Synthesis and evaluation of a series of pyridine and pyrimidine derivatives as type II c-Met inhibitors

Accepted Manuscript

Synthesis and evaluation of a series of pyridine and pyrimidine derivatives as

type II c-Met inhibitors

Yanmei Zhao, Jiankang Zhang, Rangxiao Zhuang, Ruoyu He, Jianjun Xi,

Xuwang Pan, Yidan Shao, Jinming Pan, Jingjing Sun, Zhaobin Cai, Shourong

Liu, Weiwei Huang, Xiaoqing Lv

PII:

S0968-0896(17)30319-X

http://dx.doi.org/10.1016/j.bmc.2017.04.003

BMC 13672

DOI:

Reference:

Bioorganic & Medicinal Chemistry

To appear in:

Received Date:

Accepted Date:

14 February 2017

4 April 2017

Please cite this article as: Zhao, Y., Zhang, J., Zhuang, R., He, R., Xi, J., Pan, X., Shao, Y., Pan, J., Sun, J., Cai, Z.,

Liu, S., Huang, W., Lv, X., Synthesis and evaluation of a series of pyridine and pyrimidine derivatives as type II c-

Met inhibitors, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.04.003

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers

we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and

review of the resulting proof before it is published in its final form. Please note that during the production process

errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis and evaluation of a series of pyridine and pyrimidine

derivatives as type II c-Met inhibitors

Yanmei Zhao^a, Jiankang Zhang^a, Rangxiao Zhuang^a, Ruoyu He^a, Jianjun Xi^a, Xuwang Pan^a, Yidan

Shao^a, Jinming Pan^a, Jingjing Sun^a, Zhaobin Cai^a, Shourong Liu^a, Weiwei Huang^a, Xiaoqing Lv^b*

^aDepartment of pharmaceutical Preparation, Hangzhou Xixi Hospital, Hangzhou 310023, Zhejiang

Province, China

^bCollege of medicine, Jiaxing University, Jiaxing 314001, Zhejiang Province, China

Abstract: In this study, a series of novel pyridine and pyrimidine-containing derivatives were designed,

synthesized and biologically evaluated for their c-Met inhibitory activities. In the biological evaluation,

half of the target compounds exhibited moderate to potent c-Met inhibitory activities. Among which, it

is noteworthy that compounds 13d not only showed most potent c-Met inhibitory potency but also

displayed excellent anti-proliferative activity (IC₅₀= 127 nM against EBC-1 cell line) as well as an

acceptable kinase selectivity profile. Moreover, the western blot assay indicated that 13d inhibited

c-Met phosphorylation in EBC-1 cells in a dose-dependent manner, with complete abolishment at 0.1

mM. All these experimental results suggested that 13d could be served as a promising lead compound

for the development of anticancer agents.

Keywords: c-Met; Inhibitor; Type II; Pyridine; Pyrimidine; Anti-cancer

*Corresponding author: Tel:. +86 573 8364 3848; fax: +86 573 8364 1678

E-mail address: lxqd1@126.com (X.Q. Lv)

1. Introduction

The receptor tyrosine kinase (RTK) c-Met, which is also called the hepatocyte growth factor (HGF)

receptor, plays critical roles in formation, dissemination and progression of many types of cancers, as

well as resistance.^1-3Aberrant c-Met activation via mutation, amplification, translocation, or HGF

stimulation is frequently observed in a wide variety of human cancers.^4-8For these reasons, targeting

c-Met has become an attractive strategy for cancer therapy.⁹Nowadays, various c-Met inhibitors have

been extensively studied. As reported, small c-Met inhibitors can be categorized into two classes based

on their different binding modes in the DFG motif (aspartate-phenylalanine-glycine) of the c-Met

activation loop,^10-12represented by Crizotinib (PF-02341066) (type I) and Foretinib (GSK1363089)

(type II) (Fig. 1). Type I inhibitors bind the DFG-in conformation with a U-shaped geometry to the

ATP-binding site at the entrance of a kinase pocket, while type II inhibitors bind the inactivated

DFG-out conformation that stretches from the ATP-binding site.¹³Recent research indicated that type II

inhibitors may be more effective than type I inhibitors against the mutations near the active site of

c-Met, because they not only bind to the same area occupied by the type I inhibitors but also exploit

hydrogen bonding and hydrophobic interactions with the allosteric site that beyond the entrance of

c-Met’s active site.^14,15

Figure 1. Representative type I and type II c-Met inhibitors

During the past ten years, numerous type II c-Met inhibitors have been discovered and some of

them are now in clinical trials, such as Foretinib, Cabozantinib, BMS-794833 and BMS-777607. As

described in Fig. 2, in general, the structures of type II c-Met inhibitors can be departed into four units

(bloke A-D) according to their interaction characteristics with protein molecular.^13,16In this study,

Cabozantinib and BMS-777607 are employed as lead compounds.¹⁷Cabozantinib containing a

substituted quinoline ring, potently inhibits the kinase activity of c-Met (IC₅₀= 1.3 nM), as well as

VEGFR 2 (IC₅₀= 0.035 nM), is now in phase 2 clinical trial because of its excellent in vivo efficacy

and favorable pharmacokinetic and preclinical safety profiles.^18,19The crystal structure of c-Met in

complex with Cabozantinib (or BMS-777607) showed that small molecule interact with c-Met protein

through a perfect hydrophobic pocket (Phe1134, Leu1195 and Phe1200) that adopts an inactive

DFG-out conformation, and several hydrogen bonds with key amino residues including Lys1110,

Met1160 and Asp1222, as well as ꢀ-ꢀ stacking interaction with Phe1223. BMS-777607 is an ATP

competitive c-Met inhibitor (IC₅₀= 3.9 nM) with good selectivity, which has slight activities against

VEGFR-2, Ron, Flt-1/3/4, Kit and PDGFRꢁ/ꢂ.

Hence, our strategies are application of molecular hybrid principle and bioisostere replacement to

generate novel type-II c-Met inhibitors based on the structures of Cabozantinib, BMS-777607 and

c-Met (Fig. 2). Clinical report showed that most of the type-II inhibitors elicit a certain degree of

toxicity in various organs because of VEGFR-related side effects and the more potent VEGFR activity

in some class II Met inhibitors may lead to suboptimal dosing for Met inhibition in clinical

applications.^8,20Therefore, block D in Cabozantinib which is a frequently appeared fragment in

VEGFR inhibitors was replaced with other nitrogen-containing aromatic rings in order to decrease the

VEGFR inhibitory activities and reduce the side effects. The phenyl ring (block C) and the critical

linker cyclopropane-1, 1-dicarboxamide (block B) were retained to form the ꢀ-ꢀ stacking interaction

and vital hydrogen bond with the receptor residues, respectively. Finally, different substituents were

performed on block A to investigate their influences on c-Met inhibitory activity (Fig. 2).

Figure 2. The design concept for novel type II c-Met inhibitors

2. Result and discussion

2.1 Chemistry

The synthetic route for target compounds 8a-k is outlined in Scheme 1. Firstly, cyclopropane-1,

1-dicarboxylic treated with aromatic amines in the presence of EDCI and HOBt to give the key

intermediates substituted carbamoyl cyclopropane carboxylic acid 2a-f. Treatment of

2-fluoro-4-nitrophenol with substituted dichloropyrimidines afforded aromatic ethers 4a and 4b, which

were then protected with methoxy-benzylamine to afford intermediates 5a and 5b. Reduction of

compounds 5a and 5b with Fe and NH₄Cl provided the aromatic amines 6a and 6b. Subsequently,

condensation reaction of compounds 6a and 6b with the substituted carbamoyl cyclopropane

carboxylic acid 2a-f provided compounds 7a-k, which was then deprotected with TFA to afford the

target compounds 8a-k. The target compounds 13a-d were synthesized following the route described in

Scheme 2. Commercially available pyridine carboxylic acid 9 was converted to amide 10 in the

presence of oxalyl dichloride and NH₃at room temperature. Then, nucleophilic substitution at the C-4

position of 10 with 4-amino-2-fluorophenol gave the aromatic amine 11. Coupling substituted

carbamoyl cyclopropane carboxylic acid 2a, 2c, 2d and 2e with amine 11 smoothly produced the

corresponding pyridine derivatives 12a-d. Finally, reaction of 12 with iodobenzene diacetate gave the

target compounds 13a-d.

Scheme 1. Synthesis of target compounds 8a-k. Reagents and conditions: (I) EDCI,

HOBt,cyclopropane-1, 1-dicarboxylic acid, DCM, 0 °C to rt; (II) NaOH, 2-fluoro-4-nitrophenol, H₂O,

acetone, N₂, 80 °C; (III) DMF, 100°C, 10 h; (IV) Fe, NH₄Cl, THF, H₂O, 60 °C; (V) EDCI, HOBt,

cyclopropane-1, 1-dicarboxamide, DCM, 0 °C to rt; (VI) TFA, DCM, 70 °C.

Scheme 2. Synthesis of target compounds13a-d. Reagents and conditions: (I) oxalyl dichloride, DCM,

25 °C and then NH₃, DCM, 20 °C; (II) t-BuOK, 4-amino-2-fluorophenol, DMF, rt, 30 min, then 50 °C,

3 h; (III) EDCI, HOBt, cyclopropane-1,1-dicarboxamide, DCM, Et₃N, 25 °C, 4 h; (IV) iodobenzene

diacetate, ethyl acetate , MeCN, H₂O, 0-25 °C, 2 h.

2.2 c-Met inhibitory activities

All the target compounds (8a-k and 13a-d) were screened for their c-Met inhibitory activities in vitro

by enzyme-linked immunosorbent assays (ELISAs). The results are summarized in Table 1. Half of the

target compounds (8b, 8c, 8f, 8i, 8k, 13a, 13b and 13d) exhibited moderate to potent inhibitory

activities against c-Met enzyme with IC₅₀values ranging from 17 to 457 nM. Compounds with a

substituted pyridine in the block D showed stronger kinase inhibitory activities than that of the

substituted pyrimidine (13d vs. 8f and 8k, 13b vs. 8c and 8i, 13a vs. 8a and 8g). Among the pyrimidine

derivatives (8a-k), 4-fluorophenyl derivative (8f and 8k) displayed more potent than that of compounds

with other substitution pattern in block A (8b-e and 8g-j). These data also suggested that minor changes

at block A could drastically influence the kinase inhibitory activities of the target compounds (8f vs.

8a-e). Similarly, compounds 8g-k (or 13a-d) with different substituents only at the block A position

displayed the same regularity. Additionally, replacement of the amide (12d) at R₄position with amine

(13d) caused a significantly increased in inhibitory activity. Among which, compound 13d (IC₅₀= 17

nM) showed enzymatic activity comparable to that of the positive control BMS-777607. It indicated

that this compound deserved further study with regard to its application in the treatment of cancer.

