Design, synthesis and biological evaluation of 3, 4-disubstituted-imidazolidine-2, 5-dione derivatives as HDAC6 selective inhibitors

Article abstract of DOI:10.1016/j.ejmech.2021.113526

HDAC6 isoform selective inhibitors can be pursued as an alternative to pan-HDACs inhibitors due to their therapeutic effect and low toxicity. Efforts of the structure optimization of our previous compound 10c (IC₅₀ = 4.4 nM) resulted in a new series of 3, 4-disubstituted-imidazolidine-2, 5-dione based HDAC6 inhibitors with better HDAC6 inhibitory activities and improved selectivities. The most potent compound 71 exhibited a low nanomolar HDAC6 inhibitory activity (IC₅₀ = 2.1 nM) and showed 5545-fold, 5864-fold as well as 1638-fold selectivity relative to HDAC1, HDAC2 and HDAC8, respectively. Western blot analysis further confirmed that compound 71 selectively increased the acetylation level of α-tubulin without affecting histone H3. Moreover, compound 71 also possesses good properties in term of caspase-3 activation, apoptosis induction, anti-proliferative activity, cytotoxicity and plasma stability. Therefore, compound 71 can be applied in cancer therapy or used as a lead compound to develop more potent HDAC6 selective inhibitor.

Full text of DOI:10.1016/j.ejmech.2021.113526

European Journal of Medicinal Chemistry 221 (2021) 113526
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Design, synthesis and biological evaluation of 3, 4-disubstituted-
imidazolidine-2, 5-dione derivatives as HDAC6 selective inhibitors
Tao Liang, Junxin Xue, Zefu Yao, Yang Ye, Xinying Yang, Xuben Hou, Hao Fang*
Department of Medicinal Chemistry and Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Cheeloo College of
Medicine, Shandong University, 44, West Wenhua Road, 250012, Jinan, Shandong, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
HDAC6 isoform selective inhibitors can be pursued as an alternative to pan-HDACs inhibitors due to their
therapeutic effect and low toxicity. Efforts of the structure optimization of our previous compound 10c
(IC50 ¼ 4.4 nM) resulted in a new series of 3, 4-disubstituted-imidazolidine-2, 5-dione based HDAC6
inhibitors with better HDAC6 inhibitory activities and improved selectivities. The most potent compound
71 exhibited a low nanomolar HDAC6 inhibitory activity (IC50 ¼ 2.1 nM) and showed 5545-fold, 5864-
fold as well as 1638-fold selectivity relative to HDAC1, HDAC2 and HDAC8, respectively. Western blot
Received 30 March 2021
Received in revised form
22 April 2021
Accepted 22 April 2021
Available online 7 May 2021
analysis further confirmed that compound 71 selectively increased the acetylation level of
a-tubulin
Keywords:
without affecting histone H3. Moreover, compound 71 also possesses good properties in term of caspase-
3 activation, apoptosis induction, anti-proliferative activity, cytotoxicity and plasma stability. Therefore,
compound 71 can be applied in cancer therapy or used as a lead compound to develop more potent
HDAC6 selective inhibitor.
Structure optimization
HDAC6 selective inhibitor
Anti-proliferative
Apoptosis
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
isoform, can be pursued as an alternative to pan-HDACs inhibitor
[18,19].
As an epigenetic eraser, histone deacetylases (HDACs) together
with histone acetyltransferases (HATs), maintain cellular acetyla-
tion homeostasis on histones and non-histone proteins [1,2]. Ac-
cording to the homology structure and cellular localization, 18
HDAC isoforms are phylogenetically divided into 4 classes: Class I
(HDAC1, 2, 3 and 8), Class IIa (HDAC 4, 5, 7 and 9), Class IIb (HDAC6
and 10) and Class IV (HDAC11) are zinc-dependent enzymes,
whereas Class III (SIRT 1e7) are NAD-dependent enzymes [3]. It is
believed that HDACs overexpression is associated with the cancer
initiation as well as progression and nowadays, HDACs have been
validated as clinical promising targets for the treatment of diverse
cancer [4e7]. Efforts of three decades have identified five approved
HDACs inhibitors including vorinostat, romidepsin, belinostat,
panobinostat and chidamide [8e12]. Although HDACs inhibitors
achieve a great success in cancer therapy, approved pan-HDACs
inhibitors resulted in severe side effects due to their indiscrimi-
nate inhibition toward zinc-dependent HDACs [13e17]. However,
HDAC isoform selective inhibitor, which targets specific HDAC
As a pivotal member of the HDACs family, HDAC6 is of major
significance to the progression, survival and migration of carcinoma
cells [20,21]. Accumulating evidence suggested that the inhibition
or knockout of HDAC6 could suppress the growth of carcinoma cells
[22e24], whereas no obviously defective phenotypes or lethal ef-
fects were observed in HDAC6 knockout mice [25e27]. Above
research results revealed that HDAC6 selective inhibitors could
suppress the growth of carcinoma cells while attenuating the
toxicity of current pan-HDAC inhibitors. And some HDAC6 isoform
selective inhibitors have been developed, such as Ricolinostat (ACY-
1215), Tubastatin A, NOC-7, Tubacin, Nexturastat A and HPOB
(Fig. 1A).
Recently, we developed 2,4-imidazolinedione-derived HDAC6
selective inhibitors and compound 10c showed nanomolar HDAC6
inhibitory activity (Fig. 1B, IC50 ¼ 4.4 nM) [28]. To develop more
potent and selective HDAC6 inhibitor, we optimized the structure of
previous compound 10c. Briefly, we replaced its long linker with N-
hydroxybenzamide group, which favorably accommodates
HDAC6's wide and shallow catalytic channel. Moreover, HDAC6
isoform selective inhibitors prefer large, bulky cap groups, which
are believed to be tolerated by the outer region of HDAC6 binding
pocket well. Therefore, imidazolidine-2, 5-dione scaffold with
* Corresponding author.
E-mail address: haofangcn@sdu.edu.cn (H. Fang).
https://doi.org/10.1016/j.ejmech.2021.113526
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

T. Liang, J. Xue, Z. Yao et al.
European Journal of Medicinal Chemistry 221 (2021) 113526
Fig. 1. (A) Reported selective HDAC6 inhibitors; (B) Structure optimization of previously reported compound 10c and newly developed HDAC6 inhibitors.
different aromatic rings was used as the cap group of our newly
developed HDAC6 inhibitors. Biological evaluations suggested that
the most potent compound 71 exhibited a low nanomolar HDAC6
inhibitory activity (IC50 ¼ 2.1 nM) and over 1638-fold selectivity
against HDAC1, 2 and 8 (Fig. 1B).
position of compound 3 using the N-alkylation reactions to syn-
thesize the key intermediates 4e12, which were converted into the
first series target compounds 13e21 (Scheme 1). As for the second
series of compounds, 4-(aminomethyl)benzoate and methyl 2-
amino-2-phenylacetate were used as starting material for the
synthesis of molecule 22, which was used to prepare molecule 23
with cyclization reaction. After protecting the carboxyl group of
molecule 23, the N-alkylation reactions were performed to give
molecules 25e34. Finally, molecules 25e34 were converted into
target compounds with transforming ester group into hydroxamic
acid (Scheme 2). For the third series of compounds, the cyclization
reaction of compound 45 was simply conducted to prepare inter-
mediate 46 in the presence of sodium methoxide. Compound 46
was used to synthesize compounds 60e72 by introducing various
side chains and transforming easter group into hydroxamic acid
(Scheme 3). The fourth series of compounds were synthesized in a
manner similar to the synthesis of the third series compounds
(Scheme 4).
Herein, we report the structure optimization and biological
evaluations of our newly developed HDAC6 selective inhibitors.
2. Results and discussion
2.1. Synthesis and characterization of target compounds
The synthetic methods of target compounds are displayed in
Schemes 1e4. For the first series of compounds, intermediate 1 was
prepared starting from methyl 4-(aminomethyl)benzoate and
glycine. Then intermediate 1 was transformed into intermediate 2
using cyclization reaction in the presence of hydrochloric acid.
Subsequently, different side chains were introduced into R2
2

