Design, synthesis and biological evaluation of a series of novel 2-benzamide-4-(6-oxy-N-methyl-1-naphthamide)-pyridine derivatives as potent fibroblast growth factor receptor (FGFR) inhibitors

Article abstract of DOI:10.1016/j.ejmech.2018.05.005

Starting from the phase II clinical FGFR inhibitor lucitanib (2), we conducted a medicinal chemistry approach by opening the central quinoline skeleton coupled with a scaffold hopping process thus leading to a series of novel 2-benzamide-4-(6-oxy-N-methyl-1-naphthamide)-pyridine derivatives. Compound 25a was identified to show selective and equally high potency against FGFR1/2 and VEGFR2 with IC₅₀ values less than 5.0 nM. Significant antiproliferative effects on both FGFR1/2 and VEGFR2 aberrant cancer cells were observed. In the SNU-16 xenograft model, compound 25a showed tumor growth inhibition rates of 25.0% and 81.0% at doses of 10 mg/kg and 50 mg/kg, respectively, with 5% and 10%body weight loss. In view of the synergistic potential of FGFs and VEGFs in tumor angiogenesis observed in preclinical studies, the FGFR/VEGFR2 dual inhibitor 25a may achieve better clinical benefits.

Full text of DOI:10.1016/j.ejmech.2018.05.005

European Journal of Medicinal Chemistry 154 (2018) 9e28
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis and biological evaluation of a series of novel 2-
benzamide-4-(6-oxy-N-methyl-1-naphthamide)-pyridine derivatives
as potent fibroblast growth factor receptor (FGFR) inhibitors
,
d
,
,
d
,
,
, d
Manman Wei a 1, Xia Peng b 1, Li Xing a, Yang Dai b d, Ruimin Huang c
,
Meiyu Geng b d, Ao Zhang a d, Jing Ai b **, Zilan Song a
,
,
c
,
,
d,
, d, *
a CAS Key Laboratory of Receptor Research and the State Key Laboratory for Drug Research, Shanghai Institute of Materia Medica (SIMM), Chinese Academy
of Sciences, Shanghai, 201203, China
b Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences,
Shanghai, 201203, China
c School of Life Science and Technology, ShanghaiTech University, Shanghai, 200031, China
d University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing, 100049, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Starting from the phase II clinical FGFR inhibitor lucitanib (2), we conducted a medicinal chemistry
approach by opening the central quinoline skeleton coupled with a scaffold hopping process thus leading
to a series of novel 2-benzamide-4-(6-oxy-N-methyl-1-naphthamide)-pyridine derivatives. Compound
25a was identified to show selective and equally high potency against FGFR1/2 and VEGFR2 with IC50
values less than 5.0 nM. Significant antiproliferative effects on both FGFR1/2 and VEGFR2 aberrant cancer
cells were observed. In the SNU-16 xenograft model, compound 25a showed tumor growth inhibition
rates of 25.0% and 81.0% at doses of 10 mg/kg and 50 mg/kg, respectively, with 5% and 10%body weight
loss. In view of the synergistic potential of FGFs and VEGFs in tumor angiogenesis observed in preclinical
studies, the FGFR/VEGFR2 dual inhibitor 25a may achieve better clinical benefits.
Received 5 March 2018
Received in revised form
8 April 2018
Accepted 4 May 2018
Keywords:
FGFR
VEGFR
Synergistic effect
Tumor growth inhibition
Lucitanib
© 2018 Elsevier Masson SAS. All rights reserved.
1. Introduction
translocations have been identified in a broad spectrum of human
tumors as oncogenic drivers, thus offering a new strategy to
The fibroblast growth factor receptors (FGFRs) are a family of
highly conserved transmembrane tyrosine kinases (RTKs), which
constitute four members (FGFR1-4) [1e4]. The FGF-FGFR axis is
involved in signal transduction pathways that regulate cell prolif-
eration, differentiation, embryonic development, migration, sur-
vival, angiogenesis and organogenesis [3,5e8]. Over the past
decades, several genomic alterations in the FGF-FGFR axis including
activation mutations, gene amplifications, and chromosomal
develop molecular targeted personalized medicine based on the
FGFR activation status in cancer patients [9e15]. Since FGFR, like
other RTKs (EGFR, VEGFR2), shares a ubiquitous intracellular
signaling pathway, cancer cells are prone to escape the FGFR inhi-
bition by either genomic mutations or by shifting to parallel
signaling pathways [16e20]. Accordingly, the currently approved
FGFR inhibitors are multi-target inhibitors (e.g. lenvatinib [21],
nintedanib [22], pazopanib [23]) exerting antitumor efficacy pri-
marily through targeting other RTKs rather than FGFRs [8]. There-
fore, there is a need to develop more selective inhibitors to suppress
tumors clearly through targeting FGFRs by using FGFR activation
status as a biomarker to stratify patients [24,25]. To this end, several
FGFR-selective inhibitors, including AZD4547 (1) [26] and BGJ398
(3) [27] (Fig. 1) have been in extensive clinic trials, and the outcome
of these investigations will be useful to validate FGFR inhibitors as
single anticancer therapies [28,29]. On the other hand, it is known
that tumor angiogenesis is augmented by cross-talking between
* Corresponding author. CAS Key Laboratory of Receptor Research and the State
Key Laboratory for Drug Research, Shanghai Institute of Materia Medica (SIMM),
Chinese Academy of Sciences, Shanghai, 201203, China
** Corresponding author. Division of Anti-tumor Pharmacology, State Key Labo-
ratory of Drug Research, Shanghai Institute of Materia Medica (SIMM), Chinese
Academy of Sciences, Shanghai, 201203, China.
E-mail addresses: jai@simm.ac.cn (J. Ai), songalan@simm.ac.cn (Z. Song).
These two authors contributed to this work equally.
1
https://doi.org/10.1016/j.ejmech.2018.05.005
0223-5234/© 2018 Elsevier Masson SAS. All rights reserved.

10
M. Wei et al. / European Journal of Medicinal Chemistry 154 (2018) 9e28
Fig. 1. Representative FGFR inhibitors.
FGF ligands, VEGFRs, and inflammatory mediators in tumor stroma,
and the synergistic effects of FGFs and VEGFs in tumor angiogenesis
have been observed in preclinical studies [16,30e32]. Therefore,
compared to the previous multi-target FGFR inhibitors with less
potency against FGFR, development of selective FGFR/VEGFR2 dual
inhibitors with equal or even greater potency against FGFR would
be an appealing combination [16,17].
The crystal structure of 1 [34] with FGFR1 demonstrates that the
3,5-dimethoxy-phenyl moiety occupies the hydrophobic pocket to
form a hydrogen bond with the Asp641 residue. The pyrazole core
located in the hinge region forms two hydrogen bonds with the
carbonyl of the Glu562 and the NH of the Tyr563 residues. The
substituted benzamide extends toward solvent, making two
hydrogen bonds with the carbonyl of Ala564 and the NH of Gly485.
The binding mode of another non-selective clinically being inves-
tigated FGFR inhibitor lucitanib (2) [33,34] with FGFR1 is similar to
that of 1. The N-methyl-1-naphthamide fragment forms two
hydrogen bonds with the carbonyl of the Glu531 and the NH of the
Asp641 residues, and extends to the hydrophobic pocket. The
quinoline moiety forms a hydrogen bond with the NH of the Ala564
residue in the hinge region.
In view of the similar binding patterns of 1 and 2 with FGFR1, we
envisioned that opening the quinoline fragment of 2 and intro-
ducing an amide bond as the hydrogen bond acceptor and donor
would generate structurally flexible new FGFR inhibitors retaining
similar hydrogen bonding networks (Fig. 2). Following this
approach, we identified compound SOMCL-085 [35] (25e, Fig. 1)
that showed nearly equal potency against FGFR and VEGFR2 (Fig. 2).
Encouraged by this result, a series of novel 2-benzamide-4-
aryloxypyridine derivatives were developed and a systemic SAR
study was conducted leing to the discovery of the preclinical
compound SOMCL-286 (25a). Notably, during completion of our
study, Eisai disclosed its phase I clinical compound 4 (Eꢀ7090) [36],
which also bears a 4-aryloxy-2-aminopyridine framework, but with
a different head group. This compound showed high potency
against FGFR1-3 (0.5e1.2 nM), but weaker potency against VEGFR2
(16 nM), which is different from our FGFR/VEGFR2 dual inhibitor
profile. Herein, we report the structure-activity relationship study
of our 2-benzamide-4-(6-oxy-N-methyl-1-naphthamide)pyridine
series and the pharmacological profiling of the FGFR/VEGFR dual
inhibitor 25a.
2. Chemistry
The synthesis of 2-benzamide-4-aryloxypyridine derivatives is
outlined in Schemes 1e5. Nucleophilic displacement of the C-4
fluoro atom of ethyl 4-fluorobenzoates with substituted pipera-
zines afforded compounds 6a-i in 68e89% yields. Similarly, nucle-
ophilic displacement of the C-2 bromo groups of ethyl 2-
bromothiazole-4-carboxylate (8a) and ethyl 2-bromooxazole-4-
carboxylate (8b) and the C-2 chloro group of methyl 2-
chloropyrimidine-5-carboxylate (11) afforded 9a, 9b and 12 in
70e82% yields, respectively. Hydrolysis of 6a-i, 9a, 9b and 12
afforded the corresponding acids, which were then transformed to
acyl chlorides by treating with thionyl chloride and subsequently
reacted with ammonium hydroxide to afford primary amide in-
termediates 7a-i, 10a, 10b and 13 in 33e70% yields (Scheme 1).
Condensation of 6-(2-chloropyridin-4-yloxy)-1-naphthoic acid
(14) [37] with CH3NH2 afforded intermediate 15 in 96% yield.
Compounds 19a-d were prepared by Buchwald-Hartwig cross-
coupling of 15 with benzamides 7a-d followed by deprotection
with CF3COOH in 49e58% yields for two steps. Treatment of (2-
bromoethoxy) (tert-butyl)diphenylsilane with 1H-pyrazole-4-
carboxamide afforded intermediate 18, which was converted to
compound 20 via cross-coupling with 15 followed by deprotection
with CsF in 17% overall yield. Compounds 21a-b were obtained by
cross-coupling of 15 with 10a-b in 69% and 55% yield, respectively.
Similarly, cross-coupling of 15 with 13 followed by deprotection
with CF3COOH afforded compound 22 in 51% yield for two steps
(Scheme 2).
Condensation of 6-(2-chloropyridin-4-yloxy)-1-naphthoic acid
(14) with MeOH afforded 23a in 95% yield. 2-Chloro-4-(6-oxy-
substituted)-pyridine derivatives 23b-e were synthesized by
nucleophilic substitution of 2-chloro-4-nitropyridine with the
corresponding phenol derivatives in 40e71% yields. Similarly,
nucleophilic displacement of 2,4-dichloropyridine with 2-(3,5-
dimethoxyphenyl)ethanol
[38]
or
2-(2,6-dichloro-3,5-
dimethoxyphenyl)ethanol [39] afforded 23f or 23g in 71% or 66%
yield, respectively. Intermediates 23h-i were prepared by nucleo-
philic displacement of 2,6-difluoro-3,5-dimethoxybenzyl meth-
anesulfonate
[40]
or
1-(3,5-dicholoropyridin-
4-yl)ethyl
methanesulfonate [41] with 6-chloropyridin-3-ol in 65% or 61%
yield respectively. Compounds 24a-i were prepared in 52e72%

M. Wei et al. / European Journal of Medicinal Chemistry 154 (2018) 9e28
11
Fig. 2. Design of potent FGFR/VEGFR2 dual inhibitors.
Scheme 1. Reagents and conditions: (a) K2C03, DMSO, 110 ꢁC. 75-90%; (b) (Boc)2O, NaOH, DCM/H2O (1:1), 90-98%; (c) (i) NaOH, MeOH/H2O (1:1), (ii) SOCl2. DMF, DCM, 0 ꢁC to rt; (iii)
NH4OH, DCM, rt. 33-70%: (d) K2CO3. CH3CN, 80 ꢁC, 70-76%; (e) K2CO3, DMF, rt. 75%.
yields by condensation of intermediates 7a with 23a or 23b-i fol-
lowed by deprotection with CF3COOH (Scheme 3).
dioxaspiro [2.5]octane or acryloyl chloride to afford compounds
30a,b in 32% and 80% yield, respectively (Scheme 4).
Compounds 25a-e were prepared via coupling of benzamides
7e-i with 15 in 49e74% yields. Similarly, reaction of 26 with 15
followed by deprotection with CF3COOH provided compound 27 in
52% yield, which was then reacted with 2-bromoethanol to afford
compound 28 in 62%. Meanwhile, coupling of 4-nitrobenzamide
with 15 followed by reduction with zinc powder furnished inter-
mediate 29 in 56% yield, which was then reacted with 1,6-
As shown in Scheme 5, treatment of 4-hydroxy benzamide with
1,2-dibromoethane followed by displacement with different sec-
ondary amines furnished intermediates 32a-c in 31e59% yields,
which were then reacted with 15 via cross-coupling to provide
compounds 33a-c in 57e67% yields. Similarly, reaction of ethyl 4-
hydroxybenzoate
with
tert-butyl
1-(iodomethyl)cyclo-
propylcarbamate [43,44] afforded intermediate 31, which was

12
M. Wei et al. / European Journal of Medicinal Chemistry 154 (2018) 9e28
Scheme 2. Reagents and conditions: (a) CH3NH2, EDCl. HOBt. DIPEA. DCM, rt, 95.8%; (b) K2CO3, DMF, 80 ꢁC, 63%; (c) (i) Pd2(dba)3, Xantpbos, Cs2CO3, dioxane, 100 ꢁC; (ii) CF3COOH.
DCM, 49-58% for two steps; (d) 18, Pd2(dba)3, Xantphos, CS2CO3, dioxane, 100 ꢁC; (il) CsF, DMF, 80 ꢁC, 27.3% for two steps; (e) 10a-b, Pd2(dba)3, Xantpbos, CS2CO3, dioxane, 100 ꢁC,
55-69%.
converted to benzamide 32d in 51% yield in two steps. Subsequent
coupling of 32d with 15 followed by deprotection with CF3COOH
afforded compound 33d in 55% yield.
(285 vs 12.9 nM) and in the cell (>1 mM) assays. Much worse, the
ortho-fluorinated compound 19d nearly completely lost the
biochemical activity against FGFR1. Compared to 19a, both steric
and electrostatic effects are likely responsible for the reduced po-
tency of 19b-d.
3. Results and discussion
Replacement of phenyl in the phenylpiperazine fragment with
pyrazole, thiazole, isoxazole, and pyrimidine gave compounds
20e22. All compounds retained good biochemical activity against
FGFR1, especially for compounds 21a-b, both showing IC50 values
less than 10 nM, however, we failed to obtain their cellular potency
due to poor solubility. From the results above, the 4-(3,5-
dimethylpiperazin-1-yl)benzamido moiety (as in 19a) is the best
of choice as the hydrophilic component, therefore, it was carried for
further optimization.
As shown in Figs. 1 and 2, the hydrophobic pocket in the FGFR1
interaction domain has relatively high tolerance to different het-
eroaryl rings. Since the N-methyl-1-naphthamide fragment as the
4-O-substituent of the central pyrimidine in 19a was initially taken
from the clinical compound 2 (lucitanib), we then optimized this
component to better fit the hydrophobic pocket by using various
heterocyclic bioisosteres or alkyl-aromatic rings as the replacement
(Table 2). Compared to compound 19a, replacing the naphthalene
methyl amide with corresponding ester afforded compound 24a
showing slightly reduced biochemical potency (2.1 vs 4.6 nM) but
the cellular activity was 10-fold reduced (14.9 vs 159 nM). Adding
3.1. Structure-activity relationships
All the new compounds were assayed for their biochemical ac-
tivity against FGFR1 and the potent compounds were further tested
for their antiproliferative effects in the FGFR1-translocated myeloid
leukemia KG-1cancer cells. To test our design, we first incorporated
the (3,5-dimethylpiperizin-1-yl)benzamido moiety into the 4-
aryloxy-2-aminopyrimidine core as the hydrophilic component
leading to compound 19a. As shown in Table 1, compound 19a
displayed an IC50 of 2.1 nM against FGFR1. This compound also
displayed high potency of 14.9 nM against the FGFR1-positive KG-
1cancer cell. Compared to the phase II clinical FGFR-selective in-
hibitor 1, both biochemical and cellular potency of 19a was lower,
therefore, optimization of compound 19a is needed.
We first focused on optimization of the hydrophilic component
by replacing the phenylpiperazine moiety with different sub-
stituents. Compounds 19b and 19c were obtained by introduction
of methoxy and methyl, respectively at the ortho-position of the
phenyl, both showing reduced potency either in the biochemical

