Discovery and antitumor activity of Benzo[d]imidazol-containing 2,4-diarylaminopyrimidine analogues as ALK inhibitors with mutation-combating effects

Article abstract of DOI:10.1016/j.bmc.2021.116108

To address drug resistance caused by ALK kinase mutations, a series of novel 2,4-diarylaminopyrimidine (DAAP) analogues were designed by incorporating 1H-benzo[d]imidazol motif onto the maternal framework. All compounds were efficiently synthesized and an

Full text of DOI:10.1016/j.bmc.2021.116108

Bioorg. Med. Chem. 37 (2021) 116108
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery and antitumor activity of Benzo[d]imidazol-containing 2,4-dia-
rylaminopyrimidine analogues as ALK inhibitors with
mutation-combating effects
,
,*
Zheng Lia, Ming Guob, Meng Caob, Tianming Zhaob, Mingzhu Lia *, Xin Zhaib
a Department of Integrated Traditional Chinese and Western Medicine Medical Oncology, Cancer Hospital of China Medical University, Liaoning Cancer Hospital &
Institute, Shenyang 110042, China
b Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
To address drug resistance caused by ALK kinase mutations, a series of novel 2,4-diarylaminopyrimidine (DAAP)
analogues were designed by incorporating 1H-benzo[d]imidazol motif onto the maternal framework. All com-
pounds were efficiently synthesized and antiproliferative activities against Karpas299, H2228 and A549 cell lines
were evaluated by MTT assay. Delightly, the most promising derivative H-11 was detected with IC50 values of
ALK
2,4-Diarylaminopyrimidine
Synthesis
G1202R mutant
Antitumor evaluations
0.016
μM and 0.099 μM against ALK- positive Karpas299 and H2228 cells. Meanwhile, H-11 displayed
encouraging enzymatic inhibitory potency with IC50 values of 2.7 nM, 3.8 nM and 5.7 nM toward ALKWT
,
ALKL1196M and ALKG1202R, respectively. Ultimately, the binding modes of optimal H-11 with ALK wild-type and
mutants were ideally established which further confirmed the structural basis in accordance with the SARs
analysis.
1. Introduction
the two prominent site-mutations.19,20 Accordingly, it is urgently
required to find a candidate with mutant-combating effects.
Anaplastic lymphoma kinase (ALK), a member of insulin receptor
(IR) superfamily with 1620 amino acid receptor tyrosine kinase, has
received extensive attention recently for target-based therapy.1,2 Among
nearly 30 fusion proteins that have been discovered, nucleophosmin
(NPM)-ALK in anaplastic large cell lymphoma (ALCL) and echinoderm
microtubule-associated protein-like-4 (EML4)-ALK in nonsmall-cell lung
cancer (NSCLC) are two common formalizations.3–6 The over-expression
of ALK fusion proteins could activate its related signal pathways, which
in turn induce the malignant growth of cells, the imbalance of apoptosis
regulation and tumorigenesis.7,8 Accumulating evidence suggests that
blockade of different signaling cascades of ALK are effective in inhibiting
the amplification of ALK-positive cells.9
Ceritinib, the second generation ALK inhibitor, is known to be active
against most of the Crizotinib-resistant mutants except ALKG1202R 7
.
Taking Ceritinib as the lead, a dedicated analysis to co-crystal structure
of Ceritinib with ALK protein was carried out (Fig. 2B). The results
revealed that 5-Cl-2,4-diarylaminopyrimidine (DAAP) skeleton occu-
pied the kinase domain and formed key hydrogen bonds with ALK res-
idues. Thus, as a fundamental pharmacophore, DAAPs was retained to
guarantee the on-target activity in the new design strategy. Further-
more, it had been reported that Ceritinib was ineffective for ALKG1202R
mutation due to large steric hindrance of isopropoxy moiety. In previous
study, a chemistry entry 10d was obtained by substituting isopropoxy
with tiny methoxy group and incorporating pyrrolyl motif (Fig. 2A).21
To our disappointment, 10d has been proved to be active against
ALKL1196M, but its unsatisfying potency in G1202R mutation left an
intractable problem behind. Accordingly, in order to further explore the
capacity of isopropoxy and improve functional group diversity, we
creatively removed the isopropoxy group, and instead embedded a
benzo[d]imidazole moiety to DAAP framework according to the scaffold
hopping strategy. Furthermore, the co-crystal structure of Ceritinib
Since ALK was regarded as the most promising drug target in
oncology, small-molecular inhibitors such as Crizotinib,10,11 Cer-
itinib,12,13 Alectinib,14 Brigatinib15,16 and Lorlatinib17 have been iden-
tified and launched recently (Fig. 1). Unfortunately, the inevitable
development of drug resistance mutation is the most common obstacle
to their clinical application.18 A number of mutations have been
detected in ALK kinase domain, among which L1196M and G1202R are
* Corresponding authors.
E-mail addresses: limingzhu198600@126.com (M. Li), zhaixin_syphu@126.com (X. Zhai).
https://doi.org/10.1016/j.bmc.2021.116108
Received 21 January 2021; Received in revised form 2 March 2021; Accepted 6 March 2021
Available online 14 March 2021
0968-0896/© 2021 Elsevier Ltd. All rights reserved.

Z. Li et al.
Bioorganic & Medicinal Chemistry 37 (2021) 116108
indicated that the isopropyl sulfony group ‘head’ could exhibit an
important hydrogen and hydrophobic effect, therefore, four blocks
(methylsulfonyl amino, acetyl amino, methylcarbamoyl and dimethy-
lamino acyl) were introduced in the light of bioisosterism principle to
ensure or even enrich the interaction, as shown in Fig. 2D.22 In addition,
versatile alicyclic amines as solubilizing ‘tail’ were integrated into the
benzo[d]imidazole motif to further explore the structure–activity re-
lationships (SARs).
2.2. Biological activity and discussion
2.2.1. Common properties of target compounds
Ahead of the cell-based biological evaluation, the druggability of the
compounds was assessed by the prediction of several key parameters.
Wherein, physical properties such as the lipophilic quantity (cLogP) and
the topological polar surface area (tPSA) were considered as essential
indicators for druggability.26 In this paper, cLogP and tPSA of new
synthetic chemical entities (H-1–H-28) were predicated using Molsoft
(http://molsoft.com/mprop/), and data was outlined in Table 1. As
shown, most compounds with cLogP of less than 5 and tPSA of less than
120 Å2 revealed the better feasibility. Additionally, we focus on evalu-
ating the drug-likeness score through an empirical model. Theoretically,
the drug-likeness score was expected the bigger the better. With Cer-
itinib (scored 0.55) as the reference point, we present the prediction
results of these inhibitors in Table 1, as well as the prediction specifi-
cations of the optimal H-8 (scored 1.24) and H-11 (scored 1.61) were
exhibited in Fig. 3. Overall, almost all compounds were detected >0.55
drug-likeness score which further elucidated their druggability.
In this sense, a variety of novel 2,4-diarylaminopyrimidine de-
rivatives containing benzo[d]imidazole fragment (H-1–H-28) were
designed and synthesized. All compounds were tested cytotoxicity ef-
fects on three cancer cell lines which showed a selectivity profile on
ALK-positive cell lines over other cells. Subsequently, six prominent hits
were picked into further enzymatic assays towards ALK, ALKL1196M
,
ALKG1202R, EGFR and FLT3 kinases. Ultimately, the possible binding
modes of optimal compound H-11 with ALK protein were also
performed.
2. Results and discussion
2.1. Chemistry
2.2.2. In vitro anti-proliferative activity and SARs study
The anti-proliferative activities of synthesized compounds against
three human cancer cell lines including NPM-ALK-addicted Karpas299
(Human NSCLC cell line), EML4-ALK-addicted H2228 (Human NSCLC
cell line) as well as the off-target cells A549 (Human EGFR-positive
NSCLC cell line) were evaluated by MTT assay in vitro. Ceritinib was
used as the positive control and results expressed as IC50 values were
shown in Table 1.
All compounds (H-1–H-28) were synthesized as outlined in Scheme
1. Firstly, the commercially available 2,4,5-trichloropyrimidine and
benzene-1,2-diamine were involved in N-alkylation reaction to provide
intermediate A in 84.5% yield.23 Taking the compound A as a synthon,
acylation reaction was implemented with sulfonyl chloride or acetyl
chloride to give C-1 or C-2 in satisfying results. Isatin anhydride reacted
with methylamine or dimethylamine in ethanol to yield intermediate B-
1 or B-2.24 Next, alkylation reaction of B-1 or B-2 with 2,4,5-trichloro-
pyrimidine in the presence of N,N-diisopropylethylamine (DIPEA) in
isopropanol give birth to C-3 or C-4 in yields of 85.9% and 80.2%,
repectively.
As shown in Table 1, most of target compounds displayed acceptable
cytotoxic activities against Karpas299 and H2228 with IC50 values
below 1 μM, indicating that introduce of 1H-benzo[d]imidazol instead
of isopropoxyphenyl group could produce a positive effect on anti-
proliferation of ALK-addicted cancer cell lines. Among which, chemi-
cal entity H-11 bearing dimethylamino acyl ‘head’ and 4-methylpipera-
zinyl solubilizing ‘tail’ exhibited the most promising activities against
The 4-nitrobenzene-1,2-diamine (D) was condensed with 2-chloroa-
ceic acid in the company of hydrochloric acid to afford the key inter-
mediate E, which was recrystallized from methanol in 82.3% yield.25
With the benzo[d]imidazole skeleton E in hand, a alkylation reaction
was conducted with treatment of corresponding alicyclic amines in
75–97% yields to furnish the amine products F-1–F-12. Subsequently,
the nitro group of F-1–F-12 were reduced under 80% hydrazine hydrate
in ethanol to generate pivotal amino derivatives G-1–G-12 in acceptable
yields. Eventually, G-1–G-12 were reacted with key intermediates C-
1–C-4 driven by p-TsOH to result in target compounds H-1–H-28 in
moderate yields.
