Identification of novel 1,3-diaryl-1,2,4-triazole-capped histone deacetylase 6 inhibitors with potential anti-gastric cancer activity

Article abstract of DOI:10.1016/j.ejmech.2021.113392

Histone deacetylase 6 (HDAC6) has emerged as a critical regulator of many cellular pathways in tumors due to its unique structure basis and abundant substrate types. Over the past few decades, the role played by HDAC6 inhibitors as anticancer agents has sparked great interest of biochemists worldwide. However, they were less reported for gastric cancer therapy. In this paper, with the help of bioisosteric replacement, in-house library screening, and lead optimization strategies, we designed, synthesized and verified a series of 1,3-diaryl-1,2,4-triazole-capped HDAC6 inhibitors with promising anti-gastric cancer activities. Amongst, compound 9r displayed the best inhibitory activity towards HDAC6 (IC₅₀ = 30.6 nM), with 128-fold selectivity over HDAC1. Further BLI and CETSA assay proved the high affinity of 9r to HDAC6. In addition, 9r could dose-dependently upregulate the levels of acetylated α-tubulin, without significant effect on acetylated histone H3 in MGC803 cells. Besides, 9r exhibited potent antiproliferative effect on MGC803 cells, and promoted apoptosis and suppressed the metastasis without obvious toxicity, suggesting 9r would serve as a potential lead compound for the development of novel therapeutic agents of gastric cancer.

Full text of DOI:10.1016/j.ejmech.2021.113392

European Journal of Medicinal Chemistry 218 (2021) 113392
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Identification of novel 1,3-diaryl-1,2,4-triazole-capped histone
deacetylase 6 inhibitors with potential anti-gastric cancer activity
,
,
Xin-Hui Zhang a 1, Hui-Qin Kang a 1, Yuan-Yuan Tao a, Yi-Han Li a, Jun-Ru Zhao a,
Ya-Gao a **, Li-Ying Ma a *, Hong-Min Liu a
,
,
b,
, ***
a Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan Province, Key Laboratory of Technology Drug Preparation
(Zhengzhou University), Ministry of Education of China, Key Laboratory of Henan Province for Drug Quality and Evaluation, School of Pharmaceutical
Sciences, Zhengzhou University, Zhengzhou, 450001, PR China
b China Meheco Topfond Pharmaceutical Co., Ltd, Zhumadian, 463000, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Histone deacetylase 6 (HDAC6) has emerged as a critical regulator of many cellular pathways in tumors
due to its unique structure basis and abundant substrate types. Over the past few decades, the role
played by HDAC6 inhibitors as anticancer agents has sparked great interest of biochemists worldwide.
However, they were less reported for gastric cancer therapy. In this paper, with the help of bioisosteric
replacement, in-house library screening, and lead optimization strategies, we designed, synthesized and
verified a series of 1,3-diaryl-1,2,4-triazole-capped HDAC6 inhibitors with promising anti-gastric cancer
activities. Amongst, compound 9r displayed the best inhibitory activity towards HDAC6 (IC50 ¼ 30.6 nM),
with 128-fold selectivity over HDAC1. Further BLI and CETSA assay proved the high affinity of 9r to
Received 16 February 2021
Received in revised form
14 March 2021
Accepted 16 March 2021
Available online 31 March 2021
Keywords:
HDAC6
1,2,4-Triazole
Bioisostere
Gastric cancer
HDAC6. In addition, 9r could dose-dependently upregulate the levels of acetylated a-tubulin, without
significant effect on acetylated histone H3 in MGC803 cells. Besides, 9r exhibited potent antiproliferative
effect on MGC803 cells, and promoted apoptosis and suppressed the metastasis without obvious toxicity,
suggesting 9r would serve as a potential lead compound for the development of novel therapeutic agents
of gastric cancer.
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
are classified into four classes: class I (HDAC1, 2, 3, 8), class IIa
(HDAC4, 5, 7, 9), class IIb (HDAC 6, 10), and class IV (sole HDAC11)
Histone acetylation and deacetylation are widely occurring
post-translational modification processes [1,2], which maintain the
balance between nuclear and cytoplasmic protein acetylation
levels, and are catalyzed by histone acetyltransferases (HATs) and
histone deacetylases (HDACs), respectively [3] (Fig. 1A). To date,
eighteen distinct HDACs have been identified in mammals. Ac-
cording to the sequence homology to yeast protein orthologs, they
are Zn2þ-dependent enzymes, whereas class III HDACs (sirtuins
1e7) are NADþ-dependent enzymes [4,5]. Noteworthily, the dys-
regulation of HDACs is closely related to the disruption of body
homeostasis, leading to the development of human diseases,
especially cancers [1]. HDAC inhibitors (HDACi) have been identi-
fied as effective anticancer agents by altering the acetylated status
of various cellular proteins, increasing the expression levels of p21
and other genes, and inhibiting the proliferation of tumor cells via
the induction of cellular differentiation or apoptosis [6]. Currently,
four HDAC inhibitors, including vorinostat (SAHA), belinostat,
panobinostat, and romidepsin, have been approved by FDA for the
treatment of refractory or relapsed cutaneous and peripheral T cell
lymphomas, or multiple myeloma [7], chidamide was another
potent HDACi developed and approved in China for use in periph-
eral T cell lymphomas [8]. Moreover, they are all Zn2þ-dependent
inhibitors, and their representative pharmacophores could be
divided into three domains: Cap, Linker, and a zinc-binding group
* Corresponding author. Collaborative Innovation Center of New Drug Research
and Safety Evaluation, Henan Province, Key Laboratory of Technology Drug Prep-
aration (Zhengzhou University), Ministry of Education of China, Key Laboratory of
Henan Province for Drug Quality and Evaluation, School of Pharmaceutical Sciences,
Zhengzhou University, Zhengzhou, 450001, PR China.
** Corresponding author.
*** Corresponding author.
E-mail addresses: ya_gao@zzu.edu.cn (Ya-Gao), maliying@zzu.edu.cn (L.-Y. Ma),
liuhm@zzu.edu.cn (H.-M. Liu).
1
These senior authors contribute equally to this work.
https://doi.org/10.1016/j.ejmech.2021.113392
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

X.-H. Zhang, H.-Q. Kang, Y.-Y. Tao et al.
European Journal of Medicinal Chemistry 218 (2021) 113392
Fig. 1. (A) Acetylation and deacetylation, catalyzed by HATs and HDACs respectively, occur on specific lysine residues at histones N-terminal tails. (B) Current approved HDACis for
cancer therapy in clinical, and their representative pharmacophore model: Cap, Linker and zinc-binding group (ZBG); (C) The architecture of HDAC6 functional domains. Reprinted
with permission from Journal of Medicinal Chemistry [13], Copyright 2021, American Chemical Society.
(ZBG) (Fig. 1B). However, with the exception of romidepsin (selec-
tive HDAC1/2 inhibitor), the others are all lack of isoform selectivity,
and have adverse side effects, such as fatigue, cardiotoxicity,
thrombocytopenia, and bleeding in the gastrointestinal tract
[9e11]. Therefore, interest is shifting towards the development of
isoform-selective inhibitors [7,12,13].
HDAC6, the largest member of HDAC family, contains 1215
amino acid residues, two independent catalytic domains CD1 and
CD2, which are located at the N-terminal and the central region,
respectively [14,15] (Fig. 1C). In addition, the zinc-finger ubiquitin-
binding domain (ZnF-UBP) at the C-terminal of HDAC6 mediates
the interaction with the ubiquitin proteasome and aggresome
pathway to clear misfolded proteins [16,17]. And, the nuclear export
signaling (NES) part enables HDAC6 to translocate to the cytoplasm,
in combination with other approved drugs, has sparked great in-
terest of biochemists worldwide [13,15]. However, for gastric can-
cer, which is a common malignancy as one of the leading causes of
cancer-associated death in the world [26], even though the aber-
rant overexpression of HDAC6 is critical to the process of gastric
carcinogenesis [27e29], there are fewer reports on HDAC6 in-
hibitors for the treatment of gastric cancer. Therefore, the devel-
opment of HDAC6 inhibitors targeting gastric cancer appeared to be
feasible and meaningful.
1,2,4-triazole, an integral pharmacophore of many clinically
approved drugs, showed a broad range of biological functions, such
as antibacterial, anxiolytic, antiparasitic, antitumor and antiviral
activities [30e33]. Owing to its unique dipole properties and
structural rigidity, 1,2,4-triazole exerts high affinity with the bio-
and target non-histone substrates, such as
a-tubulin [18,19], cor-
logical receptors via a variety of noncovalent interactions like p-p
tactin [20,21], heat shock protein (HSP90) [22,23] and peroxir-
edoxin [24]. Owing to its unique structure basis and abundant
substrate types, HDAC6 plays a vital role in regulating cell prolif-
eration, metastasis, invasion, and mitosis in the devlopment of
various tumors [15,25]. Over the past few decades, the role played
by selective HDAC6 inhibitors as anticancer agents, either alone or
conjugation, hydrophobicity, electrostatic interactions, hydrogen
bonding, and van der Waals force. In addition,1,2,4-triazole nucleus
is a recognized bioisostere of amides, esters, and carboxylic acids
[33,34]. Intriguingly, the widely reported HDAC6 inhibitors, such as
ricolinostat (ACY-1215), citarinostat (ACY-241), HPOB, HPB, and
Nexturastat A, all share a common amide group in their scaffolds to
2

X.-H. Zhang, H.-Q. Kang, Y.-Y. Tao et al.
European Journal of Medicinal Chemistry 218 (2021) 113392
Fig. 2. (A) Representative selective HDAC6 inhibitors; (B) Lead optimization stratey based on the hit compound screened from our in-house library.
bridge the “Linker” and “Cap” portions (Fig. 2A). Thus, it appears
that the active 1,2,4-triazole may be introduced into the backbones
of these HDAC6 inhibitors to enhance their biological activities with
the help of bioisosteric replacement strategy.
of acetic acid (AcOH) and sodium cyanoborohydride for a one-pot
reaction to obtain compound 6a-aq with the yield ranging from
65% to 75%. The intermediate ester 6a were hydrolyzed under the
action of sodium hydroxide to produce carboxylic acid product 7a
with high yield (95%), then condensed with the corresponding
primary amine fragments to form the amide product 8a-c and
compound 1 in 60%e65% yield. In addition, target compound 9a-aq
could be obtained directly from 6a-aq by hydrolysis of hydroxyl-
amine with 75%e85% yield.
Fortunately, in our previous efforts, we have successfully
developed series of 1,2,4-triazole derivatives as promising anti-
cancer agents with impressive enzymatic activity and cytotoxicity
[35e37], resulting in the building of an in-house library of com-
pounds based on 1,2,4-triazoles, by which prompted us to search
for new 1,2,4-triazole scaffolds as promising inhibitors for HDAC6.
In this work, through an in-house library compounds screening, a
hit compound 1 was picked out with potential inhibitory activity
against HDAC6 (10% inhibition at 100 nM) (Fig. 2B). Further lead
optimization strategy was adopted to design and synthesize a se-
ries of novel 1,3-diaryl-1,2,4-triazole derivatives based on com-
pound 1. Then, we performed detailed SAR studies, BLI and CETSA
assay, as well as docking analysis to investigate the affinity of target
compound with HDAC6. In addition, MTT method was used to test
the antiproliferative effect of this class of HDAC6 inhibitors on
gastric cancer cell line MGC803. Further mechanism studies
confirmed the potential anti-gastritic cancer activity of target
compound.
2.2. In vitro biological evaluations
2.2.1. Enzyme inhibition of target compounds towards HDAC6 and
HDAC1
All the synthesized 1,3-diaryl-1,2,4-triazole derivatives were
tested in vitro inhibitory activity towards HDAC6 and HDAC1. The
pan-HDACi SAHA was selected as the control, and the preliminary
evaluation showed the inhibition effect of compounds at a single
concentration of 100 nM.
In our initial attempts, we have identified compound 1 as a hit
compound from our in-house library screening, showing 10% in-
hibition towards HDAC6 at 100 nM. Meanwhile, we have reason to
believe that the hydrazide moiety was responsible for the potential
inhibitory efficacy of compound 1, since the hydrazides have good
zinc-binding ability and were widely used for the design of HDAC
inhibitors [38,39]. Inspired by this progress, we firstly tried to
optimize the “ZBG” portion of hit compound 1 by replacing with
different ZBGs to explore the effect on efficacy. As shown in Table 1,
when the hydrazide moiety was replaced with benzamide group,
which was another potent ZBG present in the scaffold of HDACi
chidamide [40], the inhibitory activity towards HDAC6 reduced
moderately, while the potency against HDAC1 increased signifi-
cantly (8c vs 1). In addition, the replacements of carboxylic acid,
amide, and N-methyl amide, all resulted in a dramatical loss of
potency (7a, 8a, 8b vs 1). Conversely, the hydroxamic acid moiety
appeared to be a better alternative to enhance the activity, namely
compound 9a, showing more than 7-fold increase in the potency
towards HDAC6, which might be attributed to the stronger zinc-
binding ability of hydroxamic acid than hydrazide [41,42].
2. Results and discussion
2.1. Chemistry
The general synthetic route of target 1,3-diaryl-1,2,4-triazole
derivatives was shown in Scheme 1. Commercially available
substituted phenylhydrazine 1a-p were mixed with aromatic
aldehyde 2a-ac in ethanol water solution to obtain intermediate
hydrazones 3a-aq with more than 90% yield. Subsequently, in the
presence of tert-butyl hydroperoxide (TBHP) and I2, 3a-aq was
involved in the ring-closure reaction with ethanolamine to get the
1,2,4-triazole scaffold 4a-aq with 75%e85% yield. Then, compound
5a-aq were produced by oxidizing 4a-aq with Dess-Martin peri-
odinane in a high yield (>95%). Next, with the help of Borch
reductive amination, compound 5a-aq were mixed with methyl 4-
aminobenzoate in methanol, followed by adding catalytic amount
3

X.-H. Zhang, H.-Q. Kang, Y.-Y. Tao et al.
European Journal of Medicinal Chemistry 218 (2021) 113392
Scheme 1. Reagents and conditions: (a) AcOH, EtOH (20% wt), 80 ꢀC; (b) ethanolamine, TBHP, I2, MeCN, reflux; (c) Dess-Martin periodinane, DCM, 0ꢀC-rt; (d) AcOH, methyl 4-
aminobenzoate, sodium cyanoborohydride, MeOH; (e) 1) sodium hydroxide, methanol, 70 ꢀC; 2) formic acid, water. (f) 1) tert-Butyl carbazate, or tert-Butyl carbamate, or
methylamine hydrochloride, or N-(tert-Butoxycarbonyl)-1,2-phenylenediamine, EDCI, HOBt, DCM, rt; 2) TFA, 1,4-dioxane; (g) 1) hydroxylamine (50% water), sodium hydroxide,
DCM: MeOH ¼ 1:2; 2) formic acid, water.
Table 1
Inhibitory effect of compounds 1, 7a, 8a-c, 9a against HDAC6 and HDAC1.
Compd.
R
HDAC6 inhibitiona @ 100 nM (%)
HDAC1 inhibitiona @ 100 nM (%)
1
-NHNH2
-OH
-NH2
10.0
ꢁ0.4
ꢁ4.4
ꢁ3.6
5.0
4.1
2.1
ꢁ2.6
ꢁ0.55
32
7a
8a
8b
8c
-NHCH3
9a
SAHA
NHOH
72.6
80.2
19
83.6
e
a
Data are represented as the mean of the inhibition rate.
Moreover, compared with SAHA, 9a displayed higher selective in-
hibition for HDAC6 (72.6% for HDAC6, and 19% for HDAC1). In this
view, compound 9a was selected as the lead compound for further
structural optimization. Likewise, the inhibition rates of derivatives
at a single concentration of 100 nM were summarized for primary
SAR studies.
As shown in Table 2, we focused on optimizing compound 9a by
modifying its capping group composed of two aromatic regions
4

