Synthesis and biological evaluation of quinazoline and quinoline bearing 2,2,6,6-tetramethylpiperidine-N-oxyl as potential epidermal growth factor receptor(EGFR) tyrosine kinase inhibitors and EPR bio-probe agents

Article abstract of DOI:10.1016/j.ejmech.2012.01.021

4-anilinoquinazoline and 4-anilinoquinoline scaffolds bearing a 2,2,6,6-tetramethylpiperidine-N-oxyl(TEMPO) have been synthesized and evaluated for their ability to inhibit EGFR tyrosine kinase and A431 cell lines. Compared to their corresponding parent c

Full text of DOI:10.1016/j.ejmech.2012.01.021

European Journal of Medicinal Chemistry 49 (2012) 271e278
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of quinazoline and quinoline bearing
2,2,6,6-tetramethylpiperidine-N-oxyl as potential epidermal growth factor
receptor(EGFR) tyrosine kinase inhibitors and EPR bio-probe agents
,b,
Siyuan Li a, Chunying Guo a, Xianqiang Sun c, Yaozong Li c, Hongli Zhao a, Dongmei Zhan a, Minbo Lan a
,
*
,
Yun Tang c
**
a Shanghai Key Laboratory of Functional Materials Chemistry, and Research Centre of Analysis and Test, East China University of Science and Technology, Meilong Road 130,
Shanghai 200237, PR China
b State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China
c Department of Pharmaceutical Science, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
4-anilinoquinazoline and 4-anilinoquinoline scaffolds bearing a 2,2,6,6-tetramethylpiperidine-N-oxy-
l(TEMPO) have been synthesized and evaluated for their ability to inhibit EGFR tyrosine kinase and A431
cell lines. Compared to their corresponding parent compounds, all of the new compounds bearing
a TEMPO showed more efficient inhibition for EGFR and A431 cells. Furthermore, we have proved that
these molecules bearing a TEMPO can exactly get into A431 cells exerting inhibitory effect that may be
used for EPR detecting. In our docking model, quinazolines bearing a TEMPO on either 6- or 3-positions
took different linking modes according to EGFR crystal structure. In contrast to their parent compounds,
these new TEMPO-derived analogues possessed compatible inhibitory effect that might be useful as
potential EGFR inhibitors and as EPR bio-probes.
Received 8 September 2011
Received in revised form
8 January 2012
Accepted 10 January 2012
Available online 24 January 2012
Keywords:
EGFR
Inhibitor
Bio-probe
Free radical
Quinazoline
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
The human EGFR is a transmembrane glycoprotein, which
consists of a single polypeptide chain of 1186 amino acids [3,4]. It
Cancer and carcinogenesis are complex pathology and
multifactor processes that require the accumulation of several
biochemical alterations in a single cell. All complex diseases are
most likely ascribed to multifactorial effect, and these factors may
interact with each other, hence inducing interdependent biological
changes in a single cell over a period of time. The interaction of the
multifactorial effects used to aggravate worsening of cancer. To
solve the extraordinary complexity of cancer and provide excellent
therapeutic drugs, several researchers have made their R&D efforts
to discover new multifunctional drugs which are able to interfere
with multiple altered pathways [1,2].
is also a receptor tyrosine kinase (RTK) that is catalytically active
and under tight regulatory control. This receptor belongs to the
ErbB/HER family of ligand-activated RTKs, which includes four
structurally similar members EGFR/HER1, ErbB2/HER2, ErbB3/
HER3 and ErbB4/HER4 [5]. EGFR and its family members play
crucial roles in regulating a number of cellular processes including
cell proliferation, survival and migration [6] and dysregulation
of their activity is, therefore, strongly associated with malignancy
of numerous types of human tumors [7]. The involvement of
increased and/or aberrant EGFR activity in human cancers is well
investigated [8,9], while cancer patients with altered EGFR
activity tend to have a more aggressive disease, associated with
a poor prognosis [5]. Considering EGFR is a rational target for anti-
tumor strategies, one of the most important methods to inhibit
EGFR is to block tyrosine kinase at ATP-binding site in cytoplasmic
domain by small-molecule inhibitors. The representative frame of
inhibitor is 4-anilinoquinazolines, such as gefitinib (ZD-1839;
Iressa) and lapatinib (GW2016; Tykerb) approved in the U.S. by
the FDA (Fig. 1).
