Synthesis and evaluation of FAK inhibitors with a 5-fluoro-7H-pyrrolo[2,3-d]pyrimidine scaffold as anti-hepatocellular carcinoma agents

European Journal of Medicinal Chemistry 223 (2021) 1 13670

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European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

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

^aDepartment of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai, 201203, China

^bKey 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

a b s t r a c t

Article history:

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.

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

1. Introduction

GSK 2256098 [22], VS-6063 [23] (also known as defactinib and PF-

04554878), and PND-1186 [24], are already undergoing clinical

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 [1 1], ovarian [12], prostate [13], and oral [14]

cancers.

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.

Therefore, FAK is a promising target for oncology [15,16], and

several FAK small molecule inhibitors have been reported and

investigated [1 7e20]. Some inhibitors, including PF-562271 [21],

* Corresponding author. ;

** Corresponding author.

E-mail addresses: jianhuang@sjtu.edu.cn (J. Huang), zhangqian5 1 1 @shmu.edu.

cn (Q. Zhang).

https://doi.org/10.1016/j.ejmech.2021.113670

H. Tan, Y. Liu, C. Gong et al.

European Journal of Medicinal Chemistry 223 (2021) 113670

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

2. Results and discussion

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 IC₅₀values 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

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

2

H. Tan, Y. Liu, C. Gong et al.

European Journal of Medicinal Chemistry 223 (2021) 113670

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

(3a and 3b) were obtained via the condensation of 6-aminouracil

(1) with 2-chloropropyl aldehyde and chloroacetone, respectively,

Kinase inhibitory activity of compounds 7 and 8 in vitro.

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-

Compd.

IC₅₀(nM)

7

8

55

206

1.5

PF-562271

addition, the pyrrole ring was oriented toward the gatekeeper

residue to form a hydrophobic interaction with Met 499, which

might contribute to the lower IC₅₀value of 7.

(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

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

3

H. Tan, Y. Liu, C. Gong et al.

European Journal of Medicinal Chemistry 223 (2021) 113670

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)₂catalyzed by Pd₂(dba)₃[38,39]. After removing the

chain at the 2-position was introduced via heating a mixture of 5e

protecting group with tetrabutylammonium ﬂuoride, another side

and 5-aminoindolin-2-one in EtOH under microwave irradiation at

4

H. Tan, Y. Liu, C. Gong et al.

European Journal of Medicinal Chemistry 223 (2021) 113670

150 ^ꢀC for 5 h according to the method described above.

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

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

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.

5

H. Tan, Y. Liu, C. Gong et al.

European Journal of Medicinal Chemistry 223 (2021) 113670

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

(

m

M)

SMMC7721 IC₅₀(mM)

7

7a

7b

7c

7d

7e

H

CH₃

H

F

Cl

H

CH₃

H

55

64

12000

22

21

13.55 2.44

12.43 2.98

>100

24.09 4.62

13.25 4.07

>100

52.79 3.05

18.70 1.46

>100

73.15 8.47

21.89 0.23

>100

CN

H

325

1.5

PF-562271

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.

6

H. Tan, Y. Liu, C. Gong et al.

European Journal of Medicinal Chemistry 223 (2021) 113670

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

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.

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

rimidine derivatives.

Compd.

Kinase assay IC₅₀(nM) YY8103 IC₅₀

(

m

M) SMMC7721 IC₅₀(mM)

7c

22

32

38

41

35

15

17

76

2098

23

21

13

26

23

22

14

12

23

24.09 4.62

>100

82.52 4.12

>100

18.25 2.49

>100

73.15 8.47

>100

27.93 2.22

>100

54.75 11.27

>100

12a

12b

12c

12d

12e

12f

12g

12h

12i

12j

12k

12l

12m

16a

16b

16c

16d

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 AUC_0-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 ¼ 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.

>100

20.54 2.79

14.44 4.23

2.39 0.59

>100

15.38 1.67

4.69 0.67

1.06 0.30

2.39 0.51

14.25 3.13

8.59 1.37

21.73 3.23

15.96 0.86

13.86 3.11

>100

15.28 3.42

10.04 2.59

18.71 3.60

10.07 1.70

33.86 6.61

23.92 3.74

PF-562271 1.5

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 IC₅₀values 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

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-

7

H. Tan, Y. Liu, C. Gong et al.

European Journal of Medicinal Chemistry 223 (2021) 113670

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.

tumor and the body weight. The tumor sizes were an average of

600.8 mm³for the vehicle and 343.4 and 295.9 mm³for soraﬁnib

and PF 562271, respectively, while those of the 16c-treated animals

were 348.3 and 153.5 mm³for 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.

