Phosphamide-containing diphenylpyrimidine analogues (PA-DPPYs) as potent focal adhesion kinase (FAK) inhibitors with enhanced activity against pancreatic cancer cell lines

Article abstract of DOI:10.1016/j.bmc.2017.09.041

A family of phosphamide-containing diphenylpyrimidine analogues (PA-DPPYs) were synthesized as potent focal adhesion kinase (FAK) inhibitors. The PA-DPPY derivatives could significantly inhibit the FAK enzymatic activity at concentrations lower than 10.69 nM. Among them, compounds 7a and 7e were two of the most active FAK inhibitors, possessing IC₅₀ values of 4.25 nM and 4.65 nM, respectively. In particular, compound 7e also displayed strong activity against AsPC cell line, with an IC₅₀ of 1.66 μM, but show low activity against the normal HPDE6-C7 cells (IC₅₀ > 20 μM), indicating its low cell cytotoxicity. Additionally, flow cytometry analysis showed that after treatment with 7e (8 μM, 72 h), both AsPC and Panc cells growth were almost totally inhibited, with a cell viability rate of 16.8% and 18.1%, respectively. Overall, compound 7e may be served as a valuable FAK inhibitor for the treatment of pancreatic cancer.

Full text of DOI:10.1016/j.bmc.2017.09.041

Accepted Manuscript
Phosphamide-Containing Diphenylpyrimidine Analogues (PA-DPPYs) as Po-
tent Focal Adhesion Kinase (FAK) Inhibitors with Enhanced Activity against
Pancreatic Cancer Cell Lines
He Liu, Bin Wu, Yang Ge, Jiaxin Xu, Shijie Song, Changyuan Wang, Jihong
Yao, Kexin Liu, Yanxia Li, Xiuli Sun, Xiaodong Ma
PII:
S0968-0896(17)31276-2
https://doi.org/10.1016/j.bmc.2017.09.041
BMC 14001
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 June 2017
25 September 2017
29 September 2017
Please cite this article as: Liu, H., Wu, B., Ge, Y., Xu, J., Song, S., Wang, C., Yao, J., Liu, K., Li, Y., Sun, X., Ma,
X., Phosphamide-Containing Diphenylpyrimidine Analogues (PA-DPPYs) as Potent Focal Adhesion Kinase (FAK)
Inhibitors with Enhanced Activity against Pancreatic Cancer Cell Lines, Bioorganic & Medicinal Chemistry (2017),
doi: https://doi.org/10.1016/j.bmc.2017.09.041
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Phosphamide-Containing Diphenylpyrimidine Analogues (PA-DPPYs)
as Potent Focal Adhesion Kinase (FAK) Inhibitors with Enhanced
Activity against Pancreatic Cancer Cell Lines
He Liua,1, Bin Wua,1, Yang Gea, Jiaxin Xua, Shijie Songa, Changyuan Wanga, Jihong Yaoa, Kexin
Liua, Yanxia Lib Xiuli Sunb and Xiaodong Maa,
a College of Pharmacy, Dalian Medical University, Dalian 116044, PR China.
b Hematology Department, Department of Respiratory Medicine, the First Affiliated Hospital of Dalian Medical
University, Dalian 116011, PR China.
1These authors contributed equally to this work.
 Corresponding author
E-mail address: xiaodong.ma@139.com (X. Ma).
ABSTRCT
A family of phosphamide-containing diphenylpyrimidine analogues (PA-DPPYs)
were synthesized as potent focal adhesion kinase (FAK) inhibitors. The PA-DPPY
derivatives could significantly inhibit the FAK enzymatic activity within
concentrations of lower than 10.69 nM. Among them, compounds 7a and 7e were two
of the most active FAK inhibitors, possessing IC50 values of 4.25 nM and 4.65 nM,
respectively. In particular, compound 7e also displayed strong activity against AsPC
cell line, with an IC50 of 1.66 μM, but show low activity against the normal
HPDE6-C7 cells (IC50 > 20 μM), indicating its low cell cytoxicity. Additionally, flow
cytometry analysis showed that after treatment of 7e (8 μM, 72 h), both AsPC and
Panc cells were almost totally inhibited, with a cell viability rate of 16.8% and 18.1%,
respectively. Overall, compound 7e may be served as a valuable FAK inhibitor for the
treatment of pancreatic cancer.
Keywords: Pancreatic Cancer; FAK; Inhibitor; Pyrimidine.
1. Introduction
Pancreatic cancer (PC), which is characterized by its invasive nature, ability to
evade aggressive therapy and frequent late stage diagnosis, leads to high mortality
rate, with an average survival of only 10 months after diagnosis.1 In 2012, PC is the
seventh most common cause of cancer deaths, resulting in 330,000 deaths
1

