PF-562271 – SYK signal

Synthesis and evaluation of FAK inhibitors with a 5-ﬂuoro-7H-pyrrolo [2,3-d]pyrimidine scaffold as anti-hepatocellular carcinoma agents
Hanyi Tan a, Yue Liu a, Chaochao Gong a, Jiawei Zhang a, Jian Huang b, **, Qian Zhang a, *
a Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai, 201203, China
b Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Centre for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai, 200240, China

A R T I C L E I N F O

Article history:
Received 16 April 2021 Received in revised form 18 June 2021
Accepted 22 June 2021
Available online 25 June 2021

Keywords:
Focal adhesion kinase (FAK) Hepatocellular carcinoma
5- Fluoro-7H-pyrrolo[2,3-d]pyrimidine derivatives
Small molecule inhibitors

A B S T R A C T

Focal adhesion kinase (FAK) is a ubiquitous intracellular non-receptor tyrosine kinase, which is involved in multiple cellular functions, including cell adhesion, migration, invasion, survival, and angiogenesis. In this study, a series of 7H-pyrrolo[2,3-d]pyrimidines were designed and synthesized according to the E- pharmacophores generated by docking a library of 667 fragments into the ATP pocket of the co-crystal complex of FAK and PF-562271 (PDB ID: 3BZ3). The 5-ﬂuoro-7H-pyrrolo[2,3-d]pyrimidine derivatives demonstrated excellent activity against FAK and the cell lines SMMC7721 and YY8103. 2-((2-((3-(Acet- amidomethyl)phenyl)amino)-5-ﬂuoro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-methylbenzamide (16c) was selected for further bioactivity evaluations in vivo, including preliminary pharmacokinetic proﬁling in rats and toxicity assays in mice, and tumor growth inhibition studies in a xenograft tumor model. The results showed that 16c did not affect the body weight gain of the animals up to a dose of 200 mg/kg, and signiﬁcantly inhibited tumor growth with a tumor growth inhibition rate of 78.6% compared with the negative control group. Furthermore, phosphoantibody array analyses of a sample of the tumor suggested that 16c inhibited the malignant proliferation of hepatocellular carcinoma (HCC) cells through decreasing the phosphorylation in the FAK cascade.
© 2021 Elsevier Masson SAS. All rights reserved.

1. Introduction

Focal adhesion kinase (FAK) is a ubiquitous intracellular non- receptor tyrosine kinase that localizes in focal adhesions, where the cell membrane attaches to the extracellular matrix [1]. FAK plays a prominent role in integrin-mediated signal transduction and is involved in multiple cellular functions, such as cell adhesion [2], migration [3], invasion [4], survival [5], and angiogenesis [6,7]. Consistent with these cellular functions, ample evidence has indi- cated that FAK is over-expressed in various types of tumors [8e10], including in thyroid [11], ovarian [12], prostate [13], and oral [14] cancers.
Therefore, FAK is a promising target for oncology [15,16], and several FAK small molecule inhibitors have been reported and investigated [17e20]. Some inhibitors, including PF-562271 [21],

* Corresponding author. ;
** Corresponding author.
E-mail addresses: [email protected] (J. Huang), [email protected]. cn (Q. Zhang).

GSK 2256098 [22], VS-6063 [23] (also known as defactinib and PF- 04554878), and PND-1186 [24], are already undergoing clinical trials (Fig. 1).
Hepatocellular carcinoma (HCC) is the third highest cause of cancer deaths worldwide, with exceedingly high rates in East and Southeast Asia [25]. Unfortunately, there are no speciﬁc drugs for liver cancer clinically available, except for soraﬁnib (Nexavar), which is a multitarget inhibitor of B-raf, C-raf, PDGFR, and VEGFR 2/ 3 and was approved for the treatment of advanced hepatocellular carcinoma by the US FDA in 2005 [26]. Fujii et al. have found that FAK mRNA was overexpressed in HCC cells compared with the corresponding non-cancerous liver tissue, and FAK can act as an independent prognostic factor [27]. FAK can promote the renewal and drug resistance of cancer stem cells (CSCs) [28]. Sun and co- workers have reported that FAK and the extracellular signal- regulated kinase (ERK1/2) pathway are involved in regulating the growth and metastasis of liver cancer stem cells (LCSCs). Using the anticancer drug salinomycin to inhibit the activity of FAK and ERK1/
2 resulted in increased stiffness of LCSCs [29,30]. Herein, we describe our attempts to develop FAK inhibitors as anti-HCC agents.

https://doi.org/10.1016/j.ejmech.2021.113670
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

Fig. 1. Structures of FAK inhibitors that have entered into phase III trials.

2. Results and discussion

2.1. Chemistry and in vitro bio-evaluations

To develop FAK inhibitors with novel scaffolds, we explored pharmacophores in the ATP pocket of the FAK protein via Glide software (Schrodinger). A library of 667 fragments was docked based on the co-crystal complex of FAK and PF-562271 (PDB ID: 3BZ3) [31], and 1700 active conformations were found. E-phar- macophores were used to generate simulated pharmacophores of the ATP pocket based on the optimal interactions between the fragments and FAK. As shown in Fig. 2, in the hinge region, three main pharmacophores were found, a hydrogen bond acceptor pharmacophore, a hydrogen bond donator pharmacophore, and a ring pharmacophore, denoted as A1, D23, and R60, respectively. A1 could overlap with the nitrogen atom at the 1-position of the py- rimidine motif of PF-562271, which is known to form a hydrogen bond interaction with Cys502. However, D23, which is located around Glu500 and the gatekeeper residue Met499, and R60, are not covered by PF-562271.
Presuming that a ﬁve-membered nitrogen-containing ring fused with pyrimidine could partially occupy the region of R60, and provide the donator pharmacophore (D23), simultaneously, two types of ring scaffolds, namely 7H-pyrrolo[2,3-d]pyrimidine and 1H-pyrazolo[3,4-d]pyrimidine, were designed, in which the hydrogen of NH could interact with Glu500, and the carbon atom at 5-position could take up the space of triﬂuoromethyl group of PF- 562271. As shown in Scheme 1, compounds 7 and 8 were

designed and prepared, which maintained the same substituent groups as PF-562271, to assess the efﬁcacy of the fused cores. 2,4- Dichloro-7H-pyrrolo[2,3-d]pyrimidine (3) was synthesized from 6-aminouracil (1), which was treated with chloroacetaldehyde to provide pyrrolo[2,3-d]pyrimidine-2,4-diol (2) in an excellent yield of 93%, and then the hydroxyl groups were replaced with chlorine atoms using phosphorus oxychloride according to the literature method [32]. 4,6-Dichloro-1H-pyrazolo[3,4-d]pyrimidine (4) is commercially available. The target compounds 7 and 8 were ob- tained through two steps of nucleophilic substitution of 3 or 4, respectively, reﬂuxing with N-(3-(aminomethyl)pyridine-2-yl)-N- methylmethanesulfonamide in EtOH, and sequentially heating
with 5-aminoindolin-2-one by microwave irradiation at 150 ◦C
[33e36].
The inhibitory activity against FAK kinase of compounds 7 and 8 was evaluated in vitro. The assay data showed that both the com- pounds had potent activity with IC50 values for compounds 7 and 8 of 55 and 206 nM, respectively (Table 1), which indicated that compound 7 was 4-fold more effective than 8.
The molecular docking simulation of 7 with the FAK kinase domain (Fig. 3) suggested that 7 possibly adopts a similar position to PF-562271 in 3BZ3. Two essential hydrogen bonds targeting the hinge region were conserved, as in the interaction of PF-562271 with the protein, which were formed by the nitrogen atom at the 1-position, and the amino group of the substituent at the 2- position, of the pyrimidine scaffold with Cys502. As expected, the hydrogen atom of NH of the introduced fused pyrrole ring formed an additional hydrogen bond with the backbone of Glu500. In

Fig. 2. (A) The co-crystal structure of PF-562271 and FAK (PDB ID: 3BZ3) and (B) the simulated pharmacophores of FAK inhibitors in the ATP pocket.

Scheme 1. Synthetic route for 7H-pyrrolo[2,3-d]pyrimidine derivative (7) and 1H-pyrazolo[3,4-d]pyrimidine derivative (8). Reagents and conditions: (a) H2O, 80 ◦C, 2 h; (b) POCl3, DIPEA, toluene, 106 ◦C, overnight. (c) N-(3-(aminomethyl)pyridine-2-yl)-N-methylmethanesulfonamide, DIPEA, EtOH, reﬂux, 1 h; (d) 5-aminoindolin-2-one, TsOH, n-BuOH, MW 150 ◦C, 3 h.

Table 1
Kinase inhibitory activity of compounds 7 and 8 in vitro.

Compd. IC50 (nM)

7 55
8 206
PF-562271 1.5

addition, the pyrrole ring was oriented toward the gatekeeper residue to form a hydrophobic interaction with Met 499, which might contribute to the lower IC50 value of 7.
Therefore, structure optimization was performed based on the 7H-pyrrolo[2,3-d]pyrimidine scaffold of 7. First, ﬁve derivatives with different substituents at the 5- or 6-position were designed and prepared to explore the impact of the groups on the pyrrole ring on the activity.
As shown in Scheme 2, the synthetic strategy was similar to the preparation of 7. First, a series of substituted 2,4-dichloro-7H-pyr- rolo[2,3-d]pyrimidines (3a-e) was synthesized. 2,4-Dichloro-5- methyl- and 2,4-dichloro-6-methyl-7H-pyrrolo[2,3-d]pyrimidine

(3a and 3b) were obtained via the condensation of 6-aminouracil
(1) with 2-chloropropyl aldehyde and chloroacetone, respectively, followed by chlorination of the hydroxyl groups by phenyl- phosphonic dichloride. 2,4-Dichloro-5-ﬂuoro- (3c), 2,4,5-trichloro- (3d), and 2,4-dichloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine (3e) were prepared by the electrophilic substitution of 2,4-dichloro-7H- pyrrolo[2,3-d]pyrimidine (3) with the halogenating agents, Selectﬂuor, N-chlorosuccinimide, and N-iodosuccinimide, respec- tively. Intermediates 3a-d were individually reﬂuxed with N-(3- (aminomethyl)pyridine-2-yl)-N-methylmethanesulfonamide in EtOH to provide 5a-d, which were reacted with 5-aminoindolin-2- one under nucleophilic substitution, microwave, or typical Buchwald-Hartwig coupling conditions to afford 7a-d [36,37].
The 5-cyano analog (7e) was synthesized using 2,4-dichloro-5- iodo-7H-pyrrolo[2,3-d]pyrimidine (3e) as the starting material (Scheme 3). The NH of the pyrrole was protected by a tosyl group, followed by substitution of the 4-chlorine atom by N-(3-(amino- methyl)pyridine-2-yl)-N-methylmethanesulfonamide to afford compound 10. The iodine atom was displaced by a cyano group

Fig. 3. Schematic representation of the proposed binding mode for compound 7 with FAK active site (PF-562271: green line; PDB ID: 3BZ3).

