Design, synthesis and bioevaluation of N-trisubstituted pyrimidine derivatives as potent aurora A kinase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2014.03.027

The design and synthesis of a new series of N-trisubstituted (at C2, C4 and C6 respectively) pyrimidine derivatives were reported, their in vitro structure-activity relationships vs. aurora A kinase were also discussed. Our results demonstrated that the introduction of characteristic N-substituted side chain at C2 of pyrimidines possessed a potent aurora A inhibitory activity, the position and the nature of the substituents on the phenyl ring of aniline side chain played key roles in cellular kinase inhibitory potency. Most tested compounds exhibited good inhibitory activities against aurora A kinase and various human tumor cell lines. Compounds 7j, 7m-n and 7p showed strong growth-inhibitory activities in the solid CNE-2 tumor cell and selectively blocked cell-cycle progression at the G₂/M phase.

Full text of DOI:10.1016/j.ejmech.2014.03.027

European Journal of Medicinal Chemistry 78 (2014) 65e71
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and bioevaluation of N-trisubstituted pyrimidine
derivatives as potent aurora A kinase inhibitors
,
,
Yu Luo a 1, Yan-Qiu Deng a 1, Jing Wang b, Zi-Jie Long b, Zheng-Chao Tu c, Wei Peng a,
**
Ji-Quan Zhang a, Quentin Liu b , Gui Lu
,
a,d,
*
a Institute of Drug Synthesis and Pharmaceutical Process, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, PR China
b State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-sen University, Guangzhou 510060, PR China
c Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, PR China
d Institute of Human Virology, Sun Yat-sen University, Guangzhou 510080, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
The design and synthesis of a new series of N-trisubstituted (at C2, C4 and C6 respectively) pyrimidine
derivatives were reported, their in vitro structureeactivity relationships vs. aurora A kinase were also
discussed. Our results demonstrated that the introduction of characteristic N-substituted side chain at C2
of pyrimidines possessed a potent aurora A inhibitory activity, the position and the nature of the sub-
stituents on the phenyl ring of aniline side chain played key roles in cellular kinase inhibitory potency.
Most tested compounds exhibited good inhibitory activities against aurora A kinase and various human
tumor cell lines. Compounds 7j, 7men and 7p showed strong growtheinhibitory activities in the solid
CNE-2 tumor cell and selectively blocked cell-cycle progression at the G2/M phase.
Received 29 November 2013
Received in revised form
18 February 2014
Accepted 9 March 2014
Available online 12 March 2014
Keywords:
Pyrimidine derivative
Small molecule inhibitor
Antitumor
Ó 2014 Elsevier Masson SAS. All rights reserved.
Aurora A kinase
Synthesis
1. Introduction
these aurora kinase inhibitors have already been under Phase I/II
evaluation for various cancers.
The aurora family of serine/threonine protein kinase is crucial
for the proper regulation of mitosis. Mammals express three aurora
kinase paralogues, known as aurora A, B and C respectively. Despite
significant sequence homology, the localization and functions of
these aurora kinases are distinct. Aurora A localizes to centrosomes
during early S phase and is involved in centrosome maturation and
separation, bipolar spindle assembly, mitotic entry, and mitotic
exit. Aurora B and C are chromosomal passenger proteins, although
aurora C kinase is less well known [1].
Substituted pyrimidine is an important structure presented in a
number of aurora kinase inhibitors (Fig. 1) [2e15]. For example, VX-
680 has been discovered as a pan-aurora selective inhibitor [2],
although the clinical research was terminated due to the safety data
(QTc prolongation was observed) [3]. ENMD-2076 selectively in-
hibits aurora A kinase with an IC50 value of 14 nM [4]. CYC116 is an
orally available aurora kinase inhibitor which is currently under-
going Phase I clinical trials [5]. AZD1152 shows selectivity for
aurora B (Ki of 0.36 nM) over aurora A (Ki of 1.37 mM) and enters
For aurora A plays an important role in cell cycle progression
and carcinogenesis, the development of small molecule inhibitors
of aurora A as potential molecular-targeted therapeutic interven-
tion against cancer has been pursued by many researchers. Some of
Phase II clinical studies [6]. MLN8054 has been proved to inhibit
aurora A selectively, and is now under Phase I clinical trials for
advanced solid tumors [7].
