Design and synthesis of diphenylpyrimidine derivatives (DPPYs) as potential dual EGFR T790M and FAK inhibitors against a diverse range of cancer cell lines

Journal Pre-proofs Design and synthesis of diphenylpyrimidine derivatives (DPPYs) as potential dual EGFR T790M and FAK inhibitors against a diverse range of cancer cell lines Min Ai, Changyuan Wang, Zeyao Tang, Kexin Liu, Xiuli Sun, Tengyue Ma, Yanxia Li, Xiaodong Ma, Lei Li, Lixue Chen PII: DOI: Reference:

S0045-2068(19)31065-X https://doi.org/10.1016/j.bioorg.2019.103408 YBIOO 103408

To appear in:

Bioorganic Chemistry

Received Date: Revised Date: Accepted Date:

5 July 2019 28 September 2019 28 October 2019

Please cite this article as: M. Ai, C. Wang, Z. Tang, K. Liu, X. Sun, T. Ma, Y. Li, X. Ma, L. Li, L. Chen, Design and synthesis of diphenylpyrimidine derivatives (DPPYs) as potential dual EGFR T790M and FAK inhibitors against a diverse range of cancer cell lines, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg. 2019.103408

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2019 Published by Elsevier Inc.

Design and synthesis of diphenylpyrimidine derivatives (DPPYs) as potential dual EGFR T790M and FAK inhibitors against a diverse range of cancer cell lines Min Ai a,1, Changyuan Wang a,1, Zeyao Tanga, Kexin Liua, Xiuli Sunb, Tengyue Mac, Yanxia Lib, Xiaodong Maa, Lei Lia, and Lixue Chena, a College

of Pharmacy, Dalian Medical University, Dalian 116044, PR China.

b Department c Dalian 1 These

of Hematology, the First Affiliated Hospital of Dalian Medical University, Dalian 116011, PR China.

Buyun Biological Technology Co., Ltd., 116085, PR China.

authors contributed equally to this work.

E-mail address: [email protected].

Abstract A new class of pyrimidine derivatives were designed and synthesized as potential dual FAK and EGFRT790M inhibitors using a fragment-based drug design strategy. This effort led to the identification of the two most active inhibitors, namely 9a and 9f, against both FAK (IC50 = 1.03 and 3.05 nM, respectively) and EGFRT790M (IC50 = 3.89 and 7.13 nM, respectively) kinase activity. Moreover, most of these compounds also exhibited strong antiproliferative activity against the three evaluated FAK-overexpressing pancreatic cancer (PC) cells (AsPC-1, BxPC-3, Panc-1) and two drug-resistant cancer cell lines (breast cancer MCF-7/adr cells and lung cancer H1975 cells) at concentrations lower than 6.936 μM. In addition, 9a was also effective in the in vivo assessment conducted in a FAK-driven human AsPC-1 cell xenograft mouse model. Overall, this study offers a new insight into the treatment of hard to treat cancers. Key Words: FAK; EGFR T790M; Inhibitor; Dual-target; Fragment-based drug design

1

1. Introduction Cancer is one of the major diseases that endangers human health and even lives worldwide today. There were 4.3 million new cancer cases and more than 2.8 million cancer deaths in China in 2015, with lung cancer having the highest incidence, and pancreatic cancer (PC) being the leading cause of cancer death [1,2]. Receptor tyrosine kinases (RTKs) and non-RTKs play important roles in cancer cell proliferation, survival and migration in response to extracellular signals. Small-molecule drugs targeting dysregulated RTKs have various clinical benefits, especially for genetically defined patient populations [3-5]. Focal adhesion kinase (FAK), one of the important non-receptor protein tyrosine kinases, is highly overexpressed in several cancers, including thyroid, brain, liver, prostate, breast, colon, head and neck. The triggering of FAK is initiated by the binding of integrin to the extracellular matrix (ECM), followed by FAK autophosphorylation at Tyr-397, which is crucial for most cell functions [6-9]. Until now, a number of small-molecule FAK inhibitors have been identified and shown to be potent inhibitors interfering tumor growth and metastasis in several preclinical and clinical models [10-13]. TAE-226 (1) is a typical FAK inhibitor (IC50=5.5 nM), with potent antiproliferative and antitumor effects in vitro and in vivo in several types of cancers [10]. Another novel FAK inhibitor, CEP-37440 (2, phase I, NCT01922752), is currently in the clinical testing stage for cancer treatment [11]. Also, VS-4718 (3, phase II, NCT02465060), significantly impacts cell viability, decreases anchorage-independent growth and motility in selected PC cells by targeting the FAK kinase activity and inhibiting Y397 phosphorylation [12,13] (Figure 1). Undoubtedly, searching for effective FAK inhibitor offers new tools to gain further understanding of the antitumor mechanisms underlying these inhibitors.

2

OH

N

A

O

N

MeHN

OMe O

N

HN

MeHN

NH

N

HN

NH

O

3 VS-4718 NMe2 O

O

N

MeN

HN OMe

R1

N

NH

N

OMe

OMe

MeN

N

N

4 WZ-4002 X=O, R1= Cl, R= Me 5 CO-1686 X=NH, R1= CF3, R= MeCO-

N

HN

HN

X

N O O S N Me Me NH

N

F3C

2 CEP-37440

R N

B

N

HN

N

Cl

1 TAE-226

N

OMe O

N

Cl

O

MeHN

N

NH N

6 AZD-9291

HN F3C

N

NH N

7 Az-DPPY

Fig. 1. Chemical structures of the novel pyrimidine derivatives as potent non-RTK (A) or RTK (B) inhibitors.

The epidermal growth factor receptor (EGFR) tyrosine kinase is another valuable clinically validated target for anticancer therapies, especially for non-small-cell lung cancer (NSCLC) treatment [14,15]. However, the T790M mutation (threonine to methionine) within the ATP site of the EGFR, occurs frequently (in approximately 60% of the patients) in NSCLC patients who develop resistance to drugs targeting EGFR, and thus leads to failure of EGFR-targeted therapy [16,17]. The pyrimidine derivatives WZ-4002 (4) [18], CO-1686 (5, Phase III) [19], and AZD-9291 (6, approved in 2015) [20,21] are novel EGFRT790M inhibitors against resistant NSCLC disease. Our considerable efforts on the structural modification of the pyrimidine scaffold also led to the identification of several promising EGFRT790M inhibitors, including Az-DPPYs (7, Figure 1) [22] and Mor-DPPYs (8, Figure 2) [23]. All these works indicated that the pyrimidine core along with the C-2,4 dianiline side chains were essential for the high anticancer activity of the inhibitors 3

[18,24,25].

