Design, synthesis and antitumor activity of novel thiophene-pyrimidine derivatives as EGFR inhibitors overcoming T790M and L858R/T790M mutations

Article abstract of DOI:10.1016/j.ejmech.2020.112511

Five series of novel thiophene-pyrimidine derivatives (9a-h, 10a-f, 11a-f, 12a-f, 13a-f) have been synthesized and tested for their anti-proliferative activity against several cancer cell lines in which EGF is highly expressed. Most of the target compounds showed excellent activity against one or more cancer cell lines. The most promising compound 13a, of which IC₅₀ values on of cell lines A549 and A431 (4.34 ± 0.60 μM and 3.79 ± 0.57 μM) were similar to the lead compound Olmutinib, showed strong activity and selectivity to EGFR^T790M and EGFR^T790M/L858R. Inhibition data of human normal hepatoma cell line LO2 indicated that most target compounds were less toxic to normal cells and had selective inhibitory effects on cancer cells. In addition, the structure-activity relationship was analyzed and the mechanism of apoptosis induced by the 13a was studied. The results showed that compound 13a induced late apoptosis of A431 cancer cells in a dose-dependent manner.

Full text of DOI:10.1016/j.ejmech.2020.112511

Journal Pre-proof
Design, synthesis and antitumor activity of novel thiophene-pyrimidine derivatives as
EGFR inhibitors overcoming T790M and L858R/T790M mutations
Zhen Xiao, Zhihui Zhou, Cilong Chu, Qian Zhang, Lingjia Zhou, Zunhua Yang, Xin Li,
Liying Yu, Pengwu Zheng, Shan Xu, Wufu Zhu
PII:
S0223-5234(20)30483-9
DOI:
https://doi.org/10.1016/j.ejmech.2020.112511
EJMECH 112511
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 September 2019
Revised Date: 1 February 2020
Accepted Date: 22 May 2020
Please cite this article as: Z. Xiao, Z. Zhou, C. Chu, Q. Zhang, L. Zhou, Z. Yang, X. Li, L. Yu, P. Zheng,
S. Xu, W. Zhu, Design, synthesis and antitumor activity of novel thiophene-pyrimidine derivatives as
EGFR inhibitors overcoming T790M and L858R/T790M mutations, European Journal of Medicinal
Chemistry, https://doi.org/10.1016/j.ejmech.2020.112511.
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Graphical Abstract for review
A series of olmutinib derivative,with the most promising compound 13a showing IC50
of 3.79 µM against A431, were designed and proved to be potent EGFR inhibitors
overcoming T790M and T790M/L858R mutations..

Design, synthesis and antitumor activity of novel thiophene-pyrimidine derivatives as EGFR inhibitors
overcoming T790M and L858R/T790M mutations
Zhen Xiao a,1, Zhihui Zhou a,1, Cilong Chu a, Qian Zhang a, Lingjia Zhou a, Zunhua Yang b, Xin Li a, Liying Yu 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 College of Pharmacy, Jiangxi University of Traditional Chinese Medicine, Nanchang, 330004, China
* Corresponding author.
Tel. & Fax: +86 791 8380-2393;
E-mail: shanxu9891@126.com (S. Xu), zhuwufu-1122@163.com (W. Zhu)
Abstract：
Five series of novel thiophene-pyrimidine derivatives (9a-h, 10a-f, 11a-f, 12a-f, 13a-f) have been synthesized and
tested for their anti-proliferative activity against several cancer cell lines in which EGF is highly expressed. Most of the
target compounds showed excellent activity against one or more cancer cell lines. The most promising compound 13a, of
which IC50 values on of cell lines A549 and A431 (4.34 ± 0.60 μM and 3.79 ± 0.57 μM) were similar to the lead
compound Olmutinib, showed strong activity and selectivity to EGFRT790M and EGFRT790M/L858R. Inhibition data of
human normal hepatoma cell line LO2 indicated that most target compounds were less toxic to normal cells and had
selective inhibitory effects on cancer cells. In addition, the structure-activity relationship was analyzed and the
mechanism of apoptosis induced by the 13a was studied. The results showed that compound 13a induced late apoptosis
of A431 cancer cells in a dose-dependent manner.
Keywords: Thiophene-pyrimidine; anti-proliferation; EGFR inhibitor; Synthesis
Corresponding author. Tel./fax: +86 791 83802393.
1 These authors contribute equally to this work.
E-mail address: shanxu9891@126.com (S. Xu), zhuwufu-1122@163.com (W. Zhu)

1. Introduction
Lung cancer has been the most common cancer disease in the world. Non-small cell lung cancer (NSCLC) accounts
for about 80% of lung cancer, and the 5-year survival rate is less than 15% [1]. With the development of molecular biology,
researchers have found that EGFR (Epidermal Growth Factor Receptor) is highly expressed in 20-80% of NSCLC
patients [2]. EGFR belongs to the receptor tyrosine kinase family, which is mainly located in the cytoplasmic membrane.
Activated by EGF and other ligands, it leads to the formation of tyrosine kinase activity in the intracellular domain
through dimerization. After activation, it conducts auto-phosphorylation, which will further activate the downstream cell
signal transduction pathway and complete the transmembrane signal transduction process [3]. The development of EGFR
inhibitors to block this process is expected to effectively inhibit the development of NSCLC. The non-conservation of
amino acids leads to EGFR mutations, such as the point mutations L858R on exon 21 and exon 19 deletion, which lead to
the activation of downstream pathway of EGFR and uncontrolled cell growth, leading to the occurrence of tumor [4]
.
The first generation of EGFR TKIs (Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors) were developed
to solve the above-mentioned mutation problems, as shown in Figure 1. Currently, the first generation on the market are
mainly Gefitinib (Iressa), Erlotinib (Tarceva) and Icotinib (Conmana), which have significant clinical response rates in
NSCLC patients. However, acquired resistance T790M and T790M/L858R occurred 9-14 months after clinical treatment,
deactivating the first-generation inhibitors [5-7]. The mutations increase the affinity of ATP for EGFR, thus increasing the
competition between ATP and reversible EGFR TKIs and restoring enzyme activity at physiologically achievable EGFR
TKIs concentrations [8]. The second generation of EGFR TKIs mainly includes Afatinib (Xovoltib) and Dacomitinib
(Vizimpro), as shown in Figure 1. These drugs contain electrophilic acrylamide that reacts irreversibly with cysteine
CYS797 to form covalent complexes, thus, overcoming resistance mediated by EGFRT790M or EGFRT790M/L858R mutation.
However, acrylamide in most drugs is so reactive that it binds to cysteine in untargeted proteins, creating toxic side
effects. The narrow therapeutic window limits its clinical application [9]. In order to solve the problem of targeted toxicity,
several third-generation inhibitors have been developed, such as WZ4002 [10], AZD9291 (Tagrisso) [11], Olmutinib (Olita)
[12], etc., as shown in Figure 1. These inhibitors not only have good anti-tumor activity, but also have good selectivity to
EGFRT790M and EGFRT790M/L858R kinase. In 2015, the US FDA approved AZD9291 to treat patients with EGFRT790M
mutations who have been treated with second-generation drugs. Another T790M selective EGFR inhibitor Olmutinib
(HM61713, BI1482694), was developed by Hanmi Pharmaceuticals and Boehringer Ingelheim. Olmutinib received
breakthrough treatment designation in the US in December 2015, and was approved for using in South Korea in May

2016 for locally advanced or metastatic EGFRT790M positive NSCLC patients. But Olmutinib is not available in more
countries because of its toxic side effects [13], such as palmoplantar keratoderma and diarrhea. Based on which, this
project analyzes and studies the molecular docking modes of Olmutinib and summarizes the structure-activity
relationship of similar drugs to optimize its structure, hoping to obtain a batch of compounds that could overcome its
toxic side effects.
Figure 1. Structures of First, Second and Third generation EGFR TKIs.
There are no reports about the co-crystalline of Olmutinib with protein in the RCSB Protein Data Bank, so we chose
EGFRT790M protein (code: 3IKA) and EGFRWT protein (code: 4ZAU) to dock with Olmutinib (Figure 2). After docking
Olmutinib with EGFRT790M protein (Figure 2A) and EGFRWT protein (Figure 2B), the following three points were found,
which were similar to the other third generation inhibitors [14]: (1) Aminopyrimidine can form two-dentate hydrogen
bonds with the amino acid residue MET793 in the hinged region; (2) Compounds bind to proteins in the form of "U".
Side chains containing aniline all point to the solvent region, while the end containing Michael receptor extends into the
back pocket (Figure 2C); (3) Olmutinib binds more tightly to EGFRT790M, and its hydrogen bond is shorter than EGFRWT
.
This may be because the T790M mutant methionine has a larger molecular structure than threonine, causing the
compound to move towards the solvent region, leading to a shorter hydrogen bond distance and the formation of
hydrogen bond between the acrylamide warhead and the nearby amino acid residue CYS797. Figure 2D shows the whole
binding site of Olmutinib in EGFRT790M protein. Based on the docking results of Olmutinib and most docking results of
the third generation EGFR TKIs, we made a spatial orientation map of Olmutinib in EGFRT790M protein (Figure 2C), and
modified the compounds on this basis.