X.-H. Zhang, H.-Q. Kang, Y.-Y. Tao et al.
European Journal of Medicinal Chemistry 218 (2021) 113392
Table 2
Inhibitory effect of compounds 9a-aq against HDAC6 and HDAC1.
Compd.
R1
R2
HDAC6 inhibitiona @ 100 nM (%)
HDAC1 inhibitiona @ 100 nM (%)
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Ph-
72.6
53.2
64.1
62.2
54.6
27.6
77.6
70.9
75.0
61.2
67.2
34.5
54.7
41.6
65.5
19.1
16.5
13.0
20.3
13.2
4.3
33.4
20.8
15.6
17.0
19.5
7.6
2-CH3-Ph-
3-CH3-Ph-
4-CH3-Ph-
4-CH2CH3-Ph-
4-CH(CH3)2-Ph-
4-OCH3-Ph-
4-F-Ph-
2-Cl-Ph-
3-Cl-Ph-
4-Cl-Ph-
3, 4-Cl-Ph-
4-Br-Ph-
13.2
4.9
26.0
4-CF3-Ph-
9p
9q
H
H
72.1
71.1
26.3
23.7
9r
9s
9t
H
79.7
79.6
39.8
53.4
59.9
60.7
55.8
58.6
57.7
30.1
38.4
8.3
4-OH
H
9u
9v
9w
9x
9y
9z
H
15.2
16.7
22.3
13.8
16.0
12.9
H
H
H
H
H
9aa
H
H
30.8
40.2
7.5
9 ab
11.2
9ac
9ad
9ae
9af
9 ag
9ah
9ai
4-F
Ph
67.6
64.6
62.3
55.4
65.9
38.9
60.7
16.3
17.0
14.7
18.0
18.2
12.8
24.6
2-Cl
3-Cl
4-Cl
4-Br
4-CF3
4-CH3
Ph-
Ph-
Ph-
Ph-
Ph-
Ph-
(continued on next page)
5

X.-H. Zhang, H.-Q. Kang, Y.-Y. Tao et al.
European Journal of Medicinal Chemistry 218 (2021) 113392
Table 2 (continued)
Compd.
R1
R2
HDAC6 inhibitiona @ 100 nM (%)
HDAC1 inhibitiona @ 100 nM (%)
9aj
2-CH3
3-CH3
Ph-
Ph-
Ph-
4-OH-Ph-
Ph-
Ph-
Ph-
Ph-
57.1
66.0
63.4
70.3
49.8
53.0
38.3
52.6
80.2
17.0
18.4
26.2
25.9
12.4
11.0
10.8
17.3
83.6
9ak
9 al
9am
9an
9ao
9ap
9aq
SAHA
4-OCH3
4-OCH3
3, 5-CH3
3, 4-CH3
4-CH2CH3
4-CH(CH3)2
e
e
a
Data are represented as the mean of the inhibition rate.
located in the N-1 and C-3 position of 1,2,4-triazole skeleton. To our
delight, all the synthetic derivatives displayed better inhibition
effect on HDAC6 than HDAC1, revealing the potential of this 1,3-
diaryl-1,2,4-triazole-based chemical architecture for the design of
selective HDAC6 inhibitors. In detailed, when the phenyl group at
C-3 position was modified with different electron-donating groups,
such as, methyl (9d), ethyl (9e), and isopropyl (9f), the activity
showed a downward trend as the steric volume of the alkyl sub-
stituents increased (down from 62.2% to 27.6%). While, the modi-
fication of methoxyl led to a modest increase in activity (9g, up to
77.6% at 100 nM). Likewise, the activity of compound 9h, 9m, 9k,
and 9n which were substituted by increasing bulky electron-
withdrawing groups F, Cl, Br, and CF3, respectively, showed a
reduced trend (down from 70.9% to 41.6%). Meanwhile, it was also
observed that double substitutions on the phenyl was unfavorable
for the efficacy (9l < 9i, 9j, 9k). In addition, we did observe that the
changes of modification sites on phenyl group have less effect on
activity, irrespective of the types of substituents (9b vs 9c vs 9d; 9i
vs 9j vs 9k). These results indicated the modification of the C-3
phenyl showed much preference to the steric hindrance of sub-
stituents, which might be explained by the reason that inhibitors
bearing properly sized capping groups could comfortably occupy
the surface groove of HDAC6 catalytic pocket well, without a steric
clash with the residues [43].
With this in mind, several aromatic heterocycle groups,
including benzothienyl, thienyl, furyl, pyridyl, and quinazolinyl,
were introduced to further explore the potential effect of modifi-
cations at C-3 position of 1,2,4-triazole. As shown in Table 2, the
potency of these heterocyclic substituents differed substantially. In
particular, thienyl appeared to be more favorable for the activity,
and most compounds exerted more than 71% inhibition rate at
100 nM towards HDAC6. Amongst, compounds 9r (79.7%) and 9s
(79.6%) bearing 5-methylthiophene-2-yl and thiophen-2-yl sub-
stituents, respectively, exhibited superior inhibition effect close to
that of SAHA (80.2% for HDAC6). However, furyl substituent did not
give too much breakthrough in activity (9v-x < 60%). In addition,
the compounds displayed diminished activity after pyridyl modi-
fications, including pyridin-2-yl (9y, 58.6%), pyridin-3-yl (9z, 57.7%),
and pyridin-4-yl (9aa, 30.8%), which might be due to the lower
hydrophobicity of pyridyl than phenyl [43]. Besides, the activity of
aforementioned C-3 phenyl modification. Likewise, double sub-
stitutions on the phenyl proved to be adverse for the activity (9ai-
ak > 9an and 9ao). In addition, the modifications of N-1 phenyl
showed little preference to the substituent sites (9ad vs 9ae, 9af;
9ai vs 9aj, 9ak). Ultimately, the inhibitory effect of compound 9a-aq
against HDAC6 at 100 nM concentration were ranked from highest
to lowest (Fig. S1 in Supporting Information). And, compounds with
>60.0% inhibition against HDAC6 were selected for further evalu-
ation of their half-maximal inhibitory concentration (IC50) values
towards HDAC6 and HDAC1, reapectively, as well as the selectivity
index. The results were summarized in Table 3.
Consistent with above SAR studies, all chosen compounds
showed potent inhibitory activity towards HDAC6 with IC50 values
at low sub-micromole levels, and almost always better selectivity
indexes than SAHA (2.7-fold). Amongst, the lead compound 9a
displayed
potent
inhibitory
activity
against
HDAC6
(IC50 ¼ 111.7 nM), showing more than 22-fold selectivity over
HDAC1. In addition, it was worth nothing that modifications at C-3
phenyl of 1,2,4-triazole did not bring much improvement in activity
(9h, IC50 ¼ 114.5 nM; 9j, IC50 ¼ 114.6 nM). In contrast, they may
cause diminished efficacy (9a vs 9c, 9d, 9g, 9i, 9k). In addition, N-1
phenyl modification appeared to be more detrimental for the ac-
tivity, resulting in weak IC50 values for most compounds (9ac-ae,
9 ag, 9ai, 9ak), and poor selectivity (Index < 10-fold). Nevertheless,
we did observe that thienyl modifications of C-3 position could
significantly enhance the activity. In particular, compounds 9p
(IC50 ¼ 30.68 nM) and 9r (IC50 ¼ 30.6 nM) exhibited more than 3-
fold increase in activity than 9a, and excellent selectivity for HDAC6
over HDAC1 (Selectivity index > 100). Notably, in comparison to the
control Tubastatin A, which is a famous selective HDAC6 inhibitor
[44], compound 9r not only exerted more potent inhibitory activity
against HDAC6, but also higher selectivity (index ¼ 128.8). Above
all, the SAR studies on the whole target derivatives could be sum-
marized in Fig. 3.
2.2.2. The selected HDAC6 inhibitors inhibit the proliferation of
MGC803 cells
As mentioned above, HDAC6 played an important role in the
process of gastric carcinogenesis [27e29], and the inhibition of
HDAC6 may trigger cell apoptosis, inhibit motility ability, and
induce senescence and mitotic catastrophe in gastric cancer cells
[45,46]. Herein, we attempted to verify the anti-gastric cancer ac-
tivities of these HDAC6 inhibitors by choosing gastric cancer cell
line MGC803 as a mode with MTT method. The results of Table 4
showed most of them could significantly inhibit the proliferation
compound
remarkably.
9 ab (40.2%) bearing quinazolinyl also attenuate
To expand the structural optimization, we further investigated
the modifications of the phenyl located in N-1 position of 1,2,4-
triazole. Unfortunately, these compounds (9ac-aq) all displayed
lower activity than lead compound 9a. Noteworthily, compared
with electron-withdrawing groups, N-1 phenyl appeared to be
more sensitive to the electron-donating substituents. For instance,
compound 9ac-ag containing F, Cl, and Br modifications showed
little difference in activity. By contrast, the activity of compounds
bearing methyl, ethyl, and isopropyl substituents presented a
of MGC803 cells with IC50 values ranging from 5.76 to 17.70 mM.
Particularly, compound 9r with the best potency and selectivity
towards HDAC6 exhibited impressive antiproliferative activity
against MGC803 cells with IC50 value of 8.45 0.93
mM, which was
somewhat comparable with SAHA (IC50 ¼ 8.67 0.93
m
M). Like-
wise, compound 9p also displayed similar inhibition effect. How-
ever, we did observe that compounds 9j, 9k, and 9q had lower
reduced trend (9ai
> 9ap > 9aq), which was similar to
6

X.-H. Zhang, H.-Q. Kang, Y.-Y. Tao et al.
European Journal of Medicinal Chemistry 218 (2021) 113392
Table 3
Inhibitory profile of selected target compounds against HDAC6 and HDAC1 (IC50, nM).
a
Compd.
R1
R2
IC50 (nM)
Selectivity index HDAC1/6
HDAC1
HDAC6
9a
9c
9d
9g
9h
9i
9j
9k
9o
H
H
H
H
H
H
H
H
H
Ph-
111.7
280.5
223.6
163.2
114.5
184.6
114.6
180.7
323.6
2488
5522
3099
2860
3400
3831
1873
2424
1457
22.3
19.7
13.9
17.5
29.7
20.8
16.3
13.4
4.5
3-CH3-Ph-
4-CH3-Ph-
4-OCH3-Ph-
4-F-Ph-
2-Cl-Ph-
3-Cl-Ph-
4-Cl-Ph-
9p
9q
H
H
30.68
54.94
3200
2084
104.3
37.9
9r
9s
H
30.6
3941
1502
128.8
44.8
4-OH
33.53
9ac
9ad
9ae
9 ag
9ai
9ak
9 al
9am
SAHA
4-F
2-Cl
3-Cl
4-Br
4-CH3
3-CH3
4-OCH3
4-OCH3
Ph-
Ph-
Ph-
Ph-
Ph-
Ph-
Ph-
4-OH-Ph-
273.6
668.9
678.3
1729
309.7
388.2
126.9
102.4
59.3
1818
3118
4396
3199
1717
3850
882.8
4303
162.7
6.6
4.7
6.5
1.9
5.5
9.9
7.0
42.0
2.7
e
e
e
e
Tubastatin A
64.04
7048
110.1
a
IC50 values for enzymatic inhibition of HDAC enzyme.
Fig. 3. The summary of SAR studies on the target compounds.
HDAC6 potency and selectivity than 9r, while much better cyto-
toxicity. In contrast, compound 9d, 9g did not show obvious inhi-
bition effect on the proliferation of MGC803 cells. On the other
hand, compound 9o, 9ac-ae, 9 ag, 9ai, 9ak, and 9 al, bearing weak
selectivity indexes that were similar to SAHA lower than 10-fold,
displayed moderate antiproliferative activity against MGC803 cells
with IC50 values ranging from 6.11 to 13.73
mM. These results sug-
gested the dual inhibition of HDAC1/6 would lead to somewhat
7

