Structure-based modification of carbonyl-diphenylpyrimidines (Car-DPPYs) as a novel focal adhesion kinase (FAK) inhibitor against various stubborn cancer cells

Article abstract of DOI:10.1016/j.ejmech.2019.04.004

A family of carbonyl-substituted diphenylpyrimidine derivatives (Car-DPPYs) with strong activity against focal adhesion kinase (FAK), were described in this manuscript. Among them, compounds 7a (IC₅₀ = 5.17 nM) and 7f (IC₅₀ = 2.58 nM) displayed equal anti-FAK enzymatic activity to the lead compound TAE226 (6.79 nM). In particular, compound 7a also exhibited strong antiproliferative activity against several stubborn cancer cells, including AsPC-1 cells (IC₅₀ = 0.105 μM), BxPC-3 cells (IC₅₀ = 0.090 μM), and MCF-7/ADR cells (IC₅₀ = 0.59 μM). Additionally, compound 7a also showed great antitumor efficacy in vivo via aAsPC-1 cancer Xenograft mouse model. The preliminary mechanism study by Western blot analysis revealed that 7a repressed FAK phosphorylation in AsPC cancer cells. Taken together, the results indicate that compound 7a may serve as a promising preclinical candidate for treatment of stubborn cancers.

Full text of DOI:10.1016/j.ejmech.2019.04.004

Accepted Manuscript
Structure-based modification of carbonyl-diphenylpyrimidines (Car-DPPYs) as a novel
focal adhesion kinase (FAK) inhibitor against various stubborn cancer cells
Luhong Wang, Min Ai, Jiawen Yu, Lingling Jin, Changyuan Wang, Zhihao Liu,
Xiaohong Shu, Zeyao Tang, Kexin Liu, Hui Luo, Wenshun Guan, Xiuli Sun, Xiaodong
Ma
PII:
S0223-5234(19)30303-4
DOI:
https://doi.org/10.1016/j.ejmech.2019.04.004
EJMECH 11239
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 October 2018
Revised Date: 1 April 2019
Accepted Date: 1 April 2019
Please cite this article as: L. Wang, M. Ai, J. Yu, L. Jin, C. Wang, Z. Liu, X. Shu, Z. Tang, K. Liu, H. Luo,
W. Guan, X. Sun, X. Ma, Structure-based modification of carbonyl-diphenylpyrimidines (Car-DPPYs) as
a novel focal adhesion kinase (FAK) inhibitor against various stubborn cancer cells, European Journal of
Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.04.004.
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ACCEPTED MANUSCRIPT
Graphical Abstract
5
0
5
y
l
G
/
8
3
4
O
n
l
G
N
Hydrogen-bond
acceptor
O
N
O
X
Asp564
R1
H
N
H
N
R
O
O
HN
Cl
N
NH
O
HN
Cl
N
NH
N
N
Cys502
TAE226
FAK inhibitor
Car-DPPYs 7a-i
Improved biological activity

ACCEPTED MANUSCRIPT
Structure-based Modification of Carbonyl-Diphenylpyrimidines
(Car-DPPYs) as a Novel Focal Adhesion Kinase (FAK) Inhibitor
against Various Stubborn Cancer Cells
Luhong Wanga,b,1, Min Aib,1, Jiawen Yua, Lingling Jinb, Changyuan Wangb, Zhihao Liub,c,
Xiaohong Shub, Zeyao Tangb, Kexin Liub, Hui Luod, Wenshun Guand , Xiuli Suna, , Xiaodong
Mab,
a Department of Hematology, the First Affiliated Hospital of Dalian Medical University, Dalian
116011, PR China.
b College of Pharmacy, College of Basic Medical Science, Dalian Medical University, Dalian
116044, PR China.
cState Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of
Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College,
Beijing 100050, PR China.
d Liaoning Ben-healthy Natural Technology and Dalian Buyun Biotechnology Co., Ltds., 116085,
PR China.
1These authors contributed equally to this work.
E-mail address: sunxl0411@163.com (X.L. Sun) and xiaodong.ma@139.com (X.D. Ma).
Abstract
A family of carbonyl-substituted diphenylpyrimidine derivatives (Car-DPPYs)
with strong activity against focal adhesion kinase (FAK), were described in this
manuscript. Among them, compounds 7a (IC50=5.17 nM) and 7f (IC50=2.58 nM)
displayed equal anti-FAK enzymatic activity to the lead compound TAE226 (6.79
nM). In particular, compound 7a also exhibited strong antiproliferative activity
against several stubborn cancer cells, including AsPC-1 cells (IC50=0.105 µM),
BxPC-3 cells (IC50= 0.090 µM), and MCF-7/ADR cells (IC50= 0.59 µM). Additionally,
compound 7a also showed great antitumor efficacy in vivo via aAsPC-1 cancer
Xenograft mouse model. The preliminary mechanism study by Western blot analysis
revealed that 7a repressed FAK phosphorylation in AsPC cancer cells. Taken together,
the results indicate that compound 7a may serve as a promising preclinical candidate
for treatment of stubborn cancers.
Keywords: Cancer; FAK; Inhibitor; Pyrimidine; Activity.
