English

• 1. INTRODUCTION

  Cancer is one of the most health problems in the world. Although many classes of drugs were used for the treatment, the needs for safe and effective anticancer compounds are still significant target^{[1, 2]}. Pyridine and pyrimidine-based compounds have been reported to show remarkable antitumor activities by means of inhibiting multiple enzymes, and some of them are already being marketed or are under clinical/preclinical studies, such as imatinib, sorafenib, crizotinib, abiraterone, BMS-794833, altiratinib, and BMS-777607^[3-6]. Among above compounds, BMS-794833, altiratinib, and BMS-777607 are representative 4-phenoxypyridine based type II c-Met kinase inhibitors^{[7, 8]}. Blocking c-Met kinase activity by small-molecule inhibitors has been identified as a promising approach for the treatment of cancers^[9].

  As described in Fig. 1, structurally, most of 4-phenoxy-pyridine type II c-Met inhibitors may be disconnected into three moieties: a 4-phenoxypyridine core (moiety A), a phenyl or substituted phenyl group (moiety B) and a linker bridge (moiety C). Moiety A and moiety B are crucial for kinase activity^[10-12]. With the goal of finding more 4-phenoxypyri-dine-based antitumor agents, N-[4-(2-fluorophenoxy)pyridin-2-yl]cyclopropanecarboxamide was used as the moiety A. Pyrimidine-2, 4-diamine was introduced into the moiety C via cyclization strategy. Furthermore, various substituents were introduced at the phenyl ring (moiety B) to investigate their effects on activities (Fig. 1). Accordingly, a series of 4-phenoxypyridine derivatives were designed, synthesized and evaluated for their in vitro antiproliferative activities against MKN-45, HT-29, A549, K562 and GIST882 cancer cell lines.

  Figure 1

  

  Figure 1.  The representative 4-phenoxypyridine type II c-Met kinase inhibitors and design strategy of the target compounds

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  2. EXPERIMENTAL

  2.1 Materials and methods

  Unless otherwise specified, all materials were obtained from commercial suppliers and were used without further purification. ¹H NMR spectra were recorded on a Bruker Biospin 600 MHz instrument using TMS as the internal standard. All chemical shifts were reported in ppm. IR spectra were recorded as KBr pellets on a Perkin-Elmer Spectrum one FT-IR spectrometer. MS spectra were obtained on an Agilent 6460 QQQ mass spectrometer (Agilent, USA) analysis system. Crystal data were obtained on a Bruker P4 X-diffractometer.

  2.2 Synthesis of N-(4-chloropyridin2-yl)-cyclopropylformamide (1)

  Cyclopropanecarbonyl chloride (9.30 g, 89.00 mmol) was dissolved in dried CH₂Cl₂ (30 mL) and dropwise added to a mixture of 4-chloropyridin-2-amine (8.80 g, 68.45 mmol), Et₃N (20.78 g, 205.35 mmol) and CH₂Cl₂ (80 mL) in an ice bath, which was then removed to raise the temperature to room temperature and stirred for 12 h. The resulting mixture was sequentially washed with 20% K₂CO₃ (50 mL × 3) and brine (50 mL × 3), and the organic phase was separated, dried over anhydrous Na₂SO₄, filtered, and the filtrate was evaporated under reduced pressure. The crude product obtained was purified by silica gel chromatography to give 9.78 g (yield: 73%) of 1 as a white solid. IR (KBr) cm^-1: 3242(-NH-), 1707(C=O), 1670, 1588(C=N), 1574(Ar), 1537(Ar), 1404, 1257, 1213, 1190, 1150, 960, 873, 824, 710; ¹H NMR (600 MHz, CDCl₃) δ 8.79 (s, 1H), 8.31 (s, 1H), 8.16 (d, J = 5.4 Hz, 1H), 7.03 (dd, J = 5.4, 1.6 Hz, 1H), 1.60~1.49 (m, 1H), 1.17~1.09 (m, 2H), 0.97~0.87 (m, 2H); MS (ESI) m/z (%): 197.1 [M+H]⁺.

