Design, synthesis, biological evaluation and molecular docking study of novel thieno[3,2-d]pyrimidine derivatives as potent FAK inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.112024

A series of 2,7-disubstituted-thieno[3,2-d]pyrimidine derivatives were designed, synthesized and evaluated as novel focal adhesion kinase (FAK) inhibitors. The novel 2,7-disubstituted-thieno[3,2-d]pyrimidine scaffold has been designed as a new kinase inhibitor platform that mimics the bioactive conformation of the well-known diaminopyrimidine motif. Most of the compounds potently suppressed the enzymatic activities of FAK and potently inhibited the proliferation of U-87MG, A-549 and MDA-MB-231 cancer cell lines. Among these derivatives, the optimized compound 26f potently inhibited the enzyme (IC₅₀ = 28.2 nM) and displayed stronger potency than TAE-226 in U-87MG, A-549 and MDA-MB-231 cells, with IC₅₀ values of 0.16, 0.27, and 0.19 μM, respectively. Compound 26f also exhibited relatively less cytotoxicity (IC₅₀ = 3.32 μM) toward a normal human cell line, HK2. According to the flow cytometry results, compound 26f induced the apoptosis of MDA-MB-231 cells in a dose-dependent manner and effectively arrested MDA-MB-231 cells in G0/G1 phase. Further investigations revealed that compound 26f potently suppressed the migration of MDA-MB-231 cells. Collectively, these data support the further development of compound 26f as a lead compound for FAK-targeted anticancer drug discovery.

Full text of DOI:10.1016/j.ejmech.2019.112024

Journal Pre-proof
Design, synthesis, biological evaluation and molecular docking study of novel
thieno[3,2-d]pyrimidine derivatives as potent FAK inhibitors
Ruifeng Wang, Sijia Yu, Xiangxin Zhao, Yixuan Chen, Bowen Yang, Tianxiao Wu,
Chenzhou Hao, Dongmei Zhao, Maosheng Cheng
PII:
S0223-5234(19)31182-1
DOI:
https://doi.org/10.1016/j.ejmech.2019.112024
EJMECH 112024
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 October 2019
Revised Date: 13 December 2019
Accepted Date: 29 December 2019
Please cite this article as: R. Wang, S. Yu, X. Zhao, Y. Chen, B. Yang, T. Wu, C. Hao, D.
Zhao, M. Cheng, Design, synthesis, biological evaluation and molecular docking study of novel
thieno[3,2-d]pyrimidine derivatives as potent FAK inhibitors, European Journal of Medicinal Chemistry
(2020), doi: https://doi.org/10.1016/j.ejmech.2019.112024.
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Graphical abstract

Design, synthesis, biological evaluation and molecular docking study of novel
thieno[3,2-d]pyrimidine derivatives as potent FAK inhibitors
Ruifeng Wanga, Sijia Yua, Xiangxin Zhaoa, Yixuan Chena, b, Bowen Yanga, Tianxiao Wua, Chenzhou Haoa,
Dongmei Zhaoa,*, Maosheng Chenga
a Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, School of Pharmaceutical
Engineering, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang, 110016, China
b The School of life Science and Biopharmaceutical, Shenyang Pharmaceutical University, 103 Wenhua Road,
Shenhe District, Shenyang, 110016, China
*Corresponding author
E-mail address: medchemzhao@163.com (D. Zhao).
Abstract
A series of 2,7-disubstituted-thieno[3,2-d]pyrimidine derivatives were designed, synthesized and evaluated as
novel focal adhesion kinase (FAK) inhibitors. The novel 2,7-disubstituted-thieno[3,2-d]pyrimidine scaffold has
been designed as a new kinase inhibitor platform that mimics the bioactive conformation of the well-known
diaminopyrimidine motif. Most of the compounds potently suppressed the enzymatic activities of FAK and
potently inhibited the proliferation of U-87MG, A-549 and MDA-MB-231 cancer cell lines. Among these
derivatives, the optimized compound 26f potently inhibited the enzyme (IC50 = 28.2 nM) and displayed stronger
potency than TAE-226 in U-87MG, A-549 and MDA-MB-231 cells, with IC50 values of 0.16, 0.27, and 0.19 µM,
respectively. Compound 26f also exhibited relatively less cytotoxicity (IC50 = 3.32 µM) toward a normal human
cell line, HK2. According to the flow cytometry results, compound 26f induced the apoptosis of MDA-MB-231
cells in a dose-dependent manner and effectively arrested MDA-MB-231 cells in G0/G1 phase. Further
investigations revealed that compound 26f potently suppressed the migration of MDA-MB-231 cells. Collectively,
these data support the further development of compound 26f as a lead compound for FAK-targeted anticancer drug
discovery.
Keywords: FAK inhibitor, Thieno[3,2-d]pyrimidine, Structure-activity relationship, Apoptosis, Migration.
1. Introduction
Focal adhesion kinase (FAK) is an intracellular non-receptor tyrosine kinase that is activated by integrins or
growth factor receptors in various types of human cancers[1]. Accumulating evidence indicates that FAK plays
significant roles in survival, motility, metastasis, adhesion, lymphangiogenesis, angiogenesis, the tumour
microenvironment, cancer stem cell functions and the epithelial-to-mesenchymal transition[2-6]. Overexpression
of FAK has been clinically observed in primary human hepatocellular carcinomas[7], human colorectal
carcinomas[8], human ovarian carcinomas[9] and human breast cancers[10], implicating a role of FAK in cancer
development. The overexpression and activation of FAK have been investigated primarily in primary or metastatic
cancers and correlate with poor clinical outcomes[11-12]. Furthermore, recent research has identified FAK as a key
mediator of the immune response in certain cancers and provides strong evidence that FAK inhibitors may trigger
immune-mediated tumour regression, revealing previously unrecognized therapeutic opportunities[13-14]. The
mechanisms of FAK activation and signalling have been extensively studied, with FAK being highlighted as a
potential target for anticancer therapeutics.
Given the upregulation of FAK in cancer, several agents are logically being developed to target FAK for
cancer therapy. In particular, many small molecule inhibitors have been designed and developed to directly block
1

ATP-kinase interactions. As shown in Figure 1, TAE226[15] exhibits antitumour activity in vitro and in vivo
against several types of malignant tumours and is often used as a positive reference compound in research. In
addition, several FAK inhibitors are being evaluated in clinical trials at different stages, including VS-4718 (2,
phase I)[16], CEP-37440 (3, phase I)[17], GSK2256098 (4, phase II)[18], PF-562271 (5, phase I)[19], and
defactinib (6, phase II)[20], among others. These compounds have exhibited potent inhibition of FAK and potent
antitumour activities in a panel of in vitro and in vivo cancer models.
Cl
N
Cl
CF3
NH
N
N
HN
N
NH
O
HN
N
NH
O
HN
O
O
N
O
O
H
N
H
N
H
N
O
N
O
N
1
2
3
TAE-226
VS-4718
Phase I
CEP-37440
Phase I
N
HO
CF3
CF3
N
N
Cl
N
HN
N
NH
HN
N
NH
HN
NH
O
N
O
N
H
N
N
N
N
N
N
O
O
NH
S
S
O
5
O
NH
O
6
O
4
GSK-2256098
Phase II
PF-562271
Phase I
Defactinib
Phase II
Figure 1. Structures of potent and selective FAK inhibitors.
Among the scaffolds of currently available FAK inhibitors, the 2,4-diaminopyridine and
2,4-diaminopyrimidine scaffolds are the major scaffolds used in compounds described by pharmaceutical
companies and academic research institutions[21-22]. A general representation of the diaminopyrimidine motif at
the ATP active site of the kinase is shown in Figure 2. The diaminopyrimidine derivatives bind to the ATP-binding
site of FAK by adopting a U-shaped ligand conformation. The compounds are anchored to the hinge region by
double-dentate hydrogen bonding of the pyrimidine nitrogen and aniline NH to the hinge region. Moreover, the
aryl residues back toward the DFG motif of activation loop and orient toward solvent region. We postulated that
we could take advantage of the active site conformation of this pharmacophore to further diversify the chemical
space by constraining the core structure and orienting the side chains into bioactive trajectories. Based on a
cyclization strategy, a novel 2,7-disubstituted-thieno[3,2-d]pyrimidine scaffold was designed as a novel kinase
inhibitor platform that mimics the bioactive conformation of the well-known diaminopyrimidine motif. To the best
of our knowledge, no thieno[3,2-d]pyrimidine derivatives serving as FAK inhibitors have been reported, and a
systematic investigation of the effects of substitutions has not yet been conducted. In addition, the SAR of these
compounds was discussed in this manuscript, and various substituents with hydrogen bond receptors were
introduced at R1 to form hydrogen bond interactions with Asp564 of the DFG motif. Based on the SAR at R1, we
decided to introduce an o-methoxyl group at the R1 position and explore the R2, R3, R4 and R5 positions to obtain a
series of potent FAK inhibitors using a structure-based drug design approach.
Figure 2. Design of the target compounds.
2

2. Chemistry
Compounds 11a-h and intermediates 14, 17, 20 and 23 were synthesized using the method depicted in
Scheme 1. Palladium-catalysed regioselective dechlorination of 2,4-dichlorothieno[3,2-d]pyrimidine (7) in the
presence of Na2CO3 occurred exclusively at the C-4 position[23], and electrophilic iodization of compound 8 using
NIS in AcOH yielded the desired C-7 iodine intermediate 9. Intermediates 10a-h were obtained in a rapid and
efficient manner via Suzuki coupling of compound 9 with the corresponding boric acid or borate.
Palladium-catalysed Buchwald cross-coupling with dimethyl(4-aminobenzyl)phosphonate afforded compounds
11a-h[24]. Acetylation of 3-bromoaniline (12) followed by Suzuki coupling with bis(pinacolato)diboron generated
intermediate 14[25]. Compound 12 was reacted with methanesulfonyl chloride to yield intermediate 15.
Subsequent methylation yielded the N-methyl sulfonamide 16. Treatment with bis(pinacolato)diboron and
Pd(dppf)Cl2 produced the required Suzuki coupling partner[26], intermediate 17. 3-Bromobenzoic acid (18) was
converted into an acid chloride and condensed with methylamine to produce intermediate 19, a subsequent Suzuki
coupling reaction with bis(pinacolato)diboron afforded intermediate 20. Commercially available
1-(chloromethyl)-4-nitrobenzene (21) was reacted with trimethyl phosphate to provide the dimethyl
(4-nitrobenzyl)phosphonate (22), which was reduced under P/C-H2 conditions to form the aniline 23.
Compounds 24a-j, 26a-i, 27a-b, and 28c-g were prepared using similar reaction conditions as depicted in
Scheme 2. The synthesis of compounds 26a-i was used as an example for illustration. Buchwald cross-coupling of
compound 10a with appropriate substituted amines in the presence of Pd(dba)2, BINAP and Cs2CO3 or Pd(AcO)2,
X-phos and Cs2CO3 yielded the intermediates 25a-i. Then, the Boc group was subsequently removed under acidic
conditions to produce compounds 26a-i.
Intermediates 31a-f, 34a-g, 36, 38 and 40 were synthesized as depicted in Scheme 3. Intermediates 31a-f and
34a-g were synthesized using a two-step synthetic approach according to our previously reported methods[27].
1-(chloromethyl)-4-nitrobenzene (21) was reacted with triethyl phosphate to provide the diethyl
(4-nitrobenzyl)phosphonate (35)[28], followed by a reduction of nitro (H2, Pd/C) to generate intermediate 36.
Additionally, compound 35 was treated with oxalyl chloride and then directly reacted with morpholine or
CH3NH2•HCl in the presence of TEA base to prepare the phosphoramide intermediates 37 and 39, which were
reduced under P/C-H2 conditions to form the anilines 38 and 40.
S
N
S
Cl
N
HN
N
S
S
S
a
b
c
d
N
N
N
Cl
N
R1
Cl
N
R1
Cl
N
Cl
N
I
10a-h
9
7
8
O
11a-h
P
O
O
Br
O
O
10a-11a: R1: 2-OCH3
B
O
10b-11b: R1: 2-COCH3
10c-11c: R1: 3-COCH3
10d-11d: R1: 3-SO2CH3
10e-11e: R1: 3-CONHCH3
10f-11f: R1: 3-NHCOCH3
f
e
g
O
N
H
Br
N
13
H
14
NH2
10g-11g: R1: 3-NCH3SO2CH3
10h-11h: R1: 4-OCH3
O
O
Br
Br
B
12
f
h
O
O
O
S
S
S
N
N
H
N
O
O
O
16
15
17
NO2
NH2
NO2
Br
Br
O
O
B
j
f
i
k
H
O
N
O
OH
H
P
O
P
O
N
O
O
O
O
Cl
19
20
O
21
18
22
23
3

