Synthesis and antiproliferative activity of novel 2-aryl-4-benzoyl- imidazole derivatives targeting tubulin polymerization

Article abstract of DOI:10.1016/j.bmc.2011.06.084

We previously reported the discovery of 2-aryl-4-benzoyl-imidazoles (ABI-I) as potent antiproliferative agents for melanoma. To further understand the structural requirements for the potency of ABI analogs, gain insight in the structure-activity relationships (SAR), and investigate metabolic stability for these compounds, we report extensive SAR studies on the ABI-I scaffold. Compared with the previous set of ABI-I analogs, the newly synthesized ABI-II analogs have lower potency in general, but some of the new analogs have comparable potency to the most active compounds in the previous set when tested in two melanoma and four prostate cancer cell lines. These SAR studies indicated that the antiproliferative activity was very sensitive to subtle changes in the ligand. Tested compounds 3ab and 8a are equally active against highly paclitaxel resistant cancer cell lines and their parental cell lines, indicating that drugs developed based on ABI-I analogs may have therapeutic advantages over paclitaxel in treating resistant tumors. Metabolic stability studies of compound 3ab revealed that N-methyl imidazole failed to extend stability as literature reported because de-methylation was found as the major metabolic pathway in rat and mouse liver microsomes. However, this sheds light on the possibility for many modifications on imidazole ring for further lead optimization since the modification on imidazole, such as compound 3ab, did not impact the potency.

Full text of DOI:10.1016/j.bmc.2011.06.084

Bioorganic & Medicinal Chemistry 19 (2011) 4782–4795
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antiproliferative activity of novel 2-aryl-4-benzoyl-imidazole
derivatives targeting tubulin polymerization
Jianjun Chen a, Chien-Ming Li b, Jin Wang a, Sunjoo Ahn b, Zhao Wang a, Yan Lu a, James T. Dalton b,
Duane D. Miller a,b, Wei Li a,
⇑
a Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, Memphis, TN 38163, USA
b GTx Inc., Memphis, TN 38163, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We previously reported the discovery of 2-aryl-4-benzoyl-imidazoles (ABI-I) as potent antiproliferative
agents for melanoma. To further understand the structural requirements for the potency of ABI analogs,
gain insight in the structure–activity relationships (SAR), and investigate metabolic stability for these
compounds, we report extensive SAR studies on the ABI-I scaffold. Compared with the previous set of
ABI-I analogs, the newly synthesized ABI-II analogs have lower potency in general, but some of the
new analogs have comparable potency to the most active compounds in the previous set when tested
in two melanoma and four prostate cancer cell lines. These SAR studies indicated that the antiprolifera-
tive activity was very sensitive to subtle changes in the ligand. Tested compounds 3ab and 8a are equally
active against highly paclitaxel resistant cancer cell lines and their parental cell lines, indicating that
drugs developed based on ABI-I analogs may have therapeutic advantages over paclitaxel in treating
resistant tumors. Metabolic stability studies of compound 3ab revealed that N-methyl imidazole failed
to extend stability as literature reported because de-methylation was found as the major metabolic path-
way in rat and mouse liver microsomes. However, this sheds light on the possibility for many modifica-
tions on imidazole ring for further lead optimization since the modification on imidazole, such as
compound 3ab, did not impact the potency.
Received 8 April 2011
Revised 24 June 2011
Accepted 28 June 2011
Available online 1 July 2011
Keywords:
2-Aryl-4-benzoyl-imidazoles
Cancer drug resistance
Melanoma
Prostate cancer
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
potent antiproliferative agents for melanoma and prostate can-
cer.10 Compared with existing tubulin-targeting agents such as
Cancer is the second leading cause of death in the world, ranked
after only heart disease.1 Of the many clinically useful anticancer
agents, drugs targeting tubulin polymerization (e.g., paclitaxel,
docetaxel, and vinblastine) have enjoyed wide success in treating
various types of cancers, including prostate cancer and mela-
noma.2–4 However, the prolonged use of those tubulin-targeting
agents often results in drug resistance in cancer cells, especially
P-glycoprotein (Pgp)-mediated multidrug resistance.5–7 The devel-
opment of multidrug resistance to tubulin inhibitors severely lim-
its their efficacy; therefore, novel tubulin inhibitors that can
effectively overcome drug resistance could significantly improve
their clinical efficacy.8,9
paclitaxel, colchicine, or vinblastine, the ABI-I compounds have
comparable in vitro and in vivo potency but can effectively circum-
vent Pgp-mediated drug resistance.10 ABI-I was designed using the
imidazole moiety as a biomimetic replacement of the central thia-
zole ring of 4-substituted methoxybenzoyl-aryl-thiazole (SMART)
(Fig. 1).10 Compared with SMART analogs,11 ABI-I compounds have
improved water solubility, pharmacokinetic (PK) properties, and
oral bioavailability without compromising activity.12 The activity
of ABI-I analogs depends strongly on the nature of the substitu-
tions on the aromatic A and C rings (Fig. 1). We also found that
the benzene sulfonyl protecting group on the N1-position of the
imidazole scaffold does not affect potency. To further understand
the structural requirements for the potency of ABI analogs, to gain
insight in the structure–activity relationships (SAR), and to investi-
gate metabolic stability for these compounds, we performed
additional SAR studies on the ABI-I scaffold.
We recently reported a series of 2-aryl-4-benzoyl-imidazole
(ABI-I) analogs targeting the colchicine binding site in tubulin as
Abbreviations: ABI, 2-aryl-4-benzoyl-imidazoles; BCRP, breast cancer resistance
protein; MDR, multidrug resistance; MRP, multidrug resistance-associated pro-
teins; Pgp, P-glycoprotein; SAR, structure–activity relationships; SMART, 4-substi-
tuted methoxybenzoyl-aryl-thiazole; TMS, tetramethylsilane.
In this paper, we report the synthesis and biological studies of
ABI-II analogs (Fig. 2). Specifically, we modified the structures of
the ABI-I by (1) converting the A ring of ABI-I from a phenyl ring
to an indole ring to understand the tolerability of the A ring
modification (ABI-II-1); (2) introducing various substituents on
⇑
Corresponding author. Tel.: +1 (901) 448 7532; fax: +1 (901) 448 6828.
E-mail address: wli@uthsc.edu (W. Li).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.06.084

J. Chen et al. / Bioorg. Med. Chem. 19 (2011) 4782–4795
4783
2. Chemistry
The synthesis of compounds 2(a, c) and 3(aa, ab, b) is summa-
rized in Scheme 1. Compound 1(a–c) was synthesized according to
our previously reported procedures.10 Treatment of 1(a, c) with
aluminum chloride provided the para-demethylated compound
2(a, c) with the 3,5-dimethoxy intact. Compound 3aa was prepared
by benzylation of the N-1 position of 1a, while methylation of the
N-1 position of 1a and 1b afforded compounds 3ab and 3b,
respectively.
Compounds 7b and 8a were synthesized following our estab-
lished method10 as outlined in Scheme 2. The substituted benzal-
dehyde compound 4(a, b) was converted to compound 5(a, b) in
the presence of ammonium hydroxide and glyoxal to construct
the imidazole scaffold. The imidazole ring of compound 5(a, b)
was protected by an appropriate phenylsulfonyl group followed
by coupling with substituted benzoyl chloride to achieve com-
pound 7(a, b). Treating compound 7a with tert-butylammonium
fluoride to remove the protecting group afforded compound 8a.
The synthesis of compounds 12(a–h) and 13(aa, ab, b), as well as
of 14(a, b), is described in Scheme 3. Protecting the indole or benz-
imidazole NH of compounds 9(a–h) with a phenylsulfonyl moiety
yielded intermediates 10(a–h), which were deprotonated by tert-
butyllithium followed by coupling with the 3,4,5-trimethoxy ben-
zoyl chloride to generate compounds 11(a–h). Removing the pro-
tecting group from 11(a–h) gave compound 12(a–h). Compounds
12a and 12b were methylated by methyliodide to obtain com-
pounds 13aa and 13b, respectively. Benzylating compound 12a
provided compound 13ab. The synthesis of compounds 15a and
15b was straightforward with a one-step reaction involved. The
commercial starting materials 14a and 14b were treated directly
by tert-butyllithium followed by benzoylation to achieve the de-
sired products 15a and 15b. Compound 15aa (see Table 1 infra
for structure) was separated as a side product from the synthesis
of compound 15a.
Figure 1. Structures of tubulin binders.
the N1-position of the imidazole ring to understand the effects of
different groups on activity (ABI-II-2); (3) demethylating the
para-methoxy group on the C ring to understand its effect on anti-
proliferative activity (ABI-II-3); (4) fusing the A and B rings to
understand the importance of relative geometry between them
(ABI-II-4), inspired by the highly active compounds (picomolar po-
tency, compound 15, Fig. 2) recently reported13 and by the same
analogs that also target tubulin with IC50 values in the nanomolar
range.14 We also conducted in vitro metabolic stability studies for
3ab (an ABI-II-2 analog), one of the best compounds in ABI-II, and
the result unveiled the vulnerable sites of ABI-II and provided
direction for future modifications on the imidazole ring to obtain
further optimized PK profiles.
The synthesis of compound 21b is outlined in Scheme 4. This
route was originally designed for the synthesis of 21a, but the
Figure 2. Design protocol for the ABI-II analogs (changes are indicated in blue).