globally. PC is also the fifth most common cause of cancer death in the United
Kingdom, and the fourth most common in the United States. The disease occurs most
often in the developed world, where about 70% of the new cases in 2012
originated.2–4 Accordingly, there is an urgent need for the development of novel
effective and safe agents for the treatment of PC.
Cl
N
F
F
HN
N
NH O
Cl
N
N
F
O
NH
HN
N
NH
O
HN
NH O
O
O
NH
NH
N
N
O
N
O
N
3
2
1
VS-4718(PND-1186)
Phase I
CEP-37440
Phase I
TAE226
Preclinical
HO
NCT01849744
NCT01922752
F
F
F
F
N
F
N
F
Cl
N
HN
N
NH
HN
N
NH
HN
NH O
N
N
NH
O
N
N
N
S
N
N
S
HN
O
O
HN
O
O
6
O
O
4
5
VS-6062(PF-562271)
Phase I
GSK2256098
Phase II
Defactinib
Phase II
NCT02523014,
NCT02428270
NCT00666926
NCT02465060
Figure 1. Chemical structures of the typical FAK inhibitors.
Focal adhesion kinase (FAK) is a cytoplasmic protein-tyrosine kinase that
modulates cell adhesion, migration, proliferation and survival in response to
extracellular signals.5,6 FAK is aberrantly overexpressed or activated in most solid
tumors, including the stubborn PC.7–9 Phosphorylation of FAK at tyrosine (Y) 397 is
required for its kinase activity to trigger down-stream events such as AKT and ERK
activation. Therefore, the level of phospho-Y397 FAK has been used as an indicator
of FAK activation.10 Small molecules targeting FAK Y397 are attractive because
blocking Y397 phosphorylation is expected to prevent uncontrolled PC growth caused
2

by FAK overexpression or hyperactivity.11
R1
O
O
O
O
P
O
O
L
G
l
HN
u
HN
5
0
6
/
G
l
R1
O
y
5
O
0
5
P
HN
N
NH
HN
N
NH
L
N
N
Asp564
R
Structure-based
Design
F
Spebrutinib
BTK Inhibitors
Pho-DPPYs
Improved anti-BTK activity
HN
O
Phase Ⅲ
O
HN
R
N
NH
N
N
Cys502
H
PA-DPPYs
N
R = Cl, NO2; R1 = EtO-, morpholine
L= -CH2- or -O(CH2)3-
O
O
HN
Cl
N
NH
N
TAE226
FAK inhibitior
Figure 2. Designed strategy of the title molecules PA-DPPY analogues.
To date, several ATP-competitive reversible FAK inhibitors have been developed to
target tumor and endothelial cells, including compounds TAE226 (1)12-14, CEP-37440
(2, phase I, NCT01922752)15,16, VS-4718 (3, PND-1186, phase I, NCT01849744)17,
VS-6062 (4, PF-562271, phase I, NCT00666926)18,19, GSK2256098 (5, phase II,
NCT02523014, NCT02428270)20,21，and defactinib (6, phase II, NCT02465060)22
(Figure 1). TAE226 is an exemplary FAK inhibitor, showing potent antiproliferative
and antitumor effects in vitro and in vivo in several types of malignancies12-14
.
GSK2256098 was introduced as a novel FAK inhibitor currently in clinical testing for
cancer treatment, displaying promising tumor regression effect in a variety of
subcutaneously-inoculated xenograft models including LoVo human colon cancer,
PC-3M prostate cancer and BxPC human PC20,21. Defactinib, a well-known FAK
inhibitor, significantly impacts cell viability, decreases anchorage-independent growth
and motility in selected PC cells by targeting FAK kinase activity and Y397
phosphorylation22. Generally, most of these potent FAK inhibitors possess a
pyrimidine template along with a N-methylforamide-substituted aniline, which are
essential to interact with amino acids Cys502, Asp564 and Leu567 in the FAK protein
3

to interfere with FAK activity23–25. Phosphoryl functional group is very effective and
widely used to improve the aqueous solubility and biological activity of anticancer
alkylating agents that require activation in vivo26–28. Our previous studies also
indicated that introduction of a phosphoryl group to the C-2 aniline side chain of the
pyrimidine scaffold enhanced the activity against the Bruton’s tyrosine kinase (BTK)
and exhibited excellent anticancer activity against B-cell leukemia cells29–32
.
According to the Fragement-based drug design strategy, a new series of
phosphamide-containing diphenylpyrimidine analogues (PA-DPPYs) were
synthesized to strengthen the anti-FAK activity for the treatment of PC in this
modification work (Figure 2). The synthesis and biological evaluation of these
newly-designed PA-DPPYs, in parallel with their putative binding mechanism with
the FAK enzyme, are also described in this manuscript.
2. Results and discussion
2.1. Chemistry
According to our reported synthetic method33–37, these newly designed PA-DPPY
analogues were synthesized as shown in Scheme 1. Commercially available
2-nitrobenzoic acid (8) was reacted with thionyl chloride, and then directly reacted
with methylamine hydrochloride in the NaHCO3 base to produce the
2-nitro-N-methylbenzamide (9). Compound
9
was then converted to
2-amino-N-methylbenzamide (10) under Fe-NH4Cl reducing condition. By region
selectively coupling with the C-2 chlorine atom in the pyrmidine reagent, compound
10 was converted to the 2-chloropyrimidine intermediate 11a,b. Meanwhile, the
chloro-substituted nitrobenzene derivatives 12a,b were reacted with triethyl
phosphate to synthesize the diethoxyphosphinyl nitrobenzenes 13a,b, which were
reduced using Fe-NH4Cl conditions to form the anilines 14a,b. Additionally,
compounds 13a,b were treated with thionyl chloride and then directly reacted with
morpholine in the NaHCO3 base to prepare the phosphoramide intermediates 16a,b.
Under the action of the Fe-NH4Cl reduction reagent, compounds 16a,b were reduced
to the anilines 17a,b. With these intermediates in hand, the desired compounds 7a-f
4

