Design, synthesis and bioevaluation of dihydropyrazolo[3,4-b]pyridine and benzo[4,5]imidazo[1,2-a]pyrimidine compounds as dual KSP and Aurora-A kinase inhibitors for anti-cancer agents

Article abstract of DOI:10.1016/j.bmc.2010.09.020

Four series of dihydropyrazolo[3,4-b]pyridines and benzo[4,5]imidazo[1,2-a] pyrimidines were designed and synthesized as dual KSP and Aurora-A kinase inhibitors for anti-cancer agents by introducing some fragments of Aurora-A kinase inhibitors into our KSP inhibitor CPUYL064. A total of 19 target compounds were evaluated by two related enzyme inhibition assays and a cytotoxicity assay in vitro. The results showed that some target compounds could inhibit both enzymes, and several of them showed significant inhibition activity against HCT116 cell line. Despite showing moderate KSP and Aurora-A kinase inhibition, the lead compounds 6a and 6e displayed significant cytotoxic activity in the micromolar range, especially against the HCT116 cell line and HepG2 cell line. The results may be useful for developing a new class of inhibitors having a dual function, KSP inhibition and Aurora-A kinase inhibition, for the treatment of cancer.

Full text of DOI:10.1016/j.bmc.2010.09.020

Bioorganic & Medicinal Chemistry 18 (2010) 8035–8043
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and bioevaluation of dihydropyrazolo[3,4-b]pyridine and
benzo[4,5]imidazo[1,2-a]pyrimidine compounds as dual KSP and Aurora-A
kinase inhibitors for anti-cancer agents
Rong-geng Fu a,b, Qi-dong You a,b, , Lei Yang c, Wu-tong Wu d, Cheng Jiang a,b, Xiao-li Xu a,b
⇑
a Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China
b Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China
c Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China
d School of Life Science and Technology, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 July 2010
Revised 6 September 2010
Accepted 8 September 2010
Available online 16 September 2010
Four series of dihydropyrazolo[3,4-b]pyridines and benzo[4,5]imidazo[1,2-a]pyrimidines were designed
and synthesized as dual KSP and Aurora-A kinase inhibitors for anti-cancer agents by introducing some
fragments of Aurora-A kinase inhibitors into our KSP inhibitor CPUYL064. A total of 19 target compounds
were evaluated by two related enzyme inhibition assays and a cytotoxicity assay in vitro. The results
showed that some target compounds could inhibit both enzymes, and several of them showed significant
inhibition activity against HCT116 cell line. Despite showing moderate KSP and Aurora-A kinase inhibi-
tion, the lead compounds 6a and 6e displayed significant cytotoxic activity in the micromolar range,
especially against the HCT116 cell line and HepG2 cell line. The results may be useful for developing a
new class of inhibitors having a dual function, KSP inhibition and Aurora-A kinase inhibition, for the
treatment of cancer.
Keywords:
Kinesin spindle protein inhibitor
Aurora-A
Kinase inhibition
Dihydropyrazolo[3,4-b]pyridine
Benzo[4,5]imidazo[1,2-a]pyrimidine
Anticancer
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
to find potential KSP inhibitors, our group recently reported a novel
kind of KSP inhibitors by rational drug design. Among them,
Uncontrolled proliferation in cancer cells is dependent upon
mitosis and its related processes. Therefore, a proven effective
strategy in cancer treatment has been to interfere with abnormal
progression of mitosis in order to halt the cell cycle in mitosis
and to induce apoptosis in tumor cells. Recently, much attention
has been focused on the new mitotic targets including the polo-like
kinases (PLKs), Aurora kinases, and kinesin motor proteins.
Kinesin spindle protein, known as KSP or Hs Eg5, has recently
emerged as a key target for selective antimitotic cancer therapy.
KSP is uniquely expressed in dividing cells, and its motor activity
has been shown to be required early in mitosis for the establish-
ment of a functional, bipolar mitotic spindle.1 So far no role for
KSP outside mitosis has been found. A range of structurally diverse
KSP inhibitors have been shown to cause cell cycle arrest, differen-
tiation and/or apoptosis of tumor cells. During the past 10 years,
the first generation of KSP inhibitors, including monastrol,2 ispine-
sib,3 CK01060234 and ARRY-5205 have been reported, and several
of these are now undergoing clinical trials.6 Monastrol (Fig. 1) is
the first specific KSP inhibitor shown to be noncompetitive with
microtubule (MT), and acting through an allosteric site. In order
CPUYL064 (Fig. 1) was found to be more potent than monastrol
in both KSP ATPase inhibitory and the ability to anti-proliferation
of human hepatocellular liver carcinoma cell line HepG2.7
On the other hand, Aurora kinases (Aurora-A, Aurora-B, and
Aurora-C) are vital component playing a critical role in regulating
many of the processes that are pivotal to mitosis, and they are re-
quired for healthy cell growth and proliferation. It was reported
that Aurora-A kinase are overexpressed in a broad range of tumor
cell lines and human primary tumors; thus, the inhibition of Aur-
ora-A kinase, by small molecules is becoming an area of major cur-
rent interest in oncology drug discovery.8 These discovery efforts
have led to several lead compounds (e.g., compound 1a9 and
1b10) and clinical candidates (e.g., VX-68011) (Fig. 1). VX-680 was
the first inhibitor of Aurora kinases and entered clinical trials.
Analysis of the crystal structure of Abl/VX-680 complexes reveals
that the aminomethylpyrazole group in VX-680 is anchored to
the hinge with three ATP-type hydrogen bonds.12 The conserved
H-bonding interactions of inhibitors with hinge region residues
are observed in most of the inhibitor-Aurora complex structures
and are essential for maintaining activity.8
Considering KSP and Aurora A kinase are two key enzymes in the
mitotic machinery,13 and KSP is one of Aurora A kinase substrates,
we proposed to design a new kind of dual inhibitors by combining
⇑
Corresponding author. Tel.: +86 25 83271351.
E-mail addresses: youqd@cpu.edu.cn, youqidong@gmail.com (Q. You).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.09.020

8036
R. Fu et al. / Bioorg. Med. Chem. 18 (2010) 8035–8043
OH
O
O
N
Cl
O
O
Cl
N
O
HN
O
N
H
N
NH
2
S
N
H
N
H
Monastrol
CPUYL064
ispinesib
O
HN
S
N
N
N
N
Cl
N
N
N
N
N
HN
N
N
H
N
H
N
N
N
N
H
1b
N
VX-680
1a
Figure 1. Structure of KSP and Aurora kinase inhibitors.
KSP inhibitor’s scaffold with Aurora-A kinase inhibitor’s fragment.
The dual targets of KSP and Aurora-A kinase may be more effective
for blocking multiple key components of the mitotic machinery
compared to separate agents and would be beneficial to enhance
antitumor activity toward a wide range of cancers. With the goal
of identifying such a new anti-cancer strategy, we devised a strategy
to derivatize CPUYL064 in such a way so as to facilitate its binding to
Aurora-A kinase while maintaining its KSP inhibitory activity.
