.2%; mp 231-233 °C. H NMR (500 MHz, CDCl3)
δ 10.6 (br, 1 H), 8.02-8.05 (m, 4 H), 7.81 (d, J = 1.0 Hz, 1 H),
7.51-7.54 (m, 3 H), 7.27 (t, J = 8.5 Hz, 2 H). MS (ESI):
calculated for C16H11FN2O, 266.1; found, 267.0 [M þ H]þ,
264.8 [M - H]-. HPLC1: tR 15.37 min, purity 98.9%.
(3-Fluorophenyl)(2-phenyl-1H-imidazol-4-yl) Methanone (5ag).
1
Yield: 23.4%; mp 212-214 °C. H NMR (500 MHz, CDCl3)
δ 8.05 (dd, J=1.5 Hz, 7.5 Hz, 2 H), 7.86 (s, 1 H), 7.84 (d, J=7.0
Hz, 1 H), 7.74 (d, J=8.5 Hz, 1 H), 7.52-7.58 (m, 4 H), 7.37 (dt,
J=2.0 Hz, 6.0 Hz, 1 H). MS (ESI): calculated for C16H11FN2O,
266.1; found, 267.0 [M þ H]þ, 264.8 [M - H]-. HPLC1: tR 15.29
min, purity >99%.
(1-(Phenylsulfonyl)-2-(2-(trifluoromethyl)phenyl)-1H-imidazol-
4-yl)(3,4,5-trimethoxyphenyl) Methanone (4ia). Yield: 25.0%;
1
mp 155-157 °C. H NMR (500 MHz, DMSO) δ 7.91 (d, J =
8.0 Hz, 1 H), 7.84 (q, J = 7.5 Hz, 5.0 Hz, 2 H), 7.77-7.80 (m,
2 H), 7.75 (s, 2 H), 7.66 (t, J=8.0 Hz, 2 H), 7.56 (d, J=7.5 Hz,
1 H), 7.18 (s, 2 H), 3.87 (s, 6 H), 3.81 (s, 3 H). MS (ESI): calculated
for C26H21F3N2O6S, 546.1; found, 569.0 [M þ Na]þ. HPLC2:
(2-Phenyl-1H-imidazol-4-yl)(p-tolyl) Methanone (5ah). Yield:
15.6%; mp 225-227 °C. 1H NMR (500 MHz, CDCl3) δ 11.1 (br,
1 H), 8.08 (d, J=7.5 Hz, 2 H), 7.93 (d, J=9.0 Hz, 2 H), 7.84 (s,
1 H), 7.48-7.52 (m, 3 H), 7.38 (d, J=10.0 Hz, 2 H), 2.50 (s, 3 H).
MS (ESI): calculated for C17H14N2O, 262.1; found, 263.0 [M þ
H]þ, 260.8 [M - H]-. HPLC2: tR 15.86 min, purity 98.7%.
(2-Phenyl-1H-imidazol-4-yl)(m-tolyl) Methanone (5ai). Yield:
20.5%; mp 168-169 °C. 1H NMR (500 MHz, CDCl3) δ 11.0 (br,
1 H), 8.09-8.11 (m, 2 H), 7.84 (d, J = 1.5 Hz, 1 H), 7.81-7.82
(m, 2 H), 7.47-7.52 (m, 5 H), 2.50 (s, 3 H). MS (ESI): calculated
for C17H14N2O, 262.1; found, 285.0 [M þ Na]þ, 260.8 [M - H]-.
HPLC2: tR 15.89 min, purity >99%.
(2-(4-Fluorophenyl)-1H-imidazol-4-yl)(3,4,5-trimethoxyphenyl)
Methanone (5ba). Yield: 12.2%; mp 176-178 °C. 1H NMR (500
MHz, CDCl3) δ 10.72 (br, 1 H), 8.02 (q, J=5.0 Hz, 2 H), 7.84
(s, 1 H), 7.19 (t, J=10.0 Hz, 2 H), 4.00 (s, 6 H), 3.97 (s, 3 H). MS
(ESI):calculatedforC19H17FN2O4, 356.1;found, 379.1[Mþ Na]þ,
354.9 [M - H]-. HPLC1: tR 17.23 min, purity >99%
t
R 16.16 min, purity 98.9%.
