Design, synthesis and biological evaluation of selective histone deacetylase 6 (HDAC6) inhibitors bearing benzoindazole or pyrazoloindazole scaffold as surface recognition motif

Article abstract of DOI:10.1016/j.bioorg.2021.104910

A series of compounds were designed and synthesized based on the compound 11i bearing phenylpyrazole scaffold with histone deacetylase 6 (HDAC6) inhibitory activity. Most of the compounds showed considerable inhibitory activity against HDAC6 and compound

Full text of DOI:10.1016/j.bioorg.2021.104910

Bioorganic Chemistry 111 (2021) 104910
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis and biological evaluation of selective histone deacetylase
6
(HDAC6) inhibitors bearing benzoindazole or pyrazoloindazole scaffold
as surface recognition motif
a
,
1
b
,
1
a
c
a
a
Qihao Xu , Yanhua Mou , Siyuan Wang , Xiaoxiao Gao , Yulong Zhang , Zhi Wang ,
a
a
a
a
a,
*
a,*
Xiangwei Xu , Yu Han , Wenlong Jia , Meihui Zhang , Linxiang Zhao , Dan Liu
a
Key Laboratory of Structure-Based Drugs Design & Discovery of Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China
Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang 110016, China
b
c
Department of Pharmacology, Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang 110016, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of compounds were designed and synthesized based on the compound 11i bearing phenylpyrazole
scaffold with histone deacetylase 6 (HDAC6) inhibitory activity. Most of the compounds showed considerable
inhibitory activity against HDAC6 and compound A16 with good inhibitory activity was found therein. We
HDAC6
Inflammatory
Neuroprotective
further found that A16 had an inhibitory effect on inflammatory mediators (NO, TNF- , IL-6) involved in in-
α
flammatory response and neuroendocrine regulation. In addition, A16 has a certain neuroprotective effect on
PC12 cells injured by hydrogen peroxide. Acute toxicity assay showed that the LD50 of A16 was 274.47 mg/kg in
mouse model. Furthermore, A16 displayed good stability properties in microsomes and plasma.
1
. Introduction
To date, eighteen mammalian histone deacetylase (HDAC) subtypes
inhibitors can stimulate the secretion of neurotrophic factors, helping
neurons and synapses remodeling, promoting nerve and angiogenesis of
ischemic tissue, and promoting regeneration and repair of damaged
tissue [6]. Notably, the selective inhibitors to HDAC6 are more effective
for ischemic stroke and less side effects than broad-spectrum HDACs
inhibitors [7].
have been identified. They are divided into four classes based on their
homology to yeast original enzymes, of which class I, class II and class IV
are classic zinc-dependent HDAC families [1,2]; while class III is nico-
tinamide adenine-dinucleotide dependent HDAC family. In class II, there
are IIa and IIb subclasses. HDAC6 belongs to the HDAC IIb subclass and
consists of 1215 amino acids.
At present, there are many HDAC6 inhibitors have been reported,
and some have entered the clinical research, such as ACY-1215, ACY-
241, KA2507, etc. But the inhibitors reported have not shown high
selectivity, also showed a certain degree of side effects. ACY-1215,
studying in clinical phase II, is only used in anti-tumor applications
with low selectivity [8–12]. Although the indications of HDAC6 in-
hibitors in clinical research have not been applied to neuroprotection or
inflammation research, HDAC6 has an important role in neuro-
protection that cannot be ignored, which has been reported in many
literatures [13–16].
Many reports have shown that the dysfunction of HDAC6 is associ-
ated with several pathology in the central nervous system [3,4]. One of
the common neurological diseases is stroke, caused by the loss of local
nerve function due to impaired blood circulation resulting in cell death
in the brain. Some researchers have proved that the expression of
HDAC6 was significantly up-regulated in both in vitro and in vivo
ischemic models. They actively introduced HDAC6 inhibitors into the
field of prevention and treatment for ischemic stroke. The perturbation
of HDAC6 in injured brain tissue resulting in increased level of histone
acetylation significantly reduced the area of cerebral infarction and
neuronal apoptosis [4,5]. In the late stage of ischemic stroke, HDAC6
In our previous work, the compound 11i bearing phenylpyrazole
scaffold was found [17], whose IC50 against HDAC6 was 20 nM. The
selective factor (SF) for HDAC1 (IC50 = 2.02
μ
M) was 101. SF represents
the selectivity of the compound to HDAC6, and its value is the ratio of
*
Corresponding authors.
E-mail addresses: linxiang.zhao@vip.sina.com (L. Zhao), sammyld@163.com (D. Liu).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2021.104910
Received 16 November 2020; Received in revised form 20 February 2021; Accepted 7 April 2021
Available online 20 April 2021
0
045-2068/© 2021 Published by Elsevier Inc.

Q. Xu et al.
Bioorganic Chemistry 111 (2021) 104910
the IC50 of HDAC1 to HDAC6. The larger the value, the higher the
selectivity. The result of molecular docking of 11i and HDAC6 showed
that the surface recognition moiety (SRM) of 11i interacted with the
amino acid residues Pro501, Phe620, Phe680 and His651 of the hy-
drophobic pocket of HDAC6 protein (Fig. 1).
In order to increase interaction between the SRM of 11i and HDAC6,
the volume of SRM of 11i was increased through the conformation
constrained strategy, then the four compounds with benzoindazole (A1-
A4) were designed and synthesized (Fig. 2).
In addition, TS-15, bearing isoxazoloindazole scaffold, was also ob-
tained in the previous work, whose IC50 against HDAC6 was 9 nM. The
Fig. 2. The design strategy of compounds A1-A4.
SF for HDAC1 (IC50 = 0.59
μ
M) was 65. We combined 11i and TS-15 for
with DMF-DMA (N, N-dimethylformamide dimethyl acetal) and hydra-
zine hydrochloride to obtain the key intermediate M2. The nuclear
substitution reaction yields intermediates 5-1, 7-1, 9-1, 11-1, and then
condensation reaction with appropriate acyl chloride, cyclization reac-
tion with hydrazine hydrate construct the key intermediates 5-3-12-3,
superimposed molecular docking and found that the part of SRM in the
two compounds could occupy the hydrophobic region of the entire
protein (Fig. 3). Therefore, we combined the SRM regions of the two
compounds to obtain a series of compounds bearing pyrazoloindazole
scaffold (Fig. 4).
1
7-3-19-3 bearing pyrazoloindazole scaffold. Nucleophilic substitution
reaction with halogenated compounds, ammonolysis reaction of esters
2
. Results and discussion
with hydroxylamine aqueous solution obtain target compounds A5-A34
(
Scheme 2).
2
.1. Chemistry
2
2
.2. Biological activity
Using 6-methoxy-1-naphthylenone (1-1) or 1-tetralone (3-1) as the
starting material, a condensation reaction with appropriate acyl chlo-
ride yields an intermediate 1-2-4-2. Then, a cyclization reaction with
hydrazine hydrate is carried out to construct a key intermediate 1-3-4-3
with benzoindazole scaffold. Nucleophilic substitution reaction with a
halide, an ammonolysis reaction of ester with aqueous hydroxylamine
solution get the final target compound A1-A4 (Scheme 1).
.2.1. Enzyme activity test of the compounds in vitro
Since the hit compounds 11i and TS-15 had no enzyme inhibitory
activity on HDAC8 and HDAC11 at the maximum tested concentration
HDAC8, 11i, IC50 > 5 M, TS-15, IC50 > 10 M; HDAC11, TS-15, IC50
10 M), we mainly performed HDAC1, 2, 3 and 6 enzyme activity tests
(
μ
μ
>
μ
on the compounds. Tubacin and ACY-1215 were used as positive con-
trols. The results indicated that all the compounds showed certain
inhibitory activity against HDAC6, and the IC50 ranged from 2.2 nM to
With 1,3-cyclohexanedione as the starting material, it was reacted
5
50 nM. Among them, A12, A17, A19, A20, A21, A23 and A30 had
single digit nanomole inhibitory activity against HDAC6, and their
inhibitory activity was also better than that of ACY-1215 (Tables 1 and
2
).
Based on the enzyme activity results, we could roughly summarize
the preliminary structure-activity relationship (SAR): a. When the tri-
cyclic system was pyrazoloindazole, the enzyme activity was better; b.
In the target compounds with pyrazoloindazole as SRM, the order in
which the R
CH CH OCH
which the R
CH
1
substituents affect the inhibitory activity of HDAC6 was: 6-
> 7-CH CH OCH > 7-CH > 7-CH CH ; the order in
substituents affect the inhibitory activity of HDAC6 was:
2
2
3
2
2
3
3
2
3
2
–
3
> –Ph>– Bn; c. When a vinyl group was inserted into the linker,
the inhibitory activity of most compounds on HDAC6 was reduced.
According to the summarized SAR, we obtained compound A16 with
good HDAC6 inhibitory activity.
2
.2.2. Evaluation of inhibitory activity of compounds against NO release
The increase of NO release is one of the important signs of neuronal
inflammation. Therefore, we evaluated the inhibitory activity of the
compounds with good inhibitory activities against HDAC6 on the release
of NO. Lipopolysaccharide (LPS) induced microglial cell inflammation
model was selected (Table 3).
The results showed that all the compounds could effectively inhibit
NO release. Compounds A10, A16 and A27 had better activity, and the
IC50 value of A16 was 0.01 M.
μ
2
.2.3. Intra-cellular target validation
We then explored whether these compounds could inhibit HDAC6 in
cellular condition. High acetylation of histone H3 was a sign of inhibi-
tion by the Class I family, and acetylation of tubulin was a sign of HDAC6
inhibition. The levels of histone H3, acetylated histone H3 and
were determined by western blot assay in SH-SY5Y cell treated with
compound A16 at different concentration and Tubacin at 1 M for 12 h.
In SH-SY5Y cell line, A16 and Tubacin promoted acetylation of
α
-tubulin
μ
Fig. 1. The possible binding mode of 11i to HDAC6 (PDB: 5EDU).
2

