Synthesis and biological evaluation of 2,4-diaminopyrimidines as selective Aurora A kinase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2015.03.044

The Aurora kinases are a family of serine/threonine kinases that interact with components of the mitotic apparatus and serve as potential therapeutic targets in oncology. Here we synthesized 15 2,4-diaminopyrimidines and evaluated their biological activities, including antiproliferation, inhibition against Aurora kinases and cell cycle effects. These compounds generally exhibited more potent cytotoxicity against tumor cell lines compared with the VX-680 control, especially compound 11c, which showed the highest cytotoxicities, with IC₅₀ values of 0.5-4.0 1/4M. Compound 11c had more than 35-fold more selectivity for Aurora A over Aurora B, and molecular docking analysis indicated that compound 11c form better interaction with Aurora A both from the perspective of structure and energy. Furthermore, compound 11c induced G2/M cell cycle arrest in HeLa cells. This series of compounds has the potential for further development as selective Aurora A inhibitors for anticancer activity.

Full text of DOI:10.1016/j.ejmech.2015.03.044

European Journal of Medicinal Chemistry 95 (2015) 174e184
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Synthesis and biological evaluation of 2,4-diaminopyrimidines as
selective Aurora A kinase inhibitors
,
,
Wen-Wen Qin a 1, Chun-Yan Sang a 1, Lin-Lin Zhang a, Wei Wei a, Heng-Zhi Tian a,
Huan-Xiang Liu a, Shi-Wu Chen a *, Ling Hui b
,
, c, **
a School of Pharmacy, Lanzhou University, Lanzhou 730000, China
b Experimental Center of Medicine, General Hospital of Lanzhou Military Command, Lanzhou 730050, China
c Key Laboratory of Stem Cells and Gene Drug of Gansu Province, General Hospital of Lanzhou Military Command, Lanzhou 730050, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The Aurora kinases are a family of serine/threonine kinases that interact with components of the mitotic
apparatus and serve as potential therapeutic targets in oncology. Here we synthesized 15 2,4-
diaminopyrimidines and evaluated their biological activities, including antiproliferation, inhibition
against Aurora kinases and cell cycle effects. These compounds generally exhibited more potent cyto-
toxicity against tumor cell lines compared with the VX-680 control, especially compound 11c, which
Received 19 November 2014
Received in revised form
17 March 2015
Accepted 19 March 2015
Available online 20 March 2015
showed the highest cytotoxicities, with IC50 values of 0.5e4.0 mM. Compound 11c had more than 35-fold
more selectivity for Aurora A over Aurora B, and molecular docking analysis indicated that compound 11c
form better interaction with Aurora A both from the perspective of structure and energy. Furthermore,
compound 11c induced G2/M cell cycle arrest in HeLa cells. This series of compounds has the potential
for further development as selective Aurora A inhibitors for anticancer activity.
Keywords:
Aurora kinase
Kinase inhibitors
Pyrimidine
© 2015 Elsevier Masson SAS. All rights reserved.
Cell cycle
1. Introduction
regulation of a mitotic checkpoint at metaphase and a role in
cytokinesis [6]. Aurora C has similar functions as Aurora B [7].
The Aurora kinase family is a subfamily of serine/threonine ki-
nases that is essential for the regulation of centrosome maturation,
mitotic spindle formation, chromosome segregation and cytoki-
nesis during mitosis [1,2]. The family includes three kinases
designated as Aurora A, B, and C, which are very closely related in
the kinase domain sequence. However, these kinases have quite
different and nonoverlapping functions during mitosis [3]. Aurora A
regulates the cell cycle and is associated with late S phase and entry
into the M phase. It associates with the spindle poles and is
involved in both centrosomal and acentrosomal spindle assembly
[4,5]. Aurora B localizes to the centromeres in prometaphase, and
then relocates to the spindle midzone at anaphase. It has functions
associated with histone phosphorylation and chromatin conden-
sation in prophase, chromosome alignment and segregation,
The expressions of Aurora A and Aurora B are elevated in a va-
riety of human cancers and are associated with poor prognosis [8].
The potential roles of Aurora kinases in regulating cell mitosis and
tumorigenesis make them attractive targets for anticancer therapy
[9]. Many Aurora kinase inhibitors have been developed and
introduced into clinical trials, including VX-680/MK-0457,
ZM447439, Hesperadin, MLN8054, MLN8237, and AZD1152
(Fig. 1) [10e12].
ZM447439 [13], Hesperadin [14] and VX-680/MK-0457 [15]
were the first generation of Aurora kinase inhibitors. These three
small molecule chemical inhibitors occupy the ATP-binding site in
Aurora kinases to inhibit catalytic activity. Unlike pan-Aurora ki-
nase inhibitors, MLN8054 and MLN8237 are ATP-competitive and
reversible Aurora A selective inhibitors, and are approximately 40-
fold and 200-fold more sensitive towards Aurora A, respectively,
compared with Aurora B [12]. MLN8237 is more potent than
MLN8054 and causes less benzodiazepine-like side effects based on
structure modulation by the addition of a methoxy group to either
end of the MLN8054 molecule [16,17]. AZD1152 is an Aurora B se-
lective inhibitor that showed 1000-fold selectivity for Aurora B over
Aurora A and a panel of 50 additional kinases in enzymatic assays
* Corresponding author. School of Pharmacy, Lanzhou University, Lanzhou
730000, China.
** Corresponding author. Experimental Center of Medicine, General Hospital of
Lanzhou Military Command, Lanzhou 730050, China.
E-mail address: chenshw@lzu.edu.cn (S.-W. Chen).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejmech.2015.03.044
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

W.-W. Qin et al. / European Journal of Medicinal Chemistry 95 (2015) 174e184
175
Fig. 1. Aurora kinase inhibitors.
[18e21]. It thus still remains uncertain how exactly aurora A and B
pan- or monospecific inhibitors induce tumor cell death and which
type of inhibitor will be preferable from a therapeutic viewpoint.
Recently, the research of novel selective Aurora inhibitors has
become a new trend, and a lot of new active compounds have been
developed [22e24].
Pyrimidine is the important pharmacology core in many Aurora
inhibitors, such as VX-680, ENMD-2076, CYC-116 and ENMD-2076
[12]. To identify additional effective Aurora inhibitors, we
protecting group provided aniline 4a in the dichloromethane so-
lution of trifluoroacetic acid [26]. To obtain compound 4b, 3-
methoxy-4-amino-benzoic acid (1b), as the raw material, the re-
action process was similar to preparing 4a. Another intermediate
aniline 6aeb was prepared by substituted reaction of 4-chloro-1-
nitrobenzene with morpholine or 4-methyl-piperazine, and the
nitro was reduced by catalytic hydrogen under the catalysis of 10%
Pd/C [27].
Our approach to the preparation of 2,4-diaminopyrimidines
based on the double SN2 displacement of pyrimidine is shown in
Scheme 3. The displacement of the 4-chloro group of 2,4-dichloro-
5-substituted pyrimidine by cyclopentyl amine, cyclopropyl amine,
and n-propylamine provided 8aed, 9 and 10, which has already
been widely reported in the literature [28]. Treatment of 8aed, 9
and 10 with the different anilines 4a,b or 7a,b in isopropanol in the
presence of hydrochloride at 80 ꢀC gave the target compounds
11aed, 12b,c and 13e19 [29]. The target compound 20 was syn-
thesized by the nitro at C-5 of 16 and reduced with hydrogen gas
under the catalysis of 10% Pd/C. The newly synthesized compounds
were characterized by physicochemical and spectral means, and
both analytical and spectral data of all the compounds were in full
agreement with the proposed structures.
designed
a series of 2,4-diaminopyrimidine compounds, our
modeling studies suggested that the pyrimidine core as well as the
secondary aromatic amine of the compounds form hydrogen bonds
with the hinge region of the kinase domain and show selectively
inhibition to Aurora A over Aurora B. Introduction of cyclopentyl
amine on the C-4 in pyrimidine can adopt a binding mode similar to
VX-680 [25]. Furthermore, the differences of F, Cl, Br and NO2 at 5-C
of pyrimidine was to investigate the effects of the electron-
withdrawing on anti-proliferation and inhibition of Aurora ki-
nase. Herein, we reported the synthesis, and evaluated their anti-
proliferation activities, inhibition of Aurora kinase and effects on
the cell cycle.
