Design, synthesis and biological activities of Nilotinib derivates as antitumor agents

Article abstract of DOI:10.1016/j.bmc.2013.02.036

A novel class of Nilotinib derivatives, B1-B20, were synthesized in high yields using various substituted anilines. All the title compounds were evaluated for their inhibitory activities against Bcr-Abl and antiproliferative effects on human leukemia cell (K562). The pharmacological results indicated that some compounds exhibited promising anticancer activity. In particular, compound B14 containing tertiary amine side chain exhibited Bcr-Abl inhibitory activity similar to that of Nilotinib. It was suggested that the introduction of the tertiary amine moiety could improve Bcr-Abl inhibitory activity and antitumor effects.

Full text of DOI:10.1016/j.bmc.2013.02.036

Bioorganic & Medicinal Chemistry 21 (2013) 2527–2534
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological activities of Nilotinib derivates
as antitumor agents
⇑
Xiaoyan Pan, Fang Wang, Yanmin Zhang, Hongping Gao, Zhigang Hu, Sicen Wang, Jie Zhang
School of Medicine, 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:
A novel class of Nilotinib derivatives, B1–B20, were synthesized in high yields using various substituted
anilines. All the title compounds were evaluated for their inhibitory activities against Bcr-Abl and
antiproliferative effects on human leukemia cell (K562). The pharmacological results indicated that some
compounds exhibited promising anticancer activity. In particular, compound B14 containing tertiary
amine side chain exhibited Bcr-Abl inhibitory activity similar to that of Nilotinib. It was suggested that
the introduction of the tertiary amine moiety could improve Bcr-Abl inhibitory activity and antitumor
effects.
Received 4 February 2013
Revised 23 February 2013
Accepted 25 February 2013
Available online 5 March 2013
Keywords:
Bcr-Abl
Tyrosine kinase
Leukemia cell
Anticancer
Nilotinib
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
the inhibitory activity. Aniline containing halogen substituents is
useful for anticancer agents design. Halogen introduction can
CML is a haematological malignancy caused by a chromosomal
rearrangement which generates fusion protein (Bcr-Abl
enhance the persistence and lipid solubility.13 Therefore, various
halogen-substituted anilines were introduced to develop novel
Bcr-Abl inhibitors. Moreover, some heterocyclic amines or anilines
containing tertiary amine side chain were used to afford new
compounds with structural diversity.
a
kinase).1,2 It arises from the juxtaposition of Abelson gene (Abl)
on chromosome 9 to the break point cluster region (Bcr) gene on
chromosome 22.3 This fusion gene encodes a chimeric Bcr-Abl
protein, in which the tyrosine kinase activity of Abl is constitu-
tively activated.4 Therefore, Bcr-Abl kinase plays an important role
in the pathogenesis of CML. It is becoming an attractive target for
CML targeted therapy.5
2. Chemistry
In the present study, the facile total synthesis of Nilotinib from
commercial available reagents was firstly performed.14 In this
study, the synthesis of one key intermediate 4-methoxyl-3-[[4-
(3-pyridyl)-2-pyrimidinyl]amino] benzoic acid was similar to that
of Nilotinib. The key intermediate (5) was prepared in five steps. At
first, carboxylate group of 3-amino-4-methoxyl benzoic acid (1)
was protected by esterizing.15 Then refluxing of (2) and acetonitrile
in the presence of concentrated HCl yielded 3-guanidino-4-meth-
oxyl benzoic acid ethyl ester nitrate (3).16,17 Reaction of (3) with
3-dimethylamino-1-(3-pyridyl)-2-propen-1-one afforded (4).18,19
This reaction was carried out in the presence of NaOH. The hydro-
lysis of 4-methoxyl-3-[[4-(3-pyridyl)-2-pyrimidinyl] amino]
benzoic acid ethyl ester (4) in NaOH aqueous afforded intermedi-
ate (5).20 Finally, compound (5) condensed with various anilines
to yield title compounds using active ester method.21
Nilotinib is a phenylamino-pyrimidine derivative which was
rationally designed as second generation Bcr-Abl inhibitor.6 It
was approved to treat adult patients in all phases of CML with
resistance to Imatinib. However, nilotibib could not suppress the
proliferation of leukemia cells harboring some Bcr/Abl mutants
(T315I).7–9 Crystallographic studies have revealed that Nilotinib
bound to Bcr-Abl through four key interactions (Fig. 1).10–12
Nilotinib was designed to fit into the ATP binding site of Bcr-Abl
which was quite conserved. The allosteric binding region was
considered as selectivity site. Therefore, the pharmacophore bound
with allosteric region was optimized so as to improve the selectiv-
ity and activity. Similar to Nilotinib, we attempted to design novel
derivatives which bind to Bcr-Abl more tightly, thereby enhancing
Twenty pyrimidin-2-ylamino-benzamides (B1–B20) were pre-
pared as Bcr-Abl inhibitors. The synthetic route was shown in
Scheme 1. Synthesis of B1–B9 was similar to that of Nilotinib.
Various heterocyclic amines were used in the synthesis of
⇑
Corresponding author. Address: College of Medicine, Xi’an Jiaotong University,
No. 76, Yanta West Road, Xi’an, Shaanxi Province 710061, PR China. Tel.: +86 29
82657740; fax: +86 29 82655451.
E-mail address: zhj8623@mail.xjtu.edu.cn (J. Zhang).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.02.036

2528
X. Pan et al. / Bioorg. Med. Chem. 21 (2013) 2527–2534
Figure 1. The interactions between Nilotinib and Bcr-Abl kinase.
