Discovery of novel Bcr-Abl inhibitors targeting myristoyl pocket and ATP site

Article abstract of DOI:10.1016/j.bmc.2014.10.030

Bcr-Abl plays an essential role in the pathogenesis and development of chronic myeloid leukaemia (CML). Inhibition of Bcr-Abl has great potential for therapeutic intervention in CML. In order to obtain novel and potent Bcr-Abl inhibitors, twenty seven 4,6-disubstituted pyrimidines were synthesized and evaluated herein. The biological results indicated that four compounds of them (C4, C5, C16, and C23) were potent Bcr-Abl inhibitors which were comparable to positive control. Moreover, C4 and C5 displayed promising antiproliferative activity against K562 cells. The results suggested that these 4,6-disubstituted pyrimidines could serve as promising leads for further optimization of Bcr-Abl inhibitors.

Full text of DOI:10.1016/j.bmc.2014.10.030

Bioorganic & Medicinal Chemistry 22 (2014) 6876–6884
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of novel Bcr–Abl inhibitors targeting myristoyl pocket
and ATP site
⇑
Jinyun Dong , Wen Lu , Xiaoyan Pan, Ping Su, Yaling Shi, Jinfeng Wang, Jie Zhang
School of Pharmacy, Health Science Center, Xi’an Jiaotong University, No. 76, Yanta West Road, Xi’an, Shaanxi Province 710061, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Bcr–Abl plays an essential role in the pathogenesis and development of chronic myeloid leukaemia
(CML). Inhibition of Bcr–Abl has great potential for therapeutic intervention in CML. In order to obtain
novel and potent Bcr–Abl inhibitors, twenty seven 4,6-disubstituted pyrimidines were synthesized and
evaluated herein. The biological results indicated that four compounds of them (C4, C5, C16, and C23)
were potent Bcr–Abl inhibitors which were comparable to positive control. Moreover, C4 and C5
displayed promising antiproliferative activity against K562 cells. The results suggested that these 4,
6-disubstituted pyrimidines could serve as promising leads for further optimization of Bcr–Abl inhibitors.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 14 October 2014
Revised 21 October 2014
Accepted 21 October 2014
Available online 27 October 2014
Keywords:
Bcr–Abl
Allosteric pocket
ATP-binding site
Myristoyl pocket
Inhibitors
1. Introduction
molecules may potentially be unaffected by mutations of kinase
domain and retain potency against resistant Bcr–Abl variants.
Chronic myeloid leukaemia (CML) is
a
myeloproliferative
Imatinib is ATP-competitive Bcr–Abl tyrosine kinase inhibitors.
Alternatively, other regulatory sites on Bcr–Abl are potential tar-
gets for small-molecule inhibitors. Myristoylation at myristoyl
binding pocket (allosteric site) of Bcr–Abl could stabilize kinase
in its inactive conformation. It is expected that small molecules
which bind to the myristoyl site could keep Bcr–Abl in inactive
configuration. GNF-2 (Fig. 2) was demonstrated to bind to the myr-
istoyl pocket which located near the carboxy terminus of ATP site.6
It could bind to myristoyl site and display potent inhibitory activity
against both wild and mutant Bcr–Abl.
disorder characterized at the molecular level by the expression of
Bcr–Abl. Bcr–Abl plays an essential role in the development of
CML. Because the Bcr–Abl tyrosine kinase is active in greater than
90% of CML cases, it has becoming an attractive target for anticancer
drugs discovery.1 Identification of the disease mechanism
prompted the first drug discovery efforts targeting the inhibition
of Bcr–Abl as means to treat CML.2 A number of potent Bcr–Abl
inhibitors have been developed and achieved clinical success in
CML patients. Imatinib (STI571) is the first drug in the family of
Bcr–Abl inhibitors while Nilotinib (AMN107) and Dasatinib (BMS-
345825) are second generation drugs. Ponatinib (AP24534) is an
orally active Bcr–Abl inhibitor while Bosutinib (SKI-606) and
Bafetinib (INNO-406) have efficacy against various point mutations
in the Bcr–Abl tyrosine kinase (Fig. 1).3 However, clinical success
was soon tempered by the emergence of drug resistance.
Bcr–Abl mutation-induced resistance remains
a
major
challenge for clinical management of CML. It was demonstrated
that combinations of GNF-2 and Imatinib could cooperate to sup-
press the emergence of resistance mutations.5 Simultaneous bind-
ing to Bcr–Abl with myristoyl site and ATP pocket could decrease
the appearance of resistance-conferring mutations and result in
the inhibition of both wild and mutant Bcr–Abl.7 Encouraged by
the clinical success of Imatinib as Bcr–Abl inhibitors for the treat-
ment of CML, we made our effort to develop new small molecules
to overcome resistance to ATP-competitive Bcr–Abl inhibitors.
GNF-5, the hydroxylethylamide analog of GNF-2, is a highly
selective non-ATP competitive Bcr–Abl inhibitor binding to myri-
stoyl pocket. Structural analysis of chemical features of GNF-5
and Imatinib revealed that they possessed similar core structures
(indicated in blue).8 Based on the similar core structures of
Imatinib and GNF-5, we designed a series of 4,6-disubstituted
All of these approved drugs compete with ATP for the binding
site of Bcr–Abl and provide significant clinical benefit.4 However,
acquired resistance has become a major challenge for treatment
of CML. An alternative strategy to discovery of new Bcr–Abl inhib-
itors involves the development of agents targeting Bcr–Abl sites
which are different with ATP site (Fig. 2).4 These novel small
⇑
Corresponding author. Tel./fax: +86 29 82655451.
E-mail address: zhj8623@mail.xjtu.edu.cn (J. Zhang).
Both authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmc.2014.10.030
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

J. Dong et al. / Bioorg. Med. Chem. 22 (2014) 6876–6884
6877
N
N
N
F
Cl
F
F
N
O
O
HN
S
N
N
HN
O
NH
N
N
H
N
N
N
N
N
H
N
N
N
H
OH
N
N
Dasatinib, BMS-345825
Imatinib, STI571
Nilotinib, AMN107
N
N
N
F
Cl
Cl
F
F
N
F
F
F
O
NH
N
O
O
O
NH
HN
O
N
N
N
H
N
N
N
N
N
N
N
N
Bafetinib, INNO-406
Bosutinib, SKI-606
Ponatinib, AP24534
Figure 1. Structures of approved Bcr–Abl inhibitors.
Figure 2. Different binding modes of inhibitors with Bcr–Abl.
pyrimidines to mimic the scaffold of Imatinib and GNF-5 (Fig. 3).
Benzoylation piperazine was attached to the scaffold to increase
the affinity with Bcr–Abl as well as the solubility.9 Here we
described a novel Bcr–Abl inhibitors design strategy, combining
the structural features of two classes of Bcr–Abl inhibitors.10 We
expect that these molecules could bind to both ATP and myristoyl
site of Bcr–Abl so as to overcome resistance to either agent alone.
Herein, we described the design, synthesis and biological
evaluation of twenty seven 4,6-disubstituted pyrimidines as
Bcr–Abl inhibitors targeting at myristoyl pocket and ATP site.