Scheme 1. Synthesis of compounds 11a-h and intermediates 14, 17, 20 and 23. Reagents and conditions: (a) Pd/C,
H2, Na2CO3, EtOH, 30 °C; (b) NIS, CH3COOH, 80 °C; (c) corresponding boric acid or borate, Pd(dppf)Cl2, K2CO3,
1,4-dioxane/H2O, 90 °C; (d) dimethyl (4-aminobenzyl)phosphonate, Pd(AcO)2, X-phos, Cs2CO3, dioxane, 90 °C;
(e) acetic anhydride, TEA, CH2Cl2, rt; (f) Bis(pinacolato)diboron, Pd(dppf)Cl2, AcOK, dioxane, 80 °C; (g)
methanesulfonyl chloride, pyridine, CH2Cl2, 0 °C; (h) CH3I, K2CO3, DMF, rt; (i) Ι) oxalyl chloride, DMF, CH2Cl2,
60 °C; ΙΙ) CH3NH2•HCl, TEA, CH2Cl2, rt; (j) trimethyl phosphite, 120 °C; and (k) Pd/C, H2, EtOH, 40 °C.
Scheme 2. Synthesis of compounds 24a-j, 26a-i, 27a-b and 28c-g. Reagents and conditions: (a) corresponding
aromatic amines, Pd(AcO)2, X-phos, Cs2CO3, dioxane, 90 °C; (b) corresponding aromatic amines, Pd(dba)2,
BINAP, Cs2CO3, dioxane, 90 °C; and (c) HCl-EA, rt.
4

Scheme 3. Synthesis of intermediates 31a-f, 34a-g, 36, 38 and 40. Reagents and conditions: (a) tert-butyl
4-aminopiperidine-1-carboxylate, HATU, DIPEA, CH2Cl2, 25–40 °C; (b) Pd/C, H2, EtOH, 40 °C; (c) R6-NH2,
HATU, DIPEA, CH2Cl2, 25–40 °C; (d) triethyl phosphite, 130 °C; (e) Ι) oxalyl chloride, DMF, CH2Cl2, 60 °C; ΙΙ)
morpholine, TEA, CH2Cl2, rt; and (f) Ι) oxalyl chloride, DMF, CH2Cl2, 60 °C; ΙΙ) CH3NH2•HCl, TEA, CH2Cl2, rt.
3.Results and discussion
3.1. Biological evaluation and analysis of the structure-activity relationship
All newly synthesized compounds were evaluated for their activities against the FAK enzyme using
homogeneous time-resolved fluorescence (HTRF) assay. Their abilities to inhibit the proliferation of U-87MG
(human glioma cancer cell), A549 (human lung cancer cell) and MDA-MB-231 (human breast cancer cell) cell
lines, which overexpress FAK [10, 29, 30], were evaluated using the MTT assay. TAE-226 was tested for
comparison.
We first focused on the R1 moiety by fixing the R2 moiety as a phosphonate group. Different fragments
containing hydrogen bond acceptors were introduced at R1 to form hydrogen bond interactions with Asp564 of the
DFG motif. As shown in Table 1, most compounds exhibited moderate inhibitory activity against FAK at
submicromolar concentrations. In particular, the inclusion of a 2-methoxyl group at R1 (11a) exhibited the
strongest inhibition of FAK (IC50 = 134.0 nM), whereas switching R1 to a 2-acetaldehyde group (11b) resulted in a
marked loss of activity (IC50 = 1083 nM). We deduced that the interaction of the C=O bond and C6 of the
thieno[3,2-d]pyrimidine resulted in an unfavourable conformation of the ligand and that this steric clash resulted in
reduced activity. The 3-substituted derivatives (11c-g) retained moderate potency against FAK, with IC50 values
5

ranging from 140.1 to 638.4 nM. However, switching the methoxyl group to the para position (11h) resulted in a
pronounced loss of activity (IC50 = 1064 nM).
S
N
HN
N
1
2
R1
3
4
O
P
O
O
Table 1 The enzymatic activity of FAK in the presence of compounds 11a-h.
Compd.
11a
R1
IC50 (nM)a
134.0
1083
Compd.
11e
R1
IC50 (nM)a
324.5
2-OCH3
2-COCH3
3-COCH3
3-SO2CH3
3-CONHCH3
3-NHCOCH3
3-NCH3SO2CH3
4-OCH3
377.0
11b
11f
351.0
140.1
6.3
638.4
11c
11g
1064
11d
11h
TAE-226
a The IC50 values are presented as the mean (nM) values from two separate experiments.
Compound 11a, which contained a 2-methoxy group at R1, exhibited stronger inhibitory activity against FAK
than compounds 11b-h. Thus, we next conducted SAR exploration at the R2 position, as shown in Table 2. The
diethyl phosphonate-substituted analogue 24a (IC50 = 156.8 nM) was equally potent as compound 11a. The
incorporation of solubilizing groups into compound 24a to produce compounds 24b-d decreased the potency of
FAK inhibition, presumably due to the suboptimal location of the methylamino group, hydroxyl group and
morpholine ring. The introduction of the N-methylformamide group (24e), which is identical to defactinib,
produced a 2-fold higher potency than exhibited by compound 11a. Because the para-substitution vector R2 points
toward the solvent region, the introduction of hydrophilic fragments at the R2 position may increase the activity. A
class of hydrophilic fragment-containing derivatives (24f-j) bearing an amide linker directly attached to the C2
phenyl moiety were designed and synthesized. Among these derivatives, the piperidine-4-yl substituted analogues
24f and 24g exhibited much higher potency (IC50 values of 38.8 and 57.7 nM, respectively) than compound 24e.
However, compounds 24h-j exhibited lower potency against FAK, with IC50 values of 143.6–199.1 nM.
In cell-based assays, most of the target compounds possessed moderate to excellent anticancer activities, with
IC50 values less than 5.0 µM. Three of these compounds (24f, 24h, and 24i) potently inhibited the proliferation of
U-87MG, A-549 and MDA-MB-231 cancer cell lines, with IC50 values ranging from 0.11 to 0.65 µM. Notably,
compound 24f, which had an IC50 value of 0.36 µM against A549 cells and of 0.11 µM against MDA-MB-231 cells,
was the strongest inhibitor of these two cancer cell lines. Since compound 24f displayed potent enzymatic and
cellular activity, it was selected for molecular docking studies with the FAK crystal structure (PDB: 2JKK). As
shown in Figure 3A, compound 24f was located deep in the ATP-binding site, and four H-bonds were observed in
the binding mode: two were established between the thieno[3,2-d]pyrimidine scaffold and Cys502 in the hinge
region of the kinase, another formed between the methoxyl group and Asp564 of the DFG motif, and the fourth
formed between NH (piperidine) and Cys427. In addition, as shown in Figure 3B, the structural model of the
drug-protein complex showed that the binding model of compound 24f to FAK was similar to that of TAE-226,
indicating that the 2,7-disubstituted-thieno[3,2-d]pyrimidine scaffold mimicked the bioactive conformation of the
well-known diaminopyrimidine motif. Compound 24f also exhibited good spatial matching with the FAK active
pocket (Figure 3C). Notably, the gatekeeper+2 residue is a smaller Leu501 in FAK (Figure 3D). Therefore, the
substituents on the 2-N-aryl moiety will occupy the lower hinge area pocket and form a hydrophobic interaction
with the side chain of Leu501, thus potentially improving the inhibition of enzyme activity.
6

Table 2 Biological activities of compounds 24a-j.
Antiproliferative activity, IC50 (µM)b
FAK,
IC50 (nM)a
Compd.
11a
R2
U-87MG
A549
MDA-MB-231
134.0
1.71 ± 0.22
1.30 ± 0.11
1.30 ± 0.07
1.95 ± 0.22
1.22 ± 0.14
0.71 ± 0.03
0.65 ± 0.07
1.06 ± 0.04
0.30 ± 0.04
0.36 ± 0.01
1.80 ± 0.24
7.81 ± 0.22
4.94 ± 0.14
1.21 ± 0.19
2.64 ± 0.23
8.35 ± 0.18
2.12 ± 0.14
0.11 ± 0.01
1.05 ± 0.11
0.44 ± 0.03
0.41 ± 0.05
156.8
183.6
321.5
351.5
79.3
0.96 ± 0.13
1.73 ± 0.04
0.35 ± 0.03
0.85 ± 0.15
0.31 ± 0.02
0.36 ± 0.06
0.45 ± 0.08
0.50 ± 0.03
0.41 ± 0.05
24a
24b
24c
24d
24e
38.8
24f
57.7
24g
24h
24i
H
N
NH
NH
167.1
199.1
O
H
N
O
143.6
6.3
0.21 ± 0.02
1.95 ± 0.26
2.77 ± 0.19
24j
1.67±0.02
1.16±0.20
4.06±0.21
TAE-226
a The IC50 values are presented as the mean (nM) values from two separate experiments.
b Concentration that inhibits the proliferation of cancer cells by 50 %. Cell proliferation was measured using the
MTT assay after incubation with the compounds for 72 h. The mean values of three independent experiments ± SE
are reported.
7

Figure 3. The predicted docked pose of compound 24f (yellow sticks) in the FAK active site (PDB: 2JKK). (A)
Detailed interactions with the protein residues. Each dashed red line represents hydrogen bonds. (B) Image
showing the overlap of compound 24f and TAE-226 (blue sticks). (C) Three-dimensional space matching diagram
of compound 24f and the active site. (D) The red frame represents the pocket in the lower hinge area.
Based on the analysis described above, we started the optimization of compound 24f by varying the
substituent on the benzene ring. As shown in Table 3, the introduction of F-, Me- or MeO- into R3 and of F- or
Me- into R4 yielded compounds 26a-c and 26f-g, respectively, which retained high potency against FAK, with IC50
values ranging from 25.7 to 48.3 nM. Further substitution of the R3 moiety to larger EtO- (26d) and CF3- (26e)
groups both resulted in a pronounced loss of activity toward FAK (IC50 = 165.6 nM and 709.0 nM, respectively).
Clearly, increasing the size of the substituent produced a significant reduction in activity, presumably due to
deleterious steric interactions with the amino acid residues of the protein skeleton. The introduction of a fluoro
group para to the methoxy substituent or a fluoro substituent on the 2-substituted aniline resulted in the potent
inhibitors 26h (IC50 = 38.2 nM) and 26i (IC50 = 24.2 nM).
In cell-based assays, most of the target compounds possessed significant anticancer activities, with IC50
values ranging from 0.19 to 3.0 µM. Analogues 26b, 26f, 26g and 26i exhibited much greater antitumour activities
than TAE-226 in U-87MG and MDA-MB-231 cells. More interestingly, the most promising compound, 26f,
displayed stronger potency than TAE-226 in U-87MG, A-549 and MDA-MB-231 cells, with IC50 values of 0.16,
0.27, and 0.19 µM, respectively. Furthermore, as shown in Figure 4,
a significant time- and
concentration-dependent decrease was observed in the viability of both A549 and MDA-MB-231 cells after
treatment with inhibitor 26f.
8

Table 3 Biological activities of compounds 26a-i.
Antiproliferative activity, IC50 (µM)b
FAK,
IC50 (nM)a
Compd.
R3
R4
R5
U-87MG
A549
MDA-MB-231
H
F
H
H
H
H
H
H
H
H
H
H
F
38.8
0.65 ± 0.07
2.37 ± 0.01
0.25 ± 0.05
6.40 ± 0.01
2.03 ± 0.14
2.09 ± 0.16
0.16 ± 0.02
0.46 ± 0.09
3.59 ± 0.33
1.48 ± 0.25
1.67±0.02
0.36 ± 0.06
2.75 ± 0.42
3.42 ± 0.17
2.88 ± 0.28
5.77 ± 0.14
4.11 ± 0.19
0.27 ± 0.05
0.87 ± 0.09
1.90 ± 0.05
5.39 ± 0.09
1.16±0.20
0.11 ± 0.01
1.61 ± 0.33
3.24 ± 0.40
2.24 ± 0.13
4.39 ± 0.13
2.75 ± 0.18
0.19 ± 0.02
0.24 ± 0.04
1.16 ± 0.05
2.57 ± 0.10
4.06±0.21
24f
26a
48.3
Me
OMe
OEt
CF3
H
H
26.5
26b
H
27.2
26c
H
165.6
709.0
28.2
26d
H
26e
F
26f
H
Me
H
25.7
26g
OMe
F
38.2
26h
H
F
24.2
26i
6.3
TAE-226
a The IC50 values are presented as the mean (nM) values from two separate experiments.
b Concentration that inhibits the proliferation of cancer cells by 50 %. Cell proliferation was measured using the
MTT assay after incubation with the compounds for 72 h. The mean values of three independent experiments ± SE
are reported.
Figure 4. The effects of treatment time and concentration of 26f on viability of A549 and MDA-MB-231 cells.
At this stage, we had identified compound 26f as displaying potent enzymatic and cellular activities. We next
addressed the effects of variation in the solubilizing group at the R6 position, the results are reported in Table 4.
Compared to the piperidine-4-yl-substituted compound 26f, the tetrahydro-pyran-4-yl analogue 27a was more than
4-fold less potent, and the 1-methylpiperidine-4-yl analogue 27b showed a 1.5-fold reduction in potency. Similarly,
a lower potency was observed for the pyrrolidin-3-yl analogues 28c-d and the piperidine-methyl-substituted
analogues 28e-g, indicating that the piperidine-4-yl moiety was an important contributor to the potency of FAK
inhibition. In cell-based assays, most compounds possessed moderate anticancer activities, with IC50 values less
than 5.0 µM. Three of these compounds (27b, 28c, and 28d) displayed much higher antiproliferative activity than
9

the others toward the A549 and MDA-MB-231 cell lines, with IC50 values in the submicromolar range.
Table 4 Biological activities of compounds 27a-b and 28c-g.
Antiproliferative activity, IC50 (µM)b
FAK,
IC50 (nM)a
Compd.
26f
R6
A549
MDA-MB-231
28.2
0.27 ± 0.05
0.19 ± 0.02
117.7
47.8
5.71±0.91
0.66±0.03
0.87±0.10
0.43±0.13
3.41±0.14
5.05±0.46
5.35±0.01
0.35±0.03
0.56±0.16
0.26±0.02
1.53±0.19
2.89±0.28
27a
27b
28c
132.3
71.3
S
28d
28e
R
120.2
111.2
28f
R
148.9
6.3
4.45±0.21
1.16±0.20
8.62±0.50
4.06±0.21
28g
S
TAE-226
a The IC50 values are presented as the mean (nM) values from two separate experiments.
b Concentration that inhibits the proliferation of cancer cells by 50 %. Cell proliferation was measured using the
MTT assay after incubation with the compounds for 72 h. The mean values of three independent experiments ± SE
are reported.
3.2. Cellular selectivity assay
We evaluated the antiproliferative activities of eight compounds toward a normal human cell line, HK2
(normal human tubular epithelial cell line), using the MTT assay to assess the cytotoxicity of the synthesized
compounds. These eight compounds potently inhibited enzyme activity and cancer cell proliferation. As shown in
Table 5, six of these compounds (24f, 24g, 24h, 26c, 26f, and 26g) displayed low cytotoxicity toward the normal
HK2 cells, indicating that these compounds exerted less of an effect on HK2 cells than on the cancer cells. Two
compounds (24e and 26h) displayed significant cytotoxicity to HK2 cells, with IC50 values < 1.0 µM.
Table 5 Antiproliferative activities of compounds toward HK2 cells
Compd.
24e
HK2 (µM)a
0.71 ± 0.02
11.39 ± 1.30
11.69 ± 0.50
Compd.
26c
HK2 (µM)a
12.21 ± 0.11
3.32 ± 0.20
4.45 ± 0.22
24f
26f
24g
26g
10

