Design, synthesis and biological evaluation of sulfonamide-substituted diphenylpyrimidine derivatives (Sul-DPPYs) as potent focal adhesion kinase (FAK) inhibitors with antitumor activity

Article abstract of DOI:10.1016/j.bmc.2017.05.044

A class of sulfonamide-substituted diphenylpyrimidines (Sul-DPPYs) were synthesized to improve activity against the focal adhesion kinase (FAK). Most of these new Sul-DPPYs displayed moderate activity against the FAK enzyme with IC₅₀ values of less than 100?nM; regardless, they could effectively inhibit several classes of refractory cancer cell lines with IC₅₀ values of less than 10?μM, including the pancreatic cancer cell lines (AsPC-1, Panc-1 and BxPC-3), the NSCLC-resistant H1975 cell line, and the B lymphocyte cell line (Ramos cells). Results of flow cytometry indicated that inhibitor 7e promoted apoptosis of pancreatic cancer cells in a dose-dependent manner. In addition, it almost completely induced the apoptosis at a concentration of 10?μM. Compound 7e may be selected as a potent FAK inhibitor for the treatment of pancreatic cancer.

Full text of DOI:10.1016/j.bmc.2017.05.044

Accepted Manuscript
Design, synthesis and biological evaluation of sulfonamide-substituted diphe-
nylpyrimidine derivatives (Sul-DPPYs) as potent focal adhesion kinase (FAK)
inhibitors with antitumor activity
Menghua Qu, Zhihao Liu, Dan Zhao, Changyuan Wang, Jianbin Zhang, Zeyao
Tang, Kexin Liu, Xiaohong Shu, Hong Yuan, Xiaodong Ma
PII:
S0968-0896(17)30434-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.05.044
BMC 13762
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
2 March 2017
17 May 2017
20 May 2017
Please cite this article as: Qu, M., Liu, Z., Zhao, D., Wang, C., Zhang, J., Tang, Z., Liu, K., Shu, X., Yuan, H., Ma,
X., Design, synthesis and biological evaluation of sulfonamide-substituted diphenylpyrimidine derivatives (Sul-
DPPYs) as potent focal adhesion kinase (FAK) inhibitors with antitumor activity, Bioorganic & Medicinal
Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.05.044
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Design, synthesis and biological evaluation of sulfonamide-
substituted diphenylpyrimidine derivatives (Sul-DPPYs) as potent
focal adhesion kinase (FAK) inhibitors with antitumor activity
Menghua Qua,1, Zhihao Liua,1, Dan Zhaoa, Changyuan Wanga, Jianbin Zhanga, Zeyao Tanga,
Kexin Liua, Xiaohong Shua, ∗, Hong Yuanb, ∗, and Xiaodong Maa, ∗
a College of Pharmacy, Dalian Medical University, Dalian 116044, PR China.
bDepartment of Laboratory, 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)
Abstract
A class of sulfonamide-substituted diphenylpyrimidines (Sul-DPPYs) were
synthesized to improve activity against the focal adhesion kinase (FAK). Most of
these new Sul-DPPYs displayed moderate activity against the FAK enzyme with IC50
values of less than 100 nM; regardless, they could effectively inhibit several classes of
refractory cancer cell lines with IC50 values of less than 10 µM, including the
pancreatic cancer cell lines (AsPC-1, Panc-1 and BxPC-3), the NSCLC-resistant
H1975 cell line, and the B lymphocyte cell line (Ramos cells). Results of flow
cytometry indicated that inhibitor 7e promoted apoptosis of pancreatic cancer cells in
a dose-dependent manner. In addition, it almost completely induced the apoptosis at a
concentration of 10 µM. Compound 7e may be selected as a potent FAK inhibitor for
the treatment of pancreatic cancer.
Key words: Cancer; FAK; inhibitor; pyrimidine; sulfonamide.

1. Introduction
Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that localizes at
sites of cell adhesion to the extracellular matrix (ECM) and mediates signaling events
downstream of the integrin engagement of the ECM.1,2 FAK played considerably
important roles in regulating cancer cell survival, proliferation, and migration. It has
also been identified as a key pathogenic mediator of various cancers. Inhibition of
FAK activity has been demonstrated to affect a number of cellular processes essential
for tumor growth and disease progression including angiogenesis and metastasis.
Thus, FAK enzyme has emerged as a promising cancer therapeutic target.3-5 Up to
present time, a couple of FAK inhibitors have been described to undergo extensively
preclinical or clinical trials for cancer therapy (Fig. 1), including TAE226 (1),6 PF-
573228 (2)7-9, defactinib (3, phase II)10, PF-562271 (4, phase I)11,12, CEP-37440 (5,
phase I)13, and VS-4718 (3, phase I)14,. Generally, most potent FAK inhibitors possess
a pyrimidine core, along with a C-4 N-methylformamide-substituted aniline side chain,
an important functionality to interact with amino acids Cys502, Asp546, and Leu553
in the FAK protein to tightly contact with FAK.15