Karpas299 and H2228 cells with the IC50 values of 0.016
μM and 0.099
μ
M, superior to Ceritinib (0.031 μM and 0.107 μM). Meanwhile, ma-
jority of derivatives has poor performances in A549 cell line as expected,
which suggested on-target potency of these compounds.
To determine effects of the R1 moiety, four different ‘head’ were
intergrated by bioisosterism principle. In view of the excellent perfor-
mances of compound 10d in previous research21, methylsulfonyl amino
was introduced into the 2,4-diarylaminopyrimidine skeleton firstly. As
shown, analogues H-1, H-5, H-9 and H-12 bearing methansulfonyl
NH
NH
O
Cl
Cl
N
N
H
N
N
N
F
N
H
N
N
H
N
Cl
O
O
S
O
O
NC
N
O
H2N
N
Crizotinib
(launched 2011)
Ceritinib
Alectinib
(launched 2014)
(launched 2014)
O
N
N
N
F
N
N
Cl
N
N
O
N
H
N
N
H
P
O
H2N
O
N
Brigatinib
(launched 2017)
Lorlatinib
(launched 2018)
Fig. 1. Structures of launched ALK inhibitors.
2

Z. Li et al.
Bioorganic & Medicinal Chemistry 37 (2021) 116108
amino motif exhibited acceptable activities against Karpass299 with
molecular H-24 against Karpas299 and H2228 cells were 22.7-fold and
5.0-fold increase, emphasizing the volume of the hydrophilic groups
may affect the activity to a certain extent. In addition, morpholine and
thiomorpholine were utilized to explore the impact of 4-position atom.
To our disappointed, compared with H-18 and H-25, H-19–H-20 and H-
26–H-27 showed depressed anti-proliferative effects with IC50 values of
IC50 values of 1.03, 0.056, 0.053 and 0.136 μM, respectively. Mean-
while, the replacement of methylsulfonyl amino with acetyl amino
group gave rise to H-2, H-6, H-10 and H-13, leading to a decrease in
activity. Sequentially, an amide-bond reversal strategy was ultilized for
the enhance activity. Delightly, compounds H-7 and H-3 bearing
methylcarbamoyl moiety displayed cytotoxicity equal or superior to
molecules bearing methylsulfonyl amido ‘head’, represented by H-1, H-
5, H-14 and H-12. To our surprise, the slightly improved potency was
observed by shift methylcarbamoyl to dimethylamino acyl group. For
instance, the potency of chemical entities H-4 and H-8 against ALK-
addicted Karpas299 were 1.3- and 1.6-fold increase of H-3 and H-7.
Given the fact that the set of compounds containing methylsulfonyl
amino and dimethylamino acyl showed better anti-proliferative potency
on the whole, both ‘head’ were used for SARs discussion of the ‘tail’
further.
0.789 μM, 1.84 μM, 0.833 μM and 1.29 μM toward ALK-addicted Kar-
pas299, demonstrated oxygen and sulfur atom of 4-position was unfit for
potency. Likewise, in H2228 cell line, this tread has also been noticed.
2.2.3. In vitro enzymatic assay
In view of the promising activity of target compounds for ALK
addicted cell lines, six optimal hits were chosen for further molecular-
level studies with ALKWT, ALKL1196M and ALKG1202R kinases in vitro. In
the meantime, the no-relative EGFR and FLT3 kinases were selected to
elucidated the kinase selectivity. Ceritinib was used as the positive
control and results were outlined in Table 2.
Owning to an occupation by R2 moiety, the solvent region tolerated
to further modification with various aliphatic amines. Pharmacological
data indicated that variation of the amino ‘tail’ did change the cyto-
toxicity corresponding. Compound H-1 bearing the 4-(2-hydroxyethyl)
piperzazinyl moiety showed decreased or even vanished activity with
As illustrated, the six tested hits showed appreciable inhibitory effect
on ALKWT with IC50 values ranging from 2.7 to 5.3 nM. Among which,
compounds H-8 and H-11 displayed potency equal or superior to posi-
tive. To our delight, after embedding 1H-benzo[d]imidazol moiety, the
tested six 2,4-diarylaminopyrimidine derivatives displayed satisfying
potency on ALKL1196M and ALKG1202R with IC50 values below 14 nM,
indicating that the application of scaffold hopping strategy is reasonable
for mutation-combating effects. Notably, H-11 exhibited prominent in-
hibitions on ALKL1196M (IC50 = 3.8 nM) and ALKG1202R (IC50 = 5.7 nM),
which were almost 2- to 30-fold enhancement contrasting to Ceritinib
(IC50 = 7.6 nM and 172 nM). In addition, all tested compounds were
detected remarkable selectivity with IC50 values over 1000 nM against
EGFR kinase. Also, the potency of tested compounds against the off-
target kinase FLT3 seems to prove this further.
IC50 values of 1 μM. Nevertheless, by removing –OH from the terminal
fragment, improvement on potency was detected. Compared to H-1 and
H-4, derivatives H-21 and H-28 bearing 4-ethylpiperazinyl moiety dis-
played IC50 values of 0.432 μM and 0.203 μM against Karpas299 cell
line, as was 2.8-fold and 1.2-fold improvement. Notably, the same trend
was also observed in chemical entities H-12 and H-22, which suggested
hydroxyl group unleash negetive effects on cytotoxicity. Furthermore,
contributed comparative high potency, compounds bearing dimethyla-
mino ‘tail’ with IC50 values ranging from 0.079
μ
M to 0.047
μM toward
Karpas299, 0.507 M to 0.127
μ
μM toward H2228 cell line, respectively.
However, decent on the antiproliferation were detected along with
magnification in size of secondray amines. As an example, derivative H-
16 was not active against three cancer cell lines. Interestingly, once the
diethylamino group was ‘closed’ to pyrrolidinyl, an obvious improve-
ment on potency was perceived. Compared to H-23, the potency of
2.3. Molecular docking studies
Simulating molecular docking could enable us to comprehend the
binding pattern between ligand and protein intuitively. To better
Fig. 2. Design strategies for target compounds (H-1–H-28) based on co-crystal structure of Ceritinib with ALKWT (PDB: 4MKC). (A) Structure of target compounds
from emergence of Ceritinib and 10d. (B) Binding mode of Ceritinib in ALK active site. (C) Overlap of target compound (pink sticks) with Ceritinib (yellow sticks) in
ALK binding site. (D) Further optimization of compounds. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version
of this article.)
3

Z. Li et al.
Bioorganic & Medicinal Chemistry 37 (2021) 116108
O
S
O
Cl
Cl
NH
N
N
Cl
Cl
C-1: R1=
C-2: R1=
a
N
b
+
NH2
N
H
N
Cl
N
H
N
Cl
N
Cl
NH2
NH2
R1
N
NH
A
C-1, C-2
O
H
Cl
N
O
N
O
B-1 C-3: R =
&
1
c
d
H
NH2
O
N
H
N
Cl
R1
R1
B-2 C-4: R =
&
1
O
N
O
B-1, B-2
C-3, C-4
R2
N
R2
N R3
NH2
NH2
Cl
R3
N
N
N
e
f
g
h
N
H
N
H
N
H
O2N
O2N
H2N
O2N
D
E
F-1~F-12
G-1~G-12
NR2R3
NR2R3
Compd.
Compd.
R2
Cl
F-1, G-1 4-(2-hydroxyethyl)piperzazinyl F-7, G-7
pyrrolidinyl
piperidinyl
N
R3
N
N
F-2, G-2
F-3, G-3
F-4, G-4
F-5, G-5
F-6, G-6
dimethylamino
4-methylpiperazinyl
4-methylpiperidinyl
diethylamino
F-8, G-8
F-9, G-9
morpholinyl
N
H
N
N
N
F-10, F-10
F-11, G-11
F-12, G-12
thiomorpholino
4-ethylpiperazinyl
4-hydroxypiperidin-1-yl
H
H
R1
diisopropylamino
H-1~H-28
Scheme 1. Reagents and conditions: (a) DIPEA, isopropanol, 80 ◦C, 3 h. (b) sulfonyl chloride or acetyl chloride, pyridine, DCM, r.t., 2.5 h. (c) CH3NH2/ (CH3)2NH
(40% in water), ethanol, 25 ◦C, 2 h. (d) 2,4,5-trichloropyrimidine, DIPEA, isopropanol, 80 ◦C, 3 h. (e) ClCH2COOH, 12 M HCl, reflux, 6 h. (f) N-aliphatic amine,
K2CO3, DMF, 60 ◦C, 3 h. (g) 80% hydrazine hydrate, FeCl3⋅6H2O, activated carbon, ethanol, reflux, 2 h. (h) C-1–C-4, TsOH, isopropanol, 80 ◦C, 6 h.
ascertain the binding mode, the molecular docking on optimal com-
pound H-11 with co-crystal structures of ALKWT (PDB: 4MKC),
ALKL1196M (PDB: 2YFX) and ALKG1202R (based on the wild-type ALK)
were conducted. Meanwhile, the conformations were analyzed using
Pymol and the 2D binding modes were generated using Discovery Studio
3.0 (Fig. 4).
the test derivatives exhibited acceptable inhibitory activities against a
panel of cancer cell lines, indicating the scaffold hopping strategy might
generate a new inspiration in the design of ALK inhibitors. The most
promising H-11 was detected optimal activities on Karpas299 (0.016
μM) and H2228 (0.099 μM) cell lines. To our delight, parallelled with
cellular results, compound H-11 showed outstanding kinase potency on
ALKWT, ALKL1196M and ALKG1202R with IC50 values of 2.7 nM, 3.8 nM
and 5.7 nM, respectively. Moreover, the tetragonal interaction mode of
H-11 with ALK protein ideally accounted for the remarkable potency of
H-11 in antiproliferative assay in vitro. Taken together, the results of
pharmacological assays together with molecular docking studies on
novel DAAP analogues identified H-11 as promising anti-mutant ALK
inhibitor.
