Novel coumarin-3-(N-aryl)carboxamides arrest breast cancer cell growth by inhibiting ErbB-2 and ERK1

Article abstract of DOI:10.1016/j.bmc.2005.02.051

A series of novel coumarin carboxamides were synthesized, and their tumor cell cytotoxic activity was investigated. These compounds specifically inhibited the growth of cancer cells that have a high level of ErbB-2 expression. Immunoprecipitation analysis

Full text of DOI:10.1016/j.bmc.2005.02.051

Bioorganic & Medicinal Chemistry 13 (2005) 3141–3147
Novel coumarin-3-(N-aryl)carboxamides arrest breast cancer
cell growth by inhibiting ErbB-2 and ERK1
Natala Srinivasa Reddy, Kiranmai Gumireddy, Muralidhar R. Mallireddigari,
Stephen C. Cosenza, Padmavathi Venkatapuram, Stanley C. Bell,
E. Premkumar Reddy and M. V. Ramana Reddy*
Fels Institute for Cancer Research, Temple University School of Medicine, 3307, North Broad Street,
Philadelphia, PA 19140-5101, USA
Received 26 January 2005; accepted 24 February 2005
Abstract—A series of novel coumarin carboxamides were synthesized, and their tumor cell cytotoxic activity was investigated. These
compounds specifically inhibited the growth of cancer cells that have a high level of ErbB-2 expression. Immunoprecipitation anal-
ysis of the cell lysates prepared from carboxamide treated cancer cells showed the inhibition of ErbB-2 phosphorylation suggesting
the interaction of these compounds with ErbB-2 receptor. The down regulation of the kinase activity was further confirmed by per-
forming in vitro kinase assay with recombinant ErbB-2 incubated with carboxamides. The inhibition of ErbB-2 phosphorylation
correlated with down-regulation of ERK1 MAP kinase activation that is involved in proliferative signaling pathway. Furthermore,
the cell-killing activity of many of these inhibitors is restricted to tumor cells with no demonstrable cytotoxicity against normal
human fibroblasts suggesting that these compounds are tumor-specific.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
as a selective anti-proliferative agent by activating p38,
stress activated protein kinase (SAPK) and the
p21WAF1/CIP1 cyclin dependent kinase inhibitor, in the
human renal cell carcinoma cell line, A-498.7,8 More re-
cently, two new naturally occurring coumarins have
been isolated and shown to inhibit the polymerization
of tubulin and arrest cells in mitotic phase by inhibiting
microtubule formation.9 These coumarin were found to
act synergistically with paclitaxel in inhibiting KB (hu-
man epidermoid carcinoma) cell proliferation.9
Coumarins are structurally simple compounds belong-
ing to a large class of molecules known as benzopyro-
nes.1 Coumarins and related compounds have diverse
biological activities, including anti-coagulant and anti-
thrombotic properties.2 Coumarin derivatives have also
been shown to be novel lipid lowering agents that pos-
sess moderate triglyceride lowering activity.3 Many cou-
marin derivatives have the unique ability to scavenge
reactive oxygen species such as hydroxyl free radicals,
superoxide radicals, or hypochlorous acid to prevent
free radical injury.4 Recently, certain coumarin deriva-
tives have been shown to function as human immunode-
ficiency virus integrase inhibitors and have been
evaluated in the treatment of HIV infection,5 whereas
others have been evaluated as anti-invasive compounds
due to their inhibitory activity against some serine pro-
teases and matrix metalloproteases (MMPs).6 Recently,
it has been shown that 6-nitro-7-hydroxy coumarin acts
Recently, we have described the growth inhibitory prop-
erties of novel coumarin sulfonamides using a panel of
cancer cell lines.10 These coumarin derivatives up-regu-
lated the activity of c-Jun NH2 terminal kinase 1
(JNK1), which is a member of the stress activated pro-
tein kinases (SAPKs) that are involved in apoptotic sig-
naling.10 Based on the diverse biological activities of
coumarins and their derivatives, we have designed and
synthesized a series of novel coumarin 3-carboxamides
and examined the cytotoxic activity of these compounds
in different cancer cell lines. Our results show that cou-
marin 3-carboxamides selectively inhibit the tumor cells
that have higher levels of epidermal growth factor recep-
tor (EGFR or erbB1) and erbB2 (HER2) receptor
expression.
Keywords: Coumarin carboxamides; ErbB-2; MAP kinase; In vitro
kinase activity.
