Synthesis and evaluation of 2-anilinopyrimidines bearing 3-aminopropamides as potential epidermal growth factor receptor inhibitors

Article abstract of DOI:10.1016/j.ejmech.2014.02.032

Novel compounds 12a-i were synthesized and biologically evaluated. Several ones exhibited stronger inhibitory activity than gefitinib against EGFR L858R/T790M and antiproliferative effects on H1975 and HCC827 cells. The 3-aminopropamide in compounds like 12h could be converted to the active acrylamide in the presence of arginine. Importantly, 12h showed improved stability relative to compound 1 whose structure is same to 12h excepting an acrylamide moiety. Interestingly, 12i, a NO donating compound of 12h, showed more potent and selective inhibition than 12h on H1975 cells. Significantly, 12i produced high levels of NO in H1975 cells but not in non-tumorous 16HBE cells, and its inhibition was diminished by NO scavenger. Furthermore, 12i dose-dependently produced inhibitory effects on EGFR downstream signaling in H1975 cells.

Full text of DOI:10.1016/j.ejmech.2014.02.032

European Journal of Medicinal Chemistry 77 (2014) 75e83
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and evaluation of 2-anilinopyrimidines bearing
3-aminopropamides as potential epidermal growth factor
receptor inhibitors
,
b
,
c
,
,
b
,
,b,
*
Chun Han a 1, Ledong Wan a 1, Hongbin Ji d, Ke Ding e, Zhangjian Huang a
,
Yisheng Lai a , Sixun Peng b, Yihua Zhang a
,
b
,
a,
,b,
*
*
a State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, PR China
b Center of Drug Discovery, China Pharmaceutical University, Nanjing 210009, PR China
c Department of Chemistry, Changzhi University, Changzhi 046011, PR China
d State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences,
Shanghai 200031, PR China
e Key Laboratory of Regenerative Biology and Institute of Chemical Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences,
Guangzhou 510530, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel compounds 12aei were synthesized and biologically evaluated. Several ones exhibited stronger
inhibitory activity than gefitinib against EGFR L858R/T790M and antiproliferative effects on H1975 and
HCC827 cells. The 3-aminopropamide in compounds like 12h could be converted to the active acryl-
amide in the presence of arginine. Importantly, 12h showed improved stability relative to compound 1
whose structure is same to 12h excepting an acrylamide moiety. Interestingly, 12i, a NO donating
compound of 12h, showed more potent and selective inhibition than 12h on H1975 cells. Significantly,
12i produced high levels of NO in H1975 cells but not in non-tumorous 16HBE cells, and its inhibition
was diminished by NO scavenger. Furthermore, 12i dose-dependently produced inhibitory effects on
EGFR downstream signaling in H1975 cells.
Received 3 January 2014
Received in revised form
5 February 2014
Accepted 13 February 2014
Available online 14 February 2014
Keywords:
EGFR inhibitor
Nitric oxide
Antiproliferative effects
3-Aminopropamides
Acrylamide
Ó 2014 Elsevier Masson SAS. All rights reserved.
Anilinopyrimidine
1. Introduction
autophosphorylation of several tyrosine residues after EGFR
dimerization [4]. Hyperactivation of EGFR signaling pathway has
Non-small cell lung cancer (NSCLC) is the leading cause of
cancer-related death worldwide [1]. The NSCLC patients at large
present with advanced, unresectable or metastatic disease, and a 5-
year survival rate of less than approximately 15% [2]. Therefore,
novel therapeutic strategies including safe and potent anti-NSCLC
drugs are urgently needed.
Epidermal growth factor receptor (EGFR) is a member of the
ErbB family of receptors [3], which is involved in various events of
cancer cell proliferation, survival, adhesion, migration, and differ-
entiation through downstream signaling pathway activation by
been found in more than half of NSCLCs due to gene amplification
or EGFR mutation [5] and plays an indispensable role in the path-
ogenesis of NSCLC [6]. In this context, EGFR tyrosine kinase in-
hibitors (TKIs) have been emerging as one of the effective and
targeting treatment strategies for NSCLC [6].
Gefitinib and erlotinib (Fig. 1), the first generation reversible
EGFR TKIs, are initially effective in the treatment of NSCLC patients
with a short in-frame deletion of exon 19 (del E746_A750) and the
L858R point mutation in exon 21 in the EGFR kinase domain [7].
However, the development of resistance has limited their clinical
efficacy, which is believed to be associated with a conversion of the
threonine gatekeeper to a methionine (T790M) in the catalytic
domain of EGFR in about 50% of cases [8,9]. This mutation restores
the affinity for ATP similar to that of wide type (WT) EGFR, and
prevents reversible inhibitors from binding at higher ATP concen-
trations [10]. The second generation irreversible EGFR TKIs include
* Corresponding authors. Center of Drug Discovery, China Pharmaceutical Uni-
versity, Nanjing 210009, PR China.
E-mail addresses: cpudahuang@163.com (Z. Huang), lcpu333@yahoo.com.cn
(Y. Lai), zyhtgd@163.com, zyhtgd@hotmail.com (Y. Zhang).
1
These two authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejmech.2014.02.032
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

76
C. Han et al. / European Journal of Medicinal Chemistry 77 (2014) 75e83
Fig. 1. Chemical structures of EGFR TKIs with a quinazoline or quinoline core scaffold.
CI-1033 [11], BIBW2992 [12], HKI-272 [13], PF00299804 [14]
(Fig. 1), etc, which are able to inhibit T790M-mutant EGFR
in vitro since an electrophilic functionality (acrylamide group) in
the molecules could undergo a Micheal addition reaction with a
conserved cysteine residue present in EGFR (Cys797) to achieve
occupancy greater than that of the reversible inhibitors [15].
Nevertheless, the dose-limiting and off-target toxicity such as
diarrhea and skin rash, which are caused by unselective inhibition
of WT EGFR, limit the clinical efficacy of these second generation
EGFR TKIs [11e14,16].
To circumvent this problem, the third generation of irreversible
EGFR TKIs such as WZ4002 (1, Scheme 1) with better selectivity
against mutant EGFR T790M over WT EGFR, was investigated [17].
