Design, synthesis and biological evaluation of 6-substituted quinolines derived from cabozantinib as c-Met inhibitors

Article abstract of DOI:10.1002/ardp.201900101

Based on the cabozantinib scaffold, novel c-Met inhibitors were rationalized from the limited knowledge of structure-activity relationships for the quinoline 6-position. Emphasis was given to modifications capable of engaging in additional polar interacti

Full text of DOI:10.1002/ardp.201900101

Received: 30 March 2019
Revised: 29 May 2019
Accepted: 18 June 2019
|
|
DOI: 10.1002/ardp.201900101
F U L L P A P E R
Design, synthesis and biological evaluation of 6‐substituted
quinolines derived from cabozantinib as c‐Met inhibitors
Vegard Torp Lien1
Dag Erlend Olberg1,3
|
|
Solveig Pettersen2
Gunhild M. Mælandsmo2,4
|
Mads Haugland Haugen2
Jo Klaveness1
|
|
1Department of Pharmacy, University of Oslo,
Oslo, Norway
Abstract
2Department of Tumor Biology, Institute for
Cancer Research, OUS Radiumhospitalet,
Oslo, Norway
Based on the cabozantinib scaffold, novel c‐Met inhibitors were rationalized from the
limited knowledge of structure‒activity relationships for the quinoline 6‐position.
Emphasis was given to modifications capable of engaging in additional polar
interactions with the c‐Met active site. In addition, ortho‐fluorinations of the terminal
benzene ring were explored. Fifteen new molecules were synthesized and evaluated
in a c‐Met enzymatic binding assay. A wide range of substituents were tolerated in
the quinoline 6‐position, while the ortho‐fluorinations performed were shown to give
considerable reductions in the c‐Met binding affinity. The antiproliferative effects of
the compounds were evaluated in the NCI60 cancer cell line panel. Most notably,
compounds 15b and 18b were able to inhibit cell proliferation more efficiently than
cabozantinib in leukemia, CNS, and breast cancer cell lines. The in vitro data agreed
well with the in silico docking results, where additional hydrogen bonding was
identified in the enzymatic pocket for the para‐amino substituted 15b and 18b.
3Norwegian Medical Cyclotron Center, Oslo,
Norway
4Institute of Medical Biology, Faculty of
Health Sciences, UiT‐Arctic University of
Norway, Tromsø, Norway
Correspondence
Vegard Torp Lien, Department of Pharmacy,
University of Oslo, Boks 1068, Blindern, 0316
Oslo, Norway.
Email: v.t.lien@farmasi.uio.no
K E Y W O R D S
cabozantinib, c‐Met, kinases, NCI60, quinolones
|
1
INTRODUCTION
molecule inhibitors are shown in Figure 1, including the regulatory
approved cabozantinib (1) and crizotinib (2).
Despite a surge in the available cancer treatments over the last
decades, drug resistance and tumor relapse remain as prominent
challenges.[1] Therefore, finding new ways of inhibiting molecular
pathways responsible for tumor cell proliferation, migration, and
invasion is a main focus in cancer research.[2,3] The tyrosine kinase c‐
Met (hepatocyte growth factor receptor) plays a central role in many
cancer diseases, and abnormal activation leads to tumor growth and
proliferation, dissociation of cells from its primary site and distant
colonization. Because of this, dysregulation of c‐Met has been
proposed as one of the primary drivers for cancer development
and metastatic processes.[4,5] Considerable efforts have been made in
developing inhibitors of c‐Met,[6–8] and some examples of small‐
Cabozantinib (1) is a multikinase inhibitor, which inhibits, among
others, the kinases c‐Met and vascular endothelial growth factor
receptor (VEGFR).[9] Cabozantinib is approved for medullary thyroid
cancer and advanced renal cell carcinoma, and several clinical studies
are currently performed for other cancer indications such as prostate
and colorectal cancer.[10] Crizotinib (2) is approved for lung cancer,
and inhibits the kinases ALK and ROS1 in addition to c‐Met.
Capmatinib (3) and AMG 337 (4) are examples of inhibitors that are
exquisitely selective for the c‐Met kinase, both currently in clinical
trials for lung cancer and metastatic solid tumors.
Extensive research has been conducted to explore the structure‒
activity relationships (SARs) for c‐Met inhibitors similar to
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
© 2019 The Authors. Archiv der Pharmazie published by Wiley‐VCH Verlag GmbH & Co. KGaA on behalf of Deutsche Pharmazeutische Gesellschaft
|
Arch Pharm Chem Life Sci. 2019;e1900101.
wileyonlinelibrary.com/journal/ardp
1 of 15
https://doi.org/10.1002/ardp.201900101

2 of 15
LIEN ET AL.
|
FIGURE 1 Examples of known inhibitors of c‐Met, including the regulatory approved inhibitors cabozantinib (1) and crizotinib (2)
cabozantinib.[11–13] For the hinge‐binding quinoline moiety, modifications
have primarily been made at the 7‐position, these frequently being
groups intended to increase solubility.[14] With other heterocycles as
hinge‐binders, substituents known to participate in more direct interac-
tions with the enzyme have been explored, exemplified with amines,
anilines, and nitrogen containing heterocycles in Figure 2.[15–18] Molecules
containing such variations have been shown to have strong interactions
with the c‐Met active site, made possible by the formation of additional
hydrogen bonds. Substitutions at the quinoline 6‐position have been less
studied, and we, therefore, sought to explore this position and whether
the introduction of functional groups capable of engaging in polar
interactions could improve the c‐Met binding affinity. This was
rationalized with the introduction of additional heteroatoms, fluorinated
groups, and hydrogen bond donors or acceptors as depicted in Figure 2,
and this strategy resulted in the target scaffold 5. In addition to the
modifications at the quinoline 6‐position, the bioisosteric replacement of
hydrogen with fluorine in ortho‐positions of the terminal benzene ring
was of interest since this would block one of the main metabolic
pathways for this compound class.[19,20] In this study, we present the
synthesis and in vitro evaluation of these novel kinase inhibitors.
|
2
RESULTS AND DISCUSSION
Chemistry
|
2.1
The two fluorinated analogs of the terminal aromatic ring, 6a and 6b,
were prepared starting from cyclopropane‐1,1‐dicarboxylic acid by
reaction with the corresponding aniline, as shown in Scheme 1.
The 6‐hydroxy‐7‐methoxyquinoline scaffold in 5 was synthesized in
a similar manner to earlier reported work,[21] although starting from 3‐
hydroxy‐4‐methoxy acetophenone, which was first benzylated to 7,
then nitrated to 8, and further reduced to the aniline 9. Cyclization into
the 4‐hydroxy quinoline 10 was achieved using ethyl formate and
further reacted with 1‐fluoro‐4‐nitrobenzene to give diaryl ether 11,
which was reduced to the aniline 12. The acids 6 were then coupled
with aniline 12 into the main scaffold 13, which could then be
FIGURE 2 Examples of known variations around the hinge binding heterocycles and our proposed target scaffold 5. Atoms with the
capability of engaging in polar interactions are displayed in red

LIEN ET AL.
3 of 15
|
SCHEME 1 Synthesis of compounds 13a,b and 14a,b. Reagents and conditions: (a) NEt3, SOCl2, THF, rt, 20 hr (6a in 30%, 6b in 41%); (b)
BnBr, K2CO3, 40°C, 18 hr, 91%; (c) HNO3, H2SO4, DCM, rt, 0.5 hr, 92%; (d) Fe, NH4Cl, EtOH, H2O, 70°C, 3 hr, 89%; (e) NaOEt, ethyl formate,
DME, rt, 24 hr, 99%; (f) 1‐fluoro‐4‐nitrobenzene, Cs2CO3, DMF, MeCN, 55°C, 24 hr, 21%; (g) Fe, NH4Cl, EtOH, H2O, 70°C, 84%; (h) 6a or 6b,
HATU, DIPEA, DMF, rt, 20 hr (13a in 61%, 13b in 62%); (i) Pd/C, 1,4‐cyclohexadiene, EtOH, 70°C, 6 hr (14a in 78%, 14b in 79%)
deprotected to the phenols 14. Cleavage of amide bonds was observed
using hydrogen gas, and 1,4‐cyclohexadiene was therefore applied as a
milder hydrogen source. Synthesis of 19 was performed in a similar
manner, using steps f–h, starting from 6,7‐dimethoxyquinolin‐4‐ol.
Further functionalization to compounds 15–18 could be achieved
as shown in Scheme 2. The phenol 14a was esterified into the para‐
substituted nitro, amino, and trifluoromethyl esters 15. The highly
fluorinated analogs 16 and 17 were synthesized using chloro
difluoromethylbenzene or 1,1,1‐trifluoro‐2‐iodoethane, respectively.
These reagents were prone to produce several side‐products, so the
yields were correspondingly low. Using 2‐chloro‐5‐nitro‐pyridine, the
nitropyridyls 18a and 18c were achieved, which then were reduced
to the corresponding aminopyridyls 18b and 18d.
The introduction of two additional fluorine atoms on the
terminal aromatic ring reduced the inhibition of c‐Met, and this is
observed for all six pairs of compounds shown in Table 1. The
increase in IC50 values resulting from this ortho‐fluorination
ranges from
a factor of about two for the amino pyridinyl
derivatives (18b/18d), to a factor of 30 for the benzyl ethers
(13a/13b). The ortho‐fluorinated analog of cabozantinib as such,
compound 19, was shown to be 27 times less potent than
cabocantinib. For the quinoline 6‐position, a range of both alkyl
and aromatic substituents are well tolerated. By comparing the
benzyl esters 15 and the pyridyls 18, it is evident that the nature
of the para‐substituent is important, with the observed affinity
trend NH2>NO2>CF3. The aniline ester 15b is the most potent
inhibitor of c‐Met in the series with an IC50 of 19 nM. Moreover,
the difluorinated benzyl ether 16 was twice as potent compared to
the unfluorinated benzyl ether 13a, while the trifluoroethyl analog
17a exhibited 50% reduced potency compared to cabozantinib.
The 6‐O‐demethylated analog 14a was equipotent to cabozantinib.
|
2.2
Biology
The novel compounds were evaluated for enzymatic c‐Met binding
affinity, and the results are reported in Table 1.