6878
J. Dong et al. / Bioorg. Med. Chem. 22 (2014) 6876–6884
N
H
H
N
N
N
N
O
N
N
H
N
N
R
N
O
Imatinib
H
N
N
N
R1
O
N
N
H
N
OH
O
GNF-5
Title Compounds
O
Figure 3. Design of 4,6-disubstituted pyrimidines as novel Bcr–Abl inhibitors.
Several title compounds displayed exclusive Bcr–Abl inhibitory
activity and antiproliferative activity against K562 cells, with
potencies similar to Imatinib. We expected to find novel lead
compounds which could display Bcr–Abl inhibitory activity
through binding to both ATP site and myristoyl pocket.
cross coupling reaction as key steps.11 4,6-Disubstituted
pyrimidines were synthesized from 4,6-dichloropyrimidine by
acid-promoted amination followed by palladium-catalyzed Suzuki
reactions with boronic acids to afforded key intermediates. Then
condensation reaction of 4,6-disubstituted pyrimidines with vari-
ous mono-benzoylation piperazines yielded title compounds.
An efficient synthesis of 4-boronobenzoic acid was developed in
three steps via a slightly modified literature procedure.12 4-Bromo-
toluene was converted to corresponding Grignard reagent, followed
by esterification in trimethylborate. Acidic hydrolysis provided
4-toluene boronic acid (1) in 60% yield after recrystallization from
2. Chemistry
The synthetic route utilized in the preparation of the title
compounds was outlined in Scheme 1. All compounds were
prepared using Grignard reaction and palladium-catalyzed Suzuki
CH3
CH3
CH3
COOH
COOH
b
a
c
O
CF3
R
MgBr
NH2
Br
Cl
B
1
B
N
e
OH
d
OH OH
Cl
OH
2
HN
N
N
N
N
O
N
NH
N
N
C24 R=3-CF3 C25 R=4-F
C26 R=2,4-diCl C27 R=3-OCH3
+
R
R
R
N
Cl
N
N
H
O
R
(4-1)~(4-19)
(3-1)~(3-19)
F3C
O
COOH
N
N
N
HN
NH
N
N
O
Cl
g
7
CF3
C20 R=3-OCH3 C21 R=3-F
C22 R=4-tBu
C23 R=2-Cl
Cl
O
COOH
Cl
f
H
N
O
N
R
+
NH
N
N
H
6
HN
N
COOH
F
O
N
N
O
F
F
N
C1 R=4-CF3
C4 R=3-F
C7 R=3-Br
C2 R=3-CF3
C5 R=2-F
C8 R=4-F
C3 R=4-tBu
C6 R=4-Br
C9 R=3-CH3CO
NH
5
C10 R=3-Cl-4-CH3 C11 R=2,4-diCl C12 R=3,4-diCl
C13 R=2-Cl C14 R=3-Cl C15 R=3,4-diF
C16 R=3,5-diCF3 C17 R=3-OCH3 C18 R=4-CH3
C19 R=3-i-Pr
Scheme 1. Reagents and conditions: (a) Mg, I2, THF, reflux, N2; (b) B(OMe)3, THF, À20 °C; (c) NaOH (1 M), KMnO4, TBAB, H2O; (d) concd H2SO4, i-PrOH, reflux; (e) Pd(Pph3)4,
Cs2CO3, CH3CN/H2O (V:V = 1:1), 90 °C; (f) CDI; THF; NaCl; piperazine/piperazine dihydrochloride (1:1); NaCl (20%), rt; (g) ClCOO-iBu; THF; TEA; 0 °C ? rt.

J. Dong et al. / Bioorg. Med. Chem. 22 (2014) 6876–6884
6879
Table 1
In vitro Bcr–Abl inhibitory and antiproliferative activity against K562 cells of title compounds (IC50
,
lM)
R1
N
N
O
N
H
N
N
R2
O
No
R1
R2
Bcr–Abl
K562
No
R1
R2
Bcr–Abl
K562
C1
C3
C5
C7
4-CF3
4-tBu
2-F
2-F
2-F
2-F
2-F
2-F
2-F
2-F
2-F
2-F
2-F
2-Cl
2-Cl
3-CF3
3-CF3
78.6
354
283
C2
C4
C6
C8
C10
C12
C14
C16
C18
C20
C22
C24
C26
3-CF3
3-F
4-Br
4-F
3-Cl-4-CH3
3,4-diCl
3-Cl
3,5-diCF3
4-CH3
3-OCH3
4-tBu
2-F
2-F
2-F
2-F
2-F
2-F
2-F
2-F
0.872
0.017
ND
ND*
17.8
306
363
>500
>500
ND
60.4
>500
102
ND
>500
14.5
>500
ND
>500
80.7
>500
ND
>500
ND
21.4
86.9
106
0.05
>500
0.385
>500
141
>500
16.1
>500
6.06
0.070
1.44
13.5
3-Br
277
C9
3-CH3CO
2,4-diCl
2-Cl
3,4-diF
3-OCH3
3-i-Pr
3-F
2-Cl
4-F
3-OCH3
>500
>500
91.8
0.130
ND
C11
C13
C15
C17
C19
C21
C23
C25
C27
2-F
2-Cl
2-Cl
3-CF3
3-CF3
ND
>500
0.563
266
3-CF3
2,4-diCl
25.9
562
4.12
Imatinib
0.014
*
ND = Not determined.
water. Oxidation of the benzylic carbon with KMnO4 in the pres-
ence of NaOH and TBAB (tetrabutylammonium bromide) afforded
4-boronobenzoic acid (2) in 50% yield over three steps. Amination
of 4,6-dichloropyrimidine with various aniline in the presence of
concd H2SO4 under refluxing in 2-propanol was performed to
obtain key intermediates (3–1)ꢀ(3–19).13 In the following step,
the palladium-catalyzed Suzuki cross coupling reactions of (3–
1)ꢀ(3–19) with 4-boronobenzoic acid afforded (4–1)ꢀ(4–19).
Mono-benzoylation piperazine derivatives (5–7) were prepared
from various substituted benzoic acid and piperazine using CDI as
condensing agent.14 Finally, the synthesis of title compounds
(C1ꢀC27) was accomplished by amide bond formation through
coupling reaction using the mixed anhydride method (isobutyl-
chloroformate) successfully.15
in biological activity. It was indicated that compounds bearing
substituents like fluorine, chlorine and trifluoromethyl on aniline
possessed potent anticancer activity. The results indicated that
the introduction of fluoro or trifluoromethyl substituent at termi-
nal aniline might increase the enzymatic and cellar activity. We
demonstrated that fluorine and trifluoromethyl were favourable
structural features for achieving potent Bcr–Abl inhibition and
antiproliferation effect against K562 cells.
Computational studies were carried out to investigate the
potential binding modes of title compounds with ATP-binding site
and myristoyl pocket of Bcr–Abl. The most potent inhibitor C4 was
selected for computational studies. Compound C4 was docked into
ATP site and myristoyl pocket of Bcr–Abl (PDB ID: 3K5V) by SYBYL-
X 2.0.17 Molecular insights based on molecular docking indicated
3. Results and discussion
The Bcr–Abl tyrosine kinase inhibitory activity of title com-
pounds were evaluated by using the well-established ADP-Glo
assays. The antiproliferative potency of them was also identified
in vitro against Bcr–Abl positive K562 cells by using MTT
method.16 Imatinib was utilized as positive control to validate
the screening conditions. The in vitro enzymatic inhibitory activity
and antiproliferative potency associated with the title compounds
were compiled in Table 1.
