Design and synthesis of new 2-anilinoquinolines bearing N-methylpicolinamide moiety as potential antiproliferative agents

Article abstract of DOI:10.1111/cbdd.12836

A series of new 2-anilinoquinolines 6a–o possessing the substantial N-methylpicolinamide motif at C5 has been designed and synthesized as sorafenib analogs. The antiproliferative activities of the target compounds were preliminarily appraised against a panel of three human cancer cell lines (MCF-7, SK-BR3, and HCT116), and a selected array was further tested over a panel of approximately 60 cancer cell lines at NCI at 10?μM concentration. Interestingly, compounds 6c, 6d, 6j, 6k, and 6l showed promising selective anticancer activities (growth inhibition >80%) toward certain cancer cells at 10?μM testing dose. Compounds 6d and 6j were advanced to five-dose testing mode to determine their GI₅₀ values and compared with our previously reported ureidoquinoline B and sorafenib as reference compounds. The 4-chloro-3-trifluoromethylaniline derivative 6j manifested superior potency than both compound B and sorafenib over eleven and eight cell lines, respectively. It showed GI₅₀ values of 0.36, 0.66, 0.68, and 0.60?μM against the breast MDA-MB-468, renal A498, and melanoma SK-MEL-5 and UACC-62 cell lines, respectively. Moreover, both 6d and 6j exerted low cytotoxic effects against HFF-1 normal cell line. Furthermore, compounds 6d and 6j were tested against both B-Raf^V600E and C-Raf kinases and displayed modest inhibitory activities, which were justified by molecular docking study. Compound 6j could serve as a promising candidate for further development of potent anticancer chemotherapeutics.

Full text of DOI:10.1111/cbdd.12836

Received Date : 24-Mar-2016
Revised Date : 10-Jun-2016
Accepted Date : 12-Jul-2016
Article type : Research Article
Design and synthesis of new 2-anilinoquinolines bearing N-methylpicolinamide moiety as
potential antiproliferative agents
a,b,c
a
a
a,b
b,d
Ashraf Kareem El-Damasy , Seon Hee Seo , Nam-Chul Cho , Ae Nim Pae , Eunice Eunkyeong Kim
a,b,
Gyochang Keum
*
a
Center for Neuro-Medicine, Brain Science Institute, Korea Institute of Science and Technology (KIST),
Hwarangro 14-gil 5, Seongbuk-gu, Seoul 136-791, Republic of Korea
b
Department of Biological Chemistry, Korea University of Science and Technology (UST), Gajungro 217,
Youseong-gu, Daejeon 305-350, Republic of Korea
c
Department of Medicinal Chemistry, Faculty of Pharmacy, University of Mansoura, Mansoura 35516,
Egypt
d
Biomedical Research Institute, KIST, Republic of Korea
Corresponding author:
Gyochang Keum
Tel.: +82-2-958-5148; fax: +82-2-958-5189; e-mail: gkeum@kist.re.kr
Abstract
A series of new 2-anilinoquinolines 6a–o possessing the substantial N-methylpicolinamide motif at C5
has been designed and synthesized as sorafenib analogues. The antiproliferative activities of the target
compounds were preliminary appraised against a panel of 3 human cancer cell lines (MCF-7, SK-BR3
and HCT116), and a selected array was further tested over a panel of approximately 60 cancer cell lines at
NCI at 10 μM concentration. Interestingly, compounds 6c, 6d, 6j, 6k and 6l showed promising selective
anticancer activities (growth inhibition > 80%) towards certain cancer cells at 10 μM testing dose.
Compounds 6d and 6j were advanced to 5-dose testing mode to determine their GI50 values, and
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1
0.1111/cbdd.12836
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compared with our previously reported ureidoquinoline B and sorafenib as reference compounds. The 4-
chloro-3-trifluoromethylaniline derivative 6j manifested superior potency than both compound B and
sorafenib over 11 and 8 cell lines, respectively. It showed GI50 values of 0.36 μM, 0.66 μM, 0.68 μM and
0
.60 μM against the breast MDA-MB-468, renal A498 and melanoma SK-MEL-5 and UACC-62 cell
lines, respectively. Moreover, both 6d and 6j exerted low cytotoxic effects against HFF-1 normal cell
V600E
line. Furthermore, compounds 6d and 6j were tested against both B-Raf
and C-Raf kinases, and
displayed modest inhibitory activities, which were justified by molecular docking study. Compound 6j
could serve as a promising candidate for further development of potent anticancer chemotherapeutics.
Keywords
Antiproliferative activity; 2-Anilinoquinoline; N-methylpicolinamide; RAF kinase
Introduction
Cancer is still one of the main leading causes of mortalities all over the globe, and researches dedicated
for the understanding of various molecular mechanisms involved in cancer and the development of new
cancer treatments still constitute an urgent research issue. In the last few decades, great efforts have been
made to circumvent the major problem of low selectivity of conventional anticancer drugs and ameliorate
their potencies (1). Few years ago, a substantial shift in anticancer drug development has been noticed,
where much attention have been paid towards the identification of new small molecules capable of
targeting specific proteins, such as kinases whose activities are more specifically correlated with
cancerous cells. A plenty of targeted anticancer agents are currently being developed in order to achieve
better anticancer activity with fewer side effects (2).
Sorafenib (Nexavar®), a diaryl urea derivative, is the first reported RAF kinase inhibitor (3–5), that has
been approved by FDA for treatment of advanced renal cell carcinoma (RCC) (6) and hepatocellular
carcinoma (7). Besides RAF suppressing activity of sorafenib, it showed multikinase inhibitory effect
over a number of oncogenic kinases, such as the proangiogenic vascular endothelial growth factor
receptor-1 (VEGFR-1), VEGFR-2, VEGFR-3, and platelet-derived growth factor receptor-β (PDGFR- β)
(
8, 9), which account for its broad spectrum anticancer activity. Nevertheless, sorafenib still has certain
drawbacks, like its poor physicochemical properties (10), weak therapeutic efficacy towards malignant
melanoma (11), and accompanying toxicities (12, 13). Therefore, extensive structural modification have
been carried out on sorafenib for both exploration and optimization purposes (14–20).
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Among these efforts, Ramurthy et al. (15) conserved the picolinamide motif of sorafenib and changed its
central ureidophenyl moiety into different bicyclic structures, exemplied by the anilinoquinazoline
derivative A (Figure 1). Such modifications resulted in potent and selective RAF kinase inhibitors on the
biochemical level, but devoid of cellular potency. On the other hand, our group has recently disclosed a
series of N-methylpicolinamide based 2-ureidoquinolines (16), represented by compound B (Figure 1),
which displayed broad spectrum anticancer activity in the cell based assay, as well as favorable RAF
kinase inhibitory effects (potency and selectivity).
Accordingly, and with the purpose to further explore the SAR of those 2,5-disubstituted quinolines (16), a
new series of 2-anilinoquinolines featuring the N-methylpicolinamide motif 6a–o has been designed and
synthesized (Figure 1). Our main objective in this study is to explore the impact of replacing the longer
spacer, urea, in compound B with the shorter amine on the cellular anticancer activity. Moreover, various
anilines were installed on C2 of quinoline to interrogate their influence on the antineoplastic activity
(
Figure 1). From another perspective, our target compounds 6a–o could be considered as positional
isomers to compound A, where the oxypicolinamide moiety is changed from C6 to C5 of quinoline. The
anticancer activities of all final compounds were initially assessed over a panel of 3 human cancer cell
lines (MCF-7, SK-BR3 and HCT116), and a selected group of 10 compounds was further examined
against a panel of approximately 60 human cancer cell lines at NCI.
Materials and Methods
Chemistry
General
All solvents and reagents were commercially available and used without further purification. IR spectra
1
(
KBr disks) were recorded with a Bruker FT-IR instrument (Bruker Bioscience, Billerica, MA, USA). H
1
3
and C NMR spectra were recorded on a Bruker Avance 300 or 400 MHz spectrometer, using
appropriate deuterated solvents, as indicated. Chemical shifts (δ) are recorded in parts per million (ppm)
upfield from tetramethylsilane (TMS) as internal standard, and s, d, t, and m are designated as singlet,
doublet, triplet and multiplet, respectively. Coupling constants (J) are reported in hertz (Hz). Mass spectra
were recorded on Waters Acquity UPLC/Synapt G2 QTOF MS mass spectrometer. The reaction progress
was monitored on TLC plate (Merck, silica gel 60 F ), and flash column chromatography was performed
2
54
using silica gel (Merck, 230–400 mesh) and the indicated solvent system. Solvents and liquid reagents
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were transferred using hypodermic syringes. Compounds 1–3 were synthesized following the literature
method (16).
General procedure for synthesis of compounds 4a–o
A mixture of compound 3 (0.2 g, 1.03 mmol) and the appropriate aniline derivative (1.03–1.55 mmol)
were fused at 160 °C for 5–60 min. The reaction mixture was cooled, and either dissolved in DCM and
concentrated under reduced pressure to afford the desired product in pure form, or diluted with water and
extracted with ethyl acetate (3×10 mL). The combined organic layers were washed with brine, dried over
Na SO , filtered and concentrated under reduced pressure. The obtained residue was purified by silica gel
2
4
chromatography using the proper eluent.
N-(3-Fluorophenyl)-5-methoxyquinolin-2-amine (4a)
The compound was purified by flash column chromatography using (Hexane:ethyl acetate, 4:1 v/v).
1
Yield: 85%, H NMR (300 MHz, CDCl ) δ 11.17 (br. s, 1H), 8.59 (d, J = 9.6 Hz, 1H), 7.69 (t, J = 8.2 Hz,
3
1
3
1
H), 7.50–7.41 (m, 2H), 7.19–7.06 (m, 4H), 6.89 (d, J =8.1 Hz, 1H), 4.02 (s, 3H); C NMR (75 MHz,
CDCl ) δ 164.86, 161.56, 156.05, 152.77, 139.57, 137.17, 134.72, 131.49, 120.51, 114.99, 113.53,
12.07, 110.36, 108.07, 105.27, 56.20.
3
1
N-(3-Trifluoromethylphenyl)-5-methoxyquinolin-2-amine (4b)
The compound was purified by flash column chromatography using (Hexane:ethyl acetate, 4:1 v/v).
1
Yield: 73%, H NMR (400 MHz, CDCl ) δ 8.42 (d, J = 8.8 Hz, 1H), 8.09 (s, 1H), 7.84 (d, J = 8.0 Hz,
3
1
6
H), 7.55 (t, J = 8.0 Hz, 1H), 7.47 (t, J = 8.8 Hz, 2H), 7.31 (t, J = 7.6 Hz, 1H), 6.89 (d, J = 8.8 Hz, 1H),
.73 (d, J = 7.6 Hz, 1H), 4.01 (s, 3H).
