Noncovalent EGFR T790M/L858R inhibitors based on diphenylpyrimidine scaffold: Design, synthesis, and bioactivity evaluation for the treatment of NSCLC

Article abstract of DOI:10.1016/j.ejmech.2021.113626

A series of diphenylpyrimidine derivatives bearing a hydroxamic acid group was designed and synthesized as noncovalent EGFR^T790M/L858R inhibitors to improve the biological activity and selectivity. One of the most promising compound 9d effectively interfered EGFR^T790M/L858R binding with ATP and suppressed the proliferation of H1975 cells with IC₅₀ values of 1.097 nM and 0.09777 μM, respectively. Moreover, compound 9d also not only exhibited a high selective index of 43.4 for EGFR^T790M/L858R over the wild-type and 10.9 for H1975 cells over A431, but also exhibited low toxicity against the normal HBE cells (IC₅₀ > 20 μΜ). In addition, the action mechanism validated that compound 9d effectively inhibited cell migration and promoted cell apoptosis by blocking cell cycle at G₂/M stage. Furthermore, the target dose-dependently downregulated the expression of p-EGFR and arrested the activation of downstream Akt and ERK in H1975. All these studies provide important clues for the discovery of potent noncovalent EGFR^T790M/L858R inhibitors.

Full text of DOI:10.1016/j.ejmech.2021.113626

European Journal of Medicinal Chemistry 223 (2021) 113626
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Noncovalent EGFR T790M/L858R inhibitors based on
diphenylpyrimidine scaffold: Design, synthesis, and bioactivity
evaluation for the treatment of NSCLC
Lixue Chen a, Yunhao Zhang a, Liangliang Tian b, Changyuan Wang a, Tuo Deng b,
,
*
Xu Zheng a, Tong Wang b, Zhen Li a, Zeyao Tang a, Qiang Meng a, Huijun Sun a, Lei Li a
,
,
, ***
Xiaodong Ma a **, Youjun Xu b
a College of Pharmacy, Dalian Medical University, Dalian, 116044, PR China
b School of Pharmaceutical Engineering, Key Laboratory of Structure-Based Drug Design & Discovery (Ministry of Education), Shenyang Pharmaceutical
University, Shenyang, 110016, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of diphenylpyrimidine derivatives bearing a hydroxamic acid group was designed and synthe-
sized as noncovalent EGFRT790M/L858R inhibitors to improve the biological activity and selectivity. One of
the most promising compound 9d effectively interfered EGFRT790M/L858R binding with ATP and sup-
Received 19 March 2021
Received in revised form
10 May 2021
Accepted 3 June 2021
Available online 16 June 2021
pressed the proliferation of H1975 cells with IC50 values of 1.097 nM and 0.09777
mM, respectively.
Moreover, compound 9d also not only exhibited a high selective index of 43.4 for EGFRT790M/L858R over
the wild-type and 10.9 for H1975 cells over A431, but also exhibited low toxicity against the normal HBE
cells (IC50 > 20 mМ). In addition, the action mechanism validated that compound 9d effectively inhibited
Keywords:
NSCLC
EGFR T790M/L858R
Resistance
Noncovalent inhibitors
cell migration and promoted cell apoptosis by blocking cell cycle at G2/M stage. Furthermore, the target
dose-dependently downregulated the expression of p-EGFR and arrested the activation of downstream
Akt and ERK in H1975. All these studies provide important clues for the discovery of potent noncovalent
EGFRT790M/L858R inhibitors.
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
an effective method to treat lung cancer driven by EGFR-sensitive
mutations. Nevertheless, the point mutation of T790 M amino
EGFR-TK (Epidermal Growth Factor Receptor-Tyrosine Kinase) is
an essential enzyme for intracellular signaling and cell trans-
formation, and abnormal activation of EGFR signaling pathway is
widespread in many types of solid tumors [1,2], especially in non-
small cell lung cancer (NSCLC). Approximately 10e15% non-Asian
and 50% Asian patients with NSCLC harbor EGFR activating muta-
tions [3,4]. The majority of EGFR mutations in NSCLC occurs in
exons 18e21 of the tyrosine kinase domain of the receptor, such as
in-frame deletions in exon 19 (among which the most prominent is
del E746_A750, also known as Del 19) or missense mutation in exon
21 (L858R) [5]. Hence, targeting the inhibition of EGFR activation is
acid enhanced the affinity of EGFR with ATP and then reduced the
sensitivity to the first-generation inhibitor (gefitinib, 1/erlotinib, 2)
(Fig. 1) [6,7]. Based on the structural framework of first-generation
inhibitor, covalent irreversible second-generation EGFR inhibitors
such as Afatinib (3) [8] and Dacomitinib (4) [9] have been devel-
oped to objectively alleviate the problem of acquired drug resis-
tance caused by T790 M mutation. The acrylamide pharmacophore
in the structure reacts with the sulfhydryl group in Cys797 residue
(Michael addition reaction) to form a covalent bond that connects
the inhibitor to the catalytic region of target enzyme. However,
because of its failure to selectively inhibit the mutant EGFR, some
side effects were observed, and the application range of second-
generation inhibitor was narrowed in clinical trial [10,11]. Excit-
ingly, the third-generation inhibitors, namely, WZ4002 (5) [12] and
AZD9291 (6) [13,14], with the 2-arylamine pyrimidine scaffold
exhibited great selective inhibitory effects on EGFRT790M/L858R over
Wild-Type EGFR. AZD9291 was approved by the FDA in 2015 for the
* Corresponding author.
** Corresponding author.
*** Corresponding author.
E-mail addresses: lilei0332@126.com (L. Li), xiaodong.ma@139.com (X. Ma),
xuyoujun@syphu.edu.cn (Y. Xu).
https://doi.org/10.1016/j.ejmech.2021.113626
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