Parameters

p.o. (10 mg/kg)

i.v. (2 mg/kg)

t_1/2(hr)

t_max(hr)

C_max(ng/mL)

AUC_0ꢁt(ng*hr/mL)

AUC_0ꢁ∞(ng*hr/mL)

Vss (mL/kg)

3.65 0.48

4.67 1.15

15.38 1.22

95.50 17.09

118.94 24.44

NA

1.46 0.26

0.08 0.00

5023.97 263.02

2298.79 115.22

2304.10 112.60

1842.6 423.51

869.41 42.87

0.51 0.01

Cl (mL/hr/kg)

MRT_0-t(hr)

MRT_0-∞(hr)

NA

4.54 0.21

6.62 0.37

0.83 0.15

0.53 0.03

Bioavailability (%)

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.

t_1/2, half-life; t_max, time to reach the maximum plasma concentration; C_max,

maximum plasma concentration; AUC, area under the plasma concentrationꢁtime

curve; MRT, mean residence time; Vss, volume of distribution at steady state; Cl,

blood clearance; NA, Not applicable; n ¼ 3, mean SEM.

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. 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 ¼ 8). All samples were injected

intraperitoneally every 3 days, after measurement of the size of the

8

H. Tan, Y. Liu, C. Gong et al.

European Journal of Medicinal Chemistry 223 (2021) 113670

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.

9

H. Tan, Y. Liu, C. Gong et al.

European Journal of Medicinal Chemistry 223 (2021) 113670

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.

10

H. Tan, Y. Liu, C. Gong et al.

European Journal of Medicinal Chemistry 223 (2021) 113670

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.

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,

4. Experimental details

400 MHz)

d

¼ 2.48 (s, 3 H), 7.11 (s, 1 H), 10.03 (brs, 1 H) ppm; ESI-MS

m/z 202.0 [MþH]^þ.

4.1. Chemistry

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.

4.1.1. 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 ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet,

m ¼ multiplet, dd ¼ double doublet and br ¼ 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.

4.1.5. 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)

1 H) ppm. ESI-MS m/z 206.0 [MþH]^þ.

d

¼ 7.75 (s, 1 H), 12.69 (s,

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

CH₂Cl₂and 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)

1 H) ppm; ESI-MS m/z 222.0 [MþH]^þ.

d

¼ 7.95 (s,1 H), 13.09 (s,

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.

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.

7H-pyrrolo[2,3-d]pyrimidine-2,4-diol (2) Light brown solid.

Yield: 93%; ESI-MS m/z 152.1 [MþH]^þ.

5-Methyl-7H-pyrrolo[2,3-d]pyrimidine-2,4-diol (2a) Light

brown solid. Yield: 79%; ESI-MS m/z 166.1 [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

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.

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)

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.

d

¼ 3.14 (s, 3 H), 3.26 (s, 3 H), 4.74 (d, J ¼ 5.40 Hz, 2 H), 6.59 (s, 1 H),

7.12 (s, 1 H), 7.41 (dd, J ¼ 7.72 Hz, J ¼ 4.72 Hz, 1 H), 7.82 (d,

J ¼ 7.64 Hz,1 H), 8.43 (d, J ¼ 4.64 Hz), 8.47 (s,1 H),11.75 (s,1 H) ppm.

ESI-MS m/z 367.1 [MþH]^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

¼ 2.38

4.1.4. General procedure of the synthesis of 3a and 3b

(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,

500 mg (3.03 mmol) of 5-methyl-7H-pyrrolo[2,3-d]pyrimidine-

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)

11

H. Tan, Y. Liu, C. Gong et al.

European Journal of Medicinal Chemistry 223 (2021) 113670

ppm. ESI-MS m/z 381.1 [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)

