Discovery of 7H-pyrrolo[2,3-d]pyridine derivatives as potent FAK inhibitors: Design, synthesis, biological evaluation and molecular docking study

Article abstract of DOI:10.1016/j.bioorg.2020.104092

Focal adhesion kinase (FAK) is an intracellular non-receptor tyrosine kinase responsible for development of various tumor types. Aiming to explore new potent inhibitors, two series of 2,4-disubstituted-7H-pyrrolo[2,3-d]pyrimidine derivatives were designed and synthesized on the base of structure-based design strategy. Biological evaluation indicated that most of these new compounds could potently inhibit FAK kinase, leading to the promising inhibitors against the proliferation of U-87MG, A-549, and MDA-MB-231 cancer cell lines. Among them, the optimized compound 18h potently inhibited the enzyme (IC₅₀ = 19.1 nM) and displayed stronger potency than TAE-226 in U-87MG, A-549 and MDA-MB-231 cells, with IC₅₀ values of 0.35, 0.24, and 0.34 μM, respectively. Compound 18h is a multi-target kinase inhibitor. Furthermore, compound 18h also exhibited relatively less cytotoxicity (IC₅₀ = 3.72 μM) toward a normal human cell line, HK2. According to the flow cytometry and wound healing assay results, compound 18h effectively induced apoptosis and G0/G1 phase arrest of MDA-MB-231 cells and suppressed the migration of U-87MG, A-549 and MDA-MB-231 cells. The docking study of compound 18h was performed to elucidate its possible binding modes and to provide a structural basis for the further structural guidance design of FAK inhibitors. Collectively, these data support the further development of compound 18h as a lead compound for FAK-targeted anticancer drug discovery.

Full text of DOI:10.1016/j.bioorg.2020.104092

BioorganicChemistry102(2020)104092
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Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Discovery of 7H-pyrrolo[2,3-d]pyridine derivatives as potent FAK inhibitors:
Design, synthesis, biological evaluation and molecular docking study
Ruifeng Wanga, Xiangxin Zhaoa, Sijia Yua, Yixuan Chena,b, Hengxian Cuia, 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
A R T I C L E I N F O
A B S T R A C T
Keywords:
Focal adhesion kinase (FAK) is an intracellular non-receptor tyrosine kinase responsible for development of
various tumor types. Aiming to explore new potent inhibitors, two series of 2,4-disubstituted-7H-pyrrolo[2,3-d]
pyrimidine derivatives were designed and synthesized on the base of structure-based design strategy. Biological
evaluation indicated that most of these new compounds could potently inhibit FAK kinase, leading to the pro-
mising inhibitors against the proliferation of U-87MG, A-549, and MDA-MB-231 cancer cell lines. Among them,
the optimized compound 18h potently inhibited the enzyme (IC50 = 19.1 nM) and displayed stronger potency
than TAE-226 in U-87MG, A-549 and MDA-MB-231 cells, with IC50 values of 0.35, 0.24, and 0.34 μM, respec-
tively. Compound 18h is a multi-target kinase inhibitor. Furthermore, compound 18h also exhibited relatively
less cytotoxicity (IC50 = 3.72 μM) toward a normal human cell line, HK2. According to the flow cytometry and
wound healing assay results, compound 18h effectively induced apoptosis and G0/G1 phase arrest of MDA-MB-
231 cells and suppressed the migration of U-87MG, A-549 and MDA-MB-231 cells. The docking study of com-
pound 18h was performed to elucidate its possible binding modes and to provide a structural basis for the further
structural guidance design of FAK inhibitors. Collectively, these data support the further development of com-
pound 18h as a lead compound for FAK-targeted anticancer drug discovery.
FAK inhibitor
7H-pyrrolo[2,3-d]pyrimidine
Anticancer
Migration
Apoptosis
Molecular docking
1. Introduction
inhibitors that bind to active forms of FAK. As shown in Fig. 1, TAE-226
(1)[15] and PF-431396 (5)[16] have shown potent antiproliferative
Focal adhesion kinase (FAK) is an intracellular non-receptor tyr-
osine kinase that is overexpressed and activated in various types of
human cancers[1,2]. FAK is indispensable to cancer cells, where it
functions to promote the overall aggressiveness of tumor growth by
controlling cellular processes such as proliferation, migration, adhesion
and survival[3–6]. Similarly, a number of subsequent studies reported
upregulation of FAK expression in a broad range of tumors including
lung pancreatic, agastric, prostate, ovarian, thyroid and breast cancers
[2,7–9]. Furthermore, FAK overexpression is highly correlated with an
invasive phenotype in these tumors[10,11] and has been correlated
with poor prognosis of cancer patients[12]. FAK regulates the DNA
damage response and its inhibition radiosensitizes mutant KRAS lung
cancer[13]. Thus, FAK should be regarded as a potential target in the
development of anti-cancer drugs[14].
and antitumor effects in vitro and in vivo in several type of malig-
nancies. And there are several inhibitors, including VS-4718 (2, phase I)
[17], GSK2256098 (3, phase II)[18], CEP-37440 (4, phase I)[19], PF-
562271 (6, phase I)[20], and defactinib (7, phase II)[21] are in clinical
development, and some have demonstrated clinically meaningful ben-
efits with manageable toxicity profiles in clinical trials.
Pharmacophore is a set of structural features responsible for the
biological activity of a molecule[22]. Herein, ligand-based pharmaco-
phore models have been summarized on ATP competitive inhibitors
(compounds 1–7) of FAK. The two main features of these pharmaco-
phore models include: double-dentate hydrogen bonding of the scaffold
to the hinge region and hydrogen bond acceptors form interactions with
Asp564 of the DFG region. This indicates the hinge region and DFG
region as the two pivotal binding sites that played important roles in
binding FAK inhibitors.
Recently, great efforts have been devoted to developing FAK in-
hibitors as anticancer treatments. Most inhibitors are ATP competitive
A general representation of the inhibitors at the ATP active site of
⁎ Corresponding author.
E-mail address: medchemzhao@163.com (D. Zhao).
https://doi.org/10.1016/j.bioorg.2020.104092
Received 5 May 2020; Received in revised form 9 July 2020; Accepted 9 July 2020
Availableonline14July2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

R. Wang, et al.
BioorganicChemistry102(2020)104092
Fig. 1. Structures of representative FAK inhibitors.
Fig. 2. Rational design of the target compounds.
the FAK is shown in Fig. 2. The diaminopyrimidine motif are anchored
to the hinge region by double-dentate hydrogen bonding of the pyr-
imidine nitrogen and aniline NH to the hinge region. Moreover, the R1
and R2 back toward the solvent region and toward DFG motif of acti-
vation loop. It was concluded that the 2,4-diaminopyridine scaffold is
the major scaffold used in the currently reported inhibitors
[15–21,23–27]. Based on the binding features analysis above, in order
to discover novel FAK inhibitors, we initiated our study of the 7H-
pyrrolo[2,3-d]pyrimidine-2,4-diamine scaffold as the hinge-binding
moiety and assumed it was feasible to introduce an extra interaction
with Glu500 by a simple addition of a hydrogen donor in this region,
which would result in hinge-binding moiety containing three donor–-
acceptor interactions instead of two in the pyrimidine case[28]. Then,
essential pharmacophores were added at the R2 position to form in-
teraction with the DFG motif. On the basis of our assumption, these two
motifs (7H-pyrrolo[2,3-d]pyrimidine and N-methyl-2-benzamide
moiety or N-methyl-N-phenylmethanesulfonamide moiety) formed our
basic molecular scaffold (Fig. 2), which was expected to fill the two
pivotal regions and provide binding affinity to FAK. In addition, in
order to extend into the solvent region surrounding the kinase hinge
segment, introduction of the N-substituted pyrazole or substituted
benzene ring, the privileged structure in the FAK inhibitor, was in-
vestigated. On the basis of above strategy, the synthesis and biological
evaluation of two series of 2,4-disubstituted-7H-pyrrolo[2,3-d]pyr-
imidine derivatives as FAK inhibitors were reported in this article.
intermediate 10. SNAr reactions between 2-fluorobenzonitrile (11) and
N-methylmethane-sulfonamide were conducted under basic conditions
to afford intermediate 12, which was then subjected to reduction by
BH3 in THF to yield intermediate 13. 2,4-dichloro-7H-pyrrolo[2,3-d]
pyrimidine (14) was protected by TsCl to afford intermediate 15, which
was equipped with intermediates 10 and 13 via SNAr reactions under
basic conditions to afford intermediates 16 and 19. Palladium-cata-
lyzed Buchwald cross-coupling with corresponding substituted anilines
afforded intermediates 17a-m and 20a-m[29], and was followed by a
deprotection step to remove the Ts-protection group to produce target
compounds 18a-m and 21a-m.
