Design, synthesis and cytotoxicity evaluation of indibulin analogs

Article abstract of DOI:10.1515/hc-2018-0016

Indibulin is one of the most potent tubulin polymerization inhibitors with minimal peripheral neuropathy. The design and synthesis of new indibulin analogs were carried out in order to investigate their anti-cancer activity. The target compounds 4a-i were synthesized in multistep reactions starting with the related indole derivatives. Compound 4f shows the highest cytotoxic activity on HT-29 and Caco-2 cell lines with the respective half maximal inhibitory concentration (IC₅₀) values of 5.1 μm and 7.3 μm. In the case of the T47-D cell line, compound 4c exerts the best cytotoxic activity with an IC₅₀ value of 11.5 μm. In the cell cycle analysis on HT-29 cells, compound 4f at 5.1 μm showed an increase in the percentage of cells in the sub-G1 phase. Altogether, nine target compounds were synthesized and characterized by infrared spectroscopy (IR), proton nuclear magnetic resonance (¹H NMR), carbon-13 nuclear magnetic resonance (¹³C NMR), mass spectrometry (MS) and elemental analysis. Some of the compounds show good cytotoxic activity against cancerous cell lines.

Full text of DOI:10.1515/hc-2018-0016

Heterocycl. Commun. 2018; aop
Ebrahim Saeedian Moghadam, Farhad Saravani, Seyednasser Ostad, Shohreh Tavajohi,
Morteza Pirali Hamedani and Mohsen Amini*
Design, synthesis and cytotoxicity evaluation of
indibulin analogs
https://doi.org/10.1515/hc-2018-0016
the ability to spread throughout the body [1, 2]. Despite
tireless efforts over the decades for prevention, diagnosis
and therapy, cancer remains a major leading cause of mor-
tality worldwide. It is the second cause of death in North
America and Europe after cardiovascular diseases [3–6].
Chemotherapy is one of the most effective techniques used
for cancer treatment. Unfortunately, the lack of selectivity
and emerging drug resistance decrease the efficacy and
potential of cancer chemotherapy, and further efforts for
finding new anti-cancer drugs are necessary [7–9].
Received January 28, 2018; accepted June 14, 2018
Abstract: Indibulin is one of the most potent tubulin poly-
merizationinhibitorswithminimalperipheralneuropathy.
The design and synthesis of new indibulin analogs were
carried out in order to investigate their anti-cancer activ-
ity. The target compounds 4a–i were synthesized in multi-
step reactions starting with the related indole derivatives.
Compound 4f shows the highest cytotoxic activity on HT-29
and Caco-2 cell lines with the respective half maximal
inhibitory concentration (IC50) values of 5.1 μm and 7.3 μm.
In the case of the T47-D cell line, compound 4c exerts the
best cytotoxic activity with an IC50 value of 11.5 μm. In the
cell cycle analysis on HT-29 cells, compound 4f at 5.1 μm
showed an increase in the percentage of cells in the sub-
G1 phase. Altogether, nine target compounds were syn-
thesized and characterized by infrared spectroscopy (IR),
proton nuclear magnetic resonance (1H NMR), carbon-13
nuclear magnetic resonance (13C NMR), mass spectrometry
(MS) and elemental analysis. Some of the compounds
show good cytotoxic activity against cancerous cell lines.
As a part of eukaryote cells, microtubules play an
important position in cell biology. Microtubules play a
role in adjusting and tuning of the various basic cellu-
lar functions including organelle transport, mitosis pro-
cesses, adjustment of motility, formation and in keeping
cell morphology and cell signaling intact [10]. During the
last few years, laboratory research and clinical investiga-
tion on anti-tubulin agents for applying these compounds
in the treatment of a large number of cancers have rapidly
increased. The rate of tubulin polymerization affects the
induction of apoptosis in cells. The main family of tubulin
polymerization inducer is taxane and its derivatives.
Paclitaxel and docetaxel exert their anti-tubulin effect by
binding to the taxol binding site of tubulin and by doing
so, they increase the rate of tubulin polymerization. In
addition to the taxane family, there is a large number of
compounds including combretastatin A4, indibulin and
nocodazole which attach to the colchicine binding site of
tubulin and cause a decrease in the tubulin polymeriza-
tion rate. With respect to the high level of cell prolifera-
tion in cancerous cells and the effect of tubulin disrupting
agents on the inhibition of the mitosis process, these com-
pounds could control cell division in cancerous cells [11].
