Synthesis and anti-breast cancer activity of novel indibulin related diarylpyrrole derivatives

Article abstract of DOI:10.1007/s40199-019-00260-9

Background: During recent years, a number of anti-tubulin agents were introduced for treatment of diverse types of cancer. Despite their potential in the treatment of cancer, drug resistance and adverse toxicity, such as peripheral neuropathy, are some of the negative effects of anti-tubulin agents. Among anti-tubulin agents, indibulin was found to cause minimal peripheral neuropathy. Thus far, however, indibulin has not entered clinical usage, caused in part by its poor aqueous solubility and other developmental problems in preclinical evaluation. Objectives: With respect to need for finding potent and safe anticancer agents, in our current research work, we synthesized several indibulin-related diarylpyrrole derivatives and investigated their anti-cancer activity. Methods: Cell cultur studies were perfomred using the MTT cell viability assay on the breast cancer cell lines MCF-7, T47-D, and MDA-MB231 and also NIH-3?T3 cells as representative of a normal cell line. The activity of some of the synthesized compounds for tubulin interaction was studied using colchicine binding and tubulin polymerization assays. The annexin V-FITC/PI method and flow cytometric analysis were used for studying apoptosis induction and cell cycle distribution. Results and conclusion: Two of the synthesized compounds, 4f and 4?g, showed high activity on the MDA-MB231 cell line (IC₅₀?= 11.82 and 13.33?μM, (respectively) and low toxicity on the normal fibroblast cells (IC₅₀?> 100?μM). All of the tested compounds were more potent on T47-D cancer cells and less toxic on NIH-3?T3 normal cells in comparison to reference compound, indibulin. The tubulin polymerization inhibition assay and [³H]colchicine binding assay showed that the main mechanism of cell death by the potent synthesized compounds was not related to an interaction with tubulin. In the annexin V/PI staining assay, the induction of apoptosis in the MCF-7 and MDA-MB231 cell lines was observed. Cell cycle analysis illustrated an increased percentage of sub-G-1 cells in the MDA-MB231 cell line as a further indication of cell death through induction of apoptosis. [Figure not available: see fulltext.].

Full text of DOI:10.1007/s40199-019-00260-9

DARU Journal of Pharmaceutical Sciences
https://doi.org/10.1007/s40199-019-00260-9
RESEARCH ARTICLE
Synthesis and anti-breast cancer activity of novel indibulin related
diarylpyrrole derivatives
1
2
3
1
Ebrahim Saeedian Moghadam & Ernest Hamel & Zahra Shahsavari & Mohsen Amini
Received: 17 November 2018 /Accepted: 6 March 2019
#
Springer Nature Switzerland AG 2019
Abstract
Background During recent years, a number of anti-tubulin agents were introduced for treatment of diverse types of cancer.
Despite their potential in the treatment of cancer, drug resistance and adverse toxicity, such as peripheral neuropathy, are some of
the negative effects of anti-tubulin agents. Among anti-tubulin agents, indibulin was found to cause minimal peripheral neurop-
athy. Thus far, however, indibulin has not entered clinical usage, caused in part by its poor aqueous solubility and other
developmental problems in preclinical evaluation.
Objectives With respect to need for finding potent and safe anticancer agents, in our current research work, we synthesized
several indibulin-related diarylpyrrole derivatives and investigated their anti-cancer activity.
Methods Cell cultur studies were perfomred using the MTTcell viability assay on the breast cancer cell lines MCF-7, T47-D, and
MDA-MB231 and also NIH-3 T3 cells as representative of a normal cell line. The activity of some of the synthesized compounds
for tubulin interaction was studied using colchicine binding and tubulin polymerization assays. The annexin V-FITC/PI method
and flow cytometric analysis were used for studying apoptosis induction and cell cycle distribution.
Results and conclusion Two of the synthesized compounds, 4f and 4 g, showed high activity on the MDA-MB231 cell line (IC =
50
1
1.82 and 13.33 μM, (respectively) and low toxicity on the normal fibroblast cells (IC > 100 μM). All of the tested compounds were
50
more potent on T47-D cancer cells and less toxic on NIH-3 T3 normal cells in comparison to reference compound, indibulin. The
3
tubulin polymerization inhibition assay and [ H]colchicine binding assay showed that the main mechanism of cell death by the potent
synthesized compounds was not related to an interaction with tubulin. In the annexin V/PI staining assay, the induction of apoptosis in
the MCF-7 and MDA-MB231 cell lines was observed. Cell cycle analysis illustrated an increased percentage of sub-G-1 cells in the
MDA-MB231 cell line as a further indication of cell death through induction of apoptosis.
.
.
.
.
Keywords Anti-cancer Synthesis MTT Apoptosis Indibulin
Introduction
*
Mohsen Amini
moamini@tums.ac.ir
Breast cancer is the most common invasive cancer in women
all over the world. Approximately one third (32%) of cancers
diagnosed in female patients are breast cancers. It is the fifth
most common cause of death related to cancer and the leading
cause of cancer death in women. Women have a 1 in 8 lifetime
risk of developing breast cancer and 1 in 35 risk of breast
cancer causing death in the US [1–4]. The rates of breast
cancers are increasing, but these rates differ by race and eth-
nicity. The disease displays a high level of complexity because
of more than 18 sub-types of breast cancer. The stages of
tumor progression are not well understood yet in breast cancer.