4 of 15
LIEN ET AL.
|
SCHEME 2 Synthesis of compounds 15‒18. Reagents and conditions: (a) 4‐nitrobenzoyl chloride, Cs2CO3, DMF, rt, 5 hr, 37%; (b) Fe, NH4Cl,
EtOH, H2O, 70°C, 3 hr, 60%; (c) 4‐(trifluoromethyl)benzoic acid, HATU, DMAP, DMA, rt, 16 hr, 53%; (d) chlorodifluoromethylbenzene, Cs2CO3,
DMF, 100°C, 20 hr, 6%; (e) 1,1,1‐trifluoro‐2‐iodoethane, Cs2CO3, DMF, 110°C, 5 hr (17a in 33%, 17b in 4%); (f) 2‐chloro‐5‐nitro‐pyridine,
Cs2CO3, DMF, rt, 1.5 hr (18a in 75%, 18c in 93%); (g) Fe, NH4Cl, EtOH, H2O, 70°C, 3 hr (18b in 54%, 18d in 59%)
In summary, substituents in the quinoline 6‐position capable of
engaging in polar interactions seem to augment c‐Met affinity.
The analogs 14a, 15a, 15b, 18b, and 18d were progressed for
further evaluation in a cancer cell proliferation inhibition assay. This
selection was based on low IC50 values for c‐Met, while simulta-
neously maintaining a structural diversity to further explore the
efficacy in cell‐based assays. These studies were performed using the
NCI60 program at the National Cancer Institute (NCI).[22,23] Here,
the compounds were tested at 10‐µM concentration in a broad range
of cell lines from nine different tumor types. The results are
presented in Table 2 as observed growth percent.
analog of cabozantinib, is seen to have a markedly reduced ability for
the inhibition of cell proliferation, even though c‐Met affinity is
comparable with cabozantinib. The importance of the para‐amino
group in 15b and 18b is evident from the notably reduced capability
of the para‐nitro analog 15a to influence the growth rates, despite
comparable c‐Met IC50 values. These observations indicate that the
6‐position on the quinoline ring is important for the interaction with
other kinases in addition to c‐Met, as can be expected for this class of
multikinase inhibitors. The same trend is also seen with the
trifluorinated compound 18d performing overall better in the cell‐
based assay than compounds 14a and 15a, despite its lower c‐Met
affinity. This observation is in compliance with known SAR on related
structures that have shown that c‐Met affinity is more sensitive to
modifications on the terminal benzene ring than is VEGFR.[24]
Compared with cabozantinib, higher growth suppressive effects are
From Table 2, it can be deduced that the tested compounds were
able to inhibit growth in a wide range of tumor cell lines. The most
potent compounds were 15b and 18b, which is consistent with the
observed trend in Table 1. The compound 14a, the 6‐O‐demethylated

LIEN ET AL.
5 of 15
|
TABLE 1 Inhibition of c‐Met enzymatic activity for the synthesized
compounds 13–19
cabozantinib, except for the TGI value for 15b. The lowest GI50
values observed were 50 and 40 nM with 18b in the HOP‐92 and
KM‐12 cell lines, respectively. The reported means in Table 3 are for
all NCI60 cancer cell lines, and the complete data are given in the
Supporting Information.
To examine whether the structural modifications affected the
kinase selectivity profile, a screen was performed on six kinases in
addition to c‐Met. Kinase selectivity was assessed for the analogs
15b, 18b, and 18d, and is presented in Table 4.
Compound
13a
R
R′
IC50 (nM)b
As can be seen in Table 4, the novel analogs have a similar kinase
selectivity profile as cabozantinib, albeit with a lower affinity toward
c‐Kit. Interestingly, 18b exhibits a stronger inhibition of ALK. In light
of the different cell proliferation results among the compounds in
Table 2, additional modes of action cannot be ruled out.
H
135
13b
F
4,074
14a
14b
15a
H
F
H
H
32
201
47
|
2.3
In silico evaluation
H
Introduction of various functionalities to the 6‐position of the
quinoline could potentially influence the binding mode of the ligands
to the active site of c‐Met. The most potent synthesized ligands,
were, therefore, further evaluated by molecular docking using
AutoDock Vina[25] via the PyRx[26] interface. The experimental
crystal structure with the c‐Met inhibitor foretinib (PDB: 3LQ8)
was employed.
15b
15c
16
H
H
H
19
394
75
By overlaying the docked structures of cabozantinib, 15b and
18b, it is seen in Figure 3a that the three ligands are well aligned
within the receptor site. The introduced 4‐amino phenyl ester in 15b
and 4‐amino pyridinyl in 18b were shown to overlap, and instead of
pointing out in the solvent‐accessible area, as is the case, for
example, the morpholine in foretinib, these groups engage in an
additional hydrogen bonding to Ala‐1226. To accommodate this
hydrogen bonding, the docked structure of 18b is shifted rightward
in Figure 3a, which may emphasize the importance of this interaction.
The interactions with the specific parts of the active site are
exemplified with 18b in Figure 3b. The nitrogen in the quinoline ring
forms a hydrogen bond with Met‐1160, while the amide linker
interacts with Lys‐1110 and Phe‐1223.
17a
17b
18a
H
F
70
1,558
324
H
18b
18c
18d
H
F
64
6,000
113
F
|
3
CONCLUSION
Rationalized from the limited knowledge of SAR around the quinoline
6‐position, novel c‐Met inhibitors were designed based on the
cabozantinib scaffold. In particular, the introduction of functional
groups capable of engaging in direct interactions with the enzyme
were emphasized. Several of the compounds displayed similar or
increased potency compared to cabozantinib in a c‐Met enzymatic
assay. Compounds 14a, 15a, 15b, 18b, and 18d evaluated in the
NCI60 program displayed high antiproliferative activity, with 15b
and 18b being the most potent, especially in leukemia, CNS, and
breast cancer cell lines. Additional hydrogen bonds to the c‐Met
active site were observed by molecular docking for the para‐amino
substituted 15b and 18b. Further on, it was shown that c‐Met affinity
19
F
Me
Me
1,078
40
Cabozantinib (1)a
H
aReference compound in the assay.
bn ≥ 2. Average values are given.
seen with 15b and 18b in several of the cell lines, particularly in cells
derived from leukemia, CNS, and breast cancer. 15b and 18b were
progressed for 5‐dose testing, and the results for selected cancer cell
lines are reported in Table 3.
The results from the 5‐dose assay corroborate compounds 15b
and 18b as potent inhibitors of cancer cell proliferation. All mean
GI50, TGI, and LC50 values for 15b and 18b are lower than for

6 of 15
LIEN ET AL.
|
TABLE 2 The effect on cell proliferation for the compounds 14a, 15a, 15b, 18b, and 18d at 10 µM on the NCI60 cell lines
Growth percent (%)
Cell line
14a
15a
15b
18b
18d
Cabozantiniba
Leukemia
CCRF‐CEM
HL‐60(TB)
K‐562
29.03
54.53
9.30
86.47
27.84
61.18
70.87
38.25
7.69
15
15
−10
22
22
8
92.86
57.69
72.20
88.82
99.25
−43.90
−20.88
3.73
22.95
3.07
MOLT‐4
44.86
52.76
41.31
29.51
48.51
11.92
38.53
50.14
58.46
RPMI‐8226
SR
10.37
9.85
Non‐small‐cell lung cancer
A549/ATCC
EKVX
56.45
71.05
57.65
33.42
67.06
57.42
87.72
48.11
68.01
99.66
81.32
88.90
32.64
83.06
71.44
100.58
95.06
87.82
33.57
25.74
11.72
−13.34
55.73
37.78
43.02
24.72
21.45
22.65
15.93
51.19
−11.47
36.46
49.31
49.41
30.99
51.26
48.02
45.20
67.73
2.04
15
40
HOP‐62
30
HOP‐92
−35
−25
30
NCI‐H226
NCI‐H23
NCI‐H322M
NCI‐H460
NCI‐H522
Colon cancer
COLO 205
HCC‐2998
HCT‐116
HCT‐15
37.70
53.55
74.83
65.43
54.96
25
10
25
91.01
79.26
52.01
61.83
95.14
22.48
34.36
103.66
108.76
94.60
94.27
96.93
18.82
76.61
−36.55
25.79
23.12
25.87
1.03
8.91
72.05
40.70
27.86
9.65
44.26
81.52
56.58
45.50
34.84
15.53
40.32
−50
38
13
20
0
HT29
KM12
12.35
33.48
3.70
10
2
SW‐620
36.75
CNS cancer
SF‐268
65.76
35.17
24.48
82.03
33.97
66.05
79.34
64.41
39.12
96.77
44.96
79.71
29.43
−74.56
−1.20
37.63
−34.57
7.81
36.82
0.16
55.46
30.58
21.15
78.74
21.75
67.30
30
−15
3
SF‐295
SF‐539
5.06
SNB‐19
54.50
−12.47
33.56
40
−5
30
SNB‐75
U251
Melanoma
LOX IMVI
MALME‐3M
M14
39.75
64.02
49.46
52.60
88.13
66.38
71.49
34.78
89.12
82.24
94.34
84.00
97.53
89.42
107.25
80.12
−51.42
25.98
10.84
33.16
31.31
33.98
33.55
40.09
10.67
43.11
39.85
3.34
49.35
58.96
56.88
3.80
13
−5
0
MDA‐MB‐435
SK‐MEL‐2
SK‐MEL‐5
UACC‐257
UACC‐62
Ovarian cancer
IGROV1
15
35
35
35
10
54.29
39.16
33.95
5.29
81.50
29.00
53.78
24.38
52.10
78.71
72.65
60.70
47.48
−24.06
28.14
75.28
0
OVCAR‐3
100.65
49.88
35
(Continues)