Using

a

fragment-based

drug

design

strategy,

herein,

a

class

of

N-alkylbenzamide-substituted diphenylpyrimidine (DPPYs) were synthesized based on the TAE-226 and Mor-DPPY scaffold (Figure 2). Fortunately, most of these compounds not only displayed strong anti-FAK activity, but also displayed high EGFRT790M inhibitory potency. These dual FAK and EGFRT790M inhibitors, their synthetic method and the characterization of their anticancer activity are described in this study. O

Me

O

06 O

O

OMe HN

N

Improved activity

Asp546 HN

NH

N

NH N

N

Cl Mor-DPPY (8) EGFRT790M inhibitor

F3C Rociletinib (5) EGFRT790M inhibitor Phase III

Fragment-based drug design

O

OMe HN

N

NH N

Cl

HN

N

NH N

Cys502 DPPYs 9a-m FAK and EGFRT790M dual-inhibitor

N

O

O R1

H N

Cl

O

MeHN

R

HN

29

N

HN

HN

4 ly

HN

/G

O

N

5 lu

O

G

N

N

TAE-226 (1) FAK inhibitor

Fig. 2. Designed strategy of the title molecules DPPYs.

2. Results and discussion 2.1 Chemistry

4

Cl

N NO2 O

NO2 O a,b

OH

NH2 O N H

10

R

c

NH2

O

14a-d

13a-d

15a-d

HN

h

NH

Br

N

NH

O

O

N H

NH

N

R1

O

NH

N

16a-d

R

Cl

R1

R1

O N H

N

f,g

NH

N

d

NH2

e R1

R

12a-d

11a-d NO2

NO2

Cl N H

O

O

9a-m

R

R = Me, Et, i-Pr, Cyclopropyl R1 = H, CH3, OCH3, Cl

Scheme 1. Synthetic route of title compounds 9a-m. Reagents and conditions: a) SOCl2, 1 h, 60 C; b) methylamine hydrochloride/ethylamine hydrochloride/isopropylamine hydrochloride/cyclopropylamine hydrochloride, NaHCO3, CH3CN, 4 h, 0 C, 91-97%; c) Fe-NH4Cl, MeOH-H2O, 2 h, 70 C, 65-78%; d) 2,4,5-trichloropyrimidine, N,N-diisopropylethylamine (DIPEA), isopropanol, 6 h, 80 C, 72-85%; e) bromoacetyl bromide, NaHCO3, CH3CN, 2 h, 0 C, 85-91%; f) morpholine, K2CO3, KI, CH3CN, 4 h, 60 C, 75-80%; g) Fe-NH4Cl, MeOH-H2O, 2 h, 70 C, 72-81%; h) 13a-d,trifluoroacetic acid, 2-BuOH, 100 C, 12 h, 11-22%.

All the newly designed DPPYs were synthesized applying the similar synthetic method illustrated in our previous work (Scheme 1) [22,26,27]. Commercially available 2-nitrobenzene acid (10) was reacted with SOCl2, and then directly reacted with alkylamine in the presence of the base NaHCO3 to produce 11a-d. The 11a-d was then reduced to give 12a-d using the Fe-NH4Cl condition.

After

region-selective

substitution

of

the

C-4

chlorine

atom

in

the

2,4,5-trichloropyrmidine reagent with arylamine, the 2,5-dichloropyrimidine intermediate 13a-d was synthesized. In addition, various C-2 aniline side chains were prepared starting from the material nitroanilines 14a-d which were reacted with bromoacetyl bromide to yield 15a-d. Compounds 15a-d were treated with morpholine, and then reduced by Fe-NH4Cl reagent to prepare the anilines 16a-d. Ultimately, the title molecules 9a-m were conveniently synthesized via the coupling reaction of anilines 16a-d with chloro-substituted pyrimidines 13a-d in the presence of trifluoroacetic acid (TFA). 2.2 Biological activity 5

Table 1. Inhibitory activity of the newly synthesized compounds against FAK and EGFRT790M kinases. Cl

N HN

N

NH

O N H

R

R1 NH

N O

O

Compound

R

R1

9a

Me

9b

Enzymatic activity(IC50, nM)a FAK

EGFRT790M

CH3

1.03±0.14

3.89±0.12

Me

OCH3

106.7±2.98

20.7±2.31

9c

Me

Cl

11.8±2.21

32.7±1.99

9d

Cyclopropyl

CH3

101.0±0.87

6.14±0.62

9e

Cyclopropyl

OCH3

125.7±3.35

41.5±1.43

9f

Et

H

3.05±0.53

7.13±1.26

9g

Et

CH3

117.7±3.39

99.1±4.92

9h

Et

OCH3

3.54±0.93

22.3±1.35

9i

Et

Cl

150.0±2.16

44.8±2.71

9j

i-Pr

H

24.9±0.73

37.6±1.20

9k

i-Pr

CH3

9.89±2.70

89.2±2.86

9l

i-Pr

OCH3

30.6±2.95

66.5±1.25

9m

i-Pr

Cl

40.0±1.34

48.4±2.68

6.8±0.79

>100

TAE-226 a

Dose-response curves were determined at five concentrations. The IC50 values are the concentrations in nanomolar needed to inhibit cell growth by 50% as determined from these curves. Table 2. Antiproliferative activity of the title molecules against various human cancer cell lines. Antiproliferative activity(IC50, μM)a

Compound AsPC-1

BxPC-3

Panc-1

Hpde6-c7

MCF/Adr

H1975

9a

0.909±0.18

0.761±0.14

1.218±0.22

1.347±0.15

0.892±0.10

0.333±0.02

9b

0.814±0.19

1.210±0.14

2.007±0.07

2.603±0.25

0.741±0.09

0.460±0.05

9c

0.909±0.18

1.510±0.19

2.084±0.06

2.738±0.27

2.143±0.17

0.882±0.07

6

9d

2.554±0.28

1.672±0.23

1.767±0.14

1.696±0.17

1.835±0.15

0.602±0.09

9e

4.134±0.32

2.972±0.19

1.695±0.09

3.561±0.28

1.874±0.03

0.950±0.09

9f

1.426±0.23

1.873±0.05

0.994±0.16

1.367±0.09

1.972±0.09

0.478±0.04

9g

1.882±0.13

1.947±0.21

1.787±0.21

2.066±0.20

0.716±0.03

0.513±0.09

9h

1.757±0.24

3.090±0.34

1.996±0.17

4.405±0.25

2.544±0.15

0.922±0.13

9i

2.436±0.38

1.699±0.24

0.856±0.20

2.447±0.18

1.383±0.27

0.983±0.14

9j

2.917±0.12

2.309±0.14

1.299±0.10

5.445±0.25

2.796±0.19

0.892±0.08

9k

3.848±0.14

2.324±0.14

1.990±0.20

3.938±0.19

3.613±0.23

0.918±0.10

9l

6.936±0.41

3.630±0.28

4.020±0.08

5.967±0.31

--

--

9m

3.795±0.29

2.477±0.12

2.134±0.08

4.985±0.27

--

--

TAE-226

6.730±0.31

12.330±0.42

>20.00

11.380±0.33

>8.00

6.79±0.17

a

Dose-response curves were determined at five concentrations. The IC50 values are the concentrations in micromolar needed to inhibit cell growth by 50% as determined from these curves.