1
Department of Medicinal Chemistry, Faculty of Pharmacy and Drug
Design & Development Research Center, The Institute of
Pharmaceutical Sciences (TIPS), Tehran University of Medical
Sciences, Tehran 1417614411, Iran
2
3
Screening Technologies Branch, Developmental Therapeutics
Program, Division of Cancer Treatment and Diagnosis, Frederick
National Laboratory for Cancer Research, National Cancer Institute,
National Institutes of Health, Frederick, MD 21702, USA
Faculty of Paramedical Sciences, Shahid Beheshti University of
Medical Sciences, Tehran, Iran

DARU J Pharm Sci
Among diverse risk factors concerning developing breast can-
cer are life style, genetic, environmental and biological fac-
tors. There are several strategies to overcome breast cancer
including surgery, radiation therapy, chemotherapy, hormonal
therapy and targeted therapy, but the morbidity and mortality
caused by breast cancer remains high [1, 3–5]. Improving the
treatment of breast cancer is thus an important scientific and
medical goal.
in the design and synthesis of new anticancer agents. We
therefore synthesized a novel series of indibulin related
diarylpyrrole derivatives and evaluated their anticancer activ-
ity on breast cancer cell lines as well as a mouse fibroblast
normal cell line to represent noncancer cells, using the 3-(4,5-
dimethylthiazoyl-2-yl) 2,5-diphenyl tetrazolium bromide
(MTT) cell viability assay. Design of the target compounds
was carried out based on development of an indibulin family,
in which the fused ring in the indole moiety was converted to a
flexible phenyl-pyrrole ring system (Fig. 2). To determine the
mechanism of cytotoxic activity of potent compounds, we
also investigated the effect of synthesized compounds on tu-
bulin polymerization, colchicine binding, cell cycle progres-
sion and apoptosis induction.
The microtubule system is an important component of eu-
karyote cells. It has critical roles in various basic cellular func-
tions, such as formation and maintaining cell morphology,
important roles in intracellular and extracellular motility, cell
division through formation of the mitotic spindle, organelle
transport and cell signaling [6]. During the last decade, atten-
tion to anti-tubulin agents and their usage in the treatment of a
large number of cancers has been expanded. Microtubule-
targeting compounds by affecting microtubule dynamics can
cause both the stabilization or destabilization of microtubules,
depending on their exact binding site in tubulin, and this dis-
ruption of microtubule dynamics invariable lead to cell death
through apoptosis. Taxoids and related compounds act in the
taxol binding site of tubulin and speed up tubulin polymeriza-
tion. With a different mechanism of action, inhibition of mi-
crotubule assembly, a large number of anti-cancer agents in-
teract with the colchicine site of tubulin. These colchicine site
compounds include combrestastatin A-4, nocodazole and
indibulin. In both cases, interaction with tubulin disrupts the
normal function of cells in mitotic processes and controls the
high cell division in cancerous cells [7]. Peripheral nephropa-
thy emerged as a major side effect of paclitaxel and of some
anti-mitotic agents that target the colchicine site. Indibulin,
representing a new generation of anti-tubulin agent, exerted
an interesting anticancer activity without leading to peripheral
nephropathy in animal models. In fact, indibulin discriminates
between mature neuronal and non-neuronal tubulin [8]. There
are reports about efforts for introducing new tubulin polymer-
ization inhibitor inspired by the structure of indibulin, several
series of related structures were synthesized and evaluated for
anti-cancer activity [9–11] (Fig. 1a). A significant tumor
growth inhibition in a mouse xenograft model of head and
neck cancer was observed for one of these new indibulin-
related structures [9] (Fig. 1b).
Materials and methods
General
All Chemical reagents and solvents were bought from Merck
(Darmstadt, Germany). Melting points were determined on a
Kofler hot-stage instrument (Reichert, Vienna, Austria). The
1
H NMR spectra were obtained on a Bruker FT-500 MHz
spectrometer (Bruker, Darmstadt, Germany) and CDCl was
3
1
3
as solvent. C NMR spectra were also taken at 125 MHz. The
spin multiplicities are written as s (singlet), d (doublet), t (trip-
let), and m (multiplet) and the Coupling constant (J) values are
given in Hertz (Hz). Mass spectra were done on an Agilent
5975B instrument (Agilent Technologies, Santa Clara, CA,
USA) with triple-axis detector. IR spectra were obtained by
a Nicolet Magna 550-FT spectrometer (Nicolet Instrument
Corporation, Madison, WI, USA). Elemental analyses were
determined using a Perkin Elmer Model 240-C instrument
(PerkinElmer, Hopkinton, MA, USA).
Chemistry
1-(4-chlorophenylpentane)-1,4-dione (2a) and 1-phenylpentane-
1,4-dione (2b) were Synthesized according to the methods re-
ported in the literature [22, 23].
Pyrrole is one of the most useful heterocycles in the syn-
thesis of new biologically active molecules. Many compounds
that include a pyrrole ring have been synthesized in drug dis-
covery laboratories. Some of these compounds have exerted
various biologic effects, including anticancer activity, and
these compounds display diverse mechanisms of action
General procedure for the preparation
of 2-methyl-1,5-diaryl-1H-pyrrole derivatives 3a-j
1,4-Diketone derivatives 2a-b (1 mmol) and the appropriate
aniline derivative (1.1 mmol) and p-tuloenesulfonic acid
(PTSA), catalytic amount, were dissolved and reacted in
refluxing ethanol for 6 h. while the reaction is completed, as
indicated by thin layer chromatography (TLC), the solvent is
evaporated and the remained residue was purified using col-
umn chromatography (hexane/ ethyl acetate 10:1 as eluent) to
obtain pyrrole derivative 3a-j.