Anti-mitotic agents such as paclitaxel and some of the
compounds that target the colchicine binding site lead to
the emergence of peripheral neuropathy as an unfortunate
side effect. Indibulin with a high level of anti-cancer activ-
ity by anti-mitotic mechanism does not exert peripheral
neuropathy, and this feature distinguishes indibulin from
other similar acting compounds. It seems that indibulin
has the ability to discriminate between mature neuronal
and non-neuronal tubulin [12]. Efforts are being continued
to find new tubulin polymerization inhibitors inspired by
the structure of indibulin [13–15] (Figure 1A). For example,
Keywords: anti-cancer; indibulin analogs; MTT; pyrrole.
Dedicated to: This manuscript is dedicated to the fond memory of
Professor Abbas Shafiee.
Introduction
Cancer is a complex group of genetic disorders involv-
ing an abnormal and uncontrolled growth of cells with
*Corresponding author: Mohsen Amini, Department of Medicinal
Chemistry, Faculty of Pharmacy and Drug Design and Development
Research Center, The Institute of Pharmaceutical Sciences (TIPS),
Tehran University of Medical Sciences, Tehran 1417614411, Iran,
e-mail: moamini@tums.ac.ir
Ebrahim Saeedian Moghadam, Farhad Saravani and Morteza Pirali
Hamedani: Department of Medicinal Chemistry, Faculty of Pharmacy
and Drug Design and Development Research Center, The Institute
of Pharmaceutical Sciences (TIPS), Tehran University of Medical
Sciences, Tehran 1417614411, Iran
Seyednasser Ostad and Shohreh Tavajohi: Department of
Toxicology and Pharmacology, Faculty of Pharmacy, Tehran
University of Medical Sciences, Tehran 1417614411, Iran
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2ꢀ
ꢀE. Saeedian Moghadam et al.: Indibulin analogs
N
HN
O
O
A
N
Indibulin
Cl
R
HN
O
O
N
R
HN
HN
O
O
N
O
O
N
R
N
R
Cl
R: alkyl, aryl, heteroaryl
B
N
O
HN
O
O
CH3
H3C
N
Squamous cell carcinoma (FaDu) LC50 = 55 nM
Tubulin polymerization IC50 = 6.6 mM
Compound A
OCH3
Figure 1ꢀPrevious structural modifications of indibulin (A), compound A with significant head and neck tumor growth inhibition (B).
a remarkable tumor growth inhibition in a mouse xeno- mouse fibroblast normal cells (NIH-3T3) as a control. The
graft model of head and neck cancer has been observed in structural modifications are explained in Figure 2.
the case of indibulin-related compound A (Figure 1B) [13].
In addition to these derivatives, a large number of other
pyrrole-based bioactive compounds have been synthesized
Results and discussion
[16–19].
In continuation of our previous efforts [20–25] in the
design and synthesis of new chemotherapeutic agents, The synthetic pathway to final compounds is shown in
in this paper we report the synthesis of a series of indibu- Scheme 1. In the first step, the Stetter reaction of ben-
lin-related N-benzylpyrrole derivatives and the evalua- zaldehyde derivatives 1a–b with methyl vinyl ketone in
tion of their anti-cancer activity on three diverse cancer the presence of a catalytic amount of sodium cyanide in
cell lines including human breast cancer tumor (T47-D), dimethylformamide (DMF) yielded 1-aryl-1,4-pentanedi-
human colon adenocarcinoma (Caco-2 and HT-29) using ones 2a–b. Then, under the Paal-Knorr conditions, diones
2a–c were allowed to react with appropriate benzylamine
derivatives in the presence of p-toluenesulfonic acid
(PTSA) as a catalyst in refluxing ethanol for 6 h to obtain
O
N
O
HN
O
HN
N
the corresponding N-benzylpyrrole derivatives 3a–i. These
pyrroles were converted into final target compounds 4a–i
by treatment with oxalyl chloride in dichloromethane in
the presence of triethylamine at room temperature, fol-
lowed by the reaction of the intermediate acid chloride
with 4-aminopyridine.
O
CH3
N
N
R1
R2
Current work structures
Cl
Indibulin
Compounds 4a–i were investigated for their cytotox-
icity against three cancer cell lines HT-29, Caco-2 and T47-D
Figure 2ꢀStructural changes on indibulin in the current work.
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ꢂ3
E. Saeedian Moghadam et al.: Indibulin analogsꢂ
O
CHO
R1
NH2
CH3
II
CH3
N
I
+
R1
O
R1
R2
R2
3a–f
1a,b
2a,b
I: methyl vinyl ketone, NaCN, DMF
II: PTSA (cat), EtOH, ∆, 6 h
III
III: 1) oxalyl chloride, TEA, DCM
2) 4-aminopyridine, TEA, N,N-dimethyl-4-aminopyridine, DCM
N
O
HN
1a, 2a: R1 = H
1b, 2b: R1 = Cl
O
3a, 4a: R1 = H, R2 = H
3b, 4b: R1 = H, R2 = Cl
3c, 4c: R1 = H, R2 = CH3
3d, 4d: R1 = Cl, R2 = H
3e, 4e: R1 = Cl, R2 = Cl
3f, 4f: R1 = Cl, R2 = CH3
CH3
N
R1
R2
4a–f
O
HN
N
O
H2N
H3C
CH3
O
N
H3C
CH3
N
I
II
CH3
H3C
+
O
X
X
X
4g–i
2c
3g–i
g: X = H
h: X = Cl
I: PTSA (cat), EtOH, ∆, 6 h
II: 1) oxalyl chloride, TEA, DCM
i: X = CH3
2) 4-aminopyridine, TEA, N,N-dimethyl-4-aminopyridine, DCM
Scheme 1ꢀSynthetic pathway to compounds 4a–i.
