Structure-based design, structure-activity relationship analysis, and antitumor activity of diaryl ether derivatives

Article abstract of DOI:10.1007/s12272-015-0578-7

To identify novel therapeutic agents to treat cancer, we synthesized a series of diaryl ether derivatives. Structure-activity relationship studies revealed that the presence of a chlorine or hydroxyl at the para-position on the phenyl ring (5h or 5k) significantly enhanced antitumor activity. Compound 5h had stronger growth inhibitory activity in HepG2, A549, and HT-29 cells than compound 5k, with IC₅₀ values of 2.57, 5.48, and 30.04 μM, respectively. Compound 5h also inhibited the growth of other cells lines, including Hep3B, PLC/PRF5, SMMC-7721, HeLa, and A375, with IC₅₀ values of 2.76, 4.26, 29.66, 18.86, and 10.21 μM, respectively. The antitumor activity of compound 5h was confirmed by a colony forming assay. Further, our results indicated that the antitumor activity of compound 5h may be mediated by enhancing expression of p21 and cl-caspase3, and leading to apoptosis of cancer cells.

Full text of DOI:10.1007/s12272-015-0578-7

Arch. Pharm. Res.
DOI 10.1007/s12272-015-0578-7
RESEARCH ARTICLE
Structure-based design, structure–activity relationship analysis,
and antitumor activity of diaryl ether derivatives
•
Shao-Mei Yang Zhi-Ning Huang Zhong-Shi Zhou
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Jin Hou Man-Yi Zheng Li-Juan Wang Yu Jiang
•
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Xin-Yi Zhou Qiu-Yue Chen Shan-Hua Li Fu-Nan Li
Received: 20 September 2014 / Accepted: 18 February 2015
Ó The Pharmaceutical Society of Korea 2015
Abstract To identify novel therapeutic agents to treat
cancer, we synthesized a series of diaryl ether derivatives.
Structure–activity relationship studies revealed that the
presence of a chlorine or hydroxyl at the para-position on
the phenyl ring (5h or 5k) significantly enhanced antitumor
activity. Compound 5h had stronger growth inhibitory ac-
tivity in HepG2, A549, and HT-29 cells than compound 5k,
with IC50 values of 2.57, 5.48, and 30.04 lM, respectively.
Compound 5h also inhibited the growth of other cells lines,
including Hep3B, PLC/PRF5, SMMC-7721, HeLa, and
A375, with IC50 values of 2.76, 4.26, 29.66, 18.86, and
10.21 lM, respectively. The antitumor activity of com-
pound 5h was confirmed by a colony forming assay. Fur-
ther, our results indicated that the antitumor activity of
compound 5h may be mediated by enhancing expression of
p21 and cl-caspase3, and leading to apoptosis of cancer
cells.
Keywords Diaryl ethers Á Structural modification Á
Antitumor
Introduction
Over the past several decades, pharmaceutical companies
and medical researchers have sought to identify novel
cancer therapeutics for drug development. Cancer is a
leading cause of death (Jemal et al. 2011), with 14 millions
new cases of cancer diagnosed each year (http://www.iarc.
fr). Thus, there is an urgent need to develop new agents to
treat cancer.
Diaryl ether is a structural feature found in many man-
made and naturally occurring organic chemicals and
medicinal agents that possesses a wide range of pharma-
cological activities (Nicolaou et al. 1999; Yang et al.
2011). The diaryl ether skeleton is considered a privileged
structure and mediates a broad range of biological effects,
including antidiabetic (Patch et al. 2011), anti-tubercular
(Yang et al. 2011), and antitumor activity (Bedos-Belval
et al. 2012); it also acts as an HIV-1 non-nucleoside reverse
transcriptase inhibitor (NNRTIs) (Tucker et al. 2008;
Kennedy-Smith et al. 2010). Sorafenib (BAY 43-9006)
(Fig. 1) is a therapeutic used to treat advanced renal cell
carcinoma (RCC) (Ratain et al. 2006), unresectable
hepatocellular carcinomas (HCC) (Abou-Alfa et al. 2006),
and thyroid cancer (Gupta-Abramson et al. 2008).
Shao-Mei Yang and Zhi-Ning Huang have contributed equally to this
work.
Electronic supplementary material The online version of this
article (doi:10.1007/s12272-015-0578-7) contains supplementary
material, which is available to authorized users.
S.-M. Yang Á Z.-N. Huang Á J. Hou Á M.-Y. Zheng Á
L.-J. Wang Á X.-Y. Zhou Á Q.-Y. Chen Á F.-N. Li (&)
School of Pharmaceutical Sciences, Xiamen University,
Xiang’an South Road, Xiamen 361102,
People’s Republic of China
e-mail: fnlee5@xmu.edu.cn
Z.-S. Zhou Á S.-H. Li
Department of Basic Medical Sciences of Medical College,
Xiamen University, Xiang’an South Road, Xiamen 361102,
People’s Republic of China
The purpose of this study was to structurally modify
Sorafenib to optimize its antitumor effect in various tumor
cell lines. In this paper, we reported the chemical synthesis
and structure–activity relationship (SAR) of a novel series
Y. Jiang
Department of Outpatient, Air Force of Chengdu Military
Command, 87 New South Road, Chengdu 610041,
People’s Republic of China
123

S.-M. Yang et al.
aqueous mixture was extracted with AcOEt. The organic
phase was combined and washed with brine, dried over
anhydrous MgSO4, filtered, and concentrated to provide
1
compound 4 (87 %) as a white solid. H NMR (400 MHz,
DMSO-d6): d 12.82 (brs, 1H), 7.95 (d, J = 8.8 Hz, 2H),
7.45 (t, J = 7.5 Hz, 2H), 7.23 (t, J = 7.3 Hz, 1H), 7.11 (d,
J = 7.7 Hz, 2H), 7.02 (d, J = 8.8 Hz, 2H); 13C NMR
(150 MHz, DMSO-d6): d 166.8, 161.0, 155.1, 131.7, 130.3,
125.2, 124.7, 119.9, 117.2.
Fig. 1 The structure of Sorafenib and potency-enhanced amide
analogues
N-(2-(Dimethylamino)ethyl)-4-phenoxybenzamide (5a)
To a solution of N,N-dimethylethylenediamine (51 lL,
0.47 mmol) and compound 4 (100 mg, 0.47 mmol) in
CH2Cl2 (5 mL) at 0 °C were added 1-hydroxybenzo-
triazole HOBt (76 mg, 0.56 mmol), 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide hydrochloride EDCI (107 mg,
0.56 mmol) and 4-methylmorpholine (155 lL, 1.41 mmol).
The reaction mixture was stirred at room temperature for
overnight, then washed with 10 % aqueous HCl, 5 %
aqueous NaHCO3, H2O and brine, dried over MgSO4, and
concentrated in vacuo. The residue was purified by column
chromatography to provide the title compound (97 %) as a
of compounds. Further, we evaluated the antitumor activity
in vitro of the target compounds and performed the cell
cycle analysis of compound 5h.
Materials and methods
Chemistry
All reagents were purchased and used without further pu-
rification unless otherwise noted. Reactions were mon-
itored by thin-layer chromatography (TLC) on YanTai
silica gel GF-254 thin-layer plates. 1H NMR and 13C NMR
spectra were determined with a Bruker Avance III
600 MHz spectrometer (400 or 600 MHz for 1H and
150 MHz for 13C). Chemical shifts are expressed in d
values (ppm), using tetramethylsilane (TMS) as the internal
standard; coupling constants (J) are given in Hz. Signal
multiplicities are characterized as singlet (s), doublet (d),
triplet (t), quartet (q), multiplet (m), broad signal (brs).