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,

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

400 MHz)

d

¼ 2.27 (s, 3 H), 3.12 (s, 3 H), 3.25 (s, 3 H), 4.70 (d,

J ¼ 5.16 Hz, 2 H), 6.22 (s, 1 H), 7.39 (dd, J ¼ 7.68 Hz, J ¼ 4.64 Hz, 1 H),

7.79 (d, J ¼ 6.52 Hz, 1 H), 8.24 (s, 1 H), 8.41 (d, J ¼ 2.80 Hz, 1 H), 11.58

(s, 1 H) ppm. ESI-MS m/z 381.1 [MþH]^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

¼ 3.10 (s, 3 H), 3.27 (s, 3 H), 4.69 (s, 2 H), 7.10 (s, 1 H),

(DMSO‑d₆, 400 MHz)

d

¼ 2.38 (s, 3 H), 3.10 (s, 3 H), 3.24 (s, 3 H), 4.75

7.38 (dd, J ¼ 7.72 Hz, J ¼ 4.68 Hz, 1 H), 7.83 (d, J ¼ 6.52 Hz, 1 H), 8.09

(brs, 1 H), 8.41 (d, J ¼ 2.92 Hz, 1 H), 11.64 (s, 1 H) ppm. ESI-MS m/z

385.1 [MþH]^þ.

(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.

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,

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

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)

(DMSO‑d₆, 400 MHz)

d

¼ 3.12 (s, 3 H), 3.28 (s, 3 H), 4.79 (d,

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

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.

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:

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.

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.

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,

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)

J ¼ 4.76 Hz, J ¼ 1.64 Hz, 1 H), 10.09 (s, 1 H), 10.99 (s, 1 H) ppm. ¹³

C

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.

NMR (DMSO‑d_6,150 MHz)

d

¼ 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

C

₂₂H₂₂N₈O₃S [MþH]^þ479.1608; 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.

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.

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,

12

H. Tan, Y. Liu, C. Gong et al.

European Journal of Medicinal Chemistry 223 (2021) 113670

J ¼ 3.20 Hz, 1 H), 10.08 (s, 1 H), 10.67 (s, 1 H) ppm. ¹³C NMR

(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.

(DMSO‑d_6,150 MHz)

d

¼ 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 [MþH]^þ493.1765; 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

209e213 ^ꢀC. ¹H NMR (DMSO‑d₆, 400 MHz)

d

¼ 3.18 (s, 3 H), 3.20 (s,

(d, J ¼ 8.12 Hz, 1 H), 7.34e7.41 (m, 2 H), 7.57 (s, 2 H), 7.80 (d,

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,

J ¼ 7.12 Hz, 1 H), 8.41 (m, 2 H), 10.10 (s, 1 H), 10.89 (brs, 1 H) ppm. ¹³

C

NMR (DMSO‑d_6,150 MHz)

d

¼ 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 [MþH]^þ493.1765; found 493.1774.

1 H), 10.88 (s, 1 H) ppm. ¹³C NMR (DMSO‑d_6,150 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

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%,

C

₂₀H₂₀FN₇O₂S [MþH]^þ442.1456; found 442.1460.

N-(3-(((5-ﬂuoro-2-((4-ﬂuorophenyl)amino)-7H-pyrrolo[2,3-

m. p. 165e168 ^ꢀC. ¹H NMR (DMSO‑d₆, 400 MHz)

d

¼ 3.16 (s, 3 H),

d]pyrimidin-4-yl)amino)methyl)pyridin-2-yl)-N-methyl-

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),

methanesulfonamide (12b) Orange solid, yield: 28%, m. p.

197e200 ^ꢀC. ¹H NMR (DMSO‑d₆, 400 MHz)

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,

d

¼ 3.19 (s, 3 H), 3.20 (s,

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),

10.79 (1 H) ppm. ¹³C NMR (DMSO‑d_6,150 MHz)

d

¼ 176.0, 156.6,

10.88 (s, 1 H) ppm. ¹³C NMR (DMSO‑d_6,150 MHz)

d

¼ 156.3, 156.1 (d,

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

(ESIþ): calcd for C₂₂H₂₁FN₈O₃S [MþH]^þ497.1514; 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.

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

[MþH]^þ. HRMS (ESIþ): calcd for C₂₀H₁₉F₂N₇O₂S [MþH]^þ460.1362;

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.