2. Results and discussion
2.2. Biological activity
2.1. Chemistry
2.2.1. Cytotoxicities of compounds 11e20
The general synthetic routes for intermediate anilines 4aeb and
7aeb are illustrated in Schemes 1 and 2, respectively. Treatment of
p-aminobenzoic acid 1a with ditertbutyl dicarbonate ((BOC)2O)
afforded 4-Boc-amino-benzoic acid 2a, and then condensation of
compound 2a with N-methyl-4-amino-piperidine generated 3a
under condensing agent tri(dimethylamino)benzotriazol-1-
yloxyphos phonium hexafluorophosphate (BOP) in the presence
of N,N-diisopropylethylamine (DIPEA). Finally, removal of the
The in vitro cytotoxicities of target compounds 11e20 were
evaluated in a panel of four human tumor cell lines (cervical car-
cinoma HeLa, lung carcinoma A-549, human colorectal adenocar-
cinoma HCT-8 and hepatic carcinoma Hep-G2 cells), with VX-680
as a reference compound. The screening procedure was based on
the standard MTT method [30], and the results are summarized in
Table 1.
All the target compounds showed better or equivalent

176
W.-W. Qin et al. / European Journal of Medicinal Chemistry 95 (2015) 174e184
Scheme 1. Synthesis of compounds 4aeb. Reagents and conditions: (i) Boc2O, NaOH, dioxane: H2O (2:1), 0 ꢀC; (ii) N-methyl-4-amino-piperidine, BOP, DIPEA, rt; (iii) TFA:
DCM ¼ 3:7, rt.
to increased or maintained cytotoxicity upon treatment of the four
cell lines. However, substitution of 4a with 7a and 7b in the C-2 of
pyrimidine resulted in decreased antiproliferation activity of the
target compounds. We next investigated the replacement of the
cyclopentyl group on the C-4 in pyrimidine by different groups,
such as cyclopropyl or n-propylamine. Unfortunately, all the syn-
thesized compounds showed equivalent or lower antiproliferation
activities.
Scheme 2. Synthesis of compounds 6aeb. Reagents and conditions: (i) morpholine or
From the results of the in vitro cytotoxic assays, we found that
compound 11c showed strong growtheinhibitory activities in the
N-methylpiperazine, K2CO3, 80 ꢀC, 5 h; (ii) H2, 10% Pd/C, 1 atm, rt.
cervical carcinoma HeLa cell line. The IC50 value of 11c was 0.9 mM,
antiproliferation activity in the four human tumor cell lines
compared with VX-680. Notably, compounds 11aed with different
substitutions (F, Cl, Br and NO2) at C-5 of pyrimidine showed
significantly different effects in regards to cytotoxicity. The anti-
proliferation activity of compounds 11b and 11c substituted with
chloride or bromide at C-5 of pyrimidine showed more potent
antiproliferation effects compared with compounds 11a and 11d, in
which C-5 in pyrimidine was substituted with fluorine and nitro,
respectively. However, we did not find any obvious differences in
cytotoxicities in compounds 12aec. The displacement of the ani-
lines from 4a to 4b in the C-2 substitute on the pyrimidine ring led
which was 30-fold lower than VX-680. Next we selected com-
pounds 11c and 12a to explore the effects on Aurora kinases and the
cell cycle in HeLa cells.
2.2.2. Compounds 11c and 12a selectively inhibit Aurora A over
Aurora B kinase in HeLa cells
Many pyrimidine compounds, such as VX-680, ENMD-2076,
CYC-116 and ENMD-2076, have entered into clinical trials as Aurora
inhibitors [12]. To explore whether 11c and 12a had similar effects
on inhibition of Aurora kinases in HeLa cells, we investigated the
Compounds
13
F
14
F
15
NO2
O
16
17
F
18
F
19
F
20
X
Y
NO2
NMe
NH2
NMe
O
NMe
O
NMe
O
R
Scheme 3. Synthetic approach of target compounds 11e20. Reagents and conditions: (i) THF, 0 ꢀC, 3 h; (ii) isopropyl alcohol, 1 drop of 37% HCl, 80 ꢀC, 18 h; (iii) ethyl acetate, H2, 10%
Pd/C, 1 atm, 60 ꢀC.

W.-W. Qin et al. / European Journal of Medicinal Chemistry 95 (2015) 174e184
177
Table 1
The cytotoxicities of compounds 11e20 at drug exposure for 48 h.
Compds
X
F
Y
R1
R2
(IC50, m
M)a
HCT-8
16.0
A-549
10.3
HeLa
45.5
Hep-G2
11a
H
10.0
2.2
2.4
5.8
4.5
9.3
4.9
11b
11c
11d
12a
12b
12c
Cl
H
4.6
3.6
1.8
0.5
1.5
0.9
Br
NO2
F
H
H
15.6
2.1
42.3
2.1
21.7
2.2
OMe
OMe
OMe
Cl
4.4
5.3
2.7
Br
12.0
2.5
3.5
13
14
15
16
17
18
19
20
F
H
H
H
H
H
H
H
H
24.0
29.0
20.0
53.7
40.3
50.0
73.9
4.2
17.3
37.2
63.7
4.6
31.4
20.0
36.4
3.8
36.5
25.0
21.9
5.2
F
NO2
NO2
F
8.7
6.0
33.0
35.0
20.0
18.5
63.4
F
5.1
14.2
7.3
F
8.8
NH2
27.0
19.4
9.7
VX-680
44.6
27.3
a
Data are the mean of three independent experiments.
effects on Aurora A and B kinases by western blot [31] and enzyme-
linked immunosorbent assay (ELISA) [32].
showed no significant changes. When the concentration of 11c was
increased to more than 10 nM, the expression levels of Aurora A and
B protein rapidly decreased in HeLa cells, and Aurora A protein was
reduced more than that of Aurora B. From Fig. 3A, we easily ob-
HeLa cells were treated with various concentrations of com-
pound 11c (10 nM, 25 nM, and 50 nM) for 12 h. The expression
levels of Aurora A and Aurora B were decreased in HeLa cells upon
exposure to 11c in a dose-dependent manner. We also observed
that the effect of 11c on Aurora A was more potent than on Aurora B,
indicating that compound 11c showed selectivity of inhibition of
Aurora A over Aurora B (Fig. 2A and C). Compound 12a showed the
same effects of 11c, but its potency was weaker than 11c (Fig. 2B
and D).
tained the IC50 values of 11c for Aurora A and B as 0.012
mM and
0.430 M, respectively. These results indicate that compound 11c
m
was more than 35-fold more selective for Aurora A compared with
Aurora B in HeLa cells. We also obtained IC50 values of 12a for
Aurora A and B of 0.043 mM and 0.395 mM, respectively, and the
selectivity of 12a for Aurora A was only 9-fold over Aurora B.
However, the IC50 values of VX-680 for Aurora A and B was
The selective effects of compounds 11c, 12a and VX-680 on
Aurora A kinase inhibition were also confirmed by ELISA, as shown
in Fig. 3. When HeLa cells were treated with less than 5 nM of
compound 11c for 12 h, the expression levels of Aurora A and B
0.261
VX-680 for Aurora
growtheinhibitory activities of compound 11c and 12a than VX-
680 were in accordance with their better selectivity of Aurora A
m
M and 0.453
m
M, respectively, and no obvious selectivity of
A
over Aurora B. Obviously, the strong

178
W.-W. Qin et al. / European Journal of Medicinal Chemistry 95 (2015) 174e184
Fig. 2. Western blot for selectivity of inhibition of Aurora A and Aurora B with compound 11c (A, C) and 12a (B, D) in HeLa cells.
Fig. 3. Compound 11c (A), 12a (B) and VX-680 (C) selectively inhibits Aurora A over Aurora B in HeLa cells.
over Aurora B.
the figures displaying the docking results were obtained using the
scientific software Pymol [35]. AutoGrid was used to produce grids
based on the position of the ligand in the proteins (PDB code 3D14
for Aurora A and 4C2V for Aurora B). In the docking process, the
protein was considered to be rigid, while the ligand was considered
flexible. The ligand 11c was docked into the appropriate binding
2.2.3. Aurora kinase binding model of compound 11c
To gain insight into the interaction of compound 11c with
Aurora A and Aurora B, docking simulation was performed using
the Autodock 4.2 [33] with Lamarckian Genetic Algorithm [34]. All

W.-W. Qin et al. / European Journal of Medicinal Chemistry 95 (2015) 174e184
179
pocket of Aurora A and Aurora B using the autodock module and the
calculated binding energy was ꢁ10.02 kcal/mol for Aurora A
and ꢁ9.05 kcal/mol for Aurora B, respectively. The resulting dock-
ing poses are shown in Fig. 4. According to the binding energy,11c is
more sensitive to Aurora A than Aurora B.
2.2.5. Compound 11c activates cyclin B1
Cyclin B is a member of the cyclin family and regulates the
progression of cells into and out of M phase [38]. Thus, we exam-
ined the effect of 11c on the expression of cyclin B in HeLa cells [39].
As shown in Fig. 6, treatment of cells with 11c resulted in an
apparent increase of cyclin B, demonstrating that compound 11c
can interfere with cell cycle progression. This conclusion is
consistent with the cell cycle analysis results.
