Design, synthesis, and biological activity of phenyl-pyrazole derivatives as BCR-ABL kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2015.04.083

Abstract 4-(Pyridin-3-yl)-1H-pyrazol-1-yl-phenyl-3-benzamide derivatives have been proposed as new BCR-ABL tyrosine kinase inhibitors by using combinational strategies of scaffold hopping and conformational constraint. In the present study, a series of 4-

Full text of DOI:10.1016/j.bmc.2015.04.083

Accepted Manuscript
Design, synthesis, and biological activity of phenyl-pyrazole derivatives as
BCR-ABL kinase inhibitors
Liming Hu, Yuyan Zheng, Zhipeng Li, Yujie Wang, Yongjuan Lv, Xuemei Qin,
Chengchu Zeng
PII:
S0968-0896(15)00404-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.04.083
BMC 12300
Reference:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
9 February 2015
28 April 2015
29 April 2015
Please cite this article as: Hu, L., Zheng, Y., Li, Z., Wang, Y., Lv, Y., Qin, X., Zeng, C., Design, synthesis, and
biological activity of phenyl-pyrazole derivatives as BCR-ABL kinase inhibitors, Bioorganic & Medicinal
Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.04.083
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
Design, synthesis, and biological activity of phenyl-pyrazole derivatives as BCR-
ABL kinase inhibitors
Liming Hu*, Yuyan Zheng, Zhipeng Li, Yujie Wang, Yongjuan Lv, Xuemei Qin, Chengchu Zeng
College of Life Science and Bioengineering, Beijing University of Technology, Beijing 100124, China
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
4-(pyridin-3-yl)-1H-pyrazol-1-yl-phenyl-3-benzamide derivatives have been proposed as new
BCR-ABL tyrosine kinase inhibitors by using combinational strategies of scaffold hopping and
conformational constraint. In the present study, a series of 4-(pyridin-3-yl)-1H-pyrazol-1-yl-
phenyl-3-benzamide derivatives were synthesized and their activities against BCR-ABL1 kinase
in vitro were evaluated by using Kinase-Glo assay. All new compounds showed moderate to
potent activities against wild-type (wt) BCR-ABL1 kinase with an IC50 range from 14.2 to 326.0
nM. Among them, seven compounds exhibited BCR-ABL1 kinase inhibitory activities with IC50
values less than 50nM. Compound 7a displayed the most potent inhibitory activity to BCR-ABL
kinase (IC50:14.2nM). Docking simulation was performed for compound 7a and 7i into the
BCR-ABL kinase structure active site to determine the probable binding model. The preliminary
structure-activity relationship was discussed. The interesting activities of these compounds may
make them promising candidates as therapeutic agents for chronic myelogenous leukemia.
Available online
Keywords:
BCR-ABL
Tyrosine kinase inhibitor
Phenyl-pyrazole
Molecular docking
2015 Elsevier Ltd. All rights reserved.
a great challenge despite of the clear success of imatinib.8 The
main mechanism of resistance is the overexpression of BCR-
1. Introduction
Chronic myeloid leukemia (CML) is clonal myeloproliferative
disorder characterized by excessive proliferation of cells of the
myeloid lineage and now represents about 20% of adult
leukemia.1 The occurrence of Philadelphia (Ph) chromosome is
characteristic of CML and is caused by the reciprocal translocation
involving chromosome 9 and chromosome 22. A segment of the
c-ABL gene on chromosome 9q34 is transposed onto the BCR
gene on chromosome 22q11, leading to the fusion of the Abelson
tyrosine kinase (ABL) gene on chromosome 9 with the
breakpoint cluster region (BCR) gene on chromosome 22 and the
ABL, mainly due to point mutations in the BCR-ABL kinase
domain. Currently, more than 100 mutations have been
discovered in patients with imatinib-resistant CML.5,9
To overcome resistances, several second-generation drugs have
been designed and synthesized, including nilotinib (AMN107)
and dasatinib (BMS-354825) to treat patients in all phases of
CML with resistances to imatinib (Figure 1).10,11 Most recently,
some third-generation BCR-ABL tyrosine kinase inhibitors were
disclosed to inhibit almost the full range of BCR-ABL kinase
domain mutations as well as the native kinase. Ponatinib
(AP24534) has been advanced for clinical investigation (Figure 1)
5. Although second-generation drugs can inhibit some imatinib-
resistant BCR-ABL mutants, there are still significant numbers
of resistant mutants which no alternative drugs could overcome.
