Design, synthesis and SAR study of 2-aminopyrimidines with diverse Michael addition acceptors for chemically tuning the potency against EGFRL858R/T790M

Article abstract of DOI:10.1016/j.bmc.2020.115680

The covalent binding nature of irreversible kinase inhibitors potentially increases the severity of “off-target” toxicity. Based on our continual strategy of chemically tuning the Michael addition acceptors, herein, we further explore the relationship amo

Full text of DOI:10.1016/j.bmc.2020.115680

Journal Pre-proofs
Design, synthesis and SAR study of 2-aminopyrimidines with diverse Mi‐
chael addition acceptors for chemically tuning the potency against EGFRL858R/
T790M
Jiaan Shao, Shuangrong Liu, Xingyu Liu, Youlu Pan, Wenteng Chen
PII:
DOI:
Reference:
S0968-0896(20)30510-1
https://doi.org/10.1016/j.bmc.2020.115680
BMC 115680
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Bioorganic & Medicinal Chemistry
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Accepted Date:
10 May 2020
22 July 2020
26 July 2020
Please cite this article as: Shao, J., Liu, S., Liu, X., Pan, Y., Chen, W., Design, synthesis and SAR study of 2-
aminopyrimidines with diverse Michael addition acceptors for chemically tuning the potency against EGFRL858R/
T790M, Bioorganic & Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.2020.115680
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Design, synthesis and SAR study of 2-aminopyrimidines
with diverse Michael addition acceptors for chemically
tuning the potency against EGFRL858R/T790M
Jiaan Shao, #, *, [a] Shuangrong Liu,#, [b] Xingyu Liu, [b] Youlu Pan, [b] Wenteng Chen,*,
[b]
a.
School of Medicine, Zhejiang University City College, Hangzhou, 310015, P. R.
China
b.
Zhejiang Province Key Laboratory of Anti-Cancer Research, College of
Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, P. R. China
# The authors contributed equally
*Corresponding authors. Email: shaojia@zucc.edu.cn (J. Shao)
wentengchen@zju.edu.cn (W. Chen)
ABSTRACT:
The covalent binding nature of irreversible kinase inhibitors potentially increases the
severity of “off-target” toxicity. Based on our continual strategy of chemically tuning
the Michael addition acceptors, herein, we further explore the relationship among the
electronic nature of Michael addition acceptors and EGFRT790M mutation selectivity as
well as “off-target” toxicity balance. By perturbing the electronic nature of acrylamide
moiety, compound 8a with a chloro-group at the α-position of the Michael addition
L858R/T790M
acceptor was identified. It was found that 8a retained the excellent EGFR
potency (IC50 = 3.9 nM) and exhibited good anti-proliferative activities against the
gefitinib-resistant NCI-H1975 cells (IC50 = 0.75 μM). Moreover, 8a displayed a
significant EGFRWT selectivity and much weaker inhibitory activity against non-EGFR
dependent SW620 cell and COS7. Preliminary study showed that 8a could arrest NCI-
H1975 cells in G0/G1 phase. This work provides a promising chemical tuned strategy
for balancing the mutant-EGFR potency and selectivity as well as “off-target” toxicity.
KEYWORDS: NSCLC; EGFR; resistant mutants; chemical tuned; Michael addition
acceptors
1. INTRODUCTION
Overexpression or abnormal activation of the epidermal growth factor receptor (EGFR)
is one of the major drivers of non-small cell lung cancer (NSCLC), which accounts for
85% of lung cancer. Approximately 50% of Asian patients and 11-16% of patients in
Western countries with NSCLC harbor mutations in EGFR. [1-2] The majority of genetic
EGFR alterations are deletions within exon 19 (60%) and L858R point mutation (30%).
[3] These sensitizing EGFR mutations pave the way for the development and therapeutic
application of EGFR TKIs. The first-generation inhibitors gefitinib, [4-5] erlotinib[6] and
icotinib [7] as well as the second-generation inhibitors afatinib [8] were approved for the
treatment of patients with the most frequent EGFR mutations (L858R and exon 19
deletions). However, patients acquired resistances and suffered a relapse within 18
months of TKIs treatment. Biopsies of resistant tumors revealed a single point mutation

of the gatekeeper residue (T790M) as the major resistant mechanism. [9] Due to the
unacceptable dose-limiting toxicity associated with quinazoline core against the wild-
type (WT) EGFR inhibition, even irreversible inhibitor afatinib did not exhibit
improved clinical efficacy for the NSCLC patients who harbor T790M mutation. [10]
Hence, third-generation inhibitors, such as rociletinib [11], osimertinib, [12] olmutinib [13]
and almonertinib (NCT03849768), structurally based on 2-aminopyrimidine were
developed to overcome the above “on-target” toxicity. In contrast to the classical
quinazoline core, the aminopyrimidine scaffold demonstrated excellent selectivity
sparing EGFR WT and good activity against the T790M resistance mutation. [14-15] Also
[16]
these EGFR inhibitors are developed with irreversible Cys797-targeting moieties.
This strategy would enable the drugs to bind the target covalently. The off-rate is
negligible compared to that of a non-covalent drug and therefore these drugs would
have a prolonged therapeutic effect and circumvent drug resistance. [17]
However, it is far from perfect that the covalent binding nature of irreversible
inhibitors potentially increases the severity of “off-target” toxicity. [18] The intrinsic
irreversible chemistry of reactive acrylamide moiety is more likely to form permanent
covalent adducts [19] with off-target proteins, especially those off-target kinases with a
[20]
homologous cysteine or unrelated targets with hyper-reactive cysteine.
How to
exploit novel covalent inhibitors for overcoming the issue of drug resistance and
reducing the risk of nonspecific covalent binding is still challenging.
Our previous work has demonstrated that fluoro-substituted olefins could retain the
mutant-EGFR inhibition and increase the safety index of irreversible inhibitors.
(Figure 1) [21] But the previous hit did not show excellent EGFR WT selectivity. To our
continued efforts, novel 2-aminopyrimidine analogues bearing α-position substituted
acrylamides, including F, Cl, Ph, CH3, were designed and synthesized in this work. We
aimed to investigate the relationship among the electronic nature of the acrylamide and
mutant-EGFR selectivity as well as the “off-target” toxicity.
Figure 1. Strategy on chemical tuning the electronic nature of the Michael addition
acceptors
2. RESULTS AND DISCUSSION
2.1 CHEMISTRY
To investigate the effect of the electronic nature of the acrylamide moiety against
EGFR mutants, a series of molecules were synthesized as shown in Scheme 2-4. The

key intermediate 3 were prepared following the procedure in Scheme 1 as previously
reported. [22] The substitution of 1-fluoro-4-nitrobenzene or 2-fluoro-5-nitropyridine 1
either with 1-methylpiperazine or N-acetyl-piperazine afforded the corresponding
products 2. The compound 3 was easily obtained by reducing the nitro group of 2 via
NaBH4 and NiCl26H2O.
R1
NO2
R1
NO2
NH2
b
a
R1
N
Z
N
Z
Z
N
N
R2
R2
F
3
2
1
(Z=C or N)
(a) amine, K2CO3, DMSO, 90 oC, 4 h, 85-92%; (b) NaBH4 and NiCl2 6H2O, DCM/MeOH, 0
Reagents and conditions:
oC, 1 h, 90-95%;
Scheme 1. Synthesis of the intermediates 3.
Tert-butyl (3-aminophenyl) carbamate was region-selectively coupled to the 4-
position of 2,4-dichloropyrimidine analogues 4 to afford 2-chloro-4-anilinopyrimidines
[23]
5 at room temperature.
Coupling reaction of 3 with 5 was accomplished in the
presence of 1N HCl under reflux, offering anilinopyrimidine 6. [24] The protection group
Boc were removed by TFA to produce 7. Acylation of amino group of 7 using diverse
acrylic acid gave compound 8a-8r, Scheme 2.
O
O
N
N
N
R3
N
R3
N
N
d
N
N
N
N
NH
N
N
NH
H
H
X
N
H
O
NH2
8
7
8q
8r
: R3=Cl, X=Cl
: R3=Cl, X=F
O
c
N
N
R3
NH
N
N
N
N
H
6
NHBoc
b
O
N
Cl
HN
NHBoc
N
R3
NH
N
R3
R3
b
a
N
N
5
N
H
N
R1
N
4
Cl
N
Cl
6
NHBoc
O
O
N
N
N
R3
N
R3
N
N
d
c
N
N
NH
N
N
NH
R1
R1
H
H
X
N
O
NH2
8
7
: R1=H, R3=Cl, X=Cl
: R1=H, R3=Cl, X=Me
: R1=H, R3=Cl, X=F
: R1=H, R3=Cl, X=Ph
H
8a
8b
8c
8d
8e
8i
8j
8k
8l
8m
8n
8o
8p
: R1=3-Me, R3=Cl, X=Cl
: R1=3-Cl, R3=Cl, X=F
: R1=3-F, R3=Cl, X=F
: R1=3-F, R3=Cl, X=Cl
: R1=2-OMe, R3=Cl, X=Me
: R1=2-OMe, R3=Cl, X=F
: R1=2-OMe, R3=CF3, X=Cl
: R1=3-CF3, R3=Cl, X=F
: R1=3-Cl, R3=Cl, X=Cl
: R1=2-Cl, R3=Cl, X=Cl
8f
8g
8h
: R1=2-OMe, R3=Cl, X=Cl
: R1=3-Me, R3=Cl, X=F
o
Reagents and conditions
3
:(a) aniline, DIEA, n-BuOH, 0 C, 2 h, 65-75%; (b) , 1N HCl, n-BuOH, reflux, 4 h, 52-60%;
(c) TFA, DCM, 0 oC, 2 h, 90-95%; (d) acrylic acid (COCl)2, TEA, DCM, 0 oC-rt, 4 h, 50-60%.
,
Scheme 2. Synthesis of the compound 8.

