Benzamides and benzamidines as specific inhibitors of epidermal growth factor receptor and v-Src protein tyrosine kinases

Article abstract of DOI:10.1016/j.bmc.2004.04.030

The benzamides 1 and the benzamidines 2 as well as the cyclic benzamidines 3 were designed and synthesized as the mimics of 4-anilinoquinazolines for an inhibitor of EGFR tyrosine kinase. The specific inhibitions of EGFR tyrosine kinase were observed in the benzamides 1c and 1d, and the benzamidine 2a, whereas the specific inhibitions of v-Src kinase were observed in the benzamide 1j and the benzamidine 2d at a 10μg/mL concentration of compounds. The cyclic benzamidines 3a and 3b showed potent kinase inhibition of EGFR at a 1.0μg/mL concentration. According to the docking simulation using the X-ray structure of EGFR kinase domain in complex with erlotinib, the LigScore2 scoring function value of erlotinib was calculated as 5.61, whereas that of the benzamide 1c was 5.05. In a similar manner, the LigScore2 value of the cyclic benzamidine 3a was calculated as 5.10.

Full text of DOI:10.1016/j.bmc.2004.04.030

Bioorganic & Medicinal Chemistry 12 (2004) 3529–3542
Benzamides and benzamidines as specific inhibitors of epidermal
growth factor receptor and v-Src protein tyrosine kinases
Toru Asano,b Tomohiro Yoshikawa,a Taikou Usui,a Hiroshi Yamamoto,a
Yoshinori Yamamoto,b Yoshimasa Ueharac and Hiroyuki Nakamuraa,*
aDepartment of Chemistry, Faculty of Science, Gakushuin University, Mejiro, Toshima-ku, 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 21 March 2004; revised 26 April 2004; accepted 26 April 2004
Available online 18 May 2004
Abstract—The benzamides 1 and the benzamidines 2 as well as the cyclic benzamidines 3 were designed and synthesized as the
mimics of 4-anilinoquinazolines for an inhibitor of EGFR tyrosine kinase. The specific inhibitions of EGFR tyrosine kinase were
observed in the benzamides 1c and 1d, and the benzamidine 2a, whereas the specific inhibitions of v-Src kinase were observed in the
benzamide 1j and the benzamidine 2d at a 10 lg/mL concentration of compounds. The cyclic benzamidines 3a and 3b showed potent
kinase inhibition of EGFR at a 1.0 lg/mL concentration. According to the docking simulation using the X-ray structure of EGFR
kinase domain in complex with erlotinib, the LigScore2 scoring function value of erlotinib was calculated as 5.61, whereas that of the
benzamide 1c was 5.05. In a similar manner, the LigScore2 value of the cyclic benzamidine 3a was calculated as 5.10.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
potent inhibitors of vascular endothelial growth factor
receptor (VEGFR),16;17 platelet-derived growth factor
receptor (PDGFR),18 c-Src,19;20 and other kinases.21
The epidermal growth factor receptor (EGFR) protein
tyrosine kinase (PTK) is one of the important kinases
that play a fundamental role in signal transduction
pathways. EGFR and its ligands (EGF, TGF-a) have
been implicated in numerous tumors of epithelial ori-
gin1;2 and proliferative disorders of the epidermis such as
psoriasis.3 Therefore, the design of inhibitors toward
EGFR-PTK is an attractive approach for the develop-
ment of new therapeutic agents.4–7
According to the crystal structure of the EGFR kinase
domain and in complex with erlotinib, which was re-
cently reported by Eigenbrot and co-workers, the in-
terplanar angle of quinazoline and aniline rings in
erlotinib is 42ꢁ and a hydrogen bonding between the
Met-769 amide nitrogen of the kinase domain and the
nitrogen (N1) of the quinazoline ring has been observed.
The other quinazoline nitrogen (N3) forms a hydrogen
bonding to the Thr-766 side chain through a water
molecule.22 We thought that the flexibility of the qui-
nazoline framework would be effective for the interac-
tion between the kinase domain and inhibitors. In this
article, we designed the benzamides 1 and the benz-
amidines 2 as the mimics of 4-anilinoquinazolines and
expected a pseudocycle formation through intramolec-
ular hydrogen bonding in the molecules.23–26 The benz-
amides 1 and the benzamidines 2 as well as the cyclic
benzamidines 3 were synthesized and examined their
biological properties:27 cytotoxicity and inhibitory
activity toward various tyrosine kinases. Further-
more, docking simulations of the benzamides 1, the
benzamidines 2, and the cyclic benzamidines 3 were
In 1994, Fry et al. discovered that the 4-anilinoquinaz-
oline (PD 153035) possess a specific inhibitory activity
toward EGFR tyrosine kinase.8 Various 4-anilinoqui-
nazoline derivatives have been synthesized so far based
upon the structure of PD 153035,9–14 and ZD1839
(Iressae)15 was found to be effective for non-small cell
lung cancer and recently approved for the clinical use in
Japan. Furthermore, 4-anilinoquinazoline derivatives
(ZD 6474, KN 1022, etc.) have been investigated as
Keywords: EGFR; Tyrosine kinase; Inhibitor.
* Corresponding author. Tel.: +81-339860221; fax: +81-359921029;
e-mail: hiroyuki.nakamura@gakushuin.ac.jp
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.04.030

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T. Asano et al. / Bioorg. Med. Chem. 12 (2004) 3529–3542
F
OMe
HN
N
HN
N
Br
O
HN
N
Cl
O
O
MeO
MeO
N
N
O
N
N
MeO
OMe
ZD 1839 (IressaTM
)
erlotinib (TarcevaTM
)
PD 153035
EGFR
EGFR
EGFR
O
H
F
Br
O
N
O
N
N
HN
N
NO2
HN
MeO
O
N
MeO
Cl
N
MeO
MeO
N
N
O
N
MeN
N
ZD 6474
VEGFR
KN 1022
PDGFR
c-Src
demonstrated using the X-ray structure of EGFR kinase
domain in complex with erlotinib (Tarcevae) and
evaluated their structure–activity relationship.
anthranilic acid 4 by two different pathways as shown in
Scheme 2. The anthranilamides 1a was synthesized from
4 through the formation of the carbamate 5.30 The ring
R
R
HN
HN
MeO
MeO
X
H
Psedocycle formation
through hydrogen bonding
N
MeO
N
H
MeO
N
H
1: X = O
2: X = NH
3: R = halogen
2. Result and discussion
opening of the carbamate 5, which was derived from 4
with ethyl chloroformate,31 with cyclohexylamine at
110 ꢁC afforded the anthranilamide 1a in 67% yield,
however, the reaction with 3-chloro-4-fluoroaniline
afforded the anthranilamide 1f in a very low yield.
Alternatively, the synthetic route using the EDCI-pro-
moted amide formation was examined. The amidation
of the N-Boc protected benzoic acid of 4 with cyclo-
hexylamine gave 6a in 78% yield, and the deprotection
of the Boc group with trifluoroacetic acid afforded the
anthranilamide 1a, quantitatively. Surprisingly, the
EDCI-promoted amidation of the N-Boc protected
benzoic acid of 4 with 3-chloro-4-fluoroaniline pro-
ceeded smoothly to afford 6f in 70% yield. The depro-
tection of the Boc group afforded the anthranilamide 1f,
quantitatively. Other amines (R1NH2) such as 1-phen-
ylethylamine, 3-bromoaniline, 3-trifluoroaniline, and 3-
methoxyaniline, also underwent the EDCI-promoted
amidation to afford the amides 6b–e, respectively, and
the deprotection of the Boc group with trifluoroacetic
acid afforded the anthranilamides 1b–e in 63–>99%
yields.
2.1. Chemistry
We first examined the synthesis of the 4,5-dimethoxy-
anthranilamides 1 from commercially available 2-ami-
no-4,5-dimethoxybenzonitrile as shown in Scheme 1.28;29
The progress of the reaction was confirmed by the
consumption of 2-amino-4,5-dimethoxybenzonitrile on
TLC. Although the reaction proceeded, separation of 1c
from the reaction mixture containing aluminum com-
plexes was not easy due to high polarity of the product.
We next examined the synthesis of the 4,5-dimethoxy-
anthranilamides 1a–f from 2-amino-4,5-dimethoxy-
HN
Br
MeO
MeO
CN
a
MeO
MeO
O
NH2
NH2
1c
Although several attempts have been made for the
synthesis of the 2-pyridylamino-4,5-dimethoxyanthra-
nilamides 1g–i, which contain pyridyl moieties at the R1
Scheme 1. Reagents and conditions: (a) 3-bromoaniline, AlCl3, neat,
180 ꢁC.