Compound 1 is built based on an anilinopyrimidine driving portion
which is different from the first and second generation EGFR TKIs
with a quinazoline or quinoline core scaffold. Accordingly, the
anilinopyrimidine is supposed to be an attracting scaffold for the
design of small molecule inhibitors against EGFR mutants. Like the
second generation inhibitors, compound 1 contains an acrylamide
group which could covalently bind to the Cys797 of EGFR T790M to
generate inhibitory activity [17]. However, the high reactivity of the
acrylamide group may cause rapid metabolism or nonspecific re-
action with off targets, lowering the bioavailability and efficiency
[18,19]. In this regard, a protection strategy over the warhead
stability towards small thiols in vitro, and importantly, could un-
dergo a -elimination (retro-Michael addition) reaction to liberate
the acrylamide which can react with thiols in the intracellular
environment [20].
b
Nitric oxide (NO), a signaling and effector molecule, plays a
pivotal role in diverse and important physiological and patho-
physiological processes [22]. It is generally believed that high levels
of NO generated from NO-donors can not only inhibit tumor cell
proliferation and survival, and induce apoptosis in sensitive tumor
cells, but also sensitize resistant tumor cells to chemotherapy, ra-
diation and immunotherapy as well as inhibit metastasis in vitro
and vivo [23]. Recently, we investigated phenylsulfonylfuroxan-
based NO-releasing compounds as potent and selective EGFR in-
hibitors for intervention of NSCLC [24,25].
As part of our ongoing program to study the novel irreversible
EGFR TKIs, we designed and synthesized a group of irreversible
EGFR inhibitors 12aeh containing 2-anilinopyrimidine as a scaffold
and 3-aminopropamide as the “protected” warhead (Scheme 1).
Additionally, the NO-donating modification strategy was applied
for compound 12h yielding 12i as comparison in efficacy. Biological
studies including kinase inhibitory activity, antiproliferative activ-
ity against several NSCLC cells, possible mechanism of action, and
NO release property for 12i, were conducted and discussed.
acrylamide could be beneficial. It is known that the
ketones could be bioconverted via a -elimination reaction to the
-unsaturated ketones, covalently modifying their biological
targets [20,21]. Accordingly, Carmi et al. masked the acrylamide
warhead of the irreversible inhibitors containing a quinazoline or
b-aminoethyl
2. Chemistry
b
a,b
Compounds 12aei were synthesized as depicted in Scheme 2.
Starting from compound 2, a methylation reaction using methyl
iodide and a following amination at 5-position by N-methyl-
piperazine furnished methylphenyl ether 4, which underwent a
quinoline driving portion to produce
a Mannich base, 3-
aminopropamide. The resulting compounds exhibited enhanced
nitro-reduction to produce aniline 5. 3-Nitrophenol
7 was
Scheme 1. Rationale for the design of 2-anilinopyrimidines 12aeh.

C. Han et al. / European Journal of Medicinal Chemistry 77 (2014) 75e83
77
Scheme 2. Synthesis of the compounds 12aei. Reagents and conditions: (a) CH3I, K2CO3, acetone, reflux, 4 h. (b) N-methyl-piperazine, K2CO3, DMSO, rt, overnight. (c) iron powder,
NH4Cl, THF/H2O (1/1, v/v), reflux, 3 h. (d) K2CO3, DMF, rt, 2 h. (e) 5, TFA, 2-BuOH, reflux, 3 h. (f) Acryloyl chloride, Et3N, anhydrous CH2Cl2, 0 ꢀC, 1 h. (g) 11aei, THF/MeOH, reflux, 4 h.
Table 1
regioselectivity
coupled
to
the
4-position
of
2,4,5-
In vitro enzymatic inhibitory activity of compounds 12aei against different types of
EGFR.
trichloropyrimidine 6 to afford aryl ether 8 at the room tempera-
ture. Coupling reaction of 5 with 8 was accomplished in the pres-
ence of trifluoroacetic acid under reflux, offering anilinopyrimidine
9. The nitro group in 9 was reduced by iron powder/NH4Cl to
produce arylamine 10. Acylation of anilino group of 10 using
acryloyl chloride gave compound 1. The Compounds 12aei were
obtained though Michael addition reaction of 1 with various sec-
ondary amine in THF/MeOH under reflux.
Compound
EGFR IC50
WT
(m
M)a
L858R
L858R/T790M
gefitinib
1
0.0001 ꢁ 0.0002
0.0044 ꢁ 0.0006
0.638 ꢁ 0.125
0.877 ꢁ 0.276
0.525 ꢁ 0.256
10 ꢁ 2.176
0.0002 ꢁ 0.0001
0.0048 ꢁ 0.0009
0.804 ꢁ 0.264
0.818 ꢁ 0.348
0.427 ꢁ 0.198
4.527 ꢁ 0.241
>10
0.941 ꢁ 0.073
0.0007 ꢁ 0.0001
0.184 ꢁ 0.087
0.181 ꢁ 0.056
0.088 ꢁ 0.024
2.177 ꢁ 0.687
>10
4.706 ꢁ 0.134
0.537 ꢁ 0.198
2.419 ꢁ 0.876
0.263 ꢁ 0.103
12a
12b
12c
12d
12e
12f
12g
12h
12i
3. Results and discussion
>10
7.442 ꢁ 2.043
0.698 ꢁ 0.324
2.388 ꢁ 0.973
0.201 ꢁ 0.099
10 ꢁ 1.923
2.385 ꢁ 0.645
5.866 ꢁ 0.003
0.262 ꢁ 0.075
3.1. Biological evaluation of 12aeh
3.1.1. Kinase inhibitory activity
a
EGFR activity assays were performed using the FRET-based Z0-Lyte assay ac-
cording to the manufacturer’s instructions. The compounds were incubated with the
kinase reaction mixture for 1.5 h before measurement. The data were means ꢁ SD of
three independent experiments.
The enzyme inhibitory activity against different types of EGFR
(EGFR WT, EGFR L858R, EGFR L858R/T790M) of the target com-
pounds 12aeh, and positive controls gefitinib and 1, were evalu-
ated using a well established FRET-based Z0-Lyte assay (Table 1)
[26,27]. Among them, compounds 12aec and 12g exhibited
reasonable and selective inhibitory activity against EGFR L858R/
T790M over WT EGFR and EGFR L858R as compound 1 did. How-
ever, their ability to inhibit EGFR L858R/T790M (IC50 ¼ 0.184, 0.181,
L858R/T790M and gefitinib-sensitive HCC827 cells bearing EGFR
del E746_A750, were next determined by a MTT assay using gefi-
tinib and 1 as positive controls. As shown in Table 2, compounds
0.088 and 0.537
mM, respectively) was lower than
1
12aeh (IC50s
proliferative activity against H1975 cells than gefitinib
(IC50 ¼ 6.917 M), but lower than 1 (IC50 ¼ 0.046 M). Among them,
compounds 12aed showed stronger inhibitory activity against
H1975 cells (IC50 ¼ 0.156, 0.166, 0.268 and 0.178 M, respectively)
and HCC827 cells (IC50s ¼ 0.210e0.337 M). One plausible expla-
nation is that the capability of different 3-aminopropamides to
¼
0.156e1.598 mM) produced stronger anti-
(IC50 ¼ 0.0007 M), but superior to gefitinib (IC50 ¼ 0.941
m
m
M).
m
m
3.1.2. Inhibitory activity on gefitinib-sensitive HCC827 and resistant
H1975 cells
The antiproliferative effects of 12aeh on EGFR mutation NSCLC
cell lines, including gefitinib-resistant H1975 cells harboring EGFR
m
m

78
C. Han et al. / European Journal of Medicinal Chemistry 77 (2014) 75e83
Table 2
Antiproliferative activity of compounds 12aei and 11i against cells harboring a different status of EGFR.