Scheme 2. Synthetic route for 5- or 6-substituted 7H-pyrrolo[2,3-d]pyrimidines 7a-d. Reagents and conditions: (a) 2-chloropropylaldehyde or chloroacetone, H2O, reﬂux, 24e72 h; (b) PhPOCl2, 165 ◦C, 12 h; (c) Selectﬂour, AcOH, MeCN, 70 ◦C, overnight; (d) NCS, DCM, THF, MW 90 ◦C, 4 h; or NIS, DCM, rt, overnight; (e) N-(3-(aminomethyl)pyridine-2- yl)-N-methylmethanesulfonamide, DIPEA, EtOH, reﬂux, overnight; (f) 5-aminoindolin-2-one, K2CO3, Pd2(dba)3, Xphos, t-BuOH, 100 ◦C, 12 h; (g) 5-aminoindolin-2-one, TsOH, n- BuOH, MW 150 ◦C, 5 h.

using Zn(CN)2 catalyzed by Pd2(dba)3 [38,39]. After removing the protecting group with tetrabutylammonium ﬂuoride, another side

chain at the 2-position was introduced via heating a mixture of 5e
and 5-aminoindolin-2-one in EtOH under microwave irradiation at

150 ◦C for 5 h according to the method described above.
The inhibitory activity against the FAK protein, and the anti- proliferative activity against two hepatic carcinoma cell lines, YY8103 and SMMC7721, of the synthesized derivatives 7a-e were evaluated using PF-562271 as reference compound and are shown in Table 2. YY8103 and SMMC7721 cell lines were chose due to that a number of literatures reported that FAK is over-expressed in SMMC7721 cells [40,41] and YY8103 cell line was established from a patient at operation in China by Dr. Ma Z. and co-workers [42]. The enzymatic data indicated that the introduction of a methyl or a halogen group to the 5-position resulted in similar or enhanced activity compared with the lead compound 7. 7a (5-methyl) was as potent as the lead compound (7), and the derivatives 7c (5-ﬂuoro) and 7d (5-chloro) both had a 2-fold higher inhibitory activity than compound 7. In contrast, the introduction of a methyl group to the 6-position (7b) resulted in a considerable decrease in activity, suggesting that the methyl group in this position is too large for the space between the 7H-pyrrolo[2,3-d]pyrimidine scaffold and the gatekeeper residue Met 499, which might lead to a steric clash with this residue. The fact that compound 7e, with a cyano group at the 5-position, showed decreased inhibitory activity compared with 7a, 7c, and 7d, implied that a hydrophobic group was more favorable than a hydrophilic group at this position.
The anti-proliferation activity was almost consistent with the
results of the FAK assay, compounds 7b and 7e both showed low activity in the cell proliferation assay. Interestingly, the inhibitory activity of 7c and 7d was approximately 14 times lower than that of PF-562271, but the cytotoxic activity was almost the same as PF- 562271. We attributed the improved cellular potency of 7, 7a, 7c and 7d to the fused pyrrole ring, which could enhance the lipid solubility and membrane permeability of these compounds.
Considering the merits of ﬂuorine for drug metabolism, further structure optimization was conducted using the 5-ﬂoro-7H-pyrrolo [2,3-d]pyrimidine (7c) scaffold with modiﬁcation of the side chains at the 2- and/or 4-position. The molecular docking study showed that 7c interacted with FAK active site in a similar orientation to

compound 7 (see Supporting Information). The side chain of 2- position, (2-oxoindolin-5-yl)amino-group, extends into the sol- vent region and pyridin-2-ylmethylamino-group at the 4-position forms a hydrogen bond interaction with Asp564. The moiety of 2- oxoindolin-5-yl is relative rigid. Herein, derivatives 12a-m were designed in order to open the lactam ring, retaining the substituent group at the 4-position of 7c at the same time. Subsequently, pyr- idin-2-ylmethylamino-group at the 4-position was replaced by a 2- (methylcarbamoyl)phenyl)amino-group for the purpose of improving the lipid solubility of the aimed compounds, which resulted in derivatives 16a-d.
As shown in Schemes 4, Derivatives 12a-m were obtained from the reaction of 5c with various anilines under Buchwald-Hartwig coupling conditions as described for the synthesis of 7c. The yields ranged from 11% to 43%. Derivatives 16a-d were synthesized from 3c using a similar procedure as above (Schemes 5), except for the protection and the deprotection steps. The nitrogen atom in the pyrrole ring of 3c was sulfonylated by stirring the substrate with p- toluenesulfonyl chloride, diethylamine, and dimethylaminopyr- idine in dichloromethane at room temperature to provide 13, which was then reacted with 2-amino-N-methylbenzamide, un- dergoing nucleophilic substitution to introduce the side chain at the 4-position (14). After removal of the protecting group by TBAF, the desired products (16a-d) were obtained via the Buchwald- Hartwig reaction of 15 with different anilines [37].
The data for the 5-ﬂuoro-7H-pyrrolo[2,3-d]pyrimidine de- rivatives 12a-m and 16a-d in the enzymatic and cellular assays are listed in Table 3. The enzyme inhibitory activity of 12a-m showed that replacement of the 5-aminoindolin-2-one of 7c with aniline (12a) or electron-withdrawing group substituted anilines (12bed) resulted in a slight decrease in activity in comparison with 7c. In contrast, when methoxy (12e-f), amido (12i-j), N-methylpiperazine (12k), or morpholine (12l-m) groups were introduced into the aniline at the 2-position, the inhibitory activity toward FAK was maintained or increased, indicating that it was necessary for potent derivatives to possess a hydrogen-bond acceptor at the meta- or

Scheme 3. Synthetic route for 5-cyan-7H-pyrrolo[2,3-d]pyrimidine (7e). Reagents and conditions: (a) TsCl, TEA, DMAP, DCM, rt, 1 h; (b) N-(3-(aminomethyl)pyridine-2-yl)-N- methylmethanesulfonamide, TEA, MeCN, rt, 2 h; (c) Zn(CN)2, Pd2(dba)3, dppf, Zn, Zn(OAc)2, 70 ◦C, 2.5 h; (d) TBAF, THF, rt, 15 min; (e) 5-aminoindolin-2-one, TsOH, n-BuOH, MW 150 ◦C, 5 h.

Table 2
Enzyme inhibition and anti-proliferative activity of 7H-pyrrolo[2,3-d]pyrimidine derivatives 7 and 7a-e.

Compd. X Y Kinase assay IC50 (nM) YY8103 IC50 (mM) SMMC7721 IC50 (mM)
7 H H 55 13.55 ± 2.44 52.79 ± 3.05
7a CH3 H 64 12.43 ± 2.98 18.70 ± 1.46
7b H CH3 12000 >100 >100
7c F H 22 24.09 ± 4.62 73.15 ± 8.47
7d Cl H 21 13.25 ± 4.07 21.89 ± 0.23
7e CN H 325 >100 >100
PF-562271 1.5 8.59 ± 1.37 23.92 ± 3.74

para-position of the aniline, which could form interactions with residues in the solvent region. The fact that the activity of the de- rivative with a m-aminomethylaniline group (12h) was reduced dramatically compared with that of the N-acetylated derivative (12i) suggested that a free aliphatic amino group is detrimental to the bioactivity possibly because the basicity of this amino group is

relative strong and could be saliﬁed as ammonium under the test conditions.
In the other series, replacing the pyridine-2-yl-N-methyl- methanesulfonamide group with 2-amino-N-methylbenzamide (compounds 16a and 16d) resulted in almost the same inhibitory activity as the corresponding compounds 7c and 12m. In addition,

Scheme 4. Synthesis of 5-ﬂuoro-7H-pyrrolo[2,3-d]pyrimidine derivatives 12a-m.

Scheme 5. Synthesis of 5-ﬂuoro-7H-pyrrolo[2,3-d]pyrimidine derivatives 16a-d. Reagents and conditions: (a) TsCl, TEA, DMAP, DCM, rt, 2 h; (b) 2-amino-N-methylbenzamide, DIPEA, MeCN, reﬂux, 40 h; (c) TBAF, THF, reﬂux, 12 h; (d) aniline, Pd2(dba)3, Xphos, K2CO3, t-BuOH, 100 ◦C, 15 h.

Table 3
Enzyme inhibition and anti-proliferative activity of 5-ﬂuoro-7H-pyrrolo[2,3-d]py- rimidine derivatives.

Compd. Kinase assay IC50 (nM) YY8103 IC50 (mM) SMMC7721 IC50 (mM)
7c 22 24.09 ± 4.62 73.15 ± 8.47
12a 32 >100 >100
12b 38 >100 27.93 ± 2.22
12c 41 82.52 ± 4.12 >100
12d 35 >100 >100
12e 15 18.25 ± 2.49 54.75 ± 11.27
12f 17 >100 >100
12g 76 >100 >100
12h 2098 >100 >100
12i 23 20.54 ± 2.79 21.73 ± 3.23
12j 21 14.44 ± 4.23 15.96 ± 0.86
12k 13 2.39 ± 0.59 13.86 ± 3.11
12l 26 >100 >100
12m 23 15.38 ± 1.67 15.28 ± 3.42
16a 22 4.69 ± 0.67 10.04 ± 2.59
16b 14 1.06 ± 0.30 18.71 ± 3.60
16c 12 2.39 ± 0.51 10.07 ± 1.70
16d 23 14.25 ± 3.13 33.86 ± 6.61
PF-562271 1.5 8.59 ± 1.37 23.92 ± 3.74

16b and 16c displayed a one-fold increase in inhibitory activity compared with the corresponding compounds 12i and 12j.
In the anti-proliferative activity assay, compounds 12k, 16a, 16b, and 16c, which had FAK IC50 values of approximately 10 nM, also showed greater anti-proliferation potency than PF-562271. How- ever, for some derivatives, such as 12e and 12f, the anti-FAK and anti-proliferative activities were not consistent. We presumed that the substituents at the 2-, and 4-position of the 5-ﬂuoro-7H-pyr- rolo[2,3-d]pyrimidine core are critical for the chemico-physical properties, particularly for the membrane permeability of the derivatives.
The molecular docking study of compounds 12k and 16c in the ATP-binding site of FAK was also performed to compare and elucidate their interaction modes. The orientation and binding model of 12k was almost as same as those of PF-562271, 7 and 7c (see Supporting Information). For 16c, three hydrogen bonds be- tween 5-ﬂuoro-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine core and the hinge motif of FAK were kept as above compounds. As shown in Fig. 4, when the side chain at the 4-position was changed as a phenylamino-group, the location of 16c was shifted a little far away

from the gatekeeper Met499, the direction of benzene ring was different from the pyridine of PF-567721 and the N-H of the methyl carbamoyl group at the benzene ring formed a hydrogen bond interaction with Glu506.

2.2. Preliminary pharmacokinetic proﬁling and toxicity study of 16c in vivo

16c was selected for further bioactivity evaluations in vivo due to its potent activity in enzymatic and cellular assays. The pharma- cokinetics and oral bioavailability of 16c were investigated in rat following single intravenous (IV) and oral gavage (PO). After the test compound was injected intravenously 2 mg/kg, the mean volume of distribution at steady state (Vss) was 1842.6 mL/kg, the mean blood clearance was 869.4 mL/min/kg, and the mean half-life was 1.46 h (Table 4). The pharmacokinetics were suboptimal but acceptable. After 16c was administrated intragastrically 10 mg/kg, the mean half-life was extended to 3.65 h, however, the mean oral bioavailability was only 0.83% estimated based on AUC0-t. The re- sults indicated that this compound is unsuitable for oral adminis- tration, and additional medicinal chemistry efforts should be made to optimize this molecule, focusing on three critical factors: metabolic stability, solubility, and/or permeability in our future research.
The acute toxicity of 16c was tested in Kunming mice via intraperitoneal administration. The animals were separated into ﬁve groups with three males and three females in each group and were administered the test compound intraperitoneally at 0, 5, 10, 50, and 200 mg/kg, respectively (n 6). After administration, the animals were observed and weighed for 10 days. The average body weight of all the groups declined slightly on the ﬁrst day, then increased gradually. At the 10th day, the average body weight values of these ﬁve groups were almost the same (Fig. 5), indicating that the test compound (16c) was safe and had low toxicity in vivo.

2.3. Tumor growth inhibition study of 16c in nude mice bearing SMMC7721 xenografts

The antitumor efﬁcacy of compound 16c was evaluated by the tumor volumes of nude mice bearing SMMC7721 xenografts (Fig. 6A). The doses of 16c were 10 and 30 mg/kg, soraﬁnib and PF-

Fig. 4. Schematic representation of the proposed binding mode for compound 16c with FAK active site (16c: yellow stick; PF-562271; green line; PDB ID: 3BZ3).

Table 4
In vivo pharmacokinetic proﬁles of 16c.

Parameters p.o. (10 mg/kg) i.v. (2 mg/kg)

t1/2 (hr) 3.65 ± 0.48 1.46 ± 0.26
tmax (hr) 4.67 ± 1.15 0.08 ± 0.00
Cmax (ng/mL) 15.38 ± 1.22 5023.97 ± 263.02
AUC0—t (ng*hr/mL) 95.50 ± 17.09 2298.79 ± 115.22
AUC0—∞ (ng*hr/mL) 118.94 ± 24.44 2304.10 ± 112.60
Vss (mL/kg) NA 1842.6 ± 423.51
Cl (mL/hr/kg) NA 869.41 ± 42.87
MRT0-t (hr) 4.54 ± 0.21 0.51 ± 0.01
MRT0-∞ (hr) 6.62 ± 0.37 0.53 ± 0.03
Bioavailability (%) 0.83 ± 0.15

t1/2, half-life; tmax, time to reach the maximum plasma concentration; Cmax, maximum plasma concentration; AUC, area under the plasma concentration—time curve; MRT, mean residence time; Vss, volume of distribution at steady state; Cl, blood clearance; NA, Not applicable; n ¼ 3, mean ± SEM.

Fig. 5. The average body weight of mice after intraperitonal injection of compound
16c.

