Design, synthesis, biological evaluation and molecular modeling of novel 2-amino-4-(1-phenylethoxy) pyridine derivatives as potential ROS1 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.11.002

With the aim of discovering potential and selective inhibitors targeting ROS1 kinase, we rationally designed, synthesized and evaluated two series of novel 2-amino-pyridine derivatives with 1-phenylethoxy at C-3 and C-4 position. The enzymic assays results indicated that six of the new compounds 13b-13d and 14a-14c showed remarkably higher inhibitory activities against ROS1 kinase. The most promising compounds, 13d and 14c displayed the most desired ROS1 inhibitory activity with IC₅₀ values of 440 nM and 370 nM respectively. Furthermore, 13d and 14c displayed ROS1 inhibitory selectivity of about 7-fold and 12-fold, relative to that of ALK sharing about 49% amino acid sequence homology in the kinase domains. They also showed good anti-proliferative effects against ROS1-addicted HCC78 cell lines with the IC₅₀ values of 8.1 μM and 65.3 μM, respectively. Moreover, molecular docking and molecular dynamics simulation studies disclosed that compound 14c and 13d shared similar binding poses with Crizotinib except the selective binding site of ROS1. It also gave a probable molecular explanation for their activity and selectivity, which the methoxyl group in benzene ring was the crucial to the selectivity to ROS1 versus ALK.

Full text of DOI:10.1016/j.ejmech.2017.11.002

Accepted Manuscript
Design, synthesis, biological evaluation and molecular modeling of novel 2-amino-4-
(1-phenylethoxy) pyridine derivatives as potential ROS1 inhibitors
Yuanxin Tian, Tingting Zhang, Lifan Long, Zhonghuang Li, Shanhe Wan, Guangfa
Wang, Yonghuan Yu, Ju Hou, Xiaoyun Wu, Jiajie Zhang
PII:
S0223-5234(17)30887-5
10.1016/j.ejmech.2017.11.002
EJMECH 9877
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 March 2017
Revised Date: 31 October 2017
Accepted Date: 1 November 2017
Please cite this article as: Y. Tian, T. Zhang, L. Long, Z. Li, S. Wan, G. Wang, Y. Yu, J. Hou, X.
Wu, J. Zhang, Design, synthesis, biological evaluation and molecular modeling of novel 2-amino-4-
(1-phenylethoxy) pyridine derivatives as potential ROS1 inhibitors, European Journal of Medicinal
Chemistry (2017), doi: 10.1016/j.ejmech.2017.11.002.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, synthesis, biological evaluation and molecular modeling
of novel 2-amino-4-(1-phenylethoxy) pyridine derivatives as
potential ROS1 inhibitors
1
1
Yuanxin Tian , Tingting Zhang , Lifan Long, Zhonghuang Li, Shanhe Wan, Guangfa
*
*
Wang, Yonghuan Yu, Ju Hou, Xiaoyun Wu , Jiajie Zhang
Guangdong Provincial Key Laboratory of New Drug Screening, School of
Pharmaceutical Science, Southern Medical University, Guangzhou 510515, PR China
*
Corresponding author:
E-mail: xywu@smu.edu.com (X.-Y. Wu), Tel.: +86-20-62789415;
E-mail: zhangjj@smu.edu.cn (J.-J. Zhang), Tel.:+86-20-62789416. Fax: +86-20-
6
1648548.
1
These authors contributed equally to this work.
Abstract
With the aim of discovering potential and selective inhibitors targeting ROS1
kinase, we rationally designed synthesized and evaluated two series of novel
-amino-pyridine derivatives with 1-phenylethoxy at C-3 and C-4 position. The
2
enzymic assays results indicated that six of the new compounds 13b-13d and 14a-14c
showed remarkably higher inhibitory activities against ROS1 kinase. The most
promising compounds, 13d and 14c displayed the most desired ROS1 inhibitory
activity with IC values of 440 nM and 370 nM respectively. Furthermore, 13d and
5
0
1
4c displayed ROS1 inhibitory selectivity of about 7-fold and 12-fold, relative to that
of ALK sharing about 49% amino acid sequence homology in the kinase domains.
They also showed good anti-proliferative effects against ROS1-addicted HCC78 cell
lines with the IC values of 8.1 ꢀM and 65.3 ꢀM, respectively. Moreover, molecular
5
0
docking and molecular dynamics simulation studies disclosed that compound 14c and
3d shared similar binding poses with Crizotinib except the selective binding site of
1
ROS1. It also gave a probable molecular explanation for their activity and selectivity,
which the methoxyl group in benzene ring was the crucial to the selectivity to ROS1
versus ALK.
1

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Keywords: ROS1 inhibitor; Pyridine derivatives; Molecular docking; Molecular
dynamics simulation.
1
.
Introduction
Receptor tyrosine kinases (RTKs) are critical players in cellular communication
network and function, including cell proliferation, migration, metabolism,
differentiation, and survival. They also are pivotal regulators of normal cellular
processes, in which their activities are normally tightly controlled and regulated [1,2].
As one of the last two orphan human RTKs and the sole member of the ROS1 family,
Proto-oncogene tyrosine-protein kinase ROS (ROS1) and its mechanism of activation
of human kinase have not been fully identified in different body tissues so far [3].
However, the ectopic expression of ROS1, as a results of gene fusion, has been
identified as another clinically actionable oncogenic driver mutation in several
cancerous diseases, including colorectal cancer [4], inflammatory myofibroblastic
tumor (IMT) [5]
, gastric adenocarcinoma [6], non small cell lung cancer (NSCLC)
[
7-9] and so on [10]. Accumulating evidence advocates that ROS1 has become a
drugable target [11,12]. Many researchers are driving efforts to develop effective
ROS1 inhibitors [13]. Until now, most of ROS1 inhibitors with excellent in vitro
inhibitory activities, including the first macrocyclic 3(rd)-generation inhibitor
lorlatinib [14], are the clinical/preclinical success of ALK inhibitors, such as
Crizotinib [15-17], Entrectinib [18] and Brigatinib [19] (their chemical structures
were listed in Figure. 1(A)), since ROS1 shares about 49% amino acid sequence
homology with ALK in the kinase domain and up to 77% of the adenosine
triphosphate(ATP)-binding region[20]. Few compounds are obtained by rational
design with active against ROS1 but inactive against ALK except a series of pyrazole
derivatives such as KIST301072 and their modification 7c (Figure. 1(A)), which
developed by S. H. Lee’s group [8,21,22]. Crizotinib, granted accelerated approval for
the first-generation ALK inhibitor by FDA on 2011, recently expanded its new
indication to the treatment of ROS1 rearranged NSCLC in March 2016 [13]. However,
the insurgence of their mutation that confer resistance [23] to the current therapy
2

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raises the need to identify new structures
.
Hence, it’s absolutely important for the
medicinal chemistry researchers to further explore and produce selective and potent
ROS1 inhibitors as therapeutic strategy for different human malignancies [24].
Figure 1.(A) The structures of clinical/preclinical ALK/ROS1 inhibitors and our
designed new compounds (framed by blue rectangle); (B) their binding poses
comparison (ALK+Crizotinib (2xp2, cyan); ALK+Entrectinib (5fto, yellow);
ALK+Brigatinib (5j7h, green); ROS1+Crizotinib (3zbf, orange); ROS1+7c (docking
structure, pink)).
As most clinical successful ALK inhibitors showed promising ROS1 activity, it
was an efficient method to explore the ROS1 inhibitor based on the ALK inhibitor
[
25] The co-crystal structures of ALK (cyan) and ROS1 (orange) complexes and the
.
docking structure of ROS1 and selective inhibitor 7c (pink) were compared to find
their subtle structure difference. As shown in Figure 1, the blue fragments can
interact with hinge region; and the green fragment can occupy the solvent area near
the hinge region. The active site occupied by red regions of 7c, which was composed
of P-loop and DFG-motif in ALK/ROS1 seems not typical interaction. Hence the
Crizotinib, Entrectinib and Brigatinib showed the dual ALK/ROS1 activities.
However, for selective inhibitor ROS1:7c complex, the red fragment in 7c displayed
additional interaction with the active site mentioned above. The interaction may
contribute to the selectivity for ROS1 versus ALK, which is in accord with the
conclusion from S. H. Lee [8]. Our results based on molecule simulation also proved
the
opinion
[26].
Combining
our
previous
studies
[27],
the
3