*
Corresponding author. Tel.: +1 215 707 7336; fax: +1 215 707
1454; e-mail: rreddy@temple.edu
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.02.051

3142
N. S. Reddy et al. / Bioorg. Med. Chem. 13 (2005) 3141–3147
NH
2
2. Chemistry
H
O
O
N
O
Although several substituted coumarins have been de-
scribed in the literature, very little information is known
about coumarin 3-carboxamides. In earlier publica-
tions,11 these compounds were synthesized in a circui-
tous way by condensing malonic esters with
salicylaldehydes, followed by hydrolysis, halogenation,
and condensation with amines as illustrated in Scheme
1.12 This method has a limitation due to a variety of sub-
stituents at different positions on both aromatic rings
and thus may not be useful in creating a large library
of coumarin 3-carboxamides. In this manuscript, we re-
port a novel route for the synthesis of coumarin-3-carb-
oxamides using the Knoevenagel type of condensation
reaction.
b
+
a
Cl
O
O
O
Y
Y
2
3
1
CHO
OH
H
N
OH
c
+
O
O
X
Y
5
4
Y
O
N
H
O
O
X
6
The reaction of methyl-3-chloro-3-oxopropionate 2 with
aromatic amines 1 in the presence of triethylamine pro-
duced methyl 3-anilino-3-oxopropionate 3, which on
hydrolysis yielded 3-anilino-3-oxopropionic acid 4. Con-
densation of 3-anilino-3-oxopropionic acid with substi-
tuted salicylaldehydes 5 in glacial acetic acid in the
presence of a catalytic amount of benzylamine produced
coumarin-3-carboxamides 6 in quantitative yields13
(Scheme 2). Alternatively, coumarin-3-carboxamides 6
were also prepared by reacting methyl 3-anilino-3-oxo-
propionate 3 with substituted salicylaldehydes 5 in the
presence of piperidine in ethanol (Scheme 3).
Scheme 2. Reagents and conditions: (a) Et3N, CH2Cl2, 0 ꢁC to rt; (b)
10% NaOH, HCl; (c) PhCH2NH2, HOAc, reflux.
Y
O
H
N
O
N
H
a
+ 5
O
O
O
O
Y
X
6
3
Scheme 3. Reagents and conditions: (a) piperidine, ethanol, reflux 1 h.
The cytotoxic properties of coumarin-3-carboxamides
(6a–l) were determined using the SKBR-3 and BT474
breast cancer cell lines as well as normal HFLs. Both
SKBR-3 and BT474 over-express ErbB-2 receptor at
about 7 · 105 receptors per cell14,15 where as HFL cells
do not express this receptor. The results showed that
coumarin carboxamides inhibited the growth of ErbB-2
over-expressing cells (SKBR-3 and BT474) more effec-
tively when compared to HFL cells that do not express
ErbB-2 receptors (Table 1). Furthermore, the carbox-
amides (6c, 6e, 6g, 6j, and 6l) exhibited more potent
cytotoxic activity (Table 1) than the coumarin carboxa-
3. Results
To determine the cytotoxic activity of these compounds,
two human breast cancer cell lines, SKBR-3, BT474
(with high expressing levels of ErbB-2 and EGFR) and
HFL cells (human lung fibroblasts that do not over-ex-
press the ErbB-2 and EGFR) were incubated in the pres-
ence of all the newly synthesized coumarin-3-
carboxamides and the cell viability determined by try-
pan blue exclusion. The results of this study are summa-
rized in Table 1.
O
CHO
O
O
a
+
OH
X
O
O
O
O
b
OH
c
O
O
O
O
O
X
X
Y
O
O
NH
2
Cl
N
H
d
+
O
O
O
X
Y
X
Scheme 1. Reagents and conditions: (a) piperidine/EtOH/HOAc; (b) HCl/H2O/EtOH; (c) SOCl2; (d) PhNH2/pyridine/dioxane.

N. S. Reddy et al. / Bioorg. Med. Chem. 13 (2005) 3141–3147
Table 1. Cell growth and ErbB-2 kinase inhibition by coumarin-3-carboxamides
3143
Y
O
N
H
O
O
X
6
Compd
X
Y
Cell line (GI50, lM)a
ErbB-2 (IC50, lM)b
BT474
SKBR-3
HFL
6a
6b
6c
6d
6e
6f
H
4-Br
4-I
120
115
21.2
87
135
110
16.3
64
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>300
>10
>10
H
8-EtO
8-EtO
8-EtO
8-EtO
6-Br
6-Cl
6-Cl
6-Cl
6-Br
6-Cl
4-Br
0.316
3-NO2, 4-Cl
3-NH2, 4-Cl
4-I
0.549
0.709
3.84
21
94
20.6
84
6g
6h
6i
4-I
4-Br
21.4
81
21.8
74
0.204
>10
>10
0.166
>10
2.17
4-I
72
75
6j
3-NO2, 4-Cl
4-Br
3-NH2, 4-Cl
20.8
63
19.4
60.5
19.4
6k
6l
20.3
a The GI50 was determined by direct extrapolation from each dose response curve.
b The IC50 of ErbB-2 kinase activity was determined using an in vitro kinase assay.
mides (6a, 6b, 6d–f, 6h, 6i) against SKBR-3 and BT474
cell lines. These results clearly show that the substitu-
tions on the coumarin ring and on the phenyl ring
substantially affect the cell-killing ability of the mole-
cules.