[12–14]. From the medicinal chemist’s viewpoint, the design
and synthesis of new compounds for breast cancer treatment is
necessary. We decided that indibulin, with its reported lack of
neurotoxicity, was a worthy target, in particular because syn-
thesis of analogues was related to our previous efforts [15–21]

DARU J Pharm Sci
Fig. 1 a Diverse structural
changes reported on indibulin
structure. b Compound A with
significant head and neck tumor
growth inhibition
a)
b)
General procedure for the preparation
of 2-(2-methyl-1,5-diaryl-1H-pyrrol-3-yl)
stirred in RT for 12 h and after completion of the reaction
(TLC), the solvent was removed using rotary evaporator and
the residue was subjected to flash chromatography (hexane/
ethyl acetate 3:1, as eluent). Final products 4a-j were obtained
in a pure crystal form.
-
2-oxo-N-(pyridin-4-yl)acetamide derivatives 4a-j
After dissolving appropriate pyrrole 3a-j (1 mmol) and
trimethylamine (TEA) (1.2 mmol) in dichloromethane
(
(
DCM) (10 mL), the solution was treated with oxalyl chloride
1.1 mmol) dropwise at 0 °C. After the addition was complet-
2-(5-(4-chlorophenyl)-2-methyl-1-phenyl-1H-pyrrol-3-yl)-2-
oxo-N-(pyridin-4-yl)acetamide (4a) Yellow solid; yield 40%;
mp 234–236 °C.
ed, the mixture was stirred at room temperature (RT) for 4 h,
then the mixture was concentrated to remove residual oxalyl
chloride under reduced pressure. The crude was dissolved in
DCM and 4-aminopyridine (1 mmol), TEA (1.2 mmol) and 4-
N,N-dimethylaminopyridine (DMAP) as a catalyst were
added to the reaction mixture. The reaction mixture was
−1
IR (KBr, cm ): 3343, 3150, 3031, 1702, 1638, 1593, 1479,
1
1415, 1327, 1146, 875, 749, 693; H NMR (500 MHz, CDCl ):
3
δ 2.49 (s, 3H, CH ), 7.00 (d, J = 8 Hz, 2H, H-Ar), 7.12–7.18 (m,
3
4H, H-Ar), 7.44 (br s, 3H, H-Ar), 7.61 (s, 1H, H-Pyrrole), 7.65
(d, J = 5 Hz, 2H, H-Pyridine), 8.58 (d, J = 5 Hz, 2H, H-Pyridine),

DARU J Pharm Sci
Fig. 2 Structural changes on
indibulin in current work
1
3
9
1
1
4
.37 (s, 1H, NH); C NMR (125 MHz, CDCl ): δ 13.8, 112.8,
2-(5-(4-chlorophenyl)-2-methyl-1-(p-tolyl)-1H-pyrrol-3-yl)-2-
oxo-N-(pyridin-4-yl) acetamide (4d) Yellow solid; yield 48%;
mp 156–158 °C.
3
13.6, 117.0, 128.1, 128.3, 129.0, 129.4, 129.5, 130.1, 133.0,
34.2, 137.0, 143.8, 144.2, 151.0, 160.9, 180.5; Mass, m/z (%):
15 (M+, 10), 294 (35), 133 (16), 103 (50), 73 (100), 51 (5);
−1
IR (KBr, cm ): 3341, 3144, 3037, 2954, 2852, 1700, 1638,
1
Anal. Calcd for C H ClN O : C, 69.31; H, 4.36; N, 10.10.
1593, 1503, 1417, 1326, 1146, 829, 754, 691; H NMR
2
4
18
3 2
Found: C, 69.21; H, 4.31; N, 10.13.
(500 MHz, CDCl ): δ 2.40 (s, 3H, CH ), 2.45 (s, 3H, CH ),
3
3
3
7.00–7.03 (m, 4H, H-Ar), 7.13 (d, J = 7.5 Hz, 2H, H-Ar), 7.22
2
3
4
-(5-(4-chlorophenyl)-1-(4-fluorophenyl)-2-methyl-1H-pyrrol-
-yl)-2-oxo-N-(pyridin-4-yl)acetamide (4b) Yellow solid; yield
3%; mp 204–206 °C.
(d, J = 7.5 Hz, 2H, H-Ar), 7.60 (s, 1H, H-Pyrrole), 7.65 (d, J =
5.5 Hz, 2H, H-Pyridine), 8.57 (d, J = 5.5 Hz, 2H, H-Pyridine),
13
9.45 (s, 1H, NH); C NMR (125 MHz, CDCl ): δ 13.7, 21.1,
3
−1
IR (KBr, cm ): 3343, 3152, 3034, 1707, 1642, 1595, 1505,
112.67, 113.67, 116.65, 127.8, 128.3, 129.3, 130.1, 130.2, 132.8,
133.9, 134.3, 139.0, 143.9, 144.3, 150.9, 161.0, 180.5; Mass, m/z
(%): 429 (M+, 16), 308 (100), 280 (12), 244 (10), 121 (19), 78
(17), 55 (15); Anal. Calcd for C H ClN O : C, 69.85; H, 4.69;
1
1415, 1326, 1227, 1145, 832, 754, 693; H NMR (500 MHz,
CDCl ): δ 2.47 (s, 3H, CH ), 6.99 (d, J = 8 Hz, 2H, H-Ar), 7.13–
3
3
7.17 (m, 6H, H-Ar), 7.61 (s, 1H, H-Pyrrole), 7.65 (d, J = 5.5 Hz,
25
20
3 2
2
H, H-Pyridine), 8.58 (d, J = 5.5 Hz, 2H, H-Pyridine), 9.41 (s,
N, 9.77. Found: C, 69.76; H, 4.65; N, 9.79.
