Design, synthesis and biological evaluation of pyridin-3-yl pyrimidines as potent Bcr-Abl inhibitors

Article abstract of DOI:10.1111/cbdd.12272

A series of pyridin-3-yl pyrimidines was synthesized and evaluated for their Bcr-Abl inhibitory and anticancer activity. The preliminary results indicated that some compounds were promising anticancer agents. Compounds A2, A8, and A9 exhibited potent Bcr-Abl inhibitory activity, suggesting that aniline containing halogen substituents might be important for biological activity. Molecular docking was carried out to investigate the binding mode of them with Bcr-Abl. Details of synthesis and SAR studies of these compounds are described. A series of phenylaminopyrimidines was designed and synthesized as potent Bcr-Abl inhibitors. The screening of these rationally designed compounds for antitumor activity had identified three candidate leads which could be further optimized to improve the anticancer activities.

Full text of DOI:10.1111/cbdd.12272

Chem Biol Drug Des 2014; 83: 592–599
Research Article
Design, Synthesis and Biological Evaluation of
Pyridin-3-yl pyrimidines as Potent Bcr-Abl Inhibitors
Nilotinib could fit into the kinase domain of Bcr-Abl, which
was quite conserved. The allosteric region, which was
adjacent to ATP-binding site, was considered as selectiv-
ity site. Therefore, the moiety interacting with allosteric
region was modified to improve the selectivity and activity.
In this article, we attempted to develop novel Bcr-Abl
inhibitors with enhanced biological activity.
Xiaoyan Pan, Jinyun Dong, Hongping Gao,
Fang Wang, Yanmin Zhang, Sicen Wang and
Jie Zhang*
School of Medicine, Xi’an Jiaotong University, No. 76,
Yanta West Road, Xi’an 710061, China
*Corresponding author: Jie Zhang,
zhj8623@mail.xjtu.edu.cn
It is well known that aniline containing halogen substitu-
ents (fluoro, chloro, brome, and iodo) is useful for anti-
cancer agents design. Halogen introduction can enhance
the persistence and lipophilicity (6). Therefore, various
halogen-substituted anilines were introduced to develop
novel Bcr-Abl inhibitors. Meanwhile, some derivatives
without halogen-substituted anilines were also prepared
to validate the effect of halogen. Moreover, some hetero-
cyclic amines and anilines containing tertiary amine side
chain were incorporated to afford adequate structural
diversity.
A series of pyridin-3-yl pyrimidines was synthesized
and evaluated for their Bcr-Abl inhibitory and antican-
cer activity. The preliminary results indicated that
some compounds were promising anticancer agents.
Compounds A2, A8, and A9 exhibited potent Bcr-Abl
inhibitory activity, suggesting that aniline containing
halogen substituents might be important for biological
activity. Molecular docking was carried out to investi-
gate the binding mode of them with Bcr-Abl. Details of
synthesis and SAR studies of these compounds are
described.
Key words: 3D-QSAR, anticancer, Bcr-Abl, leukemia cell,
Chemistry
pyridin-3-yl pyrimidines
Received 12 March 2013, revised 5 October 2013 and
accepted for publication 5 December 2013
In this study, the title compounds were prepared from
commercial available reagents (Scheme 1). One key inter-
mediates (5) was yielded in high yield. The intermediate (5)
was prepared in five steps. Firstly, carboxylate group of
3-amino-4-methylbenzoic acid (1) was protected by esterifi-
cation with ethanol (7). Then, refluxing of compound (2) and
acetonitrile in the presence of concentrated HCl afforded
3-guanidino-4-methylbenzoic acid ethyl ester nitrate (3) (8).
Recent advances in cancer therapy afford molecular
targeting agents with high efficiency and low toxicity (1).
Chronic myelogenous leukemia (CML) is hematological
malignancy caused by chromosomal rearrangement
that generates Bcr-Abl. Bcr-Abl is
a fusion protein
with deregulated tyrosine kinase activity, which is
essential for pathogenesis of CML (2). It is regarded as
highly attractive target for drug intervention. Bcr-Abl
inhibitors are the first-line therapy for most patients with
CML.