Intermediates 24, 27, 30a-d and 33 were synthesized as depicted in
Scheme 2. SNAr reactions between 1-fluoro-4-nitrobenzene (22) and 1-
methylpiperazine were conducted under basic conditions to afford in-
termediate 23, which was then subjected to reduction by Pd/C in the
presence of H2 to yield intermediate 24. 4-nitrobenzenesulfonyl
chloride (25) was reacted with ethanamine to provide the intermediate
26, which was reduced under Pd/C-H2 conditions to form the inter-
mediate 27. The starting 1H-pyrazol-4-amine (28) underwent a Mit-
sunobu reaction with corresponding alcohols to afford intermediates
29a-c, and it underwent an electrophilic substitution reaction with 1-
bromo-2-methoxyethane to afford intermediate 29d. The nitro group
was then reduced by Pd/C in the presence of H2 to yield amines 30a-d.
1-(Chloromethyl)-4-nitrobenzene (31) was reacted with triethyl phos-
phate to provide the diethyl(4-nitrobenzyl)phosphonate (32)[30],
which was reduced under Pd/C-H2 conditions to form the aniline 33.
2. Chemistry
3. Results and discussion
Intermediates 10, 13 and compounds 18a-m and 21a-m were syn-
thesized using the method depicted in Scheme 1. 2-nitrobenzoic acid
(8) was treated with oxalyl chloride and then directly reacted with
CH3NH2•HCl in the presence of NaHCO3 base to prepare the inter-
mediate 9, which was reduced under Pd/C-H2 conditions to form the
3.1. Biological evaluation and analysis of the structure-activity relationship
All the synthesized compounds were assayed for enzymatic in-
hibitory activity of FAK using
a homogeneous time resolved
2

R. Wang, et al.
BioorganicChemistry102(2020)104092
Scheme 1. Synthesis of intermediates 10, 13 and compounds 18a-m and 21a-m. Reagents and conditions: (i) Ι) oxalyl chloride, DMF, CH2Cl2, 60 °C; ΙΙ) CH3NH2•HCl,
NaHCO3, CH3CN, 60 °C; (ii) Pd/C, H2, EtOH, 40 °C; (iii) N-methylmethanesulfonamide, Cs2CO3, CH3CN, 80 °C; (iv) BH3 (2 M in THF), anhydrous THF, 60 °C; (v) Ts-
Cl, NaH, anhydrous THF, 0 °C; (vi) NH2-R2, DIPEA, n-BuOH, 100 °C; (vii) corresponding aromatic amines, Pd(AcO)2, X-phos, Cs2CO3, dioxane, 90 °C; and (viii) NaOH,
MeOH/H2O, 40 °C.
fluorescence (HTRF®) assay, as well as for their antiproliferative ac-
tivity in U-87MG (human glioma cancer cell), A549 (human lung cancer
cell) and MDA-MB-231 (human breast cancer cell) cell lines, in which
FAK has been found to be overexpressed[10,31], using the MTT assay.
TAE-226 was used as positive control.
achieving higher potency. The results indicated that most compounds
exhibited moderate inhibitory activity against the FAK, with IC50 values
ranging from 19.1 to 113.5 nM. In particular, 18h exhibited the better
activity to FAK (IC50 = 19.1 nM). Compounds 18i-m represent a series
of analogues bearing a (1-substituted)-1H-pyrazo-4-yl moiety. The
methyl-substituted compound 18i and 1,1-difluoroethyl-substituted
analog 18j were equally potent, but much higher potency
(IC50 = 54.8 nM) was observed for the methoxyethyl-substituted pyr-
azole 18k. The tetrahydro-pyran-4-yl-substituted pyrazole 18l ex-
hibited moderate potency against FAK (IC50 = 47.4 nM), and much
higher potency (IC50 = 22.3 nM) was observed for the piperidin-4-yl-
substituted analog 18m.
In the A series of compounds, we introduced the pharmacophore
fragment (N-methyl-benzamide moiety) at R2 to form hydrogen bond
interaction with Asp564 of the DFG motif. As shown in Table 1, in-
troduction of the phenyl at R1 led to compound 18a, which retained
moderate potency against FAK (IC50 = 173.9 nM). Substitution of the
phenyl moiety of compound 18a by substituted phenyl rings with
electron-donating substituents (compound 18b-c), electron-with-
drawing groups (compounds 18d-g), and 1-methyl-4-phenylpiperazine
moiety (compound 18h) gave rise to a series of analogues with struc-
tural variety that exhibited much higher potency than compound 18a,
suggesting that structural modification at this position contributes to
In cell-based assays, it can be noted that most of compounds dis-
played moderate to high potency against U-87MG, A549 and MDA-MB-
231 cells. As a general trend, IC50 values of these compounds against
there tumor cell lines ranged between 0.24 and 20.71 uM, and six of
Scheme 2. Synthesis of intermediates 24, 27, 30a-d and 33. Reagents and conditions: (i) 1-methylpiperazine, K2CO3, DMF, 80 °C; (ii) Pd/C, H2, EtOH, 40 °C; (iii)
ethanamine, Et3N, CH2Cl2, rt; (iv) for 29a-c, R3-OH, PPh3, DEAD, THF, rt; (v) for 29d, 1-bromo-2-methoxyethane, K2CO3, DMF, 60 °C; and (vi) triethyl phosphate,
130 °C.
3

R. Wang, et al.
BioorganicChemistry102(2020)104092
Table 1
Biological activities of compounds 18a-m.
Compd.
R1
FAK, IC50 (nM)a
Antiproliferative activity IC50 (μM)b
U-87MG
A549
MDA-MB-231
18a
18b
173.9
59.8
0.67
6.39
0.06
0.55
2.72
1.45
0.17
0.15
7.86
3.67
0.41
0.19
18c
66.3
6.39
0.04
0.84
0.03
1.85
0.23
18d
18e
62.9
57.9
2.10
0.10
6.19
3.39
0.14
0.15
8.50
4.43
0.51
0.27
20.71
0.38
18f
32.9
2.85
3.00
0.26
0.01
0.55
1.29
0.05
0.25
0.88
4.05
0.05
0.35
18g
113.5
18h
18i
19.1
63.2
0.35
5.77
0.05
0.15
0.24
3.86
0.01
0.50
0.34
7.72
0.04
0.17
18j
89.1
0.97
0.03
2.25
0.26
2.56
0.15
18k
18l
54.8
47.4
0.60
0.85
0.01
0.05
1.33
0.43
0.09
0.04
2.30
1.41
0.17
0.11
18m
22.3
7.0
6.59
1.57
0.05
0.11
3.16
1.60
0.28
0.03
3.92
4.84
0.10
0.35
TAE-226
a
b
The IC50 values are presented as the mean (nM) values from two separate experiments.
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 SD are reported.
them (18f, 18g, 18h, 18j, 18k and 18l) showed parallel anti-
proliferative activities against three cell lines as TAE-226. Especially,
the analogue 18h, displayed stronger potency than TAE-226 in U-
87MG, A-549 and MDA-MB-231 cells, with IC50 values of 0.35, 0.24 and
0.34 μM, respectively.
the strongest inhibitor against these two cancer cell lines.
3.2. Cellular selectivity assay
Toxicity to host cells is an important characteristic to assess the
safety of drug candidates early in the drug discovery process. In order to
evaluate whether the compounds were toxic or non-toxic to healthy
cells, the viability of normal human tubular epithelial cells (HK2) ex-
posed to each compound was assessed using MTT assay (Table 3). These
eight compounds potently inhibited enzyme activity and cancer cell
proliferation. The selectivity index (SI) values of the compounds were
also determined to compare the selectivity of the compounds. Com-
pound 18h was chosen for further studies due to their notable and se-
lective antitumor effects on three cancer cells.
In the B series of compounds, we introduced the pharmacophore
fragment (N-methyl-N-phenylmethanesulfonamide moiety) at R2 to
form hydrogen bond interaction with Asp564 of the DFG motif. As
shown in Table 2, introduction of the phenyl at R1 led to compounds
21a, which retained moderate potency against FAK (IC50 = 57.6 nM).
Similarly, substitution of the phenyl moiety of compound 21a by sub-
stituted phenyl rings with electron-donating substituents (compound
21b-c), electron-withdrawing groups (compounds 21d-g), and 1-me-
thyl-4-phenylpiperazine moiety (compound 21h) gave rise to a series of
analogues with structural variety that exhibited potent inhibitory ac-
tivity against the FAK, with IC50 values ranging from 11.3 to 43.1 nM.
Compounds 21i-m represent a series of analogues bearing a (1-sub-
stituted)-1H-pyrazo-4-yl moiety. Among them, the methoxyethyl-sub-
stituted pyrazole analog 21k and piperidin-4-yl-substituted analog 21m
exhibited potent inhibitory activity against the FAK (IC50 values of 7.1
and 10.3 nM, respectively).