To determine the mechanism by which these compounds
induce cell death, we have examined the effects of cou-
marin-3-carboxamides on the activation state of ErbB-
2, as well as downstream proliferation and survival
pathways using SKBR-3 and BT474 cells that have been
treated with epidermal growth factor (EGF). In this
study, the coumarin-3-carboxamides that showed higher
cytotoxic activity in cell culture-based assays (6c, 6e, 6g,
6j, and 6l) were added to SKBR-3 and BT474 cells that
have been treated with EGF. When whole cell extracts
derived from these cells were subjected to immunopre-
cipitation using an ErbB-2 specific antibody followed
by western blot analysis using a phosphotyrosine anti-
body, we observed a total inhibition of EGF-induced
phosphorylation of ErbB-2 by these compounds (Fig.
1). To confirm that these compounds directly inhibit
the kinase activity ErbB-2, we performed an in vitro ki-
nase assay with recombinant ErbB-2 protein using poly-
glutamine tyrosine as a substrate. These results showed
that while all of the compounds, in general, inhibit the
activity of ErbB-2, those compounds that showed higher
cytotoxic activity in the SKBR-3 and BT474 tumor cell
lines in general had the most potent ErbB-2 inhibitory
activity (Fig. 2 and Table 1).
Figure 1. Inhibition of ErbB-2 phosphorylation by coumarin-3-carb-
oxamides in SKBR-3 (A), BT474 (B) cells. Cells were treated with
50 lM carboxamide for 24 h and cell lysates were analyzed by
immunoprecipitating with an ErbB-2-specific antibody followed by
western blotting with a phosphotyrosine antibody. Data shown are
representative of three independent experiments.
3 and BT474 cells, incubated for 24 h in the presence of
coumarin-3-carboxamides 6c, 6e, 6g, 6j, and 6l, were
subjected to immunoprecipitation followed by kinase
assays (Fig. 2), we observed that these compounds
inhibited the activation of ERK1 by more than 80%.
Taken together, these results suggest that coumarin-
3-carboxamides effectively inhibit ErbB-2 mediated
signaling in cells over-expressing this protein.
4. Discussion
ErbB-2 over-expression is associated with the activation
of downstream proliferative pathways involving the
activation of ERK1/2 MAP kinases.16 It was therefore
of interest to determine whether coumarin-3-carboxam-
ide mediated inhibition of ErbB-2 affected the activation
of these proteins as well. When cell extracts from SKBR-
Our current study shows that coumarin-3-carboxamides
exert a cytotoxic effect by inhibiting ErbB-2 phosphoryl-
ation in tumor cell lines that over-express the receptor,
thereby causing growth arrest and cell death. In this
study, we investigated the effect of coumarin carbox-

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N. S. Reddy et al. / Bioorg. Med. Chem. 13 (2005) 3141–3147
125
100
75
50
25
0
6a
6b
6c
6d
6e
6f
0
-8
-7
-6
-5
-4
(A)
Inhibitor Concentration(M)
Figure 3. Inhibition of ERK1 phosphorylation by coumarin-3-carb-
oxamides in BT474 (A), SKBR-3 (B) cells. Cells were treated with
50 lM carboxamide for 24 h and cell lysates were analyzed by
immunoprecipitating with an ERK1 antibody followed by an in vitro
kinase assay using Myelin Basic Protein (MBP) as a substrate. Data
shown are representative of three independent experiments.
125
100
75
50
25
0
6g
6h
6i
6j
6k
6l
biomarker that correlates with the ability of coumarin-3-
carboxamides to inhibit tumor cell proliferation.
Results presented in this manuscript also demonstrate
that the inhibitory activity of coumarin carboxamides
can be considerably improved through substitutions on
the coumarin ring and on the phenyl ring, which in turn
results in improved tumor cell-killing ability of the mole-
cules (Table 1). The demonstration that cell-killing
activity of many of these inhibitors is restricted to tumor
cells with no demonstrable cytotoxicity against normal
human fibroblasts suggests that these compounds are tu-
mor-specific. This in turn appears to be due to the over-
expression of ErbB family of receptors on tumor cells,
which provides these cells with growth advantage.
0
-8
-7
-6
-5
-4
(B)
Inhibitor Concentration(M)
Figure 2. Inhibition of ErbB-2 kinase activity in vitro by coumarin
carboxamides. Recombinant ErbB-2 was incubated with various
concentrations of carboxamides and kinase reaction was performed
using poly(Glu:Tyr,4:1) as substrate. Data shown is a representative of
two independent experiments.
amides on downstream survival and proliferation path-
ways to characterize these compounds and elucidate
their mechanism of action.
