Novel Combretastatin-2-aminoimidazole Analogues as Potent Tubulin Assembly Inhibitors: Exploration of Unique Pharmacophoric Impact of Bridging Skeleton and Aryl Moiety

Article abstract of DOI:10.1021/acs.jmedchem.6b00101

Combretastatin A-4 (CA-4) in phosphate and serine pro-drug forms is under phase II clinical trials. With our interest of discovering CA-4 inspired new chemical entities, a novel series of 4,5-diaryl-2-aminoimidazole analogues of the compound was designed

Full text of DOI:10.1021/acs.jmedchem.6b00101

Article
pubs.acs.org/jmc
Novel Combretastatin-2-aminoimidazole Analogues as Potent
Tubulin Assembly Inhibitors: Exploration of Unique Pharmacophoric
Impact of Bridging Skeleton and Aryl Moiety
Vikas Chaudhary,†,∥ Jubina B. Venghateri,‡,∥ Hemendra P. S. Dhaked,§ Anil S. Bhoyar,†
Sankar K. Guchhait,*,† and Dulal Panda*,§
†Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, SAS Nagar,
Mohali, Punjab 160062, India
‡IITB-Monash Research Academy, Indian Institute of Technology Bombay, Mumbai 400076, India
§Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India
S
* Supporting Information
ABSTRACT: Combretastatin A-4 (CA-4) in phosphate and
serine pro-drug forms is under phase II clinical trials. With our
interest of discovering CA-4 inspired new chemical entities, a
novel series of 4,5-diaryl-2-aminoimidazole analogues of the
compound was designed and synthesized by an efficient and
diversity feasible route involving atom economical arene C−H
bond arylation. Interestingly, four compounds showed potent
cell-based antiproliferative activities in nanomolar concentra-
tions. Among the compounds, compound 12 inhibited the
proliferation of several types of cancer cells much more
efficiently than CA-4. It depolymerized microtubules, induced
spindle defects, and stalled mitosis in cells. Compound 12
bound to tubulin and inhibited the polymerization of tubulin in
vitro. In addition, podophyllotoxin and CA-4 inhibited the
binding of compound 12 to tubulin. The distinctive pharmacophoric features of the bridging motif as well as quinoline
nucleus were explored. We noted also a valuable quality of compound 12 as a potential probe in characterizing new CA-4
analogues.
Combretastatins belong to the stilbenoid phenol class of
compounds isolated from the bark of an African willow tree
named Combretum caffrum.10 In combretastatin family,
combretastatin A-4 is the most active member, which strongly
inhibits tubulin assembly by interacting at the colchicine-
binding site.11 CA-4 reversibly binds at the colchicine-binding
site of tubulin, whereas the binding of colchicine to tubulin is a
poorly reversible process.12 Importantly, most of the tubulin
inhibitors that target colchicine-binding site have been found to
display vascular disruption and CA-4 was found to be a potent
agent for rapid disruption of endothelial cell morphology in
tumor capillaries responsible for limited blood flow and thus
causing tumor cell death.13,14 CA-4 is a poorly water-soluble
compound. Its 3-O-phosphate prodrug form (CA-4-P, Zybre-
stat)15 and serine amino acid analogue of CA-4 (AVE8062)16
and combretastatin A-1 2,3-O-diphosphate (CA-1-P, OXi4503)17
(Figure 1B), which are water-soluble, are currently under
clinical trials both as a single drug and in combination for anti-
cancer therapy. CA-4 possesses a short biological half-life18
INTRODUCTION
■
Microtubules are critical elements in diverse cellular functions
such as mitosis and cell motility. Some of the microtubule-
targeting agents have been found to be clinically successful
for the treatment of cancer, and therefore, microtubules
are considered to be attractive anticancer drug targets.1−3
Microtubule perturbing agents either enhance or inhibit the
assembly of microtubules. For example, taxane ligands stimulate
microtubule polymerization whereas vinca alkaloids and
colchicine are known to induce depolymerization of micro-
tubules.4,5 Several natural ligands bind to the colchicine-binding
site of tubulin and some of them are structurally similar; for
example, combretastatin,6 podophyllotoxin,7 and steganacin8
(Figure 1A). All these agents comprise common structural
features, a 3,4,5-trimethoxyphenyl (TMP) moiety and the
cis-locking of two aryl groups, which have been found to be
crucial in inhibiting tubulin polymerization and antiprolifera-
tive activities.9 These distinctive simple structural features have
triggered many medicinal chemistry programs to focus on
structural modulation of naturally potent ligands, especially
combretastatin A-4 (CA-4), toward the discovery of potent
antitubulin agents.
Received: January 22, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jmedchem.6b00101
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 1. Structures of (A) natural products and (B) clinical trial agents which bind at the colchicine-binding site of tubulin.
Figure 2. Some representative examples for replacement of double bond: (A) heterocyclic, (B) carbocyclic, and (C) functional bridging moieties.
and undergoes isomerization of a stilbenic olefin from an active
cis-isomer to an inactive trans-isomer in the presence of light,
heat, or protic media19 and in physiological conditions.20
In recent years, efforts have been made to overcome these
limitations by derivatization of double bond and the hydroxyl
substituent in ring B. To maintain the Z-geometry of two aryl
rings, introduction of various linkers in place of double bond
have been considered, for example, heterocyclic rings including
imidazole,16 2(5H)-furanone,21 oxazolone,22 4-arylcoumarin,23
furazan,24 triazole,25 dihydroisoxazole,26 2-aminothiazole,27
tetrazole,28 and indole,29 and carbocyclic rings including cyclo-
propane30 and cyclopentenone31 (Figure 2). The maintenance
of (Z)-stilbene geometry is also known via introducing a variety
of bridging functional groups, such as a ketone,32 sulfide,33
ether,34 nitrile,35 and sulfonate36 (Figure 2). As a part of our
research activities focused on the natural product based
anticancer drug discovery,37 we developed recently novel
2-aryl-3-arylamino-imidazo-pyridine/pyrazine antitubulin anti-
cancer agents.38
In this direction, we envisaged that the replacement of the
double bond in CA-4 with a 2-aminoimidazole skeleton that
can provide additional features of multipoints hydrogen bond
donors/acceptors by C2-amino and ring N/NH functionalities
for interaction with tubulin (Figure 3). In addition, it may
provide the drug-like physicochemical properties including
aqueous solubility. Indeed, 2-aminoimidazole is a valuable
molecular motif in medicinal chemistry, as proven by its
presence in a wide range of bioactive marine natural products,39
such as oroidin,40 bromoageliferin,41 sceptrin,42 ageliferin,43
CAGE,44 and TAGE.
In the investigated 2-aminoimidazole series of compounds,
3,4,5-trimethoxyphenyl moiety as ring A (4-position) and
B
DOI: 10.1021/acs.jmedchem.6b00101
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 3. Design of novel 4,5-diaryl-2-aminoimidazoles as potential antitubulin and anticancer agents.
various relevant (hetero)aryls as ring B (5-position) were
considered. Interestingly, four of the investigated 2-amino-
imidazole analogues of CA-4 exhibited nanomolar antiprolifer-
ative activities. One compound (12) displayed significantly
more potent antiproliferative activity against human breast
adenocarcinoma (MCF-7), cervical cancer (HeLa), hepatocel-
lular carcinoma (HuH-7), highly metastatic breast cancer
(MDA-MB-231), and drug resistant mouse mammary tumor
(EMT6/AR1) than CA-4. It strongly perturbed the organ-
ization of both interphase and mitotic microtubules and
blocked the progression of the cells in mitosis. The compound
inhibited tubulin assembly via binding at the colchicine site.
Interestingly, compound 12 displayed strong fluorescence upon
binding to tubulin. Furthermore, podophyllotoxin and CA-4
were able to displace compound 12 from the tubulin−
compound 12 complex indicating that compound 12 binds to
tubulin reversibly. This provides a unique opportunity to use it
as a probe in characterizing new inhibitors of tubulin. The
structure antitubulin/proliferative activity relationship and the
important pharmacophoric features have been explored.
the methods were rarely investigated for the synthesis of 2-aryl-
imidazo[1,2-a]pyrimidine. In our initial studies for synthesizing
intermediate imidazo[1,2-a]pyrimidine, we investigated several
methods known for preparation of imidazo[1,2-a]pyridines
(Scheme 1).
In method 1, a FeCl3-catalyzed reaction50 of 2-amino-
pyrimidine with nitrostyrene was performed. However, the
formation of the desired product that required cascade
transformations of Michael addition, intramolecular nucleo-
philic substitution, and dehydronitration were not observed.
