Synthesis and biological evaluations of 1,2-diaryl pyrroles as analogues of combretastatin A-4

Article abstract of DOI:10.1111/cbdd.12617

A series of novel 1,2-diaryl pyrroles as analogues of combretastatin A-4 (CA-4, 1a) were synthesized and evaluated for their antitumour potential against three cancer cell lines. Most compounds exhibited growth inhibition against all of the cancer cell lines. Compound 7q not only exhibited prominent antitumour efficacy with IC₅₀ values of 0.390 μm in SGC-7901, 0.070 μm in HT-1080 and 0.045 μm in KB cell lines but also showed low activity with IC₅₀ values of 30.08 μm in normal L929 cell line. Moreover, compound 7q inhibited tubulin polymerization into microtubules and caused microtubule destabilization. A molecular docking study of 7q was performed to determine its binding mode at the colchicine site in the tubulin dimer. 1,2-Diaryl pyrroles as combretastatin A-4 analogues were synthesized and evaluated for anti-proliferative activities. Compound 7q exhibited antitubulin activity.

Full text of DOI:10.1111/cbdd.12617

Chem Biol Drug Des 2015; 86: 1541–1547
Research Article
Synthesis and Biological Evaluations of 1,2-Diaryl
Pyrroles as Analogues of Combretastatin A-4
1
,2
2
3
Jun Sun , Lei Chen , Chunjiang Liu ,
dividing cells in the G2/M phase of the cell cycle, resulting
in mitotic catastrophe and, ultimately, apoptotic cell death
2
3
2
Zhan Wang , Daiying Zuo , Jiatong Pan ,
3
2,4
3,
(2,3). Tubulin has at least four well-characterized binding
Huan Qi , Kai Bao , Yingliang Wu * and
2
,
sites: the laulimalide domain, the taxane domain, the vinca
domain and the colchicine domain, and many compounds
interact with tubulin at these known sites (4).
Weige Zhang *
1
Clinical Pharmacology Laboratory, Henan Province
People’s Hospital, Zhengzhou University People’s Hospital,
7
Weiwu Road, Jinshui District, Zhengzhou 450003, China
Combretastatin A-4 (Figure 1, 1a) was originally isolated
from the African willow tree combretum caffrum by Pettit
et al. in 1989; it inhibits the polymerization of tubulin by
strongly binding to the colchicine site and shows potent
cytotoxicity against a variety of human cancer cell lines
2
Key Laboratory of Structure-Based Drug Design and
Discovery, Ministry of Education, Shenyang
Pharmaceutical University, 103 Wenhua Road, Shenhe
District, Shenyang 110016, China
3
Department of Pharmacology, Shenyang Pharmaceutical
University, 103 Wenhua Road, Shenhe District, Shenyang
(5–8). The sodium phosphate of CA-4 (CA-4P, 1b), as a
water-soluble prodrug, is currently under Phase II/III clinical
evaluation as an antitumour agent. Many structure-activity
relationship (SAR) studies indicate that a 3,4,5-trimethoxy
substituted A-ring, a 4-methoxy substituted B-ring and a
cis-olefinic linker are fundamental requirements for potent
cytotoxicity (9). Up to now, several five-membered hetero-
cyclic analogues of CA-4 (1a), such as pyrazole, pyrazo-
line, cyclohexenone and oxadiazoline (10–14), have been
reported as cis-restricted, biologically active analogues of
CA-4 (1a).
1
10016, China
4
Division of Hematology/Oncology, Department of
Medicine, Beth Israel Deaconess Medical Center and
Harvard Medical School, Boston, MA 02215, USA
*
Corresponding author: Weige Zhang,
zhangweige2000@sina.com; Yingliang Wu,
yingliang_1016@163.com
A series of novel 1,2-diaryl pyrroles as analogues of
combretastatin A-4 (CA-4, 1a) were synthesized and
evaluated for their antitumour potential against three
cancer cell lines. Most compounds exhibited growth
inhibition against all of the cancer cell lines. Com-
pound 7q not only exhibited prominent antitumour
efficacy with IC50 values of 0.390 lM in SGC-7901,
Recently, several pyrrole-based CA-4 analogues, including
2,3-diaryl pyrroles (Figure 1, 2 and 3), 3,4-diaryl pyrroles (4
and 5) and 2,4-diaryl pyrroles (6), were designed, synthe-
sized and evaluated (15–17). However, there has been no
prior report of 1,2-diaryl pyrroles as analogues of CA-4 to
date. Here, to understand the SAR of pyrrole-based CA-4
analogues more clearly, we report the synthesis and
antiproliferative activity of 5-(un)substituted-1,2-diaryl pyr-
roles (Scheme 1, 7a–u) with the A-ring at the 2-position
and 1,2-diaryl pyrroles (Scheme 2, 8a–d) with the A-ring at
the 1-N position. The representative compound 7q was
investigated for the inhibition of tubulin polymerization and
in vitro immunofluorescence staining. Additionally, molecu-
lar docking studies have been performed to understand the
interaction between tubulin and the most active inhibitor 7q.