All the title molecules were evaluated for their inhibitory activity against FAK kinase with the ADP-GloTM kinase assay, as shown in Table 1[28,29]. Clearly, these newly synthesized compounds displayed strong inhibitory activity against the FAK enzyme within the drug concentrations ranging from 1.03 to 150.0 nM. Four typical molecules 9a (IC50=1.03 nM), 9f (IC50=3.05 nM), 9h (IC50=3.54 nM), and 9k (IC50=9.89 nM) remarkably interfered with the FAK enzymatic activity with IC50 values lower than 10 nM. The most active inhibitor, 9a, even displayed 6-fold higher activity than the representative FAK inhibitor TAE-226 (6.79 nM). Moreover, their activity against the EGFRT790M also showed that these molecules can exert strong inhibitory effect on this mutant kinase with the drug concentrations in the range of 3.89 to 99.1 nM. In particular, two potent FAK inhibitory compounds, 9a and 9f, also displayed strong anti-EGFRT790M activity, with IC50 values of 3.89 and 7.13 nM, respectively. Noteworthy, compound 9d significantly enhanced the inhibitory 7

activity against EGFRT790M (IC50 = 6.14 nM) despite its moderate inhibitory activity against FAK kinase (IC50 = 101 nM). These kinase-based test results revealed that the steric cyclopropyl group (R) in the C-1 aniline side chain is unfavorable to inhibit FAK enzymatic activity. The exemplary compounds 9d and 9e only exhibited moderate inhibitory activity, with IC50 values of 101.0 and 125.7 nM, respectively. In addition, compounds bearing an isopropyl group (R) also do not favor to enhance their anti-FAK and EGFR enzymatic activity, indicating that large substituent (R) is unbeneficial for these inhibitors. While a relatively small group, such as methyl or ethyl substituent is suitable. The representative compounds 9a and 9f displayed strong inhibitory effects on activity of the two kinases. The result that 9h showed strong inhibitory potency to FAK kinase, but is weak to inhibit the activity of EGFRT790M kinase, suggested that methoxy substitution (R1) is disadvantageous for identification of dual-target inhibitors. Overall, the activity in the kinase level indicated that this structural modification leads to the formation of two compounds, namely 9a and 9f, which are potent dual FAK-EGFRT790M inhibitors. Additionally, in this study, all the title molecules were also evaluated for their antiproliferative activity towards tumor cells using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay [30]. Three FAK-overexpressed PC cells (AsPC-1, BxPC-3, Panc-1) and two drug-resistant cancer cell lines (breast cancer MCF-7/adr cells and lung cancer H1975 cells) were used for the evaluation of the title molecules. TAE-226, a typical FAK inhibitor was also concurrently evaluated as a reference. The test results, shown in Table 2, revealed that these molecules generally have strong inhibitory activity toward the evaluated tumor cells, with IC50 values less than 6.936 μM (compound 9l). The representative compound 9a strongly inhibited the

8

proliferation of AsPC-1 and BxPC-3 cells at concentrations lower than 1 μM, with IC50 values of 0.909 and 0.761 μM, respectively. Moreover, compound 9a also showed antiproliferative activity against Panc-1 cells, at such low drug concentration of 1.218 μM. Evidently, the detected activity of inhibitor 9a against cancer cell is consistent with its strong anti-FAK enzymatic activity. Excitingly, these molecules also have strong anti-proliferative activity against Gefitinib-resistant H1975 cells (NSCLC), with IC50 values ranging from 0.333 to 0.918 μM. Among these molecules, the most active inhibitor 9a still showed the strongest inhibitory effect (IC50=0.333 μM) on this mutant cell line. Furthermore, compounds 9a, 9b, and 9g were also able to significantly inhibit the drug-resistant breast cancer cells (MCF-7/adr) within the concentrations of 1 μM. Notably, 9b exhibited strong anti-proliferative activity against Aspc-1 cells (IC50=0.814 μM), but weak anti-FAK activity, indicating that it might block the proliferation of cancer cells through other signaling pathway independent of FAK or had weak capability to penetrate the cell membrane. Overall, these results showed that compound 9a not only possesses strong inhibitory activity against the kinase and tumor cells, but also showed weak cytotoxicity activity to the normal human pancreatic ductal cell (Hpde6-c7) within concentration of 1.347 μM, which warrants further biological evaluation. a)

b)

Fig. 3. The effects of treating time and concentrations of inhibitor 9a on cell viability, a) AsPC-1, b) Panc-1. 9

The data shown in Figure 3 indicate that the most active inhibitor, 9a, remarkably decreased the cell viability of both AsPC-1 and Panc-1 cells with the increase of the treatment time and drug concentration. Indeed, the AsPC-1 and Panc-1 cells were almost completely inhibited after treatment with 8 μM of 9a for 24 h, with cell viability rate of 3.4 and 3.7%, respectively.

Fig. 4. Compound 9a induced AsPC-1 cell apoptosis in vitro. The cells were incubated with the indicated concentrations of 9a for 72 h, and the cells were stained with annexin V/FTIC, followed by flow cytometry analysis. One representative experiment is shown.

10

0.25μM

Blank

0.5μM

G0/G1: 66.16% S: 28.14% G2/M : 5.7 %

G0/G1: 57.38% S: 30.80% G2/M: 15.42%

G0/G1: 63.89% S: 30.39% G2/M: 5.72%

a)

1.0μM

G0/G1: 39.90% S: 32.91% G2/M: 27.19%

Cell in each phase(%)

80

G0/G1 S G2/M

60 40 20 0 B

nk la

μM 25 0.

M 5μ 0.

M 0μ 1.

Drug concentration

Fig. 5. Effects of molecule 9a on AsPC-1 cells cycle arrest detected by flow cytometry assay. Cells were treated with different concentrations of 9a 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.

In addition, the effects of compound 9a on apoptosis and cell-cycle progression in AsPC-1 cells were also explored by flow cytometry analysis [31]. As shown in Figure 4, compound 9a induced apoptosis in AsPC-1 cells in a dose- and time-dependent manner, increasing the percentages of apoptotic cells from 52.9 to 90.4% after treatment with various concentrations of 9a (from 0.25 μM to 1.0 μM) for 72 h compared with the control group. In addition, the data presented in Figure 5 revealed that the percentage of cells in the G2/M phase increased from 5.72 to 27.19%, and those in the G0/M phase decreased from 63.89 to 39.90% after treatment with 9a at concentrations from 0.25 to 1.0 μM for 72 h. In comparison, the percentage of cells in the S phase only showed minor changes. Accordingly, 9a arrested AsPC-1 cells in G2/M phase.