X.-H. Zhang, H.-Q. Kang, Y.-Y. Tao et al.
European Journal of Medicinal Chemistry 218 (2021) 113392
Table 4
shown in Fig. 4E and F, compound 9r significantly leads to the
In vitro antiproliferation assay against MGC803 cells.
accumulation of acetylated
a-tubulin (Ac-a-tubulin) in a dose
a
a
dependent manner without significantly increasing acetylation of
histone H3, a major substrate of class I HDACs in MGC803 cells.
Thus, our results indicated that 9r is a selective HDAC6 inhibitor
that selectively inactivates HDAC6 in human gastric cancer cells.
Compd.
IC50
(
m
M)
Compd.
IC50 (mM)
MGC803
MGC803
9a
9c
9d
9g
9h
9i
17.70 1.24
8.71 0.25
>20
9r
9s
8.45 0.93
>20
9ac
9ad
9ae
9 ag
9ai
9ak
9 al
9am
SAHA
10.53 1.48
9.63 0.97
13.73 1.36
9.30 1.87
12.29 0.51
13.21 2.94
7.05 0.44
>20
>20
2.2.5. Molecular docking study
8.96 0.96
>20
To get a better understanding of the behavior of the compound
9r showing impressive affinity and selectivity to HDAC6, we per-
formed molecular docking study to explore the binding models of
9r within zebrafish HDAC6 CD2 (PDB code 5EF7, 1.9 Å) by using
Molecular Operating Environment (MOE) software. As shown,
compound 9r fitted well into the hydrophobic pocket and exhibited
a unique monodentate hydroxamate-Zn2þ coordination geometry
related to phenylhydroxamate HDAC6 inhibitors (Fig. 5A and B)
[50,51]. The hydroxamate NeOꢁ group of 9r coordinated with Zn2þ
directly (Zn2þ … O distance, 1.57 Å), along with the synergistic Zn2þ
coordinations of D612, H614, and D705 residues in the bottom of
HDAC6 catalytic channel. While the C]O group formed a hydrogen
9j
5.76 0.76
5.97 0.38
6.11 0.69
8.89 0.95
6.32 0.81
9k
9o
9p
9q
8.67 0.93
a
IC50 values for proliferation inhibition of MGC803. Data are presented as the
means SDs. All experiments were carried out at least three independent times.
better cytotoxicity towards MGC803 cells compared with selec-
tively inhibiting HDAC6 alone, which might be attributed to the fact
that HDAC1 also palyed an important role in the process of gastric
cancer [47e49]. Given its superior HDAC6 potency and moderate
antiproliferative activity against MGC803 cells, compound 9r was
selected to further explore its binding affinity with HDAC6 and in-
depth anti-gastric cancer mechanism.
bond with Zn2þ-bound water molecule with O
⋯H separation of
2.06 Å. In addition, the phenyl-based linker of 9r nestled in the
hydrophobic tunnel, and was sandwiched between F583 and F643,
engaging in a
p-p stacking interaction. Notably, the NH group
formed a moderate hydrogen bonding interaction with S531 res-
idue (2.42 Å), which was confirmed to be a unique “gatekeeper”
residue located at the mouth of HDAC6 CD2 active site confering
HDAC6 affinity and selectivity, that can be used to design specific
HDAC6 inhibitors targeting this residue that is absent in other
isozymes (Fig. 5A and B) [50]. Besides, it was not observed signifi-
cant intermolecular interactions between the “Y”-shaped 1,2,4-
triazole-based capping group of 9r and the surface of HDAC6 cat-
alytic pocket, except for the phenyl group was oriented toward the
L1 loop pocket delineated by residues H463, P464, F583, and L712
(Fig. 5C), which might be explained by the fact that enzyme-
inhibitors association appeared to be driven primarily by steric
complementarity between the capping groups and the three-
dimensional contours of the loop pockets in the active site of
HDAC6 [51].
2.2.3. In vitro HDAC isoform selectivity assay for compound 9r
To further explore the HDAC isoform selective profiles of com-
pound 9r, we additionally evaluated its inhibitory activity against
HDAC2, HDAC3 and HDAC8. As shown in Table 5, compound 9r
displayed similar inhibition effect on these three isoforms, with
IC50 values of 224, 235 and 349 nM, respectively, which were all
weaker than the inhibitory activity towards HDAC6. Meanwhile, in
comparison to SAHA, 9r showed better selective inhibition effect on
HDAC6. These results suggested compound 9r showed high HDAC6
selectivity over other isoforms, especially HDAC1 (Selectivity in-
dex > 128-fold).
2.2.4. Specific binding of compound 9r to HDAC6 in vitro
Through the application of BLI, which is a new technology to
explore the interaction between biological macromolecules with
small molecules, it was fully proved that compound 9r and human
recombinant HDAC6 protein had strong binding affinity in vitro.
The equilibrium dissociation constant (Kd) of 9r to HDAC6 is
7.1 0.9 nM (Fig. 4A and B). Additionally, cell thermal migration
assay (CETSA) is a method based on the biophysical principle of
ligand induced thermal stability of target protein to detect the
binding ability of compounds to target protein in cells at the
cellular level. As shown in Fig. 4C and D, compound 9r increased the
thermal stability of HDAC6 protein in MGC803 cells, and increased
its tolerance temperature from 44 ꢀC upper than 54 ꢀC. These data
indicated that 9r could penetrate the cell membrane into the
MGC803 cells, bind with HDAC6 protein, confirming the targeting
of HDAC6. In order to discuss the inhibitory effect of 9r on HDAC6 in
more depth, we observed the changes of HDAC6 substrates by
Western blot to prove the affinity of 9r to HDAC6. The result is
2.2.6. Compound 9r induces apoptosis of MGC803 cells
In order to further explore the inhibitory activity of compound
9r on MGC803 cell growth, we first detected the effect of 9r on cell
apoptosis by flow cytometry, and treated MGC803 cells with 0, 4
and 8 mM of 9r, respectively. Flow cytometry analysis showed that
9r induced early and late apoptosis of MGC803 cells by 2.93% and
54.41% respectively, while the apoptosis rate of MGC803 cells with
DMSO was only 2.83% (Fig. 6A), indicating 9r could enhance the
apoptosis rate of MGC803 cells in a dose-dependent manner. Be-
sides, Western blot showed that with the concentration of 9r
increasing, the anti-apoptosis protein Bcl-2 was down-regulated,
and caspase-9, PARP showed a significant elevation in a dose-
dependent manner (Fig. 6B and C). These findings suggested
compound 9r induced MGC803 cell apoptosis potentially via
mitochondria-mediated pathways.
Table 5
In vitro HDAC isoform selective profiles of compound 9r.
a
Compd.
IC50 (nM)
HDAC1
Selectivity index
HDAC2
HDAC3
HDAC6
HDAC8
HDAC1/6
HDAC2/6
HDAC3/6
HDAC8/6
9r
SAHA
3941
162.7
224
27
235
41
30.6
59.3
349
645
128.8
2.7
7.3
0.45
7.7
0.69
11.4
10.8
a
IC50 values for enzymatic inhibition of HDAC enzyme.
8

X.-H. Zhang, H.-Q. Kang, Y.-Y. Tao et al.
European Journal of Medicinal Chemistry 218 (2021) 113392
Fig. 4. The specific binding of compound 9r to HDAC6 in vitro. (A, B) Measurement of affinity between 9r with HDAC6 was carried out by BLI. The dissociation constant Kd values
ꢀ
were automatically calculated by using Pall-ForteBio. (C, D) CETSA confirmed the binding capacity of 9r with HDAC6 at cellular level. It is predictable that the specific binding of
compound 9r with HDAC6 protein in MGC803 cells. (E, F) The levels of the acetylation of
a-tubulin and histone H3 after treated with 9r in MGC803 cells at different concentrations
were analyzed by Western blot. Data was shown as mean SD from three independent experiments.
2.2.7. Compound 9r inhibits cell proliferation and invasion
designed, synthesized and verified a series of phenylhydroxamate
EdU staining showed that compound 9r could significantly
inhibit the proliferation of MGC803 cells (Fig. 7A and B). Addi-
tionally, transwell assay showed that compared with the DMSO, the
treatment of 9r significantly decreased the invasion rate of
MGC803 cells in a dose-dependent manner (Fig. 7C and D).
HDAC6 inhibitors bearing 1,3-diaryl-1,2,4-triazole-derived capping
groups. SAR studies showed the embellishment of two aromatic
rings at N-1 and C-3 position of 1,2,4-triazole backbone was key for
HDAC6 potency. Amongst, compound 9r bearing
a methyl-
substituted thienyl at C-3 site displayed the best inhibitory activ-
ity towards HDAC6 (IC50 ¼ 30.6 nM), with 128-fold selectivity over
HDAC1. Docking studies disclosed the behavior of 9r showing
impressive affinity to HDAC6, that was also proved by BLI and
CETSA assay. In addition, 9r could dose-dependently upregulate the
2.2.8. Safety evaluation of compound 9r by acute toxicity assay
In order to evaluate the safety of compound 9r, acute toxicity
assay was carried out. Mice were given 2 g/kg by gavage at one time
and fed and observed normally for 14 days. During this period, not
only did the mice in the 9r treatment group survive, but they also
maintained their weight without any obvious behavioral abnor-
malities (Fig. 8A). Additionally, the staining of normal tissues of
mice in each group also showed no damage to normal tissues
(Fig. 8B). Based on these experimental results, compound 9r has no
toxic effect on tissues at 2 g/kg in vivo, which fully proves the ac-
curacy of a series of anti-tumor activity evaluation and the safety of
the experimental dose.
levels of acetylated a-tubulin, without significant effect on acety-
lated histone H3 in MGC803 cells. Cellular assay displayed 9r
exhibited potent antiproliferative effect on gastric cancer cell line
MGC803. And in-depth investigation demonstrated it was involved
in inhibiting MGC-803 cells proliferation, inducing mitochondria-
related apoptosis and suppressing the metastasis ability of
MGC803 cells. What need to be emphasized is that 9r did not show
any obvious toxicity in acute toxicity test, predicting a favorable
treatment window of 9r. Taken together, our studies provided a
series of novel HDAC6 inhibitors, especially 9r, with high selectivity
towards HDAC6 over HDAC1, showed promising anti-gastric cancer
activity, and warranted further exploration as a potential lead
compound for the development of novel therapeutic agents of
gastric cancer.
3. Conclusions
Above all, with the help of bioisosteric replacement, in-house
library screening, and lead optimization strategies, we have
9

X.-H. Zhang, H.-Q. Kang, Y.-Y. Tao et al.
European Journal of Medicinal Chemistry 218 (2021) 113392
Fig. 5. (A) Predicted binding mode of 9r docked into the active pocket of zebrafish HDAC6 CD2 (PDB code 5EF7, 1.9 Å); (B) Two-dimensional binding view of 9r in HDAC6 CD2 active
site; (C) The 1,2,4-triazole-based capping group of 9r is oriented toward the L1 loop pocket delineated by residues of H463, P464, F583, and L712 (right part, colored yellow).
Fig. 6. Compound 9r induced apoptosis in MGC803 cells. (A) MGC803 cells were treated with 0, 4 or 8 mM of 9r and then processed for FACS by using Annexin V/PI staining. (B, C)
The expression level of Bcl-2, caspase 9, PARP in mitochondria-related apoptosis pathway in MGC803 treated by compound 9r or DMSO was analyzed and GAPDH was used as
control. Data was shown as mean SD from three independent experiments. *p < 0.05, **p < 0.01 compared with the control.
10

X.-H. Zhang, H.-Q. Kang, Y.-Y. Tao et al.
European Journal of Medicinal Chemistry 218 (2021) 113392
Fig. 7. Effect of compound 9r on antiproliferation, migration and invasion ability of MGC803 cells. (A, B) Immunofluorescence staining analysis of EDUþ and quantification of EDUþ
in MGC803 cells treated with 0, 2, 4 or 8 M of 9r. (C, D) The migration and invasion ability of MGC803 cells were performed by matrigel-coated transwell assay and analyzed by
m
HCA (ThermoFisher Scientific). Data was shown as mean SD from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the control.
Fig. 8. Acute toxicity in vivo. (A) Body weight was recorded. (B) Heart, liver, spleen, lung and kidney tissue sections were fixed and embedded for HE staining. Data was shown as
mean SD from three independent experiments.
4. Experimental section
The melting points of target compounds were tested by WRS-1A
digital melting point apparatus. The NMR spectra were shown in
Supporting Information.
4.1. General
During the chemical synthesis, all of the solvents and reagents
were bought from commercial companies. The chemistry reactions
were monitored by TLC and Ultra performance liquid
chromatography-mass spectrometry (UPLC-MS, Water, Milford,
MA). Column chromatography was performed at medium pressure
with silica gel (200e300 mesh). Spectra data of 1H NMR and 13C
NMR were obtained on Bruker AVANCE III 400 M spectrometer
4.2. General process for the synthesis of the compounds (3a-aq)
To a round-bottom flask (100 mL) was added commercially
available phenylhydrazine 1a-p (10 mmoL) and 30 mL ethyl alcohol
(20% in water), then stirred at room temperature, followed by
adding aromatic aldehyde 2a-ac in portions. Then, the system was
refluxed at 80 ꢀC for 6e8 h, monitored by TLC until complete
conversion, cooled to room temperature, and filtered, the solid was
washed with cold ethyl alcohol (20% in water), dried in oven to
produce intermediate hydrazones 3a-aq with more than 90% yield.
(Bruker Instruments, Inc.), Chemical shifts (d) were reported in
parts per million (ppm) relative to tetramethylsilane (TMS) and J
values were reported in Hertz. The mass spectrum data was
recorded by Waters ACQUITY UPLC H-Class ACQUITY QDa (Water,
Milford, MA), with electrospray ionization (ESI), C18 column; col-
umn size 2.1 mm ꢂ 50 mm; mobile phase 10%e95%, acetonitrile-
water-0.1% formic acid served as a binary gradient mobile phase
with a flow rate of 0.35 mL/min. High-resolution mass spectra (HR-
MS) data were signed by Water Micromass Q-T of Micromass
spectrometer with electrospray ionization (Water, Milford, MA).
4.3. General process for the synthesis of the compounds (4a-aq)
To a stirred solution of compound 3a-aq (2.5 mmol) in aceto-
nitrile (15 mL) was added ethanolamine (7.5 mmol), TBHP
(7.5 mmol) and I2 (0.5 mmol) in turn, the syetem was refluxed at
90 ꢀC for 4e5 h. Then monitored by TLC until complete conversion,
11