OCH3
OCH3
H
OCH3
H
N
OCH3
NH2
N
N
NH2
NH2
HNO3
b
a
c
N
N
NH
COOC2H5
COOC2H5
2
COOC2H5
3
COOH
1
4
d
OCH3
H
N
B1 R=3-Cl-4-F
B2 R=3-CF3
B3 R=4-Br
N
e
N
N
B4 R=3-F
R
R
B5 R=3,5-di-trifluoromethyl
B6 R=3-Br-5-CF3
B7 R=3-Cl
B8 R=3-C(O)CH3
B9 R=3-OCH3
H2N
O
N
H
OCH3
H
N
b1-b9
N
OCH3
H
N
N
N
+
B10 R=pyridin-4-yl
COOH
B11 R=5-chloropyridin-2-yl
B12 R=pyrimidin-2-yl
B13 R=thiazol-2-yl
N
R
e
H2N
N
R
5
O
N
H
N
b10-b13
OCH3
H
N
N
B14 R=3-(dimethylamino) propyl
B15 R=2-(dimethylamino) ethyl
B16 R=2-(diethylamino) ethyl
B17 R=2-(pyrrolidin-1-yl) ethyl
B18 R=2-(piperidin-1-yl) ethyl
B19 R=2-morpholinoethyl
R
O
N
O
R
e
N
H
O
N
B20 R=3-morpholinopropyl
c14-c20
NH2
g
b14-b20
R
OH
O
f
.HCl
+
N
Cl
n
NO2
NO2
d14-d20
Scheme 1. Reagents and conditions: (a) abs EtOH, concd H2SO4, reflux, 8 h; (b) H2NCN, concd HCl, EtOH, reflux, 15 h, NH4NO3(aq); (c) 3-(dimethyl amino)-1-(3-
pyridinyl)prop-2-en-1-one, NaOH, n-BuOH, reflux, 72 h; (d) 2 M NaOH, EtOH/H2O(v:v = 1:1), 50 °C, 2 h; (e) isobutyl chloroformate, 4-methylmorpholine, 0 °C, 30 min;
substituted benzamides or substituted heterocyclic amines or compound c14–c20, 4-methylmorpholine, rt, overnight; (f) Cs2CO3, DMF, 100 °C, 2–4 h; (g) Pd/C, MeOH, 4 h.

X. Pan et al. / Bioorg. Med. Chem. 21 (2013) 2527–2534
2529
Table 2
B10–B13. Intermediates c14–c20 were firstly prepared to yield
Structure and activity of B10–B13 towards Bcr-Abl and K562 cells in vitro
B14–B20. 4-Nitrophenol reacted with different chloro-substituted
tertiary amines in DMF at 100 °C followed by the reduction of nitro
to afford c14–c20.22–24 The last procedure was completed using ac-
tive ester method.
H3CO
H
N
HN
R
O
N
N
3. Results and discussion
N
All the title compounds were evaluated for their ability to inhi-
bit Bcr-Abl with Nilotinib as positive control.25 The results were
described in Tables 1–3. Noticeably, most of them exhibited rea-
sonable inhibitory activity against Bcr-Abl. Compound B14 was
the most potent with IC50 value of 0.52 nM. Moreover, some com-
pounds (B1, B3, B4, B12, B14, B16 and B20) were also potent Bcr-
Abl inhibitors with IC50 values ranging from 1.48 to 6.34 nM. These
compounds are considered as promising leading compounds for
the development of novel Bcr-Abl inhibitors as anticancer agents.
Some title compounds were also tested for antiproliferative
activity on Bcr-Abl positive leukemia cell (K562). As shown in Ta-
bles 1–3, several compounds displayed strong growth suppression
in K562 cells. In particular, B9 and B10 exhibited potent activities
Compounds
R
Bcr-Abl
IC50 (nM)
K562 Cells
IC50 M)
(l
N
B10
B11
2754.90
ND
94.48
ND
Cl
N
N
B12
3.78
ND
N
N
S
B13
294.34
0.33
ND
with IC50 values of 93.07 and 94.48 lM, respectively. Meanwhile,
four compounds (B5, B14, B17, B18), were also more potent than
Nilotinib
—
89.13
Nilotinib.
It was found from Table 1 that compounds containing halogen
substituted anilines were more potent than that without halogen.
Many halogen substituted compounds exhibited potent antiprolif-
erative activity in K562 cells. These results indicated that halogen
introduction was essential for Bcr-Abl inhibitory activity. Among
those compounds B1, B4 and B5 were more potent than the others.
The biological activities of B10–B13 were listed in Table 2. The
replacement of anilines with heterocyclic amines led to the reduc-
tion in activities. Compound B12 displayed potent Bcr-Abl inhibi-
tory activity. It was considered to be a novel Bcr-Abl inhibitor for
further optimization.
Table 3
Structure and activity of B14–B20 towards Bcr-Abl and K562 cells in vitro
H3CO
H
N
HN
O
R
N
N
O
N
As shown in Table 3, B14–B20 showed potent Bcr-Abl inhibitory
activity. Three of them exhibited potent Bcr-Abl kinase inhibitory
activity with good K562 growth inhibitory activity. The results sug-
gested that introduction of anilines containing tertiary amine side
chain was benefit for antitumor activity. B14 was potent inhibitors
both on Bcr-Abl kinase and K562 cell, and it was a good Bcr-Abl
inhibitor hit.
