Design, synthesis and biological evaluation N2-(2-alkyoxy-6-aliphatic aminopyridin-3-yl)-2,4-diaminepyrimidine derivatives bearing acylamino or DBTD ‘head’ as potential ALK inhibitors

Article abstract of DOI:10.1016/j.bioorg.2018.09.019

Aiming to develop promising ALK inhibitors, two series of N²-(2-alkyoxy-6-aliphatic aminopyridin-3-yl)-2,4-diaminepyrimidine derivatives (22a-x and 23a-d) were designed according to scaffold hopping and bioisosterism principles. All compounds were efficiently synthesized by concise reactions and anti-proliferative activities on ALK-addicted H2228, Karpas299 cells and EGFR-expressive A549 cell were evaluated by MTT assay. Several compounds exhibited potential cytotoxic activities with IC₅₀ values below 0.10 μM. Five compounds (22g, 22h, 22l, 22s and 23a) were selected for further enzymatic determination, resulting in the discovery of 22l against ALK and ALK^L1196M with IC₅₀ values of 2.1 nM and 3.8 nM. Particularly, western blot and cell apoptosis assays identified 22l as a promising ALK inhibitor, which was capable of obviously inhibiting cellular ALK activity and inducing cell apoptosis. Eventually, molecular docking modes of 22l with ALK confirmed structural basis in accordance with the SARs analysis.

Full text of DOI:10.1016/j.bioorg.2018.09.019

Accepted Manuscript
2
Design, synthesis and biological evaluation N -(2-alkyoxy-6-aliphatic amino-
pyridin-3-yl)-2,4-diaminepyrimidine derivatives bearing acylamino or DBTD
‘
head’ as potential ALK inhibitors
Lingyun Xing, Tongfei Jing, Junlong Zhang, Ming Guo, Xiuqi Miao, Feng
Jiang, Xin Zhai
PII:
S0045-2068(18)30615-1
DOI:
https://doi.org/10.1016/j.bioorg.2018.09.019
YBIOO 2516
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
22 June 2018
29 August 2018
11 September 2018
Please cite this article as: L. Xing, T. Jing, J. Zhang, M. Guo, X. Miao, F. Jiang, X. Zhai, Design, synthesis and
2
biological evaluation N -(2-alkyoxy-6-aliphatic aminopyridin-3-yl)-2,4-diaminepyrimidine derivatives bearing
acylamino or DBTD ‘head’ as potential ALK inhibitors, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/
j.bioorg.2018.09.019
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2
Design, synthesis and biological evaluation N -(2-alkyoxy-6-aliphatic
aminopyridin-3-yl)-2,4-diaminepyrimidine
derivatives
bearing
acylamino or DBTD ‘head’ as potential ALK inhibitors
a
a
a
a
a
a
Lingyun Xing , Tongfei Jing , Junlong Zhang , Ming Guo , Xiuqi Miao , Feng Jiang ,
*,a
Xin Zhai
a
Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education,
Shenyang Pharmaceutical University, Shenyang 110016, PR China
*Corresponding author.
E-mail address: zhaixin_syphu@126.com (Xin Zhai).
Graphical abstract
Highlights
2
Twenty-eight N -(2-alkyoxy-6-aliphatic aminopyridin-3-yl)-2,4-diaminepyrimidine
derivatives (22a-x and 23a-d) bearing acylamino or DBTD ‘head’ were designed and
synthesized.
2
2l possessed remarkable antitumor potency against H2228 and Karpas299 cells with
IC values of 0.030 μM and 0.036 μM as well as excellent activity on ALK and
5
0
L1196M
ALK
with IC of 2.1 nM and 3.8 nM, respectively.
50
2
2l inhibited the cellular ALK activity and induced cell apoptosis with a
dose-dependent manner.
The molecular docking mode confirmed 22l not only formed critical hydrogen
bonding interactions and hydrophobic interactions but also overlapped well with
ceritinib in 3D model.

Abstract
2
Aiming to develop promising ALK inhibitors, two series of N -(2-alkyoxy-6-aliphatic
aminopyridin-3-yl)-2,4-diaminepyrimidine derivatives (22a-x and 23a-d) were
designed according to scaffold hopping and bioisosterism principles. All compounds
were efficiently synthesized by concise reactions and anti-proliferative activities on
ALK-addicted H2228, Karpas299 cells and EGFR-expressive A549 cell were
evaluated by MTT assay. Several compounds exhibited potential cytotoxic activities
with IC values below 0.10 μM. Five compounds (22g, 22h, 22l, 22s and 23a) were
5
0
selected for further enzymatic determination, resulting in the discovery of 22l against
L1196M
ALK and ALK
with IC values of 2.1 nM and 3.8 nM. Particularly, western
50
blot and cell apoptosis assays identified 22l as a promising ALK inhibitor, which was
capable of obviously inhibiting cellular ALK activity and inducing cell apoptosis.
Eventually, molecular docking modes of 22l with ALK confirmed structural basis in
accordance with the SARs analysis.
Keywords: ALK inhibitor; 2,4-diarylaminopyrimidine; pyridinyl group; DBTD
1
. Introduction
Anaplastic lymphoma kinase (ALK) was a single stranded transmembrane protein
belonging to the insulin receptor tyrosine kinase family [1]. The oncogenic
nucleophosmin (NPM)-ALK fusion gene was discovered as an ALK translocation
product occurring in 80% of anaplastic large-cell lymphoma [2]. Echinoderm
microtubule-associated protein-like-4 (EML4)-ALK was the first targetable fusion
kinase to be identified in 4-6% of lung adenocarcinomas [3,4]. ALK fusion genes had
generated substantial interest for the development of potent and specific ALK
inhibitors for treatment of EML4-ALK rearrangement [5]. Recently, a few ALK
inhibitors have been successfully approved or in clinical study (Fig. 1). The
first-generation ALK inhibitor crizotinib was approved in 2011 which had
demonstrated clear clinical benefits to treat ALK-positive NSCLC patients [6].
However, a majority of patients developed resistance to crizotinib treatment
successively [7,8]. Therefore, several more potent second- and third-generation
inhibitors have been identified to combat disease resistance, including ceritinib [9]
and alectinib [10], brigatinib [11], the pre-registered lorlatinib [12] and the phase III
investigational agent ensartinib [13].

Fig. 1. Structures of ALK inhibitors.
The co-crystal structure of ceritinib with ALK revealed interactions between ligand
and molecular (Fig. 2). The 4-arylaminopyrimidine and Cl atom on pyrimidine ring
were indispensable for activity which occupied the kinase domain and formed
interactions with amino acid residues [14]. Thus, this structural domain should be
remained for potent activity and selectivity [15]. While hydrophilic piperidinyl group
(‘tail’) extended to solvent and was responsible for a salt-bridge interaction with
Glu1210, tolerating to further modification with various aliphatic amines [16,17]. The
isopropoxy group on ceritinib skeleton exhibited higher kinase selectivity on account
of combination with protein pockets. To our disappointed, it had been reported that
G1202
ceritinib was ineffective for ALK
mutation duing to large steric hindrance of
isopropoxy moiety [17]. Therefore, isopropoxy group was replaced with fragments of
methoxyl, isopropylthio and hydrogen atom aiming to investigate the effect of steric
hindrance. Additionally, the replacement of benzene group with pyridinyl group in
aromatic ring system has shown many important effects on molecular which could
properly tune the lipophilicity and improve the metabolism [19-21]. Hence, pyridinyl
group as a high-impact moiety has been applied in drug design [22,23].
1
Optimization towards different R substituted (‘head’) was an effective approach to
enrich structural diversity. Beyond intramolecular hydrogen bond on isopropyl
sulfonyl group, acylamino group was regarded as a favorable design strategy which
could form hydrogen bond with receptors either [24]. Meanwhile, the acylamino
‘
head’ could enhance the affinity between molecules and enzymes to increase
inhibitory activity. In addition, the rigid
,4-dihydro-2H-benzo[1,4-b]thiazine-1,1-dioxide (DBTD) fragment was built as a
3
new structural motif by scaffold hopping [25], which could enhance the rigidity. Thus,
compounds 23a-d with DBTD ‘head’ were designed successfully to discuss the
binding ability to protein and evaluate its effectivity.