4.89 ± 0.14
8.20 ± 1.01
0.78 ± 0.04
24h
26h
TAE-226
a Concentration that inhibits the proliferation of cancer cells by 50 %. Cell proliferation was measured using the
MTT assay after incubation with the compounds for 72 h. The mean values of three independent experiments ± SE
are reported.
3.3. Kinase selectivity profile
The kinase selectivity of compound 26f was profiled against a panel of 25 kinases covering the major tumor
progression, metastasis, angiogenesis, oncogenic activation and mitogenic stimulation kinases of the human
protein kinome at a concentration of 1.0 µM, and the percent inhibition values are reported in Table 6. Compound
26f is a multi-target kinase inhibitor, with five kinases (ALK, BTK, CDK2, FAK and RET) producing greater than
80% inhibition.
Table 6 Kinase selectivity profile of compound 26f.a
Kinase
AKT1
ALK
%inhibition at 1 µM
kinase
IKBKE
IRAK4
KIT
%inhibition at 1 µM
kinase
PAK1
PIM1
%inhibition at 1 µM
15
95
51
88
77
97
38
66
72
66
72
41
27
1
67
3
BRAF
BTK
PKA
63
48
100
97
33
6
MEK1
ERK2
MET
PRKCQ
FAK
CAMKK2
CDK2
CHK1
FGFR4
HIPK3
72
13
2
RET
MKNK1
mTOR
ROCK1
ZAP70
a Selectivity profile of compound 26f measured at a concentration of 1 µM in a panel of 25 kinases generated with
the SelectScreen® Profiling Service from Life Technologies. The results represent the mean of three independent
experiments performed in triplicate.
3.4. Cell apoptosis assay
The apoptosis of MDA-MB-231 cells treated with different concentrations of the optimal derivative 26f (0.1,
0.2 and 0.4 µM) was analysed by staining cells with Annexin V-FITC and PI and performing flow cytometry to
explore whether the antiproliferative activity of compound 26f toward MDA-MB-231 cells was accompanied by
an increase in cancer cell apoptosis. As illustrated in Figure 5, compound 26f substantially increased the apoptosis
of MDA-MB-231 cells in a concentration-dependent manner, with apoptotic rates of 11.57 %, 23.33 %, and
41.82 % at concentrations of 0.1, 0.2 and 0.4 µM, respectively.
11

Figure 5. Flow cytometry analyses of apoptosis induction in MDA-MB-231 cells treated with compound 26f (0.1,
0.2 and 0.4 µM) and untreated cells (control) as a reference control for 48 h. The lower left quadrant represents live
cells, the lower right quadrant represents early apoptotic cells, the upper right quadrant represents late apoptotic
cells, and the upper left quadrant represents necrotic cells.
3.5. Cell cycle analysis
The effect of compound 26f on cell cycle progression in MDA-MB-231 cells was analysed using flow
cytometry (Figure 6). Compared with the control group, the percentages of cells in G0/G1 phase increased from
43.43 % to 56.27 %, whereas the percentage of cells in G2/M phase decreased from 17.40 % to 3.55 % after
treatment with 0.1, 0.2 or 0.4 µM compound 26f for 48 h. The percentage of cells in S phase showed only minor
changes. Evidently, derivative 26f induced the arrest of a significant percentage of cells in G0/G1 phase of the cell
cycle compared with untreated cells.
Figure 6. Effects of compound 26f on the cell cycle of MDA-MB-231 cells. Flow cytometry analysis of the cell
cycle in MDA-MB-231 cells treated with compound 26f (0.1, 0.2 or 0.4 µM) or no treatment (control) for 48 h.
3.6. Cell migration assay
Wound healing assays were performed to investigate the effect of compound 26f on the migration of
12

MDA-MB-231 cells. MDA-MB-231 cells were incubated with DMSO or compound 26f (0.15, 0.3 or 0.6 µM) for
48 h. As illustrated in the photomicrographs shown in Figure 7, untreated MDA-MB-231 cells filled most of the
wounded area 48 h after scratching the cell monolayer, whereas treatment with the indicated doses of compound
26f significantly suppressed wound healing in a time- and concentration-dependent manner. These results indicate
that compound 26f possesses a significant ability to inhibit the metastasis of MDA-MB-231 cells.
Figure 7. Compound 26f inhibited the migration of MDA-MB-231 cancer cells in a wound healing assay.
3.7. In vitro metabolic stability
We investigated the in vitro metabolic stability of compound 26f in rat liver microsomes. As shown in Table 7,
compound 26f was significantly stable in rat liver microsomes (T1/2 = 133.8 min ).
Table 7 Liver microsomal stability
Remaining (%)
T = 1 h
Remaininga (%)
CLint(mic)
(µL/min/mg)
10.4
CLint(liver)
(µL/min/mg)
18.6
parameters
in rat
T1/2 (min)
133.8
T = 1 h
72.4
92.1
a
The NADPH regeneration system (replaced with buffer) was not added to the sample during the one hour
incubation.
3.8. Molecular docking study
A docking study of compound 26f in the ATP-binding site of FAK (PDB: 2JKK) was performed to elucidate
its interaction mode. The best predicted binding mode is shown in Figure 8 and has a calculated binding energy of
-10.51 kcal/mol. Compound 26f is anchored to the hinge region via the canonical donor-acceptor hydrogen
bonding motif between the nitrogen molecules on the 2,7-disubstituted thieno[3,2-d]pyrimidine moiety and the
backbone of residue Cys502, and further stabilization may be achieved through the hydrophobic interactions of the
thieno[3,2-d]pyrimidine ring with the hydrophobic side chains of Leu553 and Ala452. Moreover, the bending of the
o-methoxy moiety back toward the activation loop region results in the formation of a hydrogen bond with Asp564
of the DFG motif. Furthermore, the water-soluble “tail” of the piperidine moiety points toward the solvent by
forming a hydrogen bond with Cys427, which is located at the edge of the active pocket, confirming that the
piperidine-4-yl moiety is an important contributor to the potency of FAK inhibition. The binding model supported
the data obtained from the biological assays described above and provides a structural basis for the further
structure-guided design of FAK inhibitors.
13

Figure 8. The predicted docked pose of compound 26f (yellow sticks) in the FAK active site (PDB: 2JKK). (A)
Detailed interactions with the protein residues. Each dashed red line represents hydrogen bonds. (B) 2D diagram of
the interaction between compound 26f and the active site cavity of FAK.
4. Conclusions
In conclusion, we designed and synthesized a novel 2,7-disubstituted-thieno[3,2-d]pyrimidine scaffold as a
constrained diaminopyrimidine pharmacophore mimic. Importantly, we validated this scaffold as a new kinase
inhibitor platform for designing FAK inhibitors. The structure-activity relationships of these compounds are
discussed in this paper from the perspective of enzymatic and cellular activities. Most compounds potently
suppressed the enzymatic activities of FAK, with IC50 values ranging from 10-7–10-8 M, and potently inhibited the
proliferation of U-87MG, A-549 and MDA-MB-231 cancer cells. In particular, the optimized compound 26f
potently inhibited the enzyme (IC50 = 28.2 nM) and displayed stronger potency than TAE-226 against U-87MG,
A-549 and MDA-MB-231 cells, with IC50 values of 0.16, 0.27, and 0.19 µM, respectively. Furthermore, compound
26f effectively induced apoptosis and arrest at the G0/G1 phase of the cell cycle in MDA-MB-231 cells and
suppressed the migration of MDA-MB-231 cells. A docking study of compound 26f was performed to elucidate its
possible binding modes and to provide a structural basis for the structure-guided design of FAK inhibitors. The
findings reveal that 2,7-disubstituted-thieno[3,2-d]pyrimidine derivatives represent a new class of FAK inhibitors
that warrant further investigation to generate potential anticancer agents.
5. Experimental section
5.1. Chemistry
Starting materials, reagents and solvents were obtained from commercial suppliers and used without further
purification unless otherwise indicated. Anhydrous solvents were dried and stored according to standard
procedures. All reactions were monitored by thin layer chromatography (TLC) on silica gel plates with
fluorescence F-254 and visualized with UV light. Column chromatography was carried out on silica gel (200-300
1
mesh). H NMR and 13C NMR spectral data were recorded in DMSO-d6, MeOD or CDCl3 on Bruker ARX-600
NMR or Bruker ARX-400 NMR spectrometers with TMS as an internal standard. High-resolution accurate mass
spectrometry (HRMS) determinations for all final target compounds were obtained on a Bruker micromass time of
flight mass spectrometer equipped with an electrospray ionization (ESI) detector. All melting points were obtained
on a Büchi melting point B-540 apparatus and are uncorrected.
5.1.1. Preparation of 2-chlorothieno[3,2-d]pyrimidine (8)
14

A solution composed of 2,4-dichlorothieno[3,2-d]pyrimidine (1 equiv) and NaCO3 (1 equiv) in EtOH was
mixed with 10 % Pd/C (0.1 equiv). The suspension was stirred at 30 °C under an atmosphere of H2 for 24 h. The
reaction mixture was filtered through a Celite pad with EtOAc washes. The filtrate was washed with H2O and
saline, dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated to obtain the crude product 8 for
the next step. 1H NMR (600 MHz, DMSO-d6) δ 9.50 (d, J = 0.5 Hz, 1H), 8.64 (d, J = 5.4 Hz, 1H), 7.63 (dd, J = 5.4,
0.6 Hz, 1H). MS (ESI) m/z(%): 170.9 [M+H]+
.
5.1.2. Preparation of 2-chloro-7-iodothieno[3,2-d]pyrimidine (9)
The intermediate 8 (1 equiv) and N-iodosuccinimide (3 equiv) were mixed in acetic acid and heated at 80 °C
for 24 h. The reaction was then partitioned between water and EtOAc, and the aqueous layer was extracted twice
with EtOAc. The combined organic fractions were washed with saline, dried over anhydrous Na2SO4, and filtered.
The filtrate was concentrated and purified using chromatography to yield the intermediate 9. 1H NMR (600 MHz,
DMSO-d6) δ 9.49 (s, 1H), 8.85 (s, 1H). MS (ESI) m/z(%): 296.5 [M+H]+.
5.1.3. General procedure for the synthesis of intermediates 10a-h.
Pd(dppf)Cl2 (0.1 equiv) was added to a solution of compound 9 (1 equiv), the corresponding boric acid or
borate (1.1 equiv), and K2CO3 (3 equiv) in 1,4-dioxane:H2O (4:1) under a nitrogen atmosphere. The mixture was
purged with nitrogen for 5 min and then heated at 90 °C until the reaction was complete. The mixture was diluted
with ethyl acetate, and the organic layer was washed with saline, dried over anhydrous Na2SO4 and filtered. The
filtrate was concentrated and purified using column chromatography to produce the coupling intermediates 10a-h.
1
5.1.3.1. 2-chloro-7-(2-methoxyphenyl)thieno[3,2-d]pyrimidine (10a). H NMR (600 MHz, DMSO-d6) δ 9.52 (s,
1H), 8.65 (s, 1H), 7.62–7.60 (m, 1H), 7.44 (t, J = 7.2 Hz, 1H), 7.19 (d, J = 8.3 Hz, 1H), 7.08 (d, J = 7.4 Hz, 1H),
3.77 (s, 3H). MS (ESI) m/z(%): 277.1 [M+H]+.
1
5.1.3.2. 2-chloro-7-(3-(methylsulfonyl)phenyl)thieno[3,2-d]pyrimidine (10d). H NMR (600 MHz, DMSO-d6) δ
9.61 (s, 1H), 9.07 (s, 1H), 8.54 (t, J = 1.5 Hz, 1H), 8.38 (d, J = 7.9 Hz, 1H), 7.99 (d, J = 8.2 Hz, 1H), 7.84 (t, J =
7.8 Hz, 1H), 3.30 (s, 3H). MS (ESI) m/z(%): 324.9 [M+H]+.
5.1.4. General procedure for the synthesis of compounds 11a-h.
Dimethyl (4-aminobenzyl)phosphonate (1.1 equiv), X-phos (0.1 equiv) and Cs2CO3 (3.0 equiv) in
1,4-dioxane, and Pd(AcO)2 (0.05 equiv) were added to solutions of compounds 10a-h (1 equiv) under a nitrogen
atmosphere. The mixture was purged with nitrogen for 5 min and then heated at 90 °C until the reaction was
complete. The mixture was diluted with ethyl acetate, and the organic layer was washed with saline, dried over
anhydrous Na2SO4, filtered and concentrated. The residue was purified using column chromatography to afford
compounds 11a-h.
5.1.4.1. dimethyl (4-((7-(2-methoxyphenyl)thieno[3,2-d]pyrimidin-2-yl)amino)benzyl)phosphonate (11a). White
1
solid; yield: 73%; mp: 139.6–141.2 °C; H NMR (600 MHz, DMSO-d6) δ 9.65 (s, 1H), 9.20 (s, 1H), 8.38 (s, 1H),
7.80 (dd, J = 7.5, 1.5 Hz, 1H), 7.77 (d, J = 8.3 Hz, 2H), 7.43 (dd, J = 11.5, 4.2 Hz, 1H), 7.19 (d, J = 8.1 Hz, 1H),
7.12–7.11 (m, 2H), 7.10–7.08 (m, 1H), 3.79 (s, 3H), 3.58 (d, J = 10.7 Hz, 6H), 3.17 (d, J = 21.1 Hz, 2H). 13C NMR
(150 MHz, DMSO-d6) δ 159.55, 157.55, 156.86, 153.62, 139.42 (d, J = 3.5 Hz), 136.10, 131.15, 131.03, 129.60 (d,
J = 6.5 Hz, 2C), 129.26, 124.04 (d, J = 9.1 Hz), 122.19, 121.80, 119.94, 118.39 (d, J = 1.9 Hz, 2C), 111.39, 55.47,
52.32 (d, J = 6.5 Hz, 2C), 30.39 (d, J = 135.2 Hz). HRMS calcd for C22H22N3O4PS, [M+Na]+, 478.0961; found
478.0946.
15