were conventionally synthesized via coupling reaction of anilines 14a,b or 17a,b with
pyrimidines 11a,b in the presence of trifluoroacetic acid (TFA) under reflux
temperature.
R
N
R
N
Cl
N
NH
O
NO2
O
NO2
O
NH2
O
Cl
N
d
Cl
a,b
c
N
OH
N
N
H
H
H
11a,b
R= Cl,NO2
O
8
10
9
O
P
NH2
L
O
NO2
O2N
L
H
N
c
e
h
O
P
O
O
O
L
O
HN
R
N
NH
P
L
13a,b
O
O
Cl
N
14a,b
7a-d
R = Cl, NO2;
L= -CH2- or -O(CH2)3-
12a,b
f
O
N
NO2
NH2
O
L
NO2
P
O
g
c
h
HN
O
L
O
P
O
L
O
L
P
P
O
HN
R
N
NH
O
Cl
N
O
N
N
O
O
7e,f
R = Cl, NO2;
16a,b
15a,b
17a,b
L= -CH2- or -O(CH2)3-
Scheme 1. Synthetic route of the title compounds 7a-f. Reagents and conditions: (a) SOCl2, 1 h, 60
C; (b) H2NMeˑHCl, NaHCO3, CH3CN, 4 h, 80 C, 95%; (c) Fe-NH4Cl, MeOH-H2O, 2 h, 70 C,
65–78%; (d) N,N-diisopropylethylamine (DIPEA), isopropanol, 6 h, 85%; (e) P(OEt)3, 5 h, 130 C,
80-91%; (f) (COCl)2, DMF, 60 C, 2 h; (g) morpholine, THF, rt., 2 h, 92%; (h) TFA, 2-BuOH, 100 C,
12 h, 12-30%.
2.2. Biological activity
All the PA-DPPY analogues were evaluated for their activity against the FAK enzyme
using the ADP-Glo™ Kinase Assay system38,39, as well as for the antiprilificative
actvity against PC cell lines (AsPC, BXPC and Panc) using the MTT assay40.
TAE226, the typical FAK inhibitor, was also tested for comparison. The test results
are shown in Table 1. Apparently, PA-DPPY analogues displayed strong anti-FAK
5

activity, with IC50 values ranging from 4.25 to 10.69 nM. Compared with TAE226,
most of the PA-DPPY analogues exhibited equivalent or stronger inhibitory activity.
In particular, compound 7a (IC50 = 4.25 nM) and 7e (IC50 = 4.65 nM), showed
stronger anti-FAK activity than TAE226 (IC50 = 6.79 nM). The kinase-based test
results indicated that the substitutens installed on the pyrimidine template did produce
a little effect on their anti-FAK activity. For example, the C-2 chlorine-substituted
analogues 7a,b are as potent as the 7c-f analogues with a nitro substitutent. Moreover,
morpholine subsitutent in analogues 7e and 7f is equivalently beneficial to the ethoxyl
group in analogues 7a-d. Additionally, inhibitors 7a, 7c and 7f , bearing a
three-carbon linker are as favorable as inhibitors 7b, 7d and 7e with a one-carbon
linker. The effects on the viability of PC cell lines showed that compounds 7a and 7e,
two of the strongest FAK inhibitors among PA-DPPYs, exhibited potent capacitcy to
interfer with the proliferaction of PC cells, especially against the BxPC-3 cells, with
IC50 values between 0.02 and 0.57 μM. Interestingly, compound 7e also displayed
strong activity against the AsPC cell line, with an IC50 of 1.66 μM, but was not active
against normal HPDE6-C7 cells at concentraion of more than 20 μM, indicating its
low cytoxicity towards normal cells. Nevertheless, most of these inhibitors are only
moderatively active against the Panc-1 cells (IC50 >10.87 μM). Furthermore, the
results in Figure 3 revealed that the cell viability was significantly decreased for both
AsPC and Panc cells treated with inhibitor 7e, in a time and concentration-dependent
manner. After treatment with 8 μM concentration of 7e for 72 h, both AsPC and Panc
cells were almost totally inhibited, with a cell viability rate of 16.8% and 18.1%,
respectively. Taken together, these results indicate that compound 7e is a potent FAK
inhibitor, nontoxic for normal cells, worthy of further research of its effects on PC
cells.
Table 1 Biological activity of the newly synthesized compounds 7a-f. a
6

