Synthesis and structure-activity relationship of 4-(2-aryl-cyclopropylamino)-quinoline-3-carbonitriles as EGFR tyrosine kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.07.071

Synthesis and structure-activity relationship of a series of 4-(2-aryl-cyclopropylamino)-quinoline-3-carbonitrile derivatives as EGFR inhibitors is described. Compounds 29 and 30 showed potent in vitro inhibitory activity in the enzymatic assay as well as in the functional cellular assay. They are moderately selective against other types of tyrosine kinases.

Full text of DOI:10.1016/j.bmcl.2007.07.071

Bioorganic & Medicinal Chemistry Letters 17 (2007) 5978–5982
Synthesis and structure–activity relationship
of 4-(2-aryl-cyclopropylamino)-quinoline-3-carbonitriles
as EGFR tyrosine kinase inhibitors
Madhavi Pannala,a Sunil Kher,a Norma Wilson,a John Gaudette,a Ila Sircar,a
Shao-Hui Zhang,b Alexei Bakhirev,b Guang Yang,b Phoebe Yuen,b
Frank Gorcsan,b Naoki Sakurai,b Miguel Barbosab and Jie-Fei Chenga,*
aDepartment of Chemistry, Tanabe Research Laboratories USA, Inc., 4540 Towne Centre Court, San Diego, CA 92121, USA
bDepartment of Biology, Tanabe Research Laboratories USA, Inc., 4540 Towne Centre Court, San Diego, CA 92121, USA
Received 22 June 2007; revised 17 July 2007; accepted 18 July 2007
Available online 21 August 2007
Abstract—Synthesis and structure–activity relationship of a series of 4-(2-aryl-cyclopropylamino)-quinoline-3-carbonitrile deriva-
tives as EGFR inhibitors is described. Compounds 29 and 30 showed potent in vitro inhibitory activity in the enzymatic assay
as well as in the functional cellular assay. They are moderately selective against other types of tyrosine kinases.
Ó 2007 Elsevier Ltd. All rights reserved.
The epidermal growth factor receptor (EGFR) is a
transmembrane glycoprotein that belongs to the erbB
family of closely related cell membrane receptors that in-
cludes EGFR (erbB-1 or HER1), erbB-2 (HER2), erbB-
3 (HER3), and erbB-4 (HER4). It is a 170 kDa protein
composed of three major functional domains: an extra-
cellular ligand-binding domain, a hydrophobic trans-
membrane domain, and a cytoplasmic tyrosine kinase
domain. Binding of ligands to EGFR leads to autophos-
phorylation of the receptor tyrosine kinase and subse-
quent activation of signal transduction pathways.
EGFR plays an important role in initiating the signaling
that directs the behavior of epithelial cells and tumors of
epithelial cell origin.1 EGFR is highly expressed in many
human cancers (e.g., bladder, cervical, head and neck,
and ovarian) and has been found to be associated with
poor prognosis and correlated with decreased survival.
EGFR is overexpressed in 40%–80% of non-small-cell
lung cancers (NSCLC), depending on histology.2a Its
pivotal role in governing cellular proliferation, survival,
and metastasis makes EGFR an attractive molecular
target, especially for the treatment of solid tumors.3
Small molecule EGFR inhibitors have been shown to be
effective antitumor agents. Iressa (1) and Tarceva (2),
two closely related quinazoline-based EGFR inhibitors,
have efficacy against several types of cancers in human
clinical trials and were approved for the treatment of
NSCLC and colon cancers. Irreversible EGFR inhibi-
tors such as EKB569 (3) and a number of small molecule
EGFR inhibitors are also undergoing clinical trials.2b In
addition, several antibodies (e.g., cetuximab, pani-
tumumab, matuzumab, and nimotuzumab that bind to
the extracellular domain of the EGFR and antisense
oligonucleotides against EGFR receptors are either
approved or undergoing clinical trials for the treatment
of cancers. Most EGFR inhibitors such as 1–3 possess
an anilinyl group at the C-4 position of the quinazoline
or quinoline-3-carbonitrile core structures. Herein, we
describe the synthesis and structure–activity relationship
of a new series of quinoline-3-carbonitrile derivatives (4)
as potent EGFR inhibitors. These compounds possess
an arylcyclopropylamino group at the C-4 position of
the quinoline-3-carbonitrile core structure. Some of the
compounds showed excellent EGFR inhibitory activity
both in enzymatic and cell-based assays (Fig. 1).