T. Liang, J. Xue, Z. Yao et al.
European Journal of Medicinal Chemistry 221 (2021) 113526
Scheme 1. Reagents and conditions: (a) (i) Triphosgene, TEA, CH2Cl2; (ii) glycine, 2 M NaOH, 0 ꢀC, 4 h, (iii) 6 M HCl, 73%; (b) Hydrochloric acid, reflux, 3 h, 80%; (c) CH3COCl, MeOH,
reflux, 5 h, 98%; (d) R'1-Br, K2CO3, KI, DMF, overnight, 75%e97%; (e) (i) LiOH, THF/H2O, rt, 6 h; (ii) isobutyl chlorocarbonate, 4-Methylmorpholine, THF, NH2OH$HCl, KOH, MeOH, rt,
6 h, 39%e81%.
Scheme 2. Reagents and conditions: (a) (i) Triphosgene, TEA, CH2Cl2; (ii) Methyl 4-(aminomethyl)benzoate, TEA, 0 ꢀC, 2 h, 74%; (b) Hydrochloric acid, reflux, 3 h, 95%; (c) CH3COCl,
MeOH, reflux, 5 h, 98%; (d) R'2-Br, K2CO3, KI, DMF, overnight, 67%e91%; (e) (i) LiOH, THF/H2O, rt, 6 h; (ii) isobutyl chlorocarbonate, 4-Methylmorpholine, THF, NH2OH$HCl, KOH,
MeOH, rt, 6 h, 52%e69%.
Scheme 3. Reagents and conditions: (a) (i) Triphosgene, TEA, CH2Cl2; (ii) Methyl 4-(aminomethyl)benzoate, TEA, 0 ꢀC, 2 h, 71%; (b) CH3ONa, THF, 1 h, 88%; (c) R'3-Br, K2CO3, KI, DMF,
overnight, 56%e95%; (d) (i) LiOH, THF/H2O, rt, 6 h; (ii) isobutyl chlorocarbonate, 4-Methylmorpholine, THF, NH2OH$HCl, KOH, MeOH, rt, 6 h, 53%e76%.
2.2. HDAC6 inhibitory activity
substituents attached to imidazolidine-2, 5-dione scaffold have
influences on HDAC6 inhibitory activities. Compounds 60e72 with
benzyl in the R1 position achieve better HDAC6 inhibitory activities
compared to compounds 78e80 with isobutyl in the R1 position.
As for the substituents in the R2 position, benzyl, 4-chloro-
benzyl or naphthyl appear to be beneficial to HDAC6 inhibitory
activities, whereas alkyl (methyl or propyl) or benzyl containing
The HDAC6 inhibitory activities of all target compounds were
evaluated in vitro using fluorescence assay with Tubastatin A and
SAHA as the positive control. As shown in Table 1, most of the target
compounds exhibited nanomolar HDAC6 inhibitory activities.
Structure and activity relationship (SAR) analysis indicated that
3

T. Liang, J. Xue, Z. Yao et al.
European Journal of Medicinal Chemistry 221 (2021) 113526
Scheme 4. Reagents and conditions: (a) (i) Triphosgene, TEA, CH2Cl2; (ii) Methyl 4-(aminomethyl)benzoate, TEA, 0 ꢀC, 2 h, 82%; (b) CH3ONa, THF, 1 h, 97%; (c) R'4-Br, K2CO3, KI, DMF,
overnight, 81%e95%; (d) (i) LiOH, THF/H2O, rt, 6 h; (ii) isobutyl chlorocarbonate, 4-Methylmorpholine, THF, NH2OH$HCl, KOH, MeOH, rt, 6 h, 58%e67%.
Table 1
HDAC6 inhibitory activities of target compounds.
Compound
R1
R2
IC50 (nM)a
13
14
15
16
17
18
19
20
21
H
H
H
H
H
H
H
H
methyl
n-propyl
Bn
4-Cl-Bn
4-CH3-Bn
4-OCH3-Bn
4-tert-butyl-Bn
phenylethyl
2-methylnaphthalen
H
H
H
H
H
H
H
118.5 6.8
53.4 1.8
12.5 0.4
8.3 0.9
17.6 1.3
28.2 5.6
40.7 3.6
21.7 3.5
13.1 1.6
74.9 2.5
50.9 3.3
71.9 9.2
54.0 3.3
82.4 5.0
72.4 4.1
114.7 5.9
24.7 4.4
91.9 3.8
74.6 2.1
107.5 4.7
56.9 6.7
21.3 1.6
6.0 0.8
37.8 1.8
102.3 15.1
139.3 13.4
20.2 0.1
28.8 2.9
25.1 1.0
4.8 1.8
2.1 0.09
13.9 2.4
251.4 10.1
174.0 11.5
129.2 6.0
27.4 0.7
26.9 3.2
H
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
(
)-35
)-36
)-37
)-38
)-39
)-40
)-41
)-42
)-43
)-44
)-60
)-61
)-62
)-63
)-64
)-65
)-66
)-67
)-68
)-69
)-70
)-71
)-72
)-78
)-79
)-80
Ph, methyl
Ph, n-propyl
Ph, Bn
Ph, 4-Cl-Bn
Ph, 4-CH3-Bn
Ph, 4-OCH3-Bn
Ph, 4-tert-butyl-Bn
Ph, phenylethyl
Ph, 1-methylnaphthalen
Ph, 2-methylnaphthalen
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Isobutyl
Isobutyl
Isobutyl
̶
̶
H
H
H
methyl
n-propyl
Bn
4-Cl-Bn
4-CH3-Bn
4-OCH3-Bn
4-tert-butyl-Bn
phenylethyl
1-methylnaphthalen
2-methylnaphthalen
2-methylpyridine
3-methylpyridine
4-methylpyridine
methyl
n-propyl
Bn
̶
̶
Tubastatin A
SAHA
a
All compounds were assayed at least two times, and the results are expressed as mean standard error of the mean (SEM).
alkyl (4-methylbenzyl, 4-methoxybenzyl or 4-tert-butylbenzyl)
impair the HDAC6 inhibitory activities. Above results indicated that
the outer surface region of HDAC6 occupied by the R2 group may be
composed of polar residues and the hydrophobic alkyl or the benzyl
containing alkyl are unable to favorably accommodate the polar
pocket, thereby reducing their contributions to HDAC6 inhibitory
activities. Therefore, we subsequently introduced pyridine ring, a
polar group, into the R2 position. In vitro evaluations suggested that
4

