Novel Quinazoline Derivatives Bearing Various 4-Aniline Moieties as Potent EGFR Inhibitors with Enhanced Activity Against NSCLC Cell Lines

Article abstract of DOI:10.1111/cbdd.12692

A class of novel quinazoline derivatives bearing various C-4 aniline moieties was synthesized and biologically evaluated as potent epidermal growth factor receptor (EGFR) inhibitors for intervention of non-small-cell lung cancer (NSCLC). Most of these inhibitors are comparable to gefitinib in inhibiting these cancer cell lines, and several of them even displayed superior inhibitory activity. In particular, analogue 5b with an IC₅₀ of 0.10 μm against the EGFR wild-type A431 cells and 5c with an IC₅₀ of 0.001 μm against the gefitinib-sensitive HCC827 cells (EGFR del E746-A750) was identified as highly active EGFR inhibitors. It was also significant that the discovered analogue 2f, not only has high potency against the gefitinib-sensitive cells (IC₅₀ = 0.031 μm), but also possesses remarkably improved activity against the gefitinib-resistant cells. In addition, the enzymatic assays and the Western blot analysis for evaluating the effects of the typical inhibitors indicated that these molecules strongly interfere with the EGFR target.

Full text of DOI:10.1111/cbdd.12692

Chem Biol Drug Des 2016; 87: 635–643
Research Letter
Novel Quinazoline Derivatives Bearing Various
4-Aniline Moieties as Potent EGFR Inhibitors with
Enhanced Activity Against NSCLC Cell Lines
Changyan Wang1, Yajun Sun1, Xingqi Zhu1,
nib (7) were the first-generation EGFR inhibitors approved
for the treatment of NSCLC in 2002 and 2004, respec-
tively. By competitively binding to the adenosine triphos-
phate (ATP)-binding pocket of the intracellular EGFR TK
Bin Wu1, Qiao Wang1, Yuhong Zhen1,
Xiaohong Shu1, Kexin Liu1, Youwen Zhou2 and
Xiaodong Ma1,*
domain, these agents effectively block the aberrant EGFR
1College of Pharmacy, Dalian Medical University, Dalian
downstream signaling essential for tumor survival and pro-
liferation (8). In general, to maintain their high antiprolifera-
tive potency, an 4-anilinoquinazoline core is essential. Until
now, a family of new quinazoline derivatives, including lap-
atinib (9), dacomitinib (10,11), and afatinib (12), has been
developed as more effective anti-NSCLC drugs. Although
these agents exhibited high response rates in the treat-
ment of NSCLC tumors, their efficacies are ultimately lim-
ited by the acquisition of drug resistance, particularly by
the mutation of the gatekeeper T790M (13–15). To over-
come the T790M mutation-related resistance, irreversible
EGFR inhibitors containing a Michael acceptor functional
group have been developed. These inhibitors form a cova-
lent bond with the Cys-797 within the EGFR active site
and have shown preclinical activity against T790M-con-
taining mutants of EGFR. However, apart from a few
examples, such as WZ4002 (16), CO-1686 (17), and
AZD9291 (18,19), these irreversible inhibitors have been
limited by associated skin rash and gastrointestinal toxicity
(20,21). Considering that covalent inhibitors cannot be dis-
placed by ATP and are not able to circumvent these
issues, the non-covalent strategy has attracted more
attention recently (22).
116044, China
2Department of Dermatology and Skin Science, University
of British Columbia, Vancouver, BC V5Z 4E8, Canada
*Corresponding author: Xiaodong Ma,
xiaodong.ma@139.com
A class of novel quinazoline derivatives bearing various
C-4 aniline moieties was synthesized and biologically
evaluated as potent epidermal growth factor receptor
(EGFR) inhibitors for intervention of non-small-cell lung
cancer (NSCLC). Most of these inhibitors are compara-
ble to gefitinib in inhibiting these cancer cell lines, and
several of them even displayed superior inhibitory
activity. In particular, analogue 5b with an IC50 of
0.10 lM against the EGFR wild-type A431 cells and 5c
with an IC50 of 0.001 lM against the gefitinib-sensitive
HCC827 cells (EGFR del E746-A750) was identified as
highly active EGFR inhibitors. It was also significant
that the discovered analogue 2f, not only has high
potency
against
the
gefitinib-sensitive
cells
(IC50 = 0.031 lM), but also possesses remarkably
improved activity against the gefitinib-resistant cells. In
addition, the enzymatic assays and the Western blot
analysis for evaluating the effects of the typical inhibi-
tors indicated that these molecules strongly interfere
with the EGFR target.