Compounds
R
Bcr-Abl
IC50 (nM)
K562 Cells
IC50 M)
(l
B14
B15
0.52
38.49
ND
N
N
N
2.34
B16
B17
B18
1.48
ND
Table 1
N
N
Structure and activity of B1–B9 towards Bcr-Abl and K562 cells in vitro
350.08
1110.42
38.61
5.15
H3CO
H
N
HN
R
N
O
B19
63.31
ND
N
N
O
O
B20
6.34
0.33
ND
N
N
Nilotinib
—
89.13
a
b
Compounds
R
Bcr-Abl IC50 (nM)
K562 Cells IC50 (lM)
B1
B2
B3
B4
3-Cl-4-F
3-CF3
4-Br
3-F
3,5-di-CF3
4.73
4125.31
4.79
3.21
NDc
ND
ND
ND
ND
4. Docking and QSAR studies
4.1. Docking
B5
4.84
B6
B7
B8
B9
3-Br-5-CF3
3-Cl
3-COCH3
3-OCH3
—
ND
34.77
ND
ND
0.33
214.82
237.47
237.47
93.07
89.13
Molecular docking study was performed to investigate the
binding mode of title compounds with Bcr-Abl. Docking was car-
ried out using Surflex-Dock Module of Sybyl-X 2.0. The small mol-
ecules and the X-ray crystal structure of Bcr-Abl in complex with
Nilotinib (PDB code: 3CS910) were imported, and Nilotinib was
used to define the binding cavity. All compounds were docked into
Nilotinib
a
b
c
ABL activity assays were performed using TTRF KinEASE-TK assay formats.
K562 cells assays were performed using MTT assays.
ND is not determined.

2530
X. Pan et al. / Bioorg. Med. Chem. 21 (2013) 2527–2534
the active site of Bcr-Abl. The binding model of B14 with Bcr-Abl
was shown in Figure 2. It was found that B14 bound to ATP pocket
of Bcr-Abl in a similar fashion to that of Nilotinib. It binds to the
kinase domain by making four hydrogen bond interactions involv-
ing the pyridyl-N and the backbone NH of Met-318, the anilino-NH
and the side chain OH of Thr-315, the amido-NH and side chain
carboxylate of Glu-286 and the amido carbonyl with the backbone
NH of the Asp-381.
874.585 and standard error of the estimate (SEE) is 0.061. The
contribution of electrostatic and steric is 53.8% and 46.2%, respec-
tively. It was found from Figure 4b that the CoMFA model could
predict test set well.
5. Conclusions
In summary, a series of novel Bcr-Abl inhibitors were prepared
and evaluated for their biological activities. Most of them exhibited
potent Bcr-Abl inhibitory activity and antiproliferation effect on
K562 cells. Compounds containing anilines were more potent than
that containing heterocyclic amines. The results revealed that the
interaction between this moiety and Bcr-Abl could mainly be
hydrophobic interaction. Moreover, the introduction of halogen
might be beneficial for Bcr-Abl inhibitory activity. Furthermore,
compounds containing tertiary amines side chain especially B14
4.2. QSAR studies
Sybyl-X 2.0 program was used to carry out QSAR studies of
these novel Bcr-Abl inhibitors. QSAR studies were performed with
the CoMFA module. The test set consisted of Nilotinib, B1, B3, B13,
B14 and B18, another 15 compounds (B1–B3, B7, B10, B12, B13,
and B14–B20) composed of the training set. The IC50 values were
converted into pIC50 according to the formula: pIC50 = log10IC50
.
could be considered as
optimization.
a novel lead compound for further
The docking conformation generated from training set with
Bcr-Abl (PDB code: 3CS9) were used. Based on the docking results,
the template molecule Nilotinib was taken and the rest of the mol-
ecules were aligned to it using structure A as scaffold by DATA-
BASE ALIGNMENT method. The aligned molecules are shown in
Figure 3.
6. Experimental
6.1. Cell growth inhibition assays
The steric and electrostatic fields were calculated at each lattice
intersection of a regularly spaced grid of 2.0 Å in all three dimen-
sions within defined region. An sp3 carbon atom with +1.00 charge
was used as probe atom. The steric and electrostatic fields were
truncated at +30.00 kcal molÀ1, and the electrostatic fields were ig-
nored at the lattice points with maximal steric interactions.
PLS method was used to linearly correlate CoMFA fields to the
inhibitory activity values. The cross-validation analysis was per-
formed using the leave one out (LOO) method in which one com-
pound is removed from the dataset and its activity is predicted
using the model derived from the rest of the dataset. The cross-val-
idated q2 (0.3) that resulted in optimum number of components
(n = 8) and lowest standard error of prediction were considered
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 Â 104 cells/well, and incubated for 24 h at 37 °C. The cells in
the wells were, respectively, treated with target compounds at var-
ious 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 super-
natant 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.
6.2. Kinase assays
for further analysis. We have evaluated different filter value
and at least selected
and reduce noise.
r
r
as 2.00 kcal molÀ1 to speed up the analysis
The kinase inhibition assay and IC50 determinations for wild
type 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
The non cross-validated r2 for the model established by the
study is 0.999. The value of the variance ratio F (n1 = 8, n2 = 7) is
Figure 2. Binding mode of Nilotinib (red) and B14 with the Bcr/Abl kinase domain.

X. Pan et al. / Bioorg. Med. Chem. 21 (2013) 2527–2534
2531
Figure 3. (a) Structure A; (b) superposition of 18 inhibitors for CoMFA construction.
Figure 4. (a) The most active molecule B14 is shown in the background. Red color represents the negative charge region, blue is the positive charge region, green is the more
bulky region, yellow is the less bulky region. (b) The predictability of the CoMFA model.