Table 1. c-Met enzymatic inhibitory activities of target compounds

Compound

R₁

X

C

N

C

-

Y

N

C

-

R₃

-

R₄

-NH₂

-CONH₂

-NH₂

-

IC₅₀(nM)^a

2140

455

8a

3-fluorophenyl

4-methylphenyl

4-chlorophenyl

4-methoxyphenyl

4-fluorophenyl

3-fluorophenyl

4-methoxyphenyl

4-chlorophenyl

4-fluorophenyl

3-fluorophenyl

4-chlorophenyl

4-fluorophenyl

-

8b

-

8c

-

457

8d

-

>10⁴

258

8e

-

8f

-

8g

H

Cl

-

2279

>10⁴

278

8h

8i

8j

8k

>10⁴

237

12d

2547

235

13a

13b

95

13c

1078

17

13d

BMS-777607

Cabozantinib

Staurosporine

12

-

2.9

-

90

^aThe values are an average of three separate determinations.

2.3 Cell proliferation assay

Based on the c-Met inhibitory activities, selected compounds (8f, 8i, 8k, 13a, 13b and 13d) were

screened for their in vitro anti-proliferative activities against two cancer cell lines (HepG2 and EBC-1)

by MTT-based assay. The results were summarized in Table 2. With the exception of compounds 13b

and 13d, all the selected compounds showed slight activities against HepG2 and EBC-1, which

indicated that introduction of pyridine ring in block D and 4-halogenated benzyl in block A is important

to maintain potent anti-proliferative activities. In additional, both of the compounds 13b and 13d

displayed the selectivity for EBC-1 cancer cell line. As expected, the most potent compound 13d

exhibited stronger activity (IC₅₀= 127.4 nM) than that of BMS-777607 against EBC-1 cell line, while

less potent than that of the dual VEGFR-2/c-Met inhibitor Cabozantinib. Thus, this compound can be

used as a promising compound for further biological evaluation.

Table 2.Anti-proliferative activities of selected compounds against HepG2 and ECB-1 cell lines

(IC₅₀, nM)^a

Compound

HepG2

>10³

12.0

ECB-1

>10³

8f

8i

8k

13a

13b

967.2

127.4

162.3

51.4

13d

BMS-777607

Cabozantinib

Doxorubicin

98.7

305.1

^aThe values are an average of three separate determinations.

2.4 Enzymatic selectivity assays

The inhibitory activities of 13d against EGFR, FLT3, VEGFR-2, KIT, PDGFRꢁ and RET kinase were

also evaluated using the homogeneous time resolved fluorescence (HTRF) method. As the data showed

in Table 3, compound 13d displayed moderate-to-good selectivity over EGFR, FLT3, KIT, PDGFRꢁ,

and RET (around 9 to 10⁴fold). Importantly, although the potency against c-Met was decreased, 13d

significantly decreased the selectivity for VEGFR-2 compared to Cabozantinib. These selectivity

profiles were consistent with the molecule’s anti-proliferative activities. It may provide an opportunity

to investigate the further selectivity and reduce VEGFR-2 related side effects.

Table 3. Kinase selectivity of compound 13d

Enzyme (IC₅₀, nM)^a

Compound

c-Met

17

EGFR

>10⁴

225

FLT3

2216

18

VEGFR-2

55

KIT

279

144

6.6

PDGFRꢁ

172

RET

387

594

10.1

4.7

13d

BMS-777607

Cabozantinib

Staurosporine

12

180

28

>10⁴

2.9

90

25

0.086

4.8

44

0.52

4.5

0.43

^aThe values are an average of three separate determinations.

2.5 Western blot analysis

To further determine whether the c-Met kinase inhibition of 13d in a cell-free system can be

recapitulated in vitro, a study was carried out to determine the effects of 13d on phosphorylation of

c-Met in EBC-1 cancer cells. As the results displayed in Fig. 3, 13d inhibits phosphorylation of c-Met

in a dose-dependent manner, and that it brings about complete inhibition of the process at 0.1 mM. The

data support the conclusion that 13d inhibits c-Met signaling and c-Met dependent cell proliferation.

Figure 3. Dose-dependent inhibition of 13d on c-Met phosphorylation in EBC-1

2.6 Docking study

To further elucidate the binding modes of target compound 13d with the active site of c-Met, docking

simulation studies were carried out by using Glidemodule of Schrödinger package. In our study, the

crystal structure of c-Met (PDB ID code: 3LQ8) was selected as the docking template. The protein

structure was prepared by Protein Preparation Wizard of Maestro, and the structure of compound 13d

was prepared by LigPrep. As depicted in Fig.4, 13d took on a similar binding mode with type II

inhibitors, occupied the ATP-binding site and formed four hydrogen bonds with residues Met1160,

Asp1222 and Lys1110, respectively. In addition, the central fluorophenyl ring (block C) could formꢀ-ꢀ

stacking with Phe1223, and the terminal 4-fluorophenyl ring (block A) insert into a hydrophobic pocket

formed by Phe1134, Leu1195 and Phe1200. These docking results gave a rational explanation for the

potency of compound 13d and it is consistent with our speculated.

Figure 4. The docking mode of 13d with c-Met. (A) ribbon show of compound 13d bound to c-Met; (B)

2D depiction of the ligand-protein interaction

3. Conclusion

A novel series of pyrimidine and pyridine-containing derivatives were designed, synthesized and

biologically evaluated for their c-Met inhibitory activities. Half of the derivatives exhibited moderate to

potent c-Met inhibitory activities. Aminopyridine derivative 13d exhibited most potent activities both

in enzyme and cell proliferation assays in vitro with an acceptable kinase selectivity profile. In

particular, compound 13d significantly decreased the selectivity for VEGFR-2 compared to

Cabozantinib, which may provide an opportunity to investigate the further selectivity and reduce

VEGFR-2 related side effects. All these results suggested that compound 13d could be a potentially

interesting lead molecule for further investigation.

4. Experimental procedures

4.1 Chemistry

Mass spectra (MS) were taken in ESI mode on Agilent 1100 LC-MS (Agilent, Palo Alto, CA, U.S.A.).

¹H NMR and ¹³C NMR spectra were recorded on Bruker 400 MHz spectrometers (Bruker Bioscience,

Billerica, MA, USA) with TMS as an internal standard and CDCl₃or DMSO-d₆as solvents. Coupling

constants (J) were reported in Hertz (Hz). Splitting patterns were designated as singlet (s), broad singlet

(brs), doublet (d), double doublet (dd), triplet (t), quartet (q), and multiplet (m). All materials were

obtained from commercial suppliers and were used without further purification. Reaction time and

purity of the products were monitored by TLC on FLUKA silica gel aluminum cards (0.2mm thickness)

with fluorescent indicator 254 nm. Column chromatography was run on silica gel (200-300 mesh) from

Qingdao Ocean Chemicals (Qingdao, Shandong, China). All yields are unoptimized and generally

represent the result of a single experiment.

4.2 General methods for the preparation of compounds 2a-f

The mixture of appropriate 1a-e (45.00 mmol), cyclopropane-1, 1-dicarboxylic acid (8.78 g, 67.49

mmol), EDCI (12.94 g, 67.49 mmol), HOBt (9.12g, 67.49 mmol) and DCM (200.00 mL) were stirred

at 25 °C for 12 hours under N₂atmosphere and monitored by TLC. The organic phase was then washed

with H₂O (100 ml × 3), dried over Na₂SO₄, filtered and concentrated under reduced pressure to give a

residue and the crude product was purified by flash chromatography (petroleum ether: ethyl acetate =

10:1 to 5:1) to give the corresponding compounds 2a-e.

4.2.1 1-(3-fluorophenylcarbamoyl)cyclopropanecarboxylic acid (2a)

White solid; Yield: 33%; ¹H-NMR (400MHz, DMSO-d₆): δ = 10.73 (s, 1H, NH), 7.61 (dt, 1H, J = 12.0,

1.6 Hz, NH), 7.35-7.30 (m, 2H, Ar-H), 6.91-6.86 (m, 1H, Ar-H), 1.42 (s, 4H, CH₂+CH₂); ESI-MS: m/z

= 223.8 [M+H]⁺.

4.2.2 1-(p-tolylcarbamoyl)cyclopropanecarboxylic acid (2b)

1

White solid; Yield: 37%; H-NMR (400MHz, DMSO-d₆): δ = 10.52 (s, 1H, NH), 7.47 (d, 2H, J = 8.4

Hz, Ar-H), 7.10 (d, 2H, J = 8.4 Hz, Ar-H), 2.25 (s, 3H, CH₃), 1.41 (s, 4H, CH₂+CH₂); ESI-MS: m/z =

219.9 [M+H]⁺.

4.2.3 1-(4-chlorophenylcarbamoyl)cyclopropanecarboxylic acid (2c)

1

White solid; Yield: 16%; H-NMR (400MHz, DMSO-d₆): δ = 10.64 (s, 1H, NH), 7.64 (d, 2H, J = 8.8

Hz, Ar-H), 7.36 (d, 2H, J = 8.8 Hz, Ar-H), 1.40 (s, 4H, CH₂+CH₂); ESI-MS: m/z = 239.8 [M+H]⁺.

4.2.4 1-(4-methoxyphenylcarbamoyl)cyclopropanecarboxylic acid (2d)

Grey solid; Yield: 23%; ¹H-NMR (400MHz, DMSO-d₆): δ = 10.43 (s, 1H, NH), 7.49 (d, 2H, J = 9.2 Hz,

Ar-H), 6.87 (dd, 2H, J = 6.8, 2.0 Hz, Ar-H), 3.72 (s, 3H, CH₃), 1.41 (s, 4H, CH₂+CH₂); ESI-MS: m/z =

235.9 [M+H]⁺.

4.2.5 1-(4-fluorobenzylcarbamoyl)cyclopropanecarboxylic acid (2e)

1

White solid; Yield: 23%; H-NMR (400MHz, DMSO-d₆): δ = 9.05 (s, 1H, NH), 7.33-7.29 (m, 2H,

Ar-H), 7.16-7.12 (m, 2H, Ar-H), 4.33 (d, 2H, J = 6.4 Hz, CH₂), 1.41 (s, 4H, CH₂+CH₂); ESI-MS: m/z =

237.8 [M+H]⁺.