4784
J. Chen et al. / Bioorg. Med. Chem. 19 (2011) 4782–4795
Scheme 1. Reagents and conditions: (a) AlCl3, THF, reflux; (b) NaH, CH3I for 3ab and 3b and BnBr for 3aa, THF, reflux.
Scheme 2. Reagents and conditions: (a) NH4OH, ethanol, glyoxal, rt; (b) NaH, substituted PhSO2Cl, THF, 0 °C–rt; (c) t-BuLi (1.7 M in pentane), substituted benzoyl chloride,
THF, À78 °C; (d) Bu4NF, rt.
Scheme 3. Reagents and conditions: (a) NaH, PhSO2Cl, THF, 0 °C–rt; (b) t-BuLi (1.7 M in pentane), benzoyl chloride, THF, À78 °C; (c) NaOH, ethanol, H2O, reflux; (d) NaH, CH3I
for 13aa and 13b or BnBr for 13ab, THF, reflux.
nonselectivity of the benzoylation at the indole-2 and imidazole-4
positions resulted in the formation of compound 21b, which is a
closely related but bulkier analog of 20a. Briefly, the indole-5-
carboxaldehyde compound 16 was protected by the phenylsulfo-
nyl group on the indole NH to afford intermediate 17.15 Compound
17 was reacted with glyoxal and ammonium hydroxide to generate
the 2-aryl-imidazole compound 18. Protecting the imidazole NH
with phenylsulfonyl gave the intermediate 19, which was coupled
with 3,4,5-trimethoxy benzoyl chloride to produce compound 20b.
Removing the protecting group from 20b provided compound 21b.
the average IC50 value obtained from all six cancer cell lines)
showed comparable activity with its chlorine counterpart com-
pound 1c (Fig. 3, 0.03 lM, all the compounds in Fig. 3 were previ-
ously reported as ABI-I analogs),10 indicating that a bromine on the
para-position of the A ring is well tolerated. Compound 21b (Table
1, 2.9 lM), with a 2-benzoylated indole as the A ring, showed mod-
erate activity, which is consistent with our observation in the pre-
vious set of ABIs that a bulky A ring reduces activity,10 suggesting
that the size of the A ring is critical for potency.
3.2. Effects of B ring modifications (ABI-II-2)
3. Antiproliferative activities of ABI-II against melanoma and
prostate cancer cells
In general, compounds with modifications on the N-1 position
in the B ring show decreased activity compared with their parental
The antiproliferative activity of these compounds was evaluated
in two human metastatic melanoma cell lines (A375 and WM164)
and four human prostate cancer cell lines (LNCaP, PC-3, Du 145,
and PPC-1) by using the methods described previously.10,16 Colchi-
cine was used as a positive control. The ability of these new analogs
to inhibit the growth of cancer cell lines is summarized in Table 1.
compounds (Table 1). Specifically, 3aa (Table 1, 0.94
90-fold decrease in potency when compared with its parent com-
pound 1a (Fig. 3, 0.01
M),10 indicating that the large benzyl group
was not well tolerated. 3ab (Table 1, 0.03 M) showed slightly de-
creased activity compared with the parent compound 1a (Fig. 3,
0.01
M),10 which may imply that a smaller group is optimal. How-
ever, compound 3b lost activity completely (Table 1, >10 M) com-
pared with its parent compound 1b (Fig. 3, 0.03
M),10 suggesting
lM) had a
l
l
l
l
3.1. Effects of A ring modifications (ABI-II-1)
l
that the binding mechanism of this compound is profoundly differ-
ent than that of its congener, compound 3aa. Compound 7b, with a
para-methoxy-phenylsulfonyl group on the N-1 position of the B
For compounds with A ring modification, compound 8a (Table
1, 0.04 lM, unless specified, the IC50 value for each compound is

Table 1
In vitro growth inhibitory effects of ABI-II compounds on melanoma and prostate cancer.
Structure
ID
R1
R2
R3
IC50 SEM (lM) (n = 4)
A375
WM164
LNCaP
PC-3
Du 145
PPC-1
R1
A
2a
2c
3aa
3ab
3b
4-Me
4-Cl
4-Me
H
H
Bn
Me
Me
3,5-(OMe)2,4-OH
3,5-(OMe)2,4-OH
3,4,5-(OMe)3
3,4,5-(OMe)3
4-F
0.11 0.02
0.68 0.15
1.06 0.22
0.03 0.01
>10
0.12 0.02
0.50 0.06
1.17 0.35
0.04 0.02
>10
0.13 0.04
0.55 0.01
0.68 0.02
0.03 0.01
>10
0.13 0.08
0.30 0.08
0.47 0.04
0.04 0.02
>10
0.17 0.07
1.00 0.21
1.50 0.52
0.03 0.01
>10
0.11 0.02
0.58 0.02
0.78 0.01
0.03 0.01
>10
4-Me
4-OMe
4-N(Me)2
4-Br
R2
N
N
B
7b
8a
(4-OMe)PhSO2
H
4-F
3,4,5-(OMe)3
0.10 0.02
0.03 0.01
0.10 0.01
0.04 0.01
0.07 0.02
0.03 0.01
0.04 0.01
0.04 0.01
0.13 0.01
0.05 0.02
0.06 0.01
0.03 0.01
O
C
R3
R1
NH
21b
3,4,5-(OMe)3-PhCO
3,4,5-(OMe)3-PhCO
1.80 0.35
1.70 0.20
1.07 0.15
2.63 0.58
5.92 0.66
4.58 0.63
HN
N
R3
R1
11b
11c
12a
12b
12c
12d
12e
12f
12h
13aa
13ab
13b
6-F
6-OMe
6-Cl
6-F
6-OMe
5-Cl
6-Me
5-F
5-OMe
6-Cl
6-Cl
PhSO2
PhSO2
H
H
H
H
H
H
NA
NA
>10
8.22 0.81
>10
>10
>10
1.47 0.37
>10
2.21 0.16
0.03 0.01
6.65 0.33
>10
>10
>10
>10
>10
>10
2.16 0.26
>10
2.26 0.45
0.04 0.01
8.08 1.21
>10
>10
>10
>10
>10
>10
>10
>10
8.63 0.59
0.05 0.02
1.04 0.25
>10
>10
2.66 0.53
>10
>10
>10
>10
>10
9.16 1.14
0.04 0.01
3.76 0.83
>10
>10
>10
>10
>10
>10
>10
>10
3.42 0.17
0.08 0.01
>10
>10
1.05 0.07
>10
>10
>10
>10
>10
1.00 0.02
0.05 0.01
4.33 0.72
>10
5
4
6
7
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
1N R2
3
2
O
H3CO
OCH3
H
OCH3
Me
Bn
Me
>10
>10
6-F
5.87 0.24
7.50 0.46
1.05 0.09
>10
8.30 1.32
12g
15a
15b
X = NH
X = S
X = O
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
N
X
O
H3CO
OCH3
OCH3
OH
S
S
N
N
15aa
NA
>10
>10
>10
>10
>10
>10
H3CO
OCH3
OCH3
Colchicine
0.02 0.01
00.03 0.02
0.02 0.01
0.01 0.01
00.01 0.01
0.02 0.01
NA—not applicable.

