Cell-based biological evaluation of a new bisamide FMS kinase inhibitor possessing pyrrolo[3,2-c]pyridine scaffold

Article abstract of DOI:10.1002/ardp.201400051

A bisamide compound 1 possessing the pyrrolo[3,2-c]pyridine nucleus was synthesized and biologically evaluated. It was tested for kinase inhibitory activity over a panel of 47 kinases, and its selectivity toward the FMS kinase was accidentally discovered. Compound 1 was tested over a panel of seven ovarian, two prostate, and six breast cancer cell lines at a single dose concentration of 10μM and showed high activity. It was further tested in a 5-dose mode to determine its IC₅₀ and total growth inhibition (TGI) values over the 15 cell lines. Compound 1 showed high potency on the submicromolar scale and good efficacy. The cytotoxic effect of compound 1 over peritoneal macrophages was also investigated. Compound 1 demonstrated higher selectivity against different cancer cell lines compared with HS-27 fibroblasts.

Full text of DOI:10.1002/ardp.201400051

Arch. Pharm. Chem. Life Sci. 2014, 347, 635–641
635
Full Paper
Cell-Based Biological Evaluation of a New Bisamide FMS
Kinase Inhibitor Possessing Pyrrolo[3,2-c]pyridine Scaffold
Mohammed I. El-Gamal1,2, Mohammed S. Abdel-Maksoud1,3, Mahmoud M. Gamal El-Din1,3
Kyung Ho Yoo4, Daejin Baek5, and Chang-Hyun Oh1,3
,
1
Center for Biomaterials, Korea Institute of Science and Technology, Seoul, Republic of Korea
2
Faculty of Pharmacy, Department of Medicinal Chemistry, University of Mansoura, Mansoura, Egypt
Department of Biomolecular Science, University of Science and Technology, Daejeon, Republic of Korea
Chemical Kinomics Research Center, Korea Institute of Science and Technology, Seoul, Republic of Korea
Department of Chemistry, Hanseo University, Seosan, Republic of Korea
3
4
5
A bisamide compound 1 possessing the pyrrolo[3,2-c]pyridine nucleus was synthesized and biologically
evaluated. It was tested for kinase inhibitory activity over a panel of 47 kinases, and its selectivity
toward the FMS kinase was accidentally discovered. Compound 1 was tested over a panel of seven
ovarian, two prostate, and six breast cancer cell lines at a single dose concentration of 10 mM and
showed high activity. It was further tested in a 5-dose mode to determine its IC50 and total growth
inhibition (TGI) values over the 15 cell lines. Compound 1 showed high potency on the submicromolar
scale and good efficacy. The cytotoxic effect of compound 1 over peritoneal macrophages was also
investigated. Compound 1 demonstrated higher selectivity against different cancer cell lines compared
with HS-27 fibroblasts.
Keywords: Anticancer / Bisamide / Diarylamide / FMS kinase / Pyrrolo[3,2-c]pyridine
Received: February 12, 2014; Revised: March 24, 2014; Accepted: April 4, 2014
DOI 10.1002/ardp.201400051
Introduction
There are direct relationships between macrophage num-
bers, angiogenesis, and malignancy progression [3]. The
growth of several tumor types is associated with over-
expression of CSF-1 and FMS receptor in cancer cells and/or
tumor stroma [4–7]. CSF-1 and FMS were found to be
important in the physiology of the breast and female
reproductive tract neoplasms. The expression of FMS in
breast cancer has been linked to poor survivability and
increased tumor size, where presumably the receptor is
involved in local invasion and metastasis [8–10]. It was also
reported that macrophages mediate hormone resistance in
prostate cancers by a nuclear receptor derepression path-
way [11]. High serum levels of CSF-1 in bone metastatic
prostate cancer have also been detected. This plasma CSF-1
may play a pivotal role in prostate cancer progression as a
clinically useful tumor marker [12].