High-resolution mass spectral (HRMS) were acquired on a
Q-Exactive series MS instrument with UV detection at
254 nm in low-resonance electrospray mode (ESI).
1
yellow oil. H NMR (400 MHz, CDCl3): d 7.80 (d, J =
8.8 Hz, 2H), 7.38 (t, J = 8.5 Hz, 2H), 7.17 (t, J = 7.5 Hz,
1H), 7.06 (d, J = 7.8 Hz, 2H), 7.01 (d, J = 8.8 Hz, 2H),
6.95 (brs, 1H), 3.54 (q, J = 5.0 Hz, 2H), 2.57 (t, J =
5.8 Hz, 2H), 2.31 (s, 6H);13C NMR (150 MHz, CDCl3): d
166.5, 159.4, 156.0, 127.4, 126.3, 126.2, 117.9, 116.7, 56.3,
45.7, 38.7; HRMS (ESI, m/z) calcd for C17H21N2O2
[M ? H]? 285.1598, found 285.1603.
N-(2-(Diethylamino)ethyl)-4-phenoxybenzamide (5b)
Titled compound 5b (88 %) was prepared by the synthetic
procedure of 5a as yellow oil. 1H NMR (400 MHz,
CDCl3): d 7.80 (d, J = 8.8 Hz, 2H), 7.37 (t, J = 7.5 Hz,
2H), 7.16 (t, J = 7.5 Hz, 1H), 7.04 (d, J = 8.5 Hz, 2H),
6.98 (d, J = 8.8 Hz, 2H), 4.89 (brs, 1H), 3.55 (q,
J = 5.3 Hz, 2H), 2.76 (t, J = 5.8 Hz, 2H), 2.68 (q,
J = 7.3 Hz, 4H),1.10 (t, J = 7.3 Hz, 6H); 13C NMR
(150 MHz, CDCl3): d 166.7, 160.2, 155.9, 129.8, 128.8,
124.1, 119.7, 117.6, 51.5, 46.8, 36.8, 11.1; HRMS (ESI, m/z)
calcd for C19H25N2O2 [M ? H]? 313.1911, found
313.1916.
4-Phenoxybenzoic acid (4)
To a solution of iodobenzene (1 mL, 8.9 mol), p-hy-
droxybenzoic acid (1.85 g, 13.4 mol), CuI (170 mg,
0.9 mol), Cs2CO3 (5.81 g, 17.8 mol) and N,N-dimethyl
glycine (276 mg, 2.7 mmol) in 1,4-dioxane under nitrogen
atmosphere at 90 °C and the mixture was stirred for 24 h.
1,4-dioxane was evaporated and the residue was extracted
with AcOEt and water. The organic phase was combined
and washed with brine, dried over anhydrous MgSO4 and
concentrated. Then the residue was purified by column
chromatography to get compounds 3 as yellow oil. Dis-
solve it in water (8 mL) and ethanol (8 mL), added NaOH
(1.82 g, 4.5 mol) and reflux 3 h. The mixture was cooled
and added 50 mL water, acidified with 2 M HCl. The
N-(2-Morpholinoethyl)-4-phenoxybenzamide (5c)
Titled compound 5c (87 %) was prepared by the synthetic
procedure of 5a as a white solid. 1H NMR (400 MHz,
CDCl3): d 7.74 (d, J = 8.8 Hz, 2H), 7.37 (t, J = 7.3 Hz,
2H), 7.16 (t, J = 7.5 Hz, 1H), 7.04 (d, J = 7.5 Hz, 2H),
123

Antitumor activity of diaryl ether derivatives
7.00 (d, J = 8.0 Hz, 2H), 6.70 (brs, 1H), 3.71 (t,
J = 4.8 Hz, 4H), 3.53 (q, J = 5.5 Hz, 2H), 2.59 (t,
J = 6.0 Hz, 2H), 2.49 (t, J = 4.5 Hz, 4H); 13C NMR
(150 Hz, CDCl3): d 166.6, 160.3, 155.9, 129.9, 128.9,
128.7, 124.2, 119.7, 117.7, 66.9, 56.9, 53.3, 36.0; HRMS
(ESI, m/z) calcd for C19H23N2O3 [M ? H]? 327.1703,
found 327.1709.
(d, J = 7.5 Hz, 2H), 7.40 (q, J = 7.3 Hz, 4H), 7.19 (t,
J = 7.3 Hz, 1H), 7.15 (t, J = 7.3 Hz, 1H), 7.08 (t,
J = 8.8 Hz, 4H); 13C NMR (150 MHz, CDCl3): d 160.5,
155.5, 129.7, 129.2, 129.1, 128.8, 128.5, 124.2, 124.1,
120.7, 120.6, 119.5, 117.3; HRMS (ESI, m/z) calcd for
C19H16NO2 [M ? H]? 290.1176, found 290.1181,
[M ? Na]? 312.0995, found 312.1000.
N-Cyclobutyl-4-phenoxybenzamide (5d)
N-(4-Fluorophenyl)-4-phenoxybenzamide (5h)
Titled compound 5d (87 %) was prepared by the synthetic
procedure of 5a as a white solid. 1H NMR (400 MHz,
CDCl3): d 7.75 (d, J = 8.8 Hz, 2H), 7.37 (t, J = 7.5 Hz,
2H), 7.16 (t, J = 7.3 Hz, 1H), 7.03 (d, J = 7.5 Hz, 2H),
6.97 (d, J = 8.8 Hz, 2H), 6.47 (brs, 1H), 4.58 (m, 1H),
2.37–2.45 (m, 2H), 1.92–2.02 (m, 2H), 1.70–1.79 (m, 2H);
13C NMR (150 MHz, CDCl3): d 165.9, 160.2, 155.9, 129.9,
129.0, 128.8, 124.1, 119.6, 117.6, 45.1, 31.1,15.1; HRMS
(ESI, m/z) calcd for C17H18NO2 [M ? H]? 268.1332,
found 268.1338, [M ? Na]? 290.1151, found 290.1157.
Titled compound 5h (47 %) was prepared by the synthetic
procedure of 5a as a white solid. 1H NMR (600 MHz,
DMSO-d6): d 10.25 (s, 1H), 8.00 (d, J = 8.6 Hz, 2H), 7.81
(q, J = 7.8 Hz, 2H), 7.44 (t, J = 7.5 Hz, 2H), 7.22 (t,
J = 7.4 Hz, 1H), 7.18 (t, J = 8.9 Hz, 2H), 7.10 (t,
J = 7.0 Hz, 4H); 13C NMR (150 MHz, DMSO-d6): d
165.1, 160.3, 156.0, 130.7, 130.4, 129.8, 124.8, 122.7,
122.6, 120.0, 117.9, 115.7, 115.5; HRMS (ESI, m/z) calcd
for C19H15FNO2 [M ? H]? 308.1081, found 308.1087,
[M ? Na]? 330.0901, found 330.0906.