Nilotinib is a potent Bcr-Abl inhibitor for the treatment for
CML (3). Crystallographic studies revealed that it could
stabilize the inactive conformation of Bcr-Abl (4). Nilotinib
interacts with Bcr-Abl through four key interactions: hydro-
gen-bond interactions with hinge region, hydrophobic
interactions with gatekeeper region, hydrogen-bond inter-
actions with DFG motif, and hydrophobic interactions with
allosteric region (Figure 1) (5).
Figure 1: The interactions between Bcr-Abl kinase and inhibitors.
ª 2013 John Wiley & Sons A/S. doi: 10.1111/cbdd.12272
592

A Series of Novel Bcr-Abl Inhibitors
Scheme 1: Reagents and conditions: (a) EtOH, concd H2SO4, reflux; (b) H2NCN, concd HCl, EtOH, reflux, NH4NO3(aq); (c) 3-(dimethyl
amino)-1-(3-pyridinyl)prop-2-en-1-one, NaOH, n-BuOH, reflux; (d) NaOH, EtOH/H2O(v:v = 1:1), 50 °C; (e) SOCl2, DMF (cat), reflux; (f)
substituted benzamides or substituted heterocyclic amines or compounds b21–b27, DIEA, DMAP (cat), DCM, r.t.; (g) Cs2CO3, DMF,
100 °C; (h) Pd/C, MeOH.
Experimental
Compound (4) was afforded by reaction of 3-dimethylami-
no-1-(3-pyridyl)-2-propen-1-one with compound (3) (9). The
hydrolysis of (4) in NaOH aqueous solution yielded
intermediate (5) (10). Finally, compound (5) was treated with
SOCl2 to form carbonyl chlorides and then reacted with
various anilines to afford title compounds (11). Various
heterocyclic aromatic amines were used in the synthesis of
A15–A20. Twenty-seven 2-pyrimidinyl-amino-benzamides
(A1–A27) were synthesized as novel Bcr-Abl inhibitors.
Cell growth inhibition assays
Growth inhibitory activities were evaluated on K562 leuke-
mia 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 9 104cells/well and incubated for
24 h at 37 °C. The cells in the wells were, respectively,
Chem Biol Drug Des 2014; 83: 592–599
593

Pan et al.
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 °C. After the supernatant
was discarded, 150 mL DMSO was added to each well,
and the absorbance values were determined by a micro-
plate reader (Bio-Rad, Hercules, CA, USA) at 490 nm.
was extracted with ethyl acetate (60 mL 9 2). The organic
phase was combined and was washed by water
(30 mL 9 2) and saturated sodium chloride (30 mL). The
organic layer was dried over Na2SO4, filtered, and distilled
under vacuum to give the crude product. The crude prod-
uct was recrystallized by ether and EtOAc, giving a white
solid (7.31 g). The total yield was 98%, mp 47–48 °C.
Kinase assays
The kinase inhibition assay and IC50 determinations for
wild-type Bcr-Abl were measured with the homogeneous
time-resolved fluorescence (HTRF) KinEASE-TK assay from
Cisbio according to the manufacturer’s instructions. Wild-
type Abl was purchased from Carna Biosciences, and
0.04 ng/lL kinase was used for test. ATP concentration
was set at its Km values (6.021 lM), and 457.7 nM substrate
was used. Kinase, substrate peptide, and inhibitors were
added in 384-well plate, and then, reaction was started by
addition of ATP. After completion of the reaction (30 min
later), an antiphosphotyrosine antibody labeled with euro-
pium cryptate and streptavidin labeled with the fluorophore
XL665 were added. The FRET between europium cryptate
and XL665 was measured to quantify the phosphorylation
of the substrate peptide. VICTOR X multilabel plate reader
was used to measure the fluorescence of the samples at
620 nm (Eu-labeled antibody) and 665 nm (XL665-labeled
streptavidin). The quotient of both intensities for reactions
made with six different inhibitor concentrations (0.032–
3.2 lM, including no inhibitor) was plotted against inhibitor
concentrations to determine IC50 values. Each reaction was
performed in duplicate, and at least two independent deter-
minations of each IC50 were made.