[25]
Similarly,
in Scheme 3, the region-selective coupling of 9 with tert-butyl (3-
hydroxyphenyl) carbamate gave the 4-position etherification product 10 at room
temperature. And the 2-chloro group was coupled with 3 to deliver 11 in the presence
of 1N HCl under reflux. The Boc group of 11 was removed by TFA to produce
arylamine 12. Acylation of amino group of 12 using diverse acrylic acid gave 13a-13f.
R2
N
Boc
N
Cl
O
Cl
O
N
H
N
Cl
Cl
b
a
N
N
N
N
R1
H
N
Cl
N
Cl
11
9
10
NH
Boc
R2
R2
N
N
N
Cl
O
N
Cl
N
N
d
c
N
N
R1
N
N
O
R1
H
H
X
13
N
H
O
12
NH2
13a: R1=2-OMe, R2=acetyl, X=F
13b: R1=2-OMe, R2=acetyl, X=Cl
13c: R1=2-OMe, R2=Me, X=F
13d: R1=2-OMe, R2=Me, X=Cl
13e: R1=H, R2=acetyl, X=Cl
13f: R1=3-CF3, R2=acetyl, X=Cl
o
Reagents and conditions:
3
(a) tert-butyl (3-hydroxyphenyl)carbamate, DIPEA, n-BuOH, 50 C, 4 h, 72%; (b) , 1N
HCl, n-BuOH, reflux, 4 h, 50-60%; (c) TFA, DCM, 0 oC, 1 h, 90-95%; (d) acrylic acid, (COCl)2, TEA, DCM, 0 oC-rt,
4 h, 40-55%.
Scheme 3. Synthesis of the compound 13
To access indole derivatives 14, commercial 2, 4, 5-trichloropyrimidine was used.
As previously reported, the introduction of the N-methylindole was achieved via AlCl3
[26]
(Scheme 4) and subsequent SNAR with 4-fluoro-3-nitroaniline to give 15. The
fluoro group of 15 was substituted with dimethylamine to afford 16. The nitro-group of
16 was reduced followed with acylation, delivering 17a-17b.
N
Cl
F
Cl
N
Cl
a
b
N
Cl
O2N
N
N
N
H
N
9
Cl
N
N
Cl
15
14
N
Cl
N
Cl
N
N
c
d,e
HN
N
N
O2N
N
N
17a: X=F
H
H
X
N
N
O
17b: X=Cl
17
16
(a) AlCl3, DCE, 80 oC, 2 h, 89%; (b) aniline, 1N HCl, n-BuOH, reflux, 4 h, 85%; (c) K2CO3,
Reagents and conditions:
(CH3)2NH, DMF, 100 oC, 2 h, 93%; (d) NaBH4 and NiCl2 6H2O, DCM/MeOH, 0 oC, 1 h, 92%; (e) acrylic acid, (COCl)2,
TEA, DCM, 0 oC-rt, 4 h, 40-55%.
Scheme 4. Synthesis of the compound 17
2.2 BIOLOGICAL EVALUATION
2.2.1 Cellular and Kinase Inhibitory Activity
The antiproliferative effects of 8a-8r, 13a-13f and 17a-17b against gefitinib-
sensitive A431 bearing EGFRWT and gefinitib-resistant NCI-H1975 bearing
EGFRL858R/T790M were evaluated using Afatinib and Rociletinib as positive controls. We

first investigated the electron nature of Michael addition receptors. As shown in Table
1, modifying the acrylamide moiety with chloro (8a, X=Cl, 0.75 μM) and fluoro (8c,
X=F, 0.34 μM) were able to retain the potencies against gefitinib-resistant NCI-H1975
cells. While replacing the α-position of acrylamides with methyl (8b, X=Me, 5.57 μM)
or phenyl (8d, X=Ph, 5.79 μM) almost decreased the potencies by 10 folds. These
seemed the strong electron-withdrawing substituents on Michael addition acceptors
would be more favor for the mutant-EGFR inhibition. Next, we examined the impacts
of R1 on the left-hand side of the NH-substituted phenyl ring. The hydrogen atom in 8a
and 8c were replaced with 2-methoxyl (8g and 8f), 3-methyl (8i and 8h), 3-fluoro (8l
and 8k), 3-chloro (8o and 8j), 2-chloro (8p) or 3-trifluromethyl (8n) groups,
respectively. The results implied that 8i and 8h (R1=3-Me) exhibited the most potent
mutant-EGFR inhibition with the IC50 values of 0.18 and 0.21 μM, respectively. Further
variants at the same position, 3-fluoro (8l and 8k), 3-chloro (8o and 8j), and 3-
trifluoromethyl (8n) did not improve the inhibitory activities. While replacing the
phenyl ring with pyridinyl ring (8q and 8r) completely abolished the activities. When
the R2 was substituted with methyl group instead of acetyl group, the resultant
compounds significantly decreased the activity by 2-4 folds. (13a vs 13c, 13b vs 13d)
Further linkers between the pyrimidine ring and 4-phenyl ring at the right-hand side
were also tested (Table 1). It was found that replacing the –NH- with –O- did not
improve the mutant inhibition. (8f vs 13a, 8g vs 13b, 8a vs 13e) Final, the substitution
position of Michael addition acceptor was investigated. When the acrylamide moiety
was migrated to the left-hand side of pyrimidine core and the aniline ring at the right-
hand side was removed and replaced with an N-methylindole, the corresponding
compounds 17a and 17b almost lost the inhibitory activity against gefitinib-resistant
NCI-H1975 cells. Importantly, selectivity indexes of inhibition against gefitinib-
sensitive A431 and gefitinib-resistance NCI-H1975 were investigated. Compared to
Afatinib (SI [IC50 (A431) / IC50 (NCI-H1975)] = 1.5) and Rociletinib (SI [IC50(A431) /
IC50(NCI-H1975)] = 11.8), some selected compounds displayed good excellent
selectivity with a SI up to 25.6-fold.
Moreover, the “off-target” toxicity of selected compounds were also evaluated. The
colon cancer cell SW620, which is not dependent on EGFR signaling for cell growth,
is used to rule out the potential activity due to non-EGFR. Compared to the inhibitory
activity against H1975 (IC50 = 0.18-0.75 μM), 8a, 8c, 8h, 8i and 8m displayed much
weaker inhibition against SW620 with IC50 value up to 29.15 μM. Besides, the monkey
fibroblast cell COS7 was selected as non-tumor cell model to test the cytotoxicity. As
shown in Table 1, the IC50 values of 8a, 8o, 8i and 8p ranged from 5.40-14.87 μM,
indicating the lower cytotoxicity comparing with those against NCI-H1975.
Table 1. Anti-proliferative activities and selectivity of compound 8, 13 and 17 against
cancer cells harboring different EGFR status and normal cell
IC50 / μM a
IC50 / μM a
SW620e COS7f
29.15±0.01 10.44±0.66
No.
SI(A431/H1975)d
A431b
H1975c
8a
8b
3.14±0.24
10.60±0.54
0.75±0.02
5.57±0.32
4.2
1.9
/
/