Compound
IC50 (m
M)a
H1975b
HCC827c
A431d
A549e
BEAS-2bf
16HBEg
gefitinib
1
6.917 ꢁ 0.657
0.046 ꢁ 0.011
0.156 ꢁ 0.034
0.166 ꢁ 0.021
0.268 ꢁ 0.043
0.178 ꢁ 0.029
1.018 ꢁ 0.125
1.042 ꢁ 0.243
1.598 ꢁ 0.296
0.931 ꢁ 0.039
0.108 ꢁ 0.029
5.656 ꢁ 0.059
0.006 ꢁ 0.002
0.008 ꢁ 0.003
0.266 ꢁ 0.015
0.249 ꢁ 0.009
0.210 ꢁ 0.023
0.337 ꢁ 0.098
0.899 ꢁ 0.078
1.497 ꢁ 0.328
0.883 ꢁ 0.067
0.799 ꢁ 0.049
0.130 ꢁ 0.008
>10
4.485 ꢁ 0.295
1.108 ꢁ 0.213
0.545 ꢁ 0.078
0.291 ꢁ 0.054
0.804 ꢁ 0.137
0.410 ꢁ 0.298
3.910 ꢁ 0.329
0.548 ꢁ 0.092
4.534 ꢁ 0.296
0.632 ꢁ 0.089
4.469 ꢁ 0.541
3.058 ꢁ 0.284
15.58 ꢁ 2.143
2.102 ꢁ 0.584
0.923 ꢁ 0.102
1.003 ꢁ 0.032
1.213 ꢁ 0.046
1.119 ꢁ 0.092
2.622 ꢁ 0.135
1.030 ꢁ 0.289
5.283 ꢁ 1.231
2.816 ꢁ 0.498
8.251 ꢁ 0.992
>10
13.94 ꢁ 1.992
1.811 ꢁ 0.253
0.615 ꢁ 0.058
0.433 ꢁ 0.106
1.888 ꢁ 0.782
0.365 ꢁ 0.069
2.269 ꢁ 0.831
0.412 ꢁ 0.145
5.779 ꢁ 1.039
1.657 ꢁ 0.394
3.368 ꢁ 1.290
19.77 ꢁ 2.018
12.71 ꢁ 3.675
1.355 ꢁ 0.321
1.143 ꢁ 0.258
0.620 ꢁ 0.182
1.129 ꢁ 0.318
0.970 ꢁ 0.047
3.596 ꢁ 1.216
1.753 ꢁ 0.152
3.705 ꢁ 0.139
2.056 ꢁ 0.284
2.876 ꢁ 0.217
7.150 ꢁ 1.219
12a
12b
12c
12d
12e
12f
12g
12h
12i
11i
a
The inhibitory effects of individual compounds on the proliferation of cancer cell lines were determined by the MTT assay. The data were means ꢁ SD from at least three
independent experiments.
b
H1975 is a human lung cancer cell line (EGFR L858R/T790M).
HCC827 is a human lung cancer cell line (EGFR del E746_A750).
A431 is human epithelial carcinoma cell line (overexpressed WT EGFR).
A549 is a human lung cancer cell line (WT EGFR/k-RAS dependent).
BEAS-2B are normal human bronchial epithelial cell lines.
16HBE are normal human bronchial epithelial cell lines.
c
d
e
f
g
liberate the acrylamide moiety in the physiological conditions
might be correlated to their anti-proliferation activity. For example,
12aed with small secondary amine moieties showed stronger
inhibitory effects than 12feh with bigger heterocycle amines, since
active compound 12a produced higher levels of compound 1,
whereas less active compound 12g produced lower levels of 1,
indicating that the ability of the tested compounds to liberate the
free warhead was positively correlated with the inhibitory activity
against H1975 cells in vitro (R ¼ 0.955, p < 0.05, determined by
Pearson’s correlation analysis).
the formers are more likely to undergo a
b-elimination reaction to
liberate the acrylamide moiety than latters.
3.1.3. Effects on the cell lines harboring WT EGFR
3.1.5. Stability and reactivity of 12h
To examine how the target compounds 12aeh affect both can-
cer cells and normal cells harboring WT EGFR, we determined their
antiproliferative activity against following cell lines: human
epithelial carcinoma cells A431 with overexpressed WT EGFR;
NSCLC cells A549 possessing WT EGFR and k-Ras mutation; human
normal bronchial epithelial cell lines BEAS-2B and 16HBE harboring
WT EGFR. As shown in Table 2, most of compounds displayed less
inhibition on the growth of the cell lines harboring WT EGFR than
inhibition on that of H1975 and HCC827 cells.
Compound 12h was selected as the prototype to test for its
chemical (buffered solutions at pH 7.4 and 9.0) and biological (50% (v/
v) rat plasma) stability, as well as for the activity against thiol nucle-
ophiles with low molecular weight (LMW) [20]. As shown in Table 4,
both of compounds 1 and 12h were stable at pH 7.4, with over 99% of
the parent compounds remaining after 24 h incubation at 37 ꢀC in the
buffer. Compound 1 was undetectable under the alkaline pH condi-
tions (pH9.0)after24h, while81% of 12h was recovered, with 1 (2.4%)
as one of the degradation products. In the presence of rat plasma,12h
showed much higher stability than 1 after incubation. The reactivity
of 1 and 12h towards thiol nucleophiles was assessed and the results
3.1.4. Ability to liberate compound 1
are summarized in Table 5. While 1 was rapidly adducted by L-
To verify whether the 3-aminopropamide moieties in the target
compounds could be converted to the active acrylamide group,
compounds 12a, 12g and 12h were selected to test the ability to
liberate compound 1 in the mimetic physiological conditions. After
cysteine, glutathione (GSH) and dithiothreitol (DTT), compound 12h
kept its integrity under the same conditions, suggesting that these
compounds with 3-aminopropamides might possess enhanced sta-
bility and reduced reactivity towards small thiol nuclephiles as
compared with 1 in vitro.
the three compounds (50 mM) were respectively incubated with
arginine (1 mM) in PBS (pH ¼ 7.4) for 72 h at 37 ꢀC, the percentages
of compound 1 were detected and determined using an HPLC-
based method. As illustrated in Table 3, all three compounds
could release compound 1 in the presence of arginine, demon-
strating that the 3-aminopropamide could be converted to acryl-
3.2. Rationale for the design of NO-donating compound 12i
Compound 12i mentioned above was designed and synthesized
based on the following reasons: i) Among the compounds 12aꢂh,
only 12h with a free hydroxyl group was suitable for further NO-
amide via a b-elimination reaction induced by a natural basic amino
acid under physiological conditions. Notably, treatment with more
Table 4
Table 3
In vitro stability studies on 1 and 12h.