11

a)

Control 9a (30mg/kg) 9a (60mg/kg)

b) 2.0

c) 2500

Tumor weight (g)

Tumor volume (mm3)

1.5 1.0 0.5

control

9a 9a (30mg/kg) (60mg/kg) Drug concentration

2000 1500 1000 500

2

4

6

8

10

Days post initial treatment (d)

Fig. 6. In vivo effects of 9a on the tumor growth of AsPC-1 cell Xenografts in nude mice. (A) Images of the tumor collected from the mice; (B) Tumor weight; (C) Tumor volume.

To further investigate the biological activity of the most active inhibitor 9a, this compound was also evaluated in vivo using a FAK-driven human AsPC-1 cell xenograft mouse model. The results shown in Figure 6 indicated that tumor growth was dose-dependently inhibited by repeated oral administration of 9a (qd) for 10 days. Specifically, the tumor growth inhibition (TGI) rates for compound 9a were 24.45 and 36.6% for the dosages of 30 and 60 mg/kg/day, respectively. 3. Conclusion A family of pyrimidine derivatives comprising potent dual FAK and EGFRT790M inhibitors was identified using a fragment-based drug design strategy. Among these inhibitors, two typical molecules, namely 9a and 9f, not only displayed strong inhibitory activity against FAK kinase (IC50 = 1.03, 3.05 nM), but also inhibited EGFRT790M mutants at concentrations of 3.89 and 7.13 nM, respectively. Additionally, these compounds also exhibited strong inhibitory activity toward the three evaluated FAK-overexpressing PC cells (AsPC-1, BxPC-3, Panc-1) and two drug-resistant 12

cancer cell lines (breast cancer MCF-7/adr cells and lung cancer H1975 cells), at concentrations below 6.936 μM. Moreover, the xenograft model study also showed the efficiency of 9a in vivo. Overall, this work identified and characterized a series of potent dual FAK and EGFRT790M inhibitors which showed great potential for the treatment of hard to treat cancers. 4. Experimental section 4.1 General methods and chemistry All solvents and chemicals were analytical reagent (AR) and used as purchased without further purification. 1H NMR and 13C NMR spectra were respectively recorded on a Bruker AV 400 MHz and AV 101 MHz spectrometer. 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 peak (d6-DMSO: 40.0, TMS: 0.0). 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), 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 title molecules 9a-m 2,5-dichloropyrimidine intermediates 13a-d were conventionally prepared as described in our previous reported method in literatures 23, 27 in addition to the adjustment of temperature. While the anilines 16a-d were generally synthesized by using nucleophilic substitution and reduction 13

reactions. as reported in literature 23 but addition of KI to promote reaction in condition f. All these intermediates were not purified and used indirectly in the following step. The prepared 2,5-dichloropyrimidine intermediate 13a-d (1.14g, 10.0 mmol) was added in one portion to 16a-d (12 mmol) and TFA (1.29 g, 10.0 mmol) in 2-butanol (20 mL). The resulting mixture was stirred at 100 °C for 12 h. Then the mixture was evaporated, and MeOH was added. The solution was poured into saturated sodium bicarbonate solution. The residue was purified by flash chromatography separations using dichloromethane/methanol as eluents MeOH/CH2Cl2 (1:30) to yield the title molecules below. (9a)2-[[5-chloro-2-(3-methy-4-((1-morpholin)acetylamino)ylanilino)pyrimidin-4-yl]amino]-N-meth ylbenzamide (Off-white power, m.p.:119.5-121.3 oC), 1H NMR (400 MHz, DMSO-d6) δ 11.61 (s, 1H), 9.39 (s, 1H), 9.27 (s, 1H), 8.78–8.73 (m, 2H), 8.22 (s, 1H), 7.76 (dd, J = 8.0, 1.6 Hz, 1H), 7.61–7.51 (m, 2H), 7.48 (td, J = 7.6, 1.5 Hz, 1H), 7.42 (dd, J = 8.6, 2.5 Hz, 1H), 7.15 (td, J = 7.6, 1.2 Hz, 1H), 3.67 (t, J = 4.5 Hz, 4H), 3.13 (s, 2H), 2.82 (d, J = 4.5 Hz, 3H), 2.56 (t, J = 4.5 Hz, 4H), 2.18 (s, 3H).

13C

NMR (101 MHz, DMSO-d6) δ 169.39, 168.09, 158.21, 155.42, 155.13, 139.80,

137.39, 131.99, 130.69, 130.68, 128.47, 123.83, 122.34, 121.69 (2C), 121.19, 118.07, 105.35, 66.77, 62.26, 53.77, 26.78, 18.37. HRMS (ESI) for C25H28ClN7O3, [M+Na]+ calcd: 532.1834; found: 532.1849.

(9b)2-[[5-chloro-2-(3-methoxy-4-((1-morpholin)acetylamino)ylanilino)pyrimidin-4-yl]amino]-N-m ethylbenzamide (Off-white power, m.p.:137.3-139.2 oC),

14

1H

NMR (400 MHz, DMSO-d6) δ 11.65 (s, 1H), 9.54 (s, 1H), 9.42 (s, 1H), 8.79‒8.75 (m, 2H), 8.23

(s, 1H), 8.03 (d, J = 8.7 Hz, 1H), 7.76 (dd, J = 7.9, 1.6 Hz, 1H), 7.44 (t, J = 7.9 Hz, 1H), 7.38 (s, 1H), 7.28 (dd, J = 8.7, 2.2 Hz, 1H), 7.14 (td, J = 7.6, 1.2 Hz, 1H), 3.78 (s, 3H), 3.66 (t, J = 4.6 Hz, 4H), 3.12 (s, 2H), 2.81 (d, J = 4.5 Hz, 3H), 2.54 (t, J = 4.6 Hz, 4H). 13C NMR (101 MHz, DMSO-d6) δ 169.39, 167.60, 158.13, 155.38, 155.03, 148.89, 139.84, 137.01, 131.90, 128.48, 122.35, 121.87, 121.62, 121.04, 119.55, 111.89, 105.55, 103.76, 66.95, 62.13, 56.29, 53.65, 26.79. HRMS (ESI) for C25H28ClN7O4, [M+H]+ calcd: 526.1891; found: 526.1978.

(9c)2-[[5-chloro-2-(3-chloro-4-((1-morpholin)acetylamino)ylanilino)pyrimidin-4-yl]amino]-N-meth ylbenzamide (Off-white power, m.p.:230.2-231.9 oC),

1H

NMR (400 MHz, DMSO-d6) δ 11.65 (s, 1H), 9.73 (s, 1H), 9.66 (s, 1H), 8.80 (q, J = 4.5 Hz, 1H),

8.71 (d, J = 8.4 Hz, 1H), 8.27 (s, 1H), 8.03–8.00 (m, 2H), 7.77 (dd, J = 7.9, 1.6 Hz, 1H), 7.55–7.50 (m, 2H), 7.17 (td, J = 7.6, 1.2 Hz, 1H), 3.68 (t, J = 4.6 Hz, 4H), 3.17 (s, 2H), 2.82 (d, J = 4.5 Hz, 3H), 2.58 (t, J = 4.6 Hz, 4H).