The chemical structure of VX-680 suggested that the replace-
ment of S-substituted side chain at C2 with N-substituted aniline
side chain should retain its affinity for aurora kinase but signifi-
cantly simplify its synthesis. ENMD-2076 inspired that the change
of styrene side chain with N-benzyl amine side chain might also be
efficient for high inhibitory activity. Moreover, it was envisaged that
further improvements in terms of both potency and physical
properties could be made by adjusting the N-substituents at C2 and
* Corresponding author.
** Corresponding author.
E-mail addresses: liuq9@mail.sysu.edu.cn (Q. Liu), lugui@mail.sysu.edu.cn
(G. Lu).
1
These authors contributed equally to this paper.
http://dx.doi.org/10.1016/j.ejmech.2014.03.027
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

66
Y. Luo et al. / European Journal of Medicinal Chemistry 78 (2014) 65e71
Fig. 1. Some pyrimidine-based aurora kinase inhibitors in clinical testing.
Fig. 2. Our designed N-trisubstituted pyrimidine derivatives.
C6 of pyrimidine (Fig. 2). Herein we described the discovery and
characterization of a new series of N-trisubstituted (at C2, C4 and
C6 respectively) pyrimidine derivatives. Their in vitro structuree
activity relationships vs. aurora A kinase and corresponding cellular
potency were also analyzed.
with 3-amino-5-methylpyrazole (2) afforded C4-substituted
pyrimidine (3) regioselectively [16,17]. Then intermediate
was generated via nucleophilic substitution at C2-position of py-
rimidine core in the presence of various aniline derivatives [18].
Microwave assisted substitution at C6-position of pyrimidine ring
gave final product 7 [19].
5
2. Results and discussions
7e (or 7f) was synthesized via nucleophilic substitution be-
tween 7b and methylsulfonyl chloride (or para-toluensulfonyl
chloride) as shown in Scheme 2. The hydrolysis of 7a via lithium
hydroxide afforded the corresponding benzoic acid 7i (Scheme 3)
[20]. 2-Benzylamine substituted pyrimidine 10 was obtained by the
reaction of intermediate 3 with benzylamine in the presence of
2.1. Chemistry
The general synthetic route for N-trisubstituted pyrimidine was
illustrated in Scheme 1. Treatment of 2,4,6-trichloropyrimidine (1)
Scheme 1. General synthesis of 2,4,6-trisubstituted pyrimidine 7.

Y. Luo et al. / European Journal of Medicinal Chemistry 78 (2014) 65e71
67
Table 1
Effects of 6-substituted pyrimidines on aurora A kinase activity.
Compound
NR1R2
Aurora A IC50 (nM)
Scheme 2. Synthesis of compounds 7e and 7f.
7a
46
7b
7c
135
25
7d
113
Scheme 3. Synthesis of compound 7i.
7e
7f
34
DIPEA [16], followed by microwave irradiated substitution with
piperazine or morpholine (Scheme 4).
573
2.2. SAR analysis
7g
7h
228
72
At first, we carried out the SAR studies on various 6-substituted
pyrimidines 7ae7h for their synthetic accessibilities (Table 1). N-
Methyl piperazine substituted compound 7a showed good inhibi-
tory activity against aurora A kinase (IC50 ¼ 46 nM). Removal of the
N-methyl (7b) caused a drop on inhibition. N-methyl piperazine
group can also be replaced by morpholine group with better ac-
tivity (46 nM of 7a vs. 25 nM of 7c). However, more bulky mor-
pholine substituents as 2,6-dimethylmorpholine (7d) lowered the
inhibitory activity. N-Mesyl piperazine substituted compound 7e
showed equal potency with 7a against aurora A, albeit N-tosyl
piperazine derivative (7f) significantly lowered the inhibitory ac-
tivity. Substituents at C6-position can also been replaced by open-
chain aliphatic amines. The activity of 7h was higher than 7g by 5-
fold, indicating that the oxygen of morpholine may also bind with
aurora kinase.