N-(3-Chlorophenyl)-5-methoxyquinolin-2-amine (4c) (21)
1
Yield: 100%, H NMR (300 MHz, CDCl
3
) δ 8.54 (d, J = 9.5 Hz, 1H), 7.67 (t, J = 8.3 Hz, 1H), 7.47–7.37
13
(
m, 3H), 7.32–7.30 (m, 2H), 7.00 (d, J = 9.5 Hz, 1H), 6.84 (d, J = 8.1 Hz, 1H), 4.01 (s, 3H). C NMR (75
MHz, CDCl ) δ 155.91, 152.82, 138.63, 138.50, 137.09, 135.34, 133.99, 130.88, 127.00, 124.03, 122.12,
3
1
13.82, 111.52, 108.64, 104.84, 56.11.
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N-(3-(Tert-butyl)phenyl)-5-methoxyquinolin-2-amine (4d) (21)
The compound was purified by flash column chromatography using (0–3% MeOH in DCM). Yield: 91%,
1
3
H NMR (400 MHz, CDCl ) δ 8.38 (dd, J = 9.2, 0.8 Hz, 1H), 7.64 (t, J = 1.6 Hz, 1H), 7.52 (t, J = 8.4 Hz,
1
H), 7.38–7.32 (m, 3H), 7.18 (dt, J = 7.4, 1.2 Hz, 2H), 7.01 (d, J = 9.2 Hz, 1H), 6.69 (dd, J = 8.0, 0.8
1
3
Hz, 1H), 4.00 (s, 3H), 1.39 (s, 9H); C NMR (100 MHz, CDCl ): δ 155.50, 155.09, 152.46, 148.75,
3
1
3
39.92, 132.45, 129.79, 128.82, 120.32, 119.21, 118.25, 117.98, 115.86, 110.23, 101.81, 55.60, 34.80,
1.36.
N-(4-Isopropylphenyl)-5-methoxyquinolin-2-amine (4e) (21)
1
Yield: 100%, H NMR (300 MHz, CDCl
3
) δ 8.30 (d, J = 9.1 Hz, 1H), 7.49–7.40 (m, 3H), 7.34 (d, J =
1
1
1
3
1.3 Hz, 1H), 7.22 (d, J = 8.4 Hz, 2H), 6.93 (d, J = 9.1 Hz, 1H), 6.78 (br. s, 1H), 6.63 (d, J = 7.8 Hz,
13
H), 3.94 (s, 3H), 2.95–2.86 (m, 1H), 1.26 (d, J = 7.0 Hz, 6H). C NMR (75 MHz, CDCl ): δ 155.53,
3
55.19, 148.80, 144.13, 137.76, 132.46, 129.78, 127.19, 121.29, 119.10, 115.86, 109.93, 101.75, 55.60,
3.62, 24.13.
N-(4-Ethylphenyl)-5-methoxyquinolin-2-amine (4f)
The compound was purified by flash column chromatography using (hexane:ethyl acetate, 3:1 v/v). Yield:
1
9
6
1
1
5%, H NMR (300 MHz, CDCl
3
) δ 8.31 (d, J = 9.0 Hz, 1H), 7.50–7.33 (m, 4H), 7.19 (d, J = 8.1 Hz, 2H),
.93 (d, J = 9.0 Hz, 1H), 6.83 (br. s, 1H), 6.64 (d, J = 7.5 Hz, 1H), 3.95 (s, 3H), 2.65 (q, J = 7.5 Hz, 2H),
13
.25 (t, J = 6.0 Hz, 3H); C NMR (75 MHz, CDCl ) δ 155.51, 155.15, 148.79, 139.53, 137.67, 132.47,
3
29.80, 128.65, 121.33, 119.11, 115.84, 109.88, 101.74, 55.61, 28.35, 15.77.
5
-Methoxy-N-(4-phenoxyphenyl)quinolin-2-amine (4g)
The compound was purified by flash column chromatography using (ethyl acetate:methanol, 9:1 v/v).
1
Yield: 71%, H NMR (300 MHz, CDCl
3
) δ 8.25 (d, J = 9.0 Hz, 1H), 7.97 (br. s, 1H), 7.52–7.26 (m, 6H),
1
3
7
1
1
.07–6.82 (m, 6H), 6.59 (d, J = 7.3 Hz, 1H), 3.88 (s, 3H); C NMR (75 MHz, CDCl ); δ 157.78, 155.55,
3
54.78, 153.00, 147.14, 135.32, 133.22, 130.39, 129.79, 123.03, 122.96, 121.16, 119.96, 118.43, 118.01,
17.27, 115.43, 110.23, 102.26, 55.66.
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N-([1,1'-Biphenyl]-4-yl)-5-methoxyquinolin-2-amine (4h)
1
Yield: 100%, H NMR (300 MHz, CDCl ) δ 10.64 (br. s, 1H), 8.49 (d, J = 9.3 Hz, 1H), 7.67–7.57 (m,
3
13
4
H), 7.46–7.38 (m, 7H), 7.03 (d, J =7.8 Hz, 1H), 6.82 (d, J =7.8 Hz, 1H), 3.99 (s, 3H); C NMR (75
MHz, CDCl ): δ 156.01, 153.02, 147.39, 146.10, 140.65, 139.71, 138.86, 137.70, 134.36, 128.98, 128.59,
27.83, 127.01, 125.17, 115.38, 113.42, 110.74, 108.50, 104.97, 56.13.
3
1
5
-Methoxy-N-(4-morpholinophenyl)quinolin-2-amine (4i)
The compound was purified by flash column chromatography using (ethyl acetate:hexane, 0–35%). Yield:
1
6
6
0%, H NMR (300 MHz, CDCl ) δ 8.27 (d, J = 9.1 Hz, 1H), 7.48–7.30 (m, 4H), 6.92 (d, J = 8.6 Hz, 3H),
3
13
.84 (d, J = 9.1 Hz, 1H), 6.62 (d, J = 7.7 Hz, 1H), 3.94 (s, 3H), 3.86 (s, 4H), 3.12 (s, 4H); C NMR (75
) δ 155.77, 155.52, 148.85, 148.03, 132.71, 132.39, 129.77, 123.46, 118.99, 116.77, 115.68,
09.64, 101.58, 66.98, 55.59, 49.91.
MHz, CDCl
3
1
N-(4-Chloro-3-(trifluoromethyl)phenyl)-5-methoxyquinolin-2-amine (4j) (21)
1
Yield: 97%, H NMR (400 MHz, CDCl ) δ 11.31 (br. s, 1H), 8.58 (d, J=9.6 Hz, 1H), 7.66 (t, J = 10.0 Hz,
3
2
1
1
H), 7.58 (d, J=8.4 Hz, 1H), 7.51 (dd, J=8.4, 2.4 Hz, 1H), 7.39 (d, J=8.4 Hz, 1H), 6.97 (d, J = 9.6 Hz,
13
H), 6.85 (d, J=8.4 Hz, 1H), 3.98 (s, 3H); C NMR (100 MHz, CDCl
3
) δ 156.07, 152.56, 140.05, 137.23,
34.99, 134.23, 133.23, 131.05, 128.97, 123.89, 123.84, 120.67, 113.80, 110.41, 107.68, 105.45, 56.21.
N-(4-Chloro-3-fluorophenyl)-5-methoxyquinolin-2-amine (4k) (21)
1
Yield: 100%, H NMR (300 MHz, CDCl
3
) δ 8.37 (d, J = 9.0 Hz, 1H), 7.94 (dd, J = 11.4, 2.1 Hz, 1H),
7
.52 (t, J = 7.5 Hz, 1H), 7.43 (d, J = 8.1 Hz, 1H), 7.29–7.25 (m, 1H), 7.16 (d, J = 8.1 Hz, 1H), 6.83–6.68
13
(
m, 3H), 3.97 (s, 3H); C NMR (75 MHz, CDCl ) δ 155.42, 153.43, 148.25, 140.55, 132.78, 130.37,
3
1
30.07, 119.58, 116.13, 115.36, 115.32, 111.06, 107.83, 107.48, 102.51, 55.66.
N-(3-Chloro-4-fluorophenyl)-5-methoxyquinolin-2-amine (4l)
1
Yield: 100%, H NMR (300 MHz, CDCl
3
) δ 8.36 (d, J = 9.0 Hz, 1H), 7.87 (dd, J = 6.3, 2.1 Hz, 1H), 7.51
(
t, J = 8.1 Hz, 1H), 7.44–7.37 (m, 2H), 7.11 (t, J = 8.7 Hz, 1H), 6.82 (d, J = 9.0 Hz, 1H), 6.70–6.50 (m,
1
3
2
1
H), 3.97 (s, 3H); C NMR (75 MHz, CDCl ) δ 155.47, 153.97, 148.29, 137.07, 132.84, 130.05, 122.05,
3
19.74, 119.65, 119.35, 116.83, 116.54, 116.05, 110.49, 102.32, 55.65.
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N-(4-Bromo-3-fluorophenyl)-5-methoxyquinolin-2-amine (4m)
The compound was purified by flash column chromatography using (ethyl acetate:hexane, 0–35%). Yield:
1
4
7%, H NMR (400 MHz, CDCl
3
) δ 8.37 (d, J = 8.7 Hz, 1H), 7.92 (dd, J = 11.1, 2.4 Hz, 1H), 7.55–7.40
13
(
m, 3H), 7.13 (dd, J = 8.4, 1.4 Hz, 1H), 6.83–6.68 (m, 3H), 3.97 (s, 3H); C NMR (100 MHz, CDCl
3
) δ
55.42, 153.39, 148.23, 141.47, 133.16, 132.79, 130.08, 119.57, 116.14, 115.78, 111.11, 107.68, 107.31,
02.54, 100.30, 55.66.
1
1
N-(3,4-Difluorophenyl)-5-methoxyquinolin-2-amine (4n) (21)
1
Yield: 98%, H NMR (400 MHz, CDCl ) δ 8.40 (d, J = 8.8 Hz, 1H), 7.85 (dq, J = 7.7, 2.4 Hz, 1H), 7.55
3
(
t, J = 8.4 Hz, 1H), 7.44 (d, J = 8.4 Hz, 1H), 7.18–7.13 (m, 2H), 6.85 (d, J = 9.2 Hz, 1H), 6.73 (d, J = 7.6
13
Hz, 1H), 4.01 (s, 3H); C NMR (100 MHz, CDCl
3
) δ 155.46, 153.81, 151.59, 147.58, 133.15, 130.30,
1
18.83, 117.38, 117.20, 115.84, 115.69, 110.50, 109.74, 109.53, 102.48, 55.66.
N-(3,4-Dimethylphenyl)-5-methoxyquinolin-2-amine (4o)
1
Yield: 100%, H NMR (300 MHz, CDCl ) δ 10.81 (br. s, 1H), 8.46 (d, J = 9.6 Hz, 1H), 7.63 (t, J = 8.3
3
Hz, 1H), 7.35 (d, J = 8.3 Hz, 1H), 7.20 (d, J = 7.8 Hz, 1H), 7.08–7.05 (m, 2H), 6.99 (d, J = 9.6 Hz, 1H),
1
3
6
1
1
3
.86 (d, J = 8.1 Hz, 1H), 3.99 (s, 3H), 2.26 (s, 6H); C NMR (75 MHz, CDCl ) δ 155.84, 152.85, 138.68,
38.52, 136.95, 136.46, 134.18, 132.26, 130.81, 125.83, 122.00, 112.90, 109.80, 108.41, 104.96, 56.12,
9.66, 19.27.