and 4g against HT-29 and compounds 4f, 4b, 4c against
Caco-2 are more active than paclitaxel, the reference drug.
In addition, all the tested compounds have lower toxicity
on NIH-3T3 normal cell line than the standard drug.
With regard to the cytotoxic activity of 4f, we further
evaluated its effect on cell cycle distribution of cultured
HT-29 cells, as determined by flow cytometry (not shown).
After treatment of cells with 4f at IC50 concentration for
24 h, this compound caused an increase in the percentage
of cells in the sub-G1 phase, representing an increase in
cell death which could be consistent with the induction
of apoptosis.
Table 1ꢀIn vitro cytotoxic activities (IC50, μm) of compounds 4a–i.
Compound
NIH-3T3
HT-29
Caco-2
T47-D
4a
32.1ꢁ ꢁ1.9
17ꢁ ꢁ2.2
16.5ꢁ ꢁ2.7
12ꢁ ꢁ1.1
25.6ꢁ ꢁ3.0
17.4ꢁ ꢁ1.7
50.6ꢁ ꢁ2.6
30.4ꢁ ꢁ3.5
65.8ꢁ ꢁ2.9
8.7ꢁ ꢁ3.1
25.3ꢁ ꢁ4.1
27.5ꢁ ꢁ1.5
31.4ꢁ ꢁ3.3
32.2ꢁ ꢁ3.5
21.2ꢁ ꢁ2.7
5.1ꢁ ꢁ1.8
49.1ꢁ ꢁ1.4
8.3ꢁ ꢁ1.9
41.1ꢁ ꢁ2.1
>100
11.5ꢁ ꢁ2.9
>100
20.5ꢁ ꢁ1.3
26.4ꢁ ꢁ1.8
40.4ꢁ ꢁ2.0
46.3ꢁ ꢁ2.2
61.1ꢁ ꢁ4.1
0.35ꢁ ꢁ0.2
4b
4c
9.8ꢁ ꢁ1.3
4d
36.3ꢁ ꢁ2.1
29.5ꢁ ꢁ2.7
7.3ꢁ ꢁ1.8
75.1ꢁ ꢁ2.0
58.2ꢁ ꢁ1.5
82.8ꢁ ꢁ2.3
17.3ꢁ ꢁ2.4
4e
4f
4g
7.7ꢁ ꢁ2.4
4h
28.7ꢁ ꢁ1.6
58.6ꢁ ꢁ3.7
19.4ꢁ ꢁ1.9
4i
Paclitaxel
with a normal cell line NIH-3T3 as control (Table 1). The
screening started with the HT-29 cell line with the tested Conclusions
compounds showing cytotoxicity with half maximal
inhibitory concentration (IC50 ) values between 5.1 and A series of indibulin-related N-benzylpyrrole derivatives
58.6 μm. The most active against HT-29 are compounds were synthesized and investigated for their cytotoxic
4f and 4g. With the Caco-2 cell line, the most active com- activity against four diverse cell lines. Compounds 4f and
pounds 4f, 4b and 4c show anti-cancer activity with an 4g show the highest cytotoxic activity against the HT-29
IC50 of less than 10 μm (7.3, 8.3 and 9.8 μm, respectively). In cell line. Cell cycle analysis shows arrest in the sub-G1
the case of T47-D cell line, compound 4c is the most active phase of cells treated with 4f which could lead to apopto-
with an IC50 value of 11.5 μm. Interestingly, compounds 4f sis induction and cell death.
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4ꢀ ꢀE. Saeedian Moghadam et al.: Indibulin analogs
2-(4-Chlorophenyl)-5-methyl-1-(4-methylbenzyl)-1H-pyrrole
(3f)ꢀLight brown oil; yield 59%; IR: 3048, 2926, 2858, 1517, 1463,
Experimental
1
1385, 1263, 1112, 912, 839, 737, 701 cm−1; H NMR: δ 2.12 (s, 3H, CH3),
Melting points were taken on a Kofler hot-stage apparatus (Reichert,
Vienna, Austria) and are uncorrected. The proton nuclear mag-
netic resonance (1H NMR) spectra (500 MHz) and carbon-13 nuclear
magnetic resonance (13C NMR) spectra (125 MHz) were recorded
on a Bruker FT-500 MHz spectrometer using CDCl3 as the solvent.