Consequently, there is still necessary to discovery new
compounds that will enrich the pool of known tyrosine kinase
inhibitors.12
generation of
a constitutively active chimeric BCR-ABL
oncogene.2,3 The encoded protein of the recombined gene is a
fusion protein having constitutively active tyrosine kinase
activity. The fusion protein BCR-ABL contains a GRB2 binding
site, SH2 and SH3 domains, and a tyrosine-kinase(TK) domain
and can activate several signaling pathways, such as the janus
kinase(JAK)/signal transduction and transcription(STAT) pathway,
phosphaditylinositide-3 (PI3) kinase pathway, RAS/mitogen-activated
protein kinase (MAPK) pathways, and the myc pathway.4 Since
the BCR-ABL protein exists in greater than 90% of CML cases,
it has become a well-validated and novel target for designing
small molecular inhibitors to treat CML.5
So far, several cocrystal structures of BCR-ABL kinase with
their inhibitors have been reported. These inhibitors possessed
some key structural elements which formed crucial interactions
between the protein and inhibitors base on of structural analysis
of the inhibitors (i.e., imatinib and ponatinib) and BCR-ABL
kinase. There are four key structural elements: (1) “head region”,
a heterocyclic moiety which binds to the adenine pocket and
forms a hydrogen bond network with residues in the hinge region;
(2) “middle part”, a methylphenyl group which occupies the
hydrophobic pocket behind the gatekeeper residue; (3) “tail
Imatinib (STI571) approved by FDA in 2001, has been a great
success for curing CML (Figure 1).6 It is now treated as a first-
line drug in conventional treatment of CML because of its high
efficacy and lower side effects.7 Surfacing resistance has become
Correspondence: huliming@bjut.edu.cn, Tel:+86 10 6739 6211
1

region”, an additional hydrophobic group which binds the pocket;
(4) “linker part”, a moderate linker between the head region and
the middle part to avoid the steric clash with the residue of BCR-
ABL.1 Basing on the analysis, a series of phenyl-pyrazole
derivatives 7 are designed as BCR-ABL kinase inhibitors in
which a moderate pyrazole linker is utilized between the head
region and the middle part ( a methylphenyl group) as shown in
Figure 2.
Figure1. Chemical structures of FDA-approved BCR-ABL inhibitors and newly developed Ponatinib.
Figure 2. Design of phenyl-pyrazole derivative as new BCR-ABL inhibitor.
2. Results and discussion
2.1. Chemistry
The synthesis of target compounds 7a-7n was outlined in
Scheme 1. Compound 2 was produced by diazotization of
aromatic amine 2-methyl-5-nitroaniline (1). 2-Methyl-5-nitro-
phenylhydrazine (3) was synthesized by reduction reaction of the
intermediate 2 with stannous chloride dihydrate. 2-bromomalon-
aldehyde was treated with 3 to produce 4-bromo-1-(2-methyl-5-
nitrophenyl)-1H-pyrazole (4) by ring-closure reaction. The
intermediate 4 reacted with 3-pyridylboronic acid to obtain 3-(1-
2-methyl-5-nirtophenyl)-1H-pyrazol-4-yl)pyridine (5) by Heck
coupling reaction under nitrogen atmosphere. 4-methyl-3-
(pyridin-3-yl) aniline (6) was produced by reduction reaction of
the intermediate 5 with hydrogen in the presence of Pd/C.
Variable substituted benzoyl chloride in anhydrous dichloro-
methane was added dropwise to compound 6 at 0 oC in the
presence of triethylamine and then the reaction mixture was
slowly warmed to temperature and stirred 4-6 h till TLC
confirmed that the reaction had finished. Then the mixture
solutions were filtered to afford the target compounds 7a-7n. The
products were purified by recrystallization in EtOH to obtain 7a-
7n.
Scheme 1. Reactions and conditions: (a) NaNO2, HCl, H2O, 0-5 oC; (b) SnCl2.H2O, HCl, H2O, 0-5 oC, 70-75 %; (c) 2-bromomalonaldehyde, EtOH, 80 oC, 2 h,
30-40 %; (d) 3-pyridylboronic acid, Pd(PPh3)4, Na2CO3, dioxane/H2O(3:1), 100oC, 6 h, 70-75%; (e) H2, Pd/C, EtOH, rt; (f) triethylamine, CH2Cl2, 0 oC, rt,4 h,
60-70 %.
2

2.2. Inhibition of BCR-ABL activity
Table 1 lists the structures of the tested compounds and their
corresponding IC50 values. All new compounds showed
moderate potent activities against wild-type (wt) BCR-ABL1
kinase with an IC50 range from 14.2 to 326.0 nM. Among them,
seven compounds exhibited BCR-ABL1 kinase inhibitory
activities with IC50 values less than 50 nM. Interestingly, 7a, 7b,
7m exhibit an IC50 value as low as 14.2 nM, 15.9 nM, 16.8 nM in
BCR-ABL kinase inhibition, about 10-fold lower than control
compound Staurosporine (STSP) respectively. Compound 7a
stands out as the most active compound in the set with an IC50
value of 14.2 nM which clearly reveals that the large
hydrophobic group is detrimental to the activities on tyrosine
kinase and its physicochemical properties to keep ideal
hydrophil-lipophilic balance of the molecules. This group was
also widely found in the chemical structures of many other
kinase inhibitors. Morever, compounds (7d, 7e, 7f and 7k) was
also about three times higher than that of control compound
STSP. These results showed that these compounds were the
novel BCR-ABL kinase inhibitors. Furthermore, antitumor
activities of the compounds 7a-7n were evaluated against BCR-
ABL positive leukemia cell in comparison with control
compound STSP. Not surprisingly, most compounds also
displayed moderate suppression on the growth of the human
chronic myelogenous leukaemia K562 cells expressing high
level of BCR-ABL protein. In particular, compound 7a potently
inhibited the growth of K562 cells with an IC50 value of 0.27 µM,
which was about hundreds of times fold lower than that of
control compound STSP (153 µM). Besides, the antitumor
activity of other compounds was higher than that of control
STSP.
Table 1. Inhibitory activities of compounds 7a-7n towards BCR-ABL kinase and K562 cells in vitro
BCR-ABL Kinase
IC50(nM)
K562 Cells IC50
Compd.