R1
O
O
L
P
HN
O
HN
R
N
NH
N
7a-f
Antiproliferative activity
Enzymatic
activity
(IC50, μM)c
Compd.
R
R1
L
(IC50, nM)b
AsPC-1
BxPC-3
Panc-1
HPDE6-C7
7a
7b
7c
7d
Cl
Cl
O
-CH2-
-O(CH2)3-
-CH2-
4.25±0.87
10.69±1.42
5.01±0.98
6.18±0.89
3.31±0.88 0.02±0.0009 14.76±2.12
5.44±1.02
4.43±0.89
4.60±0.91
1.96±0.92
O
O
O
4.93±0.49
15.19±2.31
18.26±2.15
1.04±0.23
0.82±0.11
2.04±0.38
10.87±1.05
19.19±2.01
25.38±3.12
NO2
NO2
-O(CH2)3-
N
7e
NO2
NO2
-O(CH2)3-
-CH2-
4.65±0.91
1.66±0.48
0.57±0.089
>40.00
>20.00
O
N
7f
5.69±0.35
6.79±1.42
6.42±1.03
6.73±0.99
1.02±0.11
1.03±0.35
>40.00
>20.00
8.11±0.99
O
TAE226
11.38±1.28
a Data represent the mean of at least three separate experiments.
b
Concentration needed to inhibit the autophosphorylation of the cytoplasmic domain of FAK by 50%, as
calculated using software GraphPad Prim version 5.0. Mean values of three independent experiments±SE are
reported (n=3).
c
Dose-response curves were determined at five concentrations. The IC50 values are the concentrations in
micoromolar needed to inhibit cell growth by 50%, as calculated using GraphPad Prism version 5.0.
A
B
Figure 3. The effects of treating time and concentrations of inhibitor 7e on cell viability, (A) AsPC
cells; (B) BxPC cells.
In addition, flow cytometry performance presented in Figure 4 revealed that the
7

active inhibitor 7e triggered apoptosis in AsPC cells in a dose-and time-dependent
manner31. The percentages of apoptotic cells increased from 41.7% to 91.1% after
treatment with (2.5, 5, and 10 μM) 7e for 72 h. The effect of 7e on cell-cycle
progression in AsPC cells was also investigated using by flow cytometry analysis
(Figure 5). Compared with the control group, the percentages of cells in the G2/M
phase increased from 0.89% to 22.1%, and those in the S phase decreased from 59.70%
to 37.82% after treatment with 7e at concentrations of 1, 2.5, and 5 μM for 72 h.
While the percentages of cells in the G0/M phase showed only minor changes.
Evidently, 7e significantly blocked AsPC cells in the G2/M phase.
Blank
2.5 μM
5 μM
10 μM
Figure 4. Compound 7e induced AsPC-1 cell apoptosis in vitro. The cells were incubated with the
indicated concentrations of 7e for 72 h, and the cells were stained with annexinV/FTIC, followed by
flow cytometry analysis. One representative experiment is shown.
8

Blank
2.5μM
1μM
G0/G1:38.18%
S:45.28%
G2/M:16.54%
G0/G1:39.41%
S:59.70%
G2/M:0.89%
G0/G1:38.60%
S:48.41%
G2/M:13.00%
5μM
G0/G1:40.06%
S:37.82%
G2/M:22.11%
Figure 5. Effects of inhibitor 7e on AsPC cells cycle arrest detected by flow cytometry assay. Cells
were treated with different concentration of 7e for 72 h, collected and fixed with 70% ethanol at 4 °C
overnight. Then, the cells were stained by the mixture containing 5 mL propidium iodide for 10 min at
37 °C, and the cell cycle was analyzed by a flow cytometer.
Table 2. Enzymatic activity of compound 7e against a small panel of kinasesa.
b
Kinase
FAK
%
92
71
60
48
Kinase
EGFR
%
22
45
33
26
PYK2
AXL
EGFRT790M
BTK
AURK A
JAK3
a This test was conducted by the SelectScreen™ Biochemical Profiling Lab.
bInhibiton rate at a concentration of 100 nM.
Furthermore, the typical FAK inhibitor 7e was tested against a small panel of
kinases at a concentration of 100 nM by using the SelectScreen™ Biochemical
Profiling Lab platform (Thermo-Fisher, Waltham, MA, USA). The results presented
in Table 2, revealed that, in addition to FAK, compound 7e also strongly binds to the
PYK2 and AXL kinases at a concentration of 100 nM, with inhibition rates of 71 and
60%, respectively. While for other kinase, compound 7e are not active, with inhibition
rates lower than 50%.
9