  2.3 Synthesis of N-(4-(2-fluoro-4-nitro-phenoxy)pyridin-2-yl)cyclopropylformamide (2)

  A stirring mixture of compound 8 (8.00 g, 40.68 mmol) and 2-fluoro-4-nitrophenol (15.98 g, 101.71 mmol) in chloroben-zene (100 mL) was refluxed for about 40 h. After cooling to room temperature, the reaction mixture was concentrated under reduced pressure to yield a pale solid. The solid was dissolved in CH₂Cl₂ (150 mL), washed with saturated K₂CO₃ aqueous solution (80 mL × 4) and then brine (60 mL × 4), dried over anhydrous Na₂SO₄, and finally concentrated under reduced pressure to afford a brown solid, which was purified by silica gel chromatography to give 7.13 g (yield: 55%) of 2 as a light yellow solid. IR (KBr) cm^-1: 3418(-NH-), 1687(C=O), 1617(C=N), 1529(Ar), 1493(Ar), 1354, 1300, 1272, 1180, 1070, 875, 798; ¹H NMR (600 MHz, DMSO-d₆) δ 11.00 (s, 1H), 8.43 (dd, J = 10.3, 2.2 Hz, 1H), 8.30 (d, J = 5.7 Hz, 1H), 8.19 (dd, J = 9.0, 1.9 Hz, 1H), 7.76 (d, J = 2.2 Hz, 1H), 7.61 (t, J = 8.5 Hz, 1H), 6.86 (dd, J = 5.6, 2.2 Hz, 1H), 2.04~1.95 (m, 1H), 0.78 (t, J = 6.3 Hz, 4H); MS (ESI) m/z (%): 318.1 [M+H]⁺.

  2.4 Synthesis of N-(4-(4-amino-2-fluoro-phenoxy)pyridin-2-yl)cyclopropanecarboxamide (3)

  A mixture of compound 2 (6.00 g, 18.91 mmol), iron powder (5.28 g, 94.56 mmol), acetic acid (11.36 g, 189.10 mmol), water (20 mL) and ethyl acetate (100 mL) was heated to reflux for 2 h. After completion of the reaction as indicated by TLC, the mixture was filtered immediately. The organic layer of the filtrate was separated, washed with water, dried over anhydrous Na₂SO₄ and filtered, and the filtrate was evaporated under reduced pressure when white solid appeared, which was filtered to obtain 3.51 g (yield: 65%) of 3 as a white solid. IR (KBr) cm^-1: 3411(-NH-), 2026, 1737(C=O), 1618(C=N), 1510(Ar), 1427, 1207, 1163, 993, 956, 866, 822, 610; ¹H NMR (600 MHz, DMSO-d₆) δ 10.79 (s, 1H), 8.15 (d, J = 5.7 Hz, 1H), 7.59 (s, 1H), 6.95 (t, J = 9.0 Hz, 1H), 6.67~6.61 (m, 1H), 6.49 (dd, J = 13.1, 2.2 Hz, 1H), 6.40 (d, J = 8.7 Hz, 1H), 5.44 (s, 2H), 2.03~1.88 (m, 1H), 0.76 (br, 4H); MS (ESI) m/z (%): 288.1 [M+H]⁺, 310.1 [M+Na]⁺.

  2.5 Synthesis of N-(4-(4-((2-chloropyrimidin-4-yl)amino)-2-fluorophenoxy)pyridin-2-yl)cyclopropane formamide (4)

  A mixture of compound 3 (3.00 g, 10.44 mmol), 2, 4-di-chloropyrimidine (1.87 g, 12.53 mmol), and diisopropylethylamine (1.35 g, 10.44 mmol) in isopropanol (50 mL) was heated at reflux for 25 h. Upon cooling to room temperature, the mixture evaporated under reduced pressure. The residue was dissolved in dichloromethane (80 mL), and washed with water (50 mL × 3). The organic layer was dried over anhydrous Na₂SO₄ and concentrated under reduced pressure to afford crude product, which was purified by silica gel chromatography to give 3.17 g (yield: 76%) of 4 as a white solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.90 (s, 1H), 10.34 (s, 1H), 8.25~8.22 (m, 2H), 7.88~7.60 (m, 2H), 7.50~7.30 (m, 2H), 6.85~6.71 (m, 2H), 2.03~1.92 (m, 1H), 0.84~0.72 (m, 4H); MS (ESI) m/z (%): 400.1 [M+H]⁺, 422.0 [M+Na]⁺.

  2.6 General procedure for preparing the target compounds (5a~5f)

  A mixture of compound 4 (0.20 g, 0.50 mmol), aromatic amine (1.3 equivalents) and p-toluenesulfonic acid (1.0 equivalent) in isopropanol (8.0 mL) was refluxed for about 18 h. After cooling to room temperature, the resultant precipitate was filtered and dried under vacuum to afford the target compounds 5a~5f as white solid.

  N-(4-(2-fluoro-4-((2-(phenylamino)pyrimidin-4-yl)ami- no)phenoxy)pyridin-2-yl)cyclopropanecarboxamide (5a)

  Yield: 67%; ¹H NMR (600 MHz, DMSO-d₆) δ 10.58 (d, J = 13.1 Hz, 1H), 9.37 (d, J = 11.5 Hz, 1H), 9.05~8.80 (m, 1H), 8.20~8.06 (m, 2H), 8.02 (d, J = 5.7 Hz, 1H), 7.81~7.62 (m, 3H), 7.37 (d, J = 8.4 Hz, 1H), 7.30~7.19 (m, 2H), 7.10 (t, J = 8.8 Hz, 1H), 6.91 (t, J = 7.2 Hz, 1H), 6.65~6.50 (m, 1H), 6.22 (d, J = 5.7 Hz, 1H), 2.04~1.89 (m, 1H), 0.91~0.83 (m, 2H), 0.79~0.72 (m, 2H); MS (ESI) m/z(%): 457.2 [M+H]⁺, 479.1 [M+Na]⁺.

