Design, synthesis, and biological evaluation of hydroxamic acid-substituted 2,4-diaryl aminopyrimidines as potent EGFRT790M/L858R inhibitors for the treatment of NSCLC

Article abstract of DOI:10.1016/j.bioorg.2021.105045

A series of 2,4-diarylaminopyrimidine derivatives bearing hydrophilic hydroxamic acids were designed and synthesized as potent EGFR^T790M/L858R inhibitors. Among the derivatives synthesized, 10c (IC₅₀ = 5.192 nM), 10j (IC₅₀ = 10.35 nM), and 10o (IC₅₀ = 0.3524 nM) exhibited higher potencies against EGFR^T790/M/L858R compared to the known EGFR inhibitor AZD-9291 (IC₅₀ = 20.80 nM). Moreover, 10j showed moderate activity against H1975 cells transfected with the EGFR^T790M/L858R mutant, with an IC₅₀ of 0.2113 μM over A431 (wild-type EGFR, SI = 47.3). In addition, 10j exhibited low toxicity in normal HBE cells (human bronchial epithelial cells, IC₅₀ > 40 μΜ). Analysis of the mode of action indicated that 10j effectively induced apoptosis in H1975 cells by arresting the cells in the G2/M phase. Compound 10j also demonstrated efficacy in inhibiting tumor growth in a H1975 xenograft mouse model without losing body weight or killing the mice. Taken together, these results suggested that 10j might be a promising candidate for development as a potential treatment for NSCLC harboring the EGFR^T790M/L858R mutation.

Full text of DOI:10.1016/j.bioorg.2021.105045

Journal Pre-proofs
Design, synthesis, and biological evaluation of hydroxamic acid-substituted
2,4-diaryl aminopyrimidines as potent EGFRT790M/L858R inhibitors for the
treatment of NSCLC
Lixue Chen, Yunhao Zhang, Changyuan Wang, Zeyao Tang, Qiang Meng,
Hunjun Sun, Yan Qi, Xiaodong Ma, Lei Li, Yanxia Li, Youjun Xu
PII:
S0045-2068(21)00422-3
DOI:
https://doi.org/10.1016/j.bioorg.2021.105045
YBIOO 105045
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
3 March 2021
15 May 2021
26 May 2021
Please cite this article as: L. Chen, Y. Zhang, C. Wang, Z. Tang, Q. Meng, H. Sun, Y. Qi, X. Ma, L. Li, Y. Li, Y.
Xu, Design, synthesis, and biological evaluation of hydroxamic acid-substituted 2,4-diaryl aminopyrimidines as
potent EGFRT790M/L858R inhibitors for the treatment of NSCLC, Bioorganic Chemistry (2021), doi: https://
doi.org/10.1016/j.bioorg.2021.105045
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Design, synthesis, and biological evaluation of hydroxamic acid-substituted 2,4-diaryl
aminopyrimidines as potent EGFRT790M/L858R inhibitors for the treatment of NSCLC
Lixue Chena, Yunhao Zhanga, Changyuan Wanga, Zeyao Tanga, Qiang Menga, Hunjun Suna, Yan Qia, Xiaodong
Maa, Lei Lia,, Yanxia Lib,* and Youjun Xuc,
a College of Pharmacy, Dalian Medical University, Dalian, 116044, PR China.
b
Department of Respiratory Medicine, The First Affiliated Hospital of Dalian Medical University, Dalian, PR
China.
c
School of Pharmaceutical Engineering, and Key Laboratory of Structure-Based Drug Design & Discovery
(Ministry of Education), Shenyang Pharmaceutical University, Shenyang, 110016, PR China.
* Corresponding author.
Email address: lilei0332@126.com (L. Li); liyanxia001@163.com (Y. Li); xuyoujun@syphu.edu.cn (Y. Xu).
Highlights
 Diphenylpyrimidine derivatives bearing hydroxamic acid group were designed and
synthesized as potent EGFRT790M/L858R inhibitors.
 10j demonstrated stronger inhibitory potency against EGFRT790M/L858R than that of AZD-9291.
 10j displayed extremely selective inhibitory effects on H1975 cell lines (EGFRT790M/L858R
)
over A431 cell lines (EGFRWT).
 10j could block the cell cycle of H1975 cell lines at the G2/M stage.
 10j significantly suppressed tumour growth in xenograft mouse model.
Abstract
A series of 2,4-diarylaminopyrimidine derivatives bearing hydrophilic hydroxamic acids were
designed and synthesized as potent EGFRT790M/L858R inhibitors. Among the derivatives synthesized,
10c (IC50 = 5.192 nM), 10j (IC50 = 10.35 nM), and 10o (IC50 = 0.3524 nM) exhibited higher potencies
against EGFRT790/M/L858R compared to the known EGFR inhibitor AZD-9291 (IC50 = 20.80 nM).
Moreover, 10j showed moderate activity against H1975 cells transfected with the EGFRT790M/L858R

mutant, with an IC50 of 0.2113 μM over A431 (wild-type EGFR, SI = 47.3). In addition, 10j exhibited
low toxicity in normal HBE cells (human bronchial epithelial cells, IC50 > 40 μΜ). Analysis of the
mode of action indicated that 10j effectively induced apoptosis in H1975 cells by arresting the cells
in the G2/M phase. Compound 10j also demonstrated efficacy in inhibiting tumor growth in a murine
H1975 xenograft mouse model without losing body weight or killing the mice. Taken together, these
results suggested that 10j might be a promising candidate for development as a potential treatment
for NSCLC harboring the EGFRT790M/L858R mutation.
Key words: NSCLC; EGFR T790M/L858R; resistance; inhibitors
1. Introduction
Non-small cell lung cancers (NSCLC) harboring somatic activating mutations in the tyrosine
kinase domain of the epidermal growth factor receptor (EGFR) are well-studied examples of
oncogene addiction [1]. EGFR is a transmembrane glycoprotein belonging to the human epidermal
growth factor receptor (HER) family that is essential in cell signaling pathways implicated in cell
proliferation, differentiation, and apoptosis [2,3]. EGFR mutations are the first driver alterations
characterized in NSCLC and are found in approximately 10–15% of non-Asian and 48% of Asian
patients [1,4‒5]. The most common oncogenic mutations in NSCLCs are small, in-frame deletions in
exon 19 and a L858R point mutation, both of which likely cause constitutive activation of the tyrosine
kinase domain by destabilizing the autoinhibited conformation [6‒9]. First-generation EGFR
inhibitors Gefitinib (1) [10] and Erlotinib (2) [11] (Fig. 1) were suitably efficacious in patients
suffering from NSCLC with sensitizing EGFR mutations (both exon 19 deletions and L858R).

