Synthesis, Antitumor Evaluation and Docking Study of Novel 4-Anilinoquinazoline Derivatives as Potential Epidermal Growth Factor Receptor (EGFR) Inhibitors

Full text of DOI:10.1002/cmdc.201300120

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Strike a pose! A series of 4-anilinoqui-
nazolines were designed, synthesized
and evaluated in vitro against lung and
breast cancer cell lines. Several com-
pounds were found to be endowed
with cytotoxicity in the low micromolar
range. Molecular docking suggests that
these compounds bind to EGFR in a sim-
ilar manner to known EGFR inhibitors.
G.-W. Rao,* G.-J. Xu, J. Wang, X.-L. Jiang,
H.-B. Li
&& – &&
Synthesis, Antitumor Evaluation and
Docking Study of Novel 4-
Anilinoquinazoline Derivatives as
Potential Epidermal Growth Factor
Receptor (EGFR) Inhibitors
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DOI: 10.1002/cmdc.201300120
Synthesis, Antitumor Evaluation and Docking Study of
Novel 4-Anilinoquinazoline Derivatives as Potential
Epidermal Growth Factor Receptor (EGFR) Inhibitors
Guo-Wu Rao,*[a] Geng-Jie Xu,[a] Jian Wang,[b] Xu-Liang Jiang,[b] and Hai-Bo Li[c]
Protein kinases, in particular receptor tyrosine kinases, are con-
sidered to be the second largest class of therapeutic targets by
Gray et al.[1] The most extensively studied receptor tyrosine
kinase is the epidermal growth factor receptor (EGFR).[2] Aber-
rant expression or activation of EGFR homologues has been
connected with multiple human carcinomas, and drugs target-
ing ErbB activity have been licensed for treatment of lung,
colon, breast, and head-and-neck carcinomas.[3,4] These drugs
fall into two categories: monoclonal antibodies targeting ErbB
extracellular regions, and small-molecule reversible tyrosine
kinase inhibitors, such as erlotinib, gefitinib, and lapatinib.[4,5]
These drugs are all 4-anilinoquinazoline derivatives and are
among the most potent known tyrosine kinase inhibitors.[6–10]
Numerous studies have aimed to identify novel compounds
containing the 4-anilinoquinazoline core as small-molecule in-
hibitors of EGFR.[11–15]
EGFR inhibitors, we attempted to investigate whether modifi-
cations to the 4-anilinoquinazoline structure could enhance
the antitumor activities of this compound class. The co-crystal
structure of the EGFR kinase domain in complex with lapatinib
(PDB code 1XKK) shows that the N-1 of the quinazoline ring is
hydrogen bonded to the main chain NH of Met793,[16,17] and
this interaction is presumed to exist for our design of 4-anilino-
quinazoline-containing compounds. Additionally, the 4-(3-fluo-
robenzyloxy)-3-chlorobenzenamine moiety of lapatinib lies in
a deep hydrophobic pocket at the back of the ATP binding
site.[16] A hydrogen bond interaction between the CO and NH
of the amide group with the backbone of Lys745 or Thr854
could be established by introducing an amide group on the
phenyl ring of the 4-anilinoquinazoline; the hydrophobic
amide group would presumably be accommodated in the
deep hydrophobic pocket at the back of the ATP binding site.
Taking these potential interactions into consideration, it should
be possible to design compounds with strong affinities for
EGFR and potent antitumor activities.
A novel series of 4-anilinoquinazoline derivatives were syn-
thesized and evaluated for their antitumor activity. The syn-
thetic routes are shown in Scheme 1. Early intermediates 4-
chloroquinazoline (1) and 4-chloro-6-nitroquinazoline (2) were
prepared by chlorination of 4-hydroxyquinazoline and 4-hy-
droxy-6-nitroquinazoline, respectively, according to published
methods.[18,19] Key intermediates 4-(2-(substituted-amino)aceta-
mido)aniline and 3-chloro-4-(2-(substituted-amino)acetamido)-
aniline (3a–g) were synthesized from 4-nitrobenzenamine and
2-chloro-4-nitrobenzenamine, respectively, according to a pub-
lished method.[20] Target compounds 4a–k were synthesized
from compound 3 and 1 or 2. Intermediates 5a–d were ob-
tained by the reduction of compounds 4h–k. Target com-
pounds 4l–t were prepared via the acylation of 5a–d. The full
structures of the final compounds and the yields are given in
Table 1.