F
F
F
F
F
F
Cl
N
N
N
HN
N
NH
HN
NH
N
O
HN
N
NH
N
N
O
NH
N
S
HN
O
N
O
S
HN
O
O
O
O
O
1
TAE226
3
Defactinib
Phase II
2
PF-573228
Cl
NH
N
F
F
F
F
HN
N
O
N
F
N
F
OMe
NH
HN
N
NH
HN
NH O
O
NH
N
S
N
HN
N
N
O
O
O
O
N
5
6
4
CEP-37440
Phase I
VS-4718
Phase I
HO
PF-562271
Phase I
Figure 1. Chemical structures of the novel FAK inhibitors.
The sulfonamide functional group is present in many active compounds,
exhibiting antibacterial, anti-carbonic anhydrase, diuretic, hypoglycemic, and
antithyroid properties, among other.16-18 Our previous study revealed that introducing
a functional sulfonamide into the C-2 aniline moiety of pyrimidine template could
improve the activity against the Bruton’s tyrosine kinase (BTK), which is an effective
drug target for treatment of B-cell lymphoblastic leukemia.19 According to the
structure-based drug design strategy, a family of C-2 sulfonamide-substituted
diphenylpyrimidine derivatives (Sul-DPPYs) featuring a TAE226 molecule template
were synthesized to improve inhibitory potency toward FAK kinase activity and
cancer cells in current study (Fig. 2). The synthesis and anticancer evaluation of these
newly designed compounds were also reported in this manuscript.

F
O
O
O
S O
O
O
NH
HN
HN
O
R1
O
S
O
HN
N
NH
HN
N
NH
HN
Asp546
N
N
F
Cl
Structure-based
Design
R2
H
N
Spebrutinib
BTK Inhibitors
Phase
SFA-DPPYs 10c
Improved actvity agaisnt BTK
O
HN
N
NH
O
N
R1
N
Cys502
Sul-DPPYs 7a-i
FAK inhibitor
H
N
O
O
HN
N
NH
N
Cl
TAE226
FAK inhibitior
Figure 2. Designed strategy of the title molecules Sul-DPPYs.
2. Results and discussion
2.1. Chemistry
Cl
N
H
N
H
N
Cl
Cl
O
O
OH
O
N
Cl
N
NH
O
Cl
N
a
b
c
O2N
O2N
H2N
N
H
d
11
10
8
9
R2
R2
Cl
R2
N
O
O
H2N
NO2
S
S
HN
N
NH O
O
O
R2
f
Compound 11
HN
HN
e
g
N
R1
R1
H
R2
O
S
R1
Cl
O
NH
S
R1 = H, Me, MeO
R2 = F, Me, MeO
7a-g
NH2
14a-c
NO2
13a-c
O
O
12a-c
Scheme 1. Synthetic route of the title compounds 7a-g. 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, 88%; (d) N,N-diisopropylethylamine (DIPEA), isopropanol, 6 h, 85%; (e) NaHCO3, CH3CN,
r.t, 0.5 h, 90-95%; (f) Fe-NH4Cl, MeOH-H2O, 2 h, 70 °C, 84-88%; (g) Compound 11
trifluoroacetic acid, 2-BuOH, 100 °C, 12 h, 19-33%

Cl
N
H2N
O2N
OH
HN
N
NH
O
O
O
N
H
OH
c
d
a
b
O
Compound 11
O
O
O
O
O
HN
HN
HN
NH2
S O
R
S
S O
R
R
S O
R
N
H
R = Me, CF3
7h-i
16a-b
15
17a-b
18a-b
Scheme. 2. Synthetic route of the title compounds 7h-i. Reagents and conditions: (a) CH3SO2Cl
or CF3SO2Cl, NaHCO3, CH3CN , 4 h, 70 °C, 85-90%; (b) 4-fluoronitrobenzene, KI, K2CO3, DMF,
80 °C, 80-85%; (c) Fe-NH4Cl, MeOH-H2O, 2 h, 70 °C, 70-88%; (d) Compound 11, p-
toluenesulfonic acid, 2-BuOH, 100 °C, 12 h, 15-22%.
The designed molecules 7a-i were synthesized according to the general procedures
depicted in Scheme 1 and Scheme 2.19-22 2-Nitrobenzoic acid (8) was reacted with
thionyl chloride, and then directly reacted with methylamine in the present of
NaHCO3 as base to produce 2-nitro-(N-methyl)benzamide (9). Compound 9 was
then reduced to 2-amino-substituded N-methylbenzamide (10) by using the Fe-NH4Cl
reduction reagent. Subsequently, compound 10 was region selectively coupled with
the C-4 chlorine atom in the 2,4,5-trichloropyrimidine reagent to form the 2-
chloropyrimidine scaffold 11. Aniline intermediates 14a-c were prepared from
benzenesulfonyl chloride derivatives by subsequent reaction with 4-nitroanilene and
the Fe-NH4Cl reagent. By the nucleophilic substitution reaction of 2-chlorine-
substituted pyrimidine 11 with anilines 14a-c under the p-toluenesulfonic acid
condition, the desired molecules 7a-g were synthesized. Applying a similar synthetic
strategy, another family of the title compounds 7h-i were also prepared shown in
Scheme 2. Generally, 4-aminephenol was converted to N-phenyl methanesulfonamide
intermediates 16a-b under the action of methanesulfonyl chloride or trifluoromethane
sulfonic anhydride. Then 16a-b were reacted with 4-fluoronitrobenzene to form the
intermediates 17a-b, which were conveniently converted to amine derivatives 18a-b
by using the Fe-NH4Cl reduction condition. Also under the action of p-
toluenesulfonic acid reagent, 18a-b was reacted with 2-chlorine pyrimidine 11 to
generate the title molecules 7h-i.
2.2. Biological activity

