Design, synthesis and biological evaluation of AZD9291 derivatives as selective and potent EGFRL858R/T790M inhibitors

Article abstract of DOI:10.1016/j.ejmech.2018.11.069

Third-generation epidermal growth factor receptor (EGFR)^L858R/T790M inhibitors are still the main drugs for the treatment of advanced non-small cell lung cancer (NSCLC), and these drugs have achieved remarkable clinical efficacy. However, there are still many patients suffering from drug-resistant mutations and drug side effects caused by NSCLC. In this study, guided by the molecular simulation, we applied a structure-based drug design strategy (SBDD) and optimized the structure to obtain a series of potent and selective EGFR^L858R/T790M inhibitors. The most potent compound 18e demonstrated excellent kinase inhibitory activity and selectivity for EGFR^L858R/T790M double mutants and the IC₅₀ value reached nanomolar level. The selectivity of 18e against wild-type EGFR was near to 200-fold. In addition, compound 18e also inhibited H1975 cells proliferation at G2/M phase and induced apoptosis at a concentration of 0.25 μM, which makes it more valuable for potential lung cancer research.

Full text of DOI:10.1016/j.ejmech.2018.11.069

Accepted Manuscript
Design, synthesis and biological evaluation of AZD9291 derivatives as selective and
L858R/T790M
potent EGFR
inhibitors
Bingbing Zhao, Zhen Xiao, Jianguo Qi, Rong Luo, Zhou Lan, Yanzhuo Zhang,
Xiaohan Hu, Qidong Tang, Pengwu Zheng, Shan Xu, Wufu Zhu
PII:
S0223-5234(18)31028-6
DOI:
https://doi.org/10.1016/j.ejmech.2018.11.069
EJMECH 10927
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 October 2018
Revised Date: 19 November 2018
Accepted Date: 28 November 2018
Please cite this article as: B. Zhao, Z. Xiao, J. Qi, R. Luo, Z. Lan, Y. Zhang, X. Hu, Q. Tang, P. Zheng,
S. Xu, W. Zhu, Design, synthesis and biological evaluation of AZD9291 derivatives as selective and
L858R/T790M
potent EGFR
inhibitors, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2018.11.069.
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ACCEPTED MANUSCRIPT
Design, Synthesis and Biological Evaluation of AZD9291 Derivatives as Selective and Potent EGFRL858R/T790M
Inhibitors
Bingbing Zhao a, Zhen Xiao a, Jianguo Qi b, Rong Luo c, Zhou Lan a, Yanzhuo Zhang d, Xiaohan Hu a, Qidong Tang a,
Pengwu Zheng a,*, Shan Xu a, , Wufu Zhu a,*
a Jiangxi Provincial Key Laboratory of Drug Design and Evaluation, School of Pharmacy, Jiangxi Science & Technology
Normal University, 605 Fenglin Road, Nanchang, Jiangxi 330013, China
b Henan Provincial Key Laboratory of Natural Medicine and Immunology, Jinming Road, Kaifeng, Henan 475004, China
c Jiangxi Province Institute of Materia Medica, 181 East Nanjing Road, Nanchang, Jiangxi 330000, China
d School of Pharmacy, Xuzhou Medical University, 209 Tongshan Road, Xuzhou, Jiangsu 221004, China.
* Corresponding author.
Tel. & Fax: +86 791 8380-2393;
E-mail: zhengpw@126.com (P. Zheng), zhuwufu-1122@163.com (W. Zhu)
Abstract：
Third-generation epidermal growth factor receptor (EGFR)L858R/T790M inhibitors are still the main drugs for the
treatment of advanced non-small cell lung cancer (NSCLC), and these drugs have achieved remarkable clinical efficacy.
However, there are still many patients suffering from drug-resistant mutations and drug side effects caused by NSCLC. In
this study, guided by the molecular simulation, we applied a structure-based drug design strategy (SBDD) and optimized
the structure to obtain a series of potent and selective EGFRL858R/T790M inhibitors. The most potent compound 18e
demonstrated excellent kinase inhibitory activity and selectivity for EGFRL858R/T790M double mutants and the IC50 value
reached nanomolar level. The selectivity of 18e against wild-type EGFR was near to 200-fold. In addition, compound 18e
also inhibited H1975 cells proliferation at G2/M phase and induced apoptosis at a concentration of 0.25 µM, which
makes it more valuable for potential lung cancer research.
Keywords: Epidermal growth factor receptor; Non-small cell lung cancer; Selectivity; L858R/T790M resistance
mutation
Corresponding author. Tel./fax: +86 791 83802393.
E-mail address: zhengpw@126.com (P. Zheng), shanxu9891@126.com (S. Xu), zhuwufu-1122@163.com (W. Zhu).

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1. Introduction
Nowadays, therioma is still one of the major causes of endangering human health. Of the 7 million people that die of
cancer each year, lung cancer remains the most common and deadly cancer around the world, accounting for 25% of the
total number of cancer deaths [1-3]. Non-small cell lung cancer (NSCLC) is the most prevalent form of lung cancer, with
approximately 1.5 million patients and a 5-year survival rate less than 20% [4,5]. Receptor tyrosine kinases (RTKs) are
extensively found on cell membranes of organisms. In addition, recent studies have shown that the ErbB family plays a
key role in the regulation of the physiological cycle of malignant tumors [6,7]. Epidermal growth factor receptor (EGFR)
belongs to the RTKs, is considered one of the important targets for the treatment of malignant tumors, which plays a
crucial role in the regulation of cancer cell growth, proliferation and differentiation [8-10].
Up to now, EGFR inhibitors have been developed to the third generation. The first-generation of EGFR inhibitors
are represented by gefitinib (1, Fig. 1) and erlotinib (2, Fig. 1), They are used to treat sensitive EGFR mutations caused
by the L858R mutation in exon 21 and exon 19 deletion [11,12]. In the early stage of clinical treatment, these drugs have
achieved remarkable therapeutic effect. Unfortunately, more than 50% of patients treated with first-generation EGFR
inhibitors developed resistance mutation (T790M) within 1 year [13]. The T790M mutation is thought to be more bulk and
small-polar methionine that replaces threonine in exon 20[14]. The steric hindrance of methionine hinders the binding of
drugs (gefitinib and erlotinib) to target sites in the ATP hydrophobic pocket. What’s more, study have shown that the
T790M mutation also increases the affinity of EGFR kinase for ATP [15,16]
.
Fig. 1. Chemical structures of the representative small-molecule EGFR inhibitors.

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Fig. 2. Design strategy from chemical structures of previous compounds.
Consequently, many second-generation EGFR inhibitors such as afatinib (3, Fig. 1), dacomitinib and neratinib were
designed for overcoming the resistance caused by T790M mutations [17-19]. Many of these medicines share the common
quinazoline pharmacophore and acrylamide structure (Michael Addition Receptor). The acrylamide structure can
covalently and irreversibly bind to the cysteine residue (Cys797). So, the medicine can better occupy the ATP-binding
pocket of the EGFR kinase domain, and overcome the resistance caused by the T790M mutation [20]. Unexpectedly, the
second-generation EGFR inhibitors not only inhibited mutational EGFR receptor but also the wild-type (WT) EGFR
receptor. Therefore, most patients taking the drug within a few months experienced serious side effects such as rash,
diarrhea and other side effects [21]
.
To overcome the drawbacks of acquired resistance to second-generation EGFR inhibitors, several third-generation
EGFR inhibitors have been developed. WZ4002 (4, Fig. 1) is the first reported third-generation EGFR inhibitor and the
selectivity of WZ4002 against T790M-EGFR to WT-EGFR is near to 30-100 times [22]. CO1686 (5, Fig. 1) has similar
chemical structure to WZ4002, and they all share “U-shaped” molecular structure [23]. The U. S. Food and Drug
Administration (FDA) was ended the Phase II clinical trial of CO1686, because it also inhibited insulin receptor (INSR)
and insulin-like growth factor 1 receptor (IGF1R) causing serious side effects (especially hyperglycemia) [24]
.
As the
first FDA-approved third-generation EGFR inhibitor, AZD9291 (6, Fig. 1) was marketed in the United States in
November 2015 for treatment of EGFRT790M/ L858R mutation in NSCLC patients [25]. However, the selectivity of AZD9291
to mutant kinases has not achieved the desired effect. Its binding to non-target EGFR receptors in vivo has caused serious
side effects (diarrhea, rash and cardiotoxicity), which severely limits the clinical application of AZD9291. The most
serious adverse event is dose-dependent toxicity, such as cardiotoxicity increased with escalating doses of AZD9291[26]
.
Thus, we urgently need to develop the novel EGFR inhibitors with high selectivity against WT-EGFR and low toxicity to
solve the problem of drug resistance and serious side effects.
Herein, structure-based drug design (SBDD) was applied in our compounds design strategy (Fig. 2). Docking
AZD9291 into T790M-EGFR (Fig. 3B) and WT-EGFR (Fig. 3C) revealed that the N atom in the 1-position of the
pyrimidine ring points to the hinge region residue Met793, and is adjacent to the residue Met790. The acrylamide