Overall, the binding pockets of Aurora A and Aurora B are highly
hydrophobic. Thus, hydrophobic interaction is the main driving
force for the binding of 11c to Aurora A and Aurora B. In addition to
the hydrophobic interactions, 11c can also form several important
H-bonds. For example, in Aurora A, 11c can form two H-bonds with
Glu62 and Asp155. In Aurora B, the oxygen atom in the amide group
and the nitrogen atom in the piperidine ring of 11c can form two H-
bonds with Lys122. From the distance of the two atoms that form
hydrogen bonds, Aurora A can form stronger hydrogen-bonding
interactions compared with Aurora B. Additionally, from the
shape of the binding pocket, 11c can fit much better with the
binding pocket of Aurora A. These differences may explain why
compound 11c binds better with Aurora A compared with Aurora B.
3. Conclusions
Aurora kinases have been of interest as potential therapeutic
targets in oncology. Here we describe
a series of 2,4-
diaminopyrimidine small molecule inhibitors that exert their
cytotoxic activities in human tumor cell lines through inhibition of
Aurora kinases. We specifically demonstrate that compound 11c
was selective for inhibition of Aurora A over Aurora B in HeLa cells,
and the molecular docking analysis revealed that 11c form better
interaction with Aurora A than that with Aurora B. Treatment of
HeLa cells with compound 11c also results in G2/M accumulation.
These results suggest that these compounds have potential for
further development in vivo as anticancer agents.
2.2.4. Compound 11c induces cell cycle arrest in G2/M phase
The Aurora A inhibitor induces common phenotypic effects such
as G2/M accumulation, spindle defects and chromosome
misalignment, and has enabled the identification of previously
unknown Aurora A-regulated cellular functions [36]. Many studies
have shown that VX-680 induces cell cycle arrest in the G2/M
phase. To determine whether compound 11c has similar effects on
tumor cells, we investigated its effects on cell cycle progression
using fluorescence-activated cell sorting analysis of HeLa cells
stained with propidium iodide [37] (Fig. 5).
4. Experiment
4.1. Chemistry
All starting materials and regents were purchased commercially
and used without further purified, unless otherwise stated. All re-
actions were monitored by thin layer chromatograph (TLC) on silica
gel GF254 (0.25 mm thick). Column chromatography (CC) was
performed on Silica Gel 60 (230e400 mesh, Qingdao Ocean
Chemical Ltd., China). Melting points were determined in Kofler
apparatus and were uncorrected. IR spectra were measured on a
NicoLET iS5 spectrometer on neat samples placed between KBr
plates. 1H NMR and 13C NMR spectra were recorded with a Varian
Mecury-400BB or Mecury-600BB spectrometer with TMS as an
internal standard, all chemical shift values are reported as ppm.
Mass spectra were recorded on a Bruker Dalton APEXII49e and
Esquire6000 (ESI-ION TRAP) spectrometer with ESI source as
ionization, respectively.
Treatment of HeLa cells with 11c resulted in a dose-dependent
accumulation of cells in the G2/M phase with a concomitant
decrease in the population of G1 phase cells. After 12 h of treatment
with 2 mM or 5 mM of 11c, the percentages of cells in G2/M phase
arrest were 33.2% and 43.5%, respectively, compared with 10.2% in
untreated cultures. These results demonstrate that 11c interfered
with cell proliferation by arresting the cell cycle in G2/M.
4.2. Synthesis of anilines 4a,b
4.2.1. 4-[(tert-Butoxycarbonyl)amino]benzoic acid (2a)
To a mixture of 4-aminobenzoic acid 1a (5.00 g, 36.5 mmol) in
dioxane (70 mL) and water (35 mL) were added NaOH (1.46 g,
36.5 mmol) followed by di-tert-butyl dicarbonate (11.9 g,
54.8 mmol). The reaction mixture was stirred at room temperature
for 24 h. Solvent was removed by rotary evaporation, and 3 N
aqueous hydrochloric acid was added dropwise to the residue to
adjust pH 3. A precipitate was obtained, collected, washed with
water, and dried to provide 2a (8.28 g, 96%) as a solid.
4.2.2. 4-[N-(tert-Butoxycarbonyl)amino]-N-(4-N-methyl-
piperazinyl)benzamide (3a)
To a solution of 2a (3.6 g, 15 mmol) in dry DMF (50 mL) were
added BOP (8.0 g, 18 mmol) and DIPEA (9.5 mL, 18 mmol). After
stirring at room temperature for 1 h, 4-N-methyl-piperazine
(2.5 mL, 20 mmol) was added dropwise and the reaction continued
for 24 h at room temperature under argon. The product was then
extracted with ethyl acetate (5 ꢂ 50 mL). The combined organic
extracts were washed with brine (2 ꢂ 40 mL), dried over sodium
sulfate, filtered, and the solvent removed. Purification was achieved
by flash chromatography (dichloromethane/methanol 20:1 by
Fig. 4. The binding mode of 11c with Aurora A and Aurora B. A) The interaction be-
tween 11c and Aurora A. B) The interaction between 11c and Aurora B. C) The binding
pocket of Aurora A was shown in surface. D) The binding pocket of Aurora B was shown
in surface (To show the binding pocket clearly, K103 and F104 weren't shown). The
surface is colored by gray.

180
W.-W. Qin et al. / European Journal of Medicinal Chemistry 95 (2015) 174e184
Fig. 5. Effect of 11c on cell cycle progression. (A) Control HeLa cells, (B) HeLa cells treated with 2 mM 11c for 12 h, (C) HeLa cells treated with 5 mM 11c for 12 h.
provide 4-morpholino nitrobenzene 5a (2.3 g, 40%).1H NMR
(400 MHz, CDCl3)
d
8.14 (dd, J ¼ 7.2, 2.0 Hz, 2 H), 6.84 (dd, J ¼ 7.6, 2.0
Hz, 2 H), 3.87 (t, J ¼ 4.8 Hz, 4 H), 3.41 (t, J ¼ 5.2 Hz, 4 H).
4.3.2. 4-morpholinyl aniline (6a)
Fig. 6. Western blotting analysis of expression of cyclin B1 in HeLa cells treated with
11c.
To a mixture of 5a (2.13 g, 10 mmol) in dry ethyl acetate was
added 10% Pd/C (0.5 g), following the reaction mixture was stirred
under hydrogen gas at 80 ꢀC for 12 h. The reaction mixture was
concentrated in vacuo and the crude product was purified by col-
umn chromatography (dichloromethane/methanol 15:1 by vol-
ume) to give 4-morpholinyl aniline 6a (1.5 g, yield 85%). 1H NMR
volume) to yield pure 3a (4.0 g, 80%).
4.2.3. 4-Amino-N-(4-N-methyl-piperazinyl)benzamide (4a)
(400 MHz, CDCl3)
d 6.81e6.77 (m, 2H), 6.68e6.64 (m, 2H),
A solution of 3a (2.88 g, 8.6 mmol) in 7:3 dichloromethane:tri-
fluoroacetic acid (40 mL) was stirred at room temperature for 1 h.
The solvent was evaporated in vacuo, and diethyl ether (30 mL) was
added. The precipitate was collected, washed with ether and dried
to provide white solid 4a (1.86 g, 93%). 1H NMR (400 MHz, DMSO-
3.85e3.83 (m, 4H), 3.03e3.01 (m, 4H).
4.3.3. 4-(4-methyl-piperazin-1-yl) aniline (6b)
From nitro chlorobenzene (4.7 g, 30 mmol), a similar procedure
as that described for 6a gave pure 6b (2.40 g, 42% for 2 steps) as a
white solid. 1H NMR (400 MHz, CDCl3)
d
6.81 (d, J ¼ 8.8 Hz, 2H),
d6)
d
7.74 (d, J ¼ 7.6 Hz, 1H), 7.57 (d, J ¼ 8.8 Hz, 2H), 6.52 (d,
J ¼ 8.4 Hz, 2H), 5.56 (s, 2H), 3.73e3.67 (m, 1H), 2.83 (d, J ¼ 11.6 Hz,
2H), 2.22 (s, 3H), 2.16e2.02 (m, 2H), 1.73 (d, J ¼ 10.0 Hz, 2H),
1.53e1.04 (m, 2H).
6.80 (d, J ¼ 8.2 Hz, 2H), 3.06 (t, J ¼ 4.8 Hz, 4H), 2.57 (t, J ¼ 4.8 Hz,
4H), 2.34 (s, 3H).
4.4. General synthetic procedure of compounds 8e10
4.2.4. 4-Amino-3-methoxyl-N-(4-N-methyl-piperazinyl)benzamide
(4b)
To a solution of 2,4-dichloro-5-fluoropyrimidine 7a (0.83 g,
5 mmol) in THF (10 mL) at 0 ꢀC was slowly added THF solution of
cyclopentylamine (0.74 mL, 7.5 mmol). The resulting mixture was
stirred at 0 ꢀC for 3 h (monitored by TLC). The solvent was removed
in vacuo and water (20 mL) was added and extracted with CH2Cl2
(3 ꢂ 10 mL). Combined organic phase was washed with brine, dried
over anhydrous Na2SO4, filtered and concentrated. The crude
product was purified by column chromatography on silica gel
(dichloromethane/methanol 50:1) to give 8a (0.83 g, 77%) as a solid.