* Corresponding author. Shanghai Key Laboratory of Functional Materials
Chemistry, and Research Centre of Analysis and Test, East China University of
Science and Technology, Meilong Road 130, Shanghai 200237, PR China.
Tel.: þ86 21 64253574; fax: þ86 21 64252947.
** Corresponding author. Tel.: þ86 21 64251052; fax: þ86 21 64253651.
E-mail addresses: minbolan@ecust.edu.cn (M. Lan), ytang234@ecust.edu.cn
(Y. Tang).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.01.021

272
S. Li et al. / European Journal of Medicinal Chemistry 49 (2012) 271e278
F
O
F
Cl
O
Cl
N
HN
N
HN
N
H
O
N
N
S
N
O
O
O
O
Lapatinib , GW2016
Gefitinib , ZD-1839
Fig. 1. Structure of Gefitinib and Lapatinib.
However, just inhibiting EGFR is not enough to eradicate tumors,
were synthesized by condensing with 1aec and 4-carboxy-
2,2,6,6-tetramethylpiperidine-N-oxyl (4-COOH-TEMPO). Similarly,
compounds 4aec were obtained by condensing with 2aec
and 4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl (4-NH2-TEMPO)
using EDC and HOBt in different solvents. (Scheme 1).
that is to say mono-therapeutic molecule is not sufficient for
ameliorating symptom. The limited anti-tumor efficacy and tools
make researchers try their utmost to find other better methods to
treat cancer. For example, based on multifunctional concept or
multitarget-directed drug design strategy, some researchers have
designed and synthesized several molecules which would not only
target EGFR but also intervene with alteration of cell function,
which have achieved good curative effect [10e13].
3. Results and discussion
3.1. EGFR kinase inhibitory assay
Nowadays, with the development of cancer detection tech-
niques, electron paramagnetic resonance (EPR) spin labeling using
paramagnetic molecules such as nitroxide free radicals have
gained conspicuous attention. Therefore, we get interested in
nitroxide compound which has been found to be an available agent
and its application has achieved great progress. First of all, as we
know, nitroxides have been studied as a treatment for existing
tumors [14e17]. Secondly, nitroxides are able to act as an EPR
functional bio-probe reagent [18e20]. Especially, EPR spin
labeling techniques provide a convenient method to detect can-
cers on-line. Therefore we believe that EGFR inhibitors such as
EGFR kinase inhibitory activity of 1aec, 2aec, 3aec and 4aec
was evaluated by homogeneous time-resolved fluorescence
(HTRF) KinEASE-TK assay from Cisbio according to manufacture’s
instruction. Gefitinib was chosen as a standard compound in
kinase assay and its IC50 value was 7.3 nM. As showed in Table 1,
series 1 and 3 showed the highest inhibitory activities among
these compounds, and their IC50 values were 189 nM, 53 nM and
29 nM for 1a, 1b and 1c while 65 nM, 31 nM and 15 nM for 3a, 3b
and 3c, respectively. Compounds 4aec showed the moderate
inhibitory activities and 2aec displayed the lowest inhibitory
activities. The IC50 values are different among 3aec, because
halogen is different in 4-aniline. Compound 3c is the most potent
one due to the strongest electronegativity of fluorine in 4-aniline.
4-anilinoquinazoline or 4-anilinoquinoline bearing
a nitroxyl
group are favorable to improve anticancer potency, and nitroxide
compounds may be useful as a bio-probe for EPR spin labeling of
EGFR overexpressing cancers.
For 2aec, IC50 values are close to 10
mM, possibly because the
For the present report, we choose 6-amino-4-anilinoquinazoline
and 3-carboxyl-4-anilinoquinoline, which possess similar skeleton
with Gefitinib, as our compound models. The 6-amine and
3-carboxyl are active groups which could be conjunct with other
small molecular chains. It means the two compound models are
efficient lead compounds for our purpose: enable certain anticancer
effect of EPR bio-probe agent (Fig. 2).
In this paper, a series of compounds were designed and
synthesized to show inhibitory activities against EGFR and A431
cells. These compounds were further confirmed to enter cells,
which were possible to be used as EPR bio-probe agents.
carboxyl in 3-position improves the hydrophilicity of whole
molecular that decreases the hydrophobic interaction between
ligand and EGFR protein. Furthermore, in our docking model, the
docking poses of compounds 4aec were different from 2aec. A
carboxyl was substituted on 3-position of quinoline in compounds
2aec which were less potent than series 4 compounds. The most
possible reason is that electrostatic repulsion may happen
between the carboxyl and Asp855. This can be proved in our
docking study, and the nearest distance between the two carboxyls
is only 3.72 Å.