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2
-amino-3-(1-phenylethoxy)pyridine was considered as a molecule’s skeleton of
potent ALK/ROS1 inhibitors. If the interactions between ligand and the P-loop and/or
DFG-motif (corresponding to the red region in 7c) are enhanced, the selectivity of
ligand to ROS1 versus ALK may be improved. Corresponding to the pyrimidinyl in
7
c, the benzene ring of 2-amino-3-(1-phenylethoxy) pyridine skeleton would increase
hydrophobic interactions with the hydrophobic pocket. Furthermore, as a hydrogen
bond acceptor and donator, the acylamide moiety acted as a linker because of its
flexibility that had been widely applied in drug design.
Based on the structure comparisons and binding modes analysis above, two
series of derivatives at C-3, C-4 substituted 2-amino-3-(1-phenylethoxy) pyridine and
2
-amino-4-(1-phenylethoxy) pyridine (structures shown in Figure 1(A)) were
designed and synthesized in order to improve the ROS1 inhibitory selectivity. The
1
substitutes in 2-NH position (R group) were changed to investigate the influence
2
from the solvent area near the hinge region. Then the new compounds were evaluated
the enzymic activities against ALK/ROS1 kinases and in vitro anti-proliferative
activities in their corresponding cell lines. Based on the results, two compounds were
selected for further selectivity study. Their binding modes and the selective
mechanism were disclosed by docking and MD simulation, which verified our design
strategy and displayed the structure-activity relationship.
2
. Results and discussion
2
.1. Chemistry
The target compounds were obtained by the following synthesized route with
benzoic acid derivatives illustrated in Scheme 1. Esterification of commercially
available 3-acetylbenzoic acid with concentrated sulfuric acid provided intermediate 2,
which was then subjected to reduction by sodium cyanoborohydride to offer
compound 3 as colorless liquid. Compound 5 was prepared via Mitsunobu coupling of
compound 3 with 2-bromopyridin-3-ol, treating with triphenylphosphine and diethyl
azodicarboxylate. The intermediate compound 5 followed by hydrolysis allowed
readily access to compound 6. Compound 6 was condensed with a variety of
benzylamines to acquire intermediates 7a-7g.The resulting intermediates 7a-7g was
transformed to target compounds 8a-8n of series-I with different reagents by
4

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Buchwald-Hartwig cross coupling reaction as described in Scheme 1. Target
compounds 13a-13r and 14a-14c of series-II were similarly obtained of series-I as
shown in Scheme 2.
1
13
The chemical structures of all target compounds were confirmed by H NMR, C
NMR, ESI-MS, and HRMS.
Scheme 1. Reagents and conditions: (i) H SO , methanol, reflux; (ii) Sodium cyanoborohydride,
2
4
methanol, HCl, r.t.; (iii) DEAD, PPh , THF, r.t.; (iv) methanol, NaOH, H O, rt; (v) CDI, CH Cl
3
2
2
2,
r.t.; (vi) Cs CO , Pd (dba) , BINAP, DMF, 90℃.
2
3
2
3
5

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OH
O
OH
N
9
i
Br
O
N
O
O
N
O
ii
O
OH
O
Br
Br
3
10
11
R2
R2
H
H
N
N
iii
iv
O
O
O
N
O
R1
N
Br
2a-12h
N
H
N
N
1
H N
13a-13r, 14a-14b
2
N
O
O
H
N
O
N
O
N
N
N
H
NH
14c
Scheme 2. Reagents and conditions: (i) DEAD, PPh , THF, r.t.; (ii) methanol, NaOH,
3
H O, r.t.; (iii) CDI, CH Cl , r.t.; (iv) Cs CO , Pd (dba) , BINAP, DMF, 90℃; (v)
2
2
2
2
3
2
3
acetate/CH Cl (8%-100%), r.t..
2
2
2
2
.2. Biological Activity
.2.1Kinase inhibitory activity
The kinase inhibitory activities of the target compounds were tested in duplicates
with 10 doses in 3-fold serial dilution starting at 10 mM and their inhibitory activities
were summarized in Table 1 and Table 2. As shown in this two tables, compounds
8
e-8h, 8j-8k, 8n, 13a, 13e-13j, 13m-13o and 13r exhibited moderate inhibitory
activities against ROS1 with IC50 values ranging from 1.4-9.6 ꢀM. Notably,
compound 13b-13d and 14a-14c displayed significant potency against ROS1 with
6

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IC values ranging from 370-640 nM, and others had no significant potency against
5
0
ROS1 activities (IC ＞10ꢀM). Based on the results mentioned above, we can
5
0
conclude that the substitution of C-4 position of 1-phenylethoxy is more favorable for
ROS1 inhibitory activity. In addition, it's noteworthy that 13d and 14c stood out as
the most potent ROS1 inhibitors among the two series compounds with IC values of
5
0
4
40 nM and 370 nM, respectively. Furthermore, 13d and 14c showed ROS1
inhibitory selectivity of approximately 7-fold and 12-fold relative to that of ALK.
Table 1 The kinase inhibitory activities and in vitro anti-proliferative effects of
synthesized compounds (at C-3 substituted).
1
2
Compd.
R
R
kinase
inhibitory In
vitro
a
activities IC (ꢀM)
antiproliferative
5
0
b
effects (IC , ꢀM)
5
0
ALK
8.1
ROS1
>10
H3122
43.8
HCC78
68.5
8
8
8
8
a
b
c
4-F
3-OMe
8.6
8.9
9.5
>10
>10
>10
49.4
8.3
78.6
73.9
7.8
4-OCF
3
d
3-Cl,4-F
35.7
7

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8
e
f
3-CF
3
,4-Cl 5.9
6.3
50.7
48.6
63.1
8
2-isopropyl >10
sulfonyl
8.7
>100
8
g
4-Me
4-Me
>10
>10
6.2
9.4
>100
9.1
>100
20.5
8
h
8
8
8
8
8
8
i
4-Me
4-Me
4-Me
4-Me
4-Me
3-Cl,4-F
8.0
>10
>10
8.7
7.6
8.6
>10
8.7
15.3
38.4
8.5
27.1
57.4
14.8
9.4
j
k
l
9.4
>10
>10
9.6
23.7
20.8
8.2
m
n
17.6
34.7
a
b
c
Values are the average of two independent experiments.
The date were means from at least three independent experiments.
N.D, not determined.
Table 2. The kinase inhibitory activities and in vitro anti-proliferative effects of
synthesized compounds (at C-4 substituted).
8

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1
2
Compd.
R
R
kinase
inhibitory In
vitro
a
activities IC (µM)
antiproliferative
5
0
b
effects (IC , µM)
5
0
ALK
6.1
ROS1
H3122
>100
HCC78
64.4
1
1
1
3a
3b
3c
3-OMe
1.8
3,4-di-OM 3.6
e
0.64
0.41
38.5
29.0
76.6
73.9
3,4,5-tri-
OMe
2.3
3.1
13d
13e
13f
4-Me
0.44
2.3
8.9
8.0
7.6
8.1
3-CF
,4-Cl 9.2
7.3
3
3,5- di-F
4.7
>10
6.7
2.4
13.2
1
3g
3h
3,5- di-CF
3
7.4
1.5
24.7
35.1
9.3
8.7
1
4-CN
9

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1
1
3i
3j
4-Me
6.7
1.8
7.7
8.9
4-Me
3.5
1.4
13.4
17.5
1
1
1
1
3k
4-Me
4-Me
6.8
7.0
>10
>10
9.1
>100
8.1
68.3
26.2
23.5
49.7
3l
3m
3n
3-CF
3
,4-Cl 9.7
21.7
>100
4-Me
>10
2.4
1
3o
3-CF
3
,4-Cl 9.6
8.4
38.4
>100
13p
13q
13r
4-Me
4-Me
4-Me
>10
4.7
8.7
>10
>10
2.4
9.4
>100
24.5
15.7
37.4
7.8
1
4a
4b
3,4-
1.8
0.57
14.8
9.0
di-OMe
1
3,4-
2.8
0.44
>100
>100
di-OMe
1
0

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1
4c
3,4-
4.5
0.37
47.2
0.8
65.3
2.0
di-OMe
Crizotinib
N.D
N.D
a
Values are the average of two independent experiments.
b
c
The date were means from at least three independent experiments.
N.D, not determined.
Afterwards, to further investigate the kinase selectivity of 13d and 14c, we
evaluated the inhibitory activity against a panel of 12 RTKs associated with
tumorigenesis [22,28,29] at a concentration of 1 ꢀM. As shown in the Table 3, 13d
and 14c didn’t display significant potency against the 12 RTKs (percent inhibition
values ranging from 0% to 16%).Consequently, 13d and 14c are considered as two
highly selective ROS1 inhibitors.
Table 3. Kinase selectivity profiles of compound 13d and 14c.
inhibition rate at 1 ꢀM
inhibition rate at 1 ꢀM
a
a
Kinase
(%)
Kinase
(%) )
1
3d
14c
11
13
2
13d
12
2
14c
10
3
ABL1
9
JAK2
BRAF
15
1
KIT
CDK2/cyclin A
EGFR/(ErbB1)
FGFR1
LTK(TYK1)
MAPK3
9
15
7
1
0
8
11
7
16
9
MET(c-Met)
PDGFR alpha
3
5
IGF1R
6
1
a
Values are the average of two independent experiments.
2
.2.2. In vitro anti-proliferative activity
The synthesized compounds were set to research the inhibitory effects on cell
proliferation in ALK-addicted H3122 cell and ROS1-addicted HCC78 cell by the
standard MTT colorimetric assay in vitro[30]. Crizotinib was used as positive control.
The results summarized in Table 1 and Table 2, which were derived from at least
three independent experiments. It can be noted that most of compounds displayed
moderate to high potency against HCC78 and H3122 cell, some of the compounds
showed parallel anti-proliferative activities against HCC78 cell line as Crizotinib.
1
1

