Identification of 3,5,6-substituted indolin-2-one's inhibitors of Aurora B by development of a luminescent kinase assay

Article abstract of DOI:10.1016/j.bmcl.2015.05.043

Aurora B kinase plays an important role in the cell normal mitosis and overexpresses in a variety of tumors. Inhibition of Aurora B kinase resulted in an apoptosis of cancer cells, which prevented tumor growth in xenograft models. In this Letter, we developed a luminescent kinase assay to perform high-throughput screening for identification of small molecule Aurora B inhibitors. Two 3,5,6-substituted indolin-2-one derivatives were identified within an in-house compound library. Their new derivatives were then designed and synthesized that resulting two new inhibitors of Aurora B kinase with improved potency. Docking simulation further demonstrated the proposed binding modes between indolin-2-one inhibitor and Aurora B.

Full text of DOI:10.1016/j.bmcl.2015.05.043

Bioorganic & Medicinal Chemistry Letters 25 (2015) 2937–2942
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of 3,5,6-substituted indolin-2-one’s inhibitors of
Aurora B by development of a luminescent kinase assay
Leilei Zhang a, Tianming Yang a, Xilei Xie a, Gang Liu a,b,c,
⇑
a Beijing Key Laboratory of Active Substances Discovery and Druggability Evaluation, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union
Medical College, Beijing 100050, China
b Tsinghua-Peking Center for Life Sciences, China
c Department of Pharmacology and Pharmaceutical Sciences, School of Medicine, Tsinghua University, Haidian Dist., Beijing 100084, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 February 2015
Revised 7 May 2015
Accepted 15 May 2015
Available online 2 June 2015
Aurora B kinase plays an important role in the cell normal mitosis and overexpresses in a variety of
tumors. Inhibition of Aurora B kinase resulted in an apoptosis of cancer cells, which prevented tumor
growth in xenograft models. In this Letter, we developed a luminescent kinase assay to perform high-
throughput screening for identification of small molecule Aurora B inhibitors. Two 3,5,6-substituted
indolin-2-one derivatives were identified within an in-house compound library. Their new derivatives
were then designed and synthesized that resulting two new inhibitors of Aurora B kinase with improved
potency. Docking simulation further demonstrated the proposed binding modes between indolin-2-one
inhibitor and Aurora B.
Keywords:
Aurora B
Small molecule inhibitor
Luminescent kinase assay
Indolin-2-one
Ó 2015 Elsevier Ltd. All rights reserved.
Mitosis is a key step in ensuring the genetic integrity of daugh-
ter cells. Any aberration in this process could lead to genomic
instability and the production of aneuploidy, which has long been
recognized as a frequent characteristic of cancer cells and a possi-
ble cause of tumorigenesis.1 The Aurora kinase family is a collec-
tion of highly related serine/threonine kinases playing an
important role in maintaining normal cell process,2 which was
classified three subsets of Aurora A, B and C in mammals, respec-
tively. Among them, Aurora B, known as the chromosomal passen-
ger protein, is involved in accurate chromosome segregation,
spindle-checkpoint, and cytokinesis.3 Importance of Aurora B in
cell mitosis has driven interest in development of new lead com-
pounds to develop a potential drug.
Overexpression of Aurora B at the mRNA and protein levels was
reported in different types of cancer including breast, colorectal,
kidney, lung and prostate carcinoma.4 A number of small mole-
cules inhibiting Aurora B are currently under intense clinic-phar-
macological studies, such as AZD1152, GSK1070916, AT9283,
PHA-739358, ENMD-2076,5–9 etc. In this study, we developed a
luminescent kinase assay and performed a screening of our
in-house small molecular library. Then, structure optimization
based on active compounds was conducted, and 4 new Aurora B
inhibitors of 3,5,6-substituted indolin-2-one derivatives were
eventually identified.
There are different assay technologies available for identifica-
tion of aurora kinase inhibitors, such as radiolabeled methods,10
ELISA11
(enzyme-linked
immunosorbent
assay),
DELFIA
immunoassays12 and FRET technology,13 etc. Bioluminescent
methods offer several advantages over other methods such as no
isotope contamination, antibody-free and no fluorescent tag.14 In
addition, it is automation friendly with low background and is free
from fluorescent compound interference. In this study, we devel-
oped a luminescent assay to identify small molecule inhibitors of
Aurora B.