4786
J. Chen et al. / Bioorg. Med. Chem. 19 (2011) 4782–4795
Scheme 4. Reagents and conditions: (a) (1) KOH, ethanol; (2) PhSO2Cl, acetone; (b) NH4OH, glyoxal, ethanol, rt; (c) NaH, PhSO2Cl, THF, 0 °C–rt; (d) t-BuLi (1.7 M in pentane),
benzoyl chloride, THF, À78 °C; (e) NaOH, ethanol, H2O, reflux.
position 6. This is consistent with the literature in which the
5-substitued analogues demonstrated excellent activity,14 while
6-subsitituted compounds were generally inactive. Other ring
systems such as benzoxazole (15b), benzimidazole (12g), and ben-
zothiazole (15a, 15aa) led to inactive compounds (>10 lM).
3.5. Effects of 3ab (an ABI-II-2 compound) and 8a (an ABI-II-1
compound) on multidrug resistant cell lines
Figure 3. Antiproliferative activities of ABI-I analogs.10
Pgp-mediated drug efflux represents a major drug resistant
mechanism for tumor cells to evade the buildup of effective intra-
cellular drug concentrations. The clinical use of important tubulin-
targeting drugs, such as paclitaxel and vinblastine, suffers from
multidrug resistance, with Pgp efflux as the major mechanism.18–
ring, showed comparable activity (Table 1, 0.08
lM) to the parent
compound 7c (Fig. 3, 0.06
l
M),10 which has a phenylsulfonyl group
on the N-1 position of the B ring, indicating that electronic effect
provided by the substitution group on the N-1 position of the B
ring was not significant in the binding interaction with the target.
20
Tubulin polymerization inhibitors that can effectively circum-
vent drug resistance mechanisms could significantly improve
clinical efficacy. Previously, we showed that ABI-I analogs can
overcome the Pgp-mediated mechanism.10 We hypothesized that
ABI-II analogs derived from ABI-I retain this property and may
overcome additional paclitaxel resistance mechanisms. To test this
hypothesis, we selected the most active compounds, 3ab and 8a,
and tested their activity in two separate studies. In both studies,
we included paclitaxel for comparison.
In the first study, we tested the activity of 3ab and 8a in a
Pgp-overexpressed cancer cell line (MDA-MB-435/LCC6MDR1)
and its parental, nonresistant cell line (MDA-MB-435). MDA-MB-
435 was originally designated as a breast cancer cell line, but later
it was shown to have originated from the human M14 melanoma
cell line.21 This pair of cell lines has been well validated and widely
used to assess abilities of drugs overcoming Pgp-mediated multi-
drug resistance.22 As can be seen from Table 2, compounds 3ab
and 8a demonstrated better drug resistance indices (R = 0.8 and
0.9, respectively) than does paclitaxel (R = 29).
3.3. Effects of C ring modifications (ABI-II-3)
For the C ring modification, the para-hydroxy compound was
designed to test the pharmacologic activity of this potential metab-
olite, as the 3,4,5-trimethoxy was found to be labile to demethyla-
tion under both in vitro (in the presence of liver microsomes) and
in vivo conditions.17 Unfortunately, this modification led to a com-
pound with a 10- to 20-fold decrease in activity when compared
with 2a (0.13
lM), 2c (0.62
lM) (Table 1), and their parent com-
pounds 1a (Fig. 3, 0.01
lM) and 1c (Fig. 3, 0.03
l
M).10
3.4. Effects of fused A and B rings (ABI-II-4)
Surprisingly, for the compounds with fused A and B rings, most
were inactive (Table 1). Some of them showed moderate activity
with IC50 values in the micromolar range as exemplified by com-
pounds 13aa, 13b, 12d, and 12f (1–10
13aa (N-methylated-12a, 5.6 M), 13ab (N-benzylated-12a,
>10 M), and 12a (>10 M), 13aa was the best in this series
(6-Cl-indole analogues), which may suggest that the methyl group
on the indole N-1 position of 13aa is favorable for activity while a
benzyl group on the N-1 position of 13ab diminishes activity. This
finding was further confirmed by comparing 13b (methylated-12b,
l
M). When comparing
Having confirmed that ABI-II analogs retain the activity against
Pgp-mediated drug resistance, we performed a second experiment
to test whether these compounds could be effective against other
paclitaxel-resistance mechanisms. We obtained two pairs of
paclitaxel-resistant human prostate cancer cell lines (PC-3-TxR
and DU-145-TxR) and their sensitive parental cell lines from
Dr. Evan Keller’s lab at the University of Michigan.23 Subsequent
analyses indicated that, in addition to Pgp overexpression, more
than 200 genes are upregulated in PC-3-TxR and DU145-TxR cells,
which may represent additional paclitaxel drug resistance mecha-
nisms.23 As shown in Table 2, the drug resistant indices increased
substantially for paclitaxel in these cell lines (R = 437 and 1300 for
PC-3-TxR and DU145-TxR, respectively) compared with that in
MDA-MB-435/LCC6MDR1 (R = 29), suggesting that additional
paclitaxel drug resistant mechanisms may play a role. In contrast,
both 3ab and 8a (R ꢀ 1) retained their efficacy against paclitaxel-
resistant cancer cell lines. These results suggest that ABI analogs
l
l
l
1–10
on the indole ring, may have a more favorable binding mode over
its 6-F counterpart compound 12b (>10 M), which was further
verified by comparing 5-Cl compound 12d (1.4 and 2.1 M in
A375 and WM164, respectively) and its 6-Cl counterpart com-
pound 12a (>10 M) as well as the comparison between 5-OCH3
compound 12h (0.05 M) and its 6-OCH3 counterpart compound
12c (>10 M). The substantial differences in the activity of 5- and
lM) and 12b (>10 lM). Compound 12f (4.4 lM), with a 5-F
l
l
l
l
l
6-substituted compounds suggest that the substitution on position
5 may have a favorable binding interaction with tubulin over

J. Chen et al. / Bioorg. Med. Chem. 19 (2011) 4782–4795
4787
Table 2
Antiproliferative activity of ABI-II analogs on multidrug resistant cell lines
ID
IC50 SEM (nM) (n = 4)
MDA-MB-435
MDA-MB-435/LCC6MDR1
PC-3
PC-3-TxR
Du145
DU145-TxR
Resistance index (R)*
3ab
8a
Paclitaxel
83 22
86 11
67 13
79 20
465 53
21
18
1
3
19
16
188 22
1
4
53
49
2.3 0.1
1
3
52
54
>3000
3
3
0.8/0.9/1.0
0.9/0.9/1.1
29/437/1300
16
4
0.43 0.04
*
The three different numbers in each cell of this column represent the resistance indices of MDA-MB-435/LCC6MDR1, PC-3-TxR, and Du145-TxR, respectively; Resistance
indices (R) were calculated by dividing IC50 values on multidrug resistant cell lines MDA-MB-435/LCC6MDR1, PC-3-TxR, and DU145-TxR by IC50 values on the matching
sensitive parental cell line MDA-MB-435, PC-3, and Du145, respectively. The larger the R value, the more resistant the drug.
may overcome paclitaxel-resistance mechanisms, in addition to
the well known Pgp-mediated mechanism.
shorter half-life of 3ab in comparison with that of 1a in the pres-
ence of mouse and rat live microsomes (Fig. 4). Ketone reduction
was only detected in human liver microsomes, suggesting that pre-
clinical studies may not completely reflect human metabolism.
However, we did not exhaustively test for the presence of the
ketone reduced metabolite in liver microsomes from mice, rats
and dogs. These metabolic studies revealed the labile sites of the
ABI analogs in different species and can be used to direct further
modifications of the ABI scaffold to obtain novel compounds with
improved PK and drug-like properties.
Although paclitaxel demonstrated excellent activity in nonre-
sistant cancer cell lines (0.4–17 nM), it was significantly less potent
in the MDR cancer cell lines (>400 nM). In contrast, 3ab and 8a had
essentially equivalent potency on both resistant (50–80 nM) and
nonresistant cancer cell lines (50–80 nM). These results are consis-
tent with a recent report in which a structurally similar compound
with the same mechanism of action was effective against cancer
cells that overexpress Pgp, multidrug resistance-associated pro-
teins (MRP), and breast cancer resistance protein (BCRP).24 Collec-
tively, these studies indicate that 3ab and 8a could effectively
overcome the multidrug resistance and may provide therapeutic
advantages over paclitaxel.
5. Tubulin polymerization assay on ABI-II compounds
Based on their high structure similarity, we hypothesized that
ABI-II compounds exert their effect through the inhibition of tubu-
lin polymerization, similar to that of ABI-I compounds.10 We there-
fore performed experiments to test the inhibition of tubulin
polymerization by ABI-II analogs. Bovine brain tubulin (>97% pure)
was incubated with three potent ABI-II compounds, 3aa, 3ab, and
8a, along with colchicine as a positive control, at concentrations of
4. In vitro metabolic stability studies
4.1. Hepatic stability
In our previous study, we found that compound 1a, the parent
compound of 3ab, was highly potent in vitro (Fig. 3, 0.01 lM);
5, 10, 20 lM, respectively. All tested ABI-II compounds inhibit
compound 1a showed improved oral bioavailability, presumably
due to its increased aqueous solubility over the SMART com-
pounds.12 However, further improvement of its PK properties
was needed because of high clearance. Methylation of an imidazole
ring is one potential strategy that can increase oral bioavailability
without impacting potency;25 compound 3ab was designed in this
manner. Prior to in vivo PK studies, we performed in vitro meta-
bolic stability studies with 3ab and its parent compound, 1a.
Both 3ab and 1a were examined for metabolic stability in vitro
by using liver microsomes obtained from human, rat, mouse, and
dog (Fig. 4). Both of the compounds showed species differences
in their metabolic stability. Surprisingly, compound 1a was supe-
rior to 3ab with respect to metabolic stability. In human liver
microsomes, 3ab and 1a demonstrated similar half-lives, while in
the presence of rat and mice microsome, 3ab showed a shorter
half-life (5 and 10 min in mouse and rat, respectively) than did
1a (12 and 20 min in mouse and rat, respectively). In dog, com-
pound 3ab had a slightly longer half-life than that of compound
1a. The results indicated that the methyl group on the imidazole
ring is not beneficial for improving metabolic stability, especially
in mice and rats. Therefore, further in vivo pharmacokinetic studies
were not performed for 3ab.
tubulin polymerization in a dose-dependent manner (Fig. 6). Com-
plete inhibition of tubulin polymerization was observed after
15 min of treatment with 3ab (Fig. 6A) at 20
(Fig. 6D) at 20 M, or compound 8a at both 10 and 20
(Fig. 6B). In contrast, compound 3aa (Fig. 6C) inhibited only 40%
of tubulin polymerization at the highest concentration of 20 M.
These results are consistent with the relative antiproliferative po-
tency for compound 3aa (IC50 = 0.94 M) and those of 3ab
(IC50 = 0.03 M) and 8a (IC50 = 0.04 M).
lM, colchicine
l
lM
l
l
l
l
6. Molecular modeling studies on 3aa and 3ab
Since ABI-I compounds inhibit tubulin polymerization by
binding to the colchicine site in tubulin,10 based on the tubulin
polymerization studies described above and the high structural
similarity between ABI-I and ABI-II analogs, we believe that ABI-II
compounds also bind to the colchicine binding site in tubulin.
Therefore, we examined the potential binding modes of ABI-II
and their interactions with the receptor at the colchicine site in
the tubulin dimer by using Schrodinger 2011 molecular modeling
suite (Schrodinger, Inc., New York, NY). We selected the crystal
structure of TN16-tubulin complex for these studies because the
structure of TN-16 (Fig. 1) is the closest to the structure of 3ab.26
Results for docking of 3ab and 3aa illustrate the binding mode of
ABI-II analogs (Fig. 7). Figure 7A shows the overview of the com-
4.2. Metabolic pathway of compound 3ab
Liquid chromatography–tandem mass spectrometry was used
to identify the metabolites of 3ab after incubating with liver
microsomes from the four species (Fig. 5). Hydroxylation on the
para-methyl of the A ring was a major metabolic pathway of 3ab
in all species. This finding was consistent with our previous
study.12 O-Demethylation was a minor metabolic pathway, but it
was found in each species. In mouse and rat, N-demethylation
represented the major pathway that correlated well with the
pound binding in relation to the a/b tubulin dimer, and Figure 7B
shows the details of the binding pocket, which is framed by dotted
surface. The structures of 3ab (green tube model) and TN16 (red
wire model) overlap very well in the colchicine binding pocket.
The smaller 4-methylphenyl group (A ring) of 3ab is inserted
deeply into the b-subunit, while its larger end, the 3,4,5-trim-
ethoxybenzoyl group (C ring), extends toward the
a/b interface.