5.1.4.2. dimethyl (4-((7-(2-acetylphenyl)thieno[3,2-d]pyrimidin-2-yl)amino)benzyl)phosphonate (11b). Yellow
solid; yield: 68%; mp: 171.8–172.5 ; 1H NMR (600 MHz, DMSO-d6) δ 9.55 (s, 1H), 9.18 (s, 1H), 8.25 (s, 1H),
7.89 (d, J = 7.6 Hz, 1H), 7.66 (t, J = 7.3 Hz, 1H), 7.62 (d, J = 8.3 Hz, 2H), 7.58 (t, J = 7.5 Hz, 1H), 7.54 (d, J = 7.4
Hz, 1H), 7.09 (d, J = 6.7 Hz, 2H), 3.58 (d, J = 10.7 Hz, 6H), 3.16 (d, J = 21.1 Hz, 2H), 2.32 (s, 3H). 13C NMR (150
MHz, DMSO-d6) δ 200.95, 159.00, 157.66, 153.64, 139.92, 139.19 (d, J = 3.0 Hz), 135.62, 134.34, 132.03, 131.62,
131.30, 129.65, 129.58 (d, J = 6.5 Hz, 2C), 128.12, 124.27 (d, J = 9.3 Hz), 121.94, 118.60 (d, J = 1.9 Hz, 2C),
52.31 (d, J = 6.6 Hz, 2C), 30.40 (d, J = 135.1 Hz), 28.63. HRMS calcd for C23H22N3O4PS, [M+Na]+, 490.0961;
found 490.0963.
5.1.4.3. dimethyl (4-((7-(3-acetylphenyl)thieno[3,2-d]pyrimidin-2-yl)amino)benzyl)phosphonate (11c). Light
1
yellow solid; yield: 59%; mp: 89.2–90.1 ; H NMR (600 MHz, DMSO-d6) δ 9.76 (s, 1H), 9.26 (s, 1H), 8.67 (s,
1H), 8.57 (t, J = 1.4 Hz, 1H), 8.31 (d, J = 7.8 Hz, 1H), 8.02–8.00 (m, 1H), 7.80 (d, J = 8.4 Hz, 2H), 7.68 (t, J = 7.7
Hz, 1H), 7.15 (dd, J = 8.6, 2.2 Hz, 2H), 3.59 (d, J = 10.8 Hz, 6H), 3.18 (d, J = 21.1 Hz, 2H), 2.65 (s, 3H). 13C
NMR (150 MHz, DMSO-d6) δ 197.95, 158.25, 157.75, 154.19, 139.17 (d, J = 3.4 Hz), 137.15, 135.21, 133.97,
133.28, 132.67, 129.76 (d, J = 6.4 Hz, 2C), 128.89, 127.79, 127.38, 124.43 (d, J = 9.2 Hz), 122.82, 118.63 (d, J =
1.9 Hz, 2C), 52.33 (d, J = 6.6 Hz, 2C), 30.44 (d, J = 135.2 Hz), 26.88. HRMS calcd for C23H22N3O4PS, [M+Na]+,
490.0961; found 490.0948.
5.1.4.4. dimethyl (4-((7-(3-(methylsulfonyl)phenyl)thieno[3,2-d]pyrimidin-2-yl)amino)benzyl)phosphonate (11d).
1
Light yellow solid; yield: 76%; mp: 107.0–108.1 ; H NMR (600 MHz, DMSO-d6) δ 9.76 (s, 1H), 9.27 (s, 1H),
8.75 (s, 1H), 8.50 (s, 1H), 8.46 (d, J = 7.8 Hz, 1H), 7.98 (d, J = 7.8 Hz, 1H), 7.81 (t, J = 7.8 Hz, 1H), 7.77 (d, J =
8.3 Hz, 2H), 7.22 (dd, J = 8.5, 2.1 Hz, 2H), 3.60 (d, J = 10.7 Hz, 6H), 3.29 (s, 3H), 3.19 (d, J = 21.2 Hz, 2H). 13C
NMR (150 MHz, DMSO-d6) δ 158.04, 157.82, 154.29, 141.31, 139.02 (d, J = 2.6 Hz), 136.09, 134.63, 132.92,
132.32, 129.89 (d, J = 6.5 Hz, 2C), 129.59, 125.98, 125.96, 124.51 (d, J = 9.2 Hz), 122.83, 118.77 (2C), 52.34 (d,
J = 6.8 Hz, 2C), 43.56, 30.49 (d, J = 135.2 Hz). HRMS calcd for C22H22N3O5PS2, [M+Na]+, 526.0631; found
526.0641.
5.1.4.5. dimethyl(4-((7-(3-(methylcarbamoyl)phenyl)thieno[3,2-d]pyrimidin-2-yl)amino)benzyl)phosphonate (11e).
1
Light yellow solid; yield: 83%; mp: 98.3–99.0 ; H NMR (600 MHz, DMSO-d6) δ 9.75 (s, 1H), 9.25 (s, 1H),
8.61 (s, 1H), 8.54 (q, J = 4.1 Hz, 1H), 8.46 (s, 1H), 8.21 (d, J = 7.8 Hz, 1H), 7.86 (d, J = 7.8 Hz, 1H), 7.81 (d, J =
8.4 Hz, 2H), 7.60 (t, J = 7.7 Hz, 1H), 7.15 (dd, J = 8.6, 2.2 Hz, 2H), 3.59 (d, J = 10.7 Hz, 6H), 3.17 (d, J = 21.1 Hz,
2H), 2.84 (d, J = 4.5 Hz, 3H). 13C NMR (150 MHz, DMSO-d6) δ 166.67, 158.29, 157.70, 154.14, 139.15 (d, J =
2.9 Hz), 135.01, 134.79, 133.67, 133.52, 130.51, 129.72 (d, J = 6.5 Hz, 2C), 128.40, 126.88, 126.34, 124.30 (d, J =
9.2 Hz), 122.81, 118.68 (d, J = 1.9 Hz, 2C), 52.32 (d, J = 6.5 Hz, 2C), 30.54 (d, J = 135.3 Hz), 26.31. HRMS calcd
for C23H23N4O4PS, [M+Na]+, 505.1070; found 505.1065.
5.1.4.6. dimethyl (4-((7-(3-acetamidophenyl)thieno[3,2-d]pyrimidin-2-yl)amino)benzyl)phosphonate (11f). Light
yellow solid; yield: 56%; mp: 188.7–190.9 ; 1H NMR (600 MHz, DMSO-d6) δ 10.07 (s, 1H), 9.72 (s, 1H), 9.23
(s, 1H), 8.44 (s, 1H), 8.17 (s, 1H), 7.83 (d, J = 8.3 Hz, 2H), 7.67 (d, J = 7.7 Hz, 1H), 7.64 (d, J = 8.1 Hz, 1H), 7.43
(d, J = 7.9 Hz, 1H), 7.15 (dd, J = 8.5, 2.0 Hz, 2H), 3.59 (d, J = 10.7 Hz, 6H), 3.18 (d, J = 21.1 Hz, 2H), 2.11 (s,
3H). 13C NMR (150 MHz, DMSO-d6) δ 168.40, 158.36, 157.65, 154.05, 139.45, 139.21 (d, J = 2.7 Hz), 134.39,
134.33, 133.93, 129.66 (d, J = 6.4 Hz, 2C), 128.66, 124.23 (d, J = 9.2 Hz), 123.07, 122.84, 119.06, 118.66 (d, J =
1.9 Hz, 2C), 118.63, 52.32 (d, J = 6.5 Hz, 2C), 30.50 (d, J = 135.4 Hz), 24.02. HRMS calcd for C23H23N4O4PS,
[M+Na]+, 505.1070; found 505.1081.
16

5.1.4.7.
Dimethyl (4-((7-(3-(N-methylmethylsulfonamido)phenyl)thieno[3,2-d]pyrimidin-2-yl)amino)benzyl)phosphonate
(11g). Yellow solid; yield: 53%; mp: 185.9–186.5 ; 1H NMR (600 MHz, DMSO-d6) δ 9.73 (s, 1H), 9.24 (s, 1H),
8.62 (s, 1H), 8.07 (d, J = 7.8 Hz, 1H), 8.04–8.02 (m, 1H), 7.79 (d, J = 8.3 Hz, 2H), 7.56 (t, J = 7.9 Hz, 1H), 7.48
(dd, J = 8.0, 1.2 Hz, 1H), 7.20 (dd, J = 8.6, 2.2 Hz, 2H), 3.60 (d, J = 10.8 Hz, 6H), 3.33 (s, 3H), 3.20 (d, J = 21.2
Hz, 2H), 2.98 (s, 3H). 13C NMR (150 MHz, DMSO-d6) δ 158.24, 157.76, 154.19, 142.06, 139.16 (d, J = 2.7 Hz),
135.09, 134.43, 133.25, 129.79 (d, J = 6.5 Hz, 2C), 129.11, 126.66, 126.04, 125.44, 124.52 (d, J = 9.2 Hz), 122.90,
118.79 (d, J = 1.9 Hz, 2C), 52.36 (d, J = 6.5 Hz, 2C), 37.98, 35.26, 30.45 (d, J = 135.3 Hz). HRMS calcd for
C23H25N4O5PS2, [M+H]+, 533.1077; found 533.1085；[M+Na]+, 555.0896; found 555.0891.
5.1.4.8. dimethyl (4-((7-(4-methoxyphenyl)thieno[3,2-d]pyrimidin-2-yl)amino)benzyl)phosphonate (11h). Light
yellow solid; yield: 78%; mp: 128.2–128.8 ℃; 1H NMR (600 MHz, DMSO-d6) δ 9.70 (s, 1H), 9.21 (s, 1H), 8.45 (s,
1H), 8.07–8.06 (m, 2H), 7.81 (d, J = 8.3 Hz, 2H), 7.22–7.20 (m, 2H), 7.08–7.06 (m, 2H), 3.84 (s, 3H), 3.60 (d, J =
10.7 Hz, 6H), 3.21 (d, J = 21.2 Hz, 2H). 13C NMR (150 MHz, DMSO-d6) δ 158.92, 158.41, 157.63, 153.95, 139.26
(d, J = 3.3 Hz), 133.66, 132.62, 129.70 (d, J = 6.5 Hz, 2C), 129.14 (2C), 126.06, 124.41 (d, J = 9.2 Hz), 122.83,
118.80 (d, J = 1.8 Hz, 2C), 113.80 (2C), 55.21, 52.32 (d, J = 6.5 Hz, 2C), 30.41 (d, J = 135.2 Hz). HRMS calcd for
C22H22N3O4PS, [M+Na]+, 478.0961; found 478.0954.
5.1.5. Preparation of N-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)acetamide (14)
Triethylamine (1.2 equiv) and acetic anhydride (1.5 equiv) in CH2Cl2 were added to a solution of
3-bromoaniline (1 equiv). The reaction mixture was stirred at rt. Upon completion, the mixture was diluted with
water and extracted with CH2Cl2. The organic phases were combined, washed with saline, dried over anhydrous
Na2SO4 and evaporated to afford crude product 13. Pd(dppf)Cl2 (0.05 equiv) was added to a solution of
compound 13 (1 equiv), bis(pinacolato)diboron (1.2 equiv), and AcOK (3 equiv) in dioxane. The mixture was
degassed with nitrogen and heated at 80 °C overnight. The mixture was diluted with ethyl acetate, and the organic
layer was washed with saline, dried over anhydrous Na2SO4 and filtered. The filtrate was concentrated and
purified using chromatography to provide the coupling intermediate 14. MS (ESI) m/z(%): 262.1 [M+H]+.
5.1.6.Preparation of N-methyl-N-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methanesulfonamide (17)
Pyridine (2 equiv) was added to a solution of 3-bromoaniline (1 equiv) in CH2Cl2 at 0 °C; methanesulfonyl
chloride (1 equiv) was then added in a dropwise manner. After 3 h, 1 M HCl was added, and the mixture was
diluted with CH2Cl2. The organic phases were combined, washed with saline, dried over anhydrous Na2SO4 and
evaporated to afford crude product 15. Intermediate 15 (1 equiv) and K2CO3 (3 equiv) were dissolved in DMF,
and iodomethane (1.1 equiv) was then added in a dropwise manner at rt. After 2 h, water was added in a dropwise
manner, and the solution was extracted with CH2Cl2. The organic extracts were dried and concentrated in vacuo.
The extract was purified using chromatography to produce the intermediate 16, after which treatment with
bis(pinacolato)diboron and Pd(dppf)Cl2 provided the Suzuki coupling partner intermediate 17. MS (ESI) m/z(%):
312.2 [M+H]+.
5.1.7. Preparation of N-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzamide (20)
Oxalyl chloride (3 equiv) was added to a solution of 3-bromobenzoic acid (1 equiv) in CH2Cl2, to which a
catalytic amount of DMF (2 drops) was added. After being stirred at 60 °C for 4 h, the reaction mixture was
concentrated and dried under a vacuum. A suspension of CH3NH2•HCl (1.5 equiv) and Et3N (1.5 equiv) in
17