The capacity of these newly obtained compounds for inhibiting FAK enzyme
activity was explored by ADP-Glo™ Kinase Assay,23,24 The antiproliferative activity
against cancer cells was determined by CCK-8 testing.25 The effects on cell apoptosis
were analyzed by flow cytometry. For these biological evaluation, the human
pancreatic carcinoma cell line (AsPC-1, Panc-1, BxPC-3), typical B-cell
lymphoblastic leukemia cell line (Ramos), and gefitinib-resistant lung cancer cell line
(H1975), which harbored overexpressed FAK protein, were employed. Human normal
liver cell line (LO-2) was also used as a negative selection for comparison,
Gemcitabine, a chemotherapy medication used to treat several types of cancer, was
selected as a reference compound.
Table 1. Biological activity of the newly synthesized compounds 7a-i.a
Cl
N
HN
N
NH O
N
H
R1
R2
7a-i
Antiproliferative activity (IC50, µM)
FAK
Enzymatic
activity
(IC50, nM)b
Com
pd.
R1
R2
AsPC-1
2.32
1.55
10.1
6.69
3.92
Panc-1
2.99
2.76
> 40
5.70
0.53
BxPC-3 Ramos
H1975 L0-2
NH
Me
H
84.6
23.1
19.4
22.5
31.6
6.92
20.3
6.17
12.1
24.6
2.18
9.56
7.07
8.50
27.5
28.7
> 40
7a
7b
7c
7d
7e
S
S
O
O
O
NH
82.5
O
NH
MeO
MeO
H
109.2
84.7
S
O
O
O
O
F
F
12.91 19.7
NH
S
S
O
NH
86.7
3.71
4.28
11.5
8.37
O
O
O
H
110.0
113.8
3.49
1.78
1.80
0.03
24.1
10.6
9.10
4.61
7f
NH
S
O
O
Me
4.89
2.88
7g
NH
S
O
O

O
O
O
O
S
H
H
108.8
235.9
2.75
2.39
3.61
1.75
14.2
19.8
14.4
6.29
4.10
16.5
3.37
7h
N
O
H
CF3
S
5.86
20.2
7i
N
H
O
GCTc
26.8
6.73
30.4
>20
>10
--d
>10
--
>20
TAE
226
a
6.79
1.03
5.37
b
Data represent the mean of at least three separate experiments. Dose-response curves were
determined at five concentrations. The IC50 values are the concentrations in micromolar needed to
c
inhibit cell growth by 50%, as calculated using GraphPad Prism version 5.0.
Gemcitabine. d Untested.
GCT:
AsPC cells
Panc cells
7e (µM)
7e (µM)
Figure 3. Time and concentration dependant effects of compound 7e on the antiproliferative
activity of Panc-1 and AsPC-1 cell lines. Results are representative of three separate experiments,
dates are expressed as the mean standard deviation, p < 0.05.
Sul-DPPYs were initially assessed for their ability to inhibit FAK enzymatic
activity. The in vitro IC50 values are summarized in Table 1. The kinase-based test
results indicate that all these compounds exhibit moderate inhibitory activity against
the FAK enzyme within nanomolar concentrations. Among them, 4 compounds
exhibited strong anti-FAK activity at concentrations lower than 100 nM: 7a, 7b, 7d
and 7e. Apparently, the trifluoromethanesulfonyl group is unfavorable for improving
the inhibitory activity against FAK. The exemplary compound 7i, with an IC50 value
of 235.9 nM, was considered the least active inhibitor of these compounds. In cell-
based assays, a large portion of Sul-DPPYs could effectively inhibit the proliferation
of these cancer cell lines at concentrations ranging from 0.03 to 40 µM. In particular,
compounds 7e, 7g, and 7i exhibited excellent capabilities to interfere with the
proliferation of all cancer cell lines within concentrations of 15 µM. Compounds 7e
and 7g possessed sensitivity that allows them to inhibit Panc-1 cell activity, with IC50
values of 0.53 and 0.03 µM, respectively. By contrast, the reference drug gemcitabine