In the kinase assay, compounds 3aec and 4aec, the TEMPO-
derived analogues showed the enhanced potency to EGFR in
contrast to their corresponding parent compounds. The inhibitory
activity of compounds 3aec is much higher than 4aec, and this
indirectly proved the binding mode of 3aec is much more
reasonable than 4aec in our docking experiment.
2. Chemistry
Compounds 1aec and 2aec were prepared according to the
method of Tsou et al. [21] and Wissner et al. [22]. Compounds 3aec
3.2. In vitro cytotoxicity assay
The antiproliferative activities of 3aec and 4aec were eval-
uated with human epidermoid carcinoma cells (A431)
comparing with 1aec, 2aec and TEMPO. The A431 cell lines
were known to overexpress EGFR, which was related to
abnormal activity of EGFR pathway in many carcinogenesis.
Generally, the activity of inhibiting A431 cell proliferation was
determined after 72 h of treatment with various concentrations
R
R
HN
N
HN
N
H2N
MeO
MeO
COOH
N
Fig. 2. Structure of 6-amino-4-anilinoquinazoline and 3-carboxyl-4-anilinoquinoline.
(0.1e100
mM) of the tested compounds. The cell proliferation

S. Li et al. / European Journal of Medicinal Chemistry 49 (2012) 271e278
273
R
HN
N
R
HN
N
H
H2N
O N
N
N
N
O
4-COOH-TEMPO
EDC , HOBt
1a: R = 3-Cl
3a: R = 3-Cl
3b: R = 3-Br
1b: R = 3-Br
1c: R = 3-Cl,4-F
3c: R = 3-Cl,4-F
R
R
HN
N
HN
N
O
MeO
MeO
COOH
MeO
MeO
N O
N
H
4-NH2-TEMPO
EDC , HOBt
4a: R = 3-Cl
2a: R = 3-Cl
2b: R = 3-Br
4b: R = 3-Br
4c: R = 3-Cl,4-F
2c: R = 3-Cl,4-F
Scheme 1. Syntheses of 3aec and 4aec.
was measured by MTT assay, and the results were expressed as
IC50 values (Table 2).
As showed in Table 2, treatment of A431 cells with compounds
1aec and 2aec made the cell proliferation decrease with IC50
several cancer cell lines, but it was ineffective against A431 cell
lines at our tested concentration. It seems reasonable, because
TEMPO is not an EGFR inhibitor. In the assay, antiproliferative
activities of some compounds were inconsistent with their kinase
assay data. Especially, compounds 1aec and 3aec showed anti-
proliferative potency at micromole order of magnitude even
though they had reasonable levels of target potency at the enzyme
level. Possibly, these compounds do not possess enough cell
permeable capacity.
values being 31.94 M and 26.54
m
M, 34.89
m
mM for 1aec and
97.85 M, 93.99 M and 88.83 mM for 2aec. For 2aec, the IC50
m
m
values are higher than 1aec possibly because compound 2aec bear
a carboxyl at 3-position on quinoline ring which increases their
water solubility dramatically and decreases cell permeable
capacity. As expected, compounds 3aec and 4aec bearing a TEMPO
appear more potent than their corresponding parent compounds
against A431 cells. As reported in Table 2, IC50 values of compounds
To further illustrate the effect of free radical on cell anti-
proliferation, we also carried out the control experiment by
combining compounds 1 and 2 series and TEMPO on A431 cells. As
3a, 3b, 3c decrease 5.09
1b, 1c, respectively. Extremely, IC50 values of compounds 4a, 4b and
4c decrease 52.39 M, 50.12 M and 50.69 M compared with
compounds 2a, 2b and 2c, respectively. A TEMPO on 3-position in
quinoline ring, other than a carboxyl, might help to improve
hydrophobicity to enter cells for 4aec. In parallel, we also tested
TEMPO for inhibiting A431 cells, and the IC50 value was higher than
mM, 7.51 mM and 1.84 mM in contrast to 1a,
Table 2
m
m
m
In vitro antiproliferation of compounds 1aec, 2aec, 3aec,
4aec and TEMPO in A431 cells.a
Compd
IC50 (m
M)b, A431
1a
1b
1c
2a
2b
2c
3a
3b
3c
4a
4b
4c
31.94 ꢀ 0.29
34.89 ꢀ 1.64
26.54 ꢀ 0.92d
97.85 ꢀ 0.60
93.99 ꢀ 1.81
88.83 ꢀ 1.78
26.85 ꢀ 0.38
27.38 ꢀ 1.44
24.70 ꢀ 1.02
45.46 ꢀ 2.01
43.21 ꢀ 3.41
38.14 ꢀ 2.47
＞300c
300 mM. To some extent, TEMPO showed anticancer activities on
Table 1
EGFR kinase inhibitory activities of 1aec, 2aec, 3aec, 4aec.