anhydrous CH2Cl2 was then added. The mixture was stirred at 25 °C for 4 h. The mixture was diluted with CH2Cl2,
and the organic layer was washed with saline, dried over anhydrous Na2SO4 and filtered. The filtrate was
concentrated and purified using chromatography to yield the intermediate 19, after which treatment with
bis(pinacolato)diboron and Pd(dppf)Cl2 provided the Suzuki coupling partner intermediate 20. MS (ESI) m/z(%):
262.1 [M+H]+.
5.1.8. Preparation of dimethyl (4-aminobenzyl)phosphonate (23)
To a solution of 1-(chloromethyl)-4-nitrobenzene (1 equiv) in trimethyl phosphite (1.5 equiv). The reaction
mixture was stirred at 120 °C for 10 h. Upon completion, the mixture was diluted with water and extracted with
CH2Cl2. The organic phases were combined, washed with saline, dried over anhydrous Na2SO4 and evaporated to
afford crude product 22. Intermediate 22 (1 equiv) was dissolved in ethanol, and Pd/C (0.1 equiv) was added. The
flask was flushed with H2 and stirred for 3 h at 40 °C. The reaction mixture was filtered through a Celite pad; the
1
filtrate was concentrated and purified using chromatography to produce intermediate 23. H NMR (600 MHz,
DMSO-d6) δ 6.91 (dd, J = 8.4, 2.2 Hz, 2H), 6.50 (d, J = 8.2 Hz, 2H), 4.98 (s, 2H), 3.56 (d, J = 10.7 Hz, 6H), 3.02
(d, J = 20.6 Hz, 2H). MS (ESI) m/z(%): 216.0 [M+H]+.
5.1.9. Preparation of compounds 24a-j, 26a-i, 27a-b and 28c-g.
Compounds 24a-j, 26a-i, 27a-b and 28c-g were prepared in a similar manner as compounds 11a-h.
5.1.9.1. diethyl (4-((7-(2-methoxyphenyl)thieno[3,2-d]pyrimidin-2-yl)amino)benzyl)phosphonate (24a). White
solid; yield: 67%; mp: 141.2–142.1 ; 1H NMR (600 MHz, DMSO-d6) δ 9.67 (s, 1H), 9.20 (s, 1H), 8.37 (s, 1H),
7.79 (d, J = 6.7 Hz, 3H), 7.41 (t, J = 7.6 Hz, 1H), 7.17 (d, J = 8.3 Hz, 1H), 7.13 (d, J = 7.6 Hz, 2H), 7.09 (t, J = 7.4
Hz, 1H), 3.95–3.91 (m, 4H), 3.79 (s, 3H), 3.13 (d, J = 21.1 Hz, 2H), 1.17 (t, J = 7.0 Hz, 6H). 13C NMR (150 MHz,
DMSO-d6) δ 159.54, 157.54, 156.87, 153.63, 139.37 (d, J = 3.3 Hz), 136.08, 131.16, 131.07, 129.64 (d, J = 6.4 Hz,
2C), 129.25, 124.27 (d, J = 9.2 Hz), 122.20, 121.78, 119.92, 118.22 (2C), 111.36, 61.28 (d, J = 6.5 Hz, 2C), 55.46,
31.53 (d, J = 135.7 Hz), 16.25 (d, J = 5.3 Hz, 2C). HRMS calcd for C24H26N3O4PS, [M+Na]+, 506.1274; found
506.1259.
5.1.9.2. ethyl P-(4-((7-(2-methoxyphenyl)thieno[3,2-d]pyrimidin-2-yl)amino)benzyl)-N-methylphosphonamidate
(24b). White solid; yield: 58%; mp: 116.7–117.4 ; 1H NMR (600 MHz, DMSO-d6) δ 9.63 (s, 1H), 9.19 (s, 1H),
8.37 (s, 1H), 7.81 (d, J = 7.3 Hz, 1H), 7.76 (d, J = 6.6 Hz, 2H), 7.42 (t, J = 7.7 Hz, 1H), 7.18 (d, J = 8.3 Hz, 1H),
7.12–7.08 (m, 3H), 4.34 (s, 1H), 3.88–3.83 (m, 2H), 3.79 (s, 3H), 2.97 (dd, J = 19.6, 4.6 Hz, 2H), 2.38 (dd, J =
11.3, 4.8 Hz, 3H), 1.19 (t, J = 7.0 Hz, 3H). 13C NMR (150 MHz, DMSO-d6) δ 159.56, 157.58, 156.85, 153.58,
139.04 (d, J = 2.6 Hz), 136.04, 131.14, 131.00, 129.61 (d, J = 6.1 Hz, 2C), 129.23, 125.60 (d, J = 8.0 Hz), 122.19,
121.68, 119.92, 118.18 (d, J = 1.7 Hz, 2C), 111.37, 58.70 (d, J = 6.5 Hz), 55.47, 32.81 (d, J = 123.7 Hz), 26.67,
16.36 (d, J = 6.1 Hz). HRMS calcd for C23H25N4O3PS, [M+Na]+, 491.1277; found 491.1275.
5.1.9.3. diethyl (hydroxy(4-((7-(2-methoxyphenyl)thieno[3,2-d]pyrimidin-2-yl)amino)phenyl)methyl)phosphonate
1
(24c). White solid; yield: 43%; mp: 143.2–144.1 ; H NMR (600 MHz, DMSO-d6) δ 9.70 (s, 1H), 9.21 (s, 1H),
8.38 (s, 1H), 7.83–7.78 (m, 3H), 7.45–7.41 (m, 1H), 7.26 (d, J = 7.1 Hz, 2H), 7.19 (d, J = 8.2 Hz, 1H), 7.09 (t, J =
7.4 Hz, 1H), 6.04 (dd, J = 16.0, 5.7 Hz, 1H), 4.83 (dd, J = 12.4, 5.7 Hz, 1H), 3.98–3.85 (m, 4H), 3.79 (s, 3H), 1.19
(d, J = 7.1 Hz, 3H), 1.14 (d, J = 7.1 Hz, 3H). 13C NMR (150 MHz, DMSO-d6) δ 159.51, 157.50, 156.86, 153.63,
140.24 (d, J = 2.2 Hz), 136.09, 131.14, 131.10, 130.60, 129.27 (2C), 127.53 (d, J = 6.0 Hz), 122.20, 121.85,
119.91, 117.62 (2C), 111.36, 69.09 (d, J = 163.9 Hz), 61.89 (dd, J = 48.2, 6.6 Hz, 2C), 55.45, 16.33 (dd, J = 14.3,
5.2 Hz, 2C). HRMS calcd for C24H26N3O5PS, [M+Na]+, 522.1223; found 522.1227.
18

5.1.9.4. ethyl (4-((7-(2-methoxyphenyl)thieno[3,2-d]pyrimidin-2-yl)amino)benzyl)(morpholino)phosphinate (24d).
White solid; yield: 36%; mp: 108.7–109.5 ; 1H NMR (600 MHz, DMSO-d6) δ 9.66 (s, 1H), 9.19 (s, 1H), 8.37 (s,
1H), 7.80 (t, J = 7.6 Hz, 3H), 7.42 (t, J = 7.2 Hz, 1H), 7.17 (d, J = 8.3 Hz, 1H), 7.13 (d, J = 6.9 Hz, 2H), 7.09 (t, J
= 7.4 Hz, 1H), 3.93–3.85 (m, 2H), 3.79 (s, 3H), 3.46–3.42 (m, 2H), 3.09–3.03 (m, 2H), 2.97–2.93 (m, 2H),
2.86–2.82 (m, 2H), 1.22 (d, J = 7.0 Hz, 3H). 13C NMR (150 MHz, DMSO-d6) δ 159.56, 157.58, 156.88, 153.63,
139.31 (d, J = 2.7 Hz), 136.08, 131.17, 131.08, 129.79 (d, J = 6.2 Hz, 2C), 129.28, 124.82 (d, J = 8.3 Hz), 122.22,
121.78, 119.94, 118.14 (2C), 111.37, 66.46 (d, J = 4.5 Hz, 2C), 59.09 (d, J = 6.9 Hz), 55.48, 43.77 (2C), 31.90 (d, J
= 125.7 Hz), 16.26 (d, J = 6.0 Hz). HRMS calcd for C26H29N4O4PS, [M+Na]+, 547.1539; found 547.1545.
5.1.9.5. 4-((7-(2-methoxyphenyl)thieno[3,2-d]pyrimidin-2-yl)amino)-N-methylbenzamide (24e). White solid; yield:
1
76%; mp: 210.5–211.3 ; H NMR (600 MHz, DMSO-d6) δ 9.98 (s, 1H), 9.26 (s, 1H), 8.40 (s, 1H), 8.25 (d, J =
4.4 Hz, 1H), 7.92 (d, J = 8.7 Hz, 2H), 7.77 (dd, J = 7.4, 1.3 Hz, 1H), 7.72 (d, J = 8.7 Hz, 2H), 7.47–7.44 (m, 1H),
7.21 (d, J = 8.3 Hz, 1H), 7.13 (t, J = 7.4 Hz, 1H), 3.80 (s, 3H), 2.77 (d, J = 4.4 Hz, 3H). 13C NMR (150 MHz,
DMSO-d6) δ 166.27, 159.40, 157.20, 156.86, 153.74, 143.43, 136.35, 131.26, 131.14, 129.40, 127.61 (2C), 126.53,
122.46, 122.12, 120.01, 117.14 (2C), 111.39, 55.48, 26.15. HRMS calcd for C21H18N4O2S, [M+H]+, 391.1223;
found 391.1226.
5.1.9.6. 4-((7-(2-methoxyphenyl)thieno[3,2-d]pyrimidin-2-yl)amino)-N-(piperidin-4-yl)benzamide Hydrochloride
(24f). Light yellow solid; yield: 87%; mp: 211.7–212.4 ; 1H NMR (600 MHz, DMSO-d6) δ 10.00 (s, 1H), 9.27 (s,
1H), 9.11 (s, 2H), 8.42 (s, 1H), 8.34 (d, J = 7.4 Hz, 1H), 7.91 (d, J = 8.8 Hz, 2H), 7.79–7.76 (m, 3H), 7.47–7.44 (m,
1H), 7.21 (d, J = 8.2 Hz, 1H), 7.13 (t, J = 7.4 Hz, 1H), 4.06–4.02 (m, 1H), 3.79 (s, 3H), 3.29 (d, J = 12.5 Hz, 2H),
2.97 (dd, J = 8.0, 3.6 Hz, 2H), 1.95 (d, J = 10.9 Hz, 2H), 1.83 (dd, J = 18.3, 7.9 Hz, 2H). 13C NMR (150 MHz,
DMSO-d6) δ 165.55, 159.46, 157.03, 156.88, 153.65, 143.58, 136.73, 131.24, 131.12, 129.48, 128.11 (2C), 126.37,
122.54, 122.10, 120.07, 117.05 (2C), 111.47, 55.56, 44.37, 42.16 (2C), 28.24 (2C). HRMS calcd for C25H25N5O2S,
[M+H]+, 460.1802; found 460.1798.
5.1.9.7. N-(4-((7-(2-methoxyphenyl)thieno[3,2-d]pyrimidin-2-yl)amino)phenyl)piperidine-4-carboxamide (24g).
1
Light yellow solid; yield: 83%; mp: 250.1–251.2 ; H NMR (400 MHz, DMSO-d6) δ 9.68 (s, 1H), 9.57 (s, 1H),
9.17 (s, 1H), 8.36 (s, 1H), 7.80 (d, J = 6.8 Hz, 1H), 7.74 (d, J = 8.8 Hz, 2H), 7.44 (dd, J = 14.4, 8.1 Hz, 3H), 7.18
(d, J = 8.3 Hz, 1H), 7.08 (t, J = 7.4 Hz, 1H), 3.79 (s, 3H), 2.97 (d, J = 11.7 Hz, 2H), 2.46 (d, J = 11.9 Hz, 2H),
2.40–2.33 (m, 1H), 1.96 (s, 1H), 1.64 (t, J = 11.2 Hz, 2H), 1.50 (dt, J = 12.0, 8.7 Hz, 2H). 13C NMR (150 MHz,
DMSO-d6) δ 173.24, 159.55, 157.57, 156.85, 153.57, 136.15, 135.96, 133.13, 131.16, 131.01, 129.24, 122.23,
121.52, 119.89, 119.30 (2C), 118.48 (2C), 111.35, 55.47, 45.67 (2C), 43.65, 29.54 (2C). HRMS calcd for
C25H25N5O2S, [M+H]+, 460.1802; found 460.1805.
5.1.9.8.
(S)-N-(4-((7-(2-methoxyphenyl)thieno[3,2-d]pyrimidin-2-yl)amino)phenyl)piperidine-3-carboxamide
(24h). Light yellow solid; yield: 90%; mp: 219.1–220.2 ; 1H NMR (400 MHz, DMSO-d6) δ 9.78 (s, 1H), 9.57 (s,
1H), 9.17 (s, 1H), 8.35 (s, 1H), 7.80 (d, J = 7.4 Hz, 1H), 7.74 (d, J = 8.9 Hz, 2H), 7.44 (d, J = 9.0 Hz, 3H), 7.18 (d,
J = 8.2 Hz, 1H), 7.08 (t, J = 7.4 Hz, 1H), 3.79 (s, 3H), 2.99 (d, J = 11.8 Hz, 1H), 2.84 (d, J = 11.6 Hz, 1H), 2.61 (d,
J = 10.5 Hz, 1H), 2.42–2.33 (m, 2H), 1.85 (d, J = 9.2 Hz, 1H), 1.62–1.55 (m, 2H), 1.41–1.35 (m, 1H). 13C NMR
(150 MHz, DMSO-d6) δ 172.51, 159.55, 157.57, 156.85, 153.57, 136.20, 135.97, 133.00, 131.16, 131.01, 129.25,
122.23, 121.53, 119.90, 119.33 (2C), 118.48 (2C), 111.35, 55.48, 49.08, 45.94, 44.26, 27.89, 25.31. HRMS calcd
for C25H25N5O2S, [M+H]+, 460.1802; found 460.1813.
19