Q. Xu et al.
Bioorganic Chemistry 111 (2021) 104910
Fig. 3. The structure of TS-15, 11i (green) and TS-15 (yellow) possible binding mode to HDAC6 (PDB: 5EDU). (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
nutrient solution was significantly increased. On the other hand, 0.01
μ
M of A16 could effectively reduce the NO level. The positive drug ACY-
215 also showed a strong inhibitory effect on NO. In addition, A16 can
reduce the release of TNF- and IL-6 induced by LPS, also showing a
1
α
good concentration dependence. In conclusion, A16 can generally
inhibit the synthesis and release of LPS-induced inflammatory media-
tors. MTT results showed that 10
μ
M of A16 had certain cytotoxicity, 10
μ
M of ACY-1215 also had cytotoxicity, weaker than A16. By using so-
dium nitroprusside (SNP, 25 mM) as the NO donor, it was found that
A16 and ACY-1215 could capture a certain amount of NO free radicals at
0
.01
μ
M and 0.1 M, suggesting that the effect of NO reduction was at
μ
least partly due to its direct trapping ability (Fig. 6).
2
.2.5. Evaluation of the protective effect of A16 on H
2 2
O -induced on nerve
cell line PC12
After cerebral ischemia, multiple factors such as inflammation,
apoptosis, oxidative stress, and excitotoxicity interact and affect each
other, thereby exacerbating the occurrence and development of nerve
damage [21]. Recent studies have shown that oxidative stress plays a
central role in cerebral ischemia-reperfusion injury. During cerebral
ischemia, oxidative stress and its reactive oxygen species such as su-
peroxide anion and hydroxyl radicals (reactive oxygen species) (ROS)
damage is an important mechanism of acute cerebral ischemia [22,23].
So we chose a model of hydrogen peroxide-induced neuronal death.
It was investigated whether compound A16 provided direct protec-
tion against neurons, in addition to inhibiting excessive activation of
Fig. 4. The design strategy of compounds A5-A34.
α
-tubulin but not histone H3, which proved that A16 was a selective
HDAC6 inhibitor. This finding was in accord with the enzyme-based
assay (Fig. 5).
2
.2.4. The inhibition of A16 LPS induced release of inflammatory
mediators from microglia
microglia. Using H
pre-protected with A16 for 1 h before H
them with H for 16 h. It was found that cells in the H
almost completely died, while 0.01 M A16 could significantly improve
2
O
2
-induced oxidative stress model, the cells were
stimulation and incubate
group
In the pathological mechanism of ischemic stroke, the inflammatory
response played an important role. After ischemic stroke, the microglia
inherent in the brain were activated [18]. Initial research suggested that
microglial cell would aggravate brain tissue damage in the acute phase
2 2
O
2
O
2
2 2
O
μ
the cell survival rate, reaching the normal level. Similarly, ACY-1215
can provide better protection. DPPH free radical scavenging experi-
ment showed that A16 and ACY-1215 didn’t fight against oxidative
stress through free radical scavenging, but were more likely mediated by
biological system (Fig. 7). The specific mechanism remains to be further
studied.
[
19], but as the research progressed, it was found in animal models that
the lack of microglial cell would affect the stability of microvascular
structures, thereby further increasing ischemia injury of cerebral stroke
[
20]. It could be seen that microglia played a complex and important
role in ischemic stroke.
Microglia cells were treated with LPS for 24 h and the NO level in the
◦
Scheme 1. The synthesis of A1-A4. Reagents and conditions: a. R
2
COCl, LiHMDS, dry THF, N
2
, r.t., 6 h; b. NH
2
NH
⋅H
2 2
O, AcOH, 100 C, 1 h; c. methyl 4-
◦
(
bromomethyl)benzoate, K
2
CO
3
, MeCN, 70 C, 4 h; d. NH
2
OH⋅H
2
O, NaOH, MeOH, r.t., 6 h.
3

Q. Xu et al.
Bioorganic Chemistry 111 (2021) 104910
◦
.
◦
◦
Scheme 2. The synthesis of A5-A34. Reagents and conditions: a. DMF-DMA, 95 C, 1 h; b. NH
2
NH
2
HCl, MeOH, 70 C, 3 h; c. R
1
X, K
2
CO
3
, MeCN, 70 C, 4 h; d.
◦
R
K
2
COCl, LiHMDS, dry THF, N
2
, r.t., 6 h; e. NH
2
NH
2
⋅H
2
O, AcOH, 100 C, 1 h; f. methyl 4-(bromomethyl)benzoate or (E)-methyl 3-(4-(bromomethyl)phenyl)acrylate,
◦
2
CO
3
, MeCN, 70 C, 4 h; g. NH
2
OH⋅H
2
O, NaOH, MeOH, r.t., 6 h.
mice was 274.47 mg/kg, and the 95% confidence limit of LD50 was
Table 1
1
84.56–408.17 mg/kg.
The organs of the dead mice were biopsied with doses of 500 mg/kg
and 1000 mg/kg. Observation results of pathological sections by
hematoxylin-eosin (HE) staining showed that the dose at 500 mg/kg had
a certain effect on the heart of mice. The pathological sections showed
that the muscle septum was widened, the red blood cells were hemor-
rhagic, and a few inflammatory cells were also visible, while the rest of
the organs were normal (Fig. 8).
Enzyme profiles of A1-A4 against HDAC.
Compd.
R
n
IC50
(μ
M)
HDAC1
HDAC2
HDAC3
HDAC6
The dose at 1000 mg/kg did some damage to the liver of mice. Tissue
sections showed that the liver was slightly turbid and swollen, while
other organs were normal (Fig. 9).
A1
A2
A3
A4
–OCH
3
3
0
1
1
0
–
–
–
–
>2.0
>2.0
0.27
1.27
>2.0
>2.0
>2.0
1.974
0.151
1.37
0.34
0.028
0.25
–OCH
0.18
–H
–H
–
0.27
0.086
0.12
>2.0
2.021
0.588
1.56
0.30
1
1i
1.205
0.229
0.48
0.020
0.009
0.016
0.009
3. Conclusion
TS-15
–
Tubacin
ACY-1215
–
In order to develop potent and neuroprotective selective HDAC6
inhibitor, using 11i as the hit compound, 34 compounds bearing ben-
zoindazole or pyrazoindazole scaffold were designed and synthesized
through the conformational restriction strategy. By analyzing the results
of enzyme activity and cell activity in vitro, a better active compound
A16 was obtained. Compound A16 can have a certain inhibitory effect
–
0.10
0.066
0.037
Values were the mean of two independent determinations.
2
.2.6. The stability assay of A16
To provide some basic information on drug-like properties, meta-
bolic stability studies of compounds A16 was assessed in liver micro-
somes of human and rat, Midazolam was used as a reference compound.
As shown in Table 4, A16 displayed relative stable metabolic stabilities
with T1/2 of 113.61 and 82.50 min in human and rat liver microsomes,
respectively. In addition, A16 stabilities in rat plasma were assessed. To
our delight, compound A16 was very stable in rat plasma during the
whole incubation time (see Table 5).
on some inflammatory mediators (NO, TNF- , IL-6), which is equivalent
α
to the positive control drug ACY-1215. In addition, it has been observed
that the drug has a direct neurocyte protective effect. Both A16 and
ACY-1215 can protect PC12 cell death caused by hydrogen peroxide.
Acute toxicity assay results of A16 show that when the doses reach 500
mg/kg and 1000 mg/kg, respectively, it has a certain effect on the heart
and liver of mice, and the LD50 value is 274.47 mg/kg. The stability
results of A16 show that the drug has good metabolic stability in vitro.
The above experimental results show that HDAC6 plays an important
role in the nervous system, especially in stroke, cerebral ischemia and
some neuroinflammation. Small molecule HDAC6 inhibitors have
certain development value in these aspects.
2
.2.7. Acute toxicity assay of compound A16
In order to evaluate the toxicity of A16, acute toxicity assay was
conducted. Five dose groups of Kunming male mice were designed, each
group of 3 animals, grouped as follows, 1000 mg/kg, 500 mg/kg, 250
mg/kg, 125 mg/kg, 62.5 mg/kg. It was administered at a maximum
administration volume of 0.1 ml/10 g by intraperitoneal injection (i.p.).
Fully dissolved before drug administration. After drug administration,
the mice closed their eyes and drooped, their activities were signifi-
cantly reduced, and their actions were especially slow. Their fur was
dull, not smooth, and their spirits were poor. No animals died on the day
of administration.
4. Experimental
4.1. Chemistry
All starting materials and solvents were obtained from commercial
suppliers or prepared according to known procedures without further
purification. Tubacin was purchased from Bidepharm (Shanghai, China;
CAS: 537049-40-4) with a purity of 95%. All reactions were monitored
Using the CFDA chemical drug standard method Bliss to calculate.
From the data analysis, the LD50 of compound A16 in Kunming male
4

Q. Xu et al.
Bioorganic Chemistry 111 (2021) 104910
Table 2
Enzyme profiles of A5-A34 against HDAC.
Compd.
R
1
R
2
Position
IC50
(μM)
HDAC1
HDAC2
HDAC3
HDAC6
A5
7–C
7–C
7–C
7–C
7–CH
7–CH
7–CH
7–CH
2
2
2
2
H
H
H
H
5
5
5
5
–Bn
–Bn
–Ph
–Ph
–Bn
–Bn
–Ph
–Ph
–Bn
–Bn
–Ph
–Ph
–Bn
–Bn
–Ph
–Ph
–CH
–CH
–CH
–CH
–CH
–CH
–
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
–
0.28
>2.0
0.19
>2.0
0.095
0.40
0.15
0.22
>2.0
0.81
1.01
0.43
0.78
0.82
0.34
0.29
0.12
0.16
0.12
0.25
>2.0
>2.0
2.021
0.588
1.56
0.10
>2.0
0.42
0.40
>2.0
>2.0
1.18
0.99
>2.0
>2.0
0.69
0.81
>2.0
>2.0
>2.0
0.28
0.41
1.65
0.14
0.73
0.10
>2.0
>2.0
1.974
0.151
1.37
0.066
0.28
0.16
0.35
A6
0.16
A7
0.24
0.15
A8
0.12
0.048
0.25
A9
3
3
3
3
0.11
A10
A11
A12
A13
A14
A15
A16
A17
A18
A19
A20
A29
A30
A31
A32
A33
A34
0.10
0.014
0.053
0.0022
0.11
0.39
0.094
0.74
7–C
7–C
7–C
7–C
6–C
6–C
6–C
6–C
7–C
7–C
7–C
7–C
6–C
6–C
–
2
2
2
2
2
2
2
2
2
2
2
2
2
2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
4
4
4
4
4
4
4
4
5
5
4
4
4
4
OCH
OCH
OCH
OCH
OCH
OCH
OCH
OCH
3
3
3
3
3
3
3
3
0.49
0.24
0.45
0.12
0.20
0.011
0.0044
0.31
0.54
0.65
0.093
0.10
0.0064
0.0078
0.063
0.004
0.12
3
3
3
3
3
3
0.36
0.019
0.47
OCH
OCH
OCH
OCH
3
3
3
3
0.0038
>2.0
>2.0
1.205
0.229
0.48
0.016
0.30
0.073
0.020
0.009
0.016
0.009
1
1i
TS-15
–
–
–
Tubacin
ACY-1215
–
–
–
–
–
–
0.037
Compd.
R
1
R
2
Position
IC50
(μM)
HDAC1
HDAC2
HDAC3
HDAC6
A21
A22
A23
A24
A25
A26
A27
A28
7-C
7-C
7-C
7-C
2
2
2
2
H
H
H
H
5
5
5
5
–Bn
–Bn
–Ph
–Ph
–Bn
–Bn
–Ph
–Ph
–
1
2
1
2
1
2
1
2
–
–
–
–
>2.0
0.34
0.83
0.32
0.18
>2.0
0.55
0.39
2.021
0.588
1.56
0.10
0.30
1.20
0.090
0.17
0.0078
0.31
0.16
0.057
0.14
0.0081
0.21
>2.0
0.11
7-CH
7-CH
7-CH
7-CH
–
3
3
3
3
0.048
0.17
0.16
0.77
0.55
0.23
0.073
0.11
0.14
0.71
0.085
0.020
0.009
0.016
0.009
1
1i
1.974
0.151
1.37
1.205
0.229
0.48
TS-15
–
–
Tubacin
ACY-1215
–
–
–
–
0.066
0.037
Values were the mean of two independent determinations.
by thin-layer chromatography (TLC), using silica gel plates with fluo-
rescence F254 and ultraviolet (UV) light visualization (Qingdao Haiyang
Chemical, China). Column chromatography was conducted for com-
pounds purification using silica gel (200–300 mesh). The melting points
(M.p.) were taken on an electrically heated X4 digital visual melting
point apparatus and were uncorrected. 1H NMR and C NMR were
Table 3
The anti-NO release activities of indicated compounds.
Compd.
IC50
(
μ
M)
Compd.
IC50
( M)
μ
A8
0.292
0.085
0.823
0.010
0.635
0.593
A21
0.115
0.117
0.026
0.775
0.666
16.28
13
A10
A12
A16
A19
A20
A23
A27
6
generated in DMSO‑d on Bruker spectrometers (600 MHz), using TMS
A30
as internal standard. LC-MS data were recorded on an Agilent 1100
Series LC/MSD Trap using ESI mode. High-solution mass spectra
A32
Minocycline
(
HRMS) were performed on Agilent 6540 UHD accurate mass Q-TOF MS
Values were the mean of two independent determinations
5

