Discovery of novel Bcr-AblT315I inhibitors with flexible linker. Part 1: Confirmation optimization of phenyl-1H-indazol-3-amine as hinge binding moiety

Article abstract of DOI:10.1016/j.ejmech.2019.05.091

As a continuation to our research, a series of novel Bcr-Abl inhibitors incorporated with 6-phenyl-1H-indazol-3-amine as hinge binding moiety (HBM) were developed based on confirmation analysis. Biological results indicated that these compounds exhibited an enhanced inhibition against Bcr-Abl^WT and Bcr-Abl^T315I in kinases assays, along with improved anti-proliferative activities in K562 cell assays. In particular, compound Y9 displayed comparable potency with that of imatinib. It potently inhibited Bcr-Abl^WT and Bcr-Abl^T315I kinases with IC₅₀ of 0.043 μM and 0.17 μM, respectively. Furthermore, compound Y9 inhibited the proliferation of K562 and K562R cells with IC₅₀ of 1.65 μM and 5.42 μM, respectively. Therefore, 6-phenyl-1H-indazol-3amine as HBM, combined with flexible linker, is a successful strategy contribute to research on T315I mutant resistance, and compound Y9 could be served as a starting point for further optimization.

Full text of DOI:10.1016/j.ejmech.2019.05.091

Accepted Manuscript
T315I
Discovery of novel Bcr-Abl
inhibitors with flexible linker. Part 1: Confirmation
optimization of phenyl-1H-indazol-3-amine as hinge binding moiety
Xiaoyan Pan, Liyuan Liang, Ying Sun, Ru Si, Qingqing Zhang, Jin Wang, Jia Fu,
Junjie Zhang, Jie Zhang
PII:
S0223-5234(19)30515-X
DOI:
https://doi.org/10.1016/j.ejmech.2019.05.091
EJMECH 11401
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 March 2019
Revised Date: 28 May 2019
Accepted Date: 31 May 2019
Please cite this article as: X. Pan, L. Liang, Y. Sun, R. Si, Q. Zhang, J. Wang, J. Fu, J. Zhang, J. Zhang,
T315I
Discovery of novel Bcr-Abl
inhibitors with flexible linker. Part 1: Confirmation optimization of
phenyl-1H-indazol-3-amine as hinge binding moiety, European Journal of Medicinal Chemistry (2019),
doi: https://doi.org/10.1016/j.ejmech.2019.05.091.
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ACCEPTED MANUSCRIPT
Discovery of novel Bcr-AblT315I Inhibitors with flexible Linker. Part 1:
Confirmation Optimization of Phenyl-1H-indazol-3-amine as Hinge Binding
Moiety
Xiaoyan Pana, Liyuan Lianga, Ying Suna, Ru Sia, Qingqing Zhanga, Jin Wanga, Jia Fua, Junjie
Zhangb, Jie Zhanga
aSchool of Pharmacy, Health Science Center, Xi'an Jiaotong University, Xi’an, 710061, China
bSchool of Science, Xi’an Jiaotong University, Xi’an, 710049, China
As a continuation to our previous research, a series of novel Bcr-AblT315I inhibitors
with flexible linker were developed.
Corresponding author. Tel/Fax: +86-29-82655451; E-mail: zhj8623@mail.xjtu.edu.cn (Jie Zhang).

ACCEPTED MANUSCRIPT
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Discovery of novel Bcr-AblT315I Inhibitors with flexible Linker. Part 1: Confirmation Optimization of
Phenyl-1H-indazol-3-amine as Hinge Binding Moiety
Xiaoyan Pana, Liyuan Lianga, Ying Suna, Ru Sia, Qingqing Zhanga, Jin Wanga, Jia Fua, Junjie Zhangb, Jie Zhanga,
aSchool of Pharmacy, Health Science Center, Xi'an Jiaotong University, Xi’an, 710061, China
bSchool of Science, Xi’an Jiaotong University, Xi’an, 710049, China
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
As a continuation to our research, a series of novel Bcr-Abl inhibitors incorporated
with 6-phenyl-1H-indazol-3-amine as hinge binding moiety (HBM) were
developed based on confirmation analysis. Biological results indicated that these
compounds exhibited an enhanced inhibition against Bcr-AblWT and Bcr-AblT315I
in kinases assays, along with improved anti-proliferative activities in K562 cell
assays. In particular, compound Y9 displayed comparable potency with that of
imatinib. It potently inhibited Bcr-AblWT and Bcr-AblT315I kinases with IC50 of
0.043 µM and 0.17 µM, respectively. Furthermore, compound Y9 inhibited the
proliferation of K562 and K562R cells with IC50 of 1.65 µM and 5.42 µM,
respectively. Therefore, 6-phenyl-1H-indazol-3amine as HBM, combined with
flexible linker, is a successful strategy contribute to research on T315I mutant
resistance, and compound Y9 could be served as a starting point for further
optimization.
Available online
Keywords:
CML
T315I mutant
Flexible linker
Hinge binding moiety
Phenyl-1H-indazol-3-amine
2009 Elsevier Ltd. All rights reserved.
———
Corresponding author. Tel/Fax: +86-29-82655451; E-mail: zhj8623@mail.xjtu.edu.cn (Jie Zhang).
1

[4].
1. Introduction
ACCEPTED MANUSCRIPT
Chronic myeloid leukemia (CML), characterized by the
clonal expansion of cells carrying the Philadelphia
chromosome, is a hematopoietic stem cell disease that
accounts for 15-20% of all leukemia diagnosed in adults
[1]. The t(9; 22) Philadelphia chromosome translation
fuses the BCR gene to the c-ABL proto-oncogene resulting
in a chimeric Bcr-Abl protein with constitutively activated
kinase activity [2]. By recruiting the adaptor proteins such
as Grb2, Bcr-Abl can activate several signaling pathways,
including MAPK, PI3K-Akt, and STAT5, leading to the
uncontrolled cell proliferation and CML pathogenesis [3].
Thus, Bcr-Abl represents potential therapeutic target for
the development of small molecular inhibitors. The first
approved Bcr-Abl inhibitor is imatinib which significantly
improves clinical responses and overall survival in CML
Despite the great success in clinical use of imatinib, around
15% patients can develop resistance or tolerance to this
drug due to point mutations, BCR-ABL gene
amplification, and overexpression of efflux transporter [5].
The second generation Bcr-Abl inhibitors, including
nilotinib, dasatinib and bosutinib, were then developed
(Figure 1) [6-8]. They could inhibit various imatinib-
resistance Bcr-Abl mutants except T315I mutation which
accounts for approximately 20% acquired resistance cases
[9]. Pantinib, the third generation inhibitor, has recently
achieved accelerated approval for the treatment of T315I
resistant CML, but side effects have limited its clinical
application [10, 11]. Therefore, developing novel Bcr-Abl
inhibitors on the T315I mutant is one of the top priorities
in CML research.
Figure 1. Development of Bcr-Abl inhibitors and multi-targeted tyrosine inhibitor linifanib.
As is known, the inhibitors reported above bind to the ATP
site of Bcr-Abl to prevent substrates phosphorylated, and
they belong to the type II inhibitors which target the DFG-
out confirmation of tyrosine kinases [12]. According to the
analysis of the interaction between those inhibitors and
Bcr-Abl kinase, the pharmacophore features of type II Bcr-
Abl inhibitors can be summarized as three parts, the hinge
binding moiety (HBM), the linker, the selective site (DFG-
out pocket) binding moiety (SBM) (Figure 2) [13]. Among
these three pharmacophores, HBM occupies the adenine
pocket and reacts with hinge region through hydrogen
bonds, which is quite fatal for inhibitors’ affinity. The
linker, reacting with DFG motif and surrounded by
gatekeeper residue, is very important for overcoming
T315I steric hindrance. Therefore, our efforts were focused
on the modification of the HBM and linker to design novel
Bcr-AblT315I inhibitors.
In our previous work, based on six-atom regulation,
diacylethylenediamine
and
diacylpiperazine
were
introduced as flexible linker, affording potent Bcr-Abl
inhibitors [13]. To improve the potency against Bcr-
AblT315I, 1H-indazol-3-amine, one fragment of Linifanib,
conjugated with phenyl ring, was incorporated as new
hinge binding moiety. Linfanib (Figure 1) is a multi-
targeted ATP-competitive tyrosine kinase inhibitor, and
1H-indazol-3-amine has received much attention as a novel
2