164e169 ^ꢀC. ¹H NMR (DMSO‑d₆, 400 MHz)

d

¼ 3.16 (s, 3 H), 3.18 (s,

3 H), 3.33 (s, 2 H), 4.84 (d, J ¼ 4.76 Hz, 2 H), 6.56 (d, J ¼ 8.32 Hz, 1 H),

6.94 (s, 2 H), 7.35 (d, J ¼ 8.00 Hz, 1 H), 7.40 (s, 1 H), 7.57 (s, 1 H), 7.85

(d, J ¼ 7.28 Hz, 1 H), 8.42 (s, 1 H), 8.57 (s, 1 H), 10.12 (s, 1 H), 11.31 (s,

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

1 H) ppm. ¹³C NMR (DMSO‑d_6,150 MHz)

d

¼ 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 (ESIþ): calcd for C₂₂H₂₁ClN₈O₃S [MþH]^þ513.1219; found

513.1221.

(s, 1 H), 10.97 (s, 1 H) ppm. ¹³C NMR (DMSO‑d_6,150 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

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. >

(ESIþ): calcd for

C

₂₀H₁₈ClF₂N₇O₂S [MþH]^þ494.0972; found

494.0977.

250 ^ꢀC. ¹H NMR (DMSO‑d₆, 400 MHz)

d

¼ 3.17 (s, 3 H), 3.18 (s, 3 H),

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.

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. ¹³C NMR (DMSO‑d_6,

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)

150 MHz)

d

¼ 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 [MþH]^þ504.1561; found 504.1565.

ppm. ¹³C NMR (DMSO‑d_6,150 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

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.

C

₂₁H₁₉FN₈O₂S [MþH]^þ467.1408; found 467.1411.

186e190 ^ꢀC. ¹H NMR (DMSO‑d₆, 400 MHz)

d

¼ 3.15 (s, 3 H), 3.17 (s,

N-(3-(((2-((3,4-dimethoxyphenyl)amino)-5-ﬂuoro-7H-pyr-

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.

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

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

(s, 1 H) ppm. ¹³C NMR (DMSO‑d_6,150 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,

13

H. Tan, Y. Liu, C. Gong et al.

European Journal of Medicinal Chemistry 223 (2021) 113670

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 [MþH]^þ502.1667; 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.

J ¼ 15.7 Hz), 43.2, 37.2, 36.0, 22.4 ppm. ESI-MS m/z 512.8 [MþH]^þ.

HRMS (ESIþ): calcd for C₂₃H₂₅FN₈O₃S [MþH]^þ513.1827; 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:

247e249 ^ꢀC. ¹H NMR (DMSO‑d₆, 400 MHz)

d

¼ 3.16 (s, 3 H), 3.21 (s,

11%, m. p. 188e192 ^ꢀC. ¹H NMR (DMSO‑d₆, 400 MHz)

d

¼ 2.24 (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,

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

1 H) ppm. ¹³C NMR (DMSO‑d_6,150 MHz)

d

¼ 156.26, 154.9, 152.3,

(s, 1 H) ppm. ¹³C NMR (DMSO‑d_6,150 MHz)

d

¼ 156.6, 154.9, 152.1,

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

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 [MþH]^þ540.2300; found 540.2296.

C

₂₃H₂₆FN₇O₅S [MþH]^þ532.1773; found 532.1777.

N-(3-(((5-ﬂuoro-2-((3-methoxy-2-methylphenyl)amino)-7H-

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.

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

¼ 1.97 (s, 3 H), 3.12 (s,

155e158 ^ꢀC. ¹H NMR (DMSO‑d₆, 400 MHz)

d

¼ 2.97 (m, 4 H), 3.18 (s,

3 H), 3.15 (s, 3 H), 3.75 (s, 3 H), 4.72 (d, J ¼ 5.12 Hz, 2 H), 6,63e6.65

(m, 2 H), 6.93 (t, J ¼ 7.72 Hz, 1 H), 7.05 (d, J ¼ 7.84 Hz, 1 H), 7.23 (brs,

1 H), 7.39 (t, J ¼ 5.08 Hz, 1 H), 7.70 (s, 1 H), 7.80 (d, J ¼ 7.24 Hz, 1 H),

8.42 (s, 1 H), 10.77 (s, 1 H) ppm. ¹³C NMR (DMSO‑d_6,150 MHz)

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‑d_6,150 MHz)

d

¼ 156.6, 154.9, 152.1,

d

¼ 157.5, 157.2, 154.9, 152.1, 147.8, 147.0, 142.8 (d, J ¼ 239.7 Hz),

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

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 [MþH]^þ. HRMS (ESIþ): calcd for C₂₂H₂₄FN₇O₃S [MþH]^þ

486.1718; found 486.1730.