N-Cyclopentyl-4-phenoxybenzamide (5e)
N-(4-Methylphenyl)-4-phenoxybenzamide (5i)
Titled compound 5e (90 %) was prepared by the synthetic
procedure of 5a as a white solid. 1H NMR (400 MHz,
CDCl3): d 7.73 (d, J = 8.8 Hz, 2H), 7. 37 (t, J = 7.5 Hz,
2H), 7.16 (t, J = 7.5 Hz, 1H), 7.03 (d, J = 7.8 Hz, 2H),
6.98 (d, J = 8.8 Hz, 2H), 6.18 (brs, 1H), 4.38 (m, 1H),
2.04–2.11 (m, 2H), 1.59–1.76 (m, 4H), 1.43–1.53 (m, 2H);
13C NMR (150 MHz, CDCl3): d 166.5, 160.0, 156.0, 129.8,
129.3, 128.7, 124.0, 119.5, 117.7, 51.6, 34.0, 23.7; HRMS
(ESI, m/z) calcd for C18H20NO2 [M ? H]? 282.1489,
found 282.1494, [M ? Na]? 304.1308, found 304.1313.
Titled compound 5i (51 %) was prepared by the synthetic
procedure of 5a as a white solid.1H NMR (600 MHz,
CDCl3): d 7.94 (brs, 1H), 7.84 (d, J = 8.6 Hz, 2H), 7.51
(d, J = 8.3 Hz, 2H), 7.40 (t, J = 8.0 Hz, 2H), 7.20 (t,
J = 7.4 Hz, 1H), 7.15 (d, J = 8.3 Hz, 2H), 7.07 (t,
J = 7.9 Hz, 2H), 7.02 (t, J = 8.6 Hz, 2H), 2.34 (s, 3H);
13C NMR (150 MHz, CDCl3): d 165.1, 160.7, 155.8, 135.4,
134.1, 123.0, 129.5, 129.0, 124.3, 120.4, 119.8, 117.8,
20.9; HRMS (ESI, m/z) calcd for C20H18NO2 [M ? H]?
304.1332, found 304.1338, [M ? Na]? 326.1151, found
326.1157.
N-Cyclohexyl-4-phenoxybenzamide (5f)
N-(4-Hydroxyphenyl)-4-phenoxybenzamide (5k)
Titled compound 5d (89 %) was prepared by the synthetic
procedure of 5a as a white solid. 1H NMR (400 MHz,
CDCl3): d 7.74 (d, J = 8.8 Hz, 2H), 7.38 (t, J = 7.5 Hz,
2H), 7.17 (t, J = 7.3 Hz, 1H), 7.04 (d, J = 7.5 Hz, 2H),
7.00 (d, J = 8.0 Hz, 2H), 5.96 (brs, 1H), 3.97 (m, 1H),
2.01–2.05 (m, 2H), 1.64–1.79 (m, 4H), 1.38–1.49 (m, 2H),
0.84–0.91 (m, 2H); 13C NMR (150 MHz, CDCl3): d 165.9,
160.0, 156.0, 129.8, 129.5, 128.7, 124.0, 119.5, 117.7,
48.6, 33.1, 25.5, 24.9; HRMS (ESI, m/z) calcd for
C19H22NO2 [M ? H]? 296.1645, found 296.1651,
[M ? Na]? 318.1470, found 318.1465.
Titled compound 5k (50 %) was prepared by the synthetic
procedure of 5a as a white solid. 1H NMR (600 MHz,
DMSO-d6): d 9.95 (s, 1H), 9.92 (s, 1H), 7.96 (d,
J = 8.9 Hz, 2H), 7.50 (d, J = 8.6 Hz, 2H), 7.45 (t,
J = 7.6 Hz, 2H), 7.22 (t, J = 7.3 Hz, 1H), 7.10 (d,
J = 8.6 Hz, 2H), 7.07 (d, J = 8.9 Hz, 2H), 6.73 (d,
J = 8.6 Hz, 2H); 13C NMR (150 MHz, DMSO-d6): d
164.2, 159.5, 155.7, 153.7, 130.7, 130.2, 129.8, 129.7,
124.3, 122.3, 119.5, 117.4, 115.0; HRMS (ESI, m/z) calcd
for C19H16NO3 [M ? H]? 306.1125, found 306.1130,
[M ? Na]? 328.0944, found 328.0950.
N-Phenyl-4-phenoxybenzamide (5g)
N-(4-Methoxyphenyl)-4-phenoxybenzamide (5l)
Titled compound 5f (90 %) was prepared by the synthetic
procedure of 5a as a white solid. 1H NMR (400 MHz,
CDCl3): d 7.86 (d, J = 9.0 Hz, 2H), 7.77 (brs, 1H), 7.63
Titled compound 5i (60 %) was prepared by the synthetic
procedure of 5a as a white solid. 1H NMR (600 MHz,
123

S.-M. Yang et al.
DMSO-d6): d 10.07 (brs, 1H), 7.99 (d, J = 8.6 Hz, 2H),
7.67 (d, J = 8.8 Hz, 2H), 7.45 (t, J = 7.8 Hz, 2H), 7.22 (t,
J = 7.3 Hz, 1H), 7.10 (d, J = 8.3 Hz, 2H), 7.08 (d,
J = 8.6 Hz, 2H), 6.92 (d, J = 8.8 Hz, 2H), 3.74 (s, 3H);
13C NMR (150 MHz, DMSO-d6): d 164.3, 159.6, 155.6,
155.5, 132.3, 130.2, 129.8, 129.7, 124.3, 122.0, 119.5,
117.4, 113.7, 55.1; HRMS (ESI, m/z) calcd for C20H18NO3
[M ? H]? 320.1281, found 320.1278, [M ? Na]?
342.1101, found 342.1106.
purified by column chromatography on silica gel to afford
compounds 7a (85 %) as a white solid. 1H NMR
(600 MHz, CDCl3): d 7.74 (d, J = 8.6 Hz, 2H), 7.35 (t,
J = 8.6 Hz, 2H), 7.15 (t, J = 7.3 Hz, 1H), 7.01 (d,
J = 7.9 Hz, 2H), 6.94 (d, J = 8.9 Hz, 2H), 6.68 (brs, 1H),
2.87 (m, 1H), 0.80–0.83 (m, 2H), 0.60–0.63 (m, 2H); 13C
NMR (150 MHz, CDCl3): d 168.3,160.2, 155.9, 129.9,
128.8, 128.8, 124.1, 119.6, 117.6, 23.1, 6.5; HRMS (ESI,
m/z) calcd for C16H16NO2 [M ? H]? 254.1176, found
254.1181; [M ? Na]? 276.0095, found 276.1000.
N-(4-(Trifluoromethoxyl)phenyl)-4-phenoxybenzamide
(5m)
N-(4-Chlorophenyl)-4-phenoxybenzamide (7b)
Titled compound 5m (49 %) was prepared by the synthetic
procedure of 5a as a white solid. 1H NMR (600 MHz,
DMSO-d6): d 10.37 (s, 1H), 8.00 (d, J = 9.0 Hz, 2H), 7.89
(d, J = 9.0 Hz, 2H), 7.45 (t, J = 8.6 Hz, 2H), 7.35 (d,
J = 8.0 Hz, 2H), 7.23 (t, J = 7.5 Hz, 1H), 7.11 (t,
J = 6.6 Hz, 4H); 13C NMR (150 MHz, DMSO-d6): d 164.9,
159.9, 155.5, 143.8, 138.4, 130.3, 130.0, 129.2, 124.4, 121.6,
121.4, 119.5, 117.4; HRMS (ESI, m/z) calcd for C20H15F3-
NNaO3 [M ? Na]? 396.0818, found 396.0823.