In order to search for new promising antitumor agents that
act through a mode of action similar to quinazoline-containing
[a] Dr. G.-W. Rao, G.-J. Xu
The antitumor activities of these compounds were evaluated
in vitro against the growth of human lung adenocarcinoma ep-
ithelial (A-549) and breast cancer (MCF-7) cell lines with cispla-
tin as a positive control (Table 2). The results show that several
compounds are highly effective against these two cancer cell
lines. Literature reports indicate IC50 values for erlotinib, gefiti-
nib, and lapatinib against A-549 cells to be 24.1, 11.8, and
2.80 mm, respectively;[21–23] in comparison, the IC50 values deter-
mined for compounds 4l and 4m against the same cell line
were determined to be 16.9 and 5.38 mm, respectively. IC50
values for erlotinib, gefitinib, and lapatinib against the MCF-7
cell line are reported in the literature to be 10.2, 21.6, and
College of Pharmaceutical Science
Zhejiang University of Technology
Hangzhou 310014 (P.R. China)
E-mail: rgw@zjut.edu.cn
[b] Dr. J. Wang, Dr. X.-L. Jiang
School of Pharmaceutical Engineering
Shenyang Pharmaceutical University
Shenyang 110016 (P.R. China)
[c] Dr. H.-B. Li
Laboratory of Microbiology
Nantong Centers for Disease Control and Prevention
Jiangsu 226007 (P.R. China)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201300120.
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performed using Surflex-Dock in
the Sybyl X software package.[26]
To validate our docking proto-
col, lapatinib was removed from
the PDB structure and redocked.
In agreement with the published
co-crystal structure of lapatinib
bound to the kinase domain of
EGFR,[16] the docking result illus-
trates that lapatinib binds to the
ATP binding cleft, with the qui-
nazoline ring binding to the
narrow hydrophobic pocket at
the N terminus, the N-1 of the
quinazoline ring forming a hydro-
gen bond to the main chain NH
of Met793, and the 4-(3-fluoro-
benzyloxy)-3-chlorobenzenamine
lying in
a deep hydrophobic
pocket at the back of the ATP
binding site (Figure 1a).
Compounds 4l, 4m, and 4r–t
were submitted for docking
evaluation as representative ex-
amples of active compounds
(Figure 1). The docking studies
suggest that the quinazoline
ring binds to a narrow hydro-
phobic pocket in the EGFR N-ter-
minal domain, where the N-1 of
the quinazoline ring interacts
with the backbone NH of
Met793 via a hydrogen bond,
and similarly, that the large sub-
stituted anilino group allows for
Scheme 1. Reagents and conditions: a) DMF, reflux, 12 h; b) toluene, Et3N, POCl3, reflux, 4 h; c) concd HNO3/H2SO4,
12 h; d) R2 =H, THF, 0–58C then RT, 20 h, or R2 =Cl, CH2Cl2, 0–58C then RT, 20 h; e) R2 =H, CH3OH, 508C, 20 h, or
R2 =Cl, CH2Cl2, reflux, 20 h; f) R2 =H, CH3OH, HCOONH4, 5% Pd, 508C, 4 h, or R2 =Cl, CH3CH2OH, HCl, H2O, Fe
powder, reflux, 4 h; g) CH3CH(CH3)OH, Et3N, reflux, 8 h; h) CHCl3, 4-dimethylaminopyridine, 0–58C then RT, 20 h.