3-Guanidino-4-methylbenzoic acid ethyl ester
nitrate (3)
In 100-mL round-bottomed flask, 3-amino-4-methylbenzo-
ic acid ethyl ester (2) (3.00 g, 16.76 mmol) and cyanamide
(1.68 g, 39.29 mmol) were added to ethanol (20 mL).
While being stirred, concentrated HCl (2.16 mL,
25.41 mmol) was dropped slowly into the mixture. The
mixture was heated to reflux for 15 h. Ethanol was
removed by reduced pressure distillation, and water
(20 mL) was added into the residue. Under 0 °C, NH4NO3
(2.70 g, 33.6 mmol) solution was dropped during 30 min.
Then, the mixture was stirred for 30 min and filtered, giv-
ing the crude product. The crude product need to be puri-
fied by being washed with ether, and finally a white solid
(4.66 g) was maintained. The total yield was 84.7%, mp
189–192 °C. EI-MS (m/z): 221.1 ([M]+-HNO3).
4-Methyl-3-[[4-(3-pyridyl)-2-pyrimidinyl] amino]
benzoic acid ethyl ester (4)
In
nitrate ethyl benzoate (3) (3.20 g, 11.26 mmol),
3-dimethylamino-1-(3-pyridyl)-2-propen-1-one (2.00 g,
a
round-bottomed flask, 3-guanidino-4-methyl
11.26 mmol), and sodium hydroxide (0.54 g) were dis-
solved in n-butanol (20 mL). The mixture was stirred and
heated to reflux for 72 h. Then, n-butanol was removed
completely by reduced pressure distillation. EtOAc (50 mL)
and water (30 mL) were added into the residue, and the
mixture was stirred for 20 min. The organic phase was sep-
arated from the aqueous phase, and the aqueous phase
was extracted by EtOAc (20 mL 9 2). The combined
organic phase was washed by water (15 mL) and saturated
sodium chloride (15 mL). The organic layer was dried over
Na2SO4, filtered, and distilled under vacuum to give the
crude product. The crude product was recrystallized by
ether and EtOAc, giving a white solid (2.76 g). The total
yield was 73.4%, mp 91–92 °C. EI-MS (m/z): 334.1 [M]+.
1H NMR (400 MHz, CDCl3): d = 1.40 (t, J = 7.20 Hz, 3H),
2.41 (s, 3H), 4.41 (q, J = 7.20 Hz, 14.00 Hz, 4H), 7.12 (s,
1H), 7.24 (d, J = 4.0 Hz, 1H), 7.28–7.32 (m, 2H), 7.46 (s,
1H), 7.75 (dd, J = 4.0 Hz, 8.0 Hz, 1H), 8.45 (d,
J = 8.0 Hz, 1H), 8.54 (d, J = 8.0 Hz, 1H), 8.93 (s, 1H).
Chemistry: general procedures
All solvents and reagents were purified by standard tech-
niques. Petroleum ether used refers to the fraction boiling
in the range 60–90 °C. All reactions except those in aque-
ous media were carried out by standard techniques for
moisture exclusion. Anhydrous reactions were carried out
over dried glassware under nitrogen atmosphere. Reac-
tions were monitored by TLC on 0.25-mm silica gel plates
(60GF254) and visualized with ultraviolet light. Melting points
were obtained on electrothermal melting point apparatus
and are uncorrected. 1H-NMR spectra was recorded with
a Bruker Advance 400 MHz instrument. Mass spectra
were obtained on a Shimadzu GC-MS-QP2010 instrument.