Unfortunately, after administration of the first-generation drugs for 10‒14 months, the cancers in
approximately 60% of the patients became gradually exacerbated [12, 13]. The presence of the
T790M gatekeeper mutation increased the affinity of ATP binding in the ATP-binding pocket of the
receptor, thereby outcompeting the EGFR inhibitors for binding and reducing their efficacies against
the receptor [14]. Second- and third-generation EGFR inhibitors, which functioned as irreversible
inhibitors that targeted the formation of covalent adducts with Cys797, were developed to increase
the potency of binding against the T790M/L858R mutants [15]. However, due to the narrow
selectivity window between wild-type EGFR (EGFRWT) and the mutants, patients who were
administered second-generation EGFR inhibitors, such as BIBW-2992 (3) [16] and HKI-272 (4) [17],
suffered from adverse side effects in the clinic [18]. To improve the safety profiles of the second-
generation inhibitors, third-generation EGFR inhibitors, such as WZ4002 (5) [19], AZD-9291(6) [20],
Almonertinib (7) [21], and other structurally related compounds that are selective against the
dominant mutants, have been developed in recent years. Among them, AZD-9291 (Osimertinib) was
approved by U.S. Food and Drug Administration (FDA) to be used for treating EGFR T790M/L858R
mutation NSCLC in 2015, and Almonertinib was approved by the National Medical Products
Administration (NMPA) in 2020.
NMe2
N
NMe2
F
F
O
O
O
Cl
NH
NH
Cl
NH
N
Cl
NH
N
OMe
O
O
O
NH
O
NC
NH
N
N
OMe
OMe
N
O
N
N
N
OEt
O
Erlotinib
2
)
Gefitinib
1
)
(
BIBW-2992 3
( )
(
HKI-272
4
)
(
Me
OMe
N
O
OMe
O
N
N
O
N
NMe
N
NH
NH
O
NH
O
NH
O
NH
NMe
NH
O
Me
N
Me
MeO
N
N
N
N
N
HN
N
O
HN
N
NH
Cl
N
N
NMe2
NMe2
N
N
H
N
N
H
N
H
OMe
Cl
OMe
OMe
WZ-4002
5
)
(
AZD-9291
6
)
8
9
( )
(
Almonertinib
7
(
)
(
)

Fig. 1. Chemical structures of some novel EGFR inhibitors.
Our group has spent a considerable amount of time on the discovery of novel mutant-selective
inhibitors against EGFRT790M/L858R by modifying the solution region of lead compounds such as 8
[22] and 9 [23]. The SAR results of the scaffold of these lead compounds [23, 24] indicated that the
2,4-diarylaminopyrimidine scaffold was very hydrophobic, so the introduction of hydrophilic groups
onto Ring D, as well as the extension of the linker between Rings C and D, could be effective for
improving the druglikeness and the binding activity of the scaffold. Herein, a series of 2,4-
diarylaminopyrimidine derivatives featuring hydrophilic isohydroxamic acid and carboxylic acid
groups were designed, synthesized, and evaluated by in vitro kinase enzymatic and cellular activity
assays and an in vivo xenograft mouse model in an effort to identify more effective and selective
inhibitors against EGFRT790M/L858R (Fig. 2).
O
HS Cys797-EGFRT790M
OH
N
Me
N
D
H
N
O
Linker
O
HS Cys797-EGFRT790M
N
NH
O
C
A
N
MeO
H
HN
N
NH
Cl
HN
N
O
B
N
N
Cl
, IC50 = 10.35 nM, EGFRT790M/L858R
EGFRT790M/L858R Inhibitor
Anti-NSCLC agent
WZ-4002
10j
0.21 M, H1975, SI>47.3
Fig. 2. Strategy for the design of the target 2,4-diarylaminopyrimidine derivatives.
2. Results and Discussion
2.1 Chemistry
O
NH
NHCO(CH2)2Cl
NHCO(CH2)2Cl
NH2
NHCO(CH2)2Cl
Cl
d
N
NH
R1
Cl
N
NH
R1
c
b
a
NH2
NO2
NO2
N
N
11a~11c
12a~12c
15a~15d
(R1=F, Cl)
13a~13c
14a~14d
(R1=F, Cl)
O
O
O
OEt
OEt
NHOH
N
N
N
e. reduction
N
f. hydroxamic acid
formation
16a
18a
O2N
17a
H2N
H2N
N
OEt
NHOH
O2N
H2N
O
O
16b
18b
O
O

Scheme 1. Synthesis of intermediates 15a‒d and 18a‒d.
X
O
N
NHOH
HOHN
H2N
18a
N
O
X
18b
(X=0),
(X=CH2)
O
18c
18d
(X=OCH2CH2), or
(X=NHCOCH2)
NH
g. TsOH in EtOH, 70 oC for 3 h
8.6~20.5%
O
HN
N
NH
R1
NH
N
O
O
10a~10n
(R1=F, Cl)
Cl
N
NH
15a~15d
(R1=F, Cl)
EtO
HO
N
N
X
N
R1
X
O
h. NaOH,
O
g
1,4-dioxane/H2O, rt
X
NH
NH
N
56.3~61.4%
45.3~62.1%;
OEt
H2N
17a
HN
N
NH
Cl
HN
N
NH
Cl
O
COOH
N
N
17b
(X=0),
(X=CH2)
10o~10q
10r~10t
N
O
i. NaOH, MeOH/H2O, rt,
then aq. HCl.
N
H
OMe
82.1%.
HN
N
NH
Cl
N
10u
Scheme 2. Synthesis of compounds 10a‒u.

The synthesis of the designed 2,4-diarylaminopyrimidine derivatives 10a–u is shown in
Schemes 1‒2. Briefly, the commercially available substrates 11a–c were coupled to 3-
chloropropionyl chloride via a trivial acyl substitution reaction to afford intermediates 12a–c.
Subsequently, the nitroarene group in compounds 12a–c was reduced to the corresponding aniline by
H2 in the presence of 10% Pd/C to form intermediates 13a–c, which then underwent condensation
reactions with 2,4,5-trichloropyrimidine or 2,4-dichloro-5-fluoropyrimidine to generate
intermediates 14a–d. The α, β-elimination was finally carried out to produce the key intimidates 15a–
d containing the acrylamide pharmacophore. After reduction of the NO2 group in 16a–d to the
corresponding anilines, 18a–d were synthesized by nucleophilic substitution with hydroxylamine
hydrochloride in the presence of CH3ONa. Finally, the final compounds 10a–q were synthesized by
nucleophilic aromatic substitution of the aryl chloride moiety of 15a–d with the aniline group of 17a–
b/18a–d catalyzed by TsOH. In addition, final compounds 10r–t were prepared after hydrolysis of
the ester intermediates. Lastly, the negative control 10u was synthesized by hydrolysis and Michael
addition of 10o.
2.2 Biological activity
2.2.1 EGFR inhibitory activities and selectivity assays
Table 1. EGFR tyrosine kinase inhibitory activities of 10a–u in vitroa.
R2
4'
3''
R3
1''
1'
HN 3N
NH
1N
R1
Enzymatic activity (IC50, nM)
EGFRT790M/L858R EGFRWT
SI
Compd.
R1
R2
R3

N
CONHOH
CONHOH
10a
10b
10c
10d
10e
10f
Cl
Cl
Cl
Cl
F
3-NHCOCH=CH2
3-NHCOCH=CH2
3-NHCOCH=CH2
3-NHCOCH=CH2
3-NHCOCH=CH2
3-NHCOCH=CH2
3-NHCOCH=CH2
2-NHCOCH=CH2
2-NHCOCH=CH2
2-NHCOCH=CH2
2-NHCOCH=CH2
4-NHCOCH=CH2
4-NHCOCH=CH2
4-NHCOCH=CH2
2-NHCOCH=CH2
92.67
6.376
5.192
50.29
51.03
1.471
3.343
20.74
13.88
10.35
9.199
5.198
1.636
32.31
0.3524
34.44
1.082
40.62
2.952
23.97
1.165
8.187
0.6554
17.81
65.86
6.035
9.218
0.1403
198.5
60.52
--
--
N
O
N
N
7.8
--
CONHOH
H
N
O
CONHOH
CONHOH
N
--
O
N
N
F
--
CONHOH
H
N
10g
10h
10i
F
2.4
--
O
CONHOH
CONHOH
N
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
N
CONHOH
1.3
6.4
--
O
N
N
10j
CONHOH
H
N
10k
10l
O
CONHOH
CONHOH
N
1.8
--
O
N
N
10m
10n
10o
CONHOH
H
N
6.1
171.3
O
CONHOH
COOEt
N