3.3. Cell apoptosis assay
After 48 h incubation period, the apoptotic effects of compound 18h
was analyzed for MDA-MB-231 cells based on Annexin V-PI binding
capacities in flow cytometry. As illustrated in Fig. 3, following flow
cytometric analyses, compound 18h substantially increased the apop-
tosis of MDA-MB-231 cells in a concentration-dependent manner, with
apoptotic rates of 11.89%, 22.18%, and 38.01% at concentrations of
0.15, 0.3 and 0.6 μM, respectively. Similarly, compound 18h increased
the apoptosis of A549 and U-87MG cells in a concentration-dependent
manner (Figure S1, Supporting Information).
As illustrated in Table 2, The results revealed that most of the target
compounds possess moderate anticancer activities with IC50 values
ranging from 5.78 to 35 μM. In particular, compounds 21b, 21c, 21d
and 21f exhibited favorable antiproliferative activities against three cell
lines. Among these compounds, compound 21f, which had an IC50
value of 1.89 μM against U-87MG, and 5.06 μM against A549 cells, was
4

R. Wang, et al.
BioorganicChemistry102(2020)104092
Table 2
Biological activities of compounds 21a-m.
Compd.
R1
FAK, IC50 (nM)a
Antiproliferative activity IC50 (μM)b
U-87MG
A549
MDA-MB-231
21a
21b
57.6
28.6
4.21
1.27
0.08
0.03
10.89
5.88
0.97
0.15
37.79
8.07
2.80
0.61
21c
22.8
8.55
0.34
8.84
0.24
11.12
1.06
21d
21e
22.4
23.8
7.32
7.63
0.45
0.49
7.97
0.46
12.71
29.41
1.13
0.94
11.47
0.67
0.48
0.43
21f
11.3
43.1
1.89
0.13
5.06
12.84
22.58
0.86
0.36
21g
52.35
2.28
12.65
21h
21i
25.9
15.3
12.99
8.26
1.37
0.30
0.51
7.69
0.40
0.46
35.38
43.32
1.81
4.03
12.67
21j
37.5
5.30
7.69
0.49
0.85
30.53
1.55
21k
21l
7.1
13.44
9.52
0.57
0.45
9.60
31.52
31.32
1.68
2.28
48.8
10.59
0.68
1.36
0.03
21m
10.3
44.39
1.21
0.11
34.61
1.60
51.35
4.84
3.83
TAE-226
7.0
1.57
0.35
a
b
The IC50 values are presented as the mean (nM) values from two separate experiments.
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
SD are reported.
the G2/M phase significantly decreased from 17.19% to 14.15%
(0.15 μM), 10.29% (0.3 μM), 6.80% (0.6 μM) after 48 h of incubation
with the compound 18h. Flow cytometric analysis showed that com-
pound 18h mainly arrested the MDA-MB-231 cell line in the G0/G1
stage with a concentration-dependent effect.
Table 3
Antiproliferative activities of compounds toward HK2 cells.
Compd.
HK2 (μM)a
SI valuesb
U-87MG
A549
MDA-MB-231
18b
15.23
1.75
3.72
3.97
3.15
> 50
> 50
8.20
1.48
2.38
0.61
10.62
6.61
3.71
26.46
3.72
5.22
10.50
3.18
15.50
2.98
7.32
9.88
5.21
5.13
4.14
1.98
10.94
1.72
2.23
3.89
1.59
1.69
18f
0.19
0.18
0.32
0.26
3.5. Wound healing assay
18h
18k
In order to investigate the effect of 18h on migration of the MDA-
MB-231, A549 and U-87MG cells, wound healing assays were carried
out. For this assay, the MDA-MB-231 cells were conducted with dif-
ferent concentrations of 18h (0, 0.2 and 0.6 μM) for 24 h to investigate
if compound 18h could influence the migration of the cell line after
scratching the cell monolayer (Fig. 5). As expected, breast cancer cells
in DMSO control displayed the strongest migratory ability. A time- and
concentration-dependent inhibitory effect of 18h was observed in
MDA-MB-231 cells. Similarly, compound 18h significantly inhibited the
migration of A549 and U-87MG cells in a concentration-dependent
manner (Figure S2, Supporting Information).
18l
21f
21k
TAE-226
1.01
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
SD
are reported.
b
SI = IC50 for normal cell line / IC50 for cancerous cell line. IC50 > 50 was
accepted as 50.
3.4. Cell cycle analysis
3.6. Kinase selectivity profile
The effect of compound 18h on cell cycle progression in MDA-MB-
231 cells was analysed using flow cytometry. As shown in Fig. 4, the
G0/G1 phase in MDA-MB-231 cell line significantly increased from
44.91% to 46.86% (0.15 μM), 48.44% (0.3 μM), 56.19% (0.6 μM), and
The kinase selectivity profile of 18h was assessed over 10 different
protein kinases covering the major tumor progression, angiogenesis,
metastasis and oncogenic activation kinases of the human protein
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R. Wang, et al.
BioorganicChemistry102(2020)104092
Fig. 3. Flow cytometry analyses of apoptosis in-
duction in MDA-MB-231 cells treated with com-
pound 18h (0.15, 0.3 and 0.6 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.
Fig. 4. Effects of compound 18h on the cell cycle of MDA-MB-231 cells. Flow cytometry analysis of the cell cycle in MDA-MB-231 cells treated with compound 18h
(0.15, 0.3 or 0.6 μM) or no treatment (control) for 48 h.
kinome at a concentration of 1.0 μM, and the percent inhibition values
were reported in Table 4. Compound 18h is a multi-target kinase in-
hibitor. Considering that all major human cancers do not appear to be
single but are dysregulated by several kinase pathways, multi-target
kinase inhibitors not only provide the opportunity to capture tumor
progression more effectively, but also to avoid mechanisms of re-
sistance commonly activated by the tumor to escape targeted therapies.
However, Compound 18h may also have harmful off-target side effects,
which needs to be further optimized and evaluated.
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R. Wang, et al.
BioorganicChemistry102(2020)104092
Fig. 5. (A) Inhibition of the migration of MDA-MB-231 cells treated with compound 18h for 24 h in the wound healing assay. (B) Cell migration rate was calculated
as mean
SD from three different fields.
of a hydrogen bond with Asp564 of the DFG motif. The N-methylpi-
perazine moiety appeared to point toward the solvent region. Fur-
thermore, some of hydrophobic interactions are observed between
carbon atoms in the pyrrolo[2,3-d]pyrimidine ring and Leu553 and
Ala452, but also between the carbons of the 2-aniline ring and
Gly505and Glu506. 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.
Table 4
Kinase selectivity profile of compound 18h.a
Kinase
%inhibition at 1 μM
Kinase
%inhibition at 1 μM
ABL1
ALK
96
99
23
84
35
FGFR4
FLT3
FAK
33
101
99
82
9
BRAF
BTK
KDR
CDK2
PI3K
a
Selectivity profile of compound 18h measured at a concentration of 1 μM in
a panel of 10 kinases generated with the SelectScreen® Profiling Service from
Life Technologies. The results represent the mean of three independent ex-
periments performed in triplicate.
4. Conclusions
In conclusion, based on a structure-based design strategy, two series
of FAK inhibitors bearing a 2,4-disubstituted-7H-pyrrolo[2,3-d]pyr-
imidine scaffold were designed and synthesized. Most compounds po-
tently suppressed the enzymatic activities of FAK, with IC50 values
ranging from 10−8 to 10−9 M, and potently inhibited the proliferation
of A-549, MDA-MB-231 and U-87MG cancer cells. The optimized
compound 18h potently inhibited the enzyme (IC50 = 19.1 nM) and
displayed stronger potency than TAE-226 in U-87MG, A-549 and MDA-
MB-231 cells, with IC50 values of 0.35, 0.24, and 0.34 μM, respectively.
Further investigation demonstrated that compound 18h effectively in-
duced apoptosis and G0/G1 phase arrest in MDA-MB-231 cells cells and
suppressed the migration of U-87MG, A-549 and MDA-MB-231 cells.
Taken together, these results provided a practical basis for the further
structural optimization of 2,4-disubstituted-7H-pyrrolo[2,3-d]pyr-
imidine derivatives as FAK inhibitors and antitumor agents.
3.7. Molecular docking study
A docking study of compound 18h in the ATP-binding site of FAK
(PDB: 2JKK) was performed to elucidate its interaction mode. The best
predicted binding mode is shown in Fig. 6A and has a calculated
binding energy of −10.33 kcal/mol. Compound 18h also exhibited
good spatial matching with the FAK active pocket (Fig. 6B). Three
hydrogen bonds are formed between the nitrogens in the 7H-pyrrolo
[2,3-d]pyrimidine-2,4-diamine moiety with the backbone of residues
Glu500 and Gly502 of the kinase hinge. The pyrrolo ring penetrates
deepest into the ATP binding pocket and makes van der Waals inter-
actions with the gatekeeper residue Met499. Moreover, the benzamide
moiety back toward the activation loop region results in the formation
Fig. 6. The predicted docked pose of compound 18h
(yellow sticks) in the FAK active site (PDB: 2JKK).