562271 (30 mg/kg) were used as positive controls, and the solvent was used as a negative control (n 8). All samples were injected intraperitoneally every 3 days, after measurement of the size of the

tumor and the body weight. The tumor sizes were an average of
600.8 mm3 for the vehicle and 343.4 and 295.9 mm3 for soraﬁnib and PF 562271, respectively, while those of the 16c-treated animals were 348.3 and 153.5 mm3 for the 10 and 30 mg/kg groups, respectively, on the 19th day (Fig. 6B). The tumor sizes in the mice treated with 16c were significantly reduced compared with those of the negative control (p < 0.001). The tumor growth inhibition (TGI) values of soraﬁnib, PF562271, and 16c were also calculated by empirical equation, giving values of 45.1%, 54.1%, and 78.6%, respectively, at doses of 30 mg/kg, compared with the negative control. After treatment six times, all the animals in the different groups maintained a stable body weight and no apparent toxicity was observed in these animals during the experiment, except for two animals in the PF-562271 group that died on the 11th day (Fig. 6C and D). These results indicated that 16c had relatively low toxicity in the nude mice model, as well as in normal mice, and had higher efﬁcacy in the inhibition of tumor growth in mice bearing SMMC7721 xenografts compared with soraﬁnib and PF562271, suggesting this type of FAK inhibitor has the potential to be developed as a treatment for liver cancer. 2.4. Impact of 16c on tumor signaling pathways It is important to explore the molecular mechanisms by which 16c may inhibit the malignant proliferation of HCC cells. To better delineate the signaling consequences of exposure to 16c in HCC cells, we used phosphoantibody array analyses of the test group at 30 mg/kg compared with the control group without drug treat- ment. Sample lysates were applied to the Phospho Explorer Anti- body Array, which was designed and manufactured by Full Moon Biosystems. Data were collected and analyzed by Wayen Bio- technologies. A ratio computation was used to measure the extent of protein phosphorylation. The phosphorylation ratio was calcu- lated by dividing the phosphorylated value by the non- phosphorylated value. Fig. 6. The average tumor volume and body weight of nude mice bearing SMMC7721 tumors after administration of solvent, soraﬁnib, PF-562271, and 16c. (A) Tumor volume measurements. (B) Comparison of the ﬁnal tumor volume in each group after the 19-day treatment period. *p < 0.05 and ***p < 0.001 compared with the vehicle group. (C) Body weight measurements of the nude mice. (D) Comparison of the ﬁnal body weight in each group after the 19-day treatment period. Fig. 7. Statistics of KEGG pathway enrichment analysis of differentially expressed proteins. Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to determine the enriched pathways between the test group and the negative control group. As shown in Fig. 7, several signaling pathways were inﬂuenced by this FAK inhibitor, particularly the focal adhesion, MAPK, and PI3K-Akt signaling pathways. The differences in the phosphorylation ratios between the two groups (fold change, FC) revealed signiﬁcantly changed FAK phos- phorylation and numerous other phosphorylation events classi- cally known to be downstream of FAK were also altered, including decreased phosphorylation of FAK, pyruvate dehydrogenase kinase 1 (PDK1), AKT serine/threonine kinase 1 (Akt), mechanistic target of rapamycin kinase (Homo sapiens) (mTOR), and nuclear factor NF- kappa-B (NFkB). Additional oncogenic proteins exhibiting increased phosphorylation included serine/threonine protein ki- nase 3 (GSK3), fork head box O (FOXO), and caspase 9 (Casp9) (Fig. 8). The present results suggested that 16c inhibits the malig- nant proliferation of HCC cells through decreasing the phosphor- ylation of the FAK cascade. Integrin is the primary and the most studied activator of FAK2 [3,43]. As well as integrin, FAK can also be activated by various speciﬁc growth factors and G-proteins, such as epidermal growth factor (EGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) [44]. Importantly, we found that the phosphorylation at most sites of the integrin receptor, EGFR, VEGFR, and PDGF receptor was increased dramatically, especially integrin beta-1 (phospho- Thr788, FC as 2.82), EGFR (phospho-Ser1070, phospho-Thr693, phospho-Tyr1172, and phospho-Tyr1197, FC as 3.94, 2.97, 2.02, and 2.21 respectively), and VEGFR2 (phospho-Tyr1214, phospho- Tyr951 FC as 2.67 and 2.63). These results suggested that there exist negative feedback loops between FAK and its activators, implying the rationality and possibility of using a combination of FAK inhibitors and EGFR or VEGFR inhibitors in the clinic. 3. Conclusion We have designed and synthesized 5-ﬂuoro-7H-pyrrolo[2,3-d] pyrimidine derivatives as FAK inhibitors starting from simulated pharmacophores on basis of the co-crystal complex of FAK and PF- 562271 (PDB ID: 3BZ3). Owing to its excellent enzymatic potential and anti-proliferative activity against SMMC7721 and YY8103 cells, compound 16c was selected for further biological evaluation. In vivo, the body weight of mice administered 16c intraperitoneally ranged from 0 to 200 mg/kg, and increased gradually during the observation days. Compared with soraﬁnib and PF-562271, 16c showed higher inhibition activity in nude mice bearing SMMC7721 xenografts. Treatment with compound 16c decreased the tumor size signiﬁcantly with a TGI value of 78.6% (p < 0.001) at a dose of 30 mg/kg and no apparent toxicity was observed in these animals during the experiment. Tumor samples of the 30 mg/kg treatment group and the control group without drug treatment were applied to the Phospho Explorer Antibody Array to investigate the signaling consequences of 16c exposure in HCC cells. The results suggested Fig. 8. Signaling consequences of 16c exposure in HCC cells. Schematic illustration of the signaling pathways activated after exposure to 16c; red represents proteins exhibiting increased phosphorylation and green represents proteins exhibiting decreased phosphorylation. that this FAK inhibitor can affect the phosphorylation levels of multiple signaling pathways, including down-regulating the phosphorylation of downstream proteins associated with FAK, and up-regulating the activity of several tumor suppressor genes. It is worth noting that the phosphorylation of most FAK activators was considerably up-regulated, which implied the rationality and pos- sibility of using a combination of FAK inhibitors and EGFR or VEGFR inhibitors in the clinic. 4. Experimental details 4.1. Chemistry 4.1.1. General synthetic materials and methods All commercially available solvents and reagents were used without further puriﬁcation. The synthesized compounds were chemically characterized by thin layer chromatography (TLC), proton nuclear magnetic resonance (1H NMR, 400 MHz), carbon nuclear magnetic resonance (13C NMR, 150 MHz), electrospray ionization mass spectrometry (ESI-MS), melting point and high resolution mass spectrometry (HRMS). 1H NMR and 13C NMR spectra were determined with Bruker-DPX 400 MHz or 600 MHz spectrometer. Chemical shifts are reported in parts per million (ppm) downﬁeld from tetramethylsilane (d) as the internal stan- dard in deuterated solvent and coupling constants (J) are reported in Hertz (Hz). The following abbreviations are used for spin mul- tiplicity: s singlet, d doublet, t triplet, q quartet, m multiplet, dd double doublet and br broad. ESI-MS spectra were taken on Agilent G1946D. Melting points were determined on SGWX-4 microscopic melting point apparatus. HR-MS spectra were determined on Bruker APEX III 7.0 TESLA FTMS. TLC was performed on the glass-backed silica gel sheets (silica gel 60 Å GF254). Column chromatography separations were performed on silica gel (300e400 mesh, Huanghai Chemical Ltd). Microwave assisted re- actions were carried out in a Biotage Initiator microwave synthesis instrument. 4.1.2. General procedure of the synthesis of 2, 2a and 2b A mixture of 6-aminopyrimidine-2,4(1H,3H)-dione (1) (1.0 g, 7.87 mmol) and 1.5 equivalent of 2-chloroacetaldehyde, or 2- chloropropanal, or 1-chloropropan-2-one in 8 mL water was reﬂuxed until the substrate disappeared completely. After cooled, a precipitate was ﬁltered and washed with water and acetone to afford 2, 2a and 2b correspondingly. 7H-pyrrolo[2,3-d]pyrimidine-2,4-diol (2) Light brown solid. Yield: 93%; ESI-MS m/z 152.1 [M H]þ. 5- Methyl-7H-pyrrolo[2,3-d]pyrimidine-2,4-diol (2a) Light brown solid. Yield: 79%; ESI-MS m/z 166.1 [M H]þ. 6- Methyl-7H-pyrrolo[2,3-d]pyrimidine-2,4-diol (2b) Light brown solid. Yield: 46%; ESI-MS m/z 166.1 [MþH]þ. 4.1.3. 2,4-Dichloro-7H-pyrrolo[2,3-d]pyrimidine (3) To a solution of 7H-pyrrolo[2,3-d]pyrimidine-2,4-diol (2) (2.3 g, 15.2 mmol) in 8 mL of toluene were added 4.16 mL (45.5 mmol) of phosphoryl trichloride (POCl3) and 5.18 mL (30.34 mmol) of N, N- diisopropylethylamine (DIEA). After heated at 106 ◦C for 16 h, the reaction was cooled and poured into 200 mL of ice water, following extracted by ethyl acetate for three times. The organic layer was combined, dried and concentrated to provide crude product, which was puriﬁed via column chromatography (petroleum ether/ethyl acetate: 1/1) to afford a light yellow powder (850 mg). Yield: 30%, ESI-MS m/z 187.8 [MþH]þ. 4.1.4. General procedure of the synthesis of 3a and 3b 500 mg (3.03 mmol) of 5-methyl-7H-pyrrolo[2,3-d]pyrimidine- 2,4-diol (2a) or 6-methyl-7H-pyrrolo[2,3-d]pyrimidine-2,4-diol (2b) was dissolved in 3 mL of phenylphosphonic dichloride. After heated at 160 ◦C for 12 h, the solution was cooled and poured into 200 mL of ice water, following extracted by ethyl acetate for three times. The organic layer was combined, dried and concentrated to provide crude product, which was puriﬁed via column chroma- tography (petroleum ether/ethyl acetate: 1/1) to afford 3a or 3b. 2,4-Dichloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidine (3a) Light yellow powder. Yield: 14%; m. p. >250 ◦C; 1H NMR (CDCl3, 400 MHz) d 2.48 (s, 3 H), 7.11 (s, 1 H), 10.03 (brs, 1 H) ppm; ESI-MS m/z 202.0 [M H]þ.
2,4-Dichloro-6-methyl-7H-pyrrolo[2,3-d]pyrimidine (3b)
Light yellow powder. Yield: 33%; m. p. > 250 ◦C; ESI-MS m/z 202.0 [MþH]þ.
4.1.5. 2,4-Dichloro-5-ﬂuoro-7H-pyrrolo[2,3-d]pyrimidine (3c)
A solution of 400 mg (2.14 mmol) of 2,4-dichloro-7H-pyrrolo [2,3-d]pyrimidine (3) and Selectﬂuor (1.14 g, 3.21 mmol) in 20 mL of acetonitrile and 1 mL of acetic acid was heated at 70 ◦C for 16 h.
After cooled and neutralized by saturated solution of NaHCO3, the reaction was extracted with ethyl acetate and the organic layer was combined, dried and concentrated to provide crude product, which was puriﬁed via column chromatography (petroleum ether/ethyl
acetate: 3/1) to afford a white solid (190 mg). Yield: 43%; m. p. 228e231 ◦C. 1H NMR (DMSO‑d6, 400 MHz) d ¼ 7.75 (s, 1 H), 12.69 (s,
1 H) ppm. ESI-MS m/z 206.0 [MþH]þ.
4.1.6. 2,4,5-Trichloro-7H-pyrrolo[2,3-d]pyrimidine (3d)
500 mg (2.67 mmol) of 2,4-dichloro-7H-pyrrolo[2,3-d]pyrimi- dine (3) and 428 mg (3.21 mmol) of NCS were dissolved in 5 mL of CH2Cl2 and 2 mL of THF. The reaction was heated by microwave at
90 ◦C for 4 h. After the solvent was removed in vacuum, the product
was obtain via column chromatography (petroleum ether/ethyl acetate: 1/1) as a light yellow solid (560 mg). Yield: 95%: m.p. 234e238 ◦C; 1H NMR (DMSO‑d6, 400 MHz) d ¼ 7.95 (s, 1 H), 13.09 (s,
1 H) ppm; ESI-MS m/z 222.0 [MþH]þ.
4.1.7. 2,4-Dichloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine (3e)
50 mg (0.267 mmol) of 2,4-dichloro-7H-pyrrolo[2,3-d]pyrimi- dine (3) and 60 mg (0.267 mmol) of NIS were dissolved in 5 mL of CH2Cl2. The reaction was stirred at room temperature for 15 h. After the solvent was removed in vacuum, the product was obtain via column chromatography (petroleum ether/ethyl acetate: 5/1) as a
white solid (70 mg). Yield: 84%; m. p. 229e234 ◦C; ESI-MS m/z 313.9
[MþH]þ.
4.1.8. General procedure of the synthesis of 5 and 5a-d
A mixture of 2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidines (3, or 3a-d), N-(3-(aminomethyl)pyridine-2-yl)-N-methyl- methanesulfonamide (1.2 equiv.) and DIPEA (2.0 equiv.) was reﬂuxed in EtOH (8 mL) with stirring for 6 h. The resulting mixture was cool to room temperature and ﬁltered to afford 4-substitutd compounds 5 and 5a-d correspondingly.
N-(3-(((2-chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino) methyl)pyridin-2-yl)-N-methylmethanesulfonamide (5) White solid, yield: 46%, m.p. 218-211 ◦C. 1H NMR (DMSO‑d6, 400 MHz)
d 3.14 (s, 3 H), 3.26 (s, 3 H), 4.74 (d, J 5.40 Hz, 2 H), 6.59 (s, 1 H),
7.12 (s, 1 H), 7.41 (dd, J 7.72 Hz, J 4.72 Hz, 1 H), 7.82 (d,
J 7.64 Hz, 1 H), 8.43 (d, J 4.64 Hz), 8.47 (s, 1 H), 11.75 (s, 1 H) ppm. ESI-MS m/z 367.1 [M H]þ.
N-(3-(((2-chloro-5-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)
amino)methyl)pyridin-2-yl)-N-methylmethanesulfonamide (5a) Yellow solid, yield: 66%. 1H NMR (DMSO‑d6, 400 MHz) d 2.38 (s, 3 H), 3.12 (s, 3 H), 3.28 (s, 3 H), 4.70 (s, 2 H), 6.68 (s, 1 H), 7.38 (m,
2 H), 7.85 (d, J ¼ 6.36 Hz, 1 H), 8.40 (d, J ¼ 2.96 Hz, 1 H), 11.43 (s, 1 H)