Fig. 2. Design strategies for target compounds (22a-x and 23a-d) based on co-crystal
structure of ceritinib with ALK (PDB ID code: 4MKC). (A) The co-crystal structure
of ceritinib with ALK. (B) Structure design strategies of 22a-x and 23a-d.
2
Herein,
two
series
of
N -(2-alkyoxy-6-aliphatic
aminopyridin-3-yl)-2,4-diaminepyrimidine derivatives (22a-x and 23a-d) bearing
acylamino or DBTD ‘head’ were designed and synthesized. Compounds were assayed
for the anti-proliferative activity in vitro against three cancer cell lines H2228,
Karpas299 and A549. Subsequently, five compounds were selected to test in vitro
L1196M
enzymatic evaluation on ALK and ALK
. To further study, the promising
compound 22l was evaluated for western blot assay and cell apoptosis. Finally,
molecular docking studies were carried out to identify SARs.
2
. Results and discussion
2
.1 Chemistry
The synthetic routes to compounds 22a-x and 23a-d were outlined in Scheme 1.
The commercially available 2,4,5-trichloropyrimidine (1) was reacted with
benzene-1,2-diamine (2) to provide intermediate 3 in 96.5% yield. 3 was acylated by
different acyl chloride in the presence of triethylamine to afford the key intermediates
4
a-c [26]. Meanwhile, substitution reaction of compound 2-fluoronitrobenzene (5)
with mercaptoethanol gave 6, which was chlorinated by thionyl chloride to generate
-chloroethyl-2-nitrophenyl thioether (7) as a yellow solid. 7 was oxidized by
2
hydrogen peroxide to yeild 8, which was subjected to reduction by iron powder in the
presence of hydrochloric acid to gain 9 in good yield. Intramolecular N-alkylation of 9
in dimethyl formamide gave desired DBTD analog 10, which was concentrated with
2
,4,5-trichloropyrimidine to yield the key intermediate 11
Compound 12 reacted with methanol (isopropanol or isopropyl mercaptan) in the
company of sodium hydride to obtain 13b-d without further purification.
Subsequently, amination of 13a-d by corresponding aliphatic amines with potassium
carbonate furnished target intermediates 14a-c, 14e-g, 15a-g, 16a-c or 17a-c in
satisfied yields. Treatment 14a-c, 14e-g, 15a-15g, 16a-16c or 17a-17c with
hydrochloric acid as catalysis under activated iron powder in ethanol/water (9:1) gave
rise to 18a-c, 18e-g, 19a-g, 20a-c or 21a-c, which were reacted with 4a-c and purified

on silica gel column to get target products 22a-x. Similarly, the desired compounds
3a-d were accomplished using the same method as preparation of 22a-x.
2
Scheme 1. Reagents and conditions: (i) DIPEA, ethanol, 80 °C; (ii) the acyl chloride
1
of R , triethylamine, DCM, rt; (iii) 2-mercaptoethanol, K CO , DMF, 110 °C; (iv)
2
3
thionyl chloride, toluene, 80 °C; (v) hydrogen peroxide, glacial acetic acid, 100 °C;
(
vi) iron powder, hydrochloric acid, ethanol/water(9:1), 80 °C; (vii) K CO , DMF,
2 3
1
00 °C; (viii) NaH (60%), DMF, 0~5 °C; (ix) NaH (60%), methanol(/IPA/isopropyl
mercaptan), THF, rt; (x) aliphatic amines, K CO , THF, 50 °C; (xi) iron powder,
2
3
hydrochloric acid, ethanol/water(9:1), 80 °C; (xii) 4a-d, p-toluenesulfonic acid, IPA,
8
0 °C.
2
2
.2. Biological evaluation
.2.1. In vitro anti-proliferative activity and SARs study
MTT assay was employed to evaluate the anti-proliferative characteristics of target
compounds to screen the active compounds preliminarily. Compounds 22a-x and
3a-d were investigated on cytotoxicity against ALK-addicted H2228 (Human
2
NSCLC cells), Karpas299 (Human transsexual cell lymphoma cell lines) cells and
EGFR-expressive A549 (Human EGFR-positive NSCL cell lines) cell. Using ceritinib
as the positive control, the results expressed as IC values were shown in Table 1 and
5
0
Table 2.
The pharmacological data indicated that most compounds exhibited moderate to
excellent cytotoxic activities against ALK-addicted H2228 and Karpas299 cells.
Compound 22l displayed significant activity against H2228 and Karpas299 with IC50
values of 0.030 μM and 0.036 μM. Meanwhile, 22l showed poor inhibitory activity
against A549 with IC values of 0.65 μM. Compared with 22a-x, compounds 23a-d
5
0

exhibited obviously reduced activities against H2228 and Karpas299 cells, indicating
the negative effect of DBTD group on cytotoxicity. As expected, all compounds
showed weak potency against A549 cells, suggesting the selectivity on ALK cell lines.
As shown in Table 1, anti-proliferative activity of target compounds would
preliminarily define the structure-activity relationships. A small set of compounds
3
2
2a-g with different R -moiety ‘tail’ were evaluated for cytotoxic activities, wherein
compound 22g bearing morpholinyl moiety showed the best potency with IC values
5
0
of 0.09 μM, 0.068 μM and 0.95 μM against H2228, Karpas299 and A549, which
identified polarity ‘tails’ with good hydrophilia property being beneficial for increased
potency. Compounds containing azetidin-3-ol (22d) and hydroxyethyl piperazinyl
(22e) were 2.4- and 6.3-fold more potent against H2228 than those inserting methyl
group in piperazinyl fragment (22f). Meanwhile, significant potency loss was detected
on compounds 22a-c with diethylamino, piperidyl and pyrrolidinyl groups due to the
lack of hydrophilicity.
1
As a general trend, among diverse chain R -moiety, compounds bearing sulfonyl
(
22l) and acetyl (22g) showed better activities against H2228 with IC values less
50
than 0.1 μM than compound bearing butyl-2-acrylamide substitution (22h). Generally,
introduction of butyl-2-acrylamide moiety would result in unsatisfactory activities as
indicated in 22h-k, which illustrated the effect of steric on cellular potency. Notably,
it was found that compound 22l bearing sulfonyl ‘head’ increased ALK potency
significantly.
2
The IC values of 22l-x showed that variations of R group had a marked impact
5
0
2
on activity. The R moiety was introduced with lipophilic substituents which occupied
the ALK protein lipophilic pocket to improve the activity. Derivatives 22m-o with
isopropyl provided moderate potency, however, shifting isopropyl to bulk
isopropylthio led to poor activity deducing to the steric hindrance within the
hydrophobic pocket. For example, the inhibitory activities of 22q and 22r were
decreased extremely with IC50 values more than 1 μM against tested cell lines.
Replacement of isopropyl with hydrogen atom resulted in compounds 22s-x, which
2
decreased activity, indicating R -hydrophobic pocket should be inserted the motif
2
with appropriate volume. Thus, methoxyl on R moiety (22l) was regarded as the
optimal substituent.
To integrate above factors and process further explore, the effect of rigid sulfonyl
‘head’ group was studied by scaffold hopping principle. As illustrated in Table 2,
compounds with rigid DBTD ‘head’ were inferior to corresponding compounds
bearing acylamino ‘head’. Extremely, 23d demonstrated dramatic poor effect on
H2228 and Karpas299 cells with IC values more than 5 μM. Further analysis on
5
0
SARs would be detailed description on molecular docking studies.
Table 1. Cytotoxicity of 22a-x against H2228, Karpas299 and A549 cell lines in vitro.

IC (µM)
5
0
1
2
3
Compd.
R
R
R
H2228
Karpas299
A549
2
2a
2b
0.92
0.16
2.16
2
0.51
0.80
0.62
0.24
1.51
0.09
0.19
0.98
0.78
0.37
0.030
0.054
0.65
0.74
0.66
1.12
1.47
0.094
0.19
0.06
0.052
1.28
0.068
0.09
0.23
0.12
0.08
0.036
0.04
0.87
1.66
0.84
1.19
1.08
1.06
1.23
0.54
0.18
3.11
0.95
1.12
1.88
0.97
0.66
0.65
0.32
0.62
2.51
3.23
5.22
4.69
2
2c
2d
2e
2f
2
2
2
2
2g
2h
2
2
2i
2j
2k
2l
2m
2
2
2
2
2
2n
2o
2p
2q
2
2
2
22r

2
2s
2t
H
H
H
H
H
H
0.20
0.68
0.94
0.92
0.67
0.038
0.37
0.287
1.21
2.81
0.58
0.66
2
2
2u
2v
0.61
2
0.11
2
2w
2x
ceritinib
0.096
2
1.26
1.79
4.23
0.79
0.032
0.048
Table 2. Cytotoxicity of 23a-d against H2228, Karpas299 and A549 cell lines in vitro.
IC (µM)
5
0
2
3
Compd.
R
R
H2228
Karpas299
A549
2
3a
3b
2.05
0.82
1.62
2
2.33
3.41
1.28
2.68
2.84
0.44
2
3c
2d
ceritinib
2
>5
>5
2.26
0.79
0.032
0.048
2
.2.2 In vitro enzymatic assays
Based on the cellular assays, further enzymatic assays in vitro were of particular
importance to evaluate antitumor potential. Five representative compounds (22g, 22h,
L1196M
2
2l, 22s and 23a) were selected against ALK and ALK
inhibitory studies. The
results were summarized in Table 3, which were comparable to the positive control
ceritinib.
As shown in Table 3, compared with 22l, compound 23a was observed a loss in
L1196M
activity against ALK and ALK
, which indicated that DBTD group was
1
unfavorable to inhibitory activity. However, optimal compounds with chain R -moiety
displayed good inhibitory potency against ALK with IC values ranging from 2.1 to
5
0
8
.3 nM. Notably, compound 22l turned out to be superior to ceritinib (IC = 2.3 nM)
50

with IC50 value of 2.1 nM. Likewise, compounds 22g and 22l showed significant
L1196M
inhibitory activity to ceritinib (IC = 8.9 nM) against ALK
with IC values 5.4
50
5
0
nM and 3.8 nM, respectively. Paralleling with cellular results, 22l was found to be
.4- and 2.3-fold more potent than 22h with butyl-2-acrylamide ‘head’ and 22s with
1
L1196M
hydrogen atom substitution on pyridinyl group against ALK
that appropriate steric hindrance was favor to improve the activities.
, which revealed
L1196M
Table 3. Enzymatic inhibition of five compounds against ALK and ALK
in
vitro.
IC (nM)
5
0
Compd.
L1196M
ALK
3.6
ALK
5.4
22g
2
2h
5.8
12.8
3.8
2
2l
2.1
2
2s
8.3
20,3
>3000
8.9
23a
>3000
2.3
ceritinib
2
.2.3. In western blot assay
Western blot assay was an important method to elucidate the modulation of target
phosphorylation effect of 22l in the in vitro assay. Using ceritinib as the positive
control, compound 22l and ceritinib were examined to determine the abilities to
inhibit Karpas299 cell lysates (Fig. 3).
As demonstrated in Fig. 3, 22l suppressed ALK expression and reduced
phosphorylation of ALK in a dose-dependent suppression manner from 25 nM to 100
nM, which was comparable to ceritinib. Slight inhibition of ALK expression and
phosphorylation could be observed at a dose of 50 nM, meanwhile, obvious
inhibitions were discovered a concentration of 100 nM. These results addressed 22l
did potently inhibit protein activity.
Fig. 3. Mechanism of action of compound 22l and ceritinib in Karpas299 cell lines.
Cells were treated with 22l or ceritinib at the indicated concentrations for 6 h and the
levels of protein were evaluated by western blot analysis of cell lysates using specific
antibodies.
2
.2.4. Apoptosis analysis.