Q. Xu et al.
Bioorganic Chemistry 111 (2021) 104910
in ESI mode with Agilent 1290 infinity HPLC. All the target compounds
1
13
were characterized by H NMR, C NMR, LC-MS and HRMS.
4
.1.1. General procedure for the synthesis of A1-A4
Material 1-1 (0.5 g, 2.84 mmol) was dissolved in 40 ml THF, adding
◦
lithium bis(trimethylsilyl)amide (5.7 ml, 5.68 mmol) at 0 C. Benzoyl
chloride (0.4 ml, 3.41 mmol) was added into the mixture at the same
temperature. The mixture was stirred at room temperature for 6 h. The
resulting solution was dried directly in vacuum to give 1-2 as yellow
solid.
To a solution of 1-2 in 50 ml acetic acid was added 80% hydrazine
◦
hydrate (0.23 ml, 3.58 mmol). The reaction was warmed to 100 C for 1
h. 150 ml water was added into the resulting solution, and extracted
with ethyl acetate (30 ml × 3). The organic layers were combined,
2 4
washed with saturated sodium bicarbonate, dried with Na SO and
Fig. 5. Validation of HDAC6 inhibition in SH-SY5Y cells after 12 h of treatment
evaporated. Finally, the resulting residue was purified by column
chromatography on silica gel as indicated to give 1-3 as white solid.
To a solution of 1-3 (0.3 g, 1.09 mmol) in 50 ml acetonitrile was
with compound A16 at 0.125, 0.25, 0.5, 1
μ
M and Tubacin at 1 M.
μ
2 3
added methyl 4-(bromomethyl)benzoate (0.5 g, 2.18 mmol) and K CO
Fig. 6. The decrease LPS-induced inflamma-
tory mediator release of A16 and ACY-1215 in
N9 microglia cells. NO: compound A16
decreased LPS-induced NO release. Compound
A16 decreased LPS-induced TNF-
α
(TNF- ) and
α
IL-6 (IL-6) production in N9 microglia. MTT: The
effect of compound A16 and ACY-1215 on cell
viability. SNP: compound A16 had NO radical
scavenging activity. Data were shown as mean ±
#
###
SD. p < 0.05,
p < 0.001 compared with
*
*
***
control group and *p < 0.05, p < 0.01 and
0.001 compared with model group.
p
<
6

Q. Xu et al.
Bioorganic Chemistry 111 (2021) 104910
Fig. 7. The H -induced cell death protec-
2
O
2
tion of A16 and ACY-1215 in PC12 cells. MTT:
PC12 cells were pre-treated with A16 and ACY-
1
2 2
215 for 1 h, and then H O was added and
incubated for 16 h. Cells were added with 5 mg/
ml MTT and incubated for additional 4 h. Data
#
##
were shown as mean
compared with control group and
compared with H group. DPPH: A16 had no
±
SD.
p
<
0.001
*
**
p < 0.001
2 2
O
DPPH free radical scavenging activity. *p < 0.05
compared with control group.
1
5
27.2, 126.7, 126.5, 126.3, 119.9, 115.7, 115.2, 114.3, 112.6, 112.0,
Table 4
5.4, 52.2, 32.7, 30.9, 29.7, 19.0. HRMS (ESI+) m/z calcd for
The metabolic stability study of A16.
ꢀ
C
27
H
25
N
3
O
3
[Mꢀ H] 438.1896 found: 438.1821.
Compd.
Species
T
1/2 (minute)
Clint (mL/min/kg)
A16
Human
Rat
113.61
82.50
6.35
15.30
30.11
4.1.1.3. N-hydroxy-4-[(3-benzyl-4,5-dihydro-1H-benzo[g]indazol-2-yl)
◦
methyl]benzamide (A3). Yield: 38.7%. Mp: 140–144 C. ESI-MS: m/z,
Midazolam
Human
Rat
273.91
969.83
ꢀ
1
4
1
4
08.1 [Mꢀ H] . H NMR (600 MHz, DMSO‑d
6
) δ: 11.15 (s, 1H), 9.01 (s,
2.56
H), 7.66–7.62 (m, 2H), 7.26 (m, 2H), 7.24–7.14 (m, 5H), 7.14–7.08 (m,
Values were the mean of two independent determinations.
H), 5.33 (s, 2H), 4.03 (s, 2H), 2.86 (t, J = 7.3 Hz, 2H), 2.57 (t, J = 8.1
1
3
Hz, 2H). C NMR (151 MHz, DMSO) δ: 164.2, 146.9, 141.2, 138.3,
1
37.8, 137.3, 136.6, 132.1, 130.1, 128.9, 128.7, 127.4, 127.2, 127.0,
Table 5
1
26.8, 126.5, 121.9, 115.2, 54.0, 52.3, 32.6, 29.3, 18.9. HRMS (ESI+)
The stability study of A16 in rat plasma.
ꢀ
m/z calcd for C26
H
23
N
3
O
2
[Mꢀ H] 408.1790 found: 408.1699.
Compd.
Species
T1/2 (minute)
A16
Rat
>120
4
.1.1.4. N-hydroxy-4-[(3-phenyl-4,5-dihydro-1H-benzo[g]indazol-2-yl)
◦
methyl]benzamide (A4). Yield: 42.2%. Mp: 187–192 C. ESI-MS: m/z,
Values were the mean of two independent determinations.
ꢀ
1
3
94.3 [Mꢀ H] . H NMR (600 MHz, DMSO‑d
1H), 7.71–7.69 (m, 2H), 7.50–7.45 (m, 5H), 7.40–7.36 (m, 4H),
.21–7.18 (m, 2H), 5.81 (s, 2H), 2.92 (t, J = 8.2 Hz, 2H), 2.69 (t, J = 7.3
6
) δ: 11.18 (s, 1H), 9.06 (s,
◦
0.27 g, 2.18 mmol). The reaction was warmed to 70 C for 4 h. Finally,
(
7
the resulting residue was purified by column chromatography on silica
gel as indicated to give 1-4 as white solid.
1
3
Hz, 2H). C NMR (151 MHz, DMSO) δ: 163.0, 146.3, 145.5, 138.9,
1
38.1, 136.5, 135.8, 132.7, 128.4, 128.3, 128.0, 127.1, 126.9, 126.1,
To a solution of 1-4 (0.2 g, 0.47 mmol) in methanol (20 ml) was
1
25.5, 121.5, 115.4, 53.4, 51.7, 29.6, 28.3, 19.4, 18.2. HRMS (ESI+) m/
added NaOH (2 mol/L, 4 ml) and NH
2
OH (50 wt% in water, 3 ml)
ꢀ
◦
z calcd for C25
H
21
N
3
O
2
[Mꢀ H] 394.1634 found: 394.1567.
dropwise successively at 0 C. The reaction was warmed to room tem-
perature for 6 h. The mixture solution was concentrated to dryness, and
the obtained solid was dissolved in water (10 ml). The resulting solution
was adjusted to neutral with 1 mol/L aqueous solution of HCl. Finally,
the resulting residue was purified by column chromatography on silica
gel as indicated to give A1 as white solid.
4
.1.2. Procedure for the synthesis of 1,5,6,7-tetrahydro-4H-indazol-4-one
M2)
Material 1,3-Cyclohexanedione (10.0 g, 89.2 mmol) was dissolved
(
in 30 ml N, N-dimethylformamide dimethyl acetal (DMF-DMA). The
◦
reaction was warmed to 95 C for 1 h. The resulting solution was dried
Compounds A2-A4 were synthesized by the synthetic method from 1
to 1 to A1 as white solid.
directly in vacuum to give M1 as reddish brown solid.
To a solution of M1 (14.9 g) in 150 ml methanol was added hydra-
zine hydrochloride (11.2 g, 107.0 mmol). The reaction was warmed to
4
.1.1.1. N-hydroxy-4-[(7-methoxy-3-phenyl-4,5-dihydro-1H-benzo[g]
◦
6
5 C for 3 h. The resulting solution was dried directly in vacuum to give
◦
indazol-2-yl)methyl]benzamide (A1). Yield: 53.1%. Mp: 194–197 C. ESI-
ꢀ
1
the yellow solid. The solid was dripped washing with 60 ml dichloro-
MS: m/z, 424.3 [Mꢀ H] . H NMR (600 MHz, DMSO‑d
6
) δ: 11.16 (s, 1H),
.02 (s, 1H), 7.63 (d, J = 8.0 Hz, 2H), 7.46–7.42 (m, 5H), 7.39–7.30 (m,
H), 7.07 (d, J = 8.1 Hz, 2H), 5.39 (s, 2H), 3.77 (s, 3H), 2.90 (t, J = 7.9
methane and dried to give M2 as yellowish solid.
9
3
1
3
4.1.3. General procedure for the synthesis of A5-A34
Hz, 2H), 2.67 (t, J = 8.1 Hz, 2H). C NMR (151 MHz, DMSO) δ: 164.2,
To a solution of M2 (5.8 g, 42.6 mmol) in 150 ml acetonitrile was
1
1
1
59.1, 147.3, 146.4, 141.6, 141.2, 139.8, 138.6, 133.8, 132.2, 129.9,
29.3, 129.0, 127.5, 127.1, 126.9, 123.4, 119.6, 115.2, 114.6, 114.3,
added EtBr (6.4 ml, 85.2 mmol) and K
2
CO
3
(11.8 g, 85.2 mmol). The
◦
reaction was warmed to 70 C for 4 h. Finally, the resulting residue was
purified by column chromatography on silica gel as indicated to give 5-1
as yellowish solid.
12.7, 55.5, 29.7, 20.5, 19.3. HRMS (ESI+) m/z calcd for C26
23 3 3
H N O
ꢀ
[
Mꢀ H] 424.1739 found: 424.1654.
Material 5-1 (0.5 g, 3.0 mmol) was dissolved in 50 ml THF, adding
4
.1.1.2. N-hydroxy-4-[(3-benzyl-7-methoxy-4,5-dihydro-1H-benzo[g]
◦
lithium bis(trimethylsilyl)amide (6.0 ml, 6.0 mmol) at 0 C. Phenyl-
◦
indazol-2-yl)methyl]benzamide (A2). Yield: 26.1%. Mp: 108–113 C. ESI-
acetyl chloride (0.8 ml, 4.5 mmol) was added into the mixture at the
same temperature. The mixture was stirred at room temperature for 6 h.
The resulting solution was dried directly in vacuum to give 5-2 as yellow
solid.
ꢀ
1
MS: m/z, 438.3 [Mꢀ H] . H NMR (600 MHz, DMSO‑d
6
) δ: 11.14 (s, 1H),
.00 (s, 1H), 7.63 (d, J = 8.0 Hz, 2H), 7.28 (d, J = 7.1 Hz, 2H), 7.13–7.08
m, 5H), 6.92–6.75 (m, 3H), 5.30 (s, 2H), 4.01 (s, 2H), 3.76 (s, 3H), 2.84
9
(
(
1
3
t, J = 7.3 Hz, 2H), 2.55 (t, J = 7.4 Hz, 2H). C NMR (151 MHz, DMSO)
To a solution of 5-2 in 50 ml acetic acid was added 80% hydrazine
δ: 164.2, 158.9, 146.9, 140.3, 139.4, 138.4, 137.6, 128.9, 128.7, 127.4,
◦
hydrate (0.25 ml, 5.0 mmol). The reaction was warmed to 100 C for 1 h.
7