FMS, first discovered as the oncogene responsible for feline
McDonough sarcoma, is a type III receptor tyrosine kinase
that acts as exclusive receptor for the macrophage or
monocyte colony-stimulating factor (M-CSF or CSF-1). Signal
transduction through CSF-1/FMS or M-CSF/FMS binding
results in survival, proliferation, and differentiation of
monocyte/macrophage lineage [1, 2]. Over-expression of
CSF-1 and/or FMS has been implicated in a number of disease
states such as the growth of metastasis of certain types of
cancer, in promoting osteoclast proliferation in bone
osteolysis, and in many inflammatory disorders. Inhibition
of CSF-1 and/or FMS may help treat these pathological
conditions.
There are many reported FMS kinase inhibitors possessing a
diversity of chemical scaffolds. We have recently reviewed and
classified them according to their chemotypes [13]. And we
have reported a selective and potent bisamide FMS kinase
inhibitor (compound 1, Fig. 1) [14]. Due to the relationship
Correspondences: Dr. Chang-Hyun Oh, Center for Biomaterials, Korea
Institute of Science and Technology, P.O. Box 131, Cheongryang, Seoul
130-650, Republic of Korea
E-mail: choh@kist.re.kr
Fax: þ82 2 958 5308
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636
M. I. El-Gamal et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 635–641
O
CF3
O
H3C
O
Cl
N
N
H
O
N
H
N
CF3
N
H
N
H
HN
O
N
N
O
Sorafenib
1
Figure 1. Structures of the target compound 1
and sorafenib.
between FMS kinase and macrophage, and female reproduc-
tive system, prostate, and breast cancers, we studied the
antiproliferative activities of compound 1 over 15 cancer cell
lines of those three cancer types and HS-27 fibroblasts, and its
cytotoxicity against macrophages in the present investiga-
tion. The results and experimental procedures are reported
in detail.
6 using Pd–C in hydrogen atmosphere gave the corresponding
amino compound, N-(1-(3-aminophenyl)-1H-pyrrolo[3,2-c]-
pyridin-4-yl)benzamide (7). Condensation of compound 7
with 4-morpholino-3-(trifluoromethyl)benzoic acid in the
presence of HOBt/EDCI/triethylamine furnished the target
compound 1 [14].
Biology
Results and discussion
Compound 1 was tested at a single-dose concentration of
1 mM over a panel of 47 kinases. It inhibited the activities of
FMS and KDR kinases by 90 and 71%, respectively (Fig. 2).
Compound 1 was further tested in a 10-dose testing mode in
order to determine its IC50 values over FMS and KDR kinases
(Table 1). The results showed that compound 1 is 11 times
more selective toward FMS kinase than KDR kinase [14].
The colony stimulating factor-1 (CSF-1) and its receptor FMS
(encoded by the c-fms proto-oncogene) constitute a reciprocal
system that has been linked to several human epithelial
cancers including ovarian, breast, and prostate cancers [7, 19–
21]. Compound 1 was tested for antiproliferative activities
over a panel of seven ovarian, two prostate, and six breast
cancer cell lines at the National Cancer Institute (NCI,
Bethesda, Maryland, USA) [22]. The results are shown in
Table 2 and Fig. 3. At a single-dose concentration of 10 mM,
compound 1 exerted more than 100% growth inhibition over
SK-OV-3 ovarian cancer cell line and MDA-MB-468 breast
cancer cell line. The %inhibition was >90% in cases of IGROV1
Chemistry
Synthesis of the target compound 1 was successfully achieved
as illustrated in Scheme 1. 7-Hydroxy-1H-pyrrolo[2,3-b]pyridi-
nium 3-chlorobenzoate (3) was prepared by reacting 7-
azaindole (2) with 3-chloroperbenzoic acid [15, 16]. Com-
pound 3 was then heated with phosphorus oxychloride to
produce 4-chloro-7-azaindole (4) [16]. Compound 4 was
prepared according to the literature procedure [14, 17, 18].