Yields are given in Table 1.
a
deeper
interaction
and
a better fit in the hydrophobic
pocket at the back of the ATP
binding site.[16,27]
6.60 mm, respectively,[6,21,24] and the IC50 values for compound
4l, 4m, 4r, 4s, and 4t against MCF-7 cells were determined to
be in a comparable range (7.83, 4.18, 9.38, 9.77, and 7.45 mm,
respectively). Compound 4m displayed similar cytotoxic activi-
ties against the both cancer cell lines. In contrast, compounds
4l–t, which contain nucleophilic substituents at the 6-position
of the anilinoquinazoline scaffold, showed moderate to excel-
lent in vitro antitumor activity and potential selectivity for
breast over lung cancer, indicating that this type of substitu-
tion is beneficial for activity. However, compounds containing
electrophilic substituents at the 6-position of the anilinoquina-
zoline core (4h–k) exhibit decreased activity, suggesting that
electrophilic substituents at the 6-position are unfavorable.[25]
A docking analysis was carried out in an attempt to rational-
ize the observed biological results and to predict the potential
binding modes of active compounds with their putative intra-
cellular target, EGFR. Docking of the inhibitors into the crystal
structure of the EGFR kinase domain (PDB code 1XKK)[16,17] was
Docking poses for compounds 4l and 4m predicted the for-
mation of only one hydrogen-bonding interaction between
the N-1 of the quinazoline ring and Met793 (2.10 and 2.01 ꢁ,
respectively), and the model also suggests that the 4-(2-(dieth-
yl amino)acetamido)anilino group of both 4l and 4m would
probably also lie in a deep hydrophobic pocket at the back of
the ATP binding site, in a manner similar to that of lapatinib
(Figure 1b,c).
The docking poses generated predict hydrogen-bonding in-
teractions between the CO groups at the 6-position of 4r,s,t
and Cys797 (2.16, 2.07 and 2.03 ꢁ, respectively) (Figure 1d–f).
Furthermore, interactions were also predicted to form between
the CO and NH groups at the 4-position of 4s,t and Lys745
(2.67 and 2.62 ꢁ, respectively) and Thr854 (2.55 and 2.57 ꢁ, re-
spectively) (Figure 1e,f). Both models for compound 4s and 4t
predicted the formation of a hydrogen bond with Met793
(2.05 and 2.08 ꢁ, respectively). Finally, the docked pose for
compound 4r indicates the formation of hydrogen bonds with
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Table 1. Isolated yields and melting point ranges for synthesized com-
pounds 4a–t.
Table 2. Antitumor activities of 4-anilinoquinazolines 4a–t against
a human lung adenocarcinoma epithelial (A-549) and a breast cancer
(MCF-7) cell line in vitro.[a]
Compd
IC50 [mm]
Compd
IC50 [mm]
A-549 MCF-7
A-549
MCF-7
4a
4b
4c
4d
4e
4 f
4g
4h
4i
>100
>100
>100
79.4
>100
68.6
>100
>100
>100
114
>100
>100
>100
175
55.4
43.0
>100
>100
>100
49.1
4m
4n
4o
4p
4q
4r
4s
4t
Cisplatin
Erlotinib[21]
Gefitinib[22,24]
Lapatinib[6,23]
5.38
4.18
94.6 77.5
44.0
>100
22.3
34.1
18.2
9.38
9.77
7.45
23.0
10.2
21.6
52.6
30.3
54.6
Compd R1
R2 NR3R4
Yield[a] [%] Mp[b] [8C]
>100
4a
4b
4c
4d
4e
4 f
4g
4h
4i
H
H
H
H
H
H
H
NO2
H
H
H
H
N(CH3)2
morpholino
m-CH3C6H4NH 61
N(CH2CH3)2
46
43
207–210
222–224
221–223
145–148
208–210
113–115
184–186
235–238
218–220
174–176
224–226
179–181
216–218
235–238
272–274
105–107
191–193
241–243
112–114
116–118
20.3
24.1
11.8
4j
4k
4l
89.4
16.9
36.8
7.83
66
76
2.80
6.60
Cl N(CH2CH3)2
H
N(CH2CH2CH3)2 89
[a] IC50 values were determined as described in the Experimental Section.
Cl N(CH2CH2CH3)2 91
H
N(CH2CH3)2
78
90
NO2
Cl N(CH2CH3)2
4j
NO2
H
N(CH2CH2CH3)2 89
lowed by compounds 4m, 4r, 4l, 4s and 4t (Table 3); these
compounds are calculated to have high C-scores, which is in
keeping with their observed potent antitumor activity in vitro.