0
.070 lM in HT-1080 and 0.045 lM in KB cell lines but
also showed low activity with IC50 values of 30.08 lM
in normal L929 cell line. Moreover, compound 7q inhib-
ited tubulin polymerization into microtubules and
caused microtubule destabilization. A molecular dock-
ing study of 7q was performed to determine its binding
mode at the colchicine site in the tubulin dimer.
Key words: 1,2-diaryl pyrroles, antitumour, combretastatin
A-4, molecular docking study, tubulin polymerization
Received 20 May 2015, revised 13 July 2015 and accepted
for publication 14 July 2015
Experimental
Microtubules, which are primarily composed of the
a,b-heterodimeric protein tubulin, are a component of the
mitotic spindle and are essential to the mitotic division of
cells (1). Interfering with either the assembly or disassem-
bly of microtubules and spindle poisons and arrests
Chemistry
General
Unless otherwise indicated, all solvents and organic
reagents were obtained from commercially available
ª 2015 John Wiley & Sons A/S. doi: 10.1111/cbdd.12617
1541

Sun et al.
Figure 1: Structure of CA-4 and some
pyrrole-based CA-4 analogues.
sources and were used without purification. The reaction
process was monitored using TLC with silica gel plates
excess CH
water, dried over anhydrous Na
under vacuum.
2
Cl
2
. The solution was washed with deionized
2 4
SO , filtered and dried
(thickness 0.25 mm, Indicator F-254) under UV light. The
purification of the products was performed using column
ꢀ
chromatography (60 A, 200–300 mesh; Qingdao Ocean
Chemicals, Qingdao, China) or silica gel plates (0.25 mm
layer; Qingdao Ocean Chemicals) with the designated
solvents. The mass spectra (MS) were measured using
an Agilent 1100-sl mass spectrometer with an electro-
spray ionization (ESI) source from Agilent Co., Ltd (Santa
Clara, CA, USA). High resolution accurate mass determi-
nations (HRMS) for all final target compounds were
obtained using a Bruker Micromass Time of Flight mass
Biochemistry methods
Cell culture
The gastric adenocarcinoma SGC-7901 cells, fibrosar-
coma HT-1080 cells and mouth epidermal carcinoma KB
cells were cultured in RPMI-1640 medium containing 10%
FBS, 100 U/mL penicillin and 100 U/mL streptomycin at
3
2
7 °C in humidified atmosphere with 5% CO . All of the
1
spectrometer equipped with ESI. H NMR was taken in
cells were purchased from the American Type Culture Col-
lection (ATCC, Manassas, VA, USA).
3
CDCl solution on Bruker ARX-300, Bruker AV-400, and
Bruker AV-600 spectrometers with TMS as an internal
standard. The chemical shifts were reported in p.p.m.
downfield from tetramethylsilane and proton-proton cou-
1
Antiproliferative activity assay
pling constants (J) in Hz. For
H
NMR: CDCl
3
1
3
13
The in vitro antiproliferative activities of CA-4 (1a) and all
of the target compounds were determined using an
MTT (Sigma-Aldrich, St. Louis, MO, USA) assay. Briefly,
7
.26 p.p.m.; for
C NMR:
CDCl3 77.0 p.p.m. The
melting points were measured using a X-4 Micro-Melting
point detector form Beijing Tech Instrument Co., Ltd
the cells were seeded into 96-well plates at a density of
(Beijing, China), without correction.