has an IC50 value of more than 20 µM against the AsPC-1, Panc-1, and BxPC-3 cell
lines. This finding suggests that Sul-DPPYs are more active than gemcitabine against
pancreatic cancer. All Sul-DPPYs also showed low cytotoxic activity against the
normal liver cell line LO-2, indicating that Su-DPPYs exert less effect on LO-2 cells
than the cancer cells. Typically, compound 7e, still possesses low cytotoxicity against
normal LO-2 cells (IC50=11.5 µM). Although compound 7e is less active against FAK
enzyme, it possesses strong potency to inhibit pancreatic carcinoma cell lines, and low
toxicity against LO-2 cells. Therefore, inhibitor 7e may be selected as a new valuable
lead compound to discover more effective drugs for treatment of pancreatic cancer.
Compound 7e, the active inhibitor, was selected to evaluate the inhibition of cell
viability in Panc-1 and AsPC-1 cell lines. As presented in Figure 3, compound 7e
apparently inhibited cell viability for Panc-1 at concentrations ranging from 0.5 µM to
8 µM and for Aspc-1 from 1.25 to 10 µM. In particular, cell viability was inhibited up
to 50% at 72 h for all the concentrations. With increases in time and concentration,
the viability rate was suppressed by 82.8% for Panc-1 cells and 76.1% for AsPC-1
cells.
Concentration (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 annexin V/FTIC, followed
by flow cytometry analysis. One representative experiment is shown.
2.3. Flow cytometry

To further investigate the antiproliferative mechanism of these inhibitors on
pancreatic cancer cells, the effects of inhibitor 7e on apoptosis in the AsPC-1 cell line
was evaluated by flow cytometry (Fig. 4). Compound 7e strongly induced the
apoptosis of AsPC-1 cells, with apoptotic rates of 55.8%, 74.9%, and 87.3% at
concentrations of 1, 5, and 10 µmol/L, respectively. These results indicated that the
percentages of cell apoptosis correspond to the concentrations of the compounds,
which confirmed that compound 7e could induce apoptosis in a dose-dependent
manner.
2.4. Molecular simulations
To explore the putative interaction mechanism of Sul-DPPYs with FAK enzyme,
the active inhibitor 7e and the less active inhibitor 7i were docked into the binding
pocket of the FAK enzyme (PDB code: 2JKK) by using Autodock4.2,26 respectively.
The docking models are shown in Figure 5. AutoDock 4.2 with its default parameters
was used.27,28 The novel FAK inhibitor TAE226 was also analyzed using the same
procedure for comparison (Fig. 5A).
A
Glu500
Cys502
Cys427
Met499
Ile428
Leu553
Glu506
Asp564
Gly429
Leu567
Glu430
C
B
Glu500
Glu500
Cys502
Cys502
Cys427
Cys427
Ile428
Met499
Met499
Ile428
Leu553
Leu553
Glu506
Gly429
Glu506
Gly429
Asp564
Asp564
Leu567
Leu567
Glu430
Glu430
Figure 5. Predicted binding pose of the typical inhibitors in FAK enzyme (PDB code: 2JKK), A:
TAE226, B: inhibitor 7e; C:inhibitor 7i.

The binding model clearly indicated that inhibitors 7e (Fig. 5B) and 7i (Fig. 5C)
interact with the FAK enzyme similarly to TAE226 (Fig. 5A). All of them formed
several important contacts with the FAK protein below: (1) hydrogen-bond forces
between the N-1 atom of the pyrimidine core with the amino acid Cys502 for TAE226
and inhibitor 7i, whereas the hydrogen bond comes from the NH group in amide
functionality with Leu553 for 7e; (2) van der Waal contacts produced by the C-5
chlorine atom with the amino acid Met499; (3) dipole-induced dipole interactions
formed by the hydrophilic section of the binding pocket surrounded with amino acids
Ile428 and Glu429 with the morpholine substituent in TAE226, and the sulfonamide
group in inhibitor 7e. However, for inhibitor 7i, these polar forces disappeared
because the sulfonamide substituent moved far away from the hydrophilic binding
section. Instead, the hydrophobic phenyl ring in molecules 7e and 7i occupied the
hydrophilic section, which possesses unfavorable characteristics that hinder
interaction with the FAK enzyme. To summarize, this binding model exactly
explained their anti-FAK activity.
3. Conclusion
A new class of diphenylpyrimidine derivatives with a sulfonamide functionality
were synthesized and biologically evaluated as potent FAK inhibitors. The typical
inhibitor 7e effectively inhibited FAK activity with an IC50 value of 86.7 nM. Notably,
inhibitor 7e significantly suppressed the proliferation of the pancreatic cancer cell
lines AsPC-1and Panc-1 at concentrations of 3.92 and 0.53 µM, respectively. Flow
cytometry results indicated that 7e promoted the apoptotic rate of AsPC cells, which
is 87.3% at a concentration of 10 µM. In addition, molecular simulation results
suggested that 7e binds tightly with the FAK enzyme. These results provided a
theoretical basis for the further structural optimization of sulfonamide-substituted
derivatives as FAK inhibitors and demonstrated that compound 7e could potentially
inhibit FAK for the treatment of pancreatic cancer.
4. Experimental section
4.1. General methods and chemistry
Reagents were obtained from commercial suppliers and used without further
purification. Solvents were purified and stored according to standard procedures. All
reactions were monitored by TLC, using silica gel plates with fluorescence F254