L. Chen, Y. Zhang, L. Tian et al.
European Journal of Medicinal Chemistry 223 (2021) 113626
Fig. 1. Chemical structures of some EGFR inhibitors.
treatment of EGFRT790M/L858R mutant lung cancer. With further
development of clinical application, approximately 40% [15] of the
patients treated with AZD9291 developed a tertiary Cys797 to Ser
797 (C797S) point mutation, thus hindering the covalent bond
formation from irreversible inhibitors binding to Cys797 [16,17].
Loss of covalent interaction resulted by C797S mutation causes a
significant decrease in the effects of treatment, leading to the
development of resistance. Hence, based on the novel 2-arylamine
pyrimidine template, it is essential to develop reversible EGFR in-
hibitors that not only exhibit excellent inhibitory effects on the
acquired drug-resistant T790M mutation but also remain potent in
other activating mutants [18,19].
reduced in Argon atmosphere to produce the corresponding
products 14a-k using ZneNH4Cl. Moreover, regioselective substi-
tution of the C-2 chlorine atom in 12a with NH2 intermediates 14a-
h using TsOH afforded the corresponding products 15a-h. Finally,
the targets 9a-h were obtained by nucleophilic substitution with
hydroxylamine hydrochloride using CH3ONa. Compounds 9i-k
were also produced using similar methods and conditions. Com-
pound 9l with two hydroxamic acid groups was obtained by pro-
longing the reaction time of preparing 9g. In addition, compound
9m was synthesized by the hydrolysis reaction of compound 15g.
2.2. Biological activity
Our previous study also validated that this 2-arylamine pyrim-
idine skeleton has strong activity against a panel of kinases,
including FAK [20], JAK3 [21], and BTK [22]. We also synthesized a
class of N-alkylbenzamide-substituted diphenylpyrimidines based
on TAE-226 (FAK inhibitor) [23] and Mor-DPPY (7, anti-EGFRT790M/
L858R) [24] using the fragment-based drug design strategy. Inter-
estingly, the representative compound 8 [20] showed improved
inhibitory activity against EGFRT790M/L858R than AZD9291. Although
compound 8 only showed moderate antiproliferative activity
2.2.1. EGFR inhibitory and selective activity assay
Activities of all the targets against EGFRWT and EGFRT790M/L858R
were evaluated using ADP-Glo™ kinase assay system. One positive
control, AZD9291, was also investigated under the same conditions.
The results shown in Table 1 indicate that installing hydroxamic
acid on C-200 position of ring A was an effective method to design
noncovalent anti-EGFRT790M/L858R inhibitors. The enzyme-based
results show that the designed noncovalent targets displayed
strong inhibitory activities against the EGFRT790M/L858R with IC50
ranging from 0.2446 to 198.5 nM. Among them, compounds 9d
(IC50 ¼ 1.097 nM) and 9e (IC50 ¼ 6.820 nM) showed stronger affinity
to EGFRT790M/L858R than AZD9291 (IC50 ¼ 20.80 nM). Some com-
pounds 9d and 9g showed selective inhibitory effects on
EGFRT790M/L858R over Wild-Type EGFR, indicating their weak
interference with the expression of EGFRWT. It was observed that
the linker installed between rings C and D clearly effected the ac-
tivities; the substitution of linear carbon units was the most helpful
in inhibiting the EGFRT790M/L858R binding with ATP, such as com-
pounds 9d and 9e. Compared with N-methylpiperazinyl group,
compound 9d substituted with a morpholinyl group exhibited
excellent selectivity with an SI of 47. When a hydroxamic acid group
was installed on ring D, compounds 9i, 9j, and 9k showed weak
inhibitory activities against EGFRT790M/L858R in spite of replacing the
linker with 0e3 carbon units. Compound 9l with two hydroxamic
acid groups exhibited moderate anti-EGFRT790M/L858R activity
without selectivity. Notably, the ester-substituted compound
against H1975 (IC50 of 0.333
mM), it provided valuable clues to
design potent noncovalent inhibitors against EGFRT790M/L858R. In
addition, molecular docking studies suggested that double
hydrogen bonding interactions were formed between the 200-amide
group of compound 8 and the residues of Asp800. Accordingly,
further structural optimization of the hit compound 8 was carried
out by introducing a hydroxamic acid group instead of an amide
group with an aim to improve the anticancer activity in this study
(Fig. 2).
2. Results and discussion
2.1. Chemistry
All the newly designed targets were synthesized as shown in
Scheme 1. Commercially available 3-chloropropionyl chloride was
reacted with 2-aminobenzoic acid ethyl ester in the presence of a
base, DIPEA, to afford 12a and 12b. Compounds 13a-k were then
2

L. Chen, Y. Zhang, L. Tian et al.
European Journal of Medicinal Chemistry 223 (2021) 113626
Fig. 2. Design strategy of title compounds.
Scheme 1. Synthetic route of targets 9a‒m, 15g.
(prodrug form of carboxylic acid) 15g also showed strong kinase
inhibitory activity, and it is worthy for further evaluation.
lines A431-EGFRWT, H1975-EGFRT790M/L858R, A549-KRASG12S, and
H23 KRASmutant, one human metastatic pancreatic adenocarcinoma
cell line (Aspc-1), and one normal cell line (HBE). The results shown
in Table 2 indicate that all the targets showed strong inhibitory
2.2.2. Cellular inhibitory and selective activity assay
The antiproliferative activities of the targets and positive control
AZD9291 were studied by conducting a CCK-8 assay in NSCLC cell
activity against H1975 with IC50 < 1.11
mM. Compounds 9b and 9c
(IC50 0.0781 M) showed more activity than AZD9291
<
m
3

L. Chen, Y. Zhang, L. Tian et al.
European Journal of Medicinal Chemistry 223 (2021) 113626
Table 1
activity against H1975, with IC50 values of 0.1291
0.3846 M, respectively. It is also not conducive for antitumor ac-
tivity when ring D is replaced with a hydroxamic acid group (9l,
IC50 ¼ 1.110 M). In addition, the yield of targets 9m (IC50 > 10 M)
and 15g (IC50 ¼ 2.905 M) without the substitution of hydroxamic
mM and
EGFR tyrosine kinase inhibitory activities of 9a-m, 15g in vitroa.
m
m
m
m
acid group at the C-200 position of ring A showed a significant
decrease against inhibiting the proliferation of cells, further con-
firming the rationality of the design strategy. Notably, compounds
9b or 9c showed strong inhibitory activity and selectivity against
H1975, but their hepatotoxicity prevented the possibility of further
study of the target. According to the test results of activity, selec-
tivity, and toxicity, compound 9d was selected as the candidate for
further mechanism research.
.
Compd.
R1
R2
Enzymatic activity (IC50, nM)
EGFRT790M/L858R
EGFRWT
9a
9b
9c
9d
-CONHOH
-CONHOH
-CONHOH
-CONHOH
116.5 5.68
168.7 7.21
68.81 2.31
98.78 1.56
1.097 0.47
6.564 1.23
4.495 0.56
47.66 3.20
2.2.3. Migration inhibition assay
A wound-healing assay, as shown in Fig. 3, was preformed to
evaluate the inhibition of migration of H1975. After scratching the
cell monolayer, H1975 cells were exposed to different concentra-
tions of 0.5 and 2 mM for 0, 24, and 48 h. The negative control group
9e
-CONHOH
6.82 1.14
0.7632 0.04
demonstrated the strongest migratory ability, and the width of the
scratch was significantly narrowed. As expected, the transfer of cell
numbers to the scratch in the compound 9d-treated groups was
obviously less than the negative control group. The evaluation of
wound-healing assay indicated that the migration and invasion of
H1975 cells were effectively inhibited by compound 9d.
9f
-CONHOH
-CONHOH
64.79 4.69
16.04 1.56
1.246 0.48
57.31 2.35
9g
9h
-CONHOH
113.5 6.51
126.0 5.24
2.2.4. Morphological staining analysis
DAPI staining was carried out to observe the morphological
changes in H1975 cells exposed with compound 9d in different
concentrations of 0.5 and 2.0 mM for 48 h. As shown in Fig. 4A,
subsequent to DAPI staining, chromatin shrinks as well as gathering
towards the nuclear membrane were clearly observed. With the
increase in concentration, the nucleus was disintegrated to from
fragments. Under natural lights, the proportion of abnormal cells
such as fusiform and sickle type increased significantly (Fig. 4B). All
the phenomena indicate the characteristics of apoptotic pro-
grammed cell death.
9i
9j
-CONHMe
-CONHMe
-CONHMe
49.94 5.65
117.5 3.21
44.96 2.50
118.7 4.52
1.095 0.26
46.69 4.32
9k
9l
-CONHOH
-COOH
65.41 3.22
198.5 6.78
50.22 5.20
81.40 2.31
9m
15g
-COOEt
0.2446 0.04
32.00 1.58
2.2.5. Flow cytometry analysis
To gain an in-depth understanding of mechanism of action, the
apoptosis and cycle of H1975 cells treated with compound 9d were
evaluated by flow cytometry analysis. The analysis results show
that after exposure with compound 9d in different concentrations
AZD9291
e
e
20.80 2.22
567.5 6.57
a
Dose-response curves were determined at three concentrations. The IC50 values
are the concentrations in nanomolar level needed to inhibit the cell growth by 50%
as determined from these curves.
of 0.125, 0.5, and 2 mM for 48 h, the apoptosis rate of H1975 cells
increased from 4.31% to 91.22% in a dose-dependent manner
(Fig. 5A). Correspondingly, the effect of compound 9d on cell cycle
progression is shown in Fig. 5B. When exposed with the dosage of
(IC50
(IC50 ¼ 0.09777
¼
0.091
m
M). Compounds 9a (IC50
¼
0.1093) and 9d
0.125 mM, the percentage of G1/G0 phase significantly decreased
mM) showed equivalent potency against prolifera-
from 58.05% to 20.25%. The percentage of G2/M phase increased
from 30.23% to 64.66%, and the percentage of S phase increased
from 11.72% to 15.08% as well. With the increase in concentration to
0.5 mM, conversely, the proportion of S phase decreased to 9.86%,
and the proportion of G2/M phase further increased up to 70.99%.
Notably, when the concentration of exposure was further increased
tion compared with the positive drug. Several of the targets, 9a, 9c,
and 9d, showed high selective antiproliferative activity against
H1975 over A431 (SI > 10), indicating their weak side effects.
Meanwhile, the targets also showed strong inhibitory activity
against A549, H23 bearing k-ras mutant tumor cells, and ASCP-1
with the values of IC50 in nanomolar level. However, these in-
hibitors are not sensitive to the normal HBE cells with concentra-
to 2.0 mM, the machine failed to capture the process of blocking cell
division due to a large number of dead cells left (Table 3). Flow
cytometric analysis indicated that compound 9d induced the
apoptosis of H1975 by arresting the cell cycle at the G2/M stage.
tions of up to 8.566 mM, indicating their low cytotoxicity. The SAR
analysis of targets indicates that when ring D was substituted with
a morpholinyl group, one carbon unit linker between rings C and D
was the most effective for the activity and selectivity than by
2.2.6. Effects of the inhibitors on EGFRT790M/L858R activation and
downstream signaling
extending or shortening the linker (9c, IC50 < 0.0781
mM). The
substitution of ring D with an N-methylpiperazinyl group, 9b, with
rings C and D connected directly, showed the highest activity
With strong kinase and cellular inhibitory activities, compound
9d was further validated by evaluating its potential suppression on
the activity of EGFRT790M/L858R and downstream signaling in
H1975 cells. The cells were exposed with compound 9d in various
(IC50 < 0.0781
mM). When replacing the linker with an acetamide
linker, compounds 9g and 9h exhibited moderate inhibitory
4