Carna Biosciences and 0.04 ng/
concentration was set at its Km values (6.021
l
L kinase were used for test. ATP
6.3.1. 3-Amino-4-methoxyl-benzoic acid ethyl ester (2)
3-Amino-4-methoxyl benzoic acid (6.01 g, 36 mmol) was dis-
solved in 100 mL dehydrated ethanol. Under 0 °C, 2 mL concen-
trated H2SO4 was dropped slowly 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, 100 mL water was added, and the solution was ad-
justed to pH 6–7 with sodium bicarbonate. Then the aqueous
was extracted with ethyl acetate (50 mL Â 2). The organic phase
was combined, and was washed by water (30 mL Â 2), saturated
sodium chloride (30 mL). The organic layer was dried over Na2SO4,
filtered, and distilled under vacuum to give the crude product. The
crude product was purified by chromatography (petroleum ether/
ethyl acetate = 1:1), giving a white solid (6.87 g). The total yield
was 98%, mp 55–57 °C. EI-MS (m/z): 195.1 ([M]+). 1H NMR
(400 MHz, CDCl3): d = 1.39 (t, J = 6.0 Hz, 3H), 3.92 (s, 3H), 4.34 (q,
J = 6.0 Hz, 14.00 Hz, 2H), 6.81 (d, J = 8.0 Hz, 1H), 7.42 (s, 1H), 7.50
(d, J = 8.0 Hz, 1H).
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 anti-
phosphotyrosine antibody labeled with europium 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. A Tecan
i-control infinite 500 was used to measure the fluorescence of
the samples at 620 nm (Eu-labeled antibody) and 665 nm (XL665
labeled streptavidin) 500
of both intensities for reactions made with six different inhibitor
concentrations (0.032 nM–3.2 M, including no inhibitor) was
ls after excition at 320 nm. The quotient
l
plotted against inhibitor concentrations to determine IC50 values.
Each reaction was performed in duplicate, and at least two inde-
pendent determinations of each IC50 were made.
6.3. Chemistry: general procedures
6.3.2. 3-Guanidino-4-methoxyl benzoic acid ethyl ester nitrate
(3)
All solvents and reagents were purified by standard techniques.
Petroleum ether (PE) used refers to the fraction boiling in the range
60–90 °C. All reactions except those in aqueous media were carried
out by standard techniques for moisture exclusion. Anhydrous
reactions were carried out over dried glassware under nitrogen
atmosphere. Reactions were monitored by TLC on 0.25 mm silica
gel plates (60GF254) and visualized with ultraviolet light. Melting
points were obtained on electrothermal meliting 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.
In 100 mL round bottom flask, 3-amino-4-methoxyl-benzoic
acid ethyl ester (2) (3.00 g, 16.56 mmol), and cyanamide (1.59 g,
38.09 mmol) were added to ethanol (20 mL). While being stirred,
concentrated HCl (2.1 mL, 24.84 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 (2.64 g,
33.12 mmol) solution was dropped during 30 min. Then the mix-
ture was stirred for 30 min, and filtered, giving the crude product.
The crude product need to be purified by being washed with ether,

2532
X. Pan et al. / Bioorg. Med. Chem. 21 (2013) 2527–2534
and finally a white solid (4.01 g) was maintained. The total yield
was 84.7%, mp 172–174 °C. EI-MS (m/z): 237.1 ([M]+ÀHNO3). 1H
NMR (400 MHz, D2O): d = 1.22 (t, J = 8.0 Hz, 3H), 3.81 (s, 3H),
4.21 (q, J = 6.0 Hz, 14.00 Hz, 2H), 7.09 (d, J = 12.0 Hz, 1H), 7.80 (s,
1H), 7.93 (d, J = 8.0 Hz, 1H).
CDCl3): d = 4.04 (s, 3H), 7.02 (d, J = 8.0 Hz, 1H), 7.16 (t, J = 8.0 Hz,
1H), 7.26 (d, J = 8.0 Hz, 1H), 7.45 (d, J = 4.0 Hz, 1H), 7.56–7.58 (m,
1H), 7.63 (d, J = 8.0 Hz, 1H), 7.85 (d, J = 4.0 Hz, 1H), 7.99 (d,
J = 12.0 Hz, 2H), 8.47 (d, J = 8.0 Hz, 1H), 8.60 (d, J = 4.0 Hz, 1H),
8.77 (s, 1H).
Compounds B2–B13 were prepared by using the general proce-
dure described above.
6.3.3. 4-Methoxyl-3-[[4-(3-pyridyl)-2-pyrimidinyl] amino]
benzoic acid ethyl ester (4)
In a round-bottom flask, 3-guanidino-4-methoxyl nitrate ethyl
benzoate (3) (3.00 g, 10 mmol), 3-dimethylamino-1-(3-pyridyl)-
2-propen-1-one (1.76 g, 10 mmol), sodium hydroxide (0.48 g,
12 mmol) was dissolved in n-butanol (20 mL). The mixture was
stirred and heated to reflux for 48 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 separated from the aque-
ous phase, and the aqueous phase was extracted by EtOAc
(20 mL Â 2). The combined organic phase was washed by water
(15 mL), 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 purified by recrystalliza-
tion from ether and EtOAc, giving a white solid (2.57 g). The total
yield was 73.4%, mp 99–100 °C. EI-MS (m/z): 350.1 [M]+. 1H NMR
(400 MHz, DMSO-d6): d = 1.34 (t, J = 8.0 Hz, 3H), 3.96 (s, 3H), 4.34
(q, J = 8.0 Hz, 16.0 Hz, 2H), 7.19 (d, J = 8.0 Hz, 1H), 7.57–7.59 (m,
1H), 7.61 (d, J = 4.0 Hz, 1H), 7.71 (d, J = 8.0 Hz, 1H), 8.37 (s, 1H),
8.55 (d, J = 8.0 Hz, 1H), 8.64 (d, J = 8.0 Hz, 1H), 8.75 (d, J = 8.0 Hz,
1H), 9.03 (s, 1H).