Fusion of 4 with m-nitroaniline neat led to nucleophilic
displacement of the 4-chloro group by the aromatic amino
group, followed by rearrangement of the resulting secondary
amine to give the amine hydrochloride salt 5. Reaction of
the amino group of 1-(3-nitrophenyl)-1H-pyrrolo[3,2-c]pyridin-
4-amine hydrochloride (5) with benzoyl chloride in the
presence of diisopropylamine as a base produced the
benzamido derivative, N-(1-(3-nitrophenyl)-1H-pyrrolo[3,2-c]-
pyridin-4-yl)benzamide (6). Reduction of the nitro group of
Cl
O
NO2
N
b
H2N
c
a
O
HCl
N
H
N
H
N
N
N
N
OH
N
O
H
Cl
N
2
4
3
5
CF3
H
N
NO2
NH2
H
N
H
N
H
N
e
N
d
f
N
N
O
O
N
O
N
O
N
7
1
6
Scheme 1. Reagents and conditions: (a) 3-chloroperoxybenzoic acid, DME/heptane (1:2), rt, 2.5 h, 89.2%; (b) POCl3, 55°C, then rt, then
85–90°C, 18 h, 80%; (c) m-nitroaniline, 180°C, 2–5 h, 9.5%; (d) benzoyl chloride, diisopropylamine, CH3CN, rt, 8 h, 15%; (e) Pd/C, H2, THF,
rt, 2 h, 32%; (f) 4-morpholino-3-(trifluoromethyl)benzoic acid, HOBt, EDCI, TEA, DMF, 80°C, 12 h, 16%.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 635–641
Cell-Based Biological Evaluation of a New Bisamide Derivative
637
100
80
60
40
20
0
-20
-40
Kinase
Figure 2. Percentages of enzymatic inhibitions exerted by compound 1 over 47 kinases.
Table 1. IC50 of compound 1 over FMS and KDR kinases.
in submicromolar scale over all the tested cell lines. Of special
interest, it exhibited 2-digit nanomolar IC50 values over five
ovarian, two prostate, and three breast cancer cell lines.
In addition to its strong potency, compound 1 showed high
efficacy also. Its TGI values were less than 100 mM against 12
cancer cell lines. The highest efficacy was expressed against
OVCAR-3 ovarian cancer cell line and DU-145 prostate cancer
cell line with 100% growth inhibition at a concentration as
small as 45 and 98 nM, respectively.
The highest percent inhibition was reported against
SK-OV-3 ovarian cancer cell line (106.21%). The IC50 and TGI
values of compound 1 over this cell line were 33 nM and
3.60 mM, respectively. Woo et al. reported high expression of
CSF-1 mRNA level and high level of secreted CSF-1 protein level
Kinase enzyme
IC50 (nM)
FMS
KDR
96
1058
ovarian cancer cell line and T-47D breast cancer cell line, and
more than 73% over another eight cell lines of the three tested
cancer types.
Compound 1 was further tested in a 5-dose testing mode in
order to determine its IC50 and total growth inhibition (TGI)
concentration values. It showed high potency with IC50 values
Table 2. Inhibition percentages at a single-dose of 10 mM, and IC50 and TGI values in mM of compound 1 and sorafenib over ovarian,
prostate, and breast cancer cell lines.
5-Dose results of compound 1
5-Dose results of sorafenib
%Inhibition
(1-dose)
a)
a)
Cell line
IC50
TGIb)
IC50
TGIb)
Ovarian cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
PC-3
DU-145
MCF7
91.01
73.13
48.34
58.93
83.66
85.72
106.21
76.33
81.33
86.03
79.25
44.47
74.84
92.43
102.66
0.30
0.057
0.022
0.239
0.200
0.056
0.036
0.033
0.055
0.036
0.040
0.158
0.990
0.203
0.084
0.029
–
56.9
2.51
3.16
3.16
3.16
3.16
2.51
2.51
2.00
3.16
2.51
1.26
2.51
3.16
1.58
2.00
–
7.94
10.00
25.10
15.85
19.95
15.85
6.31
0.045
50.0
>100
>100
26.4
3.60
>100
0.098
54.0
3.42
97.2
45.8
7.46
Prostate cancer
Breast cancer
7.94
12.59
10.00
3.98
19.95
7.94
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
MDA-MB-468
–
6.31
7.94
0.10
7.80
HS-27 fibroblasts
a)
IC50 is the concentration producing 50% inhibition. The results are expressed as means of duplicate experiments.