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J. Chen et al. / Bioorg. Med. Chem. 19 (2011) 4782–4795
Figure 4. In vitro metabolic profiles of 3ab and 1a.
Figure 5. In vitro metabolism of 3ab and 1a. H, M, R, and D indicate human, mouse, rat, and dog microsomes, respectively. Bold denotes major pathway.
Interestingly, when the B ring N-substituent switches from a
methyl moiety in 3ab to a benzyl group in 3aa, the corresponding
binding mode turns upside down. The bulky side of 3aa, which
consists of the B ring N-benzyl moiety and A ring 4-methylphenyl
group, is forced to occupy the wide upper location of the pocket.
Additionally, the C ring of 3aa shifts up from the narrow bottom
of the lower pocket region. As a result, the hydrophobic interac-
tions and likely formation of hydrogen bonds between the ligand
and residues of tubulin, such as TYR202 in S6, LEU242 in H7, and
MET259 in H8, may have been reduced greatly and thus lead to
the decreased in vitro activity from 3ab to 3aa.
It is noteworthy to mention that the best binding poses sug-
gested by molecular modeling for the colchicine binding site seem
to be strongly influenced by the choice of the starting ligand–tubu-
lin complex. We previously used the DAMA-colchicine in the tubu-
lin complex for studying potential binding pose of ABI-I analogs.10
When we used a different ligand–tubulin complex in which the li-
gand TN-16 (Fig. 1) is more similar to ABI analogs, we obtained the

J. Chen et al. / Bioorg. Med. Chem. 19 (2011) 4782–4795
4789
Figure 6. Effect of ABI-II compounds on tubulin polymerization in vitro. Tubulin (0.4 mg/assay) was exposed to 5, 10, 20
lM ABI-II compounds (vehicle control, 5% DMSO)
3ab (panel A), 8a (panel B), 3aa (panel C) or colchicine (positive control, panel D). Absorbance at 340 nm was monitored at 37 °C every minute for 15 min.
Figure 7. Binding modes of 3aa and 3ab in the colchicine binding site of tubulin.
alternative best binding poses described above. The major differ-
ence is that the overall ligand geometry is more linear and pene-
trates deeper into the binding pocket of the b-tubulin monomer
(Fig. 7). Because of the difficulty and complexity of tubulin crystal
structures, all existing tubulin complexes have very poor
resolution (RMS >3 Å). Both of the binding poses are theoretically
possible, but the true binding pose can be obtained only by solving
ABI-tubulin crystal structures in the future.
cancer agent. Therefore, the metabolic pathway of 3ab not only
revealed the labile sites of ABI analogs but also laid the foundation
for further modification of the ABI analogs toward a more drugga-
ble direction.
8. Experimental section
8.1. General
7. Conclusions
All reagents were purchased from Sigma–Aldrich Chemical Co.
(St. Louis, MO), 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 in an argon atmosphere. Routine thin-layer chromatography
(TLC) was performed on aluminum-backed Uniplates (Analtech,
Newark, DE). Melting points were measured with the Fisher-Johns
melting point apparatus (uncorrected). Nuclear magnetic reso-
nance spectra were obtained on a Varian Inova-500 spectrometer
(Santa Clara CA) or a Bruker AX 300 (Billerica, MA) spectrometer.
In summary, we synthesized ABI-II compounds and performed
extensive SAR studies. Although most of the ABI-II compounds
are less potent than are ABI-I compounds, they provide deep in-
sight into the pharmacophore of ABI analogues. The in vitro meta-
bolic studies on 3ab showed that N-methylation on the imidazole
ring is not beneficial for improving metabolic stability; however, a
better pharmacokinetic profile may be achieved upon appropriate
modification on the imidazole NH, thus providing a better opportu-
nity for the ABI analogs to be developed into a more drug-like anti-

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J. Chen et al. / Bioorg. Med. Chem. 19 (2011) 4782–4795
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 examined via re-
verse phase high performance liquid chromatography RP-HPLC
on a Waters 2695 HPLC system equipped with a photodiode array
detector (Milford, MA). Two RP-HPLC methods were conducted
NaHCO3 solution (aqueous) and extracted by ethyl acetate
(150 ml). The organic layer was dried over magnesium sulfate
and concentrated. The residue was purified by flash column chro-
matography (hexane/ethyl acetate, 4:1) or recrystallized from
water and methanol to give a white solid. Yield: 80%.
8.5. General procedure for the synthesis of 2-aryl-1-(phenyl-
sulfonyl)-1H-imidazole (6a–b, 19), 1-(phenylsulfonyl)-1H-
indole (10a–f, 10h), and 1-(phenylsulfonyl)-1H-benzo[d]-
imidazole (10g)
using
a
Supelco Ascentis™
5
l
M
RP-Amide column (250 Â
4.6 mm) from Sigma–Aldrich (St. Louis, Mo) at ambient tempera-
ture, and a flow rate of 0.7 mL/min. HPLC1: Gradient: Solvent A
(water) and Solvent B (methanol): 0–15 min 40–100% B (linear gra-
dient), 15–25 min 100% B. HPLC2: Gradient: Solvent A (water) and
To a solution of imidazoles (5a, 5b, 18), indoles (9a–f, 9h), or
benzimidazole (9g) (1.3–3.5 g, 10 mmol) in anhydrous THF
(200 ml) at 0 °C was added sodium hydride (60% dispersion in
mineral oil, 48 mg, 12 mmol) and stirred for 20 min. 4-Methoxy-
benzenesulfonyl chloride (2.5 g, 12 mmol) (for 6b) or benzenesul-
fonyl chloride (2.1 g, 12 mmol) (for others) was added, and the
reaction mixture was stirred overnight. After dilution by 200 ml
of saturated NaHCO3 solution (aqueous), the reaction mixture
was extracted by ethyl acetate (600 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: 40–95%.
Solvent
B (methanol): 0–5 min 10–40% B (linear gradient),
5–15 min 40–100% B, 15–30 min 100% B. UV detection at
254 nm. Purity of the compounds was established by careful inte-
gration of areas for all peaks detected and is reported for each com-
pound in Section 8.2.
8.2. General procedure for the synthesis of (2-aryl-1H-imidazol-
4-yl)methanone (2a, 2c, 3aa, 3ab, 3b) and (1-substituted-indol-
2-yl)(3,4,5-trimethoxyphenyl)methanone (13aa, 13ab, 13b)
8.2.1. Compounds 2a and 2c
To a solution of ABI-I (1a and 1c, 200–210 mg, 0.57 mmol) in
THF (20 mL) was added aluminum chloride (758 mg, 5.7 mmol).
The reaction mixture was stirred overnight. Water was added fol-
lowed by extraction with ethyl acetate (150 ml). The organic layer
was dried over magnesium sulfate and concentrated. The residue
was subjected to flash column chromatography (hexane/ethyl ace-
tate, 1:1) to give a white-yellowish solid. Yield: 60–80%.
8.6. General procedure for the synthesis of aryl (2-aryl-1-
(phenylsulfonyl)-1H-imidazol-4-yl)methanone (7a–b, 20b),
(1-(phenylsulfonyl)-1H-indol-2-yl)(3,4,5-trimethoxyphenyl)-
methanone (11a–f, 11h), (1-(phenylsulfonyl)-1H-benzo[d]-
imidazol-2-yl)(3,4,5-trimethoxyphenyl)methanone (11g),
benzo[d]thiazol-2-yl(3,4,5-trimethoxyphenyl)methanone
(15a), and benzo[d]oxazol-2-yl(3,4,5-trimethoxyphenyl)-
methanone (15b)
8.2.2. Compounds 3aa, 3ab, 3b, 13aa, 13ab, 13b
To a solution of ABI-I (1a and 1b) or (1H-indol-2-yl)(3,4,5-tri-
methoxyphenyl)methanone (12a, 12b) (100–150 mg, 0.4 mmol)
in THF (10 mL) in ice-bath was added sodium hydride (60% disper-
sion in mineral oil, 28 mg, 0.60 mmol) followed by adding methyl
iodide (85 mg, 0.60 mmol) (for 3ab, 3b, 13aa, 13b) or benzyl bro-
mide (102 mg, 0.60 mmol) (for 3aa, 13ab). The resulting reaction
mixture was stirred for 5 h 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 (hexane/ethyl ace-
tate, 2:1) to give a white solid. Yield: 50–98%.
To a solution of 2-aryl-1-(phenylsulfonyl)-1H-imidazole (6a–b,
19), 1-(phenylsulfonyl)-1H-indole (10a–f, 10h), or 1-(phenylsulfo-
nyl)-1H-benzo[d]imidazole (10g) or benzothiazole (14a), benzox-
azole (14b) (1.6–2.3 g, 5.0 mmol) in anhydrous THF (30 ml) at
À78 °C was added 1.7 M tert-butyllithium in pentane (3.5 mL,
6.0 mmol) and stirred for 10 min. 3,4,5-Trimethoxybenzoyl chlo-
ride (1.4 g, 6.0 mmol) was added at À78 °C and stirred overnight.
The reaction mixture was diluted with 100 ml of saturated NaHCO3
solution (aqueous) and extracted by ethyl acetate (300 ml). The or-
ganic layer was dried over magnesium sulfate and concentrated.
The residue was purified by flash column chromatography
(hexane/ethyl acetate, 3:1) to give a white solid. Yield: 5–45%.
8.3. General procedure the synthesis of 2-aryl-1H-imidazole
(18, 5a and 5b)
8.7. General procedure for the synthesis of (1H-benzo[d]-
imidazol-2-yl)(3,4,5-trimethoxyphenyl)methanone (12g),
(1H-indol-2-yl)(3,4,5-trimethoxyphenyl)methanone (12a–f,
12h), and (2-aryl-1H-imidazol-4-yl)methanone (21b)
To a solution of appropriate benzaldehyde (17, 4a, and 4b,
14–28 g, 100 mmol) in ethanol (400 ml) at 0 °C was added a solu-
tion of 40% oxalaldehyde in water (16 g, 110 mmol) and a solution
of 29% ammonium hydroxide in water (120 g, 1000 mmol). 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: 10–30%.
To a solution of (1-(phenylsulfonyl)-1H-indol-2-yl)(3,4,5-trime-
thoxyphenyl)methanone (11a–f, 11h), (1-(phenylsulfonyl)-1H-
benzo[d]imidazol-2-yl)(3,4,5-trimethoxyphenyl)methanone (11g),
or (1-(phenylsulfonyl)-2-(1-(phenylsulfonyl)-2-(3,4,5-trimethoxy-
benzoyl)-1H-indol-5-yl)-1H-imidazol-4-yl)(3,4,5-trimethoxyphen-
yl)methanone (20b) (450–850 mg, 1.0 mmol) in ethanol (20 ml)
was added sodium hydroxide (400 mg, 10 mmol) and stirred over-
night in darkness. The reaction mixture was diluted by 50 ml of
water and extracted by ethyl acetate (250 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: 30–95%.
8.4. General procedure for the synthesis of (2-aryl-1H-imidazol-
4-yl)methanone (8a)
To a solution of aryl (2-aryl-1-(phenylsulfonyl)-1H-imidazol-4-
yl) methanone (7a, 1.1 g, 2.0 mmol) in THF (25.0 ml) was added
1.0 M tetrabutyl ammonium fluoride (4 mL, 4.0 mmol) and stirred
overnight. The reaction mixture was diluted by 60 ml of saturated