X.-H. Zhang, H.-Q. Kang, Y.-Y. Tao et al.
European Journal of Medicinal Chemistry 218 (2021) 113392
cooled to room temperature, and added 10 mL water, followed by
extracting with ethyl acetate (15 mL * 3). Then, the organic layer
was combined, and washed with saturated sodium chloride solu-
tion (25 mL). Finally, the solvent was concentrated, and the pre-
cipitate was purified by column chromatography over silica gel to
give the compound 4a-aq with 75%e85% yield.
Then water (10 mL) was added to quench the system, and extracted
with dichloromethane (15 mL * 3). Next, the organic layer was
combined, and washed with saturated sodium chloride solution
(25 mL). Finally, the solvent was concentrated and purified by
column chromatography over silica gel to get the Boc-protected
amide product, then this solid was dissolved into 1,4-dioxane, fol-
lowed by the addition of trifluoroacetic acid (TFA) to remove the
Boc protecting group. When complete conversion, the solvent was
removed under reduced pressure to get compound 1 as white solid.
Likewise, tert-Butyl carbamate, methylamine hydrochloride, and N-
(tert-Butoxycarbonyl)-1,2-phenylenediamine was used to produce
compounds 8a-c, respectively, the yield ranged from 60% to 65%.
4.4. General process for the synthesis of the compounds (5a-aq)
Compound 4a-aq (1 mmol) was dissolved in dichloromethane
(8 mL), then kept stirring in an ice-bath, followed by adding Dess-
Martin periodinane in portions (1.5 mmol). After stirred at room
temperature for 12 h, the system was quenched with a solution of
saturated sodium thiosulfate and sodium bicarbonate, followed by
extracting with ethyl acetate (15 mL * 3). Then, the organic layer
was combined, and washed with saturated sodium chloride solu-
tion (25 mL). The organic solvent was removed under vacuum to
get compound 5a-aq used directly for next step without
purification.
4.7.1. 4-(((1,3-diphenyl-1H-1,2,4-triazol-5-yl)methyl)amino)
benzohydrazide (1)
White solid, yield: 70%. Mp: 191e192 ꢀC. 1H NMR (400 MHz,
DMSO‑d6, ppm)
d 9.34 (s, 1H, NH, D2O exchangeable), 8.06 (d,
J ¼ 6.9 Hz, 2H, AreH), 7.70 (d, J ¼ 7.6 Hz, 2H, AreH), 7.61 (dd, J ¼ 9.5,
5.1 Hz, 3H, AreH), 7.56 (d, J ¼ 10.2 Hz, 2H, AreH), 7.48 (dt, J ¼ 6.9,
4.8 Hz, 3H, AreH), 6.75 (t, J ¼ 5.5 Hz, 1H, NH, D2O exchangeable),
6.58 (d, J ¼ 8.6 Hz, 2H, AreH), 4.54 (d, J ¼ 5.5 Hz, 2H,eCH2e), 4.32
(s, 2H, NH2, D2O exchangeable). 13C NMR (101 MHz, DMSO‑d6, ppm)
4.5. General process for the synthesis of the compounds (6a-aq)
d
166.19, 160.35, 154.33, 150.24, 136.90, 130.41, 129.54, 129.44,
Compound 5a-aq (0.5 mmol) was dissolved in methanol (5 mL),
then stirred at room temperature, and added methyl 4-
aminobenzoate (0.45 mmol), followed by the addition of catalytic
amount of acetic acid (0.05 mmol). After stirred for 0.5 h, the so-
dium cyanoborohydride (0.55 mmol) was added in portions, then
stirred for another 12 h. Water (10 mL) was added to quench the
system, and extracted with ethyl acetate (15 mL * 3). Then, the
organic layer was combined, and washed with saturated sodium
chloride solution (25 mL). Finally, removed the solvent and purified
by column chromatography over silica gel to give the compound
6a-aq with 65%e75% yield.
129.06, 128.81, 128.26, 125.87, 124.63, 120.94, 111.22. HR-MS(ESI),
calcd. C22H20N6O, [M þ H]þ m/z: 385.1777, found: 385.1770.
4.7.2. 4-(((1,3-diphenyl-1H-1,2,4-triazol-5-yl)methyl)amino)
benzamide (8a)
White solid, yield: 68%. Mp: 220e221 ꢀC. 1H NMR (400 MHz,
DMSO‑d6, ppm)
d
8.08e8.04 (m, 2H, AreH), 7.70 (d, J ¼ 7.4 Hz, 2H,
AreH), 7.62 (dd, J ¼ 7.7, 4.2 Hz, 3H, AreH), 7.57 (dd, J ¼ 10.2, 5.4 Hz,
2H, AreH), 7.52e7.41 (m, 3H, AreH), 6.90 (s, 1H, NH, D2O
exchangeable), 6.75 (s, 1H, NH, D2O exchangeable), 6.58 (d,
J ¼ 8.6 Hz, 2H), 4.54 (d, J ¼ 4.9 Hz, 2H,eCH2e). 13C NMR (101 MHz,
DMSO‑d6, ppm)
d 167.83, 160.36, 154.35, 150.35, 136.91, 130.44,
4.6. General process for the synthesis of the compounds (7a)
129.53, 129.43, 129.05, 128.93, 128.81, 125.87, 124.63, 121.99, 111.09,
38.87. HR-MS(ESI), calcd. C22H19N5O, [M þ H]þ m/z: 370.1668,
found: 370.1662.
To a mixture of compound 6a (0.5 mmol) in methanol (8 mmol)
was added sodium hydroxide (2.5 mmol), the system was kept
refluxing at 70 ꢀC for 5e6 h, then monitored by TLC until complete
conversion, then cooled to room temperature, and the solvent was
removed under reduced pressure. Subsequently, the precipitate
was dissolved in water (5 mL), followed by adding formic acid to
adjust pH to 5e6, the solid was filtered, and washed with water,
dried in oven to get compound 7a as white solid with 95% yield.
4-(((1,3-diphenyl-1H-1,2,4-triazol-5-yl)methyl)amino)benzoic
acid (7a).
4.7.3. 4-(((1,3-diphenyl-1H-1,2,4-triazol-5-yl)methyl)amino)-N-
methylbenzamide (8b)
White solid, yield: 75%. Mp: 221e222 ꢀC. 1H NMR (400 MHz,
DMSO‑d6, ppm)
d
8.08e8.04 (m, 2H, AreH), 8.00 (d, J ¼ 4.5 Hz, 1H,
NH, D2O exchangeable), 7.70 (d, J ¼ 7.6 Hz, 2H, AreH), 7.64e7.54 (m,
5H, AreH), 7.53e7.42 (m, 3H, AreH), 6.73 (t, J ¼ 5.5 Hz, 1H, NH, D2O
exchangeable), 6.58 (d, J ¼ 8.7 Hz, 2H), 4.54 (d, J ¼ 5.5 Hz,
2H,eCH2e), 2.71 (d, J ¼ 4.5 Hz, 3H,eCH3e). 13C NMR (101 MHz,
White solid, yield: 68.8%. Mp: 188e189 ꢀC. 1H NMR (400 MHz,
DMSO‑d6, ppm)
d
12.08 (s, 1H, OH, D2O exchangeable), 8.06 (d,
DMSO‑d6, ppm) d 166.51, 160.35, 154.36, 150.16, 136.92, 130.44,
J ¼ 7.9 Hz, 2H, AreH), 7.73e7.68 (m, 3H, AreH), 7.66 (s, 1H, AreH),
7.62 (d, J ¼ 7.6 Hz, 1H, AreH), 7.60e7.53 (m, 2H, AreH), 7.50 (t,
J ¼ 7.0 Hz, 2H, AreH), 7.45 (dd, J ¼ 10.4, 3.8 Hz, 1H, AreH), 7.01 (t,
J ¼ 5.3 Hz, 1H, NH, D2O exchangeable), 6.61 (d, J ¼ 8.4 Hz, 2H,
AreH), 4.57 (d, J ¼ 5.3 Hz, 2H,eCH2e). 13C NMR (101 MHz,
129.53, 129.43, 129.05, 128.80, 128.35, 125.86, 124.64, 122.35, 111.17,
38.87, 26.05. HR-MS(ESI), calcd.
384.1824, found: 384.1818.
C
23H21N5O, [M
þ
H]þ m/z:
4.7.4. N-(2-aminophenyl)-4-(((1,3-diphenyl-1H-1,2,4-triazol-5-yl)
methyl)amino) ben-zamide(8c)
DMSO‑d6, ppm)
d 167.35, 160.39, 154.14, 151.65, 136.88, 130.98,
White solid, yield: 63%. Mp: 240e241 ꢀC. 1H NMR (400 MHz,
130.42, 129.54, 129.43, 129.08, 125.88, 124.69, 38.73. HR-MS(ESI),
calcd. C22H18N4O2, [MþH]þ m/z: 371.1508, found: 371.1502.
DMSO‑d6, ppm).
d 9.29 (s, 1H, NH, D2O exchangeable), 8.27e7.99
(m, 2H, AreH), 7.75 (d, J ¼ 8.7 Hz, 2H, AreH), 7.73e7.69 (m, 2H,
AreH), 7.62 (t, J ¼ 7.4 Hz, 2H, AreH), 7.57 (d, J ¼ 7.2 Hz, 1H, AreH),
7.50 (s, 1H, AreH), 7.49e7.41 (m, 2H, AreH), 7.13 (dd, J ¼ 7.8, 1.2 Hz,
1H, AreH), 6.94 (dd, J ¼ 10.9, 4.4 Hz, 1H, AreH), 6.85 (t, J ¼ 5.5 Hz,
1H, NH, D2O exchangeable), 6.76 (dd, J ¼ 7.9, 1.2 Hz, 1H, AreH), 6.63
(d, J ¼ 8.8 Hz, 2H, AreH), 6.61e6.55 (m, 1H, AreH), 4.81 (s, 2H, NH,
D2O exchangeable), 4.58 (d, J ¼ 5.6 Hz, 2H,eCH2e). 13C NMR
4.7. General process for the synthesis of the compounds (1, 8a-c)
To a mixture of compound 7a (0.3 mmol) in dichloromethane
(10 mL) was added EDCI (0.9 mmol) and HOBt (0.3 mmol), then
stirred at room temperature for 0.5 h. Subsequently, tert-Butyl
carbazate (0.6 mmol), one of involved amine fragments, was added.
The system was kept stirring at room temperature for another 12 h.
(101 MHz, DMSO‑d6, ppm)
d 164.96, 160.38, 154.35, 150.56, 142.94,
12

X.-H. Zhang, H.-Q. Kang, Y.-Y. Tao et al.
European Journal of Medicinal Chemistry 218 (2021) 113392
136.92, 130.44, 129.56, 129.21, 125.87, 124.64, 124.05, 116.30, 116.16,
111.17. HR-MS(ESI), calcd. C28H24N6O, [M þ H]þ m/z: 461.2090,
found: 461.2083.
136.85, 129.52, 129.37, 129.02, 128.15, 127.61, 125.82, 124.58, 120.14,
111.26, 20.91. HR-MS (ESI), calcd.
400.1773, found: 400.1767.
C
23H21N5O2, [MþH]þ m/z:
4.8. General process for the synthesis of the compounds (9a-aq)
4.8.5. 4-(((3-(4-ethylphenyl)-1-phenyl-1H-1,2,4-triazol-5-yl)
methyl)amino)-N-hydroxybenzamide (9e)
To a mixture of compound 6a-aq (0.25 mmol) in DCM:
MeOH ¼ 1:2 (8 mL) was added hydroxylamine (50% in water,
5 mmol), then stirred at room temperature for 0.5 h, followed by
adding sodium hydroxide (1 mmol). The system was kept stirring
for another 0.5e1 h, monitored by TLC until complete conversion.
Then the solvent was removed under vacuum, and the precipitate
was dissolved in water (5 mL), followed by the addition of formic
acid to adjust pH to 5e6, the solid was filtered, and washed with
water, dried in oven to get target compounds 9a-9aq with 75%e85%
yield.
Dark yellow solid, yield: 81%. Mp: 227e228 ꢀC. 1H NMR
(400 MHz, DMSO‑d6, ppm)
d 10.84 (s, 1H, OH, D2O exchangeable),
8.78 (s, 1H, NH, D2O exchangeable), 7.97 (d, J ¼ 8.0 Hz, 2H, AreH),
7.68 (d, J ¼ 7.8 Hz, 2H, AreH), 7.66e7.48 (m, 5H, AreH), 7.33 (d,
J ¼ 8.0 Hz, 2H, AreH), 6.75 (t, J ¼ 5.6 Hz,1H, NH, D2O exchangeable),
6.58 (t, J ¼ 7.3 Hz, 2H, AreH), 4.54 (dd, J ¼ 9.0, 5.4 Hz, 2H,eCH2e),
2.66 (q, J ¼ 7.6 Hz, 2H, eCH2-),1.21 (t, J ¼ 7.6 Hz, 3H, eCH3). 13C NMR
(101 MHz, DMSO‑d6, ppm)
d 160.42, 154.12, 150.28, 145.21, 136.85,
130.93, 129.52, 129.01, 128.17, 127.86, 125.91, 124.62, 124.57, 120.14,
111.26, 27.97, 15.30. LC-MS tR ¼ 4.748 min; calcd. C24H23N5O2,
[MþH]þ m/z: 414.20, found: 414.23.
4.8.1. 4-(((1,3-diphenyl-1H-1,2,4-triazol-5-yl)methyl)amino)-N-
hydroxybenzamide (9a)
4.8.6. N-hydroxy-4-(((3-(4-isopropylphenyl)-1-phenyl-1H-1,2,4-
triazol-5-yl)methyl)amino)benzamide (9f)
White solid, yield: 85%. Mp: 213e214 ꢀC. 1H NMR (400 MHz,
DMSO‑d6, ppm)
d
10.84 (s, 1H, OH, D2O exchangeable), 8.78 (s, 1H,
Yellowish brown solid, yield: 86%. Mp: 227e228 ꢀC. 1H NMR
NH, D2O exchangeable), 8.05 (d, J ¼ 7.5 Hz, 2H, AreH), 7.69 (d,
J ¼ 7.8 Hz, 2H, AreH), 7.61 (td, J ¼ 14.5, 13.6, 6.3 Hz, 4H, AreH), 7.50
(h, J ¼ 7.7, 6.9 Hz, 4H, AreH), 6.76 (t, J ¼ 5.7 Hz, 1H, NH, D2O
exchangeable), 6.59 (t, J ¼ 7.9 Hz, 2H, AreH), 4.55 (dd, J ¼ 9.5,
(400 MHz, DMSO‑d6, ppm)
d
7.97 (d, J ¼ 8.0 Hz, 2H, AreH), 7.68 (d,
J ¼ 7.3 Hz, 3H, AreH), 7.65e7.47 (m, 4H, AreH), 7.36 (d, J ¼ 7.9 Hz,
2H, AreH), 6.74 (t, J ¼ 5.6 Hz, 1H, OH, D2O exchangeable), 6.55 (dd,
J ¼ 15.3, 8.3 Hz, 2H, AreH), 4.52 (t, J ¼ 4.6 Hz, 2H, eCH2-), 2.94 (h,
J ¼ 7.0 Hz, 1H, eCHe), 1.23 (d, J ¼ 6.8 Hz, 6H,-CH3).13C NMR
5.5 Hz, 2H, eCH2-). 13C NMR (101 MHz, DMSO‑d6, ppm)
d 160.33,
154.30, 154.15, 150.28, 136.82, 130.95, 130.32, 129.54, 129.47, 129.09,
128.82, 128.15, 125.85, 124.67, 124.61, 120.15, 111.26. HR-MS (ESI),
calcd. C22H19N5O2, [MþH]þ m/z: 386.1617, found: 386.1610.
(101 MHz, DMSO‑d6, ppm) d 160.41, 154.20, 154.13, 150.26, 149.80,
136.87, 129.52, 129.01, 128.10, 128.01, 126.71, 125.94, 124.56, 111.27,
33.25, 23.67. LC-MS tR ¼ 5.000 min; calcd. C25H25N5O2, [MþH]þ m/
z: 428.21, found: 428.23.
4.8.2. N-hydroxy-4-(((1-phenyl-3-(o-tolyl)-1H-1,2,4-triazol-5-yl)
methyl)amino)benzamide (9b)
4.8.7. N-hydroxy-4-(((3-(4-methoxyphenyl)-1-phenyl-1H-1,2,4-
triazol-5-yl)methyl)amino)benzamide (9g)
Off-white solid, yield: 81%. Mp: 224e225 ꢀC. 1H NMR (400 MHz,
DMSO‑d6, ppm)
d
10.80 (s, 1H, OH, D2O exchangeable), 8.71 (s, 1H,
Light yellow solid, yield: 81%. Mp: 210e211 ꢀC. 1H NMR
NH, D2O exchangeable), 7.97 (d, J ¼ 7.0 Hz, 1H, AreH), 7.71 (d,
J ¼ 7.6 Hz, 2H, AreH), 7.61 (t, J ¼ 7.5 Hz, 2H, AreH), 7.54 (dd, J ¼ 12.0,
8.0 Hz, 3H, AreH), 7.37e7.26 (m, 3H, AreH), 6.74 (t, J ¼ 5.5 Hz, 1H,
NH, D2O exchangeable), 6.62 (d, J ¼ 8.7 Hz, 2H, AreH), 4.56 (d,
J ¼ 5.5 Hz, 2H, eCH2-), 2.61 (s, 3H, eCH3). 13C NMR (101 MHz,
(400 MHz, DMSO‑d6, ppm) d 10.80 (s, 1H, OH, D2O exchangeable),
8.71 (s, 1H, NH, D2O exchangeable), 7.98 (d, J ¼ 8.4 Hz, 2H, AreH),
7.68 (d, J ¼ 7.4 Hz, 2H, AreH), 7.59 (t, J ¼ 7.2 Hz, 2H, AreH), 7.52 (d,
J ¼ 8.5 Hz, 3H), 7.04 (d, J ¼ 8.4 Hz, 2H, AreH), 6.75 (s, 1H, NH, D2O
exchangeable), 6.58 (d, J ¼ 8.4 Hz, 2H, AreH), 4.51 (d, J ¼ 4.5 Hz, 2H,
DMSO‑d6, ppm)
d
161.12, 153.29, 150.40, 136.99, 136.39, 131.23,
eCH2-), 3.80 (s, 3H, eCH3). 13C NMR (101 MHz, DMSO)
d 164.77,
129.55, 128.94, 128.13, 125.84, 124.41, 120.27, 111.36, 21.66. HR-MS
160.30, 160.20, 154.03, 150.33, 136.97, 129.51, 128.92, 128.17, 127.38,
124.56, 123.00, 120.26, 114.18, 111.28, 55.18. HR-MS(ESI), calcd.
C
(ESI), calcd. C23H21N5O2, [MþH]þ m/z: 400.1773, found: 400.1767.
23H21N5O3, [MþH]þ m/z: 416.1722, found: 416.1716.
4.8.3. N-hydroxy-4-(((1-phenyl-3-(p-tolyl)-1H-1,2,4-triazol-5-yl)
methyl)amino)benzamide (9c)
4.8.8. 4-(((3-(4-fluorophenyl)-1-phenyl-1H-1,2,4-triazol-5-yl)
methyl)amino)-N-hydroxybenzamide (9h)
Brown solid, yield: 82%. Mp: 224e225 ꢀC. 1H NMR (400 MHz,
DMSO‑d6, ppm)
d
7.92e7.81 (m, 2H, AreH), 7.68 (d, J ¼ 7.9 Hz, 2H,
Gray solid, yield: 84%. Mp: 214e215 ꢀC. 1H NMR (400 MHz,
AreH), 7.65e7.48 (m, 5H, AreH), 7.38 (t, J ¼ 7.6 Hz, 1H, AreH), 7.27
(d, J ¼ 7.7 Hz, 1H, AreH), 6.75 (t, J ¼ 5.7 Hz, 1H, NH, D2O
exchangeable), 6.55 (dd, J ¼ 11.3, 8.4 Hz, 2H, AreH), 4.53 (d,
DMSO‑d6, ppm)
d
8.09 (dd, J ¼ 8.6, 5.6 Hz, 2H, AreH), 7.69 (d,
J ¼ 7.6 Hz, 2H, AreH), 7.65e7.48 (m, 5H, AreH), 7.33 (t, J ¼ 8.7 Hz,
2H, AreH), 6.75 (t, J ¼ 5.6 Hz, 1H, NH, D2O exchangeable), 6.56 (dd,
J ¼ 13.2, 8.5 Hz, 2H, AreH), 4.53 (d, J ¼ 5.5 Hz, 2H, eCH2-). 13C NMR
J
¼
5.5 Hz, 2H, eCH2-), 2.38 (s, 3H, eCH3). 13C NMR
(101 MHz,DMSO‑d6, ppm)
d
160.41, 154.20, 150.25, 137.99, 136.83,
(101 MHz, DMSO‑d6, ppm) d 164.02, 161.57, 159.56, 154.43, 150.24,
130.76, 130.26, 130.11, 129.53, 129.06, 128.73, 128.13, 126.33, 124.58,
123.05, 120.16, 111.25, 20.96. HR-MS (ESI), calcd. C23H21N5O2,
[MþH]þ m/z: 400.1773, found: 400.1767.
136.75, 130.76, 129.54, 129.13, 128.13, 128.05, 126.91, 124.62, 120.17,
115.93, 115.71, 111.27,110.97. LC-MS tR
¼
4.322min；calcd.
C
22H18FN5O2, [MþH]þ m/z: 404.15, found: 404.15.
4.8.4. N-hydroxy-4-(((1-phenyl-3-(p-tolyl)-1H-1,2,4-triazol-5-yl)
methyl)amino)benzamide (9d)
4.8.9. 4-(((3-(2-chlorophenyl)-1-phenyl-1H-1,2,4-triazol-5-yl)
methyl)amino)-N-hydroxybenzamide (9i)
Light yellow solid, yield: 83%. Mp: 224e225 ꢀC.1H NMR
Gray solid, yield: 81%. Mp: 214e215 ꢀC.1H NMR (400 MHz,
(400 MHz, DMSO‑d6, ppm)
d
10.85 (s, 1H, OH, D2O exchangeable),
DMSO‑d6, ppm) d 10.80 (s, 1H, OH, D2O exchangeable), 8.70 (s, 1H,
8.80 (s, 1H, NH, D2O exchangeable), 7.95 (d, J ¼ 7.9 Hz, 2H, AreH),
7.68 (d, J ¼ 7.6 Hz, 2H, AreH), 7.64e7.48 (m, 5H, AreH), 7.30 (d,
J ¼ 8.0 Hz, 2H, AreH), 6.75 (t, J ¼ 5.6 Hz, 1H), 6.58 (d, J ¼ 8.6 Hz, 2H,
AreH), 4.53 (d, J ¼ 5.8 Hz, 2H, eCH2-), 2.36 (s, 3H,-CH3). 13C NMR
NH, D2O exchangeable), 8.00e7.83 (m,1H, AreH), 7.71 (d, J ¼ 7.7 Hz,
2H, AreH), 7.66e7.54 (m, 4H, AreH), 7.53e7.44 (m, 4H, AreH), 6.74
(s,1H, NH, D2O exchangeable), 6.60 (d, J ¼ 8.4 Hz, 2H, AreH), 4.57 (s,
2H,eCH2e).13C NMR (101 MHz, DMSO‑d6, ppm)
d 164.74, 158.90,
(101 MHz, DMSO‑d6, ppm)
d
164.79, 160.42, 154.12, 150.28, 138.99,
153.70, 150.34, 136.82, 131.56, 131.27, 130.71, 130.65, 129.56, 129.37,
13