M. Wei et al. / European Journal of Medicinal Chemistry 154 (2018) 9e28
13
Scheme 3. Reagents and conditions: (a) for 23b-e, phenol derivatives, Cs2CO3, DMSO, rt, 40-71%; (b) for 23f, 23g, phenylethanol derivatives, t-BuOK, t-BuOH, reflux, 66-71%; (c)
methane sulfonates, K2CO3. DMF, 60 ꢁC, 61-65%; (d) 7a, Pd2(dba)3. Xantphos, Cs2CO3. dioxane, 100 ꢁC; (ii) CF3COOH. DCM, rt, 52-72% for two steps; (e) SOCl2. MeOH, reflux, 95%.
another methoxy substituent yielded compound 24b, which also
retained good biochemical activity (8.2 nM), but the cellular po-
tency was much reduced (118 nM). Saturation of the N-methyl-1-
naphthamide to N-ethyl 3,4-dihydroquinoline-1(2H)-carboxamide
24c led to significantly reduced potency both in the biochemical
(132 nM) and cellular (118 nM) assays. Replacement of the naphthyl
amide with naphthyl urea yielded compound 24d, which retained
moderate potency against FGFR1, whereas the antiproliferative ef-
fect was significantly reduced. Similar results were obtained by
replacing the naphthyl amide moiety with phenylurea (24e).
We also attempted to replace the naphthamide moiety in 19a
with other hydrophobic functionalities, including 3,5-
various heterocyclic amines including substituted piperazines,
morpholine, substituted or unsubstituted piperidines were tested.
All these compounds (25a-e, 27, 28) displayed excellent potency
against FGFR1 with IC50 values of less than 2.0 nM, although the
antiproliferative effects were significantly different. The sub-
stituents on these heterocyclic amino moieties have no effects on
the biochemical activity but significantly impaired the cellular
potency. For example, the unsubstituted piperazine 25a has similar
biochemical activity to the 3,5-dimethylpiperazine 19a (1.0 vs
2.1 nM), whereas the antiproliferative potency of 25a is 4-fold more
potent than 19a (0.5 vs 14.9 nM). Similar enzymatic activity was
also observed for morpholine analog 25a and the 3,5-
dimethylmorpholine 25c (1.2 vs 1.9 nM), however, their cellular
potency was 9.3 nM and 154 nM, respectively. This discrepancy
may reflect the capability of cell permeability of these substituted
or unsubstituted heterocyclic analogues. Further, replacement of
the 3,5-dimethylpiperizin-1-yl moiety in 19a with a Michael
acceptor acrylamido moiety afforded compound 30b retaining
good biochemical potency against FGFR1 (12.8 nM), but the anti-
proliferative effect was significantly reduced. Compounds 35a-
d bearing a flexible alkyloxy moiety as the solvent interaction group
also displayed good to high potency both in the biochemical
(3.6e7.4 nM) and cellular (4.1e54 nM) assays. Based on the results
above, several compounds showed high potency in the biochemical
and cell proliferation assays with IC50 values compatible to that of
the clinical FGFR1 inhibitor 1. To evaluate the developability of this
dimethoxyphenethyl
as
in
1
and
3,5-dimethoxy-2,6-
dichlorophenethyl as in 3. The corresponding compounds 24f and
24g completely lost the potency against FGFR1, indicating the H-
bonding networks in the amido moiety of 19a are critical. Inter-
estingly, replacement of the naphthamido moiety with 3,5-
dimethoxy-2,6-difluorobenzyl and shifting its location from C-4
to C-5 position of the central pyrimidine ring led to compound 24h
retaining moderate potency both in the biochemical (22.2 nM) and
cellular (116 nM) assays. However, further substitution on this
compound yielded compound 24i that lost FGFR1 potency.
Since modification of the naphthamide moiety did not yield
more potent compounds than compound 19a. We decided to
further modify the solvent interaction region that was occupied by
the 3,5-dimethylpiperizin-1-yl moiety in 19a. As shown in Table 3,

14
M. Wei et al. / European Journal of Medicinal Chemistry 154 (2018) 9e28
Scheme 4. Reagents and conditions: (a) Pd2(dba)3, Xantphos, Cs2CO3, dioxane, 100 ꢁC, 49-74%; (b) CF3COOH, DCM, 96%; (c) 2-bromoethanol, K2CO3, DMF, 80 ꢁC, 62%: (d) Zn, NH4Cl,
THF/MeOH (1:1), 73.5%: (e) for 30a 1,6-dioxaspiro[2.5]octane. DIPEA, EtOH, 32%; for 30b, acryloyl chloride, DIPEA, dry DCM, 80%.
class compounds, we selected compound 25a for further profiling
due to its highest potency in both enzymatic and cellular assays
among this series.
17.5 nM, 82.5 nM and 237.5 nM) [33], or the new released structure
of the phase I clinical compound 4 (FGFR1-3 IC50: 0.5e1.2 nM;
VEGFR2 IC50: 16 nM) [36], compound 25a showed nearly equally
high potency against FGFR1, FGFR2 and VEGFR-2 with IC50 values of
1.0, 4.5 and 2.9 nM, respectively (Table 4). Much lower potency was
observed for other kinases tested in our experiments. The result
shows that the new inhibitor 25a is a highly potent and selective
FGFR1/VEGFR2 inhibitor.
3.2. Molecular docking of 25a
To elucidate the interaction mode of 2-benzamide-4-(6-oxy-N-
methyl-1-naphthamide)-pyridine 25a with FGFR, we conducted
molecular docking analysis using a reported crystal structure of
FGFR1 (PDB ID: 4RWL) [38]. As illustrated in Fig. 3, 25a has a similar
binding mode with lucitanib (2) and maintains the key H-bonding
network observed in the complex of FGFR1 with 2. The N-methyl-1-
naphthamide fragment forms two hydrogen bonds with the
carbonyl of the Glu531 and the NH of the Asp641 residues, and
extends to the hydrophobic pocket. The 2-benzamide pyridine
moiety forms a H-bonding with the NH of the Ala564 residue in the
hinge region. The N-methyl piperazine group extends to the solvent
region and forms an additional H-bonding with the carbonyl of the
Glu571 upon protonizing in physiological conditions.
3.4. Antiproliferative activity study of 25a
To evaluate further the antiproliferative effect of 25a in FGFR
mediated cancer cells, five aberrant FGFR cell lines were tested,
including FGFR1-amplified H1581 cells, FGFR1-translocated KG1
cells, FGFR2-amplified SNU-16 cells, FGFR3-amplified RT112 cells
and FGFR3-mutated UMUC14 cells. As shown in Table 5, 25a
demonstrated greater antiproliferative effects in all these FGFR1-3
subtype-selective cell lines than the selective FGFR inhibitor 1
(AZD-4547). As a FGFR/VEGFR2 dual inhibitor, 25a also inhibited
the proliferation of BAF3/VEGFR2 cell lines with an IC50 less than
1.5 nM.
3.3. Kinase inhibition profile of 25a
The selectivity profile of 25a was evaluated against a panel of
3.5. Pharmacokinetical study of compound 25a
tyrosine kinases closely related to FGFR. Compared to VEGFR/FGFR
dual inhibitor 2 (VEGFR1-2 IC50: 7 nM and 25 nM; FGFR1-3 IC50
:
The pharmacokinetical profile (PK) of the FGFR/VEGFR2 dual

M. Wei et al. / European Journal of Medicinal Chemistry 154 (2018) 9e28
15
Scheme 5. Reagents and conditions: (a) 1,2-dibromoethane, K2CO3, acetone, 50 ꢁC, N2, 51%; (b) amines, K2CO3, CH3CN, reflux, 55-62%; (c) tert-butyl 1-(iodomethyl)cyclo-
propylcarbamate. K2CO3, acetone, rt, 64%; (d) (i) NaOH, MeOH/H2O (1:1), 60 ꢁC; (ii) SOCl2, DMF, DCM, 0 ꢁC to rt; (iii) NH4OH, DCM, rt, 51%; (e) for 33a-c, 15, Pd2(dba)3, Xantphos,
Cs2CO3, dioxane, 100 ꢁC; for 33d, (i) 15, Pd2(dba)3, Xantphos, Cs2CO3, dioxane, 100 ꢁC; (ii) CF3COOH, DCM, rt, 55-67%.
inhibitor 25a was tested in Sprague-Dawley (SD) rats dosed iv
(1 mg/kg) and orally (3 mg/kg). As shown in Table 6, compound 25a
showed an acceptable half-time of 1.85 h (T1/2) and relatively
moderate oral bioavailability of 14.9%. The plasma exposure of this
compound is also relatively low after both intravenous and oral
administration. However, by making compound 25a as a hydro-
chloride salt (with two molecular of HCl), this compound is aque-
ously soluble, and readily used for in vivo study.
This compound as its hydrochloride salt is now under our early
preclinical investigation.
5. Experimental section
5.1. General methods
NMR spectral data were recorded on a Varian Mercury 300, 400
or 500 NMR spectrometer. Low-resolution mass spectra (MS) and
high-resolution mass spectra (HRMS) were recorded at anionizing
voltage of 70 eV on a Finnigan/MAT95 spectrometer. Column
chromatography was carried out on silica gel (200e300 mesh). All
reactions were monitored using thin layer chromatography (TLC)
on silica gel plates. All the reagents were obtained from commercial
sources, and used without further purification unless otherwise
stated.
3.6. In vivo antitumor activity of compound 25a
Finally, the in vivo antitumor activity of 25a was evaluated in
SNU-16 xenograft model. Compound 25a was administered via
intraperitoneal injection at doses of 10 mg/kg or 50 mg/kg once
daily for 21 consecutive days. The results were demonstrated in
Fig. 4. 25a was found to suppress tumor growth in a dose-
dependent manner, with tumor growth inhibition rate (TGI) of
25.0% and 81.0% at doses of 10 mg/kg and 50 mg/kg, respectively.
There is no obvious body weight loss during the experiment.
5.1.1. 6-(2-(4-((3R,5S)-3,5-dimethylpiperazin-1-yl)benzamido)
pyridin-4-yloxy)-N-methyl-1-naphthamide (19a)
To a mixture of 5a (5.0 g, 32.44 mmol, 1 equiv) and (2S,6R)-2,6-
dimethylpiperazine (3.7 g, 32.44 mmol, 1 equiv) in 60 mL DMSO
was added K2CO3 (8.97 g, 64.88 mmol, 2 equiv). The mixture was
heated at 110 ꢁC for 12 h. The reaction mixture was diluted with
120 mL ice water. Ethyl acetate was added and the reaction mixture
was partitioned between ethyl acetate and water. The organic
phase was washed with water and brine, then dried over anhydrous
Na2SO4, filtered and concentrated. The resulting residue was
further purified via silica gel chromatography to give ethyl 4-
4. Conclusion
In summary, starting from the phase II clinical FGFR inhibitor
lucitanib (2), a medicinal chemistry approach was conducted by
opening the quinoline fragment coupled with a scaffold hopping
process thus leading a series of novel 2-benzamide-4-(6-oxy-N-
methyl-1-naphthamide)-pyridine derivatives. Compound 25a was
identified to show equally high potency against FGFR1/2 and
VEGFR2 with IC50 values less than 5.0 nM. It also suppressed the
proliferation of both FGFR1/2 and VEGFR2 addictive cancer cells.
In vivo in SNU-16 xenograft model, compound 25a showed tumor
growth inhibition rates of 25.0% and 81.0% at doses of 10 mg/kg and
50 mg/kg, respectively, without obvious body weight loss. In view
of the synergistic effects of FGFs and VEGFs in tumor angiogenesis
observed in preclinical studies, the selective and equally potent
FGFR/VEGFR2 dual inhibitor 25a might have better clinical benefit.
((3R,5S)-3,5-dimethylpiperazin-1-yl)benzoate as
a
white solid
(7.25 g, 90%). NMR (300 MHz, CDCl3)
d
7.85 (d, J ¼ 9.0 Hz, 2H), 6.79
(d, J ¼ 9.0 Hz, 2H), 4.26 (q, J ¼ 7.1 Hz, 2H), 3.60 (d, J ¼ 12.0 Hz, 2H),
2.96e2.88 (m, 2H), 2.32 (t, J ¼ 11.3 Hz, 2H), 1.30 (t, J ¼ 7.1 Hz, 3H),
1.08 (d, J ¼ 6.3 Hz, 6H).
The obtained methyl 4-((3R,5S)-3,5-dimethylpiperazin-1-yl)
benzoate was dissolved in dichloromethane (DCM) and stirred for
15 min after adding 1 N NaOH (aq) (32.12 mL, 32.12 mmol, 1.1

16
M. Wei et al. / European Journal of Medicinal Chemistry 154 (2018) 9e28
Table 1
phase was washed with saturated NaHCO3 (aq) and brine, then
dried over anhydrous Na2SO4, filtered and concentrated. The
resulting residue was further purified via silica gel chromatography
to give 6a as colorless oil. (10.00 g, 98%). 1H NMR (300 MHz, CDCl3)
Biochemical and cellular activities of new compounds against FGFR1.
d
7.77 (d, J ¼ 8.8 Hz, 2H), 6.68 (d, J ¼ 8.7 Hz, 2H), 4.20e4.08 (m, 4H),
3.44 (d, J ¼ 12.5 Hz, 2H), 2.87 (dd, J ¼ 12.4, 3.8 Hz, 2H), 1.35 (s, 9H),
1.20 (t, J ¼ 7.1 Hz, 3H), 1.14 (d, J ¼ 6.8 Hz, 6H).
To a solution of 6a (10.00 g, 28.70 mmol, 1 equiv) dissolved in
MeOH was added 1 N NaOH (aq) (57.40 mL, 57.40 mmol, 2 equiv).
The mixture was heated at 60 ꢁC for 4 h and allowed to cool down
to room temperature. The reaction mixture was acidified with 2 N
HCl (aq) to adjust PH to 5e6. The resulting precipitate was collected
by filtration, washed with water and dried to a constant weight to
afford 4-((3R,5S)-4-(tert-butoxycarbonyl)-3,5-dimethylpiperazin-
Compound
Ar
FGFR1 IC50 (nM)a KG1 cell IC50 (nM)a
2.10 0.90 14.9 0.8
19a
1-yl)benzoic acid (9.79 g, 98%). 1H NMR (300 MHz, DMSO)
d 7.76 (d,
J ¼ 8.7 Hz, 2H), 6.97 (d, J ¼ 8.9 Hz, 2H), 4.18e4.05 (m, 2H), 3.75 (d,
J ¼ 12.8 Hz, 2H), 2.98 (dd, J ¼ 12.9, 4.3 Hz, 2H), 1.42 (s, 9H), 1.19 (d,
J ¼ 6.7 Hz, 6H).
19b
285 12.7
NTb
The obtained acid (9.79 g, 29.28 mmol, 1 equiv) was dissolved in
dry DCM and allowed to cool down to 0 ꢁC. DMF (22.57 mL,
292.8 mmol, 10equiv) and thionyl chloride (4.25 mL, 58.56 mmol, 2
equiv) dissolved in dry DCM were added dropwise to the mixture
above. Then the reaction mixture was allowed to warm up to room
temperature slowly and left to stir for 2 h. The solvent was removed
under reduced pressure at room temperature. Then the residue was
redissolved in dry DCM, and was added to the DCM solution of
NH4OH (11.28 mL 292.8 mmol,10 equiv), which was cooled down to
0 ꢁC already. The ice-bath was removed and the reaction mixture
was stirred at room temperature for 4 h. The reaction mixture was
then extracted with DCM and the organic phase was washed with
saturated NaHCO3 (aq) and brine, then dried over anhydrous
Na2SO4, filtered and concentrated. The resulting residue was
further purified via silica gel chromatography to give 7a as a white
19c
19d
12.9 0.50
>1000
>1000
NTb
solid (4.88 g, 50%). 1H NMR (300 MHz, CDCl3)
d
7.67 (d, J ¼ 8.5 Hz,
20
23.5 3.20
278 11.2
2H), 6.81 (d, J ¼ 8.7 Hz, 2H), 5.87 (s, 2H), 4.29e4.10 (m, 2H), 3.50 (d,
J ¼ 12.3 Hz, 2H), 2.97 (dd, J ¼ 12.3, 3.8 Hz, 2H), 1.43 (s, 9H), 1.25 (d,
J ¼ 6.8 Hz, 6H).
14 (8.0 g, 26.69 mmol, 1 equiv), methylamine hydrochloride
(2.7 g, 40.04 mmol, 1.5 equiv), EDCI (7.68 g, 40.04 mmol, 1.5 equiv),
HOBt (3.61 g, 26.69 mmol, 1.0 equiv) and DIPEA (11.02 mL,
66.72 mmol, 2.5 equiv) were dissolved in dry DCM and the reaction
mixture was left to stir at room temperature overnight. The reac-
tion mixture was then extracted with DCM and the organic phase
was washed with saturated NaHCO3 (aq) and brine, then dried over
anhydrous Na2SO4, filtered and concentrated. The resulting residue
was further purified via silica gel chromatography to give 15 as
21a
7.60 3.50
NTb
21b
2.80 0.10
NTb
white solid (8.0 g, 95.8%). 1H NMR (300 MHz, CDCl3)
d 8.40 (d,
J ¼ 9.2 Hz,1H), 8.18 (d, J ¼ 5.5 Hz,1H), 7.81 (d, J ¼ 8.3 Hz,1H), 7.55 (d,
J ¼ 6.8 Hz, 1H), 7.51e7.42 (m, 2H), 7.24 (dd, J ¼ 9.3, 2.3 Hz, 1H), 6.78
(d, J ¼ 7.6 Hz, 2H), 6.05 (d, J ¼ 4.0 Hz, 1H), 3.05 (d, J ¼ 4.9 Hz, 3H).
7a (100 mg, 0.30 mmol, 1 equiv) and 15 (112.56 mg, 0.36 mmol,
1.2 equiv) were dissolved in 10 mL dry dioxane. Then Pd2(dba)3
(27.47 mg, 0.03 mmol, 0.1 equiv), Xantphos (34.72 mg, 0.06 mmol,
0.2 equiv) and Cs2CO3 (195.49 mg, 0.60 mmol, 2 equiv) were added
to the mixture above. The reaction mixture was left to stir at 110 ꢁC
for 5 h under nitrogen atmosphere. The reaction mixture was then
extracted with DCM and the organic phase was washed with
saturated NaHCO3 (aq) and brine, then dried over anhydrous
Na2SO4, filtered and concentrated. The resulting residue was
further purified via silica gel chromatography to give (2S,6R)-tert-
butyl 2,6-dimethyl-4-(4-(4-(5-(methylcarbamoyl)naphthalen-2-
yloxy)pyridin-2-ylcarbamoyl)phenyl)piperazine-1-carboxylate,
which was redissolved in methanol solution of 2 N HCl. The reac-
tion mixture was left to stir at room temperature for 2 h. Then the
reaction mixture was basified with sodium bicarbonate to adjust
22
10.2 3.20
30.3 5.90
1(AZD-4547)
e
0.9 0.3
8.7 1.0
a
Values are the mean SD of two independent assays.
NT ¼ not tested.
b
equiv). Then a solution of di-tert-butyl dicarbonate ester in DCM
was added dropwise. The reaction mixture was stirred at room
temperature until the starting material was consumed completely.
The reaction mixture was then extracted with DCM and the organic