Introduction
Cancer remains a major public health problem worldwide. In 2018, 1,735,350
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new cancer cases and 609,640 cancer deaths are projected to occur in the United
States [1]. Small-molecule targeted therapy drugs, which are generally inhibitors of
enzymatic domains on mutated, overexpressed, or otherwise critical proteins within
the cancer cells, have had a significant impact on the treatment of various cancers
[2-5]. Focal adhesion kinase (FAK)，a cytoplasmic protein tyrosine kinase，
overexpressed and activated in several advanced-stage solid cancers [6,7]. FAK
promotes tumor progression and metastasis through effects on cancer cells, as well as
stromal cells of the tumor microenvironment. Targeted inhibition of the FAK pathway
is a promising therapeutic approach to broadly suppress various tumor-promoting
signaling pathways that converge on FAK activation [8-11]. FAK therefore represents
an important target for the development of anti-neoplastic and anti-metastatic drugs.
Small-molecule FAK inhibitors are designed to bind residues that surround the
ATP-binding pocket of kinases, including TAE226 (1) [12,13], PF562271(2) [14],
Defactinib (3) [15], CEP-37440(4) [16], and pyrimidines (5,6) [17,18] etc. (Figure 1).
Generally, these novel FAK inhibitors are comprised of a pyrimidine core, along with
a N-methylbenzamide functional group. In cell culture and animal models, they
effectively decrease FAK Y397 autophosphorylation and prevent cell migration, but
do not necessarily induce cell apoptosis in adherent culture conditions [19,20].
TAE226 is a well-characterized cellular-active and selective nanomolar affinity FAK
inhibitors, showing effective inhibitory activity against growth and invasion of glioma
and ovarian cancer cells. In addition, TAE226 can also significantly increase the
survival rate of animals with glioma Xenografts or ovarian tumor cell implants
[12,13]. Recently, our efforts on structural modification of pyrimidine scaffold also
led to the identification of several more potent FAK inhibitors than TAE226, such as
compounds 5 and 6 [17,18]. The co-crystal structure of FAK in complex with TAE226
indicated that the hydrophilic functional group at the C-2 aniline side chain is
favorable to strengthen the contacts with the amino acids Gln438 and Gly505 in the
binding pocket of the FAK protein [21,22]. Our previous studies on structural
modifications also revealed that H-bond, such as sulfonate and phosphate groups are
favorable to increase the binding affinity with FAK kinase [17,18]. Accordingly, with
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the aim of producing additional kinase inhibitors with strong hydrogen-bonding
affinity for FAK protein, a N-morpholine formamide functional group was introduced
into the aniline side chain at the C-2 position of the pyrimidine core in this study
(Figure 2). Herein, the design strategy, synthesis and biological activity of these
inhibitors in vivo and in vitro are all described in detail.
F
F
F
Cl
F
F
N
F
N
N
HN
N
NH
HN
N
NH O
HN
N
NH
N
N
O
NH
N
S
N
S
N
HN
N
O
HN
O
O
O
O
O
O
3
2
1
Defactinib
Phase II
PF-562271
TAE226
Phase I
Cl
Cl
NO2
NH
N
N
N
HN
N
O
NH
O
HN
N
O
NH O
HN
N
O
N
H
N
N
H
H
O
O
O
HN
S
N
P
O
N
N
O
F
HO
4
6
5
CEP-37440
Phase I
Figure 1. Chemical structures of the novel pyrimidines as FAK inhibitors.
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O
N
X
Hydrogen-bond
acceptor
O
O
Asp564
N
R1
H
N
H
R
N
O
O
HN
Cl
N
NH
O
HN
Cl
N
NH
N
N
Cys502
Car-DPPYs 7a-i
FAK inhibitor
TAE226
Figure 2. Design strategy of the title molecules Car-DPPYs.
2. Chemistry
Cl
NH
N
Cl
Cl
NH2
N
NO2
O
NO2 O
OH
Cl
N
N
O
N
d
Cl
CH3
a,b
c
O
CH3
N
H
HN
HN
CH3
9
8
10
11
Cl
NO2
NH2
HN
N
O
NH
O
NO2
N
H
c
11
a,b
O
O
e
O
N
N
N
O
OH
12
O
13
O
14
7a
Scheme 1. Synthetic route of the title compound 7a. Reagents and conditions:(a)SOCl2, 60 , 1h;
(b)H2NMeˑHCl or morpholine, NaHCO3, CH3CN, 80 , 4h, 85-95%; (c)Fe-NH4Cl, MeOH-H2O,
70 , 2h, 72%; (d)N,N-Diisopropylethylamine(DIPEA), isopropanol, 100 , 6h, 85%; (e)trifluo
-roacetic acid, 2-BuOH, 100 , 12 h, 38%.
4

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Cl
N
NO2
HN
N
NH
O
NO2
Br
R1
NO2
OH
N
R
H
O
b,c,d
EtO
e,f
11
O
O
R
N
O
O
R
N
a
O
O
R
OEt
O
O
7b-e
16a-c
17a-c
15
Scheme 2. Synthetic route of the title compounds 7b-e. Reagents and conditions:(a)K2CO3, KI,
CH3CN, 80 , 4h, 75-80%; (b)KOH, MeOH-H2O, 87-91%; (c)SOCl2, 60 , 1h; (d)morpholine,
K2CO3, KI, CH3CN, 80 , 4h, 72-84%; (e)Fe-NH4Cl, MeOH-H2O, 70 , 2h, 71-75%; (f)tri
-fluoroacetic acid, 2-BuOH, 100 , 12 h, 28-41%.