13C

NMR (101 MHz, DMSO-d6): δ 169.37, 168.33, 157.78, 155.48,

155.07, 139.63, 137.92, 132.18, 128.65, 128.54, 124.07, 122.75, 122.55, 121.63, 121.34, 119.49, 118.91, 106.11, 66.88(2C), 62.02, 53.65(2C), 26.79. HRMS (ESI) for C24H25Cl2N7O3, [M+H]+ calcd:

530.1396;

found:530.1476.

(9d)2-[[5-chloro-2-(3-methy-4-((1-morpholin)acetylamino)ylanilino)pyrimidin-4-yl]amino]-N-cycl opropylbenzamide (Off-white power, m.p.:209.0-210.3 oC), 1H NMR (400 MHz, DMSO-d6) δ 11.50 (s, 1H), 9.42 (s, 1H), 9.29 (s, 1H), 8.76 (d, J = 4.3 Hz, 1H), 8.71 (d, J = 8.4 Hz, 1H), 8.23 (s, 1H), 7.72 (dd, J = 7.9, 1.6 Hz, 1H), 7.56 (d, J = 2.4 Hz, 1H), 7.54–7.46 (m, 2H), 7.42 (dd, J = 8.7,

15

2.4 Hz, 1H), 7.14 (td, J = 7.6, 1.2 Hz, 1H), 3.67 (t, J = 4.6 Hz, 4H), 3.13 (s, 2H), 3.00–2.72 (m, 1H), 2.56 (t, J = 4.6 Hz, 4H), 2.18 (s, 3H), 0.89–0.68 (m, 2H), 0.66–0.53 (m, 2H). 13C NMR (101 MHz, DMSO-d6): δ 170.31, 168.11, 158.20, 155.42, 155.17, 139.61, 137.40, 132.06, 130.72, 130.66, 128.81, 123.86, 122.32, 121.72, 121.62, 121.32, 118.00, 105.32, 66.77(2C), 62.24, 53.77(2C), 23.58, 18.39, 6.20(2C). HRMS (ESI) for C27H30ClN7O3, [M+H]+ calcd: 536.2099; found: 536.2191. (9e)2-[[5-chloro-2-(3-methoxy-4-((1-morpholin)acetylamino)ylanilino)pyrimidin-4-yl]amino]-N-cy clopropylbenzamide (Off-white power, m.p.:223.1-224.6 oC), 1H NMR (400 MHz, DMSO-d6) δ 11.54 (s, 1H), 9.55 (s, 1H), 9.43 (s, 1H), 8.86–8.59 (m, 2H), 8.24 (s, 1H), 8.04 (d, J = 8.7 Hz, 1H), 7.73 (dd, J = 7.9, 1.6 Hz, 1H), 7.45 (t, J = 7.9 Hz, 1H), 7.39 (s, 1H), 7.29 (dd, J = 8.7, 2.2 Hz, 1H), 7.13 (td, J = 7.6, 1.2 Hz, 1H), 3.78 (s, 3H), 3.67 (t, J = 4.6 Hz, 4H), 3.12 (s, 2H), 2.98–2.81 (m, 1H), 2.55 (t, J = 4.7 Hz, 4H), 0.76–0.71 (m, 2H), 0.66–0.52 (m, 2H).

13C

NMR (101 MHz, DMSO-d6):

δ 170.33, 167.61, 158.14, 155.40, 155.06, 148.90, 139.67, 137.02, 131.95, 128.82, 122.31, 121.87, 121.65, 121.18, 119.56, 111.87, 105.55, 103.73, 66.95(2C), 62.14, 56.30, 53.65(2C), 23.58, 6.19(2C). HRMS (ESI) for C27H30ClN7O4, [M+H]+ calcd: 552.2048; found: 552.2136. (9f)2-[[5-chloro-2-(4-((1-morpholin)acetylamino)ylanilino)pyrimidin-4-yl]amino]-N-ethylbenzamid e (Off-white power, m.p.:198.6-199.4 oC), 1H NMR (400 MHz, DMSO-d6) δ 11.52 (s, 1H), 9.65 (s, 1H), 9.43 (s, 1H), 8.80 (t, J = 5.6 Hz, 1H), 8.75–8.69 (m, 1H), 8.21 (s, 1H), 7.77 (dd, J = 7.9, 1.6 Hz, 1H), 7.59 (d, J = 9.1 Hz, 2H), 7.53 (d, J = 9.1 Hz, 2H), 7.51–7.45 (m, 1H), 7.16 (td, J = 7.6, 1.2 Hz, 1H), 3.65 (t, J = 4.6 Hz, 4H), 3.34–3.28 (m, 2H), 3.12 (s, 2H), 2.55–2.51 (m, 4H), 1.15 (t, J = 7.2 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 168.70, 168.06, 158.20, 155.42, 155.09, 139.71, 136.34, 133.34, 131.79, 128.54, 122.37, 121.90, 121.57, 120.40 (2C), 120.32 (2C), 105.27, 66.55 (2C),

16

62.53, 53.69 (2C), 34.58, 14.97.

HRMS (ESI) for C25H28ClN7O3, [M+H]+ calcd: 510.1942; found:

510.2042. (9g)2-[[5-chloro-2-(3-methy-4-((1-morpholin)acetylamino)ylanilino)pyrimidin-4-yl]amino]-N-ethyl benzamide (Off-white power, m.p.:209.0-211.0 oC), 1H NMR (400 MHz, DMSO-d6) δ 11.54 (s, 1H), 9.42 (s, 1H), 9.29 (s, 1H), 8.80 (t, J = 5.5 Hz, 1H), 8.73 (d, J = 8.5 Hz, 1H), 8.22 (s, 1H), 7.77 (dd, J = 7.9, 1.6 Hz, 1H), 7.56 (d, J = 2.4 Hz, 1H), 7.56–7.44 (m, 2H), 7.42 (dd, J = 8.7, 2.5 Hz, 1H), 7.16 (td, J = 7.6, 1.2 Hz, 1H), 3.67 (t, J = 4.6 Hz, 4H), 3.31 (td, J = 7.3, 5.5 Hz, 2H), 3.13 (s, 2H), 2.56 (t, J = 4.6 Hz, 4H), 2.18 (s, 3H), 1.15 (t, J = 7.2 Hz, 3H). 13C NMR (101 MHz, DMSO-d6): δ 168.74, 168.11, 158.20, 155.41, 155.15, 139.72, 137.41, 131.96, 130.71, 130.67, 128.58, 123.86, 122.37, 121.70, 121.64, 121.58, 118.01, 105.31, 66.77(2C), 62.25, 53.77(2C), 34.59, 18.39, 14.97. HRMS (ESI) for C26H30ClN7O3, [M+H]+ calcd: 524.2099; found: 524.2190.