(A) Detailed interactions with the protein residues.
Each dashed red line represents hydrogen bonds. (B)
Three-dimensional space matching diagram of
compound 18h and the active site. (For interpreta-
tion of the references to colour in this figure legend,
the reader is referred to the web version of this ar-
ticle.)
7

R. Wang, et al.
BioorganicChemistry102(2020)104092
5. Experimental section
5.1.5. General procedure for the synthesis of intermediates 17a-m and
20a-m
5.1. Chemistry
To solutions of 16 or 19 (1 equiv), corresponding substituted ani-
lines (1.1 equiv), X-phos (0.1 equiv) and Cs2CO3 (3.0 equiv) in 1,4-
dioxane, Pd(AcO)2 (0.05 equiv) was added under a nitrogen atmo-
sphere. The mixture was purged with nitrogen for 10 min and then
heated at 90 °C until completion of the reaction. The mixture was di-
luted with ethyl acetate, and the organic layer was washed with brine,
dried over anhydrous Na2SO4 and filtered. The filtrate was con-
centrated and purified by chromatography to give the coupling inter-
mediates 17a-m or 20a-m.
Starting materials, reagents and solvents were obtained from com-
mercial suppliers and used without further purification unless other-
wise indicated. Anhydrous solvents were dried and stored according to
standard procedures. All reactions were monitored by thin layer chro-
matography (TLC) on silica gel plates with fluorescence F-254 and vi-
sualized with UV light. Column chromatography was carried out on
silica gel (200–300 mesh). 1H 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.
5.1.6. General procedure for the synthesis of compounds 18a-m and 21a-
m.
Intermediates 17a-m or 20a-m were dissolved in methanol (5 mL),
and a 2 N sodium hydroxide solution (1.0 mL) was added to the mix-
ture. The mixture was heated to 40 °C and stirred for 10 h. The majority
of the solvent was removed under vacuum, and the residue was parti-
tioned between ethyl acetate and water. The organic layer was dried
over anhydrous Na2SO4 and concentrated. The residue was purified by
column chromatography to afford compounds 18a-m and 21a-m.
5.1.1. Preparation of 2-amino-N-methylbenzamide (10)
Oxalyl chloride (3 equiv) was added to a solution of 2-nitrobenzoic
acid (1 equiv) in anhydrous CH2Cl2, to which a catalytic amount of
DMF (1 drops) was added. After being stirred at 60 °C for 6 h, the
reaction mixture was concentrated and dried under a vacuum. A sus-
pension of CH3NH2•HCl (1.5 equiv) and NaHCO3 (1.5 equiv) in acet-
onitrile was then added. The mixture was stirred at 60 °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 inter-
mediate 9. Intermediate 9 (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 10. MS (ESI)
5.1.6.1. N-methyl-2-((2-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)
amino)benzamide (18a). White solid, yield: 76%; 1H NMR (600 MHz,
DMSO‑d6) δ 11.73 (s, 1H), 11.38 (s, 1H), 9.07 (d, J = 8.3 Hz, 1H), 8.99
(s, 1H), 8.75 (d, J = 4.1 Hz, 1H), 7.84 (d, J = 7.9 Hz, 2H), 7.76 (d,
J = 7.7 Hz, 1H), 7.50 (t, J = 7.7 Hz, 1H), 7.26 (t, J = 7.7 Hz, 2H), 7.05
(t, J = 7.5 Hz, 1H), 7.01 (s, 1H), 6.89 (t, J = 7.2 Hz, 1H), 6.34 (s, 1H),
2.84 (d, J = 4.3 Hz, 3H). 13C NMR (150 MHz, DMSO‑d6) δ 169.56,
155.53, 152.89, 152.17, 141.64, 141.18, 131.83, 128.29 (2C), 127.94,
120.55, 120.45, 120.22, 120.19, 118.95, 118.42 (2C), 99.06, 97.32,
26.35. HRMS calcd for
359.1619.
C
20H18N6O, [M+H]+
, 359.1615; found
m/z(%): 151.1 [M+H]+
.
5.1.2. Preparation of N-(2-(aminomethyl)phenyl)-N-methylmethanesulfonamide
(13)
5.1.6.2. 2-((2-((3,4-dimethoxyphenyl)amino)-7H-pyrrolo[2,3-d]
pyrimidin-4-yl)amino)-N-methylbenzamide (18b). White solid, yield:
67%; 1H NMR (600 MHz, DMSO‑d6) δ 11.74 (s, 1H), 11.31 (s, 1H),
9.09 (d, J = 8.4 Hz, 1H), 8.79 (s, 1H), 8.75 (d, J = 4.5 Hz, 1H), 7.76
(dd, J = 7.9, 1.1 Hz, 1H), 7.52 (d, J = 2.0 Hz, 1H), 7.47 (t, J = 7.8 Hz,
1H), 7.35 (dd, J = 8.6, 2.2 Hz, 1H), 7.04 (t, J = 7.2 Hz, 1H), 6.99 (dd,
J = 3.2, 2.4 Hz, 1H), 6.86 (d, J = 8.7 Hz, 1H), 6.31 (dd, J = 3.3,
1.9 Hz, 1H), 3.74 (s, 3H), 3.72 (s, 3H), 2.84 (d, J = 4.5 Hz, 3H). 13C
NMR (150 MHz, DMSO‑d6) δ 169.59, 155.82, 152.84, 152.26, 148.68,
143.04, 141.32, 135.57, 131.85, 127.94, 120.33, 120.31, 119.92,
118.71, 112.50, 110.44, 104.57, 98.80, 97.28, 56.05, 55.38, 26.35.
HRMS calcd for C22H22N6O3, [M+H]+, 419.1826; found 419.1828.
To a solution of N-methyl-methanesulfonamide (1 equiv), 2-fluoro-
benzonitrile (1 equiv), and cesium carbonate (1.2 equiv) in acetonitrile.
The reaction mixture was stirred at 80 °C for 10 h. Upon completion,
the mixture was diluted with water and extracted by CH2Cl2. The or-
ganic phases were combined, washed by brine, dried over anhydrous
Na2SO4 and evaporated to afford crude product 12. Intermediate 12 (1
equiv) was dissolved in anhydrous tetrahydrofuran and protected with
argon. BH3 (in 2 M THF) (1.5 equiv) was then added, and the mixture
was stirred at 60 °C until completion of the reaction. The reaction was
quenched with H2O and extracted thrice with CH2Cl2, and the organic
layer was washed with brine, dried over anhydrous Na2SO4, and
filtered. The filtrate was concentrated and purified by chromatography
5.1.6.3. N-methyl-2-((2-((3,4,5-trimethoxyphenyl)amino)-7H-pyrrolo
[2,3-d]pyrimidin-4-yl)amino)benzamide (18c). White solid, yield: 73%;
1H NMR (600 MHz, DMSO‑d6) δ 11.77 (s, 1H), 11.35 (s, 1H), 9.11 (d,
J = 8.4 Hz, 1H), 8.89 (s, 1H), 8.76 (d, J = 4.2 Hz, 1H), 7.76 (d,
J = 7.7 Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.28 (s, 2H), 7.07–7.02 (m,
2H), 6.33 (s, 1H), 3.76 (s, 6H), 3.63 (s, 3H), 2.84 (d, J = 4.2 Hz, 3H).
13C NMR (150 MHz, DMSO‑d6) δ 169.59, 155.49, 152.80, 152.61 (2C),
152.01, 141.30, 137.85, 131.86, 131.41, 127.97, 120.35, 120.25,
120.14, 118.73, 98.98, 97.30, 96.40 (2C), 60.16, 55.68 (2C), 26.36.
HRMS calcd for C23H24N6O4, [M+H]+, 449.1932; found 449.1923.
to afford intermediate 13. MS (ESI) m/z(%): 215.3 [M+H]+
.
5.1.3. Preparation of 2,4-dichloro-7-tosyl-7H-pyrrolo[2,3-d]pyrimidine
(15)
NaH (1.1 equiv) was slowly added to a THF solution of 2,4-dichloro-
7H-pyrrolo[2,3-d]pyrimidineat (1 equiv) 0 °C. The reaction mixture was
stirred at 0 °C for 0.5 h. p-Toluensulfonyl chloride (1 equiv) was then
added and it was stirred at 0 °C for 4 h. The solvent was evaporated to
dryness, and the crude product was diluted with H2O and filtered. Filter
cake is the crude product 15 for the next step.