3-Amino-4-methylbenzoic acid ethyl ester (2)
3-Amino-4-methylbenzoic acid (6.30 g, 41.72 mmol) was
dissolved in 140 mL dehydrated ethanol. Under 0 °C,
4 mL concentrated H2SO4 was dropped to the above
solution, and then, the solution was heated to reflux for
12 h. After the reaction was completed, ethanol was
removed by reduced pressure distillation. Under 0 °C,
200 mL water was added, and the solution was adjusted
to pH 6–7 with sodium bicarbonate. Then, the aqueous
4-Methyl-3-[[4-(3-pyridyl)-2-pyrimidinyl] amino]
benzoic acid (5)
In a 250-mL flask, 4-methyl-3-[[4-(3-pyridyl-2-pyrimidinyl)
amino] ethyl benzoate (4) (5.18 g, 15.5 mmol) was
dissolved into the mixed solution of ethanol/water (60/
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A Series of Novel Bcr-Abl Inhibitors
60 mL). While being stirred, NaOH (2 M, 25 mL) was
drooped in the mixture. Then, the mixture was heated to
45–50 °C and was maintained at this temperature for
4 h. Ethanol was removed by reduced pressure distilla-
tion, and then, EtOAc (20 mL) was added. Then, the
organic phase was abandoned. The aqueous phase was
adjusted to pH 7–8 using HCl (2 M), and yellow solid
was precipitated. The suspension was filtered, giving the
crude product (4.10 g). The total yield was 87%, mp
258–260 °C. EI-MS (m/z): 306.1 [M]+. 1H NMR
(400 MHz, DMSO-d6): d = 2.31 (s, 3H), 7.12 (s, 1H),
7.30 (d, J = 8.0 Hz, 1H), 7.52 (d, J = 4.0 Hz, 1H), 7.60–
7.66 (m, 3H), 8.30 (s, 1H), 8.57 (d, J = 4.0 Hz, 2H),
8.76 (s, 1H), 9.13 (s, 1H), 9.32 (s, 1H).
0 °C, the CH2Cl2 (10 mL) solution of N, N’-diisopropyl eth-
ylamine (4.0 mL) and 4-(dimethylamino) pyridine (cat) was
dropped slowly into the above suspension. After that, the
mixture was reacted at r.t. overnight. The mixture was
diluted with CH2Cl2 (20 mL) and was washed with satu-
rated NaCl solution (10 mL 9 2), saturated NaHCO3 solu-
tion (10 mL 9 3), and water (10 mL 9 2). Then, the
organic phase was dried by Na2SO4 and filtered, giving
the crude product. The crude product was purified by
chromatography (ethyl acetate: methanol = 3:1), giving a
solid (0.68 g). The total yield was 56.7%.
Mp 234–237 °C, EI-MS (m/z): 382.1[M]+, 1H NMR
(400 MHz, CDCl3): d = 2.46 (s, 3H), 7.26 (d, J = 4.0 Hz,
1H), 7.36–7.38 (m, 3H), 7.44–7.46 (m, 1H), 7.60 (d, J =
8.0 Hz, 1H), 8.29 (s, 1H), 8.39–8.46 (m, 3H), 8.55 (d, J =
4.0 Hz, 1H), 8.74–8.75 (m, 2H), 8.85 (s, 1H), 9.29 (s, 1H).
4-Methyl-3-[[4-(3-pyridyl)-2-pyrimidinyl] amino]
benzoyl chloride (6)
In a 50-mL flask, 4-methyl-3-[[4-(3-pyridyl)-2-pyrimidinyl]
amino] benzoic acid (5) (0.96 g, 3.14 mmol) was added in
anhyd SOCl2 (10 mL). Then, in the suspension, DMF was
added 3–4 drops. The mixture was heated to reflux for
1.5 h. Then, SOCl2 was removed completely by reduced
pressure distillation. The residue was reserved for the next
step.
Compounds A16–A20 were prepared using the general
procedure described above.
N, N-dimethyl-3-(4-nitrophenoxy) propan-1-amine
(c21)
In 100-mL flask, 4-nitrophenol (1.39 g, 10 mmol) was
dissolved in 20 mL anhydrous DMF. Then, 3-chloro-N,
N-dimethylpropan-1-amine hydrochloride (d21) (1.57 g,
10 mmol), and Cs2CO3 (5.29 g, 15 mmol) were added to
the solution. Under N2, the mixture was heated to 100 °C
and was reacted for 2.5 h. The mixture was filtered, and
the filtrate was poured to ice water (200 mL). The aqueous
phase was extracted by EtOAc (30 mL 9 3). The com-
bined organic phase was washed by Na2CO3
(10 mL 9 4), water (10 mL 9 2), and saturated sodium
chloride (15 mL). The organic layer was dried over Na2SO4
to give the crude product.