2.3. Molecular modeling analysis
A
B
Arg505
Arg505
Leu567
Leu567
H2O
Glu506
H2O
Glu506
Gly505
Asp564
Asp564
Gly505
Cys502
Leu501
Cys502
Leu501
Gln438
Gln438
Arg505
Arg505
C
D
Leu567
Leu567
H2O
Glu506
H2O
Glu506
Gly505
Asp564
Gly505
Asp564
Cys502
Leu501
Cys502
Gln438
Gln438
Leu501
Figure 6. Predicted binding models of the typical PA-DPPY analogues in FAK enzyme (PDB code:
2JKK), A: TAE226; B: inhibitor 7e; C: inhibitor 7d; D: inhibitor 7f.
To investigate the putative interaction mechanism of the PA-DPPY derivatives
with the FAK enzyme, three representative molecules 7d,7e, and 7f were individually
docked into the ATP binding pocket of the FAK enzyme (PDB: 2JKK).41 The lead
compound TAE226 was also analyzed for comparison (Figure 6A). The program
AutoDock 4.2 with its default parameters was used.42,43 All the docking results are
presented in Figure 6, indicating that PA-DPPY derivatives could tightly contact with
the FAK enzyme as to lead compound TAE226. Notably, PA-DPPY analogues formed
three strong hydrogen bonds, which are produced by the N-1 atom in the pyrimidine
core with amino acid Cys502, the carbonyl group in N-methylbenzamide with the
amino acid Asp564, and the nitrogen atom in N-methylbenzamide with Arg505,
Gly506 through a water molecule. From the docking results, it can be seen that only
10

little differences in the hydrophilic binding sites surrounded by the amino acids
Gly505 and Glu506 appeared for PA-DPPYs. Significantly, in this binding sections,
the morpholine substituent and the ethoxyl group in PA-DPPYs formed strong
contacts with the amino acid Gly505 and Glu506 through the polar induced forces. In
addition, the C-5 substituents in the pyrimidine core, including the nitro and chlorine
groups produced equivalent interaction forces with the amino acid Gln438. Due to the
similar binding interaction forces, all these inhibitors displayed potent inhibitory
potency against the FAK kinase activity. Overall, this putative binding models exactly
explain their biological activity data.
3. Conclusion
A new class of pyrimidine derivatives featuring a phosphamide functional group were
designed and synthesized as potent FAK inhibitors for the treatment of PC.Most of
these compounds displayed strong activity against the FAK kinase at concentrations
lower than 10.69 nM. Compounds 7a and 7e, the two most active inhibitors, could
inhibit FAK activity at concentrations of 4.25 nM and 4.65 nM, respectively. The two
inhibitors are clearly stronger than the novel FAK inhibitors TAE226 (IC50 = 6.79
nM). Compound 7e also displayed strong activity against AsPC cell line, with an IC50
of 1.66 μM, but show low activity against normal HPDE6-C7 cells at a concentration
higher than 20 μM, indicating its low cell cytoxicity. In addition, inhibitor 7e also
induced apoptosis in AsPC cells in a dose and time-dependent manner. In summary,
this contribution provided an effective FAK inhibitor, namely 7e, which may serve as
a potential lead compound toward pancreatic cancer.
4. Experimental section
4.1. General methods and chemistry
Unless otherwise noted, all solvents and chemicals were used as purchased
1
without further purification. H NMR and 13C NMR spectra were recorded on a
Brucker AV 400 MHz spectrometer and Brucker AV 100 MHz spectrometer,
respectively. Coupling constants (J) are expressed in hertz (Hz). Chemical shifts (δ)
of NMR are reported in parts per million (ppm) units relative to the residual solvent
11

1
peak (d6-DMSO: 39.0, TMS: 0.0). H NMR spectra are reported in the following
order: multiplicity, approximate coupling constant (J value) in hertz, and number of
protons; signals are characterized as s (singlet), d (doublet), dd (doublet of doublets), t
(triplet), m (multiplet), and br (broad signal). High resolution ESI-MS was performed
on an AB Sciex TripleTOF® 4600 LC/MS/MS system. All reactions were monitored
by TLC, using silica gel plates with fluorescence F254 (TLC Silica gel 60 F254,
Merck) and UV light visualization. Flash chromatography separations were obtained
on Silica Gel (300–400 mesh) using dichloromethane/methanol as eluents.
4.2. Preparation of the 2-chloropyrimidine intermediates 11a,b
2-Amino-N-methylbenzamide 10 was prepared according to the general
procedure in literatures 33 to 35. The prepared 2-amino-N-methylbenzamide 10 (1.00
g, 6.67 mmol) was added in one portion to 2,4,5-trichloropyrimidine (1.22 g, 6.67
mmol) or 2,4-dichloro-5-nitropyrimidine (1.29 g, 6.67 mmol) and DIPEA (1.29 g,
10.0 mmol) in isopropanol (100 mL). The resulting mixture was stirred at 85 °C for 6
h. The mixture was evaporated to dryness, and the residue was recrystallised from
MeCN/water
20:1
to
yield
2-(2-chloro-5-substitudedpyrimidin-4-ylamino)-N-methylbenzamide (1.69 g, 85 %).
4.3. Preparation of the title molecules 7a-f
Phosphoryl-substituted anilines 14a,b and 17a,b were prepared according to the
literature 37. The synthesized anilines 14a,b or 17a,b (1.00 mmol), 11a,b (1.00
mmol), TFA (1.50 mmol), and 2-BuOH (20 mL) was mixed in around-bottom flask.
The slurry was heated to 100 °C for 12 h. The reaction mixture was allowed to cool to
room temperature and was neutralized with a saturated aqueous sodium bicarbonate
solution. The aqueous mixture was then extracted with CH2Cl2 (3× 20 mL) three
times. The organic layer was dried over anhydrous sodium sulfate and concentrated in
vacuo to obtain a crude product. The residue was purified by column chromatography
on silica gel (elution with 3% methanol /dichloromethane) to give the title products.
N-(3-((5-Chloro-2-((4-((diethoxyphosphoryl)methy))amino)phenylamino-4-pyrim
idinyl)amino)phenyl)-N-methylbenzamide (7a)
12