  N-(4-(2-fluoro-4-((2-((4-fluorophenyl)amino)pyri- midin-4-yl)amino)phenoxy)pyridin-2-yl)cyclo- propanecarboxamide (5b)

  Yield: 59%; ¹H NMR (600 MHz, DMSO-d₆) δ 10.74~10.55 (m, 1H), 9.53~9.32 (m, 1H), 9.16~8.87 (m, 1H), 8.20~7.92 (m, 3H), 7.70 (d, J = 13.4 Hz, 3H), 7.41~7.28 (m, 1H), 7.19~7.04 (m, 1H), 7.04~6.88 (m, 2H), 6.64~6.50 (m, 1H), 6.31~6.12 (m, 1H), 5.86~5.82 (m, 1H), 2.03~1.88 (m, 1H), 0.96~0.58 (m, 4H); IR (KBr, cm^-1): 3258(-NH-), 2355, 1678(C=O), 1591, 1508(Ar), 1422(Ar), 1312, 1207, 982, 820; MS (ESI) m/z (%): 475.2 [M+H]⁺, 497.1 [M+Na]⁺.

  N-(4-(4-((2-((4-chlorophenyl)amino)pyrimidin- 4-yl)amino)-2-fluorophenoxy)pyridin- 2-yl)cyclopropanecarboxamide (5c)

  Yield: 55%; ¹H NMR (600 MHz, DMSO-d₆) δ 10.67~10.45 (m, 1H), 9.50~9.31 (m, 1H), 9.18~8.92 (m, 1H), 8.26~7.96 (m, 3H), 7.79~7.67 (m, 3H), 7.35 (d, J = 7.3 Hz, 1H), 7.26~7.00 (m, 3H), 6.59 (dd, J = 5.7, 2.3 Hz, 1H), 6.24 (d, J = 5.7 Hz, 1H), 2.07~1.86 (m, 1H), 0.90~0.83 (m, 2H), 0.79~0.73 (m, 2H); IR (KBr, cm^-1): 3281(-NH-), 2353, 1672(C=O), 1585, 1416(Ar), 1308, 1204, 1094, 978, 804, 687; MS (ESI) m/z (%): 491.1 [M+H]⁺, 513.1 [M+Na]⁺.

  N-(4-(4-((2-((3-chlorophenyl)amino)pyrimidin- 4-yl)amino)-2-fluorophenoxy)pyridin-2- yl)cyclopropanecarboxamide (5d)

  Yield: 57%; ¹H NMR (600 MHz, DMSO-d₆) δ 10.51 (br, 1H), 9.36 (br, 1H), 9.17~9.04 (m, 1H), 8.13 (d, J = 5.7 Hz, 1H), 8.04 (d, J = 5.7 Hz, 1H), 7.95 (s, 1H), 7.92~7.83 (m, 1H), 7.74 (d, J = 1.9 Hz, 1H), 7.57 (d, J = 8.2 Hz, 1H), 7.41 (d, J = 8.8 Hz, 1H), 7.18 (t, J = 8.1 Hz, 1H), 7.12 (t, J = 8.8 Hz, 1H), 6.87 (d, J = 7.9 Hz, 1H), 6.60~6.55 (m, 1H), 6.31~6.22 (m, 1H), 2.03~1.84 (m, 1H), 0.91~0.86 (m, 2H), 0.79~0.74 (m, 2H); IR (KBr, cm^-1): 3305(-NH-), 2357, 1679(C=O), 1619, 1416, 1314, 1207, 1105, 990, 808; MS (ESI) m/z (%): 491.1 [M+H]⁺, 513.1 [M+Na]⁺.

  N-(4-(4-((2-((3-chloro-4-fluorophenyl)amino)pyri- midin-4-yl)amino)-2-fluorophenoxy)pyridin- 2-yl)cyclopropanecarboxamide (5e)

  Yield: 45%; ¹H NMR (600 MHz, DMSO-d₆) δ 10.76~10.50 (m, 1H), 9.48 (d, J = 13.0 Hz, 1H), 9.25 (d, J = 13.9 Hz, 1H), 8.17~8.10 (m, 1H), 8.06~7.97 (m, 3H), 7.71 (d, J = 3.4 Hz, 1H), 7.58 (br, 1H), 7.45~7.33 (m, 1H), 7.20~7.03 (m, 2H), 6.64~6.54 (m, 1H), 6.26 (t, J = 6.8 Hz, 1H), 1.97 (d, J = 4.1 Hz, 1H), 0.92~0.71 (m, 4H); IR (KBr, cm^-1): 3269(-NH-), 2349, 1678(C=O), 1591, 1413(Ar), 1310, 1259, 1207, 1101, 990, 808; MS (ESI) m/z (%): 509.1 [M+H]⁺, 531.1 [M+Na]⁺.