Cl
N
HN
N
NH
O
NO2
NO2
CH3
N
OEt
H
O
NO2
b,c,d
HS
e,f
S
S
S
a
O
N
O
OEt
O
N
F
O
O
18
7f
19
20
Scheme 3. Synthetic route of title compound 7f. Reagents and conditions: (a)K2CO3, KI, CH3CN,
80 , 4h, 72%; (b)KOH, MeOH-H2O, 83%; (c)SOCl2, 60 , 1h; (d)morpholine, K2CO3, KI,
CH3CN, 80 , 4h, 82%; (e)Fe-NH4Cl, MeOH-H2O, 70 , 2h, 78%; (f)trifluoroacetic acid,
2-BuOH, 100 , 12 h, 23%.
All the title molecules were generally synthesized using the procedures shown in
schemes 1 to 3 [17,18,23]. The key 2-chloropyrimidine intermediate 11 was prepared
according to our previously reported method via subsequent formylation, reduction,
and nucleophilic substitution reactions [17,18]. Commercially available 4-nitrobenzac
-etic acid (12) was reacted with morpholine in the presence of the activating reagent
ethyl(dimethylaminopropyl) carbodiimide (EDC) to prepare the N-morpholine
formamide derivative 13. After reducing the nitro group in compound 13 with
Fe-NH4Cl, the amino derivative 14 was synthesized. Finally, the title molecule 7a is
produced by substituting the chloride atom at the C-2 position of the pyrimidine
intermediate 11 with an amino group in compound 14. For the synthesis of the title
molecule 7b-e, 4-nitrophenol was reacted with α-bromoacetate to prepare the
5

ACCEPTED MANUSCRIPT
intermediates 16a-c in the first step. Then, 16a-c was reacted with morpholine to
produce compounds 17a-c. Also, compounds 17a-c were conveniently converted to
the title molecule 7b-e via reduction and nucleophilic substitution reactions. To
synthesize the title molecule 7f, 4-nitrofluoro was reacted with 2-mercapto acetate.
The ester group in compound 19 was then hydrolyzed to produce intermediate 20.
Compound 20 was reduced to amino-substituted 21, which was coupled with
intermediate 11 in the presence of trifluoroacetic acid (TFA) to prepare the title
molecule 7f.
Table 1. Inhibition of FAK enzymatic activity of the newly synthesized compounds
7a-f.
Cl
N
HN
R
N
NH O
R1
N
H
FAK Enzymatic
activity
Compound
R
R1
CLogPb
(IC50, nM)a
O
5.17 0.75
7a
7b
CH3
CH3
±
5.319
4.425
4.779
4.734
5.263
4.853
4.965
N
O
O
O
O
7.28 1.21
±
N
O
O
O
75.8 7.52
7c
±
N
N
O
O
Ο
38.0 5.39
7d
CH3
CH3
CH3
±
Ο
Ο
N
N
25.6 4.86
7e
±
O
O
S
2.58 0.83
7f
±
O
Tae226
6.79±0.58
a Concentration to inhibit by 50% FAK tyrosine kinase activity measured by ADP-GloTM kinase
6

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b
assay. Mean values of the three independent experiments ± SE are reported. CLogP value was
calculated by using ChemDraw Ultra software® (Cambridge Park Drive 2003 version).
All the title molecules were evaluated for their biological activity against FAK
kinase by using the ADP-GloTM assay (Promega, Madison, WI, USA) [25,26]. As
shown in Table 1, these newly synthesized compounds significantly inhibited FAK
enzymatic activity at concentrations ranging from 2.58 to 75.8 nM. Two typical
molecules 7a and 7f exhibited equal inhibitory potency to TAE226 (6.79 nM), with
IC50 values of 5.17 and 2.58 nM, respectively. The kinase-based test result revealed
that the bulky cyclopropyl group is less beneficial than the small methyl group at the
formamide part. For instance, the methyl-substituted compound 7b (7.28 nM) had
almost 10 times higher anti-FAK activity than compound 7c (75.8 nM), which
possesses a cyclopropyl substituent. Furthermore, introducing an additional methyl
(7d), or ethyl (7e) group into the C-2 aniline side chain is also unfavorable. The
exemplary compounds 7d (38.0 nM) and 7e (25.6 nM) have remarkably reduced
inhibitory potency compared with 7b. However, when replacing the oxygen group in
the aniline side chain at the C-2 position of the pyrimidine core with a sulfur atom (7f),
or removing the oxygen atom (7a), the anti-FAK activity of the desired compounds
slightly improved. For the title molecules, their ClogP values could produce
significant effect on the capability to penetrate cancer cell memberance, and thus
affecting their anti-proliferative activity of cancer cells. Generally, the ClogP values is
more high, the activity against cancer cell is more strong [27-29]. Accordingly, we
also explored their ClogP values by using ChemDraw Ultra software®, and found that
compound 7a, not only displayed strong inhibitory potency to FAK enzymatic activity,
but also has the highest ClogP value in this class of inhibitors. Overall, this work led
to the identification of two potent FAK inhibitors, namely 7a and 7f, which are
worthy of further biological evaluation.