Three employed Aurora B peptidic substrates (Table 1)15–17
were synthesized via a standard Fmoc peptide chemistry. All
obtained crude peptides were subsequently purified by a prepara-
tive chromatography and analyzed with a fast liquid chromato-
graphic–mass spectrometry (LC–MS) system, which purities were
greater than 82% (see in the Supplementary data).
The peptidic substrates (100
with Aurora B at 0, 100 and 200 ng/well, respectively, in a 50
final volume containing 25 M ATP. After certain reaction times,
lM) were preliminarily incubated
l
L
l
the phosphorylation of peptides was measured using the Kinase-
Glo reagent, which can determine kinase activity by quantitating
the amount of ATP remaining in solution following a kinase
assay.14 Figure 1 demonstrated that all three peptides showed
strikingly less luminescence, indicating that all three peptides
were able to be phosphorylated in the course of the Aurora B
⇑
Corresponding author. Tel./fax: +86 10 63167165.
E-mail address: gangliu27@yahoo.com (G. Liu).
http://dx.doi.org/10.1016/j.bmcl.2015.05.043
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

2938
L. Zhang et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2937–2942
Table 1
Aurora B peptidic substrates
No.
Sequences
Purity (%)
Calcd (MS, [M+nH]n+
)
Found (MS, [M+nH]n+
)
SB1
SB2
SB3
ARTKQTARKSTGGKAPRKQLAGCG15
LRRLSLG LRRLSLGLRRLSLGLRRLSLG16
LRRASLG17
82
97
99
824.5 (n = 3)
1067.7 (n = 3)
772.5 (n = 1)
824.8 (n = 3)
1068.0 (n = 3)
772.4 (n = 1)
reaction and the ATP was consumed. At the highest concentration
(200 ng/well), Aurora B phosphorylated peptide SB1 was a similar
extent to peptide SB2, but phosphorylated peptide SB3 was to a
lesser extent. SB2 was finally selected to use as the peptide sub-
strate in further screen of inhibitors against Aurora B, because
SB2 was more sensitive to phosphorylation than SB1.
The luminescent kinase assay conditions were optimized with
respects to the amount of ATP, kinase, and peptidic substrate.
Figure 2A illustrated that serial twofold dilutions of ATP resulted
in a linear correlation of luminescence intensity with the amount
of ATP depletion. Thus, 25
of concentrations, and excess peptide SB2 (200
in 50 L reaction final volume to determine the optimal Aurora B
lM ATP, the middle in the linear range
lM) were set up
l
concentration. With the observation in Figure 2B, decreasing RLU
readings of Aurora B in a concentration dependent way led to
determine 300 ng/well of Aurora B as the optimal concentration
for inhibitor screening assay. Additionally, decreased ATP’s amount
was also observed as consumption of SB2 increased in a 50
tions with 25 M ATP and 300 ng/well of Aurora B (Fig. 2C). The
luminescence changed most dramatically when SB2 was at
25 M. Collectively, the optimal conditions were finally set up
lL reac-
l
Figure 1. Peptide substrates were phosphorylated by kinase Aurora B in a dose-
dependent manner.
l
Figure 2. Optimization of luminescent kinase assay. (A) RLU readings linearly correlated with ATP concentrations. (B) Aurora B affects the change of luminescence in a
concentration dependent manner in kinase reactions. (C) ATP depletion increased as SB2 increased. (D) Z0 values for kinase assay, Z0 values higher than 0.5 indicate that the
assay is good.

L. Zhang et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2937–2942
2939
O
O
O
O
N
H
N
N
O
O
F
N
H
HN
N
H
HN
F
N
H
O
N
H
O
13-15 (IC50 = 8.2 uM)
(IC50 = 3.3 uM)
13-39
N
H
N
H
N
N
F
O
O
F
F
O
N
N
H
HN
H
N
H
N
O
H
(IC50 = 2.5 uM)
6h
(IC50 = 1.8 uM)
6i
Figure 3. 3,5,6-Substituted indolin-2-ones that inhibited Aurora B kinase activity.