(TLC Silica gel 60 F254, Merck) and UV light visualization. High resolution ESI-MS
were performed on an AB Sciex TripleTOF® 4600 LC/MS/MS system. 1H 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), dt (doublet of triplets), q
(quartet), m (multiplet) and br (broad signal). Flash chromatography separations were
obtained on silica gel (300–400 mesh) using dichloromethane/methanol as eluents.
4.2.1. Preparation of the 2-chlorine pyrimidine 11.19,20
2-Amino-N-methylbenzamide 10 was prepared according to the general procedure in
literature 19. 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 80 °C for 6 hours. The mixture was evaporated to
dryness. The organic phase was evaporated to dryness and the residue was
recrystallised from MeCN/water 20:1 to yield 2-(2,5-dichloropyrimidin-4-ylamino)-
N-methylbenzamide 16.87 g, 85 %.
4.2.2. Preparation of the title molecules7a-h.21,22
Anilines 14a-c and 18a-b were synthesized using the reported method in literatures 21.
The obtained compounds 14a-c or 18a-b (1 mmol), 11 (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 5 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.
N-(3-((5-Nitro-2-(4-(3-(4-
methylbenzenesulfonicamide)methyl)phenylamino)-4-pyrimidinyl)amino)phenyl)-N-
methylbenzamide (10a). Yield: 25.3%; off-brown solid; 1H NMR (400 MHz, DMSO-
d6): δ 1.90 (s, 3H), 2.37 (s, 3H), 2.80 (d, J = 4.0 Hz, 3H), 6.80 (d, J = 12.0 Hz, 1H),
7.12 (t, J = 8.0 Hz, 1H), 7.31-7.36 (m, 4H), 7.42 (s, 1H), 7.53 (d, J = 8.0 Hz, 2H),
7.75 (d, J = 8.0 Hz, 1H), 8.20 (s, 1H), 8.65 (d, J = 8.0 Hz, 1H), 8.78 (d, J = 4.0 Hz,
1H), 9.35 (s, 1H), 9.44 (s, 1H), 11.61 (s, 1H); 13C NMR (100 MHz, DMSO-d6): δ
18.42, 21.48, 26.79, 105.6, 117.9, 121.2, 121.7 (2C), 122.3, 127.1 (2C), 127.8, 128.5,
128.8, 130.0 (2C), 131.8, 135.4, 138.3, 139.1, 139.7, 143.3, 151.1, 153.4, 158.0, 169.3;
HRMS (ESI) for C26H25ClN6O3S; [M+H]+ calcd: 537.1470; found: 537.1443.
4.2.2.2.
N-(3-((5-Nitro-2-(4-
(4-methylbenzenesulfonicamide)phenylamino)-4-
pyrimidinyl)amino)phenyl)-N-methylbenzamide (10b). Yield: 27.5%; off-brown solid;
1H NMR (400 MHz, DMSO- d6): δ 2.35 (s, 3H), 2.81 (d, J = 4.0 Hz, 3H), 6.99 (d, J =
12.0 Hz, 2H), 7.16 (t, J = 8.0 Hz, 1H), 7.31-7.37 (m, 3H), 7.50 (d, J = 8.0 Hz, 2H),
7.64 (d, J = 8.0 Hz, 2H), 7.78 (d, J = 8.0 Hz, 1H), 8.20 (s, 1H), 8,67 (d, J = 4.0 Hz,
1H), 8.81 (d, J = 4.0 Hz, 1H), 9.43 (s, 1H), 10.0 (s, 1H), 11.64 (s, 1H); 13C NMR (100
MHz, DMSO-d6): δ 21.44, 26.79, 105.5, 121.0, 121.1, 121.8, 122.1 (2C), 122.4, 126.0,
127.2 (2C), 128.5, 130.0 (2C), 131.7, 132.0. 137.2, 137.4, 139.8, 143.5, 155.1, 155.4,
158.1, 169.4; HRMS (ESI) for C25H23ClN6O3S; [M+H]+ calcd: 523.1314; fou
methoxyl nd: 523.1281.
4.2.2.3.
N-(3-((5-Nitro-2-(4-(3-(4-
methylbenzenesulfonicamide)methoxyl)phenylamino)-4-pyrimidinyl)amino)phenyl)-
1
N-methylbenzamide (10c). Yield: 32.8%; off-brown solid; H NMR (400 MHz,
DMSO- d6): δ 2.37 (s, 3H), 2.82 (d, J = 4.0 Hz, 3H), 3.33 (s, 3H),7.07 (d, J = 8.0 Hz,
1H), 7.15 (t, J = 8.0 Hz, 1H), 7.23 (t, J = 8.0 Hz, 2H), 7.35 (d, J = 8.0 Hz, 3H), 7.59
(d, J = 8.0 Hz, 2H), 7.79 (d, J = 8.0 Hz, 1H), 8.24 (s, 1H), 8.71-8.83 (m, 2H), 9.26
13
(s,1H), 9.48 (s, 1H), 11.68 (s, 1H); C NMR (100 MHz, DMSO-d6): δ 21.44, 26.79,
55.56, 104.0, 105.8, 111.8, 119.3, 121.1, 121.6, 122.4, 126.9, 127.3 (2C), 128.5, 129.6
(2C), 131.9, 138.4, 139.8, 139.9, 143.0, 153.6, 155.0, 155.4, 158.0, 169.4; HRMS
(ESI) for C26H25ClN6O4S; [M+H]+ calcd: 553.1419; found: 553.1382.
4.2.2.4
N-(3-((5-Chloro-2-(4-(3-(4-
fluorinebenzenesulfonicamide)methoxyl)phenylamino)-4-pyrimidinyl)amino)phenyl)-
1
N-methylbenzamide (10d). Yield: 25.9%; off-brown solid; H NMR (400 MHz,
DMSO- d6): δ 2.82 (d, J = 4.0 Hz, 3H), 3.30 (s, 3H), 7.07 (d, J = 8.0 Hz, 1H), 7.13 (t,