General procedure for synthesis of compounds 5a–o
To a stirred solution of the appropriate methoxyquinoline 4 (1.55 mmol) in anhydrous DCM (18 mL) at
0
° C, 1M solution of BBr in DCM (5.5 mL, 4.65 mmol) was added dropwise. After complete addition,
3
the reaction mixture was gradually warmed to rt and stirred for 9 h. Water was added and the resulting
solution was neutralized with saturated solution of NaHCO . The aqueous layer was extracted with ethyl
acetate (3×50 mL), the organic layers were combined, washed with brine, dried over Na SO , filtered and
3
2
4
concentrated under reduced pressure to afford the desired product, unless stated otherwise, in pure form.
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N-(3-Fluorophenyl)-5-hydroxyquinolin-2-amine (5a)
1
Yield: 69%, H NMR (400 MHz, Acetone-d ) δ 9.10 (br. s, 1H), 8.38 (d, J = 9.0 Hz, 1H), 8.32 (dt, J =
6
1
2.4, 2.2 Hz, 1H), 7.55 (dd, J = 8.8, 1.2 Hz, 1H), 7.43 (t, J = 8.2 Hz, 1H), 7.33 (t, J = 8.0 Hz, 2H), 7.04
(
d, J = 9.1 Hz, 1H), 6.81 (dd, J = 7.6, 0.7 Hz, 1H), 6.74 (dt, J = 8.3, 2.0 Hz, 1H).
N-(3-Trifluoromethylphenyl)-5-hydroxyquinolin-2-amine (5b)
The compound was purified by flash column chromatography (hexane:ethyl acetate, 3:1 then
1
hexane:ethyl acetate, 1:2 v/v). Yield: 100%, H NMR (300 MHz, Acetone-d ) δ 9.01 (br. s, 2H), 8.75 (s,
6
1
7
1
1
H), 8.38 (d, J = 9.0 Hz, 1H), 8.15 (d, J = 7.8 Hz, 1H), 7.54 (t, J = 7.8 Hz, 1H), 7.43 (t, J = 8.1 Hz, 1H),
13
3
.33–7.27 (m, 2H), 7.03 (d, J = 9.0 Hz, 1H), 6.78 (dd, J = 7.5, 0.6 Hz, 1H); C NMR (75 MHz, CDCl ) δ
54.18, 153.24, 148.47, 142.52, 132.04, 129.81, 129.36, 121.62, 118.30, 117.06, 115.06, 114.70, 112.11,
06.48.
N-(3-Chlorophenyl)-5-hydroxyquinolin-2-amine (5c) (21)
1
Yield; 93.6%; H NMR (300 MHz, Acetone-d ) δ 8.90 (br. s, 2H), 8.47 (t, J = 2.0 Hz, 1H), 8.38 (d, J =
6
9
1
1
.0 Hz, 1H), 7.80 (dt, J = 8.2, 1.2 Hz, 1H), 7.46–7.28 (m, 3H), 7.02–6.97 (m, 2H), 6.79 (d, J = 7.5 Hz,
13
H); C NMR (75 MHz, CDCl
3
): δ 154.21, 153.24, 148.57, 143.22, 133.78, 131.96, 129.85, 129.78,
20.64, 118.33, 118.12, 116.74, 115.04, 112.14, 106.45.
N-(3-(Tert-butyl)phenyl)-5-hydoxyquinolin-2-amine (5d) (21)
The compound was purified by flash column chromatography using (0–2% methanol in DCM). Yield:
1
4
8%; H NMR (400 MHz, Acetone-d
6
) δ 8.55 (br. s, 1H), 8.33 (dd, J = 9.0, 0.7 Hz, 1H), 8.21 (t, J = 2.0
Hz, 1H), 7.77 (dq, J = 8.0, 1.0 Hz, 1H), 7.39 (t, J = 7.7 Hz, 1H), 7.30–7.24 (m, 2H), 7.07 (dq, J = 7.8, 1.0
13
Hz, 1H), 7.01 (d, J = 9.0 Hz, 1H), 6.75 (dd, J = 7.6, 1.0 Hz, 1H), 1.40 (s, 9H ); C NMR (100 MHz,
Acetone-d ): δ 154.76, 153.19, 151.44, 148.99, 141.47, 131.46, 129.40, 128.08, 118.33, 118.25, 116.35,
6
1
16.12, 114.85, 111.89, 106.01, 34.41, 30.83.
N-(4-Isopropylphenyl)-5-hydroxyquinolin-2-amine (5e) (21)
1
Yield: 81%, H NMR (300 MHz, Acetone-d ) δ 8.43 (br. s, 1H), 8.15 (d, J = 9.1 Hz, 1H), 7.74 (d, J = 7.9
6
Hz, 2H), 7.20 (t, J = 7.5 Hz, 1H), 7.12 (d, J = 8.4 Hz, 1H), 7.04 (d, J = 7.9 Hz, 2H), 6.82 (d, J = 9.1 Hz,
13
1
H), 6.56 (d, J = 7.5 Hz, 1H), 2.76–2.67 (m, 1H), 1.07 (d, J = 6.9 Hz, 6H); C NMR (75 MHz, Acetone-
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d6): δ 154.78, 153.26, 149.02, 141.74, 139.45, 131.56, 129.51, 126.35, 119.12, 118.16, 114.77, 111.86,
1
05.92, 33.37, 23.66.
2
-((4-Ethylphenyl)amino)quinolin-5-ol (5f)
The compound was purified by flash column chromatography (hexane-ethyl acetate, 3:1 v/v). Yield: 77%,
1
H NMR (300 MHz, Acetone-d ) δ 8.76 (br. s, 2H), 8.38 (d, J = 9.0 Hz, 1H), 7.93 (d, J = 8.5 Hz, 2H),
6
7
.43–7.32 (m, 2H), 7.17 (d, J = 8.4 Hz, 2H), 7.01 (d, J = 9.1 Hz, 2H), 6.76 (dd, J = 7.3, 1.1 Hz, 1H), 2.62
13
(
q, J = 7.7 Hz, 2H), 1.24 (t, J = 7.6 Hz, 3H); C NMR (75 MHz, Acetone-d
6
) δ 154.86, 153.35, 149.05,
1
39.33, 137.25, 131.72, 129.62, 127.93, 119.33, 118.15, 114.87, 111.84, 106.03, 28.04, 15.53.
2
-((4-Phenoxyphenyl)amino)quinolin-5-ol (5g)
The compound was purified by flash column chromatography (hexane-ethyl acetate, 3:1 v/v). Yield: 38%,
1
6
H NMR (300 MHz, Acetone-d ) δ 8.80 (br. s, 2H), 8.35 (d, J = 9.0 Hz, 1H), 8.08 (d, J = 9.0 Hz, 2H),
1
3
7
1
1
.41–7.29 (m, 4H), 7.10–6.98 (m, 6H), 6.75 (d, J = 7.5 Hz, 1H); C NMR (75 MHz, Acetone-d ) δ
6
58.51, 154.61, 153.25, 150.87, 148.88, 137.84, 131.69, 129.72, 129.59, 122.50, 120.34, 119.72, 118.21,
17.70, 114.84, 111.92, 106.06.
N-([1,1'-Biphenyl]-4-yl)-5-hydroxyquinolin-2-amine (5h)
The compound was purified by flash column chromatography (hexane-ethyl acetate, 1:2 v/v). Yield:
1
9
8
0.5%, H NMR (400 MHz, Acetone-d ) δ 8.88 (br. s, 2H), 8.36 (dd, J = 8.8, 0.4 Hz, 1H), 8.18 (dt, J =
6
.8, 2.4 Hz, 2H), 7.70–7.65 (m, 4H), 7.48–7.40 (m, 3H), 7.36–7.30 (m, 2H), 7.04 (d, J = 8.8 Hz, 1H), 6.77
13
(
dd, J = 7.2, 0.8 Hz, 1H); C NMR (100 MHz, CDCl
3
) δ 154.50, 153.23, 148.88, 141.29, 140.89, 133.60,
1
31.67, 129.60, 128.76, 126.94, 126.50, 126.26, 118.97, 118.31, 114.87, 112.15, 106.12.
5
-Hydroxy-N-(4-morpholinophenyl)quinolin-2-amine (5i)
The compound was purified by flash column chromatography (hexane-ethyl acetate, 1:2 v/v). Yield:
1
6
2
1
7.3%, H NMR (300 MHz, Acetone-d ) δ 8.90 (br. s, 1H), 8.27 (d, J = 9.1 Hz, 1H), 7.89 (d, J = 9.0 Hz,
6
H), 7.35 (t, J = 8.3 Hz, 1H), 7.23 (d, J = 8.3 Hz, 1H), 6.95 (t, J = 9.0 Hz, 3H), 6.70 (dd, J = 7.6, 0.8 Hz,
H), 3.79 (t, J = 4.7 Hz, 4H), 3.09 (t, J = 4.8 Hz, 4H).
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N-(4-Chloro-3-(trifluoromethyl)phenyl)-5- hydroxyquinolin -2-amine (5j) (21)
1
Yield: 99%, H NMR (300 MHz, Acetone-d ) δ 9.10 (br. s, 2H), 8.84 (s, 1H), 8.41(d, J = 8.9 Hz, 1H),
6
13
8
.21 (d, J = 7.8 Hz, 1H), 7.54–7.32 (m, 3H), 7.03 (d, J = 9.0 Hz, 1H), 6.82 (d, J = 7.4 Hz, 1H); C NMR
(
75 MHz, Acetone-d ): δ 153.89, 153.30, 148.17, 141.06, 132.32, 131.57, 129.99, 125.16, 122.67, 121.91,
6
1
18.21, 117.28, 117.21, 115.19, 112.09, 106.76.
2
-((4-Chloro-3-fluorophenyl)amino)quinolin-5-ol (5k) (21)
The compound was purified by flash column chromatography using (hexane:ethyl acetate, 3:1 v/v). Yield:
1
5
8
7
1
4%, H NMR (400 MHz, Acetone-d ) δ 9.02 (br. s, 2H), 8.57 (dd, J = 12.8, 2.4 Hz, 1H), 8.39 (dd, J =
6
.8, 0.4 Hz, 1H), 7.58 (dq, J = 8.8, 0.8 Hz, 1H), 7.47–7.36 (m, 3H), 7.01 (d, J = 9.2 Hz, 1H), 6.81 (dd, J =
13
.2, 1.2 Hz, 1H); C NMR (100 MHz, Acetone-d ) δ 153.98, 153.24, 148.42, 142.42, 142.31, 132.09,
6
29.96, 129.86, 118.35, 115.10, 115.08, 115.05, 112.13, 106.60, 106.32.