TGI is the concentration producing 100% inhibition.
b)
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Arch. Pharm. Chem. Life Sci. 2014, 347, 635–641
Figure 3. Dose–response curves of compound 1 over the ovarian, prostate, and breast cancer subpanels.
in SK-OV-3 cell line [23]. So it can be assumed that the
anticancer activity of compound 1 against this cell line is
related to CSF-1 and FMS inhibition.
sorafenib may be responsible for its enhanced selectivity
toward FMS kinase. On the other hand, sorafenib is a multiple-
kinase inhibitor.
Sorafenib is a standard anticancer agent. The N-phenyl-
pyrrolo[3,2-c]pyridine nucleus of compound 1 is considered
as a conformationally restricted analog of the 4-phenoxy-
pyridine moiety of sorafenib (Fig. 1). Due to the structural
similarity between compound 1 and sorafenib, and the
potential anticancer activity of sorafenib, it was utilized as a
reference compound in this work to compare the activity
of compound 1 with its activity. Upon comparison, it was
found that compound 1 showed superior potencies over 14
tested cell lines (in submicromolar range) to those of
sorafenib (micromolar scale). The efficacy of compound 1
was higher than that of sorafenib against OVCAR-3 and
SK-OV-3 ovarian cancer cell lines and MDA-MB-231/ATCC
breast cancer cell line. Sorafenib is a well-known B-RAF
kinase inhibitor. But the target compound 1 did not show
significant B-RAF kinase inhibitory effect (IC50 > 2 mM). The
restricted conformation of compound 1 compared with
In order to compare the selectivity of compound 1 and
sorafenib toward cancer cells compared with normal cells,
they were tested against HS-27 fibroblasts. Their IC50 values
against HS-27 were 0.30 and 7.8 mM, respectively (Table 2). It
was found that compound 1 exerted superior selectivity
indices against nine cell lines to sorafenib (Table 3).
FMS kinase binds to macrophage or monocyte colony-
stimulating factor (M-CSF or CSF-1). Signal transduction
through that binding results in survival, proliferation, and
differentiation of monocyte/macrophage lineage [1, 2]. So
compound 1 was also tested for inhibitory effect over
peritoneal macrophage cells in a 5-dose testing mode
(Fig. 4). At 1 mM concentration, it inhibited 52% of macro-
phage cell viability. Bingle et al. [3] reported a direct
relationship between macrophage numbers, angiogenesis
and malignancy progression. So we can conclude that the
inhibitory effect of compound 1 against macrophage cells
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Arch. Pharm. Chem. Life Sci. 2014, 347, 635–641
Cell-Based Biological Evaluation of a New Bisamide Derivative
639
Table 3. Selectivity indices of compound 1 toward cancer cell
lines over HS-27 fibroblasts.a)
sorafenib against 14 cell lines, and more efficacious over three
cell lines. Compound 1 demonstrated higher selectivity
indices than sorafenib against nine cancer cell lines. It also
showed inhibitory effect against peritoneal macrophage
cellular viability. So it can be concluded that the strong
antiproliferative activity of compound 1 may be mainly due to
FMS kinase inhibition.
Cell line
Compound 1
Sorafenib
IGROV1
5.26
13.64
1.26
1.50
5.36
8.33
9.09
5.45
8.33
7.50
1.90
0.30
1.48
3.57
10.34
3.11
2.47
2.47
2.47
2.47
3.11
3.11
3.90
2.47
3.11
6.19
3.11
2.47
4.94
3.90
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
PC-3
DU-145
MCF7
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
MDA-MB-468
Experimental
General
Melting points were obtained on a Walden Precision Apparatus
Electrothermal 9300 apparatus and are uncorrected. Nuclear
magnetic resonance (NMR) spectral analyses were performed
using a Bruker ARX-300, 300 MHz spectrometer and a Bruker ARX-
400, 400 MHz spectrometer (Bruker Bioscience, Billerica, MA,
USA) with tetramethylsilane (TMS) as an internal standard. Mass
spectra (MS) and purity of the target compound (>96%) were
determined by LC-MS analysis using the following system: Waters
2998 photodiode array detector, Waters 3100 mass detector,
Waters SFO system fluidics organizer, Waters 2545 binary
gradient module, Waters reagent manager, Waters 2767 sample
manager, SunfireTM C18 column (4.6 mm ꢀ 50 mm, 5 mm particle
size); solvent gradient ¼ 95% A at 0 min, 1% A at 5 min; solvent A:
0.035% trifluoroacetic acid (TFA) in water; solvent B: 0.035% TFA
in CH3OH; flow rate ¼ 3.0 mL/min; the AUC was calculated using
Waters MassLynx 4.1 software. All reagents and solvents were
purchased from Aldrich Chemical Co. and Tokyo Chemical
Industry (TCI) Co., and used without further purification.
a)
Selectivity index was calculated by dividing the IC50 value
against HS-27 by the IC50 value against the cell line (Table 2).