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Eight compounds (8d, 8l, 13d-13e, 13g-13i and 14a) displayed significant potency
with IC values of 7.3-9.4 ꢀM, which turned out to be similar to that of Crizotinib
5
0
(
IC =2.00 ꢀM). Nevertheless, the cellular responses of some synthesized compounds
50
were not consistent with the enzymatic assays absolutely. 13b-13c and 14b-14c
exhibited good potency against ROS1 kinase, however, gave the weak potency
against HCC78 cell line, which expressed the SLC34A2-ROS1 fusion protein. 8d,
8
h-8i, 8k, 8l-8m, 13e-13f, 13g, 13i, 13l-13m and 13q-13r showed poor activities
against ROS1 kinase, but they displayed anti-proliferative activities against HCC78
cell line at IC ＜10ꢀM. We supposed that the different sensitivity to ROS1 fusion
5
0
genes induced by synthesized compounds led to different response in HCC78 cell line,
and some compounds could induce cell apoptosis, or accelerate cell metabolism, or
have some other mechanism [31].
2
.3. Preliminary structure-activity relationships
Based on the biological activities listed in Table 1 and Table 2, the preliminary
structure-activity relationships of the synthesized compounds against ROS1 were
summarized in Figure 2. In general, the 1-phenylethoxy substitution at C-4 position
1
of the 2-amino-pyridine ring displayed better activity than that at C-3 position. For R
group, one of hydrogen atoms of 2-NH , replaced by hydrophilic group, would be in
2
favor of activity. Among the substitution groups, 1-(piperidin-4-yl)-1H-pyrazol-4-yl
was the optimal group, then followed by 1-(1-methylpiperidin-4-yl)-1H-pyrazol-4-yl,
pyrimidin-2-yl, 1,3-dimethyl-1H-pyrazol-4-yl and 1-methyl-1H-pyrazol-4-yl. ROS1
1
2
activities were relatively poor when R was other heterocyclics and benzenes. For R
group, the methoxyl groups on the benzene ring of the synthesized compounds were
in favor of the activity. Among these groups, 3,4,5-trimethoxyl was the optimal group,
then followed by 3,4-dimethoxyl, 3-methoxyl, 4-methyl and 4-CN. For example,
compound 13c (IC =410 nM), possessing three favorable methoxyl groups,
5
0
maintained significant potency against ROS1 kinase than the relevant two and
mono-substituted analogs 13b (IC =640 nM) and 13a (IC =1.8 µM). Furthermore,
5
0
50
the compounds 14a-14c with methoxyl in benzene ring also showed promising
1
2

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activities against ROS1 kinase. The results demonstrated that the methoxyl may be
crucial to the selectivity of ROS1 versus ALK.
Figure 2.Preliminary structure-activity relationships of ROS1 kinase inhibitory
2
.4 Molecular modeling:
In order to investigate the probable binding conformations of the new compounds,
docking experiments were performed with the Surflex-Dock program from Sybyl7.3.
The crystal structure of ROS1 (PDB entry: 3zbf) and the compounds are pretreated
according to our previous work [32]. The docking results indicated that the 13a-13r
and 14a-14c adopted a similar binding mode to the Crizotinib. However, the
compounds 8a-8n couldn’t fit the binding site of ROS1 well because of the steric
1
hindrance effect. Due to the same R group that extended to the solvent region near
the hinge region, the compounds 13d, 13e and 14c were chosen as examples to
illustrate the binding modes (displayed in Figure 3).
1
3

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Figure 3. Superimposition of docking poses for the new compound 14c (blue), 13e
(magenta),13d (yellow) and Crizotinib (gray) based on docking.
As we expected, the 2-amine-pyridine moiety can formed double H-bonds with the
Met2029 from hinge region. Comparing Crizotinib, the difference between the two
binding mode was that Crizotinib could form double H-bonds with Glu2027 and
Met2029. The interaction with hinge regions is a typical characteristic of the dual
ALK/ROS1 inhibitors. Our designed group (red group in Figure 1) extended to
another active site composed of P-loop and DFG-motif. The NH from acylamino
made H-bond to Asp2102 from DFG-motif. The 14c with -OCH group formed an
3
additional H-bond with Lys1980 from beta-sheet, which could explain that the
compounds with -OCH group (13a-13c, 14a-14c) displayed the lower inhibitory
3
activity against ROS1 and better selectivity to ALK. Moreover, the benzamide
(ph-CO-NH) linker could match the hydrophobic pocket and increased the
hydrophobic interactions, which was favorable for the activity and selectivity.
Although compound 14c showed the relative selectivity and activity as we
expected, it was not desired. Moreover, the difference between 13e and 14c is only the
2
R group, while the activities showed great difference. We noticed that the compounds
with 3-CF -4-Cl group have poorer selectivity, such as 8e, 13e, 13m and 13o. In order
3
2
to further explore the contribution of R group in details, molecular dynamic (MD)
1
4

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simulations were carried out on the active compound 14c. The compound 13e with the
1
same R group and less activity was chosen as a comparison.
Figure 4. (A)The conformation changes of 14c (initial: blue; after MD: orange) and
1
3e (initial: magenta; after MD: green) after 20 ns Molecular Dynamic simulation; (B)
View of superimposed conformational change of the ligand 14c. The OCH(CH3)
group turned from inner to outer of the ATP-binding site (shown in ball); (C) View of
superimposed conformational change of the ligand 13e.
The docking structures as the initial structures were processed in explicit aqueous
solution. After 20 ns MD simulation, the stability of each system is evaluated by the
root-mean-square deviation (RMSD) of the backbone atoms against their initial
structures (shown in Figure S1 of supporting information). From the Figure S1, we
could see the 14c had larger RMSD values than 13e although the RMSD values of
protein backbone atoms were relatively stable, indicating there was larger
conformation changes of 14c than that of 13e. In fact, compound 14c really displayed
larger conformation changes (shown in Figure 4). In initial docking structure, the
amino-pyridine of two compounds can interact with Met2029 from the hinge region
by double H-bonds and the acylamide linker formed H-bonds with Asp2102 from
DFG-motif. Compound 14c has an additional H-bond between Lys1980 and -OCH3
group. During 20ns MD simulation, the H-bonds mentioned above in 13e were
1
5

ACCEPTED MANUSCRIPT
weakened and gradually disappeared because of another H-bond between C=O and
Asn2084. Although the conformation had little changes, the key interaction for
activities was disappeared and there were no other interactions in selective active site
between the ligand and protein, which directly led to the low activity of ROS1. For
compound 14c, because of the H-bonds made by -OCH and Lys1980, the
3
conformation displayed larger changes to fit the selective active site composed by the
DFG-motif and P-loop. The ethoxyl group was almost displayed 180 degree turned
from the inner to outer of hydrophobic pocket (shown in Figure 4B). This
conformation change directly led to the disappearance of hydrogen bonds in the hinge
region. The details of the hydrogen bonds of 14c and 13e are listed in Table 4.
Table 4. Hydrogen bonds analysis of 14c and 13e during the 20ns MD simulation.
13e (2.3ꢀM)
14c (0.37ꢀM)
donor
acceptor
occupancy donor
acceptor
occupancy
ASN151-Side-ND2 MOL298-Side-O2
89.85%
12.28%
4.87%
2.12%
1.54%
1.08%
MOL298-Side-N2 MET96-Main-O 32.95%
MET96-Main-N MOL298-Side-N6 22.51%
MOL298-Side-N1 ASP169-Side-OD1 18.67%
MOL298-Side-N1 ASP169-Side-OD2 4.57%
MET96-Main-N
MOL298-Side-N6
HIE144-Side-NE2
HIE144-Side-NE2
HIE144-Side-NE2
MOL298-Side-N1
ASP169-Side-OD1
MOL298-Side-F1
MOL298-Side-F2
MOL298-Side-F3
LYS47-Side-NZ
LYS47-Side-NZ
MOL298-Side-O2 0.07%
MOL298-Side-N1 0.02%
From the Table 4, we could see the occupancy of H-bonds from the hinge region
and DFG-motif in 14c were 55.46% and 23.24%, which is corresponding in 13e were
1
2.28% and 4.87%, while another H-bonds between Asn151(Asn2084) and C=O
showed the high occupancy 89.85% in 13e. Moreover, because of the H-bond from
Lys1980, the 3,4-dimethoxypheny group could match the hydrophilic pocket made by
P-loop and DFG-motif, which benefited the lower activity to ROS1. This
phenomenon happened to in accord with the interaction of compound 7c, one of the
selective ROS1 from S. H. Lee’s group. Therefore, the 14c displayed the relative
selective to ROS1 versus ALK. On account of the reduction of key interaction in the
hinge region and the selective site, 14c didn’t show promising activity. Further
modification of 14c is underway based on our simulation results.
1
6

