Design, synthesis and biological evaluation of novel indazole-based derivatives as potent HDAC inhibitors via fragment-based virtual screening

Article abstract of DOI:10.1016/j.ejmech.2020.112189

Based on fragment-based virtual screening and bioisoterism strategies, novel indazole and pyrazolo[3,4-b] pyridine derivatives as HDACs inhibitors were designed, synthesized and evaluated. Most of these compounds displayed good to excellent inhibitory activities against HDACs, especially compounds 15k and 15m were identified as potent inhibitors of HDAC1 (IC₅₀ = 2.7 nM and IC₅₀ = 3.1 nM), HDAC2 (IC₅₀ = 4.2 nM and IC₅₀ = 3.6 nM) and HDAC8 (IC₅₀ = 3.6 nM and IC₅₀ = 3.3 nM). Further anti-proliferation assays revealed that compounds 15k and 15m showed better anti-proliferative activities against HCT-116 and HeLa cells than positive control SAHA. The western blot analysis results indicated that compounds 15k and 15m noticeably up-regulated the level of acetylated α-tubulin and histone H3. In addition, the two compounds 15k and 15m could arrest cell cycle in G2/M phase and promote cell apoptosis, which was similar as the reference compound SAHA. Through the molecular docking and dynamic studies, the potent HDAC inhibitory activities mainly caused by van der Waals and electrostatic interactions with the HDACs.

Full text of DOI:10.1016/j.ejmech.2020.112189

Journal Pre-proof
Design, synthesis and biological evaluation of novel indazole-based derivatives as
potent HDAC inhibitors via fragment-based virtual screening
Jian Liu, Jingxian Zhou, Fengjun He, Liang Gao, Yu Wen, Lina Gao, Ping Wang, Di
Kang, Lihong Hu
PII:
S0223-5234(20)30156-2
DOI:
https://doi.org/10.1016/j.ejmech.2020.112189
EJMECH 112189
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 January 2020
Revised Date: 21 February 2020
Accepted Date: 24 February 2020
Please cite this article as: J. Liu, J. Zhou, F. He, L. Gao, Y. Wen, L. Gao, P. Wang, D. Kang, L. Hu,
Design, synthesis and biological evaluation of novel indazole-based derivatives as potent HDAC
inhibitors via fragment-based virtual screening, European Journal of Medicinal Chemistry (2020), doi:
https://doi.org/10.1016/j.ejmech.2020.112189.
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Design, Synthesis and Biological Evaluation
of Novel Indazole-Based Derivatives as
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Jian Liu a, #, *, Jingxian Zhou a,#, Fengjun He a, #, Liang Gao a, Yu Wen a, Lina Gao a, Ping Wang a, Di Kang a,*, Lihong Hu a,*
Fragment Library

European Journal of Medicinal Chemistry
Journal homepage: www.elsevier.com
Design, Synthesis and Biological Evaluation of Novel Indazole-Based Derivatives as
potent HDAC Inhibitors via Fragment-Based Virtual Screening
Jian Liu a, #, *, Jingxian Zhou a,#, Fengjun He a, #, Liang Gao a, Yu Wen a, Lina Gao a, Ping Wang a, Di Kang
a,*, Lihong Hu a,*
a Jiangsu Key Laboratory for Functional Substance of Chinese Medicine, Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources
Industrialization, Stake Key Laboratory Cultivation Base for TCM Quality and Efficacy, School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing,
210023, PR China
*Corresponding author: liujian623@njucm.edu.cn (Jian Liu); kangdi@njucm.edu.cn (Di Kang); lhhu@njucm.edu.cn (Lihong Hu)
# Jian Liu, Jingxian Zhou and Fengjun He contributed equally.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Based on fragment-based virtual screening and bioisoterism strategies, novel indazole and
pyrazolo[3,4-b] pyridine derivatives as HDACs inhibitors were designed, synthesized and
evaluated. Most of these compounds displayed good to excellent inhibitory activities against
HDACs, especially compounds 15k and 15m were identified as potent inhibitors of HDAC1
(IC50 = 2.7 nM and IC50 = 3.1 nM), HDAC2 (IC50 = 4.2 nM and IC50 = 3.6 nM) and HDAC8
(IC50 = 3.6 nM and IC50 = 3.3 nM). Further anti-proliferation assays revealed that compounds
15k and 15m showed better anti-proliferative activities against HCT-116 and HeLa cells than
positive control SAHA. The western blot analysis results indicated that compounds 15k and
15m noticeably up-regulated the level of acetylated α-tubulin and histone H3. In addition, the
two compounds 15k and 15m could arrest cell cycle in G2/M phase and promote cell apoptosis,
which was similar as the reference compound SAHA. Through the molecular docking and
dynamic studies, the potent HDAC inhibitory activities mainly caused by van der Waals and
electrostatic interactions with the HDACs.
Available online
Keywords:
HDAC
HDAC inhibitors
Fragment-based drug design
Indazole scaffold
Antitumor activity
2019 Elsevier Ltd. All rights reserved.
1.Introduction
lysine residues located on the amino-terminal tails of histone
protein, subsequently the deacetylation process induces
Cancer is characterized by the uncontrolled growth and spread
of abnormal cells, and it is a complex life-threatening disease that
can appear in different tissues and almost everywhere in the body
[1]. At present, cancer has been a leading cause of death and a
major public health problem with increasing incidence and
mortality. Apart from the use of radiotherapy and surgery,
targeted therapies have revolutionized the treatment of cancers
over the last two decades and have been the cornerstone of
personalized medicine, because of lower systemic toxicity and
adverse side effects [2, 3]. Nowadays, it is generally believed that
widespread of epigenetic disorders interact with potential gene
mutations lead to the tumorigenesis [4]. Consequently,
epigenetics is such a rapidly evolving field, which providing a
great quantity of opportunities for developing targeted cancer
therapies [5].
condensed and transcriptionally inactive heterochromatin [9].
However, the HATs have the opposite effect, which relate to the
histone acetylation and relax chromatin structure resulting in
enhancing transcription [10]. It exists a balance between histone
acetylation and deacetylation via the regulation of HATs and
HDACs in normal cells, therefore interference with the
acetylation/deacetylation process has been linked to cancer
development [11-14]. HDAC-mediated histone deacetylation
plays a major role in the regulation of cell differentiation,
proliferation and survival [15, 16]. Overexpressed HDACs can
lead to chromatin condensation and cause down-regulation of
tumor suppressor genes [17], therefore the inhibition of HDACs
has become an important target in cancer research.
According to the sequence homology and catalytic
mechanisms, the HDACs family, which consists of 18 isoforms,
are divided into four classes of subfamilies: class I (HDAC 1, 2,
3 and 8), class II (HDAC 4, 5, 6, 7, 9 and 10), class III (sirtuin 1,
2, 3, 4, 5, 6 and 7) and class IV (HDAC 11). The class I, class II
and class IV HDACs are Zn2+-dependent enzymes, that class I
HDACs are most present in the nucleus and class II HDACs
shuttle between cytoplasm and nucleus, whereas class III are
The acetylation and deacetylation of lysine belongs to crucial
post-translational modifications in the epigenetic field [6, 7].
Histone deacetylases (HDACs) and histone acetyltransferases
(HATs) significantly affects the epigenetic regulation of gene
expression and has pleiotropic effects at the cellular and systemic
levels [8]. The HDACs are responsible for the deacetylation of

NAD+-dependent [18]. Till date, several HDAC inhibitors
(HDACIs) were addressed for cancer treatment. Vorinostat (1,
SAHA) was the first HDAC inhibitor approved by U.S. Food and
Drug Administration in 2006 for the treatment of cutaneous T-
cell lymphoma [19, 20]. Then, Romidespin (2, FK228),
Belinostat (3, PXD101) and panobinostat (4, LBH589) have
been successively approved by FDA and Chidamide (5, HBI-
8000) has been approved by CFDA for the treatment of specific
lymphoma and myeloma diseases (Figure 1) [21-24].
Meanwhile, there are several HDACIs under clinical trial for the
treatment of various cancers, for example Abexinostat (PCI-
24781), Resminostat (RAS-2410), Givinostat (ITF-2357) and
Pracinostat (SB-939), etc [25]. Due to the diversity of HDACIs
scaffolds, discovering novel potential HDACIs bearing novel
scaffolds are still in great demand.
Fragment-based drug design has become an important and
powerful tool for the discovery and optimization of new drug
leads [26-28]. While screening low molecular weight fragment
library as a potential source of small, ligand efficient substances
has become an important operation in many drug discoveries.
Fragment screening is very important for fragment-based drug
design, and several fragment discovery techniques have been
reported, such as X-ray crystallographic screening, NMR-based
methods, molecular docking, or biophysical approaches, etc [29-
32]. It is general accepted that docking fragments render crystal
structure data is critical for ligand design, however, it exists a
challenge that small fragments are harder to dock relative to
larger drug sized molecules. Another challenge of fragment-
based drug design was that when the active fragment was
discovered, how to optimize and combine the various fragment,
then eventually enhanced the compound activities and found the
hit compound.
Our research group has been interested in the design, synthesis
and biological evaluation of anti-tumor agents. In our previous
study, with the drug repurposing strategy, we combined
pharmacophore modeling, 3D-QSAR, docking studies and
molecular dynamics simulation to identify a potential HDACIs
from DrugBank database [33]. The built pharmacophore model,
molecular docking and dynamic simulation methods could be
utilized to explore other potential HDACIs with novel scaffolds.
Herein, with the aim of exploring novel HDACIs scaffolds, a
fragment library was virtual screened via the previously
established theoretical calculation model. As a result, based on
the rational fragment-growing strategy, several HDAC inhibitors
were designed, synthesized and evaluated bearing novel indazole
and pyrazolo[3,4-b] pyridine scaffolds. Most of these compounds
demonstrated outstanding potencies against HDACs and
displayed powerful anti-proliferative activities toward cancer
lines in vitro. Additionally, the molecular docking and molecular
dynamic simulation studies were carried out to investigate the
receptor-ligand interaction.
Fig. 1. Structures of representative HDAC inhibitors.
plays a key factor in protein-ligand binding (Figure 2B).
Meanwhile, the cap region corresponds to a hydrophobic center,
which occupies the hydrophobic region and forms hydrophobic
interaction. Besides, the ZBG and cap region connect by a linear
or cyclic linker, the linker needs to fit a certain space distance
and the amide group often forms H-bond with target protein.
Compared to the ZBG and linker region, there are numerous
research work about the cap region structural modification, and
several HDAC inhibitors have been found [34-37]. Therefore,
herein only the fragments in cap area are filtered by the fragment-
based virtual screening, with the aim of exploring novel scaffolds
and enhancing the inhibitory activity.
2.Results and discussion
2.1 Fragment-based drug design
The common pharmacophore models of HDACIs often
consist of three parts: (1) a zinc-binding group (ZBG), such as
hydroxamic acid and 2-aminobenzamide; (2) a cap group,
generally a hydrophobic and aromatic group; (3) a linker portion,
typically linear or cyclic structures (Figure 2A). The generated
HDAC pharmacophore model contained four chemical features
[33], the zinc-binding group contain a H-bond donor and a H-
bond receptor, the interaction between ZBG and zinc or histidine