R1
R2
(µM)
7a
7b
7c
7d
7e
7f
3-CF3
3-Cl
H
H
H
14.20
15.90
213.5
41.02
43.27
46.20
0.27
1.32
12.49
6.52
0.76
18.3
4-F
H
4-CH2CH3
4-CF3
4-OCH3
H
H
7g
7h
H
H
50.60
178.0
1.67
21.5
7i
7j
H
H
H
326.0
129.1
26.58
136.0
3.26
8.65
N
7k
N
7l
H
H
H
117.4
16.87
2.74
2.33
7m
7n
175.3
121.8
98.3
STSP
153.0
2.3. Docking Studies
3

B
A
Figure 3. A and B show the binding model of inhibitor 7a and 7i to BCR-ABL kinase. Oxygen atom (red stick), nitrogen atom (blue stick). H-bond (green dash
line). pi-pi stack (purple dash line).
To investigate the possible bonding mechanism of these com-
pounds, compound 7a and 7i were selected for docking studies.
The binding mode of selected molecule was studied by
Autodock 4.0 with the help of AutodockTools. BCR-ABL
tyrosine kinase (PDB:3cs9) was selected as the receptor for
docking study. The best docking conformation was selected for
the analysis and the docking result is shown in Figure 2. The
pyridazine moiety in 7a and 7i could formed an essential
hydrogen bond with the NH of Met318 in the hinge region of
Bcr-Abl. Morever, the amide also formed two hydrogen bonds
with Glu286 and Asp381. However, the pyrazole linker in 7a
could form pi-pi stacking interaction with Thr315 of BCR-ABL,
which may cause that inhibitory activities of compound 7a
towards BCR-ABL kinase is stronger than compound 7i.
was purified by column chromatography (silica gel, petroleum
ether:ethyl acetate = 4:1) to give the solid (2.3 g). Yellow solid;
yield: 70.0%; H NMR (DMSO-d6, 400 MHz, δ ppm): 7.84 (d, J
= 2.4 Hz, 1H), 7.37 (dd, J = 8.1, 2.4 Hz, 1H), 7.16 (d, J = 8.1 Hz,
1H), 6.88 (s, 1H), 4.22 (s, 2H), 2.13 (s, 3H). MS (ESI): m/z
168.1 [M+H]+.
1
4.1.2. 4-bromo-1-(2-methyl-5-nitrophenyl)-1H-pyrazole (4)
2-bromomalonaldehyde (2.4 g, 16.0 mmol) in EtOH (15 mL)
was treated with (2-methyl-5-nitrophenyl) hydrazine (4.0 g, 23.9
mmol) and refluxed for 1 h. The the reaction mixture was
concentrated under reduced pressure and the residue was purified
by column chromatography (silica gel, petroleum ether:ethyl
1
acetate=12:1) to the solid (1.4 g). Yellow solid; yield: 30%; H
NMR (CDCl3, 400 MHz, δ ppm): 8.36-8.10 (m, 2H), 7.74 (d, J
= 8.2 Hz, 2H), 7.53 (d, J = 8.7 Hz, 1H), 2.43 (s, 3H). MS (ESI):
m/z 281.4 [M+H]+.
3. Conclusion
In summary, a series of novel phenyl-pyrazole BCR-ABL
tyrosine kinase inhibitors were synthesized and identified as
BCR-ABL tyrosine kinase. The result indicates that compound
7a exhibited an IC50 value as low as 14.2 nM and showed highly
inhibition to BCR-ABL tyrosine kinase. 7a may be proved as a
new promising lead compound for the further development of
new BCR-ABL inhibitors overcoming the clinical acquired
resistance.
4.1.3. 3-(1-(2-methyl-5-nitrophenyl)-1H-pyrazol-4-yl)pyridine (5)
The boronic acid (1.8 g, 14.6mmol), the arylhalide (2.0 g, 7.1
mmol) and Pd(PPh3)4 (0.4 g, 0.35 mmol) were sequentially
added to degassed 1,4-dioxane (10 mL) and the mixture was
o
stirred at 25 C for 10 min. Degassed aqueous Na2CO3 solution
(1 M, 14.2 mmol) was added and the reaction mixture was
heated under argon at reflux for 6 h. The mixture was filtered
and concentrated under reduced pressure and the residue was
purified by column chromatography (silica gel, petroleum
ether:ethyl acetate = 1:1) to give the solid (1.5 g). Light yellow
4. Experimental section
4.1. Chemistry
1
solid; yield: 72%; H NMR (CDCl3, 400 MHz, δ ppm): 8.87 (s,
Unless otherwise noted, all materials were obtained from
commercial suppliers and dried and purified by standard
procedures. Melting points were determined on a Beijing Keyi
elec-opti instrument factory melting point apparatus. Proton
nuclear magnetic resonance (1H NMR) spectra were recorded
with a Bruker Avance DRX400 spectrometer with CDCl3 or
DMSO-d6 as the solvent and tetramethyl-silane (TMS) as the
internal standard. The chemical shifts were reported in δ (ppm).
Mass spectra (MS) data were obtained using Esquire6000 Mass
Spectrometer. Petroleum ether used for column chromatography
had a boiling range of 60-90 oC.13
1H), 8.56 (d, J = 3.9 Hz, 1H), 8.31 (d, J = 2.2 Hz, 1H), 8.24 (dd,
J = 8.5, 2.2 Hz, 1H), 8.10 (s, 1H), 8.00 (s, 1H), 7.87 (d, J = 7.9
Hz, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.28 (s, 1H), 2.06 (s, 3H). MS
(ESI): m/z: 281.1 [M+H]+.