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warhead interacts with mutation gatekeeper site residue Cys797 via a hydrogen bond. The indole ring points to the
Asp-Phe-Gly (DFG) motif. Furthermore, the entire binding site (Fig. 3D) is far away from the L858R mutant region so
that the L858R mutation does not affect the binding site. Based on previous research [25,27], we first introduced small
molecule substituents at the end of the acrylamide side chain to change the reactivity of the acrylamide warhead with
residue Cys797. According to previous research, the two reported compounds have attracted our attention. Compound 17
was reported by Finlay, M. [26] and had good kinase inhibitory activity with an IC50 value of 0.9 nM for EGFRT790M/L858R
kinase. Compound 23 was reported by Gao HY.[28], and its IC50 value for H1975 cells was 15 nM. Therefore, on the basis
of these two compounds, we designed the next two series of compounds. The second series of compounds with different
substituents at the 5-position or 6-position of the pyrimidine ring was designed by the ring expansion and splitting
principle based on reported compound 17. Finally, in view of the good enzymatic and cellular inhibitory of published
compound 23, we introduced fluorine into the indole ring, which presumably increases the polar interaction between the
ligand and the DFG motif residue. Moreover, the F-atom can weaken the demethylation ability of the indole ring and
enhances the anti-metabolism ability of AZD9291. Thus, we suspected that this design strategy may help us to discover
novel inhibitors for overcoming EGFRL858R/T790M double mutations.
2. Results and discussion
2.1 Chemistry
Scheme 1. Synthetic route of the compounds 16a-h, 17b-e, 18a-e. Reagents and conditions: (a) CH3I, NaH, THF, rt., 1.5
h; (b) AlCl3, DME, 80
110 , 2.5 h; (e) Zn, NH4Cl, C2H5OH, H2O, 105
The synthetic route of the target compounds 16a-h, 17b-e, 18a-e was shown in Scheme 1. Commercially available
ºC, 3 h; (c) TsOH, 1,4-dioxane, 85
ºC, 3 h; (d) N,N,N-trimethylethylenediamine, DIPEA, DMA,
ºC
ºC, 3 h; (f) NaHCO3, DCM, 0
ºC, 0.5-2 h.
starting material indole 7 reacted with methyl iodide to get 8. Commercially available 11a,11b and 11c were reacted with
8 to obtain 12a-c. Next, 12a-c were coupled with commercially available 7 to obtain 13a-c. 15a-c was obtained by the
reaction of amination and reduction from 14a-c with 13a-c as raw material. Corresponding acid chlorides were obtained

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from different substituted acrylic acids by the chlorination reaction, and finally reacted with 15a-c to provide the target
compounds 16a-h, 17b-e, 18a-e. The synthetic route of 23b-d, 24a-d, 25a-d was outlined in Scheme 2, which was
basically same as the route of compounds 16a-h, 17b-e and 18a-e, except that the 5-fluoroindole 9 was used as the
starting material. Finally, 22a-c reacted with corresponding acid chlorides yielding compounds 23b-d, 24a-d and 25a-d.
Scheme 2. Synthetic route of the compounds 23b-d, 24a-d, 25a-d. Reagents and conditions: (a) CH3I, NaH, THF, rt., 1.5
h; (b) AlCl3, DME, 80
110 , 2.5 h; (e) Zn, NH4Cl, C2H5OH, H2O, 105
2.2 Binding site analysis of AZD9291
º
C
, 3 h; (c) TsOH, 1,4-dioxane, 85
º
C
, 3 h; (d) N,N,N’-trimethylethylenediamine, DIPEA, DMA,
ºC
º
C
, 3 h; (f) NaHCO3, DCM, 0 ºC
, 0.5-2 h.
Before conducting the experiment, we analyzed the binding site of AZD9291 with WT-EGFR (PDB code:4ZAU)
and T790M-EGFR (PDB code:3IKA), respectively. The crystal complex of AZD9291 binding to WT-EGFR was
available from the PDB library (http://www.rcsb.org/pdb/home/home.do). As shown in Fig. 3C, bidentate hydrogen bond
was formed with the methionine residue Met793 between the polar hydrogen atom in the imine bridge and the N atom in
the 1-position of the pyrimidine ring. In addition, cysteine residue Cys797 also formed strong hydrogen binding force
with the oxygen atoms in the acrylamide side chains (bond length 2.5Å). There is no crystal complex of AZD9291 binds
to T790M EGFR kinase domain in the PDB library, we simulated the binding model of published T790M structure (PDB
code: 3IKA) to AZD9291 by AutoDock 4.2 software (The Scripps Research Institute, USA). From Fig. 3 B and Fig. 3 D,
we can know that AZD9291 formed strong hydrogen binding force with T790M-EGFR kinase domain on the outer edge
of the ATP hydrophobic pocket. Two hydrogen bonds were formed between hinge region residue Met793 (show blue in
the Fig. 3 B) and the pyrimidine core. In addition, the L858R mutation site (show red in the Fig. 3 B) was far away from
the small molecule ligand AZD9291 so that the firs-generation EGFR mutation would not affect the binding site.
Furthermore, in our expectation, the hydrogen binding force formed between the side chain containing the Michael
Addition Receptor and the Cys797 residue (show green in the Fig. 3 B) is stronger than that of WT-EGFR (4ZAU).

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A
B
C
D
Fig. 3. Binding site of AZD9291. (A) Chemical structure of AZD9291 and its depicted binding mode with T790M-EGFR.
(B) The binding model of AZD9291 (green) bound to T790M-EGFR (PDB code:3IKA). (C) The binding model of
AZD9291 (green) bound to WT-EGFR (PDB code:4ZAU). (D) Overview of the binding site of AZD9291 with
T790M-EGFR (PDB code:3IKA).
2.3. Modification of acrylamide side chain
It can be seen from the results of molecular simulate docking, acrylamide side chain containing Michael Addition
Receptors directly extend to the Cys797 residue at the edge of the ATP hydrophobic pocket and formed stronger
hydrogen binding force. Moreover, inspired by the research reported by Mark E. [27] and Xia G. [29], the introduction of
small molecule alkyl side chains at the bottom of the acrylamide warhead regulates the reactivity of the olefine with
residue Cys797 and impairs the irreversible binding of drug to non-targeting EGFR protein, thereby reducing the side
effects of the drug. To conduct this strategy, we designed and synthesized the first series of AZD9291 derivatives with
different sizes of substituents at the ortho position of the acrylamide warhead (16a-h) and evaluated them in enzymatic
inhibition and antiproliferation activity assays.
The enzymatic and cellular activities of compounds 16a-h were show in Table 1. As predicted, when we introduced
strong electron withdrawing groups (-F, -Cl) into the acrylamide warhead, 16a, 16b, and 16c exhibited lower
antiproliferation activities against H1975 cells, and partially lost kinase activity against both EGFRT790M/L858R and
EGFRWT. The most gratifying result is that the kinase selectivity of all compounds against EGFRT790M/L858R to EGFRWT
has been improved. The results are consistent with our expectations. We discovered compounds with high EGFR mutated
kinase selectivity by modifying the structure of acrylamide. However, with the increasing of the substituted alkyl side
chain, the inhibitory activity of the compounds (16f, 16g and 16h) on H1975 cells and the enzymatic inhibition against

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EGFR kinase gradually increased, but it was still weaker than AZD9291. We will solve this problem in the next
experiment. Accordingly, we concluded that changing the acrylamide carbon chain length csan modulate the cellular and
enzymatic activity of compounds. Only when the carbon chain length of the acrylamide warhead is more than four can
the cellular and enzymatic activity be improved.
Table 1
In vitro inhibitory activities of compounds 16a-h.
16a-h
EGFR IC50 (nM)a
Cellular activity IC50 (µM)
Selectivity
(WT/TL)
b
Compd.
R1
LogD7.4
WT
65
TL
6
H1975
4.229
A549
2.462
HepG2
3.794
10.8
7.8
-
2.9
3.4
4.1
16a
16b
16c
257
33
1.289
>50
7.524
>50
1.391
>50
>2000 >2000
395
1282
52
7.6
6.1
-
4.543
5.606
1.819
0.660
4.096
0.612
6.934
5.663
4.391
3.4
3.6
4.1
16d
16e
16f
209
397
>2000
1277
1502
16
177
203
8
7.2
7.4
2.0
3.862
1.219
0.073
1.533
0.808
0.615
6.674
0.431
1.605
3.8
4.2
3.0
16g
16h
AZD9291
aEnzymatic inhibitory activities assays were examined by using the ELISA-based EGFR-tyrosine kinase (TK) assay.
bLogD7.4 values calculated by the chemaxon website (www.chemaxon.com).
2.4 Structure-based design of pyrimidine ring targeting Met793
From the results of our previous molecular docking (Fig. 3 B), we found that the pyrimidine ring of AZD9291
formed a bidentate hydrogen bond with hinge region residue Met793, with the bond lengths of 2.0Å and 2.6Å,
respectively. The hydrogen bond interaction is moderate. Guided by the docking result (Fig. 3), we hypothesized that
expanding the spatial structure of the pyrimidine ring would result in a fully occupied hinge region protein cavity.
Furthermore, the -CN at the 5-position of the pyrimidine ring in compound 17 directly points to gatekeeper Met790, thus

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increasing the binding capacity of protein with ligand. Consequently, based on the results of 16a-h, we retained the
alkyl-substituted acrylamide warhead and introduced small molecule substituents (-CN, -CH3) into the pyrimidine ring to
obtain compound 17b-e and 18a-e.
Table 2
In vitro Enzymatic inhibitory activities of compounds 17b-e and 18a-e.
17b-e, 18a-e
EGFR IC50 (nM)a
Cellular activityIC50 (µM)
Selectivity
(WT/TL)
b
Compd.
R1’
R2
R3
LogD7.4
WT
311
TL
25
H1975
1.187
A549
0.308
HepG2
0.768
-H
-H
-CN
-CN
-CN
-CN
-CN
-H
12.4
6.9
2.8
3.2
3.7
3.9
4.1
3.5
3.5
3.9
4.2
4.2
3.0
17
17b
137
387
20
16
219
37
93
10
18
38
8
1.622
1. 151
1.107
0.823
4.812
2.231
1.993
1.227
0.254
0.073
0.133
0.162
1.101
0.166
2.907
1.731
1.085
1.07
0.115
1.359
1.811
1.685
12.611
3.641
2.053
3.546
0.365
1.605
-H
24.2
4.5
17c
-H
985
17d
-H
377
10.2
14.5
8.3
17e
-CH3
-CH3
-CH3
-CH3
-CH3
-H
1336
83
18a
-H
18b
-H
1255
>2000
1531
16
69.7
-
18c
-H
18d
-H
191.3
2.0
0.329
0.615
18e
-H
8
AZD9291
aEnzymatic inhibitory activities assays were examined by using the ELISA-based EGFR-tyrosine kinase (TK) assay.
bLogD7.4 values calculated by the chemaxon website (www.chemaxon.com).
2.4.1 Structure-activity relationship studies of target compounds 17b-e and 18a-e.
The enzymatic and cellular inhibitory activities of 17b-e and 18a-e were shown in Table 2. In general, the
introduction of -CN and -CH3 into the pyrimidine ring notably increased the selectivity for WT-EGFR against
double-mutant EGFR, with the selectivity from 5 to 200-fold. Moreover, the cellular anti-proliferative activity of
compounds 17b-e and 18a-e is more potent than that of compounds 16a-h. Particularly, compounds 18a-e, which
introduced the -CH3 moiety, dramatically changed both the double-mutant EGFR enzymatic inhibitory activity (the IC50