8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
6.50±0.35
6.16±0.52
2.37±0.12
1.58±0.13
4.44±0.98
5.37±0.13
1.30±0.08
7.13±1.21
7.09±0.49
1.14±0.14
3.68±0.33
2.86±0.33
0.14±0.06
1.83±0.11
0.87±0.38
0.34±0.12
5.79±0.31
3.82±0.16
1.82±0.28
3.12±0.19
0.21±0.04
0.18±0.05
4.18±0.57
6.56±0.43
0.42±0.42
0.20±0.02
9.88±0.14
1.02±0.17
1.72±0.23
1.64±0.40
19.1
1.1
0.6
0.9
1.4
25.6
7.2
1.7
1.1
2.7
18.4
0.3
0.1
1.1
0.5
0.9
/
0.8
0.7
0.1
0.5
0.6
/
0.1
1.5
11.8
3.61±1.26
/
/
/
/
/
/
/
/
/
/
2.33±0.07
2.07±0.17
5.40±0.04
/
/
/
/
/
/
/
3.99±0.31
/
/
2.34±0.46
3.02±1.08
2.41±0.84
7.22±0.56
14.87±1.32
8q
8r
/
/
/
/
/
/
/
/
18.09±0.97 20.54±0.24
>50
1.89±0.19
/
/
/
/
13a
13b
13c
13d
13e
13f
17a
17b
Afatinib
Rociletinib
7.03±0.18
3.32±0.51
10.98±0.13 16.72±0.64
1.06±0.20
0.62±0.14
1.47±0.16
>50
2.20±0.24
1.40±0.01
6.01±0.10
13.57±0.25
1.15±0.13
2.50±0.18
>50
16.88±1.29
0.92±0.01
0.51±0.01
/
4.60±0.40
/
/
/
/
/
/
2.13±0.25
10.50±0.50
7.39±1.11
a.The inhibitory effects of individual compounds on the proliferation of cancer cell lines
were determined by the MTT assay. The data reported are the mean values from two
b.
c.
independent experiments. A431 is human epithelial carcinoma cell line (EGFRWT).
H1975 is a human non-small-cell lung cancer cell line (EGFRL858R/T790M). d. ratio of the IC50
values of [A431] vs [H1975]. e. SW620 is human colon carcinoma cell line (EGFR negative).
f. COS7 is monkey fibroblast cell (used as normal cell).
Encouraged by the good anti-proliferative activity against gefitinib-resistant cell
NCI-H1975, the kinase inhibition against EGFRs including wild-type and
L858R/T790M mutant were measured. As presented in Table 2, compounds (8a, 8i, 8o
and 8q) bearing a α-chloroacrylamide group showed more excellent inhibitory
activities against double-mutant kinase EGFRL858R/T790M with the IC50 values ranging
from 2.6-7.5 nM. The different activity between chloro-substituted 8a and fluoro-
substituted 8c could be explained by the calculated charge distribution. (Figure S1 in
supporting information) Moreover, the compounds exhibited a better selectivity
between the wild-type and double-mutant EGFRs. For example, the IC50 value of 8a
against EGFRWT was 70 nM, which was 17.9-fold of that against EGFRL858R/T790M (3.9
nM). Similarly, the IC50 value of 8i against EGFRWT was 51 nM, which was 19.6-fold

of that against EGFRL858R/T790M (2.6 nM). These data demonstrated that the
incorporation of chloro group on acrylamide group could retain the inhibitory activity
against EGFRL858R/T790M while with a good EGFRWT selectivity.
Table 2. Inhibitory activity of selected compounds against different types of EGFRs
EGFR IC50/nMa
No.
SI(WT/DM)b
WT
70
>1000
>1000
51
257
53
301
130
130
DM(L858R/T790M)
8a
8c
8h
8i
8m
8o
8p
8q
13e
3.9
213
174
2.6
16
3.2
47
7.5
13
17.9
/
/
19.6
16.1
16.6
6.4
17.3
10.0
Afatinib
1.2
21
0.06
Rociletinib
76
1.8
42.2
a EGFR activity assays were performed using HTRF method. The
data reported are the mean values from duplicated wells. b. ratio
of the IC50 values of [WT] vs [DM]
2.2.2 8a Induces Cell Cycle Arrest in NCI-H1975
In order to better verify the effect of compound 8a on cell cycle, flow cytometry was
performed on NCI-H1975 cells treated with various concentrations of 8a. As shown in
Figure 2, the percentage of cells in the G0/G1 phase increased from 41.92% to 58.39%,
and those in the G2/M phase decreased from 34.04% to 22.18% after treatment with 8a
at the concentrations from 0.25 μM to 1.0 μM for 24 h. Accordingly, 8a arrested NCI-
H1975 cells in G0/G1 phase.
Figure 2. Effects of 8a on H1975 cells cycle arrest detected by flow cytometry assay.
Cells were treated with different concentrations of 8a for 24 h, collected and fixed with

70% ethanol at 4 oC overnight. Then, the cells were stained by the mixture containing
o
propidium iodide for 30 min at 37 C, and the cell cycle was analyzed by a flow
cytometer.
2.3 Molecular Docking Study of 8a
8a was initially docked to the EGFR binding pocket based on the co-crystal sturcture
of the Rociletinib-EGFRT790M (PDB ID: 5XDK) and the resulting simulated complex
of 8a-EGFRT790M was shown in Figure 3. The docking study revealed that 8a occupied
the binding pocket of kinase in a similar pattern to Rociletinib. The aminopyrimidine
moiety of 8a binds to Met793 residue at the hinge region through two strong hydrogen
bonding interactions. The warhead, chloro-substituted acrylamide moiety, oriented to
the thiol group of Cys797 with a measured distance of 1.70 Å, indicating the possibility
of forming a covalent bond.
Figure 3. Putative binding mode of 8a within the active pocket of the EGFR [T790M]
3. CONCLUSION
On the basis of our previous work, herein we further explored the relationship among
electronic nature of Michael addition acceptor and EGFRT790M mutation selectivity as
well as “off-target” toxicity balance. The cellular-based anti-proliferative inhibition and
kinase inhibitory assay demonstrated that the chemical perturbation of acrylamide with
chloro- group at α-position could retain the good inhibitory activity against gefitinib-
resistant NCI-H1975 cells with EGFRL858R/T790M. This led to the identification of 8a,
which was found to effectively inhibit NCI-H1975 (IC50 = 0.75 μM) and EGFR
L858R/T790M
(IC50 = 3.9 nM) as well as display excellent wild-type and mutant EGFRs
selectivity. Moreover, 8a exhibited a weaker inhibition against EGFR-independent

colon cancer cell line SW620 and non-tumor monkey fibroblast cell COS7 with IC50
values up to 10 μM. In all, this work provided a promising chemical tuned strategy for
balancing the mutant-EGFR potency and selectivity as well as “off-target” toxicity.
4. EXPERIMENTAL SECTIONS
4.1 GENERAL METHODS
Unless otherwise noted, all solvents and chemicals were used as purchased without
further purification. All reported yields are isolated yields after column
1
chromatography. H NMR spectra were recorded on a Bruker DRX-500 [Bruker
Biospin, Germany]. Chemical shifts are reported in ppm relative to the residual solvent
peak (CDCl3, TMS: 0.00). Multiplicity was indicated as follows: s (singlet); d (doublet);
t (triplet); q (quartet); m (multiplet); dd (doublet of doublet); dt (triplet of doublet); td
(doublet of triplet); brs (broad singlet) etc. Intermediates were purified by column
chromatography on silica gel (200-300 mesh). HRMS of all biologically evaluated
compounds was confirmed on a Agilent 1290 HPLC-6224 Time of Fight Mass
Spectrometer using Phenomenex Luna 5l C18, 100 Å, 150-4.60 mm 5 micron column
at a flow rate of 0.5 mL/min using liner gradients buffer B in A (B: CH3OH containing
0.1% formic acid, A: H2O containing 0.1% formic acid). Mobile phase B was increased
linearly from 5% to 95% over 7 min and 95% over the next 2 min, after which the
column was equilibrated to 5% for 1 min.
4.2 SYNTHESIS OF INTERMEDIATES
Synthesis and Characterization of Compounds 2a, 3a, 5a, 6a, 7a, 10, 14, 15 and 16
1-(4-(4-nitrophenyl) piperazin-1-yl) ethan-1-one (2a). [27] Organic solid. Yield=92%.
1H NMR (500 MHz, DMSO-d6) δ 8.08-8.02 (m, 2H), 7.05-6.99 (m, 2H), 3.44 (t, J =
5.1 Hz, 4H), 2.42 (t, J = 5.1 Hz, 4H), 2.22 (s, 3H).
1-(4-(4-aminophenyl) piperazin-1-yl) ethan-1-one (3a). To a solution of 2a (1.0
mmol) and NiCl26H2O (2.0 mmol) in 10 mL of MeOH/DCM (V/V=1:4) was added
o
o
NaBH4 (4.0 mmol) at 0 C. The mixture was stirred at 0 C for 30 min then at room
temperature for 30 min. The mixture was filtered, and the filtrate was evaporated to
dryness under reduced pressure. The crude product was purified by flash column
chromatography on silica gel. Brown solid in 90% yield. 1H NMR (500 MHz, DMSO-
d6) δ 6.71 (d, J = 8.0 Hz, 2H), 6.50 (d, J = 8.0 Hz, 2H), 4.70 (s, 2H), 3.55-3.51 (m, 4H),
2.85 (dt, J = 33.0, 5.0 Hz, 4H), 2.02 (s, 3H).
[28]
tert-butyl (3-((2,5-dichloropyrimidin-4-yl) amino) phenyl) carbamate (5a).
Yellow solid. Yield: 72%. 1H NMR (500 MHz, DMSO-d6) δ 9.54 (s, 1H), 9.46 (s, 1H),
8.37 (s, 1H), 7.71 (t, J = 2.0 Hz, 1H), 7.28-7.15 (m, 3H), 1.48 (s, 9H).
tert-butyl
(3-((2-((4-(4-acetylpiperazin-1-yl)-2-methoxyphenyl)
amino)-5-
chloropyrimidin-4-yl) amino) phenyl) carbamate (6a). [29] Yellow solid. Yield: 52%.
1H NMR (500 MHz, DMSO-d6) δ 8.41 (s, 1H), 8.01 (s, 1H), 7.70-7.68 (m, 2H), 6.95-
6.92 (m, 2H), 6.77-6.75 (m, 1H), 6.65 (d, J = 2.5 Hz, 1H), 6.44 (dd, J = 9.0, 2.5 Hz,
1H), 6.33-6.31 (m, 1H), 4.94 (s, 2H), 3.79 (s, 3H), 3.60-3.56 (m, 4H), 3.07 (dt, J = 29.0,
5.0 Hz, 4H), 2.05 (s, 3H).
1-(4-(4-((4-((3-aminophenyl)
amino)-5-chloropyrimidin-2-yl)
amino)-3-
methoxyphenyl) piperazin-1-yl) ethanone (7a). To a solution of 6a (1.0 mmol) in
DCM (5 mL) was added TFA (10.0 mmol) at ice-bath. The mixture was stirred at room