5.1.6.4. 2-((2-((4-cyanophenyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)
amino)-N-methylbenzamide (18d). Light yellow solid, yield: 68%; 1H
NMR (600 MHz, DMSO‑d6) δ 11.78 (s, 1H), 11.59 (s, 1H), 9.63 (s, 1H),
8.98 (d, J = 8.2 Hz, 1H), 8.77 (d, J = 4.5 Hz, 1H), 8.05 (d, J = 8.8 Hz,
2H), 7.78 (dd, J = 7.9, 1.2 Hz, 1H), 7.69 (d, J = 8.8 Hz, 2H), 7.58–7.53
(m, 1H), 7.11–7.07 (m, 2H), 6.38 (dd, J = 3.3, 1.9 Hz, 1H), 2.83 (d,
J = 4.5 Hz, 3H). 13C NMR (150 MHz, DMSO‑d6) δ 169.49, 154.41,
152.93, 151.58, 145.95, 140.86, 132.83 (2C), 131.90, 128.02, 121.14,
5.1.4. General procedure for the synthesis of intermediates 16 and 19
To a solution of 15 (1 equiv) in n-BuOH, DIPEA (1.1 equiv) and
intermediate 10 or 13 (1.2 equiv) were added. The mixture was stirred
at 100 °C for 5 h. The mixture was diluted with EtOAc and washed
successively with water and brine. The organic layer was dried over
Na2SO4, filtered, and evaporated. The resulting intermediate was pur-
ified by chromatography.
8

R. Wang, et al.
BioorganicChemistry102(2020)104092
120.85, 120.66, 119.94, 119.40, 117.65 (2C), 100.86, 99.83, 97.48,
calcd for C18H18N8O, [M+H]+, 363.1676; found 363.1667.
26.35. HRMS calcd for
406.1389.
C
21H17N7O, [M+Na]+
, 406.1387; found
5.1.6.10. 2-((2-((1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)amino)-7H-
pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-methylbenzamide
(18j). Light
5.1.6.5. 2-((2-((4-acetamidophenyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-
4-yl)amino)-N-methylbenzamide (18e). Light yellow solid, yield: 65%;
1H NMR (600 MHz, DMSO‑d6) δ 11.70 (s, 1H), 11.31 (s, 1H), 9.76 (s,
1H), 9.05 (d, J = 8.4 Hz, 1H), 8.89 (s, 1H), 8.74 (d, J = 4.5 Hz, 1H),
7.76 (dd, J = 7.9, 1.3 Hz, 1H), 7.71 (d, J = 8.9 Hz, 2H), 7.49–7.42 (m,
3H), 7.07–7.03 (m, 1H), 6.99 (dd, J = 3.3, 2.3 Hz, 1H), 6.32 (dd,
J = 3.3, 1.9 Hz, 1H), 2.83 (d, J = 4.5 Hz, 3H), 2.02 (s, 3H). 13C NMR
(150 MHz, DMSO‑d6) δ 169.55, 167.65, 155.70, 152.85, 152.29,
141.22, 137.08, 132.46, 131.76, 127.93, 120.55, 120.38, 120.05,
119.46 (2C), 118.89 (2C), 98.88, 97.29, 26.35, 23.86. HRMS calcd
for C22H21N7O2, [M+Na]+, 438.1649; found 438.1654.
yellow solid, yield: 52%; 1H NMR (400 MHz, MeOD) δ 8.86 (d,
J = 8.4 Hz, 1H), 8.04 (s, 1H), 7.69–7.64 (m, 2H), 7.49 (ddd,
J = 8.7, 7.4, 1.6 Hz, 1H), 7.06 (td, J = 7.9, 1.1 Hz, 1H), 6.90 (d,
J = 3.6 Hz, 1H), 6.45 (d, J = 3.6 Hz, 1H), 4.48 (td, J = 14.3, 3.9 Hz,
2H), 2.93 (s, 3H). 13C NMR (150 MHz, MeOD) δ 172.24, 157.55,
155.26, 154.06, 142.20, 133.35, 132.92, 130.85, 128.87, 126.76,
122.68, 122.63, 122.22, 120.42, 115.25 (t, J = 242.2 Hz), 100.16,
98.94, 54.71 (t, J = 27.1 Hz), 26.82. HRMS calcd for C19H18F2N8O, [M
+H]+, 413.1644; found 413.1643.
5.1.6.11. 2-((2-((1-(2-methoxyethyl)-1H-pyrazol-4-yl)amino)-7H-pyrrolo
[2,3-d]pyrimidin-4-yl)amino)-N-methylbenzamide (18k). White solid,
yield: 63%; 1H NMR (400 MHz, MeOD) δ 8.84 (d, J = 8.4 Hz, 1H),
7.95 (s, 1H), 7.65 (dd, J = 7.9, 1.4 Hz, 1H), 7.60 (s, 1H), 7.49 (ddd,
J = 8.6, 7.4, 1.5 Hz, 1H), 7.06 (dd, J = 11.0, 4.1 Hz, 1H), 6.89 (d,
J = 3.6 Hz, 1H), 6.45 (d, J = 3.6 Hz, 1H), 4.23 (t, J = 5.3 Hz, 2H),
3.73 (t, J = 5.3 Hz, 2H), 2.93 (s, 3H). 13C NMR (100 MHz, MeOD) δ
172.21, 157.69, 155.29, 153.95, 142.14, 132.93, 132.28, 128.84,
125.79, 122.73, 122.69, 122.25, 121.99, 120.36, 100.10, 98.98,
5.1.6.6. 2-((2-((4-(N-ethylsulfamoyl)phenyl)amino)-7H-pyrrolo[2,3-d]
pyrimidin-4-yl)amino)-N-methylbenzamide (18f). White solid, yield:
59%; 1H NMR (600 MHz, DMSO‑d6) δ 11.76 (s, 1H), 11.51 (s, 1H),
9.52 (s, 1H), 9.00 (d, J = 8.3 Hz, 1H), 8.76 (d, J = 4.5 Hz, 1H), 8.02 (d,
J = 8.8 Hz, 2H), 7.77 (dd, J = 7.9, 1.1 Hz, 1H), 7.65 (d, J = 8.8 Hz,
2H), 7.54–7.50 (m, 1H), 7.30 (t, J = 5.8 Hz, 1H), 7.11–7.07 (m, 2H),
6.37 (dd, J = 3.3, 1.9 Hz, 1H), 2.83 (d, J = 4.5 Hz, 3H), 2.79–2.74 (m,
2H), 0.98 (t, J = 7.2 Hz, 3H) 13C NMR (150 MHz, DMSO‑d6) δ 169.50,
154.69, 152.91, 151.74, 145.22, 140.93, 131.79, 130.65, 128.01,
127.37 (2C), 120.94, 120.77, 120.70, 119.35, 117.33 (2C), 99.66,
72.34, 59.09, 53.03, 26.81. HRMS calcd for C20H22N8O2, [M+H]+
407.1938; found 407.1937.
,
97.45, 37.51, 26.35, 14.70. HRMS calcd for C22H23N7O3S, [M+Na]+
488.1475; found 488.1457.
,
5.1.6.12. N-methyl-2-((2-((1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazol-4-
yl)amino)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)benzamide
(18l). Light yellow solid, yield: 78%; 1H NMR (400 MHz, DMSO‑d6) δ
11.62 (s, 1H), 11.20 (s, 1H), 9.01 (s, 1H), 8.81–8.68 (m, 2H), 8.02 (s,
1H), 7.76 (d, J = 7.1 Hz, 1H), 7.55 (s, 1H), 7.49 (t, J = 7.5 Hz, 1H),
7.05 (t, J = 7.4 Hz, 1H), 6.95 (dd, J = 3.0, 2.5 Hz, 1H), 6.31 (dd,
J = 3.2, 1.8 Hz, 1H), 4.36–4.26 (m, 1H), 4.00–3.92 (m, 2H), 3.47 (td,
5.1.6.7. diethyl(4-((4-((2-(methylcarbamoyl)phenyl)amino)-7H-pyrrolo
[2,3-d]pyrimidin-2-yl)amino)benzyl)phosphonate (18g). Light yellow
solid, yield: 53%; 1H NMR (600 MHz, DMSO‑d6) δ 11.73 (s, 1H),
11.37 (s, 1H), 9.06 (d, J = 8.3 Hz, 1H), 8.97 (s, 1H), 8.76 (d,
J = 4.1 Hz, 1H), 7.76 (t, J = 7.9 Hz, 3H), 7.50 (t, J = 7.6 Hz, 1H),
7.16 (d, J = 6.9 Hz, 2H), 7.05 (t, J = 7.4 Hz, 1H), 7.00 (s, 1H), 6.33 (s,
1H), 3.97–3.90 (m, 4H), 3.14 (d, J = 21.0 Hz, 2H), 2.84 (d, J = 4.2 Hz,
3H), 1.18 (t, J = 7.0 Hz, 6H). 13C NMR (150 MHz, DMSO‑d6) δ 169.55,
155.57, 152.87, 152.22, 141.19, 140.13 (d, J = 3.4 Hz), 131.81,
129.61 (d, J = 6.5 Hz, 2C), 127.94, 123.47 (d, J = 9.2 Hz), 120.55,
120.42, 120.19, 118.92, 118.51 (d, J = 1.6 Hz, 2C), 99.00, 97.31,
61.28 (d, J = 6.5 Hz, 2C), 31.59 (d, J = 135.9 Hz), 26.35, 16.27 (d,
J = 5.3 Hz, 2C). HRMS calcd for C25H29N6O4P, [M+Na]+, 531.1880;
found 531.1881.