(9h)2-[[5-chloro-2-(3-methoxy-4-((1-morpholin)acetylamino)ylanilino)pyrimidin-4-yl]amino]-N-et hylbenzamide (Off-white power, m.p.:128.9-130.1 oC), 1H NMR (400 MHz, DMSO-d6) δ 11.58 (s, 1H), 9.55 (s, 1H), 9.43 (s, 1H), 8.81 (t, J = 5.5 Hz, 1H), 8.76 (d, J = 7.5 Hz, 1H), 8.23 (s, 1H), 8.04 (d, J = 8.7 Hz, 1H), 7.77 (dd, J = 8.0, 1.6 Hz, 1H), 7.45 (t, 1H), 7.41–7.37 (m, 1H), 7.29 (dd, J = 8.7, 2.2 Hz, 1H), 7.15 (td, J = 7.6, 1.2 Hz, 1H), 3.78 (s, 3H), 3.67 (t, J = 4.5 Hz, 1H), 3.36–3.23 (m, 1H), 3.12 (s, 2H), 2.55 (t, J = 4.6 Hz, 4H), 1.15 (t, J = 7.2 Hz, 3H).

13C

NMR (101 MHz, DMSO-d6) δ

168.73, 167.60, 158.13, 155.38, 155.03, 148.89, 139.77, 137.01, 131.84, 128.58, 122.35, 121.87, 121.63, 121.42, 119.55, 111.88, 105.53, 103.74, 66.95 (2C), 62.13, 56.30, 53.65 (2C), 34.59, 14.96. HRMS (ESI) for C26H30ClN7O4, [M+H]+ calcd: 540.2048; found: 540.2149.

17

(9i)2-[[5-chloro-2-(3-chloro-4-((1-morpholin)acetylamino)ylanilino)pyrimidin-4-yl]amino]-N-ethyl benzamide (Off-white power, m.p.:234.4-235.6 oC), 1H NMR (400 MHz, DMSO-d6) δ 11.56 (s, 1H), 9.73 (s, 1H), 9.66 (s, 1H), 8.82 (t, J = 5.5 Hz, 1H), 8.76–8.62 (m, 1H), 8.27 (s, 1H), 8.11–7.89 (m, 2H), 7.78 (dd, J = 7.9, 1.6 Hz, 1H), 7.70–7.33 (m, 2H), 7.17 (td, J = 7.6, 1.1 Hz, 1H), 3.68 (t, J = 4.5 Hz, 4H), 3.37–3.24 (m, 2H), 3.17 (s, 2H), 2.58 (t, J = 4.5 Hz, 4H), 1.15 (t, J = 7.2 Hz, 3H). 13C

NMR (101 MHz, DMSO-d6) δ 168.69, 168.30, 157.77, 155.47, 155.06, 139.53, 137.91, 132.09,

128.62 (2C), 124.05, 122.73, 122.54, 121.75, 121.65, 119.46, 118.88, 106.07, 66.87 (2C), 62.01, 53.64 (2C), 34.59, 14.96. HRMS (ESI) for C25H27Cl2N7O3, [M+H]+ calcd: 544.1552; found: 544.1640.

(9j)2-[[5-chloro-2-(4-((1-morpholin)acetylamino)ylanilino)pyrimidin-4-yl]amino]-N-isopropylbenz amide (Off-white power, m.p.:222.4-224.1 oC), 1H NMR (400 MHz, DMSO-d6) δ 11.38 (s, 1H), 9.65 (s, 1H), 9.43 (s, 1H), 8.69 (d, J = 8.4 Hz, 1H), 8.56 (d, J = 7.7 Hz, 1H), 8.21 (s, 1H), 7.77 (dd, J = 7.8, 1.6 Hz, 1H), 7.59 (d, J = 9.1 Hz, 2H), 7.53 (d, J = 9.1 Hz, 2H), 7.51–7.45 (m, 1H), 7.16 (td, J = 7.5, 1.2 Hz, 1H), 4.38–3.95 (m, 1H), 3.65 (t, J = 4.6 Hz, 4H), 3.12 (s, 2H), 2.58–2.51 (m, 4H), 1.19 (s, 3H), 1.18 (s, 3H).

13C

NMR (101 MHz, DMSO-d6) δ 168.07, 158.22, 155.45, 155.10,

139.51, 136.36, 133.34, 131.68, 128.76, 122.40, 122.15, 121.97, 120.34 (2C), 120.33(2C), 105.23, 66.56(2C), 62.53, 53.69(3C), 41.59, 22.55. HRMS (ESI) for C26H30ClN7O3, [M+H]+ calcd: 524.2099; found: 524.2189.

(9k)2-[[5-chloro-2-(3-methy-4-((1-morpholin)acetylamino)ylanilino)pyrimidin-4-yl]amino]-N-isopr opylbenzamide (Off-white power, m.p.:220.0-222.0 oC), 1H NMR (400 MHz, DMSO-d6) δ 11.39 (s,

18

1H), 9.42 (s, 1H), 9.29 (s, 1H), 8.68 (d, J = 8.4 Hz, 1H), 8.57 (d, J = 7.7 Hz, 1H), 8.22 (s, 1H), 7.77 (dd, J = 7.9, 1.6 Hz, 1H), 7.57 (d, J = 2.4 Hz, 1H), 7.54–7.45 (m, 2H), 7.42 (dd, J = 8.7, 2.4 Hz, 1H), 7.16 (td, J = 7.5, 1.2 Hz, 1H), 4.43–3.95 (m, 1H), 3.67 (t, J = 4.6 Hz, 4H), 3.13 (s, 2H), 2.56 (t, J = 4.7 Hz, 4H), 2.17 (s, 3H), 1.19 (s, 3H), 1.18 (s, 3H).

13C

NMR (100 MHz, DMSO-d6) δ 168.09,

158.19, 155.41, 155.13, 139.50, 137.40, 131.83, 130.70, 130.63, 128.79, 123.85, 122.37, 122.13, 121.74, 121.58, 117.96, 105.25, 66.76(2C), 62.24, 53.76(2C), 41.58, 22.55(2C). HRMS (ESI) for C27H32ClN7O3, [M+H]+ calcd: 538.2255; found:538.2348.

(9l)2-[[5-chloro-2-(3-methoxy-4-((1-morpholin)acetylamino)ylanilino)pyrimidin-4-yl]amino]-N-iso propylbenzamide (Off-white power, m.p.:197.1-199.0 oC), 1H NMR (400 MHz, DMSO-d6) δ 11.45 (s, 1H), 9.55 (s, 1H), 9.43 (s, 1H), 8.72 (d, J = 8.3 Hz, 1H), 8.57 (d, J = 7.7 Hz, 1H), 8.23 (s, 1H), 8.03 (d, J = 8.7 Hz, 1H), 7.77 (dd, J = 7.9, 1.6 Hz, 1H), 7.45 (t, J = 7.9 Hz, 1H), 7.39 (s, 1H), 7.29 (dd, J = 8.7, 2.2 Hz, 1H), 7.15 (td, J = 7.5, 1.2 Hz, 1H), 4.33–4.08 (m, 1H), 3.78 (s, 3H), 3.67 (t, J = 4.5 Hz, 4H), 3.12 (s, 2H), 2.55 (t, J = 4.6 Hz, 4H), 1.19 (s, 3H), 1.18 (s, 3H). 13C NMR (101 MHz, DMSO-d6): δ 168.11, 167.62, 158.16, 155.41, 155.05, 148.91, 139.59, 137.04, 131.75, 128.81, 122.36, 121.97, 121.87, 121.67, 119.56, 111.86, 105.49, 103.73, 66.96(2C), 62.14, 56.31, 53.66(2C), 41.59, 22.55(2C). HRMS (ESI) for C27H32ClN7O4, [M+H]+ calcd: 554.2204; found: 554.2285.