13
1H, NH); C NMR (125 MHz, CDCl ): δ 13.7, 112.7, 113.6,
3
1
8
1
3
16.6 (d, J = 22.5 Hz), 116.8, 128.4, 129.4, 129.8 (d, J =
.75 Hz), 132.9, 133.2, 133.9, 143.8, 144.1, 150.8, 160.8,
62.3 (d, J = 248.75 Hz), 180.6; Mass, m/z (%): 433 (M+, 12),
2-(5-(4-chlorophenyl)-1-(4-methoxyphenyl)-2-methyl-1H-
pyrrol-3-yl)-2-oxo-N-(pyridin-4-yl) acetamide (4e) Yellow sol-
id; yield 51%; mp 162–164 °C.
−
1
12 (100), 248 (7), 128 (8), 51 (5); Anal. Calcd for
IR (KBr, cm ): 3338, 3037, 2962, 2840, 1701, 1638,
1
C H ClFN O : C, 66.44; H, 3.95; N, 9.69. Found: C, 66.58;
1587, 1507, 1416, 1327, 1249, 1030, 832, 755; H NMR
24
17
3 2
H, 3.93; N, 9.68.
(500 MHz, CDCl ): δ 2.46 (s, 3H, CH ), 3.84 (s, 3H,
3
3
OCH ), 6.93 (d, J = 8 Hz, 2H, H-Ar), 7.02 (d, J = 8 Hz, 2H,
3
2
-(1,5-bis(4-chlorophenyl)-2-methyl-1H-pyrrol-3-yl)-2-oxo-N-
H-Ar), 7.05 (d, J = 8 Hz, 2H, H-Ar), 7.15 (d, J = 8 Hz, 2H, H-
Ar), 7.60 (s, 1H, H-Pyrrole), 7.65 (d, J = 5 Hz, 2H, H-
(
pyridin-4-yl)acetamide (4c) Yellow solid; yield 44%; mp
2
13–215 °C.
Pyridine), 8.58 (d, J = 5 Hz, 2H, H-Pyridine), 9.41 (s, 1H,
−
1
13
IR (KBr, cm ): 3342, 2925, 2854, 1695, 1644, 1592,
491, 1415, 1326, 1161, 1093, 830, 753, 669; H NMR
NH); C NMR (125 MHz, CDCl ): δ 13.7, 55.4, 112.57,
3
1
1
113.6, 114.8, 116.5, 128.3, 129.1, 129.4, 129.5, 130.2,
132.9, 134.0, 143.9, 144.5, 150.9, 159.7, 161.0, 180.4;
Mass, m/z (%): 445 (M+, 17), 324 (90),135 (31), 84 (100),
51 (34); Anal. Calcd for C H ClN O : C, 67.34; H, 4.52; N,
(
500 MHz, CDCl ): δ 2.48 (s, 3H, CH ), 7.00 (d, J = 8 Hz,
3 3
2
H, H-Ar), 7.09 (d, J = 8 Hz, 2H, H-Ar), 7.17 (d, J = 8 Hz, 2H,
H-Ar), 7.42 (d, J = 8 Hz, 2H, H-Ar), 7.61 (s, 1H, H-Pyrrole),
.64 (d, J = 5 Hz, 2H, H-Pyridine), 8.58 (d, J = 5 Hz, 2H, H-
2
5
20
3 3
7
9.42. Found: C, 67.45; H, 4.50; N, 9.43.
1
3
Pyridine), 9.36 (s, 1H, NH); C NMR (125 MHz, CDCl ): δ
3
1
1
3.7, 112.9, 113.6, 116.9, 128.5, 129.4, 129.5, 129.8, 131.8,
33.2, 134.0, 135.1, 135.4, 144.0, 144.1, 151.0, 160.9, 180.6;
2-(2-methyl-1,5-diphenyl-1H-pyrrol-3-yl)-2-oxo-N-(pyridin-4-
yl) acetamide (4f) Yellow solid; yield 54%; mp 216–218 °C.
−
1
Mass, m/z (%): 449 (M+, 7), 328 (60), 149 (25), 100 (90), 72
IR (KBr, cm ): 3335, 3063, 2855, 1697, 1634, 1582,
1
(
100); Anal. Calcd for C H Cl N O : C, 64.01; H, 3.81; N,
1497, 1410, 1328, 1146, 876, 766, 695; H NMR
2
4
17
2 3 2
9
.33. Found: C, 64.19; H, 3.84; N, 9.35.
(500 MHz, CDCl ): δ 2.49 (s, 3H, CH ), 7.00–7.15 (m, 7H,
3
3

DARU J Pharm Sci
H-Ar), 7.41 (br s, 3H, H-Ar), 7.64 (br s, 3H, H-Pyrrole & H-
Pyridine), 8.58 (d, J = 5 Hz, 2H, H-Pyridine), 9.38 (s, 1H,
m/z (%): 395 (M+, 20), 274 (100), 231 (8), 128 (7), 91 (5);
Anal. Calcd for C H N O : C, 75.93; H, 5.35; N, 10.63.
2
5 21 3 2
1
3
NH); C NMR (125 MHz, CDCl ): δ 13.8, 112.4, 113.6,
Found: C, 75.99; H, 5.38; N, 10.61.