ppm. ESI-MS m/z 381.1 [M H]þ.
N-(3-(((2-chloro-6-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)
amino)methyl)pyridin-2-yl)-N-methylmethanesulfonamide (5b) White solid, yield: 32%, m.p. 243e245 ◦C. 1H NMR (DMSO‑d6, 400 MHz) d 2.27 (s, 3 H), 3.12 (s, 3 H), 3.25 (s, 3 H), 4.70 (d,
J 5.16 Hz, 2 H), 6.22 (s, 1 H), 7.39 (dd, J 7.68 Hz, J 4.64 Hz, 1 H),
7.79 (d, J 6.52 Hz, 1 H), 8.24 (s, 1 H), 8.41 (d, J 2.80 Hz, 1 H), 11.58 (s, 1 H) ppm. ESI-MS m/z 381.1 [M H]þ.
N-(3-(((2-chloro-5-ﬂuoro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)
amino)methyl)pyridin-2-yl)-N-methylmethanesulfonamide (5c) White solid, yield: 45%, m. p. 243e246 ◦C. 1H NMR (DMSO‑d6, 400 MHz) d 3.10 (s, 3 H), 3.27 (s, 3 H), 4.69 (s, 2 H), 7.10 (s, 1 H),
7.38 (dd, J 7.72 Hz, J 4.68 Hz, 1 H), 7.83 (d, J 6.52 Hz, 1 H), 8.09
(brs, 1 H), 8.41 (d, J 2.92 Hz, 1 H), 11.64 (s, 1 H) ppm. ESI-MS m/z
385.1 [M H]þ.
N-(3-(((2,5-dichloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)
amino)methyl)pyridin-2-yl)-N-methylmethanesulfonamide (5d) White solid, yield: 66%, m. p. 234e238 ◦C. 1H NMR (DMSO‑d6, 400 MHz) d ¼ 3.11 (s, 3 H), 3.27 (s, 3 H), 4.75 (d, J ¼ 5.12 Hz, 2 H),
7.34 (d, J ¼ 2.52 Hz, 1 H), 7.39 (dd, J ¼ 7.72 Hz, J ¼ 4.72 Hz, 1 H), 7.68
(t, J ¼ 5.84 Hz, 1 H), 7.83 (d, J ¼ 7.44 Hz, 1 H), 8.41 (d, J ¼ 2.84 Hz, 1 H), 12.12 (s, 1 H) ppm. ESI-MS m/z 401.0 [MþH]þ.
4.1.9. N-(3-(((6-chloro-1H-pyrazolo[3,4-d]pyrimidin-4-yl)amino) methyl)pyridin-2-yl)-N-methylmethanesulfonamide (6)
A mixture of 4,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (4) (90 mg, 0.459 mmol), N-(3-(aminomethyl)pyridine-2-yl)-N-meth-
ylmethanesulfonamide (113 mg, 0.527 mmol) and DIPEA (0.15 mL, 0.878 mmol) was reﬂuxed in EtOH (8 mL) with stirring for 6 h. The resulting mixture was cool to room temperature and ﬁltered to
afford compound 6 as white solid. Yield: 37%, m. p. 131e135 ◦C. 1H
NMR (DMSO‑d6, 400 MHz) d ¼ 3.12 (s, 3 H), 3.23 (s, 3 H), 4.76 (d,
J ¼ 5.60 Hz, 2 H), 7.42 (dd, J ¼ 7.72 Hz, J ¼ 4.84 Hz, 1 H), 7.85 (d,
J ¼ 7.72 Hz, 1 H), 8.13 (s, 1 H), 8.44 (d, J ¼ 3.12 Hz, 1 H), 9.20 (t,
J ¼ 5.64 Hz, 1 H) ppm; ESI-MS m/z 367.9 [MþH]þ.
4.1.10. 2,4-Dichloro-5-iodo-7-tosyl-7H-pyrrolo[2,3-d]pyrimidine (9)
To a solution of 455 mg (1.454 mmol) of 2,4-dichloro-5-iodo- 7H-pyrrolo[2,3-d]pyrimidine (3e), trimethylamine (0.61 mL,
4.36 mmol) and DMAP (17 mg, 0.15 mmol) in 20 mL of CH2Cl2, 333 mg (1.74 mmol) of p-toluensulfonyl chloride was added. The reaction was stirred at room temperature for 1 h, then poured into 50 mL of water and extracted with dichloromethane. The organic layer was combined, dried and concentrated to provide crude product, which was puriﬁed via column chromatography
(dichloromethane) to afford a light yellow solid (520 mg). Yield: 77%, m. p. 189e193 ◦C. 1H NMR (CDCl3-d1, 400 MHz) d ¼ 2.44 (s,
3 H), 7.37 (d, J ¼ 8.04 Hz, 2 H), 7.91 (s, 1 H), 8.10 (d, J ¼ 8.28 Hz, 2 H)
ppm. ESI-MS m/z 467.9 [MþH]þ.
4.1.11. N-(3-(((2-chloro-5-iodo-7-tosyl-7H-pyrrolo[2,3-d] pyrimidin-4-yl)amino)methyl)pyridin-2-yl)-N- methylmethanesulfonamide (10)
A mixture of compound 9 (520 mg, 1.11 mmol), N-(3-(amino- methyl)pyridine-2-yl)-N-methylmethanesulfonamide (289 mg,
1.34 mmol) and TEA (0.46 mL, 3.34 mmol) was stirred in MeCN (10 mL) at rt for 2 h. The resulting mixture concentrated in vacuo and puriﬁed by chromatography on silica gel (CH2Cl2: MeOH, 150:
1) to afford compound 10. White solid, yield: 67%, m. p. 193e197 ◦C.
1H NMR (DMSO‑d6, 400 MHz) d ¼ 2.35 (s, 3 H), 3.09 (s, 3 H), 3.23 (s,
3 H), 4.78 (d, J ¼ 5.28 Hz, 2 H), 7.36 (dd, J ¼ 7.60 Hz, J ¼ 4.72 Hz, 1 H),
7.45 (d, J ¼ 8.08 Hz, 2 H), 7.50 (brs, 1 H), 7.81 (d, J ¼ 6.76 Hz, 1 H), 7.85
(s, 1 H), 7.96 (d, J ¼ 8.28 Hz, 2 H), 8.40 (d, J ¼ 3.08 Hz, 1 H) ppm. ESI- MS m/z 647.0 [MþH]þ.

4.1.12. N-(3-(((2-chloro-5-cyano-7-tosyl-7H-pyrrolo[2,3-d] pyrimidin-4-yl)amino)methyl)pyridin-2-yl)-N- methylmethanesulfonamide (11)
A mixture of compound 10 (520 mg, 1.11 mmol), Zn(CN)2 (11 mg, 0.0929 mmol), Pd2(dba)3 (7 mg, 0.007774 mmol), dppf (9 mg,
0.0155 mmol), zinc powder (0.25 mg, 0.00387 mmol) and Zn(OAc)2 (0.75 mg, 0.00387 mmol) was stirred in DMF (2 mL) at 70 ◦C for
2.5 h under a nitrogen atmosphere. The resulting mixture was poured into ice-cold water (50 mL), ﬁltered. The solid was puriﬁed by chromatography on silica gel (PE: EtOAc, 1: 1) to afford com-
pound 11 as white solid. Yield: 36%, m. p. 190e192 ◦C. 1H NMR
(DMSO‑d6, 400 MHz) d ¼ 2.38 (s, 3 H), 3.10 (s, 3 H), 3.24 (s, 3 H), 4.75
(d, J ¼ 5.24 Hz), 7.36 (dd, J ¼ 7.68 Hz, J ¼ 4.72 Hz, 1 H), 7.50 (d,
J ¼ 8.12 Hz, 2 H), 7.84 (d, J ¼ 6.68 Hz, 1 H), 7.96 (brs, 1 H), 8.02 (d,
J ¼ 8.32 Hz, 2 H), 8.41 (d, J ¼ 3.04 Hz, 1 H), 8.72 (s, 1 H) ppm. ESI-MS
m/z 546.1 [MþH]þ.
4.1.13. N-(3-(((2-chloro-5-cyano-7H-pyrrolo[2,3-d]pyrimidin-4-yl) amino)methyl)pyridin-2-yl)-N-methylmethanesulfonamide (5e)
A mixture of compound 11 (450 mg, 0.826 mmol) and TBAF (260 mg, 1.00 mmol) was stirred in THF (20 mL) at rt for 15 min. The resulting mixture was concentrated in vacuo and puriﬁed by chromatography on silica gel (CH2Cl2: MeOH, 50: 1) to afford
compound 5e as white solid. Yield: 47%, m. p. > 250 ◦C. 1H NMR
(DMSO‑d6, 400 MHz) d ¼ 3.12 (s, 3 H), 3.28 (s, 3 H), 4.79 (d,
J ¼ 5.24 Hz, 2 H), 7.40 (dd, J ¼ 7.64 Hz, J ¼ 4.54 Hz, 1 H), 7.70 (t,
J ¼ 5.84 Hz, 1 H), 7.86 (d, J ¼ 6.16 Hz, 1 H), 8.20 (s, 1 H), 8.43 (d,
J ¼ 2.92 Hz, 1 H), 12.92 (s, 1 H) ppm. ESI-MS m/z 392.1 [MþH]þ.
4.1.14. General procedure of the synthesis of 7, 7a-e and 8
Method A: A mixture of compound 5, 5b, 5e, or 6 (0.204 mmol), 5-aminoindolin-2-one (30 mg, 0.204 mmol) and p-methyl benze- nesulfonic acid (70 mg, 0.408 mmol) was heated to 150 ◦C in n-
BuOH (1 mL) by microwave synthesis instrument and stirred for 5 h. The resulting mixture was concentrated in vacuo and puriﬁed by chromatography on silica gel (CH2Cl2: MeOH, 20: 1) to afford aimed compounds 7, 7b, 7e and 8.
Method B: A mixture of compound 5a, 5c, or 5d (0.130 mmol), 5-aminoindolin-2-one (20 mg, 0.130 mmol), K2CO3 (54 mg,
0.391 mmol), Pd2(dba)3 (6 mg, 0.0065 mmol) and Xphos (6.2 mg,
0.013 mmol) was stirred in t-BuOH (3 mL) at 100 ◦C for 12 h under a nitrogen atmosphere. The resulting mixture was poured into ice- cold water and extracted twice with ethyl acetate. The organic layers was concentrated in vacuo and puriﬁed by chromatography on silica gel (CH2Cl2: MeOH, 30: 1) to afford aimed compounds 7a, 7c, and 7d.
N-methyl-N-(3-(((2-((2-oxoindolin-5-yl)amino)-7H-pyrrolo [2,3-d]pyrimidin-4-yl)amino)methyl)pyridin-2-yl)meth- anesulfonamide (7) Method A, white solid, yield: 26%, m. p.
172e176 ◦C. 1H NMR (DMSO‑d6, 400 MHz) d ¼ 3.16 (s, 3 H), 3.18 (s,
3 H), 3.31 (s, 2 H), 4.79 (d, J ¼ 5.80 Hz, 2 H), 6.41 (s, 1 H), 6.54 (d,
J ¼ 8.36 Hz, 1 H), 6.76 (t, J ¼ 2.36 Hz, 1 H), 7.38e7.41 (m, 2 H), 7.61 (s,
1 H), 7.72 (s, 1 H), 7.82 (d, J ¼ 6.80 Hz, 1 H), 8.39 (s, 1 H), 8.41 (dd,
J ¼ 4.76 Hz, J ¼ 1.64 Hz, 1 H), 10.09 (s, 1 H), 10.99 (s, 1 H) ppm. 13C NMR (DMSO‑d6, 150 MHz) d 176.0, 156.2, 156.0, 152.1, 147.1, 137.7, 136.3, 136.2, 134.9, 125.4, 123.9, 117.9, 116.8, 115.2, 108.4, 98.7, 96.9,
37.2, 36.0 ppm. ESI-MS m/z 478.8 [MþH]þ. HRMS (ESIþ): calcd for C22H22N8O3S [M H]þ 479.1608; found 479.1622.
N-methyl-N-(3-(((5-methyl-2-((2-oxoindolin-5-yl)amino)-
7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)methyl)pyridin-2-yl) methanesulfonamide (7a) Method B, brown solid, yield: 40%, m. p. 166e172 ◦C. 1H NMR (DMSO‑d6, 400 MHz) d ¼ 2.31 (s, 3 H), 3.17 (s,
6 H), 3.28 (s, 2 H), 4.80 (d, J ¼ 5.44 Hz, 2 H), 6.51 (m, 2 H), 6.72 (brs,
1 H), 7.33 (d, J ¼ 8.36 Hz, 1 H), 7.38 (dd, J ¼ 7.64 Hz, J ¼ 4.72 Hz, 1 H),
7.55 (s, 1 H), 7.85 (d, J ¼ 7.08 Hz, 1 H), 8.33 (s, 1 H), 8.40 (d,