J = 8.0 Hz, 1H), 7.21-7.27 (m, 2H), 7.39 (t, J = 8.0 Hz, 3H), 7.71-7.78 (m, 3H), 8.23
(s, 1H), 8.70 (d, J = 8.0 Hz, 1H), 8.80 (d, J = 4.0 Hz, 1H) 9.42 (s, 1H), 9.48 (s, 1H),
13
11.66 (s, 1H); C NMR (100 MHz, DMSO-d6): δ 26.79, 55.44, 103.8, 105.9, 111.8,
116.1, 116.4, 118.7, 121.1, 121.7, 122.4, 127.9, 128.5, 130.2 (d, J = 40 Hz, 1C), 131.9,
137.6 (d, J = 12.0 Hz, 1C), 139.8, 140.3, 153.9, 155.0, 155.4, 157.9, 163.3, 165.8,
169.4; HRMS (ESI) for C25H22ClFN6O4S; [M+H]+ calcd: 557.1169; found: 557.1147.
4.2.2.5 N-(3-((5-Chloro-2-(3-(4-fluorinebenzenesulfonicamide) phenylamino)-4-
pyrimidinyl)amino)phenyl)-N-methylbenzamide (10e). Yield:31.8%; off-brown solid;
1H NMR (400 MHz, DMSO- d6): δ 2.82 (d, J = 4.0 Hz, 3H), 6.97 (d, J = 8.0 Hz, 2H),
7.14 (t, J = 8.0 Hz, 1H), 7.31 (t, J = 8.0 Hz, 1H), 7.41 (t, J = 8.0 Hz, 2H), 7.51 (d, J =
8.0 Hz, 2H), 7.75-7.80 (m, 3H), 8.19 (s, 1H), 8.66 (d, J = 4.0 Hz, 1H), 8.78 (d, J = 4.0
Hz, 1H), 9.44 (s, 1H), 10.06 (s, 1H), 11.61 (s, 1H); 13C NMR (100 MHz, DMSO-d6):
δ 26.79, 105.5, 116.7, 117.0, 120.9, 121.2, 121.8, 122.4, 122.5 (2C), 135.4, 128.5,
130.22 (d, J = 40 Hz, 1C), 131.5, 131.7, 136.3 (d, J = 12 Hz, 1C), 137.8, 139.7, 155.1,
155.4, 158.1, 163.5, 166.0, 169.3; HRMS (ESI) for C24H20ClFN6O3S; [M+H]+ calcd:
527.1063; found: 527.1044.
4.2.2.6
N-(3-((5-Nitro-2-(4-(3-(4-
methoxylbenzenesulfonicamide)methyl)phenylamino)-4-pyrimidinyl)amino)phenyl)-
N-methylbenzamide (7f) Yield: 29.9%; off-brown solid; 1HNMR (400 MHz, DMSO-
d6): δ 1.93 (s, 3H), 2.51 (s, 3H), 2.82 (d, J = 4.0 Hz, 3H), 6.80 (d, J = 8.0 Hz, 1H),
7.10-7.14 (m, 3H), 7.30-7.36 (m, 2H), 7.43(s, 1H), 7.58 (d, J = 8.0 Hz, 2H), 7.77 (d, J
= 8.0 Hz, 1H), 8.22 (s, 1H), 8.66 (d, J = 8.0 Hz, 1H), 8.81 (d, J = 4.0 Hz, 1H), 9.28
(s,1H), 9.45 (s, 1H), 11.62 (s, 1H); 13C NMR (400 MHz, DMSO-d6): δ 18.44, 26.79,
56.11, 105.6, 114.7 (2C), 117.9, 121.2, 121.7, 122.3, 127.8, 128.5, 129.0, 129.3 (2C),
131.9, 132.8, 135.4, 139.0, 139.7, 155.2, 155.4, 158.0, 162.7, 169.4; HRMS (ESI) for
C26H25ClN6O4S; [M+H]+ calcd: 553.1419; found: 553.1411.
4.2.2.7
N-(3-((5-Chloro-2-(4-(4-methoxylbenzenesulfonicamide)phenylamino)-4-
pyrimidinyl)amino)phenyl)-N-methylbenzamide (10g). Yield: 19.7%; off-yellow solid;
1H NMR (400 MHz, DMSO- d6): δ 2.52 (s, 3H), 2.82 (d, J = 4.0 Hz, 3H), 6.99 (d, J =
8.0 Hz, 2H), 7.08 (d, J = 8.0 Hz, 2H), 7.16 (t, J = 4.0 Hz, 1H), 7.30-7.34 (m, 1H),
7.50 (d, J = 8.0 Hz, 2H), 7.68 (d, J = 8.0 Hz, 2H), 7.79 (d, J = 8.0 Hz, 1H), 8.20 (s,
1H), 8.68 (d, J = 8.0 Hz, 1H), 8.83 (s,1H), 9.43 (s, 1H), 9.97 (s, 1H), 11.68 (s, 1H);
13C NMR (100 MHz, DMSO-d6): δ 26.78, 56.06, 105.4, 114.7 (2C), 121.0, 121.11,
121.8, 122.0 (2C), 122.3, 128.5, 129.4 (2C), 131.7, 131.8 (2C), 132.2, 137.28, 139.75,