Apoptosis assay elucidated the process for the observed induction of cellular death,
so it played a necessary role to assess the potency of compound 22l. The cytotoxicity
of 22l was further confirmed by an acridine orange (AO)/ethidium bromide (EB)
fluorescent staining assay against H2228 and Karpas299 cell lines (Fig. 4). This
staining solution was a mixture of AO and EB, in which AO was used for live cell
identification and EB for identification of necrotic cells. The AO permeated into the
intact cell membrane and thus stains live cells to appear green, whereas, EB stained
necrotic cells to appear orange when visualized by fluorescent microscopy due to
disruption of membrane integrity. Therefore, early apoptotic cells contained bright
green condensed bodies and later apoptotic cells presented colored orange nuclei
indicating.
As shown in Fig. 4, H2228 and Karpas299 cells were observed cell apoptosis in 22l
treated groups. It was observed that control cells displayed normal cell morphological
features with uniformly stained green nuclei, while compound 22l at the concentration
of 160 nM started to induce morphological changes. At the concentration of 320 nM,
the number of green cells was reduced and orange cells were increased indicating
cells underwent apoptotic stage. Simultaneously, apoptotic cells were clearly observed
at 640 nM. These results explored 22l induced dose-dependent apoptosis of H2228
and Karpas299 cells as evident by observed hall marks of apoptosis.
Fig. 4. AO/EB stained apoptosis of H2228 and Karpas299 cell lines. H2228 and
Karpas299 cell were treated with different concentrations of 22l for 24 h, and then
examined by fluorescence microscope.
2
.2.5. Molecular docking studies
Computer aided molecular docking was used to simulate interactions and evaluate
active sites. In order to detail the binding mode, molecular dockings of 22l and 23a
were performed based on co-crystal structures of ALK (PDB ID code: 4MKC) by
Discovery Studio 2017 (Fig. 5).
As shown in Fig. 5A, 22l occupied the same kinase domain with ceritinib. Both
cerinitib and 22l were found to form two hydrogen bonds via the amino and

pyrimidine nitrogen atoms onto the backbone oxygen and nitrogen of Met1199 (Fig.
B and Fig. 5C), which were essential for binding to ALK. The Cl atom on
5
pyrimidine ring of 22l formed hydrophobic interactions with Leu1196 and Leu1256.
Regardless of the size of the aromatic ring, cerinitib and 22l formed alkyl interactions
with Leu1122. Meanwhile, the acylamino group of compound 22l not only formed
hydrogen bond interactions with Lys1150, but also carbon hydrogen bond interactions
with Gly1125 (Fig. 5D), as illustrated the reasonability of the chain ‘head’. The
water-soluble ‘tail’ of 22l was engaged in carbon hydrogen bond interactions with
Asp1203 and Leu1122, which was similar to ceritinib. These simulation models
proved that 22l had familiar contact to cerinitib with ALK.
It was available from Fig. 5E and Fig. 5F that 23a vanished interactions with
Lys1150 and Gly1125 deducing to the rigid DBTD ‘head’, which explained the reason
of poor activities and the important of the chain ‘head’. Hence, the results from the
docking study were in accordance with the SARs analysis.
Fig. 5. The binding poses of cerinitib, 22l and 23a with ALK (PDB ID code: 4MKC).
(A) Composite model of cerinitib (red sticks) and 22l (gray sticks) with ALK. (B)
Docking model of cerinitib with ALK. (C) Docking model of compound 22l with
ALK. (D) 2D diagram of the interaction between 22l and ALK. (E) Docking model of
2
3a (purple sticks) with ALK. (F) 2D diagram of the interaction between 23a and
ALK.
3
. Conclusions
In this
2
paper,
two
series
of
N -(2-alkyoxy-6-aliphatic
aminopyridin-3-yl)-2,4-diaminepyrimidine derivatives (22a-x and 23a-d) bearing
acylamino or DBTD ‘head’ were designed, synthesized and evaluated for biological
activities. Compared with compounds 22a-x bearing acylamino group, compounds
with rigid DBTD ‘head’ (23a-d) decreased the potency relative. Importantly, 22l
possessed remarkable antitumor potency against H2228 and Karpas299 cells with IC50

values of 0.030 μM and 0.036 μM as well as excellent activity on ALK and
L1196M
ALK
with IC of 2.1 nM and 3.8 nM, respectively. Consistently, western blot
50
and cell apoptosis tests illustrated 22l could inhibite the cellular ALK activity and
induce cell apoptosis in a dose-dependent manner. Moreover, molecular docking
modes detailed that 22l could not only form critical hydrogen bonding interactions
and hydrophobic interactions but also overlap well with ceritinib in 3D model, which
was consistent with observed SARs for this class of compounds. In conclusion, the
target compound 22l was identified as a promising ALK inhibitor for cancer
treatments.
Experimental section
4
.1. Chemistry
All melting points were obtained on a Büchi Melting Point B-540 apparatus (Büchi
Labortechnik, Flawil, Switzerland) and were uncorrected. Mass spectra (MS) were
1
taken in ESI mode on Agilent 1100 LC-MS (Agilent, palo Alto, CA, USA). H NMR
1
3
and C NMR spectra were performed using Bruker spectrometers (Bruker Bioscience,
respectively, Billerica, MA, USA) with TMS as an internal standard. Column
chromatography was run on silica gel (200-300 mesh) from Qingdao Ocean
Chemicals (Qingdao, Shandong, China). Unless otherwise noted, all materials were
obtained from commercially available sources and were used without further
purification.
1
4
2
.1.1 Preparation of N -(2,5-dichloropyrimidin-4-yl)benzene-1,2-diamine (3)
,4,5-trichloropyrimidine 1 (30.0 g, 0.16 mol), benzene-1,2-diamine 2 (17.7 g, 0.16
mol) and N,N-diisopropylethylamine (54.0 mL, 0.33 mol) was added to ethanol (300
mL). The resulting solution was stirred at 80 °C for 2 h. The reaction mixture was
evaporated and the residue was stirred with aqueous HCl (0.1M, 10 mL) for 30
minutes. The solid was collected by filtration and dried under vacuum. The crude
solid was triturated with dichloromethane to give a solid which was collected by
filtration and dried under vacuum to afford a white solid 3 in 96.5% yield. MS (ESI)
+
m/z: [M+H] 255.0.
4
.1.2 General procedure for preparation of compounds (4a-c)
Acetylchloride, methylsulfonyl chloride or (E)-but-2-enoyl chloride (0.094 mol) was
added to a mixture of 3 (20.0 g, 0.079 mol) and triethylamine (33.0 mL, 0.24 mol) in
DCM (100 mL) at 0 °C for 30 min, then stirred at room temperature for 2 h. The
reaction mixture was evaporated and the residue was stirred with water (200.0 mL).
The solid was collected by filtration and dried under vacuum to give 4a-c.
4
.1.2.1 N-(2-((2,5-dichloropyrimidin-4-yl)amino)phenyl)acetamide (4a)
+
Yield: 89.2%; MS (ESI) m/z: 297.0 [M+H] .

