Discovery of novel 2-aryl-4-benzoyl-imidazole (ABI-III) analogues targeting tubulin polymerization as antiproliferative agents

Article abstract of DOI:10.1021/jm300564b

Novel ABI-III compounds were designed and synthesized based on our previously reported ABI-I and ABI-II analogues. ABI-III compounds are highly potent against a panel of melanoma and prostate cancer cell lines, with the best compound having an average IC<

Full text of DOI:10.1021/jm300564b

Brief Article
pubs.acs.org/jmc
Discovery of Novel 2‑Aryl-4-benzoyl-imidazole (ABI-III) Analogues
Targeting Tubulin Polymerization As Antiproliferative Agents
Jianjun Chen,† Sunjoo Ahn,‡ Jin Wang,† Yan Lu,† James T. Dalton,‡ Duane D. Miller,†,‡ and Wei Li*,†
†Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, Memphis, Tennessee
38163, United States
‡GTx Inc., Memphis, Tennessee 38163, United States
S
* Supporting Information
ABSTRACT: Novel ABI-III compounds were designed and synthesized based on our previously reported ABI-I and ABI-II
analogues. ABI-III compounds are highly potent against a panel of melanoma and prostate cancer cell lines, with the best
compound having an average IC50 value of 3.8 nM. They are not substrate of Pgp and thus may effectively overcome Pgp-
mediated multidrug resistance. ABI-III analogues maintain their mechanisms of action by inhibition of tubulin polymerization.
compounds with substitution on the position-6 of the indole
ring, while compounds with substitution on position-5 showed
moderate activity.
INTRODUCTION
■
Because of its critical role in mitosis and cell division, tubulin
polymerization represents an excellent cancer drug target, with
several clinical drugs developed (e.g., paclitaxel, docetaxe,l and
vinblastine).1,2 However, one major problem for current
tubulin inhibitors is the development of drug resistance in
cancer patients. P-glycoprotein (Pgp)-mediated multidrug
resistance is one major reason for failure of treatment by
paclitaxel and vinblastine.3,4 Accordingly, the search for novel
tubulin inhibitors that can overcome multidrug resistance has
intensified in recent years; and as a result, a number of active
compounds have been identified.5−13 Our search has focused
on the discovery and optimization of novel tubulin inhibitors
with high potency and acceptable pharmacologic and
pharmacokinetic properties.9,10,12−16
Since indole is a very important building block for many
biological active agents including tubulin inhibitors,17−19 after
analyzing the structure−activity relationships (SAR) of ABI-I
and ABI-II analogues, we designed and attempted to synthesize
compound 21a (Figure 1) with an unsubstituted indole as the
A ring. However, because of the lack of selectivity with the
benzoylation reaction, we only obtained 21b with an extra
3′,4′,5′-trimethoxyphenyl on the indole 2-position.13 This
compound showed only moderate activity, probably due to
the extra bulkiness from the 3′,4′,5′-trimethoxyphenyl group in
the indole 2-position, which may not fit into the binding pocket
of tubulin. Our continued efforts resulted in the discovery of
compound 6a with a 3-indole as the A ring (Figure 1). We
found that 6a demonstrated significantly improved potency
with an average IC50 value of 3.8 nM in five tested cancer cell
lines. Encouraged by this discovery, we performed a focused
SAR study on the indole ring (Figure 1, ABI-III) by (a) varying
the X in the indole A-ring (X = NH, O, S )or (b) retaining the
indole nitrogen but introducing a methyl or a bulkier
phenylsulfonyl group on the indole nitrogen atom. In this
article, we report the synthesis and biological testing for these
highly potent compounds on both resistant and parental cancer
cell lines. We confirmed that the mechanism of action of these
novel compounds is through the inhibition of tubulin
polymerization. In addition, we performed in vitro assays of
Pgp function to further demonstrate that these new analogues
are not a substrate of Pgp and thus can effectively overcome
Pgp-mediated multidrug resistance that are common to several
existing antimitotic drugs.