Based on the pharmacophore we have identified and the bind-
ing mode analysis for KSP inhibitors,14,15 we mapped the structure
of CPUYL064 with the pharmacophore of KSP inhibitor, which con-
sists of four chemical features (Fig. 2): one aromatic ring (part 1),
one hydrophobic group (part 2), a side chain (part 3) and a flexible
heterocyclic ring (part 4). Substituted pyrazoles are common fea-
ture of Aurora-A kinase inhibitor scaffolds which formatted three
ATP-type hydrogen bonds to hinge residues.8 In order to incorpo-
rating Aurora-A kinase inhibitory functionality into the scaffold
of the KSP, our strategy is to utilize dihydropyrazolo[3,4-b]pyridine
moiety as the core structure in which the pyrazole ring was sup-
posed to perform as a hinge region-interacting group while the
dihydropyridine ring maintained in CPUYL064 as a KSP scaffold.
Thus, the structure of CPUYL064 was modified by replacing the
dihydropyridine group with dihydropyrazolo[3,4-b]pyridine moi-
ety and altering the benzene ring with different heterocyclic sys-
tems to afford three series of target compounds (series 1–3).
Some compounds demonstrated significant KSP inhibitory activi-
ties along with modest Aurora-A kinase inhibition. To optimize fur-
ther and determine the optimal hinge region-interacting group, we
carried out a more extensive exploration of this site by replacing
the pyrazole group with the benzimidazole moiety to afford the
target compounds (series 4), which utilized benzo[4,5]imi-
dazo[1,2-a]pyrimidine moiety as the core structure.
O
series 1
series 2
N
H
part 1
R
R
R
HN
HN
HN
part 1
N
N
N
N
H
part 3
Cl
part 3
part 2
O
O
part 2
part 4
S
N
H
O
O
N
H
N
H
part 4
Hinge
fused pharmacophore
N
H
N
O
HN
N
N
NH
NH
series 3
series 4
N
H
N
H
Hinge
Hinge
O
O
N
N
R
H
N
N
H
Figure 2. The design of the dual inhibitors of KSP and Aurora-A kinase.

R. Fu et al. / Bioorg. Med. Chem. 18 (2010) 8035–8043
8037
O
O
2a R = 4-OCH
NH
2
2
3
HN
2b R = 4-NO
2
2
a
2c R = 4-CF
2
3
2d R = 3-Cl-4-F
2
R
2
R
2e R = 4-F
2
2
2a-2e
R
O
3a R =phenyl
1
R = 4-OCH
2 3
1
3b R =phenyl
R = 4-NO
1
2
2
b
N
H
R
3c R =phenyl
1
R = 4-CF
HN
2
2
3
3d R =phenyl
R = 3-Cl-4-F
N
1
2
N
H
3e R =phenyl
R = 4-F
2
1
series 1
R
O
1
4a R =thienyl
1
R = 4-OCH
2
3
CHO
N
2a-2e
NH
2
4b R =thienyl
R = 4-NO
R
b
1
2
2
HN
HN
2
HN
H
R
N
1
4c R =thienyl
1
R = 4-CF
N
2
3
N
H
4d R =thienyl
R = 3-Cl-4-F
1
2
R = phenyl, thienyl, furyl
1
series 2
5a R =furyl
R = 4-OCH
2 3
1
R
O
1
5b R =furyl
R = 4-NO
1
2
2
N
H
5c R =furyl
1
R = 4-CF
b
2
3
R
2
5d R =furyl
R = 3-Cl-4-F
1
2
N
N
H
5e R =furyl
R = 4-F
2
1
series 3
6a R =furyl
1
R = 4-OCH
2
3
R
O
1
N
c
6b R =furyl
R = 4-NO
CHO
1
2
2
2a-2e
NH
2
N
N
H
6c R =furyl
R = 4-CF
R
1
2 3
N
H
1
R
2
6d R =furyl
R = 3-Cl-4-F
2
1
N
N
H
R = furyl
1
6e R =furyl
1
R = 4-F
2
series 4
Scheme 1. General route for the synthesis of dihydropyrazolo[3,4-b]pyridines. Reagents and conditions: (a) CH3COCH2COOC2H5, toluene, 10 h, reflex; (b) FeCl3, C2H5OH, 1–3 h,
50 °C; (c) CAN, C2H5OH, 1–3 h, 50 °C.
detection of a malachite green-phosphate complex.18 The IC50 val-
2. Chemistry
ues of the target compounds against KSP were determined by mea-
suring the activities of microtubule-activated ATPase activity.
Monastrol, CPUYL064 and CK0106023 were synthesized19,20 and
used as positive controls. In order to obtain potential compounds
with specific inhibitory effect against Aurora-A kinase, we used
the Aurora-A kinase activity assay kit developed by Cell Signaling
Technology. Compound 1a was synthesized21 and used as positive
controls. The anti-proliferative cytotoxic activity was also evalu-
ated by using HCT116 tumor cell line.
The synthesis of the dihydropyrazolo[3,4-b]pyridines (3a–3e,
4a–4d and 5a–5e) is presented in Scheme 1. N-Alkyl acetylaceta-
mides 2a–2e were prepared by ester/amide exchange reaction. The
one-pot reaction involves a three-component condensation reaction
of 3(5)-aminepyrazole, N-alkyl acetylacetamides 2a–2e and the
appropriate aromatic aldehyde in the presence of FeCl3 as a catalyst
at ambient temperature.
Several methods are available for the synthesis of benzo[4,5]imi-
dazo[1,2-a]pyrimidines.16,17 Despite related reactions utilizing var-
ious b-dicarbonyl compounds have been reported by several groups,
our investigation represents the precursors of the use of N-alkyl-3-
oxobutanamide and ceric ammonium nitrate (CAN) as catalyst in
this process. Compounds (6a–6e) were prepared according to the
procedures describedin Scheme1. In a typical experiment, 2-amino-
benzimidazole, N-alkyl acetylacetamides, 2-furaldehyde and ceric
ammonium nitrate were dissolved in absolute ethanol. The reaction
mixture was maintained for 1–3 h at ambient temperature followed
by removal of the solvent under vacuum and subsequent purifica-
tion by recrystallization from ethanol to yield the product (6a–6e).
The benzo[4,5]imidazo[1,2-a]pyrimidines and dihydropyrazol-
o[3,4-b]pyridines were all synthesized as racemic mixtures.
The data in Table 1 clearly showed that all dihydropyrazolo[3,4-
b]pyridine compounds exhibited significant inhibiting activities to-
ward KSP with the IC50 values range in the domains of 0.001–
10.4 lM. Given the IC50 for CPUYL064 is 0.18 lM, the introduction
of the pyrazole Aurora-A inhibiting pharmacophore at the dihydro-
pyridine is comparably tolerated but gradually decreases KSP
inhibitory in these three series of dihydropyrazolo[3,4-b]pyridines.
With the exception of compounds 3d, 3e, 4a and 5a, all the other
dihydropyrazolo[3,4-b]pyridines were weaker than CPUYL064,
generally inhibiting KSP at nanomolar to low micromolar levels.