Gefitinib, as a reversible EGFR inhibitor, is broadly used in
the treatment of NSCLC patients (5,6). The co-crystal
structure of gefitinib complexed with the mutant EGFR
T790M (PDB code: 4I22) indicates that the C-4 aniline side
chain lies in a deep hydrophobic pocket at the back of the
ATP-binding site (Figure 1) (23–25). Previous structure and
activity relationship (SAR) explorations disclosed that modi-
fying the aniline moiety is sensitive to the key mutant
T790M, the inhibition of which will substantially block the
drug resistance (26–31). On the basis of the quinazoline
backbone, we focused on the aniline section and synthe-
sized a number of novel 4-anilinoquinazoline derivatives
bearing various new C-4 aniline moieties (2a–i, 3a–g, 4a–
c, and 5a–c, Figure 2). All these compounds were evalu-
ated for their activities against various NSCLC cell lines
Key words: EGFR, inhibitor, NSCLC, quinazoline, T790M
Received 1 September 2015, revised 8 November 2015 and
accepted for publication 12 November 2015
The EGFR is an important member of the erbB receptor
family (erbB1/EGFR, erbB2/HER2, erbB3/HER3, and
erbB4/HER4) of receptor tyrosine kinases (TKs), and often
overexpressed in several human solid tumors, in particular,
colorectal, pancreatic, bladder, lung, etc. (1,2) Compre-
hensive clinical studies showed that inhibiting EGFR family
receptor TKs represents a major advance in the treatment
of solid tumors (3,4). Gefitinib (1, Figure 1) (5,6) and erloti-
(A431WT
,
HCC827delE746-A750
,
H1975L858R/T790M
,
and
ª 2015 John Wiley & Sons A/S. doi: 10.1111/cbdd.12692
635

Wang et al.
A549k-Ras mutation) as well as the MCF-7 breast cancer
cell line and the SGC-7901 stomach cancer cell. In addi-
tion, enzymatic assays and Western blot analysis were per-
formed to evaluate their effects, while molecular simulation
was also carried out in terms of the excellent inhibitors.
Title compounds 3a–g were synthesized starting from the
material 10a, b (Scheme 2). Treatment of 10a, b with ethyl
bromoacetate prepared compounds 11a, b. After simple
transformations of 11a, b, the nitrophenyl intermediates
12a–g were prepared in good yields (42–73%). Using the
Fe-NH4Cl condition, intermediates 12a–g were reduced to
aniline derivatives 13a–g in good yields. Also by coupling
the 4-chloroquinazoline core with the 4-substituted anilines
12a–g, the title compounds 3a–g were conveniently pro-
duced.
Chemistry
As illustrated in Scheme 1, amidation reaction of anilines
6a–b and bromoacetyl bromide afforded the intermediate
7a–b. Coupling 7a–b with substituted phenol produced
the nitrophenyl analogues 8a–i. After reducing the nitro
group in compounds 8a–i, the key aniline intermediates
9a–i were prepared. Finally, treating 9a–i with 4-chloroqui-
nazoline core in methanol solvents provided the title com-
pounds 2a–i with yields of 66–86%.
With the aim to synthesize the title compounds 4a–c and
5a–c, key intermediates N-phenyl benzenesulfonamides
15a–c and 18a–c were prepared in the first step
(Schemes 3 and 4). By reducing the nitro group in com-
pounds 15a–c and 18a–c, aniline analogues 16a–c and
19a–c were prepared, respectively. After installing the N-
phenyl benzenesulfonamide moieties with the 4-chloroqui-
nazoline core, the title compounds 4a–c (Scheme 3) and
5a–c (Scheme 4) were provided in yields of 63–85% and
67–81%, respectively.