Percentages of 1 liberated from 12a, 12g and 12h.
Condition
Percent (%) of compound remaining (50 m
M, 24 h)a
Compound
Percent (%) of compound 1a
1
12 h
12a
12g
12h
18.52 ꢁ 0.42
2.41 ꢁ 0.80
5.44 ꢁ 0.37
pH ¼ 7.4
101.02 ꢁ 1.42
99.48 ꢁ 2.93
81.39 ꢁ 1.59
104.9 ꢁ 3.07
pH ¼ 9.0
Below the limit of detection
Below the limit of detection
50% (v/v) rat plasma
a
The compound 1 was detected by LC-UV after incubation in the
presence of arginine (1 mM) at 37 ꢀC for 72 h. Means ꢁ SD, n ¼ 3. The
peak corresponding to 1 was collected and verified by HRMS.
a
Percentages of test compounds remaining, detected by LC-UV after incubation
for 24 h at 37 ꢀC (see the Experimental Section). Means ꢁ SD, n ¼ 3.

C. Han et al. / European Journal of Medicinal Chemistry 77 (2014) 75e83
79
Table 5
0.130 mM) over H1975 and HCC827 cells, respectively, suggesting
In vitro reactivity studies on compounds 1 and 12h.
the synergic effects of anilinopyrimidine and NO donor moieties. To
verify the contribution of NO to the inhibitory activity, the levels of
NO released by compounds 12i, 11i and gefitinib were detected
in vitro. NSCLC cells H1975 and normal 16HBE cells were respec-
Condition
Percent (%) of compound remaining
(50
m
M, 1 h) a
1
12 h
tively exposed to 100 mM of 12i, 11i or gefitinib for the same in-
GSH
DTT
73.52 ꢁ 3.80
11.32 ꢁ 2.03
12.21 ꢁ 2.67
100.07 ꢁ 0.23
100.92 ꢁ 1.96
101.19 ꢁ 2.19
cubation time. The levels of NO produced by individual compounds
in the cells are presented as that of nitrite, which was produced in
the lysates of these cells and determined by the Griess assay
(Fig. 2a) [32]. As expected, treatment with gefitinib resulted in little
NO in H1975 and 16HBE cells. Treatment with 12i promoted high
levels of NO in H1975 cells, which was even greater than the group
of furoxan compound 11i. In contrast, NO was almost not detected
in the cell lysates of 16HBE cells treated with 12i. One plausible
explanation was that better recognition capacity of 12i to the EGFR
mutation relative to 11i could be helpful for cell penetration,
resulting in selective and high NO production in H1975 cells.
Next, 12i was tested on its antiproliferative effects in the pres-
ence or absence of a NO scavenger, hemoglobin. H1975 and HCC827
cells were pre-treated with, or without, different concentrations of
L
-cysteine
a
Percentages of test compounds remaining, detected by LC-UV after incubation
in the presence of LMW thiols (2 mM) for 1 h at pH 7.4, 37 ꢀC (see the Experimental
Section). Means ꢁ SD, n ¼ 3.
donating modification; ii) Although 12h did not exhibit the stron-
gest antiproliferative activity against H1975 and HCC827 cell lines
relative to other compounds, it showed relatively low cell growth
inhibitory effects on normal cells with WT EGFR (Table 2). Given
that cancer cells are more sensitive to the high levels of NO
compared to normal cells [28e31], we hypothesized such furoxan-
based 3-aminopropamide 12i may display strong antiproliferative
activity selectively against H1975 and HCC827 cell lines.
hemoglobin for 1 h and then treated with 5 mM of 12i. The effects of
different treatments on the growth of H1975 and HCC827 cells
were determined by the MTT assay (Fig. 2b). It was observed that
treatment with 12i alone remarkably inhibited the growth of
H1975 and HCC827 cells and this inhibitory effect was diminished
by pretreatment with hemoglobin in a dose-dependent manner.
These results indicated that NO produced by 12i played a significant
role in the inhibition on NSCLC cell growth in vitro.
3.3. Biological evaluation of 12i
3.3.1. Kinase and cellular inhibitory activities of 12i
Kinase and cellular inhibitory activities of 12i were assayed and
the results are summarized in Tables 1 and 2, respectively. It
exhibited more potent enzyme inhibition over EGFR L858R/T790M
(IC50 ¼ 0.263
m
m
M) than gefitinib (IC50 ¼ 0.941
M). Furthermore, 12i displayed stronger activity
M) than its precursor 12h (IC50 ¼ 0.931 and
mM) and 12h
(IC50 ¼ 2.419
(IC50 ¼ 0.108 and 0.130
m
3.3.3. Effects on EGFR activation and downstream signaling
0.799
exhibited lower cell growth inhibitory activity against cells with
WT EGFR relative to 12h (IC50s range 2.876e8.251 M for 12i,
0.632e2.816 M for 12h, respectively), suggested that 12i might
mM) against H1975 and HCC827 cell lines. Interestingly, 12i
To investigate the mechanisms underlying the activity of NO-
releasing 3-aminopropamide-based EGFR inhibitor, we examined
the inhibitory effects of 12i on EGFR activation and the downstream
signal in H1975 cell line harboring EGFR L858R/T790M using gefi-
tinib as a control. H1975 cells were treated with vehicle control,
indicated doses of 12i, or gefitinib. The expression and activation of
EGFR-related signal events, Akt and ERK were determined by
immunoblotting assays, and the results are shown in Fig. 3. Com-
pound 12i inhibited the phosphorylation of EGFR in a dose-
m
m
possess a higher safety index.