Synthesis
7-Hydroxy-1H-pyrrolo[2,3-b]pyridinium 3-chlorobenzoate
(3) [15, 16]
To a solution of 7-azaindole (2, 19.83 g, 167.9 mmol) in DME/
heptane (1:2, 294 mL) was added 3-chloroperbenzoic acid (85 wt%,
46.2 g, 194.9 mmol) portionwise at 8–26°C. Precipitation oc-
curred after half of the 3-chloroperbenzoic acid was added.
The slurry was stirred at room temperature for 2.5h. The precipitate
was filtered and washed with DME/heptane (1:2, 100 mL). The
product was dried to yield an off-white solid (43.58 g, 89.2%). m.p.:
141–143°C (Lit. m.p.: 144.1–146°C) [16].
Figure 4. Effect of compound 1 on peritoneal macrophage cell
viability.
4-Chloro-7-azaindole (4) [16]
may contribute, at least in part, to its anticancer activities
To compound 3 (43.4 g, 149.3 mmol) was added POCl3 (170.4 g) at
room temperature. The solution was heated to 55°C, and then
heating was removed. The temperature slowly went up to 74°C
in about 1 h without external heating. The mixture was further
heated to 85–90°C for 18 h. Caution: Two exothermic events were
observed, one at 50–60°C and the other at 105–110°C. To prevent a
runaway reaction on scale, the solution was first heated to 55°C and then
heated slowly to 85–90°C. The solution was cooled to 50°C, and
POCl3 was distilled off in vacuo. The residue was dissolved in
acetonitrile (100 mL) and quenched by slow addition of water
(100 mL) while keeping the temperature under 50°C. The mixture
was basified to pH 9 with 50% NaOH solution. The slurry was
allowed to cool to room temperature, and the precipitates were
filtered. The wet cake was reslurried with H2O (200 mL), filtered,
and dried to afford the product (18.15 g, 80%). m.p.: 176–177°C
(Lit. m.p.: 175.3–177°C) [16].
in vivo.
Conclusion
A bisamide compound 1 containing pyrrolo[3,2-c]pyridine
scaffold was synthesized and reported to possess selective FMS
kinase inhibitory activity. It was tested over a panel of seven
ovarian, two prostate, and six breast cancer cell lines at the
NCI, USA. It exerted strong potency with submicromolar
IC50 values against all the tested cell lines, and high efficacy
over most of them. Its IC50 and TGI values were in 2-digit
nanomolar scale (22 and 45 nM, respectively) on OVCAR-3
ovarian cancer cell line. Compound 1 was more potent than
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Arch. Pharm. Chem. Life Sci. 2014, 347, 635–641
1-(3-Nitrophenyl)-1H-pyrrolo[3,2-c]pyridin-4-amine
hydrochloride (5)
mixture was cooled and then partitioned between water and
ethyl acetate and the organic layer was separated. The aqueous
layer was then extracted with ethyl acetate and the combined
organic extracts were washed with brine and dried over
anhydrous Na2SO4. After evaporation of the organic solvent,
the residue was purified by column chromatography (silica gel,
hexane/ethyl acetate 1:1v/v) to yield compound 1 as a yellow
crystalline solid (5.0 mg, 16%); m.p. 130–132°C; 1H NMR (300 MHz,
DMSO-d6) d 10.85 (brs, 1H), 10.65 (brs, 1H), 8.28–8.27 (m, 1H), 8.13–
8.09 (m, 4H), 7.88–7.83 (m, 1H), 7.75 (d, J ¼ 3.3 Hz, 2H), 7.67–7.54
(m, 6H), 7.41–7.37 (m, 1H), 6.69–6.67 (m, 1H), 3.75–3.72 (m, 4H),
2.97–2.89 (m, 4H); 13C NMR (100 MHz, DMSO-d6) d 163.3, 158.2,
141.0, 139.5, 133.3, 133.1, 132.1, 129.3, 128.5, 128.1, 127.8, 127.3,
126.9, 126.1, 125.3, 124.5, 124.0, 123.5, 123.3, 123.0,121.4, 120.8,
119.6, 116.9, 114.4, 104.5, 67.4, 53.6; IR (KBr) 3418, 2919, 1607,
1496, 1323, 1121, 850, 709 cmꢁ1; MS m/z 586.2 (Mþ1)þ.