In conclusion, a novel series of 4-anilinoquinazoline deriva-
tives has been synthesized and tested for their antitumor activ-
ities against A-549 and MCF-7 cell lines. The results showed
several compounds to be endowed with cytotoxicity in the
low micromolar range. Compounds 4l, 4m, 4r–t exhibited
potent antitumor activity with IC50 values in the range of 4.18–
9.77 mm against a human breast cancer (MCF-7) cell line. Mo-
lecular docking studies supported the postulation that the
active compounds bind to EGFR in the same manner as known
EGFR inhibitors.[27] Together, these results will facilitate and
guide the design of novel and more potent quinazoline deriva-
tives.
4k
4l
NO2
Cl N(CH2CH2CH3)2 83
(CH3)2CHCH2O
cyclohexylmethoxyl
(CH3)2CHCH2O
cyclohexylmethoxyl Cl N(CH2CH3)2
(CH3)2CHCH2O
(CH3)2CH
(CH3)2CHCH2O
CH3CH2
(CH3)2CH
H
H
N(CH2CH3)2
N(CH2CH3)2
40
46
65
71
4m
4n
4o
4p
4q
4r
Cl N(CH2CH3)2
H
H
N(CH2CH2CH3)2 63
N(CH2CH2CH3)2 60
Cl N(CH2CH2CH3)2 88
Cl N(CH2CH2CH3)2 73
Cl N(CH2CH2CH3)2 74
4s
4t
[a] Isolated yield of the final step, determined after purification by column
chromatography. [b] Melting point (mp) values were determined on a X-4
melting point apparatus and uncorrected.
Met793 and Thr854 (1.99 ꢁ and 2.40 ꢁ, respectively) (Fig-
ure 1d).
In the computational study, stronger binding energies were
calculated for compounds 4m and 4t than for compounds 4l,
4r, and 4s. Calculated Helmholtz free energies of interactions
for protein–ligand atom pairs rank 4r over lapatinib followed
by 4m and 4t. Charge and van der Waals interactions between
the protein and ligand suggest that 4s and 4t are superior li-
gands for EGFR than compounds
Experimental Section
Chemistry
Full details about the instruments and reagents used are given in
the Supporting Information along with characterisation data for all
4l, 4m, and 4r. Scoring of the
compounds with respect to the
Table 3. Surflex dock scores of compounds 4l, 4m, 4r–t, and reference agent lapatinib.
reward for hydrogen bonding,
lipophilic contact, and rotational
entropy, along with an intercept
terms revealed that compound
4s and 4t are calculated to have
more interactions with EGFR
than 4l, 4m, and 4r. The con-
sensus score (C-score) summariz-
es all of the calculated forces of
interaction between the ligand
and receptor (EGFR); both the C-
score and crash score predict la-
patinib to be the best ligand, fol-
Compd
C-score[a] Crash score[b] Polar score[c] G-score[d] PMF score[e] D-score[f] Chem score[g]
Lapatinib
4l
4m
4r
4s
4t
7.43
5.21
6.08
5.66
5.35
5.40
À0.89
À1.37
À0.94
À1.27
À1.84
À1.88
2.13
1.12
1.13
2.30
2.18
2.22
À237
À203
À224
À197
À199
À212
À10.4
À2.63
À14.7
À15.1
À9.17
À11.7
À699
À645
À675
À663
À687
À690
À43.8
À33.5
À35.7
À39.5
À40.3
À40.1
[a] Consensus score (C-score) reports the output of the total score. [b] Crash score reveals inappropriate pene-
tration into the binding site. [c] Polar region of the ligand. [d] G-score shows hydrogen bonding, complex
(ligand–protein), and internal (ligand–ligand) energies. [e] Potential of mean force (PMF) score indicates the
Helmholtz free energies of interactions for protein–ligand atom pairs. [f] D-score for charge and van der Waals
interactions between the protein and ligand. [g] Chem score points to hydrogen bonding, lipophilic contact,
and rotational entropy, along with an intercept term.