3.3.2. Effects of NO on antiproliferative activity
From Table 2 it can be seen that 12h (IC50 ¼ 0.931 and 0.799
mM)
and 11i (IC50 ¼ 5.656 and >10
mM), which are two moieties of 12i,
exhibited much less inhibitory activity than 12i (IC50 ¼ 0.108 and
dependent manner, and the treatment of 12i at 0.1 mM
Fig. 2. Effects of NO production by the target compounds on NSCLC cell proliferation. (a) Variable levels of NO (presented as nitrite) produced by the indicated compounds. H1975
and 16HBE cells were treated with individual compounds at 100 M for 150 min, and the concentrations of nitrite in the cell lysates were determined by Griess assay. The individual
values were determined by measuring absorbance at 540 nm and calculated according to the standard curve. (b) Effects of hemoglobin on the antiproliferative effect of 12i. H1975
and HCC827 cells were pretreated with the indicated concentrations of hemoglobin (Hb) (0, 1, 5, 10 or 20 M) for 1 h and treated with 5 M of 12i for 24 h. The results were
expressed as percent of cell growth inhibition relative to control cells. Data were expressed by means ꢁ SD.
m
m
m

80
C. Han et al. / European Journal of Medicinal Chemistry 77 (2014) 75e83
performed on silica gel GF/UV 254, and the chromatograms were
conducted on silica gel (100e200 mesh) and visualized under UV
light at 254 and 365 nm. All solvents were reagent grade and, when
necessary, were purified and dried by standards methods. Organic
solutions were dried over anhydrous sodium sulfate. Compounds
secondary amines and alkylol amines were commercially available.
Compound 1 was synthesized as described in literature [17]. 11i
were synthesized as previously described [28e31].
5.1.1. General procedure for the preparation of 12aei
Compound 1 was dissolved in MeOH/THF (5/30 mL), and the
required amine base (2e3.6 equiv) was added. The mixture was
heated under reflux for 4 h prior to the solvent being removed, 5%
NH3 (aq) (50 mL) was added and the mixture was extracted with
CH2Cl2 (50 ꢃ 3 mL). The organic layers were collected, washed with
brine and dried over anhydrous sodium sulfate. After removal all
solvent, the residue was purified using flash chromatography with
dichloromethane- methanol- triethylamine to obtain the target
compounds.
Fig. 3. Compound 12i inhibit the activation of EGFR and downstream signaling in
H1975 NSCLC cells harboring EGFR L858R/T790M.
significantly reduced the expression of phosphor-EGFR in our
experimental condition. Importantly, the treatment of 12i (1
completely suppressed the expression of phosphor-EGFR, -Akt and
-Erk, while gefitinib (1 M) didn’t show significant suppressive
mM)
5.1.1.1. N-(3-((5-Chloro-2-((2-methoxy-4-(4-methylpiperazin-1-yl)
phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-3-(dimethylamino)prop-
anamide (12a). The title compound was obtained starting from 11a
and 1. As a yellow solid, 50% yield. mp 95e97 ꢀC. Analytical data for
m
effects as compared to both vehicle control and 12i.
12a: 1H NMR (300 MHz, CDCl3):
d
2.37 (s, 9H, 3 ꢃ NCH3), 2.53 (t, 2H,
4. Conclusions
COCH2, J ¼ 6.06 Hz), 2.62 (t, 4H, 2 ꢃ NCH2CH2N, J ¼ 4.41 Hz), 2.68 (t,
2H, NCH2, J ¼ 5.82 Hz), 3.13 (t, 4H, 2 ꢃ NCH2CH2N, J ¼ 4.5 Hz), 3.81
(s, 3H, OCH3), 6.23 (dd,1H, ArH, J ¼ 7.2 Hz), 6.45 (s,1H, ArH), 6.93 (d,
1H, ArH, J ¼ 8.07 Hz), 7.31 (s, 1H, ArH), 7.39 (t, 1H, ArH, J ¼ 8.1 Hz),
7.44 (brs, 1H, ArH), 7.62 (d, 1H, ArH, J ¼ 8.16 Hz), 7.65 (brs, 1H,
PhNH), 8.22 (s, 1H, ArH), 11.01 (s, 1H, PhNHCO); 13C NMR (75 MHz,
In summary, a series of irreversible EGFR inhibitors 12aei con-
taining 2-anilinopyrimidine as a scaffold and 3-aminopropamide as
a “protected” warhead have been designed and synthesized.
Several compounds exhibited stronger enzyme inhibitory activity
against EGFR L858R/T790M and antiproliferative effects on H1975
and HCC827 cells than gefitinib. It was observed that compounds
12a, 12g and 12h could be converted to the active compound 1 in
the presence of natural basic amino acid arginine under physio-
logical PBS conditions, and the amounts of compound 1 liberated
by these compounds were positively correlated with their inhibi-
tory effects on H1975 cells in vitro. Importantly, 12h showed
improved stability towards small thiol nuclephiles relative to 1
whose structure is same to 12h excepting an acrylamide moiety.
Interestingly, 12i, a NO donating analogue of 12h, showed more
potent and selective inhibition than 12h on EGFR mutant H1975
cells but only moderate anti-proliferation activity against cells with
WT EGFR (A431, A549, BEAS-2B and 16HBE cells), suggesting that it
might possess better selectivity and safety index. Significantly, 12i
produced high levels of NO in H1975 cells but not in non-tumorous
16HBE cells, and its antiproliferative activity was diminished by NO
scavenger hemoglobin. Furthermore, 12i dose-dependently pro-
duced inhibitory effects on EGFR and downstream signaling in
H1975 cells. Therefore, our novel findings provide a proof of prin-
ciple in design of new EGFR TKIs. Further investigation on the
in vivo growth inhibitory effects of human cancer cells and the
mechanisms underlying the action of these hybrids are undergoing,
and the results will be reported in the due course.
DMSO-d6):
d 170.05, 163.88, 158.57, 158.01, 152.13, 151.50, 148.38,
140.39, 129.68, 122.91, 119.52, 116.36, 116.07, 112.28, 106.40, 103.93,
99.86, 55.48, 54.59, 54.52, 48.48, 45.60, 44.48, 34.13; ESI-MS: m/z
540 [M þ H]þ; ESI-HRMS (m/z): [M þ H]þ calcd for C27H34ClN7O3,
540.2490; obsd 540.2493.
5.1.1.2. N-(3-((5-Chloro-2-((2-methoxy-4-(4-methylpiperazin-1-yl)
phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-3-(diethylamino)prop-
anamide (12b). The title compound was obtained starting from 11b
and 1. As a yellow solid, 55% yield. mp 79e81 ꢀC. Analytical data for
12b: 1H NMR (300 MHz, CDCl3):
d
1.11 (t, 6H, 2 ꢃ NCH2CH3, J ¼ 7.14
Hz), 2.37 (s, 3H, NCH3), 2.61 (t, 4H, 2 ꢃ NCH2CH2N, J ¼ 4.83 Hz), 2.69
(t, 4H, 2 ꢃ NCH2CH3, J ¼ 7.14 Hz), 2.83 (t, 4H, COCH2CH2N, J ¼ 6.09
Hz), 3.13 (t, 4H, 2 ꢃ NCH2CH2N, J ¼ 4.5 Hz), 3.81 (s, 3H, OCH3), 6.22
(dd, 1H, ArH, J ¼ 7.11 Hz), 6.45 (d, 1H, ArH, J ¼ 2.25 Hz), 6.92 (d, 1H,
ArH, J ¼ 7.92 Hz), 7.37 (t, 1H, ArH, J ¼ 8.13 Hz), 7.40 (s, 2H, ArH), 7.57
(d, 1H, ArH, J ¼ 7.95 Hz), 7.64 (brs, 1H, PhNH), 8.22 (s, 1H, ArH),11.29
(s, 1H, PhNHCO); 13C NMR (75 MHz, DMSO-d6):
d 175.42, 168.91,
163.49, 163.19, 157.41, 156.50, 153.24, 145.66, 134.94, 128.46, 124.80,
121.58, 121.29, 117.51, 111.66, 109.20, 105.12, 60.75, 59.78, 53.75,
53.20, 51.41, 50.85, 38.69, 16.33; ESI-MS: m/z 568 [M þ H]þ; ESI-
HRMS (m/z): [M þ H]þ calcd for C29H38ClN7O3, 568.2803; obsd
568.2805.