(1-(Phenylsulfonyl)-2-(2-(trifluoromethyl)phenyl)-1H-imidazol-
4-yl)(4-fluorophenyl) Methanone (4ib). Yield: 25.0%; mp 151-
153 °C. 1H NMR (500 MHz, CDCl3) δ 8.03 (q, J=5.5 Hz, 3.0
Hz, 2 H), 7.90 (d, J=8.0 Hz, 2 H), 7.80 (d, J=8.0 Hz, 1 H), 7.69
(q, J=7.0 Hz, 6.5 Hz, 2 H), 7.61 (t, J=8.0 Hz, 1 H), 7.52 (t, J=
8.0 Hz, 2 H), 7.34-7.36 (m, 2 H), 7.23 (t, J=8.5 Hz, 2 H). MS
(ESI): calculated for C23H14F4N2O3S, 474.1; found, 497.0 [M þ
Na]þ. HPLC2: tR 16.80 min, purity 98.2%.
(2-(4-(Benzyloxy)phenyl)-1-(phenylsulfonyl)-1H-imidazol-4-yl)-
(4-fluorophenyl) Methanone (4jb). Yield: 22.3.0%; mp 149-
1
151 °C. H NMR (500 MHz, CDCl3) δ 8.09 (q, J = 5.5 Hz,
3.5 Hz, 2 H), 7.82 (d, J=7.5 Hz, 2 H), 7.63 (t, 7.5 Hz, 1 H), 7.36-
7.50 (m, 10 H), 7.25 (t, J=8.5 Hz, 2 H), 6.98 (d, J=8.0 Hz, 2 H),
5.17 (s, 2 H). MS (ESI): calculated for C29H21FN2O4S, 512.1;
found, 535.0 [M þ Na]þ. HPLC2: tR 18.35 min, purity 95.1%.

Article
(2-(4-Methoxyphenyl)-1H-imidazol-4-yl)(3,4,5-trimethoxyphenyl)
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7425
(2-(3,4-Dimethoxyphenyl)-1H-imidazol-4-yl)(3,4,5-trimethoxy-
Methanone (5ca). Yield: 10.2%; mp 220-222 °C. 1H NMR (300
MHz, CDCl3) δ 10.24 (br, 1 H), 7.93 (d, J=14.5 Hz, 2 H), 7.81
(s, 1 H), 7.24 (s, 2 H), 7.03 (d, J=14.5 Hz, 2 H), 3.97 (s, 3 H), 3.95
(s, 6 H), 3.90 (s, 3 H). MS (ESI): calculated for C20H20N2O5,
368.1; found, 391.0 [M þ Na]þ, 367.0 [M - H]-. HPLC2: tR
14.46 min, purity 98.4%.
phenyl) Methanone (5ha). Yield: 85.0%; mp 100-102 °C. H
1
NMR (500 MHz, CDCl3) δ 10.19 (br, 1 H), 7.81 (s, 1 H), 7.58 (d,
J=1.5 Hz, 1 H), 7.48 (d, J=8.0 Hz, 1 H), 7.25 (s, 2 H), 6.97 (d,
J=8.5 Hz, 1 H), 4.00 (s, 3 H), 3.96 (s, 6 H), 3.95 (s, 6 H). MS (ESI):
calculated for C21H22N2O6, 398.2; found, 399.1 [M þ H]þ, 397.0
[M - H]-. HPLC2: tR 13.73 min, purity >99%.