6,7-Dialkoxyquinoline-3-carbonitrile derivatives 7 were
prepared as shown in Scheme 1. Thus, 4-chloro-7-
hydroxy-6-methoxy-quinoline-3-carbonitrile (5)4 under-
went Mitsunobu reaction with a series of substituted
Keywords: EGFR inhibitor; Quinoline-3-carbonitrile.
*
Corresponding author. Tel.: +1 858 622 7029; fax: +1 858 558
9383; e-mail: jcheng@trlusa.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.07.071

M. Pannala et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5978–5982
5979
F
O
HN
N
Cl
HN
N
N
O
O
O
O
N
O
O
N
Tarceva (2)
Iressa (1)
R5
R4
R3
F
HN
N
Cl
H
HN
N
CN
N
R1
R2
CN
O
O
N
4
EKB-569 (3)
Figure 1. EGFR inhibitors.
Cl
HN
CN
Cl
NH
N
MeO
R1O
CN
MeO
R1O
MeO
HO
CN
MeO
MeO
CN
a
b
N
N
N
7a
7
5
6
Scheme 1. Reagents and conditions: (a) R1OH, PPh3P, DIAD DCM, 0 °C, rt; (b) ( )-2-trans-phenylcyclopropyl amine, 2-ethoxyethanol, pyridine
hydrochloride, reflux, 3 h.
OH
OEt
a
CN
b
COOEt
Br
Br
+
COOEt
CN
Br
N
NH2
CN
N
H
11
8
10
9
Cl
N
CN
HN
HN
c
e
d
Br
CN
CN
Br
R
N
N
14
12
13
Scheme 2. Reagents and conditions: (a) Toluene, reflux, 2 h; (b) Dowtherm A, reflux, 10 h; (c) Oxalyl chloride, DCM, rt, 48 h; (d) ( )-2-trans-
phenylcyclopropylamine, 2-ethoxyethanol, pyridine hydrochloride, reflux, 4 h; (d) RB(OH)2, Pd(PPh3)4, aq K2CO3; DME, reflux 12 h.
COOEt
COOH
a
b
R
R
R
16
NHBoc
17
NH2
15
d
c
R
R
18
19
Scheme 3. Reagents and conditions: (a) Ethyl diazoacetate, Cuacac, 40 °C, 12 h; (b) 5 N NaOH, dioxane, reflux, 1 h; (c) DPPA, Et3N, cyclohexane,
t-BuOH, 70 °C, 18 h, (Boc)2O, 3 h; (d) 1 N HCl in ether, 6 h.

5980
M. Pannala et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5978–5982
alcohols to give the 6-methoxy-7-alkoxy-4-chloroquino-
line-3-carbonitrile intermediate (6). Amination of 6 with
trans-2-phenylcyclopropylamine furnished the desired
4-(2-phenylcyclopropylamino)-6,7-dialkoxyquinoline-
3-carbonitrile derivatives (7). The similar sequence
of synthetic steps yielded 6-alkoxy-7-methoxy analogs
from the corresponding 4-chloro-6-hydroxy-7-methoxy-
quinoline-3-carbonitrile precursor. Reaction of 1-phenyl-
cyclopropylamine with 4-chloro-6,7-dimethoxy-quino-
line-3-carbonitrile gave compound 7a in good yield.