2
-((3-Chloro-4-fluorophenyl)amino)quinolin-5-ol (5l)
The compound was purified by flash column chromatography (hexane:ethyl acetate, 3:1 v/v). Yield: 40%,
1
H NMR (400 MHz, Acetone-d ) δ 8.92 (br. s, 2H), 8.57 (dd, J = 6.8, 2.4 Hz, 1H), 8.38 (dd, J = 8.8, 0.4
6
Hz, 1H), 7.82 (dq, J = 4.0, 2.4 Hz, 1H), 7.43 (t, J = 7.6 Hz, 1H), 7.34 (dt, J = 8.0, 0.8 Hz, 1H), 7.24 (t, J =
1
3
8
1
1
.8 Hz, 1H), 6.98 (d, J = 8.8 Hz, 1H), 6.79 (dd, J = 7.6, 0.8 Hz, 1H); C NMR (100 MHz, Acetone-d ) δ
6
54.12, 153.26, 148.48, 138.95, 132.01, 129.80, 119.92, 118.44, 118.38, 118.24, 116.36, 116.14, 115.04,
11.99, 106.44.
N-(4-Bromo-3-fluorophenyl)-5-hydoxyquinolin-2-amine (5m)
1
Yield: 100%, H NMR (300 MHz, Acetone-d ) δ 9.05 (br. s, 1H), 8.55 (d, J = 11.2 Hz, 1H), 8.37 (d, J =
6
9
.2 Hz, 1H), 7.54–7.52 (m, 3H), 7.43–7.33 (m, 2H), 7.02 (d, J = 9.0 Hz, 1H), 6.80 (d, J = 7.9 Hz, 1H).
-((3,4-Difluorophenyl)amino)quinolin-5-ol (5n) (21)
2
The compound was purified by flash column chromatography (hexane:ethyl acetate, 3:1 v/v). Yield: 40%,
1
H NMR (400 MHz, Acetone-d ) δ 8.95 (br. s, 2H), 8.54 (dq, J = 10.0, 2.4 Hz, 1H), 8.37 (dd, J = 8.8, 0.4
6
Hz, 1H), 7.55-7.51 (m, 1H), 7.43 (t, J = 8.0 Hz, 1H), 7.35 (d, J = 8.0 Hz, 1H), 7.25 (q, J = 8.8 Hz, 1H),
1
3
6
1
1
.98 (d, J = 8.8 Hz, 1H), 6.79 (dd, J = 7.2, 0.8 Hz, 1H); C NMR (100 MHz, Acetone-d ) δ 154.14,
6
53.25, 148.49, 131.97, 129.79, 118.26, 116.86, 116.67, 115.02, 114.26, 114.24, 112.00, 107.52, 107.29,
06.43.
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2
-((3,4-Dimethylphenyl)amino)quinolin-5-ol (5o)
The compound was purified by flash column chromatography (hexane:ethyl acetate, 3:1 v/v). Yield: 55%,
1
6
H NMR (300 MHz, Acetone-d ) δ 8.61 (br. s, 2H), 8.35 (d, J = 9.1 Hz, 1H), 7.77 (dd, J = 8.0, 1.9 Hz,
1
H), 7.68 (d, J = 1.4 Hz, 1H), 7.41–7.29 (m, 2H), 7.09 (d, J = 8.1 Hz, 1H), 7.02 (d, J = 9.1 Hz, 1H), 6.75
13
(
dd, J = 7.4, 0.9 Hz, 1H), 2.26 (s, 3H), 2.21 (s, 3H ); C NMR (75 MHz, Acetone-d ) δ 154.89, 153.36,
6
1
1
48.71, 139.17, 136.42, 131.84, 129.70, 129.59, 120.84, 117.80, 117.11, 114.72, 111.74, 106.06, 19.32,
8.32.
General procedure for synthesis of compounds 6a–n
2 3
A suspension of the appropriate 5-hydroxyl anilinoquinoline 5 (0.185 mmol) and anhydrous K CO (0.56
mmol) in DMF (2 mL) was stirred at r.t. for 30 minutes. Then, a solution of 4-(3-
chloropropyl)morpholine (0.030 g, 0.185 mmol) or 4-(3-chloropropyl)methyl piperazine (0.063 g, 0.36
mmol) in DMF (1 mL) was added, followed by NaI (0.67 mmol). The reaction mixture was heated at 95
°C for 4–24 h, then cooled down to r.t. Water was added, and the aqueous layer was extracted with ethyl
acetate (3×30 mL). The combined organic layers were washed with brine, dried over Na SO , filtered,
2
4
concentrated under reduced pressure and the resultant residue was purified by flash column
chromatography using the appropriate eluent.
4
-((2-((3-Fluorophenyl)amino)quinolin-5-yl)oxy)-N-methylpicolinamide (6a)
The compound was purified by flash column chromatography using (0–35% ethyl acetate in hexane) and
then crystallized from aq. MeOH to afford the entitled compound as pure yellowish white solid (11%
−
1
1
yield); mp 265–266 °C, IR (KBr) ν/cm : 3393, 3327 (2NH), 1668 (C=O), H NMR (400 MHz, DMSO-
) δ 9.83 (s, 1H), 8.80 (q, J = 4.8 Hz, 1H), 8.55 (d, J = 5.6 Hz, 1H), 8.22 (dd, J = 4.0, 12.5 Hz, 1H), 8.03
d, J = 9.1 Hz, 1H), 7.75–7.69 (m, 2H), 7.56 (d, J = 7.8 Hz, 1H), 7.41–7.34 (m, 2H), 7.22–7.18 (m, 2H),
d
6
(
13
7
.10 (d, J = 9.0 Hz, 1H), 6.81 (dt, J = 8.5, 2.5 Hz, 1H), 2.79 (d, J = 4.7 Hz, 3H); C NMR (100 MHz,
DMSO-d ) δ 166.41, 164.18, 154.88, 153.08, 151.07, 149.20, 148.83, 143.35, 143.24, 131.23, 130.60,
6
1
30.40, 125.07, 117.31, 115.55, 114.95, 114.81, 114.42, 109.36, 105.72, 105.46, 26.47, HRMS (ESI-TOF)
+
m/z calcd for C H FN O [M-H] : 387.1258, found: 387.1255.
2
2
16
4
2
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N-Methyl-4-((2-((3-(trifluoromethyl)phenyl)amino)quinolin-5- yl)oxy picolin- amide (6b)
The compound was purified by flash column chromatography using (0–35% EA in Hex) to afford the
1
entitled compound as pure yellowish white solid (20.2% yield); mp 235–236 °C, H NMR (400 MHz,
CDCl
.4 Hz, 1H), 7.76 (d, J = 8.5 Hz, 1H), 7.60 (t, J = 7.8 Hz, 1H), 7.50 (t, J = 7.9 Hz, 1H), 7.35 (d, J = 7.7
Hz, 1H), 7.12 (br. s, 1H), 7.03 (dd, J = 7.6, 0.8 Hz, 1H), 7.00 (q, J = 2.6 Hz, 1H), 6.87 (d, J = 9.0 Hz, 1H),
3
) δ 8.43 (d, J = 5.6 Hz, 1H), 8.16 (s, 1H), 8.06–8.01 (m, 2H), 7.92 (d, J = 8.1 Hz, 1H), 7.79 (d, J =
2
1
3
3
1
.05 (d, J = 5.1 Hz, 3H). C NMR (100 MHz, CDCl ) δ 166.56, 164.48, 153.88, 152.50, 149.90, 148.91,
3
31.79, 129.82, 129.54, 125.09, 122.58, 119.24, 117.75, 116.35, 114.39, 113.85, 112.90, 110.33, 26.15.
4
-((2-((3-Chlorophenyl)amino)quinolin-5-yl)oxy)-N-methylpicolinamide (6c)
The compound was purified by flash column chromatography using (0–1% MeOH in DCM) and then
crystallized from aq. MeOH to afford the entitled compound as pure yellow solid (27.5% yield); mp 246–
−
1
1
2
8
47 °C, IR (KBr) ν/cm : 3360, 3323 (2NH), 1667 (C=O), H NMR (400 MHz, DMSO-d
6
) δ 9.80 (s, 1H),
.80 (q, J = 4.4 Hz, 1H), 8.55 (d, J = 5.6 Hz, 1H), 8.33 (t, J = 2.0 Hz, 1H), 8.03 (d, J = 8.8 Hz, 1H), 7.81
(
dq, J = 8.0, 0.4 Hz, 1H), 7.72 (m, 2H), 7.42 (d, J = 2.4 Hz, 1H), 7.37 (t, J = 8.0 Hz, 1H), 7.22–7.18 (m,
13
2
H), 7.10 (d, J = 9.2 Hz, 1H), 7.04 (ddd, J = 7.6, 0.8 Hz, 1H), 2.79 (d, J = 4.8 Hz, 3H), C NMR (100
) δ 166.40, 164.17, 154.83, 153.09, 151.07, 149.22, 148.80, 142.99, 133.53, 131.28,
30.73, 130.43, 125.01, 121.34, 118.28, 117.40, 117.34, 115.55, 114.97, 114.42, 109.37, 26.47, HRMS
MHz, DMSO-d
6
1
+
(
ESI-TOF) m/z calcd for C H ClN O Na [M+Na] : 427.0938, found: 427.0937.
22 17 4 2
4
-((2-((3-(Tert-butyl)phenyl)amino)quinolin-5-yl)oxy)-N-methylpicolinamide (6d)
The compound was purified by flash column chromatography using (0–50% EA in Hex) to afford the
entitled compound as pure yellow solid (33% yield); mp 92–95 °C, IR (KBr) ν/cm−1: 3380, 3323 (2NH),
1
1
668 (C=O), H NMR (400 MHz, CDCl3) δ 8.41 (d, J = 5.5 Hz, 1H), 8.06 (q, J = 4.9 Hz, 1H), 7.99 (dd, J
=
=
9.2, 0.5 Hz, 1H ), 7.81 (d, J = 2.4 Hz, 1H ), 7.71 (d, J = 8.5 Hz, 1H ), 7.64 (t, J = 1.8 Hz, 1H), 7.59 (t, J
7.7 Hz, 1H ), 7.39 (dq, J = 8.0, 1.2 Hz, 1H ), 7.33 (t, J = 7.7 Hz, 1H ), 7.20 (dt, J = 7.7, 1.6 Hz, 1H ),
1
3
7
1
1
:
3
.00–6.96 (m, 3H), 3.04 (d, J = 5.1 Hz, 3H), 1.37 (s, 9H); C NMR (100 MHz, CDCl ) δ 166.52, 164.48,
55.07, 152.60, 149.89, 149.41, 139.09, 131.92, 129.96, 128.91, 123.96, 121.03, 118.66, 118.46, 117.33,
+
13.87, 113.80, 112.17, 110.48, 34.81, 31.33, 26.16; HRMS (ESI-TOF) m/z calcd for C H N O [M-H]
26
25
4
2
425.1978, found: 425.1977.