Compd
aInhibitory activity at 10
m
M
aIC50 (nM)
1a
1b
1c
2a
2b
2c
3a
3b
98.9 ꢀ 0.5
99.3 ꢀ 0.1
99.5 ꢀ 0.2
63.4 ꢀ 2.1
60.1 ꢀ 1.3
66.5 ꢀ 0.7
99.4 ꢀ 0.2
99.4 ꢀ 0.4
99.5 ꢀ 0.3
88.3 ꢀ 0.4
90.3 ꢀ 1.2
92.8 ꢀ 0.8
99.6 ꢀ 0.2
189 ꢀ 18
53 ꢀ 8
29 ꢀ 4
9021 ꢀ 124
8719 ꢀ 38
9284 ꢀ 231
65 ꢀ 6
Tempo
a
The values are the mean ꢀ SD of at least three inde-
pendent experiments.
b
Concentration of compound resulting in 50% inhibi-
31 ꢀ 2
tion of A431 cell proliferation. The cell proliferation in
A431 cells was determined by the MTT assay, after 72 h of
3c
4a
4b
15 ꢀ 6
623 ꢀ 79
342 ꢀ 27
422 ꢀ 18
7.3 ꢀ 0.2
incubation with compounds (0.1e100 mM).
c
IC50 was not determined because less than 50%
inhibition was observed at the highest tested concen-
4c
Gefitinibb
tration (300
m
M).
Reported activity in reference.IC50 25e30
A431 [24].
a
d
The values are the mean ꢀ SD of at least two independent experiments.
IC50 value of Gefitinib is 3.1 nM determined by Klutchko et al. [23].
mM against
b

274
S. Li et al. / European Journal of Medicinal Chemistry 49 (2012) 271e278
Table 3
3.4. Molecular docking
IC50
(mM) of combination of compounds 1/2 and TEMPO on A431 cell
antiproliferation.
Many EGFR crystal structures have been reported from previous
Compd
TEMPO (1 eq.)
TEMPO (2 eq.)
TEMPO (5 eq.)
studies [26,27]. Among all the structures, 1XKK was selected for our
docking investigation because of its high resolution and the high
similarity of our compounds and lapatinib in the crystal structure.
Comparing the 4-anilinoquinazoline with the corresponding frag-
ment of lapatinib in the binding pocket, all of the ligands took the
rational binding pose in our study. Furthermore, the ring-Oꢁ1 state
possesses more similar electrostatics with the radical state than the
ring-OH state, thus it is reasonable to use the ring-Oꢁ1 state as
a substitute.
Compound 3c binds in the ATP-binding cleft in a fashion
similar to what observed in other kinase-quinazoline crystal
structure (Fig.4C) [26,27]. A hydrogen bond forms between the N1
of the quinazoline and the main chain NH of Met793, and the
distance is 2.31 Å while the angle value is 173ꢂ. The 3-chloro-
4-fluorophenylamino was deeply in the back of the ATP-binding
site and makes predominantly hydrophobic interactions with
the protein. Increasing this hydrophobic interaction might
increase the activity slightly. That is the possible reason why 3c
was more potent towards EGFR than its corresponding ligands in
series a and b in our kinase assay. The N in tetramethylpiperidine
in 3c formed a hydrogen bond with carboxyl in Asp800. The
distance and the angle of this hydrogen bond were 1.84Å and
152ꢂ. Thus, we supposed that positive charged in this region
might increase the activity. Compound 1c took almost the same
binding mode with compound 3c except for the tetramethylpi-
peridine regions. The 1-N of quinazoline is located 1.98Å from the
backbone NH of Met793, and the angle of this hydrogen bond is
164ꢂ. Detailed binding mode of compound 1c to EGFR can be seen
in Fig.4A.