The fragment-based virtual screening protocol can be seen in
Figure 2C. The fragment library was provided by enamine
fragments (molecular weight < 300, clogP ≤ 3, and number of
rotatable bonds ≤ 3). The database screening was performed
(https://enamine.net/index.php),
which
contained
11809
(A).
(B).
O
H
N
N
H
OH
SAHA
Linker
O
Zn2+
Cap
(C).
(D).
(E).
H
N
N
R
H
N
O
N
O
HN
molecular hybridization
Indazole
OH
HN
N
linker
H
Cap
ZBG
O
H
bioisosterism
N
OH
N
H
O
SAHA
R
H
O
N
OH
N
N
H
N
HN
O
Fig. 2. (A). The pharmacophore of HDACIs; (B). The built pharmacophore model of HDACIs; (C). The protocol of fragment-based virtual
screening; (D). The binding mode of indazole scaffold with HDAC2; (E). Design strategy of the target compounds;
using the Ligand Pharmacophore Mapping protocol in Discovery
Studio 4.0. The built HDACIs pharmacophore model were
defined as a query for pharmacophore screening. The fit values
were calculated based on the chemical substructures map the
location constraints of the hydrophobic features and their
distance deviation from the hydrophobic centers. Finally, only
the compounds showed good fit values in pharmacophore model
could enter the further molecular docking study.
Subsequently, the HDAC2 protein (4LXZ) was selected in the
molecular docking procedure. In the docking process, with
position constraint docking method, the spatial position of cap
group in endogenous ligand (SAHA) was relatively fixed. Thus,
the phenyl ring of SAHA merely formed hydrophobic interaction
with the lipophilic amino acid residues, such as Pro34, Phe210,
Leu276 and Tyr308. While the fragments with excellent docking
score and similar binding pattern with SAHA could be retained,
and 8 fragments were finally retained (Table 1). Considering the
synthesis feasibility and draggability, indazole scaffold had good
superior physicochemical properties and was often utilized in
medicinal chemistry research (Figure 2D) [38-40], therefore it
had become the first choice for our compound design. According
to the molecular hybridization strategy, the phenyl ring of SAHA
could be substituted by indazole scaffold, and a series of indazole
derivatives were designed, synthesized and biological evaluated.
Meanwhile, with the aim of enriching the structure-activity
relationship, several pyrazolo[3,4-b] pyridine derivatives were
also designed, which was the bioisoterism of indazole.
Table 1. The Fragment-Based Virtual Screening Results
Fragments
Fit value
Glide
8.33
F1
F2
6.47
5.98
7.99
F3
F4
F5
5.42
5.60
5.45
7.85
7.63
6.36
F6
F7
5.13
4.88
6.09
5.92

intermediates 11a-e were obtained. Treatment of the intermediate
11a-e with various substituted-phenylboronic acid catalyzed by
Pd(dppf)Cl2 and Cs2CO3 in refluxing dioxane/water afforded key
intermediate 12a-t in 69.2%-78.4% yield. Then the intermediate
12a-t were successively ester hydrolyzed, condensed with
NH2OTHP, and removed the protected group BOC and THP. As
a result, the target indazole derivatives were obtained in 54.2%-
68.4% yield. According to the similar synthetic route, 1H-
pyrazolo[3,4-b] pyridine derivatives 21a-b were synthesized
from the starting material 2,6-dichloronicotinonitrile (16) through
several steps (Scheme 2).
F8
4.65
5.76
2.2 Chemistry
The preparation of indazole derivatives 15a-t were described
in Scheme 1. Compounds 15a-t were synthesized from starting
material hydrazine and 4-bromo-2-fluorobenzonitrile (6) through
nine steps. The indazole intermediate (8) was condensed with
acyl chloride (10) in the presence of DIPEA, which the
Scheme 1. Reagents and conditions: (a) hydrazine hydrate, anhydrous alcohol, 85 , 86.2%; (b) Boc2O, DMAP, THF, 70.2%;(c) Oxalyl
chloride, DMF, DCM, rt; (d) DIPEA, DCM, rt, 46.3%-57.2%; (e) Pd(dppf)2Cl2, K2CO3, Aryl boric acid, 80 , 69.2%-78.4%; (f) TFA, THF,
rt, 75.4%-84.1%; (g) LiOH, NaOH, Methanol, 55.2%-61.5%; (h) NH2OTHP, K2CO3, HATU, DMF，41.2%-56.8%; (i) HCl▪EtOAc, dioxane，
54.2%-68.4%
Scheme 2. Reagents and conditions: (a) Pd(Ph3)2Cl2, K2CO3, 3-Methoxybenzeneboronic acid or 4-Methoxybenzeneboronic acid, 100 ℃,
70.5%-76.3%; (b) hydrazine hydrate, n-butyl alcohol, reflux, 72.2%-73.5%; (c) DIPEA, DCM, rt, 46.3%-57.2%; (d) LiOH, NaOH, Methanol,
rt, 68.3%-72.4%; (e) NH2OTHP, K2CO3, HATU, DMF, rt, 65.4%-67.2%; (f) HCl▪EtOAc, dioxane, rt, 58.3%-61.2%;

2.3 In vitro HDAC inhibitory activity
15o
15p
15q
15r
15s
15t
C
C
C
C
C
C
N
N
23.0
36.2
76
25.9
31.6
168
62
24.5
32.5
54
Table 2. Structures and in vitro inhibitory activity against HDACs of
all compounds
HDAC1
HDAC2
IC50(nM) IC50(nM)
HDAC8
Cpd
R
X
C
Y
IC50(nM)a
13
41
15a
9.6
1.9
2.1
13.1
3.9
12.6
3.0
2.6
6.3
4.5
3340
3.3
7.2
44
15b
15c
15d
15e
15f
C
C
C
C
C
C
C
C
C
C
C
C
C
2425
2.4
4630
5.3
4.2
3.4
21a
21b
3.7
2.7
3.4
3.0
3.1
1.8
4.6
2.7
2.5
3.1
4.6
4.0
5.2
4.6
3.5
6.0
4.4
5.4
4.2
5.0
3.6
4.8
3.8
4.0
3.2
4.9
4.4
2.4
4.2
3.6
4.1
3.3
4.4
6.0
9.0
SAHA
13
70
a The IC50 values are shown as the average values from two separate
experiments.
15g
15h
15i
Firstly, to evaluate the inhibitory activity of HDAC inhibitors,
all these compound potencies against the recombinant human
HDAC1, HDAC2 and HDAC8 isoforms were interrogated using
SAHA as the reference compound. The results of the mean
values of two experiments, expressed as half maximal inhibitory
concentration (IC50) values were listed in Table 2.
As shown in Table 2, the effects of regiochemical
substitution and electronics on the phenyl ring of indazole
derivatives were explored. Compared to positive control SAHA,
most of the compounds exhibited excellent inhibitory activity
against HDAC1, HDAC2 and HDAC8 isoforms which
demonstrated the 6-phenyl-1H-indazole (F1) was an excellent
capping scaffold possessing strong binding affinity with the
HDACs. The electron cloud density of the functional groups at
phenyl C-2 position have a notable influence on inhibitory
activity, as exemplified by 15e (2-Cl, HDAC1 IC50 = 2.7 nM;
HDAC2 IC50 = 5.2 nM; HDAC6 IC50 = 4.0 nM) and 15i (2-F,
15j
O
15k
15l
HDAC1 IC50 = 1.8 nM; HDAC2 IC50 = 4.4 nM; HDAC6 IC50
=
2.4 nM), which is better than 15j (2-OMe, HDAC1 IC50 = 4.6
nM; HDAC2 IC50 = 5.4 nM; HDAC6 IC50 = 4.2 nM).
Meanwhile, the size of substituents at phenyl 3-position also
influenced the HDAC inhibitory activities. For example,
compound with meta-methoxy (15b) exhibited best activities
15m
15n
(HDAC1 IC50 = 1.9 nM; HDAC2 IC50 = 3.9 nM; HDAC6 IC50
=
3.0 nM), while replacing with bulkier ethoxy group (15c) and
small size hydrogen atoms (15a) led to the loss of activity. As for
the meta-chlorine atom (15d) and meta-trifluoromethyl
substituent (15l), it also led to slight reductions of the inhibition
potencies. Besides, both para-electron-withdrawing and para-