5. Experimental protocols
5.1.1.3. N-(3-((5-Chloro-2-((2-methoxy-4-(4-methylpiperazin-1-yl)
phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-3-(pyrrolidin-1-yl)prop-
anamide (12c). The title compound was obtained starting from 11c
and 1. As a yellow solid, 60% yield. mp 129e131 ꢀC. Analytical data
5.1. Chemistry
1H and 13C NMR spectra were recorded with a Bruker Avance
300 MHz spectrometer at 300 K, using TMS as an internal standard.
MS spectra were recorded on a Mariner Mass Spectrum (ESI) and
HRMS on Agilent technologies LC/MSD TOF. Melting points of in-
dividual compounds were determined on a Mel-TEMP II melting
point apparatus and uncorrected. TLCs and preparative TLC were
for 12c: 1H NMR (300 MHz, CDCl3):
d
1.93 (m, 4H, 2 ꢃ CH2), 2.43 (s,
3H, NCH3), 2.71 (m, 4H, 2 ꢃ NCH2CH2N), 2.84 (t, 2H, COCH2CH2N,
J ¼ 5.97 Hz), 2.98 (m, 4H, 2 ꢃ NCH2), 3.15 (m, 2H, COCH2CH2N), 3.17
(m, 4H, 2 ꢃ NCH2CH2N), 3.81 (s, 3H, OCH3), 6.22 (dd,1H, ArH, J ¼ 7.8
Hz), 6.43 (s, 1H, ArH), 6.92 (d, 1H, ArH, J ¼ 7.86 Hz), 7.37 (t, 1H, ArH,
J ¼ 8.04 Hz), 7.44 (s, 1H, ArH), 7.56 (d, 3H, 2 ꢃ ArH, PhNH,

C. Han et al. / European Journal of Medicinal Chemistry 77 (2014) 75e83
81
J ¼ 7.95 Hz), 8.21 (s, 1H, ArH), 10.78 (s, 1H, PhNHCO); 13C NMR
2.52 (t, 6H, 2 ꢃ NCH2CH2N, COCH2CH2N, J ¼ 4.02 Hz), 2.58 (t, 4H,
2 ꢃ NCH2CH2N, J ¼ 4.08 Hz), 2.72 (t, 2H, COCH2CH2N, J ¼ 5.7 Hz),
3.11 (t, 4H, 2 ꢃ NCH2CH2N, J ¼ 4.53 Hz), 3.52 (t, 4H, 2 ꢃ NCH2CH2N,
J ¼ 4.53 Hz), 3.82 (s, 3H, OCH3), 4.29 (q, 2H, COONCH2CH3, J ¼ 7.08
Hz), 6.22 (dd, 1H, ArH, J ¼ 7.77 Hz), 6.45 (s, 1H, ArH), 6.95 (d, 1H,
ArH, J ¼ 7.26 Hz), 7.30 (s, 1H, ArH), 7.40 (t, 2H, ArH, J ¼ 8.07 Hz), 7.62
(d, 2H, ArH, PhNH, J ¼ 7.44 Hz), 8.23 (s, 1H, ArH), 10.7 (s, 1H,
(75 MHz, DMSO-d6): d 169.06, 163.65, 158.26, 157.92, 152.11, 151.90,
148.24, 140.30, 129.68, 122.91, 119.60, 116.50, 116.17, 112.35, 106.45,
103.94, 99.90, 55.50, 54.30, 53.15, 50.37, 48.26, 45.26, 33.84, 22.86;
ESI-MS: m/z 566 [M þ H]þ; ESI-HRMS (m/z): [M þ H]þ calcd for
C
29H36ClN7O3, 566.2646; obsd 566.2644.
5.1.1.4. N-(3-((5-Chloro-2-((2-methoxy-4-(4-methylpiperazin-1-yl)
phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-3-(piperidin-1-yl)prop-
anamide (12d). The title compound was obtained starting from 11d
and 1. As a yellow solid, 61% yield. mp 190e192 ꢀC. Analytical data
PhNHCO); 13C NMR (75 MHz, DMSO-d6):
d 170.25, 163.66, 158.22,
157.92, 154.51, 152.13, 151.10, 148.39, 140.41, 129.68, 122.56, 119.50,
116.32, 116.09, 112.32, 106.39, 103.92, 99.80, 60.63, 55.47, 54.61,
53.60, 52.12, 48.59, 45.73, 43.27, 34.09, 14.52; ESI-MS: m/z 653
[M þ H]þ; ESI-HRMS (m/z): [M þ H]þ calcd for C32H41ClN8O5,
653.2967; obsd 653.2969.
for 12d: 1H NMR (300 MHz, CDCl3):
d 1.54 (m, 2H, CH2), 1.73 (m, 4H,
2 ꢃ CH2), 2.42 (s, 3H, NCH3), 2.71 (m, 10H, 2 ꢃ NCH2CH2N,
COCH2CH2N, 2 ꢃ NCH2), 2.91 (m, 2H, COCH2CH2N), 3.16 (m, 4H,
2 ꢃ NCH2CH2N), 3.81 (s, 3H, OCH3), 6.22 (dd, 1H, ArH, J ¼ 6.96 Hz),
6.44 (s, 1H, ArH), 6.92 (d, 1H, ArH, J ¼ 7.59 Hz), 7.38 (m, 1H, ArH),
7.46 (s,1H, ArH), 7.57 (m, 3H, 2 ꢃ ArH, PhNH), 8.21 (s,1H, ArH),11.08
5.1.1.8. N-(3-((5-Chloro-2-((2-methoxy-4-(4-methylpiperazin-1-yl)
phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-3-(4-(2-hydroxyethyl)
piperazin-1-yl)propanamide (12h). The title compound was ob-
tained starting from 11h and 1. As a yellow solid, 49% yield. mp 99e
(s, 1H, PhNHCO); 13C NMR (75 MHz, DMSO-d6):
d 169.98, 163.65,
158.23, 157.92, 152.13, 151.89, 148.40, 140.41, 129.68, 123.31, 119.57,
116.34, 116.07, 112.30, 106.42, 103.94, 99.86, 55.49, 54.44, 53.68,
53.25, 48.41, 45.47, 33.26, 24.78, 23.31; ESI-MS: m/z 580 [M þ H]þ;
ESI-HRMS (m/z): [M þ H]þ calcd for C30H38ClN7O3, 580.2803; obsd
580.2806.