We previously reported the discovery of ABI-I/II (2-aryl-4-
benzoyl-imidazole, Figure 1) analogues with potent antiproli-
Figure 1. Structures of ABI-I/II/III.
ferative activity against a panel of melanoma and prostate
cancer cell lines.12,13 We modified the A and B rings of these
analogues by introducing different functional groups to explore
their effects on the activity.13 The A ring modification with a
bulky 2-(3′,4′,5′-trimethoxyphneyl)-indole ring (Figure 1, 21b)
displacing a phenyl ring led to significantly decreased activity;
B-ring modification by methylation or benzylation on the
imidazole NH resulted in similar or decreased activity for N-
methylated and N-benzylated compounds, respectively. Fused
A/B ring (Figure 1, ABI-II) resulted in much lower activity for
RESULTS AND DISCUSSION
■
Chemistry. The synthesis of compound 6a−c is outlined in
Scheme 1. Briefly, the indole-3-carboxaldehyde compound 1
Received: April 24, 2012
© XXXX American Chemical Society
A
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Journal of Medicinal Chemistry
Brief Article
Scheme 1. Synthesis of 6a−c
Reagents and conditions: (a) 1. KOH, ethanol; 2. PhSO2CI, acetone, RT; (b) NH4OH, glyoxal, ethanol, RT; (c) NaH, PhSO2CI, THF, 0 °C to RT;
(d) t-BuLi (1.7 M in pentane), 3,4,5-trimethoxybenzoyl chloride, THF, −78 °C; (e) NaOH, ethanol, H2O, reflux; (f) TBAF, THF, RT; (g) NaH,
CH3I, THF.
was protected by the phenylsulfonyl group on the indole NH to
afford N-protected indole-3-carboxaldehyde 2. Compound 2
was reacted with glyoxal and ammonium hydroxide to generate
the 2-aryl-1H-imidazole compound 3. Protection of the
imidazole NH on compound 3 with phenylsulfonyl provided
the intermediate 4, which was coupled with 3,4,5-trimethoxy
benzoyl chloride in the presence of tert-butyl lithium to
produce compound 5. The benzoylation occurred in imidazole
position-4 because of less steric hindrance in the two tautomers
as confirmed by 2D NMR studies (Supporting Information
S8−15). Removal of the protecting group from compound 5 by
sodium hydroxide or by tert-butylammonium fluoride (TBAF)
yielded compound 6a and 6b, respectively. Compound 6a was
further methylated by methyl iodide to produce the
dimethylated compound 6c.
trimethoxy benzoyl chloride followed by treating with tert-
butylammonium fluoride to remove the protecting group.
Antiproliferative Activities of ABI-III in Melanoma and
Prostate Cancer Cells. The antiproliferative activity of these
compounds was evaluated in two human metastatic melanoma
cell lines (A375 and WM164) and three human prostate cancer
cell lines (LNCaP, PC-3, and Du 145) using the methods
described previously.12,20 Colchicine was used as a positive
control. The ability of these new analogues to inhibit the
growth of cancer cell lines is summarized in Table 1. The 3-
indole compound 6a showed high activity (3.8 nM; unless
specified, the IC50 value for each compound is the average of all
five cancer cell lines), identifying this compound as the most
potent compound in the ABI family (I/II/III) to date. The
bulky phenylsulfonyl group in the 3-indole ring (6b) resulted in
50-fold lower activity (196.2 nM), suggesting the size of the
substituent on the indole NH may play an important role in
determining activity. Consistent with this hypothesis, the
dimethylated 3-indole compound 6c showed very good
potency (39.8 nM), indicating that a smaller group on either
the imidazole- or indole-NH was well tolerated. The
benzofuran compound 6d and benzothiophene compound 6e
showed relatively strong activity (41.2 nM, 35.0 nM for 6d and
6e, respectively), although it is about 10-fold less active than
the 3-indole compound 6a, implying that the replacement of
the 3-indole NH with either an oxygen or a sulfur does not
provide any beneficial effect on the activity. Resistance indices
(RI) were calculated by dividing IC50 values on paclitaxel
resistant cell lines PC-3/TxR by IC50 values on the matching
sensitive parental cell line PC-3. The larger the RI value, the
more resistant the drug.