The highest activity was observed for compound 5a, with IC50 of
1.1 nM, while 5e being the lowest with 10.36
substituted compound 3a inhibited the KSP activity at concentra-
tions up to 2.82 M, while the other compounds 3b–3e demon-
lM. The phenyl-
l
strated more potent inhibitory effects than the corresponding
thienyl or furyl-substituted compounds, with the IC50 values in
3. Results and discussion
the submicromolar range (from 0.14 to 0.4 lM). Generally, when
the R1 group was furyl or thienyl, the IC50 values increased with
the electrophilic group or halogen substitution and decreased with
KSP inhibitory activity was assessed by measuring the release of
inorganic phosphate from ATP hydrolysis through absorbance

8038
R. Fu et al. / Bioorg. Med. Chem. 18 (2010) 8035–8043
ties in the preliminary screening, but their IC50 values were all less
potent than 10 M. It has been observed from Table 1 that most of
Table 1
Bioactivity and lipophilicity of the synthesized dihydropyrazolo[3,4-b]pyridines
l
a
the dihydropyrazolo[3,4-b]pyridines with halogen substituent
groups at the side chain R2 group such as 3-chloro-4-fluoro espe-
cially, have significantly higher cytotoxicities than those with elec-
tron contributing groups or electron-withdrawing groups.
Compound 5a exhibited the most potent inhibitory activity for KSP
and moderate activity for Aurora-A kinase; however, the cytotoxic-
ity dramatically decreased. The expanded discrepancy might be
come from the poor lipophilicity of the compounds. In addition to
the anti-cancer activity, the relationships between the cytotoxic ef-
fects on HCT116 cells (shown by the IC50 values) and the lipophilicity
(shown by mi log P values23) of these three series compounds were
determined and shown in Table 1. The mi log P values for these three
series compounds are in the range 2.6–4.3. The compounds with mi
log P valuesbetween 4.0 and4.3 weremoreactive forHCT116cancer
cells. The cytotoxicity gradually rise with increasing the lipophilicity
caused by the introduction of different halogen substituents at R2
group. Evidenced by the lower mi log P value that indicates lipophil-
icity for those compounds such as 5a, we proposed that the disap-
pointing cytotoxicities of those compounds could be attributed to
the relatively poor membrane permeability of them. By increasing
lipophilicity of these three series compounds, we could expect an in-
crease in the affinity of the compounds for the lipid bilayer of the cell
membrane and thus an easier penetration into the cell.
Compound
R2
KSP IC50
M)
Aurora-A
mi
HCT116
c
(
l
IC50
(
lM)
log Pb
IC50
(lM)
3a
3b
3c
3d
3e
4a
4b
4c
4d
5a
5b
5c
5d
5e
4-OCH3
4-NO2
4-CF3
3-Cl-4-F
4-F
4-OCH3
4-NO2
4-CF3
3-Cl-4-F
4-OCH3
4-NO2
4-CF3
3-Cl-4-F
4-F
2.82
0.20
0.40
0.17
0.14
0.0025
0.82
0.32
0.44
0.0011
2.76
1.06
0.40
>50
>50
>50
>50
>50
>50
>50
>50
>50
9.25
>50
>50
15.00
8.10
>50
40.34
>50
3.456
3.358
4.294
4.169
3.563
3.355
3.257
4.193
4.068
2.713
2.615
3.552
3.426
2.820
2.409
5.746
5.943
3.572
>50
33.76
4.97
2.36
42.68
>50
21.02
11.56
25.00
>50
>50
>50
19.76
39.79
>50
12.70
0.94
10.36
5.47
0.18
0.029
40.18
Monastrol
CPUYL064
CK0106023
1a
/
/
/
/
4.97
4.71
a
The inhibitory activities against KSP were determined by measuring the
MT-activated ATPase activity.
b
Lipophilicity was evaluated by in silico calculation developed at Molinspiration.
Calculation was performed using Molinspiration online property calculation toolkit
(http://www.molinspiration.com).
c
IC50 values were derived from single dose–response curves generated from
triplicate data points.
From our structure–activity relationship (SAR) studies as dis-
cussed above, it can be concluded that the phenyl or thienyl series
are generally preferred for the inhibition of KSP whereas furyl ser-
ies leads to potent inhibition of Aurora-A kinase. To optimize fur-
ther and determine the optimal hinge region-interacting group,
we align the scaffold of compound 5e to the known Aurora-A ki-
nase inhibitor 1b. The molecular alignment of compounds 1b and
5e is shown in Figure 3. With the aim of increasing hydrophobic
interaction with the hinge region of the Aurora kinase and retain-
ing the global shape of 5e, we kept the furyl substituent on the R1
group and replaced the pyrazole group with the benzimidazole
moiety to afford compounds 6a–6e.
As shown in Table 2, it was observed that replacement of the pyr-
azole group by the benzimidazole moiety for compounds 5a–5e
leads to enhancement of the inhibitory kinase activity for Aurora-
A. Unlike Aurora-A kinase inhibition, the benzo[4,5]imidazo[1,2-
a]pyrimidine derivatives (compounds 6a–6e) decreases some
potency of KSP inhibition. Although compounds 6a–6e were moder-
ately potent KSP and Aurora-A kinase inhibitory fused-molecules,
most compounds displayed more balanced inhibitory activities
against two targets compare to dihydropyrazolo[3,4-b]pyridines
and showed more potent anti-proliferative activities against the
the incorporation of the methoxy group at the side chain R2 group.
These results suggest that the substituent groups of R1 play an
important role with regard to KSP inhibitory. And more clearly,
the phenyl or thienyl series are generally preferred for the inhibi-
tion of KSP.
For Aurora-A kinase, the inhibition activities of the dihydropy-
razolo[3,4-b]pyridines were relatively weak compared with that
against KSP. The changed R1 group of these three series compounds
affected the inhibition activity towards Aurora-A kinase. When the
R1 group was phenyl and thienyl, compounds 3a–3e and 4a–4d re-
sulted in a substantial loss in potency for Aurora-A kinase. How-
ever it was interesting that compounds 5a, 5d and 5e with the R1
group being furyl showed better inhibition activities against Aur-
ora-A kinase with the IC50 values of 9.25, 15.00 and 8.10 lM,
respectively, compared with the phenyl and thienyl substituted
compounds. The introduction of phenyl and thienyl at R1 group
cause significant potency loss in Aurora-A kinase inhibitory, pre-
sumably due to unfavorable steric interactions affecting the bind-
ing motif of the molecule in the Aurora-A kinase hinge site.
These results indicate that the furyl substituent on the R1 group
proved to be important for Aurora-A kinase inhibition in these
dihydropyrazolo[3,4-b]pyridines.
All the dihydropyrazolo[3,4-b]pyridines were evaluated for cyto-
toxicity to cancer HCT116 cell line by means of the 3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay as
described elsewhere.22 As shown in Table 1, these three series of
dihydropyrazolo[3,4-b]pyridine compounds were mostly only mod-
erately to weakly potent growth inhibition of HCT116 cell line, while
compound 3c and 3d was found to have more potency against
HCT116 cell line with IC50 ranging from 4.9 to 11.5 lM, which were
comparable to the positive control CPUYL064. Compound 6a exhib-
ited significantly inhibition on the growth of the HCT116 cells, much
more active than its counterpart compound 5a. Correspondingly, mi
log P value for compound 6a (mi log P = 4.177) was higher than for
compounds 5a (mi log P = 2.713) indicating better membrane pene-
tration which could at least partially account for the good cytotoxic-
ity obtained despite weaker KSP inhibition and similar Aurora-A
kinase inhibition. Compounds 6b and 6d show similar inhibitory
activity against Aurora-A kinase and similar anti-proliferative po-
tency against HCT116 cells despite the KSP component for com-
pound 6d, which is nearly 20-fold higher than that of 6b. These
results indicate that the inhibition mechanisms of compounds 6b
and 6d were mainly dominated by the inhibition of Aurora-A kinase.