4.2.6 1-(4-fluorophenylcarbamoyl)cyclopropanecarboxylic acid (2f)

1

White solid; Yield: 22%; H-NMR (400MHz, DMSO-d₆): δ = 10.57 (s, 1H, NH), 7.79-7.44 (m, 2H,

Ar-H), 7.25-6.94 (m, 2H, Ar-H), 1.40 (s, 4H, CH₂+CH₂); ESI-MS: m/z = 224.0 [M+H]⁺.

4.3 Preparation of compounds 4a and 4b

A solution of 2-fluoro-4-nitrophenol (15.00 g, 95.48 mmol), compound 3a or 3b (95.48 mmol) and

NaOH (5.73 g, 143.22 mmol) in acetone (200.00 mL) and H₂O (200.00 mL) was stirred at 80 °C for 12

hours under N₂atmosphere. Upon completion of the reaction, the mixture was concentrated under

reduced pressure to remove the solvent. The residue was washed with H₂O (100 mL) and then filtered

and dried to obtain the desired target compounds 4a or 4b.

4.3.1 2-chloro-4-(2-fluoro-4-nitrophenoxy)pyrimidine (4a)

Yellow solid; Yield: 35%; ¹H-NMR (400MHz, DMSO-d₆): δ = 8.77 (d, 1H, J = 6.0 Hz, pyrimidine-H),

8.43 (dd, 1H, J = 10.0, 2.4 Hz, Ar-H), 8.24 (d, 1H, J = 8.8 Hz, Ar-H), 7.81(t, 1H, J = 4.4 Hz, Ar-H),

7.41 (dd, 1H, J = 5.6, 0.8Hz, pyrimidine-H); ESI-MS: m/z = 269.8 [M+H]⁺.

4.3.2 4-chloro-6-(2-fluoro-4-nitrophenoxy)pyrimidine (4b)

Brown solid; Yield: 47%; ¹H-NMR (400MHz, DMSO-d₆): δ = 8.71 (s, 1H, NH), 8.42 (dd, 1H, J = 10.0,

2.4 Hz, pyrimidine-H), 8.24-8.21 (dq, 1H, J = 8.8, 1.2 Hz, pyrimidine-H), 7.80-7.74 (m, 2H, Ar-H);

ESI-MS: m/z = 269.8 [M+H]⁺.

4.4 General procedure for the synthesis of key intermediates 5a and 5b

To a solution of (4-methoxyphenyl)methanamine (5.09 g, 37.09 mmol), an intermediate 4a or 4b

(10.00 g, 37.09 mmol) in DMF (100.00 mL) was added and stirred at 100°C for 10 hours under N₂

atmosphere. Once the reaction was completed, the mixture was diluted with H₂O (100 ml) and

extracted with ethyl acetate (200 mL × 2). The combined organic layers were washed with brine, dried

over with Na₂SO₄, filtered and concentrated under reduced pressure to give a residue. The residue was

purified by flash chromatography to get the title compounds 5a and 5b.

4.4.1 4-(2-fluoro-4-nitrophenoxy)-N-(4-methoxybenzyl)pyrimidin-2-amine (5a)

1

Yield: 53%; H-NMR (400MHz, DMSO-d₆): δ = 8.19-8.16 (m, 1H, Ar-H), 8.01-7.98 (m, 1H, Ar-H),

7.69-7.59 (m, 2H, Ar-H), 7.24 (d, 1H, J = 8.8 Hz, pyrimidine-H), 7.14 (t, 1H, J = 8.8 Hz, Ar-H), 6.90 (d,

1H, J = 4.8 Hz, pyrimidine-H), 6.49-6.33 (m, 2H, Ar-H), 4.41 (d, 1H, J = 5.2 Hz, CH₂), 4.29 (d, 1H, J=

6.0 Hz, CH₂), 3.73 (s, 3H, CH₃); ESI-MS: m/z = 370.9 [M+H]⁺.

4.4.2 6-(2-fluoro-4-nitrophenoxy)-N-(4-methoxybenzyl)pyrimidin-4-amine (5b)

1

Yield: 58%; H-NMR (400MHz, DMSO-d₆): δ = 8.32 (d, 1H, J = 8.0 Hz, pyrimidine-H), 8.14 (d, 1H, J

= 9.2, Ar-H), 7.62 (t, 2H, J = 8.4 Hz, Ar-H), 7.26 (d, 2H, J = 7.6 Hz, pyrimidine-H), 6.12 (s, 2H, Ar-H);

ESI-MS: m/z = 370.9 [M+H]⁺.

4.5 General procedure for the preparation of 6a and 6b

In a dry flask under N₂atmosphere, compound 5a or 5b (3.00 g, 8.10 mmol), Fe (2.26 g, 40.50 mmol),

NH₄Cl (2.17 g, 40.50 mmol) in THF (25.00 mL) and H₂O (25.00 mL) was stirred at 60°C for 12 h.

After the reaction was completed, the mixture was filtered and concentrated under reduced pressure to

give a residue and then diluted with H₂O (50 mL) and extracted with ethyl acetate (50 mL × 2). The

combined organic layers were washed with brine (50 mL × 2), dried over Na₂SO₄and then

concentrated under reduced pressure to give compound 6a or 6b.

4.5.1 4-(4-amino-2-fluorophenoxy)-N-(4-methoxybenzyl)pyrimidin-2-amine (6a)

1

Yield: 91%; H-NMR (400MHz, DMSO-d₆): δ = 8.30 (s, 1H, Ar-H), 7.91-7.89 (m, 1H, Ar-H), 7.24 (d,

2H, J = 8.4 Hz, Ar-H), 6.91-6.89 (m, 4H, Ar-H), 6.49 (d, 2H, J = 5.6 Hz, Ar-H), 4.42 (d, 2H, J = 5.2 Hz,

CH₂), 3.73-3.72 (m, 5H, NH₂and CH₃); ESI-MS: m/z = 341.0 [M+H]⁺.

4.5.2 6-(4-amino-2-fluorophenoxy)-N-(4-methoxybenzyl)pyrimidin-4-amine (6b)

1

Yield: 73%; H-NMR (400MHz, DMSO-d₆): δ = 8.10 (s, 1H, pyrimidine-H), 7.80 (s, 1H, Ar-H), 7.21

(d, 2H, J = 6.4 Hz, Ar-H), 6.89 (s, 3H, Ar-H), 6.47-6.39 (m, 2H, Ar-H), 5.79 (s, 1H, pyrimidine-H),

4.40 (s, 2H, CH₂), 3.72 (s, 3H, CH₃); ESI-MS: m/z = 341.0 [M+H]⁺.

4.6 General procedure for the key intermediates 7a-k

The mixture of an appropriate 6a or 6b (300.00 mg, 0.88 mmol), 2a-f (0.88 mmol), EDCI (202.77 mg,

1.06mmol), HOBt (142.92 mg, 1.06 mmol) and DCM (10.0 mL) were stirred at 20°C for 12 hours

under N₂atmosphere. The organic phase was then concentrated under reduced pressure to remove

solvent, washed with H₂O (10 ml), extracted with ethyl acetate (10 mL × 2), dried over Na₂SO₄, filtered

and concentrated under reduced pressure to give a residue. The crude product was purified by flash C₁₈

silica gel chromatography (ISCO®; 4g SepaFlash® C₁₈Silica Flash Column, Eluent of 0~70%

MeCN/H₂O @ 60mL/min) to give the corresponding compounds 7a-k.

4.6.1

N-(3-fluoro-4-(2-(4-methoxybenzylamino)pyrimidin-4-yloxy)phenyl)-N-(3-fluorophenyl)cyclopro

pane-1,1-dicarboxamide (7a)

1

Yield: 28%; H-NMR (400MHz, DMSO-d₆): δ = 10.22 (s, 2H, Ar-H), 8.15-8.08 (m, 1H, Ar-H),

7.80-7.74 (m, 2H, Ar-H), 7.62 (d, 1H, J = 12.0 Hz, Ar-H), 7.38-7.10 (m, 6H, Ar-H), 6.90-6.84 (m, 4H,

Ar-H), 4.34-4.26 (m, 1H, CH₂), 3.68 (s, 3H, CH₃), 1.46 (s, 4H, CH₂+CH₂); ESI-MS: m/z = 545.9

[M+H]⁺.

4.6.2

N-(3-fluoro-4-(2-(4-methoxybenzylamino)pyrimidin-4-yloxy)phenyl)-N-p-tolylcyclopropane-1,1-d

icarboxamide (7b)

1

Yield: 34%; H-NMR (400MHz, DMSO-d₆): δ = 10.28 (s, 1H, NH), 9.88 (s, 1H, COOH), 8.15 (s, 1H,

Ar-H), 7.77 (s, 2H, Ar-H), 7.48(d, 2H, J = 8.4 Hz, Ar-H), 7.24(t, 1H, J = 8.4 Hz, Ar-H), 7.10 (d, 2H, J =

8.0, Ar-H), 6.79 (s, 3H, Ar-H), 6.22 (s, 1H, Ar-H), 4.32 (s, 1H, CH₂), 3.99 (s, 1H, CH₂), 3.69 (s, 3H,

CH₃), 2.25 (s, 3H, CH₃), 1.46 (d, 4H, J = 6.4 Hz, CH₂+CH₂); ESI-MS: m/z = 541.9 [M+H]⁺.

4.6.3

N-(4-chlorophenyl)-N-(3-fluoro-4-(2-(4-methoxybenzylamino)pyrimidin-4-yloxy)phenyl)cyclopro

pane-1,1-dicarboxamide (7c)

1

Yield: 31%; H-NMR (400MHz, DMSO-d₆): δ = 10.24 (s, 1H, NH), 10.12 (d, 1H, J = 4.8 Hz, Ar-H),

8.16 (s, 1H, Ar-H), 7.80 (s, 1H, Ar-H), 7.65 (d, 2H, J = 8.8 Hz, Ar-H), 7.35 (d, 3H, J = 8.8 Hz, Ar-H),

7.27-7.18 (m, 2H, Ar-H), 6.86 (d, 1H, J = 6.3 Hz, Ar-H), 6.23 (s, 1H, Ar-H), 4.35 (t, 1H, J = 5.2 Hz,

CH₂), 4.27 (d, 1H, J = 6.0 Hz, CH₂), 3.70 (d, 3H, J = 11.6 Hz, CH₃), 1.46 (d, 4H, J = 4.4 Hz,

CH₂+CH₂); ESI-MS: m/z = 561.9 [M+H]⁺.