It was indicated from Table 1 that some of them displayed mod-
erate to high inhibitory activity against Bcr–Abl. Further evaluation
suggested that these compounds could potent inhibit the prolifer-
ation of Bcr–Abl positive K562 cells. Four compounds (C4, C5, C16,
C23) displayed potent enzymatic inhibitory activity with IC50 val-
ues at nanomolar ranges which were comparable to positive con-
trol. The most potent compounds (C4 and C5) strongly inhibited
Bcr–Abl with IC50 values of 0.017 and 0.050 lM, respectively. Some
of them exhibited promising antiproliferative activity against K562
cells. In particularly, compounds (C4 and C5) could also signifi-
cantly suppress Bcr–Abl dependent K562 cells proliferation with
IC50 values of 17.8 and 14.5 lM, respectively. In general, the anti-
proliferative results were consistent with the Bcr–Abl inhibitory
assays. It was indicated that inhibition of Bcr–Abl might be one
of the basis for anticancer activity. We found that the variety and
position of halogen-substitution on aniline played important role
Figure 4. The predicted binding modes of C4 with ATP-binding site of Bcr–Abl.

6880
J. Dong et al. / Bioorg. Med. Chem. 22 (2014) 6876–6884
Imatinib and might be
a promising new candidate Bcr–Abl
inhibitor. Moreover, some of them exhibited potent antiprolifera-
tive activities against K562 cell line. Molecular docking results
indicated that these 4,6-disubstituted pyrimidines could
interact more tightly with both ATP site and myristoyl pocket of
Bcr–Abl.
5. Experimental
5.1. Chemistry: general procedure18
All solvents and reagents were obtained from commercial
suppliers and purified according to standard procedure. All non-
aqueous reactions were performed over dried glassware under
nitrogen atmosphere. All reactions were monitored by TLC on
0.25-mm silica gel plates (fluorescence F254) and visualized with
UV light. Petroleum ether used refers to the fraction boiling in
the range 60–90 °C. Melting points were determined on electro-
thermal melting point apparatus and are uncorrected. 1H NMR
spectra were measured on Bruker Advance (400 MHz) in CDCl3 or
DMSO-d6. Coupling constants (J) are expressed in hertz (Hz) and
chemical shifts (d) of NMR are described in parts per million
(ppm) units relative to internal control (TMS). Mass spectra were
obtained on Shimadzu HPLC-MS-QP2010 instrument.
Figure 5. The predicted binding modes of C4 with myristoyl pocket of Bcr–Abl.
favorable binding mode of C4 with both pockets. The results indi-
cated that the representative compound C4 could tightly bind to
both ATP site and myristoyl pocket of Bcr–Abl. In the minimized
complex of C4 with ATP site of Bcr–Abl (Fig. 4), the ligand adopted
the same conformation as Imatinib. In particular, there was a
hydrogen bond formed between nitrogen atom of pyrimidine ring
and Met 337 of ATP biding site with distance of 2.72 Å.
The myristoyl pocket is a cylindrical shaped cavity located at
the C-terminus of Bcr–Abl. In the minimized complex of C4 with
myristoyl pocket of Bcr–Abl (Fig. 5), the ligand bound to receptor
in a similar mode as GNF-2. Specially, the 2-fluorobenzoyl moiety
might occupy a deeper position in the binding pocket than GNF-2.
The proposed binding mode of C4 may account for the potential of
inhibitory activity against mutants of Bcr–Abl.
These 4,6-disubstituted pyrimidines did not resemble the typi-
cal ATP-competitive Bcr–Abl inhibitors in structure.18 Hence they
are expected to target both ATP-binding site and myristoyl pocket.
They could provide the potential to be unaffected by mutations in
the ATP site which make resistant of CML to ATP-competitive
inhibitors such as Imatinib.
5.1.1. Preparation of (4-methylphenyl)boronic acid (1)19
A 250 mL, two-necked, round-bottomed flask containing dry
magnesium (3.50 g, 150 mmol) and iodine (trace) is equipped with
two rubber septum stirred under the protection of nitrogen. The
solution of 4-bromotoluene (100 mmol) in 60 mL anhydrous THF
was added slowly into the flask through a syringe at a rate to main-
tain reflux. After addition finishing, the resulting mixture was kept
in refluxing for 5 h with stirring. The mixture is cooled to À30 °C,
then the trimethylborate (14.1 g, 150 mmol) in 100 mL anhydrous
THF was added slowly through a syringe. After addition finishing,
the resulting mixture is allowed to stir at room temperature for
overnight and then treated with HCl (2 mol/L, 100 mL), stirred
1 h at rt THF was removed by rotary evaporation. The aqueous
layer was extracted with ethyl acetate (50 mL Â 3). The combined
organic layer was dried over Na2SO4, filtered and concentrated by
rotary evaporation to give the yellow crude product. The crude
product was purified by recrystallization from water and obtained
a white solid (8.40 g, 62%).
4. Conclusion
5.1.2. Preparation of 4-carboxyphenylboronic acid (2)20
A
solution of (4-methylphenyl)boronic acid (1) (6.80 g,
In summary, a series of 4,6-disubstituted pyrimidines were
developed as novel Bcr–Abl inhibitors based on Imatinib and
GNF-5. Some of them displayed potent Bcr–Abl inhibition and anti-
proliferative potency against Bca–Abl positive leukemia cell line
K562. This study demonstrated that a variety of structures can
effectively target both ATP site and myristoyl pocket and provided
new lead compounds for developing novel Bcr–Abl inhibitors. Fur-
ther structural optimization of these promising anticancer agents
will be reported in due course.
50 mmol) in 150 mL of 1 mol/L aqueous NaOH was added in one
portion to a mixture of KMnO4 (23.6 g, 150 mmol) and 0.50 g tetra-
butylammoniumbromide in 500 mL water. The mixture was stir-
red at room temperature for overnight. 100 mL ethanol was
added and stirred 1 h. The dark residue was filtered off and the
clear filtrate was acidified with conc. HCl. The resulting precipitate
was filtered out and dried under vacuum to obtain a 6.80 g white
solid, yield 82%.
In recent studies, combinations of Imatinib and GNF-5 dis-
played potent Bcr–Abl inhibitory activity and antiproliferative
potency against K562 cells. We proved that 4,6-disubstituted
pyrimidine derivatives could be further modified as novel Bcr–
Abl inhibitors targeting ATP site and myristoyl pocket. Many title
compounds displayed varied degree of anticancer potency and
Bcr–Abl inhibitory activity comparable to positive control. The
results indicated that fluorine and trifluoromethyl were more suit-
able for their potency. Three compounds (C4, C5, C23) exhibited
significant Bcr–Abl inhibitory activities, with IC50 values below
70 nM. Among them, C4 exhibited potency comparable with
5.1.3. 6-Chloro-N-[4-(trifluoromethyl)phenyl]pyrimidin-4-
amine (3–1)21
The 4-(trifluoromethyl)aniline (3.22 g, 20 mmol) and 4,6-
dichloropyrimidine (3.88 g, 26 mmol)were dissolved in 30 mL of
2-propanol. Concd H2SO4 (1.50 mL) was added slowly under stir-
ring at room temperature. The mixture was reacted under reflux
and was monitored by TLC. The mixture was stored at 4 °C over-
night. The precipitated product was filtered off, washed with a
small amount of ice-cold 2-propanol, and dried in vacuum to afford
a solid 4.90 g, yield 90%.