4.1.4. 4-methyl-3-(4-(pyridin-3-yl)-1H-pyrazol-1-yl)aniline (6)
Compounds 5 (1.0 g, 3.6 mmol) in EtOH (10 mL) was
hydrogenated over Pd/C (0.1 g) under balloon pressure over
night. The mixture was filtered and the filtrate was under
reduced pressure to the next step directly.
4.1.5. General procedure for the synthesis of derivatives 7.
4.1.1. (2-methyl-5-nitrophenyl)hydrazine (3)
The appropriate Benzoyl chloride (1.2 mmol) in CH2Cl2 (10
mL) was added to an ice cooled solution of compound 6 ( 1.2
mmol), Et3N (0.2 mL), and CH2Cl2 (5 mL). The solution was
allowed to stir at room temperature for 3 h. A large amount of
solid precipitated and was filtered. The residue was purified by
recrystallization in EtOH.
2-methyl-5-nitroaniline (3.0 g, 19.7 mmol) was dissolved in a
mixture of H2O (5 mL) and 37% HCl (5 mL). A solution of
NaNO2 (1.4 g, 20.3 mmol) in H2O (15 mL) was added dropwise
at 0 oC, followed by the addition of SnCl2·H2O (9.0 g, 39.8
mmol) in 37% HCl (7 mL) at that temperature. After stirring at 0
oC for 0.5 h, the reaction mixture was neutralized with 1M
NaOH to pH 7-8, and extracted with DCM. The organic phase
was separated and dried over anhydrous MgSO4. The organic
phase was concentrated under reduced pressure and the residue
4.1.5.1 N-(4-methyl-3-(4-(pyridin-3-yl)-1H-pyrazol-1-yl)phenyl)-
3-(trifluoromethyl)benzamide (7a)
4

White solid; yield: 65.0%; m.p. 178-179 oC; 1H NMR
(DMSO-d6, 400 MHz, δ ppm): 10.68 (s, 1H), 9.02 (s, 1H), 8.71
(s, 1H), 8.48 (d, J = 4.1 Hz, 1H), 8.43-8.23 (m, 3H), 8.16 (t, J =
17.2 Hz, 1H), 7.97 (d, J = 10.7 Hz, 2H), 7.80 (dd, J = 15.3, 7.9
Hz, 2H), 7.48 (dt, J = 12.1, 6.0 Hz, 1H), 7.42 (d, J = 8.4 Hz, 1H),
2.27 (s, 3H). 13C NMR (DMSO-d6, 100 MHz, δ ppm): 164.52,
147.04, 146.16, 139.76, 138.46, 137.92, 135.94, 133.67, 132.33,
131.91, 130.23, 129.86, 129.54, 129.44, 128.90, 128.73, 128.72,
128.29, 124.66, 120.75, 119.90, 118.16, 17.93. HRMS (ESI):
m/z 423.1433 calcd for C23H17F3N4O [M+H]+, found 423.1432.
1-yl)phenyl)benzamide (7f)
White solid; yield: 70.0%; m.p. 208-209 oC; 1H NMR
(DMSO-d6, 400 MHz, δ ppm): 10.25 (s, 1H), 8.99 (d, J = 1.9 Hz,
1H), 8.67 (s, 1H), 8.45 (d, J = 4.6 Hz, 1H), 8.31 (s, 1H), 8.10 (d,
J = 7.9 Hz, 1H), 8.02-7.91 (m, 3H), 7.78 (dt, J = 11.7, 5.8 Hz,
1H), 7.50-7.32 (m, 2H), 7.07 (d, J = 8.7 Hz, 2H), 3.84 (s, 3H),
2.25 (s, 3H). 13C NMR (DMSO-d6, 100 MHz, δ ppm): 165.41,
162.50, 147.85, 147.00, 139.76, 138.47, 138.31, 132.79, 131.76,
130.05, 129.24, 129.11, 128.54, 127.63, 127.16, 127.16, 124.31,
120.47, 120.11, 117.90, 114.13, 55.91, 17.87. HRMS (ESI): m/z
385.1665 calcd for C23H20N4O2 [M+H]+, found 385.1667.
4.1.5.2 3-chloro-N-(4-methyl-3-(4-(pyridin-3-yl)-1H-pyrazol-1-
yl)phenyl)benzamide (7b)
4.1.5.7 N-(4-methyl-3-(4-(pyridin-3-yl)-1H-pyrazol-1-yl)phenyl)
-4-((4-methylpiperazin-1-yl)methyl)benzamide (7g)
White solid; yield: 70.0%; m.p. 160-164 oC; 1H NMR
(DMSO-d6, 400 MHz, δ ppm): 10.50 (s, 1H), 8.99 (s, 1H), 8.68
(s, 1H), 8.45 (d, J = 4.7 Hz, 1H), 8.32 (s, 1H), 8.10 (d, J = 7.9 Hz,
1H), 8.03 (s, 1H), 7.99-7.90 (m, 2H), 7.79 (d, J = 8.3 Hz, 1H),
7.67 (d, J = 7.9 Hz, 1H), 7.58 (t, J = 7.9 Hz, 1H), 7.42 (t, J = 6.7
Hz, 2H), 2.26 (s, 3H). 13C NMR (DMSO-d6, 100 MHz, δ ppm):
164.55, 147.88, 147.02, 139.80, 138.38, 137.97, 137.07, 133.74,
132.80, 131.98, 131.89, 130.90, 129.25, 128.51, 128.20, 127.85 ,
126.94, 124.31, 120.58, 120.17, 118.03, 17.93. HRMS (ESI):
m/z 389.1169 calcd for C22H17ClN4O [M+H]+, found 389.1167.