As exhibited in Fig. 4A and 4B, H-11 formed two principle hydrogen
bonds with residues Met1199 whereby amine and pyrimidine nitrogen
atom in very similar pattern with Ceritinib in ALKWT as expected, as also
proved on-target activity of title compounds. In the meantime, the 5-Cl
atom of the pyrimidine group also forms two alkyl interactions with
Ala1148 and Leu1196 and compound H-11 is anchored to the hinge
region, indicating that 2,4-diaminopyrimidine skeleton is essential for
ALK inhibitory potency. Subsequently, as shown in Fig. 4C and 4D, in
the ALKL1196M binding model, a halogen interaction was detected be-
tween chlorine atom in H-11 and L1196M, which may account for the
combating effect of ALKL1196M. Notably, 1H-benzo[d]imidazol moiety
forms Pi-Alkyl interactions with Leu1122 in ALKWT and ALKL1196M
mutant, which emphasized the implement of scaffold hopping strategy is
reasonable. Interestingly, in the G1202R binding model, the third
hydrogen bond was present between Leu1122 and the NH of the 1H-
benzo[d]imidazol belongs to H-11 (Fig. 4F). It can be boldly speculated
that the third hydrogen bond was responsible for the improvement of
G1202R-overcoming effect. All in all, the results of molecular docking
verified the accuracy of cellular and enzymatic data, indicating that H-
11 is very likely to be a candidate for combating ALKG1202R mutant.
4. Experimental procedures
4.1. Chemistry
Unless noted, all materials were gains from commercially available
sources and were put in use without further purification. Melting points
of all compounds were obtained on a Büchi Melting Point B-540 appa-
ratus (BüchiLabortechnik, Switzerland) which were uncorrected. Mass
spectra (MS) were performed in ESI mode on agilent 1100 LC-MS
(Agilent, palo Alto, USA). 1H NMR and 13C NMR spectra were taken
using Brüker spectrometers (Bruker Bioscience, Billerica, USA) with
TMS as an internal standard. Column chromatography was run on silica
gel (200–300 mesh)from Qingdao Ocean Chemical (Qingdao, China).
3. Conclusion
4.1.1. General procedure for preparation of compounds (B-1–B-2)
To the solution of isatoic anhydride (A, 30.0 g, 0.18 mol) in the
ethanol (0.15 L) was added 40% methylamine or dimethylamine
aqueous solution (0.24 mol) slowly. The reaction mixture was heated to
25 ◦C and stirred for 2 h. The reaction mixture was poured into saturated
brine and stirred for 0.5 h. Then the mixture was extracted with ethyl
With the aim to develop novel ALK inhibitors with mutation-
combating effects, scaffold hopping strategy and bioisosterism princi-
ple were implemented by incorporating the 1H-benzo[d]imidazol motif
onto the 2,4-diarylaminopyrimidine skeleton, which furnished twenty
eight new compounds (H-1–H-28). The MTT assay discovered most of
4

Z. Li et al.
Bioorganic & Medicinal Chemistry 37 (2021) 116108
Table 1
Structures, anti-proliferative activities and physical properties of compounds H-1–H-28.
Compd.
R1
NR2R3
IC50
(
μ
M) ± SDa,b
Physical properties
Karpas299
H2228
A549
cLogPc
tPSAd
Druglikenessscore
1.12
H-1
1.03 ± 0.10
1.72 ± 0.12
>10
2.62
123.86
H-2
1.35 ± 0.12
2.36 ± 0.15
>10
2.42
105.26
1.31
H-3
H-4
0.391 ± 0.03
0.249 ± 0.02
1.31 ± 0.12
1.29 ± 0.11
>10
>10
2.72
2.63
107.53
98.69
1.66
1.69
H-5
H-6
0.056 ± 0.002
0.079 ± 0.003
0.326 ± 0.02
0.507 ± 0.02
3.01 ± 0.21
>10
3.33
3.14
103.57
84.97
0.65
0.83
H-7
H-8
0.062 ± 0.006
0.047 ± 0.005
0.338 ± 0.03
0.127 ± 0.02
4.79 ± 0.13
>10
3.43
3.34
87.24
78.40
1.21
1.24
H-9
0.053 ± 0.002
0.107 ± 0.02
0.244 ± 0.03
0.230 ± 0.02
2.77 ± 0.11
>10
3.12
2.92
106.89
88.29
1.05
1.24
H-10
H-11
H-12
0.016 ± 0.006
0.136 ± 0.02
0.099 ± 0.02
0.770 ± 0.06
2.03 ± 0.07
>10
3.13
4.70
81.72
1.61
1.05
103.42
H-13
0.193 ± 0.03
0.975 ± 0.05
6.03 ± 0.21
4.50
84.81
1.23
H-14
H-15
0.077 ± 0.002
0.079 ± 0.002
0.632 ± 0.07
1.23 ± 0.11
>10
>10
4.79
4.09
87.08
1.58
0.49
103.12
H-16
H-17
H-18
H-19
H-20
H-21
2.01 ± 0.15
3.79 ± 0.15
0.347 ± 0.02
0.429 ± 0.04
1.07 ± 0.10
3.23 ± 0.21
1.03 ± 0.11
5.37 ± 0.11
1.86 ± 0.04
6.01 ± 0.13
>10
5.22
3.95
4.35
3.24
3.57
3.37
103.05
103.78
103.42
111.32
103.42
106.95
0.57
0.90
0.99
0.77
0.80
0.062 ± 0.004
0.068 ± 0.007
0.798 ± 0.05
1.84 ± 0.12
>10
0.432 ± 0.03
>10
0.92
(continued on next page)
5

Z. Li et al.
Bioorganic & Medicinal Chemistry 37 (2021) 116108
Table 1 (continued)
Compd.
R1
NR2R3
IC50
(
μ
M) ± SDa,b
Physical properties
Karpas299
H2228
A549
cLogPc
tPSAd
Druglikenessscore
H-22
H-23
1.29 ± 0.10
1.23 ± 0.14
1.30 ± 0.11
1.45 ± 0.12
>10
2.93
4.10
119.14
77.95
1.06
1.13
2.93 ± 0.09
H-24
H-25
H-26
H-27
H-28
0.054 ± 0.004
0.057 ± 0.003
0.833 ± 0.03
1.29 ± 0.13
0.291 ± 0.01
0.337 ± 0.01
1.27 ± 0.11
2.35 ± 0.16
0.583 ± 0.02
0.107 ± 0.03
>10
3.96
4.36
3.25
3.98
3.38
5.48
78.61
78.25
86.16
78.25
81.78
86.08
1.47
1.56
1.36
1.39
1.50
0.55
1.87 ± 0.09
>10
7.36 ± 0.26
>10
0.203 ± 0.02
Ceritinib e
–
–
0.031 ± 0.007
>10
a
b
c
Values are the means of at least three independent experiments.
SD: standard deviation.
cLogP measured between 1-octanol/water phosphate buffered.
Topological polar surface area.
d
e
Used as positive control.
Fig. 3. Representative drug-likeness model score results of Ceritinib, H-8 and H-11. (The green curve indicates that a compound is possibly not a drug, while the blue
curve suggests the compound may possess drug-likeness properties.) (For interpretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)
acetate (100.0 mL) for 3 times and the combined organic layers were
Table 2
evaporated under reduced pressure to give B-1–B-2.
Inhibitory effects of selected compounds against ALK, ALK secondary mutations
(L1196M and G1202R), EGFR and FLT3.
Compd.
4.1.1.1. 2-amino-N-methylbenzamide (B-1). Beige solid; Yield: 95.7%;
IC50 (nM) a
ALKWT
MS (ESI) m/z: 151.2 [M + H]+.
ALKL1196M
ALKG1202R
EGFR
FLT3
4.1.1.2. 2-amino-N,N-dimethylbenzamide (B-2). Claybank oil; Yield:
99.4%; MS (ESI) m/z: 165.4 [M + H]+.
H-5
4.0
3.2
3.6
2.7
5.3
3.4
3.2
3.7
3.8
4.9
3.8
8.4
5.3
7.6
8.2
6.9
7.0
5.7
7.1
13.3
172
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>200
>200
134.7
>200
185.5
103.1
86.4
H-8
H-9
H-11
H-18
H-24
Ceritinib
4.1.2. General procedure for preparation of compounds (C-3–C-4)
To the solution of B-1 or B-2 (0.15 mol) in the isopropanol (0.20 L)
was added 2,4,5-trichloropyrimidine(27.5 g, 0.15 mol) and DIPEA(49.5
mL, 0.30 mol). The reaction mixture was heated and stirred for 3 h at
80 ◦C. The mixture was filtered when TLC showed the reaction was
completed. 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 solid was collected to give C-3–C-4 after trituration in
dichloromethane and filtration.
a
Values are the means of at least two independent experiments.
6

Z. Li et al.
Bioorganic & Medicinal Chemistry 37 (2021) 116108
Fig. 4. The binding models of H-11 with ALKWT, ALKL1196M and ALKG1202R. (A) Binding mode of H-11 (pink sticks) with adjacent residues in the ALKWT model and
overlapping with Ceritinib (yellow sticks). (B) Predicted binding conformation for H-11 in the binding site cavity of ALKWT. (C) Predicted binding conformation for
H-11 in the binding site cavity of ALKL1196M. (D) 2D diagram of the interaction between H-11 and ALKL1196M. (E) Predicted binding conformation for H-11 in the
binding site cavity of ALKG1202R. (F) 2D diagram of the interaction between H-11 and ALKG1202R. (For interpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article.)