J. Dong et al. / Bioorg. Med. Chem. 22 (2014) 6876–6884
6881
The intermediate compounds (3–1)ꢀ(3–19) were prepared by
5.1.7. 6-(4-{[4-(2-Fluorobenzoyl)piperazin-1-yl]carbonyl}
using the general procedure described above.
phenyl)-N-[3-(trifluoromethyl)phenyl]pyrimidin-4-amine (C2)
This compound was prepared from compounds (4–2) and (5)
using the same procedure as described above: Yield 18%;
mp = 265–267 °C; EI-MS (m/z): 548.9 (M+); 1H NMR (DMSO-d6,
400 MHz): d 10.11 (s, 1H), 8.83 (s, 1H), 8.29 (s, 1H), 8.06–8.17
(m, 2H), 7.95 (d, J = 8.0 Hz, 1H), 7.58–7.66 (m, 3H), 7.49–7.56 (m,
1H), 7.42–7.48 (m, 1H), 7.38 (d, J = 8.0 Hz, 1H), 7.21–7.36 (m,
3H), 3.39À3.82 (m, 8H)
5.1.4. 4-(6-{[4-(Trifluoromethyl)phenyl]amino}pyrimidin-4-yl)
benzoic acid (4–1)22
Under a nitrogen atmosphere, 6-chloro-N-[4-(trifluoromethyl)
phenyl]pyrimidin-4-amine (3–1) (4.10 g, 10 mmol), 4-carbo-
xyphenylboronic acid (2) (2.00 g, 12 mmol), Pd(PPh3)4 (0.60 g,
0.50 mmol) and Cs2CO3 (13.0 g, 30 mmol) were suspended in a
mixture of CH3CN/H2O (100 mL, V:V = 1:1). The mixture was
heated under reflux for 48 h at 90 °C. The hot suspension was fil-
tered and the filtrate distilled by rotary evaporation to remove ace-
tonitrile. Water was added, and the mixture was extracted three
times with EtOAc (3 Â 30 ml). The aqueous layer was acidified with
conc. HCl and extracted two more times with EtOAc. The combined
organic layers were washed with brine and dried over Na2SO4.
Evaporation of the solvent gave the crude product, which was puri-
fied by flash column chromatography to afford the product (2.90 g,
81%).
5.1.8. N-(4-tert-Butylphenyl)-6-(4-{[4-(2-fluorobenzoyl)
piperazin-1-yl]carbonyl}phenyl)pyrimidin-4-amine (C3)
This compound was prepared from compounds (4–3) and (5)
using the same procedure as described above: Yield 15%;
mp = 209–211 °C; EI-MS (m/z): 537.3 (M+); 1H NMR (DMSO-d6,
400 MHz): d 9.67 (s, 1H), 8.70 (s, 1H), 7.99–8.16 (m, 2H), 7.60
(m, J = 8.0 Hz,4H), 7.49–7.56 (m, 1H), 7.42–7.48 (m, 1H),7.375 (d,
J = 12.0 Hz, 2H), 7.27–7.35 (m, 2H), 7.25 (s, 1H), 3.40–3.85 (m,
8H), 1.29 (m, 9 H).
The key intermediate Compounds (4–1)ꢀ(4–19) were prepared
5.1.9. 6-(4-{[4-(2-Fluorobenzoyl)piperazin-1-yl]carbonyl}phenyl)
-N-(3-fluorophenyl)pyrimidin-4-amine (C4)
by using the general procedure described above.
This compound was prepared from compounds (4–4) and (5)
using the same procedure as described above: Yield 16%;
mp = 274–275 °C; EI-MS (m/z): 498.9 (M+); 1H NMR (DMSO-d6,
400 MHz): d 9.98 (s, 1H), 8.80 (s, 1H), 8.05–8.16 (m, 2H),7.865 (d,
J = 12.0 Hz, 1H), 7.61 (d, J = 8.0 Hz, 2H), 7.49–7.56 (m, 1H),
7.42–7.48 (m, 1H), 7.36–7.41 (m, 2H), 7.25–7.35 (m, 3H),
6.82–6.90 (t, J = 8.0 Hz, 1H), 3.38À3.83 (m, 8 H).
5.1.5. Preparation of 1-(2-fluorobenzoyl)piperazine (5)
A solution of 2-fluorobenzoic acid (7.00 g, 0.05 mol) and CDI
(8.90 g, 0.055 mol) was stirred in dry THF (30 mL) at room temper-
ature for 30 min. In a separate round bottom flask add piperazine
(10.76 g, 125 mmol) and piperazine dihydrochloride (20.0 g,
125 mmol) in 60 mL of water. Stir the reaction mixture for 5 min
and add 14.0 g of NaCl. Add this brine solution to the round bottom
flask containing acyl imidazole. Stir the reaction mixture for 5 hour.
The mixture was filtered and the filtrate distilled by rotary evapo-
ration to remove THF. The aqueous layer was washed with ethyl
acetate (3 Â 10 mL) to remove diacylated product. The PH of the
aqueous layer was adjusted to about 9 using saturated solution of
NaOH and washed with ethyl acetate (4 Â 30 mL). The aqueous
layer was discarded. The organic layer was washed with water
(4 Â 25 mL), dried over anhydrous Na2SO4 and concentrated by
rotary evaporation and purified by flash chromatography to afford
1-(2-fluorobenzoyl)piperazine as colourless solid (4.90 g, 48%).
The other intermediates 1-(2-chlorobenzoyl)piperazine (6) and
1-[3-(trifluoromethyl)benzoyl]piperazine (7) were prepared by
using the general procedure described above.
5.1.10. 6-(4-{[4-(2-Fluorobenzoyl)piperazin-1-yl]carbonyl}
phenyl)-N-(2-fluorophenyl)pyrimidin-4-amine (C5)
This compound was prepared from compounds (4–5) and (5)
using the same procedure as described above: Yield 18%;
mp = 198–199 °C; EI-MS (m/z): 498.9 (M+); 1H NMR (DMSO-d6,
400 MHz): d 9.475 (d, J = 12 Hz, 1H), 8.695 (d, J = 12 Hz, 1H),
8.11–8.02 (m, 2H), 7.66–7.53 (m, 6H), 7.36–7.25 (m, 3H), 7.24–
7.12 (m, 2H), 3.84–3.43 (m, 8H).
5.1.11. N-(4-Bromophenyl)-6-(4-{[4-(2-fluorobenzoyl)piperazin-
1-yl]carbonyl}phenyl)pyrimidin-4-amine (C6)
This compound was prepared from compounds (4–6) and (5)
using the same procedure as described above: Yield 18%;
mp = 288–290 °C; EI-MS (m/z): 561.1 (M+); 1H NMR (DMSO-d6,
400 MHz): d 9.90 (s, 1H), 8.77 (s, 1H), 8.10 (d, J = 8.0 Hz, 1H),
7.73 (d, J = 8.0 Hz,2H), 7.58 (d, J = 8.0 Hz,2H), 7.53 (d,
J = 8.0 Hz,2H), 7.40–7.47 (m, 1H), 7.23–7.39 (m, 4H), 6.79 (d,
J = 8.0 Hz, 1H), 3.54–3.82 (m, 8 H).