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values from 8 to 93 nM) and the selectivity of the double-mutant EGFR against wild-type EGFR (the selectivity from
8-fold to 200-fold). We conjectured that the electron-donating group -CH3 increased the electron cloud density of the
pyrimidine ring. Hence, it enhanced the hydrogen bond reactivity between the adjacent N and the Met793 residue. We
observed that 17b was obtained by introducing a chlorine atom at the ortho-position of acrylamide in 17, similar to 18a
and 18b. However, the enzymatic activity and selectivity of both 17b and 18b for the double-mutant EGFR were
moderately decreased; their IC50 values were in the range of 10 to 20 nM, and the selectivity for wild-type EGFR did not
exceed 10 times. However, 17b and 18b also showed moderate cellular anti-proliferative activity over H1975 cells, with
IC50 values of 1.622 µM and 2.231 µM, respectively. As the acrylamide carbon chain length increased, the enzymatic
activity and selectivity underwent tremendous change. When the carbon chain length of the acrylamide warhead was 5,
the IC50 values of 17c and 18c against wild-type EGFR were 387 nM and 1255 nM, respectively. When the carbon chain
length was 6, the enzymatic inhibitory activity of compound 17d and 18d against the wild-type EGFR kinase was
decreased, but the inhibitory activity against the double-mutant EGFR was retained (with IC50 values of 219 nM and 38
nM, respectively). Therefore, we concluded that properly increasing the acrylamide side chain length can increase the
selectivity of compound to EGFRT790M/L858R mutant kinases and strengthen the inhibitory activity of compound to H1975
cells. In addition, by appropriately increasing the volume of pyrimidine ring, the compounds fully occupied the target
protein cavity of hinge region. Therefore, it can increase the affinity of compound with target protein.
2.5 Structural modification based on the molecular simulation
2.5.1 Binding mode study of potential compounds 17c and 18e.
To further investigate the binding mode of compounds with EGFR, we conducted a molecular simulation of
EGFRT790M with representative compounds 17c and 18e (Fig. 4). The binding models of compounds 17c and 18e with
EGFR protein were the same as that of our previous hypothesis. Compound 17c formed three hydrogen bonds with
EGFR protein (The bule dashed), wherein the pyrimidine ring formed a bidentate hydrogen bond with hinge residue
Met793. In addition, the -CN group at the 5-position of the pyrimidine ring directly pointed to the gatekeeper residue
Met790 and adjoined the residue Thr854 with a length of 2.5Å (The red dashed). The entire conformation sufficiently
occupied the protein hydrophobic cavity. The binding mode of 18e was the same as that of 17c. The 6-methyl-substituted
pyrimidine ring was connected to the hinge residue Met793 via a bidentate hydrogen bond. In addition, the
alkyl-substituted acrylamide side chain was adjacent to residue Cys797(with the hydrogen bond length of 2.0Å). The
hydrogen binding forces of 17c and 18e with hinge region residue Met793 are stronger than those of AZD9291.

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A
B
C
D
Fig. 4. Docking mode of 17c and 18e.
(A) Binding mode of compound 17c bind to T790M-EGFR. (B) Overview of the binding site of compound 17c with
T790M-EGFR. (C) Overview of the binding site of compound 18e with T790M-EGFR (D) Binding model of compound
18e bind to T790M-EGFR.
2.5.2 Explore 5-position fluorin substitution of indole ring extend to ATP hydrophobic pocket
Since AZD9291 was approved by the FDA, many researchers are devoted to studying the in vivo metabolism and
metabolites of AZD9291 to gain inspiration for the study of more potent EGFR inhibitors. Finlay MR and his co-worker
were the first reported the major metabolic processes and metabolites of AZD929 in the body [26]. N-demethylation is the
main metabolism way of AZD9291 in the body, produced two metabolites AZ5104 and AZ7550. Moreover, based upon
the docking results of compound 17c and 18e with T790M active domain, indole ring burried into ATP-binding pocket
and was close to DFG motif. Consequently, on the one hand, we introduced F-atom into the indole ring and amplify the
volume of the indole ring to enhance the binding interaction and selectivity of the inhibitors with T790M-EGFR. On the
other hand, the introduction of F-atom can weaken the demethylation ability of the indole ring and enhances the
anti-metabolism ability of AZD9291c derivatives. Moreover, the strategy of introducing fluorine atom is commonly used
in the development of new drugs, and compounds containing fluorine account for a significant proportion of the drugs

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used for clinical treatment [30,31]. According to the metabolites of AZD9291 in vivo, Gao H. and co-workers [28] also
designed and synthesized a series of derivatives of AZD9291, and conducted a detailed analysis of the results of in vivo
experiments. According to their experimental results, compound 23 contained a fluorine-substituted indole ring structure
exhibited EGFRT790M/L858R inhibitory activity.
In order to implement our design strategy, we retained –CH3 and –CN on the pyrimidine ring and designed three
series of fluorine-substituted derivatives at the 5-position of the indole ring (23b-d, 24a-d, and 25a-d). As shown in
Table 3, all compounds with fluorine substituted at the 5-position of the indole ring showed obvious decreases in
enzymatic activity. Accordingly, the cellular anti-proliferative activity of compounds 23b-d, 24a-d, and 25a-d also
moderately changed compared to compounds 17b-e. In particular, compounds 25a-d showed the highest decrease in both
enzymatic activity and cellular activity, with IC50 values of H1975 cells ranging from 3.8 to 12.2 µM. On the one hand,
the introduction of the strong electron-withdrawing group fluorine moiety into the indole ring that was buried in the DFG
motif may greatly reduce the hydrophobic interaction between the indole and the residues of the DFG motif. On the other
hand, based on the research of Gao H. [28], the introduction of fluorine reduced both the electron cloud density of the
indole ring and the dealkylation ability of N-methyl indole. Thus, it can change the stability of drug structure and caused
toxicity of the drug. In addition, it is also possible that the introduction of F-atom causes the compound to miss the target,
and we will conduct in-depth research in future experiments. LogD7.4 values were predicted to explain the results, and the
logD7.4 values of fluorinated substitutes were 0.2 to 0.5 higher than compounds without fluorinated substitution. Most
fluorinated substitutes have logD7.4 values greater than 4.0. These results indicated that the poor solubility caused the
decrease in the inhibitory activity, because the moderately lipid-soluble permeability of molecules is favorable for drugs
to penetrate cell membranes.
Table 3
In vitro Enzymatic inhibitory activities of compounds 23b-d, 24a-d and 25a-d
23b-d, 24a-d, 25a-d
EGFR IC50 (nM)
Cellular activity IC50 (µM)
Selectivity
(WT/TL)
b
Compd.
R1’
R2
R3
LogD7.4
WT
8.7
TL
H1975
0.254
A549
0.329
HepG2
0.429
-H
-H
-H
-H
0.07
124
1.8
3.1
3.9
23
684
388
2.596
3.602
5.285
23b

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-H
-H
-H
977
>2000
7.359
2.734
11.404
10.209
1.127
2.421
3.833
10.440
8.520
6.144
1.034
9.703
4.725
0.185
0.741
1.489
9.152
9.083
3.923
3.399
17.549
5.881
2.837
2.014
4.281
10.984
13.998
12.116
1.605
4.2
4.4
3.0
3.8
4.1
4.2
3.6
4.1
4.4
4.5
3.0
23c
23d
/
1.6
/
-H
12
7.5
-H
-CN
-CN
-CN
-CN
-H
>2000
>2000
415
>2000
>2000
360
24a
-H
24b
/
-H
1.2
5.3
1.9
/
24c
-H
1064
1094
>2000
>2000
>2000
16
201
24d
-CH3
-CH3
-CH3
-CH3
-H
591
25a
-H
998
25b
-H
892
25c
/
-H
923
/
12.232 13.258
0.073 0.615
25d
-H
8
2.0
AZD9291
aEnzymatic inhibitory activities assays were examined by using the ELISA-based EGFR-tyrosine kinase (TK) assay.
bLogD7.4 values calculated by the chemaxon website (www.chemaxon.com).
To further investigate whether the introduction of a fluorine atom can increase the hydrogen bonding capacity, we
performed molecular docking to predict the binding modes of compounds 24d and 25b. The docking results in Figure 5
show that compounds 24d (Fig. 5 A) and 25b (Fig.5 B) all formed weak hydrogen bond with the Cys797 residue (24d
and 25b with length of 2.3Å and 2.2Å, respectively). In addition, we can see that the -CN group of the compound 24d
lost hydrogen binding force with the residue Thr854. Neither 24d nor 25b engaged in hydrogen binding interactions with
the residues in the DFG motif. We speculated that the F-atom is a strong electron-withdrawing group, which has a large
influence on the electron cloud density of the entire molecular skeleton, and even changes the electron cloud distribution
of the entire molecule. At the same time, from the LogD7.4 values, we can see that the polarity of this series of
compounds does increases, which is not conducive to molecular transmembrane absorption, resulting in great changes in
their activity and selectivity. We will conductced another in-depth study on F-substituted compounds.

