Discovery of novel inhibitors of Aurora kinases with indazole scaffold: In silico fragment-based and knowledge-based drug design

Article abstract of DOI:10.1016/j.ejmech.2016.08.026

Aurora kinases have emerged as important anticancer targets so that there are several inhibitors have advanced into clinical study. Herein, we identified novel indazole derivatives as potent Aurora kinases inhibitors by utilizing in silico fragment-based approach and knowledge-based drug design. After intensive hit-to-lead optimization, compounds 17 (dual Aurora A and B), 21 (Aurora B selective) and 30 (Aurora A selective) possessed indazole privileged scaffold with different substituents, which provide sub-type kinase selectivity. Computational modeling helps in understanding that the isoform selectivity could be targeted specific residue in the Aurora kinase binding pocket in particular targeting residues Arg220, Thr217 or Glu177.

Full text of DOI:10.1016/j.ejmech.2016.08.026

Accepted Manuscript
Discovery of novel inhibitors of Aurora kinases with indazole scaffold: In silico
fragment-based and knowledge-based drug design
Chun-Feng Chang, Wen-Hsing Lin, Yi-Yu Ke, Yih-Shyan Lin, Wen-Chieh Wang,
Chun-Hwa Chen, Po-Chu Kuo, John T.A. Hsu, Biing-Jiun Uang, Hsing-Pang Hsieh
PII:
S0223-5234(16)30669-9
10.1016/j.ejmech.2016.08.026
EJMECH 8822
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 May 2016
Revised Date: 12 August 2016
Accepted Date: 13 August 2016
Please cite this article as: C.-F. Chang, W.-H. Lin, Y.-Y. Ke, Y.-S. Lin, W.-C. Wang, C.-H. Chen, P.-C.
Kuo, J.T.A. Hsu, B.-J. Uang, H.-P. Hsieh, Discovery of novel inhibitors of Aurora kinases with indazole
scaffold: In silico fragment-based and knowledge-based drug design, European Journal of Medicinal
Chemistry (2016), doi: 10.1016/j.ejmech.2016.08.026.
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ACCEPTED MANUSCRIPT
Graphical abstract
New series of indazole-based derivatives were synthesized and structure-activity
relationship was constructed. Hit compound from sub-structure search, followed by in
silico fragment-based drug design and knowledge-based drug design to discover
compound 17 as a potent novel Aurora kinases inhibitor.

ACCEPTED MANUSCRIPT
Submitted to EJMC as Original Research Article
Discovery of novel inhibitors of Aurora kinases with indazole scaffold: in silico
fragment-based and knowledge-based drug design
a,b
a
a
a
a
Chun-Feng Chang , Wen-Hsing Lin , Yi-Yu Ke , Yih-Shyan Lin , Wen-Chieh Wang ,
a
a
a
b,∗
Chun-Hwa Chen , Po-Chu Kuo , John T. A. Hsu , Biing-Jiun Uang , and Hsing-Pang
a,b,∗
Hsieh
Abstract
Aurora kinases have emerged as important anticancer targets so that there are several
inhibitors have advanced into clinical study. Herein, we identified novel indazole
derivatives as potent Aurora kinases inhibitors by utilizing in silico fragment-based
approach and knowledge-based drug design. After intensive hit-to-lead optimization,
compounds 17 (dual Aurora A and B), 21 (Aurora B selective) and 30 (Aurora A
selective) possessed indazole privileged scaffold with different substituents, which
a
Institute of Biotechnology and Pharmaceutical Research, National Health Research
Institutes, 35 Keyan Road, Zhunan, Miaoli County 35053, Taiwan, ROC
.
b
Department of Chemistry, National Tsing Hua University, No. 101, Section 2,
Kuang-Fu Road, Hsinchu 30013, Taiwan, ROC.
∗
To whom correspondence should be addressed: Phone, +886-37-246-166 ext. 35708;
fax, +886-37-586-456; hphsieh@nhri.org.tw and Phone, +886-3-5715131 ext. 33410;
bjuang@mx.nthu.edu.tw
1

ACCEPTED MANUSCRIPT
provide sub-type kinase selectivity. Computational modeling helps in understanding
that the isoform selectivity could be targeted specific residue in the Aurora kinase
binding pocket in particular targeting residues Arg220, Thr217 or Glu177.
Key words: Aurora kinases inhibitor, Indazole, Ligand efficiency, Isoform selectivity,
Structure-activity relationship (SAR)
2

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1
. Introduction
Protein kinases catalyze the transfer of a phosphate group from adenosine
triphosphate (ATP) to serine, threonine or tyrosine residue of target proteins. They play
a key role in various cellular signaling pathways, including cellular differentiation,
proliferation and migration [1]. Abnormal protein kinases expression is implicated in a
diverse range of serious diseases including diabetes, inflammation and cancers.
Nowadays, more than 500 kinases have been identified of human genome [2].
Imatinib (Gleevec) was the first tyrosine kinase inhibitor approved by the US Food and
Drug Administration (FDA) for the treatment of chronic myeloid leukemia [3], and
kinases have become an attractive target for the development of anti-malignant agents.
To date, 30 more kinase inhibitors have achieved FDA approval and currently a
number of experimental kinase inhibitors are enrolled in clinical trials [4, 5].
Aurora kinases belong to the serine/threonine kinase family, and are critical
regulators of mitosis. Three human paralogues, Aurora A, B, and C, have been
identified, each with a distinct subcellular location and function [6, 7]. Aurora A is
involved in chromosome maturation, bipolar spindle assembly and orientation in the
late S phase and early M phase. Aurora B acts primarily during the M phase and is
required for proper chromosome segregation and cytokinesis [8]. Overexpression of
both Aurora A and Aurora B was found in various human malignancies, e.g. glioma,
breast, ovarian and thyroid cancers [9, 10] . Currently, several Aurora inhibitors, such
3