Light yellow solid; yield: 80.0%; m.p. 209-211 oC; 1H NMR
(DMSO-d6, 400 MHz, δ ppm): 10.32 (d, J = 48.9 Hz, 1H), 8.94
(d, J = 36.3 Hz, 1H), 8.69 (s, 1H), 8.44 (d, J = 4.6 Hz, 1H), 8.32
(s, 1H), 8.10 (d, J = 7.9 Hz, 1H), 7.98 (d, J = 1.8 Hz, 1H), 7.92 (d,
J = 8.1 Hz, 2H), 7.81-7.66 (m, 2H), 7.48-7.41 (m, 2H), 3.82 (s,
2H), 3.36 (s, 8H), 2.50 (s, 3H), 2.25 (s, 3H). 13C NMR (CDCl3,
100 MHz, δ ppm): 165.78, 147.76, 146.94, 142.69, 139.57,
137.83, 136.80, 133.33, 132.82, 131.77, 129.28, 129.20, 128.29,
127.71, 127.22, 123.78, 120.55, 120.25, 118.07, 62.41, 54.95,
52.87, 45.84, 17.75. HRMS (ESI): m/z 467.2559 calcd for
C28H30N6O [M+H]+, found 467.2556.
4.1.5.3 4-fluoro-N-(4-methyl-3-(4-(pyridin-3-yl)-1H-pyrazol-1-
yl)phenyl)benzamide (7c)
White solid; yield: 75.0%; m.p. 186-190 oC; 1H NMR
(DMSO-d6, 400 MHz, δ ppm): 10.51 (s, 1H), 9.13 (s, 1H), 8.79
(s, 1H), 8.57 (d, J = 4.8 Hz, 1H), 8.50-8.30 (m, 2H), 8.08 (dt, J =
18.1, 9.0 Hz, 2H), 8.02 (d, J = 1.6 Hz, 1H), 7.79 (dd, J = 8.3, 1.7
Hz, 1H), 7.68 (dd, J = 7.9, 5.2 Hz, 1H), 7.38 (dd, J = 17.8, 8.8
Hz, 3H), 2.26 (s, 3H). 13C NMR (DMSO-d6, 100 MHz, δ ppm):
164.96, 144.48, 143.58, 139.61, 138.63, 138.23, 136.22, 131.85,
131.51, 131.49, 130.96, 130.87, 130.10, 129.98, 127.95, 125.69,
120.74, 119.09, 117.98, 115.92, 115.71, 17.89. HRMS (ESI):
m/z 373.1465 calcd for C22H17FN4O [M+H]+, found 373.1462.
4.1.5.8 4-methoxy-N-(4-methyl-3-(4-(pyridin-3-yl)-1H-pyrazol-1-
yl)phenyl)benzamide (7h)
White solid; yield: 82.0%; m.p. 197-199 oC; 1H NMR
(CDCl3, 400 MHz, δ ppm): 8.79 (s, 1H), 8.63 (s, 1H), 8.47 (s,
1H), 7.96 (d, J = 17.9 Hz, 1H), 7.91 (s, 1H), 7.88-7.76 (m, 4H),
7.55 (d, J = 8.1 Hz, 1H), 7.41 (d, J = 7.8 Hz, 2H), 7.35-7.20 (m,
2H), 3.67 (t, J = 21.6 Hz, 4H), 3.55 (s, 2H), 2.45 (s, 4H), 2.24 (s,
3H); 13C NMR (CDCl3, 100 MHz, δ ppm): 165.77, 147.64,
146.82, 141.91, 139.55, 137.84, 136.77, 133.56, 132.90, 131.76,
129.36, 129.24, 127.72, 127.27, 123.83, 120.60, 120.23, 118.13,
66.79, 62.78, 53.52, 17.70. HRMS (ESI): m/z 454.2243 calcd for
C27H27NO2 [M+H]+, found 454.2243.
4.1.5.4 4-fluoro-N-(4-methyl-3-(4-(pyridin-3-yl)-1H-pyrazol-1-
yl)phenyl)benzamide (7d)
White solid; yield: 76.0%; m.p. 186-190 oC; 1H NMR
(DMSO-d6, 400 MHz, δ ppm): 10.34 (s, 1H), 8.99 (s, 1H), 8.68
(s, 1H), 8.44 (d, J = 3.8 Hz, 1H), 8.31 (s, 1H), 8.10 (d, J = 7.8
Hz, 1H), 7.99 (s, 1H), 7.91 (d, J = 8.0 Hz, 2H), 7.72 (ddd, J =
18.1, 17.3, 5.9 Hz, 3H), 7.45-7.32 (m, 4H), 3.35 (s, 3H), 2.50 (s,
2H), 2.23 (t, J = 20.4 Hz, 3H). 13C NMR (DMSO-d6, 100 MHz, δ
ppm): 165.93, 148.35, 147.85, 146.99, 139.75, 138.34, 132.78,
132.55, 132.18, 132.09, 131.96, 131.80, 129.25, 129.12, 128.53,
128.25, 127.75, 124.32, 120.45, 120.10, 117.86, 28.54, 19.11,
17.91. HRMS (ESI): m/z 383.1872 calcd for C24H22N4O2
[M+H]+, found 383.1869.