fragment to interact with the hinge region of tyrosine
that hypothesis, the phenyl ring should be far away from
indazole, making the angle smaller and more adjustable.
Therefore, in order to enhance the potency, 6-phenyl-1H-
indazol-3-amine was introduced as new HBM, adapting
small spatial confirmation to enter into the ATP pocket
more easily. Furthermore, we explore the effect of terminal
phenyl ring with various para-substituents especially
halogen substituents, as SBM, on inhibitors’ activity.
In summary, Bcr-AblT315I mutation is still a major
challenge for CML treatment. Herein, we described the
design, synthesis and biological evaluation of a novel class
of Bcr-Abl inhibitors bearing 6-phenyl-1H-indazol-3-
amine as HBM and para-substituted phenyl ring as SBM.
Moreover, N,N'-diacylpiperazine or N,N'-diacylethylene-
diamine was still applied as flexible linker to reduce steric
ACCEPTED MANUSCRIPT
kinase [14]. Consequently, 1H-indazol-3-amine conjugated
with phenyl ring was validated as HBM of Bcr-Abl
inhibitor (Figure 2), which enhanced the potency against
Bcr-AblT315I for the inhibitors with diacylethylenediamine
or diacylpiperazine as flexible linker [15].
As a continuation to our previous research, we investigate
the effect of phenyl-1H-indazol-3-amine’s confirmation on
the affinity with Bcr-Abl. As shown in Figure 2, the
phenyl ring is on 4-position of 1H-indazol-3-amine, and
the angle between them is almost 90 degree, making this
HBM in large spatial conformation. Since the adenine
pocket of Bcr-Abl prone to be a little narrow, the
inhibitors, with 4-phenyl-1H-indazol-3-amine as HBM,
may enter into the ATP pocket with high energy barrier,
which may affect the activity towards Bcr-Abl. Based on
clash of Bcr-AblT315I
.
Figure 2. Design strategy and structure of novel Bcr-Abl inhibitors as anti-leukemia agents.
intermediate
4
was obtained from 4-bromo-2-
2. Chemistry
The general synthetic procedure for the target compounds
was outlined in Scheme 1. There were two types of key
intermediates employed to afford the title compounds. One
was halogenated indazolamide (3 and 4). Another was
diaryl ethylenediamine (2a-2e) or diaryl piperazine (6a-
fluorobenzonitrile reacting with hydrazine monohydrate in
refluxing 1-butanol [17]. For the synthesis of intermediates
(2a-2e) and (6a-6e), monoacylation of symmetric amines
was used as the key step. In our previous work, benzoic
acid was activated by CDI under solvent-free condition,
followed by reaction with ethylenediamine or piperazine to
generated monoacylated products [18]. This method was
good for benzoic acid with low melting point. When it
comes to benzoic acid with high melting point, especially
6e). The key intermediate
3 was prepared from
commercially available 2-fluoro-6-iodobenzonitrile by
using ten equiv of hydrazine monohydrate in refluxing
ethanol in the presence of NaHCO3 [16]. While
3

presence of triethylamine. The anhydrides were further
benzoic acid with substituents at para posiAtioCn,CthEisPmTeEthDod MANUSCRIPT
can’t work very well or even had no product. Therefore, in
order to expand the benzoic acid diversity and simplify the
experiment operation when doing monoacylation of
symmetric amines, new synthesis methods were developed
with more stable and universal reaction condition. In terms
of monoacylation of ethylenediamine, CDI was continued
to be used as activating reagents, but various reaction
conditions were explored with different solvents such as
THF, DCM, ACN and so on. Consequently, acetonitrile
was the best reaction solvents with high yield of product
(1a-1e) and minimal bisadduct [19]. For piperazine
monoacylation, trimethylacetyl chloride was used as
treated with piperazine in ethanol to provide monoacylated
products (5a-5e) [20]. Next, compounds (1a-1e) or (5a-5e)
reacted with 4-boronobenzoic acid, using PyBOP as
condensation reagents and DMF as solvents, to get the key
intermediates (2a-2e) and (6a-6e). Finally, aminoindazole
3 or 4 was coupled with diaryl ethylenediamine (2a-2e) or
diaryl piperazine (6a-6e), using classical Pd-catalyzed
Suzuki coupling reaction [21], to afford the title
compounds Y1-Y5, Y6-Y10, Y11-Y15 and Y16-Y20. All
1
the title compounds were characterized by H NMR, 13C
NMR, mass spectroscopy, melting point, MS, and HRMS
data. Detailed synthetic procedures are described in the
Experimental Section.
activating
reagent,
to
generate
trimethylacetic
arylcarboxylic anhydride from various benzoic acids in the
Scheme 1. Synthesis route of the title compounds (Y1-Y20). Reagents and conditions: a. CDI, CH3CN, r.t.; b. PyBOP, TEA, DMF; c.
NH2NH2•H2O, NaHCO3, EtOH, reflux; d. NH2NH2•H2O, 1-butanol, reflux; e. Et3N, CH2Cl2, EtOH, r.t.; f. Pd(PPh3)4, Cs2CO3, CH3CN/H2O
(V:V= 3:2), 90ºC.
In vitro kinase inhibition and antiproliferative potency of
all the title compounds were depicted in Table 1.
3. Results and discussion
All the title compounds were evaluated for their enzymatic
inhibition against both Bcr-AblWT and Bcr-AblT315I as well
as K562 cellular activities. The tyrosine kinase inhibitory
potency was assayed by using the well-established ADP-
Glo assays [22]. The antiproliferative potency was
identified in vitro against Bcr-Abl positive K562 cells by
using MTT method with imatinib as positive control [23].
According to our previous research, with 4-phenyl-1H-
indazol-3-amine as HBM, the o- or p- substituents on the
terminal phenyl ring was favorable for compounds’
activity. Taken steric hindrance into consideration, p-
substituents were further diversified and investigated (Y1-
Y5 and Y11-Y15). As shown in Table 1, most of the title
4

values confirmed an improved potency of most compounds
compounds with 4-phenyl-1H-indazol-3-AamCinCe EaPs THEBDM MANUSCRIPT
could retain comparable potency against Bcr-AblWT and
Bcr-AblT315I when alkyl and halogen substituents were
introduced to p- position on terminal phenyl ring.
Specially, compounds Y3 and Y12 displayed good potency
against both WT and T315I Bcr-Abl kinases. For Bcr-
AblWT inhibitory assays, compounds Y4 and Y15 were the
most potent in each series with IC50 values of 0.11 and
4.28 µM, respectively. When it comes to Bcr-AblT315I
assays, most compounds exhibited moderate to good
activity. In particular, compounds Y3, Y12 and Y14 had
good inhibition potency with IC50 values under micromolar
level. Overall, biological evaluation indicated that, with
1H-indazol-3-amine as hinge binding fragment, the
activities of this chemotypes was durable to the
diversification of p-substituents on terminal phenyl ring.
Importantly, the further modification was focus on the
confirmation of phenyl-1H-indazol-3-amine as HBM, and
the phenyl ring was changed from 4-position to 6-position
in indazole, with p-substituents invariant (Y6-Y10 and
Y16-Y20). As depicted in Table 1, the observed IC50
with respect to both Bcr-AblWT and Bcr-AblT315I. In
particular, compound Y9 displayed comparable potency
with that of imatinib. With ethylenediamine as linker,
compounds Y6-Y10 had better inhibition potency than
other three series against Bcr-AblWT, with IC50 value from
0.043 to 3.45 µM. Besides, compound Y9 and Y10
displayed excellent activity against Bcr-AblT315I
,
comparable to that of imatinib. For compounds Y16-Y20,
with piperazine as linker, all compounds had IC50 values
under micromolar level in kinase inhibitory assays, except
for compound Y18 toward Bcr-Abl and compound Y17
against T315I mutant. Consequently, in comparison to Y1-
Y5 and Y11-Y15, these title compounds with 6-phenyl-
1H-indazol-3-amine as HBM exhibited 10- to 100- fold
increase in potency against Bcr-AblWT and Bcr-AblT315I
kinases. In addition, halogen substituent 4-Cl-3-CF3 was
the most beneficial group for enzymatic inhibition against
WT and T315I Bcr-Abl kinases.
Table 1 Structures and biological activities of title compounds (IC50, µM).
AblT315I
IC50(µM)
Compd
Linker
Het
R
AblWT IC50(µM)
K562 IC50(µM)
Y1
Y2
4-F
1.35
38.53
94.84
9.49
4-Br
31.61
>150
Y3
Y4
Y5
4-CH3
0.52
0.11
5.75
5.17
5.93
4-Cl-3-CF3
4-N(CH3)2
20.00
76.76
17.08
>150
Y6
Y7
4-F
2.45
2.16
0.38
0.043
3.45
61.20
2.68
4.30
0.17
0.29
13.42
14.04
1.45
4-Br
Y8
4-CH3
Y9
4-Cl-3-CF3
4-N(CH3)2
1.65
Y10
7.52
5