4.6.4

N-(3-fluoro-4-(2-(4-methoxybenzylamino)pyrimidin-4-yloxy)phenyl)-N-(4-methoxyphenyl)cyclop

ropane-1,1-dicarboxamide (7d)

1

Yield: 33%; H-NMR (400MHz, DMSO-d₆): δ = 10.35-10.30 (m, 1H, NH), 9.80 (d, 1H, J = 5.2 Hz,

Ar-H), 8.15 (s, 1H, Ar-H), 7.80-7.71 (m, 2H, Ar-H), 7.49 (d, 2H, J = 8.8 Hz, Ar-H), 7.27-7.18 (m, 2H,

Ar-H), 6.88-6.86 (m, 4H, Ar-H), 6.23 (s, 1H, Ar-H), 4.27 (d, 1H, J = 5.6 Hz, CH₂), 3.72-3.68 (m, 7H,

CH₃+CH₂), 1.46-1.44 (m, 4H, CH₂+CH₂); ESI-MS: m/z = 557.9 [M+H]⁺.

4.6.5

N-(3-fluoro-4-(2-(4-methoxybenzylamino)pyrimidin-4-yloxy)phenyl)-N-(4-fluorobenzyl)cycloprop

ane-1,1-dicarboxamide (7e)

1

Yield: 22%. H NMR (400MHz, DMSO-d₆): δ = 8.53-8.34 (m, 1H, NH), 8.19-7.97 (m, 1H, NH),

7.91-7.64 (m, 2H, Ar-H+pyrimidine-H), 7.21 (s, 1H, Ar-H), 7.30-7.07 (m, 6H, Ar-H), 6.85 (d, 1H, J =

6.8 Hz, pyrimidine-H), 6.82-6.70 (m, 1H, Ar-H), 6.23 (s, 1H, Ar-H), 4.35-4.25 (m, 3H, CH₂+CH), 3.99

(s, 1H, CH), 3.71 (d, 3H, J = 10.4 Hz, CH₃), 1.39 (s, 4H, CH₂+CH₂); ESI-MS: m/z = 559.9 [M+H]⁺.

4.6.6

N-(3-fluoro-4-(2-(4-methoxybenzylamino)pyrimidin-4-yloxy)phenyl)-N-(4-fluorophenyl)cyclopro

pane-1,1-dicarboxamide (7f)

Yield: 28%. ¹H NMR (400MHz, DMSO-d₆): δ = 10.31 (s, 1H, NH), 10.03 (s, 1H, NH), 8.16 (d, 1H, J =

4.0 Hz, Ar-H), 7.81-7.63 (m, 4H, Ar-H), 7.45 (s, 1H, Ar-H), 7.26-7.14 (m, 4H, Ar-H), 6.82-6.77 (m, 3H,

Ar-H), 6.25 (d, 1H, J = 5.2 Hz, Ar-H), 3.69 (s, 3H, CH₃), 3.42 (s, 2H, CH₂), 1.47 (s, 4H, CH₂+CH₂);

ESI-MS: m/z = 544.4 [M+H]⁺.

4.6.7

N-(3-fluoro-4-(6-(4-methoxybenzylamino)pyrimidin-4-yloxy)phenyl)-N-(3-fluorophenyl)cyclopro

pane-1,1-dicarboxamide (7g)

Yield: 25%. ¹H NMR (400MHz, DMSO-d₆): δ = 10.21 (s, 1H, NH), 10.20 (s, 1H, NH), 8.16 (d, 1H, J =

4.0 Hz, Ar-H), 7.73 (d, 1H, J = 13.2 Hz, Ar-H), 7.63 (d, 1H, J = 11.6 Hz, , Ar-H), 7.43-7.28 (m, 3H,

Ar-H), 7.23 (d, 3H, J = 8.4 Hz, Ar-H), 6.88 (d, 3H, J = 8.4 Hz, Ar-H), 5.92 (s, 1H, pyrimidine-H), 4.42

(s, 2H, CH₂), 3.72 (s, 3H, CH₃), 1.45 (s, 4H, CH₂+CH₂); ESI-MS: m/z = 545.9 [M+H]⁺.

4.6.8

N-(3-fluoro-4-(6-(4-methoxybenzylamino)pyrimidin-4-yloxy)phenyl)-N-(4-methoxyphenyl)cyclop

ropane-1,1-dicarboxamide (7h)

1

Yield: 32%. H NMR (400MHz, DMSO-d₆): δ = 10.33 (s, 1H, NH), 9.80 (s, 1H, NH), 8.09 (s, 1H,

pyrimidine-H), 7.74 (d, 1H, J = 12.4 Hz, Ar-H), 7.50 (d, 2H, J = 8.8 Hz, Ar-H), 7.37 (d, 1H, J = 8.4 Hz,

Ar-H), 7.23 (d, 3H, J = 8.4 Hz, Ar-H), 6.98-6.78 (m, 4H, Ar-H), 5.92 (s, 1H, pyrimidine-H), 4.43 (s, 2H,

CH₂), 3.72 (d, 6H, J = 1.2 Hz, CH₃+CH₃), 1.55-1.34 (m, 4H, CH₂+CH₂); ESI-MS: m/z = 557.9

[M+H]⁺.

4.6.9

N-(4-chlorophenyl)-N-(3-fluoro-4-(6-(4-methoxybenzylamino)pyrimidin-4-yloxy)phenyl)cyclopro

pane-1,1-dicarboxamide (7i)

1

Yield: 23%. H NMR (400MHz, DMSO-d₆): δ = 10.23 (s, 1H, NH), 10.12 (s, 1H, NH), 8.09 (s, 1H,

pyrimidine-H), 7.80-7.57 (m, 3H, Ar-H), 7.36 (d, 3H, J = 8.8 Hz, Ar-H) 7.24 (d, 3H, J = 8.0 Hz, Ar -H),

6.89 (d, 2H, J = 8.4 Hz, Ar -H), 5.92 (s, 1H, pyrimidine-H), 4.43 (s, 2H, CH₂), 3.73 (s, 3H, CH₃), 1.45

(d, 4H, J = 2.4 Hz, CH₂+CH₂); ESI-MS: m/z = 561.9 [M+H]⁺.

4.6.10

N-(3-fluoro-4-(6-(4-methoxybenzylamino)pyrimidin-4-yloxy)phenyl)-N-(4-fluorobenzyl)cycloprop

ane-1,1-dicarboxamide (7j)

1

Yield: 26%. H NMR (400MHz, DMSO-d₆): δ = 10.74 (s, 1H, NH), 8.47 (t, J = 5.6 Hz, 1H, NH), 8.09

(s, 1H, pyrimidine-H), 7.85 (s, 1H, Ar-H), 7.74 (d, 1H, J = 11.6 Hz, Ar-H), 7.39-7.27 (m, 3H, Ar-H),

7.23 (d, 3H, J = 8.2 Hz, Ar-H), 7.13 (t, 2H, J = 8.8 Hz, Ar-H), 6.88 (d, 2H, J = 8.4 Hz, Ar-H), 5.92 (s,

1H, CH₂), 4.43 (s, 1H, CH₂), 4.30 (d, 2H, J = 5.6 Hz, CH₂), 3.72 (s, 3H, CH₃), 1.37 (s, 4H, CH₂+CH₂);

ESI-MS: m/z = 559.9 [M+H]⁺.

4.6.11

N-(3-fluoro-4-(6-(4-methoxybenzylamino)pyrimidin-4-yloxy)phenyl)-N-(4-fluorophenyl)cyclopro

pane-1,1-dicarboxamide (7k)

1

Yield: 29%. H NMR (400MHz, DMSO-d₆): δ = 9.52 (s, 1H, NH), 8.54 (s, 1H, NH), 8.21 (s, 1H,

pyrimidine-H), 7.67 (dd, 1H, J = 9.2, 1.6 Hz, Ar-H), 7.46-7.43 (m, 2H, Ar-H), 7.36 (d, 2H, J = 7.2 Hz,

Ar-H), 7.18-7.11 (m, 2H, Ar-H), 7.07-7.03 (m, 2H, Ar-H), 6.90 (d, 2H, J = 6.8 Hz, Ar -H), 5.84 (s, 1H,

pyrimidine-H), 4.41 (s, 2H, CH₂), 3.81 (s, 3H, CH₃), 1.71-1.61 (m, 4H, CH₂+CH₂); ESI-MS: m/z =

544.4 [M+H]⁺.

4.7 General methods for preparation of pyrimidine-containing target compounds 8a-k

An appropriate intermediates 7a-k (0.27 mmol) and TFA (3.14 g, 27.50 mmol) were dissolved in DCM

(10.00 mL), and the reaction mixture was stirred at 70°C for 24 hours under N₂atmosphere. The

solution was concentrated in vacuum and the residue was purified by prep-HPLC (TFA condition) to

give desired compounds 8a-k.

4.7.1

N-(4-(2-aminopyrimidin-4-yloxy)-3-fluorophenyl)-N-(3-fluorophenyl)cyclopropane-1,1-dicarboxa

mide (8a)

1

Yield: 56%. Purity: 97%. H NMR (400MHz, DMSO-d₆): δ = 10.27 (d, 2H, J = 5.2 Hz, NH), 8.19 (d,

1H, J = 5.6 Hz, pyrimidine-H), 7.79 (dd, 1H, J = 13.2, 2.4 Hz, Ar-H), 7.69 (dd, 1H, J = 11.8, 2.0 Hz,

Ar-H), 7.49-7.36 (m, 3H, Ar-H), 7.36-7.26 (m, 1H, Ar-H), 7.01-6.90 (m, 1H, Ar-H), 6.72 (s, 2H, NH₂),

6.28 (d, 1H, J = 5.6 Hz, Ar-H), 1.52 (d, 4H, J = 3.2 Hz, CH₂+CH₂); ¹³C NMR (100 MHz, d₆-DMSO): δ

= 169.38, 168.59, 168.56, 164.03, 162.47 (d, J_C-F= 239.6 Hz), 160.58, 153.82 (d, J_C-F= 242.8 Hz),

141.17 (d, J_C-F= 11.0 Hz), 137.84 (d, J_C-F= 10.0 Hz), 135.24 (d, J_C-F= 12.7 Hz), 130.54 (d, J_C-F= 9.4

Hz), 124.42 (d, J_C-F= 2.0 Hz), 117.05 (d, J_C-F= 3.0 Hz), 116.36 (d, J_C-F= 3.0 Hz), 110.41 (d, J_C-F= 20.9

Hz), 109.27 (d, J_C-F= 23.0 Hz), 107.51 (d, J_C-F= 26.1 Hz), 95.89, 32.58, 15.73; ESI-MS: m/z =

425.9[M+H]⁺.

