Acryloylamino-salicylanilides as EGFR PTK inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.06.088

A series of acryloylamino-salicylanilides were synthesized as inhibitors of EGFR PTK. A strategy of pseudo six-membered ring formed through intramolecular hydrogen bonding in salicylanilides is employed to mimic the planar pyrimidine ring of quinazoline E

Full text of DOI:10.1016/j.bmcl.2005.06.088

Bioorganic & Medicinal Chemistry Letters 16 (2006) 469–472
Acryloylamino-salicylanilides as EGFR PTK inhibitors
Wei Deng, Zongru Guo,* Yanshen Guo, Zhiqiang Feng, Yi Jiang and Fengming Chu
Department of Synthetic Medicinal Chemistry, Institute of Materia Medica, Chinese Academy of Medical
Sciences and Peking Union Medical College, Beijing 100050, PR China
Received 11 April 2005; revised 21 June 2005; accepted 27 June 2005
Available online 3 November 2005
Abstract—A series of acryloylamino-salicylanilides were synthesized as inhibitors of EGFR PTK. A strategy of pseudo six-mem-
bered ring formed through intramolecular hydrogen bonding in salicylanilides is employed to mimic the planar pyrimidine ring
of quinazoline EGFR inhibitors. Acrylamido moiety is incorporated to target the Cys-773 of EGFR specifically. Some of the
obtained compounds exhibited good activity as EGFR inhibitors.
Ó 2005 Elsevier Ltd. All rights reserved.
The protein tyrosine kinase (PTK) plays critical roles in
many of the signal transduction processes that control
cell growth, differentiation, mitosis and apoptosis. The
epidermal growth factor receptor (EGFR) belongs to
the family of transmembrane growth factor receptor
PTKs. The EGFR and its ligands (EGF, TGF-a) have
been implicated in various tumors of epithelial origin
(e.g., aquamous cell carcinoma, breast, ovarian and
NSC lung cancer).1,2 Thus, inhibitors of the EGFR have
emerged as promising anticancer agents and two main
approaches, humanized monoclonal antibodies and
tyrosine kinase inhibitors, have been developed.
the backbone NH of Met-769 via a hydrogen bond,
and similarly, a water molecule-mediated hydrogen
bonding interaction is observed between the N-3 of the
quinazoline ring and the Thr-766 side chain. These inter-
actions underscore the importance of both nitrogen
atoms for binding and the subsequent inhibitory capac-
ity. In fact, replacement of a carbon atom for either
nitrogen atom results in drastic loss in inhibitory activi-
ty. (3) The aniline moiety lies in a deep and hydrophobic
pocket. (4) The acrylamido moiety at C-6 or C-7 of the
quinazoline ring conveys an irreversible inhibition and a
more potent antitumour activity as compared to the
congeners in the absence of this unique moiety. Michael
addition occurred between the acrylamido moiety of the
inhibitor and sulfhydryl group of the Cys-773 residue is
believed to be responsible for the distinct activity profile.
In the last few years, a large structural variety of com-
pounds, such as 4-anilinoquinazolines,3–5 4-anilinopy-
razolo[3,4-d]pyrimidines,6 4-anilinoquinoline-3-carbonitr-
iles,7 4-anilinopyrazolo- and 4-anilinopyrroloquinazo-
lines,8 were reported as EGFR tyrosine kinase
inhibitors. Representative inhibitors of the quinazoline
series are illustrated in Figure 1.9
Herein, we report the synthesis and biological activity of
a series of EGFR PTK inhibitors with an acrylamido
group at the 4- or 5-position of salicylanilides. A dock
model and preliminary SAR based on in vitro enzyme
assay will also be discussed.