5.1.6. 6-(4-{[4-(2-Fluorobenzoyl)piperazin-1-yl]carbonyl}
phenyl)-N-[4-(trifluoromethyl)phenyl]pyrimidin-4-amine (C1)
Isobutyl chloroformate (1.5 mL, 10 mmol) and triethylamine
4 mL were dissolved in 20 mL of THF and cooled to 0 °C, with vig-
orous stirring for 10 min.
A solution of 4-(6-{[4-(trifluoro-
methyl)phenyl]amino}pyrimidin-4-yl)benzoic acid (4–1) (1.79 g,
5 mmol) and 2.5 mL triethylamine in 50 mL of THF was added
slowly, maintaining the temperature at room temperature. The
system was stirred for overnight and a solution of 1-(2-fluor-
obenzoyl)piperazine (5) in 10 mL THF was added. After 24 h, the
mixture was filtered and the filtrate was concentrated by rotary
evaporation to remove THF. The residue was poured into water
and extracted with ethyl acetate. The organic layer was dried over
anhydrous Na2SO4, filtered and the ethyl acetate was removed by
rotary evaporation. The production was purified by flash chroma-
tography on silica gel using 1:5 EtOAc/petroleum ether as eluant.
A single targeted compound (0.40 g) with write solid was obtained
in 15% yield. Mp = 325–326 °C ;EI-MS (m/z): 549.0 (M+); 1H NMR
(DMSO-d6, 400 MHz): d 10.2 (s, 1H), 8.9 (s, 1H), 8.05–8.19 (m,
2H), 7.98 (d, J = 8.0 Hz, 2H), 7.71 (d, J = 8.0 Hz, 2H), 7.58–7.67 (m,
2H), 7.49–7.56 (m, 1H), 7.39–7.48 (m, 1H), 7.40 (s, 1H),
7.19–7.35 (m, 2H), 3.39À3.91 (m, 8 H).
5.1.12. N-(3-bromophenyl)-6-(4-{[4-(2-fluorobenzoyl)piperazin-
1-yl]carbonyl}phenyl)pyrimidin-4-amine (C7)
This compound was prepared from compounds (4–7) and (5)
using the same procedure as described above: Yield 20%;
mp = 246–248 °C; 1H NMR (DMSO-d6, 400 MHz): d 9.94 (s, 1H),
8.81 (s, 1H), 8.14–8.21 (m,1H), 8.06–8.13 (m, 2H), 7.57–7.67 (m,
3H), 7.48–7.56 (m, 1H), 7.38–7.47 (m, 1H), 7.32 (t, J = 8.0 Hz, 4H),
7.22 (d, J = 8.0 Hz, 1H), 3.37–3.86 (m, 8 H).
5.1.13. 6-(4-{[4-(2-Fluorobenzoyl)piperazin-1-yl]carbonyl}
phenyl)-N-(4-fluorophenyl)pyrimidin-4-amine (C8)
This compound was prepared from compounds (4–8) and (5)
using the same procedure as described above: Yield 19%;
mp = 285–286 °C; EI-MS (m/z): 498.9 (M+); 1H NMR (DMSO-d6,
400 MHz): d 9.77 (d, J = 8.0 Hz, 1H), 8.17 (d, J = 8.0 Hz, 1H), 8.00–
8.17 (m, 2H), 7.66–7.80 (m, 2H), 7.40–7.65 (m, 4H), 7.13–7.38
(m, 5H), 3.42–3.90 (m, 8 H).

6882
J. Dong et al. / Bioorg. Med. Chem. 22 (2014) 6876–6884
5.1.14. 1-(3-{[6-(4-{[4-(2-Fluorobenzoyl)piperazin-1-yl]carbonyl}
phenyl)pyrimidin-4-yl]amino}phenyl)ethanone (C9)
This compound was prepared from compounds (4–9) and (5)
using the same procedure as described above: Yield 17%;
mp = 244–246 °C; EI-MS (m/z): 523.1 (M+); 1H NMR
(DMSO-d6, 400 MHz): d 9.98 (s, 1H), 8.79 (s, 1H), 8.28 (s, 1H),
8.17–8.02 (m, 3H), 7.69–7.58 (m, 3H), 7.57–7.49 (m, 2H), 7.48–
7.41 (m, 1H), 7.40–7.25 (m, 3H), 3.85–3.37 (m, 8 H), 2.62–2.58
(s,3H).
5.1.21. N-[3,5-Bis(trifluoromethyl)phenyl]-6-(4-{[4-(2-fluoro
benzoyl)piperazin-1-yl]carbonyl}phenyl)pyrimidin-4-amine
(C16)
This compound was prepared from compounds (4–16) and (5)
using the same procedure as described above: Yield 24%;
mp = 256–257 °C; EI-MS (m/z): 617.0 (M+); 1H NMR (DMSO-d6,
400 MHz): d 10.45 (s, 1H), 8.90 (s, 1H), 8.48 (s, 2H), 8.18–8.09
(m, 2H), 7.69 (s, 1H),7.64–7.61 (m, 2H), 7.58–7.55 (m, 1H), 7.48–
7.41 (m, 1H), 7.38–7.25 (m, 3H), 3.85–3.36 (m, 8H).
5.1.15. N-(3-Chloro-4-methylphenyl)-6-(4-{[4-(2-fluorobenzoyl)
piperazin-1-yl]carbonyl}phenyl)pyrimidin-4-amine (C10)
This compound was prepared from compounds (4–10) and (5)
using the same procedure as described above: Yield 18%;
mp = 258–261 °C; EI-MS (m/z): 529.1 (M+); 1H NMR (DMSO-d6,
400 MHz): d 9.85 (s, 1H), 8.78 (s, 1H), 8.13–8.00 (m, 3H), 7.60 (d,
J = 8.0 Hz, 2H), 7.48–7.40 (m, 2H), 7.37–7.23 (m, 5 H), 3.84–3.49
(m, 8 H), 2.34–2.25 (s, 3H).
5.1.22. 6-(4-{[4-(2-Fluorobenzoyl)piperazin-1-yl]carbonyl}
phenyl)-N-(3-methoxyphenyl)pyrimidin-4-amine (C17)
This compound was prepared from compounds (4–17) and (5)
using the same procedure as described above: Yield 22%;
mp = 215–216 °C; EI-MS (m/z): 511.2 (M+); 1H NMR (DMSO-d6,
400 MHz): d 9.75 (s, 1H), 8.75 (s, 1H), 8.09 (d, J = 8.0 Hz, 2H),
7.64–7.57 (m, 2H),7.55–7.48 (m, 1H), 7.47–7.40 (m, 2H), 7.36–
7.23 (m, 5H), 6.645 (d, J = 4.0 Hz, 1H), 3.77 (s, 3H), 3.75–3.39 (m,
8H).
5.1.16. N-(2,4-Dichlorophenyl)-6-(4-{[4-(2-fluorobenzoyl)
piperazin-1-yl]carbonyl}phenyl)pyrimidin-4-amine (C11)
This compound was prepared from compounds (4–11) and (5)
using the same procedure as described above: Yield 15%;
mp = 226–228 °C; EI-MS (m/z): 549.0 (M+); 1H NMR (DMSO-d6,
400 MHz): d 9.34 (s, 1H), 8.69 (s, 1H), 8.16–8.05 (m, 2H), 7.97 (d,
J = 8.0 Hz, 1H),7.72 (d, J = 2.0 Hz, 1H), 7.65 (s, 1H), 7.595 (d,
J = 4.0 Hz, 2H), 7.49–7.42 (m, 2H), 7.36–7.27 (m, 1H), 7.02 (s, 2H),
3.83–3.43 (m, 8H).