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A
B
Fig. 5. Docking mode of 24d and 25b.
(A) The binding model of representative compounds 24d bound to T790M-EGFR (PDB code:3IKA). (B) The binding
model of representative compounds 25b bound to T790M-EGFR (PDB code:3IKA).
2.6 Fluorescent staining is used to analyze cell morphological changes of the H1975 cells.
Fluorescence staining experiments were selected to validate and evaluate the antiproliferation by 18e on H1975 cells.
The results were shown in Fig. 6. AO staining fluorescence analysis results show that compared to the control group,
compound 18e significantly inhibited H1975 cells proliferation and induced apoptosis at the concentration of 0.25 µM.
Moreover, after treated with compound 18e, the chromatin of H1975 cells obviously shrink, and the cell edge were sharp.
Nextly, we conducted H33342 fluorescence staining assay to further verify that compound 18e induced H1975 cell
apoptosis. As illustrated in Fig. 6, the control group cells structures were complete, only a small amount of dyes into cells
and the nucleus showed weak blue fluorescence under inverted fluorescence microscope. While, after treatment with 0.25
µM compound 18e, H1975 cells structures were destroyed, chromatin was shrunk, and a large amount of the dye entered
cells. Hence, the dense staining was observed under an inverted fluorescence microscope and the fluorescence intensity
were significantly enhanced. Those phenomenons suggested that 18e induced apoptosis. Therefore, based on the results
of AO staining and H33342 staining, we can draw a conclusion that compound 18e inhibited H1975 cell proliferation and
induced apoptosis at lower concentration.

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FIGURE 6. 18e suppressed cell proliferation (acridine orange, AO) and induced apoptosis (Hoechst 33342) in H1975
cells.
2.7 Effect on cell cycle progression.
To investigate the mode of cell proliferation inhibiton by compound 18e, cell cycle distribution analysis was
performed on A549 cells. The obtained results were presented in Fig. 7, which shown that 18e induced apoptosis in A549
cells at low concentration. As the concentration of compound 18e increasing, 18e significantly depressed the G0/G1
phase population from 56.78% to 39.55%. The number of apoptotic cells gradually increased, and the cell cycle was
blocked in G2/M phase, while no significant change in S phase was observed. These findings indicated that 18e
successfully inhibited cell proliferation in a dose-dependent manner.

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FIGURE 7. Flow cytometry analysis of A549 cells treated with compound 18e for 24 h.
3. Conclusions
In summary, based on potent inhibitors 17 and 23, three series of EGFRT790M/L858R inhibitors were designed and
synthesized. The changes in reactivity of the acrylamide warhead reduced the affinity of the medicine to non-target
proteins. With the help of computer-aided drug design, we increased the spatial volume of the pyrimidine ring to enhance
the selectivity of compounds for EGFRT790M/L858R. Furthermore, based on the metabolites of AZD9291, we introduced
fluorine atom into indole ring, which resulted in the compound losing its kinase selectivity. Finally, we replaced
acrylamide with hexenamide and introduced methyl group at the 6-position of pyrimidine ring to obtain the optimized
compound 18e without fluorine substitution, whose physiological activity suggested that 18e can be used as a potent
EGFRT790M/L858R inhibitor for further study.
4. Experimental section
4.1 Chemistry
Unless otherwise required, all reagents used in the experiments were purchased from commercial analytical grade
and used without further purification. Frequently used solvents (Ethanol, petroleum ether, ethyl acetate and
dichloromethane, etc.) were absolutely anhydrous. All actions were monitored through GF254 thin-layer chromatography
plate and spots were visualized with iodine or light (in 254 nm or 365 nm). The structure of the target compound was
confirmed by 1H NMR and 13C NMR spectra at room temperature on Bruker 400 MHz spectrometer (Bruker Bioscience,

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Billerica, MA, USA) with tetramethylsilane (TMS) as an internal standard. Mass spectrometry (MS) was performed on
Waters High Resolution Quadrupole Time of Flight Tandem Mass Spectrometry (QTOF). The purity of the compound
was determined by Agilent 1260 liquid chromatograph fitted with an Inertex-C18 column. All target compounds had
purity of ≥95%.
4.1.1 General procedure for the synthesis of 16a-h
We used indole 7 as starting material to synthesize interediates 15a-c by the method reported in the literature [32]
Different substituted acrylic acids were chlorinated by POCl3 to obtain the corresponding acyl chloride at 80 C. Under
an ice bath, differently substituted acryl chloride was dissolved in dichloromethane and stirred at 0 for 10-30 min.
.
º
ºC
Another intermediates 15a-c were added to the above acryl chloride solution, and then added NaHCO3 solid
powder, stirred at 0 ºC for 0.5-3 h. After the reaction was completed, the reaction mixture was filtered and solvent
was distilled off under reduced pressure. The crude product was purified using flash chromatography with
dichloromethane/methanol (v/v, from 50:1 to 20:1) as eluents.
4.1.1.1 N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amin
amino)phenyl)-2-fluoroacrylamid (16a)
Light yellow solid. 37.9% yield, m.p: 190.7-192.1 ºC. 1H NMR (400 MHz, DMSO-d6) δ 10.04 (s, 1H), 8.92 (s, 1H),
8.54 (s, 1H), 8.31 (dd, J = 17.9, 6.6 Hz, 2H), 7.96 (s, 1H), 7.53 (d, J = 8.1 Hz, 1H), 7.29–7.20 (m, 2H), 7.17 (t, J = 7.4 Hz,
1H), 7.07 (s, 1H), 5.46–5.41 (m, 1H), 5.08 (d, J = 13.4 Hz, 1H), 3.91 (s, 3H), 3.89 (s, 3H), 3.27–3.22 (m, 2H), 2.87 (s,
2H), 2.65 (s, 3H), 2.58 (s, 6H); 13C NMR (400 MHz, DMSO-d6) δ 169.87, 164.85, 161.25, 158.98, 157.65, 144.25,
136.88, 134.21, 129.45, 128.96, 122.54, 122.21, 121.84, 119.98, 114.12, 111.53, 110.11, 109.86, 104.99, 99.32, 95.02,
59.23, 58.45, 56.88, 47.25×2, 42.23, 34.58. TOF MS ES+ (m/z): (M + H)+, calcd for C28H32FN7O2: 517.2602, found,
518.2680.
4.1.1.2 2-chloro-N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((4-(1-methyl-1H-indol-3-yl) pyrimidin
-2-yl)amino)phenyl)acrylamide (16b)
Light yellow solid. 32.1% yield, m.p: 191.3-192.4 ºC. 1H NMR (400 MHz, DMSO-d6) δ 10.25 (s, 1H), 9.40 (s, 1H),
8.90 (s, 1H), 8.66 (t, J = 7.7 Hz, 2H), 8.33 (s, 1H), 7.86 (d, J = 8.2 Hz, 1H), 7.55 (tt, J = 15.0, 7.3 Hz, 3H), 7.37 (s, 1H),
6.22 (s, 1H), 5.99 (s, 1H), 4.23 (s, 3H), 4.19 (s, 3H), 3.23 (t, J = 4.8 Hz, 4H), 3.06 (s, 3H), 2.69 (t, J = 5.1 Hz, 2H), 2.59 (s,
6H). TOF MS ES+ (m/z): (M + H)+, calcd for C28H32ClN7O2: 533.2306, found, 534.2384.
4.1.1.3 2,3,3-Trichloro-N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((4-(1-methyl-1H-indol-3-yl)
pyrimidin-2-yl)amino)phenyl)acrylamide (16c)
Light yellow solid. 36.8% yield, m.p: 287.7-288.4 ºC. 1H NMR (400 MHz, DMSO-d6) δ 11.36 (s, 1H), 9.07 (d, J =

ACCEPTED MANUSCRIPT
7.3 Hz, 1H), 8.96 (s, 1H), 8.18 (s, 1H), 7.85 (d, J = 7.4 Hz, 1H), 7.56 (d, J = 8.2 Hz, 1H), 7.35 (s, 1H), 7.25 (dd, J = 15.6,
8.0 Hz, 3H), 6.92 (s, 1H), 3.90 (s, 3H), 3.67 (s, 3H), 3.20–3.02 (m, 2H), 2.89 (s, 3H), 2.26 (s, 6H). TOF MS ES+ (m/z):
(M + H)+, calcd for C28H30Cl3N7O2: 601.1527, found, 602.1605.
4.1.1.4
(Z)-N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)
amino)phenyl)but-2-enamide (16d)
White solid. 33.2% yield, m.p: 159.7-161.2 ºC. 1H NMR (400 MHz, DMSO-d6) δ 9.93 (s, 1H), 9.06 (s, 1H), 8.65 (s,
1H), 8.32 (d, J = 5.3 Hz, 1H), 8.25 (d, J = 7.9 Hz, 1H), 8.19 (s, 1H), 7.90 (s, 1H), 7.52 (d, J = 8.2 Hz, 1H), 7.23 (dd, J =
12.1, 6.3 Hz, 2H), 7.14 (t, J = 7.5 Hz, 1H), 7.02 (s, 1H), 6.81 (dq, J = 13.8, 6.9 Hz, 1H), 3.91 (s, 3H), 3.85 (s, 3H), 2.94 (d,
J = 5.2 Hz, 2H), 2.69 (s, 3H), 2.46 (d, J = 5.3 Hz, 2H), 2.31 (s, 6H), 1.90 (d, J = 6.6 Hz, 3H); 13C NMR (400 MHz,
DMSO-d6) δ 163.48, 162.12, 160.39, 158.12, 139.49, 138.17, 136.48, 133.29, 129.87, 128.24, 125.82, 122.46, 121.85,
121.35, 119.45, 112.91, 110.97, 108.79, 108.65, 106.42, 102.59, 59.71, 56.51, 55.87, 45.05×2, 43.12, 33.44, 18.04. TOF
MS ES+ (m/z): (M + H)+, calcd for C29H35N7O2: 513.2852, found, 514.2925.
4.1.1.5
N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)
amino)phenyl)-3-methylbut-2-enamide (16e)
1
Light yellow solid. 40.2% yield, m.p: 178.2-179.1 ºC. H NMR (400 MHz, DMSO-d6) δ 9.88 (s, 1H), 9.03 (s, 1H),
8.53 (s, 1H), 8.29 (t, J = 7.7 Hz, 2H), 7.96 (s, 1H), 7.49 (d, J = 8.2 Hz, 1H), 7.18 (tt, J = 15.0, 7.3 Hz, 3H), 7.00 (s, 1H),
5.85 (s, 1H), 5.62 (s, 1H), 3.86 (s, 3H), 3.82 (s, 3H), 2.86 (s, 2H), 2.69 (s, 3H), 2.32 (t, J = 5.1 Hz, 2H), 2.22 (s, 6H), 2.13
(s, 3H), 1.87 (s, 3H). TOF MS ES+ (m/z): (M + H)+, calcd for C30H37N7O2: 527.3009, found, 528.3087.
4.1.1.6
(Z)-N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)
amino)phenyl)-4-methylpent-2-enamide (16f)
White solid. 35.7% yield, m.p: 208.2-209.6 ºC. 1H NMR (400 MHz, DMSO-d6) δ 10.04 (s, 1H), 9.13 (s, 1H), 8.67 (s,
1H), 8.32 (d, J = 5.2 Hz, 1H), 8.23 (d, J = 7.8 Hz, 1H), 7.92 (d, J = 24.3 Hz, 1H), 7.53 (d, J = 8.1 Hz, 1H), 7.23 (t, J = 6.5
Hz, 2H), 7.15 (t, J = 7.4 Hz, 1H), 7.02 (s, 1H), 6.80 (d, J = 6.5 Hz, 1H), 6.09 (d, J = 14.9 Hz, 1H), 3.91 (s, 3H), 3.85 (s,
3H), 2.89 (s, 2H), 2.71 (d, J = 9.7 Hz, 3H), 2.41 (dd, J = 17.0, 10.3 Hz, 3H), 2.26 (s, 6H), 1.07 (d, J = 6.7 Hz, 5H); 13C
NMR (400 MHz, DMSO-d6) δ 168.74, 166.08, 159.98, 157.21, 151.27, 142.89, 136.24, 134.05, 129.87, 128.45, 123.21,
122.23, 121.98, 120.87, 119.82, 113.52, 111.25, 109.87, 109.42, 104.71, 98.87, 59.78, 56.82, 55.49, 46.02x2, 42.08,
33.89, 25.09, 20.92×2. TOF MS ES+ (m/z): (M + H)+, calcd for C31H39N7O2: 541.3165, found, 542.3242.
4.1.1.7
(Z)-N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)
amino)phenyl)pent-2-enamide (16g)
Light yellow solid. 37.4% yield, m.p: 245.6-247.1 ºC. 1H NMR (400 MHz, DMSO-d6) δ 10.72 (s, 1H), 9.73 (s, 1H),