N-(3-chloro-4-fluorophenyl)-4-methyl-3-(4-(pyridin-
3-yl)pyrimidin-2-ylamino)benzamide (A1)
In a 50 mL flask, 4-methyl-3-[[4-(3-pyridyl)-2-pyrimidinyl]
amino] benzoyl chloride (6) was dissolved into CH2Cl2
(20 mL). Under 0 °C, the CH2Cl2 (10 mL) solution of 3-
Chloro-4-fluoroaniline (0.46 g, 3.14 mmol), N, N0-diisopro-
pyl ethylamine (4.0 mL) and 4-(dimethylamino) pyridine
(cat.) was dropped slowly into the above suspension. After
that, the mixture was reacted at r.t. overnight. The mixture
was diluted with CH2Cl2 (20 mL), and was washed with
saturated NaCl solution (10 mL 9 2), saturated NaHCO3
solution (10 mL 9 3), water (10 mL 9 2). Then the organic
phase was dried by Na2SO4, and filtered, giving the crude
product. The crude product was purified by chromato-
4-(3-(Dimethylamino) propoxy) benzenamine (b21)
N, N-dimethyl-3-(4-nitrophenoxy) propan-1-amine (c21)
(3.92 mmol) was dissolved in 30 mL anhydro-methanol,
and then, 0.1 g Pd/C(5%) was added to this solution. After
that, the mixture was reacted at r.t. under N2 for 4 h. The
mixture was filtered, and Pd/C was washed with methanol
for 3–4 times. The organic phases were combined, and
methanol was removed by reduced pressure distillation to
give the crude product. The residue was reserved for the
next step.
graphy (ethyl acetate:methanol = 5:1), giving
a solid
(0.36 g). The total yield was 27%. Mp 196–198 °C, EI-MS
(m/z): 432.9[M]+, 1H NMR (400 MHz, CDCl3): d = 2.21 (s,
3H), 7.12–7.56 (m, 4H), 7.69 (s, 1H), 7.88 (d, J = 8.0 Hz,
1H), 8.24 (d, J = 8.0 Hz, 1H), 8.43 (d, J = 8.0 Hz, 1H),
8.56 (d, J = 4.0 Hz, 1H), 8.64 (d, J = 4.0 Hz, 1H), 8.85 (s,
1H), 9.19 (s, 1H), 9.30 (s, 1H), 10.18 (s, 1H).
Compounds A2–A14 were prepared using the general
Compounds b21–b27 were prepared using the general
procedure described above.
procedure described above.
4-Methyl-3-(4-(pyridin-3-yl) pyrimidin-2-ylamino)-N-
(pyridin-4-yl) benzamide (A15)
In a 50-mL flask, 4-methyl-3-[[4-(3-pyridyl)-2-pyrimidinyl]
amino] benzoyl chloride (6) and pyridin-4-amine (0.35 g,
3.72 mmol) was dissolved into CH2Cl2 (20 mL). Under
N-(4-(3-(dimethylamino)propoxy) phenyl)-4-methyl-
3-(4-(pyridin-3-yl) pyrimidin-2-ylamino) benzamide
(A21)
In a 50-mL flask, 4-methyl-3-[[4-(3-pyridyl)-2-pyrimidinyl]
amino] benzoyl chloride (6) was dissolved into CH2Cl2
Chem Biol Drug Des 2014; 83: 592–599
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Pan et al.
Table 2: Structure and activity of A15–A20
Bcr-Abl
(20 mL). Under 0 °C, the CH2Cl2 (10 mL) solution of
4-(3-(dimethylamino) propoxy)benzenamine (b21) (3.92 mmol),
N, N’-diisopropyl ethylamine (5.0 mL), and 4-(dimethylami-
no) pyridine (cat) was dropped slowly into the above sus-
pension. After that, the mixture was reacted at r.t.