Table 2. Antiproliferative activity of the title compounds 7a-f against cancer cells.a
Antiproliferation activity(IC50, µM)b
Compo
LO2
(normal
cells)
Pancreatic cancer cells
Drug-resistant cells
und
MCF-7/AD
1975
AsPC-1
BxPC-3
PANC-1
4.12±1.10
>10.0
HPDEC
R
0.105±0.0 0.090±0.0
39 22
1.75±0.45 5.99±1.13
6.62±1.33
1.17±0.46
0.59±0.085
3.70±0.86
7a
7b
0.160±0.0
25
0.594±0.08
5
4.79±1.02
2.93±0.96
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0.514±0.0
0.910±0.09
>10.0
>10.0
>10.0
16.7±2.01
1.04±0.33
2.17±0.38
1.99±0.42
11.4±1.35
>5.00
1.44±0.408
2.10±0.95
1.82±0.36
>5.00
7c
7d
91
4
0.495±0.1
0.821±0.07
2.87±0.88 5.34±0.99
2.60±0.75 8.73±0.98
1.22±0.59 2.89±0.67
1.54±0.46
0.75±0.11
5.51±0.54
>10.0
08
8
0.444±0.0
0.674±0.09
7e
99
9
0.413±0.0
4.33±0.607
7f
82
6.73±0.66
>10.0
>2.00
5.37±0.908
Tae226
a
Concentration that inhibits by 50% the proliferation of cancer cells. The cell proliferation was
determined by the MTT assay after 72 h of incubation with compounds (0.625 – 10 µM). Mean
values of three independent experiments ± SE are reported.
To investigate the effects of these newly synthesized molecules on the
proliferation of cancer cells in vitro, several stubborn cancer cell lines were used for
biological evaluation, including pancreatic cancer (PC) cell lines (AsPC-1, BxPC-3,
PANC-1 cells), and mutant H1975 cells (a gefitinib-resistant non-small cell cancer
lung (NSCLC) cell line) and MCF-7/ADR cells (Adriamycin-Resistant human breast
carcinoma cells). In addition, two human normal human cell lines LO2 (normal
human hepatocyte cells) and HPDEC (pancreatic ductal epithelial cells) were also
used to test their cytotoxicity. The novel FAK inhibitor TAE226 was also tested as a
positive control. As shown in Table 2, most of these compounds displayed strong
inhibitory potency against these stubborn cancer cells at drug concentrations lower
than 10 µM. Compared with TAE226, this class of compounds have significantly
enhanced anticancer activity. In particular, compound 7a, which has an IC50 value of
0.105 µM against AsPC-1, and 0.090 µM against BxPC-3 cells, was the strongest
inhibitor against PC cell lines. In contrast, compound 7c bearing a cyclopropyl
substituent was ineffective in blocking the proliferation of PC cells, with IC50 values
of more than 10 µM. This finding suggests that the bulky group at the C-2 aniline side
chain (compound 7c) is less favorable than the small methyl substituent. Possibly, the
cyclopropyl group produced steric hindrance in the FAK binding pocket, thus
reducing the binding affinity. Generally, their in vitro activity against PC cells in
accordance with the inhibitory potency to FAK kinase. In spite of its similar inhibitory
potency to TAE226 against FAK kinase, compound 7a exhibited improved anti-PC
cells activity possibly because of its higher ClogP (5.319) value than TAE226 (4.965)
(Table 1). Or possibly for the reason that most of the kinases have many subtypes
(Type 1, Type 2, Type 3, etc.), such as PYK2 and FAK, which have 73% similarity in
8

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the catalytic domains [30,31], compound 7a may interfer with other subtypes or other
tumor signal transduction pathways, while TAE226 has no. These further studies on
the mechanism of action of kinase will be performed in our future work.
Remarkably, most of these molecules are also quite effective in interfering with
the proliferation of the drug-resistant cancer cell lines, with IC50 values ranging from
0.59 to 2.93 µM against MCF-7/ADR cells, and from 0.160 to 1.17 µM against
NSCLC-resistant H1975 cells. Among these molecules, compound 7a possess the
strongest ability to inhibit MCF-7/ADR cells (IC50= 0.59 µM), while compound 7b,
exhibited the highest inhibitory potency against H1975 cells (IC50= 0.190 µM). These
newly obtained FAK inhibitors provide a new insight to overcome the stubborn
drug-resistance cancer cells.
In addition, the potent inhibitor 7a also has very low cytotoxicity toward the
normal HPDEC cells (IC50= 6.62 µM) and LO2 cells (IC50= 3.70 µM). The data
shown in Figure 3 reveals that compound 7a markedly inhibited the proliferations of
AsPC-1 cells with increasing drug concentration from 125 to 1,000 nM, and treatment
time from 24 to 72 h. The results presented in Figure 4 revealed that 7a could induce
striking nuclear condensation, membrane blebbing, nuclear fragmentation, and
apoptotic bodies, all of which are characteristics of apoptotic programmed cell death.
Thus, taken together, these findings indicate that compound 7a may serve as a new
promising agent for the treatment of cancers that are hard to treat.
150
150
100
50
100
50
0
0
control
125
500
1000
control
125
500
1000
Drug concentration (nM)
TAE226
Drug concentration (nM)
7a
Figure 3. The effects of treating time and concentrations of 7a on AsPC-1 cell viability.