101 ꢀC. Analytical data for 12h: 1H NMR (300 MHz, DMSO):
d 2.38
(s, 3H, NCH3), 2.51e2.66 (m, 16H, 6 ꢃ NCH2CH2N, COCH2CH2N), 3.15
(t, 2H, NCH2, J ¼ 3.87 Hz), 3.17 (brs, 4H, 2 ꢃ NCH2CH2N), 3.56 (brs,
2H, HOCH2), 3.74 (s, 3H, OCH3), 6.20 (brs, 1H, ArH), 6.38 (s, 1H, ArH),
6.93 (d, 1H, ArH, J ¼ 7.17 Hz), 7.26 (d, 1H, ArH, J ¼ 7.25 Hz), 7.38 (t,
1H, ArH, J ¼ 7.35 Hz), 7.47 (d, 1H, ArH, J ¼ 8.01 Hz), 7.58 (s, 1H, ArH),
8.10 (s, 1H, PhNH), 8.34 (s, 1H, ArH),10.35 (s, 1H, PhNHCO); 13C NMR
5.1.1.5. N-(3-((5-Chloro-2-((2-methoxy-4-(4-methylpiperazin-1-yl)
p h e n y l ) a m i n o ) p y r i m i d i n - 4 - y l ) o x y ) p h e n y l ) - 3 -
morpholinopropanamide (12e). The title compound was obtained
starting from 11e and 1. As a yellow solid, 64% yield. mp 103e
(75 MHz, DMSO-d6): d 170.97, 164.45, 159.00, 158.70, 152.89, 151.90,
148.86, 141.26, 130.42, 123.65, 120.48, 117.08, 116. 89, 113.10, 107. 29,
104.76, 100.73, 60.22, 58.32, 56.31, 54.94, 54.09, 53.22, 52.19, 48.89,
46.19, 34.65; ESI-MS: m/z 625 [M þ H]þ; ESI-HRMS (m/z): [M þ H]þ
calcd for C31H41ClN8O4, 625.3018; obsd 625.3020.
105 ꢀC. Analytical data for 12e: 1H NMR (300 MHz, CDCl3):
d
2.37 (s,
3H, NCH3), 2.53e2.60 (m, 10H, 2 ꢃ NCH2CH2N, COCH2CH2N,
NCH2CH2O), 2.71 (m, 2H, COCH2CH2N), 3.11 (m, 4H,
2
ꢃ
2 ꢃ NCH2CH2N), 3.72 (m, 4H, 2 ꢃ OCH2CH2N), 3.82 (s, 3H, OCH3),
6.21 (dd, 1H, ArH, J ¼ 6.96 Hz), 6.45 (s, 1H, ArH), 6.95 (d, 1H, ArH,
J ¼ 7.8 Hz), 7.33 (s, 1H, ArH), 7.41 (t, 2H, ArH, J ¼ 8.1 Hz), 7.63 (d, 2H,
5.1.1.9. 4-(2-(4-(3-((3-((5-Chloro-2-((2-methoxy-4-(4-
methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)
amino)-3-oxopropyl)piperazin-1-yl)ethoxy)-3-(phenylsulfonyl)-
1,2,5-oxadiazole 2-oxide (12i). The title compound was obtained
starting from 11i and 1. As a yellow solid, 60% yield. mp 132e134 ꢀC.
ArH, PhNH, J ¼ 7.11 Hz), 8.23 (s, 1H, ArH), 10.93 (s, 1H, PhNHCO); 13
C
NMR (75 MHz, DMSO-d6):
d 170.30, 163.66, 158.23, 157.91, 152.14,
151.90, 148.38, 140.43, 129.69, 122.91, 119.50, 116.32, 116.08, 112.32,
106.39, 103.93, 99.79, 66.12, 55.46, 54.60, 54.06, 53.00, 48.57, 45.72,
33.95; ESI-MS: m/z 582 [M þ H]þ; ESI-HRMS (m/z): [M þ H]þ calcd
for C29H36ClN7O4, 582.2596; obsd 582.2599.
Analytical data for 12i: 1H NMR (300 MHz, CDCl3):
d 2.35 (s, 3H,
NCH3), 2.57 (m, 14H, 6 ꢃ NCH2CH2N, COCH2CH2N), 2.69 (t, 2H,
COCH2CH2N, J ¼ 5.1 Hz), 2.82 (t, 2H, NCH2, J ¼ 5.28 Hz), 3.09 (brs,
4H, 2 ꢃ NCH2CH2N), 3.76 (s, 3H, OCH3), 4.51 (t, 2H, OCH2, J ¼ 5.22
Hz), 6.23 (d, 1H, ArH, J ¼ 6.54 Hz), 6.45 (s, 1H, ArH), 6.94 (d, 1H, ArH,
J ¼ 7.95 Hz), 7.31(s, 1H, ArH), 7.40 (t, 2H, ArH, J ¼ 8.16 Hz), 7.60 (t,
2H, ArH, J ¼ 7.41 Hz), 7.67 (d, 1H, ArH, J ¼ 6.93 Hz), 7.75 (t, 2H, ArH,
PhNH, J ¼ 7.41 Hz), 8.05 (d, 2H, ArH, J ¼ 7.32 Hz), 8.23 (s, 1H, ArH),
5.1.1.6. N-(3-((5-Chloro-2-((2-methoxy-4-(4-methylpiperazin-1-yl)
phenyl)amino)pyrimidin-4-yl)oxy)phenyl)-3-(4-methylpiperazin-1-
yl)propanamide (12f). The title compound was obtained starting
from 11f and 1. As a yellow solid, 55% yield. mp 173e175 ꢀC.
11.05 (s, 1H, PhNHCO); 13C NMR (75 MHz, DMSO-d6):
d 170.89,
Analytical data for 12f: 1H NMR (300 MHz, CDCl3):
d
2.29 (s, 3H,
164.15, 159.87, 159.21, 158.44, 152.65, 151.90, 140.94, 137.82, 136.57,
130.50, 130.21, 128.65, 127.82, 123.65, 119.99, 116.79, 116.53, 112.77,
106.86, 104.76, 100.32, 69.84, 56.06, 55.98, 55.12, 54.13, 53.28,
52.99, 49.08, 46.24, 34.69; ESI-MS: m/z 849 [M þ H]þ; ESI-HRMS
(m/z): [M þ H]þ calcd for C39H45ClN10O8S, 849.2909; obsd 849.2911.