Increasing the catalyst quantity and variation in solvent did not
promote the reaction, plausibly due to the poorly nucleophilic
nature of 2-aminopyrimidine. In method 2, a reaction of
2-aminopyrimidine with 2-bromoacetophenone in the presence
of sodium bicarbonate was done.51 The desired product
formed, but the conversion was found to be less and side
products formed on prolonging the reaction. In method 3, the
cyclo-condensation reaction of 2-aminopyrimidine with aceto-
phenone via imine formation followed by tandem intra-
molecular C−H amination catalyzed by Cu(OAc)2 and ZnI2
was done following a method reported for 2-aminopyridine.52
The desired product formed in moderate yield, although the
reaction was incomplete. Variation in Cu catalyst, solvent, and
temperature did not improve the yield considerably. In the next
step, selective C-3 arylation of imidazo[1,2-a]pyrimidine was
investigated with bromobenzene. The Pd-catalyzed selective
direct C3−H arylation of 2-unsubstituted imidazo [1,2-
a]pyridines with aryl halides is known.53 We followed the
method for the direct C3-arylation of C-2 substituted imidazo
[1,2-a]pyrimidine (route 1). However, much less conversion
was observed. With increased quantity of Pd(OAc)2-precatalyst
(5 mol %), the desired product was obtained in high yield.
In the next step, 2,3-diaryl-imidazopyrimidine was subjected to
cleavage of fused pyrimidine ring by using 20% ethanolic
hydrazine under microwave-assisted dielectric heating.
Following this protocol, final compounds 1, 2, 3, 4, 5, 6, 8,
10, 11, 12, and 13 with relevant (hetero) aryl substitutions
were synthesized (Figure 4).
RESULTS AND DISCUSSION
■
Chemistry. Our designed 2-amino-imidazole class of
compounds possess relevant functionalized/substituted two
aryl moieties at 4 and 5 positions, and one of the aryls is 3,4,5-
trimethoxyphenyl. A survey of the literature revealed that
4,5-disubstituted 2-amino-imidazoles were, in general, prepared
by a classical reaction of benzil45 or benzoin46 with guanidine.
The reaction47 of α-haloketone with N-acetylguanidine
followed by deacetylation, and the reaction of α-aminoketone
with cyanamide leading to 2-amino-imidazoles are also
known.48 However, all these methods are limited to prepara-
tion of symmetrical 4,5-diaryl or 4,5-aryl/alkyl substituted
2-aminoimidazoles only. In addition, to prepare our designed
compounds in these methods, the starting compounds would
be benzil/benzoin, α-haloketone, or α-aminoketone and should
possess 3,4,5-trimethoxyphenyl at one end and relevant
substituted aryl group at other, which are difficult to access.
These constraints impelled development of a convenient and
diversely feasible method for preparation of our designed
compounds. Recently, hydrazine-mediated cleavage of imidazo-
[1,2-a]pyrimidine scaffold leading to the formation of
2-aminoimidazole has been demonstrated.49 This encouraged
us to envisage a new route to access our desired set of
unsymmetrical relevant 4,5-diaryl substituted 2-aminoimida-
zoles. The preparation of 2-aryl-imidazo[1,2-a]pyridine by
cyclocondensation of 2-aminopyridine with versatile bis/
monoelectrophilic reactants are widely known, but surprisingly,
While this route was applied to the synthesis of compounds
7, 9, and 14, corresponding arylation reactions were found
to be low-yielding. Therefore, they were prepared by another
method (route 2, Scheme 1). In this method (route 2,
Scheme 1), C3 bromination of imidazo[1,2-a]pyrimidine by
NBS followed by Suzuki coupling with arylboronic acids were
done, which provided the desired products 2,3-diarylimidazo-
pyrimidine in comparative yield. For the synthesis of com-
pound 15, a direct C3 arylation of 2-(3,4,5-trimethoxyphenyl)-
imidazopyrimidine with 5-bromo-2-methoxyphenol using
C
DOI: 10.1021/acs.jmedchem.6b00101
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Scheme 1. Synthesis of Investigated Compounds (1−15)
Figure 4. Synthesized 4,5-biarylated-2-aminoimidazoles (1−15).
1
optimized reaction conditions did not occur. The protection−
deprotection chemistry was then applied. The hydroxyl group
of 5-bromo-2-methoxyphenol was protected as a benzyloxy
moiety by reaction with benzyl bromide.54 After several
experiments, the arylation with 2-(benzyloxy)-4-bromo-1-
methoxybenzene was found to be successful with a reported
method55 with some modifications such as an increase in the
catalyst, base, and ligand. After C3-arylation, the hydrazine
mediated ring cleavage and subsequent debenzylation by
classical Pd−C catalyzed hydrogenation provided desired
compound (15) (Scheme 1). The structures of the compounds
(1−15) were characterized by H and 13C NMR (Supporting
Information).
Biological Studies. Cell-Based Screening of Combretas-
tatin Analogues for Their Antiproliferative Activity. Com-
bretastatin analogues (1−15) were screened in HeLa and
MCF-7 cell lines using sulforhodamine assay (Table 1). CA-4
was used as a control to make a comparison of the potency of
the synthesized combretastatin analogues. The half-maximal
inhibitory concentration (IC50) for all the compounds (1−15)
tested against MCF-7 and HeLa cells is shown in Table 1. Of
the enlisted compounds, four compounds, namely compound
D
DOI: 10.1021/acs.jmedchem.6b00101
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 1. Half-Maximal Inhibitory Concentration of
Synthesized Combretastatin Analogues in MCF-7 and HeLa
Cell Line
combretastatin analogues
IC50 in MCF-7 (nM)
187
IC50 in HeLa (nM)
197
1
5
3
2
(27 12) × 103
(12 4) × 103
(8 2) × 103
(3 1) × 103
(34 14) × 103
(8 7.7) × 103
(3 2) × 103
3
4
5
(3.4 3) × 103
6
174
7
166
9
7
(53 24) × 103
(18 7) × 103
(18 12) × 103
(14 7) × 103
8
9
71
6
68
2
10
11
12
13
14
15
CA-4
(21 3) × 103
(13 6) × 103
(18 2) × 103
(4 1) × 103
3
2
10
1
(8 6) × 103
(6 4) × 103
220 12
(3 2) × 103
(8 3) × 103
390 21
18
2
25
2
Figure 5. Compound 12 and CA-4 inhibited the proliferation of
MCF-7 and HeLa cells. Effects of compound 12 in MCF-7 (A) and
HeLa (B) and the effects of CA-4 in MCF-7 (C) and HeLa (D) are
shown.
12, 9, 6, and 1, were found to potently inhibit the proliferation
of MCF-7 and HeLa cells. Because compound 12 was found to
be the most potent among the tested combretastatin analogues,
and therefore the antiproliferative activity of compound 12 was
further characterized.
A structure−activity relationship was established based
on the variation of the ring B in compounds (1−15) tested
for antiproliferative activities in MCF-7 and HeLa cell line.
The presence of 4-methoxy- or 3,4-dimethoxy-phenyl as
ring B provided very good antiproliferative activities. On the
other hand, 2-aminopyridine or pyridine as ring B showed
poor activities. Moderate activities were shown by com-
pounds that contain ring B moieties, 3,4,5-trimethoxyphenyl,
4-chloro/fluoro-phenyl, 3-methoxyphenyl, aniline, or indole.
The compound containing quinoline as ring B was found to
be most potent antiproliferative and superior effective than
CA-4.
Effect of Compound 12 on the Cell Proliferation in Cancer
Cell Lines. Compound 12 inhibited the proliferation of MCF-7
and HeLa cells with a half-maximal inhibitory concentration
(IC50) at 3 2 and 10 1 nM, respectively (Figure 5A,B).
CA-4 inhibited the proliferation of MCF-7 and HeLa cells with
an IC50 value of 18 2 and 25 2 nM, respectively (Figure 5C
and 5D). The results indicated that compound 12 displayed
more potent antiproliferative effects against MCF-7 and HeLa
cells than CA-4. Compound 12 also inhibited the proliferation
of highly metastatic breast cancer cells (MDA-MB-231), drug
resistant mouse mammary cancer cells (EMT6/AR1), and
hepatocarcinoma cells (HuH-7) more potently than CA-4
(Table 2). On the basis of structure−activity relationships and
the superior antiproliferative activities (Tables 1 and 2) of most
of the investigated class of compounds compared to CA-4 in
multiple cancer cell lines (MCF-7, HeLa, MDA-MB-231,
EMT6/AR1, and HuH-7), it is evident that the 2-amino-
imidazole skeleton bridging two aryls (A and B), as
replacement of double bond in the CA-4, and the quinoline
as ring B are important pharmacophoric features in addition to
trimethoxyphenyl as ring A. This may be plausibly due to the
biological target−ligand interaction stabilized by hydrogen
bonding with ring N/NH and C2-amine functionalities of
2-aminoimidazole and quinoline N in the ligand.