Q. Xu et al.
Bioorganic Chemistry 111 (2021) 104910
A
B
D
F
2
0
m
20 m
C
20
m
20 m
E
20
m
20 m
G
20 m
Fig. 8. The effect of dosage of 500 mg/kg on pathology of mouse organs (400 magnification). Scale bar = 20
D) brain; (E) spleen; (F) kidney; (G) heart.
μ
m. (A) lung; (B) liver; (C) brain (hippocampus);
(
1
50 ml water was added into the resulting solution, and extracted with
ethyl acetate (30 ml × 3). The organic layers were combined, washed
with saturated sodium bicarbonate, dried with Na SO and evaporated.
as indicated to give 5-4a and 5-4b as white solid.
To a solution of 5-4a (0.08 g, 0.19 mmol) or 5-4b (0.08 g, 0.19
2
4
mmol) in methanol (10 ml) was added NaOH (2 mol/L, 3 ml) and
◦
Finally, the resulting residue was purified by column chromatography
on silica gel as indicated to give 5-3 as white solid.
NH
2
OH (50 wt% in water, 2 ml) dropwise successively at 0 C. The re-
action was warmed to room temperature for 6 h. The mixture solution
was concentrated to dryness, and the obtained solid was dissolved in
water (10 ml). The resulting solution was adjusted to neutral with 1
mol/L aqueous solution of HCl. Finally, the resulting residue was puri-
fied by column chromatography on silica gel as indicated to give A5 or
To a solution of 5-3 (0.4 g, 1.4 mmol) in 50 ml acetonitrile was added
methyl 4-(bromomethyl)benzoate (0.48 g, 2.1 mmol) and K
2
CO
3
(0.34
◦
g, 2.8 mmol). The reaction was warmed to 70 C for 4 h. Finally, the
resulting residue was purified by column chromatography on silica gel
8

Q. Xu et al.
Bioorganic Chemistry 111 (2021) 104910
A
C
E
B
D
F
2
0
m
20
m
20
m
20 m
20
m
20 m
Fig. 9. The effect of dosage of 1000 mg/kg on pathology of mouse organs (400 magnification). Scale bar = 20
kidney; (F) heart.
μ
m. (A) lung; (B) liver; (C) brain; (D) spleen; (E)
A6 as white solid.
135.2, 132.3, 128.8, 128.7, 127.5, 127.2, 126.2, 123.0, 112.0, 108.2,
Compounds A7-A34 were synthesized by the synthetic method from
M2 to A5 or A6 as white solid.
52.7, 46.5, 32.9, 22.0, 19.5, 15.8. HRMS (ESI+) m/z calcd for
ꢀ
C
25
H
25
N
5
O
2
[Mꢀ H] 426.2008 found: 426.1938.
4
.1.3.1. N-hydroxy-4-[(3-benzyl-7-ethyl-4,5-dihydropyrazolo[3,4-e]inda-
4.1.3.3. N-hydroxy-4-[(7-ethyl-3-phenyl-4,5-dihydropyrazolo[3,4-e]inda-
◦
◦
zol-1(7H)-yl)methyl]benzamide (A5). Yield: 1.1%. Mp: 135–138 C. ESI-
MS: m/z, 425.9 [Mꢀ H] . H NMR (600 MHz, DMSO‑d
zol-1(7H)-yl)methyl]benzamide (A7). Yield: 1.5%. Mp: 164–166 C. ESI-
ꢀ
1
ꢀ
1
6
) δ: 11.18 (s, 1H),
MS: m/z, 412.3 [Mꢀ H] . H NMR (600 MHz, DMSO‑d
6
) δ: 11.15 (s, 1H),
9
2
2
3
1
1
4
.01 (s, 1H), 7.95 (s, 1H), 7.68 (d, J = 8.0 Hz, 2H), 7.26 (d, J = 7.3 Hz,
8.93 (s, 1H), 8.03 (s, 1H), 7.70 (d, J = 6.1 Hz, 2H), 7.68 (d, J = 6.8 Hz,
H), 7.24–7.13 (m, 5H), 5.41 (s, 2H), 4.04 (q, J = 7.2 Hz, 2H), 3.88 (s,
H), 2.69 (t, J = 7.4 Hz, 2H), 2.55 (t, J = 7.7 Hz, 2H), 1.33 (t, J = 7.2 Hz,
2H), 7.47–7.24 (m, 5H), 5.52 (s, 2H), 4.09 (q, J = 7.2 Hz, 2H), 2.99 (t, J
1
3
= 7.6 Hz, 2H), 2.81 (t, J = 7.6 Hz, 2H), 1.36 (t, J = 7.2 Hz, 3H). C NMR
(151 MHz, DMSO) δ: 163.6, 149.4, 147.2, 136.0, 134.2, 128.9, 127.6,
127.5, 127.3, 127.0, 123.4, 114.1, 111.3, 108.0, 53.1, 46.6, 22.2, 20.8,
1
3
H). C NMR (151 MHz, DMSO) δ: 149.4, 147.7, 140.3, 135.2, 128.8,
28.7, 127.2, 126.2, 123.0, 111.9, 108.2, 52.7, 46.5, 32.9, 22.0, 19.5,
5.8. HRMS (ESI+) m/z calcd for C25
H
25
5
N O
2
[Mꢀ H]ꢀ 426.2008 found:
15.9. HRMS (ESI+) m/z calcd for C24
H
23
N
5
O
2
[Mꢀ H]ꢀ 412.1852 found:
26.1924.
412.1746.
4
.1.3.2. N-hydroxy-4-((3-benzyl-7-ethyl-4,5-dihydropyrazolo[3,4-e]inda-
4.1.3.4. N-hydroxy-4-[(7-ethyl-3-phenyl-4,5-dihydropyrazolo[3,4-e]inda-
◦
◦
zol-2(7H)-yl)methyl)benzamide (A6). Yield: 1.2%. Mp: 132–136 C. ESI-
zol-2(7H)-yl)methyl]benzamide (A8). Yield: 1.8%. Mp: 205–207 C. ESI-
ꢀ
1
ꢀ
1
MS: m/z, 426.4 [Mꢀ H] . H NMR (600 MHz, DMSO‑d
6
) δ: 11.12 (s, 1H),
MS: m/z, 412.3 [Mꢀ H] . H NMR (600 MHz, DMSO‑d
6
) δ: 11.14 (s, 1H),
9
2
2
3
.01 (s, 1H), 7.95 (s, 1H), 7.68 (d, J = 7.9 Hz, 2H), 7.26 (d, J = 7.4 Hz,
9.00 (s, 1H), 7.89 (s, 1H), 7.64 (d, J = 8.0 Hz, 2H), 7.47–7.37 (m, 5H),
7.04 (d, J = 7.9 Hz, 2H), 5.31 (s, 2H), 4.10 (q, J = 7.2 Hz, 2H), 2.76 (d, J
H), 7.24–7.14 (m, 5H), 5.42 (s, 2H), 4.04 (q, J = 7.3 Hz, 2H), 3.88 (s,
H), 2.69 (t, J = 7.7 Hz, 2H), 2.55 (t, J = 7.9 Hz, 2H), 1.33 (t, J = 7.2 Hz,
1
3
= 5.9 Hz, 2H), 2.72 (d, J = 6.0 Hz, 2H), 1.38 (t, J = 7.2 Hz, 3H). C NMR
(151 MHz, DMSO) δ: 164.3, 149.8, 143.9, 141.7, 139.9, 132.1, 130.0,
1
3
H). C NMR (151 MHz, DMSO) δ: 164.3, 149.4, 147.7, 141.3, 140.3,
9