4
.1.2.2 N-(2-((2,5-dichloropyrimidin-4-yl)amino)phenyl)methanesulfonamide (4b)
+
Yield: 85.8%; MS (ESI) m/z: 333.2 [M+H] .
4
.1.2.3 (E)-N-(2-((2,5-dichloropyrimidin-4-yl)amino)phenyl)but-2-enamide (4c)
+
Yield: 91.1%; MS (ESI) m/z: 322.9 [M+H] .
4
.1.3 Preparation of 2-((2-nitrophenyl)thio)ethan-1-ol (6)
To the solution of mercaptoethanol (16.6 g, 0.21 mol) in dry DMF (1 L) was added
-fluoronitrobenzene 5 (30.0 g, 0.21 mol) and K CO (58.7 g, 0.42 mol). The reaction
2
2
3
mixture was heated with stirring at 110 °C for 6 h. After reacting at room temperature,
the reaction mixture was poured into water and extracted with AcOEt (3×100 mL).
The combined organic layer was washed with brine, dried over MgSO , filtered, and
4
concentrated under reduced pressure to yield a yellow solid 6 in 94.2% yield. MS
+
(
ESI) m/z(%): 200.1 [M+H] .
4
.1.4 Preparation of 2-chloroethyl-2-nitrophenyl thioether (7)
Thionyl chloride (17.85 g, 0.15 mol) was added dropwise to a solution of 6 (10.0 g,
.05 mol) in toluene (100 mL) at 50 °C for 1 h, then refluxed for 10 h. After cooling,
0
the reaction mixture was concentrated under reduced pressure. The residue was stirred
with water. The solid was collected by filtration and dried under vacuum to gain 7 as a
+
yellow solid in 93.4% yield. MS (ESI) m/z(%): 218.1 [M+H] .
4
.1.5 Preparation of 1-((2-chloroethyl)sulfonyl)-2-nitrobenzene (8)
To the solution of 7 (10.9 g, 0.05 mol) in CH COOH (100 mL), 30% H O (30.5 mL,
3
2
2
0
.16 mol) was added dropwise while maintaining the temperature below 50 ºC. After
the addition was completed, the mixture was heated to 100 ºC for another 13 h. After
cooling, the reaction mixture was poured into water to give a light yellow precipitate.
This was collected by filtration and air-dried to give 8 as a light yellow solid in 89.0%
+
yield. MS (ESI) m/z(%): 250.0 [M+H] .
4
.1.6 Preparation of 2-((2-chloroethyl)sulfonyl)aniline (9)
To the solution of 8 (12.9 g, 0.05 mol) in ethanol/water (9:1, 60 mL) was added
hydrochloric acid (7.0 mL) and iron powder (11.6 g, 0.2 mol). The resulting mixture
was heated at 80 °C for 3 h, filtered through celite and concentrated to remove ethanol.
The aqueous solution was extracted with ethyl acetate (3×100 mL) and the combined
organic layers were washed with water, brine, dried over Na SO and concentrated to
2
4
+
gain 9 in 67.3% yield. MS (ESI) m/z(%): 220.6 [M+H] .
4
.1.7 Preparation of 3,4-dihydro-2H-benzo[1,4-b]thiazine-1,1-dioxide (10)
To the solution of 9 (10.0 g, 0.05 mol) in DMF (100 mL), K CO (18.9 g, 0.14 mol)
2
3
was added. The mixture was stirred at 100 °C for 10 h. After cooling, the reaction

mixture was poured into water to give a light grey precipitate. This was collected by
filtration and air-dried to give 10 as a light grey solid in 55.4% yield. MS (ESI)
+
m/z(%): 184.0 [M+H] .
4
4
.1.8
Preparation
of
-(2,5-dichlorophenyl)-3,4-dihydro-2H-benzo[1,4-b]thiazine-1,1-dioxide (11)
To the suspension of 60% NaH (0.66 g, 0.016 mol) in DMF (20 mL) was added 10
1.0 g, 0.005 mol) at 0 °C. The mixture was stirred for 30 min, and then 1 (1.2 g,
.007 mol) diluted in DMF (6 mL) was added slowly. The mixture was warmed to
(
0
room temperature and stirred overnight. The reaction mixture was poured into water
to give a yellow precipitate which was collected by filtration. The crude product
purified by column chromatography using dichloromethane/methanol as an eluent to
+
1
afford 11 a white solid in 41.8% yield. MS (ESI) m/z(%): 329.8 [M+H] ; H NMR
(
(
(
400 MHz, DMSO-d ) δ : 9.61 (s, 1H), 8.53 (d, J = 8.4 Hz, 1H), 8.30 (s, 1H), 8.01
6 H
dd, J = 8.0, 1.4 Hz, 1H), 7.77 – 7.69 (m, 1H), 7.33 (dd, J = 17.7, 9.8 Hz, 1H), 6.60
dd, J = 16.5, 9.4 Hz, 1H), 6.50 (d, J = 16.5 Hz, 1H), 6.04 (d, J = 9.4 Hz, 1H).
4
.1.9 General procedure for preparation of compounds (13b-d)
To the suspension of 60% NaH (4.6 g, 0.11 mol) in THF (20 mL) was added 12 (20.0
g, 0.1 mol) at 0 °C. The mixture was stirred at 0 °C for 30 min, and then methanol,
IPA or isopropyl mercaptan (0.1 mol) was added slowly. The mixture was warmed to
room temperature and stirred for 6 h. The reaction mixture was poured into water to
give a yellow precipitate which was collected by filtration to afford 13b-d.
4
.1.9.1 6-chloro-2-methoxy-3-nitropyridine (13b)
+
Yield: 72.7%; MS (ESI) m/z: 188.9 [M+H] .
4
.1.9.2 6-chloro-2-isopropoxy-3-nitropyridine (13c)
+
Yield: 69.1%; MS (ESI) m/z: 217.1 [M+H] .
4
.1.9.3 6-chloro-2-(isopropylthio)-3-nitropyridine (13d)
+
Yield: 78.2%; MS (ESI) m/z: 233.0 [M+H] .
4
.1.10 General procedure for preparation of compounds (14a-c, 14e-g, 15a-g, 16a-c
and 17a-c)
A solution of 13a-d (0.005 mol) in THF (20 mL) was added K CO (0.011 mol) and
2
3
various secondary amines (0.011 mol), which was stirred at 50 °C. After cooling to
room temperature, the reaction mixture was poured into water. The aqueous solution
was extracted with ethyl acetate (3×100 mL) and the combined organic layers were
washed with water, brine, dried over Na SO and concentrated to gain 14a-c, 14e-g,
2
4
1
5a-g, 16a-c or 17a-c.

4
.1.10.1 4-(5-nitropyridin-2-yl)morpholine (14a)
+
Yield: 89.3%; MS (ESI) m/z: 210.1 [M+H] .
4
.1.10.2 5-nitro-2-(piperidin-1-yl)pyridine (14b)
+
Yield: 91.0%; MS (ESI) m/z: 208.1 [M+H] .
4
.1.10.3 N,N-diethyl-5-nitropyridin-2-amine (14c)
+
Yield: 87.6%; MS (ESI) m/z: 196.3 [M+H] .
4
.1.10.4 1-(5-nitropyridin-2-yl)azetidin-3-ol (14e)
+
Yield: 89.0%; MS (ESI) m/z: 196.1 [M+H] .
4
.1.10.5 2-(4-(5-nitropyridin-2-yl)piperazin-1-yl)ethan-1-ol (14f)
+
Yield: 83.4%; MS (ESI) m/z: 253.2 [M+H] .
4
.1.10.6 3-methyl-1-(5-nitropyridin-2-yl)piperazine (14g)
+
Yield: 86.7%; MS (ESI) m/z: 222.2 [M+H] .
4
.1.10.7 4-(6-methoxy-5-nitropyridin-2-yl)morpholine (15a)
+
Yield: 90.1%; MS (ESI) m/z: 240.0 [M+H] .
4
.1.10.8 2-methoxy-3-nitro-6-(piperidin-1-yl)pyridine (15b)
+
Yield: 89.4%; MS (ESI) m/z: 238.1 [M+H] .
4
.1.10.9 N,N-diethyl-6-methoxy-5-nitropyridin-2-amine (15c)
+
Yield: 78.6%; MS (ESI) m/z: 226.0 [M+H] .
4
.1.10.10 2-methoxy-3-nitro-6-(pyrrolidin-1-yl)pyridine (15d)
+
Yield: 84.2%; MS (ESI) m/z: 224.2 [M+H] .
4
.1.10.11 1-(6-methoxy-5-nitropyridin-2-yl)azetidin-3-ol (15e)
+
Yield: 80.5%; MS (ESI) m/z: 226.0 [M+H] .
4
.1.10.12 2-(4-(6-methoxy-5-nitropyridin-2-yl)piperazin-1-yl)ethan-1-ol (15f)
+
Yield: 79.3%; MS (ESI) m/z: 283.3 [M+H] .
4
.1.10.13 1-(6-methoxy-5-nitropyridin-2-yl)-3-methylpiperazine (15g)
+
Yield: 71.6%; MS (ESI) m/z: 253.1 [M+H] .
4
.1.10.14 4-(6-isopropoxy-5-nitropyridin-2-yl)morpholine (16a)
+
Yield: 89.5%; MS (ESI) m/z: 268.1 [M+H] .

4
.1.10.15 2-isopropoxy-3-nitro-6-(piperidin-1-yl)pyridine (16b)
+
Yield: 88.4%; MS (ESI) m/z: 266.0 [M+H] .
2
2
4
.1.10.16 N ,N -diethyl-6-isopropoxy-5-nitropyridin-2-amine (16c)
+
Yield: 85.1%; MS (ESI) m/z: 254.1 [M+H] .
4
.1.10.17 4-(6-(isopropylthio)-5-nitropyridin-2-yl)morpholine (17a)
+
Yield: 82.9%; MS (ESI) m/z: 284.1 [M+H] .
4
.1.10.18 2-(isopropylthio)-3-nitro-6-(piperidin-1-yl)pyridine (17b)
+
Yield: 86.2%; MS (ESI) m/z: 282.1 [M+H] .
2
2
4
.1.10.19 N ,N -diethyl-6-(isopropylthio)-5-nitropyridin-2-amine (17c)
+
Yield: 86.8%; MS (ESI) m/z: 270.1 [M+H] .
4
.1.11 General procedure for preparation of compounds (18a-c, 18d-g, 19a-g, 20a-c
and 21a-c)
To the solution of 14a-c, 14e-g, 15a-g, 16a-c or 17a-c (0.005 mol) in ethanol/water
(9:1, 30 mL) was added hydrochloric acid (1.0 mL) and iron powder (1.12 g, 0.02
mol). The resulting mixture was heated at 80 °C for 3 h, filtered through celite and
concentrated to remove ethanol. The aqueous solution was extracted with ethyl
acetate (3×100 mL) and the combined organic layers were washed with water, brine,
dried over Na SO and concentrated to gain 18a-c, 18e-g, 19a-g, 20a-c or 21a-c.
2
4
4
.1.11.1 6-morpholinopyridin-3-amine (18a)
+
Yield: 79.3%; MS (ESI) m/z: 180.1 [M+H] .
4
.1.11.2 6-(piperidin-1-yl)pyridin-3-amine (18b)
+
Yield: 92.6%; MS (ESI) m/z: 178.1 [M+H] .
2
2
4
.1.11.3 N ,N -diethylpyridine-2,5-diamine (18c)
+
Yield: 87.4%; MS (ESI) m/z: 166.1 [M+H] .
4
.1.11.4 1-(5-aminopyridin-2-yl)azetidin-3-ol (18e)
+
Yield: 87.3%; MS (ESI) m/z: 166.1 [M+H] .
4
.1.11.5 2-(4-(5-aminopyridin-2-yl)piperazin-1-yl)ethan-1-ol (18f)
+
Yield: 80.7%; MS (ESI) m/z: 223.2 [M+H] .
4
.1.11.6 6-(3-methylpiperazin-1-yl)pyridin-3-amine (18g)
+
Yield: 82.4%; MS (ESI) m/z: 192.2 [M+H] .