4
1
–3 9 10 /well (depending on the cell growth rate) and
cultured for 24 h. Then, triplicate wells were treated with
the media and reagents. After 72 h of incubation at
General procedure for the synthesis of the amides
compounds 12a–o, 12r–s and 15a–d)
The arylamine (0.48 mmol), 4-oxobutanoic
0.48 mmol), DCC (0.96 mmol) and DMAP (0.096 mmol)
were stirred in CH Cl (10 mL) at room temperature for
3
2
7 °C in 5% CO , the drug containing medium was
(
removed and replaced with 100 lL of fresh medium
containing 5 mg/mL MTT solution. Four hours later, the
medium with MTT was removed, and 100 lL of DMSO
were added to each well. The plates were gently agi-
tated until the purple formazan crystals were dissolved,
and OD490 was determined using a microplate reader
acid
(
2
2
1
.5 h; the mixture was poured into water and extracted
with CH Cl . The combined organic layers were washed
2
2
2 4
with water, dried over anhydrous Na SO , and the solvent
(MK3; Thermo, Waltham, MA, USA). The data were cal-
was then evaporated under reduced pressure.
culated and plotted as per cent viability compared to
the control. The 50% inhibitory concentration (IC50) was
defined as the concentration that reduced the absor-
bance of the untreated wells by 50% of the vehicle in
the MTT assay.
General procedure for the synthesis of
compounds 7a–o, 7r–s and 8a–d
The amide (0.27 mmol) and Lawesson’s reagent
(0.27 mmol) in anhydrous toluene (7 mL) were heated to
reflux under a nitrogen atmosphere. After 0.5 h, the sol-
vent was removed, and the residue was dissolved in
Cytotoxic potential of 7q and 8b against L929 cell were
carried out in the similar way.
1
542
Chem Biol Drug Des 2015; 86: 1541–1547

Combretastatin A-4 Analogues
Scheme 1: Reagents and conditions: (i)
MeLi, anhydrous tetrahydrofuran, ꢀ20 °C,
2
for 2 h; (ii) OHC-COOH.H O, AcOH, reflux
for 12 h, then Zn powder, AcOH, r.t., for
h; (iii) arylamine, DCC, DMAP, CH Cl , r.t.,
for 0.5 h; (iv) Lawesson’s reagent,
anhydrous toluene, N , reflux for 0.5 h; (v)
2
2
2
2
Zn powder, AcOH, r.t., for 1 h; (vi) POCl3,
anhydrous DMF, 0 °C, then 60 °C, for 3 h.
Scheme 2: Reagents and conditions: (i)
OHC-COOH.H
then Zn powder, AcOH, r.t., for 2 h; (ii)
arylamine, DCC, DMAP, CH Cl , r.t., for
.5 h; (iii) Lawesson’s reagent, anhydrous
toluene, N , reflux for 0.5 h.
2
O, AcOH, reflux for 12 h,
2
2
0
2
In vitro tubulin polymerization assay
The effect of compounds 7q and CA-4 (1a) on the
The control and treated cells were fixed with 4% formalde-
hyde in PBS for 30 min at ꢀ20 °C, then washed twice
with PBS and permeabilized with 0.1% (v/v) Triton X-100
in PBS for 5 min. Then, the cells were blocked with 5%
bovine serum albumin (BSA) in PBS for 10 min. The pri-
mary a-tubulin antibody (Santa Cruz, CA) was diluted
(1:100) with 2% BSA in PBS and incubated overnight at
4 °C. The cells were washed with PBS to remove
unbound primary antibody and then the cells were incu-
bated with FITC-conjugated anti-mouse secondary anti-
body, diluted (1:1000) with 2% BSA in PBS, for 3 h at
37 °C. The cells were washed with PBS to remove
unbound secondary antibody, nucleus was stained with
4,6-diamino-2-phenolindol dihydrochloride (DAPI), and
then, the immunofluorescence was detected using a fluo-
rescence microscope (Olympus, Tokyo, Japan).
polymerization of tubulin was determined using
fluorescence-based tubulin polymerization assay kit (Cat.
BK011P; Cytoskeleton, Denver, CO, USA) according to
the manufacturer’s protocol. Tubulin was resuspended in
ice cold G-PEM buffer (80 mM PIPES, 2 mM MgCl
.5 mM EGTA and 1 mM GTP, 20% (v/v) glycerol) and
a
#
2
,
0
added to the wells on a 96-well plate containing the desig-
nated concentration of 7q, CA-4 (1a) or the vehicle. The
samples were mixed well, and the tubulin assembly was
monitored (emission wavelength is 450 nm; excitation
wavelength is 360 nm) at 1 min intervals for 90 min at
37 °C using a plate reader (FASCalibur; BD Biosciences,
Franklin Lakes, NJ, USA), and the increasing absorbance
was transformed into the extent of polymerization of tubu-
lin (%) using computer calculations, while IC50 values were
calculated using GRAPHPAD PRISM software.