2.4 Anti-proliferation of all compounds against HCT-116, MCF-
7 and HeLa cells
electron-donating groups on the phenyl ring could inhibit
HDACs, for example compound 15k (4-OMe) and 15m (4-Cl)
exhibited good enzyme inhibitory activity and potent cellular
anti-proliferative activities (Table 3). With the aim of enhancing
enzyme potency, several disubstituted compounds were also
synthesized. However, compound with 3,4-dimethoxy (15f), 3,5-
dimethoxy (15n), 2-fluoro-5-methoxy (15g) and 2-fluoro-5-
methoxy (15h) decreased the enzymatic inhibitory activity
against HDAC1, HDAC2 and HDAC8.
Additionally, the linker between cap group and zinc-binding
group also had effects on the inhibitory activities against
HDACs. It was because the linker might affect the orientation of
cap group and zinc-binding group, then influenced the binding
affinity between protein and ligand. We examined different
lengths of methylene (15q-15s, 15a) and cyclohexyl (15t). In
general, compound 15t with cycloalkyl linker showed weaker
activities than compounds 15a, 15q-15s which with linear alkyl
linker. Furthermore, better inhibitory activity showed by
increasing length of linker, and as a result, the methylene with six
carbon atoms was the optimal compound (15a). Meanwhile, for a
better understanding of cap group, different aromatic ring
substituents (15o and 15q) had also been synthesized to enhance
the hydrophobic interactions. Compared to compound with
pyridine substituent (15o) and thiophene substituent (15p), the
compound with phenyl substituent manifested the best enzymatic
inhibitory activity. Moreover, with the bioisoterism strategy,
several pyrazolo[3,4-b] pyridine derivatives (21a-21b) were also
designed and synthesized. While the indazole scaffold substituted
by pyrazolo[3,4-b] pyridine, the HDACs inhibitory activities
slightly decreased (15b vs 21a, 15k vs 21b), thus the 6-phenyl-
1H-indazole (F1) scaffold was essential.
Furthermore, the anti-proliferative activities of the new
compounds along with positive control SAHA were assayed with
against three solid cancer cell lines including HCT116 (human
colorectal cancer cells), MCF-7 (human breast cancer cells) and
HeLa (human cervical cancer cells). As summarized in Table 3,
the majority of compounds exhibited good anti-proliferative
activity. Coincidentally with the enzymatic assay results, with the
shortening of linker length, the cellular activities of the
compounds 15a, 15s and 15r was sharply decreased, and the anti-
proliferative activity of compound 15q almost lost. Besides, the
activity of the di-methoxy substituted compounds were relatively
weaker than the mono-methoxy substituted compounds (15f: 3,4-
dimethoxy; 15n: 3,5-dimethoxy vs 15b: 3-methoxy; 15j: 2-
methoxy; 15k: 4-methoxy). Therefore, considering the ligand
efficiency, mono-substituted compounds would be preferred.
Additionally, Compounds 15k (IC50 = 0.9 µM), 15m (IC50 = 1.0
µM) and 21b (IC50 = 1.2 µM) demonstrated similar anti-
proliferative activities against MCF-7 cells with the reference
compound SAHA (IC50 = 0.8 µM). Furthermore, as far as HCT-
116 and HeLa cells concerned, compounds 15k, 15m and 21b
showed better anti-proliferative activities than SAHA,
respectively. Consequently, considering the results of enzymatic
inhibitory and anti-proliferative activity, the compounds 15k,
15m and 21b would be further evaluated.
Table 3.In vitro anti-proliferative activity against HCT-116, MCF-7 and HeLa cells of all compounds.
IC50(µM)a
IC50(µM)
MCF-7
Cpd
Cpd
HCT-116
10.2
MCF-7
5.5
HeLa
12.5
HCT-116
8.1
HeLa
3.0
15a
15c
15b
15d
15f
19.1
27.5
10.9
2.0
4.2
2.6
2.4
2.2
4.9
2.5
0.9
1.0
40.1
41.5
7.4
8.6
0.8
10.8
8.1
41.8
14.6
7.6
0.2
21.3
9.8
15e
15g
7.5
3.3
15h
15j
15l
15i
4.1
13.8
4.4
2.7
5.7
5.9
15k
15m
15o
2.1
2.9
47.3
55.2
8.3
6.0
3.9
4.8
15n
15p
15r
15t
21b
9.5
7.3
12.3
NAb
10.6
2.7
6.9
7.5
29.5
5.8
15q
15s
NA
20.1
3.6
23.5
47.4
4.1
4.4
NA
1.2
NA
4.8
21a
SAHA
4.9
5.0
a The IC50 values are shown as the average values from two separate experiments.
b ‘NA’ means IC50 > 50 µM
2.5Analysis Ac-tubulin and Ac-histone H3 levels in HCT-116 cells by Western bloting

Fig. 3. Western blot analysis of Ac-tubulin and Ac-histone H3 from HCT-116 cell.
In an effort to further validate the target in the these novel
compound 15k and 15m led to more pronounced upregulation of
acetylated histone H3 and acetylated tubulin than the
pyrazolo[3,4-b] pyridine inhibitor 21b, which was consistent
with the result of HDAC enzymatic inhibition and cancer cells
anti-proliferative activities. Moreover, the western blot analysis
indicated that our designed compounds, like the positive control
SAHA, were pan-HDACs inhibitors.
indazole and pyrazolo[3,4-b] pyridine series, compounds 15k,
15m and 21b were evaluated for the expression of histone H3
and α-tubulin acetylation HCT-116 cell line, which were
important biomarkers associated with intracellular HDAC
inhibition. For this assay, SAHA was also used as the positive
control. As shown in Figure 3, the result revealed that indazole
Fig. 4. Cell cycle analysis through PI/RNase staining with flow cytometry for the cells after different treatments of DMSO as (A) control, (B)
15k, (C) 15m, (D) 21b, (E) SAHA, n = 3; and (F) the quantitative measurement of cell cycle phase.
2.6 Cell cycle analysis
21b
50.0
26.2
26.1
29.4
23.9
44.2
SAHA
To further determine whether synthesized inhibitors had an effect
on cell cycle, the compounds 15k, 15m and 21b were evaluated at
least three times using HCT-116 colon cancer cell line for cell cycle
detection after 24 hours treatment. The results were shown in Table
4, which indicated that compounds 15k and 15m evidently arrested
HCT-116 in G2/M phase like positive control SAHA, because the
percentage of cell in G2/M phase for both compounds 15k and 15m
was increased from about 26% to 44%. Whereas no increasing
percentage of G2/M phase cells treated with 21b for 24 hours
indicate that the compound 21b had no significant effect on cell
cycle against HCT-116 cells (Figure 4). Thus, from the results of
cell cycle, the indazole inhibitors 15k and 15m could arrest cell
cycle in G2/M phase, which was similar as the reference compound
SAHA.
a
The values represent means of three separate experiments with
SD less than 10%
2.7 Cell apoptosis assay
Furthermore, promoting cell apoptosis is an efficient way to
eliminate tumor cells. HCT-116 cells were used to evaluate the
apoptotic effect of compounds. Consistent with the cell cycle
effect, flow cytometry analysis show that compounds 15k and
15m induced cell apoptosis in a dose-dependent manner (Figure
5). The percentage of Annexin+/PI+ cells treated by compounds
15k and 15m was increased to 54%, 66%, 76% and 48%, 71%,
74% at 1µM, 3µM or 10µM concentration respectively, with no
significant difference from SAHA treatment group which was
increased to 66%, 70% and 76% at the same concentration.
Additionally, similar with the cell cycle analysis, the compound
21b had a weak effect on HCT-116 cells apoptosis due to the tiny
increasing double positive cells (17%) at 10 µM concentration in
comparison to DMSO group (7%). Thus, the indazole
compounds 15k and 15m also had similar effect cell apoptosis as
reference compounds SAHA.
Table 4. Cell cycle analysis of HCT116
Proportion of HCT-116 cells at 3 µM concentration
Compound
G0/G1 (%)
48.4
S (%)
25.2
G2/M (%)a
26.4
control
15k
29.8
26.9
43.3
15m
26.2
29.7
44.1
2.8 Molecular docking

Table 5.
docked fitted as well as the co-crystal (Fig 6a). As for the
docking conformation of SAHA (Table 5), the distances between
hydroxamic acid group (ZBG) and zinc iron were 2.4 Å and 1.9
Å, respectively, which demonstrated that the re-docked ligand
tightly bound with Zn2+ and the docking method was reliable.
Additionally, the hydroxamic acid group (ZBG) of
compounds 15k, 15m and 21b also entered into the active site by
chelating the essential catalytic zinc ion (Figure 6b-d, Table 5).
The hydroxamic acid group of these compounds also formed
hydrogen binding interactions with His145 and His146. And the
indazole scaffold was close to a hydrophobic area and formed
hydrophobic interactions with the Pro34, Phe210, Leu276 and
Tyr308. Most importantly, both of the compounds 15k, 15m and
21b exhibited similar docking conformations to positive control
SAHA, it might be explained that the novel indazole derivatives
15k, 15m and pyrazolo[3,4-b]pyridine derivative 21b
demonstrated potent HDAC1, HDAC2 and HDAC8 inhibition.
Besides, through the docking scoring results, the compound 15m
revealed stronger binding affinity with target protein and had
higher docking scores than compound 15k and 21b (docking
score 15m: -7.266 vs 15k: -6.622; 21b: -5.919). Therefore,
among these three compounds, compound 15m exhibited the best
the enzyme inhibitory against HDAC1, HDAC2 and HDAC8.
The docking result of compound SAHA, 15k, 15m and 21b
Compound Docking score
The distance between
Zn2+ and -CO-NH-OH
SAHA
15k
-6.138
-6.622
-7.266
-5.919
2.4 Å; 1.9 Å
2.4 Å; 2.5 Å
15m
21b
2.3 Å; 2.0 Å
2.8 Å; 22 Å
To better understand the possible binding modes between
potent inhibitors and HDAC target, we performed molecular
docking analysis of SAHA, 15k, 15m and 21b in the binding-site
of HDAC2 (PDB ID: 4LXZ), respectively. The docking
simulation was carried out by the Glide module of Schrodinger
Maestro 2019, meanwhile the image files were generated using
pymol 1.1.
Firstly, the reference compound SAHA was re-docked into
the HDAC2 protein, to verify that whether the docking software
was suitable for HDAC2 docking and whether the docking
parameters were reasonable. As we expected, the RMSD value
calculated between the co-crystal and re-docked ligand was 0.61
Å, meanwhile the docking orientations and interactions of re-
Fig. 5. Effects of HDAC inhibitors on cell apoptosis in HCT-116 cells after incubation with 15k, 15m, 21b and SAHA for 24 h. (A)
Representative flow cytometric histograms of control group, (B) 15k, 15m, 21b and SAHA. (C) The percentage of Annexin+/PI+ cells
determined by flow cytometry. Data are expressed as the mean ± SD. *P<0.05, **P<0.01, ***P<0.001, ns, no significance, n = 3.
(A).
(B).