Table 2. Half-Maximal Inhibitory Concentration of
Compound 12 and CA-4 in Different Tumor Cell Lines
cell lines
12 (nM)
CA-4 (nM)
MDA-MB-231
EMT6/AR1
HuH-7
96 13
331 32
495 11
350
7
335 10
430
9
Compound 12 Depolymerized Microtubules and Caused
Mitotic Block in MCF-7 Cells. Combretastatins are known to
depolymerize cellular microtubules. Compound 12 also found
to depolymerize microtubules in a concentration-dependent
manner. For example, 5 nM compound 12 depolymerized
interphase microtubules moderately whereas 10 nM compound
12 induced strong depolymerization of interphase microtubules
in MCF-7 cells (Figure 6).
Further, compound 12 also perturbed the mitotic spindles.
At concentrations above 5 nM of compound 12, monopolar
spindles were observed and the cells were found to be blocked
in mitosis (Figure 7). A flow cytometry analysis of MCF-7 cells
treated with 5 and 10 nM compound 12 indicated that 40
and 74% of the cells were in the G2/M phase, respectively
(Table 3). Whereas in comparison, 20 and 46% of the cells
were in the G2/M phase in the presence of 5 and 10 nM CA-4,
respectively (Figure 8). To further corroborate the results,
phosphohistone H3 (Ser-10) staining of the cells was per-
formed. Phosphohistone H3 (Ser-10) is a mitotic marker of
cells. The number of phosphohistone-stained cells increased in
a concentration-dependent manner as compared to the vehicle-
treated cells (Figure 9). The percentage of the mitotic cells was
determined to be 8 1, 15 2, and 26 1% in the absence
and presence of 5 and 10 nM compound 12, respectively.
Compound 12 Inhibited the Migration of MDA-MB-231
Cells. Combretastatins are well-known antivascular agents.
Therefore, the effect of compound 12 on the wound closure
of migratory tumor cells, MDA-MB-231 was evaluated.
E
DOI: 10.1021/acs.jmedchem.6b00101
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 6. Compound 12 showed stronger depolymerizing effects on the interphase microtubule network than CA-4. MCF- 7 cells were incubated in
the absence and presence of 5 and 10 nM of compound 12 (A) and 5, 10, and 50 nM of CA-4 (B) for 24 h. Cells were fixed and stained for α-tubulin
(green) and nuclear stain, Hoechst (blue). Scale bar is 1 μm.
Figure 7. Effects of compound 12 and CA-4 on the mitotic spindles in MCF-7 cells. (A) MCF-7 cells were incubated either with the vehicle or with
5 and 10 nM compound 12 (A) and 5, 10, and 50 nM CA-4 (B) for 24 h and stained for α-tubulin (green) and nuclear stain, Hoechst (blue). Scale
bar is 1 μm.
Compound 12 Inhibited the Polymerization of Tubulin in
Vitro. Because compound 12 depolymerized microtubules in
cells, the effect of compound 12 on the assembly kinetics
of tubulin was analyzed in vitro. Compound 12 inhibited the
rate and extent of the assembly of tubulin in a concentration-
dependent manner (Figure 11A) and the half-maximal
inhibition (IC50) of tubulin assembly occurred in the presence
of 1.6 0.7 μM of the compound. The results indicated that
compound 12 is a potent inhibitor of tubulin assembly.
Compounds 9 and 6 were found to inhibit the tubulin assembly
Table 3. Percentage of MCF-7 Cells after Treatment with
Compound 12 and CA-4 and Analyzed by Flow Cytometry
compd
control
G1
S
G2/M
49
34
16
51
41
14
35
25
10
23
16
23
16
40
74
26
43
63
12 (5 nM)
12 (10 nM)
CA-4 (5 nM)
CA-4 (10 nM)
CA-4 (50 nM)
with an IC50 of 11
1 and 13
2 μM, respectively
(Figure 11B,C). Under similar conditions, CA-4 inhibited the
assembly of tubulin with an IC50 of at 1.7 0.1 μM (Figure 11D).
Compound 1 (20 μM) did not have a significant inhibitory
effect on the assembly of tubulin.
The wound was completely closed after 18 h in control cells
(Figure 10). CA-4 (50 nM) showed a minimal (8 1%)
inhibitory effect on cell migration, while 50 nM compound 12
inhibited the wound healing by 56 3% and 150 nM of CA-4
Determination of the Dissociation Constant of Com-
pound 12 to Tubulin in Vitro. Compound 12 inhibited the
assembly of tubulin. Therefore, we analyzed its binding with
inhibited the wound healing by 52
cated that compound 12 strongly inhibits the migration of
MDA-MB-231cells than CA-4.
4%. The finding indi-
F
DOI: 10.1021/acs.jmedchem.6b00101
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 8. Compound 12 increased the percentage of cells in the G2/M phase. MCF-7 cells were incubated with either in the absence (A) or in the
presence of 5 (B) and 10 (C) nM compound 12 and 5 (D), 10 (E), and 50 (F) nM CA-4 for 24 h. The flow cytometry analysis was performed by
staining the DNA with propidium iodide.
Figure 9. Compound 12 increased the number of phosphohistone
stained cells. MCF-7 cells were incubated in the absence and presence
of 5 and 10 nM compound 12 for 24 h and stained for phosphohistone
(green) and nuclear stain Hoechst (blue). Scale bar is 10 μm.
Figure 10. Compound 12 inhibited MDA-MB-231 cell migration.
Effects of compound 12 and CA-4 on the wound closure are shown.
Scale bar is 1 μm.
intensity in aqueous solution (Figure 13). Interestingly, the
fluorescence intensity of compound 12 increased several fold in
the presence of tubulin, indicating that it formed a complex
with tubulin (Figure 13).
Podophyllotoxin and CA-4 are known to bind to the
colchicine site on tubulin.56,57 Therefore, the binding site of
compound 12 on tubulin was monitored by performing a
competition experiment using podophyllotoxin as well as CA-4.
The preincubation of podophyllotoxin with tubulin reduced the
development of compound 12 fluorescence intensity in a
tubulin. Compound 12 quenched the fluorescence intensity
of tubulin in a concentration-dependent fashion, indicating
that it interacts with tubulin (Figure 12A). The fluorescence
intensity changes were fitted in a binding isotherm to obtain
a dissociation constant (Kd) for the binding of compound 12
to tubulin (Figure 12 B). The Kd was calculated to be 1.9
0.3 μM.
Determination of the Binding Site of Compound 12 on
Tubulin. Compound 12 displayed very low fluorescence
G
DOI: 10.1021/acs.jmedchem.6b00101
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 11. Compounds 12, 9, 6, and CA-4 inhibited the assembly of tubulin in vitro. Tubulin (12 μM) was polymerized in the presence of 1 mM
■
▼
□
●
○
▲
▼
GTP and 1 M glutamate without ( ) or with different concentrations 0.5 ( ), 1 ( ), 2 ( ), 3 ( ), and 5 ( ) μM of compound 12 (A), different
●
▲
●
▲
▼
concentrations 5 ( ), 10 ( ), 15 ( ), and 20 (◀) μM of compound 9 (B), different concentrations 5( ), 10( ), 15( ), 20 (◀) and 30 (▶)
◆
●
▲
▼
and 40 μM ( ) of compound 6 (C), and different concentrations 0.5( ), 1( ), 2( ), 3 (◀), and 5 (▶) μM of CA-4 (D). Three independent
sets of experiments were performed for each compound. One of the sets is shown of all the experiments.
■
Figure 12. Compound 12 reduced the intrinsic tryptophan fluorescence of tubulin. (A) Tubulin (2 μM) was incubated without ( ) and with
◆
●
○
▲
▼
different concentrations of 1 ( ), 2 ( ), 5 ( ), 10 (Δ), 15 ( ) and 20 ( ) μM of compound 12 in 25 mM PIPES buffer, pH 6.8, for 10 min at
25 °C. (B) The change in fluorescence intensity of tubulin upon binding with compound 12 was plotted. The experiment was conducted four times.
One of the four sets is shown.
concentration-dependent manner (Figure 14A). A Ki value was
determined to be 0.3 0.14 μM (Figure 14B). Similarly, CA-4
reduced the binding of compound 12 to tubulin in a concen-
tration dependent fashion (Figure 14C), and the Ki value was
found to be 0.68 0.7 μM (Figure 14D).