L. Chen, Y. Zhang, L. Tian et al.
European Journal of Medicinal Chemistry 223 (2021) 113626
Table 2
Cellular antiproliferative activities of 9a-m and 15g (IC50, mM).
Compd.
Anti-proliferative activity (IC50
,
m
M)
ASPC-1
SI
H1975
A431
A549
HBE
H23
L-02
9a
9b
9c
9d
9e
9f
9g
9h
9i
0.1093 0.09
<0.0781
<0.0781
1.581 0.22
0.2532 0.07
2.427 0.12
1.066 0.10
0.6916 0.06
1.404 0.11
0.6698 0.03
1.532 0.11
0.1271 0.06
1.156 0.12
0.4482 0.04
2.065 0.23
e
0.1093 0.13
>40
22.59 1.58
29.11 1.23
23.61 0.78
29.83 1.25
26.51 2.21
20.21 0.78
>40
12.65 0.23
15.93 0.32
8.566 0.58
>40
1.410 0.23
<0.156
<0.156
0.8007 0.05
0.2173 0.05
0.5537 0.06
0.6249 0.09
0.6425 0.07
0.665 0.09
0.8195 0.06
1.74 0.06
1.541 0.07
8.425 1.10
2.114 0.21
6.397 0.98
e
2.191 0.23
<1.25
14.46
>3.24
>31.08
10.90
0.84
4.38
5.19
3.98
0.39
4.72
2.88
1.86
e
<0.078
<0.078
<1.25
0.09777 0.02
0.8218 0.05
0.3209 0.07
0.1291 0.07
0.3846 0.06
0.3289 0.03
0.245 0.04
0.1556 0.07
1.11 0.11
>10
0.0977 0.03
0.2028 0.01
0.3209 0.05
0.1291 0.06
0.3846 0.07
0.3289 0.07
0.245 0.09
0.1556 0.02
1.11 0.09
e
0.1691 0.05
0.2442 0.05
1.229 0.12
0.5302 0.06
0.7498 0.04
0.4994 0.02
0.3741 0.02
<0.156
3.052 0.56
1.9494 0.97
1.733 0.55
4.814 0.87
2.114 0.56
1.733 0.55
1.278 0.80
<1.25
7.869 0.40
15.51 1.25
30.22 2.32
e
9j
9k
9l
1.577 0.36
e
9m
15g
AZD9291
e
2.905 0.23
0.091 0.01
e
e
e
e
e
e
1.226 0.12
e
e
e
e
e
a Dose-response curves were determined at five concentrations. The IC50 values are the concentrations in micromolar level needed to inhibit the cell growth by 50% as
determined from these curves.
concentrations (0.5 and 1.0
mM) for 24 h, and then the levels of p-
at concentrations from 0.5 to 1.0 mM. These results indicated that 9d
could block the autophosphorylation of EGFR and then inhibited
the activation of the downstream signaling kinases.
EGFRT790M/L858R, p-AKT and p-ERK were determined by immuno-
blotting analysis. The results shown in Fig. 6 show that the
expression of p-EGFRT790 M/L858R decreased after administration
of 9d with the concentration of 0.5
phosphorylation of AKT and ERK, which acted downstream of the
signaling pathway, was also inhibited significantly. When the
dosage was increased to 1.0 mM, the expression of p-EGFR as well as
p-Akt and p-ERK further decreased in a dose-dependent manner.
Similar results were obtained in H1975 cells treated with AZD9291
mM. Correspondingly, the
2.2.7. Pharmacokinetic studies
After intravenous injection of 9d in SD rats, the pharmacokinetic
parameters and curve of the blood drug concentrationetime and
are shown in Table 4 and Fig. 7. The drug concentration declined
rapidly within 12 h, the elimination half-life was 9.51 h. Strikingly,
the area under the drug time curve of 9d (AUC(0-∞)
) was
Fig. 3. Representative images of H1975 cells treated with different concentrations of 9d for 0, 24, and 48 h by the wound-healing assay.
5

