Discovery of 2-aminopyridines bearing a pyridone moiety as potent ALK inhibitors to overcome the crizotinib-resistant mutants

Article abstract of DOI:10.1016/j.ejmech.2019.111734

Despite the initial benefit demonstrated in clinical setting with ALK inhibitors, the challenging resistant mutants (F1174L, L1196M and G1202R) invariably developed. In this work, a series of 2-aminopyridine derivatives were designed and synthesized by C-5 position incorporation of a 2-pyridone moiety and bioisosteric replacement of the C-3 position linkers. Optimization of the 2-aminopyridine derivatives led to the identification of hit 18d displaying a significant growth inhibition against a variety of ALK-addicted cancer cells. Especially in the case of ALK-positive Karpas-299 cell, 18d exhibited excellent anti-proliferative potency with an IC₅₀ value of about 40 nM. Moreover, 18d demonstrated encouraging activities against wild-type ALK (19 nM), ROS1 (2.3 nM) as well as challenging crizotinib-resistant ALK^L1196M and ALK^G1202R mutants with IC₅₀ values of 45 nM and 22 nM, respectively. Additionally flow cytometric analysis indicates that 18d inhibited Karpas-299 cell viability via G1 phase arrest. Taken together, this work provided a promising ALK inhibitor to circumvent the clinical crizotinib-resistant mutants.

Full text of DOI:10.1016/j.ejmech.2019.111734

European Journal of Medicinal Chemistry 183 (2019) 111734
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Discovery of 2-aminopyridines bearing a pyridone moiety as potent
ALK inhibitors to overcome the crizotinib-resistant mutants
Wenteng Chen 1, Xiao Guo 1, Can Zhang, Di Ke, Guolin Zhang*, Yongping Yu**
Zhejiang Province Key Laboratory of Anti-Cancer Research, College of Pharmaceutical Sciences, Zhejiang University, 310058, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Despite the initial benefit demonstrated in clinical setting with ALK inhibitors, the challenging resistant
mutants (F1174L, L1196M and G1202R) invariably developed. In this work, a series of 2-aminopyridine
derivatives were designed and synthesized by C-5 position incorporation of a 2-pyridone moiety and
bioisosteric replacement of the C-3 position linkers. Optimization of the 2-aminopyridine derivatives led
to the identification of hit 18d displaying a significant growth inhibition against a variety of ALK-addicted
cancer cells. Especially in the case of ALK-positive Karpas-299 cell, 18d exhibited excellent anti-
proliferative potency with an IC50 value of about 40 nM. Moreover, 18d demonstrated encouraging ac-
tivities against wild-type ALK (19 nM), ROS1 (2.3 nM) as well as challenging crizotinib-resistant
ALKL1196M and ALKG1202R mutants with IC50 values of 45 nM and 22 nM, respectively. Additionally flow
cytometric analysis indicates that 18d inhibited Karpas-299 cell viability via G1 phase arrest. Taken
together, this work provided a promising ALK inhibitor to circumvent the clinical crizotinib-resistant
mutants.
Received 26 June 2019
Received in revised form
22 September 2019
Accepted 23 September 2019
Available online 24 September 2019
Keywords:
ALK inhibitors
Crizotinib-resistance
Solvent-front mutation
Pyridone
Linker
© 2019 Published by Elsevier Masson SAS.
1. Introduction
approved next generation of ALK inhibitors, ceritinib (2, Novartis)
[18e20], brigatinib (3, Takeda) [21e24], alectinib (4, Roche)
Anaplastic lymphoma kinase (ALK) is a transmembrane receptor
tyrosine kinase (RTK) belonging to the insulin receptor superfamily
[1]. Deregulation of ALK gene is involved in the carcinogenesis
process of various human cancers such as anaplastic large cell
lymphomas (ALCL) [2], non-small cell lung cancer (NSCLC) [3],
neuroblastoma [4] and inflammatory myofibroblastic tumors (IMT)
[5], frequently in the forms of fusion with other oncogenes (NMP,
EML4, TIM, etc) [6], mutation [7], gene amplification [8,9] or protein
overexpression [10]. Hence, ALK becomes a promising drug target
for cancer therapy [11,12]. To date, a respectable number of small
molecule inhibitors were approved for modulating ALK activity.
Despite the demonstrated clinical efficacy of 1st generation ALK
inhibitor crizotinib (1，Pfizer) for the treatment of advanced
NSCLC, patients develop disease relapse within one year post-
treatment [13]. The short-lived clinical outcome is partly due to
emergence of point mutations within the ALK kinase domain
(F1174L, F1174C, L1196M, G1202R, etc) [14e17]. The recently
[25e27] and lorlatinib (5, Pfizer) [28e30] exhibited a promising
clinical efficacy against the crizotinib-resistant mutants including
the gatekeeper mutation L1196M. However, the frequency of the
G1202R mutation was found an increase to 21e43% in patients
during the treatment of two already launched inhibitors, alectinib
and ceritinib [31]. Therefore, there is still room to develop new ALK
inhibitors, particularly ones capable of combating the challenging
L1196M and G1202R mutations (see Fig. 1).
Generally, many currently available ALK inhibitors are ATP-
competitive type I TKIs, which feature an ATP adenine equivalent
kinase hinge binder with Met1199 and Glu1197. Additionally, an aryl
fragment extended into the inside hydrophobic pocket, a functional
linker for conformational interaction with the gate-keeper and an
extra motif extended to the solvent area [32e34]. We conducted a
molecular modeling on crizotinib with ALKG1202R/L1196M by incor-
L1196M
porating the Arg1202 mutation using ALK
-crizotinib co-
crystal structure (PDB 2YFX) as a template [32]. As shown in
Fig. 2A, the solvent-exposure motif of crizotinib sits atop and in
close proximity to the residue Arg1202, which possibly accounts for
the moderate potency of crizotinib on G1202R mutation. Thus, it is
questioned whether the exploration of a new “tail” for the solvent-
front region might give rise to new inhibitors for combating
* Corresponding author.
** Corresponding author.
E-mail addresses: guolinzhang@zju.edu.cn (G. Zhang), yyu@zju.edu.cn (Y. Yu).
These two authors contributed equally to this work.
1
https://doi.org/10.1016/j.ejmech.2019.111734
0223-5234/© 2019 Published by Elsevier Masson SAS.

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W. Chen et al. / European Journal of Medicinal Chemistry 183 (2019) 111734
Fig. 1. The structure of ALK inhibitors in clinic.
Fig. 2. Design strategies for target compounds. (A) Binding conformation of crizotinib with ALK G1202R/L1196M. (B) The design of new ALK inhibitor analogues with pyridone moiety.
solvent-front mutation ALKG1202R. Based on this hypothesis, a new
“tail” pyridone motif was designed and proposed to trigger addi-
tional interaction with the solvent-front mutation G1202R (Fig. 2B,
analogue 8). Furthermore, the linkers at the C-3 position of the 2-
aminopyridine core were also investigated. A flexible sp3 (O-
linker and S-linker, analogue 18 and 25) and rigid sp2 (amide-linker,
analogue 13) were designed for the compatibility of conformational
tuning the inhibitors and gate-keeper mutation L1196M.
commercially available alkyl halides, to give the intermediates 3 as
shown in Scheme 1. The benzyl alcohol derivatives 4 were easily
converted to ether derivatives 5 using classical Mitsunobu coupling
[34]. Chemo-selective reduction of the nitro group with iron and
acetic acid (HOAc) afforded amine 6, and regioselective bromina-
tion gave the desired product 7 [35,36]. Then palladium-catalyzed
boronation of 7 followed by a sequential Suzuki coupling with
aryl bromides 2 or 3 delivered compounds 8a-8l.
The construction of 2-amino-5-bromo-N-(1-(2, 6-dichloro-3-
fluorophenyl) ethyl) nicotinamide 12 began with 1-(2, 6-dichloro-
3-fluorophenyl) ethan-1-ol 4d, as presented in Scheme 2. The
mesylate 9 was easily obtained from benzyl alcohol 4d via MsCl
protection [37]. Substitution of 9 with sodium azide, followed by
reduction of the resulting azides with Zn/NH4Cl [38], gave the
desired amine 11. Condensation of amine 11 with 2-amino-5-
2. Results and discussion
2.1. Chemistry
The synthesis commenced with hydrolysis of 2-amino-4-
bromopyridine 1, followed by substitution with various

W. Chen et al. / European Journal of Medicinal Chemistry 183 (2019) 111734
3
Scheme 1. Synthesis of compounds 8a-8l.
Scheme 2. Synthesis of compounds 13a-13b.
bromonicotinic acid delivered the amide 12. Next, introduction of
the pyridone fragments 2 or 3 at the C-5 position of 12 was ach-
ieved by similar Suzuki coupling shown in Scheme 1. Compound 12
was subsequently transformed to products 13a-13b.
The chiral enantiomer 18a-18g were similarly obtained as
shown in Scheme 1 And (S)-1-(2, 6-dichloro-3-fluorophenyl)
ethan-1-ol 14 was utilized as the alternative starting material. A
Mitsunobu reaction of chiral alcohol (S)-14 and 3-hydroxy-2-
nitropyridine proceeded in good yield with complete inversion of
stereochemistry to give the desired ether (R)-15 [35]. Similarly,
subsequent chemo-selective reduction of the nitro group and
region-selective bromination gave the key intermediate 17 [35,36].