In addition, we checked whether CA-4 and podophyllotoxin
could displace tubulin bound compound 12. Tubulin was first
incubated with compound 12 for 30 min and then either CA-4
or podophyllotoxin was added to the reaction milieu for further
30 min. The addition of CA-4 and podophyllotoxin reduced
the fluorescence of tubulin−compound 12 complex, indicating
that the binding of compound 12 to tubulin is reversible
(Supporting Information, Figure S1). Because the fluorescence
of compound 12 strongly increases upon binding to tubulin and
compound 12 reversibly binds to tubulin, compound 12 can be
used as a probe to evaluate tubulin inhibitors that bind to the
CA-4 site in tubulin. Then we checked whether vinblastine and
paclitaxel could inhibit the binding of compound 12 with
tubulin. Tubulin was incubated with either vinblastine or
paclitaxel for 10 min. Then compound 12 was added to the
reaction mixtures. Vinblastine and paclitaxel could not reduce
the development of the fluorescence intensity of compound 12,
indicating that these agents did not inhibit the binding of
compound 12 to tubulin (Supporting Information, Figure S2).
The results together indicated that compound 12 binds at
the colchicine-binding site of tubulin. Further, the findings
H
DOI: 10.1021/acs.jmedchem.6b00101
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
CONCLUSION
■
In conclusion, we designed a new series of analogues of
combretastatin A-4, comprising 2-aminoimidazole as a bridging
skeleton replacing a double bond in CA-4, 3,4,5-trimethox-
yphenyl in ring A and various relevant (hetero) arenes in ring
B. Fifteen compounds were synthesized by a diversity feasible
and atom-economical route involving arene C−H bond
arylation. Four compounds exhibited antiproliferative activities
in nanomolar concentrations. 5-Quinolin-3-yl and 4-(3,4,5-tri-
methoxyphenyl) substituted imidazol-2-amine (compound 12)
was most potent. Compound 12 showed higher antiprolifer-
ative activity than CA-4 against five different cancer cell lines
including a drug resistant cell line. In addition, compound 12
exerted stronger inhibitory effect on the migration of highly
metastatic MDA-MB-231 than CA-4, indicating its strong anti-
metastatic potential. Compound 12 was found to cause a
significant depolymerization and perturbation of the interphase
and mitotic microtubule network and induced mitotic arrest in
MCF-7 cells. Like CA-4, compound 12 inhibited the rate and
extent of an in vitro assembly of purified tubulin with an IC50 of
1.6 μM. A dissociation constant of 1.9 μM is indicative of a
strong binding affinity of compound 12 to tubulin. Importantly,
compound 12 when bound to tubulin showed an increase in
the fluorescence intensity, indicating its potential use as a probe
Figure 13. The fluorescence intensity of compound 12 increased in
the presence of tubulin. Tubulin (2 μM) was incubated without ( )
■
●
▲
and with 5 ( ) and 10 μM ( ) of compound 12 in 25 mM PIPES
▼
○
buffer for 10 min at 25 °C. The spectra of 5 ( ) and 10 ( ) μM
compound 12 were also monitored using 350 nm as the excitation
wavelength.
indicated that compound 12 may be used to screen agents that
bind to the colchicine site on tubulin in general and particularly
for CA-4 analogues.
■
Figure 14. Compound 12 bound at the colchicine site on tubulin. (A) Tubulin (3 μM) was incubated without ( ) or with different concentrations
◆
●
▲
▼
○
□
1 ( ), 3 ( ), 5 ( ), 10 ( ), 15 ( ), and 20 μM ( ) of podophyllotoxin in 25 mM PIPES for 15 min at 25 °C. Then, the reaction mixtures were
◇
incubated with 6 μM compound 12 for an additional 15 min at 25 °C. The spectrum of 6 μM ( ) compound 12 in the absence of tubulin is also
shown. The experiment was performed three times. (B) The changes in the fluorescence intensities were plotted against different concentrations of
podophyllotoxin. (C) Tubulin (3 μM) was incubated without ( ) or with different concentrations of 1 ( ), 3 ( ), 5 ( ), 10 ( ), 15 ( ), and
20 ( ) μM of CA-4 in 25 mM PIPES for 15 min at 25 °C. Subsequently, the reaction mixtures were incubated with 6 μM compound 12 for another
■
□
●
○
▲
▼
◆
15 min at 25 °C. Three sets of experiments were performed. One of the sets is shown. (D) The change in fluorescence intensity was plotted against
CA-4 concentrations.
I
DOI: 10.1021/acs.jmedchem.6b00101
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(198 mg, 1.35 equiv) was done following a reported method55 with
use of increased quantities of cesium carbonate (360 mg, 2.2 equiv),
palladium acetate (33 mg, 15 mol %), and triphenylphosphine (40 mg,
30 mol %). Subsequently, the ring cleavage reaction was performed as
described above. Finally, the benzyl deprotection was done in the
presence of Pd−C in MeOH under H2 gas. The crude reaction
mixture was subjected to column chromatography on silica gel
(60−120 mess size). Compound 15 was obtained in 21% overall yield
starting from intermediate 2-(3,4,5-trimethoxyphenyl)imidazo[1,2-
a]pyrimidine. The structures of investigated compounds (1−15)
were characterized by 1H and 13C NMR, IR, and confirmed by HRMS
(ESI). Their purity tested by HPLC were found to be >95%.
Materials and Methods. Reagents. Sulforhodamine B (SRB),
mouse monoclonal, anti-α tubulin IgG as primary antibody, DABCO
(1,4-diazabicyclo[2.2.2]octane), FITC conjugated antimouse IgG
secondary antibody, and CA-4, Ethylene glycol-bis(2aminoethyl
ether)-N,N,N′,N′-tetraacetic acid (EGTA) was purchased from
Sigma (St Louis, MO, USA). Podophyllotoxin was procured from
MP Biomedicals, India. Dulbecco’s phosphate buffered saline (DPBS),
Hoechst 33258, bovine serum albumin (BSA), piperazine-1,4-bis-2-
ethanesulfonic acid (PIPES), magnesium chloride (MgCl2), trichloro-
acetic acid (TCA), dimethyl sulfoxide, sodium bicarbonate, an
antibiotic−antimycotic solution comprising of streptomycin, ampho-
tericin B, and penicillin, and minimal essential media were purchased
from HiMedia, Mumbai (India). Fetal bovine serum used for cell
culture media was procured from Invitrogen. Phosphohistone H3
(Ser-10) primary antibody was purchased from Santa Cruz Biotech.
All the reagents utilized for the experiments were of analytical grade.
Cell Culture. MCF-7, HeLa and MDA-MB-231 were purchased
from the cell repository of National Centre for Cell Science, Pune,
India. EMT6/AR1 cell line was purchased from Sigma, and HuH-7
cells were obtained from the laboratory of Dr. S. Das (Indian Institute
of Science, Bangalore, India). HeLa and MCF-7 cells were cultured in
minimal essential media (MEM), whereas MDA-MB-231, HuH-7, and
EMT6AR1 was cultured in Dulbecco’s Modified Eagle’S Medium
supplemented with 2.2 g/L of sodium bicarbonate, 10% (v/v) fetal
bovine serum (FBS: US origin), and 1% antibiotic−antimycotic
solution composed of streptomycin, amphotericin B, and penicillin.
Cell lines were cultured at 37 °C in a humidified incubator (Sanyo,
Tokyo, Japan) with 5% carbon dioxide.
Cell-Based Screening Assay. Synthesized combretastatin analogues,
(1−15) and CA-4 used in the study were dissolved in 100% cell
culture grade DMSO. The compounds were serially diluted in minimal
essential media (MEM) to maintain the final concentration of DMSO
as <0.1% for testing on cancer cell lines. A concentration of 200 nM of
synthesized combretastatin analogues (1−15) and CA-4 were initially
used for screening the potency of compounds in HeLa cell line.
Half-Maximal Inhibition of Tumor Cell Growth by Combretas-
tatin Analogues. MCF-7 and HeLa cells (1 × 105 cells/mL) were
seeded in 96-well plates and incubated for 24 h for attachment. The
cells were then incubated with different concentrations of synthesized
combretastatin analogues and incubated for 48 h for MCF-7 and 24 h
for HeLa. CA-4 was used as a control for comparing the potencies of
the synthesized combretastatin analogues. MDA-MB-231, EMT6AR1,
and HuH-7 cells were incubated with compound 12 and CA-4 for
24 h. Subsequently, cells were fixed with 50% TCA and the half-
maximal inhibitory concentration (IC50) for the respective compounds
was determined by sulforhodamine B assay.58 Three independent sets
of experiments were performed.
in the characterization of new antitubulin agents. Competition
studies with CA-4 and podophyllotoxin for tubulin-binding
signified that compound 12 binds at the colchicine site on
tubulin. The results indicated the importance of 2-amino-
imidazole as bridging skeleton and quinoline motif in ring B in
the structural modulation of CA-4 for generating a new class of
antitubulin agents.