The low anti-proliferative activity of compound 6c might be attrib-
uted to the poor aqueous solubility, a phenomenon that was
observed in our synthetic efforts. Interestingly, compound 6e is
30-fold less potent an inhibitor of KSP than CPUYL064 and about
1.2-fold less potent than 1a as Aurora-A kinase inhibitor. It was
HCT116 cell line with the IC50 values of 4.97 and 2.36
tively, compared with that of the positive control CPUYL064’s
12.70 M. Compounds with thienyl group at the R1 group, such as
4b, 4c and 4d, showed moderate anti-proliferative cytotoxic activi-
ties with an IC50 in the 11–25 M range. At enzymatic level com-
lM, respec-
l
l
pounds 3a–3e and 4a–4d showed more potent inhibition of KSP
than Aurora-A kinase. Thus, the potential effect of Aurora-A kinase
inhibition on proliferation of HCT116 cells may be negligible. The
furyl derivatives 5a–5e also showed some anti-proliferation activi-

R. Fu et al. / Bioorg. Med. Chem. 18 (2010) 8035–8043
8039
N
N
N
N
N
N
N
H
1b
Hinge
O
O
Alignment
F
N
O
N
N
O
N
N
N
H
N
N
H
R
2
HN
O
F
N
N
N
N
H
N
O
H
6a-6e
N
H
HN
N
N
H
5e
Hinge
Figure 3. The design of the compounds 6a–6e.
In attempts to better understand the observed SARs, docking
studies with the representative compound 6e was performed to
suggest possible binding modes of the chimeras on KSP (Fig. 4)
and Aurora-A kinase (Fig. 5). As shown in Figure 4, compound 6e
Table 2
Bioactivity and lipophilicity of the synthesized benzo[4,5]imidazo[1,2-a]pyrimidines
a
c
Compound
R2
KSP IC50
M)
Aurora-A
mi log Pb
HCT116 IC50
(lM)
(
l
IC50
(lM)
interacted with
a cage formed by Glu116, Gly117, Glu118,
6a
6b
6c
6d
4-OCH3
4-NO2
4-CF3
3-Cl-4-F
2.46
20.85
3.32
0.96
5.65
5.47
0.18
0.029
40.18
7.58
3.00
1.32
5.73
6.15
>50
40.34
>50
4.97
4.177
4.079
5.016
4.890
4.284
2.409
5.746
5.943
3.572
8.71
11.49
>50
10.90
4.96
>50
12.70
0.94
4.71
Trp127, Ala133, Ile136, Pro137, Tyr211, Leu214 and Glu215, and
formed one hydrogen bonds with the main chain oxygen of residue
Gly117. In addition, the furan group of compound 6e interacted
with the cooperative minor pocket mainly surrounded by Arg221
and Ala218. As shown in Figure 5, the benzo[4,5]imidazo[1,2-
a]pyrimidine core of 6e fitted into the hinge region of Aurora-A ki-
nase, and the benzo[4,5]imidazo[1,2-a]pyrimidine core N@C and
NH atoms formed two H-bonds with the main chain of Ala213. In
addition, the benzo[4,5]imidazo[1,2-a]pyrimidine core also formed
hydrophobic interactions with the surrounding residues, including
Leu139, Val147, Ala160, and Leu263. The large amide substituents
of 6e extend to the outer portion of the binding pocket through a
region adjacent to Thr217.
6e
4-F
Monastrol
CPUYL064
CK0106023
1a
/
/
/
/
a
The inhibitory activities against KSP were determined by measuring the MT-
activated ATPase activity.
b
Lipophilicity was evaluated by in silico calculation developed at Molinspiration.
Calculation was performed using Molinspiration online property calculation toolkit
(http://www.molinspiration.com).
c
IC50 values were derived from single dose–response curves generated from
triplicate data points.
Finally, the most promising compounds representing different
structural series were selected for further evaluation for their anti-
proliferative cytotoxic activity against other human tumor cell lines,
such as HepG2 (hepatocarcinoma) and A2780 (ovarian cancer). As
shown in Table 3, most tested compounds exhibited inhibitions on
the growth of selected tumor cell lines, especially on HCT116 cell
line. Althoughit was not easy to obtainan obvious structure–activity
relationshipfromthe Table3, wecouldconcludethatthesubstituent
groups of the side chain play an important role with regard to
cytotoxicity and the replacement of pyrazole group by the
found to inhibit proliferation of HCT116 cells at a concentration
(IC50 = 4.96
pound6e’s anti-proliferativeactivitywasnearly 2.5-foldhigherthan
that of CPUYL064 (IC50 = 12.70 M). These results suggest that at
lM) similar to that of 1a (IC50 = 4.71 lM), while com-
l
least for compound 6e the two components, KSP and Aurora-A ki-
nase inhibition, both contribute to the anti-proliferative activity
and the inhibition mechanism of compound 6e was mainly domi-
nated by the inhibition of Aurora-A kinase.
Figure 4. Model of compound 6e bound to the KSP. Docking simulation was performed using the program GOLD and the crystal structure of KSP (PDB code 1Q0B). For clarity,
only interacting residues are displayed. (a) Compound 6e is depicted by sticks and balls. (b) The KSP is represented by molecular surface.

8040
R. Fu et al. / Bioorg. Med. Chem. 18 (2010) 8035–8043
Figure 5. Model of compound 6e bound to the Aurora-A kinase. Docking simulation was performed using the program GOLD and the crystal structure of Aurora-A kinase (PDB
code 2BMC). For clarity, only interacting residues are displayed. (a) Compound 6e is depicted by sticks and balls. (b) The Aurora-A kinase is represented by molecular surface.
recorded on a Nicolet Impact 410 instrument (KBr pellet). 1H
Table 3
Cellular IC50
(
lM) of selected compounds
NMR spectra were recorded with a Bruker Avance 300 MHz spec-
trometer at 300 K, using TMS as an internal standard. MS spectra
were recorded on a Shimadzu GC–MS 2010 (EI) or a Mariner Mass
Spectrum (ESI), or a LC/MSD TOF HRMS Spectrum. All compounds
were routinely checked by TLC and 1H NMR. TLCs and preparative
thin-layer chromatography were performed on silica gel GF/UV
254, and the chromatograms were performed on silica gel (200–
300 mesh) visualized under UV light at 254 and 365 nm. All sol-
vents were reagent grade and, when necessary, were purified and
dried by standards methods. Concentration of solutions after reac-
tions and extractions involved the use of a rotary evaporator oper-
ating at a reduced pressure of ca. 20 Torr. Organic solutions were
dried over anhydrous sodium sulfate. Analytical results are within
(0.50% of the theoretical values).