Table 2
Information of 3,5,6-substituted indolin-2-one derivatives
Entry
R1
R2
R3/R4
% inhibition*
R3
R1
O
R2
N
N
H
H
5
*
O
O
5a
5b
H
NA#
NA
S
*
O
*
O
S
F
O
*
*
*
O
F3C
NH
O
O
O
5c
NA
NA
*
*
*
H
N
*
O
*
5d
*
S
*
NH
F
F
F
N
5e
5f
NA
NA
NA
*
*
*
O
NH
*
N
N
O
O
H
O
*
NH
*
5g
O
O
*
N
*
NH
S
5h
35
*
(continued on next page)

2940
L. Zhang et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2937–2942
Table 2 (continued)
Entry
R1
R2
R3/R4
% inhibition*
O
O
8
R3
R1
O
R2
N
H
N
H
O
*
*
N
N
*
8a
8b
NA
NA
*
NH
O
N
N
*
F
*
R4
O
R1
R2
N
H
N
H
6
O
O
n-C7H15 NH
O
*
*
N
*
N
*
*
*
6a
6b
NA
31
N
H
H
N
O
O
N
S
O
*
N
O
O
6c
*
NA
*
*
O
*
*
*
N
N
O
O
6d
6e
NA
65
O
F
NH
O
F
*
*
O
N
*
6f
NA
NA
*
*
S
*
O
N
*
6g
*
N
O
N
N
*
*
*
6h
6i
83
97
N
*
H
F
F
F
*
NH
N
H
*
O
F
F
N
N
*
*
NH
*
6j
47
S
F
*
O
O
*
*
6k
NA
*
Percentage inhibition was tested under the concentration of 10
NA: percentage inhibition under the tested concentration was less than 30%.
l
M, and the values were the average of duplicate measurements.
#
25
l
M ATP, 300 ng/well of Aurora B and 25
l
M SB2 in a 50
l
L final
program aiming at developing benzofused heterocyclic privileged
structures with drug-like properties.18–29 Thirteen compounds
were greater than 60% inhibition of Aurora B activity under the
volume. Notably, we found that less than 2% DMSO in volume
could confer a negligible influence on activity of Aurora B. Under
the optimal assay condition, the Z0-factor was further analyzed
with running total of 48 positive wells in the presence of Aurora
B and 48 negative wells in absence of Aurora B. Calculations based
on these data provided a Z0 score of 0.77, indicating that the devel-
oped luminescence-based assay was an excellent assay (Fig. 2D).
Three thousand small molecular compounds in our stock com-
pound solution were screened by the developed assay (complete
experimental details see in the Supplementary data). Our com-
pound collection were assembled through a scaffold-directed
concentration of 10
were at 3.3 M (13–39) and 8.2
of Aurora B (Fig. 3).
l
M in the primary screening. Two of them
l
lM (13–15) with 50% inhibition
The two active compounds identified in the preliminary screen-
ing belong to indolin-2-one derivatives which have multiple bio-
logical activities.30–32 Table
2 indicated the new designed
derivatives of 13–39 and 13–15 because we were looking for more
potent inhibitors against Aurora B. Phenoxyl, dipropylamino,
diethylamino, pyrrolidin, piperazin, morpholino were introduced

L. Zhang et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2937–2942
2941
O
O
O
R
3
F
F
R
1
F
a
b
R
1
O
2
O N
2
NO
2
O N
2
NO
2
O N
2
NO
3
2
1
R
4
R
R
3
3
R
1
d
R
R
c
1
1
e
O
O
O
N
N
N
H
H N
2
HN
R
HN
R
H
H
2
2
5a-h
4
6a-k
Or
O
O
O
O
R
3
R
R
3
g
f
1
R
1
O
O
N
H N
N
H
2
HN
H
R
2
7
8a-b
Scheme 1. Synthesis of compounds 5a–h, 6a–k, 8a–b. Reagents and conditions: R1–R4 was defined in Table 2 (a) RR0NH, DIPEA or ROH, K2CO3; (b) RCH(COOC2H5)2, NaF, THF;
(c) Sn, HCl (aq), reflux; (d) (RCO)2O, or RNCO, or RNCS, or RSO2Cl; (e) When R3 = H, R4CHO, pyridine; (f) Pd/C, HCOONH4, rt; (g) (RCO)2O, or RNCO, or RNCS, or RSO2Cl.