The ErbB-2 family of growth factors receptors, EGFR
and ErbB-2/HER2 in particular, are known to play
important roles in breast cancer pathogenesis.17 In order
to develop a targeted drug, it is important to have a
clear understanding of the growth factor receptor-
dependent pathways that are activated in breast tumor
cells. Most mitogenic signals transduced through growth
factor receptor activation ultimately converge on com-
mon downstream effectors, the ERK1/2 MAP kinases.18
Activated ERK1/2 control many processes that are cen-
tral to tumor cell proliferation, survival, and tumor pro-
gression.19 Increased expression of activated ERK1/2
has been demonstrated in a number of human malignan-
cies20 and over-expression of ErbB-2 in tumor cell lines
results in the up-regulation of activated ERK1/2.21 Our
data show that coumarin carboxamides inhibited ERK1
activation in EGFR/ErbB-2-over-expressing tumor cell
lines (Fig. 3).
5. Conclusions
We have identified a new series of compounds, couma-
rin-3-(N-aryl) carboxamides that are novel classes of po-
tent ErbB-2 and ERK1 MAP kinase inhibitors. The
compounds, 6c, 6e, 6g, 6j, and 6l in particular, demon-
strated a 4–5-fold increase in cytotoxic activity as com-
pared to 6a and 6b. These results show that
substitutions on the coumarin ring and also on the
phenyl group can drastically alter the cytotoxic property
of the compounds, and suggest that a coumarin carbox-
amide with increased potency can be designed and syn-
thesized by analyzing structure–activity relationship.
Such a molecule is likely to have much higher ErbB-2
and ERK1 MAPK inhibitory activities, which in turn
could result in enhanced anti-tumor activity.
6. Experimental
6.1. Chemistry
Furthermore, although EGF stimulated the activation
of ErbB-2 and ERK1 in SKBR-3 and BT474 breast can-
cer cells, exposure to coumarin carboxamides com-
pletely inhibited these effects (Fig. 2). These results
highlight the importance of ligand induced growth ef-
fects on the level of receptor activation in tumor cells.
They also show that ERK1 may be a useful downstream
6.1.1. General information. Unless otherwise noted,
chemicals were commercially available and used as re-
ceived without further purification. Thin-layer chroma-
tography was performed on precoated silica gel F254

N. S. Reddy et al. / Bioorg. Med. Chem. 13 (2005) 3141–3147
3145
plates (Sigma–Aldrich) with a UV indicator. Silica gel
column chromatography was performed using silica
gel 60A (Aldrich 70–230 mesh). Melting points were
determined in open glass capillaries using Mel-Temp
tents for 1 h. The precipitated product that comes out
after cooling was filtered, washed with 2-propanol
(3 · 20 mL) and diethyl ether (2 · 20 mL) to obtain a
pure product (68–88%).
1
apparatus and are uncorrected. H NMR spectra were
recorded using a Brucker 400 MHz spectrometer in
DMSO-d6 or CDCl3 as a solvent and TMS as an inter-
nal standard. Chemical shifts (d) are given in parts per
million relative to TMS. Elemental analyses for these
compounds were performed by Quantitative Technolo-
gies Inc., Whitehouse, New Jersey, USA, and are within
0.4% of theoretical values.
6.1.3.3. Coumarin 3-(N-4-bromophenyl)carboxamide
(6a). Coumarin 3-(N-4-bromophenyl)carboxamide (6a)
was prepared according to method A starting from N-
(4-bromophenyl)-3-oxopropionic acid (2.0 g, 7.75
mmol) and salicylaldehyde (0.946 g, 7.75 mmol), affor-
ded 2.03 g (76%) of (6a): mp 269–271 ꢁC; 1H NMR
(400 MHz, DMSO): d 10.67 (br s, 1H), 8.88 (s, 1H),
7.98 (dd, J = 8.0, 1.5 Hz, 1H), 7.76 (m, 1H), 7.69 (d,
J = 7.0 Hz, 2H), 7.56 (d, J = 7.0 Hz, 2H), 7.53 (m,
1H), and 7.45 (t, J = 7.0 Hz, 1H). Anal. (C16H10NO3Br)
C, H, N.
6.1.2. General procedure for preparation of N-aryl 3-oxo
propionic acids (4a–c). Methyl-3-chloro-3-oxopropio-
nate (100 mmol) was added dropwise to a solution of
dichloromethane (100 mL) containing substituted ani-
line (100 mmol) and triethylamine (100 mmol) at 0 ꢁC
stirred for an additional 5 h. The resulting reaction mix-
ture was washed with cold water (3 · 50 mL), dried
(Na2SO4), filtered, and the organic layer evaporated un-
der reduced pressure to obtain methyl N-aryl 3-oxoprop-
ionate (3) quantitatively. Methyl N-aryl 3-oxo-
propionate (3) upon hydrolysis with a 10% KOH solu-
tion followed by neutralization with dil HCl produced
N-aryl 3-oxo propionic acid in 78–86% yield.