Experimental Section
General procedure for the preparation of compound
N-[4-(N, N-dibutylsulfonamide)phenyl]-7-methoxy-6-[3-(4-
morpholinyl)propoxy]-4-quinazolinamine(5c): To
a stirred
mixture of aniline intermediates 19a–c (3 mmol) in MeOH
(10 mL), 4-chloroquinazoline derivatives (3.3 mmol) were
added. The solution was refluxed for 2 h and then poured
into H2O (100 mL) and extracted with EtOAc
(3 9 100 mL). The organic layer was collected and
washed with brine. The organic layer was dried over
sodium sulfate and concentrated in vacuo to obtain a
crude product. The residue was purified by column chro-
matography on silica gel (elution with 5% DCM/MeOH) to
give a white solid product. Yield 55.3%; mp 204.7–
206.3 °C; 1H NMR ([D]DMSO): d = 1.02 (t, 6H, 2CH3),
1.35 (m, 4H, 2CH2), 1.47 (m, 4H, 2CH2), 1.93 (m, 2H,
CH2), 2.35–2.40 (m, 6H, 3NCH2), 3.21(t, 4H, 2NCH2),
3.70(t, 4H, 2OCH2), 3.75(s, 3H, OCH3), 3.791 (t, 2H,
Figure 1: Structure of gefitinib and its binding sites with EGFR
(PDB code: 4I22) (23).
Figure 2: Structures of the designed compounds bearing various 4-aniline moieties.
636
Chem Biol Drug Des 2016; 87: 635–643

Quinazoline Derivatives with Enhanced Anti-Cancer Activity
R2
R2
R1
R1
R1
R1
O
O
O
O
O
Br
c
a
b
NH
NH
NH
NH2
O2N
H2N
O
O2N
O2N
9a–i
7a–b
8a–i
6a–b
R1
O
R2
4NH
O
Cl
N
N
O
O
3
1
O
HN
N
N
O
N
O
N
d
2a–i
2
a
b
R1
R2
ArOCH2CONH
3-position
2
f
R1
H
R2
ArOCH2CONH
4-position
Scheme 1: Synthetic route of the title
compounds 2a–i. Reagents and conditions:
(a) BrCH2COBr, NaHCO3, CH3CN, r.t, 0.5 h.
(b) Substituted phenol, K2CO3, KI, CH3CN,
2 h, 80 °C. (c) Fe-NH4Cl, EtOH-H2O, 2 h,
80 °C. (d) MeOH, reflux, 2 h.
H
H
2-Me
2-Cl
2-Me
3-position
g
H
4-t-Bu
4-position
c
d
e
H
H
H
4-t-Bu
4-Cl
3-position
3-position
3-position
h
i
H
4-Cl
4-position
4-position
MeO
2,4-DiMe2
2,4-DiMe2
R1
R1
O
O
R1
R1
O
O
OH
O
a
b,c
e
O
O
NH
NH
d
O2N
O2N
O2N
H2N
R2
R2
13a–g
12a–g
11a,b
10a,b
R1
O
R2
O
N
H
O
Cl
O
HN
N
O
O
N
N
O
N
N
O
N
Scheme 2: Synthetic route of the title
compounds 3a–g. Reagents and conditions:
(a) BrCH2COOMe, K2CO3, KI, CH3CN, 2 h,
80 °C. (b) NaOH, H2O-MeOH, 2 h, 60 °C.
(c) (COCl)2, 1 h, 60 °C. (d) aniline, NaHCO3,
CH3CN, 2 h, 60 °C. (e) Fe-NH4Cl, EtOH-
H2O, 2 h, 80 °C. (f) MeOH, reflux, 2 h.
f
3a–g
3
R1
R2
3
e
R1
R2
a
b
c
d
H
H
H
H
3-CF3
4-Cl
2-Cl
2-Me
H
4-Me
f
g
H
Cl
3-Cl-4-F
H
OCH2), 4.02(br, H, NH), 7.65–7.31 (m, 6H, ArH), 8.41(s,
H, N=CH); 13C NMR ([D]DMSO): d = 15.1(2C), 20.3(2C),
28.1, 29.3(2C), 49.3(2C), 50.2, 53.1(2C), 57.2, 67.1, 67.4
(2C), 100.1, 110.2, 113.2, 117.5(2C), 129.1(2C), 129.9,
145.9, 146.7, 152.3, 154.9, 159.1, 170.1; MS (ESI+) m/z
586 [M + H]+; Anal. calcd for C30H43N5O5S: C 61.49, H
7.38, N 11.95, O 13.69, S 5.49.