Figure 2. Binding site of Olmutinib. (A) The binding model of Olmutinib bound to T790M-EGFR (PDB
code:3IKA). (B) The binding model of Olmutinib bound to EGFRWT (PDB code:4ZAU). (C) The spatial orientation of
Olmutinib in EGFRT790M. (D) Overview of the binding site of Olmutinib with EGFRT790M
.
[15]
[16]
On the one hand, Pfizer's Cheng H
and Planken S
teams have reported the development process of the third
generation EGFR inhibitors PF-06459988 and PF-06747775 (shown in Figure 1), whose structures are very similar to
Olmutinib's. In their reports, the original compound containing phenylpiperazine had poor permeability. Replacing the
phenylpiperazine with N1-methylpyrazole group could improve ligand efficiency to a certain extent. This point was used
for reference in our design strategy. By changing the length of the side chain and the electrophilicity of the group in the
deep solvent region, we explored which type of side chain was effective for the activity of the compounds. On the other
hand, according to previous studies [17-19], although covalent inhibitors have advantages over their reversible, noncovalent
conjugated counterparts, such as improving biochemical efficiency, prolonging the action time, and avoiding the potential
of some drug-resistant mechanisms, when covalent inhibitors are highly reactive and / or lack of specificity, covalent
modifications in proteins may participate in immunotoxicity and specific hypersensitivity. In previously published
literature [20-22], the introduction of small substituents at the end of the acrylamide side chain can change the activity of the
reaction between the acrylamide warhead and the amino acid residue CYS797 in the back pocket. Therefore, we

introduced side chains of different lengths at the end of acrylamide to investigate the effect of side chain length and
whether the side chain contains branched chains on the activity of the compounds. The design strategy is shown in Figure
3. However, in this study, the activity of compounds containing N1-methylpyrazole group was decreased, possibly
because the chain length was too short to allow the compounds’ side chain to penetrate into the solvent zone well.
Inspired by WZ4002, we introduced a side chain containing methoxide and planned to extend the side chain
appropriately on the basis of N1-methylpyrazole group, so the fennel amine structure was adopted. The activity was
significantly increased as expected. We also explored the effect of methoxy’s position in anisidine and whether there
being a nitrogen in the benzene ring on the activity of the compounds. After discovering that the methoxy group in
anisidine has the best activity at the 3 positions of the benzene ring, we considered to introduce the electron-withdrawing
group, namely the cyano group, at the 3 positions of the benzene ring to explore the changing rules of the activity of the
compound. Finally, compounds 9a-h, 10a-f, 11a-f, 12a-f, 13a-f were obtained.
Figure 3. Design strategy of target compounds.
2. Results and discussion
2.1. Chemistry
The synthetic route of the target compounds 9a-h, 10a-f, 11a-f, 12a-f, 13a-f was shown in Scheme 1. Taking
commercially available methyl 3-aminothiophene-2-carboxylate (1) as starting material, thieno[3,2-d]pyrimidine-2,4-diol
(2) was synthesized by mechanical stirring with urea at 120 °C for 4 h. 2 was treated by POCl3 via chlorination reaction
with a few drops of DMF as catalyst to yield 2,4-dichlorothieno[3,2-d]pyrimidine (3). 3 and m-Nitrophenol were added
to 1,4-dioxane, and caesium carbonate was used as catalyst to obtain 2-chloro-4-(3-nitrophenoxy)thieno[3,2-d]pyrimidine

(4) by nucleophilic substitution reaction. Through dissolving 4 in acetonitrile and adding TsOH and side chain amine
(5a-e) in turn, the key intermediates 6a-e were obtained. 6a-e were reduced to obtain 7a-e by hydrazine hydrate. Finally,
7a-e were reacted with corresponding acid chlorides to yield target compounds 9a-h, 10a-f, 11a-f, 12a-f, 13a-f.
Scheme 1. Synthetic route of the target compounds.
Reagents and conditions: (a) urea, N2, 120 , 4 h; (b) POCl3, DMF, 120 , 3 h; (c) m-Nitrophenol, 1,4-dioxane,
CS2CO3, r.t., 4 h; (d)TsOH, acetonitrile, 100 , 3 h; (e) FeCl3.6H2O, activated carbon1, hydrazine, ethanol4-dioxane,
80 , 4 h; (f) DIPEA, DCM, 0 , 0.5-2 h.
2.2. Biological evaluation
2.2.1 In vitro antiproliferative activity against cancer cell lines of compounds 9a-h, 10a-f, 11a-f, 12a-f, 13a-f
The antiproliferative activity test of the target compounds 9a-h were carried out in parallel against four human
cancer cell lines A549 (human lung cancer cell), A431 (epidermal squamous cell), Hela (cervical cancer cell), MCF-7
(breast cancer cell) and LO2 (human normal liver cell) by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) method, compound Olmutinib being the reference. In addition, we calculated the partition coefficient LogD7.4
of all the target compounds. Results were summarized in Table 1 and the values were the average of at least three
independent experiments. There was a tendency that the activity of the compounds decreased with the increased of the
chain length of the side chain R1. 9f with hexenamide group on R1 got lower IC50 values of A431 and Hela cancer cells
than 9a that contained acrylamide group on R1. In addition, the antiproliferative activities of these compounds against
A431 and A549 were generally better than those against MCF-7 and Hela. The high level of EGFR expression in A431
and A549 cells indicated that the compounds mainly target the EGFR signaling pathway, which was in line with the
expected effect. It was obvious that compound 9g containing fluorine atom was almost inactive. The activity of all
compounds became worse when R2 was 5a, which might be caused by the fact that the chain of 5a was too short to
extend well into the solvent region.
Table 1
Structures and activity of target compounds 9a-h.

IC50(μM)a
Hela
Compd.
R1
LogD7.4c
A549
A431
MCF-7
9a
9b
9c
9d
26.51 ± 1.49 13.86 ± 1.81 17.33 ± 1.29
29.53± 1.92 33.31 ± 1.99 43.46 ± 1.12
31.87 ± 1.16 38.91 ± 1.76 39.62 ± 1.37
37.64± 1.14
4.51
4.89
5.14
5.34
>50
43.87± 1.26
>50
25.61± 1.39
36.23± 1.99
49.41± 2.22
9e
9f
29.94± 1.07 46.14 ± 1.21 48.84 ± 1.37
>50
39.13± 2.96
>50
5.62
5.78
4.43
26.34± 2.01
>50
23.39± 1.99
>50
32.17± 2.43
>50
9g
9h
>50
>50
>50
>50
4.90
Olmutinib b
AZD9291 b
-
-
4.29 ± 0.21
5.29 ± 0.59
9.27 ± 0.76
6.90 ± 0.93
4.81
3.00
0.665 ± 0.08 0.771 ± 0.11 1.827 ± 0.14
3.665 ± 0.21
aThe values are an average of three separate determinations.
bUsed as the positive control.
cLogD7.4 values were calculated by the chemaxon website (http://www.chemaxon.com).
Table 1 showed that when the side chain is 5a, the activity is generally poor, so we looked for new directions to
solve the problem. At the same time, we abandoned the introduction of fluorine atom because the activity of the
introduction of fluorine was worse. Inspired by WZ4002 and AZD9291, we introduced methoxy group into the side chain
and replaced N1-methylpyrazole group with anisidine structure, which also lengthened the side chain length in the
solvent region appropriately, and explored the effect of the methoxy group location of anisidine on the activity of the
compound. Most target compounds were less toxic to normal cells and had selective inhibitory effects on cancer cells, as
shown in Table 2. Compounds 10a-f, 11a-f and 12a-f exhibited better antiproliferative activity than 9a-h. The IC50 of
compound 11a to A549 and A431 was the same as that of the positive compound Olmutinib, which was 4.29 ± 0.21 μM

and 4.79 ± 0.47 μM, respectively. The activity of Olmutinib to the two cells was 4.29 ± 0.21 μM and 5.29 ± 0.59 μM,
respectively. The antitumor activity of AZD9291 was generally higher than that of Olmutinib and target compounds. The
introduction of flexible chains of different lengths at R1 had little effect on the activity, such as compounds 10a and 10d,
12a and 12b. Changing the chain length in a small range had little effect on the activity of compounds, but the effect of
changing the chain length in a large range on the activity needs further confirmation. If there were branched chains in
flexible chains, the activity of compounds would be reduced, such as compounds 10d and 10e. The IC50 values against
A549, A431 and Hela cell lines of 10d contained pentenamide group were 16.09 ± 1.41, 15.09 ± 1.20 and 36.13 ± 1.57
μM, respectively, which were significantly higher than those of 10e with 4-methylpent-2-enamide group on R1. The IC50
values of the latter against the three cell lines were 37.93 ± 1.45, 25.18 ± 1.02, 45.06 ± 1.12 μM, respectively. The same
phenomenon could be found by comparing the activity of 11b and 11c. This may be due to the fact that R1 with branched
chain does not have an effective group which can form hydrogen bond, and occupied a large space structure, which made
it difficult to interact with the surrounding amino acid residues. In addition, the activity of 11a-f was general higher than
that of 10a-f and 12a-f. It can be concluded that when the methoxy group was in metaposition, the activity would
generally increase, which might be due to the enhancement of the conjugation of side-chain benzene rings. In order to
investigate the effect of electrophilicity and repulsion of phenyl ring meta-group on its activity, a series of compounds
13a-f have been synthesized by replacing methoxy group (electron-donating group) with cyano group (electron-accepting
group), and the activity is shown in Table 3. The results showed that the introduction of electron-withdrawing can slightly
improve the activity on the whole. In addition, 13a-f showed lower toxic effect on normal cell line LO2 than Olmutinib,
which was in line with our expectation. Among them, the most promising compound 13a showed superior activity
against A549, A431 and Hela, with the IC50 values of 4.34 ± 0.60 μM, 3.79 ± 0.57 μM and 6.39 ± 0.94 μM, which was
almost equal to that of the lead compound Olmutinib. Moreover, according to the relationship of compounds’ acrylamide
side chain, activity and LogD7.4, it can be seen that the longer the flexible chain of acrylamide is, the worse the general
activity is, and the higher the LogD7.4 value is, such as 11a and 11d, 12a and 12e. The compounds with higher LogD7.4
value have poor solubility. Thus, the inhibition activity decreased greatly.
Table 2
Structures and activity of target compounds 10a-f, 11a-f and 12a-f.