N
N
COOEt
COOEt
COOH
COOH
COOH
COOH
10p
10q
Cl
Cl
Cl
Cl
Cl
Cl
2-NHCOCH=CH2
3-NHCOCH=CH2
2-NHCOCH=CH2
2-NHCOCH=CH2
3-NHCOCH=CH2
6.863
5.602
165.9
7.401
117.5
>500
20.80
1.517
45.44
2.081
326.0
189.2
>500
567.5
--
8.1
--
N
10r
N
10s
44.1
1.6
--
N
10t
3-
N
10u
NHCO(CH2)2OMe
AZD-9291
27.3
a Dose-response curves were determined at three different concentrations. The IC50 values are the concentrations in
nanomolar needed to inhibit cell growth by 50% as determined from these curves.
All the targets were evaluated for their activities against EGFRWT and EGFRT790M/L858R using the
ADP-GloTM kinase assay system, with AZD-9291 functioning as the control. The results represented
in Table 1 indicated that most of the targets displayed strong inhibitory activities against
EGFRT790M/L858R, with IC50 values ranging from 0.3524 nM to 165.9 nM. Several of the targets, such
as 10c (IC50 = 5.192 nM), 10j (IC50 = 10.35 nM), and 10o (IC50 = 0.3524 nM), exhibited tighter
binding to EGFRT790M/L858R than the EGFR inhibitor AZD-9291 as well as higher selectivity for
EGFRT790M/L858R over EGFRWT. Moreover, compounds 10h–k, which featured the acrylamide
pharmacophore at the C''-2 position on Ring A, inhibited EGFRT790M/L858R with IC50 values ranging
from 9.199 nM to 20.74 nM. The 2,4-diarylaminopyrimidine derivatives bearing ester groups as
prodrugs of the carboxylic acids on Ring D had stronger potencies compared to the corresponding

carboxylic acids. However, replacement of the 5-chlorine on the pyrimidine core with a fluorine
substituent (10e–g) was ineffective for improving the potency against EGFRT790M/L858R and selectivity
over EGFRWT. Notably, 10u, which did not contain an acrylamide group, weakly inhibited both
EGFRT790M/L858R and EGFRWT, suggesting that the acrylamide moiety was necessary for activity in
this scaffold.
2.2.2 Cellular inhibition and selectivity assays
Table 2. Cellular anti-proliferative activities of 10a–u (IC50, μM)a.
Anti-proliferative activity (IC50, μM)
Compd.
SI
H1975
2.537
5.962
4.255
>40
A431
7.31
>10
A549
>1.25
>1.25
>1.25
>1.25
>1.25
>1.25
>1.25
0.6543
0.5411
0.409
>1.25
>1.25
>1.25
>1.25
HBE
14.7
ASPC-1
2.245
3.483
4.23
H23
7.204
6.708
7.775
25.27
23.91
13.06
25.67
0.6715
<0.3125
0.5251
4.382
16.43
6.928
16.35
L-02
2.42
10a
10b
2.88
>1.68
>2.35
--
8.348
7.724
15.43
7.812
5.249
11.14
>40
2.863
2.767
11.07
4.922
2.661
23.66
2.529
4.364
3.058
22.09
9.293
4.759
14.00
10c
>10
10d
>10
>5
10e
9.556
5.079
>40
>10
>5
>1.05
1.23
--
10f
6.24
>10
>5
10g
>5
10h
0.3653
0.3114
0.2113
0.3698
>40
>10
0.8001
0.3594
0.5203
0.7571
>5
>27.4
22.0
>47.3
>27.0
--
10i
6.856
>10
24.5
10j
>40
10k
>10
>40
10l
9.947
13.97
>10
6.252
6.775
37.65
10m
10n
7.261
>10
4.533
>5
1.92
--
AZD-9291
0.091
1.226
a Dose-response curves were determined at five different concentrations. The IC50 values are the concentrations in
micromolar needed to inhibit cell growth by 50% as determined from these curves.

Table 3. Cellular anti-proliferative activities of 10o–u (IC50, μM)a.
Anti-proliferative activity
Compounds
(IC50, μM)
H1975
L-02
10o
10p
0.6884
19.52
0.5731
0.8183
3.145
1.154
>40
4.909
4.171
13.31
9.511
19.52
--
10r
10s
10t
10u
AZD-9291
0.091
a Dose-response curves were determined at five different concentrations. The IC50 values are the concentrations in
micromolar needed to inhibit cell growth by 50% as determined from these curves.
The antiproliferative activities of 10a–n and the positive control AZD-9291 were investigated
in the NSCLC cell lines A431 EGFRWT, H1975 EGFRT790M/L858R, A549KRAS G12S and H23KRAS mutant
,
as well as a human metastatic pancreatic adenocarcinoma cell line (Aspc-1) and a normal cell line
(HBE), using the CCK-8 assay (Table 2). The results of the cell-based anti-proliferative activity
assays indicated that most of the 2,4-diarylaminopyrimidine derivatives strongly inhibited the
proliferation of the H1975 cells, with the IC50 values of some of these derivatives in the nanomolar
regime, such as 10h (IC50 = 0.3653 μM), 10i (IC50 = 0.3114 μM), 10j (IC50 = 0.2113 μM), and 10k
(IC50 = 0.3698 μM). These four analogs were also highly selective for EGFRT790M/L858R in the H1975
cells over EGFRWT in the A431 cell line, with selectivity indices (SI) > 22, all of which were higher
than the positive control. Higher selectivities indicated that the inhibitors more weakly inhibited
normal cell EGFR expression compared to the mutants, which is advantageous for mitigating the side
effects associated with targeting the entire EGFR network.

The most obvious SAR trend of the 2,4-diarylaminopyrimidines obtained by the cell-based
activity assays was that the substitution of the acrylamide pharmacophore on Ring A noticeably
improved the anti-proliferation activities of the derivatives. When the acrylamide remained in the 3-
position of Ring A, extending the linker between Rings C and D was able to effectively increase the
anti-proliferative activity of the derivatives in the H1975 cell line. For example, substitution of the
group with an ethoxy piperidine linker was the most effective for enhancing the anti-proliferative
activity, such as in 10c (IC50 = 4.255 μM), 10f (IC50 = 5.079 μM), and 10j (IC50 = 0.2113 μM).
However, when this linker was an acetamide, such as in compounds 10d, 10g, and 10n, the
compounds exhibited reduced activity against H1975. Meanwhile, the 2,4-diarylaminopyrimidines
analogs featuring esters and carboxylic acid (10o–u) inhibited the proliferation of the H1975 and L-
02 cell lines. For example, compounds 10o (IC50 = 0.6884 μM) and 10p (IC50 = 0.5731 μM) strongly
inhibited cellular proliferation in H1975 cells and did not demonstrate selectivity for the kinase
domain; however, the substitution of these groups with hydroxamic acid groups led to an
enhancement in the anti-proliferative activity of this scaffold (Table 3). Measuring the toxicity of
drugs in host cells is important for assessing the safety of the candidates early in the drug discovery
process. Most of the 2,4-diarylaminopyrimidines analogs exhibited weak inhibition against normal
HBE and L-02 cells. In particular, the most active inhibitor 10j had the highest selectivity index (SI >
47.3) of all the analogs, and it also exhibited weak inhibition against the proliferation of HBE (IC50 >
40 μM) and L-02 (IC50 = 3.058 μM) cells, indicating potential for further development. Meanwhile,
10h, 10i, and 10j also strongly inhibited the proliferation of the KRAS mutant lung cancer A549 and