ACCEPTED MANUSCRIPT
Y11
Y12
Y13
Y14
Y15
Y16
Y17
Y18
4-F
17.56
20.86
1.50
101.83
9.25
4-Br
7.12
4-CH3
5.85
178.66
2.08
>150
4.90
4-Cl-3-CF3
4-N(CH3)2
4-F
10.66
4.28
86.06
8.60
>150
39.11
11.30
59.3
2.34
4-Br
6.28
31.90
3.72
4-CH3
32.25
1.99
Y19
Y20
4-Cl-3-CF3
4-N(CH3)2
5.33
4.84
2.54
6.23
61.16
4.26
imatinib
0.054
0.28
Furthermore, we investigated the kinase selectivity of the
most potent compound (Y9) against other three kinases
including Src, Hck and p38α for its selective profile. The
results were summarized in Figure 3. The results revealed
that compound Y9 showed less potency against Hck, while
it exhibited some inhibitory activity against p38α and Src
kinases with IC50 value of 34.5 µM and 15.1 µM,
respectively. It was demonstrated that this inhibitor
exhibited a good selectivity for Bcr-Abl relative to other
three kinases. In addition, because of the inhibiton potency
of compound Y9 against p38α, especially Src kinases, we
speculated that it may cause some inconsistency of Bcr-
Abl inhibition and CML cells growth inhibition in the
following anti-proliferation assays.
Figure 3. Kinases selectivity profile of compound Y9 (IC50, µM).
Next, we investigated the growth inhibition of these title
compounds against Bcr-Abl positive K562 cells, and the
results were also presented in Table 1. The majority of
them displayed moderate to high antiproliferative
activities. Specially, compounds Y8 and Y9 were more
potent than imatinib with IC50 values of 1.45 and 1.65 µM,
respectively. Meanwhile, the activities of compounds Y14
and Y19 were comparable to that of imatinib, with IC50
values of 4.90 µM and 4.84 µM. In accordance with the
kinase inhibitory results against Bcr-Abl, compounds with
phenyl ring on 6-position of indazole exhibited better
antiproliferative activities than that of those corresponding
compounds with phenyl ring on 4-position. Interestingly, it
was found that compounds bearing N(CH3)2 substituent
(Y5, Y15 and Y20) displayed little inhibitory activities
toward K562 cells, although they had good potency against
Bcr-Abl kinases. We presumed that the N(CH3)2 group had
been protonated in buffer, resulting in poor membrane
permeability for these compounds in cell assays.
Consequently, their poor permeability lead to little effect
6

on K562 cells proliferation. Above all, phenyl ring on 6-
antiproliferative activities than 4-position.
ACCEPTED MANUSCRIPT
position of indazole was more favorable for
Figure 4. Anti-proliferative activity of
compound Y9 against K562R cells (A)
expressing T315I mutation, and cytotoxicity
of compound Y9 toward two normal human
cells, including Hek293 cells (B) and
EA.hy926 cells (C).
Having demonstrated the potent inhibition of Abl kinases
and anti-proliferative activity, compound Y9 was further
evaluated for its activity toward K562R cells expressing
T315I mutation [24], with Imatinib as positive control. The
results was displayed in Figure 4A, and compound Y9 was
found to potently inhibit the growth of K562R cells with
IC50 value of 5.42 µM, while Imatinib has an IC50 value of
90.00 µM toward K562R cells. In addition, we identified
the cytotoxicity of compound Y9 on two normal cells,
including Hek293 (human embryonic kidney 293 cells)
and EA.hy 926 (human vascular endothelial cells), with
Imatinib as control. As shown in Figure 4B and 4C,
compound Y9 exhibited less toxicity toward these two cell
lines when its concentration increased up to 150 µM. In
summary, compound Y9 displayed selectivity growth
inhibitory activity against Bcr-Abl positive K562 and
K562R cells compared to normal human cell lines.
findings suggested that the influence of compound Y9 on
cell viability of K562 might be attributed to the down
regulation of Bcr-Abl phosphorylation. In addition,
although compound Y9 exhibit potent inhibitory activity
against K562 cells compared with imatinib, it just lead a
modest decrement of Bcr-Abl phosphorylated level in this
assay. This finding suggested that compound Y9 might
take effect through other signal pathway besides targeting
Bcr-Abl, which need to be further explored in our
undergoing work.
Figure 5. The effect of compound Y9 on the phosphorylated level of
We further evaluated the effect of compound Y9 on the
expression level and phosphorylation of Bcr-Abl in K562
cells using western blot assay. The results was shown in
Figure 5. It was found that compound Y9 does-
dependently decreased the phosphorylation of Bcr-Abl in
K562 cells compared with the negative control group,
while the expression level of Bcr-Abl was unchanged. Our
Bcr-Abl in K562 cells.
To gain a better understanding of the interaction between
compound Y9 and kinases protein, molecular docking
studies was conducted using Sybyl-X (Version 2.0, Tripos
Inc. St. Louis, MO) [25]. Crystal structures of Bcr-AblWT
(PDB IP: 1IEP) and Bcr-AblT315I (PDB IP: 3QRJ) [26, 27],
7

obtained from Protein Data Bank, were applied to do the
depicted in Figure 6.
ACCEPTED MANUSCRIPT
docking as target protein. The docking results were
Figure 6. The binding mode of compound Y9 with Bcr-AblWT (A) and Bcr-AblT315I (B), imatinib (A, cyan) and panitinib (B, cyan) were used
as control molecule for Bcr-AblWT and Bcr-AblT315I docking, respectively.
As shown in Figure 6A, compound Y9 bound across the
ATP-binding site of Bcr-AblWT through five hydrogen
bonds. The N-H2 of 1H-indazol-3-amine, as hydrogen
bond donor, formed three hydrogen bonds with hinge
region residues. One N-H formed two hydrogen bonds
with Thr319 and Met318 with length of 2.70Å and 1.95Å,
respectively. The other N-H formed one hydrogen bond
with Thr319 with distance of 2.74Å. The conversed
Asp381 of DGF motif generated one hydrogen bond with
the terminal carbonyl oxygen in linker part with the length
of 2.09Å. Furthermore, Glu286 of α-helix, as receptor,
formed one hydrogen bond with the N-H of terminal amide
group, and the distance was 1.76Å. As for Bcr-AblT315I
binding, the results was displayed in Figure 6B. The two
hydrogen atom of amine in indazole interacted with
Thr319 to generate two hydrogen bonds, and the bond
length was 2.27Å and 2.36Å, respectively. Other two
hydrogen bonds were same as that of its binding to wild
type. One hydrogen bond was generated between the
carbonyl group of Glu286 and the N-H of terminal amide
in linker part, and its distance was 1.63Å. The other
hydrogen bond was formed between the N-H in backbone
of Asp381 and the carbonyl of terminal amide group with
bond length of 2.07Å. Based on above docking analysis, it
was found that the binding mode of compound Y9 with
Abl kinases were similar as imatinib binding to Bcr-AblWT
and panitinib to Bcr-AblT315I, respectively. The terminal
amide group could generated hydrogen bonds with
conserved Glu286 and Asp381. Moreover, the indazole
formed hydrogen bond network with hinge region residues.
That may explain the potent activities of compound Y9
toward Abl kinases.
For compounds with piperazine as linker, we also did in
silico docking to rationalize its lower potency against Abl
kinases compared with those compounds with ethylene-
diamine as linker. The most potent compound Y19 was
used to do this study. For Bcr-AblWT docking, the results
was depicted in Figure 7A, and there were two hydrogen
bonds between them. The hydrogen atom of amine in
indazole ring formed one hydrogen bond with Thr319, and
the other one with Met318, for the bond length of 2.72Å
and 1.92Å, respectively. For Bcr-AblT315I, the binding
interactions of compound Y19 was shown in Figure 7B,
with two hydrogen bonds as follows: 1) the first forming
between N atom of indazole ring and NH2 group of
Met318, the distance was 1.78Å, 2) the second forming
between N-H inside indazole ring and carbonyl group in
backbone of Glu316 with length of 2.18Å. Overall, the
binding mode of compound Y19 were similar as imatinib
with Bcr-AblWT and panitinb with Bcr-AblT315I
,
respectively. However, compared with Y9 docking results,
there was no hydrogen bonds between terminal amide of
8

compound Y19 and DFG motif or conserved Glu286,
as linker had lower kinase activities.
ACCEPTED MANUSCRIPT
which may explain why these compounds with piperazine
Figure 7. The binding mode of compound Y19 with Bcr-AblWT (A) and Bcr-AblT315I (B), imatinib (A, cyan) and panitinib (B, cyan) were used
as control molecule for Bcr-AblWT and Bcr-AblT315I docking, respectively.
Furthermore, in order to investigate the effect of phenyl
position on the binding of 1H-indazol-3-amine with hinge
region, compounds Y4 and Y9 were performed docking
using Surflex-dock module of Sybyl-X 2.0, with Bcr-
AblWT as target protein. As shown in Figure 8A, both
compounds applied similar mode to bind with Bcr-Abl
kinase, and 1H-indazol-3-amine in both core structures
generated hydrogen bonds with hinge region residues.
However, the pyrazoles’ direction differed between
compounds Y4 and Y9 under the effect of phenyl ring
position. We speculated that this may lead to different
binding situations from perspective of energy crash. To
visualize the energy situation between molecules and
kinase protein, Glide docking module of Schrödinger was
applied to do the analysis [28]. As depicted in Figure 8B,
4-phenyl-1H-indazol-3-amine in compound Y4 had a
certain degree of steric crashes with surrounding residues
including Leu248, Tyr253 and Met318. As for compound
Y9, from Figure 8C, 6-phenyl-1H-indazol-3-amine have
little crash with Leu248, even no crash with Tyr253
especially Met318 in hinge region. Through the
comparison between two energy situations of compounds
Y4 and Y9 in the hinge region, 6-phenyl-1H-indazol-3-
amine was more favorable structure as HBM for these
chemotype inhibitors with flexible linker.
9