4
-((2-((4-Isopropylphenyl)amino)quinolin-5-yl)oxy)-N-methylpicolinamide (6e)
The compound was purified by flash column chromatography using (0–50% EA in hexane) to afford the
−
1
entitled compound as pure yellow solid (36% yield); mp 165–166 °C, IR (KBr) ν/cm : 3392, 3332
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(
2NH), 1672 (C=O), 1H NMR (400 MHz, CDCl ) δ 8.40 (d, J = 5.6 Hz, 1H), 8.06 (q, J = 5.0 Hz, 1H),
3
7
7
3
1
1
.94 (dd, J = 9.1, 0.6 Hz, 1H), 7.81 (d, J = 2.4 Hz, 1H), 7.69 (d, J = 8.5 Hz, 2H), 7.57 (t, J = 7.7 Hz, 1H),
.50 (d, J = 8.5 Hz, 2H), 7.25 (d, J = 8.4 Hz, 2H), 7.14 (br. s, 1H), 6.98–6.93 (m, 3H), 3.03 (d, J = 5.1 Hz,
13
3
H), 2.97–2.90 (m, 1H), 1.29 (d, J = 6.9 Hz, 6H); C NMR (100 MHz, CDCl ) δ 166.62, 164.53, 155.31,
52.46, 149.86, 149.44, 149.33, 144.45, 137.32, 131.45, 129.61, 127.18, 124.57, 121.38, 117.47, 113.73,
13.67, 112.10, 110.47, 33.61, 26.16, 24.09.
4
-((2-((4-Ethylphenyl)amino)quinolin-5-yl)oxy)-N-methylpicolinamide (6f)
The compound was purified by flash column chromatography (silica gel, hexane-ethyl acetate, 2:1 then
−
1
1
3
:2 v/v) to afford the entitled compound as yellow solid (56% yield); mp 158–159 °C, IR (KBr) ν/cm :
1
394, 3336 (2NH), 1674 (C=O), H NMR (400 MHz, CDCl ) δ 8.38 (d, J = 5.6 Hz, 1H), 8.09 (q, J = 5.0
3
Hz, 1H), 7.92 (d, J = 9.1 Hz, 1H), 7.82 (d, J = 2.5 Hz, 1H ), 7.68 (d, J = 8.5 Hz, 1H), 7.56–7.49 (m, 3H),
7
3
1
1
.33 (br. s, 1H), 7.21 (d, J = 8.4 Hz, 2H), 6.96–6.91 (m, 3H), 3.03 (d, J = 9.2 Hz, 1H), 2.80 (d, J = 5.1 Hz,
13
H), 2.66 (q, J = 7.6 Hz, 2H), 1.27 (t, J = 7.6 Hz, 3H), C NMR (100 MHz, CDCl ) δ 166.62, 164.56,
3
55.37, 152.46, 149.87, 149.46, 149.31, 139.68, 137.37, 131.36, 129.56, 128.58, 124.58, 121.36, 117.43,
13.72, 113.61, 112.24, 110.47, 28.33, 26.18, 15.72.
N-Methyl-4-((2-((4-phenoxyphenyl)amino)quinolin-5-yl)oxy)picolinamide (6g)
The compound was purified by flash column chromatography (silica gel, hexane:ethyl acetate, 2:1 then
−
1
1
3
:3 v/v) to afford the entitled compound as yellow solid (44% yield); mp 190–191 °C, IR (KBr) ν/cm :
1
383, 3334 (2NH), 1671 (C=O), H NMR (400 MHz, CDCl ) δ 8.40 (d, J = 5.5 Hz, 1H), 8.06 (q, J = 5.0
3
Hz, 1H), 7.96 (dd, J = 9.0, 0.5 Hz, 1H), 7.81 (d, J = 2.4 Hz, 1H), 7.70 (d, J = 8.5 Hz, 1H), 7.63–7.55 (m,
3
1
1
1
H), 7.39–7.34 (m, 2H), 7.14-7.10 (m, 2H), 7.08–7.04 (m, 4H), 6.99–6.96 (m, 2H), 6.88 (d, J = 9.0 Hz,
13
H), 3.03 (d, J = 5.1 Hz, 3H), C NMR (100 MHz, CDCl ) δ 166.61, 164.53, 157.74, 155.11, 152.98,
3
52.47, 149.87, 149.34, 149.32, 135.38, 131.51, 129.73, 129.67, 124.65, 122.99, 122.96, 119.96, 118.39,
17.49, 113.80, 113.78, 112.27, 110.41, 26.17.
4
-((2-([1,1'-Biphenyl]-4-ylamino)quinolin-5-yl)oxy)-N-methylpicolinamide (6h)
The compound was purified by flash column chromatography using (0–2% MeOH in DCM) and then
crystallized from aq. MeOH to afford the entitled compound as pure light yellow solid (41% yield); mp
−
1
1
2
1
1
6
24–226 °C, IR (KBr) ν/cm : 3397, 3330 (2NH), 1670 (C=O), H NMR (400 MHz, DMSO-d ) δ 9.75 (s,
H), 8.82 (q, J = 4.6 Hz, 1H), 8.56 (d, J = 5.6 Hz, 1H), 8.12 (d, J = 8.7 Hz, 2H), 8.00 (d, J = 9.1 Hz,
H), 7.71–7.69 (m, 6H), 7.47 (t, J = 7.5 Hz, 2H), 7.42 (d, J = 2.5 Hz, 1H), 7.33 (t, J = 7.3 Hz, 1H), 7.22
(
dd, J = 5.6, 2.6 Hz, 1H), 7.17 (dd, J = 6.9, 1.8 Hz, 1H), 7.12 (d, J = 9.2 Hz, 1H), 2.79 (d, J = 4.8 Hz,
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1
3
3
1
1
H), C NMR (100 MHz, DMSO-d ) δ 166.45, 164.17, 155.05, 153.07, 151.07, 149.18, 149.13, 141.03,
6
40.46, 133.48, 130.97, 130.30, 129.36, 127.34, 127.16, 126.53, 124.97, 119.42, 117.19, 115.64, 114.66,
14.42, 109.31, 26.48.
N-Methyl-4-((2-((4-morpholinophenyl)amino)quinolin-5-yl)oxy)picolinamide (6i)
The compound was purified by flash column chromatography using (0–2.5% MeOH in DCM) to afford
−
1
the entitled compound as pure yellow solid (65% yield); mp 112–115 °C, IR (KBr) ν/cm : 3318 (NH),
1
1
668 (C=O), H NMR (300 MHz, CDCl
3
) δ 8.37 (d, J = 5.4 Hz, 1H), 8.02 (d, J = 4.5 Hz, 1H), 7.94 (dd, J
=
=
9.1, 0.6 Hz, 1H), 7.81 (d, J = 2.4 Hz, 1H), 7.69 (d, J = 8.5 Hz, 2H), 7.90 (d, J = 9.0 Hz, 1H), 7.76 (d, J
2.1 Hz, 1H), 7.64 (d, J = 8.4 Hz, 1H), 7.54 (t, J = 7.5 Hz, 1H), 7.43–7.40 (m, 3H), 6.94–6.92 (m, 4H),
13
6
.84 (d, J = 9.0 Hz, 1H), 3.87 (t, J = 4.5 Hz, 4H), 3.14 (t, J = 4.2 Hz, 4H), 3.00 (d, J = 4.8 Hz, 3H); C
NMR (75 MHz, CDCl ) δ 166.64, 164.53, 155.88, 152.45, 149.86, 149.49, 149.37, 148.32, 132.36,
32.09, 131.51, 129.68, 124.38, 123.66, 123.53, 117.39, 116.70, 113.56, 111.56, 110.43, 66.94, 49.81,
3
1
2
6.18.
4
-((2-((4-Chloro-3-(trifluoromethyl)phenyl)amino)quinolin-5-yl)oxy)-N-methylpicolinamide (6j)
The compound was purified by flash column chromatography using (0–30% EA in Hex) to afford the
−
1
entitled compound as pure yellow solid (43.2% yield); mp 220–223 °C, IR (KBr) ν/cm : 3392, 3316
1
(
2NH), 1665 (C=O), H NMR(400 MHz, DMSO-d ) δ 10.09 (s, 1H), 8.80 (q, J = 4.9 Hz, 1H), 8.76 (d, J =
6
2
.6 Hz, 1H), 8.55 (d, J = 5.6 Hz, 1H), 8.22 (dd, J = 8.8, 2.5 Hz, 1H ), 8.07 (d, J = 9.2 Hz, 1H ), 7.76–7.67
(
m, 3H), 7.42 (d, J = 2.6 Hz, 1H ), 7.23–7.21(m, 2H), 7.11 (d, J = 9.2 Hz, 1H), 2.79 (d, J = 4.8 Hz, 3H);
1
3
6
C NMR (100 MHz, DMSO-d ) δ 166.37, 164.15, 154.57, 153.09, 151.07, 149.23, 148.53, 140.98,
1
1
32.34, 131.59, 130.61, 127.21, 126.90, 125.00, 123.40, 121.80, 117.50, 117.36, 115.50, 115.28, 114.43,
+
09.36, 26.47; HRMS (ESI-TOF) m/z calcd for C H ClF N O [M-H] : 471.0836, found: 471.0832.
23
15
3
4
2
4
-((2-((4-Chloro-3-fluorophenyl)amino)quinolin-5-yl)oxy)-N-methylpicolinamide (6k)
The compound was purified by flash column chromatography (silica gel, hexane-ethyl acetate, 1:1 then
−
1
2
3
1
7
:1 v/v) to afford the entitled compound as yellow solid (32% yield); mp 278–280 °C, IR (KBr) ν/cm :
1
367, 3320 (2NH), 1665 (C=O); H NMR (300 MHz, DMSO-d ) δ 9.94 (br. s, 1H), 8.78 (d, J = 4.6 Hz,
6
H), 8.53 (d, J = 5.6 Hz, 1H), 8.43 (d, J = 12.8 Hz, 1H), 8.04 (d, J = 9.0 Hz, 1H), 7.77–7.68 (m, 2H),
.60–7.48 (m, 2H), 7.40 (br. s, 1H), 7.20 (d, J = 5.8 Hz, 2H), 7.09 (d, J = 9.0 Hz, 1H), 2.78 (d, J = 4.6
1
3
Hz, 3H); C NMR (75 MHz, DMSO-d6) δ 166.38, 164.15, 155.93, 154.62, 153.09, 151.07, 149.19,
1
1
48.66, 142.19, 142.04, 131.44, 130.69, 130.49, 125.12, 117.43, 115.89, 115.51, 115.17, 114.43, 110.98,
+
10.75, 26.47; HRMS (ESI-TOF) m/z calcd for C H ClFN O Na [M+Na] : 445.0844, found: 445.0841.
22
16
4
2
This article is protected by copyright. All rights reserved.