T. Liang, J. Xue, Z. Yao et al.
European Journal of Medicinal Chemistry 221 (2021) 113526
these compounds containing a pyridine ring (compounds 70, 71
and 72) achieve better HDAC6 inhibitory activities. In our present
study, the most potent compound 71 exhibits a low nanomolar
HDAC6 inhibition (IC50 ¼ 2.17 nM) and its HDAC6 inhibitory activity
is 12-fold better than that of Tubastatin A (IC50 ¼ 27.44 nM).
Subsequently, computational modelling of compound 71 was
employed to investigate its good HDAC6 inhibitory activity. After
protein preparation and grid generation, (R)-71 or (S)-71 was
calculated into the HDAC6 (PDB ID: 5EDU) binding site by means of
GLIDE (Glide, version 11.5, Schrodinger). Molecular docking studies
suggested that the hydroxamate moiety of (R)-71 or (S)-71 chelates
zinc ion well and the pyridine group of (R)-71 or (S)-71 forms
hydrogen bond with H651 residue (Fig. 2). The presence of H651
residue at the outer surface region of HDAC6 explains why the
introduction of the pyridine group could improve the HDAC6
inhibitory activity, while the introduction of alkyl or benzyl con-
taining alkyl impair HDAC6 inhibitory activity.
they could attenuate the toxicity of current pan-HDACs inhibitors
while remaining therapeutic effect. Therefore, we evaluated the
anti-proliferative activity of compound 71. As the results summa-
rized in Table 3, compound 71 shows low micromolar anti-
proliferative activities against various human leukemia cell lines
(KG1, IC50 ¼ 7.04
m
M; Jurkat, IC50 ¼ 9.29
M) and human multiple myeloma
M). Notably, compound 71 possessed
m
M; HEL, IC50 ¼ 4.58
mM
and HL-60, IC50 ¼ 3.23
m
(NCIeH929, IC50 ¼ 7.61
m
better anti-proliferation activity than Tubastatin A.
2.5. Western blot
To confirm that the anti-proliferative activities of compound 71
are the effect of HDAC6 inhibition instead of HDAC1 or HDAC2 in-
hibition, Western blot analysis was performed. As shown in Fig. 3,
compound 71, strongly increase the acetylation level of a-tubulin (a
major substrate for HDAC6) in a time-dependent and dose-
dependent manner, whereas it has only a slight effect on the
acetylation of histone H3 (a major substrate of HDAC1 and HDAC2).
The results indicated that our HDAC6 inhibitor 71 could suppress
the growth of carcinoma cells by inhibiting HDAC6 instead of
HDAC1 or HDAC2.
2.3. HDAC isoform selectivity
For potent compounds (compounds 16, 63, 70 and 71) with a
low nanomolar HDAC6 inhibitory activities, we subsequently
investigated their HDAC isoform selectivities against HDAC1,
HDAC2 and HDAC8 (Table 2). The results indicated that R1 group
plays an essential role in HDAC6 isoform selectivity. Despite com-
pound 16 with no substituent in R1 position shows good HDAC6
isoform selectivity (103-fold, 130-fold and 119-fold selectivity to-
ward HDAC1, HDAC2 and HDAC8, respectively), the introduction of
benzyl group into R1 position of compound 16 improves its HDAC6
isoform selectivity greatly (compound 63, 1362-fold, 1377-fold and
664-fold selectivity against HDAC1, HDAC2 and HDAC8,
respectively).
In addition, the most potent compound 71 possesses good
HDAC6 isoform selectivity and it displays 5545-fold selectivity
against HDAC1, 5864-fold selectivity against HDAC2 as well as
1638-fold selectivity relative to HDAC8. Given the good HDAC6
inhibitory activity and HDAC6 isoform selectivity of compound 71,
it was chosen for further biological evaluations.
2.6. Mitochondrial depolarization assay (mtDJ) and Caspase-3
activation assay in HL-60 cells
Literature revealed that HDAC6 inhibition induces the acetyla-
tion of Ku70, thus regulating the interaction between Ku70 and Bax,
and initiates the dissociation of Bax from Ku70, followed by mito-
chondrial outer membrane permeabilization (MOMP), caspase
cascade events and apoptosis [29e33]. Therefore, we explored the
mitochondrial potential changes and caspase-3 activation in HL-
60 cells treated with compound 71.
Following incubation with 3 mM compound 71, the mitochon-
drial potential change was determined using tetramethylrhod-
amine ethyl ester (TMRE). As shown in Fig. 4A, the significant
mitochondrial potential loss was observed and compound 71
induced mitochondrial depolarization in
manner.
a time-dependent
2.4. In vitro anti-proliferative activity
As for caspase-3 activation, incubating compound 71 signifi-
cantly increased the amount of activated caspase-3 in HL-60 cells
compared with vehicle (0.2% DMSO). As the incubation time in-
creases, more and more caspase-3 was activated. It is worth
HDAC6 isoform selective inhibitors have emerged as an alter-
native to pan-HDACs inhibitor for the anti-cancer therapy because
Fig. 2. (A) Proposed binding mode of (R)-71 (depicted in gray) in the HDAC6 binding site; (B) Proposed binding mode of (S)-71 (depicted in gray) in the HDAC6 binding site.
Hydrogen bonds were shown as yellow dotted line and the zinc ion was shown as a gray sphere.
5

T. Liang, J. Xue, Z. Yao et al.
European Journal of Medicinal Chemistry 221 (2021) 113526
Table 2
HDACs isoform profiles of potent target compounds.
a
Compound
IC50 (nM)
HDAC1
HDAC2
HDAC6
HDAC8
16
63
70
71
862.5 17.5
1082.0 145.0
8263.9 451.6
5081.0 178.9
12314.4 501.0
8674.5 1119.5
112.5 2.5
8.3 0.9
6.0 0.8
4.8 1.8
2.1 0.09
27.4 0.7
26.9 3.2
994.0 18.1
8176.5 1095.5
3422.0 235.0
11645.0 1295.0
7582.5 145.5
45.5 0.5
3987.5 298.5
7826.0 98.1
3441.0 307.0
1899.0 105.0
1279.3 32.9
Tubastatin A
SAHA
a
The results are expressed as mean SEM (n ꢁ 2).
Table 3
Anti-proliferative activity of compound 71.
Compound
IC50
KG1
(m
M)a
HEL
Jurkat
HL-60
NCIeH929
71
7.04 1.03
8.82 0.36
4.58 0.06
8.09 0.24
9.29 1.61
14.58 1.03
3.23 0.09
4.09 0.09
7.61 0.76
15.42 1.48
Tubastatin A
a
The results are expressed as mean SEM (n ꢁ 3).
Fig. 3. The acetylation of a-tubulin and histone H3 induced by compound 71 or Tubastatin A for 12 h (A) and 24 h (B) in HL-60 cells.
mentioning that compound 71 exhibits the better property in term
of caspase-3 activation compared with Tubastatin A (Fig. 4B).
Compound 71 and Tubastatin A exhibit acceptable cytotoxicity to-
ward HUVECs (Fig. 7A) and GEC-1 cells (Fig. 7B). Notably, our
compound 71 could kill HL-60 cells selectively and spare normal-
tissue cell lines.
2.7. TUNEL assay on HL-60 cells
Subsequently, TUNEL assay was designed to investigate the
ability of compound 71 to induce apoptosis in HL-60 cells. As
illustrated in Fig. 5, weak green fluorescence was observed in HL-
60 cells incubated Tubastatin A, whereas the HL-60 cells treated
with compound 71 were labeled with stronger green fluorescence.
In other words, compound 71 has the stronger ability to induce
apoptosis compared to Tubastatin A in HL-60 cells.
2.10. Stability of compound 71 in rabbit plasma
Finally, we preliminarily investigated the plasma stability of
compound 71. Upon treatment compound 71 with rabbit plasma
for indicated times (0, 0.5, 1, 3, 6, 9, and 12 h), the recovered
compound was measured using HPLC and a small amount of
compound 71 was degraded (Fig. 8). The result indicates that
compound 71 possesses good plasma stability.
2.8. Flow cytometry analysis on HL-60 cells
3. Conclusion
To further determine the apoptotic rate of HL-60 cells in
response to compound 71 or Tubastatin A, flow cytometry analysis
was performed (Fig. 6). Upon treatment with Tubastatin A or
compound 71 for 48 h, HL-60 cells displayed modest apoptosis
(apoptotic rates are 12.36% and 20.50%, respectively). The results
are consistent of TUNEL assay.
Efforts of the structure optimization of previous compound 10c
(IC50 ¼ 4.4 nM) resulted in a series of more potent and selective
HDAC6 inhibitors. Compound 71, the most potent molecule, has a
low nanomolar inhibitor activity for HDAC6 (IC50 ¼ 2.1 nM), and
improved selectivity against HDAC1, HDAC2 and HDAC8, respec-
tively. Western blot analysis further confirmed that compound 71
2.9. Cytotoxicity studies
selectively increased the acetylation level of a-tubulin with a slight
effect on the acetylation of histone H3. Moreover, compound 71 can
induce mitochondrial outer membrane permeabilization (MOMP),
caspase-3 activation and apoptosis. In the other hand, compound
We also evaluated the cytotoxicity of compound 71 and
Tubastatin A toward normal-tissue cells (HUVECs and GEC-1 cells).
6

T. Liang, J. Xue, Z. Yao et al.
European Journal of Medicinal Chemistry 221 (2021) 113526
Fig. 4. Mitochondrial potential changes (A) and caspase-3 activation (B) in HL-60 cells treated compound 71 or Tubastatin A for indicated time.
Fig. 5. TUNEL assay in HL-60 cells treated with compound 71 and Tubastatin A.
71 also possesses good properties in term of anti-proliferative ac-
evaluated using a fluorescence assay with Tubastatin A as the
positive control. Generally, HDAC6 enzyme was incubated with a
range of concentrations of test compounds for 10 min at 37 ꢀC,
followed by the addition of fluorogenic substrates (Ac-LeuGlyLy-
s(Ac)-AMC). After incubating for 2 h, the reaction was quenched
by developer containing TSA and trypsin. Incubate the mixture at
37 ꢀC for another 30 min, and measure the fluorescence intensity
with the excitation and emission wavelength of 390 nm and
460 nm using a Thermo Varioskan microtiter plate reader. The
data was analyzed to calculate the inhibition ratios and IC50
values.
tivities, plasma stability and cytotoxicity. In summary, our com-
pound 71 demonstrates a paradigm for anti-cancer therapy using
HDAC6 selective inhibitor and it can also be used as lead compound
to develop more potent and selective HDAC6 inhibitor.
4. Materials and methods
4.1. Biological evaluations
4.1.1. HDAC6 inhibitory activities
The HDAC6 inhibitory activities of all target compounds were
7