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as pan-Aurora inhibitor VX-680 [11], PHA-739358 [12] and AT-9283 [13],
Aurora-A-selective MLN-8237 [14] and MK-5108 [15], and Aurora-B-selective
AZD-1152 [16] are in clinical development to treat variety of cancers (Fig. 1).
O
N
N
NH
N
N
N
CH3
HN
N
OMe
H
O
NH
O
HN
N
N
O
NH
N
N
NH
O
N
S
N
N
N
N
H3C
H
H
MK-0457 (VX-680)
Aurora A Ki = 0.7 nM
Aurora B Ki = 18 nM
Aurora C Ki = 4.6 nM
PHA-739358 (Danusertib)
Aurora A IC50 = 13 nM
Aurora B IC50 = 79 nM
Aurora C IC50 = 61 nM
AT-9283
Aurora A IC50 = 3 nM
Aurora B IC50 = 3 nM
F
NH
N
N
Cl
O
OH
OMe
O
OH
COOH
N
COOH
OMe
P
NH
N
NH
N
O
O
S
N
N
N
F
F
H
N
O
N
N
H
Cl
MK-5108 (VX-689)
MLN-8237 (Alisertib)
Aurora A IC50 = 1.2 nM
Aurora B IC50 = 396 nM
AZD-1152 (Barasertib)
Aurora A IC50 = 1368 nM
Aurora B IC50 = 0.37 nM
Aurora A IC50 = 0.046 nM
Aurora B IC50 = 14 nM
Aurora C IC50 = 12 nM
Fig. 1. Chemical structures of the preventative Aurora kinases inhibitors.
Fragment-based drug discovery (FBDD) has been widely adopted by the
pharmaceutical industry over the past decades [17]. This method allows the
identification of small fragments which bind only weakly to the biological targets and
postulates that further high-affinity leads could be generated via the combination of the
small fragments. In contrast to high-throughput screening (HTS), which heavily relies
on the screening of a great quantity of chemical libraries, FBDD is a more efficient
methodology in designing drug-like molecules [18]. Ligand efficiency (LE) is a
4

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measurement of ligand activity normalized by the number of heavy atom (HA), defined
as the ratio of Gibbs free energy (ꢀG) to HA of the compound: ꢀG/ HA (where ꢀG =
-
RTlnKi or –RTlnIC ). LE values can be used to prioritize the lead compounds with
50
favorable ligand efficiency as well as pharmacological properties. However, LE
values were noted to be higher on average for smaller molecules than for the larger
ones [19, 20]. Accordingly, fit quality (FQ) was therefore proposed to transform LE
into metrics that are more reliable within a broader range of molecule sizes. FQ values
are obtained by normalizing the LE with scaled values defined as the ratio of LE to
LE_Scale of the compound: LE/ LE_Scale. FQ score could be a size-independent
parameter to assess suitable fragments, where FQ near to 1 indicated an optimal
ligand binding [21-23].
Herein, we report the discovery of a series of indazole derivatives as novel Aurora
kinase inhibitors using knowledge-based drug design and in silico FBDD. Screening of
our in-house database by sub-structure screening, identified compound 8 which showed
weak inhibition against Aurora A (IC = 13 µM). Compound 9a was further designed
5
0
using in silico techniques, and resulting in 10-fold potency improvement. Finally, two
additional binding regions were explored, which resulted in compound 17, a potent
Aurora A inhibitor (IC = 26 nM) (Scheme 1).
5
0
5

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Scheme 1. Flowchart depicting the process of discovery novel indazole-based Aurora
kinase A inhibitor, 17.
2
. Chemistry
The preparation of hit compound 8 and its derivatives is illustrated in Scheme 2.
Iodination of commercially available indazole 6 with iodine and potassium hydroxide
afforded 3-iodo-indazole 7, which underwent Suzuki coupling with
3
-aminophenylboronic acid to give aniline 8 in a good yield. Amide bond formation
through HOBt-mediated coupling or acylation was carried out with aniline 8 to give the
desired analogues 9a-f as well as 9g (from the hydrolysis of 9d under basic
condition).
6

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Scheme 2. Reaction conditions and reagents : (i) I , KOH, DMF, rt, 4 h; (ii)
2
3
-Aminophenylboronic acid, Pd(dppf)Cl , 2M Na CO3(aq), 1,4-dioxane, microwave
2 2
o
irradiation: 120 C, 30 min; (iii) (a) RCOOH, EDC, HOBt, DIPEA, DMF, rt, 12 h or (b)
cyclic anhydride, pyr, DCM, rt, 12 h ; (iv) 1N NaOH , MeOH, rt, 2 h.
(aq)
The preparation of C-5 substituted indazoles is outlined in Scheme 3. Intermediate
1 was prepared from 5-nitroindazole as described [24]. The amino group of
1
3
-iodo-5-amino indazole 11 was reacted with various electrophiles to provide a series
C-5 substituted 3-iodo-indazoles 12a-j. Palladium catalyzed Suzuki coupling was
performed between 3-aminophenylboronic acid and 3-iodo-indazoles 12a-j to give the
corresponding aniline analogues 13a-j, which were further reacted with maleic
anhydride to afford the final compounds 14-23. Analogues 24-30 of compound 17 were
prepared either by HOBt-mediated amide-bond formation of 13d with the
7