4
-((2-((3-Chloro-4-fluorophenyl)amino)quinolin-5-yl)oxy)-N-methylpicolinamide (6l)
The compound was purified by flash column chromatography (silica gel, hexane:ethyl acetate, 1:1 then
−
1
2
3
1
:1 v/v) to afford the entitled compound as yellow solid (33% yield); mp 259–260 °C, IR (KBr) ν/cm :
1
380, 3317 (2NH), 1667 (C=O) H NMR (300 MHz, DMSO-d
6
) δ 9.79 (br. s, 1H), 8.78 (d, J = 4.7 Hz,
H), 8.54 (d, J = 5.4 Hz, 1H), 8.44 (d, J = 3.8 Hz, 1H), 8.01 (d, J = 9.3 Hz, 1H), 7.81–7.69 (m, 3H), 7.41
1
3
(
(
t, J = 9.1 Hz, 2H), 7.19 (d, J = 4.1 Hz, 2H), 7.06 (d, J = 8.9 Hz, 1H), 2.78 (d, J = 4.4 Hz, 3H); C NMR
75 MHz, DMSO-d ) δ 166.40, 164.15, 154.71, 153.09, 151.07, 149.21, 148.73, 138.80, 131.32, 130.45,
6
1
24.95, 120.00, 119.52, 119.16, 119.07, 117.34, 117.10, 115.41, 114.95, 114.42, 109.33, 26.46.
4
-((2-((4-Bromo-3-fluorophenyl)amino)quinolin-5-yl)oxy)-N-methylpicolinamide (6m)
The compound was purified by flash column chromatography using (0–50% EA in Hex) to afford the
1
entitled compound as pure yellow solid (17.7% yield); mp 283–285 °C, H NMR (400 MHz, DMSO-d ) δ
6
9
.97 (s, 1H), 8.80 (q, J = 4.9 Hz, 1H), 8.55 (d, J = 5.6 Hz, 1H), 8.42 (dd, J = 12.2, 2.4 Hz, 1H), 8.05 (d, J
9.1 Hz, 1H), 7.78–7.70 (m, 2H), 7.63 (t, J = 8.6 Hz, 1H), 7.54 (dd, J = 8.8, 2.3 Hz, 1H), 7.41 (d, J = 2.6
=
13
Hz, 1H), 7.23–7.21 (m, 2H), 7.10 (d, J = 9.2 Hz, 1H), 2.79 (d, J = 4.9 Hz, 3H); C NMR (100 MHz,
DMSO-d ) δ 166.40, 164.16, 154.64, 153.18, 151.17, 149.19, 148.59, 142.64, 133.48, 131.57, 130.51,
6
1
25.19, 117.24, 116.46, 116.39, 115.54, 115.00, 114.44, 109.35, 106.83, 106.04, 26.47, HRMS (ESI-
+
TOF) m/z calcd for C H BrFN O [M-H] : 465.0363, found: 465.0370.
22
15
4
2
4
-((2-((3,4-Difluorophenyl)amino)quinolin-5-yl)oxy)-N-methylpicolinamide (6n)
The compound was purified by flash column chromatography (silica gel, hexane-ethyl acetate, 1:1 then
2
3
1
7
4
1
1
4
:1 v/v) to afford the entitled compound as yellow solid (40% yield); mp 248–251 °C, IR (KBr) ν/cm−1:
1
381, 3325 (2NH), 1667 (C=O); H NMR (300 MHz, DMSO-d ) δ 9.82 (br. s, 1H), 8.78 (d, J = 4.7 Hz,
6
H), 8.54 (d, J = 5.6 Hz, 1H), 8.39 (dq, J = 7.4, 2.5 Hz, 1H), 8.00 (d, J = 9.1 Hz, 1H), 7.74–7.66 (m, 2H),
.52 (br. s, 1H), 7.44–7.35 (m, 2H), 7.19 (dt, J = 8.6, 1.5 Hz, 2H), 7.06 (d, J = 9.2 Hz, 1H), 2.78 (d, J =
13
.7 Hz, 3H); C NMR (75 MHz, DMSO-d ) δ 166.40, 164.16, 154.72, 153.09, 151.06, 149.19, 148.75,
6
38.76, 138.61, 131.29, 130.41, 125.02, 117.85, 117.61, 117.33, 115.41, 114.95, 114.42, 109.33, 107.80,
+
07.80, 107.50, 26.46. HRMS (ESI-TOF) m/z calcd for C H F N O Na [M+Na] : 429.1139, found:
22
16
2
4
2
29.1142.
4
-((2-((3,4-Dimethylphenyl)amino)quinolin-5-yl)oxy)-N-methylpicolinamide (6o)
The compound was purified by flash column chromatography (hexane-ethyl acetate, 2:1 then 1:2 v/v) to
−
1
afford the entitled compound as yellow solid (52% yield); mp 207–208 °C, IR (KBr) ν/cm : 3390, 3338
This article is protected by copyright. All rights reserved.

1
(
2NH), 1672 (C=O), H NMR (400 MHz, DMSO-d ) δ 9.41 (s,1H), 8.79 (q, J = 4.6 Hz, 1H), 8.54 (d, J =
6
5
.6 Hz, 1H), 7.93 (d, J = 9.2 Hz, 1H), 7.77 (dd, J = 8.1, 2.0 Hz, 1H ), 7.68–7.65 (m, 3H), 7.41 (d, J = 2.6
Hz, 1H ), 7.20 (q, J = 2.6 Hz, 1H ), 7.12–7.10 (m, 2H), 7.05 (d, J = 9.2 Hz, 1H), 2.80 (d, J = 4.8 Hz, 3H),
1
3
2
1
1
4
6
.26 (s, 3H), 2.21 (s, 3H), C NMR (100 MHz, DMSO-d ) δ 166.46, 164.18, 155.30, 153.07, 151.02,
49.31, 149.19, 139.20, 136.57, 130.66, 130.09, 130.05, 129.65, 124.83, 120.65, 116.96, 116.90, 115.41,
+
14.38, 114.25, 109.32, 26.46, 20.27, 19.21, HRMS (ESI-TOF) m/z calcd for C H N O Na [M+Na] :
24
22
4
2
21.1641, found: 421.1634.
Preliminary anticancer screening over 3 human cancer cell lines
The antineoplastic activity of the target compounds was examined over 3 human cancer cell lines using
the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium (MTT) assay. HCT-116
(
Human colorectal carcinoma), MCF-7 (breast cancer cells) and SK-BR3 (breast cancer cells) were
supplied from the Korea Cell Line Bank (KCLB). All cell lines were grown in RPMI 1640/DMEM
Gibco BRL) supplemented with 10% (v/v) heat inactivated Fetal Bovine Serum (FBS) and maintained at
7 °C in a humidified atmosphere with 5% CO . The cells (5 × 104 cells/mL) were seeded into 96-well
(
3
2
plate. Various concentrations of samples were added to each well in duplicate, then incubated at 37 °C
with 5% CO for 2 days such that time cells are in the exponential phase of growth at the time of
2
compound addition. Add 15 μ of the Dye Solution (Promrga, Cell Titer 96) to each well. Incubate the
plate at 37 °C for up to 4 h in a humidified, 5 % CO atmosphere. After incubation, add 100 μ of the
2
solubilization Solution/Stop Mix (Promrga, Cell Titer 96) to each well. Allow the plate to stand overnight
in a sealed container with a humidified atmosphere at room temperature to completely solubilize the
formazan crystals. The optical density was measured using a microplate reader (Versamax, Molecular
Devices) with a 570 nm wavelength.
NCI anticancer screening over 60 human cancer cell lines
The cancer cell screening over full panel of 60 human cancer cell lines was applied at the National Cancer
Institute (NCI), Bethesda, Maryland, USA (22) following the standard procedure (23).
In vitro cytotoxicity evaluation against HFF-1 cell line
It was conducted using MTT assay following the same protocol described above.
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In vitro enzyme assay for compounds 6d and 6j
SM
Reaction Biology Corporation Kinase HotSpot service (24) was used for screening of compounds 6d
and 6j. The assay protocol is briefly described as following: In a final reaction volume of 25 μL, kinase
(
5–10 mU) is incubated with 25 mM Tris pH 7.5, 0.02 mM ethylene glycol tetracetic acid (EGTA),
3
3
0
5
.66 mg/mL myelin basic protein, 10 μM magnesium acetate and [γ P-ATP] (specific activity approx.
00 cpm/pmol, concentration as required). The reaction is initiated by the addition of the Mg-ATP mix.
After incubation for 40 min at room temperature, the reaction is stopped by the addition of 5 μL of a 3%
phosphoric acid solution. 10 μL of the reaction solution is then spotted onto a P30 filtermat and washed
three times for 5 min in 75 mM phosphoric acid and once in methanol prior to drying and scintillation
counting.
Molecular docking
V600E
The docking study was performed using the X-ray co-crystal structure of B-Raf
, in its DFG-out
conformation, with sorafenib (PDB: 1UWJ) (25) and C-Raf homology model. For docking study with
DFG-out conformation of C-Raf (amino acid code in Uniprot: P04049) , the homology model of C-Raf
copying with sorafenib was generated from a B-RAFV600E X-ray crystal structure using Modeller
module implemented in Discovery Studio program. The homology model was selected with low PDF
energy, and the proteins and ligands were respectively prepared using Protein Prepare Wizard and
Ligprep module in Maestro 9.7 (Schrödinger, LLC, New York). After adding hydrogens, the protein was
neutralized and then optimized with energy minimization for hydrogens only. The ligand was prepared
through protonation at pH value of 7.4 and energy minimization. Gold suite ver. 5.2 (CCDC, Cambridge,
V600E
UK) with gold score was employed to predict the binding mode of the ligand into B-Raf
and C-Raf.
The poses with the highest gold score were selected as the best docking poses and used for analysis of
binding mode.
Results and Discussion
Synthesis
Synthesis of the key intermediate 3
The synthetic pathway for preparation of compound 3 was reported and discussed in details (Scheme 1)
(
16).
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Synthesis of the new intermediates 4a–o and 5a–o and the target compounds 6a–o
As depicted in Scheme 2, the introduction of aniline motif at C2 of quinoline was the first step for the
preparation of the target compounds 6a–o. Nucleophilic substitution of 2-chloro-5-methoxyquinoline (3)
with various substituted anilines was efficiently achieved under neat conditions at 160 °C for 5–60
minutes to afford the 5-methoxyanilinoquinolines 4a–o in moderate to excellent yield (47–100%) (21).
Demethylation of the produced methoxy derivatives 4a–o with boron tribromide in DCM at 0 °C
furnished the corresponding hydroxyl derivatives 5a–o (21). The installation of the second functionality,
N-methylpicolinamide, at C5 of quinoline skeleton was accomplished through O-arylation of the
hydroxyquinolines 5a–o with 4-chloro-N-methylpicolinamide utilizing cesium carbonate as a base in
DMSO at 135 °C for 4 hours.