9

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7a
Control
300
150
600(nM)
600(nM)
0
Tae226
150
300
Figure 4. Morphological changes of AsPC-1 cells (100 ×, final magnification, green images:
AO/EB (acridine orange (AO)/ethidium bromide (EB) double fluorescent staining of AsPC-1 cells
treated with different concentrations of 7a and TAE226 for 72h, blue images).
7a
TAE226
Control
10 50 100 100 (nM)
FAK
p-FAK
MEK1
p-MEK1
ERK1/2
p=ERK1/2
β-actin
Figure 5. Compound 7a inhibited the activation of FAK and downstream signaling in AsPC-1
cells.
To investigate the effects of compound 7a on FAK activation and downstream
signaling in AsPC-1 cells, the expression and activation of proteins involved in FAK
mediated MEK pathways were assessed via immunoblotting analysis (Figure 5)
[32,33]. The analysis demonstrated that treatment with 7a significantly repressed
FAK phosphorylation in a dose-dependent manner. For compound 7a, complete
inhibition was achieved at a concentration of 50 nM for 72 h, while for TAE226, it
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was achieved at a concentration of 100 nM, which showed the superior anticancer
potency of compound 7a. However, compound 7a produced little effect on the
expression of MEK and ERK proteins, suggesting that compound 7a may interfere
with other downstream signaling proteins of the FAK pathway. The extensive
mechanism of action of the signaling pathway will be explored in our subsequent
work.
C)
B)
A)
2.0
1.5
1.0
0.5
0
2000
control
30mg/kg
60mg/kg
1500
control
1000
500
7a
(30mg/kg)
7a
(60mg/kg)
0
30mg/kg
60mg/kg
control
0
2
4
6
8
Figure 6. In vivo antitumor effect of compound 7a in a human AsPC-1 Xenograt nude mouse
model. (A) Images of the tumor collected from the mice; (B) Tumor weight; (C) Tumor volume;
Data are presented as mean ± SD (n = 3).
2.4 In vivo antitumor activity
To analyze whether treatment with 7a can modulate tumor growth in vivo, we
treated mice bearing human AsPC-1 colon cancer Xenografts with 7a or vehicle as
controls (Figure 6) [34]. The assay results shown in Figure 6 clearly demonstrated
that tumor growth could be significantly inhibited by repeated oral administration of
7a (qd) for 7 days, with tumor growth inhibition (TGI) values of 67 and 87% at
dosages of 60 and 30 mg/kg/day, respectively.
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B)
A)
Gly550
Gly550
Gln438
Gln438
Gly505
Asp564
Asp564
Gly505
Cys502
Cys502
Gln438
D)
C)
E)
Gly550
Gly550
Gly550
Gln438
Asp564
Asp564
Asp564
Gln438
Gly505
Cys502
Gly505
Gly505
Cys502
Cys502
Figure 7. Putative binding models of Car-DPPYs in FAK enzyme (PDB code: 2JKK) [21], A:
TAE226; B: inhibitor 7a, C: inhibitor 7c; D: inhibitor 7d; E: inhibitor 7f.
Moreover, several representative Car-DPPYs were also separately docked into the
into the ATP binding pocket of the FAK enzyme (PDB: 2JKK) to establish their
putative interaction mechanism with FAK kinase (Figure 7) [21]. The crystal
structure of TAE226 in complex with FAK was also examined and is displayed in
Figure 7A for comparison. The docking performance was evaluated with the program
AutoDock 4.2 using its default parameters [35, 36]. The results clearly revealed that
both the two potent inhibitors 7a (Energy score: -16.5776 KJ·mol-1) (Figure 7B) and
7f (Energy score: -16.9000 KJ·mol-1) (Figure 7E) could tightly bind to the FAK
kinase in similar manner as TAE226 (Energy score: -15.2683 KJ·mol-1). All of them
formed a strong hydrogen bond between the oxygen atom in the amide group and
amino acid Asp564. Beyond that, our synthesized compounds 7a and 7f formed a new
strong hydrogen bond through the introduced carbonyl group with amino acid Gln438.
Moreover, the most potent inhibitor 7f also produced an additional hydrogen bond
between the N-1 atom of pyrimidine core with the amide acid Cys502 in FAK protein.
However, for the less potent molecules 7c (Energy score: -14.2728 KJ·mol-1) (Figure
7C) and 7d (Energy score: -14.9905 KJ·mol-1) (Figure 7D), this strong hydrogen
12

ACCEPTED MANUSCRIPT
bond disappeared due to the steric hindrance caused by cyclopropyl or methyl group.
It is possible that these reduced binding contacts eventually result in the loss of
anti-FAK enzymatic activity. In brief, these binding models are in accordance with
their biological activity data.
Conclusion
A new class of Car-pyrimidines were synthesized and biologically evaluated as
potent FAK inhibitors. Two of them displayed equal anti-FAK enzymatic activity
against FAK to the lead compound TAE226, with IC50 values of 5.17 nM (7a), and
2.58 nM (7f). In particular, compound 7a could also significantly block the
proliferation of AsPC-1 (IC50= 0.105 nM), BxPC-3 (IC50= 0.090 nM), and
MCF-7/ADR cells (IC50= 0.59 nM). The Western blot analysis demonstrated that
treatment with 7a significantly repressed FAK phosphorylation at a very low
concentration in AsPC cells. In vivo antitumor efficacy using AsPC-1 cancer
Xenograft mouse model showed that tumor growth was greatly inhibited by repeated
oral administration of 7a. All these evaluations suggested that inhibitor 7a may serve
as a promising preclinical candidate for treatment of stubborn cancer disease.