LIEN ET AL.
7 of 15
|
TABLE 2 (Continued)
Growth percent (%)
Cell line
14a
15a
15b
51.72
18b
29.88
18d
Cabozantiniba
OVCAR‐4
OVCAR‐5
OVCAR‐8
NCI/ADR‐RES
SK‐OV‐3
Renal cancer
786‐0
37.08
80.78
73.73
71.74
86.52
75.47
101.37
88.98
85.76
108.17
28.41
70.03
69.69
82.31
81.28
25
13
38
20
25
30.98
51.86
35.52
69.65
40.01
47.12
73.79
61.33
96.20
59.38
67.80
78.05
58.46
30.54
76.75
54.41
106.39
89.70
87.50
93.52
83.78
53.95
104.78
71.15
−17.36
64.04
39.53
20.86
−10.38
27.61
72.19
9.18
39.15
−20.88
18.58
43.54
11.48
14.83
36.33
19.76
96.45
−6.52
50.87
65.58
65.77
30.60
84.53
45.26
25
−35
20
A498
ACHN
CAKI‐1
10
RXF 393
SN12C
−15
12
TK‐10
12
UO‐31
0
Prostate cancer
PC‐3
57.10
77.70
79.70
97.70
25.44
45.34
24.46
49.53
51.54
80.74
20
30
DU‐145
Breast cancer
MCF7
51.21
71.02
42.71
79.90
55.32
86.40
59.53
82.13
96.91
52.64
90.40
88.82
111.19
84.46
23.78
−1.25
−16.93
36.09
19.45
59.28
19.99
9.52
42.69
7.33
31.74
65.87
25.79
74.18
29.00
33.34
49.57
25
15
0
MDA‐MB‐231/ATCC
HS 578T
BT‐549
61.89
5.99
40
25
27
13.7
T‐47D
MDA‐MB‐468
Meanc
31.72
28.71
Note: Data are presented as growth percent; 100 is no change (as for the control), 0 is no growth (same number of cells), and below 0 is lethality
(reduction in number of cells).
aValues extracted from the NCI60 database and included for comparison.
bBold values indicate better‐observed effect than with cabozantinib.
cMean growth observed.
dNCI database #: 807002 (14a), 806999 (15a), 807003 (15b), 807000 (18b), 807001 (18d).
was more negatively affected than the antiproliferative properties by
ortho‐fluorinations performed on the terminal benzene ring. A similar
kinase selectivity profile as for cabozantinib was observed for 15b,
18b, and 18d. In conclusion, new SAR knowledge for the 6‐position of
the quinoline ring has been obtained, indicating that such modifica-
tions are generally well tolerated. Further evaluation of 15b and 18b
as new anticancer agents are warranted.
reactions were performed under argon atmosphere with dried
solvents and reagents. Thin‐layer chromatography was performed
on Merck silica gel 60 F254 plates, and visualized using UV light at
312 or 365 nm, a phosphomolybdic acid solution (12 g phosphomo-
lybdic acid in 250 ml EtOH) or a potassium permanganate (1.5 g
KMnO4, 10 g K2CO3, 2.5 ml 5 M NaOH/H2O, 200 ml H2O) solution
for detection. Column chromatography was performed with silica gel
(pore size 60 Å, 230–400 mesh particle size) purchased from Fluka.
1H and 13C NMR spectra were obtained on a Bruker AVIII HD 400
instrument (400/101 MHz). Chemical shifts (δ) are reported in parts
per million, and coupling constants are reported in Hertz (Hz). The
residual proton solvent resonance in 1H NMR (CDCl3 at δ 7.27,
DMSO‐d6 at δ 2.50) and the residual carbon solvent resonance in
13C NMR (CDCl3 at δ 77.16 ppm and DMSO‐d6 at δ 39.52) are used
as reference (please see the Supporting Information for the original
spectra). Accurate mass determination (HRMS) in positive or
|
4
EXPERIMENTAL
Chemistry
|
4.1
|
4.1.1
General
All chemicals were purchased from Sigma‐Aldrich or Fluorochem and
used without further purification. Air and/or moisture sensitive

8 of 15
LIEN ET AL.
|
TABLE 3 GI50, TGI, and LC50 values for the compounds 15b and 18b in the NCI60 panel
15b
GI50
18b
Cabozantinib
Cell line
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
Leukemia
K‐562
1.10
0.63
4.90
>100
>100
0.89
3.24
>100
>100
>100
>100
0.25
2.00
3.16
>100
>100
SR
>100
25.12
CNS cancer
SF‐539
0.25
0.59
2.04
>100
>100
0.85
2.82
4.79
56.23
>100
1.58
3.98
12.59
>100
>100
>100
U251
>100
16.60
Melanoma
LOX IMVI
1.91
2.40
5.37
>100
>100
1.74
2.51
3.47
6.46
6.92
2.51
7.94
15.85
25.12
>100
>100
SK‐MEL‐2
14.45
45.71
Breast cancer
MDA‐MB‐231/ATCC
HS 578T
2.00
0.29
1.91
6.46
2.09
38.02
60.26
>100
91.20
2.45
1.66
2.09
8.51
7.41
23.99
>100
>100
87.10
3.16
0.79
2.58
39.81
10.00
35.40
>100
>100
97.80
Mean (all 60 cell lines)
Note: Values are in µM.
aGI50, TGI, and LC50; concentrations where 50% growth inhibition, total growth inhibition, and 50% cell lethality are observed.
negative mode was performed on a Waters Prospec Q instrument,
ionized by electrospray (ESI). Liquid chromatography‐mass spectro-
metry was performed on a Thermo Finnigan LCQ Deca XP Plus using
a gradient from 10 to 90% acetonitrile in water over 10 min and
preparative high‐performance liquid chromatography (HPLC) was
performed on a Waters Delta Prep 4000, using a gradient from 20 to
80% acetonitrile in water, collecting fractions of 10 ml/min. Chemical
purity was >95% for the biologically tested structures.
stirred at ambient temperature for 20 hr. The reaction mixture was
quenched with NaOH (30 ml, 1 M) and diluted with EtOAc (10 ml).
The phases were separated, and the organic phase was extracted
with NaOH (2 × 10 ml, 1 M). The combined basic extracts were then
acidified to pH 1–2 with HCl (1 M), and the title compound was
achieved by suction filtration as a white solid (0.41 g, 41%). 1H NMR
(400 MHz, DMSO‐d6): δ 10.71 (s, 1H), 7.60–7.57 (m, 2H), 7.15–7.10
(m, 2H), 1.39 (s, 4H).[24]
The InChI codes of the investigated compounds together with
some biological activity data are provided as Supporting Information.
|
4.1.3
Synthesis of 1‐((2,4,6‐trifluorophenyl)‐
carbamoyl)cyclopropanecarboxylic acid (6b)
|
4.1.2
Synthesis of 1‐((4‐fluorophenyl)carbamoyl)‐
The title compound was achieved in a similar manner as 6a using
cyclopropane‐1,1‐dicarboxylic acid (363 mg, 2.79 mmol) and 2,4,6‐
trifluoroaniline (476 mg, 3.24 mmol) and obtained as a white solid
(0.217 g, 30%). 1H NMR (400 MHz, DMSO‐d6): δ 10.25 (s, 1H),
7.22–7.17 (m, 2H), 1.50–1.46 (m, 2H), 1.44–1.40 (m, 2H). 13C NMR
(101 MHz, CDCl3): δ 173.64, 167.83, 159.09, 156.60, 111.52, 100.77,
27.15, 18.22. HRMS (ESI‒) m/z calcd. for C11H7F3NO3 [M‒H]−:
258.0384, found 258.0382.
cyclopropanecarboxylic acid (6a)
Triethylamine (0.68 ml, 4.88 mmol) was added via syringe to a
solution of cyclopropane‐1,1‐dicarboxylic acid (579 mg, 4.45 mmol)
in THF (10 ml) at 0°C. The solution was stirred for 15 min at 0°C
before SOCl2 (0.33 ml, 4.54 mmol) was added via syringe. After
another 15 min of stirring, 4‐fluoroaniline (0.574 mg, 5.17 mmol) in
THF (5 ml) was added via cannula at 0°C, and the solution was then
TABLE 4 Percent inhibition of the kinases at 1 µM of 15b, 18b, and 18d
Percent inhibition at 1 µM
Compound
15b
RET
97
ALK
39
ROS
90
c‐Met
95
VEGFR2
97
EGFR
22
c‐Kit
61
18b
99
80
97
99
100
98
28
75
18d
92
28
96
97
18
71
Cabozantinib, 1
99
38
91
99
93
19
94
Note: Cabozantinib was included as a reference.