Compound 4c was another TEMPO bearing ligand with a certain
activity in EGFR kinase assay. The 3-chloro-4-fluorophenylamino in
compound 4c occupied the same position as compound 3c (Fig.4D).
However, the quinazoline ring was binding differently. A hydrogen
bond was formed between the N in 4-aniline and the Asp855
carboxyl. The oxygen in methoxyl of dimethoxyquinoline occupied
the N1 position of the quinazoline in compound 3c, and this oxygen
in methoxyl formed a hydrogen bond with the main chain NH of
Met793. An Asp855 and Met793 across binding pose formed.
However, compound 2c was binding in a different binding mode
compared with the back bone of compound 4c (Fig.4B). The
dimethoxyquinoline took a 180ꢂ spin and the hydrogen bond with
Met793 was broken by this rotation.
1a
1b
1c
2a
2b
2c
32.54 ꢀ 0.78
34.43 ꢀ 1.20
28.51 ꢀ 0.48
96.65 ꢀ 0.40
92.21 ꢀ 1.21
87.55 ꢀ 1.54
31.55 ꢀ 1.02
33.21 ꢀ 0.98
24.74 ꢀ 1.21
98.52 ꢀ 0.91
91.90 ꢀ 1.93
88.11 ꢀ 2.91
32.33 ꢀ 0.34
33.45 ꢀ 1.42
26.12 ꢀ 1.27
97.40 ꢀ 0.33
92.29 ꢀ 0.17
87.23 ꢀ 3.32
shown in Table 3, the potency was altered not too much even
though the ratio (TEMPO/1 or 2) was up to 5. It clearly indicates
combination TEMPO with inhibitor does not exhibit synergistic
effect on antiproliferation of A431 cell lines. Conjugation inhibitor
with TEMPO, only in this way, could display the more favorable
antiproliferation on A431 cells as the data shown in Table 2 and
Table 3. Although intracellular TEMPO does not attack a specific
molecular target, it may alter the entire physicochemical characters
of the molecule. Hydrophobicity, for example, is helpful in entering
into cells to some extent for 4aec.
3.3. EPR detecting
Free radical like TEMPO and other small molecules can readily
enter cells [25], but it is not clear that whether a molecule bearing
a TEMPO such as 3c and 4c can enter cells. Considering compounds
3aec and 4aec are likely to be a bio-probe for cancer which needs
to enter cancer cells, we tested 3c and 4c entering A431 cells using
EPR techniques. As a result, EPR signals of intracellular 3c and 4c
could be detected, which proved these compounds can get into
cells (Fig. 3).
As shown in Fig. 3, EPR signals of 3c and 4c in DMSO were
smoothly triple peaks. Treating A431 cells with 3c and 4c, removing
3c and 4c in the medium, 3c and 4c inside cells can be detected
with EPR spectroscopy which displays a triple peak (3c-cell and
4c-cell). Despite little higher noise in signals caused by cells in the
DMSO solution, it indicated that the 3c and 4c were able to enter
A431 cells. As we know the EGFR is characterized by three main
domains: an extracellular ligandbinding domain, a transmembrane
domain and a cytoplasmic domain containing the tyrosine kinase
[5,25]. EGFR inhibitors must get into cytoplasmic domain to bind
with tyrosine kinase. In MTT assays, 3c and 4c achieved favorable
antiproliferation against A431 cell lines compared with 1aec and
2aec, which also proved 3c and 4c can enter cells. Considering
kinase inhibitory activity of 3aec (IC50 ¼ 65, 31, 15 nM) is compa-
rable to 1aec (IC50 ¼ 189, 53, 29 nM), compounds 3aec are more
favorable to be candidates of EGFR inhibitor and bio-probes for EPR
spin labeling of EGFR overexpressing cancers.