Biological evaluation
In vitro anticancer activity evaluation of the target compounds against 3 human cancer cell
lines
All final compounds were subjected to preliminary assessment for their antiproliferative activity against
three cancer cell lines, MCF-7 & SK-BR3 (breast cancer cells) and HCT-116 (Human colorectal
carcinoma) by MTT assay method, using sorafenib as a reference compound. The compounds were tested
at two concentrations of 100 μM & 10 μM, and the percentage of growth inhibition (GI) was recorded as
listed in Table 1. The results revealed that all compounds showed comparable (slightly inferior or
superior) anticancer activity at 100 μM. Of special interest, compounds 6d and 6j, that displayed high GI
values against all cell lines (82.77–86.71 and 76.25–90.81 %, respectively), similar to sorafenib.
Antiproliferative activity of the target compounds over NCI-60 cell line panel
The primary screening demonstrated the anticancer potential of our compounds that is worthy to be
broadly scouted. Therefore, their structures were submitted to National Cancer Institute (NCI) (22), and
1
0 compounds were selected on the basis of degree of structural variation and computer modeling
techniques for assessment of their in vitro antineoplastic activity against a panel of 60 cancer cell lines
representing 9 different tissues (blood, lung, colon, CNS, skin, ovary, kidney, prostate and breast). The
compounds were tested at a single dose concentration of 10 μM, and the mean percentages of growth
(
%G), GI, range of growth, as well as number of responsive cells were determined as shown in Table 2.
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Based on the overall anticancer activity parameter, mean GI%, the potencies of the tested compounds
could be generally ranked in the following order; 3,4-disubstituted anilines compounds, 6j–l (41.1–
4
4.9%) > 3-substituted anilines, 6c and 6d (36.7–37.7%) > 4-substituted anilines, 6e–h (19.2–31.3%).
The only exception to this order is the 3,4-difluoroaniline derivative, 6n, that showed mean GI value of
2.3%. In terms of the positive cytostatic activity parameter, it is noticed that all of the tested compounds
3
possess selective cytostatic anticancer activities (blocking tumor cell proliferation) towards certain cell
lines, in contrast to the known broad cytotoxic effects (killing of cancer cell) of sorafenib. For example,
compound 6f demonstrated remarkable GI (>50%) against 2 cell lines out of 59, while compound 6k
showed strong growth inhibitory effects over 17 cell lines out of 58.
Regarding the anticancer activity towards individual cell lines (Table 3), the prostate PC-3 cell line was
found to be highly susceptible (%G < 40%) to most of the tested compounds. Concerning the 3-
substituted anilines 6c and 6d showed selective potency against leukemia cancer cell MOLT-4 with GI
values of 67.57 and 65.56%, respectively. Meanwhile, colon cancer cell HT29 proved to be selectively
sensitive to 6d (GI = 80.3%). Moreover, compound 6c, with chlorine moiety, exerted significant activity
towards the colon cancer cells HCT-15 and SW-620, melanoma cells SK-MEL-5 and UACC-62, and
breast cancer cell MDA-MB-468 with GI values of 71.32, 79.25, 80.48, 75.30 and 82.61 %, respectively.
On the other hand, the 4-substituted anilines, 6e–h, displayed weak to moderate inhibitory activity against
most of the cell lines. It seems that the variation of substituents at 4-position of aniline has not shown
remarkable change in the antitumor activity over the individual cell lines. In respect to the 3,4-
disubstitiuted anilines, compounds 6j with 4-chloro-3-trifluoromethylphenyl, and 6l with 3-chloro-4-
fluorophenyl exerted selective potency against the leukemia K-562, melanoma SK-MEL-5, prostate PC-3
and breast MDA-MB-468 cell lines, with GI values of 69.32–74.00, 87.69–90.31, 69.22–84.70 and
8
9.47–93.49 %, respectively. It is interesting to find that the reversal of substituents of compound 6l, as in
its positional isomer 6k led to noticeable shift of the anticancer activity toward different cell lines.
Compound 6k exerted lethal effect (GI = 111.72%) against the CNS cancer cell SNB-75, beside its
pronounced inhibitory effect (GI values of 90.52, 98.64, 91.89 and 91.81%) over the lung cancer HOP-92,
melanoma MALME-3M, ovarian OVCAR-4 and renal RXF-393 cell lines, respectively. The full growth
inhibitory activities of both 6d and 6j over 58 cell lines is illustrated in Figure 2.
After this initial single dose screening of the 10 selected compounds, compounds 6d and 6j that satisfied
the threshold inhibition criteria of NCI were further tested in a five-dose testing mode to determine their
GI values (the molar concentration causing half-maximal growth inhibition). The GI values of 6d and
50
50
6
j in comparison to both compound B (16) and sorafenib (26) are showed in Table 4.
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As shown in Table 4, most of the GI data for both 6d and 6j were less than 10 μM. In comparison to
50
sorafenib, it was found that compound 6d is less potent than sorafenib over all cell lines. On the contrary,
compound 6j (with 4-chloro-3-trifluoromethylphenyl moiety) displayed superior anticancer potency than
both compound B and sorafenib over 11 and 8 cell lines, respectively, with GI values of 0.356–1.81μM
50
(
Figure 3). Among those sensitive cell lines are the melanoma cells SK-MEL-5, UACC-257 and UACC-
V600E
6
2, in which the mutant B-Raf
is overexpressed (27, 28). Towards them, compound 6j showed
GI values of 0.678 μM, 1.71 μM and 0.6 μM, respectively. Such interesting finding was an apparent
50
V600E
indication that 6j may possess B-Raf
inhibitory effect. Moreover, compound 6j displayed potent
antiproliferative activity against the breast cancer cell MDA-MB-468 with GI value of 0.356 μM, being
50
1
0 and 6 times more potent than both B and sorafenib, respectively. In respect to the other cell lines, both
d and 6j seemed to be less potent than compound B and sorafenib. From these results, we can conclude
6
that either structural rigidification of sorafenib or spacer change of compound B from urea to amine
resulted in generation of selective anticancer agents, which target only certain cell lines with better
potency.
Evaluation of cytotoxicity against the HFF-1 normal cell
To get some insights about the differential cytotoxicity of this new set of compounds, the most active
members 6d and 6j have been tested against the human foreskin fibroblast (HFF-1) normal cell line using
the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium (MTT) assay (Table 5).
Interestingly, both compounds 6d and 6j showed low growth inhibitory effects as indicated by their high
GI50 values (> 10.0 μM), which point out their selective growth inhibitory activities towards human
cancer cells rather normal cell lines.
In vitro kinase inhibitory activity of compounds 6d and 6j
In order to explore the impact of our structural modifications on the RAF kinase inhibitory activity,
V600E
compounds 6d and 6j were tested against both B-Raf
and C-RAF kinases (Table 6). Unfortunately,
both 6d and 6j showed modest inhibitory activity. Upon comparison with the ureidoquinoline B (16), we
can conclude that replacement of the urea linker with amine has a negative impact on RAF kinase
inhibition. Such findings reflect the significance of urea motif at C2 position of quinoline scaffold in
terms of RAF kinase activity.
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Moreover, kinase screening of both compounds 6d and 6j over a number of receptor and non-receptor
protein kinases was performed (Table 7). The results revealed that 6d and 6j do not possess considerable
inhibitory effects against the tested kinases. Relatively, compound 6j exhibited a moderate inhibitory
activity against Pim3 kinase with 38.9% inhibition at 10 μM. These biochemical assay results denote that
the selective cellular potency of our anilinoquinolines is not due to protein kinase inhibition, which
suggest the existence of other leading mechanism(s) responsible for this antineoplastic effect.
Accordingly, further screening of compound 6j over a broad array of potential molecular targets is
currently under processing.
Molecular docking study
In attempt to warrant the modest RAF kinase inhibitory activities of this type of compounds, represented
by compound 6j, a molecular docking study was performed. The anilinoquinoline 6j as well as its
V600E
ureidoquinoline homolog B were docked in the catalytic kinase domain of B-Raf
(PDB accession
code 1UWJ) (24), and C-Raf homology model. As shown in Figure 4, both compounds 6j and B could
V600E
bind with the hinge region residue Cys532 (B-Raf
)/Cys424 (C-Raf) by two hydrogen bonds via their
picolinamide moiety. Also, their quinoline scaffold was engaged in multiple hydrophobic and electrostatic
V600E
interactions with various amino acid residues (like Lys483 and Thr529 (B-Raf
)) in the gatekeeper
region. However, within the DFG loop, only the ureidoquinoline B was able to bind with the backbone
V600E
NH of Asp594 (B-Raf
)/Asp486 (C-Raf) via its carbonyl oxygen of urea, while compound 6j missed
this key interaction. Also, an additional difference between the binding modes of compounds 6j and B
was observed in the allosteric site of RAF kinase. The urea spacer of compound B enabled its terminal 4-
chloro-3-trifluoromethylphenyl group to be deeply inserted within the hydrophobic allosteric site adjacent
to the ATP binding site. Such hydrophobic interactions were lost in case of the anilinoquinoline
derivative 6j, mainly because of its short amine spacer. The aforementioned observations could provide
some insights about the unpretentious RAF kinase inhibitory effects of compound 6j, as an example for
this new series of 2-anilinoquinolines.
Conclusion
In conclusion, a series of novel 2-anilinoquinolines possessing 5-oxypicolinamide moiety has been
designed, synthesized, and evaluated for its anticancer activity over a panel of 60 cancer cell lines. All of
the tested anilinoquinolines displayed selective antineoplastic activities against a number of cancer cells.
Compound 6j proved to be the most active member in this series with superior potency than its
corresponding ureidoquinoline B and sorafenib across certain cell lines, with submicromolar or single
This article is protected by copyright. All rights reserved.

digit micro-molar GI values. In addition, 6j showed low cytotoxic activity against the HFF-1 normal cell
50
line. The modest RAF inhibitory activity of these derivatives suggest the presence of other underlying
mechanism(s) responsible for their anticancer activities. Accordingly, further screening of compound 6j
over a large group of prospective molecular targets is currently in progress.
Acknowledgments
This research was supported by the Korea Institute of Science and Technology (KIST) Institutional
Program (2E26650 and 2E26663). We would like to express our sincere gratitude and appreciation to the
National Cancer Institute (NCI, Bethesda, Maryland, USA) for conducting the anticancer evaluation of
the new compounds.
Supplementary data
1
13
Supplementary data ( H NMR, C NMR, and HRMS data for representative compounds and NCI cell
based assays) associated with this article was submitted.
Conflict of interest
The authors declare no conflict of interest.
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Table 1. Preliminary antiproliferative activity of the target compounds against a panel of 3 human cancer
a
cell lines.
%
Growth inhibition
MCF-7
Compound
No.