(C).
(D).
Fig. 6. (A). The re-dock result of SAHA with HDAC2 (4LXZ): experiment (blue), calculated (yellow); (B). The docking mode of compound
15k with HDAC2: 15k (magentas), SAHA (blue); (C). The docking mode of compound 15m with HDAC2: 15m (pink), SAHA (blue); (D).
The docking mode of compound 21b with HDAC2: 21b (orange), SAHA (blue);
2.9 Molecular dynamic simulation
fluctuation above 2.0 Å. The loop regions were found to highly
fluctuate and the active site residues remained less fluctuated.
The average RMSF per residue for the 15k, 15m, 21b and SAHA
complexes were 0.73, 0.77, 0.78 and 0.81 Å, respectively. The
relatively larger RMSF per residue of 21b complex also might be
explained by the relatively higher RMSD fluctuations and weaker
binding of 15k and 15m. As mentioned above, some important
residues in the binding pocket kept a small RMSF value, such as
Pro34, His140, His141, Phe210, Leu276 and Tyr308, indicating
the binding pockets were kept stable.
The binding-free energies were calculated by using MM-
GBSA method based on the equilibrium trajectories to reflect the
binding affinity between the protein and ligand (Table 6).
Overall, the predicted binding free energy was less than zero,
implying the binding interaction between HDAC and the
compounds could be spontaneous. As a result, compound 15k
had the lowest binding free energy (-73.71 kcal·mol-1), indicating
a more stable ligand conformation and a better binding-affinity
than 15m (-72.36 kcal·mol-1), 21b (-71.11 kcal·mol-1) and SAHA
(-69.41 kcal·mol-1). Furthermore, the contributions favoring
ligand binding were van der Waals energy (-47.56 kcal·mol-) and
electrostatic interaction energy (-37.69 kcal·mol-1), the nonpolar
solvation interaction was also beneficial for ligand binding (-
30.98 kcal·mol-1), whereas the polar solvation energy (40.93
kcal·mol-1) was detrimental to bind with HDAC. Therefore, the
inhibitors 15k, 15m, 21b and SAHA both had strong van der
Waals and electrostatic interactions with the HDACs, then the
compounds tightly bound to the protein.
The understanding of protein-ligand binding mode is of
critical importance for biomedical research, yet the process itself
had been very difficult to study because of its intrinsically
dynamic character. Thus, in order to obtain the stable binding
conformation and further validate the docking results, the
compounds 15k, 15m, 21b and SAHA in complex with HDAC2
(4LXZ) were carried out molecular dynamic simulation for 100
ns, using TIP3P water mode. The RMSD values of protein
backbone atoms relative to the initial structure were calculated to
examine the protein stability over the course of simulation
period. As a result, the RMSD values for these three novel
inhibitors did not fluctuate significantly, and it were converged to
1.6 Å for the 100 ns, which was clearly identified the three
inhibitors have more stability in HDAC2 protein (Figure 7A).
Especially, the RMSD values of Zn2+ and nearby residues were
less than 0.1 Å. Therefore, these results demonstrated that the
stabilities of the dynamic equilibrium for the complex systems
were reliable and that the trajectories could be useful for the
subsequent analysis.
The RMSF value of amino acid residues is another important
basis for investigating the stability of protein-ligand complexes
(Figure 7B). The RMSF value reflecting the flexibility of each
residue pointed out their relative vibrational motion during
simulations, and the residues with higher RMSF values revealed
they were more flexible. All the systems displayed similar RMSF
fluctuation trends, which the majority of RMSF values were less
than 1.0 Å and fewer residues in all the systems showed high
3
15k
15m
2
1
0
0
20
80
100
40Time (6n0s)
Figure 7. Dynamics of compounds bound to HDAC. (A) The backbone RMSD of the protein along the simulation time. (B) RMSF for protein-
inhibitor during MD simulation.
Table 6. Binding free energy (kcal·mol-1) of inhibitor-protein complexes along with the individual energy components (kcal·mol-1)
contributions.
Contribution
15k
15m
21b
SAHA
a
△GVDW
-47.56± 2.86
-37.69± 1.54
-42.11± 2.04
-41.60± 1.92
-43.09± 2.86
-41.98± 2.25
-32.23± 1.75
-52.31± 2.48
b
△Gele

c
△
GGB
40.93± 3.68
-30.98± 2.34
-73.71± 4.02
42.66± 3.12
-31.78± 1.35
-72.36± 3.54
42.96± 2.98
-29.66± 1.78
-71.11± 2.62
38.77± 2.75
-24.15± 1.18
-69.41± 3.33
d
△GGA
e
△
Gbind
a Contribution to the free energy of binding from the van der Waals energy.
b Contribution to the free energy of binding from the electrostatic energy.
c Contribution to the free energy of binding from the polar solvation energies.
d Contribution to the free energy of binding from the non-polar solvation energies.
e Free energy of binding.
4.1.2 tert-Butyl- 3-amino-6-bromo-1H-indazole-1-carboxylate (8)
3. Conclusions
DMAP (100.0 mg) and Boc2O (566.1mg, 2.6 mmol) were
added to the solution of building block 6 (500.0 mg, 2.4 mmol) in
THF (10 mL). The reaction mixture was stirred for 1 h and
monitored by TLC. After concentrated, the residue was dissolved
in EtOAc (100 mL) and washed with 1M HCl (20 mL × 2),
NaHCO3 (20 mL ×2) and brine (20 mL × 2), dried over Na2SO4,
concentrated in vacuo. The residue was purified by
chromatography on silica gel Petroleum-EtOAc (1:1) to give 8 as
In summary, we designed and synthesized a novel series of
HDAC inhibitors bearing indazole scaffold via fragment-based
virtual screening, and based on the bioisoterism strategy, several
pyrazolo[3,4-b] pyridine derivatives were also designed and
synthesized. The addressed compounds were evaluated for their
in vitro biological activities, especially compounds 15k, 15m and
21b exhibited significant antitumor activities in vitro.
Importantly, the indazole inhibitor 15k and 15m possessed
relatively better inhibitory activities than pyrazolo[3,4-b]
pyridine 21b. The western blot analysis results showed that
compounds 15k and 15m caused a substantial increase in the
level of acetylated α-tubulin and histone H3 in a dose-dependent
manner. Besides, the two compounds 15k and 15m could arrest
cell cycle in G2/M phase and promote cell apoptosis, which was
similar as the reference compound SAHA. Meanwhile, the
molecular docking and dynamic simulation studies were
performed to investigate the putative interaction mechanism with
the HDAC target. As a result, the potent HDAC inhibitory
activities were caused by the inhibitors which showed strong van
der Waals and electrostatic interactions with the HDAC and
tightly bound to the target. Additionally, the highlighted
compounds 15k and 15m showed attractive and practical
applications in the field of cancer therapy. Further studies on the
structural optimization and biological evaluation of 15k and 15m
are currently underway in our laboratory, and our study will
provide a basis for discovering novel HDAC inhibitors.
1
white solid (653.7 mg, 88.8%). Mp 202.5-204.0 °C. H NMR
(400 MHz, DMSO-d6) δ: 8.12 (s, 1H), 7.80 (d, J = 8.4 Hz, 1H),
7.45 (dd, J = 8.4, 1.7 Hz, 1H), 6.44 (s, 2H), 1.57 (s, 9H).
4.1.3 methyl 8-chloro-8-oxooctanoate (10)
The oxalyl chloride (0.91 mL, 10.8 mmol) was added into the
solution of methyl 8-chloro-8-oxooctanoate (9, 680 mg, 3.6
mmol) and dry DMF (0.15 mL) in dry DCM (20 mL), then the
mixture stirred at room temperature for 4 h. The excess oxalyl
chloride and solvent were evaporated under vacuum. The crude
product (10) of monomethyl acid chloride was directly used for
further reaction without any purification.
4.1.4 tert-butyl-6-bromo-3-(8-methoxy-8-oxooctanamido)-1H-
indazole-1-carboxylate (11a)
The previous monomethyl acid chloride (10, 741.9 mg, 3.6
mmol) was dissolved in dry DCM (20 mL) with DIPEA (1.4 mL,
10.8 mmol), and the mixture was stirred for 30 min. Then
compound 8 (863.2mg, 2.77 mmol) was added into the mixture
and stirred for 1 h at room temperature. The solvent was
evaporated under vacuum, and residue was purified by
chromatography on silica gel Petroleum-EtOAc (2:1) to give 11a
4.Experimental section
4.1 Chemistry
1
as white solid (810.0 mg, 46.8%). Mp 222.3-204.6 °C. H NMR
Solvents were distilled under the positive pressure of dry
argon before use and dried using standard methods. Chemicals
were obtained from local suppliers and were used without further
purification. All reactions were monitored by thin-layer
chromatography (silica gel 60 F254 glass plates). NMR spectra
were recorded on Bruker 400 MHz instruments, and the chemical
shifts were presented in terms of parts per million with TMS as
the internal reference. Electron-spray ionization mass spectra in
positive mode (ESI-MS) data were obtained with a Bruker
Esquire 3000+ spectrometer. Flash column chromatography was
performed on silica gel (200-300 mesh, Adamas, China).
(500 MHz, DMSO-d6) δ 11.03 (s, 1H), 8.26 (s, 1H), 7.96 (d, J =
8.6 Hz, 1H), 7.52 (dd, J = 8.7, 1.7 Hz, 1H), 3.81 (s, 3H), 2.40 (t,
J = 7.4 Hz, 2H), 1.96 (t, J = 7.4 Hz, 2H), 1.63 (s, 11H), 1.51-1.56
(m, 2H), 1.26-1.40 (m, 4H). ESI-MS (m/z): [M+H]+=483.2
(Calcd:483.40).
General method for preparation of compounds 15a-t (exemplified
by 15a)
4.1.5 tert-butyl 3-(8-methoxy-8-oxooctanamido)-6-phenyl-1H-
indazole-1-carboxylate (12a)
The intermediate 11a (790 mg, 1.64 mmol) was dissolved in
dioxane (10 mL) and water (5 mL). Then, Phenylboronic acid
(300 mg, 1.97 mmol), Pd(dppf)2Cl2 (60 mg), and K2CO3 (650
mg, 4.92 mmol). The reaction mixture was heated at 80 for 1 h
, then the mixture cooled to rt. The mixture was concentrated and
extracted with Ethyl acetate. The residue was washed with H2O
(30 mL), and brine (30 mL), dried over Na2SO4, and concentrated
in vacuo. The residue was purified by chromatography on silica
gel Petroleum-EtOAc (2:1) to give 12a as white solid (591.5 mg,
4.1.1 Bromo-1H-indazol-3-amine (7)
4-bromo-2-fluorobenzonitrile (6, 5.0 g, 25.1 mmol) was
dissolved in 1-butanol (20 mL), then followed by the addition of
NH2NH2(1.0 mL, 50.3 mmol). The reaction mixture was heated
to reflux for 4 h. Then cooled to rt, filtered, washed with n-
hexane and dried to give 7 as white solid (4.8 g, 85.7%). Mp
237.5-238.4 °C. 1H NMR (400 MHz, DMSO-d6) δ: 11.52 (s, 1H),
7.64 (d, J = 8.5 Hz, 1H), 7.37-7.52 (m, 1H), 7.02 (dd, J = 8.5, 1.6
Hz, 1H), 5.47 (s, 2H)..
1
75.2%). Mp 231.0-234.3 °C. H NMR (500 MHz, DMSO-d6) δ