ACCEPTED MANUSCRIPT
8.70 (s, 1H), 8.26 (s, 2H), 7.54 (s, 1H), 7.36–7.14 (m, 4H), 7.03–6.75 (m, 3H), 3.91 (s, 3H), 3.85 (s, 3H), 2.73 (s, 6H),
2.62 (s, 3H), 2.20 (s, 2H), 1.35–1.20 (m, 4H), 1.04 (s, 3H). TOF MS ES+ (m/z): (M + H)+, calcd for C30H37N7O2:
527.3009, found, 528.3087.
4.1.1.8
(Z)-N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)
amino)phenyl)hex-2-enamide (16h)
White solid. 34.1% yield, m.p: 185.2-186.7 ºC. 1H NMR (400 MHz, DMSO-d6) δ 10.02 (s, 1H), 9.13 (s, 1H), 8.68 (s,
1H), 8.32 (d, J = 5.3 Hz, 1H), 8.24 (d, J = 7.8 Hz, 1H), 7.90 (s, 1H), 7.53 (d, J = 8.2 Hz, 1H), 7.24 (t, J = 6.7 Hz, 2H),
7.15 (t, J = 7.4 Hz, 1H), 7.03 (s, 1H), 6.81 (dtd, J = 11.0, 6.9, 3.8 Hz, 2H), 6.11 (d, J = 15.3 Hz, 1H), 5.80–5.72 (m, 1H),
3.92 (s, 3H), 3.85 (s, 3H), 2.90 (s, 2H), 2.71 (s, 3H), 2.36 (s, 2H), 2.24 (d, J = 7.5 Hz, 6H), 2.14 (dd, J = 13.7, 6.5 Hz, 2H),
1.50–1.42 (m, 2H), 0.93 (dd, J = 13.5, 6.2 Hz, 3H); 13C NMR (400 MHz, DMSO-d6) δ 167.72, 163.33, 160.35, 158.17,
148.83, 146.23, 144.00, 138.18, 134.35, 128.31, 125.90, 125.81, 125.77, 122.78, 122.42, 121.78, 121.35, 112.94, 110.97,
107.59, 105.68, 57.16, 56.50, 55.89, 45.51×2, 43.01, 33.83, 21.66, 21.30, 13.96. TOF MS ES+ (m/z): (M + H)+, calcd for
C31H39N7O2: 541.3165, found, 543.3245.
4.1.2 General procedure for the synthesis of 17b-e
The synthetic method of compounds 17b-e are similarly to 16a-h. The difference is that we need to replace the
pyrimidine with cyan-substituted pyrimidine and the rest of the synthesis conditions are the same.
4.1.2.1
2-chloro-N-(5-((5-cyano-4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)-2-((2-(dimethylamino)ethyl)
(methyl)amino)-4-methoxyphenyl)acrylamide (17b)
1
Pure white solid. 38.9% yield, m.p: 152.3-153.1 ºC. H NMR (400 MHz, DMSO-d6) δ 10.03 (s, 1H), 9.45 (s, 1H),
8.69 (s, 1H), 8.50 (s, 1H), 8.24 (s, 1H), 7.53 (d, J = 7.6 Hz, 1H), 7.33–7.20 (m, 1H), 7.14 (s, 1H), 6.47 (s, 1H), 6.29 (s,
1H), 6.07 (s, 1H), 5.86 (s, 1H), 3.92 (s, 3H), 3.78 (s, 3H), 3.26–3.19 (m, 2H), 2.76 (s, 2H), 2.72 (s, 3H), 2.47 (s, 6H); 13C
NMR (400 MHz, DMSO-d6) δ 173.87, 165.21, 164.87, 163.52, 143.78, 137.85, 136.53, 133.89, 130.08, 129.32, 123.54,
122.32, 121.38, 121.05, 119.87, 113.92, 113.27, 111.23, 109.88, 109.26, 98.53, 96.73, 59.81, 58.08, 55.78, 46.18×2,
41.25, 35.09. TOF MS ES+ (m/z): (M + H)+, calcd for C29H31ClN8O2: 558.2259, found, 559.2337.
4.1.2.2
(Z)-N-(5-((5-cyano-4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)-2-((2-(dimethylamino)ethyl)(methyl)
amino)-4-methoxyphenyl)pent-2-enamide (17c)
Pure white solid. 44.1% yield, m.p: 128.5-130.1 ºC. 1H NMR (400 MHz, DMSO-d6) δ 9.92 (s, 1H), 9.41 (d, J = 9.0
Hz, 1H), 8.67 (s, 1H), 8.48 (s, 1H), 8.38 (s, 1H), 7.51 (d, J = 7.5 Hz, 1H), 7.23 (s, 1H), 7.06 (s, 1H), 6.91–6.73 (m, 2H),
6.05 (d, J = 15.4 Hz, 1H), 5.74 (d, J = 15.6 Hz, 1H), 3.91 (s, 3H), 3.72 (s, 3H), 2.91 (s, 2H), 2.74 (d, J = 10.9 Hz, 3H),
2.39 (s, 2H), 2.23 (d, J = 5.8 Hz, 6H), 2.20–2.14 (m, 2H), 1.02 (dd, J = 9.8, 7.5 Hz, 3H). TOF MS ES+ (m/z): (M + H)+,