L. Chen, Y. Zhang, L. Tian et al.
European Journal of Medicinal Chemistry 223 (2021) 113626
Fig. 4. Morphological changes in H 1975 cells (100 ꢀ , final magnification). A): DAPI staining of H 1975 cells treated with different concentrations of compound 9d for 72 h. B):
Under natural light, H1975 cells treated with different concentrations of compound 9d for 72 h.
45192.58
9d was 13.13 h.
m
g/L*h. Meanwhile, the mean residence time MRT(0-∞) of
catalytic site of EGFRT790M/L858R kinase as a model, the binding
mode of compound 9d with EGFRT790M/L858R, was elucidated using
AutoDock 4.2 software. The results of docking study indicate that
compound 9d exactly occupied the site of ATP-binding pocket in a
U-shape, but the binding conformation of compound 9d was
reversed to that of WZ4002. The interactions between compound
2.3. Molecular modelling analysis
The crystal complex (PDB code: 3IKA) of WZ4002 binding to the
6

L. Chen, Y. Zhang, L. Tian et al.
European Journal of Medicinal Chemistry 223 (2021) 113626
Fig. 5. A) Compound 9d induced H1975 cells apoptosis in vitro. B) Effects of compound 9d on H1975 cycle arrest were detected by flow cytometry assay.
7

L. Chen, Y. Zhang, L. Tian et al.
European Journal of Medicinal Chemistry 223 (2021) 113626
Table 3
The change of cell cycle and apoptotic proportion of H1975 cells after administration of 9d (%).
G1/G0
S
G2/M
Necrotic cells
Viable apoptosis
Non-viable apoptosis
CON.
0.125
58.05
20.25
19.16
57.28
11.72
15.08
9.86
30.23
64.66
70.99
31.90
2.99
0.01
0.07
0.14
0.73
0.31
0.56
0.67
1.39
4.00
15.53
90.55
mM
0.5
2.0
m
M
mM
10.83
affinity of enzyme binding the target over ATP. 2) Several van der
Waals interactions existed between compound 9d and the residues
of protein, such as ring A with Leu 718 and ring C and D with Leu
844 (Fig. 8). The docking model combined with the data obtained
from the biological assays provided a structural basis for further
development of noncovalent EGFRT790M/L858R inhibitors.
3. Conclusion
Replacement of the acrylamide group with an N-methylformyl
group installed on the C-200 position of 4-aniline in WZ4002 pro-
vided a significant method for designing noncovalent EGFRT790M/
L858R
inhibitors and a desirable starting point for further optimi-
zation.
A class of diphenylpyrimidine derivatives bearing a
hydroxamic acid functionality was synthesized as effective non-
covalent EGFRT790M/L858R inhibitors. Among them, several com-
pounds not only showed strong inhibitory activity against
EGFRT790M/L858R and selectivity of binding the receptor, but also
displayed high antiproliferative activity against H1975 cells.
Furthermore, these inhibitors induced remarkable apoptosis in
H1975 cells by blocking the cell cycle at the G2/M stage, as well as
inhibited the activation of EGFR and its downstream pathways.
Overall, all these studies provide a new insight to discover novel
noncovalent inhibitors for the treatment of NSCLC harboring
EGFRT790M/L858R mutation.
Fig. 6. Compound 9d inhibited the autophosphorylation of EGFRT790M/L858R and acti-
vation of its downstream signaling proteins (A) and grayscale analysis (B) in
H1975 cells.
Table 4
Pharmacokinetic parameters of 9d after intravenous administration.
9d
AUC(0-t)
AUC(0-∞) (mg/L*h)
MRT(0-t) (h)
MRT(0-∞) (h)
t1/2z (h)
(
m
g/L*h)
37632.19 1244.71
45192.58 2100.40
8.196 0.213
13.13 0.75
4. Experimental section
9.51 0.48
4.1. General methods and chemistry
CLz (L/h/kg)
0.554 0.03
Unless otherwise noted, commercial solvents and reagents were
used without further purifications. High resolution ESI-MS was
performed on an Agilent 1100 HPLC/MS system. 1H NMR and 13C
NMR spectra on a Brucker AV 400 MHz spectrometer were recorded
in DMSO‑d6. Coupling constants (J) are expressed in hertz (Hz).
Chemical shifts (d) of NMR are reported in parts per million (ppm)
units relative to internal control (TMS). All reactions were moni-
tored by TLC, using silica gel plates with fluorescence GF254 and UV
light visualization. Flash chromatography separations were ob-
tained on Silica Gel (300e400 mesh) using dichloromethane/
methanol as eluents.
4.2. General procedure for the synthesis of 9a-m
11a-b (50 mmol), 2,4,5-trichloropyrimidine (50 mmol) and
DIPEA (100 mmol) in acetonitrile, it was gradually heated to 80 ꢁ
C
and stirred for 10 h. The precipitate was filtrated and washed with
water, and the acquired production could directly be used without
further purification. 13a-k was prepared according to the reported
process [20e22,24]. Ar atmosphere, a mixture of 13a-k (15 mmol),
ammonium chloride (20 mmol) and zinc powder (120 mmol) in
ethanol/water (100/20 mL) was reacted at 70 ꢁC for 1 h. After the
reaction, the solvent was distilled off and the residue was dispersed
in dichloromethane. The zinc powder was removed by filtration,
and the organic layer was washed with dilute ammonia and dried
over anhydrous sodium sulfate. The organic was vacuum distilled to
Fig. 7. Drug concentration-time curve of 9d.
9d and EGFRT790M/L858R included two parts: 1) The hydroxamic acid
functional group of the molecule formed two hydrogen bonds with
Asp800 residue, and another strong hydrogen bond was estab-
lished between the 2-amino group in the pyrimidine core of
compound 9d and Met 793 in the kinase hinge. The hydrogen
bonding forces were envisioned to be important for improving the
8