M. Wei et al. / European Journal of Medicinal Chemistry 154 (2018) 9e28
17
Table 2
Biochemical and cellular activities of new compounds against FGFR1.11
Compound
4-
5-
H
FGFR1 IC50 (nM)a
4.6 0.4
KG1 cell IC50 (nM)a
24a
159 27.4
24b
H
8.2 2.5
>1000
24c
H
132 9.4
118 19.7
24d
H
67.5 31.3
504 279
24e
24f
H
H
36.1 3.8
548 303
>1000
NTb
24g
24h
H
>1000
NTb
H
22.2 0.7
116 9.5
(continued on next page)

18
M. Wei et al. / European Journal of Medicinal Chemistry 154 (2018) 9e28
Table 2 (continued )
Compound
24i
4-
H
5-
FGFR1 IC50 (nM)a
>1000
KG1 cell IC50 (nM)a
NTb
a
Values are the mean SD of two independent assays.
NT ¼ not tested.
b
pH to 7e8. The reaction mixture was then extracted with DCM and
the organic phase was washed with saturated NaHCO3 (aq) and
brine, then dried over anhydrous Na2SO4, filtered and concentrated.
The resulting residue was further purified via silica gel chroma-
tography to give 19a as white solid (106 mg, 58% for two steps). 1H
(t, J ¼ 11.3 Hz, 2H), 1.10 (d, J ¼ 6.3 Hz, 6H). 13C NMR (126 MHz, CDCl3)
d
174.85, 172.52, 170.53, 157.50, 156.45, 155.94, 152.96, 143.04,
138.64, 138.46, 133.92, 132.92, 132.10, 131.80, 129.82, 128.80, 125.33,
121.58, 121.55, 116.06, 112.92, 106.45, 58.31, 54.34, 33.58, 30.49,
24.69, 22.91. MS (ESI) m/z 524.20 [M þ H]þ. HRMS: calcd for
NMR (300 MHz, CDCl3)
d
8.45 (s,1H), 8.34 (d, J ¼ 9.2 Hz,1H), 8.09 (d,
C
31H34N5O3 [M þ H]þ, 524.2656; found 524.2670.
J ¼ 5.7 Hz, 1H), 7.97 (d, J ¼ 1.3 Hz, 1H), 7.79 (d, J ¼ 8.1 Hz, 1H), 7.70 (d,
J ¼ 8.6 Hz, 2H), 7.50 (s, 2H), 7.41 (t, J ¼ 7.6 Hz, 1H), 7.29 (dd, J ¼ 9.1,
1.7 Hz, 1H), 6.83 (d, J ¼ 8.7 Hz, 2H), 6.60 (dd, J ¼ 5.6, 1.5 Hz, 1H), 6.07
(d, J ¼ 4.3 Hz, 1H), 3.61 (d, J ¼ 11.1 Hz, 2H), 3.03 (d, J ¼ 4.7 Hz, 3H),
2.94 (s, 2H), 2.35 (t, J ¼ 11.2 Hz, 2H), 1.10 (d, J ¼ 6.2 Hz, 6H). 13C NMR
5.1.4. 6-(2-(4-((3R,5S)-3,5-dimethylpiperazin-1-yl)-2-
fluorobenzamido)pyridin-4-yloxy)-N-methyl-1-naphthamide (19d)
7d was obtained according to the similar procedure of preparing
7a starting from 5d. White solid; yield 37%; 1H NMR (300 MHz,
(126 MHz, CDCl3)
d 170.06, 166.33, 165.30, 153.75, 153.64, 152.24,
CDCl3)
d
8.78 (s, 1H), 8.11e8.01 (m, 1H), 6.88 (d, J ¼ 9.6 Hz, 2H), 5.97
149.15, 134.78, 134.76, 129.99, 128.82, 128.27, 127.90, 125.76, 124.52,
122.66, 121.49, 117.56, 113.95, 108.94, 102.31, 54.34, 50.44, 26.83,
19.68. MS (ESI) m/z 510.40 [M þ H]þ. HRMS: calcd for C30H32N5O3
[M þ H]þ, 510.2500; found 510.2513.
(s, 1H), 4.32e4.16 (m, 2H), 2.87 (dt, J ¼ 11.6, 7.9 Hz, 4H), 1.43 (s, 9H),
1.32 (d, J ¼ 7.0 Hz, 6H).
19d was obtained according to the similar procedure of pre-
paring 19a using 15 and 7d. White solid; yield 58%; 1H NMR
(300 MHz, DMSO)
d
12.56 (s, 1H), 8.54 (d, J ¼ 4.6 Hz, 1H), 8.35 (d,
5.1.2. 6-(2-(4-((3R,5S)-3,5-dimethylpiperazin-1-yl)-2-
methoxybenzamido)pyridin-4-yloxy)-N-methyl-1-naphthamide
(19b)
J ¼ 9.2 Hz, 1H), 8.29 (d, J ¼ 5.7 Hz, 1H), 8.01 (dd, J ¼ 15.5, 8.2 Hz, 2H),
7.89 (s, 1H), 7.83 (d, J ¼ 1.9 Hz, 1H), 7.67e7.55 (m, 2H), 7.46 (dd,
J ¼ 9.3, 2.1 Hz, 1H), 7.26 (dd, J ¼ 10.8, 2.0 Hz, 1H), 7.09 (t, J ¼ 8.3 Hz,
1H), 6.83 (dd, J ¼ 5.8, 1.9 Hz, 1H), 3.22 (s, 2H), 3.00 (d, J ¼ 10.1 Hz,
2H), 2.87 (d, J ¼ 3.5 Hz, 3H), 2.44 (t, J ¼ 10.6 Hz, 2H), 0.99 (d,
7b was obtained according to the similar procedure of preparing
7a starting from 5b. White solid; yield 39%; 1H NMR (300 MHz,
CDCl3)
d
8.09 (d, J ¼ 8.9 Hz, 1H), 7.57 (s, 1H), 6.56 (d, J ¼ 8.9 Hz, 1H),
J ¼ 6.1 Hz, 6H). 13C NMR (126 MHz, DMSO)
d 168.83, 165.60,164.70
6.35 (s, 1H), 5.64 (s, 1H), 4.34e4.19 (m, 2H), 3.96 (s, 3H), 3.57 (d,
J ¼ 12.5 Hz, 2H), 3.08 (dd, J ¼ 12.5, 4.3 Hz, 2H), 1.49 (s, 9H), 1.32 (d,
J ¼ 6.8 Hz, 6H).
(d, J ¼ 252 Hz), 163.27, 153.78 (d, J ¼ 8.8 Hz), 153.69, 151.42, 150.15,
134.64 (d, J ¼ 84.4 Hz), 133.41 (d, J ¼ 10.1 Hz), 129.44, 128.44, 127.60,
126.11, 124.92, 123.38 (d, J ¼ 2.5 Hz), 121.27, 117.62, 111.72 (d,
J ¼ 21.4 Hz), 109.16 (d, J ¼ 23.9 Hz), 108.86, 101.42, 59.38, 50.00,
26.22, 18.84. MS (ESI) m/z 528.30 [M þ H]þ. HRMS: calcd for
19b was obtained according to the similar procedure of pre-
paring 19a using 15 and 7b. White solid; yield 49%; 1H NMR
(300 MHz, CDCl3)
d
10.28 (s, 1H), 8.37 (d, J ¼ 9.1 Hz, 1H), 8.17 (d,
C
30H31FN5O3 [M þ H]þ, 528.2405; found 528.2418.
J ¼ 5.7 Hz, 1H), 8.10 (d, J ¼ 1.9 Hz,1H), 7.99 (d, J ¼ 8.9 Hz,1H), 7.83 (d,
J ¼ 8.1 Hz, 1H), 7.54 (d, J ¼ 6.5 Hz, 2H), 7.44 (t, J ¼ 7.6 Hz, 1H), 7.34
(dd, J ¼ 9.3, 2.1 Hz,1H), 6.63 (dd, J ¼ 5.6, 2.0 Hz,1H), 6.53 (dd, J ¼ 9.2,
1.4 Hz, 1H), 6.37 (s, 1H), 6.24 (d, J ¼ 5.0 Hz, 1H), 4.06 (s, 3H), 3.64 (d,
J ¼ 10.1 Hz, 2H), 3.06 (d, J ¼ 4.8 Hz, 3H), 3.03e2.93 (m, 2H), 2.41 (t,
J ¼ 11.2 Hz, 2H), 1.15 (d, J ¼ 6.3 Hz, 6H). 13C NMR (126 MHz, CDCl3)
5.1.5. 1-(2-hydroxyethyl)-N-(4-(5-(methylcarbamoyl)naphthalen-
2-yloxy)pyridin-2-yl)-1H-pyrazole-4-carboxamide (20)
16 (362 mg, 1.00 mmol, 2 equiv), 17 (56 mg, 0.50 mmol, 1 equiv)
and K2CO3 (138 mg, 1.00 mmol, 2 equiv) dissolved in DMF were
heated at 60 ꢁC overnight. The reaction mixture was then extracted
with ethyl acetate and the organic phase was washed with satu-
rated NaHCO3 (aq) and brine, then dried over anhydrous Na2SO4,
filtered and concentrated. The resulting residue was further puri-
fied via silica gel chromatography to give 18 as white solid (124 mg,
d
170.28, 166.25, 163.95, 159.26, 155.23, 154.30, 152.54, 149.36,
134.90, 133.71, 130.09, 128.32, 127.95, 125.87, 124.63, 121.65, 117.58,
110.93, 108.86, 107.68, 103.07, 97.04, 56.15, 54.43, 50.65, 26.97,
19.76. MS (ESI) m/z 540.30 [M þ H]þ. HRMS: calcd for C31H34N5O4
[M þ H]þ, 540.2605; found 540.2610.
63%). 1H NMR (300 MHz, CDCl3)
d 7.94 (s,1H), 7.79 (s,1H), 7.52e7.50
(m, 4H), 7.42e7.33 (m, 6H), 5.61 (s, 2H), 4.24 (t, J ¼ 4.9 Hz, 2H), 3.98
5.1.3. 6-(2-(4-((3R,5S)-3,5-dimethylpiperazin-1-yl)-2-
(t, J ¼ 5.0 Hz, 2H), 1.00 (s, 9H).
methylbenzamido)pyridin-4-yloxy)-N-methyl-1-naphthamide (19c)
7c was obtained according to the similar procedure of preparing
7a starting from 5c. White solid; yield 33%; 1H NMR (300 MHz,
18 (116 mg, 0.30 mmol, 1 equiv) and 15 (112 mg, 0.36 mmol, 1.2
equiv) were dissolved in 10 mL dry dioxane. Then Pd2(dba)3 (27 mg,
0.03 mmol, 0.1 equiv), Xantphos (34 mg, 0.06 mmol, 0.2 equiv) and
Cs2CO3 (195 mg, 0.60 mmol, 2 equiv) were added to the mixture
above. The reaction mixture was left to stir at 110 ꢁC for 5 h under
nitrogen atmosphere. The reaction mixture was then extracted
with DCM and the organic phase was washed with saturated
NaHCO3 (aq) and brine, then dried over anhydrous Na2SO4, filtered
and concentrated. The resulting residue was further purified via
silica gel chromatography to give 1-(2-(tert-butyldiphenylsilyloxy)
ethyl)-N-(4-(5-(methylcarbamoyl)naphthalen-2-yloxy)pyridin-2-
yl)–pyrazole-4-carboxamide as white solid (131 mg, 65%). 1H NMR
CDCl3)
d
7.44 (d, J ¼ 8.6 Hz, 1H), 6.70 (d, J ¼ 7.2 Hz, 2H), 5.69 (s, 2H),
4.31e4.19 (m, 2H), 3.49 (d, J ¼ 11.9 Hz, 2H), 2.96 (dd, J ¼ 12.3, 4.1 Hz,
2H), 2.52 (s, 3H), 1.49 (s, 9H), 1.32 (d, J ¼ 6.8 Hz, 6H).
19c was obtained according to the similar procedure of pre-
paring 19a using 15 and 7c. White solid; yield 52%; 1H NMR
(300 MHz, CDCl3)
d
8.28 (d, J ¼ 9.2 Hz, 1H), 8.04 (d, J ¼ 5.8 Hz, 1H),
7.89 (s, 1H), 7.80 (d, J ¼ 8.1 Hz, 1H), 7.53 (d, J ¼ 6.9 Hz, 2H), 7.42 (dd,
J ¼ 13.3, 7.9 Hz, 2H), 7.30 (s, 1H), 6.64 (dd, J ¼ 13.9, 7.0 Hz, 3H), 3.57
(d, J ¼ 10.9 Hz, 2H), 2.99 (s, 3H), 2.96e2.91 (m, 2H), 2.40 (s, 3H), 2.30