Q. Xu et al.
Bioorganic Chemistry 111 (2021) 104910
1
1
4
29.3, 128.9, 127.4, 126.9, 123.3, 113.6, 111.3, 52.4, 46.5, 22.1, 19.8,
2H), 7.05 (d, J = 7.9 Hz, 2H), 5.23 (s, 2H), 4.20 (t, J = 5.2 Hz, 2H), 4.00
(s, 2H), 3.66 (t, J = 5.2 Hz, 2H), 3.23 (s, 3H), 2.73 (t, J = 7.3 Hz, 2H),
6.0. HRMS (ESI+) m/z calcd for C24
H
23
5
N O
2
[Mꢀ H]ꢀ 412.1852 found:
1
3
12.1756.
2.62 (t, J = 7.3 Hz, 2H). C NMR (151 MHz, DMSO) δ: 149.9, 143.3,
1
38.4, 137.6, 128.9, 128.6, 128.5, 127.2, 126.7, 125.0, 124.2, 114.0,
4
.1.3.5. N-hydroxy-4-[(3-benzyl-7-methyl-4,5-dihydropyrazolo[3,4-e]
113.2, 111.6, 70.9, 58.3, 52.0, 51.2, 29.8, 22.1, 19.4. HRMS (ESI+) m/z
ꢀ
◦
indazol-1(7H)-yl)methyl]benza-mide (A9). Yield: 2.9%. Mp: 162–167 C.
calcd for C26
H
27
N
5
O
3
[Mꢀ H] 456.2114 found: 456.2030.
ꢀ
1
ESI-MS: m/z, 412.3 [Mꢀ H] . H NMR (600 MHz, DMSO‑d
H), 9.01 (s, 1H), 7.90 (s, 1H), 7.67 (d, J = 8.1 Hz, 2H), 7.27 (t, J = 7.5
Hz, 2H), 7.23–7.19 (m, 4H), 7.17 (t, J = 7.3 Hz, 1H), 5.41 (s, 2H), 3.88
6
) δ: 11.14 (s,
1
4.1.3.11. N-hydroxy-4-[(7-(2-methoxyethyl)-3-phenyl-4,5-dihydropyr-
azolo[3,4-e]indazol-1(7H)-yl)methyl]benzamide (A15). Yield: 0.8%. Mp:
1
3
◦
ꢀ
1
(
s, 2H), 3.75 (s, 3H), 2.68 (t, J = 7.6 Hz, 2H), 2.55 (t, J = 7.6 Hz, 2H).
C
202–208 C. ESI-MS: m/z, 441.9 [Mꢀ H] . H NMR (600 MHz,
DMSO‑d
NMR (151 MHz, DMSO) δ: 164.5, 149.7, 147.8, 141.2, 140.1, 135.1,
6
) δ: 11.14 (s, 1H), 9.01 (s, 1H), 7.99 (s, 1H), 7.69 (t, J = 7.5 Hz,
1
5
32.1, 128.8, 128.7, 127.5, 127.2, 126.3, 124.4, 114.2, 112.1, 108.3,
4H), 7.44 (t, J = 7.5 Hz, 2H), 7.33 (t, J = 7.3 Hz, 1H), 7.28 (d, J = 7.9 Hz,
2H), 5.53 (s, 2H), 4.20 (t, J = 5.1 Hz, 2H), 3.67 (t, J = 5.1 Hz, 2H), 3.21
2.7, 38.7, 32.8, 21.8, 19.4. HRMS (ESI+) m/z calcd for C24
23 5 2
H N O
ꢀ
13
[
Mꢀ H] 412.1852 found: 412.1764.
(s, 3H), 2.99 (t, J = 7.5 Hz, 2H), 2.81 (t, J = 7.6 Hz, 2H). C NMR (151
MHz, DMSO) δ: 149.6, 149.5, 148.5, 147.2, 146.5, 135.9, 135.3, 134.4,
4
.1.3.6. N-hydroxy-4-[(3-benzyl-7-methyl-4,5-dihydropyrazolo[3,4-e]
134.2, 128.9, 128.9, 128.6, 127.3, 127.0, 127.0, 114.1, 111.4, 111.1,
indazol-2(7H)-yl)methyl]benza-mide
(A10). Yield:
3.7%.
Mp:
108.3, 108.0, 70.7, 58.3, 53.1, 51.4, 22.1, 20.8. HRMS (ESI+) m/z calcd
ꢀ
◦
ꢀ
1
1
58–160 C. ESI-MS: m/z, 412.4 [Mꢀ H] . H NMR (600 MHz,
for C25
H
25
5
N O
3
[Mꢀ H] 442.1957 found: 442.1894.
DMSO‑d
6
) δ: 11.15 (s, 1H), 9.00 (s, 1H), 7.75 (s, 1H), 7.62 (d, J = 8.1 Hz,
2
2
2
H), 7.25 (t, J = 7.5 Hz, 2H), 7.18 (t, J = 7.3 Hz, 1H), 7.11 (d, J = 7.4 Hz,
H), 7.06 (d, J = 8.1 Hz, 2H), 5.24 (s, 2H), 4.00 (s, 2H), 3.79 (s, 3H),
4.1.3.12. N-hydroxy-4-[(7-(2-methoxyethyl)-3-phenyl-4,5-dihydropyr-
azolo[3,4-e]indazol-2(7H)-yl)methyl]benzamide (A16). Yield: 0.6%. Mp:
1
3
◦
ꢀ
1
.71 (t, J = 7.3 Hz, 2H), 2.61 (t, J = 7.2 Hz, 2H). C NMR (151 MHz,
191–193 C. ESI-MS: m/z, 441.8 [Mꢀ H] . H NMR (600 MHz,
DMSO‑d
DMSO) δ: 164.5, 150.0, 143.3, 141.4, 138.3, 137.8, 131.9, 128.9, 128.6,
6
) δ: 11.15 (s, 1H), 8.99 (s, 1H), 7.85 (s, 1H), 7.64 (d, J = 7.8 Hz,
1
27.3, 126.8, 124.5, 113.3, 111.6, 52.1, 38.6, 29.8, 21.9, 19.3. HRMS
2H), 7.49 (t, J = 7.4 Hz, 2H), 7.44 (t, J = 7.0 Hz, 1H), 7.38 (d, J = 7.9 Hz,
2H), 7.04 (d, J = 7.9 Hz, 2H), 5.32 (s, 2H), 4.23 (t, J = 5.1 Hz, 2H), 3.68
(
ESI+) m/z calcd for C24
H
23
N
5
O
2
[Mꢀ H]ꢀ 412.1852 found: 412.1759.
(
t, J = 5.1 Hz, 2H), 3.25 (s, 3H), 2.77 (t, J = 7.2 Hz, 2H), 2.72 (t, J = 7.0
13
4
.1.3.7. N-hydroxy-4-[(7-methyl-3-phenyl-4,5-dihydropyrazolo[3,4-e]
Hz, 2H). C NMR (151 MHz, DMSO) δ: 164.3, 150.1, 143.8, 141.7,
indazol-1(7H)-yl)methyl]benza-mide
(A11). Yield:
2.6%.
Mp:
139.9, 132.1, 130.0, 129.3, 128.9, 127.5, 126.9, 124.5, 113.6, 111.3,
◦
ꢀ
1
1
48–152 C. ESI-MS: m/z, 397.7 [Mꢀ H] . H NMR (600 MHz,
70.9, 58.3, 52.4, 51.3, 22.1, 19.8. HRMS (ESI+) m/z calcd for
ꢀ
DMSO‑d
6
) δ: 11.14 (s, 1H), 9.02 (s, 1H), 7.98 (s, 1H), 7.69 (t, J = 8.5 Hz,
C
25
H
25
N
5
O
3
[Mꢀ H] 442.1957 found: 442.1877.
4
2
H), 7.43 (t, J = 7.7 Hz, 2H), 7.33 (t, J = 7.4 Hz, 1H), 7.28 (d, J = 8.3 Hz,
H), 5.52 (s, 2H), 3.80 (s, 3H), 2.99 (t, J = 7.6 Hz, 2H), 2.80 (t, J = 7.6
4.1.3.13. N-hydroxy-4-[(3-benzyl-6-(2-methoxyethyl)-4,5-dihydropyr-
1
3
Hz, 2H). C NMR (151 MHz, DMSO) δ: 164.3, 149.6, 147.3, 140.8,