5.1.9.9. (R)-N-(4-((7-(2-methoxyphenyl)thieno[3,2-d]pyrimidin-2-yl)amino)phenyl)piperidine-3-carboxamide (24i).
Light yellow solid; yield: 82%; mp: 220.0–220.8 ; 1H NMR (600 MHz, MeOD) δ 8.97 (s, 1H), 8.23 (s, 1H), 7.87
(dd, J = 7.5, 1.6 Hz, 1H), 7.75 (d, J = 9.0 Hz, 2H), 7.42–7.36 (m, 3H), 7.13 (d, J = 8.1 Hz, 1H), 7.08 (td, J = 7.5,
0.9 Hz, 1H), 3.82 (s, 3H), 3.10 (dd, J = 12.4, 3.1 Hz, 1H), 2.97 (d, J = 12.4 Hz, 1H), 2.84–2.78 (m, 1H), 2.62 (td, J
= 12.5, 2.7 Hz, 1H), 2.54–2.49 (m, 1H), 2.02–1.97 (m, 1H), 1.78–1.70 (m, 2H), 1.60–1.54 (m, 1H). 13C NMR (150
MHz, MeOD) δ 175.08, 161.46, 159.06, 158.65, 154.13, 138.58, 136.80, 133.50, 133.03, 132.52, 130.27, 123.83,
123.71, 121.83 (2C), 121.21, 120.01 (2C), 112.24, 56.03, 46.71, 45.37, 29.06, 25.97. HRMS calcd for
C25H25N5O2S, [M+H]+, 460.1802; found 460.1810.
5.1.9.10. 2-amino-N-(4-((7-(2-methoxyphenyl)thieno[3,2-d]pyrimidin-2-yl)amino)phenyl)acetamide (24j). Light
yellow solid; yield: 81%; mp: 205.4–206.3 ; 1H NMR (400 MHz, DMSO-d6) δ 9.60 (s, 1H), 9.17 (s, 1H), 8.36 (s,
1H), 7.81–7.75 (m, 3H), 7.49 (d, J = 8.9 Hz, 2H), 7.43 (dd, J = 11.2, 4.4 Hz, 1H), 7.18 (d, J = 8.2 Hz, 1H), 7.08 (t,
J = 7.4 Hz, 1H), 3.79 (s, 3H), 3.24 (s, 2H), 1.94 (s, 2H). 13C NMR (150 MHz, DMSO-d6) δ 171.48, 159.56, 157.57,
156.85, 153.58, 136.37, 135.98, 132.46, 131.16, 131.01, 129.24, 122.21, 121.56, 119.91, 119.28 (2C), 118.65 (2C),
111.35, 55.47, 45.44. HRMS calcd for C21H19N5O2S, [M+Na]+, 428.1152; found 428.1150.
5.1.9.11.
3-fluoro-4-((7-(2-methoxyphenyl)thieno[3,2-d]pyrimidin-2-yl)amino)-N-(piperidin-4-yl)benzamide
Hydrochloride (26a). White solid; yield: 79%; mp: 222.5–223.3 ; 1H NMR (600 MHz, DMSO-d6) δ 9.32 (s, 1H),
9.28 (s, 1H), 9.09 (s, 2H), 8.50 (d, J = 7.4 Hz, 1H), 8.45 (s, 1H), 8.28 (t, J = 8.4 Hz, 1H), 7.79 (dd, J = 12.3, 1.9 Hz,
1H), 7.77 (dd, J = 7.5, 1.7 Hz, 1H), 7.66 (dd, J = 8.5, 1.7 Hz, 1H), 7.43–7.40 (m, 1H), 7.18 (d, J = 7.9 Hz, 1H),
7.07 (td, J = 7.5, 0.9 Hz, 1H), 4.08–4.02 (m, 1H), 3.79 (s, 3H), 3.30 (d, J = 12.7 Hz, 2H), 3.01–2.96 (m, 2H), 1.95
(dd, J = 13.4, 2.8 Hz, 2H), 1.86–1.80 (m, 2H). 13C NMR (150 MHz, DMSO-d6) δ 164.31, 159.52, 156.91, 156.84,
153.71, 152.51 (d, J = 245.3 Hz), 137.12, 131.07, 131.00, 130.97, 129.39, 128.42 (d, J = 6.2 Hz), 123.53 (d, J =
1.9 Hz), 123.13, 121.92, 121.23, 120.05, 114.24 (d, J = 21.1 Hz), 111.45, 55.56, 44.52, 42.12 (2C), 28.14 (2C).
HRMS calcd for C25H24FN5O2S, [M+H]+, 478.1708; found 478.1709.
5.1.9.12. 4-((7-(2-methoxyphenyl)thieno[3,2-d]pyrimidin-2-yl)amino)-3-methyl-N-(piperidin-4-yl)benzamide (26b).
1
Light yellow solid; yield: 82%; mp: 224.6–225.1 ; H NMR (400 MHz, DMSO-d6) δ 9.10 (s, 1H), 8.33 (s, 1H),
7.96 (t, J = 9.5 Hz, 2H), 7.81 (dd, J = 7.6, 1.7 Hz, 1H), 7.67 (d, J = 1.3 Hz, 1H), 7.58 (dd, J = 8.5, 1.9 Hz, 1H),
7.37 (dd, J = 11.2, 4.5 Hz, 1H), 7.14 (d, J = 8.1 Hz, 1H), 7.02 (td, J = 7.5, 0.8 Hz, 1H), 3.83–3.78 (m, 4H), 2.95 (d,
J = 11.6 Hz, 2H), 2.48–2.45 (m, 2H), 2.29 (s, 3H), 1.72 (dd, J = 11.7, 2.1 Hz, 2H), 1.46–1.35 (m, 2H). 13C NMR
(150 MHz, DMSO-d6) δ 165.54, 159.73, 156.84, 156.60, 152.82, 140.57, 138.49, 131.07, 130.90, 129.49, 129.44,
129.07, 128.79, 125.37, 122.28, 121.76, 121.50, 120.06, 111.46, 55.57, 44.42, 42.15 (2C), 28.21 (2C), 18.19.
HRMS calcd for C26H27N5O2S, [M+H]+, 474.1958; found 474.1958.
5.1.9.13.
3-methoxy-4-((7-(2-methoxyphenyl)thieno[3,2-d]pyrimidin-2-yl)amino)-N-(piperidin-4-yl)benzamide
1
(26c). Yellow solid; yield: 86%; mp: 109.1–111.0 ; H NMR (600 MHz, DMSO-d6) δ 9.26 (s, 1H), 8.53 (d, J =
8.4 Hz, 1H), 8.44 (s, 1H), 8.15 (s, 1H), 8.13 (d, J = 7.9 Hz, 1H), 7.79 (dd, J = 7.5, 1.3 Hz, 1H), 7.51 (s, 1H),
7.48–7.44 (m, 1H), 7.42 (dd, J = 8.5, 1.3 Hz, 1H), 7.21 (d, J = 8.3 Hz, 1H), 7.13 (t, J = 7.4 Hz, 1H), 3.95 (s, 3H),
3.86–3.77 (m, 4H), 2.96 (d, J = 12.1 Hz, 2H), 2.51–2.48 (m, 2H), 1.73 (d, J = 9.8 Hz, 2H), 1.46–1.40 (m, 2H). 13
C
NMR (150 MHz, DMSO-d6) δ 164.85, 159.37, 156.82, 156.71, 153.96, 147.00, 136.80, 131.63, 131.10, 131.06,
129.65, 129.46, 127.47, 122.97, 121.96, 120.05, 116.48, 111.46, 109.28, 56.09, 55.53, 47.44, 45.46 (2C), 33.16
(2C). HRMS calcd for C26H27N5O3S, [M+H]+, 490.1907; found 490.1911.
20