4.7.2

N-(4-(2-aminopyrimidin-4-yloxy)-3-fluorophenyl)-N-p-tolylcyclopropane-1,

1-dicarboxamide (8b)

1

Yield: 50%. Purity: 99%. H NMR (400MHz, DMSO-d₆): δ = 10.25 (s, 1H, NH), 9.87 (s, 1H, NH),

8.11 (d, 1H, J = 5.6 Hz, Ar-H), 7.77-7.66 (m, 1H, Ar-H), 7.48 (d, 2H, J = 8.4 Hz, Ar-H), 7.37 (d, 1H, J

= 8.8 Hz, Ar-H), 7.28-7.19 (m, 1H, Ar-H), 7.09 (d, 2H, J = 8.4 Hz, Ar-H), 6.64 (s, 2H, NH₂), 6.20 (d,

1H, J = 5.6 Hz, Ar-H), 2.24 (s, 3H, CH₃), 1.49-1.38 (m, 4H, CH₂+CH₂); ¹³C NMR (100 MHz,

d₆-DMSO): δ = 169.39, 168.95, 168.29, 164.04, 160.57, 153.83 (d, J_C-F= 242.9 Hz), 137.81 (d, J_C-F

=

10.0 Hz), 136.72, 135.23 (d, J_C-F= 13.0 Hz), 133.08, 129.34, 124.42 (d, J_C-F= 2.0 Hz), 121.01, 117.01

(d, J_C-F= 3.0 Hz), 109.25 (d, J_C-F= 23.0 Hz), 95.89, 32.10, 20.91, 15.82; ESI-MS: m/z = 421.9 [M+H]⁺.

4.7.3

N-(4-(2-aminopyrimidin-4-yloxy)-3-fluorophenyl)-N-(4-chlorophenyl)cyclopropane-1,1-dicarboxa

mide (8c)

1

Yield: 30%. Purity: 98%. H NMR (400MHz, DMSO-d₆): δ = 10.22 (s, 1H, NH), 10.11 (s, 1H, NH),

8.11 (d, 1H, J = 5.6 Hz, Ar-H), 7.71 (dd, 1H, J = 13.2,2.4 Hz, Ar-H), 7.64 (d, 2H, J = 8.8 Hz, Ar-H),

7.40-7.32 (m, 3H, Ar-H), 7.29-7.19 (m, 1H, Ar-H), 6.64 (s, 2H, NH₂), 6.20 (d, 1H, J = 5.6 Hz, Ar-H),

1.43 (d, 4H, J = 6.0 Hz, CH₂+CH₂); ¹³C NMR (100 MHz, d₆-DMSO): δ = 169.38, 168.64, 168.47,

164.03, 160.58, 153.83 (d, J_C-F= 243.0 Hz), 138.33, 137.84 (d, J_C-F= 9.9 Hz), 135.23 (d, J_C-F= 12.8

Hz),128.85, 127.67, 124.41 (d, J_C-F= 2.5 Hz), 122.40, 117.01 (d, J_C-F= 3.0 Hz), 109.24 (d, J_C-F= 22.9

Hz), 95.89, 32.46, 15.75; ESI-MS: m/z = 441.9 [M+H]⁺.

4.7.4

N-(4-(2-aminopyrimidin-4-yloxy)-3-fluorophenyl)-N-(4-methoxyphenyl)cyclopropane-1,1-dicarbo

xamide (8d)

1

Yield: 46%. Purity: 98%. H NMR (400MHz, DMSO-d₆): δ = 10.39 (s, 1H, NH), 9.86 (s, 1H, NH),

8.18 (d, 1H, J = 5.6 Hz, Ar-H), 7.80 (dd, 1H, J = 13.2, 2.0 Hz, Ar-H), 7.56 (d, 2H, J = 9.2 Hz, Ar-H),

7.45 (d, 1H, J = 9.2 Hz, Ar-H), 7.36-7.26 (m, 1H, Ar-H), 6.94 (d, 2H, J = 9.2 Hz, Ar-H), 6.72 (s, 2H,

NH₂), 6.27 (d, 1H, J = 5.6 Hz, Ar-H), 3.78 (s, 3H, CH₃), 1.60-1.40 (m, 4H, CH₂+CH₂); ¹³C NMR (100

MHz, d₆-DMSO): δ = 169.39, 168.93, 168.27, 164.02, 160.58, 156.10, 153.83 (d, J_C-F= 242.9 Hz),

137.81 (d, J_C-F= 9.8 Hz), 135.20 (d, J_C-F= 12.6 Hz), 132.22, 124.42 (d, J_C-F= 2.0 Hz), 122.76, 116.97

(d, J_C-F= 3.0 Hz), 114.10, 109.19 (d, J_C-F= 23.1 Hz), 95.89, 55.65, 31.91, 15.81; ESI-MS: m/z = 437.9

[M+H]⁺.

4.7.5

N-(4-(2-aminopyrimidin-4-yloxy)-3-fluorophenyl)-N-(4-fluorobenzyl)cyclopropane-1,1-dicarboxa

mide (8e)

1

Yield: 42%. Purity: 98%. H NMR (400MHz, DMSO-d₆): δ = 10.72 (s, 1H, NH), 8.47 (t, 1H, J = 5.6

Hz, NH), 8.12 (d, 1H, J = 5.6 Hz, Ar-H), 7.74 (dd, 1H, J = 12.8, 2.0 Hz, Ar-H), 7.44-7.22 (m, 4H,

Ar-H), 7.14 (t, 2H, J = 8.8 Hz, Ar-H), 6.65 (s, 2H, NH₂), 6.21 (d, 1H, J = 5.6 Hz, Ar-H), 4.31 (d, 2H, J

= 5.6 Hz, CH₂), 1.39 (s, 4H, CH₂+CH₂); ¹³C NMR (100 MHz, d₆-DMSO): δ = 170.75, 169.38, 168.79,

164.02, 161.63 (d, J_C-F= 240.7 Hz), 160.58, 153.89 (d, J_C-F= 243.0 Hz), 137.66 (d, J_C-F= 9.8 Hz),

135.98 (d, J_C-F= 2.9 Hz), 135.19 (d, J_C-F= 12.5 Hz), 129.60 (d, J_C-F= 8.1 Hz), 124.41 (d, J_C-F= 2.0 Hz),

116.68 (d, J_C-F= 3.0 Hz), 115.42 (d, J_C-F= 21.1 Hz), 108.91 (d, J_C-F= 23.1 Hz), 95.87, 42.44, 30.51,

16.09; ESI-MS: m/z = 439.9 [M+H]⁺.

4.7.6

N-(4-(2-aminopyrimidin-4-yloxy)-3-fluorophenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxa

mide (8f)

1

Yield: 49%. Purity: 95%. H NMR (400MHz, DMSO-d₆): δ = 10.27 (s, 1H, NH), 10.01 (s, 1H, NH),

8.13 (d, 1H, J = 4.4 Hz, Ar-H), 7.75 (dd, 1H, J = 10.0, 0.8 Hz, Ar-H), 7.64 (dd, 1H, J = 6.8, 4.0 Hz,

Ar-H), 7.39 (d, 1H, J = 6.8 Hz, Ar-H), 7.27 (t, J = 7.2 Hz, 1H, Ar-H), 7.15 (t, J = 6.8 Hz, 2H, Ar-H),

6.65 (s, 1H, Ar-H), 6.22 (d, 1H, J = 4.4 Hz, Ar-H), 1.46 (d, 4H, J = 7.6 Hz, CH₂+CH₂); ¹³C NMR (100

MHz, d₆-DMSO): δ = 169.08, 168.74, 168.45, 166.29, 158.77 (d, J_C-F= 238.6 Hz), 158.30, 153.90 (d,

J_C-F= 242.8 Hz), 137.83 (d, J_C-F= 9.8 Hz), 135.62 (d, J_C-F= 2.8 Hz), 135.48 (d, J_C-F= 12.4 Hz), 124.37,

122.93 (d, J_C-F= 7.8 Hz), 116.94 (d, J_C-F= 3.2 Hz), 115.47 (d, J_C-F= 22.0 Hz), 109.13 (d, J_C-F= 23.1

Hz), 86.26, 32.18, 15.8; ESI-MS: m/z = 426.0 [M+H]⁺.

4.7.7

N-(4-(6-aminopyrimidin-4-yloxy)-3-fluorophenyl)-N-(3-fluorophenyl)cyclopropane-1,1-dicarboxa

mide (8g)

1

Yield: 53%. Purity: 99%. H NMR (400MHz, DMSO-d₆): δ = 10.21 (d, 2H, J = 5.2 Hz, NH), 8.04 (s,

1H, Ar-H), 7.75 (dd, 1H, J = 13.2, 2.4 Hz, Ar-H), 7.63 (dd, 1H, J = 11.2, 2.0 Hz, Ar-H), 7.44-7.29 (m,

3H, Ar-H), 7.24 (t, 1H, J = 8.8 Hz, Ar-H), 7.01-6.78 (m, 3H, NH₂+Ar-H), 5.82 (s, 1H, Ar-H), 1.46 (s,

4H, CH₂+CH₂); ¹³C NMR (100 MHz, d₆-DMSO): δ = 169.08, 168.59, 168.56, 166.50, 162.46 (d, J_C-F

=

239.6 Hz), 158.50, 153.91 (d, J_C-F= 242.9 Hz), 141.16 (d, J_C-F= 11.1 Hz), 137.79 (d, J_C-F= 9.7 Hz),

135.55 (d, J_C-F= 12.7 Hz), 130.53 (d, J_C-F= 9.4 Hz), 124.38 (d, J_C-F= 2.0 Hz), 117.00 (d, J_C-F= 2.5 Hz),

116.43 (d, J_C-F= 2.5 Hz), 110.42 (d, J_C-F= 20.9 Hz), 109.20 (d, J_C-F= 23.1 Hz), 107.57 (d, J_C-F= 26.0

Hz), 86.19, 32.53, 15.8; ESI-MS: m/z = 425.9 [M+H]⁺.

4.7.8

N-(4-(6-aminopyrimidin-4-yloxy)-3-fluorophenyl)-N-(4-methoxyphenyl)cyclopropane-1,1-dicarbo

xamide (8h)

1

Yield: 58%. Purity: 99%. H NMR (400MHz, DMSO-d₆): δ = 10.34 (s, 1H, NH), 9.81 (s, 1H, NH),

8.04 (s, 1H, Ar-H), 7.76 (dd, 1H, J = 13.2, 2.0 Hz, Ar-H), 7.51 (d, 2H, J = 8.8 Hz, Ar-H), 7.39 (d, 1H, J

= 9.2 Hz, Ar-H), 7.30-7.14 (m, 1H, Ar-H), 7.02-6.78 (m, 4H, NH₂+Ar-H), 5.83 (s, 1H, Ar-H), 3.72 (s,

3H, CH₃), 1.64-1.34 (m, 4H, CH₂+CH₂); ¹³C NMR (100 MHz, d₆-DMSO): δ = 169.09, 168.91, 168.27,

166.43, 158.43, 156.10, 153.92 (d, J_C-F= 242.8 Hz), 137.79 (d, J_C-F= 9.9 Hz), 135.48 (d, J_C-F= 12.5

Hz), 132.22, 124.40, 122.82, 116.93 (d, J_C-F= 3.0 Hz), 114.08, 109.12 (d, J_C-F= 23.0 Hz), 86.20, 55.64,

31.87, 15.88; ESI-MS: m/z = 437.9[M+H]⁺.