4
.1.11.7 2-methoxy-6-morpholinopyridin-3-amine (19a)
+
Yield: 90.1%; MS (ESI) m/z: 210.1 [M+H] .
4
.1.11.8 2-methoxy-6-(piperidin-1-yl)pyridin-3-amine (19b)
+
Yield: 89.5%; MS (ESI) m/z: 208.1 [M+H] .
2
2
4
.1.11.9 N ,N -diethyl-6-methoxypyridine-2,5-diamine (19c)
+
Yield: 87.3%; MS (ESI) m/z: 196.1 [M+H] .
4
.1.11.10 2-methoxy-6-(pyrrolidin-1-yl)pyridin-3-amine (19d)
+
Yield: 86.1%; MS (ESI) m/z: 194.1 [M+H] .
4
.1.11.11 1-(5-amino-6-methoxypyridin-2-yl)azetidin-3-ol (19e)
+
Yield: 80.6%; MS (ESI) m/z: 196.0 [M+H] .
4
.1.11.12 2-(4-(5-amino-6-methoxypyridin-2-yl)piperazin-1-yl)ethan-1-ol (19f)
+
Yield: 79.4%; MS (ESI) m/z: 253.1 [M+H] .
4
.1.11.13 2-methoxy-6-(3-methylpiperazin-1-yl)pyridin-3-amine (19g)
+
Yield: 82.1%; MS (ESI) m/z: 223.2 [M+H] .
4
.1.10.142-isopropoxy-6-morpholinopyridin-3-amine (20a)
+
Yield: 86.4%; MS (ESI) m/z: 238.2 [M+H] .
4
.1.11.15 2-isopropoxy-6-(piperidin-1-yl)pyridin-3-amine (20b)
+
Yield: 84.2%; MS (ESI) m/z: 236.2 [M+H] .
2
2
4
.1.11.16 N ,N -diethyl-6-isopropoxypyridine-2,5-diamine (20c)
+
Yield: 85.7%; MS (ESI) m/z: 224.0 [M+H] .
4
.1.11.17 2-(isopropylthio)-6-morpholinopyridin-3-amine (21a)
+
Yield: 86.1%; MS (ESI) m/z: 254.1 [M+H] .
4
.1.11.18 2-(isopropylthio)-6-(piperidin-1-yl)pyridin-3-amine (21b)
+
Yield: 82.2%; MS (ESI) m/z: 252.2 [M+H] .
2
2
4
.1.11.19 N ,N -diethyl-6-(isopropylthio)pyridine-2,5-diamine (21c)
+
Yield: 86.5%; MS (ESI) m/z: 240.2 [M+H] .
4
.1.12 General procedure for preparation of compounds (22a-x and 23a-d)
To the solution of 18a-c, 18e-g, 19a-g, 20a-c or 21a-c (0.005 mol) in isopropanol was
added 4a-c or 11(0.005mol) and p-toluenesulfonic (0.005 mol). The resulting mixture

was heated at 80 °C for 24 h. After completion of the reaction as indicated by TLC,
the mixture was filtered immediately. Then the residue was stirred with aqueous
NaHCO . The aqueous solution was extracted with DCM (3×50 mL) and combined
3
organic layers. The mixture was evaporated and purified on silica gel (200-300 mesh)
using dichloromethane/methanol (2500:1). Then evaporate the mixture. The solid was
dried under vacuum and gained pure compounds 22a-x and 23a-d.
4
.1.12.1
N-(2-((5-chloro-2-((4-(diethylamino)-2-methoxyphenyl)amino)pyrimidin-4-yl)amino)
phenyl)acetamide (22a)
-
1
Yield: 82.0%; m.p.: 172.3-178.6 °C; MS (ESI) m/z: 454.4 [M-H] ; H NMR(400 MHz,
DMSO-d ) δ : 10.06 (s, 1H), 8.23 (s, 1H), 7.99 (s, 1H), 7.78 (s, 1H), 7.63 (d, J = 8.2
6
H
Hz, 1H), 7.58 (d, J = 8.6 Hz, 1H), 7.24 (dd, J = 8.0, 2.1 Hz, 1H), 7.30-7.14 (m, 2H),
6
6
.20 (d, J = 8.4 Hz, 1H), 3.84 (d, J = 2.1 Hz, 4H), 3.77 (s, 3H), 2.06 (s, 3H), 1.27 (s,
H).
4
.1.12.2
N-(2-((5-chloro-2-((2-methoxy-4-(piperidin-1-yl)phenyl)amino)pyrimidin-4-yl)amino
phenyl)acetamide (22b)
)
+
1
Yield: 84.5%; m.p.: 178.3-179.6 °C; MS (ESI) m/z: 468.4 [M+H] ; H NMR (400
MHz, DMSO-d ) δ : 10.03 (s, 1H), 8.36 (s, 1H), 8.02 (s, 1H), 7.81 (s, 1H), 7.73 (d, J
6
H
=
8.0 Hz, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.25 (dd, J = 7.2, 2.0 Hz, 1H), 7.22-7.08 (m,
H), 6.18 (d, J = 8.5 Hz, 1H), 3.77 (s, 3H), 3.44 (d, J = 5.1 Hz, 4H), 2.09 (s, 3H), 1.57
s, 6H).
2
(
4
.1.12.3
N-(2-((5-chloro-2-((2-methoxy-4-(pyrrolidin-1-yl)phenyl)amino)pyrimidin-4-yl)amin
o)phenyl)acetamide (22c)
+
1
Yield: 80.4%; m.p.: 183.8-185.6 °C; MS (ESI) m/z: 454.4 [M+H] ; H NMR (400
MHz, DMSO-d ) δ : 10.08 (s, 1H), 8.32 (s, 1H), 8.00 (s, 1H), 7.80 (s, 1H), 7.77-7.72
6
H
(m, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.29 – 7.21 (m, 1H), 7.17 – 7.10 (m, 2H), 5.84 (d, J
=
8.2 Hz, 1H), 3.79 (s, 3H), 3.36 (s, 4H), 2.09 (s, 3H), 1.94 (s, 4H).
4
.1.12.4
N-(2-((5-chloro-2-((4-(3-hydroxyazetidin-1-yl)-2-methoxyphenyl)amino)pyrimidin-4-
yl)amino)phenyl)acetamide (22d)
Yield: 85.2%; m.p.: 183.8-190.6 °C; MS (ESI) m/z: 454.4 [M-H] ; 1H NMR (400
-
MHz, DMSO-d ) δ : 10.01 (s, 1H), 8.31 (s, 1H), 7.99 (s, 1H), 7.79 (s, 1H), 7.73 (s,
6
H
1
H), 7.41 (d, J = 8.3 Hz, 1H), 7.24 (d, J = 9.3 Hz, 1H), 7.18 – 7.06 (m, 2H), 6.36 (t, J
6.0 Hz, 1H), 5.33 (s, 1H), 3.89 (s, 1H), 3.77 (s, 3H), 3.66 (dd, J = 11.1, 4.5 Hz, 1H),
=