line and SGC-7901 cell line were also chosen for further
biological investigation. All theses cell-based evaluations
were performed using the MTT assay (32). The test results
are summarized in Table 1. In addition, the analogues 2a,
2g, 3a, and 5c which showed promising activity against
the NSCLC cell lines were also investigated for their inter-
ference effects on the EGFR enzymatic activity. The enzy-
matic activity data are shown in Table 2. As a control, the
effect of gefitinib was also evaluated. The results from
these assays revealed that the quinazoline derivatives syn-
thesized by us were as potent as gefitinib in inhibiting the
proliferation of the tested cell lines, and several analogues
even displayed higher antitumor activity than the lead
compound gefitinib. Importantly, analogues 2f–h and 5c
also showed enhanced antigefitinib-resistant H1975 cells,
Results and Discussion
To evaluate the bioactivity of the synthesized compounds,
the typical NSCLC cell lines A431WT, HCC827delE746-A750
,
mutant
H1975L858R/T790M, and A549K-ras
were used in this
study. Additionally, the EGFR-overexpressing MCF-7 cell
Chem Biol Drug Des 2016; 87: 635–643
637

Wang et al.
R1
R1
O
O
R2
b
R2
H
N
H
N
S
R1
NH2
R2
Cl
S
S
O2N
O
O
O
O
a
O2N
H2N
HN
14a,b
16a–c
15a–c
R1
4 H
N
R2
O
Cl
N
S
3
1
O
N
O
O
O
O
N
N
O
O
N
N
d
4a–c
ArSO2NH
4
R1
R2
a
b
c
MeO
MeO
H
3-MeO
4-Cl
4-position
4-position
3-position
Scheme 3: Synthetic route of the title
compounds 4a–c. Reagents and conditions:
(a) ArSO2Cl, NaHCO3, CH3CN, reflux, 2 h.
(b) Fe-NH4Cl, EtOH-H2O, 2 h, 80 °C. (c)
MeOH, reflux, 2 h.
3-MeO
R1
R1
O
O
H2N
R1
O2N
R3
S
R3
a
Cl
b
N
S
R2
N
R3
S
R2
NO2
R2
NH
O
O
O
O
17a,b
18a–c
19a–c
O
S
O
R1
R2
N
R3
O
HN
O
Cl
N
N
O
O
N
N
O
N
Scheme 4: Synthetic route of the title
compounds 5a–c. Reagents and conditions:
(a) RNH2, NaHCO3, CH3CN, reflux, 2 h. (b)
Fe-NH4Cl, EtOH-H2O, 2 h, 80 °C. (c)
MeOH, reflux, 2 h.
N
O
5a R1 = H, R2 = 4-ClPh, R3 = H
5b R1 = Me, R2 = 2-ClPh, R3 = H
5c R1 = H, R2 = R3 = n-Butyl
c
with IC50 values of 17.9, 12.5, 18.3 and 18.9 lM, respec-
tively.
other hand, fortunately, the set of analogues 5a–c bearing
an N-substituent benzenesulfonamide moiety displayed a
powerful inhibitory potency (IC50 < 0.70 lM). The peculiar
analogue 5b (IC50 = 0.07 lM) bearing an N-(2-chlorophe-
nyl)-sulfonamide substituent showed approximately 19
times higher activity than gefitinib. In contrast, replacing
the 2-chlorophenyl group with a dibutyl substituent (ana-
logue 5c) resulted in about a 10-fold loss of anti-wild-type
potency.
Inhibitory activity on cancer cells
Regarding the effects on the wild-type A431 cells, more
than half of the tested molecules have IC50 values in the
range of 0.07–7.01 lM. Obviously, introducing a 2-phenox-
yacetamide molecule into the 4-aniline ring does not favor
the improvement of the activity against the A431 cells.