4.7.9

N-(4-(6-aminopyrimidin-4-yloxy)-3-fluorophenyl)-N-(4-chlorophenyl)cyclopropane-1,1-dicarboxa

mide (8i)

1

Yield: 44%. Purity: 99%. H NMR (400MHz, DMSO-d₆): δ = 10.23 (s, 1H, NH), 10.11 (s, 1H, NH),

8.01 (s, 1H, NH), 7.73 (dd, 1H, J = 12.8, 2.0 Hz, Ar-H), 7.65 (d, 2H, J = 8.8 Hz, Ar-H), 7.43-7.30 (m,

3H, Ar-H), 7.22 (t, 1H, J = 8.8 Hz, Ar-H), 6.89 (s, 2H, NH₂), 5.80 (s, 1H, Ar-H), 1.43 (d, 4H, J = 2.4

Hz, CH₂+CH₂); ¹³C NMR (100 MHz, d₆-DMSO): δ = 169.09, 168.63, 168.48, 166.50, 158.50, 153.91

(d, J_C-F= 242.9 Hz), 138.32, 137.80 (d, J_C-F= 10.0 Hz), 135.53 (d, J_C-F= 12.5 Hz), 128.83, 127.68,

124.40 (d, J_C-F= 2.0 Hz), 122.47, 116.96 (d, J_C-F= 3.0 Hz), 109.17 (d, J_C-F= 23.1 Hz),, 86.19, 32.41,

15.83; ESI-MS: m/z = 441.8 [M+H]⁺.

4.7.10

N-(4-(6-aminopyrimidin-4-yloxy)-3-fluorophenyl)-N-(4-fluorobenzyl)cyclopropane-1,1-dicarboxa

mide (8j)

1

Yield: 86%. Purity: 99%. H NMR (400MHz, DMSO-d₆): δ = 10.74 (s, 1H, NH), 8.48 (t, 1H, J = 6.0

Hz, NH), 8.03 (s, 1H, Ar-H), 7.76 (dd, 1H, J = 13.2, 2.4 Hz, Ar-H) , 7.39-7.28 (m, 3H, Ar-H), 7.27-7.20

(m, 1H, Ar-H), 7.14 (t, 2H, J = 8.8 Hz, Ar-H), 6.8 (s, 2H, NH₂), 5.82 (s, 1H, Ar-H), 4.31 (d, 2H, J = 6.0

Hz, CH₂), 1.38 (s, 4H, CH₂+CH₂); ¹³C NMR (100 MHz, d₆-DMSO): δ = 170.72, 169.07, 168.73,

166.50, 161.63 (d, J_C-F= 240.7 Hz), 158.49, 153.98 (d, J_C-F= 242.9 Hz), 137.64 (d, J_C-F= 9.9 Hz),

136.00 (d, J_C-F= 3.0 Hz), 135.48 (d, J_C-F= 12.5 Hz), 129.59 (d, J_C-F= 8.0 Hz), 124.48 (d, J_C-F= 2.0 Hz),

116.58 (d, J_C-F= 3.0 Hz), 115.41 (d, J_C-F= 21.1 Hz), 108.81 (d, J_C-F= 23.1 Hz),, 86.20, 42.45, 30.52,

16.11; ESI-MS: m/z = 439.9[M+H]⁺.

4.7.11

N-(4-(6-aminopyrimidin-4-yloxy)-3-fluorophenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxa

mide (8k)

Yield: 41%. Purity: 95%. ¹H NMR (400MHz, DMSO-d₆): δ = 9.69 (s, 1H, NH), 8.31 (s, 1H, NH), 8.24

(s, 1H, NH), 7.71 (dd, 1H, J = 9.6, 2.0 Hz, Ar-H), 7.46-7.43 (m, 2H, Ar-H), 7.08 (t, 2H, J = 6.8 Hz,

Ar-H), 5.98 (s, 1H, Ar-H), 1.77 (dd, 2H, J = 6.4, 4.0 Hz, CH₂), 1.63 (dd, 2H, J = 6.4, 4.0 Hz, CH₂); ¹³C

NMR (100 MHz, d₆-DMSO): δ = 169.39, 168.72, 168.43, 164.02, 160.57, 158.77 (d, J_C-F= 238.9 Hz),

153.83 (d, J_C-F= 242.9 Hz), 137.84 (d, J_C-F= 9.9 Hz), 135.64 (d, J_C-F= 2.4 Hz), 135.22 (d, J_C-F= 12.1

Hz), 124.43, 122.86 (d, J_C-F= 7.8 Hz), 116.98 (d, J_C-F= 3.9 Hz), 115.50 (d, J_C-F= 22.1 Hz), 109.21 (d,

J_C-F= 23.0 Hz), 95.89, 32.23, 15.75; ESI-MS: m/z = 426.2 [M+H]⁺.

4.8 Synthesis of 3, 4-dichloropicolinamide (10)

To a solution of 3, 4-dichloropicolinic acid (1.50 g, 7.81 mmol) in DCM (100.00 mL) was added

oxalyldichloride (991.64 mg, 7.81 mmol) and stirred at 25°C for 3 h. The reaction mixture was

concentrated under reduced pressure to remove solvent and then NH₃was bubbled into the solution of

3, 4-dichloropyridine-2-carbonyl chloride (80.00 mg, 0.38 mmol) in DCM (30.00 mL) at 20°C for 30

minutes. Then the reaction mixture was concentrated under reduced pressure to remove the DCM to

afford the desired compound 10 (60.00 mg, 0.27 mmol, 85% purity) as a grey solid.

Yield: 70%; ESI-MS: m/z = 190.9[M+H]⁺.

4.9 Preparation of 4-(4-amino-2-fluorophenoxy)-3-chloropicolinamide (11)

4-amino-2-fluorophenol (2.39 g, 18.84 mmol) and t-BuOK (2.82 g, 25.12 mmol) were added into DMF

(50.00 mL). After stirring at rt for 30 min, compound 10 (2.40 g, 12.56 mmol) was added. The reaction

mixture was stirred at 50°C for 3 h. The mixture was diluted with ethyl acetate and washed with

saturated aqueous sodium bicarbonate solution. The organic phases was concentrated under reduced

pressure to remove solvent and the residue was purified by chromatography to afford compound 11

(2.83 g, 8.54 mmol) as a white solid.

1

Yield: 68%. H NMR (400MHz, DMSO-d₆): δ = 8.31 (d, 1H, J = 5.6 Hz, Ar-H), 8.03 (s, 1H, Ar-H),

7.73 (s, 1H, Ar-H), 7.04 (t, 1H, J = 9.2 Hz, Ar-H), 6.73 (d, 1H, J = 5.6 Hz, Ar-H), 6.54 (dd, 1H, J =

13.2, 2.4 Hz, NH₂), 6.45 (dd, 1H, J = 8.8, 2.0 Hz, NH₂), 5.55 (s, 2H, NH₂). ESI-MS: m/z = 281.9

[M+H]⁺.

4.10 General methods for the synthesis of key intermediates 12a-d

The mixture of an appropriate 2a-f (1.07 mmol), EDCI (307.68 mg, 1.61 mmol), HOBt (216.87 mg,

1.61 mmol) and DCM (10.0 mL) were stirred at 25°C for 2 h. Then, Et₃N (0.5 mL) was added,

followed by compound 11 (300.00 mg, 1.07 mmol), the mixture stirred for another 2 h at 25°C. The

mixture was then concentrated under reduced pressure to remove solvent, washed with H₂O (10ml),

extracted with ethyl acetate (10 mL × 2), dried over Na₂SO₄, filtered and concentrated under reduced

pressure to give a residue and the crude product was purified by flash C₁₈silica gel chromatography

(ISCO®; 12g SepaFlash® C₁₈Silica Flash Column, Eluent of 0~70% MeCN/H₂O @ 60mL/min) to

give the corresponding compounds 12a-d.

4.10.1

N-(4-(2-carbamoyl-3-chloropyridin-4-yloxy)-3-fluorophenyl)-N-(3-fluorophenyl)cyclopropane-1,1

-dicarboxamide (12a)

1

Yield: 47%. H NMR (400MHz, DMSO-d₆): δ = 10.45-10.09 (m, 2H, Ar-H), 8.34 (d, 1H, J = 5.2 Hz,

Ar-H), 8.06 (s, 1H, NH), 7.90 (dd, 1H, J = 13.2, 2.0 Hz, Ar-H), 7.77 (s, 1H, NH), 7.68-7.58 (m, 1H,

Ar-H), 7.55-7.47 (m, 1H, Ar-H), 7.43-7.30 (m, 3H, Ar-H+NH₂), 6.94-6.85 (m, 1H, Ar-H), 6.80 (d, 1H,

J = 5.6 Hz, Ar-H), 1.55-1.36 (m, 4H, CH₂). ESI-MS: m/z = 487.0 [M+H]⁺.

4.10.2

N-(4-(2-carbamoyl-3-chloropyridin-4-yloxy)-3-fluorophenyl)-N-(4-chlorophenyl)cyclopropane-1,1

-dicarboxamide (12b)

Yield: 49%. ¹H NMR (400MHz, DMSO-d₆): δ = 10.66 (s, 1H, Ar-H), 10.44 (d, 1H, J = 12.0 Hz, Ar-H),

8.40-8.26 (m, 1H, Ar-H), 8.06 (s, 1H, NH), 7.95-7.82 (m, 2H, Ar-H), 7.76 (s, 1H, NH), 7.40-7.32 (m,

5H, Ar-H+CH₂), 6.85-6.80 (m, 1H, Ar-H), 1.49-1.40 (m, 4H, CH₂). ESI-MS: m/z = 502.9 [M+H]⁺.

4.10.3

N-(4-(2-carbamoyl-3-chloropyridin-4-yloxy)-3-fluorophenyl)-N-(4-fluorobenzyl)cyclopropane-1,1

-dicarboxamide (12c)

Yield: 17%; ESI-MS: m/z = 500.8[M+H]⁺.