Orange-yellow solid (164 mg, 12%), Purity (HPLC): 98.9%, 1H NMR (400 MHz,
DMSO-d6): δ 11.61 (s, 1H), 9.46 (s, 1H), 8.73-8.77 (m, 2H), 8.22 (s, 1H), 7.8-7.67 (m,
1H), 7.59 (d, J = 8.4 Hz, 2H), 7.48-7.52 (m, 1H), 7.23-7.08 (m, 3H), 3.90-3.97 (m,
4H), 3.18（s, 1H）, 3.13 (s, 1H), 2.81 (d, J = 4.4 Hz, 3H), 1.17 (t, J = 7.2 Hz, 6H); 13C
NMR (100 MHz, DMSO-d6): δ 169.16, 157.96, 155.23, 154.90, 139.55, 138.95,
131.70, 129.98 (2C), 128.25, 125.50, 122.19, 121.71, 120.99, 119.85 (2C), 105.28,
61.58
(2C),
32.51,
31.16,
16.53
(2C);
HRMS
(ESI) m/z
calcd
for C23H27ClN5O4P [M+H]+ 504.1489, found 504.1568.
N-(3-((5-Chloro-2-((4-((diethoxyphosphoryl)propoxyl))amino)phenylamino-4-pyr
imidinyl)amino)phenyl)-N-methylbenzamide (7b)
1
Orang-yellow solid (165 mg, 18%), Purity (HPLC): 97.2%, H NMR (400 MHz,
DMSO-d6): δ 11.60 (s, 1H), 9.30 (s, 1H), 8.76 (d, J = 4.0 Hz, 2H), 8.18 (s, 1H), 7.75
(d, J = 8.0 Hz, 1H), 7.62-7.40 (m, 3H), 7.13 (t, J = 8.0 Hz, 1H), 6.87 (d, J = 8.0 Hz,
2H), 3.97-4.0 (m, 6H), 2.81 (d, J = 4.0 Hz, 3H), 1.89 (s, 4H), 1.22 (t, J = 8.0 Hz, 6H);
13C NMR (100 MHz, DMSO-d6): δ 169.40, 158.43, 155.41, 155.12, 154.11, 139.86,
133.83, 131.95, 128.43, 122.31, 122.04 (2C), 121.77, 121.03, 114.78 (2C), 104.96,
67.58, 61.45 (2C), 22.78, 22.31, 20.92, 16.74 (2C); HRMS(ESI) m/z calcd
for C25H31ClN5O5P
[M+H]+ 548.1751,
found 548.1832.
N-(3-((5-Nitro-2-((4-((diethoxyphosphoryl)methyl))amino)phenylamino-4-pyrimi
dinyl)amino)phenyl)-N-methylbenzamide (7c)
1
Orang-yellow solid (100 mg, 29%), Purity (HPLC): 98.5%, H NMR: (400 MHz,
DMSO-d6) δ 12.05 (s, 1H), 10.50 (s, 1H), 9.12 (s, 1H), 8.65 (d, J = 4.4 Hz, 1H), 8.18
(d, J = 6.8 Hz, 1H), 7.80-7.37 (m, 4H), 7.36-7.01 (m, 3H), 4.27-3.65 (m, 4H), 3.21 (s,
13
1H), 3.15 (s, 1H), 2.77 (d, J = 4.4 Hz, 3H), 1.17 (t, J = 8.0 Hz, 6H); C NMR: (100
MHz, DMSO-d6) δ 168.30, 159.92, 158.40, 154.08, 137.36, 136.32, 130.69, 130.29,
130.22, 128.35, 127.98, 127.89, 127.11, 126.07, 124.90, 121.60, 121.14, 61.78 (2C),
32.74,
31.40,
16.69
(2C);
HRMS (ESI) m/z
found
calcd
for C23H27N6O6P [M+H]+ 515.1730,
515.1813.
N-(3-((5-Nitro-2-((4-((diethoxyphosphoryl)propoxyl))amino)phenylamino-4-pyri
midinyl)amino)phenyl)-N-methylbenzamide (7d)
13