Similar procedure as that described for 8a gave pure 8bed, 9
and 10 as a solid.
From compound 1b (1.67 g, 10 mmol), a similar procedure as
that described for 4a gave pure 4b (2.02 g, 77% for 3 steps) as a
white solid. 1H NMR (400 MHz, DMSO-d6)
d
7.79 (d, J ¼ 8.0 Hz, 1H),
7.29 (dd, J ¼ 5.6 Hz, 2H), 6.59 (dd, J ¼ 5.6 Hz, 1H), 5.20 (s, 2H), 3.80
(s, 3H), 3.73e3.65 (m, 1H), 3.75 (d, J ¼ 11.6 Hz, 2H), 2.15 (s, 1H),
1.91e1.88 (m, 2H), 1.72 (d, J ¼ 10.2 Hz, 2H), 1.57e1.51 (m, 2H).
4.3. Synthesis of anilines 6a,b
4.3.1. 4-morpholino nitrobenzene (5a)
To a mixture of nitro chlorobenzene (4.7 g, 30 mmol), mor-
pholine (4.25 mL, 48 mmol) in dioxane (50 mL) and water (25 mL)
were added potassium carbonate (3.6 g, 35.9 mmol). The reaction
mixture was stirred at 80 ꢀC for 5 h. White solid was removed by
filtration, the solvent was removed in vacuo to provide yellow solid.
Solid was purified by column chromatography (pure CH2Cl2) to
4.4.1. General synthetic procedure of compounds 11e19
4.4.1.1. 4-((4-(cyclopentylamino)-5-fluoro-2-yl)amino)-N-(1-
methylpiperidin-4-yl) benzamidepyrimidine (11a). A mixture of 8a
(130 mg, 0.6 mmol), 4a (115 mg, 0.5 mmol), 5 mL isopropyl alcohol,
and 1 drop of 37% HCl was refluxed at 80 ꢀC for 18 h. Solvents were

W.-W. Qin et al. / European Journal of Medicinal Chemistry 95 (2015) 174e184
181
evaporated in vacuo and the residue purified by column chroma-
tography on silica gel (dichloromethane/methanol 20:1) gave
desired product 11a 172 mg. Yield: 83%; White solid; m.p.:
190e192 ꢀC; IR (KBr, cmꢁ1) 3456, 3287, 2945,1635,1611,1543,1489,
J ¼ 3.6 Hz, 1H), 7.57 (s, 1H), 7.52e7.47 (m, 3H), 4.35e4.29 (m, 1H),
3.92 (s, 3H), 3.76e3.68 (m, 1H), 2.76 (d, J ¼ 11.2 Hz, 2H), 2.15 (s, 3H),
1.99e1.88 (m, 4H), 1.75e1.72 (m, 4H), 1.63e1.55 (m, 6H); 13C NMR
(100 MHz, DMSO-d6)
d
165.7, 155.4, 152.4 (d, J ¼ 12.0 Hz, 1C), 146.9,
1377, 1347, 852, 770; 1H NMR (400 MHz, DMSO-d6)
d
9.41 (s, 1H),
141.8 (d, J ¼ 245.0 Hz, 1C), 138.9 (d, J ¼ 19.0 Hz, 1C), 132.7, 127.1,
120.7, 116.1, 109.7, 56.5, 54.9, 52.3, 46.8, 46.1, 41.8, 32.5, 31.8, 24.1,
19.0, 11.6; HRMS (ESI) 443.2571 for [MþH]þ (calcd 443.2565 for
7.97 (d, J ¼ 7.6 Hz, 1H), 7.87 (d, J ¼ 4.0 Hz, 1H), 7.80 (d, J ¼ 8.8 Hz,
2H), 7.73 (d, J ¼ 9.2 Hz, 2H), 7.40 (d, J ¼ 6.8 Hz, 1H), 4.37e4.30 (m,
1H), 3.74e3.66 (m, 1H), 2.75 (d, J ¼ 11.6 Hz, 2H), 2.14 (s, 3H),
2.01e1.88 (m, 4H), 1.74e1.71 (m, 4H), 1.63e1.51 (m, 6H); 13C NMR
C23H32FN6O2).
(100 MHz, DMSO-d6)
d
165.8, 155.9, 152.2 (d, J ¼ 12.0 Hz, 1C), 144.4,
4.4.1.6. 4-((5-chloro-4-(cyclopentylamino)-2-yl)amino)-3-methoxy-
N-(1-methylpiperidin-4-yl)benzamidepyrimidine (12b). From com-
pound 8b (138 mg, 0.6 mmol), 4b (130 mg, 0.49 mmol), a similar
procedure as that described for 11a gave pure 12a 70 mg. Yield:
31%; White solid; m.p.: 197e199 ꢀC; IR (KBr, cmꢁ1) 3409, 2947,
1629, 1511, 1477, 1378, 1027, 767, 683, 638; 1H NMR (400 MHz,
141.5 (d, J ¼ 244.0 Hz,1C),138.9 (d, J ¼ 19.0 Hz,1C),128.3 (2C),126.6,
117.0 (2C), 55.1 (2C), 52.3, 46.8, 46.4, 32.5, 32.1, 24.1; HRMS (ESI)
413.2472 for [MþH]þ (calcd 413.2460 for C22H30FN6O).
4.4.1.2. 4-((5-chloro-4-(cyclopentylamino)-2-yl)amino)-N-(1-
methylpiperidin-4-yl) benzamidepyrimidine (11b). From compound
8b (105 mg, 0.5 mmol), 4a (105 mg, 0.5 mmol), a similar procedure
as that described for 11a gave pure 11b as a white solid 156 mg.
Yield: 72%; White solid; m.p.: 184e186 ꢀC; IR (KBr, cmꢁ1) 3409,
3287, 2935, 1628, 1576, 1526, 1508, 1380, 848, 771; 1H NMR
DMSO-d6)
d
8.41 (d, J ¼ 9.2 Hz, 1H), 8.08 (d, J ¼ 7.6 Hz, 1H), 7.97 (s,
1H), 7.70 (s, 1H), 7.49 (d, J ¼ 1.6 Hz, 1H), 7.47 (s, 1H), 6.97 (d,
J ¼ 7.2 Hz, 1H), 4.40e4.33 (m, 1H), 3.92 (s, 3H), 3.77e3.67 (m, 1H),
2.76 (d, J ¼ 11.6 Hz, 2H), 2.15 (s, 3H), 2.01e1.89 (m, 4H), 1.76e1.66
(m, 4H), 1.64e1.54 (m, 6H); 13C NMR (100 MHz, DMSO-d6)
d 165.5,
(400 MHz, DMSO-d6)
d
9.47 (s, 1H), 8.15 (d, J ¼ 8.0 Hz, 1H), 7.98 (s,
157.7, 157.6, 153.4, 147.3, 132.2, 127.7, 120.4, 117.0, 109.8, 105.1, 56.5,
55.1, 52.8, 47.0, 46.4, 32.3, 32.0, 24.1; HRMS (ESI) 459.2281 for
[MþH]þ (calcd 459.2270 for C23H32ClN6O2).
1H), 7.80 (d, J ¼ 8.8 Hz, 2H), 7.74 (d, J ¼ 8.8 Hz, 2H), 6.88 (d,
J ¼ 7.2 Hz, 1H), 4.42e4.35 (m, 1H), 3.75e3.66 (m, 1H), 2.75 (d,
J ¼ 12.0 Hz, 2H), 2.14 (s, 3H), 2.02e1.88 (m, 4H), 1.74e1.71 (m, 4H),
1.66e1.59 (m, 6H); 13C NMR (100 MHz, DMSO-d6)
d
165.7, 158.1,
4.4.1.7. 4-((5-bromo-4-(cyclopentylamino)-2-yl)amino)-3-methoxy-
N-(1-methylpiperidin-4-yl)benzamidepyrimidine (12c). From com-
pound 8c (145 mg, 0.53 mmol), 4b (130 mg, 0.49 mmol), a similar
procedure as that described for 11a gave pure 12c 75 mg. Yield:
31%; White solid; m.p.: 204e206 ꢀC; IR (KBr, cmꢁ1) 3395, 2947,
1632, 1612, 1597, 1572, 1505, 1378, 777, 767, 671; 1H NMR (400 MHz,
157.6, 153.4,144.0, 128.3 (2C), 127.0, 117.6 (2C), 104.6, 55.1 (2C), 52.8,
46.8, 46.4, 32.4, 32.0, 24.1; HRMS (ESI) 429.2174 for [MþH]þ (calcd
429.2164 for C22H30ClN6O).