Among the analogues 2a–i, the most active inhibitor 2b
(IC50 = 0.42 lM) is still less potent than the lead com-
pound gefitinib. Also, the benzenesulfonamide substituent
(analogues 4a–c, IC50 > 9.31 lM) did not favor the sup-
pression of the proliferation of the wild-type cells. On the
In the particular case of the activity against the gefitinib-
sensitive cells, all the tested molecules were able to
suppress HCC827 cells proliferation at nanomolar concen-
trations. The most active inhibitors, namely 2c and 5c,
showed the same IC50 value of 0.001 lM. Admittedly, the
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Chem Biol Drug Des 2016; 87: 635–643

Quinazoline Derivatives with Enhanced Anti-Cancer Activity
Table 1: Cellular antiproliferative activities of the title compoundsa
Cellular antiproliferative activity (IC50, lM)
Compound
A431
HCC827
H1975
A549
MCF-7
SGC-7901
2a
2b
2c
2d
2e
2f
6.69 Æ 0.12
4.97 Æ 0.56
19.7 Æ 1.1
>10.0
7.01 Æ 0.74
6.42 Æ 0.32
17.8 Æ 1.60
>10.0
5.80 Æ 0.45
2.85 Æ 0.65
0.33 Æ 0.74
0.42 Æ 0.42
1.54 Æ 0.33
1.03 Æ 0.51
8.33 Æ 0.21
0.10 Æ 0.01
>10.0
9.31 Æ 0.33
10.30 Æ 0.70
0.20 Æ 0.02
0.07 Æ 0.01
0.70 Æ 0.02
1.33 Æ 0.54
0.003 Æ 0.0009
0.018 Æ 0.0001
0.001 Æ 0.0006
>0.040
>0.040
>0.040
31.4 Æ 3.3
19.5 Æ 5.2
29.0 Æ 3.4
26.3 Æ 2.3
26.0 Æ 6.1
17.9 Æ 3.3
12.5 Æ 4.3
18.3 Æ 5.5
19.7 Æ 3.3
29.7 Æ 4.6
34.01 Æ 4.1
24.7 Æ 3.2
31.1 Æ 4.2
38.2 Æ 9.1
29.7 Æ 6.3
33.2 Æ 7.1
28.5 Æ 8.1
39.4 Æ 9.1
>40.0
35.1 Æ 3.4
34.2 Æ 4.6
38.3 Æ 8.1
24.5 Æ 5.5
28.27 Æ 4.3
36.5 Æ 7.7
39.3 Æ 6.4
39.1 Æ 7.2
>40.0
18.3 Æ 3.5
26.7 Æ 4.6
25.3 Æ 3.3
24.7 Æ 4.5
23.0 Æ 5.1
26.3 Æ 6.1
25.8 Æ 4.1
38.6 Æ 6.2
>40.0
11.9 Æ 2.2
12.5 Æ 4.2
17.3 Æ 1.2
15.1 Æ 2.1
10.5 Æ 4.1
17.8 Æ 2.2
18.7 Æ 4.1
15.2 Æ 1.1
18.3 Æ 3.3
6.8 Æ 1.1
24.5 Æ 2.1
29.6 Æ 5.1
14.3 Æ 2.1
>40.0
13.2 Æ 3.1
18.6 Æ 1.2
13.7 Æ 2.2
14.2 Æ 3.3
30.8 Æ 8.1
22.0 Æ 6.4
23.8 Æ 4.4
9.7 Æ 1.2
13.1 Æ 4.1
10.0 Æ 3.1
18.1 Æ 1.2
14.2 Æ 1.1
20.1 Æ 6.1
14.5 Æ 2.0
19.6 Æ 7.2
17.9 Æ 3.3
16.7 Æ 1.2
15.2 Æ 3.1
5.7 Æ 1.0
12.1 Æ 1.1
15.2 Æ 2.3
14.5 Æ 2.0
17.5 Æ 3.1
16.9 Æ 4.3
13.7 Æ 2.0
15.6 Æ 1.2
13.5 Æ 2.0
>40.0
2g
2h
2i
0.031 Æ 0.007
>0.040
>0.040
3a
3b
3c
3d
3e
3f
0.005 Æ 0.001
0.006
0.006 Æ 0.0008
0.009 Æ 0.0005
0.006 Æ 0.0001
0.004 Æ 0.0004
0.006 Æ 0.0009
0.005 Æ 0.0007
0.017 Æ 0.0021
0.017 Æ 0.0010
0.033 Æ 0.0074
0.020 Æ 0.0010
0.001 Æ 0.0004
0.013 Æ 0.07
3g
4a
4b
4c
5a
5b
5c
Gefitinib
32.5 Æ 5.4
32.2 Æ 6.1
>40.0
35.9 Æ 3.5
35.4 Æ 4.3
18.9 Æ 5.5
26.9 Æ 8.7
13.1 Æ 2.1
38.4 Æ 6.2
6.4 Æ 1.1
27.6 Æ 4.2
22.7 Æ 5.6
39.7 Æ 9.1
aThe IC50 values were obtained by the Logit method based on the data obtained from three separate experiments and expressed as
mean Æ SD (p < 0.05).