ACCEPTED MANUSCRIPT
corresponding carboxylic acids or monoethyl fumarate following by hydrolysis and
hydrogenation. In similar manner, synthesis of the 6-substituted analogue 33 was
achieved as depicted in Scheme 4.
o
Scheme 3. Reaction conditions and reagents : (i) (a) I , KOH, DMF, 65 C, 1 h; (b) 57
2
o
o
%
HI (aq), 90 C, 3 h; (ii) PhNCO, pyr, DCM, 0 C , 10 min; (iii) PhCOCl, pyr, 10 min;
o
(
iv) PhCHO, EtOH, then NaBH , cat. HOAc, 0 C, 4 h; (v) R -PhSO Cl, pyr, DCM, 0
4
1
2
o
C, 30 min; (vi) 3-Aminophenylboronic acid, Pd(dppf)Cl , 2M Na CO3(aq), 1,4-dioxane,
2
2
o
microwave irradiation: 120 C, 30 min; (vii) maleic anhydride, pyr, DCM, rt, 12 h; (viii)
(
a) R COOH, EDC, HOBt, DIPEA, DMF, rt, 12 h; or (b) anhydride, pyr, DCM, rt, 12 h;
2
(
ix) monoethyl fumarate, EDC, HOBt, DIPEA, DMF, rt, 12 h; then 1N NaOH ,
(aq)
MeOH, rt, 12 h or H (1 atm), Pd/C, MeOH, rt, 12 h.
2
8

ACCEPTED MANUSCRIPT
o
Scheme 4. Reaction conditions and reagents : (i) (a) I , KOH, DMF, 65 C, 1 h; (b) 57
2
o
o
%
HI (aq), 90 C, 3 h; (c) PhSO Cl, py, DCM, 0 C, 30 min; (ii) (a) Pd(dppf)Cl , 2M
2 2
o
Na CO , 3-Aminophenylboronic acid, 1,4-dioxane, microwave irradiation: 120 C,
2
3(aq)
3
0 min; (b) maleic anhydride, pyr, DCM, rt, 12 h.
3
. Results and Discussion
3
.1. Identification of hit compound 8 via sub-structure screening
Indazole is considered to be a privileged scaffold for many kinase inhibitors, as
illustrated in Fig. 2. For example, Axitinib (1), a potent vascular endothelial growth
factor receptor (VEGFR) kinase inhibitor, was first discovered in 2002 and later
approved for the treatment of renal cell carcinoma (RCC) in 2012 [25]. Moreover,
6
-anilinoindazole 2, developed by AstraZeneca, was demonstrated as a selective
inhibitor of c-Jun N-terminal kinase 3 (JNK3) (IC = 3 nM) [26]. Kusakabe et al.
5
0
discovered 5,6-disubstituted indazole 3, which exhibited excellent activity against
monopolar spindle 1 kinase (Mps1) (IC50 = 10.4 nM) [27]. 6-Pyrazolylindazole 4
reported by Pastor et al. showed potent activity against interleukin-2 inducible T cell
kinase (ITK) and was considered as a potential candidate for the treatment of asthma
9

ACCEPTED MANUSCRIPT
[
28]. Recently, compound 5 (CFI-400945), a novel polo-like kinase 4 (PLK4) inhibitor,
was identified as a clinical candidate for cancer therapy [29]. All of the aforementioned
share a common indazole scaffold, which presumably plays a role in forming pivotal
interactions with hinge residues. This finding inspired us to employ the indazole
scaffold in the search of novel Aurora A inhibitors. A total of 17 compounds were
selected from our in-house library and evaluated for Aurora A inhibitory activity (Table
S1, supplemental information). We identified indazole derivative 8 (Scheme. 1) as an
initial hit (IC = 13 µM) for further hit-to-lead optimization.
5
0
Fig. 2. Indazole-based privileged scaffold as kinase inhibitors 1-5.
1
0

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3
.2. Hit to Lead Optimization
Dock studies of compound 8 to Aurora A crystal structure (PDB ID: 2W1C) was
undertaken to investigate the binding orientation of 8 in the binding pocket. After
simple energy minimization, the binding model reveals the interactions between
indazole and hinge residues. The N1-H atom was found to form hydrogen bond with
the carboxyl group of Glu211, whereas the N2 atom is hydrogen bonded to the
backbone of Ala213 (Fig. 3). This model also suggested that additional interactions
between substituents on the amino group of the aniline and binding pocket might be
possible. Accordingly, with in silico FBDD approach (Discovery studio 2.5), a total
2
25 fragments were suggested by with Ludi algorism (Table S2, supplemental
information). The acryloyl fragment with the highest Ludi_3_score was selected to
synthesize. In order to value the relation of functional group and activities, the LE and
FQ calculation was applied to guide the compound optimization. Of these, the
acryloyl analogs 9a and 9b showed the highest LE and FQ scores. As expected, these
two compounds exhibited improved inhibition activity against Aurora A with IC50
values of 1.66 µM and 0.79 µM, respectively, compared to initial hit 8 (IC = 13 µM)
50
(Table 1). The Ludi_3_Score also showed a high correlation with the inhibitory activity
2
of Aurora A with the R = 0.7776. This result had also implied the accuracy of docking
conformation of the hit 8. As shown in Table 1, the negatively charged carboxylic
11