  N-(4-(2-fluoro-4-((2-(p-tolylamino)pyrimidin- 4-yl)amino)phenoxy)pyridin-2-yl)cyclo- propanecarboxamide (5f)

  Yield: 62%; ¹H NMR (600 MHz, DMSO-d₆) δ 10.63 (s, 1H), 9.38 (s, 1H), 8.85 (s, 1H), 8.16~8.08 (m, 2H), 8.00 (d, J = 0.6 Hz, 1H), 7.72 (s, 1H), 7.57 (d, J = 8.3 Hz, 2H), 7.37 (d, J = 8.7 Hz, 1H), 7.10 (t, J = 8.9 Hz, 1H), 7.07~7.01 (m, 2H), 6.59 (dd, J = 5.7, 2.3 Hz, 1H), 6.19 (d, J = 5.7 Hz, 1H), 2.27 (s, 3H), 2.05~1.90 (m, 1H), 0.89~0.82 (m, 2H), 0.79~0.72 (m, 2H); IR (KBr, cm^-1): 3196(-NH-), 3017, 2363, 1680(C=O), 1586, 1524(Ar), 1420, 1308, 1206, 1101, 986, 804, 700; MS (ESI) m/z (%): 471.2 [M+H]⁺, 493.2 [M+Na]⁺.

  2.7 X-ray data collection and structure refinement

  The white powder of compounds 5d and 5e was dissolved in ethanol/ethyl acetate/tetrahydrofuran = 5:2:3 (V/V/V) and 5:3:1 (V/V/V) mixed solvents, respectively. After slowly evaporating the solvents for several days, some single crystals suitable for X-ray analysis were obtained. The X-ray crystallography data for two crystals were collected on a Bruker APEX-II CCD automatic diffractometer with graphite-monochromatized MoKa radiation (λ = 0.71073 Å) using the φ and ω-scan mode at 296(2) K. The structure was solved by direct methods and refined with the SHELX crystallographic software package^[13] and expanded by Fourier technique. The non-hydrogen atoms were refined anisotropically. The hydrogen atoms bound to carbon were determined with theoretical calculations and those attached to nitrogen and oxygen were determined with successive difference Fourier syntheses. Empirical absorption correction was applied. The structure was solved by direct methods using SADABS^[14]. The hydrogen atoms were placed at the calculated positions and refined as riding atoms with isotropic displacement parameters^[15]. Crystallographic data and experimental details of structural analyses for 5d and 5e are summarized in Table 1. The hydrogen bonds of the title compound are listed in Table 2, and geometric parameters for it can be found in Table 3.

  Table 1

  

  Table 1.  Crystal Data of Compounds 5d and 5e

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  Compound	5d	5e
  CCDC	2088950	2088951
  Empirical formula	C25H20ClFN6O2	C25H19ClF2N6O2
  Formula weight	490.91	508.91
  Crystal size (mm 3)	0.23 × 0.20 × 0.16	0.28 × 0.25 × 0.22
  Crystal system	Monoclinic	Monoclinic
  Space group	P21/c	P21/c
  a (Å)	11.0500(10)	10.9998(18)
  b (Å)	18.3045(17)	18.517(3)
  c (Å)	13.5646(9)	13.6355(16)
  α (°)	90	90
  β (°)	122.806(5)	123.315(9)
  γ (°)	90	90
  Volume (Å3)	2306.1(4)	2320.9(6)
  Z	4	4
  Dc (g·cm-3)	1.411	1.456
  Index ranges	–9≤h≤13 –21≤k≤21 –16≤l≤14	–12≤h≤13 –22≤k≤14 –16≤l≤15
  Absorption coefficient (μ)	0.210	0.218
  F(000)	1012	1048
  Theta range for data collection (o)	2.104 to 24.998	2.099 to 24.998
  Reflections collected	11625	11670
  Independent reflections	4051	4072
  Rint	0.0273	0.0437
  Goodness-of-fit on F2	1.060	1.054
  R, wR(I > 2σ(I))	R = 0.0606, wR = 0.2068	R = 0.0495, wR = 0.1139
  R, wR(all data)	R = 0.0866, wR = 0.2263	R = 0.0943, wR = 0.1282
  R = ∑(||Fo|–|Fc||)/∑|Fo|, wR = [∑w(Fo2–Fc2)2/∑w(Fo2)2]1/2

  Table 2

  

  Table 2.  Hydrogen Bond Lengths (Å) and Bond Angles (°) of 5d and 5e

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  5d	D–H···A	d(D–H)			d(H···A)	d(D···A)	∠DHA
  	N(1)–H(1)···N(5)		0.86	2.26		3.084(4)	160
  	N(4)–H(4)···O(2)#1		0.86	2.06		2.871(4)	157
  5e	N(1)–H(1)···N(5)#1		0.86	2.24		3.051(3)	159
  	N(4)–H(4)···O(2)#2		0.86	2.06		2.874(2)	157
  Symmetry codes: 5d: #1: –x, y+1/2, –z+1/2; 5e: #1: –x, –y, –z; #2: x, –y–1/2, z–1/2