1
Orang-yellow solid (120 mg, 20%), Purity (HPLC): 96.7%, H NMR: (400 MHz,
DMSO-d6) δ 12.05 (s, 1H), 10.39 (s, 1H), 9.09 (s, 1H), 8.63 (d, J = 4.0 Hz, 1H), 8.15
(d, J = 8.0 Hz, 1H), 7.63 (d, J = 8.0 Hz, 1H), 7.51 (d, J = 12 Hz, 3H), 7.27 (s, 1H),
6.81 (d, J = 8.0 Hz, 2H), 3.97-4.02 (m, 6H), 2.77 (d, J = 4.0 Hz, 3H), 1.89 (s, 4H),
13
1.23 (t, J = 8.0 Hz, 6H); C NMR: (400 MHz, DMSO-d6) δ 168.30, 159.90, 158.37,
155.43, 154.11, 136.34, 131.90, 130.73, 128.29, 126.93, 125.94, 124.86, 123.13 (2C),
121.30, 114.73 (2C), 67.74, 61.40 (2C), 26.62, 22.29, 20.89, 16.80 (2C);
HRMS (ESI) m/z
calcd
for C25H31N6O7P [M+H]+ 559.1992, found 559.2080.
N-(3-((5-Nitro-2-((4-(((1-morpholino)ethoxyphosphinyl)propoxyl))amino)phenyl
amino-4-pyrimidinyl)amino)phenyl)-N-methylbenzamide (7e)
1
Orang-yellow solid (124 mg, 23%), Purity (HPLC): 98.1%, H NMR: (400 MHz,
DMSO-d6) δ 12.05 (s, 1H), 10.39 (s, 1H), 9.09 (s, 1H), 8.63 (d, J = 4.6 Hz, 1H), 8.16
(d, J = 8.2 Hz, 1H), 7.63 (d, J = 8.0 Hz, 1H), 7.56-7.41 (m, 3H), 7.27 (t, J = 8.0 Hz,
1H), 6.82 (d, J = 8.0 Hz, 2H), 4.10-3.75 (m, 4H), 3.52 (t, J = 4.4 Hz, 4H), 3.01 (d, J =
2.6 Hz, 4H), 2.77 (d, J = 4.0 Hz, 3H), 1.81-1.88 (m, 4H), 1.21 (t, J = 8.0 Hz, 3H); 13C
NMR: (100 MHz, DMSO-d6) δ 168.30, 159.90, 158.38, 155.45, 154.12, 136.35,
131.87, 130.75, 128.30, 126.93, 125.94, 124.87, 123.139 (2C), 121.30, 114.74 (2C),
67.96, 67.02 (2C), 59.26, 43.98 (2C), 26.62, 22.55, 20.97, 16.70; HRMS (ESI) m/z
calcd for C27H34N7O7P [M+H]+ 600.2257, found 600.2340.
N-(3-((5-Nitro-2-((4-(((1-morpholino)ethoxyphosphinyl)methy))amino)phenylami
no-4-pyrimidinyl)amino)-N-methylbenzamide (7f)
1
Orang-yellow solid (307 mg ,25%), Purity (HPLC): 98.5%, H NMR: (400 MHz,
DMSO-d6) δ 12.04 (s, 1H), 10.51 (s, 1H), 9.12 (s, 1H), 8.65 (s, 1H), 8.18 (d, J = 4.0
Hz, 1H), 7.80-7.42 (m, 4H), 7.29 (s, 1H), 7.15 (s, 2H), 4.06-3.68 (m, 2H), 3.42 (d, J =
12.4 Hz, 4H), 3.11 (d, J = 12.0 Hz, 2H), 3.01-2.66 (m, 7H), 1.21 (t, J = 6.8 Hz, 3H);
13C NMR: (100 MHz, DMSO-d6) δ 168.31, 159.88, 158.37, 154.14, 137.27, 136.32,
130.74, 130.41, 130.35, 128.42, 128.37, 127.18, 126.10, 124.95, 121.58, 120.95 (2C),
66.92 (2C), 59.61, 44.18 (2C), 33.04, 31.80, 16.73; HRMS (ESI) m/z calcd
for C25H30N7O6P [M+H]+ 556.1995, found 556.2077.
14

4.4. In vitro kinase enzymatic assay
FAK enzyme system (Catalog. V9301, Promega) was used for in vitro enzymatic
assay. Cncentrations consisting of suitable levels from 1 to 200 nM were used for all of
the tested compunds. The test was performed in a 384-well plate. Detailed and
complete protocols can be founded in the ADP-GloTM kinase Assay Technical Manual
available
at:
https://cn.promega.com/resources/protocols/product-information-sheets/n/fak-kinase-
enzyme-system-protocol/. Briefly, procedures include: (1) perform a 5 μL kinase
reaction using 1× kinase buffer (e.g., 1× reactionbuffer A), (2) incubate at room
temperature for 60 minutes, (3) add 5 μL of ADP-Glo™ Reagent to stop the kinase
reaction and deplete theunconsumed ATP, leaving only ADP and a very low
background of ATP, (4) incubate at room temperature for 40 minutes, (5) add 10 μL of
Kinase Detection, (6) reagent to convert ADP to ATP andintroduce luciferase and
luciferin to detect ATP, (7) incubate at room temperature for 30 minutes, (8) plate was
measured on TriStar® LB942 Multimode Microplate Reader (BERTHOLD) to detect
the luminescence (Integration time 0.5-1 second). Curve fitting and data presentations
were performed using GraphPad Prism version 5.0.
4.5. Cellular activity assay
AsPC-1, Panc-1, BxPC-3, and HPDE6-C7 cells were purchased from Fuheng
Biology Company (Shanghai, China). Methylthiazolyldiphenyl-tetrazolium bromide
(MTT) reagent was purchased from Biotool Company (Switzerland). The Annexin
V-FITC Apoptosis Detection Kit and Cell Cycle Assay were purchased from
Beyotime Company (China). All cell lines were grown in DMEM (Gibco®, USA)
supplemented with 10% FBS (Gibco®, USA), 1% penicillin-streptomycin (Beyotime
Company, China). The cells were maintained and propagated as monolayer cultures at
37 °C in humidified 5% CO2 incubator.
Cells were seeded in a 96-well plate at a density of 4,000 to 5,000 cells/well and
were maintained at 37 C in a 5% CO2 incubator in DMEM containing 10% FBS for
one day. Cells were then exposed to the synthesiaed inhibitors for 72 h, and the
15