A mixture of compound 4 (230 mg, 2.0 mmol) and 3-nitroaniline
(1.38 g, 10.0 mmol) was fused at 180°C for 2–5 h with stirring. The
reaction mixture was cooled to room temperature and dissolved
in ethanol (150 mL). The resulting suspension was filtered to
remove the insoluble material, and the filtrate was evaporated
under reduced pressure. The residue was purified by column
chromatography (silica gel, ethyl acetate/methanol 10:1 v/v then
switching to ethyl acetate/methanol 5:1 v/v) to obtain the desired
purified product. Yield 9.5%; 1H NMR (300 MHz, CD3OD) d 8.33 (t,
1H, J ¼ 2.0 Hz), 8.25 (dd, 1H, J ¼ 1.1, 6.1 Hz), 7.94–7.91 (m, 1H),
7.81–7.76 (m, 1H), 7.63 (d, 1H, J ¼ 2.2 Hz), 7.45 (d, 1H, J ¼ 3.4 Hz),
6.90 (d, 1H, J ¼ 3.4 Hz), 6.85 (d, 1H, J ¼ 6.3 Hz); 13C NMR (75 MHz,
CD3OD)
d 155.1, 150.4, 144.5, 140.3, 132.2, 131.0, 127.2,
122.6, 119.7, 115.0, 113.3, 104.5, 98.6; MS m/z: 255.95 (Mþ2)þ,
254.65 (M)þ.
Biological screening
N-(1-(3-Nitrophenyl)-1H-pyrrolo[3,2-c]pyridin-4-yl)-
benzamide (6)
Cancer cell line screening at the NCI
Screening against the 15 cancer cell lines was carried out at the
National Cancer Institute (NCI), Bethesda, Maryland, USA [22]
applying the standard protocol of the NCI [24, 25].
To a stirred solution of compound 5 (0.465 g, 1.6 mmol) in
acetonitrile (25 mL) at room temperature, diisopropylamine
(0.7 mL, 4.0 mmol) was slowly added under nitrogen atmosphere.
Benzoyl chloride (0.2 mL, 1.9 mmol) was slowly added and the
reaction mixture was stirred at room temperature for 8 h. The
reaction mixture was concentrated under reduced pressure, and
then water (20 mL) and CH2Cl2 (20 mL) were added to the residue.
The organic layer was separated, and the aqueous layer was
extracted with CH2Cl2 (2 ꢀ 20 mL). The combined organic layer
extracts were washed with brine, 1 N HCl, and then aqueous
NaHCO3, dried over anhydrous MgSO4, and filtered. The organic
solvent was evaporated under reduced pressure, and the residue
was purified by column chromatography (silica gel, hexane/ethyl
acetate 3:1 v/v) to give the target product. Yield 15%; 1H NMR
(300 MHz, DMSO-d6) d 10.90 (brs, 1H), 8.41 (t, 1H, J ¼ 2.1 Hz), 8.30
(d, 1H, J ¼ 8.0 Hz), 8.16–8.09 (m, 4H), 7.93–7.82 (m, 2H), 7.62–7.52
(m, 4H), 6.74 (brs, 1H); MS m/z: 359.11 (Mþ1)þ, 358.24 (M)þ.
MTT assay for peritoneal macrophage cell viability
Peritoneal macrophages were plated at a density of 105 cells/well
in 96-well plates. Cytotoxicity studies were performed 24 h after
treating cells with various concentrations of the test compound 1.