T. Asano et al. / Bioorg. Med. Chem. 12 (2004) 3529–3542
3531
O
MeO
O
a
c
b
d
MeO
N
H
O
R1
O
5
HN
MeO
MeO
CO2H
NH2
MeO
MeO
R1
NH2
4
HN
1a-f
MeO
MeO
O
NHBoc
6a-f
Scheme 2. Reagents and conditions: (a) i. ethyl chloroformate, THF, reflux; ii. PBr3, Et2O, reflux; (b) R1NH2, DMF, 100 ꢁC; (c) i. (Boc)2O, NaOH,
H2O; ii. R1NH2, EDCI, HOBt, DMF; (d) TFA, CH2Cl2.
group, in a similar manner, the amide bond formations
did not take place, probably due to the weak nucleo-
philicity of pyridylamines in comparison with cyclohexyl
and phenethyl amines, and anilines. We succeeded in the
synthesis of the 2-pyridylamino-4,5-dimethoxyanthra-
nilamides 1g–i from vanillic acid 7 through the amide
formation of pyridyl amines with the acid halide as
shown in Scheme 3. The vanillic acid 7 was converted to
the methyl ester in MeOH at reflux and then methylated
with iodomethane in acetone to give methyl 3,4-di-
methoxybenzoate 8. Selective nitration at the C6 posi-
tion of 8 with 70% nitric acid in acetic acid at 50–60 ꢁC
followed by hydrolysis of a methyl ester afforded the 2-
nitrobenzoic acid 9. The 2-nitrobenzoic acid 9 was
treated with thionyl chloride in refluxing CH2Cl2 to
convert the corresponding acid chloride, which under-
went the amide bond formation with pyridylamines to
give the 2-nitrobenzylamides 10g–i. The reduction of
10g–i with tin(II) chloride in MeOH afforded the
2-pyridylamino-4,5-dimethoxyanthranilamides 1g–i in
good yields.32
DCC and HOBt to give 4,5-dimethoxyanthranilamide
11, which was converted to the protected amide 12. The
treatment of 12 with Lawesson’s reagent at 80 ꢁC
followed by methylation with iodomethane afforded the
thioamidate 13. The nucleophilic substitution of the
methanethiol group in 13 by 3-bromoaniline in MeOH
resulted in the formation of the undesired product,
4,6,7-trimethoxy-2,2-dimethyl-1,2-dihydro-quinazoline,
which was generated by the nucleophilic attack of
methanol instead of 3-bromoanilin. The substitution
reactions of the thioamidate 13 were also examined in
the different solvents, such as THF, DMF, and MeCN,
however, 12, which was obtained by hydrolysis of 13,
was observed in all cases. After several attempts, we
found that the substitution reactions of the thioamidate
13 by anilines proceeded without any solvents at 110 ꢁC
to give the corresponding cyclic benzamidines 3a–d. The
deprotection of 3a–d was carried out under the acidic
condition to give the 2-amino-4,5-dimethoxyphenylam-
idines 2a–d.
We next designed 2,3,4-trimethoxyanthranilamide 1j as
the mimics of 4-anilinoquinazoline, where the rigid
conformation would be expected through the double
intramolecular hydrogen bondings: one would be
formed between an oxygen of the 2-methoxy group and
As illustrated in Scheme 4, the 2-amino-4,5-dimethoxy-
phenylamidines 2a–d were synthesized from 4,5-di-
methoxyanthranilic acid 4 by six steps.33 The amidation
reaction of 4 with ammonia proceeded in the presence of
MeO
MeO
CO2H
NO2
MeO
HO
CO2H
MeO
MeO
CO2Me
b
a
9
7
8
R2
O
R2
O
HN
HN
MeO
MeO
MeO
MeO
c
d
NO2
NH2
10g-i (R2= 2-4-pyridyl)
1g-i
Scheme 3. Reagents and conditions: (a) i. concd H2SO4, MeOH, reflux; ii. MeI, K2CO3, acetone, reflux; (b) i. HNO3, AcOH, 50–60 ꢁC; ii. NaOH,
THF; (c) i. SOCl2, CH2Cl2, reflux; ii. pyridylamine, CH2Cl2; (d) SnCl2, MeOH.

3532
T. Asano et al. / Bioorg. Med. Chem. 12 (2004) 3529–3542
O
a
MeO
MeO
b
MeO
MeO
CONH2
NH2
c
NH
4
N
H
12
11
R3
N
R3
SMe
N
HN
HN
MeO
MeO
MeO
MeO
MeO
MeO
e
d
NH
N
H
N
H
NH2
13
3a-d
2a-d
Scheme 4. Reagents and conditions: (a) NH3, DCC, HOBt, THF; (b) acetone, p-TsOH, reflux; (c) i. Lawesson’s reagent, 80 ꢁC; ii. MeI; (d) amine,
neat, 110 ꢁC; (e) HCl, reflux.
a hydrogen of the amide group, and the other would be
formed between a hydrogen of amino group and a
carbonyl oxygen, as shown in Figure 1. The synthesis of
2,3,4-trimethoxyanthranilamide 1j was accomplished
from 2,3,4-trimethoxyanthranilic acid 14 as shown in
Scheme 5. The nitration of 2,3,4-trimethoxy benzoic
acid 14 with 70% nitric acid at 0 ꢁC afforded the corre-
sponding 6-nitrobenzoic acid 15.34 The 6-nitrobenzoic
acid 15 was treated with thionyl chloride to generate the
acid chloride, which underwent the amide formation
with 3-bromoaniline to give the 6-nitrobenzamide 16.
The hydrogenation of 16 in the presence of Pd/C in
MeOH afforded 2,3,4-trimethoxyanthranilamide 1j,
quantitatively.
2.2. Biological evaluation
The cytotoxicity of the benzamides 1a–j, the benzami-
dines 2a–d, and 3a–d toward A43135 human epidermoid
carcinoma cells was determined. The concentration of
compounds, which exhibited the 50% cell growth inhi-
bition, is shown as an IC50 value in Table 1. The benz-
amides 1c–e and 1j, which have a substituent at the meta
position on the aniline group in the molecules, exhibited
the cell growth inhibition and the IC50 values are 0.34,
0.47, 0.20, and 0.92 mM, respectively, although the 50%
cell growth inhibition was not observed at a 1.0 mM
concentration of the benzamides 1a–b and 1f–i. The
benzamidines 2a–d exhibited the similar cell growth
inhibitions as the corresponding benzamides and the
IC50 values are 0.20–0.46 mM. Interestingly, the cyclic
benzamidines 3a–d exhibited relatively higher cell
growth inhibitions in comparison with the correspond-
ing benzamidines 2a–d and the IC50 values are 0.09–
0.32 mM.
Hydrogen Bonding
H
MeO
N
Br
MeO
MeO
O
H
N
H
Hydrogen Bonding
1j
Figure 1. A rigid conformation through intramolecular double
hydrogen bondings.
MeO
MeO HN
Br
MeO
a
MeO
MeO
CO2H
NO2
MeO
MeO
b
MeO
MeO
CO2H
O
NO2
14
15
16
MeO HN
Br
MeO
MeO
c
O
NH2
1j
Scheme 5. Reagents and conditions: (a) 70% HNO3, 0 ꢁC; (b) i. SOCl2, CH2Cl2, reflux; ii. 3-bromoaniline; (c) H2, Pd/C, MeOH.

T. Asano et al. / Bioorg. Med. Chem. 12 (2004) 3529–3542
3533
Table 1. Cytotoxicity of the benzamides 1a–j, and the benzamidines 2a–d and 3a–d toward A431 cells
R
N
R
O
R
O
R
HN
HN
HN
HN
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
NH
N
H
NH2
NH2
NH2
1j
1
2
3
R
IC50 (mM)a
R
IC50 (mM)
0.30
1a
1b
1c
>1
2a
2b
2c
Br
Cl
Me
>1
0.46
0.34
0.34
Br
OMe
F
1d
1e
0.47
0.20
2d
3a
0.20
0.13
CF
3
Cl
Br
OMe
F
1f
>1
>1
3b
3c
0.20
0.32
Cl
Cl
1g
N
OMe
F
1h
1i
1j
>1
3d
0.09
N
N
Cl
>1
0.92
Br
a An IC50 of >1 indicates that no curve was noted in the dose response up to 1 mM.
The benzamides 1a–j, the benzamidines 2a–d and 3a–d,
were tested for the inhibitory activity of EGF-stimulated
EGFR tyrosine kinase phosphorylation according to
assay conditions described in detail in the literature.36
AG1478 and ZD1839 were also tested as a control
inhibitor for the kinase assay. As a preliminary experi-
ment, the inhibition assay of EGFR tyrosine kinase was
carried out at a 10 lg/mL concentration of compounds as
shown in Figure 2. The maximum phosphorylation
activity to the peptide substrate by the EGF-stimulated
EGFR tyrosine kinase is plotted as 100% activity after
reduction of the kinase activity without stimulated by
EGF. Among the compounds synthesized, the cyclic
benzamidines 3a and 3b exhibited inhibitory activities
higher than 50% at a 10 lg/mL concentration, therefore
the kinase assay was carried out at lower concentrations
(1.0 and 0.1 lg/mL). AG1478 and ZD1839, which have
been reported to be potent inhibitors of EGFR tyrosine
kinase,5–7 showed high inhibition of the kinase activity:
The phosphorylation activities were 10% and 35% at a
0.1 lg/mL concentration of compounds, respectively.
Although the inhibitory activity of the compounds 1a–j,
3c, and 2a–d did not show significant kinase inhibition at
a 10 lg/mL concentration, the only pseudocycle 1d, which
has trifluoromethyl group at a meta position on the
aniline moiety, exhibited a high inhibitory activity at a
10 lg/mL concentration. However, the pseudocycle 1d
did not show the expected kinase inhibition at a 1.0 lg/mL
concentration. Interestingly, the cyclic benzamidines 3a
and 3b, which have the cyclic framework by conjunction
with a ketal formation in the molecule, showed high
inhibitions at 10 and 1.0 lg/mL concentrations.
Inhibition specificity of the compounds 1c, 1d, 1j, 2a, 2d,
and 3a was investigated semi-quantitatively using PKA,
PKC, v-Src, eEF2K, and Flt-1 (Table 2).37–43 The indi-
cations ++, +, and ) mean >80%, 50–80%, and <50%
kinase inhibition activities in each kinase assay, respec-
tively. The benzamide 1c, which has a bromo group at a
meta position on the aniline moiety, exhibited inhibitory