temperature for 1 h. The reaction solution was neutralized with saturated aqueous
NaHCO3, and extracted with DCM (3 X 15 mL). The organic layer was washed with
saturated aqueous NaCl, dried over anhydrous Na2SO4, and concentrated in vacuo to
obtain a brown solid in 90% yield. 1H NMR (500 MHz, DMSO-d6) δ 8.51 (s, 1H), 8.10
(s, 1H), 7.82-7.72 (m, 2H), 7.04-7.00 (m, 2H), 6.84 (d, J = 7.9 Hz,1H), 6.74 (d, J = 2.4
Hz,1H), 6.54-6.52 (m, 1H), 6.41-6.40 (m, 1H), 5.04 (s, 2H), 3.88 (s, 3H), 3.69-3.65(m,
4H), 3.20-3.12 (m, 4H), 2.14 (s, 3H).
tert-butyl (3-((2,5-dichloropyrimidin-4-yl) oxy) phenyl) carbamate (10). [30] Yellow
solid. Yield: 72%. 1H NMR (500 MHz, DMSO-d6) δ 9.64 (s, 1H), 8.83 (s, 1H), 7.47 (t,
J = 2.0 Hz, 1H), 7.38-7.32 (m, 2H), 6.91-6.89 (m, 1H), 1.48 (s, 9H).
3-(2-chloropyrimidin-4-yl)-1-methyl-1H-indole (14)
A
suspension of 2,4-
dichloropyrimidine (1.0 mmol) and aluminum chloride (1.0 mmol) in DME was stirred
at ambient temperature for 5 min. To this was added N-methylindole (1.0 mmol), and
the mixture was heated to 80 °C for 2 h. The cool reaction mixture was added dropwise
to vigorously stirring water over 5 min. Upon complete addition the mixture was stirred
for 30 min, filtered and the solid washed with water. The crude product was purified by
flash silica chromatography, eluting with DCM. Pure fractions were evaporated to
dryness to afford 14 as a yellow solid. Yield: 89%. 1H NMR (500 MHz, DMSO-d6) δ
8.80 (s, 1H), 8.73 (s, 1H), 8.56 (d, J = 7.6 Hz, 1H), 7.62 (d, J = 8.0 Hz, 1H), 7.38-7.34
(m, 1H), 7.34-7.30 (m, 1H), 3.96 (s, 3H). HRMS (ESI) calcd. for C13H10Cl2N3 [M+H]+=
278.0246, found 278.0247.
5-chloro-N-(4-fluoro-3-nitrophenyl)-4-(1-methyl-1H-indol-3-yl)
pyrimidin-2-
amine (15) 1N HCl (1.2 equiv) was added in one portion to 3-(2-chloropyrimidin-4-yl)-
1-methylindole (1.0 mmol) and 4-fluoro-5-nitroaniline (1.0 equiv) in n-BuOH. The
resulting mixture was stirred at 120 °C for 4 h. The mixture was cooled to room
temperature. The precipitate was collected by filtration, washed with n-BuOH, and
1
dried under vacuum to afford 15 as a yellow solid. Yield: 85%. HNMR (500 MHz,
DMSO-d6) δ 10.11 (s, 1H), 8.70-8.68 (m, 1H), 8.62-8.60 (m, 2H), 8.55 (s, 1H), 8.11-
8.08 (m, 1H), 7.58-7.52 (m, 2H), 7.34-7.30 (m, 1H), 7.22-7.19 (m, 1H), 3.94 (s, 3H).
HRMS (ESI) calcd. for C19H14ClFN5O2 [M+H]+=398.0815, found 398.0816.
N4-(5-chloro-4-(1-methyl-1H-indol-3-yl) pyrimidin-2-yl)-N1, N1-dimethyl-2-
nitrobenzene-1, 4-diamine (16) To a solution of N-(4-fluoro-5-nitrophenyl)-4-(1-
methylindol-3-yl)pyrimidin-2-amine (1.0 mmol) and K2CO3 (2.0 equiv) in DMF was
added N,N-dimethylamine (1.0 equiv). The mixture was heated at 100 °C for 2 h. Then
the reaction was evaporated and purified by silica chromatography to afford red solid.
Yield: 92.6%. 1HNMR (500 MHz, DMSO-d6) δ 9.76 (s, 1H), 8.58 (s, 2H), 8.48 (s, 1H),
8.32 (d, J = 2.3 Hz, 1H), 7.81 (dd, J = 9.0, 2.1 Hz, 1H), 7.56 (d, J = 8.2 Hz, 1H), 7.30
(t, J = 7.5 Hz, 1H), 7.25 (d, J = 9.1 Hz, 1H), 7.17 (t, J = 7.3 Hz, 1H), 3.93 (s, 3H), 2.77
(s, 6H). HRMS (ESI) calcd. for C21H20ClN6O2 [M+H]+= 423.1331, found 423.1336.
4.3 Synthesis of final compounds
To a solution of acrylic acid (4.0 mmol, 4.0 equiv) in anhydrous DCM (8 mL) was
added 3 drops of DMF at 0 oC and oxalyl chloride (3.47 mmol, 3.47 equiv) in DCM (2
mL) was added dropwise. Then the mixture was stirred at 0-10 oC for 20 min and at 25
oC for 2h, then the temperature of reaction mixture is adjusted to 45 oC for 5 min. Next,