Elemental analyses were carried out using a Perkin Elmer Model
240-C apparatus. Infrared (IR) spectra were recorded using KBr pellets
on a Nicolet Magna 550-FT spectrometer. Mass spectra were recorded
on an Agilent 5975C spectrometer. 1-Phenylpentane-1,4-dione (2a) and
1-(4-chlorophenylpentane)-1,4-dione (2b) were prepared using the
Stetter reaction as previously reported [26, 27]. Compound 2c was
commercially available.
2.30 (s, 3H, CH3), 5.04 (s, 2H, CH2), 6.02 (s, 1H, pyrrole), 6.19 (s, 1H,
pyrrole), 6.78 (d, Jꢁ=ꢁ7.5 Hz, 2H, Ar), 7.08 (d, Jꢁ=ꢁ7.5 Hz, 2H, Ar), 7.18 (d,
Jꢁ=ꢁ7.5 Hz, 2H, Ar), 7.21 (d, Jꢁ=ꢁ7.5 Hz, 2H, Ar); 13C NMR: δ 12.4, 20.9, 47.2,
107.2, 108.2, 125.3, 128.4, 129.4, 129.5, 130.7, 132.2, 132.3, 133.1, 135.6,
136.5; MS: m/z 295 (15), 197 (11), 149 (14), 111 (30), 97 (52), 83 (54), 57
(100%). Anal. Calcd for C19H18ClN: C, 77.15; H, 6.13; N, 4.74. Found: C,
77.06; H, 6.17; N, 4.72.
General procedure for the preparation of substituted
acetamides 4a–i
A solution of pyrrole 3a–i (1 mmol) and triethylamine (1.2 mmol) in
dichloromethane (10 mL) was treated dropwise at 0°C with a solu-
tion of oxalyl chloride (1.1 mmol) in dichloromethane (5 mL). The
mixture was stirred at room temperature for 4 h and then concen-
trated [33]. The residue was dissolved in dichloromethane and the
solution was treated with triethylamine (1.2 mmol), 4-aminopyridine
and a catalytic amount of N,N-dimethyl-4-aminopyridine. The mix-
ture was stirred at room temperature for 12 h, concentrated and the
residue was subjected to silica gel column chromatography eluting
with hexane/ethyl acetate (3:1) to obtain the analytically pure prod-
uct 4a–i. Compounds 4b and 4h have been mentioned previously in
the literature but not fully characterized [34].
General procedure for the preparation of N-benzylpyrrole
derivatives 3a–i
A mixture of appropriate diketone 2a–c (1 mmol), a benzylamine deriv-
ative (1.1 mmol) and a catalytic amount of PTSA in ethanol was heated
under reflux for 6 h. After the completion of the reaction, the mixture
was concentrated and the residue of 3a–i was purified by flash chro-
matography (hexane/ethyl acetate 10:1 as an eluent) [28]. Compounds
3a, 3d, 3g, 3h and 3i have been reported previously [29–32].
1-(4-Chlorobenzyl)-2-methyl-5-phenyl-1H-pyrrole (3b)ꢀLight yel-
low solid; yield 51%; mp 83–84°C; IR: 3063, 3027, 2972, 2935, 1599,
2-(1-Benzyl-2-methyl-5-phenyl-1H-pyrrol-3-yl)-2-oxo-N-(pyridin-
4-yl)acetamide (4a)ꢀYellow solid; yield 41%; mp 161–163°C;
IR: 3280, 2925, 2858, 1697, 1641, 1581, 1502, 1475, 1135, 995, 833,
1
1513, 1489, 1447, 1400, 1312, 1088, 1011, 816, 750, 699 cm−1; H NMR:
δ 2.13 (s, 3H, CH3), 5.08 (s, 2H, CH2), 6.04 (s, 1H, pyrrole), 6.21 (s, 1H,
pyrrole), 6.83 (d, Jꢁ=ꢁ7.5 Hz, 2H, Ar), 7.21–7.29 (m, 7H, Ar); 13C NMR: 12.6,
47.0, 107.5, 108.3, 126.8, 127.0, 128.5, 128.6, 128.9, 130.2, 132.8, 133.7,
134.6, 137.5; MS: m/z 281 (84), 156 (100), 125 (84), 89 (16%). Anal.
Calcd for C18H16ClN: C, 76.72; H, 5.72; N, 4.97. Found: C, 76.61; H, 5.66;
N, 5.03.