Docking studies
The docking studies were performed using ACCELRYS DISCOV-
ERY STUDIO 3.0. The X-ray crystal structures of tubulin in com-
plex with DMDA-colchicine (PDB code: 1SA0) were
downloaded from the Protein Data Bank (http://
www.rcsb.org/pdb) for possible use in the modelling studies
and were also minimized using the CHARMm force field
(19). In the docking process, chain A and chain B of the
crystal structure were used, and the protein protocol was
Immunofluorescence staining
Immunofluorescence studies were performed to detect
microtubule-associated tubulin protein after exposure to
CA-4 (1a) and the selected compounds 7q (18). The
HT-1080 cells were seeded at 1 9 10 per well and
grown for 24 h. The cells were treated with the vehicle or
4
twice the IC50 concentration of CA-4 (1a) or 7q for 48 h.
Chem Biol Drug Des 2015; 86: 1541–1547
1543

Sun et al.
prepared via several operations. Then, the receptor model
was minimized using the CHARMm force field, and a bind-
12r–s, which were treated with Lawesson’s reagent to
afford 1,2-diaryl pyrroles 7a–o and 7r–s (23). The reduction
of the nitro derivatives 7n and 7o with Zn powder in AcOH
gave the amino derivatives 7q and 7p (24). Vilsmeier-Haack
ꢀ
ing sphere with a radius of 9.0 A was defined using the orig-
inal ligand (DAMA-colchicine) as the binding site. The amino
acid residues in this area were mainly considered. In this
binding domain, there was no ion to be considered. CA-4
formylation of 7r and 7s with DMF/POCl
myl-1,2-diaryl pyrroles 7u and 7t (25).
3
produced 5-for-
(
1a) and 7q were drawn with Chemdraw and fully minimized
using the CHARMm force field. Finally, they were docked
into the binding site using the CDOCKER protocol with the
default settings.
Target compounds 8a–d were synthesized using the same
methods with different starting materials (Scheme 2).
Biological studies
Results and Discussion
In vitro anti-proliferative activity
Chemistry
The target compounds and reference compound CA-4
were screened for their anti-proliferative activity against
three human cancer cell lines: gastric adenocarcinoma
SGC-7901 cells, fibrosarcoma HT-1080 cells and mouth
epidermal carcinoma KB cells, using a standard MTT
assay. The drug concentration required to inhibit cell
growth by 50% (IC50) was determined, and the values are
shown in Table 1.
The compounds 7a–u were prepared from 3,4,5-
trimethoxybenzoic acid (9) following the synthetic routes
summarized in Scheme 1. First, acetophenone 10 was syn-
thesized by treating 9 with MeLi in anhydrous tetrahydro-
furan (20). An aldol condensation of 10 was performed in
the presence of an excess of glyoxylic acid monohydrate in
refluxing acetic acid for at least 12 h to prepare the acrylic
acid, which was subsequently reduced to 11 using Zn pow-
der (21,22). 4-Oxobutanoic acid, 11, reacted with diverse
arylamines to give the corresponding amides 12a–o and
Most of the target compounds showed moderate-to-excel-
lent antiproliferative activity against different cancer cells
a
Table 1: Anti-proliferative activities of tar-
get compounds as measured using the
MTT test in three human tumour cell lines
IC50 (lM)
Compounds
SGC-7901
HT-1080
KB
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
8
8
8
8
a
b
c
d
e
f
>100
>100
>100
16.61 ꢁ 2.85
46.13 ꢁ 4.41
13.89 ꢁ 1.73
92.45 ꢁ 9.48
70.19 ꢁ 6.32
>100
13.00 ꢁ 0.86
32.73 ꢁ 2.24
>100
6.46 ꢁ 0.59
>100
>100
>100
>100
5.41 ꢁ 0.50
11.26 ꢁ 1.03
33.61 ꢁ 5.02
9.37 ꢁ 0.96
>100
3.68 ꢁ 0.44
7.38 ꢁ 0.88
12.03 ꢁ 1.12
>100
>100
>100
0.39 ꢁ 0.02