ACCEPTED MANUSCRIPT
group of 9b plays a key role in its activity. Ethyl amide 9c was inactive. E-geometry
analogs 9d, 9e and 9g as well as saturated derivative 9f were very poor Aurora A
inhibitors, suggesting the Z-geometry to be crucial.
Fig. 3. Compound 8 in the Aurora A docking model (PDB ID: 2W1C). Green lines
represent hydrogen bonding interaction with distances indicated. In step I, the
fragment-based drug design procedural was performed to synthesize 9a-d. Finally,
combine the knowledge-based and structure-based approach to optimize the hit 8.
Table 1.
IC , LE and FQ of compounds 8, 9a-g to Aurora A
5
0
1
2

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Aurora A
c
d
e
Compd
R
Ludi_3_Score
-
HA
LE
FQ
IC ( µM )
5
0
8
H
13.56
1.66
16
20
0.41
0.39
0.74
0.84
O
2
2
79
47
9
a
9
b
0.79
2.86
9.61
25
25
26
0.36
0.30
0.26
0.86
0.77
0.69
9
c
215
163
9
d
a
b
9
e
12.9 %
ND
ND
ND
ND
ND
ND
a
9
f
42.8 %
32.3 %
ND
ND
ND
ND
ND
ND
a
9
g
a
b
c
d
e
Percentage inhibition at a compound concentration of 1 µM.
ND : Not determined.
Heavy atom count (HA).
Ligand efficiency (LE) was calculated as –RTlnIC /HAC.
50
Fit quality was calculated as Ref. [22].
Based on the structure of the previously reported, indazole-based kinase inhibitors
(Fig. 2) as well as computer-modeling of compound 8, we hypothesized that addition
of a phenyl ring onto the indazole scaffold might increase the hydrophobic interaction
for potency improvement. Taken into consideration for additional interaction to
binding pocket, we started to introduce various substituents into the C-5 or C-6
1
3

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position of indazole 9b analogues as shown in Table 2. Derivatives bearing phenyl
urea 14, phenyl amide 15 and benzylamine 16 moieties showed equal or slightly better
potency compared to 9b. More importantly, compared to non-substituted 9b, C-5
substituted phenyl sulfonamide 17 displayed a 30-fold improvement in potency against
Aurora A (IC = 26 nM) and a high FQ value (0.99). On the contrary, analog 33
5
0
bearing a phenyl sulfonamide on the C-6 position (33) displayed 10-fold less potency
than 17, reflecting the hydrophobic preference of optimization on the C-5 position in
this indazole series.
Table 2.
SAR investigation of substituents to indazole scaffold.
Aurora A
a
b
c
Compd
R
HA
LE
FQ
IC ( µM )
5
0
9
b
H
0.79
23
34
0.36
0.26
0.86
0.86
1
1
1
4
5
6
5-PhNHCONH
0.26
0.95
0.79
5-PhCONH
32
31
0.26
0.27
0.79
0.82
5-PhCH NH
2
1
4

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1
3
7
3
5-PhSO
2
2
NH
NH
0.026
0.26
33
33
0.31
0.27
0.99
0.87
6-PhSO
a
b
c
Heavy atom count (HA).
Ligand efficiency (LE) was calculated as –RTlnIC /HAC.
Fit quality was calculated as Ref. [22].
50
Compound 17 by was further optimized by the addition of substituents to the
pare-position of the phenylsulfonyl group (Table 3); the methyl (18), methoxy (21), and
nitro (22) analogues were roughly equipotent, suggesting the electronic properties of
the substituent at that position to have little effect on Aurora A inhibition. However,
the phenyl- and t-butyl-modified compounds 19 and 22 were inactive against Aurora A
kinase, suggesting that the back-pocket was not large enough to accommodate bulky
substituents. That the potency of fluoro analog 23 was close to 17 revealed that the
inhibitory activity of substituent at para-position is size-dependent.
A variety of enoic acid analogues were also investigated, E-geometry compound 24,
saturated acid 25 and amide analog 26 exhibited comparable activity to Z-geometry 17;
whereas saturated esters 27-29 showed slightly less activity. Interestingly, the butynoic
amide 30 was roughly as active against Aurora A as 17, suggesting that the
phenylsulfonyl group of 30 dominates the inhibitory activity for this series of
compounds. The anti-proliferative effects of 17-30 on the colon cancer cell line
HCT-116 were also evaluated (Table 3). Although 27, 28, and 30 showed slightly
1
5