1
757, 698 cm−1; H NMR: δ 2.54 (s, 3H, CH3), 5.18 (s, 2H, CH2), 6.93 (d,
Jꢁ=ꢁ7.0 Hz, 2H, Ar), 7.24–7.34 (m, 8H, Ar), 7.49 (s, 1H, pyrrole), 7.79 (d,
Jꢁ=ꢁ5.0 Hz, 2H, pyridine), 8.57 (d, Jꢁ=ꢁ5.0 Hz, 2H, pyridine), 9.65 (s, 1H,
NH); 13C NMR: δ 13.0, 47.8, 112.4, 113.9, 116.3, 125.4, 127.6, 128.1, 128.5,
129.0, 129.1, 131.5, 135.6, 136.4, 143.4, 145.8, 148.5, 161.2, 179.7; MS: m/z
395 (21), 274 (100), 91 (72), 57 (10%). Anal. Calcd for C25H21N3O2: C,
75.93; H, 5.35; N, 10.63. Found: C, 75.80; H, 5.32; N, 10.69.
2-Methyl-1-(4-methylbenzyl)-5-phenyl-1H-pyrrole(3c)ꢀLightbrown
solid; yield 57%; mp 61–62°C; IR: 3054, 3024, 2922, 2860, 1601, 1513,
1444, 1400, 1353, 1311, 1025, 799, 751, 700 cm−1; 1H NMR: δ 2.09 (s, 3H,
CH3), 2.26 (s, 3H, CH3), 5.04 (s, 2H, CH2), 6.02 (s, 1H, pyrrole), 6.21 (s,
1H, pyrrole), 6.79 (d, Jꢁ=ꢁ7.5 Hz, 2H, Ar), 7.04 (d, Jꢁ=ꢁ7.5 Hz, 2H, Ar), 7.14 (t,
Jꢁ=ꢁ7.0 Hz, 1H, Ar), 7.21 (t, Jꢁ=ꢁ7.0 Hz, 2H, Ar), 7.27 (d, Jꢁ=ꢁ7.0 Hz, 2H, Ar); 13C
NMR: 12.6, 21.1, 47.5, 107.2, 108.0, 125.6, 126.6, 128.4, 128.6, 129.4, 130.4,
133.9, 134.6, 136.0, 136.5; MS: m/z 261 (40), 156 (30), 125 (17), 105 (100),
77 (13%). Anal. Calcd for C19H19N: C, 87.31; H, 7.33; N, 5.36. Found: C,
87.39; H, 7.27; N, 5.34.
2-(1-(4-Chlorobenzyl)-2-methyl-5-phenyl-1H-pyrrol-3-yl)-2-oxo-
N-(pyridin-4-yl)acetamide (4b)ꢀYellow solid; yield 39%; mp
195–197°C; IR: 3335, 2923, 2852, 1708, 1632, 1593, 1506, 1473, 1350, 813,
763, 698, 556 cm−1; 1H NMR: δ 2.54 (s, 3H, CH3), 5.14 (s, 2H, CH2), 6.85
(d, Jꢁ=ꢁ8.0 Hz, 2H, Ar), 7.25–7.38 (m, 7H, Ar), 7.52 (s, 1H, pyrrole), 7.63 (d,
Jꢁ=ꢁ5.5 Hz, 2H, pyridine), 8.56 (d, Jꢁ=ꢁ5.5 Hz, 2H, pyridine), 9.40 (s, 1H,
NH); 13C NMR: δ 12.8, 47.2, 112.7, 113.6, 116.6, 126.9, 128.1, 128.6, 129.1,
129.2, 131.5, 133.5, 135.1, 135.3, 142.7, 143.9, 150.7, 150.9, 161.0, 180.4; MS:
m/z 429 (11), 308 (54), 236 (11), 125 (59), 103 (53), 73 (100), 51 (7%).
Anal. Calcd for C25H20ClN3O2: C, 69.85; H, 4.69; N, 9.77. Found: C, 69.89;
H, 4.60; N, 9.74.
1-(4-Chlorobenzyl)-2-(4-chlorophenyl)-5-methyl-1H-pyrrole
(3e)ꢀLight brown oil; yield 50%; IR: 3053, 2927, 2854, 1511, 1492,
1466, 1265, 1095, 1014, 832, 739, 706 cm−1; 1H NMR: δ 2.09 (s, 3H, CH3),
5.02 (s, 2H, CH2), 6.03 (s, 1H, pyrrole), 6.19 (s, 1H, pyrrole), 6.79 (d,
Jꢁ=ꢁ8.0 Hz, 2H, Ar), 7.14 (d, Jꢁ=ꢁ8.0 Hz, 2H, Ar), 7.20–7.25 (m, 4H, Ar);
13C NMR: 12.4, 46.9, 107.7, 108.6, 126.8, 128.5, 128.9, 129.6, 130.5, 132.0,
132.6, 132.9, 133.1, 137.2; MS: m/z 315 (10), 190 (16), 125 (40), 100 (67),
57 (100%). Anal. Calcd for C18H15Cl2N: C, 68.37; H, 4.78; N, 4.43. Found:
C, 68.28; H, 4.73; N, 4.46.