ACCEPTED MANUSCRIPT
calcd for C31H36N8O2 :552.6830, found, 553.3048.
4.1.2.3
(Z)-N-(5-((5-cyano-4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)-2-((2-(dimethylamino)ethyl)(methyl)
amino)-4-methoxyphenyl)-4-methylpent-2-enamide (17d)
Pure white solid. 41.2% yield, m.p: 160.3-162.1 ºC. 1H NMR (400 MHz, CDCl3) δ 9.35 (s, 1H), 9.26 (s, 1H), 8.65 (s,
1H), 8.49 (s, 2H), 7.78 (s, 1H), 7.36 (d, J = 7.3 Hz, 1H), 7.33–7.27 (m, 2H), 6.92 (d, J = 16.8 Hz, 2H), 6.72 (s, 1H), 3.91
(d, J = 6.6 Hz, 6H), 3.29 (s, 2H), 3.01 (s, 2H), 2.74 (d, J = 11.2 Hz, 9H), 2.53 (d, J = 6.8 Hz, 1H), 1.10 (d, J = 6.8 Hz, 6H);
13C NMR (400 MHz, DMSO-d6) δ 174.31, 166.25, 164.28, 162.87, 151.82, 142.98, 136.58, 133.52, 129.85, 128.32,
122.53, 121.38, 121.07, 119.88, 119.05, 114.08, 113.87, 111.84, 109.86, 109.43, 98.72, 96.02, 59.87, 57.98, 57.03,
47.86×2, 41.05, 34.08, 24.87, 22.23×2. TOF MS ES+ (m/z): (M + H)+, calcd for C32H38N8O2: 566.3118, found,
567.3203 .
4.1.2.4
(Z)-N-(5-((5-cyano-4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)-2-((2-(dimethylamino)ethyl) (methyl)
amino)-4-methoxyphenyl)hex-2-enamide (17e)
1
Pure white solid. 39.1% yield, m.p: 158.1-160.6 ºC. H NMR (400 MHz, DMSO-d6) δ 9.92 (s, 1H), 9.40 (s, 1H),
8.66 (s, 1H), 8.47 (s, 1H), 8.37 (s, 1H), 7.51 (d, J = 8.2 Hz, 1H), 7.23 (s, 1H), 7.05 (s, 1H), 6.82–6.69 (m, 2H), 6.07 (d, J
= 15.3 Hz, 1H), 5.75 (d, J = 15.5 Hz, 1H), 3.90 (s, 3H), 3.72 (s, 3H), 2.91 (s, 2H), 2.74 (s, 3H), 2.41 (d, J = 5.8 Hz, 2H),
2.23 (s, 6H), 2.17–2.13 (m, 2H), 1.45–1.41 (m, 2H), 0.89–0.87 (m, 3H). TOF MS ES+ (m/z): (M + H)+, calcd for
C32H38N8O2: 556.3118, found, 567.3192.
4.1.3 General procedure for the synthesis of 18a-e.
The synthetic method of compounds 18a-e are similarly to 16a-h. The difference is that we need to replace the
pyrimidine with methyl-substituted pyrimidine and the rest of the synthesis conditions are the same.
4.1.3.1 N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((4-methyl-6-(1-methyl-1H-indol-3-yl) pyrimidin
-2-yl) amino)phenyl)acrylamide (18a)
Light yellow solid. 31.9% yield, m.p: 292.7-293.4 ºC. 1H NMR (400 MHz, DMSO-d6) δ 10.90 (s, 1H), 9.89 (s, 1H),
9.05 (s, 1H), 8.64 (s, 1H), 8.22 (s, 1H), 7.52 (d, J = 7.0 Hz, 1H), 7.35 (d, J = 10.9 Hz, 1H), 7.27–7.14 (m, 3H), 6.95 (s,
1H), 6.24 (d, J = 18.6 Hz, 1H), 5.68 (d, J = 10.2 Hz, 1H), 3.90 (d, J = 3.3 Hz, 3H), 3.89 (s, 3H), 3.29 (s, 3H), 3.15 (s, 2H),
2.73 (s, 6H), 2.59 (s, 2H), 2.49 (s, 3H). TOF MS ES+ (m/z): (M + H)+, calcd for C29H35N7O2 :513.2852, found, 514.2931.
4.1.3.2
pyrimidin-2-yl)amino)phenyl)acrylamide (18b)
Light yellow solid. 31.5% yield, m.p: 164.5-165.7 ºC. H NMR (400 MHz, DMSO-d6) δ 9.94 (s, 1H), 9.12 (s, 1H),
2-chloro-N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((4-methyl-6-(1-methyl-1H-indol-3-yl)
1

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8.53 (s, 1H), 8.16 (d, J = 8.0 Hz, 1H), 7.69 (s, 1H), 7.43 (d, J = 8.1 Hz, 1H), 7.15 (t, J = 7.6 Hz, 1H), 7.09 (t, J = 7.8 Hz,
2H), 6.99 (s, 1H), 6.49 (d, J = 2.1 Hz, 1H), 6.03 (d, J = 2.1 Hz, 1H), 3.83 (s, 3H), 3.81 (s, 3H), 3.18 (t, J = 7.0 Hz, 2H),
2.82 (t, 2H), 2.57 (s, 3H), 2.40 (s, 6H), 2.29 (s, 3H). TOF MS ES+ (m/z): (M + H)+, calcd for C29H34ClN7O2: 547.2463,
found, 548.2544.
4.1.3.3
(Z)-N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((4-methyl-6-(1-methyl-1H-indol-3-yl)
pyrimidin-2-yl)amino)phenyl)pent-2-enamide (18c)
1
Light yellow solid. 34.4% yield, m.p: 189.7-191.2 ºC. H NMR (400 MHz, DMSO-d6) δ 9.83 (s, 1H), 9.15 (s, 1H),
8.57 (s, 1H), 8.03 (d, J = 8.0 Hz, 1H), 7.54 (s, 1H), 7.33 (d, J = 8.1 Hz, 1H), 7.09–7.01 (m, 1H), 6.97 (t, J = 7.2, 5.5 Hz,
2H), 6.82 (s, 1H), 6.69 (ddt, J = 21.8, 15.5, 6.3 Hz, 2H), 3.73 (s, 3H), 3.67 (s, 3H), 2.70 (t, 2H), 2.36–2.28 (m, 3H), 2.19
(s, 3H), 2.16 (t, J = 6.2 Hz, 2H), 2.06 (s, 6H), 2.01–1.95 (m, 2H), 0.88 (t, J = 7.4 Hz, 3H); 13C NMR (400 MHz,
DMSO-d6) δ 168.72, 168.06, 165.84, 160.89, 145.03, 142.56, 136.52, 133.24, 129.55, 128.31, 123.08, 121.85, 120.63,
119.51, 119.75, 113.82, 111.21, 109.58, 109.01, 104.53, 98.27, 58.86, 58.32, 56.81, 47.23×2, 41.45, 33.87, 25.24, 19.85,
14.68. TOF MS ES+ (m/z): (M + H)+, calcd for C31H39N7O2: 576.2854, found, 577.2935.
4.1.3.4
(Z)-N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((4-methyl-6-(1-methyl-1H-indol-3-yl)
pyrimidin-2-yl)amino)phenyl)-4-methylpent-2-enamide (18d)
1
Light yellow solid. 38.2% yield, m.p: 172.3-173.5 ºC. H NMR (400 MHz, DMSO-d6) δ 9.86 (s, 1H), 9.20 (s, 1H),
8.58 (s, 1H), 8.03 (d, J = 8.0 Hz, 1H), 7.53 (s, 1H), 7.33 (d, J = 8.2 Hz, 1H), 7.04 (t, J = 7.7 Hz, 1H), 6.98 (d, J = 4.6 Hz,
2H), 6.82 (s, 1H), 6.63 (ddd, J = 6.5, 15.5, 30.9 Hz, 2H), 3.74 (s, 3H), 3.68 (s, 3H), 2.69 (t, J = 5.7 Hz, 2H), 2.34 (t, J =
11.4, 13.2 Hz, 3H), 2.17 (d, J = 22.2 Hz, 6H), 2.06 (s, 6H), 0.89 (d, J = 6.7 Hz, 6H). TOF MS ES+ (m/z): (M + H)+, calcd
for C32H41N7O2: 555.3322, found, 556.3405.
4.1.3.5
(Z)-N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((4-methyl-6-(1-methyl-1H-indol-3-yl)
pyrimidin-2-yl)amino)phenyl)hex-2-enamide (18e)
1
Light yellow solid. 39.4% yield, m.p: 129.4-131.1 ºC. H NMR (400 MHz, DMSO-d6) δ 9.80 (s, 1H), 9.15 (s, 1H),
8.57 (s, 1H), 8.04 (d, J = 8.0 Hz, 1H), 7.54 (s, 1H), 7.33 (d, J = 8.1 Hz, 1H), 7.04 (t, J = 7.4 Hz, 1H), 7.00–6.94 (m, 2H),
6.83 (s, 1H), 6.66–6.56 (m, 2H), 3.74 (s, 3H), 3.68 (s, 3H), 2.72 (t, J = 5.7 Hz, 2H), 2.31 (t, J = 1.9 Hz, 3H), 2.20 (d, J =
4.5 Hz, 5H), 2.09 (s, 6H), 2.05–2.01 (m, 2H), 1.31 (q, J = 7.4 Hz, 2H), 0.75 (t, J = 7.4 Hz, 3H); 13C NMR (400 MHz,
DMSO-d6) δ 167.89, 166.97, 165.84, 160.25, 143.84, 139.54, 136.25, 133.34, 129.28, 128.65, 122.87, 122.23, 120.87,
120.08, 119.25, 113.87, 111.08, 109.87, 109.23, 104.87, 98.27, 59.21, 57.53, 55.82, 46.25×2, 41.87, 34.08, 28.27, 24.32,
21.89, 16.78. TOF MS ES+ (m/z): (M + H)+, calcd for C32H41N7O2: 555.3322, found, 556.3400.
4.1.4 General procedure for the synthesis of 23b-d