(9m)2-[[5-chloro-2-(3-chloro-4-((1-morpholin)acetylamino)ylanilino)pyrimidin-4-yl]amino]-N-isop ropylbenzamide (Off-white power, m.p.:258.2-260.0 oC), 1H NMR (400 MHz, DMSO-d6) δ 11.42 (s, 1H), 9.72 (s, 1H), 9.66 (s, 1H), 8.64 (d, J = 8.2 Hz, 1H), 8.58 (d, J = 7.7 Hz, 1H), 8.27 (s, 1H),

19

8.09–7.87 (m, 2H), 7.77 (dd, J = 7.9, 1.5 Hz, 1H), 7.62–7.40 (m, 2H), 7.18 (t, J = 7.7 Hz, 1H), 4.17–4.08 (m, 1H), 3.68 (t, J = 4.5 Hz, 4H), 3.17 (s, 2H), 2.58 (t, J = 4.6 Hz, 4H), 1.19 (s, 3H), 1.17 (s, 3H). 13C NMR (101 MHz, DMSO-d6): δ 168.30, 168.04, 157.77, 155.49, 155.06, 139.31, 137.91, 131.98, 128.83, 128.61, 124.05, 122.73, 122.56, 122.31, 121.71, 119.43, 118.85, 106.01, 66.87(2C), 62.01, 53.64(2C), 41.59, 22.54(2C). HRMS (ESI) for C26H29Cl2N7O3, [M+H]+ calcd: 558.1709; found: 558.1799.

4.3 In vitro kinase enzymatic assay The ADP-GloTM Kinase enzyme system (FAK Catalog. V9301;

EGFRT790M/L858R Catalog.

V5324 Promega), purchased from Promega Corporation (USA), was used for in vitro enzymatic assay. Concentrations consisting of suitable levels from 1 nM, 10 nM, 100 nM, 500 nM to 1 μM 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-syste m-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 min, (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. 20

4.4 Cellular activity assay All the cancer cell lines 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 3,000 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 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. The IC50 values were calculated using GraphPad Prim version 5.0. 4.5 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 9a (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).

21

4.6 Mouse tumour xenograft efficacy study The female BALB/cJNju-Foxn1nu/Nju mice weighing 20–25 g in 5–6 week-old were purchased from the Model Animal Research Center of Nanjing University, Nanjing, China. All animals were housed in a controlled environment at 23±2 °C 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 0.1mL of PBS and injected subcutaneously into the right oxter region of the mice. Three days after implantation, the mice were randomly divided into seven groups in which the mice in control group were received by oral 25% PEG-400, and the mice in other six groups were oral administrated with 9a at the doses of 30 and 60 mg/kg for 10 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. Acknowledgement We are grateful to the National Natural Science Foundation of China (No. 81603186), National Natural Science Foundation of Liaoning (No. 3512942), Research Fund of Higher Education of Liaoning province（LQ2017039, LQ2017008）for the financial support of this research. Reference [1] Q.J. Lin, F. Yang, C. Jin, D.L. Fu, Current status and progress of pancreatic cancer in China, World J. Gastroenterol. 21 (2015) 7988−8003. [2] W. Chen, K. Sun, R. Zheng, H. Zeng, S. Zhang, C. Xia, Z. Yang, H. Li, X. Zou, J. He, Cancer 22

incidence and mortality in China, 2014, Chin. J. Cancer Res. 30 (2018) 1−12. [3] J.J. Cui, A new challenging and promising era of tyrosine kinase inhibitors, ACS Med. Chem. Lett. 5 (2014) 272−274. [4] S.H. Raval, R.D. Singh, D.V. Joshi, H.B. Patel, S.K. Mody, Recent developments in receptor tyrosine kinases targeted anticancer therapy, Vet. World 9 (2016) 80−90. [5] G. Manning, D.B. Whyte, R. Martinez, T. Hunter, S. Sudarsanam, The protein kinase complement of the human genome, Science 298 (2002) 1912−1934. [6] V.M. Golubovskaya, Targeting FAK in human cancer: from finding to first clinical trials, Front Biosci (Landmark Ed). 19 (2014) 687−706. [7] M. Roy-Luzarraga, K. Hodivala-Dilke, Molecular pathways: Endothelial cell FAK-A target for cancer treatment, Clin. Cancer Res. 22 (2016) 3718−3724. [8] Y.L. Tai, L.C. Chen, T.L. Shen, Emerging roles of focal adhesion kinase in cancer, Biomed Res. Int. 2015 (2015) 1−13. [9] F.J. Sulzmaier, C. Jean, D.D. Schlaepfer, FAK in cancer: mechanistic findings and clinical applications, Nat. Rev. Cancer 14 (2014) 598−610. [10] Q. Shi, A.B. Hjelmeland, S.T. Keir, L. Song, S. Wickman, D. Jackson, O. Ohmori, D.D. Bigner, H.S. Friedman, J.N. Rich, A novel low ‐ molecular weight inhibitor of focal adhesion kinase, TAE226, inhibits glioma growth, Mol. Carcinog. 46 (2007) 488−496. [11] G.R. Ott, M. Cheng, K.S. Learn, J. Wagner, D.E. Gingrich, J.G. Lisko, M. Curry, E.F. Mesaros, A.K. Ghose, M.R. Quail, Discovery of clinical candidate CEP-37440, a selective inhibitor of focal adhesion kinase (FAK) and anaplastic lymphoma kinase (ALK), J. Med. Chem. 59 (2016)