10.96 (s, 1H), 8.07 (d, J = 8.6 Hz, 1H), 7.74 (d, J = 7.7 Hz, 2H),
7.64 (dd, J = 8.5, 1.6 Hz, 1H), 7.52 (t, J = 7.7 Hz, 1H), 7.36-7.46
(m, 3H), 3.58 (s, 3H), 2.46 (t, J = 7.6 Hz, 2H), 2.30 (t, J = 7.4 Hz,
2H), 1.65 (s, 11H), 1.52-1.57 (m, 2H), 1.28-1.38 (m, 4H). ESI-
MS (m/z): [M+H]+=480.3 (Calcd:480.59).
NMR (500 MHz, DMSO-d6) δ 12.68 (s, 1H), 10.34 (s, 2H), 8.67
(s, 1H), 7.84 (d, J = 8.6 Hz, 1H), 7.63 (s, 1H), 7.41 (t, J = 7.9 Hz,
1H), 7.36 (d, J = 8.7 Hz, 1H), 7.29 (d, J = 7.7 Hz, 1H), 7.24 (t, J
= 2.1 Hz, 1H), 3.85 (s, 3H), 2.40 (t, J = 7.4 Hz, 2H), 1.96 (t, J =
7.3 Hz, 2H), 1.64 (t, J = 7.2 Hz, 2H), 1.52 (t, J = 7.2 Hz, 2H),
1.27-1.40 (m, 4H). 13C NMR (126 MHz, DMSO-d6) δ 171.75,
169.58, 160.18, 142.54, 142.11, 140.84, 138.97, 130.46, 123.38,
119.95, 119.67, 116.11, 113.56, 113.07, 108.14, 55.62, 35.7,
32.74, 28.90, 25.55. ESI-MS (m/z): [M+H]+=411.1
(Calcd:411.20). HRMS: calcd for C22H27N4O4 (M+H)+411.2032,
found 411.2016.
4.1.6 8-oxo-8-((6-phenyl-1H-indazol-3-yl) amino) octanoic acid
(14a)
The solution of DCM (15 mL) with 11a (479.0 mg, 1 mmol)
was added TFA (5.0 mL) dropwise. The reaction mixture was
stirred at room temperature for 2h. After concentrated, TFA was
removed. Then, the residue was dissolved with DCM (20 mL)
and neutralized to pH 7-8 with saturated aqueous NaHCO3. The
organic phase was concentrated and purified chromatography on
silica gel Petroleum-EtOAc (1:1) to give 13a as colorless oil
4.1.9 N1-(6-(3-ethoxyphenyl)-1H-indazol-3-yl)-N8-
hydroxyoctanediamide (15c)
white solid: 98.3 mg, yield: 68.4%. Mp 226.9-229.5 °C. HPLC
1
(591.5 mg, 75.2%). ESI-MS
(Calcd:380.59).
(m/z): [M+H]+=380.1
analysis: MeOH-H2O (80: 20), 6.88 min, 97.2% purity. H NMR
(500 MHz, DMSO-d6) δ 12.69 (s, 1H), 10.36 (s, 2H), 8.70 (s,
1H), 7.83 (d, J = 8.5 Hz, 1H), 7.62 (s, 1H), 7.34-7.40 (m, 2H),
7.27 (d, J = 7.7 Hz, 1H), 7.22 (s, 1H), 6.95 (dd, J = 8.0, 2.5 Hz,
1H), 4.12 (q, J = 6.9 Hz, 2H), 2.40 (t, J = 7.4 Hz, 2H), 1.96 (t, J =
7.4 Hz, 2H), 1.61-1.67 (m, 2H), 1.49-1.55 (m, 2H), 1.26-1.41 (m,
7H). 13C NMR (126 MHz, DMSO-d6) δ 171.75, 169.55, 159.44,
142.51, 142.11, 140.82, 138.99, 130.46, 123.39, 119.83, 119.66,
116.07, 63.50, 35.74, 32.72, 28.90, 25.55, 15.17. ESI-MS (m/z):
[M+H]+=425.2 (Calcd:425.22). HRMS: calcd for C23H29N4O4
(M+H)+ 425.2189, found 425.2172.
The 13a (274.0 mg, 0.72 mmol) was dissolved with solution of
MeOH (10 mL) and water (4 mL) and added with LiOH (85 mg,
3.6 mmol). The reaction mixture was stirred at room temperature
for 4h. The mixture was concentrated, and the residue was
neutralized to pH 2-3 with 1M aqueous hydrochloric acid at 0 °C.
A solid was precipitated which was filtered, washed with cold
water, and dried in vacuum to afford 14a as a white solid (262.8
mg, 60.5%). Mp 214.3-216.7 °C. 1H NMR (500 MHz, DMSO-d6)
δ 12.59 (s, 1H), 11.87 (s, 1H), 10.35 (s, 1H), 8.10 (d, J = 8.4 Hz,
1H), 7.85 (d, J = 8.5 Hz, 1H), 7.78 (d, J = 7.4 Hz, 2H), 7.63 (dd,
J = 8.0, 1.6 Hz, 1H), 7.50 (t, J = 7.1 Hz, 1H), 7.34-7.45 (m, 2H),
2.41 (t, J = 7.4 Hz, 2H), 2.22 (t, J = 7.3 Hz, 2H), 1.64 (s, 11H),
1.53 (t, J = 7.2 Hz, 2H), 1.29-1.42 (m, 4H). ESI-MS (m/z): [M-
H]-=364.0 (Calcd:364.43).
4.1.10 N1-(6-(3-chlorophenyl)-1H-indazol-3-yl)-N8-
hydroxyoctanediamide (15d)
white solid: 75.2 mg, yield: 63.1%. Mp 220.1-223.6 °C. HPLC
1
analysis: MeOH-H2O (80: 20), 7.26 min, 96.2% purity. H NMR
4.1.7 N1-hydroxy-N8-(6-phenyl-1H-indazol-3-yl)-octanediamide
(15a)
(500 MHz, DMSO-d6) δ 12.78 (s, 1H), 10.40 (s, 2H), 8.72 (s, 1H),
7.87 (d, J = 8.5 Hz, 1H), 7.79 (s, 1H), 7.64-7.73 (m, 2H), 7.51 (t,
J = 7.8 Hz, 1H), 7.45 (d, J = 7.9 Hz, 1H), 7.37 (d, J = 8.6 Hz,
1H), 2.41 (t, J = 7.4 Hz, 2H), 1.97 (t, J = 7.4 Hz, 2H), 1.61-1.67
(m, 2H), 1.49-1.55 (m, 2H), 1.27-1.38 (m, 4H). 13C NMR (126
MHz, DMSO-d6) δ 171.79, 169.58, 143.19, 142.03, 140.88,
137.45, 134.18, 131.22, 127.76, 127.31, 126.34, 123.64, 119.45,
116.32, 108.48, 35.75, 32.74, 28.90, 25.54. ESI-MS (m/z):
[M+H]+=415.2 (Calcd:415.15). HRMS: calcd for C21H24ClN4O3
(M+H)+ 415.1537, found 415.1518.
To a stirred solution of NH2OTHP (300 mg, 2.55 mmol),
HATU (290 mg, 0.77 mmol) and K2CO3 (210 mg, 1.53 mmol) in
dry DMF (10 mL) was added 14a (200 mg, 0.51 mmol), and the
resulting mixture was heated at 40 °C for overnight. The reaction
solution was added brine (25 mL), and extracted with Ethyl
acetate (30 mL). The organic phase was washed with brine
(20mL), dried over Na2SO4, and concentrated in vacuo. The
crude product was directly used for further reaction without any
purification. The crude product (110 mg, 0.22 mmol) was
dissolved in saturated hydrochloric acid in1,4-dioxne (20 mL).
The reaction mixture was stirred at room temperature for 3h. The
solvent was removed, and the residue was stirred with saturated
aqueous NaHCO3 (15 mL) for 1h. The mixture was filtered, and
the residue was washed with cold water, DCM, MeOH and dried
in vacuum to afford the desired product 15a as a white solid
(122.6 mg, 63.2%). Mp 241.2-244.4 °C. HPLC analysis: MeOH-
H2O (80: 20), 6.75 min, 96.8% purity. 1H NMR (500 MHz,
DMSO-d6) δ 12.71 (s, 1H), 10.37 (s, 2H), 8.69 (s, 1H), 7.85 (d, J
= 8.5 Hz, 1H), 7.73 (d, J = 7.5 Hz, 2H), 7.62 (s, 1H), 7.50 (t, J =
7.6 Hz, 2H), 7.44-7.31 (m, 2H), 2.40 (t, J = 7.4 Hz, 2H), 1.96 (t,
J = 7.4 Hz, 2H), 1.61-1.67 (m, 2H), 1.49-1.55 (m, 2H), 1.26-1.40
(m, 4H). 13C NMR (126 MHz, DMSO-d6) δ 171.75, 169.57,
142.18, 141.00, 140.84, 139.07, 129.42, 127.94, 127.60, 123.47,
119.58, 116.01, 107.99, 35.75, 32.74, 28.90, 25.55. ESI-MS
(m/z): [M+H]+=381.2 (Calcd:381.19). HRMS: calcd for
C21H25N4O3 (M+H)+ 381.1927, found 381.1913.
4.1.11 N1-(6-(2-chlorophenyl)-1H-indazol-3-yl)-N8-
hydroxyoctanediamide (15e)
white solid: 80.3 mg, yield: 65.4%. Mp 226.9-229.5 °C. HPLC
1
analysis: MeOH-H2O (80: 20), 6.95 min, 97.4% purity. H NMR
(500 MHz, DMSO-d6) δ 12.70 (s, 1H), 10.37 (s, 2H), 8.67 (s,
1H), 7.82 (d, J = 8.5 Hz, 1H), 7.59 (dd, J = 7.2, 2.1 Hz, 1H),
7.41-7.50 (m, 4H), 7.09 (d, J = 8.5 Hz, 1H), 2.40 (t, J = 7.4 Hz,
2H), 1.96 (t, J = 7.4 Hz, 2H), 1.61-1.67 (s, 2H), 1.50-1.54 (m,
2H), 1.26-1.38 (m, 4H). 13C NMR (126 MHz, DMSO-d6) δ
171.76, 169.55, 142.08, 140.88, 139.80, 136.67, 132.82, 129.36,
123.64, 119.35, 116.15, 108.12, 35.74, 32.73, 28.90, 25.54. ESI-
MS (m/z): [M+H]+=415.1 (Calcd:415.15). HRMS: calcd for
C21H24ClN4O3 (M+H)+ 415.1537, found 415.1521.
4.1.12 N1-(6-(3,4-dimethoxyphenyl)-1H-indazol-3-yl)-N8-
hydroxyoctanediamide (15f)
white solid: 87.8 mg, yield: 63.6%. Mp 236.3-238.7 °C. HPLC
1
analysis: MeOH-H2O (80: 20), 7.42 min, 97.3% purity. H NMR
4.1.8 N1-hydroxy-N8-(6-(3-methoxyphenyl)-1H-indazol-3-
yl)octanediamide (15b)
(500 MHz, DMSO-d6) δ 12.62 (s, 1H), 10.35 (s, 1H), 10.34 (s,
2H), 8.68 (s, 1H), 7.81 (d, J = 8.5 Hz, 1H), 7.59 (s, 1H), 7.35 (d,
J = 8.6 Hz, 1H), 7.21-7.30 (m, 2H), 7.06 (d, J = 8.3 Hz, 1H), 3.87
(s, 3H), 3.81 (s, 3H), 2.40 (t, J = 7.4 Hz, 2H), 1.96 (t, J = 7.4 Hz,
2H), 1.62-1.65 (m, 2H), 1.48-1.55 (m, 2H), 1.28-1.40 (m, 4H).
White solid: 245 mg， yield: 60.5%. Mp 233.8-236.3 °C.
HPLC analysis: MeOH-H2O (80: 20), 7.16 min, 96.6% purity. 1H