3534
T. Asano et al. / Bioorg. Med. Chem. 12 (2004) 3529–3542
Figure 2. Inhibition of EGF-stimulated EGFR phosphorylation by compounds. EGFR tyrosine kinase activities are expressed as a percentage of the
maximal phosphorylation induced by EGF.
activities toward EGFR and Flt-1 at a 10 lg/mL con-
centration. The benzamide 1d, the benzamidine 2a, and
the cyclic benzamidine 3a exhibited selective inhibitory
activities toward EGFR at a 10 lg/mL concentration.
Moreover, the cyclic benzamidine 3a showed a high
inhibition of EGFR tyrosine kinase even at a 1.0 lg/mL
concentration. Although the benzamide 1j and the
benzamidine 2d did not show the inhibition of EGFR
kinase, the specific inhibition activity was observed in
the case of v-Src at a 10 lg/mL concentration.
possible binding modes of the pseudocycle 1c and the
cyclic benzamidine 3a, we performed molecular docking
experiments of 1c and 3a with the ligand binding site of
EGFR kinase (PDB code 1M17). The protein backbone
was taken from the X-ray structure of EGFR kinase
domain in complex with erlotinib (Tarcevae), which has
been currently studied in Phase III clinical trials as
anticancer agent. Docking calculations were carried out
by replacing the erlotinib with compounds 1c and 3a.44–46
Figure 3a shows the docking mode of the benzamide 1c
overlaid with the binding conformation of erlotinib into
the active site of EGFR kinase domain. Superimposition
of the benzamide 1c with the control ligand, erlotinib,
shows a slightly different binding mode. Although we
expected the pseudocycle formation of the benzamide 1c
through an intramolecular hydrogen bonding in the
binding site of EGFR-PTK, the formation of the rot-
amer 1c0 would be more suitable to be positioned in the
binding site according to the docking calculation, as
shown in Scheme 6. A hydrogen bonding was observed
between the nitrogen (N1) of the quinazoline ring and the
amide nitrogen of Met-769 of the EGFR kinase domain
in the crystal structure complexed with erlotinib, whereas
a hydrogen bonding was observed between the amine
hydrogen atom of the benzamide 1c and the carbonyl
oxygen of Met-769 in the ligand binding pocket. A little
difference in the formation of hydrogen bonding between
the inhibitors and the kinase domain may affect the
inhibition of EGFR kinase activity. The LigScore2
scoring function gives us an index of the binding affinity
of protein–ligand complexes and the higher value of the
LigScore indicates the higher binding affinity of a pro-
tein–ligand complex.47 The LigScore2 value of erlotinib
was calculated as 5.61, whereas that of the benzamide 1c
was 5.05. These docking calculations also support the
results obtained from the EGFR kinase assay that erl-
otinib possesses a higher inhibition of the EGFR kinase
activity in comparison with the benzamide 1c.
2.3. Molecular modeling of inhibitors in the ligand binding
site of EGFR-PTK
To better understand the structure–activity relationship
between the EGFR-PTK inhibitory activity and the
Table 2. Inhibition specificity of the compounds 1c, 1d, 1j, 2a, 2d, and
3a
Com-
Concn PKA PKC v-Src eEF2K EGFR Flt-1
pounds (lg/
mL)
1c
1d
1j
10
1
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
+
)
)
)
)
+
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
)
+
)
+
)
)
)
+
)
)
)
)
++
+
)
+
)
)
)
)
)
)
)
)
)
)
)
)
)
10
1
10
1
2a
10
1
0.1
2d
3a
10
1
10
1
0.1
++: inhibition more than 80%, +: inhibition of 50–80%, ): inhibition
under 50%.

T. Asano et al. / Bioorg. Med. Chem. 12 (2004) 3529–3542
3535
Figure 3. Docking modes of the benzamide 1c (a) and the cyclic benzamidine 3a (b) overlaid with the binding conformation of erlotinib into the
active site of EGFR kinase domain. Docking model was calculated by the DS modeling based on the X-ray analysis data of EGFR-PTK with
erlotinib (PDB code: 1M17).22 The docked molecules are indicated by gray thick sticks and the binding orientation of erlotinib is drawn by orange
sticks. Hydrogen bonds are shown as a green dotted line.
HN
Br
NH2
N
MeO
MeO
MeO
MeO
O
Br
NH2
NH2
1c
1c'
Scheme 6. A equilibrium between the benzamide 1c and 1c0.
Figure 3b shows the docking mode of the cyclic benz-
amidine 3a superimposed on the binding conformation
of erlotinib into the active site of EGFR kinase domain.
According to the docking simulation, the cyclic benz-
amidine 3a showed a quite different binding mode from
erlotinib. A hydrogen bonding was observed between
the methoxy oxygen on the benzene ring and the amide
nitrogen of Met-769 of EGFR kinase domain and the
similar hydrogen binding was observed in the crystal
structure of EGFR kinase domain complexed with erl-
otinib. The LigScore2 value of the cyclic benzamidine 3a
was calculated as 5.10. This value indicates that the
cyclic benzamidine 3a is higher potent for the inhibition
of the EGFR kinase activity in comparison with 1c.
potent kinase inhibition of EGFR at a 1.0 lg/mL con-
centration. The structure–activity relationship of the
benzamide 1c by the docking calculation revealed that
the formation of the rotamer 1c0 would be more suitable
to be positioned in the binding site, although the
pseudocycle formation of a quinazoline ring through the
intramolecular hydrogen bonding was expected. This
stable formation of 1c0 resulted in the lower inhibition of
EGFR tyrosine kinase in comparison with 4-anilino-
quinazolines such as AG 1478 and ZD 1839. The
docking simulation of the cyclic benzamidine 3a, which
showed the highest inhibition of EGFR tyrosine kinase
among the compounds synthesized in this paper, re-
vealed the possibility of the different binding mode from
the benzamide 1c or erlotinib. We believe that the cur-
rent information would be very useful for designs of
protein tyrosine kinase-targeted new therapeutic agents.
3. Conclusion
We succeeded in the synthesis of the benzamides 1 and
the benzamidines 2 as the mimics of 4-anilinoquinazo-
lines. The specific inhibitions of EGFR tyrosine kinase
were observed in the benzamides 1c and 1d, and the
benzamidine 2a, whereas the specific inhibitions of v-Src
kinase were observed in the benzamide 1j and the
benzamidine 2d at a 10 lg/mL concentration of com-
pounds. The cyclic benzamidines 3a and 3b showed a
4. Experimental
4.1. Chemistry
1H NMR and 13C NMR spectra were measured on a
JEOL JNM-AL300 (300 MHz) and VARIAN UNITY-
INOVA 400 (400 MHz) spectrometers. Chemical shifts