According to the crystal structure of the AQ4774
10
TM
(Tarceva )–EGFR complex (Fig. 2) and previous
structure–activity relationship (SAR) of 4-anilinoqui-
nazolines,3–8,11–19 pivotal interactions between the
receptor and the inhibitors have been revealed as
follows: (1) The quinazoline ring binds to a narrow
hydrophobic pocket in the N-terminal domain of EGFR
TK. (2) The N-1 of the quinazoline ring interacts with
The salicylanilide molecules may assume alternative
conformations via either NHꢀ ꢀ ꢀO hydrogen bonding
(I) or OHꢀ ꢀ ꢀO@C hydrogen bonding (II). Intramolecu-
lar hydrogen bonding stabilizes both conformations
and makes the molecule considerably rigid. By NMR
spectroscopy, Suezawa et al. have shown that almost
all salicylanilide derivatives without 3- or 20(60)-substitu-
ent form a pseudo six-membered ring via a strong
hydrogen bond between OH and O@C, and take a fairly
rigid and planar conformation (II) (Fig. 3).20 The con-
formational similarity of II to quinazoline led to the
Keywords: EGFR; Inhibitors; Salicylanilides.
*
Corresponding author. Tel.: +86 10 631 65249; fax: +86 10 831
55752; e-mail: zrguo@imm.ac.cn
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.06.088

470
W. Deng et al. / Bioorg. Med. Chem. Lett. 16 (2006) 469–472
F
Cl
HN
O
HN
N
O
O
O
O
N
O
O
N
N
N
IressaTM
TarcevaTM
F
O
Br
HN
N
Cl
HN
H
HN
N
N
N
O
O
O
N
N
Figure 1. Representative EGFR PTK inhibitors.
TM
Figure 2. Crystal structure of AQ4774 (Tarceva )–EGFR complex.
6'
6'
from the sulfhydryl group of Cys-773, which is favorable
for the occurrence of the Michael addition.
O
HN
HN
N
5
4
2'
N
H
5
4
6
7
O
H
N
2'
O
H
Most of the 5-substituted salicylanilides (5–14) listed in
Table 1 were obtained from 2-methoxyl-5-nitro-benzoic
acid. The acid was transformed into the corresponding
acid chloride using SOCl2, which reacted with substituted
aniline to give the corresponding anilide. Ether cleavage
with BCl3 gave 5-nitro-salicylanilide, and subsequent
reduction of the nitro group and acylation of the resulting
amino group with acryloyl chloride or propionyl chloride
yielded 5-acryloylamino-salicylanilides (5, 7, 9–14). To
identify the role of the o-hydroxyl group, 5-acryloylami-
no-2-methoxyl-benzanilides (6 and 8) were prepared.
The intermediate 5-nitro-salicylanilide could also be syn-
thesized through the reaction of the corresponding sali-
cylic acid with substituted aniline with phosphorus
trichloride in boiling toluene (1 and 3) (Scheme 1).
O
3
3
salicylanilide
quinazoline
Figure 3. Salicylanilide and quinazoline.
hypothesis that the pseudo six-membered ring may
function as a mimic of the pyrimidine ring of the quin-
azolines. The acrylamide side chain at the 5- or 4- posi-
tion of salicylanilide corresponds to that of the 6- or
7-position of the quinazoline core. The presence of the
acrylamido moiety favors the trapping of the Cys-773
of EGFR.
Docking studies were performed to fit salicylanilides to
the enzyme. The structure of salicylanilides and Tarc-
TM
eva are in good spatial match (Fig. 4). The pseudo
To explore the regioisomeric effect of the 4-acryloylami-
no moiety on the activity, 4-substituted salicylanilides (2
and 4) were prepared in a different approach as shown in
Scheme 2. p-Aminosalicylic acid (PAS) reacting with
acryloyl chloride gave 4-acryloylamino-salicylic acid,
which was converted to 4-acryloylamino-salicylanilide
by acylation with substituted aniline.
six-membered ring and pyrimidine ring orient in a simi-
lar manner and superpose at the same site. The oxygen
atom of the phenolic group forms a hydrogen bond with
the backbone NH of Met-769 as the N1 atom of the qui-
nazoline does. The oxygen of the carbonyl group binds
to a structural water molecule via hydrogen bonding
similar to the way N3 atom of the quinazoline functions.