T. Liang, J. Xue, Z. Yao et al.
European Journal of Medicinal Chemistry 221 (2021) 113526
Fig. 6. Flow cytometry analysis of HL-60 cells treated with compound 71 or Tubastatin A.
Fig. 7. Cytotoxicity of compound 71 and Tubastatin A against HUVECs (A) and GEC-1 cells (B).
tested compounds for 10 min, and the fluorogenic substrate (Ac-
LeuGlyLys(Ac)-AMC substrate for HDAC1 and 2; Ac-LeuGlyLys(tfa)-
AMC substrate for HDAC 8) were added, followed by incubation for
1 h. After quenching the reaction with developer, incubate the
mixture at 37 ꢀC for another 30 min. Subsequently, measure the
fluorescence intensity with a Thermo microtiter plate reader with
an excitation of 390 nm and an emission wavelength of 460 nm.
Then Varioskan Prism Graph Pad software was used to calculate the
IC50 values.
4.1.3. In vitro Antiproliferative activities
All of the cell lines were cultured with RPMI1640 medium (10%
FBS) at 37 ꢀC in a 5% CO2 humidified incubator. After seeding the
cells in 96-well plates at 3500e4000 cells per well (100
the cells were cultured overnight, followed by treatment with the
indicated dilutions of compound 71 (100 L/well) for 48 h. Add
10 L 0.5% MTT to each well and incubate the mixture for another
4 h. Subsequently, centrifuge and remove the supernatant carefully,
followed by the addition of 150 L DMSO. A Thermo Varioskan
mL/well),
m
m
Fig. 8. The stability of compound 71 in rabbit plasma.
m
microtiter-plate reader was used to analyze the absorbance at
570 nm. The inhibition ratios and IC50 values were calculated with
Prism Graph Pad software.
4.1.2. HDACs isoform selectivity
HDAC1, 2 and 8 were incubated with a serial dilutions of the
8

T. Liang, J. Xue, Z. Yao et al.
European Journal of Medicinal Chemistry 221 (2021) 113526
4.1.4. Western blot
at a flow rate of 1 mL/min. The mobile phase consists of 42%
Treated HL-60 cells were harvested (800 rpm, 5 min) and lysed
with 1 ꢂ RIPA buffer containing 1 mM PMSF on ice for 30 min.
Collect the supernatant (4 ꢀC, 14000 rpm, 10 min) and boil it in
loading buffer. Then samples were electrophoretically separated on
12.5% SDS PAGE gel and transferred to PVDF membrane, followed
by being blocked with 5% nonfat dry milk for 1 h. Subsequently, the
membranes were washed with TBST and incubated with primary
acetonitrile and 58% H2O containing 0.1% formic acid.
4.1.10. Cytotoxicity studies
Normal-tissue cells (HUVECs and GEC-1 cells) was selected to
evaluate the cytotoxicity of tested compounds. HUVECs were
cultured with RPMI1640 medium containing 10% FBS as well as
0.1% heparin and GEC-1 cell lines were cultured with RPMI1640
medium containing 10% FBS. HUVECs and GEC-1 cells were seeded
in a 96-well plate for 8 h and then treated with the indicated
dilution of tested compounds for 48 h. Following incubation with
0.5% MTT for 4 h, the supernatant was removed and DMSO was
added. Finally, the absorbance was determined using a Thermo
Varioskan microtiter-plate reader at 570 nm.
anti-bodies of Ac- a
-tubulin Ac-H3 and GAPDH overnight at 4 ꢀC.
Finally, wash the membranes with TBST for another three times and
incubate them with the secondary antibody for 1 h, followed by ECL
reaction. Images of the member were monitored with an Amer-
sham Imager 680.
4.1.5. TUNEL assay
TUNEL assay was designed to detect apoptotic HL-60 cells. HL-
4.1.11. Molecular docking
60 cells (1 ꢂ 106 cells/well) in 6-well clear bottom plates were
The crystal structure of human HDAC6 (PDB ID: 5EDU) was
selected for molecular docking studies using GLIDE (Glide, version
11.5, Schrodinger). After protein preparation and grid generation,
(S)-71 or (R)-71 was calculated into HDAC6 binding site and the top
scored binding poses were chosen as proposed binding modes.
incubated with 3 mM compound 71, 3 mM Tubastatin A or vehicle
(0.2% DMSO) for 48 h. Above cells were washed with pre-chilled
PBS and incubated with paraformaldehyde for 30 min. Then they
were treated with 0.5% Tritonx-100 in PBS for 5 min and washed
with cold PBS. After adding FITC-12-Dutp and terminal deoxy-
nucleotidyl transferase, the cells were incubated for 60 min. Wash
the cells two times with cold PBS buffer to eliminate background
fluorescence and the green fluorescence was monitored with a
Zeiss Axio Observer A1 fluorescence microscope.
4.2. Chemistry
All chemical reagents are analytical grade and used without
further purification. Reactions were monitored by thin-layer
chromatography (TLC) on 0.25 mm silica gel plates (60 GF-254).
Melting points were determined by the RY-1 electrothermal
melting point apparatus. 1H NMR and 13C NMR spectra were ob-
tained on a Brucker DRX spectrometer at 400 MHz. High-resolution
mass spectra (HRMS) were conducted on an Agilent 6510 quadru-
pole time-off light liquid chromatography/mass spectrometer (LC/
MS) delivered with electrospray ionization (ESI). The purities of
target compounds were determined by reversed-phase (RP)-HPLC
and they achieved a minimum of 95% purity.
4.1.6. Flow-cytometry analysis
Flow-cytometry analysis was performed on HL-60 cells. Cells
(1 ꢂ 106 cells/well) in 6-well clear bottom plates were incubated
with indicated dilutions of tested compounds for 48 h and then
cells were collected and washed with PBS. Subsequently, resuspend
cells with 195
addition of 5
m
L Annexin V-FITC binding buffer, followed by the
m
L propidium iodide (PI) and 2.5 L Annexin V-FITC.
m
After incubating for 15 min, the samples were analyzed with flow
cytometry.
4.2.1. ((4-(methoxycarbonyl)benzyl)carbamoyl)glycine (1)
4.1.7. Mitochondrial depolarization assay (mtDJ
)
To the solution of triphosgene (23.74 g, 80 mmol) in CH2Cl2, the
solution of methyl 4-(aminomethyl)benzoate (33.04 g, 200 mmol)
with triethylamine (55.60 mL, 400 mmol) was added slowly under
ice bath. Stir the mixture for 30 min at 0 ꢀC and then the solvent
was removed to give methyl 4-(isocyanatomethyl)benzoate.
Glycine (30.03 g, 400 mmol) was dissolved with 2 M NaOH
(200 mL) and the solution of methyl 4-(isocyanatomethyl)benzoate
in toluene (200 mL) was added. After stirring the mixture for 3 h,
the water phase was acidified (pH ¼ 2) with 6 M HCl and the
product was obtained by filtration. White solid, yield: 73%, mp
1 ꢂ 106 HL-60 cells were seeded in 6-well plate and then they
were cultured overnight. Following treatment with compound 71,
Tubastatin A or vehicle (0.2% DMSO) for indicated time, cells were
incubated with tetramethylrhodamine ethyl ester (100 nM) for
30 min at 37 ꢀC. Subsequently, collect and wash cells with PBS for
three times and then transfer them into a black 96-well plate, fol-
lowed by the analysis of the fluorescence intensity (Ex: 540 nm,
Em: 579 nm).
4.1.8. Caspase 3 activation assay
191e192 ꢀC. 1H NMR (400 MHz, DMSO‑d6)
d 12.21 (s, 1H), 7.91 (d,
The caspase-3 activation in treated HL-60 cells was measured
using GreenNuc™ Caspase-3 Assay Kit for Live Cells. HL-60 cells
were seeded in 6-well clear bottom plate for 8 h. Upon treatment
with indicated concentrations of tested compounds or vehicle (0.2%
DMSO) for different time (6 h, 12 h, 18 h and 24 h), cells were
harvested and washed with PBS buffer. After incubating with
GreenNuc™ Caspase-3 substrate for 30 min, transfer the cells into a
black 96-well plate and measure the fluorescence intensity (Ex:
485 nm, Em: 515 nm). The results are expressed as fold increase in
caspase activity of apoptotic cells over that of non-induced cells.
J ¼ 8.2 Hz, 2H), 7.39 (d, J ¼ 8.1 Hz, 2H), 6.78 (t, J ¼ 5.9 Hz,1H), 6.29 (t,
J ¼ 5.6 Hz, 1H), 4.29 (d, J ¼ 5.9 Hz, 2H), 3.84 (s, 3H), 3.72 (d,
J ¼ 5.8 Hz, 2H).
4.2.2. 4-((2,5-dioxoimidazolidin-1-yl)methyl)benzoic acid (2)
The intermediate 1 (39.93 g, 150 mmol) was dissolved using
hydrochloric acid (300 mL), followed by refluxing for 3 h. After
cooling the reaction solution down, the white crystal precipitation
was isolated to give compound 2. White solid, yield: 80%,
mp > 300 ꢀC. 1H NMR (400 MHz, DMSO‑d6)
d 12.95 (s, 1H), 8.18 (s,
1H), 7.91 (d, J ¼ 8.4, 2.0 Hz, 2H), 7.38 (d, J ¼ 8.2 Hz, 2H), 4.60 (s, 2H),
4.1.9. Stability of compound 71 in rat plasma
4.01 (s, 2H).
200
pound 71 (50
0.5 h, 1 h, 3 h, 6 h, 9 h and 12 h). Add 600
the suspension was centrifuged and filtered. HPLC was performed
to analyze the samples with a C18 column (150 mm ꢂ 4.6 mm, 5 m)
m
L rabbit plasma was incubated with the solution of com-
L, 2 mg/mL in DMSO) at 37 ꢀC for indicated time (0 h,
L acetonitrile and then
m
4.2.3. Methyl 4-((2,5-dioxoimidazolidin-1-yl)methyl)benzoate (3)
To the solution of compound 2 (32.78 g, 140 mmol) in MeOH,
acetyl chloride (25.2 mL, 350 mmol) was slowly added at 0 ꢀC and
the mixture was refluxed for 5 h. Cool the reaction solution down
m
m
9