M. Wei et al. / European Journal of Medicinal Chemistry 154 (2018) 9e28
19
at 80 ꢁC overnight. The reaction mixture was then extracted with
ethyl acetate and the organic phase was washed with saturated
NaHCO3 (aq) and brine, then dried over anhydrous Na2SO4, filtered
and concentrated. The resulting residue was further purified via
silica gel chromatography to give 20 as white solid (35 mg, 42%). 1H
Table 3
Biochemical and cellular activities of new compounds against FGFR1.21
NMR (300 MHz, DMSO)
d
10.59 (s, 1H), 8.53 (d, J ¼ 4.6 Hz, 1H), 8.43
(s, 1H), 8.33 (d, J ¼ 9.3 Hz, 1H), 8.27 (d, J ¼ 5.6 Hz, 1H), 8.09 (s, 1H),
8.02 (dd, J ¼ 7.1, 1.5 Hz, 1H), 7.82 (d, J ¼ 8.3 Hz, 2H), 7.64e7.53 (m,
2H), 7.45 (dd, J ¼ 9.2, 1.7 Hz, 1H), 6.80 (d, J ¼ 5.7 Hz, 1H), 4.14 (t,
J ¼ 5.4 Hz, 2H), 3.72 (t, J ¼ 5.2 Hz, 2H), 2.86 (d, J ¼ 4.1 Hz, 3H). 13C
NMR (126 MHz, DMSO)
d 169.29, 165.84, 161.59, 154.72, 151.93,
149.99, 139.94, 135.43, 134.75, 133.57, 129.86, 128.80, 127.97, 126.52,
125.30, 121.73, 117.99, 117.89, 108.98, 102.12, 60.09, 54.93, 26.66. MS
(ESI) m/z 432.30 [M þ H]þ. HRMS: calcd for C23H22N5O4 [M þ H]þ,
432.1666; found 432.1668.
Compound
X
FGFR1 IC50, nMa KG1 cell IC50, nM (%)a
25a
1.0 0.3
1.2 0.3
1.9 0.7
<0.5
25b
25c
9.3 4.9
154 35.6
5.1.6. N-(4-(5-(methylcarbamoyl)naphthalen-2-yloxy)pyridin-2-
yl)-2-(4-methylpiperazin-1-yl)thiazole-4-carboxamide (21a)
8a (888 mg, 4.00 mmol, 1 equiv), 1-methylpiperazine (0.66 mL,
6.00 mmol, 1.5 equiv) and K2CO3 (828 mg, 6.00 mmol, 1.5 equiv)
dissolved in CH3CN were heated at 80 ꢁC for 3 h. The reaction
mixture was then extracted with DCM and the organic phase was
washed with saturated NaHCO3 (aq) and brine, then dried over
anhydrous Na2SO4, filtered and concentrated. The resulting residue
was further purified via silica gel chromatography to give 9a as a
yellowish-brown grease (710 mg, 70%). 1H NMR (300 MHz, CDCl3)
25d
25e
2.0 0.5
1.9 0.3
<4.1
5.8 0.3
d
8.11 (s, 1H), 4.28 (q, J ¼ 7.1 Hz, 2H), 3.64e3.50 (m, 4H), 2.58e2.44
(m, 4H), 2.34 (s, 3H), 1.33 (t, J ¼ 7.1 Hz, 3H).
27
28
1.6 0.6
1.2 0.2
72.2 7.2
To a solution of 9a (690 mg, 2.88 mmol, 1 equiv) dissolved in
MeOH was added 1 N NaOH (aq) (5.76 mL, 5.76 mmol, 2 equiv). The
reaction mixture was heated at 60 ꢁC for 2 h and was allowed to
cool down to room temperature. The residue was acidified with 2 N
HCl (aq) to adjust PH to 5e6. Then the solvent was removed under
reduced pressure to obtain the crude acid for next step. The ob-
tained acid (653 mg in theory, 2.88 mmol, 1 equiv) was dissolved in
dry DCM and was allowed to cool down to 0 ꢁC. DMF (2.22 mL,
28.8 mmol, 10equiv) and thionyl chloride (0.82 mL, 11.52 mmol, 4
equiv) dissolved in dry DCM were added dropwise to the mixture
above. Then the reaction mixture was allowed to warm up to room
temperature slowly and was left to stir for 2 h. The solvent was
removed under reduced pressure at room temperature. Then the
residue was redissolved in dry DCM, and was added to the DCM
solution of NH4OH (1.11 mL, 28.8 mmol, 10 equiv), which was
allowed to cool down to 0 ꢁC already. The ice-bath was removed
and the reaction mixture was left to stir at room temperature for
4 h. The reaction mixture was then extracted with DCM and the
organic phase was washed with saturated NaHCO3 (aq) and brine,
then dried over anhydrous Na2SO4, filtered and concentrated. The
resulting residue was further purified via silica gel chromatography
<4.1
30a
6.2 0.5
<4.1
30b
33a
12.8 2.5
4.1 0.8
369 31.5
<4.1
33b
6.1 1.4
<4.1
33c
33d
3.6 0.1
7.4 0.6
5.1 0.5
to give 10a (456 mg, 70%). 1H NMR (300 MHz, CDCl3)
d 7.42 (s, 1H),
7.01 (s, 1H), 5.73 (s, 1H), 3.58e3.44 (m, 4H), 2.57e2.46 (m, 4H), 2.35
(s, 3H).
54.5 8.6
21a was obtained according to the similar procedure of pre-
paring 19a using 15 and 10a. White solid; yield 69%; 1H NMR
a
Values are the mean SD of two independent assays.
(300 MHz, CDCl3)
d
9.61 (s, 1H), 8.40 (d, J ¼ 9.0 Hz, 1H), 8.19 (d,
J ¼ 5.6 Hz,1H), 8.02 (d, J ¼ 1.8 Hz,1H), 7.85 (d, J ¼ 8.2 Hz,1H), 7.57 (d,
J ¼ 7.0 Hz, 2H), 7.52e7.44 (m, 1H), 7.42 (s, 1H), 7.35 (dd, J ¼ 9.2,
2.3 Hz, 1H), 6.67 (dd, J ¼ 5.7, 2.1 Hz, 1H), 6.08 (d, J ¼ 4.1 Hz, 1H),
(300 MHz, CDCl3)
d
8.35 (d, J ¼ 9.2 Hz, 1H), 8.20 (s, 1H), 8.09 (d,
J ¼ 5.5 Hz, 1H), 7.93 (s, 2H), 7.86e7.76 (m, 2H), 7.52 (d, J ¼ 6.5 Hz,
2H), 7.45 (d, J ¼ 7.1 Hz, 4H), 7.42e7.24 (m, 8H), 6.60 (d, J ¼ 5.7 Hz,
1H), 6.07 (d, J ¼ 5.1 Hz, 1H), 4.18 (t, J ¼ 4.7 Hz, 2H), 3.92 (t, J ¼ 4.8 Hz,
2H), 3.04 (d, J ¼ 4.8 Hz, 3H), 0.93 (s, 9H).
3.63e3.51 (m, 4H), 3.10 (d, J ¼ 4.9 Hz, 3H), 2.58e2.48 (m, 4H). 13
C
NMR (126 MHz, CDCl3)
d 170.40, 170.03, 166.28, 159.65, 153.10,
152.25, 149.39, 145.86, 134.79, 130.06, 128.27, 127.92, 125.77, 124.49,
121.55, 117.58, 113.38, 109.10, 102.30, 54.07, 48.29, 46.17, 26.87. MS
(ESI) m/z 503.32 [M þ H]þ. HRMS: calcd for C26H27N6O3S [M þ H]þ,
503.1860; found 503.1872.
The intermediate obtained above (131 mg, 0.20 mmol, 1 equiv)
and CsF (89 mg, 0.59 mmol, 3 equiv) dissolved in DMF were heated

20
M. Wei et al. / European Journal of Medicinal Chemistry 154 (2018) 9e28
Fig. 3. The molecular docking study of FGFR1 with 25a (A) and 2 (B). The atoms of 25a are colored as follows: carbon, light blue; oxygen, red; nitrogen, blue; hydrogen, white.
Hydrogen bonds are shown as red dashed lines to the key residues (Glu531, Asp641, Ala564 and Glu571). The protein is shown as a cartoon and key residues are shown as sticks. The
atoms of compounds are colored as follows: carbon, grey; oxygen, red; nitrogen, blue; hydrogen, white. (For interpretation of the references to color in this figure legend, the reader
is referred to the Web version of this article.)
Table 4
preparing 19a using 15 and 10b. Light yellow solid; yield 55%; 1H
NMR (300 MHz, CDCl3)
Kinase-selectivity profile of 25a.
d
9.20 (s, 1H), 8.35 (d, J ¼ 9.2 Hz, 1H), 8.13 (d,
Tyrosine kinasea
IC50 (nM)b
Tyrosine kinasea
IC50 (nM)b
J ¼ 5.6 Hz, 1H), 7.92 (d, J ¼ 1.7 Hz, 1H), 7.79 (d, J ¼ 8.2 Hz, 1H), 7.71 (s,
1H), 7.52 (d, J ¼ 7.1 Hz, 2H), 7.46e7.38 (m, 1H), 7.29 (dd, J ¼ 9.3,
2.1 Hz, 1H), 6.61 (dd, J ¼ 5.8, 2.0 Hz, 1H), 6.01 (d, J ¼ 3.5 Hz, 1H),
3.55e3.44 (m, 4H), 3.04 (d, J ¼ 4.8 Hz, 3H), 2.47e2.37 (m, 4H), 2.29
FGFR1
FGFR2
FGFR3
FGFR4
VEGFR2
VEGFR1
PDGFR
PDGFR
c-KIT
1.0 0.3
4.5 0.7
10.6 0.4
>1000
FLT-3
EGFR
ErbB2
ErbB4
c-Src
Bcr-Abl
EPH-A2
IGF1R
e
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
e
(s, 3H). 13C NMR (126 MHz, CDCl3)
d 170.01, 166.26, 160.66, 159.65,
2.9 0.4
152.84,152.20,149.40,135.59,135.55,134.78,130.05,128.28,127.93,
125.77, 124.50, 121.54, 117.60, 109.14, 102.31, 54.04, 46.21, 45.53,
26.87. MS (ESI) m/z 487.35 [M þ H]þ. HRMS: calcd for C26H27N6O4
[M þ H]þ, 487.2088; found 487.2078.
79.3%@10 nM
44.1%@10 nM
59.5%@100 nM
54.7%@10 nM
a
b
a
EGFR, epidermal growth factor receptor; VEGFR, vascular endothelial growth
factor; PDGFR, platelet derived growth factor receptor; c-Kit, stem cell factor re-
ceptor; Flt, fms-like tyrosine kinase; c-Src, cellular Src kinase; Bcr-Abl, breakpoint
cluster region-abelson; EPHA, erythropoietin-producing hepatocellular carcinoma
receptor.
5.1.8. 2-((3R,5S)-3,5-dimethylpiperazin-1-yl)-N-(4-(5-
(methylcarbamoyl)naphthalene-2-yloxy)pyridin-2-yl)pyrimidine-
5-carboxamide (22)
13 was obtained according to the similar procedure of preparing
7a starting from 11 and (2R,6S)-2,6-dimethylpiperazine. Light yel-
b
Values are the mean SD of three independent assays.
low solid; yield 44%; 1H NMR (300 MHz, CDCl3)
d 8.72 (s, 2H), 5.68
(s, 2H), 4.71 (d, J ¼ 13.2 Hz, 2H), 4.39e4.21 (m, 2H), 3.17 (dd, J ¼ 13.0,
Table 5
Antiproliferative activity of 25a on FGFR addicted cells and BAF3/VEGFR-2 cells.
4.5 Hz, 2H), 1.49 (s, 9H), 1.19 (d, J ¼ 6.9 Hz, 6H).
22 was obtained according to the similar procedure of preparing
19a using 15 and 13. Light yellow solid; yield 51%; 1H NMR
Cell Lines
IC50 (nM)a
25a (SOMCL-286)
1 (AZD4547)
(300 MHz, CDCl3)
d
8.76 (s, 2H), 8.55 (s, 1H), 8.39 (d, J ¼ 9.2 Hz, 1H),
H1581 (FGFR1)
KG1 (FGFR1)
SNU16 (FGFR2)
RT112 (FGFR3)
UMUC14 (FGFR3)
BAF3/VEGFR-2
22.3 0.0
<0.5
<0.5
52.6 28.1
1.7 0.2
<1.5
40.0 2.8
1.9 0.0
6.2 1.4
83.8 12.5
26.9 0.2
222 33.8
8.13 (d, J ¼ 5.7 Hz, 1H), 7.96 (d, J ¼ 1.8 Hz, 1H), 7.84 (d, J ¼ 8.1 Hz, 1H),
7.56 (d, J ¼ 6.3 Hz, 2H), 7.51e7.43 (m, 1H), 7.32 (dd, J ¼ 9.2, 2.3 Hz,
1H), 6.67 (dd, J ¼ 5.7, 2.1 Hz, 1H), 6.20 (d, J ¼ 4.9 Hz, 1H), 4.76 (d,
J ¼ 11.6 Hz, 2H), 3.08 (d, J ¼ 4.8 Hz, 3H), 2.87 (s, 2H), 2.59e2.47 (m,
2H), 1.15 (d, J ¼ 6.2 Hz, 6H). 13C NMR (126 MHz, CDCl3)
d 170.17,
a
166.54, 163.04, 161.86, 157.80, 153.39, 152.32, 149.36, 134.93, 130.21,
128.50, 128.10, 125.97, 124.69, 121.61, 117.74, 115.29, 109.42, 102.85,
50.94, 50.57, 27.05, 19.61. MS (ESI) m/z 512.30 [M þ H]þ. HRMS:
calcd for C28H30N7O3 [M þ H]þ, 512.2405; found 512.2398.
Values are the mean SD of two independent assays.
5.1.7. N-(4-(5-(methylcarbamoyl)naphthalen-2-yloxy)pyridin-2-
yl)-2-(4-methylpiperazin-1-yl)oxazole-4-carboxamide (21b)
10b was obtained according to the similar procedure of pre-
paring 10a starting from 8b. White solid; yield 40%; 1H NMR
5.1.9. Methyl 6-(2-(4-((3R,5S)-3,5-dimethylpiperazin-1-yl)
benzamido)pyridin-4- yloxy)-1-naphthoate (24a)
(300 MHz, CDCl3)
(m, 4H), 2.52e2.42 (m, 4H), 2.33 (s, 3H).
d 7.76 (s, 1H), 6.71 (s, 1H), 5.70 (s, 1H), 3.56e3.49
14 (100 mg, 0.33 mmol, 1 equiv) and thionyl chloride (0.05 mL,
0.66 mmol, 2 equiv) were dissolved in anhydrous MeOH. The re-
action mixture was refluxed overnight. Then the solvent was
21b was obtained according to the similar procedure of
Table 6
Pharmacokinetics profile of 25a in SD rats.a
Route
T1/2 (h)
AUClast (h.ng/mL)
AUCINF_obs (h.ng/mL)
CL_obs (mL/min/kg)
Vss_obs (mL/kg)
F (%)
14.9
Po (3 mg/kg)
Iv (1 mg/kg)
1.85
0.897
21.4
47.9
22.7
49.0
e
e
346
16,694
a
Values are the average of three runs. Vehicle: PO: DMSO/0.5%HPMC (5/95, v/v); iv: EtOH/PEG300/NaCl (10/40/50, v/v/v). CL, clearance; Vss, volume of distribution; T1/2
half-life; AUC, area under the plasma concentration time curve; F, oral bioavailability.
,