(4-Fluorophenyl)(2-(4-methoxyphenyl)-1H-imidazol-4-yl)
Methanone (5cb). Yield: 15.2%; mp 245-247 °C. 1H NMR (500
MHz, CDCl3) δ 10.20 (br, 1 H), 7.93-7.96 (m, 2 H), 7.85 (d, J=
5.0 Hz, 2 H), 7.68 (s, 1 H), 7.15-7.17 (m, 2 H), 6.95 (d, J=6.0
Hz, 2 H), 3.82 (s, 3 H). MS (ESI): calculated for C17H13FN2O2,
296.1; found, 319.1 [M þ Na]þ, 294.9 [M - H]-. HPLC2:
tR 15.40 min, purity 98.8%.
(2-(3,4-Dimethoxyphenyl)-1H-imidazol-4-yl)(4-fluorophenyl)
Methanone (5hb). Yield: 78.3%; mp 174-176 °C. 1H NMR (500
MHz, CDCl3) δ 8.02 (t, J =9.0 Hz, 2 H), 7.75 (s, 1 H), 7.57 (s,
1 H), 7.48 (d, J=8.5 Hz, 1 H), 7.23 (t, J=8.5 Hz, 2 H), 6.95 (d,
J=8.5 Hz, 1 H), 3.99 (s, 3 H), 3.96 (s, 3 H). MS (ESI): calculated
for C18H15FN2O3, 326.1; found, 349.0 [M þ Na]þ, 324.9 [M -
H]-. HPLC2: tR 14.65 min, purity >99%.
(2-(p-Tolyl)-1H-imidazol-4-yl)(3,4,5-trimethoxyphenyl) Metha-
none (5da). Yield: 48.5%; mp 201-203 °C. 1H NMR (500 MHz,
CDCl3) δ 10.40 (br, 1 H), 7.88 (d, J=8.0 Hz, 2 H), 7.82 (s, 1 H),
7.31 (d, J = 8.0 Hz, 2 H), 7.24 (s, 2 H), 3.96 (s, 3 H), 3.94 (s,
6 H), 2.43 (s, 3 H). MS (ESI): calculated for C20H20N2O4,
352.1; found, 375.2 [M þ Na]þ. HPLC2: tR 15.45 min, purity
97.4%.
(2-(2-(Trifluoromethyl)phenyl)-1H-imidazol-4-yl)(3,4,5-tri-
methoxyphenyl) Methanone (5ia). Yield: 83.8%; mp 75-77 °C.
1H NMR(500MHz, CDCl3) δ10.37 (br, 1 H), 8.00-8.02 (m, 1 H),
7.87 (s, 1 H), 7.82-7.85 (m, 1 H), 7.69-7.74 (m, 1 H), 7.62-7.66
(m, 1 H), 7.25 (s, 2 H), 3.99 (s, 3 H), 3.98 (s, 6 H). MS (ESI):
calculated for C20H17F3N2O4, 406.1; found, 429.1 [M þ Na]þ,
405.0 [M - H]-. HPLC2: tR 13.98 min, purity >99%.
(4-Fluorophenyl)(2-(2-(trifluoromethyl)phenyl)-1H-imidazol-
4-yl) Methanone (5ib). Yield: 91.1%; mp 152-154 °C. 1H NMR
(500 MHz, CDCl3) δ 8.12-8.14 (m, 2 H), 7.97 (d, J = 7.5 Hz,
1 H), 7.82-7.85 (m, 2 H), 7.69 (t, J=7.5 Hz, 1 H), 7.61 (t, J=8.0
Hz, 1 H), 7.22 (t, J = 9.0 Hz, 2 H). MS (ESI): calculated for
C17H10F4N2O, 334.1; found, 357.1 [M þ Na]þ, 332.9 [M - H]-.
HPLC2: tR 15.10 min, purity >99%.
(4-Fluorophenyl)(2-(p-tolyl)-1H-imidazol-4-yl) Methanone (5db).