T. Liang, J. Xue, Z. Yao et al.
European Journal of Medicinal Chemistry 221 (2021) 113526
and the solvent was evaporated under vacuum. The crude product
was washed with n-hexane to give a white solid, yield: 98%, mp
(bromomethyl)-4-(tert-butyl)benzene in a manner similar to that
described for the preparation of 4. White solid, yield: 95%, mp
178e180 ꢀC. 1H NMR (400 MHz, CDCl3)
d
7.99 (d, J ¼ 8.3 Hz, 2H),
119e120 ꢀC. 1H NMR (400 MHz, CDCl3)
d
8.00 (d, J ¼ 8.2 Hz, 2H),
7.45 (d, J ¼ 8.3 Hz, 2H), 6.10 (s, 1H), 4.71 (s, 2H), 4.01 (d, J ¼ 1.1 Hz,
7.47 (d, J ¼ 8.2 Hz, 2H), 7.37 (d, J ¼ 8.1 Hz, 2H), 7.17 (d, J ¼ 8.1 Hz, 2H),
2H), 3.91 (s, 3H).
4.72 (s, 2H), 4.53 (s, 2H), 3.91 (s, 3H), 3.76 (s, 2H), 1.31 (s, 9H).
4.2.4. Methyl 4-((3-methyl-2,5-dioxoimidazolidin-1-yl)methyl)
benzoate (4)
4.2.11. Methyl 4-((2,5-dioxo-3-phenethylimidazolidin-1-yl)methyl)
benzoate (11)
Compound 4 was mixed with K2CO3 (2.48 g, 10 mmol) and
iodomethane (0.74 g, 12 mmol) in 50 mL DMF for 6 h. Poured the
mixture into cool water and extracted three times with ethyl ace-
tate. Dry the organic phases over with MgSO4 and then the organic
solvent was concentrated to give the crude product, which was
purified by silica gel column chromatography. White solid, yield:
The title compound was synthesized from 3 and (2-bromoethyl)
benzene in a manner similar to that described for the preparation of
4. White solid, yield: 83%, mp 88e90 ꢀC. 1H NMR (400 MHz, CDCl3)
d
7.97 (dd, J ¼ 13.7, 8.2 Hz, 2H), 7.44e7.35 (m, 2H), 7.35e7.27 (m,
3H), 7.19e7.14 (m, 2H), 4.66 (d, J ¼ 2.4 Hz, 2H), 4.52 (s, 1H), 3.91 (s,
2H), 3.66 (s, 3H), 3.08 (d, J ¼ 6.9 Hz, 1H), 2.89 (t, J ¼ 7.1 Hz, 2H).
97%, mp 119e121 ꢀC. 1H NMR (400 MHz, CDCl3)
d
7.99 (d, J ¼ 8.3 Hz,
2H), 7.46 (d, J ¼ 8.2 Hz, 2H), 4.69 (s, 2H), 3.98e3.84 (m, 5H), 3.00 (s,
4.2.12. Methyl 4-((3-(naphthalen-1-ylmethyl)-2,5-
dioxoimidazolidin-1-yl)methyl) benzoate (12)
3H).
The title compound was synthesized from 3 and 1-(bromo-
methyl)naphthalene in a manner similar to that described for the
preparation of 4. White solid, yield: 82%, mp 197e199 ꢀC. 1H NMR
4.2.5. Methyl 4-((2,5-dioxo-3-propylimidazolidin-1-yl)methyl)
benzoate (5)
The title compound was synthesized from 3 and 1-iodopropane
in a manner similar to that described for the preparation of 4. White
(400 MHz, CDCl3)
d
8.13e8.06 (m, 1H), 8.00 (d, J ¼ 8.1 Hz, 2H),
7.93e7.84 (m, 2H), 7.58e7.52 (m, 2H), 7.44 (dd, J ¼ 10.7, 8.2 Hz, 3H),
7.38 (dd, J ¼ 7.0, 1.4 Hz, 1H), 5.02 (s, 2H), 4.72 (s, 2H), 3.91 (s, 3H),
3.61 (s, 2H).
solid, yield: 75%, mp 73e74 ꢀC. 1H NMR (400 MHz, DMSO‑d6)
d 7.93
(d, J ¼ 8.2 Hz, 2H), 7.41 (d, J ¼ 8.2 Hz, 2H), 4.62 (s, 2H), 4.06 (s, 2H),
3.84 (s, 3H), 3.26 (s, 2H), 1.52 (q, J ¼ 7.3 Hz, 2H), 0.85 (t, J ¼ 7.4 Hz,
3H).
4.2.13. N-hydroxy-4-((3-methyl-2,5-dioxoimidazolidin-1-yl)
methyl)benzamide (13)
4.2.6. Methyl 4-((3-benzyl-2,5-dioxoimidazolidin-1-yl)methyl)
benzoate (6)
To the solution of compound 4 (1.57 g, 6 mm0l) in THF, the
aqueous solution of LiOH was added and the mixture was stirred for
6 h. After removing THF, the water phase was acidified, followed by
extracting three times using ethyl acetate. Then, the organic solvent
was evaporated under vacuum to give the hydrolysate without
further purification.
Subsequently, the hydrolysate (0.74 g, 3 mmol) was dissolved
with THF and then isobutyl chloroformate (0.42 mL, 3.3 mmol) as
well as N-methylmorpholine (0.43 mL, 3.9 mmol) were added. After
stirring the mixture for 30 min, the freshly prepared hydroxyl-
amine (0.40 g, 12 mmol) in methanol was added and the reaction
solution was stirred for another 6 h at room temperature. The
solvent was concentrated and the residue was dissolved in 50 ml
ethyl acetate, followed by washing with 1 M HCl and saturated
brine. The crude product was purified by silica gel column chro-
matography to give a white solid, yield: 39%, mp 194e196 ꢀC. 1H
The title compound was synthesized from 3 and benzyl bromide
in a manner similar to that described for the preparation of 4. White
solid, yield: 94%, mp 90e92 ꢀC. 1H NMR (400 MHz, CDCl3)
d 8.00 (d,
J ¼ 8.2 Hz, 2H), 7.48 (d, J ¼ 8.2 Hz, 2H), 7.40e7.32 (m, 3H), 7.24 (dd,
J ¼ 7.9, 1.7 Hz, 2H), 4.73 (s, 2H), 4.56 (s, 2H), 3.91 (s, 3H), 3.76 (s, 2H).
4.2.7. Methyl 4-((3-(4-chlorobenzyl)-2,5-dioxoimidazolidin-1-yl)
methyl)benzoate (7)
The title compound was synthesized from 3 and 1-(bromo-
methyl)-4-chlorobenzene in a manner similar to that described for
the preparation of 4. White solid, yield: 94%, mp 121e123 ꢀC. 1H
NMR (400 MHz, CDCl3)
d
8.00 (d, J ¼ 8.3 Hz, 2H), 7.47 (d, J ¼ 8.3 Hz,
2H), 7.33 (d, J ¼ 8.4 Hz, 2H), 7.18 (d, J ¼ 8.4 Hz, 2H), 4.72 (s, 2H), 4.53
(s, 2H), 3.91 (s, 3H), 3.75 (s, 2H).
NMR (400 MHz, DMSO‑d6)
J ¼ 8.2 Hz, 2H), 7.33 (d, J ¼ 8.0 Hz, 2H), 4.58 (s, 2H), 4.04 (s, 2H), 2.87
(s, 3H). 13C NMR (101 MHz, DMSO)
170.75, 164.45, 156.71, 140.23,
d 11.18 (s, 1H), 9.02 (s, 1H), 7.70 (d,
4.2.8. Methyl 4-((3-(4-methylbenzyl)-2,5-dioxoimidazolidin-1-yl)
methyl)benzoate (8)
d
The title compound was synthesized from 3 and 1-(bromo-
methyl)-4-methylbenzene in a manner similar to that described for
the preparation of 4. White solid, yield: 95%, mp 132e134 ꢀC. 1H
132.37, 127.79, 127.55, 51.88, 41.63, 29.71. HRMS (AP-ESI) m/z,
calculated for C12H13N3O4, ([M þ H]þ): 264.0979, found: 264.0981.
HPLC tR ¼ 5.33 min (97.12% purity).
NMR (400 MHz, CDCl3)
2H), 7.14 (q, J ¼ 8.1 Hz, 4H), 4.72 (s, 2H), 4.51 (s, 2H), 3.91 (s, 3H),
d
8.00 (d, J ¼ 8.3 Hz, 2H), 7.47 (d, J ¼ 8.3 Hz,
4.2.14. 4-((2,5-dioxo-3-propylimidazolidin-1-yl)methyl)-N-
hydroxybenzamide (14)
3.73 (s, 2H), 2.34 (s, 3H).
The title compound was synthesized from 5 in a manner similar
to that described for the preparation of 13. White solid, yield: 81%,
4.2.9. Methyl 4-((3-(4-methoxybenzyl)-2,5-dioxoimidazolidin-1-yl)
methyl)benzoate (9)
mp 182e184 ꢀC. 1H NMR (400 MHz, DMSO‑d6)
d 11.18 (s,1H), 9.03 (s,
The title compound was synthesized from 3 and 1-(bromo-
methyl)-4-methoxybenzene in a manner similar to that described for
the preparation of 4. White solid, yield: 94%, mp 129e131 ꢀC. 1H
1H), 7.70 (d, J ¼ 8.2 Hz, 2H), 7.32 (d, J ¼ 8.2 Hz, 2H), 4.58 (s, 2H), 4.06
(s, 2H), 3.25 (t, J ¼ 7.2 Hz, 2H), 1.53 (p, J ¼ 7.3 Hz, 2H), 0.85 (q, J ¼ 7.4,
6.0 Hz, 3H). 13C NMR (101 MHz, DMSO)
d 170.86, 164.37, 156.52,
NMR (400 MHz, CDCl3)
d
8.00 (d, J ¼ 8.3 Hz, 2H), 7.47 (d, J ¼ 8.1 Hz,
140.23, 132.39, 127.76, 127.56, 49.84, 44.23, 41.59, 20.91, 11.50.
HRMS (AP-ESI) m/z, calculated for C14H17N3O4, ([M þ H]þ):
292.1292, found: 292.1289. HPLC tR ¼ 7.32 min (99.43% purity).
2H), 7.17 (d, J ¼ 8.4 Hz, 2H), 6.88 (d, J ¼ 8.4 Hz, 2H), 4.71 (s, 2H), 4.49
(s, 2H), 3.91 (s, 3H), 3.80 (s, 3H), 3.73 (s, 2H).
4.2.10. Methyl 4-((3-(4-(tert-butyl)benzyl)-2,5-dioxoimidazolidin-
1-yl)methyl)benzoate (10)
4.2.15. 4-((3-benzyl-2,5-dioxoimidazolidin-1-yl)methyl)-N-
hydroxybenzamide (15)
The title compound was synthesized from
3
and 1-
The title compound was synthesized from 6 in a manner similar
10