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Figure 1. The docking models of 4-anilinoquinazoline inhibitors a) lapatinib, b) 4m, c) 4l, d) 4r, e) 4s, and f) 4t with EGFR. Inhibitors are shown in stick model
colored by atom type. Hydrogen bonds are depicted as yellow dashed lines. The receptor is shown in thin stick and ribbon style (green).
intermediates and additional final compounds. Given below are
two representative protocols for the synthesis of 4 f and 4o. Com-
pounds 4a–k and 4l–t were synthesized in the same manner as
4 f and 4o, respectively. 4-Chloroquinazoline (1) was prepared
from 4-hydroxyquinazoline according to a literature procedure,[18,19]
and 4-(2-(dipropylamino)acetamido) aniline (3e) was prepared as
described by Moses et al.[20]
19.9, 56.5, 58.5, 115.1, 119.1, 122.9, 123.1, 126.1, 127.7, 132.8, 134.4,
134.5, 149.6, 154.5, 157.7, 169.4 ppm; IR (KBr): n˜ =3381, 3256, 2958,
1667, 1618, 1605, 682 cmÀ1; HRMS (ESI): m/z [M+H]+ calcd for
C22H28N5O: 378.2288, found: 378.2291; HPLC: tR =5.27 min (>95%).
4-(3-Chloro-4-(2-(diethylamino)acetamido)anilino)-6-(cyclohexyl-
methoxylformamido)quinazoline (4o): 4-(3-Chloro-4-(2-(diethyl-
amino)acetamido)anilino)-6-nitroquinazoline (4i, 0.80 g, 1.9 mmol)
was dissolved in anhydrous EtOH (30 mL) with stirring. Fe powder
(2.00 g, 35.7 mmol), concd HCl (0.5 mL) and H2O (2.0 mL) were
added to the mixture. The reaction was stirred at RT for 30 min,
then heated at reflux for 4 h and monitored to completion by TLC
(petroleum ether/EtOAC, 1:1). Upon completion, the reaction mix-
ture was filtered under suction. The filtrate was neutralized with sa-
turated aq Na2CO3 and then filtered again. The filtrate was concen-
trated in vacuo, and the residue was dissolved in EtOAc (30 mL)
and then extracted with 3% aq NH3 (30 mL). The organic phase
was dried over anhydrous MgSO4, filtered, and concentrated in
vacuo to give compound 5b as a yellow solid (0.58 g, 77.9%).
4-(4-(2-(Dipropylamino)acetamido)anilino)quinazoline (4 f):
A
mixture of 1 (0.59 g, 3.6 mmol), 3e (1.00 g, 4.0 mmol), and Et3N
(0.61 g, 6.0 mmol) was dissolved in isopropanol (20 mL), and the
mixture was heated at reflux for 8 h with stirring. The solvent was
removed in vacuo, and the residue was purified by column chro-
matography (petroleum ether/EtOAc, gradient elution, 8:1, 4:1, 2:1,
1:1, 0:1) to give 4 f as an off-white solid (1.20 g, 88.7%): mp: 113–
115 8C; 1H NMR (500 MHz, [D6]DMSO): d=0.89 (t, J=7.4 Hz, 6H),
1.44–1.52 (m, 4H), 2.49–2.52 (m, 4H), 3.18 (s, 2H), 7.61–7.66 (m,
3H), 7.77–7.79 (m, 3H), 7.84–7.87 (m, 1H), 8.54–8.56 (m, 2H), 9.59
(s, 1H), 9.79 ppm (s, 1H); 13C NMR (125 MHz, [D6]DMSO): d=11.7,
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Compound 5b (0.26 g, 0.65 mmol) and DMAP (0.15 g, 1.2 mmol)
were dissolved in CHCl3 (10 mL) with stirring. A solution of cyclo-
hexylmethyl chloroformate (0.70 g, 4.0 mmol) and CHCl3 (5 mL)
was added dropwise to the mixture at 0–58C. The mixture was
stirred at 0–58C for 20 min and then stirring was continued at RT
while monitoring by TLC (petroleum ether/EtOAc, 1:1). Upon com-
pletion, the reaction mixture was basified with 3% aq NH3 in an
ice bath. The water phase was extracted with CH2Cl2 (3ꢂ10 mL),
and the combined organic phase was washed with water and
dried over anhydrous MgSO4. The solvent was removed in vacuo,
and the yellow residue was further purified by column chromatog-