1
Yield: 56.3%; mp 229-231 °C. H NMR (500 MHz, CDCl3)
δ 10.50 (br, 1 H), 7.99-8.02 (m, 2 H), 7.88 (d, J=8.0 Hz, 2 H),
7.60 (d, J=1.0 Hz, 1 H), 7.30 (d, J=8.0 Hz, 2 H), 7.23 (t, J=9.0
Hz, 2 H), 2.43 (s, 3 H). MS (ESI): calculated for C17H13FN2O,
280.1; found, 281.0 [M þ H]þ, 278.9 [M - H]-. HPLC2: tR 16.31
min, purity >99%.
(3,4,5-Trimethoxyphenyl)(2-(3,4,5-trimethoxyphenyl)-1H-
imidazol-4-yl) Methanone (5ea). Yield: 86.8%; mp 196-198 °C. 1H
NMR (500 MHz, DMSO) δ 13.3 (br, 0.47 H), 13.50 (br, 0.52 H),
8.19 (s, 0.49 H), 7.90 (s, 1 H), 7.83 (s, 0.5 H), 7.59 (s, 1 H), 7.40 (s,
1 H), 7.18 (s, 1 H), 3.89 (s, 6 H), 3.86 (s, 6 H), 3.77 (s, 3 H), 3.72 (s,
3 H). MS (ESI): calculated for C22H24N2O7, 428.2; found, 451.1
[M þ Na]þ, 426.9 [M - H]-. HPLC2: tR 14.49 min, purity >99%.
(4-Fluorophenyl)(2-(3,4,5-trimethoxyphenyl)-1H-imidazol-4-yl)
Methanone (5eb). Yield: 90.2%; mp 153-155 °C. 1H NMR (500
MHz, CDCl3) δ 10.42 (br, 1 H), 8.00 (q, J=5.5 Hz, 3.0 Hz, 2 H),
7.76 (s, 1H), 7.23 (t, J=8.5 Hz, 2 H), 7.19 (s, 2 H), 3.94 (s, 3 H),
3.92 (s, 3 H). MS (ESI): calculated for C19H17FN2O4, 356.1;
found, 379.0 [M þ Na]þ, 354.9 [M - H]-. HPLC2: tR 15.31 min,
purity >99%.
(2-(4-(Benzyloxy)phenyl)-1H-imidazol-4-yl)(3,4,5-trimethoxy-
phenyl) Methanone (5ja). Yield: 16.5%; mp 191-193 °C. 1H
NMR (500 MHz, CDCl3) δ 10.22 (br, 1 H), 7.93 (d, J=9.0 Hz,
2 H), 7.81 (s, 1 H), 7.37-7.47 (m, 5 H), 7.24 (s, 2 H), 7.11 (d, J=
8.5 Hz, 2 H), 5.16 (s, 2 H), 3.97 (s, 3 H), 3.95 (s, 6 H). MS (ESI):
calculated for C26H24N2O5, 444.2; found, 467.1 [M þ Na]þ,
442.9 [M - H]-. HPLC2: tR 17.36 min, purity 95.5%.
(2-(4-(Benzyloxy)phenyl)-1H-imidazol-4-yl)(4-fluorophenyl)
Methanone (5jb). Yield: 84.7%; mp 212-214 °C. 1H NMR (300
MHz, CDCl3) δ 10.28 (br, 1 H), 799-8.04 (m, 2 H), 7.92-7.95
(m, 2 H), 7.76 (d, J = 1.5 Hz, 1 H), 7.38-7.48 (m, 5 H), 7.20-
7.25 (m, 2 H), 7.09-7.12 (m, 2 H), 5.16 (s, 2 H). MS (ESI):
calculated for C23H17FN2O2, 372.1; found, 395.1 [M þ Na]þ.
HPLC2: tR 17.97 min, purity 97.8%.
(2-(4-Chlorophenyl)-1H-imidazol-4-yl)(3,4,5-trimethoxyphenyl)
Methanone (5fa). Yield: 36.9%; mp 193-195 °C. 1H NMR (500
MHz, CDCl3) δ 10.75 (br, 1 H), 7.96 (d, J = 8.5 Hz, 2 H), 7.83
(s, 1 H), 7.47 (d, J=9.0 Hz, 2 H), 7.23 (s, 2 H), 3.97 (s, 3 H), 3.94
(s, 6 H), 2.43 (s, 3 H). MS (ESI): calculated for C19H17ClN2O4,
372.1; found, 395.1 [M þ Na]þ, 370.9 [M - H]-. HPLC2: tR 16.36
min, purity >99%.