Titled compound 7b (59 %) was prepared by the synthetic
procedure of 7a as a white solid. 1H NMR (600 MHz,
DMSO-d6): d 10.31 (s, 1H), 8.00 (d, J = 8.6 Hz, 2H), 7.81
(d, J = 7.9 Hz, 2H), 7.45 (t, J = 7.8 Hz, 2H), 7.40 (d,
J = 8.8 Hz, 2H), 7.23 (t, J = 7.3 Hz, 1H), 7.10 (t,
J = 8.4 Hz, 4H); 13C NMR (150 MHz, DMSO-d6): d
164.8, 159.9, 155.5, 138.2, 130.2, 130.0, 129.2, 126.5,
127.1, 124.4, 121.8, 119.5, 117.4; HRMS (ESI, m/z) calcd
for C19H15ClNO2 [M ? H]? 324.0786, found 324.0791,
[M ? Na]? 346.0605, found 346.0611.
N-(4-tert-Butylphenyl)-4-phenoxybenzamide (5n)
Titled compound 5n (67 %) was prepared by the synthetic
procedure of 5a as a white solid. 1H NMR (600 MHz,
CDCl3): d 8.09 (brs, 1H), 7.81 (d, J = 8.8 Hz, 2H), 7.55
(d, J = 8.6 Hz, 2H), 7.36 (t, J = 7.5 Hz, 2H), 7.33 (d,
J = 8.6 Hz, 2H), 7.16 (t, J = 7.5 Hz, 1H), 7.03 (d,
J = 7.7 Hz, 2H), 6.96 (d, J = 8.8 Hz, 2H), 1.30 (s, 9H);
13C NMR (150 MHz, CDCl3): d 165.2, 160.6, 155.8, 147.4,
135.4, 129.9, 130.0, 129.0, 125.8, 124.3, 120.1, 119.7,
117.7, 34.3, 31.3; HRMS (ESI, m/z) calcd for C23H24FNO2
[M ? H]? 346.1802, found 346.1807, [M ? Na]?
368.1621, found 368.1626.
N-(4-Bromophenyl)-4-phenoxybenzamide (7c)
Titled compound 7c (51 %) was prepared by the synthetic
procedure of 7a as a white solid. 1H NMR (600 MHz,
DMSO-d6): d 10.30 (s, 1H), 7.99 (d, J = 8.6 Hz, 2H), 7.75
(d, J = 8.8 Hz, 2H), 7.53 (d, J = 8.8 Hz, 2H), 7.45 (t,
J = 7.9 Hz, 2H), 7.23 (t, J = 7.4 Hz, 1H), 7.10 (t,
J = 9.1 Hz, 4H); 13C NMR (150 MHz, DMSO-d6): d
164.8, 159.9, 155.5, 138.6, 131.4, 130.3, 130.0, 129.2,
124.4, 122.2, 119.6, 117.4, 115.2; HRMS (ESI, m/z) calcd
for C19H15BrNNaO2 [M ? Na]? 390.0106, found
390.0106.
N-Cyclopropyl-4-phenoxybenzamide (7a)
N-(4-Cyanophenyl)-4-phenoxybenzamide (7d)
To a solution of compound 4 (100 mg, 0.47 mmol) in
CHCl3 (5 mL) was added slowly thionyl dichloride (42 lL,
0.56 mmol) under nitrogen atmosphere at 0 °C. The reac-
tion mixture was stirred at 100 °C for 2 h, and evaporated
to get compound 6, which was used to next reaction im-
mediately because of its high instability.
Titled compound 7d (57 %) was prepared by the synthetic
procedure of 7a as a yellow oil. 1H NMR (600 MHz,
CDCl3): d 8.22 (d, J = 8.8 Hz, 2H), 8.08 (d, J = 8.4 Hz,
2H), 7.54 (t, J = 7.9 Hz, 1H), 7.46 (d, J = 9.0 Hz, 2H),
7.43 (d, J = 8.4 Hz, 2H), 7.26 (t, J = 7.3 Hz, 1H), 7.13 (d,
J = 8.8 Hz, 2H), 7.10 (d, J = 8.8 Hz, 2H); 13C NMR
(150 MHz, CDCl3): d 164.2, 162.2, 154.7, 143.4, 133.2,
130.3, 128.9, 128.8, 125.4, 124.9, 120.6, 118.3, 117.5,
108.4; HRMS (ESI, m/z) calcd for C20H15N2O2 [M ? H]?
315.1128, found 315.1134, [M ? Na]? 337.0949, found
337.0953.
To a solution of cyclopropylamine (33 lL, 0.47 mmol)
was added Et3N (200 lL, 1.41 mmol), followed by drop
wise addition of acid chloride in anhydrous CH2Cl2. The
solution was stirred for 2 h at room temperature, then it
was washed successively with 10 % HCl, 5 % NaOH,
water and brine, dried over anhydrous MgSO4, filtered and
concentrated under reduced pressure. The residue was
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Antitumor activity of diaryl ether derivatives
N-(2,4-Difluorophenyl)-4-phenoxybenzamide (7e)
Titled compound 7e (48 %) was prepared by the synthetic
1
procedure of 7a as a yellow solid. H NMR (600 MHz,
N-(2-Cyano-4-fluorophenyl)-4-phenoxybenzamide (7i)
Titled compound 7i (33 %) was prepared by the synthetic
1
procedure of 7a as a yellow solid. H NMR (600 MHz,
CDCl3): d 8.40 (t, J = 9.2 Hz, 1H), 7.87 (d, J = 8.8 Hz,
3H), 7.41 (t, J = 8.0 Hz, 2H), 7.21 (t, J = 7.5 Hz, 1H),
7.08 (t, J = 7.5 Hz, 4H), 6.89–6.96 (m, 2H); 13C NMR
(150 MHz, CDCl3): d 163.8, 160.2, 154.6, 129.0, 128.1,
127.4, 123.5, 119.0, 116.8, 110.4, 110.3, 110.2, 102.7,
102.6, 102.4; HRMS (ESI, m/z) calcd for C19H14F2NO2
[M ? H]? 326.0987, found 326.0993, [M ? Na]?
348.0807, found 348.0812.
DMSO-d6): d 8.28 (d, J = 5.9, 2.8 Hz, 1H), 8.07 (t,
J = 7.7 Hz, 1H), 8.00 (d, J = 8.8 Hz, 2H), 7.54 (t,
J = 9.2 Hz, 1H), 7.46 (t, J = 8.1 Hz, 2H), 7.24 (t,
J = 7.5 Hz, 1H), 7.13 (d, J = 8.6 Hz, 2H), 7.12 (d,
J = 8.9 Hz, 2H); 13C NMR (150 MHz, DMSO-d6): d
165.5, 160.6, 157.9, 155.9, 136.8, 130.8, 130.5, 129.1,
128.1, 124.9, 124.6, 120.1, 117.9, 117.4, 114.5, 100.3;
HRMS (ESI, m/z) calcd for C20H14FN2O2 [M ? H]?
333.1034, found 333.1039, [M ? Na]? 355.0853, found
355.0859.
N-(2-Chloro-4-fluorophenyl)-4-phenoxybenzamide (7f)
Titled compound 7f (47 %) was prepared by the synthetic
1
procedure of 7a as a yellow solid. H NMR (600 MHz,
N-(3,4-Difluorophenyl)-4-phenoxybenzamide (7j)
CDCl3): d 8.49 (d, J = 9.2, 5.7 Hz, 2H), 8.26 (brs, 1H),
7.89 (d, J = 8.8 Hz, 2H), 7.41 (t, J = 8.1 Hz, 2H), 7.21 (t,
J = 7.3 Hz, 1H), 7.42 (d, J = 7.9, 2.8 Hz, 1H), 7.10 (d,
J = 7.5 Hz, 2H), 7.08 (d, J = 8.8 Hz, 2H); 13C NMR
(150 MHz, CDCl3): d 164.5, 161.2, 155.6, 130.0, 129.0,
124.5, 124.4, 122.8, 122.7, 120.0, 117.8, 116.3, 116.2,
114.8, 114.6; HRMS (ESI, m/z) calcd for C19H14ClFNO2
[M ? H]? 342.0692, found 342.0697, [M ? Na]?