13C NMR (126 MHz, DMSO-d6) δ 171.72, 169.57, 149.51,
149.04, 142.25, 140.82, 139.07, 133.72, 123.22, 119.77, 119.56,
115.70, 112.66, 111.28, 107.46, 56.06, 35.76, 32.73, 28.90,
25.54. ESI-MS (m/z): [M+H]+=441.1 (Calcd:441.21). HRMS:
calcd for C23H29N4O5 (M+H)+ 441.2138, found 441.2121.
25.55. ESI-MS (m/z): [M+H]+=411.3 (Calcd: 411.20). HRMS:
calcd for C22H27N4O4 (M+H)+ 411.2032, found 411.2017.
4.1.17 N1-hydroxy-N8-(6-(4-methoxyphenyl)-1H-indazol-3-
yl)octanediamide (15k)
4.1.13 N1-(6-(2-fluoro-5-methoxyphenyl)-1H-indazol-3-yl)-N8-
hydroxyoctanediamide (15g)
white solid: 70.5 mg, yield: 55.2%. Mp 218.9-221.2 °C. HPLC
analysis: MeOH-H2O (80: 20), 7.36 min, 96.7% purity. H NMR
1
(500 MHz, DMSO-d6) δ 12.64 (s, 1H), 10.36 (s, 1H), 10.33 (s,
1H), 8.69 (1H), 7.81 (d, J = 8.6 Hz, 1H), 7.67 (d, J = 8.7 Hz, 2H),
7.55 (s, 1H), 7.32 (d, J = 8.6 Hz, 1H), 7.05 (d, J = 8.7 Hz, 2H),
3.81 (s, 3H), 2.39 (t, J = 7.4 Hz, 1H), 1.96 (t, J = 7.4 Hz, 2H),
1.61-1.67 (m, 2H), 1.49-1.55 (m, 2H), 1.28-1.37 (m, 4H). 13C
NMR (126 MHz, DMSO-d6) δ 171.73, 169.55, 159.41, 142.30,
140.81, 138.75, 133.30, 128.65, 123.34, 119.35, 115.63, 114.85,
107.21, 55.65, 35.76, 32.73, 28.90, 25.55. ESI-MS (m/z):
[M+H]+=411.1 (Calcd: 411.20). HRMS: calcd for C21H27N4O4
(M+H)+ 411.2032, found 411.2016.
white solid: 79.7 mg, yield: 64.9%. Mp 232.3-234.8 °C. HPLC
analysis: MeOH-H2O (80: 20), 6.90 min, 96.6% purity. H NMR
1
(500 MHz, DMSO-d6) δ 12.74 (s, 1H), 10.38 (s, 1H), 10.35 (s,
1H) 8.68 (s, 1H), 7.82 (d, J = 8.6 Hz, 1H), 7.57 (s, 1H), 7.22-7.29
(m, 2H), 7.10 (dd, J = 6.4, 3.2 Hz, 1H), 6.96-7.00 (m, 1H), 3.81
(s, 1H), 2.40 (t, J = 7.4 Hz, 2H), 1.96 (t, J = 7.4 Hz, 2H), 1.61-
1.67 (m, 2H), 1.49-1.55 (m, 2H), 1.28-1.37 (m, 4H). 13C NMR
(126 MHz, DMSO-d6) δ 171.80, 169.57, 156.12, 154.88, 152.98,
141.60, 140.88, 133.63, 129.61, 129.49, 123.03, 121.11, 117.37,
117.17, 116.12, 115.97, 114.90, 110.59, 56.15, 35.74, 32.73,
28.90, 25.54. ESI-MS (m/z): [M+H]+=429.3 (Calcd:429.19).
HRMS: calcd for C22H26FN4O4 (M+H)+ 429.1938, found
429.1924.
4.1.18 N1-hydroxy-N8-(6-(3-(trifluoromethyl)phenyl)-1H-indazol-
3-yl)octanediamide (15l)
white solid: 68.4 mg, yield: 54.3%. Mp 232.7-235.0 °C. HPLC
1
4.1.14 N1-(6-(2-fluoro-3-methoxyphenyl)-1H-indazol-3-yl)-N8-
analysis: MeOH-H2O (80: 20), 7.26 min, 96.4% purity. H NMR
(500 MHz, DMSO-d6) δ 12.79 (s, 1H), 10.40 (s, 2H), 8.69 (s,
1H), 8.06 (d, J = 7.2 Hz, 1H), 8.03 (s, 1H), 7.89 (d, J = 8.6 Hz,
1H), 7.72-7.78 (m, 3H), 7.43 (dd, J = 8.5, 1.5 Hz, 1H), 2.41 (t, J
= 7.4 Hz, 2H), 1.96 (t, J = 7.4 Hz, 2 H), 1.61-1.67 (m, 2H), 1.49-
1.55 (m, 2H), 1.28-1.35(m, 4H). 13C NMR (126 MHz, DMSO-d6)
δ 171.80, 169.56, 142.06, 140.90, 137.37, 131.77, 130.53,
130.35, 130.10, 125.79, 124.54, 124.00, 123.75, 123.6, 119.48,
116.39, 108.73, 35.74, 32.73, 28.90, 25.54. ESI-MS (m/z):
[M+H]+=449.1 (Calcd: 449.20). HRMS: calcd for C22H24F3N4O4
(M+H)+449.1801, found 449.1788.
hydroxyoctanediamide (15h)
white solid: 77.6 mg, yield: 58.2%. Mp 230.7-233.2 °C. HPLC
analysis: MeOH-H2O (80: 20), 7.05 min, 96.5% purity. H NMR
1
(500 MHz, DMSO-d6) δ 12.75 (s, 1H), 10.38 (s, 1H), 10.36 (s,
1H), 7.84 (d, J = 8.5 Hz, 1H), 7.53 (s, 1H), 7.17-7.27 (m, 3H),
7.11 (t, J = 7.1 Hz, 1H), 3.89 (s, 3H), 2.40 (t, J = 7.4 Hz, 2H),
1.96 (t, J = 7.4 Hz, 2H), 1.61-1.67 (m, 2H), 1.49-1.54 (m, 2H),
1.25-1.36 (m, 4H). 13C NMR (126 MHz, DMSO-d6) δ 171.81,
169.46, 150.17, 148.29, 148.20, 141.60, 140.85, 133.47, 129.73,
124.98, 123.06, 122.34, 121.12, 116.08, 113.37, 110.58, 56.55,
35.73, 32.70, 28.89, 25.56. ESI-MS (m/z): [M+H]+=429.4
(Calcd:429.19). HRMS: calcd for C22H26FN4O4 (M+H)+
429.1938, found 429.1922.
4.1.19 N1-(6-(4-chlorophenyl)-1H-indazol-3-yl)-N8-
hydroxyoctanediamide (15m)
white solid: 65.5 mg, yield: 63.4%. Mp 216.5-218.8 °C. HPLC
1
4.1.15 N1-(6-(2-fluorophenyl)-1H-indazol-3-yl)-N8-
analysis: MeOH-H2O (80: 20), 6.96 min, 95.8% purity. H NMR
(500 MHz, DMSO-d6) δ 12.73 (s, 1H), 10.37 (s, 1H), 10.35 (s,
1H), 8.67 (s, 1H), 7.86 (d, J = 8.6 Hz, 1H), 7.76 (d, J = 8.6 Hz,
2H), 7.63 (s, 1H), 7.55 (d, J = 8.5 Hz, 2H), 7.35 (d, J = 8.5 Hz,
1H), 2.40 (t, J = 7.4 Hz, 2H), 1.96 (t, J = 7.4 Hz, 2H), 1.60-1.69
(m, 2H), 1.48-1.56 (m, 2H), 1.27-1.40 (m, 4H). 13C NMR (126
MHz, DMSO-d6) δ 171.76, 169.55, 142.08, 140.88, 139.80,
137.67, 132.82, 129.36, 123.64, 119.35, 116.15, 108.12, 35.74,
32.73, 28.90, 25.54. ESI-MS (m/z): [M+H]+=415.3 (Calcd:
415.15). HRMS: calcd for C21H24ClN4O3 (M+H)+ 415.1537,
found 415.1522.
hydroxyoctanediamide (15i)
white solid: 80.6 mg, yield: 64.5%. Mp 227.5-229.3 °C. HPLC
analysis: MeOH-H2O (80: 20), 6.84 min, 96.1% purity. H NMR
1
(500 MHz, DMSO-d6) δ 12.74 (s, 1H), 10.38 (s, 1H), 10.35 (s,
1H) 8.67 (s, 1H), 7.85 (d, J = 8.5 Hz, 1H), 7.61 (td, J = 7.9, 1.8
Hz, 1H), 7.56 (s, 1H), 7.43-7.47 (m, 1H), 7.32-7.36 (m, 2H), 7.22
(d, J = 8.5 Hz, 1H), 2.40 (t, J = 7.4 Hz, 2H), 1.96 (t, J = 7.4 Hz,
2H), 1.61-1.67 (m, 2H), 1.49-1.55 (m, 2H), 1.28-1.37 (m, 4H).
13C NMR (126 MHz, DMSO-d6) δ 171.80, 169.57, 160.60,
158.65, 141.64, 140.88, 133.58, 131.59, 130.07, 129.03, 128.92,
125.43, 123.09, 121.13, 116.69, 116.51, 116.07, 110.53, 35.74,
32.74, 28.90, 25.54. ESI-MS (m/z): [M+H]+=399.4
(Calcd:399.18). HRMS: calcd for C21H24FN4O3 (M+H)+
399.1832, found 399.1802.
4.1.20 N1-(6-(3,5-dimethoxyphenyl)-1H-indazol-3-yl)-N8-
hydroxyoctanediamide (15n)
white solid: 73.6 mg, yield: 62.4%. Mp 241.2-243.6 °C. HPLC
1
analysis: MeOH-H2O (80: 20), 7.42 min, 97.1% purity. H NMR
4.1.16 N1-hydroxy-N8-(6-(2-methoxyphenyl)-1H-indazol-3-
yl)octanediamide (15j)
(500 MHz, DMSO-d6) δ 12.68 (s, 1H), 10.36 (s, 2H), 8.67 (s,
1H), 7.83 (d, J = 8.5 Hz, 1H), 7.62 (s, 1H), 7.35 (d, J = 9.4 Hz,
1H), 6.84 (d, J = 2.2 Hz, 2H), 6.53 (t, J = 2.2 Hz, 1H), 3.83 (s,
6H), 2.40 (t, J = 7.4 Hz, 2H), 1.96 (t, J = 7.4 Hz, 2H), 1.62-1.65
(m, 2H), 1.50-1.53 (m, 2H), 1.30-1.35 (m, 4H). 13C NMR (126
MHz, DMSO-d6) δ 171.74, 169.55, 161.27, 143.23, 142.02,
140.82, 139.03, 123.31, 119.70, 116.18, 108.21, 105.73, 99.87,
55.76, 35.74, 32.73, 28.90, 25.54. ESI-MS (m/z): [M+H]+=441.3
(Calcd: 441.21). HRMS: calcd for C23H29N4O5 (M+H)+ 441.2138,
found 441.2123.
white solid: 82.7 mg, yield: 64.9%. Mp 220.6-222.5 °C. HPLC
1
analysis: MeOH-H2O (80: 20), 7.10 min, 95.9% purity. H NMR
(500 MHz, DMSO-d6) δ 12.61 (s, 1H), 10.35 (s, 1H), 10.32 (s,
1H), 8.68 (s, 1H), 7.74 (d, J = 8.5 Hz, 1H), 7.46 (s, 1H), 7.34-
7.39 (m, 2H), 7.14 (d, J = 8.3 Hz, 2H), 7.06 (td, J = 7.5, 1.1 Hz,
1H), 3.78 (s, 3H), 2.39 (t, J = 7.4 Hz, 2H), 1.96 (t, J = 7.4 Hz,
1H), 1.62-1.65 (m, 2H), 1.49-1.53 (m, 2H), 1.30-1.35 (m, 4H).
13C NMR (126 MHz, DMSO-d6) δ 171.75, 169.58, 160.18,
142.54, 142.11, 140.84, 138.97, 130.46, 123.38, 119.95, 119.67,
116.11, 113.56, 113.07, 108.14, 55.62, 35.75, 32.74, 28.90,
4.1.21 N1-hydroxy-N8-(6-(pyridin-4-yl)-1H-indazol-3-
yl)octanediamide (15o)