NCH3), 2.38 (s, 3H, NCH3), 2.52 (t, 8H, 4 ꢃ NCH2CH2N, J ¼ 5.76 Hz),
2.61 (t, 6H, COCH2CH2N, 2 ꢃ NCH2CH2N, J ¼ 4.41 Hz), 2.71 (t, 2H,
COCH2CH2N, J ¼ 5.7 Hz), 3.13 (t, 4H, 2 ꢃ NCH2CH2N, J ¼ 4.62 Hz),
3.82 (s, 3H, OCH3), 6.23 (dd, 1H, ArH, J ¼ 7.92 Hz), 6.45 (s, 1H, ArH),
6.95 (d, 1H, ArH, J ¼ 8.07 Hz), 7.40 (t, 2H, ArH, J ¼ 8.16 Hz), 7.46 (s,
1H, ArH), 7.62 (d, 2H, ArH, PhNH, J ¼ 7.41 Hz), 8.23 (s,1H, ArH),11.06
(s, 1H, PhNHCO); 13C NMR (75 MHz, DMSO-d6): 170.33, 163.66,
158.23, 157.92, 152.14, 151.11, 148.31, 140.46, 129.67, 122.81, 119.55,
116.27, 116.05, 112.29, 106.05, 103.94, 99.84, 55.49, 54.52, 54.41,
53.49, 51.97, 48.49, 45.59, 45.27, 34.10; ESI-MS: m/z 595 [M þ H]þ;
ESI-HRMS (m/z): [M þ H]þ calcd for C30H39ClN8O3, 595.2912; obsd
595.2914.
5.2. In vitro stability and reactivity assays
Chemical stability was tested under physiological (0.01 M PBS,
pH 7.4), and alkaline (0.01 M borate buffer, pH 9.0) pH conditions, at
37 ꢀC. 1 mM Stock solutions of 12h and 1 were prepared in DMSO,
and each sample was incubated at a final concentration of 50 mM in
prewarmed buffered solutions (final concentration of DMSO: 5% v/
v). At the regular time points, solutions were sampled with same
volume of acetonitrile and immediately injected into the HPLC
system. For rat plasma stability assays, quickly-thawed rat plasma
was diluted to 50% (v/v) with 0.1 M PBS, pH 7.4. And then com-
pound stock solution in DMSO was added (final compound con-
5.1.1.7. Ethyl-4-(3-((3-((5-chloro-2-((2-methoxy-4-(4-
methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)oxy)phenyl)
amino)-3-oxopropyl)piperazine-1-carboxylate (12g). The title com-
pound was obtained starting from 11g and 1. As a yellow solid, 59%
yield. mp 98e100 ꢀC. Analytical data for 12g: 1H NMR (300 MHz,
centration: 50
mM, DMSO concentration: 5% v/v) and maintained at
CDCl3):
d
1.27 (t, 3H, COONCH2CH3, J ¼ 7.11 Hz), 2.36 (s, 3H, NCH3),
37 ꢀC. At specific time, two volumes of acetonitrile were added and

82
C. Han et al. / European Journal of Medicinal Chemistry 77 (2014) 75e83
the mixture was centrifuged (4 ꢀC, 14,000 g, 15 min) and analyzed
by RP HPLC. The reactivity of 12h and 1 toward dithiothreitol (DTT),
production, while sodium nitrate at different concentrations was
used as the positive control for the standard curve.
L-cysteine and reduced GSH was evaluated by adding a freshly
prepared thiol solution (2 mM) to PBS (pH 7.4) containing the test
5.2.6. Western blotting analysis
compound (1 mM). Formation of conjugates with LMW thiols after
The H1975 cells were incubated with 0.01, 0.1 or 1
vehicle control (0.1% DMSO) and 0.1 or 1 M gefitinib for 24 h.
Following incubation, the cells were harvested and lyzed. The cell
lysates (50 g/lane) were separated by sodium dodecyl sulfate-
mM 12i or
1 h of incubation at 37 ꢀC was measured by HPLCeUV.
m
5.2.1. Releasing ability of compound 1
m
The releasing ability of parent compound 1 was tested in PBS
(0.01 M, PH 7.4) solution with arginine (1 mM) at 37 ꢀC. 1 mM Stock
solutions of 12a, 12g and 12h were prepared in DMSO. Each sample
polyacrylamide gel electrophoresis (7.5% gel) and transferred onto
nitrocellulose membranes. After they were blocked with 5% fat-free
milk, the target proteins were probed with anti-EGFR, anti-
phospho-EGFR (Tyr1068), anti-Akt, antiphospho-Akt (Ser473),
with a final concentration of 50 mM was incubated in prewarmed
buffered solutions (final concentration of DMSO: 5% v/v). At the
regular time points, solutions were mixtured with same volume of
acetonitrile and immediately injected into HPLC system.
anti-ERK, antiphospho-ERK (Thr202/Tyr204), and anti-b-actin an-
tibodies (Cell Signaling, Boston, MA), respectively. The bound an-
tibodies were detected by horseradish peroxidase (HRP)-
conjugated second antibodies and visualized using the enhanced
chemiluminescent reagent.
The HPLC analysis was performed on a Shimadzu 2010-20AT
HPLC system on
a
C18 reversed-phase column (5
mm,
150 mm ꢃ 4.6 mm) by a gradient elution using (A) 0.05 M of
phosphate buffer (pH ¼ 3); (B) acetonitrile as the mobile phase
0 min (27% B) /12.5 min (55% B) /15 min (90% B), and then
returning to initial conditions after 4 min, followed by 5 min re-
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equilibration time. Injection volume: 10
mL. The flow rate was
1.0 mL/min, and ultraviolet detection was at 281 nm.
5.2.2. Cell lines and reagents
H1975 (NSCLC, EGFR L858R/T790M), HCC827 (NSCLC, EGFR del
E746_A750), A431 (epidermoid carcinoma, EGFR overexpression),
A549 (NSCLC, EGFR wild type), BEAS-2B and 16HBE (human
bronchial epithelial cells) cells were obtained from ATCC. The cells
were maintained at 37 ꢀC in a 5% CO2 incubator in DMEM or RPMI
1640 (Hyclone) containing 10% fetal bovine serum (FBS, Biochrom,
AG).
5.2.3. In vitro enzymatic activity assay
Wild type and EGFR mutants (L858R, L858R/T790M) and the Z0-
Lyte Kinase Kit were purchased from Invitrogen. Ten concentration
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