Synthesis and biological evaluation of benzamides and benzamidines: Structural requirement of a pyrimidine ring for inhibition of EGFR tyrosine kinase

Article abstract of DOI:10.1016/j.bmcl.2004.02.001

The benzamides 1 and the benzamidines 2-3 were synthesized as the mimics of 4-anilinoquinazolines, which possess inhibition of epidermal growth factor receptor (EGFR) tyrosine kinase, and tested for cytotoxicity toward A431 and inhibitory activity toward

Full text of DOI:10.1016/j.bmcl.2004.02.001

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2299–2302
Synthesis and biological evaluation of benzamides and
benzamidines: structural requirement of a pyrimidine ring for
inhibition of EGFR tyrosine kinase
Toru Asano,b Tomohiro Yoshikawa,a Hiroyuki Nakamura,a,* Yoshimasa Ueharac
and Yoshinori Yamamotob
aDepartment of Chemistry, Faculty of Science, Gakushuin University, Tokyo 171-8588, Japan
bDepartment of Chemistry, Graduate School of Science, Tohoku University, Sendai 980-8588, Japan
cDepartment of Bioactive Molecules, National Institute of Infectious Diseases, Tokyo 162-8640, Japan
Received 5 December 2003; revised 30 January 2004; accepted 31 January 2004
Abstract—The benzamides 1 and the benzamidines 2–3 were synthesized as the mimics of 4-anilinoquinazolines, which possess
inhibition of epidermal growth factor receptor (EGFR) tyrosine kinase, and tested for cytotoxicity toward A431 and inhibitory
activity toward autophosphorylation by the enzyme assay. High cell growth inhibition was observed in a series of the cyclic benz-
amides 3: the IC50 values are 0.09–0.32 mM. The benzamidines 3a and 3b exhibited high inhibition of EGFR tyrosine kinase at a
1.0 lM concentration, although the benzamides 1 and the benzamidines 2 did not show significant kinase inhibition at a 10 lM
concentration.
Ó 2004 Elsevier Ltd. All rights reserved.
Since the discovery of the 4-anilinoquinazoline (PD
153035), which possesses a specific inhibitory activity
toward the epidermal growth factor receptor (EGFR)
tyrosine kinase,1;2 various 4-anilinoquinazoline deriva-
tives have been synthesized.3 Among them, ZD1839
(Iressae) was found to be effective for lung cancer
patients and approved for the clinical use in 2002.4
Furthermore, inhibitors of vascular endothelial growth
factor receptor tyrosine kinases have also been observed
in 4-anilinoquinazolines.5 According to the crystal
structure of the EGFR kinase domain and in complex
with erlotinib, the interplanar angle of quinazoline and
aniline rings in erlotinib is 42° and hydrogen bonding
between the Met769 amide nitrogen of the kinase domain
and the nitrogen (N1) of the quinazoline ring has been
observed.6 We thought that the flexibility of the qui-
nazoline framework would be effective for the interac-
tion between the kinase domain and inhibitors, and
therefore designed the benzamides 1 and the benzam-
idines 2 as the mimics of 4-anilinoquinazolines, in which
the formation of a pseudocycle7 of the quinazoline ring
through the intramolecular hydrogen bonding would be
expected. Herein we report the synthesis and biological
evaluation of the benzamides 1 and the benzamidines 2
as well as the protected benzamidines 3.
The 4,5-dimethoxyanthranilamides 1a–f were synthe-
sized from 4,5-dimethoxyanthranilic acid 4 by two dif-
ferent pathways (Scheme 1). The EDCI-promoted
amidation of the N-protected benzoic acid 4 with amines
(R1NH2) afforded the amides 5, and the deprotection of
the Boc group with trifluoroacetic acid afforded the
anthranilamides 1, quantitatively. In an alternative
pathway,8 the ring opening of the carbamates 69 with
amines (R1NH2) afforded the anthranilamides 1. The 2-
pyridylamino-4,5-dimethoxyanthranilamides 1g–i were
synthesized from 4,5-dimethoxy 2-nitrobenzoic acid 7
(Scheme 2). The 2-nitrobenzoic acid 7 was treated with
thionyl chloride followed by the amide formation with
pyridylamines to give the corresponding 2-nitro-
benzylamides 8g–i. The reduction of 8 with tin(II)
chloride in MeOH afforded 1g–i in good yields.
The 2-amino-4,5-dimethoxyphenylamidines 2a–d were
synthesized from 2-amino-4,5-dimethoxybenzoic acid 4
(Scheme 3).10 The dimethoxyanthranilamide 9 derived
Keywords: EGFR; Inhibitor; Benzamidines; Tyrosine kinase.
* Corresponding author. Tel.: +81-339860221; fax: +81-359921029;
e-mail: hiroyuki.nakamura@gakushuin.ac.jp
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.02.001