C

₂₄H₂₇FN₈O₃S [MþH]^þ527.1984; found 527.1980.

N-(3-(((5-ﬂuoro-2-((2-methoxy-4-morpholinophenyl)

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.

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

¼ 3.03

178e183 ^ꢀC. ¹H NMR (DMSO‑d₆, 400 MHz)

d

¼ 3.11 (s, 3 H), 3.14 (s,

(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‑d_6,

3 H), 3.78 (s, 2 H), 4.80 (d, J ¼ 5.84 Hz, 2 H), 7.00 (d, J ¼ 6.64 Hz, 1 H),

7.15e7.23 (m, 4 H), 7.40 (dd, J ¼ 7.80 Hz, J ¼ 4.76 Hz, 1 H), 7.57e7.64

(m, 3 H), 7.73 (d, J ¼ 7.00 Hz, 1 H), 7.93 (s, 1 H), 8.41 (d, J ¼ 2.8 Hz,

1 H), 9.51 (s, 1 H) ppm. ¹³C NMR (DMSO‑d_6,150 MHz)

d

¼ 157.8 (d,

150 MHz)

d

¼ 156.3, 154.6, 152.0, 148.6, 147.3, 147.0, 146.1, 142.8 (d,

J ¼ 30.36 Hz), 156.1, 155.0, 152.1, 147.2, 147.1, 142.8 (d, J ¼ 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 [MþH]^þ. HRMS (ESIþ): calcd for C₂₁H₁₉FN₈O₂S [MþH]^þ

471.1721; found 471.1736.

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

C

₂₅H₂₉FN₈O₄S [MþH]^þ557.2089; found 557.2085.

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.

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

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‑d_6,150 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

(ESIþ): calcd for C₂₂H₂₃FN₈O₃S [MþH]^þ499.1671; 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.

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‑d_6,

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‑d_6,376 MHz)

[MþH]^þ.

d

¼ 164.64 ppm. ESI-MS m/z 359.9

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,

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

1 H) ppm. ¹³C NMR (DMSO‑d_6,150 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,

14

H. Tan, Y. Liu, C. Gong et al.

European Journal of Medicinal Chemistry 223 (2021) 113670

afford compound 14. White solid, yield: 54%, m. p. >250 ^ꢀC. ¹H NMR

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

(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

C

₂₂H₂₀FN₇O₂[MþH]^þ434.1735; found 434.1738.

(d, J ¼ 5.60 Hz, 1 H), 8.81 (d, J ¼ 2.84 Hz, 1 H), 12.19 (s, 1 H) ppm. ¹³

C

2-((5-ﬂuoro-2-((2-methoxy-4-morpholinophenyl)amino)-

NMR (DMSO‑d_6,150 MHz)

d

¼ 168.6, 154.5, 152.3, 146.8, 146.3, 143.4

7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-methylbenzamide

(d, J ¼ 252.6 Hz), 138.2, 133.6, 130.1, 127.9, 127.6, 122.9, 121.3, 107.1

(16d) Brown solid, yield: 17%, m. p. >250 ^ꢀC. ¹H NMR (DMSO‑d₆,

(d, J ¼ 28.4 Hz), 96.4 (d, J ¼ 17.5 Hz), 26.1, 21.0 ppm. ¹⁹F NMR

400 MHz)

d

¼ 2.78 (d, J ¼ 4.44 Hz, 3 H), 3.08 (brs, 4 H), 3.74 (brs,

(DMSO‑d_6,376 MHz)

d

¼ 162.38 ppm. ESI-MS m/z 473.8 [MþH]^þ.

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

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)

(DMSO‑d_6,150 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

C

₂₅H₂₆FN₇O₃[MþH]^þ492.2154; found 492.2156.

d

¼ 2.76 (d, J ¼ 4.32 Hz, 3 H), 7.11 (t, J ¼ 7.40 Hz, 1 H), 7.26 (s, 1 H),

7.52 (t, J ¼ 7.56 Hz,1 H), 7.72 (d, J ¼ 7.40 Hz,1 H), 8.68 (d, J ¼ 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.