3536
1
T. Asano et al. / Bioorg. Med. Chem. 12 (2004) 3529–3542
of H NMR and 13C NMR were expressed in parts per
million. IR spectra were measured on a Shimadzu
FTIR-8200A Spectrometer. Analytical thin layer chro-
matography (TLC) was performed on a glass plates
(Merck Kieselgel 60 F254, layer thickness 0.2 mm or RP-
18 F254s, layer thickness 0.2 mm). Visualization was
accompanied by UV light (254 nm), anisaldehyde and
KMnO4. Column chromatography was performed on
silica gel (Merck Kieselgel 70–230 mesh). All reactions
were carried out under argon atmosphere using stan-
dard Schlenk techniques. Most chemicals and solvents
were analytical grade and used without further purifi-
cation.
and 1-phenylethylamine (1.3 mL, 10 mmol), a similar
procedure as that described for 6a gave 6b (0.64 g, 79%)
as a solid: 1H NMR (CDCl3, 400 MHz) d 10.4 (br s, 1H),
8.01 (s, 1H), 7.38–7.27 (m, 5H), 6.87 (s, 1H), 5.28 (quint,
J ¼ 7:0 Hz, 1H), 3.94 (s, 3H), 3.84 (s, 3H), 1.60 (d,
J ¼ 7:0 Hz, 3H), 1.50 (s, 9H). IR (KBr) 1652, 1523,
1247, 1026, 869, 815, 769 cmꢀ1. HRMS (ESI) calcd for
C22H29N2O5, m=z 401.2071 (M+Hþ); found, m=z
401.2073.
4.1.4. [4,5-Dimethoxy-2-(3-bromophenylcarbamoyl)-phen-
yl]-carbamic acid tert-butyl ester (6c). From the N-Boc
anthranilic acid (0.6 g, 2 mmol), EDCI (0.6 g, 3 mmol),
HOBt (0.4 g, 3 mmol), triethylamine (1.4 mL, 10 mmol),
and 3-bromoaniline (1.1 mL, 10 mmol), a similar pro-
cedure as that described for 6a gave the crude product of
6c, which was used for the next N-Boc deprotection
without further purification.
4.1.1. N-Boc protection of 2-amino-4,5-dimethoxyanthra-
nilic acid (4). To a mixture of 2-amino-4,5-dimethoxy-
benzoic acid 4 (1.4 g, 7 mmol) in dioxane (15 mL) and
water (7.5 mL) were added triethylamine (1.4 mL,
10 mmol) followed by di-tert-butyl dicarbonate (2.4 mL,
10 mmol). The reaction mixture was stirred at room
temperature for 24 h. Solvent was removed by rotary
evaporation, and 3 N aqueous hydrochloric acid was
added dropwise to the residue. A precipitate was ob-
tained, collected, washed with water, and dried to pro-
vide the N-Boc anthranilic acid (2.0 g, >99%) as a solid;
1H NMR (CDCl3, 400 MHz) d 9.97 (s, 1H), 8.08 (s, 1H),
7.59 (s, 1H), 7.50 (d, 2H), 7.36 (t, 2H), 7.17 (d, 1H), 6.98
(s, 1H), 3.96 (s, 3H), 3.88 (s, 3H), 1.50 (s, 9H). 13C NMR
(CDCl3, 100 MHz) d 167.2, 153.2, 152.8, 143.3, 137.3,
136.3, 129.1, 124.9, 121.1, 111.4, 109.9, 103.5, 80.3, 56.6,
56.0, 28.3.
4.1.5. [4,5-Dimethoxy-2-(3-trifluoromethyl-phenylcarb-
amoyl)-phenyl]-carbamic acid tert-butyl ester (6d).
From the N-Boc anthranilic acid (0.6 g, 2 mmol),
EDCI (0.6 g, 3 mmol), HOBt (0.4 g, 3 mmol), triethyl-
amine (1.4 mL, 10 mmol), and 3-trifluoromethylaniline
(1.25 mL, 10 mmol), a similar procedure as that
described for 6a gave 6d (0.3 g, 35%) as a white solid: 1H
NMR (CDCl3, 300 MHz) d 9.88 (s, 1H), 7.61 (dt,
J ¼ 2:1, 7.5 Hz, 1H), 7.47 (s, 1H), 7.28 (s, 1H), 7.26 (d,
J ¼ 5:1 Hz, 1H), 6.86 (s, 1H), 3.92 (s, 3H), 3.90 (s, 3H),
1.50 (s, 9H). 13C NMR (CDCl3, 75 MHz) d 153.1, 153.0,
143.3, 138.0, 136.3, 132.0, 131.6, 131.2, 130.8, 129.6,
123.9, 121.2, 117.8, 117.5, 111.2, 111.0, 110.0, 103.7,
80.5, 56.7, 56.1, 28.4. IR (KBr) 3321, 1685, 1517, 1163,
4.1.2. [4,5-Dimethoxy-2-cyclohexylcarbamoylphenyl]-car-
bamic acid tert-butyl ester (6a). To a solution of the
N-Boc anthranilic acid (0.6 g, 2 mmol) in dry DMF
(6 mL) were added EDCI (0.58 g, 3 mmol), HOBt
(0.41 g, 3 mmol) and triethylamine (1.39 mL, 10 mmol).
After stirring at room temperature for 24 h, cyclohexyl-
amine (1.1 mL, 10 mmol) was added dropwise and the
reaction continued for 48 h at room temperature under
argon. Water was then added and the mixture stirred
for 5 min. The product was then extracted with ethyl
acetate. The combined organic extracts were washed
with brine, dried over sodium sulfate, filtered, and the
solvent removed. Purification was achieved by flash
chromatography (ethyl acetate/hexane 1:5 by volume) to
yield 6a (0.59 g, 78%) as a solid: 1H NMR (CDCl3,
400 MHz) d 10.4 (br s, 1H), 8.07 (s, 1H), 6.85 (s, 1H),
5.98 (s, 1H), 3.94 (s, 3H), 3.90 (t, J ¼ 11:2 Hz, 1H), 3.84
(s, 3H), 2.0–1.65 (m, 5H), 1.50 (s, 9H), 1.45–1.16 (m,
5H). 13C NMR (CDCl3, 400 MHz) d 167.7, 153.2, 152.4,
143.0, 136.1, 111.0, 110.1, 103.3, 103.2, 79.9, 56.8, 56.6,
55.9, 55.8, 48.7, 48.6, 33.0, 28.3, 25.1, 24.9. IR (KBr)
3323, 2979, 1716, 1654, 1092 cmꢀ1. MS (EI) m=z 378
(Mþ), 278 (Mþ)t-BuOCO), 263 (Mþ)t-BuOCONH).
858, 795, 694 cmꢀ1
C21H23F3N2O5, m=z 441.1632 (M+Hþ); found, m=z
441.1633.
.
HRMS (ESI) calcd for
4.1.6. [4,5-Dimethoxy-2-(3-methoxy-phenylcarbamoyl)-
phenyl]-carbamic acid tert-butyl ester (6e). From the N-
Boc anthranilic acid (0.6 g, 2 mmol), EDCI (0.6 g,
3 mmol), HOBt (0.4 g, 3 mmol), triethylamine (1.4 mL,
10 mmol), and 3-trifluoromethylaniline (1.25 mL,
10 mmol), a similar procedure as that described for 6a
1
gave 6e (0.1 g, 18%) as a white solid: H NMR (CDCl3,
300 MHz) d 9.97 (br s, 1H), 8.07 (s, 1H), 7.80 (br s, 1H),
7.27 (t, J ¼ 6:0 Hz, 1H), 7.22 (m, 1H), 7.05 (ddd,
J ¼ 0:8, 0.8, 7.8 Hz, 1H), 6.72 (dd, J ¼ 2:4, 8.4 Hz, 1H),
3.95 (s, 3H), 3.85 (s, 3H), 3.82 (s, 3H), 1.49 (s, 9H). 13C
NMR (CDCl3, 75 MHz) d 167.1, 160.0, 153.1, 152.6,
143.2, 138.5, 136.1, 129.7, 113.1, 111.4, 110.4, 109.9,
106.8, 103.5, 80.3, 56.5, 56.0, 55.3, 28.4. IR (KBr) 3354,
2979, 1720, 1525, 1157, 860, 767, 685 cmꢀ1. HRMS
(ESI) calcd for C21H29N2O6, m=z 403.1864 (M+Hþ);
found, m=z 403.1866.
4.1.3. [4,5-Dimethoxy-2-(1-phenyl-ethylcarbamoyl)-phen-
yl]-carbamic acid tert-butyl ester (6b). From the N-Boc
anthranilic acid (0.6 g, 2 mmol), EDCI (0.6 g, 3 mmol),
HOBt (0.4 g, 3 mmol), triethylamine (1.4 mL, 10 mmol),
4.1.7.
[4,5-Dimethoxy-2-(3-chloro-4-fluorophenylcarb-
amoyl)-phenyl]-carbamic acid tert-butyl ester (6f). From
the N-Boc anthranilic acid (0.6 g, 2 mmol), EDCI (0.6 g,