LIEN ET AL.
9 of 15
|
FIGURE 3 (a) Overlaid structures of cabozantinib (blue), 15b (white), and 18b (pink) in the active site of c‐Met. (b) Main polar interactions
with the enzyme active site exemplified with 18b. Some protein residues are removed for clarity
|
|
4.1.4
Synthesis of 1‐(3‐(benzyloxy)‐4‐
4.1.6
Synthesis of 1‐(2‐amino‐5‐(benzyloxy)‐4‐
methoxyphenyl)ethanone (7)
methoxyphenyl)ethanone (9)
Benzyl bromide (10.7 ml, 90.1 mmol) was added to a stirred solution
of 3‐hydroxy‐4‐methoxy acetophenone, 6 (13.5 g, 81.4 mmol) and
K2CO3 (18.6 g, 134.6 mmol) in DMF (100 ml). The solution was
stirred at 40°C for 18 hr, and then diluted with EtOAc (80 ml) and
water (80 ml). The crude mixture was extracted with EtOAc
(2 × 50 ml), washed with water (4 × 50 ml) and brine (50 ml), dried
over MgSO4, and concentrated on a rotary evaporator. The title
compound was achieved as a white solid (18.9 g, 91%) and used
without further purification. 1H NMR (400 MHz, CDCl3): δ 7.60–7.56
(m, 2H), 7.46 (d, 2H, J = 7.2 Hz), 7.38 (t, 2H, J = 7.6 Hz), 7.3 (t, 1H,
J = 7.3 Hz), 6.91 (d, 1H, J = 9.2 Hz), 5.19 (s, 2H), 3.94 (s, 3H), 2.52
(s, 3H). 13C NMR (101 MHz, CDCl3) δ 196.82, 154.05, 148.16,
136.69, 130.48, 128.73, 128.17, 127.66, 123.66, 112.85, 110.54,
71.08, 56.22, 26.34.[27]
Iron (17 g, 304.4 mmol), NH4Cl (19 g, 312.5 mmol), and 8 (20.4 g,
67.6 mmol) were weighed out in a round‐bottom flask and water
(150 ml) and EtOH (200 ml) were added. The reaction mixture was
stirred for 3 hr at 70°C and then cooled and filtered through Celite,
which was then washed with EtOAc (150 ml). The filtrate was then
washed with water (150 ml) and brine (100 ml), dried over MgSO4,
and concentrated on a rotary evaporator. The crude mixture was
purified by column chromatography (heptane/EtOAc, 2:1). The title
compound was achieved as an off‐white solid (16.4 g, 89%). 1H NMR
(400 MHz, CDCl3): δ 7.47–7.43 (m, 5H), 7,16 (s, 1H), 6.17 (s, 1H), 5.07
(s, 2H), 3.90 (s, 3H), 2.41 (s, 3H). 13C NMR (101 MHz, CDCl3):
δ 198.66, 156.57, 147.53, 138.97, 137.40, 128.67, 128.13, 127.87,
119.19, 111.08, 99.65, 73.12, 55.95, 27.81. HRMS (ESI+) m/z calcd.
for C16H17NNaO3 [M+Na]+: 294.1101, found 294.1102.
|
|
4.1.5
Synthesis of 1‐(5‐(benzyloxy)‐4‐methoxy‐2‐
4.1.7
Synthesis of 6‐(benzyloxy)‐7‐
nitrophenyl)ethanone (8)
methoxyquinolin‐4‐ol (10)
7 (18.9 g, 73.8 mmol) was dissolved in DCM (250 ml) and cooled to
0°C on an ice bath. HNO3 (10 ml, 223.8 mmol) was slowly added over
10 min, before H2SO4 (8 ml, 150.1 mmol) was added over 10 min. The
solution was then stirred at ambient temperature for 15 min before it
was washed with water (100 ml) and saturated NaHCO3 solution
(100 ml) until neutral. The organic phase was dried over MgSO4 and
concentrated on a rotary evaporator. The title compound was
achieved as a light yellow solid (20.4 g, 92%). 1H NMR (400 MHz,
CDCl3): δ 7.62 (s, 1H), 7.47–7.34 (m, 5H), 6.81 (s, 1H), 5.22 (s, 2H),
3.98 (s, 3H), 2.45 (s, 3H). 13C NMR (100 MHz, CDCl3): δ 199.0, 153.2,
150.3, 138.9, 135.2, 132.6, 129.0, 128.7, 127.6, 110.7, 107.3, 71.7,
56.7, 30.4. HRMS (ESI+) m/z calcd. for C16H15NNaO5 [M+Na]+:
324.0842, found 324.0843.
9 (16.4 g, 60.3 mmol) and sodium ethoxide (17 g, 250 mmol) were
weighed out in a round‐bottom flask, put under argon, dissolved in
DME (200 ml) and stirred for 30 min. Ethyl formate was added via
syringe, and the mixture was stirred for 24 hr at room temperature.
The solution was then made neutral using 1 M HCl, and the solids
formed were filtered off and washed with water (100 ml). The filtrate
was extracted with EtOAc (2 × 80 ml), washed with water (100 ml)
and brine (100 ml), dried over MgSO4, and concentrated on a rotary
evaporator. The crude product was purified by column chromato-
graphy (heptane/EtOAc, 1:1, heptane/EtOAc/MeOH, 5:5:2), and the
product was achieved as a light brown solid (16.9 g, 99%). 1H NMR
(400 MHz, methanol‐d4): δ 7.90 (d, 1H, J = 7.2 Hz), 7.70 (s, 1H), 7.49
(d, 2H, J = 7.6 Hz), 7.38 (t, 2H, J = 7.6 Hz), 7.32 (d, 1H, J = 7.2 Hz), 7.04

10 of 15
LIEN ET AL.
|
(s, 1H), 6.33 (d, 1H, J = 6.8 Hz), 5.18 (s, 2H), 3.97 (s, 3H). 13C NMR
(100 MHz, methanol‐d4): δ 175.2, 157.2, 149.6, 140.9, 138.2, 137.7,
129.6, 129.2, 128.9, 119.1, 107.4, 105.7, 100.0, 72.0, 56.9. HRMS
(ESI+) m/z calcd. for C17H15NNaO3 [M+Na]+: 304.0944, found
304.0944.
4.99 mmol) was added. Aniline 12 (0.814 g, 2.19 mmol) dissolved in
DMF (40 ml) was added after 10 min. The mixture was stirred for
20 hr at ambient temperature, and then diluted with EtOAc (60 ml),
washed with water (4 × 30 ml) and brine (2 × 30 ml), dried over
MgSO4, and concentrated on a rotary evaporator. The solid was
further purified by column chromatography (heptane/EtOAc, 1:1,
heptane/EtOAc/MeOH, 5:5:2), and the title compound was achieved
as a white solid (0.767 g, 61%). 1H NMR (400 MHz, DMSO‐d6):
δ 10.17 (s, 1H), 10.04 (s, 1H), 8.49 (d, 1H, J = 5.2 Hz), 7.76 (d, 2H,
J = 8.8 Hz), 7.66 (s, 1H), 7.65–7.62 (m, 2H), 7.52, d, 2H, J = 6.8 Hz),
7.42 (s, 1H), 7.42 (t, 2H, J = 7.6 Hz), 7.38–7.33 (m, 1H), 7.22, (d, 2H,
J = 9.2 Hz), 7.15 (t, 2H, J = 8.8 Hz), 6.45 (d, 1H, J = 5.6 Hz), 5.26 (s, 2H),
3.96 (s, 3H), 1.48 (s, 4H). 13C NMR (101 MHz, DMSO‐d6) δ 168.2,
168.1, 160.1, 157.1, 152.8, 149.5, 148.8, 148.4, 136.5, 136.6, 135.1,
128.4, 128.0, 122.4, 122.4, 122.2, 121.1, 115.1, 115.1, 114.9, 107.8,
103.2, 100.6, 70.0, 55.8, 31.5, 15.9. HRMS (ESI+) m/z calcd. for
C34H29FN3O5 [M+H]+: 578.2086, found 578.2086.
|
4.1.8
Synthesis of 6‐(benzyloxy)‐7‐methoxy‐4‐(4‐
nitrophenoxy)quinoline (11)
10 (16.9 g, 60.2 mmol) and Cs2CO3 (39.1 g, 120 mmol) were weighed
out in
a round‐bottom flask, dissolved in DMF (200 ml) and
acetonitrile (150 ml), and stirred for 20 min. 1‐Fluoro‐4‐nitrobenzene
(21.67 g, 153.6 mmol) was then added over 5 min via syringe. The
mixture was stirred at 55°C for 24 hr, then diluted with EtOAc
(200 ml) and washed with water (4 × 150 ml) and brine (100 ml), dried
over MgSO4, and concentrated on a rotary evaporator. The crude
mixture was purified by column chromatography (heptane/EtOAc,
4:1, heptane/EtOAc/MeOH, 5:5:2), and the title compound was
achieved as an yellow solid (5 g, 21%). 1H NMR (400 MHz, CDCl3): δ
8.62 (d, 1H, J = 4.8 Hz), 8.28 (d, 2H, J = 9.2 Hz), 7.55 (s, 1H), 7.42 (d,
2H, J = 5.6 Hz), 7.37 (s, 1H), 7.35–7.28 (m, 3H), 7.18 (d, 2H, J = 9.2 Hz),
6.72 (d, 1H, J = 5.6 Hz), 5.25 (s, 2H), 4.07 (s, 3H). 13C NMR (101 MHz,
CDCl3): δ 160.6, 158.7, 154.4, 149.7, 148.0, 144.5, 136.0, 128.8,
128.3, 127.5, 126.4, 119.6, 116.7, 107.6, 106.5, 101.2, 71.1, 56.5.
HRMS (ESI+) m/z calcd. for C23H19N2O5 [M+H]+: 403.1288, found
403.1287.
|
4.1.11
Synthesis of N‐(4‐((6‐(benzyloxy)‐7‐
methoxyquinolin‐4‐yl)oxy)phenyl)‐N‐(2,4,6‐
trifluorophenyl)cyclopropane‐1,1‐dicarboxamide
(13b)
6b (0.616 g, 2.38 mmol), HATU (1.8 g, 4.73 mmol), and DMF (40 ml)
were placed in a round‐bottom flask, and then DIPEA (0.87 ml,
4.99 mmol) was added. Aniline 12 (0.811 g, 2.18 mmol) dissolved in
DMF (40 ml) was added after 10 min. The mixture was stirred for
20 hr at room temperature. The mixture was then diluted with EtOAc
(60 ml), washed with water (4 × 30 ml) and brine (2 × 30 ml), dried
over MgSO4, and concentrated on a rotary evaporator. The solid was
further purified by column chromatography (heptane/EtOAc, 1:1,
heptane/EtOAc/MeOH, 5:5:2). The title compound was achieved as a
white solid (0.827 g, 62%). 1H NMR (400 MHz, DMSO‐d6): δ 10.39 (s,
1H), 9.64 (s, 1H), 8.48 (d, 1H, J = 5.2 Hz), 7.78 (d, 2H, J = 8.8 Hz), 7.66
(s, 1H), 7.51 (d, 2H, J = 6.8 Hz), 7.42 (s, 1H), 7.42 (t, 2H, J = 7.6 Hz),
7.37–7.33 (m, 1H), 7.28 (t, 2H, J = 8.4 Hz), 7.22 (d, 2H, J = 9.0 Hz),
6.44 (d, 1H, J = 5.2 Hz), 5.26 (s, 2H), 3.95 (s, 3H), 1.59–1.50 (m, 4H).
13C NMR (101 MHz, DMSO‐d6) δ 169.9, 167.7, 159.9, 156.9, 152.7,
149.6, 148.9, 148.3, 146.5, 136.5, 136.1, 128.4, 128.0, 122.0, 121.1,
115.1, 111.3, 108.0, 103.1, 101.1, 100.8, 100.6, 100.5, 70.0, 55.8,
30.3, 16.2. HRMS (ESI+) m/z calcd. for C34H27F3N3O5 [M+H]+:
614.1897, found 614.1896.
|
4.1.9
Synthesis of 4‐((6‐(benzyloxy)‐7‐
methoxyquinolin‐4‐yl)oxy)aniline (12)
Iron (6.6 g, 118.2 mmol), NH4Cl (6.5 g, 107.7 mmol), and 11 (5 g,
12.5 mmol) were weighed out in a round‐bottom flask, and water
(80 ml) and EtOH (100 ml) were added. The reaction mixture was
stirred for 3 hr at 70°C, and then cooled and filtered through Celite,
which was then washed with EtOAc (150 ml). The organic filtrate was
then washed with water (150 ml) and brine (100 ml), dried over
MgSO4, and concentrated on
a rotary evaporator. The title
compound was achieved as a light brown solid (3.89 g, 84%), and
used without any further purification. 1H NMR (400 MHz, CDCl3): δ
8.45 (d, 1H, J = 5.2 Hz), 7.67 (s, 1H), 7.52 (d, 2H, J = 7.2), 7.43 (s, 1H),
7.40 (t, 2H, J = 7.6 Hz), 7.35–7.31 (m, 1H), 6.97 (d, 2H, J = 8.4 Hz),
6.75 (d, 2H, J = 8.8 Hz), 6.42 (d, 1H, J = 5.2 Hz), 5.29 (s, 2H), 4.04
(s, 3H), 3.71 (bs, 2 H). 13C NMR (101 MHz, CDCl3) δ 161.9, 153.5,
148.8, 148.7, 146.6, 146.2, 144.3, 136.5, 128.7, 128.2, 127.8, 122.3,
116.4, 116.0, 107.8, 102.8, 101.6, 71.0, 56.3. HRMS (ESI+) m/z calcd.
for C23H21N2O3 [M+H]+: 373.1547, found 373.1546.
|
4.1.12
Synthesis of N‐(4‐fluorophenyl)‐N‐(4‐((6‐
hydroxy‐7‐methoxyquinolin‐4‐yl)oxy)phenyl)‐
cyclopropane‐1,1‐dicarboxamide (14a)
13a (0.174 g, 0.301 mmol) was placed under argon in a round‐bottom
flask and dissolved in dry ethanol (6 ml). Pd/C (50% water content,
10% loading, 67 mg, 0.0315 mmol Pd) was added under an argon
atmosphere, before 1,4‐cyclohexadiene (0.28 ml, 3.01 mmol) was
added via syringe. The reaction mixture was heated for 6 hr at 80°C
and then filtered through Celite, which was then washed with EtOAc
|
4.1.10
Synthesis of N‐(4‐((6‐(benzyloxy)‐7‐
methoxyquinolin‐4‐yl)oxy)phenyl)‐N‐(4‐fluorophenyl)‐
cyclopropane‐1,1‐dicarboxamide (13a)
6a (0.560 g, 2.51 mmol), HATU (1.28, 3.37 mmol), and DMF (40 ml)
were placed in a round‐bottom flask, and then DIPEA (0.87 ml,