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W. Chen et al. / European Journal of Medicinal Chemistry 183 (2019) 111734
Finally, treatment of 17 with aryl bromides 2 or 3 under Suzuki
coupling conditions furnished the desired products 18a-18g
(Scheme 3).
Compared to the ether 5 and 15, the thioether 22 was not ob-
tained from 2-nitropyridine-3-thiol via conventional Mitsunobu
reaction. As shown in Scheme 4, intermediate 22 was obtained via
dichloro-3-fluorophenyl group was kept as the R2 substituent
while the stereochemistry of the O-linker and modification on the
N-position of pyridone ring (R1 part) were further investigated. The
results are shown in Table 2. Compared to the racemates (8g, 8h
and 8l), the corresponding R-enantiomers 18a-18c displayed 2- to
5- fold more activity against Karpas-299 (8g vs 18a, 8h vs 18b and 8l
vs 18c). The encouraging results suggested the R-configuration is
more effective in affecting the proliferation of Karpas-299.
Appending alkanes at the N-position of pyridone rings (18b, 18e-
18g) displayed good tolerance in anti-proliferative activities
hydrolysis of the dimethylcarbamothioate 21 followed by
a
sequential substitution with mesylate 19 [37,39]. And the dime-
thylcarbamothioate 21 was prepared from 3-hydroxyl-2-
nitropyridine with dimethylthiocarbamoyl chloride and DABCO in
DMF following with a tautomerism [37]. Next, reduction of the
nitro group and bromination of 23 delivered key intermediate 24.
Compound 24 was subsequently transformed to target products
25a-25d in a similar manner as described in Scheme 1.
(0.04e0.21 mM), albeit the carbon chain length slightly decreased
the cellular potency. Moreover, incorporation of basic substitution
(N, N-dimethylethyl, 18d) displayed more effective potency and the
IC50 value reached to 0.04
mM, while introducing a bulky benzyl
group resulted in a slight decrease in potency (18c, IC50 ¼ 0.13
m
M).
In consideration of bio-isosteres, a S-linker was also evaluated.
However, the compounds with a S-linker (25a-25d) exhibited
moderate anti-proliferative activities even with the N, N-dime-
2.2. Biological evaluation
2.2.1. In vitro inhibitory activities against ALK-dependent cancer
cells
Activities of target compounds (8a-8l, 13a-13b, 18a-18g and
25a-25d) against tumor cells expressing NMP-ALK are demon-
strated in ALCL cell Karpas-299 (Tables 1e3).
In order to identify potent ALK inhibitors, we initially selected
pyridin-2(1H)-one at C-5 position and retained the oxygen as a
linker while varying the aryl fragment (R2 part). As shown in
Table 1, the incorporation of phenyl (8a), 4-methylphenyl (8c), 4-
chlorophenyl (8e), 2-pyridinyl (8i), 3-pyridinyl (8j) and 4-
pyridinyl (8l) as the R2 substitution resulted in a significant loss
of activity. While the 2, 6-dichloro-3-fluorophenyl substitution (8g,
thylethyl group (25d, IC50 ¼ 0.28
mM). It therefore appears in this
case the O-linker is more favor for the cellular potency.
Givin their promising anti-proliferactive activities against
Karpas-299, 18a-18g were selected for further evaluation in other
ALK-addicted cancer cells, including ALCL (Karpas-299 (NPM-ALK)),
neuroblastoma (SH-SY5Y (F1174L), SK-N-BE2 (wt)) and NSCLC
(NCIeH2228 (EML4-ALK)). The results are summerized in Table 3,
most of the compounds displayed moderate to high potency across
the entire panel of these ALK-addicted cell lines. This likely reflects
that anti-proliferative activity of compound 18 is “on-target” to
ALK.
0.12
(0.03
m
m
M) is tolerated with a comparable IC50 value to crizotinib
M). This indicated that 2, 6-dichloro-3-fluorophenyl group
2.2.2. In vitro inhibitory activities against ALK wild type and
resistant mutants
would be beneficial for a hydrophobic interaction. The activities did
not significantly change with the introduction of methyl (8h,
0.20 mM) or benzyl (8l, 0.32 mM) as R1 substitutions. However,
simply replacing the O-linker with another rigid amide-linker (13)
caused a complete loss of activity. 13a and 13b displayed no activity
up to 20 mM, suggesting that the rigidity of the amide-linker would
Based on the SAR above, 18a-18e with representative structural
divesity were chosen for further evaluation. As shown in Table 4,
18a-18e showed good ALKWT potency that is consistent with the
potency against the ALK-addicited cancer cells. Next, their inhibi-
tory activities against the most common and challenging crizotinib-
resistant mutants including gatekeeper mutation L1196M and
solvent-front mutant G1202R were tested. Compared to crizotinib
be detrimental for the interaction with the target protein.
With the expectation of an improvement in potency, 2, 6-
Scheme 3. Synthesis of compounds 18a-18g.

W. Chen et al. / European Journal of Medicinal Chemistry 183 (2019) 111734
5
Scheme 4. Synthesis of compounds 25a-25d.
(IC50 ¼ 366 nM), 18a, 18b and 18d showed excellent inhibitory po-
tencies against the crizotinib-resistant mutation ALKL1196M with
IC50 values of 66, 85 and 45 nM, respectively. Notably, 18d exhibited
excellent inhibitory activity with a competitive IC50 value (22 nM)
against ALK G1202R, the most refractory mutation accounting for
drug resistance of the 2nd generation ALK inhibitors. Additionally,
18d displayed an excellent effect against ROS1 (2.3 nM). These
indicated 18d might be a promising ALK/ROS1 inhibitor for over-
coming the clinical crizotinib-resistant mutants.
with Arg1202, which possibly explains the potency of 18d to
overcome G1202R mutant.
3. Conclusion
The emergence of on-target kinase domain mutations (L1196M)
and solvent-front mutations (G1202R) are among the most recal-
citrant of crizotinib-resistance. Based on the built ALKL1196M/G1202R
model, the piperidinyl-pyrzaole ring of crizotinib was altered with a
new 2-pyridone motif as well as a flexible sp3 (S-linker) and rigid
sp2 (amide-linker) functionalities were introduced to replace the
known O-linker. Optimization of 2-aminopyridine derivatives led
to the identification of hit 18d. 18d displayed remarkable anti-
proliferative potencies against ALK-addicted cancer cell lines with
2.2.3. 18d induces G1-phase cell cycle arrest in Karpas-299 cell
To better understand the possible mechanism associated with
anti-proliferative activity, the effect of 18d on the cell cycle distri-
bution in Karpas-299 was explored. After cells were treated with
vehicle or 1
mM of 18d for 48 h, DNA content in the control group
the IC50 values ranging from 0.04 mM to 1.67 mM. Moreover, 18d
displayed a typical histogram observed in exponentially growing
cells, while 18d induced G1-phase cell cycle arrest in Karpas-299
(81.5%). (Fig. 3). This result indicated that the cell cycle arrest
contributed to the anti-proliferative activity of 18d.
exhibited encouraging activities against the most common gate-
keeper mutant L1196M (45 nM), the challenging resistant mutant
G1202R (22 nM) and ROS1 (2.3 nM), which are superior to crizoti-
nib. Additionally, 18d inhibited Karpas-299 cell viability via G1
phase arrest. These findings show that 18d would be a promising
ALK inhibitor for overcoming the clinical crizotinib-resistant
mutants.
2.3. Molecular docking
In order to better understand the binding mode, molecular
docking model of 18d were performed based upon the cocrystal
4. Experimental section
L1196M
structure of ALK
with crizotinib (PDB 2YFX). As shown in
Fig. 4A, 18d occupies the kinase domain in a similar manner to
crizotinib. Interestingly, the basic “tail”, 1-(2-(dimethylamino)
ethyl)pyridine-2(1H)-one moiety, was engaged in hydrogen bond
interactions with Asp1203 and Ser1206/Gly1202, possibly vali-
dating our hypothesis. Furthermore, in the G1202R/L1196M mutant
model (Fig. 4B), the“tail” of 18d was involved in a hydrogen bond
4.1. Chemistry
Unless otherwise noted, all solvents and chemicals were used as
purchased without further purification. Purity of all final com-
pounds was 95% or higher. All reported yields are isolated yields
after column chromatography. 1H NMR spectra were recorded on a

Table 1
In vitro anti-proliferative activities of compound 8 and 13.
Compound
R1
R2
Karpass299
IC50
Ma
/
m
8a
8b
8c
H
4.40 0.29
2.86 0.24
10.17 1.39
CH3
H
8d
8e
8f
CH3
H
7.78 0.20
17.80 1.28
20.92 1.41
CH3
8g
H
0.12 0.004
8h
8i
CH3
CH3
CH3
CH3
0.20 0.05
11.91 0.08
11.17 1.12
>20
8j
8k
8l
Bn
0.32 0.19
13a
13b
crizotinib
H
CH3
e
e
e
e
>20
>20
0.03 0.004
a
The inhibitory effects of individual compounds on the proliferation of cancer cell were determined by the MTT assay. The data reported are the mean values from two independent experiments.