4.2. Biological assay

4.2.1. In vitro kinase assay

The 50

10 mM MgCl₂, 0.1 mg/mL BSA, 1 mM DTT, 240 ng FAK, 0.2 mg/mL

Poly (Glu, Tyr) and 10 M ATP. The compounds were diluted in 10%

DMSO and 5 L of the dilution was added to a 50 L reaction so that

mL assay reaction mixture contains 40 mM Tris, pH 7.4,

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.

m

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

IC₅₀values were calculated using nonlinear regression with

normalized doseꢁresponse ﬁt using Prism GraphPad software.

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

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 ꢂ 10³cells 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-

(DMSO‑d_6,150 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

C

₂₂H₁₈FN₇O₂[MþH]^þ432.1579; found 432.1580.

2-((2-((3-(acetamidomethyl)phenyl)amino)-5-ﬂuoro-7H-

trations (0.1, 1,10, 20, 50 and 100

for 48 h. After 10 L 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 L DMSO to every well to dissolve the purple precipitate.

mmol/L) in the presence of 10% FBS

pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-methylbenzamide (16b)

m

White solid, yield: 25%, m. p. >250 ^ꢀC. ¹H NMR (DMSO‑d₆, 400 MHz)

d

¼ 1.88 (s, 3 H), 2.81 (d, J ¼ 4.40 Hz, 3 H), 4.21 (d, J ¼ 5.64 Hz, 2 H),

m

6.80 (d, J ¼ 7.32 Hz,1 H), 6.88 (s,1 H), 7.09 (t, J ¼ 7.24 Hz,1 H), 7.21 (t,

J ¼ 7.84 Hz, 1 H), 7.50 (t, J ¼ 7.32 Hz, 1 H), 7.55 (s, 1 H), 7.73 (d,

J ¼ 6.92 Hz, 1 H), 7.83 (d, J ¼ 8.36 Hz, 1 H), 8.31 (s, 1 H), 8.71 (d,

J ¼ 4.20 Hz, 1 H), 9.03 (d, J ¼ 8.56 Hz, 1 H), 9.10 (s, 1 H), 11.10 (s, 1 H),

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.

11.50 (s, 1 H) ppm. ¹³C NMR (DMSO‑d_6,150 MHz)

d

¼ 169.0, 168.9,

155.9, 151.6, 147.7, 142.2 (d, J ¼ 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 [MþH]^þ. HRMS (ESIþ): calcd for C₂₃H₂₂FN₇O₂[MþH]^þ

448.1892; found 448.1896.

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.

2-((2-((3-acetamidophenyl)amino)-5-ﬂuoro-7H-pyrrolo[2,3-

d]pyrimidin-4-yl)amino)-N-methylbenzamide (16c) White solid,

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

yield: 27%, m. p. >250 ^ꢀC. ¹H NMR (DMSO‑d₆, 400 MHz)

d

¼ 2.01 (s,

3 H), 2.78 (d, J ¼ 4.36 Hz, 3 H), 6.84 (s, 1 H), 7.03e7.16 (m, 3 H), 7.42

(t, J ¼ 8.24 Hz,1 H), 7.60 (d, J ¼ 7.92 Hz,1 H), 7.69 (d, J ¼ 7.88 Hz,1 H),

7.77 (s, 1 H), 8.67 (d, J ¼ 4.16 Hz, 1 H), 9.05 (d, J ¼ 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‑d_6,150 MHz)

d

¼ 169.0, 167.9, 156.0, 151.6, 147.7, 142.2 (d,

15

H. Tan, Y. Liu, C. Gong et al.

European Journal of Medicinal Chemistry 223 (2021) 113670

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.

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

4.2.4. Acute toxicity study

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.

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 ¼ 6). After administration, the animals were

observed and weighed every day for 10 days.

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.

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 ꢂ 10⁷cells 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 ¼ (length ꢂ width [2])/2. Tumor growth inhibition

(TGI) was calculated using the following formula: TGI

(%) ¼ [1 ꢁ (T ꢁ T₀)/(C ꢁ C₀)] ꢂ 100, where T and T₀are 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 C₀are the mean tumor volume for the control group.

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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17

Article Doi

Synthesis and evaluation of FAK inhibitors with a 5-fluoro-7H-pyrrolo[2,3-d]pyrimidine scaffold as anti-hepatocellular carcinoma agents

DOI: 10.1016/j.ejmech.2021.113670

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