EXPERIMENTAL SECTION
■
Chemistry. General Considerations. All reactants and reagents
were obtained from the commercial source and used without further
purification. The NMR spectra were recorded on a Bruker Avance
DPX 400 MHz spectrometer in CDCl3/DMSO-d6/CD3OD solvents
using TMS as an internal standard. J values are given in Hz. HRMS
(ESI) were recorded with Bruker−Maxis mass spectrometers. IR
spectra were recorded on a Nicolet FT−IR Impact 410 instrument as a
thin film (neat). The reactions were monitored by TLC (Merck, Silica
gel 60 F254). The purity of synthesized compounds was analyzed by
HPLC (Shimadzu LC-6AD system), Phenomenex RP-C18 column
(250 mm × 4.60 mm), particle size 5 μm, flow rate 1 mL/min, using
water−acetonitrile. 2-(Benzyloxy)-4-bromo-1-methoxybenzene was
prepared following a known method.54
Synthesis of Compounds (1−15). Representative Experimental
Procedure for the Synthesis of Compound 2. A mixture of
2-aminopyrimidine (114 mg, 1.2 mmol), 3,4,5-trimethoxyacetophe-
none (210 mg, 1.0 mmol), Cu(OAc)2·H2O (20 mg, 0.1 mmol), 1,10-
phenanthroline (18 mg, 0.1 mmol), and zinc iodide (32 mg,
0.1 mmol) in 1,2-dichlorobenzene (2 mL) was stirred at 120 °C.
After maximal conversion (24 h, monitored by TLC), the resultant
mixture was washed by hexane to remove the solvent. The column
chromatographic purification of crude mass on neutral alumina
provided the intermediate 2-(3,4,5-trimethoxyphenyl)imidazo[1,2-a]-
pyrimidine (157 mg, 55%). For C-3 direct arylation (route 1),
intermediate 2-(3,4,5-trimethoxyphenyl)imidazo[1,2-a]pyrimidine
(342 mg, 1.2 mmol) was stirred with bromobenzene (157 mg,
1 mmol), KOAc (196 mg, 2 mmol), and Pd(OAc)2 (11.2 mg, 5 mol %)
at 150 °C in DMAc (4 mL). After maximal conversion (24 h, moni-
tored by TLC), solvent was evaporated under vacuum. The crude
reaction mixture was extracted with EtOAc (2 × 40 mL) and washed
with water (2 × 20 mL). The organic layer was dried with anhyd
Na2SO4 and concentrated under vacuum, which provided intermediate
2,3-diarylated imidazopyrimidine. Then the resultant crude mixture
without further purification was subjected to ring cleavage by heating
with hydrazine hydrate (20% in methanol, 3 mL) under microwave
irradiation at 120 °C. After completion of the reaction (15 min,
monitored by TLC), the solvent was evaporated under vacuum. The
column chromatographic purification of crude mass on silica gel
provided compound 2 in 36% isolated yield (starting from
intermediate 2-(3,4,5-trimethoxyphenyl)imidazo[1,2-a]pyrimidine).
Compounds 1, 2, 3, 4, 5, 6, 8, 10, 11, 12, and 13 were prepared by
following this procedure. For the synthesis of compounds 7, 9, and 14,
an alternative process (route 2) was followed. For bromination
at C-3 position, 2-(3,4,5-trimethoxyphenyl)imidazo[1,2-a]pyrimidine
(285 mg, 1 mmol) in methanol (2 mL) was stirred at 27−29 °C with
small quantitywise addition of NBS (178 mg, 1 mmol). After complete
conversion (1 h, monitored by TLC), the solvent was evaporated
under vacuum. To this crude mixture were added DMAc−H2O (1:1,
4 mL), arylboronic acid (1.3 equiv), Na2CO3 (530 mg, 5 equiv),
TBAB (322 mg, 1 equiv), and Pd(PPh3)4 (115 mg, 0.1 mmol). The
mixture was then refluxed with stirring at 105 °C. After completion of
the reaction (monitored by TLC), a solvent mixture was evaporated
under vacuum. The resultant mixture was extracted with EtOAc
(2 × 40 mL) and washed with water (2 × 20 mL). The organic layer
was dried with anhyd Na2SO4, and concentrated under vacuum. Then
the resultant crude mixture of intermediate 2,3-diarylated imidazopyr-
imidine without further purification was subjected to ring cleavage fol-
lowing the process mentioned above. For the synthesis of compound 15,
arylation of 2-(3,4,5-trimethoxyphenyl)imidazo[1,2-a]pyrimidine
(0.50 mmol, 145 mg) with 2-(benzyloxy)-4-bromo-1-methoxybenzene
Immunostaining and Microscopy. MCF-7 cells (0.5 × 105 cells/mL)
were seeded on glass coverslips in a 24-well tissue culture plate for
24 h and then incubated without or with different concentrations of
compound 12 for 24 h. The cells were fixed with 3.7% formaldehyde
and stained with α-tubulin (IgG) or phosphohistone H3 (Ser-10)
antibodies for 1 h. Then, FITC conjugated IgG secondary antibody
were added onto the cells and incubated for 1 h and washed with
DPBS three times. The cells were finally stained with (20 μg/mL) of
Hoechst 33258. The images were captured using an Eclipse TE 2000U
microscope (Nikon, Tokyo, Japan).
J
DOI: 10.1021/acs.jmedchem.6b00101
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Cell Migration Assay. MDA-MB-231 cells were grown in 35 mm
culture dishes until 90% confluency. Subsequently, the media was
aspirated and a wound was created in the middle of the dish by
scratching on the confluent cells using a 10 μL microtip.59 The cells
were then incubated in the absence and presence of different
concentrations of compound 12 and CA-4. The wound closure was
monitored at different time points (0, 4, and 18 h). Images were
captured and the percentage of wound closure was calculated using
Image Pro-Plus software.
Cell Cycle Analysis by Flow Cytometry. MCF-7 cells were
incubated in the absence and presence of compound 12 and CA-4
for 24 h. Subsequently, the cells were fixed with 70% alcohol in DPBS.
The fixed cells were incubated with RNase (1 μg/mL) and propidium
iodide (50 μg/mL) for 2 h. The flow cytometry analysis were
performed using BD FACS Aria special order system (Becton
Dickinson, San Jose, CA, USA), and the obtained data were fitted
using a software, Modfit LT version 3.2 (Verity Software House, ME,
USA).60
The Effects of Compounds 12, 9, and 6 on the Assembly Kinetics
of Tubulin in Vitro. Tubulin was purified from goat brains using 1 M
glutamate as described previously.61 The purity of tubulin was
estimated to be >97% using Coomassie Brilliant Blue stained
SDS-PAGE. Tubulin (12 μM) was resuspended in a buffer containing
25 mM PIPES, 1 mM EGTA, and 3 mM MgCl2 with 1 M glutamate
and was incubated without or with different concentrations (0.5−5 μM)
of compound 12, (0.5 to 20 μM) of compound 9, (5−40 μM) of
compound 6, and (0.5−5 μM) of CA-4 for 10 min on ice. After
10 min, 1 mM GTP was added to the reaction mixtures, and
subsequently, the assembly kinetics of tubulin was monitored at
350 nm (37 °C) for 20 min using Spectramax M2e. Similarly,
inhibition of the assembly kinetics of tubulin was tested using CA4 for
comparison of the potency of compound 12. Three independent
experiments were performed.
Determination of the Dissociation Constant (Kd) of the Binding
of Compound 12 and Tubulin. Tubulin (2 μM) was incubated in the
absence and presence of different concentrations (1−20 μM) of
compound 12 for 10 min at 25 °C. Tryptophan fluorescence of tubulin
in the presence and absence of compound 12 was monitored using an
excitation wavelength of 295 nm in a 0.3 cm path length cuvette
(FP-6500 spectrofluorometer JASCO, Tokyo, Japan). A dissociation
constant for the binding interaction of compound 12 and tubulin
additional 30 min. The spectra were monitored using 350 nm as the
excitation wavelength.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acs.jmedchem.6b00101.
Spectral data of intermediates and compounds 1−15,
1
scanned copies of H NMR and 13C NMR of inter-
mediates and 1−15, reversible binding of compound 12
to tubulin, and effects of vinblastine and paclitaxel on the
binding of 12 to tubulin (PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Authors
■
*For S.K.G.: phone, +91 (0)172 2214683; fax, +91 (0)
1722214692; E-mail, skguchhait@niper.ac.in, skguchhait@
gmail.com.