Table 2: Inhibitory activity of compounds 2a, 2g, 3a, and 5c
butylphenoxy)acetamide-substituted 2g (IC50 = 12.5 lM),
against wild-type and mutant EGFR receptor kinasesa
and the 2-(4-chlorophenoxy)acetamide-substituted 2h
(IC50 = 18.3 lM). Among the four series of analogues, the
class of inhibitors 2a–i bearing a 2-phenoxyacetamide
EGFR IC50 [nM]
Compound
WT
L858R/T790M
moiety displayed considerable activity against the H1975
cells. Notably, the moiety at the para position of the 4-ani-
line group (analogues 2f–g) is actually advantageous, indi-
cating that a substituent at the para position will be in
close contact with key mutant amino acid T790M and thus
form strong binding forces.
2a
2g
11.49 Æ 0.23
4.51 Æ 0.07
6.53 Æ 1.10
8.06 Æ 0.78
5.94 Æ 1.11
10.79 Æ 0.65
5.32 Æ 1.02
3a
5c
11.52 Æ 0.96
71.08 Æ 1.56
832.3 Æ 101.7
Gefitinib
aIC50 values were obtained by the Logit method based on the
data obtained from three separate experiments and expressed as
mean Æ SD (p < 0.05).
To further evaluate their bioactivity, the A549 cells, the
MCF-7 cells, and the SGC-7901 cells were also used in
the subsequent biological evaluation. Regarding the
effects on the EGFR-negative A549 cells, most of the
synthesized compounds had IC50 values ranging from 20
to 40 lM. Moreover, these molecules also showed
approximately twofold increase in inhibitory activity
against the MCF-7 and SGC-7901 cells proliferation. In
particular, the 2,4-dimethylphenoxyacetamide-subsituted
2i and the N,N-dibutyl-benzenesulfonamide-substituted
5c exhibited a superior potency for inhibiting both the
MCF-7 cells and SGC-7901 cells at concentrations of
9.7 lM and below.
four designed moieties appear to be greatly effective.
Notably, HCC827 cells are far sensitive to the class of
compounds 2a–i bearing a 2-phenoxyacetamide moiety,
and a small structural change in the 2-phenoxyacetamide
moiety did significantly affect its capability. Also as indi-
cated by the activity data of compounds 2a–d and 2h–i
(IC50 > 0.040 lM), a small substituent, such as Cl, and
Me, at the C-4 position of the 2-phenoxyacetamide moiety
is not suitable.
As expected, by modifying the 4-aniline group, we pro-
duced several inhibitors that exhibited improved activity
against drug-resistant cells, such as the 2-(2-methylphe-
noxy)acetamide-substituted 2f (IC50 = 17.9 lM), the 2-(4-t-
Kinase inhibitory activity
The 4-aniline quinazoline scaffold has long been recog-
nized as a classical EGFR kinase inhibitor motif (3–6,
Chem Biol Drug Des 2016; 87: 635–643
639

Wang et al.
Figure 3: Compound 2f, 2g, 3a, and 5c inhibited the activation of EGFR and downstream signaling in H1975 NSCLC cell harboring
EGFR L858R/T790M.
9–12). To further confirm the targeted active site of our
synthesized compounds, the activities of the selected
representative inhibitors compounds 2a, 2g, 3a, and 5c
against both the wild-type EGFR and the mutant
EGFRT790M enzymes were evaluated. The results,
shown in Table 2, revealed that all the four active inhibi-
tors were able to inhibit EGFR enzymes at nanomolar
concentrations. Specifically, compound 2g which bears a
bulky t-butyl group was very effective, showing a stron-
ger activity against the wild-type EGFR kinase than gefi-
tinib. However, an even more exciting result was the
inhibitory activity it displayed against the drug-resistant
mutant cells with the EGFR harboring the T790M muta-
tion. Compared with the control (IC50 = 832.3 nM), all the
selected compounds (IC50 < 71.08 nM) showed greatly
improved potency against the cells expressing
EGFRT790M. Additionally, the t-butyl-substituted ana-
logue 2g was the most potent inhibitor against the
A
C
D
B
D
E
Figure 4: (A) Putative binding mode of
analogue 2c within the active pocket of the
EGFR T790M (PDB code: 4I22). (B) Putative
binding mode of 2g within the active pocket
of the EGFRT790M (PDB code: 4I22). (C)
Putative binding mode of 2i within the active
pocket of the EGFRT790M (PDB code:
4I22). (D) Putative binding mode of 3a within
the active pocket of the EGFRT790M (PDB
code: 4I22). (E) Putative binding mode of 4a
within the active pocket of the EGFRT790M
(PDB code: 4I22). (F) Putative binding mode
of 5c within the active pocket of the
EGFRT790M (PDB code: 4I22) (23).