IC50(μM)a
Compd.
R1
LogD7.4c
A549
A431
Hela
MCF-7
LO2
10a
10b
10c
10d
8.61 ± 1.43
9.30 ± 1.43
26.09 ± 1.41
15.99 ± 1.20
>50
5.36
5.75
5.99
6.19
13.71 ± 1.02 11.75 ± 1.07 45.11 ± 2.05
18.09 ± 1.51 17.62 ± 0.98 28.75 ± 1.41
16.09 ± 1.41 15.09 ± 1.20 36.13 ± 1.57
>50
46.12 ± 1.19
>50
>50
48.87 ± 1.49
>50
10e
37.93 ± 1.45 25.18 ± 1.02 45.06 ± 1.12
>50
Nd
6.48
10f
11a
11b
11c
11d
5.21 ± 0.71
4.34 ± 0.50
4.22 ± 0.62
4.79 ± 0.47
31.44 ± 1.61
16.39 ± 0.98
35.01 ± 1.51
18.99 ± 1.31
14.56± 1.19
35.83 ± 1.64
43.44± 1.47
>50
30.28 ± 1.31
46.04 ± 1.62
Nd
6.64
5.36
5.75
5.99
6.19
10.48 ± 1.17 13.09 ± 1.17 20.06 ± 1.39
27.69 ± 1.24 25.79 ± 1.38 26.09 ± 1.41
19.61± 1.82
37.10± 1.91
5.56± 0.81
29.23± 1.40
23.38± 1.49
8.09 ± 0.71
29.41± 1.29
27.04± 1.63
7.73± 1.01
>50
11e
43.85± 1.29
Nd
6.48
11f
12a
12b
12c
12d
5.02± 0.66
>50
>50
>50
6.64
4.74
5.12
5.37
5.57
20.39 ± 1.76 27.81 ± 1.46 47.13 ± 1.67
31.87 ± 1.78 37.17 ± 1.80
>50
>100
>50
>50
40.12 ± 1.94
>50
>100
>50
36.93 ± 1.44
>50
25.77 ± 1.29 26.21± 1.66
49.91± 2.82
12e
12f
>50
>50
>50
>50
>50
Nd
5.86
6.01
21.54± 1.47
4.29 ± 0.21
30.34± 1.09
5.29 ± 0.59
>50
41.65 ± 1.49
Olmutinib b
AZD9291 b
-
-
9.27 ± 0.76
6.90 ± 0.93
25.48± 1.31
>50
4.81
3.00
0.665 ± 0.08 0.771 ± 0.11 1.827 ± 0.14
3.665 ± 0.21
aThe values are an average of three separate determinations.

bUsed as the positive control.
cLogD7.4 values were calculated by the chemaxon website (http://www.chemaxon.com).
Nd: Not done.
Table 3
Structures and activity of target compounds 13a-f.
IC50(μM)a
Compd.
R1
LogD7.4c
A549
A431
Hela
MCF-7
LO2
13a
13b
13c
13d
4.34 ± 0.60
3.79 ± 0.57
6.39 ± 0.94
18.99 ± 1.71
>50
5.38
5.76
6.01
6.21
11.34 ± 0.94 20.09 ± 1.54 23.61 ± 1.77 33.10 ± 0.98 47.85± 1.67
16.89 ± 1.15 28.28 ± 1.72 19.83 ± 1.81 41.05 ± 1.84
>50
>50
14.90± 1.07
19.01± 1.34
36.23± 1.39
24.12± 1.28
69.41± 2.12
36.49± 1.55
>50
>50
13e
>50
>50
6.49
6.65
13f
16.72± 1.51
4.29 ± 0.21
20.39± 1.94
5.29 ± 0.59
42.11± 2.05
9.27 ± 0.76
49.13± 2.13
6.90 ± 0.93
Olmutinib b
AZD9291 b
-
-
25.48± 1.31
>50
4.81
3.00
0.665 ± 0.08 0.771 ± 0.11 1.827 ± 0.14 3.665 ± 0.21
aThe values are an average of three separate determinations.
bUsed as the positive control.
cLogD7.4 values were calculated by the chemaxon website (http://www.chemaxon.com).
2.2.2 In vitro H1975 cell viability of partially preferred compounds
In order to investigate target compounds’ inhibition potency against dual-mutation EGFRT790M/L858R, an experiment
against cell line H1975 (EGFRT790M/L858R) was carried out with five preferred compounds (10a, 10f, 11a. 11f, 13a) being
the test objects. The result is showed in Table 4. The activity of AZD9291 was higher than Olmutinib and target
compounds, which was consistent with data of Table 2-3. Most compounds had greater inhibition than Olmutinib. It is
interesting that anisodine compounds 10a and 11a exhibited lower IC50 than 13a that containing cyano group. The result

suggests that the anisodine group could improve the inhibition to EGFRT790M/L858R, which gave us a new way to explore
dual-mutation inhibitors, and we would expand on this in the next phase of our project. Although the activity of 13a with
IC50 of 699.2 nM against H1975 was lower than AZD9291, it was almost equal to Olmutinib’, which indicated that 13a
had the potential to inhibit dual-mutation T790M/L858R.
Table 4
Activity of partially preferred compounds against cancer cell H1975.
% Inhibitiona
Compd.
IC50 (nM)a
10 μM
1 μM
83.81
94.26
86.80
14.60
79.52
74.57
-
59.10
3.25
50.61
-0.18
44.31
6.08
-
206.6
2139.0
418.0
Nd
699.2
520.5
73.4
10a
10f
11a
11f
13a
Olmutinib b
AZD9291 b
a The values are an average of three separate determinations.;
b Used as a positive control;
Nd: Not done.
2.2.3 Kinase inhibitory activity of compounds 10f, 11a, 11f, 13a
Compounds 10f, 11a, 11f, 13a with excellent antiproliferative activity were further screened out for kinase
inhibition test. The results exhibited that compounds 11a and 13a got IC50 values of nanomolar level, and had strong
EGFRT790M and EGFRT790M/ L858R activity and selectivity. The results are shown in Table 5. The mutation selectivity of
11a was 13 times higher than that of EGFRWT, and that of 13a was 15 times higher. Besides, compounds 11a and 13a
exhibited an extremely strong inhibitory activity to EGFRT790M/L858R with the inhibition rate of over 100%. However, 10f
and 11f showed poor kinase activity, and their inhibition activity to H1975 with double mutation EGFRT790M/ L858R were
also poor, both of which were compounds with long alkyl side chains at R1. Perhaps these compounds have an
antiproliferative effect on cells by inhibiting other pathways rather than EGFR pathway.
In order to examine whether compounds 11a and 13a were selective EGFR inhibitors, they were also evaluated
against other tyrosine kinases. The results were shown in Figure 4 with clear bar charts. Compounds 11a and 13a had
little activity on c-Met, KDR, and mTOR, and the inhibition rate was between - 0.2% and 9.7%. However, the
inhibition rate of PI3K was about 31.5%. It suggested that the compounds 11a and 13a had slight inhibition on
PI3K, but it is far lower than EGFRT790M and EGFRT790M/L858R. The promising compounds 11a and 13a could
inhibit EGFRT790M kinase by 94.8% and 95.9% at the concentration of 1µM, and 103.6% at the same concentration when
inhibited EGFRT790M/L858R kinase. These data demonstrated that compounds 11a and 13a were selective EGFR inhibitors
overcoming T790M and T790M/L858R mutations.

Table 5
Activity of partially preferred compounds against EGFRT790M/L858R, EGFRT790M, EGFRWT, PI3K, mTOR, c-met and KDR.
Compd.
10f
11a
11f
13a
Olmutinibb
EGFRT790M
21.7
Nd
7.4
Nd
Nd
Nd
Nd
94.8
103.6
24.0
31.6
9.7
24.5
Nd
4.0
Nd
Nd
Nd
Nd
95.9
103.6
23.5
31.5
9.3
98.1
Nd
EGFRT790M/L858R
EGFRWT
PI3K
20.3
Nd
% Inhibition
(1µM)a
Nd
mTOR
0.5
6.0
7.1
Nd
c-Met
KDR
-0.2
1.1
EGFRT790M
EGFRT790M/L858R
EGFRWT
>1000 76.6 >1000 65.0
Nd 15.3 Nd 13.6
>1000 >1000 >1000 >1000
48.0
12.7
>1000
>20
IC50 (nM)a
IC50 (EGFRWT ) / IC50 (EGFRT790M
IC50 (EGFRWT ) / IC50 (EGFRT790M//L858R
)
-
-
>13
>65
-
-
>15
>73
>78
)
aThe values are an average of three separate determinations.
bUsed as a positive control.
Nd: Not done.
Figure 4. Inhibition of several kinases by compounds 11a and 13a at 1 µM
2.2.4 Morphology of cells by fluorescence microscopy of 13a.
Fluorescence staining was performed to verify and evaluate the anti-proliferation and apoptosis effect on A431 cells
of 13a which was screened according to anti - proliferation and kinase activities. The results were shown in Figure 5, the

control group cell (Fig. 5A) was treated with nothing and showed normal A431 cell shape. Compound 13a (Fig. 5B) in a
concentration of 0.33µM significantly inhibited the proliferation of A431 cells compared with the control group. After
treatment with 13a, the number of A431 cells decreased, the fluorescence intensity of A431 cells increased significantly,
the chromatin contracted significantly, and the cell edge was sharp. Therefore, according to the results of acridine orange
staining, we can draw the conclusion that compound 13a could inhibit the proliferation and induce apoptosis of A431
cells at a low concentration, which suggested that 13a was worth further study.
Figure 5. Morphology of cells by fluorescence microscopy of 13a.
2.2.5 Apoptosis result analysis of 13a.
In order to better verify the effect and mechanism of 13a inducing apoptosis, cytofluorimetric analysis of apoptotic
events about 13a was carried out through the exposure of Annexin V marker in the A431 cell line. Figure 6 shows that
treatment with 13a at different concentrations (0.2 μM, 4 μM, 8 μM) for 48 h stimulated late apoptotic in a
dose-dependent manner. In addition, compared with the Olmutinib, which got a percent of 14.78% in the stage of late
apoptotic control drug at a concentration of 5 μM, 13a at concentration of 4 μM exhibited higher percent of 16.41%. The
results were consistent with the previous pharmacological experiments, indicating that the most promising compound 13a
was a potential EGFR inhibitor.