LIEN ET AL.
11 of 15
|
(20 ml). The organic filtrate was washed with water (10 ml) and brine
(10 ml), dried over MgSO4, and concentrated on a rotary evaporator.
The crude was further purified by column chromatography (heptane/
EtOAc, 1:1, heptane/EtOAc/MeOH, 5:5:2), and the title compound
was achieved as a white solid (0.114 g, 78%). 1H NMR (400 MHz,
DMSO‐d6): δ 11.08 (s, 1H), 10.32 (s, 1H), 10.05 (s, 1H), 8.72 (d, 1H,
J = 6 Hz), 7.84 (d, 2H, J = 8 Hz), 7.71 (s, 1H), 7.67–7.61 (m, 2H), 7.62
(s, 1H), 7.34 (d, 2H, J = 8.4 Hz), 7.15 (t, 2H, J = 8 Hz), 6.74 (d, 1H,
J = 6 Hz), 4.03 (s, 3H), 1.49 (s, 4H). 13C NMR (101 MHz, DMSO‐d6)
δ 168.24, 168.16, 164.87, 155.79, 149.68, 148.02, 142.41, 137.66,
135.19, 122.48, 122.41, 122.30, 121.30, 115.75, 115.16, 114.94,
103.27, 102.79, 100.32, 56.43, 31.72, 15.41. HRMS (ESI+) m/z calcd.
for C27H23FN3O5 [M+H]+: 488.1616, found 488.1615.
(d, 1H, J = 5.6 Hz), 8.42 (d, 2H, J = 8.8 Hz), 8.37 (d, 2H, J = 8.8 Hz), 8.15
(s, 1H), 7.63 (d, 2H, J = 8.8 Hz), 7.62 (s, 1H), 7.48–7.44 (m, 2H), 7.15
(d, 2H, J = 9.2 Hz), 7.04 (t, 2H, J = 8.8 Hz), 6.50 (d, 1H, J = 5.2 Hz), 3.98
(s, 3H), 1.74–1.62 (m, 4H). 13C NMR (101 MHz, CDCl3) δ 169.41,
168.87, 163.19, 162.11, 159.95 (d, J = 245 Hz), 153.94, 151.28,
151.16, 150.54, 149.45, 140.35, 135.31, 134.58, 133.19 (d, J = 3 Hz),
131.63, 123.90, 122.90, (d, J = 8 Hz), 122.64, 121.75, 116.03, 115.81,
114.88, 108.87, 103.27, 56.42, 29.34, 17.72. HRMS (ESI+) m/z calcd.
for C34H25FN4O8 [M+H]+: 637.1729, found 637.1726.
|
4.1.15
Synthesis of 4‐(4‐(1‐((4‐fluorophenyl)‐
carbamoyl)cyclopropane‐1‐carboxamido)phenoxy)‐
7‐methoxyquinolin‐6‐yl 4‐aminobenzoate (15b)
Iron (21 mg, 0.286 mmol), NH4Cl (26 mg, 0.486 mmol), and 15a
(21 mg, 0.033 mmol) were weighed out in a round‐bottom flask, and
water (1 ml) and EtOH (2 ml) were added. The reaction mixture was
stirred for 3 hr at 70°C, and then cooled and filtered through Celite,
which was then washed with EtOAc (10 ml). The organic filtrate was
then washed with water (10 ml) and brine (10 ml), dried over MgSO4,
and concentrated on a rotary evaporator. The crude was further
purified by column chromatography (heptane/EtOAc, 1:1, heptane/
EtOAc/MeOH, 5:5:2), and the title compound was achieved as a
white solid (12 mg, 60%). 1H NMR (400 MHz, CDCl3): δ 9.29 (s, 1H),
9.02 (s, 1H), 8.56 (d, 1H, J = 5.6 Hz), 8.09 (s, 1H), 8.04 (d, 2H,
J = 8.8 Hz), 7.59 (d, 2H, J = 9.2 Hz), 7.55 (s, 1H), 7.49–7.45 (m, 2H),
7.12 (d, 2H, J = 9.2 Hz), 7.03 (t, 2H, J = 8.8 Hz), 6.71 (d, 2H, J = 8.8 Hz),
6.45 (d, 1H, J = 5.2 Hz), 4.20 (bs, 2H) 3.96 (s, 3H), 1.72–1.64 (m, 4H).
13C NMR (101 MHz, CDCl3) δ 169.19, 169.13, 165.13, 162.06,
159.85 (d, J = 245 Hz), 158.63, 154.78, 151.80, 150.64, 141.22,
135.11, 133.32 (d, J = 3 Hz), 132.76, 122.80 (d, J = 7 Hz), 122.67,
121.73, 118.21, 115.99, 115.83, 115.76, 115.02, 114.03, 108.40,
103.05, 56.35, 29.24, 17.80. HRMS (ESI+) m/z calcd. for
C34H27FN4O6 [M+H]+: 607.1987, found 607.1984.
|
4.1.13
Synthesis of N‐(4‐((6‐hydroxy‐7‐
methoxyquinolin‐4‐yl)oxy)phenyl)‐N‐(2,4,6‐
trifluorophenyl)cyclopropane‐1,1‐dicarboxamide
(14b)
13b (0.186 g, 0.303 mmol) was placed under argon in a round‐bottom
flask and dissolved in dry ethanol (15 ml). Pd/C (50% water content,
10% loading, 60 mg, 0.028 mmol Pd) was added under an argon
atmosphere, before 1,4‐cyclohexadiene (0.28 ml, 3.01 mmol) was
added via syringe. The mixture was heated for 6 hr at 80°C and then
filtered through Celite, which was then washed with EtOAc (30 ml).
The organic filtrate was washed with water (15 ml) and brine (15 ml),
dried over MgSO4, and concentrated on a rotary evaporator. The
crude was further purified by column chromatography (heptane/
EtOAc, 1:1, heptane/EtOAc/MeOH, 5:5:2), and the title compound
was achieved as a white solid (0.125 g, 79%). 1H NMR (400 MHz,
DMSO‐d6): δ 10.36 (s, 1H), 9.94 (s, 1H), 9.64 (s, 1H), 8.43 (d, 1H,
J = 5.2 Hz), 7.74 (d, 2H, J = 8.8 Hz), 7.45 (s, 1H), 7.37 (s, 1H), 7.28
(t, 2H, J = 8.4 Hz), 7.19 (d, 2H, J = 8.8 Hz), 6.42 (d, 1H, J = 5.2 Hz), 3.95
(s, 3H), 1.58–1.49 (m, 4H). 13C NMR (101 MHz, DMSO‐d6) δ 169.8,
167.6, 159.5, 159.3, 152.3, 149.9, 147.9, 147.4, 145.9, 135.9, 122.1,
120.7, 115.8, 107.8, 103.2, 102.4, 101.1, 100.8, 100.5, 55.6, 30.3,
16.2. HRMS (ESI+) m/z calcd. for C27H21F3N3O5 [M+H]+: 524.1428,
found 524.1428.
|
4.1.16
Synthesis of 4‐(4‐(1‐((4‐fluorophenyl)‐
carbamoyl)cyclopropane‐1‐carboxamido)phenoxy)‐
7‐methoxyquinolin‐6‐yl 4‐(trifluoromethyl)benzoate
(15c)
|
4.1.14
Synthesis of 4‐(4‐(1‐((4‐fluorophenyl)‐
14a (36 mg, 0.0738 mmol), HATU (50 mg, 0.131 mmol), DMAP
(10 mg, 0.0819 mmol), and 4‐(trifluoromethyl)benzoic acid (36 mg,
0.189 mmol) were weighed out in a round‐bottom flask and dissolved
in THF (2 ml) and DMA (1 ml), and the reaction mixture was stirred
overnight at ambient temperature. The mixture was then diluted with
water (5 ml) and EtOAc (5 ml), extracted with EtOAc (2 × 5 ml),
washed with water (4 × 5 ml) and brine (5 ml), dried over MgSO4, and
concentrated on a rotary evaporator. The crude was further purified
by column chromatography (heptane/EtOAc, 1:1, heptane/EtOAc/
MeOH, 5:5:2), and the title compound was achieved as a white solid
(26 mg, 53%). 1H NMR (400 MHz, DMSO‐d6): δ 10.19 (s, 1H), 10.05
(s, 1H), 8.65 (d, 1H, J = 5.2 Hz), 8.37 (d, 2H, J = 8 Hz), 8.29 (s, 1H), 8.01
(d, 2H, J = 8.4 Hz), 7.76 (d, 2H, J = 8.8 Hz), 7.63 (s, 1H), 7.66–7.62
carbamoyl)cyclopropane‐1‐carboxamido)phenoxy)‐
7‐methoxyquinolin‐6‐yl 4‐nitrobenzoate (15a)
14a (51 mg, 0.105 mmol) and Cs2CO3 (85 mg, 0.261 mmol) were
weighed out in a round‐bottom flask, dissolved in DMF (3 ml) and
stirred for 10 min. 4‐Nitrobenzoyl chloride was then added, and the
resulting mixture was stirred for 5 hr at ambient temperature. The
solution was diluted with EtOAc (10 ml) and water (10 ml), extracted
with EtOAc (2 × 10 ml), washed with NaOH (3 × 10 ml, 1 M), water
(4 × 10 ml), and brine (10 ml), and then dried over MgSO4 and
concentrated on a rotary evaporator. No further purification was
needed, and the title compound was achieved as a white solid (25 mg,
37%). 1H NMR (400 MHz, CDCl3): δ 9.48 (s, 1H), 8.76 (s, 1H), 8.63