X.-H. Zhang, H.-Q. Kang, Y.-Y. Tao et al.
European Journal of Medicinal Chemistry 218 (2021) 113392
129.07, 128.13, 127.25, 124.53, 120.30, 111.35. HR-MS (ESI), calcd.
C
D2O exchangeable), 6.56 (d, J ¼ 8.3 Hz, 2H, AreH), 4.53 (d,
22H18ClN5O2, [MþNa]þ m/z: 442.1047, found:442.1040.
J ¼ 5.5 Hz, 2H, eCH2-). 13C NMR (101 MHz, DMSO‑d6, ppm)
d 164.73,
158.33, 154.91, 150.24, 136.67, 132.01, 131.72, 131.35, 130.97, 129.57,
129.29, 128.18, 127.33, 125.90, 124.68, 120.35, 111.29, 38.87. LC-MS
tR ¼ 5.083 min; calcd. C23H18F3N5O2, [MþH]þ m/z: 454.15, found:
454.07.
4.8.10. 4-(((3-(3-chlorophenyl)-1-phenyl-1H-1,2,4-triazol-5-yl)
methyl)amino)-N-hydroxybenzamide (9j)
Brown solid, yield: 83%. Mp: 214e215 ꢀC. 1H NMR (400 MHz,
DMSO‑d6, ppm)
d 10.80 (s, 1H, OH, D2O exchangeable), 8.71 (s, 1H,
NH, D2O exchangeable), 8.21e7.94 (m, 2H, AreH), 7.71 (d, J ¼ 7.2 Hz,
2H, AreH), 7.61 (t, J ¼ 7.3 Hz, 2H, AreH), 7.57 (d, J ¼ 7.0 Hz, 1H,
AreH), 7.56e7.52 (m, 3H, AreH), 7.51 (s, 1H, AreH), 6.75 (t,
J ¼ 5.4 Hz, 1H, NH, D2O exchangeable), 6.58 (d, J ¼ 8.7 Hz, 2H,
AreH), 4.54 (d, J ¼ 5.5 Hz, 1H, eCH2-). 13C NMR (101 MHz, DMSO‑d6,
4.8.15. 4-(((3-(benzo[b]thiophen-2-yl)-1-phenyl-1H-1,2,4-triazol-
5-yl)methyl)amino)-N-hydroxybenzamide (9o)
Dark brown solid, yield: 84%. Mp: 231e232 ꢀC. 1H NMR
(400 MHz, DMSO‑d6, ppm)
d 10.80 (s, 1H, OH, D2O exchangeable),
8.71 (s, 1H, NH, D2O exchangeable), 8.01 (d, J ¼ 6.5 Hz, 2H, AreH),
7.93 (s, 1H, AreH), 7.71 (d, J ¼ 7.6 Hz, 2H, AreH), 7.66e7.55 (m, 3H,
AreH), 7.52 (d, J ¼ 8.3 Hz, 2H, AreH), 7.45e7.38 (m, 2H, AreH), 6.79
(s, 1H, NH, D2O exchangeable), 6.57 (d, J ¼ 8.5 Hz, 2H, AreH), 4.54
(d, J ¼ 5.5 Hz, 2H, eCH2-). 13C NMR (101 MHz, DMSO‑d6, ppm)
ppm) d 164.72, 159.07, 154.70,150.26, 136.75, 133.60, 132.44, 130.95,
129.56, 129.30, 129.22, 128.17, 125.36, 124.67, 124.42, 120.33, 111.28.
HR-MS (ESI), calcd. C22H18ClN5O2, [MþNa]þ m/z: 442.1047, found:
442.1040.
d
164.75, 162.28, 156.74, 154.81, 150.25, 139.56, 139.17, 136.61,
4.8.11. 4-(((3-(4-chlorophenyl)-1-phenyl-1H-1,2,4-triazol-5-yl)
methyl)amino)-N-hydroxybenzamide (9k)
133.06,129.59,129.31, 128.20, 125.40, 124.84,124.80, 124.37,122.88,
122.61, 120.33, 111.26, 38.78. HR-MS (ESI), calcd. C24H19N5O2S,
[MþNa]þ m/z: 464.1157, found: 464.1150.
Dark brown solid, yield: 82%. Mp: 214e215 ꢀC.1H NMR
(400 MHz, DMSO‑d6, ppm)
d 10.79 (s, 1H, OH, D2O exchangeable),
8.70 (s, 1H, NH, D2O exchangeable), 8.05 (d, J ¼ 8.2 Hz, 2H, AreH),
7.69 (d, J ¼ 7.7 Hz, 2H, AreH), 7.61 (t, J ¼ 7.3 Hz, 2H, AreH),
7.58e7.54 (m, 3H, AreH), 7.51 (d, J ¼ 8.3 Hz, 2H, AreH), 6.74 (d,
J ¼ 5.9 Hz, 1H, NH, D2O exchangeable), 6.57 (d, J ¼ 8.4 Hz, 2H,
AreH), 4.53 (d, J ¼ 5.4 Hz, 2H, eCH2-). 13C NMR (101 MHz, DMSO‑d6,
4.8.16. N-hydroxy-4-(((3-(3-methylthiophen-2-yl)-1-phenyl-1H-
1,2,4-triazol-5-yl)methyl)amino)benzamide (9p)
Gray solid, yield: 81%. Mp: 231e232 ꢀC.1H NMR (400 MHz,
DMSO‑d6, ppm)
d 10.80 (s, 1H, OH, D2O exchangeable), 8.71 (s, 1H,
NH, D2O exchangeable), 7.68 (d, J ¼ 7.7 Hz, 2H, AreH), 7.60 (t,
J ¼ 7.3 Hz, 2H, AreH), 7.55 (d, J ¼ 7.0 Hz, 1H, AreH), 7.53e7.46 (m,
3H, AreH), 7.01 (d, J ¼ 4.8 Hz, 1H, AreH), 6.76 (s, 1H, NH, D2O
exchangeable), 6.56 (d, J ¼ 8.3 Hz, 2H, AreH), 4.51 (s, 2H, eCH2-),
ppm)
d 164.75, 159.44, 154.59, 150.29, 136.80, 134.05, 129.55,
129.29, 129.15, 128.96, 128.17, 127.60, 124.65, 120.30, 111.28. HR-MS
(ESI), calcd.
420.1040.
C
22H18ClN5O2, [MþNa]þ m/z: 442.1047, found:
2.54 (s, 3H, eCH3-). 13C NMR (101 MHz, DMSO‑d6, ppm)
d 164.73,
157.38, 153.59, 150.28, 137.06, 136.79, 131.60, 129.55, 129.04, 128.14,
126.73, 125.91, 124.59, 120.28, 111.28, 38.70, 15.46. HR-MS (ESI),
calcd. C21H19N5O2S, [MþNa]þ m/z: 428.1157, found: 428.1150.
4.8.12. 4-(((3-(3,4-dichlorophenyl)-1-phenyl-1H-1,2,4-triazol-5-yl)
methyl)amino)-N-hydroxybenzamide (9l)
Gray solid, yield: 81%. Mp: 214e215 ꢀC.1H NMR (400 MHz,
DMSO‑d6, ppm)
d
10.80 (s, 1H, OH, D2O exchangeable), 8.71 (s, 1H,
4.8.17. 4-(((3-(3-bromothiophen-2-yl)-1-phenyl-1H-1,2,4-triazol-
5-yl)methyl)amino)-N-hydroxybenzamide (9q)
NH, D2O exchangeable), 8.18 (s, 1H, AreH), 8.00 (d, J ¼ 8.4 Hz, 1H,
AreH), 7.77 (d, J ¼ 8.4 Hz, 1H, AreH), 7.71 (d, J ¼ 7.8 Hz, 2H, AreH),
7.66e7.56 (m, 3H, AreH), 7.52 (d, J ¼ 8.4 Hz, 2H, AreH), 6.75 (t,
J ¼ 5.2 Hz, 1H, NH, D2O exchangeable), 6.58 (d, J ¼ 8.5 Hz, 2H,
AreH), 4.54 (d, J ¼ 5.3 Hz, 2H, eCH2-). 13C NMR (101 MHz, DMSO‑d6,
Dark brown solid, yield: 83%. Mp: 231e232 ꢀC. 1H NMR
(400 MHz, DMSO‑d6, ppm)
d 10.79 (s, 1H, OH, D2O exchangeable),
7.78 (d, J ¼ 1.6 Hz, 1H, NH, D2O exchangeable), 7.69e7.64 (m, 2H,
AreH), 7.60 (d, J ¼ 2.0 Hz, 2H, AreH), 7.58 (t, J ¼ 3.0 Hz, 2H, AreH),
7.51 (s, 1H, AreH), 7.49 (s, 1H, AreH), 6.74 (s, 1H, NH, D2O
exchangeable), 6.53 (d, J ¼ 8.5 Hz, 2H, AreH), 4.50 (s, 2H,eCH2e).
ppm)
d 164.72, 158.33, 154.91, 150.24, 136.67, 132.01, 131.73, 131.36,
130.96, 129.57, 125.90, 124.68, 120.34, 111.28. HR-MS (ESI), calcd.
22H17Cl2N5O2, [MþNa]þ m/z: 476.0657, found: 476.0650.
C
13C NMR (101 MHz, DMSO‑d6, ppm)
d 164.73, 155.60, 154.71, 150.17,
136.51, 134.71, 129.57, 129.33, 128.18, 127.89, 125.09, 124.77, 120.34,
111.23, 109.54, 38.72. HR-MS (ESI), calcd. C20H16BrN5O2S, [MþNa]þ
m/z: 492.0106, found: 4692.0099.
4.8.13. 4-(((3-(4-bromophenyl)-1-phenyl-1H-1,2,4-triazol-5-yl)
methyl)amino)-N-hydroxybenzamide (9m)
White gray solid, yield: 84%. Mp: 214e215 ꢀC. 1H NMR
(400 MHz, DMSO‑d6, ppm)
d
10.80 (s, 1H, OH, D2O exchangeable),
4.8.18. N-hydroxy-4-(((3-(5-methylthiophen-2-yl)-1-phenyl-1H-
1,2,4-triazol-5-yl)methyl)amino)benzamide (9r)
8.70 (s, 1H, NH, D2O exchangeable), 7.98 (d, J ¼ 8.2 Hz, 2H, AreH),
7.69 (d, J ¼ 8.3 Hz, 4H, AreH), 7.60 (t, J ¼ 7.5 Hz, 2H, AreH), 7.56 (d,
J ¼ 7.0 Hz, 1H, AreH), 7.51 (d, J ¼ 8.4 Hz, 2H, AreH), 6.75 (s, 1H, NH,
D2O exchangeable), 6.57 (d, J ¼ 8.4 Hz, 2H, AreH), 4.52 (d, J ¼ 4.1 Hz,
White yellow solid, yield: 84%. Mp: 231e232 ꢀC. 1H NMR
(400 MHz, DMSO‑d6, ppm)
d 10.79 (s, 1H, OH, D2O exchangeable),
8.70 (s, 1H, NH, D2O exchangeable), 7.65 (d, J ¼ 7.3 Hz, 2H, AreH),
7.58 (dd, J ¼ 14.5, 6.7 Hz, 3H, AreH), 7.50 (d, J ¼ 8.3 Hz, 2H, AreH),
7.43 (d, J ¼ 3.2 Hz, 1H, AreH), 6.85 (d, J ¼ 3.4 Hz, 1H, AreH), 6.75 (t,
J ¼ 5.5 Hz, 1H, NH, D2O exchangeable), 6.53 (d, J ¼ 8.2 Hz, 2H,
AreH), 4.48 (d, J ¼ 5.2 Hz, 2H, eCH2-), 2.48 (s, 3H, eCH3). 13C NMR
2H, eCH2-). 13C NMR (101 MHz, DMSO‑d6, ppm)
d 164.75, 159.50,
154.61, 150.29, 136.80, 131.88, 129.63, 129.55, 129.16, 128.17, 127.86,
124.65, 122.77, 120.29, 111.27. HR-MS (ESI), calcd. C22H18BrN5O2,
[MþNa]þ m/z: 486.0542, found: 464.0535.
(101 MHz, DMSO‑d6, ppm)
d 164.74, 156.83, 154.15, 150.23, 140.98,
4.8.14. N-hydroxy-4-(((1-phenyl-3-(4-(trifluoromethyl)phenyl)-
1H-1,2,4-triazol-5-yl)methyl)amino)benzamide (9n)
136.68, 130.65, 129.53, 129.08, 128.16, 126.41, 126.29, 124.67, 120.27,
111.22, 38.69, 15.00. HR-MS (ESI), calcd. C21H19N5O2S, [MþNa]þ m/
z: 428.1157, found: 406.1150.
Pale pink solid, yield: 85%. Mp: 214e215 ꢀC. 1H NMR (400 MHz,
DMSO‑d6, ppm)
d 10.79 (s, 1H, OH, D2O exchangeable), 8.70 (s, 1H,
NH, D2O exchangeable), 8.18 (d, J ¼ 2.1 Hz, 1H, AreH), 8.00 (dd,
J ¼ 8.5, 2.1 Hz, 1H, AreH), 7.77 (d, J ¼ 8.4 Hz, 1H, AreH), 7.70 (d,
J ¼ 7.6 Hz, 2H, AreH), 7.62 (d, J ¼ 7.5 Hz, 2H, AreH), 7.60e7.54 (m,
2H, AreH), 7.51 (d, J ¼ 8.4 Hz, 2H, AreH), 6.74 (d, J ¼ 5.7 Hz, 1H, NH,
4.8.19. N-hydroxy-4-(((1-(4-hydroxyphenyl)-3-(thiophen-2-yl)-
1H-1,2,4-triazol-5-yl)methyl)amino)benzamide (9s)
White solid, yield: 82%. Mp: 231e232 ꢀC. 1H NMR (400 MHz,
DMSO‑d6, ppm) d 10.79 (s, 1H, OH, D2O exchangeable), 10.03 (s, 1H,
14