6.1.3.4. Coumarin 3-(N-4-iodophenyl)carboxamide (6b).
Coumarin 3-(N-4-iodophenyl)carboxamide (6b) was
prepared according to method B starting from methyl
N-(4-iodophenyl)-3-oxopropionate (2.47 g, 7.75 mmol)
and salicylaldehyde (0.946 g, 7.75 mmol), afforded
2.67 g (88%) of (6b): mp 281–282 ꢁC; 1H NMR
(400 MHz, DMSO): d 10.89 (br s, 1H), 9.09 (s, 1H),
8.20 (d, J = 7.7 Hz, 1H), 8.08 (t, J = 8.5 Hz, 1H), 7.92
(d, J = 8.6 Hz, 2H), 7.77 (d, J = 8.6 Hz, 2H), 7.75 (m,
1H), and 7.64 (t, J = 8.5 Hz, 1H). Anal. (C16H10NO3I)
C, H, N.
6.1.2.1. 4-Iodoanilino-3-oxopropionic acid (4a). Yield
82%, mp 162–164 ꢁC; 1H NMR (400 MHz, DMSO):
10.64 (br s, 1H), 7.58 (d, J = 9.0 Hz, 2H), 7.35 (d,
J = 9.0 Hz, 2H), 3.13 (s, 2H).
6.1.3.5. 8-Ethoxycoumarin 3-(N-4-bromophenyl)carb-
oxamide (6c). 8-Ethoxycoumarin 3-(N-4-bromophenyl)-
carboxamide (6c) was prepared according to method A
starting from N-(4-bromophenyl)-3-oxopropionic acid
(2.0 g, 7.75 mmol) and 3-ethoxysalicylaldehyde (1.29 g,
7.75 mmol), afforded 1.80 g (60%) of (6c): mp 242–
244 ꢁC; 1H NMR (400 MHz, DMSO): d 10.71 (br s,
1H), 8.83 (s, 1H), 7.68 (d, J = 8.9 Hz, 2H), 7.53 (d,
J = 8.9 Hz, 2H), 7.49 (d, J = 7.8 Hz, 1H), 7.41 (d,
J = 7.6 Hz, 1H), 7.34 (t, J = 7.8 Hz, 1H), 4.23 (q,
J = 7.0 Hz, 2H), and 1.38 (t, J = 6.9 Hz, 3H). Anal.
(C18H14NO4Br) C, H, N.
6.1.2.2. 4-Bromoanilino-3-oxopropionic acid (4b).
Yield 86% mp 130–132 ꢁC; 1H NMR (400 MHz,
DMSO): 10.48 (br s, 1H), 7.50 (d, J = 9.0 Hz, 2H),
7.42 (d, J = 9.0 Hz, 2H), 3.14 (s, 2H).
6.1.2.3. 4-Chloro-3-nitroanilino 3-oxopropionic acid
1
(4c). Yield 78%, mp 118–120 ꢁC; H NMR (400 MHz,
DMSO):11.5 (br s, 1H), 8.36 (s, 1H), 7.70 (d,
J = 8.5 Hz, 1H), 7.62 (d, J = 8.5 Hz, 1H), 3.15 (s, 2H).
6.1.3.6. 8-Ethoxycoumarin 3-(N-4-chloro 3-nitrophenyl)-
carboxamide (6d). 8-Ethoxycoumarin 3-(N-4-chloro 3-
nitrophenyl)carboxamide (6d) was prepared according
6.1.3. General procedure for the preparation of coumarin
3-carboxamides (6a–l)
6.1.3.1. Method A. To a solution of N-(aryl)-3-oxo-
propionic acid (7.75 mmol) and salicylaldehyde
(7.75 mmol) in 12 mL glacial acetic acid was added cata-
lytic amount of benzyl amine (300 lL) and refluxed for
5–8 h. Most of the times the condensed product comes
out of the solution, which was filtered, washed with 2-
propanol (3 · 20 mL) to get the pure product. Some
cases where solid is not formed, the reaction mixture
was diluted with 75 mL diethyl ether, washed with satu-
rated NaHCO3 (2 · 25 mL), 5% HCl (2 · 20 mL), water
(2 · 25 mL), and brine (2 · 25 mL), dried (Na2SO4), fil-
tered, and evaporated in vacuo to get pure (60–80%)
of the title compounds.
to method
A starting from N-(4-chloro 3-nitro-
phenyl)-3-oxopropionic acid (2.0 g, 7.75 mmol) and 3-
ethoxysalicylaldehyde (1.29 g, 7.75 mmol), afforded
2.41 g (80%) of (6d): mp 260–262 ꢁC; 1H NMR
(400 MHz, DMSO): d 11.08 (br s, 1H), 8.90 (s, 1H),
8.41 (d, J = 2.5 Hz, 1H), 7.71 (dd, J = 8.8, 2.5 Hz, 1H),
7.27–7.15 (m, 3H), 7.43 (d, J = 8.8 Hz, 1H), 4.14 (q,
J = 7.0 Hz, 2H), and 1.46 (t, J = 7.0 Hz, 3H). Anal.