Next, the influences of C2 substituents of pyrimidine on aurora A
kinase activities were analyzed and the results were presented in
Table 2. 4-Aminobenzoates (7a and 7j) and 4-aminobenzoic acid
(7i) exhibited comparable abilities to inhibit aurora A kinase, while
corresponding benzamide (7k) reduced the activity by 2 fold.
Benzamide (7k) and acetamide (7r) possessing similar structures
but opposite electron properties, showed comparable activities
against aurora kinase. Steric hindrance played an important role in
inhibition, meta- and para-substituents led to remarkable
improvement comparing with ortho-substituents (7men vs. 7l,
7peq vs. 7o). 4-Substituted 7t also caused sharp decline in inhib-
itory activity due to the bulky 2,5-piperazinedione substituent.
Strong electron-withdrawing groups as 4-nitro (7u), 4-cyano (7w)
and 4-sulfonamide (7x) gave best activities. We also synthesized
3,4-disubstituted compound 7z with a bit higher activity than 7a
and 7m. Compounds 10a and 10b with prolonged C2 side chain
showed declined activities.
2.3. Cell assay
Then fifteen compounds, whose IC50 values were lower than
50 nM, were further evaluated for in vitro anticancer screening.
Using VX-680 as positive control, their IC50 values against various
human cancer cell lines (including breast cancer cell lines as MCF-7,
SK-BR-3, MDA-MD-231, leukemia cell lines as K562, U937, HL-60,
MOLT-4, human pancreas cell lines as PNAC-1, human lung
adenocarcinoma cell lines as A549 and A431, prostate cancer cell
lines as DU145 and PC3, cervical cancer cell lines as Hela, the gastric
cancer cell lines as NCI-N87, human colon cancer cell lines as HCT-
116) were tested and listed in Tables 3 and 4. Most tested com-
pounds showed potent antiproliferative effects (except benzoic
acid derivative 7i), particularly for breast cancer and leukemia cell
lines.
Scheme 4. Synthesis of compound 10.

68
Y. Luo et al. / European Journal of Medicinal Chemistry 78 (2014) 65e71
Table 2 (continued)
2.4. Cell cycle arrest induced by the compounds
Compound
Ar
NR1R2
Aurora A IC50 (nM)
8.0
Cancer progression has been proposed to involve the loss of cell-
cycle checkpoint controls which regulate the passage through the
cell-cycle. These checkpoints monitor the integrity of the DNA and
ensure that genes are expressed in a coordinated manner [21]. By
7w
7x
7y
7z
5.0
11
Table 2
Effects of 2-substituted pyrimidines on aurora A kinase activity.
7.7
10a
10b
127
97
supplementing existing cell cycle machinery with extrinsic cell-
cycle regulators, it may be possible to block initiation or progres-
sion of cancer. For example, several flavonoids have been proved to
inhibit tumor growth in animal models, and they were also able to
interrupt cell cycle progression in either G1 or G2 [22e24].
Compound
Ar
NR1R2
Aurora A IC50 (nM)
46
7a
In our study, cell cycle analysis was conducted in the solid CNE-2
tumor cells by flow cytometry as shown in Fig. 3. C represents the
G0/G1 phase of the cell cycle, D represents S phase, E represents the
G2/M phase and F represents polyploid period. The corresponding
percentages expressed the distribution of the cell cycle phase
following treatment with compounds. CTR was the negative control
experiments, which indicated growth arrest of cells in the G2/M
phase of the cell cycle in a concentration-dependent manner
(Fig. 3a). Treatment with high concentrations of compounds 7m
7i
32
7j
22
7k
7l
101
200
22
(1.0
mM) can induce a remarkable G2/M arrest in CNE-2 cells
comparing to a relatively low concentration of 7m (0.01 and 0.1 mM,
Fig. 3b).