4. Experimential 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 on a Brucker AV 400
MHz spectrometer were recorded in [d]DMSO. Coupling constants (J) are expressed
in hertz (Hz). Chemical shifts (δ) of NMR are reported in parts per million (ppm)
units relative to internal control (TMS). 1H 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), q (quartet), m (multiplet), and br s (broad signal). High resolution ESI-MS
were 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
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were obtained on Silica Gel (300–400 mesh) using dichloromethane/methanol as
eluents.
4.2.1. Preparation of the 2-chlorine pyrimidine 11.
2-Amino-N-methylbenzamide 10 was prepared according to the general
procedure in literatures 17 and 18. The prepared 2-amino-N-methylbenzamide 10
(10.00 g, 66.67 mmol) was added in one portion to 2,4,5-trichloropyrimidine (12.23 g,
66.67 mmol) and N,N-diisopropylethylamine (DIPEA) (12.93 g, 100 mmol) in
isopropanol (100 mL). The resulting mixture was stirred at 100 °C for 6 hours. The
mixture was evaporated to dryness and the residue was recrystallised from
MeCN/water 20:1 to yield 2-(2,5-dichloropyrimidin-4-yl amino)-N-methylbenzamide
16.87 g, 85 %.
4.2.2. Preparation of the title molecules7a-f.
Various morpholine-substituted anilines 14 and 17a-c were prepared according to
the literatures 23 and 24. The synthesized aniliens 7a or 7b-e (1 mmol), 7f (1 mmol),
TFA (1.5 mmol), and 2-BuOH (20 mL) was mixed in a round-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 (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 5% methanol/dichloromethane) to give the title product.
4.2.2.1 (7a)2-[[5-chloro-2-[3-[(4-(1-ethyl-4-phenylamino))-2-formyl]morpholine]
-pyrimidin-4-yl]amino]-N-methylbenzamide
Yield 38.0%; gray white solid; 1H NMR (400 MHz, DMSO-d6): δ 11.59 (s, 1H),
9.43 (s, 1H), 8.75 (s, 2H), 8.21 (s, 1H), 7.75 (dd, J = 8.0, 1.4 Hz, 1H), 7.59 (d, J = 8.0
Hz, 2H), 7.52–7.40 (m, 1H), 7.21–7.05 (m, 3H), 3.66 (s, 2H), 3.53 (t, J = 4.6 Hz,
13
2H), 3.50–3.43 (m, 6H), 2.81 (s, 3H); C NMR (400 MHz, DMSO-d6): δ 169.78,
169.38, 158.23, 155.46, 155.10, 139.77, 139.02, 131.88, 129.38, 129.35(3C), 128.46,
122.41, 121.94, 121.26, 120.21(2C), 105.47, 66.55(2C), 46.44, 42.14, 26.79; HRMS
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(ESI) for C24H25ClN6O3, [M+H]+ calcd: 481.1749; found: 481.1719.
4.2.2.2 (7b)2-[[5-chloro-2-[4-[2-(4-(1-oxygen-4-phenylamino))hydrogen-3-formyl]
-morpholine]pyrimidin-4-yl]amino]-N-methylbenzamide
Yield 32.5%; gray white solid; 1H NMR (400 MHz, DMSO-d6): δ 11.61 (s, 1H),
9.30 (s, 1H), 8.75-8.77 (t, J = 4.0 Hz, 2H), 8.17 (s, 1H), 7.73-7.75 (d, J = 8.0 Hz, 1H),
7.49-7.52 (d, J = 12.0 Hz, 2H), 7.45-7.47 (d, J = 8.0 Hz, 1H), 7.10-7.14 (t, J = 8.0 Hz,
1H), 6.86-6.88 (d, J = 8.0 Hz, 2H), 4.79 (s, 2H), 3.56-3.61 (dd, J = 4.0, 16.0 Hz, 4H),
3.47 (br, 4H), 2.80-2.81 (d, J = 4.0 Hz, 3H); 13C NMR (400 MHz, DMSO-d6): δ
169.45, 166.75 (2C), 158.51, 155.45, 155.18, 153.79, 139.91, 134.09, 132.05, 128.48,
122.33, 122.17, 121.83, 121.03, 114.95 (3C), 105.03, 66.67, 66.61, 45.34, 42.13,
26.84; ESI-MS m/z 571.1 [M–H]–.