M. Wei et al. / European Journal of Medicinal Chemistry 154 (2018) 9e28
21
Fig. 4. The inhibitory effect of 25a on tumor growth in SNU-16 xenograft model. Results are expressed as the mean SEM (n ¼ 6 for the inhibitor-treated group, n ¼ 12 for the
vehicle control group). ***p < 0.001. vs vehicle group, as determined using a t-test.
removed under reduced pressure, and the residue was redissolved
in DCM and was washed with saturated NaHCO3 (aq) and brine,
then dried over anhydrous Na2SO4, filtered and concentrated. The
resulting residue was further purified via silica gel chromatography
to give 23a as a white solid (98 mg, 95%). 1H NMR (300 MHz, CDCl3)
for next step.
Crude
7-(benzyloxy)-6-methoxyquinoline-4-carbonitrile
(183 mg, 0.63 mmol, 1 equiv) and NaOH (126 mg, 3.15 mmol, 5
equiv) were dissolved in EtOH: H2O (1:1). The reaction mixture was
refluxed for 6 h. Then the solvent was removed to minor amount
under reduced pressure. The mixture was acidified with 2 N HCl
(aq) to adjust PH to 5e6. The resulting precipitate was collected by
filtration, washed with water and dried to a constant weight to
afford 7-(benzyloxy)-6-methoxyquinoline-4-carboxylic acid. Then
the crude acid obtained was dissolved in DCM, and methylamine
hydrochloride (63.81 mg, 0.95 mmol, 1.5 equiv), EDCI (182 mg,
0.95 mmol, 1.5 equiv), HOBt (85 mg, 0.63 mmol, 1.0 equiv) and
DIPEA (0.26 mL, 1.58 mmol, 2.5 equiv) were added successively. The
reaction mixture was left to stir at room temperature overnight.
The reaction mixture was then washed with saturated NaHCO3 (aq)
and brine, then dried over anhydrous Na2SO4, filtered and
concentrated. The resulting residue was further purified via silica
gel chromatography to give 7-(benzyloxy)-6-methoxy-N-methyl-
quinoline-4-carboxamide (171 mg, 84%). 1H NMR (300 MHz, CDCl3)
d
9.06 (d, J ¼ 9.4 Hz, 1H), 8.24 (dd, J ¼ 12.0, 6.5 Hz, 2H), 7.98 (d,
J ¼ 8.1 Hz, 1H), 7.59e7.54 (m, 2H), 7.36 (d, J ¼ 9.2 Hz, 1H), 6.91e6.81
(m, 2H), 4.02 (s, 3H).
24a was obtained according to the procedure of preparing 19a
using 23a and 7a. White solid; yield 52%; 1H NMR (300 MHz, CDCl3)
d
9.02 (d, J ¼ 9.4 Hz, 1H), 8.58 (s, 1H), 8.16 (dd, J ¼ 10.2, 6.5 Hz, 2H),
8.08 (d, J ¼ 2.0 Hz, 1H), 7.96 (d, J ¼ 8.2 Hz, 1H), 7.78 (d, J ¼ 8.8 Hz,
2H), 7.58 (d, J ¼ 2.3 Hz, 1H), 7.52 (t, J ¼ 7.8 Hz, 1H), 7.42 (dd, J ¼ 9.4,
2.4 Hz, 1H), 6.89 (d, J ¼ 8.8 Hz, 2H), 6.66 (dd, J ¼ 5.7, 2.1 Hz, 1H), 4.01
(s, 3H), 3.66 (d, J ¼ 12.0 Hz, 2H), 3.03e2.97 (m, 2H), 2.40 (t,
J ¼ 11.3 Hz, 2H), 1.15 (d, J ¼ 6.3 Hz, 6H). 13C NMR (126 MHz, CDCl3)
d
167.95, 166.35, 165.42, 153.98, 153.77, 152.26, 149.31, 135.15,
132.97, 130.00, 129.17, 129.01, 128.78, 127.37, 125.69, 122.92, 122.25,
117.71, 114.11, 109.01, 102.72, 54.48, 52.41, 50.59, 19.83. MS (ESI) m/z
511.30 [M þ H]þ. HRMS: calcd for C30H31N4O4 [M þ H]þ, 511.2340;
found 511.2343.
d
8.66 (d, J ¼ 4.5 Hz, 1H), 7.62 (s, 1H), 7.49 (d, J ¼ 7.2 Hz, 2H), 7.44 (s,
1H), 7.40e7.29 (m, 3H), 7.23 (s, 1H), 6.21 (s, 1H), 5.30 (s, 2H), 4.00 (s,
3H), 3.09 (d, J ¼ 4.9 Hz, 3H).
7-(benzyloxy)-6-methoxy-N-methylquinoline-4-carboxamide
(171 mg, 0.53 mmol, 1 equiv), 10% Pd/C (20 mg) and potassium
formate (134 mg, 1.59 mmol, 3 equiv) were dissolved in 17 mL
MeOH and 10 mL H2O. The reaction mixture was left to stir at room
temperature overnight. The reaction mixture was then extracted
with DCM and the organic phase was washed with saturated
NaHCO3 (aq) and brine, then dried over anhydrous Na2SO4, filtered
and concentrated. The resulting residue was further purified via
silica gel chromatography to give 7-hydroxy-6-methoxy-N-meth-
ylquinoline-4-carboxamide (107 mg, 87%). 1H NMR (300 MHz,
5.1.10. 7-(2-(4-((3R,5S)-3,5-dimethylpiperazin-1-yl)benzamido)
pyridin-4-yloxy)-6-methoxy-N-methylquinoline-4-carboxamide
(24b)
7-(benzyloxy)-4-chloro-6-methoxyquinoline
(200 mg,
0.70 mmol, 1 equiv), Zn (CN)2 (165 mg, 1.40 mmol, 2equiv) and
Pd(PPh3)4 (81 mg, 0.07 mmol, 0.1 equiv) were dissolved in DMF and
the reaction mixture was heated at 160 ꢁC overnight. The reaction
mixture was then extracted with ethyl acetate and the organic
phase was washed with saturated NaHCO3 (aq) and brine, then
dried over anhydrous Na2SO4, filtered and concentrated to give the
crude product 7-(benzyloxy)-6-methoxyquinoline-4-carbonitrile
CDCl3)
d
8.77 (d, J ¼ 4.8 Hz, 1H), 7.49 (s, 1H), 7.32 (d, J ¼ 4.3 Hz, 1H),

22
M. Wei et al. / European Journal of Medicinal Chemistry 154 (2018) 9e28
6.17 (s, 1H), 4.81 (s, 1H), 4.01 (s, 3H), 3.11 (d, J ¼ 4.8 Hz, 3H).
2-chloro-4-nitropyridine (200 mg, 1.26 mmol, 1.2 equiv), 7-
hydroxy-6-methoxy-N-methylquinoline-4-carboxamide (244 mg,
1.05 mmol, 1 equiv) and Cs2CO3 (684 mg, 2.10 mmol, 2 equiv) were
dissolved in DMSO. The reaction mixture was left to stir at room
temperature overnight. The mixture was diluted with ice water. The
reaction mixture was then extracted with ethyl acetate and the
organic phase was washed with saturated NaHCO3 (aq) and brine,
then dried over anhydrous Na2SO4, filtered and concentrated. The
resulting residue was further purified via silica gel chromatography
to give 23b as light yellow solid (227 mg, 63%). 1H NMR (300 MHz,
1.05 (d, J ¼ 6.0 Hz, 6H). 13C NMR (126 MHz, DMSO)
d 165.91, 165.85,
155.98, 154.89, 153.37, 151.79, 149.93, 136.13, 135.32, 130.01, 127.57,
124.94, 123.78, 122.45, 122.10, 120.24, 118.02, 116.72, 113.63, 108.98,
102.25, 53.27, 50.56, 34.57, 19.04, 15.89. MS (ESI) m/z 539.44 [M þ
H]þ. HRMS: calcd for C31H35N6O3 [M þ H]þ, 539.2765; found
539.2776.
5.1.13. 4-((3R,5S)-3,5-dimethylpiperazin-1-yl)-N-(4-(4-(3-
ethylureido)-2-methoxyphenoxy)pyridin-2-yl)benzamide (24e)
23e was obtained according to the similar procedure of pre-
paring 23b starting from 1-ethyl-3-(4-hydroxy-3-methoxyphenyl)
CDCl3)
d
8.84 (d, J ¼ 4.4 Hz, 1H), 8.24 (d, J ¼ 6.2 Hz, 1H), 7.83 (d,
urea. Grey solid; yield 71%. 1H NMR (300 MHz, CDCl3)
d 8.19 (d,
J ¼ 5.8 Hz, 2H), 7.44 (d, J ¼ 4.4 Hz, 1H), 6.82 (d, J ¼ 4.8 Hz, 2H), 6.17
(s, 1H), 3.93 (s, 3H), 3.14 (d, J ¼ 4.9 Hz, 3H).
J ¼ 6.1 Hz, 1H), 7.43 (s, 1H), 6.99 (d, J ¼ 8.8 Hz, 1H), 6.74 (s, 2H), 6.69
(d, J ¼ 8.8 Hz, 1H), 6.37 (s, 1H), 4.67 (s, 1H), 3.79 (s, 3H), 3.34 (dd,
J ¼ 15.8, 8.6 Hz, 2H), 1.21 (t, J ¼ 7.2 Hz, 3H).
24b was obtained according to the procedure of preparing 19a
using 7a and 23b. White solid; yield 59%; 1H NMR (300 MHz, CDCl3)
24e was obtained according to the procedure of preparing 19a
using 23e and 7a. White solid; yield 55%; 1H NMR (300 MHz,
d
8.75 (d, J ¼ 4.4 Hz, 1H), 8.67 (s, 1H), 8.15 (d, J ¼ 5.7 Hz, 1H), 7.95 (d,
J ¼ 2.1 Hz, 1H), 7.83e7.70 (m, 4H), 7.36 (d, J ¼ 4.4 Hz, 1H), 6.88 (d,
J ¼ 8.8 Hz, 2H), 6.71 (dd, J ¼ 5.7, 2.3 Hz, 1H), 6.53 (d, J ¼ 4.6 Hz, 1H),
3.93 (s, 3H), 3.72 (d, J ¼ 10.7 Hz, 2H), 3.22 (s, 2H), 3.09 (d, J ¼ 4.8 Hz,
3H), 2.80 (t, J ¼ 11.7 Hz, 2H), 1.35 (d, J ¼ 6.3 Hz, 6H). 13C NMR
DMSO)
d
10.37 (s, 1H), 8.73 (s, 1H), 8.16 (d, J ¼ 5.6 Hz, 1H), 7.90 (d,
J ¼ 8.3 Hz, 2H), 7.67 (s, 1H), 7.46 (s, 1H), 7.05e6.89 (m, 4H), 6.59 (d,
J ¼ 5.6 Hz, 1H), 6.23 (t, J ¼ 5.6 Hz, 1H), 3.80 (d, J ¼ 11.4 Hz, 2H), 3.69
(s, 3H), 3.17e3.07 (m, 2H), 2.90 (s, 2H), 2.33 (t, J ¼ 11.3 Hz, 2H),
(126 MHz, DMSO)
d
167.52, 165.85, 165.61, 154.79, 152.98, 151.59,
1.14e1.01 (m, 9H). 13C NMR (126 MHz, DMSO)
d 166.65, 165.74,
149.77, 149.02, 145.75, 144.81, 140.53, 130.04, 123.89, 122.94, 121.47,
119.93, 113.87, 108.24, 105.93, 101.42, 56.49, 52.07, 50.73, 26.66,
17.88. MS (ESI) m/z 541.34 [M þ H]þ. HRMS: calcd for C30H33N6O4
[M þ H]þ, 541.2558; found 541.2549.
155.62, 154.63, 153.39, 151.68, 149.34, 140.08, 135.46, 113.60, 110.14,
107.59, 103.53, 100.86, 55.96, 50.57, 34.42, 19.18, 15.96. MS (ESI) m/z
519.41 [M þ H]þ. HRMS: calcd for C28H35N6O4 [M þ H]þ, 519.2714;
found 519.2725.
5.1.11. 6-(2-(4-((3R,5S)-3,5-dimethylpiperazin-1-yl)benzamido)
pyridin-4-yloxy)-N-ethyl-3,4-dihydroquinoline-1(2H)-carboxamide
(24c)
23c was obtained according to the similar procedure of pre-
paring 23b starting from N-ethyl-6-hydroxy-3,4-dihydroquinoline-
1(2H)-carboxamide. White solid, yield 40%; 1H NMR (300 MHz,
5.1.14. N-(4-(3,5-dimethoxyphenethoxy)pyridin-2-yl)-4-((3R,5S)-
3,5-dimethylpiperazin-1-yl)benzamide (24f)
2,4-dichloropyridine (200 mg, 1.35 mmol, 1.1 equiv), 2-(3,5-
dimethoxyphenyl)ethanol (224 mg, 1.23 mmol, 1 equiv) and t-
BuOK (276 mg, 2.46 mmo, 2 equiv) were refluxed in t-BuOH over-
night. The reaction mixture was filtered and concentrated under
reduced pressure. The residue was redissolved in DCM and washed
with brine, then dried over anhydrous Na2SO4, filtered and
concentrated. The resulting residue was further purified via silica
gel chromatography to give 23f as white solid (257 mg, 71%). 1H
CDCl3)
d
8.22 (d, J ¼ 5.5 Hz, 1H), 7.40 (d, J ¼ 9.1 Hz, 1H), 6.92e6.85
(m, 2H), 6.83e6.77 (m, 2H), 5.05 (s, 1H), 3.79e3.70 (m, 2H),
3.39e3.26 (m, 2H), 2.74 (t, J ¼ 6.7 Hz, 2H), 1.94 (dt, J ¼ 12.8, 6.6 Hz,
2H), 1.16 (t, J ¼ 7.2 Hz, 3H).
24c was obtained according to the procedure of preparing 19a
using 23c and 7a. White solid; yield 57%; 1H NMR (300 MHz, CDCl3)
NMR (300 MHz, CDCl3)
d
7.98 (d, J ¼ 5.5 Hz, 1H), 6.85e6.77 (m, 1H),
6.69 (s, 1H), 6.38 (d, J ¼ 1.6 Hz, 2H), 6.29 (s, 1H), 4.46 (t, J ¼ 7.0 Hz,
d
8.51 (s, 1H), 8.14 (d, J ¼ 5.6 Hz, 1H), 7.98 (s, 1H), 7.79 (d, J ¼ 8.4 Hz,
2H), 3.72 (s, 6H), 2.96 (t, J ¼ 7.0 Hz, 2H).
2H), 7.34 (d, J ¼ 8.2 Hz, 1H), 6.93 (t, J ¼ 8.0 Hz, 4H), 6.64 (d,
J ¼ 5.7 Hz, 1H), 5.10 (s, 1H), 3.77 (t, J ¼ 5.7 Hz, 2H), 3.69 (d,
J ¼ 12.0 Hz, 2H), 3.39e3.26 (m, 2H), 3.05 (s, 2H), 2.76 (t, J ¼ 6.6 Hz,
2H), 2.49 (s, 2H), 1.99e1.88 (m, 2H), 1.22e1.14 (m, 9H). 13C NMR
24f was obtained according to the similar procedure of pre-
paring 19a using 23f and 7a. White solid; yield 68%; 1H NMR
(300 MHz, CDCl3)
d
8.05 (d, J ¼ 5.7 Hz, 1H), 7.79e7.68 (m, 3H), 7.13
(dd, J ¼ 5.8, 1.5 Hz, 1H), 7.10 (s, 1H), 6.91 (d, J ¼ 8.9 Hz, 2H), 6.46 (d,
J ¼ 2.0 Hz, 2H), 6.33 (s, 1H), 4.51 (t, J ¼ 6.9 Hz, 2H), 3.78 (s, 6H), 3.68
(d, J ¼ 10.2 Hz, 2H), 3.03 (t, J ¼ 6.9 Hz, 4H), 2.42 (t, J ¼ 11.2 Hz, 2H),
(126 MHz, CDCl3)
d 166.60, 165.25, 156.68, 153.65, 153.42, 150.23,
149.03, 136.59, 134.06, 128.86, 124.70, 123.13, 121.34, 118.79, 114.23,
108.82, 101.97, 53.89, 50.74, 43.45, 35.78, 27.27, 23.60, 19.08, 15.35.
MS (ESI) m/z 529.50 [M þ H]þ. HRMS: calcd for C30H37N6O3 [M þ
H]þ, 529.2922; found 529.2931.
1.16 (d, J ¼ 6.3 Hz, 6H). 13C NMR (126 MHz, CDCl3)
d 165.65, 165.20,
160.90, 153.84, 147.74, 147.67, 141.12, 128.95, 123.00, 114.19, 108.47,
107.22, 99.85, 98.61, 66.64, 55.45, 54.48, 50.61, 36.03, 19.82. MS
(ESI) m/z 491.30 [M þ H]þ. HRMS: calcd for C28H35N4O4 [M þ H]þ,
491.2653; found 491.2659.
5.1.12. 4-((3R,5S)-3,5-dimethylpiperazin-1-yl)-N-(4-(5-(3-
ethylureido)naphthalen-2-yloxy)pyridin-2-yl)benzamide (24d)
23d was obtained according to the similar procedure of pre-
paring 23b starting from 1-ethyl-3-(6-hydroxynaphthalen-1-yl)
5.1.15. N-(4-(2,6-dichloro-3,5-dimethoxyphenethoxy)pyridin-2-yl)-
4-((3R,5S)-3,5-dimethylpiperazin-1-yl)benzamide (24g)
urea. White solid, yield 55%; 1H NMR (300 MHz, CDCl3)
d
8.21 (d,
23g was obtained according to the similar procedure of pre-
paring 23f starting from 2-(2,6-dichloro-3,5-dimethoxyphenyl)
J ¼ 6.2 Hz, 1H), 8.13 (d, J ¼ 9.1 Hz, 1H), 7.81 (d, J ¼ 7.0 Hz, 1H), 7.59 (s,
1H), 7.56e7.43 (m, 3H), 7.16 (dd, J ¼ 9.1, 2.3 Hz, 1H), 6.82e6.80 (m,
2H), 3.28 (dd, J ¼ 12.7, 6.7 Hz, 2H), 1.12 (t, J ¼ 7.3 Hz, 3H).
ethanol. White solid, yield 66%. 1H NMR (300 MHz, CDCl3)
d 8.02 (d,
J ¼ 5.5 Hz, 1H), 6.85 (d, J ¼ 5.5 Hz, 1H), 6.75 (s, 1H), 6.50 (s, 1H), 4.51
24d was obtained according to the procedure of preparing 19a
using 23d and 7a. White solid; yield 65%; 1H NMR (300 MHz,
(t, J ¼ 7.1 Hz, 2H), 3.91 (s, 6H), 3.48 (t, J ¼ 7.1 Hz, 2H).
24g was obtained according to the similar procedure of pre-
paring 19a using 23g and 7a. White solid; yield 72%; 1H NMR
DMSO)
d 10.48 (s, 1H), 8.62 (s, 1H), 8.30e8.19 (m, 2H), 8.00 (d,
J ¼ 7.4 Hz, 1H), 7.88 (d, J ¼ 10.9 Hz, 3H), 7.70 (s, 1H), 7.56 (d,
J ¼ 8.0 Hz, 1H), 7.45 (t, J ¼ 10.6 Hz, 2H), 6.93 (d, J ¼ 8.6 Hz, 2H),
6.83e6.76 (m, 1H), 6.61 (s, 1H), 3.76 (d, J ¼ 11.6 Hz, 2H), 3.24e3.11
(m, 2H), 2.84 (s, 2H), 2.26 (t, J ¼ 10.9 Hz, 2H), 1.11 (t, J ¼ 7.2 Hz, 3H),
(300 MHz, CDCl3)
d
8.04 (d, J ¼ 5.7 Hz, 1H), 7.83 (s, 1H), 7.74 (d,
J ¼ 8.7 Hz, 2H), 7.20 (d, J ¼ 5.6 Hz,1H), 7.03 (s,1H), 6.89 (d, J ¼ 8.7 Hz,
2H), 6.48 (s, 1H), 4.49 (t, J ¼ 7.2 Hz, 2H), 3.90 (s, 6H), 3.67 (d,
J ¼ 10.7 Hz, 2H), 3.49 (t, J ¼ 7.2 Hz, 2H), 3.08e2.94 (m, 2H), 2.41 (t,