ACCEPTED MANUSCRIPT
3
. Conclusion
In summary, in order to explore selective ROS1 inhibitor, two series of new
-amino-pyridine derivatives with 1-phenylethoxy at C-3 and C-4 position
2
processing amide linker were designed, synthesized and evaluated for their
biological activities against ROS1 and ALK. The results indicated that most
compounds, especially C4-substituted derivatives, exhibited moderate enzyme
activities of ROS1 and ALK (with the IC50 below 10 ꢀM). Especially, the
compounds 13d and 14c stood out the promising ROS1 inhibitory activities with the
IC50 values of 440 nM and 370 nM. Moreover, they also displayed the ROS1
inhibitory selectivity of about 10-fold relative to that of ALK in enzyme domain. The
molecular docking demonstrated that the binding poses of 13d and 14c with ROS1
were similar to Crizotinib except the selective binding site. Molecular dynamic
simulation revealed that the distinct conformation changes of 14c lead to the better
selectivity and the unsatisfactory activity to ROS1. The 2-amino-pyridine derivatives
with 1-phenylethoxy at C-3 and C-4 position of our studies had not been reported,
which had broken the limits of several existing ROS1 inhibitors. The further
modifications of 13d and 14c to improve the selectivity and activity to ROS1 are
underway based on our results.
4
. Experimental
4
.1. Chemistry
All chemicals were purchased from commercial vendors and were used without
1
13
further purification unless otherwise stated. The H and C NMR spectra were
generated on a BRUKER AVIII 400 MHz and 101 MHz spectrometer with
tetramethylsilane (TMS) as the internal standard. Mass spectra were performed on
WATERS ZQ4000 with electrospray ionization source. High resolution mass spectra
(
HRMS) were recorded on a Thermo Fisher Scientific Orbitrap Fusion Tribrid
Q-OT-qIT) mass spectrometer (Thermo Fisher Scientific, Bremen, German). The
(
CHN elemental analyses of all the target compounds were recorded by LECO
1
7

ACCEPTED MANUSCRIPT
TRUSPEC CHN analyzer. All reactions were assayed by thin-layer chromatography
(
TLC). Flash column chromatography separations were performed on silica gel
200-300 mesh, Qingdao Ocean Chemicals, China) by using Yamazen AI-580 flash
(
chromatography. Melting points were obtained on an X-4 Melting-point Apparatus
with Microscope (Gongyi City Yuhua Instrument Co., Ltd., Henan, China) and were
uncorrected.
4
.1.1. methyl 3-acetylbenzoate (2)
To a solution of 3-acetylbenzoic acid (1) (5.00 g, 30.7 mmol) in MeOH (147 mL)
added into 10 mL concentrated H SO . The reaction mixture was stirred at reflux for
2
4
2
4h. After removal of the solvent, the residue was poured into H O (80 mL) and
2
extracted with EtOAc. The organic layer was washed with H O and brine and dried
2
over Na SO . Filtration and concentration in vacuo to give 2 as light yellow liquid
2
4
1
(
4.54 g, 83%). H NMR (400 MHz, CDCl ) δ 8.60 (s, ArH, 1H), 8.24 (t, J = 7.5 Hz,
3
ArH, 1H), 8.17 (d, J = 7.5 Hz, ArH, 1H), 7.57 (t, J = 7.7 Hz, ArH, 1H), 3.96 (s, OCH3,
+
3
H), 2.66 (s, CH , 3H). ESI-MS m/z: 179 [M+H] .
3
4
.1.2. methyl 3-(1-hydroxyethyl)benzoate (3)
To a solution of methyl 3-acetylbenzoate 2 (4.00 g, 22.5 mmol) in MeOH (104
mL) was added sodium cyanoborohydride slowly (2.84 g, 45.0 mmol). The mixture
was adjusted pH to 3, and stirred at ambient temperature for overnight. After removal
of the solvent, the residue was poured into H O (65 mL) and extracted with EtOAc.
2
The organic layer was washed with H O and brine and dried over MgSO , The
2
4
concentrates was purified by flash chromatography on silica gel eluting with EtOAc/
1
petroleum ether (1%-10%) to give 3 as pale yellow liquid (3.24 g, 80%). H NMR
(
400 MHz, DMSO-d ) δ 7.98 (s, ArH, 1H), 7.82 (d, J = 7.6 Hz, ArH, 1H), 7.61 (d, J =
6
7
.6 Hz, ArH, 1H), 7.47 (t, J = 7.6 Hz, ArH, 1H), 5.31 (d, J = 4.4 Hz, OH, 1H), 4.80 (q,
J = 6.0 Hz, CH, 1H), 3.86 (s, OCH , 3H), 1.34 (d, J = 6.4 Hz, CH , 3H). ESI-MS m/z:
3
3
+
1
81[M+H] .
4
.1.3. methyl 3-(1-((2-bromopyridin-3-yl)oxy)ethyl)benzoate (5)
To a stirred solution of triphenylphosphine (6.31 g, 24.1 mmol) and DEAD (4.49,
1
8

ACCEPTED MANUSCRIPT
2
5.8 mmol) in THF (30 mL) at 0 ℃was added a solution of 3 (3.10 g, 17.2 mmol) and
-bromopyridin-3-ol 4 (3.44 g, 19.8 mmol) in THF (20 mL). The resulting luminous
2
yellow solution was stirred under a nitrogen atmosphere at ambient temperature for 24
h at which all starting materials had been consumed. The solvent was removed, the
residue was purified by flash chromatography on silica gel eluting with EtOAc/
1
petroleum ether (1%-20%) to give 5 as light yellow liquid (4.97 g, 86%). H NMR
(
400 MHz, DMSO-d ) δ 8.06 (s, ArH, 1H), 7.92 (dd, J = 1.2, ArH, 4.4 Hz, 1H), 7.88
6
(d, J = 7.6 Hz, ArH, 1H), 7.72 (d, J = 7.6 Hz, ArH, 1H), 7.54 (t, J = 7.8 Hz, ArH, 1H),
7
6
3
.45 (dd, J = 1.2, 8.0 Hz, ArH, 1H), 7.29 (dd, J = 4.8, 8.0 Hz, ArH, 1H), 5.84 (q, J =
.4 Hz, CH, 1H), 3.86 (s, OCH , 3H), 1.62 (d, J = 6.4 Hz, CH , 3H). ESI-MS m/z:
3
3
+
36.6 and 338.5 [M+H] .
4
.1.4. 3-(1-((2-bromopyridin-3-yl)oxy)ethyl)benzoic acid (6)
To a solution of 5 (4.80 g, 14.29 mmol) in MeOH (60mL) was added sodium
hydroxide (1.42 g, 35.5mmol) in water (21mL), the mixture was stirred at ambient
temperature for 1.5h, after the complete conversion of the starting material, the
solution was acidified to pH=5 with 1M hydrochloric acid. The mixture was filtered
1
and condensed to give 6 as white solid (4.14 g, 90%). H NMR (400 MHz, DMSO-d )
6
δ 8.03 (s, ArH, 1H), 7.92 (dd, J = 1.2, 4.4 Hz, ArH, 1H), 7.86 (d, J = 7.6 Hz, ArH,
1
8
1
H), 7.68 (d, J = 8.0 Hz, ArH, 1H), 7.51 (t, J = 7.6 Hz, ArH, 1H), 7.44 (dd, J = 0.8,
.0 Hz, ArH, 1H), 7.29 (dd, J = 4.8, 8.8 Hz, ArH, 1H), 5.82 (q, J = 6.4 Hz, CH, 1H),
-
.62 (d, J = 6.4 Hz, CH , 3H). ESI-MS m/z: 320.2 and 322.3[M-H] .
3
4
.1.5. 3-(1-((2-bromopyridin-3-yl)oxy)ethyl)-N-(4-fluorobenzyl)benzamide (7a)
CDI (112 mg, 0.69 mmol) was added under nitrogen to a solution of 6 (200 mg,
0
.62 mmol) in dry methylene chloride (15 mL). The solution was stirred at room
temperature for 1 h, then (4-fluorophenyl)methanamine (116 mg, 0.93 mmol) was
added, and stirred at room temperature under nitrogen or 24 h. After the complete
conversion of the starting material, the reaction was quenched with 1 M HCl. The
aqueous phase was extracted with methylene chloride (3 × 50 mL). The combined
organic phases were washed with brine until neutral pH, dried on Na SO , filtered,
2
4
1
9