2-(2-Methyl-1-(4-methylbenzyl)-5-phenyl-1H-pyrrol-3-yl)-2-oxo-
N-(pyridin-4-yl)acetamide (4c)ꢀYellow solid; yield 34%; mp
145–147°C; IR: 3279, 2920, 1704, 1636, 1605, 1512, 1416, 1354, 1135, 828,
804, 695, 569 cm−1; 1H NMR: δ 2.33 (s, 3H, CH3), 2.54 (s, 3H, CH3), 5.14
(s, 2H, CH2), 6.82 (d, Jꢁ=ꢁ8.0 Hz, 2H, Ar), 7.12 (d, Jꢁ=ꢁ8.0 Hz, 2H, Ar),
7.27–7.36 (m, 5H, Ar), 7.51 (s, 1H, pyrrole), 7.69 (d, Jꢁ=ꢁ5.5 Hz, 2H, pyri-
dine), 8.56 (d, Jꢁ=ꢁ5.5 Hz, 2H, pyridine), 9.50 (s, 1H, NH); 13C NMR: δ
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E. Saeedian Moghadam et al.: Indibulin analogsꢂ ꢂ5
13C NMR: δ 12.1, 12.5, 46.3, 110.9, 113.5, 115.7, 126.9, 129.2, 129.3, 133.6, 134.4,
141.7, 144.0, 150.7, 150.8, 161.3, 180.0; MS: m/z 367 (8), 342 (28), 246 (67),
125 (100), 89 (11), 57 (10%). Anal. Calcd for C20H18ClN3O2: C, 65.31; H, 4.93;
N, 11.42. Found: C, 65.22; H, 4.90; N, 11.36.
13.0, 21.0, 47.6, 112.5, 113.7, 116.4, 125.4, 128.0, 128.5, 129.2, 129.6, 131.7,
133.5, 135.6, 137.3, 143.3, 144.7, 149.8, 149.9, 161.2, 180.1; MS: m/z 409
(25), 308 (20), 288 (90), 183 (8), 125 (18), 105 (100%). Anal. Calcd
for C26H23N3O2: C, 76.26; H, 5.66; N, 10.26. Found: C, 76.20; H, 5.61;
N, 10.27.
2-(2,5-Dimethyl-1-(4-methylbenzyl)-1H-pyrrol-3-yl)-2-oxo-
N-(pyridin-4-yl)acetamide (4i)ꢀYellow solid; yield 35%; mp
142–143°C; IR: 3340, 2922, 2855, 1708, 1618, 1594, 1502, 1476, 1411,
1036, 824, 753, 550 cm−1; 1H NMR: δ 2.17 (s, 3H, CH3), 2.33 (s, 3H, CH3),
2.55 (s, 3H, CH3), 5.06 (s, 2H, CH2), 6.81 (d, Jꢁ=ꢁ7.5 Hz, 2H, Ar), 7.13 (d,
Jꢁ=ꢁ7.5 Hz, 2H, Ar), 7.18 (s, 1H, pyrrole), 7.66 (d, Jꢁ=ꢁ5.0 Hz, 2H, pyridine),
8.56 (d, Jꢁ=ꢁ5.0 Hz, 2H, pyridine), 9.47 (s, 1H, NH); 13C NMR: δ 12.1, 12.6,
21.0, 46.7, 110.5, 113.6, 115.4, 125.4, 129.5, 129.6, 132.8, 137.4, 142.2, 144.4,
150.2, 161.4, 179.6; MS: m/z 347 (7), 322 (16), 226 (52), 121 (6), 105 (100),
79 (10%). Anal. Calcd for C21H21N3O2: C, 72.60; H, 6.09; N, 12.10. Found:
C, 72.69; H, 6.14; N, 12.09.