T. Asano et al. / Bioorg. Med. Chem. 12 (2004) 3529–3542
3537
3 mmol), HOBt (0.4 g, 3 mmol), triethylamine (1.4 mL,
10 mmol), and 3-chloro-4-fluoroaniline (1.46 g,
125.0, 123.0, 120.7, 116.0, 113.1, 106.4, 57.3, 56.6. IR
(KBr) 2940, 1664, 1587, 1205, 995, 837, 781, 721 cmꢀ1
.
10 mmol), a similar procedure as that described for 6a
gave 6f (0.59 g, 70%) as a white solid: 1H NMR (CDCl3,
400 MHz) d 9.90 (br s, 1H), 8.03 (s, 1H), 7.91 (s, 1H),
7.72 (dd, J ¼ 6:4, 2.8 Hz, 1H), 7.38 (m, 1H), 7.13 (t,
J ¼ 8:8 Hz, 1H), 6.96 (s, 1H), 3.93 (s, 3H), 3.82 (s, 3H),
1.50 (s, 9H). 13C NMR (CDCl3, 75 MHz) d 167.1, 156.3,
153.2, 143.4, 136.4, 134.1, 123.2, 120.7, 116.7, 116.5,
110.9, 110.1, 103.8, 80.5, 56.7, 56.1, 28.3. IR (KBr) 3323,
2980, 1716, 1655, 1092 cmꢀ1. MS (EI) m=z 424 (Mþ), 324
(Mþ)t-BuOCO), 180 (Mþ)t-BuOCO)C6H3ClF).
HRMS (EI) calcd for C15H15N2O3, m=z 272.1155
(Mþ)Br); found, m=z 272.1157.
4.1.11. 2-Amino-4,5-dimethoxy-N-(3-trifluoromethyl-phen-
yl)-benzamide (1d). From compound 6d (0.2 g,
0.5 mmol), a similar procedure as that described for 1a
provided white solid 1d (0.1 g, 63%): mp 158–162 ꢁC; 1H
NMR (CD3OD, 300 MHz) d 8.13 (s, 1H), 7.91 (d,
J ¼ 7:8 Hz, 1H), 7.54 (t, J ¼ 7:8 Hz, 2H), 7.50 (s, 1H),
7.42 (d, J ¼ 7:8 Hz, 1H), 6.84 (s, 1H), 3.92 (s, 3H), 3.90
(s, 3H). 13C NMR (CD3OD, 75 MHz) d 167.8, 154.2,
147.3, 140.4, 132.1, 131.7, 130.5, 127.2, 125.3, 123.6,
121.6, 120.0, 118.7, 113.0, 57.3, 56.6. IR (KBr) 2955,
1668, 1525, 1330, 1207, 797 cmꢀ1. HRMS (EI) calcd for
C16H15N2O3, m=z 340.1029 (Mþ); found, m=z 340.1030.
4.1.8. 2-Amino-N-cyclohexyl-4,5-dimethoxy-benzamide
(1a). (A) The procedure from the carbamate 5: A mix-
ture of cyclohexylamine (0.3 mL, 3 mmol) and the car-
bamate 5 (0.6 g, 3 mmol) in DMF (5 mL) was stirred at
100 ꢁC for 24 h. The solvent was evaporated off under
reduced pressure to give a residue, which was dissolved
in EtOAc and washed with saturated aqueous ammo-
nium chloride. The solution was dried over Na2SO4 and
filtered and the solvent was evaporated off under re-
duced pressure to yield the crude product. The crude
product was purified by column chromatography (ethyl
acetate/hexane 1:5 by volume) to yield pure 1a (0.6 g,
67%). (B) The N-Boc deprotection from 6a. A solution
of 6a (0.38 g, 1 mmol) in 6:1 dichloromethane/trifluoro-
acetic acid (4.9 mL) was stirred at room temperature for
2 h. The solvent was evaporated in vacuo, and diethyl
ether was added. The precipitate was collected, washed
with ether, and dried to provide white solid 1a: 1H NMR
(CD3OD, 300 MHz) d 7.06 (s, 1H), 6.37 (s, 1H), 3.79 (s,
3H), 3.76 (s, 3H), 1.97–1.64 (m, 5H), 1.45–1.16 (m, 5H).
13C NMR (CD3OD, 75 MHz) d 170.3, 154.4, 146.3,
141.5, 114.0, 109.0, 101.9, 57.8, 56.1, 50.0, 33.9, 26.7,
26.6. IR (KBr) 3354, 2932, 2480, 1626, 1541, 1265, 858,
827, 771 cmꢀ1. HRMS (ESI) calcd for C15H22N2O3, m=z
279.1703 (M+Hþ); found, m=z 279.1704.
4.1.12. 2-Amino-4,5-dimethoxy-N-(3-methoxy-phenyl)-
benzamide (1e). From compound 6e (0.4 g, 1 mmol), a
similar procedure as that described for 1a provided
1
white solid 1e (0.3 g, >99%): mp 162–164 ꢁC; H NMR
(CDCl3, 400 MHz) d 8.18 (s, 1H), 7.21 (s, 1H), 7.17 (t,
J ¼ 8:0 Hz, 1H), 7.01 (d, J ¼ 8:0 Hz, 1H), 6.90 (s, 1H),
6.67 (dd, J ¼ 2:0, 8.0 Hz, 1H), 3.92 (s, 3H), 3.81 (s, 3H),
3.75 (s, 3H). 13C NMR (CDCl3, 75 MHz) d 164.7, 159.8,
153.3, 149.1, 138.6, 138.0, 129.5, 127.0, 112.3, 110.5,
109.9, 106.9, 105.8, 56.7, 55.3. IR (KBr) 3315, 1651,
1608, 1522, 1339, 1285, 1053, 880, 762, 683 cmꢀ1
.
HRMS (EI) calcd for C16H18N2O4, m=z 302.1261 (Mþ);
found, m=z 302.1263.
4.1.13. 2-Amino-N-(4-chloro-3-fluoro-phenyl)-4,5-benz-
amide (1f). From compound 6e (0.4 g, 1 mmol), a simi-
lar procedure as that described for 1a provided the white
1
solid 1f (0.2 g, 10%): mp 162–165 ꢁC; H NMR (CDCl3,
400 MHz) d 8.05 (s, 1H), 7.69 (dd, J ¼ 2:8, 6.6 Hz, 1H),
7.35 (ddd, J ¼ 2:6, 4.0, 9.1 Hz, 1H), 7.07 (t, J ¼ 8:8 Hz,
1H), 6.93 (s, 1H), 6.18 (s, 1H), 3.83 (s, 3H), 3.78 (s, 3H).
13C NMR (CDCl3, 75 MHz) d 156.7, 154.1, 144.6, 141.7,
135.9, 130.2, 121.7, 116.8, 116.5, 112.5, 108.3, 101.9,
57.3, 56.1. IR (KBr) 3341, 1655, 1502, 1394, 1242, 997,
826, 769 cmꢀ1. HRMS (EI) calcd for C15H14ClFN2O3,
m=z 324.0671 (Mþ); found, m=z 324.0673.
4.1.9. 2-Amino-4,5-dimethoxy-N-(1-phenyl-ethyl)-benz-
amide (1b). From compound 6b (0.4 g, 1 mmol), a simi-
lar procedure as that described for 1a provided the white
solid 1b (0.3 g, >99%): mp 147–149 ꢁC; 1H NMR
(CDCl3, 400 MHz) 7.51 (s, 1H), 7.38–7.32 (m, 4H), 7.27
(t, J ¼ 6:4 Hz, 1H), 6.77 (s, 1H), 5.25 (t, J ¼ 7:2 Hz,
1H), 3.90 (s, 3H), 3.89 (s, 3H), 1.58 (d, J ¼ 6:8 Hz, 3H).
13C NMR (CDCl3, 75 MHz) d 165.6, 153.2, 148.9, 142.3,
142.2, 138.2, 128.5, 127.2, 126.3, 110.1, 106.9, 56.6, 56.4,
49.6, 21.1. IR (KBr) 3231, 1638, 1522, 1277, 1223, 868,
772, 704 cmꢀ1. HRMS (EI) calcd for C17H20N2O3, m=z
300.1468 (Mþ); found, m=z 300.1467.
4.1.14. 2-Nitro-4,5-dimethoxy-N-pyridin-2-yl-benzamide
(10g). A suspension of benzoic acid 9 (0.2 g, 1 mmol),
15 mL of dry CH2Cl2, and thionyl chloride (0.4 mL,
4 mmol) was allowed to reflux for 3 h. The solvent was
removed under vacuum. To a suspension of the acid
chloride in 5 mL of dry CH2Cl2 was added 2-amino-
pyridine (0.09 g, 1 mmol), and the mixture was allowed
to stir at room temperature for 4 h. The solvent was
removed under reduced pressure. The residue was trea-
ted with ice-water, and the pH of the slurry was adjusted
to a value of 7 by adding 1 N NaOH. This solution was
partitioned between EtOAc and 1 N NaOH. The layers
were shaken, and the organics were separated and wa-
shed additionally with H2O. The organic layer was
4.1.10. 2-Amino-N-(3-bromo-phenyl)-4,5-dimethoxy-benz-
amide (1c). From compound 6c (0.4 g, 1 mmol), a similar
procedure as that described for 1a provided white solid
1c (0.3 g, >99%): mp >200 ꢁC; 1H NMR (CD3OD,
300 MHz) d 7.99 (s, 1H), 7.61 (dt, J ¼ 2:1, 7.5 Hz, 1H),
7.47 (s, 1H), 7.28 (s, 1H), 7.26 (d, J ¼ 5:1 Hz, 1H), 6.86
(s, 1H), 3.92 (s, 3H), 3.90 (s, 3H). 13C NMR (CD3OD,
75 MHz) d 167.6, 154.2, 147.1, 141.1, 133.4, 131.1, 128.2,