Analogs with substitutions at either the C-6 or C-7 posi-
tion were synthesized as shown in Scheme 2. Reaction of
3- or 4-bromoaniline (8) with 2-cyano-3-ethoxy-acrylic
acid ethyl ester (9) provided the corresponding 2-cya-
no-3-(phenylamino)acrylate derivatives (10), which gave
Table 1. 4-(2-Phenylcyclopropylamino)-6- or 7-alkoxyquinoline-3-carbonitrilesa
HN
R1
R2
CN
N
Compound
R1
R2
Ki (nM)
IC50 (cell, nM)
20
21
22
OMe
H
OMe
OMe
OMe
H
24
180
360
90
2580
8270
O
23
24
OMe
OMe
120
64
1610
405
O
N
N
O
O
O
25
26
27
28
29
30
31
OMe
OMe
52
68
na
68
O
N
O
OMe
220
60
173
52.8
5
N
O
OMe
OMe
OMe
OMe
O
O
N
O
8.4
3
O
N
O
5.7
360
N
N
H3C
O
360
H3C
32
33
34
35
H
340
40
5260
2100
2530
234
N
H N
2
H
H
O
O
400
80
N
H
N
a Ki value was measured based on an 11-point curve, performed in duplicate. IC50 value was an average of two or more independent experiments.6,7

M. Pannala et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5978–5982
5981
rise to 4-hydroxyl-quinoline-3-carbonitriles under ther-
mal cyclization (Dowtherm) condition.4 Chlorination
with oxalyl chloride followed by amination furnished
brominated intermediates (13). Finally, Suzuki coupling
with appropriate boronic acids from commercial sources
provided the desired 6- or 7-substituted products (14,
32–35).
pylamines (19). Coupling of these amines with 4-
chloro-3-cyanoquinolines provided ( )-2-trans-phenyl-
cyclopropylamino derivatives.
The compounds prepared as above were tested in enzy-
matic assay using fluorescent polarization.6 Selected
compounds were further profiled on their ability to inhi-
bit the EGF-stimulated autophosphorylation at Y1068
in A431 cells.7 The potency of 4-(2-phenylcyclopropyla-
mino)-6,7-dialkoxyquinoline-3-carbonitriles is similar to
that of 4-anilino-6,7-dialkoxyquinoline-3-carbonitriles3
or quinazoline-based EGFR inhibitors.4 For example,
6,7-dimethoxy-4-(2-phenylcyclopropylamino)-quino-
line-3-carbonitrile (20) showed a Ki at 24 nM, which is
comparable to 4-(3-methylanilino)-6,7-dimethoxyquino-
line-3-carbonitrile (Ki: 44 nM, data not shown).4 How-
ever, the closely related analog 7a is inactive,
indicating the orientation of aryl group at C-4 position
plays a key role for the activity.
Scheme 3 depicts the synthesis of substituted trans-phe-
nylcyclopropylamino derivatives.5 Thus, substituted
vinyl benzenes (15) were converted into trans-2-phenyl-
cycloproanecarboxylic acid esters (16). Hydrolysis
followed by Curtius rearrangement gave rise to the
Boc protected amines (17), which were deprotected to
give the desired substituted ( )-2-trans-phenylcyclopro-
Table 2. Substituted 4-(2-phenylcyclopropylamino)-6, 7-dimethoxy-
quinoline-3-carbonitrilesa
R5
R4
The effects of substitutions at C-6 and C-7 positions
were first investigated. Removal of one of the two meth-
oxy groups at 6 or 7 positions (21 and 22) led to a dra-
matic reduction of kinase inhibitory activity and
significant loss of cellular activities indicating that both
the alkoxy groups at 6 and 7 positions are required for
potency. With this result, we investigated other alkoxy
groups to find a replacement of the methoxy group that
would improve other properties such as cellular activity
while retaining the potency in the enzymatic assay.