white solid: 62.3 mg, yield 60.3%. Mp 230.9-232.7 °C. HPLC
analysis: MeOH-H2O (80: 20), 7.14 min, 96.6% purity. H NMR
2H), 1.97 (t, J = 7.2 Hz, 2H), 1.63-1.67 (m, 2H), 1.52-1.58 (m,
2H), 1.30-1.36 (m, 2H). 13C NMR (126 MHz, DMSO-d6) δ
171.71, 169.53, 160.18, 142.53, 142.11, 140.80, 139.00, 130.46,
123.42, 119.94, 119.68, 116.06, 113.55, 113.08, 108.16, 55.61,
35.66, 32.66, 28.75, 25.41. ESI-MS (m/z): [M+H]+=397.2 (Calcd:
397.19). HRMS: calcd for C21H25N4O4 (M+H)+397.1876, found
397.1864.
1
(500 MHz, DMSO-d6) δ 12.86 (s, 1H), 10.42 (s, 1H), 10.36 (s, 1
H), 8.68 (s, 1H), 8.63-8.67 (m, 2H), 7.91 (d, J = 8.5 Hz, 1H),
7.80 (s, 1H), 7.76-7.79 (m, 2H), 7.47 (dd, J = 8.5, 1.5 Hz, 1H),
2.40 (t, J = 7.4 Hz, 2H), 1.96 (t, J = 7.4 Hz, 2H), 1.63-1.66 (m,
2H), 1.52-1.53 (m, 2H), 1.28-1.35 (m, 4H). 13C NMR (126 MHz,
DMSO-d6) δ 171.75, 169.57, 142.18, 140.99, 140.84, 139.07,
129.42, 127.94, 127.60, 123.47, 119.58, 116.01, 107.99, 35.75,
32.74, 28.90, 25.55. ESI-MS (m/z): [M+H]+=382.3 (Calcd:
382.19). HRMS: calcd for C20H24N5O3 (M+H)+ 382.1879, found
382.1865.
4.1.26 N1-hydroxy-N4-(6-(3-methoxyphenyl)-1H-indazol-3-yl)-
cyclohexane-1,4-dicarboxamide (15t)
white solid 69.4 mg; yield 68.5%. Mp 218.2-221.0 °C. HPLC
1
analysis: MeOH-H2O (80: 20), 7.03 min, 96.0% purity. H NMR
(500 MHz, DMSO-d6) δ 12.73 (s, 1H), 10.44 (s, 1H), 10.33 (s,
1H), 8.75 (s, 1H), 7.82 (d, J = 8.5 Hz, 1H), 7.63 (s, 1H), 7.40 (t, J
= 7.9 Hz, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.28 (d, J = 8.3 Hz, 1H),
6.97 (dd, J = 8.2, 2.6 Hz, 1H), 3.84 (s, 3H), 1.89-2.10 (m, 4H),
1.76 (s, 2H), 1.45-1.58 (m, 4H). 13C NMR (126 MHz, DMSO-d6)
δ 174.53, 172.36, 160.18, 142.53, 142.10, 140.79, 138.96,
130.46, 123.28, 119.96, 119.64, 116.12, 113.53, 113.10, 108.17,
55.62, 43.61, 28.87, 28.74. ESI-MS (m/z): [M+H]+=409.2 (Calcd:
409.19). HRMS: calcd for C22H25N4O4 (M+H)+ 409.1876, found
409.1863.
4.1.22 N1-hydroxy-N8-(6-(thiophen-3-yl)-1H-indazol-3-
yl)octanediamide (15p)
white solid: 84.5 mg, yield 70.3%. Mp 227.5-239.4 °C. HPLC
analysis: MeOH-H2O (80: 20), 7.36 min, 97.4% purity. H NMR
1
(500 MHz, DMSO-d6) δ 12.67 (s, 1H), 10.34 (s, 2H), 8.71 (s,
1H), 7.95 (s, 1H), 7.80 (d, J = 8.5 Hz, 1H), 7.63-7.69 (m, 3H),
7.44 (d, J = 8.6 Hz, 1H), 2.40 (t, J = 7.4 Hz, 2H), 1.97 (t, J = 7.4
Hz, 2H), 1.61-1.67 (m, 2H), 1.51-1.55 (m, 2H), 1.28-1.38 (m,
4H).13C NMR (126 MHz, DMSO-d6) δ 171.73, 169.56, 142.15,
142.11, 140.90, 133.85, 127.56, 126.95, 123.37, 121.89, 119.16,
115.78, 107.11, 35.76, 32.74, 28.90, 25.55. ESI-MS (m/z):
[M+H]+=387.3 (Calcd: 387.15). HRMS: calcd for C19H22N4O3S
(M+H)+ 387.1491, found 387.1478.
4.1.27 N1-hydroxy-N8-(6-(3-methoxyphenyl)-1H-pyrazolo[3,4-b]
pyridin-3-yl) octane-diamide (21a)
white solid 70.7 mg; yield 61.5%. Mp 231.9-234.3 °C. HPLC
1
4.1.23 N1-hydroxy-N5-(6-(3-methoxyphenyl)-1H-indazol-3-yl)-
analysis: MeOH-H2O (85: 15), 7.36 min, 97.4% purity. H NMR
(500 MHz, DMSO-d6) δ 13.20 (s, 1H), 10.64 (s, 1H), 10.38 (s,
1H), 8.70 (s, 1H), 8.45 (d, J = 8.6 Hz, 1H), 7.73 (t, J = 8.4Hz,
2H), 7.70 (t, J = 2.1Hz, 1H), 7.45 (t, J = 7.9 Hz, 1H), 7.06 (dd, J
= 8.2, 2.7 Hz, 1H), 3.86 (s, 3H), 2.41 (t, J = 7.4 Hz, 2H), 1.96 (t,
J = 7.4 Hz, 2H), 1.61-1.67 (m, 2H), 1.48-1.54 (m, 2H), 1.27-1.36
(m, 4H). 13C NMR (126 MHz, DMSO-d6) δ 171.75, 169.57,
142.18, 140.99, 140.84, 139.07, 129.42, 127.94, 127.60, 123.47,
119.58, 116.01, 107.99, 66.81, 35.75, 32.74, 28.90, 25.55. ESI-
MS (m/z): [M+H]+=412.5 (Calcd: 412.20). HRMS: calcd for
C21H26N5O4 (M+H)+ 412.1985, found 412.1973.
glutaramide (15q)
white solid: 73.0 mg, yield 63.2%. Mp 210.5-213.3 °C. HPLC
analysis: MeOH-H2O (80: 20), 6.50 min, 96.2% purity. H NMR
1
(500 MHz, DMSO-d6) δ 12.70 (s, 1H), 10.44 (s, 1H), 10.39 (s,
1H), 8.73 (s, 1H), 7.86 (d, J = 8.5 Hz, 1H), 7.63 (s, 1H), 7.41 (t, J
= 7.9 Hz, 1H), 7.36 (d, J = 8.6 Hz, 1H), 7.29 (d, J = 7.7 Hz, 1H),
7.24 (s, 1H), 6.97 (dd, J = 8.2, 2.5 Hz, 1H), 3.85 (s, 3H), 2.42 (t,
J = 7.4 Hz, 2H), 2.07 (t, J = 7.5 Hz, 2H), 1.84-1.90 (m, 2H). 13C
NMR (126 MHz, DMSO-d6) δ 171.26, 169.19, 160.18, 142.54,
142.11, 140.79, 138.98, 130.46, 123.44, 119.95, 119.65, 116.09,
113.55, 113.09, 108.14, 55.61, 35.07, 32.15, 21.71. ESI-MS
(m/z): [M+H]+=369.4 (Calcd: 369.16). HRMS: calcd for
C19H21N4O4 (M+H)+ 369.1563, found 369.1552.
4.1.27 N1-hydroxy-N8-(6-(4-methoxyphenyl)-1H-pyrazolo[3,4-b]-
pyridin-3-yl)- octanediamide (21b)
white solid 72.9 mg; yield 55.2%. Mp 225.4-228.5 °C. HPLC
1
4.1.24 N1-hydroxy-N6-(6-(3-methoxyphenyl)-1H-indazol-3-yl)-
analysis: MeOH-H2O (85: 15), 7.52 min, 96.3% purity. H NMR
(500 MHz, DMSO-d6) δ 13.10 (s, 1H), 10.63 (s, 2H), 8.40 (d, J =
8.5 Hz, 1H), 8.12 (d, J = 8.8 Hz, 2H), 7.67 (d, J = 8.7 Hz, 1H),
7.08 (d, J = 8.5 Hz, 2H), 3.84 (s, 3H), 2.40 (t, J = 7.3 Hz, 1H),
1.95 (t, J = 7.4 Hz, 2H), 1.59-1.67 (m, 2H), 1.49-1.53 (m, 2H),
1.25-1.40 (m, 4H). 13C NMR (126 MHz, DMSO-d6) δ 171.73,
169.55, 159.41, 142.30, 140.81, 138.75, 133.30, 128.65, 123.34,
119.35, 115.63, 114.85, 107.21, 55.65, 35.76, 32.73, 28.90, 25.55.
ESI-MS (m/z): [M+H]+=412.4 (Calcd: 412.20). HRMS: calcd for
C21H26N5O4 (M+H)+ 412.1985, found 412.1981.
adipamide (15r)
white solid: 58.8 mg, yield 67.2%. Mp 216.4-218.7 °C. HPLC
analysis: MeOH-H2O (80: 20), 6.66 min, 96.4% purity. H NMR
1
(500 MHz, DMSO-d6) δ 12.70 (s, 1H), 10.39 (s, 1H), 10.37 (s,
1H), 8.71 (s, 1H), 7.84 (d, J = 8.5Hz, 1H), 7.63 (s, 1H), 7.41 (t, J
= 7.9 Hz, 1H), 7.36 (d, J = 8.6 Hz, 1H), 7.29 (d, J = 7.8 Hz, 1H),
7.24 (s, 1H), 6.97 (dd, J = 8.2, 2.4 Hz, 1H), 3.85 (s, 3H), 2.41 (t,
J = 7.2 Hz, 2H), 2.02 (t, J = 7.0 Hz, 2H), 1.57-1.66 (m, 4H). 13C
NMR (126 MHz, DMSO-d6) δ 176.34, 174.19, 164.94, 147.28,
146.86, 145.57, 143.73, 135.22, 128.21, 124.70, 124.43, 120.84,
118.31, 117.83, 112.95, 60.37, 40.34, 37.42, 30.12. ESI-MS
(m/z): [M+H]+=383.4 (Calcd: 383.17). HRMS: calcd for
C20H23N4O4 (M+H)+383.1719, found 383.1706.
4.2 HDAC inhibition assay
For the HDACs assay, the recombinant human HDAC1, 2 and
8 were purchased from BPS Bioscience (USA). All reactions
were performed in the black half area 96-well microplates. A
serial dilution of the inhibitors and enzymes were pre-incubated
at 25 for 15 min, and then fluorogenic substrate Boc-Lys(Ac)-
AMC (for HDAC1 and HDAC2) or Boc-Lys(TFA)-AMC (for
HDAC8) was added. After incubation at 37 for 60 min, the
mixture was stopped by the addition of 25 µL of developer
containing trypsin and SAHA for 10 min. Fluorescence intensity
was measured using the Thermo Scientific Varioskan Flash
Station at excitation and emission wavelengths of 355 and 460
4.1.25 N1-hydroxy-N7-(6-(3-methoxyphenyl)-1H-indazol-3-yl)-
heptanediamide (15s)
white solid 65.2 mg; yield 62.5%. Mp 222.7-225.4 °C. HPLC
analysis: MeOH-H2O (80: 20), 6.72 min, 96.8% purity. H NMR
(500 MHz, DMSO-d6) δ 12.71 (s, 1H), 10.38 (s, 2H), 7.85 (d, J =
8.5 Hz, 1H), 7.63 (s, 1H), 7.41 (t, J = 7.9 Hz, 1H), 7.36 (d, J =
8.5 Hz, 1H), 7.29 (d, J = 7.9 Hz, 1H), 7.24 (t, J = 2.1 Hz, 1H),
6.97 (dd, J = 8.2, 1.6 Hz, 0H), 3.85 (s, 3H), 2.40 (t, J = 7.4 Hz,
1