4.2.2.3 (7c) 2-[[5-chloro-2-[4-[2-(4-(1-oxygen-4-phenylamino))hydrogen-3-formyl]
morpholine]pyrimidin-4-yl]amino]-N-cyclopropylbenzamide
Yield 28.7%; gray white solid; 1H NMR (400 MHz, DMSO-d6): δ 11.48 (s, 1H),
9.30 (s, 1H), 8.74-8.75 (d, J = 4.0 Hz, 1H), 8.70 (br, 1H), 8.18 (s, 1H), 7.69-7.72 (dd,
J = 4.0, 8.0 Hz, 1H), 7.49-7.52 (d, J = 12.0 Hz, 2H), 7.45-7.47 (d, J = 8.0 Hz, 1H),
7.09-7.12 (t, J = 4.0, 8.0 Hz, 1H), 6.85-6.88 (d, J = 12.0 Hz, 2H), 4.79 (s, 2H),
3.56-3.61 (dd, J = 4.0, 16.0 Hz, 4H), 3.47 (br, 4H), 2.85-2.92 (m, 1H), 0.70-0.75 (m,
2H), 0.59-0.62 (m, 2H); 13C NMR (400 MHz, DMSO-d6): δ 166.4, 165.4, 160.5,
155.7, 155.0, 153.2, 149.0, 147.9, 145.4, 144.6, 137.0, 128.6, 116.0, 110.2, 108.7,
107.4, 106.7, 102.4, 66.6, 64.0, 56.6, 54.4, 51.9, 36.6, 29.4, 23.7; ESI-MS m/z 571.1
[M–H]–.
4.2.2.4 (7d)2-[[5-chloro-2-[4-[2-(4-(1-oxygen-4-phenylamino))methyl-3-formyl]
-morpholine]pyrimidin-4-yl]amino]-N-methylbenzamide
Yield 36.4%; gray white solid; 1H NMR (400 MHz, DMSO-d6): δ 11.57 (s, 1H),
9.29 (s, 1H), 8.75 (s, 2H), 8.17 (s, 1H), 7.75 (dd, J = 8.0, 1.4 Hz, 1H), 7.51 (d, J = 9.0
Hz, 3H), 7.13 (t, J = 7.6 Hz, 1H), 6.80 (d, J = 9.0 Hz, 2H), 5.19 (q, J = 6.4 Hz, 1H),
3.66–3.40 (m, 8H), 2.81 (s, 3H), 1.42 (d, J = 6.4 Hz, 3H); 13C NMR (400 MHz,
DMSO-d6): δ 169.41, 169.38, 158.43, 155.43, 155.13, 152.77, 139.83, 134.12, 131.89,
128.44, 122.29, 122.13, 121.87, 121.14, 115.20(3C), 105.00, 71.30, 66.67, 45.73,
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42.43, 26.78, 18.03(2C); HRMS (ESI) for C25H27ClN6O4, [M+H]+ calcd: 511.1855;
found: 511.1819.
4.2.2.5 (7e)2-[[5-chloro-2-[4-[2-(4-(1-oxygen-4-phenylamino))ethyl-3-formyl]
-morpholine]pyrimidin-4-yl]amino]-N-methylbenzamide
Yield 41.0%; gray white solid; 1H NMR (400 MHz, DMSO-d6): δ 11.57 (s, 1H),
9.29 (s, 1H), 8.75 (s, 2H), 8.17 (s, 1H), 7.75 (d, J = 7.8 Hz, 1H), 7.55–7.37 (m, 3H),
7.12 (d, J = 7.6 Hz, 1H), 6.79 (d, J =9.0 Hz, 2H), 4.95 (d, J = 6.4 Hz, 1H), 3.71–3.41
13
(m, 8H), 2.81 (s, 3H), 1.81 (dd, J = 10.4, 7.0 Hz, 2H), 1.00 (t, J = 7.4 Hz, 3H); C
NMR (400 MHz, DMSO-d6): δ 169.38, 168.88, 158.44, 155.43, 155.15, 153.19,
139.83, 134.13, 131.88, 128.45, 122.28, 122.10, 121.88, 121.16, 115.23(3C), 104.99,
76.79, 66.75, 45.74, 42.45, 26.78, 25.47, 10.28(2C); HRMS (ESI) for C26H29ClN6O4,
[M+H]+ calcd: 525.2012; found: 525.1977.
4.2.2.6 (7f)2-[[5-chloro-2-[4-[2-(4-(1-sulphur-4-phenylamino))hydrogen-3-formyl]
morpholine]pyrimidin-4-yl]amino]-N-methylbenzamide
Yield 39.3%; gray white solid; 1H NMR (400 MHz, DMSO-d6): δ 11.61 (s, 1H),
9.55 (s, 1H), 8.76 (d, J = 4.0 Hz, 2H), 8.24 (s, 1H), 7.76 (dd, J = 8.0, 2.0 Hz, 1H), 7.65
(d, J = 8.0 Hz, 2H), 7.54 (t, J = 8.0 Hz, 1H), 7.34(d, J = 8.0 Hz, 2H), 7.16 (t, J = 8.0
Hz, 1H), 3.86 (s, 2H), 3.60–3.50 (m, 4H), 3.49–3.39 (m, 4H), 2.81 (s, 3H); 13C
NMR (400 MHz, DMSO-d6): δ 169.37, 167.18, 157.96, 155.50, 155.05, 139.90,
139.68, 131.99, 131.60(2C), 128.48, 126.83, 122.55, 121.95, 121.35, 120.37(2C),
105.88, 66.54(2C), 46.63, 42.27, 37.02, 26.79; HRMS (ESI) for C24H25ClN6O3S,
[M+H]+ calcd: 513.1470; found: 513.1440.