J. Chen et al. / Bioorg. Med. Chem. 19 (2011) 4782–4795
4791
8.8. Synthesis of 1-(phenylsulfonyl)-1H-indole-3-carboxal-
dehyde (17)
6H). MS (ESI): calcd for C11H13N3, 187.1, found 187.9 [M+H]+,
185.8 [MÀH]À.
To
a
solution of indole-3-carboxaldehyde (16, 28.5 g,
8.16. 2-(4-Bromophenyl)-1-(phenylsulfonyl)-1H-imidazole (6a)
100 mmol) in ethanol (500 mL) at room temperature was added
potassium hydroxide (6.2 g, 110 mmol); the mixture was stirred
until total solubilization. The ethanol was completely removed in
a vacuum and acetone (250 mL) was added followed by benzene-
sulfonyl chloride (19.4 g, 110 mmol). The precipitate was filtered
off, and the filtrate was concentrated and recrystallized from meth-
anol to give a white solid. Yield: 40%.
Yield: 61.2%. 1H NMR (CDCl3, 500 MHz) d 7.71 (d, J = 2.0 Hz, 1H),
7.64 (t, J = 7.0 Hz, 1H), 7.57 (d, J = 9.0 Hz, 2H), 7.49 (d, J = 7.0 Hz,
2H), 7.45 (t, J = 9.0 Hz, 2H), 7.34 (d, J = 8.5 Hz, 2H), 7.18 (d,
J = 1.5 Hz, 1H). MS (ESI) calcd for C15H11BrN2O2S 362.0, found
363.0 [M+H]+.
8.17. 4-(1-((4-Methoxyphenyl)sulfonyl)-1H-imidazol-2-yl)-N,N-
dimethylaniline (6b)
8.9. (4-Hydroxy-3,5-dimethoxyphenyl)(2-(p-tolyl)-1H-imidazol-
4-yl)methanone (2a)
Yield: 61.5%. 1H NMR (500 MHz, CDCl3) d 7.58 (d, J = 1.5 Hz, 1H),
7.36 (t, J = 8.43 Hz, 4H), 7.03–7.09 (m, 1H), 6.80 (d, J = 9.0 Hz, 2H),
6.69 (d, J = 8.8 Hz, 2H), 3.84 (s, 3H), 3.05 (s, 6H). MS (ESI): calcd for
Yield: 56.8%; mp 220–222 °C. 1H NMR (CDCl3, 500 MHz) d 8.02
(d, J = 8.0 Hz, 2H), 7.91 (s, 1H), 7.39 (s, 2H), 7.28 (d, J = 7.5 Hz, 2H),
4.00 (s, 6H), 2.44 (s, 3H). MS (ESI) calcd for C19H18FN2O4 338.1,
found 339.1 [M+H]+. HPLC1: tR 3.91 min, purity 96.5%.
C
18H19N3O3S, 357.1, found 358.2 [M+H]+.
8.18. (2-(4-Bromophenyl)-1-(phenylsulfonyl)-1H-imidazol-4-
yl)(3,4,5-trimethoxyphenyl)methanone (7a)
8.10. (2-(4-Chlorophenyl)-1H-imidazol-4-yl)(4-hydroxy-3,5-
dimethoxyphenyl)methanone (2c)
Yield: 32.6% 1H NMR (CDCl3, 500 MHz) d 8.06 (d, J = 8.0 Hz, 2H),
7.88 (d, J = 8.5 Hz, 1H), 7.77 (t, J = 7.0 Hz, 1H), 7.54–7.63 (m, 4H),
7.31–7.36 (m, 4H), 4.04 (s, 3H), 4.01 (s, 6H). MS (ESI) calcd for
Yield: 80.2%; mp 216–218 °C. 1H NMR (CD3OD, 500 MHz) d 8.06
(d, J = 8.5 Hz, 2H), 7.99 (s, 1H), 7.61 (d, J = 8.0 Hz, 2H), 7.52 (s, 2H),
4.01 (s, 6H). MS (ESI) calcd for C18H15ClN2O4 358.1, found 359.1
[M+H]+. HPLC2: tR 4.12 min, purity >99%.
C
25H21BrN2O6S 556.0, found 557.0 [M+H]+.
8.19. (2-(4-(Dimethylamino)phenyl)-1-((4-methoxyphenyl)
sulfonyl)-1H-imidazol-4-yl)(4-fluorophenyl)methanone (7b)
8.11. (1-Benzyl-2-(p-tolyl)-1H-imidazol-4-yl)(3,4,5-trimethoxy-
phenyl)methanone (3aa)
Yield: 34.1%; mp 147–149 °C. 1H NMR (CDCl3, 500 MHz) d 8.07
(q, J = 8.5 Hz, 5.5 Hz, 2H), 7.78 (d, J = 9.0 Hz, 2H), 7.41 (d, J = 8.5 Hz,
2H), 7.39 (s, 1H), 7.23 (t, J = 8.5 Hz, 2H), 6.91 (d, J = 9.0 Hz, 2H), 6.68
(d, J = 9.0 Hz, 2H), 3.89 (s, 3H), 3.08 (s, 6H). MS (ESI) calcd for
Yield: 92.8%; mp 135–137 °C. 1H NMR (CDCl3, 500 MHz) d 7.81
(s, 1H), 7.80 (d, J = 6.5 Hz, 2H), 7.58 (d, J = 8.0 Hz, 2H), 7.41–7.45 (m,
3H), 7.31–7.33 (m, 2H), 7.20 (d, J = 7.0 Hz, 2H), 5.33 (s, 2H), 3.99 (s,
3H), 3.98 (s, 6H), 2.47 (s, 3H). MS (ESI) calcd for C27H26N2O4 442.2,
found 443.1 [M+H]+. HPLC1: tR 4.28 min, purity >99%.
C
25H22FN3O4S 479.1, found 502.0 [M+Na]+. HPLC2: tR 18.0 min,
purity >99%.
8.12. Methyl-2-(p-tolyl)-1H-imidazol-4-yl)(3,4,5-trimethoxy-
8.20. (2-(4-Bromophenyl)-1H-imidazol-4-yl)(3,4,5-trimeth-
phenyl)methanone (3ab)
oxyphenyl)methanone (8a)
Yield: 87.4%; mp 110–112 °C. 1H NMR (CDCl3, 500 MHz) d 7.87
(s, 2H), 7.86 (d, J = 8.0 Hz, 1H), 7.65 (d, J = 10 Hz, 2H), 7.37 (d,
J = 10 Hz, 2H), 4.01 (s, 6H), 4.00 (s, 3H), 3.90 (s, 3H), 2.53 (s, 3H).
MS (ESI) calcd for C21H22N2O4 366.2, found 367.2 [M+H]+. HPLC1:
tR 4.23 min, purity >99%.
Yield: 25.6%; mp 190–192 °C. 1H NMR (CDCl3, 500 MHz) d 7.99
(d, J = 8.5 Hz, 2H), 7.92 (s, 1H), 7.70 (d, J = 8.5 Hz, 2H), 7.32 (s, 2H),
4.03 (s, 3H), 4.00 (s, 6H). MS (ESI) calcd for C19H17BrN2O4 416.0,
found 417.0 [M+H]+. HPLC2: tR 4.24 min, purity 98.8%.
8.21. 6-Chloro-1-(phenylsulfonyl)-1H-indole (10a)
8.13. (4-Fluorophenyl)(2-(4-methoxyphenyl)-1-methyl-1H-imi-
dazol-4-yl)methanone (3b)
Yield: 82.3%. 1H NMR (500 MHz, CDCl3) d 8.09 (s, 1H), 7.95 (d,
J = 7.8 Hz, 2H), 7.59–7.66 (m, 2H), 7.46–7.58 (m, 3H), 7.24–7.30
(m, 1H), 6.70 (d, J = 3.7 Hz, 1H). MS (ESI) calcd for C14H10ClNO2S
291.0, found 314.0 [M+Na]+.
Yield: 90.2%; mp 148–150 °C. 1H NMR (CDCl3, 500 MHz) d 8.45
(q, J = 8.5 Hz, 5.5 Hz, 2H), 7.79 (s, 1H), 7.63 (d, J = 8.5 Hz, 2H), 7.16
(t, J = 8.5 Hz, 2H), 7.03 (d, J = 9.0 Hz, 2H), 3.89 (s, 3H), 3.82 (s, 3H).
MS (ESI) calcd for C18H15 FN2O2 310.1, found 311.0 [M+H]+. HPLC2:
tR 4.01 min, purity 97.6%.
8.22. 6-Fluoro-1-(phenylsulfonyl)-1H-indole (10b)
Yield: 92.5%. 1H NMR (500 MHz, CDCl3) d 7.95 (d, J = 7.8 Hz, 2H),
7.80 (dd, J = 2.0, 9.6 Hz, 1H), 7.58–7.66 (m, 2H), 7.47–7.57 (m, 3H),
7.06 (dt, J = 2.44 Hz, 8.9 Hz, 1H), 6.70 (d, J = 3.6 Hz, 1H). MS (ESI)
calcd for C14H10FNO2S 275.1, found 298.1 [M+Na]+.
8.14. 2-(4-Bromophenyl)-1H-imidazole (5a)
Yield: 19.5%. 1H NMR (CDCl3, 300 MHz) d 12.59 (s, 1H), 7.87 (d,
J = 8.1 Hz, 2H), 7.64 (d, J = 8.1 Hz, 2H), 7.27 (s, 1H), 7.04 (s, 1H). MS
(ESI) calcd for C9H7BrN2 222.0, found 222.8 [M+H]+.
8.23. 6-Methoxy-1-(phenylsulfonyl)-1H-indole (10c)
Yield: 93.8%. 1H NMR (500 MHz, CDCl3) d 7.93 (d, J = 9.0 Hz, 2H),
7.58–7.61 (m, 2H), 7.49–7.52 (m, 3H), 7.45 (d, J = 9.0 Hz, 1H), 6.93
(dd, J = 9.5 Hz, 2.0 Hz, 1H), 6.65 (d, J = 3.0 Hz, 1H), 3.94 (s, 3H). MS
(ESI) calcd for C15H13NO3S 287.1, found 310.0 [M+Na]+.
8.15. 4-(1H-Imidazol-2-yl)-N,N-dimethylaniline (5b)
Yield: 16.9%. 1H NMR (300 MHz, CDCl3) d 7.70 (dd, J = 7.0 Hz,
2.0 Hz, 2H), 7.10 (s, 2H), 6.75 (dd, J = 9.0 Hz, 2.0 Hz, 2H), 3.02 (s,