5.1.9.14. 3-ethoxy-4-((7-(2-methoxyphenyl)thieno[3,2-d]pyrimidin-2-yl)amino)-N-(piperidin-4-yl)benzamide (26d).
1
Light yellow solid; yield: 78%; mp: 200.9–201.7 ; H NMR (600 MHz, DMSO-d6) δ 9.27 (s, 1H), 8.55 (d, J =
8.4 Hz, 1H), 8.45 (s, 1H), 8.12–8.09 (m, 2H), 7.79 (dd, J = 7.5, 1.6 Hz, 1H), 7.50 (d, J = 1.4 Hz, 1H), 7.48–7.45
(m, 1H), 7.41 (dd, J = 8.5, 1.4 Hz, 1H), 7.22 (d, J = 8.2 Hz, 1H), 7.14 (t, J = 7.4 Hz, 1H), 4.20 (q, J = 6.9 Hz, 2H),
3.83–3.80 (m, 4H), 2.96 (d, J = 11.3 Hz, 2H), 2.50–2.46 (m, 2H), 1.73 (d, J = 10.7 Hz, 2H), 1.47–1.41 (m, 5H).
13C NMR (100 MHz, DMSO-d6) δ 164.79, 159.32, 156.81, 156.60, 153.92, 146.04, 136.75, 131.67, 131.49, 131.11,
131.03, 129.44, 127.36, 122.96, 121.95, 120.02, 116.30, 111.46, 110.08, 64.30, 55.50, 47.47, 45.48 (2C), 33.19
(2C), 14.60. HRMS calcd for C27H29N5O3S, [M+H]+, 504.2064; found 504.2064.
5.1.9.15.
4-((7-(2-methoxyphenyl)thieno[3,2-d]pyrimidin-2-yl)amino)-N-(piperidin-4-yl)-3-(trifluoromethyl)benzamide
(26e). Light yellow solid; yield: 84%; mp: 99.3–100.5 ; 1H NMR (400 MHz, CDCl3) δ 8.95 (s, 1H), 8.89 (d, J =
8.7 Hz, 1H), 8.19 (s, 1H), 8.01 (s, 1H), 7.86 (t, J = 8.0 Hz, 2H), 7.72 (s, 1H), 7.38 (t, J = 7.5 Hz, 1H), 7.11–7.01 (m,
2H), 6.36 (d, J = 7.4 Hz, 1H), 4.08–4.02 (m, 1H), 3.81 (s, 3H), 3.08 (d, J = 12.0 Hz, 2H), 2.69 (d, J = 11.5 Hz, 2H),
2.00 (d, J = 10.8 Hz, 2H), 1.47–1.40 (m, 2H). 13C NMR (150 MHz, DMSO-d6) δ 163.71, 159.50, 157.67, 156.82,
154.00, 140.25, 137.03, 131.72, 131.48, 131.05, 130.86, 129.50, 129.29, 125.52, 125.31, 123.94 (q, J = 273.3 Hz),
123.39, 121.89, 120.01, 111.47, 55.54, 47.01, 44.70 (2C), 31.97 (2C). HRMS calcd for C26H24F3N5O2S, [M+H]+,
528.1676; found 528.1685.
5.1.9.16. 2-fluoro-4-((7-(2-methoxyphenyl)thieno[3,2-d]pyrimidin-2-yl)amino)-N-(piperidin-4-yl)benzamide (26f).
Light yellow solid; yield: 88%; mp: 131.0–132.1 ; 1H NMR (400 MHz, DMSO-d6) δ 10.17 (s, 1H), 9.28 (s, 1H),
8.41 (s, 1H), 8.09 (d, J = 13.8 Hz, 1H), 7.80 (dd, J = 7.7, 3.1 Hz, 1H), 7.74 (dd, J = 7.5, 1.7 Hz, 1H), 7.51–7.42 (m,
3H), 7.19 (d, J = 8.2 Hz, 1H), 7.11 (td, J = 7.5, 0.7 Hz, 1H), 3.81–3.77 (m, 4H), 2.94 (d, J = 12.0 Hz, 2H),
2.53–2.47 (m, 2H), 1.77–1.72 (m, 2H), 1.43–1.35 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 162.67 (d, J = 1.6
Hz), 159.78 (d, J = 245.2 Hz), 159.25, 156.92, 156.81, 153.83, 144.42 (d, J = 12.2 Hz), 136.53, 131.41, 131.00,
130.16 (d, J = 4.4 Hz), 129.44, 122.90, 122.07, 120.10, 115.61 (d, J = 14.2 Hz), 113.51, 111.41, 104.41 (d, J = 29.4
Hz), 55.41, 47.25, 45.11 (2C), 32.83 (2C). HRMS calcd for C25H24FN5O2S, [M+H]+, 478.1708; found 478.1716.
5.1.9.17. 4-((7-(2-methoxyphenyl)thieno[3,2-d]pyrimidin-2-yl)amino)-2-methyl-N-(piperidin-4-yl)benzamide (26g).
Light yellow solid; yield: 83%; mp: 193.0–193.8 ; 1H NMR (400 MHz, CDCl3) δ 8.93 (s, 1H), 8.19 (s, 1H), 7.97
(d, J = 7.3 Hz, 1H), 7.69 (s, 1H), 7.47–7.38 (m, 3H), 7.28 (d, J = 8.6 Hz, 1H), 7.12 (t, J = 7.4 Hz, 1H), 7.06 (d, J =
8.3 Hz, 1H), 5.74 (d, J = 7.9 Hz, 1H), 4.10–4.01 (m, 1H), 3.84 (s, 3H), 3.12 (d, J = 12.2 Hz, 2H), 2.76 (t, J = 11.3
Hz, 2H), 2.41 (s, 3H), 2.05 (d, J = 10.6 Hz, 2H), 1.42 (d, J = 8.8 Hz, 2H). 13C NMR (150 MHz, DMSO-d6) δ
168.22, 159.44, 157.38, 156.83, 153.70, 141.63, 136.26, 135.82, 131.08, 130.97, 129.66, 129.25, 127.69, 122.23,
122.15, 120.14, 119.48, 114.90, 111.51, 55.50, 45.87, 44.09 (2C), 31.24 (2C), 20.10. HRMS calcd for
C26H27N5O2S, [M+H]+, 474.1958; found 474.1913.
5.1.9.18.
2-fluoro-5-methoxy-4-((7-(2-methoxyphenyl)thieno[3,2-d]pyrimidin-2-yl)amino)-N-(piperidin-4-yl)benzamide
Hydrochloride (26h). Light yellow solid; yield: 87%; mp: 207.9–208.5 ; 1H NMR (400 MHz, DMSO-d6) δ 9.33
(s, 1H), 9.13–9.03 (m, 2H), 8.48 (t, J = 6.7 Hz, 2H), 8.32 (s, 1H), 8.21 (dd, J = 7.2, 1.9 Hz, 1H), 7.73 (dd, J = 7.5,
1.6 Hz, 1H), 7.49–7.46 (m, 1H), 7.23–7.16 (m, 2H), 7.10 (t, J = 7.4 Hz, 1H), 4.05–4.01 (m, 1H), 3.93 (s, 3H), 3.79
(s, 3H), 3.27 (d, J = 12.5 Hz, 2H), 2.99 (d, J = 11.2 Hz, 2H), 2.02–1.95 (m, 2H), 1.83–1.73 (m, 2H). 13C NMR (150
21

MHz, DMSO-d6) δ 163.09, 159.35, 156.77, 155.68, 155.31 (d, J = 234.2 Hz), 153.64, 143.30, 137.96, 132.04 (d, J
= 12.6 Hz), 131.42, 130.97, 129.65, 123.43, 121.81, 120.20, 114.69 (d, J = 15.5 Hz), 111.49, 110.51 (d, J = 3.8 Hz),
104.88 (d, J = 32.4 Hz), 56.54, 55.45, 44.40, 41.95 (2C), 28.03 (2C). HRMS calcd for C26H26FN5O3S, [M+H]+,
508.1813; found 508.1828.
5.1.9.19.
2,5-difluoro-4-((7-(2-methoxyphenyl)thieno[3,2-d]pyrimidin-2-yl)amino)-N-(piperidin-4-yl)benzamide
1
Hydrochloride (26i). Light yellow solid; yield: 80%; mp: 235.4–236.6 ; H NMR (600 MHz, DMSO-d6) δ 9.49
(s, 1H), 9.33 (s, 1H), 9.12–9.00 (m, 2H), 8.46 (s, 1H), 8.41 (dd, J = 13.0, 6.6 Hz, 1H), 8.35 (d, J = 7.1 Hz, 1H),
7.71 (dd, J = 7.5, 1.7 Hz, 1H), 7.46–7.41 (m, 2H), 7.18 (d, J = 7.9 Hz, 1H), 7.08 (td, J = 7.5, 0.8 Hz, 1H),
4.04–3.98 (m, 1H), 3.79 (s, 3H), 3.26 (d, J = 12.7 Hz, 2H), 2.98 (q, J = 12.0 Hz, 2H), 1.97 (dd, J = 13.4, 3.0 Hz,
2H), 1.80–1.73 (m, 2H). 13C NMR (150 MHz, DMSO-d6) δ 162.22, 159.20, 156.79, 156.49, 155.21 (d, J = 243.7
Hz), 153.99, 147.83 (d, J = 241.8 Hz), 137.10, 131.59 (t, J = 12.5 Hz), 131.42, 131.01, 129.54, 123.65, 121.95,
120.17, 115.95 (dd, J = 16.7, 6.0 Hz), 115.37 (dd, J = 22.9, 4.3 Hz), 111.44, 107.84 (d, J = 31.9 Hz), 55.45, 44.33,
41.89 (2C), 27.93 (2C). HRMS calcd for C25H23F2N5O2S, [M+H]+, 496.1613; found 496.1617.
5.1.9.20.
2-fluoro-4-((7-(2-methoxyphenyl)thieno[3,2-d]pyrimidin-2-yl)amino)-N-(tetrahydro-2H-pyran-4-yl)benzamide
(27a). Light yellow solid; yield: 88%; mp: 230.1–231.0 ; 1H NMR (400 MHz, DMSO-d6) δ 10.16 (s, 1H), 9.28 (s,
1H), 8.41 (s, 1H), 8.09 (d, J = 14.4 Hz, 1H), 7.90 (d, J = 5.3 Hz, 1H), 7.73 (d, J = 7.1 Hz, 1H), 7.53–7.43 (m, 3H),
7.20 (d, J = 7.9 Hz, 1H), 7.11 (t, J = 7.1 Hz, 1H), 3.97 (s, 1H), 3.86 (d, J = 10.4 Hz, 2H), 3.79 (s, 3H), 3.42–3.38
(m, 2H), 1.76 (d, J = 11.2 Hz, 2H), 1.58–1.50 (m, 2H). 13C NMR (150 MHz, DMSO-d6) δ 162.86, 159.80 (d, J =
245.8 Hz), 159.26, 156.91, 156.82, 153.86, 144.52 (d, J = 12.2 Hz), 136.57, 131.45, 131.01, 130.19 (d, J = 4.1 Hz),
129.48, 122.94, 122.08, 120.13, 115.45 (d, J = 14.2 Hz), 113.55, 111.44, 104.40 (d, J = 29.4 Hz), 66.04 (2C),
55.43, 45.63, 32.34 (2C). HRMS calcd for C25H23FN4O3S, [M+Na]+, 501.1369; found 501.1362.
5.1.9.21.
2-fluoro-4-((7-(2-methoxyphenyl)thieno[3,2-d]pyrimidin-2-yl)amino)-N-(1-methylpiperidin-4-yl)benzamide (27b).
Yellow solid; yield: 83%; mp: 222.5–223.2 ; 1H NMR (600 MHz, DMSO-d6) δ 10.17 (s, 1H), 9.29 (s, 1H), 8.41
(s, 1H), 8.09 (d, J = 14.1 Hz, 1H), 7.87 (d, J = 5.4 Hz, 1H), 7.73 (dd, J = 7.4, 1.1 Hz, 1H), 7.51–7.43 (m, 3H), 7.20
(d, J = 8.3 Hz, 1H), 7.11 (t, J = 7.4 Hz, 1H), 3.79 (s, 3H), 3.76–3.72 (m, 1H), 2.82 (d, J = 10.7 Hz, 2H), 2.23 (s,
3H), 2.10 (d, J = 9.0 Hz, 2H), 1.79 (d, J = 10.7 Hz, 2H), 1.61–1.55 (m, 2H). 13C NMR (150 MHz, DMSO-d6) δ
163.00, 159.79 (d, J = 245.4 Hz), 159.27, 156.92, 156.82, 153.89, 144.50 (d, J = 12.4 Hz), 136.61, 131.45, 131.03,
130.18 (d, J = 4.0 Hz), 129.49, 122.94, 122.08, 120.13, 115.52 (d, J = 14.1 Hz), 113.53, 111.44, 104.40 (d, J = 29.2
Hz), 55.44, 54.00 (2C), 45.98, 45.45, 30.93 (2C). HRMS calcd for C26H26FN5O2S, [M+H]+, 492.1864; found
492.1874.
5.1.9.22. (S)-2-fluoro-4-((7-(2-methoxyphenyl)thieno[3,2-d]pyrimidin-2-yl)amino)-N-(pyrrolidin-3-yl)benzamide
(28c). Light yellow solid; yield: 76%; mp: 162.8–163.6 ; 1H NMR (600 MHz, DMSO-d6) δ 10.18 (s, 1H), 9.28 (s,
1H), 8.41 (s, 1H), 8.09 (d, J = 14.4 Hz, 1H), 7.98 (d, J = 4.8 Hz, 1H), 7.73 (d, J = 7.2 Hz, 1H), 7.50 (d, J = 8.3 Hz,
1H), 7.47–7.44 (m, 2H), 7.20 (d, J = 8.3 Hz, 1H), 7.11 (t, J = 7.4 Hz, 1H), 4.31–4.27 (m, 1H), 3.79 (s, 3H), 2.97
(dd, J = 11.1, 6.6 Hz, 1H), 2.91 (dd, J = 11.8, 5.1 Hz, 1H), 2.75 (dd, J = 11.5, 5.0 Hz, 1H), 2.66 (dd, J = 11.1, 3.8
Hz, 1H), 2.01–1.95 (m, 1H), 1.63–1.60 (m, 1H). 13C NMR (150 MHz, DMSO-d6) δ 163.30, 159.86 (d, J = 245.6
Hz), 159.28, 156.93, 156.84, 153.91, 144.56 (d, J = 12.0 Hz), 136.62, 131.48, 131.04, 130.24 (d, J = 4.0 Hz),
129.52, 122.97, 122.09, 120.15, 115.37 (d, J = 14.2 Hz), 113.56, 111.44, 104.41 (d, J = 29.6 Hz), 55.44, 52.89,
22