4.10.4

N-(4-(2-carbamoyl-3-chloropyridin-4-yloxy)-3-fluorophenyl)-N-(4-fluorophenyl)cyclopropane-1,1

-dicarboxamide (12d)

1

Yield: 19%. H NMR (400MHz, DMSO-d₆): δ = 10.42 (s, 1H, Ar-H), 10.18-9.96 (m, 1H, Ar-H),

8.35-8.30 (m, 1H, Ar-H), 8.07-7.88 (m, 2H, Ar-H), 7.77-7.75 (m, 1H, NH), 7.65-7.62 (m, 2H,

NH+Ar-H), 7.54-7.49 (m, 1H, Ar-H), 7.19-7.14 (m, 3H, Ar-H+CH₂), 6.81-6.77 (m, 1H, Ar-H),

1.49-1.38 (m, 4H, CH₂); ¹³C NMR (100 MHz, d₆-DMSO): δ = 168.82, 168.36, 166.98, 160.51, 158.79

(d, J_C-F= 238.7 Hz), 154.69, 153.27 (d, J_C-F= 243.7 Hz), 149.23, 138.89 (d, J_C-F= 9.8 Hz), 135.62 (d,

J_C-F= 2.8 Hz), 135.45 (d, J_C-F= 12.2 Hz), 123.76, 122.96 (d, J_C-F= 7.7 Hz), 117.48 (d, J_C-F= 4.5 Hz),

116.91, 115.49 (d, J_C-F= 22.0 Hz), 111.13, 109.50 (d, J_C-F= 22.8 Hz), 32.34, 15.81; ESI-MS: m/z =

486.9 [M+H]⁺.

4.11 General methods for preparation of pyrimidine-contained target compounds (13a-d)

12a-d (0.20 mmol) in ethyl acetate (2.0 mL), acetonitrile (2.0 mL) and water (1.0 mL) at 0°C was

added iodobenzene diacetate (82 mg, 0.26 mmol) and the mixture was stirred for 2 hours at 25°C. Then,

saturated NaHCO₃(3 mL) was added, followed by additional 30 mL of ethyl acetate. The mixture was

filtered, and the filtrate was washed with brine and evaporated under reduced pressure. The crude

product was purified by chromatography on silica gel to give the corresponding compounds 13a-d.

4.11.1

N-(4-(2-amino-3-chloropyridin-4-yloxy)-3-fluorophenyl)-N-(3-fluorophenyl)cyclopropane-1,1-dic

arboxamide (13a)

1

Yield: 68%. Purity: 96%. H NMR (400MHz, d₄-MeOD): δ = 7.88 (dd, 1H, J = 8.8, 2.4 Hz, Ar-H),

7.84 (d, 1H, J = 7.2 Hz, Ar-H), 7.52-7.63 (m, 1H, Ar-H), 7.46 (d, 1H, J = 8.0 Hz, Ar-H), 7.40-7.25 (m,

3H, Ar-H), 6.98-6.81 (m, 1H, Ar-H), 6.42 (d, 1H, J = 6.8 Hz, Ar-H), 1.66 (s, 4H, CH₂); ¹³C NMR (100

MHz, d₆-DMSO): δ = 168.80, 168.54, 163.20, 162.45 (d, J_C-F= 239.4 Hz), 154.23, 153.11 (d, J_C-F

244.9 Hz), 141.15 (d, J_C-F= 11.0 Hz), 139.99, 139.19 (d, J_C-F= 12.2 Hz), 135.13 (d, J_C-F= 12.2 Hz)，

130.52 (d, J_C-F= 9.5 Hz), 123.74, 117.44 (d, J_C-F= 3.0 Hz), 116.45 (d, J_C-F= 2.2 Hz), 110.44 (d, J_C-F

=

21.0 Hz), 109.43 (d, J_C-F= 22.3 Hz), 107.58 (d, J_C-F= 26.2 Hz), 102.62, 101.37, 32.78, 15.76; ESI-MS:

m/z = 458.9 [M+H]⁺.

4.11.2

N-(4-(2-amino-3-chloropyridin-4-yloxy)-3-fluorophenyl)-N-(4-chlorophenyl)cyclopropane-1,1-dic

arboxamide (13b)

1

Yield: 61%. Purity: 95%. H NMR (400MHz, d₄-MeOD): δ = 7.88 (dd, 1H, J = 12.8, 2.4 Hz, Ar-H),

7.84 (d, 1H, J = 7.2 Hz, Ar-H), 7.62-7.53 (m, 2H, Ar-H), 7.48-7.40 (m, 1H, Ar-H), 7.37-7.30 (m, 3H,

Ar-H), 6.38 (d, 1H, J = 7.2 Hz, Ar-H), 1.64 (s, 4H, CH₂); ESI-MS: m/z = 474.8 [M+H]⁺.

4.11.3

N-(4-(2-amino-3-chloropyridin-4-yloxy)-3-fluorophenyl)-N-(4-fluorobenzyl)cyclopropane-1,1-dica

rboxamide (13c)

1

Yield: 55%. Purity: 98%. H NMR (400MHz, d₄-MeOD): δ = 7.86 (dd, 1H, J = 12.8, 2.4 Hz, Ar-H),

7.82 (d, 1H, J = 7.2 Hz, Ar-H), 7.44-7.22 (m, 4H, Ar-H), 7.04 (t, 2H, J = 8.8 Hz, Ar-H), 6.34 (d, 1H, J

= 6.8 Hz, Ar-H), 4.50-4.31 (m, 2H, CH₂), 1.72-1.37 (m, 4H, CH₂);¹³C NMR (100 MHz, d₆-DMSO): δ

= 170.70, 168.93, 161.62 (d, J_C-F= 240.6 Hz), 162.67, 154.86, 153.24 (d, J_C-F= 243.0 Hz), 141.27,

138.83 (d, J_C-F= 9.4 Hz), 136.00 (d, J_C-F= 2.9 Hz), 135.28 (d, J_C-F= 13.3 Hz), 129.61 (d, J_C-F= 8.1 Hz),

123.82, 117.10 (d, J_C-F= 3.2 Hz), 115.41 (d, J_C-F= 21.1 Hz), 109.11 (d, J_C-F= 22.5 Hz), 102.28, 101.22,

42.43, 30.58, 16.18; ESI-MS: m/z = 472.9 [M+H]⁺.

4.11.4

N-(4-(2-amino-3-chloropyridin-4-yloxy)-3-fluorophenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dic

arboxamide (13d)

1

Yield: 60%. Purity: 97%. H NMR (400MHz, d₄-MeOD): δ = 10.44 (s, 1H, NH), 9.99 (s, 1H, NH),

7.95-7.80 (m, 2H, Ar-H), 7.64-7.60 (m, 2H, Ar-H), 7.51-7.48 (m, 1H, Ar-H), 7.38 (t, 1H, J = 8.8 Hz,

Ar-H), 7.13 (t, 2H, J = 8.8 Hz, Ar-H), 6.22 (d, 1H, J = 6.8 Hz, Ar-H), 1.50-1.42 (m, 4H, CH₂); ¹³C

NMR (100 MHz, d₆-DMSO): δ = 176.11, 168.76, 168.37, 160.16, 158.78 (d, J_C-F= 238.7 Hz), 157.97,

154.35, 153.39 (d, J_C-F= 243.5 Hz), 147.69, 138.21 (d, J_C-F= 9.2 Hz), 136.29 (d, J_C-F= 12.1 Hz),

135.61 (d, J_C-F= 3.7 Hz), 123.57, 122.95 (d, J_C-F= 8.0 Hz), 117.25 (d, J_C-F= 3.7 Hz), 115.47 (d, J_C-F

=

22.0 Hz), 109.40 (d, J_C-F= 23.0 Hz), 100.77, 100.60, 79.08,32.27, 28.53, 15.80; ESI-MS: m/z = 458.9

[M+H]⁺.

4.12 Pharmacology

4.12.1 c-Met kinase assay

The effects of indicated compound on the activities of c-Met kinases were evaluated using

enzyme-linked immunosorbent assays (ELISAs) with purified recombinant proteins.^{21, 22}Briefly, 20

ꢃg/mL poly (Glu, Tyr) 4:1 (Sigma, St. Louis, MO, USA) was preloated in 96-well plates as a substrate.

Then 50 ꢃL aliquot of 10 mM ATP solution diluted in kinase reaction buffer (50 mM HEPES [pH 7.4],

50 mM MgCl₂, 0.5 mM MnCl₂, 0.2 mM Na₃VO₄, and 1 mM DTT) was added to each well. After this, 1

ꢃL of various concentrations of indicated compound diluted in 1% DMSO (v/v) (Sigma) were added to

each reaction well. DMSO (1%, v/v) was used as the negative control. The kinase reaction was initiated

by the addition of purified c-Met tyrosine kinase proteins diluted in 49 ꢃL of kinase reaction buffer

solution. After incubation for 45 min at 25 °C, 100 ꢃL anti-phosphotyrosine (PY99) antibody (1:500,

diluted in 5 mM BSAT-PBS) was was added. After 30 min incubation at 37 °C, the plate was washed

three times, and 100 ꢃL horseradish peroxidase-conjugated goat anti-mouse IgG (1:2000, diluted in 5

mM BSAT-PBS) was added. The plate was then incubated at 37 °C for 30 min and washed 3 times. A

100 ꢃL aliquot of a solution containing 0.03% H₂O₂and 2 mM o-phenylenediamine in 0.1 mol/L citrate

buffer (pH 5.5) was added. The reaction was terminated by the addition of 50 ꢃL of 2 mol/L H₂SO₄as

the color changed, and the plate was analyzed using a multi-well spectrophotometer (SpectraMAX 190,

Molecular Devices, Sunnyvale, CA, USA) at 490 nm. The inhibition rate (%) was calculated using the

following equation: [1- (A490/A490 control)] × 100%. The IC₅₀values were calculated from the

inhibition curves in two separate experiments.

4.12.2 MTT assay In vitro

The anti-proliferative activities of compounds were evaluated against HepG2 and EBC-1 cell lines

using the standard CTG assay in vitro. The cancer cell lines were cultured in EMEM supplement with

10% fetal bovine serum (FBS). Approximate 4 × 10³cells, suspended in EMEM medium, were plated

onto each well of a 96-well plate and incubated in 5% CO₂at 37 for 24 h. The tested compounds at

the indicated final concentrations were added to the culture medium and the cell cultures were

continued for 72 h. Cell titer-Glo reagent was added to each well, and incubated with cells at room

temperature for 10 minutes, and the luminescence was measured with Envision. All compounds were

tested in each of the cell lines. The results expressed as IC₅₀(inhibitory concentration 50%) of

determination and calculated by using the GraphPad Prism 5.