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J. Chen et al. / Bioorg. Med. Chem. 19 (2011) 4782–4795
8.24. 5-Chloro-1-(phenylsulfonyl)-1H-indole (10d)
8.32. (6-Chloro-1-(phenylsulfonyl)-1H-indol-2-yl)(3,4,5-tri-
methoxyphenyl)methanone (11d)
Yield: 96.3%. 1H NMR (500 MHz, CDCl3) d 7.99 (d, J = 9.0 Hz, 1H),
7.92 (d, J = 7.8 Hz, 2H), 7.68–7.59 (m, 2H), 7.56 (d, J = 1.7 Hz, 1H),
7.47–7.55 (m, 2H), 7.31–7.36 (m, 1H), 6.67 (d, J = 3.4 Hz, 1H).
MS (ESI) calcd for C14H10ClNO2S 291.0, found 314.0 [M+Na]+.
Yield: 39.6%. 1H NMR (500 MHz, CDCl3) d 8.08–8.20 (m, 3H),
7.65–7.72 (m, 1H), 7.56–7.65 (m, 3H), 7.45–7.53 (m, 1H), 7.28–
7.35 (m, 2H), 6.92 (s, 1H), 4.02 (s, 3H), 3.94 (s, 6H). MS (ESI) calcd
for C24H20ClNO6S 485.1, found 508.1 [M+Na]+.
8.25. 6-Methyl-1-(phenylsulfonyl)-1H-indole (10e)
8.33. (6-Methyl-1-(phenylsulfonyl)-1H-indol-2-yl)(3,4,5-tri-
Yield: 95.1%. 1H NMR (500 MHz, CDCl3) d 7.94 (d, J = 7.8 Hz, 2H),
7.88 (s, 1H), 7.57–7.63 (m, 1H), 7.56 (d, J = 3.7 Hz, 1H), 7.44–7.53
(m, 3H), 7.12 (d, J = 7.8 Hz, 1H), 6.68 (d, J = 3.7 Hz, 1H), 2.54 (s,
3H). MS (ESI) calcd for C15H13NO2S 271.1, found 294.0 [M+Na]+.
methoxyphenyl)methanone (11e)
Yield: 26.4%. 1H NMR (500 MHz, CDCl3) d 8.19 (d, J = 7.8 Hz, 2H),
8.01 (s, 1H), 7.65–7.70 (m, 1H), 7.48–7.63 (m, 3H), 7.33 (s, 2H), 7.21
(d, J = 8.1 Hz, 1H), 6.98 (s, 1H), 4.02 (s, 3H), 3.92–3.97 (m, 6H), 2.61
(s, 3H). MS (ESI) calcd for C25H23NO6S 465.1, found 488.0 [M+Na]+.
8.26. 5-Fluoro-1-(phenylsulfonyl)-1H-indole (10f)
8.34. (5-Fluoro-1-(phenylsulfonyl)-1H-indol-2-yl)(3,4,5-tri-
methoxyphenyl)methanone (11f)
Yield: 88.6%. 1H NMR (500 MHz, CDCl3) d 8.00 (dd, J = 4.4,
9.0 Hz, 1H), 7.92 (d, J = 7.6 Hz, 2H), 7.67 (d, J = 3.7 Hz, 1H), 7.62
(t, J = 7.4 Hz, 1H), 7.51 (t, J = 7.8 Hz, 2H), 7.24 (dd, J = 2.4, 8.8 Hz,
1H), 7.11 (dt, J = 2.4 Hz, 9.0 Hz, 1H), 6.69 (d, J = 3.7 Hz, 1H). MS
(ESI) calcd for C14H10FNO2S 275.1, found 298.1 [M+Na]+.
Yield: 41.5%. 1H NMR (500 MHz, CDCl3) d 8.15 (d, J = 7.8 Hz, 3H),
7.68 (t, J = 7.3 Hz, 1H), 7.59 (t, J = 7.7 Hz, 2H), 7.22–7.34 (m, 4H),
6.95 (s, 1H), 3.99–4.05 (m, 3H), 3.95 (s, 6H). MS (ESI) calcd for
C
24H20FNO6S 469.1, found 492.0 [M+Na]+.
8.27. 1-(Phenylsulfonyl)-1H-benzo[d]imidazole (10g)
8.35. (1-(Phenylsulfonyl)-1H-benzo[d]imidazol-2-yl)(3,4,5-tri-
methoxyphenyl)methanone (11g)
Yield: 99%. 1H NMR (500 MHz, CDCl3) d 8.46 (s, 1H), 8.07 (d,
J = 7.6 Hz, 2H), 7.94 (d, J = 8.3 Hz, 1H), 7.84 (d, J = 7.6 Hz, 1H),
7.70 (t, J = 7.6 Hz, 1H), 7.59 (t, J = 7.8 Hz, 2H), 7.40–7.51 (m, 2H).
MS (ESI) calcd for C13H10N2O2S 258.1, found 259.0 [M+H]+.
Yield: 15.2%. 1H NMR (500 MHz, CDCl3) d 8.36 (d, J = 7.6 Hz, 2H),
8.14 (d, J = 8.3 Hz, 1H), 8.07 (d, J = 7.8 Hz, 1H), 7.91 (d, J = 8.1 Hz,
1H), 7.50–7.80 (m, 5H), 7.09 (s, 1H), 4.03 (s, 3H), 3.96 (s, 6H). MS
(ESI) calcd for C23H20N2O6S 452.1, found 475.0 [M+Na]+.
8.28. 5-Methoxy-1-(phenylsulfonyl)-1H-indole (10h)
Yield: 92.6%. 1H NMR (500 MHz, CDCl3) d 7.84–7.90 (m, 3H),
7.51–7.53 (m, 2H), 7.43 (t, J = 8.0 Hz, 2H), 6.98 (d, J = 2.0 Hz, 1H),
6.93 (dd, J = 9.0 Hz, 2.5 Hz, 1H), 6.60 (d, J = 3.5 Hz, 1H), 3.82 (s,
3H). MS (ESI) calcd for C15H13NO3S 287.1, found 310.0 [M+Na]+.
8.36. (5-Methoxy-1-(phenylsulfonyl)-1H-indol-2-yl)(3,4,5-tri-
methoxyphenyl)methanone (11h)
Yield: 32.1%; 1H NMR (500 MHz, CDCl3) d 8.03–8.07 (m, 4H),
7.60 (t, J = 7.5 Hz, 1H), 7.51 (t, J = 7.5 Hz, 2H), 7.25 (s, 1H), 7.09
(dd, J = 9.0 Hz, 2.0 Hz, 1H), 7.00 (d, J = 2.5 Hz, 1H), 6.89 (s, 1H),
3.97 (s, 3H), 3.90 (s, 6H), 3.85 (s, 3H). MS (ESI) calcd for C25H23NO7S
481.1, found 504.1 [M+Na]+.
8.29. (6-Chloro-1-(phenylsulfonyl)-1H-indol-2-yl)(3,4,5-
trimethoxyphenyl)methanone (11a)
Yield: 34.7%. 1H NMR (500 MHz, CDCl3) d 8.17–8.25 (m, 3H),
7.69–7.75 (m, 1H), 7.60–7.66 (m, 2H), 7.58 (d, J = 8.3 Hz, 1H),
7.37 (dd, J = 1.5 Hz, 8.3 Hz, 1H), 7.32 (d, J = 9.3 Hz, 2H), 6.96 (s,
1H), 4.02 (s, 3H), 3.95 (s, 6H). MS (ESI) calcd for C24H20ClNO6S
485.1, found 508.0 [M+Na]+.
8.37. (6-Chloro-1H-indol-2-yl)(3,4,5-trimethoxyphenyl)meth-
anone (12a)
Yield: 76.8%; mp 207–209 °C. 1H NMR (CDCl3, 500 MHz) d 9.37
(s, 1H), 7.72 (d, J = 8.5 Hz, 1H), 7.56 (s, 1H), 7.32 (s, 2H), 7.21–7.23
(m, 2H), 4.03 (s, 3H), 4.02 (s, 6H). MS (ESI) calcd for C18H16ClNO4
345.1, found 368.0 [M+Na]+. HPLC1: tR 4.32 min, purity >99%.
8.30. (6-Fluoro-1-(phenylsulfonyl)-1H-indol-2-yl)(3,4,5-
trimethoxyphenyl)methanone (11b)
8.38. (6-Fluoro-1H-indol-2-yl)(3,4,5-trimethoxyphenyl)meth-
anone (12b)
Yield: 34.7%; mp 171–173 °C. 1H NMR (500 MHz, CDCl3) d 8.22
(d, J = 7.5 Hz, 2H), 7.93 (dd, J = 1.8 Hz, 9.8 Hz, 1H), 7.67–7.75 (m,
1H), 7.57–7.67 (m, 3H), 7.29–7.34 (s, 2H), 7.15 (dt, J = 2.20 Hz,
8.91 Hz, 1H), 6.98 (s, 1H), 4.02 (s, 3H), 3.95 (s, 6H). MS (ESI) calcd
for C24H20FNO6S 469.1, found 492.0 [M+Na]+. HPLC1: tR 4.01 min,
purity >99%.
Yield: 82.5%; mp 202–204 °C. 1H NMR (CDCl3, 500 MHz) d 9.32
(s, 1H), 7.74 (q, J = 5.0 Hz, 1H), 7.32 (s, 2H), 7.20–7.23 (m, 2H), 7.03
(dt, J = 9.0 Hz, 2.0 Hz, 1H), 4.03 (s, 3H), 4.02 (s, 6H). MS (ESI) calcd
for C18H16FNO4 329.1, found 352.0 [M+Na]+, 327.9 [MÀH]À. HPLC1:
tR 4.27 min, purity 98.7%.
8.31. (6-Methoxy-1-(phenylsulfonyl)-1H-indol-2-yl)(3,4,5-tri
methoxyphenyl)methanone (11c)
8.39. (6-Methoxy-1H-indol-2-yl)(3,4,5-trimethoxyphenyl)-
methanone (12c)
Yield: 26.4%; mp 156–158 °C. 1H NMR (500 MHz, CDCl3) d 8.18
(d, J = 7.8 Hz, 2H), 7.73 (s, 1H), 7.71–7.65 (m, 1H), 7.57–7.63 (m,
2H), 7.53 (d, J = 8.5 Hz, 1H), 7.32 (s, 2H), 7.01 (dd, J = 1.8 Hz,
8.7 Hz, 1H), 6.99 (s, 1H), 4.01 (s, J = 5.9 Hz, 6H), 3.95 (s, 6H). MS
(ESI) calcd for C25H23NO7S 481.1, found 504.1 [M+Na]+. HPLC1: tR
3.99 min, purity 98.3%.
Yield: 86.9%; mp 167–169 °C. 1H NMR (CDCl3, 500 MHz) d 9.06
(s, 1H), 7.52 (d, J = 9.0 Hz, 1H), 7.17 (s, 2H), 7.06 (s, 1H), 6.77–6.80
(m, 2H), 3.88 (s, 3H), 3.87 (s, 6H), 3.82 (s, 3H). MS (ESI) calcd for
C
19H19NO5 341.1, found 364.1 [M+Na]+. HPLC2: tR 4.21 min, purity
97.4%.