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cellular potency, most of the analogues lacked cellular activity, presumably due to the
inherently high polarity and poor cell permeability of these inhibitors.
Table 3.
The IC50 of the compounds 17-30 against Aurora A, Aurora B and cellular
anti-proliferative activities
a
Aurora A
Aurora B
HCT-116
1
2
Compd
R
R
IC ( µM ) IC ( µM )
GI ( µM )
5
0
50
50
17
18
19
20
21
22
23
24
25
26
H
CH
Ph
0.026
0.31
> 1
0.015
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
0.032
3
b
ND
tBu
> 1
ND
OCH
0.48
0.48
0.06
0.051
0.074
0.066
0.031
0.026
0.030
ND
3
NO
2
F
H
H
0.028
0.049
H
1
6

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27
28
29
30
H
H
H
H
0.083
0.208
0.314
0.085
0.048
0.064
0.220
> 1
12
1.9
> 20
3.6
a
b
Carried out by Invitrogen.
ND : Not determined.
3
.3. Kinome profiling of compound 17
The selectivity profile of compound 17 was determined by measuring the inhibition
efficiency against a panel of 24 kinases at 1 µM (Table 4). Significant inhibition (> 80%)
by 17 was only observed for fms-related tyrosine kinase 3 (FLT3) and tyrosine
threonine kinase (TTK) as well as Aurora family (A, B, and C). Accordingly,
compound 17 is said to display a good overall selectivity profile.
Table 4.
a
Kinase Profiling of compound 17
Kinase tested
% inhibition at 1
µM
Kinase tested
% inhibition at 1 µM
ABL1
31
86
89
KDR (VEGFR2)
KIT
48
-4
AURKA (Aurora A)
AURKB (Aurora B)
NTRK1 (TRKA)
PDGFRA
58
AURKC (Aurora C)
94
24
(PDGFR alpha)
1
7

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PDGFRB
21
CDK1/cyclin B
CDK2/cyclin A
CHEK1 (CHK1)
-2
1
(PDGFR beta)
PIK3C2A
17
16
(
PI3K-C2 alpha)
PIK3C2B
20
(PI3K-C2 beta)
CHEK2 (CHK2)
FLT1 (VEGFR1)
FLT3
8
RET
61
10
8
1
ROCK1
ROCK2
TEK (Tie2)
TTK
85
64
39
FLT4 (VEGFR3)
JAK1
48
93
a
Carried out by Invitrogen.
3
.4. Binding Model Study
Molecular docking analysis was applied to investigate the binding information of
1
7 was conducted (Fig. 4). Noteworthy findings were: (1) The indazole core was found
to form hydrogen bonds with the hinge residues (Glu211 and Ala213); (2) In the
back-pocket, Lys141 was observed to form hydrogen bonds with sulfonyl group of 17;
H-bond interaction with Lys141 contributed significantly to inhibit Aurora A may
explain the high potency of 17 in the comparison of bond difference including
sulfonamide, amide, urea and N-alkyl bond; (3) The carboxylic acid of 17 is positioned
within an appropriate distance (3.03 Å and 2.85 Å) to make hydrogen bonds with
1
8

ACCEPTED MANUSCRIPT
Thr217 and Arg 220. These two amino acids are unique to type A kinase among Aurora
family. The sub-type selectivity may be achieved by engaging these interactions.
Fig. 4. Docking analysis for compound 17 with Aurora A (PDB ID: 2W1C). Green
lines represent hydrogen bonding interaction; and red lines represent hydrophobic
interaction between the inhibitor and protein, with the distances indicated.
Inspired by these observations, the Aurora B inhibitory activity of compounds 17-30
have been examined. It was found that compounds 18, 21 and 22 significantly
inhibited Aurora B with 10-fold selectivity between Aurora A and Aurora B. Butynoic
amide 30 was found to be an Aurora-A-selective inhibitor with an IC over 1 µM
5
0
against Aurora B (Table 3). More importantly, the molecular docking studies of 21
1
9

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(
IC of Aurora B > IC of Aurora A) and 30 (IC of Aurora A > IC of Aurora B)
5
0
50
50
50
were conducted to further investigate the isoform selectivity (Fig. 5). It is observed
that the methoxy group of compound 21 possessed a steric repulsion to Asp274
(Aurora A residue) in the back pocket of Aurora A, resulting in less Aurora A activity.
On the other hand, the alkyne group of compound 30 lacked a hydrogen bond
interaction with Glu177 (Aurora B residue), which led to a significant loss in
inhibitory activity of Aurora B. Overall, the sub-type selectivity could be influenced by
the unique residues such as Arg220, Thr217, Asp274 in Aurora A and Glu177 in Aurora
B.
Fig. 5. (A) Docking analysis for compound 21 with Aurora B (PDB ID: 4C2V), the
wire mode is the Asp 274 surface area of Aurora A. (B) Docking analysis for
compound 30 with Aurora A (PDB ID: 2W1C), the wire mode is the Glu177 surface
area of Aurora B. Green lines represent hydrogen bonding interaction and purple lines
represent hydrophobic interaction between the inhibitor and protein.
2
0

ACCEPTED MANUSCRIPT
4
. Conclusion
In summary, a series of indazole-based derivatives were synthesized and SAR
studies conducted, with view to the development of a novel Aurora kinases inhibitor.
The carboxylic acid group extending from the C-3 position of the aniline and
substituted groups at the C-5 position of the indazole core are two newly identified
pharmoacophore to interact with binding region for potency improvement,
respectively. More importantly, we combined knowledge-based substructure search
and in silico fragment-based approach to rapidly identify a novel series of Aurora
kinases inhibitors such as the dual Aurora A and B inhibitor (compound 17), selective
Aurora B inhibitor (compound 21), and selective Aurora A inhibitor (compound 30).
Computational modeling was used to understand how isoform selectivity could be
affected by targeting specific residue in the Aurora kinases binding pocket in particular
targeting residues Arg220, Thr217 or Glu177. Overall, the excellent enzymatic
activity of compound 17 together with the high isoform selectivity of compound 30
validated our SAR findings, and should greatly inform future design of Aurora
kinases inhibitors.
5
. Experimental
5
.1 Chemistry
2
1