Figure 6. Apoptosis result analysis of 13a. (Q2-1: mechanical damage; Q2-2: late apoptotic and death; Q2-3: living cells;
Q2-4: early apoptotic).
2.3. Docking study
To explore the binding mode of target compound 13a with the active site of EGFRT790M, molecular docking
simulation studies were carried out by the PYMOL software. The results of docking were presented in Figure 7, indicating
that the compound showed U-shape in the kinase domain and combined with EGFR kinase. Aminopyrimidine formed
double hydrogen bonds with amino acid residue MET790 in the hinged region and side chain amine penetrated into the
solvent region, which were similar to Olmutinib. The difference was that the amide structure interacted with ARG841
rather than CYS797. The docking results showed that pyrimidine amino group was indispensable for the formation of
double hydrogen bond with MET793, and the transformation direction of side chain was of certain significance. In
addition, the hydrogen bond distances between Olmutinib and EGFRT790M were 2.7 Å, 2.1 Å, 2.6 Å in turn, while those
between 13a and EGFRT790M were shorter (2.2 Å, 1.9 Å, 2.0 Å in turn), which indicated that 13a was more closely bound
to EGFRT790M
.

Figure 7. Binding site of 13a. (A) The binding model of 13a bound to EGFRT790M (PDB code:3IKA). (B) Overview of
the binding site of 13a with EGFRT790M
.
3. Conclusions
We designed and synthesized five series of thiophene-pyrimidine derivatives in this paper. The most promising
compound showed superior activity against A549, A431 and Hela, with the IC50 values of 4.34 ± 0.60 μM, 3.79 ± 0.57
μM and 6.39 ± 0.94 μM, which was almost equal to that of the lead compound Olmutinib. Especially 13a exhibited
nanomolar level activity against H1975cell line, of which the IC50 value was 699.2nM. The IC50 of most target
compounds to the normal cell line LO2 are more than 50, indicating that they can selectively inhibit cancer cells. In the
experiment of kinase activity, the selected compounds have strong selectivity to EGFR, little inhibition to KDR and
C-Me, and slight activity to PI3K and mTOR downstream of EGFR signal pathway. Especially 11a and 13a can inhibit
EGFRT790M/L858R by 103.6%. Moreover, Annexin V-FITC staining and acridine orange staining tests suggested that 13a
could induce the late apoptosis of A431 by a dose - dependent manner. The whole result showed that compound 13a was
a potential EGFRT790M/L858R inhibitor, which would be further studied in the future in our group.
4. Experimental section
4.1. General Information
Unless otherwise required, all reagents used in the experiment 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 TLC plate and spots were
observed with iodine or light under 254 nm or 365 nm. Tetramethylsilane (TMS) being an internal standard, the
structures of the target compounds were confirmed by 1H NMR and 13C NMR spectra on Bruker 400 MHz spectrometer

(Bruker Bioscience Billerica, MA, USA) at room temperature. Waters High Resolution Quadrupole Time of Flight
Tandem Mass Spectrometry (QTOF) was studied by Mass spectrometry (MS). The purity of the compounds was
determined by Agilent 1260 liquid chromatograph fitted with an Inertex-C18 column. All target compounds have purity
greater than or equal to 95%. The conditions of HPLC are as follows: (1) Chromatographic column：Dikma Luna C18; (2)
Mobile phase: V (0.02 mol / L potassium dihydrogen phosphate (2 mol / L sodium hydroxide solution adjusted to pH
6.0)): V (acetonitrile): 50:50; (3) Column temperature: 30 ℃; (4) Detection wavelength: 240 nm, 270 nm, 254 nm; (5)
Flow rate: 1.0 mL/min.
4.2. Procedure for the Preparation of thieno[3,2-d] pyrimidine-2,4-diol (2)
Methyl 3-aminothiophene-2-carboxylate (1,125.0 g, 159.2 mmol) and urea (48.0 g, 792.1 mmol) were packed in a
500 mL flask. The reaction system was protected by N2 and heated to 120 °C and stirred for 4 h. After the reaction was
completed, the above mixed solution was added to 1000 mL aqueous solution, stirred at room temperature for 0.5 h, and
then a large number of white solids were precipitated. After filtering and drying the solids, 18.4 g of white solid was
obtained, the yield was 68.5%, m.p.138.6-140.8 °C. 1H NMR (400 MHz, DMSO-d6) δ 11.38 (d, J = 5.4 Hz, 2H), 8.04 (d,
J = 5.2 Hz, 1H), 6.90 (d, J = 5.2 Hz, 1H).
4.3. Procedure for the Preparation of 2,4-dichlorothieno[3,2-d] pyrimidine (3)
Compound 2 (18.0 g, 107.0 mmol) was dissolved in 100 mL phosphorus oxychloride. The reaction was catalyzed by
3 mL N, N-dimethylformamide and stirred for 3 h at 120 °C. After the reaction was completed, the reaction mixture was
cooled to room temperature, then slowly added to 500 mL ice water and stirred vigorously, with solid precipitation. Wash
filter cake with distilled water. After filtering and drying the solids, 17.5 g of light grey solid was obtained, the yield was
79.6%, m.p.121.5-129.1 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.70 (d, J = 5.4 Hz, 1H), 7.74 (d, J = 5.4 Hz, 1H).
4.4. Procedure for the Preparation of 2-chloro-4-(3-nitrophenoxy) thieno [3,2-d] pyrimidine (4)
Compound 3 (17.0 g, 82.9 mmol) was dissolved in 120 mL 4-dioxane, then added m-nitrophenol (12.0 g, 86.3
mmol), cesium carbonate (32.0 g, 99.5 mmol) into the solvent and stirred at room temperature for 4 h. A large number of
solids precipitated during the reaction. After the reaction was completed, the reaction liquid was poured into 250 mL
water and stirred for 10 min. After filtering and drying the solids, 25.2 g of white solid was obtained with a yield of
98.7%, m.p.158.3-162.7°C. 1H NMR (400 MHz, DMSO-d6) δ 8.51 (d, J = 5.4 Hz, 1H), 8.28 (s, 1H), 8.16 (d, J = 8.2 Hz,
1H), 7.85 (d, J = 8.2 Hz, 1H), 7.74 (t, J = 8.2 Hz, 1H), 7.59 (d, J = 5.4 Hz, 1H)
4.5. General Procedure for the Preparation of Compounds 6a-e
.
Compound 4 (5 g, 16.2 mmol) was dissolved with 60 mL acetonitrile, then p-toluene sulfonic acid (5.6 g, 32.5 mmol)
and 5a-e (16.2 mmol) were added to the above solution in turn. The reaction was completed after stirring for 4-5 h at

100 °C. After cooling to room temperature, acetonitrile was concentrated by 50% under vacuum and a large amount of
solid precipitated after adding twice the amount of water. The solid was filtered at atmospheric pressure and dried to
obtain key intermediates 6a-e.
4.5.1. N-(1-methyl-1H-pyrazol-3-yl)-4-(3-nitrophenoxy)thieno[3,2-d]pyrimidin-2-amine (6a).
1
A faint yellow solid; Yield: 75.6%; m.p.: 165.2–167.4 °C; H NMR (400 MHz, DMSO-d6) δ 8.37 (s, 1H), 8.26 –
8.20 (m, 2H), 7.95 – 7.88 (m, 2H), 7.81 (dd, J = 8.0, 5.2 Hz, 1H), 7.68 (d, J = 5.4 Hz, 1H), 7.34 (d, J = 5.4 Hz, 2H), 3.65
(s, 3H)
.
4.5.2 N-(4-methoxyphenyl)-4-(3-nitrophenoxy)thieno[3,2-d]pyrimidin-2-amine (6b).
A pale white solid; Yield: 86.1%; m.p.: 173.6–179.1 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.54 (s, 1H), 8.35 (d, J =
5.4 Hz, 1H), 8.32 (s, 1H), 8.23 (d, J = 8.2 Hz, 1H), 7.90 (d, J = 8.2 Hz, 1H), 7.81 (t, J = 8.2 Hz, 1H), 7.50 (d, J = 7.8 Hz,
1H), 7.37 (d, J = 5.4 Hz, 1H), 7.12 (d, J = 7.8 Hz, 1H), 6.71 (d, J = 7.8 Hz, 2H), 3.68 (s, 3H).
4.5.3 N-(3-methoxyphenyl)-4-(3-nitrophenoxy)thieno[3,2-d]pyrimidin-2-amine (6c).
A light yellow solid; Yield: 90.1%; m.p.: 169.5.2–172.7 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.51 (s, 1H), 8.35 (s,
2H), 8.25 (d, J = 8.0 Hz, 1H), 7.90 (d, J = 7.8 Hz, 1H), 7.80 (t, J = 8.1 Hz, 1H), 7.40 (d, J = 5.0 Hz, 1H), 7.23 (s, 1H),
7.08 (d, J = 7.8 Hz, 1H), 7.00 (t, J = 8.0 Hz, 1H), 6.44 (d, J = 8.0 Hz, 1H), 3.62 (s, 3H).
4.5.4 N-(6-methoxypyridin-3-yl)-4-(3-nitrophenoxy)thieno[3,2-d]pyrimidin-2-amine (6d).
A light yellow solid; Yield: 78.6%; m.p.: 159.1–162.5 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.38 (s, 1H), 8.32 (d, J
= 5.6 Hz, 2H), 8.22 (d, J = 8.2 Hz, 1H), 7.89 (d, J = 8.4 Hz, 2H), 7.83 – 7.75 (m, 2H), 7.38 (d, J = 5.4 Hz, 1H), 6.61 (d, J
= 10.0 Hz, 1H), 3.77 (s, 3H).
4.5.5 3-((4-(3-nitrophenoxy)thieno[3,2-d]pyrimidin-2-yl)amino)benzonitrile (6e).
A pale white solid; Yield: 80.7%; m.p.: 176.1–179.3 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.94 (s, 1H), 8.39 (d, J =
5.6 Hz, 1H), 8.35 (s, 1H), 8.23 (d, J = 8.2 Hz, 1H), 8.00 – 7.91 (m, 3H), 7.84 (d, J = 8.2 Hz, 1H), 7.45 (d, J = 5.4 Hz, 1H),
7.36 – 7.31 (m, 1H), 7.28 (d, J = 7.6 Hz, 1H).
4.6. General Procedure for the Preparation of Compounds 7a-e
Compounds 6a-e (13.0 mmol) were dissolved in 60 mL ethanol. Ferric chloride hexahydrate (15.6 mmol) and
activated carbon (91.0 mmol) were added in turn. After heating up to 80 °C, hydrazine hydrate (130.0 mmol) was mixed
with 10 mL ethanol and added to the above solution. The mixture was refluxed and stirred for 4 h. After the reaction, the
solids in the reaction system were removed by filtration. Then wash the cake with 10ml anhydrous ethanol and collect the
filtrate. Most of the solvents were removed by vacuum distillation. Adding saturated sodium bicarbonate aqueous
solution 60 mL and stirring vigorously to precipitate solid. Compounds 7a-e were obtained by filtration and cake drying.