*For D.P.: phone, +91 222 576 7838; fax, +91 222 572 3480;
E-mail, panda@iitb.ac.in.
Author Contributions
∥V.C. and J.B.V. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge the financial support from DAE-
SRC, DST, and CSIR, Government of India, for this
investigation. D.P. thanks DAE-SRC fellowship. V.C. is thankful
to CSIR, Government of India for his fellowship. A.S.B. is
thankful to NIPER for his fellowship.
ABBREVIATIONS USED
■
NBS, N-bromosuccinimide; EtOAc, ethyl acetate; KOAc,
potassium acetate; Pd(OAc)2, palladium acetate; DMAc,
dimethylacetamide; Cu(Ac)2, copper(II) acetate; TBAB,
tetrabutylammonium bromide; Pd(PPh3)4, tetrakis-
(triphenylphosphine)palladium(0); Pd/C, palladium on car-
bon; MeOH, methanol; DMSO, dimethyl sulfoxide; PIPES,
piperazine-N,N′-bis(ethanesulfonic acid); EGTA, ethylene
glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid;
GTP, guanosine-5′-triphosphate; DPBS, Dulbecco’s phosphate
buffered saline; TLC, thin layer chromatography:
was calculated by fitting the change in fluorescence data in an
ΔFmax ×L
equation ΔF =
using Graph Pad Prism software version 5.
Kd + L
Four independent sets of experiments were performed.
Characterization of the Binding of Compound 12 on Tubulin.
Tubulin (2 μM) was incubated without and with different
concentrations of compound 12 (5 and 10 μM) in 25 mM PIPES
buffer, pH 6.8, for 10 min at 25 °C. The fluorescence spectra
(420−520 nm) were recorded using an excitation wavelength of
350 nm. The experiments were repeated thrice.
Determination of the Binding Site of Compound 12 on Tubulin.
Tubulin (3 μM) was incubated without or with different
concentrations (1−20 μM) of podophyllotoxin in 25 mM PIPES,
pH 6.8, for 15 min at 25 °C. Subsequently, compound 12 (6 μM) was
added to all the reaction mixtures and incubated for 15 min at 25 °C.
Similar experiments were performed using tubulin (3 μM) with
different concentrations of (1−20 μM) of CA-4, vinblastine (10 and
30 μM), and paclitaxel (10 and 30 μM) in 25 mM PIPES, pH 6.8, for
15 min at 25 °C. Subsequently, the reaction mixtures were incubated
with 6 μM compound 12 for 15 min at 25 °C. After the incubation, the
fluorescence spectra (420−520 nm) were recorded using an excitation
wavelength of 350 nm. The inhibition constant was calculated using an
equation.
REFERENCES
■
(1) Jordan, M. A.; Wilson, L. Microtubules as a target for anticancer
drugs. Nat. Rev. Cancer 2004, 4, 253−265.
(2) Li, Q.; Sham, H. L. Discovery and development of antimitotic
agents that inhibit tubulin polymerisation for the treatment of cancer.
Expert Opin. Ther. Pat. 2002, 12, 1663−1702.
(3) Mukhtar, E.; Adhami, V. M.; Mukhtar, H. Targeting microtubules
by natural agents for cancer therapy. Mol. Cancer Ther. 2014, 13, 275−
284.
(4) Nogales, E.; Whittaker, M.; Milligan, R. A.; Downing, K. H. High-
Resolution model of the microtubule. Cell 1999, 96, 79−88.
(5) Ravelli, R. B.; Gigant, B.; Curmi, P. A.; Jourdain, I.; Lachkar, S.;
Sobel, A.; Knossow, M. Insight into tubulin regulation from a complex
with colchicine and a stathmin-like domain. Nature 2004, 428, 198−
202.
EC50
Ki =
L
1 +
Kd
In a separate experiment, tubulin was first incubated with
compound 12 for 30 min. Then the reaction mixture was incubated
without or with either 50 μM podophyllotoxin or 50 μM CA-4 for an
(6) Nam, N. H. Combretastatin A-4 analogues as antimitotic
antitumor agents. Curr. Med. Chem. 2003, 10, 1697−1722.
K
DOI: 10.1021/acs.jmedchem.6b00101
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(7) Bohlin, L.; Rosen, B. Podophyllotoxin derivatives: drug discovery
and development. Drug Discovery Today 1996, 1, 343−351.
(8) Kupchan, S. M.; Britton, R. W.; Ziegler, M. F.; Gilmore, C. J.;
Restivo, R. J.; Bryan, R. F. Steganacin and steganangin, novel
antileukemic lignan lactones from Steganotaenia araliacea. J. Am.
Chem. Soc. 1973, 95, 1335−1336.
(22) Nam, N.-H.; Kim, Y.; You, Y.-J.; Hong, D.-H.; Kim, H.-M.; Ahn,
B.-Z. Combretoxazolones: synthesis, cytotoxicity and antitumor
activity. Bioorg. Med. Chem. Lett. 2001, 11, 3073−3076.
(23) Bailly, C.; Bal, C.; Barbier, P.; Combes, S.; Finet, J. − P.;
Hildebrand, M. − P.; Peyrot, V.; Wattez, N. Synthesis and biological
evaluation of 4-arylcoumarin analogues of combretastatins. J. Med.
Chem. 2003, 46, 5437−5444.
(9) (a) Jordan, A.; Hadfield, J. A.; Lawrence, N. J.; McGown, A. T.
Tubulin as a target for anticancer drugs: agents which interact with
mitotic spindle. Med. Res. Rev. 1998, 18, 259−296. (b) Alvarez, C.;
(24) Tron, G. C.; Pagliai, F.; Del Grosso, E.; Genazzani, A. A.; Sorba,
G. Synthesis and cytotoxic evaluation of combretafurazans. J. Med.
Chem. 2005, 48, 3260−3268.
́ ́
Alvarez, R.; Corchete, P.; Perez-Melero, C.; Pelaez, R.; Medarde, M.
(25) Pati, H. N.; Wicks, M.; Holt, H. L., Jr.; LeBlanc, R.; Weisbruch,
P.; Forrest, L.; Lee, M. Synthesis and biological evaluation of cis-
combretastatin analogs and their novel 1,2,3-triazole derivatives.
Heterocycl. Commun. 2005, 11, 117−120.
(26) Simoni, D.; Grisolia, G.; Giannini, G.; Roberti, M.; Rondanin,
R.; Piccagli, L.; Baruchello, R.; Rossi, M.; Romagnoli, R.; Invidiata, F.
P.; Grimaudo, S.; Jung, M. K.; Hamel, E.; Gebbia, N.; Crosta, L.;
Abbadessa, V.; Di Cristina, A.; Dusonchet, L.; Meli, M.; Tolomeo, M.
Heterocyclic and phenyl double-bond-locked combretastatin ana-
logues possessing potent apoptosis-inducing activity in HL60 and in
MDR cell lines. J. Med. Chem. 2005, 48, 723−736.
(27) Romagnoli, R.; Baraldi, P. G.; Brancale, A.; Ricci, A.; Hamel, E.;
Bortolozzi, R.; Basso, G.; Viola, G. Convergent synthesis and biological
evaluation of 2-amino-4- (3′,4′,5′-trimethoxyphenyl)-5-aryl thiazoles
as microtubule targetingagents. J. Med. Chem. 2011, 54, 5144−5153.
(28) Beale, T. M.; Allwood, D. M.; Bender, A.; Bond, P. J.; Brenton,
J. D.; Charnock-Jones, D. S.; Ley, S. V.; Myers, R. M.; Shearman, J. W.;
Temple, J.; Unger, J.; Watts, C. A.; Xian, J. A-ring dihalogenation
increases the cellular activity of combretastatin-templated tetrazoles.
ACS Med. Chem. Lett. 2012, 3, 177−181.
(29) La Regina, G.; Bai, R.; Coluccia, A.; Famiglini, V.; Pelliccia, S.;
Passacantilli, S.; Mazzoccoli, C.; Ruggieri, V.; Verrico, A.; Miele, A.;
Monti, L.; Nalli, M.; Alfonsi, R.; Di Marcotullio, L.; Gulino, A.; Ricci,
B.; Soriani, A.; Santoni, A.; Caraglia, M.; Porto, S.; Da Pozzo, E.;
Martini, C.; Brancale, A.; Marinelli, L.; Novellino, E.; Vultaggio, S.;
Varasi, M.; Mercurio, C.; Bigogno, C.; Dondio, G.; Hamel, E.; Lavia,
P.; Silvestri, R. New indole tubulin assembly inhibitors cause stable
arrest of mitotic progression, enhanced stimulation of natural killer cell
cytotoxic activity and repression of Hedgehog-dependent cancer. J.