ACCEPTED MANUSCRIPT
All reagents were purchased from commercial suppliers and were used without
further purification unless otherwise noted. All reactions were carried out under dry
nitrogen atmosphere and were monitored by TLC using Merck 60 F254 silica gel glass
backed plates (5 cm x 10 cm); zones were detected visually under UV irradiation (254
nm) or by spraying with phosphomolybdic acid reagent (Aldrich) followed by heating
8
0 ℃. Microwave reactions were performed in CEM Discover SP System. Flash
column chromatography using silica gel (Merck Kiesegel 60, no. 9385, 230-400 mesh
ASTM). Proton NMR and carbon NMR spectra were recorded on a Varian
Mercury-300 or Varian Mercury-400 spectrometers, and the chemical shifts were
recorded in parts per million (ppm, δ) and reported relative to TMS or the solvent peak.
Low-resolution mass spectra (LRMS) data were obtained from Agilent MSD-1100
ESI-MS/ MS system and Agilent 1200 series APCI-ESI-MS/ MS system.
5
.2. 3-(1H-indazol-3-yl)aniline (8)
A mixture of 3-iodo-1H-indazole(7) [30] (1 g, 4.1 mmol) and 3-aminophenylboronic
acid (786.0 mg, 5.74 mmol) in dioxane (10.2 mL) was added Pd(dppf)Cl (300 mg,
2
0
.41 mmol) and 2M Na CO (10.2 mL). The mixture was irradiated by microwave at
2 3
1
20 ℃ for 30 minutes. After completion, the reaction mixture was cooled to room
temperature, added with water, and extracted with ethyl acetate. The organic layer was
separated, dried over Na SO , concentrated under vacuum, and purified by silica gel
2
4
2
2

ACCEPTED MANUSCRIPT
column chromatography using a mixture of hexane/ethyl acetate (1:1) to afford 8 (421
1
mg, 49%); H NMR (400 MHz, DMSO-d ) δ 13.09 (s, 1H), 8.02 (d, J = 8.4 Hz, 1H),
6
7
.55 (d, J = 8.4 Hz, 1H), 7.37 (t, J = 7.6 Hz, 1H), 7.24 (s, 1H), 7.17 (t, J = 6.8 Hz, 1H),
13
7
.14-7.10 (m, 2H), 6.58 (dt, J = 6.8, 2.0 Hz, 1H), 5.19 (bs, 2H); C NMR (100 MHz,
CD OD) δ 149.39, 146.78, 143.30, 135.63, 130.69, 127.93, 122.31, 122.28, 121.97,
3
+
1
18.70, 116.53, 115.67, 111.44; LCMS (ESI) m/z: 210.1 [M+H] .
5
.3. General procedure for the syntheses of compounds 9a-f
Method (i): In a glass vial, a mixture of compound 8 (0.34 mmol), carboxylic acid
(0.17 mmol), EDCI (0.26 mmol) and HOBt (0.26 mmol) in anhydrous DMF (4.0 mL)
was stirred under argon atmosphere for 16 h at room temperature. After completion, the
reaction was added with water and extracted with ethyl acetate. The organic layer was
washed with brine, dried over anhydrous Na SO , and concentrated under vacuum. The
2
4
residue was then purified by silica gel column chromatography using the appropriate
mobile phase to give the title compounds 9a or 9c-9f. Method (ii): To a solution of
compound 8 (0.34 mmol) in CH Cl was added pyridine (5 eq) and appropriate cyclic
2
2
anhydride. The resulting mixture was stirred under nitrogen atmosphere for 6 h at room
temperature. After completion of the reaction, the product was precipitated out and
collected by filtration to give the title compounds 9b or 9g.
5
.3.1. N-[3-(1H-Indazol-3-yl)phenyl]-2-propenamide (9a)
2
3