4.48 ꢁ 0.56
>100
1.24 ꢁ 0.16
17.05 ꢁ 1.37
70.76 ꢁ 6.54
g
h
25.23 ꢁ 2.15
10.31 ꢁ 1.26
8.15 ꢁ 0.86
2.16 ꢁ 0.20
2.28 ꢁ 0.19
76.23 ꢁ 7.25
i
j
>100
16.34 ꢁ 1.51
2.20 ꢁ 0.21
2.63 ꢁ 0.31
85.63 ꢁ 9.37
k
l
m
n
o
p
q
r
>100
>100
>100
>100
56.01 ꢁ 3.46
20.10 ꢁ 2.93
0.070 ꢁ 0.005
6.44 ꢁ 0.68
0.045 ꢁ 0.004
2.44 ꢁ 0.27
s
t
>100
>100
>100
>100
>100
>100
u
a
b
c
d
55.21 ꢁ 7.83
5.23 ꢁ 4.72
2.13 ꢁ 0.24
0.20 ꢁ 0.03
10.36 ꢁ 1.43
3.23 ꢁ 3.46
0.024 ꢁ 0.002
0.70 ꢁ 0.07
1.95 ꢁ 0.24
2.15 ꢁ 1.95
1.53 ꢁ 1.39
0.016 ꢁ 0.002
1.56 ꢁ 0.18
1.01 ꢁ 0.09
10.93 ꢁ 9.57
2.16 ꢁ 1.92
b
CA-4
0.0073 ꢁ 0.0040
Bold values show the IC50 values of target compounds which have excellent activity.
a
IC50: concentration of the compound (lM) that produces 50% cell growth inhibition after
7
2 h of drug exposure, as determined using the MTT assay. Each experiment was per-
formed in triplicate.
b
Used as a positive control.
1
544
Chem Biol Drug Des 2015; 86: 1541–1547

Combretastatin A-4 Analogues
0
.8
with potencies in the single-digit lM range, indicating that
the introduction of the pyrrole moiety as a suitable mimic for
the cis-olefin present in CA-4 maintained potent anti-prolif-
erative activity. Compound 7q, which contains a methoxy
group at C4 and an amino group at C3 of the B-ring,
showed the most potent activity against SGC-7901, HT-
0.7
Control
0.6
0.5
0.4
(
1a)
7q
7q
7
7
q
q
1
0
080 and KB cells, with IC50 values of 0.39, 0.070 and
0.3
.045 lM, respectively. The activity decreased 9–50-fold
0
.2
.1
2
when the 3-NH was changed to 3-OMe (7k) or 3-OH (7l),
0
the antiproliferative activity of compound 7s was almost lost
0
10
20
30
40
50
60
70
when 3-NH was removed. When the electron-withdrawing
Time (minutes)
2
was used in place of the original electron-donating, the
compounds, such as 7b, 7e, 7f, 7i, 7j, 7r, displayed a dra-
matic decrease in their biological activities, some of them
lost completely. In addition, a similar phenomenon was
observed for compound 7u, which contains a formyl at the
C5 position of the pyrrole moiety. When the A-ring and B-
ring changed positions, the results also indicated that the
electron-donating ability was stronger than the electron-
withdrawing. Compounds 8a and 8b were better than com-
pounds 8c and 8d. Consequently, this finding indicates that
the combination of the 3,4,5-trimethoxyphenyl with the 3-
amino-4-methoxyphenyl is the most favourable modification
for the anti-proliferative activity of 1,2-diaryl pyrroles.
Figure 2: Effects of 7q on tubulin polymerization. Tubulin had
been preincubated for 1 min with 7q at 2.5 lM, 5.0 lM, 10.0 lM
and 15.0 lM, CA-4 at 2.0 lM, Taxol at 5.0 lM or vehicle DMSO at
room temperature before GTP was added to start the tubulin
polymerization reactions. The reaction was monitored at 37 °C.
Taxol and CA-4 were included as positive controls.
stained with a tubulin antibodies indicated a well-organized
microtubular network in the control cells (Figure 3). In addi-
tion to CA-4, compound 7q could significantly impact the
microtubule network in HT-1080 cells and induce cell
rounding, detachment and a loss of the radial distribution of
structured microtubules at a concentration that was twice
the IC50. These results indicate that compound 7q can
destabilize microtubules.
The therapeutic window is of great significance for anti-
cancer agents. For further understanding the cytotoxic
potential of these target compounds for normal human
cells, two of the most active compounds, 7q and 8b, were
assayed in vitro against L929 cell, a normal fibroblast cell.