W. Chen et al. / European Journal of Medicinal Chemistry 183 (2019) 111734
7
Table 2
In vitro anti-proliferative activities of compound 18 and 25.
Compound
R1
X
Karpass299
IC
50 mMa
/
18a
18b
18c
18d
H
Me
Bn
O
O
O
O
0.05 0.03
0.04 0.00
0.13 0.01
0.04 0.01
18e
O
0.06 0.01
18f
O
O
0.21 0.06
0.11 0.01
18g
25a
25b
25c
25d
H
S
S
S
S
e
0.72 0.02
0.40 0.17
0.79 0.10
0.28 0.18
0.03 0.004
Me
Bn
fx21
e
crizotinib
Table 3
Anti-proliferative activities of selected compounds against a panel of ALK-addicted cell linesa.
Compd.
Cell line/IC50(mM)
Karpas-299 (NPM-ALK)
SH-SY5Y (F1174L)
SK-N-BE2 (WT)
NCIeH2228 (EML4-ALK)
18a
18b
18c
18d
18e
18f
18g
crizotinib
0.05 0.03
0.04 0.00
0.13 0.01
0.04 0.01
0.06 0.01
0.21 0.06
0.11 0.01
0.03 0.004
2.07 0.19
1.87 0.35
1.35 0.28
1.67 0.25
2.51 0.08
1.85 0.05
2.15 0.42
0.85 0.08
0.75 0.23
0.83 0.03
1.02 0.08
0.71 0.11
1.44 0.02
1.23 0.19
1.43 0.01
1.33 0.25
0.64 0.06
0.69 0.05
1.30 0.21
0.44 0.28
1.20 0.06
3.32 0.28
1.22 0.03
0.64 0.03
a
The inhibitory effects of individual compounds on the proliferation of cancer cell lines were determined by the MTT assay. The data reported are the mean values from two
independent experiments.
4.1.1. General procedure for the synthesis of 4-bromopyridin-2(1H)-
one (2)
Table 4
Inhibitory activities of selected compounds against different status of ALKs and
ROS1.a.
2-amino-4-bromopyridine (3.5 mmol) was dissolved in 20 mL
2M H2SO4 in an ice-bath. A saturated NaNO2 (4.0 mmol) solution
was added dropwise into the above solution. After the completion
of addition overnight, the precipitate was collected for further re-
action without any purification. Light yellow solid. Yield: 72%. 1H
Compd.
Kinase/IC50 (nM)
ALK (WT)
ALK(L1196M)
ALK(G1202R)
ROS1
18a
18b
18c
18d
18e
crizotinib
12
21
43
19
21
30
66
85
393
45
136
366
43
44
e
e
e
NMR (500 MHz, CDCl3)
d
13.07 (s, 1H), 7.22 (d, J ¼ 7.0 Hz, 1H), 6.83
e
(d, J ¼ 1.5 Hz, 1H), 6.46 (dd, J ¼ 7.0, 2.0 Hz, 1H). HRMS (ESI) m/z calcd
22
e
2.3
e
for C5H4BrNO [MþH]þ:173.9549, found: 173.9551.
>500
6.5
a
The data reported are the mean values from two duplicated wells.
4.1.2. General procedure for the synthesis of 3
To an ice-cold solution of 2-hydroxy-4-bromopyridine
(5.75 mmol) in THF was added NaH (5.75 mmol) portion-wise.
The reaction mixture was stirred in an ice-bath for 15 min fol-
lowed by addition of halide or iodine (17.24 mmol). The resulting
reaction mixture was stirred at room temperature for 16 h. After the
completion of the reaction, 20 mL of H2O was added and the re-
action mixture was extracted with EtOAc 3 times. The combined
organic layer was collected and rinsed with brine. The mixture was
evaporated to obtain the crude product and purified by silica gel for
3a-3d.
Bruker DRX-500 [Bruker Biospin, Germany]. Chemical shifts are
reported in ppm relative to the residual solvent peak (CDCl3, TMS:
0.00). Multiplicity was indicated as follows: s (singlet); d (doublet);
t (triplet); q (quartet); m (multiplet); dd (doublet of doublet); dt
(triplet of doublet); td (doublet of triplet); brs (broad singlet) etc.
Intermediates were purified by column chromatography on silica
gel (200e300 mesh). HPLC analysis and the HRMS of all biologically
evaluated compounds was confirmed on a Agilent 1290 HPLC-6224
Time of Fight Mass Spectrometer using PhenomenexLuna 5
m C18,
100 Å, 150 ꢀ 4.60 mm 5
m
m column at a flow rate of 0.5 mL/min
using liner gradients buffer B in A (B: CH3OH containing 0.1% formic
acid, A: H2O containing 0.1% formic acid). Mobile phase B was
increased linearly from 5% to 95% over 7 min and 95% over the next
2 min, after which the column was equilibrated to 5% for 1 min.
4.1.2.1. 4-bromo-1-methylpyridin-2(1H)-one (3a): [40]. Yellow
solid. Yield: 87%. HRMS (ESI) m/z calcd for C6H7BrNO
[MþH]þ:187.9706, found: 187.9702.

8
W. Chen et al. / European Journal of Medicinal Chemistry 183 (2019) 111734
Fig. 3. Cell cycle arrest induced by 18d in Karpas-299 cell.
Fig. 4. The binding models of 18d with ALK. (A) Predicted binding conformation of 18d in the binding site cavity of ALKL1196M (PDB 2YFX). (B) Predicted binding conformation of 18d
in the ATP binding site of ALKG1202R/L1196M
.
4.1.2.2. 4-bromo-1-ethylpyridin-2(1H)-one (3b): [41]. Yellow solid.
Yield: 81%. HRMS (ESI) m/z calcd for C7H9BrNO [MþH]þ: 201.9862,
found: 201.9872.
4.1.3. General procedure for the synthesis of 2-nitro-5-(1-(pyridin-
2-yl)ethoxy)pyridine (5i)
1-(pyridin-2-yl)ethan-1-ol (20 mmol), PPh3 (30 mmol) and 2-
nitro-3-hydroxyl pyridine (20 mmol) were dissolved in 30 mL THF
under the N2 atmosphere. When the reaction was cooled to 0 ꢁC,
DEAD (30 mmol) was added dropwise. Then the reaction was
warmed to room temperature for 3 h. The reaction mixture was
evaporated and crystallized by EtOH-Pet. Yellow solid. Yield:
4.1.2.3. 1-benzyl-4-bromopyridin-2(1H)-one (3c): [41]. White solid.
Yield: 65%. HRMS (ESI) m/z calcd for
264.0019, Found:264.0026.
C
12H11BrNO [MþH]þ:
85%.1H NMR (500 MHz, DMSO‑d6)
d 8.62-8.55 (m, 1H), 8.08 (dd,
4.1.2.4. 4-bromo-1-(2-(dimethylamino)ethyl)pyridin-2(1H)-one
(3d). White solid. Yield: 60%.1H NMR (500 MHz, CDCl3)
7.19 (d,
J ¼ 5.0, 1.5 Hz, 1H), 7.86-7.83 (m, 2H), 7.66 (dd, J ¼ 8.5, 4.5 Hz, 1H),
7.45 (d, J ¼ 8.0 Hz,1H), 7.36-7.33 (m,1H), 5.82 (q, J ¼ 6.5 Hz,1H),1.62
(d, J ¼ 6.5 Hz, 3H). HRMS (ESI) m/z calcd for C12H12N3O3 [MþH]þ:
246.0873, Found: 246.0880.
d
J ¼ 7.5 Hz, 1H), 6.80 (d, J ¼ 2.0 Hz, 1H), 6.29 (dd, J ¼ 7.0, 2.0 Hz, 1H),
3.96 (t, J ¼ 6.0 Hz, 2H), 2.59 (t, J ¼ 6.0 Hz, 2H), 2.26 (s, 6H). HRMS
(ESI) m/z calcd for C9H14BrN2O [MþH]þ: 245.0284, Found:
245.0282.