M. Wei et al. / European Journal of Medicinal Chemistry 154 (2018) 9e28
23
J ¼ 11.2 Hz, 2H), 1.16 (d, J ¼ 6.3 Hz, 6H). 13C NMR (126 MHz, CDCl3)
153.49, 152.03, 148.99, 134.61, 134.58, 129.80, 128.66, 128.12, 127.73,
125.62, 124.41, 122.89, 121.32, 117.42, 113.87, 108.79, 102.16, 54.52,
47.19, 45.87, 26.68. MS (ESI) m/z 496.30 [M þ H]þ. HRMS: calcd for
d
165.72, 165.11, 154.47, 153.79, 147.71, 136.21, 128.97, 122.98, 115.85,
114.13, 108.54, 99.82, 96.20, 63.79, 56.66, 54.47, 50.59, 31.59, 19.83.
MS (ESI) m/z 559.20 [M þ H]þ. HRMS: calcd for C28H33Cl2N4O4 [M þ
H]þ, 559.1873; found 559.1882.
C
29H30N5O3 [M þ H]þ, 496.2343; found 496.2355.
5.1.19. N-Methyl-6-(2-(4-morpholinobenzamido)pyridin-4-yloxy)-
1-naphthamide (25b)
5.1.16. N-(5-(2,6-difluoro-3,5-dimethoxybenzyloxy)pyridin-2-yl)-
4-((3R,5S)-3,5-dimethylpiperazin-1-yl)benzamide (24h)
7f was obtained according to the similar procedure of preparing
7a starting from ethyl 4-fluorobenzoate and morpholine. White
6-chloropyridin-3-ol (356 mg, 2.76 mmol, 1.2 equiv), 2,6-
difluoro-3,5-dimethoxybenzyl
methanesulfonate
(649 mg,
solid; yield 60%; 1H NMR (300 MHz, DMSO)
d
7.76 (d, J ¼ 8.9 Hz,
2.30 mmol, 1 equiv) and K2CO3 (795 mg, 5.75 mmol, 2 equiv) dis-
solved in DMF were heated at 60 ꢁC overnight. The reaction mixture
was then extracted with ethyl acetate and the organic phase was
washed with saturated NaHCO3 (aq) and brine, then dried over
anhydrous Na2SO4, filtered and concentrated. The resulting residue
was further purified via silica gel chromatography to give 23h
2H), 7.70 (s, 1H), 7.04 (s, 1H), 6.94 (d, J ¼ 8.9 Hz, 2H), 3.79e3.66 (m,
4H), 3.25e3.15 (m, 4H).
25b was obtained according to the similar procedure of pre-
paring 19a using 15 and 7f. White solid; yield 74%; 1H NMR
(300 MHz, CDCl3)
d
8.51 (s, 1H), 8.41 (d, J ¼ 9.2 Hz, 1H), 8.16 (d,
J ¼ 5.7 Hz, 1H), 8.04 (s, 1H), 7.83 (dd, J ¼ 19.0, 8.2 Hz, 3H), 7.58 (d,
J ¼ 6.8 Hz, 2H), 7.48 (t, J ¼ 7.7 Hz, 1H), 7.36 (d, J ¼ 9.1 Hz, 1H), 6.90 (d,
J ¼ 8.4 Hz, 2H), 6.67 (d, J ¼ 5.8 Hz, 1H), 6.09 (s, 1H), 3.90e3.80 (m,
4H), 3.33e3.24 (m, 4H), 3.10 (d, J ¼ 4.7 Hz, 3H). 13C NMR (126 MHz,
(759 mg, 65%). 1H NMR (300 MHz, CDCl3)
7.25e7.16 (m, 2H), 6.62 (t, J ¼ 8.1 Hz, 1H), 5.11 (s, 2H), 3.83 (s, 6H).
24h was obtained according to the similar procedure of pre-
paring 19a using 23h and 7a. White solid; yield 67%; 1H NMR
d
8.07 (d, J ¼ 2.7 Hz, 1H),
CDCl3)
d 170.22, 166.55, 165.39, 153.99, 153.81, 152.38, 149.30,
(300 MHz, CDCl3)
d
8.47 (s, 1H), 8.33 (d, J ¼ 9.1 Hz, 1H), 8.04 (s, 1H),
134.94, 130.19, 128.99, 128.45,128.08, 125.95, 124.70,123.80, 121.68,
117.76, 114.04, 109.16, 102.50, 66.75, 47.87, 27.04. MS (ESI) m/z
483.30 [M þ H]þ. HRMS: calcd for C28H27N4O4 [M þ H]þ, 483.2027;
found 483.2024.
7.82 (d, J ¼ 8.7 Hz, 2H), 7.41 (dd, J ¼ 9.0, 2.6 Hz, 1H), 6.92 (d,
J ¼ 8.7 Hz, 2H), 6.67 (t, J ¼ 8.1 Hz, 1H), 5.17 (s, 2H), 3.89 (s, 6H), 3.68
(d, J ¼ 11.4 Hz, 2H), 3.01 (s, 2H), 2.41 (t, J ¼ 11.2 Hz, 2H), 1.16 (d,
J ¼ 6.3 Hz, 6H). 13C NMR (126 MHz, CDCl3)
d 165.07, 153.66, 151.59,
146.41, 145.06 (dd, J ¼ 244.2, 5.7 Hz), 143.69 (dd, J ¼ 11.4, 3.8 Hz),
135.91, 128.90, 125.14, 123.34, 114.63, 114.22, 113.61 (t, J ¼ 17.3 Hz),
101.98, 59.64, 57.44, 54.58, 50.60, 19.81. MS (ESI) m/z 513.30 [M þ
H]þ. HRMS: calcd for C27H31F2N4O4 [M þ H]þ, 513.2308; found
513.2309.
5.1.20. 6-(2-(4-(2,6-dimethylmorpholino)benzamido)pyridin-4-
yloxy)-N-methyl-1-naphthamide (25c)
7g was obtained according to the similar procedure of preparing
7a
dimethylmorpholine. White solid; yield 55%; 1H NMR (300 MHz,
CDCl3)
starting
from
ethyl
4-fluorobenzoate
and
2,6-
d
7.67 (d, J ¼ 8.7 Hz, 2H), 6.82 (d, J ¼ 8.7 Hz, 2H), 5.68 (s, 2H),
5.1.17. N-(5-(1-(3,5-dicholoropyridin-4-yl)ethoxy)pyridin-2-yl)-4-
((3R,5S)-3,5-dimethylpiperazin-1-yl)benzamide (24i)
23i was obtained according to the similar procedure of prepar-
ing 23h starting from 1-(3,5-dicholoropyridin-4-yl)ethyl meth-
anesulfonate. White solid, yield 61%. 1H NMR (300 MHz, CDCl3)
3.82e3.64 (m, 2H), 3.52 (d, J ¼ 11.6 Hz, 2H), 2.44 (t, J ¼ 11.4 Hz, 2H),
1.21 (d, J ¼ 6.3 Hz, 6H).
25c was obtained according to the similar procedure of pre-
paring 19a using 15 and 7g. White solid; yield 61%; 1H NMR
(300 MHz, CDCl3)
d
8.48 (s, 1H), 8.41 (d, J ¼ 9.2 Hz, 1H), 8.16 (d,
d
8.44 (s, 1H), 7.97 (d, J ¼ 2.4 Hz, 1H), 7.18e7.09 (m, 1H), 6.01e5.91
J ¼ 5.8 Hz, 1H), 8.04 (d, J ¼ 1.6 Hz, 1H), 7.86 (d, J ¼ 8.3 Hz, 1H), 7.78
(d, J ¼ 8.8 Hz, 2H), 7.58 (d, J ¼ 7.0 Hz, 2H), 7.52e7.44 (m, 1H), 7.36
(dd, J ¼ 9.2, 2.0 Hz, 1H), 6.89 (d, J ¼ 8.7 Hz, 2H), 6.67 (dd, J ¼ 5.7,
1.8 Hz, 1H), 6.07 (d, J ¼ 5.5 Hz, 1H), 3.83e3.71 (m, 2H), 3.59 (d,
J ¼ 11.4 Hz, 2H), 3.11 (d, J ¼ 4.8 Hz, 3H), 2.52 (t, J ¼ 11.3 Hz, 2H), 1.28
(m, 1H), 1.79 (d, J ¼ 6.7 Hz, 3H).
24i was obtained according to the similar procedure of prepar-
ing 19a using 23i and 7a. White solid; yield 61%; 1H NMR (300 MHz,
CDCl3)
d
8.44 (s, 2H), 8.42 (s, 1H), 8.24 (d, J ¼ 9.0 Hz, 1H), 7.91 (d,
J ¼ 2.2 Hz, 1H), 7.78 (d, J ¼ 8.7 Hz, 2H), 7.23 (d, J ¼ 9.3 Hz, 1H), 6.90
(d, J ¼ 8.8 Hz, 2H), 5.98 (q, J ¼ 6.4 Hz, 1H), 3.67 (d, J ¼ 10.9 Hz, 2H),
3.08e2.97 (m, 2H), 2.43 (t, J ¼ 11.2 Hz, 2H), 1.79 (d, J ¼ 6.6 Hz, 3H),
(d, J ¼ 6.2 Hz, 6H). 13C NMR (126 MHz, CDCl3)
d 170.24, 166.54,
165.42, 153.84, 153.60, 152.34, 149.26, 134.91, 130.16, 129.17, 129.00,
128.44, 128.06, 125.94, 124.71, 123.34, 121.66, 117.76, 113.98, 109.13,
102.46, 71.57, 53.15, 27.01, 19.16. MS (ESI) m/z 511.50 [M þ H]þ.
HRMS: calcd for C30H31N4O4 [M þ H]þ, 511.2340; found 511.2337.
1.17 (d, J ¼ 6.3 Hz, 6H). 13C NMR (126 MHz, CDCl3)
d 164.98, 153.52,
150.00, 149.07, 146.40, 143.59, 135.62, 131.21, 128.87, 125.23, 123.42,
114.71, 114.37, 72.65, 54.26, 50.78, 29.84, 19.41, 18.78. MS (ESI) m/z
500.30 [M þ H]þ. HRMS: calcd for C25H28Cl2N5O2 [M þ H]þ,
500.1615; found 500.1622.
5.1.21. N-Methyl-6-(2-(4-(3-methylpiperazin-1-yl)benzamido)
pyridin-4-yloxy)-1-naphthamide (25d)
7h was obtained according to the similar procedure of preparing
7a starting from ethyl 4-fluorobenzoate and 2-methylpiperazine.
5.1.18. N-Methyl-6-(2-(4-(4-methylpiperazin-1-yl)benzamido)
pyridin-4-yloxy)-1-naphthamide (25a)
White solid; yield 43%; 1H NMR (300 MHz, CDCl3)
d 7.72 (d,
7e was obtained according to the similar procedure of preparing
7a starting from ethyl 4-fluorobenzoate and 1-methylpiperazine.
J ¼ 8.5 Hz, 2H), 6.83 (d, J ¼ 8.5 Hz, 2H), 5.81 (s, 2H), 4.33 (s, 1H), 3.93
(d, J ¼ 13.0 Hz, 1H), 3.64 (d, J ¼ 12.3 Hz, 1H), 3.51 (d, J ¼ 12.4 Hz, 1H),
3.34e3.23 (m, 1H), 3.12 (dd, J ¼ 12.5, 3.7 Hz, 1H), 2.92 (td, J ¼ 11.9,
3.8 Hz, 1H), 1.48 (s, 9H), 1.24 (s, 3H).
White solid; yield 54%; 1H NMR (300 MHz, CDCl3)
d 7.72 (d,
J ¼ 8.8 Hz, 2H), 6.89 (d, J ¼ 8.8 Hz, 2H), 5.76 (s, 2H), 3.42e3.26 (m,
4H), 2.68e2.49 (m, 4H), 2.35 (s, 3H).
25d was obtained according to the similar procedure of pre-
25a was obtained according to the similar procedure of pre-
paring 19a using 15 and 7e. White solid; yield 70%; 1H NMR
paring 19a using 15 and 7h. White solid; yield 49%; 1H NMR
(300 MHz, DMSO)
d
10.50 (s, 1H), 8.53 (d, J ¼ 4.7 Hz, 1H), 8.34 (d,
(300 MHz, CDCl3)
d
8.59 (s, 1H), 8.38 (d, J ¼ 9.2 Hz, 1H), 8.13 (d,
J ¼ 9.4 Hz, 1H), 8.28 (d, J ¼ 5.6 Hz, 1H), 8.02 (dd, J ¼ 6.9, 1.9 Hz, 1H),
7.90 (s, 1H), 7.88e7.83 (m, 2H), 7.81 (d, J ¼ 2.0 Hz, 1H), 7.65e7.54 (m,
2H), 7.45 (dd, J ¼ 9.2, 2.3 Hz, 1H), 6.94 (d, J ¼ 9.1 Hz, 2H), 6.81 (dd,
J ¼ 5.8, 2.1 Hz, 1H), 3.74 (t, J ¼ 10.2 Hz, 2H), 3.01 (d, J ¼ 10.6 Hz, 1H),
2.86 (s, 3H), 2.84e2.64 (m, 3H), 2.42e2.32 (m, 1H), 1.06 (d,
J ¼ 5.6 Hz,1H), 8.02 (s,1H), 7.83 (d, J ¼ 8.0 Hz,1H), 7.75 (d, J ¼ 8.3 Hz,
2H), 7.54 (d, J ¼ 7.7 Hz, 2H), 7.44 (t, J ¼ 7.6 Hz, 1H), 7.33 (d, J ¼ 9.1 Hz,
1H), 6.88 (d, J ¼ 8.4 Hz, 2H), 6.65 (d, J ¼ 5.6 Hz, 1H), 6.28 (d,
J ¼ 3.9 Hz, 1H), 3.34 (s, 4H), 3.05 (d, J ¼ 4.6 Hz, 3H), 2.56 (s, 4H), 2.35
(s, 3H). 13C NMR (126 MHz, CDCl3)
d
169.93, 166.18, 165.16, 153.56,
J ¼ 6.3 Hz, 3H). 13C NMR (126 MHz, DMSO)
d 169.29, 165.90, 165.79,