3
1
1
16.7, 127.0, 128.1, 128.2, 128.3, 128.8, 129.4, 131.6,
35.2, 137.3, 143.9, 144.1, 150.8, 161.0, 180.6; Mass, m/z
2-(1-(4-methoxyphenyl)-2-methyl-5-phenyl-1H-pyrrol-3-yl)-2-
oxo-N-(pyridin-4 yl)acetamide (4j) Yellow solid; yield 52%;
mp 134–136 °C.
(
7
%): 381 (M+, 20), 324 (12), 260 (100), 217 (10), 128 (8),
7 (9); Anal. Calcd for C H N O : C, 75.57; H, 5.02; N,
2
4 19 3 2
−
1
11.02. Found: C, 75.66; H, 5.05; N, 11.01.
IR (KBr, cm ): 3338, 3039, 2927, 2837, 1700, 1636,
1
1
594, 1503, 1478, 1411, 833, 754, 694; H NMR
2
-(1-(4-fluorophenyl)-2-methyl-5-phenyl-1H-pyrrol-3-yl)-2-
(500 MHz, CDCl ): δ 2.47 (s, 3H, CH ), 3.83 (s, 3H,
3 3
oxo-N-(pyridin-4-yl) acetamide (4 g) Yellow solid; yield 49%;
mp 205–207 °C.
OCH ), 6.91 (d, J = 8 Hz, 2H, H-Ar), 7.06 (d, J = 8 Hz, 2H,
H-Ar), 7.10 (d, J = 6 Hz, 2H, H-Ar), 7.12–7.19 (m, 3H, H-Ar),
3
−
1
IR (KBr, cm ): 3336, 3043, 2915, 1701, 1633, 1500,
7.61 (s, 1H, H-Pyrrole), 7.65 (d, J = 5 Hz, 2H, H-Pyridine),
1
13
1
477, 1411, 1329, 1145, 966, 875, 766, 695; H NMR
8.57 (d, J = 5 Hz, 2H, H-Pyridine), 9.41 (s, 1H, NH);
C
(
500 MHz, CDCl ): δ 2.46 (s, 3H, CH ), 7.06 (d, J = 7 Hz,
NMR (125 MHz, CDCl ): δ 13.8, 55.4, 112.2, 113.6, 114.2,
3
3
3
2
7
H, H-Ar), 7.08–7.19 (m, 7H, H-Ar), 7.59 (s, 1H, H-Pyrrole),
.65 (d, J = 5.5 Hz, 2H, H-Pyridine), 8.56 (d, J = 5.5 Hz, 2H,
116.5, 126.9, 128.0, 128.3, 129.1, 129.8, 131.7, 135.3, 143.9,
144.3, 150.9, 159.5, 161.1, 180.5; Mass, m/z (%): 411 (M+,
19), 290 (100), 236 (25), 152 (15), 111 (30), 83 (51), 57 (60);
Anal. Calcd for C H N O : C, 72.98; H, 5.14; N, 10.21.
1
3
H-Pyridine), 9.49 (s, 1H, NH); C NMR (125 MHz, CDCl3):
δ 13.7, 112.4, 113.6, 116.4 (d, J = 22.5 Hz), 116.7, 127.1,
2
5 21 3 3
1
1
28.1, 128.3, 129.8 (d, J = 8.75 Hz), 131.4, 133.1, 135.2,
43.7, 143.9, 150.9, 161.0, 162.2 (d, J = 248.5 Hz), 180.7;
Found: C, 72.87; H, 5.10; N, 10.23.
Biological evaluation
Cell culture
Mass, m/z (%): 399 (M+, 17), 278 (100), 129 (12), 95 (10), 55
(
6); Anal. Calcd for C H FN O : C, 72.17; H, 4.54; N,
24 18 3 2
1
0.52. Found: C, 72.10; H, 4.55; N, 10.45.
2
-(1-(4-chlorophenyl)-2-methyl-5-phenyl-1H-pyrrol-3-yl)-2-
For cell culture, appropriate cell lines, MCF-7, MDA-MB231,
T47-D (human breast cancer cell lines) and NIH-3 T3 (mouse
fibroblast cell line) were purchased from the Pasteur Institute
(Tehran, Iran). RPMI-1640 medium (Sigma-Aldrich, St.
Louis, MO, USA) was used for cell culturing, supplemented
with penicillin (100 U/mL), 10% heat-inactivated fetal bovine
serum (Gibco, Grand Island, NY, USA), and (100 μg/mL)
streptomycin (Roche, Mannheim, Germany) in a humidified
oxo-N-(pyridin-4-yl)acetamide (4 h) Yellow solid; yield 46%;
mp 215–217 °C.
−
1
IR (KBr, cm ): 3258, 3144, 3058, 2923, 1708, 1648,
1
1
583, 1503, 1412, 1329, 1149, 829, 744, 697; H NMR
(
500 MHz, CDCl ): δ 2.47 (s, 3H, CH ), 7.06–7.12 (m, 4H,
3 3
H-Ar), 7.18–7.22 (m, 3H, H-Ar), 7.38 (d, J = 7.5 Hz, 2H, H-
Ar), 7.60 (s, 1H, H-Pyrrole), 7.65 (d, J = 5 Hz, 2H, H-
Pyridine), 8.57 (d, J = 5 Hz, 2H, H-Pyridine), 9.46 (s, 1H,
incubator with 5% CO at 37 °C.