23

7478−7496. [12] T. Shimizu, K. Fukuoka, M. Takeda, T. Iwasa, T. Yoshida, J. Horobin, M. Keegan, L. Vaickus, A. Chavan, M. Padval, K. Nakagawa, A first-in-Asian phase 1 study to evaluate safety, pharmacokinetics and clinical activity of VS-6063, a focal adhesion kinase (FAK) inhibitor in Japanese patients with advanced solid tumors, Cancer Chemother. Pharmacol. 77 (2016) 997−1003. [13] Y. Kang, W. Hu, C. Ivan, H.J. Dalton, T. Miyake, C.V. Pecot, B. Zand, T. Liu, J. Huang, N.B. Jennings, Role of focal adhesion kinase in regulating YB–1–mediated paclitaxel resistance in ovarian cancer, J. Natl. Cancer Inst. 105 (2013) 1485−1495. [14] S. Fang, Z. Wang, EGFR mutations as a prognostic and predictive marker in non-small-cell lung cancer, Drug Des. Devel. Ther. 8 (2014) 1595−1611. [15] R.M. Jotte, D.R. Spigel, Advances in molecular‐based personalized non‐small‐cell lung cancer therapy: targeting epidermal growth factor receptor and mechanisms of resistance, Cancer Med. 4 (2015) 1621−1632. [16] S. Kobayashi, T.J. Boggon, T. Dayaram, P.A. Jänne, O. Kocher, M. Meyerson, B.E. Johnson, M.J. Eck, D.G. Tenen, B. Halmos, EGFR mutation and resistance of non–small-cell lung cancer to gefitinib, N. Engl. J. Med. 352 (2005) 786−792. [17] E.L. Kwak, R. Sordella, D.W. Bell, N. Godin-Heymann, R.A. Okimoto, B.W. Brannigan, P.L. Harris, D.R. Driscoll, P. Fidias, T.J. Lynch, Irreversible inhibitors of the EGF receptor may circumvent acquired resistance to gefitinib, Proc. Natl. Acad. Sci. U S A. 102 (2005) 7665−7670. [18] W. Zhou, D. Ercan, L. Chen, C.H. Yun, D. Li, M. Capelletti, A.B. Cortot, L. Chirieac, R.E. Iacob, R. Padera, Novel mutant-selective EGFR kinase inhibitors against EGFR T790M, Nature

24

462 (2009) 1070−1074. [19] A.O. Walter, R.T. Sjin, H.J. Haringsma, K. Ohashi, J. Sun, K. Lee, A. Dubrovskiy, M. Labenski, Z. Zhu, Z. Wang, Discovery of a mutant-selective covalent inhibitor of EGFR that overcomes T790M-mediated resistance in NSCLC, Cancer Discov. 3 (2013) 1404−1415. [20] D.A. Cross, S.E. Ashton, S. Ghiorghiu, C. Eberlein, C.A. Nebhan, P.J. Spitzler, J.P. Orme, M.R. Finlay, R.A. Ward, M.J. Mellor, AZD9291, an irreversible EGFR TKI, overcomes T790M-mediated resistance to EGFR inhibitors in lung cancer, Cancer Discov. 4 (2014) 1046−1061. [21] M.R. Finlay, M. Anderton, S. Ashton, P. Ballard, P.A. Bethel, M.R. Box, R.H. Bradbury, S.J. Brown, S. Butterworth, A. Campbell, Discovery of a potent and selective EGFR inhibitor (AZD9291) of both sensitizing and T790M resistance mutations that spares the wild type form of the receptor, J. Med. Chem. 57 (2014) 8249−8267. [22] Z. Song, Y. Jin, Y. Ge, C. Wang, J. Zhang, Z. Tang, J. Peng, K. Liu, Y. Li, X. Ma, Synthesis and biological evaluation of azole-diphenylpyrimidine derivatives (AzDPPYs) as potent T790M mutant form of epidermal growth factor receptor inhibitors, Bioorg. Med. Chem. 24 (2016) 5505−5512. [23] Z. Song, S. Huang, H. Yu, Y. Jiang, C. Wang, Q. Meng, X. Shu, H. Sun, K. Liu, Y. Li, Synthesis and biological evaluation of morpholine-substituted diphenylpyrimidine derivatives (Mor-DPPYs) as potent EGFR T790M inhibitors with improved activity toward the gefitinib-resistant non-small cell lung cancers (NSCLC), Eur. J. Med. Chem. 133 (2017) 329−339. [24] Z. Song, Y. Ge, C. Wang, S. Huang, X. Shu, K. Liu, Y. Zhou, X. Ma, Challenges and

25

Perspectives on the Development of Small-Molecule EGFR Inhibitors against T790M-Mediated Resistance in Non-Small-Cell Lung Cancer, J. Med. Chem. 59 (2016) 6580−6594. [25] X. Lu, L. Yu, Z. Zhang, X. Ren, J.B. Smaill, K. Ding, Targeting EGFRL858R/T790M and EGFRL858R/T790M/C797S resistance mutations in NSCLC: Current developments in medicinal chemistry, Med. Res. Rev. 38 (2018) 1550−1581. [26] Y. Ge, C. Wang, S. Song, J. Huang, Z. Liu, Y. Li, Q. Meng, J. Zhang, J. Yao, K. Liu, X. Ma, X. Sun, Identification of highly potent BTK and JAK3 dual inhibitors with improved activity for the treatment of B-cell lymphoma, Eur. J. Med. Chem. 143 (2018) 1847−1857. [27] M. Qu, Z. Liu, D. Zhao, C. Wang, J. Zhang, Z. Tang, K. Liu, X. Shu, H. Yuan, X. Ma, Design, synthesis and biological evaluation of sulfonamide-substituted diphenylpyrimidine derivatives (Sul-DPPYs) as potent focal adhesion kinase (FAK) inhibitors with antitumor activity, Bioorg. Med. Chem. 25 (2017) 3989−3996. [28] R. Somberg, B. Pferdehirt, K. Kupcho, Kinase-Glo Luminescent Kinase Assay: Detect virtually any kinase, Cell Notes 5 (2003) 5−8. [29] H. Zegzouti, M. Zdanovskaia, K. Hsiao, S.A. Goueli, ADP-Glo: a Bioluminescent and homogeneous ADP monitoring assay for kinases, Assay Drug Dev. Technol. 7 (2009) 560−572. [30] L. Śliwka, K. Wiktorska, P. Suchocki, M. Milczarek, S. Mielczarek, K. Lubelska, T. Cierpiał, P. Łyżwa, P. Kiełbasiński, A. Jaromin, The comparison of MTT and CVS assays for the assessment of anticancer agent interactions, PloS one. 11 (2016) e0155772.

26

Graphical Abstract

6 50

lu

G

O N HN

N

O

HN

Asp546 HN

N

NH Fregment-based drug design

N

Cl Mor-DPPY EGFR

T790M

R

HN

O

HN Cl

N

OMe

Cl

N

N

NH N

Cys502 DPPYs 9a-m FAK and EGFRT790M dual inhibitor

O

HN

O R1

H N

inhibitor

FAK binding sites MeHN

9 42 ly

O

O

O

/G

EGFRT790M binding sites

NH N

TAE226 FAK inhibitor

27

Highlights  Novel Pyrimidines as potential dual FAK and EGFRT790M inhibitors.  9a, 9f were the strongest FAK and EGFRT790M inhibitors (IC50 < 7.13 nM)  9a exhibited strong antiproliferative activity against various cancer cells.  9a was effective in the in vivo assessment conducted in AsPC-1 cell xenograft mouse model.

28

We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.

29