azolo[3,4-e]indazol-1(6H)-yl)methyl]benzamide (A17). Yield: 0.7%. Mp:
◦
ꢀ
1
1
5
3
36.0, 134.2, 132.4, 128.9, 127.6, 127.3, 127.0, 124.7, 111.3, 108.1,
159–164 C. ESI-MS: m/z, 456.0 [Mꢀ H] . H NMR (600 MHz,
DMSO‑d
) δ: 11.14 (s, 1H), 9.00 (s, 1H), 7.67 (d, J = 8.0 Hz, 2H), 7.60 (s,
1H), 7.28 (t, J = 7.5 Hz, 2H), 7.23 (t, J = 6.4 Hz, 4H), 7.18 (t, J = 6.5 Hz,
H), 5.47 (s, 2H), 4.18 (t, J = 5.0 Hz, 2H), 3.89 (s, 2H), 3.60 (t, J = 5.0
Hz, 2H), 3.16 (s, 3H), 2.82 (t, J = 8.0 Hz, 2H), 2.57 (t, J = 7.9 Hz, 2H).
3.0, 22.1, 20.8. HRMS (ESI+) m/z calcd for C23
H
21
N
5
O
2
[Mꢀ H]ꢀ
6
98.1695 found: 398.1622.
1
4
.1.3.8. N-hydroxy-4-[(7-methyl-3-phenyl-4,5-dihydropyrazolo[3,4-e]
1
3
indazol-2(7H)-yl)methyl]benza-mide
(A12). Yield:
5.7%.
Mp:
C NMR (151 MHz, DMSO) δ: 164.4, 147.5, 141.4, 141.1, 140.3, 135.6,
◦
ꢀ
1
1
55–158 C. ESI-MS: m/z, 397.7 [Mꢀ H] . H NMR (600 MHz,
132.3, 132.0, 130.0, 128.8, 128.7, 127.5, 127.1, 126.3, 110.4, 108.9,
DMSO‑d
6
) δ: 11.15 (s, 1H), 9.00 (s, 1H), 7.84 (s, 1H), 7.64 (d, J = 8.3 Hz,
71.0, 58.4, 52.9, 49.1, 33.0, 20.5, 19.1. HRMS (ESI+) m/z calcd for
ꢀ
2
7
2
1
1
H), 7.49 (t, J = 7.3 Hz, 2H), 7.44 (t, J = 7.9 Hz, 1H), 7.39–7.36 (m, 2H),
C
26
H
27
N
5
O
3
[Mꢀ H] 456.2114 found: 456.2034.
.04 (d, J = 8.3 Hz, 2H), 5.31 (s, 2H), 3.81 (s, 3H), 2.75 (t, J = 6.6 Hz,
1
3
H), 2.70 (t, J = 6.8 Hz, 2H). C NMR (151 MHz, DMSO) δ: 164.3,
4.1.3.14. N-hydroxy-4-[(3-benzyl-6-(2-methoxyethyl)-4,5-dihydropyr-
50.0, 143.8, 139.9, 132.1, 130.0, 129.3, 129.3, 128.9, 127.5, 126.9,
azolo[3,4-e]indazol-2(6H)-yl)methyl]benzamide (A18). Yield: 0.5%. Mp:
◦
ꢀ
1
24.7, 113.6, 111.5, 52.4, 38.8, 22.0, 19.8. HRMS (ESI+) m/z calcd for
202–204 C. ESI-MS: m/z, 455.9 [Mꢀ H] . H NMR (600 MHz,
DMSO‑d
) δ: 11.18 (s, 1H), 9.00 (s, 1H), 7.63 (d, J = 8.2 Hz, 2H), 7.54 (s,
ꢀ
C
23
H
21
N
5
O
2
[Mꢀ H] 398.1695 found: 398.1591.
6
1
H), 7.25 (t, J = 7.5 Hz, 2H), 7.18 (t, J = 7.3 Hz, 1H), 7.12 (d, J = 7.3 Hz,
4
.1.3.9. N-hydroxy-4-[(3-benzyl-7-(2-methoxyethyl)-4,5-dihydropyrazolo
2H), 7.06 (d, J = 8.2 Hz, 2H), 5.23 (s, 2H), 4.22 (t, J = 5.3 Hz, 2H), 4.00
(s, 2H), 3.64 (t, J = 5.3 Hz, 2H), 3.20 (s, 3H), 2.86 (t, J = 7.6 Hz, 2H),
[
3,4-e]indazol-1(7H)-yl)methyl]benzamide (A13). Yield: 1.0%. Mp:
ꢀ
1
1
3
◦
1
45–148 C. ESI-MS: m/z, 455.8 [Mꢀ H] . H NMR (600 MHz,
) δ: 11.15 (s, 1H), 9.03 (s, 1H), 7.90 (s, 1H), 7.68 (d, J = 8.0 Hz,
H), 7.27 (t, J = 7.5 Hz, 2H), 7.21 (d, J = 7.6 Hz, 4H), 7.17 (t, J = 7.2 Hz,
H), 5.41 (s, 2H), 4.16 (t, J = 5.1 Hz, 2H), 3.89 (s, 2H), 3.63 (t, J = 5.2
2.65 (t, J = 7.6 Hz, 2H). C NMR (151 MHz, DMSO) δ: 164.2, 143.7,
DMSO‑d
6
141.2, 138.4, 137.6, 132.3, 128.9, 128.6, 127.4, 127.2, 126.7, 112.1,
2
1
71.2, 58.5, 51.9, 48.9, 29.9, 20.4, 19.0. HRMS (ESI+) m/z calcd for
ꢀ
C
26
H
27
N
5
O
3
[Mꢀ H] 456.2114 found: 456.2028.
Hz, 2H), 3.18 (s, 3H), 2.69 (t, J = 7.5 Hz, 2H), 2.55 (t, J = 7.6 Hz, 2H).
1
3
C NMR (151 MHz, DMSO) δ: 164.5, 149.6, 147.7, 141.2, 140.3, 135.1,
4.1.3.15. N-hydroxy-4-[(6-(2-methoxyethyl)-3-phenyl-4,5-dihydropyr-
1
5
32.3, 128.8, 128.7, 127.5, 127.2, 126.2, 124.3, 112.0, 108.2, 70.7,
azolo[3,4-e]indazol-1(6H)-yl)methyl]benzamide (A19). Yield: 0.7%. Mp:
◦
ꢀ
1
8.3, 52.7, 51.4, 32.9, 22.0, 19.4. HRMS (ESI+) m/z calcd for
130–134 C. ESI-MS: m/z, 442.0 [Mꢀ H] . H NMR (600 MHz,
DMSO‑d
) δ: 11.14 (s, 1H), 9.01 (s, 1H), 7.69 (m, 5H), 7.44 (t, J = 7.5 Hz,
H), 7.33 (t, J = 7.3 Hz, 1H), 7.30 (d, J = 7.9 Hz, 2H), 5.58 (s, 2H), 4.24
ꢀ
C
26
H
27
5
N O
3
[Mꢀ H] 456.2114 found: 456.2036.
6
2
4
.1.3.10. N-hydroxy-4-[(3-benzyl-7-(2-methoxyethyl)-4,5-dihydropyr-
(t, J = 5.0 Hz, 2H), 3.64 (t, J = 5.1 Hz, 2H), 3.19 (s, 3H), 3.03 (t, J = 7.7
13
azolo[3,4-e]indazol-2(7H)-yl)methyl]benzamide (A14). Yield: 0.7%. Mp:
Hz, 2H), 2.95 (t, J = 7.8 Hz, 2H). C NMR (151 MHz, DMSO) δ: 147.0,
◦
ꢀ
1
1
61–166 C. ESI-MS: m/z, 455.8 [Mꢀ H] . H NMR (600 MHz,
DMSO‑d
) δ: 11.15 (s, 1H), 9.00 (s, 1H), 7.76 (s, 1H), 7.62 (d, J = 7.8 Hz,
H), 7.25 (t, J = 7.3 Hz, 2H), 7.18 (t, J = 7.2 Hz, 1H), 7.11 (d, J = 7.5 Hz,
141.3, 136.4, 134.3, 132.2, 128.9, 127.6, 127.2, 127.0, 109.8, 108.7,
71.0, 58.5, 53.3, 49.2, 20.7, 20.5. HRMS (ESI+) m/z calcd for
6
ꢀ
2
C
25
H
25
N
5
O
3
[Mꢀ H] 442.1957 found: 442.1875.
1
0