ACCEPTED MANUSCRIPT
Figure 8. The docking results of compound Y4 and Y9 with Bcr-Abl (A), and the visualized energy situation of compounds Y4 (B) and Y9 (C)
with surrounding residues. The orange dash bonds represent bad crash, and the red dash bonds correspond to ugly crash.
of Bcr-AblWT and Bcr-AblT315I with five or four hydrogen
4. Conclusion
Herein, we described the continuous optimization of
previously disclosed Bcr-Abl inhibitors bearing 4-phenyl-
1H-indazol-3-amine as HBM. The effort was focus on
confirmation optimization of phenyl-1H-indazol-3-amine
interacting with the hinge region. The strategy was to
transfer phenyl ring from 4-position to 6-position of
indazole, far away from pyrazole, resulting in the structure
more adjustable to generate favorable binding mode with
lower energy barrier. Finally, a series of compounds with
6-phenyl-1H-indazol-3-amine as HBM, and corresponding
compounds with previous reported indazol-3-amine
structure used as comparison, were synthesized and
evaluated. The biological results indicated that most
designed compounds had an enhanced effect in kinase and
cell assays, especially that compound Y9 displayed potent
bonds, respectively. Furthermore, visualized energy
situations analysis between phenyl-1H-indazol-3-amines
and hinge region residues indicated that phenyl ring at 6-
position led to lower crash with surroundings, more
favorable for binding in ATP pocket. Besides, as described
previously, diacylpiperazine especially diacylenediamine
reduce the steric clash with gatekeeper residues of Bcr-
AblT315I
.
Therefore, 6-phenyl-1H-indazol-3-amine as
HBM, combined with flexible linker, is a successful
strategy contribute to the research on T315I mutant
resistance. Finally, compound Y9, displaying promising
enzymatic inhibition as well as antiproliferative potency,
could be considered as valuable starting point for further
effort.
5. Experimental section
inhibition against both Bcr-AblWT and Bcr-AblT315I
,
5.1. Chemistry: General procedure
comparable to that of imatinib. Meanwhile, it exhibited the
excellent antiproliferative activity toward Bcr-Abl positive
K562 and K562R cells, better than imatinib. Moreover,
using molecular modeling, it was rationalized that
modification of phenyl-1H-indazol-3-amine confirmation
could improve the affinity of Bcr-Abl inhibitors with hinge
region. In particular, compound Y9 fit well to ATP pocket
Reagents and solvents were purified according to the
standard procedure. The reactions except those in aqueous
were performed by standard techniques for the exclusion of
moisture. Reactions were monitored by thin layer
chromatography (TLC) on 0.25-mm silica gel plates
(GF254) and visualized with UV light. Melting points were
determined on electrothermal melting point apparatus and
10

1
were uncorrected. H NMR spectra was measured at 400
boronic acid (3a)
ACCEPTED MANUSCRIPT
MHz on a Bruker Advance AC 400 instrument with TMS
4-carboxyphenylboronic acid (0.996 g, 6 mmol), PyBOP
(3.43 g, 6.6 mmol) were dissolved in 10 mL anhydrous
DMF, and anhydrous triethylamine (1.8 mL, 12 mmol) was
added dropwise to the solution. After stirring at room
temperature for 20 min, N-(2-aminoethyl)-4-fluorobenz-
amide (2a) was added and the reaction mixture was stirred
overnight at room temperature. The mixture was dissolved
in 100 mL H2O and then extracted with EtOAc (50 mL×3).
The combined organic layer was washed with water (20
mL×3) and brine (30 mL), dried over Na2SO4 for
overnight, filtered, and concentrated in vacuo. The crude
product was purified by flash chromatography with
PE/EtOAc (v/v = 1:5) as eluent to afford (3a) as white
solid (1.1 g, 55%). Mp 326 327ºC, EI -MS (m/z): 329.00
[M-H]-.
as an internal standard. Mass spectra was measured using
gas or liquid chromatography mass spectrometry (GC/LC-
MS) with electrospray ionization (ESI). HRMS was
measured using AXIMA-CFRTM MALDI-TOF-MS or
micrOTOF-Q . All compounds were purified by flash
chromatography with silica gel.
5.1.1. 4-iodo-1H-indazol-3-amine (1)
2-fluoro-6-iodobenzonitrile (5.0 g, 10.1 mmol), NaHCO3
(2.6 g, 31 mmol) and hydrazine monohydrate (5.0 g, 99.9
mmol) were dissolved in ethanol 60 mL. The resulting
mixture was heated to reflux for 8 h. After cooling to room
temperature, 50 mL water was added and the reaction
mixture was allowed to stir for another 2 h at room
temperature. The product was collected by filtration and
dried under vacuum to give (1) as slight yellow solid (4.8
g, 92%).
The intermediate compounds 3b-3e were prepared using
the same procedure as described above.
5.1.2. N-(2-aminoethyl)-4-fluorobenzamide (2a)
5.1.4. (4-((2-(4-bromobenzamido)ethyl)carbamoyl)phenyl)
boronic acid (3b)
4-fluorobenzoic acid (15 mmol), carbonyldiimidazole
(2.92 g, 18 mmol) were dissolved in 60 mL anhydrous
ACN, and was stirred for 1 h at room temperature. In a
separate dry 50 mL round bottom flask, fitted with an
addition funnel, was placed 10 mL (150 mmol) of
ethylenediamine. The activated 4-fluorobenzoic acid was
transferred to the addition funnel and added dropwise to
the ethylenediamine. The reaction was stirred at room
temperature for 3h. Then CH3CN was evaporated, and 40
mL HCl solution (6 mol/L) was added to the residue. The
mixture was extracted with CH2Cl2 (10 mL) to remove
diacylated product. The aqueous layer was adjust to PH
8~9 with 10 g NaOH, and extracted with CH2Cl2 (30
mL×3). Then the organic solution was combined, dried
over Na2SO4 overnight, concentrated by rotary evaporation
to afford the crude product which can be directly used in
the next step. EI-MS (m/z) 183[M+H]+.
N-(2-aminoethyl)-4-bromobenzamide (2b) was used as
starting material to obtain 3b (2.0 g, 86%). Mp
318 320ºC, EI-MS (m/z): 389.05 [M-H]-.
5.1.5. (4-((2-(4-methylbenzamido)ethyl)carbamoyl)phenyl)
boronic acid (3c)
N-(2-aminoethyl)-4-methylbenzamide (2c) was used as
starting material to obtain 3c (1.69 g, 87%). Mp
330 331ºC, EI-MS (m/z): 325.05 [M-H]-.
5.1.6. (4-((2-(4-chloro-3-(trifluoromethyl)benzamido)ethyl)
carbamoyl)phenyl)boronic acid (3d)
N-(2-aminoethyl)-4-chloro-3-(trifluoromethyl)benzamide
(2d) was used as starting material to obtain 3d (2.18 g,
88%). Mp 125 127ºC, EI-MS (m/z): 415.05 [M+H]+.
5.1.7. (4-((2-(4-(dimethylamino)benzamido)ethyl)carbamoyl)
phenyl)boronic acid (3e)
N-(2-aminoethyl)-4-(dimethylamino)benzamide (2e) was
used as starting material to obtain 3e (1.48 g, 69%). Mp
180 182ºC, EI-MS (m/z): 356.05 [M+H]+, 354.10 [M-H]-.
5.1.8. 4-(3-amino-1H-indazol-4-yl)-N-(2-(4-fluorobenz-amido)
ethyl)benzamide (Y1)
The intermediates 2b-2e were prepared by ethylenediamine
and commercially benzoic acids with different substituents
using the same procedure as described above.
5.1.3. (4-((2-(4-fluorobenzamido)ethyl)carbamoyl)phenyl)
11