W. Chen et al. / European Journal of Medicinal Chemistry 183 (2019) 111734
9
4.1.4. General procedure for the synthesis of 5-(1-(pyridin-2-yl)
ethoxy)pyridin-2-amine (6i)
4.1.6.3. 6-Amino-5-(1-(p-tolyl)ethoxy)-[3,40-bipyridin]-20(10H)-one
(8c). Yellow solid; m.p. 178.1e179.2 ꢁC; Yield: 71%. HPLC: 100%,
The 5i (16 mmol) and Fe (48 mmol) were dissolved in 100 mL
EtOH and 25 mL AcOH. The reaction was refluxed for 2 h. After the
completion of the reaction, the pH was adjusted to a basic condition
with Na2CO3 solution. The mixture was extracted with EtOAc and
the combined organic layer was evaporated to get the crude
product. The final product was crystallized with EtOAc/Pet. Yield:
tR ¼ 4.507 min 1H NMR (500 MHz, DMSO‑d6)
d 11.40 (s, 1H), 7.88 (s,
1H), 7.55 (d, J ¼ 8.2 Hz, 2H), 7.40 (d, J ¼ 8.2 Hz, 2H), 7.33 (d,
J ¼ 6.8 Hz, 1H), 7.26 (s, 1H), 6.47-6.38 (m, 2H), 6.28 (s, 2H), 5.84-5.74
(m, 1H), 2.32 (s, 3H), 1.57 (d, J ¼ 6.3 Hz, 3H). 13C NMR (500 MHz,
DMSO‑d6) d 162.7, 152.6, 149.6, 139.6139.2, 137.4, 136.8, 135.1, 129.0,
125.8, 120.6, 115.5, 113.2, 102.9, 74.6, 23.9, 20.7. HRMS (ESI) m/z
88%. 1H NMR (500 MHz, DMSO‑d6)
d 8.55-8.54 (m, 1H), 7.78 (td,
calcd for C19H20N3O2 [MþH]þ: 322.1550, Found: 322.1552.
J ¼ 7.5, 1.5 Hz, 1H), 7.50 (d, J ¼ 8.0 Hz, 1H), 7.46 (dd, J ¼ 5.0, 1.5 Hz,
1H), 7.31-7.28 (m, 1H), 6.83 (dd, J ¼ 8.0, 1.5 Hz, 1H), 6.35 (dd, J ¼ 8.0,
5.0 Hz, 1H), 5.77 (s, 2H), 5.43 (q, J ¼ 6.5 Hz, 1H), 1.60 (d, J ¼ 6.5 Hz,
3H). HRMS (ESI) m/z calcd for C12H14N3O [MþH]þ: 216.1131, Found:
216.1129.
4.1.6.4. 6-Amino-10-methyl-5-(1-(p-tolyl) ethoxy)-[3,40-bipyridin]-
20(10H)-one (8d). Yellow solid; m.p. 186e187 ꢁC; Yield: 76%. HPLC:
95.89%, tR ¼ 4.620 min 1H NMR (500 MHz, CDCl3)
d 7.84 (s, 1H), 7.24
(t, J ¼ 7.1 Hz, 3H), 7.16 (d, J ¼ 7.9 Hz, 2H), 6.95 (d, J ¼ 1.3 Hz, 1H), 6.52
(d, J ¼ 1.5 Hz, 1H), 6.24 (d, J ¼ 7.0 Hz, 1H), 5.33 (q, J ¼ 6.4 Hz, 1H),
5.26 (s, 2H), 3.52 (s, 3H), 2.32 (s, 3H), 1.67 (d, J ¼ 6.4 Hz, 3H). 13C
4.1.5. General procedure for the synthesis of 3-bromo-5-(1-
(pyridin-2-yl)ethoxy)pyridin-2-amine (7i)
NMR (500 MHz, DMSO‑d6)
d 162.1, 152.6, 148.6, 139.6, 139.3, 139.3,
137.4, 136.8, 129.0, 125.8, 120.3, 115.5, 112.2, 103.0, 74.7, 36.2, 23.8,
20.7. HRMS (ESI) m/z calcd for C20H22N3O2 [MþH]þ: 336.1707,
Found: 336.1702.
The 6i (15 mmol) was dissolved in 50 mL MeCN and 25 mL
CH2Cl2, NBS (15 mmol) was added to the above mixture portion-
wise in an ice-bath. After the reaction was stirred for 15 min,
5 mL H2O was added to quench the reaction. The mixture was
extracted with DCM and the combined organic layer was evapo-
rated to get the crude product. The final product was crystallized
4.1.6.5. 6-Amino-5-(1-(4-chlorophenyl) ethoxy)-[3, 40-bipyridin]-
20(10H)-one (8e). Black solid; m.p. 189e192 ꢁC; Yield: 62%. HPLC:
100%, tR ¼ 4.693 min 1H NMR (500 MHz, DMSO‑d6)
d 11.41 (s, 1H),
with MeOH. Yield: 78%. 1H NMR (500 MHz, DMSO‑d6)
d 8.57-8.55
7.89 (s, 1H), 7.54 (d, J ¼ 8.3 Hz, 2H), 7.41 (d, J ¼ 8.3 Hz, 2H), 7.33 (d,
J ¼ 6.8 Hz, 1H), 7.26 (s, 1H), 6.47-6.38 (m, 2H), 6.28 (s, 2H), 5.84-5.74
(m, 1H), 1.57 (d, J ¼ 6.3 Hz, 3H). 13C NMR (500 MHz, DMSO‑d6)
(m, 1H), 7.81 (td, J ¼ 7.5, 1.5 Hz, 1H), 7.53-7.51 (m, 2H), 7.33-7.31 (m,
1H), 7.02 (d, J ¼ 2.0 Hz, 1H), 6.07 (s, 2H), 5.51 (q, J ¼ 6.5 Hz, 1H), 1.60
(d, J ¼ 6.5 Hz, 3H). HRMS (ESI) m/z calcd for C12H13BrN3O [MþH]þ:
294.0237, Found: 294.0233.
d162.7, 152.6, 149.5, 141.7, 139.0, 137.7, 135.1, 132.1, 128.5, 127.9,
120.6, 115.6, 113.2, 102.9, 74.0, 23.6. HRMS (ESI) m/z calcd for
C
18H17ClN3O2 [MþH]þ: 342.1004, Found: 342.1001.
4.1.6. General procedure for the synthesis of 8
To a 50 mL round-bottom flask under a nitrogen atmosphere 7
(10.0 mmol), bispinacolatodiboron (10.0 mmol), PdCl2(dppf)2
(0.5 mmol), potassium acetate (15 mmol) and 1, 4-dioxane (20 mL)
were added. The reaction mixture was stirred at 80 ꢁC for 8 h. After
the completion of the reaction, Cs2CO3 (15 mmol), PdCl2(dppf)2
(0.5 mmol), 3 (11.0 mmol) and 0.5 mL H2O were added to the above
mixture. The reaction was stirred at 110 ꢁC overnight. After the
completion of the reaction, 20 mL of H2O was added and the re-
action mixture was extracted with DCM for 3 times. The combined
organic layer was collected and rinsed with brine. The mixture was
evaporated to obtain the crude product and purified by silica gel for
8a-8l.
4.1.6.6. 6-Amino-5-(1-(4-chlorophenyl) ethoxy)-10-methyl-[3, 40-
bipyridin]-20(10H)-one (8f). Grey solid; m.p. 257e259 ꢁC; Yield: 67%.
HPLC: 100%, tR ¼ 4.807 min.1H NMR (500 MHz, CDCl3)
d 7.85 (d,
J ¼ 1.1 Hz, 1H), 7.33 (dd, J ¼ 20.9, 8.5 Hz, 4H), 7.27 (d, J ¼ 7.1 Hz, 1H),
6.92 (d, J ¼ 1.3 Hz, 1H), 6.55 (d, J ¼ 1.5 Hz, 1H), 6.26 (dd, J ¼ 7.0,
1.7 Hz, 1H), 5.36 (dd, J ¼ 12.6, 6.1 Hz, 3H), 3.55 (s, 3H), 1.69 (d,
J ¼ 6.4 Hz, 3H). 13C NMR (500 MHz, DMSO‑d6)
d 162.1, 152.5, 148.4,
141.6, 139.3, 139.0, 137.5, 132.1, 128.4, 127.9, 120.2,115.6, 112.2, 102.9,
74.0, 36.2, 23.5. HRMS (ESI) m/z calcd for C19H19ClN3O2 [MþH]þ:
356.1160, Found: 356.1157.
4.1.6.7. 6-Amino-5-(1-(2, 6-dichloro-3-fluorophenyl)ethoxy)-[3,40-
bipyridin]-20(10H)-one (8g). Yellow solid; m.p. 250e252 ꢁC; Yield:
71%. HPLC: 99.58%, tR ¼ 5.027 min 1H NMR (500 MHz, DMSO‑d6)
4.1.6.1. 6-Amino-5-(1-phenylethoxy)-[3,
40-bipyridin]-20(10H)-one
d
11.43 (s, 1H), 7.92 (d, J ¼ 1.8 Hz, 1H), 7.57 (dd, J ¼ 9.0, 4.9 Hz, 1H),
(8a). Grey solid; m.p. 231e233 ꢁC; Yield: 71%. HPLC: 98.77%,
7.46 (t, J ¼ 8.7 Hz, 1H), 7.34 (d, J ¼ 6.9 Hz, 1H), 6.94 (d, J ¼ 1.6 Hz, 1H),
6.32 (dd, J ¼ 6.9, 1.7 Hz, 1H), 6.27 (d, J ¼ 1.5 Hz, 1H), 6.21 (s, 2H), 6.16
(t, J ¼ 6.6 Hz, 1H), 1.81 (d, J ¼ 6.6 Hz, 3H). 13C NMR (500 MHz,
tR ¼ 4.073 min 1H NMR (500 MHz, DMSO‑d6)
d 11.39 (s, 1H), 7.85 (d,
J ¼ 1.6 Hz, 1H), 7.48 (d, J ¼ 7.4 Hz, 2H), 7.32 (dd, J ¼ 15.4, 7.6 Hz, 3H),
7.22 (dd, J ¼ 15.3, 4.4 Hz, 2H), 6.37 (d, J ¼ 4.8 Hz, 2H), 6.24 (s, 2H),
5.73 (q, J ¼ 6.2 Hz, 1H), 1.57 (d, J ¼ 6.3 Hz, 3H). 13C NMR (500 MHz,
DMSO‑d6)
d
162.6, 156.8 (d, J ¼ 246.3 Hz), 152.1, 149.3, 138.5, 138.1,
136.7, 135.3, 130.6 (d, J ¼ 5.0 Hz), 128.7 (d, J ¼ 2.5 Hz), 121.0 (d,
J ¼ 18.8 Hz), 120.5117.5 (d, J ¼ 23.8 Hz), 114.2, 113.1, 102.6 72.1, 18.7.
HRMS (ESI) m/z calcd for C18H15Cl2FN3O2 [MþH]þ: 394.0520,
Found: 394.0516.
DMSO‑d6) d162.8, 152.5, 149.6, 142.6, 139.2, 137.4, 135.1, 128.5, 127.6,
125.9, 120.6, 115.5, 113.2, 103.0, 74.8, 23.8. HRMS (ESI) m/z calcd for
C
18H18N3O2 [MþH]þ: 308.1394, Found: 308.1394.
4.1.6.2. 6-Amino-10-methyl-5-(1-phenylethoxy)-[3,40-bipyridin]-
4.1.6.8. 6-Amino-5-(1-(2, 6-dichloro-3-fluorophenyl) ethoxy)-10-
methyl-[3, 40-bipyridin]-20(10H)-one (8h). Yellow solid; m.p.
130e133 ꢁC; Yield: 78%. HPLC: 95.73%, tR ¼ 5.193 min 1H NMR
20(10H)-one (8b). Black solid; m.p. 205e207 ꢁC; Yield: 65%. HPLC:
100%, tR ¼ 4.240 min 1H NMR (500 MHz, DMSO‑d6)
d 7.87 (d,
J ¼ 1.3 Hz, 1H), 7.61 (d, J ¼ 7.1 Hz, 1H), 7.48 (d, J ¼ 7.4 Hz, 2H), 7.32 (t,
J ¼ 7.5 Hz, 2H), 7.23 (dd, J ¼ 8.1, 4.5 Hz, 2H), 6.47 (d, J ¼ 1.5 Hz, 1H),
6.46-6.37 (m, 1H), 6.25 (s, 2H), 5.74 (q, J ¼ 6.2 Hz, 1H), 3.35 (s, 3H),
(500 MHz, CDCl3)
d
7.92 (d, J ¼ 1.7 Hz, 1H), 7.35 (dd, J ¼ 8.9, 4.8 Hz,
1H), 7.09 (dd, J ¼ 8.8, 8.0 Hz, 1H), 6.99 (d, J ¼ 1.8 Hz, 1H), 6.60 (d,
J ¼ 2.0 Hz, 1H), 6.27 (dd, J ¼ 7.1, 2.1 Hz, 1H), 6.13 (q, J ¼ 6.7 Hz, 1H),
5.07 (d, J ¼ 23.9 Hz, 2H), 3.56 (s, 3H), 1.88 (d, J ¼ 6.7 Hz, 3H). 13C
1.57 (d, J ¼ 6.3 Hz, 3H). 13C NMR (500 MHz, DMSO‑d6)
d 162.1, 152.5,
148.5, 142.6, 139.3, 139.2, 137.4, 128.5, 127.6, 125.9, 120.2, 115.5,
112.2, 102.9, 74.8, 36.2, 23.8. HRMS (ESI) m/z calcd for C19H20N3O2
[MþH]þ: 322.1550, Found: 322.1554.
NMR (500 MHz, DMSO‑d6)
d
161.9, 156.8 (d, J ¼ 245.0 Hz), 152.1,
148.3, 139.6, 138.5, 138.1, 136.7, 130.6 (d, J ¼ 8.8 Hz), 128.7 (d,
J ¼ 3.8 Hz), 121.0 (d, J ¼ 18.8 Hz), 120.2, 117.5 (d, J ¼ 23.8 Hz), 114.1,