ACCEPTED MANUSCRIPT
1
and the solvent was eliminated under reduced pressure to give 7a (240 mg, 90%). H
NMR (400 MHz, DMSO-d ) δ 9.07 (t, J = 5.8 Hz, NH, 1H), 7.96 (s, ArH, 1H), 7.91
6
(d, J = 4.0 Hz, ArH, 1H), 7.80 (d, J = 8.0 Hz, ArH, 1H), 7.58 (d, J = 7.6 Hz, ArH, 1H),
7
2
6
.47 (t, J = 7.6 Hz, ArH, 1H), 7.42 (d, J = 7.6 Hz, ArH, 1H), 7.38 – 7.35 (m, ArH,
H), 7.29 (dd, J = 4.4, 8.0 Hz, ArH, 1H), 7.16 (t, J = 8.6 Hz, ArH, 2H), 5.75 (q, J =
.4 Hz, CH, 1H), 4.46 (d, J = 5.6 Hz, CH , 2H), 1.63 (d, J = 6.0 Hz, CH , 3H).
2
3
-
ESI-MS m/z: 427.4 and 429.5[M-H] .
4
.1.6. 3-(1-((2-bromopyridin-3-yl)oxy)ethyl)-N-(3-methoxybenzyl)benzamide (7b)
Compound 6 (200 mg, 0.62 mmol) and (3-methoxyphenyl)methanamine (127
mg, 0.93 mmol) following the similar procedure described for the preparation of 7a
1
afforded 7b (251 mg, 92%). H NMR (400 MHz, CDCl ) δ 7.88 (d, J = 8.0 Hz, ArH,
3
2
H), 7.71 (d, J = 8.0 Hz, ArH, 1H), 7.53 (d, J = 7.6 Hz, ArH, 1H), 7.39 (t, J = 7.6 Hz,
ArH, 1H), 7.24 (t, J = 7.8 Hz, ArH, 1H), 7.03 – 7.00 (m, ArH, 1H), 6.96 – 6.88 (m,
ArH, 4H), 6.82 (d, J = 8.0 Hz, ArH, 1H), 5.36 (q, J = 6.2 Hz, CH, 1H), 4.59 (d, J =
5
4
.6 Hz, CH , 2H), 3.77 (s, OCH , 3H), 1.69 (d, J = 6.4 Hz, CH , 3H). ESI-MS m/z:
2 3 3
+
41.5 and 443.6[M+H] .
4
3
.1.7.
-(1-((2-bromopyridin-3-yl)oxy)ethyl)-N-(4-(trifluoromethoxy)benzyl)benzamide (7c)
Compound 6 (200 mg, 0.62 mmol) and (4-(trifluoromethoxy)phenyl) methane
mine (177 mg, 0.93 mmol) following the similar procedure described for the
1
preparation of 7a afforded 7c (264 mg, 86%). H NMR (400 MHz, DMSO-d ) δ 9.11
6
(t, J = 5.6 Hz, NH, 1H), 7.97 (s, ArH, 1H), 7.91 (d, J = 4.4 Hz, ArH, 1H), 7.81 (d, J =
7
7
.6 Hz, ArH, 1H), 7.59 (d, J = 7.6 Hz, ArH, 1H), 7.50 – 7.38 (m, ArH, 4H), 7.36 –
.27 (m, ArH, 3H), 5.76 (q, J = 6.2 Hz, CH, 1H), 4.50 (d, J = 5.6 Hz, CH , 2H), 1.64
2
-
(
d, J = 6.4 Hz, CH , 3H). ESI-MS m/z: 493.3 and 495.4[M-H] .
3
4
(
.1.8. 3-(1-((2-bromopyridin-3-yl)oxy)ethyl)-N-(3-chloro-4-fluorobenzyl)benzamide
7d)
Compound 6 (200 mg, 0.62 mmol) and (3-chloro-4-fluorophenyl) methanamine
(100 mg, 0.93 mmol) following the similar procedure described for the preparation of
2
0

ACCEPTED MANUSCRIPT
1
7
a afforded 7d (261 mg, 91%). H NMR (400 MHz, DMSO-d ) δ 9.10 (t, J = 6.0 Hz,
6
NH, 1H), 7.96 (s, ArH, 1H), 7.91 (dd, J = 1.6, 4.4 Hz, ArH, 1H), 7.80 (d, J = 7.8 Hz,
ArH, 1H), 7.59 (d, J = 8.0 Hz, ArH, 1H), 7.53 (dd, J = 2.0, 7.2 Hz, ArH, 1H), 7.48 (t,
J = 7.8 Hz, ArH, 1H), 7.42 (dd, J = 1.6, 8.0 Hz, ArH, 1H), 7.36 – 7.33 (m, ArH, 2H),
7
.29 (dd, J = 4.4, 8.0 Hz, ArH, 1H), 5.76 (q, J = 6.2 Hz, CH, 1H), 4.46 (d, J = 6.0 Hz,
+
CH , 2H), 1.63 (d, J = 6.4 Hz, CH , 3H). ESI-MS m/z: 463.5 and 465.6[M+H] .
2
3
4
3
.1.9.
-(1-((2-bromopyridin-3-yl)oxy)ethyl)-N-(4-chloro-3-(trifluoromethyl)benzyl)benzami
de (7e)
Compound 6 (200 mg, 0.62 mmol) and (4-chloro-3-(trifluoromethyl)phenyl)
methanamine (194 mg, 0.93 mmol) following the similar procedure described for the
1
preparation of 7a afforded 7e (276 mg, 87%). H NMR (400 MHz, DMSO-d ) δ 9.16
6
(
t, J = 5.8 Hz, NH, 1H), 7.96 (s, ArH, 1H), 7.91 (dd, J = 1.2, 4.4 Hz, ArH, 1H), 7.82 –
.79 (m, ArH, 2H), 7.70 (d, J = 8.4 Hz, ArH, 1H), 7.65 – 7.59 (m, ArH, 2H), 7.49 (t, J
7.6 Hz, ArH, 1H), 7.42 (dd, J = 1.6, 8.4 Hz, ArH, 1H), 7.28 (dd, J = 4.8, 8.4 Hz,
7
=
ArH, 1H), 5.76 (q, J = 6.4 Hz, CH, 1H), 4.54 (d, J = 6.0 Hz, CH , 2H), 1.63 (d, J =
2
-
6
.0 Hz, CH , 3H). ESI-MS m/z: 511.8 and 513.7[M-H] .
3
4
.1.10. 3-(1-((2-bromopyridin-3-yl)oxy)ethyl)-N-(2-(isopropylsulfonyl)
benzyl)benzamide (7f)
Compound 6 (200 mg, 0.62 mmol) and (2-(isopropylsulfonyl)phenyl)
methanamine (198 mg, 0.93 mmol) following the similar procedure described for the
1
preparation of 7a afforded 7f (278 mg, 87%). H NMR (400 MHz, DMSO-d ) δ 9.23
6
(
t, J = 5.6 Hz, NH, 1H), 7.99 (s, ArH, 1H), 7.92 (d, J = 3.6 Hz, ArH, 1H), 7.87 (dd, J
8.0, 14.0 Hz, ArH, 2H), 7.72 (t, J = 7.6 Hz, ArH, 1H), 7.61 (d, J = 4.4 Hz, ArH, 2H),
.57 – 7.49 (m, ArH, 2H), 7.43 (d, J = 8.0 Hz, ArH, 1H), 7.32 (dd, J = 4.8, 8.4 Hz,
ArH, 1H), 5.78 (q, J = 6.4 Hz, CH, 1H), 4.85 (m, CH , 2H), 3.75 (m, SCH, 2H), 1.65
=
7
2
(
d, J = 6.4 Hz, OCHCH , 3H), 1.23 (d, J = 6.8 Hz, CH , 3H), 1.22 (d, J = 6.8 Hz, CH ,
3 3 3
+
3
H). ESI-MS m/z: 517.5 and 519.6[M+H] .
4
.1.11. 3-(1-((2-bromopyridin-3-yl)oxy)ethyl)-N-(4-methylbenzyl)benzamide (7g)
Compound 6 (200 mg, 0.62 mmol) and p-tolylmethanamine (111 mg, 0.93 mmol)
2
1