  Table 3

  

  Table 3.  Geometric Parameters of 5d and 5e

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  Bond	Dist. for 5d	Dist. for 5e		Angle	5d (o)	5e (o)
  C(1)–N(1)	1.397(4)	1.409(3)		C(6)–C(1)–N(1)	124.3(3)	122.9(3)
  C(1)–C(2)	1.403(5)	1.387(4)		C(6)–C(1)–C(2)	118.6(3)	119.2(3)
  C(3)–Cl(1)	1.745(4)	1.728(3)		N(2)–C(7)–N(3)	127.8(3)	127.5(2)
  C(7)–N(2)	1.334(4)	1.341(3)		N(3)–C(7)–N(1)	114.5(3)	114.8(2)
  C(7)–N(1)	1.381(4)	1.364(3)		C(8)–C(9)–C(10)	116.6(3)	116.2(3)
  C(8)–C(9)	1.365(5)	1.358(4)		C(12)–C(11)–N(4)	119.8(3)	121.1(2)
  C(10)–N(4)	1.357(4)	1.361(3)		C(18)–C(17)–C(21)	120.4(3)	120.1(3)
  						To be continued
  C(11)–C(12)	1.388(5)	1.396(4)		N(5)–C(19)–N(6)	114.7(3)	114.5(2)
  C(11)–N(4)	1.401(4)	1.405(3)		C(22)–C(23)–C(24)	117.5(3)	115.9(3)
  C(14)–O(1)	1.382(4)	1.398(3)		C(24)–C(23)–C(25)	58.5(3)	58.6(2)
  C(17)–C(18)	1.377(4)	1.377(4)		C(7)–N(1)–C(1)	127.6(3)	127.6(2)
  C(17)–O(1)	1.379(4)	1.379(3)		C(7)–N(2)–C(10)	116.0(3)	115.9(2)
  C(19)–N(5)	1.333(4)	1.338(3)		C(7)–N(3)–C(8)	113.8(3)	113.9(2)
  C(19)–N(6)	1.399(4)	1.398(3)		C(22)–N(6)–C(19)	126.2(3)	126.2(2)
  C(22)–O(2)	1.226(4)	1.230(3)		C(17)–O(1)–C(14)	117.7(2)	116.8(2)
  C(22)–N(6)	1.362(4)	1.358(3)		N(1)–C(1)–C(2)–C(3)	–177.3(3)	175.3(2)
  C(22)–C(23)	1.471(5)	1.464(4)		O(1)–C(14)–C(15)–C(16)	178.5(3)	178.3(2
  C(23)–C(24)	1.507(5)	1.509(4)		N(2)–C(7)–N(1)–C(1)	–7.6(5)	6.1(4)
  C(24)–C(25)	1.477(6)	1.469(4)		N(6)–C(22)–C(23)–C(25)	–133.3(3)	–163.5(2)

  2.8 In vitro anticancer activity test of the target compounds

  The antiproliferative activities of compounds 5a~5f were evaluated against human gastric cancer cell lines MKN-45, human colon cancer cell lines HT-29, human lung adenocarcinoma cell lines A549, human chronic myeloid leukemia cell lines K562 and human gastrointestinal stromal tumor cell lines GIST882 using the standard MTT assay in vitro, with Foretinib as the positive control. The cancer cell lines were cultured in minimum essential medium (MEM) supplement with 10% fetal bovine serum (FBS). Approximate 4 × 10³ cells, suspended in MEM medium, were plated onto each well of a 96-well plate and incubated in 5% CO₂ at 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. Fresh MTT was added to each well at a terminal concentration of 5 μg/mL, and incubated with cells at 37 ℃ for 4 h. The formazan crystals were dissolved in 100 mL DMSO each well, and the absorbency at 492 nm (for absorbance of MTT formazan) and 630 nm (for the reference wavelength) was measured with an ELISA reader. All compounds were tested three times in each cell line. The results expressed as IC₅₀ (inhibitory concentration 50%) were the averages of three determinations and calculated by using the Bacus Laboratories Incorporated Slide Scanner (Bliss) software.

  3. RESULTS AND DISCUSSION

  3.1 Synthesis

  The syntheses of target compounds 5a~5f were outlined in Scheme 1. Commercially available 4-chloropyridin-2-amine was condensed with cyclopropanecarbonyl chloride in the presence of Et₃N to provide 1, which underwent a nucleophilic substitution with 2-fluoro-4-nitrophenol to give the desired intermediate 2. Reduction of the nitro group of 2 with iron powder and acetic acid in ethyl acetate/water (10:1 v/v) provided aniline compound 3. 3 reacted with 2, 4-dichloro-pyrimidine in refluxing i-PrOH to provide key intermediate 4 under the catalysis of DIPEA. 4 reacted with different substituted anilines in the presence of p-toluenesulfonic acid in refluxing i-PrOH to yield the target compounds 5a-5f, and the their structures were confirmed by ¹H NMR, IR and ESI-MS.