ACCEPTED MANUSCRIPT
Column chromatography was carried out using a mixture of DCM/MeOH (30:1) to
1
give 9a. Yield: 70%; H NMR (400 MHz, CD OD) δ 8.35 (s, 1H), 8.09 (d, J = 8.4 Hz,
3
1
H), 7.71 (d, J = 7.6 Hz, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.48 (t, J = 8.0 Hz, 1H), 7.43
(t, J = 8.0 Hz, 1H), 7.23 (t, J = 6.8 Hz, 1H), 6.52-6.37 (m, 2H), 5.80 (dd, J = 9.0, 2.0
13
Hz, 1H); C NMR (100 MHz, CD OD) δ 166.48, 145.77, 143.41, 140.42, 135.71,
3
1
1
5
32.65, 130.56, 128.14, 128.03, 124.45, 122.62, 122.11, 121.89, 120.83, 120.44,
+
11.59; LCMS (ESI) m/z: 264.1 [M+H] .
.3.2. (Z)-4-((3-(1H-Indazol-3-yl)phe nyl)amino)-4-oxobut-2-enoic acid (9b)
Compound 9b was purified by precipitation from the reaction mixture. Yield: 70%;
1
H NMR (300 MHz, CD OD) δ 8.30 (s, 1H), 8.07 (d, J = 8.1 Hz, 1H), 7.77 (d, J = 7.5
3
Hz, 1H), 7.69 (d, J = 8.1 Hz, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.51 (t, J = 7.8 Hz, 1H),
7
.44 (t, J = 8.1 Hz, 1H), 7.24 (t, J = 7.8 Hz, 1H), 6.63 (d, J = 12.6 Hz, 1H), 6.35 (d, J
13
=
12.6 Hz, 1H); C NMR (100 MHz, CD OD) δ 168.57 166.33, 145.47, 143.45,
3
1
1
5
39.58, 135.83, 134.95, 132.52, 130.66, 128.04, 125.22, 122.66, 122.00, 121.83,
+
21.21, 120.70, 111.66; LCMS (ESI) m/z: 308.1 [M+H] .
1
4
.3.3. N -(3-(1H-Indazol-3-yl)phenyl)-N -ethylmaleamide (9c)
Column chromatography was carried out using a mixture of ethyl acetate/hexane
1
(
2:1) to give 9c. Yield: 55%; H NMR (400 MHz, DMSO-d ) δ 13.25 (s, 1H), 11.40 (s,
6
1
H), 8.37 (s, 1H), 8.08 (d, J = 8.4 Hz, 1H), 7.71 (d, J = 7.6 Hz, 1H), 7.63-7.59 (m,
2
4

ACCEPTED MANUSCRIPT
2
H), 7.48-7.39 (m, 2H), 7.21 (t, J = 7.6 Hz, 1H), 6.29 (d, J = 12.4 Hz, 2H), 3.22-3.17
1
3
(
m, 2H), 1.08 (t, J = 7.2 Hz, 3H); C NMR (100 MHz, CD OD) δ 167.30, 165.40,
3
1
1
[
5
45.56, 143.33, 140.13, 135.60, 134.00, 132.95, 130.49, 127.90, 124.46, 122.51,
22.04, 121.78, 120.73, 120.28, 111.58, 35.60, 14.54; LCMS (ESI) m/z: 335.1
+
M+H] .
.3.4. (E)-Ethyl 4-((3-(1H-indazol-3-yl)phenyl)amino)-4-oxobut-2-enoate (9d)
Column chromatography was carried out using a mixture of ethyl acetate/hexane
1
(
1:1) to give 9d. Yield: 61%; H NMR (300 MHz, CD OD) δ 8.38 (t, J = 1,8 Hz, 1H),
3
8
.08 (dt, J = 8.1, 0.9 Hz, 1H), 7.76-7.67 (m, 2H), 7.57 (d, J = 8.1 Hz, 1H), 7.51 (d, J =
.1 Hz, 1H), 7.46-7.40 (m, 1H), 7.27-7.22 (m, 2H), 6.87 (d, J = 15.3 Hz, 1H), 4.27 (q,
8
1
3
J = 6.9 Hz, 2H), 1.33 (t, J = 6.9 Hz, 3H); C NMR (100 MHz, CD OD) δ 166.96,
3
1
1
[
5
64.24, 145.55, 143.45, 140.14, 138.32, 135.80, 131.84, 130.62, 128.02, 124.82,
22.64, 122.07, 121.86, 120.74, 120.31, 111.65, 62.47, 14.59; LCMS (ESI) m/z: 336.1
+
M+H] .
.3.5. N-(3-(1H-Indazol-3-yl)phenyl)cinnamamide (9e)
Column chromatography was carried out using a mixture of ethyl acetate/hexane
1
(
1:2) to give 9e. Yield: 61%; H NMR (400 MHz, CD OD) δ 8.39 (s, 1H), 8.11 (d, J =
3
8
.4 Hz, 1H), 7.73-7.69 (m, 3H), 7.63 (d, J = 7.2 Hz, 2H), 7.56 (d, J = 8.4 Hz, 1H),
13
7
.52-7.38 (m, 5H), 7.25 (t, J = 7.2 Hz, 1H), 6.85 (d, J = 11.6, 2.4 Hz, 1H); C NMR
2
5