J. Chen et al. / Bioorg. Med. Chem. 19 (2011) 4782–4795
4793
8.40. (5-Chloro-1H-indol-2-yl)(3,4,5-trimethoxyphenyl)-
methanone (12d)
4.02 (s, 3H), 3.97 (s, 6H). MS (ESI) calcd for C19H18FNO4 343.1,
found 344.0 [M+H]+. HPLC2: tR 4.19 min, purity >99%.
Yield: 87.2%; mp 252–254 °C. 1H NMR (DMSO-d6, 500 MHz) d
12.20 (s, 1H), 7.83 (s, 1H), 7.56 (d, J = 8.5 Hz, 1H), 7.36 (d,
J = 8.5 Hz, 1H), 7.27–7.30 (m, 3H), 3.93 (s, 6H), 3.84 (s, 3H). MS
(ESI) calcd for C18H16ClNO4 345.1, found 368.1 [M+Na]+. HPLC1:
tR 17.3 min, purity >99%.
8.48. Benzo[d]thiazol-2-yl(3,4,5-trimethoxyphenyl)methanone
(15a)
Yield: 8.4%; mp 144–146 °C. 1H NMR (CDCl3, 500 MHz) d 8.29
(d, J = 8.0 Hz, 1H), 8.09 (d, J = 7.5 Hz, 1H), 8.03 (s, 2H), 7.63–7.67
(m, 2H), 4.06 (s, 6H), 4.04 (s, 3H). MS (ESI) calcd for C17H15N2O4S
329.1, found 352.1 [M+Na]+. HPLC1: tR 17.5 min, purity 95.2%.
8.41. (6-Methyl-1H-indol-2-yl)(3,4,5-trimethoxyphenyl)meth-
anone (12e)
8.49. Bis(benzo[d]thiazol-2-yl)(3,4,5-trimethoxyphenyl)-
methanol (15aa)
Yield: 90.1%; mp 179–181 °C. 1H NMR (CDCl3, 500 MHz) d 9.37
(s, 1H), 7.67 (d, J = 8.5 Hz, 1H), 7.32–7.33 (m, 3H), 7.20 (s, 1H), 7.08
(d, J = 8.5 Hz, 1H), 4.02 (s, 3H), 4.01 (s, 6H), 2.57 (s, 3H). MS (ESI)
calcd for
4.14 min, purity >99%.
Yield: 40.5%; mp 50–52 °C. 1H NMR (CDCl3, 500 MHz) d 8.10 (d,
J = 8.0 Hz, 2H), 7.95 (d, J = 8.0 Hz, 2H), 7.55 (t, J = 7.0 Hz, 2H), 7.46
(t, J = 7.5 Hz, 2H), 7.23 (s, 2H), 6.22 (s, 1H), 3.90 (s, 6H), 3.88 (s,
3H). MS (ESI) calcd for C24H20N2O4S2 464.1, found 487.1 [M+Na]+.
HPLC2: tR 3.99 min, purity 98.8%.
C
19H19NO4 345.1, found 368.0 [M+Na]+. HPLC1: tR
8.42. (5-Fluoro-1H-indol-2-yl)(3,4,5-trimethoxyphenyl)meth-
anone (12f)
8.50. Benzo[d]oxazol-2-yl(3,4,5-trimethoxyphenyl)meth-
anone (15b)
Yield: 69.8%; mp 196–198 °C. 1H NMR (CDCl3, 500 MHz) d 9.34
(s, 1H), 7.50 (q, J = 4.5 Hz, 1H), 7.43 (dd, J = 8.5 Hz, 2.0 Hz, 1H), 7.33
(s, 2H), 7.20–7.24 (m, 2H), 4.03 (s, 3H), 4.02 (s, 6H). MS (ESI) calcd
for C18H16FNO4 329.1, found 352.0 [M+Na]+, 327.9 [MÀH]À. HPLC1:
tR 4.10 min, purity >99%.
Yield: 22.5%; mp 89–91 °C. 1H NMR (CDCl3, 500 MHz) d 7.81–
7.83 (m, 1H), 7.48–7.58 (m, 3H), 7.32–7.43 (m, 2H), 4.06 (s, 3H),
4.01 (s, 6H). MS (ESI) calcd for C17H15NO5 313.1, found 336.1
[M+Na]+. HPLC2: tR 4.13 min, purity >97.2%.
8.43. (1H-Benzo[d]imidazol-2-yl)(3,4,5-trimethoxyphenyl)-
methanone (12g)
8.51. 1-(Phenylsulfonyl)-1H-indole-5-carbaldehyde (17)
Yield: 12.7%; mp 191–193 °C. 1H NMR (CDCl3, 500 MHz) d 10.43
(s, 1H), 8.20 (s, 2H), 7.99 (br, 1H), 7.68 (br, 1H), 7.49 (s, 2H), 4.07 (s,
6H), 4.04 (s, 3H). MS (ESI) calcd for C17H16N2O4 312.1, found 310.9
[MÀH]À. HPLC1: tR 4.30 min, purity >99%.
1H NMR (500 MHz, CDCl3) Yield: 32.6% d 10.17 (s, 1H), 8.25–
8.39 (m, 2H), 7.97–8.09 (m, 3H), 7.69 (t, J = 7.33 Hz, 1H), 7.59 (t,
J = 7.5 Hz, 2H), 7.39–7.54 (m, 2H). MS (ESI) calcd for C15H11NO3S
285.1, found 286.0 [M+H]+.
8.44. (5-Methoxy-1H-indol-2-yl)(3,4,5-trimethoxyphenyl)-
methanone (12h)
8.52. 5-(1H-Imidazol-2-yl)-1-(phenylsulfonyl)-1H-indole (18)
1H NMR (500 MHz, DMSO-d6) Yield: 12.0% d 8.33 (d, J = 2.9 Hz,
2H), 8.13 (d, J = 7.8 Hz, 2H), 7.98–8.04 (m, 1H), 7.62–7.67 (m,
1H), 7.55 (d, J = 7.82 Hz, 2H), 7.22–7.34 (m, 4H). MS (ESI) calcd
for C17H13N3O2S 323.1, found 324.0 [M+H]+.
Yield: 96.3%; mp 215–217 °C. 1H NMR (DMSO, 500 MHz) d
11.81 (s, 1H), 7.89 (d, J = 9.0 Hz, 1H), 7.22 (s, 2H), 7.15–7.16 (m,
2H), 6.97 (dd, J = 9.0 Hz, 2.5 Hz, 1H), 3.88 (s, 6H), 3.78 (s, 3H),
3.77 (s, 3H). MS (ESI) calcd for C19H19NO5 341.1, found 339.9
[MÀH]À. HPLC2: tR 4.18 min, purity >99%.
8.53. 1-(Phenylsulfonyl)-5-(1-(phenylsulfonyl)-1H-imidazol-
2-yl)-1H-indole (19)
8.45. (6-Chloro-1-methyl-1H-indol-2-yl)(3,4,5-trimethoxy-
phenyl)methanone (13aa)
Yield: 23.6%. 1H NMR (CDCl3, 500 MHz) d 8.01 (d, J = 8.5 Hz, 1H),
7.95 (d, J = 7.5 Hz, 2H), 7.73 (d, J = 1.0 Hz, 1H), 7.70 (d, J = 4.0 Hz,
1H), 7.63–7.66 (m, 2H), 7.52–7.56 (m, 3H), 7.31–7.34 (m, 3H),
7.22 (t, J = 8.5 Hz, 2H), 7.17 (s, 1H), 6.14 (d, J = 3.5 Hz, 1H). MS
(ESI) calcd for C23H17N3O4S2 463.1, found 464.0 [M+H]+.
Yield: 85.3%; mp 123–125 °C. 1H NMR (CDCl3, 500 MHz) d 7.67
(d, J = 8.0 Hz, 1H), 7.51 (s, 1H), 7.33 (s, 2H), 7.21 (d, J = 8.0 Hz, 1H),
7.08 (s, 1H), 4.12 (s, 3H), 4.02 (s, 3H), 3.98 (s, 6H). MS (ESI) calcd for
C
19H18ClNO4 359.1, found 382.0 [M+Na]+. HPLC2: tR 9.3 min, purity
98.6%.
8.54. (1-(Phenylsulfonyl)-2-(1-(phenylsulfonyl)-2-(3,4,5-
trimethoxybenzoyl)-1H-indol-5-yl)-1H-imidazol-4-yl)(3,4,5-
trimethoxyphenyl)methanone (20b)
8.46. (1-Benzyl-6-chloro-1H-indol-2-yl)(3,4,5-trimethoxy-
phenyl)methanone (13ab)
Yield: 73.4%; liquid. 1H NMR (CDCl3, 500 MHz) d 7.70 (d,
J = 8.0 Hz, 1H), 7.48 (s, 1H), 7.41–7.43 (m, 2H), 7.28–7.37 (m, 2H),
7.17–7.23 (m, 5H), 5.83 (s, 2H), 4.02 (s, 3H), 3.96 (s, 6H). MS
(ESI) calcd for C25H22ClNO4 435.1, found 458.0 [M+Na]+. HPLC1:
tR 10.2 min, purity >99%.
Yield: 15.9%. 1H NMR (500 MHz, CDCl3) d 8.18–8.25 (m, 3H),
8.04 (d, J = 8.1 Hz, 2H), 7.70–7.78 (m, 2H), 7.61–7.69 (m, 3H),
7.55 (t, J = 7.7 Hz, 3H), 7.50 (s, 1H), 7.38 (s, 2H), 7.34 (s, 2H), 6.94
(s, 1H), 3.99–4.06 (m, 12H), 3.94–3.99 (m, 6H). MS (ESI) calcd for
C
43H37N3O12S2 851.2, found 852.1 [M+H]+.
8.47. (6-Fluoro-1-methyl-1H-indol-2-yl)(3,4,5-trimethoxy-
phenyl)methanone (13b)
8.55. (5-(4-(3,4,5-Trimethoxybenzoyl)-1H-imidazol-2-yl)-1H-
indol-2-yl)(3,4,5-trimethoxyphenyl)methanone (21b)
Yield: 50.8%; mp 98–100 °C. 1H NMR (CDCl3, 500 MHz) d 7.69
(q, J = 8.5 Hz, 1H), 7.26 (s, 2H), 7.02–7.17 (m, 3H), 4.11 (s, 3H),
Yield: 45.9%; mp 239–241 °C. 1H NMR (CDCl3, 500 MHz) d 10.45
(s, 1H), 9.44 (s, 1H), 8.41 (s, 1H), 8.04 (d, J = 8.5 Hz, 1H), 7.86 (s, 1H),