(2-(4-Chlorophenyl)-1H-imidazol-4-yl)(4-fluorophenyl) Metha-
none (5fb). Yield: 83.7%; mp 232-234 °C. 1H NMR (500 MHz,
CDCl3) δ 10.78 (br, 1 H), 8.00 (q, J=5.5 Hz, 3.0 Hz, 2 H), 7.96
(d, J=9.0 Hz, 2 H), 7.78 (s, 1 H), 7.47 (d, J=8.0 Hz, 2 H), 7.24
(t, J = 8.5 Hz, 2 H). MS (ESI): calculated for C16H10ClFN2O,
300.1; found, 323.0 [M þ Na]þ, 298.8 [M - H]-. HPLC2:
tR 17.08 min, purity >99%.
(2-(4-Hydroxyphenyl)-1H-imidazol-4-yl)(3,4,5-trimethoxyphenyl)
Methanone (5ka). Yield: 72.3%. mp 191-193 °C. 1H NMR (500
MHz, CD3OD) δ8.31 (s, 1 H), 7.90 (d, J=8.5 Hz, 2 H), 7.31 (s, 2 H),
7.05 (s, 2 H), 3.95 (s, 6 H), 3.88 (s, 3 H). MS (ESI): calculated
for C19H18N2O5, 354.1; found, 355.1 [M þ H]þ, 352.9 [M - H]-.
HPLC2: tR 12.25 min, purity 98.7%.
(2-(4-(Hydroxyphenyl)-1H-imidazol-4-yl)(4-fluorophenyl)
Methanone (5kb). Yield: 89.0%; mp 276-278 °C. 1H NMR (500
MHz, CDCl3) δ 8.31 (s, 1 H), 8.13 (q, J=5.5 Hz, 3.0 Hz, 2 H),
7.93 (d, J=8.5 Hz, 2 H), 7.38 (t, J=8.5 Hz, 2 H), 7.07 (d, J=8.5
Hz, 2 H). MS (ESI): calculated for C16H11FN2O2, 282.1; found,
283.0 [M þ H]þ, 280.9 [M - H]-. HPLC2: tR 13.46 min, purity
97.7%.
(2-(4-(Trifluoromethyl)phenyl)-1H-imidazol-4-yl)(3,4,5-tri-
methoxyphenyl) Methanone (5la). Yield: 85.3%; mp 195-196 °C.
1H NMR (CDCl3, 500 MHz) δ 8.22 (d, J=8.5 Hz, 2 H), 7.96 (s,
1 H), 7.83 (d, J=8.5 Hz, 2 H), 7.34 (s, 2 H), 4.04 (s, 3 H), 4.00 (s,
6 H). MS (ESI) calcd for C20H17F3N2O4, 406.1; found, 407.1 [M þ
H]þ. HPLC2: tR 18.00 min, purity >99%.
(2-(4-(Dimethylamino)phenyl)-1H-imidazol-4-yl)(3,4,5-tri-
methoxyphenyl) Methanone (5ga). Yield: 91.2%; mp 195-197 °C.
1H NMR (500 MHz, CDCl3) δ 10.39 (br, 1 H), 7.87 (d, J=8.5 Hz,
2 H), 7.80 (s, 1 H), 7.23 (s, 2 H), 6.75(d, J=9.0 Hz, 2 H), 3.95 (s,
3 H), 3.94 (s, 6 H), 3.05 (s, 6 H). MS (ESI): calculated for C21H23-
N3O4, 381.2; found, 404.2 [M þ Na]þ, 380.0 [M - H]-. HPLC2:
(2-Phenyl-1H-imidazol-1-yl)(3,4,5-trimethoxyphenyl) Methanone
1
tR 15.20 min, purity 95.8%.