3538
T. Asano et al. / Bioorg. Med. Chem. 12 (2004) 3529–3542
dried over Na2SO4 and filtered and the solvent was
removed under reduced pressure. After drying, the solid
was recrystallized from the proper solvents to give 10g
(0.2 g, 50%) as a white solid: 1H NMR (CDCl3,
400 MHz) d 9.84 (s, 1H), 8.29 (d, 1H), 7.77 (d, 1H), 7.73
(t, 1H), 7.56 (s, 1H), 7.01 (s, 1H), 6.92 (q, 1H), 3.95 (s,
3H), 3.92 (s, 3H). 13C NMR (CDCl3, 75 MHz) d 165.1,
153.5, 151.6, 149.5, 146.9, 138.8, 138.6, 127.0, 119.9,
115.1, 110.1, 107.0, 56.8, 56.6. IR (KBr) 1653, 1578
procedure as that described for 1g provided white solid
1
1h (0.2 g, 70%): H NMR (CDCl3, 400 MHz) d 8.35 (d,
1H), 8.16 (d, 1H), 7.91 (s, 1H), 7.27 (q, 1H), 6.98 (s, 1H),
6.23 (s, 1H), 5.30 (s, 2H), 3.87 (s, 3H), 3.85 (s, 3H). 13C
NMR (CDCl3, 75 MHz) d 170.0, 155.8, 148.4, 144.8,
143.1, 141.7, 138.0, 130.3, 125.1, 114.0, 107.2, 101.6,
57.8, 56.1. IR (KBr) 1653, 1516, 1418, 1207, 895, 795,
700 cmꢀ1. HRMS (EI) calcd for C14H15N3O3, m=z
273.1108 (Mþ); found, m=z 273.1108.
1510, 1470, 1435, 1393, 1315, 1225, 1053, 875, 783 cmꢀ1
.
HRMS (ESI) calcd for C14H13N3O5, m=z 304.0928
(M+Hþ); found, m=z 304.0927.
4.1.19. 2-Amino-4,5-dimethoxy-N-pyridin-4-yl-benzamide
(1i). From compound 10i (0.2 g, 1 mmol), a similar
procedure as that described for 1g provided white solid
1
4.1.15. 2-Nitro-4,5-dimethoxy-N-pyridin-3-yl-benzamide
(10h). From compound 9 (0.2 g, 1 mmol), a similar
procedure as that described for 10g provided the white
1i (0.4 g, 73%): H NMR (CDCl3, 400 MHz) d 8.42 (d,
2H), 7.82 (d, 2H), 7.31 (s, 1H), 6.47 (s, 1H), 3.4:5 (s, 3H),
3.43 (s, 3H). 13C NMR (CDCl3, 75 MHz) d 167.3, 154.4,
150.5, 145.5, 145.4, 141.3, 114.0, 111.3, 106.8, 101.2,
1
solid 10h (0.2 g, 67%): H NMR (CD3OD, 400 MHz) d
8.82 (d, 1H), 8.35 (d, 1H), 8.23 (d, 1H), 7.79 (s, 1H), 7.48
(q, 1H), 7.26 (s, 1H), 4.02 (s, 3H), 4.00 (s, 3H). 13C NMR
(CD3OD, 75 MHz) d 168.0, 155.3, 151.3, 145.7, 142.2,
140.1, 137.5, 129.4, 128.0, 125.4, 111.7, 108.5, 57.3, 57.0.
IR (KBr) 1661, 1508, 1281, 1227, 1055, 880, 710,
665 cmꢀ1. HRMS (EI) calcd for C14H13N3O5, m=z
303.0850 (Mþ); found, m=z 303.0851.
57.2, 55.8. IR (KBr) 1699, 1583, 1508, 1331, 1285 cmꢀ1
.
HRMS (ESI) calcd for C14H15N3O3, m=z 273.1108 (Mþ);
found, m=z 273.1107.
4.1.20. 6,7-Dimethoxy-2,2-dimethyl-2,3-dihydro-1H-qui-
nazolin-4-one (12). To a stirring solution of anthramide
11 (2.9 g, 15 mmol) in acetone (23 mL) was added p-
toluenesulfonic acid monohydrate (0.01 g, 0.09 mmol),
and the resulting homogeneous mixture was refluxed for
1 h. The solvent was subsequently cooled to ambient
temperature and removed under reduced pressure. The
resulting solid was partitioned between EtOAc and
saturated aqueous NaHCO3. The layers were shaken,
and the organics were separated and washed addition-
ally with H2O. The organic layer was dried over Na2SO4
and filtered and the solvent was evaporated off under
reduced pressure to yield 12 (2.5 g, 71%) as a white solid:
1H NMR (CD3OD, 400 MHz) d 7.27 (s, 1H), 6.33 (s,
1H), 3.87 (s, 3H), 3.81 (s, 3H), 1.55 (s, 6H). 13C NMR
(CD3OD, 75 MHz) d 166.5, 156.3, 144.8, 143.1, 111.3,
106.4, 99.0, 68.7, 57.0, 56.3, 28.9. IR (KBr) 3449, 2928,
1614, 1510, 1394, 1231, 1105, 1001 cmꢀ1. HRMS (EI)
calcd for C12H16N2O3, m=z 236.1155 (Mþ); found, m=z
236.1156.
4.1.16. 2-Nitro-4,5-dimethoxy-N-pyridin-4-yl-benzamide
(10i). From compound 9 (0.2 g, 1 mmol), a similar pro-
cedure as that described for 10g provided the white solid
1
10i (0.2 g, 51%): H NMR (CDCl3, 400 MHz) d 8.52 (d,
2H), 7.73 (s, 1H), 7.65 (s, 1H), 7.52 (d, 2H), 6.97 (s, 1H),
3.98 (s, 6H). 13C NMR (CDCl3, 75 MHz) d 165.4, 153.8,
150.6, 149.9, 145.0, 138.4, 126.4, 113.9, 109.9, 107.1,
56.8, 56.7. IR (KBr) 1699, 1583, 1508, 1331, 1285, 1051,
876, 787 cmꢀ1. HRMS (EI) calcd for C14H13N3O5, m=z
303.0850 (Mþ); found, m=z 303.0853.
4.1.17. 2-Amino-4,5-dimethoxy-N-pyridin-2-yl-benzamide
(1g). A solution of nitrobenzamide 10g (0.6 g, 2 mmol)
and SnCl2 (1.9 g, 10 mmol) in MeOH (90 mL) was ref-
luxed for 3 h. After solvent removal in vacuo and
digestion in CH2Cl2, saturated aqueous NaHCO3 was
added and the mixture was stirred for 16 h at room
temperature. The mixture was then filtered through
Celite and the organic layer separated. The aqueous
layer was extracted with CH2Cl2 and the combined or-
ganic layer were washed with water, dried (MgSO4), and
evaporated in vacuo to give the amine 1g (0.5 g, 89%) as
4.1.21. 6,7-Dimethoxy-2,2-dimethyl-4-methylsulfanyl-1,2-
dihydro-quinazoline (13). To a stirring solution of 12
(2.5 g, 11 mmol) in THF (22 mL) at 40 ꢁC was added
Lawesson’s reagent (2.4 g, 6 mmol). The resulting mix-
ture was heated to 80 ꢁC for 3 h, cooled to ambient
temperature and solvent removed in vacuo. The result-
ing foamy residue was triturated with CH2Cl2, and the
solid was filtered to yield product. The filtered solid
dried in a Buchner funnel overnight to afford 1.68 g of
the corresponding thioamide as a yellow solid: 1H NMR
(CDCl3, 400 MHz) d 7.71 (s, 1H), 6.20 (s, 1H), 3.82 (s,
3H), 3.78 (s, 3H), 1.47 (s, 6H). 13C NMR (CDCl3,
75 MHz) d 189.0, 157.2, 143.2, 141.0, 115.2, 112.7, 98.3,
68.6, 56.9.
1
a white solid: H NMR (CD3OD, 400 MHz) d 8.81 (s,
1H), 8.27 (s, 1H), 8.23 (d, J ¼ 4:8 Hz, 1H), 7.70 (t,
J ¼ 7:2 Hz, 1H), 7.00 (q, J ¼ 0:8, 4.8, 7.2 Hz, 1H), 6.96
(s, 1H), 6.19 (s, 1H), 5.51 (s, 2H), 3.84 (s, 3H), 3.78 (s,
3H). 13C NMR (CD3OD, 75 MHz) d 169.3, 155.7, 153.6,
148.8, 148.4, 141.9, 139.5, 120.6, 116.2, 113.3, 107.2,
101.8, 57.6, 56.1. IR (KBr) 3340, 2950, 1653, 1506, 1304,
1205, 1165, 999 cmꢀ1
.
HRMS (EI) calcd for
C14H15N3O3, m=z 273.1108 (Mþ); found, m=z 273.1109.
To a stirring solution of the thioamide (2.5 g, 10 mmol)
in MeOH (20 mL) was added MeI (1.87 mL, 30 mmol).
The resulting dark yellow solution was stirred for 2 h,
4.1.18. 2-Amino-4,5-dimethoxy-N-pyridin-3-yl-benzamide
(1h). From compound 10h (0.2 g, 1 mmol), a similar