(C18H14N2O6Cl) C, H, N.
6.1.3.7. 8-Ethoxycoumarin 3-(N-4-chloro 3-aminophen-
yl)carboxamide (6e). A solution of compound 6d (1.0 g,
2.57 mmol) in 1:2 water/acetone (30 mL) mixture was
heated at 50 ꢁC for 30 min and sodium hydrosulfite
(8.95 g, 51.4 mmol) was added and maintained at
50 ꢁC for another 30 min. After cooling to room temper-
ature, the reaction mixture was diluted with 50 mL of
6.1.3.2. Method B. To a solution of salicylaldehyde
(7.75 mmol) in 30 mL warm ethanol, was added methyl
N-(aryl)-3-oxopropionate (7.75 mmol) and catalytic
amount of piperidine (0.75 mmol) and refluxed the con-

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N. S. Reddy et al. / Bioorg. Med. Chem. 13 (2005) 3141–3147
water and extracted with ethyl acetate (100 mL). The
aqueous phase was further extracted with ethyl acetate
(2 · 50 mL). The combined organic layer was washed
successively with water (3 · 50 mL), brine (2 · 25 mL)
and dried over anhydrous sodium sulfate. Removal of
the solvent on a rotavapor yielded a crude product that
was further purified on silica gel column using petro-
leum ether and ethyl acetate mixture (9:1) afforded
6.1.3.12. 6-Chlorocoumarin 3-(N-4-chloro 3-nitrophenyl)-
carboxamide (6j). 6-Chlorocoumarin 3-(N-4-chloro 3-
nitrophenyl)carboxamide (6j) was prepared according
to A starting from N-(4-chloro, 3-nitrophenyl)-3-oxo-
propionic acid (2.0 g, 7.75 mmol) and 5-chlorosalicylal-
dehyde (1.21 g, 7.75 mmol), afforded 2.35 g (80%) of
1
(6j): mp 293–295 ꢁC; H NMR (400 MHz, DMSO): d
11.03 (br s, 1H), 8.92 (s, 1H), 8.64 (d, J = 2.5 Hz, 1H),
8.21 (d, J = 2.5 Hz, 1H), 8.05 (dd, J = 8.8, 2.5 Hz, 1H),
7.89 (dd, J = 8.8, 2.5 Hz, 1H), 7.67 (d, J = 8.0 Hz, 1H),
and 7.44 (d, J = 8.0 Hz, 1H). Anal. (C16H8N2O5Cl2) C,
H, N.
1
0.276 g (30%) of pure product 6e: mp 182–186 ꢁC; H
NMR (400 MHz, DMSO): d 10.21 (br s, 1H), 8.20 (s,
1H), 8.11 (d, J = 2.5 Hz, 1H), 7.71 (dd, J = 8.8, 2.5 Hz,
1H), 7.27–7.15 (m, 3H), 5.36 (br s, 2H),7.43 (d,
J = 8.8 Hz, 1H), 4.14 (q, J = 7.0 Hz, 2H), and 1.46 (t,
J = 7.0 Hz, 3H). Anal. (C18H16N2O4Cl) C, H, N.
6.1.3.13. 6-Bromocoumarin 3-(N-4-bromophenyl)carb-
oxamide (6k). 6-Bromocoumarin 3-(N-4-bromophenyl)-
carboxamide (6k) was prepared according to method
6.1.3.8. 8-Ethoxycoumarin 3-(N-4-iodophenyl)carbox-
amide (6f). 8-Ethoxycoumarin 3-(N-4-iodophenyl)carb-
oxamide (6f) was prepared according to method B
starting from methyl N-(4-iodophenyl)-3-oxopropionate
(2.47 g, 7.75 mmol) and 3-ethoxysalicylaldehyde (1.29 g,
7.75 mmol), afforded 2.29 g (68%) of (6f): mp 252–
255 ꢁC; 1H NMR (400 MHz, DMSO): d 10.73 (br s,
1H), 8.89 (s, 1H), 7.75 (d, J = 8.7 Hz, 2H), 7.59 (d,
J = 8.7 Hz, 2H), 7.56 (d, J = 7.8 Hz, 1H), 7.48 (d,
J = 8.2 Hz, 1H), 7.30 (t, J = 7.6 Hz, 1H), 4.25 (q,
J = 7.0 Hz, 2H), and 1.45 (t, J = 7.0 Hz, 3H). Anal.