(5aaa). Yield: 39.8%; mp 113-115 °C. H NMR (500 MHz,
(2-(4-(Dimethylamino)phenyl)-1H-imidazol-4-yl)(4-fluorophenyl)
Methanone (5gb). Yield: 86.7%; mp 278-280 °C. 1H NMR (500
MHz, CDCl3) δ 10.21 (br, 1 H), 7.98 (q, J=5.0 Hz, 3.5 Hz, 2 H),
7.84 (d, J=8.5 Hz, 2 H), 7.72 (s, 1 H), 7.20 (t, J=8.5 Hz, 2 H),
6.76 (t, J=9.0 Hz, 2 H), 3.06 (s, 6 H). MS (ESI): calculated for
C18H16FN3O, 309.1; found, 332.1 [M þ Na]þ, 307.9 [M - H]-.
HPLC2: tR 16.06 min, purity 95.6%.
CDCl3) δ 7.53 (q, J=5.0 Hz, 3.0 Hz, 2 H), 7.41 (d, J=1.0 Hz,
1 H), 7.33-7.35 (m, 3 H), 7.23 (d, J=1.0 Hz, 1 H), 7.03 (s, 2 H),
3.93 (s, 3 H), 3.85 (s, 6 H). MS (ESI): calculated for C19H18N2O4,
338.1; found, 339.1 [M þ H]þ. HPLC2: tR 13.8 min, purity 95.6%.
(4-Methoxyphenyl)(2-phenyl-1H-imidazol-1-yl) Methanone
(5aba). Yield: 56.3%; mp 68-70 °C. 1H NMR (500 MHz, CDCl3)
δ 7.78(d, J=9.0 Hz, 2 H), 7.54-7.56(m, 2H), 7.32-7.34 (m, 4 H),

7426 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20
Chen et al.
7.21 (d, J=1.0 Hz, 1 H), 6.93 (d, J=8.5 Hz, 2 H), 3.90 (s, 3 H).
MS (ESI): calculated for C17H14N2O2, 278.1; found, 301.0 [M þ
Na]þ, 276.8 [M - H]-. HPLC2: tR 14.72 min, purity 95.7%.
(4-Fluorophenyl)(2-(p-tolyl)-1H-imidazol-4-yl) Methanone HCl
growth by 50% of untreated control) values were obtained by
nonlinear regression analysis using GraphPad Prism (GraphPad
Software, San Diego, CA).
Animals. Female C57/BL mice, age 4-6 weeks, were pur-
chased from Harlan Laboratories (Harlan Laboratories Inc.,
Indianapolis, IN). Our animal housing met the Association for
Assessment and Accreditation and Laboratory Animal Care
specifications. All of the procedures were conducted in accor-
dance with guidelines of our Institutional Animal Care and Use
Committee.
1
Salt (5db-HCl). Yield: 95%; mp 115-117 °C. H NMR (500
MHz, DMSO) δ 8.20-8.23 (m, 2 H), 8.18 (s, 1 H), 8.04 (d, J=
6.5 Hz, 2 H), 7.42 (t, J=8.0 Hz, 2 H), 7.37 (d, J=7.0 Hz, 2 H),
2.38 (s, 3 H). MS (ESI): calculated for C17H14FClN2O, 316.1;
found, 281.0 [M - HCl þ H]þ. HPLC2: tR 17.16 min, purity
>99%.
(3,4,5-Trihydroxyphenyl)(2-(3,4,5-trihydroxyphenyl)-1H-imidazol-
4-yl) Methanone (6ea). Yield: 66.1%; mp 294-296 °C. 1H NMR
(500 MHz, CD3OD) δ 8.07 (s, 1 H), 7.07 (s, 2 H), 7.02 (s, 2 H).