Yield 0.43 g, 69%. Mp 232 234ºC, EI-MS (m/z): 414.15
In a 100 mL round bottom flask with an aAcoCnCdeEnsPerTtEubDe, MANUSCRIPT
4-iodo-1H-indazol-3-amine (1) (0.39 g, 1.5 mmol), (4-((2-
(4-fluorobenzamido)ethyl)carbamoyl)phenyl)boronic acid
(3a) (1.8 mmol), Cs2CO3 (1.46 g, 4.5 mmol), Pd(PPh3)4
(0.09 g, 0.075 mmol) was dissolved in 50 mL ACN/H2O
(v/v = 3: 2). Then the reaction mixture was degassed for 3
times, heated at 90 ºC in an oil bath and stirred under
nitrogen for 24 h. The mixture was cooled to room
temperature, filtered, and evaporated to remove ACN. The
residue was diluted with 30 mL H2O and then extracted
with ethyl acetate (30 mL×3). The combined organic layer
was washed with brine, dried over Na2SO4 for overnight,
filtered, and concentrated in vacuo to give the crude
product, which was isolated by flash chromatography on
silica gel (EtOAc) to obtain the title compound (0.12 g,
19%). Mp 291 292ºC, EI-MS (m/z): 418.15 [M+H]+,
416.10 [M-H]-. 1H NMR (400 MHz, DMSO-d6) δ 11.83 (s,
1H), 7.99 (d, J = 8.2 Hz, 2H), 7.96 - 7.92 (m, 2H), 7.58 (d,
J = 8.2 Hz, 2H), 7.35 - 7.28 (m, 4H), 6.85 (dd, J = 5.4, 2.4
Hz, 1H), 4.32 (s, 2H), 3.47 (s, 4H). 13C NMR (101 MHz,
DMSO-d6) δ 166.71, 165.92, 163.05, 148.44, 142.49,
142.39, 135.20, 133.97, 131.48, 130.36, 130.27, 129.25,
127.79, 126.70, 119.85, 115.76, 115.54, 110.69, 109.86.
The compounds Y2-Y5 were prepared using the same
procedure as described above, with 4-iodo-1H-indazol-3-
amine (1) (1.5 mmol) and various boronic acid (3b-3e) (1.8
mmol) as starting materials.
[M+H]+, 412.15 [M-H]-. 1H NMR (400 MHz, DMSO-d6) δ
11.83 (s, 1H), 7.99 (d, J = 8.1 Hz, 2H), 7.77 (d, J = 8.0 Hz,
2H), 7.57 (d, J = 8.0 Hz, 2H), 7.30 (dd, J = 17.4, 6.6 Hz,
4H), 6.85 (dd, J = 5.2, 2.1 Hz, 1H), 4.32 (s, 2H), 3.47 (s,
4H), 2.36 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ
166.89, 166.70, 148.41, 142.50, 142.38, 141.44, 135.20,
133.96, 132.19, 129.25, 127.79, 127.70, 126.71, 119.86,
110.69, 109.86, 21.42.
5.1.11. N-(2-(4-(3-amino-1H-indazol-4-yl)benzamido) ethyl)-
4-chloro-3(trifluoromethyl)benzamide (Y4)
Yield 0.61 g, 81%. Mp 284 286ºC, EI-MS (m/z): 502.10
[M+H]+, 500.10 [M-H]-. 1H NMR (400 MHz, DMSO-d6) δ
11.83 (s, 1H), 8.31 (s, 1H), 8.16 (d, J = 8.4 Hz, 1H), 7.98
(d, J = 8.3 Hz, 2H), 7.89 (d, J = 8.4 Hz, 1H), 7.57 (d, J =
8.3 Hz, 2H), 7.36 - 7.28 (m, 2H), 6.85 (dd, J = 5.4, 2.4 Hz,
1H), 4.32 (s, 2H), 3.49 (s, 4H). 13C NMR (101 MHz,
DMSO-d6) δ 166.73, 164.68, 148.43, 142.49, 142.42,
135.19, 134.32, 133.94, 133.40, 132.37, 129.24, 127.79,
126.88, 126.71, 124.51, 121.79, 119.85, 110.69, 109.87,
22.98.
5.1.12. 4-(3-amino-1H-indazol-4-yl)-N-(2-(4-(dimethylamino)
benzamido)ethyl)benzamide (Y5)
Yield 0.18 g, 27%. Mp 279 281ºC, EI-MS (m/z): 443.15
[M+H]+, 441.10 [M-H]-. HRMS (ESI): calcd for [M+H]+
1
C25H27N6O2: 443.2195, found 443.0052. H NMR (400
MHz, DMSO-d6) δ 11.84 (s, 1H), 7.99 (d, J = 8.0 Hz, 2H),
7.74 (d, J = 8.8 Hz, 3H), 7.57 (d, J = 8.1 Hz, 2H), 7.32 (d,
J = 5.2 Hz, 2H), 6.88 - 6.83 (m, 1H), 6.71 (d, J = 8.8 Hz,
2H), 4.33 (s, 2H), 3.44 (s, 4H), 2.96 (s, 6H).
5.1.9. 4-(3-amino-1H-indazol-4-yl)-N-(2-(4-bromobenz-amido)
ethyl)benzamide (Y2)
Yield 0.20 g, 28%. Mp 300~302ºC; EI-MS (m/z)
1
476.05[M-H]-. H NMR (400 MHz, DMSO-d6) δ 11.83 (s,
5.1.13. 6-bromo-1H-indazol-3-amine (4)
1H), 7.98 (d, J = 8.2 Hz, 2H), 7.81 (d, J = 8.5 Hz, 2H),
7.70 (d, J = 8.5 Hz, 2H), 7.57 (d, J = 8.2 Hz, 2H), 7.35 -
7.27 (m, 2H), 6.85 (dd, J = 5.4, 2.4 Hz, 1H), 4.32 (s, 2H),
3.47 (s, 4H). 13C NMR (101 MHz, DMSO-d6) δ 166.71,
166.05, 148.42, 142.50, 142.39, 135.21, 134.13, 133.97,
131.77, 129.84, 129.25, 127.80, 126.71, 125.34, 119.85,
110.69, 109.86.
4-bromo-2-fluorobenzonitrile (5.0 g, 25.1 mmol) was
dissolved in 1-butanol (20 mL), then followed by the
addition of hydrazine monohydrate (1.0 mL, 50.3 mmol).
The reaction mixture was heated to reflux for 4 h. Then
cooled to room temperture, filtered, washed with n-hexane
and dried to give 4 as white solid (4.77 g, 85%).
The compounds Y6-Y10 were prepared using the same
5.1.10. 4-(3-amino-1H-indazol-4-yl)-N-(2-(4-methylbenzamido) procedure as compound Y1, with 6-bromo-1H-indazol-3-
ethyl)benzamide (Y3) amine (4) (1.5 mmol) and various boronic acids (3a-3e)
12

(1.8 mmol) as starting materials.
5.43 (s, 2H), 3.44 (s, 4H), 2.97 (s, 6H).
ACCEPTED MANUSCRIPT
5.1.14. 4-(3-amino-1H-indazol-6-yl)-N-(2-(4-fluorobenzamido) 5.1.19. (4-fluorophenyl)(piperazin-1-yl)methanone (5a)
ethyl)benzamide (Y6)
Trimethylacetyl chloride (2.42 mL, 20 mmol) and Et3N
(4.60 mL, 30 mmol) were added to a mixture of 4-
fluorobenzoic acid (20 mmol) in dry CH2Cl2 (80 mL). The
resulting mixture was stirred at room temperature for 0.5-1
h until a clear solution was observed. A solution of
piperazine (3.44 g, 40 mmol) in EtOH (80 mL) was added,
and the mixture was further stirred for 3 h. Then the
reaction solution was evaporated to remove solvents. The
residue was dissolved in 20 mL H2O, and concd HCl (4
mL) was added. The resulting mixture was extracted with
CH2Cl2 (5 mL) to remove bisadduct. NaOH (8 g) was
added to the aqueous solution and then extracted with
CH2Cl2 (30 mL × 3). The organic extract was further
washed with H2O (30 mL) and then dried over Na2SO4.
Evaporation of the solvent at reduced pressure yielded the
residue which was further used in next step. EI-MS (m/z)
208[M]+.
Yield 0.24 g, 38%. Mp 277 279ºC, HRMS (ESI): calcd
for [M+H]+ C23H21FN5O2: 418.1679, found 418.1669. H
1
NMR (400 MHz, DMSO-d6) δ 11.52 (s, 1H), 8.68 (d, J =
3.6 Hz, 2H), 7.93 - 7.97 (m, 4H), 7.83 - 7.75 (m, 2H), 7.50
(s, 1H), 7.38 - 7.20 (m, 3H), 5.43 (s, 2H), 3.47 (s, 4H).
5.1.15. 4-(3-amino-1H-indazol-6-yl)-N-(2-(4-bromobenzamido)
ethyl)benzamide (Y7)
Yield 0.20 g, 29%. Mp 239 241ºC, HRMS (ESI): calcd
for [M-H]- C23H19BrN5O2: 476.0722, found 476.9787. 1H
NMR (400 MHz, DMSO-d6) δ 9.96 (s, 1H), 8.69 (s, 1H),
8.36 (s, 1H), 7.81 (s, 1H), 7.78 (s, 1H), 7.73 (s, 1H), 7.70
(d, J = 2.8 Hz, 2H), 7.68 (s, 1H), 6.80 (s, 1H), 6.78 (s, 1H),
3.42 - 3.39 (m, 4H).
5.1.16. 4-(3-amino-1H-indazol-6-yl)-N-(2-(4-methylbenzamido)
ethyl)benzamide (Y8)
Yield 0.14 g, 22%. Mp 172 174ºC, HRMS (ESI): calcd
1
for [M+H]+ C24H24N5O2: 414.1930, found 414.1925. H
The intermediates 5b-5e were prepared by commercially
benzoic acids bearing different substituents and piperazine
using the same method as described above.
NMR (400 MHz, DMSO-d6) δ 8.54 (d, J = 5.5 Hz, 1H),
8.22 (s, 1H), 7.83 (dd, J = 21.8, 8.1 Hz, 2H), 7.71 - 7.76
(m, 4H), 7.27 (d, J = 8.0 Hz, 2H), 6.79 (d, J = 8.7 Hz, 1H),
3.40 - 3.44 (m, 4H), 2.35 (s, 3H).
The intermediates 6a-6e was obtained using the same
method as described for the preparation of compound 3a,
with 4-carboxyphenylboronic acid (6 mmol) and
corresponding monoacylated piperazine 5a-5e (7.2 mmol)
as starting materials.
5.1.17. N-(2-(4-(3-amino-1H-indazol-6-yl)benzamido) ethyl)-
4-chloro-3(trifluoromethyl)benzamide (Y9)
Yield 0.29 g, 38%. Mp 256 258ºC, HRMS (ESI): calcd
for [M+H]+ C24H20ClF3N5O2: 502.1258, found 502.1285.
1H NMR (400 MHz, DMSO-d6) δ 11.52 (s, 1H), 8.99 (s,
1H), 8.70 (s, 1H), 8.31 (s, 1H), 8.16 (d, J = 7.3 Hz, 1H),
7.95 (d, J = 7.0 Hz, 2H), 7.89 (d, J = 7.8 Hz, 1H), 7.80 (d,
J = 6.2 Hz, 3H), 7.50 (s, 1H), 7.27 (d, J = 8.2 Hz, 1H), 5.43
(s, 2H), 3.49 (s, 4H).
5.1.20. (4-(4-(4-fluorobenzoyl)piperazine-1-carbonyl)phenyl)
boronic acid (6a)
(4-fluorophenyl)(piperazin-1-yl)methanone (5a) as starting
materials. Yield 1.28 g, 60%. Mp 224 226ºC, EI-MS
(m/z): 357.15 [M+H]+, 355.10 [M-H]-.
5.1.21. (4-(4-(4-bromobenzoyl)piperazine-1-carbonyl)phenyl)
boronic acid (6b)
5.1.18. 4-(3-amino-1H-indazol-6-yl)-N-(2-(4-(dimethylamino)
benzamido)ethyl)benzamide (Y10)
(4-bromophenyl)(piperazin-1-yl)methanone
(5b)
as
Yield 0.26 g, 40%. Mp 271 273ºC, HRMS (ESI): calcd
starting materials. Yield 2.04 g, 82%. Mp 220 222ºC, EI-
MS (m/z): 415.05 [M-H]-。
1
for [M+H]+ C25H27N6O2: 443.2195, found 443.2189. H
NMR (400 MHz, DMSO-d6) δ 7.96 (d, J = 8.4 Hz, 2H),
7.78 - 7.81 (m, 3H), 7.74 (d, J = 9.0 Hz, 2H), 7.50 (s, 1H),
7.27 (dd, J = 8.4, 1.4 Hz, 1H), 6.71 (d, J = 9.0 Hz, 2H),
5.1.22. (4-(4-(4-methylbenzoyl)piperazine-1-carbonyl)phenyl)
boronic acid (6c)
piperazin-1-yl(p-tolyl)methanone (5c) as starting materials.
13