4.1.2.1. 2-((2,5-dichloropyrimidin-4-yl)amino)-N-methylbenzamide(C-
3). White solid; Yield: 85.9%; MS (ESI) m/z: 297.3 [M + H]+.
4.1.4.6. N-isopropyl-N-((6-nitro-1H-benzo[d]imidazol-2-yl)methyl)
propan-2-amine (F-6). DeepBrown solid; Yield: 62.3%; MS (ESI) m/z:
277.3 [M + H]+.
4.1.2.2. 2-((2,5-dichloropyrimidin-4-yl)amino)-N,N-dimethylbenzamide
(C-4). White solid; Yield: 80.2%; MS (ESI) m/z: 311.2 [M + H]+.
4.1.4.7. 6-nitro-2-(pyrrolidin-1-ylmethyl)-1H-benzo[d]imidazole (F-7).
Wheat solid; Yield: 91.7%; MS (ESI) m/z: 247.4 [M + H]+.
4.1.3. Preparation of 2-(chloromethyl)-6-nitro-1H-benzo[d]imidazole (E)
To the solution of 4-nitrobenzene-1,2-diamine (D, 10.0 g, 0.065 mol)
in 12 M HCl (133 mL, 24.6 mol) was added 2-chloroacetic acid (12.3 g,
0.131 mol), then refluxed for 6 h. After the reaction completed, the re-
action mixture was adjusted pH to 9 with ammonium hydroxide at ice-
salt baths. The precipitate was collected by filtration and recrystallized
in absolute methanol to give E as a wheat solid in a overall yield of
82.3%. MS (ESI) m/z: 212.3 [M + H]+.
4.1.4.8. 6-nitro-2-(piperidin-1-ylmethyl)-1H-benzo[d]imidazole (F-8).
Wheat solid; Yield: 93.0%; MS (ESI) m/z: 261.4 [M + H]+.
4.1.4.9. 4-((6-nitro-1H-benzo[d]imidazol-2-yl)methyl)morpholine (F-9).
Brown solid; Yield: 79.6%; MS (ESI) m/z: 263.4 [M + H]+.
4.1.4.10. 4-((6-nitro-1H-benzo[d]imidazol-2-yl)methyl)thiomorpholine
(F-10). Brown solid; Yield: 75.1%; MS (ESI) m/z: 279.7 [M + H]+.
4.1.4. General procedure for preparation of compounds (F-1–F-12)
To the solution of E (5.0 g, 0.024 mol) in DMF (25 mL) was added
K2CO3 (11.5 g, 0.083 mol) and N-aliphatic amines (0.047 mol). The
resulting solution was stirred at 60 ◦C for 3 h. When the completion of
the reaction, the reaction mixture was poured into water and stirred for
another 1 h. The precipitate was collected by filtration and air-dried to
give F-1–F-12.
4.1.4.11. 2-((4-ethylpiperazin-1-yl)methyl)-6-nitro-1H-benzo[d]imidazole
(F-11). Goldenrod solid; Yield: 83.0%; MS (ESI) m/z: 289.4 [M + H]+.
4.1.4.12. 1-((6-nitro-1H-benzo[d]imidazol-2-yl)methyl)piperidin-4-ol (F-
12). Brown solid; Yield: 97.0%; MS (ESI) m/z: 277.3 [M + H]+.
4.1.5. General procedure for preparation of compounds (G-1–G-12)
To a mixture of F-1–F-12 (11 mmol), activated carbon (0.04 g, 3.2
mmol) and FeCl3▪ 6H2O (0.44 g, 1.6 mmol) in ethanol (30 mL) was
dropwise added 80% hydrazine hydrate (30 mL) while maintaining the
temperature below 60 ◦C. After the addition completed, the mixture was
warmed to reflux and stirred for 2 h. When completion of the reaction as
indicated by TLC, the mixture was filtered immediately. The filtrate was
concentrated to about 15 mL under reduced pressure, which was
resuspended in water (15 mL) after cooling. The mixture was extracted
with ethyl acetate (0.1 L) for 3 times and the combined organic layers
were evaporated under reduced pressure to give G-1–G-12.
4.1.4.1. 2-(4-((6-nitro-1H-benzo[d]imidazol-2-yl)methyl)piperazin-1-yl)
ethan-1-ol (F-1). Goldenrod solid; Yield: 97.8%; MS (ESI) m/z: 306.4
[M + H]+.
4.1.4.2. N,N-dimethyl-1-(6-nitro-1H-benzo[d]imidazol-2-yl)methanamine
(F-2). Brown solid; Yield: 96.2%; MS (ESI) m/z: 221.4 [M + H]+.
4.1.4.3. 2-((4-methylpiperazin-1-yl)methyl)-6-nitro-1H-benzo[d]imid-
azole (F-3). Brown solid; Yield: 91.7%; MS (ESI) m/z: 276.5 [M + H]+.
4.1.4.4. 2-((4-methylpiperidin-1-yl)methyl)-6-nitro-1H-benzo[d]imidazole
(F-4). Brown solid; Yield: 76.9%; MS (ESI) m/z: 276.6 [M + H]+.
4.1.5.1. 2-(4-((6-amino-1H-benzo[d]imidazol-2-yl)methyl)piperazin-1-yl)
ethan-1-ol (G-1). Light brown solid; Yield: 97.8%; MS (ESI) m/z: 276.4
[M + H]+.
4.1.4.5. N-ethyl-N-((6-nitro-1H-benzo[d]imidazol-2-yl)methyl)ethan-
amine (F-5). Deep orange solid; Yield: 85.1%; MS (ESI) m/z: 249.4 [M
+ H]+.
7

Z. Li et al.
Bioorganic & Medicinal Chemistry 37 (2021) 116108
4.1.5.2. 2-((dimethylamino)methyl)-1H-benzo[d]imidazol-6-amine
(G-
J = 7.5 Hz, 1H), 4.44 (s, 1H), 3.66 (s, 2H), 3.51 (s, 2H), 3.48 (d, J = 6.0
Hz, 2H), 2.49 – 2.45 (m, 4H), 2.45 – 2.39 (m, 4H), 2.10 (s, 3H). Anal.
calcd for C26H30ClN9O2 (%): C, 58.26; H, 5.64; N,23.52. Found (%): C,
58.30; H, 5.66; N, 23.50.
2). Light brown solid; Yield: 90.7%; MS (ESI) m/z: 191.4 [M + H]+.
4.1.5.3. 2-((4-methylpiperazin-1-yl)methyl)-1H-benzo[d]imidazol-6-
amine (G-3). Gray solid; Yield:93.5%; MS (ESI) m/z: 246.4 [M + H]+.
4.1.6.3. 2-((5-chloro-2-((2-((4-(2-hydroxyethyl)piperazin-1-yl)methyl)-
1H-benzo[d]imidazol-6-yl)amino)pyrimidin-4-yl)amino)-N-methyl-
benzamide (H-3). White solid; Yield: 72.3%; m.p.:251.2–253.0 ◦C; MS
(ESI) m/z: 534.40 [Mꢀ H]-. 1H NMR (400 MHz, DMSO‑d6) δ 12.16 (s,
1H), 11.69 (s, 1H), 9.38 (s, 1H), 8.83 (s, 1H), 8.78 (s, 1H), 8.20 (s, 1H),
7.83 (s, 1H), 7.75 (d, J = 7.6 Hz, 1H), 7.39 (s, 2H), 7.33 (d, J = 8.7 Hz,
1H), 7.09 (t, J = 7.4 Hz, 1H), 5.70 (d, J = 7.7 Hz, 1H), 3.68 (s, 2H), 3.50
(d, J = 4.7 Hz, 2H), 3.47 (d, J = 6.1 Hz, 2H), 2.81 (d, J = 4.1 Hz, 3H),
2.47 (s, 4H), 2.38 (t, J = 6.1 Hz, 4H). Anal. calcd for C26H30ClN9O (%):
C, 58.26; H, 5.64; N,23.52. Found (%): C, 58.31; H, 5.68; N, 23.50.
4.1.5.4. 2-((4-methylpiperidin-1-yl)methyl)-1H-benzo[d]imidazol-6-
amine (G-4). Brown solid; Yield: 85.9%; MS (ESI) m/z: 245.5 [M + H]+.
4.1.5.5. 2-((diethylamino)methyl)-1H-benzo[d]imidazol-6-amine (G-5).
Brown solid; Yield: 81.3%; MS (ESI) m/z: 219.4 [M + H]+.
4.1.5.6. 2-((diisopropylamino)methyl)-1H-benzo[d]imidazol-6-amine (G-
6). Black solid; Yield: 65.2%; MS (ESI) m/z: 247.5 [M + H]+.
4.1.5.7. 2-(pyrrolidin-1-ylmethyl)-1H-benzo[d]imidazol-6-amine (G-7).
Brown solid; Yield:86.6%; MS (ESI) m/z: 217.5 [M + H]+.
4.1.6.4. 2-((5-chloro-2-((2-((4-(2-hydroxyethyl)piperazin-1-yl)methyl)-
1H-benzo[d]imidazol-6-yl)amino)pyrimidin-4-yl)amino)-N,N-dime-
thylbenzamide (H-4). Light Gray solid; Yield: 65.4%; m.
p.:253.7–255.8 ◦C; MS (ESI) m/z: 548.46 [Mꢀ H]-. 1H NMR (400 MHz,
DMSO‑d6) δ 12.14 (s, 1H), 9.30 (s, 1H), 9.18 (s, 1H), 8.38 (d, J = 8.0 Hz,
1H), 8.17 (s, 1H), 7.80 (s, 1H), 7.42 (d, J = 7.6 Hz, 2H), 7.37 (d, J = 12.9
Hz, 1H), 7.33 (s, 1H), 7.15 (t, J = 7.2 Hz, 1H), 4.66 (s, 1H), 3.67 (s, 2H),
3.50 (d, J = 5.8 Hz, 2H), 3.47 (d, J = 6.0 Hz, 2H), 2.99 (s, 6H), 2.46 (s,
4H), 2.40 – 2.35 (m, 4H). Anal. calcd for C27H32ClN9O (%): C, 58.96; H,
5.86; N,22.92. Found (%): C, 58.98; H, 5.88; N, 22.90.