5.1.23. 6-(4-{[4-(2-Fluorobenzoyl)piperazin-1-yl]carbonyl}
phenyl)-N-(4-methylphenyl)pyrimidin-4-amine (C18)
This compound was prepared from compounds (4–18) and (5)
using the same procedure as described above: Yield 19%;
mp = 248–250 °C; EI-MS (m/z): 495.1 (M+); 1H NMR (DMSO-d6,
400 MHz): d 9.645 (d, J = 12.0 Hz, 1H), 8.70 (d, J = 8.0 Hz, 1H),
8.14–8.00 (m, 2H), 7.70–7.50 (m, 5H), 7.47–7.39 (m, 1H), 7.37–
7.25 (m, 3H), 7.21–7.11 (m, 2H), 3.85–3.48 (m, 8H), 2.285 (d,
J = 12.0 Hz, 3H).
5.1.17. N-(3,4-Dichlorophenyl)-6-(4-{[4-(2-fluorobenzoyl)
piperazin-1-yl]carbonyl}phenyl)pyrimidin-4-amine (C12)
This compound was prepared from compounds (4–12) and (5)
using the same procedure as described above: Yield 17%;
mp = 288–289 °C; EI-MS (m/z): 549.1 (M+); 1H NMR (DMSO-d6,
400 MHz): d 10.06 (s, 1H), 8.83 (s, 1H), 8.26 (s, 1H), 8.11 (s, 2H),
7.65–7.58 (m, 4H), 7.52 (s, 1H), 7.45 (s, 1H), 7.36–7.27 (m, 2H),
7.02 (s, 1H), 3.83–3.35 (m, 8 H).
5.1.24. 6-(4-{[4-(2-Fluorobenzoyl)piperazin-1-yl]carbonyl}
phenyl)-N-(3-isopropylphenyl)pyrimidin-4-amine (C19)
This compound was prepared from compounds (4–19) and (5)
using the same procedure as described above: Yield 21%;
mp = 196–197 °C; EI-MS (m/z):523.1 (M+); 1H NMR (400 MHz,
DMSO) d 9.71 (s, 1H), 8.73 (s, 1H), 8.08 (s, 2H), 7.61 (m, 2H), 7.51
(m, 2H), 7.45 (m, 1H), 7.39–7.22 (m, 5H), 6.94 (d, J = 7.6 Hz, 1H),
3.58 (m, 8H), 2.89 (h, J = 6.8 Hz, 1H), 1.23 (d, J = 6.9 Hz, 6H).
5.1.18. N-(2-Chlorophenyl)-6-(4-{[4-(2-fluorobenzoyl)piperazin
-1-yl]carbonyl}phenyl)pyrimidin-4-amine (C13)
5.1.25. 6-(4-{[4-(2-Chlorobenzoyl)piperazin-1-yl]carbonyl}
phenyl)-N-(3-methoxyphenyl)pyrimidin-4-amine (C20)
This compound was prepared from compounds (4–13) and (5)
using the same procedure as described above: Yield 20%;
mp = 202–204 °C; EI-MS (m/z): 515.1 (M+); 1H NMR (DMSO-d6,
400 MHz): d 9.30 (s, 1H), 8.67 (s, 1H), 8.03–8.14 (m, 2H), 7.89 (d,
J = 8.0 Hz, 1H),7.64–7.51 (m, 4H), 7.49–7.42 (m, 1H), 7.41–7.26
(m, 4H), 7.22 (t, J = 6.0 Hz, 1H), 3.83–3.39 (m, 8 H).
This compound was prepared from compounds (4–2) and (6)
using the same procedure as described above: Yield 21%;
mp = 227–229 °C; EI-MS (m/z): 527.3 (M+); 1H NMR (DMSO-d6,
400 MHz): d 9.75 (s, 1H), 8.75 (s, 1H), 8.15–8.02 (m, 2H), 7.65 (s,
1H), 7.62–7.56 (m, 2H), 7.50–7.39 (m, 4H), 7.265 (d, J = 4.0 Hz,
2H), 7.02 (s, 1H), 6.68–6.60 (m, 1H), 3.77 (s, 3H), 3.75–3.37 (m, 8H).
5.1.19. N-(3-Chlorophenyl)-6-(4-{[4-(2-fluorobenzoyl)piperazin
-1-yl]carbonyl}phenyl)pyrimidin-4-amine (C14)
5.1.26. 6-(4-{[4-(2-Chlorobenzoyl)piperazin-1-yl]carbonyl}
phenyl)-N-(3-fluorophenyl)pyrimidin-4-amine (C21)
This compound was prepared from compounds (4–8) and (6)
using the same procedure as described above: Yield 20%;
mp = 252–255 °C; EI-MS (m/z): 515.3 (M+); 1H NMR (DMSO-d6,
400 MHz): d 9.99 (s, 1H), 8.80 (s, 1H), 8.16–8.03 (m, 2H), 7.865
(d, J = 12.0 Hz, 1H), 7.68 (s, 1H), 7.63––7.54 (m, 2H), 7.48–7.38
(m, 4H), 7.33–7.28 (m, 1H), 7.04 (s, 1H), 6.92–6.81 (m, 1H), 3.83–
3.39 (m, 8H).
This compound was prepared from compounds (4–14) and (5)
using the same procedure as described above: Yield 18%;
mp = 268–270 °C; EI-MS (m/z): 515.1 (M+); 1H NMR (DMSO-d6,
400 MHz): d 9.96 (s, 1 H), 8.81 (s, 1H), 8.10 (s, 2H), 8.07 (s, 1H),
7.68–7.59 (m, 2H), 7.58–7.54 (m, 1H), 7.53–7.48 (m, 1H), 7.47–
7.41 (m, 1H), 7.38 (t, J = 6.0 Hz, 1H), 7.35–7.25 (m, 3H), 7.09 (d,
J = 8.0 Hz, 1H), 3.84–3.35 (m, 8H).
5.1.20. N-(3,4-Difluorophenyl)-6-(4-{[4-(2-fluorobenzoyl)
piperazin-1-yl]carbonyl}phenyl)pyrimidin-4-amine (C15)
This compound was prepared from compounds (4–15) and (5)
using the same procedure as described above: Yield 20%;
mp = 276–278 °C; EI-MS (m/z): 517.1 (M+); 1H NMR (DMSO-d6,
400 MHz): d 9.97 (s, 1H), 8.79 (s, 1H), 8.15–8.08 (m, 2H), 7.64 (s,
1H), 7.61 (d, J = 8.0 Hz, 2H), 7.56–7.48 (m, 1H), 7.45 (d, J = 8.0 Hz,
2H), 7.39 (s, 1H), 7.33–7.25 (m, 2H), 7.02 (s, 1H), 3.79–3.48
(m, 8H).