J ¼ 3.20 Hz, 1 H), 10.08 (s, 1 H), 10.67 (s, 1 H) ppm. 13C NMR
(DMSO‑d6, 150 MHz) d 176.0, 156.8, 155.7, 152.4, 152.1, 146.9, 137.8,
136.3, 136.2, 135.1, 125.4, 123.9, 116.8, 115.7, 115.2, 108.6, 108.4, 97.0,
37.1, 36.1, 35.9, 12.2 ppm. ESI-MS m/z 493.2 [MþH]þ. HRMS (ESIþ): calcd for C23H24N8O3S [M H]þ 493.1765; found 493.1769.
N-methyl-N-(3-(((6-methyl-2-((2-oxoindolin-5-yl)amino)-
7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)methyl)pyridin-2-yl) methanesulfonamide (7b) Method A, gray solid, yield: 14%, m. p.
>250 ◦C. 1H NMR (DMSO‑d6, 400 MHz) d ¼ 2.22 (s, 3 H), 3.16 (s, 3 H), 3.17 (s, 3 H), 3.30 (s, 2 H), 4.78 (d, J ¼ 5.32 Hz, 2 H), 6.06 (s, 1 H), 6.54
(d, J ¼ 8.12 Hz, 1 H), 7.34e7.41 (m, 2 H), 7.57 (s, 2 H), 7.80 (d,
J ¼ 7.12 Hz, 1 H), 8.41 (m, 2 H), 10.10 (s, 1 H), 10.89 (brs, 1 H) ppm. 13C NMR (DMSO‑d6, 150 MHz) d 176.0, 155.3, 152.0, 147.0, 137.6, 136.4,
134.9, 128.0, 127.9, 125.4, 125.3, 123.9, 116.9, 115.2, 108.4, 97.2, 95.9,
37.1, 36.0, 28.8, 13.1 ppm. ESI-MS m/z 493.3 [MþH]þ. HRMS (ESIþ): calcd for C23H24N8O3S [M H]þ 493.1765; found 493.1774.
N-(3-(((5-ﬂuoro-2-((2-oxoindolin-5-yl)amino)-7H-pyrrolo
[2,3-d]pyrimidin-4-yl)amino)methyl)pyridin-2-yl)-N-methyl- methanesulfonamide (7c) Method B, faint yellow solid, yield: 31%, m. p. 165e168 ◦C. 1H NMR (DMSO‑d6, 400 MHz) d ¼ 3.16 (s, 3 H),
3.17 (s, 3 H), 3.31 (s. 2H), 4.79 (d, J ¼ 5.56 Hz, 2 H), 6.54 (d,
J ¼ 8.36 Hz, 1 H), 6.68 (s, 1 H), 7.23 (brs, 1 H), 7.34 (d, J ¼ 7.72 Hz, 1 H),
7.39 (dd, J ¼ 7.60 Hz, J ¼ 4.60 Hz, 1 H), 7.56 (s, 1 H), 7.82 (d,
J ¼ 7.76 Hz, 1 H), 8.41 (d, J ¼ 3.00 Hz, 1 H), 8.51 (s, 1 H), 10.11 (s, 1 H), 10.79 (1 H) ppm. 13C NMR (DMSO‑d6, 150 MHz) d ¼ 176.0, 156.6,
154.9, 152.0, 147.4, 147.0, 142.9 (d, J ¼ 239.7 Hz), 137.7, 136.8, 135.7,
134.8, 125.4, 123.9, 117.3, 115.7, 108.4, 100.0 (d, J ¼ 25.7 Hz), 86.9 (d,
J ¼ 15.6 Hz), 37.1, 36.0, 35.9 ppm. ESI-MS m/z 497.1 [MþH]þ. HRMS (ESI ): calcd for C22H21FN8O3S [M H]þ 497.1514; found 497.1517.
N-(3-(((5-chloro-2-((2-oxoindolin-5-yl)amino)-7H-pyrrolo
[2,3-d]pyrimidin-4-yl)amino)methyl)pyridin-2-yl)-N-methyl- methanesulfonamide (7d) Method B, red solid, yield: 16%, m. p. 164e169 ◦C. 1H NMR (DMSO‑d6, 400 MHz) d 3.16 (s, 3 H), 3.18 (s,
3 H), 3.33 (s, 2 H), 4.84 (d, J 4.76 Hz, 2 H), 6.56 (d, J 8.32 Hz, 1 H),
6.94 (s, 2 H), 7.35 (d, J 8.00 Hz, 1 H), 7.40 (s, 1 H), 7.57 (s, 1 H), 7.85
(d, J 7.28 Hz, 1 H), 8.42 (s, 1 H), 8.57 (s, 1 H), 10.12 (s, 1 H), 11.31 (s, 1 H) ppm. 13C NMR (DMSO‑d6, 150 MHz) d 176.0, 156.4, 155.7,
152.2, 150.8, 147.1, 137.9, 136.8, 135.6, 134.8, 125.5, 123.9, 117.4, 115.7,
115.5, 108.4, 101.3, 94.2, 37.1, 35.9 ppm. ESI-MS m/z 513.1 [MþH]þ. HRMS (ESI ): calcd for C22H21ClN8O3S [M H]þ 513.1219; found 513.1221.
N-(3-(((5-cyano-2-((2-oxoindolin-5-yl)amino)-7H-pyrrolo [2,3-d]pyrimidin-4-yl)amino)methyl)pyridin-2-yl)-N-methyl- methanesulfonamide (7e) Method A, white solid, yield: 9%, m. p. >
250 ◦C. 1H NMR (DMSO‑d6, 400 MHz) d ¼ 3.17 (s, 3 H), 3.18 (s, 3 H),
3.31 (s, 2 H), 4.85 (d, J ¼ 5.60 Hz, 2 H), 6.58 (d, J ¼ 8.56 Hz, 1 H), 6.84
(brs, 1 H), 7.35 (d, J ¼ 8.24 Hz, 1 H), 7.41 (dd, J ¼ 7.64 Hz, J ¼ 4.84,
1 H), 7.55 (s, 1 H), 7.84e7.88 (m, 2 H), 8.44 (d, J ¼ 2.92 Hz, 1 H), 8.75
(s, 1 H), 10.15 (s, 1 H), 12.15 (s, 1 H) ppm. C NMR (DMSO‑d6,
150 MHz) d 176.0, 155.9, 152.1, 147.2, 145.3, 137.7, 137.6, 134.0,
130.2, 127.9, 125.8, 125.3, 123.9, 117.2, 115.6, 108.6, 95.2, 82.8, 37.0,
35.9, 35.8, 20.6 ppm. ESI-MS m/z 504.2 [MþH]þ. HRMS (ESIþ): calcd for C23H21N9O3S [M H]þ 504.1561; found 504.1565.
N-methyl-N-(3-(((6-((2-oxoindolin-5-yl)amino)-1H-pyrazolo
[3,4-d]pyrimidin-4-yl)amino)methyl)pyridin-2-yl)meth- anesulfonamide (8) Method A, white solid, yield: 48%, m. p. 186e190 ◦C. 1H NMR (DMSO‑d6, 400 MHz) d ¼ 3.15 (s, 3 H), 3.17 (s,
3 H), 3.34 (s, 2 H), 4.80 (d, J ¼ 5.60 Hz, 2 H), 6.59 (d, J ¼ 8.32 Hz, 1 H),
7.39e7.43 (m, 2 H), 7.60 (s, 1 H), 7.85 (d, J ¼ 7.20 Hz, 1 H), 7.90 (s,
1 H), 8.35 (s, 1 H), 8.43 (dd, J ¼ 4.60 Hz, J ¼ 1.60 Hz, 1 H), 8.77 (s, 1 H),
10.15 (s, 1 H), 12.76 (s, 1 H) ppm. ESI-MS m/z 479.9 [MþH]þ.
4.1.15. General procedure of the synthesis of 12a-m
A mixture of compound 5c (50 mg, 0.130 mmol), corresponding anilines (0.130 mmol), K2CO3 (54 mg, 0.391 mmol), Pd2(dba)3