X.-H. Zhang, H.-Q. Kang, Y.-Y. Tao et al.
European Journal of Medicinal Chemistry 218 (2021) 113392
OH, D2O exchangeable), 8.71 (s, 1H, NH, D2O exchangeable), 7.62 (d,
J ¼ 4.3 Hz, 2H, AreH), 7.51 (d, J ¼ 8.4 Hz, 2H, AreH), 7.44 (d,
J ¼ 8.5 Hz, 2H, AreH), 7.15 (t, J ¼ 4.3 Hz, 1H, AreH), 6.97e6.89 (m,
2H, AreH), 6.74 (t, J ¼ 5.7 Hz, 1H, NH, D2O exchangeable), 6.54 (d,
J ¼ 8.4 Hz, 2H, AreH), 4.42 (d, J ¼ 5.6 Hz, 2H, eCH2-). 13C NMR
4.8.24. 4-(((3-(5-bromofuran-2-yl)-1-phenyl-1H-1,2,4-triazol-5-yl)
methyl)amino)-N-hydroxybenzamide (9x)
Off-white solid, yield: 82%. Mp: 237e238 ꢀC. 1H NMR (400 MHz,
DMSO‑d6, ppm).
d 10.79 (s, 1H, OH, D2O exchangeable), 8.70 (s, 1H,
NH, D2O exchangeable), 7.67 (d, J ¼ 7.3 Hz, 2H, AreH), 7.63e7.55 (m,
3H, AreH), 7.50 (d, J ¼ 8.6 Hz, 2H, AreH), 7.04 (d, J ¼ 3.5 Hz, 1H,
AreH), 6.77 (d, J ¼ 3.5 Hz, 1H, AreH), 6.75 (s, 1H, NH, D2O
exchangeable), 6.53 (d, J ¼ 8.7 Hz, 2H, AreH), 4.52 (d, J ¼ 3.7 Hz, 2H,
(101 MHz, DMSO‑d6, ppm)
d 164.77, 158.09, 156.45, 154.17, 150.26,
133.36, 128.17, 128.06, 128.00, 127.23, 126.50, 125.99, 120.21, 115.78,
111.20, 38.47. HR-MS (ESI), calcd. C20H17N5O3S, [MþNa]þ m/z:
430.0950, found: 430.0943.
eCH2-). 13C NMR (101 MHz, DMSO‑d6, ppm)
d 165.22, 155.03,
153.48, 150.70, 148.20, 137.06, 130.07, 129.77, 128.68, 125.19, 123.35,
120.87, 114.34, 112.68, 111.75, 39.26. HR-MS (ESI), calcd.
4.8.20. 4-(((3-(5-chlorothiophen-2-yl)-1-phenyl-1H-1,2,4-triazol-
5-yl)methyl)amino)-N-hydroxybenzamide (9t)
C
20H16BrN5O3, [MþH]þ m/z: 454.0515, found: 454.0508.
Off-white solid, yield: 84%. Mp: 236e237 ꢀC. 1H NMR (400 MHz,
4.8.25. N-hydroxy-4-(((1-phenyl-3-(pyridin-2-yl)-1H-1,2,4-triazol-
5-yl)methyl)amino)benzamide (9y)
DMSO‑d6, ppm).
d 10.79 (s, 1H, OH, D2O exchangeable), 8.70 (s, 1H,
NH, D2O exchangeable), 7.66 (d, J ¼ 6.9 Hz, 2H, AreH), 7.59 (dd,
J ¼ 16.2, 8.2 Hz, 3H, AreH), 7.50 (d, J ¼ 7.6 Hz, 3H, AreH), 7.20 (d,
J ¼ 3.5 Hz, 1H, AreH), 6.75 (s, 1H, NH, D2O exchangeable), 6.53 (d,
J ¼ 8.3 Hz, 2H, AreH), 4.50 (d, J ¼ 4.8 Hz, 2H, eCH2-). 13C NMR
Bright yellow solid, yield: 85%. Mp: 210e211 ꢀC.1H NMR
(400 MHz, DMSO‑d6, ppm).
d 10.80 (s, 1H, OH, D2O exchangeable),
8.71 (s, 1H, NH, D2O exchangeable), 8.68 (d, J ¼ 4.4 Hz, 1H, AreH),
8.10 (d, J ¼ 7.9 Hz, 1H, AreH), 7.98e7.90 (m, 1H, AreH), 7.71 (d,
J ¼ 7.4 Hz, 2H, AreH), 7.62 (t, J ¼ 7.4 Hz, 2H, AreH), 7.57 (d,
J ¼ 7.1 Hz, 1H, AreH), 7.53 (t, J ¼ 7.4 Hz, 2H, AreH), 7.46 (dd, J ¼ 6.9,
5.2 Hz, 1H, AreH), 6.77 (t, J ¼ 5.4 Hz, 1H, NH, D2O exchangeable),
(101 MHz, DMSO‑d6, ppm)
d 165.24, 156.29, 155.15, 150.68, 137.02,
132.53, 130.07, 129.79, 128.67, 128.55, 126.41, 125.28, 120.86, 111.74,
100.00, 39.20. HR-MS (ESI), calcd. C20H16ClN5O2S, [MþH]þ m/z:
426.0791, found: 426.0785.
6.59 (d, J ¼ 8.6 Hz, 2H, AreH), 4.57 (d, J ¼ 5.5 Hz, 2H, eCH2-). 13
C
NMR (101 MHz, DMSO‑d6, ppm)
d 165.27, 160.93, 155.01, 150.82,
4.8.21. N-hydroxy-4-(((1-phenyl-3-(thiophen-3-yl)-1H-1,2,4-
triazol-5-yl)methyl)amino)benzamide (9u)
150.18, 149.67, 137.59, 137.38, 130.06, 129.67, 128.67, 125.15, 124.73,
122.21, 120.80, 111.80, 39.38. HR-MS (ESI), calcd. C21H18N6O2,
[MþH]þ m/z: 387.1569, found: 387.1563.
Yellow-white solid, yield: 82%. Mp: 231e232 ꢀC. 1H NMR
(400 MHz, DMSO‑d6, ppm).
d 10.79 (s, 1H, OH, D2O exchangeable),
8.70 (s, 1H, NH, D2O exchangeable), 8.03 (t, J ¼ 18.9 Hz, 1H, AreH),
7.67 (d, J ¼ 6.5 Hz, 3H, AreH), 7.60 (t, J ¼ 6.8 Hz, 3H, AreH), 7.55 (d,
J ¼ 7.1 Hz, 1H, AreH), 7.51 (d, J ¼ 8.6 Hz, 2H, AreH), 6.75 (s, 1H, NH,
D2O exchangeable), 6.56 (t, J ¼ 8.9 Hz, 2H, AreH), 4.51 (s, 2H, eCH2-
4.8.26. N-hydroxy-4-(((1-phenyl-3-(pyridin-3-yl)-1H-1,2,4-triazol-
5-yl)methyl)amino)benzamide (9z)
Dark yellowish white solid, yield: 83%. Mp: 210e211 ꢀC. 1H NMR
(400 MHz, DMSO‑d6, ppm).
d 10.80 (s, 1H, OH, D2O exchangeable),
). 13C NMR (101 MHz, DMSO‑d6, ppm)
d
165.27, 158.16, 154.50,
9.22 (s, 1H, NH, D2O exchangeable), 8.71 (s, 2H, AreH), 8.37 (d,
J ¼ 7.9 Hz, 1H, AreH), 7.71 (d, J ¼ 7.3 Hz, 2H, AreH), 7.62 (t,
J ¼ 7.3 Hz, 2H, AreH), 7.58 (d, J ¼ 7.0 Hz,1H, AreH), 7.53 (dd, J ¼ 11.0,
7.0 Hz, 3H, AreH), 6.76 (s, 1H, NH, D2O exchangeable), 6.58 (d,
J ¼ 8.6 Hz, 2H, AreH), 4.55 (d, J ¼ 3.8 Hz, 2H, eCH2-). 13C NMR
150.78, 137.35, 130.02, 129.52, 128.67, 127.91, 126.36, 125.17, 124.83,
120.79, 111.75, 39.31. HR-MS (ESI), calcd. C20H17N5O2S, [MþH]þ m/
z: 392.1181, found: 392.1175.
(101 MHz, DMSO‑d6, ppm)
d 165.27, 158.76, 155.30, 150.86, 150.77,
4.8.22. 4-(((3-(furan-2-yl)-1-phenyl-1H-1,2,4-triazol-5-yl)methyl)
amino)-N-hydroxybenzamide (9v)
147.39, 137.24, 133.78, 130.08, 129.77, 128.68, 126.80, 125.24, 124.53,
120.84, 111.80. HR-MS (ESI), calcd. C21H18N6O2, [MþH]þ m/z:
387.1569, found: 387.1564.
Light red solid, yield: 82%. Mp: 236e237 ꢀC. 1H NMR (400 MHz,
DMSO‑d6, ppm).
d 10.80 (s, 1H, OH, D2O exchangeable), 8.72 (s, 1H,
NH, D2O exchangeable), 7.83 (s, 1H, AreH), 7.68 (d, J ¼ 7.7 Hz, 2H,
AreH), 7.60 (t, J ¼ 7.4 Hz, 2H, AreH), 7.56 (d, J ¼ 7.1 Hz, 1H, AreH),
7.51 (d, J ¼ 8.6 Hz, 2H, AreH), 7.00 (d, J ¼ 3.3 Hz, 1H, AreH), 6.76 (t,
J ¼ 5.5 Hz, 1H, NH, D2O exchangeable), 6.65 (dd, J ¼ 3.2, 1.7 Hz, 1H,
AreH), 6.55 (d, J ¼ 8.6 Hz, 2H, AreH), 4.56 (t, J ¼ 20.4 Hz, 2H, eCH2-
4.8.27. N-hydroxy-4-(((1-phenyl-3-(pyridin-4-yl)-1H-1,2,4-triazol-
5-yl)methyl)amino)benzamide (9aa)
White solid, yield: 83%. Mp: 210e211 ꢀC. 1H NMR (400 MHz,
DMSO‑d6, ppm).
d 10.80 (s, 1H, OH, D2O exchangeable), 8.71 (d,
J ¼ 4.2 Hz, 1H, NH, D2O exchangeable), 8.70 (d, J ¼ 4.2 Hz, 2H,
AreH),7 0.96 (d, J ¼ 5.4 Hz, 2H, AreH), 7.71 (d, J ¼ 7.3 Hz, 2H, AreH),
7.65e7.56 (m, 3H, AreH), 7.52 (d, J ¼ 8.4 Hz, 2H, AreH), 6.76 (t,
J ¼ 5.1 Hz, 1H, AreH), 6.57 (d, J ¼ 8.5 Hz, 2H), 4.56 (d, J ¼ 5.3 Hz, 2H,
). 13C NMR (101 MHz, DMSO‑d6, ppm)
d 154.57, 154.52, 153.48,
150.75, 144.66, 137.23, 130.03, 129.55, 128.66, 125.07, 120.80, 111.75,
111.14, 108.39, 39.27, 13.83. HR-MS (ESI), calcd.
C20H17N5O3,
[MþNa]þ m/z: 398.1229, found: 398.1223.
eCH2-). 13C NMR (101 MHz, DMSO‑d6, ppm)
d 165.25, 158.96,
155.64, 150.98, 150.74, 137.97, 137.17, 130.10, 129.91, 128.68, 125.30,
120.87, 120.52, 111.78. HR-MS (ESI), calcd. C21H18N6O2, [MþH]þ m/
z: 387.1569, found: 387.1563.
4.8.23. N-hydroxy-4-(((3-(5-methylfuran-2-yl)-1-phenyl-1H-1,2,4-
triazol-5-yl)methyl)amino)benzamide (9w)
White solid, yield: 83%. Mp: 236e237 ꢀC. 1H NMR (400 MHz,
DMSO‑d6, ppm).
d
10.80 (s, 1H, OH, D2O exchangeable), 8.72 (s, 1H,
4.8.28. N-hydroxy-4-(((1-phenyl-3-(quinolin-6-yl)-1H-1,2,4-
triazol-5-yl)methyl)amino)benzamide (9 ab)
NH, D2O exchangeable), 7.66 (d, J ¼ 7.4 Hz, 2H, AreH), 7.60 (t,
J ¼ 7.4 Hz, 2H, AreH), 7.55 (d, J ¼ 7.0 Hz, 1H, AreH), 7.50 (d,
J ¼ 8.5 Hz, 2H, AreH), 6.87 (d, J ¼ 3.1 Hz, 1H, AreH), 6.75 (t,
J ¼ 5.3 Hz, 1H, NH, D2O exchangeable), 6.54 (d, J ¼ 8.6 Hz, 2H,
AreH), 6.25 (d, J ¼ 2.4 Hz, 1H, AreH), 4.51 (d, J ¼ 5.5 Hz, 2H, eCH2-),
Light yellow solid, yield: 85%. Mp: 210e211 ꢀC. 1H NMR
(400 MHz, DMSO‑d6, ppm).
d 10.79 (s, 1H, OH, D2O exchangeable),
8.94 (d, J ¼ 2.7 Hz, 1H, NH, D2O exchangeable), 8.70 (s, 2H, AreH),
8.55 (d, J ¼ 8.5 Hz, 1H, AreH), 8.42 (dd, J ¼ 8.8, 1.7 Hz, 1H, AreH),
8.13 (d, J ¼ 8.8 Hz, 1H, AreH), 7.74 (d, J ¼ 7.5 Hz, 2H, AreH), 7.63 (t,
J ¼ 7.5 Hz, 2H, AreH), 7.59 (dd, J ¼ 7.5, 3.1 Hz, 2H, AreH), 7.52 (d,
J ¼ 8.6 Hz, 2H, AreH), 6.79 (s, 1H, NH, D2O exchangeable), 6.60 (d,
J ¼ 8.7 Hz, 2H, AreH), 4.58 (d, J ¼ 5.2 Hz, 1H, eCH2-). 13C NMR
2.35 (s, 3H, eCH3). 13C NMR (101 MHz, DMSO)
d 165.26, 154.76,
154.42, 150.75, 146.22, 144.54, 137.19, 130.05, 129.64, 128.67, 125.13,
120.83, 112.25, 111.76, 110.17, 39.27. HR-MS (ESI), calcd. C21H19N5O3,
[MþH]þ m/z: 390.1566, found: 390.1560.
15