Compound
HCT116
A2780
HepG2
3c
3d
4c
5d
6a
6b
6d
6e
4.97
2.36
11.56
19.76
8.71
11.49
10.90
4.96
>50
29.40
42.38
>50
20.00
10.12
48.49
9.34
3.05
>50
>50
3.00
4.26
>50
4.97
13.54
25.98
7.38
2.10
5.85
CK0106023
1a
0.94
4.71
benzimidazole moiety for compounds 5a–5e leads to an increased
cytotoxicity to the human tumor cell lines. Compound 6a exhibited
good anti-proliferative cytotoxicity against several tumor cell lines.
Specifically, it had an IC50 value of 4.97
lM against human hepato-
5.2. General procedure for the synthesis of compounds 2a–2e
cellular liver carcinoma cell line HepG2, and this was similar to the
anti-proliferative cytotoxicity of CK0106023 and compound 6e with
Ethyl acetoacetate (40 mmol) and aniline (40 mmol) was dis-
solved in toluene (100 mL) under nitrogen atmosphere. The mix-
ture was stirred at refluxing for 10 h, then remove of the major
part of solvent in vacuo. The mixture was concentrated and puri-
fied by column chromatography on silica gel by eluting with a mix-
ture of petroleum ether/ethyl acetate (1:2) to give the desired
product.
an IC50 value of 2.10 and 7.38
found to have a similar potency against A2780 cell line with an
IC50 value of 3.05 M compared with that of CK0106023’s 3.00 M.
lM, respectively. Compound 6b was
l
l
Further evaluation of benzo[4,5]imidazo[1,2-a]pyrimidine com-
pound’s anti-cancer mechanism was on the way.
4. Conclusions
5.2.1. N-(4-Methoxyphenyl)acetylacetamide (2a)
Yield 16.6%, white powdery crystal, mp 112–114 °C. 1H NMR
(300 MHz, CDCl3): d 2.33 (s, 3H), 3.58 (s, 2H), 3.79 (s, 3H), 6.86
(d, 2H, J = 9.0 Hz), 7.44 (d, 2H, J = 9.0 Hz), 8.93 (s, 1H). EI-MS (m/
z): 207 (M+).
The correlation between KSP inhibition and suppression of tumor
growth has brought the design of KSP inhibitors to the forefront of
cancer research and is validated as an important cancer target. By
combining structural features from known inhibitors of KSP and
Aurora-A, we have been able to design, synthesize and evaluate
two types of dual inhibitors of KSP and Aurora-A based on a
CPUYL064 core structure. Although, compounds 6a and 6e identified
through this new design approach were moderately potent KSP and
Aurora-A kinase inhibitory fused-molecules, these two compounds
displayed significant cytotoxic activity in the micromolar range,
especially against the HCT116 cell line and HepG2 cell line. Taken to-
gether these results suggest that the compounds described in this
study can serve as lead analogs for further development of more po-
tent dual KSP and Aurora-A inhibitors in single molecules.
5.2.2. N-(4-Nitrophenyl)acetylacetamide (2b)
Yield 76.1%, yellow powdery crystal, mp 103–106 °C. 1H NMR
(300 MHz, CDCl3): d 2.40 (s, 3H), 3.65 (s, 2H), 7.48 (d, 2H,
J = 9.0 Hz), 8.22 (d, 2H, J = 9.0 Hz), 9.75 (s, 1H). EI-MS (m/z): 222
(M+).
5.2.3. N-(4-Trifluoromethylphenyl)acetylacetamide (2c)
Yield 36.1%, white powdery crystal, mp 92–95 °C. 1H NMR
(300 MHz, CDCl3): d 2.35 (s, 3H), 3.63 (s, 2H), 7.58 (d, 2H,
J = 8.4 Hz), 7.69 (d, 2H, J = 8.7 Hz), 9.46 (s, 1H). EI-MS (m/z): 245
(M+).
5. Experimental
5.1. General
5.2.4. N-(3-Chloro-4-fluorophenyl)acetylacetamide (2d)
Yield 62.3%, white powdery crystal, mp 65–66 °C. 1H NMR
(300 MHz, CDCl3): d 3.34 (s, 3H), 3.60 (s, 2H), 7.08 (t, 1H,
J = 9.0 Hz), 7.44 (m, 1H), 7.75 (m, 1H), 9.33 (s, 1H). EI-MS (m/z):
229 (M+).
Melting points were determined on a Mel-TEMP II melting point
apparatus and are uncorrected. Infrared (IR) spectra (KBr) were

R. Fu et al. / Bioorg. Med. Chem. 18 (2010) 8035–8043
8041
5.2.5. N-(4-Fluorophenyl)acetylacetamide (2e)
(300 MHz, DMSO-d6): d 2.09 (s, 3H), 3.69 (s, 3H), 5.67 (s, 1H),
6.80 (m, 4H), 7.20 (d, 1H), 7.43 (s, 1H), 7.45 (d, 2H, J = 8.4 Hz),
8.72 (s, 1H), 9.25 (s, 1H), 11.94 (s, 1H). EI-MS (m/z): 366 (M+).
HRMS (ESI): Calcd for (C19H18N4O2S)Na+: 389.1048. Found:
389.1063 [M+Na]+.
Yield 62.3%, white powdery crystal, mp 98–100 °C. 1H NMR
(300 MHz, CDCl3): d 2.33 (s, 3H), 3.59 (s, 2H), 7.02 (t, 2H,
J = 8.4 Hz), 7.51 (m, 2H), 9.15 (s, 1H). EI-MS (m/z): 195 (M+).
5.3. General procedure for the synthesis of compounds 3a–3e,
4a–4d and 5a–5e
5.3.7. 4-Thiophene-6-methyl-N-(4-nitrophenyl)-4,7-dihydro-
2H-pyrazole[3,4-b]pyridine-5-carboxamide (4b)
Yield 19%, mp 231–233 °C. IR (cmꢀ1): 3377, 3298, 3080, 1638,
1525, 1496, 1343, 1252, 1158, 843, 719. 1H NMR (300 MHz,
DMSO-d6): d 2.15 (s, 3H), 5.76 (s, 1H), 6.77–6.82 (m, 2H), 7.20 (d,
1H, J = 4.8 Hz), 7.39 (s, 1H), 7.81 (d, 2H, J = 9.3 Hz), 8.14 (d, 2H,
J = 9.3 Hz), 9.10 (s, 1H), 9.96 (s, 1H), 12.05 (s, 1H, NH). EI-MS (m/
z): 381 (M+). HRMS (ESI): Calcd for (C18H15N5O3S)Na+: 404.0794.
Found: 404.0788 [M+Na]+.
A solutionof 5-aminopyrazole (1.2 mmol), N-alkyl-3-oxobutana-
mides (1.2 mmol) and aldehyde (1.2 mmol) in absolute ethanol
(5 mL) was heated to 50 °C with stirring under nitrogen atmosphere.
After stirred for 60 min, the mixture was added a catalytic amount of
ferric chloride. The precipitation was occurred in 60–180 min, fil-
trated to yield the crude product, which was purified by recrystalli-
zation from 95% EtOH to give the desired compounds.