Q. Xu et al.
Bioorganic Chemistry 111 (2021) 104910
4
.1.3.16. N-hydroxy-4-[(6-(2-methoxyethyl)-3-phenyl-4,5-dihydropyr-
Hz, 2H), 7.20–7.16 (m, 3H), 6.43 (d, J = 15.6 Hz, 1H), 5.38 (s, 2H), 3.87
1
3
azolo[3,4-e]indazol-2(6H)-yl)methyl]benzamide (A20). Yield: 0.5%. Mp:
(s, 2H), 3.76 (s, 3H), 2.68 (t, J = 7.5 Hz, 2H), 2.56 (t, J = 7.3 Hz, 2H).
C
◦
ꢀ
1
1
59–166 C. ESI-MS: m/z, 441.8 [Mꢀ H] . H NMR (600 MHz,
NMR (151 MHz, DMSO) δ: 163.2, 149.7, 147.7, 140.1, 139.4, 138.4,
135.1, 134.3, 128.8, 128.7, 128.1, 127.7, 126.3, 124.4, 119.2, 112.0,
DMSO‑d
6
) δ: 11.15 (s, 1H), 8.99 (s, 1H), 7.67–7.60 (m, 3H), 7.49 (t, J =
7
.4 Hz, 2H), 7.44 (t, J = 7.1 Hz, 1H), 7.38 (d, J = 7.5 Hz, 2H), 7.04 (d, J
108.3, 52.7, 38.7, 32.8, 21.8, 19.4. HRMS (ESI+) m/z calcd for
ꢀ
=
8.0 Hz, 2H), 5.31 (s, 2H), 4.24 (t, J = 5.0 Hz, 2H), 3.65 (t, J = 5.0 Hz,
C
26
H
25
N
5
O
2
[Mꢀ H] 438.2008 found: 438.1926.
1
3
2
H), 3.20 (s, 3H), 2.91 (t, J = 7.5 Hz, 2H), 2.76 (t, J = 7.6 Hz, 2H).
C
NMR (151 MHz, DMSO) δ: 164.3, 144.3, 141.8, 141.4, 139.8, 132.5,
4.1.3.22. (E)-N-hydroxy-3-{4-[(3-benzyl-7-methyl-4,5-dihydropyrazolo
1
4
4
32.1, 130.1, 129.3, 128.9, 127.5, 126.8, 112.3, 111.9, 71.2, 58.5, 52.4,
[3,4-e]indazol-2(7H)-yl)methyl]phenyl}acrylamide (A26). Yield: 2.3%.
8.9, 29.1, 20.5, 19.4. HRMS (ESI+) m/z calcd for C25
25 5 3
H N O
[Mꢀ H]ꢀ
ꢀ
1
Mp: 188–190 ◦C. ESI-MS: m/z, 438.3 [Mꢀ H] . H NMR (600 MHz,
42.1957 found: 442.1865.
DMSO‑d ) δ: 10.74 (s, 1H), 9.02 (s, 1H), 7.75 (s, 1H), 7.43 (d, J = 7.8 Hz,
6
2
H), 7.39 (d, J = 15.7 Hz, 1H), 7.24 (t, J = 5.6 Hz, 2H), 7.18 (t, J = 7.2
4
.1.3.17. (E)-N-hydroxy-3-{4-[(3-benzyl-7-ethyl-4,5-dihydropyrazolo
Hz, 1H), 7.11 (d, J = 7.5 Hz, 2H), 7.03 (d, J = 7.8 Hz, 2H), 6.40 (d, J =
15.8 Hz, 1H), 5.21 (s, 2H), 4.00 (s, 2H), 3.79 (s, 3H), 2.71 (t, J = 5.3 Hz,
[
3,4-e]indazol-1(7H)-yl)methyl]phenyl}acrylamide (A21). Yield: 9.5%.
Mp: 164–168 ◦C. ESI-MS: m/z, 452.3 [Mꢀ H] . H NMR (600 MHz,
ꢀ
1
2H), 2.60 (t, J = 5.3 Hz, 2H). C NMR (151 MHz, DMSO) δ: 163.1,
13
DMSO‑d
6
) δ: 10.76 (s, 1H), 9.03 (s, 1H), 7.94 (s, 1H), 7.50 (d, J = 8.1 Hz,
149.9, 143.2, 139.7, 138.4, 137.5, 134.2, 128.9, 128.6, 127.9, 126.7,
3
6
2
3
1
1
H), 7.42 (d, J = 15.8 Hz,1H), 7.27 (t, J = 7.5 Hz, 3H), 7.17 (m, 3H),
124.4, 119.3, 113.2, 111.8, 52.1, 38.7, 29.8, 22.1, 19.4. HRMS (ESI+)
ꢀ
.42 (d, J = 15.8 Hz, 1H), 5.39 (s, 2H), 4.05 (q, J = 7.3 Hz, 2H), 3.88 (s,
H), 2.69 (t, J = 7.6 Hz, 2H), 2.54 (t, J = 7.6 Hz, 2H), 1.33 (t, J = 7.2 Hz,
m/z calcd for C26
H
25
N
5
O
2
[Mꢀ H] 438.2008 found: 438.1905.
1
3
H). C NMR (151 MHz, DMSO) δ: 163.0, 149.4, 147.6, 140.3, 139.5,
35.2, 134.3, 128.8, 128.7, 128.1, 127.8, 126.2, 123.0, 119.4, 111.9,
4.1.3.23. (E)-N-hydroxy-3-{4-[(7-methyl-3-phenyl-4,5-dihydropyrazolo
[3,4-e]indazol-1(7H)-yl)methyl]phenyl}acrylamide (A27). Yield: 2.9%.
Mp: 201–204 ◦C. ESI-MS: m/z, 423.7 [Mꢀ H] . H NMR (600 MHz,
ꢀ
1
08.2, 52.7, 46.5, 32.9, 22.0, 19.5, 15.8. HRMS (ESI+) m/z calcd for
ꢀ
C
27
H
27
5
N O
2
[Mꢀ H] 452.2165 found: 452.2079.
DMSO‑d ) δ: 10.76 (s, 1H), 9.12 (s, 1H), 7.98 (s, 1H), 7.68 (d, J = 7.4 Hz,
6
2
H), 7.52 (d, J = 7.9 Hz, 2H), 7.43 (t, J = 7.6 Hz, 2H), 7.38 (d, J = 15.8
4
.1.3.18. (E)-N-hydroxy-3-{4-[(3-benzyl-7-ethyl-4,5-dihydropyrazolo
Hz, 1H), 7.33 (t, J = 7.3 Hz, 1H), 7.25 (d, J = 7.9 Hz, 2H), 6.41 (d, J =
15.8 Hz, 1H), 5.49 (s, 2H), 3.80 (s, 3H), 2.98 (t, J = 7.6 Hz, 2H), 2.80 (t,
[
3,4-e]indazol-2(7H)-yl)methyl]phenyl}acrylamide (A22). Yield: 3.7%.
Mp: 206–208 ◦C. ESI-MS: m/z, 452.3 [Mꢀ H] . H NMR (600 MHz,
ꢀ
1
J = 7.6 Hz, 2H). C NMR (151 MHz, DMSO) δ: 162.9, 149.6, 147.2,
13
DMSO‑d
6
) δ: 10.75 (s, 1H), 9.02 (s, 1H), 7.81 (s, 1H), 7.44 (d, J = 8.0 Hz,
139.0, 135.9, 134.2, 128.9, 128.1, 127.9, 127.6, 127.0, 124.7, 119.5,
2
H), 7.40 (d, J = 15.8 Hz, 1H), 7.24 (t, J = 7.5 Hz, 2H), 7.18 (d, J = 7.2
111.3, 108.2, 53.1, 38.8, 22.1, 20.8. HRMS (ESI+) m/z calcd for
ꢀ
Hz, 1H), 7.11 (d, J = 7.3 Hz, 2H), 7.03 (d, J = 8.0 Hz, 2H), 6.40 (d, J =
C
25
H
23
N
5
O
2
[Mꢀ H] 424.1852 found: 424.1768.
1
5.8 Hz, 1H), 5.21 (s, 2H), 4.08 (q, J = 7.2 Hz, 2H), 4.00 (s, 2H), 2.72 (t,
13
J = 7.3 Hz, 2H), 2.61 (t, J = 7.3 Hz, 2H), 1.36 (t, J = 7.2 Hz, 3H).
C
4.1.3.24. (E)-N-hydroxy-3-{4-[(7-methyl-3-phenyl-4,5-dihydropyrazolo
NMR (151 MHz, DMSO) δ: 163.1, 149.7, 143.3, 139.7, 138.4, 138.3,
[3,4-e]indazol-2(7H)-yl)methyl]phenyl}acrylamide (A28). Yield: 4.7%.
Mp: 181–186 ◦C. ESI-MS: m/z, 423.7 [Mꢀ H] . H NMR (600 MHz,
ꢀ
1
1
37.5, 134.2, 128.9, 128.6, 127.9, 126.7, 123.0, 119.3, 113.2, 111.6,
5
2.1, 46.4, 29.8, 22.2, 19.4, 16.0. HRMS (ESI+) m/z calcd for
DMSO‑d ) δ: 10.78 (s, 1H), 9.02 (s, 1H), 7.84 (s, 1H), 7.51–7.42 (m, 6H),
6
ꢀ
C
27
H
27
5
N O
2
[Mꢀ H] 452.2165 found: 452.2099.
7.38 (d, J = 7.0 Hz, 2H), 7.01 (d, J = 7.9 Hz, 2H), 6.42 (d, J = 15.8 Hz,
1
H), 5.28 (s, 2H), 3.81 (s, 3H), 2.75 (t, J = 6.5 Hz, 2H), 2.70 (t, J = 6.7
13
4
.1.3.19. (E)-N-hydroxy-3-{4-[(7-ethyl-3-phenyl-4,5-dihydropyrazolo
Hz, 2H). C NMR (151 MHz, DMSO) δ: 163.1, 150.0, 143.7, 139.8,
[
3,4-e]indazol-1(7H)-yl)methyl]phenyl}acrylamide (A23). Yield: 6.9%.
138.2, 134.2, 130.0, 129.3, 129.3, 128.9, 128.0, 127.5, 124.7, 119.4,
Mp: 168–170 ◦C. ESI-MS: m/z, 438.3 [Mꢀ H] . H NMR (600 MHz,
ꢀ
1
113.6, 111.5, 52.5, 38.8, 22.0, 19.8. HRMS (ESI+) m/z calcd for
ꢀ
DMSO‑d
6
) δ: 10.74 (s, 1H), 9.02 (s, 1H), 8.03 (s, 1H), 7.68 (d, J = 7.2 Hz,
C
25
H
23
N
5
O
2
[Mꢀ H] 424.1852 found: 424.1770.
2
8
2
3
1
5
H), 7.52 (d, J = 8.1 Hz, 2H), 7.42 (m, 3H), 7.32 (m, 1H), 7.27 (d, J =
.1 Hz, 2H), 6.42 (d, J = 15.8 Hz, 1H), 5.50 (s, 2H), 4.09 (q, J = 7.2 Hz,
H), 2.99 (t, J = 7.6 Hz, 2H), 2.81 (t, J = 7.6 Hz, 2H), 1.37 (t, J = 7.3 Hz,
4.1.3.25. N-hydroxy-4-[(7-ethyl-3-methyl-4,5-dihydropyrazolo[3,4-e]
indazol-1(7H)-yl)methyl]benzamide
(A29). Yield:
3.2%.
Mp:
1
3
ꢀ
1
◦
H). C NMR (151 MHz, DMSO) δ: 163.0, 149.4, 147.2, 139.1, 136.0,
137–140 C. ESI-MS: m/z, 350.2 [Mꢀ H] . H NMR (600 MHz,
34.2, 128.9, 128.1, 127.9, 127.6, 127.0, 123.4, 119.5, 111.3, 108.0,
DMSO‑d ) δ: 11.14 (s, 1H), 9.00 (s, 1H), 7.94 (s, 1H), 7.67 (d, J = 8.1 Hz,
6
3.1, 46.6, 22.2, 20.8, 15.9. HRMS (ESI+) m/z calcd for C26
H
25
N
5
O
2
[M-
2H), 7.20 (d, J = 8.1 Hz, 2H), 5.36 (s, 2H), 4.05 (q, J = 7.1 Hz, 2H), 2.74
ꢀ
H] 438.2008 found: 438.1928.
(t, J = 7.6 Hz, 2H), 2.65 (t, J = 7.4 Hz, 2H), 2.10 (s, 3H), 1.34 (t, J = 7.2
Hz, 3H). 13C NMR (151 MHz, DMSO) δ: 164.4, 149.5, 144.4, 134.8,
132.2, 127.5, 127.3, 123.0, 112.0, 108.3, 52.6, 46.5, 22.1, 19.3, 15.8,
4
.1.3.20. (E)-N-hydroxy-3-{4-[(7-ethyl-3-phenyl-4,5-dihydropyrazolo
[
3,4-e]indazol-2(7H)-yl)methyl]phenyl}acrylamide (A24). Yield: 4.0%.
12.0. HRMS (ESI+) m/z calcd for C19
H
21
N
5
O
2
[Mꢀ H]ꢀ 350.1695 found:
Mp: 164–168 ◦C. ESI-MS: m/z, 438.3 [Mꢀ H] . H NMR (600 MHz,
ꢀ
1
350.1612.
6
DMSO‑d ) δ: 10.75 (s, 1H), 9.02 (s, 1H), 7.89 (s, 1H), 7.46 (m, 5H), 7.39
(
t, 3H), 7.01 (d, J = 8.1 Hz, 2H), 6.40 (d, J = 15.8 Hz, 1H), 5.29 (s, 2H),
4.1.3.26. N-hydroxy-4-[(7-ethyl-3-methyl-4,5-dihydropyrazolo[3,4-e]
4
1
1
1
.10 (q, J = 7.2 Hz, 2H), 2.76 (t, J = 6.7 Hz, 2H), 2.71 (t, J = 6.8 Hz, 2H),
indazol-2(7H)-yl)methyl]benzamide
(A30). Yield:
2.7%.
Mp:
1
3
ꢀ
1
◦
.38 (t, J = 7.2 Hz, 3H). C NMR (151 MHz, DMSO) δ: 163.2, 149.9,
204–207 C. ESI-MS: m/z, 350.3 [Mꢀ H] . H NMR (600 MHz,
43.8, 139.9, 138.4, 134.1, 130.0, 129.3, 128.9, 128.0, 127.5, 123.3,
DMSO‑d ) δ: 11.16 (s, 1H), 9.01 (s, 1H), 7.79 (s, 1H), 7.69 (d, J = 8.3 Hz,
6
19.3, 113.6, 111.3, 52.5, 46.5, 22.1, 19.8, 16.0. HRMS (ESI+) m/z
2H), 7.17 (d, J = 8.3 Hz, 2H), 5.28 (s, 2H), 4.07 (q, J = 7.2 Hz, 2H), 2.73
ꢀ
calcd for C26
H
25
5
N O
2
[Mꢀ H] 438.2008 found: 438.1921.
(t, J = 7.2 Hz, 2H), 2.64 (t, J = 7.3 Hz, 2H), 2.14 (s, 3H), 1.36 (t, J = 7.3
Hz, 3H). 13C NMR (151 MHz, DMSO) δ: 164.4, 149.8, 143.0, 141.6,
134.8, 132.1, 127.5, 127.2, 123.0, 112.5, 111.6, 51.9, 46.4, 22.1, 19.2,
4
.1.3.21. (E)-N-hydroxy-3-{4-[(3-benzyl-7-methyl-4,5-dihydropyrazolo
[
3,4-e]indazol-1(7H)-yl)methyl]phenyl}acrylamide (A25). Yield: 2.5%.
16.0, 9.6. HRMS (ESI+) m/z calcd for C19
H
21
N
5
O
2
[Mꢀ H]ꢀ 350.1695
Mp: 165–168 ◦C. ESI-MS: m/z, 438.4 [Mꢀ H] . H NMR (600 MHz,
ꢀ
1
found: 350.1602.
DMSO‑d
6
) δ: 10.78 (s, 1H), 9.02 (s, 1H), 7.90 (s, 1H), 7.50 (d, J = 7.8 Hz,
2
H), 7.40 (d, J = 16.0 Hz, 1H), 7.27 (t, J = 5.7 Hz, 2H), 7.22 (t, J = 6.1
1
1