4.1.5.9 4-((1H-imidazol-1-yl)methyl)-N-(4-methyl-3-(4-(pyridin-
3-yl)-1H-pyrazol-1-yl)phenyl)benzamide (7i)
White solid; yield: 75.0%; m.p. 162-164 oC; 1H NMR
(CDCl3, 400 MHz, δ ppm): 8.84 (s, 1H), 8.58-8.45 (m, 2H), 8.02
(s, 1H), 7.96 (s, 1H), 7.87 (dd, J = 19.8, 8.2 Hz, 4H), 7.61 (dd, J
= 13.6, 5.2 Hz, 2H), 7.34 (dd, J = 11.8, 6.6 Hz, 2H), 7.23 (d, J =
8.0 Hz, 2H), 7.13 (s, 1H), 6.94 (s, 1H), 5.21 (s, 2H), 2.31 (s, 3H).
13C NMR (DMSO-d6, 100 MHz, δ ppm): 165.69 (s), 147.86 (s),
146.98 (s), 141.76 (s), 139.75 (s), 138.36 (s), 138.30 (s), 138.21
(s), 137.97 (s), 134.54 (s), 132.80 (s), 131.85 (s), 129.27 (s),
128.76 (s), 128.58 (s), 128.54 (s), 127.91 (s), 124.34 (s), 120.50
(s), 120.37 (s), 120.29 (s), 120.12 (s), 117.89 (s), 101.10 (s),
49.70 (s), 17.93 (s). HRMS (ESI): m/z 435.1933 calcd for
C26H22N6O [M+H]+, found 435.1935.
4.1.5.5 N-(4-methyl-3-(4-(pyridin-3-yl)-1H-pyrazol-1-yl)phenyl)
-4-(trifluoromethyl)benzamide (7e)
White solid; yield: 68.0%; m.p. 225-226 oC; 1H NMR
(DMSO-d6, 400 MHz, δ ppm): 10.64 (s, 1H), 8.99 (d, J = 1.7 Hz,
1H), 8.69 (s, 1H), 8.45 (dd, J = 4.7, 1.4 Hz, 1H), 8.33 (s, 1H),
8.16 (t, J = 10.6 Hz, 2H), 8.13-8.06 (m, 1H), 7.98 (t, J = 5.9 Hz,
1H), 7.93 (d, J = 8.3 Hz, 2H), 7.79 (dd, J = 8.3, 2.0 Hz, 1H), 7.42
(dd, J = 6.3, 2.3 Hz, 2H), 2.27 (s, 3H). 13C NMR (DMSO-d6, 100
MHz, δ ppm): 164.88, 147.87, 147.00, 139.81, 138.89, 138.40,
137.91, 132.79, 131.94, 129.27, 129.05, 128.50, 128.50, 128.30,
128.30, 125.92 , 125.89, 125.72, 124.32, 120.59, 120.17, 118.02,
17.95. HRMS (ESI): m/z 423.1433 calcd for C23H17F3N4O
[M+H]+, found 423.1425.
4.1.5.10 4-((diethylamino)methyl)-N-(4-methyl-3-(4-(pyridin-3-
yl)-1H-pyrazol-1-yl)phenyl)benzamide (7j)
White solid; yield: 80.0%; m.p. 169-172 oC; 1H NMR
(CDCl3, 400 MHz, δ ppm): 8.81 (t, J = 11.5 Hz, 1H), 8.49 (dt, J
= 13.3, 6.6 Hz, 1H), 8.01 (s, 2H), 7.95 (s, 1H), 7.85-7.78 (m, 4H),
7.57 (dd, J = 8.3, 2.3 Hz, 1H), 7.48 (d, J = 8.1 Hz, 2H), 7.32 (dd,
J = 8.2, 4.2 Hz, 2H), 2.63-2.48 (m, 4H), 2.29 (s, 3H), 1.06 (t, J =
7.1 Hz, 6H); 13C NMR (CDCl3, 100 MHz, δ ppm): 165.94,
147.73, 146.93, 144.19, 139.53, 137.80, 136.87, 133.12, 132.81,
131.72, 129.17, 129.05, 128.31, 127.72, 127.19, 123.76, 120.64 ,
4.1.5.6 4-methoxy-N-(4-methyl-3-(4-(pyridin-3-yl)-1H-pyrazol-
5

120.23, 118.14, 57.20, 46.87, 17.68, 11.61. HRMS (ESI): m/z
440.2450 calcd for C27H29N5O [M+H]+, found 440.2451.
DMSO as 5mM stock solution. Then, the stock solution was
diluted to 1000 µM with DMSO and transferred to the dose plate.
The compound was serially diluted with DMSO in 5-fold. Then,
each concentration was diluted 10-fold with reaction buffer to
4.1.5.11 4-((4-methyl-1,4-diazepan-1-yl)methyl)-N-(4-methyl-3-
(4-(pyridin-3-yl)-1H-pyrazol-1-yl)phenyl)benzamide (7k)
obtain
a 10X final concentration. The compounds were
White solid; yield: 79.0%; m.p. 178-180 oC; 1H NMR
(CDCl3, 400 MHz, δ ppm): 8.78 (d, J = 1.7 Hz, 1H), 8.74 (s, 1H),
8.46 (dd, J = 4.8, 1.4 Hz, 1H), 7.97 (s, 1H), 7.91 (s, 1H), 7.87-
7.76 (m, 4H), 7.56 (dd, J = 8.3, 2.0 Hz, 1H), 7.40 (d, J = 8.1 Hz,
2H), 7.34-7.26 (m, 1H), 7.24 (d, J = 8.4 Hz, 1H), 3.64 (s, 2H),
2.72-2.63 (m, 6H), 2.60 (d, J = 5.7 Hz, 2H), 2.36 (s, 3H), 2.23 (s,
3H), 1.89-1.72 (m, 2H). 13C NMR (CDCl3, 100 MHz, δ ppm):
165.95, 147.73, 146.92, 144.05, 139.52, 137.80, 136.88, 133.19,
132.80, 131.73, 129.14, 128.91, 128.29, 127.73, 127.24, 123.78,
120.62 , 120.21, 118.13, 62.38, 57.34, 56.62, 54.31, 46.86, 27.27,
17.72. HRMS (ESI): m/z 481.2716 calcd for C29H32N6O [M+H]+,
found 481.2716 .