4.7. General Procedure for the Preparation of Target Compounds 9a-h, 10a-f, 11a-f, 12a-f, 13a-f
Compounds 7a-e (1.5 mmol) were dissolved in 30 mL dichloromethane and DIPEA (3.0 mmol) was added into it.
The solution was stirred in ice bath for 5 min, then the diluted amide (8a-h, 3.0 mmol) with equal amount of
dichloromethane was dripped slowly into the above solution. The reaction was completed within 2 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 70:1 to 30:1) as eluents.
4.7.1 N-(3-((2-((1-methyl-1H-pyrazol-3-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)acrylamide (9a).
A faint yellow solid; Yield: 32.6%; m.p.: 197.2–199.4 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.33 (d, J = 26.6 Hz,
1H), 8.38 (d, J = 4.2 Hz, 1H), 7.81 – 7.73 (m, 1H), 7.65 (d, J = 14.4 Hz, 1H), 7.58 (d, J = 8.4 Hz, 1H), 7.47 (dd, J = 16.5,
9.0 Hz, 3H), 7.10 (t, J = 7.3 Hz, 1H), 6.43 (dd, J = 16.6, 9.8 Hz, 1H), 6.26 (d, J = 17.2 Hz, 1H), 6.09 (d, J = 9.4 Hz, 1H),
5.83 (dd, J = 37.2, 10.4 Hz, 1H), 3.76 (s, 3H). TOF MS ES + (m/z): (M + H)+, calcd for C19H16N6O2S: 393.1134, found,
393.1134.
4.7.2 (E)-N-(3-((2-((1-methyl-1H-pyrazol-3-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)but-2-enamide (9b).
A faint yellow solid; Yield: 31.1%; m.p.: 201.2–205.9 °C; 1H NMR 400 MHz, DMSO-d6) δ 9.87 (s, 2H), 8.30 (d, J =
5.4 Hz, 2H), 7.41 (s, 1H), 7.37 (d, J = 5.2 Hz, 2H), 7.32 (s, 1H), 6.86 (s, 3H), 6.12 (d, J = 4.4 Hz, 1H), 3.70 (s, 3H), 3.63
(s, 3H). TOF MS ES + (m/z): (M + H)+, calcd for C20H18N6O2S: 407.1289, found, 407.1290.
4.7.3 3-methyl-N-(3-((2-((1-methyl-1H-pyrazol-3-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)but-2-enamide (9c).
1
A faint yellow solid; Yield: 29.8%; m.p.: 215.5–218.7 °C; H NMR 400 MHz, DMSO-d6) δ 10.03 (s, 1H), 8.36 (s,
1H), 7.50 (d, J = 8.8 Hz, 2H), 7.43 (d, J = 15.6 Hz, 2H), 7.25 (s, 3H), 6.78 (d, J = 18.8 Hz, 1H), 6.10 (s, 1H), 3.72 (d, J =
4.8 Hz, 3H), 2.50 (s, 6H). TOF MS ES + (m/z): (M + H)+, calcd for C21H20N6O2S: 421.1447, found, 421.1451.
4.7.4 (E)-N-(3-((2-((1-methyl-1H-pyrazol-3-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)pent-2-enamide (9d).
1
A faint yellow solid; Yield: 39.0%; m.p.: 223.1–225.4 °C; H NMR 400 MHz, DMSO-d6) δ 10.55 (s, 1H), 9.58 (s,
1H), 7.83 (d, J = 4.6 Hz, 2H), 7.76 (s, 1H), 7.62 (d, J = 7.8 Hz, 1H), 7.46 (d, J = 7.6 Hz, 1H), 7.13 (s, 1H), 7.07 (d, J =
8.0 Hz, 1H), 6.88 – 6.79 (m, 1H), 6.45 (s, 1H), 6.40 (d, J = 7.8 Hz, 1H), 3.67 (s, 3H), 2.19 (dd, J = 13.8, 6.8 Hz, 2H),
1.02 (t, J = 7.6 Hz, 3H). TOF MS ES + (m/z): (M + H)+, calcd for C21H20N6O2S: 421.1447, found, 421.1448.
4.7.5 3-methyl-N-(3-((2-((1-methyl-1H-pyrazol-3-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)but-2-enamide (9e).
1
A faint yellow solid; Yield: 33.1%; m.p.: 231.7–234.3 °C; H NMR 400 MHz, DMSO-d6) δ 10.25 (s, 1H), 9.34 (s,
1H), 7.94 (d, J = 7.2 Hz, 2H), 7.60 – 7.54 (m, 1H), 7.22 (d, J = 4.6 Hz, 3H), 7.01 (d, J = 5.0 Hz, 3H), 6.38 (d, J = 15.0 Hz,
1H), 3.76 (s, 3H), 1.07 (m, 1H), 0.93 (d, J = 7.2 Hz, 6H). TOF MS ES + (m/z): (M + H)+, calcd for C22H22N6O2S:
435.1634, found, 435.1631.
4.7.6 (E)-N-(3-((2-((1-methyl-1H-pyrazol-3-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)hex-2-enamide (9f).

1
A faint yellow solid; Yield: 31.7%; m.p.: 222.9–226.8 °C; H NMR 400 MHz, DMSO-d6) δ 10.98 (s, 1H), 9.64 (s,
1H), 8.27 (d, J = 5.4 Hz, 2H), 7.75 (d, J = 7.2 Hz, 2H), 7.51 – 7.44 (m, 1H), 7.32 (d, J = 4.8 Hz, 3H), 7.16 (d, J = 8.0 Hz,
1H), 5.68 (d, J = 15.4 Hz, 1H), 3.66 (s, 3H), 1.34 – 1.27 (m, 4H), 0.87 (d, J = 7.2 Hz, 3H). TOF MS ES + (m/z): (M +
H)+, calcd for C22H22N6O2S: 435.1634, found, 435.1627.
4.7.7 2-fluoro-N-(3-((2-((1-methyl-1H-pyrazol-3-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)acrylamide (9g).
A faint yellow solid; Yield: 28.4%; m.p.: 209.6–211.4 °C; 1H NMR 400 MHz, DMSO-d6) δ 9.58 (s, 1H), 8.23 (d, J =
5.2 Hz, 1H), 7.35 (s, 1H), 7.29 (d, J = 5.2 Hz, 1H), 7.09 (t, J = 7.8 Hz, 1H), 6.87 (s, 1H), 6.65 (s, 1H), 6.50 (d, J = 8.0 Hz,
1H), 6.48 – 6.38 (m, 2H), 5.31 (s, 2H), 3.67 (s, 3H). TOF MS ES + (m/z): (M + H)+, calcd for C19H15FN6O2S: 411.1039,
found, 411.0941.
4.7.8 N-(3-((2-((1-methyl-1H-pyrazol-3-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)methacrylamide (9h).
1
A faint yellow solid; Yield: 32.5%; m.p.: 217.9–219.1 °C; H NMR 400 MHz, DMSO-d6) δ 9.74 (s, 1H), 8.33 (s,
1H), 7.64 (d, J = 1.8 Hz, 1H), 7.56 (d, J = 7.6 Hz, 1H), 7.43 (t, J = 8.0 Hz, 4H), 7.12 (d, J = 8.0 Hz, 1H), 6.86 (s, 1H),
6.11 (s, 2H), 3.73 (s, 3H), 2.18 (s, 3H). TOF MS ES + (m/z): (M + H)+, calcd for C20H18N6O2S: 407.1426, found,
407.1433.
4.7.9 N-(3-((2-((4-methoxyphenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)acrylamide (10a).
A faint yellow solid; Yield: 33.6%; m.p.: 224.9–226.1 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.35 (s, 1H), 9.28 (s,
1H), 8.26 (d, J = 5.4 Hz, 1H), 7.73 (s, 1H), 7.60 (d, J = 8.2 Hz, 1H), 7.47 (dd, J = 16.4, 8.2 Hz, 3H), 7.33 (d, J = 5.2 Hz,
1H), 7.07 (d, J = 9.4 Hz, 1H), 6.71 (d, J = 8.6 Hz, 2H), 6.44 (dd, J = 16.8, 10.1 Hz, 1H), 6.27 (d, J = 18.4 Hz, 1H), 5.80 –
5.74 (m, 1H), 3.67 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 170.04, 168.86, 168.59, 163.13, 159.19, 157.45, 145.54,
138.93, 136.85, 135.17, 132.57, 128.40, 125.54（2C）, 122.26, 121.85, 118.65（2C）, 118.23, 60.31（2C）, 26.29. TOF
MS ES + (m/z): (M + H)+, calcd for C22H18N4O3S: 419.1178, found, 419.1230.
4.7.10 (E)-N-(3-((2-((4-methoxyphenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)but-2-enamide (10b).
1
A white solid; Yield: 40.1%; m.p.: 202.4–203.7 °C; H NMR 400 MHz, DMSO-d6) δ 10.37 (s, 1H), 9.38 (s, 1H),
8.46 (s, 1H), 8.22 (d, J = 5.0 Hz, 1H), 7.86 (d, J = 10.8 Hz, 1H), 7.74 (s, 1H), 7.54 (d, J = 5.8 Hz, 1H), 7.39 (t, J = 8.4 Hz,
1H), 7.26 (d, J = 5.6 Hz, 1H), 7.04 (d, J = 8.2 Hz, 1H), 6.80 (td, J = 13.6, 6.4 Hz, 2H), 6.61 (d, J = 9.2 Hz, 1H), 6.13 (d, J
= 15.4 Hz, 1H), 3.78 (s, 3H), 1.96 (d, J = 6.8 Hz, 3H). TOF MS ES + (m/z): (M + H)+, calcd for C23H20N4O3S: 433.1378,
found, 433.1386.
4.7.11 N-(3-((2-((4-methoxyphenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)-3-methylbut-2-enamide (10c).
1
A faint yellow solid; Yield: 28.1%; m.p.: 198.7–199.8 °C; H NMR 400 MHz, DMSO-d6) δ 10.05 (s, 1H), 9.29 (s,
1H), 7.71 (s, 1H), 7.51 (s, 4H), 7.43 – 7.38 (m, 1H), 7.33 (s, 1H), 7.00 (d, J = 6.6 Hz, 1H), 6.71 (s, 2H), 5.87 (s, 1H), 3.67
(s, 3H), 2.13 (s, 3H), 1.86 (s, 3H). TOF MS ES + (m/z): (M + H)+, calcd for C24H22N4O3S: 447.1345, found, 447.1339.