J = 11.4, 2.2 Hz, 2H), 2.84 (d, J = 4.4 Hz, 3H), 2.00–1.87 (m, 4H). 13
C
NMR (100 MHz, DMSO‑d6) δ 169.53, 155.76, 153.07, 152.80, 141.30,
131.73, 129.76, 127.98, 124.16, 120.39, 120.33, 119.54, 119.04,
117.08, 98.24, 97.29, 65.98 (2C), 57.12, 33.10 (2C), 26.33. HRMS
calcd for C22H24N8O2, [M+H]+, 433.2095; found 433.2100.
5.1.6.13. N-methyl-2-((2-((1-(piperidin-4-yl)-1H-pyrazol-4-yl)amino)-
7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)benzamide (18m). White solid,
yield: 85%; 1H NMR (600 MHz, DMSO‑d6) δ 11.63 (s, 1H), 11.22 (s,
1H), 9.01 (s, 1H), 8.82–8.72 (m, 2H), 7.97 (s, 1H), 7.76 (d, J = 7.5 Hz,
1H), 7.55 (s, 1H), 7.48 (t, J = 7.6 Hz, 1H), 7.04 (t, J = 7.4 Hz, 1H),
6.94 (s, 1H), 6.29 (s, 1H), 4.21 (d, J = 6.4 Hz, 1H), 3.19 (d,
J = 12.3 Hz, 2H), 2.82 (dd, J = 18.3, 8.1 Hz, 5H), 2.04 (d,
J = 12.4 Hz, 2H), 1.92–1.86 (m, 2H). 13C NMR (150 MHz, DMSO‑d6)
δ 169.53, 155.73, 153.05, 152.77, 141.33, 131.76, 131.54, 128.68,
127.99, 124.12, 120.33, 119.56, 118.36, 117.10, 98.24, 97.27, 57.62,
5.1.6.8. N-methyl-2-((2-((4-(4-methylpiperazin-1-yl)phenyl)amino)-7H-
pyrrolo[2,3-d]pyrimidin-4-yl)amino)benzamide (18h). White solid, yield:
82%; 1H NMR (600 MHz, DMSO‑d6) δ 11.71 (s, 1H), 11.28 (s, 1H), 9.08
(d, J = 8.4 Hz, 1H), 8.75 (q, J = 4.0 Hz, 1H), 8.71 (s, 1H), 7.75 (dd,
J = 7.9, 1.3 Hz, 1H), 7.64 (d, J = 8.9 Hz, 2H), 7.50–7.45 (m, 1H),
7.06–7.01 (m, 1H), 6.95 (dd, J = 3.3, 2.3 Hz, 1H), 6.88 (d, J = 9.1 Hz,
2H), 6.30 (dd, J = 3.4, 1.9 Hz, 1H), 3.09–3.05 (m, 4H), 2.83 (d,
J = 4.5 Hz, 3H), 2.53–2.50 (m, 4H), 2.25 (s, 3H). 13C NMR (150 MHz,
DMSO‑d6) δ 169.58, 156.04, 152.86, 152.45, 145.45, 141.32, 134.04,
131.84, 127.90, 120.44, 120.28, 120.02 (2C), 119.78, 118.70, 116.01
(2C), 98.68, 97.26, 54.70 (2C), 49.06 (2C), 45.66, 26.35. HRMS calcd
for C25H28N8O, [M+H]+, 457.2459; found 457.2460.
44.15 (2C), 31.95 (2C), 26.34. HRMS calcd for C22H25N9O, [M+H]+
432.2255; found 432.2261.
,
5.1.6.14. N-methyl-N-(2-(((2-(phenylamino)-7H-pyrrolo[2,3-d]
pyrimidin-4-yl)amino)methyl)phenyl)methanesulfonamide (21a). White
solid, yield: 68%; 1H NMR (600 MHz, CDCl3) δ 9.63 (s, 1H), 7.62 (d,
J = 7.7 Hz, 3H), 7.35–7.30 (m, 2H), 7.29–7.24 (m, 3H), 7.03 (s, 1H),
6.94 (t, J = 7.3 Hz, 1H), 6.45 (d, J = 3.1 Hz, 1H), 6.18 (d, J = 3.1 Hz,
1H), 5.93 (t, J = 5.3 Hz, 1H), 5.21 (s, 1H), 4.66 (s, 1H), 3.27 (s, 3H),
3.00 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 156.56, 155.80, 151.50,
140.71, 139.90, 139.84, 131.50, 129.51, 128.96 (2C), 128.91, 126.65,
121.68, 119.33 (2C), 118.46, 98.83, 97.85, 40.81, 39.66, 36.12. HRMS
calcd for C21H22N6O2S, [M+H]+, 423.1598; found 423.1604.
5.1.6.9. N-methyl-2-((2-((1-methyl-1H-pyrazol-4-yl)amino)-7H-pyrrolo
[2,3-d]pyrimidin-4-yl)amino)benzamide (18i). White solid, yield: 75%;
1H NMR (600 MHz, DMSO‑d6) δ 11.63 (s, 1H), 11.21 (s, 1H), 9.02 (s,
1H), 8.77 (s, 1H), 8.74 (d, J = 4.5 Hz, 1H), 7.90 (s, 1H), 7.78–7.72 (m,
1H), 7.49 (dd, J = 10.4, 3.0 Hz, 2H), 7.04 (t, J = 7.5 Hz, 1H), 6.94 (dd,
J = 3.2, 2.3 Hz, 1H), 6.29 (dd, J = 3.3, 1.9 Hz, 1H), 3.80 (s, 3H), 2.83
(d, J = 4.5 Hz, 3H). 13C NMR (150 MHz, DMSO‑d6) δ 169.53, 155.72,
153.05, 152.79, 141.31, 131.73, 129.80, 127.97, 124.48, 120.47,
120.37, 120.08, 119.55, 118.95, 98.22, 97.26, 38.67, 26.35. HRMS
5.1.6.15. N-(2-(((2-((3,4-dimethoxyphenyl)amino)-7H-pyrrolo[2,3-d]
pyrimidin-4-yl)amino)methyl)phenyl)-N-methylmethanesulfonamide
(21b). White solid, yield: 72%; 1H NMR (400 MHz, DMSO‑d6) δ 10.96
9

R. Wang, et al.
BioorganicChemistry102(2020)104092
(s, 1H), 8.32 (s, 1H), 7.57 (t, J = 5.3 Hz, 1H), 7.55–7.50 (m, 2H), 7.45
(dd, J = 6.7, 2.5 Hz, 1H), 7.37–7.30 (m, 2H), 7.22 (dd, J = 8.7, 2.3 Hz,
1H), 6.78 (dd, J = 3.2, 2.3 Hz, 1H), 6.73 (d, J = 8.8 Hz, 1H), 6.44 (s,
1H), 4.86 (d, J = 18.3 Hz, 2H), 3.67 (s, 3H), 3.66 (s, 3H), 3.23 (s, 3H),
3.11 (s, 3H). 13C NMR (150 MHz, DMSO‑d6) δ 156.52, 156.14, 151.80,
148.60, 142.46, 140.37, 139.73, 136.14, 128.53, 128.29, 127.80,
127.00, 117.91, 112.63, 109.70, 103.93, 98.93, 97.14, 56.05, 55.27,
38.79, 36.09. HRMS calcd for C23H26N6O4S, [M+H]+, 483.1809;
found 483.1812.
127.73, 127.03, 122.53, 118.17, 117.69 (2C), 98.96, 97.32, 61.23 (d,
J = 6.4 Hz, 2C), 38.80, 36.13, 31.56 (d, J = 135.7 Hz), 16.25 (d,
J = 5.3 Hz, 2C). HRMS calcd for C26H33N6O5PS, [M+Na]+, 595.1863;
found 595.1869.