5.1.27. N-(4-tert-Butylphenyl)-6-(4-{[4-(2-chlorobenzoyl)
piperazin-1-yl]carbonyl}phenyl)pyrimidin-4-amine (C22)
This compound was prepared from compounds (4–11) and (6)
using the same procedure as described above: Yield 20%;
mp = 209–210 °C; EI-MS (m/z): 553.3 (M+); 1H NMR (DMSO-d6,
400 MHz): d 9.67 (s, 1H), 8.70 (s, 1H), 8.16–8.00 (m, 2H), 7.65–
7.53 (m, 5H), 7.52–7.41 (m, 3H), 7.375 (d, J = 12.0 Hz, 2H), 7.17
(s, 1H), 3.84–3.35 (m, 8H), 1.29 (s, 9H).

J. Dong et al. / Bioorg. Med. Chem. 22 (2014) 6876–6884
6883
5.1.28. 6-(4-{[4-(2-Chlorobenzoyl)piperazin-1-yl]carbonyl}
phenyl)-N-(2-chlorophenyl)pyrimidin-4-amine (C23)
This compound was prepared from compounds (4–17) and (6)
using the same procedure as described above: Yield 21%;
mp = 237–239 °C; EI-MS (m/z): 531.1 (M+); 1H NMR (400 MHz,
DMSO) d 9.96 (s, 1H), 8.81 (s, 1H), 8.17–8.02 (m, 4H), 7.64–7.53
(m, 4H), 7.52–7.40 (m, 2H), 7.38 (t, J = 8.1 Hz, 1H), 7.30 (s, 1H),
7.09 (d, J = 6.9 Hz, 1H), 3.68 (m, 8H).
of ATP were left. Then the mixture was incubated at room temper-
ature for 40 min and added 10 L of Kinase Detection Reagent to
l
convert ADP to ATP and introduced luciferase and luciferin to
detect ATP. At last, the mixture was incubated at room tempera-
ture for 30–60 min and measured the luminescence with a plate-
reading luminometer. The signal was correlated with the amount
of ATP present in the reaction and was inversely correlated with
the kinase activity.
5.1.29. 6-[4-({4-[3-(Trifluoromethyl)benzoyl]piperazin-1-yl}
carbonyl)phenyl]-N-[3-(trifluoromethyl)phenyl]pyrimidin-4-
amine (C24)
This compound was prepared from compounds (4–2) and (7)
using the same procedure as described above: Yield 24%;
mp = 220–222 °C; ESI-MS (m/z): 600.1 (M+H)+; 1H NMR (DMSO-
d6, 400 MHz): d 10.11 (s, 1H), 8.83 (s, 1H), 8.29 (s, 1H), 8.125 (d,
J = 4.0 Hz,2H), 7.95 (d, J = 8.0 Hz, 1H), 7.855 (d, J = 4.0 Hz, 1H),
7.80 (s, 1H), 7.78–7.72 (m, 1H), 7.64–7.56 (m,4H), 7.38 (d,
J = 8.0 Hz,1H), 7.32 (s, 1H), 3.83–3.38 (m, 8H)
5.3. Cell growth inhibitory activity in cancer cells25
The title compounds were evaluated for their antiproliferative
potency against Bcr–Abl positive K562 cells using MTT method.
Exponentially growing cells were harvested and plated in 96-well
plates at a concentration of 1 Â 104 cells/well, and then incubated
for 24 h at 37 °C. The cells in the wells were treated with the title
compounds respectively 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 °C. Supernatant was discarded, and 150 mL DMSO was
added to each well. Absorbance values were determined by a
microplate reader (Bio-Rad Instruments) at 570 nm. The IC50 val-
ues were calculated according to inhibition ratios.
5.1.30. N-(4-Fluorophenyl)-6-[4-({4-[3-(trifluoromethyl)benzoyl]
piperazin-1-yl}carbonyl)phenyl]pyrimidin-4-amine (C25)
This compound was prepared from compounds (4–8) and (7)
using the same procedure as described above: Yield 19%;
mp = 193–195 °C; EI-MS (m/z): 549.4 (M+); 1H NMR (DMSO-d6,
400 MHz): d 9.77 (s, 1H), 8.72 (s, 1H), 8.095 (d, J = 4.0 Hz, 2H),
7.91–7.83 (m, 1H), 7.80 (s, 1H), 7.76–7.70 (m, 3H), 7.59 (d,
J = 8.0 Hz, 2H), 7.29–7.15 (m, 3H), 7.02 (s, 1H), 3.85–3.37 (m, 8H).
5.4. Molecular docking26
In order to understand the binding mode of the title compounds
with both ATP site and myristoyl pocket of Bcr–Abl, we performed
a molecule docking using Sybyl/Surflex-dock based on crystal
structures of Bcr–Abl. The inhibitors were docked into the active
site using ligand-based mode. The crystal structure of Bcr–Abl
complex with Imatinib and GNF-2 was obtained from RCSB Protein
Data Bank (PDB ID: 3K5V). The ligands and water molecules were
removed and hydrogen was added and minimized using Tripos
force field and Pullman charges. The residues in a radius 5.0 Å
around ligands were selected as active site. C4 was depicted with
Sybyl/Sketch module (Tripos Inc.) and optimized applying Powell’s
method with Tripos force field with convergence criterion set at
0.05 kcal/(Å mol), and assigned with Gasteiger–Hückel method.
Other docking parameters were kept at default.
5.1.31. N-(2,4-Dichlorophenyl)-6-[4-({4-[3-(trifluoromethyl)benzoyl]
piperazin-1-yl}carbonyl)phenyl]pyrimidin-4-amine (C26)
This compound was prepared from compounds (4–11) and (7)
using the same procedure as described above: Yield 22%;
mp = 152–155 °C; EI-MS (m/z): 599.3 (M+); 1H NMR (DMSO-d6,
400 MHz): d 9.34 (s, 1H), 8.69 (s, 1H), 8.105 (d, J = 4.0 Hz, 2H),
7.97 (d, J = 8.0 Hz, 1H), 7.88–7.82 (m, 1H), 7.80 (s, 1H), 7.77–7.04
(m, 1H), 7.715 (d, J = 4.0 Hz, 2H), 7.63–7.55 (m, 2H), 7.50–7.41
(m,2H), 3.85–3.42 (m, 8H).
5.1.32. N-(3-Methoxyphenyl)-6-[4-({4-[3-(trifluoromethyl)
benzoyl] piperazin-1-yl}carbonyl)phenyl]pyrimidin-4-amine (C27)
This compound was prepared from compounds (4–17) and (7)
using the same procedure as described above: Yield 25%;
mp = 84–86 °C; EI-MS (m/z): 561.3 (M+); 1H NMR (400 MHz,
DMSO) d 9.80 (s, 1H), 8.76 (s, 1H), 8.10 (d, J = 6.9 Hz, 2H), 7.87–
7.74 (m, 3H), 7.61–7.56 (m, 2H), 7.44 (s, 1H), 7.33–7.20 (m, 4H),
6.64 (d, J = 6.2 Hz, 1H), 3.77 (s, 3H), 3.66–3.32 (m, 8H).
Acknowledgment
This work was supported by the National Natural Science Foun-
dation of China (Grant NO. 81302641 and 81302737), and the Fun-
damental Research Funds for the Central Universities (xjj2014140).
Supplementary data
5.2. Bcr–Abl inhibitory activity assays23
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.10.030.