Met793 was proved to be an important residue in ligand binding
[28]. Both compounds 3c and 4c hydrogen bonded to this residue
and showed favorable biological activity for EGFR and A431 cell
lines, and 3aec might be used as an inhibitor with bio-probe
function. Our theoretical results were in conformity with the
4c-cel
l
3c-cell
4c
3c
Fig. 3. The EPR signals of 3c and 4c in DMSO and cells, respectively.a

S. Li et al. / European Journal of Medicinal Chemistry 49 (2012) 271e278
275
Fig. 4. Compound 1c, 2c, 3c and 4c binding to the ATP-binding pocket of EGFR. The carbon in EGFR was colored in green, ligand carbons were colored in pink. The important
residues in EGFR and all the ligands were represented with the sticks style. Hydrogen bonds were represented with yellow discontinuous line. The left top sign A, B, C and D
corresponding to compounds 1c, 2c, 3c and 4c, respectively. All graphical pictures were made using the Pymol program. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
cellular and the molecular experimental data. The model will be
helpful for our further structural elaboration of the novel TEMPO
beard EGFR inhibitors.
EPR spin labeling of EGFR overexpressing cancers as a bio-probe
agent. Further work will be focused on improving cell membrane
permeability of these TEMPO-derived analogues.
4. Conclusion
5. Experimental
In this paper, we synthesized a series of 4-anilinoquinazoline
and 4-anilinoquinoline derivatives bearing a TEMPO. We also
evaluated these compounds and their corresponding parent
compounds in vitro with EGFR tyrosine kinase and A431 cell lines.
All of the compounds bearing TEMPO showed more favorable effect
than their parent compounds in EGFR kinase assay and anti-
proliferative assay. Among of them, series 3 showed the higher
potency to EGFR kinase. Furthermore, we have proved that inhib-
itor bearing a TEMPO can enter cells to exert inhibitory effect. It is
concluded that inhibitor bearing TEMPO can increase anti-
proliferative potency against A431 cells, such as series 3 which are
more favorable to be EGFR inhibitors and can possibly be used for
5.1. Chemistry
Melting points were determined in open capillary tubes on an
SGW X-4 micro melting point apparatus and were uncorrected.
Electron impact and high-resolution mass spectra were obtained
on a Micromass GCT spectrometer. Infrared spectra were recor-
ded on a Nicolet FT-IR series using KBr cell. Conventional 1H
NMR spectra of nitroxide-containing molecules are quite broad
which are not used for identify the molecular structure during
a chemical synthesis [29], while 13C NMR spectra are sufficiently
legible and used by researchers [30]. 13C NMR spectra were
measured on Brucker 400 MHz in
d scale. The spectra were

276
S. Li et al. / European Journal of Medicinal Chemistry 49 (2012) 271e278
obtained with solutions of DMSO-d6 or CH3OH-d4 with TMS as
internal standard, and the values of the chemical shifts ( ) were
given in ppm. Elemental analyses were carried out at VARIO ELZ
5.1.2.2. 4-(3-Bromophenylamino)-6,7-dimethoxy-N-[(2,2,6,6-tetramethyl-
1-oxyl)piperidin-4-yl]quinoline-3-carboxamide (4b). Pink solid, 17%
yield. mp 138e140 ꢂC. IR (KBr) cmꢁ1 3362(NH), 3325 3062(NH,
acylamide), 2975(CH3), 2931(CH2), 1628(CO), 1365(NO). 13C NMR
d
apparatus.
(400 MHz, CH3OH-d4): d 155.2,151.2,148.8,147.6,146.4,146.0,131.9,
5.1.1. General procedures for the synthesis of 3aec
126.3, 123.7, 123.3, 119.0, 118.0, 108.1, 103.3, 56.8. HRMS for
C27H32N4O4Br, Calcd: 555.1607, Found: 555.1625. Anal. Calc. for
C27H32BrN4O4: C, 58.28; H, 5.80; N, 10.07. Found: C, 57.98; H, 5.51;
N, 9.89.
To a stirred solution of 1a (1.00 g, 3.70 mmol) and 4-carboxy-
2,2,6,6-tetramethylpiperidine-N-oxyl (4-COOH-TEMPO) (1.48 g,
7.40 mmol) in 150 mL dichloromethane at 0 ꢂC, tetrahydrofunan
solution 50 mL containing HOBt (1.00 g, 7.40 mmol) was added.
After being stirred at 0 ꢂC for 30 min, EDC (1.42 g, 7.40 mmol) was
added. Then the temperature of the reaction warmed to room
temperature naturally, and the reaction mixture was kept stirring
overnight before evaporating under reduced pressure. The residue
dissolved in 200 mL chloroform, was rinsed three times
with NaHCO3 saturated solution, when the organic layer dried
over anhydrous MgSO4, the solvent removed. The crude
material was purified by silica gel chromatography (eluent
CH3CO2C2H5:THF ¼ 5:2), and generated pure desired product.