5.1.6.21. N-methyl-N-(2-(((2-((4-(4-methylpiperazin-1-yl)phenyl)amino)-
7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)methyl)phenyl)
methanesulfonamide (21h). White solid, yield: 78%; 1H NMR (600 MHz,
DMSO‑d6) δ 10.98 (s, 1H), 8.25 (s, 1H), 7.64 (s, 1H), 7.53 (d,
J = 8.1 Hz, 3H), 7.41 (d, J = 6.6 Hz, 1H), 7.35–7.30 (m, 2H), 6.73
(d, J = 9.3 Hz, 3H), 6.45 (s, 1H), 4.82 (d, J = 76.6 Hz, 2H), 3.22 (s,
3H), 3.12 (s, 3H), 3.01 (s, 4H), 2.55–2.51 (m, 4H), 2.26 (s, 3H). 13C
NMR (150 MHz, DMSO‑d6) δ 156.49, 156.26, 151.92, 144.63, 140.44,
139.66, 134.81, 128.29, 128.25, 127.68, 127.03, 119.06 (2C), 117.75,
116.12 (2C), 98.98, 97.08, 54.50 (2C), 48.96 (2C), 45.37, 38.79, 36.20.
HRMS calcd for C26H32N8O2S, [M+H]+, 521.2442; found 521.2449.
5.1.6.16. N-methyl-N-(2-(((2-((3,4,5-trimethoxyphenyl)amino)-7H-
pyrrolo[2,3-d]pyrimidin-4-yl)amino)methyl)phenyl)methanesulfonamide
(21c). White solid, yield: 71%; 1H NMR (600 MHz, DMSO‑d6) δ 11.01
(s, 1H), 8.46 (s, 1H), 7.63 (s, 1H), 7.54–7.50 (m, 1H), 7.47 (dd, J = 7.2,
1.8 Hz, 1H), 7.34 (ddd, J = 7.9, 7.1, 1.7 Hz, 2H), 7.23 (s, 2H),
6.84–6.80 (m, 1H), 6.45 (s, 1H), 4.87 (d, J = 49.3 Hz, 2H), 3.67 (s, 6H),
3.57 (s, 3H), 3.23 (s, 3H), 3.11 (s, 3H). 13C NMR (150 MHz, DMSO‑d6) δ
156.53, 155.85, 152.51 (2C), 151.49, 140.32, 139.81, 138.23, 130.97,
128.80, 128.34, 127.94, 127.01, 118.16, 98.99, 97.29, 95.87 (2C),
60.09, 55.55 (2C), 38.83, 36.01. HRMS calcd for C24H28N6O5S, [M
+H]+, 513.1915; found 513.1924.
5.1.6.22. N-methyl-N-(2-(((2-((1-methyl-1H-pyrazol-4-yl)amino)-7H-
pyrrolo[2,3-d]pyrimidin-4-yl)amino)methyl)phenyl)methanesulfonamide
(21i). White solid, yield: 76%; 1H NMR (600 MHz, DMSO‑d6) δ 10.87
(s, 1H), 8.33 (s, 1H), 7.70 (s, 1H), 7.58 (s, 1H), 7.54 (d, J = 6.8 Hz, 1H),
7.39 (d, J = 7.2 Hz, 1H), 7.36–7.30 (m, 3H), 6.74–6.73 (m, 1H), 6.41
(s, 1H), 4.84 (d, J = 5.0 Hz, 2H), 3.67 (s, 3H), 3.25 (s, 3H), 3.12 (s, 3H).
13C NMR (150 MHz, DMSO‑d6) δ 156.84, 156.02, 152.21, 140.30,
139.66, 129.66, 129.33, 128.25, 127.72, 126.89, 124.90, 119.36,
117.56, 98.91, 96.54, 38.77, 38.52, 35.97. HRMS calcd for
5.1.6.17. N-(2-(((2-((4-cyanophenyl)amino)-7H-pyrrolo[2,3-d]
pyrimidin-4-yl)amino)methyl)phenyl)-N-methylmethanesulfonamide
(21d). White solid, yield: 63%; 1H NMR (600 MHz, DMSO‑d6) δ 11.24
(s, 1H), 9.33 (s, 1H), 8.01–7.82 (m, 3H), 7.53 (s, 2H), 7.43–7.32 (m,
3H), 6.90 (s, 1H), 6.53 (s, 1H), 5.75 (s, 1H), 4.89 (s, 2H), 3.24 (s, 3H),
3.13 (s, 3H). 13C NMR (150 MHz, DMSO‑d6) δ 156.60, 154.90, 151.13,
146.38, 139.97, 139.73, 132.64 (2C), 128.34, 128.20, 127.85, 127.02,
120.06, 119.10, 117.24 (2C), 100.06, 99.09, 98.10, 38.79, 36.04.
HRMS calcd for C22H21N7O2S, [M+H]+, 448.1550; found 448.1548.
C
19H22N8O2S, [M+H]+, 427.1659; found 427.1666.
5.1.6.23. N-(2-(((2-((1-(2,2-difluoroethyl)-1H-pyrazol-4-yl)amino)-7H-
pyrrolo[2,3-d]pyrimidin-4-yl)amino)methyl)phenyl)-N-
methylmethanesulfonamide (21j). Light yellow solid, yield: 58%; 1H
NMR (600 MHz, DMSO‑d6) δ 10.92 (s, 1H), 8.47 (s, 1H), 7.84 (s, 1H),
7.63 (s, 1H), 7.54 (d, J = 7.6 Hz, 1H), 7.50 (s, 1H), 7.39 (d, J = 7.3 Hz,
1H), 7.35–7.31 (m, 2H), 6.75 (d, J = 2.3 Hz, 1H), 6.43 (s, 1H), 4.85 (s,
2H), 4.43 (t, J = 13.5 Hz, 2H), 3.25 (s, 3H), 3.11 (s, 3H). 13C NMR
(150 MHz, DMSO‑d6) δ 156.89, 155.63, 151.79, 140.15, 139.68,
131.02, 129.65, 128.26, 127.77, 126.87, 125.30, 119.59, 117.76,
114.21 (t, J = 241.5 Hz), 99.00, 96.63, 52.84 (t, J = 26.2 Hz),
38.77, 35.90. HRMS calcd for C20H22F2N8O2S, [M+H]+, 477.1627;
found 477.1637.
5.1.6.18. N-(4-((4-((2-(N-methylmethylsulfonamido)benzyl)amino)-7H-
pyrrolo[2,3-d]pyrimidin-2-yl)amino)phenyl)acetamide
(21e). Light
yellow solid, yield: 72%; 1H NMR (400 MHz, DMSO‑d6) δ 10.98 (s,
1H), 9.66 (s, 1H), 8.46 (s, 1H), 7.66–7.57 (m, 3H), 7.52 (dd, J = 7.3,
1.8 Hz, 1H), 7.41 (dd, J = 6.8, 2.4 Hz, 1H), 7.36–7.31 (m, 3H), 7.30 (s,
1H), 6.79 (dd, J = 3.3, 2.3 Hz, 1H), 6.45 (s, 1H), 4.85 (d, J = 29.0 Hz,
2H), 3.24 (s, 3H), 3.12 (s, 3H), 1.99 (s, 3H). 13C NMR (150 MHz,
DMSO‑d6) δ 167.49, 156.52, 155.97, 151.75, 140.29, 139.66, 137.60,
131.74, 128.25, 128.21, 127.71, 127.01, 119.45 (2C), 118.03, 117.95
(2C), 98.94, 97.21, 38.79, 36.13, 23.80. HRMS calcd for C23H25N7O3S,
[M+H]+, 480.1812; found 480.1822.
5.1.6.24. N-(2-(((2-((1-(2-methoxyethyl)-1H-pyrazol-4-yl)amino)-7H-
pyrrolo[2,3-d]pyrimidin-4-yl)amino)methyl)phenyl)-N-
methylmethanesulfonamide (21k). White solid, yield: 51%; 1H NMR
(600 MHz, DMSO‑d6) δ 10.90 (s, 1H), 8.36 (s, 1H), 7.78 (s, 1H), 7.59
(s, 1H), 7.53 (d, J = 7.4 Hz, 1H), 7.41 (d, J = 5.9 Hz, 2H), 7.36–7.31
(m, 2H), 6.77–6.73 (m, 1H), 6.42 (s, 1H), 4.85 (d, J = 3.7 Hz, 2H), 4.08
(s, 2H), 3.55 (s, 2H), 3.26 (s, 3H), 3.17 (s, 3H), 3.11 (s, 3H). 13C NMR
(150 MHz, DMSO‑d6) δ 156.82, 156.07, 152.17, 140.34, 139.68,
129.65, 128.33, 128.24, 127.73, 126.87, 124.71, 118.97, 117.53,
98.89, 96.52, 70.81, 57.93, 51.08, 38.79, 35.93. HRMS calcd for
5.1.6.19. N-ethyl-4-((4-((2-(N-methylmethylsulfonamido)benzyl)amino)-
7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)benzenesulfonamide (21f). White
solid, yield: 53%; 1H NMR (400 MHz, DMSO‑d6) δ 11.15 (s, 1H), 9.15
(s, 1H), 7.86 (d, J = 8.8 Hz, 2H), 7.78 (s, 1H), 7.55 (dd, J = 7.4,
1.5 Hz, 1H), 7.49 (d, J = 8.9 Hz, 2H), 7.42 (dd, J = 7.0, 2.2 Hz, 1H),
7.34 (qd, J = 7.4, 3.4 Hz, 2H), 7.20 (t, J = 5.8 Hz, 1H), 6.88 (dd,
J = 3.2, 2.4 Hz, 1H), 6.51 (s, 1H), 4.88 (s, 2H), 3.23 (s, 3H), 3.12 (s,
3H), 2.77–2.68 (m, 2H), 0.95 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz,
DMSO‑d6) δ 156.56, 155.12, 151.30, 145.63, 140.06, 139.71, 129.87,
128.30, 128.20, 127.79, 127.27 (2C), 126.99, 118.87, 116.72 (2C),
99.03, 97.92, 38.74, 37.43, 36.05, 14.67. HRMS calcd for
C
21H26N8O3S, [M+H]+, 471.1921; found 471.1933.