364.0511, found 364.0517.
Titled compound 7j (53 %) was prepared by the synthetic
1
procedure of 7a as a yellow solid. H NMR (600 MHz,
CDCl3): d 7.98 (brs, 1H), 7.82 (d, J = 8.6 Hz, 2H), 7.72
(m, 1H), 7.41 (t, J = 8.4 Hz, 2H), 7.21 (t, J = 7.3 Hz,
2H), 7.12 (q, J = 8.8 Hz, 1H), 7.07 (d, J = 7.7 Hz, 2H),
7.03 (d, J = 8.8 Hz, 2H); 13C NMR (150 MHz, CDCl3): d
165.1, 161.1, 155.6, 130.1, 129.0, 128.4, 124.5, 120.0,
117.8, 117.3, 117.1, 115.9, 115.8, 110.2, 110.0; HRMS
(ESI, m/z) calcd for C19H14F2NO2 [M ? H]? 326.0987,
found 326.0993, [M ? Na]? 348.0807, found 348.0812.
N-(2-Bromo-4-fluorophenyl)-4-phenoxybenzamide (7g)
Titled compound 7g (44 %) was prepared by the synthetic
1
procedure of 7a as a yellow solid. H NMR (600 MHz,
N-(2,5-Difluorophenyl)-4-phenoxybenzamide (7k)
Titled compound 7k (27 %) was prepared by the synthetic
1
procedure of 7a as a yellow solid. H NMR (600 MHz,
CDCl3): d 8.37 (d, J = 9.2, 5.5 Hz, 1H), 8.17 (s, 1H), 7.80
(d, J = 8.8 Hz, 2H), 7.31 (t, J = 8.4 Hz, 2H), 7.23 (d,
J = 7.8, 2.8 Hz, 1H), 7.11 (t, J = 7.4 Hz, 1H), 6.94–7.02
(m, 5H); 13C NMR (150 MHz, CDCl3): d 163.5, 160.3,
154.6, 129.0, 128.1, 127.4, 123.5, 121.9, 121.8, 119.0,
118.4, 118.2, 116.8, 114.4, 114.2; HRMS (ESI, m/z) calcd
for C19H14BrFNO2 [M ? H]? 386.0186, found 386.0192,
[M ? Na]? 408.0006, found 408.0011.
CDCl3): d 8.34 (m, 1H), 8.03 (brs, 1H), 7.87 (d,
J = 8.6 Hz, 2H), 7.42 (t, J = 8.3 Hz, 2H), 7.22 (t,
J = 7.6 Hz, 1H), 7.06–7.11 (m, 5H), 6.77 (m, 1H); 13C
NMR (150 MHz, CDCl3): d 164.6, 161.3, 155.5, 130.0,
129.1, 128.1, 124.5, 120.0, 117.7, 115.2, 114.1, 110.2,
110.0, 108.9, 108.7; HRMS (ESI, m/z) calcd for C19H14-
F2NO2 [M ? H]? 326.0990, found 326.0993, [M ? Na]?
348.0808, found 348.0812.
N-(4-Fluoro-2-iodophenyl)-4-phenoxybenzamide (7h)
Titled compound 7 h (38 %) was prepared by the synthetic
1
procedure of 7a as a yellow solid. H NMR (600 MHz,
N-(3,5-Difluorophenyl)-4-phenoxybenzamide (7l)
Titled compound 7l (34 %) was prepared by the synthetic
1
procedure of 7a as a yellow solid. H NMR (600 MHz,
CDCl3): d 8.37 (d, J = 9.2, 5.6 Hz, 1H), 8.10 (brs, 1H),
7.94 (d, J = 8.9 Hz, 2H), 7.56 (m, 1H), 7.42 (t,
J = 8.1 Hz, 2H), 7.22 (t, J = 7.3 Hz, 1H), 7.16 (m, 1H),
7.10 (t, J = 6.6 Hz, 4H); 13C NMR (150 MHz, CDCl3): d
164.6, 161.3, 159.5, 155.6, 134.9, 130.1, 129.2, 128.4,
125.5, 124.6, 122.8, 120.1, 117.9, 116.2, 89.9; HRMS (ESI,
m/z) calcd for C19H14FINO2 [M ? H]? 434.0048, found
434.0053, [M ? Na]? 455.9867, found 455.9873.
CDCl3): d 7.84 (brs, 1H), 7.83 (d, J = 8.6 Hz, 2H), 7.41 (t,
J = 7.9 Hz, 2H), 7.26 (d, J = 8.9 Hz, 1H), 7.20-7.23 (m,
2H), 7.08 (d, J = 7.6 Hz, 2H), 7.06 (d, J = 8.6 Hz, 2H),
6.60 (t, J = 8.9 Hz, 1H); 13C NMR (150 MHz, CDCl3): d
165.3, 164.1, 164.0, 162.4, 161.3, 155.6, 130.1, 129.1,
128.3, 124.6, 120.0, 117.8, 103.3, 103.1, 99.7; HRMS (ESI,
m/z) calcd for C19H13F2NaO2 [M ? Na]? 348.0819, found
348.0812.
123

S.-M. Yang et al.
N-(2,4,5-Trifluorophenyl)-4-phenoxybenzamide (7m)
Ethyl 4-(4-phenoxybenzamido)benzoate (8)
Titled compound 7m (10 %) was prepared by the synthetic
1
procedure of 7a as a yellow solid. H NMR (400 MHz,
The compound 4 (107 mg, 0.5 mmol), p-amino benzoic
acid ethyl ester (83 mg, 0.5 mmol) and Et3N (140 lL,
1.0 mmol) were dissolved in MeCN (8 mL), and O-(7-az-
abenzotriazol-1-yl)-N,N,N’,N’-te-tramethyluronium hex-
afluorophosphate HATU (380 mg, 1.0 mmol) was added to
the solution. After 12 h the reaction was completed. 10 mL
of a saturated NaCl solution was then added and the pro-
duct extracted with EtOAc (3 9 20 mL). The combined
organics were washed with 2 N HCl, H2O, 5 % NaHCO3,
and then H2O. The organics were dried over MgSO4, fil-
tered, and concentrated under reduced pressure. The resi-
due was purified by flash chromatography on silica gel to
CDCl3): d 8.46 (m, 1H), 7.19 (brs, 1H), 7.85 (d,
J = 9.0 Hz, 2H), 7.42 (t, J = 7.5 Hz, 2H), 7.22 (t,
J = 7.5 Hz, 1H), 7.08 (t, J = 8.8 Hz, 4H), 7.02 (m, 1H);
13C NMR (150 MHz, CDCl3): d 164.4, 161.5, 155.5, 130.1,
129.1, 128.0, 124.7, 120.1, 117.9, 110.6, 110.5, 105.0,
104.8; HRMS (ESI, m/z) calcd for C19H13F3NO2
[M ? H]? 344.0893, found 344.0898, [M ? Na]?
366.0718, found 366.0708.