3
.56 (dd, J = 11.0, 5.7 Hz, 1H), 3.41 (dd, J = 13.0, 6.5 Hz, 1H), 3.24 (dd, J = 13.3, 5.9
Hz, 1H), 2.09 (s, 3H).
4
.1.12.5
N-(2-((5-chloro-2-((4-(4-(2-hydroxyethyl)piperazin-1-yl)-2-methoxyphenyl)amino)py
rimidin-4-yl)amino)phenyl)acetamide (22e)
Yield: 79.8%; m.p.:150.1-155.5°C; MS (ESI) m/z: 513.4 [M+H] ; 1H NMR (400
+
MHz, DMSO-d ) δ : 10.01 (s, 1H), 8.37 (s, 1H), 8.01 (s, 1H), 7.83 (s, 1H), 7.73 (d, J
6
H
=
6.2 Hz, 1H), 7.61 (d, J = 8.4 Hz, 1H), 7.26 (d, J = 7.8 Hz, 1H), 7.16 (s, 2H), 6.19 (d,
J = 8.3 Hz, 1H), 4.46 (s, 1H), 3.78 (s, 3H), 3.54 (t, J = 5.8 Hz, 2H), 3.40 (s, 4H), 2.53
1
3
(
s, 4H), 2.45 (t, J = 6.0 Hz, 2H), 2.09 (s, 3H). C-NMR (100 MHz, DMSO-d ) δ :
6 C
1
1
2
70.07, 159.21, 156.06, 154.95, 154.42, 135.09, 132.32, 131.14, 126.42, 125.74,
25.15 (2C), 112.98, 104.01, 98.18, 60.77, 58.95, 53.34 (2C), 53.09, 45.63 (2C),
9.46, 23.46.
4
.1.12.6
N-(2-((5-chloro-2-((2-methoxy-4-(3-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-
yl)amino)phenyl)acetamide (22f)
Yield: 84.5%; m.p.:164.1-164.8°C; MS (ESI) m/z: 483.4 [M+H] ; 1H NMR (400
+
MHz, DMSO-d ) δ : 10.03 (s, 1H), 8.35 (s, 1H), 8.12 (s, 1H), 7.87 (s, 1H), 7.72 (d, J
6
H
=
6.2 Hz, 1H), 7.52 (d, J = 8.4 Hz, 1H), 7.22 (d, J = 6.9 Hz, 1H), 7.18 (s, 2H), 6.23 (d,
J = 8.4 Hz, 1H), 3.78 (s, 3H), 3.40 (s, 4H), 2.53 (s, 4H), 2.09 (s, 3H) 1.39 (s, 3H).
4
.1.12.7
N-(2-((5-chloro-2-((2-methoxy-4-morpholinophenyl)amino)pyrimidin-4-yl)amino)ph
enyl)acetamide (22g)
-
1
Yield: 81.3%; m.p.:154.1-159.5°C; MS (ESI) m/z: 468.4 [M-H] ; H-NMR (400 MHz,
DMSO-d ) δ : 10.04 (s, 1H), 8.39 (s, 1H), 8.03 (s, 1H), 7.85 (s, 1H), 7.72 (d, J = 8.2
6
H
Hz, 1H), 7.65 (d, J = 8.4 Hz, 1H), 7.27 (dd, J = 7.2 Hz, 2.1HZ, 1H), 7.23-7.09 (m, 2H),
6
3
.20 (d, J = 8.5 Hz, 1H), 3.79 (s, 3H), 3.72-3.69 (m, 4H), 3.37-3.34 (m, 4H), 2.09 (s,
H).
4
.1.12.8
(E)-N-(2-((5-chloro-2-((2-methoxy-4-morpholinophenyl)amino)pyrimidin-4-yl)amino
)
phenyl)but-2-enamide (22h)
+
1
Yield: 78.3%; m.p.:172.3-178.6°C; MS (ESI) m/z: 496.6 [M+H] ; H-NMR (400
MHz, DMSO-d ) δ : 10.07 (s, 1H), 8.51 (s, 1H), 8.01 (s, 1H), 7.83 (s, 1H), 7.75-7.69
6
H
(m, J = 13.9, 1H), 7.65 (d, 1H), 7.38-7.27 (m, J = 9.0 Hz, 1H), 7.24-7.14 (m, 2H),
6
4
.87 (dq, J = 13.9, 6.9 Hz, 1H), 6.19 (d, J = 8.8 Hz, 2H), 3.79 (s, 3H), 3.76-3.67 (m,
H), 3.39-3.33 (m, 4H), 1.23 (t, J = 7.2 Hz, 3H).

4
.1.12.9
E)-N-(2-((5-chloro-2-((6-(diethylamino)-2-methoxypyridin-3-yl)amino)pyrimidin-4-
yl)amino)phenyl)but-2-enamide (22i)
(
-
1
Yield: 84.8%; m.p.:193.8-194.6°C; MS (ESI) m/z: 480.5 [M-H] ; H-NMR (400 MHz,
DMSO-d ) δ : 10.07 (s, 1H), 8.33 (s, 1H), 7.90 (s, 1H), 7.83 (s, 1H), 7.75-7.67 (m,
6
H
1
H), 7.56 (d, J = 8.4 Hz, 1H), 7.38-7.27 (m, J = 8.7 Hz, 1H), 7.24-7.14 (m, 2H), 6.87
(dq, J = 12.9, 6.9 Hz, 1H), 6.19 (d, J = 8.4 Hz, 2H), 3.79 (s, 3H), 3.67-3.56 (m, 4H),
1
.26 (t, 6H), 1.43 (t, J = 6.9 Hz, 3H).
.1.12.10
4
(E)-N-(2-((5-chloro-2-((6-(3-hydroxyazetidin-1-yl)-2-methoxypyridin-3-yl)amino)pyr
imidin-4-yl)amino)phenyl)but-2-enamide (22j)
Yield: 82.5%; m.p.:161.0-162.1°C; MS (ESI) m/z: 482.4 [M+H] ; H-NMR (400
+
1
MHz, DMSO-d ) δ : 10.01 (s, 1H), 8.31 (s, 1H), 7.99 (s, 1H), 7.73 (s, 1H), 7.75-7.67
6
H
(m, 1H), 7.56 (d, J = 8.4 Hz, 1H), 7.38-7.27 (m, J = 8.4 Hz, 1H), 7.24-7.14 (m, 2H),
6
3
.87 (dq, J = 13.3, 6.3 Hz, 1H), 6.19 (d, J = 8.5 Hz, 2H), 5.33 (s, 1H), 4.33 (m, 1H),
.98 (dd, J = 11.1, 4.5 Hz, 2H), 3.79 (s, 3H), 3.68 (dd, J = 11.1, 4.5 Hz, 2H), 1.52 (t, J
=
6.8 Hz, 3H).
4
.1.12.11
E)-N-(2-((5-chloro-2-((6-(4-(2-hydroxyethyl)piperazin-1-yl)-2-methoxypyridin-3-yl)
amino)pyrimidin-4-yl)amino)phenyl)but-2-enamide (22k)
(
+
1
Yield: 81.6%; m.p.:161.0-162.1°C; MS (ESI) m/z: 539.4 [M+H] ; H-NMR (400
MHz, DMSO-d ) δ : 10.02 (s, 1H), 8.33 (s, 1H), 7.83 (s, 1H), 7.77 (s, 1H), 7.75-7.67
6
H
(m, J = 13.8, 1H), 7.56 (d, J = 8.4 Hz, 1H), 7.38-7.27 (m, 1H), 7.24-7.18 (m, 2H),
6
3
6
.86 (dq, J = 13.8, 6.9 Hz, 1H), 6.19 (d, J = 8.4 Hz, 2H), 4.46 (s, 1H), 3.79 (s, 3H),
.55 (t, J = 5.8 Hz, 2H), 3.40 (s, 4H), 2.62 (s, 4H), 2.43 (t, J = 5.8 Hz, 2H), 1.52 (t, J =
.9 Hz, 3H).
4
.1.12.12
N-(2-((5-chloro-2-((2-methoxy-4-morpholinophenyl)amino)pyrimidin-4-yl)amino)ph
enyl)methanesulfonamide (22l)
+
1
Yield: 85.3%; m.p.: 181.0-182.1°C; MS (ESI) m/z: 506.4 [M+H] ; H-NMR (400
MHz, DMSO-d ) δ : 9.32 (s, 1H), 8.46 (s, 1H), 8.12 (s, 1H), 8.08 (s, 1H), 7.98 (s, 1H),
6
H
7
8
.60 (d, J = 8.3 Hz, 1H), 7.34 (d, J = 8.1 Hz, 1H), 7.18 (t, J = 7.8 Hz, 2H), 6.25 (d, J =
1
3
.4 Hz, 1H), 3.78 (s, 3H), 3.75-3.69 (m, 4H), 3.53 (s, 4H), 2.96 (s, 3H). C-NMR
(
100 MHz, DMSO-d ) δ : 159.44, 155.80, 155.58, 155.05, 154.85, 135.97, 128.89,
6 C
1
2
27.52, 127.31, 124.80, 124.53, 113.25, 104.06, 98.16, 66.41 (2C), 53.13, 46.05 (2C),
9.16.