Q. Xu et al.
Bioorganic Chemistry 111 (2021) 104910
4
.1.3.27. N-hydroxy-4-[(7-(2-methoxyethyl)-3-methyl-4,5-dihydropyr-
determinations.
azolo[3,4-e]indazol-1(7H)-yl)methyl]benzamide (A31). Yield: 0.9%. Mp:
◦
ꢀ
1
1
87–190 C. ESI-MS: m/z, 380.3 [Mꢀ H] . H NMR (600 MHz,
DMSO‑d
) δ: 11.07 (s, 1H), 9.03 (s, 1H), 7.90 (s, 1H), 7.67 (d, J = 8.1 Hz,
H), 7.20 (d, J = 7.9 Hz, 2H), 5.36 (s, 2H), 4.17 (t, J = 5.2 Hz, 2H), 3.65
t, J = 5.2 Hz, 2H), 3.20 (s, 3H), 2.74 (t, J = 7.5 Hz, 2H), 2.66 (t, J = 7.4
4.2.3. Nitrite determination assay
6
N9 cells were incubated for 24 h by adherent culture. Cells were
incubated with 1 g/ml LPS (Sigma-Aldrich) and different concentra-
tions (0.01 M, 0.1 M, 1 M and 10 M) of compounds in serum-free
2
μ
(
μ
μ
μ
μ
1
3
Hz, 2H), 2.10 (s, 3H). C NMR (151 MHz, DMSO) δ: 171.9, 149.9,
44.4, 134.8, 130.0, 128.6, 127.5, 127.4, 124.4, 124.4, 112.1, 108.4,
medium for 24 h. Nitrite concentration (as a measure of NO produc-
tion) in the supernatant was determined by using Griess reagents. The
absorbance was measured at 540 nm using a microplate reader (Syn-
ergy-HT, Biotek). The IC50 values were the mean of two independent
determinations.
1
7
0.8, 58.4, 52.7, 51.4, 23.0, 19.3, 12.1. HRMS (ESI+) m/z calcd for
ꢀ
C
20
H
23
N
5
O
3
[Mꢀ H] 380.1801 found: 380.1720.
4
.1.3.28. N-hydroxy-4-[(7-(2-methoxyethyl)-3-methyl-4,5-dihydropyr-
azolo[3,4-e]indazol-2(7H)-yl)methyl]benzamide (A32). Yield: 0.8%. Mp:
4.2.4. NO capture ability determination assay
◦
ꢀ
1
1
96–199 C. ESI-MS: m/z, 380.3 [Mꢀ H] . H NMR (600 MHz,
DMSO‑d
) δ: 11.16 (s, 1H), 9.00 (s, 1H), 7.75 (s, 1H), 7.69 (d, J = 8.0 Hz,
H), 7.17 (d, J = 8.0 Hz, 2H), 5.29 (s, 2H), 4.20 (t, J = 5.2 Hz, 2H), 3.67
t, J = 5.2 Hz, 2H), 3.24 (s, 3H), 2.73 (t, J = 7.2 Hz, 2H), 2.65 (t, J = 7.2
SNP (Sodium Nitroprusside) was dissolved in PBS to prepare the 100
mM stock solution. In this experiment, SNP solution of 25
μ
L was added
6
2
to 975 L PBS solution which included different concentration of tested
μ
(
compounds. After 60 min under room temperature condition, the con-
1
3
2ꢀ
Hz, 2H), 2.14 (s, 3H). C NMR (151 MHz, DMSO) δ: 164.5, 150.1,
centration of NO was tested using Griess assay. The inhibitions of
1
42.8, 141.6, 134.9, 132.1, 127.5, 127.2, 124.2, 112.6, 111.6, 70.9,
serial concentrations of compounds were calculated and the IC50 were
estimated using Logit method. The data (mean ± SD) were calculated
according to inhibition ratios from three independent tests.
5
8.3, 51.9, 51.2, 22.1, 19.1, 9.6. HRMS (ESI+) m/z calcd for
ꢀ
C
20
H
23
5
N O
3
[Mꢀ H] 380.1801 found: 380.1702.
4
.2.5. Western blot assay
4
.1.3.29. N-hydroxy-4-[(6-(2-methoxyethyl)-3-methyl-4,5-dihydropyr-
The human neuroblastoma cell line SH-SY5Y cells were incubated in
azolo[3,4-e]indazol-1(6H)-yl)methyl]benzamide (A33). Yield: 0.5%. Mp:
◦
ꢀ
1
presence of the test compound A16 and Tubacin (in 0.5% DMSO) for 12
h, harvested, and rinsed with ice-cold PBS. Total protein extracts (40 µg)
were prepared by lysing cells in NP40 buffer (50 mM Tris-HCl, pH 8.0
1
76–179 C. ESI-MS: m/z, 379.6 [Mꢀ H] . H NMR (600 MHz,
DMSO‑d
6
) δ: 11.14 (s, 1H), 8.98 (s, 1H), 7.90 (s, 1H), 7.68–7.65 (m, 2H),
7
5
2
1
5
.21 (t, J = 8.1 Hz, 2H), 5.36 (s, 2H), 4.17 (t, J = 5.3 Hz, 2H), 3.65 (t, J =
0
.5% sodium deoxycholate, 100 µM leupeptin, 2 µg/mL aprotinin,150
.3 Hz, 2H), 3.19 (s, 3H), 2.74 (t, J = 7.5 Hz, 2H), 2.66 (t, J = 7.5 Hz,
H), 2.10 (s, 3H). 13C NMR (151 MHz, DMSO) δ: 164.4, 149.8, 144.5,
34.7, 132.1, 127.6, 127.4, 127.3, 124.3, 112.2, 110.6, 108.4, 70.8,
8.4, 52.7, 51.4, 22.2, 19.3, 12.1. LC-MS m/z 379.6 [Mꢀ H]ꢀ .
mM NaCl, 1% NP-40, 0.1% SDS and 1 mM phenylmethylsulfonyl fluo-
ride). Protein concentrations in the lysates were determined using a Bio-
Rad protein assay kit (Bio–Rad Laboratories Inc., Hercules, CA, USA)
according to the manufacturer’s instructions. The samples were sepa-
rated on SDS polyacrylamide gels and then transferred to nitrocellulose
membranes and blocked with 5% nonfat dried milk. The membranes
4
.1.3.30. N-hydroxy-4-[(6-(2-methoxyethyl)-3-methyl-4,5-dihydropyr-
azolo[3,4-e]indazol-2(6H)-yl)methyl]benzamide (A34). Yield: 0.6%. Mp:
◦
ꢀ
1
were incubated with antibodies to H3, Ac-H3, -tubulin, Ac-tubulin,
α
1
81–183 C. ESI-MS: m/z, 379.6 [Mꢀ H] . H NMR (600 MHz,
DMSO‑d
) δ: 11.15 (s, 1H), 9.00 (s, 1H), 7.69 (d, J = 8.0 Hz, 2H), 7.53 (s,
H), 7.17 (d, J = 8.0 Hz, 2H), 5.28 (s, 2H), 4.22 (t, J = 5.3 Hz, 2H), 3.64
t, J = 5.3 Hz, 2H), 3.20 (s, 3H), 2.86 (t, J = 7.6 Hz, 2H), 2.68 (t, J = 7.6
HDAC6, and β-actin overnight at 4 ◦C. The immune-complexes were
visualized using enhanced chemiluminescence western blot detection
reagents (Amersham Biosciences Inc., Piscataway, NJ).
6
1
(
1
3
Hz, 2H), 2.14 (s, 3H). C NMR (151 MHz, DMSO) δ: 164.5, 150.4,
4
.2.6. Evaluation of inflammatory mediator assay
1
43.4, 141.8, 134.7, 132.4, 127.6, 127.2, 124.2, 112.4, 111.3, 71.3,
8.6, 51.9, 48.9, 20.6, 18.9, 9.8. LC-MS m/z 379.6 [Mꢀ H]ꢀ .
The secreted levels of TNF and IL-6 were measured using Mouse Th1/
5
Th2/Th17 Cytokine Kit. It was purchased from BD Pharmingen. After
specific time in the LPS condition, the supernatants were collected and
analyzed by a flow cytometer (BD FACSCalibur). The data was analyzed
by FCAP Array v3.0. IL-2, IL-4, IL-10, IL-17A and IFN-γ were not
detected in the experiment. The data (mean ± SD) were calculated ac-
cording to inhibition ratios from three independent tests.
4
4
.2. Biological assays
.2.1. Cell culture
The mouse N9 microglial cultures were placed into IMDM medium
supplemented 5% FBS for 24 h. PC12 cells were maintained in
RPMI1640 supplemented with 10% FBS, 100 units/ml Penicillin G and
4.2.7. MTT assay
◦
4
1
00 g/ml streptomycin. All the cells were incubated at 37 C in hu-
N9 Cells were seeded in 96-well plates at 50 × 10 /ml (100
μL/well).
midified air containing with 5% CO
2
.
After 24 h incubation, 4 different concentrations (final concentrations
were 0.01, 0.1, 1 and 10 M) of tested compounds were added into the
wells. Cells were incubated for the continuous 24 h. After treatment,
MTT was added into the cells at a final concentration of 500 g/ml, and
the plates were incubated for an additional 4 h. The resultant formazan
crystals were dissolved in 200 L of DMSO, and the absorbance was
μ
4
.2.2. HDAC inhibition assay
The general procedure for the enzyme-based assay was carried out
μ
according to published protocols [24] with slight modification. The re-
combinant human HDAC1, 2, 3 and 6 were purchased from BPS
Bioscience (USA). All reactions were performed in the black half area 96-
μ
measured at 570 nm using a microplate reader (Synergy HT, Bio-Tek).
The data (mean ± SD) were calculated according to inhibition ratios
from three independent tests.
well plates. A serial dilution of the inhibitors (5
μ
L/well) and enzymes
◦
(
5
μ
L/well) were pre-incubated in HDAC buffer (10
μ
L/well) at 25 C for
1
5 min, then fluorogenic substrate (5
μL/well) Boc–Lys(Ac)–AMC was
◦
added. After incubation at 37 C for 60 min, the mixture was stopped by
the addition of developer (25 L/well) for 10 min. Fluorescence in-
4.2.8. The protection of A16 on H
2 2
O -induced PC12 injury assay
μ
MTT assay was used to determine the cell viability. PC12 cells were
4
tensity was measured using the Thermo Scientific Varioskan Flash Sta-
tion at excitation and emission wavelengths of 355 and 460 nm,
respectively. The IC50 values were extracted by curve fitting the dose/
response slopes. The IC50 values were the mean of two independent
seeded at 20 × 10 /ml (100
were pretreated with A16 and ACY-1215 for 1 h, and then challenge
(500 M) and incubated for 16 h. Cells were added with 5 mg/ml
MTT and incubated for additional 4 h. The optimal density was detected
μ
L/well) in 96-well plate. After 24 h, cells
H
2
O
2
μ
1
2

Q. Xu et al.
Bioorganic Chemistry 111 (2021) 104910
by the microplate reader at a wavelength of 570 nm. The data (mean ±
SD) were calculated according to inhibition ratios from three indepen-
dent tests.
Acknowledgement
This work was supported by the National Natural Science Foundation
of China (No.81473086), the Natural Science Foundation of Liaoning
4
.2.9. DPPH scanvenging capacity assay
Province,
China
(No.2015020728-301)
and
LiaoNing -
The radical scavenging activity of compounds was determined by
Revitalization Talents Program (XLYC1902089).
using DPPH assay. In the experiment, a DPPH ethanol solution with a
concentration of 1 × 10ꢀ M was prepared, and placed on a shaker away
4
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Declaration of Competing Interest
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The authors declare that they have no known competing financial
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1
3