H23 cell lines and the pancreatic cancer ASCP-1 cell line, suggesting that the targets might have
multi-target effects.
2.2.3 Migration inhibition assay
After evaluation of the EGFR inhibitory activities and selectivity of the 2,4-
diarylaminopyrimidines analogs 10a–u, 10j was selected for further studies. Wound-healing assays
were conducted to determine if different concentrations (0.25, 1, and 4 µM) of 10j could influence
the migration of H1975 cells 0, 24, 48 h after scratching the cell monolayer (Fig. 3). As expected, the
DMSO control vehicle promoted the strongest migration of the H1975 cells. Under the same
conditions, H1975 cell migration in the 10j-treated groups was significantly lower than in the control
group. Therefore, the results of the wound healing assay indicated that 10j could effectively inhibit
the migration and invasion of H1975 cells.
Control
10j (4 M)
10j (1 M)


0 h
24 h
48 h

Fig. 3. Representative images of the H1975 cells treated with different concentrations of 10j for 0,
24, and 48 h during the wound-healing assay.
2.2.4 Morphological staining analysis
The analysis of the morphology change of H1975 cells were carried out using DAPI staining.
As shown in Fig. 4A, H1975 bearing EGFR T790M/L858R-mutated exposed with 10j, chromatin
condenses and nuclear disintegration were observed subsequent to DAPI staining. Under natural light,
the proportion of abnormal cells, such as fusiform and sickle type, increased significantly (Fig. 4B).
All of the phenomena was the characteristics of apoptotic programmed cell death.
Control
10j (0.25 M)

A)
10j (1 M)
10j (4 M)



Control
10j (0.25 M)

B)
10j (1 M)
10j (4 M)


Fig. 4. Morphological changes of the H1975 cells (100x final magnification). A): DAPI staining of
the H1975 cells treated with different concentrations (0.25, 1, and 4 µM) of 10j for 72 h; B): Under
nature light, H1975 cells treated with different concentrations (0.25, 1, and 4 µM) of 10j for 72 h.
2.2.5 Flow cytometry analysis
To characterize the propensity of 10j to induce apoptosis, flow cytometry was performed on the
H1975 cell lines using Annexin V-PI as the indicator of apoptosis. After incubating the H1975 cells
with 10j at concentrations ranging from 0.5 to 2.0 μM for 48 h, the percentage of apoptotic cells
dramatically increased from 15.99% to 21.29% (Fig. 5A). Correspondingly, compared to the control

group, the percentage of the H1975 cells in G0/G1 phase significantly decreased from 40.32% to
18.38% in a dose-dependent manner, while the percentage of cells in the G2/M phase increased from
46.38% to 65.85% at a concentration of 0.15 μM). However, the proportion of H1975 cells in S phase
essentially remained unchanged in the concentration range tested (Fig. 5B). Therefore, the flow
cytometry results indicated that 10j mainly arrested the H1975 cells in the G2/M stage in a
concentration-dependent manner.
10j ( M)

Control
A
)
0.5
0.5
10j ( M)

1.0
2.0
10j ( M)

Control
B
)
0.5

Fig. 5. A) 10j-induced H1975 cell apoptosis. B) Effects of 10j on H1975 cell-cycle arrest determined
by flow cytometry.
2.2.6 In vivo antitumor activity
Due to its potent inhibitory activity against EGFRT790M/L858R and H1975 proliferation, as well as
a low toxicity in normal cells, 10j was further evaluated for antitumor efficacy in vivo using a
xenograft mouse model of EGFRT790M/L858R-driven human NSCLC cells. Severe combined
immunodeficient (SCID) mice harboring established H1975 tumor xenografts were orally dosed with
10j at 50 mg/kg daily over a 14-day period. Tumor growth slowed down noticeably at this dosage,
such that the tumor weight decreased by ~80% after the 14 days (Fig. 6). In addition, there was no
mortality or significant loss of body weight (< 5% relative to the vehicle-matched controls) during
treatment.

B
)
A
)
Fig. 6. In vivo effects of 10j on the tumor growth of H1975 cell xenografts in nude mice. (A) Images
of the tumors collected from the mice; (B) tumor weight; data are presented as the mean ± SD (n =
4). *p < 0.05 and **p < 0.01 compared to the control group.
2.3 Molecular modeling
To better understand the interactions between 10j and EGFRT790M/L858R, molecular docking
studies of 10j in the ATP-binding site of EGFRT790M/L858R (PDB: 3IKA) were conducted using the
software AutoDock 4.2. As shown in Fig. 7, both 10j and the known EGFR inhibitor WZ4002
exhibited tight interactions with the residues in the active site of EGFRT790M/L858R in a U-shape, while
the binding conformation of 10j was reversed compared to WZ4002. The C''-2 acrylamide
functionality on Ring A of 10j formed hydrogen bond interactions with Cys797 (closer in space),
which enabled Michael additions of the Cys797 sulfhydryl group onto the acrylamide to form a
covalent adduct with the receptor. The docking studies revealed that acrylamide pharmacophore in
10h, 10i, 10j, and 10k was essential for promoting the inhibition of cellular proliferation by
irreversibly inhibiting the EGFRT790M/L858R receptor. In addition, strong hydrogen bonding
interactions were observed between the N-1 of the pyrimidine core of WZ4002 and Met793 in the
kinase hinge of the receptor as well as between the 2-aminopyrimidine NH of 10j and Met793.

Furthermore, hydrophobic interactions between Leu718 and Rings B and C of 10j, as well as between
Leu844 and Rings C and D of 10j, were observed. These molecular docking results were consistent
with the empirical data obtained from the biological assays, and they provided a structural basis for
further rational design of EGFRT790M/L858R inhibitors.
A)
B)
Fig. 7. Putative binding mode of 10j with EGFRT790M/L858R. (A) Detailed interactions with the protein
residues. Each dashed yellow line represents hydrogen bonds. (B) Three-dimensional space matching
diagram of 10j and the active site.
Fig. 7. Putative binding mode of 10j in the ATP-binding site of EGFRT790M/L858R. (A) Detailed
interactions with the residues in the active site of the receptor. Each dashed yellow line represents
hydrogen bonds. (B) Three-dimensional space matching diagram of 10j in the active site.
3. Conclusion
A series of diphenylpyrimidine derivatives bearing hydroxamic acid groups was designed and
synthesized as potent and selective inhibitors of EGFRT790M/L858R over EGFRTM. Among them, 10j
was identified as the most promising lead compound and was found to strongly inhibit the
EGFRT790M/L858R mutated kinase with an IC50 value of 10.35 nM. In addition, it moderately suppressed

the proliferation of H1975 cells transfected with the EGFRT790M/L858R mutant with an IC50 value of
0.2113 μM over A431 (EGFRWT, IC50 > 10 μM). Meanwhile, 10j also exhibited lower toxicity in
normal HBE and L-02 cells, indicating an improved administration safety margin. Moreover, 10j
induced apoptosis by arresting the H1975 cells in the G2/M phase. The lead 10j also displayed anti-
cancer efficacy in an H1975-driven xenograft mouse model in vivo. Overall, these results suggested
that 10j might be promising as a potent and selective EGFRT790M/L858R inhibitor for further
development.
4. Experimental section
4.1 General methods and chemistry
Unless otherwise noted, commercial solvents and reagents were used without further
1
purifications. High resolution ESI-MS was performed on an Agilent 1100 HPLC/MS system. H
13
NMR and C NMR spectra on a Brucker AV 400 MHz spectrometer were recorded in DMSO-d.
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 monitored by TLC,
using silica gel plates with fluorescence GF254 and UV light visualization. Flash chromatography
separations were obtained on Silica Gel (300~400 mesh) using dichloromethane/methanol as eluents.
4.2 General procedure for the synthesis of 10a~u
4.2.1 General procedure for the synthesis of 10a~n
A mixture of 11a~c (50 mmol) and potassium carbonate (50 mmol) in acetonitrile was gradually
added 3-chloropropannoyl chloride (100 mmol) under argon. After the addition, it was allowed to