6.3.6. 4-Methoxyl-3-(4-(pyridin-3-yl) pyrimidin-2-ylamino)-N-
(3-(trifluoro methyl) phenyl) benzamide (B2)
Mp 244–246 °C, EI-MS(m/z): 465.0[M]+, 1H NMR (400 MHz,
CDCl3): d = 4.04 (s, 3H), 7.02 (d, J = 8.0 Hz, 1H), 7.26 (d, J = 8.0 Hz,
1H), 7.43–7.46 (m, 1H), 7.50 (d, J = 8.0 Hz, 2H), 7.61 (d, J = 8.0 Hz,
2H), 7.63 (d, J = 4.0 Hz, 1H), 7.97 (s, 2H), 8.50 (d, J = 8.0 Hz, 1H),
8.60 (d, J = 4.0 Hz, 1H), 8.76 (d, J = 4.0 Hz, 1H).
6.3.7. N-(4-Bromophenyl)-4-methoxyl-3-(4-(pyridin-3-
yl)pyrimidin-2-ylamino) benzamide (B3)
Mp 220–223 °C, EI-MS (m/z): 474.9[MÀ1]+, 1H NMR (400 MHz,
CDCl3): d = 4.05 (s, 3H), 7.04 (d, J = 12.0 Hz, 1H), 7.26 (s, 1H),
7.42–7.46 (m, 2H), 7.52 (t, J = 8.0 Hz, 1H), 7.66 (d, J = 8.0 Hz, 1H),
7.96 (s, 2H), 7.99 (s, 1H), 8.10 (s, 1H), 8.50 (d, J = 8.0 Hz, 1H), 8.61
(d, J = 4.0 Hz, 1H), 8.76 (d, J = 8.0 Hz, 1H).
6.3.8. N-(3-Fluorophenyl)-4-methoxyl-3-(4-(pyridin-3-
yl)pyrimidin-2-yl amino) benzamide (B4)
Mp 220–221 °C, EI-MS (m/z): 415.1[M]+, 1H NMR (400 MHz,
CDCl3): d = 4.03 (s, 3H), 6.85–6.88 (m, 1H), 7.00 (d, J = 8.0 Hz, 1H),
7.25 (d, J = 8.0 Hz, 1H), 7.32 (s, 2H), 7.42–7.45 (m, 1H), 7.62 (d,
J = 8.0 Hz, 1H), 7.67 (d, J = 8.0 Hz, 1H), 8.08 (s, 1H), 8.50 (d,
J = 8.0 Hz, 1H), 8.59 (d, J = 8.0 Hz, 1H), 8.76 (d, J = 4.0 Hz, 1H).
6.3.4. 4-Methoxyl-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) (6.70 g, 19.14 mmol) was dissolved into
the mixed solution of ethanol/water (60/60 mL). While being stir-
red, NaOH (2 mol/L, 25 mL) was drooped in the mixture. Then the
mixture was heated to 45–50 °C and was maintained at this tem-
perature overnight. Ethanol was removed by reduced pressure dis-
tillation, and then EtOAc (20 mL) was added. Then the organic
phase was abandoned. The aqueous phase was adjusted pH to 7–
8 using HCl (2 mol/L), and yellow solid was precipitated. The sus-
pension was filtered, giving the crude product (5.36 g). The total
yield was 87%, mp 340–342 °C. EI-MS (m/z): 305.1 [MÀOH]+. 1H
NMR (400 MHz, DMSO-d6): d = 3.86 (s, 3H), 6.96 (d, J = 8.0 Hz,
1H), 7.50 (d, J = 4.0 Hz, 1H), 7.55–7.58 (m, 1H), 7.62 (d, J = 8.0 Hz,
1H), 8.24 (s, 1H), 8.57 (d, J = 8.0 Hz, 1H), 8.60 (d, J = 8.0 Hz, 1H),
8.68 (s, 1H), 8.72 (d, J = 4.0 Hz, 1H).
6.3.9. N-(3,5-Bis(trifluoromethyl)phenyl)-4-methoxyl-3-(4-
(pyridin-3-yl) pyrimidin-2-ylamino) benzamide (B5)
Mp 241–243 °C, EI-MS (m/z): 533.1[M]+, 1H NMR (400 MHz,
CDCl3): d = 4.06 (s, 3H), 7.05 (d, J = 8.0 Hz, 1H), 7.44–7.47 (m, 1H),
7.66 (s, 1H), 7.68–7.72(m, 1H), 8.01 (d, J = 8.0 Hz, 1H), 8.19 (s,
1H), 8.27(s, 2H), 8.34 (s, 1H), 8.46 (d, J = 8.0 Hz, 1H), 8.62 (d,
J = 8.0 Hz, 1H), 8.76 (s, 1H).