in R1 position, respectively. Different substituted amides, sulfon-
amides, ureas and thioureas formed in R2. Meanwhile, R3 included
benzyl, allyl, pyrrol, furan, thiophen, pyridine groups, etc. Above
introduced groups are all common functional groups or pharma-
cophore in drugs. The preparation of target compounds in
Table 2 followed the previously published protocols23 and was
described in Scheme 1. Selective displacement of one of fluorine
atoms of DFDNB by secondary amines produced compound 2.
Substitution of another fluorine atom with the anion of diethyl
malonate gave compound 3. 3 was converted to indolin-2-one
(compound 4) with tin powder and hydrochloric acid under reflux
condition in a high yield, which is the essential intermediate in
synthetic route. Subsequent reaction with anhydride, sulfonyl
chloride, isocyanate, or isothiocyanate of 4 produced compound
5a–h. Further condensation of intermediate 5 (when R3 = H) with
an organic base provided compound 6a–k. In addition, compound
3 (when R3 – H) could be reduced to compound 7 under Pd-
C/HCOONH4, which was subsequently converted to compound
8a–b by anhydride, sulfonyl chloride, isocyanate, or isothiocyanate
(R2).
The luminescent assay described above was used to screen
these 21 compounds at initial concentration of 10
Eventually, 2 new active compounds (6h and 6i) were identified.
Their IC50 values were 2.5 M and 1.8 M, respectively (Fig. 3).
lM.
l
l
Due to limited data, it is difficult to deduce a full scape of SAR
from the results. However, it did reveal some SAR trends. The sub-
stitution at 30 position is very important for inhibition effect on
Aurora B. When R1 and R2 were respectively substituted with mor-
pholino and fluorophenyl-acetamide, hydrogen or butyl with R3
resulted in significant loss of potency (5f, 5g). When R1 and R2
were respectively substituted with diethylamino and acetamide,
it showed little effect on Aurora B when R3 was substituted with
thiophen, pyridine and propane (6f, 6g, 6k). When R1 and R2 were
respectively substituted with pyrrolidin, trifluomethylphenyl acet-
amide, thiophen with R3 led to reduced activity (6j). Therefore,
double bond and aromatic ring or pyrrol ring at 30 position are nec-
essary for inhibition activity.
Figure 4. The interactions between 6h and Aurora B. (A) Docking of the 6h into the
active site of Aurora B. (B) The 2D plot of the interactions between 6h and Aurora B.
carbonyl group of the residue Ala157 or Tyr156 in Aurora B was
able to form a hydrogen bond with amino group of indol in 6h.
The residue Ala157 or Tyr156 was the hydrogen bond receptor or
donor in the hinge region of the kinase. Furthermore, the backbone
amino group of the residue Ala157 was also able to give an addi-
tional hydrogen bond with carbonyl group of 6h. Therefore, good
binding pattern shown in the docking study proved that indolin-
2-ones have great potential to be new human Aurora B inhibitors.
Molecular modeling was further performed by docking com-
pounds 6h into the ATP binding site of Aurora B.33 Figure 4 showed
that 6h can properly occupy ATP binding pocket via docking of 6h
to human Aurora B kinase by using LibDock module of Discovery
Studio 2.5 software (Libdock score 95.9467). The backbone

2942
L. Zhang et al. / Bioorg. Med. Chem. Lett. 25 (2015) 2937–2942
Sarpong, M. A.; Sasaki, K.; Schmidt, S. J.; Silva, D. J.; Sutton, D.; Tang, J.;
In this study, a luminescent kinase assay using Kinase-Glo
Thompson, C. S.; Tummino, P. J.; Wang, J. C.; Xiang, H.; Yang, J.; Dhanak, D. J.