This water molecule, in turn, associates with the OH of
Thr-766 by another hydrogen bond. The aniline moiety
situates into the same hydrophobic pocket. The acry-
lamido side chain extrudes from the enzyme surface to-
The synthesized target compounds were tested for their
inhibitory activity toward the EGFR tyrosine kinase.
The results are listed in Table 1. Inhibitory activities
are given as percentage inhibition at four geometric con-
centrations of 10, 1.0, 0.1, and 0.01 lM.
˚
wards the solvent. The c-carbon is positioned 4.11 A

W. Deng et al. / Bioorg. Med. Chem. Lett. 16 (2006) 469–472
471
Figure 4. Compound 3 docked (gray) and X-ray crystallographic conformations of AQ4774 (red).
Table 1. Inhibitory activity of salicylanilides toward EGFR
R1
HN
O
R3
R2
Compound
R1
R2
R3
EGFR PTK (lM)
10
1
0.1
0.01
1
2
3-Cl
3-Cl
OH
OH
OH
OH
OH
OMe
OH
OMe
OH
OH
OH
OH
OH
OH
5-CH2@CHC(O)NH
4-CH2@CHC(O)NH
5-CH2@CHC(O)NH
4-CH2@CHC(O)NH
5-CH2@CHC(O)NH
5-CH2@CHC(O)NH
5-CH2@CHC(O)NH
5-CH2@CHC(O)NH
5-CH2@CHC(O)NH
5- CH3CH2C(O)NH
5-CH2@CHC(O)NH
5-CH3CH2C(O)NH
5-CH2@CHC(O)NH
5-CH2@CHC(O)NH
62.6
73.1
91.3
93.0
91.2
46.3
38.3
89.9
90.6
89.3
—
21.6
23.3
88.2
89.5
86.6
—
—
—
3
3-Br
3-Br
76.0
52.9
18.8
—
4
5
3-PhO
3-PhO
6
1.59
93.5
5.84
7
3-Ph(OH)CH
3-Ph(OH)CH
3-Et2NC(O)
3-Et2NC(O)
4-PhO
91.7
—
84.8
—
49.3
—
8
9
63.9
46.6
62.2
48.5
91.0
91.9
54.4
—
40.4
—
14.1
—
10
11
12
13
14
28.1
—
0.8
0
—
4-PhO
3-(CH2)4NC(O)
3-(CH2)5NC(O)
—
88.6
91.2
84.2
88.0
26.0
75.1
Enzyme tests were performed using ELISA. Ô—Õ not determined.
O
O
O
O
O
O
Ar
O2N
O2N
Ar
O2N
b
a
e
O2N
OH
N
N
OH
H
H
ArNH2
ArNH2
OH
OH
O
c
c
d
O
O
O
H
H
d
Ar
Ar
N
R
N
Ar
H2N
N
H
N
R
N
R
or
H
H
O
O
OH
OH
Scheme 1. Reagents and conditions: (a) SOCl2, reflux, then ArNH2, Et3N, THF, rt; (b) BCl3, dichloromethane, rt; (c) H2, Raney-Ni, methanol,
1 atm, rt; (d) Acryloyl chloride or propionyl chloride, Et3N, THF, rt; (e) PCl3, toluene, reflux.
O
O
O
Ar
b
a
OH
OH
O
O
N
OH
OH
H
ArNH2
N
N
OH
H2N
H
H
Scheme 2. Reagents and conditions: (a) Acryloyl chloride, NaOH (aqueous), rt; (b) PCl3, toluene, reflux.
The data presented in Table 1 clearly show that most
compounds exhibit high activities toward EGFR. The
percentage inhibitions of the three compounds (3, 4
and 14) at a concentration of 0.01 lM are more than
50%. When the phenolic hydroxyl groups of the active
compounds 5 and 7 are methylated, the resulting mole-
cules 6 and 8 are abolished from the ability to form the
intramolecular hydrogen bond and are devoid of the

472
W. Deng et al. / Bioorg. Med. Chem. Lett. 16 (2006) 469–472
Zhou, H.; Showalter, H. D. H.; Winters, R. T.; Leopold,
W. R.; Fry, D. W.; Nelson, J. M.; Slintak, V.; Elliott, W.
L.; Roberts, B. J.; Vincent, P. W.; Patmore, S. J. J. Med.