50.57, 45.40, 32.61. HRMS calcd for C24H22FN5O2S, [M+H]+, 464.1551; found 464.1561.
5.1.9.23. (R)-2-fluoro-4-((7-(2-methoxyphenyl)thieno[3,2-d]pyrimidin-2-yl)amino)-N-(pyrrolidin-3-yl)benzamide
1
(28d). Light yellow solid; yield: 81%; mp: 161.9–162.8 ; H NMR (400 MHz, MeOD) δ 9.04 (s, 1H), 8.25 (s,
1H), 8.15 (d, J = 15.1 Hz, 1H), 7.79 (d, J = 7.2 Hz, 1H), 7.62 (t, J = 8.6 Hz, 1H), 7.41–7.34 (m, 2H), 7.18–7.04 (m,
2H), 4.50 (s, 1H), 3.82 (s, 3H), 3.23 (dd, J = 11.6, 6.9 Hz, 1H), 3.18–3.10 (m, 1H), 3.04–2.88 (m, 2H), 2.23 (dt, J =
14.4, 7.6 Hz, 1H), 1.85 (dd, J = 12.0, 5.8 Hz, 1H). 13C NMR (150 MHz, MeOD) δ 166.63 (d, J = 1.7 Hz), 162.39
(d, J = 245.9 Hz), 161.07, 158.59, 158.32, 154.34, 147.05 (d, J = 12.9 Hz), 137.08, 133.39, 132.35, 131.64 (d, J =
3.5 Hz), 130.43, 125.04, 123.71, 121.41, 114.93, 114.85, 112.31, 105.87 (d, J = 30.4 Hz), 55.99, 53.22, 52.11,
46.33, 33.22. HRMS calcd for C24H22FN5O2S, [M+H]+, 464.1551; found 464.1560.
5.1.9.24. 2-fluoro-4-((7-(2-methoxyphenyl)thieno[3,2-d]pyrimidin-2-yl)amino)-N-(piperidin-4-ylmethyl)benzamide
(28e). Yellow solid; yield: 91%; mp: 182.5–183.2 ; 1H NMR (600 MHz, DMSO-d6) δ 9.05 (s, 1H), 8.27 (s, 1H),
7.94 (d, J = 16.2 Hz, 1H), 7.87 (d, J = 7.4 Hz, 1H), 7.67 (s, 1H), 7.45 (t, J = 9.0 Hz, 1H), 7.43–7.39 (m, 1H), 7.30
(d, J = 8.6 Hz, 1H), 7.17 (d, J = 7.8 Hz, 1H), 7.08 (td, J = 7.5, 0.9 Hz, 1H), 3.80 (s, 3H), 3.10 (s, 2H), 2.90 (d, J =
12.0 Hz, 2H), 2.39 (t, J = 11.3 Hz, 2H), 1.85–1.82 (m, 1H), 1.57 (d, J = 10.2 Hz, 2H), 1.01 (qd, J = 12.6, 3.7 Hz,
2H). 13C NMR (150 MHz, DMSO-d6) δ 163.69 (d, J = 2.0 Hz), 160.53 (d, J = 243.9 Hz), 160.11, 159.87, 156.75,
153.08, 134.87, 131.02, 130.97, 129.74 (d, J = 4.3 Hz), 129.01, 122.54, 119.99, 119.79, 116.05, 111.57, 111.32,
104.97 (d, J = 27.0 Hz), 55.41, 45.99 (2C), 45.26, 36.54, 31.12 (2C). HRMS calcd for C26H26FN5O2S, [M+H]+,
492.1864; found 492.1876.
5.1.9.25.
(R)-2-fluoro-4-((7-(2-methoxyphenyl)thieno[3,2-d]pyrimidin-2-yl)amino)-N-(piperidin-3-ylmethyl)benzamide (28f).
Light yellow solid; yield: 82%; mp: 213.1–214.3 ; 1H NMR (600 MHz, CDCl3) δ 8.96 (s, 1H), 8.19 (s, 1H), 8.12
(d, J = 15.0 Hz, 1H), 7.97 (t, J = 8.8 Hz, 1H), 7.90 (d, J = 6.5 Hz, 1H), 7.77 (s, 1H), 7.44 (t, J = 7.3 Hz, 1H),
7.17–7.14 (m, 2H), 7.08 (d, J = 8.3 Hz, 1H), 6.83–6.78 (m, 1H), 3.84 (s, 3H), 3.42–3.38 (m, 1H), 3.36–3.31 (m,
1H), 3.18 (d, J = 11.7 Hz, 1H), 3.07 (d, J = 11.9 Hz, 1H), 2.63 (t, J = 10.8 Hz, 1H), 2.47 (t, J = 11.0 Hz, 1H),
1.91–1.86 (m, 2H), 1.74 (d, J = 13.5 Hz, 1H), 1.59–1.53 (m, 1H), 1.23–1.19 (m, 1H). 13C NMR (150 MHz, CD3Cl)
δ 163.78 (d, J = 3.4 Hz), 161.60 (d, J = 243.7 Hz), 160.09, 157.04, 156.67, 152.89, 144.81 (d, J = 13.5 Hz), 135.97,
132.32 (d, J = 3.4 Hz), 131.98, 131.41, 129.66, 124.16, 122.02, 120.62, 114.05, 113.24 (d, J = 12.3 Hz), 111.27,
104.89 (d, J = 32.4 Hz), 55.65, 50.15, 46.49, 43.43, 36.99, 28.83, 25.27. HRMS calcd for C26H26FN5O2S, [M+H]+,
492.1864; found 492.1876.
5.1.9.26.
(S)-2-fluoro-4-((7-(2-methoxyphenyl)thieno[3,2-d]pyrimidin-2-yl)amino)-N-(piperidin-3-ylmethyl)benzamide
(28g). Light yellow solid; yield: 85%; mp: 214.2–215.0 ; 1H NMR (600 MHz, DMSO-d6) δ 10.18 (s, 1H), 9.29 (s,
1H), 8.42 (s, 1H), 8.08 (dd, J = 14.5, 1.3 Hz, 1H), 7.97 (dd, J = 9.1, 5.3 Hz, 1H), 7.73 (dd, J = 7.5, 1.6 Hz, 1H),
7.51 (t, J = 8.5 Hz, 1H), 7.48–7.43 (m, 2H), 7.20 (d, J = 8.2 Hz, 1H), 7.11 (t, J = 7.4 Hz, 1H), 3.79 (s, 3H), 3.08 (t,
J = 6.4 Hz, 2H), 2.90 (d, J = 9.6 Hz, 1H), 2.80 (d, J = 11.6 Hz, 1H), 2.40 (td, J = 11.4, 2.4 Hz, 1H), 2.19–2.15 (m,
1H), 1.74–1.70 (m, 1H), 1.64–1.61 (m, 1H), 1.55 (dd, J = 10.6, 4.4 Hz, 1H), 1.31–1.28 (m, 1H), 1.07–1.01 (m, 1H).
13C NMR (150 MHz, DMSO-d6) δ 163.42, 159.84 (d, J = 245.0 Hz), 159.27, 156.93, 156.83, 153.90, 144.52 (d, J
= 12.3 Hz), 136.62, 131.44, 131.05, 130.27 (d, J = 4.3 Hz), 129.48, 122.94, 122.09, 120.13, 115.28 (d, J = 14.2
Hz), 113.61, 111.44, 104.41 (d, J = 29.4 Hz), 55.44, 50.58, 46.56, 43.04, 36.78, 29.09, 25.71. HRMS calcd for
C26H26FN5O2S, [M+H]+, 492.1864; found 492.1875.
23

5.1.10. General procedure for the synthesis of intermediates 31a-f and 34a-g.
HATU (1.2 equiv) and DIEA (1.5 equiv) were added to a solution of 2-fluoro-4-nitrobenzoic acid (1 equiv)
and R6-NH2 (1 equiv) in CH2Cl2 at rt. The resulting mixture was heated to 25–40 °C and stirred until the reaction
was complete. The mixture was diluted with ethyl acetate, and the organic layer was washed with saline, dried over
anhydrous Na2SO4 and filtered. The filtrate was concentrated and purified using chromatography to yield the
intermediates 33a-g. Intermediates 33a-g (1 equiv) were dissolved in ethanol, and Pd/C (0.1 equiv) was added. The
flask was flushed with H2 and stirred for 6 h at 40 °C. The reaction mixture was filtered through a Celite pad and
the filtrate was concentrated to dryness, yielding intermediates 34a-g.
5.1.11. Preparation of diethyl (4-aminobenzyl)phosphonate (36)
Intermediate 36 was prepared in a similar manner as intermediate 23. MS (ESI) m/z(%): 244.0 [M+H]+
5.1.12. General procedure for the synthesis of intermediates 38 and 40.
To a solution of 1-(chloromethyl)-4-nitrobenzene (1 equiv) in triethyl phosphite (1.5 equiv). The reaction
mixture was stirred at 130 °C for 10 h. Upon completion, the mixture was diluted with water and extracted with
CH2Cl2. The organic phases were combined, washed with saline, dried over anhydrous Na2SO4 and evaporated to
afford crude product 35. Oxalyl chloride (3 equiv) was added to a solution of compound 35 (1 equiv) in
CH2Cl2, to which a catalytic amount of DMF (2 drops) was added. The reaction was stirred at 60 °C for 4 h. The
reaction mixture was concentrated and dried under a vacuum. A suspension of CH3NH2•HCl (1.2 equiv) or
morpholine (1.2 equiv) and Et3N (1.5 equiv) in anhydrous CH2Cl2 was then added. The mixture was stirred at
25 °C for 4 h and then diluted with CH2Cl2, and the organic layer was washed with saline, dried over anhydrous
Na2SO4 and filtered. The filtrate was concentrated and purified using chromatography to yield intermediate 37 or
39. Intermediate 37 or 39 (1 equiv) was dissolved in ethanol, and Pd/C (0.1 equiv) was added. The flask was
flushed with H2 and stirred for 6 h at 40 °C. The reaction mixture was filtered through a Celite pad, and the filtrate
was concentrated to dryness, yielding intermediate 38 or 40. 38: MS (ESI) m/z(%): 285.1 [M+H]+; 40: MS (ESI)
m/z(%): 229.1 [M+H]+.
5.2. Pharmacological assay
5.2.1. FAK HTRF assay
The FAK kinase assay was performed using the HTRF® KinEASE™-TK kit (Cisbio Bioassays, France) in
white 384-well small volume plates with a total working volume of 20 µL. The purified FAK enzyme was
purchased from Carna Biosciences (Japan). Compounds were diluted with kinase reaction buffer in a stepwise
manner from a concentrated stock of 8 mM in 100 % DMSO. The IC50 measurements were performed in replicates.
For each assay, 4 µL of dispensed compounds, 4 µL of mix 1 (ATP +Substrate TK) and 2 µL of the kinase (0.111
ng/µL) were added to the assay wells. The assay plates were incubated at 25 °C for 50 min, and reactions were
terminated by adding 10 µL of mix 2 (Sa-XL665+TK-Antibody-Cryptate). After a final incubation (60 min at
room temperature), HTRF signals were obtained by measuring the fluorescence of the plates at 620 nM (Cryptate)
and 665 nM (XL665) using an Infinite® F500 microplate reader (Tecan, Switzerland). A ratio was calculated
(665/620) for each well. For IC50 measurements, values were normalized and fitted with Prism software (GraphPad
software).
5.2.2. Cell proliferation assay
U-87MG, A549, MDA-MB-231 and HK2 cells were cultured in a 96-well plate at a density of 4000–5000
cells/well and were maintained at 37 °C in a humidified atmosphere of 5 % CO2 for 24 h. The tested compounds
24

were added to the culture medium at the indicated final concentrations and incubated for 72 h. Fresh MTT was
added to each well at a final concentration of 5 mg/mL in phosphate-buffered saline (PBS), and the cells were then
incubated at 37 °C for 4 h. The formazan crystals in each well were dissolved in 150 mL DMSO, and the
absorbance of each test well was measured at λ490nm using a Thermo reader (Multiskan GO).
5.2.3. Cell apoptosis analysis
The ability of compound 26f to induce the apoptosis of MDA-MB-231 cells was quantified by performing
annexin V and PI staining and flow cytometry. Briefly, after treatment with compound 26f for 48 h, cells were
harvested, washed twice with PBS, and subjected to annexin V and propidium iodide staining using the annexin V
FITC apoptosis kit (US Everbright® Inc.) according to the manufacturer's protocol. After staining, flow cytometry
(Becton-Dickinson FACSCalibur) was performed to quantify the number of apoptotic cells.
5.2.4. Cell cycle analysis
MDA-MB-231 cells (1×106 cells) were seeded in six-well plates and cultured for 24 h. Then, the cells were
treated with DMSO or one of several concentrations of compound 26f for 48 h. After incubation, cells were
collected, washed twice with cold PBS and then fixed with ethanol (70 %) at 4 °C overnight. The cells were
centrifuged to remove the fixative solution and washed twice with cold PBS. Finally, the cells were stained with PI
at 4 °C in the dark for 30 min. The cells were analysed using a flow cytometer with the Cell Quest acquisition and
analysis software program (Becton-Dickinson FACSCalibur).
5.2.5. Wound healing assay
MDA-MB-231 cells (2×106/well) were seeded in six-well plates and grown to approximately 100 %
confluence in culture medium. Subsequently, a cell-free line was manually created by scratching the confluent cell
monolayers with a 200 µL pipette tip. The wounded cell monolayers were washed three times with
phosphate-buffered saline (PBS) and incubated with serum-free medium. Then, cells were treated with different
concentrations of compound 26f, incubated for 48 h, and photographed at 24 and 48 h with an inverted
microscope.
5.2.6. Liver microsomal stability assay
The liver microsomal stability assay was performed by incubating with microsomes (rat microsome,
Biopredic, Lot No. MIC254034) (0.5 mg/mL) at 37 °C with compound 26f at a final concentration of 1 µM in
potassium phosphate buffer (pH 7.4, 100 mM with 10 mM MgCl2). The incubation was initiated by the addition of
prewarmed cofactors (1 mmol NADPH). After incubation at 37 °C for different times (0, 5, 10, 20, 30, and 60 min),
the protein was precipitated by the addition of cold acetonitrile. Then, the precipitated proteins were then removed
by centrifugation , and the supernatants were injected into an LC-MS/MS system. The metabolic stability tests of
rat livers are fully in accordance with the Guide for the Care and Use of Laboratory Animals.
5.2.7. Molecular docking study
Ligand structures were prepared using Maestro 9.0 within the Schrödinger package. The crystal structures of
FAK (PDB ID: 2JKK) were retrieved from the RCSB Protein Data Bank (http://www.pdb.org) and prepared for
molecular docking using Protein Preparation Wizard. The ligand structures were optimized with the Maestro
Ligprep module, regulated to a protonated state of pH 7.4, and minimized with an OPLS 2005 force field to
produce low-energy conformers. Compounds were docked into binding sites with the Glide module within the
Schrödinger package using the united-atom scoring function. For all docking simulations, the grid centre was
25

placed on the centroid of the included ligand binding site, and a 20×20×20 Å grid box size was used. The docking
simulation was performed twenty times to provide a sufficient number of constellation groups, and the output was
characterized by the favourable binding affinity value. In addition, the figures were prepared using PyMOL.
Acknowledgments
We gratefully acknowledge the financial support from the Program for Innovative Research Team of the
Ministry of Education and Program for Liaoning Innovative Research Team in University.
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