raphy (petroleum ether/EtOAc, gradient elution, 8:1, 4:1, 2:1, 1:1,
0:1) to give 4o as a light yellow solid (0.25 g, 71.1%): mp: 272–
The ProtoMol was generated using standard fully automated Sur-
flex-Dock procedures to characterize the surface properties of the
EGFR active site, including steric effects, hydrogen bond acceptor
groups, and hydrogen bond donor groups. During docking, ligands
were aligned to the ProtoMol based on surface shape, with each
pose bedding scored based on hydrophobic (HY) and polar con-
tacts between atoms. The reference ligand was redocked into the
binding pocket to reproduce the binding mode observed in the
crystal structure; afterwards, the molecules in the data set were
docked into the active site to investigate the binding modes and
affinities. Docked poses in the active site were visualized using
Sybyl X.[26]
1
2748C; H NMR (500 MHz, [D6]DMSO): d=1.04–1.05 (m, 2H), 1.07 (t,
J=7.1 Hz, 6H), 1.21–1.26 (m, 3H), 1.66–1.78 (m, 6H), 2.64 (q, J=
7.1 Hz, 4H), 3.20 (s, 2H), 3.98 (d, J=6.6 Hz, 2H), 7.74–7.77 (m, 3H),
8.15 (d, J=2.4 Hz, 1H), 8.30 (d, J=9.0 Hz, 1H), 8.54 (d, J=6.3 Hz,
2H), 9.86 (s, 1H), 9.95 (s, 1H), 10.01 ppm (s, 1H); 13C NMR
(125 MHz, [D6]DMSO): d=12.3, 25.1, 25.9, 29.1, 36.9, 48.1, 57.9,
69.3, 110.4, 115.5, 120.6, 121.5, 121.9, 122.4, 126.6, 128.4, 129.9,
136.0, 137.1, 146.1, 152.8, 153.9, 157.1, 169.6 ppm; IR (KBr): n˜ =
3428, 3287, 2970, 2927, 1726, 1681, 1633, 1609, 693 cmÀ1; HRMS
(ESI): m/z [M+H]+ calcd for C28H36ClN6O3: 539.2532, found:
539.2534; HPLC: tR =33.10 min (>99%).
Acknowledgements
The authors are very grateful for financial support from the Na-
tional Natural Science Foundation of China (grant no. 20802069),
the Natural Science Foundation of Zhejiang Province (P.R. China)
(grant no. Y2090985), and the Education Department of Liaoning
Province (P.R. China) (grant no. L2011174). The authors also
thank Tripos, Inc. (St. Louis, USA) for kindly providing free access
to their software package for evaluation.
Keywords: 4-anilinoquinazolines
epidermal growth factor receptor (EGFR) · molecular docking
·
antitumor agents
·
Biological evaluation
The cytotoxic activities of compounds 4a–t were determined
against A-549 and MCF-7 cell lines, obtained from the Shanghai In-
stitutes for Biological Science, Chinese Academy of Sciences, using
an MTT assay.[28] In brief, tumor cells were cultivated at 378C, 5%
CO2 in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supple-
mented with 100UmLÀ1 penicillin, 0.1mgmLÀ1 streptomycin, 10%
v/v fetal bovine serum for 3–5 d. Then, tumor cells were treated
with trypsin–EDTA solution and then seeded into 96-well plates at
5ꢂ103 cells/well and incubated in a 5% CO2 incubator at 378C for
24 h. The cells were treated with the synthesized compounds at
different concentrations in DMEM for 72 h. Mitochondrial metabo-
lism was measured as a marker for cell growth by adding 10 mL/
well MTT (5 mgmLÀ1 in medium, Sigma) with 3 h of incubation at
378C. Crystals formed were dissolved in 150 mL DMSO. The absorb-
ance was determined using a microplate reader at 490 nm. The ab-
sorbance data were converted into a cell proliferation percentage,
compared with DMSO-only treated control cells, to determine
growth inhibition. Each assay was performed in triplicate.
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Received: March 13, 2013
Published online on && &&, 0000
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