6.3.10. N-(3-Bromo-5-(trifluoromethyl)phenyl)-4-methoxyl-3-
(4-(pyridin-3-yl) pyrimidin-2-ylamino) benzamide (B6)
Mp 227–230 °C, EI-MS (m/z): 544.0[M]+, 1H NMR (400 MHz,
CDCl3): d = 4.05 (s, 3H), 7.04 (d, J = 8.0 Hz, 1H), 7.27 (s, 1H), 7.44–
7.47 (m, 1H), 7.55 (s, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.98 (d,
J = 12.0 Hz, 2H), 8.20 (d, J = 4.0 Hz, 2H), 8.46 (d, J = 8.0 Hz, 1H),
8.62 (d, J = 4.0 Hz, 1H), 8.80 (s, 1H).
6.3.5. N-(3-Chloro-4-fluorophenyl)-4-methoxyl-3-(4-(pyridin-3-
yl)pyrimidin-2-ylamino) benzamide (B1)
6.3.11. N-(3-Chlorophenyl)-4-methoxyl-3-(4-(pyridin-3-
yl)pyrimidin-2-ylamino) benzamide (B7)
In a 100 ml flask, 4-methoxyl-3-[[4-(3-pyridyl)-2-pyrimidinyl]
amino] benzoic acid (5) (1.00 g, 3.10 mmol) and 4-methylmorpho-
line (1 mL, 9.3 mmol) were added in CHCl2 (15 mL). Under 0 °C,
the CH2Cl2 (8 mL) solution of isobutyl chloroformate (0.6 mL,
4.65 mmol) was dropped slowly into the above suspension. Then
the mixture was reacted under 0 °C for 30 min.
After that, the CH2Cl2 (10 mL) solution of 3-chloro-4-fluoro ani-
line (0.54 g, 3.10 mmol), isobutyl chloroformate (0.6 mL,
4.65 mmol) was dropped slowly into the above suspension. Then
the ice bath was removed and the mixture was reacted at rt over-
night. The mixture was diluted with CH2Cl2 (20 mL), and was
washed with saturated water (10 mL Â 2), saturated Na2CO3 solu-
tion (10 mL Â 3), NaCl solution (10 mL). Then the organic phase
was dried by Na2SO4, and filtered, giving the crude product. The
crude product was purified by chromatography (ethyl acetate/
methanol = 1:1), giving a solid (0.69 g). The total yield was 34%,
mp 226–227 °C, EI-MS (m/z): 449.0[M]+, 1H NMR (400 MHz,
Mp 211–213 °C, EI-MS (m/z): 431.0[M]+, 1H NMR (400 MHz,
CDCl3): d = 4.04 (s, 3H), 7.03 (d, J = 8.0 Hz, 1H), 715 (d, J = 8.0 Hz,
1H), 7.27 (d, J = 8.0 Hz, 1H), 7.30–7.34 (m, 1H), 7.44–7.47 (m,
1H), 7.60 (d, J = 8.0 Hz, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.77 (s, 1H),
7.99 (d, J = 4.0 Hz, 2H), 8.51 (d, J = 8.0 Hz, 1H), 8.62 (d, J = 4.0 Hz,
1H), 8.78 (d, J = 4.0 Hz, 1H).
6.3.12. N-(3-Acetylphenyl)-4-methxol-3-(4-(pyridin-3-
yl)pyrimidin-2-ylamino) benzamide (B8)
Mp 191–195 °C, EI-MS (m/z): 439.1[M]+, 1H NMR (400 MHz,
DMSO-d6): d = 2.60 (s, 3H), 3.97 (s, 3H), 7.23 (d, J = 8.0 Hz, 1H),
7.50–7.53 (m, 2H), 7.59 (d, J = 8.0 Hz, 1H), 7.72 (d, J = 8.0 Hz, 1H),
7.81 (d, J = 8.oHz, 1H), 8.12 (d, J = 8.0 Hz, 1H), 8.39 (s, 1H), 8.49
(s, 1H), 8.58 (d, J = 8.0 Hz, 1H), 8.64 (d, J = 4.0 Hz, 1H), 8.71 (d,
J = 8.0 Hz,), 8.91 (s, 1H).

X. Pan et al. / Bioorg. Med. Chem. 21 (2013) 2527–2534
2533
6.3.13. N-(3-Methoxyphenyl)-4-methoxyl-3-(4-(pyridin-3-yl)
pyrimidin-2-yl amino) benzamide (B9)
6.3.20. N-(4-(3-(Dimethylamino) propoxy) phenyl)-4-methoxyl-
3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)benzamide (B14)
In a 100 mL flask, 4-methoxyl-3-[[4-(3-pyridyl)-2-pyrimidinyl]
amino] benzoic acid (5) (1.00 g, 3.10 mmol) and 4-methylmorpho-
line (1 mL, 9.3 mmol) were added in CHCl2 (15 mL). Under 0 °C, the
CH2Cl2 (8 mL) solution of isobutyl chloroformate (0.6 mL,
4.65 mmol) was dropped slowly into the above suspension. Then
the mixture was reacted under 0 °C for 30 min.
Mp 208–210 °C, EI-MS (m/z): 427.0[M]+, 1H NMR (400 MHz,
CDCl3): d = 3.85 (s, 3H), 4.02 (s, 3H), 6.91–6.95 (m, 2H), 7.00 (d,
J = 8.0 Hz, 1H), 7.25 (d, J = 4.0 Hz, 1H), 7.40–7.43 (m, 1H), 7.50 (d,
J = 12.0 Hz, 1H), 7.58 (d, J = 12.0 Hz, 1H), 7.61–7.65 (m, 1H), 7.90
(s, 1H), 7.96 (d, J = 8.0 Hz, 1H), 8.51 (d, J = 8.0 Hz, 1H), 8.59 (d,
J = 8.0 Hz, 1H), 8.74 (s, 1H).