T. Asano et al. / Bioorg. Med. Chem. 12 (2004) 3529–3542
3539
and the solvent was removed under reduced pressure.
The residue was partitioned between EtOAc and satu-
rated aqueous NaHCO3. The layers were shaken, the
organic layer separated, dried over Na2SO4, and solvent
removed in vacuo. The residue was recrystallized from
C19H23N3O3, m=z 342.1812 (M+Hþ); found, m=z
342.1818.
4.1.25. (3-Chloro-4-fluoro-phenyl)-(6,7-dimethoxy-2,2-di-
methyl-1,2-dihydro-quinazolin-4-yl)-amine (3d). From a
thiomethylether 13 (0.3 g, 1 mmol), a similar procedure
as that described for 3a provided a yellow solid 3d (0.1 g,
38%): 1H NMR (CD3OD, 400 MHz) d 7.55 (s, 1H), 7.10
(t, J ¼ 8:8 Hz, 1H), 7.01 (d, J ¼ 4:8 Hz, 1H), 6.81 (br s,
1H), 6.19 (s, 1H), 3.88 (s, 3H), 3.86 (s, 3H), 1.47 (s, 6H).
1
hexane to yield 13 (2.6 g, 98%) as a yellow solid: H
NMR (CDCl3, 400 MHz) d 6.96 (s, 1H), 6.16 (s, 1H),
3.84 (s, 3H), 3.83 (s, 3H), 2.46 (s, 3H), 31.47 (s, 6H). 13C
NMR (CDCl3, 75 MHz) d 159.2, 153.1, 141.3, 138.7,
109.1, 108.4, 98.0, 70.5, 56.6, 55.8, 29.3, 12.3. IR (KBr)
3283, 1626, 1506, 1267, 1031, 876, 837 cmꢀ1. HRMS (EI)
calcd for C13H18N2O2S, m=z 252.0927 (Mþ)CH2);
found, m=z 252.0927.
13C NMR (CD3OD, 68 MHz) d 155.5, 153.9, 143.5,
:
143.5, 125.7, 123.9, 123.8, 121.8, 118.2, 117.9, 110.8,
107.2, 99.8, 98.5, 67.9, 57.1, 56.3, 28.4. IR (KBr) 3568,
3255, 1618, 1508, 1281, 1093, 781. HRMS (ESI) calcd
for C19H19ClN3O2, m=z 364.1223 (M+Hþ); found, m=z
364.1223.
4.1.22. (3-Bromo-phenyl)-(6,7-dimethoxy-2,2-dimethyl-
1,2-dihydro-quinazolon-4-yl)-amine (3a). To a thiometh-
ylether 13 (0.3 g, 1 mmol) was added 3-bromoaniline
(0.5 mL, 5 mmol). The resulting homogeneous solution
was heated overnight, cooled to ambient temperature,
and the mixture was purified by preparative thin layer
chromatography (2:1 hexane/EtOAc) to yield pure 3a
(0.2 g, 46%) as a yellow solid: 1H NMR (CD3OD,
400 MHz) d 7.48 (br s, 1H), 7.29–7.20 (m, 3H), 7.04 (d,
J ¼ 6:4 Hz, 1H), 6.38 (s, 1H), 3.88 (s, 3H), 3.84 (s, 3H),
1.43 (s, 6H). 13C NMR (CD3OD, 75 MHz) d 155.2,
153.3, 143.3, 143.2, 131.7, 126.7, 126.6, 123.7, 122.5,
110.6, 107.1, 99.7, 67.9, 657.3, 57.1, 56.2, 28.5. IR (KBr)
3336, 1614, 1506, 1277, 1008, 853 cmꢀ1. HRMS (ESI)
calcd for C19H20BrN3O2, m=z 390.0812 (M+Hþ); found,
m=z 390.0812. Anal. Calcd for C19H20BrN3O2: C, 55.40;
H, 5.17; N, 10.77; Br, 20.47. Found: C, 55.254; H, 5.474;
N, 10.617; Br, 20.65.
4.1.26. 2-Amino-N-(3-bromo-phenyl)-4,5-dimethoxy-benz-
amidine (2a). The acetonide 3a (0.8 g, 2 mmol) was
heated at reflux in 6 N HCl (12.5 mL) overnight and
cooled to ambient temperature. The solution was
evaporated at reduced pressure. The residue was taken
up in isopropyl alcohol and sonicated. The solid was
filtered and dried in vacuo to afford 0.2 g of 2a as a white
1
solid: mp 170–174 ꢁC; H NMR (CD3OD, 400 MHz) d
7.85 (s, 1H), 7.71 (d, J ¼ 8:4 Hz, 1H), 7.57 (m, 2H), 7.36
(s, 1H), 7.01 (s, 1H), 3.98 (s, 3H), 3.95 (s, 3H). 13C NMR
(CD3OD, 75 MHz) d 163.1, 155.2, 148.6, 136.7, 133.1,
132.7, 129.5, 125.3, 124.2, 114.4, 107.4, 107.2, 57.2, 56.9.
IR (KBr) 3015, 2835, 2606, 1670, 1521, 1232, 1074,
1000 cmꢀ1. HRMS (EI) calcd for C15H16N3O2, m=z
349.0420 (Mþ); found, m=z 349.0422.
4.1.23. (3-Chloro-phenyl)-(6,7-dimethoxy-2,2-dimethyl-
1,2-dihydro-quinazolon-4-yl)-amine (3b). From a thio-
methylether 13 (0.3 g, 1 mmol), a similar procedure as
that described for 3a provided a yellow solid 3b (0.2 g,
56%): 1H NMR (CD3OD, 400 MHz) d 7.48 (s, 1H), 7.33
(t, J ¼ 7:6 Hz, 1H), 7.11 (br s, 1H), 7.07 (d, J ¼ 7:6 Hz,
1H), 7.01 (br s, 1H), 6.37 (s, 1H), 3.88 (s, 3H), 3.85 (s,
3H), 1.43 (s, 6H). 13C NMR (CD3OD, 75 MHz) d 155.0,
153.1, 143.2, 135.6, 131.4, 123.5, 121.9, 110.6, 107.4,
99.7, 67.9, 57.1, 56.2, 28.5. IR (KBr) 2964, 2931, 1629,
1504, 1278, 1082, 864, 827, 785. HRMS (ESI) calcd for
C18H20ClN3O2, m=z 346.1317 (M+Hþ); found, m=z
346.1317.
4.1.27. 2-Amino-N-(3-chloro-phenyl)-4,5-dimethoxy-benz-
amidine (2b). From a acetonide 3b (0.3 g, 1 mmol), a
similar procedure as that described for 2a provided a
white solid 2b (0.2 g, 61%): mp 173–176 ꢁC; H NMR
(CD3OD, 400 MHz) d 7.75 (s, 1H), 7.61 (m, 3H), 7.48 (s,
1H), 7.19 (s, 1H), 4.01 (s, 3H), 3.99 (s, 3H). 13C NMR
(CD3OD, 75 MHz) d 163.1, 154.7, 149.8, 136.7, 132.7,
130.4, 126.8, 125.1, 115.3, 114.6, 108.5, 57.2, 57.1. IR
(KBr) 3042, 2816, 2608, 1665, 1593, 1371, 1234, 1076,
989, 868 cmꢀ1. HRMS (EI) calcd for C15H16ClN3O2, m=z
305.0926 (Mþ); found, m=z 305.0928.
1
4.1.24. (6,7-Dimethoxy-2,2-dimethyl-1,2-dihydro-quinaz-
olin-4-yl)-(3-methoxy-phenyl)-amine (3c). From a thio-
methylether 13 (0.3 g, 1 mmol), a similar procedure as
that described for 3a provided a yellow solid 3c (0.09 g,
4.1.28. 2-Amino-4,5-dimethoxy-N-(3-methoxy-phenyl)-
benzamidine (2c). From a acetonide 3c (0.3 g, 1 mmol),
a similar procedure as that described for 2a provided a
white solid 2c (0.2 g, 81%): mp >200 ꢁC; 1H NMR
(CD3OD, 400 MHz) d 7.53 (t, J ¼ 7:6 Hz, 1H), 7.42 (s,
1H), 7.23 (s, 1H), 7.18 (m, 2H), 7.11 (s, 2H), 4.00 (s, 3H),
3.98 (s, 3H), 3.92 (s, 3H). 13C NMR (CD3OD, 75 MHz)
d 162.9, 162.2, 155.1, 147.9, 136.2, 132.0, 118.0, 115.8,
114.3, 112.8, 111.8, 106.9, 57.3, 57.0, 56.2. IR (KBr)
2835, 1655, 1524, 1234, 989, 869 cmꢀ1. HRMS (EI) calcd
for C16H19N3O3, m=z 301.1421 (Mþ); found, m=z
301.1424.
26%): 1H NMR (CD3OD, 400 MHz)
d 7.46 (t,
J ¼ 8:4 Hz, 1H), 7.40 (s, 1H), 6.99 (dd, J ¼ 2:4, 9.2 Hz,
1H), 6.94 (d, J ¼ 3:6 Hz, 1H), 6.93 (s, 1H), 6.44 (s, 1H),
3.93 (s, 3H), 3.87 (s, 3H), 1.58 (s, 6H). 13C NMR
(CD3OD, 75 MHz) d 180.4, 162.4, 154.8, 147.8, 147.3,
134.4, 131.8, 117.6, 114.4, 114.2, 113.2, 111.5, 106.7,
103.4, 67.7, 56.3, 55.9, 55.6, 27.3, 24.2. IR (KBr) 3460,
1601, 1508, 1042, 700. HRMS (ESI) calcd for