overnight. The mixture was diluted with CH2Cl2 (20 mL)
and was washed with saturated NaCl solution (10 mL 9 2)
and saturated NaHCO3 solution (10 mL 9 3). Then, the
organic phase was dried by Na2SO4 and filtered, giving the
crude product. The crude product was purified by chroma-
tography (ethyl acetate: methanol = 1:1), giving a solid
K562 cells
IC50 (lM)
Compounds
R
IC50 (lM)
N
N
A15
A16
A17
A18
A19
A20
2.190
88.682
10.274
260.148
6.087
4.025
3.578
0.305
0.210
3.578
0.214
H
N
HN
R
N
O
N
N
CI
N
N
N
(0.52 g). The total yield was 34%, mp 133–136 °C, EI-MS
23.957
5.936
+
(m/z): 437.3[M-NC2H6+H]
;
1H NMR (400 MHz, CDCl3):
N
S
d = 2.29–2.33 (m, 2H), 2.45 (s, 3H), 2.74 (s, 6H), 3.09 (t,
J = 8.0 Hz, 2H), 4.07 (t, J = 6.0 Hz, 2H), 6.88 (d,
J = 8.0 Hz, 2H), 7.25 (d, J = 4.0 Hz, 1H), 7.35 (d,
J = 8.0 Hz, 1H), 7.41–7.44 (m, 1H), 7.57 (d, J = 8.0 Hz,
3H), 7.98 (s, 1H), 8.44 (d, J = 8.0 Hz, 1H), 8.55 (d,
J = 8.0 Hz, 1H), 8.74 (d, J = 4.0 Hz, 1H), 8.81 (s, 1H),
9.27 (s, 1H).
N
Nilotinib
–
3.538
Table 3: Structure and activity of A21–A27
Bcr-Abl
K562 cells
Compounds
R
IC50 (lM)
IC50 (lM)
Compounds A22–A27 were prepared using the general
procedure described above.
N
A21
A22
A23
A24
A25
A26
A27
13.193
2.162
6.332
N
20.998
>100
>100
>100
0.010
>100
0.214
The melting point and spectral data of compounds
(A2–A14, A16–A20, and A22–A27) are provided as
Appendix S1 and S2.
N
2.273
H
N
HN
O
R
O
N
N
N
N
17.262
2.621
N
Results and Discussion
N
26.906
10.695
3.538
O
O
All the title compounds were evaluated for their Bcr-Abl
inhibitory activity (12). The results were summarized in
Tables 1, 2 and 3. Most of them exhibited potent Bcr-Abl
inhibitory activity. Compound (A26) was the most potent
with IC50 value of 10.15 nM. Moreover, four compounds
(A2, A8, A9, and A19) were also potent Bcr-Abl inhibitors
with IC50 values ranging from 135.56 to 258.57 nM. These
N
Nilotinib
–
Table 1: Structure and activity of A1–A14
Bcr-Abl
K562 cells
Compounds
R
IC50 (lM)
IC50 (lM)
A1
A2
3-Cl-4-F
3-CF3
3,4- di-Cl
4-OCH3
4-Br
2-F
3-F
3,5-di-CF3
3-Br-5-CF3
4-CH3
9.569
0.136
1.513
0.924
0.483
0.569
1.069
0.259
0.219
1.105
NDa
0.407
2.167
0.391
5.855
0.439
5.934
6.404
0.218
0.258
1.984
1.283
0.704
4.370
16.783
3.538
A3
A4
H
N
A5
A6
HN
R
O
N
N
A7
A8
N
A9
A10
A11
A12
A13
A14
Figure 2: Binding mode of nilotinib (red) and A26 with Bcr-Abl.
3-Cl
2,6-di-CH3
3-C(O)CH3
3-OCH3
–
0.956
0.542
5.243
0.214
compounds could be considered as promising candidates
for the development of novel Bcr-Abl inhibitors.
Nilotinib
All the compounds were evaluated for antitumor activities
against Bcr-Abl positive leukemia cell (K562). Most of them
aND is not determined.
596
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A Series of Novel Bcr-Abl Inhibitors
A
B
Figure 3: (A) Structure A; (B)
Superposition of 18 inhibitors for
CoMFA construction.
exhibited potent antiproliferative activity. In particular, com-
pounds (A8) and (A9) displayed potent activities with IC50
values of 217.65 and 258.32 nM, respectively. Five com-
pounds (A1, A3, A5, A12, and A18) were more potent
than nilotinib. The anticancer activities of A2, A10, A11,
A21, A22, and A25 were comparable with that of nilotinib.
ingly, A26 was more potent than nilotinib, exhibiting excel-
lent inhibitory activity against Bcr-Abl kinase. It might be
considered as a potent Bcr-Abl inhibitor.