(C18H14NO4I) C, H, N.
A starting from N-(4-bromophenyl)-3-oxopropionic
acid (2.0 g, 7.75 mmol) and 5-bromosalicylaldehyde
(1.56 g, 7.75 mmol), afforded 2.36 g (72%) of 6k: mp
1
253–256 ꢁC; H NMR (400 MHz, DMSO): d 10.65 (br
s, 1H), 8.81 (s, 1H), 8.24 (d, J = 2.5 Hz, 1H), 7.89 (dd,
J = 9.0, 2.5 Hz, 1H), 7.67 (d, J = 9.5 Hz, 2H), 7.52 (d,
J = 9.5 Hz, 2H), and 7.50 (d, J = 8.5 Hz, 1H). Anal.
(C16H9NO3Br2) C, H, N.
6.1.3.14. 6-Chlorocoumarin 3-(N-4-chloro 3-aminophen-
yl)carboxamide (6l). To a solution of the compound 6j
(0.974 g, 2.57 mmol) in 1:2 water/acetone (30 mL), at
50 ꢁC for 30 min, sodium hydrosulfite (8.95 g,
51.4 mmol) was added and continued at 50 ꢁC for an-
other 30 min. After cooling to room temperature,
50 mL of water was added and extracted with ethyl ace-
tate (100 mL). The aqueous layer was further extracted
with ethyl acetate (2 · 50 mL). The combined organic
phase was washed successively with water (3 · 50 mL),
brine (2 · 25 mL), dried over sodium sulfate, and evapo-
rated to obtain crude product, which was further puri-
fied on silica gel column using petroleum ether and
ethyl acetate mixture (9:1) afforded 0.27 g (30%) pure
product 6l: mp 186–190 ꢁC; 1H NMR (400 MHz,
DMSO): d 10.21 (br s, 1H), 8.60 (s, 1H), 8.41 (d,
J = 2.5 Hz, 1H), 7.71 (dd, J = 8.8, 2.5 Hz, 1H), 7.27–
7.15 (m, 3H), 5.46 (br s, 2H), and 7.43 (d, J = 8.8 Hz,
1H). Anal. (C16H10N2O3Cl2) C, H, N.
6.1.3.9. 6-Bromocoumarin 3-(N-4-iodophenyl)carbox-
amide (6g). 6-Bromocoumarin 3-(N-4-iodophenyl)carb-
oxamide (6g) was prepared according to method B
starting from methyl N-(4-iodophenyl)-3-oxopropionate
(2.47 g, 7.75 mmol) and 5-bromosalicylaldehyde (1.56 g,
7.75 mmol), afforded 2.73 g (75%) of (6g): mp 258–
260 ꢁC; 1H NMR (400 MHz, DMSO): d 10.52 (br s,
1H), 8.70 (s, 1H), 8.14 (d, J = 2.6 Hz, 1H), 7.79 (dd,
J = 8.9, 2.2 Hz, 1H), 7.59 (d, J = 8.5 Hz, 2H), 7.43 (d,
J = 8.6 Hz, 2H), and 7.39 (d, J = 8.9 Hz, 1H). Anal.
(C16H9NO3BrI) C, H, N.
6.1.3.10. 6-Chlorocoumarin 3-(N-4-bromophenyl)carb-
oxamide (6h). 6-Chlorocoumarin 3-(N-4-bromophenyl)-
carboxamide (6h) was prepared according to method
A starting from N-(4-bromophenyl)-3-oxopropionic
acid (2.0 g, 7.75 mmol) and 5-chlorosalicylaldehyde
(1.21 g, 7.75 mmol), afforded 2.03 g (69%) of (6h): mp
6.1.4. Cell lines. HFL human lung fibroblasts, BT474 and
SKBR-3 breast carcinoma cells were grown in DulbeccoÕs
Modified Eagles Medium (DMEM) supplemented with
10% fetal bovine serum (FBS), 2 mM L-glutamine,
100 U/mL penicillin, and 0.01 mg/mL streptomycin at
37 ꢁC under humidified conditions in 5% CO2.
1
168–172 ꢁC; H NMR (400 MHz, DMSO): 12.74 (br s,
1H), 9.01 (s, 1H), 7.84 (d, J = 2.6 Hz, 1H), 7.79 (dd,
J = 8.9, 2.2 Hz, 1H), 7.72 (d, J = 8.5 Hz, 2H), 7.43 (d,
J = 8.6 Hz, 2H), and 7.09 (d, J = 8.9 Hz, 1H). Anal.
(C16H9NO3BrCl) C, H, N.
6.1.3.11. 6-Chlorocoumarin 3-(N-4-iodophenyl)carbox-
amide (6i). 6-Chlorocoumarin 3-(N-4-iodophenyl)carb-
oxamide (6i) was prepared according to method B
starting from methyl N-(4-iodophenyl)-3-oxopropionate
(2.47 g, 7.75 mmol) and 5-chlorosalicylaldehyde (1.21 g,
7.75 mmol), afforded 2.37 g (72%) of (6i): mp 267–
269 ꢁC; 1H NMR (400 MHz, DMSO): d 10.52 (br s,
1H), 8.71 (s, 1H), 8.01 (d, J = 2.5 Hz, 1H), 7.68 (dd,
J = 8.9, 2.5 Hz, 1H), 7.59 (d, J = 8.8 Hz, 2H), 7.46 (d,
J = 9.0 Hz, 2H), and 7.43 (d, J = 8.8 Hz, 1H). Anal.
(C16H9NO3ClI) C, H, N.
6.1.5. In vitro cytotoxicity. Cells lines (1 · 105) were
seeded into six well dishes and 24 h later, each com-
pound was added at five different concentrations over
a 2 log dilution (1–100 mM). The total number of viable
cells was determined 96 h post-treatment by trypan blue
exclusion. The percentage of viable cells remaining was
calculated as follows: # viable cells (compound trea-
ted)/# viable cells (DMSO treated) 100. Dose response
*
curves were generated by plotting the percentage of
cells at each concentration versus the concentration
tested.

N. S. Reddy et al. / Bioorg. Med. Chem. 13 (2005) 3141–3147
3147
6.1.6. Analysis of ErbB-2 tyrosine phosphorylation by
Acknowledgements
immunoprecipitation followed by western blotting. Immu-
noprecipitation and western blot analysis was performed
according to the procedure as described by VaraPrasad
et al.22 Briefly, SKBR-3/BT474 cells were grown in
100 mm plates to 80% confluence and serum starved
for 18 h. The cells were incubated with the appropriate
compound (50 lM) for 2 h and stimulated with 10 ng/
mL EGF. After 15 min, the cells were washed twice with
ice cold PBS and lysed in lysis buffer.22 Two hundred
micrograms of the lysate was immunoprecipitated with
an ErbB-2 antibody at 4 ꢁC overnight in the presence
of 50 lL protein A beads. The immune complex was
washed twice with PBS, resuspended in 50 lL of Lae-
mmli buffer, and boiled for 3 min. The samples were
electrophoresed on a 10% polyacrylamide gel and trans-
ferred onto a PVDF membrane. The membrane was
blocked with 3% milk and the level of tyrosine phos-
phorylated ErbB-2 was determined using an anti-phos-
photyrosine antibody (clone 4G10; Upstate Cell
Signaling Solutions, Lake Placid, NY).
This work was supported by grants from the Fels Foun-
dation and Onconova Therapeutics, Inc. New Jersey.
The authors thank Dr. Stacey J. Baker for editing this
manuscript.
References and notes
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6.1.7. In vitro kinase kinase assay for ErbB-2 inhibition.
Recombinant ErbB-2 was expressed in baculovirus-in-
fected Sf9 cells and purified according to the supplierÕs
instructions (BD Biosciences). One hundred nanograms
of purified ErbB-2 was incubated with various concen-
trations of coumarin carboxamides for 30 min at room
temperature. After incubation, kinase reactions were
performed at 30 ꢁC for 20 min in the presence of
100 lM ATP, 40 lCi 32P-cATP, and 0.25 lg/mL poly-
(Glu:Tyr,4:1), (PGT; Sigma Chemical Co). The reaction
was terminated by addition of 0.25 mM EDTA and
spotted onto P81 phosphocellulose filters. The filters
were washed thrice for 5 min each with 0.75% phospho-
ric acid, once with acetone and the level of radioactivity
determined using a liquid scintillation counter. Kinase
assays were performed in triplicate and represent an
average of two independent experiments. Non-specific
binding was determined by conducting the assay in
the absence of enzyme and the value subtracted from
each of the experimental values. The level of kinase
activity is expressed as a percent of the maximal kinase
activity.
6.1.8. In vitro kinase assay for ERK1 inhibition. SKBR-3
and BT474 cell lysates were prepared as mentioned
above and immunoprecipitated using an ERK1/2 anti-
body for 2 h at 4 ꢁC. The immune complexes were
washed and incubated in kinase reaction buffer contain-
ing 100 lM ATP, 40 lCi 32P-cATP, and 2.5 lg MBP at
30 ꢁC for 20 min. The reactions were terminated by the
addition of Laemmli buffer, boiled for 5 min, and elec-
trophoresed on a 15% sodium dodecyl sulfate–poly-
acrylamide gel followed by autoradiography.
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