(6 mg, 0.0065 mmol) and Xphos (6.2 mg, 0.013 mmol) was stirred in t-BuOH (3 mL) at 100 ◦C for 12 h under a nitrogen atmosphere. The resulting mixture was poured into ice-cold water and extracted
twice with ethyl acetate. The organic layers was concentrated in vacuo and puriﬁed by chromatography on silica gel (CH2Cl2: MeOH, 30: 1) to afford compounds 12a-m.
N-(3-(((5-ﬂuoro-2-(phenylamino)-7H-pyrrolo[2,3-d]pyr- imidin-4-yl)amino)methyl)pyridin-2-yl)-N-methyl- methanesulfonamide (12a) Faint yellow solid, yield: 34%, m. p.
209e213 ◦C. 1H NMR (DMSO‑d6, 400 MHz) d ¼ 3.18 (s, 3 H), 3.20 (s,
3 H), 4.83 (d, J ¼ 5.28 Hz, 2 H), 6.74 (s, 1 H), 6.78 (t, J ¼ 7.24 Hz, 1 H),
7.09 (t, J ¼ 7.48 Hz, 2 H), 7.31 (brs, 1 H), 7.41 (t, J ¼ 4.76 Hz, 1 H), 7.62
(d, J ¼ 7.72 Hz, 2 H), 7.85 (d, J ¼ 7.28 Hz, 1 H), 8.43 (s, 1 H), 8.70 (s,
1 H), 10.88 (s, 1 H) ppm. 13C NMR (DMSO‑d6, 150 MHz) d ¼ 156.3,
154.9, 152.1, 147.3, 147.0, 142.8 (d, J ¼ 240.0 Hz), 141.4, 137.5, 134.7,
128.0, 123.9, 119.8, 117.8, 100.3 (d, J ¼ 25.5 Hz), 87.2 (d, J ¼ 15.6 Hz),
37.1 , 36.0 ppm. ESI-MS m/z 441.9 [MþH]þ. HRMS (ESIþ): calcd for C20H20FN7O2S [M H]þ 442.1456; found 442.1460.
N-(3-(((5-ﬂuoro-2-((4-ﬂuorophenyl)amino)-7H-pyrrolo[2,3-
d]pyrimidin-4-yl)amino)methyl)pyridin-2-yl)-N-methyl- methanesulfonamide (12b) Orange solid, yield: 28%, m. p. 197e200 ◦C. 1H NMR (DMSO‑d6, 400 MHz) d 3.19 (s, 3 H), 3.20 (s,
3 H), 4.82 (s, 2 H), 6.74 (s, 1 H), 6.93 (m, 2 H), 7.30 (brs, 1 H), 7.41 (brs,
1 H), 7.63 (m, 2 H), 7.84 (d, J ¼ 7.32 Hz, 1 H), 8.44 (s, 1 H), 8.76 (s, 1 H),
10.88 (s, 1 H) ppm. 13C NMR (DMSO‑d6, 150 MHz) d ¼ 156.3, 156.1 (d,
J ¼ 235.0 Hz), 155.0, 152.1, 147.2, 147.0, 142.8 (d, J ¼ 240.0 Hz), 137.8,
137.6, 134.7, 123.9, 119.3 (d, J ¼ 6.9 Hz) 114.4 (d, J ¼ 21.7 Hz), 100.3 (d, J ¼ 25.7 Hz), 87.2 (d, J ¼ 15.9 Hz), 37.1, 36.0 ppm. ESI-MS m/z 459.8 [M H]þ. HRMS (ESI ): calcd for C20H19F2N7O2S [M H]þ 460.1362;
found 460.1365.
N-(3-(((2-((3-chloro-4-ﬂuorophenyl)amino)-5-ﬂuoro-7H- pyrrolo[2,3-d]pyrimidin-4-yl)amino)methyl)pyridin-2-yl)-N- methylmethanesulfonamide (12c) Gray solid, yield: 31%, m. p.
213e218 ◦C. 1H NMR (DMSO‑d6, 400 MHz) d ¼ 3.15 (s, 3 H), 3.18 (s,
3 H), 4.81 (d, J ¼ 5.40 Hz, 2 H), 6.75 (s, 1 H), 7.12 (t, J ¼ 9.16 Hz, 1 H),
7.32 (brs, 1 H), 7.39 (dd, J ¼ 7.72 Hz, J ¼ 4.68 Hz, 1 H), 7.55 (brs, 1 H),
7.83 (d, J ¼ 7.84 Hz, 1 H), 7.99 (d, J ¼ 5.12 Hz, 1 H), 8.41 (s, 1 H), 8.95 (s, 1 H), 10.97 (s, 1 H) ppm. 13C NMR (DMSO‑d6, 150 MHz) d ¼ 155.8, 155.0, 152.1, 151.0 (d, J ¼ 237.4 Hz), 147.1, 146.9, 142.8 (d,
J ¼ 240.3 Hz), 138.8, 137.7, 134.6, 123.9, 118.6, 118.4 (d, J ¼ 17.9 Hz),
117.7 (d, J ¼ 5.7 Hz), 116.1 (d, J ¼ 21.1 Hz), 100.7 (d, J ¼ 25.6 Hz), 87.4 (d, J ¼ 15.9 Hz), 37.2, 36.0 ppm. ESI-MS m/z 493.8 [MþH]þ. HRMS
(ESI ): calcd for C20H18ClF2N7O2S [M H]þ 494.0972; found
494.0977.
N-(3-(((2-((3-cyanophenyl)amino)-5-ﬂuoro-7H-pyrrolo[2,3- d]pyrimidin-4-yl)amino)methyl)pyridin-2-yl)-N-methyl- methanesulfonamide (12d) Brown solid, yield: 33%, m. p. >250 ◦C. 1H NMR (DMSO‑d6, 400 MHz) d ¼ 3.18 (s, 3 H), 3.22 (s, 3 H), 4.85 (d,
J ¼ 5.68 Hz, 3 H), 6.81 (s, 1 H), 7.23 (d, J ¼ 7.68 Hz, 1 H), 7.31 (t,
J ¼ 7.92 Hz, 2 H), 7.40e7.43 (m, 2 H), 7.86 (d, J ¼ 7.72 Hz, 1 H), 7.92 (d,
J ¼ 7,96 Hz, 1 H), 8.24 (s, 1 H), 8.44 (s, 1 H), 9.16 (s, 1 H), 11.07 (s, 1 H) ppm. 13C NMR (DMSO‑d6, 150 MHz) d ¼ 155.7, 155.1, 152.1, 147.1,
146.7, 142.7 (d, J ¼ 240.0 Hz), 142.3, 137.7, 134.6, 129.4, 123.9, 123.1,
122.2, 120.0, 119.1, 111.0, 100.9 (d, J ¼ 25.8 Hz), 87.6 (d, J ¼ 15.6 Hz),
37.2 , 35.9 ppm. ESI-MS m/z 466.8 [MþH]þ. HRMS (ESIþ): calcd for C21H19FN8O2S [M H]þ 467.1408; found 467.1411.
N-(3-(((2-((3,4-dimethoxyphenyl)amino)-5-ﬂuoro-7H-pyr-
rolo[2,3-d]pyrimidin-4-yl)amino)methyl)pyridin-2-yl)-N-meth- ylmethanesulfonamide (12e) Yellow solid, yield: 28%, m. p. 115e118 ◦C. 1H NMR (DMSO‑d6, 400 MHz) d ¼ 3.14 (s, 3 H), 3.18 (s,
3 H), 3.64 (s, 3 H), 3.65 (s, 3 H), 4.80 (d, J ¼ 5.76 Hz, 2 H), 6.67e6.69
(m, 2 H), 7.13 (d, J ¼ 8.28 Hz, 1 H), 7.21 (brs, 1 H), 7.37e7.39 (m, 2 H),
7.84 (d, J ¼ 7.08 Hz, 1 H), 8.40 (d, J ¼ 3.04 Hz, 1 H), 8.45 (s, 1 H), 10.79 (s, 1 H) ppm. 13C NMR (DMSO‑d6, 150 MHz) d ¼ 156.5, 154.9, 152.1, 148.4, 147.4, 147.1, 142.9 (d, J ¼ 239.9 Hz), 142.7, 137.8, 135.3, 134.8,

123.9, 112.2, 109.9, 104.1, 100.0 (d, J ¼ 25.8 Hz), 86.9 (d, J ¼ 15.7 Hz),
55.8, 55.1, 37.2, 35.9 ppm. ESI-MS m/z 501.9 [MþH]þ. HRMS (ESIþ): calcd for C22H24FN7O4S [M H]þ 502.1667; found 502.1672.
N-(3-(((5-ﬂuoro-2-((3,4,5-trimethoxyphenyl)amino)-7H-pyr-
rolo[2,3-d]pyrimidin-4-yl)amino)methyl)pyridin-2-yl)-N-meth- ylmethanesulfonamide (12f) Yellow solid, yield: 43%, m. p. 247e249 ◦C. 1H NMR (DMSO‑d6, 400 MHz) d ¼ 3.16 (s, 3 H), 3.21 (s,
3 H), 3.58 (s, 3 H), 3.68 (s, 6 H), 4.85 (d, J ¼ 5.84 Hz, 2 H), 6.75 (s, 1 H),
7.18 (s, 2 H), 7.24 (m, 1 H), 7.41 (dd, J ¼ 7.56 Hz, J ¼ 4.64 Hz, 1 H), 7.90
(d, J ¼ 7.28 Hz, 1 H), 8.42 (d, J ¼ 3.04 Hz, 1 H), 8.57 (s, 1 H), 10.85 (s, 1 H) ppm. 13C NMR (DMSO‑d6, 150 MHz) d ¼ 156.26, 154.9, 152.3,
152.1, 147.2, 147.1, 142.8 (d, J ¼ 241.1 Hz), 138.0, 137.5, 134.8, 131.1,
124.0, 100.2 (d, J ¼ 25.8 Hz), 96.1, 87.1 (d, J ¼ 15.6 Hz), 59.9, 55.4,
37.2, 35.8 ppm. ESI-MS m/z 531.8 [MþH]þ. HRMS (ESIþ): calcd for C23H26FN7O5S [M H]þ 532.1773; found 532.1777.
N-(3-(((5-ﬂuoro-2-((3-methoxy-2-methylphenyl)amino)-7H-
pyrrolo[2,3-d]pyrimidin-4-yl)amino)methyl)pyridin-2-yl)-N- methylmethanesulfonamide (12g) Yellow solid, yield: 29%, m. p. 107e112 ◦C. 1H NMR (DMSO‑d6, 400 MHz) d 1.97 (s, 3 H), 3.12 (s,
3 H), 3.15 (s, 3 H), 3.75 (s, 3 H), 4.72 (d, J 5.12 Hz, 2 H), 6,63e6.65
(m, 2 H), 6.93 (t, J 7.72 Hz, 1 H), 7.05 (d, J 7.84 Hz, 1 H), 7.23 (brs,
1 H), 7.39 (t, J 5.08 Hz, 1 H), 7.70 (s, 1 H), 7.80 (d, J 7.24 Hz, 1 H),
8.42 (s, 1 H), 10.77 (s, 1 H) ppm. 13C NMR (DMSO‑d6, 150 MHz)
d 157.5, 157.2, 154.9, 152.1, 147.8, 147.0, 142.8 (d, J 239.7 Hz),
139.6, 137.7, 134.8, 125.2, 123.8, 119.0, 116.7, 105.3, 99.8 (d,
J ¼ 25.4 Hz), 86.9 (d, J ¼ 15.9 Hz), 55.2, 37.0, 36.0, 10.4 ppm. ESI-MS m/z 485.9 [M H]þ. HRMS (ESI ): calcd for C22H24FN7O3S [M H]þ 486.1718; found 486.1730.
N-(3-(((2-((3-(aminomethyl)phenyl)amino)-5-ﬂuoro-7H-pyr- rolo[2,3-d]pyrimidin-4-yl)amino)methyl)pyridin-2-yl)-N-meth- ylmethanesulfonamide (12h) Faint yellow solid, yield: 25%, m. p.
178e183 ◦C. 1H NMR (DMSO‑d6, 400 MHz) d 3.11 (s, 3 H), 3.14 (s,
3 H), 3.78 (s, 2 H), 4.80 (d, J 5.84 Hz, 2 H), 7.00 (d, J 6.64 Hz, 1 H),
7.15e7.23 (m, 4 H), 7.40 (dd, J 7.80 Hz, J 4.76 Hz, 1 H), 7.57e7.64
(m, 3 H), 7.73 (d, J 7.00 Hz, 1 H), 7.93 (s, 1 H), 8.41 (d, J 2.8 Hz, 1 H), 9.51 (s, 1 H) ppm. 13C NMR (DMSO‑d6, 150 MHz) d 157.8 (d, J 30.36 Hz), 156.1, 155.0, 152.1, 147.2, 147.1, 142.8 (d, J 240.15 Hz),
141.7, 137.7, 134.7, 133.7, 128.5, 123.9, 119.8, 118.4, 117.9, 100.5 (d, J ¼ 25.65 Hz), 87.26 (d, J ¼ 15.74 Hz), 42.5, 37.1, 35.9 ppm. ESI-MS m/ z 470.9 [M H]þ. HRMS (ESI ): calcd for C21H19FN8O2S [M H]þ 471.1721; found 471.1736.
N-(3-((5-ﬂuoro-4-(((2-(N-methylmethylsulfonamido)pyr- idin-3-yl)methyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-2-yl) amino)phenyl)acetamide (12i) Yellow solid, yield: 34%, m. p.
138e143 ◦C. 1H NMR (DMSO‑d6, 400 MHz) d ¼ 2.00 (s, 3 H), 3.17 (s,
3 H), 3.19 (s, 3 H), 4.83 (d, J ¼ 4.72 Hz, 2 H), 6.72 (s, 1 H), 6.98 (t,
J ¼ 8.08 Hz, 1 H), 7.05 (d, J ¼ 7.52 Hz, 1 H), 7.26 (brs, 1 H), 7.39 (dd,
J ¼ 7.60 Hz, J ¼ 4.76 Hz, 1 H), 7.45 (d, J ¼ 7.44 Hz, 1 H), 7.64 (s, 1 H),
7.86 (d, J ¼ 7.48 Hz, 1 H), 8.41 (s, 1 H), 8.70 (s, 1 H), 9.74 (s, 1 H), 10.84 (s, 1 H) ppm. 13C NMR (DMSO‑d6, 150 MHz) d ¼ 167.8, 156.3, 154.9, 152.1, 147.3, 147.0, 142.8 (d, J ¼ 239.9 Hz), 141.5, 139.0, 137.7, 134.8,
128.0, 123.9, 113.5, 111.7, 109.7, 100.2 (d, J ¼ 25.7 Hz), 87.2 (d,
J ¼ 15.6 Hz), 37.1, 36.0, 23.8 ppm. ESI-MS m/z 498.8 [MþH]þ. HRMS (ESI ): calcd for C22H23FN8O3S [M H]þ 499.1671; found 499.1675.
N-(3-((5-ﬂuoro-4-(((2-(N-methylmethylsulfonamido)pyr-
idin-3-yl)methyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-2-yl) amino)benzyl)acetamide (12j) Orange solid, yield: 30%, m. p. 119e122 ◦C. 1H NMR (DMSO‑d6, 400 MHz) d ¼ 1.85 (s, 3 H), 3.18 (s,
3 H), 3.20 (s, 3 H), 4.12 (d, J ¼ 5.52 Hz, 1 H), 4.83 (d, J ¼ 5.52 Hz, 2 H),
6.69 (d, J ¼ 7.28 Hz, 1 H), 6.74 (s, 1 H), 7.03 (t, J ¼ 7.72 Hz, 1 H), 7.27
(brs, 1 H), 7.39e7.42 (m, 2 H), 7.67 (d, J ¼ 8.36 Hz, 1 H), 7.86 (d,
J ¼ 7.28 Hz, 1 H), 8.23 (brs, 1 H), 8.42 (s, 1 H), 8.71 (s, 1 H), 10.86 (s, 1 H) ppm. 13C NMR (DMSO‑d6, 150 MHz) d ¼ 168.8, 156.3, 154.9,
152.1, 147.3, 147.1, 142.8 (d, J ¼ 239.9 Hz), 141.4, 139.3, 137.7, 134.7,
128.0, 123.9, 118.8, 117.0, 116.4, 100.3 (d, J ¼ 25.3 Hz), 87.2 (d,