155.1, 155.4, 158.1, 162.7, 169.4; HRMS (ESI) for C25H23ClN6O4S; [M+H]+ calcd:
539.1263; found: 539.1249.
4.2.2.8
N-(3-((5-Chloro-2-(4-(4-methylsalfonamido)phenoxy)-4-
pyrimidinyl)amino)phenyl)-N-methylbenzamide (10h). Yield: 25.3%; off-brown solid;
1H NMR (400 MHz, DMSO- d6): δ 2.51 (s. 3H), 2.81 (d, J = 4.0 Hz, 3H), 6.97 (dd, J
= 4.0 Hz, 12.0 Hz, 4H), 7.13 (t, J = 8.0 Hz, 1H), 7.22 (d, J = 12.0 Hz, 2H), 7.46 (t, J =
8 Hz, 1H), 7.66 (d, J = 8.0 Hz, 2H), 7.77 (d, J = 8.0 Hz, 1H), 8.22 (s, 1H), 8.73-8.82
13
(m, 2H), 9.49 (s, 1H), 9.62 (s, 1H), 11.63 (s, 1H); C NMR (100 MHz, DMSO-d6): δ
26.79 (2C), 105.5, 119.1 (2C), 119.5 (2C), 121.2, 121.9, 122.0, 122.4, 133.2 (2C),
128.5, 130.1, 131.9, 133.6, 136.6 139.8, 151.5, 154.8, 155.1, 155.5, 158.2, 169.4;
HRMS (ESI) for C25H23ClN6O4S; [M+H]+ calcd: 539.1263; found: 539.1229.
4.2.2.9
N-(3-((5-Chloro-2-(4-(4-trifluoromethylsalfonamido)phenoxy)-4-
pyrimidinyl)amino)phenyl)-N-methylbenzamide (10i). Yield: 21.8%; off-brown solid;
1H NMR (400 MHz, DMSO- d6): δ 2.81 (d, J = 4.0 Hz, 3H), 6.90 (d, J = 4.0 Hz, 2H),
6.96 (d, J = 4.0 Hz, 2H), 7.13 (d, J = 8.0 Hz, 4H), 7.45 (t, J = 8.0 Hz, 1H), 7.65 (d, J =
8.0 Hz, 2H), 7.77 (d, J = 8.0 Hz, 1H), 8.21 (s, 1H), 8.73 (d, J = 4.0 Hz, 1H), 8.79 (d, J
13
= 8.0 Hz, 1H) 9.48 (s, 1H), 11.61 (s, 1H); C NMR (100 MHz, DMSO-d6): δ 26.78,
105.4, 118.7 (2C), 118.8, 119.4 (2C), 119.7, 121.2, 121.9, 122.0, 122.4, 122.9, 125.6
(2C), 128.5, 131.9, 136.5, 139.7, 151.6, 154.4, 155.1, 155.5, 158.3, 169.4; HRMS
(ESI) for C26H20ClF3N6O4S; [M+H]+ calcd: 593.0980; found: 593.0960.
4.3. In vitro kinase enzymatic assay
The FAK kinase enzyme system (Catalog. V9301) , purchased from Promega
Corporation (USA), was used to test the activity according to the instructions of the
manufacturer. Concentrations consisting of suitable levels from 10 to 500 nM were
used for all of the tested compunds. The more detailed and complete protocols can be
founded in the ADP-GloTM kinase Assay Technical Manual available at:
http://cn.promega.com/resources/protocols/product-information-sheets/n/btk-kinase-
enzyme-system-protocol/. The test was performed in a 384-well plate, and includes
the major steps below: (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
Ramos, AsPC-1, Panc-1, BxPC-3, H1975, and L0-2 cells were purchased from
Fuheng Biology Company (Shanghai, China). The Cell Counting Kit-8 (CCK-8)
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 RPMI-1640 (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.
4.4.2. Cellular activity assay
CCK-8 method assay method was used to test the cell viability. 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 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.
Gemcitabine was employed as the positive control. The IC50 values were calculated
using GraphPad Prim version 5.0.
4.5. Cell Apoptosis Assay