T. Liang, J. Xue, Z. Yao et al.
European Journal of Medicinal Chemistry 221 (2021) 113526
to that described for the preparation of 13. White solid, yield: 48%,
2H), 4.01 (s, 2H), 3.56 (t, J ¼ 7.2 Hz, 2H), 2.84 (t, J ¼ 7.2 Hz, 2H). 13
C
mp 188e189 ꢀC. 1H NMR (400 MHz, DMSO‑d6)
d
11.21 (s, 1H), 9.05
NMR (101 MHz, DMSO) d 170.62, 164.42, 156.28, 140.15, 139.06,
(s, 1H), 7.72 (d, J ¼ 8.2 Hz, 2H), 7.45e7.34 (m, 4H), 7.30 (td, J ¼ 6.2,
132.36, 129.16, 128.89, 127.63, 127.55, 126.82, 49.94, 43.88, 41.52,
33.65. HRMS (AP-ESI) m/z, calculated for C19H19N3O4, ([M þ H]þ):
354.1448, found: 354.1443. HPLC tR ¼ 19.14 min (98.23% purity).
1.7 Hz, 3H), 4.63 (s, 2H), 4.52 (s, 2H), 3.98 (s, 2H). 13C NMR (101 MHz,
DMSO)
128.10,128.00,127.82,127.60, 50.09, 46.42, 41.80. HRMS (AP-ESI) m/
z, calculated for 18H17N3O4, ([M
H]þ): 340.1292, found:
d 170.56, 164.44, 156.80, 140.15, 136.86, 132.44, 129.15,
C
þ
4.2.21. N-hydroxy-4-((3-(naphthalen-2-ylmethyl)-2,5-
dioxoimidazolidin-1-yl)methyl)benzamide (21)
340.1283. HPLC tR ¼ 9.24 min (99.27% purity).
The title compound was synthesized from 12 in a manner
similar to that described for the preparation of 13. White solid,
4.2.16. 4-((3-(4-chlorobenzyl)-2,5-dioxoimidazolidin-1-yl)methyl)-
N-hydroxybenzamide (16)
yield: 57%, mp 183e185 ꢀC. 1H NMR (400 MHz, DMSO‑d6)
d 11.20 (s,
The title compound was synthesized from 7 in a manner similar
to that described for the preparation of 13. White solid, yield: 51%,
1H), 9.03 (s, 1H), 8.14 (d, J ¼ 7.9 Hz, 1H), 8.05e7.85 (m, 2H), 7.72 (d,
J ¼ 7.9 Hz, 2H), 7.58 (q, J ¼ 6.1, 4.5 Hz, 2H), 7.50 (d, J ¼ 5.1 Hz, 2H),
mp 186e188 ꢀC. 1H NMR (400 MHz, DMSO‑d6)
d
11.20 (s, 1H), 9.04
7.36 (d, J ¼ 7.9 Hz, 2H), 4.99 (s, 2H), 4.65 (s, 2H), 3.92 (s, 2H). 13
C
(s, 1H), 7.72 (d, J ¼ 8.2 Hz, 2H), 7.42 (d, J ¼ 8.2 Hz, 2H), 7.40e7.30 (m,
NMR (101 MHz, DMSO) d 170.44, 164.45, 156.60, 140.16, 133.94,
4H), 4.62 (s, 2H), 4.51 (s, 2H), 4.00 (s, 2H). 13C NMR (101 MHz,
132.46, 132.06, 131.30, 129.18, 128.95, 127.78, 127.62, 127.11, 127.06,
126.53, 125.98, 123.61, 50.02, 44.57, 41.79. HRMS (AP-ESI) m/z,
calculated for C22H19N3O4, ([M þ H]þ): 390.1448, found: 390.1444.
HPLC tR ¼ 4.82 min (98.89% purity).
DMSO)
d 170.56, 164.45, 156.84, 140.12, 135.97, 132.62, 132.44,
130.03, 129.07, 127.82, 127.61, 50.14, 45.77, 41.81. HRMS (AP-ESI) m/
z, calculated for C18H16ClN3O4, ([M þ H]þ): 374.0902, found:
374.0899. HPLC tR ¼ 19.05 min (99.87% purity).
4.2.22. Methyl 4-((3-(2-methoxy-2-oxo-1-phenylethyl)ureido)
methyl)benzoate (22)
4.2.17. N-hydroxy-4-((3-(4-methylbenzyl)-2,5-dioxoimidazolidin-
1-yl)methyl)benzamide (17)
To the solution of triphosgene (23.74 g, 80 mmol) in CH2Cl2, the
solution of methyl 2-amino-2-phenylacetate (33.02 g, 200 mmol)
with triethylamine (55.60 mL, 400 mmol) was added slowly. When
the titration was completed, stir the mixture for 30 min at 0 ꢀC.
Subsequently, methyl 4-(aminomethyl)benzoate (33.02 g,
200 mmol) in CH2Cl2 was added and the reaction solution was
stirred for another 30 min. Wash the organic phase with 1 M HCl as
well as saturated brine and the solvent was concentrated to give the
title compound. White solid, yield: 80%, mp 144e146 ꢀC. 1H NMR
The title compound was synthesized from 8 in a manner similar
to that described for the preparation of 13. White solid, yield: 44%,
mp 192e193 ꢀC. 1H NMR (400 MHz, DMSO‑d6)
d 11.18 (s, 1H), 9.02
(s, 1H), 7.83e7.62 (d, J ¼ 8.2 Hz, 2H), 7.35 (d, J ¼ 8.2 Hz, 2H),
7.27e7.08 (m, 4H), 4.62 (s, 2H), 4.46 (s, 2H), 3.95 (s, 2H), 2.29 (s, 3H).
13C NMR (101 MHz, DMSO)
d 170.54, 164.43, 156.72, 140.16, 137.20,
133.76, 132.45, 129.71, 128.16, 127.81, 127.60, 49.93, 46.14, 41.77,