Cell viabilities were determined using colorimetric MTT assays, as
described previously [26].
Evaluation of the antiproliferative activity against HS-27
fibroblasts
HS-27 fibroblasts were purchased from American Type Culture
Collection (ATCC, Rockville, MD, USA) and maintained in
Dulbecco’s modified Eagle medium (DMEM; Welgene, Daegu,
Republic of Korea) supplemented with bovine calf serum (BCS;
Welgene) and 1% penicillin/streptomycin (Welgene) in a humidi-
fied atmosphere with 5% CO2 at 37°C. HS-27 fibroblasts were
taken from culture substrate with 0.05% trypsin–0.02% EDTA and
plated at a density of 5 ꢀ 103 cells/well in 96-well plates and then
incubated at 37°C for 24 h in a humidified atmosphere with 5%
CO2 prior to treatment with various concentrations (threefold
serial dilution, 12 points) of the tested compounds. The cells were
incubated for 48 h after treatment with the test compounds. The
HS-27 cell viability was assessed by the conventional 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) re-
duction assay. MTT assays were carried out with CellTiter 961
(Promega) according to the manufacturer’s instructions. The
absorbance at 590 nm was recorded using EnVision 2103 (Perkin
Elmer, Boston, MA, USA). The IC50 values were calculated using
GraphPad Prism 4.0 software. Triplicate testing was performed
for each test compound.
N-(1-(3-Aminophenyl)-1H-pyrrolo[3,2-c]pyridin-4-yl)-
benzamide (7)
A mixture of compound 6 (0.466 g, 1.3 mmol) and Pd/C (10%) in
anhydrous THF (20 mL) was stirred in hydrogen atmosphere at
room temperature for 2 h. The reaction mixture was filtered
through celite, and the filtrate was concentrated under reduced
pressure. The residue was purified by column chromatography
(silica gel, hexane/ethyl acetate 1:2 v/v) to give the purified
1
desired product. Yield 32%; H NMR (300 MHz, DMSO-d6) d 10.84
(brs, 1H), 8.11–8.09 (m, 3H), 7.62 (d, 2H, J ¼ 3.2 Hz), 7.54 (d, 2H,
J ¼ 7.5 Hz), 7.42 (brs, 1H), 7.22 (t, 1H, J ¼ 7.9 Hz), 6.77 (brs, 1H),
6.69–6.62 (m, 3H), 5.50 (brs, 2H); MS m/z: 330.24 (Mþ2)þ, 329.21
(Mþ1)þ.
N-(3-(4-Benzamido-1H-pyrrolo[3,2-c]pyridin-1-yl)phenyl)-
4-morpholino-3-(trifluoromethyl)benzamide (1)
This work was supported by Korea Institute of Science and Technology
(KIST), KIST Project (2E23720). We would like to thank the National
Cancer Institute (NCI), Bethesda, Maryland, USA, for performing the
anticancer testing of the target compound over 15 cancer cell lines.
A mixture of compound 7 (17.0 mg, 0.05 mmol), 3-(trifluoro-
methyl)-4-morpholinobenzoic acid (28.5 mg, 0.1 mmol), HOBt
(15.4 mg, 0.11 mmol), and EDCI (24.83 mg, 0.13 mmol) in dry
DMF (1.0 mL) was cooled to 0°C under nitrogen atmosphere. To
the reaction mixture, TEA (0.02 mL, 0.01 mmol) was added at 0°C.
The mixture was then stirred at 80°C for 12 h. The reaction
The authors have declared no conflict of interest.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2014, 347, 635–641
Cell-Based Biological Evaluation of a New Bisamide Derivative
641
References
[14] M. I. El-Gamal, M.-H. Jung, C.-H. Oh, Bioorg. Med. Chem. Lett.
2010, 20, 3216–3218.
[1] J. Woolford, V. Rothwell, L. Rohrschneider, Mol. Cell Biol. 1985,
5, 3458–3466.
[15] C.-C. Cheng, C.-P. Chang, W.-S. Yu, F.-T. Hung, Y.-I. Liu,
G.-R. Wu, P.-T. Chou, J. Phys. Chem. A 2003, 107, 1459–1471.