X.-H. Zhang, H.-Q. Kang, Y.-Y. Tao et al.
European Journal of Medicinal Chemistry 218 (2021) 113392
(101 MHz, DMSO‑d6, ppm)
136.86, 136.65, 129.63, 129.57, 129.19, 128.34, 128.21, 127.94, 127.04,
125.30, 124.71, 122.07, 120.30, 111.26, 38.93. HR-MS (ESI), calcd.
d
159.90, 154.72, 151.14, 150.30, 148.05,
J ¼ 8.2 Hz, 2H, AreH), 7.53 (s, 1H, AreH), 7.50 (d, J ¼ 5.9 Hz, 2H,
AreH), 7.47 (d, J ¼ 3.0 Hz, 2H, AreH), 6.59 (d, J ¼ 8.3 Hz, 2H, AreH),
4.56 (s, 2H, eCH2-). 13C NMR (101 MHz, DMSO‑d6, ppm)
d 164.75,
C
25H20N6O2, [MþH]þ m/z: 437.1726, found: 437.1719.
160.49, 154.57, 150.26, 136.20, 132.45, 130.27, 129.52, 128.82, 128.16,
126.60, 125.92, 121.94, 120.37, 111.33, 38.97. HR-MS (ESI), calcd.
4.8.29. 4-(((1-(4-fluorophenyl)-3-phenyl-1H-1,2,4-triazol-5-yl)
methyl)amino)-N-hydroxybenzamide (9ac)
C
22H18BrN5O2, [MþNa]þ m/z: 486.0542, found: 486.0535.
Off-white solid, yield: 81%. Mp: 214e215 ꢀC. 1H NMR (400 MHz,
4.8.34. N-hydroxy-4-(((3-phenyl-1-(4-(trifluoromethyl)phenyl)-
1H-1,2,4-triazol-5-yl)methyl)amino)benzamide (9ah)
DMSO‑d6, ppm)
d 10.78 (s, 1H, OH, D2O exchangeable), 8.69 (s, 1H,
NH, D2O exchangeable), 8.05 (d, J ¼ 7.2 Hz, 2H, AreH), 7.74 (dd,
J ¼ 8.8, 4.7 Hz, 2H, AreH), 7.53 (s, 1H, AreH), 7.50 (d, J ¼ 5.8 Hz, 2H,
AreH), 7.48e7.45 (m, 2H, AreH), 7.43 (d, J ¼ 8.4 Hz, 2H, AreH), 6.70
(t, J ¼ 5.7 Hz, 1H, NH, D2O exchangeable), 6.57 (d, J ¼ 8.3 Hz, 2H,
AreH), 4.53 (d, J ¼ 5.3 Hz, 2H, eCH2-). 13C NMR (101 MHz, DMSO‑d6,
Light brown solid, yield: 82%. Mp: 224e225 ꢀC. 1H NMR
(400 MHz, DMSO‑d6, ppm)
d 10.79 (s, 1H, OH, D2O exchangeable),
8.69 (s, 1H, NH, D2O exchangeable), 8.12e8.01 (m, 2H, AreH), 7.95
(d, J ¼ 2.6 Hz, 4H, AreH), 7.53 (d, J ¼ 2.4 Hz, 1H, AreH), 7.51 (s, 2H,
AreH), 7.48 (d, J ¼ 2.7 Hz, 2H, AreH), 6.73 (t, J ¼ 5.7 Hz, 1H, NH, D2O
exchangeable), 6.60 (d, J ¼ 8.3 Hz, 2H, AreH), 4.64 (d, J ¼ 5.4 Hz, 2H,
ppm) d 164.76, 163.09, 160.65, 160.33, 154.54, 150.26, 133.37, 133.34,
130.38, 129.45, 128.80, 128.16, 127.19, 127.10, 125.88, 120.32, 116.50,
116.27, 111.28, 38.83. HR-MS (ESI), calcd. C22H18FN5O2, [MþH]þ m/
z: 404.1523, found: 404.1516.
eCH2-). 13C NMR (101 MHz, DMSO)
d 164.74, 160.70, 154.89, 150.24,
140.14, 130.12, 129.65, 128.85, 128.15, 126.72, 125.98, 125.05, 122.47,
120.43, 111.37, 39.19. HR-MS (ESI), calcd. C23H18F3N5O2, [MþH]þ m/
z: 454.1491,found: 454.1484.
4.8.30. 4-(((1-(2-chlorophenyl)-3-phenyl-1H-1,2,4-triazol-5-yl)
methyl)amino)-N-hydroxybenzamide (9ad)
4.8.35. N-hydroxy-4-(((3-phenyl-1-(p-tolyl)-1H-1,2,4-triazol-5-yl)
methyl)amino)benzamide (9ai)
White solid, yield: 81%. Mp: 214e215 ꢀC. 1H NMR (400 MHz,
DMSO‑d6, ppm)
d
10.76 (s, 1H, OH, D2O exchangeable), 8.67 (s, 1H,
Pink solid, yield: 81%. Mp: 224e225 ꢀC. 1H NMR (400 MHz,
NH, D2O exchangeable), 8.16e7.97 (m, 2H, AreH), 7.75 (d, J ¼ 8.1 Hz,
1H, AreH), 7.68 (d, J ¼ 7.7 Hz, 1H, AreH), 7.62 (d, J ¼ 7.8 Hz, 1H,
AreH), 7.56 (d, J ¼ 7.6 Hz, 1H, AreH), 7.49 (d, J ¼ 1.9 Hz, 2H, AreH),
7.47 (s, 3H, AreH), 6.63 (s, 1H, NH, D2O exchangeable), 6.49 (d,
J ¼ 8.4 Hz, 2H, AreH), 4.39 (s, 2H, eCH2-). 13C NMR (101 MHz,
DMSO‑d6, ppm) d 10.78 (s, 1H, OH, D2O exchangeable), 8.69 (s, 1H,
NH, D2O exchangeable), 8.05 (d, J ¼ 7.3 Hz, 2H, AreH), 7.59e7.55 (m,
2H, AreH), 7.53 (d, J ¼ 2.0 Hz, 1H, AreH), 7.52e7.49 (m, 2H, AreH),
7.48e7.44 (m, 2H, AreH), 7.40 (d, J ¼ 8.0 Hz, 2H, AreH), 6.74 (d,
J ¼ 6.7 Hz, 1H, NH, D2O exchangeable), 6.68e6.46 (m, 2H, AreH),
4.50 (d, J ¼ 4.2 Hz, 2H, eCH2-), 2.41 (s, 3H, eCH3). 13C NMR
DMSO)
d 164.76, 160.74, 155.84, 150.15, 134.28, 131.91, 130.66,
130.33, 129.47, 128.82, 128.36, 128.10, 125.88, 120.25, 111.15, 38.59.
HR-MS (ESI), calcd. C22H18ClN5O2, [MþNa]þ m/z: 442.1047, found:
442.1040.
(101 MHz, DMSO‑d6, ppm) d 164.79, 160.26, 154.22, 150.34, 138.75,
134.49,130.52, 129.92, 129.36, 128.78, 128.17,125.85,124.52,120.29,
111.29, 38.81, 20.68. HR-MS (ESI), calcd. C23H21N5O2, [MþH]þ m/z:
400.1773, found: 400.1767.
4.8.31. 4-(((1-(3-chlorophenyl)-3-phenyl-1H-1,2,4-triazol-5-yl)
methyl)amino)-N-hydroxybenzamide (9ae)
4.8.36. N-hydroxy-4-(((3-phenyl-1-(o-tolyl)-1H-1,2,4-triazol-5-yl)
methyl)amino)benzamide (9aj)
White solid, yield: 84%. Mp: 214e215 ꢀC.1H NMR (400 MHz,
DMSO‑d6, ppm)
d
10.81 (s, 1H, OH, D2O exchangeable), 8.70 (s, 1H,
White solid, yield: 85%. Mp: 224e225 ꢀC. 1H NMR (400 MHz,
NH, D2O exchangeable), 8.15e7.95 (m, 2H, AreH), 7.82 (s, 1H,
AreH), 7.70 (s, 1H, AreH), 7.63 (d, J ¼ 4.5 Hz, 2H, AreH), 7.55 (s, 1H,
AreH), 7.52 (d, J ¼ 6.7 Hz, 2H, AreH), 7.49 (d, J ¼ 2.8 Hz, 2H, AreH),
6.73 (t, J ¼ 5.7 Hz,1H, NH, D2O exchangeable), 6.61 (d, J ¼ 8.3 Hz, 2H,
AreH), 4.60 (d, J ¼ 5.4 Hz, 2H, eCH2-).13C NMR (101 MHz, DMSO‑d6,
DMSO‑d6, ppm) d 10.85 (s, 1H, OH, D2O exchangeable), 8.79 (s, 1H,
NH, D2O exchangeable), 8.03 (dd, J ¼ 8.1, 1.6 Hz, 2H, AreH), 7.51 (s,
2H, AreH), 7.49 (d, J ¼ 3.4 Hz, 2H, AreH), 7.47 (d, J ¼ 7.3 Hz, 3H,
AreH), 7.45e7.43 (m, 1H, AreH), 7.40 (dd, J ¼ 7.4, 1.8 Hz, 1H, AreH),
6.68 (t, J ¼ 5.8 Hz, 1H, NH, D2O exchangeable), 6.57e6.39 (m, 2H,
AreH), 4.35 (d, J ¼ 5.7 Hz, 2H, eCH2-), 2.03 (s, 3H, eCH3). 13C NMR
ppm)
d 164.76, 160.51, 154.70, 150.26, 138.12, 133.71, 131.13, 130.23,
129.56, 128.98, 128.82, 128.17, 125.95, 124.55, 123.30, 120.37, 111.33,
38.99. HR-MS (ESI), calcd. C22H18ClN5O2, [MþNa]þ m/z: 442.1047,
found: 442.1040.
(101 MHz, DMSO‑d6, ppm) d 164.74, 160.38, 155.20, 150.26, 135.81,
135.13, 131.19, 130.58, 130.07, 129.35, 128.80, 128.13, 127.18, 126.89,
125.80, 120.05, 111.07, 38.30, 16.95. HR-MS (ESI), calcd. C23H21N5O2,
[MþH]þ m/z: 400.1773, found: 400.1767.
4.8.32. 4-(((1-(4-chlorophenyl)-3-phenyl-1H-1,2,4-triazol-5-yl)
methyl)amino)-N-hydroxybenzamide (9af)
4.8.37. N-hydroxy-4-(((3-phenyl-1-(m-tolyl)-1H-1,2,4-triazol-5-yl)
methyl)amino)benzamide (9ak)
White solid, yield: 84%. Mp: 214e215 ꢀC.1H NMR (400 MHz,
DMSO‑d6, ppm)
d
10.79 (s, 1H, OH, D2O exchangeable), 8.70 (s, 1H,
Off-white solid, yield: 83%. Mp: 224e225 ꢀC. 1H NMR (400 MHz,
NH, D2O exchangeable), 8.09e7.98 (m, 2H, AreH), 7.72 (d,
J ¼ 8.2 Hz, 2H, AreH), 7.66 (d, J ¼ 8.3 Hz, 2H, AreH), 7.53 (s, 1H,
AreH), 7.52e7.48 (m, 2H, AreH), 7.47 (d, J ¼ 3.2 Hz, 2H, AreH), 6.72
(s, 1H, NH, D2O exchangeable), 6.60e6.47 (m, 2H, AreH), 4.56 (d,
J ¼ 5.4 Hz, 2H, eCH2-). 13C NMR (101 MHz, DMSO- d6, ppm)
DMSO‑d6, ppm)
d
8.05 (dd, J ¼ 8.2, 1.7 Hz, 2H, AreH), 7.53 (s, 1H,
AreH), 7.51 (s, 2H, AreH), 7.48 (d, J ¼ 2.4 Hz, 3H, AreH), 7.47 (d,
J ¼ 1.7 Hz, 2H, AreH), 7.35 (s, 1H, AreH), 6.76 (t, J ¼ 5.6 Hz, 1H, NH,
D2O exchangeable), 6.61e6.55 (m, 2H, AreH), 4.53 (d, J ¼ 5.6 Hz,
2H, eCH2-), 2.38 (s, 3H, eCH3). 13C NMR (101 MHz, DMSO‑d6, ppm)
d
164.74, 160.46, 154.59, 150.25, 135.78, 133.46, 130.27, 129.52,
d
164.62, 160.27, 154.30, 150.27, 139.29, 136.83, 130.45, 129.64,
128.83, 128.16, 126.37, 125.91, 120.34, 111.30, 38.94. HR-MS (ESI),
129.40, 129.27, 128.80, 128.13, 125.85, 125.10, 121.62, 120.31, 111.27,
calcd. C22H18ClN5O2, [MþH]þ m/z: 442.1047, found: 442.1040.
38.84, 20.80. HR-MS (ESI), calcd.
400.1773, found: 400.1767.
C
23H21N5O2, [MþH]þ m/z:
4.8.33. 4-(((1-(3-chlorophenyl)-3-phenyl-1H-1,2,4-triazol-5-yl)
methyl)amino)-N-hydroxybenzamide (9 ag)
4.8.38. N-hydroxy-4-(((1-(4-methoxyphenyl)-3-phenyl-1H-1,2,4-
triazol-5-yl)methyl)amino)benzamide (9 al)
Dark brown solid, yield: 81%. Mp: 214e215 ꢀC. 1H NMR
(400 MHz, DMSO‑d6, ppm)
d
10.78 (s, 1H, OH, D2O exchangeable),
Flesh red solid, yield: 84%. Mp: 210e211 ꢀC. 1H NMR (400 MHz,
8.05 (d, J ¼ 7.2 Hz, 2H, AreH), 7.79 (d, J ¼ 8.2 Hz, 2H, AreH), 7.65 (d,
DMSO‑d6, ppm)
d 10.81 (s, 1H, OH, D2O exchangeable), 8.72 (s, 1H,
16