CA-4 (1a) was also used as a positive control. The IC50
values were found to be 30.08, 0.75 and 0.82 lM, respec-
tively. It was noticeable that IC50 values were 77- to 668-
fold higher than that against the tested tumour cells for
Molecular modelling
To better understand the interaction of the newly synthe-
sized compounds with tubulin, the potential binding mode
7q, 0.38- to 3.75-fold higher for 8b and 34- to 112-fold
higher for CA-4 (1a), thus compound 7q showed more
selective antiproliferative activity than CA-4 (1a) and 8b.
Inhibition of tubulin polymerization
To confirm that the antiproliferative activities of these
compounds were related to the microtubule system, com-
pound 7q, one of the most active compounds in the series
of CA-4 analogues, was evaluated for its inhibition of
tubulin polymerization (Figure 2). Compound 7q exhibited
antitubulin activity with an IC50 value of 10.06 lM, which is
less potent than that of CA-4 (IC50 = 0.92 lM). Further-
more, compound 7q inhibited the polymerization of tubulin
in a concentration-dependent manner. Treatment with 2.5,
5.0, 10.0 and 15.0 lM of compound 7q, inhibited tubulin
polymerization by 17.5%, 35.2%, 48.0% and 60.5%,
respectively.
Figure 3: Effects of CA-4 and 7q on the microtubule network of
HT-1080 cancer cells. Untreated cells (control) and cells treated
with CA-4 or 7q at concentration of twice the IC50 for 12 h were
fixed and stained with anti-a-tubulin-FITC specific antibodies
followed by DAPI (4 ,6-diamidino-2-phenylindole). Microtubules
and unassembled tubulin are shown in green, and nuclei stained
with DAPI are shown in blue.
Analysis of immunofluorescence staining
We evaluated the activity of compound 7q on the micro-
tubule network via tubulin immunostaining to further identify
the cellular changes that are possibly relevant to its antipro-
liferative activity. A microscopic analysis of HT-1080 cells
0
Chem Biol Drug Des 2015; 86: 1541–1547
1545

Sun et al.
A
B
Figure 4: (A) Predicted modes for compound 7q (purple) and CA-4 (orange) binding in the colchicine-binding site of tubulin (PDB code:
SA0), and overlapping with DAMA-colchicine (cyan, the native ligand of 1SA0); (B) Docking conformation of compound 7q (purple) in the
1
colchicine-binding site of tubulin. The dashed green lines represent hydrogen bonds, and the distance between the ligands and the
ꢀ
protein is <2.8 A. The surrounding amino acid side chains are shown in grey stick format and labelled.
of compound 7q at the colchicine site in the tubulin dimer
was investigated using the CDOCKER program in DISCOV-
ERY STUDIO 3.0 (Accelrys, San Diego, CA, USA) software
with the tubulin crystal structure (PDB: 1SA0), as in our
former study (26). Both compound 7q and CA-4 (1a) were
docked into tubulin. From Figure 4A, we know that com-
pound 7q (purple) could be perfectly superimposed with
CA-4 (orange) and DAMA-colchicine (cyan) in their binding
orientations. Hydrogen bonds were present between
Asnb258 of tubulin and the amino group of compound 7q,
the hydroxyl group of CA-4 (1a) and the carbonyl group
on ring-C of DAMA-colchicine. Additionally, the trimethoxy-
benzene rings of these compounds are well overlapped
with each other, and hydrogen bonds formed between the
oxygen atoms of two methoxyl groups and the key residue
in the active site (Cysb241). In the quantitative docking
studies, the CDOCKER docking score of 7q is ꢀ11.87,
while that of CA-4 (1a) is ꢀ24.58. This result demon-
strated that compound 7q could bind tightly, but less than
CA-4 (1a). Moreover, compound 7n, one of the lowest
active compounds, was evaluated by using the same
score function. The CDOCKER docking score of 7n
was found to be ꢀ2.14, which is much lower than that of
the 2-position and 3-amino-4-methoxyphenyl (B-ring) at
the 1-N position, showed the highest activities with IC50
values at the sub-micromolar level and more selective
antiproliferative activity than CA-4 (1a) and 8b when they
were assayed in vitro against L929 cell, a normal fibrob-
last cell. In vitro immunofluorescence staining and tubulin
polymerization assays indicated that compound 7q
caused microtubule destabilization and inhibited tubulin
polymerization into microtubules. All of these results are
well-supported by molecular modelling studies, where it
was observed that 7q and CA-4 superimposed well with
each other and with DAMA-colchicine in their binding ori-
entations. These compounds can be considered more
stable analogues of combretastatin A-4 in further biologi-
cal evaluation.