  Scheme 1

  

  Scheme 1.  Synthetic route and structure of target compounds

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  3.2 Crystal structures

  The structures of compounds 5d and 5e were further confirmed by single-crystal X-ray diffraction analysis. The molecular structures of compounds 5d and 5e with atom-numbering are shown in Fig. 2. Both crystal structures of 5d and 5e crystallize in monoclinic space group P2₁/c. Their crystal structures show that the two molecules have a 2, 4-diarylaminopyrimidine skeleton, in which all bond lengths and bond angles fall in normal ranges. Both of the crystal structures consist of five rings: a three-membered cyclopropane (A), a pyrimidine ring (B), a pyridine ring (C) and two benzene rings (D, E). For compound 5d, the molecule is not coplanar because the dihedral angles between rings A, B and C are 75.44° (A/C), 78.30° (A/B), 61.80° (A/D) and 33.91° (A/E), indicating rings B and C are almost perpendicular to ring A. For compound 5e, the molecule is also not coplanar because the dihedral angles between rings A, B, C, D and E are 75.12° (A/C), 77.42° (A/B), 61.87° (A/D) and 47.67° (A/D), so similar perpendicular conformation is also found. For compounds 5d and 5e, the bond lengths of C(22)–O(2) are 1.226(4) and 1.230(3) Å belonging to the typical C=O double bond^[16]. Meanwhile, hydrogen bonding interactions play a significant role in the crystal packing of 5d and 5e (Table 2). A mass of intermolecular hydrogen bonds N(4)–H(4A)⋅⋅⋅O(2), N(3)–H(6A)⋅⋅⋅N(6) and N(6)–H(6)⋅⋅⋅N(3) found in the two compounds play a major role in stabilizing the molecule. And the molecular structures of compounds 5d and 5e are shown in Fig. 3, depicting the molecular packing and hydrogen bonds in a unit cell. Within the molecule, the bond lengths and bond angles present no unusual features.

  Figure 2

  

  Figure 2.  Molecular structure of compound 5d and 5e in a single crystal at 296 K

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  Figure 3

  

  Figure 3.  Crystal packing diagrams of compounds 5d and 5e

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  3.3 Biological activity analysis

  The IC₅₀ values of 5a~5f and Foretinib against MKN-45, HT-29, A549, K562 and GIST882 cancer cells are presented in Table 4, in which all the tested compounds show excellent antiproliferative activities against different cancer cells with IC₅₀ values ranging from 0.38 to 6.29 μM, indicating that newly synthesized 4-phenoxypyridine derivatives maintained antitumor activities. Notably, compounds 5a (R₁ = H), 5c (R₁ = 4-Cl) and 5e (R₁ = 3-Cl-4-F) exhibited remarkable antiproliferative activities against GIST882, K562 and A549 cell lines with IC₅₀ values of 0.68 μM (foretinib: 0.75 μM), 0.38 μM (foretinib: 0.39 μM) and 0.60 μM (foretinib: 0.94 μM), respectively, which were all better than the positive control foretinib.

  Table 4

  

  Table 4.  Antiproliferative Activities of Target Compounds against Some Cancer Cells in vitro

  DownLoad: CSV

  Compd.	R1	IC50 (μM)
  		MKN-45	HT-29	A549	K562	GIST882
  5a	H	2.38	3.51	2.34	0.95	0.68
  5b	4-F	1.26	2.09	2.25	0.61	1.92
  5c	4-Cl	0.62	1.05	1.50	0.38	1.54
  5d	3-Cl	1.36	2.36	2.23	1.01	2.85
  5e	3-Cl-4-F	0.75	2.92	0.60	0.65	2.44
  5f	4-CH3	3.34	6.29	3.62	1.98	2.39
  Foretinib	---	0.032	0.86	0.94	0.39	0.75

  3.4 Physicochemical and ADME parameters

  Furthermore, some physicochemical and ADME properties of the synthesized compounds and positive controls were predicted using the SwissADME (a free web tool to evaluate pharmacokinetics, drug likeness and medicinal chemistry friendliness of small molecules) for their adaptability with Lipinski's rule of five^[17-19]. Compounds obeying at least three of the four criteria are considered to adhere to Lipinski Rule.

  As demonstrated in Table 5, the most active compounds show variable permeability based on gastrointestinal absorption (GI), according to the BOILED-Egg predictive model (Brain Or IntestinaL EstimateD permeation method). All predicted compounds showed high gastrointestinal absorption. With respect to oral bioavailability, it is expected 0.55 of probability of oral bioavailability score > 10% in the rat for all compounds, the same as the control drug foretinib (0.55). Compound 5a~5f exhibited potent in vitro antitumor activity, low toxicity and reasonable physicochemical properties, because suitable flexible and size in the bioavailability radar map (Fig. 4). All these data suggests that compound 5a~5f could be considered as a candidate for further research.