Replacement of 6-methoxy with a benzyloxy (23)
resulted in a fivefold decrease in activity. Other basic
nitrogen-containing side chains, notably 2-(1-morpholi-
nyl) ethoxy (24), 3-(1-morpholinyl) propyloxy (25) or
3-(3-pyridinyl) propyloxy (26), at C-6 position retained
the in vitro potency. The same side chains tended to
increasethe activity when placed at C-7 position as com-
pared to the C-6 position. For example, 3-(1-morpholi-
nyl) propyloxy at C-7 position (29) dramatically
increased potency with Ki of 8.4 nM and showed excellent
cellular activity with IC50 at ꢀ5 nM. 4-[(trans-2-Phenyl-
cyclopropyl)amino]-6-methoxy-7-[3-(4-methylpiperazinyl)
propoxy]quinoline-3-carbonitrile (30) was the most
R3
HN
MeO
MeO
CN
N
Compound
R3
R4
R5
Ki (nM)
36
37
H
H
H
OMe
Cl
OMe
44
n.a.
O
38
39
H
H
OMe
H
n.a.
80
O
40
41
42
43
H
H
H
F
H
H
Br
H
Br
OMe
H
220
n.a
52
H
36
a Ki value was measured based on an 11-point curve, performed in
duplicate.
Table 3. Selectivity profile for selected compoundsa
Compound
EGFR Ki (lM)
ABL Ki (lM)
SYK Ki (lM)
Flt-1 Ki (lM)
KDR Ki (lM)
SRC Ki (lM)
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
0.024
0.18
0.36
0.12
0.064
0.052
0.068
0.22
0.06
0.008
0.003
0.36
0.34
0.04
0.4
0.95
>10
>10
>10
>10
1.8
0.25
>10
>10
>10
>10
>10
>10
0.14
0.16
>10
0.041
>10
>10
0.45
>10
0.3
1.6
2.1
0.15
0.17
0.21
0.33
>10
>10
>10
>10
>10
0.91
1.71
1.9
>10
>10
2.6
0.15
0.56
0.65
0.099
0.14
0.42
0.3
>10
>10
1.8
0.3
0.049
0.11
0.045
0.027
>10
0.39
0.2
2.9
0.35
0.11
1.5
0.044
>10
>10
0.77
>10
0.3
>10
>10
1.5
>10
1.4
0.28
0.086
0.57
0.14
>10
0.92
>10
0.17
0.08
a Ki value was measured based on an 11-point curve, performed in duplicate at ATP concentrations equal to respective Km value of the enzymes.

5982
M. Pannala et al. / Bioorg. Med. Chem. Lett. 17 (2007) 5978–5982
Miller, K.; Powell, D. W.; Yaczko, D.; Young, M.;
Tischler, M.; Arndt, K.; Discafani, C.; Etienne, C.;
Gibbons, J.; Grod, J.; Lucas, J.; Weber, J. M.; Boschelli,
F. J. Med. Chem. 2001, 44, 3965–3977; (c) Wissner, A.;
Overbeek, E.; Reich, M. F.; Floyd, M. B.; Johnson, B. D.;
Mamuya, N.; Rosfjord, E. C.; Discafani, C.; Davis, R.; Shi,
X.; Rabindran, S. K.; Gruber, B. C.; Ye, F.; Hallett, W. A.;
Nilakantan, R.; Shen, R.; Wang, Y.-F.; Greenberger, L. M.
H. -R. J. Med. Chem. 2003, 46, 49–63.
potent EGFR inhibitor in this chemical class. It is inter-
esting to note that compounds 32–35 that contain no
alkoxy groups at C-6 or C-7 positions showed reason-
able in vitro activity but there was a >50-times shift in
cellular activity, especially for those compounds with
C-6 substitutions (Table 1).
Effect of substituents on the phenyl ring of the phenylcy-
clopropylamine moiety on the activity was explored next
and is summarized in Table 2. 6,7-Dimethoxyquinoline-
3-carbonitrile was chosen as the core structure for explo-
ration due to its ease of synthesis. Substitutions at C-50
position (R5), except halogens, are less tolerated
especially when the C-40 position was simultaneously
substituted (cf. 37, 38, and 41). Halogen atoms fluorine,
chlorine, and bromine are well tolerated in all three
positions (36, 40, 42, and 43).