12 of 15
LIEN ET AL.
|
(m, 2H), 7.25 (d, 2H, J = 8.8 Hz), 7.15 (t, 2H, J = 9.2 Hz), 6.51 (d, 1H,
J = 5.2 Hz), 3.95 (s, 3H), 1.47 (s, 4H). 13C NMR (101 MHz, DMSO‐d6):
δ 168.12 (merged C=O), 163.20, 160.94, 158.26 (d, J = 241 Hz),
153.30, 151.80, 149.29, 149.13, 139.79, 136.60, 135.16 (d, J = 2 Hz),
132.25, 130.80, 126.09, 126.05, 122.45, 122.37, 122.21, 121.08,
115.12, 114.90, 114.60 (d, J = 20 Hz), 109.14, 103.03, 56.38, 31.55,
15.38. HRMS (ESI+) m/z calcd. for C35H26F4N3O6 [M+H]+: 660.1752,
found 660.1750.
(m, 2H). 13C NMR (CDCl3, 101 MHz): δ 169.53, 168.86, 161.86,
161.18, 158.75, 153.90, 150.49, 147.75, 135.46, 133.18, 128.86,
127.80, 123.00, 122.92, 122.64, 121.77, 116.04, 115.82, 104.18,
67.18 (d, J = 35 Hz), 56.47, 31.06, 29.30, 17.86. HRMS (ESI+) m/z
calcd. for C29H24F4N3O5 [M+H]+: 570.1647, found 570.1646.
|
4.1.19
Synthesis of N‐(4‐((7‐methoxy‐6‐(2,2,2‐
trifluoroethoxy)quinolin‐4‐yl)oxy)phenyl)‐N‐(2,4,6‐
trifluorophenyl)cyclopropane‐1,1‐dicarboxamide
(17b)
|
4.1.17
Synthesis of N‐(4‐((6‐(difluoro(phenyl)‐
methoxy)‐7‐methoxyquinolin‐4‐yl)oxy)phenyl)‐N‐
(4‐fluorophenyl)cyclopropane‐1,1‐dicarboxamide (16)
14b (75 mg, 0.144 mmol) and Cs2CO3 (94 mg, 0.289 mmol) were
weighed out in a round‐bottom flask, dissolved in DMF (1 ml), and
stirred for 10 min. 1,1,1‐Trifluoro‐2‐iodoethane (0.03 ml, 0.304 mmol)
in DMF (1 ml) was then added via syringe, and the mixture was stirred
for 5 hr at 110°C. The mixture was then diluted with water (5 ml) and
EtOAc (5 ml) and the phases were separated. The aqueous phase was
extracted with EtOAc (2 × 5 ml), and the combined organic phases
were washed with water (4 × 5 ml) and brine (5 ml), dried over MgSO4
and concentrated on a rotary evaporator. The crude product was
further purified by column chromatography (heptane/EtOAc, 1:1,
heptane/EtOAc/MeOH, 5:5:2), and the title compound was achieved
as a white solid (4 mg, 5%). 1H NMR (600 MHz, CDCl3): δ 9.87 (s, 1H),
8.51 (d, 1H, J = 5.3 Hz), 7.77 (s, 1H), 7.66 (d, 2H, J = 8.9 Hz), 7.56 (s, 1H),
7.15 (d, 2H, J = 8.9 Hz), 6.78 (t, 2H, J = 7.5 Hz), 6.49 (d, 1H, J = 5.5 Hz),
4.55 (t, 2H, J = 8.1 Hz), 4.05 (s, 3H), 1.89–1.86 (m, 2H), 1.71–1.68
(m, 2H). 13C NMR (151 MHz, CDCl3): δ 171.36, 168.01, 162.24,
160.51, 159.33, 157.55, 149.75, 148.36, 136.23, 127.76, 124.17,
122.88, 122.59, 122.11, 121.72, 121.28, 115.62, 109.86, 107.29,
103.89, 103.06, 100.96, 66.94, 56.88, 28.52, 19.07. HRMS (ESI+) m/z
calcd. for C29H22F6N3O5 [M+H]+: 606.1458, found 606.1457.
14a (50 mg, 0.103 mmol) and Cs2CO3 (50 mg, 0.150 mmol) were
weighed out in a round‐bottom flask, dissolved in DMF (2 ml) and
stirred for 10 min. Chloro difluoromethylbenzene (0.02 ml,
0.152 mmol) was then added via syringe, and the mixture was stirred
for 20 hr at 100°C. The mixture was then diluted with water (5 ml)
and EtOAc (5 ml), extracted with EtOAc (2 × 5 ml), washed with
water (4 × 5 ml) and brine (5 ml), dried over MgSO4, and concentrated
on a rotary evaporator. The crude was further purified by column
chromatography (heptane/EtOAc, 1:1, heptane/EtOAc/MeOH, 5:5:2),
and the title compound was achieved as a white solid (4 mg, 6%). 1H
NMR (600 MHz, CDCl3): δ 9.51 (s, 1H), 8.65 (s, 1H), 8.57 (d, 1H,
J = 5.6 Hz), 8.3 (s, 1H), 7.83 (d, 2H, J = 6.8 Hz), 7.67 (d, 3H, J = 8.8 Hz),
7.55–7.45 (m, 5H), 7.19 (d, 2H, J = 8.8 Hz), 7.06 (t, 2H, J = 8.8 Hz),
6.52 (d, 1H, J = 5.6 Hz), 4.05 (s, 3H), 1.87–1.65 (m, 4H). 13C NMR
(151 MHz, CDCl3): δ 170.10, 168.69, 167.56, 160.89, 159.27, 158.94,
148.34, 143.27, 142.41, 137.34, 132.93, 131.57, 128.79, 125.82,
123.27, 123.22, 122.87, 121.67, 116.06, 115.91, 115.15, 114.96,
102.44, 101.90, 57.56, 29.83, 18.33. HRMS (ESI+) m/z calcd. for
C34H27F3N3O5 [M+H]+: 614.1897, found 614.1896.
|
4.1.20
Synthesis of N‐(4‐fluorophenyl)‐N‐(4‐((7‐
|
4.1.18
Synthesis of N‐(4‐fluorophenyl)‐N‐(4‐((7‐
methoxy‐6‐((5‐nitropyridin‐2‐yl)oxy)quinolin‐4‐yl)oxy)‐
phenyl)cyclopropane‐1,1‐dicarboxamide (18a)
methoxy‐6‐(2,2,2‐trifluoroethoxy)quinolin‐4‐yl)oxy)‐
phenyl)cyclopropane‐1,1‐dicarboxamide (17a)
2‐Chloro‐5‐nitropyridine (35 mg, 0.221 mmol) was added to
a
14a (50 mg, 0.103 mmol) and Cs2CO3 (69 mg, 0.212 mmol) were
weighed out in a round‐bottom flask, dissolved in DMF (1 ml),
and stirred for 10 min. 1,1,1‐Trifluoro‐2‐iodoethane (82.5 mg,
0.393 mmol) in DMF (1 ml) was then added via syringe, and the
mixture was stirred for 5 hr at 110°C. The mixture was then diluted
with water (5 ml) and EtOAc (5 ml) and the phases were separated.
The aqueous phase was extracted with EtOAc (2 × 5 ml), and the
combined organic phases were washed with water (4 × 5 ml) and
brine (5 ml), dried over MgSO4 and concentrated on a rotary
evaporator. The crude product was further purified by column
chromatography (heptane/EtOAc, 1:1, heptane/EtOAc/MeOH, 5:5:2),
and the title compound was achieved as a white solid (19 mg, 33%).
1H NMR (CDCl3, 400 MHz): δ 9.58 (s, 1H), 8.76 (s, 1H), 8.51 (s, 1H),
7.67 (s, 1H), 7.66 (d, 2H, J = 8.8 Hz), 7.55 (s, 1H), 7.49–7.46 (m, 2H),
7.16 (d, 2H, J = 8.8 Hz), 7.04 (t, 2H, J = 8.8 Hz), 6.49 (d, 1H, J = 5.1 Hz),
4.55 (q, 2H, J = 8.1 Hz), 4.04 (s, 3H), 1.78–1.72 (m, 2H), 1.70–1.65
solution of 14a (45 mg, 0.0922 mmol) and Cs2CO3 (91 mg,
0.279 mmol) in DMF (3 ml). The mixture was stirred for 1.5 hr at
ambient temperature, and the mixture was then diluted with water
(5 ml) and EtOAc (5 ml), extracted with EtOAc (2 × 5 ml), washed with
water (4 × 5 ml) and brine (5 ml), dried over MgSO4, and concentrated
on a rotary evaporator. The crude was further purified by column
chromatography (heptane/EtOAc, 1:1, heptane/EtOAc/MeOH, 5:5:2),
and the title compound was achieved as a light yellow solid (44 mg,
79%). 1H NMR (400 MHz, DMSO‐d6): δ 10.21 (s, 1H), 10.05 (s, 1H),
8.98 (d, 1H, J = 2.8 Hz), 8.67–8.64 (m, 1H), 8.62 (d, 1H, J = 2.8 Hz),
8.08 (s, 1H), 7.76 (d, 2H, J = 8.8 Hz), 7.66–7.62 (m, 2H), 7.62 (s, 1H),
7.37 (d, 1H, J = 8.8 Hz), 7.24 (d, 2H, J = 8.8 Hz), 7.14 (t, 2H, J = 8.8 Hz),
6.52 (d, 1H, J = 5.2 Hz), 3.87 (s, 3H), 1.48 (s, 4H). 13C NMR (101 MHz,
DMSO‐d6): δ 168.12, 168.11, 166.04, 161.13, 158.24 (d, J = 240 Hz),
153.99, 151.21, 149.03, 148.56, 144.48, 141.84, 140.63, 136.62,
135.79, 135.13 (d, J = 2 Hz), 122.38 (d, J = 8 Hz), 122.16, 121.06,