Acknowledgments
We gratefully acknowledge the National S&T Major Project
(2012ZX09103101-060), the National Natural Science
Foundation of China (30973614) and the Natural Science
Foundation of Liaoning Province (2013010434-401) for
generous financial support. This work was also supported
by the Program for Innovative Research Team of the Min-
istry of Education and Program for Liaoning Innovative
Research Team in University.
7
q. The results of the computational modelling analysis
further support the results of the inhibition of tubulin
polymerization.
Conclusions
References
In summary, we have synthesized new 1,2-diaryl pyrroles
as analogues of CA-4, where the olefinic core structure
of CA-4 was replaced by 5-(un)substituted-pyrrole. The
evaluation of the antiproliferative activities of these new
compounds against three human cancer cell lines (SGC-
1. Downing K.H. (2000) Structural basis for the interac-
tion of tubulin with proteins and drugs that affect
microtubule dynamics. Annu Rev Cell Dev Biol;16:89–
111.
7
7
901, HT-1080 and KB) demonstrated that compounds
k, 7q, 8a and 8b exhibited significant antiproliferative
2. Salum L.B., Altei W.F., Chiaradia L.D., Cordeiro
M.N.S., Canevarolo R.R., Melo C.P., Winter S.E. et al.
(2013) Cytotoxic 3,4,5-trimethoxychalcones as mitotic
activities. 7q, with the 3,4,5-trimethoxyphenyl (A-ring) at
546
1
Chem Biol Drug Des 2015; 86: 1541–1547

Combretastatin A-4 Analogues
cytotoxic agents. Bioorg Med
arresters and cell migration inhibitors. Eur J Med
Chem;63:501–510.
totic
and
Chem;14:4627–4638.
3
4
5
. Wienecke A., Bacher G. (2009) Indibulin, a novel micro-
tubule inhibitor, discriminates between mature neuronal
and nonneuronal tubulin. Cancer Res;69:171–177.
. Lu Y., Chen J., Xiao M., Li W., Miller D.D. (2012) An
overview of tubulin inhibitors that interact with the col-
chicine binding site. Pharm Res;29:2943–2971.
. Pettit G.R., Singh S.B., Niven M.L., Hamel E., Schmidt
J.M. (1987) Isolation, structure, and synthesis of com-
bretastatins A-1 and B-1, potent new inhibitors of
microtubule assembly, derived from Combretum caf-
frum. J Nat Prod;50:119–131.
. Dorr R.T., Dvorakova K., Snead K., Alberts D.S., Sal-
mon S.E., Pettit G.R. (1996) Antitumor activity of com-
bretastatin-A4 phosphate, a natural product tubulin
inhibitor. Invest New Drugs;14:131–137.
. Pettit G.R., Singh S.B., Hamel E., Lin C.M., Alberts
D.S., Garcia-Kendall D. (1989) Isolation and structure
of the strong cell growth and tubulin inhibitor combre-
tastatin A-4. Experientia;45:209–211.
16. van Loon A.A., Holton M.K., Downey C.R., White T.M.,
Rolph C.E., Bruening S.R., Li G., Delaney K.M., Pelkey
S.J., Pelkey E.T. (2014) Preparation of dibenzo[e, g]
isoindol-1-ones via Scholl-type oxidative cyclization
reactions. J Org Chem;79:8049–8058.
17. Hong D., Zhu Y., Li Y., Lin X., Lu P., Wang Y. (2011)
Three-component synthesis of polysubstituted pyrroles
from a-diazoketones, nitroalkenes, and amines. Org
Lett;13:4668–4671.
18. Qiao F., Zuo D., Shen X., Qi H., Wang H., Zhang W.,
Wu Y. (2012) DAT-230, a novel microtubule inhibitor,
exhibits potent anti-tumor activity by inducing G2/M
phase arrest, apoptosis in vitro and perfusion decrease
in vivo to HT-1080. Cancer Chemother Pharma-
col;70:259–270.
19. Ravelli R.B., Gigant B., Curmi P.A., Jourdain I., Lach-
kar S., Sobel A., Knossow M. (2004) Insight into tubu-
lin regulation from a complex with colchicine and a
stathmin-like domain. Nature;428:198–202.