10
W. Chen et al. / European Journal of Medicinal Chemistry 183 (2019) 111734
112.1, 102.6, 72.1, 36.2, 18.7. HRMS (ESI) m/z calcd for
C
4.1.8. General procedure for the synthesis of 13
According to the synthetic procedure of 4.1.6.
19H17Cl2FN3O2 [MþH]þ: 408.0676, Found: 408.0672.
4.1.6.9. 6-Amino-10-methyl-5-(1-(pyridin-2-yl) ethoxy)-[3, 40-bipyr-
4.1.8.1. 6-Amino-N-(1-(2, 6-dichloro-3-fluorophenyl) ethyl)-20-oxo-
10, 20-dihydro-[3, 40-bipyridine]-5-carboxamide (13a). Brown solid;
m.p. 288e290 ꢁC; Yield: 69%. HPLC: 95.62%, tR ¼ 5.960 min 1H NMR
idin]-20(10H)-one (8i). Black solid; m.p. 204e207 ꢁC; Yield: 46%.
HPLC: 100%, tR ¼ 3.360 min 1H NMR (500 MHz, DMSO‑d6)
d 8.53 (d,
J ¼ 4.2 Hz, 1H), 7.89 (d, J ¼ 1.8 Hz, 1H), 7.78 (td, J ¼ 7.7, 1.6 Hz, 1H),
7.62 (d, J ¼ 7.0 Hz, 1H), 7.53 (d, J ¼ 7.9 Hz, 1H), 7.28 (dd, J ¼ 6.6,
4.9 Hz, 1H), 7.20 (d, J ¼ 1.7 Hz, 1H), 6.49e6.35 (m, 2H), 6.27 (s, 2H),
5.68 (q, J ¼ 6.4 Hz, 1H), 3.37 (s, 3H), 1.61 (d, J ¼ 6.4 Hz, 3H). 13C NMR
(500 MHz, DMSO‑d6)
d
11.52 (s, 1H), 9.23 (d, J ¼ 5.0 Hz, 1H), 8.48 (s,
1H), 8.40 (d, J ¼ 13.0 Hz, 1H), 7.50e7.45 (m, 1H), 7.42 (d, J ¼ 6.8 Hz,
1H), 7.34 (dd, J ¼ 17.3, 8.6 Hz, 3H), 6.75 (s, 1H), 6.59 (d, J ¼ 6.5 Hz,
1H), 5.69-5.54 (m, 1H), 1.60 (d, J ¼ 7.3 Hz, 3H). 13C NMR (500 MHz,
(500 MHz, DMSO‑d6)
d
162.0, 161.3, 152.5, 148.9, 148.5, 139.4, 139.2,
DMSO‑d6)
d
171.0, 166.5, 162.8, 159.3, 156.6 (d, J ¼ 242.5 Hz), 149.9,
137.7, 137.2, 122.9, 120.3, 120.0, 115.5, 112.2, 102.9, 76.3, 36.2, 21.8.
HRMS (ESI) m/z calcd for C18H19N4O2 [MþH]þ: 323.1503, Found:
323.1507.
148.8, 140.1, 135.5, 135.1, 119.9, 116.0, 115.8, 113.7, 108.3, 102.9, 47.6,
17.0. HRMS (ESI) m/z calcd for C19H16Cl2FN4O2 [MþH]þ: 421.0629,
Found: 421.0627.
4.1.6.10. 6-Amino-10-methyl-5-(1-(pyridin-3-yl)
ethoxy)-[3,
40-
4.1.8.2. 6-Amino-N-(1-(2,
6-dichloro-3-fluorophenyl)
ethyl)-10-
bipyridin]-20(10H)-one (8j). Black solid; m.p. 203e205 ꢁC; Yield:
41%. HPLC: 100%, tR ¼ 2.567 min 1H NMR (500 MHz, DMSO‑d6)
methyl-20-oxo-10, 20-dihydro-[3, 40-bipyridine]-5-carboxamide (13b).
Yellow solid; m. p. 290e293 ꢁC; HPLC: 96.50%, tR ¼ 6.087 min 1H
d
8.72 (s, 1H), 8.54-8.38 (m, 1H), 7.92 (ddd, J ¼ 8.6, 5.2, 1.9 Hz, 2H),
NMR (500 MHz, DMSO‑d6)
d
9.22 (d, J ¼ 5.0 Hz,1H), 8.51 (s,1H), 8.41
7.63 (d, J ¼ 7.1 Hz, 1H), 7.42-7.32 (m, 2H), 6.54 (d, J ¼ 2.0 Hz, 1H),
6.48 (dd, J ¼ 7.2, 2.1 Hz, 1H), 6.31 (s, 2H), 5.86 (q, J ¼ 6.3 Hz, 1H), 3.39
(d, J ¼ 10.3 Hz, 3H), 1.59 (d, J ¼ 6.4 Hz, 3H). 13C NMR (500 MHz,
(s, 1H), 7.76 (d, J ¼ 7.0 Hz, 1H), 7.48-7.46 (m, 1H), 7.37-7.34 (m, 3H),
6.86 (s, 1H), 6.67 (d, J ¼ 7.0 Hz, 1H), 5.68-5.58 (m, 1H), 3.44 (s, 3H),
1.62 (d, J ¼ 7.5 Hz, 3H). 13C NMR (125 MHz, DMSO‑d6)
d 170.3, 166.4,
DMSO‑d6)
d
162.1, 152.7, 148.9, 148.4, 147.8, 139.3, 138.8, 137.8, 133.7,
162.2, 159.3, 156.6 (d, J ¼ 244 Hz), 149.8, 147.7, 140.1, 139.7, 135.0,
130.1 (d, J ¼ 8.8 Hz), 119.5, 115.8 (d, J ¼ 23.8 Hz), 112.6, 108.2, 102.8,
59.7, 36.3, 17.1. HRMS (ESI) m/z calcd for C20H18Cl2FN4O2 [MþH]þ:
435.0785, Found: 435.0783.
123.7, 120.2, 115.8, 112.2, 102.9, 72.8, 36.2, 23.2. HRMS (ESI) m/z
calcd for C18H19N4O2 [MþH]þ: 323.1503, Found: 323.1510.
4.1.6.11. 6-Amino-10-methyl-5-(1-(pyridin-4-yl)
bipyridin]-20(10H)-one (8k). Yellow oil; Yield: 38%. HPLC: 100%,
ethoxy)-[3,
40-
4.1.9. General procedure for the synthesis of (R)-3-(1-(2,6-dichloro-
3-fluorophenyl) ethoxy)-2-nitropyridine (15)
tR ¼ 2.287 min.1H NMR (500 MHz, CDCl3)
d
8.61 (d, J ¼ 5.8 Hz, 2H),
7.91 (d, J ¼ 1.6 Hz,1H), 7.28 (d, J ¼ 5.9 Hz, 2H), 7.25 (d, J ¼ 7.2 Hz,1H),
6.85 (d, J ¼ 1.5 Hz, 1H), 6.52 (d, J ¼ 1.7 Hz, 1H), 6.22 (dd, J ¼ 7.1,
1.9 Hz, 1H), 5.36 (q, J ¼ 6.4 Hz, 1H), 5.09 (s, 2H), 3.53 (s, 3H), 1.71 (d,
(S)-1-(2,6-dichloro-3-fluorophenyl) ethanol (20 mmol), PPh3
(30 mmol) and 2-nitro-3-hydroxyl pyridine (20 mmol) were dis-
solved in 30 mL THF under the N2 atmosphere. When the reaction
was cooled to 0 ꢁC, the DEAD (30 mmol) was added dropwise. Then
the reaction was warmed to room temperature for 3 h. The reaction
mixture was evaporated and crystallized by EtOH-Pet. Light yellow
J ¼ 6.5 Hz, 3H). 13C NMR (500 MHz, DMSO‑d6)
d 162.1, 152.4, 151.6,
151.4, 149.7, 139.4, 139.0, 121.0, 120.2, 115.6, 112.4, 102.9, 73.5, 36.2,
22.9. HRMS (ESI): m/z calcd for C18H19N4O2 [MþH]þ: 323.1503,
Found: 323.1500.
solid. Yield: 88%. 1H NMR (500 MHz, CDCl3)
d
8.01 (dd, J ¼ 4.5,
1.5 Hz, 1H), 7.38-7.35 (m, 1H), 7.31-7.28 (m, 1H), 7.21 (dd, J ¼ 8.5,
1.0 Hz, 1H), 7.07 (dd, J ¼ 9.0, 8.0 Hz, 1H), 6.09 (q, J ¼ 7.0 Hz, 1H), 1.83
(d, J ¼ 6.5 Hz, 3H). HRMS (ESI) m/z calcd for C13H10Cl2FN2O3
[MþH]þ: 331.0047, Found: 331.0046.
4.1.6.12. 6-Amino-10-benzyl-5-(1-(2,
ethoxy)-[3,40-bipyridin]-20(10H)-one
6-dichloro-3-fluorophenyl)
(8l). Yellow
solid;
m.p.
89e91 ꢁC; Yield: 62%. HPLC: 96.62%, tR ¼ 6.160 min 1H NMR
(500 MHz, CDCl3)
d
7.93 (d, J ¼ 1.8 Hz, 1H), 7.36 (tt, J ¼ 8.5, 6.0 Hz,
6H), 7.27 (d, J ¼ 7.2 Hz, 1H), 7.09 (dd, J ¼ 8.8, 8.0 Hz, 1H), 6.99 (d,
J ¼ 1.8 Hz, 1H), 6.64 (d, J ¼ 2.0 Hz, 1H), 6.28 (dd, J ¼ 7.2, 2.1 Hz, 1H),
6.13 (q, J ¼ 6.7 Hz, 1H), 5.17 (s, 2H), 5.06 (s, 2H), 1.88 (d, J ¼ 6.7 Hz,
4.1.10. General procedure for the synthesis of (R)-3-(1-(2,6-
dichloro-3-fluorophenyl) ethoxy) pyridin-2-amine (16)
The 15 (16 mmol) and Fe (48 mmol) were dissolved in 100 mL
EtOH and 25 mL AcOH. The reaction was refluxed for 2 h. After the
completion of reaction, the pH was adjusted to basic condition with
Na2CO3 solution. The mixture was extracted with EtOAc and the
combined organic layer was evaporated to get the crude product.
The final product was crystallized with EtOAc/Pet [23]. Yield: 91%.
HRMS (ESI) m/z calcd for C13H12Cl2FN2O [MþH]þ: 301.0305, Found:
301.0306.
3H). 13C NMR (500 MHz, DMSO‑d6)
d
161.5, 156.8 (d, J ¼ 245 Hz),
152.3, 148.4, 138.9, 138.5, 138.3, 137.5, 136.6, 130.5, 128.7 (d,
J ¼ 3.75 Hz), 128.5, 127.8, 127.5, 121.0 (d, J ¼ 20 Hz), 120.0, 117.5 (d,
J ¼ 22.5 Hz), 114.0, 112.6, 103.2, 72.1, 50.7, 18.6. HRMS (ESI) m/z calcd
for C25H21Cl2FN3O2 [MþH]þ: 484.0989, Found: 484.0992.
4.1.7. General procedure for the synthesis of 2-amino-5-bromo-N-
(1-(2,6-dichloro-3-fluorophenyl)ethyl)nicotinamide (12)
To an ice-cold solution of 2-amino-5-bromonicotinic acid
(0.6 mmol) in anhydrous DMF (5 mL), HATU (0.66 mmol), DIPEA
(0.72 mmol) and 1-(2,6-dichloro-3-fluorophenyl)ethan-1-amine
(0.72 mmol) was added portion-wise. The reaction mixture was
warmed to room temperature and stirred at rt for 6 h. After the
completion of the reaction, 20 mL of H2O was added and the re-
action mixture was extracted with EtOAc for 3 times. The combined
organic layer was collected and rinsed with brine. The mixture was
evaporated to obtain the crude product and purified by silica gel for
4.1.11. General procedure for the synthesis of (R)-5-bromo-3-(1-
(2,6-dichloro-3-fluorophenyl) ethoxy) pyridin-2-amine (17)
The 16 (15 mmol) was dissolved in 50 mL MeCN and 25 mL
CH2Cl2, NBS (15 mmol) was added to the above mixture portion-
wise at ice-bath. After the reaction was stirred for 15 min,
5 mL H2O was added to quench the reaction. The mixture was
extracted with DCM and the combined organic layer was evapo-
rated to get the crude product. The final product was crystallized
with MeOH. Yield: 81%. 1H NMR (500 MHz, CDCl3)
d 7.66 (d,
12. Yield: 84%. 1H NMR (500 MHz, CDCl3)
d
8.17 (d, J ¼ 2.5 Hz, 1H),
J ¼ 2.0 Hz, 1H), 7.32-7.29 (m, 1H), 7.07 (dd, J ¼ 8.5, 8.0 Hz, 1H), 6.83
(d, J ¼ 2.0 Hz, 1H), 5.98 (q, J ¼ 7.0 Hz, 1H), 4.85 (s, 2H), 1.81 (d,
J ¼ 7.0 Hz, 3H). HRMS (ESI) m/z calcd for C13H11BrCl2FN2O [MþH]þ:
378.9410, Found: 378.9400.
7.71 (d, J ¼ 2.5 Hz, 1H), 7.29 (s, 1H), 7.07- 6.97 (m, 2H), 6.33 (s, 2H),
6.10-6.00 (m, 1H), 1.67 (d, J ¼ 7.5 Hz, 3H). HRMS (ESI) m/z calcd for
C
14H12BrCl2FN3O, [MþH]þ: 405.9519, Found: 405.9519.