2-(1-Benzyl-5-(4-chlorophenyl)-2-methyl-1H-pyrrol-3-yl)-2-oxo-
N-(pyridin-4-yl) acetamide (4d)ꢀYellow solid; yield 37%; mp
132–134°C; IR: 3353, 2923, 2846, 1703, 1634, 1589, 1503, 1416, 1136, 824,
1
724, 661 cm−1; H NMR: δ 2.55 (s, 3H, CH3), 5.14 (s, 2H, CH2), 6.91 (d,
Jꢁ=ꢁ7.0 Hz, 2H, Ar), 7.21 (d, Jꢁ=ꢁ7.5 Hz, 2H, Ar), 7.29 (d, Jꢁ=ꢁ7.5 Hz, 2H, Ar),
7.30–7.35 (m, 3H, Ar), 7.52 (s, 1H, pyrrole), 7.63 (d, Jꢁ=ꢁ5.5 Hz, 2H, pyri-
dine), 8.56 (d, Jꢁ=ꢁ5.5 Hz, 2H, pyridine), 9.43 (s, 1H, NH); 13C NMR: δ
12.8, 47.7, 113.0, 113.6, 116.5, 125.4, 127.7, 128.7, 129.0, 130.1, 130.4, 131.4,
134.1, 136.3, 143.2, 143.9, 150.7, 150.9, 161.1, 180.3; MS: m/z 429 (17), 308
(100), 125 (88), 89 (11%). Anal. Calcd for C25H20ClN3O2: C, 69.85; H,
4.69; N, 9.77. Found: C, 69.74; H, 4.65; N, 9.79.
2-(1-(4-Chlorobenzyl)-5-(4-chlorophenyl)-2-methyl-1H-pyrrol-
3-yl)-2-oxo-N-(pyridin-4-yl) acetamide (4e)ꢀYellow solid; yield
38%; mp 157–159°C; IR: 3343, 2928, 1693, 1649, 1590, 1500, 1406, 1137,
1091, 809, 750, 557 cm−1; 1H NMR: δ 2.53 (s, 3H, CH3), 5.10 (s, 2H, CH2),
6.83 (d, Jꢁ=ꢁ8.4 Hz, 2H, Ar), 7.18 (d, Jꢁ=ꢁ8.4 Hz, 2H, Ar), 7.26–7.32 (m,
4H, Ar), 7.49 (s, 1H, pyrrole), 7.70 (d, Jꢁ=ꢁ6.1 Hz, 2H, pyridine), 8.56 (d,
Jꢁ=ꢁ6.1 Hz, 2H, pyridine), 9.51 (s, 1H, NH); 13C NMR: δ 12.8, 47.1, 113.0,
113.7, 116.5, 126.7, 128.8, 129.3, 129.9, 130.3, 133.6, 134.0, 134.3, 134.7,
143.0, 144.7, 149.8, 161.0, 180.1; MS: m/z 463 (13), 435 (5), 342 (77), 125
(100), 89 (11%). Anal. Calcd for C25H19Cl2N3O2: C, 64.67; H, 4.12; N, 9.05.
Found: C, 64.57; H, 4.10; N, 9.03.
Cell lines
Four cell lines, namely human breast adenocarcinoma (T47-D),
human colon adenocarcinoma (Caco-2 and HT-29) and mouse embryo
fibroblast (NIH-3T3), were obtained from the Pasteur Institute
(Tehran, Iran). The cells were cultured in RPMI-1640 medium (Sigma)
supplemented with 10% heat-inactivated fetal bovine serum (FBS;
Gibco, USA), penicillin (100 U/mL) and streptomycin (100 μg/mL)
(Roche, Germany) at 37°C in a humidified incubator with 5% CO2. In
the case of the Caco-2 cell line, the final cell culture medium con-
tained RPMI-1640 (55%) and Dulbecco’s Modified Eagle Medium:
Nutrient Mixture F-12 (DMEM-F12) (35%) plus the supplements men-
tioned above.
2-(5-(4-Chlorophenyl)-2-methyl-1-(4-methylbenzyl)-1H-pyrrol-
3-yl)-2-oxo-N-(pyridin-4-yl) acetamide (4f)ꢀYellow solid; yield
33%; mp 150–151°C; IR: 3353, 2920, 2849, 1700, 1638, 1593, 1505, 1477,
1
1129, 827, 805, 751, 658 cm−1; H NMR: δ 2.33 (s, 3H, CH3), 2.55 (s, 3H,
Cytotoxicity evaluation by the MTT assay
CH3), 5.11 (s, 2H, CH2), 6.80 (d, Jꢁ=ꢁ7.5 Hz, 2H, Ar), 7.13 (d, Jꢁ=ꢁ7.5 Hz,
2H, Ar), 7.22 (d, Jꢁ=ꢁ8.0 Hz, 2H, Ar), 7.29 (d, Jꢁ=ꢁ8.0 Hz, 2H, Ar), 7.52 (s,
1H, pyrrole), 7.63 (d, Jꢁ=ꢁ5.2 Hz, 2H, pyridine), 8.56 (d, Jꢁ=ꢁ5.2 Hz, 2H,
pyridine), 9.41 (s, 1H, NH); 13C NMR: δ 12.8, 21.0, 47.6, 112.9, 113.6,
116.4, 125.3, 128.7, 129.7, 130.2, 130.3, 133.2, 134.0, 134.1, 137.4, 143.3,
143.9, 150.7, 150.9, 161.1, 180.2; MS: m/z 443 (28), 322 (90), 218 (7), 105
(100), 79 (18%). Anal. Calcd for C26H22ClN3O2: C, 70.35; H, 5.00; N, 9.47.