LIEN ET AL.
13 of 15
|
115.09, 114.87, 114.04, 111.12, 108.91, 102.99, 56.25, 31.51, 15.39.
HRMS (ESI+) m/z calcd. for C32H25FN5O7 [M+H]+: 610.1730, found
610.1729.
HRMS (ESI+) m/z calcd. for C32H23F3N5O7 [M+H]+: 646.1544, found
646.1540.
|
4.1.23
Synthesis of N‐(4‐((6‐((5‐aminopyridin‐2‐yl)‐
|
4.1.21
Synthesis of N‐(4‐((6‐((5‐aminopyridin‐2‐yl)‐
oxy)‐7‐methoxyquinolin‐4‐yl)oxy)phenyl)‐N‐(2,4,6‐
trifluorophenyl)cyclopropane‐1,1‐dicarboxamide
(18d)
oxy)‐7‐methoxyquinolin‐4‐yl)oxy)phenyl)‐N‐(4‐
fluorophenyl)cyclopropane‐1,1‐dicarboxamide (18b)
Iron (58 mg, 1.03 mmol), NH4Cl (44 mg, 0.823 mmol), and 18a (29 mg,
0.048 mmol) were weighed out in a round‐bottom flask, and water
(2 ml) and EtOH (3 ml) were added. The reaction mixture was stirred
for 3 hr at 70°C, and then cooled and filtered through Celite, which
was then washed with EtOAc (10 ml). The organic filtrate was then
washed with water (10 ml) and brine (10 ml), dried over MgSO4, and
concentrated on a rotary evaporator. The crude was further purified
by column chromatography (heptane/EtOAc, 1:1, heptane/EtOAc/
MeOH, 5:5:2), and the title compound was achieved as a white solid
(15 mg, 54%). 1H NMR (400 MHz, DMSO‐d6): δ 10.18 (s, 1H), 10.05
(s, 1H), 8.57, (d, 1H, J = 5.6 Hz), 7.74 (d, 2H, J = 8.8 Hz), 7.65 (s, 1H),
7.65–7.62 (m, 2H), 7.53 (s, 1H), 7.48 (d, 1H), 7.20 (d, 2H, J = 8.8 Hz),
7.14 (t, 2H, J = 8.8 Hz), 7.13–7.10 (m, 1H), 6.85 (d, 1H, J = 8.4 Hz),
6.46 (d, 1H, J = 5.2 Hz), 3.90 (s, 3H), 1.48 (s, 4H). 13C NMR (101 MHz,
DMSO‐d6): δ 168.15 (merged C=O), 160.75, 158.27 (d, J = 241 Hz),
154.39, 153.87, 149.93, 149.16, 147.33, 146.07, 141.63, 136.56,
135.16 (d, J = 2 Hz), 132.00, 125.72, 122.42 (d, J = 8 Hz), 122.17,
121.07, 115.12, 114.90, 111.80, 110.46, 108.26, 102.89, 55.99,
31.56, 15.41. HRMS (ESI+) m/z calcd. for C32H27FN5O5 [M+H]+:
580.1991, found 580.1988.
Iron (27 mg, 0.483 mmol), NH4Cl (31 mg, 0.58 mmol), and 18c (32 mg,
0.050 mmol) were weighed out in a round‐bottom flask, and water
(2 ml) and EtOH (3 ml) were added. The reaction mixture was stirred
for 3 hr at 70°C, and then cooled, filtered through Celite, which was
then washed with EtOAc (10 ml). The organic filtrate was then
washed with water (10 ml) and brine (10 ml), dried over MgSO4, and
concentrated on a rotary evaporator. The crude was further purified
by column chromatography (heptane/EtOAc, 1:1, heptane/EtOAc/
MeOH, 5:5:2), and the title compound was achieved as a white solid
(18 mg, 59%). 1H NMR (600 MHz, DMSO‐d6): δ 10.39 (s, 1H), 9.64
(s, 1H), 8.55 (d, 1H, J = 5.4 Hz), 7.74 (d, 2H, J = 9 Hz), 7.63 (s, 1H), 7.51
(s, 1H), 7.46 (d, 1H, J = 3 Hz), 7.28 (t, 2H, J = 7.8 Hz), 7.20 (d, 2H,
J = 8.4 Hz), 7.10 (dd, 1H, J = 3, 9 Hz), 6.84 (d, 1H, J = 9 Hz), 6.43 (d, 1H,
J = 5.4 Hz), 3.89 (s, 3H), 1.55–1.50 (m, 4H). 13C NMR (151 MHz,
DMSO‐d6): δ 169.87, 167.75, 160.28, 159.17, 157.39, 154.31, 153.93,
150.45, 149.51, 148.02, 144.98, 141.70, 136.32, 131.89, 125.74,
122.09, 121.12, 114.97, 111.82, 110.46, 108.92, 102.97, 100.90,
56.06, 30.53, 16.45. HRMS (ESI+) m/z calcd. for C32H25F3N5O5
[M+H]+: 616.1802, found 616.1800.
|
|
4.1.22
Synthesis of N‐(4‐((7‐methoxy‐6‐((5‐
4.1.24
Synthesis of N‐(4‐((6,7‐dimethoxyquinolin‐
nitropyridin‐2‐yl)oxy)quinolin‐4‐yl)oxy)phenyl)‐
N‐(2,4,6‐trifluorophenyl)cyclopropane‐1,1‐
dicarboxamide (18c)
4‐yl)oxy)phenyl)‐N‐(2,4,6‐trifluorophenyl)‐
cyclopropane‐1,1‐dicarboxamide (19)
6b (0.167 g, 0.64 mmol), HATU (0.424 g, 1.12 mmol), and DMF (5 ml)
were placed in a round‐bottom flask, and then DIPEA (0.26 ml,
1.49 mmol) was added. 4‐((6,7‐Dimethoxyquinolin‐4‐yl)oxy)aniline[28]
(0.156 g, 0.53 mmol) dissolved in DMF (5 ml) was added after 10 min.
The mixture was stirred for 20 hr at room temperature. The mixture
was then diluted with EtOAc (20 ml), washed with water (4 × 10 ml)
and brine (2 × 10 ml), dried over MgSO4, and concentrated on a
rotary evaporator. This solid was further purified on a preparative
HPLC, and the title compound was achieved as a white solid (0.128 g,
45%). 1H NMR (600 MHz, DMSO‐d6): δ 10.55 (s, 1H), 9.64 (s, 1H),
8.79 (d, 1H, J = 6.4 Hz), 7.86 (d, 2H, J = 8.9 Hz), 7.73 (s, 1H), 7.62
(s, 1H), 7.38 (d, 2H, J = 8.8 Hz), 7.29 (t, 2H, J = 8.5 Hz), 6.79 (d, 1H,
J = 6.5 Hz), 4.03 (s, 3H), 4.03 (s, 3H), 1.59–1.53 (m, 4H). 13C NMR
2‐Chloro‐5‐nitropyridine (29 mg, 0.183 mmol) was added to
a
solution of 14b (46 mg, 0.0879 mmol) and Cs2CO3 (77 mg,
0.236 mmol) in DMF (3 ml). The mixture was stirred for 1.5 hr at
ambient temperature, and the mixture was then diluted with water
(5 ml) and EtOAc (5 ml), extracted with EtOAc (2 × 5 ml), washed with
water (4 × 5 ml) and brine (5 ml), dried over MgSO4, and concentrated
on a rotary evaporator. The crude was further purified by column
chromatography (heptane/EtOAc, 1:1, heptane/EtOAc/MeOH, 5:5:2),
and the title compound was achieved as a light yellow solid (53 mg,
93%). 1H NMR (400 MHz, DMSO‐d6): δ 10.42 (s, 1H), 9.65 (s, 1H),
8.98 (d, 1H, J = 2.8 Hz), 8.66–8.64 (m, 1H), 8.62 (d, 1H, J = 2.8 Hz),
8.06 (s, 1H), 7.76 (d, 2 H, J = 8.8 Hz), 7.62 (s, 1H), 7.36 (d, 1H,
J = 8.8 Hz), 7.30–7.23 (m, 4H), 6.51 (d, 1H, J = 5.6 Hz), 3.87 (s, 3H),
1.56–1.51 (m, 4H). 13C NMR (101 MHz, DMSO‐d6): δ 169.84, 167.68,
166.08, 160.93, 160.1 (dt, J = 245, 15 Hz), 158.1 (ddd, J = 249, 7 Hz),
153.89, 151.41, 149.19, 148.88, 144.50, 141.80, 140.64, 136.38,
135.79, 122.03, 121.14, 114.93, 113.99, 111.4 (dd, J = 17.0, 4.9 Hz),
111.13, 109.18, 103.00, 100.8 (dt, J = 26.4, 2.5), 56.24, 30.34, 16.24.
(151 MHz, DMSO‐d6):
δ 169.99, 167.83, 165.19, 160.2 (ddd,
J = 249.16, 8 Hz), 158.2 (dt, J = 246, 15 Hz), 155.82, 150.99, 148.12,
143.56, 137.88, 137.45, 122.16, 121.48, 115.35, 111.4 (t, J = 27 Hz),
103.20, 100.9 (td, J = 17, 5 Hz), 100.57, 100.26, 56.56, 56.42, 30.48,
16.32. HRMS (ESI+) m/z calcd. for C28H23F3N3O5 [M+H]+: 538.1584,
found 538.1585.