5.3.1. 4-Phenyl-6-methyl-N-(4-methoxyphenyl)-4,7-dihydro-
2H-pyrazole[3,4-b]pyridine-5-carboxamide (3a)
5.3.8. 4-Thiophene-6-methyl-N-(4-trifluoromethylphenyl)-4,7-
dihydro-2H-pyrazole[3,4-b]pyridine-5-carboxamide (4c)
Yield 29%, mp 246–248 °C. IR (cmꢀ1): 3384, 3285, 3082, 1644,
1600, 1523, 1403, 1332, 1256, 1110, 1064, 829, 715. 1H NMR
(300 MHz, DMSO-d6): d 2.13 (s, 3H), 5.73 (s, 1H), 6.78 (s, 1H),
6.81 (t, 1H, J = 3.3 Hz), 7.20 (d, 1H, J = 4.8 Hz), 7.37 (s, 1H), 7.58
(d, 2H, J = 8.7 Hz), 7.78 (d, 2H, J = 8.7 Hz), 8.95 (s, 1H), 9.72 (s,
1H), 12.00 (s, 1H). EI-MS (m/z): 404 (M+). HRMS (ESI): Calcd for
(C19H15F3N4OS)Na+: 427.0817. Found: 427.0809 [M+Na]+.
Yield 30%, mp 155–157 °C. IR (cmꢀ1): 3411, 3292, 1643, 1598,
1510. 1H NMR (300 MHz, DMSO-d6): d 2.11 (s, 3H), 3.67 (s, 3H),
5.32 (s, 1H), 6.77 (d, 2H, J = 8.7 Hz), 7.07 (m, 1H), 7.15–7.22 (m,
5H), 7.47 (d, 2H, J = 9.0 Hz), 8.62 (s, 1H), 9.15 (s, 1H), 11.81 (s,
1H). EI-MS (m/z): 360 (M+). HRMS (ESI): Calcd for (C21H20N4O2)-
Na+: 383.1484. Found: 383.1467 [M+Na]+.
5.3.2. 4-Phenyl-6-methyl-N-(4-nitrophenyl)-4,7-dihydro-2H-
pyrazole[3,4-b]pyridine-5-carboxamide (3b)
Yield 15.5%, mp 224–226 °C. IR (cmꢀ1): 3414, 3293, 3195, 1644,
1598, 1510, 1408, 1242, 1172, 1031, 826, 785. 1H NMR (300 MHz,
DMSO-d6): d 2.17 (s, 3H), 5.42 (s, 1H), 7.20–7.21 (m, 6H), 7.75 (d,
2H, J = 9.0 Hz), 8.10 (d, 2H, J = 9.0 Hz), 9.00 (s, 1H), 9.87 (s, 1H),
11.92 (s, 1H). EI-MS (m/z): 375 (M+). HRMS (ESI): Calcd for
(C20H17N5O3)Na+: 398.1229. Found: 398.1226 [M+Na]+.
5.3.9. 4-Thiophene-6-methyl-N-(3-chloro-4-fluorophenyl)-4,7-
dihydro-2H-pyrazole[3,4-b]pyridine-5-carboxamide (4d)
Yield 45%, mp 241–243 °C. IR (cmꢀ1): 3390, 3275, 3198, 3083,
1640, 1600, 1500, 1388, 1243, 1132, 1054, 858, 716. 1H NMR
(300 MHz, DMSO-d6): d 2.11 (s, 3H), 5.70 (s, 1H), 6.78 (d, 1H,
J = 2.7 Hz), 6.81 (t, 1H, J = 3.6 Hz), 7.20 (q, 1H, J = 5.1 Hz), 7.29 (t,
1H, J = 9.0 Hz), 7.36 (s, 1H), 7.47 (m, 1H), 7.88 (q, 1H, J = 6.9 Hz),
8.91 (s, 1H), 9.57 (s, 1H), 12.00 (s, 1H). EI-MS (m/z): 388 (M+).
HRMS (ESI): Calcd for (C18H14ClFN4OS)Na+: 411.0459. Found:
411.0466 [M+Na]+.
5.3.3. 4-Phenyl-6-methyl-N-(4-trifluoromethylphenyl)-4,7-
dihydro-2H-pyrazole[3,4-b]pyridine-5-carboxamide (3c)
Yield 27%, mp 259–260 °C. IR (cmꢀ1): 3401, 3315, 3198, 3081,
1644, 1598, 1523, 1403, 1332, 1254, 1111, 1064, 827, 705. 1H
NMR (300 MHz, DMSO-d6): d 2.15 (s, 3H), 5.40 (s, 1H), 7.07 (m,
1H), 7.20–7.22 (m, 5H), 7.55 (d, 2H, J = 8.7 Hz), 7.68 (d, 2H,
J = 8.1 Hz), 8.87 (s, 1H), 9.63 (s, 1H), 11.89 (s, 1H). EI-MS (m/z):
398 (M+). HRMS (ESI): Calcd for (C21H17F3N4O)Na+: 421.1252.
Found: 421.1269 [M+Na]+.
5.3.10. 4-Furan-6-methyl-N-(4-methoxyphenyl)-4,7-dihydro-
2H-pyrazole[3,4-b]pyridine-5-carboxamide (5a)
Yield 21%, mp 261–263 °C. IR (cmꢀ1): 3425, 3289, 3196, 1643,
1599, 1511, 1409, 1243, 1173, 1031, 826, 734. 1H NMR
(300 MHz, DMSO-d6): d 2.08 (s, 3H), 3.69 (s, 3H), 5.43 (s, 1H),
6.01 (d, 1H, J = 3.0 Hz), 6.24 (d, 1H, J = 3.0 Hz), 6.82 (d, 2H,
J = 9.0 Hz), 7.34 (s, 1H), 7.43 (s, 1H), 7.46 (d, 2H, J = 9.0 Hz), 8.66
(s, 1H), 9.33 (s, 1H), 11.92 (s, 1H). EI-MS (m/z): 350 (M+). HRMS
(ESI): Calcd for (C19H18N4O3)Na+: 373.1277. Found: 373.1290
[M+Na]+.
5.3.4. 4-Phenyl-6-methyl-N-(3-chloro-4-fluorophenyl)-4,7-
dihydro-2H-pyrazole[3,4-b]pyridine-5-carboxamide (3d)
Yield 23%, mp 222–224 °C. IR (cmꢀ1): 3401, 3308, 3201, 3084,
1641, 1500, 1391, 1251, 1149, 1073, 808, 701. 1H NMR
(300 MHz, DMSO-d6): d 2.14 (s, 3H), 5.36 (s, 1H), 7.08 (m, 1H),
7.18–7.28 (m, 6H), 7.40 (m, 1H), 7.80 (m, 1H), 8.81 (s, 1H), 9.48
(s, 1H), 11.87 (s, 1H). EI-MS (m/z): 382 (M+). HRMS (ESI): Calcd
for (C20H16ClFN4O)H+: 383.1077. Found: 383.1070 [M+H]+.