ACCEPTED MANUSCRIPT
following the similar procedure described for the preparation of 7a afforded 7g (244
1
mg, 93%). H NMR (400 MHz, DMSO-d ) δ 9.01 (t, J = 6.0 Hz, NH, 1H), 7.96 (s,
6
ArH, 1H), 7.91 (dd, J = 1.6, 4.4 Hz, ArH, 1H), 7.79 (d, J = 7.6 Hz, ArH, 1H), 7.57 (d,
J = 8.0 Hz, ArH, 1H), 7.47 (t, J = 7.8 Hz, ArH, 1H), 7.42 (dd, J = 1.6, 8.4 Hz, ArH,
1
H), 7.28 (dd, J = 4.8, 8.4 Hz, ArH, 1H), 7.21 (d, J = 8.0 Hz, ArH, 2H), 7.15 – 7.13
(
m, ArH, 2H), 5.75 (q, J = 6.2 Hz, CH, 1H), 4.43 (d, J = 6.0 Hz, CH , 2H), 2.28 (s,
2
+
ArCH , 3H), 1.63 (d, J = 6.4 Hz, CH , 3H). ESI-MS m/z: 425.4 and 427.5[M+H] .
3
3
4
.1.12. methyl 3-(1-((2-bromopyridin-4-yl)oxy)ethyl)benzoate (10)
Compound 3 (3.10 g, 17.2 mmol) and 2-bromopyridin-4-ol 9 (3.44 g, 19.8 mmol)
following the similar procedure described for the preparation of 5 afforded 10 (4.80 g,
1
8
3%). H NMR (400 MHz, DMSO) δ 8.14 (d, J = 5.6 Hz, ArH, 1H), 8.03 (s, ArH,
H), 7.90 (d, J = 7.6 Hz, ArH, 1H), 7.72 (d, J = 7.6 Hz, ArH, 1H), 7.55 (t, J = 7.6 Hz,
1
ArH, 1H), 7.23 (d, J = 1.6 Hz, ArH, 1H), 7.03 (dd, J = 2.0, 6.0 Hz, ArH, 1H), 5.87 (q,
J = 6.2 Hz, CH, 1H), 3.86 (s, OCH , 3H), 1.58 (d, J = 6.0 Hz, CH , 3H),ESI-MS m/z:
3
3
+
3
36.6 and 338.5 [M+H] .
.1.13. 3-(1-((2-bromopyridin-4-yl)oxy)ethyl)benzoic acid (11)
Compound 10 (4.80 g, 14.29 mmol) and sodium hydroxide (1.42 g, 35.5mmol)
4
following the similar procedure described for the preparation of 6 afforded 11 (3.6 g,
1
8
0%). H NMR (400 MHz, DMSO-d ) δ 13.06 (s, COOH, 1H), 8.14 (d, J = 5.6 Hz,
6
ArH, 1H), 8.01 (s, ArH, 1H), 7.88 (d, J = 7.6 Hz, ArH, 1H), 7.68 (d, J = 7.6 Hz, ArH,
1
5
3
H), 7.52 (t, J = 7.6 Hz, ArH, 1H), 7.23 (s, ArH, 1H), 7.03 (d, J = 5.6 Hz, ArH, 1H),
.86 (q, J = 6.4 Hz, CH, 1H), 1.58 (d, J = 6.0 Hz, CH , 3H), ESI-MS m/z: 320.2 and
3
－
22.3 [M−H] .
4
.1.14. 3-(1-((2-bromopyridin-4-yl)oxy)ethyl)-N-(3-methoxybenzyl)benzamide (12a)
Compound 11 (200 mg, 0.62 mmol) and (3-methoxyphenyl)methanamine (125
mg, 0.93 mmol) following the similar procedure described for the preparation of 7a
1
afforded 12a (224 mg, 82%). H NMR (400 MHz, CDCl ) δ 8.01 (d, J = 5.6 Hz, ArH,
3
1
H),7.86 (d, J = 8.0 Hz, ArH, 1H), 7.71 (d, J = 8.0 Hz, ArH, 1H), 7.46 (d, J = 7.6 Hz,
ArH, 1H), 7.39 (t, J = 7.6 Hz, ArH, 1H), 7.24 (t, J = 7.8 Hz, ArH, 1H), 6.96 – 6.88 (m,
ArH, 3H), 6.82 (d, J = 8.0 Hz, ArH, 1H), 6.68 (d, J = 8.0 Hz, ArH, 1H), 5.36 (q, J =
2
2

ACCEPTED MANUSCRIPT
6
.2 Hz, CH, 1H), 4.59 (d, J = 5.6 Hz, CH , 2H), 3.77 (s, OCH , 3H), 1.69 (d, J = 6.4
2
3
+
Hz, CH , 3H). ESI-MS m/z: 441.5 and 443.6[M+H] .
3
4
(
.1.15. 3-(1-((2-bromopyridin-4-yl)oxy)ethyl)-N-(3,4-dimethoxybenzyl)benzamide
12b)
Compound 11 (200 mg, 0.62 mmol) and (3,4-dimethoxyphenyl)methanamine
(155 mg, 0.93 mmol) following the similar procedure described for the preparation of
1
7
a afforded 12b (251 mg, 86%). H NMR (400 MHz, DMSO-d ) δ 8.97 (t, J = 5.8 Hz,
6
NH, 1H), 8.13 (d, J = 6.0 Hz, ArH, 1H), 7.94 (s, ArH, 1H), 7.81 (d, J = 7.6 Hz, ArH,
H), 7.58 (d, J = 8.0 Hz, ArH, 1H), 7.48 (t, J = 7.6 Hz, ArH, 1H), 7.20 (d, J = 2.4 Hz,
1
ArH, 1H), 7.01 (dd, J = 2.4, 5.6 Hz, ArH, 1H), 6.95 (d, J = 1.6 Hz, ArH, 1H), 6.90 (d,
J = 8.0 Hz, ArH, 1H), 6.84 (dd, J = 2.0, 8.4 Hz, ArH, 1H), 5.79 (q, J = 6.4 Hz, CH,
1
H), 4.41 (d, J = 6.0 Hz, CH , 2H), 3.74 (s, OCH , 3H), 3.72 (s, OCH , 3H), 1.60 (d, J
2 3 3
+
=
6.4 Hz, CH , 3H). ESI-MS m/z: 471.3 and 473.2[M+H] .
3
4
(
.1.16. 3-(1-((2-bromopyridin-4-yl)oxy)ethyl)-N-(3,4,5-trimethoxybenzyl)benzamide
12c)
Compound 11 (200 mg, 0.62 mmol) and (3,4,5-trimethoxyphenyl) meth anamine
(183 mg, 0.93 mmol) following the similar procedure described for the preparation of
1
7
a afforded 12c (276 mg, 89%). H NMR (400 MHz, DMSO-d ) δ 8.99 (t, J = 5.8 Hz,
6
NH, 1H), 8.13 (d, J = 5.6 Hz, ArH, 1H), 7.94 (s, ArH, 1H), 7.82 (d, J = 7.6 Hz, ArH,
H), 7.59 (d, J = 7.6 Hz, ArH, 1H), 7.49 (t, J = 7.6 Hz, ArH, 1H), 7.20 (d, J = 2.0 Hz,
1
ArH, 1H), 7.01 (dd, J = 2.4, 5.6 Hz, ArH, 1H), 6.65 (s, ArH, 2H), 5.80 (q, J = 6.2 Hz,
CH, 1H), 4.42 (d, J = 6.0 Hz, CH , 2H), 3.75 (s, 2×OCH , 6H), 3.64 (s, OCH , 3H),
2
3
3
+
1
.60 (d, J= 6.4 Hz, CH , 3H). ESI-MS m/z: 500.4 and 502.5[M+H] .
3
4
.1.17. 3-(1-((2-bromopyridin-4-yl)oxy)ethyl)-N-(4-methylbenzyl)benzamide (12d)
Compound 11 (200 mg, 0.62 mmol) and p-tolylmethanamine (111 mg, 0.93 mmol)
following the similar procedure described for the preparation of 7a afforded 12d (231
1
mg, 88%). H NMR (400 MHz, DMSO-d ) δ 9.02 (s, NH, 1H), 8.13 (d, J = 5.2 Hz,
6
ArH, 1H), 7.95 (s, ArH, 1H), 7.82 (d, J = 7.2 Hz, ArH, 1H), 7.58 (d, J = 7.6 Hz, ArH,
1
H), 7.48 (t, J = 7.6 Hz, ArH, 1H), 7.20 (s, ArH, 3H), 7.14 (d, J = 7.2 Hz, ArH, 2H),
2
3

ACCEPTED MANUSCRIPT
7
2
4
.02 (d, J = 5.2 Hz, ArH, 1H), 5.79 (q, J = 6.0 Hz, CH, 1H), 4.44 (d, J = 4.8 Hz, CH2,
H), 2.28 (s, ArCH , 3H), 1.60 (d, J = 5.6 Hz, CH , 3H). ESI-MS m/z: 425.4 and
3
3
+
27.5[M+H] .
4
.1.18. 3-(1-((2-bromopyridin-4-yl)oxy)ethyl)-N-(4-chloro-3-(trifluoromethyl)benzyl)
benzamide (12e)
Compound 11 (200 mg, 0.62 mmol) and (4-chloro-3-(trifluoromethyl)phenyl)
methanamine (194 mg, 0.93 mmol) following the similar procedure described for the
1
preparation of 7a afforded 12e (265 mg, 82%). H NMR (400 MHz, DMSO-d ) δ 9.16
6
(t, J = 5.8 Hz, NH, 1H), 7.97 (s, ArH, 1H), 7.92 (d, J = 4.4 Hz, ArH, 1H), 7.81 (d, J =
9
.2 Hz, ArH, 2H), 7.70 (d, J = 8.4 Hz, ArH, 1H), 7.62 (dd, J = 8.4, 16.0 Hz, ArH, 2H),
.49 (t, J = 7.8 Hz, ArH, 1H), 7.42 (d, J = 8.0 Hz, ArH, 1H), 7.28 (dd, J = 4.4, 8.0 Hz,
7
ArH, 1H), 5.76 (q, J = 6.4 Hz, CH, 1H), 4.54 (d, J = 6.0 Hz, CH , 2H), 1.64 (d, J =
2
-
6
.4 Hz, CH , 3H). ESI-MS m/z: 511.8 and 513.7[M-H] .
3
4
.1.19. 3-(1-((2-bromopyridin-4-yl)oxy)ethyl)-N-(3,5-difluorobenzyl)benzamide
(12f)
Compound 11 (200 mg, 0.62 mmol) and (3,5-difluorophenyl)methanamine (132
mg, 0.93 mmol) following the similar procedure described for the preparation of 7a
1
afforded 12f (234 mg, 83%). H NMR (400 MHz, DMSO-d ) δ 9.14 (t, J = 5.8 Hz,
6
NH, 1H), 8.13 (d, J = 5.6 Hz, ArH, 1H), 7.97 (s, ArH, 1H), 7.85 (d, J = 7.6 Hz, ArH,
1
7
1
H), 7.61 (d, J = 7.6 Hz, ArH, 1H), 7.50 (t, J = 7.6 Hz, ArH, 1H), 7.21 (s, ArH, 1H),
.10 (t, J = 9.2 Hz, ArH, 1H), 7.03 (t, J = 7.2 Hz, ArH, 3H), 5.81 (q, J = 6.0 Hz, CH,
H), 4.51 (d, J = 5.6 Hz, CH , 2H), 1.60 (d, J = 6.4 Hz, CH , 3H). ESI-MS m/z: 447.5
2
3
+
and 449.6[M+H] .
4
.1.20.
N-(3,5-bis(trifluoromethyl)benzyl)-3-(1-((2-bromopyridin-4-yl)oxy)ethyl)benzamide
12g)
Compound 11 (200 mg, 0.62 mmol) and (3,5-bis(trifluoromethyl)-phenyl)
(
methanamine (226 mg, 0.93 mmol) following the similar procedure described for the
1
preparation of 7a afforded 12f (288 mg, 85%). H NMR (400 MHz, DMSO-d ) δ 9.23
6
(t, J = 5.8 Hz, NH, 1H), 8.13 (d, J = 6.0 Hz, ArH, 1H), 8.02 (d, J = 7.2 Hz, ArH, 3H),
2
4