J ¼ 15.7 Hz), 43.2, 37.2, 36.0, 22.4 ppm. ESI-MS m/z 512.8 [MþH]þ. HRMS (ESI ): calcd for C23H25FN8O3S [M H]þ 513.1827; found 513.1834.
N-(3-(((5-ﬂuoro-2-((4-(4-methylpiperazin-1-yl)phenyl) amino)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)methyl)pyr- idin-2-yl)-N-methylmethanesulfonamide (12k) Red solid, yield:
11%, m. p. 188e192 ◦C. 1H NMR (DMSO‑d6, 400 MHz) d 2.24 (s,
3 H), 2.50 (s, 4 H), 3.00 (s, 4 H), 3.17 (s, 3 H), 3.19 (s, 3 H), 4.80 (d,
J ¼ 5.08 Hz, 2 H), 6.68 (s, 1 H), 6.72 (d, J ¼ 8.52 Hz, 2 H), 7.22 (brs,
1 H), 7.42e7.45 (m, 3 H), 7.84 (d, J ¼ 7.64 Hz, 1 H), 8.41 (s, 2 H), 10.78 (s, 1 H) ppm. 13C NMR (DMSO‑d6, 150 MHz) d ¼ 156.6, 154.9, 152.1, 147.6, 147.0, 144.9, 142.9 (d, J ¼ 240.0 Hz), 137.6, 134.8, 133.8, 123.9,
119.4, 115.8, 99.8 (d, J ¼ 25.9 Hz), 86.9 (d, J ¼ 16.0 Hz), 54.4, 48.8,
45.4, 37.1, 36.0 ppm. ESI-MS m/z 539.9 [MþH]þ. HRMS (ESIþ): calcd for C25H30FN9O2S [M H]þ 540.2300; found 540.2296.
N-(3-(((5-ﬂuoro-2-((4-morpholinophenyl)amino)-7H-pyr-
rolo[2,3-d]pyrimidin-4-yl)amino)methyl)pyridin-2-yl)-N-meth- ylmethanesulfonamide (12l) Gray solid, yield: 24%, m. p. 155e158 ◦C. 1H NMR (DMSO‑d6, 400 MHz) d ¼ 2.97 (m, 4 H), 3.18 (s,
3 H), 3.19 (s, 3 H), 3.72 (m, 4 H), 4.80 (d, J ¼ 5.52 Hz, 2 H), 6.68 (s,
1 H), 6.73 (d, J ¼ 8.96 Hz, 2 H), 7.23 (brs, 1 H), 7.39e7.42 (m, 1 H),
7.46 (d, J ¼ 8.72 Hz, 2 H), 7.84 (d, J ¼ 6.84 Hz, 1 H), 8.43 (s, 2 H), 10.78 (s, 1 H) ppm. 13C NMR (DMSO‑d6, 150 MHz) d ¼ 156.6, 154.9, 152.1, 147.5, 147.0, 145.0, 142.9 (d, J ¼ 240.0 Hz), 137.6, 134.8, 134.1, 123.9,
119.3, 115.5, 99.9 (d, J ¼ 25.8 Hz), 86.9 (d, J ¼ 15.9 Hz), 66.0, 49.3,
37.1, 36.0 ppm. ESI-MS m/z 526.8 [MþH]þ. HRMS (ESIþ): calcd for C24H27FN8O3S [M H]þ 527.1984; found 527.1980.
N-(3-(((5-ﬂuoro-2-((2-methoxy-4-morpholinophenyl)
amino)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)methyl)pyr- idin-2-yl)-N-methylmethanesulfonamide (12m) Brown solid, yield: 18%, m. p. 138e141 ◦C. 1H NMR (DMSO‑d6, 400 MHz) d 3.03
(s, 4 H), 3.16 (s, 3 H), 3.26 (s, 3 H), 3.73 (s, 4 H), 3.80 (s, 3 H), 4.74 (d,
J ¼ 4.72 Hz, 2 H), 6.34 (d, J ¼ 7.20 Hz, 1 H), 6.60 (s, 1 H), 6.71 (s, 1 H),
7.03 (s, 1 H), 7.40 (m, 2 H), 7.85 (d, J ¼ 7.28 Hz, 1 H), 8.00 (d, J ¼ 8.72 Hz, 1 H), 8.42 (s, 1 H), 10.87 (s, 1 H) ppm. 13C NMR (DMSO‑d6, 150 MHz) d ¼ 156.3, 154.6, 152.0, 148.6, 147.3, 147.0, 146.1, 142.8 (d,
J ¼ 240.1 Hz), 137.5, 135.0, 123.9, 122.4, 119.2, 106.5, 100.1 (d,
J ¼ 25.9 Hz), 99.7, 87.1 (d, J ¼ 15.9 Hz), 66.0, 55.5, 49.3, 37.0,
35.8 ppm. ESI-MS m/z 556.8 [MþH]þ. HRMS (ESIþ): calcd for C25H29FN8O4S [MþH]þ 557.2089; found 557.2085.
4.1.16. 2,4-Dichloro-5-ﬂuoro-7-tosyl-7H-pyrrolo[2,3-d]pyrimidine (13)
To a solution of 920 mg (4.49 mmol of 2,4-dichloro-5-ﬂuoro-7H- pyrrolo[2,3-d]pyrimidine (3c), trimethylamine (1.87 mL,
13.47 mmol) and DMAP (55 mg (0.45 mmol)) in 30 mL of CH2Cl2,
1.03 g (5.39 mmol) of p-toluensulfonyl chloride was added. The reaction was stirred at room temperature for 2 h, then poured into 50 mL of water and extracted with dichloromethane. The organic layer was combined, dried and concentrated to provide crude product, which was puriﬁed via column chromatography (dichloromethane) to afford a white solid (1.15 g). Yield: 71%; 1H NMR (DMSO‑d6, 400 MHz) d ¼ 2.37 (s, 3 H), 7.49 (d, J ¼ 7.68 Hz, 2 H),
7.99 (d, J ¼ 8.16 Hz, 2 H), 8.26 (s, 1 H) ppm. 13C NMR (DMSO‑d6,
150 MHz) d ¼ 152.9, 151.0, 147.5, 146.8, 141.7 (d, J ¼ 254.6 Hz), 133.1,
130.3, 127.8, 112.4 (d, J ¼ 28.4 Hz), 109.1 (d, J ¼ 16.1 Hz), 21.0 ppm.
19F NMR (DMSO‑d6, 376 MHz) d ¼ 164.64 ppm. ESI-MS m/z 359.9 [MþH]þ.
4.1.17. 2-((2-Chloro-5-ﬂuoro-7-tosyl-7H-pyrrolo[2,3-d]pyrimidin- 4-yl)amino)-N-methylbenzamide (14)
A mixture of compound 13 (1150 mg, 3.20 mmol), 2-amino-N- methylbenzamide (577 mg, 3.84 mmol) and DIPEA (1.1 mL,
6.40 mmol) was reﬂuxed in MeCN (30 mL) with stirring for 40 h. The resulting mixture was cool to room temperature and ﬁltered to

afford compound 14. White solid, yield: 54%, m. p. >250 ◦C. 1H NMR (DMSO‑d6, 400 MHz) d ¼ 2.39 (s, 3 H), 2.79 (d, J ¼ 2.96 Hz, 3 H), 7.20
(t, J ¼ 5.04 Hz, 1 H), 7.50 (d, J ¼ 5.44 Hz, 2 H), 7.58 (t, J ¼ 5.32 Hz, 1 H),
7.78 (d, J ¼ 5.20 Hz, 1 H), 7.84 (s, 1 H), 7.99 (d, J ¼ 5.48 Hz, 2 H), 8.56
(d, J ¼ 5.60 Hz, 1 H), 8.81 (d, J ¼ 2.84 Hz, 1 H), 12.19 (s, 1 H) ppm. 13C NMR (DMSO‑d6, 150 MHz) d ¼ 168.6, 154.5, 152.3, 146.8, 146.3, 143.4 (d, J ¼ 252.6 Hz), 138.2, 133.6, 130.1, 127.9, 127.6, 122.9, 121.3, 107.1
(d, J ¼ 28.4 Hz), 96.4 (d, J ¼ 17.5 Hz), 26.1, 21.0 ppm. 19F NMR (DMSO‑d6, 376 MHz) d ¼ 162.38 ppm. ESI-MS m/z 473.8 [MþH]þ.
4.1.18. 2-((2-Chloro-5-ﬂuoro-7H-pyrrolo[2,3-d]pyrimidin-4-yl) amino)-N-methylbenzamide (15)
A mixture of compound 14 (480 mg, 1.01 mmol) and TBAF (400 mg, 1.52 mmol) was reﬂuxed in THF (20 mL) with stirring for
12 h. The resulting mixture was concentrated in vacuo, then washed with water and MeOH to afford compound 15 as a gray solid. Yield: 87%, m. p. >250 ◦C. 1H NMR (DMSO‑d6, 400 MHz)
d 2.76 (d, J 4.32 Hz, 3 H), 7.11 (t, J 7.40 Hz, 1 H), 7.26 (s, 1 H),
7.52 (t, J 7.56 Hz, 1 H), 7.72 (d, J 7.40 Hz, 1 H), 8.68 (d, J 8.48 Hz,
1 H), 8.75 (s, 1 H), 11.89 (s, 1 H), 11.94 (s, 1 H) ppm. ESI-MS m/z 319.9 [MþH]þ.
4.1.19. General procedure of the synthesis of 16a-d
A mixture of compound 15 (42 mg, 0.130 mmol), anilines (0.130 mmol), K2CO3 (54 mg, 0.391 mmol), Pd2(dba)3 (6 mg, 0.0065 mmol) and Xphos (6.2 mg, 0.013 mmol) was stirred in t-
BuOH (3 mL) at 100 ◦C for 12 h under a nitrogen atmosphere. The
resulting mixture was poured into ice-cold water and extracted twice with ethyl acetate. The organic layers was concentrated in vacuo and puriﬁed by chromatography on silica gel (CH2Cl2: MeOH, 30: 1) to afford compounds 16a-d respectively.
2-((5-ﬂuoro-2-((2-oxoindolin-5-yl)amino)-7H-pyrrolo[2,3-d] pyrimidin-4-yl)amino)-N-methylbenzamide (16a) Faint yellow solid, yield: 13%, m. p. >250 ◦C. 1H NMR (DMSO‑d6, 400 MHz)
d ¼ 2.75 (d, J ¼ 3.6 Hz, 3 H), 3.38 (s, 2 H), 6.66 (d, J ¼ 8.20 Hz, 1 H),
6.78 (s, 1 H), 7.03 (t, J ¼ 7.16 Hz, 1 H), 7.39e7.41 (m, 2 H), 7.66 (d,
J ¼ 8.00 Hz, 1 H), 7.70 (s, 1 H), 8.64 (s, 1 H), 8.84 (s, 1 H), 8.90 (d,
J ¼ 8.20 Hz, 1 H), 10.17 (s, 1 H), 10.97 (s, 1 H), 11.36 (s, 1 H). 13C NMR (DMSO‑d6, 150 MHz) d ¼ 176.1, 168.9, 156.3, 151.6, 147.9, 142.3 (d,
J ¼ 240.7 Hz), 140.1, 137.3, 135.3, 131.3, 127.7, 125.6, 121.4, 120.8,
120.0, 118.2, 116.3, 108.5, 101.2 (d, J ¼ 25.3 Hz), 88.3 (d, J ¼ 15.6 Hz),
36.0, 26.1 ppm. ESI-MS m/z 431.9 [MþH]þ. HRMS (ESIþ): calcd for C22H18FN7O2 [M H]þ 432.1579; found 432.1580.
2-((2-((3-(acetamidomethyl)phenyl)amino)-5-ﬂuoro-7H-
pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-methylbenzamide (16b) White solid, yield: 25%, m. p. >250 ◦C. 1H NMR (DMSO‑d6, 400 MHz) d 1.88 (s, 3 H), 2.81 (d, J 4.40 Hz, 3 H), 4.21 (d, J 5.64 Hz, 2 H),
6.80 (d, J 7.32 Hz, 1 H), 6.88 (s, 1 H), 7.09 (t, J 7.24 Hz, 1 H), 7.21 (t,
J 7.84 Hz, 1 H), 7.50 (t, J 7.32 Hz, 1 H), 7.55 (s, 1 H), 7.73 (d,
J 6.92 Hz, 1 H), 7.83 (d, J 8.36 Hz, 1 H), 8.31 (s, 1 H), 8.71 (d,
J 4.20 Hz, 1 H), 9.03 (d, J 8.56 Hz, 1 H), 9.10 (s, 1 H), 11.10 (s, 1 H), 11.50 (s, 1 H) ppm. 13C NMR (DMSO‑d6, 150 MHz) d 169.0, 168.9,
155.9, 151.6, 147.7, 142.2 (d, J 240.6 Hz), 141.1, 140.1, 139.4, 131.4,
128.1, 127.7, 121.3, 120.9, 119.9, 119.4, 117.5, 117.1, 101.6 (d,
J ¼ 25.5 Hz), 88.6 (d, J ¼ 15.6 Hz), 42.3, 26.1, 22.5 ppm. ESI-MS m/z
447.9 [M H]þ. HRMS (ESI ): calcd for C23H22FN7O2 [M H]þ 448.1892; found 448.1896.
2-((2-((3-acetamidophenyl)amino)-5-ﬂuoro-7H-pyrrolo[2,3- d]pyrimidin-4-yl)amino)-N-methylbenzamide (16c) White solid, yield: 27%, m. p. >250 ◦C. 1H NMR (DMSO‑d6, 400 MHz) d 2.01 (s,
3 H), 2.78 (d, J 4.36 Hz, 3 H), 6.84 (s, 1 H), 7.03e7.16 (m, 3 H), 7.42
(t, J 8.24 Hz, 1 H), 7.60 (d, J 7.92 Hz, 1 H), 7.69 (d, J 7.88 Hz, 1 H),
7.77 (s, 1 H), 8.67 (d, J 4.16 Hz, 1 H), 9.05 (d, J 8.64 Hz, 1 H), 9.07
(s, 1 H), 9.80 (s, 1 H), 11.05 (s, 1 H), 11.49 (s, 1 H) ppm. 13C NMR
(DMSO‑d6, 150 MHz) d ¼ 169.0, 167.9, 156.0, 151.6, 147.7, 142.2 (d,