3.51 (m, 8H), 2.35 (s, 3H). 13C NMR (101 MHz, DMSO-
Yield 2.0 g, 80%. Mp 209 211ºC, EI-MAS C(mC/zE):P3T5E1.D15 MANUSCRIPT
[M-H]-.
d6) δ 171.94, 169.84, 169.53, 148.38, 142.54, 140.86,
139.88, 135.24, 135.06, 133.13, 129.40, 127.66, 126.72,
119.89, 110.65, 109.79, 28.47, 22.98, 21.38.
5.1.23. (4-(4-(4-chloro-3-(trifluoromethyl)benzoyl) piperazine-1-
carbonyl)phenyl)boronic acid (6d)
(4-chloro-3-(trifluoromethyl)phenyl)(piperazin-1-yl) meth-
aneone (5d) as starting materials. Yield 2.19 g, 83%. Mp
156 158ºC, EI-MS (m/z): 441.10 [M+H]+.
5.1.28. (4-(4-(3-amino-1H-indazol-4-yl)benzoyl)piperazin-
1-yl)(4-chloro-3-(trifluoromethyl)phenyl)Methanone (Y14)
Yield 0.30 g, 38%. Mp 253 255ºC, EI-MS (m/z): 528.10
[M+H]+, 526.15 [M-H]-. 1H NMR (400 MHz, DMSO-d6) δ
11.83 (s, 1H), 7.92 (s, 1H), 7.85 - 7.77 (m, 2H), 7.56 (s,
4H), 7.32 (s, 1H), 7.31 (d, J = 2.2 Hz, 1H), 6.85 (s, 1H),
4.35 (s, 2H), 3.90 - 3.39 (m, 8H). 13C NMR (101 MHz,
DMSO-d6) δ 169.57, 167.28, 148.45, 142.54, 140.97,
135.71, 135.19, 135.01, 133.17, 132.38, 129.41, 127.67,
127.17, 126.72, 124.38, 121.67, 119.89, 110.65, 109.80,
60.30, 28.47, 21.24, 14.55, 12.93.
5.1.24. (4-(4-(4-(dimethylamino)benzoyl)piperazine-1-carbonyl)
phenyl)boronic acid (6e)
(4-(dimethylamino)phenyl)(piperazin-1-yl)methanone (5e)
as starting materials. Yield 1.48 g, 65%. Mp 208 210ºC,
EI-MS (m/z): 382.10 [M+H]+, 380.05 [M-H]-.
The compounds Y11-Y15 were prepared using the same
procedure as compound Y1, with 4-iodo-1H-indazol-3-
amine (1) (1.5 mmol) and various boronic acid (6a-6e)
(1.8 mmol) as starting materials.
5.1.29. (4-(4-(3-amino-1H-indazol-4-yl)benzoyl)piperazin-
1-yl)(4-(dimethylamino)phenyl)methanone (Y15)
5.1.25. (4-(4-(3-amino-1H-indazol-4-yl)benzoyl)piperazin-
1-yl)(4-fluorophenyl)methanone (Y11)
Yield 0.20 g, 28%. Mp 282 284ºC, EI-MS (m/z): 469.29
[M+H]+, 467.15 [M-H]-. HRMS (ESI): calcd for [M+H]+
Yield 0.31 g, 47%. Mp 283 285ºC, EI-MS (m/z): 444.02
[M+H]+, 442.15 [M-H]-. 1H NMR (400 MHz, DMSO-d6) δ
11.83 (s, 1H), 7.56 (s, 4H), 7.54 - 7.51 (m, 2H), 7.32 (s,
1H), 7.32 - 7.30 (m, 2H), 7.29 (d, J = 3.1 Hz, 1H), 6.88 -
6.82 (m, 1H), 4.36 (s, 2H), 3.68 - 3.49 (m, 8H). 13C NMR
(101 MHz, DMSO-d6) δ 169.55, 168.83, 164.32, 161.87,
148.45, 142.55, 140.95, 135.24, 135.04, 132.45, 130.21,
130.13, 129.41, 127.73, 126.72, 119.90, 116.03, 115.81,
110.65, 109.80, 60.37, 28.54, 21.53, 14.53.
1
C27H29N6O2: 469.2352, found 469.0079. H NMR (400
MHz, DMSO-d6) δ 11.83 (s, 1H), 7.56 (s, 4H), 7.33 (d, J =
7.4 Hz, 4H), 6.85 (s, 1H), 6.72 (d, J = 8.2 Hz, 2H), 4.36 (s,
2H), 3.59 (s, 8H), 2.96 (s, 6H).
The compounds Y16-Y20 were prepared using the same
procedure as compound Y1, with 6-bromo-1H-indazol-3-
amine (4) (1.5 mmol) and various boronic acid (6a-6e) (1.8
mmol) as starting materials.
5.1.26. (4-(4-(3-amino-1H-indazol-4-yl)benzoyl)piperazin-
1-yl)(4-bromophenyl)methanone (Y12)
5.1.30. (4-(4-(3-amino-1H-indazol-6-yl)benzoyl)piperazin-
1-yl)(4-fluorophenyl)methanone (Y16)
Yield 0.18 g, 24%. Mp 277 279ºC, EI-MS (m/z): 504.10
[M+H]+, 502.15 [M-H]-. 1H NMR (400 MHz, DMSO-d6) δ
11.83 (s, 1H), 7.72 - 7.63 (m, 2H), 7.56 (s, 4H), 7.41 (d, J
= 7.7 Hz, 2H), 7.32 (d, J = 2.2 Hz, 2H), 6.87 - 6.83 (m,
1H), 4.36 (s, 2H), 3.65 - 3.49 (m, 8H).
Yield 0.24 g, 36%. Mp 257 259ºC, HRMS (ESI): calcd
1
for [M+H]+ C25H23FN5O2: 444.1836, found 444.1831. H
NMR (400 MHz, DMSO-d6) δ 11.52 (s, 1H), 7.78 (d, J =
8.2 Hz, 3H), 7.63 - 7.42 (m, 5H), 7.36 - 7.16 (m, 3H), 5.42
(s, 2H), 3.49 - 3.67 (m, 8H).
5.1.27. (4-(4-(3-amino-1H-indazol-4-yl)benzoyl)piperazin-
1-yl)(p-tolyl)methanone (Y13)
5.1.31. (4-(4-(3-amino-1H-indazol-6-yl)benzoyl)piperazin-
1-yl)(4-bromophenyl)methanone (Y17)
Yield 0.57 g, 86%. Mp 259 261ºC, EI-MS (m/z): 440.20
[M+H]+, 438.20 [M-H]-. 1H NMR (400 MHz, DMSO-d6) δ
11.82 (s, 1H), 7.56 (s, 4H), 7.38 - 7.30 (m, 4H), 7.27 (d, J
= 7.7 Hz, 2H), 6.87 - 6.82 (m, 1H), 4.35 (s, 2H), 3.65 -
Yield 0.30 g, 40%. Mp 134 136ºC, HRMS (ESI): calcd
1
for [M+H]+ C25H23BrN5O2: 504.1035, found 504.1029. H
NMR (400 MHz, DMSO-d6) δ 7.78 (d, J = 7.1 Hz, 3H),
7.68 (d, J = 5.9 Hz, 3H), 7.53 (d, J = 8.5 Hz, 2H), 7.47 (s,
14