o
the mixture was cooled to 0 C. A solution of the aniline 7, 12 or 16 (1.0 mmol) and
Et3N (5.0 mmol, 5.0 equiv) in anhydrous DCM-DMF (6 mL/1 mL) was added dropwise
to the above mixture. The mixture was stirred at room temperature for another 3-4 h.
The mixture was quenched with saturated Na2CO3 solution, extracted with EtOAc (3 X
20 mL) and dried over anhydrous sodium sulfate. The combined organic layer was
evaporated to dryness under reduced pressure. The residue was purified through silica
gel to give pure product.
Characterization of the final compounds
N-(3-((2-((4-(4-acetylpiperazin-1-yl) phenyl) amino)-5-chloropyrimidin-4-yl)
amino) phenyl)-2-chloroacrylamide (8a)
Light yellow solid, m.p.: 123-125 oC. 1H NMR (500 MHz, DMSO-d6) δ 10.22 (s, 1H),
9.15 (s, 1H), 8.90 (s, 1H), 8.10 (s, 1H), 7.93 (s, 1H), 7.47 (t, J = 9.5 Hz, 3H), 7.42-7.26
(m, 2H), 6.75 (d, J = 8.5 Hz, 2H), 6.42 (d, J = 2.5 Hz, 1H), 6.10 (d, J = 3.0 Hz, 1H),
3.55 (dd, J = 10.0, 8.0 Hz, 4H), 3.06-2.86 (m, 4H), 2.03 (s, 3H). 13C NMR (125 MHz,
CDCl3) 169.2, 158.8, 158.0, 155.6, 154.4, 147.0, 138.8, 137.5, 132.8, 132.2, 129.5,
124.1, 121.6, 118.1, 117.5, 115.8, 113.2, 104.8, 50.5, 50.1, 46.4, 41.5, 21.5. HRMS
(m/z): [M+H] + calcd for C25H25Cl2N7O2:526.1520, found: 526.1521.
N-(3-((2-((4-(4-acetylpiperazin-1-yl) phenyl) amino)-5-chloropyrimidin-4-yl)
amino) phenyl) methacrylamide (8b)
o
1
Light yellow solid, m. p.: 153-155 C. H NMR (500 MHz, CDCl3) δ 8.04 (m, 2H),
7.54 (s, 1H), 7.44 (d, J = 8.5 Hz, 2H), 7.41-7.39 (m, 1H), 7.30 (d, J = 6.0 Hz, 2H), 7.10
(s, 1H), 7.06 (s, 1H), 6.86 (d, J = 8.5 Hz, 2H), 5.78 (s, 1H), 5.47 (s, 1H), 3.78-3.76 (m,
2H), 3.62-3.60 (m, 2H), 3.10-3.07 (m, 4H), 2.14 (s, 3H), 2.05 (s, 3H). 13C NMR (125
MHz, CDCl3)  169.2, 166.7, 158.1, 155.6, 154.5, 146.9, 140.8, 138.7, 138.5, 132.9,
129.4, 121.5, 120.3, 117.5, 117.3, 115.5, 113.0, 104.9, 50.5, 50.2, 46.4, 41.5, 21.5, 18.9.
HRMS (m/z): [M+H] + calcd for C26H28ClN7O2:506.2066, found: 506.2072.
N-(3-((2-((4-(4-acetylpiperazin-1-yl) phenyl) amino)-5-chloropyrimidin-4-yl)
amino) phenyl)-2-fluoroacrylamide (8c)
White solid, m.p.: 115-117oC. 1H NMR (500 MHz, CDCl3) δ 8.09 (s, 1H), 8.05 (s, 1H),
7.95 (s, 1H), 7.43-7.40 (m, 3H), 7.35-7.32 (m, 2H), 7.11 (s, 1H), 7.04 (s, 1H), 6.87 (d,
J = 8.0 Hz, 2H), 5.84 (d, J = 48.5 Hz, 1H), 5.27 (d, J = 14.5 Hz, 1H), 3.77 (m, 2H), 3.61
+
(m, 2H), 3.08-3.10 (m, 4H), 2.14 (s, 3H). HRMS (m/z): [M+H]
C25H25ClFN7O2: 510.1815, found: 510.1814.
calcd for
N-(3-((2-((4-(4-acetylpiperazin-1-yl) phenyl) amino)-5-chloropyrimidin-4-yl)
amino) phenyl)-2-phenylacrylamide (8d)
White solid, m.p.: 107-108 oC. 1H NMR (500 MHz, DMSO-d6) δ 10.28 (s, 1H), 9.11
(s, 1H), 8.89 (s, 1H), 8.09 (s, 1H), 7.97 (s, 1H), 7.48-7.47 (m, 5H), 7.37-7.35 (m, 5H),
6.76 (d, J = 7.0 Hz, 2H), 5.95 (s, 1H), 5.73 (s, 1H), 3.52 (m, 4H), 2.98-2.92 (m, 4H),
2.02 (s, 3H). 13C NMR (125MHz, CDCl3)  169.1, 165.4, 158.0, 155.7, 154.2, 147.0,
145.1, 138.7, 138.3, 136.6, 132.9, 129.4, 129.1, 129.0, 128.6, 128.4, 128.2, 123.6, 121.5,
117.8, 117.6, 115.5, 113.1, 104.8, 50.6, 50.2, 46.4, 41.5, 21.5. HRMS (m/z): [M+H] +
calcd for C31H30ClN7O2: 568.2222, found: 568.2212
N-(3-((2-((4-(4-acetylpiperazin-1-yl)-2-methoxyphenyl)
chloropyrimidin-4-yl) amino) phenyl) methacrylamide (8e)
amino)-5-

White solid, m.p.: 117-119 oC. 1H NMR (500 MHz, CDCl3) δ 8.14 (d, J = 9.0 Hz, 1H),
8.06 (s, 1H), 7.94 (s, 1H), 7.56 (s, 1H), 7.43-7.40 (m, 3H), 7.33 (t, J = 8.0 Hz, 1H), 7.09
(s, 1H), 6.52 (d, J = 2.5 Hz, 1H), 6.41 (d, J = 8.5 Hz, 1H), 5.79 (s, 1H), 5.47 (s, 1H),
3.87 (s, 3H), 3.78-3.76 (m, 2H), 3.62-3.60 (m, 2H), 3.10-3.06 (m, 4H), 2.15 (s, 3H),
+
2.05 (s, 3H). HRMS (m/z): [M+H] calcd for C27H30ClN7O3: 536.2171, found:
536.2174.
N-(3-((2-((4-(4-acetylpiperazin-1-yl)-2-methoxyphenyl)
amino)-5-
chloropyrimidin-4-yl) amino) phenyl)-2-fluoroacrylamide (8f)
White solid, m.p. 223-223 oC. 1H NMR (500 MHz, CDCl3) δ 8.11 (d, J = 8.5 Hz, 1H),
8.07 (s, 1H), 8.00 (s, 1H), 7.96 (d, J = 4.0 Hz, 1H), 7.44 (t, J = 7.0 Hz, 2H), 7.37-7.34
(m, 2H), 7.10 (s, 1H), 6.54 (d, J = 2.0 Hz, 1H), 6.42 (d, J = 8.5 Hz, 1H), 5.85 (dd, J =
48.0, 3.5 Hz, 1H), 5.26 (dd, J = 15.0, 3.5 Hz, 1H), 3.88 (s, 3H), 3.79-3.77 (m, 2H), 3.63
-3.61 (m, 2H), 3.11-3.06 (m, 4H), 2.15 (s, 3H). 13C NMR (125 MHz, CDCl3)  169.1，
157.8，157.3，156.3 (d, J = 301.3 Hz), 155.6, 154.6, 149.4, 147.0, 138.9, 137.2,
129.6, 123.0, 120.2, 118.3, 115. 8, 113.3, 108.5, 104.7, 101.4, 100.3(d, J = 15.0 Hz),
+
55.8, 50.8, 50.6, 46.5, 41.6, 21.5. HRMS (m/z): [M+H] calcd for C26H27ClFN7O3:
540.1921, found: 540.1928.
N-(3-((2-((4-(4-acetylpiperazin-1-yl)-2-methoxyphenyl)
amino)-5-
chloropyrimidin-4-yl) amino) phenyl)-2-chloroacrylamide (8g)
Yellow solid, m.p.: 192-193 oC. 1H NMR (500 MHz, CDCl3) δ 8.36 (s, 1H), 8.12 (d, J
= 8.5 Hz, 1H), 8.06 (s, 1H), 8.02-8.00 (m, 1H), 7.46 (d, J = 7.5 Hz, 1H), 7.42 (d, J =
8.0 Hz, 2H), 7.37 (d, J = 8.0 Hz, 1H), 7.11 (s, 1H), 6.74 (d, J = 1.5 Hz, 1H), 6.53 (d, J
= 2.0 Hz, 1H), 6.42 (d, J = 8.5 Hz, 1H), 5.93 (d, J = 1.5 Hz, 1H), 3.88 (s, 3H), 3.78 (t,
J = 4.5Hz, 2H), 3.62 (t, J = 5.0 Hz, 2H), 3.11-3.07 (m, 4H), 2.15 (s, 3H). HRMS (m/z):
[M+H] + calcd for C26H27Cl2N7O3: 556.1625, found: 556.1632.
N-(3-((2-((4-(4-acetylpiperazin-1-yl)-3-methylphenyl) amino)-5-chloropyrimidin-
4-yl) amino) phenyl)-2-fluoroacrylamide (8h)
White solid, m.p.: 96-97 oC. 1H NMR (500 MHz, CDCl3) δ 8.09 (s, 1H), 8.07 (s, 1H),
7.95 (s, 1H), 7.49 (d, J = 7.5 Hz, 1H), 7.35-7.32 (m, 4H), 7.11 (s, 1H), 6.96 (s, 1H),
6.92 (d, J = 9.0 Hz, 1H), 5.85 (dd, J = 48.0, 3.5 Hz, 1H), 5.28 (dd, J = 15.0, 3.5 Hz,
1H), 3.75-3.71 (m, 2H), 3.60-3.58 (m, 2H), 2.87-2.81 (m, 4H), 2.28 (s, 3H), 2.15 (s,
3H). HRMS (m/z): [M+H]+ calcd for C26H27ClFN7O2: 524.1972, found: 524.1978.
N-(3-((2-((4-(4-acetylpiperazin-1-yl)-3-methylphenyl) amino)-5-chloropyrimidin-
4-yl) amino) phenyl)-2-chloroacrylamide (8i)
o
1
White solid, m.p.: 110-111 C. H NMR (500 MHz, CDCl3) δ 8.36 (s, 1H), 8.06 (m,
2H), 7.52 (d, J = 8.0 Hz, 1H), 7.35-7.32 (m, 3H), 7.28 (s, 1H), 7.12 (s, 1H), 6.99 (s,
1H), 6.91 (d, J = 8.5 Hz, 1H), 6.74 (d, J = 1.0 Hz, 1H), 5.93 (d, J = 1.0 Hz, 1H), 3.75-
13
3.71 (m, 2H), 3.60-3.58 (m, 2H), 2.87-2.81 (m, 4H), 2.27 (s, 3H), 2.15 (s, 3H). C
NMR (125 MHz, CDCl3) 169.3, 158.8, 158.0, 155.6, 154.5, 146.3, 138.9, 137.5, 135.3,
133.6, 132.1, 129.6, 124.2, 122.8, 119.7, 118.3, 118.2, 115.7, 113.2, 105.0, 52.4, 52.0,
47.1, 42.2, 21.6, 18.0. HRMS (m/z): [M+H]+ calcd for C26H27Cl2N7O2: 540.1676, found:
540.1682.
N-(3-((2-((4-(4-acetylpiperazin-1-yl)-3-chlorophenyl) amino)-5-chloropyrimidin-
4-yl) amino) phenyl)-2-fluoroacrylamide (8j)