4.1.5.8. 2-(piperidin-1-ylmethyl)-1H-benzo[d]imidazol-6-amine (G-8).
Brown solid; Yield: 91.7%; MS (ESI) m/z: 231.4 [M + H]+.
4.1.5.9. 2-(morpholinomethyl)-1H-benzo[d]imidazol-6-amine
(G-9).
Brown solid; Yield: 80.1%; MS (ESI) m/z: 233.4 [M + H]+.
4.1.5.10. 2-(thiomorpholinomethyl)-1H-benzo[d]imidazol-6-amine
(G-
10). Deep brown solid; Yield: 75.3%; MS (ESI) m/z: 249.6[M + H]+.
4.1.6.5. N-(2-((5-chloro-2-((2-((dimethylamino)methyl)-1H-benzo[d]imi-
dazol-6-yl)amino)pyrimidin-4-yl)amino)phenyl)methanesulfonamide (H-
5). White solid; Yield: 95.9%; m.p.:235.7–237.0 ◦C; MS (ESI) m/z:
487.27 [M + H]+. 1H NMR (400 MHz, DMSO‑d6) δ 12.14 (s, 1H), 9.27 (s,
1H), 8.57 (s, 1H), 8.16 (s, 1H), 8.15 (s, 1H), 7.74 (s, 1H), 7.36 (d, J = 7.8
Hz, 2H), 7.33 (s, 1H), 7.29 (d, J = 9.7 Hz, 1H), 7.25 (d, J = 7.7 Hz, 1H),
7.16 (t, J = 7.1 Hz, 1H), 3.63 (s, 3H), 2.94 (s, 2H), 2.24 (s, 6H).Anal.
calcd for C21H23ClN8O2S (%): C, 51.80; H, 4.76; N,23.01. Found (%): C,
51.83; H, 4.78; N, 22.08.
4.1.5.11. 2-((4-ethylpiperazin-1-yl)methyl)-1H-benzo[d]imidazol-6-
amine (G-11). Brown solid; Yield: 89.9%; MS (ESI) m/z: 260.4[M +
H]+.
4.1.5.12. 1-((6-amino-1H-benzo[d]imidazol-2-yl)methyl)piperidin-4-ol
(G-12). Brown solid; Yield: 85.7%; MS (ESI) m/z: 247.4[M + H]+.
4.1.6. General procedure for preparation of compounds (H-1–H-28)
To a solution of G-1–G-12 (3 mmol) in isopropanol(15.0 mL) was
added p-methylbenzenesulfonic acid(0.52 g, 3 mmol) and C-1–C-4 (3
mmol) under stirring. After the reaction mixture heated to 80 ◦C for 6 h,
it was hot filtered to give the crude product which was resuspended in
water and adjusted the pH to 8 with sodium bicarbonate. Then the
mixture was extracted with ethyl acetate (20.0 mL) for 3 times and the
combined organic layers were evaporated under reduced pressure. The
solid was collected after purifying through column chromatography
using dichloromethane/methanol as an eluent. H-1–H-28 were obtained
in satisfactory yields which were purified by column chromatography
with the eluting system of DCM and methanol in different ratios.
4.1.6.6. N-(2-((5-chloro-2-((2-((dimethylamino)methyl)-1H-benzo[d]imi-
dazol-6-yl)amino)pyrimidin-4-yl)amino)phenyl)acetamide (H-6). White
solid; Yield: 85.5%; m.p.:232.9–234.2 ◦C; MS (ESI) m/z: 449.34 [Mꢀ H]-.
1H NMR (400 MHz, DMSO‑d6) δ 12.08 (s, 1H), 10.13 (s, 1H), 9.18 (s,
1H), 8.43 (s, 1H), 8.12 (s, 1H), 7.85 (d, J = 7.7 Hz, 1H), 7.70 (d, J = 36.2
Hz, 1H), 7.38 – 7.30 (m, 2H), 7.28 (d, J = 7.6 Hz, 2H), 7.17 (t, J = 7.5
Hz, 1H), 3.61 (s, 2H), 2.23 (s, 6H), 2.11 (s, 3H).13C NMR (101 MHz,
DMSO‑d6) δ 170.14, 158.61, 156.19, 154.80, 152.00, 139.03, 135.48,
134.87, 132.29, 126.98, 125.99, 125.33, 125.09, 118.07, 115.42,
104.29, 102.66, 99.98, 57.42, 45.59 (2C), 23.49.Anal. calcd for
C
22H23ClN8O (%): C, 58.60; H, 5.14; N,24.85. Found (%): C, 58.58; H,
4.1.6.1. N-(2-((5-chloro-2-((2-((4-(2-hydroxyethyl)piperazin-1-yl)
methyl)-1H-benzo[d]imidazol-6-yl)amino)pyrimidin-4-yl)amino)phenyl)
5.16; N, 24.87.
methanesulfonamide
(H-1). Beige
solid;
Yield:
80.5%;
m.
4.1.6.7. 2-((5-chloro-2-((2-((dimethylamino)methyl)-1H-benzo[d]imida-
zol-6-yl)amino)pyrimidin-4-yl)amino)-N-methylbenzamide (H-7). White
solid; Yield: 50.0%; m.p.:221.6–222.9 ◦C; MS (ESI) m/z: 449.56 [Mꢀ H]-.
1H NMR (400 MHz, DMSO‑d6) δ 12.22 (s, 1H), 11.69 (s, 1H), 9.38 (s,
1H), 8.85 (d, J = 6.2 Hz, 1H), 8.78 (d, J = 4.0 Hz, 1H), 8.20 (s, 1H), 7.82
(s, 1H), 7.76 (d, J = 7.7 Hz, 1H), 7.39 (d, J = 12.0 Hz, 2H), 7.35 (s, 1H),
7.10 (t, J = 7.4 Hz, 1H), 3.65 (s, 2H), 2.82 (d, J = 4.2 Hz, 3H), 2.25 (s,
6H).13C NMR (101 MHz, DMSO‑d6) δ 169.45, 158.70, 155.40, 155.03,
152.53, 152.45, 140.01, 134.99, 132.07, 128.38, 125.98, 122.10,
121.59, 120.70, 116.54, 105.10, 105.05, 57.41, 45.59(2C), 26.78. Anal.
calcd for C22H23ClN8O (%): C, 58.60; H, 5.14; N,24.85. Found (%): C,
58.63; H, 5.16; N, 24.88.
p.:261.2–264.1 ◦C; MS (ESI) m/z: 572.51 [M + H]+. 1H NMR (400 MHz,
DMSO‑d6) δ 12.05 (s, 1H), 9.27 (s, 1H), 8.63 (s, 1H), 8.18 (d, J = 7.8 Hz,
1H), 8.15 (s, 1H), 7.76 (s, 1H), 7.35 (d, J = 7.9 Hz, 2H), 7.32 (s, 2H),
7.19 (t, J = 7.4 Hz, 1H), 7.12 (t, J = 7.0 Hz, 1H), 4.38 (s, 1H), 3.66 (s,
2H), 3.50 (d, J = 4.0 Hz, 2H), 3.47 (d, J = 6.1 Hz, 2H), 2.92 (s, 3H), 2.47
(s, 4H), 2.39 (t, J = 6.2 Hz, 4H). Anal. calcd for C25H30ClN9O3S (%): C,
52.49; H, 5.29; N,22.04. Found (%): C, 52.52; H, 5.31; N, 22.06.
4.1.6.2. N-(2-((5-chloro-2-((2-((4-(2-hydroxyethyl)piperazin-1-yl)
methyl)-1H-benzo[d]imidazol-6-yl)amino)pyrimidin-4-yl)amino)phenyl)
acetamide (H-2). Light yellow solid; Yield: 92.0%; m.p.:257.4–259.2 ◦C;
MS (ESI) m/z: 536.02 [M + H]+. 1H NMR (400 MHz, DMSO‑d6) δ 12.00
(s, 1H), 10.09 (s, 1H), 9.18 (s, 1H), 8.42 (s, 1H), 8.11 (s, 1H), 7.84 (d, J
= 7.2 Hz, 1H), 7.69 (s, 1H), 7.31 (s, 2H), 7.28 (d, J = 7.9 Hz, 2H), 7.17 (t,
4.1.6.8. 2-((5-chloro-2-((2-((dimethylamino)methyl)-1H-benzo[d]imida-
zol-6-yl)amino)pyrimidin-4-yl)amino)-N,N-dimethylbenzamide
(H-8).
8

Z. Li et al.
Bioorganic & Medicinal Chemistry 37 (2021) 116108
White solid; Yield: 69.3%; m.p.:222.9–225.3 ◦C; MS (ESI) m/z: 463.39
[Mꢀ H]-. 1H NMR (400 MHz, DMSO‑d6) δ 12.20 (s, 1H), 9.29 (s, 1H),
9.17 (s, 1H), 8.38 (d, J = 8.6 Hz, 1H), 8.17 (s, 1H), 7.79 (s, 1H), 7.44 –
7.39 (m, 2H), 7.34 (s, 2H), 7.15 (t, J = 7.4 Hz, 1H), 3.62 (s, 2H), 2.99 (s,
6H), 2.23 (s, 6H). Anal. calcd for C23H25ClN8O (%): C, 59.41; H, 5.42;
N,24.10. Found (%): C, 59.38; H, 5.39; N, 24.13.