2
1
3
NH); C NMR (125 MHz, CDCl ): δ 13.7, 112.60, 113.64,
3
1
1
(
16.9, 127.2, 128.2, 128.3, 129.4, 129.6, 131.3, 134.7, 135.1,
35.7, 143.6, 143.9, 150.9, 160.9, 180.7; Mass, m/z (%): 415
M+, 13), 294 (100), 230 (9), 128 (12), 78 (5); Anal. Calcd for
C H ClN O : C, 69.31; H, 4.36; N, 10.10. Found: C, 69.39;
Cytotoxicity evaluation by the MTT assay
MTT assay for all compounds was done according to method
reported in our previous work [24].
2
4
18
3 2
H, 4.38; N, 10.14.
Colchicine binding inhibition and in vitro inhibitory tubulin
polymerization assay
2
-(2-methyl-5-phenyl-1-(p-tolyl)-1H-pyrrol-3-yl)-2-oxo-N-
(
pyridin-4-yl)acetamide (4i) Yellow solid; yield 39%; mp 212–
2
14 °C.
In the present study, purified bovine brain tubulin was used in
the tubulin polymerization and colchicine binding assays, pre-
pared as reported previously [25]. The experimental method
−
1
IR (KBr, cm ): 3347, 3153, 3034, 2919, 2864, 1708,
637, 1593, 1500, 1409, 1325, 1144, 823, 764, 698; H
1
1
3
NMR (500 MHz, CDCl ): δ 2.38 (s, 3H, CH ), 2.46 (s, 3H,
for measuring [ H]colchicine binding inhibition to tubulin
3
3
CH ), 7.02 (d, J = 7.5, 2H, H-Ar), 7.07–7.17 (m, 5H, H-Ar), 7.
was reported in detail previously [26]. Briefly, the ligand bind-
ing assay was performed in 0.1 mL reaction volumes. Each
reaction mixture contained 0.1 mg/mL (1.0 μM) tubulin,
3
2
5
9
0 (d, J = 7.5, 2H, H-Ar), 7.60 (s, 1H, H-Pyrrole), 7.65 (d, J =
Hz, 2H, H-Pyridine), 8.57 (d, J = 5 Hz, 2H, H-Pyridine),
1
3
3
.47 (s, 1H, NH); C NMR (125 MHz, CDCl ): δ 13.8, 21.1,
5.0 μM [ H]colchicine and 5.0 μM of potential inhibitory
3
1
1
12.2, 113.6, 116.6, 126.9, 127.8, 128.0, 128.2, 129.9, 131.7,
34.5, 135.2, 138.7, 143.9, 144.1, 150.9, 161.1, 180.5; Mass,
compound. Combretastatin-A4 (CA-4), a highly potent inhib-
itor of colchicine binding to tubulin, was used as a positive

DARU J Pharm Sci
Scheme 1 Synthetic pathway of desired compounds 4a-j
standard [27]. Reaction mixtures were incubated for 10 min at
concentration for 24 h). After treatment, cells were washed
twice with phosphate buffered saline (PBS), mixed with
500 μL of binding buffer, and stained with 5 μL of annexin
V-fluorescein isothiocyanate (FITC) and 5 μL of PI (PI 50 μg/
mL) for 10 min at RT in the dark. Apoptotic cells were quan-
tified by a FACS Calibur flow cytometer (BD Biosciences,
3
7 °C, the time that the reaction in control reaction mixtures is
about 40–60% complete. At this time, reaction mixtures were
diluted and stopped with water at 0 °C, and the mixtures were
filtered through Whatman DEAE-cellulose filters. The filters
then washed with ice-cold water. Radiolabel bound to the
filters was measured using a liquid scintillation counter. For
investigation of tubulin polymerization inhibitory potential of
selected compounds, 1.0 mg/mL (10 μM) purified tubulin was
treated without Guanosine-5′-triphosphate (GTP) with various
concentrations of synthesized compound at 30 °C for 15 min.
Then reaction mixtures were placed on ice, and GTP (10 μL
of 10 μM) was added. Reaction mixtures were transferred to
cuvettes held at 0 °C in recording spectrophotometers. After
baselines were recorded at 350 nm at 0 °C, the temperature
was increased to 30 °C rapidly (over about 30 s) and change in
turbidity was followed over 20 min. Compound concentra-
tions that resulted in a 50% decrease in turbidity (IC ) were
a,
b
Table 1 In vitro cytotoxic activities (IC50
compounds 4a-j
)
of synthesized
Compounds
MCF-7
T47-D
MDA-
MB231
NIH-
3 T3
4
4
4
4
4
4
4
4
4
4
a
b
c
d
e
f
>10
34.04 ± 2.9
41.95 ± 2.1
40.56 ± 3.6
43.28 ± 1.7
45.49 ± 2.3
16.6 ± 1.5
15.61 ± 2.1
13.49 ± 2.7
12.19 ± 1.4
23.17 ± 1.9
>100
33.79 ± 1.8
33.63 ± 3.2
37.11 ± 2.8
28.69 ± 1.6
10.55 ± 1.3
11.82 ± 2.2
13.33 ± 1.5
26.02 ± 3.1
28.06 ± 3.3
32.52 ± 2.4
9.0 ± 0.6
99.1 ± 1.4
21.0 ± 2.3
76.2 ± 3.5
53.5 ± 1.7
47.4 ± 1.9
>100
7.5 ± 0.5
>10
>10
>10
5
0
>10
then determined [28].
g
>10
>100
h
>10
35.5 ± 2.8
37.9 ± 1.8
49.3 ± 2.1
11.2 ± 1.6
Annexin V-FITC/PI assay
i
>10
j
>10
To quantify the cell death modality, the annexin V/propidium
Indibulin
>100
5
iodide (PI) staining assay was used. Briefly, 3 × 10 cells
a
(
MCF-7 for 4b and MDA-MB231 for 4e) were plated and
IC50 values are in μM
b
then the cells were treated with 4b or 4e (at the IC50
IC50, compound concentration required to inhibit cell proliferation by 50%

DARU J Pharm Sci
4
San Jose, CA); 1 × 10 cells were counted for each sample.