640
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Quinazoline Derivatives with Enhanced Anti-Cancer Activity
EGFRT790M enzyme, exhibiting an IC50 value of 5.32 nM.
These results directly corroborated that these analogues
are effective EGFR inhibitors.
group of quinazoline core and the amino acid Met793
(NH), and the N-3 group of quinazoline core and the
amino acid Thr854 (NH) disappeared in the active inhibi-
tors. Nevertheless, there are several new interactions, such
as the carbonyl group (C=O) with MET793 (NH), a water
molecule-mediated hydrogen bond between quinazoline
N3 and the side chain of Pro794. Taken together, the
results of all these analyses were consistent with the test
results.
Effects on EGFR activation and downstream
signaling
To get an insight into the mechanisms underlying the
activity of these inhibitors, the four active representative
compounds 2a, 2g, 3a, and 5c were assessed for their
inhibitory effects on the activation of EGFR and the down-
stream signaling in H1975 cell. The expression and activa-
tion of proteins involved in the EGFR-mediated AKT
pathway were assessed by immunoblotting, and the
results are presented in Figure 3. These assessments
demonstrated that treatment with these inhibitors greatly
repressed the phosphorylation of the EGFR and down-
stream signaling proteins of the AKT pathway in a dose-
dependent manner achieving complete inhibition at a
concentration of 1.0 lM, while the total protein levels of
EGFR, AKT, and b-actin remain unchanged.
Conclusion
In summary, a series of 4-anilinoquinazoline derivatives
containing various C-4 aniline moieties were synthesized
and biologically evaluated. Most of these compounds
exhibited moderate to good inhibitory effects toward the
EGFR-overexpressing cell lines, and several analogues
even demonstrated higher potency than gefitinib. Further-
more, we also identified four analogues 2f, 2g, 2h, and 5c
with enhanced inhibitory activity against drug-resistant can-
cer cells. Western blot analysis indicated that the com-
pounds 2f, 2g, 3a, and 5c inhibited EGFR activation and
downstream signaling in H1975 cells at a concentration of
0.5 lM. Overall, this study provided a number of new valu-
able 4-anilinoquinazoline derivatives worthy of further devel-
opment as potentially more effective EGFR inhibitors.
Molecular simulation
In addition, a set of typical analogues 2g, 2i, and 5c,
which were effective at inhibiting the H1975 cells, were
chosen to dock into the ATP-binding site in the model of
EGFR kinase harboring the T790M mutation (PDB code:
4I22). The program AUTODOCK 4.2 in parallel with the default
parameters was applied (33,34). For comparison, three
less potent inhibitors 2c, 3a, and 4a were also evaluated
using same procedure. The results revealed that all the
selected compounds were able to interact with the ATP-
binding cleft of the enzyme through hydrophobic forces
and/or hydrogen bond forces, as shown in Figure 4.
Clearly, in the less potent compounds 2c, 3a, and 4a, the
newly introduced 4-aniline groups were so bulky that they
were squeezed out of the hydrophilic pocket containing
amino acids Lys745, Asp855, Thr854, Leu788, while they
remained close to the amino acids Phe795, Cys796,
Asp800, and Cys797. Instead, the hydrophilic morphine
side chain fully occupied the hydrophobic region of the
pocket. Consistent with the less potent activity, this bind-
Acknowledgments
This work was supported in part by grants from the
National Natural Science Foundation of China (No.
81402788), and the PhD Start-up Fund of Natural Science
Foundation of Liaoning Province, China (No. 20141115).
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