4.1.6.14. 2-((5-chloro-2-((2-((4-methylpiperidin-1-yl)methyl)-1H-benzo
[d]imidazol-6-yl)amino)pyrimidin-4-yl)amino)-N-methylbenzamide
(H-
14). White solid; Yield: 73.6%; m.p.:232.6–234.9 ◦C; MS (ESI) m/z:
505.42 [M + H]+. 1H NMR (600 MHz, DMSO‑d6) δ 12.17 (s, 1H), 11.68
(s, 1H), 9.37 (s, 1H), 8.84 (s, 1H), 8.79 (s, 1H), 8.19 (s, 1H), 7.82 (s, 1H),
7.75 (d, J = 7.7 Hz, 1H), 7.39 (s, 2H), 7.33 (d, J = 8.3 Hz, 1H), 7.08 (t, J
= 7.4 Hz, 1H), 3.66 (s, 2H), 2.83 (s, 3H), 2.81 (d, J = 4.1 Hz, 4H), 2.04 (t,
J = 11.1 Hz, 2H), 1.59 (d, J = 12.5 Hz, 2H), 1.19 (dd, J = 11.7, 2.0 Hz,
1H), 0.90 (d, J = 6.4 Hz, 3H). Anal. calcd for C26H29ClN8O (%): C, 61.84;
H, 5.79; N,22.19. Found (%): C, 61.88; H, 5.77; N, 22.16.
4.1.6.9. N-(2-((5-chloro-2-((2-((4-methylpiperazin-1-yl)methyl)-1H-
benzo[d]imidazol-6-yl)amino)pyrimidin-4-yl)amino)phenyl)meth-
anesulfonamide (H-9). Creamy white solid; Yield: 71.0%; m.
p.:248.7–250.2 ◦C; MS (ESI) m/z: 540.33 [Mꢀ H]-. 1H NMR (400 MHz,
DMSO‑d6) δ 9.27 (s, 1H), 8.74 (s, 1H), 8.21 (d, J = 7.6 Hz, 1H), 8.15 (s,
1H), 7.78 (s, 1H), 7.35 (d, J = 5.8 Hz, 2H), 7.33 (s, 2H), 7.13 (d, J = 7.0
Hz, 1H), 7.09 (dd, J = 7.5, 1.7 Hz, 2H), 3.67 (s, 2H), 2.89 (s, 3H), 2.50 –
2.42 (m, 3H), 2.38 (s, 4H), 2.18 (s, 4H).13C NMR (101 MHz, DMSO‑d6) δ
167.74, 158.69, 155.74, 154.69, 151.82, 135.15, 134.50, 130.99,
130.78, 130.32, 130.05, 125.93, 125.77, 124.63, 124.08, 116.09,
104.44, 56.17, 54.99(2C), 53.06(2C), 46.09, 29.46. Anal. calcd for
4.1.6.15. N-(2-((5-chloro-2-((2-((diethylamino)methyl)-1H-benzo[d]imi-
dazol-6-yl)amino)pyrimidin-4-yl)amino)phenyl)methanesulfonamide (H-
15). Gray solid; Yield: 61.2%; m.p.:243.4–245.9 ◦C; MS (ESI) m/z:
513.49 [Mꢀ H]-. 1H NMR (400 MHz, DMSO‑d6) δ 11.96 (s, 1H), 9.68 (s,
1H), 9.23 (s, 1H), 9.05 (s, 1H), 8.33 (d, J = 7.7 Hz, 1H), 8.11 (s, 1H),
7.82 (s, 1H), 7.34 (s, 2H), 7.26 (d, J = 7.8 Hz, 2H), 6.93 (t, J = 7.1 Hz,
1H), 3.75 (s, 2H), 2.78 (s, 3H), 2.58 – 2.52 (m, 4H), 1.03 (t, J = 7.1 Hz,
6H).Anal. calcd for C23H27ClN8O2S (%): C, 53.64; H, 5.28; N,21.76.
Found (%): C, 53.66; H, 5.24; N, 21.77.
C
24H28ClN9O2S (%): C, 53.18; H, 5.21; N,23.26. Found (%): C, 53.15; H,
5.21; N, 23.28.
4.1.6.10. N-(2-((5-chloro-2-((2-((4-methylpiperazin-1-yl)methyl)-1H-
benzo[d]imidazol-6-yl)amino)pyrimidin-4-yl)amino)phenyl)acetamide (H-
10). White solid; Yield: 64.8%; m.p.:247.2–250.0 ◦C; MS (ESI) m/z:
506.36 [M + H]+. 1H NMR (400 MHz, DMSO‑d6) δ 9.28 (s, 1H), 8.63 (s,
1H), 8.18 (d, J = 8.3 Hz, 1H), 8.16 (s, 1H), 7.77 (s, 1H), 7.36 (d, J = 7.8
Hz, 1H), 7.32 (s, 1H), 7.23 – 7.17 (m, 1H), 7.13 (dd, J = 13.7, 7.8 Hz,
2H), 6.61 (s, 1H), 6.46 (d, J = 8.4 Hz, 1H), 3.57 (s, 2H), 2.93 (s, 3H),
2.85 (dd, J = 14.4, 7.2 Hz, 3H), 2.44 (s, 4H), 2.34 (dt, J = 13.8, 7.0 Hz,
4H). Anal. calcd for C25H28ClN9O (%): C, 59.34; H, 5.58; N,24.91. Found
(%): C, 59.30; H, 5.55; N, 24.90.
4.1.6.16. N-(2-((5-chloro-2-((2-((diisopropylamino)methyl)-1H-benzo[d]
imidazol-6-yl)amino)pyrimidin-4-yl)amino)phenyl)methanesulfonamide
(H-16). Gray solid; Yield: 49.3%; m.p.:246.7–248.9 ◦C; MS (ESI) m/z:
543.51 [M + H]+. 1H NMR (400 MHz, DMSO‑d6) δ 11.56 (s, 1H), 9.26 (s,
1H), 8.69 (s, 1H), 8.21 (d, J = 6.1 Hz, 1H), 8.14 (s, 1H), 7.78 (s, 1H),
7.34 (d, J = 6.6 Hz, 2H), 7.30 (s, 2H), 7.12 (dd, J = 11.6, 8.1 Hz, 2H),
3.79 (s, 2H), 3.04 (d, J = 6.1 Hz, 2H), 2.91 (s, 3H), 1.03 (d, J = 5.8 Hz,
12H).13C NMR (101 MHz, DMSO‑d6) δ 158.74, 156.46, 155.72, 154.63,
134.80, 134.45, 131.21, 130.05, 125.69, 125.60, 125.56, 124.53,
123.99, 123.97, 115.76, 115.68, 104.39, 49.22(2C), 44.03, 29.48, 20.84
(4C). Anal. calcd for C25H31ClN8O2S (%): C, 55.29; H, 5.75; N,20.63.
Found (%): C, 55.33; H, 5.70; N, 20.65.
4.1.6.11. 2-((5-chloro-2-((2-((4-methylpiperazin-1-yl)methyl)-1H-benzo
[d]imidazol-6-yl)amino)pyrimidin-4-yl)amino)-N,N-dimethylbenzamide
(H-11). Beige solid; Yield: 74.5%; m.p.:252.1–254.2 ◦C; MS (ESI) m/z:
518.48 [Mꢀ H]-. 1H NMR (400 MHz, DMSO‑d6) δ 9.29 (s, 1H), 9.16 (s,
1H), 8.37 (d, J = 7.8 Hz, 1H), 8.17 (s, 1H), 7.80 (s, 1H), 7.41 (d, J = 7.4
Hz, 2H), 7.34 (d, J = 8.7 Hz, 1H), 7.14 (t, J = 7.5 Hz, 1H), 6.07 (s, 1H),
5.64 (s, 1H), 3.67 (s, 2H), 3.51 (s, 4H), 2.98 (s, 4H), 2.36 (s, 6H), 2.16 (s,
3H). Anal. calcd for C26H30ClN9O (%): C, 60.05; H, 5.81; N,24.24. Found
(%): C, 60.10; H, 5.78; N, 24.23.
4.1.6.17. N-(2-((5-chloro-2-((2-(pyrrolidin-1-ylmethyl)-1H-benzo[d]imi-
dazol-6-yl)amino)pyrimidin-4-yl)amino)phenyl)methanesulfonamide (H-
17). White solid; Yield: 85.6%; m.p.:243.7–245.5 ◦C; MS (ESI) m/z:
511.38 [Mꢀ H]-. 1H NMR (600 MHz, DMSO‑d6) δ 12.08 (s, 1H), 9.26 (s,
1H), 8.65 (s, 1H), 8.19 (d, J = 7.5 Hz, 1H), 8.15 (s, 1H), 7.75 (s, 0H),
7.34 (d, J = 7.5 Hz, 2H), 7.33 (s, 2H), 7.18 (t, J = 7.1 Hz, 1H), 7.11 (t, J
= 7.5 Hz, 1H), 3.79 (s, 2H), 2.92 (s, 3H), 2.54 (s, 4H), 1.73 (s, 4H). Anal.
calcd for C23H25ClN8O2S (%): C, 53.85; H, 4.91; N,21.84. Found (%): C,
53.90; H, 4.88; N, 21.82.
4.1.6.12. N-(2-((5-chloro-2-((2-((4-methylpiperidin-1-yl)methyl)-1H-
benzo[d]imidazol-6-yl)amino)pyrimidin-4-yl)amino)phenyl)meth-
anesulfonamide
(H-12). White
solid;
Yield:
77.3%;
m.