Found: C, 70.46; H, 5.03; N, 9.49.
All the final compounds were investigated for their antiprolifera-
tive potential at 0.01–100 μm concentrations. Two controls were
prepared within each 96-well plate: a solvent [dimethyl sulfoxide
(DMSO)] control and a positive control (paclitaxel). Briefly, appro-
priate cells were seeded in 96-well plates (Nunc, Roskilde, Den-
mark) at a density of 10 000 viable cells per well and incubated
for 24 h to allow for cell attachment. Solutions of the compounds
were prepared by serial dilution from the stock solution and added
to each well. Cells were then incubated with the compounds for
another 48 h. The response of cells to compounds was evaluated
by determining the cell survival using 3-(4,5-dimethylthiazoyl-
2-yl) 2,5-diphenyl tetrazolium bromide (MTT, Carl Roth, Karlsruhe,
Germany). At first, cells were washed with phosphate buffered
saline (PBS) and then 20 μL of the MTT reagent (5 mg/mL) in
PBS was added to each well. After 4 h of incubation at 37°C, the
medium was discarded, DMSO (100 μL) was added to each well and
the plates were vigorously shaken to dissolve the purple tetrazo-
lium crystals in DMSO. The absorbance of each well was measured
using a plate reader (Anthous 2020; Austria) at a test wavelength
of 550 nm against a standard reference solution at 690 nm. Assays
were performed in triplicate in three independent experiments, and
the concentration required for 50% inhibition of cell viability (IC50)
was calculated by plotting the percentage cytotoxicity versus the
concentration on a logarithmic graph [35].
2-(1-Benzyl-2,5-dimethyl-1H-pyrrol-3-yl)-2-oxo-N-(pyridin-4-yl)
acetamide (4g)ꢀYellow solid; yield 31%; mp 150–152°C; IR: 3365,
2923, 2851, 1703, 1634, 1577, 1499, 1412, 1356, 1137, 823, 752, 660 cm−1;
1H NMR: δ 2.16 (s, 3H, CH3), 2.55 (s, 3H, CH3), 5.09 (s, 2H, CH2), 6.92
(d, Jꢁ=ꢁ7.5 Hz, 2H, Ar), 7.19 (s, 1H, pyrrole), 7.24–7.34 (m, 3H, Ar), 7.63
(d, Jꢁ=ꢁ5.5 Hz, 2H, pyridine), 8.55 (d, Jꢁ=ꢁ5.5 Hz, 2H, pyridine), 9.43 (s,
1H, NH); 13C NMR: δ 12.1, 12.6, 46.9, 110.7, 113.6, 115.6, 125.4, 127.7, 129.5,
129.6, 135.9, 142.0, 144.1, 150.6, 150.8, 161.4, 179.9; MS: m/z 333 (12), 212
(100), 184 (4), 121 (6), 91 (80%). Anal. Calcd for C20H19N3O2: C, 72.05;
H, 5.74; N, 12.60. Found: C, 72.12; H, 5.80; N, 12.61.
2-(1-(4-Chlorobenzyl)-2,5-dimethyl-1H-pyrrol-3-yl)-2-oxo-N-
(pyridin-4-yl)acetamide (4h)ꢀYellow solid; yield 27%; mp 129–131°C;
IR: 3350, 2921, 2852, 1707, 1594, 1479, 1416, 1131, 879, 824, 754, 553 cm−1;
1H NMR: δ 2.15 (s, 3H, CH3), 2.53 (s, 3H, CH3), 5.06 (s, 2H, CH2), 6.85 (d,
Jꢁ=ꢁ8.1 Hz, 2H, Ar), 7.19 (s, 1H, pyrrole), 7.30 (d, Jꢁ=ꢁ8.1 Hz, 2H, Ar), 7.63 (d,
Jꢁ=ꢁ6.2 Hz, 2H, pyridine), 8.56 (d, Jꢁ=ꢁ6.2 Hz, 2H, pyridine), 9.41 (s, 1H, NH);
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ꢀꢀꢀꢁ
6ꢀ ꢀE. Saeedian Moghadam et al.: Indibulin analogs
potent anti-breast cancer agents. Eur. J. Med. Chem. 2014, 86,
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Flow cytometric analysis of cell cycle distribution
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trifugation, for cell fixation, 0.7 mL of cold 75% ethanol was added
to samples on ice for at least 3 h. Cells were washed with PBS and
resuspended in 0.25 mL of PBS, with 5 μL of 10 mg/mL RNase A and
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by the Research Council of Tehran University of Medical
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