W. Chen et al. / European Journal of Medicinal Chemistry 183 (2019) 111734
11
4.1.12. General procedure for the synthesis of 18
calcd for C20H19Cl2FN3O2 [MþH]þ: 422.0833, Found: 422.0831.
According to the synthetic procedure of 4.1.6.
4.1.12.6. Ethyl (R)-2-(6-amino-5-(1-(2, 6-dichloro-3-fluorophenyl)
ethoxy)-20-oxo-[3, 40-bipyridin]-10(20H)-yl)acetate (18f). Yellow
4.1.12.1. (R)-6-Amino-5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)-
[3,40-bipyridin]-20(10H)-one (18a). Yellow solid; m.p. 265e267 ꢁC;
solid; m.p. 142e145 ꢁC; Yield: 57%. [
a
]25D ¼ þ 34 (c ¼ 0.5, MeOH);
Yield: 69%. [
a
]25D ¼ þ 10 (c ¼ 0.5, MeOH); HPLC: 97.39%,
HPLC: 96.82%, tR ¼ 5.467 min 1H NMR (500 MHz, DMSO‑d6)
d 7.95
tR ¼ 5.020 min 1H NMR (500 MHz, DMSO‑d6)
d
11.44 (s, 1H), 7.92 (s,
(d, J ¼ 1.7 Hz, 1H), 7.63 (d, J ¼ 7.2 Hz, 1H), 7.55 (dd, J ¼ 8.9, 4.9 Hz,
1H), 7.43 (t, J ¼ 8.7 Hz, 1H), 6.94 (d, J ¼ 1.5 Hz, 1H), 6.43 (dd, J ¼ 7.2,
1.9 Hz, 1H), 6.36 (d, J ¼ 1.8 Hz, 1H), 6.26 (s, 2H), 6.14 (q, J ¼ 6.6 Hz,
1H), 4.63 (s, 2H), 4.12 (q, J ¼ 7.1 Hz, 2H), 1.79 (d, J ¼ 6.6 Hz, 3H), 1.19
1H), 7.57 (dd, J ¼ 8.9, 4.9 Hz, 1H), 7.45 (t, J ¼ 8.7 Hz, 1H), 7.35 (d,
J ¼ 6.9 Hz, 1H), 6.94 (s, 1H), 6.35-6.30 (m, 1H), 6.28 (s, 1H), 6.22 (s,
2H), 6.15 (q, J ¼ 6.5 Hz, 1H), 1.81 (d, J ¼ 6.6 Hz, 3H). 13C NMR
(500 MHz, DMSO‑d6)
d
162.6, 156.8 (d, J ¼ 246.3 Hz), 152.2, 149.4,
(t, J ¼ 7.1 Hz, 3H). 13C NMR (500 MHz, DMSO‑d6)
d168.2, 161.5, 156.9
138.5, 138.1, 136.7, 135.3, 130.6, 128.7 (d, J ¼ 3.8 Hz), 121.0 (d,
J ¼ 20.0 Hz), 120.5, 117.5 (d, J ¼ 23.8 Hz), 114.2, 113.1, 102.7, 72.1, 18.7.
HRMS (ESI) m/z calcd for C18H15Cl2FN3O2 [MþH]þ: 394.0520,
Found: 394.0527.
(d, J ¼ 243.8 Hz), 152.3, 149.1, 139.4, 138.5, 138.4, 136.7, 130.6, 128.7
(d, J ¼ 3.8 Hz),121.0 (d, J ¼ 18.8 Hz),119.9,117.6 (d, J ¼ 22.5 Hz),114.1,
112.0, 103.0, 72.1, 61.0, 49.7, 18.7, 14.1. HRMS (ESI): m/z calcd for
C
22H21Cl2FN3O4 [MþH]þ: 480.0888, Found: 480.0882.
4.1.12.2. (R)-6-Amino-5-(1-(2, 6-dichloro-3-fluorophenyl) ethoxy)-
10-methyl-[3, 40-bipyridin]-20(10H)-one (18b). Yellow solid; m.p.
4.1.12.7. (R)-6-Amino-5-(1-(2, 6-dichloro-3-fluorophenyl) ethoxy)-
10-propyl-[3, 40-bipyridin]-20(10H)-one (18g). Yellow solid; m.p.
109e111 ꢁC; Yield: 75%. [
a
]25D ¼ þ 8 (c ¼ 0.5, MeOH); HPLC:
157e159 ꢁC; Yield: 65%. [
a
]25D ¼ þ 16 (c ¼ 0.5, MeOH); HPLC:
95.83%, tR ¼ 5.160 min 1H NMR (500 MHz, DMSO‑d6)
d
7.94 (s, 1H),
95.72%, tR ¼ 5.820 min.1H NMR (500 MHz, DMSO‑d6)
d 7.92 (d,
7.66 (d, J ¼ 6.8 Hz, 1H), 7.57 (dd, J ¼ 8.8, 4.8 Hz, 1H), 7.46 (t,
J ¼ 8.6 Hz, 1H), 6.95 (s, 1H), 6.37 (d, J ¼ 6.5 Hz, 2H), 6.21 (s, 2H), 6.18-
6.01 (m,1H), 3.39 (s, 3H),1.81 (d, J ¼ 6.5 Hz, 3H). 13C NMR (500 MHz,
J ¼ 1.8 Hz, 1H), 7.62 (d, J ¼ 7.1 Hz, 1H), 7.54 (dd, J ¼ 8.9, 4.9 Hz, 1H),
7.42 (t, J ¼ 8.7 Hz, 1H), 6.92 (d, J ¼ 1.8 Hz, 1H), 6.38-6.30 (m, 2H),
6.21 (s, 2H), 6.12 (q, J ¼ 6.6 Hz, 1H), 3.81-3.70 (m, 2H), 1.78 (d,
J ¼ 6.6 Hz, 3H), 1.61 (dt, J ¼ 14.6, 7.3 Hz, 2H), 0.83 (t, J ¼ 7.4 Hz, 3H).
DMSO‑d6)
d
161.9, 156.8 (d, J ¼ 246.3 Hz), 152.1, 148.3, 139.6, 138.5,
137.9, 136.6, 130.6, 128.7 (d, J ¼ 2.5 Hz), 121.0 (d, J ¼ 20.0 Hz), 120.2,
117.5 (d, J ¼ 22.5 Hz), 114.2, 112.1, 102.6, 72.1, 36.2, 18.7. HRMS (ESI)
m/z calcd for C19H17Cl2FN3O2 [MþH]þ: 408.0676, Found: 408.0672.
13C NMR (500 MHz, DMSO‑d6)
d
161.5, 156.8 (d, J ¼ 246.3 Hz), 152.2,
148.1, 139.0, 138.5, 138.1, 136.7, 130.6, 128.7 (d, J ¼ 3.8 Hz), 121.0 (d,
J ¼ 18.8 Hz), 120.1, 117.5 (d, J ¼ 22.5 Hz), 114.1, 112.4, 102.7, 72.1, 49.6,
22.0, 18.7, 10.9. HRMS (ESI) m/z calcd for C21H21Cl2FN3O2 [MþH]þ:
436.0989 Found: 436.0989.
4.1.12.3. (R)-6-Amino-10-benzyl-5-(1-(2, 6-dichloro-3-fluorophenyl)
ethoxy)-[3,40-bipyridin]-20(10H)-one (18c). Yellow solid; m.p.
112e114 ꢁC; Yield: 66%. [
a
]25D ¼ þ 43 (c ¼ 0.5, MeOH); HPLC:
4.1.13. General procedure for the synthesis of (R)-5-bromo-3-((1-
(2,6-dichloro-3-fluorophenyl) ethyl) thio) pyridin-2-amine (24)
The general synthesis was according to the literature [25]. Yield:
100%, tR ¼ 6.160 min 1H NMR (500 MHz, DMSO‑d6)
d 7.96 (s, 1H),