White solid, m.p.: 176-177 oC; 1H NMR (500 MHz, DMSO-d6) δ 10.31 (s, 1H), 9.44
(s, 1H), 8.38 (s, 1H), 7.86 (s, 1H), 7.37 (m, 2H), 7.14 (m, 3H), 5.73 (d, J = 47.5 Hz,
1H), 5.52-5.43 (m, 2H), 5.33 (d, J = 14.0Hz, 1H), 3.60 (s, 4H), 2.97 (d, J = 31.5 Hz,
4H), 2.05 (s, 3H). HRMS (m/z): [M+H]+ calcd for C25H24Cl2FN7O2: 544.1425, found:
544.1429.
N-(3-((2-((4-(4-acetylpiperazin-1-yl)-3-fluorophenyl) amino)-5-chloropyrimidin-
4-yl) amino) phenyl)-2-fluoroacrylamide (8k)
Yellow solid, m.p.: 106-107 oC. 1H NMR (500 MHz, CDCl3) δ 8.15 (s, 1H), 8.07 (d, J
= 2.0 Hz, 1H), 8.03 (s, 1H), 7.58 (d, J = 14.5 Hz, 1H), 7.41-7.35 (m, 2H), 7.29 (d, J =
8.0 Hz, 1H), 7.18 (s, 1H), 7.13 (s, 1H), 7.03 (d, J = 8.5 Hz, 1H), 6.85-6.82 (m, 1H),
5.84 (d, J = 48.0 Hz, 1H), 5.27 (d, J = 15.0 Hz, 1H), 3.78 (m, 2H), 3.62 (m, 2H), 2.99
(d, J = 17.5 Hz, 4H), 2.14 (d, J = 2.0 Hz, 3H). HRMS (m/z): [M+H]+ calcd for
C25H24ClF2N7O2: 528.1721, found: 528.1725.
N-(3-((2-((4-(4-acetylpiperazin-1-yl)-3-fluorophenyl) amino)-5-chloropyrimidin-
4-yl) amino) phenyl)-2-chloroacrylamide (8l)
o
1
Yellow solid, m.p.: 100-101 C. H NMR (500 MHz, CDCl3) δ 8.40 (s, 1H), 8.14 (s,
1H), 8.08 (s, 1H), 7.60 (dd, J = 14.5, 2.5Hz, 1H), 7.44 (d, J = 9.0 Hz, 1H), 7.37 (t, J =
8.0 Hz, 1H), 7.13 (m, 1H), 7.08 (m, 1H), 7.02 (d, J = 8.5 Hz, 1H), 6.85-6.82 (m, 1H),
6.74 (d, J = 1.5 Hz, 1H), 5.93 (d, J = 1.0 Hz, 1H), 3.78 (t, J = 5.0 Hz, 2H), 3.62 (t, J =
5.0 Hz, 2H), 3.01 (t, J = 5.0 Hz, 2H), 2.98 (t, J = 5.0 Hz, 2H), 2.14 (s, 3H). HRMS
(m/z): [M+H]+ calcd for C25H24Cl2FN7O2: 544.1425, found: 544.1431.
N-(3-((2-((4-(4-acetylpiperazin-1-yl)-2-methoxyphenyl) amino)-5-(trifluoromethyl)
pyrimidin-4-yl) amino) phenyl)-2-chloroacrylamide (8m)
o
Yellow solid, m.p.: 113-114 C.1H NMR (500 MHz, CDCl3) δ 8.40 (s, 1H), 8.14 (s,
1H), 8.08 (s, 1H), 7.60 (dd, J = 14.5, 2.5 Hz, 1H), 7.44 (d, J = 9.0 Hz, 1H), 7.37 (t, J =
8.0 Hz, 1H), 7.13 (m, 1H), 7.08 (m, 1H), 7.02 (d, J = 8.5 Hz, 1H), 6.85-6.82 (m, 1H),
6.74 (d, J = 1.5 Hz, 1H), 5.93 (d, J = 1.0 Hz, 1H), 3.78 (t, J = 5.0 Hz, 2H), 3.62 (t, J =
5.0 Hz, 2H), 3.01 (t, J = 5.0 Hz, 2H), 2.98 (t, J = 5.0 Hz, 2H), 2.14 (s, 3H), 1.65 (s, 3H).
HRMS (m/z): [M+H]+ calcd for C27H27ClF3N7O3: 590.1889, found: 590.1990.
N-(3-((2-((4-(4-acetylpiperazin-1-yl)-3-(trifluoromethyl)
phenyl)
amino)-5-
chloropyrimidin-4-yl) amino) phenyl)-2-fluoroacrylamide (8n)
White solid, m.p.: 112-113 oC. 1H NMR (500 MHz, DMSO-d6) δ 10.32 (s, 1H), 9.57
(s, 1H), 9.02 (s, 1H), 8.19 (s, 1H), 8.03-7.99 (m, 2H), 7.81 (s, 1H), 7.55 (d, J = 6.5 Hz,
1H), 7.35-7.33 (m, 2H), 7.25 (d, J = 8.0 Hz, 1H), 5.69 (dd, J = 48.0, 3.0 Hz, 1H), 5.42
13
(dd, J = 15.5, 3.0 Hz, 1H), 3.51 (m, 4H), 2.75-2.70 (m, 4H), 2.04 (s, 3H). C NMR
(125 MHz, CDCl3) δ 169.3, 157.5 (q, J = 30 Hz), 157.3, 156.1 (d, J = 267.5 Hz), 155.8,
153.9, 146.0, 138.5, 137.3, 137.1, 129.7, 128.1 (q, J = 28.75 Hz), 124.8, 124.2 (q, J =
161.3 Hz), 123.5, 118.2, 118.1(q, J = 7.0 Hz), 116.0, 113.5, 106.0, 100.4 (d, J = 15 Hz),
53.9, 53.2, 47.1, 42.1, 21.6. HRMS (m/z): [M+H]+ calcd for C26H24ClF4N7O2: 578.1689,
found: 578.1680.
N-(3-((2-((4-(4-acetylpiperazin-1-yl)-3-chlorophenyl) amino)-5-chloropyrimidin-
4-yl) amino) phenyl)-2-chloroacrylamide (8o)
White solid, m.p.: 140-141 oC. 1H NMR (500 MHz, DMSO-d6) δ 10.20 (s, 1H), 9.41
(s, 1H), 8.98 (s, 1H), 8.16 (s, 1H), 7.92 (s, 1H), 7.74 (s, 1H), 7.53-7.51 (m, 2H), 7.35