MS (ESI): calculated for C16H12N2O7, 344.1; found, 345.0 [M þ
H]þ, 342.9 [M - H]-. HPLC2: tR 3.62 min, purity 97.9%.
(2-(4-Chlorophenyl)-1H-imidazol-4-yl)(3,4,5-trihydroxyphenyl)
Methanone (6fa). Yield: 79.3%; mp>300 °C. 1H NMR (500 MHz,
CD3OD) δ 8.02 (d, J=8.5 Hz, 2 H), 7.77 (s, 1 H), 7.54 (d, J=8.5
Hz, 2 H), 7.14 (s, 2 H). MS (ESI): calculated for C16H11ClN2O4,
330.0; found, 331.1 [M þ Na]þ, 328.9 [M - H]-. HPLC2: tR 11.9
min, purity 95.6%.
(2-(3,4-Dihydroxyphenyl)-1H-imidazol-4-yl)(3,4,5-trihydroxy-
phenyl) Methanone (6ha). Yield: 62.2%; mp>300 °C. 1H NMR
(500 MHz, CD3OD) δ 8.11 (s, 1 H), 7.46 (d, J = 2.0 Hz, 1 H),
7.42 (dd, J=8.5 Hz, 2.0 Hz, 1 H), 7.10 (s, 2 H), 7.02 (d, J=8.5
Hz, 1 H). MS (ESI): calculated for C16H12N2O6, 328.1; found,
329.0 [M þ H]þ, 326.9 [M - H]-. HPLC2: tR 3.64 min, purity
97.9%.
Determination of Aqueous Solubility. The aqueous solubility
of selected compounds was estimated using a miniaturized
shake-flask method.32 Approximately 1 mg of each compound
was suspended in either 1 mL water or pH 7.0 buffer in a glass
vial and shaken at 450 rpm for 24 h at room temperature. The
resulting mixture was centrifuged at 21000g for 10 min, and the
concentration in the supernatant was measured by Acquity LC-
MS/MS consisting of triple quadrupole mass spectrometer
(Waters, Milford, MA) that was operated in positive ion mode
with electrospray ionization. Chromatographic separation of
the analytes was performed using a C6-phenyl column (50 mmꢀ
2.1 mm i.d, 3.5 μM) (Phenomenex, Torrance, CA) with guard
column, applying isocratic elution with water (10%) and ace-
tonitrile (90%). The flow rate was set to 0.5 mL/min.
In Vivo Evaluation of Efficacy. Mouse melanoma B16-F1
cells were prepared in FBS-free DMEM medium (Cellgro
Mediatech) at a concentration of 5 ꢀ 106 viable cells/mL. The
cell suspension (100 μL) was injected subcutaneously in the right
dorsal flank of each mouse. When tumor size reached about
100-150 mm3, about 7 days after cell inoculation, all mice
bearing tumors were divided into control and treatment groups
based on tumor size (n=5 per group). Each group had similar
average tumor size. Mice in control groups (negative control)
were injected intraperitoneally with 50 μL of vehicle solution
only or DTIC at 60 mg/kg (positive control) once daily.24
Tumor volume was measured every 2 days with a Traceable
electronic digital caliper (Fisher Scientific, Inc., Pittsburgh, PA)
and calculated using the formula a ꢀ b2 ꢀ 0.5, where a and b
represented the larger and smaller diameters, respectively.33
Tumor volume was expressed in cubic millimeters. Data were
expressed as mean ( SE for each group and plotted as a function
of time. Percentage tumor reduction at the conclusion of the
experiment (14 days after starting treatment) was calculated
with the formula 100-100ꢀ[(T - T0)/(C - C0)], where T repre-
sents mean tumor volume of a treated group on a specific day,
T0 represents mean tumor volume of the same group on the first
day of treatment, C represents mean tumor volume of a control
on a specific day, and C0 represents mean tumor volume of the
same group on the first day of treatment. Animal activity and
average body weight of each group were monitored during the
entire experiment period to assess compound toxicity. At the
end of treatment, all mice were euthanized by CO2 followed
by cervical dislocation, and tumors were harvested for further
studies.