5.1.2.3. 4-(3-Chloro-4-fluorophenylamino)-6,7-dimethoxy-N-[(2,2,6,6-
tetramethyl-1-oxyl)piperidin-4-yl]quinoline-3-carboxamide (4c). Pink
solid, 12% yield. mp 148e150 ꢂC. IR (KBr) cmꢁ1 3336(NH), 3066(NH,
acylamide), 2973(CH3), 2934(CH2), 1624(CO), 1368(NO). 13C NMR
(400 MHz, CH3OH-d4): d 155.2, 151.2, 148.8, 147.5, 146.7, 141.8, 123.0,
121.4,117.6,108.1,103.1, 56.8. HRMSfor C27H32N4O4FCl (M þ H), Calcd:
530.2096, Found: 530.2103. Anal. Calc. for C27H31ClFN4O4: C, 61.19; H,
5.90; N, 10.57. Found: C, 61.46; H, 6.11; N, 10.52.
5.2. Kinase assay
5.1.1.1. 2,2,6,6-Tetramethyl-1-oxyl-N-[4-(3-chlorophenylamino)qui-
nazolin-6-yl]piperidine-4-carboxamide (3a). Red solid, 23% yield.
mp 206e208 ꢂC. IR (KBr) cmꢁ1 3282(NH), 3199 3144 3087 3065
(NH, acylamide), 2977(CH3), 2926(CH2), 1694(CO), 1363(NO). 13C
The kinase inhibition assay and IC50 determinations for wild
type EGFR were measured with the homogeneous time-resolved
fluorescence (HTRF) KinEASE-TK assay from Cisbio according to
the manufacturer’s instructions. Wild type EGFR was purchased
NMR (400 MHz, DMSO-d6):
d 157.4, 154.2, 146.8, 141.2, 137.8, 133.0,
131.0, 129.3, 127.8, 124.0, 121.9, 120.9, 115.6, 112.2. HRMS for
C24H27N5O2Cl, Calcd: 452.1853, Found: 452.1855. Anal. Calc. for
C24H27ClN5O2: C, 63.64; H, 6.01; N, 15.46. Found: C, 63.52; H, 5.63;
N, 15.12.
from Carna Biosciences and 0.09 ng/
ATP concentration were set at its Km values (23
m
L kinase were used for test.
mM), and 180 nM
substrate were used. Kinase, substrate peptide and inhibitors were
added in 384 well plate, and then reaction was started by addition
of ATP. After completion of the reaction (50 min later), an anti-
phosphotyrosine antibody labeled with europium cryptate and
streptavidin labeled with the fluorophore XL665 were added. The
FRET between europium cryptate and XL665 was measured to
quantify the phosphorylation of the substrate peptide. A Tecan
i-control infinite 500 was used to measure the fluorescence of the
samples at 620 nm (Eu-labeled antibody) and 665 nm (XL665
5.1.1.2. 2,2,6,6-Tetramethyl-1-oxyl-N-[4-(3-bromophenylamino)qui-
nazolin-6- yl]piperidine-4-carboxamide (3b). Red solid, 30% yield.
mp 216e218 ꢂC. IR (KBr) cmꢁ1 3281(NH), 3241 3143 3085 3040(NH,
acylamide), 2974(CH3), 2928(CH2), 1693(CO), 1362(NO). 13C NMR
(400 MHz, DMSO-d6): d 157.3, 154.0, 146.8, 141.3, 137.8, 131.2, 129.1,
127.7, 126.7, 124.7, 121.8, 121.4, 115.6, 112.1. HRMS for C24H27N5O2Br,
Calcd: 496.1348, Found: 496.1349. Anal. Calc. for C24H27BrN5O2: C,
57.95; H, 5.47; N, 14.08. Found: C, 57.83; H, 5.51; N,13.87.
labeled streptavidin) 500
of both intensities for reactions made with ten different inhibitor
concentrations (0.01 nMe10 M, including no inhibitor) was
ms after excition at 320 nm. The quotient
m
5.1.1.3. 2,2,6,6-Tetramethyl-1-oxyl-N-[4-(3-chloro-4-fluorophenylamino)
quinazolin-6-yl]piperidine-4-carboxamide (3c). Red solid, 18%
yield. mp 200e202 ꢂC. IR (KBr) cmꢁ1 3531(NH), 3433 3331 3213
3108(NH, acylamide), 2978(CH3), 2935(CH2), 1661(CO), 1366(NO).
plotted against inhibitor concentrations to determine IC50 values.