7m
We also analyzed other potent compounds for their cell cycle
effects. Treatment of CNE-2 cells with compounds 7m, 7n and 7p at
1.0 mM concentration arrested a majority of cell population in G2/M
7n
7o
7p
34
phase (92.5%, 90.6% and 90.5%), compounds 7a and 7j caused a
slightly lower distribution in G2/M phase (84.3% and 89.0%
respectively), other compounds showed little or no effects on the
cell cycle arrest (Table 5). Hence, we concluded that compounds 7j,
7men and 7p showed strong growtheinhibitory activities in the
solid CNE-2 tumor cell and selectively blocked cell-cycle
147
10
Table 3
Relative IC50
lines.
7q
7r
13
(mM) values for compounds against leukemia and breast tumor cell
118
Compound HL-60 K562 U937
MOLT-4 MCF-7 MDA-MD-231 SK-BR-3
VX-680
7a
7c
7e
7i
0.0382 0.0791 0.0360 0.0212 1.30
0.127
0.359
9.99
8.65
3.18
5.85
NA
4.02
11.3
14.7
2.29
1.61
0.437
2.18
0.883
15.9
0.960
2.16
0.203 0.359 0.119
0.750 0.537 0.447
0.604 0.556 0.233
0.161
0.302
0.215
NA
1.78
0.938 0.571
1.99
NA
7s
7t
112
549
0.716
NA
0.162
NA
NA
NA
7j
0.181 0.307 0.103
0.397 0.509 0.126
0.335 0.271 0.0923 0.116
0.0941 1.32
0.192
7m
7n
7p
7q
7u
7v
7w
7x
7y
0.933 0.240
2.75 0.243
0.131 0.0972 0.0217 0.0201 0.436 0.0564
0.0834 0.192 0.0554 0.0531 0.840 0.138
0.0698 0.137 0.0433 0.0181 0.424 0.0844
0.0909 0.138 0.0252 0.0260 0.642 0.114
0.0218 0.0581 0.00903 0.0136 0.372 0.0757
7u
7v
12
16
0.213 1.43
0.100
0.648
2.68
1.21
0.204
1.60 0.355 0.210
0.0607 1.07
7z
0.129 0.137 0.0550 0.0429 0.813 0.124

Y. Luo et al. / European Journal of Medicinal Chemistry 78 (2014) 65e71
69
Table 4
Table 5
Relative IC50
(
m
M) values for compounds against other tumor cell lines.
Distribution of cell cycle phases following treatment with compounds in the solid
CNE-2 tumor cells.