In Vitro Microtubule Polymerization Assay. Bovine brain
tubulin (0.4 mg) (Cytoskeleton, Denver, CO) was mixed with
10 μM of the test compound and incubated in 110 μL of general
tubulin buffer (80 mM PIPES, 2.0 mM MgCl2, 0.5 mM EGTA,
and 1 mM GTP) at pH 6.9. The absorbance at 340 nm was
monitored every 1 min for 15 min by the SYNERGY 4 micro-
plate reader (Bio-Tek Instruments, Winooski, VT). The spectro-
photometer was set at 37 °C for tubulin polymerization.
Competitive Colchicine Binding Assay. Each test compound
was prepared at 20ꢀ concentration in G-PEM buffer (Cytoskeleton
Inc., Denver, CO) followed by pipetting 10 μL of test compound
into the 96-well plates. Ten μL of tritiated labeled colchicine
(Perkin-Elmer, Waltham, MA) was added to each testing well.
Subsequently, 180 μL of bead/tubulin (GE Healthcare Bio-
Sciences Corp., Piscataway, NJ) suspension was added into
each well. The plate was incubated for 45 min at 37 °C before
it was read by a Topcount NXT plate reader (Perkin-Elmer,
Waltham, MA). We included nonradiolabeled “cold” colchicine
as a positive control and paclitaxel as a negative control because
paclitaxel binds to a different site in tubulin and does not com-
pete for the colchicine site binding. Data were processed using
GraphPad Prism software.
Biology
Cell Culture and Cytotoxicity Assay. We examined the anti-
proliferative activity of the ABI compounds in three melanoma
cell lines (A375 and WM-164, human melanoma cell line;
B16-F1, mouse melanoma cell line) 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) except the PPC-1 cell line, which was
kindly provided by Dr. Mitchell Steiner at the University of
Tennessee Health Science Center. MDA-MB-435 and MDA-
MB-435/LCCMDR1 cells were kindly provided by Dr. Robert
Clarke at Georgetown University School of Medicine, Washington,
DC. 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) supple-
mented with 10% FBS (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 differ-
ent 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 treat-
ment 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
Molecular Modeling. All molecular modeling studies were
performed with Schrodinger Molecular Modeling Suite 2009
(Schrodinger LLC, New York, NY), running on a Dell Linux
workstation. Because the size of ABI compounds is much closer
to that of ABT-751, rather than DAMA-colcochine, we selected
tubulin complex with ABT-751 (PDB code: 3KHC) as our model-
ing system. ABIs were built and prepared using the Ligprep
module, and they were docked into the ABT-751 site using the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7427
Glide module in Schrodinger Suite. The best docking complexes
were subject to restricted molecular dynamics to release any
strains using Macromodel module with OPLS-2005 force field.
The ligand and its surrounding residues within 15 A were
˚
allowed to move freely, while residues outside the 15 A radius
were kept rigid.
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Acknowledgment. The project was supported by grant no.
1R15CA125623-01A2 from NIH/NCI. Its contents are solely
the responsibility of the authors and do not necessarily
represent the official views of the NIH. Additional support
comes from the Van Vleet Endowed Professorship (DDM),
Division of Pharmaceutics, The Ohio State University, and
GTx, Inc. Zhao Wang acknowledges the support from the
Alma & Hal Reagan Fellowship. We thank Drs. Vivian S.
Loveless and Christina Barrett for their help in the tubulin
colchicine site binding assay and Dr. Ryan Yates for provid-
ing access to an HPLC instrument. We also thank Dr. David
Armbruster, University of Tennessee Health Science Center
library, for his editorial assistance.
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Supporting Information Available: Dose-response curves in
Pgp-mediated multidrug resistant and their parental sensitive
melanoma cell lines for compounds 5cb, 4cb, and 4fb; competi-
tive binding at the colchicine site in tubulin for compound 5cb;
analytical and spectroscopic data for all final compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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