4.7.12 (E)-N-(3-((2-((4-methoxyphenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)pent-2-enamide (10d).
1
A faint yellow solid; Yield: 30.9%; m.p.: 189.7–192.1 °C; H NMR 400 MHz, DMSO-d6) δ 10.14 (s, 1H), 9.36 (s,
1H), 8.40 (s, 1H), 8.36 (d, J = 5.2 Hz, 1H), 7.83 (m, J = 8.2 Hz, 2H), 7.55 (s, 1H), 7.46 (d, J = 6.6 Hz, 1H), 7.39 – 7.31
(m, 2H), 7.32 (d, J = 5.6 Hz, 1H), 6.77 (d, J = 9.8 Hz, 1H), 6.36 (d, J = 5.8 Hz, 1H) 5.97 (s, 1H), 3.63 (s, 3H), 2.11 (m,
2H), 1.06 (m, 3H). TOF MS ES + (m/z): (M + H)+, calcd for C24H22N4O3S: 447.1345, found, 447.1343.
4.7.13 (E)-N-(3-((2-((4-methoxyphenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)-4-methylpent-2-enamide (10e).
1
A white solid; Yield: 38.3%; m.p.: 199.7–201.3 °C; H NMR 400 MHz, DMSO-d6) δ 10.08 (s, 1H), 9.62 (s, 1H),
8.31 (d, J = 5.4 Hz, 1H), 7.83 (s, 1H), 7.57 (d, J = 7.8 Hz, 1H), 7.41 (t, J = 7.8 Hz, 2H), 7.21 (dd, J = 16.2, 7.0 Hz,, 2H),
6.98 (d, J = 6.8 Hz, 1H), 6.53 (dd, J = 12.4, 5.8 Hz, 2H), 6.31 (d, J = 7.8 Hz, 1H), 6.11 (d, J = 8.6 Hz, 1H), 3.83 (s, 3H),
2.34 (d, J = 13.8 Hz, 1H), 1.17 (d, J = 6.4 Hz, 6H). TOF MS ES + (m/z): (M + H)+, calcd for C25H24N4O3S: 461.1647,
found, 461.1648.
4.7.14 (E)-N-(3-((2-((4-methoxyphenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)hex-2-enamide (10f).
A faint yellow solid; Yield: 28.7%; m.p.: 240.7–243.5 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.34 (s, 1H), 9.51 (s,
1H), 8.43 (s, 1H), 8.30 (d, J = 5.8 Hz, 1H), 7.83 (d, J = 8.0 Hz, 2H), 7.55 (t, J = 8.0 Hz, 1H), 7.41 – 7.36 (t, J = 5.4 Hz,
2H), 7.14 (dd, J = 7.8, 1.6 Hz, 1H), 6.71 – 6.63 (m, 3H), 6.03 (d, J = 14.8 Hz, 1H), 3.88 (s, 3H), 2.18 – 2.11 (m, 2H), 2.03
– 1.97 (m, 2H), 1.21 (s, 3H). TOF MS ES + (m/z): (M + H)+, calcd for C25H24N4O3S: 461.1647, found, 461.1649.
4.7.15 N-(3-((2-((3-methoxyphenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)acrylamide (11a).
1
A white solid; Yield: 30.1%; m.p.: 215.2–217.1 °C; H NMR (400 MHz, DMSO-d6) δ 10.36 (s, 1H), 9.48 (s, 1H),
8.31 (d, J = 4.4 Hz, 1H), 7.73 (s, 1H), 7.59 (d, J = 8.4 Hz, 1H), 7.48 – 7.42 (m, 1H), 7.40 (d, J = 5.2 Hz, 1H), 7.34 (s, 1H),
7.23 (d, J = 8.2 Hz, 1H), 7.08 (d, J = 8.0 Hz, 1H), 7.02 (t, J = 7.8 Hz, 1H), 6.48 – 6.38 (m, 2H), 6.26 (d, J = 16.6 Hz, 1H),
5.78 (d, J = 10.4 Hz, 1H), 3.63 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 164.98, 163.96, 163.79, 159.84, 158.06,
152.62, 142.07, 140.78, 137.32, 132.02, 130.34, 129.30, 127.66, 123.64, 117.28, 117.05, 113.12, 111.57, 108.37, 106.88,
105.04, 55.16. TOF MS ES + (m/z): (M + H)+, calcd for C22H18N4O3S: 419.1178, found, 419.1195.
4.7.16 (E)-N-(3-((2-((3-methoxyphenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)but-2-enamide (11b).
A faint yellow solid; Yield: 29.9%; m.p.: 222.9.2–226.4 °C; 1H NMR 400 MHz, DMSO-d6) δ 10.27 (s, 1H), 9.46 (s,
1H), 8.30 (s, 1H), 7.85 (s, 1H), 7.68 (d, J = 8.8 Hz, 1H), 7.38 (d, J = 5.2 Hz, 4H), 7.02 (s, 3H), 6.45 (s, 2H), 3.63 (s, 3H),
1.35 – 1.17 (m, 3H). 13C NMR (101 MHz, DMSO-d6) δ 164.12, 159.84, 158.06, 152.61, 151.89, 142.08, 141.10, 139.96,
137.30, 130.23, 129.28, 126.19, 123.89, 123.64, 116.90, 112.95, 112.76, 111.57, 106.90, 105.02, 55.16, 30.80, 17.86.
LC-MS for C23H20N4O3S: m/z=433.1330 [M+H]+. TOF MS ES + (m/z): (M + H)+, calcd for C23H20N4O3S: 433.1334,
found, 433.1330.
4.7.17 N-(3-((2-((3-methoxyphenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)-3-methylbut-2-enamide (11c).