5.1.6.25. N-methyl-N-(2-(((2-((1-(tetrahydro-2H-pyran-4-yl)-1H-
pyrazol-4-yl)amino)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)methyl)
phenyl)methanesulfonamide (21l). White solid, yield: 75%; 1H NMR
(600 MHz, MeOD) δ 7.94 (s, 1H), 7.53 (d, J = 7.5 Hz, 1H), 7.49 (d,
J = 7.7 Hz, 1H), 7.42 (s, 1H), 7.37–7.31 (m, 2H), 6.77 (d, J = 3.5 Hz,
1H), 6.39 (d, J = 3.5 Hz, 1H), 4.98 (d, J = 36.4 Hz, 2H), 4.21–4.15 (m,
1H), 3.96 (d, J = 11.6 Hz, 2H), 3.46 (t, J = 10.3 Hz, 2H), 3.29 (s, 3H),
3.09 (s, 3H), 1.93–1.81 (m, 4H). 13C NMR (150 MHz, MeOD) δ 159.06,
157.36, 152.08, 141.68, 141.23, 131.05, 130.29, 129.83, 129.24,
127.93, 125.45, 119.70, 119.20, 99.90, 98.32, 67.92 (2C), 59.19,
41.86, 39.48, 36.29, 34.27 (2C). HRMS calcd for C23H28N8O3S, [M
+H]+, 497.2078; found 497.2082.
C
23H27N7O4S2, [M+H]+, 530.1639; found 530.1640.
5.1.6.20. diethyl(4-((4-((2-(N-methylmethylsulfonamido)benzyl)amino)-
7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)benzyl)phosphonate (21g). Light
yellow solid, yield: 48%; 1H NMR (600 MHz, DMSO‑d6) δ 11.03 (s, 1H),
8.55 (s, 1H), 7.69–7.61 (m, 3H), 7.53 (d, J = 7.9 Hz, 1H), 7.41 (d,
J = 6.5 Hz, 1H), 7.33 (dt, J = 13.1, 6.4 Hz, 2H), 7.01 (d, J = 7.0 Hz,
2H), 6.80 (s, 1H), 6.46 (s, 1H), 4.85 (d, J = 54.3 Hz, 2H), 3.92 (dd,
J = 12.6, 6.7 Hz, 4H), 3.23 (s, 3H), 3.12 (s, 3H), 3.08 (s, 1H), 3.05 (s,
1H), 1.16 (t, J = 7.0 Hz, 6H). 13C NMR (150 MHz, DMSO‑d6) δ 156.54,
155.87, 151.62, 140.59, 140.26, 139.69, 129.53, 129.48, 128.27 (2C),
10

R. Wang, et al.
BioorganicChemistry102(2020)104092
5.1.6.26. N-methyl-N-(2-(((2-((1-(piperidin-4-yl)-1H-pyrazol-4-yl)
amino)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)methyl)phenyl)
and the filtrate was purified using chromatography to produce inter-
mediate 33. MS (ESI) m/z(%): 244.0 [M+H]+
.
methanesulfonamide (21m). Light yellow solid, yield: 86%; 1H NMR
(600 MHz, DMSO‑d6) δ 10.88 (s, 1H), 8.32 (s, 1H), 7.78 (s, 1H), 7.58 (s,
1H), 7.54 (d, J = 7.4 Hz, 1H), 7.42 (d, J = 7.0 Hz, 1H), 7.38–7.30 (m,
3H), 6.73 (s, 1H), 6.41 (s, 1H), 4.84 (d, J = 3.6 Hz, 2H), 3.91 (s, 1H),
3.25 (s, 3H), 3.13 (s, 3H), 2.96 (d, J = 11.5 Hz, 2H), 2.51 (s, 2H), 1.76
(s, 2H), 1.61 (d, J = 7.5 Hz, 2H). 13C NMR (150 MHz, DMSO‑d6) δ
156.89, 156.18, 152.36, 140.44, 139.64, 128.91, 128.35, 128.24,
127.70, 126.87, 124.45, 117.51, 116.33, 98.87, 96.49, 58.82, 45.20
(2C), 38.78, 36.05, 33.56 (2C). HRMS calcd for C23H29N9O2S, [M
+H]+, 496.2238; found 496.2241.
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).
5.1.7. Preparation of 4-(4-methylpiperazin-1-yl)aniline (24)
To a solution of 1-fluoro-4-nitrobenzene (1 equiv), 1-methylpiper-
azine (1.1 equiv), potassium carbonate (3 equiv) in N,N-di-
methylformamide. The reaction mixture was stirred at 80 °C. 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 23.
Intermediate 23 (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 purified using chromatography to produce intermediate 24.
5.2.2. Cell proliferation assay
MS (ESI) m/z(%): 192.3 [M+H]+
.
A549, MDA-MB-231, U-87MG 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 were added to the culture medium at the indicated final
concentrations and incubated for 72 h. Fresh MTT solution was added
and incubated for 4 h. The formazan crystals in each well were dis-
solved in 150 μL DMSO, and the absorbance of each test well was
measured at λ490nm using a Thermo reader (Multiskan GO).
5.1.8. Preparation of 4-amino-N-ethylbenzenesulfonamide (27)
To a solution of 4-nitrobenzenesulfonyl chloride (1 equiv), ethyla-
mine hydrochloride (1.1 equiv), triethylamine (3 equiv) in anhydrous
CH2Cl2. The reaction mixture was stirred at 25 °C. Upon completion,
the mixture was diluted with water and extracted with CH2Cl2. The
organic phases were combined, washed with saline, dried over anhy-
drous Na2SO4 and purified by column chromatography to give inter-
mediate 26. Intermediate 26 (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 intermediate 27.
5.2.3. Cell apoptosis analysis
The ability of compound 18h 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 18h 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
according to the manufacturer's protocol. After staining, flow cytometry
(Becton-Dickinson FACSCalibur) was performed to quantify the number
of apoptotic cells.
MS (ESI) m/z(%): 201.4 [M+H]+
.
5.1.9. General procedure for the synthesis of intermediates 30a-d.
4-nitro-1H-pyrazole (1 equiv), triphenylphosphine (1.5 equiv) and
corresponding alcohols (1 equiv) were dissolved in THF, and di-tert-
butyl azodicarboxylate (1.5 equiv) was added over approximately
15 min. The resulting mixture was stirred at 25 °C. Upon completion,
the residue was concentrated to dryness and purified by column chro-
matography to give intermediates 29a-c. A solution of 4-nitro-1H-pyr-
azole (1 equiv), bromo(methoxy)methane (1.1 eq) and potassium car-
bonate (1 equiv) in acetonitrile was heated at 60 °C for 6 h. The mixture
was diluted with ethyl acetate, and the organic layer was washed with
brine, dried over anhydrous Na2SO4 and filtered. The filtrate was
concentrated and purified by chromatography to give intermediates
29d. Intermediates 29a-d (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 30a-d.
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 18h 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
5.1.10. Preparation of diethyl (4-aminobenzyl)phosphonate (33)
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 18h, incubated for 24 h, and
photographed at 12 and 24 h with an inverted microscope. Migration
rate = [1 − (current wound size / initial wound size)] * 100%.
To a solution of 1-(chloromethyl)-4-nitrobenzene (1 equiv) in trie-
thyl phosphate (2 equiv). The reaction mixture was stirred at 130 °C for
12 h. Upon completion, the mixture was diluted with water and ex-
tracted with CH2Cl2. The organic phases were combined, washed with
saline, dried over anhydrous Na2SO4 and evaporated to afford crude
product 32. Intermediate 32 (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
11

R. Wang, et al.
BioorganicChemistry102(2020)104092
5.2.6. Molecular docking study
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Declaration of Competing Interest
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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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Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.104092.
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