Compounds 6d and 6e were synthesized following the route
outlined in Scheme 2. The benzofuran (8d) or benzothiophene
Scheme 2. Synthesis of 6d and 6e
Reagents and conditions: (a) NH4OH, glyoxal, ethanol, RT; (b) NaH,
PhSO2CI, THF, 0 °C to RT; (c) t-BuLi (1.7 M in pentane), 3,4,5-
trimethoxybenzoyl chloride, THF, −78 °C; (d) TBAF, THF, RT.
compound (8e) was converted to compound 9(d,e) in the
presence of ammonium hydroxide and glyoxal to construct the
imidazole scaffold. The imidazole ring of compound 9(d,e) was
protected by a phenylsulfonyl group to achieve N-protected
compound 10(d,e). Finally, compound 6(d,e) was generated
through a one-pot reaction by coupling 10(d,e) with 3,4,5-
Effects of ABI-III Compound on Paclitaxel Resistant
Cell Line. Paclitaxel resistance is a major mechanism
accounting for therapeutic failures for the clinical use of
Table 1. In Vitro Growth Inhibitory Effects of ABI-III Compounds on Melanoma and Prostate Cancer Cells
B
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Journal of Medicinal Chemistry
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Table 2. Antiproliferative Activity of ABI-III Analogues on Paclitaxel Resistant Cell Line
IC50 SEM (nM)(n = 4)
ID
PC-3
6a
6b
6c
6d
6e
paclitaxel
3.7 0.3
3.7 0.8
1
33.3 2.1
27.6 9.8
0.9
20.8 0.5
22.4 5.1
1.1
40.7 5.1
42.8 11.9
1
105.0 2.1
98.6 6.3
0.9
0.4 0.1
188.0 22.0
437
PC-3/TxR
RI*
paclitaxel and vinblastine.21−23 Our previous ABI-I/II analogues
were shown to effectively overcome paclitaxel resistance.13 We
hypothesized the ABI-III analogues modified based on ABI-I/II
scaffold would retain the ability to circumvent paclitaxel
resistance mechanisms. To support our hypothesis, we
examined the antiproliferative activity of compounds 6a−e in
a paclitaxel resistance cell line: PC-3/TxR, with paclitaxel as
positive control.24 In the PC-3/TxR cell line, more than 200
genes are upregulated in addition to Pgp overexpression, which
may represent additional paclitaxel drug resistance mecha-
nisms.24 As indicated in Table 2, compounds 6a−e showed
equal potency on both the parental (PC-3) and its paclitaxel
resistant cell line (PC-3/TxR) with resistance indices ranging
from 0.9 to 1.1. While paclitaxel showed pico-molar activity
(0.4 nM) in the parental PC-3 cells, it demonstrated
significantly lesser potency (188.0 nM) in the PC-3/TxR cell
line with a resistance index of 437. These results clearly
indicated that ABI-III analogues can overcome paclitaxel-
resistance mechanisms and suggest that they may provide a
therapeutic advantage over paclitaxel.
Figure 2. Effect of 6a on Pgp ATPase activity. Change in luminescence
compared to 100 μM Na3VO4 treated samples was plotted (mean
SD, n = 3). Compound 6a showed a similar effect as vehicle control on
Pgp ATPase activity, indicating 6a is not the substrate for Pgp. *, p <
0.05.
ABI-III Compound 6a Is Not a Substrate of Pgp. The
results from paclitaxel resistant cells not only confirmed our
hypothesis that the ABI-III compounds can overcome paclitaxel
resistance but also guided us to perform the Pgp-glo assay to
examine the effect of compound 6a on Pgp ATPase. In the Pgp-
glo assay, ATP was incubated with recombinant human Pgp
and then unmetabolized ATP in the presence of test compound
was detected as a luciferase-generated luminescent signal. The
decreases in luminescence after drug treatment reflect ATP
consumption by Pgp. ATP consumption by Na3VO4 is
attributed to minor non-Pgp ATPase activities in this system.
The changes of luminescence of compound treated samples
from the one of Na3VO4 treated samples were plotted to
illustrate the stimulation or inhibition of Pgp ATPase activity by
compound treatment (Figure 2). Verapamil, a well-known Pgp
substrate, stimulated Pgp ATPase activity resulting in
significantly decreased luminescence (P < 0.05). However, no
significant difference in luminescence changes was observed
between the vehicle control treated group and 6a treated
groups (up to 1 μM), suggesting that 6a is neither a stimulator
nor an inhibitor for Pgp ATPase. The result from Pgp-glo assay
strongly indicates that 6a is not a substrate of Pgp and can at
least partially explain the ability of ABI-III compounds on
overcoming paclitaxel resistance.
Tubulin Polymerization Assay on ABI-III Compounds.