4.4.1.3. 4-((5-bromo-4-(cyclopentylamino)-2-yl)amino)-N-(1-
methylpiperidin-4-yl) benzamidepyrimidine (11c). From compound
8c (154 mg, 0.56 mmol), 4a (130 mg, 0.56 mmol), a similar proce-
dure as that described for 11a gave pure 11c 166 mg. Yield: 63%;
White solid; m.p.: 194e196 ꢀC; IR (KBr, cmꢁ1) 3407, 3218, 2950,
1630, 1604, 1554, 1510, 1494, 1336, 853, 762; 1H NMR (400 MHz,
DMSO-d6)
d
8.39 (d, J ¼ 8.8 Hz, 1H), 8.09 (d, J ¼ 7.6 Hz, 1H), 8.05 (s,
1H), 7.72 (s, 1H), 7.49 (d, J ¼ 1.6 Hz, 1H), 7.47 (s, 1H), 6.66 (d,
J ¼ 7.2 Hz, 1H), 4.39e4.32 (m, 1H), 3.92 (s, 3H), 3.77e3.68 (m, 1H),
2.76 (d, J ¼ 11.6 Hz, 2H), 2.15 (s, 3H), 2.01e1.89 (m, 4H), 1.76e1.65
(m, 4H), 1.63e1.53 (m, 6H); 13C NMR (100 MHz, DMSO-d6)
d 165.6,
DMSO-d6)
d
9.46 (s, 1H), 8.05 (s, 1H), 8.00 (d, J ¼ 7.6 Hz, 1H), 7.80 (d,
158.4, 158.2, 156.3, 147.5, 132.2, 127.8, 120.5, 117.2, 109.8, 94.3, 56.5,
55.1, 53.1, 47.1, 46.5, 32.4, 32.1, 24.1, 19.0; HRMS (ESI) 503.1776 for
[MþH]þ (calcd 503.1765 for C23H32BrN6O2).
J ¼ 8.8 Hz, 2H), 7.75 (d, J ¼ 8.8 Hz, 2H), 6.60 (d, J ¼ 3.2 Hz, 1H),
4.42e4.35 (m, 1H), 3.75e3.66 (m, 1H), 2.75 (d, J ¼ 11.6 Hz, 2H), 2.14
(s, 3H), 2.02e1.88 (m, 4H), 1.74e1.71 (m, 4H), 1.66e1.59 (m, 6H); 13
C
NMR (100 MHz, DMSO-d6)
d
165.9, 158.4, 157.6, 153.4, 144.0, 128.2
4.4.1.8. N4-cyclopentyl-5-fluoro-N2-(4-morpholinophenyl)-2,4-
diaminepyrimidine (13). From compound 8a (89 mg, 0.5 mmol), 6a
(143 mg, 0.75 mmol), a similar procedure as that described for 11a
gave pure 13 120 mg. Yield: 75%; White solid; m.p.: 164e166 ꢀC; IR
(KBr, cmꢁ1), 3316, 2968, 1627, 1586, 1542, 1542, 1516, 1375, 1226,
(2C), 127.0, 117.6 (2C), 104.6, 55.2, 52.9, 46.8, 46.4, 32.4, 32.0, 24.1;
HRMS (ESI) 473.1673 for [MþH]þ (calcd 473.1659 for C22H30BrN6O).
4.4.1.4. 4-((4-(cyclopentylamino)-5-nitro-2-yl)amino)-N-(1-
methylpiperidin-4-yl) benzamidepyrimidine (11d). From compound
8d (120 mg, 0.5 mmol), 4a (95 mg, 0.5 mmol), a similar procedure
as that described for 11a gave pure 11d 90 mg. Yield: 60%; yellow
solid; m.p.: 198e200 ꢀC; IR (KBr, cmꢁ1) 3423, 2968, 1616, 1585,
1543, 1503, 1418, 1386, 1227, 787, 768; 1H NMR (600 MHz, DMSO-
774, 740, 655; 1H NMR (400 MHz, DMSO-d6)
d 8.71 (s, 1H), 7.76 (d,
J ¼ 3.6 Hz, 1H), 7.56 (d, J ¼ 9.2 Hz, 2H), 7.21 (d, J ¼ 7.2 Hz, 1H), 6.82
(d, J ¼ 9.2 Hz, 2H), 4.32e4.29 (m, 1H), 3.71 (t, J ¼ 4.8 Hz, 4H), 2.98 (t,
J ¼ 4.8 Hz, 4H), 1.98e1.91 (m, 2H), 1.70e1.67 (m, 2H), 1.57e1.53 (m,
4H); 13C NMR (100 MHz, DMSO-d6)
d
156.2,151.9 (d, J ¼ 12.0 Hz,1C),
d6)
d
10.39 (s, 1H), 9.05 (s, 1H), 8.31 (d, J ¼ 6.6 Hz, 1H), 7.92e7.80 (m,
145.6,140.8 (d, J ¼ 242.0 Hz,1C),138.5 (d, J ¼ 18.0 Hz,1C),119.5 (2C),
115.8 (2C), 66.3, 56.2, 55.0, 51.8, 49.7, 49.2, 32.2, 23.7, 18.6; HRMS
(ESI) 358.2047 for [MþH]þ (calcd 358.2038 for C19H25FN5O).
2H), 7.76 (s, 1H), 7.73 (d, J ¼ 8.4 Hz, 1H), 3.71e3.67 (m, 1H),
3.60e3.56 (m, 1H), 2.98 (d, J ¼ 6.6 Hz, 4H), 2.37 (s, 3H), 1.90e1.82
(m, 2H),1.76e1.68 (m, 2H),1.30e1.28 (m, 2H),1.18e1.13 (m, 6H); 13
C
NMR (150 MHz, DMSO-d6)
d
165.0, 159.5, 158.0, 153.1, 139.9, 127.9,
4.4.1.9. N4-cyclopentyl-5-fluoro-N2-(4-(4-methylpiperazin-1-yl)
phenyl)- 2,4-diaminepyrimidine (14). From compound 8a (150 mg,
0.7 mmol), 6b (100 mg, 0.55 mmol), a similar procedure as that
described for 11a gave pure 14 62 mg. Yield: 31%; White solid; m.p.:
168e170 ꢀC; IR (KBr, cmꢁ1) 3436, 3265, 2925,1675, 1616, 1517, 1475,
127.8, 123.1, 121.6 (2C), 119.8, 53.5 (2C), 45.5, 42.7, 42.3, 30.3, 30.2,
21.5; HRMS (ESI) 440.2417 for [MþH]þ (calcd 440.2405 for
C
22H30N7O3).
4.4.1.5. 4-((4-(cyclopentylamino)-5-fluoro-2-yl)amino)-3-methoxy-
N-(1-methylpiperidin- 4-yl)benzamidepyrimidine (12a). From com-
pound 8a (85 mg, 0.4 mmol), 4b (86 mg, 0.33 mmol), a similar
procedure as that described for 11a gave pure 12a 61 mg. Yield:
42%; White solid; m.p.: 194e196 ꢀC; IR (KBr, cmꢁ1) 3391, 2947,
1632, 1611, 1596, 1566, 1499, 1477, 776, 661; 1H NMR (400 MHz,
1442, 808, 566; 1H NMR (400 MHz, DMSO-d6)
d 8.69 (s, 1H), 7.75 (d,
J ¼ 3.6 Hz, 1H), 7.56 (d, J ¼ 8.8 Hz, 2H), 7.20 (d, J ¼ 7.2 Hz, 1H), 6.81
(d, J ¼ 8.8 Hz, 2H), 4.32e4.25 (m, 1H), 3.06 (s, 4H), 2.63 (s, 4H), 2.33
(s, 3H), 1.97e1.91 (m, 2H), 1.69e1.65 (m, 2H), 1.58e1.50 (m, 4H); 13
C
NMR (150 MHz, DMSO-d6)
d
158.7, 156.3, 152.1 (d, J ¼ 12.0 Hz, 1C),
145.3, 140.9 (d, J ¼ 242.6 Hz, 1C), 138.5, 134.4, 119.7, 116.4 (2C), 56.4,
DMSO-d6)
d
8.46 (d, J ¼ 8.8 Hz, 1H), 8.10 (d, J ¼ 7.6 Hz, 1H), 7.89 (d,
54.4, 52.0, 48.9, 48.7, 45.0, 32.3, 23.8, 18.7; HRMS (ESI) 371.2360 for

182
W.-W. Qin et al. / European Journal of Medicinal Chemistry 95 (2015) 174e184
[MþH]þ (calcd 371.2354 for C20H28FN6).