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T. Asano et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2299–2302
F
OMe
OMe
HN
N
O
HN
N
Cl
HN
N
Br
O
O
N
O
MeO
MeO
N
N
N
MeO
erlotinib
PD 153035
ZD 1839 (IressaTM
)
R
R
HN
HN
MeO
MeO
MeO
MeO
Pseudocycle formation
X
H
N
through hydrogen bonding
N
H
N
H
3: X = halogen
1: X = O
2: X = NH
from 4 with ammonia by DCC coupling was converted
to 10 in the presence of a catalytic amount of p-tolu-
enesulfonic acid (p-TsOH) in acetone, and 10 was trea-
ted with LawessonÕs reagent followed by methylation
with iodomethane afforded the thioamidate 11. Dis-
placement of methanethiol by amines gave the protected
R1
HN
MeO
a
b
O
R1
O
MeO
NHBoc
HN
MeO
MeO
CO2H
NH2
MeO
MeO
5
NH2
O
MeO
MeO
1a-f
O
4
d
c
N
H
O
6
Scheme 1. Reagents and conditions: (a) i. (Boc)2O, NaOH, H2O; ii R1NH2, EDCI, HOBt, DMF, 35–71%; (b) TFA, CH2Cl2, >99%; (c) i. ethyl
chloroformate, THF; ii. PBr3, Et2O, >99%; (d) R1NH2, DMF, 100 °C, 10–80%.
R1
O
R1
O
HN
HN
MeO
MeO
CO2H
NO2
MeO
MeO
MeO
MeO
a
b
NO2
8g-i (R1 = 2-4-pyridyl)
NH2
7
1g-i
Scheme 2. Reagents and conditions: (a) i. SOCl2, CH2Cl2; ii. pyridylamine, CH2Cl2, 10–67%; (b) SnCl2, MeOH, 70–89%.
O
MeO
MeO
CONH2
NH2
MeO
MeO
c
a
b
NH
4
N
H
9
10
R3
N
SMe
N
R3
HN
HN
MeO
MeO
e
d
MeO
MeO
MeO
MeO
NH
N
H
N
H
NH2
2a-d
3a-d
11
Scheme 3. Reagents and conditions: (a) NH3, DCC, HOBt, THF, 97%; (b) acetone, p-TsOH, 71%; (c) i. LawessonÕs reagent, 60%; ii. MeI, 98%;
(d) amine, 110 °C, 26–56%; (e) HCl, reflux, 34–82%.

T. Asano et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2299–2302
2301
Table 1. Cytotoxicity of the benzamides 1a–i, and the benzamidines 2a–d and 3a–d toward A431 cells
R
R
N
R
HN
HN
HN
MeO
MeO
MeO
MeO
MeO
MeO
O
NH
NH2
N
H
NH2
1
2
3
R
IC50 (mM)a
>1
R
IC50 (mM)
0.30
1a
2a
Br
Cl
Me
1b
1c
>1
2b
2c
0.46
0.34
0.34
Br
OMe
F
1d
1e
0.47
0.20
2d
3a
0.20
0.13
CF3
Cl
Br
OMe
F
1f
>1
>1
3b
3c
3d
0.20
0.32
0.09
Cl
Cl
1g
N
OMe
F
1h
1i
>1
>1
N
N
Cl
a An IC50 of >1 indicates that no curve was noted in the dose response up to 1 mM.
100
80
60
40
20
0
AG1478
0.1
conc. (µm/gl)
ZD1839
1c
1d
1i
1
2a
2b
2c
10
3a
3b
compounds
3d
Figure 1. Inhibition of EGF-stimulated EGFR phosphorylation by compounds. EGFR tyrosine kinase activities are expressed as a percentage of
the maximal phosphorylation induced by EGF.

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T. Asano et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2299–2302
benzamidines 3a–d. The deprotection of 3a–d was car-
ried out under the acidic condition to give the 2-amino-
4,5-dimethoxyphenylamidines 2a–d.
References and notes
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mides 1c–e, which have an aniline group in the
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the kinase activity: the phosphorylation activities were
10% and 35% at a 0.1 lg/mL concentration of com-
pounds, respectively. The compounds 1a–i, 3c, and 2a–d
did not show significant tyrosine kinase inhibition at a
10 lg/mL concentration, unexpectedly. Interestingly, the
compounds 3a,b, which have the cyclic framework by
conjunction with a ketal formation between an amine
and an amidine moiety in the molecule, showed a high
inhibitory activity at 10 and 1.0 lg/mL.
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Acknowledgement
Part of this work was supported by the Naito Founda-
tion, Japan.