24
M. Wei et al. / European Journal of Medicinal Chemistry 154 (2018) 9e28
154.94,153.73,152.02,149.97,135.44,134.76,130.00,129.86,128.82,
127.95, 126.52, 125.28, 122.53, 121.69, 117.88, 113.61, 109.05, 102.31,
54.14, 50.49, 47.00, 45.17, 26.66, 19.28. MS (ESI) m/z 496.30 [M þ
H]þ. HRMS: calcd for C29H30N5O3 [M þ H]þ, 496.2343; found
496.2336.
NMR (126 MHz, DMSO) d 169.27, 166.38, 165.87, 154.61, 151.99,
150.66, 150.10, 135.43, 134.76, 132.22, 129.87, 128.85, 128.69, 127.98,
127.13, 126.54, 125.31, 121.67, 117.91, 109.41, 102.52, 60.48, 58.40,
54.16, 41.57, 32.47, 26.66. MS (ESI) m/z 525.40 [M þ H]þ. HRMS:
calcd for C31H33N4O4 [M þ H]þ, 525.2496; found 525.2490.
5.1.22. 6-(2-(4-(4-(2-hydroxyethyl)piperazin-1-yl)benzamido)
pyridin-4-yloxy)-N-methyl-1-naphthamide (25e)
5.1.25. 6-(2-(4-((4-hydroxytetrahydro-2H-pyran-4-yl)
methylamino)benzamido)pyridin-4-yloxy)-N-methyl-1-
7i was obtained according to the similar procedure of preparing
7a starting from ethyl 4-fluorobenzoate and 2-(piperazin-1-yl)
naphthamide (30a)
4-nitrobenzamide (166 mg, 1.00 mmol, 1 equiv) and 15 (406 mg,
1.3 mmol, 1.3 equiv) were dissolved in 10 mL dry dioxane. Then
Pd2(dba)3 (92 mg, 0.10 mmol, 0.1 equiv), Xantphos (116 mg,
0.20 mmol, 0.2 equiv) and Cs2CO3 (652 mg, 2.00 mmol, 2 equiv)
were added to the mixture above. Then the reaction mixture was
left to stir at 110 ꢁC for 5 h under nitrogen atmosphere. The reaction
mixture was then extracted with DCM and the organic phase was
washed with saturated NaHCO3 (aq) and brine, then dried over
anhydrous Na2SO4, filtered and concentrated. The resulting residue
was further purified via silica gel chromatography to give N-
methyl-6-(2-(4-nitrobenzamido)pyridin-4-yloxy)-1-naphthamide
as light yellow solid (340 mg, 76.8%). 1H NMR (300 MHz, DMSO)
ethanol. White solid; yield 48%; 1H NMR (300 MHz, DMSO)
d 7.73
(d, J ¼ 8.7 Hz, 2H), 7.69 (s, 1H), 7.01 (s, 1H), 6.92 (d, J ¼ 8.5 Hz, 2H),
4.44 (t, J ¼ 5.5 Hz, 1H), 3.53 (q, J ¼ 5.9 Hz, 2H), 3.23 (s, 3H), 2.53 (s,
3H), 2.43 (t, J ¼ 6.1 Hz, 2H).
25e was obtained according to the similar procedure of pre-
paring 19a using 15 and 7i. White solid; yield 51%; 1H NMR
(300 MHz, CDCl3)
d
8.46 (s, 1H), 8.34 (d, J ¼ 9.2 Hz, 1H), 8.10 (d,
J ¼ 5.7 Hz,1H), 7.98 (d, J ¼ 2.0 Hz,1H), 7.80 (d, J ¼ 8.8 Hz,1H), 7.72 (d,
J ¼ 8.8 Hz, 2H), 7.52 (d, J ¼ 6.7 Hz, 2H), 7.45e7.39 (m, 1H), 7.29 (dd,
J ¼ 9.4, 2.2 Hz, 1H), 6.84 (d, J ¼ 8.9 Hz, 2H), 6.61 (dd, J ¼ 5.5, 2.0 Hz,
1H), 6.04 (d, J ¼ 2.0 Hz, 1H), 3.62 (t, J ¼ 5.1 Hz, 2H), 3.35e3.25 (m,
4H), 3.04 (d, J ¼ 4.9 Hz, 3H), 2.65e2.59 (m, 4H), 2.58e2.53 (m, 2H).
d
11.28 (s, 1H), 8.52 (d, J ¼ 3.1 Hz, 1H), 8.38e8.24 (m, 4H), 8.16 (d,
13C NMR (126 MHz, CDCl3)
d
170.71, 166.77, 165.84, 153.79, 153.73,
J ¼ 8.7 Hz, 2H), 8.03 (d, J ¼ 6.9 Hz, 1H), 7.83 (d, J ¼ 5.9 Hz, 2H), 7.60
(d, J ¼ 6.9 Hz, 2H), 7.46 (d, J ¼ 10.0 Hz, 1H), 6.88 (d, J ¼ 5.6 Hz, 1H),
2.86 (d, J ¼ 4.4 Hz, 3H).
152.04,148.99,134.79, 134.75, 130.09, 128.99, 128.23, 127.98,125.97,
124.93, 122.98, 121.58, 117.88, 114.21, 109.11, 102.38, 59.86, 58.14,
52.87, 47.44, 26.76. MS (ESI) m/z 526.30 [M þ H]þ. HRMS: calcd for
N-methyl-6-(2-(4-nitrobenzamido)pyridin-4-yloxy)-1-
C
30H32N5O4 [M þ H]þ, 526.2449; found 526.2460.
naphthamide (340 mg, 0.77 mmol,1 equiv), Zn (251 mg, 3.84 mmol,
5 equiv) and NH4Cl (82 mg, 1.54 mmol, 2 equiv) were dissolved in
15 mL MeOH and 15 mL THF. The reaction mixture was left to stir at
room temperature overnight. After completion, the mixture was
filtered and the filtrate was concentrated and further purified via
silica gel chromatography to give 29 as white solid (233 mg, 73.5%).
5.1.23. N-Methyl-6-(2-(4-(piperazin-1-yl)benzamido)pyridin-4-
yloxy)-1-naphthamide (27)
26 was obtained according to the similar procedure of preparing
7a starting from tert-butyl 4-(4-(ethoxycarbonyl)phenyl)piperi-
dine-1-carboxylate [42]. White solid; yield 54%; 1H NMR (300 MHz,
1H NMR (300 MHz, CDCl3)
d
8.47 (s, 1H), 8.40 (d, J ¼ 9.7 Hz, 1H), 8.15
CDCl3)
d
7.70 (d, J ¼ 8.1 Hz, 2H), 7.22 (d, J ¼ 9.2 Hz, 2H), 5.97 (s, 1H),
(d, J ¼ 5.9 Hz, 1H), 8.03 (s, 1H), 7.85 (d, J ¼ 8.6 Hz, 1H), 7.70 (d,
J ¼ 7.6 Hz, 2H), 7.58 (d, J ¼ 7.8 Hz, 2H), 7.47 (t, J ¼ 7.9 Hz, 1H), 7.35 (d,
J ¼ 8.5 Hz, 1H), 6.68 (d, J ¼ 7.7 Hz, 3H), 6.06 (s, 1H), 4.05 (s, 2H), 3.10
(d, J ¼ 4.3 Hz, 3H).
5.64 (s, 1H), 4.20 (d, J ¼ 10.0 Hz, 2H), 2.84e2.57 (m, 3H), 1.77 (d,
J ¼ 12.8 Hz, 2H), 1.65e1.58 (m, 1H), 1.55 (d, J ¼ 4.5 Hz, 1H), 1.42 (s,
9H).
27 was obtained according to the similar procedure of preparing
29 (82 mg, 0.20 mmol, 1 equiv), 1,6-dioxaspiro[2.5]octane
19a using 15 and 26. White solid; yield 52%; 1H NMR (300 MHz,
(67 mg, 0.60 mmol, 3 equiv) and DIPEA (0.17 mL, 1.00 mmol, 5
equiv) were heated at 75 ꢁC in EtOH for 4 d. The solvent was
removed under reduced pressure, and the residue was redissolved
in DCM. The mixture was washed with saturated NH4Cl (aq) and
brine, then dried over anhydrous Na2SO4, filtered and concentrated.
The resulting residue was further purified via silica gel chroma-
tography to give 30a as white solid (34 mg, 32%). 1H NMR
DMSO)
d
10.83 (s, 1H), 8.55 (d, J ¼ 4.7 Hz, 1H), 8.33 (dd, J ¼ 10.3,
7.5 Hz, 2H), 8.03 (dd, J ¼ 7.2, 1.9 Hz, 1H), 7.95 (d, J ¼ 8.2 Hz, 2H), 7.86
(d, J ¼ 2.1 Hz, 1H), 7.82 (d, J ¼ 2.2 Hz, 1H), 7.64e7.55 (m, 2H), 7.46
(dd, J ¼ 9.2, 2.4 Hz, 1H), 7.33 (d, J ¼ 8.2 Hz, 2H), 6.85 (dd, J ¼ 5.7,
2.2 Hz, 1H), 3.36 (s, 2H), 3.04e2.89 (m, 3H), 2.86 (d, J ¼ 4.5 Hz, 3H),
1.96e1.77 (m, 4H). 13C NMR (126 MHz, DMSO)
d 169.28, 166.29,
165.89, 154.58, 151.96, 150.12, 149.42, 135.41, 134.76, 132.59, 129.88,
128.86, 127.99, 127.00, 126.54, 125.34, 121.68, 117.94, 109.44, 102.53,
43.90, 39.41, 29.61, 26.66. MS (ESI) m/z 481.30 [M þ H]þ. HRMS:
calcd for C29H29N4O3 [M þ H]þ, 481.2234; found 481.2247.
(300 MHz, DMSO)
d
10.29 (s, 1H), 8.53 (d, J ¼ 5.3 Hz, 1H), 8.33 (d,
J ¼ 9.2 Hz, 1H), 8.26 (d, J ¼ 5.8 Hz, 1H), 8.02 (d, J ¼ 7.6 Hz, 1H), 7.79
(dd, J ¼ 16.6, 10.5 Hz, 3H), 7.64e7.53 (m, 2H), 7.45 (d, J ¼ 9.1 Hz, 1H),
6.78 (d, J ¼ 5.0 Hz,1H), 6.66 (d, J ¼ 8.2 Hz, 2H), 6.18 (t, J ¼ 5.6 Hz,1H),
4.54 (s, 1H), 3.61 (d, J ¼ 6.8 Hz, 4H), 3.07 (d, J ¼ 5.6 Hz, 2H), 2.86 (d,
J ¼ 4.1 Hz, 3H), 1.64e1.54 (m, 2H), 1.49e1.40 (m, 2H). 13C NMR
5.1.24. 6-(2-(4-(1-(2-hydroxyethyl)piperidin-4-yl)benzamido)
pyridin-4-yloxy)-N-methyl-1-naphthamide (28)
(126 MHz, DMSO) d 169.29, 166.02, 165.73, 155.10, 153.25, 152.05,
27 (52 mg, 0.11 mmol,
1
equiv), 2-bromoethanol (24
m
L,
149.92, 135.44, 134.75, 130.10, 129.85, 128.79, 127.93, 126.51, 125.26,
121.70, 119.97, 117.85, 111.38, 108.85, 102.16, 68.62, 63.31, 53.62,
35.61, 26.65. MS (ESI) m/z 527.30 [M þ H]þ. HRMS: calcd for
0.33 mmol, 3 equiv) and K2CO3 (30 mg, 0.22 mmol, 2 equiv) were
heated at 80 ꢁC in DMF for 5 h. The reaction mixture was then
extracted with ethyl acetate and the organic phase was washed
with saturated NaHCO3 (aq) and brine, then dried over anhydrous
Na2SO4, filtered and concentrated. The resulting residue was
further purified via silica gel chromatography to give 28 as white
C
30H31N4O5 [M þ H]þ, 527.2289; found 527.2294.
5.1.26. 6-(2-(4-acrylamidobenzamido)pyridin-4-yloxy)-N-methyl-
1-naphthamide (30b)
solid (35 mg, 62%). 1H NMR (300 MHz, DMSO)
d
10.78 (s, 1H), 8.53
29 (94 mg, 0.23 mmol, 1 equiv) and DIPEA (76
equiv) were dissolved in dry DCM and allowed to cool down to 0 ꢁC.
Acryloyl chloride (24 L, 0.30 mmol,1.3 equiv) was added dropwise.
mL, 0.46 mmol, 2
(d, J ¼ 4.5 Hz, 1H), 8.32 (dd, J ¼ 11.0, 7.5 Hz, 2H), 8.03 (dd, J ¼ 7.0,
2.1 Hz, 1H), 7.91 (d, J ¼ 8.2 Hz, 2H), 7.86 (d, J ¼ 2.0 Hz, 1H), 7.81 (d,
J ¼ 2.1 Hz, 1H), 7.65e7.55 (m, 2H), 7.46 (dd, J ¼ 9.3, 2.4 Hz, 1H), 7.34
(d, J ¼ 8.2 Hz, 2H), 6.84 (dd, J ¼ 5.7, 2.3 Hz, 1H), 3.56 (s, 2H), 3.08 (s,
m
The reaction mixture was left to stir at room temperature over-
night. The mixture was washed with saturated NH4Cl (aq) and
brine, then dried over anhydrous Na2SO4, filtered and concentrated.
2H), 2.86 (d, J ¼ 3.4 Hz, 3H), 2.59 (s, 2H), 2.27 (s, 3H), 1.77 (s, 4H). 13
C