L. Chen, Y. Zhang, L. Tian et al.
European Journal of Medicinal Chemistry 223 (2021) 113626
Fig. 8. Putative binding mode of 9d with EGFRT790M/L858R. (A) Detailed interactions with the protein residues. Each dashed yellow line represents hydrogen bonds. (B) Three-
dimensional space matching diagram of 9d and the active site.
give the crude which was purified by silica-gel column separation
to get the 14a-k.
128.08, 122.65, 122.19, 120.09 (2C), 119.84, 105.39, 66.68 (2C), 62.58,
53.58 (2C). HRMS (ESIþ) for C22H23ClN6O3, [MþH]þ calcd: 455.1593,
found: 455.1564.
A mixture of 12a-b (5.0 mmol), 14a-k (5.0 mmol), and p-TsOH
(7.5 mmol) in ethyl alcohol was gradually heated to 70 ꢁC and
stirred for 3 h. After reaction, the solvent was vacuum distilled and
the residuum was dispersed with saturated sodium bicarbonate/
ethyl acetate. The crude product was purified using flash chroma-
tography with dichloromethane/methanol as eluents.
4.2.4. 2-((5-chloro-2-((4-(2-morpholinoethoxy)phenyl)amino)
pyrimidin-4-yl)amino)-N-hydroxy benzamide (9d)
Yield 61.2%; off-white solid; dichloromethane/methanol: 12/1
(v/v). 1H NMR (400 MHz, DMSO‒d6):
d 11.51 (s, 1H), 11.16 (s, 1H),
Ar atmosphere,15a-k (2.0 mmol) and hydroxylamine (40 mmol)
in anhydrous methanol, sodium methoxide (25 mmol) in methanol
solution was slowly added to the mixture. After the addition, the
reaction was allowed to warm room temperature and stirred for
2 h. Adjusted pH to 6 with dilute hydrochloric acid, the inorganic
salt was removed by filtration and the rest of organic phase was
9.35 (s, 1H), 9.30 (s, 1H), 8.69 (s, 1H), 8.18 (s, 1H), 7.61 (d, J ¼ 7.68 Hz,
1H), 7.51 (d, J ¼ 8.52 Hz, 2H), 7.48 (t, J ¼ 8.28 Hz, 1H), 7.11 (t,
J ¼ 7.32 Hz, 1H), 6.87 (d, J ¼ 8.94 Hz, 2H), 4.06 (t, J ¼ 5.70 Hz, 2H),
3.59 (t, J ¼ 3.96 Hz, 4H), 2.71e2.68 (m, 2H), 2.50e2.42 (m, 4H); 13
C
NMR (100 MHz, DMSO‒d6):
d 166.23, 158.45, 155.37, 155.17, 154.18,
139.54, 133.77, 131.92, 128.02, 122.50, 122.06 (2C), 122.01, 119.63,
114.80 (2C), 104.80, 66.55 (2C), 65.85, 57.53, 54.07 (2C). HRMS
(ESIþ) for C23H25ClN6O4, [MþH]þ calcd: 485.1699, found: 485.1673.
~
vacuum distilled to produce the 9a k.
4.2.1. 2-((5-chloro-2-((4-morpholinophenyl)amino)pyrimidin-4-yl)
amino)-N-hydroxy benzamide (9a)
4.2.5. 2-((5-chloro-2-((4-(2-(4-methylpiperazin-1-yl)ethoxy)
phenyl)amino)pyrimidin-4-yl)amino)-N-hydroxybenzamide (9e)
Yield 62.3%; off-white solid; dichloromethane/methanol: 10/1
Yield 65.2%; off-white solid; dichloromethane/methanol: 10/1
(v/v). 1H NMR (400 MHz, DMSO‒d6):
d 11.51 (s, 1H), 11.17 (s, 1H),
(v/v). 1H NMR (400 MHz, DMSO‒d6):
d 11.16 (s,1H), 9.29 (s,1H), 8.68
9.34 (s, 1H), 9.26 (s, 1H), 8.71 (s, 1H), 8.17 (s, 1H), 7.61(dd, J ¼ 7.84,
1.14 Hz, 1H), 7.49e7.47 (m, 3H), 7.12(td, J ¼ 7.83, 0.84 Hz, 1H), 6.89
(d, J ¼ 8.89 Hz, 2H), 3.74 (t, J ¼ 4.92 Hz, 4H), 3.05 (t, J ¼ 4.62 Hz, 4H);
(s, 1H), 8.18 (s, 1H), 7.61 (d, J ¼ 7.62 Hz, 1H), 7.51 (d, J ¼ 8.28 Hz, 2H),
7.48 (t, J ¼ 7.68 Hz, 1H), 7.11 (t, J ¼ 7.44 Hz, 1H), 6.86 (d, J ¼ 8.82 Hz,
2H), 4.03 (t, J ¼ 5.76 Hz, 2H), 3.42e3.40 (m, 4H), 2.66 (t, J ¼ 5.70 Hz,
2H), 2.38e2.27 (m, 4H), 2.16 (s, 3H); 13C NMR (100 MHz, DMSO‒d6):
13C NMR (100 MHz, DMSO‒d6):
d 166.34, 158.48, 155.36, 155.14,
147.01, 139.61, 132.92, 131.98, 128.01, 122.48, 121.99, 121.75 (2C),
119.54, 115.97 (2C), 104.65, 66.65, 49.72. HRMS (ESIþ) for
d
166.20, 158.46, 155.37, 155.16, 154.25, 139.53, 133.72, 131.89,
C
21H21ClN6O3, [MþH]þ calcd: 441.1436, found: 441.1409.
128.01,122.49,122.09 (2C),122.01,119.66,114.78 (2C),104.79, 66.19,
57.14, 55.13 (2C), 53.43 (2C), 46.13. HRMS (ESIþ) for C24H28ClN7O3,
[MþH]þ calcd: 498.2015, found: 498.1997.
4.2.2. 2-((5-chloro-2-((4-(4-methylpiperazin-1-yl)phenyl)amino)
pyrimidin-4-yl)amino)-N-hydroxy benzamide (9b)
Yield 62.3%; off-white solid; dichloromethane/methanol: 10/1
4.2.6. 2-((5-chloro-2-((4-(3-morpholinopropoxy)phenyl)amino)
pyrimidin-4-yl)amino)-N-hydroxy benzamide (9f)
(v/v). 1H NMR (400 MHz, DMSO‒d6):
d 11.17 (s, 1H), 9.23 (s,1H), 8.71
(s, 1H), 8.17 (s,1H), 7.61(dd, J ¼ 7.75, 0.96 Hz,1H), 7.48e7.46 (m, 3H),
7.11(td, J ¼ 7.59, 0.78 Hz, 1H), 6.87 (d, J ¼ 9.00 Hz, 2H), 3.08 (t,
J ¼ 4.62 Hz, 4H), 2.49 (t, J ¼ 4.60 Hz, 4H), 2.24 (s, 3H); 13C NMR
Yield 55.0%; off-white solid; dichloromethane/methanol: 12/1
(v/v). 1H NMR (400 MHz, DMSO‒d6):
d 11.51 (s, 1H), 11.16 (s, 1H),
9.37 (s, 1H), 9.29 (s, 1H), 8.69 (s, 1H), 8.18 (s, 1H), 7.61 (d, J ¼ 7.62 Hz,
1H), 7.50 (d, J ¼ 8.52 Hz, 2H), 7.47 (t, J ¼ 7.62 Hz, 1H), 7.11 (t,
J ¼ 7.56 Hz, 1H), 6.85 (d, J ¼ 8.94 Hz, 2H), 3.97 (t, J ¼ 6.30 Hz, 2H),
3.57 (t, J ¼ 4.14 Hz, 4H), 2.42 (t, J ¼ 7.08 Hz, 2H), 2.39e2.36 (m, 4H),
(100 MHz, DMSO‒d6):
d 166.24, 158.52, 155.34, 155.16, 146.98,
139.60, 132.61, 131.93, 127.99, 122.43, 121.95 (2C), 121.77, 119.51,
116.20 (2C), 104.58, 55.07 (2C), 49.19 (2C), 46.10. HRMS (ESIþ) for
22H24ClN7O2, [MþH]þ calcd: 454.1753, found: 454.1730.
1.88e1.84 (m, 2H); 13C NMR (100 MHz, DMSO‒d6):
d 166.22, 158.48,
C
155.37, 155.17, 154.41, 139.55, 133.65, 131.90, 128.02, 122.48, 122.14
(2C), 122.00, 119.62, 114.76 (2C), 104.80, 66.63 (2C), 66.40, 55.38,
53.82 (2C), 26.39. HRMS (ESIþ) for C24H27ClN6O4, [MþH]þ calcd:
499.1855, found: 499.1837.
4.2.3. 2-((5-chloro-2-((4-(morpholinomethyl)phenyl)amino)
pyrimidin-4-yl)amino)-N-hydroxy benzamide (9c)
Yield 56.4%; off-white solid; dichloromethane/methanol: 12/1
(v/v). 1H NMR (400 MHz, DMSO‒d6):
d 11.18 (s,1H), 9.46 (s, 1H), 8.70
(s, 1H), 8.22 (s, 1H), 7.63e7.59 (m, 3H), 7.47 (t, J ¼ 7.27 Hz, 1H), 7.18
4.2.7. 2-((5-chloro-2-((4-(2-morpholino-2-oxoethoxy)phenyl)
amino)pyrimidin-4-yl)amino)-N-hydroxybenzamide (9g)
Yield 56.3%; off-white solid; dichloromethane/methanol: 12/1
(d, J ¼ 7.92 Hz, 2H), 7.13 (t, J ¼ 7.26 Hz, 1H), 3.57e3.54 (m, 4H), 3.39
(s, 2H), 2.36e2.31 (m, 4H); 13C NMR (100 MHz, DMSO‒d6):
d 166.23,
158.28, 155.44, 155.16, 139.57, 139.49, 131.87, 131.38, 129.62 (2C),
(v/v). 1H NMR (400 MHz, DMSO‒d6):
d 11.50 (s, 1H), 11.17 (s, 1H),
9