7.77 (d, J ¼ 7.1 Hz,1H), 7.57 (dd, J ¼ 8.9, 4.9 Hz,1H), 7.45 (t, J ¼ 8.7 Hz,
1H), 7.37-7.27 (m, 5H), 6.97 (s, 1H), 6.43 (d, J ¼ 7.2 Hz, 1H), 6.40 (s,
1H), 6.25 (s, 2H), 6.16 (q, J ¼ 6.5 Hz, 1H), 5.07 (s, 2H), 1.81 (d,
58%. 1H NMR (500 MHz, CDCl3)
d
8.03 (t, J ¼ 3.0 Hz, 1H), 7.51 (dd,
J ¼ 9.5, 2.5 Hz, 1H), 7.29 (dd, J ¼ 9.0, 5.0 Hz, 0.5H), 7.20 (dd, J ¼ 9.0,
5.0 Hz, 0.5H), 7.01 (t, J ¼ 8.0 Hz, 1H), 5.17 (s, 2H), 5.05-4.97 (m, 1H),
1.82 (dd, J ¼ 7.5, 4.5Hz, 3H).
J ¼ 6.6 Hz, 3H). 13C NMR (125 MHz, DMSO)
d 161.5, 156.8 (d,
J ¼ 245.0 Hz), 152.3, 148.4, 138.9, 138.5, 138.3, 137.5, 136.6, 130.5,
128.7, 128.5, 127.7, 127.5, 121.0 (d, J ¼ 18.8 Hz), 120.0, 117.5 (d,
J ¼ 22.5 Hz), 114.1, 112.6, 103.2, 72.1, 50.7, 18.6. HRMS (ESI) m/z calcd
for C25H21Cl2FN3O2 [MþH]þ: 484.0989, Found: 484.0987.
4.1.14. General procedure for the synthesis of 25
The general synthesis was according to the procedure for 4.1.6.
4.1.12.4. (R)-6-Amino-5-(1-(2, 6-dichloro-3-fluorophenyl) ethoxy)-
4.1.14.1. (R)-6-Amino-5-((1-(2, 6-dichloro-3-fluorophenyl) ethyl)
thio)-[3, 40-bipyridin]-20(10H)-one (25a). Yellow solid; m.p.
1'-(2-(dimethylamino)ethyl)-[3,40-bipyridin]-20(10H)-one
(18d).
Yellow solid; m.p. 198e201 ꢁC; Yield: 53%. [
a
]25D ¼ þ 27 (c ¼ 0.5,
230e233 ꢁC; Yield: 75%. [
a
]25D ¼ þ 134 (c ¼ 0.25, MeOH); HPLC:
MeOH); HPLC: 95.78%, tR ¼ 4.127 min 1H NMR (500 MHz, DMSO‑d6)
100%, tR ¼ 6.220 min 1H NMR (500 MHz, DMSO‑d6)
d 11.44 (s, 1H),
d
7.95 (s, 1H), 7.65-7.54 (m, 2H), 7.45 (t, J ¼ 8.6 Hz, 1H), 6.95 (s, 1H),
8.32 (s, 1H), 7.46 (dd, J ¼ 17.6, 2.0 Hz, 1H), 7.38-7.32 (m, 2H), 6.64 (d,
J ¼ 5.0 Hz, 2H), 6.39 -6.14 (m, 2H), 5.07 (d, J ¼ 7.2 Hz, 1H), 2.09-1.64
6.37 (dd, J ¼ 23.1, 16.1 Hz, 2H), 6.22 (s, 2H), 6.15 (dd, J ¼ 12.9, 6.3 Hz,
1H), 3.93 (t, J ¼ 5.9 Hz, 2H), 2.49 (d, J ¼ 6.1 Hz, 2H), 2.18 (s, 6H), 1.81
(m, 3H). 13C NMR (500 MHz, DMSO‑d6)
d162.6, 161.0, 160.9, 157.2(d,
(d, J ¼ 6.5 Hz, 3H). 13C NMR (500 MHz, DMSO‑d6)
d
161.5, 156.8 (d,
J ¼ 243.8 Hz), 155.9 (d, J ¼ 245.0 Hz), 148.5, 147.9, 147.8, 142.1, 142.0,
139.1, 139.0, 135.4, 131.1, 131.0, 129.6 (d, J ¼ 2.5 Hz), 129.2 (d,
J ¼ 2.5 Hz),129.1,129.0,121.9,121.8,121.5 (d, J ¼ 17.5 Hz),121.3,116.3
(d, J ¼ 23.8 Hz), 116.2 (d, J ¼ 22.5 Hz), 113.2, 110.4, 110.3, 102.6, 43.9,
43.1, 18.3, 18.2. HRMS (ESI) m/z calcd for C18H15Cl2FN3OS [MþH]þ:
410.0291, Found: 410.0296.
J ¼ 245.0 Hz), 152.2, 148.2, 139.3, 138.5, 138.1, 130.6, 1128.7 (d,
J ¼ 2.5 Hz), 121.0 (d, J ¼ 18.8 Hz), 120.1, 117.5 (d, J ¼ 23.8 Hz), 114.1,
112.3, 102.5, 72.1, 57.5, 45.7, 45.3, 18.7. HRMS (ESI) m/z calcd for
C
22H24Cl2FN4O2 [MþH]þ: 465.1255, Found:465.1258.
4.1.12.5. (R)-6-Amino-5-(1-(2, 6-dichloro-3-fluorophenyl) ethoxy)-
10-propyl-[3, 40-bipyridin]-20(10H)-one (18e). White solid; m.p.
4.1.14.2. (R)-6-Amino-5-((1-(2, 6-dichloro-3-fluorophenyl) ethyl)
thio)-10-methyl-[3,40-bipyridin]-20(10H)-one (25b). Yellow solid;
184e186 ꢁC; Yield: 68%. [
a
]25D ¼ þ 13 (c ¼ 0.5, MeOH); HPLC:
96.96%, tR ¼ 5.447 min 1H NMR (500 MHz, DMSO‑d6)
d
7.91 (d,
m.p. 194e197 ꢁC; Yield: 77%. [
a
]25D ¼ þ 134 (c ¼ 0.5, MeOH);
J ¼ 1.9 Hz, 1H), 7.69-7.62 (m, 1H), 7.55 (dd, J ¼ 9.0, 4.9 Hz, 1H), 7.44
(t, J ¼ 8.7 Hz, 1H), 6.92 (d, J ¼ 1.9 Hz, 1H), 6.36 (dd, J ¼ 7.2, 2.1 Hz,
1H), 6.32 (d, J ¼ 2.0 Hz, 1H), 6.21 (s, 2H), 6.13 (q, J ¼ 6.6 Hz, 1H), 3.85
(q, J ¼ 7.1 Hz, 2H), 1.18 (t, J ¼ 7.1 Hz, 3H). 13C NMR (500 MHz,
HPLC: 95.81%, tR ¼ 6.327 min 1H NMR (500 MHz, DMSO‑d6)
d 8.34
(dd, J ¼ 3.6, 2.5 Hz, 1H), 7.67 (d, J ¼ 7.1 Hz, 1H), 7.50 (dd, J ¼ 15.7,
2.4 Hz, 1H), 7.36 (dt, J ¼ 9.6, 2.9 Hz, 1H), 6.65 (s, 2H), 6.37 (ddd,
J ¼ 8.3, 7.3, 1.6 Hz, 2H), 5.12-5.01 (m, 1H), 3.40 (d, J ¼ 0.9 Hz, 3H),
DMSO‑d6)
d
161.3, 156.8 (d, J ¼ 245.0 Hz), 152.2, 148.2, 138.5, 138.1,
1.81 (dd, J ¼ 7.4, 1.9 Hz, 3H). 13C NMR (500 MHz, DMSO‑d6)
d 161.9,
136.7,130.6,128.7 (d, J ¼ 3.8 Hz),121.0 (d, J ¼ 18.8 Hz),120.1,117.5 (d,
161.0, 160.9, 157.1 (d, J ¼ 245.0 Hz), 155.9 (d, J ¼ 245.0 Hz), 147.8,
J ¼ 22.5 Hz), 114.1, 112.4, 102.9, 72.1, 43.1, 18.7, 14.5. HRMS (ESI) m/z
147.7, 147.4, 142.0, 141.9. 139.7, 139.6, 139.2, 139.1, 131.0, 130.9, 129.6,

