4
Y. Zheng et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
Table 1 (continued)
Compd
R1
R2
IC50 (l
M)a
U937
K562
A549
LoVo
HT29
O
N
S
O
N
7m
>100
8.808
0.472
0.950
0.553
0.789
N
Cl
AT-9283
6.700
1.600
0.512
0.383
a
Cellular proliferation was determined by MTT assay.
Table 2
Aurora A/B inhibitory activity of pyrazole–benzimidazole derivatives
The coordinate for the Aurora A (PDB ID: 2W1G) and Aurora B
(PDB ID: 2VGO) structures were obtained from the RCSB Protein
Data Bank. Protein structures were prepared using Glide software
package. In the case of compound 7k, the X-ray crystal structure
showed the ligand sitting deeply in the ATP-binding site of Aurora
A/B (Fig. 4A and B). In Aurora A, the benzimidazole motif binds in
the cleft defined by Ala213, Pro214, Leu215, and Gly216. The li-
gand participates in hydrogen-bonding interactions with NH of
Arg137 and also to the backbone NH and carbonyl of Ala213 of
the protein hinge region (Fig. 4A). In addition, there have no hydro-
gen bond to backbone carbonyl of Glu211 and also to the backbone
carbonyl of Lys162 compared with binding mode of AT-9283 with
Aurora A (Fig. 4C). In Aurora B, the pyrazole ring of compound 7k
makes two hydrogen bonds to NH of Ala173 and also to the back-
bone carbonyl of Glu171 of the protein hinge region (Fig. 4B). As
unexpected, the pyrimidine ring of the compound 7k make two ex-
tra hydrogen bonds to the backbone NH of Lys122 compared with
binding mode of AT-9283 with Aurora B (Fig. 4D). This is in agree-
ment with the Aurora A/B kinase inhibitory activity of compound
7k (IC50 of Aurora A and B is 28.9 and 2.2, respectively).
Compd
IC50(nM)a
Aurora A
Aurora B
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
68.5
71.9
214.9
124.9
91.1
321.3
1134
NTb
86.1
248.8
28.9
45.1
77.9
3.3
91.8
143.9
412.3
191.0
138.6
231.3
NT
1201
98.8
424.1
2.2
7k
7l
7m
AT-9283
2.3
392.3
2.7
a
Typically average of at least two experiments.
Not tested.
b
inhibitory activity (Table 2). It has been observed from Table 2 that
the derivatives such as 7a, 7i, 7k and 7l with small group at 2-po-
sition of pyrimidine ring such as methyl, methylthio and methyl
sulfonyl, have significantly Aurora A/B kinase inhibitory activity
(IC50 ranging from 2.2 to 100 nM). Especially, compound 7k and
7l have similar inhibitory activity (IC50 = 2.2 and 2.3 nM, respec-
As an attempt to gain a better insight into the molecular struc-
tures of compounds under investigation, conformational analysis
has been performed by use of the MM+ force field (calculations
in vacuo, bond dipole option for electrostatics, Polak–Ribiere algo-
rithm, and RMS gradient of 0.01 kcal/Å mol) as implemented in
HyperChem 7.0.21 The most stable conformer was fully optimized
with DFT calculation by Gaussian 03W software22 using the
B3LYP hybrid functional23–26 and standard 6-31G (d) basis set.
The calculation results showed that the lowest energy-minimized
structures of the compounds under investigation exhibited a copla-
nar arrangement of the benzimidazole and pyrazole moiety. Such
arrangement was stabilized by intramolecular hydrogen bond be-
tween the NH group and the adjacent nitrogen atom in imidazole
ring (Fig. 5).
Electronic effects have also been shown to control the pharma-
cological activities of drugs.27 Quantum chemical parameters in
structure–activity relationships (SAR) are reported to yield promis-
ing results for correlation of biological activity.28–30 At the molec-
ular level, the reactivity of a molecule is dominated by the
frontier molecular orbitals (FMO), namely the highest occupied
molecular orbital (HOMO) and the lowest unoccupied molecular
orbital (LUMO). According to the FMO concept,31 the HOMO and
LUMO of a molecule play important roles in intermolecular inter-
actions. Extending the concept to binding in drug–receptor sys-
tems, the major contribution to binding involves the interaction
between the HOMO of the drug with the LUMO of the receptor
and that between LUMO of the drug with the HOMO of the recep-
tor.32 The extents of these stabilizing interactions are inversely re-
lated to the energy gap between the interacting orbitals. Higher
HOMO energy and lower LUMO energy in the drug molecule result
in larger stabilizing interactions and, hence, binding with the
receptor. The orbital energies of both HOMO and LUMO and their
gaps, were calculated for all the molecules and are reported in
tively) on Aurora
B compared with AT-9283(IC50 = 2.7 nM).
Although there is an extra hydrogen bond between the pyrimidine
ring of the compounds and binding site, the compounds 7c, 7g and
7j, which also have pyrimidine ring, have poor Aurora A/B kinase
affinity due to the steric effect. In addition, the compounds that
have morpholine ring at the side chain demonstrated evident Aur-
ora A/B kinase inhibitory activity.
In vitro kinase assays revealed that 7k inhibited the enzyme
activities of Aurora A and B kinase. The cellular activity of 7k
was also tested by immunofluorescence. Histone H3, a direct sub-
strate of Aurora B kinase, is phosphorylated on Serine 10 residue by
Aurora B kinase, which becomes activated in mitosis cells.18 Thus,
detection of histone H3 phosphorylation (pHisH3) on Serine 10 re-
flects the activity of Aurora B kinase in cancer cells. As shown in
Fig. 2, in A549 cells, the decreasing fluorescence indicated that
the activity of Aurora B was inhibited by 7k in a concentration-
dependent manner.
Aurora B is a chromosomal passenger protein that is associated
with the centromeres in the early stages of mitosis, later localizes
to the spindle midzone and the midbody of mitosis cells. It plays a
role in chromosome condensation and cytokinesis.19 Therefore, 7k
may disturb the mitotic progression of human carcinoma cells. The
morphological changes of cell nucleus were examined by using
immunofluorescence staining (Fig. 3). As expected, nuclei of vari-
ous sizes and shapes were also observed in 7k-treated cells. These
phenotypes are thought to be consistent with Aurora B functional
repression.20