N-(3,4,5-Trifluorophenyl)-4-phenoxybenzamide (7n)
1
afford compound 8 (51 %) as a yellow solid. H NMR
(600 MHz, CDCl3): d 8.05 (d, J = 8.8 Hz, 2H), 7.85 (d,
J = 8.8 Hz, 2H), 7.73 (d, J = 8.6 Hz, 2H), 7.40 (t,
J = 8.0 Hz, 2H), 7.20 (t, J = 7.4 Hz, 2H), 7.06 (d,
J = 8.6 Hz, 2H), 7.04 (d, J = 8.8 Hz, 2H), 4.37 (q,
J = 7.3 Hz, 2H), 1.39 (t, J = 7.2 Hz, 3H); 13C NMR
(150 MHz, DMSO-d6): d 166.2, 165.1, 161.2, 155.6, 142.2,
130.8, 130.1, 129.1, 128.7, 126.1, 124.6, 120.0, 119.2,
117.8, 60.9, 14.4; HRMS (ESI, m/z) calcd for C22H20NO4
[M ? H]? 362.1387, found 362.1392.
Titled compound 7n (27 %) was prepared by the synthetic
1
procedure of 7a as a yellow solid. H NMR (600 MHz,
CDCl3): d 7.82 (d, J = 8.9 Hz, 2H), 7.79 (brs, 1H), 7.41 (t,
J = 7.6 Hz, 2H), 7.35-7.38 (m, 2H), 7.22 (t, J = 7.3 Hz,
1H), 7.08 (d, J = 7.6 Hz, 2H), 7.05 (d, J = 8.9 Hz, 2H);
13C NMR (150 MHz, CDCl3): d 165.1, 161.0, 155.9, 130.2,
129.7, 128.8, 124.5, 119.8, 117.5, 104.3, 104.2, 104.1,
104.0; HRMS (ESI, m/z) calcd for C19H13F3NO2
[M ? H]? 344.0893, found 344.0898, [M ? Na]?
366.0718, found 366.0708.
Cell growth inhibition rate (MTT assay)
The compounds were evaluated for their antiproliferative
activity on cancer cell lines like HepG2 (liver hepatoma
cells), A549 (lung cancer), HT-29 (colon adenocarcinoma
cancer). All the cell lines were incubated at 37 °C in 5 %
CO2. The culture medium used was the DMEM supple-
mented with 10 % of fetal bovine serum (FBS) (except
HT-29 cell line for RPMI-1640).
N-(2,3,4-Trifluorophenyl)-4-phenoxybenzamide (7o)
Titled compound 7o (14 %) was prepared by the synthetic
1
procedure of 7a as a yellow solid. H NMR (400 MHz,
CDCl3): d 8.13 (m, 1H), 7.86 (d, J = 8.8 Hz, 3H), 7.42 (t,
J = 7.9 Hz, 2H), 7.22 (t, J = 7.3 Hz, 1H), 7.06–7.10 (m,
4H), 7.02 (q, J = 10.0 Hz, 1H); 13C NMR (150 MHz,
CDCl3): d 164.9, 161.4, 155.5, 130.0, 129.1, 127.9, 124.6,
123.9, 123.8, 120.0, 117.8, 116.2, 116.1, 111.7, 111.6;
HRMS (ESI, m/z) calcd for C19H13F3NO2 [M ? H]?
344.0893, found 344.0889, [M ? Na]? 366.0718, found
366.0707.
The cells were seeded on 96-well plate at 5000 cells/
well and incubated overnight. The cells were treated with
compounds at 20 lM concentration, while control cells
were treated with equal DMSO. After 48 h treatment,
75 lL MTT solution (5 mg/mL was added to each well,
and incubated for 4 h, then the culture medium were re-
moved and dissolved in 100 lL DMSO. After 10 min, the
OD (optical density) was detected at 490 nm on microplate
reader. If the inhibitory rate of tested compounds >50 %,
we evaluated their IC50 values.
N-(4-Bromo-3,5-difluorophenyl)-4-phenoxybenzamide (7p)
Titled compound 7p (15 %) was prepared by the synthetic
1
procedure of 7a as a yellow solid. H NMR (600 MHz,
CDCl3): d 8.06 (brs, 1H), 7.84 (d, J = 8.6 Hz, 2H), 7.42 (t,
J = 7.6 Hz, 4H), 7.22 (t, J = 7.3 Hz, 1H), 7.08 (d,
J = 7.6 Hz, 2H), 7.05 (d, J = 8.9 Hz, 2H); 13C NMR
(150 MHz, CDCl3): d 164.1, 160.5, 159.7, 158.2, 154.5,
137.8, 129.1, 128.1, 126.9, 123.7, 119.1, 116.7, 102.8;
HRMS (ESI, m/z) calcd for C19H13BrF2NO2 [M ? H]?
404.0092, found 404.0098, [M ? Na]? 425.9912, found
425.9917.
Colony forming assay
The cells were seeded on 6-well plate at 1000 cells/well
and incubated in different concentrations of compound 5h
for 1 week. Then the cells were washed with PBS, fixed by
70 % ethanol and stained with 0.25 % crystal violet. The
number of colonies was analyzed. These experiments were
performed in triplicate.
123

Antitumor activity of diaryl ether derivatives
Scheme 1 Reagents and
conditions: (a) CuI, N,N-
dimethylglycine, Cs2CO3, 1,4-
dioxane, 90 °C; (b) NaOH,
EtOH/H2O, reflux
Scheme 2 Reagents and
conditions: (a) EDCI, HOBt,
Et3N, CH2Cl2, 0 °C to rt,
overnight
Western blotting
cells were gated to include the apoptotic and necrotic cells.
A map was set to the linear model, and a horizontal his-
togram for PI in the third fluorescence channel (FL3,
610 nm) was obtained. Finally, a log ploidy determination
of the diploid and tetraploid cells was performed. A tube of
unstained cells was used as a blank control to adjust the test
conditions.
Cells were lysed with SDS buffer containing 200 mM Tris
(pH 6.8), 40 % glycerol, and 10 % mercaptoethanol. Pro-
tein (30 lg) obtained from cell extracts were separated on
10 or 15 % SDS/polyacrylamide gel, and then transferred
to Immobilon-P membrane. Membranes were blocked with
10 % nonfat milk in Tris-buffered saline containing 0.1 %
Tween-20 for 1 h at room temperature and then incubated
overnight at 4 °C with respective primary antibodies di-
luted 1:1000–2000 in TTBS containing 5 % nonfat milk.
After three times washing with TTBS, membranes were
incubated with a horseradish peroxidase-conjugated anti-
rabbit, anti-mouse serum were used as a secondary anti-
bodies (diluted 1:10,000 in TTBS containing 5 % nonfat
milk) for 1 h at room temperature, and the antigen–anti-
body complexes were visualized using the ECL kit. b-actin
was analyzed as a loading control.
Results and discussion
Chemistry
All of the target compounds were prepared from the gen-
eral intermediate 4-phenoxybenzoic acid (4), which was
obtained through an Ullmann coupling reaction (Ma and
Cai 2003). Iodobenzene was subjected to a condensation
reaction with compound 2 to obtain compound 3. Com-
pound 3 was further hydrolyzed to generate compound 4,
with a yield of 57 % (Scheme 1).
Flow cytometric analysis of apoptosis
The target compounds were obtained via the three routes
outlined in Schemes 2, 3, 4. Compounds 5a–n were ob-
tained by following condensation reaction of commercially
available amines and intermediate 4 using 1.1–1.2 equiv of
HOBt, 1.0 equiv of EDCI, and 3.0 equiv of triethylamine
(Scheme 2).