1H), 7.41 (d, J = 6.4 Hz, 2H), 5.41 (s, 2H), 3.68 - 3.44 (m,
this step, the plate was incubated at room temperature for
30 min, and the luminescence was measured by VICTOX
X multiple plate reader. The luminescence signal is
proportional to the amount of ATP present and inversely
correlated to kinase activity.
ACCEPTED MANUSCRIPT
8H).
5.1.32. (4-(4-(3-amino-1H-indazol-6-yl)benzoyl)piperazin-
1-yl)(p-tolyl)methanone (Y18)
Yield 0.21 g, 32%. Mp 244 246ºC, HRMS (ESI): calcd
1
for [M+H]+ C26H26N5O2: 440.2087, found 440.2085. H
5.3 Cell growth inhibition assays
NMR (400 MHz, DMSO-d6) δ 11.52 (s, 1H), 7.78 (d, J =
8.2 Hz, 3H), 7.53 (d, J = 8.0 Hz, 2H), 7.47 (s, 1H), 7.34 (d,
J = 7.8 Hz, 2H), 7.30 - 7.22 (m, 3H), 5.42 (s, 2H), 3.50 -
3.65 (m, 8H), 2.35 (s, 3H).
The MTT assays were performed to evaluate the
antiproliferative activity and identify the cytotoxicity of the
title compounds in vitro. Imatinib was used as the positive
control. The K562R cells expressing T315I mutation was
kindly provided by Professor Libo Yao (Fourth Military
Medical University, Xi’an, China). The Hek293 cells was
provided by Dr. Tao Zhang (Xi’an Jiaotong University,
Xi’an, China). The EA.hy926 cells was provided by Dr.
Wen Lu (Xi’an Jiaotong University, Xi’an, China). The
cancer cell lines (K562 and K562R cells) were cultured in
RPMI 1640 medium with 10% fetal bovine serum (FBS).
The two normal human cells (Hek293 and EA.hy926 cells)
were cultured in DMEM medium with 10% FBS.
5.1.33. (4-(4-(3-amino-1H-indazol-6-yl)benzoyl)piperazin-
1-yl)(4-chloro-3-(trifluoromethyl)phenyl)methanone (Y19)
Yield 0.16 g, 20%. Mp 224 226ºC, HRMS (ESI): calcd
1
for [M]+ C26H21ClF3N5O2: 527.1336, found 527.0687. H
NMR (400 MHz, DMSO-d6) δ 8.45 (s, 1H), 8.30 (s, 1H),
8.18 (d, J = 8.2 Hz, 1H), 7.90 - 7.75 (m, 5H), 7.37 (d, J =
8.0 Hz, 2H), 3.61 - 3.37 (m, 8H).
5.1.34. (4-(4-(3-amino-1H-indazol-6-yl)benzoyl)piperazin-
1-yl)(4-(dimethylamino)phenyl)methanone (Y20)
Yield 0.29 g, 41%. Mp 200 203ºC, HRMS (ESI): calcd
The cells were seeded into 96-well microtiter plates (2500
cells in 180 µl of nutrient medium per well), and incubated
in 5% CO2 at 37 °C for 12 h. The tested compounds (20 µl)
at the indicated final concentrations were added to the
culture medium and incubated for 48 h. Fresh MTT was
added to each well at the final concentration of 0.5 mg/mL,
and incubated with cells at 37°C for 4 h. Then aspirate the
supernatant, and add 150 µL DMSO to each well. The
absorbance values were determined by a microplate reader
(Bio-Rad, Hercules, CA, USA) at 490 nm. The IC50 values
were calculated according to inhibition ratios.
1
for [M+H]+ C27H29N6O2: 469.2352, found 469.2342. H
NMR (400 MHz, DMSO-d6) δ 7.78 (d, J = 8.2 Hz, 3H),
7.53 (d, J = 8.3 Hz, 2H), 7.47 (s, 1H), 7.32 (d, J = 8.8 Hz,
2H), 7.24 (dd, J = 8.4, 1.3 Hz, 1H), 6.72 (d, J = 8.9 Hz,
2H), 5.42 (s, 2H), 3.575 - 3.59 (m, 8H), 2.96 (s, 6H).
5.2. In vitro kinase assays
Kinase inhibition assays were performed using ADP-GloTM
kinase assay kit from Promega (Madison, WI) according to
the manufacturer’s instructions, with Imatinib as the
positive control. General procedures were as following:
Kinase was incubated with the corresponding substrate
/ATP mixture and inhibitors in a final buffer of Tris 40
mM (pH 7.4), MgCl2 10 mM, BSA 0.1mg/mL, DTT 1mM
with a total volume of 5 µL in a 384-well plate, and the
kinase reaction proceeded at 30 for 60 min. Then 5 µL
ADP-GloTM reagent was added and incubated at room
temperature for 40 min, in order to terminate the kinase
reaction and consume the remaining ATP. Next, 10 µL
kinase detection reagent was added to convert ADP to
ATP, and introduced luciferase/luciferin to detect ATP. At
5.4 Western blot assay
K562 cells were treated with vehicle (DMSO) or drug at
desired concentration for 4 h. The cells were lysed in SDS
sample buffer, collected, and normalized using BCA
protein assay kit before being diluted in SDS loading
buffer. Then the samples containing equal amounts of
protein were separated by SDS-PAGE. After
electrophoresis, proteins were transferred to PVDF
membranes and blocked with 5% nonfat milk in Tris-
buffered saline with 0.1% Tween-20. Membranes were
15

Passerini, N.H. Russell, J.J. Reiffers, T.C. Shea, B.
incubated with antibodies Bcr-Abl, p-Bcr-AAbCl,CanEdPaTctEinDat MANUSCRIPT
4
overnight. The membranes were washed with PBS
Chapuis, S. Cout, S. Tura, E. Morra, R.A. Larson, A.
Saven, C. Peschel, A. Gratwohl, F. Mandelli, M. Ben-
Am, I. Gathmann, R. Capdeville, R.L. Paquette, B.J.
Druker, Imatinib induces hematologic and cytogenetic
responses in patients with chronic myelogenous
leukemia in myeloid blast crisis: results of a phase II
study, Blood 99(2002) 3530-3539.
three times, and incubated with the appropriate anti-HRP
secondary antibodies for 2 h at room temperature. Finally,
immunoreactive proteins were visualized using the
enhanced chemiluminescence system from Pierce
Chemical.
5.5 Molecular docking study
In order to investigate the binding mode of the title
2. E.K. Greuber, P. Smith-Pearson, J. Wang, A.M.
Pendergast, Role of ABL family kinases in cancer:
from leukaemia to solid tumors, Nat. Rev. Cancer
13(2013) 559-571.
compounds with both Bcr-AblWT and Bcr-AblT315I
,
molecule docking was performed using Surflex-Dock
Module of Sybyl-X 2.0. The molecules were drawn with
Sketch and minimized by Powell's method for 1000
iterations under Tripos Force field with Gasteiger-Huckel
charge. Crystal structures of Bcr-AblWT (PDB ID: 1IEP)
and Bcr-AblT315I (PDB ID: 3QRJ) were imported, and
corresponding ligand was used to define the binding cavity
and generate the promotal. The ligands and water
molecules were removed and hydrogen was added and
minimized using Tripos force field and Pullman charges.
The residues in a radius 5.0Å around ligands were selected
as active site. The other docking parameters were kept at
default. The inhibitors were docked into the active site
using ligand-based mode.
3. A. Quintas-Cardama, J. Cortes, Molecular biology of
bcr-abl1-positive chronic myeloid leukemia, Blood
113(2009) 1619-1630.
4. R. Capdeville, E. Buchdunger, J. Zimmermann, A.
Matter, Glivec (STI571, Imatinib), a rationally
developed, targeted anticancer drug, Nat. Rev. Drug
Discov. 1(2002) 493-502.
5. P. Gupta, R.J. Kathawata, L. Wei, F. Wang, X. Wang,
B.J. Druker, L.W. Fu, Z.S. Chen, PBA2, a novel
inhibitor of imatinib-resistant BCR-ABL T315I
mutation in chronic myeloid leukemia, Cancer Lett.
383(2016), 220-229.
6. J.Y. Blay, M. von Mehren, Nilotinib: a novel,
selective tyrosine kinase inhibitor, Semin. Oncol.
38(2011) S3-S9.
Acknowledgment
Thanks for Professor Dehua Pei at The Ohio State
University to provide Schrödinger software for docking
analysis. This work was supported by the National Natural
Science Foundation of China (NSFC, No. 81602965 and
No. 81573285), the Natural Science Basic Research Plan in
Shaanxi Province of China (No.2017JQ8002 and No.
2018JM7071), the China Postdoctoral Science Foundation
(No. 2016M602837), and the Shaanxi Province
Postdoctoral Science Foundation.
7. N.P. Shah, C. Tran, F.Y. Lee, P. Chen, D. Norris, C.L.
Sawyers, Overriding imatinib resistance with a novel
ABL kinase inhibitor, Science 305(2004) 399-401.
8. L. Stansfield, T.E. Hughes, T.L. Walsh-Chocolaad,
Bosutinib:
a
second-generation tyrosine kinase
inhibitor for chronic myelogenous leukemia, Ann.
Pharmacother 47(2013) 1703-1711.
9. H.N. Banayath, O.P. Sharma, M.S. Kumar, R.
Baskaran, identification of novel tyrosine kinase
inhibitors for drug resistant T315I mutant BCR-ABL:
References
1. C.L. Sawyers, A. Houchhaus, E. Feldman, J.M.
Goldman, C.B. Miller, O.G. Ottmann, C.A. Schiffer,
M. Taipaz, F. Guilhot, M.W. Deininger, T. Fischer,
S.G. O’Brien, R.M. Stone, C.B. Gambacorti-
a
virtual screening and molecular dynamics
simulations study, Sci. Rep. 4(2014) 6948.
10. T. O’Hare, W.C. Shakespeare, X. Zhu, C.A. Eide,
16