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The synthetic method of compounds 23b-d are similarly to 16a-h. The difference is that we need to replace the
indole with fluorine-substituted indole and the rest of the synthesis conditions are the same.
4.1.1.1
(Z)-N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-5-((4-(5-fluoro-1-methyl-1H-indol-3-yl)pyrimidin-2-yl)
amino)-4-methoxyphenyl)pent-2-enamide (23b)
White solid. 35.1% yield, m.p: 194.6-195.7 ºC. 1H NMR (400 MHz, DMSO-d6) δ 9.87 (s, 1H), 8.71 (s, 1H), 8.61 (s,
1H), 8.29 (d, J = 4.7 Hz, 1H), 8.05 (d, 1H), 7.54 (s, 1H), 7.23 (d, J = 5.7 Hz, 1H), 7.09 (d, J = 7.4 Hz, 3H), 6.58 (s, 1H),
6.09 (s, 1H), 3.91–3.87 (m, 7H), 3.18–3.13 (m, 2H), 2.73 (s, 9H), 2.66 (s, 2H); 13C NMR (400 MHz, DMSO-d6) δ 170.82,
166.45, 160.21, 157.01, 156.87, 144.89, 143.21, 133.77, 132.12, 129.88, 128.96, 122.53, 122.31, 115.03, 113.26, 112.87,
112.43, 111.12, 108.98, 104.53, 98.23, 58.67, 56.78, 55.96, 47.18×2, 41.25, 33.68, 20.05, 13.89. TOF MS ES+ (m/z): (M
+ H)+, calcd for C30H36FN7O2: 545.2915, found, 546.2995.
4.1.4.2
(Z)-N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-5-((4-(5-fluoro-1-methyl-1H-indol-3-yl)pyrimidin-2-yl)
amino)-4-methoxyphenyl)-4-methylpent-2-enamide (23c)
White solid. 34.3% yield, m.p: 123.7-125.1 ºC. 1H NMR (400 MHz, DMSO-d6) δ 9.83 (s, 1H), 8.57 (s, 1H), 8.03 (d,
J = 8.0 Hz, 1H), 7.54 (s, 1H), 7.33 (d, J = 8.1 Hz, 1H), 7.07–7.01 (m, 1H), 6.97 (q, J = 7.2, 5.5 Hz, 3H), 6.82 (s, 1H), 6.68
(tt, J = 15.5, 6.5 Hz, 2H), 3.73 (s, 3H), 3.67 (s, 3H), 2.73–2.68 (m, 2H), 2.35–2.29 (m, 3H), 2.15 (s, 2H), 2.06 (s, 6H),
2.01–1.95 (m, 2H), 0.80 (t, J = 7.4 Hz, 3H). TOF MS ES+ (m/z): (M + H)+, calcd for C31H38FN7O2 :559.3071, found,
560.3161.
4.1.4.3
(Z)-N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-5-((4-(5-fluoro-1-methyl-1H-indol-3-yl)pyrimidin-2-yl)
amino)-4-methoxyphenyl)hex-2-enamide (23d)
White solid. 36.2% yield, m.p: 211.9-213.4 ºC. 1H NMR (400 MHz, DMSO-d6) δ 9.93 (s, 1H), 8.92 (s, 1H), 8.62 (s,
1H), 8.28 (d, J = 5.3 Hz, 1H), 8.09 (s, 1H), 8.02 (d, J = 10.7 Hz, 1H), 7.53 (dd, J = 9.0, 4.5 Hz, 1H), 7.17 (d, J = 5.3 Hz,
1H), 7.12–7.04 (m, 1H), 7.01 (s, 1H), 6.78 (dd, J = 15.5, 6.1 Hz, 1H), 6.11 (d, J = 15.4 Hz, 1H), 2.95 (s, 2H), 2.69 (s, 3H),
2.53 (s, 3H), 2.35 (s, 6H), 1.07 (s, 3H), 1.05 (s, 3H); 13C NMR (400 MHz, DMSO-d6) δ 169.71, 166.58, 159.83, 157.98,
157.56, 143.96, 139.84, 133.54, 132.02, 130.25, 128.87, 122.53,122.05, 114.93, 113.81, 112.59, 112.31, 111.23, 109.25,
104.36, 98.63, 59.27, 58.63, 56.87, 48.03×2, 40.82, 34.25, 28.76, 22.56, 15.28. TOF MS ES+ (m/z): (M + H)+, calcd for
C31H38FN7O2 :559.3071, found, 560.3150.
4.1.5 General procedure for the synthesis of 24a-d
The synthetic method of compounds 24a-d are similarly to 16a-h. The difference is that we need to replace the
indole and pyrimidine with fluorine-substituted indole and cyan-substituted pyrimidine, respectively. The rest of the
synthesis conditions are the same.

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4.1.5.1
N-(5-((5-cyano-4-(5-fluoro-1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)-2-((2-(dimethylamino)ethyl)
(methyl)amino)-4-methoxyphenyl)acrylamide (24a)
White solid. 39.3% yield, m.p: 155.8-159.3 ºC. 1H NMR (400 MHz, DMSO-d6) δ 10.70 (s, 1H), 9.97 (s, 1H), 8.78 (s,
1H), 8.25 (s, 2H), 7.58 (d, J = 8.0 Hz, 1H), 7.37 (d, J = 6.6 Hz, 1H), 7.27 (d, J = 6.3 Hz, 2H), 7.01 (s, 1H), 6.20 (d, J =
17.0 Hz, 1H), 5.69 (d, J = 10.3 Hz, 1H), 3.92 (s, 3H), 3.85 (s, 3H), 3.34 (s, 2H), 2.75 (s, 3H), 2.64 (s, 2H), 2.50 (s, 6H).
TOF MS ES+ (m/z): (M + H)+, calcd for C29H31FN8O2: 542.2554, found, 543.2632.
4.1.5.2
(Z)-N-(5-((5-cyano-4-(5-fluoro-1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)-2-((2-(dimethylamino)ethyl)
(methyl)amino)-4-methoxyphenyl)pent-2-enamide (24b)
White solid. 40.8% yield, m.p: 135.1-136.7 ºC. 1H NMR (400 MHz, DMSO-d6) δ 9.66 (s, 1H), 9.42 (s, 1H), 8.51 (s,
1H), 8.35 (s, 1H), 8.05 (s, 1H), 7.35 (s, 1H), 6.94 (s, 2H), 6.32 (s, 1H), 6.09 (s, 1H), 5.87 (s, 1H), 3.72 (s, 3H), 3.63 (s,
3H), 3.19 (t, J = 7.0 Hz, 2H), 2.88 (t, J = 7.3 Hz, 2H), 2.54 (d, J = 14.9 Hz, 3H), 2.50 (s, 6H), 2.33 (s, 2H), 1.02 (t, J = 9.3
Hz, 3H); 13C NMR (400 MHz, DMSO-d6) δ 164.40, 163.70, 162.53, 161.39, 158.87, 151.59, 141.98, 137.07, 135.81,
134.14, 133.23, 123.40, 122.12, 120.36, 119.71, 112.12, 112.02, 111.62, 111.36, 111.05, 106.33, 101.98, 56.37, 54.66,
50.43, 43.82, 43.08×2, 34.14, 29.47, 14.37. TOF MS ES+ (m/z): (M + H)+, calcd for C31H35FN8O2: 570.2867, found,
571.2950.
4.1.5.3
(Z)-N-(5-((5-cyano-4-(5-fluoro-1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)-2-((2-(dimethylamino)ethyl)
(methyl)amino)-4-methoxyphenyl)-4-methylpent-2-enamide (24c)
White solid. 44.1% yield, m.p: 179.8-181.2 ºC. 1H NMR (400 MHz, DMSO-d6) δ 9.66 (s, 1H), 9.42 (s, 1H), 8.51 (s,
1H), 8.35 (s, 1H), 8.05 (s, 1H), 7.35 (s, 1H), 6.94 (s, 2H), 6.32 (s, 1H), 6.09 (s, 1H), 5.87 (s, 1H), 5.62 (s, 1H), 3.72 (s,
3H), 3.63 (s, 3H), 3.19 (d, J = 7.1 Hz, 2H), 2.88 (d, J = 7.2 Hz, 2H), 2.52 (s, 3H), 2.50 (s, 6H), 2.33 (s, 2H), 1.01 (s, 3H);
13C NMR (400 MHz, DMSO-d6) δ 164.23, 163.75, 162.64, 161.66, 155.15, 150.80, 142.55, 135.85, 134.26, 127.12,
123.03, 122.84, 120.95, 120.02, 119.90, 112.21, 112.11, 111.72, 111.45, 111.23, 109.05, 106.29, 56.30, 55.82, 44.39,
42.56, 34.25, 30.63, 29.58, 22.02×2, 21.67. TOF MS ES+ (m/z): (M + H)+, calcd for C32H37FN8O2: 584.3204, found,
585.3102.
4.1.5.4
(Z)-N-(5-((5-cyano-4-(5-fluoro-1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)-2-((2-(dimethylamino)ethyl)
(methyl)amino)-4-methoxyphenyl)hex-2-enamide (24d)
White solid. 43.2% yield, m.p: 141.7-142.5 ºC. 1H NMR (400 MHz, DMSO-d6) δ 9.71 (s, 1H), 9.57 (s, 1H), 8.66 (s,
1H), 8.53 (s, 1H), 8.25 (s, 1H), 7.53 (s, 1H), 7.05 (d, J = 25.2 Hz, 2H), 6.75 (ddd, J = 31.4, 15.5, 6.3 Hz, 2H), 6.34 (d, J =
15.9 Hz, 1H), 3.90 (s, 3H), 3.74 (s, 3H), 3.10 (s, 2H), 2.86 (s, 2H), 2.68 (s, 3H), 2.50 (s, 6H), 2.44–2.40 (m, 1H), 1.20 (s,
6H);
13C NMR (400 MHz, DMSO-d6) δ 163.75, 163.19, 162.02, 160.98, 159.63, 157.29, 150.86, 144.29, 142.14, 135.28,