Med. Chem. 2015, 58, 5789−5807.
Exploring the effect of 2,3,4-trimethoxy-phenyl moiety as a component
of indolephenstatins. Eur. J. Med. Chem. 2010, 45, 588−597.
(c) Brancale, A.; Silvestri, R. Indole, a core nucleus for potent
inhibitors of tubulin polymerization. Med. Res. Rev. 2007, 27, 209−238.
(d) Ray, K.; Bhattacharyya, B.; Biswas, B. B. Role of B-ring of
colchicine in its binding to tubulin. J. Biol. Chem. 1981, 256, 6241−
6244.
(10) Pettit, G. R.; Singh, S. B.; Hamel, E.; Lin, C. M.; Alberts, D. S.;
Garcia-Kendal, D. Isolation and structure of the strong cell growth and
tubulin inhibitor combretastatin A-4. Experientia 1989, 45, 209−211.
(11) (a) Hamel, E. Antimitotic natural products and their
interactions with tubulin. Med. Res. Rev. 1996, 16, 207−231. (b) Jordan,
M. A.; Wilson, L. Microtubules as a target for anticancer drugs. Nat.
Rev. Cancer 2004, 4, 253−265. (c) Woods, J. A.; Hadfield, J. A.; Pettit,
G. R.; Fox, B. W.; McGown, A. T. The interaction with tubulin of a
series of stilbenes based on combretastatin A-4. Br. J. Cancer 1995, 71,
705−711.
(12) Lin, C. M.; Ho, H. H.; Pettit, G. R.; Hamel, E. Antimitotic
natural products combretastatin A-4 and combretastatin A-2: studies
on the mechanism of their inhibition of the binding of colchicine to
tubulin. Biochemistry 1989, 28, 6984−6991.
(13) Patterson, D. M.; Rustin, G. J. S. Vascular damaging agents. Clin.
Oncol. 2007, 19, 443−456.
(14) Nagaiah, G.; Remick, S. C. CA-4 phosphate: a novel vascular
disrupting agent. Future Oncol. 2010, 6, 1219−1228.
(15) (a) Dowlati, A.; Robertson, K.; Cooney, M.; Petros, W. P.;
Stratford, M.; Jesberger, J.; Rafie, N.; Overmoyer, B.; Makkar, V.;
Stambler, B.; Taylor, A.; Waas, J.; Lewin, J. S.; McCrae, K. R.; Remick,
S. C. A phase I pharmacokinetic and translational study of the novel
vascular targeting agent combretastatin A-4 phosphate on a single-dose
intravenous schedule in patients with advanced cancer. Cancer Res.
2002, 62, 3408−3416. (b) Rustin, G. J.; Galbraith, S. M.; Anderson,
H.; Stratford, M.; Folkes, L. K.; Sena, L.; Gumbrell, L.; Price, P. M.
Phase I clinical trial of weekly CA-4 phosphate: clinical and
pharmacokinetic results. J. Clin. Oncol. 2003, 21, 2815−2822.
(16) Hori, K.; Saito, S. Microvascular mechanisms by which the
combretastatin A-4 derivative AC7700 (AVE8062) induces tumour
blood flow stasis. Br. J. Cancer 2003, 89, 1334−1344.
(30) (a) Chen, H.; Li, Y.; Sheng, C.; Lv, Z.; Dong, G.; Wang, T.; Liu,
J.; Zhang, M.; Li, L.; Zhang, T.; Geng, D.; Niu, C.; Li, K. Design and
synthesis of cyclopropylamide analogues of combretastatin-A4 as novel
microtubule-stabilizing agents. J. Med. Chem. 2013, 56, 685−699.
(b) Jonnalagadda, S. S.; ter Haar, E.; Hamel, E.; Lin, C. M.; Magarian,
R. A.; Day, B. W. Synthesis and biological evaluation of 1,1-dichloro-
2,3-diarylcyclopropanes as antitubulin and anti-breast cancer agents.
Bioorg. Med. Chem. 1997, 5, 715−722.
(17) Hua, J.; Sheng, Y.; Pinney, K. G.; Garner, C. M.; Kane, R. R.;
Prezioso, J. A.; Pettit, G. R.; Chaplin, D. J.; Edvardsen, K. Oxi4503, a
novel vascular targeting agent: effects on blood flow and antitumor
activity in comparison to combretastatin A-4 phosphate. Anticancer
Res. 2003, 23, 1433−1440.
(18) Wang, L.; Woods, K. W.; Li, Q.; Barr, K. J.; McCroskey, R. W.;
Hannick, S. M.; Gherke, L.; Credo, R. B.; Hui, Y.-H.; Marsh, K.;
Warner, R.; Lee, J. Y.; Zielinski-Mozng, N.; Frost, D.; Rosenberg, S.
H.; Sham, H. L. Potent, orally active heterocycle-based combretastatin
A-4 analogues: synthesis, structure-activity relationship, pharmacoki-
netics, and in vivo antitumor activity evaluation. J. Med. Chem. 2002,
45, 1697−1711.
(31) Nam, N. − H.; Kim, Y.; You, Y. − J.; Hong, D. − H.; Kim, H. −
M.; Ahn, B. − Z. Synthesis and anti-tumor activity of novel
combretastatins: combretocyclopentenones and related analogues.
Bioorg. Med. Chem. Lett. 2002, 12, 1955−1958.
(32) (a) Nien, C.-Y.; Chen, Y.-C.; Kuo, C.-C.; Hsieh, H.-P.; Chang,
C.-Y.; Wu, J.-S.; Wu, S.-Y.; Liou, J.-P.; Chang, J.-Y. 5-Amino-2-
aroylquinolines as highly potent tubulin polymerization inhibitors. J.
Med. Chem. 2010, 53, 2309−2313. (b) Pettit, G. R.; Toki, B.; Herald,
D. L.; Verdier-Pinard, P.; Boyd, M. R.; Hamel, E.; Pettit, R. K.
Antineoplastic agents. 379. Synthesis of phenstatin phosphate. J. Med.
Chem. 1998, 41, 1688−1695. (c) Liou, J. P.; Chang, C. W.; Song, J.
W.; Yang, Y. N.; Yeh, C. F.; Tseng, H. Y.; Lo, Y. K.; Chang, Y. L.;
Chang, C. M.; Hsieh, H. P. Synthesis and struture-reactivity
relationship of 2-aminobenzophenone derivatives as antimitotic agents.
J. Med. Chem. 2002, 45, 2556−2562.
(19) Tron, G. C.; Pirali, T.; Sorba, G.; Pagliai, F.; Busacca, S.;
Genazzani, A. A. Medicinal chemistry of CA-4: present and future
directions. J. Med. Chem. 2006, 49, 3033−3044.
(20) Aprile, S.; Del Grosso, E.; Tron, G. C.; Grosa, G. In vitro
metabolism study of combretastatin A-4 in rat and human liver
microsomes. Drug Metab. Dispos. 2007, 35, 2252−2261.
(21) Kim, Y.; Nam, N.-H.; You, Y.-J.; Ahn, B.-Z. Synthesis and
cytotoxicity of 3,4-diaryl-2(5H)-furanones. Bioorg. Med. Chem. Lett.
2002, 12, 719−722.
(33) La Regina, G.; Edler, M. C.; Brancale, A.; Kandil, S.; Coluccia,
A.; Piscitelli, F.; Hamel, E.; De Martino, G. D.; Matesanz, R.; Díaz, J.
F.; Scovassi, A. I.; Prosperi, E.; Lavecchia, A.; Novellino, E.; Artico, M.;
Silvestri, R. Arylthioindole inhibitors of tubulin polymerization. 3.
Biological evaluation, structure-activity relationships and molecular
modeling studies. J. Med. Chem. 2007, 50, 2865−2874.
L
DOI: 10.1021/acs.jmedchem.6b00101
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(34) Lawrence, N. J.; Rennison, D.; Woo, M.; McGown, A. T.;
Hadfield, J. A. Antimitotic and cell growth inhibitory properties of
combretastatin A-4-like ethers. Bioorg. Med. Chem. Lett. 2001, 11,
51−54.
(35) Ohsumi, K.; Nakagawa, R.; Fukuda, Y.; Hatanaka, T.; Morinaga,
Y.; Nihei, Y.; Ohishi, K.; Suga, Y.; Akiyama, Y.; Tsuji, T. Novel
combretastatin analogues effective against murine solid tumors: Design
and structure-activity relationships. J. Med. Chem. 1998, 41, 3022−
3032.