Compound PNAC-1 A549 A431 DU145 PC3
HeLa NCI-N87 HCT-116
Compound
Distribution [%]a
VX-680
7a
7c
7e
7i
4.13
1.08
1.59
1.22
39.3
1.32
0.693
1.12
0.351
0.455
0.442
0.457
0.435
8.76
3.05 0.240 0.400 5.81 2.93 11.6
1.83 0.532 0.798 1.50 1.27 17.8
1.59 0.832 0.611 1.09 1.89 3.23
4.32
0.672
1.04
1.24
NA
1.66
0.923
2.24
0.736
1.37
1.63
1.82
0.650
12.2
2.60
1.59
G1
S
G2/M
F
CTR
7a
7c
7j
7l
7m
7n
7p
10a
52.5
1.7
52.9
1.9
38.7
3.2
3.4
14.7
2.2
7.1
1.7
8.2
1.7
2.2
1.7
6.2
38.2
84.3
35.7
89.0
49.0
92.5
90.6
90.5
35.3
2.9
1.73 1.61
19.7 NA
0.791 2.06 1.70 10.9
NA NA NA NA
11.2
4.0
7.0
3.7
2.4
2.4
3.9
3.0
7j
1.22 0.440 0.499 1.36 0.985 6.32
7m
7n
7p
7q
7u
7v
7w
7x
7y
1.69 0.332 2.65
2.65 0.385 5.05
0.723 0.0875 3.09
0.631 0.162 2.24
0.723 0.150 1.20
0.926 0.166 1.34
0.785 0.140 1.27
1.08 0.546 7.19
2.00 0.867 18.9
0.573 0.425 1.77
1.48 0.619 7.47
1.09 0.331 3.88
0.962 1.11 4.32
0.977 0.331 5.78
3.3
55.4
a
CNE-2 cells were treated with compound (1.0 mM) for 24 h and subjected to cell
16.7 2.76
0.926 4.38 3.06 24.0
cycle analysis (by flow cytometry) following staining with PI.
0.580
0.508
1.04 0.331 0.631 0.967 0.607 17.5
1.59 0.0833 0.900 1.38 0.322 3.62
7z
respectively. The enzyme surface model of 7c was showed in Fig. 4c,
which revealed that the molecule was well embedded in the active
pocket. This molecular docking result, along with the biological
assay data, suggested that compound 7c was a potential inhibitor of
aurora A kinase.
progression at the G2/M phase. The studies on more cancer cell lines
were underway in our laboratory.
For compound 7f, its bulky N-tosyl piperazine moiety influenced
the binding affinity, hence showed a lower activity against aurora A
when compared with 7e (Fig. 4d vs. e). We also found that com-
pound 10a formed only one hydrogen bond with residue K162 due
to its prolonged C2-side chain, the reorientation of the molecule
significantly decreased the inhibitory activity (Fig. 4b).
Furthermore, the energy analysis [26] also indicates the insta-
bility of aurora A-7f complex in contrast to aurora A-7x complex
(Table 6).
2.5. Molecular docking
To gain better understanding of the potency of these derivatives
and guide further SAR studies, we also examined the interactions of
several compounds with aurora A crystal structure (2BMC.pdb).
The molecular docking was performed utilizing C-DOCKER pro-
gram within Discovery Studio 2.5 [25].
The binding modes of compound 7c and aurora A kinase were
depicted in Fig. 4a. Compound 7c was potently bound to the ATP
binding site of aurora A kinase via hydrophobic interactions and the
binding was stabilized by three hydrogen bonds interactions. The
carboxylic oxygen of 7c formed one hydrogen bond with residue
K162, the C2-substituted amino and the amino of pyrazole formed
another two hydrogen bonds with residues A213 and L139
3. Conclusions
In summary, a new series of N-trisubstituents pyrimidine de-
rivatives were designed and synthesized as potential aurora A
Fig. 3. Cell cycle distribution of CNE-2 cells after compounds treatment for 24 h. (a) Cells treated with CTR were collected, fixed with 70% ethanol and then stained with PI. (b) Cells
treated with 7m. Flow cytometric analysis was conducted to determine the cell cycle distribution.

70
Y. Luo et al. / European Journal of Medicinal Chemistry 78 (2014) 65e71
Fig. 4. The binding mode of compounds 7c (a,c), 10a (b), 7e (d) and 7f (e) with residues in active site of aurora A kinase.
Table 6
Appendix A. Supplementary data
The CDOCK energy and the IC50 value of compounds.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.03.027.
Compound
Aurora A IC50 (nM)
CDOCK energy (kcal/mol)
7a
7c
7x
7e
7f
46
25
5.0
34
573
127
ꢀ36.61
ꢀ33.11
ꢀ39.74
ꢀ35.21
ꢀ26.66
ꢀ31.67
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