After that, the CH2Cl2 (10 mL) solution of 4-(3-(dimethyl-
amino)propoxy)benzenamine (c14) (3.92 mmol), isobutyl chloro-
formate (0.6 mL, 4.65 mmol) was dropped slowly into the above
suspension. Then the ice bath was removed and the mixture was
reacted at rt overnight. The mixture was diluted with CH2Cl2
(20 mL), and was washed with saturated water (10 mL Â 2), satu-
rated Na2CO3 solution (10 mL Â 3), NaCl solution (10 mL). Then
the organic phase was dried by Na2SO4, and filtered, giving the
crude product. The crude product was purified by chromatography
(ethyl acetate/methanol = 1:1), giving a solid (0.4 g). The total yield
was 26%, mp 133–136 °C, EI-MS (m/z): 498.3[M]+, 1H NMR
(400 MHz, DMSO-d6): d = 1.83–1.90 (m, 2H), 2.21 (s, 6H), 2.43 (t,
J = 8.0 Hz, 2H), 3.95 (s, 3H), 3.99 (t, J = 6.0 Hz, 2H), 6.92 (d,
J = 8.0 Hz, 2H), 7.20 (d, J = 8.0 Hz, 1H), 7.50–7.53 (m, 1H), 7.58 (d,
J = 8.0 Hz, 1H), 7.68 (d, J = 8.0 Hz, 2H), 7.74 (d, J = 12.0 Hz, 1H),
8.47 (s, 1H), 8.57 (d, J = 8.0 Hz, 1H), 8.63 (d, J = 4.0 Hz, 1H), 8.72
(d, J = 4.0 Hz, 1H), 8.85 (s, 1H).
6.3.14. 4-Methoxyl-3-(4-(pyridin-3-yl) pyrimidin-2-ylamino)-N-
(pyridin-4-yl) benzamide (B10)
Mp 241–243 °C, EI-MS (m/z): 398.1[M]+, 1H NMR (400 MHz,
DMSO-d6): d = 3.98 (s, 3H), 7.24 (d, J = 8.0 Hz, 1H), 7.50–7.55 (m,
1H), 7.59 (d, J = 8.0 Hz, 1H), 7.79 (d, J = 8.0 Hz, 1H), 7.82 (d,
J = 8.0 Hz, 2H), 8.47 (s, 3H), 8.57 (d, J = 8.0 Hz, 1H), 8.64 (d,
J = 8.0 Hz, 1H), 8.72 (d, J = 8.0 Hz, 1H), 8.93 (s, 1H).
6.3.15. N-(5-Chloropyridin-2-yl)-4-methoxyl-3-(4-(pyridin-3-
yl)pyrimidin-2-ylbamino) benzamide (B11)
Mp 216–218 °C, EI-MS (m/z): 432.0[M]+, 1H NMR (400 MHz,
DMSO-d6): d = 3.97 (s, 3H), 7.19 (d, J = 8.0 Hz, 1H), 7.53–7.56 (m,
1H), 7.60 (d, J = 8.0 Hz, 1H), 7.69 (d, J = 8.0 Hz, 1H), 7.88 (d,
J = 8.0 Hz, 1H), 7.98 (d, J = 8.0 Hz, 1H), 8.28 (d, J = 12.0 Hz, 1H),
8.45 (d, J = 8.0 Hz, 1H), 8.59 (d, J = 8.0 Hz, 1H), 8.64 (t, J = 4.0 Hz,
1H), 8.73 (t, J = 6.0 Hz, 1H), 8.97 (d, J = 8.0 Hz, 1H).
Compounds B15–B20 were prepared by using the general pro-
cedure described above.
6.3.16. 4-Methoxyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)-N-
(pyrimidin-2-yl) benzamide (B12)
Mp 187–190 °C, EI-MS (m/z): 399.1[M]+, 1H NMR (400 MHz,
DMSO-d6): d = 3.96 (s, 3H), 7.19 (d, J = 12.0 Hz, 1H), 7.6 (m, 1H),
7.51–7.54 (m, 1H), 7.59 (d, J = 4.0 Hz, 1H), 7.79 (d, J = 8.0 Hz, 1H),
8.47 (s, 1H), 8.59 (d, J = 8.0 Hz, 1H), 8.63 (d, J = 8.0 Hz, 1H), 8.72
(d, J = 8.0 Hz, 1H), 8.75 (d, J = 4.0 Hz, 2H), 8.89 (s, 1H).
6.3.21. N-(4-(2-(Dimethylamino)ethoxy)phenyl)-4-methoxyl-3-
(4-(pyridin-3-yl)pyrimidin-2-ylamino)benzamide (B15)
Mp 149–151 °C, EI-MS (m/z): 484.2[M]+, 1H NMR (400 MHz,
DMSO-d6): d = 2.22 (s, 6H), 2.62 (t, J = 6.0 Hz, 2H), 3.95 (s, 3H),
4.04 (t, J = 6.0 Hz, 2H), 6.94 (d, J = 8.0 Hz, 2H), 7.20 (d, J = 8.0 Hz,
1H), 7.50–7.53 (m, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.68 (d, J = 8.0 Hz,
2H), 7.74 (d, J = 8.0 Hz, 1H), 8.47 (s, 1H), 8.57 (d, J = 8.0 Hz, 1H),
8.63 (d, J = 4.0 Hz, 1H), 8.72 (d, J = 4.0 Hz, 1H), 8.85 (s, 1H).