warm to room temperature, and continuously stirred for 60 min. The mixture was diluted with ethyl
acetate and brine, the separated organic layer was washed with brine and dried over anhydrous
Na2SO4. After removing the organic solvent under vacuum, the rest of organic substance was
dispersed with petroleum ether to give the crude. 12a~c (40 mmol), Pd‒C (5% w/w) were added to
methanol, catalytic hydrogenation was carried out at room temperature for 10 hours. Pd‒C was
filtered out and the organic phase was vacuum distilled to obtain 13a~c. 13a~c (30 mmol), 2,4,5-
trichloropyrimidine/2,4-dichloro-5-fluoropyrimidine (30 mmol) and DIPEA (60 mmol) in
o
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.
Finally, a mixture of 14a~d (25 mmol), TEA (200 mmol) and MeCN was stirred under refluxing for
6 h. Then water was gradually added to the cooled mixture and stirred for 30 min to allow the final
target 15a~d precipitated from the mixture.
16a~d and the corresponding reduced amine intermediate were prepared according to the
reported process [25‒28]. Argon atmosphere, 17a~d (25 mmol) and hydroxylamine (0.125 mol) in
anhydrous methanol, sodium methoxide (0.15 mol) 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 vacuum distilled to produce the 18a~d.
A mixture of 15a~d (3.0 mmol), 18a~d (1.5 mmol), and p-TsOH (4.5 mmol) in n-BuOH was
gradually heated to 70 oC and stirred for 3 h. After reaction, the solvent was vacuum distilled and the

residuum was dispersed with saturated sodium bicarbonate/ethyl acetate. The organic was vacuum
distilled to give the crude which was purified by (Prep-HPLC) to get the 10a~n.
4.2.1.1 1-(4-((4-((3-acrylamidophenyl)amino)-5-chloropyrimidin-2-yl)amino)phenyl)-N-hydroxy
piperidine-4-carboxamide (10a)
1
Yield 19.6%; off-white solid. H NMR(400 MHz, DMSO‒d6): δ 10.46(s, 1H), 10.19(s, 1H),
9.08(s, 1H), 8.85(s, 1H), 8.73(s, 1H), 8.09(s, 1H), 7.86(s, 1H), 7.55(d, J=7.08 Hz, 1H), 7.44(d, J=8.34
Hz, 2H), 7.30(t, J=7.74 Hz, 2H), 6.71(d, J=7.02 Hz, 2H), 6.47(dd, J=16.86, 10.14 Hz, 1H), 6.28(d,
J=16.86 Hz, 1H), 5.77(d, J=10.14 Hz, 1H), 3.53–3.51(m, 2H), 2.50–2.48(m, 2H), 2.11–2.06(m, 1H),
1.71–1.66(m, 4H); 13C NMR(100 MHz, DMSO‒d6): δ 171.86, 163.56, 158.28, 156.54, 155.25,
146.68, 139.45, 139.33, 132.94, 132.40, 129.03, 127.32, 120.60(2C), 119.47, 116.78(2C), 115.88,
115.75, 103.42, 49.74(2C), 40.67, 28.61(2C). HRMS (ESI+) for C25H26ClN7O3, [M+Na]+ calcd:
530.1678, found: 530.1666.
4.2.1.2 1-(4-((4-((3-acrylamidophenyl)amino)-5-chloropyrimidin-2-yl)amino)benzyl)-N-hydroxy
piperidine-4-carboxamide (10b)
1
Yield 10.2%; off-white solid. H NMR(400 MHz, DMSO‒d6): δ 10.40(s, 1H), 10.24(s, 1H),
9.34(s, 1H), 8.93(s, 1H), 8.69(s, 1H), 8.14(s, 1H), 7.93(s, 1H), 7.57‒7.53(m, 3H), 7.32‒7.30(m, 2H),
7.04(d, J=7.45 Hz, 2H), 6.48(dd, J=16.84, 10.27 Hz, 1H), 6.27(d, J=16.84 Hz, 1H), 5.77(d, J=10.27
Hz, 1H), 3.35(s, 2H), 2.81(d, J=10.02 Hz, 2H), 1.99‒1.93(m, 1H), 1.93‒1.87(m, 2H), 1.63‒1.57(m,
13
4H); C NMR(100 MHz, DMSO‒d6): δ 171.89, 163.63, 158.12, 156.61, 155.22, 139.82, 139.59,
139.30, 132.41, 130.52, 129.55(2C), 129.00, 127.28, 119.61, 119.07(2C), 115.88, 115.67, 104.22,

62.21, 52.77(2C), 40.00, 28.61(2C). HRMS (ESI+) for C26H28ClN7O3, [M+H]+ calcd: 522.2015,
found: 522.2054.
4.2.1.3 1-(2-(4-((4-((3-acrylamidophenyl)amino)-5-chloropyrimidin-2-yl)amino)phenoxy)ethyl)-N-
hydroxypiperidine-4-carboxamide (10c)
1
Yield 15.1%; off-white solid. H NMR(400 MHz, DMSO‒d6): δ 10.39(s, 1H), 10.20(s, 1H),
9.14(s, 1H), 8.88(s, 1H), 8.69(s, 1H), 8.09(s, 1H), 7.89(s, 1H), 7.50(d, J=8.36 Hz, 1H), 7.48(d, J=8.52
Hz, 2H), 7.30(t, J=7.61 Hz, 1H), 7.30(d, J=7.61 Hz, 2H), 6.69(d, J=7.62 Hz, 2H), 6.46(dd, J=16.91,
10.22 Hz, 1H), 6.27(dd, J=16.91, 1.26 Hz, 1H), 5.76(dd, J=10.22, 1.26 Hz, 1H), 3.93(t, J=5.40 Hz,
13
2H), 2.93(d, J=11.09 Hz, 2H), 2.62(t, J=5.40 Hz, 2H), 1.99‒1.94(m, 3H), 1.64‒1.58(m, 4H); C
NMR(100 MHz, DMSO‒d6): δ 172.00, 163.59, 158.24, 156.58, 155.23, 153.67, 139.48, 139.33,
134.02, 132.35, 129.03, 127.32, 120.98(2C), 119.56, 115.90, 115.73, 114.56(2C), 103.68, 66.15,
57.39, 53.59(2C), 40.00, 28.91(2C). HRMS (ESI+) for C27H30ClN7O4, [M+Na]+ calcd: 574.1940,
found: 574.1984.
4.2.1.4 1-(2-((4-((4-((3-acrylamidophenyl)amino)-5-chloropyrimidin-2-yl)amino)phenyl)amino)-2-
oxoethyl)-N-hydroxypiperidine-4-carboxamide (10d)
1
Yield 11.6%; off-white solid. H NMR(400 MHz, DMSO‒d6): δ 10.42(s, 1H), 10.18(s, 1H),
9.44(s, 1H), 9.26(s, 1H), 8.90(s, 1H), 8.70(s, 1H), 8.13(s, 1H), 7.96(d, J=8.56 Hz, 2H), 7.90(s, 1H),
7.49(d, J=7.74 Hz, 1H), 7.38(d, J=8.59 Hz, 2H), 7.36(d, J=7.57 Hz, 1H), 7.30(t, J=7.96 Hz, 1H),
6.46(dd, J=16.92, 10.14 Hz, 1H), 6.26(dd, J=16.92, 1.68 Hz, 1H), 5.75(dd, J=10.14, 1.68 Hz, 1H),
3.04(s, 2H), 2.88(d, J=10.96 Hz, 2H), 2.10(t, J=11.21 Hz, 2H), 1.99‒1.95(m, 1H), 1.76‒1.70(m, 2H),