AsPC-1 cells (2 to 5 ×105 cells/well) incubated in 6-well plates were treated with
solvent control (DMSO), or compound 7e (1, 5 or 10 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 assay (Becton-
Dickinson, USA).
4.6. 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 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 4 was used in the docking
studies. The 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 7e. 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 China (No.
81402788) and the Cultivation Project of Youth Techstars, Dalian (2016RQ043) for
the financial support of this research.
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Graphical Abstract
Design,
synthesis
and
biological
evaluation
of
sulfonamide-substituted diphenylpyrimidine derivatives
(Sul-DPPYs) as potent focal adhesion kinase (FAK)
inhibitors with antitumor activity
Menghua Qua,1, Zhihao Liua,1, Dan Zhaoa, Changyuan Wanga, Jianbin Zhanga, Zeyao Tanga,
Kexin Liua, Xiaohong Shua, ∗, Hong Yuanb, ∗, and Xiaodong Maa, ∗
a College of Pharmacy, Dalian Medical University, Dalian 116044, PR China.
bDepartment of Laboratory, 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)

F
O
O
O
S O
F
O
O
O
NH
S
O
HN
HN
O
HN
Asp546
R2
H
N
HN
N
NH
HN
N
NH
N
N
O
HN
R1
N
NH
F
Cl
Structure-based
Design
Spebrutinib
BTK Inhibitors
Phase
SFA-DPPYs 10c
Improved actvity agaisnt BTK
N
Cys502
O
Sul-DPPYs 7e
FAK inhibitor
IC50 = 3.92 (AsPC cells)
IC50 = 0.53 (Panc cells)
IC50 = 2.18 (Ramos cells)
IC50 = 3.71 (H1975 cells)
N
H
N
O
O
HN
N
NH
N
Cl
TAE226
FAK inhibitior
F
Glu506/Gly429
O
S
O
O
N
HN
Asp546
H
N
H
N
O
HN
R1
N
NH
O
O
HN
N
NH
N
Cys502
N
Sul-DPPYs 7e
Cl
IC50 = 3.92 (AsPC cells)
IC50 = 0.53 (Panc cells)
IC50 = 2.18 (Ramos cells)
IC50 = 3.71 (H1975 cells)
TAE226
FAK inhibitors

Highlights
ꢀ
ꢀ
ꢀ
ꢀ
Sulfonamide-substituted diphenylpyrimidines (Sul-DPPYs) were synthesized as
FAK inhibitors
Sul-DPPYs could effectively inhibit pancreatic cancer cell lines and NSCLC-
resistant H1975 cell line (IC50 < 10µM).
Compound 7e promoted apoptosis of pancreatic cancer cells in a dose-
dependent manner.
Compound 7e may be selected asis a potential FAK inhibitor for treatment of
pancreatic cancer.