4.1.6.18. N-(2-((5-chloro-2-((2-(piperidin-1-ylmethyl)-1H-benzo[d]imi-
dazol-6-yl)amino)pyrimidin-4-yl)amino)phenyl)methanesulfonamide (H-
18). Pink solid; Yield: 78.3%; m.p.:252.3–255.0 ◦C; MS (ESI) m/z:
525.46 [Mꢀ H]-. 1H NMR (400 MHz, DMSO‑d6) δ 12.04 (s, 1H), 9.26 (s,
1H), 8.63 (s, 1H), 8.18 (d, J = 8.0 Hz, 1H), 8.15 (s, 1H), 7.75 (s, 1H),
7.35 (dd, J = 7.8, 1.2 Hz, 2H), 7.32 (s, 2H), 7.19 (t, J = 7.4 Hz, 1H), 7.11
(t, J = 7.1 Hz, 1H), 3.63 (s, 2H), 2.92 (s, 3H), 2.41 (s, 4H), 1.55 – 1.52
(m, 4H), 1.40 (d, J = 4.4 Hz, 2H). Anal. calcd for C24H27ClN8O2S (%): C,
54.69; H, 5.16; N,21.26. Found (%): C, 54.72; H, 5.18; N, 21.23.
p.:248.6–249.7 ◦C; MS (ESI) m/z: 539.38 [Mꢀ H]-. 1H NMR (600 MHz,
DMSO‑d6) δ 12.07 (s, 1H), 9.24 (s, 1H), 8.98 (s, 1H), 8.30 (s, 1H), 8.12 (s,
1H), 7.80 (s, 1H), 7.34 (s, 2H), 7.27 (d, J = 7.8 Hz, 2H), 6.95 (t, J = 7.3
Hz, 1H), 6.89 (s, 1H), 3.65 (s, 2H), 2.83 (s, 3H), 2.80 (s, 4H), 2.04 (t, J =
11.1 Hz, 2H), 1.59 (d, J = 11.9 Hz, 2H), 1.19 (dd, J = 10.6, 1.4 Hz, 1H),
0.90 (d, J = 6.5 Hz, 3H). Anal. calcd for C25H29ClN8O2S (%): C, 55.50; H,
5.91; N,20.71. Found (%): C, 55.53; H, 5.88; N, 20.74.
4.1.6.13. N-(2-((5-chloro-2-((2-((4-methylpiperidin-1-yl)methyl)-1H-
benzo[d]imidazol-6-yl)amino)pyrimidin-4-yl)amino)phenyl)acetamide (H-
13). White solid; Yield: 80.6%; m.p.:253.2–256.7 ◦C; MS (ESI) m/z:
541.34 [M + H]+. 1H NMR (400 MHz, DMSO‑d6) δ 12.04 (s, 1H), 10.15
(s, 1H), 9.16 (s, 1H), 8.43 (s, 1H), 8.11 (s, 1H), 7.84 (d, J = 7.9 Hz, 1H),
7.69 (s, 1H), 7.30 (d, J = 7.1 Hz, 2H), 7.27 (d, J = 7.6 Hz, 2H), 7.15 (t, J
= 7.5 Hz, 1H), 3.63 (s, 2H), 2.81 (d, J = 11.2 Hz, 2H), 2.10 (s, 3H), 1.56
(t, J = 20.4 Hz, 2H), 1.23 (s, 4H), 1.17 (s, 1H), 0.89 (d, J = 6.3 Hz, 3H).
Anal. calcd for C26H29ClN8O1(%): C, 61.84; H, 5.79; N,22.19. Found
(%): C, 61.85; H, 5.77; N, 22.14.
4.1.6.19. N-(2-((5-chloro-2-((2-(morpholinomethyl)-1H-benzo[d]imida-
zol-6-yl)amino)pyrimidin-4-yl)amino)phenyl)methanesulfonamide
(H-
19). Light yellow solid; Yield: 80.1%; m.p.:254.8–257.7 ◦C; MS (ESI) m/
z: 527.44 [Mꢀ H]-. 1H NMR (400 MHz, DMSO‑d6) δ 12.08 (s, 1H), 9.27
(s, 1H), 8.71 (s, 1H), 8.21 (d, J = 7.3 Hz, 1H), 8.15 (s, 1H), 7.79 (s, 1H),
7.33 (s, 2H), 7.33 (s, 22H), 7.17 – 7.05 (m, 2H), 3.68 (s, 2H), 3.62 – 3.60
(m, 4H), 2.90 (s, 3H), 2.46 (s, 4H).Anal. calcd for C23H25ClN8O3S (%): C,
52.22; H, 4.76; N,21.18. Found (%): C, 52.20; H, 4.78; N, 21.22.
4.1.6.20. N-(2-((5-chloro-2-((2-(thiomorpholinomethyl)-1H-benzo[d]imi-
dazol-6-yl)amino)pyrimidin-4-yl)amino)phenyl)methanesulfonamide (H-
9

Z. Li et al.
Bioorganic & Medicinal Chemistry 37 (2021) 116108
20). Light yellow solid; Yield: 67.3%; m.p.:252.2–254.6 ◦C; MS (ESI) m/
z: 543.38 [Mꢀ H]-. 1H NMR (400 MHz, DMSO‑d6) δ 11.44 (s, 1H), 9.37
(s, 1H), 8.98 (s, 1H), 8.84 – 8.56 (m, 1H), 8.43 (s, 1H), 8.23 (s, 1H), 8.08
(d, J = 12.0 Hz, 1H), 7.53 (d, J = 6.9 Hz, 1H), 7.34 (d, J = 47.6 Hz, 2H),
7.12 (d, J = 51.4 Hz, 2H), 3.51 (s, 2H), 3.02 (d, J = 3.5 Hz, 4H), 2.92 (s,
34H), 2.51 (s, 4H). Anal. calcd for C23H25ClN8O3S2 (%): C, 50.68; H,
4.62; N,20.56. Found (%): C, 50.71; H, 4.65; N, 20.54.
[Mꢀ H]-. 1H NMR (400 MHz, DMSO‑d6) δ 12.17 (s, 1H), 9.31 (s, 1H),
9.18 (s, 1H), 8.38 (d, J = 8.4 Hz, 1H), 8.17 (s, 1H), 7.82 (s, 1H), 7.42 (d,
J = 7.6 Hz, 2H), 7.34 (t, J = 7.4 Hz, 2H), 7.15 (t, J = 7.4 Hz, 1H), 3.69 (s,
2H), 3.63 – 3.60 (m, 4H), 2.98 (d, J = 8.1 Hz, 6H), 2.46 (s, 4H).Anal.
calcd for C25H27ClN8O2 (%): C, 59.23; H, 5.37; N,22.10. Found (%): C,
59.26; H, 5.39; N, 22.04.
4.1.6.27. 2-((5-chloro-2-((2-(thiomorpholinomethyl)-1H-benzo[d]imida-
zol-6-yl)amino)pyrimidin-4-yl)amino)-N,N-dimethylbenzamide (H-27).
Light yellow solid; Yield: 48.0%; m.p.: 235.1–238.6 ◦C; MS (ESI) m/z:
521.75 [Mꢀ H]-. 1H NMR (400 MHz, DMSO‑d6) δ 12.11 (s, 1H), 9.34 (s,
1H), 9.18 (s, 1H), 8.37 (d, J = 8.5 Hz, 1H), 8.18 (s, 1H), 7.86 (s, 1H),
7.42 (d, J = 6.4 Hz, 2H), 7.37 (t, J = 7.7 Hz, 2H), 7.16 (t, J = 7.4 Hz, 1H),
3.80 (s, 2H), 2.99 (s, 6H), 2.78 (d, J = 3.0 Hz, 4H), 2.68 (d, J = 3.9 Hz,
4H).Anal. calcd for C25H27ClN8OS (%): C, 57.41; H, 5.20; N,21.42.
Found (%): C, 57.35; H, 5.16; N, 21.40.
4.1.6.21. N-(2-((5-chloro-2-((2-((4-ethylpiperazin-1-yl)methyl)-1H-
benzo[d]imidazol-6-yl)amino)pyrimidin-4-yl)amino)phenyl)meth-
anesulfonamide
(H-21). White
solid;
Yield:
71.2%;
m.p.:
250.7–253.2 ◦C; MS (ESI) m/z: 556.06 [M + H]+. 1H NMR (400 MHz,
DMSO‑d6) δ 12.03 (s, 1H), 9.28 (s, 1H), 8.64 (s, 1H), 8.18 (d, J = 7.9 Hz,
1H), 8.16 (s, 1H), 7.77 (s, 1H), 7.35 (d, J = 7.7 Hz, 2H), 7.32 (s, 1H),
7.19 (dd, J = 15.2, 7.3 Hz, 2H), 7.15 – 7.09 (m, 1H), 3.57 (s, 2H), 2.93 (s,
3H), 2.85 (dd, J = 14.3, 7.1 Hz, 2H), 2.44 (s, 4H), 2.40 – 2.27 (m, 4H),
0.99 – 0.97 (m, 3H).13C NMR (101 MHz, DMSO‑d6) δ 158.69, 155.80,
154.76, 151.81, 149.45, 147.63, 144.62, 135.15, 134.74, 130.45,
130.05, 126.43, 126.33, 124.75, 124.44, 115.95, 111.27, 53.15 (2C),
52.61, 52.03, 46.05, 29.46, 12.33(2C). Anal. calcd for C25H30ClN9O2S
(%): C, 54.00; H, 5.44; N,22.67. Found (%): C, 54.05; H, 5.45; N, 22.69.
4.1.6.28. 2-((5-chloro-2-((2-((4-ethylpiperazin-1-yl)methyl)-1H-benzo[d]
imidazol-6-yl)amino)pyrimidin-4-yl)amino)-N,N-dimethylbenzamide (H-
28). Light yellow solid; Yield: 61.2%; m.p.: 242.3–245.8 ◦C; MS (ESI)
m/z: 534.68 [M + H]+. 1H NMR (400 MHz, DMSO‑d6) δ 9.40 (s, 1H),
9.21 (s, 1H), 8.36 (d, J = 7.9 Hz, 1H), 8.19 (s, 1H), 8.01 (d, J = 49.2 Hz,
1H), 7.50 (d, J = 7.7 Hz, 2H), 7.43 (d, J = 8.6 Hz, 2H), 7.12 (d, J = 7.7
Hz, 2H), 3.89 (s, 2H), 3.10 (s, 2H), 2.99 (s, 6H), 2.52 (s, 4H), 2.29 (s,
4H), 1.17 (t, J = 7.1 Hz, 3H).Anal. calcd for C27H32ClN9O (%): C, 60.72;
H, 6.04; N,23.60. Found (%): C, 60.77; H, 6.00; N, 23.61.