ACCEPTED MANUSCRIPT
7
.95 (s, ArH, 1H), 7.83 (d, J = 7.6 Hz, ArH, 1H), 7.62 (d, J = 7.6 Hz, ArH, 1H), 7.51
(t, J = 7.8 Hz, ArH, 1H), 7.20 (d, J = 2.4 Hz, ArH, 1H), 7.01 (dd, J = 2.4, 6.0 Hz, ArH,
1
H), 5.81 (q, J = 6.4 Hz, CH, 1H), 4.66 (d, J = 6.0 Hz, CH , 2H), 1.60 (d, J = 6.4 Hz,
2
+
CH , 3H).ESI-MS m/z: 547.5 and 549.6[M+H] .
3
4
.1.21. 3-(1-((2-bromopyridin-4-yl)oxy)ethyl)-N-(4-cyanobenzyl)benzamide (12h)
Compound 11 (200 mg, 0.62 mmol) and 4-(aminomethyl)benzonitrile (122 mg,
0
.93 mmol) following the similar procedure described for the preparation of 7a
1
afforded 12h (240 mg, 89%). H NMR (400 MHz, DMSO-d ) δ 9.19 (t, J = 5.6 Hz,
6
NH, 1H), 8.14 (d, J = 5.6 Hz, ArH, 1H), 7.97 (s, ArH, 1H), 7.84(d, J = 7.6, ArH, 1H),
7
3
4
4
.81 (d, J = 7.6, ArH, 2H), 7.61 (d, J = 7.6 Hz, ArH, 1H), 7.52 (d, J = 7.6 Hz, ArH,
H), 7.21 (s, ArH, 1H), 7.02 (d, J = 5.6 Hz, ArH, 1H), 5.81 (q, J = 6.0 Hz, CH, 1H),
.56 (d, J = 5.6 Hz, CH , 2H), 1.60 (d, J = 6.0 Hz, CH , 3H). ESI-MS m/z: 436.5 and
2
3
+
38.5[M+H] .
4
.1.22.
N-(4-fluorobenzyl)-3-(1-((2-((1-methyl-1H-pyrazol-4-yl)amino)pyridin-3-yl)oxy)ethyl)
benzamide (8a)
A mixture of compound 7a (212 mg, 0.50 mmol), 1-methyl-1H-pyrazol-4-amine (97
mg, 1.00 mmol), Pd (dpa) (30 mg, 0.03 mmol), BINAP (60 mg, 0.10 mmol) and
2
3
Cs CO (446 mg, 1.40 mmol) in DMF (15 mL) was degassed and charged with
2
3
nitrogen for three times and then heated in an oil bath at 90 ℃ for 7 h under the
protection of nitrogen. The reaction mixture was diluted with water (30 mL) and
extracted with EtOAc (30 mL × 3). The combined organic layer was dried over
anhydrous Na SO , filtered, and concentrated. The concentrates was purified by flash
2
4
chromatography on silica gel eluting with EtOAc/CH Cl (8%-100%) to give 8a (90
2
2
1
mg, 45%). mp 105-107 ℃. H NMR (400 MHz, CDCl ) δ 8.08 (s, ArH, 1H), 7.87 (s,
3
ArH, 1H), 7.76 (dd, J = 1.2, 4.8 Hz, ArH, 1H), 7.68 (d, J = 7.6 Hz, ArH, 1H), 7.53 –
7
.51 (m, ArH, 2H), 7.44 (t, J = 7.6 Hz, ArH, 1H), 7.36 – 7.32 (m, ArH, 2H), 7.05 (t, J
8.8 Hz, ArH, 2H), 6.85 (s, ArNH, 1H), 6.73 (dd, J = 1.2, 8.0 Hz, ArH, 1H), 6.49 –
.45 (m, ArH, 1H), 5.39 (q, J = 6.4 Hz, CH, 1H), 4.62 (d, J = 5.6 Hz, CH , 2H), 3.91
=
6
2
2
5

ACCEPTED MANUSCRIPT
1
3
(
s, NCH , 3H), 1.75 (d, J = 6.4 Hz, CH , 3H). C NMR (101 MHz, CDCl ) δ 166.9,
3 3 3
1
1
63.4, 146.7, 142.9, 140.0, 138.7, 134.9, 133.9, 130.6, 129.6, 129.5, 129.2, 128.5,
26.1, 124.4, 123.2, 121.2, 117.3, 115.7, 115.5, 112.9, 76.5, 43.4, 39.1, 24.3. ESI-MS
+
+
+
m/z: 446.6[M+H] . HRMS, ESI , m/z: Calcd. for C H FN O [M+H] , 446.1990;
25
24
5
2
found, 446.1992. Anal. Calcd. for C H FN O : C, 67.40; H, 5.43; N, 15.72; found:
2
5
24
5
2
C, 67.14; H, 5.57; N, 15.89.
4
.1.23.
N-(3-methoxybenzyl)-3-(1-((2-((1-methyl-1H-pyrazol-4-yl)amino)pyridin-3-yl)oxy)eth
yl)benzamide (8b)
Compound 7b (225 mg, 0.50 mmol) and 1-methyl-1H-pyrazol-4-amine (97 mg,
1
.00 mmol) following the similar procedure described above for the preparation of 8a
1
afforded 8b (118 mg, 52%). mp 90-92 ℃. H NMR (400 MHz, DMSO-d ) δ 9.00 (t,
6
J = 6.0 Hz, NH, 1H), 8.20 (s, ArH, 1H), 8.08 (s, ArH, 1H), 8.01 (s, ArH, 1H), 7.78 (d,
J = 7.6 Hz, ArH, 1H),7.65 – 7.62 (m, ArH, 3H), 7.45 (t, J = 7.8 Hz, ArH, 1H), 7.24 (t,
J = 8.0 Hz, ArH, 1H), 6.98 (dd, J =1.2, 8.0 Hz, ArH, 1H), 6.89 (d, J = 8.0 Hz, ArNH,
1
1
3
H), 6.88 (s, ArH, 1H), 6.83 – 6.80 (m, ArH, 1H), 6.47 (dd, J = 5.2, 8.0 Hz, ArNH,
H), 5.63 (q, J = 6.4 Hz, CH, 1H), 4.45 (d, J = 6.0 Hz, CH , 2H), 3.81 (s, CH , 3H),
2
3
1
3
.73 (s, CH , 3H), 1.67 (d, J = 6.4 Hz, CHCH , 3H). C NMR (101 MHz, DMSO-d )
3
3
6
δ 166.5, 159.7, 147.0, 143.2, 141.6, 140.1, 138.5, 135.0, 130.6, 129.8, 129.0, 128.9,
1
26.8, 125.5, 124.2, 120.8, 119.8, 117.7, 113.4, 112.7, 112.5, 75.5, 55.4, 43.0, 39.0,
+
+
+
2
4.3. ESI-MS m/z: 458.6 [M+H] . HRMS, ESI , m/z: Calcd. for C H N O [M+H] ,
26 27 5 3
4
58.2185; found, 458.2190. Anal. Calcd. for C H N O : C, 68.25; H, 5.95; N, 15.31;
26 27 5 3
found: C, 68.08; H, 6.12; N, 15.13.
4
3
.1.24.
-(1-((2-((1-methyl-1H-pyrazol-4-yl)amino)pyridin-3-yl)oxy)ethyl)-N-(3-(trifluoromet
hoxy)benzyl)benzamide (8c)
Compound 7c (248 mg, 0.50 mmol) and 1-methyl-1H-pyrazol-4-amine (97 mg, 1.00
mmol) following the similar procedure described above for the preparation of 8a
1
afforded 8c (122 mg, 48%). mp 102-105 ℃. H NMR (400 MHz, CDCl ) δ 8.08 (s,
3
ArH, 1H), 7.89 (s, ArH, 1H), 7.76 (d, J = 5.2 Hz, ArH, 1H), 7.69 (d, J = 7.6 Hz, ArH,
2
6