1.60‒1.58(m, 2H); 13C NMR(100 MHz, DMSO‒d6): δ 171.96, 168.33, 163.63, 158.10, 156.55,
155.19, 139.60, 139.34, 136.59, 132.74, 132.38, 128.95, 127.30, 120.32(2C), 119.60, 119.52(2C),
115.75, 115.38, 104.11, 62.59, 53.32(2C), 40.49, 28.80(2C). HRMS (ESI+) for C27H29ClN8O4,
[M+Na]+ calcd: 587.1893, found: 587.1926.
4.2.1.5 1-(4-((4-((3-acrylamidophenyl)amino)-5-fluoropyrimidin-2-yl)amino)benzyl)-N-hydroxy
piperidine-4-carboxamide (10e)
1
Yield 8.6%; off-white solid. H NMR(400 MHz, DMSO‒d6): δ 10.39(s, 1H), 10.17(s, 1H),
9.43(s, 1H), 9.17(s, 1H), 8.69(s, 1H), 8.10(d, J=3.48 Hz, 1H), 7.97(s, 1H), 7.61(d, J=7.98 Hz, 2H),
7.49(d, J=7.07 Hz, 1H), 7.46(d, J=8.14 Hz, 1H), 7.28(t, J=8.04 Hz, 1H), 7.08(d, J=7.74 Hz, 2H),
6.47(dd, J=16.93, 10.15 Hz, 1H), 6.27(dd, J=16.93, 1.56 Hz, 1H), 5.77(dd, J=10.15, 1.56 Hz, 1H),
3.35(s, 2H), 2.85‒2.77(m, 2H), 1.99‒1.91(m, 1H), 1.91‒1.78(m, 2H), 1.64‒1.57(m, 4H); 13C
NMR(100 MHz, DMSO‒d6): δ 171.93, 163.60, 155.95(d, J=1.73 Hz), 150.25(d, J=10.66 Hz),
141.24(d, J=19.15 Hz), 141.07(d, J=245.44 Hz), 140.21, 139.56, 139.49, 132.42, 129.51(2C), 129.07,
127.25, 118.76(2C), 117.89, 115.29, 113.95, 62.32, 52.83(2C), 40.00, 28.69(2C). HRMS (ESI+) for
C26H28FN7O3, [M+H]+ calcd: 506.2310, found: 506.2327.
4.2.1.6 1-(2-(4-((4-((3-acrylamidophenyl)amino)-5-fluoropyrimidin-2-yl)amino)phenoxy)ethyl)-N-
hydroxypiperidine-4-carboxamide (10f)
1
Yield 11.3%; off-white solid. H NMR(400 MHz, DMSO‒d6): δ 10.41(s, 1H), 10.14(s, 1H),
9.38(s, 1H), 8.98(s, 1H), 8.72(s, 1H), 8.07(d, J=3.12 Hz, 1H), 7.94(s, 1H), 7.54(d, J=8.52 Hz, 2H),
7.49(d, J=7.17 Hz, 1H), 7.43(d, J=7.61 Hz, 1H), 7.28(t, J=7.98 Hz, 1H), 6.76(d, J=8.50 Hz, 2H),

6.47(dd, J=16.88, 10.15 Hz, 1H), 6.28(dd, J=16.88 Hz, 1H), 5.77(dd, J=10.15 Hz, 1H), 3.97(t, J=5.64
Hz, 2H), 2.97‒2.95(m, 2H), 2.69‒2.63(m, 2H), 2.02‒1.97(m, 3H), 1.63‒1.59(m, 4H); 13C NMR(100
MHz, DMSO‒d6): δ 171.92, 163.57, 156.17(d, J=1.80 Hz), 153.49, 150.23(d, J=10.78 Hz), 141.27(d,
J=19.08 Hz),140.50(d, J=245.93 Hz), 139.55, 139.48, 134.54, 132.38, 129.09, 127.27, 120.75(2C),
117.84, 115.28, 114.67(2C), 113.96, 66.13, 57.36, 53.55(2C), 40.00, 28.83(2C). HRMS (ESI+) for
C27H30FN7O4, [M+Na]+ calcd: 558.2236, found: 558.2260.
4.2.1.7 1-(2-((4-((4-((3-acrylamidophenyl)amino)-5-fluoropyrimidin-2-yl)amino)phenyl)amino)-2-
oxoethyl)-N-hydroxypiperidine-4-carboxamide (10g)
1
Yield 15.1%; off-white solid. H NMR(400 MHz, DMSO‒d6): δ 10.42(s, 1H), 10.17(s, 1H),
9.46(s, 1H), 9.41(s, 1H), 9.12(s, 1H), 8.70(s, 1H), 8.10(d, J=3.48 Hz, 1H), 7.99(s, 1H), 7.61(d, J=8.78
Hz, 2H), 7.56(d, J=8.49 Hz, 1H), 7.45‒7.42(m, 3H), 7.28(t, J=8.05 Hz, 1H), 6.49(dd, J=16.84, 10.00
Hz, 1H), 6.28(dd, J=16.84, 1.21 Hz, 1H), 5.77(dd, J=10.00, 1.21 Hz, 1H), 3.07(s, 2H), 2.91(d,
J=10.35 Hz, 2H), 2.13(t, J=10.61 Hz, 2H), 2.02‒1.96(m, 1H), 1.79‒1.70(m, 2H), 1.62‒1.59(m, 2H);
13C NMR(100 MHz, DMSO‒d6): δ 171.96, 168.25, 163.61, 155.95(d, J=1.80 Hz), 150.20(d, J=10.65
Hz), 141.15(d, J=19.79 Hz), 140.96(d, J=245.15 Hz), 139.60(2C), 137.11, 132.48, 132.44, 129.03,
127.19, 120.38(2C), 119.21(2C), 117.73, 115.12, 113.61, 62.54(2C), 53.31, 40.91, 28.78(2C). HRMS
(ESI+) for C27H29FN8O4, [M+Na]+ calcd: 571.2188, found: 571.2191.
4.2.1.8 1-(4-((4-((2-acrylamidophenyl)amino)-5-chloropyrimidin-2-yl)amino)phenyl)-N-hydroxy
piperidine-4-carboxamide (10h)
1
Yield 20.5%; off-white solid. HNMR (400 MHz, DMSO‒d6): δ 10.46(s, 1H), 10.23(s, 1H),