  Table 5

  

  Table 5.  Physicochemical Properties and ADME Properties of Target Compounds

  DownLoad: CSV

  Compd.	MW (g/mol) < 500	H-bond acceptors < 10	H-bond donors < 5	Log P o/w < 5	Violation Lipinski Rule of 5	aPSA (Ų) ≤140	Rotatable bonds < 10	bBBB	cGI	dBS
  5a	456.47	6	3	3.94	0	101.06	9	NO	Low	0.55
  5b	474.46	7	3	4.13	0	101.06	9	NO	Low	0.55
  5c	490.92	6	3	4.46	0	101.06	9	NO	Low	0.55
  5d	490.92	6	3	4.41	0	101.06	9	NO	Low	0.55
  5e	508.91	7	3	4.66	1	101.06	9	NO	Low	0.55
  5f	470.50	6	3	4.12	0	101.06	9	NO	Low	0.55
  Foretinib	632.65	10	2	4.80	1	111.25	14	NO	Low	0.55
  aPSA – polar surface area. bBBB – blood-brain barrier. cGI – gastrointestinal absorption. dBS – bioavailability score.

  Figure 4

  

  Figure 4.  The bioavailability radar enables a first glance at the drug-likeness of target compounds and Foretinib. The pink area represents the optimal range for each properties (lipophilicity: XLOGP3 between –0.7 and +5.0, size: MW between 150 and 500 g/mol, polarity: TPSA between 20 and 130 Ų, solubility: logS not higher than 6, saturation: fraction of carbons in the sp³ hybridization not less than 0.25, and flexibility: no more than 9 rotatable bonds.

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  Supplementary data associated with this article have been deposited in the ESI.
  Electronic supplementary information
  
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• 

  Figure 1  The representative 4-phenoxypyridine type II c-Met kinase inhibitors and design strategy of the target compounds

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  Scheme 1  Synthetic route and structure of target compounds

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  Figure 2  Molecular structure of compound 5d and 5e in a single crystal at 296 K

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  Figure 3  Crystal packing diagrams of compounds 5d and 5e

  下载: 全尺寸图片 幻灯片
  

  Figure 4  The bioavailability radar enables a first glance at the drug-likeness of target compounds and Foretinib. The pink area represents the optimal range for each properties (lipophilicity: XLOGP3 between –0.7 and +5.0, size: MW between 150 and 500 g/mol, polarity: TPSA between 20 and 130 Ų, solubility: logS not higher than 6, saturation: fraction of carbons in the sp³ hybridization not less than 0.25, and flexibility: no more than 9 rotatable bonds.

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  Table 1.  Crystal Data of Compounds 5d and 5e

  Compound	5d	5e
  CCDC	2088950	2088951
  Empirical formula	C25H20ClFN6O2	C25H19ClF2N6O2
  Formula weight	490.91	508.91
  Crystal size (mm 3)	0.23 × 0.20 × 0.16	0.28 × 0.25 × 0.22
  Crystal system	Monoclinic	Monoclinic
  Space group	P21/c	P21/c
  a (Å)	11.0500(10)	10.9998(18)
  b (Å)	18.3045(17)	18.517(3)
  c (Å)	13.5646(9)	13.6355(16)
  α (°)	90	90
  β (°)	122.806(5)	123.315(9)
  γ (°)	90	90
  Volume (Å3)	2306.1(4)	2320.9(6)
  Z	4	4
  Dc (g·cm-3)	1.411	1.456
  Index ranges	–9≤h≤13 –21≤k≤21 –16≤l≤14	–12≤h≤13 –22≤k≤14 –16≤l≤15
  Absorption coefficient (μ)	0.210	0.218
  F(000)	1012	1048
  Theta range for data collection (o)	2.104 to 24.998	2.099 to 24.998
  Reflections collected	11625	11670
  Independent reflections	4051	4072
  Rint	0.0273	0.0437
  Goodness-of-fit on F2	1.060	1.054
  R, wR(I > 2σ(I))	R = 0.0606, wR = 0.2068	R = 0.0495, wR = 0.1139
  R, wR(all data)	R = 0.0866, wR = 0.2263	R = 0.0943, wR = 0.1282
  R = ∑(||Fo|–|Fc||)/∑|Fo|, wR = [∑w(Fo2–Fc2)2/∑w(Fo2)2]1/2

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  Table 2.  Hydrogen Bond Lengths (Å) and Bond Angles (°) of 5d and 5e

  5d	D–H···A	d(D–H)			d(H···A)	d(D···A)	∠DHA
  	N(1)–H(1)···N(5)		0.86	2.26		3.084(4)	160
  	N(4)–H(4)···O(2)#1		0.86	2.06		2.871(4)	157
  5e	N(1)–H(1)···N(5)#1		0.86	2.24		3.051(3)	159
  	N(4)–H(4)···O(2)#2		0.86	2.06		2.874(2)	157
  Symmetry codes: 5d: #1: –x, y+1/2, –z+1/2; 5e: #1: –x, –y, –z; #2: x, –y–1/2, z–1/2