3540
T. Asano et al. / Bioorg. Med. Chem. 12 (2004) 3529–3542
4.1.29. 2-Amino-N-(4-chloro-3-fluoro-phenyl)-4,5-dimeth-
oxy-benzamidine (2d). From a acetonide 3d (0.3 g,
1 mmol), a similar procedure as that described for 2a
provided a white solid 2d (0.3 g, 82%): mp >200 ꢁC; H
NMR (CD3OD, 300 MHz) d 7.84 (br s, 1H), 7.56 (br s,
1H), 7.47 (t, J ¼ 9:0 Hz, 1H), 7.39 (s, 1H), 3.95 (s, 3H),
3.93 (s, 3H). 13C NMR (CD3OD, 75 MHz) d 163.3,
160.9, 157.6, 155.0, 149.4, 131.9, 129.6, 127.6, 123.4,
119.3, 119.0, 114.4, 108.2, 57.2, 57.0. IR (KBr) 3044,
2835, 1670, 1523, 1236, 1076 cmꢀ1. HRMS (EI) calcd for
C15H15ClFN3O2, m=z 323.0831 (Mþ); found, m=z
323.0833.
FCS. In Falcon 3072 96-well culture plate, the cells
(0.5 · 104 cells/well) were cultured on seven wells with
the medium containing the compounds at various con-
centrations (2–2000 ppm), and incubated for 3 days at
37 ꢁC in CO2 incubator. It is known that DMSO is
nontoxic at the concentration lower than 0.5%. We also
confirmed by the control experiment that DMSO was
nontoxic at the concentrations shown above. The med-
ium was removed, and the cells were washed three times
with PBS()) and then dyed by crystal violet (0.4% in
MeOH) for counting cells on Microplate Reader. The
results are presented as the concentration of agents that
resulted in 50% of the cell number of untreated cultures
(IC50).
1
4.1.30.
6-Nitro-N-(3-bromo-phenyl)-2,3,4-trimethoxy-
benzamide (16). A suspension of benzoic acid 15 (0.8 g,
3 mmol), 15 mL of dry CH2Cl2, and thionyl chloride
(0.7 mL, 9 mmol) was allowed to reflux for 2 h. The
solvent was removed under vacuum. To a suspension of
the acid chloride in 15 mL of dry CH2Cl2 was added 3-
bromoaniline (0.3 mL, 3 mmol), and the mixture was
allowed to stir at room temperature for 12 h. The solvent
was removed under reduced pressure. The residue was
treated with ice-water, and the pH of the slurry was
adjusted to a value of 7 by adding 1 N NaOH. This
solution was partitioned between EtOAc and 1 N
NaOH. The layers were shaken, and the organics were
separated and washed additionally with H2O. The or-
ganic layer was dried over Na2SO4 and filtered and the
solvent was removed under reduced pressure. The resi-
due was purified by flash layer chromatography (5:1
hexane/EtOAc) to yield the pure 16 (1.0 g, 78%) as a
brown solid: 1H NMR (CDCl3, 300 MHz) d 7.82 (s, 1H),
7.71 (br s, 1H), 7.51 (s, 1H), 7.48 (d, J ¼ 8:0 Hz, 1H),
7.29 (d, J ¼ 8:0 Hz, 1H), 7.22 (t, J ¼ 8:0 Hz, 1H), 3.99
(s, 3H), 3.97 (s, 3H), 3.93 (s, 3H). 13C NMR (CDCl3,
75 MHz) d 161.7, 153.9, 150.7, 147.4, 140.9, 138.8, 130.4,
127.8, 123.2, 122.7, 121.1, 108.8, 104.2, 104.1, 62.6, 61.3,
56.6. IR (KBr) 3257, 1654, 1591, 1522, 1400, 1338, 1112,
966, 920, 775. HRMS (ESI) calcd for C16H15BrN2O6,
m=z 411.0186 (M+Hþ); found, m=z 411.0187.
4.2.1. EGFR tyrosine kinase assay. For EGF receptor
tyrosine kinase assay, membrane fractions were pre-
pared from A431 cells (20 mM Hepes, pH 7.4, 0.2%
Triton X-100) by the method of Hanai et al.36 and the
kinase assay by the method of Bertics et al.37 Briefly,
15 lL of an assay mixture (20 mM Hepes, pH 7.4, 5 mM
MnCl2) containing a membrane fraction of 10 lg protein
and 2 lg/mL EGF and 1.0 mg/mL angiotensin II as
substrate were preincubated at 25 ꢁC for 30 min with a
sample, and kinase reaction was started by adding 5 lL
of [c-32P]ATP (1 lM, 5 lCi/assay). After 15 min incu-
bation at 0 ꢁC, reaction was stopped with ice cold 20%
trichloroacetic acid (TCA), the supernatant was spotted
on P81 cellulose paper (Whatmann), washed and the
radioactivities remaining were measured by scintillation
counter.
4.2.2. VEGFR tyrosine kinase assay. VEGFR tyrosine
kinase activity was measured using membrane fractions
derived from Flt-1 overexpressing Tn5 cells as
described.37 Briefly, membrane fractions (2 lg) were
incubated with kinase assay buffer containing Hepes
(50 mM, pH 7.4), Triton X-100 (0.1%), MnCl2 (10 mM),
MgCl2 (2 mM), DTT (1 mM), NaF (1 mM), Na3VO4
(0.1 mM), and [c-32P]ATP (2.5 lCi, 10 lM) at 25 ꢁC for
10 min and the reaction was terminated by addition of
SDS loading buffer. After boiling for 3 min, the reaction
mixture was fractionated by SDS-PAGE (7.5% poly-
acrylamide gel) and bands of phosphorylated Flt-1 were
visualized by autoradiography.
4.1.31. 6-Amino-N-(3-bromo-phenyl)-2,3,4-trimethoxy-
benzamide (1j). A solution of the benzamide 16 (1.0 g,
2.3 mmol) in methanol was stirred with 60 mg of 10%
Pd–C under hydrogen for 4 h. The catalyst was filtered
off. The solvent was then stripped off on a rotary evap-
orator to provide 0.9 g (>99%) of 1j as a white solid: mp
185–187 ꢁC; 1H NMR (CDCl3, 300 MHz) d 10.27 (s, 1H),
7.57 (d, J ¼ 9:9 Hz, 2H), 7.36 (t, J ¼ 7:5 Hz, 2H), 7.18 (t,
J ¼ 7:2 Hz, 1H), 4.06 (s, 3H), 4.01 (s, 3H), 3.90 (s, 3H).
13C NMR (CDCl3, 75 MHz) d 164.1, 156.4, 152.6, 142.1,
136.4, 128.9, 128.8, 125.3, 121.1, 110.3, 107.6, 62.5, 61.1,
57.0. IR (KBr) 2800, 1622, 1541, 1339, 1119, 806,
743 cmꢀ1. HRMS (EI) calcd for C16H17BrN2O4, m=z
380.0372 (Mþ); found, m=z 380.0366.
4.2.3. Multiple protein kinase assay. Kinase activities of
PKA, PKC, v-Src, and eEF2K were assessed as
described.38;39 NIH 3T3 cells transformed with v-Src
were suspended in a hypotonic buffer containing 1 mM
Hepes-NaOH (pH 7.4), 5 mM MgCl2, and 25 lg/mL
each of antipain, leupeptin, and pepstatin A and allowed
to stand on ice for 10 min. After vigorous mixing for
1 min with a vortex mixer, the concentration of Hepes-
NaOH (pH 7.4) was made equal to 20 mM. Then the
lysate was centrifuged at 500g for 5 min as 4 ꢁC to obtain
the supernatant postnuclear fraction, which included
protein kinases and their substrate proteins. Additions
were made to the supernatant to give a final concen-
tration of 1 mg/mL of protein/20 mM Hepes-NaOH
4.2. Cytotoxicity
The compounds (10 mg) were dissolved in 0.1 mL of
DMSO, and the resulting solution was diluted with 10%

T. Asano et al. / Bioorg. Med. Chem. 12 (2004) 3529–3542
3541
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114.
(pH 7.4)/10 mM MgCl2/0.1 mM Na3VO4/1 mM NaF/
20 lM cAMP/100 lM CaCl2. DMSO solution of protein
kinase inhibitors (3 lL) was added to 22 lL of post-
nuclear fraction and the mixture was incubated for
10 min on ice. The kinase reaction was initiated by
adding 5 lL of [c-32P]ATP (75 lM, 10 lCi) and the
mixture was incubated for 20 min at 25 ꢁC. At the end of
the reaction 10 lL of fourfold concentrated SDS-PAGE
sample buffer was added to the mixture and phosphor-
ylated protein was separated by SDS-PAGE (9% w/v
gel). To detect PTK activity, the gel was further treated
with NaOH (1 N) for 2 h at 55 ꢁC. The results were
visualized by autoradiography. Position of the substrate
protein band of each kinase was identified according to
the sensitivity to various protein kinase inhibitors or
activators and phosphoamino acid analysis as previ-
ously described.
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4.3. Docking methods
EGFR kinase domain/erlotinib complex X-ray structure
(PDB code 1M17)22 was taken from the Protein Data
Bank. The study of complexes formed by EGFR kinase
domain with 1c, 3a, and ATP was performed using the
DS-Modeling-SBD program. Initially, hydrogen atoms
were added to the protein, considering all the residues in
their neutral form. Water molecules were removed and
the added hydrogens were minimized while keeping all
the heavy atoms fixed. The minimization followed
steepest descent and conjugate gradient algorithms for
interaction each using CHARMM force field in DS-
Modeling-SBD. The EGFR active site was defined using
the inhibitor, erlotinib [6,7-bis(2-methoxy-ethoxy)qui-
nazoline-4-yl]-(3-ethynylphenyl)amine and included all
ꢀ
amino acid residues within 10 A radius from the center
of the ligand. Docking was performed using the auto-
matic calculations. The protein active site conforma-
tions for the some conformations of the ligand were
retrieved for the further analysis.46
17. Hennequin, L. F.; Stokes, E. S. E.; Thomas, A. P.;
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18. Matsuno, K.; Ichimura, M.; Nakajima, T.; Tahara, K.;
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A.; Scarborough, R. M.; Lokker, N. A.; Yu, J.-C.; Irie, Y.;
Tsukuda, E.; Ide, S.; Oda, S.; Nomoto, Y. J. Med. Chem.
2002, 45, 3057–3066.
Acknowledgements
We thank Prof. M. Shibuya (The University of Tokyo)
for providing a vaculovirus vector carrying human flt-1.
We also thank the Screening Committee of New Anti-
cancer Agents supported by Grant-in-Aid for Scientific
Research on Priority Area ‘Cancer’ from the Ministry of
Education, Science, Sports, Culture and Technology,
Japan for the protein kinase assay. Part of this work was
supported by the Naito Foundation, Japan.
19. Osherov, N.; Levitzki, A. Eur. J. Biochem. 1994, 225,
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20. Boschelli, D. H.; Ye, F. e.; Wang, Y. D.; Dutia, M.; John-
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pKi ¼ 0:517527 ꢀ 0:043650ðvdW Grid SoftÞ
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þ 0:143901ðC þ polÞ0:00099039ðTotpol^2Þ
ð1Þ
where vdW_Grid_Soft is the grid-based soft van der
Waals energy of interaction between the ligand and the
protein. C þ pol is the total surface area of the ligand
involved in attractive polar interactions with the protein
2
2
and Totpol^2 is ðC þ polÞ þ ðC ꢀ polÞ where C þ pol
is the total surface area of the ligand involved in repulsive
polar interactions with the protein.