9.05(s, 1H), 8.73(s, 1H), 8.45(s, 1H), 8.07(s, 1H), 7.78(d, J=7.81 Hz, 1H), 7.44(d, J=7.80 Hz, 1H),
7.38(d, J=8.32 Hz, 2H), 7.33(t, J=7.69 Hz, 1H), 7.26(t, J=7.50 Hz, 1H), 6.74(d, J=8.35 Hz, 2H),
6.52(dd, J=17.01, 10.21 Hz, 1H), 6.34(dd, J=17.01, 1H), 5.81(dd, J=10.21 Hz, 1H), 3.57(d, J=11.91
Hz, 2H), 2.54(td, J=10.75, 3.84 Hz, 1H), 2.13‒2.09(m, 2H), 1.74‒1.68(m, 4H); 13C NMR(100 MHz,
DMSO‒d6): δ 171.87, 164.74, 158.35, 156.41, 154.90, 146.88, 132.96, 132.27, 131.60, 131.44,
128.15, 127.63, 126.07, 125.77, 125.00, 120.66(2C), 116.78(2C), 103.80, 49.77(2C), 40.00,
28.60(2C). HRMS (ESI+) for C25H26ClN7O3, [M+Na]+ calcd: 530.1678, found: 530.1687.
4.2.1.9 1-(4-((4-((2-acrylamidophenyl)amino)-5-chloropyrimidin-2-yl)amino)benzyl)-N-hydroxy
piperidine-4-carboxamide (10i)
1
Yield 11.0%; off-white solid. H NMR(400 MHz, DMSO‒d6): δ 10.38(s, 1H), 10.20(s, 1H),
9.27(s, 1H), 8.68(s, 1H), 8.52(s, 1H), 8.12(s, 1H), 7.76(d, J=7.60 Hz, 1H), 7.48(d, J=8.16 Hz, 3H),
7.32(t, J=7.20 Hz, 1H), 7.28(t, J=7.56 Hz, 1H), 7.03(d, J=8.28 Hz, 2H), 6.52(dd, J=16.92, 10.12 Hz,
1H), 6.34(dd, J=16.92, 1.36 Hz, 1H), 5.82(dd, J=10.12, 1.36 Hz, 1H), 3.33(s, 2H), 2.80(d, J=10.76
Hz, 2H), 1.98‒1.93(m, 1H), 1.86‒1.82(m, 2H), 1.63‒1.56(m, 4H); 13C NMR(100 MHz, DMSO‒d6):
δ 172.04, 164.74, 158.22, 156.58, 154.95, 139.61, 132.50, 131.95, 131.47, 131.37, 129.25(2C),
128.20, 127.96, 125.97(2C), 124.94, 119.14(2C), 104.45, 62.43, 52.97(2C), 40.00, 28.89(2C).
4.2.1.10 1-(2-(4-((4-((2-acrylamidophenyl)amino)-5-chloropyrimidin-2-yl)amino)phenoxy)ethyl)-N-
hydroxypiperidine-4-carboxamide (10j)
1
Yield 13.3%; off-white solid. H NMR(400 MHz, DMSO‒d6): δ 10.39(s, 1H), 10.22(s, 1H),
9.12(s, 1H), 8.70(s, 1H), 8.47(s, 1H), 8.08(s, 1H), 7.75(d, J=7.76 Hz, 1H), 7.46(d, J=7.86 Hz, 1H),

7.43(d, J=8.04 Hz, 2H), 7.34(t, J=7.49 Hz, 1H), 7.27(t, J=7.56 Hz, 1H), 6.72(d, J=8.05 Hz, 2H),
6.52(dd, J=16.88, 10.00 Hz, 1H), 6.34(d, J=16.88 Hz, 1H), 5.82(d, J=10.00 Hz, 1H), 3.98(t, J=5.23
Hz, 2H), 2.95(d, J=10.71 Hz, 2H), 2.63(t, J=5.23 Hz, 2H), 1.99‒1.96(m, 3H), 1.63‒1.58(m, 4H); 13C
NMR(100 MHz, DMSO‒d6): δ 171.99, 164.73, 158.30, 156.48, 153.67, 134.04, 132.17, 131.76,
131.44, 128.18, 127.79, 126.06, 125.89, 124.95, 120.93(2C), 114.62(2C), 103.97, 66.22, 57.42,
53.62(2C), 40.00, 28.93(2C). HRMS (ESI+) for C27H30ClN7O4, [M+Na]+ calcd: 574.1940, found:
574.1954.
4.2.1.11 1-(2-((4-((4-((2-acrylamidophenyl)amino)-5-chloropyrimidin-2-yl)amino)phenyl)amino)-2-
oxoethyl)-N-hydroxypiperidine-4-carboxamide (10k)
1
Yield 12.9%; off-white solid. H NMR(400 MHz, DMSO‒d6): δ 10.42(s, 1H), 10.20(s, 1H),
9.52(s, 1H), 9.25(s, 1H), 8.71(s, 1H), 8.51(s, 1H), 8.11(s, 1H), 7.77(d, J=7.43 Hz, 1H), 7.48(d, J=7.70
Hz, 3H), 7.38(d, J=8.59 Hz, 2H), 7.30(t, J=8.48 Hz, 2H), 6.34(dd, J=16.97, 10.24 Hz, 1H), 6.53(dd,
J=16.97 Hz, 1H), 5.81(d, J=10.24 Hz, 1H), 3.11(s, 2H), 2.93‒2.90(d, J=9.61 Hz, 2H), 2.16(t, J=11.53
Hz, 2H), 2.01‒1.98(m, 1H), 1.80‒1.70(m, 2H), 1.63‒1.60(m, 2H); 13C NMR(100 MHz, DMSO‒d6):
δ 171.92, 168.12, 164.74, 158.14, 156.56, 154.93, 136.63, 132.72, 132.13, 131.91, 131.45, 128.19,
127.88, 125.95(2C), 124.97, 120.24(2C), 119.38(2C), 104.31, 62.34, 53.25(2C), 39.21, 28.64(2C).
HRMS (ESI+) for C27H29ClN8O4, [M+Na]+ calcd: 587.1893, found: 587.1908.
4.2.1.12 1-(4-((4-((4-acrylamidophenyl)amino)-5-chloropyrimidin-2-yl)amino)benzyl)-N-hydroxy
piperidine-4-carboxamide (10l)
1
Yield 9.4%; off-white solid. H NMR(400 MHz, DMSO‒d6): δ 10.38(s, 1H), 10.17(s, 1H),

9.27(s, 1H), 8.80(s, 1H), 8.70(s, 1H), 8.11(s, 1H), 7.68(d, J=8.82 Hz, 2H), 7.62(d, J=8.22 Hz, 2H),
7.55(d, J=8.10 Hz, 2H), 7.08(d, J=8.16 Hz, 2H), 6.47(dd, J=16.92, 10.06 Hz, 1H), 6.29(dd, J=16.92,
1.74 Hz, 1H), 5.77(dd, J=10.06, 1.74 Hz, 1H), 3.33(s, 2H), 2.79(d, J=10.98 Hz, 2H), 1.96‒1.91(m,
1H), 1.84‒1.81(m, 2H), 1.59‒1.55(m, 4H);13C NMR(100 MHz, DMSO‒d6): δ 172.03, 163.46, 158.20,
156.42, 154.83, 139.67, 135.62, 134.66, 132.41, 131.43, 129.33(2C), 127.11, 124.20(2C), 119.84(2C),
119.28(2C), 104.23, 62.51, 52.96(2C), 40.00, 28.89(2C). HRMS (ESI+) for C26H28ClN7O3, [M+H]+
calcd: 522.2015, found: 522.2050.
4.2.1.13 1-(2-(4-((4-((4-acrylamidophenyl)amino)-5-chloropyrimidin-2-yl)amino)phenoxy)ethyl)-N-
hydroxypiperidine-4-carboxamide (10m)
1
Yield 14.3%; off-white solid. H NMR(400 MHz, DMSO‒d6): δ 10.39(s, 1H), 10.17(s, 1H),
9.12(s, 1H), 8.77(s, 1H), 8.69(s, 1H), 8.07(s, 1H), 7.66(d, J=8.64 Hz, 2H), 7.59(d, J=7.15 Hz, 2H),
7.47(d, J=8.30 Hz, 2H), 6.77(d, J=8.58 Hz, 2H), 6.45(dd, J=16.94, 10.16 Hz, 1H), 6.28(dd, J=16.94,
1.14 Hz, 1H), 5.77(dd, J=10.16, 1.14 Hz, 1H), 3.98(t, J=5.70 Hz, 2H), 2.94(d, J=11.16 Hz, 2H), 2.63(t,
13
J=5.70 Hz, 2H), 1.98‒1.93(m, 3H), 1.62‒1.57(m, 4H); C NMR(100 MHz, DMSO‒d6): δ 171.96,
163.43, 158.32, 156.42, 154.92, 153.78, 135.57, 134.70, 134.05, 132.39, 127.09, 124,29(2C),
121.26(2C), 119.84(2C), 114.60(2C), 103.61, 66.16, 57.48, 53.62(2C), 40.00, 28.93(2C). HRMS
(ESI+) for C27H30ClN7O4, [M+H]+ calcd: 552.2121, found: 552.2138.
4.2.1.14 1-(2-((4-((4-((4-acrylamidophenyl)amino)-5-chloropyrimidin-2-yl)amino)phenyl)amino)-2-
oxoethyl)-N-hydroxypiperidine-4-carboxamide (10n)
1
Yield 12.5%; off-white solid. H NMR(400 MHz, DMSO‒d6): δ 10.43(s, 1H), 10.18(s, 1H),