HCT116
SK-BR-3
Ar
1
00 μM 10 μM 100 μM 10 μM 100 μM 10 μM
6
6
a
3-F-C
3-CF
6
H
4
71.30
73.20
69.04
85.58
79.33
67.55
64.39
53.64
69.55
90.81
55.91
68.33
75.67
71.61
26.48
97.32
60.28
20.17
30.51
8.83
48.71
33.05
52.38
82.77
83.74
60.27
55.30
50.03
57.94
77.45
46.08
58.54
37.37
56.09
69.16
96.06
17.17
39.08
21.06
-2.02
11.91
13.85
10.21
10.58
7.96
66.61
58.31
70.63
86.71
83.68
66.99
65.51
46.63
65.77
76.25
64.87
79.33
59.48
68.98
72.68
93.12
12.66
45.58
17.56
6.78
b
3
-C
6
H -
4
6
c
3-Cl-C
6
H
4
-
6
d
3-(CH
3
)
3
2
C-C
6
H
4
-
6
e
4-(CH
3
)
CH-C
6
H
4
-
-16.00
29.08
1.03
12.05
5.20
6
f
4-Et-C
4-OPh-C
4-Ph-C
4-Morpholin-C
4-Cl-3-CF -C
H -
6 4
6
6
g
6
H
4
-
21.58
16.66
12.72
24.53
56.38
69.53
35.94
61.85
24.43
48.87
h
6
H
4
-
15.65
-5.49
57.52
22.31
25.18
52.84
30.74
13.85
48.41
6
i
6 4
H -
6
j
3
6
H
3
-
24.85
34.04
50.08
33.18
49.53
17.69
40.45
6
k
4-Cl-3-F-C
3-Cl-4-F-C
6
6
H
H
3
3
-
-
6
l
6
m
4-Br-3-F-C
3,4-F -C
3,4-(CH
6
H
3
-
6
n
o
2
H -
6 3
6
3
)
2
-C
6
H
3
-
Sorafenib
a
Compounds were tested in duplicate mode at two concentrations of 100 μM and 10 μM.
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Table 2. Overview of the preliminary anticancer assay at single dose concentration of 10 μM.
6
0 cell lines assay in single dose (10 μM)
Compound
No.
NSC
Mean
Mean
Range of
growth %
17.4 to 97.7
The most sensitive
cell line
Positive
cytostatic effect
16/57
a
code
growth % Growth inhibition %
6
c
778307
776931
776933
780447
780448
776934
776932
780446
782196
782194
63.3
62.3
80.8
79.8
68.7
75.6
57.7
58.9
55.1
67.7
36.7
37.7
19.2
20.2
31.3
24.4
42.3
41.1
44.9
32.3
MDA-MB-468 (breast)
6d
19.7 to 100.7 HT29 (colon)
31.5 to 122.3 PC-3 (prostate)
37.7 to 102.9 SR (leukemia)
18/58
6
e
3/58
6f
2/59
6g
30.2 to 99.9
SR (leukemia)
9/59
6h
32.5 to 104.9 PC-3 (prostate)
4/58
6
j
9.69 to 93.7
SK-MEL-5 (melanoma)
20/58
6k
−11.7 to 99.5 SNB-75 (CNS)
17/59
6
l
6.51 to 97.0
29.4 to 98.5
MDA-MB-468 (breast)
OVCAR-4 (ovarian)
23/58
6n
11/60
a
The ratio between number of cell lines with percent growth from 0 to 50 and total number of cell lines.
Table 3. The growth inhibition percentages of the tested compounds over the most sensitive cell lines at 10 μM
a,b
concentration.
%
Growth inhibition
6g 6h
778307 776931 776933 780447 780448 776934 776932 780446 782196 782194
Cell line/
6c
6d
6e
6f
6j
6k
6l
6n
Cancer type
NSC code
K-562
57.16
67.57
67.56
14.87
22.33
67.38
71.32
36.54
79.25
15.33
61.64
54.87
80.48
73.24
65.56
60.25
51.66
40.85
61.88
50.70
80.30
37.81
15.49
48.75
19.35
52.66
57.37
46.80
57.60
19.36
23.57
30.74
41.21
37.07
–
39.95
49.08
62.28
–
59.02
57.33
69.77
28.76
37.33
52.0
51.15
42.85
55.30
28.54
40.25
36.09
31.28
17.98
3.33
69.32
66.34
66.04
25.61
55.37
63.75
49.21
35.08
52.69
38.16
51.81
45.69
90.31
16.24
30.79
37.79
90.52
47.32
54.61
40.68
17.47
18.02
111.72
46.74
98.64
55.24
74.00
NT
50.73
47.80
37.50
25.72
39.01
64.49
68.47
37.80
42.49
56.76
55.79
37.98
48.06
Leukemia
MOLT-4
SR
NT
Lung Cancer
Colon Cancer
HOP-92
NCI-H522
HCT-116
HCT-15
HT29
22.12
60.04
82.44
83.55
18.63
79.67
12.44
73.25
59.75
87.69
32.18
32.5
48.06
27.64
–
45.3
37.51
12.22
32.19
34.42
21.77
63.74
SW-620
SNB-75
U251
CNS Cancer
Melanoma
20.80
33.26
10.05
21.26
25.6
27.95
11.26
37.63
21.58
33.63
13.65
29.02
MALME-3M
SK-MEL-5
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UACC-62
OVCAR-4
RXF 393
UO-31
75.30
NT
36.83
39.83
15.49
57.82
76.37
54.66
55.11
37.85
17.26
–
39.72
25.26
10.01
23.52
55.84
38.95
23.44
37.55
47.94
–
46.68
29.07
–
64.88
51.61
22.19
38.60
84.70
41.71
89.47
47.69
91.89
91.81
18.38
33.91
48.68
48.72
74.73
59.90
24.74
31.31
69.22
34.04
93.49
56.19
70.65
17.32
17.32
62.65
19.92
32.41
Ovarian Cancer
Renal Cancer
27.48
18.51
61.05
25.26
82.61
22.60
68.53
21.26
27.17
42.07
64.59
56.72
36.33
39.38
67.54
42.78
33.09
Prostate Cancer PC-3
Breast Cancer
T-47D
MDA-MB-468
a
Bold figures indicate GI > 60%.
b
–
; GI < 10%, NT; not tested.
a,b
Table 4. GI50 values (μM) of compounds 6d, 6j, B and sorafenib over NCI-60 cell line panel.
Cell lines
GI50 value (μM)
Cell lines
GI50 value (μM)
6
d
6j
B
Sorafenib
6d
6j
B
Sorafenib
Leukemia
CCRF-CEM
HL-60(TB)
K-562
Melanoma
M14
3.44 4.44 >100
6.04 50.8 NT
3.97 6.28 >100
3.33 1.74 >100
3.86 5.32 NT
3.80 3.97 3.22
2.0
3.24 6.46
4.60 3.94
5.00
3.12
7.18
4.59
2.0
1.58
1.58
2.0
1.58
3.16
3.16
1.58
3.16
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
NT
NT
NT
NT
MOLT-4
RPMI-8226
SR
3.51 0.678 2.62
4.39 1.71 >100
3.93 0.60 1.97
1.58
2.0
Non-Small Cell Lung Cancer
2.32
A549/ATCC
EKVX
5.00 6.59 2.83
NT NT NT
3.16
2.51
2.0
Ovarian Cancer
IGROV1
12.8 17.9
6.26 6.70
7.70 3.39
12.3
2.84
1.17
3.85
3.47
3.88
3.78
2.51
3.16
3.16
3.16
2.51
2.51
2.51
HOP-62
30.7 13.9 2.76
2.45 3.81 1.51
7.58 1.22 3.73
7.07 6.66 4.81
> 100 23.7 3.91
4.01 5.36 2.79
9.84 6.04 14.7
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
HOP-92
1.58
2.0
NCI-H226
NCI-H23
NT
NT
2.0
8.28 14.6
NCI-H322M
NCI-H460
NCI-H522
Colon Cancer
COLO 205
HCC-2998
HCT-116
2.51
2.51
2.0
NCI/ADR-RES 5.98 10.6
SK-OV-3
Renal Cancer
786-0
49.1 19.1
3.80 11.6
1.89
1.36
1.66
4.28
3.16
2.51
3.16
3.16
NT
NT
NT 4.36
NT >100
2.0
A498
6.17 0.66
3.16
1.58
ACHN
NT
NT
3.17 1.81 2.28
CAKI-1
5.85 16.8
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HCT-15
HT29
3.04 6.47 NT
3.26 8.54 2.04
2.51
2.0
RXF 393
SN12C
4.94 7.13
4.66 18.9
16.9 12.6
3.46 25.4
1.89
2.62
2.17
2.62
3.16
2.51
3.98
2.51
KM12
NT
NT 9.94
1.58
2.51
TK-10
SW-620
CNS Cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
3.86 3.02 NT
UO-31
Prostate Cancer
PC-3
12.8 14.7 3.84
3.90 8.66 2.16
8.35 14.3 2.60
7.61 3.70 5.06
5.15 4.97 1.09
2.51
1.58
1.58
3.16
3.16
3.05 1.22
57.7
4.74
2.0
DU-145
14.7 15.7
3.16
Breast Cancer
MCF7
2.83 2.75
3.50 4.97
5.91
2.51
1.26
MDA-MB-
1
.67
2
31/ATCC
U251
12.8 14.7 3.12
2.0
HS 578T
BT-549
15.8 9.24
3.28 5.43
3.59 4.79
1.73
4.67
1.47
2.51
3.16
1.58
2.0
Melanoma
LOX IMVI
MALME-3M
4.21 9.84 2.32
3.18 9.01 2.61
1.58
2.0
T-47D
MDA-MB-468
2.46 0.356 3.56
a
Bold figures indicate superior potency than compound B and sorafenib, bold underlined figures refer to submicromolar GI50
b
values. NT, not tested.
a
Table 5. Cytotoxicity evaluation of compounds 6d and 6j against HFF-1 normal cell line.
Tested
% Growth inhibition
concentrations
6d
6j
1
0 μM
17.17±4.44
30.27±2.18
1
0
.0 μM
.1 μM
5.28±4.62
6.88±4.41
> 10.0
7.94±0.57
2.66±0.83
> 10.0
GI (μM)
5
0
a
%
Growth inhibition was recorded for each compound at 3 different concentrations.
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V600E
a
Table 6. In vitro enzymatic activity of compounds 6d, 6j and B over B-Raf
and C-Raf kinases.
V600E
Compound No.
BRAF
C-RAF
a
b
a
b
%
Inhibition IC50 (μM) % Inhibition IC50 (μM)
6
6
d
13.9
9.09
81.8
> 100
> 100
0.316
37.3
−5.83
96.3
92.6
> 100
0.061
j
c
B
a
Compounds were tested in single dose duplicate mode at a concentration of 10 μM.
Compounds were tested in a 10-dose IC50 mode with 3-fold serial dilution starting at 100 μM.
The assay results were retrieved from reference 16.
b
c
a
Table 7. In vitro enzymatic screening of compounds 6d and 6j against certain protein kinases.
Kinases
ABL1
% Enzymatic inhibition
6
d
6j
−7.77
NT
5.14
T315I
ABL
22.6
c-Kit
NT
−1.04
3.52
FLT3
−2.94
9.13
NT
FMS
−0.87
−36.0
−8.77
3.93
P38a/MAPK14
LCK
NT
VEGFR2
Pim3
−65.4
−74.6
12.3
38.9
TrkA
6.39
a
Compounds were tested in single dose duplicate mode at a concentration of 10 μM.
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