V.M. Rivera, F. Wang, L.T. Adrian, T. Zhou, W.S.
development of a safe, practical synthesis of ABT-
869, Org. Process Res. Dev. 13(2009) 1419-1425.
17. W.G. Kong, Y. Zhou, Q.L. Song, Lewis-acid
promoted chemoselective condensation of 2-
aminobenzimi-dazoles or 3-aminoindazoles with 3-
ethoxycyclo-butanones to construct fused nitrogen
heterocycles, Adv. Synth. Catal. 360(2018) 1943-
1948.
ACCEPTED MANUSCRIPT
Huang, Q. Xu, C.A. 3rd Metcalf, J.W. Tyner, M.M.
Loriaux, A.S. Corbin, S. Wardwell, Y. Ning, J.A.
Keats, Y. Wang, R. Sundaramoorthi, M. Thomas, D.
Zhou, J. Snodgrass, L. Commodore, T.K. Sawyer,
D.C. Dalgarno, M.W. Deininger, B.J. Druker, T.
Clackson, AP24534, a pan-BCR-ABL inhibitor for
chronic myeloid leukemia, potently inhibits the T315I
mutant and overcomes mutation-based resistance,
Cancer Cell 16(2009) 401-412.
18. S.K. Verma, R. Ghorpade, A. Pratap, M.P. Kaushik,
CDI-mediated monoacylation of symmetrical
diamines and selective acylation of primary amines of
unsymmetrical diamines, Green Chem. 14(2012) 326-
329.
11. U.S. Food and Drug Administration. FDA Drug
Safety Communication: FDA asks manufacturer of the
leukemia drug Iclusig (ponatinib) to suspend
marketing
and
sales.
19. D.W. 2nd Zehnder, D.B. Smithrud, Facile synthesis of
rotaxanes through condensation reactions of DCC-
[2]rotaxanes, Org. Lett. 3(2001) 2485-2487.
http://www.fda.gov/Drugs/DrugSafety/ucm
373040.htm (accessed May 26, 2015).
12. B. Okram, A. Nagle, F.J. Adrian, C. Lee, P. Ren, X.
Wang, T. Sim, Y. Xie, X. Wang, G. Xia, G. Spraggon,
M. Warmuth, Y. Liu, N.S. Gray, A general strategy for
creating “inactive-confirmation” abl inhibitors, Chem.
Biol. 13(2006) 779-786.
20. L. Lai, E. Wang, B.J. Luh, Reaction of piperazine with
trimethylacetic
arylcarboxylic
anhydride;
A
convenient method for preparing monoacylated
piperazine derivatives, Synthesis 3(2001) 361-363.
21. X. Pan, F. Wang, Y. Zhang, H. Gao, Z. Hu, S. Wang, J.
Zhang, Design, synthesis and biological activities of
Nilotinib derivates as antitumor agents, Bioorg. Med.
Chem. 21(2013) 2527-2534.
13. X. Pan, J. Dong, Y. Shi, R. Shao, F. Wei, J. Wang, J.
Zhang, Discovery of novel Bcr-abl inhibitors with
diacylated piperazine as the flexible linker, Org.
Biomol. Chem, 13(2015) 7050-7066.
22. H. Zegzouti, M. Zdanovskaia, K. Hsiao, S.A. Goueli,
ADP-Glo: a Bioluminescent and homogeneous ADP
monitoring assay for kinases, Assay Drug Dev.
Technol. 7(2009) 560-572.
14. Y. Luo, F. Jiang, T.B. Cole, V.P. Hradil, D. Reuter, A.
Chakravartty, D.H. Albert, S.K. Davidsen, B.F. Cox,
E.M. McKeegan, G.B. Fox, A novel multi-targeted
tyrosine kinase inhibitor, linifanib (ABT-869),
produces functional and structural changes in tumor
vasculature in an orthotopic rat glioma model, Cancer
Chemother. Pharmacol. 69(2012) 911-921.
23. J. Dong, X. Pan, J. Wang, P. Su, L. Zhang, F. Wei, J.
Zhang, Synthesis and biological evaluation of novel
aromatic-heterocyclic biphenyls as potent anti-
leukemia agents, Eur. J. Med. Chem. 101(2015) 780-
789.
15. Y. Shan, J. Dong, X. Pan, L. Zhang, J. Zhang, Y.
Dong, M. Wang, Expanding the structural diversity of
Bcr-Abl inhibitors: Dibenzoylpiperazin incorporated
with 1H-indazol-3-amine, Eur. J. Med. Chem.
104(2015) 139-147.
24. Y. Ru, Q. Wang, X. Liu, M. Zhang, D. Zhong, M. Ye,
Y. Li, H. Han, L. Yao, X. Li, The chimeric ubiquitin
ligase SH2-U-box inhibits the growth of imatinib-
sensitive and resistant CML by targeting the native
and T315I-mutant BCR-ABL, Sci. Rep. 6 (2016):
28352.
16. A.W. Kruger, M.J. Rozema, A. Chu-kung, J.
Gandarilla, A.R. Haight, B.J. Kotecki, S.M. Richter,
A.M. Schwartz, Z. Wang, The discovery and
25. H.M. Revankar, M.V. Kulkarni, S.D. Joshi, U.A.
17

More, Synthesis, biological evaluation and docking
ACCEPTED MANUSCRIPT
studies of 4-aryloxymethyl coumarins derived from
substructures and degradation products of
vancomycin, Eur. J. Med. Chem. 70(2013) 750-757.
26. B. Nagar, W.G. Bornman, P. Oellicena, T. Schindler,
D.R. Veach, W.T. Miller, B. Clarkson, J. Kuriyan,
Crystal structures of the kinase domain of c-Abl in
complex with the small molecule inhibitors
PD173955 and imatinib (STI-571), Cancer Res.
62(2002) 4236-4243.
27. W.W. Chan, S.C. Wise, M.D. Kaufman, Y.M. Ahn,
C.L. Ensinger, T. Haack, M.M. Hood, J. Jones, J.W.
Lord, W.P. Lu, D. Miller, W.C. Patt, B.D. Smith, P.A.
Petillo, T.J. Rutkoski, H. Telikepalli, L. Vogeti, T. Yao,
L. Chun, R. Clark, P. Evangelista, L.C. Gavrilescu, K.
Lazarides, V.M. Zaleskas, L.J. Stewart, R.A. Van
Etten, D.L. Flynn, Conformational control inhibition
of the BCR-ABL 1 tyrosine kinase, including the
gatekeeper T315I mutant, by the switch-control
inhibitor DCC-2036, Cancer Cell 19(2011) 556-568.
28. C.A. Rhodes, P.G. Dougherty, J.K. Cooper, Z. Qian,
S. Lindert, Q.E. Wang, D. Pei, Cell-permeable
bicyclic peptidyl inhibitors against NEMO-IκB kinase
interaction directly from a combinatorial library, J.
Am. Chem. Soc. 140(2018) 12102-12110.
18

ACCEPTED MANUSCRIPT
Highlights
‹ Novel Bcr-Abl inhibitors with flexible linker to reduce steric clash with Ile315 in
Bcr-AblT315I kinase.
‹ 6-phenyl-1H-indazol-3-amine is a promising hinge binding moiety.
‹ Compound Y9 exhibited potent activity comparable to that of imatinib.