Each reaction was performed in duplicate, and at least two inde-
pendent determinations of each IC50 were made.
13C NMR (400 MHz, DMSO-d6):
d
157.3, 153.9, 152.1, 146.6, 137.7,
5.3. Biology. In vitro evaluation assay
136.8, 129.1, 127.6, 124.1, 122.9, 119.0, 117.5, 115.4, 111.9. HRMS
for C24H27N5O2FCl (M þ H), Calcd: 473.1808, Found: 473.1825. Anal.
Calc. for C24H26ClFN5O2: C, 61.21; H, 5.56; N, 14.87. Found: C, 61.01;
H,5.89; N,14.56.
Human epidermoid carcinoma (A431) cells were routinely
grown at 37 ꢂC in a humidified incubator with 5% CO2 in DMEM
medium supplemented with 10% fetal bovine serum (FBS), 2 mM
glutamine, 50 U/mL penicillin and 50
mg/mL streptomycin. To
5.1.2. General procedures for the synthesis of 4aec
determine the cell proliferation, the A431 cells were seeded in 96-
well plates at 1 ꢃ 104 cells/well. All experiments were performed
after 24 h of incubation at 37 ꢂC in 5% CO2.
The synthesis of 4aec was performed using similar protocols for
the synthesis of 3aec except using appropriate 4-anilinoquinoline
and 4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl (4-NH2-TEMPO)
instead of appropriate 4-anilinoquinazoline and 4-carboxy-
2,2,6,6-tetramethylpiperidine-N-oxyl (4-COOH-TEMPO).
The cell proliferation was evaluated with the MTT(3-(4,5-
dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay,
as described by Mosmann [31]. Briefly, A431 cells were treated with
compounds (0.1e100 m
M) for 72 h at 37 ꢂC in 5% CO2. After that, the
5.1.2.1. 4-(3-Chlorophenylamino)-6,7-dimethoxy-N-[(2,2,6,6-tetramethyl-
1-oxyl)piperidin-4-yl]quinoline-3-carꢁbo1xamide (4a). Pink solid, 15%
yield. mp 139e141 ꢂC. IR (KBr) cm 3282(NH), 3065(NH, acyla-
mide), 2969(CH3), 2931(CH2), 1628(CO), 1365(NO). 13C NMR
cells were washed with phosphate buffered saline (PBS) and then
incubated with MTT (5 mg/mL) in DMEM medium for 4 h. After
removal of MTT and further washing, the formazan crystals were
dissolved with DMSO. The amount of formazan was measured
(492 nm, 630 nm) with a spectrophotometer.
(400 MHz, DMSO-d6): d 152.6, 148.9,148.1, 146.5, 145.2, 142.9, 133.3,
130.6, 120.9, 120.8, 117.9, 116.6, 116.3, 108.3, 102.3, 56.2, 56.1. HRMS
for C27H32N4O4Cl, Calcd: 511.2112, Found: 511.2122. Anal. Calc. for
C27H32ClN4O4: C, 63.34; H, 6.30; N, 10.94. Found: C, 63.01; H, 6.49;
N, 11.12.
The cell viability was expressed as percentage of control cells
and calculated by the formula Ft/Fnt ꢃ 100, where Ft ¼ absorbance
of treated cells and Fnt ¼ absorbance of untreated cells. At least
three independent doseeresponse curves were done and the

S. Li et al. / European Journal of Medicinal Chemistry 49 (2012) 271e278
277
concentration of compound resulting in 50% inhibition of cell
proliferation (IC50) was calculated.
and No. 09520703200) and the National Special Fund for State Key
Laboratory of Bioreactor Engineering (No. 2060204).
5.4. EPR detecting
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lines as cell model same as MTT assays. Concisely, A431 cells were
seeded in 6-well plates at 1 ꢃ 106 cells/well. After 24 h, the cells
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Acknowledgements
This work was supported by the Science and Technology
Commission of Shanghai Municipality (STCSM, No. 10dz2220500

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