As described above, cells in the exponential growth phase
were treated with compound 5h for 24 h, and cell death or
apoptosis was evaluated. Cell suspensions (0.3 mL,
1 9 106 cells/mL) were placed in the flow cytometry ap-
paratus. Before detection, the horizontal coordinate was set
for propidium iodide (PI) and the vertical coordinate was
set for SSC (side scatter, side light scatter). The PI-positive
Compounds 5a–n were obtained via Scheme 3. Com-
pound 4 was treated with thionyl chloride to obtain the
123

S.-M. Yang et al.
Scheme 3 Reagents and
conditions: (a) SOCl2, CHCl3,
reflux; (b) amine, CH2Cl2, Et3N,
rt
Scheme 4 Reagents and
conditions: (a) 4-aminobenzoic
acid ethyl ester, HATU,
CH3CN, rt
Table 1 Inhibition of growth by diaryl ether derivatives in HepG2, A549, and HT-29 cells
Compounds
R
HepG2a
A549a
HT-29a
IR@20 lM (%)b
IC50 (lM)
NT
IR@20 lM (%)
IC50 (lM)
NT
IR@20 lM (%)
56.35
IC50 (lM)
47.69
5a
5b
17.54
19.16
29.30
28.5
NT
NT
85.65
41.68
5c
15.13
NT
10.60
NT
8.18
NT
7a
5d
5e
14.53
12.48
16.95
NT
NT
NT
10.90
21.50
55.70
NT
NT
NT
32.96
62.44
69.96
NT
35.88
25.65
5f
31.77
NT
35.47
NT
87.89
16.22
5g
5h
54.39
85.92
17.32
2.57
69.50
79.48
24.37
5.48
77.47
52.60
8.75
30.04
7b
7c
5i
67.53
46.78
42.66
54.66
9.15
NT
NT
NT
86.43
90.91
64.96
63.39
2.03
48.70
71.47
50.43
76.91
NT
10.48
13.07
7.28
3.49
16.16
17.30
5j
123

Antitumor activity of diaryl ether derivatives
Table 1 continued
Compounds
R
HepG2a
A549a
HT-29a
IR@20 lM (%)b
IC50 (lM)
IR@20 lM (%)
IC50 (lM)
IR@20 lM (%)
IC50 (lM)
5k
5l
75.92
39.65
8.00
98.78
76.35
3.43
96.65
64.07
0.03
NT
8.82
4.94
5m
7d
43.85
19.36
NT
NT
88.78
23.16
3.07
NT
57.47
36.74
5.35
NT
5n
32.34
NT
41.48
NT
85.61
2.46
8
35.46
25.13
NT
NT
40.36
NE
NT
NT
37.21
44.70
NT
NT
7e
7f
3.48
NT
NE
NT
45.98
17.10
13.20
76.84
NT
NT
NT
7.88
7g
7h
7i
10.68
24.71
70.00
NT
NE
NT
NT
NE
NT
16.82
72.78
18.45
7j
28.01
17.95
NT
NT
14.87
NE
NT
NT
61.36
63.71
10.33
22.53
7k
7l
46.21
14.79
NT
NT
51.74
NE
15.91
NT
63.63
33.67
4.39
NT
7m
7n
7o
7p
54.71
NE
15.59
NT
67.40
7.60
21.56
NT
49.89
20.48
66.99
NT
NT
53.28
18.45
52.00
19.23
17.79
123

S.-M. Yang et al.
Table 1 continued
Compounds
R
HepG2a
A549a
HT-29a
IR@20 lM (%)b
IC50 (lM)
IR@20 lM (%)
IC50 (lM)
IR@20 lM (%)
IC50 (lM)
Sorafenib
65.18
9.61
NT
13.15c
NT
9.37c
NT means not tested, NE means no effect
a
Each compound was tested in triplicate
b
IR is the mean inhibitory rate calculated from three independent experiments measured at 48 h after treatment with the test compound at the
concentration of 20 lM
c
Huang et al. (2012); Tang et al. (2014)
Fig. 2 Compound 5h inhibits
proliferation of various cancer
cells. a IC50 of compound 5h
against various cancer cells.
b The inhibitory curves of
various cancer cells treatment
with compound 5h. c The
growth curves of HepG2 cells
treatment with different dose
compound 5h. d Colony
forming assay performed by
different dose compound 5h
treatment and control HepG2
cells (n = 3)
corresponding acyl chloride 6, which was reacted with var-
ious amines in the presence of triethylamine in anhydrous
dichloromethane to obtain the target compounds 7a–v.
Compound 8 was prepared from intermediate 4 and
4-aminobenzoic acid ethyl ester, which yielded the corre-
sponding acid (Scheme 4).
human tumor cell lines. The results are summarized in
Table 1, and revealed that most of the compounds dis-
played antitumor activity.
We initially investigated a series of N-alkyl-4-phe-
noxybenzamide derivatives (5a–f and 7a) and found
that these compounds displayed less potent antitumor
activity than sorafenib (Table 1). Further, the N-(2-sub-
stituent)ethyl-4-phenoxybenzamide derivatives (5a–c)
were the least potent as antitumor agents. Along with an
increase in membered rings,the inhibitory activity of
compounds 7a and 5d–f were improved when the
In vitro antitumor activity
In this study, the antitumor activity in vitro of the prepared
diaryl ether derivatives was evaluated in three different
123

Antitumor activity of diaryl ether derivatives
Fig. 3 Compound 5h promotes
apoptosis of hepatoma
carcinoma cell by inducing the
expression of p21 and cl-
caspase3. a, b Expression was
evaluated in a time-dependent
manner in HepG2 cells after
incubation with 10 lM
compound 5h. c, d Expression
was evaluated in a dose-
dependent manner in HepG2
cells after incubation with
compound 5h for 8 h. e Flow
cytometry detection of
compound 5h promotes
hepatoma carcinoma HepG2
cell apoptosis
cyclohexyl group was replaced with a phenyl group
(compound 5g). Compound 5g had better antitumor ac-
tivity than compound 5f in HepG2, A549, and HT-29 cells
with IC50 values of 17.32, 24.37, and 8.75 lM, respec-
tively. We then studied the influence of the 4-substitution
on the phenyl ring. Compared with compound 5g, which
has no substituents on the C ring, compounds with elec-
tron-donating groups (5i–n) and halogen groups (5h, 7b
and 7c) in the para positon of C ring exhibited excellent
good inhibitory activity. On one hand, among the com-
pounds with electron-donating groups, compound 5k
exhibited the strongest inhibitory activity on HepG2, A549,
and HT-29 cells, with IC50 values of 8.00, 3.43, and
0.03 lM, respectively. On the other hand compound 5h
showed the strongest inhibitory activity than other com-
pounds with halogen groups on HepG2, A549, and HT-29
cells, with IC50 values of 2.57, 5.48, and 30.04 lM, re-
spectively. Meantime, with electron-withdrawing groups,
compounds 7d and 8 reduced the inhibition. Considering
the ease of synthesis, we chose compound 5h to continue
the structure modification studies (7e–p). We found that
inserting two or three halogen groups at C ring (7e–7p)
123

S.-M. Yang et al.
couldn’t increase the antitumor activities against HepG2
cells perhaps because of the change from spatial structure
and election cloud density. Even some compounds (7e–
h and 7k) had no inhibitory activity against A549 cells at
up to 20 lM concentrations. Hence, further in vitro studies
were performed with compound 5h.
compound 5h played its own role in promoting apoptosis in
cancer cells.
Acknowledgments This work was supported by the National Nat-
ural Science Foundation of China (NSFC-81472230), and the Fun-
damental Research Funds for the Xiamen Universities (CXB-
2014011).