4
.1.12.13
N-(2-((5-chloro-2-((2-isopropoxy-4-morpholinophenyl)amino)pyrimidin-4-yl)amino)
phenyl)methanesulfonamide (22m)
-
1
Yield: 78.5%; m.p.: 176.7-178.2°C; MS (ESI) m/z: 532.5 [M-H] ; H-NMR (400 MHz,
DMSO-d ) δ : 9.34 (s, 1H), 8.48 (s, 1H), 8.08 (s, 1H), 7.93 (d, J = 6.6 Hz, 1H), 7.87
6
H
(s, 1H), 7.64 (d, J = 8.4 Hz, 1H), 7.35 (dd, J = 7.4, 1.9 Hz, 1H), 7.21-7.15 (m, 2H),
6
4
.19 (d, J = 8.5 Hz, 1H), 5.14 (dt, J = 12.3, 6.2 Hz, 1H), 3. 37-3.70 (m, 4H), 3.36 (s,
H), 2.96 (s, 3H), 1.22 (d, J = 6.2 Hz, 6H).
4
.1.12.14
N-(2-((5-chloro-2-((2-isopropoxy-4-(piperidin-1-yl)phenyl)amino)pyrimidin-4-yl)ami
no)phenyl)methanesulfonamide (22n)
-
1
Yield: 84.9%; m.p.: 211.0-212.1°C; MS (ESI) m/z: 530.5 [M-H] ; H-NMR (400 MHz,
DMSO-d ) δ : 9.33 (s, 1H), 8.59 (s, 1H), 8.08-8.03 (m, 1H), 7.99 (d, J = 5.8 Hz, 1H),
6
H
7
2
2
.82 (s, 1H), 7.58 (d, J = 8.5 Hz, 1H), 7.31 (dd, J = 7.3, 2.0 Hz, 1H), 7.10-7.05 (m,
H), 6.19 (d, J = 8.5 Hz, 1H), 5.13 (dt, J = 12.2, 6.2 Hz, 1H), 3.44 (d, J = 5.1 Hz, 4H),
.91 (s, 3H), 1.58 (s, 6H), 1.23 (d, J = 4.0 Hz, 6H).
4
.1.12.15
N-(2-((5-chloro-2-((4-(diethylamino)-2-isopropoxyphenyl)amino)pyrimidin-4-yl)ami
no)phenyl)methanesulfonamide (22o)
-
1
Yield: 82.2%; m.p.: 196.3-202.8°C; MS (ESI) m/z: 518.5 [M-H] ; H-NMR (400 MHz,
DMSO-d ) δ : 9.29 (s, 1H), 8.42 (s, 1H), 8.05 (s, 1H), 8.00 (s, 1H), 7.86 (s, 1H), 7.45
6
H
(d, J = 8.5 Hz, 1H), 7.33 (m, 1H), 7.13 (m, J = 6.8 Hz, 2H), 5.97 (d, J = 8.5 Hz, 1H),
5
1
.17-5.07 (m, 1H), 3.44 (q, J = 6.9 Hz, 4H), 2.96 (s, 3H), 1.20 (d, J = 6.2 Hz, 6H),
.13 (t, J = 6.9 Hz, 6H).
4
.1.12.16
N-(2-((5-chloro-2-((2-(isopropylthio)-4-morpholinophenyl)amino)pyrimidin-4-yl)ami
no)phenyl)methanesulfonamide (22p)
-
1
Yield: 80.7%; m.p.: 166.9-168.3°C; MS (ESI) m/z: 548.5 [M-H] ; H-NMR (400 MHz,
DMSO-d ) δ : 9.32 (s, 1H), 8.41 (s, 2H), 8.05 (s, 2H), 7.32 (t, J = 8.5 Hz, 2H), 7.11 (s,
6
H
2
4
H), 6.52 (d, J = 8.7 Hz, 1H), 3.83-3.75 (dt, J = 13.6, 6.8 Hz, 1H), 3.73 (m, J = 13.6,
H), 3.44 (m, 4H), 2.98 (s, 3H), 1.28 (d, J = 6.8 Hz, 6H).
4
.1.12.17
N-(2-((5-chloro-2-((2-(isopropylthio)-4-(piperidin-1-yl)phenyl)amino)pyrimidin-4-yl)
amino)phenyl)methanesulfonamide (22q)
+
1
Yield: 86.4%; m.p.: 186.4-192.1°C; MS (ESI) m/z: 548.4 [M+H] ; H-NMR (400

MHz, DMSO-d ) δ : 9.79 (s, 1H), 8.69 (s, 1H), 8.29 (s, 1H), 8.08 (s, 1H), 8.00 (s, 1H),
6
H
7
3
6
.26 (t, J = 9.5 Hz, 2H), 6.96 (t, J =7.1 Hz, 1H), 6.85 (s, 1H), 6.51 (d, J = 8.7 Hz, 1H),
.82 (dt, J = 13.6, 6.9 Hz, 1H), 3.55 (d, J = 5.0 Hz, 4H), 2.86 (s, 3H), 1.71-1.50 (m,
H), 1.29 (d, J = 6.8 Hz, 6H).
4
.1.12.18
N-(2-((5-chloro-2-((4-(diethylamino)-2-(isopropylthio)phenyl)amino)pyrimidin-4-yl)a
mino)phenyl)methanesulfonamide (22r)
+
1
Yield: 82.3%; m.p.: 173.0-176.2°C; MS (ESI) m/z: 536.2 [M+H] ; H-NMR (400
MHz, DMSO-d ) δ : 9.29 (s, 1H), 9.08 (s, 1H), 8.49 (s, 1H), 8.18 (s, 1H), 8.12 (s, 1H),
6
H
7
.99 (d, J = 7.9 Hz, 1H), 7.70 (d, J = 9.4 Hz, 1H), 7.39 (dd, J = 7.8, 1.4 Hz, 1H), 7.31
(td, J = 7.8, 1.4 Hz, 1H), 7.22 (td, J = 7.7, 1.4 Hz, 1H), 6.69 (d, J = 8.4 Hz, 1H),
3
.43-3.40 (m, 4H), 2.96 (s, 3H), 1.64-1.49 (m, 6H), 1.23 (s, 6H).
.1.12.19
4
N-(2-((5-chloro-2-((4-morpholinophenyl)amino)pyrimidin-4-yl)amino)phenyl)methan
esulfonamide (22s)
+
1
Yield: 83.4%; m.p.: 201.2-202.8°C; MS (ESI) m/z: 476.3 [M+H] ; H-NMR (400
MHz, DMSO-d ) δ : 9.29 (s, 1H), 9.15 (s, 1H), 8.51 (s, 1H), 8.24 (s, 1H), 8.13 (s, 1H),
6
H
7
.97 (d, J = 7.8 Hz, 1H), 7.76 (d, J = 9.9 Hz, 1H), 7.39 (dd, J = 7.9, 1.4 Hz, 1H), 7.32
(
t, J = 7.1 Hz, 1H), 7.23 (td, J = 7.7, 1H), 6.69 (d, J = 9.1 Hz, 1H), 3.70 (m, 4H), 3.32
s, 4H), 2.95 (s, 3H).
(
4
.1.12.20
N-(2-((5-chloro-2-((4-(piperidin-1-yl)phenyl)amino)pyrimidin-4-yl)amino)phenyl)me
thanesulfonamide (22t)
Yield: 82.6%; m.p.: 164.3-167.3°C; MS (ESI) m/z: 474.4 [M+H] ; H-NMR (400
+
1
MHz, DMSO-d ) δ : 9.30 (s, 1H), 9.07 (s, 1H), 8.50 (s, 1H), 8.18 (d, 1H), 8.11 (s,
6
H
1
H), 8.00 (s, 1H), 7.68 (dd, J = 9.3, 1.5 Hz, 1H), 7.37 (dd, J = 7.8, 1.3 Hz, 1H), 7.27 (t,
J = 7.2 Hz, 1H), 7.19 (t, J = 7.8 Hz, 1H), 6.67 (d, J = 8.9 Hz, 1H), 3.41 (d, J = 5.4 Hz,
4
H), 2.95 (s, 3H), 1.55 (m, 6H).
.1.12.21
4
N-(2-((5-chloro-2-((4-(diethylamino)phenyl)amino)pyrimidin-4-yl)amino)phenyl)met
hanesulfonamide (22u)
+
1
Yield: 81.4%; m.p.: 183.6-186.1°C; MS (ESI) m/z: 462.4 [M+H] ; H-NMR (400
MHz, DMSO-d ) δ : 8.95 (s, 1H), 8.56 (s, 1H), 8.05 (m, 3H), 7.60 (d, J = 8.5 Hz, 1H),
6
H
7
3
.36 (dd, J = 7.7, 1.5 Hz, 1H), 7.16 (m, 2H), 7.12 (d, 1H), 6.46 (d, J = 9.1 Hz, 1H),
.45 (q, J = 7.0 Hz, 4H), 2.93 (s, 3H), 1.09 (t, J = 7.0Hz, 6H).

4
.1.12.22
N-(2-((5-chloro-2-((6-(3-hydroxyazetidin-1-yl)pyridin-3-yl)amino)pyrimidin-4-yl)ami
no)phenyl)methanesulfonamide (22v)
+
1
Yield: 84.3%; m.p.: 169.5-172.2°C; MS (ESI) m/z: 462.0 [M+H] ; H-NMR (400
MHz, DMSO-d ) δ : 8.92 (s, 1H), 8.59 (s, 1H), 8.34 – 7.84 (m, 3H), 7.61 (d, J = 9.1
6
H
Hz, 1H), 7.35 (dd, J = 9.5 Hz, 4.5 Hz, 1H), 7.27 – 7.05 (m, 2H), 7.15 (d, 1H), 6.46 (d,
J = 3.1 Hz, 1H), 5.38 (s, 1H), 4.43 (m, 1H), 3.98 (dd, J = 8.3, 4.5 Hz, 2H), 3.68 (dd, J
=6.8, 2H), 2.93 (s, 3H).
4
.1.12.23
N-(2-((5-chloro-2-((6-(4-(2-hydroxyethyl)piperazin-1-yl)pyridin-3-yl)amino)pyrimidi
n-4-yl)amino)phenyl)methanesulfonamide (22w)
Yield: 83.9%; m.p.: 181.0-185.1°C; MS (ESI) m/z: 519.4 [M+H] ; H-NMR (400
+
1
MHz, DMSO-d ) δ : 9.25 (s, 1H), 8.86 (s, 1H), 8.34 – 7.84 (m, 3H), 7.60 (d, J = 9.1
6
H
Hz, 1H), 7.35 (dd, J = 7.0, 1.5 Hz, 1H), 7.27 – 7.05 (m, 2H), 7.17 (d, 1H), 6.46 (d, J =
2
3
.8 Hz, 1H), 4.46 (s, 1H), 3.45 (t, J = 6.2 Hz, 2H), 3.36 (s, 4H), 2.62 (s, 4H), 2.93 (s,
H), 2.42 (t, J = 5.6 Hz, 2H).
4
.1.12.24
N-(2-((5-chloro-2-((6-(3-methylpiperazin-1-yl)pyridin-3-yl)amino)pyrimidin-4-yl)ami
no)phenyl)methanesulfonamide (22x)
+
1
Yield: 81.8%; m.p.: 193.2-199.2°C; MS (ESI) m/z: 489.4 [M+H] ; H-NMR (400
MHz, DMSO-d ) δ : 8.95 (s, 1H), 8.56 (s, 1H), 8.34 – 7.84 (m, 3H), 7.61 (d, J = 10.0
6
H
Hz, 1H), 7.35 (dd, J = 8.3, 2.6 Hz, 1H), 7.27 – 7.05 (m, 2H), 7.17 (d, 1H), 6.46 (d, J =
1
1
.8 Hz, 1H), 3.25 (s, 1H), 3.10 (s, 1H), 2.93 (s, 3H), 2.88 (s, 1H), 2.79 (s, 1H), 2.73 (s,
H), 2.72 (s, 1H), 2.68 (s, 1H), 1.31 (s, 3H), 1.07(s, 1H).
4
4
.1.12.25
-(5-chloro-2-((6-(4-(2-hydroxyethyl)piperazin-1-yl)-2-methoxypyridin-3-yl)amino)p
yrimidin-4-yl)-3,4-dihydro-2H-benzo[1,4- b]thiazine-1,1-dioxide (23a)
+
1
Yield: 78.4%; m.p.:190.8-196.0°C; MS (ESI) m/z: 546.4 [M+H] ; H NMR (400 MHz,
CDCl ) δ : 9.13 (s, 1H), 8.47 (d, J = 8.3 Hz, 1H), 8.17 (d, J = 7.5 Hz, 1H), 8.11 (s,
3
H
1
1
2
H), 8.00 (d, J = 8.3 Hz, 1H), 7.62 (t, J = 7.5 Hz, 1H), 7.28 (d, J = 6.1 Hz, 1H), 7.03 (s,
H), 6.51 (m, 2H), 6.08 (dd, J = 6.1 Hz, 4.9Hz, 2H), 4.02 (s, 1H), 3.94 (s, 3H), 3.74 (s,
1
3
H), 3.55 (s, 4H), 2.76 (s, 4H), 2.71 (d, J = 4.9 Hz, 2H). C-NMR (100 MHz,
DMSO-d ) δ : 159.66, 156.37, 155.90, 155.35, 154.90, 137.78, 137.38, 135.20,
6
C
1
29.98, 129.84, 126.46, 123.92, 123.48, 112.09, 98.28, 70.26, 60.77, 58.96, 53.38
(2C), 53.06, 45.51 (2C), 29.47.
4
4
.1.12.26
-(5-chloro-2-((2-methoxy-6-(piperidin-1-yl)pyridin-3-yl)amino)pyrimidin-4-yl)-3,4-