ACCEPTED MANUSCRIPT
1
H), 7.53 (d, J = 4.8 Hz, ArH, 2H), 7.45 (t, J = 7.6 Hz, ArH, 1H), 7.4 (d, J = 8.0 Hz,
ArH, 3H), 7.21 (d, J = 8.0 Hz, ArH, 2H), 6.90 (s, ArH, 1H), 6.74 (d, J = 7.6 Hz, ArH,
1
1
H), 6.59 (s, ArNH, 1H), 6.47 (dd, J = 5.2, 7.6 Hz, ArH, 1H), 5.40 (q, J = 6.2 Hz, CH,
H), 4.66 (d, J = 5.6 Hz, CH , 2H), 3.91 (s, NCH , 3H), 1.76 (d, J = 6.4 Hz, CH , 3H).
2
3
3
1
3
C NMR (101 MHz, CDCl ) δ 166.9, 148.5, 146.7, 142.9, 140.0, 138.8, 136.9, 134.7,
3
1
3
30.6, 129.3, 129.2, 128.5, 126.2, 124.6, 124.4, 123.3, 121.2, 117.3, 112.9, 76.5, 43.3,
+
+
9.1, 24.4. ESI-MS m/z: 512.6 [M+H] . HRMS, ESI , m/z: Calcd. for
+
C H F N O [M+H] , 512.1906; found, 512.1909. Anal. Calcd. for C H F N O : C,
26
24
3
5
3
26 24
3
5
3
6
1.05; H, 4.73; N, 13.69; found: C, 61.40; H, 4.56; N, 13.37.
4
.1.25.
N-(3-chloro-4-fluorobenzyl)-3-(1-((2-((1-methyl-1H-pyrazol-4-yl)amino)pyridin-3-yl)
oxy)ethyl)benzamide (8d)
Compound 7d (232 mg, 0.50 mmol) and 1-methyl-1H-pyrazol-4-amine (97 mg, 1.00
mmol) following the similar procedure described above for the preparation of 8a
1
afforded 8d (151 mg, 63%). mp 99-101 ℃ . H NMR (400 MHz, CDCl ) δ 8.08 (s, ArH,
3
1
1
H), 7.88 (s, ArH, 1H), 7.76 (dd, J = 1.2, 5.2 Hz, ArH, 1H), 7.70 (d, J = 7.6 Hz ArH,
H), 7.53 (d, J = 7.6 Hz, ArH, 1H), 7.52 (s, ArH, 1H), 7.46 – 7.39 (m, ArH, 2H), 7.23
(
m, ArH, 1H), 7.11 (t, J = 8.6 Hz, ArH, 1H), 6.85 (s, ArH, 1H), 6.73 (dd, J = 7.6, 1.2
Hz, ArH, 1H), 6.61 (s, ArNH, 1H), 6.47 (dd, J = 5.2, 7.6 Hz, ArH, 1H), 5.40 (q, J =
.4 Hz, CH, 1H), 4.59 (d, J = 6.0 Hz, CH , 2H), 3.90 (s, NCH , 3H), 1.76 (d, J = 6.4
6
2
3
13
Hz, CH , 3H). C NMR (101 MHz, CDCl ) δ 166.9, 158.7, 156.2, 146.7, 142.9,
3
3
1
40.0, 138.8, 135.3, 134.6, 130.6, 130.0, 129.2, 128.6, 127.6, 126.2, 124.4, 121.2,
+
1
17.3, 116.8, 116.6, 112.9, 76.5, 42.9, 39.1, 24.4. ESI-MS m/z: 480.9 [M+H] . HRMS,
+
+
ESI , m/z: Calcd. for C H ClFN O [M+H] , 480.1600; found, 480.1603. Anal.
25
23
5
2
Calcd. for C H ClFN O : C, 62.56; H, 4.83; N, 14.59; found: C, 62.25; H, 4.58; N,
25
23
5
2
1
4.35.
4
.1.26.
N-(4-chloro-3-(trifluoromethyl)benzyl)-3-(1-((2-((1-methyl-1H-pyrazol-4-yl)amino)py
ridin-3-yl)oxy)ethyl)benzamide (8e)
2
7

ACCEPTED MANUSCRIPT
Compound 7e (257 mg, 0.50 mmol) and 1-methyl-1H-pyrazol-4-amine (97 mg,
1
.00 mmol) following the similar procedure described above for the preparation of 8a
1
afforded 8e (111 mg, 42%). mp 123-125 ℃. H NMR (400 MHz, DMSO-d ) δ 9.13 (t,
6
J = 5.8 Hz, CONH, 1H), 8.20 (s, ArH, 1H), 8.08 (s, ArH, 1H), 8.00 (s, ArH, 1H), 7.79
(
dd, J = 1.2, 10.8 Hz, ArH, 2H), 7.69 – 7.61 (m, ArH, 4H), 7.46 (t, J = 7.8 Hz, ArH,
H), 6.97 (dd, J = 1.2, 8.0 Hz, ArH, 1H), 6.47 (dd, J = 4.8, 7.6 Hz, ArH, 1H), 5.63 (q,
J = 6.2 Hz, CH, 1H), 4.53 (d, J = 6.0 Hz, CH , 2H), 3.80 (s, NCH , 3H), 1.67 (d, J =
1
2
3
1
3
6
1
1
.4 Hz, CH , 3H). C NMR (101 MHz, DMSO-d ) δ 166.7, 147.0, 146.0, 143.3, 140.2,
3
6
40.1, 138.5, 138.3, 135.4, 134.7, 133.5, 132.0, 130.6, 129.4, 129.1, 126.8, 125.5,
+
24.2, 123.8, 120.8, 117.7, 112.7, 75.5, 42.2, 39.0, 24.3. ESI-MS m/z: 531.0 [M+H] .
+
+
HRMS, ESI , m/z: Calcd. for C H ClF N O [M+H] , 530.1573; found, 530.1570.
2
6
23
3
5
2
Anal. Calcd. for C H ClF N O : C, 58.93; H, 4.37; N, 13.22; found: C, 58.75; H,
26
23
3
5
2
4
.13; N, 13.07.
4
.1.27.
N-(2-(isopropylsulfonyl)benzyl)-3-(1-((2-((1-methyl-1H-pyrazol-4-yl)amino)pyridin-3
-yl)oxy)ethyl)benzamide (8f)
Compound 7f (259 mg, 0.50 mmol) and 1-methyl-1H-pyrazol-4-amine (97 mg,
1
.00 mmol) following the similar procedure described above for the preparation of 8a
1
afforded 8f (123 mg, 46%). mp 156-158 ℃. H NMR (400 MHz, CDCl ) δ 8.09 (s,
3
ArH, 1H), 7.98 (dd, J = 1.2, 8.0 Hz, ArH, 1H), 7.85 (s, ArH, 1H), 7.81 (dd, J = 0.8,
7
–
0
1
1
.6 Hz, ArH, 1H), 7.74 (dd, J = 1.6, 5.2 Hz, ArH, 1H), 7.67 – 7.63 (m, ArH, 2H), 7.54
7.47 (m, ArH, 3H), 7.40 (t, J = 7.6 Hz, ArH, 1H), 6.85 (s, ArNH, 1H), 6.71 (dd, J =
.8, 7.6 Hz, ArH, 1H), 6.46 (dd, J = 5.2, 8.0 Hz, ArH, 1H), 5.37 (q, J = 6.4 Hz, CH,
H), 4.88 (d, J = 6.4 Hz, CH , 2H), 3.92 (s, NCH , 3H), 3.38 – 3.23 (m, CH(CH ) ,
2
3
3 2
H), 1.72 (d, J = 6.4 Hz, OCHCH , 3H), 1.38 (d, J = 6.8 Hz, CH(CH ) , 3H) , 1.37 (d,
3
3 2
1
3
J = 6.8 Hz, CH(CH ) , 3H). C NMR (101 MHz, DMSO-d ) δ 166.9, 147.0, 143.3,
3
2
6
1
1
42.4, 140.9, 140.6, 140.1, 134.9, 134.6, 134.4, 131.1, 130.7, 129.2, 129.1, 129.1,
27.8, 126.9, 125.6, 120.9, 117.8, 112.7, 75.5, 54.4, 39.0, 24.3, 15.4, 15.3. ESI-MS
+
+
+
m/z: 534.5 [M+H] . HRMS, ESI , m/z: Calcd. for C H N O S [M+H] , 534.2169;
2
8
31
5
4
2
8