We hypothesized that ABI-III compounds maintain their
mechanism of action by inhibiting tubulin polymerization
based on the structure similarity of ABI-III and ABI-I/II
analogues. To confirm our hypothesis, we conducted in vitro
tubulin polymerization assays on ABI-III analogues. Bovine
brain tubulin (>97% pure) was incubated with compounds 6a−
e, at concentrations of 5 and 10 μM. Colchicine at 5 μM was
used as a positive control. Compounds 6a−e inhibited tubulin
polymerization in a dose-dependent manner (Figure 3).
Complete inhibition of tubulin polymerization was observed
Figure 3. Effect of ABI-III compounds on tubulin polymerization in
vitro. Tubulin (0.4 mg/assay) was exposed to 5, 10 μM ABI-III
compounds (vehicle control, 5% DMSO): 6a (A), 6b (B), 6c (C), 6d
(D), 6e (E), or colchicine (positive control, at 5 μM). Absorbance at
340 nm was monitored at 37 °C every minute for 20 min.
after 20 min of treatment with 6a (Figure 3A) at 10 μM, while
70% inhibition was achieved by 6a at 5 μM. Compound 6b
(Figure 3B) inhibited tubulin polymerization by 67% and 85%
at 5 and 10 μM, respectively. Compound 6c (Figure 3C)
inhibited 65% and 80% of tubulin polymerization at 5 and 10
μM, respectively, while 30% and 60% inhibition of tubulin
polymerization was observed for compound 6d (Figure 3D) at
C
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Journal of Medicinal Chemistry
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5 and 10 μM, respectively. Compound 6e (Figure 3E) at 10
μM inhibited tubulin polymerization to an extent of 30% but
had no effect on tubulin polymerization 5 μM (same as control
DMSO). These results clearly indicate that the ABI-III
compounds are strong inhibitors of tubulin polymerization
and the inhibitory effects are roughly proportional to the
relative antiproliferative potency for 6a (IC50 = 3.8 nM), 6b
(IC50 = 196.2 nM), 6c (IC50 = 39.8 nM), 6d (IC50 = 41.2 nM),
and 6e (IC50 = 35.0 nM).
Molecular Modeling Studies on 6a. Tubulin polymer-
ization assay suggests that ABI-III compounds exert their effects
by inhibiting tubulin polymerization. To better understand how
ABI-III analogues interact with tubulin, we investigated the
theoretical binding mode of 6a at colchicine binding site in
tubulin dimer using Schrodinger 2011 molecular modeling suite
(Schrodinger, Inc., New York, NY). We tried to dock
compound 6a and 21b into two different tubulin crystal
structures (PDB ID code: 1SA0 or 3HKD). However,
compound 21b did not fit the binding pockets in either crystal
structures due to the extra bulkiness provided by the 3′,4′,5′-
trimethoxyphenyl on the indole 2-position, which may explain
the low activity of this compound (2.9 μM).13 Compound 6a
demonstrated excellent binding in these models with glide
scores of −7.42 and −10.50 for 1SA0 and 3HKD, respectively.
This result is consistent with our previous observation that
TN16-tubulin complex (3HKD) fits ABI scaffold better than
the colchicine-tubulin complex (1SA0).13
interaction provided by the indole A ring and phenyl C ring,
can at least partially explain the high potency of 6a.
CONCLUSIONS
■
Novel ABI-III analogues were synthesized, and a focused SAR
study was performed. The ABI-III compounds were highly
potent against melanoma and prostate cancers in vitro. The 3-
indole compound 6a was identified as the most potent ABI
analogue. The ABI-IIIs were not substrates of Pgp, and
demonstrated equal potency on both a paclitaxel resistant
cancer cell line and its matching parental cell line. Thus, they
can effectively overcome Pgp-mediated multidrug resistance
and paclitaxel resistance. The mechanism of the action studies
suggests that the ABI-III compounds exert their effect by
inhibiting tubulin polymerization. Molecular modeling provides
insights into the binding of ABI-III analogues in tubulin. In
summary, novel ABI-III analogues represent a potent, new class
of tubulin inhibitors that hold great potential for development
into more effective therapeutics for treatment of paclitaxel
resistant cancers.