X.-H. Zhang, H.-Q. Kang, Y.-Y. Tao et al.
European Journal of Medicinal Chemistry 218 (2021) 113392
NH, D2O exchangeable), 8.11e7.94 (m, 2H, AreH), 7.62e7.58 (m, 2H,
AreH), 7.53 (s, 1H, AreH), 7.51 (s, 1H, AreH), 7.48 (d, J ¼ 2.0 Hz, 1H,
AreH), 7.47e7.40 (m, 2H, AreH), 7.18e7.05 (m, 2H, AreH), 6.75 (t,
J ¼ 5.6 Hz, 1H, NH, D2O exchangeable), 6.57 (d, J ¼ 8.5 Hz, 2H,
AreH), 4.47 (d, J ¼ 5.5 Hz, 2H, eCH2-), 3.84 (s, 3H, eCH3). 13C NMR
4.8.43. N-hydroxy-4-(((1-(4-isopropylphenyl)-3-phenyl-1H-1,2,4-
triazol-5-yl)methyl)amino)benzamide (9aq)
Off-white solid, yield: 81%. Mp: 227e228 ꢀC. 1H NMR (400 MHz,
DMSO‑d6, ppm)
d 10.80 (s, 1H, OH, D2O exchangeable), 8.70 (s, 1H,
NH, D2O exchangeable), 8.07 (t, J ¼ 20.6 Hz, 2H, AreH), 7.60 (d,
J ¼ 8.2 Hz, 2H, AreH), 7.52 (d, J ¼ 8.6 Hz, 2H, AreH), 7.47 (t,
J ¼ 7.2 Hz, 5H, AreH), 6.76 (s, 1H, NH, D2O exchangeable), 6.59 (d,
J ¼ 8.6 Hz, 2H, AreH), 4.50 (d, J ¼ 5.0 Hz, 2H,eCH2e), 3.00 (dt,
J ¼ 13.6, 6.7 Hz, 1H, eCHe), 1.26 (d, J ¼ 6.9 Hz, 6H, eCH3). 13C NMR
(101 MHz, DMSO‑d6, ppm)
d 164.77, 160.11, 159.51, 154.25, 150.33,
130.55, 129.78, 129.33, 128.78, 128.17, 126.33, 125.81, 120.20, 114.56,
111.25, 55.53, 38.67. HR-MS (ESI), calcd. C23H21N5O3, [MþH]þ m/z:
416.1772, found: 416.1716.
(101 MHz, DMSO)
d 164.76, 160.24, 154.17, 150.34, 149.43, 134.72,
4.8.39. N-hydroxy-4-(((3-(4-hydroxyphenyl)-1-(4-
methoxyphenyl)-1H-1,2,4-triazol-5-yl)methyl)amino)benzamide
(9am)
130.50, 129.38, 128.80, 128.16, 127.34, 125.83, 124.59, 120.25, 111.30,
38.86, 33.12, 23.71. HR-MS (ESI), calcd. C25H25N5O2, [MþH]þ m/z:
428.2086, found: 428.2080.
Orange-red solid, yield: 81%. Mp: 210e211 ꢀC. 1H NMR
(400 MHz, DMSO‑d6, ppm)
d
10.79 (s, 1H, OH, D2O exchangeable),
4.9. Cell line and reagents
10.01 (s, 1H, OH, D2O exchangeable), 8.70 (s, 1H, NH, D2O
exchangeable), 8.01e7.84 (m, 2H, AreH), 7.53e7.49 (m, 2H, AreH),
7.46e7.42 (m, 2H, AreH), 7.28 (d, J ¼ 7.8 Hz, 2H, AreH), 6.96e6.84
(m, 2H, AreH), 6.71 (s, 1H, NH, D2O exchangeable), 6.57 (d,
J ¼ 8.3 Hz, 2H, AreH), 4.48 (dd, J ¼ 48.9, 5.5 Hz, 2H, eCH2-), 2.34 (s,
Human gastric cancer cell line (MGC803), obtained from the Cell
Bank of Shanghai, was grown in grown in Dulbecco’s modified
eagle medium (DMEM, Gibico, US), supplemented with penicillin G
(100 UI/mL), streptomycin (100 mg/mL) and 10% fetal calf serum at
37 ꢀC in 5% CO2 atmosphere.
3H, eOCH3). 13C NMR (101 MHz, DMSO‑d6, ppm)
d 164.78, 160.05,
157.97, 153.98, 150.35, 138.71, 129.32, 128.37, 128.16, 127.93, 126.39,
125.74, 120.16, 115.75, 111.23, 38.60, 20.92. LC-MS tR ¼ 4.229 min;
calcd. C23H21N5O3, [MþH]þ m/z: 416.18, found: 416.21.
4.10. Cell viability
For cell viability of compounds, MGC803 was seeded at a density
of 2500 cells per well in 96-well plate at 37ꢀС in 5% CO2 atmo-
sphere. Cells were then allowed to adhere for 24 h, medium in the
wells was replaced with fresh medium containing compound
which was diluted in 7 dilution steps covering the range from
4.8.40. 4-(((1-(3,5-dimethylphenyl)-3-phenyl-1H-1,2,4-triazol-5-
yl)methyl)amino)-N-hydroxybenzamide (9an)
White solid, yield: 82%. Mp: 227e228 ꢀC. 1H NMR (400 MHz,
DMSO‑d6, ppm)
d 10.82 (s, 1H, OH, D2O exchangeable), 8.74 (s, 1H,
NH, D2O exchangeable), 8.03 (dd, J ¼ 8.2, 1.5 Hz, 2H, AreH), 7.52 (s,
1H, AreH), 7.51e7.46 (m, 3H, AreH), 7.44 (d, J ¼ 6.8 Hz, 1H, AreH),
7.26 (s, 2H, AreH), 7.16 (s, 1H, AreH), 6.74 (t, J ¼ 5.5 Hz, 1H, NH, D2O
exchangeable), 6.57 (d, J ¼ 8.4 Hz, 2H), 4.52 (d, J ¼ 5.6 Hz, 2H, eCH2-
50
m
M to 0.78
m
M. And DMSO was used as negative control. After
72 h incubation, 20
mL of MTT (Meilune, China) solution was added
to each well. After 4 h, the absorbance of each well was measured at
wavelength of 490 nm on spectrophotometer. Data was corrected
for background. IC50 was calculated by GraphPad Prism 6.0 [52].
), 2.33 (s, 6H, eCH3). 13C NMR (101 MHz, DMSO‑d6, ppm)
d 164.79,
160.19, 154.26, 150.32, 138.97, 136.73, 130.44, 130.36, 129.39, 128.80,
128.15, 125.84, 122.17, 120.14, 111.29, 38.76, 20.71. HR-MS (ESI),
calcd. C24H23N5O2, [MþH]þ m/z: 414.1930, found: 414.1923.
4.11. Analysis of apoptosis
Flow cytometric analysis based on the fluorochrome propidium
iodide was performed as described previously. MGC803 was seeded
at a density of 0.4 ꢂ 106 cells/well into 24-well plates and treated
with induced concerntration for 48 h, then cells were harvested
and washed by PBS, and stained by Annenxin V-FITC (Dojindo,
Japan), PI (Dojindo, Japan) at room temperature. Then cells were
analyzed by flow cytometry [53].
4.8.41. 4-(((1-(3,4-dimethylphenyl)-3-phenyl-1H-1,2,4-triazol-5-
yl)methyl)amino)-N-hydroxybenzamide (9ao)
Light red solid, yield: 83%. Mp: 227e228 ꢀC. 1H NMR (400 MHz,
DMSO‑d6, ppm)
d 8.13e7.98 (m, 2H, AreH), 7.53 (s, 1H, AreH), 7.50
(dd, J ¼ 8.6, 2.2 Hz, 3H, AreH), 7.46 (d, J ¼ 5.0 Hz, 2H, AreH), 7.44 (d,
J ¼ 2.4 Hz, 1H, AreH), 7.35 (d, J ¼ 3.7 Hz, 1H, AreH), 6.76 (t,
J ¼ 5.6 Hz, 1H, NH, D2O exchangeable), 6.58 (d, J ¼ 8.5 Hz, 2H,
AreH), 4.50 (d, J ¼ 5.6 Hz, 2H, eCH2-), 2.30 (d, J ¼ 7.1 Hz, 6H, eCH3).
4.12. In vitro assay on HDAC1, HDAC6
13C NMR (101 MHz, DMSO‑d6, ppm)
d 164.68, 160.16, 154.18, 150.30,
137.82, 137.52, 134.60, 130.49, 130.21, 129.36, 128.79, 128.13, 125.82,
125.49, 121.85, 120.22, 111.28, 38.78, 19.29, 19.04. HR-MS (ESI),
calcd. C24H23N5O2, [MþH]þ m/z: 414.1930, found: 414.1923.
OptiPlate-96 black microplates (PerkinElmer) were used for
HDAC1 and HDAC6 enzyme activity assay in vitro. A series of
compounds, SAHA were diluted with HDAC assay buffer containing
1% DMSO. Then human recombinant HDAC6 (BPS Bioscience, USA,
catalog no. 50006) in incubation buffer, BSA, the fluorogenic sub-
strate (Z-(Ac)-Lys-AMC) and the tested compounds were incubated
in 96-well black plates at 37 ꢀCfor 40 min. After incubation, trypsin
was added to release the fluorescent fragment of AMC for 15 min.
The fluorescence intensity was measured with an excitation
wavelength of 390 nm and emission wavelength of 460 nm. The
IC50 of the compounds was calculated and fitted by GraphPad prism
6.0 [54].
4.8.42. 4-(((1-(4-ethylphenyl)-3-phenyl-1H-1,2,4-triazol-5-yl)
methyl)amino)-N-hydroxybenzamide (9ap)
Light yellow solid, yield: 84%. Mp: 227e228 ꢀC. 1H NMR
(400 MHz, DMSO‑d6, ppm)
d 10.79 (s, 1H, OH, D2O exchangeable),
8.70 (d, J ¼ 3.9 Hz, 1H, NH, D2O exchangeable), 8.13e7.86 (m, 2H,
AreH), 7.59 (d, J ¼ 7.8 Hz, 2H, AreH), 7.51 (dd, J ¼ 5.3, 3.3 Hz, 2H,
AreH), 7.50e7.45 (m, 2H, AreH), 7.43 (d, J ¼ 8.0 Hz, 3H, AreH), 6.75
(dd, J ¼ 7.6, 4.8 Hz, 1H, NH, D2O exchangeable), 6.59 (dd, J ¼ 8.8,
2.8 Hz, 2H, AreH), 4.50 (d, J ¼ 5.4 Hz, 2H, eCH2-), 2.71 (q, J ¼ 7.6 Hz,
2H, eCH2-), 1.23 (t, J ¼ 7.6 Hz, 3H, eCH3). 13C NMR (101 MHz,
4.13. BioLayer interferometry (BLI)
DMSO‑d6, ppm)
d 164.76, 160.25, 154.20, 150.34, 144.89, 134.66,
130.50, 129.37, 128.79, 128.77, 128.16, 125.84, 124.59, 120.26, 111.29,
38.87, 27.75, 15.38. HR-MS (ESI), calcd. C24H23N5O2, [MþH]þ m/z:
414.1930, found: 414.1923.
HDAC6 (treated with NHS-LC-Biotin) at a concentration of 1 mg/
mL was immobilized on SA sensors in kinetics buffer in 96-well
ꢀ
black microplates (ForteBio, USA). The experiment was performed
17

X.-H. Zhang, H.-Q. Kang, Y.-Y. Tao et al.
European Journal of Medicinal Chemistry 218 (2021) 113392
on an Octet96 instrument (Pall, USA) at 25 ꢀC. Activation was 50 s,
immobilization of HDAC6 600 s, followed by washing for 300 s and
then binding the compound 9r for 300 s. Data was analyzed by the
or medium containing 20% FBS (lower chambers). Meanwhile
compound 9r was added into the chamber. With 72 h incubation,
the medium was abandoned, and the chambers were washed by
PBS. Cell was fixed by 4% paraformaldehyde solution and stained
with DAPI, and washed by PBS for three times. The chamber was
photographed and analyzed by Molecular Devices (Thermo Fisher
Scientific, USA).
ꢀ
ꢀ
ForteBio Data Analysis 8.1 software (ForteBio, USA).
4.14. Cellular thermal shift assay (CETSA)
The interaction between HDAC6 and compound 9r in vitro was
further tested by the cellular thermal shift assay (CETSA). The
technique can verify the binding of small molecules to corre-
sponding target proteins for cellular level. Briefly, MGC803 was
cultured in 6-well plate for 24 h and then were incubated with
4.19. Acute toxicity
8-10-week-old C57BL/6 mice (20 females and 20 males) were
divided into two groups, respectively. One group served as negative
control treated with corresponding solvent (0.5% CMC-Na). Another
group was orally administrated compound 9r on 2000 mg/kg a
single time. For the observation of the following 14 days, the signs
of toxicity (body weight, mortality, behavior) were recorded. Acute
toxicity caused a set of experimental animals with 50% subject
compounds dose was calculated for quantitative evaluation of
death (LD50). And H&E staining of the lung, heart, liver, spleen and
kidney tissue were assessed [54,55].
media containing DMSO or compound 9r (4 mM) for 6 h. Cells were
resuspended in ice-cold PBS and divided equally into 6 PCR tubes
and lysed by repeated freezing and thawing heated. The lysate was
heated at a temperature gradient from 44 ꢀC to 54 ꢀC for 3 min.
Subsequently, the supernatant of the cells was analyzed by a
Western blot assay.
4.15. Molecular docking study
All animals were maintained in accordance with the Regulations
of Experimental Animal Administration issued by the State Com-
mittee of Science and Technology of People’s Republic of China.
The 3D structures of the compounds for docking were built
using MOE 2015, and energy minimization was processed using the
force field AMBER 10: EHT. The X-ray crystal structures of HDAC6
(PDB code 5EF7, 1.9 Å) was obtained from PDB database, and pre-
pared with the QuickPrep module of MOE 2015 using the default
parameters. The co-crystalized ligand of the protein was used to
define the active site for docking. The default Triangle Matcher
placement method was employed for docking, and GBVI/WSA dG
scoring function was used to assess the free energy of binging of
compound 9r from a given pose and rank the final poses.
4.20. Statistical analysis
All data are reported as means SD. Differences were analyzed
by Student’s t-test (with 95% confidence interval). p values < 0.05
were considered as statistically significant.
Declaration of competing interest
4.16. Western blot analysis
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 MGC803 was treated with the induced concentration of
compound 9r for 48 h. Then cells were lysed with loading buffer
(0.1% SDS 150 mM NaCl, 25 mM Tris-HCl, 1% deoxycholic acid so-
dium salt, 1% NP-40, 1% PMSF). After centrifuge, supernatant was
collected and the protein concentration was quantized by the BCA
Protein Quantitation Kit (Beyotime Biotechnology, China). Each
sample was separated by 8e10% SDS/PAGE gels and transferred to a
0.22 mm PVDF membrane. After blocking in 5% non-fat dry milk at
room temperature for 2 h, the membrane was incubated with the
specific peroxidase-conjugated primary antibodies independently,
Ac-a-tubulin (CST, USA), a-tubulin (CST, USA), Ac-H3 (Abcam, UK),
H3 (Abcam, UK), Bcl-2 (Abcam, UK), Caspase 9 (Abcam, UK),
Cleaved-Caspase 9 (Abcam, UK), PARP (Abcam, UK), Cleaved-PARP
(Abcam, UK), GAPDH (Abcam, UK) overnight at 4 ꢀC and followed
by incubation with horseradish peroxidase conjugated secondary
antibodies (Abcam, UK), and the ECL chemiluminescence detection
kit (Meilune, China) was used and the chemiluminescence signal is
then observed and collected in the dark using photosensitive film.
Acknowledgement
This work was supported by National Natural Science Founda-
tion of China (81703328 by Liying Ma; 81430085 and 81773562 by
Hongmin Liu); National Key Research Program (2018YFE0195100
and 2016YFA0501800 by Hongmin Liu, China); Scientific Program
of Henan Province (182102310070 by Liying Ma, China); China
Postdoctoral Science Foundation (2020M672249 by Liying Ma);
Henan Scientific Innovation Talent Team, Department of Education
(19IRTSTHN001 by Zhao Wen, China).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113392.
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