6
7
8
9
. Chaudari A., Pandeya S.N., Kumar P., Sharma P.P.,
Gupta S., Soni N., Verma K.K., Bhardwaj G. (2007)
Combretastatin a-4 analogs as anticancer agents. Mini
Rev Med Chem;12:1186–1205.
. Hsieh H.P., Liou J.P., Mahindroo N. (2005) Pharma-
ceutical design of antimitotic agents based on combre-
tastatins. Curr Pharm Des;11:1655–1677.
20. Genna D.T., Posner G.H. (2011) Cyanocuprates con-
vert carboxylic acids directly into ketones. Org
Lett;13:5358–5361.
21. Li X., Liu N., Zhang H., Knudson S.E., Li H.J., Lai C.T.,
Simmerling C., Slayden R.A., Tonge P.J. (2011) CoA
adducts of 4-oxo-4-phenylbut-2-enoates: inhibitors of
MenB from the M. tuberculosis menaquinone biosyn-
thesis pathway. ACS Med Chem Lett;2:818–823.
22. Kameo K., Ogawa K., Takeshita K., Nakaike S., Tomi-
sawa K., Sota K. (1988) Studies on antirheumatic
agents: 3-benzoylpropionic acid derivatives. Chem
Pharm Bull;36:2050–2060.
23. Nishio T. (1998) Sulfur-containing heterocycles derived
by reaction of x-Keto amides with Lawesson’s
reagent. Helv Chim Acta;81:1207–1214.
24. Jia Y., Bois-Choussy M., Zhu J. (2007) Synthesis of
DEFG ring of complestatin and chloropeptin I:
highly atropdiastereoselective macrocyclization by
intramolecular Suzuki-Miyaura reaction. Org Lett;9:
2401–2404.
25. Raposo M.M.M., Sousa A.M.R.C., Fonseca A.M.C.,
Kirsch G. (2006) Synthesis of formyl-thienylpyrroles:
versatile building blocks for NLO materials. Tetrahe-
dron;62:3493–3501.
26. Guan Q., Yang F., Guo D., Xu J., Jiang M., Liu C.,
Bao K., Wu Y., Zhang W. (2014) Synthesis and biologi-
cal evaluation of novel 3,4-diaryl-1,2,5-selenadiazol
analogues of combretastatin A-4. Eur J Med Chem;87:
1–9.
1
1
1
0. LeBlanc R., Dickson J., Brown T., Stewart M., Pati
H.N., VanDerveer D., Arman H., Harris J., Pennington
W., Holt H.L. Jr., Lee M. (2005) Synthesis and cytotox-
icity of epoxide and pyrazole analogs of the combre-
tastatins. Bioorg Med Chem;13:6025–6034.
1. Johnson M., Younglove B., Lee L., LeBlanc R., Holt H.
Jr., Hills P., Mackay H., Brown T., Mooberry S.L., Lee
M. (2007) Design, synthesis, and biological testing of
pyrazoline derivatives of combretastatin-A4. Bioorg
Med Chem Lett;17:5897–5901.
2. Ruprich J., Prout A., Dickson J., Younglove B., Nolan
L., Baxi K., LeBlanc R., Forrest L., Hills P., Holt H. Jr.,
Mackay H., Brown T., Mooberry S.L., Lee M. (2007)
Design, synthesis and biological testing of cyclo-
hexenone derivatives of combretastatin-A4. Lett Drug
Des Discov;4:144–148.
1
3. Lee L., Robb L.M., Lee M., Davis R., Mackay H.,
Chavda S., Babu B., O’Brien E.L., Risinger A.L.,
Mooberry S.L., Lee M. (2010) Design, synthesis, and
biological evaluations of 2,5-diaryl-2,3-dihydro-1,3,4-
oxadiazoline analogs of combretastatin-A4. J Med
Chem;53:325–334.
1
4. Tron G.C., Pirali T., Sorba G., Pagliai F., Busacca S.,
Genazzani A.A. (2006) Medicinal chemistry of combre-
tastatin A4: present and future directions. J Med
Chem;49:3033–3044.
Supporting Information
Additional Supporting Information may be found in the
online version of this article:
1
5. Banwell M.G., Hamel E., Hockless D.C., Verdier-Pi-
nard P., Willis A.C., Wong D.J. (2006) 4,5-Diaryl-1H-
pyrrole-2-carboxylates as combretastatin A-4/lamel-
larin T hybrids: synthesis and evaluation as antimi-
Appendix S1. Supplementary information.
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