Chem. 1999, 42, 1803.
activity (inactive at 10 lM), indicating that the pseudo
six-membered ring formed through the intramolecular
hydrogen bond between OH and O@C in the salicylic
acid part is important for the inhibition.
5. Tsou, H. R.; Mamuya, N.; Johnson, B. D.; Reich, M. F.;
Gruber, B. C.; Ye, F.; Nilakantan, R.; Shen, R.; Disca-
fani, C.; DeBlanc, R.; Davis, R.; Koehn, F. E.; Green-
berger, L. M.; Wang, Y. F.; Wissner, A. J. Med. Chem.
2001, 44, 2719.
6. Traxeler, P.; Bold, G.; Frei, J.; Lang, M.; Lydon, N.;
Mett, H.; Buchdunger, E.; Meyer, T.; Mueller, M.; Furet,
P. J. J. Med. Chem. 1997, 40, 3601.
7. Wissner, A.; Overbeek, E.; Reich, M. F.; Floyd, M. B.;
Johnson, B. D.; Mamuya, N.; Rosfjord, E. C.; Discafani,
C.; Davis, R.; Shi, X. Q.; Rabindran, S. K.; Gruber, B. C.;
Ye, F.; Hallett, W. A.; Nilakantan, R.; Shen, R.; Wang, Y.
F.; Greenberger, L. M.; Tsou, H. R. J. Med. Chem. 2003,
46, 49.
The presence of acrylamido group at position 4 or 5 of
the salicylic acid moiety is another prerequisite for the
inhibition, as shown in compounds 1–5, 7, 9, 11, 13,
and 14. However, the replacement of a saturated propi-
onamido group (compounds 10 and 12) for the acrylam-
ido group (compounds 9 and 11) leads to significant
decrease in activity, which agrees with the hypothesis
that acrylamido group is critical for the inhibitory activ-
ity by alkylation reaction with Cys-773 as Michael
acceptor.
Compounds with bulky substituents such as phenoxy
and benzyl in the 3-position of the aniline moiety (com-
pounds 5, 7, 9, 11, 13, and 14) were shown to be active,
suggesting that the hydrophobic pocket of the kinase
can accommodate large groups.
8. Palmer, B. D.; Trumpp-Kallmeyer, S.; Fry, D. W.;
Nelson, J. M.; Showalter, H. D. H.; Denny, W. A. J.
Med. Chem. 1997, 40, 1519.
9. Fry, D. W.; Bridges, A. J.; Denny, W. A.; Doherty, A.;
Gries, K. D.; Hicks, J. L.; Hook, K. E.; Keller, P. R.;
Leopold, W. R.; Loo, J. A.; McNamara, D. J.; Nelson, J.
M.; Sherwood, V.; Smaill, J. B.; Trumpp-Kallmeyer, S.;
Dobrusin, E. M. Proc. Natl. Acad. Sci. U.S.A. 1998, 95,
12022.
10. Stanos, J.; Sliwkowski, M. X.; Eigenbrot, C. J. Biol.
Chem. 2002, 277, 46265.
11. Rewcastle, G. W.; Denny, W. A.; Bridges, A. J.; Zhou, H.;
Cody, D. R.; McMichael, A.; Fry, D. W. J. Med. Chem.
1996, 38, 3482.
12. Thompson, A. M.; Bridges, A. J.; Fry, D. W.; Kraker, A.
J.; Denny, W. A. J . Med. Chem. 1996, 38, 3780.