Med. Chem. 2010, 53, 3973.
7. Shah, M.; Gallipoli, P.; Lyons, J.; Holyoake, T.; Jørgensen, H. Blood Cell Mol. Dis.
2012, 48, 199.
8. Cohen, R. B.; Jones, S. F.; Aggarwal, C.; von Mehren, M.; Cheng, J.; Spigel, D. R.;
Greco, F. A.; Mariani, M.; Rocchetti, M.; Ceruti, R.; Comis, S.; Laffranchi, B.; Moll,
J.; Burris, H. A. Clin. Cancer Res. 2009, 15, 6694.
9. How, J.; Yee, K. Expert Opin. Invest. Drug. 2012, 21, 717.
10. Lang, Q.; Zhang, H.; Li, J.; Xie, F.; Zhang, Y.; Wan, B.; Yu, L. Mol. Biol. Rep. 2010,
37, 1577.
11. Fu, D. H.; Jiang, W.; Zheng, J. T.; Zhao, G. Y.; Li, Y.; Yi, H.; Li, Z. R.; Jiang, J. D.;
Yang, K. Q.; Wang, Y.; Si, S. Y. Mol. Cancer Ther. 2008, 7, 2386.
12. Howard, S.; Berdini, V.; Boulstridge, J. A.; Carr, M. G.; Cross, D. M.; Curry, J.;
Devine, L. A.; Early, T. R.; Fazal, L.; Gill, A. L.; Heathcote, M.; Maman, S.;
Matthews, J. E.; McMenamin, R. L.; Navarro, E. F.; O’Brien, M. A.; O’Reilly, M.;
Rees, D. C.; Reule, M.; Tisi, D.; Williams, G.; Vinkovic´, M.; Wyatt, P. G. J. Med.
Chem. 2009, 52, 379.
reagent was developed for high-throughput identification of novel
Aurora B inhibitors. In the first round of screening, a total of 3000
compounds were screened and 2 active 3,5,6-substituted indolin-
2-ones were identified. Furthermore, twenty-one 3,5,6-substituted
indolin-2-one derivatives were designed and synthesized that gave
2 new active compounds with improved inhibitory potency of
2.5 lM (6h) and 1.8 lM (6i), respectively. Docking study mimicked
the binding pattern of 3,5,6-substituted indolin-2-one with Aurora
B indicating that two hydrogen bonds occurred between the resi-
due Ala157 or Tyr156 in Aurora B and amino group of indol in
6h with great potential to be new human Aurora B inhibitors.
Acknowledgments
13. Xie, F.; Zhu, H.; Zhang, H.; Lang, Q.; Tang, L.; Huang, Q.; Yu, L. Eur. J. Med. Chem.
2015, 89, 310.
We thank Dr. Yu Long and Li Jie at the Fudan University for
kindly providing recombinant Aurora B as well as Dr. Yang Ying
at the Institute of Materia Medica, Chinese Academy of Medical
Sciences and Peking Union Medical College for great help in dock-
ing studies.
14. www.promega.com.
15. Aurora B Kinase Assay. www.perkinelmer.com.
16. Bain, J.; Plater, L.; Elliott, M.; Shpiro, N.; Hastie, C. J.; McLauchlan, H.; Klevernic,
I.; Arthur, J. S.; Alessi, D. R.; Cohen, P. Biochem. J. 2007, 408, 297.
17. Shiao, H. Y.; Coumar, M. S.; Chang, C. W.; Ke, Y. Y.; Chi, Y. H.; Chu, C. Y.; Sun, H.
Y.; Chen, C. H.; Lin, W. H.; Fung, K. S.; Kuo, P. C.; Huang, C. T.; Chang, K. Y.; Lu, C.
T.; Hsu, J. T.; Chen, C. T.; Jiaang, W. T.; Chao, Y. S.; Hsieh, H. P. J. Med. Chem.
2013, 56, 5247.