4.12.3 Western blot analysis

EBC-1 cells were treated with the increasing dose of indicated compound for 2 h at 37

and then

lysed in 1 × SDS sample buffer. The cell lysates were subsequently resolved by 10% SDS-PAGE and

transferred to nitrocellulose membranes. The membranes were probed with the appropriate primary

antibodies and then with horseradish peroxidase-conjugated anti-rabbit or antimouseIgG. The

immunoreactive proteins were detected using an enhanced chemiluminescence detection reagent

(Thermo Fisher).

4.12.4 Docking studies

The docking simulation studies were carried out by using Glidemodule of Schrödinger package and the

crystal structure of c-Met (PDB ID code: 3LQ8) was selected as the docking template. The protein

structure was prepared by Protein Preparation Wizard of Maestro, and the structure of compound 13d

was prepared by LigPrep. The image files were generated using pymol system.

Conflict of interest

The authors confirm that this article content has no conflict of interest.

Acknowledgments

This study was supported by Grants from the Health care and key specialist scientific research project

of Hangzhou Administration of Science and Technology (Grants 20140733Q42), the Major science and

technology innovation program of Hangzhou (Grant 20142013A60 and Grant 20152013A03).

References:

1. Peruzzi, B.; Bottaro, D. P. Targeting the c-Met signaling pathway in cancer. Clin. Cancer Res. 2006,

12, 3657-3660.

2. Gherardi, E.; Birchmeier, W.; Birchmeier, C.; Vande Woude, G. Targeting MET in cancer: rationale

and progress. Nat. Rev. Cancer. 2012, 12, 89-103.

3. Sierra, J. R.; Tsao, M. S. c-MET as a potential therapeutic target and biomarker in cancer. Ther. Adv.

Med. Oncol. 2011, 3, 21-35.

4. Straussman, R.; Morikawa, T.; Shee, K.; Barzily-Rokni, M.; Qian, Z. R.; Du, J.; Davis, A.; Mongare,

M. M.; Gould, J.; Frederick, D. T.; Cooper, Z. A.; Chapman, P. B.; Solit, D. B.; Ribas, A.; Lo, R. S;

Flaherty, K. T.; Ogino, S.; Wargo, J. A.; Golub, T. R. Tumour micro-environment elicits innate

resistance to RAF inhibitors through HGF secretion. Nature. 2012, 487, 500-504.

5. Cecchi, F.; Rabe, D. C.; Bottaro, D. P. Targeting the HGF/Met signalling pathway in cancer. Eur. J.

Cancer. 2010, 46, 1260-1270.

6. Eder, J. P.; Vande Woude, G. F.; Boerner, S. A.; LoRusso, P. M. Novel therapeutic inhibitors of the

c-Met signaling pathway in cancer. Clin. Cancer Res. 2009, 15, 2207-2214.

7. Liu, X.; Yao, W.; Newton, R. C.; Scherle, P. A. Targeting the c-MET signaling pathway for cancer

therapy. Expert Opin. Investig. Drugs. 2008, 17, 997-1011.

8. Cui, J. J. Targeting receptor tyrosine kinase MET in cancer: small molecule inhibitors and clinical

progress. J. Med. Chem. 2014, 57, 4427-4453.

9. Comoglio, P. M.; Giordano, S.; Trusolino, L. Drug development of MET inhibitors: targeting

oncogene addiction and expedience. Nat. Rev. Drug Discov. 2008, 7, 504-516.

10. Dussault, I.; Bellon, S. F. From concept to reality: the long road to c-Met and RON receptor

tyrosine kinase inhibitors for the treatment of cancer. Anticancer Agents Med. Chem. 2009, 9, 221-229.

11. Bellon, S. F.; Kaplan-Lefko, P.; Yang, Y.; Zhang, Y.; Moriguchi, J.; Rex, K.; Johnson, C. W.; Rose, P.

E.; Long, A. M.; O'Connor, A. B.; Gu, Y.; Coxon, A.; Kim, T. S.; Tasker, A.; Burgess, T. L.; Dussault, I.

c-Met inhibitors with novel binding mode show activity against several hereditary papillary renal cell

carcinoma-related mutations. J. Biol. Chem. 2008, 283, 2675-2683.

12. Underiner, T. L.; Herbertz, T.; Miknyoczki, S. J. Discovery of small molecule c-Met inhibitors:

Evolution and profiles of clinical candidates. Anticancer Agents Med. Chem. 2010, 10, 7-27.

13. Schroeder, G. M.; An, Y.; Cai, Z. W.; Chen, X. T.; Clark, C.; Cornelius, L. A.; Dai, J.; Gullo-Brown,

J.; Gupta, A.; Henley, B.; Hunt, J. T.; Jeyaseelan, R.; Kamath, A.; Kim, K.; Lippy, J.; Lombardo, L. J.;

Manne, V.; Oppenheimer, S.; Sack, J. S.; Schmidt, R. J.; Shen, G.; Stefanski, K.; Tokarski, J. S.; Trainor,

G. L.; Wautlet, B. S.; Wei, D.; Williams, D. K.; Zhang, Y.; Zhang, Y.; Fargnoli, J.; Borzilleri, R. M.

Discovery

of

N-(4-(2-amino-3-chloropyridin-4-yloxy)-3-fluorophenyl)-4-ethoxy-1-(4-fluorophenyl)-2-oxo-1,2-dihyd

ropyridine-3-carboxamide (BMS-777607), a selective and orally efficacious inhibitor of the Met kinase

superfamily. J. Med. Chem. 2009, 52, 1251-1254.

14. Dussault, I.; Bellon, S. F. c-Met inhibitors with different binding modes: two is better than one. Cell

Cycle. 2008, 7, 1157-1160.

15. Berthou, S.; Aebersold, D. M.; Schmidt, L. S.; Stroka, D.; Heigl, C.; Streit, B.; Stalder, D.; Gruber,

G.; Liang, C.; Howlett, A. R.; Candinas, D.; Greiner, R. H.; Lipson, K. E.; Zimmer, Y. The Met kinase

inhibitor SU11274 exhibits a selective inhibition pattern toward different receptor mutated variants.

Oncogene. 2004, 23, 5387-5393.

16. Li, S.; Huang, Q.; Liu, Y.; Zhang, X.; Liu, S.; He, C.; Gong, P. Design, synthesis and antitumour

activity of bisquinoline derivatives connected by 4-oxy-3-fluoroaniline moiety. Eur. J. Med. Chem.

2013, 64, 62-73.

17. Qian, F.; Engst, S.; Yamaguchi, K.; Yu, P.; Won, K. A.; Mock, L.; Lou, T.; Tan, J.; Li, C.; Tam, D.;

Lougheed, J.; Yakes, F. M.; Bentzien, F.; Xu, W.; Zaks, T.; Wooster, R.; Greshock, J.; Joly, A. H.

Inhibition of tumor cell growth, invasion, and metastasis by EXEL-2880 (XL880, GSK1363089), a

novel inhibitor of HGF and VEGF receptor tyrosine kinases. Cancer Res. 2009, 69, 8009-8016.

18. Buchanan, S. G.; Hendle, J.; Lee, P. S.; Smith, C. R.; Bounaud, P. Y.; Jessen, K. A.; Tang, C. M.;

Huser, N. H.; Felce, J. D.; Froning, K. J.; Peterman, M. C.; Aubol, B. E.; Gessert, S. F.; Sauder, J. M.;

Schwinn, K. D.; Russell, M.; Rooney, I. A.; Adams, J.; Leon, B. C.; Do, T. H.; Blaney, J. M.;

Sprengeler, P. A.; Thompson, D. A.; Smyth, L.; Pelletier, L. A.; Atwell, S.; Holme, K.; Wasserman, S.

R.; Emtage, S.; Burley, S. K.; Reich, S. H. SGX523 is an exquisitely selective, ATP-competitive

inhibitor of the MET receptor tyrosine kinase with antitumor activity in vivo. Mol. Cancer Ther. 2009 ,

8, 3181-3190.

19. Liu, J.; Nie, M.; Wang, Y.; Hu, J.; Zhang, F.; Gao, Y.; Liu, Y.; Gong, P. Design, synthesis and

structure-activity relationships of novel 4-phenoxyquinoline derivatives containing 1,2,4-triazolone

moiety as c-Met kinase inhibitors. Eur. J. Med. Chem. 2016, 123, 431-446.

20. She, N.; Zhuo, L.; Jiang, W.; Zhu, X.; Wang, J.; Ming, Z.; Zhao, X.; Cong, X.; Huang, W. Design,

synthesis and evaluation of highly selective pyridone-based class II MET inhibitors. Bioorg. Med.

Chem. Lett. 2014, 24, 3351-3355.

21. Albrecht, B. K.; Harmange, J. C.; Bauer, D.; Berry, L.; Bode, C.; Boezio, A. A.; Chen, A.;

Choquette, D.; Dussault, I.; Fridrich, C.; Hirai, S.; Hoffman, D.; Larrow, J. F.; Kaplan-Lefko, P.; Lin, J.;

Lohman, J.; Long, A. M.; Moriguchi, J.; O'Connor, A.; Potashman, M. H.; Reese, M.; Rex, K.;

Siegmund, A.; Shah, K.; Shimanovich, R.; Springer, S. K.; Teffera, Y.; Yang, Y.; Zhang, Y.; Bellon, S. F.

Discovery and optimization of triazolopyridazines as potent and selective inhibitors of the c-Met kinase.

J. Med. Chem. 2008, 51, 2879-2882.

22. Liu, L.; Siegmund, A.; Xi, N.; Kaplan-Lefko, P.; Rex, K.; Chen, A.; Lin, J.; Moriguchi, J.; Berry, L.;

Huang, L.; Teffera, Y.; Yang, Y.; Zhang, Y.; Bellon, S. F.; Lee, M.; Shimanovich, R.; Bak, A.;

Dominguez, C.; Norman, M. H.; Harmange, J. C.; Dussault, I.; Kim, T. S. Discovery of a potent,

selective,

and

orally

bioavailable

c-Met

inhibitor:

1-(2-hydroxy-2-methylpropyl)-N-(5-(7-methoxyquinolin-4-yloxy)pyridin-2-yl)-5-methyl-3-oxo-2-phen

yl-2,3-dihydro-1H-pyrazole-4-carboxamide (AMG 458). J. Med. Chem. 2008, 51, 3688-3691.

Graphical abstract

Article Doi

Synthesis and evaluation of a series of pyridine and pyrimidine derivatives as type II c-Met inhibitors

DOI: 10.1016/j.bmc.2017.04.003

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.bmc.2017.04.003

Full text of DOI:10.1016/j.bmc.2017.04.003

Products guided by the article

R&D Labs maybe for 868733-36-2

Relevant to this article

Hot Product