21.16. HRMS (AP-ESI) m/z, calculated for C19H19N3O4, ([M þ H]þ):
354.1448, found: 354.1446. HPLC tR ¼ 16.08 min (98.49% purity).
(400 MHz, DMSO‑d6)
d
7.91 (d, J ¼ 8.3 Hz, 2H), 7.47e7.30 (m, 7H),
6.98 (d, J ¼ 7.7 Hz,1H), 6.65 (t, J ¼ 6.1 Hz,1H), 5.30 (d, J ¼ 7.7 Hz,1H),
4.2.18. N-hydroxy-4-((3-(4-methoxybenzyl)-2,5-
dioxoimidazolidin-1-yl)methyl)benzamide (18)
4.31 (d, J ¼ 6.1 Hz, 2H), 3.84 (s, 3H), 3.62 (s, 3H).
The title compound was synthesized from 9 in a manner similar
to that described for the preparation of 13. White solid, yield: 61%,
4.2.23. 4-((2,5-dioxo-4-phenylimidazolidin-1-yl)methyl)benzoic
acid (23)
The title compound was synthesized from compound 22 in a
manner similar to that described for the preparation of compound
2. White solid, yield: 95%, mp 230e231 ꢀC. 1H NMR (400 MHz,
mp 183e185 ꢀC. 1H NMR (400 MHz, DMSO‑d6)
d
11.19 (s, 1H), 9.03
(s, 1H), 7.71 (d, J ¼ 8.2 Hz, 2H), 7.35 (d, J ¼ 8.2 Hz, 2H), 7.22 (d,
J ¼ 8.1 Hz, 2H), 6.92 (d, J ¼ 8.1 Hz, 2H), 4.61 (s, 2H), 4.44 (s, 2H), 3.93
(s, 2H), 3.74 (s, 3H). 13C NMR (101 MHz, DMSO)
d
170.54, 164.41,
DMSO‑d6)
d
12.94 (s, 1H), 8.86 (s, 1H), 7.91 (d, J ¼ 8.2 Hz, 2H),
159.20, 156.64, 140.15, 132.43, 129.65, 128.64, 127.81, 127.59, 114.54,
7.55e7.23 (m, 7H), 5.34 (s, 1H), 4.64 (s, 2H).
55.55, 49.81, 45.83, 41.75. HRMS (AP-ESI) m/z, calculated for
C
19H19N3O5, ([M
þ
H]þ): 370.1398, found: 370.1406. HPLC
4.2.24. Methyl 4-((2,5-dioxo-4-phenylimidazolidin-1-yl)methyl)
benzoate (24)
The title compound was synthesized from compound 23 in a
manner similar to that described for the preparation of compound
3. White solid, yield: 98%, mp 178e180 ꢀC. 1H NMR (400 MHz,
tR ¼ 9.38 min (97.30% purity).
4.2.19. 4-((3-(4-(tert-butyl)benzyl)-2,5-dioxoimidazolidin-1-yl)
methyl)-N-hydroxybenzamide (19)
The title compound was synthesized from 10 in a manner
similar to that described for the preparation of 13. White solid,
DMSO‑d6)
d
8.87 (s, 1H), 7.93 (d, J ¼ 8.3 Hz, 2H), 7.53e7.37 (m, 5H),
7.37e7.30 (m, 2H), 5.34 (s, 1H), 4.65 (s, 2H), 3.84 (s, 3H).
yield: 55%, mp 142e144 ꢀC. 1H NMR (400 MHz, DMSO‑d6)
d 11.20 (s,
1H), 9.03 (s, 1H), 7.72 (d, J ¼ 8.0 Hz, 2H), 7.37 (t, J ¼ 8.8 Hz, 4H), 7.21
4.2.25. Methyl 4-((4-methyl-2,5-dioxo-4-phenylimidazolidin-1-yl)
methyl)benzoate (25)
The title compound was synthesized from compound 24 and
iodomethane in a manner similar to that described for the prepa-
ration of compound 4. White solid, yield: 79%, mp 228e230 ꢀC. 1H
(d, J ¼ 8.1 Hz, 2H), 4.62 (s, 2H), 4.47 (s, 2H), 3.98 (s, 2H), 1.27 (s, 9H).
13C NMR (101 MHz, DMSO)
d
170.52, 164.45, 156.72, 150.39, 140.15,
133.83, 132.45, 127.93, 127.86, 127.63, 125.89, 50.06, 46.07, 41.80,
34.66, 31.57. HRMS (AP-ESI) m/z, calculated for C22H25N3O4, ([M þ
H]þ): 396.1918, found: 396.1913. HPLC tR ¼ 10.06 min (98.89%
purity).
NMR (400 MHz, DMSO‑d6)
d
9.09 (s, 1H), 7.91 (d, J ¼ 8.3 Hz, 2H),
7.52e7.45 (m, 2H), 7.45e7.37 (m, 3H), 7.34 (dd, J ¼ 7.7, 3.0 Hz, 2H),
4.64 (s, 2H), 3.84 (s, 3H), 1.72 (s, 3H).
4.2.20. 4-((2,5-dioxo-3-phenethylimidazolidin-1-yl)methyl)-N-
hydroxybenzamide (20)
4.2.26. Methyl 4-((2,5-dioxo-4-phenyl-4-propylimidazolidin-1-yl)
methyl)benzoate (26)
The title compound was synthesized from compound 24 and 1-
iodopropane in a manner similar to that described for the prepa-
ration of compound 4. White solid, yield: 67%, mp 70e72 ꢀC. 1H
The title compound was synthesized from 11 in a manner
similar to that described for the preparation of 13. White solid,
yield: 67%, mp 188e190 ꢀC. 1H NMR (400 MHz, DMSO‑d6)
d
11.19 (s,
1H), 9.02 (s, 1H), 7.69 (d, J ¼ 8.1 Hz, 2H), 7.39e7.13 (m, 7H), 4.54 (s,
11