Supplementary data
18. Zhao, H. Y.; Liu, G. J. Comb. Chem. 2007, 9, 1164.
19. Li, L.; Wang, Z. G.; Chen, Y. Y.; Yuan, Y. Y.; Liu, G. J. Comb. Chem. 2007, 9, 959.
20. Zhao, H. Y.; Liu, G. J. Comb. Chem. 2007, 9, 756.
21. Wang, Z. G.; Yuan, Y. Y.; Chen, Y. Y.; Sun, G. C.; Wu, X. H.; Zhang, S. M.; Han, C.
Y.; Wang, G. X.; Li, L.; Liu, G. J. Comb. Chem. 2007, 9, 652.
22. Yuan, Y. Y.; Liu, G.; Li, L.; Wang, Z. G.; Wang, L. J. Comb. Chem. 2007, 9, 158.
23. Yang, T. M.; Liu, G. J. Comb. Chem. 2007, 9, 86.
24. Liu, G.; Li, L.; Kou, B. B.; Zhang, S. D.; Zhang, L.; Yuan, Y. Y.; Ma, T.; Shang, Y.; Li,
Y. C. J. Comb. Chem. 2007, 9, 70.
Supplementary data (all target compounds were characterized
by 1H NMR and mass spectra analyses. Supplementary information
on general peptide substrate synthesis, mass spectrometric data of
peptide substrates and luminescent assay protocols) associated
with this article can be found, in the online version, at http://
dx.doi.org/10.1016/j.bmcl.2015.05.043.
25. Kou, B. B.; Zhang, F.; Yang, T. M.; Liu, G. J. Comb. Chem. 2006, 8, 841.
26. Zhang, J.; Zhang, L.; Zhang, S. D.; Liu, G. J. Comb. Chem. 2005, 7, 657.
27. Li, L.; Liu, G. J. Comb. Chem. 2004, 6, 811.
References and notes
28. Zhang, L.; Liu, G. J. Comb. Chem. 2004, 6, 431.
29. Wu, X. H.; Liu, G. Mol. Divers. 2004, 8, 165.
1. Weaver, B. A.; Cleveland, D. W. Cancer Cell 2005, 8, 7.
30. Eastwood, P.; González, J.; Gómez, E.; Vidal, B.; Caturla, F.; Roca, R.; Balagué, C.;
Orellana, A.; Domínguez, M. Bioorg. Med. Chem. Lett. 2011, 21, 4130.
31. Schrader, T.; Sage, C.; Gao, Y.; Edwards, J.; Barden, J.; Thatte, J.; Fu, L.; Solomon,
M.; Liu, L.; Al-Shamma, H.; Gatlin, J.; Le, M.; Xing, C.; Espinola, S.; Jones, R. M.
Bioorg. Med. Chem. Lett. 2011, 21, 6013.
32. Yue, X.; Varga, E. V.; Stropova, D.; Vanderah, T. W.; Yamamura, H. I.; Roeske, W.
R. Eur. J. Pharmacol. 2006, 540, 57.
33. Elkins, J. M.; Santaguida, S.; Musacchio, A.; Knapp, S. J. Med. Chem. 2012, 55,
7841.
2. Carvajal, R. D.; Tse, A.; Schwartz, G. K. Clin. Cancer Res. 2006, 12, 6869.
3. Murata-Hori, M.; Tatsuka, M.; Wang, Y. L. Mol. Biol. Cell 2002, 13, 1099.
4. Marumoto, T.; Zhang, D.; Saya, H. Nat. Rev. Cancer 2005, 5, 42.
5. Mori, N.; Ishikawa, C.; Senba, M.; Kimura, M.; Okano, Y. Biochem. Pharmacol.
2011, 81, 1106.
6. Adams, N. D.; Adams, J. L.; Burgess, J. L.; Chaudhari, A. M.; Copeland, R. A.;
Donatelli, C. A.; Drewry, D. H.; Fisher, K. E.; Hamajima, T.; Hardwicke, M. A.;
Huffman, W. F.; Koretke-Brown, K. K.; Lai, Z. V.; McDonald, O. B.; Nakamura, H.;
Newlander, K. A.; Oleykowski, C. A.; Parrish, C. A.; Patrick, D. R.; Plant, R.;