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133.67, 126.59, 126.48, 124.70, 122.55, 119.26, 111.69, 111.60, 111.14, 110.88, 110.53, 105.56, 66.28, 55.76, 54.25,
42.84, 42.44, 33.68, 33.38, 28.96, 21.08, 13.55. TOF MS ES+ (m/z): (M + H)+, calcd for C32H37FN8O2: 584.3204, found,
585.3107.
4.1.6 General procedure for the synthesis of 25a-d
The synthetic methods of compound 25a-d are similarly to 16a-h. The difference is that we need to replace the
indole and pyrimidine with fluorine-substituted indole and methyl-substituted pyrimidine, respectively. The rest of the
synthesis conditions are the same.
4.1.6.1 N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-5-((4-(5-fluoro-1-methyl-1H-indol-3-yl)-6-methylpyrimidin-2-
yl)amino)-4-methoxyphenyl)acrylamide (25a)
1
Light yellow solid. 50.3% yield, m.p: 245.1-246.3 ºC. H NMR (400 MHz, DMSO-d6) δ 9.32 (s, 1H), 8.73 (s, 1H),
7.86 (s, 1H), 7.50–7.43 (m, 2H), 7.42 (s, 1H), 7.35 (dd, J = 7.5, 4.9 Hz, 1H), 7.03 (td, J = 7.8, 1.5 Hz, 1H), 6.38 (s, 1H),
6.31 (t, J = 13.4 Hz, 1H), 5.90 (dd, J = 13.6, 3.1 Hz, 1H), 5.83 (dd, J = 13.4, 3.1 Hz, 1H), 3.84 (s, 3H), 3.77 (s, 3H), 3.69
(t, J = 7.1 Hz, 2H), 3.00 (s, 3H), 2.74 (t, J = 7.1 Hz, 2H), 2.42 (s, 3H), 2.33 (s, 6H); 13C NMR (400 MHz, DMSO-d6) δ
170.02, 169.32, 168.72, 162.03, 158.4, 144.21, 134.65, 132.52, 131.45, 131.23, 129.87, 126.98, 122.83, 115.54, 113.98,
112.98, 112.65, 111.53, 110.23, 104.97, 98.98, 58.96, 57.25, 56.38, 45.85×2, 42.08, 34.85, 25.35. TOF MS ES+ (m/z):
(M + H)+, calcd for C29H34FN7O2: 531.2758, found, 532.2836.
4.1.6.2 (Z)-N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-5-((4-(5-fluoro-1-methyl-1H-indol-3-yl)-6-methylpyrimidin
-2-yl)amino)-4-methoxyphenyl)pent-2-enamide (25b)
1
Light yellow solid. 52.5% yield, m.p: 239.7-240.1 ºC. H NMR (400 MHz, DMSO-d6) δ 9.24 (s, 1H), 8.73 (s, 1H),
7.86 (s, 1H), 7.51–7.40 (m, 3H), 7.34 (dd, J = 7.5, 4.9 Hz, 1H), 7.04 (td, J = 7.7, 1.5 Hz, 1H), 6.38 (s, 1H), 6.22–6.15 (m,
2H), 3.84 (s, 3H), 3.77 (s, 3H), 3.69 (t, J = 7.1 Hz, 2H), 3.00 (s, 3H), 2.74 (t, J = 7.1 Hz, 2H), 2.45–2.36 (m, 5H), 2.33 (s,
6H), 1.08(m, 3H). TOF MS ES+ (m/z): (M + H)+ , calcd for C31H38FN7O2 :559.3071, found, 560.3145.
4.1.6.3 (Z)-N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-5-((4-(5-fluoro-1-methyl-1H-indol-3-yl)-6-methylpyrimidin
-2-yl)amino)-4-methoxyphenyl)-4-methylpent-2-enamide (25c)
1
Light yellow solid. 46.3% yield, m.p: 208.5-209.4 ºC. H NMR (400 MHz, DMSO-d6) δ 9.35 (s, 1H), 8.70 (s, 1H),
7.86 (s, 1H), 7.52 (s, 1H), 7.48 (m, 2H), 7.35 (dd, J = 7.5, 5.0 Hz, 1H), 7.04 (td, J = 7.8, 1.5 Hz, 1H), 6.38 (s, 1H), 6.22
(dd, J = 11.0, 0.9 Hz, 1H), 6.02 (m, 1H), 3.84 (s, 3H), 3.77 (s, 3H), 3.69 (t, J = 7.1 Hz, 2H), 3.00 (s, 3H), 2.74 (t, J = 7.1
Hz, 2H), 2.66 (m, 1H), 2.42 (s, 3H), 2.34 (s, 6H), 1.04 (dd, J = 6.8, 1.1 Hz, 6H); 13C NMR (400 MHz, DMSO-d6) δ
170.21, 169.53, 167.24, 161.56, 157.98, 151.93, 143.99, 133.98, 132.57, 130.81, 129.39, 122.86, 119.84, 115.53, 113.88,
112.98, 112.57, 111.53, 109.88, 105.23, 99.02, 59.23, 57.69, 56.89, 46.92×2, 42.12, 35.08, 24.98, 24.32, 22.46×2. TOF

ACCEPTED MANUSCRIPT
MS ES+ (m/z): (M + H)+, calcd for C32H40FN7O2: 573.3228, found, 574.3306.
4.1.6.4 (Z)-N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-5-((4-(5-fluoro-1-methyl-1H-indol-3-yl)-6-methylpyrimidin
-2-yl)amino)-4-methoxyphenyl)hex-2-enamide (25d)
1
Light yellow solid. 58.7% yield, m.p: 195.7-196.6 ºC. H NMR (400 MHz, DMSO-d6) δ 9.21 (s, 1H), 8.70 (s, 1H),
7.86 (s, 1H), 7.52 (s, 1H), 7.48 – 7.40 (m, 2H), 7.35 (dd, J = 7.5, 5.0 Hz, 1H), 7.04 (td, J = 7.8, 1.5 Hz, 1H), 6.38 (s, 1H),
6.28 (dt, J = 10.8, 6.1 Hz, 1H), 6.20–6.14 (m, 1H), 3.84 (s, 3H), 3.77 (s, 3H), 3.69 (t, J = 7.1 Hz, 2H), 3.00 (s, 3H), 2.74 (t,
J = 7.1 Hz, 2H), 2.42 (s, 3H), 2.35–2.27 (m, 8H), 1.49 (m, 2H), 0.89 (t, J = 8.0 Hz, 3H). TOF MS ES+ (m/z): (M + H)+,
calcd for C32H40FN7O2: 573.3228, found, 574.3312.
4.2 Molecular docking study
All the molecular docking simulation were carried out by the AutoDock 4.2 software (The Scripps Research
Institute, USA).
The docking tutorial we used and the detailed AutoDock basic operation methods can be found at:
http://autodock.scripps.edu/faqs-help/tutorial. The protein preparation process of flexible docking mainly includes fixing
the exact residues, adding hydrogen atoms, removing irrelevant water molecules and adding charges, etc. The crystal
structure (PDB: 4ZAU, http://www.pdb.org/) of WT-EGFR bind to inhibitor 6 and the crystal structure (PDB: 3IKA,
http://www.pdb.org/) of T790M-EGFR bind to inhibitor 4 were used in the docking studies. We first removed inhibitors 4
and 6 from their crystal structure, then put target compounds in the binding site. Finally, we used genetic algorithm to
optimize energy. Only the best-scoring ligand-protein complexe was used for the binding site analysis. All the docking
results were processed and modified in PyMOL 1.8.x software (https://pymol.org).
4.3 In vitro enzymatic activity assay
All compounds we designed and synthesized were tested for their activity against EGFRWT and EGFRT790M/L858R by
mobility shift assay with ATP concentration at Km. Compounds were diluted with 100% DMSO to require maximum
inhibitory concentration, and then transfer 100 µL of the compound dilution to a well in a 96-well plate. For all
compounds, they were tested from 1µM, 3-fold dilution for 5 points. The kinase reaction contained 0.04 ng/µL EGFRWT
or 0.02 ng/µL EGFRT790M/L858R, 0.5 µM substrate-biotin, 10 µM ATP, 1 mM DTT, 1 mM MnCl2 and 5 mM MgCl2. Kinase
react and stop incubate at 28ºC for specified period of time. Add 25 µL stop buffer to stop reaction, then collect data on
Caliper and copy conversion data from Caliper program. Percent inhibition = (max-conversion)/(max-min) ×100.“max”
stands for DMSO control; “min” stands for low control.
4.4 In vitro anti-proliferative activity assay
H1975, HepG2 and A549 cell were obtained from the Cell Cultures Collection of Chinese Academy of Sciences
(Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences). We tested the anti-proliferative effect of all

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compounds against above cancer cell lines by the standard MTT assay in vitro, with EGFR inhibitor AZD9291 as
positive control. All cells were maintained and propagated as monolayer at 37 ºC for 24 h in 5% CO2 incubator in
DMEM or RPMI1640 containing 10% fetalbovine serum. A certain concentration of test compounds were added to the
culture medium for further 72 hours and fresh MTT reagent (Thiazolyl blue tetrazolium bromide) was added to each well
at a concentration of 5 µg/mL and incubated with cells for 4 hours at 37 ºC. The formazan crystals were dissolved in 100
µL DMSO each well. The absorbency at 492 nm (for absorbance of MTT formazan) and 630 nm (for the reference
wavelength) was measured with the ELISA reader. All of compounds were tested three times in each of cell lines. The
results expressed as inhibition rates or IC50 (half-maximal inhibitory concentration) were the averages of two
determinations and calculated by using the Bacus Laboratories Incorporated Slide Scanner (Bliss) software.
4.5 Cell cycle progression assay
A549 cells were seeded in a 6-well plate at 1×105 cells per well and incubated for 24 h. Then cells were treated with
vehicle (0.1% DMSO) or different concentrations of 18e for 24 h. Cells were harvested and fixed with ice-cold 70%
ethanol at 4ºC for 12 h. Ethanol was removed and the cells were washed with cold PBS. Then cells were incubated in 0.5
mL of PBS containing 1 mg/mL Rnase for 30 min at 37ºC. Then the cells were stained with propidium iodide (PI) in the
dark for 30 min. The DNA contents was then measured by flow cytometer.
5. Acknowledgements
We gratefully acknowledge the generous support provided by The National Natural Science Funds of China (No.
21662014), Jiangxi Outstanding Youth Talent Support Program (20171BCB23078), Natural Science Foundation of
Jiangxi, China (20171ACB21052, 20181BBG70003, 20181ACB20025), Jiangxi Science and Technology Normal
University Innovative Research Team (2017CXTD002).
6. References
1. Song Z, Ge Y, Wang C, et al. Challenges and perspectives on the development of small-molecule EGFR inhibitors
against T790M-mediated resistance in non-small-cell lung cancer[J]. Journal of Medicinal Chemistry,
2016,59(14):6580-6594
2. Gschwind A, Fischer OM, Ullrich A. et al. The discovery of receptor tyrosine kinases: targets for cancer therapy[J].
Nature Reviews Cancer, 2004, 4(5):361-370.
3. Chico L K, Van Eldik L J, Watterson D M. et al. Targeting protein kinases in central nervous system disorders[J].
Nature Reviews Drug Discovery, 2009, 8(11):892-909.
4. Torre L A, Bray F, Siegel R L, et al. Global cancer statistics, 2012[J]. Ca A Cancer Journal for Clinicians, 2015,
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Table of Contents
Based on the structure-activity relationship of AZD9291, three series of AZ9291 derivatives bearing
Pyrimidine phenyl acrylamide structure were designed, synthesized. Moreover, antitumour activity,
enzyme-based selectivity, AO staining, H33342 staining, cell cycle progression and docking study were
carried out.

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Research Highlights
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Three series of potent and selective EGFRL858R/T790M inhibitors were designed and characterized.
Most of the synthesized compounds showed moderate to significant antitumor activity.
The selectivity of 18e against wild-type EGFR was near to 200-fold. 18e arrest efficiently the cell cycle
progression in G2/ M phase of A549 cells.
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Docking study was investigated to explore the binding modes of compounds with EGFRT790M
.