ACCEPTED MANUSCRIPT
(
100 MHz, CD OD) δ 164.04, 143.16, 141.83, 140.69, 139.92, 134.85, 134.43, 130.06,
3
1
1
5
29.64, 129.23 (CHx2), 127.99 (CHx2), 126.41, 122.34, 121.91, 121.36, 120.74,
+
20.25, 118.61, 117.76, 110.93; LCMS (ESI) m/z: 340.1 [M+H] .
.3.6. 4-((3-(1H-Indazol-3-yl)phenyl)amino)-4-oxobutanoic acid (9f)
Compound 9f was purified by precipitation from the reaction mixture. Yield: 61%;
1
H NMR (300 MHz, DMSO-d ) δ 13.23 (s, 1H), 10.14 (s, 1H), 8,32 (s, 1H), 8.07 (d, J
6
=
8.1 Hz, 1H), 7.67-7.58 (m, 3H), 7.44-7.38 (m, 2H), 7.22 (t, J = 8.1 Hz, 1H),
1
3
2
1
1
5
.62-2.53 (m, 4H); C NMR (100 MHz, DMSO-d ) δ 173.89, 170.29, 142.91, 141.62,
6
39.76, 134.13, 129.23, 126.11, 121.17, 121.05, 120.53, 120.02, 117.98, 117.14,
+
10.71, 31.16, 28.82; LCMS (ESI) m/z: 310.1 [M+H] .
.4 (E)-4-((3-(1H-indazol-3-yl)phenyl)amino)-4-oxobut-2-enoic acid (9g)
A solution of compound 9d (50 mg, 0.16 mmol) in MeOH (1.6 ml) was added 1N
NaOH aqueous solution (1.6 ml), and then stirred for 2 h to complete the reaction. The
mixture was concentrated to remove MeOH. The residue was partitioned between ethyl
acetate and water. The water layer was acidified by 2N HCl. The resulting solid was
1
filtered and dried to afford 9g. Yield (40 mg, 82%); H NMR (400 MHz, DMSO-d ) δ
6
1
3.28 (s, 1H), 10.67 (s, 1H), 8.43 (d, J = 1.6 Hz, 1H), 8.09 (d, J = 8.4 Hz, 1H),
.76-7.70 (m, 2H), 7.61 (d, J = 8.4 Hz, 1H), 7.49 (td, J = 8.0, 2.0 Hz, 1H), 7.42 (td, J
7
=
8.0, 1.2 Hz, 1H), 7.24 (d, J = 6.8 Hz, 1H), 7.18 (dd, J = 15.6, 2.0 Hz, 1H), 6.71 (dd,
2
6

ACCEPTED MANUSCRIPT
1
3
J = 15.6, 2.0 Hz, 1H); C NMR (100 MHz, DMSO-d ) δ 166.41, 161.84, 142.71,
6
1
1
5
41.64, 139.11, 137.10, 134.34, 131.00, 129.49, 126.17, 122.26, 121.15, 120.49,
+
20.02, 118.51, 117.63, 110.76; LCMS (ESI) m/z: 308.0 [M+H] .
.5. General procedure for the syntheses of compounds 12a-j
Method (i): To a solution of compound 11 [24] (600 mg, 2.31 mmol) in CH Cl
2
2
(11.55 ml) was added pyridine (0.93 ml, 11.55 mmol). Phenyl isocyanate (0.51 ml, 4.62
mmol) was then added to the mixture under ice-bath, and stirred for 10 minute to
complete the reaction. The reaction was quenched by adding water. The solvent was
removed by vacuum. The resulting mixture was extracted by ethyl acetate. The
combined organic layers were washed with brine, dried over anhydrous Na SO and
2
4
concentrated under vacuum. The residue was then purified by silica gel column
chromatography using the appropriate mobile phase to give the title compounds 12a.
Method (ii): To a solution of compound 11 (300 mg, 1.16 mmol) in pyridine (0.47 ml)
was added benzoyl chloride (0.16 ml, 1.39 mmol) under ice-bath. The mixture was
stirred for 10 minutes, and then added with ethyl acetate and water. The organic layer
was washed with brine, dried over anhydrous Na SO , and concentrated under vacuum.
2
4
The residue was then purified by silica gel column chromatography using the
appropriate mobile phase to give the title compounds 12b. Method (iii): A mixture of
compound 11 (500 mg, 1.93 mmol) and benzaldehyde (0.39 ml, 3.86 mmol) in ethanol
2
7

ACCEPTED MANUSCRIPT
(9.65 ml) was stirred for 10 minutes. Catalytic amount of acetic acid was ejected to the
mixture under ice-bath. A portion of NaBH (146 mg, 3.86 mmol) was slow added to
4
the reaction. After stirred for 4 h, water was added to quench the reaction. The solution
was then removed by vacuum. The resulting mixture was extracted by ethyl acetate.
The combined organic layers were washed with brine, dried over anhydrous Na SO
2
4
and concentrated under vacuum. The residue was then purified by silica gel column
chromatography using the appropriate mobile phase to give the title compounds 12c.
Method (iv): To a stirred solution of compound 11 (500 mg, 1.93 mmol) and pyridine
(
0.78 ml, 2.90 mmol) in CH Cl (9.65 ml) was added dropwise benzenesulfonyl
2 2
chloride under ice-bath for 10 minutes. The mixture was quenched with water, and
concentrated under vacuum. The resulting mixture was extracted by ethyl acetate. The
combined organic layers were washed with brine, dried over anhydrous Na SO and
2
4
concentrated under vacuum. The residue was then purified by silica gel column
chromatography using the appropriate mobile phase to give the title compounds 12d-j.
5
.5.1. 1-(3-Iodo-1H-indazol-5-yl)-3-phenylurea (12a)
Column chromatography was carried out using a mixture of ethyl acetate/hexane
1
(
1:1) to give 12a. Yield: 92%; H NMR (400 MHz, DMSO-d ) δ 13.36 (s, 1H), 8.81 (s,
6
1
H), 8.69 (s, 1H), 7.74 (d, J = 1.2 Hz, 1H), 7.49-7.46 (m, 3H), 7.31-7.26 (m, 3H),
13
6
.96 (td, J = 7.6, 1.2 Hz, 1H); C NMR (100 MHz, DMSO-d ) δ 152.90, 139.77,
6
2
8