9.52(s, 1H), 9.25(s, 1H), 8.72(s, 1H), 8.71(s, 1H), 8.12(s, 1H), 7.69‒7.64(m, 4H), 7.55(d, J=8.63 Hz,
2H), 7.44(d, J=8.60 Hz, 2H), 6.47(dd, J=16.90, 10.00 Hz, 1H), 6.27(dd, J=16.90, 2.10 Hz, 1H),
5.76(dd, J=10.00, 2.10 Hz, 1H), 3.09(s, 2H), 2.90(d, J=11.0 Hz, 2H), 2.14(t, J=11.4 Hz, 2H),
2.02‒1.94(m, 1H), 1.75(qd, J=12.2 Hz, 2H), 1.60(dd, J=11.66 Hz, 2H); 13C NMR(100 MHz,
DMSO‒d6): δ 171.96, 168.29, 163.45, 158.15, 156.29, 154.81, 136.73, 135.54, 134.70, 132.78,
132.42, 127.11, 123.89(2C), 120.43(2C), 119.84(2C), 119.59(2C), 104.17, 62.46, 53.30(2C), 39.28,
28.72(2C). HRMS (ESI+) for C27H29ClN8O4, [M+Na]+ calcd: 587.1893, found: 587.1940.
4.2.2 General procedure for the synthesis of 10o~q
A mixture of 15a~d (3.0 mmol), 18a~d (1.5 mmol), and p-TsOH (4.5 mmol) in ethyl alcohol
was gradually heated to 70 oC 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 chromatography with dichloromethane/methanol as eluents.
4.2.1.15 ethyl 1-(4-((4-((2-acrylamidophenyl)amino)-5-chloropyrimidin-2-yl)amino)phenyl)
piperidine-4-carboxylate (10o)
1
Yield 60.2%; off-white solid. H NMR(400 MHz, DMSO‒d6): δ 10.23(s, 1H), 9.07(s, 1H),
7.46(s, 1H), 8.07(s, 1H), 7.78(d, J=7.92 Hz, 1H), 7.44(d, J=7.56 Hz, 1H), 7.38(d, J=8.40 Hz, 2H),
7.34(td, J=7.86, 1.20 Hz, 1H), 7.27(td, J=7.98, 1.08 Hz, 1H), 6.73(d, J=8.76 Hz, 2H), 6.52(dd,
J=16.98, 10.20 Hz, 1H), 6.33(dd, J=16.98, 1.62 Hz, 1H), 5.81 (dd, J=10.20, 1.62 Hz, 1H), 4.08(q,
J=7.14 Hz, 2H), 3.47(dt, J=12.42, 3.17 Hz, 2H), 2.64(td, J=12.06, 2.28 Hz, 2H), 2.43(tt, J=11.22,
3.84 Hz, 1H), 1.89(dd, J=13.20, 2.94 Hz, 2H), 1.66(qd, J=11.64, 3.84 Hz, 2H), 1.19(t, J=7.14 Hz,

3H); 13C NMR(100 MHz, DMSO‒d6): δ 174.76, 164.72, 158.34, 156.40, 154.89, 146.60, 133.06,
132.25, 131.61, 131.44, 128.16, 127.65, 126.05, 125.76, 124.98, 120.61, 116.85, 103.82, 60.32, 49.45,
40.56, 28.17, 14.57. HRMS (ESI+) for C27H29ClN6O3, [M+Na]+ calcd: 543.1882, found: 543.1881.
4.2.1.16 ethyl 1-(4-((4-((2-acrylamidophenyl)amino)-5-chloropyrimidin-2-yl)amino)benzyl)
piperidine-4-carboxylate (10p)
1
Yield 56.3%; off-white solid. H NMR(400 MHz, DMSO‒d6): δ 10.21(s, 1H), 9.28(s, 1H),
8.52(s, 1H), 8.12(s, 1H), 7.74(dd, J=7.98, 1.21 Hz, 1H), 7.47(dd, J=7.98, 1.21 Hz, 1H), 7.46(d, J=8.42
Hz, 2H), 7.32(td, J=7.51, 1.40 Hz, 1H), 7.27(td, J=7.51, 1.40 Hz, 1H), 7.00(d, J=8.42 Hz, 2H),
6.51(dd, J=17.02, 10.21 Hz, 1H), 6.32(dd, J=17.02, 1.74 Hz, 1H), 5.80(dd, J=10.21, 1.74 Hz, 1H),
4.05(q, J=7.08 Hz, 2H), 3.31(s, 2H), 2.70(d, J=10.74 Hz, 2H), 2.26(tt, J=11.06, 3.84 Hz, 1H), 1.92(t,
J=10.80 Hz, 2H), 1.77(d, J=12.96 Hz, 2H), 1.53(qd, J=11.22, 3.42 Hz, 2H),1.19(t, J=7.08 Hz, 3H);
13C NMR(100 MHz, DMSO‒d6): δ 174.90, 164.70, 158.19, 156.56, 154.95, 132.03, 131.95, 131.45,
129.23(2C), 128.21, 127.98, 125.96(2C), 124.89, 119.08(2C), 104.41, 62.38, 60.24, 52.55(2C), 40.81,
28.46(2C), 14.57. HRMS (ESI+) for C28H31ClN6O3, [M+H]+ calcd: 535.2219, found: 535.2220.
4.2.1.17 ethyl 1-(4-((4-((3-acrylamidophenyl)amino)-5-chloropyrimidin-2-yl)amino)benzyl)
piperidine-4-carboxylate (10q)
1
Yield 61.4%; off-white solid. H NMR(400 MHz, DMSO‒d6): δ 10.18(s, 1H), 9.30(s, 1H),
8.94(s, 1H), 8.13(s, 1H), 7.89(s, 1H), 7.55‒7.52(m, 3H), 7.33‒7.30(m, 2H), 7.00 (d, J=8.28 Hz, 2H),
6.45(dd, J=16.95, 10.14 Hz, 1H), 6.26(dd, J=16.95, 1.80 Hz, 1H), 5.74(dd, J=10.14, 1.80 Hz, 1H),
4.04(q, J=7.08 Hz, 2H), 3.28(s, 2H), 2.69(d, J=10.86 Hz, 2H), 2.24(tt, J=11.22, 3.84 Hz, 1H), 1.89(t,