L. Chen, Y. Zhang, L. Tian et al.
European Journal of Medicinal Chemistry 223 (2021) 113626
9.34 (s, 1H), 9.30 (s, 1H), 8.70 (s, 1H), 8.18 (s, 1H), 7.61 (d, J ¼ 7.62 Hz,
1H), 7.51e7.46 (m, 3H), 7.10 (t, J ¼ 7.55 Hz, 1H), 6.86 (t, J ¼ 8.90 Hz,
2H), 4.79 (s, 2H), 3.61 (t, J ¼ 4.30 Hz, 2H), 3.57 (t, J ¼ 4.30 Hz, 2H),
(2C), 26.78, 26.82. HRMS (ESIþ) for C27H32ClN7O4, [MþH]þ calcd:
554.2277, found: 554.2272.
3.49e3.45 (m, 4H); 13C NMR (100 MHz, DMSO‒d6):
d
166.69,
4.2.12. 2-((5-chloro-2-((4-(2-(hydroxyamino)-2-oxoethoxy)
166.28, 158.48, 155.37, 155.16, 153.76, 139.54, 134.04, 131.92, 128.00,
122.44 (2C), 122.12, 122.00, 119.60, 114.92 (2C), 104.84, 70.25, 66.71,
66.55, 45.32, 42.09. HRMS (ESIþ) for C23H23ClN6O5, [MþNa]þ calcd:
521.1311, found: 521.1288.
phenyl)amino)pyrimidin-4-yl)amino)-N-hydroxybenzamide (9l)
Yield 69.6%; off-white solid. Ar atmosphere, 15g (1.0 mmol) and
hydroxylamine (20 mmol) in anhydrous methanol, sodium meth-
oxide (25 mmol) in methanol solution was slowly added to the
mixture. After the addition, the reaction was allowed to warm room
temperature and stirred for 12 h. Adjusted pH to 6 with dilute
hydrochloric acid, the inorganic salt was removed by filtration and
the rest of organic phase was vacuum distilled to produce the 9l. 1H
4.2.8. 2-((5-chloro-2-((4-(2-morpholinoacetamido)phenyl)amino)
pyrimidin-4-yl)amino)-N-hydroxy benzamide (9h)
Yield 60.3%; off-white solid; dichloromethane/methanol: 12/1
(v/v). 1H NMR (400 MHz, DMSO‒d6):
d
11.52 (s, 1H), 11.17 (s, 1H),
NMR (400 MHz, DMSO‒d6): d 11.52 (s, 1H), 11.15 (s, 1H), 10.84 (s,
9.64 (s,1H), 9.43 (s,1H), 9.35 (s,1H), 8.70 (s,1H), 8.22 (s,1H), 7.62 (d,
J ¼ 7.68 Hz, 1H), 7.58 (d, J ¼ 8.52 Hz, 2H), 7.52 (t, J ¼ 8.70 Hz, 2H),
7.49 (t, J ¼ 7.86 Hz, 1H), 7.14 (t, J ¼ 7.49 Hz, 1H), 3.65 (t, J ¼ 4.02 Hz,
4H), 3.11 (s, 2H), 2.53e2.51 (m, 4H); 13C NMR (100 MHz, DMSO‒d6):
1H), 9.36 (s, 1H), 9.32 (s, 1H), 8.98 (s, 1H), 8.68 (s, 1H), 8.18 (s, 1H),
7.61 (d, J ¼ 7.56 Hz, 1H), 7.52 (d, J ¼ 8.28 Hz, 2H), 7.49 (t, J ¼ 7.44 Hz,
1H), 7.12 (t, J ¼ 7.26 Hz,1H), 6.89 (t, J ¼ 8.76 Hz, 2H), 4.43 (s, 2H); 13
C
NMR (100 MHz, DMSO‒d6): d 166.37, 165.00, 158.48, 155.44, 155.23,
d
168.07, 166.33, 158.27, 155.42, 155.20, 139.51, 136.36, 133.40,
153.58, 139.54, 134.36, 132.04, 128.07, 122.63, 122.10, 122.04 (2C),
119.73, 115.08 (2C), 104.95, 66.76. HRMS (ESIþ) for C19H17ClN6O5,
[M ꢂ H]- calcd: 443.0876, found: 443.0874.
131.90, 128.09, 122.63, 122.18, 120.49 (2C), 120.39 (2C), 119.83,
105.20, 66.56 (2C), 62.50, 53.71 (2C). HRMS (ESIþ) for C23H24ClN7O4,
[MþNa]þ calcd: 520.1471, found: 520.1448.
4.2.13. 2-((5-chloro-2-((4-(2-morpholino-2-oxoethoxy)phenyl)
amino)pyrimidin-4-yl)amino)benzoic acid (9m)
4.2.9. 1-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)
pyrimidin-2-yl)amino)phenyl)-N-hydroxypiperidine-4-
carboxamide (9i)
Yield 62.1%; off-white solid. 15g (100 mg) and 10% aq. Sodium
hydroxide in 1,4-dioxane/water, the mixture was stirred at room
temperature for 5 h. After reaction, adjusted pH to 6 with dilute
hydrochloric acid and distilled out 1,4-dioxane. Then, the precipi-
tate was filtrated and washed with water to obtain 9m which could
directly be used without further purification. 1H NMR (400 MHz,
Yield 58.2%; off-white solid; dichloromethane/methanol: 8/1 (v/
v). 1H NMR (400 MHz, DMSO‒d6):
d 11.60 (s, 1H), 10.47 (s, 1H), 9.21
(s, 1H), 8.76e8.73 (m, 3H), 8.16 (s, 1H), 7.74 (d, J ¼ 7.80 Hz, 1H),
7.48e7.45 (m, 3H), 7.12 (t, J ¼ 7.62 Hz, 1H), 6.88 (d, J ¼ 8.88 Hz, 2H),
3.63 (d, J ¼ 12.24 Hz, 2H), 2.80 (d, J ¼ 4.44 Hz, 3H), 2.60(td, J ¼ 11.94,
2.52 Hz, 2H), 2.16e2.11 (m, 1H), 1.76e1.68 (m 4H); 13C NMR
DMSO‒d6): d 11.48 (s, 1H), 9.33 (s, 1H), 8.92 (s, 1H), 8.21 (s, 1H), 8.03
(d, J ¼ 7.52 Hz, 1H), 7.55 (t, J ¼ 7.60 Hz, 1H), 7.51 (d, J ¼ 8.76 Hz, 2H),
7.12 (t, J ¼ 7.60 Hz, 1H), 6.90 (d, J ¼ 8.77 Hz, 2H), 4.80 (s, 2H),