(36) (a) Gwaltney, S. L., II; Imade, H. M.; Barr, K. J.; Li, Q.; Gehrke,
L.; Credo, R. B.; Warner, R. B.; Lee, J. Y.; Kovar, P.; Wang, J.; Nukkala,
M. A.; Zielinski, N. A.; Frost, D.; Ng, S.-C.; Sham, H. L. Novel
sulfonate analogues of combretastatin A-4: potent antimitotic agents.
Bioorg. Med. Chem. Lett. 2001, 11, 871−874. (b) Lee, H.-Y.; Chang, J.-
Y.; Nien, C.-Y.; Kuo, C.-C.; Shih, K.-H.; Wu, C.-H.; Chang, C.-Y.; Lai,
W.-Y.; Liou, J.-P. 5-Amino-2-aroylquinolines as highly potent tubulin
polymerization inhibitors. part 2. The impact of bridging groups at
position C-2. J. Med. Chem. 2011, 54, 8517−8525.
(37) Kandekar, S.; Preet, R.; Kashyap, M.; M U, R. P.; Mohapatra, P.;
Das, D.; Satapathy, S. R.; Siddharth, S.; Jain, V.; Choudhuri, M.;
Kundu, C. N.; Guchhait, S. K.; Bharatam, P. V. Structural elaboration
of a natural product: identification of 3,3′-diindolylmethane amino-
phosphonate and urea derivatives as potent anticancer agents.
ChemMedChem 2013, 8, 1873−1884.
producing klebsiella pneumonia. ACS Med. Chem. Lett. 2012, 3, 357−
361.
(49) Ermolat’ev, D. S.; Svidritsky, E. P.; Babaev, E. V.; Van der
Eycken, E. Microwave-assisted synthesis of substituted 2-amino-1H-
imidazoles from imidazo[1,2- a]pyrimidines. Tetrahedron Lett. 2009,
50, 5218−5220.
(50) Santra, A.; Bagdi, A. K.; Majee, A.; Hajra, A. Iron(III)-catalyzed
cascade reaction between nitroolefines and 2-aminopyridines: Syn-
thesis of imidazo [1,2-a] pyridines and easy access towards zolimidine.
Adv. Synth. Catal. 2013, 355, 1065−1070.
(51) Song, G.; Zhang, Y.; Li, X. Rhodium and Iridium complexes of
abnormal N-heterocyclic carbenes derived from Imidazo[1,2-a]-
pyridine. Organometallics 2008, 27, 1936−1943.
(52) Bagdi, A. K.; Rahman, M.; Santra, S.; Majee, A.; Hajra, A.
Copper-catalyzed synthesis of imidazo [1,2-a]pyridines through
tandem imine formation-oxidative cyclization under ambient air:
One-step synthesis of zolimidine on a gram-scale. Adv. Synth. Catal.
2013, 355, 1741−1747.
(53) Fu, H. Y.; Chen, L.; Doucet, H. Phosphine-free palladium-
catalyzed direct arylation of imidazo [1,2-a] pyridines with aryl
bromides at low catalyst loading. J. Org. Chem. 2012, 77, 4473−4478.
(54) Wang, H.; Ma, Y.; Tian, H.; Yu, A.; Chang, J.; Wu, Y. Tetrabutyl
ammonium bromide-mediated benzylation of phenols in water under
mild condition. Tetrahedron 2014, 70, 2669−2673.
́
(55) Ermolat’ev, D. S.; Gimenez, V. N.; Babaev, E. V.; Van der
(38) Sanghai, N.; Jain, V.; Preet, R.; Kandekar, S.; Das, S.; Trivedi,
N.; Mohapatra, P.; Priyadarshani, G.; Kashyap, M.; Das, D.; Satapathy,
S. R.; Siddharth, S.; Guchhait, S. K.; Kundu, C. N.; Bharatam, P. V.
Combretastatin A-4 inspired novel 2-aryl-3-arylamino-imidazo-pyr-
idines/pyrazines as tubulin polymerization inhibitors, antimitotic and
anticancer agents. Med. Chem. Commun. 2014, 5, 766−782.
(39) Al-Mourabit, A.; Zancanella, M. A.; Tilvi, S.; Romo, D.
Biosynthesis, asymmetric synthesis, and pharmacology, including
cellular targets, of the pyrrole-2-aminoimidazole marine alkaloids.
Nat. Prod. Rep. 2011, 28, 1229−1260.
Eycken, E. Efficient Pd(0)-mediated microwave-assisted arylation of 2-
substituted imidazo[1,2-a]pyrimidines. J. Comb. Chem. 2006, 8, 659−
663.
(56) Cortese, F.; Bhattacharyya, B.; Wolff, J. Podophyllotoxin as a
probe for the colchicine binding site of tubulin. J. Biol. Chem. 1977,
252, 1134−1140.
(57) Sackett, D. L. Podophyllotoxin, steganacin and combretastatin:
natural products that bind at the colchicine site of tubulin. Pharmacol.
Ther. 1993, 59, 163−228.
(58) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.;
Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. New
colorimetric cytotoxicity assay for anticancer-drug screening. J. Natl.
Cancer. Inst. 1990, 82, 1107−1112.
(59) Kapoor, S.; Panda, D. Kinetic stabilization of microtubule
dynamics by indanocine perturbs EB1 localization, induces defects in
cell polarity and inhibits migration of MDA-MB-231 cells. Biochem.
Pharmacol. 2012, 83, 1495−1506.
(60) Venghateri, J. B.; Gupta, T. K.; Verma, P. J.; Kunwar, A.; Panda,
D. Ansamitocin P3 depolymerizes microtubules and induces apoptosis
by binding to tubulin at the vinblastine site. PLoS One 2013, 8, e75182.
(61) Hamel, E.; Lin, C. M. Glutamate-induced polymerization of
tubulin: characteristics of the reaction and application to the large-scale
purification of tubulin. Arch. Biochem. Biophys. 1981, 209, 29−40.
̌
̌
(40) Zidar, N.; Montalvao, S.; Hodnik, Z.; Nawrot, D. A.; Zula, A.;
̃
̌ ̌ ̌
Ilas, J.; Kikelj, D.; Tammela, P.; Masic, L. P. Antimicrobial activity of
the marine alkaloids, clathrodin and oroidin, and their synthetic
analogues. Mar. Drugs 2014, 12, 940−963.
(41) Keifer, P. A.; Schwartz, R. E.; Koker, M. E. S.; Hughes, J. R. G.;
Rittschof, D.; Rinehart, K. L. Bioactive bromopyrrole metabolites from
the caribbean sponge Agelas conifera. J. Org. Chem. 1991, 56, 2965−
2975.
(42) Walker, R. P.; Faulkner, D. J.; Van Engen, D. V.; Clardy, J.
Sceptrin, an antimicrobial agent from the sponge Agelas sceptrum. J.
Am. Chem. Soc. 1981, 103, 6772−6773.
(43) Kobayashi, J.; Tsuda, M.; Murayama, T.; Nakamura, H.;
Ohizumi, Y.; Ishibashi, M.; Iwamura, M.; Ohta, T.; Nozoe, S.
Ageliferins, potent actomyosin Atpase activators from the Okinawan
marine sponge. Tetrahedron 1990, 46, 5579−5586.
(44) Huigens, R. W., III; Richards, J. J.; Parise, G.; Ballard, T. E.;
Zeng, W.; Deora, R.; Melander, C. Inhibition of pseudomonas
aeruginosa biofilm formation with bromoageliferin analogues. J. Am.
Chem. Soc. 2007, 129, 6966−6967.
(45) Nishimura, T.; Kitajima, K. Reaction of guanidines with a-
diketones. Syntheses of 4,5-disubstituted-2- aminoimidazoles and 2,6-
unsymmetrically substituted imidazo[4,5-d]imidazoles. J. Org. Chem.
1979, 44, 818−824.
(46) Bhaskar, V. H.; Sarath, K.; Kumar, M. Synthesis and
antiinflammatory activity of some 2-(substituted phenyl)-3-(4,5-
diphenyl-1H-imidazol-2-yl)-1,3-thiazolidin-4-one. Asian J. Chem.
2008, 20, 5133−5138.
(47) (a) Soh, C. H.; Chui, W. K.; Lam, Y. An efficient and
expeditious synthesis of di- and monosubstituted 2-aminoimidazoles. J.
Comb. Chem. 2008, 10, 118−122. (b) Little, T. L.; Webber, S. E. A
simple and practical synthesis of 2-aminoimidazoles. J. Org. Chem.
1994, 59, 7299−7305.
(48) Worthington, R. J.; Bunders, C. A.; Reed, C. S.; Melander, C.
Small molecule suppression of carbapenem resistance in NDM-1
M
DOI: 10.1021/acs.jmedchem.6b00101
J. Med. Chem. XXXX, XXX, XXX−XXX