1
A white solid; Yield: 33.6%; m.p.: 198.4–201.7 °C; H NMR 400 MHz, DMSO-d6) δ 10.99 (s, 1H), 9.48 (s, 1H),
8.32 (d, J = 5.3 Hz, 1H), 7.76 (d, J = 9.0 Hz, 2H), 7.53 – 7.45 (m, 2H), 7.40 (d, J = 5.2 Hz, 2H), 7.17 (d, J = 8.8 Hz, 2H),
7.03 (d, J = 8.0 Hz, 1H), 6.45 (d, J = 6.8 Hz, 1H), 3.62 (s, 3H), 2.06 (s, 3H), 1.81 (s, 3H). TOF MS ES + (m/z): (M + H)+,
calcd for C24H22N4O3S: 447.1491, found, 447.1484.
4.7.18 (E)-N-(3-((2-((3-methoxyphenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)pent-2-enamide (11d).
1
A faint yellow solid; Yield: 40.4%; m.p.: 235.2–239.4 °C; H NMR 400 MHz, DMSO-d6) δ 10.01 (s, 1H), 9.43 (s,
1H), 8.22 (d, J = 5.2 Hz, 1H), 7.71 (d, J = 9.0 Hz, 2H), 7.43 – 7.32 (m, 3H), 7.28 (d, J = 5.2 Hz, 2H), 7.11 (d, J = 8.6 Hz,
2H), 6.83 (d, J = 8.0 Hz, 1H), 6.44 (d, J = 6.8 Hz, 1H), 3.66 (s, 3H), 2.02 (dd, J = 13.8, 6.4 Hz, 2H), 1.01 (t, J = 7.6 Hz,
3H). TOF MS ES + (m/z): (M + H)+, calcd for C24H22N4O3S: 447.1491, found, 447.1488.
4.7.19 (E)-N-(3-((2-((3-methoxyphenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)-4-methylpent-2-enamide (11e).
1
A faint yellow solid; Yield: 32.6%; m.p.: 246.7–249.7 °C; H NMR 400 MHz, DMSO-d6) δ 10.40 (s, 1H), 9.47 (s,
1H), 8.31 (d, J = 5.4 Hz, 1H), 7.73 (s, 1H), 7.57 (d, J = 8.2 Hz, 1H), 7.41 – 7.34 (m, 3H), 7.23 (d, J = 6.4 Hz, 1H), 7.04
(d, J = 6.8 Hz, 2H), 6.80 (dd, J = 15.4, 4.8 Hz, 1H), 6.45 (d, J = 7.8 Hz, 1H), 6.10 (d, J = 15.6 Hz, 1H), 3.63 (s, 3H), 2.87
(d, J = 13.8 Hz, 1H), 1.04 (d, J = 6.6 Hz, 6H). TOF MS ES + (m/z): (M + H)+, calcd for C25H24N4O3S: 461.1647, found,
461.1651.
4.7.20 (E)-N-(3-((2-((3-methoxyphenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)hex-2-enamide (11f).
A faint yellow solid; Yield: 37.7%; m.p.: 232.2–238.4 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.18 (s, 1H), 9.47 (s,
1H), 8.32 (s, 1H), 7.71 (s, 1H), 7.55 (d, J = 7.9 Hz, 1H), 7.41 (d, J = 10.1 Hz, 2H), 7.34 (s, 1H), 7.22 (s, 1H), 7.09 – 6.98
(m, 2H), 6.81 (s, 1H), 6.45 (s, 1H), 6.10 (d, J = 15.1 Hz, 1H), 3.62 (s, 3H), 2.18 (d, J = 6.3 Hz, 2H), 1.45 (d, J = 6.7 Hz,
2H), 0.90 (t, J = 7.3 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 165.04, 164.19, 164.05, 159.89, 158.12, 152.64, 145.39,
142.16, 141.17, 137.49, 130.40, 129.41, 124.95, 123.77, 117.05, 116.97, 112.99, 111.59, 108.41, 106.89, 104.97, 55.23,
33.85, 21.45, 14.03. TOF MS ES + (m/z): (M + H)+, calcd for C25H24N4O3S: 461.1647, found, 461.1643.
4.7.21 N-(3-((2-((6-methoxypyridin-3-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)acrylamide (12a).
A faint yellow solid; Yield: 44.4%; m.p.: 185.7–187.4 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 9.38 (s,
1H), 8.39 (s, 1H), 8.28 (d, J = 5.4 Hz, 1H), 7.93 (d, J = 10.6 Hz, 1H), 7.74 (d, J = 7.2 Hz, 1H), 7.57 (d, J = 8.4 Hz, 1H),
7.44 (t, J = 8.2 Hz, 1H), 7.36 (d, J = 5.4 Hz, 1H), 7.07 (dd, J = 8.2, 1.6 Hz, 1H), 6.61 (d, J = 8.4 Hz, 1H), 6.44 (dd, J =
17.0, 10.2 Hz, 1H), 6.26 (dd, J = 17.0, 1.8 Hz, 1H), 5.77 (dd, J = 10.2, 1.8 Hz, 1H), 3.77 (s, 3H). TOF MS ES + (m/z):
(M + H)+, calcd for C21H17N5O3S: 420.1130, found, 420.1123.
4.7.22 (E)-N-(3-((2-((6-methoxypyridin-3-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)but-2-enamide (12b).
1
A white solid; Yield: 42.6%; m.p.: 196.3–199.1 °C; H NMR 400 MHz, DMSO-d6) δ 10.17 (s, 1H), 9.38 (s, 1H),
8.39 (s, 1H), 8.29 (d, J = 5.2 Hz, 1H), 7.93 (d, J = 10.4 Hz, 1H), 7.72 (s, 1H), 7.54 (d, J = 8.0 Hz, 1H), 7.42 (t, J = 8.4 Hz,

1H), 7.36 (d, J = 5.2 Hz, 1H), 7.04 (d, J = 8.0 Hz, 1H), 6.80 (td, J = 13.8, 6.6 Hz, 1H), 6.61 (d, J = 9.2 Hz, 1H), 6.13 (d, J
= 15.4 Hz, 1H), 3.78 (s, 3H), 1.86 (d, J = 6.8 Hz, 3H). TOF MS ES + (m/z): (M + H)+, calcd for C22H19N5O3S: 434.1287,
found, 434.1286.
4.7.23 N-(3-((2-((6-methoxypyridin-3-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)-3-methylbut-2-enamide (12c).
1
A white solid; Yield: 38.5%; m.p.: 207.9–209.5 °C; H NMR 400 MHz, DMSO-d6) δ 10.04 (s, 1H), 9.38 (s, 1H),
8.38 (s, 1H), 8.28 (d, J = 5.4 Hz, 1H), 7.93 (d, J = 8.1 Hz, 1H), 7.72 (s, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.42 – 7.33 (m, 2H),
7.01 (d, J = 7.8 Hz, 1H), 6.61 (d, J = 9.8 Hz, 1H), 5.87 (s, 1H), 3.78 (s, 3H), 2.12 (s, 3H), 1.86 (s, 3H). 13C NMR (101
MHz, DMSO-d6) δ 170.01, 169.89, 168.96, 163.76, 163.12, 157.33, 157.14, 146.17, 142.55, 142.08, 136.74, 136.45,
134.97, 128.48, 124.20, 121.58, 121.48, 117.67, 114.48, 113.04, 58.14, 32.23, 24.75. TOF MS ES + (m/z): (M + H)+,
calcd for C23H21N5O3S: 448.1443, found, 448.2701.
4.7.24 (E)-N-(3-((2-((6-methoxypyridin-3-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)pent-2-enamide (12d).
1
A white solid; Yield: 36.6%; m.p.: 219.2–223.4 °C; H NMR 400 MHz, DMSO-d6) δ 10.14 (s, 1H), 9.38 (s, 1H),
8.37 (s, 1H), 8.26 (d, J = 5.2 Hz, 1H), 7.83 (d, J = 8.2 Hz, 1H), 7.65 (s, 1H), 7.47 (d, J = 6.8 Hz, 1H), 7.33 – 7.24 (m, 2H),
7.31 (d, J = 7.4 Hz, 1H), 6.77 (d, J = 9.8 Hz, 1H), 6.36 (d, J = 5.8 Hz, 1H) 5.97 (s, 1H), 3.73 (s, 3H), 2.09 (m, 2H), 1.01
(m, 3H). TOF MS ES + (m/z): (M + H)+, calcd for C23H21N5O3S: 448.1443, found, 448.1456.
4.7.25 (E)-N-(3-((2-((6-methoxypyridin-3-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)-4-methylpent-2-enamide
(12e).
1
A faint yellow solid; Yield: 32.6%; m.p.: 240.1–242.6 °C; H NMR 400 MHz, DMSO-d6) δ 10.48 (s, 1H), 9.57 (s,
1H), 8.47 (d, J = 5.4 Hz, 1H), 7.82 (s, 1H), 7.57 (d, J = 8.2 Hz, 1H), 7.41 – 7.33 (m, 2H), 7.21 (d, J = 6.4 Hz, 1H), 7.01
(d, J = 6.8 Hz, 2H), 6.72 (dd, J = 12.4, 4.6 Hz, 1H), 6.46 (d, J = 7.4 Hz, 1H), 6.11 (d, J = 8.6 Hz, 1H), 3.73 (s, 3H), 2.57
(d, J = 13.8 Hz, 1H), 1.07 (d, J = 6.6 Hz, 6H). TOF MS ES + (m/z): (M + H)+, calcd for C24H23N5O3S: 462.1600, found,
462.1597.
4.7.26 (E)-N-(3-((2-((6-methoxypyridin-3-yl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)hex-2-enamide (12f).
1
A white solid; Yield: 29.4%; m.p.: 247.2–249.2 °C; H NMR (400 MHz, DMSO-d6) δ 10.29 (s, 1H), 9.39 (s, 1H),
8.39 (s, 1H), 8.29 (d, J = 5.6 Hz, 1H), 8.09 (s, 1H), 7.93 (d, J = 8.0 Hz, 1H), 7.55 (d, J = 8.2 Hz, 1H), 7.40 (d, J = 3.8 Hz,
1H), 7.36 (d, J = 5.4 Hz, 1H), 7.04 (dd, J = 7.8, 1.8 Hz, 1H), 6.84 – 6.74 (m, 2H), 6.13 (d, J = 14.6 Hz, 1H), 3.78 (s, 3H),
2.20 – 2.15 (m, 2H), 2.02 – 1.96 (m, 2H), 1.30 (s, 3H). TOF MS ES + (m/z): (M + H)+, calcd for C24H23N5O3S: 462.1600,
found, 462.1599.
4.7.27 N-(3-((2-((3-cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)acrylamide (13a).
A faint yellow solid; Yield: 37.8%; m.p.: 186.2–188.7 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.71 (s, 1H), 9.91 (s,
1H), 8.35 (d, J = 5.4 Hz, 1H), 8.05 (s, 1H), 7.88 (d, J = 8.0 Hz, 1H), 7.82 (s, 1H), 7.58 (d, J = 7.0 Hz, 1H), 7.45 (dd, J =