Docking and SAR Studies
As shown from Table 1, compounds with halogen-substi-
tuted anilines were more potent than the others. It was
suggested that incorporation of halogen could improve
their biological activity. The results indicated that halogen
played important role in Bcr-Abl inhibitory activity. Higher
potency of A2, A8, and A9 indicated that trifluoromethyl
group may be of benefit to anticancer activity.
Docking
To investigate the interactions between inhibitors and Bcr-
Abl, molecular docking was carried out using Surflex-Dock
Mode of SYBYL-X 2.0 program package (Tripos, St. Louis,
MO, USA). The small molecules and the X-ray crystal
structure of Bcr-Abl (PDB ID: 3CS9) were imported. Niloti-
nib was used to define the binding cavity (13). All com-
pounds were used to perform docking, and the docking
result of A26 was shown in Figure 2. It binds to active site
of Bcr-Abl in a similar fashion to that of nilotinib. It inter-
acts with receptor through five hydrogen bonds involving
the pyridyl-N and the backbone NH of Met-318, the anili-
no-NH and the OH of Thr-315, the amido-NH and side
chain carboxylate of Glu-286, the amido carbonyl and the
backbone NH of the Asp-381 and the morpholine-O with
side chain guanidyl of Arg-362.
The biological activities of A15–A20 were listed in Table 2.
Replacement of anilines with heterocyclic aromatic amines
led to reduction in activity. Compound A18 was more
potent than A17, which suggested that incorporation of
halogen into heterocyclic amines could improve Bcr-Abl
inhibitory activity and antitumor effects. Moreover, A19 dis-
played similar Bcr-Abl inhibitory activity compared with nil-
otinib and could be considered as a novel lead compound
for further optimization.
As shown in Table 3, A21–A23 displayed potent antiprolif-
erative activity against K562 cells. However, A24, A25,
and A27 exhibited poor Bcr-Abl inhibitory activity. Surpris-
QSAR studies
3D-QSAR studies were performed with CoMFA module of
SYBYL-X 2.0. The test set consisted of nilotinib, A6, A8,
A
B
Figure 4: (A) The most active molecule A26 is shown in the background. Red color represents the negative charge region, blue is the
positive charge region, green is the more bulky region, and yellow is the less bulky region. (B) the predictability of the CoMFA model.
Chem Biol Drug Des 2014; 83: 592–599
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Pan et al.
A14, A17, and A26, the other 18 compounds (A1, A4–A6,
A8, A10, A12, A14–A20, A24, A26, and A27) composed
of the training set. The IC50 values were converted into
Acknowledgment
This work was supported by the National Natural Science
Foundation (NNSF) of China (Grant No. 81302737,
81302641, and 81230079), the Fundamental Research
Funds for the Central Universities, and Natural Science
Basic Research Plan in Shaanxi Province of China.
pIC50 according to the formula: pIC50 = log10IC50
.
The conformations of training set generated from docking
study were used. Based on the docking results, the
template molecule nilotinib was taken and the others
were aligned to it using structure A as scaffold by DATA-
BASE ALIGNMENT method in the SYBYL-X 2.0. The
aligned molecules are shown in Figure 3.
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In this study, a series of novel Bcr-Abl inhibitors was
designed and synthesized. The moiety interacting with
allosteric region of Bcr-Abl was modified to improve the
selectivity and biological activity. Most of them exhibited
potent Bcr-Abl inhibitory activity and antiproliferation
against K562 cells. The results indicated that incorporation
of halogenated-aniline or anilines containing tertiary amine
moiety could improve Bcr-Abl inhibitory activity and antitu-
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compounds gave a contribution to the exploration of Bcr-
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Chem Biol Drug Des 2014; 83: 592–599

A Series of Novel Bcr-Abl Inhibitors
Appendix S2. GC-MS and 1H NMR of compounds A1–
A27.
Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Appendix S1. Melting points and spectral data of com-
pounds (A2–A14, A16–A20, A22–A27).
Chem Biol Drug Des 2014; 83: 592–599
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