The Bcr–Abl inhibitory activity assay was performed using ADP-
Glo™ Kinase assay kit (Promega, catalog: V9101 and Abl Kinase
Enzyme System (Promega, catalog: V1901) according to the manu-
facturer’s instructions. The Abl1 reaction utilizes ATP and gener-
ates ADP. Then the ADP-Glo™ reagent is added to simultaneously
terminate the kinase reaction and deplete the remaining ATP.
Finally, the Kinase Detection Reagent is added to convert ADP to
ATP and the newly synthesized ATP is converted to light using
the luciferase reaction.
Reference
1. Mughal, A.; Aslam, H. M.; Khan, A. M.; Saleem, S.; Umah, R.; Saleem, M. Infect.
Agent. Cancer 2013, 8, 23.
2. Lambert, G. K.; Duhme-Klair, A. K.; Morgan, T.; Ramjee, M. K. Drug Discovery
Today 2013, 18, 992.
3. Cui, J. J. ACS Med. Chem. Lett. 2014, 5, 272.
4. Quintás-Cardama, A.; Kantarjian, H.; Cortes, J. Nat. Rev. Drug Disc. 2007, 6, 834.
5. Adrián, F. J.; Ding, Q.; Sim, T.; Velentza, A.; Sloan, C.; Liu, Y.; Zhang, G.; Hur, W.;
Ding, S.; Manley, P.; Mestan, J.; Fabbro, D.; Gray, N. S. Nat. Chem. Biol. 2006, 2,
95.
6. Deng, X.; Okram, B.; Ding, Q.; Zhang, J.; Choi, Y.; Adrián, F. J.; Wojciechowski, A.;
Zhang, G.; Che, J.; Bursulaya, B.; Cowan-Jacob, S. W.; Rummel, G.; Sim, T.; Gray,
N. S. J. Med. Chem. 2010, 53, 6934.
7. Zhang, J.; Adrián, F. J.; Jahnke, W.; Cowan-Jacob, S. W.; Li, A. G.; Iacob, R. E.; Sim,
T.; Powers, J.; Dierks, C.; Sun, F.; Guo, G. R.; Ding, Q.; Okram, B.; Choi, Y.;
Wojciechowski, A.; Deng, X.; Liu, G.; Fendrich, G.; Strauss, A.; Vajpai, N.;
Grzesiek, S.; Tuntland, T.; Liu, Y.; Bursulaya, B.; Azam, M.; Manley, P. W.; Engen,
J. R.; Daley, G. Q.; Warmuth, M.; Gray, N. S. Nature 2010, 463, 501.
Abl was incubated with substrates, inhibitors and ATP in a final
buffer of 25 mM HEPES (pH 7.4), 10 mM MgCl2, 0.01% Triton X-100,
100
ume of 10
in two steps once the kinase reaction is complete.24 Subsequently,
L ADP-Glo Reagent was added to stop the kinase reaction and
deplete the unconsumed ATP. Only ADP and a very low background
l
g/mL BSA, 2.5 mM DTT in 384-well plate with the total vol-
lL. Then the ADP-Glo™ Kinase Assay was performed
5
l

6884
J. Dong et al. / Bioorg. Med. Chem. 22 (2014) 6876–6884
8. Li, Y.; Shen, M.; Zhang, Z.; Luo, J.; Pan, X.; Lu, X.; Long, H.; Wen, D.; Zhang, F.;
Leng, F.; Li, Y.; Tu, Z.; Ren, X.; Ding, K. J. Med. Chem. 2012, 55, 10033.
9. Capdeville, R.; Buchdunger, E.; Zimmermann, J.; Matter, A. Nat. Rev. Drug Disc.
2002, 1, 493.
10. Mahboobi, S.; Dove, S.; Sellmer, A.; Winkler, M.; Eichhorn, E.; Pongratz, H.;
Ciossek, T.; Baer, T.; Maier, T.; Beckers, T. J. Med. Chem. 2009, 52, 2265.
11. Ren, X.; Pan, X.; Zhang, Z.; Wang, D.; Lu, X.; Li, Y.; Wen, D.; Long, H.; Luo, J.;
Feng, Y.; Zhuang, X.; Zhang, F.; Liu, J.; Leng, F.; Lang, X.; Bai, Y.; She, M.; Tu, Z.;
Pan, J.; Ding, K. J. Med. Chem. 2013, 56, 879.
18. Reddy, M. V.; Pallela, V. R.; Cosenza, S. C.; Mallireddigari, M. R.; Patti, R.;
Bonagura, M.; Truongcao, M.; Akula, B.; Jatiani, S. S.; Reddy, E. P. Bioorg. Med.
Chem. 2010, 18, 2317.
19. Pan, X.; Wang, F.; Zhang, Y.; Gao, H.; Hu, Z.; Wang, S.; Zhang, J. Bioorg. Med.
Chem. 2013, 21, 2527.
20. Zhang, Q.; Rich, J. O.; Cotterill, I. C.; Pantaleone, D. P.; Michels, P. C. J. Am. Chem.
Soc. 2005, 127, 7286.
21. Pan, X.; Dong, J.; Gao, H.; Wang, F.; Zhang, Y.; Wang, S.; Zhang, J. Chem. Biol.
Drug Des. 2014, 83, 592.
12. Rosen, B.; Wilson, D.; Wilson, C. J.; Peterca, M.; Won, B. C.; Huang, C.; Lipski, L.
R.; Zeng, X.; Ungar, G.; Heiney, P. A.; Percec, V. J. Am. Chem. Soc. 2009, 131,
17500.
13. Hartung, C. G.; Backes, A. C.; Felber, B.; Missio, A.; Philipp, A. Tetrahedron 2006,
62, 10055.
14. Verma, S. K.; Ghorpade, R.; Pratap, A.; Kaushik, M. P. Green Chem. 2012, 14, 326.
15. Liu, F. Z.; Fang, H.; Zhu, H. W.; Wang, Q.; Yang, Y.; Xu, W. F. Bioorg. Med. Chem.
2008, 16, 578.
22. Wang, C.; Dong, J.; Zhang, Y.; Wang, F.; Gao, H.; Li, P.; Wang, S.; Zhang, J.
MedChemComm 2013, 4, 1434.
23. Wang, C.; Gao, H.; Dong, J.; Zhang, Y.; Su, P.; Shi, Y.; Zhang, J. Bioorg. Med. Chem.
2014, 22, 277.
24. Zhang, C.; Tan, C.; Zu, X.; Zhai, X.; Liu, F.; Chu, B.; Ma, X.; Chen, Y.; Gong, P.;
Jiang, Y. Eur. J. Med. Chem. 2011, 46, 1404.
25. Lu, W.; Wang, F.; Zhang, T.; Dong, J.; Gao, H.; Su, P.; Shi, Y.; Zhang, J. Bioorg.
Med. Chem. 2014, 22, 2707.
16. Asaki, T.; Sugiyama, Y.; Hamamoto, T.; Higashioka, M.; Umehara, M.; Naito, H.;
Niwa, T. Bioorg. Med. Chem. Lett. 2006, 16, 1421.
26. Zhang, J.; Shan, Y.; Pan, X.; Wang, C.; Xu, W.; He, L. Chem. Biol. Drug. Des. 2011,
78, 709.
17. Zhang, J.; Zhang, Y.; Shan, Y.; Li, N.; Ma, W.; He, L. Eur. J. Med. Chem. 2010, 45,
2798.