(d, J = 5.0 Hz, 2H), 6.91 (d, J = 8.5 Hz, 1H), 6.40 (d, J = 2.0 Hz, 1H), 6.08 (d, J = 2.5
13
Hz, 1H), 3.56-3.54 (m, 4H), 2.86-2.84 (m, 2H), 2.80-2.78 (m, 2H), 2.03 (s, 3H). C
NMR (125 MHz, CDCl3) 169.3, 158.8, 157.6, 155.7, 154.4, 143.6, 138.6, 137.6, 136.0,
132.1, 129.8, 129.3, 124.2, 121.7, 120.6, 118.8, 118.3, 116.0, 113.4, 105.6, 51.9, 51.4,
46.8, 41.9, 21.6. HRMS (m/z): [M+H]+ calcd for C25H24Cl3N7O2: 560.1130, found:
560.1139.
N-(3-((2-((4-(4-acetylpiperazin-1-yl)-2-chlorophenyl) amino)-5-chloropyrimidin-
4-yl) amino) phenyl)-2-chloroacrylamide (8p)
White solid, m.p.: 166-167 oC. 1H NMR (500 MHz, DMSO-d6) δ 10.13 (s, 1H), 8.82
(s, 1H), 8.37 (s, 1H), 8.06 (s, 1H), 7.91 (s, 1H), 7.48 (d, J = 9.0 Hz, 1H), 7.43 (d, J =
7.5 Hz, 1H), 7.34 (d, J = 8.5 Hz, 1H), 7.18 (t, J = 8.0 Hz, 1H), 7.01 (d, J = 2.5 Hz, 1H),
6.81 (dd, J = 9.0, 2.5 Hz, 1H), 6.43 (d, J = 2.5 Hz, 1H), 6.10 (d, J = 2.5 Hz, 1H), 3.57-
13
3.56 (m, 4H), 3.15-3.06 (m, 4H), 2.05 (s, 3H). C NMR (125 MHz, CDCl3) 169.1,
158.7, 157.7, 155.6, 154.6, 147.1, 138.8, 137.5, 132.1, 129.6, 129.3, 124.6, 124.2, 122.7,
118.1, 117.5, 115.9, 115.8, 113.2, 105.6, 50.0, 49.7, 46.2, 41.4, 21.5. HRMS (m/z):
[M+H]+ calcd for C25H24Cl3N7O2: 560.1130, found: 560.1143.
N-(3-((2-((6-(4-acetylpiperazin-1-yl) pyridin-3-yl) amino)-5-chloropyrimidin-4-yl)
amino) phenyl)-2-chloroacrylamide (8q)
White solid, m.p.: 115-116 oC. 1H NMR (500 MHz, DMSO-d6) δ 10.18 (s, 1H), 9.14
(s, 1H), 8.90 (s, 1H), 8.27 (s, 1H), 8.10 (s, 1H), 7.92 (m, 1H), 7.84 (dd, J = 9.0, 2.5 Hz,
1H), 7.46 (d, J = 7.5 Hz, 1H), 7.37 (s, 1H), 7.31 (t, J = 8.0 Hz, 1H), 6.66 (d, J = 8.0 Hz,
1H), 6.41 (d, J = 2.5 Hz, 1H), 6.10 (d, J = 2.5 Hz, 1H), 3.54-3.51(m, 4H), 3.41-3.39 (m,
2H), 3.34-3.31 (m, 2H), 2.04 (s, 3H). HRMS (m/z): [M+H]+ calcd for C24H24Cl2N8O2:
527.1472, found: 527.1480.
N-(3-((2-((6-(4-acetylpiperazin-1-yl) pyridin-3-yl) amino)-5-chloropyrimidin-4-yl)
amino) phenyl)-2-fluoroacrylamide (8r)
Yellow solid, m.p.: 135-136 oC. 1H NMR (500 MHz, CDCl3) δ 8.27 (d, J = 2.5 Hz, 1H),
8.14 (s, 1H), 8.04 (s, 1H), 7.97 (d, J = 2.5 Hz, 1H), 7.82 (dd, J = 9.0, 2.5 Hz, 1H), 7.36
-7.30 (m, 3H), 7.11 (s, 1H), 6.89 (s, 1H), 6.63 (d, J = 9.0 Hz, 1H), 5.84 (dd, J = 47.5,
3.5 Hz, 1H), 5.27 (dd, J = 15.5, 3.5 Hz, 1H), 3.78-3.74 (m, 2H), 3.9-3.56 (m, 4H), 3.45-
3.43 (m, 2H), 2.15 (s, 3H). HRMS (m/z): [M+H]+ calcd for C24H24ClFN8O2: 511.1768,
found: 511.1760.
N-(3-((2-((4-(4-acetylpiperazin-1-yl)-2-methoxyphenyl)
amino)-5-
chloropyrimidin-4-yl) oxy) phenyl)-2-fluoroacrylamide (13a)
White solid, m.p.: 94-95 oC. 1H NMR (500 MHz, CDCl3) δ 8.25 (s, 1H), 8.03 (d, J =
4.5 Hz, 1H), 7.66 (d, J = 7.0 Hz, 2H), 7.49-7.45 (m, 3H), 7.05 (dd, J = 8.0, 1.5Hz, 1H),
6.46 (d, J = 2.0 Hz, 1H), 6.19 (s, 1H), 5.83 (dd, J = 48.0, 3.5 Hz, 1H), 5.27 (dd, J =
15.0, 3.5 Hz, 1H), 3.76 (t, J = 5.0 Hz, 2H), 3.61 (t, J = 5.0 Hz, 2H), 3.06-3.02 (m, 4H),
13
2.14 (s, 3H), 1.64 (s, 3H). C NMR (125 MHz, CDCl3) δ169.1, 164.6, 157.9, 157.5,
157.5, 157.3, 156.0 (d, J = 270 Hz), 153.0, 148.8, 146.8, 137.9, 130.2, 122.7, 119.2,
117.5, 114.4, 108.6, 101.1, 100.5 (d, J = 15.0 Hz), 55.8, 50.8, 50.7, 46.4, 41.6, 21.5,
HRMS (m/z): [M+H]+ calcd for C26H26ClFN6O4: 541.1761, found: 541.1763.
N-(3-((2-((4-(4-acetylpiperazin-1-yl)-2-methoxyphenyl)
amino)-5-
chloropyrimidin-4-yl) oxy) phenyl)-2-chloroacrylamide (13b)

White solid, m.p.: 98-99 oC. 1H NMR (500 MHz, CDCl3) δ 8.47 (s, 1H), 8.24 (s, 1H),
7.66 (d, J = 7.5 Hz, 1H), 7.51-7.47 (m, 3H), 7.05 (dd, J = 8.0, 1.5 Hz, 1H), 6.72 (d, J =
1.5 Hz, 1H), 6.46 (d, J = 2.5 Hz, 1H), 6.20 (s, 1H), 5.93 (d, J = 1.0 Hz, 1H), 3.82 (s,
3H), 3.76 (d, J = 5.0 Hz, 2H), 3.62-3.60 (m, 2H), 3.07-3.01 (m, 4H), 2.14 (s, 3H). 13C
NMR (125MHz, CDCl3) 169.1, 164.6, 158.7, 157.9, 157.5, 153.0, 148.7, 146.8, 138.2,
132.0, 130.1, 124.3, 122.6, 122.1, 119.2, 117.5, 114.4, 108.6, 105.8, 101.0, 55.8, 50.8,
50.6, 46.4, 41.5, 21.5. HRMS (m/z): [M+H]+ calcd for C26H26Cl2N6O4: 557.1465, found:
557.1467.
N-(3-((5-chloro-2-((2-methoxy-4-(4-methylpiperazin-1-yl)
pyrimidin-4-yl) oxy) phenyl)-2-fluoroacrylamide (13c)
phenyl)
amino)
White solid, m.p.: 174-175 oC. 1H NMR (500 MHz, CDCl3) δ 8.23 (s, 1H), 8.02 (s, 1H),
7.63 (d, J = 7.0 Hz, 1H), 7.51 (s, 1H), 7.46 (t, J = 8.0 Hz, 2H), 7.05 (d, J = 7.5 Hz, 1H),
6.45 (s, 1H), 6.20 (s, 1H), 5.83 (dd, J = 48.0, 3.0 Hz, 1H), 5.26 (dd, J = 15.0, 3.0 Hz,
1H), 3.81 (s, 3H), 3.11 (m, 3H), 2.58 (m, 4H), 2.36 (s, 3H). HRMS (m/z): [M+H]+ calcd
for C25H26ClFN6O3: 513.1812, found: 513.1819.
2-chloro-N-(3-((5-chloro-2-((2-methoxy-4-(4-methylpiperazin-1-yl) phenyl) amino)
pyrimidin-4-yl) oxy) phenyl)acrylamide (13d)
White solid, m.p.: 106-107 oC. 1H NMR (500 MHz, CDCl3) δ 8.43 (s, 1H), 8.23 (s, 1H),
7.63 (d, J = 7.5 Hz, 1H), 7.53 (t, J = 2.0 Hz, 1H), 7.46 (t, J = 8.5 Hz, 2H), 7.05 (dd, J =
8.0, 1.5 Hz, 1H), 6.73 (d, J = 1.5 Hz, 1H), 6.45 (d, J = 2.0 Hz, 1H), 6.21 (s, 1H), 5.92
(d, J = 1.5 Hz, 1H), 3.82 (s, 3H), 3.13-3.11 (m, 4H), 2.60-2.58 (m, 4H), 2.37 (s, 3H).
13C NMR (125MHz, CDCl3) 164.5, 158.7, 157.9, 157.6, 153.4, 153.0, 148.8, 147.0,
138.1, 132.0, 130.1, 124.3, 121.9, 119.3, 119.2, 117.4, 114.4, 108.1, 100.4, 55.7, 55.1,
49.9, 46.0. HRMS (m/z): [M+H]+ calcd for C25H26Cl2N6O3: 529.1516, found: 529.1519.
N-(3-((2-((4-(4-acetylpiperazin-1-yl) phenyl) amino)-5-chloropyrimidin-4-yl) oxy)
phenyl)-2-chloroacrylamide (13e)
White solid, m.p.: 185-186 oC. 1H NMR (500 MHz, DMSO-d6) δ 10.38 (s, 1H), 9.56
(s, 1H), 8.42 (s, 1H), 7.70 (d, J = 8.0 Hz, 1H), 7.62 (d, J = 2.0 Hz, 1H), 7.48 (t, J = 8.5
Hz, 1H), 7.25 (m, 2H), 7.07 (dd, J = 8.0, 2.0 Hz, 1H), 6.69 (m, 2H), 6.43 (d, J = 3.0 Hz,
1H), 6.12 (d, J = 2.5 Hz, 1H), 3.56-3.53 (m, 4H), 2.99-2.93 (m, 4H), 2.03 (s, 3H). 13C
NMR (125MHz, CDCl3) 169.1, 164.5, 158.8, 157.9, 157.7, 152.9, 146.9, 138.1, 132.4,
131.9, 130.0, 124.4, 120.5, 119.1, 117.5, 117.4, 114.4, 106.2, 50.5, 50.1, 46.4, 41.5,
21.5. HRMS (m/z): [M+H]+ calcd for C25H24Cl2N6O3: 527.1360, found: 527.1363.
N-(3-((2-((4-(4-acetylpiperazin-1-yl)-3-(trifluoromethyl)
phenyl)
amino)-5-
chloropyrimidin-4-yl) oxy) phenyl)-2-chloroacrylamide (13f)
White solid, m.p.:145-146 oC. 1H NMR (500 MHz, DMSO-d6) δ 10.39 (s, 1H), 9.96 (s,
1H), 8.53 (s, 1H), 7.73 (m, 1H), 7.70-7.66(m, 2H), 7.65-7.64 (m, 1H), 7.47 (t, J = 8.0
Hz, 1H), 7.22-7.21 (m, 1H), 7.08-7.07 (m, 1H), 6.43 (d, J = 3.0 Hz, 1H), 6.11 (d, J =
2.5 Hz, 1H), 3.51-3.50 (m, 4H), 2.75-2.73 (m, 2H), 2.70-2.68(m, 2H), 2.04 (s, 3H).
HRMS (m/z): [M+H]+ calcd for C26H23Cl2F3N6O3: 595.1234, found: 595.1240.
N-(5-((5-chloro-4-(1-methyl-1H-indol-3-yl)
pyrimidin-2-yl)
amino)-2-
(dimethylamino) phenyl)-2-fluoroacrylamide (17a)
White solid, m.p：204-205 oC. 1H NMR (500 MHz, CDCl3) δ 9.53 (s, 1H), 8.54 (d, J
= 8.0 Hz,1H), 8.51 (d, J = 2.5 Hz,1H), 8.36 (s,1H), 8.29 (s,1H), 7.70 (dd, J1 = 8.5 Hz,