4.1.6.22. N-(2-((5-chloro-2-((2-((4-hydroxypiperidin-1-yl)methyl)-1H-
benzo[d]imidazol-6-yl)amino)pyrimidin-4-yl)amino)phenyl)meth-
anesulfonamide
(H-22). White
solid;
Yield:
80.0%;
m.p.:
257.3–259.2 ◦C; MS (ESI) m/z: 541.46 [Mꢀ H]-. 1H NMR (400 MHz,
DMSO‑d6) δ 12.09 (s, 1H), 9.29 (s, 1H), 8.50 (s, 1H), 8.18 (s, 1H), 8.13
(d, J = 7.8 Hz, 1H), 7.77 (s, 1H), 7.39 (d, J = 7.8 Hz, 2H), 7.33 (s, 2H),
7.30 (d, J = 8.5 Hz, 1H), 7.19 (t, J = 7.1 Hz, 1H), 4.66 (s, 1H), 3.77 (s,
1H), 2.97 (s, 3H), 2.81 (s, 2H), 2.30 (s, 2H), 1.76 (d, J = 11.2 Hz, 2H),
1.48 (d, J = 9.2 Hz, 2H). Anal. calcd for C24H27ClN8O3S (%): C, 53.08; H,
5.01; N,20.63. Found (%): C, 53.05; H, 5.04; N, 20.65.
4.2. Biological section
4.2.1. Physical properties and MTT assay
Physical properties such as cLogP, topological polar surface area
(TPSA) and drug-likeness scores were calculated for each compounds
using Molsoft (http://molsoft.com/mprop/).
The cancer cell lines were cultured in minimum essential medium
(MEM) supplement with 10%fetal bovine serum (FBS). The cells in the
logarithmic growth phase were seeded in a 96-well plate at a rate of
about 5 × 103 cells/well and incubated under standard conditions for
24 h. Triplicate wells were treated with media and various concentra-
tions of tested compounds and incubated for 72 h. Then, the compound-
4.1.6.23. 2-((5-chloro-2-((2-((diethylamino)methyl)-1H-benzo[d]imida-
zol-6-yl)amino)pyrimidin-4-yl)amino)-N,N-dimethylbenzamide (H-23).
Light yellow solid; Yield: 59.1%; m.p.: 237.1–239.6 ◦C; MS (ESI) m/z:
491.68 [Mꢀ H]-. 1H NMR (400 MHz, DMSO‑d6) δ 12.02 (s, 1H), 9.31 (s,
1H), 9.18 (s, 1H), 8.38 (d, J = 8.5 Hz, 1H), 8.18 (s, 1H), 7.84 (s, 1H),
7.42 (dd, J = 7.6, 1.5 Hz, 2H), 7.33 (dd, J = 8.6, 1.7 Hz, 2H), 7.15 (t, J =
7.5 Hz, 1H), 3.87 (s, 2H), 2.99 (s, 6H), 2.64 (d, J = 6.9 Hz, 4H), 1.06 (t, J
= 7.1 Hz, 6H).Anal. calcd for C25H29ClN8O (%): C, 60.91; H, 5.93;
N,22.73. Found (%): C, 60.95; H, 5.94; N, 22.70.
containing anchorage-dependent cell medium was replaced by 100
fresh medium with 5 mg/mL MTT solution. After 4 h incubation, 100
μL
L
μ
DMSO was added to each MTT removed well and placed the 39-well
plate on a micro-oscillator for 5 min until the purple formazan crystals
were dissolved. The absorbency at 492 nm (for absorbance of MTT
formazan) and 630 nm (for the reference wavelength) was measured
with an ELISA reader. The results, expressed as IC50 (inhibitory con-
centration 50%), were the averages of three determinations and calcu-
lated relative to the vehicle (DMSO) control by the Bacus Laboratories
Incorporated Slide Scanner (Bliss) software.
4.1.6.24. 2-((5-chloro-2-((2-(pyrrolidin-1-ylmethyl)-1H-benzo[d]imida-
zol-6-yl)amino)pyrimidin-4-yl)amino)-N,N-dimethylbenzamide (H-24).
Light yellow solid; Yield: 60.3%; m.p.: 238.6–2340.9 ◦C; MS (ESI) m/z:
489.54 [Mꢀ H]-. 1H NMR (400 MHz, DMSO‑d6) δ 12.20 (s, 1H), 9.32 (s,
1H), 9.17 (s, 1H), 8.37 (d, J = 8.4 Hz, 1H), 8.18 (s, 1H), 7.85 (s, 1H),
7.47 – 7.41 (m, 2H), 7.37 (s, 2H), 7.16 (t, J = 7.3 Hz, 1H), 4.05 (s, 2H),
2.99 (s, 6H), 2.81 (s, 4H), 1.81 (s, 4H). Anal. calcd for C25H27ClN8O (%):
C, 61.16; H, 5.54; N,22.82. Found (%): C, 61.13; H, 5.50; N, 22.85.
4.2.2. In vitro enzymatic assay
Mobility shift assay was used to evaluate enzymatic activities of
compounds. The mixture of peptide substrates, appropriate kinase
(Carna), ATP, and different concentrations of compounds were
converged with the kinase reaction buffer (50 mM HEPES, pH = 7.5,
0.0015% Brij-35, 10 mM MgCl2, 2 mM DTT), with DMSO solution as the
negative group. The kinase reaction was initiated by the addition of
tyrosine kinase proteins diluted in 39 µL of kinase reaction buffer solu-
tion and incubated for 1 h at 28 ◦C. And then 25 µL of stop buffer
(100mMHEPES, pH = 7.5, 0.015% Brij-35, 0.2% Coating Reagent#3,
50mMEDTA) was added to stop the reaction. The data were collected on
Caliper at 320 nm and 615 nm and converted to inhibition values. IC50
was presented in MS Excel and the curves fitted by XLfit excel add-
inversion 4.3.1.
4.1.6.25. 2-((5-chloro-2-((2-(piperidin-1-ylmethyl)-1H-benzo[d]imidazol-
6-yl)amino)pyrimidin-4-yl)amino)-N,N-dimethylbenzamide (H-25). Light
yellow solid; Yield: 67.2%; m.p.: 239.9–242.7 ◦C; MS (ESI) m/z: 503.76
[Mꢀ H]-. 1H NMR (400 MHz, DMSO‑d6) δ 12.09 (s, 1H), 9.29 (s, 1H),
9.16 (s, 1H), 8.38 (d, J = 8.2 Hz, 1H), 8.17 (s, 1H), 8.09 (d, J = 15.0 Hz,
1H), 7.80 (s, 1H), 7.41 (d, J = 7.6 Hz, 2H), 7.39 (s, 1H), 7.32 (s, 1H),
3.66 (s, 2H), 2.99 (s, 6H), 2.44 (s, 4H), 1.54 (d, J = 5.1 Hz, 4H), 1.40 (s,
2H).Anal. calcd for C26H29ClN8O (%): C, 61.84; H, 5.79; N,22.19. Found
(%): C, 61.88; H, 5.83; N, 22.15.
4.1.6.26. 2-((5-chloro-2-((2-(morpholinomethyl)-1H-benzo[d]imidazol-6-
yl)amino)pyrimidin-4-yl)amino)-N,N-dimethylbenzamide
(H-26). Gray
solid; Yield: 50.3%; m.p.: 237.6–240.1 ◦C; MS (ESI) m/z: 505.74
10

Z. Li et al.
Bioorganic & Medicinal Chemistry 37 (2021) 116108
7 Friboulet L, Li NX, Katayama R, et al. The ALK inhibitor ceritinib overcomes
crizotinib resistance in non-small cell lung cancer. Cancer Discov. 2014;4(6):662–673.
8 Coluccia AML, Gunby RH, Tartari CJ, et al. Anaplastic lymphoma kinase and its
signalling molecules as novel targets in lymphoma therapy. Expert Opin Ther Targets.
2005;9(3):515–532.
4.3. Molecular modeling
The molecular docking was performed with Accelrys Discovery
Studio 3.0. The protein files (PDB 4MKC and 2YFX) were obtained from
Protein Data Bank (http://www.rcsb.org/pdb/). The ALKG1202R model
was built using 4MKC as a template. 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 pro-
tein model was typed with the CHARM mforce field and a binding sphere
within 50 Å radius was defined as the binding site around the reference
ligand (ceritinib). Ceritinib and H-11 were drawn with ChemBio Draw
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 (Biovia, http://accelrys.com) and
the2D binding mode figure was generated from it. All 3D figures were
generated from Pymol (The Pymol Molecular Graphics System, Version
1.4.1. Schrodinger, LLC).
9 Wang L, Jiang ZL, Huang DL, et al. JAK/STAT3 regulated global gene expression
dynamics during late-stage reprogramming process. BMC Genomics. 2018;19(1):183.
10 Christensen JG, Zou HY, Arango ME, et al. Cytoreductive antitumor activity of PF-
2341066, a novel inhibitor of anaplastic lymphoma kinase and c-Met, in
experimental models of anaplastic large-cell lymphoma. Mol Cancer Ther. 2007;6:
3314–3322.
´
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Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
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Acknowledgments
This work was supported by National Natur-
al Science Foundation of China (No. 81673308) and Development Proj-
ect of Ministry of Education Innovation Team (No. IRT1073).
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Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmc.2021.116108.
22 Cao M, Chen YX, Zhao TM, et al. Pyrroformyl-containing 2,4-diaminopyrimidine
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