Both early apoptotic (annexin V-positive, PI-negative) and
late apoptotic (double positive of annexin Vand PI) cells were
detected. The percentages of cells in each quadrant were ana-
lyzed using the Flowing Software version 2.4.1 (Turku Centre
for Biotechnology University of Turku, Finland). The exper-
iments were repeated at least three times.
of 50 μg/mL PI was added. The fluorescence of the cells was
determined with a FACS-Calibur flow cytometer
(BDBiosciences, San Jose, CA) [29].
Results and discussion
Chemistry
Flow cytometric analysis of cell cycle distribution
The synthetic pathway of novel target compounds is outlined in
Scheme 1. In the first step, appropriate derivatives of benzalde-
hyde, 1a or 1b, were treated with methyl vinyl ketone in the
presence of sodium cyanide as catalyst and N,N-
dimethylformamide (DMF) as solvent to yield 1 aryl-1,4-
pentanediones 2a-b. Then, diones 2a-b were refluxed with ani-
line derivatives using a catalytic amount of PTSA in ethanol for
6 h to give corresponding 1,5-diarylpyrrole derivatives 3a-j. The
compounds 4a-j were prepared through two continuous
6
For analysis of DNA content using flow cytometry, 1 × 10
MCF-7 cells, in a 6-well plate, were treated with compound 4e
at the IC50 concentration) for 24 h. For cell fixation, cells
were centrifuged and 0.7 ml of cold 75% ethanol was added
to samples, and the samples were placed on ice for at least 3 h.
After washing with PBS, cells were resuspended in 0.25 mL
of PBS, with 5 μL of 10 mg/mL RNase A and Triton X-100
(
(
0.1%). Then, cells were incubated at 37 °C for 1 h, and 10 μL
Fig. 3 4b induced cell death
modes in the MCF-7 cell line. a
4
b (7.5 μM) treated MCF-7 cells
for 24 h. b After 24 h of incuba-
tion with 4b, both the percentages
of early apoptotic cells and late
apoptotic cells (stained with both
annexin V and PI) increased sig-
nificantly. Data are representative
of three independent experiments.
*
P < 0.05 denotes a mean sig-
nificantly different from untreated
cells. 4e induced cell death mo-
dalities in the MDA-MB-231 cell
line. c 4e (10.55 μM) treated
MDA-MB-231 for 24 h. d After
2
4 h of incubation with 4e, the
percentages of early and late apo-
ptotic cells (stained with both
annexin V and PI) increased sig-
nificantly. Data are representative
of three independent experiments.
*
P < 0.05 denotes a mean sig-
nificantly different from untreated
cells

DARU J Pharm Sci
Fig. 3 (continued)
reactions. In the first step, reaction of oxalyl chloride with pyrrole
derivatives in the presence of dry TEA in DCM at RT gave the
related acyl chloride. Then, the mixture was concentrated under
reduced pressure to remove excess oxalyl chloride. The residue
was dissolved in dry DCM and TEA, a catalytic amount of
DMAP and 3-aminopyridine were added and stirred at RT for
12 h. The mixture was concentrated and the final products 4a-j
were purified using column chromatography (hexane/ethyl
Fig. 4 4e induces cell cycle arrest
in the MDA-MB-231 cells. a
Typical DNA content histograms
of cells treated with 4e
(10.55 μM) for 24 h

DARU J Pharm Sci
acetate 3:1, as eluent). Altogether, ten target compounds were
synthesized in good to moderate yields. Chemical structure of
all the final compounds was confirmed by H NMR, C NMR,
MS, IR and elemental analysis.
Colchicine binding and tubulin polymerization inhibition
1
13
Tubulin protein dynamics (polymerization and depolymeriza-
tion) is one of the most important and attractive targets for
designing anticancer agents. To investigate the ability of com-
pounds 4a-j to bind to the colchicine site of tubulin, we tested
Biological evaluation
their inhibition potential at 5 μM on the binding of 5 μM
3
[
H]colchicine to 1 μM tubulin. None of the tested com-
Anticancer activity
pounds had a significant inhibitory effect in this assay (results
not shown), while the potent colchicine site inhibitor CA-4
was strongly active (98 ± 0.2% inhibition of colchicine bind-
ing, with an IC50 of 0.73 ± 0.04 μM for inhibition of tubulin
assembly). Furthermore, we evaluated the effects of 4b and
In the first step of biological investigation, our target
compounds 4a-j were tested for their cytotoxicity against
three breast cancer cell lines (MCF-7, T47-D and MDA-
MB231) as well as a mouse embryonic fibroblast cell line
4
g on the polymerization of purified tubulin, using the highly
(
NIH-3 T3) as a normal cell line, using the MTT assay.
potent CA-4 as a reference standard. Inhibition was not ob-
served at 20 μM, the highest concentration examined. Tubulin
polymerization inhibitory effect of indibulin (IC50 =
0.30 μM) was also reported in the literature [30]. These data
indicate that an interaction with tubulin is not the main cause
of anticancer activity of the effective compounds in this series.
Thus, we investigated other cell death mechanisms.