DMSO-d6)
d
156.1, 152.2 (d, J ¼ 12.0 Hz, 1C), 145.5, 140.7 (d,
J ¼ 241.0 Hz, 1C), 138.4 (d, J ¼ 17.0 Hz, 1C), 134.1, 119.4 (2C), 115.7
(2C), 66.3 (2C), 49.6 (2C), 41.7, 22.2, 11.5; HRMS (ESI) 332.1889 for
[MþH]þ (calcd 332.1881 for C17H23FN5O).
4.4.1.10. N4-cyclopentyl-N2-(4-morpholinophenyl)-5-nitro-2,4-
diaminepyrimidine (15). From compound 8d (89 mg, 0.5 mmol), 6a
(161 mg, 0.75 mmol), a similar procedure as that described for 11a
gave pure 15 120 mg. Yield: 62%; White solid; m.p.: 169e171 ꢀC; IR
(KBr, cmꢁ1) 3438, 3370, 2958, 1730, 1628, 1587, 1548, 1513, 1270,
4.4.1.15. N4-cyclopentyl-N2-(4-(4-methylpiperazin-1-yl)phenyl)-
2,4,5-triaminepyrimidine (20). Compound 16 (225 mg, 0.56 mmol)
was dissolved in dry ethyl acetate (10 mL), added 0.1 g 10% of Pd/C.
The reaction was stirred for 14 h under hydrogen gas at 60 ꢀC, then
Pd/C was filtered, the filtrate was evaporated and the solid was
786, 642; 1H NMR (600 MHz, DMSO-d6)
d 10.28 (s, 1H), 8.92 (s, 1H),
8.50 (d, J ¼ 6.6 Hz, 1H), 7.66 (d, J ¼ 9.6 Hz, 2H), 6.91 (d, J ¼ 9.6 Hz,
2H), 4.44e4.42 (m, 1H), 3.72 (t, J ¼ 4.8 Hz, 4H), 3.05 (t, J ¼ 4.2 Hz,
4H), 2.04e2.01 (m, 2H), 1.71e1.61 (m, 2H), 1.35e1.21 (m, 4H); 13C
purified
by column
chromatography
(dichloromethane:
NMR (150 MHz, DMSO-d6)
d
166.9, 158.9, 157.2, 155.1, 147.4, 131.6,
methanol ¼ 15:1) to give 20 160 mg. Yield: 80%; pink solid; m.p.:
121.6, 115.1, 114.7, 66.8 (2C), 52.5, 48.7 (2C), 32.3 (2C), 23.2 (2C);
159e161 ꢀC; IR (KBr, cmꢁ1) 3354, 3198, 2965,1731, 1659,1582,1559,
HRMS (ESI) 385.1993 for [MþH]þ (calcd 385.1983 for C19H25N6O3).
1513, 1470, 1357, 826, 764, 652; 1H NMR (600 MHz, DMSO-d6)
d 8.15
(s, 1H), 7.56 (d, J ¼ 9.0 Hz, 2H), 7.31 (s, 1H), 6.77 (d, J ¼ 9.0 Hz, 2H),
6.08 (d, J ¼ 6.6 Hz,1H), 2.98 (t, J ¼ 4.8 Hz, 4H), 2.43 (t, J ¼ 4.2 Hz, 6H),
2.19 (s, 3H), 2.00e1.97 (m, 1H), 1.88 (brs, 2H), 1.72e1.70 (m, 2H),
1.58e1.55 (m, 2H), 1.51e1.46 (m, 2H); 13C NMR (150 MHz, DMSO-
4.4.1.11. N4-cyclopentyl-N2-(4-(4-methylpiperazin-1-yl)phenyl)-5-
nitro- 2,4-diaminepyrimidine (16). From compound 8d (145 mg,
0.6 mmol), 6b (92 mg, 0.47 mmol), a similar procedure as that
described for 11a gave pure 16 166 mg. Yield: 89%; White solid;
m.p.: 173e175 ꢀC; IR (KBr, cmꢁ1) 3469, 3346, 2954,1615,1589,1536,
d6)
d 153.8, 144.9, 138.3, 135.6, 129.1, 120.1, 118.6 (2C), 116.6 (2C),
55.3 (2C), 52.3, 49.8 (2C), 46.2, 32.9 (2C), 22.8 (2C); HRMS (ESI)
1512, 1498, 1361, 826, 783; 1H NMR (600 MHz, DMSO-d6)
d
10.27 (s,
368.2570 for [MþH]þ (calcd 368.2557 for C20H30N7).
1H), 8.93 (s, 1H), 8.49 (d, J ¼ 6.6 Hz, 1H), 7.66 (d, J ¼ 9.0 Hz, 2H), 6.91
(d, J ¼ 9.0 Hz, 2H), 4.44e4.42 (m, 1H), 3.09 (s, 4H), 2.47 (s, 4H), 2.23
4.5. Biology
(s, 3H), 2.10e2.02 (m, 2H), 1.75e1.69 (m, 2H), 1.65e1.54 (m, 4H); 13
C
NMR (150 MHz, DMSO-d6)
d
158.9, 157.1, 155.1, 147.2, 130.6, 121.1
4.5.1. Cytotoxicity assays
(2C), 119.8, 115.3 (2C), 54.5 (2C), 52.5, 48.2, 45.6, 32.3 (2C), 23.5;
Cells were incubated at 37 ꢀC in a 5% CO2 atmosphere. The MTT
assay were used to determined growth inhibition. The synthetic
compounds and reference compound VX-680 were dissolved in
saline for five concentrations (0.001e100 mM). The A-549, HepG2,
HCT-8 and HeLa cells were plated in 96-well plates and allowed to
attach for 4e6 h, then exposed in quadplex well for 48 h. The media
HRMS (ESI) 398.2308 for [MþH]þ (calcd 398.2299 for C20H28N7O2).
4.4.1.12. N4-cyclopropyl-5-fluoro-N2-(4-morpholinophenyl)-2,4-
diaminepyrimidine (17). From compound 9 (140 mg, 0.75 mmol),
6a (89 mg, 0.5 mmol), a similar procedure as that described for 11a
gave pure 17 95 mg. Yield: 58%; White solid; m.p.: 163e165 ꢀC; IR
(KBr, cmꢁ1) 3274, 2953, 1627, 1588, 1541, 1516, 1377, 1229, 773, 636;
was aspirated, and 10 mL of 5 mg/mL MTT solution (dilute in sterile
PBS) diluted in serum-free media was added to each well. After 4 h
of incubation, the solution was centrifuged for 10 min under
1H NMR (400 MHz, DMSO-d6)
d
7.77 (d, J ¼ 3.6 Hz, 1H), 7.57 (d,
J ¼ 8.8 Hz, 2H), 6.92 (s, 1H), 6.90 (d, J ¼ 9.2 Hz, 2H), 5.16 (brs, 1H),
3.87 (t, J ¼ 4.8 Hz, 4H), 3.11 (t, J ¼ 4.8 Hz, 4H), 2.86e2.80 (m, 1H),
0.91e0.86 (m, 2H), 0.66e0.63 (m, 2H); 13C NMR (150 MHz, CDCl3)
2000 rpm. The supernatant was mixed with 150 mL DMSO, and
shaken on an oscillator. The absorbance at l490 was determined on
a plate reader. IC50 values were determined from a log plot of
percent of control versus concentration [36].
d
156.1, 153.6 (d, J ¼ 10.9 Hz, 1C), 146.5, 141.9 (d, J ¼ 242.5 Hz, 1C),
138.8 (d, J ¼ 18.9 Hz,1C),133.5,120.2 (2C),116.6 (2C), 67.0, 66.9, 51.1,
50.3 (2C), 23.4, 7.2; HRMS (ESI) 330.1729 for [MþH]þ (calcd
330.1725 for C17H21FN5O).
4.5.2. Western blot analysis
6
HeLa cells (1 ꢂ 10 cells) exposed to compound 11c were
collected into tubes and then washed with PBS. Cell pellets were
lysed with lyses buffer (50 mM TriseHCl, pH 7.5, 10 mM EDTA, 0.2 M
NaCl, 1.5 mM PMSF and 1% SDS). Cell lysates were boiled for 10 min,
centrifuged and stored at ꢁ20 ꢀC. Cell lysates containing
4.4.1.13. 5-fluoro-N2-(4-(4-methylpiperazin-1-yl)phenyl)-N4-
propyl- 2,4-diaminepyrimidine (18). From compound 10 (113 mg,
0.6 mmol), 6b (95 mg, 0.5 mmol), a similar procedure as that
described for 11a gave pure 18 92 mg. Yield: 70%; White solid; m.p.:
166e168 ꢀC; IR (KBr, cmꢁ1) 3385, 2965,1683,1618,1519,1440, 1379,
10e20
mm g protein were separated and transferred to nitro-
cellulosefilters. The blots were incubated with the corresponding
antibodies and developed [36].