14 of 15
LIEN ET AL.
|
|
4.1.25
Synthesis of N‐(4‐((6,7‐dimethoxyquinolin‐
(PDB: 3LQ8). This was prepared for docking using AutoDock Tools
(ligand and water removed and polar hydrogens added). The ligands
were build using Avogadro,[30] and initial geometrical optimization
was done using the same software. After docking, visualization of the
conformations and binding interactions were done in PyMol.[31]
Initially, the performance of the docking method was validated by the
redocking of the experimental ligand.
4‐yl)oxy)phenyl)‐N‐(4‐fluorophenyl)cyclopropane‐1,1‐
dicarboxamide (1, cabozantinib)
6a (0.146 g, 0.654 mmol), HATU (0.415 g, 1.09 mmol), and DMF (5 ml)
were placed in a round‐bottom flask, and then DIPEA (0.26 ml,
1.49 mmol) was added. 4‐((6,7‐Dimethoxyquinolin‐4‐yl)oxy)aniline
(0.157 g, 0.53 mmol) dissolved in DMF (5 ml) was added after
10 min. The mixture was stirred for 20 hr at room temperature.
The mixture was then diluted with EtOAc (20 ml), washed with water
(4 × 10 ml) and brine (2 × 10 ml), dried over MgSO4, and concentrated
CONFLICT OF INTERESTS
The authors declare that there are no conflicts of interests.
on
a rotary evaporator. This solid was further purified using
preparative HPLC. The title compound was achieved as a white solid
(0.112 g, 42%). 1H NMR (CDCl3, 400 MHz): δ 10.20 (s, 1H), 8.74 (s,
1H), 8.46 (d, 1H, J = 6.4 Hz), 7.99 (s, 1H), 7.79 (d, 2H, J = 8.8 Hz), 7.63
(s, 1H), 7.50–7.47 (m, 2 H), 7.18 (d, 2H, J = 8.8 Hz), 7.02 (t, 2H,
J = 8.8 Hz), 6.68 (d, 1H, J = 6.4 Hz), 4.13 (s, 3H), 4.09 (s, 3H), 1.83–1.81
(m, 2H), 1.69–1.67 (m, 2H). 13C NMR (101 MHz, CDCl3): δ 170.08,
169.04, 165.83, 159.99 (d, J = 246 Hz), 156.86, 151.90, 148.70,
141.46, 138.70, 137.12, 133.15 (d, J = 3 Hz), 123.24 (d, J = 8 Hz),
122.95, 121.65, 116.20, 115.87 (d, J = 23 Hz), 102.67, 101.13,
100.05, 57.37, 56.74, 29.21, 18.23. MS (ESI+) m/z calcd. for
C28H24FN3O5 [M+H]+: 502.2, found 502.3.
ORCID
Vegard Torp Lien
http://orcid.org/0000-0001-8830-9077
REFERENCES
[1] M. Huang, A. Shen, J. Ding, M. Geng, Trends Pharmacol. Sci. 2014, 35,
41.
[2] D. Hanahan, R. A. Weinberg, Cell 2011, 144, 646.
[3] S. A. Eccles, D. R. Welch, Lancet 2007, 369, 1742.
[4] F. Cecchi, D. C. Rabe, D. P. Bottaro, Expert Opin. Ther. Targets 2012,
16, 553.
[5] J. R. Sierra, M.‐S. Tsao, Ther. Adv. Med. Oncol. 2011, 3, S21.
[6] J. J. Cui, J. Med. Chem. 2014, 57, 4427.
|
4.2
Biological assays
[7] P. Lv, Z. Wang, H. Zhu, Curr. Med. Chem. 2016, 2, 1.
[8] T. Underiner, T. Herbertz, S. Miknyoczki, Anticancer Agents Med.
Chem. 2010, 10, 7.
|
4.2.1
Enzymatic c‐Met assay
[9] F. M. Yakes, J. Chen, J. Tan, K. Yamaguchi, Y. Shi, P. Yu, F. Qian, F.
Chu, F. Bentzien, B. Cancilla, J. Orf, A. You, A. D. Laird, S. Engst, L.
Lee, J. Lesch, Y. C. Chou, A. H. Joly, Mol. Cancer Ther. 2011, 10, 2298.
[10] U.S. National Library of Medicine, https://clinicaltrials.gov/ct2/results?
cond=&term=cabozantinib&cntry=&state=&city=&dist= (Viewed May
2019).
The enzymatic c‐Met assay was purchased from Cyclex and used
following the manufacturer’s instructions. Briefly, while placed on ice,
recombinant c‐Met was added to wells precoated with a substrate,
and the reaction was started by adding buffer containing the
inhibitors in appropriate dilutions. The plate was incubated at 30°C
for 60 minutes. After washing with buffer, a horseradish peroxidase‐
conjugated detection antibody PY‐39 was added to each well, and
then incubated at ambient temperature for 60 min. The TMB
substrate was added after another round of washing and then
incubated at ambient temperature for 10 min. Stop solution was then
added, and absorbance was measured using a spectrophotometric
plate reader (Perkin Elmer VICTOR™ X3). The results were analyzed
using GraphPad Prism 7.04.
[11] L. Liu, M. H. Norman, M. Lee, N. Xi, A. Siegmund, A. A. Boezio, S.
Booker, D. Choquette, N. D. D’Angelo, J. Germain, K. Yang, Y. Yang,
Y. Zhang, S. F. Bellon, D. A. Whittington, J. C. Harmange, C.
Dominguez, T. S. Kim, I. Dussault, J. Med. Chem. 2012, 55, 1868.
[12] M. Mannion, S. Raeppel, S. Claridge, N. Zhou, O. Saavedra, L.
Isakovic, L. Zhan, F. Gaudette, F. Raeppel, R. Déziel, N. Beaulieu, H.
Nguyen, I. Chute, C. Beaulieu, I. Dupont, M. F. Robert, S. Lefebvre, M.
Dubay, J. Rahil, J. Wang, H. Ste‐Croix, A. Robert Macleod, J. M.
Besterman, A. Vaisburg, Bioorg. Med. Chem. Lett. 2009, 19, 6552.
[13] X. Wang, N. Jiang, S. Zhao, S. Xi, J. Wang, T. Jing, W. Zhang, M. Guo,
P. Gong, X. Zhai, Bioorg. Med. Chem. 2016, 25, 886.
[14] P. K. Parikh, M. D. Ghate, Eur. J. Med. Chem. 2017, 143, 1103.
[15] G. M. Schroeder, X. T. Chen, D. K. Williams, D. S. Nirschl, Z. W. Cai, D.
Wei, J. S. Tokarski, Y. An, J. Sack, Z. Chen, T. Huynh, W. Vaccaro, M.
Poss, B. Wautlet, J. Gullo‐Brown, K. Kellar, V. Manne, J. T. Hunt, T.
W. Wong, L. J. Lombardo, J. Fargnoli, R. M. Borzilleri, Bioorganic Med.
Chem. 2008, 18, 1945.
|
4.2.2
Cell proliferation
Testing was performed by the Developmental Therapeutics Program,
Division of Cancer Treatment and Diagnosis, National Cancer
Institute. The studies were performed using the NCI60 panel and
performed according to their internal procedures.[29]
[16] Z. W. Cai, D. Wei, G. M. Schroeder, L. Cornelius, K. Kim, X. T. Chen, R.
J. Schmidt, D. K. Williams, J. S. Tokarski, Y. An, J. S. Sack, V. Manne,
A. Kamath, Y. Zhang, P. Marathe, J. T. Hunt, L. J. Lombardo, J.
Fargnoli, R. M. Borzilleri, Bioorg. Med. Chem. Lett. 2008, 18, 3224.
[17] G. M. Schroeder, Y. An, Z. W. Cai, X. T. Chen, C. Clark, L. Cornelius, J.
Dai, J. Gullo‐Brown, A. Gupta, B. Henley, J. T. Hunt, R. Jeyaseelan, A.
Kamath, K. Kim, J. Lippy, L. J. Lombardo, V. Manne, S. Oppenheimer,
J. S. Sack, R. J. Schmidt, G. Shen, K. Stefanski, J. S. Tokarski, G. L.
Trainor, B. S. Wautlet, D. Wei, D. K. Williams, Y. Zhang, Y. Zhang, J.
Fargnoli, R. M. Borzilleri, J. Med. Chem. 2009, 52, 1251.
|
4.3
Molecular docking
Dockings were performed using AutoDock Vina[25] via the PyRX[26]
interface. The experimental crystal structure of foretinib in the
enzymatic site of c‐Met was downloaded from the Protein Data Bank

LIEN ET AL.
15 of 15
|
[18] X. Jiang, H. Liu, Z. Song, X. Peng, Y. Ji, Q. Yao, M. Geng, J. Ai, A.
Zhang, Bioorg. Med. Chem. 2015, 23, 564.
[30] M. D. Hanwell, D. E. Curtis, D. C. Lonie, T. Vandermeersch, E. Zurek,
G. R. Hutchison, J. Cheminform. 2012, 4, 1.
[19] S. Lacy, B. Hsu, D. Miles, D. Aftab, R. Wang, L. Nguyen, Drug Metab.
Dispos. 2015, 43, 1190.
[31] The PyMOL Molecular Graphics System. Version 1.8 Schrödinger.
[20] D. T. Aftab, S. Naganathan, W. Xu, S. Lacy, L. Nguyen, WO 2014/
145693, 2014.
SUPPORTING INFORMATION
[21] L. Bannen, D. S.‐M. Chan, J. Chen, D. L. Esther, WO 2005/030140,
2004.
Additional supporting information may be found online in the
Supporting Information section.
[22] National Cancer Institute, https://dtp.cancer.gov/discovery_develop-
ment/nci‐60/default.htm (Viewed May 2019).
[23] R. H. Shoemaker, Nat. Rev. Cancer 2006, 6, 813.
[24] Z. Zhan, J. Ai, Q. Liu, Y. Ji, T. Chen, Y. Xu, M. Geng, W. Duan, ACS
Med. Chem. Lett. 2014, 5, 673.
How to cite this article: Lien VT, Pettersen S, Haugen MH,
Olberg DE, Mælandsmo GM, Klaveness J. Design, synthesis
and biological evaluation of 6‐substituted quinolines derived
from cabozantinib as c‐Met inhibitors. Arch Pharm Chem Life
Sci. 2019;e1900101.
[25] O. Trott, A. J. Olson, J. Comput. Chem. 2010, 31, 455.
[26] S. Dallakyan, A. J. Olson, Methods Mol. Biol. 2015, 1263, 243.
[27] P. Bose, J. Banerji, Phytochemistry 1991, 30, 2438.
[28] V. T. Lien, J. Klaveness, D. E. Olberg, J. Label. Compd. Radiopharm.
2017, 61, 11.
https://doi.org/10.1002/ardp.201900101
[29] National Cancer Institute, https://dtp.cancer.gov/discovery_develop-
ment/nci‐60/methodology.htm (Viewed May 2019).