6.3.17. 4-Methoxyl-3-(4-(pyridin-3-yl) pyrimidin-2-ylamino)-N-
(thiazol-2-yl) benzamide (B13)
Mp 205–208 °C, EI-MS (m/z): 403.9[M]+, 1H NMR (400 MHz,
DMSO-d6): d = 3.97 (s, 3H), 7.22 (d, J = 8.0 Hz, 1H), 7.28 (d,
J = 4.0 Hz, 1H), 7.58 (d, J = 4.0 Hz, 2H), 7.60 (d, J = 4.0 Hz, 1H),
7.96 (d, J = 8.0 Hz, 1H), 8.45 (s, 1H), 8.60 (d, J = 8.0 Hz, 1H), 8.65
(d, J = 4.0 Hz, 1H), 8.74 (d, J = 4.0 Hz, 1H), 9.03 (s, 1H).
6.3.22. N-(4-(2-(Diethylamino)ethoxy)phenyl)-4-methoxyl-3-
(4-(pyridin-3-yl)pyrimidin-2-ylamino)benzamide (B16)
Mp 149–152 °C, EI-MS (m/z): 512.3[M]+, 1H NMR (400 MHz,
DMSO-d6): d = 0.98 (t, d = 8.0 Hz, 6H), 2.55 (q, J = 6 Hz, 14 Hz, 4H),
2.77 (t, J = 6.0 Hz, 2H), 3.95 (s, 3H), 4.00 (t, J = 6.0 Hz, 2H), 6.93
(d, J = 8.0 Hz, 2H), 7.20 (d, J = 8.0 Hz, 1H), 7.50–7.53 (m, 1H), 7.58
(d, J = 8.0 Hz, 1H), 7.68 (d, J = 8.0 Hz, 2H), 7.74 (d, J = 8.0 Hz, 1H),
8.47 (s, 1H), 8.57 (d, J = 8.0 Hz, 1H), 8.63 (d, J = 4.0 Hz, 1H), 8.72
(d, J = 4.0 Hz, 1H), 8.85 (s, 1H).
6.3.18. N,N-Dimethyl-3-(4-nitrophenoxy) propan-1-amine (d14)
In 100 mL flask, 4-nitrophenol (1.39 g, 10 mmol) was dissolved
in 20 mL anhydro-DMF. Then 3-chloro- N,N-dimethylpropan-1-
amine hydrochloride (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 fil-
tered, and the filtrate was poured to ice water (200 mL). The aque-
ous phase was extracted by EtOAc (30 mL Â 3). The combined
organic phase was washed by Na2CO3 (10 mL Â 4), water
(10 mL Â 2), saturated sodium chloride (15 mL). The organic layer
was dried over Na2SO4, filtered, and distilled under vacuum to give
the crude product.
6.3.23. 4-Methoxyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)-N-
(4-(2-(pyrrolidin-1-yl) ethoxy) phenyl) benzamide (B17)
Mp 118–121 °C, EI-MS (m/z): 510.2[M]+, 1H NMR (400 MHz,
DMSO-d6): d = 1.70 (m, 4H), 2.55 (m, 4H), 2.82 (t, J = 6.0 Hz, 2H),
3.95 (s, 3H), 4.06 (t, J = 6.0 Hz, 2H), 6.94 (d, J = 8.0 Hz, 2H), 7.20
(d, J = 8.0 Hz, 1H), 7.50–7.53 (m, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.68
(d, J = 8.0 Hz, 2H), 7.75 (d, J = 8.0 Hz, 1H), 8.47 (s, 1H), 8.57 (d,
J = 8.0 Hz, 1H), 8.63 (d, J = 4.0 Hz, 1H), 8.72 (d, J = 4.0 Hz, 1H),
8.86 (s, 1H).
6.3.19. 4-(3-(Dimethylamino) propoxy) benzenamine (c14)
N,N-Dimethyl-3-(4-nitrophenoxy) propan-1-amine (d14)
(3.92 mmol) was dissolved in 30 mL anhydrous methanol, and
then 0.1 g Pd/C (5%) was added to this solution. After that, the mix-
ture was reacted at room temperature under N2 for 4 h. The mix-
ture was filtered, and Pd/C was washed with methanol for 3–4
times. The organic phases were combined and methanol was re-
moved by reduced pressure distillation to give the crude product.
The residue was reserved for the next step.
6.3.24. 4-Methoxyl-N-(4-(2-(piperidin-1-yl)ethoxy) phenyl)-3-
(4-(pyridin-3-yl) pyrimidin-2-ylamino) benzamide (B18)
Mp 141–144 °C, EI-MS (m/z): 524.2[M]+, 1H NMR (400 MHz,
CDCl3): d = 2.10 (m, 2H), 2.60 (m, 8H), 3.06 (t, J = 4.0 Hz, 2H), 4.03
(s, 3H), 4.27 (t, J = 4.0 Hz, 2H), 6.95 (d, J = 8.0 Hz, 2H), 7.01 (d,
J = 8.0 Hz, 1H), 7.41–7.45 (m, 1H), 7.59 (d, J = 8.0 Hz, 2H), 7.63 (d,
J = 8.0 Hz, 1H), 7.90 (s, 1H), 7.98 (s, 1H), 8.51 (d, J = 8.0 Hz, 1H),
8.60 (d, J = 4.0 Hz, 1H), 8.76 (s,1H).
Compounds c14–c20 were prepared by using the general proce-
dure described above.

2534
X. Pan et al. / Bioorg. Med. Chem. 21 (2013) 2527–2534
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Acknowledgments
This work was supported by the National Natural Science Foun-
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Supplementary data
Supplementary data associated with this article can be found, in
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