[2] C. J. Sherr, Biochim. Biophys. Acta 1988, 948, 225–243.
[16] X. Wang, B. Zhi, J. Baum, Y. Chen, R. Crockett, L. Huang,
S. Eisenberg, J. Ng, R. Larsen, M. Martinelli, P. Reider, J. Org.
Chem. 2006, 71, 4021–4023.
[3] L. Bingle, N. J. Brown, C. E. Lewis, J. Pathol. 2002, 196, 254–
265.
[4] A. Janowska-Wieczorek, A. R. Belch, A. Jacobs, D. Bowen,
R. Padua, E. Paietta, E. R. Stanley, Blood 1991, 77, 1796–
1803.
[17] N. S. Girgis, S. B. Larson, R. K. Robins, H. B. Cottam, J.
Heterocyclic Chem. 1989, 26, 317–325.
[18] M. I. El-Gamal, M.-H. Jung, W. S. Lee, T. Sim, K. H. Yoo,
[5] E. P. Toy, J. T. Chambers, B. M. Kacinski, M. B. Flick,
C.-H. Oh, Eur. J. Med. Chem. 2011, 46, 3218–3226.
S. K. Chambers, Gynecol. Oncol. 2001, 80, 194–200.
[19] B. M. Kacinski, D. Carter, K. Mittal, L. D. Yee, K. A. Scata,
L. Donofrio, S. K. Chambers, K. I. Wang, T. Yang-Feng, L. R.
Rohrschneider, V. M. Rothwell, Am. J. Pathol. 1990, 137, 135–147.
[6] H. Ide, D. B. Seligson, S. Memarzadeh, L. Xin, S. Horvath,
P. Dubey, M. B. Flick, B. M. Kacinski, A. Palotie, O. N. Witte,
Proc. Natl. Acad. Sci. USA 2002, 99, 14404–14409.
[20] B. M. Kacinski, K. A. Scata, D. Carter, L. D. Yee, E. Sapi, B. L.
King, S. K. Chambers, M. A. Jones, M. H. Pirro, E. R. Stanley,
et al., Oncogene 1991, 6, 941–952.
[7] E. Sapi, Exp. Biol. Med. 2004, 229, 1–11.
[8] L. D. Yee, L. Liu, Anticancer Res. 2000, 20, 4379–4383.
[21] A. E. Filderman, A. Bruckner, B. M. Kacinski, N. Deng,
[9] E. Y. Lin, A. V. Nguyen, R. G. Russell, J. W. Pollard, J. Exp. Med.
2001, 193, 727–739.
H. G. Remold, Cancer Res. 1992, 52, 3661–3666.
[22] NCI website: <www.dtp.nci.nih.gov>.
[10] H. M. Kluger, M. Dollad-Filhart, S. Rodov, B. M. Kacinski, R. L.
Camp, D. L. Rimm, Clin. Cancer Res. 2004, 10, 173–177.
[23] H.-H. Woo, C. F. Laszlo, S. Greco, S. K. Chambers, Mol. Cancer
2012, 11, 58.
[11] P. Zhu, S. H. Baek, E. M. Bourk, K. A. Ohgi, I. Garcia-Bassets,
H. Sanjo, S. Akira, P. F. Kotol, C. K. Glass, M. G. Rosenfeld,
D. W. Rose, Cell 2006, 124, 615–629.
[24] DTP Data Search: <http://dtp.nci.nih.gov/dtpstandard/dwindex/
index.jsp>.
[12] H. Ide, K. Hatake, Y. Terado, H. Tsukino, T. Okegawa, K. Nutahara,
[25] DTP Human Tumor Cell Line Screen Process: <http://www.
dtp.nci.nih.gov/branches/btb/ivclsp.html>.
E. Higashihara, S. Horis, Hum. Cell 2008, 21, 1–6.
[13] M. I. El-Gamal, H. S. Anbar, K. H. Yoo, C.-H. Oh, Med. Res. Rev.
2013, 33, 599–636.
[26] J. Y. Kim, S. J. Park, K. J. Yun, Y. W. Cho, H. J. Park, K.-T. Lee,
Eur. J. Pharmacol. 2008, 584, 175–184.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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