5.3.11. 4-Furan-6-methyl-N-(4-nitrophenyl)-4,7-dihydro-2H-
pyrazole[3,4-b]pyridine-5-carboxamide (5b)
Yield 15%, mp 229–231 °C. IR (cmꢀ1): 3373, 3289, 3196, 3077,
1643, 1593, 1495, 1342, 1252, 1147, 1006, 842, 748. 1H NMR
(300 MHz, DMSO-d6): d 2.08 (s, 3H), 5.53 (s, 1H), 5.97 (d, 1H,
J = 3.0 Hz), 6.24 (q, 1H, J = 3.0 Hz), 7.40 (s, 1H), 7.43 (m, 1H), 7.82
(d, 2H, J = 9.0 Hz), 8.15 (d, 2H, J = 9.0 Hz), 9.03 (s, 1H), 10.02 (s,
1H), 12.02 (s, 1H). EI-MS (m/z): 365 (M+). HRMS (ESI): Calcd for
(C18H15N5O4)Na+: 388.1022. Found: 388.1023 [M+Na]+.
5.3.5. 4-Phenyl-6-methyl-N-(4-fluorophenyl)-4,7-dihydro-2H-
pyrazole[3,4-b]pyridine-5-carboxamide (3e)
Yield 25%, mp 232–234 °C. IR (cmꢀ1): 3404, 3301, 3084, 2360,
1642, 1600, 1509, 1400, 1221, 1149, 820, 702. 1H NMR
(300 MHz, DMSO-d6): d 2.12 (s, 3H), 5.34 (s, 1H), 7.02–7.25 (m,
8H), 7.48 (d, 1H, J = 2.1 Hz), 7.50 (d, 1H, J = 2.1 Hz), 8.68 (s, 1H),
9.31 (s, 1H), 11.85 (s, 1H). EI-MS (m/z): 348 (M+). EI-MS (m/z):
348 (M+). HRMS (ESI): Calcd for (C20H17FN4O)H+: 349.1466. Found:
349.1455 [M+H]+.
5.3.12. 4-Furan-6-methyl-N-(4-trifluoromethylphenyl)-4,7-
dihydro-2H-pyrazole[3,4-b]pyridine-5-carboxamide (5c)
Yield 17%, mp 248–250 °C. IR (cmꢀ1): 3388, 3293, 3084, 1649,
1602, 1524, 1403, 1332, 1257, 1111, 1008, 832, 748. 1H NMR
(300 MHz, DMSO-d6): d 2.12 (s, 3H), 5.50 (s, 1H), 5.99 (d, 1H,
J = 3.0 Hz), 6.24 (q, 1H, J = 3.0 Hz), 7.40 (s, 1H), 7.42 (d, 1H,
J = 0.9 Hz), 7.60 (d, 2H, J = 8.7 Hz), 7.79 (d, 2H, J = 8.7 Hz), 8.89 (s,
1H), 9.78 (s, 1H), 11.99 (s, 1H). EI-MS (m/z): 388 (M+). HRMS
5.3.6. 4-Thiophene-6-methyl-N-(4-methoxyphenyl)-4,7-
dihydro-2H-pyrazole[3,4-b]pyridine-5-carboxamide (4a)
Yield 56%, mp 223–225 °C. IR (cmꢀ1): 3385, 3318, 1640, 1602,
1514, 1405, 1317, 1244, 1132, 1030, 826, 723. 1H NMR

8042
R. Fu et al. / Bioorg. Med. Chem. 18 (2010) 8035–8043
(ESI): Calcd for (C19H15F3N4O2)Na+: 411.1045. Found: 411.1051
[M+Na]+.
6.99–7.91 (m, 8H), 9.95 (s, 1H), 10.39 (s, 1H). EI-MS: (m/z) 422
(M+). Anal. Calcd for C22H16ClFN4O2: C, 62.49; H, 3.81; N, 13.25.
Found: C, 62.21; H, 3.97; N, 13.58.
5.3.13. 4-Furan-6-methyl-N-(3-chloro-4-fluorophenyl)-4,7-
dihydro-2H-pyrazole[3,4-b]pyridine-5-carboxamide (5d)
Yield 34%, mp 239–241 °C. IR (cmꢀ1): 3388, 3280, 3085, 1643,
1602, 1503, 1387, 1246, 1148, 1004, 921, 787. 1H NMR
(300 MHz, DMSO-d6): d 2.10 (s, 3H), 5.46 (s, 1H), 5.97 (d, 1H,
J = 3.0 Hz), 6.25 (q, 1H), 7.30 (t, 1H), 7.38 (s, 1H), 7.43 (d, 1H,
J = 0.9 Hz), 7.49 (m, 1H), 7.91 (m, 1H), 8.85 (s, 1H), 9.64 (s, 1H),
11.98 (s, 1H, NH). EI-MS (m/z): 372 (M+). HRMS (ESI): Calcd for
(C18H14ClFN4O2)Na+: 395.0687. Found: 395.0697 [M+Na]+.
5.4.5. N-(4-Fluorobenzyl)-4-furan-2-methyl-1,4-dihydro-
benzo[4,5]imidazo[1,2-a]pyrimidine-3-carboxamide (6e)
Yield 34%, white powdery crystal, mp 275–276 °C (dec). IR
(cmꢀ1): 3421, 2924, 2973, 2360, 1625, 1509, 1460. 1H NMR
(300 MHz, DMSO-d6): d 2.05 (s, 3H), 6.34 (s, 1H), 6.52 (s, 1H),
6.79 (s, 1H), 7.01–7.60 (m, 9H), 9.83 (s, 1H), 10.33 (s, 1H). EI-MS:
(m/z) 388 (M+). Anal. Calcd for C22H17FN4O2: C, 68.03; H, 4.41; N,
14.43. Found: C, 67.64; H, 4.50; N, 14.36.
5.3.14. 4-Furan-6-methyl-N-(4-fluorophenyl)-4,7-dihydro-2H-
pyrazole[3,4-b]pyridine-5-carboxamide (5e)
5.5. Bioactivity test
Yield 30%, mp 228–230 °C. IR (cmꢀ1): 3384, 3289, 3084, 1643,
1604, 1509, 1396, 1220, 1150, 1005, 823, 746. 1H NMR
(300 MHz, DMSO-d6): d 2.09 (s, 3H), 5.45 (s, 1H), 6.00 (d, 1H,
J = 3.0 Hz), 6.24 (t, 1H, J = 2.7 Hz), 7.07 (t, 2H, J = 9.0 Hz), 7.35 (s,
1H), 7.43 (s, 1H), 7.58 (q, 2H, J = 9.0 Hz), 8.73 (s, 1H), 9.50 (s, 1H),
11.94 (s, 1H). EI-MS (m/z): 338 (M+). HRMS (ESI): Calcd for
(C18H15FN4O2)H+: 339.1259. Found: 339.1268 [M+H]+.