800, 722; 1H NMR (600 MHz, DMSO-d6)
d
8.73 (s, 1H), 7.77 (d,
J ¼ 4.2 Hz, 1H), 7.55 (d, J ¼ 9.0 Hz, 2H), 7.36 (s, J ¼ 4.8 Hz, 1H), 6.83
(d, J ¼ 9.6 Hz, 2H), 3.330 (dd, J ¼ 13.8, 6.0 Hz, 2H), 3.15e3.10 (m, 4H),
2.78e2.72 (m, 4H), 2.42 (s, 3H),1.60e1.55 (m, 2H), 0.88 (t, J ¼ 7.2 Hz,
4.5.3. Analysis of cell cycle by flow cytometry
For cell cycle analysis, we used the cervical carcinoma HeLa cell
line grown in RPMI-1640 supplemented with 10% (v/v) heati-
nactivated fetal calf serum, 2 mM L-glutamine, 100 units/mL
3H); 13C NMR (150 MHz, DMSO-d6)
d156.1, 152.1 (d, J ¼ 11.4 Hz, 1C),
144.8, 140.6 (d, J ¼ 242.5 Hz, 1C), 138.3 (d, J ¼ 17.3 Hz, 1C), 134.2,
119.3 (2C),116.1 (2C), 53.8 (2C), 48.1 (2C), 44.4, 41.7, 22.1,11.4; HRMS
(ESI) 345.2206 for [MþH]þ (calcd 345.2197 for C18H26FN6).
penicillin, and 24 m
g/mL gentamicin and incubated at 37 ꢀC in a
humidified atmosphere of 5% CO2 and 95% air. Untreated and
drugtreated cells ((3e5) ꢂ 105) were harvested and fixed overnight
in 70% ethanol at 4 ꢀC. Cells were then washed three times with
PBS, incubated for 1 h with 1 mg/mL RNase A and 20 mg/mL pro-
pidium iodide at room temperature, and analyzed with a flow cy-
4.4.1.14. 5-fluoro-N2-(4-morpholinophenyl)-N4-propyl-2,4-
diaminepyrimidine (19). From compound 10 (113 mg, 0.6 mmol), 6a
(89 mg, 0.5 mmol), a similar procedure as that described for 11a
gave pure 19 166 mg. Yield: 96%; White solid; m.p.: 162e163 ꢀC; IR
(KBr, cmꢁ1) 3341, 2960, 1630, 1589, 1544, 1517, 1372, 1226, 776, 659;
tometer (COULTER EPICS XL, USA) as described previously [35].
1H NMR (400 MHz, DMSO-d6)
d
8.73 (s, 1H), 7.77 (d, J ¼ 3.6 Hz, 1H),
4.5.4. ELISA experiment
7.56 (d, J ¼ 9.2 Hz, 2H), 7.37 (t, J ¼ 5.2 Hz, 1H), 6.82 (d, J ¼ 8.8 Hz,
2H), 3.71 (t, J ¼ 4.8 Hz, 4H), 3.33e3.27 (m, 2H), 2.98 (t, J ¼ 4.8 Hz,
4H), 1.62e1.53 (m, 2H), 0.89 (t, J ¼ 7.2 Hz, 3H); 13C NMR (100 MHz,
Use Purified Human Aurora A or B antibody to coat microtiter
plate wells, make solid-phase antibody, then add Aurora A or B to
wells, Combined Aurora A which With HRP labeled, become

W.-W. Qin et al. / European Journal of Medicinal Chemistry 95 (2015) 174e184
183
maintaining the spindle assembly checkpoint, J. Cell Biol. 161 (2003)
281e294.
antibody-antigen-enzyme-antibody complex, after washing
Completely, Add TMB substrate solution, TMB substrate becomes
blue color At HRP enzyme-catalyzed, reaction is terminated by the
addition of a sulphuric acid solution and the color change is
measured spectrophotometrically at a wavelength of 450 nm [32].
[15] E.A. Harrington, D. Bebbington, J. Moore, R.K. Rasmussen, A.O. Ajose-Adeogun,
T. Nakayama, J.A. Graham, C. Demur, T. Hercend, A. Diu-Hercend, M. Su,
J.M. Golec, K.M. Miller, VX-680, a potent and selective small-molecule in-
hibitor of the aurora kinases, suppresses tumor growth in vivo, Nat. Med. 10
(2004) 262e267.
[16] M.G. Manfredi, J.A. Ecsedy, K.A. Meetze, S.K. Balani, O. Burenkova, W. Chen,
K.M. Galvin, K.M. Hoar, J.J. Huck, P.J. LeRoy, E.T. Ray, T.B. Sells, B. Stringer,
S.G. Stroud, T.J. Vos, G.S. Weatherhead, D.R. Wysong, M. Zhang, J.B. Bolen,
C.F. Claiborne, Antitumor activity of MLN8054, an orally active small-molecule
4.5.5. Molecular docking study
The docking simulation was performed using the Aurodock 4.2
software. Before docking, the protein structure was minimized
firstly. The crystal waters were removed and the Kollman united
atom charges and polar hydrogen was added to the two proteins.
The ligand in the crystal structure was used to determine the
location of a docking grid box and was then removed prior to grid
generation in next step. Gasteiger charges were assigned to the new
constructed structures in Autodock. At the same time, the Non-
polar hydrogen atoms were merged and the rotatable bonds were
defined. Based on the ligand in the crystal structure, the grid maps
of the protein were produced using AutoGrid module embedded in
Autodock software. As result, a grid size of 60 ꢂ 60 ꢂ 66 Å points
and 0.375 Å spacing were generated. Each docking process was
performed in 250,000 energy evaluation with 10 conformations
kept and the most favorable pose of each compound was displayed.
inhibitor of aurora
A kinase, Proc. Natl. Acad. Sci. U. S. A. 104 (2007)
4106e4111.
[17] J.M. Maris, C.L. Morton, R. Gorlick, E.A. Kolb, R. Lock, H. Carol, S.T. Keir,
C.P. Reynolds, M.H. Kang, J. Wu, M.A. Smith, P.J. Houghton, Initial testing of the
aurora kinase a inhibitor MLN8237 by the pediatric preclinical testing pro-
gram (PPTP), Pediatr. Blood Cancer 55 (2010) 26e34.
[18] A.A. Mortlock, K.M. Foote, N.M. Heron, F.H. Jung, G. Pasque, J.J. Lohmann,
N. Warin, F. Renaud, C. De Savi, N.J. Roberts, T. Johnso, C.B. Dousson, G.B. Hill,
D. Perkins, G. Hatter, R.W. Wilkinson, S.R. Wedge, S.P. Heaton, R. Odedra,
N.J. Keen, C. Crafter, E. Brown, K. Thompson, S. Brightwell, L. Khatri,
M.C. Brady, S. Kearney, D. McKillop, S. Rhead, T. Parry, S. Green, Discovery,
synthesis, and in vivo activity of a new class of pyrazoloquinazolines as se-
lective inhibitors of aurora B kinase, J. Med. Chem. 50 (2007) 2213e2224.
[19] M. Grundy, C. Seedhouse, S. Shang, J. Richardson, N. Russell, M. Pallis, The FLT3
internal tandem duplication mutation is a secondary target of the aurora B
kinase inhibitor AZD1152-HQPA in acute myelogenous leukemia cells, Mol.
Cancer Ther. 9 (2010) 661e672.
[20] A.A. Mortlock, F.T. Boyle, S. Green, AZD1152, a selective inhibitor of Aurora B
kinase, inhibits human tumor xenograft growth by inducing apoptosis, Clin.
Cancer Res. 13 (2007) 3682e3688.
[21] Y. Tao, P. Zhang, F. Girdler, V. Frascogna, M. Castedo, J. Bourhis, G. Kroemer,
E. Deutsch, Enhancement of radiation response in p53-deficient cancer cells
by the Aurora-B kinase inhibitor AZD1152, Oncogene 27 (2008) 3244e3255.
[22] J. Li, H.-R. Hu, Q.-Y. Lang, H.-X. Zhang, Q. Huang, Y.-Y. Wu, L. Yu,
A thienopyrimidine derivative induces growth inhibition and apoptosis in
Acknowledgments
This work was Financially supported by NSFC (21372110,
81372177), and the Fundamental Research Funds for the Central
Universities, Lanzhou University (lzujbky-2014-k20).
human cancer cell lines via inhibiting Aurora
B kinase activity original
research article, Eur. J. Med. Chem. 65 (2014) 151e157.
[23] A.-D. Jagtap, P.-T. Chang, J.-R. Liu, H.-C. Wang, N.-B. Kondekar, L.-J. Shen, H.-
W. Tseng, G.-S. Chen, J.-W. Chern, Novel acylureidoindolin-2-one derivatives
as dual Aurora B/FLT3 inhibitors for the treatment of acute myeloid leukemia,
Eur. J. Med. Chem. 85 (2014) 268e288.
[24] H.-C. Wang, A.-D. Jagtap, P.-T. Chang, J.-R. Liu, C.-P. Liu, H.-W. Tseng, G.-
S. Chen, J.-W. Chern, Bioisosteric replacement of an acylureido moiety
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.03.044.
attached to an indolin-2-one scaffold with
pyridinoylamido moiety produces a selectively potent Aurora-B inhibitor,
Eur. J. Med. Chem. 84 (2014) 312e334.
a malonamido or a 2/4-
References
[25] T. Sandra, N.G. Jorg, J. Petra, B. Andreas, G. Christian, R.S. Jeffrey, G. Oliver,
E.B. Michael, T. Klaus, R. Daniel, H.S. Peter, Identification of Ustilago maydis
Aurora kinase as a novel antifungal target, ACS Chem. Biol. 6 (2011) 926e933.
[26] F. Mu, S.L. Coffing, D.J. Riese II, R.L. Geahlen, P. Verdier-Pinard, E. Hamel,
J. Johnson, M. Cushman, Design synthesis and biological evaluation of a series
of lavendustin A analogues that inhibit EGFR and Syk tyrosine kinases, as well
as tubulin polymerization, J. Med. Chem. 44 (2001) 441e452.
[27] S. Perez Silanes, L. Orus, A.M. Oficialdegui, J. Martınez Esparza, B. Lasheras,
J. del Rıo2, A. Monge, New 3-[4-(3-substituted phenyl) piperazin-1-yl]-l-
(benzo[b]thiophen-3-yl)-propanol derivatives with dual action at 5-HT1A
serotonin receptors and serotonin transporter as a new class of antidepres-
sants, Pharmazie 57 (2002) 515e518.
[28] H.R. Lawrence, M.P. Martin, Y. Luo, R. Pireddu, H. Yang, H. Gevariya, S. Ozcan,
J.Y. Zhu, R. Kendig, M. Rodriguez, R. Elias, J.Q. Cheng, S.M. Sebti, E. Schonbrunn,
N.J. Lawrence, Development of o-chlorophenyl substituted pyrimidines as
exceptionally potent aurora kinase inhibitors, J. Med. Chem. 55 (2012)
7392e7416.
[1] J. Fu, M. Bian, Q. Jiang, C. Zhang, Roles of aurora kinases in mitosis and
tumorigenesis, Mol. Cancer Res. 5 (2007) 1e10.
[2] M. Carmena, W.C. Earnshaw, The cellular geography of Aurora kinases, Nat.
Rev. Mol. Cell. Biol. 4 (2003) 842e854.
[3] G. Vader, S. M.A Lens, The Aurora kinase family in cell division and cancer,
Biochim. Biophys. Acta 1786 (2008) 60e72.
[4] T. Marumo, D. Zhang, H. Saya, Aurora-A: a guardian of poles, Nat. Rev. Cancer
5 (2005) 42e50.
[5] T. Saeki, M. Ouchi, T. Ouchi, Physiological and oncogenic Aurora-A pathway,
Int. J. Biol. Sci. 5 (2009) 758e762.
[6] G. Vader, R.H. Medema, S.M. Lens, The chromosomal passenger complex:
guiding Aurora-B through mitosis, J. Cell Biol. 173 (2006) 833e837.
[7] K. Sasai, H. Katayama, D.L. Stenoien, S. Fujii, R. Honda, M. Kimura, Y. Okano,
M. Tatsuka, F. Suzuki, E.A. Nigg, W.C. Earnshaw, W.R. Brinkley, S. Sen, Aurora-C
kinase is
a novel chromosomal passenger protein that can complement
Aurora-B kinase function in mitotic cells, Cell Motil. Cytoskelet. 59 (2004)
249e263.
[29] Y. Luo, Y.-Q. Deng, J. Wang, Z.-J. Long, Z.-C. Tu, W. Peng, J.-Q. Zhang, Q. Liu,
G. Lu, Design, synthesis and bioevaluation of N-trisubstituted pyrimidine
derivatives as potent aurora A kinase inhibitors, Eur. J. Med. Chem. 78 (2014)
65e71.
[30] J.-F. Liu, C.-Y. Sang, X.-H. Xu, L.-L. Zhang, L. Hui, J.-B. Zhang, S.-W. Chen,
Synthesis and cytotoxic activity on human cancer cells of carbamate de-
[8] C.N. Landen Jr., Y.G. Lin, A. Immaneni, M.T. Deavers, W.M. Merritt,
W.A. Spannuth, D.C. Bodurka, D.M. Gershenson, W.R. Brinkley, A.K. Sood,
Overexpression of the centrosomal protein Aurora-A kinase is associated with
poor prognosis in epithelial ovarian cancer patients, Clin. Cancer Res. 13
(2007) 4098e4104.
[9] O. Gautschi, J. Heighway, P.C. Mack, P.R. Purnell Jr., P.N. lara, D.R. Gandara,
Aurora kinases as anticancer drug targets, Clin. Cancer Res. 14 (2008)
1639e1648.
rivatives of
4b-(1,2,3-triazol-1-yl)podophyllotoxin, Eur. J. Med. Chem. 64
(2013) 621e628.
[31] H. Yang, C.C. On, R.I. Feldman, S.V. Nicosia, P.A. Kruk, J.Q. Cheng, Aurora-A
kinase regulates telomerase activity through c-Myc in human ovarian and
breast epithelial cells, Cancer Res. 64 (2004) 463e467.
[10] N. Keen, S. Taylor, Aurora-kinase inhibitors as anticancer agents, Nat. Rev.
Cancer 4 (2004) 927e936.
[11] H. Katayama, S. Sen, Aurora kinase inhibitors as anticancer molecules, Bio-
chim. Biophys. Acta 1799 (2010) 829e839.
[12] J.R. Pollard, M. Mortimore, Discovery and development of Aurora kinase in-
hibitors as anticancer agents, J. Med. Chem. 52 (2009) 2629e2651.
[13] C. Ditchfield, V.L. Johnson, A. Tighe, R. Ellston, C. Haworth, T. Johnson,
A. Mortlock, N. Keen, S.S. Taylor, Aurora B couples chromosome alignment
with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores, J. Cell
Biol. 161 (2003) 267e280.
[14] S. Hauf, R.W. Cole, S. LaTerra, C. Zimmer, G. Schnapp, R. Walter, A. Heckel,
J. van Meel, C.L. Rieder, J.M. Peters, The small molecule hesperadin reveals a
role for Aurora B in correcting kinetochore-microtubule attachment and in
[32] S. Leng, J. McElhaney, J. Walston, D. Xie, N. Fedarko, G. Kuchel, Elisa and
multiplex technologies for cytokine measurement in inflammation and aging
research, J. Gerontol. A Biol. Sci. Med. Sci. 63 (2008) 879e884.
[33] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell,
A.J. Olson, AutoDock4 and AutoDockTools4: automated docking with selective
receptor flexibility, J. Comput. Chem. 30 (2009) 2785e2791.
[34] G.M. Morris, D.S. Goodsell, R.S. Halliday, R. Huey, W.E. Hart, R.K. Belew,
A.J. Olson, Automated docking using a lamarckian genetic algorithm and an
empirical binding free energy function, J. Comput. Chem. 19 (1998)
1639e1662.

184
W.-W. Qin et al. / European Journal of Medicinal Chemistry 95 (2015) 174e184
[35] S.C. DeLano Scientific, The PyMOL molecular graphics system, 2002.
[36] C. Cheng, Z.G. Liu, H. Zhang, J.D. Xie, X.G. Chen, X.Q. Zhao, F. Wang, Y.J. Liang,
L.K. Chen, S. Singh, J.J. Chen, T.T. Talele, Z.S. Chen, F.T. Zhong, L.W. Fu,
Enhancing chemosensitivity in ABCB1- and ABCG2-overexpressing cells and
cancer stem-like cells by an Aurora kinase inhibitor CCT129202, Mol. Pharm. 9
(2012) 1971e1982.
inducer of caspase-3 and -7, Eur. J. Med. Chem. 49 (2012) 48e54.
[38] N. Asli, O. Ozlem, B.T. Neslihan, D. Beyhan, Cyclin A and cyclin B1 over-
expression in differentiated thyroid carcinoma, Med. Oncol. 29 (2012)
294e300.
[39] A.M. Egloff, J. Weissfeld, S.R. Land, O.J. Finn, Evaluation of anticyclin B1 serum
antibody as a diagnostic and prognostic biomarker for lung cancer, Ann. N. Y.
Acad. Sci. 1062 (2005) 29e40.
[37] W.-T. Huang, J. Liu, J.-F. Liu, L. Hui, Y.-L. Ding, S.-W. Chen, Synthesis and
biological evaluation of conjugates of deoxypodophyllotoxin and 5-FU as