dihydro-2H-benzo[1,4-b]thiazine-1,1-dioxide (23b)
Yield: 77.8 %; m.p.:158.8-166.8°C; MS (ESI) m/z: 501.3 [M+H] ; H NMR (400
+
1
MHz, CDCl ) δ : 9.17 (s, 1H), 8.46 (s, 1H), 8.00 (m, 4H), 7.60 (s, 1H), 7.15 (s, 1H),
3
H
6
2
.55 (s, 2H), 6.27 (s, 1H), 6.03 (s, 1H), 3.94 (s, 3H), 3.46 (s, 4H), 1.71 (s, 4H), 1.26 (s,
H).
4
4
.1.12.27
-(5-chloro-2-((2-methoxy-6-(pyrrolidin-1-yl)pyridin-3-yl)amino)pyrimidin-4-yl)-3,4
-
dihydro-2H-benzo[1,4-b]thiazine-1,1-dioxide (23c)
+
1
Yield: 80.5%; m.p.:178.5-181.2°C; MS (ESI) m/z: 487.4 [M+H] ; H NMR (400
MHz, CDCl ) δ : 9.57 (s, 1H), 8.40 (d, J = 8.1 Hz, 1H), 8.01(m, 2H), 7.72 (s, 1H),
3
H
7
1
.51 (s, 1H), 7.42 (m, 1H), 7.30 (m, 2H), 6.58 – 6.50 (m,, 2H), 6.06 (d, J = 9.0 Hz,
H), 5.87 (s, 1H), 3.89 (s, 3H), 3.45 (s, 4H), 2.02 (s, 4H).
4
4
4
.1.12.28
-(5-chloro-2-((2-isopropoxy-6-(piperidin-1-yl)pyridin-3-yl)amino)pyrimidin-4-yl)-3,
-dihydro-2H-benzo[1,4- b]thiazine-1,1-dioxide (23d)
+
1
Yield: 75.3%; m.p.:162.3.5-169.2°C; MS (ESI) m/z: 529.4 [M+H] ; H NMR (400
MHz, CDCl ) δ : 9.81 (s, 1H), 8.30 (s, 1H), 8.17 (s, 1H), 8.08 (s, 2H), 7.80 (s, 1H),
3
H
7
1
1
1
.54 (m, 2H), 6.55 – 6.52 (m, 2H), 6.05 (s, 2H), 3.56 – 3.49 (m, 7H), 2.23 (s, 4H),
1
3
.90 (s, 2H), 1.72 (s, 4H). C-NMR (100 MHz, DMSO-d ) δ : 159.62, 158.74,
6
C
55.88, 155.21, 154.89, 137.77, 136.87, 135.10, 129.98, 129.81, 126.56, 123.95,
23.64, 111.64, 97.85, 67.88, 51.84, 46.43 (2C), 44.64, 25.39 (2C), 24.75, 22.43 (2C).
4
.2. MTT assay in vitro
The cytotoxic activities of compounds 22a-x and 23a-d were evaluated against H2228,
Karpas299 and A549 by the standard MTT assay in vitro, with ceritinib as the
positive control. The cancer cell lines were cultured in minimum essential medium
(MEM) supplement with 10% fetal bovine serum (FBS). Approximate 4 × 103 cells,
suspended in MEM medium, were plated into each well of a 96-well plate and
o
incubated in 5% CO at 37 C for 24 h. The tested compounds at the indicated final
2
concentrations were added to the culture medium and incubated for 72 h. Fresh MTT
was added to each well at the terminal concentration of 5 μg/mL, and incubated with
o
cells at 37 C for 4 h. The formazan crystals in each well were dissolved in 100 μL
DMSO, and the absorbency at 492 nm (for absorbance of MTT formazan) and 630
nm (for the reference wavelength) was measured with an ELISA reader. All of the
compounds were tested three times in each of the cell lines. The results, expressed as
IC (inhibitory concentration 50%), were the averages of three determinations and
5
0
calculated relative to the vehicle (DMSO) control by the Bacus Laboratories
Incorporated Slide Scanner (Bliss) software.

4
.3. In vitro enzymatic assays
L1196M
The in vitro enzymatic assays versus ALK and ALK
were evaluated by
homogeneous time-resolved fluorescence (HTRF) assay. In enzymatic assay, the
solution of peptide substrates, ATP, appropriate kinase, and diluted compound was
mixed with the kinase reaction buffer (50 mM HEPES, pH 7.5, 0.0015% Brij-35, 10
mM MgCl , 2 mM DTT), with blank DMSO solution as the negative control. The
2
kinase reaction was initiated by the addition of tyrosine kinase proteins diluted in 39
o
μL of kinase reaction buffer solution and incubated at 28 C for 60 min. And then add
2
5 μL of stop buffer (100 mM HEPES, pH 7.5, 0.015% Brij-35, 0.2% Coating
Reagent #3, 50 mM EDTA) to stop reaction. The plate was read by Caliper at 320 nm
and 615 nm. IC values were calculated from the inhibition curves.
5
0
4
.4. Western blotting
Western blotting analysis was performed as described Karpas299 cells were treated
with 22l or ceritinib. After treatment for 24 h, cells were collected. The protein in the
cytoplasm was extracted, quantified with protein assay reagent and electrophoresed
on SDS-polyacrylamide gel until the proteins of different molecular weights were
separated. Then, the different proteins contained in the SDS-polyacrylamide gel were
transferred to polyvinylidene difluoride (PVDF) membranes. After incubated
o
overnight at 4 C with the respective primary antibodies, horseradish peroxidase
(HRP)-conjugated secondary antibodies (1: 3000) was added. The Enhanced
chemiluminescence reagent was used to identify the specific bands. Densitometry
analysis on immunoblots were performed using ImageJ2 software.
4
.5. Morphology assays of apoptotic cells
Apoptotic morphological changes of H2228 and Karpas299 cells were detected by
AO/EB staining. Briefly, cells were seeded in six-well plates for 24 h, then treated
with ceritinib or different concentrations of 22l for 48 h. The cells were washed with
phosphate buffer saline (PBS) and then stained with AO/EB mixed solution (AO: EB
¼
1: 1) for 15 min. The stained cells were washed twice with PBS and observed by
fluorescence microscope.
4
.6. Molecular docking
The molecular modeling studies were performed with Discovery Studio 2017. The
protein coordinates (PDB ID code: 4MKC) were downloaded from the Protein Data
Bank (http://www.rcsb.org/ pdb/). In the docking process, the protein protocol was
prepared by several operations, including standardization of atom names and insertion
of missing atoms in residues. Then, the receptor model was typed with the CHARMm
force field and a binding sphere with radius of 12 Å was defined through the original
ligand (ceritinib) as the binding site. The ceritinib, 22l and 23a were drawn with
ChemSketch and fully minimized using the CHARMm force field. Finally, they were
docked into the binding site using the CDOCKER protocol with the default settings.

Conflict of interest
The authors have declared no conflict of interest.
Acknowledgements
This work was supported by National Natural Science Foundation of China
(No.81673308), Natural Science Foundation of Liaoning Province (No.201602703)
and Development Project of Ministry of Education Innovation Team (No. IRT1073).
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