M. Wei et al. / European Journal of Medicinal Chemistry 154 (2018) 9e28
25
The resulting residue was further purified via silica gel chroma-
tography to give 30b as white solid (86 mg, 80%). 1H NMR
(300 MHz, DMSO)
C
29H26F2N4NaO4 [M þ Na]þ, 555.1814; found 555.1824.
d
10.76 (s, 1H), 10.41 (s, 1H), 8.53 (d, J ¼ 4.0 Hz,
5.1.29. 6-(2-(4-(2-(3-fluoroazetidin-1-yl)ethoxy)benzamido)
pyridin-4-yloxy)-N-methyl-1-naphthamide (33c)
1H), 8.41e8.25 (m, 2H), 8.03 (dd, J ¼ 6.6, 2.5 Hz, 1H), 7.97 (d,
J ¼ 8.5 Hz, 2H), 7.83 (dd, J ¼ 8.8, 2.3 Hz, 2H), 7.75 (d, J ¼ 8.3 Hz, 2H),
7.65e7.55 (m, 2H), 7.46 (dd, J ¼ 8.8, 1.6 Hz, 1H), 6.84 (d, J ¼ 5.6 Hz,
1H), 6.46 (dd, J ¼ 17.4, 9.9 Hz, 1H), 6.29 (d, J ¼ 16.3 Hz, 1H), 5.81 (d,
32c was obtained according to the similar procedure of pre-
paring 32a using 3-fluoroazetidine hydrochloride. White solid;
yield 59%; 1H NMR (300 MHz, CDCl3)
d
7.77 (d, J ¼ 8.6 Hz, 2H), 6.92
J ¼ 10.7 Hz, 1H), 2.86 (s, 3H). 13C NMR (126 MHz, DMSO)
d 169.28,
(d, J ¼ 8.6 Hz, 2H), 5.77 (s, 2H), 5.28e5.21 (m, 0.5H), 5.09e5.02 (m,
0.5H), 4.04 (t, J ¼ 5.2 Hz, 2H), 3.85e3.73 (m, 2H), 3.38e3.21 (m, 2H),
2.93 (t, J ¼ 5.2 Hz, 2H).
165.88, 163.97, 154.64, 151.96, 150.01, 142.84, 135.44, 134.76, 132.05,
129.87, 129.56, 128.85, 128.14, 127.98, 126.54, 125.31, 121.69, 118.92,
117.94, 109.36, 102.46, 26.66. MS (ESI) m/z 467.30 [M þ H]þ. HRMS:
calcd for C27H22N4NaO4 [M þ Na]þ, 489.1533; found 489.1535.
33c was obtained according to the similar procedure of pre-
paring 19a using 15 and 32c. White solid; yield 57%; 1H NMR
(300 MHz, CDCl3)
d
8.59 (s, 1H), 8.40 (d, J ¼ 9.2 Hz, 1H), 8.14 (d,
5.1.27. N-methyl-6-(2-(4-(2-(pyrrolidin-1-yl)ethoxy)benzamido)
pyridin-4-yloxy)-1-naphthamide (33a)
J ¼ 5.7 Hz, 1H), 8.03 (s, 1H), 7.91e7.77 (m, 3H), 7.61e7.53 (m, 2H),
7.46 (t, J ¼ 7.6 Hz,1H), 7.34 (dd, J ¼ 9.1, 2.1 Hz,1H), 6.93 (d, J ¼ 8.7 Hz,
2H), 6.66 (dd, J ¼ 5.6, 2.0 Hz, 1H), 6.16 (d, J ¼ 4.5 Hz, 1H), 5.28e5.19
(m, 1H), 5.09e5.00 (m, 1H), 4.04 (t, J ¼ 5.2 Hz, 2H), 3.85e3.72 (m,
2H), 3.33 (dd, J ¼ 8.2, 6.0 Hz, 1H), 3.25 (dd, J ¼ 8.2, 6.1 Hz, 1H), 3.08
(d, J ¼ 4.8 Hz, 3H), 2.93 (t, J ¼ 5.1 Hz, 2H). 13C NMR (126 MHz, CDCl3)
4-hydroxybenzamide (4.80 g, 35 mmol,
1
equiv), 1,2-
dibromoethane (18 mL, 210 mmol, 6 equiv) and K2CO3 (5.33 g,
38.5 mmol, 1.1 equiv) were heated at 50 ꢁC in acetone under nir-
togen atomasphere. After completion, the mixture was filtered and
the filtrate was concentrated and further purified via silica gel
chromatography to give 4-(2-bromoethoxy)benzamide (4.36 g,
d
170.22, 166.54, 165.39, 162.11, 153.69, 152.33, 149.36, 134.92,
51%). 1H NMR (300 MHz, CDCl3)
d
7.79 (d, J ¼ 7.7 Hz, 2H), 6.95 (d,
130.17, 129.32, 128.47, 128.08, 126.53, 125.96, 124.72, 121.63, 117.75,
114.63, 109.25, 102.61, 82.66 (d, J ¼ 205.4 Hz), 67.17, 62.71(d,
J ¼ 20.2 Hz), 58.19, 27.02. MS (ESI) m/z 515.30 [M þ H]þ. HRMS:
calcd for C29H27FN4NaO4 [M þ Na]þ, 537.1909; found 537.1917.
J ¼ 7.8 Hz, 2H), 5.74 (s, 2H), 4.35 (t, J ¼ 6.1 Hz, 2H), 3.66 (t, J ¼ 6.2 Hz,
2H).
4-(2-bromoethoxy)benzamide (1.00 g, 4.10 mmol,
1 equiv),
pyrrolidine (321 mg, 4.51 mmol, 1.10 equiv) and K2CO3 (1.13 g,
8.20 mmol, 2 equiv) were refluxed in CH3CN overnight. After
completion, the mixture was filtered and the filtrate was concen-
trated and further purified via silica gel chromatography to give 32a
5.1.30. 6-(2-(4-((1-aminocyclopropyl)methoxy)benzamido)
pyridin-4-yloxy)-N-methyl-1-naphthamide (33d)
Ethyl 4-hydroxybenzoate (322 mg, 1.50 mmol, 1 equiv), tert-
butyl 1-(iodomethyl)cyclopropylcarbamate (668 mg, 2.25 mmol,
1.5 equiv) and K2CO3 (621 mg, 4.50 mmol, 3 equiv) in acetone were
refluxed overnight. After completion, the mixture was filtered and
the filtrate was concentrated and further purified via silica gel
chromatography to give compound 31 (322 mg, 64%). 1H NMR
as white solid (595 mg, 62%). 1H NMR (300 MHz, CDCl3)
d 7.76 (d,
J ¼ 8.8 Hz, 2H), 6.93 (d, J ¼ 8.8 Hz, 2H), 6.00 (s, 2H), 4.14 (t, J ¼ 5.9 Hz,
2H), 2.91 (t, J ¼ 5.9 Hz, 2H), 2.63 (s, 4H), 1.80 (dt, J ¼ 6.5, 3.2 Hz, 4H).
33a was obtained according to the similar procedure of pre-
paring 19a using 15 and 32a. White solid; yield 67%; 1H NMR
(300 MHz, CDCl3)
d
8.56 (s, 1H), 8.41 (d, J ¼ 9.2 Hz, 1H), 8.15 (d,
(300 MHz, CDCl3)
d
7.92 (d, J ¼ 8.7 Hz, 2H), 6.84 (d, J ¼ 8.7 Hz, 2H),
J ¼ 5.7 Hz,1H), 8.03 (d, J ¼ 1.9 Hz,1H), 7.84 (t, J ¼ 9.4 Hz, 3H), 7.58 (d,
J ¼ 6.0 Hz, 2H), 7.52e7.44 (m, 1H), 7.35 (dd, J ¼ 9.2, 2.3 Hz, 1H), 6.97
(d, J ¼ 8.7 Hz, 2H), 6.67 (dd, J ¼ 5.7, 2.1 Hz, 1H), 6.13 (d, J ¼ 3.6 Hz,
1H), 4.16 (t, J ¼ 5.8 Hz, 2H), 3.10 (d, J ¼ 4.9 Hz, 3H), 2.92 (t, J ¼ 5.8 Hz,
4.28 (q, J ¼ 7.1 Hz, 2H), 3.96 (s, 2H), 1.37 (s, 9H), 1.32 (t, J ¼ 7.2 Hz,
3H), 0.86 (d, J ¼ 8.4 Hz, 4H).
32d was obtained according to the similar procedure of pre-
paring 7a starting from 31. White solid; yield 51%; 1H NMR
2H), 2.64 (s, 4H), 1.82 (s, 4H). 13C NMR (126 MHz, CDCl3)
d 170.19,
(300 MHz, DMSO)
d
7.81 (d, J ¼ 8.7 Hz, 3H), 7.30 (s, 1H), 7.14 (s, 1H),
166.51, 165.36, 162.26, 153.66, 152.33, 149.33, 134.90, 130.16, 129.25,
128.43, 128.05, 126.32, 125.92, 124.67, 121.63, 117.72, 114.73, 109.22,
102.55, 67.40, 54.98, 54.87, 27.00, 23.62. MS (ESI) m/z 511.30 [M þ
H]þ. HRMS: calcd for C30H31N4O4 [M þ H]þ, 511.2340; found
511.2332.
6.94 (d, J ¼ 8.5 Hz, 2H), 4.02 (s, 2H), 1.36 (s, 9H), 0.75 (d, J ¼ 15.7 Hz,
4H).
33d was obtained according to the similar procedure of pre-
paring 19a using 15 and 32d. White solid; yield 55%; 1H NMR
(300 MHz, CDCl3)
d
8.54 (s, 1H), 8.41 (d, J ¼ 9.0 Hz, 1H), 8.16 (d,
J ¼ 5.7 Hz, 1H), 8.03 (s, 1H), 7.84 (t, J ¼ 8.4 Hz, 3H), 7.57 (s, 2H), 7.47
(t, J ¼ 7.4 Hz,1H), 7.35 (d, J ¼ 9.1 Hz, 1H), 6.95 (d, J ¼ 8.4 Hz, 2H), 6.67
(d, J ¼ 5.4 Hz, 1H), 6.10 (s, 1H), 3.89 (s, 2H), 3.10 (d, J ¼ 4.6 Hz, 3H),
5.1.28. 6-(2-(4-(2-(3,3-difluoroazetidin-1-yl)ethoxy)benzamido)
pyridin-4-yloxy)-N-methyl-1-naphthamide (33b)
0.77 (s, 2H), 0.65 (s, 2H). 13C NMR (126 MHz, CDCl3)
d 170.20,166.53,
32b was obtained according to the similar procedure of pre-
paring 32a using 3,3-difluoroazetidine hydrochloride. White solid;
165.34, 162.52, 153.66, 152.34, 149.33,134.92, 130.19, 129.32, 128.47,
128.08, 126.44, 125.95, 124.69, 121.65, 117.75, 114.73, 109.30, 102.61,
76.65, 27.02, 13.01. MS (ESI) m/z 483.20 [M þ H]þ. HRMS: calcd for
yield 55%; 1H NMR (300 MHz, CDCl3)
d
7.77 (d, J ¼ 8.7 Hz, 2H), 6.92
(d, J ¼ 8.7 Hz, 2H), 5.78 (s, 2H), 4.09 (t, J ¼ 5.1 Hz, 2H), 3.73 (t,
J ¼ 12.0 Hz, 4H), 3.00 (t, J ¼ 5.0 Hz, 2H).
C
28H27N4O4 [M þ H]þ, 483.2027; found 483.2036.
33b was obtained according to the similar procedure of pre-
paring 19a using 15 and 32b. White solid; yield 63%; 1H NMR
(300 MHz, CDCl3)
d
8.60 (s, 1H), 8.40 (d, J ¼ 9.2 Hz, 1H), 8.14 (d,
5.2. Molecular docking
J ¼ 5.7 Hz, 1H), 8.03 (s, 1H), 7.83 (t, J ¼ 8.6 Hz, 3H), 7.62e7.53 (m,
2H), 7.46 (t, J ¼ 7.6 Hz, 1H), 7.34 (dd, J ¼ 9.3, 2.0 Hz, 1H), 6.92 (d,
J ¼ 8.6 Hz, 2H), 6.67 (dd, J ¼ 5.5, 1.8 Hz, 1H), 6.15 (d, J ¼ 4.5 Hz, 1H),
4.08 (t, J ¼ 5.0 Hz, 2H), 3.73 (t, J ¼ 12.0 Hz, 4H), 3.08 (d, J ¼ 4.8 Hz,
Docking study was performed using Maestro 9.3. X-ray crystal
structure of FGFR1 (PDB ID: 4RWL) was downloaded from RCSB
Protein Date Bank and prepared by Protein Preparation Wizard
Workflow in the schrӧdinger program suite. Compound 25a was
prepared by Ligand Preparation and docked into the defined
binding site without constraint. Based on the Glide-score, top
ranking compounds were submitted. The final result for molecular
docking was visualized by using PyMol.
3H), 2.99 (t, J ¼ 4.7 Hz, 2H). 13C NMR (126 MHz, CDCl3)
d 170.31,
166.67,165.42,162.06,153.76,152.45,149.46,135.04,130.30,129.46,
128.60, 128.21, 126.79, 126.07, 124.82, 121.76, 117.87, 116.95 (t,
J ¼ 275.9 Hz), 114.71, 109.39, 102.73, 67.74, 66.02, (t, J ¼ 22.7 Hz),
57.46, 27.14. MS (ESI) m/z 555.30 [M þ Na]þ. HRMS: calcd for

26
M. Wei et al. / European Journal of Medicinal Chemistry 154 (2018) 9e28
5.3. ELISA kinase assay
guidelines on animal care and approved by the Institute Animal
Care and Use Committee at Shanghai Institute of Materia Medica.
The effects of indicated compounds on the activities of various
tyrosine kinases were determined using enzyme-linked immuno-
sorbent assays (ELISAs) with purified recombinant proteins. Briefly,
5.6. In vivo antitumor activity assay
20
m
g/mL poly (Glu, Tyr)
(Sigma, St Louis, MO, USA) was pre-
4:1
Female nude mice (4e6 weeks old) were housed and main-
tained under specific pathogen-free conditions. Animal procedures
were performed according to institutional ethical guidelines of
coated in 96-well plates as a substrate. A 50-
ATP solution diluted in kinase reaction buffer (50 mM HEPES [pH
7.4], 50 mM MgCl2, 0.5 mM MnCl2, 0.2 mM Na3VO4, and 1 mM DTT)
mL aliquot of 10 mM
animal care. The tumor cells at a density of 5 ꢂ 106 in 200
mL were
was added to each well; 1 mL of various concentrations of indicated
injected subcutaneously (s.c.) into the right flank of nude mice and
then allowed to grow to 700e800 mm3, which was defined as a
well-developed tumor. Subsequently, the well-developed tumors
were cut into 1-mm [3] fragments and transplanted s. c. into the
right flank of nude mice using a trocar. When the tumor volume
reached 100e150 mm3, the mice were randomly assigned into a
vehicle control group (n ¼ 12) and treatment groups (n ¼ 6 per
group). The control groups were given vehicle alone, and the
treatment groups received 25a at the indicated doses via oral
administration once daily for 3 weeks. The sizes of the tumors were
measured twice per week using a microcaliper. Tumor volume
(TV) ¼ (length ꢂ width [2])/2, and the individual relative tumor
volume (RTV) was calculated as follows: RTV ¼ Vt/V0, where Vt is
the volume on a particular day and V0 is the volume at the begin-
ning of the treatment. The RTV was shown on indicated days as the
median RTV SEM indicated for groups of mice. Percent (%) inhi-
bition (TGI) values were measured on the final day of study for the
drug-treated mice compared with vehicle-treated mice and were
calculated as 100 ꢂ {1 - [(VTreated Final day - VTreated Day 0)/(VControl Final
day - VControl Day 0)].
compounds diluted in 1% DMSO (v/v) (Sigma, St Louis, MO, USA)
were then added to each reaction well. 1% DMSO (v/v) was used as
the negative control. The kinase reaction was initiated by the
addition of purified tyrosine kinase proteins diluted in 49 mL of
kinase reaction buffer. After incubation for 60 min at 37 ꢁC, the
plate was washed three times with phosphate-buffered saline (PBS)
containing 0.1% Tween 20 (T-PBS). Anti-phosphotyrosine (PY99)
antibody (100
added. After a 30-min incubation at 37 ꢁC, the plate was washed
three times, and 100 L horseradish peroxidase-conjugated goat
mL; 1:500, diluted in 5 mg/mL BSA T-PBS) was then
m
anti-mouse IgG (1:1000, diluted in 5 mg/mL BSA T-PBS) was added.
The plate was then incubated at 37 ꢁC for 30 min and washed 3
times. A 100-
2 mg/mL o-phenylenediamine in 0.1 M citrate buffer (pH 5.5) was
added. The reaction was terminated by the addition of 50 L of
mL aliquot of a solution containing 0.03% H2O2 and
m
2 M H2SO4 as the color changed, and the plate was analyzed using a
multi-well spectrophotometer (SpectraMAX190, from Molecular
Devices, Palo Alto, CA, USA) at 490 nm. The inhibition rate (%) was
calculated using the following equation: [1-(A490/A490 con-
trol)] ꢂ 100%. The IC50 values were calculated from the inhibition
curves in two separate experiments.
All animal experiments were performed according to the insti-
tutional ethical guidelines on animal care and approved by the
Institute Animal Care and Use Committee at Shanghai Institute of
Materia Medica (2017-04-DJ-26).
5.4. Cell proliferation assay
Human lung cancer cell line NCI-H1581, human acute myelog-
enous leukemia cell line KG-1, human gastric cancer cell line SNU-
16 were purchased from American Type Culture Collection (ATCC,
Manassas, VA, USA). Human bladder cancer cell line RT-112 and
Mouse Pre-B cell line BaF3 were purchased from DSMZ-German
collection of microorganisms and cell cultures. Human bladder
cancer cell line UMUC-14 was purchased from European Collection
of Cell Cultures (ECACC). All the cell lines were routinely main-
tained in media according to the suppliers' recommendations and
authenticated via short tandem repeats analysis by Genesky Bio-
pharma Technology (last tested in 2016).
Cells were seeded in 96-well cell culture plates. On the day
when seeding, the cells were exposed to various concentrations of
compounds and further cultured for 72 h at 37 ꢁC. Cell proliferation
was then determined using Cell Counts Kit-8 (CCK8) or the thiazolyl
blue tetrazolium bromide (MTT, from Sigma-Aldrich, St. Louis, MO,
USA) assay. The IC50 values were calculated by concentration-
response curve fitting using the four-parameter method.
5.7. Statistical analysis
Data from in vitro assays are presented as the mean SD,
whereas data from in vivo efficacy evaluations are presented as the
mean SE. Significance was determined by Student's t-test, and
differences were considered statistically significant at *p < 0.05, **p
< 0.01, ***p < 0.001.
Acknowledgement
This work was supported by grants from National Natural Sci-
ence Foundation of China (81703327, 81773565, 81430080,
81773762). Supporting from the International Cooperative Program
(GJHZ1622) and Key Program of the Frontier Science (160621) of
the Chinese Academy of Sciences, the Shanghai Commission of
Science and Technology (16XD1404600, 14431900400), the
“Personalized Medicines – Molecular Signature-based Drug Dis-
covery and Development”, Strategic Priority Research Program of
the Chinese Academy of Sciences (No. XDA12020000) are also
highly appreciated.
5.5. Pharmacokinetic parameters in rats
Compound 25a was administrated to male Sprague-Dawley (SD)
rats (n ¼ 3) either as a solution of 1 mg/kg in EtOH/PEG300/NaCl
(10/40/50, v/v/v) intravenously or as a suspension of 3 mg/kg in
DMSO/0.5% HPMC (5/95, v/v) orally by gavage. Blood samples were
collected at 0.25, 0.5, 1, 2, 4, 6, 8 and 24 h following oral dosing. The
blood samples were placed on wet ice, and serum was collected
after 2 centrifugation. Serum samples were frozen and stored
at ꢀ20 ꢁC. The serum samples were analyzed utilizing HPLC-
coupled tandem mass spectrometry (LC-MS/MS). All animal ex-
periments were performed according to the institutional ethical
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.ejmech.2018.05.005.
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