J ¼ 240.7 Hz), 141.2, 140.2, 139.1, 131.4, 128.1, 127.6, 121.4, 120.8,
119.7, 114.3, 112.2, 110.5, 101.5 (d, J ¼ 25.5 Hz), 88.6 (d, J ¼ 15.4 Hz),
26.1, 23.8 ppm. ESI-MS m/z 433.9 [MþH]þ. HRMS (ESIþ): calcd for C22H20FN7O2 [M H]þ 434.1735; found 434.1738.
2-((5-ﬂuoro-2-((2-methoxy-4-morpholinophenyl)amino)- 7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-methylbenzamide (16d) Brown solid, yield: 17%, m. p. >250 ◦C. 1H NMR (DMSO‑d6,
400 MHz) d ¼ 2.78 (d, J ¼ 4.44 Hz, 3 H), 3.08 (brs, 4 H), 3.74 (brs,
4 H), 3.79 (s, 3 H), 6.47 (d, J ¼ 8.72 Hz, 1 H), 6.63 (s, 1 H), 6.79 (s, 1 H),
7.03 (t, J ¼ 7.04 Hz, 1 H), 7.40 (t, J ¼ 7.48 Hz, 1 H), 7.51 (s, 1 H), 7.68 (d,
J ¼ 6.72 Hz, 1 H), 7.79 (d, J ¼ 8.76 Hz, 1 H), 8.67 (s, 1 H), 8.83 (d,
J ¼ 8.12 Hz, 1 H), 10.98 (s, 1 H), 11.39 (s, 1 H) ppm. 13C NMR (DMSO‑d6, 150 MHz) d ¼ 168.9, 156.9, 151.7, 150.9, 148.2, 147.6, 142.3 (d, J ¼ 240.4 Hz), 140.1, 131.3, 127.6, 123.0, 121.6, 121.3, 120.8, 119.8,
106.4, 101.1 (d, J ¼ 25.7 Hz), 99.8, 88.3 (d, J ¼ 15.5 Hz), 66.0, 55.4,
49.2, 26.1 ppm. ESI-MS m/z 491.9 [MþH]þ. HRMS (ESIþ): calcd for C25H26FN7O3 [MþH]þ 492.2154; found 492.2156.
4.2. Biological assay

4.2.1. In vitro kinase assay
The 50 mL assay reaction mixture contains 40 mM Tris, pH 7.4, 10 mM MgCl2, 0.1 mg/mL BSA, 1 mM DTT, 240 ng FAK, 0.2 mg/mL Poly (Glu, Tyr) and 10 mM ATP. The compounds were diluted in 10% DMSO and 5 mL of the dilution was added to a 50 mL reaction so that the ﬁnal concentration of DMSO is 1% in all of reactions. All of the enzymatic reactions were conducted at 30 ◦C for 40 min. The assay was performed using Kinase-Glo Plus luminescence kinase assay
kit. It measures kinase activity by quantitating the amount of ATP remaining in solution following a kinase reaction. The luminescent signal from the assay is correlated with the amount of ATP present and is inversely correlated with the amount of kinase activity. The IC50 values were calculated using nonlinear regression with normalized dose—response ﬁt using Prism GraphPad software.
4.2.2. Cell culture and cell proliferation assay
The cell lines YY8103 and SMMC7721 were maintained as a monolayer culture in DMEM, supplemented with 10% FBS in a humidiﬁed atmosphere (5% CO2) at 37 ◦C.
Cell proliferation was determined using MTT (Adamas-Beta) according to manufacture instructions. 3-5 × 103 cells per well
were seeded in a 96-well plate, grown at 37 ◦C for 12 h. Subse-
quently, cells were treated with compounds at increasing concen- trations (0.1, 1, 10, 20, 50 and 100 mmol/L) in the presence of 10% FBS for 48 h. After 10 mL MTT dye (5 mg/mL) was added to each well,
cells were incubated at 37 ◦C for 4 h. Then, Remove the medium and add 200 mL DMSO to every well to dissolve the purple precipitate.
The absorbance (OD) was read on a SpectraMax M5 microplate reader (Molecular Devices) at 490 nm. Percent inhibition of IC50 values of compounds were calculated by comparison with DMSO- treated control wells. All the experiments were performed three times.

4.2.3. In vivo pharmacokinetic assay
All animal experiments were carried out in accordance with the National Institutes of Health guide for the care and use of laboratory animals.
The pharmacokinetics and oral bioavailability of 16c were investigated in rat following single intravenous and oral gavage. The tested sample was dissolved in DMSO/Solutol/saline (5/10/85). Six healthy male SD rats, which were purchased from Shanghai Sippr-BK laboratory animal Co. Ltd. (Shanghai, China), 6e8 weeks old and weighed 220 ± 30 g, were separated into 2 groups: three for
p.o. and three for i.v. Blood samples were collected at times of 0, 0.0833, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h after administration 2 mg/kg

of the tested compound for the i.v. group and at times of 0, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 h after administration 10 mg/kg of the test compound for the p.o. group. The blood were taken via jugular vein,
0.2 mL/time point. Sample were placed in tubes containing K2- EDTA and stored on ice until centrifuged. The blood samples were centrifuged at 6800 g for 6 min at 2e8 ◦C within 1 h after collected,
stored frozen at approximately 80 ◦C and analyzed by LCMS/MS. Pharmacokinetic parameters shown are the mean ± SEM.

4.2.4. Acute toxicity study
30 normal mice (Kunming mice, 18e22 g) were purchased from Shanghai Sippr-BK laboratory animal Co. Ltd. (Shanghai, China) and separated into 5 groups with half male and half female in each one. Tested compound was solved in DMSO/propylene glycol/PBS (2/5/ 3) and administrated intraperitoneally with 0, 5, 10, 50 and 200 mg/ kg respectively (n 6). After administration, the animals were observed and weighed every day for 10 days.

4.2.5. Mouse tumor xenograft efﬁcacy study
45 Six-week-old male BALB/c nude mice were purchased from Shanghai Sippr-BK laboratory animal Co. Ltd. (Shanghai, China). PF- 562271 and Soraﬁnib were purchased from Selleck Chemicals (Shanghai, China). A mixture solvent, DMSO/propylene glycol/PBS with a proportion of 2:5:3, was employed as negative control and the solvent of all tested compounds. SMMC 7721 hepatoma carci- noma xenografts were established by 1.0 107 cells subcutane- ously inoculated in nude mice. Treatments were initiated when tumors reached a mean group size of 25e35 mm3. All mice were randomized to 5 groups as control (solvent, 1.0 mL/kg, ip admin- istration), PF-562271 (30 mg/kg, ip administration), soraﬁnib (30 mg/kg, ip administration), 16c (10 mg/kg, ip administration) and 16c (30 mg/kg, ip administration) group. The size of tumors and the body weights of mice were measured and recorded individually every 3 days before every administration with microcalipers and scale respectively. An exponential rapid increase is observed in the tumor volume for the control group. Tumor volume (V) was calculated as V (length width [2])/2. Tumor growth inhibition (TGI) was calculated using the following formula: TGI (%) [1 (T T0)/(C C0)] 100, where T and T0 are the mean tumor volume on a specific experimental day and on the first day of treatment, respectively, for the experimental groups, and likewise, where C and C0 are the mean tumor volume for the control group.

4.2.6. Cancer signaling phosphoprotein proﬁling using antibody array
The Phospho Explorer Antibody Array, PEX100, was designed and manufactured by Full Moon Biosystems, Inc. (Sunnyvale, CA, USA), containing 1318 antibodies against the phosphorylation sites of 584 proteins. Each antibody has two replicates that are printed on a coated glass microscope slide, along with multiple positive and negative controls. Lysates were obtained from the samples of the test group with 30 mg/kg treatment and the negative control group and were used in the antibody array experiment. The experiment was performed by Wayen Biotechnology (Shanghai, China) ac- cording to an established protocol. The ﬂuorescence signal of each antibody was obtained from the ﬂuorescence intensity of the antibody spot. A ratio computation was used to measure the extent of protein phosphorylation. The phosphorylation ratio was calcu- lated as follows: phosphorylation ratio phospho value/unphos- pho value.

4.3. Molecular docking study

The published FAK crystal structure (PDB entry 3BZ3; Cancer Res. 2008, 68, 1935e1944) was used as a basis set for the FAK

structure. The binding region was deﬁned as all of the atoms that are 10 Å around the ligand in the crystal structure. Docking runs performed using the program Schrodinger, and all calculations utilized the standard default settings. Types of interaction of the complex and ligand were analyzed by PyMOL molecular graphics system after the molecular docking.

Declaration of competing interest

The authors declare that they have no known competing ﬁnancial interests or personal relationships that could have appeared to inﬂuence the work reported in this paper.

Acknowledgements

This work was supported ﬁnancially by Shanghai Committee of Science and Technology of China (No. 14431900600, 15431902900 and 19431901800). Victoria Muir, PhD, from Liwen Bianji, Edanz Group China (www.liwenbianji.cn/ac), edited the English text of a draft of this manuscript.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ejmech.2021.113670.

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