transferred with their concentrations ranging from 100 µM to
0.006 µM to an assay plate for the BCR-ABL activity test with a
volume of 1 µL/well. The positive compound STSP, with a stock
concentration of 10 mM in DMSO, was diluted with DMSO to
100 µM and then 5-fold serially diluted with DMSO. Each
concentration in DMSO is 10-fold diluted with reaction buffer to
obtain 10X final concentration. The STSP in concentrations
ranging from 10 µM to 0.00064 µM was transferred to an assay
plate for the BCR-ABL activity test with 1 µL/well. For HPE (no
kinase and no compound, but containing ATP, substrate and 1%
DMSO) and ZPE (no compound but containing kinase, ATP,
substrate and 1% DMSO) well, 2 µL DMSO 10-fold was diluted
with reaction buffer (containing 25 mM HEPES, 10 mM MgCl2,
100 µg/mL BSA, 0.01% TritonX-100, 2.5 mM DTT and adjusted
pH to 7.4) to obtain 10% DMSO solution. It was then transferred
to the assay plate at 1 µL/well. The procedure of kinase reaction
is: 1) Add 10× compound to the assay plate in a 384-well plate
layout, 1 µL/well. For the HPE and ZPE well, equal volume (1
µL/well) of 10% DMSO was added to the 384-well assay plate; 2)
Add 2.5× kinase ABL1 into the assay plate as 384-well plate
layout, 4µL/well. For HPE wells, equal volume (4 µL/well) of
assay buffer was added to the 384-well assay plate; 3) Centrifuge
the assay plate with 1000 rpm for 1 min to mix them; 4) Pre-
incubate the assay plate at 30 °C for 30 min; 5) Mix an equal
volume of 4× ATP and 4 X substrate to obtain 2× ATP-substrate
mixtures. 2 X ATP-ABL tide mixture is the reaction mixture for
kinase ABL1. 6) Add 2×ATP-substrate mixture to the assay plate,
5 µL /well; 7) Centrifuge the assay plate with 1000 rpm for 1
4.1.5.12 N-(4-methyl-3-(4-(pyridin-3-yl)-1H-pyrazol-1-yl)phenyl)
-4-(pyrrolidin-1-ylmethyl)benzamide (7l)
White solid; yield: 70.0%; m.p. 179-180 oC; 1H NMR
(CDCl3, 400 MHz, δ ppm): 8.79 (d, J = 1.6 Hz, 1H), 8.69 (s,
1H), 8.47 (dd, J = 4.7, 1.2 Hz, 1H), 7.97 (s, 1H), 7.88 (d, J = 11.8
Hz, 1H), 7.86-7.74 (m, 4H), 7.53 (dd, J = 8.3, 2.0 Hz, 1H), 7.39
(d, J = 8.1 Hz, 2H), 7.34-7.26 (m, 1H), 7.22 (d, J = 8.4 Hz, 1H),
3.63 (s, 2H), 2.48 (s, 4H), 2.22 (s, 3H), 1.77 (s, 4H). 13C NMR
(CDCl3, 100 MHz, δ ppm): 165.92, 147.76, 146.94, 143.91,
139.50, 137.80, 136.81, 133.11, 132.79, 131.71, 129.19, 128.97,
128.29, 127.71, 127.21, 123.77, 120.68 , 120.23, 118.20, 60.24,
54.20, 23.49, 17.69. HRMS (ESI): m/z 438.2294 calcd for
C27H27N5O [M+H]+, found 438.2292 .
4.1.5.13 4-((4-ethylpiperazin-1-yl)methyl)-N-(4-methyl-3-(4-(py-
ridin-3-yl)-1H-pyrazol-1-yl)phenyl)benzamide (7m)
o
min to mix them; 8) Incubate the plate for 1 hour at 30 C; 9)
White solid; yield: 82.0%; m.p. 168-170 oC; 1H NMR
(CDCl3, 400 MHz, δ ppm): 8.80 (d, J = 1.4 Hz, 1H), 8.61 (s,
1H), 8.49 (d, J = 4.3 Hz, 1H), 7.99 (s, 1H), 7.94 (s, 1H), 7.84 (t, J
= 10.2 Hz, 4H), 7.61 (d, J = 8.1 Hz, 1H), 7.38 (t, J = 11.9 Hz,
2H), 7.36-7.24 (m, 2H), 3.57 (s, 2H), 2.65 (dd, J = 19.3, 12.1 Hz,
8H), 2.27 (s, 3H), 1.43 (s, 2H), 1.17 (t, J = 7.2 Hz, 3H). 13C
NMR (CDCl3, 100 MHz, δ ppm): 165.75, 147.74, 146.92,
142.07, 139.60, 137.84, 136.83, 133.57, 132.84, 131.79, 129.29,
129.23, 128.30, 127.71, 127.32, 123.79, 120.55, 120.27, 118.05,
62.10, 52.19, 51.75, 26.90, 17.73, 10.88. HRMS (ESI): m/z
481.2716 calcd for C29H32N6O [M+H]+, found 481.2716.