5. Meyer, O. G. J.; Frohlich, R.; Haufe, G. Synthesis 2000, 10,
1479–1490.
6. The protein tyrosine kinase activity of EGFR was moni-
tored by measuring the initial velocity of turnover of a
peptide substrate pp60v-src (Biosource). Fluorescence polar-
ization (FP), in which the size and shape of a fluorescent
molecule is determined by the ratio of fluorescence intensity
from parallel and polarized light (anisotropy), was used to
evaluate the phosphorylation of pp60v-src (phospho-pp60v-src
)
(Turek et al., (2001) Analytical Biochemistry 299, 45–53).
The anisotropic change was recorded continuously for
20 minutes to monitor the progression of an EGFR
reaction. Since the anisotropy change does not linearly
correspond to the concentration of the phosphorylated
pp60v-src (phospho-pp60v-src), an authentic reaction product
peptide, phospho-pp60v-src (Biosource), was used to convert
the time-courses of anisotropy changes to the time-courses
of EGFR reactions. The initial velocities of EGFR
reactions were obtained by linear regressional fitting of
the converted reaction time-courses. All the assays were
performed in 96-well microtiter plates (LJL, HE, black 96-
well plates) using recombinant human EGFR (Invitrogen).
In a typical 96-well plate assay, the decrease in the
anisotropy (kex = 485 nm, kem = 530 nm) (increase in the
concentration of phospho-pp60v-src) of a 12 lL assay
solution in each well was monitored continuously using
an Analyst HT or GT multi-well plate reader (Molecular
Devices). Each 12 lL assay solution contained 10 mM
HepesÆNa+ (pH 7.5), 2.5 mM MgCl2, 1 mM MnCl2,
0.5 mM DTT, 1 nM Invitrogen EGFR, 1X antibody/tracer
detection solution (Panvera/Invitrogen), 10 lM ATP, 5 lM
pp60v-src peptide, 1% DMSO, and 0.0098–10 lM testing
compound. Assays were initiated with the addition of the
ATP substrate. Data were fitted to the Dixon competitive
inhibition equation using Grafit 5.0 (Erithacus Software).
7. Cell-based EGFR autophosphorylation (Y1068) was per-
formed with A431 cells in a 96-well format. Briefly, A431 cells
(60,000 per well) were incubated at 37 °C overnight in 200 lL
of Dulbecco’s Modified Eagle’s Medium (DMEM, GIB-
COBRL, Cat#11995-073) containing 10% FBS. After
removal of the medium, EGFR inhibitor in 100 lL DMEM
was added and incubated for 60 min at 37 °C. Human EGF
(40 ng/well) in DMEM was added and incubated for addi-
tional 20 min. Routine wash and 60 lL PIPA-2 buffer were
added to lyse the cells. After centrifugation, the supernatant
of the cell lysates was detected with ELISA kit for pY1068
according to the manufacturer’s instruction (Biosource).
In conclusion, a series of 4-(2-phenylcyclopropylamino)-
quinoline-3-carbonitriles were prepared and their activity
as EGFR inhibitors was determined. Alkoxy substitu-
ents at both C-6 and C-7 positions enhanced the activity.
Compounds with basic amine-containing groups at C-7
position showed single digit nM potency in the cellular
phosphorylation assay. Most reported EGFR inhibitors
with quinazoline or quinoline-3-carbonitrile core struc-
tures contain a substituted aniline moiety at the C-4
position. We have shown that a phenylcyclopropyl-
amine moiety could be an excellent surrogate for the
aniline moiety, leading to the identification of current
compound series. All these compounds showed moder-
ate to excellent selectivity over other kinases listed in
Table 3.
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