13. Bridges, A. J.; Zhou, H.; Cody, D. R.; Rewcastle, G. W.;
McMichael, A.; Showalter, H. D. H.; Fry, D. W.; Kraker,
A. J.; Denny, W. A. J. Med. Chem. 1996, 39, 267.
14. Rewcastle, G. W.; Palmer, B. D.; Bridges, A. J.; Show-
alter, H. D. H.; Sun, L.; Nelson, J.; McMichael, A.;
Kraker, A. J.; Fry, D. W.; Denny, W. A. J. Med. Chem.
1996, 39, 918.
15. Rewcastle, G. W.; Palmer, B. D.; Thompson, A. M.;
Bridges, A. J.; Cody, D.; Zhou, H.; Fry, D. W.;
McMichael, A.; Denny, W. A. J. Med. Chem. 1996, 39,
1823.
The structural features of acryloylamino-salicylanilides
as EGFR PTK inhibitors include: (1) Salicylanilide is
implicitly necessary as the structure framework, and
the phenolic hydroxyl group is very important for the
pseudo six-membered ring. (2) The acrylamido side
chain as Michael acceptor increases the inhibitory activ-
ity. (3) The substituent at the aniline moiety is favorable
for activity.
It could be verified that the pseudo six-membered ring
formed by the intramolecular hydrogen bond in salicy-
lanilides can isosterically replace pyrimidine ring of the
quinazolines. Acrylamido side chain enhances inhibition
activity of EGFR PTK. Not only halogen but bulky
substituents at the aniline moiety are beneficial for the
increase in the inhibitory activity.
Acknowledgments
16. Thompson, A. M.; Murray, D. K.; Elliott, W. L.; Fry,
D. W.; Nelson, J. A.; Showalter, H. D. H.; Roberts, B.
J.; Vincent, P. W.; Denny, W. A. J. Med. Chem. 1997,
40, 3915.
17. Rewcastle, G. W.; Murray, D. K.; Elliott, W. L.; Fry, D.
W.; Howard, C. T.; Nelson, J. M.; Roberts, B. J.; Vincent,
P. W.; Showalter, H. D. H.; Winters, R. T.; Denny, W. A.
J. Med. Chem. 1998, 41, 742.
We thank Professors Jian Ding and Liping Lin of
Shanghai Institute of Materia Medica, Chinese Acade-
my of Sciences for the test of EGFR inhibitory activity
in vitro. We also thank the analytical division of Insti-
tute of Materia Medica for the spectroscopic data and
HR-MS data.
18. Wissner, A.; Berger, D. M.; Boschelli, D. H.; Floyd, M.
B., Jr.; Greenberger, L. M.; Gruber, B. C.; Johnson, B. D.;
Mamuya, N.; Nilakantan, R.; Reich, M. F.; Shen, R.;
Tsou, H. R.; Upeslacis, E.; Wang, Y. F.; Wu, B.; Ye, F.;
Zhang, N. J. Med. Chem. 2000, 43, 3244.
References and notes
1. Aaronson, S. A. Science 1991, 254, 1146.
2. Ullrich, A.; Schlessinger, J. Cell 1990, 61, 203.
3. Smaill, J. B.; Rewcastle, G. W.; Loo, J. A.; Greis, K. D.;
Chan, O. H.; Reyner, E. L.; Lipka, E.; Showalter, H. D.
H.; Vincent, P. W.; Elliott, W. L.; Denny, W. A. J. Med.
Chem. 2000, 43, 1380.
19. Smaill, J. B.; Showalter, H. D. H.; Zhou, H.; Bridges, A.
J.; McNamara, D. J.; Fry, D. W.; Nelson, J. M.;
Sherwood, V.; Vincent, P. W.; Roberts, B. J.; Elliott, W.
L.; Denny, W. A. J. Med. Chem. 2001, 44, 429.
20. Suezawa, H.; Hirota, M.; Yuzuri, T.; Hamada, Y.;
Takeuchi, I.; Sugiura, M. Bull. Chem. Soc. Jpn. 2000, 73,
2335.
4. Smaill, J. B.; Palmer, B. D.; Rewcastle, G. W.; Denny, W.
A.; McNamara, D. J.; Dobrsin, E. M.; Bridges, A. J.;