number of cells used per experiment for each cell lines was adjusted to obtain an
absorbance of 0.5 to 1.2 at 570 nm with a microplate reader (Thermo, USA).
Compounds were tested at appropriate concentrations (1.25 to 40 μmol/L), with each
concentration duplicated five times. TAE226 was employed as the positive control.
The IC50 values were calculated using GraphPad Prim version 5.0.
4.6. Cell apoptosis assay
AsPC-1 cells (3 to 5×105 cells/well) incubated in 6-well plates were treated with
solvent control (DMSO), or compound 7e (1, 1.2 or 5 μM) in medium containing 5%
FBS for 72 h. Then, collected and fixed with 70% ethanol at 4 °C overnight. After
being fixed with 75% ethanol at 4 °C for 24 h, the cells were stained with Annexin
V-FITC (5 μL)/propidium iodide (5 μL), and analyzed by flow cytometry
(Becton-Dickinson, USA).
4.7. Molecular docking study
The AutoDock 4.2 software with its default parameters was used to perform the
docking exploration. Detailed tutorials that guide users through basic AutoDock usage,
docking with flexible rings, and virtual screening with AutoDock can be founded at:
http://autodock.scripps.edu/faqs-help/tutorial/using-autodock-4-with-autodocktools.
Generally, the crystal structure (PDB: 2JKK) of the kinase domain of FAK enzyme
bound to inhibitor TAE226 was used in the docking studies. Enzyme preparation and
the hydrogen atoms adding was performed in the prepared process. The whole EGFR
enzyme was defined as a receptor and the site sphere was selected on the basis of the
binding location of TAE226. By moving TAE226 and the irrelevant water molecule,
the typical inhibitor 7e was docked into the pocket. The binding interaction energy
was calculated to include Van der Waals, electrostatic, and torsional energy terms
defined in the tripos force field. The structure optimization was performed using a
genetic algorithm, and only the best-scoring ligand protein complexes were kept for
analyses.
Acknowledgement
We are grateful to the National Natural Science Foundation of China (No.
16

81402788, 81672945), the project of family noninvasive mechanical ventilation
system construction and promotion for AECOPD patients: Health and Family
Planning Commission of Liaoning Province (No. 2015B004), and the Cultivation
Project of Youth Techstars, Dalian, China (2016RQ043) for the financial support of
this research.
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20

Graphical Abstract
Phosphamide-Containing Diphenylpyrimidine Analogues (PA-DPPYs) as
Potent Focal Adhesion Kinase (FAK) Inhibitors with Enhanced Activity
against Pancreatic Cancer Cell Lines
He Liua,1, Bin Wua,1, Yang Gea, Jiaxin Xua, Shijie Songa, Changyuan Wanga, Jihong Yaoa, Kexin
Liua, Yanxia Lib Xiuli Sunb and Xiaodong Maa,
a College of Pharmacy, Dalian Medical University, Dalian 116044, PR China.
b Hematology Department, Department of Respiratory Medicine, the First Affiliated Hospital of Dalian Medical
University, Dalian 116011, PR China.
1These authors contributed equally to this work.
 Corresponding author
E-mail address: xiaodong.ma@139.com (X. Ma).
G
l
u
5
0
6
/
G
R1
O
O
l
y
O
O
O
5
0
P
5
O
O
N
L
HN
O
P
O
HN
HN
N
NH
HN
N
NH
O
Asp564
N
N
R
F
Structure-based
Design
Spebrutinib
BTK Inhibitors
Pho-DPPYs
HN
O
R = Cl, NO2; R1 = EtO-, morpholine
L= -CH2- or -O(CH2)3-
Improved anti-BTK actvity
O
Phase Ⅲ
HN
N
NH
N
O2N
Cys502
N
PA-DPPY 7e
IC50 = 4.65 (FAK kinase)
IC50 = 1.66 (AsPC cells)
IC50 = 0.57 (BxPC cells)
IC50 > 20 (HPDE6-C7 cells)
H
N
O
O
HN
N
NH
N
Cl
TAE226
FAK inhibitior
21

Highlights
 PA-DPPYs could significantly inhibit FAK activity at concentrations ranged from
4.25 to 4.65 nM.
 PA-DPPYs displayed improved activity against pancreatic cancer cell lines
compared with TAE226..

The typical compound 7e triggered apoptosis in AsPC cells in a dose and
time-dependent manner.
22