3.63e3.52 (m, 4H), 3.51e3.45 (m 4H); 13C NMR (100 MHz, DMSO‒
(100 MHz, DMSO‒d6):
d 171.90, 169.44, 158.54, 155.42, 155.13,
147.23, 139.94, 132.52, 131.99, 128.41, 122.29, 121.84 (2C), 121.77,
120.97, 116.80 (2C), 104.73, 49.67 (2C), 39.65, 28.58 (2C), 26.81.
HRMS (ESIþ) for C24H26ClN7O3, [MþH]þ calcd: 496.1842, found:
496.1858.
d6):
d 170.50, 166.82, 158.57, 155.51, 155.31, 153.79, 142.16, 134.40,
134.12,131.78,123.57,122.56 (2C),121.11,116.36,114.88 (2C),105.29,
66.80, 66.67 (2C), 45.43, 42.22. HRMS (ESIþ) for C23H22ClN5O5,
[M ꢂ H]- calcd: 482.1237, found: 482.1225.
4.2.10. 1-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)
pyrimidin-2-yl)amino)benzyl)-N-hydroxypiperidine-4-
carboxamide (9j)
4.2.14. Ethyl 2-((5-chloro-2-((4-(2-morpholino-2-oxoethoxy)
phenyl)amino)pyrimidin-4-yl)amino) benzoate (15g)
Yield 61.7%; off-white solid; dichloromethane/methanol: 8/1 (v/
Yield 55.3%; off-white solid. 1H NMR (400 MHz, DMSO‒d6):
v). 1H NMR (400 MHz, DMSO‒d6):
d
11.60 (s, 1H), 10.39 (s, 1H), 9.44
d
10.98 (s,1H), 9.38 (s,1H), 8.88 (s,1H), 8.22 (s,1H), 8.03(dd, J ¼ 7.92,
(s, 1H), 8.78e8.74 (m, 2H), 8.68 (s, 1H), 8.21 (s, 1H), 7.75 (d,
J ¼ 7.44 Hz, 1H), 7.58 (d, J ¼ 7.15 Hz, 2H), 7.46 (t, J ¼ 7.30 Hz, 1H), 7.17
(d, J ¼ 7.63 Hz, 2H), 7.14 (t, J ¼ 7.32 Hz, 1H), 3.38 (s, 2H), 2.84e2.82
(m, 2H), 2.81 (s, 3H), 1.98e1.93 (m, 1H), 1.86 (t, J ¼ 10.25 Hz, 2H),
1.24 Hz, 1H), 7.59 (t, J ¼ 7.96 Hz, 1H), 7.50 (d, J ¼ 8.68 Hz, 2H), 7.15 (t,
J ¼ 7.68 Hz, 1H), 6.88 (d, J ¼ 8.68 Hz, 2H), 4.80 (s, 2H), 4.36 (q,
J ¼ 7.04 Hz, 2H), 3.63e3.54 (m, 4H), 3.50e3.44(m 4H), 1.33 (t,
J ¼ 7.04 Hz, 3H); 13C NMR (100 MHz, DMSO‒d6):
d 168.07, 166.71,
1.63e1.56 (m 4H); 13C NMR (100 MHz, DMSO‒d6):
d
172.04, 169.40,
158.49, 155.41 (2C), 153.91, 141.64, 134.54, 133.96, 131.26, 122.39
(3C), 121.60, 116.14, 114.97 (2C), 105.06, 66.77, 66.56 (2C), 61.81,
45.33, 42.10, 14.46. HRMS (ESIþ) for C25H26ClN5O5, [MþNa]þ calcd:
534.1515, found: 534.1522.
158.28, 155.47, 155.12, 139.79, 139.41, 132.21, 131.88, 129.39 (2C),
128.46, 122.42, 121.94, 121.23, 120.13 (2C), 105.46, 62.48, 53.04 (2C),
40.00, 28.95 (2C), 26.81. HRMS (ESIþ) for C25H28ClN7O3, [MþH]þ
calcd: 532.1834, found: 532.1815.
4.3. Kinase enzymatic assays
4.2.11. 1-(3-(4-((5-chloro-4-((2-(methylcarbamoyl)phenyl)amino)
pyrimidin-2-yl)amino)phenoxy) propyl)-N-hydroxypiperidine-4-
carboxamide (9k)
The ADP-Glo™ system (EGFRWT, Catalog. V9261; EGFRT790M/
L858R, Catalog. V5325) were purchased from Promega Corporation
(USA) and were used to perform the enzymatic assays. The exper-
iments were performed according to the instructions of the
manufacturer. The more detailed and complete protocols, and the
active kinase data were available at: https://www.promega.com.cn/
resources/protocols/product-information-sheets/n/EGFR-kinase-
enzyme-system-protocol/. For all of the tested targets, concentra-
tions consisting of suitable levels from 0.1 to 1000 nM were used.
The test was performed in a 384-well plate, and includes the major
Yield 69.8%; off-white solid; dichloromethane/methanol: 10/1
(v/v). 1H NMR (400 MHz, DMSO‒d6):
d 11.59 (s, 1H), 10.39 (s, 1H),
9.27 (s, 1H), 8.77e8.67 (m, 3H), 8.17 (s, 1H), 7.74 (d, J ¼ 7.14 Hz, 1H),
7.51 (d, J ¼ 7.02 Hz, 2H), 7.46 (t, J ¼ 7.25 Hz, 1H), 7.13 (d, J ¼ 6.60 Hz,
2H), 6.85 (d, J ¼ 8.22 Hz, 2H), 3.96 (t, J ¼ 5.70 Hz, 2H), 2.90 (d,
J ¼ 5.70 Hz, 2H), 2.80 (d, J ¼ 2.82 Hz, 3H), 2.43e2.39 (m, 2H),
1.98e1.94 (m, 1H), 1.88e1.83 (m, 4H), 1.64e1.56 (m 4H); 13C NMR
(100 MHz, DMSO‒d6):
d 171.79, 169.40, 158.48, 155.41, 155.11,
154.42, 139.86, 133.65, 131.91, 128.41, 122.30, 122.17 (2C), 121.77,
121.04, 114.74 (2C), 104.93, 66.48, 55.16, 53.30 (2C), 40.00, 28.90
steps below: (1) perform 5
buffer (e.g., 1 ꢀ reaction buffer A), (2) incubate at room temperature
m
L kinase reaction using 1 ꢀ kinase
10