4.3. In vitro kinase enzymatic assay
FAK kinase enzyme system (Catalog. V1971, Promega) was used for in vitro
kinase enzymatic assay. Concentrations 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://www.promega.com.cn/Products/Cell-Signaling
/Kinase-Assays-and-Kinase-Biology/FAK-Kinase-Enzyme-System/?fq=FAK&catNu
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m=V1971. Brefiely, procedures inclued: (1) perform a 5 µL kinase reaction using 1×
kinase buffer (e.g., 1× reaction buffer A), (2) incubate at room temperature for 60
minutes, (3) add 5 µL of ADP-Glo™ Reagent to stop the kinase reaction and deplete
the unconsumed 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 and introduce 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.
4.4 . Cellular activity assay
4.4.1. Cell culture and reagents
AsPC-1, BxPC-3, PANC-1, HPDEC, H1975, MCF7/ADR and LO2 cells were
purchased from Fuheng Biology Company (Shanghai, China). 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).
AsPC-1, BxPC-3, PANC-1, HPDEC, LO2 Cells were grown in RPMI-DMEM
(Gibco®, USA) and H1975, Mcf-7/ADR cells were grown in RPMI-1640 (Gibco®,
USA), all of them were 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.
4.4.2. Cellular activity assay
Cells were seeded in 96-well plates at a density of 3,000 to 5,000 cells/well and
were maintained at 37 °C in a 5% CO2 incubator in RPMI1640 or DMEM containing
10% FBS for one day. Cells were then exposed to the compouds for 72 h, and the
number of cells used per experiment for each cell lines was adjusted to obtain an
absorbance of 0.5 to 1.2 at 450 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
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IC50 values were calculated using GraphPad Prim version 5.0.
4.5.AO/EB staining assay
Approximately 2×105 cells/well of AsPC-1 cells in 6-well plate were incubated
in an incubator for 12 h, then treated with different concentrations of inhibitors for 72
h. After incubation, the cells were washed with PBS for two times. Then, total of 20
µL of the solution containing the AO/EB acridine orange (AO)/ethidium bromide (EB)
dye mix (1.0 µg/mL of AO and 1.0 µg/mL of EB in PBS) was added to the cells. The
apoptotic, necrotic and live cells were observed and counted under the fluorescent
inverted microscope (OLYMPUS, Japan).
4.6. Western blotting assay
The lysates from Namalwa cells in different groups were extracted and
centrifuged at 14,000 g for 15 min at 4 ℃, then the total proteins were obtained. An
aliquot (50 µg protein) was loaded onto a 8%-12% SDS-PAGE gels and separated
electrophoretically. Then the target proteins were transferred to a PVDF membrane
(Millipore, USA). After blocking the PVDF membrane in 5% dried skim milk (Boster
Biological Technology, China) for 2 h at room temperature, the membrane was
incubated overnight at 4 ℃ with primary antibodies for 12 h. Protein detection was
performed based on an enhanced chemiluminescence (ECL) method and
photographed by using a BioSpectrum Gel Imaging System (HR410, UVP,USA). In
order to eliminate the variations, data were adjusted to β-Actin expression: IOD of
objective protein versus IOD of β-Actin expression.
4.7. Mouse tumor Xenograft efficacy study
The female BALB/cJNju-Foxn1nu/Nju mice weighing 20-25 g in 5-6 week-old
were provided by the Model Animal Research Center of Nanjing University (Nanjing,
China) (SCXK: 2016-0001). All animals were housed in a controlled environment at
23 ± 2 ℃ under a 12-h dark/light cycle with free access to food and water. The
animal maintenance and experiments were performed in accordance with the
guidelines of the Animal Care and Use Committee. AsPC-1 cells (3 × 106) were
suspended in 0.1 mL of PBS and injected subcutaneously into the right oxter region of
the mice. Three days after implantation, the mice were randomly divided into three
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groups in which the mice in control group were received by oral 25% PEG-400, and
the mice in other two groups were oral administrated with 7a at the doses of 30 and 60
mg/kg for 7 days. In the process, the side length of the tumor was measured to
calculate the tumor volume based on the formula: V = A × B2/2, where A means the
lager perpendicular diameters and B is the smaller perpendicular diameters. At the end
of the test, the animals were sacrificed and the tumors were obtained, photographed
and weighted.
4.8. Molecular docking study
The AutoDock 4.2 software 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 may be found at: http://autodock.scripps.edu/
faqs-help/tutorial. Generally, the crystal structure (PDB: 2JKK) of the kinase domain
of FAK bound to inhibitor 7a was used in the docking studies.The enzyme preparation
and the hydrogen atoms adding was performed in the prepared process. The whole
FAK 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 7a. 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.
Acknowledgments
We are grateful to the National Natural Science Foundation of Liaoning province
(20180530066), the National Natural Science Foundation of China (No. 81603186,
81672945), the Research Fund of Higher Education of Liaoning province （
LQ2017039, LQ2017008）for the financial support of this research.
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Highlights
‹ Car-DPPYs were identified as potent FAK inhibitors.
‹ Compounds 7a and 7f were equal to TAE226 for inhibiting FAK enzymatic
activity.
‹
Compound 7a not only exhibited strong antiproliferative activity against
AsPC-1 cells, BxPC-3 cells and MCF-7/ADR cells, but also showed great
antitumor efficacy in vivo via AsPC-1 Xenograft mouse model.
‹ Western blot analysis revealed that 7a repressed FAK phosphorylation in
cancer cells.