Kinase-glo was added to each well (10 µL/well), and then
incubated for 20 min at 27 C; 10) Read luminescence signal
with Envision. The raw data were analyzed by Prism 5.0, and the
inhibitory rate was calculated by the following formula:
Compound inhibitory rate = (“compound” reading-ZPE) /
(HPE-ZPE)×100%
o
4.3. K562 Cells grouth inhibitory assay
Growth inhibitory activities were evaluated on K562
leukemia cancer cell lines. The effects of the compounds on cell
viability were evaluated using the MTT assay. Exponentially
growing cells were harvested and plated in 96-well plates at a
concentration of 1×104 cells/well, and incubated for 24 h at 37 oC.
The cells in the wells were, respectively, treated with target
compounds at various concentrations for 48 h. Then, 20 mL
MTT (5 mg/mL) was added to each well and incubated for 4 h at
37 oC. After the supernatant was discarded, 150 mL DMSO was
added to each well, and the absorbance values were determined
by a microplate reader (Bio-Rad Instruments) at 490 nm. The
IC50 values were calculated according to inhibition ratios.
4.1.5.14 4-((4-(hydroxymethyl)piperazin-1-yl)methyl)-N-(4-me-
thyl-3-(4-(pyridin-3-yl)-1H-pyrazol-1-yl)phenyl)benzamide (7n)
White solid; yield: 70.0%; m.p. 172-175 oC; 1H NMR
(CDCl3, 400 MHz, δ ppm): 8.80 (d, J = 1.4 Hz, 1H), 8.61 (s, 1H),
8.49 (d, J = 4.3 Hz, 1H), 7.99 (s, 1H), 7.94 (s, 1H), 7.84 (t, J =
10.2 Hz, 4H), 7.61 (d, J = 8.1 Hz, 1H), 7.38 (t, J = 11.9 Hz, 2H),
7.36-7.24 (m, 2H), 3.57 (s, 2H), 2.65 (dd, J = 19.3, 12.1 Hz, 8H),
2.27 (s, 3H), 1.43 (s, 2H), 1.17 (t, J = 7.2 Hz, 3H). 13C NMR
(CDCl3, 100 MHz, δ ppm): 165.75, 147.74, 146.92, 142.07,
139.60, 137.84, 136.83, 133.57, 132.84, 131.79, 129.29, 129.23,
128.30, 127.71, 127.32, 123.79, 120.55, 120.27, 118.05, 62.10,
52.19, 51.75, 26.90, 17.73, 10.88. HRMS (ESI): m/z 497.2665
calcd for C29H32N6O2 [M+H]+, found 497.2662.
4.4. Molecular docking methods
4.4.1. BCR-ABL kinase core initial model building
The BCR-ABL kinase core model was generated by
Discovery Studio 4.0 (DS4.0) based on the 3cs9 protein
crystallographic structure. Unwanted water and ligands were
removed by the DS4.0, and the lost atoms such as hydrogen were
added to build the initial receptor structure for docking.14
4.2. BCR-ABL kinase inhibitory assay
The Kinase-Glo luminescent kinase assay is a homogeneous
non-radioactive method for determining the activity of purified
kinases by quantifying the amount of ATP remaining in solution
following a kinase reaction. Compound 7a-7n was dissolved in
4.4.2. Small molecular preparation
6

12. M. Mojzych; V. Subertov; A. Bielawska; K. Bielawski; V.
Bazgier; K. Berka; T. Gucký; E. Fornal; V. Krystof. E. J. Med.
Chem. 2014, 78, 217.
13. L.F. Yang; X.M. Xu; Y.L. Huang; B. Zhang; C.C. Zeng; H.Q. He;
C.X. Wang; L.M. Hu. Bioorg. Med. Chem. Lett., 2010, 20, 5496.
14. Accelrys Software Inc. Discovery Studio Modeling Environment,
Release 4.0; San Diego: Accelrys Software Inc. Eur. J. Med.
Chem. 2013.
The structure of 7a and 7i was drawn by Gaussian03
software, then optimized the molecules to the minimum energy
conformation used the semi-empirical AM1 method.
4.4.3. Docking protocol
Docking procedure was performed by AutoDock 4 software
with the help of Autodock Tools. Firstly, the no-polar hydrogen
of receptor and ligands were removed, and then add the
Computer Gasteiger charge for receptor and ligands. The grid
maps were calculated using AutoGrid4 for three dockings, a grid
map with 70×80×70 points and a grid-point spacing of 0.375 Å
was applied. In order to fully explore the possible binding
conformations, 100 conformations were generated using the
Lamarckian Genetic Algorithm (LGA). For other docking
parameters, standard values were used as software default.
Cluster analysis was performed on the results using a root mean
square (RMS) tolerance of 2.0 Å. The conformations and
binding energy for further analysis were obtained from the
average of the biggest cluster.15
15. G. M. Morris; D. S. Goodsell; R. S. Halliday; R.Huey; W. E. Hart;
R. K. Belew and A. J. Olson. J. Comput. Chem., 1998, 19, 1639.
Acknowledgments
The authors would like to acknowledge financial support
from the Chinese Natural Science Foundation Project
(21272020), the Project of Construction of Innovative Teams
and Teacher Career Development for Universities and
Colleges Under Beijing Municipality (IDHT20140504).
References and notes
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Graphical abstract
8