L. Chen, Y. Zhang, L. Tian et al.
European Journal of Medicinal Chemistry 223 (2021) 113626
for 60 min, (3) add 5
reaction and deplete the unconsumed ATP leaving only ADP and a
very low background of ATP, (4) incubate at room temperature for
m
L of ADP-Glo™ reagent to stop the kinase
by Annexin V-propidium iodide assay. Briefly, after treatment with
different concentrations of compounds for 48 h, the cells were
collected and washed twice with ice-cold PBS, cells then resus-
pended in binding buffer with Annexin-V and PI and incubated at
room temperature for 15 min in the dark. The stained cells were
analyzed using flow cytometry (Beckman, USA). The H1975
(1 ꢀ 106 cells/well) incubated in 6-well plates were treated with
solvent control (DMSO), 9d in medium containing 5% FBS for 72 h.
Then, collected and fixed with 70% ethanol at 4 ꢁC overnight. After
being fixed with 70% ethanol at 4 ꢁC, the cells were stained with
40 min, (5) add 10 mL of kinase detection, (6) reagent to convert ADP
to ATP and introduce luciferase and luciferin to detect ATP, (7)
incubate at room temperature for 30 min, (8) plate was measured
on TriStar® LB942 Multimode Microplate Reader (BERTHOLD) to
detect the luminescence (Integration time 0.5e1 s). Curve fitting
and data presentations were performed using GraphPad Prism
version 5.0.
Annexin V-FITC (5
mL)/propidium iodide (5 mL), and analyzed by
4.4. Cell activity assay
flow cytometry assay (Becton-Dickinson, USA).
Cell cycle distribution was evaluated by flow cytometry. The
cells at a density of approximately 2 ꢀ 105 cells/well were incu-
bated in 6-well plates, treated with different concentrations of 9d
for 48 h, collected and fixed with 70% ice-cold ethanol at 4 ꢁC
A 431, A 549, Aspc-1, and H 23 cells were obtained from the
American Type Culture Collection. H 1975, HBE and L-02 cells were
purchased from Fuheng Biology Company (Shanghai, China). H
1975, Aspc-1, H 23, and A 549 cells were grown in RPMI-1640
(Gibco®, USA) supplemented with 10% FBS (Gibco®, USA), 1%
penicillin-streptomycin (Beyotime Company, China). A 431, HBE
and L-02 cells were grown in DMEM (Gibco®, USA) supplemented
with 10% FBS (Gibco®, USA), 1% penicillin-streptomycin (Beyotime
Company, China). All cells were maintained and propagated as
monolayer cultures at 37 ꢁC in humidified 5% CO2 incubator. The
Cell Counting Kit-8 (CCK-8) reagent was purchased from Biotool
Company (Switzerland). Cell viability was assessed by the CCK-8
assay based on the reduction of CCK-8 by succinate de-
hydrogenases of viable cells to a yellow formazan product. Cells
were grown in 96-well culture plates (3000e4000/well) for 12 h
overnight. Following this, cells were stained with 500 mL PBS con-
taining 50 mg/mL propidium iodide and 0.5 mg/mL RNase for
30 min at 37 ꢁC. The DNA content was analyzed on a flow cytometer
(Beckman, USA).
4.8. Western blotting assay
The H1975 (2 ꢀ 105 cells/well) were plated in 6-well plates and
treated with 9d (0.5 and 1 mM) for 48 h. Then, the cells were washed
twice times with PBS and lysed in a RIPA lysis buffer containing
phenylmethanesulfonyl fluoride (PMSF). The lysate was centrifuged
at 14,000 rpm for 15 min at 4 ꢁC. The proteins were separated by
SDS-PAGE (10e15%) and electrophoretically transferred to PVDF
membranes. The blots were incubated in 1 ꢀ TBST containing 5%
non-fat dry milk for 2 h to block nonspecific binding sites and
incubated overnight at 4 ꢁC with primary antibodies. Then the blots
were incubated with horseradish peroxidase-conjugated anti-
bodies (1:4000 dilution) for 1 h at room temperature after washing.
Proteins were determined based on the enhanced chem-
iluminescence (ECL) method and images were taken using the Bio-
Spectrum Gel Imaging System (UVP, USA).
before compounds of various concentrations (1.25e40
added. Cell proliferation was determined after treatment with
compounds for 72 h. Subsequently, 10 L of CCK-8 reagent (Biotool
mM) were
m
Company, Switzerland, 5.0 mg/mL) dissolved in the medium was
added and the cells were incubated for another 1e2 h at 37 ꢁC. The
absorbance was measured at 450 nm with a microplate reader
(Thermo Fisher, USA). All doses were tested in triplicates and the
experiment was repeated at least three times. IC50 values were
calculated using GraphPad Prism version 5.0.
4.5. Wound-healing assay
The cancer cells were cultured in 6-well plates for 48 h at 37 ꢁC.
The injury lines were created in the cell monolayer and washed
with PBS to remove cell debris, then the cells were treated with 9d
(0.5 and 2 mM) for 0, 24, and 48 h. After that, the dead cells were
washed away with PBS, and the images were photographed by the
4.9. Pharmacokinetic
Male Sprague-Dawley (SD) rats, weighing 200 20 g, were used
to study in vivo pharmacokinetics. All animal experiments were
approved by the Animal Care and Use Committee of Dalian Medical
University. The animals were maintained at 25 2 ꢁC and 50e60%
relative humidity (RH) under natural light/dark conditions for 1
week before the experiment. Six rats were administered by tail vein
injection (9d, 25 mg/kg). Blood samples were collected from the
retro-orbital plexus under mild anesthesia into micro centrifuge
tubes containing heparin (40 IU/ml blood) at different time points
(10 min, 20 min, 40 min, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h and 24 h).
Plasma was separated by centrifuging the blood samples at
7000 rpm for 5 min and stored at ꢂ20 ꢁC prior to analysis.
fluorescence microscope (OLYMPUS, Tokyo, Japan).
4.6. DAPI staining assays
Approximately 2 ꢀ 105 cells/well of H1975 cells in 6-well plates
were incubated in an incubator for 12 h, then treated with different
concentrations of inhibitors (0.125, 0.5, and 2 mM) for 72 h. After
incubation, the cells were washed with PBS twice. DAPI staining
was performed after being treated as mentioned above. The cells
plated in 6-well plates were washed twice with PBS and fixed with
10% formaldehyde for 10 min, then washed with PBS three times.
The content of 9d in rat plasma was measured with a LC-MS/MS
system. The plasma samples (200 mL) were mixed with ethyl ace-
Cells were subsequently incubated in DAPI (1.0
room temperature for 10 min, washed with PBS and examined
under a fluorescence microscope (OLYMPUS, Tokyo, Japan).
m
g/mL) solution at
tate for extraction. The mixture was vortexed for 3 min and
centrifuged at 4500 rpm for 5 min. The supernatant was dried
under nitrogen, and the residue was re-dissolved with mobile
phase. The de-clustering potential (DP) and the entrance potential
(EP) were set at 55 V and 10 V. The collision voltage (CE) and the
collision exit potential (CXP) were set at 30 eV and 40 V, respec-
tively. Pharmacokinetic parameters were obtained using the DAS
2.1.1 software (Shanghai, China). The mass transition ion-pair was
selected as m/z 485.3 / 452.3.
4.7. Flow cytometry assay
The Annexin V-FITC Apoptosis Detection Kit and Cell Cycle Assay
were all purchased from Beyotime Company (Shanghai, China).
Compounds-induced apoptotic cell death was further quantitated
11

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European Journal of Medicinal Chemistry 223 (2021) 113626
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The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by the "innovative research team in
SYPHU by the supporting fund for universities from the Chinese
Central Government (51150039)", Research Fund of Higher Edu-
cation of Liaoning Province (LQ2019004), Science and Technology
Innovation Fund of Dalian, Liaoning province, China
(2020JJ27SN072), “Xing Liao” Talents Project of Liaoning Province,
China (XLYC1807011), Liaoning Province Ph.D. Research Start-up
Fund (2020-BS-199), and Scientific Research Fund of Liaoning
Province Education Administration (LZ2020041).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113626.
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