14.8, 6.7 Hz, 2H), 7.35 – 7.26 (m, 2H), 7.10 (d, J = 8.0 Hz, 1H), 6.49 (dd, J = 16.8, 10.2 Hz, 1H), 6.24 (d, J = 17.6 Hz,
1H), 5.97 (dd, J = 17.6, 10.0 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 164.77, 164.15, 163.86, 157.51, 152.42, 141.85,
140.94, 137.72, 132.20, 130.30, 129.94, 127.39, 124.78, 123.66, 123.37, 121.09, 119.34, 117.40, 117.22, 113.14, 111.67,
109.09. TOF MS ES + (m/z): (M + H)+, calcd for C22H15N5O2S: 414.1026, found, 414.1024.
4.7.28 (E)-N-(3-((2-((3-cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)but-2-enamide (13b).
A faint yellow solid; Yield: 40.1%; m.p.: 189.6–191.4 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.26 (s, 1H), 9.87 (s,
1H), 8.33 (d, J = 4.5 Hz, 1H), 8.03 (s, 1H), 7.86 (s, 1H), 7.78 (s, 1H), 7.52 (d, J = 11.2 Hz, 1H), 7.42 (d, J = 9.6 Hz, 2H),
7.29 (d, J = 12.5 Hz, 2H), 7.06 (d, J = 6.1 Hz, 1H), 6.78 (s, 1H), 6.13 (d, J = 14.5 Hz, 1H), 1.83 (d, J = 7.7 Hz, 3H). 13C
NMR (101 MHz, DMSO-d6) δ 164.73, 164.15（2C）, 157.50, 152.40, 141.86, 141.20, 140.50, 137.67, 130.21, 129.92,
126.32, 124.76, 123.65, 123.35, 121.09, 119.36, 117.23, 116.88, 112.98, 111.66, 109.09, 17.85. TOF MS ES + (m/z): (M
+ H)+, calcd for C23H17N5O2S: 428.1179, found, 428.1181.
4.7.29 N-(3-((2-((3-cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)-3-methylbut-2-enamide (13c).
A faint yellow solid; Yield: 32.5%; m.p.: 187.5–190.7 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.05 (s, 1H), 9.89 (s,
1H), 8.35 (d, J = 5.4 Hz, 1H), 8.05 (s, 1H), 7.89 (d, J = 8.6 Hz, 1H), 7.77 (s, 1H), 7.45 (dd, J = 7.6, 6.0 Hz, 3H), 7.30 (t, J
= 9.1 Hz, 2H), 7.06 (d, J = 8.4 Hz, 1H), 5.86 (s, 1H), 2.12 (s, 3H), 1.85 (s, 3H). TOF MS ES + (m/z): (M + H)+, calcd for
C24H19N5O2S: 442.1338, found, 442.1339.
4.7.30 (E)-N-(3-((2-((3-cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)pent-2-enamide (13d).
A faint yellow solid; Yield: 37.3%; m.p.: 215.3–218.4 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.21 (s, 1H), 9.88 (s,
1H), 8.35 (d, J = 5.4 Hz, 1H), 8.04 (s, 1H), 7.88 (d, J = 7.7 Hz, 1H), 7.77 (s, 1H), 7.52 (d, J = 8.2 Hz, 1H), 7.45 (dd, J =
10.6, 6.6 Hz, 2H), 7.35 – 7.28 (m, 2H), 7.08 (d, J = 6.2 Hz, 1H), 6.89 – 6.81 (m, 1H), 6.10 (d, J = 15.4 Hz, 1H), 2.26 –
2.17 (m, 2H), 1.03 (t, J = 7.4 Hz, 3H). TOF MS ES + (m/z): (M + H)+, calcd for C24H19N5O2S: 442.1338, found,
442.1343.
4.7.31 (E)-N-(3-((2-((3-cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)-4-methylpent-2-enamide (13e).
A faint yellow solid; Yield: 36.9%; m.p.: 230.1–231.7 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.19 (s, 1H), 9.88 (s,
1H), 8.35 (d, J = 3.8 Hz, 1H), 8.04 (s, 1H), 7.88 (d, J = 6.8 Hz, 1H), 7.77 (s, 1H), 7.52 (d, J = 7.8 Hz, 1H), 7.46 – 7.42 (m,
2H), 7.31 (d, J = 9.8 Hz, 2H), 7.08 (d, J = 7.8 Hz, 1H), 6.79 (dd, J = 15.2, 6.6 Hz, 1H), 6.05 (d, J = 15.4 Hz, 1H), 2.45
(dd, J = 13.6, 6.8 Hz, 1H), 1.03 (d, J = 5.6 Hz, 6H). TOF MS ES + (m/z): (M + H)+, calcd for C25H21N5O2S: 456.1494,
found, 456.1503.
4.7.32 (E)-N-(3-((2-((3-cyanophenyl)amino)thieno[3,2-d]pyrimidin-4-yl)oxy)phenyl)hex-2-enamide (13f).
A faint yellow solid; Yield: 35.9%; m.p.: 221.8–224.4 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.18 (s, 1H), 9.88 (s,
1H), 8.35 (d, J = 5.4 Hz, 1H), 8.04 (s, 1H), 7.87 (d, J = 7.8 Hz, 1H), 7.77 (s, 1H), 7.52 (d, J = 7.4 Hz, 1H), 7.45 (dd, J =

10.4, 6.8 Hz, 2H), 7.31 (dd, J = 18.2, 8.0 Hz, 2H), 7.09 (d, J = 7.8 Hz, 1H), 6.83 – 6.74 (m, 1H), 6.10 (d, J = 15.6 Hz,
1H), 2.17 (dd, J = 13.6, 7.0 Hz, 2H), 1.45 (dd, J = 14.6, 7.2 Hz, 2H), 0.90 (t, J = 7.4 Hz, 3H). TOF MS ES + (m/z): (M +
H)+, calcd for C25H21N5O2S: 456.1494, found, 456.1508.
4.8. In vitro anti-proliferative activity assay
The target compounds (9a-h, 10a-f, 11a-f, 12a-f, 13a-f) were evaluated for cytotoxic activity against A549, A431,
Hela, MCF-7 and LO2 by 3-(4,5-Dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) method, with
Olmutinib being the positive control according to the reported procedures [23]
.
4.9. In vitro enzymatic activity assay
The potent compounds 10f, 11a, 11f and 13a were tested for their activities against different enzymes using
Kinase-Glo Luminescent Kinase Assay (Promega, Madison, WI, USA), with Olmutinib as positive control according to
the reported procedures [24]
.
4.10. Acridine orange staining
The potent compounds 13a was tested for its effect on cell apoptosis, with Olmutinib as positive control. The cells
were seeded evenly in a 24-well plate. 24 h later, compound 13a was added to 24-well plate at a concentration of 333
μg/mL. Plate was incubated at 37 °C in a 5% CO2 incubator for 6 h. After 6 h, the cells were washed 3 times with buffer
solution PBS, followed by acridine orange stain. After 15 min of avoiding light staining, the cells were observed under a
microscope.
4.11. Annexin V-FITC staining
The potent compounds 13a was tested for its effect on cell cycle progression, with Olmutinib as positive control.
The specific method of experiment referred to the previous method of our research group [20]
4.12. Molecular docking studies
.
For docking purposes, the three-dimensional structure of the EGFRT790M (PDB code: 3IKA) and EGFRWT (PDB
code: 4ZAU) were obtained from RCSB Protein Data Bank. Hydrogen atoms were added to the structure allowing for
appropriate ionization at physiological pH and the water was removed. The protonated state of several important residue
was adjusted and docking was carried out by using AutoDock in favor of forming reasonable hydrogen bond with the
ligand. And using PyMol 1.8.6 to analyzing the results.
5. Acknowledgements
This work was supported by The National Natural Science Funds (No.21662014, No.21967009), Natural Science

Foundation of Jiangxi, China (20171BCB23078, 20181BBG70003, 20192BAB215061), Science and Technology Project
Founded by the Education Department of Jiangxi Province, China (GJJ180628).
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Table 1
Structures and activity of target compounds 9a-h.
IC50(μM)a
Hela
Compd.
R1
LogD7.4c
A549
A431
MCF-7
9a
9b
9c
9d
26.51 ± 1.49 13.86 ± 1.81 17.33 ± 1.29
29.53± 1.92 33.31 ± 1.99 43.46 ± 1.12
31.87 ± 1.16 38.91 ± 1.76 39.62 ± 1.37
37.64± 1.14
4.51
4.89
5.14
5.34
>50
43.87± 1.26
>50
25.61± 1.39
36.23± 1.99
49.41± 2.22
9e
9f
29.94± 1.07 46.14 ± 1.21 48.84 ± 1.37
>50
39.13± 2.96
>50
5.62
5.78
4.43
26.34± 2.01
>50
23.39± 1.99
>50
32.17± 2.43
>50
9g
9h
>50
>50
>50
>50
4.90
Olmutinib b
AZD9291 b
-
-
4.29 ± 0.21
5.29 ± 0.59
9.27 ± 0.76
6.90 ± 0.93
4.81
3.00
0.665 ± 0.08 0.771 ± 0.11 1.827 ± 0.14
3.665 ± 0.21
aThe values are an average of three separate determinations.
bUsed as the positive control.
cLogD7.4 values were calculated by the chemaxon website (http://www.chemaxon.com).

Table 2
Structures and activity of target compounds 10a-f, 11a-f and 12a-f.
IC50(μM)a
Compd.
R1
LogD7.4c
A549
A431
Hela
MCF-7
LO2
10a
10b
10c
10d
8.61 ± 1.43
9.30 ± 1.43
26.09 ± 1.41
15.99 ± 1.20
>50
5.36
5.75
5.99
6.19
13.71 ± 1.02 11.75 ± 1.07 45.11 ± 2.05
18.09 ± 1.51 17.62 ± 0.98 28.75 ± 1.41
16.09 ± 1.41 15.09 ± 1.20 36.13 ± 1.57
>50
46.12 ± 1.19
>50
>50
48.87 ± 1.49
>50
10e
37.93 ± 1.45 25.18 ± 1.02 45.06 ± 1.12
>50
Nd
6.48
10f
11a
11b
11c
11d
5.21 ± 0.71
4.34 ± 0.50
4.22 ± 0.62
4.79 ± 0.47
31.44 ± 1.61
16.39 ± 0.98
35.01 ± 1.51
18.99 ± 1.31
14.56± 1.19
35.83 ± 1.64
43.44± 1.47
>50
30.28 ± 1.31
46.04 ± 1.62
Nd
6.64
5.36
5.75
5.99
6.19
10.48 ± 1.17 13.09 ± 1.17 20.06 ± 1.39
27.69 ± 1.24 25.79 ± 1.38 26.09 ± 1.41
19.61± 1.82
37.10± 1.91
5.56± 0.81
29.23± 1.40
23.38± 1.49
8.09 ± 0.71
29.41± 1.29
27.04± 1.63
7.73± 1.01
>50
11e
43.85± 1.29
Nd
6.48
11f
12a
12b
5.02± 0.66
>50
>50
>50
>50
6.64
4.74
5.12
20.39 ± 1.76 27.81 ± 1.46 47.13 ± 1.67
31.87 ± 1.78 37.17 ± 1.80
>50
>50