Indibulin was used as positive control. In the MCF-7 cell
line, except compound 4b, none of the target compounds
showed an IC50 < 10 μM. 4b had an IC50 of around 7.5 ±
0
.5 μM. In the case of the T47-D cell line, target com-
pounds exerted good to moderate cytotoxicity, with IC50
values between 12.19 and 45.49 μM. Compounds 4i
(
(
IC5 0 :12.19 μM), 4 h (IC5 0 :13.49 μM), 4 g
IC :15.61 μM) and 4f (IC :16.60 μM) showed the best
5
0
50
cytotoxic activity. For the MDA-MB231 cell line, results
of the cytotoxicity assay were similar to those obtained
with the T47-D cell line. In this cell line, the range of
cytotoxic activity (IC50) was between 10.55 and
Annexin V/ PI apoptosis assay
In order to examine whether the anticancer effects of drugs on the
cells were associated with the induction of apoptosis or not, FITC
conjugated annexin V and PI staining was used to distinguish
apoptotic cells. MCF-7 and MDA-MB231 cells were incubated
with 4b and 4e at their respective IC concentration for 24 h, and
3
7.11 μM. Compounds 4e, 4f and 4 g were the most
potent and showed IC50 values of 10.55, 11.82 and
3.33 μM, respectively. Compounds 4f and 4 g had no
1
50
cytotoxicity even at 100 μM concentration against the
normal cell line, and their selectivity towards the breast
cancer cells make these two derivatives especially inter-
esting for future investigations. In addition we should
note that none of the target compounds had any cytotoxic
effect on the normal NIH-3 T3 cell line, while indibulin
did. Results of the MTT assay are shown in Table 1.
the percentages of apoptotic cells were measured. Control cells
were negative for both annexin V-FITC and PI. Treatment of
both the MCF-7 and MDA-MB231 cell lines with either 4b or
4e increased the percentage of both early (very significantly) and
late apoptotic cells (Fig. 3). With respect to these results, it seems
that early apoptosis induction is the major cause of cell death
with these compounds.
Fig. 5 Possible mode of interaction between tubulin active site and 4f, 4 g and indibulin structures

DARU J Pharm Sci
Cell cycle analysis
Compounds 4b and 4e showed the highest cytotoxic activity
on the MCF-7 and MDA-MB231 cell lines, respectively. Both
of the compounds induced apoptosis in tested cell lines.
Compounds 4f and 4 g not only showed high toxicity on the
MDA-MB231 cancer cell line but also showed very low tox-
icity on normal cells (IC50 above 100 μM). This is an ideal
choice to continue further pharmacological investigation on
these two derivatives for finding their exact mechanism of
action. A ligand binding assay plus a tubulin polymerization
inhibitory assay showed that interaction of active compounds
with tubulin was not the cause of the anticancer activity by the
synthesized compounds and that other important factors may
trigger apoptosis induction and finally cell death. Our data
indicate that it will be beneficial to investigate other deriva-
tives with the 1,5-diaryl pyrrole scaffold in a search for more
potent and safer anticancer agents.
With regard to cytotoxic activity and apoptosis induction abil-
ity of 4e, we evaluated the effect of 4e on cell cycle distribu-
tion of cultured MDA-MB231 cell line using flow cytometric
cell cycle analysis. After treatment of cells with compound 4e
at the IC50 concentration for 24 h, there was an increased
percentage of cells in the sub-G1 region, representing an in-
crease in cell death consistent with the induction of apoptosis
shown in Fig. 4.
Docking study
With respect to the results of tubulin polymerization inhibi-
tion, we decided to study the possible mode of interactions of
4
f and 4 g with tubulin protein using molecular docking soft-
ware to understand the reason of inactivity of synthesized
compounds in inhibiting the tubulin polymerization.
Indibulin was also docked in tubulin active site for compari-
son as a reference. Conversion of rigid and flat indole part of
indibulin to flexible 2-arylpyrrole and substituting the N-4-
chlorobenzyl part with N-aryl moiety, drastically decreased
the effect of synthesized compounds on tubulin polymeriza-
tion in comparison with indibulin, and the interaction of these
parts with active site of tubulin is the key cause in showing
activity or inactivity of tested compounds on tubulin polymer-
ization. in both 4f and 4 g, phenyl ring attached to C-5 of
pyrrole ring showed interactions with VAL176, SER177 and
TYR223 residues. These interactions exactly repeated be-
tween the above resides and six-membered ring of indole of
indibulin. SER177 has another interaction with five-
membered ring of indole of indibulin, which this interaction
is absent between SER177 and pyrrole ring of 4f and 4 g.
Because of the ability of phenyl ring attached to C-5 of pyrrole
ring in 4f and 4 g, it seems the interactions of the phenyl ring
in these compounds is not as strong as the interactions in
indole of indibulin with the mentioned residues. The N-4-
chlorobenzyl moiety of indibulin has interactions with
ALA787, LEU681 and CYS674, but these interactions are
completely absent for the N-aryl part of the 4f and 4 g. there
is also some other differences in mode of interactions between
the residues in active site and structures of 4f, 4 g and
indibulin. Altogether, it seems these differences in interac-
tions, especially presented for N-4-chlorobenzyl moiety of
indibulin, are the cause of inactivity of 4f and 4 g in tubulin
polymerization inhibition (Fig. 5).
Acknowledgements This work was financially supported by Research
Council of Tehran University of Medical Sciences.
The content of this paper is solely the responsibility of the authors and
does not necessarily reflect the official views of the National Institutes of
Health.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
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