EXPERIMENTAL PROCEDURES
■
All reagents for the synthesis were purchased from commercial sources
and were used without further purification. Moisture-sensitive
reactions were carried out under an argon atmosphere. NMR spectra
were obtained on an Agilent Inova-500 MHz spectrometer (Agilent
Technologies Inc., Santa Clara, CA). Mass spectral data was collected
on a Bruker ESQUIRE-LC/MS system (Bruker Daltonics, Billerica,
MA) equipped with an ESI source. The purity of the final compounds
was analyzed by an Agilent 1100 HPLC system (Santa Clara, CA).
HPLC conditions: 90% methanol at flow rate of 1.0 mL/min using a
Luna-PFP 5 μM column (250 × 4.6 mm) purchased from
Phenomenex (Torrance, CA) at ambient temperature. UV detection
was set at 280 nm. Purities of the compounds were established by
careful integration of areas for all peaks detected and ≥95%.
The overview of the binding site of 6a and TN-16 in 3HKD
is shown in Figure 4A in which the mesh indicates residue
2-(1H-Indol-3-yl)-1H-imidazol-4-yl)(3,4,5-trimethoxyphenyl)
Methanone (6a). To a solution of (1-(phenylsulfonyl)-2-(1-(phenyl-
sulfonyl)-1H-indol-3-yl)-1H-imidazol-4-yl)(3,4,5-trimethoxyphenyl)-
methanone 5 (0.66 g, 1 mmol) in ethanol (40 mL) and water (4 mL)
was added sodium hydroxide (0.4 g, 10 mmol) and stirred overnight
under refluxing conditions in darkness. The reaction mixture was
diluted by 50 mL of water 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 1:1) to give a yellow solid. Yield: 60%. Mp 210−212 °C.
1H NMR (CD3OD, 500 MHz) δ 8.31 (d, J = 6.5 Hz, 1 H), 7.99 (s, 1
H), 7.90 (s, 1 H), 7.48−7.52 (m, 3 H), 7.24−7.28 (m, 2 H), 4.00 (s, 6
H), 3.93 (s, 3 H). MS (ESI) calcd for C21H19N3O4, 377.1; found,
400.1 [M + Na]+. HPLC: tR 4.33 min, purity >99%.
The synthesis and chromatographic data for 6b−d and their
intermediates are detailed in the Supporting Information.
Figure 4. Binding modes of 6a in the colchicine binding site of tubulin.
surface within 4 Å from TN-16. This binding pocket is located
on the interface between the α- and β-subunits of the tubulin
dimer and extended inside to the nucleoside-binding domain of
the β-subunit.25,26 Figure 4B illustrated the close view of the
potential binding pose. Generally, 6a (orange tube model)
overlapped very well with TN-16 (green wire model). The 3-
indole group of 6a penetrates deeply into the bottom of the
pocket. The interaction was strongly stabilized by two
hydrogen bonds: the first one provided by the indole NH
and ASN167 in β-S5; the second formed between imidazole
NH and VAL238 in β-H7, while the native ligand, TN-16, has
only one hydrogen bond with VAL238 in β-H7 in this
modeling study. The 3,4,5-trimethoxybenzoyl group (C ring) of
6a extends toward the α/β interface, similar to the mode of the
active ABI-II compound.13 The C ring moiety contributes to
the binding interaction by forming the third hydrogen bond
between one of its methoxyl groups and the hydroxyl group of
THR197 in α-T5, which further enhances the interaction
between 6a and the tubulin dimer. The three observed
hydrogen bonds along with the possible hydrophobic
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthesis of 6b−e; NMR and LC-MS. Procedures for cell
culture, cytotoxicity assay, Pgp ATPase assay, in vitro
microtubule polymerization assay, and molecular modeling.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: (901)448-7532. Fax: (901)448-6828. E-mail: wli@uthsc.
edu.
D
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Journal of Medicinal Chemistry
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The project was supported by Grant Number R01CA148706
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. Bob Moore and Ms. Peihong Guan from the
University of Tennessee Health Science Center for providing
access for the use of the Synergy 2 plate reader. We thank Dr.
Michael Mohler for the editorial work.
ABBREVIATIONS USED
■
ABI, 2-aryl-4-benzoyl-imidazoles; MDR, multidrug resistance;
Pgp, P-glycoprotein; SAR, structure−activity relationships;
TMS, tetramethylsilane; TBAF, tert-butylammonium fluoride;
RT, room temperature
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