5.5.1. Preparation of Eg5
Coding regions were PCR amplified from a template (obtained in
our lab) containing full-length human Eg5. The primers used were,
forward 50-TATAGG GCG AAT TCC GCC ATG GCG TCG CAG CCA-30
and reverse 50-ACG GGC TGC AGC AAG CTC GAG TTT TAAACG TTC
TAT-30. The region encoding residues 2–386 was subcloned into
pET28a (NOVAGEN). Protein expression in Escherichia coli cells was
inducedwith 0.5 mM IPTG. Cells were harvestedafter 20 h of growth
at 20 °C and then disrupted by sonication. The soluble lysate was
clarified by centrifugation and applied to a SP-Sepharose column
(Amersham Pharmacia Biotech) in a buffer A (20 mM Na-PIPES, pH
6.3; 20 mM NaCl; 1 mM MgCl2; 1 mM Na-EGTA). Protein was eluted
with a linear gradient of 20–1000 mM NaCl. Eg5 was identified by
SDS–PAGE, and then applied to Mono-Q columns (Amersham Phar-
macia Biotech) in a buffer B (20 mM Tris–HCl, pH 8.8; 1 mM MgCl2;
1 mM Na-EGTA). A gradient from 0 to 1000 mM NaCl was used to
elute Eg5. Fractions were analyzed by SDS–PAGE. The most concen-
tratedfractionwas dialyzedagainst ATPasebuffer(20 mM Na-PIPES,
pH 7.5; 1 mM MgCl2; 1 mM Na-EGTA) and then aliquoted, frozen in
liquid nitrogen, stored at ꢀ80 °C.
5.4. General procedure for the synthesis of compounds 6a–6e
A solution of 2-aminobenzimidazole (1.2 mmol), N-alkyl-3-
oxobutanamide (1.2 mmol), 2-furaldehyde (1.2 mmol) and ceric
ammonium nitrate (0.06 mmol) in absolute ethanol (2 mL) was
heated to 50 °C with stirring. The precipitation was occurred in
60–180 min and the reaction mixture was cooled to room temper-
ature. The mixture was filtered to give the crude product, which
was further purified by recrystallization from 95% EtOH to give
the desired compounds.
5.4.1. N-(4-Methoxybenzyl)-4-furan-2-methyl-1,4-dihydro-
benzo[4,5]imidazo[1,2-a]pyrimidine-3-carboxamide (6a)
Yield 35%, white powdery crystal, mp 248–250 °C (dec). IR
(cmꢀ1): 3302, 2836, 2973, 1627, 1512, 1460, 1250. 1H NMR
(300 MHz, DMSO-d6): d 2.24 (s, 3H), 3.71 (s, 3H), 6.34 (s, 1H),
6.61 (s, 1H), 6.78 (s, 1H), 6.85–7.50 (m, 9H), 9.62 (s, 1H), 10.24
(s, 1H). EI-MS: (m/z) 400 (M+). Anal. Calcd for C23H20N4O3: C,
68.64; H, 5.51; N, 13.92. Found: C, 68.85; H, 5.13; N, 14.07.
5.5.2. ATPase activity assay
All experiments were done at room temperature. The reagents
were added to wells of a 96-well clear plate and the final reaction
of the assay contained 20 mM PIPES, pH 7.5, 5.0 mM MgCl2, 1 mM
EGTA, 10 mM paclitaxel, 0.6 mM tubulin (MT), 0.5 mM ATP, 2%
DMSO containing inhibitors in a reaction volume of 100 mL. Reac-
tions were started by adding ATP. The plates were incubated at
37 °C for 30 min. Following incubation the malachite-green based
reagents were added to detect the release of inorganic phosphate.
The plates were incubated for an additional 5 min at the room tem-
perature, and then 34% sodium citrate of 10 mL was added. The
absorbance at 610 nm was determined using Multiskan Spectrum
Microplate Spectrophotometer (Thermo Electron Corporation).
The controls without Eg5 or MTs are the background and should
be subtracted from all values. The controls with MTs but without
Eg5 give the nucleotide hydrolysis by MTs and should be sub-
tracted from corresponding values with Eg5 and the same concen-
tration of MTs. The data were analyzed using Microsoft Excel to
obtain the IC50 of the test compounds.
5.4.2. N-(4-Nitrobenzyl)-4-furan-2-methyl-1,4-dihydro-
benzo[4,5]imidazo[1,2-a]pyrimidine-3-carboxamide (6b)
Yield 32%, yellow powdery crystal, mp 255–256 °C (dec). IR
(cmꢀ1): 3396, 2844, 1626, 1502, 1332, 1249. 1H NMR (300 MHz,
DMSO-d6): d 2.29 (s, 3H), 6.33 (s, 1H), 6.50 (s, 1H), 6.85 (s, 1H),
6.85–8.23 (m, 9H), 10.32 (s, 1H),10.50 (s, 1H). EI-MS: (m/z) 415
(M+). Anal. Calcd for C22H17N5O4ꢁ0.3H2O: C, 62.79; H, 4.21; N,
16.64. Found: C, 62.44; H, 4.16; N, 17.14.
5.4.3. N-(4-Trifluoromethylbenzyl)-4-furan-2-methyl-1,4-dihy
dro-benzo[4,5]imidazo[1,2-a]pyrimidine-3-carboxamide (6c)
Yield 35%, yellow powdery crystal, mp 273–274 °C (dec). IR
(cmꢀ1): 3290, 2850, 2360, 1629, 1522, 1407, 1331. 1H NMR
(300 MHz, DMSO-d6): d 2.28 (s, 3H), 6.33 (s, 1H), 6.50 (s, 1H),
6.83 (s, 1H), 6.99–7.82 (m, 9H), 10.10 (s, 1H), 10.42 (s, 1H). EI-
MS: (m/z) 438 (M+). Anal. Calcd for C23H17F3N4O2: C, 63.01; H,
3.91; N, 12.78. Found: C, 62.61; H, 3.93; N, 12.88.
5.5.3. In vitro Aurora-A kinase activity assays
To determine the inhibition of Aurora-A kinase activity by target
compounds, the HTScanÒ Aurora-A kinase assay kit (Cell Signaling
Technology) was used according to the manufacturer’s instruction.
Briefly, 20 ng purified recombinant human Aurora-A kinase was
5.4.4. N-(3-Chloro-4-fluorobenzyl)-4-furan-2-methyl-1,4-dihydro-
benzo[4,5]imidazo[1,2-a]pyrimidine-3-carboxamide (6d)
Yield 30%, yellow powdery crystal, mp 273–274 °C (dec). IR
(cmꢀ1): 3268, 2845, 1626, 1500, 1460, 1258. 1H NMR (300 MHz,
DMSO-d6): d 2.26 (s, 3H), 6.34 (s, 1H), 6.51 (s, 1H), 6.79 (s, 1H),
added to a 50
200
l
L reaction mixture containing 1ꢂ kinase buffer and
l
mol/L cold ATP in the presence of different concentrations of
test compounds (ranging from 10ꢀ4 to 10ꢀ9 mol/L). After incubation
at room temperature for 15 min, biotinylated peptide substrate (Cell
Signaling Technology) was added to each reaction mixture at a final

R. Fu et al. / Bioorg. Med. Chem. 18 (2010) 8035–8043
8043
concentration of 1.5 lmol/L, and the mixtures were further incu-
References and notes
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Acknowledgments
Acknowledgments are made to the National Natural Science
Foundation of China (Grant Nos. 30801437 and 30772624). Special-
ized Research Fund for the Doctoral Program of Higher Education
(20090096110003), and the National Major Science and Technology
Project of China (Innovation and Development of New Drugs)
(2008ZX09401-001, 2009ZX09501-003) for financial supports.