4794
J. Chen et al. / Bioorg. Med. Chem. 19 (2011) 4782–4795
7.61 (d, J = 8.5 Hz, 1H), 7.26–7.29 (m, 5H), 3.99 (s, 3H), 3.95–3.97
(m, 15H). MS (ESI) calcd for C31H29N3O8 571.2, found 572.2
[M+H]+. HPLC2: tR 4.09 min, purity 96.3%.
8.59. Molecular modeling
All molecular modeling studies were performed with Schro-
dinger Molecular Modeling Suite 2010 (Schrodinger LLC, New York,
NY) running on a Dell Linux workstation. We selected tubulin com-
plex with TN16 (PDB code: 3HKD) as our modeling system because
of the structure similarity between ABI-II analogs and TN16. ABI-IIs
were built and prepared using the Ligprep module, and they were
docked into the TN16 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 Macromod-
el module with OPLS-2005 force field. The ligand and its surround-
ing residues within 15 Å were allowed to move freely, while
residues outside the 15 Å radius were kept rigid.
8.56. Cell culture and cytotoxicity assay
We examined the antiproliferative activity of the ABI-II com-
pounds in two melanoma cell lines (A375 and WM-164) and four
human prostate cancer cell lines (LNCaP, DU 145, PC-3, and
PPC-1). 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. The Pgp overexpressing multi-
drug resistant cell line MDA-MB-435/LCC6MDRI and matching
sensitive parent cell line were kindly provided by Dr. Robert Clarke
(Georgetown University, Washington, DC). Paclitaxel-resistant PC-
3-TxR, DU145-TxR, and their parental cell lines were gifts from Dr.
Evan Keller at the University of Michigan. Melanoma cells were
cultured in DMEM (Cellgro Mediatech, Inc., Herndon, VA), and
prostate cancer cells were cultured in RPMI 1640 (Cellgro Media-
tech, Inc.,) supplemented with 10% FBS (Cellgro Mediatech).
Cultures were maintained at 37 °C in a humidified atmosphere
containing 5% CO2. Cells (1000–5000) 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 mel-
anoma cells) or 96 h (slow growing prostate cancer cells) in three
to 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).
Acknowledgements
The project was supported by Grant Number 1R01CA148706-
01A1 from NIH/NCI. The contents are solely the responsibility of
the authors and do not necessarily represent the official views of
the NIH. Additional support came from GTx, Inc. We thank Dr. Ryan
Yates for providing access to an HPLC instrument. We also thank
Dr. David Armbruster, University of Tennessee Health Science Cen-
ter, and Dr. Michael L. Mohler from GTx, Inc., for editorial
assistance.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.06.084.
References and notes
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8.57. In vitro metabolic stability
Metabolic stability studies were conducted by incubating
0.5 lM of test compounds in a total reaction volume of 1 ml con-
taining 1 mg/mL microsomal protein in reaction buffer [0.2 M of
phosphate buffer solution (pH 7.4), 1.3 mM NADP+, 3.3 mM glu-
cose-6-phosphate, and 0.4 U/mL glucose-6-phosphate dehydroge-
nase] at 37 °C in a shaking water bath. The compound 3ab, at
50 lM concentration, with the above mentioned conditions, was
used for metabolite identification studies. The NADPH regenerating
system (solutions A and B) was obtained from BD Biosciences
(Bedford, MA). The total DMSO concentration in the reaction solu-
tion was approximately 0.5% (v/v). Aliquots (100
tion mixtures used to determine metabolic stability were sampled at
5, 10, 20, 30, 60, and90 min. Acetonitrile(150 L)containing200 nM
of the internal standard was added to quench the reaction and to
precipitate the proteins. Samples were then centrifuged at 4000g
for 30 min at room temperature, and the supernatant was analyzed
directly by liquid chromatography–tandem mass spectrography.
lL) from the reac-
l
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8.58. In vitro microtubule polymerization assay
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mixed with 5, 10, 20
sitive control) and incubated in 110
l
M of the test compounds or colchicine (po-
L of general tubulin buffer
l
(80 mM PIPES, 2.0 mM MgCl2, 0.5 mMEGTA, 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 microplate reader (Bio-Tek Instru-
ments, Winooski, VT). The spectrophotometer was set at 37 °C
for tubulin polymerization.

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