12
W. Chen et al. / European Journal of Medicinal Chemistry 183 (2019) 111734
129.2 (d, J ¼ 3.8 Hz), 129.1, 129.0, 121.0, 120.9, 116.3 (d, J ¼ 23.8 Hz),
116.2 (d, J ¼ 22.5 Hz), 112.1, 110.4, 102.5, 43.9, 43.2, 36.2, 18.3, 18.2.
HRMS (ESI) m/z calcd for C19H17Cl2FN3OS [MþH]þ: 424.0448,
Found: 424.0446.
the fixed cells were stained with propidium iodide (10 mg/mL) for
30 min at 37 ꢁC and cell cycle variables analyzed by fluorescence-
activated cell sorting analysis (BD).
4.3. Computational methods for molecular docking
4.1.14.3. (R)-6-Amino-10-benzyl-5-((1-(2, 6-dichloro-3-fluorophenyl)
ethyl)thio)-[3,40-bipyridin]-20(10H)-one (25c). Yellow solid; m.p.
The molecular modeling simulations were performed using
64e68 ꢁC; Yield: 67%. [
a
]25D ¼ þ 93 (c ¼ 0.25, MeOH); HPLC:
Tripos Sybyl x1.3 molecular modeling package [42]. The co-crystal
95.25%, tR ¼ 6.967 min 1H NMR (500 MHz, CDCl3)
d 8.28 (t,
[L1196M]
structure of ALK
in complex with crizotinib was obtained
J ¼ 2.2 Hz, 1H), 7.64 (d, J ¼ 2.3 Hz, 1H), 7.38-7.30 (m, 6H), 7.02 (dd,
J ¼ 8.8, 8.0 Hz, 1H), 6.63 (dd, J ¼ 9.3, 1.9 Hz, 1H), 6.26 (td, J ¼ 7.1,
2.1 Hz, 1H), 5.48 (d, J ¼ 5.0 Hz, 2H), 5.17 (s, 2H), 5.06 (ddt, J ¼ 8.8, 7.4,
4.4 Hz, 1H), 1.86 (dd, J ¼ 7.3, 4.8 Hz, 3H). 13C NMR (500 MHz,
from the RSC Protein Data Bank (http://www.rcsb.org) (PDB code:
2YFX). The initial structure of 18d was generated by Sketch module
in Sybyl x1.3. The geometries of the compound were subsequently
optimized using the Tripos force field with Gasteiger-Hückel
charges. The produced conformation of 18d was then inserted into
the binding pocket of ALK [L1196M] to replace ligand crizotinib for the
DMSO‑d6)
d
161.4, 161.1, 161.0, 157.2 (d, J ¼ 243.8 Hz), 155.9 (d,
J ¼ 245.0 Hz), 148.0, 147.9, 147.6, 142.1, 142.0, 139.2, 139.1, 139.0,
137.5, 131.1, 131.0, 129.6 (d, J ¼ 3.8 Hz), 129.2 (d, J ¼ 2.5 Hz), 129.1,
129.0, 128.5, 127.7, 127.7, 127.5, 121.8 (d, J ¼ 20.0 Hz), 121.5 (d,
J ¼ 17.5 Hz), 120.9, 120.8, 116.3 (d, J ¼ 22.5 Hz), 116.2 (d, J ¼ 22.5 Hz),
112.8, 110.5, 103.2, 50.7, 43.9, 43.2, 18.4, 18.3. HRMS (ESI) m/z calcd
for C25H21Cl2FN3OS [MþH]þ: 500.0761, Found: 500.0758.
initial structural model of 18d binding to ALK [L1196M]. Molecular
[L1196M]
docking studies of 18d with the ALK
binding pocket was
performed with FlexiDock module in Sybyl x1.3 [43]. The docked
complexes of inhibitoreenzyme were selected according to the
criteria of interacting energy combined with geometrical matching
quality. These complexes were used as the starting conformation
4.1.14.4. (R)-6-Amino-5-((1-(2, 6-dichloro-3-fluorophenyl) ethyl)
thio)-1'-(2-(dimethylamino)ethyl)-[3,40-bipyridin]-20(10H)-one (25d).
for the geometrical optimization to achieve the final models of 18d
[G1202R/L1196M]
binding to ALK [L1196M]. The ALK
using 2YFX as a template.
model was built
Brown solid; m.p. 192e195 ꢁC; Yield: 61%. [
a
]25D ¼ þ 157 (c ¼ 0.1,
MeOH); HPLC: 96.29%, tR ¼ 5.100 min 1H NMR (500 MHz, DMSO‑d6)
d
8.34 (dd, J ¼ 3.6, 2.5 Hz, 1H), 7.61 (d, J ¼ 7.1 Hz, 1H), 7.50 (dd,
Declaration of competing interest
The authors declare no conflict of interest.
Acknowledgments
J ¼ 13.3, 2.4 Hz, 1H), 7.43-7.23 (m, 2H), 6.63 (s, 2H), 6.40-6.28 (m,
2H), 5.12-5.01 (m, 1H), 3.95 (t, J ¼ 6.2 Hz, 2H), 2.55- 2.51 (m, 2H),
2.20 (s, 6H), 1.81 (dd, J ¼ 7.4, 2.3 Hz, 3H). 13C NMR (500 MHz,
DMSO‑d6)
d
161.6, 161.1, 161.0, 157.2 (d, J ¼ 245.0 Hz), 155.9 (d,
J ¼ 245.0 Hz), 148.0, 147.9, 147.6, 142.1, 142.0, 139.4, 139.2, 139.1,
131.1, 131.0, 129.6 (d, J ¼ 3.8 Hz), 129.2 (d, J ¼ 3.8 Hz), 129.1, 129.0,
121.8 (d, J ¼ 18.8 Hz), 121.5 (d, J ¼ 17.5 Hz), 121.0, 120.9, 116.3 (d,
J ¼ 22.5 Hz), 116.2 (d, J ¼ 22.5 Hz), 112.4, 110.5, 102.8, 56.9, 45.2,
44.6, 44.0, 43.2, 18.4, 18.3. HRMS (ESI) m/z calcd for C22H24Cl2FN4OS
[MþH]þ: 481.1026, Found: 481.1020.
We thank Marc A. Giulianotti in Torry Pines Institute for Mo-
lecular Studies, USA, for critical reading this manuscript. This
project was supported by Zhejiang Provincial Natural Science
Foundationof China (No. LY18H300001) to W. Chen; National Key
R&D Program of China (No. 2017YFE0102200) and Osteoporosis &
Breast Cancer Research Center, USA to Y. Yu. We thank Jianyang Pan
(Research and Service Center, College of Pharmaceutical Sciences,
Zhejiang University) for performing NMR spectrometry for struc-
tural elucidation.
4.2. Biological evaluation
4.2.1. Kinase inhibitory assay
The assays were performed in vitro using Homogeneous time
resolved fluorescence (HTRF) method (Cisbio). ALK, ALK L1196M, ALK
Appendix A. Supplementary data
G1202R
and ROS1 were purchased from Carna. The kinases and
substrates were incubated first with synthesized analogues for
5 min in enzymatic buffer. Then ATP (Sigma) was added into the
reaction mixture to start the enzyme reaction. The ATP concen-
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2019.111734.
trations used in each enzyme reaction were 24 mM (ALK) and 37 mM
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