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  Table 3.  Geometric Parameters of 5d and 5e

  Bond	Dist. for 5d	Dist. for 5e		Angle	5d (o)	5e (o)
  C(1)–N(1)	1.397(4)	1.409(3)		C(6)–C(1)–N(1)	124.3(3)	122.9(3)
  C(1)–C(2)	1.403(5)	1.387(4)		C(6)–C(1)–C(2)	118.6(3)	119.2(3)
  C(3)–Cl(1)	1.745(4)	1.728(3)		N(2)–C(7)–N(3)	127.8(3)	127.5(2)
  C(7)–N(2)	1.334(4)	1.341(3)		N(3)–C(7)–N(1)	114.5(3)	114.8(2)
  C(7)–N(1)	1.381(4)	1.364(3)		C(8)–C(9)–C(10)	116.6(3)	116.2(3)
  C(8)–C(9)	1.365(5)	1.358(4)		C(12)–C(11)–N(4)	119.8(3)	121.1(2)
  C(10)–N(4)	1.357(4)	1.361(3)		C(18)–C(17)–C(21)	120.4(3)	120.1(3)
  						To be continued
  C(11)–C(12)	1.388(5)	1.396(4)		N(5)–C(19)–N(6)	114.7(3)	114.5(2)
  C(11)–N(4)	1.401(4)	1.405(3)		C(22)–C(23)–C(24)	117.5(3)	115.9(3)
  C(14)–O(1)	1.382(4)	1.398(3)		C(24)–C(23)–C(25)	58.5(3)	58.6(2)
  C(17)–C(18)	1.377(4)	1.377(4)		C(7)–N(1)–C(1)	127.6(3)	127.6(2)
  C(17)–O(1)	1.379(4)	1.379(3)		C(7)–N(2)–C(10)	116.0(3)	115.9(2)
  C(19)–N(5)	1.333(4)	1.338(3)		C(7)–N(3)–C(8)	113.8(3)	113.9(2)
  C(19)–N(6)	1.399(4)	1.398(3)		C(22)–N(6)–C(19)	126.2(3)	126.2(2)
  C(22)–O(2)	1.226(4)	1.230(3)		C(17)–O(1)–C(14)	117.7(2)	116.8(2)
  C(22)–N(6)	1.362(4)	1.358(3)		N(1)–C(1)–C(2)–C(3)	–177.3(3)	175.3(2)
  C(22)–C(23)	1.471(5)	1.464(4)		O(1)–C(14)–C(15)–C(16)	178.5(3)	178.3(2
  C(23)–C(24)	1.507(5)	1.509(4)		N(2)–C(7)–N(1)–C(1)	–7.6(5)	6.1(4)
  C(24)–C(25)	1.477(6)	1.469(4)		N(6)–C(22)–C(23)–C(25)	–133.3(3)	–163.5(2)

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  Table 4.  Antiproliferative Activities of Target Compounds against Some Cancer Cells in vitro

  Compd.	R1	IC50 (μM)
  		MKN-45	HT-29	A549	K562	GIST882
  5a	H	2.38	3.51	2.34	0.95	0.68
  5b	4-F	1.26	2.09	2.25	0.61	1.92
  5c	4-Cl	0.62	1.05	1.50	0.38	1.54
  5d	3-Cl	1.36	2.36	2.23	1.01	2.85
  5e	3-Cl-4-F	0.75	2.92	0.60	0.65	2.44
  5f	4-CH3	3.34	6.29	3.62	1.98	2.39
  Foretinib	---	0.032	0.86	0.94	0.39	0.75

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  Table 5.  Physicochemical Properties and ADME Properties of Target Compounds

  Compd.	MW (g/mol) < 500	H-bond acceptors < 10	H-bond donors < 5	Log P o/w < 5	Violation Lipinski Rule of 5	aPSA (Ų) ≤140	Rotatable bonds < 10	bBBB	cGI	dBS
  5a	456.47	6	3	3.94	0	101.06	9	NO	Low	0.55
  5b	474.46	7	3	4.13	0	101.06	9	NO	Low	0.55
  5c	490.92	6	3	4.46	0	101.06	9	NO	Low	0.55
  5d	490.92	6	3	4.41	0	101.06	9	NO	Low	0.55
  5e	508.91	7	3	4.66	1	101.06	9	NO	Low	0.55
  5f	470.50	6	3	4.12	0	101.06	9	NO	Low	0.55
  Foretinib	632.65	10	2	4.80	1	111.25	14	NO	Low	0.55
  aPSA – polar surface area. bBBB – blood-brain barrier. cGI – gastrointestinal absorption. dBS – bioavailability score.

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•