J2 = 2.5 Hz, 1H), 7.39-7.38 (m,1H), 7.33 (t, J = 7.0 Hz, 1H), 7.24-7.20 (m, 1H), 7.22-
7.20 (m,2H), 5.81 (dd, J = 47.5, 3.0 Hz, 1H), 5.24(dd, J = 15.0, 3.0 Hz, 1H), 3.90 (s,
3H), 2.67 (s, 6H). 13C NMR (125 MHz, CDCl3) δ 158.7, 158.2, 157.4, 157.3(d, J =
28.75 Hz), 156.8(d, J = 270 Hz), 138.5, 137.0, 136.8, 134.4, 132.8, 127.2, 123.6, 123.0,
121.4, 120.8, 116.9, 116.4, 111.3, 111.2, 109.6, 99.6(d, J = 16.25 Hz), 45.3, 33.6.
HRMS (m/z): [M + H]+ calcd for C24H22ClFN6O: 465.1600; found,465.1605.
2-chloro-N-(5-((5-chloro-4-(1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino)-2-
(dimethylamino) phenyl) acrylamide (17b)
Yellow solid, M.p：101-102 oC. 1H NMR (500 MHz, CDCl3) δ 10.01 (s, 1H), 8.55 (d,
J = 8.0 Hz, 1H), 8.50 (d, J = 2.5 Hz, 1H), 8.37 (s, 1H), 8.28 (s, 1H), 7.70 (dd, J = 8.5,
2.5 Hz, 1H), 7.38 (d, J = 8.0 Hz, 1H), 7.34-7.31 (m, 1H), 7.26-7.23 (m, 1H), 7.20 (d, J
= 9.0 Hz, 1H), 6.71 (d, J = 1.5 Hz, 1H), 5.91 (d, J = 1.5 Hz, 1H), 3.89 (s, 3H), 2.69 (s,
13
6H). C NMR (125MHz, CDCl3) 158.7, 158.6, 158.2, 157.4, 138.6, 137.0, 136.7,
134.4, 133.2, 133.1, 127.2, 123.6, 123.3, 123.0, 121.4, 120.7, 116.9, 116.3, 111.2, 111.0,
109.5, 45.2, 33.6, 29.8. HRMS (m/z): [M + H]+ calcd for C24H22Cl2N6O: 481.1305;
found, 481.1311.
4.4 BIOLOGICAL EVALUATION
Cell proliferation assay
The human epidermal carcinoma cell line A431 and human non-small cell lung
cancer cell line NCI-H1975 were used to evaluate the potency of synthesized analogues
in cell-based level. The human colon carcinoma cell line SW620 and monkey fibroblast
cell COS7 were used as control cell lines to evaluate the cytotoxicity. A431 and SW620
was cultured with RPMI 1640 (GIBCO). COS7 and NCI-H1975 was cultured with
Dulbecco's Modified Eagle's Medium (GIBCO). Both mediums were supplemented
with penicillin, streptomycin and 10% fetal bovine serum. A431 and NCI-H1975 cells
were seeded in density of 2000 cells/well and 2500 cells/well, respectively, in 96-well
plates (Corning) for 24 hours. Duplicate wells were treated with test or reference
compounds for 48 hours at various concentrations or DMSO (Sigma) as control. Plates
were incubated at 37 oC in 5% CO2 atmosphere. Cell proliferation was determined by
using MTT assay. The IC50 was calculated using GraphPad Prism 4.0.
Kinase inhibition assay
The assays were performed in vitro using Homogeneous time-resolved fluorescence
(HTRF) method (Cisbio). EGFR was purchased from Sigma. The kinases and
substrates were incubated first with synthesized analogues for 5 minutes in enzymatic
WT
buffer (for EGFR
and EGFR L858R/T790M). Then ATP (Sigma) was added into the
reaction mixture to start the enzyme reaction. The ATP concentrations used in each
enzyme reaction were 1.65 μM, equivalent to the Km of ATP for the corresponding
enzyme in this assay condition. The assays were conducted at room temperature for 30
minutes and stopped by detection reagents which contain EDTA. The detection step
lasted for 1 hour. The IC50 was calculated using GraphPad Prism 4.0.
Cell cycle analysis
NCI-H1975 cells in 6-well plates were treated with either DMSO or compound 8a at
o
indicated concentrations and cultured at 37 C and 5% CO2. After 2 d, the cells were
trypsinized, washed in cold PBS, and centrifuged. Cell pellets were re-suspended with

RNase and stained with PI. Samples were read in a flow cytometry using CytoFLEX
LX (BECKMAN COULTER).
Docking study
The X-ray structure (PDB 5XDK) was downloaded from the Protein Data Bank
(PDB, http://www.pdb.org) as a template. The molecular modeling simulations were
[31]
performed using Sybyl-X 1.3 software package.
The initial structure of 8a was
generated by Sketch module in Sybyl-X 1.3. The geometries of the compound were
subsequently optimized using the Tripos force field with Gasteiger-Hückel charges.
The produced conformation of 8a was then inserted into the binding pocket of
EGFRT790M to replace the Rociletinib for the initial structural model of 8a binding to
EGFRT790M. Molecular docking studies of 8a with the EGFRT790M binding pocket was
performed with FlexiDock module in Sybyl-X 1.3 while all of the parameters were
default. [32] The docked complexes of inhibitor-enzyme were selected according to the
criteria of interacting energy combined with geometrical matching quality.
ACKNOWLEDGEMENTS
We thank Dr. Youlu Pan for performing the charge distribution calculation in this work.
This project was supported by the Zhejiang Provincial Natural Science Foundation of
China under grant No. LY20H300001 and Medical Health Science and Technology
Project of Zhejiang Provincial Health Commission under grant No. 2020KY681 to J.
Shao; Zhejiang Provincial Natural Science Foundation of China under grant No.
LY18H300001 to W. Chen.
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Declaration of interests
☒
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.
☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:
 The electronic nature of warhead acrylamide moiety was investigated.

 8a retained the excellent EGFRL858R/T790M potency and good anti-proliferative
activities against the NCI-H1975 cells.
 8a also displayed a significant EGFRWT selectivity and weaker inhibition against
non-EGFR dependent SW620 and COS7 cell.
 Preliminary study showed that 8a could arrest NCI-H1975 cells in G0/G1 phase.