nm, respectively. The IC50 values were extracted by curve fitting
the dose/response slopes.
Molecular docking of compounds 15k, 15m and 21b into the
three-dimensional X-ray structure of HDAC2 (PDB ID: 4LXZ)
was carried out using the Glide-dock module of the Schrodinger
Maestro 2019. The three-dimensional structure of compound was
constructed using ChemBio 3D Ultra software, then it was
energetically minimized by using MMFF94 with 5000 iterations
and minimum RMS gradient 0.05. Furthermore, the ligands were
also prepared by Ligand Prepare module of Schrodinger Maestro
2019. The possible states of ligands were calculated by Epik
considering metal binding states of the ligand. The protein was
prepared by the Protein preparation wizard of Schrodinger
Maestro 2019. The waters were eliminated from the protein and
the polar hydrogen was added, and the protonation states of
charged residues in HDAC2 were also processed. Receptor grids
were generated using Receptor Grid Generation. The generated
binding site was just the zinc-binding pocket of HDAC2,
including several key amino acid residues.
4.3 Cell antiproliferative assay
In vitro anti-proliferative activity of synthesized compounds
was evaluated by MTT method on HCT-116, MCF-7 and HeLa
cells. Briefly, cells (5 × 104 cells/well) were seeded into 96-well
plates and incubated in 1640 (or DEME) culture medium
containing 100 IU/mL penicillin, 100 µg/mL streptomycin and
10% FBS under 37 °C, 5%CO2 overnight. Various concentrations
of compounds were introduced into wells and incubated for 72 h.
After incubation, 10 µL MTT solution (5 mg/mL) was added into
each well and incubated for another 4 h. Finally, the formazan
crystal was dissolved with dimethyl sulfoxide (DMSO) and the
absorbance was read at 490 nm using a microplate reader (Tecan
Infinite 200 PRO, Mannedorf, Switzerland). The IC50 was
calculated by GraphPad Prism 7.0 based on the ratio of the
absorbance values of treated and untreated cells.
4.8 Molecular dynamic simulation
4.4 Western blot analysis
To assess the binding stability of identified inhibitors against
HDAC2 active site, a 100 ns long molecular dynamics simulation
was carried out, which performed with Desmond in Schrodinger
Maestro 2019. Simulation procedure and parameter settings of
the MD simulations were similar to the previous works [33]. The
model was solvated in a truncated octahedron box of the
transferable interaction potential (TIP3P) water molecules with a
margin distance of 10 Å. Sodium ions were added to neutralize
the systems. The particle mesh Ewald (PME) method was used
for long-range electrostatic interactions with a cutoff distance of
10 Å. Next, the entire setup was subjected to a stepwise
equilibration procedure: The first minimization with 2500 steps
of steepest descent and 2500 steps of conjugate gradient was
performed with the protein, Zn2+ and ligand fixed by 10.0
kcal/(mol·Å2). Then, 5000 steps of steepest descent and 15000
steps of the conjugate gradient were employed in the whole
system with the constraints switched off. After energy
minimization, the whole system was gradually heated from 0K to
300 K for 60 ps under NVT, followed by a 600 ps NPT
simulation at 1 atm, with harmonic restraints of 2 kcal/(mol·Å2)
on the complex. Subsequently, 100 ns MD simulations were
performed in NPT ensemble for each system. The temperature
and pressure were kept constant using a Langevin thermostat and
a Langevin barostat, respectively. All hydrogen atoms were
constrained by the SHAKE algorithm. The resulting trajectories
were analyzed with the Desmond module. Subsequently, the
binding free energies (∆Gbind) for the complexes were calculated
by using MM-GBSA programs.
The HCT116 cells treated with each compound or DMSO at
various concentration for eight hours were washed twice with
PBS. Appropriate amount of RIPA (Beyotime) lysis buffer with
protease and phosphatase inhibitors (ThermoFisher) were added,
mixed thoroughly and lysed on ice for 30 minutes, and then fully
disrupted by an ultrasonic disrupter for 10 seconds. Protein
quantification was performed by BCA Protein Concentration
Determination Kit (Beyotime) following centrifugation at 12000
rpm for 15 minutes at 4 . The 40 ug of cell lysates were
separated by SDS-PAGE at 100V constant voltage for 2 hours,
and transferred to PVDF membranes at 40V for 70 minutes.
After 5% skim milk was blocked for 1 hour, the primary antibody
was added and incubated overnight before being washed three
times with PBST. The secondary antibody was incubated for 2
hours at room temperature the next day and subsequently washed
three times with PBST. Then the signal was detected with ECL
(ThermoFisher) using Tanon 5200.
The primary antibodies from Abcam company used for
western blot were rabbit anti-Histone H3, rabbit anti-Histone
H3(acetyl K9+K14+K18+K23+K27), rabbit anti- Tubulin (acetyl
K40) and rabbit anti- Tubulin. The secondary antibodies was
anti-rabbit HRP (1:10000, Jackson ImmunoResearch).
4.5 Cell cycle analysis
HCT116 cells were seeded into 6-well plates at 1×106 cells
per well with complete medium overnight. The old cultured
medium was replaced by fresh medium containing each
compound of 3 µM concentrations. After 24 hours incubation,
cells were collected and rinsed twice with PBS, then fixed with
75% cold ethanol at -20 overnight and stained for 30min with
PI/RNase staining buffer (BD Pharmingen). Flow cytometry was
performed and analyzed by ACEA NovoCyte.
Disclosure statement
There are no conflicts to declare.
Acknowledgment
4.6 Cell apoptosis analysis
This work was supported by the National Natural Science
Foundation of China (grants 81703342, 81473110, 81773596),
Natural Science Foundation of Jiangsu (grants BK20161050),
Natural Science Foundation of Jiangsu Higher Education
Institutions (grant: 17KJA360004), Postgraduate Research &
Practice Innovation Program of Jiangsu Province (grants
SJCX18_0448, KYCX18_1614).
HCT116 (1×106 cells/well) were seeded into 6-well plates
with complete medium overnight. The old cultured medium was
removed and fresh medium containing each compound of various
concentrations was then added into each well immediately. After
24 hours incubation, cells were rinsed twice with PBS and
detached by EDTA-free trypsin. Flowing annexin V-FITC/PI
staining for 10min using Annexin V-FITC/PI Apoptosis
Detection Kit (A211 Vazyme), Cell apoptosis was performed by
ACEA NovoCyte and analyzed by FlowJo_V10.
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Highlights
(1)、Based on fragment-drug design strategies, a novel series of indazole derivatives
were designed and synthesized as histone deacetylases (HDACs) inhibitors.
(2)、Representative compounds 15k and 15m exhibited potent HDAC inhibitory
activities against HDAC1, 2 and 8.
(3)、15k and 15m exhibited higher antiproliferative activities than the positive drug
SAHA toward HCT-116 and HeLa.
(4)、15k and 15m arrested cell cycle in G2/M phase and promoted cell apoptosis,
which was similar as SAHA.

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: