Structure-based design of 2,4-diaminopyrimidine derivatives bearing a pyrrolyl group as ALK and ROS1 inhibitors

Article abstract of DOI:10.1039/c9nj05980f

In order to develop potent ALK and ROS1 dual inhibitors, twenty-eight 2,4-diaminopyrimidine derivatives (9a-9n and 10a-10n) bearing a pyrrolyl moiety were designed and synthesized based on the co-crystal structure of ceritinib with ALK^wt protein. Most compounds displayed considerable activity against ALK and ROS1 addicted cells; meanwhile, compound 10d showed excellent activity against Karpas299, H2228 and HCC78 with IC₅₀ values of 0.01, 0.08 and 0.042 μM. Subsequently, seven compounds were selected for kinase studies in vitro, resulting in the discovery of 10d with IC₅₀ values of 1.8, 4.3 and 3.6 nM against ALK, ALK^L1196M and ROS1, respectively. Furthermore, the biological assays revealed that compound 10d induced cell apoptosis in a dose-dependent manner. Ultimately, molecular docking studies presented reasonable and optimal binding interactions with ALK^wt and ROS1.

Full text of DOI:10.1039/c9nj05980f

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DOI: 10.1039/C9NJ05980F
Structure-based design of 2,4-diaminopyrimidine derivatives bearing
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pyrrolyl group as ALK & ROS1 inhibitors
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Jie Wanga, Shangfei Weia, Tong Lia, Lingyun Xing a, Meng Caoa, Nan Jianga, Ming
Guoa, Daiying Zuob, *, Xin Zhaia,*
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a
Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education,
Shenyang Pharmaceutical University, Shenyang 110016, PR China
b Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang 110016, China.
*Corresponding author.
E-mail address: zhaixin_syphu@126.com (Xin Zhai), zuodaiying@163.com (Daiying. Zuo).
Abstract:
In order to develop potent ALK and ROS1 dual inhibitors, twenty-eight
2,4-diaminopyrimidine derivatives (9a-9n and 10a-10n) bearing pyrrolyl moiety were
designed and synthesized based on co-crystal structure of ceritinib with ALKwt
protein. Most compounds displayed considerable activity against ALK and ROS1
addicted cells, meanwhile, compound 10d showed excellent activity against
Karpas299, H2228 and HCC78 with IC50 values of 0.01, 0.08 and 0.042 μM.
Subsequently, seven compounds were selected for kinase studies in vitro, resulting in
the discovery of 10d with IC50 values of 1.8, 4.3 and 3.6 nM against ALK, ALKL1196M
and ROS1, respectively. Furthermore, the biological assays revealed that compound
10d induced cell apoptosis in a dose-dependent manner. Ultimately, molecular
docking studies presented the reasonable and optimal binding interactions with ALKwt
and ROS1.
Keywords: synthesis; 2,4-diaminopyrimidine; ALK&ROS1; anti-proliferation
activities

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1. Introduction
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Anaplastic lymphoma kinase (ALK) receptor tyrosine kinase and its ligand have
received extensive attention recently among the target-based therapy.1 In contrast to
dormancy in normal tissues, abnormal ALK gene in a variety of tumor cells was
mainly represented by gene fusion,2 overexpression and point mutation.3 Moreover,
the transforming echinoderm microtubule associated protein like 4-ALK
(EML4-ALK) fusion gene was recognized as the driver gene for non-small cell lung
cancer (NSCLC).4 Similar to ALK, c-ros oncogene 1 (ROS1), an orphan receptor
tyrosine kinase, has been identified in the expression of oncogenic fusion proteins in a
subset of NSCLC patients.5-6 Based on approximately 49% amino acid homology of
ALK and ROS1,7 a majority of ALK inhibitors had shown outstanding efficacy in
ROS1 positive NSCLC patients, greatly accelerating the clinical development of ALK
and ROS1 dual inhibitors.8
Crizotinib was the first ALK and ROS1 dual inhibitor approved in 2011 based on
the impressive objective response rate and progression-free survival times as the
standard therapy for ALK-positive lung cancer patients.9-10 However, resistance to
crizotinib was tested in patients within about 1 year of treatment, which exhibited
tumor relapse and regrowth.11-12 As a consequence, several crizotinib-resistant ALK
inhibitors have been reported, including ceritinib,13 alectinib,14 brigatinib,15
lorlatinib16 and TPX-00L05 17 (Fig. 1).
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Cl
N
O
NH
F
O
HN
N
NH
N
O
N
Cl
O
N
P
O
H
N
N
N
N
F
N
O
N
Cl
N
H2N
N
O
H2N
N
N
N
N
Lorlatinib
(launched 2018)
Crizotinib
(launched 2011)
Brigatinib
(launched 2017)
Alectinib
(launched 2014)
F
O
NH
hydrophilic
"tail"
Linker
Cl
Cl
N
N
NH
N
O
N
N
N
O
N
N
N
N
H
H
H
H
NH
O
S
O
O
R1
N
O
Target Compounds
Ceritinib
(launched 2014)
TPX-0005
(phase II)
Fig. 1 Structures of ALK inhibitors and design strategies of target compounds.
The co-crystal structure of ceritinib with ALKwt (Fig. 2A)18-19 indicated that
ceritinib as the lead compound embedded the surface of ALK protein completely,
meanwhile, the key fragments of 2,4-diarylaminopyrimidine and 5-Cl atom were
retained to assume the kinase hinge binding moiety and the essential protein
interactions in the ATP binding site.20 Then, for the sake of improving the potency
and diversity, primary structural modification was mainly focused on the optimization

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of aromatic group substituents. 21 As shown in Fig. 2B and 2C, the isopropyl DsOuIl:f1o0.n10y3l9/C9NJ05980F
motif could form important hydrogen and hydrophobic effect, therefore, sulfonamido
and acetamido segments were chosen to guarantee or even enrich the interaction
manners.22-23 In term of the effect of steric hindrance, methoxyl group was disclosed
as an effective isopropoxyl surrogate.24
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Fig. 2 Design strategy for target compounds based on co-crystal structure of ceritinib
with ALKwt (PDB ID code: 4MKC). (A) Binding conformation of ceritinib (blue
sticks) in the ATP binding site of ALKwt and the existence of ceritinib on the surface
of ALKwt protein. (B) The general structural formula of target compounds and
hydrophobic diagram of the “tail”. (C) 2D diagram of the interaction between pyrrolyl
moiety and ALKwt.
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Recently, extensive biological activities such as anti-ulcer,25 antitumor
anti-inflammatory
and
27
of pyrrolyl-containing compounds have received the
consideration in the field of drug design and development. As expected, 2D diagram
(Fig. 2C) showed that the pyrrolyl fragment formed new π-anion and carbon
hydrogen bond interactions, indicating the introduction of pyrrolyl moiety was
considerable to improve the structural optimization. Thus, pyrrolyl segment was
accessed as a linker to improve the affinity with the biological target. On the other
hand, simulation hydrophobic diagram of the “tail” revealed that hydrophilic “tail”
was located in a strong hydrophilic region. For further verification, different aliphatic
and alicyclic amines would be brought to discuss the influence of hydrophilic
moieties in solvent-interaction region.28
Accordingly, twenty-eight 2,4-diaminopyrimidine compounds were subjected to
design, synthesis and prediction for their physical properties, followed by their
anti-proliferative activities in vitro assay against five human cancer cell lines to study
the structure-activity relationships. In addition, seven compounds were further
evaluated for their activity towards the ALK, ALKL1196M, ROS1 and EGFR kinase in
vitro. Especially, the cell apoptosis assay and molecular docking mode analysis of the
most potent compound 10d were also performed.
2. Chemistry
Target compounds 9a-9n and 10a-10n were synthesized as illustrated in Scheme 1.
Substitution of commercially available 2,4,5-trichloropyrimidine (1) with
bnzene-1,2-diamine (2) afforded intermediate 3, which was acylated by acetyl
chloride or methanesulfonyl chloride in the presence of triethylamine without further

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purification to bring the key intermediates 4a and 4b in high yields.29 Meanwhile,
treatment of 4-fluoro-2-methoxy-1-nitrobenzene (5) with 1H-pyrrole (6) in DMF at
100 °C for 8 h gave rise to 7 as a yellow solid. Refluxing in ethanol, the intermediate
8 was obtained via reduction of compound 7 with iron powder and catalytic amounts
of ammonium chloride.30 Subsequently, 4a and 4b were reacted with 8 in refluxing
isopropanol and purified on the silica gel column to arrive at critical compounds 9a
and 10a. Eventually, Mannich reaction of the resultant 9a and 10a with appropriate
aliphatic amines and 37% formaldehyde in the presence of acetic acid at room
temperature provided target compounds 9b-9n and 10b-10n in satisfied yields.
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Cl
Cl
NH2
Cl
N
N
N
i
ii
N
N
Cl
Cl
N
Cl
NH2
N
N
Cl
H
H
NH
NH2
R1
4a-4b
2
3
1
Cl
N
N
v
N
N
N
H
H
F
N
N
NH
O
R1
H
N
iv
9a; 10a
iii
O2N
H2N
O2N
O
O
,
-
: R1 = acetyl
: R1 = methanesulfonyl
4b 10a 10n
O
4a 9a 9n
6
5
7
8
,
-
: R2 = 4-ethylpiperazinyl
: R2 = morpholinyl
: R2 = 1,1-dioxidothiomorpholinyl
: R2 = 2-hydroxyethylaminyl
9b, 10b
9c, 10c
9d, 10d
9e, 10e
9f, 10f
9i, 10i
9j, 10j
Cl
N
: R2 = 4-methylpiperazinyl
: R2 = piperidinyl
: R2 = diethylaminyl
N
9k, 10k
9l, 10l
vi
: R2 = 4-(2-hydroxyethyl)piperazinyl
R2
N
N
N
: R2 = 4-methylpiperdinyl
: R2 = dimethylaminyl
: R2 = azetidin-3-olyl
9m, 10m
9n, 10n
H
H
NH
O
R1
9g, 10g
: R2 = pyrrolidinyl
9b-9n; 10b-10n
: R2 = thiomorpholinyl
9h, 10h
Scheme 1. Reagents and conditions: (i) DIPEA, isopropanol, 80 °C; (ii) the acyl
chloride of R1, TEA, DCM, rt; (iii) K2CO3, DMF, 100 °C; (iv) iron powder,
hydrochloric acid, ethanol/water (9:1), 80 °C; (v) IPA, 80 °C; (vi) aliphatic amines,
acetic acid, formaldehyde (37%), rt.
3. Results and discussion
3.1. Physical properties, in vitro anti-proliferative activity and SARs study
The physical properties were listed to assess the “drug-likeness” of compounds
9a-9n and 10a-10n according to the Lipinski rule.31 The predicted results of cLogP
and TPSA were shown in Table 1. The lipophilic quantity cLogP is the logarithm of
the partition coefficient between 1-octanol and water and the topological polar surface
area (TPSA) is defined as the surface sum over all polar atoms for the optimisation of
cell permeability. Most compounds with cLogP of less than 5 and TPSA of less than
140 Ų revealed the better druggability which were essential to execute further in vitro
assays.
Table 1. Physical properties of 9a-9n and 10a-10n.
a cLogP
3.75
b TPSA (Ų)
93.10
TPSA (Ų)
Compd.
Compd.
cLogP
9a
9b
9c
10a
10b
10c
3.36
3.43
3.19
118.55
125.03
131.02
3.81
99.58
3.56
105.57

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9d
3.53
4.41
4.67
4.14
4.12
3.31
3.08
4.35
3.08
3.79
3.06
5.62
99.58
96.34
10d
10e
3.20
3.99
4.33
3.73
3.65
2.92
2.63
3.97
2.68
3.20
2.59
5.62
125.03
9e
121.79
121.79
121.79
147.09
164.31
150.81
121.79
145.26
121.79
142.02
113.62
9f
96.34
10f
9g
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9h
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138.86
125.36
96.34
10h
9i
10i
9j
10j
9k
9l
10k
119.81
96.34
10l
9m
10m
10n
9n
116.57
113.62
cCeritinib
Ceritinib
a cLogP measured between 1-octanol/water phosphate buffered.
b TPSA defined the surface sum over all polar atoms.
c Used as positive control.
The MTT assay in vitro was adopted to evaluate the anti-proliferation activities of
all the synthesized compounds 9a-9n and 10a-10n. Taking ceritinib as the positive
control, target compounds were investigated the potency against NPM-ALK-addicted
Karpas299 (Human anaplastic large cell lymphoma cell line) cell,
EML4-ALK-addicted H2228 (Human non-small-cell lung cancer cell line) cell and
ROS1-addicted HCC78 (Human lung adenocarcinoma cell line) cell as well as the
off-target cells, namely H460 (Human large cell lung cancer cell line) and A549
(Human EGFR-positive non-small-cell lung cancer cell line). The results of IC50
values were shown in Table 2, as mean values of experiments performed in triplicate.
As illustrated in Table 2, satisfactory data were obtained that most compounds
exhibited moderate to excellent cytotoxic activity and marked selectivity. As a general
trend, the title compounds showed more potent on Karpas299, H2228 and HCC78
cells superior to H460 and A549 cell lines. Moreover, introduction of hydrophilic
group bending down into the solvent area displayed higher anti-proliferation activities
compared to compounds 9a and 10a. Among target compounds, the most promising
compound 10d showed excellent activity against Karpas299, H2228 and HCC78 with
the IC50 values of 0.01, 0.08 and 0.042 μM, which were 2.7-, 1.2- and 1.8-fold more
active than ceritinib (0.027, 0.099 and 0.074 μM).
The initial SARs study was commenced by the introduction of diverse R1 at the
“head” of the benzene ring. It was worth noting that sulfonamido group compounds
10a-10n turned out to significantly enhance the potency as compared with compounds
9a-9n bearing acetamido, suggesting that sulfonamido group was necessary to the
anti-proliferation effect. Among the target-addicted cells, most of the compounds
showed similar activity trends.

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Further analysis revealed that different hydrophilic R2 had a systematic influence
on their potency. Compounds 9i and 10i bearing 1,1-dioxidothiomorpholinyl moiety
showed decreased or even vanished activity with IC50 of more than 1 μM against
Karpas299, H2228 and HCC78. The loss in activity might be due to the steric clash.
Notably, the same trend was observed among compounds 10k bearing diethylaminyl
and 10m bearing dimethylaminyl. Interestingly, in Karpas299 cells, mono-substituted
compounds 9j (IC50 = 0.23 μM) and 10j (IC50 = 0.09 μM) displayed moderate potency
as compared to other compounds bearing double-substituted group. Meanwhile, in
H2228 cells, the morpholinyl 10c (IC50 = 0.37 μM) and 4-methylpiperazinyl 10d (IC50
= 0.08 μM) afforded greater activity towards piperidinyl 10e (IC50 = 0.55 μM) and
4-methylpiperdinyl 10f (IC50 = 0.64 μM), indicating the embedded of
electron-withdrawing group was advantageous to potency. In addition, incorporation
of a polar moiety (hydroxyl group) on 10b gave rise to compound 10l which achieved
2.1-, 1.1- and 1.7-fold activities against Karpas299, H2228 and HCC78 cell lines,
respectively.
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Table 2. Cytotoxicity of 9a-9n and 10a-10n against Karpas299, H2228, HCC78,
H460 and A549 cell lines in vitro.
Cl
N
N
R2
N
N
N
H
H
NH
O
R1
aIC50 (μM) ± SD
Compd.
9a
R1
R2
Karpas299
H2228
HCC78
H460
A549
O
-
0.56±0.02
1.01±0.31
0.54±0.022
0.86±0.013
0.12±0.006
0.81±0.04
0.59±0.012
0.83±0.12
0.72±0.08
0.89±0.06
0.13±0.01
0.66±0.08
0.54±0.03
0.59±0.25
0.48±0.07
O
O
O
O
O
O
O
N
N
9b
9c
9d
9e
9f
0.48±0.06
0.51±0.018
0.046±0.001
0.60±0.021
0.42±0.005
1.77±0.43
2.58±0.67
1.37±0.47
2.3±0.39
>10
O
N
1.38±0.02
1.98±0.09
1.69±0.22
0.75±0.04
N
N
N
N
1.54±0.25
N
9g
9h
0.71±0.007
0.86±0.004
0.78±0.03
1.82±0.40
1.03±0.18
1.74±0.35
1.23±0.47
0.80±0.09
5.47±0.34
6.17±0.21
S
N

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aIC50 (μM) ± SD
Compd.
R1
R2
Karpas299
H2228
HCC78
H460
A549
6
7
8
9
O
O
O
O
O
O
O
O
S
N
9i
1.23±0.05
0.24±0.08
0.62±0.09
0.26±0.01
0.18±0.041
0.36±0.04
0.16±0.04
0.18±0.01
0.14±0.03
0.01±0.006
0.20±0.02
0.32±0.03
0.36±0.04
0.44±0.017
1.04±0.16
4.31±0.72
0.60±0.34
1.06±0.04
0.84±0.06
0.37±0.08
0.66±0.07
0.71±0.07
0.24±0.02
0.37±0.06
0.08±0.003
0.55±0.09
0.64±0.01
0.70±0.013
1.29±0.21
3.21±0.73
3.46±0.18
0.27±0.02
0.92±0.08
0.78±0.04
0.43±0.06
0.63±0.03
0.51±0.11
0.44±0.03
0.36±0.08
0.042±0.01
0.48±0.02
0.31±0.04
0.22±0.01
0.82±0.13
1.87±0.21
1.01±0.03
1.08±0.07
0.12±0.01
4.22±0.28
0.68±0.16
4.19±0.45
0.13±0.03
3.38±0.03
2.68±0.04
2.50±0.06
2.67±0.02
3.10±0.05
1.63±0.18
1.41±0.33
>10
3.46±0.23
3.56±0.39
0.93±0.24
0.78±0.02
2.11±0.31
3.86±0.09
2.62±0.09
1.20±0.07
3.41±0.03
1.09±0.08
1.56±0.23
2.96±0.68
3.4±0.06
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H
N
HO
9j
N
9k
N
N
9l
HO
N
9m
9n
HO
N
O
S
O
O
O
O
O
O
O
O
O
10a
10b
10c
10d
10e
10f
10g
10h
10i
-
O
S
N
N
O
S
O
N
O
S
N
N
O
S
N
O
S
N
O
S
N
O
S
S
N
2.26±0.09
7.00±1.04
O
S
O
S
N
O

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aIC50 (μM) ± SD
Compd.
10j
R1
R2
Karpas299
H2228
HCC78
H460
A549
O
S
O
O
O
O
O
H
N
HO
0.09±0.008
0.27±0.06
0.093±0.02
1.87±0.05
1.56±0.07
0.71±0.03
0.94±0.08
1.21±0.25
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O
S
N
10k
10l
0.23±0.005
0.084±0.009
0.059±0.004
0.50±0.07
0.21±0.03
0.18±0.05
0.29±0.04
0.26±0.021
0.12±0.07
2.91±0.30
0.44±0.04
3.12±0.24
O
S
N
N
HO
O
S
N
10m
O
S
HO
N
10n
0.12±0.008
0.36±0.02
0.18±0.03
1.28±0.42
0.20±0.03
1.05±0.16
0.79±0.07
bCeritinib
0.027±0.002
0.099±0.01
0.074±0.01
a Data presented is mean ± SD value of three independent experiments.
b Used as positive control.
3.2. In vitro enzymatic assays
In view of the promising activity of target compounds on ALK and ROS1 addicted
cells, seven optimized compounds were selected for further molecular-level studies
with ALK, ALKL1196M, ROS1 and EGFR kinases in vitro. The non-related EGFR
kinase was selected to verify the kinase selectivity. Taking ceritinib as the positive
control, the results expressed as IC50 were summarized in Table 3.
As illustrated in Table 3, most compounds (9d, 10b, 10d and 10m) performed
potent inhibitory activities against ALK, ALKL1196M and ROS1 as dual kinases
inhibitors with IC50 below 30 nM, parallelled to the cellular activity. Contrastly,
compound 9i bearing acetamido (R1) and thiomorpholine 1,1-dioxide (R2) fragment
(IC50 > 100 nM) extinguished the activity dramatically. Additionally, both tested
compounds and ceritinib revealed no activity against EGFR with IC50 value over 100
nM. To our delight, compound 10d was distinguished with IC50 value of 1.8, 4.3 and
3.6 nM against ALK, ALKL1196M and ROS1 which was comparable to that of ceritinib
(IC50 = 2.3, 8.9 and 2.7 nM). Thus, compound 10d was preferred for further
evaluation in forthcoming biological and molecular docking studies.
Table 3. Enzymatic inhibition of selected compounds 9d, 9i, 9l, 10b, 10d, 10g and
10m against ALK, ALKL1196M, ROS1 and EGFR in vitro.
IC50 (nM)
Compd.
ALK
4.5
ALKL1196M
9.4
ROS1
4.1
EGFR
>100
>100
>100
9d
9i
>100
16.2
>100
>100
23.6
9l
28.9

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10b
10d
7.2
1.8
7.4
28
9.0
4.3
6.3
3.6
9.2
5.6
2.7
>100
>100
>100
>100
>100
10g
18.2
7.8
10m
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aCeritinib
2.3
8.9
a Used as positive control.
3.3. Apoptosis analysis
To further determine the promising anti-proliferation potency, compound 10d was
monitored the apoptotic morphological changes with Hoechst 33258 or AO/EB
staining for 48 h in Karpas299 cells; ceritinib was tested for comparison.32
As shown in the upper part in Fig. 3, control cells group showed uniform blue
fluorescence by Hoechst 33258 staining. Nuclear shrinkage and fragmentation were
performed on 10d or ceritinib treated cells. Meanwhile, the number and degree of
apoptosis cells were increased obviously on 10d of 50 nM treatment group. As shown
in the lower part in Fig. 3, control cells group showed homogeneous green
fluorescence and normal structure by AO/EB staining. At the concentration of 25 nM,
10d or ceritinib treated cells increased the number of early apoptosis cells with
enhanced green fluorescence. Moreover, the terminal apoptosis cells accompanied by
the emergence of irregular orange-red fluorescence were increased markedly on 10d
of 50 nM treatment group. In terms of the similar phenomena observed in Hoechst
33258 or AO/EB staining, 10d was revealed to promote Karpas299 cell apoptosis in a
dose-dependent manner.
Fig. 3 10d induces apoptosis in Karpas299 cells. Karpas299 cells were treated with
10d (25 and 50 nM) or ceritinib (25 nM) for 24 h, then stained with Hoechst 33258 or
AO/EB and examined by fluorescence microscope.
3.4. Molecular docking studies
Simulating molecular dockings could comprehend the binding pattern between
ligand and protein intuitively. Based on the co-crystal structure of ceritinib with
ALKwt (PDB ID code: 4MKC)
19
and ROS1 (PDB ID code: 3ZBF),33 the

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conformations were conducted using Pymol and the 2D binding modes were
generated from Discovery Studio 2017 (Fig. 4).
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8
9
Structural comparisons in Fig. 4A exhibited that 10d and ceritinib occupied the
same active site as expected. From the 2D diagram of Fig. 4B and 4D, the structural
domain of 5-chloro-2,4-diaminopyrimidine skeleton formed hydrogen bond with
Met1199, hydrophobic interaction with Leu1196 and linked up to Val1130 and
Leu1256 by aryl interactions. Moreover, 10d containing pyrrolyl group increased the
effect of π-anion interaction with Asp1203. Compared with 9d bearing acetamido
group (Fig. 4E), the sulfonamido compound 10d engaged a new hydrogen bond onto
the backbone nitrogen of Lys1150.
As demonstrated in Fig. 4F, the main binding driving force was thought to be
matching between 10d and ROS1. Concretely, 10d was involved in the hydrophobic
interactions with Ala1978, Val1959, Leu2026 and Lys1980. In particular, the
pyrimidine and 4-NH formed hydrogen interaction with Met2029, as might be the
reason that 10d could enhance the ROS1 enzyme activity. Thus, the results from the
docking study revealed that compound 10d was in accordance with the SARs analysis
as an ALK and ROS1 inhibitor.
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Fig. 4 The binding models of ceritinib, 10d and 9d with ALK & ROS1. (A) Predicted
binding conformation for 10d (pink sticks) in the binding site cavity of ALKwt (PDB
ID code: 4MKC) and overlapping with ceritinib (blue sticks). (B) 2D diagram of the
interaction between ceritinib and ALK. (C) Predicted binding conformation of 10d in
the ATP binding site of ALK. (D) 2D diagram of the interaction between 10d and
ALK. (E) 2D diagram of the interaction between 9d and ALK. (F) Predicted binding
conformation of 10d in the ATP binding site of ROS1 (PDB ID code: 3ZBF).
4. Conclusion
In this paper, twenty-eight 2,4-diaminopyrimidine compounds (9a-9n and 10a-10n)
bearing pyrrolyl moiety were designed and synthesized as ALK and ROS1 dual

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inhibitors. Most compounds displayed promising predicted drug-likeness properties,
and exhibited moderate to excellent activity against Karpas299, H2228 and HCC78,
as well as off-target potency toward H460 and A549. The preliminary exploration on
a panel of cell lines indicated that compound 10d bearing sulfonamide (R1) and
4-methylpiperazinyl (R2) moiety was of great promising among the compounds with
IC50 values of 0.01, 0.08 and 0.042 μM against Karpas299, H2228 and HCC78. To
our delight, paralleling with cellular results, compound 10d showed outstanding
kinase potency on ALK, ALKL1196M and ROS1 with IC50 of 1.8, 4.3 and 3.6 nM,
respectively. Moreover, the Hoechst 33258 and AO/EB assay proved clearly that 10d
induced the cell apoptosis in a dose-dependent manner. Finally, molecular dockings
exemplified that 10d could foster potent affinity by forming significant hydrogen and
hydrophobic interactions with ALKwt (PDB ID code: 4MKC) and ROS1 (PDB ID
code: 3ZBF). In summary, compound 10d was regarded as a new potential inhibitor
and further studies would be discussed and reported in the future.
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5. Experimental procedures
5.1. 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 LCeMS (Agilent, Palo Alto, CA, USA). The H
and 13C NMR were performed using Bruker ARX-400 or ARX-600 spectrometers
(Bruker Bioscience, 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.
5.1.1. Preparation of N1-(2,5-dichloropyrimidin-4-yl)benzene-1,2-diamine (3)
Benzene-1,2-diamine (17.7 g, 0.16 mol) together with 2,4,5-trichloropyrimidine
(30.0 g, 0.16 mol) and DIPEA (54.0 mL, 0.33 mol) was added in isopropanol (300
mL). The reaction mixture was heated and stirred for 2 h at 80 °C. The mixture was
filtered immediately. The cake was resuspended in water and adjusted the pH to 7
with hydrochloric acid. The mixture was filtered after stirring for 30 min. The white
solid was collected to give 3 in a 96% yield after trituration in dichloromethane and
filtration. MS (ESI) m/z: 255.0 [M+H]+.
5.1.2. General procedure for preparation of compounds (4a-4b)
To a solution of 3 (20.0 g, 0.079 mol) in dichloromethane (100 mL) was added
trimethylamine (33.4 mL, 0.24 mol), and the mixture was reacted 2.5 h at room
temperature, after which substituted acyl chloride or sulfonyl chloride (7 mL, 0.094
mol) was added dropwise. The reaction mixture was evaporated under reduced
pressure whereafter water (200 mL) was poured into the mixture and stirring for 0.5 h.
The resulting solids were isolated by filtration and washed with water to give 4a-4b.
5.1.2.1. N-(2-((2,5-dichloropyrimidin-4-yl)amino)phenyl)acetamide (4a)

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Yield: 89.0%; MS (ESI) m/z: 297.0 [M+H]+.
5
6
7
5.1.2.2. N-(2-((2,5-dichloropyrimidin-4-yl)amino)phenyl)methanesulfonamide (4b)
Yield: 85.0%; MS (ESI) m/z: 333.2 [M+H]+.
8
9
5.1.3. Preparation of 1-(3-methoxy-4-nitrophenyl)-1H-pyrrole (7)
To a solvent of 4-fluoro-2-methoxy-1-nitrobenzene (20.0 g, 0.12 mol) in
N,N-dimethylformamide (100 mL) was added 1H-pyrrole (9 mL, 0.14 mol) and
K2CO3 (50.0 g, 0.36 mol). The reaction mixture was heated and stirred for 8 h at
100 °C. After cooling to room temperature, the reaction mixture was poured into
water (500 mL) and stirred for 1 h. The resulting solid was collected by filtration and
washed with water to obtain yellow solid 7 in an 75% yield. MS (ESI) m/z: 219.2
[M+H]+.
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5.1.4. Preparation of 2-methoxy-4-(1H-pyrrol-1-yl)aniline hydrochloride (8)
Iron powder (15.7 g, 0.28 mol) and hydrogen chloride (3.0 mL, 0.2 V/m) was
added to ethanol (95%, 200 mL). The resulting solution was heated to 70 °C and
stirred for 0.5 h. 7 was added to the suspension. The mixture was stirred for 4 h at
80 °C. After completion of reaction indicated by TLC, the mixture was filtered
through diatomite, and the filtrate was removed under reduced pressure. The residue
was added water (100 mL) and adjusted pH to 8 with sodium bicarbonate. The
solution was extracted with ethyl acetate two times (2×100 mL). The combined
organic layers were added 1,4-dioxane of hydrogen chloride and filtered to give the
white solid 8 yield 86%. MS (ESI) m/z: 189.1 [M+H]+.
5.1.5. General procedure for preparation of compounds (9a and 10a)
A mixture of 4a or 4b (0.03 mol) and 8 (9.0 g, 0.04 mol) in isopropanol (100 mL)
was refluxed for 24 h. The mixture was filtered immediately when TLC showed the
reaction was completed. The cake was resuspended in water and adjusted the pH to 7
with sodium bicarbonate. The mixture was extracted with DCM (2×100 mL). The
combined organic layers were evaporated under reduced pressure and purified by
column chromatography to gain the solid 9a and 10a.
5.1.5.1.
N-(2-((5-chloro-2-((2-methoxy-4-(1H-pyrrol-1-yl)phenyl)amino)pyrimidin-4-yl)amino
)phenyl)acetamide (9a)
Yield: 73.0%; M.p.: 202-205 ºC; MS (ESI) m/z: 449.6 [M+H]+; 1H-NMR (600 MHz,
DMSO-d6) δH: 10.03 (s, 1H), 8.55 (s, 1H), 8.14 (s, 1H), 7.90 (d, J = 8.5 Hz, 1H), 7.81
(s, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.35 (d, J = 7.9 Hz, 1H), 7.33 – 7.28 (m, 3H), 7.24
(dd, J = 11.1, 4.1 Hz, 1H), 7.16 (d, J = 2.4 Hz, 1H), 6.91 (dd, J = 8.6, 2.1 Hz, 1H),
6.25 (t, J = 2.1 Hz, 2H), 3.90 (s, 3H), 2.11 (s, 3H).
5.1.5.2.
N-(2-((5-chloro-2-((2-methoxy-4-(1H-pyrrol-1-yl)phenyl)amino)pyrimidin-4-yl)amino
)phenyl)methanesulfonamide (10a)
Yield: 69.0%; M.p.: 206-207 ºC; MS (ESI) m/z: 485.6 [M+H]+; 1H-NMR (600 MHz,
DMSO-d6) δH: 9.29 (s, 1H), 8.56 (s, 1H), 8.16 (s, 1H), 8.00 (s, 1H), 7.94 (d, J = 7.9

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Hz, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.40 (dd, J = 7.9, 1.3 Hz, 1H), 7.36 – 7.33 (m, 2H),
7.31 (d, J = 7.7 Hz, 1H), 7.26 – 7.21 (m, 1H), 7.17 (d, J = 2.3 Hz, 1H), 6.94 (dd, J =
8.6, 2.0 Hz, 1H), 6.27 – 6.22 (m, 2H), 3.89 (s, 3H), 2.96 (s, 3H) ; C-NMR (100
MHz, DMSO- d6) δc: 158.35, 156.36, 155.03, 151.02, 136.25, 134.74, 129.77, 127.50,
127.39, 126.29, 126.03, 125.68, 122.53, 119.73(2C), 111.31, 110.47(2C), 104.90,
103.85, 56.51, 39.52.
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5.1.6. General procedure for preparation of compounds (9b-9n and 10b-10n)
Various aliphatic amines (0.2 mmol) and formaldehyde (37%, 0.02 g, 0.3 mmol) in
acetic acid (5 mL) were stirred 0.5 h at room temperature. Then, 9a or 10a which was
solved in acetic acid (5 mL) was added slowly to the reaction mixture. After reacting
for 1 h, the solvent was evaporated under reduced pressure. The residue was adjusted
the pH to 8 with sodium bicarbonate. The mixture was extracted with DCM (2×100
mL). The combined organic layers were evaporated under reduced pressure and
purified by column chromatography to gain the solid 9b-9n and 10b-10n.
5.1.6.1.
N-(2-((5-chloro-2-((4-(2-((4-ethylpiperazin-1-yl)methyl)-1H-pyrrol-1-yl)-2-methoxyp
henyl)amino)pyrimidin-4-yl)amino)phenyl)acetamide (9b)
1
Yield: 59.0%; M.p.: 107-109 ºC; MS (ESI) m/z: 573.9 [M-H]-; H-NMR (400 MHz,
DMSO- d6) δH: 10.02 (s, 1H), 8.59 (s, 1H), 8.16 (s, 1H), 7.96 (d, J = 8.6 Hz, 1H), 7.78
(s, 1H), 7.72 (d, J = 7.3 Hz, 1H), 7.42 – 7.35 (m, 2H), 7.31 – 7.21 (m, 2H), 6.93 (s,
1H), 6.86 (d, J = 8.5 Hz, 1H), 6.13 (d, J = 3.2 Hz, 2H), 3.86 (s, 3H), 3.46 – 3.29 (m,
4H), 2.33 (s, 8H), 2.10 (s, 3H), 0.99 (t, J = 7.1 Hz, 3H); 13C-NMR (100 MHz, DMSO-
d6) δc: 170.09, 157.89, 156.57, 154.96, 148.96, 134.79, 132.11, 131.76, 128.86,
127.52, 127.48, 125.91, 125.64, 124.95, 122.76, 120.05, 116.66, 111.87, 108.58,
108.27, 105.17, 56.50, 53.75, 53.01, 52.91, 52.71, 52.66, 52.04, 23.58, 12.46.
5.1.6.2.
N-(2-((5-chloro-2-((2-methoxy-4-(2-(morpholinomethyl)-1H-pyrrol-1-yl)phenyl)amin
o)pyrimidin-4-yl)amino)phenyl)acetamide (9c)
1
Yield: 67.0%; M.p.: 189-191 ºC; MS (ESI) m/z: 546.8 [M-H]-; H-NMR (400 MHz,
DMSO- d6) δH: 10.02 (s, 1H), 8.60 (s, 1H), 8.16 (s, 1H), 7.97 (d, J = 8.5 Hz, 1H), 7.79
(s, 1H), 7.72 (d, J = 7.8 Hz, 1H), 7.39 (s, 1H), 7.37 (s, 1H), 7.28 (dd, J = 11.4, 3.9 Hz,
1H), 7.23 (t, J = 6.9 Hz, 1H), 6.94 (s, 1H), 6.85 (d, J = 8.4 Hz, 1H), 6.14 (d, J = 1.7
Hz, 2H), 3.86 (s, 3H), 3.56 (s, 4H), 3.28 (s, 2H), 2.36 (s, 4H), 2.10 (s, 3H); 13C-NMR
(100 MHz, DMSO- d6) δc: 170.09, 157.89, 156.59, 154.96, 148.97, 134.74, 132.15,
131.75, 128.38, 127.60, 127.50, 125.90, 125.63, 124.94, 122.92, 120.02, 116.74,
111.99, 108.61, 108.27, 105.18, 66.72 (2C), 56.49, 54.10, 53.27 (2C), 23.58.
5.1.6.3.
N-(2-((5-chloro-2-((2-methoxy-4-(2-((4-methylpiperazin-1-yl)methyl)-1H-pyrrol-1-yl)
phenyl)amino)pyrimidin-4-yl)amino)phenyl)acetamide (9d)
1
Yield: 49.0%; M.p.: 119-121 ºC; MS (ESI) m/z: 559.4 [M-H]-; H-NMR (400 MHz,
DMSO- d6) δH: 10.02 (s, 1H), 8.58 (s, 1H), 8.16 (d, J = 6.3 Hz, 1H), 7.95 (d, J = 8.6
Hz, 1H), 7.78 (s, 1H), 7.72 (d, J = 7.8 Hz, 1H), 7.38 (s, 1H), 7.36 (s, 1H), 7.28 (t, J =

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7.0 Hz, 1H), 7.23 (t, J = 7.2 Hz, 1H), 6.93 (s, 1H), 6.85 (d, J = 8.6 Hz, 1H), 6.16 –
6.09 (m, 2H), 3.84 (d, J = 9.0 Hz, 3H), 3.25 (s, 2H), 2.33 (s, 8H), 2.15 (s, 3H), 2.10 (s,
3H); 13C-NMR (100 MHz, DMSO- d6) δc: 170.93, 158.70, 157.38, 155.77, 149.78,
135.61, 132.91, 132.58, 129.68, 128.32, 128.29, 126.74, 126.46, 125.78, 123.57,
120.87, 117.47, 112.71, 109.37, 109.09, 105.96, 57.31, 56.09 (2C), 54.54, 53.45 (2C),
46.99, 24.37.
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5.1.6.4.
N-(2-((5-chloro-2-((2-methoxy-4-(2-(piperidin-1-ylmethyl)-1H-pyrrol-1-yl)phenyl)ami
no)pyrimidin-4-yl)amino)phenyl)acetamide (9e)
1
Yield: 63.0%; M.p.: 108-110 ºC; MS (ESI) m/z: 544.5 [M-H]-; H-NMR (400 MHz,
DMSO- d6) δH: 10.02 (s, 1H), 8.59 (s, 1H), 8.15 (s, 1H), 7.96 (d, J = 8.6 Hz, 1H), 7.78
(s, 1H), 7.71 (d, J = 7.9 Hz, 1H), 7.39 (d, J = 7.0 Hz, 1H), 7.37 (d, J = 7.8 Hz, 1H),
7.27 (dd, J = 11.4, 3.8 Hz, 1H), 7.22 (t, J = 6.9 Hz, 1H), 6.92 (s, 1H), 6.85 (dd, J =
8.6, 1.8 Hz, 1H), 6.14 – 6.08 (m, 2H), 3.86 (s, 3H), 3.22 (s, 2H), 2.33 (s, 4H), 2.09 (s,
3H), 1.47 (s, 4H), 1.39 (s, 2H).
5.1.6.5.
N-(2-((5-chloro-2-((2-methoxy-4-(2-((4-methylpiperidin-1-yl)methyl)-1H-pyrrol-1-yl)
phenyl)amino)pyrimidin-4-yl)amino)phenyl)acetamide (9f)
1
Yield: 73.0%; M.p.: 111-113 ºC; MS (ESI) m/z: 558.4 [M-H]-; H-NMR (400 MHz,
DMSO- d6) δH: 10.02 (s, 1H), 8.59 (s, 1H), 8.15 (s, 1H), 7.96 (d, J = 8.6 Hz, 1H), 7.78
(s, 1H), 7.72 (d, J = 7.0 Hz, 1H), 7.40 – 7.35 (m, 2H), 7.28 (td, J = 7.7, 1.4 Hz, 1H),
7.22 (td, J = 7.6, 1.3 Hz, 1H), 6.94 – 6.91 (m, 1H), 6.85 (dd, J = 8.6, 1.8 Hz, 1H), 6.15
– 6.08 (m, 2H), 3.85 (s, 3H), 3.24 (s, 2H), 2.83 (d, J = 10.6 Hz, 2H), 2.10 (s, 3H), 1.90
– 1.81 (m, 2H), 1.58 (d, J = 11.3 Hz, 2H), 1.32 (s, 1H), 1.08 – 1.01 (m, 2H), 0.89 (d, J
13
= 6.5 Hz, 3H); C-NMR (100 MHz, DMSO- d6) δc: 170.09, 157.90, 156.58, 154.96,
148.98, 134.81, 132.12, 131.78, 128.37, 127.52, 127.47, 125.88, 125.62, 124.96,
122.72, 120.05, 116.73, 111.84, 108.63, 108.27, 105.17, 65.37, 56.41, 54.12, 53.31,
34.46, 30.80, 23.57, 22.24, 15.63.
5.1.6.6.
N-(2-((5-chloro-2-((2-methoxy-4-(2-(pyrrolidin-1-ylmethyl)-1H-pyrrol-1-yl)phenyl)a
mino)pyrimidin-4-yl)amino)phenyl)acetamide (9g)
1
Yield: 67.0%; M.p.: 95-98 ºC; MS (ESI) m/z: 530.4 [M-H]-; H-NMR (400 MHz,
DMSO- d6) δH: 10.02 (s, 1H), 8.58 (s, 1H), 8.15 (s, 1H), 7.96 (d, J = 8.6 Hz, 1H), 7.77
(s, 1H), 7.71 (d, J = 7.9 Hz, 1H), 7.46 (s, 1H), 7.37 (d, J = 7.8 Hz, 1H), 7.27 (dd, J =
10.9, 4.4 Hz, 1H), 7.22 (t, J = 7.5 Hz, 1H), 6.93 – 6.89 (m, 1H), 6.86 (dd, J = 8.6, 1.9
Hz, 1H), 6.15 – 6.10 (m, 2H), 3.83 (s, 3H), 3.41 (s, 2H), 2.45 (s, 4H), 2.09 (s, 3H),
13
1.70 (s, 4H); C-NMR (100 MHz, DMSO- d6) δc: 170.08, 157.88, 156.59, 154.96,
148.97, 134.75, 132.15, 131.75, 130.43, 127.56, 127.42, 125.89, 125.61, 124.93,
122.31, 120.05, 116.44, 110.77, 108.51, 108.33, 105.12, 56.21, 53.65 (2C), 51.10,
23.57, 23.52 (2C).

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5.1.6.7.
N-(2-((5-chloro-2-((2-methoxy-4-(2-(thiomorpholinomethyl)-1H-pyrrol-1-yl)phenyl)a
6
7
8
mino)pyrimidin-4-yl)amino)phenyl)acetamide (9h)
1
Yield: 59.0%; M.p.: 96-98 ºC; MS (ESI) m/z: 562.4 [M-H]-; H-NMR (400 MHz,
DMSO- d6) δH: 10.01 (s, 1H), 8.59 (s, 1H), 8.15 (s, 1H), 7.96 (d, J = 8.6 Hz, 1H), 7.79
(s, 1H), 7.71 (d, J = 7.7 Hz, 1H), 7.39 – 7.35 (m, 1H), 7.28 (dd, J = 11.4, 3.8 Hz, 1H),
7.26 – 7.21 (m, 2H), 6.94 – 6.89 (m, 1H), 6.85 (dd, J = 8.6, 2.0 Hz, 1H), 6.14 – 6.10
(m, 2H), 3.86 (s, 3H), 3.30 (s, 2H), 2.57 (s, 4H), 2.55 (s, 4H), 2.09 (s, 3H); 13C-NMR
(100 MHz, DMSO- d6) δc: 170.09, 157.89, 156.59, 154.97, 148.97, 134.78, 132.15,
131.75, 128.70, 127.66, 127.51, 125.88, 125.64, 124.93, 123.03, 119.95, 117.07,
111.90, 108.71, 108.20, 105.16, 56.56, 54.48 (2C), 54.39, 27.77 (2C), 23.57.
9
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59
60
5.1.6.8.
N-(2-((5-chloro-2-((4-(2-((1,1-dioxidothiomorpholino)methyl)-1H-pyrrol-1-yl)-2-met
hoxyphenyl)amino)pyrimidin-4-yl)amino)phenyl)acetamide (9i)
1
Yield: 61.0%; M.p.: 144-147 ºC; MS (ESI) m/z: 594.5 [M-H]-; H-NMR (400 MHz,
DMSO- d6) δH: 9.99 (s, 1H), 8.60 (s, 1H), 8.16 (s, 1H), 7.97 (d, J = 8.5 Hz, 1H), 7.83
(s, 1H), 7.70 (d, J = 7.3 Hz, 1H), 7.38 (d, J = 7.7 Hz, 1H), 7.29 (t, J = 6.8 Hz, 1H),
7.26 – 7.21 (m, 1H), 6.97 (d, J = 1.9 Hz, 1H), 6.74 (d, J = 8.4 Hz, 1H), 6.08 (s, 2H),
3.83 (s, 3H), 3.35 (s, 2H), 2.94 (s, 4H), 2.69 (s, 4H), 2.09 (s, 3H).
5.1.6.9.
N-(2-((5-chloro-2-((4-(2-(((2-hydroxyethyl)amino)methyl)-1H-pyrrol-1-yl)-2-methoxy
phenyl)amino)pyrimidin-4-yl)amino)phenyl)acetamide (9j)
1
Yield: 71.0%; M.p.: 94-96 ºC; MS (ESI) m/z: 520.7 [M-H]-; H-NMR (400 MHz,
DMSO- d6) δH: 10.02 (s, 1H), 8.58 (s, 1H), 8.15 (s, 1H), 7.95 (d, J = 8.5 Hz, 1H), 7.79
(s, 1H), 7.71 (d, J = 7.8 Hz, 1H), 7.36 (d, J = 7.8 Hz, 1H), 7.33 (d, J = 2.1 Hz, 1H),
7.28 (dd, J = 11.4, 3.9 Hz, 1H), 7.23 (t, J = 6.9 Hz, 1H), 6.90 – 6.87 (m, 1H), 6.83
(dd, J = 8.6, 2.0 Hz, 1H), 6.15 (d, J = 1.5 Hz, 1H), 6.12 (t, J = 3.1 Hz, 1H), 4.51 (s,
1H), 3.85 (s, 3H), 3.60 (s, 2H), 3.45 (d, J = 4.6 Hz, 2H), 2.59 (t, J = 5.6 Hz, 2H), 2.09
(s, 3H).
5.1.6.10.
N-(2-((5-chloro-2-((4-(2-((diethylamino)methyl)-1H-pyrrol-1-yl)-2-methoxyphenyl)a
mino)pyrimidin-4-yl)amino)phenyl)acetamide (9k)
1
Yield: 53.0%; M.p.: 79-81 ºC; MS (ESI) m/z: 532.6 [M-H]-; H-NMR (400 MHz,
DMSO- d6) δH: 10.02 (s, 1H), 8.59 (s, 1H), 8.15 (s, 1H), 7.96 (d, J = 8.6 Hz, 1H), 7.78
(s, 1H), 7.71 (d, J = 7.8 Hz, 1H), 7.40 – 7.33 (m, 1H), 7.31 – 7.18 (m, 3H), 6.90 –
6.83 (m, 2H), 6.16 – 6.08 (m, 2H), 3.84 (s, 3H), 3.39 (s, 2H), 2.45 (d, J = 6.4 Hz, 4H),
2.09 (s, 3H), 0.85 (t, J = 7.1 Hz, 6H).
5.1.6.11.
N-(2-((5-chloro-2-((4-(2-((4-(2-hydroxyethyl)piperazin-1-yl)methyl)-1H-pyrrol-1-yl)-
2-methoxyphenyl)amino)pyrimidin-4-yl)amino)phenyl)acetamide (9l)
1
Yield: 63.0%; M.p.: 191-194 ºC; MS (ESI) m/z: 589.8 [M-H]-; H-NMR (400 MHz,
DMSO- d6) δH: 10.02 (s, 1H), 8.58 (s, 1H), 8.15 (s, 1H), 7.95 (d, J = 8.6 Hz, 1H), 7.78

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(s, 1H), 7.71 (d, J = 7.8 Hz, 1H), 7.39 – 7.34 (m, 2H), 7.28 (dd, J = 11.4, 3.8 Hz, 1H),
7.23 (dd, J = 11.3, 3.8 Hz, 1H), 6.95 – 6.90 (m, 1H), 6.85 (dd, J = 8.6, 1.9 Hz, 1H),
6.16 – 6.07 (m, 2H), 4.38 (s, 1H), 3.86 (s, 3H), 3.48 (dd, J = 11.0, 5.8 Hz, 2H), 3.25
(s, 2H), 2.46 – 2.29 (m, 10H), 2.09 (s, 3H).
9
5.1.6.12.
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14
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N-(2-((5-chloro-2-((4-(2-((dimethylamino)methyl)-1H-pyrrol-1-yl)-2-methoxyphenyl)
amino)pyrimidin-4-yl)amino)phenyl)acetamide (9m)
1
Yield: 43.0%; M.p.: 206-207 ºC; MS (ESI) m/z: 504.7 [M-H]-; H-NMR (400 MHz,
DMSO- d6) δH: 10.02 (s, 1H), 8.58 (s, 1H), 8.15 (s, 1H), 7.96 (d, J = 8.6 Hz, 1H), 7.78
(s, 1H), 7.71 (d, J = 6.8 Hz, 1H), 7.40 – 7.34 (m, 2H), 7.28 (td, J = 7.6, 1.4 Hz, 1H),
7.23 (td, J = 7.6, 1.4 Hz, 1H), 6.93 (t, J = 2.3 Hz, 1H), 6.87 (dd, J = 8.6, 2.0 Hz, 1H),
6.15 – 6.10 (m, 2H), 3.85 (s, 3H), 3.22 (s, 2H), 2.15 (s, 6H), 2.09 (s, 3H).
5.1.6.13.
N-(2-((5-chloro-2-((4-(2-((3-hydroxyazetidin-1-yl)methyl)-1H-pyrrol-1-yl)-2-methoxy
phenyl)amino)pyrimidin-4-yl)amino)phenyl)acetamide (9n)
1
Yield: 64.0%; M.p.: 114-117 ºC; MS (ESI) m/z: 532.6 [M-H]-; H-NMR (400 MHz,
DMSO- d6) δH: 10.03 (s, 1H), 8.58 (s, 1H), 8.15 (s, 1H), 7.94 (d, J = 8.5 Hz, 1H), 7.79
(s, 1H), 7.71 (d, J = 7.6 Hz, 1H), 7.37 (d, J = 7.7 Hz, 1H), 7.34 (d, J = 1.8 Hz, 1H),
7.31 – 7.27 (m, 1H), 7.26 – 7.21 (m, 1H), 6.91 – 6.89 (m, 1H), 6.85 (dd, J = 8.6, 1.7
Hz, 1H), 6.13 – 6.08 (m, 2H), 5.28 (d, J = 6.3 Hz, 1H), 4.19 (dd, J = 12.0, 6.0 Hz,
1H), 3.85 (s, 3H), 3.46 (t, J = 6.6 Hz, 2H), 3.40 (s, 2H), 2.75 (t, J = 6.5 Hz, 2H), 2.10
(s, 3H).
5.1.6.14.
N-(2-((5-chloro-2-((4-(2-((4-ethylpiperazin-1-yl)methyl)-1H-pyrrol-1-yl)-2-methoxyp
henyl)amino)pyrimidin-4-yl)amino)phenyl)methanesulfonamide (10b)
Yield: 45.0%; M.p.: 196-198 ºC; MS (ESI) m/z: 609.5 [M-H]-; 1H-NMR (400 MHz,
DMSO- d6) δH: 9.30 (s, 1H), 8.66 (s, 1H), 8.18 (s, 1H), 7.95 (s, 1H), 7.92 (d, J = 7.8
Hz, 1H), 7.87 (d, J = 8.6 Hz, 1H), 7.43 – 7.37 (m, 2H), 7.26 (dd, J = 11.5, 3.9 Hz,
1H), 7.24 – 7.19 (m, 1H), 6.97 – 6.93 (m, 1H), 6.89 (dd, J = 8.6, 1.9 Hz, 1H), 6.15 –
6.10 (m, 2H), 3.85 (s, 3H), 3.28 (s, 2H), 2.93 (s, 3H), 2.37 – 2.32 (m, 10H), 0.99 (t, J
= 7.1 Hz, 3H); 13C-NMR (100 MHz, DMSO- d6) δc: 158.12, 156.47, 155.02, 149.66,
135.24, 134.26, 130.78, 128.79, 127.41, 126.74 (2C), 126.11, 125.78, 122.81, 121.01,
116.60, 111.99, 108.65, 108.32, 105.16, 108.20, 105.16, 65.38, 56.47, 55.14 (2C),
53.68, 52.43 (2C), 45.94.
3.1.6.15.
5.1.6.15.
N-(2-((5-chloro-2-((2-methoxy-4-(2-(morpholinomethyl)-1H-pyrrol-1-yl)phenyl)amin
o)pyrimidin-4-yl)amino)phenyl)methanesulfonamide (10c)
1
Yield: 73.0%; M.p.: 199-202 ºC; MS (ESI) m/z: 582.4 [M-H]-; H-NMR (400 MHz,
DMSO- d6) δH: 9.30 (s, 1H), 8.62 (s, 1H), 8.19 (s, 1H), 7.95 (s, 1H), 7.92 – 7.84 (m,
2H), 7.44 – 7.39 (m, 2H), 7.31 (t, J = 7.0 Hz, 1H), 7.24 (t, J = 6.9 Hz, 1H), 6.95 (t, J =
2.2 Hz, 1H), 6.88 (dd, J = 8.6, 1.8 Hz, 1H), 6.16 – 6.11 (m, 2H), 3.85 (s, 3H), 3.55 (s,

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4H), 3.29 (s, 2H), 2.95 (s, 3H), 2.36 (s, 4H); C-NMR (100 MHz, DMSOD-OdI:61)0.1δ0c3:9/C9NJ05980F
158.08, 156.57, 155.08, 149.61, 135.18, 134.42, 130.36, 128.33, 127.44, 127.25,
127.14, 126.55, 125.93, 122.93, 120.91, 116.64, 112.08, 108.67, 108.31, 105.14,
66.71(2C), 56.44(2C), 54.11 , 53.27(2C).
9
5.1.6.16.
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N-(2-((5-chloro-2-((2-methoxy-4-(2-((4-methylpiperazin-1-yl)methyl)-1H-pyrrol-1-yl)
phenyl)amino)pyrimidin-4-yl)amino)phenyl)methanesulfonamide (10d)
1
Yield: 53.0%; M.p.: 191-193 ºC; MS (ESI) m/z: 595.4 [M-H]-; H-NMR (400 MHz,
DMSO- d6) δH: 9.32 (s, 1H), 8.65 (s, 1H), 8.18 (s, 1H), 7.95 (s, 1H), 7.92 (d, J = 7.7
Hz, 1H), 7.87 (d, J = 8.6 Hz, 1H), 7.43 – 7.37 (m, 2H), 7.27 (dd, J = 12.3, 5.8 Hz,
1H), 7.22 (t, J = 6.9 Hz, 1H), 6.95 (d, J = 1.9 Hz, 1H), 6.92 – 6.84 (m, 1H), 6.17 –
6.13 (m, 2H), 3.85 (s, 3H), 3.27 (s, 2H), 2.93 (s, 3H), 2.36 (s, 8H), 2.18 (s, 3H).
5.1.6.17.
N-(2-((5-chloro-2-((2-methoxy-4-(2-(piperidin-1-ylmethyl)-1H-pyrrol-1-yl)phenyl)ami
no)pyrimidin-4-yl)amino)phenyl)methanesulfonamide (10e)
1
Yield: 51.0%; M.p.: 178-181 ºC; MS (ESI) m/z: 580.4 [M-H]-; H-NMR (400 MHz,
DMSO- d6) δH: 9.29 (s, 1H), 8.62 (s, 1H), 8.18 (s, 1H), 7.95 (s, 1H), 7.91 (d, J = 7.9
Hz, 1H), 7.87 (d, J = 8.6 Hz, 1H), 7.44 – 7.37 (m, 2H), 7.29 (dd, J = 11.5, 3.9 Hz,
1H), 7.25 – 7.20 (m, 1H), 6.96 – 6.92 (m, 1H), 6.88 (dd, J = 8.6, 2.0 Hz, 1H), 6.16 –
6.09 (m, 2H), 3.85 (s, 3H), 3.26 (s, 2H), 2.94 (s, 3H), 2.34 (s, 4H), 1.46 (d, J = 4.6 Hz,
4H), 1.39 (s, 2H); 13C-NMR (100 MHz, DMSO- d6) δc: 158.10, 156.51, 155.05,
149.62, 135.24, 134.38, 130.49, 128.90, 127.41, 127.05, 126.99, 126.33, 125.83,
122.78, 120.95, 116.69, 111.99, 108.73, 108.31, 105.15, 65.38, 56.39, 54.39,
53.87(2C), 25.94 (2C), 24.41.
5.1.6.18.
N-(2-((5-chloro-2-((2-methoxy-4-(2-((4-methylpiperidin-1-yl)methyl)-1H-pyrrol-1-yl)
phenyl)amino)pyrimidin-4-yl)amino)phenyl)methanesulfonamide (10f)
Yield: 67.0%; M.p.: 106-108 ºC; MS (ESI) m/z: 596.8 [M+H]+; 1H-NMR (400 MHz,
DMSO- d6) δH: 9.29 (s, 1H), 8.62 (s, 1H), 8.18 (s, 1H), 7.96 (s, 1H), 7.91 (d, J = 7.9
Hz, 1H), 7.87 (d, J = 8.5 Hz, 1H), 7.44 – 7.38 (m, 2H), 7.29 (t, J = 7.0 Hz, 1H), 7.23
(t, J = 6.9 Hz, 1H), 6.94 (d, J = 2.1 Hz, 1H), 6.88 (dd, J = 8.6, 1.9 Hz, 1H), 6.16 –
6.09 (m, 2H), 3.85 (s, 3H), 3.27 (s, 2H), 2.94 (s, 3H), 2.84 (d, J = 11.3 Hz, 2H), 1.88
(t, J = 12.0 Hz, 2H), 1.58 (d, J = 11.8 Hz, 2H), 1.32 (s, 1H), 1.09 – 1.00 (m, 2H), 0.88
(d, J = 6.5 Hz, 3H).
5.1.6.19.
N-(2-((5-chloro-2-((2-methoxy-4-(2-(pyrrolidin-1-ylmethyl)-1H-pyrrol-1-yl)phenyl)a
mino)pyrimidin-4-yl)amino)phenyl)methanesulfonamide (10g)
1
Yield: 37.0%; M.p.: 97-99 ºC; MS (ESI) m/z: 566.4 [M-H]-; H-NMR (400 MHz,
DMSO- d6) δH: 9.38 (s, 1H), 8.65 (s, 1H), 8.18 (s, 1H), 7.95 (s, 1H), 7.90 (d, J = 7.8
Hz, 1H), 7.87 (d, J = 8.6 Hz, 1H), 7.48 (d, J = 2.0 Hz, 1H), 7.41 (dd, J = 7.7, 1.4 Hz,
1H), 7.28 (td, J = 7.7, 1.4 Hz, 1H), 7.22 (td, J = 7.6, 1.4 Hz, 1H), 6.95 – 6.92 (m, 1H),

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6.88 (dd, J = 8.6, 2.0 Hz, 1H), 6.19 – 6.09 (m, 2H), 3.83 (s, 3H), 3.46 (s, 2H),D2O.I:9140.1(0s3,9/C9NJ05980F
3H), 2.48 (s, 4H), 1.70 (s, 4H).
6
7
5.1.6.20.
8
9
N-(2-((5-chloro-2-((2-methoxy-4-(2-(thiomorpholinomethyl)-1H-pyrrol-1-yl)phenyl)a
mino)pyrimidin-4-yl)amino)phenyl)methanesulfonamide (10h)
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11
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1
Yield: 42.0%; M.p.: 106-109 ºC; MS (ESI) m/z: 598.4 [M-H]-; H-NMR (400 MHz,
DMSO- d6) δH: 9.30 (s, 1H), 8.62 (s, 1H), 8.19 (s, 1H), 7.96 (s, 1H), 7.90 (d, J = 8.4
Hz, 1H), 7.87 (d, J = 8.6 Hz, 1H), 7.41 (dd, J = 7.8, 1.3 Hz, 1H), 7.31 (t, J = 7.0 Hz,
1H), 7.28 – 7.21 (m, 2H), 6.95 – 6.92 (m, 1H), 6.87 (dd, J = 8.6, 1.9 Hz, 1H), 6.15 –
6.09 (m, 2H), 3.86 (s, 3H), 2.95 (s, 3H), 2.58 (d, J = 5.4 Hz, 4H), 2.55 (s, 4H).
5.1.6.21.
N-(2-((5-chloro-2-((4-(2-((1,1-dioxidothiomorpholino)methyl)-1H-pyrrol-1-yl)-2-met
hoxyphenyl)amino)pyrimidin-4-yl)amino)phenyl)methanesulfonamide (10i)
Yield: 79.0%; M.p.: 116-119 ºC; MS (ESI) m/z: 654.8 [M+Na]+; 1H-NMR (400 MHz,
DMSO- d6) δH: 9.30 (s, 1H), 8.62 (s, 1H), 8.18 (s, 1H), 7.97 (s, 1H), 7.92 – 7.84 (m,
2H), 7.41 (dd, J = 7.8, 1.3 Hz, 1H), 7.32 (t, J = 7.0 Hz, 1H), 7.26 (dd, J = 10.8, 4.4
Hz, 1H), 7.20 (d, J = 2.1 Hz, 1H), 6.97 – 6.93 (m, 1H), 6.87 (dd, J = 8.6, 1.9 Hz, 1H),
6.18 (d, J = 1.6 Hz, 1H), 6.15 (t, J = 3.1 Hz, 1H), 3.86 (s, 3H), 3.51 (s, 2H), 3.02 (s,
4H), 2.95 (s, 3H), 2.83 (s, 4H).
5.1.6.22.
N-(2-((5-chloro-2-((4-(2-(((2-hydroxyethyl)amino)methyl)-1H-pyrrol-1-yl)-2-methoxy
phenyl)amino)pyrimidin-4-yl)amino)phenyl)methanesulfonamide (10j)
1
Yield: 64.0%; M.p.: 159-162 ºC; MS (ESI) m/z: 556.7 [M-H]-; H-NMR (400 MHz,
DMSO- d6) δH: 8.65 (s, 1H), 8.18 (s, 1H), 7.96 (s, 1H), 7.92 – 7.85 (m, 2H), 7.43 –
7.38 (m, 2H), 7.30 – 7.25 (m, 1H), 7.23 (t, J = 7.4 Hz, 1H), 6.96 (s, 1H), 6.93 – 6.89
(m, 1H), 6.18 (d, J = 1.6 Hz, 1H), 6.15 (t, J = 3.1 Hz, 1H), 4.21 (s, 2H), 3.84 (s, 3H),
13
3.65 (t, J = 6.8 Hz, 2H), 3.51 (s, 2H), 2.97 – 2.91 (m, 5H); C-NMR (100 MHz,
DMSO- d6) δc: 158.09, 156.55, 155.02, 149.67, 135.08, 134.24, 130.80, 130.24,
127.51, 126.85, 126.84, 126.74, 126.33, 125.86, 122.94, 120.98, 116.64, 111.25,
108.72, 108.41, 105.16, 65.38, 62.83, 56.38, 51.74, 48.84.
5.1.6.23.
N-(2-((5-chloro-2-((4-(2-((diethylamino)methyl)-1H-pyrrol-1-yl)-2-methoxyphenyl)a
mino)pyrimidin-4-yl)amino)phenyl)methanesulfonamide (10k)
1
Yield: 81%; M.p.: 138-141 ºC; MS (ESI) m/z: 568.3 [M-H]-; H-NMR (400 MHz,
DMSO- d6) δH: 9.31 (s, 1H), 8.63 (s, 1H), 8.18 (s, 1H), 7.95 (s, 1H), 7.91 – 7.86 (m,
2H), 7.44 – 7.38 (m, 1H), 7.32 – 7.19 (m, 3H), 6.91 – 6.86 (m, 2H), 6.15 – 6.12 (m,
2H), 3.84 (s, 3H), 3.45 (s, 2H), 2.94 (s, 3H), 2.50 – 2.43 (m, 4H), 0.85 (t, J = 7.1 Hz,
6H).
5.1.6.24.
N-(2-((5-chloro-2-((4-(2-((4-(hydroxymethyl)piperazin-1-yl)methyl)-1H-pyrrol-1-yl)-2
-methoxyphenyl)amino)pyrimidin-4-yl)amino)phenyl)methanesulfonamide (10l)

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1
Yield: 48%; M.p.: 109-112 ºC; MS (ESI) m/z: 625.8 [M-H]-; H-NMR (400 MHz,
DMSO- d6) δH: 9.30 (s, 1H), 8.64 (s, 1H), 8.18 (s, 1H), 7.95 (s, 1H), 7.91 (d, J = 7.8
Hz, 1H), 7.87 (d, J = 8.5 Hz, 1H), 7.43 – 7.38 (m, 2H), 7.31 – 7.25 (m, 1H), 7.23 (dd,
J = 12.0, 4.5 Hz, 1H), 6.96 – 6.93 (m, 1H), 6.89 (dd, J = 8.6, 1.9 Hz, 1H), 6.15 – 6.10
(m, 2H), 4.40 (s, 1H), 3.85 (s, 3H), 3.49 (s, 2H), 3.27 (s, 2H), 2.94 (s, 3H), 2.46 – 2.35
(m, 10H); 13C-NMR (100 MHz, DMSO- d6) δc: 158.11, 156.49, 155.03, 149.64,
135.23, 134.30, 130.68, 128.80, 127.41, 126.84, 126.21, 125.82, 122,80, 120.97,
116.61, 111.95, 108.67, 108.32, 105.16, 65.38, 65.56, 58.77, 56.48(2C), 53.73, 53.66
(2C), 52.55.
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5.1.6.25.
N-(2-((5-chloro-2-((4-(2-((dimethylamino)methyl)-1H-pyrrol-1-yl)-2-methoxyphenyl)
amino)pyrimidin-4-yl)amino)phenyl)methanesulfonamide (10m)
1
Yield: 58%; M.p.: 165-168 ºC; MS (ESI) m/z: 540.8 [M-H]-; H-NMR (400 MHz,
DMSO- d6) δH: 9.14 (s, 1H), 8.16 – 8.08 (m, 2H), 8.02 – 7.94 (m, 2H), 7.44 (d, J = 2.1
Hz, 1H), 7.26 (dd, J = 7.9, 1.1 Hz, 1H), 7.03 – 6.91 (m, 3H), 6.82 (t, J = 7.2 Hz, 1H),
6.17 – 6.09 (m, 2H), 3.86 (s, 3H), 3.24 (s, 2H), 2.15 (s, 6H), 1.89 (s, 3H).
5.1.6.26.
N-(2-((5-chloro-2-((4-(2-((3-hydroxyazetidin-1-yl)methyl)-1H-pyrrol-1-yl)-2-methoxy
phenyl)amino)pyrimidin-4-yl)amino)phenyl)methanesulfonamide (10n)
1
Yield: 53%; M.p.: 232-235 ºC; MS (ESI) m/z: 568.6 [M-H]-; H-NMR (400 MHz,
DMSO- d6) δH: 9.33 (s, 1H), 8.63 (s, 1H), 8.18 (s, 1H), 7.97 (s, 1H), 7.90 (d, J = 7.5
Hz, 1H), 7.85 (d, J = 8.5 Hz, 1H), 7.41 (d, J = 7.7 Hz, 1H), 7.35 (d, J = 1.8 Hz, 1H),
7.30 (t, J = 7.1 Hz, 1H), 7.24 (t, J = 7.0 Hz, 1H), 6.92 (s, 1H), 6.88 (dd, J = 8.6, 1.7
Hz, 1H), 6.16 – 6.09 (m, 2H), 5.30 (d, J = 6.3 Hz, 1H), 4.22 – 4.14 (m, 1H), 3.85 (s,
3H), 3.52 – 3.43 (m, 4H), 2.94 (s, 3H), 2.79 (t, J = 6.7 Hz, 2H).
5.2. Physical properties and MTT assay in vitro
Physical properties such as Log P and topological polar surface area (TPSA) were
calculated for each compounds using Discovery Studio 2017 and SwissADME
(http://swissadme.ch/index.php#).
The cytotoxic activities of compounds 9a-9n and 10a-10n were evaluated against
Karpas299, H2228, HCC78, A549 and H460 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 incubated in 5% CO2 at 37 °C for 24 h. The tested
compounds at the indicated final 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 μM, and incubated with 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 IC50 (inhibitory concentration 50%),

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were the averages of three determinations and calculated relative to theDOvIe: 1h0i.1c0l3e9/C9NJ05980F
(DMSO) control by the Bacus Laboratories Incorporated Slide Scanner (Bliss)
software.
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5.3. In vitro enzymatic assays
The in vitro enzymatic assays versus ALK, ALKL1196M, ROS1 and EGFR 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 MgCl2, 2 mM DTT), with blank DMSO solution as the
negative control. The kinase reaction was initiated by the addition of tyrosine kinase
proteins diluted in 39 μL of kinase reaction buffer solution and incubated at 28 °C for
1 h. And then add 25 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. IC50 values were calculated from the inhibition curves.
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5.4. Morphology analysis of apoptotic cells and nuclear morphology assays
Apoptotic morphological changes of Karpas299 cells was detected by AO/EB
staining. Briefly, cells were seeded in six-well plates for 24 h, and then treated with
ceritinib (25 nM) or different concentrations (25 nM and 50 nM) of 10d 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 a quarter. The stained cells were washed twice
with PBS and observed by fluorescence microscope.
To investigate the apoptotic morphological changes of nuclear chromatin in
Karpas299 cells, Hoechst 33258 staining was performed. In brief, cells were seeded in
six-well plates overnight, and then treated with ceritinib (50 nM) or different
concentrations (25 nM and 50 nM) of 10d. After 48 h, the medium was discarded and
the cells were washed with PBS. After that, the cells were stained with Hoechst 33258
solution for 15 min. The stained cells were washed twice with PBS and observed by
fluorescence microscope.
5.5. Molecular docking
The molecular docking was performed with Accelrys Discovery Studio 2017. The
protein files (PDB ID code: 4MKC and 3ZBF) were obtained from Protein Data Bank
(http://www.rcsb.org/pdb/). During the docking process, the protein was prepared
through several steps, such as standardization of atom names and insertion of missing
atoms in residues. Then, the protein model was typed with the CHARMm force field
and a binding sphere within 50 Å radius was defined as the binding site around the
reference ligand (ceritinib). Ceritinib, and were drawn with ChemBioDraw 3D and
fully minimized using the CHARMm force field. Finally, they were searching for
possible conformations in the binding site using the CDOCKER protocol with default
settings. The binding results were viewed using Discovery Studio 2017 and the 2D
binding mode figures were generated from it. All 3D figures were generated from
Pymol (The Pymol Molecular Graphics System, Version 1.4.1. Schrodinger, LLC).

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Conflict of interest
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The authors have declared no conflict of interest.
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Acknowledgements
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This work was supported by National Natural Science Foundation of China
(No.81673308), Youth Backbone Talent Training Project of Shenyang
Pharmaceutical University (No. ZQN2018008) and Development Project of Ministry
of Education Innovation Team (No. IRT1073).
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