The ability of compound 5h to inhibit the growth of
various cell lines from hepatoma (HepG2, Hep3B, PLC/
PRF/5, and SMMC-7721), lung cancer (A549), colon
adenocarcinoma (HT-29), cervical cancer (HeLa), and
human skin melanoma (A375) cell lines was examined,
and the results were presented in Fig. 2a and b. As noted,
compound 5h potently inhibited the growth of liver cancer
cell lines, including HepG2, Hep3B, PLC/PRF/5, and
SMMC-7721 cells, with IC50 values of 2.57, 2.76, 4.26,
and 29.66 lM respectively. Compound 5h also inhibited
the growth of A549 cells, with an IC50 value of 5.48 lM.
Further, it exhibited weaker inhibitory activity against
References
Abou-Alfa, G.K., L. Schwartz, S. Ricci, D. Amadori, A. Santoro, A.
Figer, J. De Greve, J.Y. Douillard, C. Lathia, B. Schwartz, I.
Taylor, M. Moscovici, and L.B. Saltz. 2006. Phase II study of
sorafenib in patients with advanced hepatocellular carcinoma.
Journal of Clinical Oncology 24: 4293–4300.
´
Bedos-Belval, F., A. Rouch, C. Vanucci-Bacque, and M. Baltas.
2012. Diaryl ether derivatives as anticancer agents–a review.
MedChemComm 3: 1356–1372.
Gupta-Abramson, V., A.B. Troxel, A. Nellore, K. Puttaswamy, M.
Redlinger, K. Ransone, S.J. Mandel, K.T. Flaherty, L.A.
Loevner, P.J. O’dwyer, and M.S. Brose. 2008. Phase II trial of
sorafenib in advanced thyroid cancer. Journal of Clinical
Oncology 26: 4714–4719.
Huang, T.T., Y.C. Huang, X.Y. Qing, Y. Xia, X. Luo, T.H. Ye, and
L.T. Yu. 2012. Synthesis and biological evaluation of novel N-
methyl-picolinamide-4-thiol derivatives as potential antitumor
agents. Molecules 17: 6317–6330.
Jemal, A., F. Bray, M.M. Center, J. Ferlay, E. Ward, and D. Forman.
2011. Global cancer statistics. CA: A Cancer Journal for
Clinicians 61: 69–90.
Kennedy-Smith, J.J., N. Arora, J. Roland Billedeau, J. Fretland, J.Q.
Hang, G.M. Heilek, S.F. Harris, D. Hirschfeld, H. Javanbakht, Y.
Li, W. Liang, R. Roetz, M. Smith, G. Su, J.M. Suh, A.G.
Villasen˜or, J. Wu, D. Yasuda, K. Klumpp, and Z.K. Sweeney.
2010. Synthesis and biological activity of new pyridonediaryl
ether non-nucleoside inhibitors of HIV-1 reverse transcriptase.
MedChemComm 1: 79–83.
several other cell lines, including HT-29 (IC50
=
=
30.04 lM), HeLa (IC50 = 18.86 lM) and A375 (IC50
10.21 lM). In particular, the Compound 5h exhibited
higher potency than our template Sorafenib in HepG2 and
A549 (2.57 vs. 16.30, 5.48 vs. 13.15). These results
indicated that compound 5h was a relatively selective
inhibitor of human liver cancer cell lines. Both results of
the proliferation and colony forming assay confirmed that
compound 5h significantly inhibited the proliferation of
hepatoma cells (Fig. 2c, d).
Exposed to compound 5h, HepG2 cells increased the
expression of two important apoptotic markers, p21 and cl-
caspase, in a time- and dose-dependent manner (Fig. 3a–d).
We also took Sorafenib as a positive control. However, the
results showed that Sorafenib had no regulation on the
expression of p21 and cl-caspase which meant the more
potent of compound 5h than Sorafenib (Fig. S A, B). Flow
cytometry analysis of apoptosis confirmed that compound
5h could significantly promote HepG2 cell apoptosis
(Fig. 3e). Taken together, these data suggest that com-
pound 5h exerted its antitumor activity by promoting
apoptosis in cancer cells.
Ma, D., and Q. Cai. 2003. N,N-dimethyl glycine-promoted Ullmann
coupling reaction of phenols and aryl halides. Organic Letters 5:
3799–3802.
¨
Nicolaou, K.C., C.N. Boddy, S. Brase, and N. Winssinger. 1999.
Chemistry, biology, and medicine of the glycopeptide antibi-
otics. Angewandte Chemie International Edition 38: 2096–2152.
Patch, R.J., L.L. Searle, A.J. Kim, D. De, X. Zhu, H.B. Askari, J.C.
O’neill, M.C. Abad, D. Rentzeperis, J. Liu, M. Kemmerer, L.
Lin, J. Kasturi, J.G. Geisler, J.M. Lenhard, M.R. Player, and
M.D. Gaul. 2011. Identification of diaryl ether-based ligands for
estrogen-related receptor alpha as potential antidiabetic agents.
Journal of Medicinal Chemistry 54: 788–808.
Ratain, M.J., T. Eisen, W.M. Stadler, K.T. Flaherty, S.B. Kaye, G.L.
Rosner, M. Gore, A.A. Desai, A. Patnaik, H.Q. Xiong, E.
Rowinsky, J.L. Abbruzzese, C. Xia, R. Simantov, B. Schwartz,
and P.J. O’dwyer. 2006. Phase II placebo-controlled randomized
discontinuation trial of sorafenib in patients with metastatic renal
cell carcinoma. Journal of Clinical Oncology 24: 2505–2512.
Tang, K., C. Luo, Y. Li, C. Lu, W. Zhou, H. Huang, and X. Chen.
2014. The study of a novel sorafenib derivative HLC-080 as an
antitumor agent. PLoS ONE 9(7): e101889.
In conclusion, we designed and synthesized diaryl ether
derivatives of sorafenib to identify new antitumor agents,
then evaluated and characterized the antitumor effect
in vitro of these compounds through SAR and some bio-
logical experiments. The synthesis methods and purifica-
tion of these compounds were relatively simple, and their
yields were also pretty high. Under the treatment of com-
pound 5h, cell growth and cell cycle pathways were acti-
vated in cancer cells. Our results showed that compound 5h
(IC50 = 2.57 lM) inhibited HepG2 cell proliferation more
effectively than sorafenib (IC50 = 9.61 lM). Western
blotting results indicated that compound 5h could induce
the expression of p21 and cl-caspase3, which meant that
Tucker, T.J., S. Saggar, J.T. Sisko, R.M. Tynebor, T.M. Williams, P.J.
Felock, J.A. Flynn, M.T. Lai, Y. Liang, G. Mcgaughey, M. Liu,
M. Miller, G. Moyer, V. Munshi, R. Perlow-Poehnelt, S. Prasad,
R. Sanchez, M. Torrent, J.P. Vacca, B.L. Wan, and Y. Yan.
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Antitumor activity of diaryl ether derivatives
2008. The design and synthesis of diaryl ether second generation
HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs)
with enhanced potency versus key clinical mutations. Bioorganic
& Medicinal Chemistry Letters 18: 2959–2966.
evaluation of novel molecules with a diphenyl ether nucleus as
potential antitubercular agents. Bioorganic & Medicinal Chem-
istry Letters 22: 954–957.
Yang, Y., Z. Wang, J. Yang, T. Yang, W. Pi, W. Ang, Y. Lin, Y. Liu,
Z. Li, Y. Luo, and Y. Wei. 2011. Design, synthesis and
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