T. Liang, J. Xue, Z. Yao et al.
European Journal of Medicinal Chemistry 221 (2021) 113526
NMR (400 MHz, CDCl3)
d
7.95 (d, J ¼ 8.3 Hz, 2H), 7.52e7.47 (m, 2H),
NMR (400 MHz, CDCl3) d 8.03e7.84 (m, 2H), 7.44e7.41 (m, 2H),
7.41e7.31 (m, 5H), 6.37 (s, 1H), 4.73e4.62 (m, 2H), 3.90 (s, 3H),
2.22e1.98 (m, 2H), 1.33e1.21 (m, 1H), 1.19e1.08 (m, 1H), 0.88 (t,
J ¼ 7.3 Hz, 3H).
7.40e7.32 (m, 5H), 7.21 (ddd, J ¼ 11.2, 5.3, 3.3 Hz, 3H), 7.10e7.06 (m,
1H), 7.03 (dd, J ¼ 6.9, 1.8 Hz, 1H), 4.80e4.71 (m, 2H), 3.90 (s, 3H),
3.13 (ddd, J ¼ 14.1, 9.3, 6.7 Hz, 1H), 2.96 (ddd, J ¼ 13.4, 9.5, 6.7 Hz,
1H), 1.78e1.67 (m, 1H), 1.51e1.38 (m, 1H).
4.2.27. Methyl 4-((4-benzyl-2,5-dioxo-4-phenylimidazolidin-1-yl)
methyl)benzoate (27)
4.2.33. Methyl 4-((4-(naphthalen-1-ylmethyl)-2,5-dioxo-4-
phenylimidazolidin-1-yl) methyl) benzoate (33)
The title compound was synthesized from compound 24 and
(bromomethyl)benzene in a manner similar to that described for
the preparation of compound 4. White solid, yield: 91%, mp
The title compound was synthesized from compound 24 and 1-
(bromomethyl)naphthalene in a manner similar to that described for
the preparation of compound 4. White solid, yield: 82%, mp
172e174 ꢀC. 1H NMR (400 MHz, DMSO‑d6)
d 9.22 (s, 1H), 7.77e7.66
(m, 4H), 7.52e7.43 (m, 2H), 7.39 (s, 1H), 7.33e7.20 (m, 5H), 6.75 (d,
J ¼ 8.3 Hz, 2H), 4.40 (d, J ¼ 16.1 Hz,1H), 4.32 (d, J ¼ 16.1 Hz,1H), 3.84
(s, 3H), 3.57 (d, J ¼ 13.5 Hz, 1H), 3.07 (d, J ¼ 13.6 Hz, 1H).
167e169 ꢀC. 1H NMR (400 MHz, DMSO‑d6)
d 9.26 (s, 1H), 8.33e8.18
(d, J ¼ 7.2 Hz, 1H), 7.93e7.88 (m, 1H), 7.87e7.78 (m, 3H), 7.60e7.54
(m, 2H), 7.55e7.45 (m, 5H), 7.45e7.34 (m, 2H), 6.50 (d, J ¼ 8.2 Hz,
2H), 4.32 (d, J ¼ 16.0 Hz, 1H), 4.22 (d, J ¼ 13.6 Hz, 2H), 3.85 (s, 3H),
3.52 (d, J ¼ 14.2 Hz, 1H).
4.2.28. Methyl 4-((4-(4-chlorobenzyl)-2,5-dioxo-4-
phenylimidazolidin-1-yl)methyl) benzoate (28)
The title compound was synthesized from compound 24 and 1-
4.2.34. Methyl 4-((4-(naphthalen-2-ylmethyl)-2,5-dioxo-4-
phenylimidazolidin-1-yl) methyl)benzoate (34)
(bromomethyl)-4-chlorobenzene in
described for the preparation of compound 4. White solid, yield:
80%, mp 184e186 ꢀC. 1H NMR (400 MHz, DMSO‑d6)
9.23 (s, 1H),
a manner similar to that
The title compound was synthesized from compound 24 and 2-
(bromomethyl)naphthalene in a manner similar to that described for
the preparation of compound 4. White solid, yield: 85%, mp
d
7.79 (d, J ¼ 8.3 Hz, 2H), 7.72e7.66 (m, 2H), 7.47 (td, J ¼ 7.3, 6.3,
1.4 Hz, 2H), 7.42e7.37 (m, 1H), 7.25 (d, J ¼ 8.0 Hz, 2H), 7.20 (d,
J ¼ 8.0 Hz, 2H), 6.80 (d, J ¼ 8.2 Hz, 2H), 4.38 (d, J ¼ 15.3 Hz, 2H), 3.84
(s, 3H), 3.55 (d, J ¼ 13.5 Hz, 1H), 3.05 (d, J ¼ 13.6 Hz, 1H).
233e235 ꢀC. 1H NMR (400 MHz, DMSO‑d6)
d 9.39 (s, 1H), 7.90 (d,
J ¼ 8.1 Hz, 1H), 7.85e7.69 (m, 5H), 7.55 (ddd, J ¼ 8.2, 6.8, 1.3 Hz, 1H),
7.52e7.47 (m, 3H), 7.43e7.40 (m, 2H), 7.13 (d, J ¼ 8.3 Hz, 2H), 6.44
(d, J ¼ 8.2 Hz, 2H), 4.42e4.23 (m, 2H), 3.82 (s, 3H), 3.74 (d,
J ¼ 13.5 Hz, 1H), 3.25 (d, J ¼ 13.5 Hz, 1H).
4.2.29. Methyl 4-((4-(4-methylbenzyl)-2,5-dioxo-4-
phenylimidazolidin-1-yl)methyl) benzoate (29)
The title compound was synthesized from compound 24 and 1-
(bromomethyl)-4-methylbenzene in a manner similar to that
described for the preparation of compound 4. White solid, yield:
4.2.35. N-hydroxy-4-((4-methyl-2,5-dioxo-4-phenylimidazolidin-
1-yl)methyl)benzamide (35)
The title compound was synthesized from compound 25 in a
manner similar to that described for the preparation of compound
13. White solid, yield: 59%, mp > 300 ꢀC. 1H NMR (400 MHz,
86%, mp 199e201 ꢀC. 1H NMR (400 MHz, DMSO‑d6)
d 9.20 (s, 1H),
7.77e7.64 (m, 4H), 7.46 (dd, J ¼ 8.4, 6.8 Hz, 2H), 7.42e7.35 (m, 1H),
7.09 (d, J ¼ 7.8 Hz, 2H), 7.01 (d, J ¼ 7.8 Hz, 2H), 6.75 (d, J ¼ 8.1 Hz,
2H), 4.41 (d, J ¼ 16.0 Hz, 1H), 4.32 (d, J ¼ 16.0 Hz, 1H), 3.85 (s, 3H),
3.50 (d, J ¼ 13.6 Hz, 1H), 3.00 (d, J ¼ 13.5 Hz, 1H), 2.29 (s, 3H).
DMSO‑d6)
d
11.16 (s, 1H), 9.07 (s, 1H), 9.02 (s, 1H), 7.68 (d, J ¼ 8.2 Hz,
2H), 7.48 (dt, J ¼ 6.4, 1.4 Hz, 2H), 7.44e7.38 (m, 2H), 7.37e7.31 (m,
1H), 7.25 (d, J ¼ 8.2 Hz, 2H), 4.59 (s, 2H), 1.71 (s, 3H). 13C NMR
(101 MHz, DMSO)
d 175.63, 164.34, 155.86, 140.17, 139.90, 132.46,
4.2.30. Methyl 4-((4-(4-methoxybenzyl)-2,5-dioxo-4-
phenylimidazolidin-1-yl)methyl) benzoate (30)
129.09, 128.52, 127.67, 127.50, 125.84, 63.47, 41.40, 25.42. HRMS
(AP-ESI) m/z, calculated for C18H17N3O4, ([M þ H]þ): 340.1292,
found: 340.1291. HPLC tR ¼ 32.41 min (96.68% purity).
The title compound was synthesized from compound 24 and 1-
(bromomethyl)-4-methoxybenzene in a manner similar to that
described for the preparation of compound 4. White solid, yield:
4.2.36. 4-((2,5-dioxo-4-phenyl-4-propylimidazolidin-1-yl)methyl)-
N-hydroxybenzamide (36)
82%, mp 220e222 ꢀC. 1H NMR (400 MHz, DMSO‑d6)
d 9.24 (s, 1H),
7.69 (t, J ¼ 7.8 Hz, 4H), 7.46 (t, J ¼ 7.7 Hz, 2H), 7.41e7.36 (m, 1H), 7.15
(d, J ¼ 8.5 Hz, 2H), 6.79 (d, J ¼ 8.6 Hz, 2H), 6.67 (d, J ¼ 8.1 Hz, 2H),
4.43 (d, J ¼ 16.2 Hz,1H), 4.32 (d, J ¼ 16.1 Hz,1H), 3.85 (s, 3H), 3.75 (s,
3H), 3.48 (d, J ¼ 13.7 Hz, 1H), 3.00 (d, J ¼ 13.9 Hz, 1H).
The title compound was synthesized from compound 26 in a
manner similar to that described for the preparation of compound
13. White solid, yield: 52%, mp 148e150 ꢀC. 1H NMR (400 MHz,
DMSO‑d6)
d
11.18 (s, 1H), 9.14 (s, 1H), 9.03 (s, 1H), 7.69 (d, J ¼ 7.9 Hz,
2H), 7.52 (d, J ¼ 7.7 Hz, 2H), 7.40 (t, J ¼ 7.6 Hz, 2H), 7.33 (t, J ¼ 7.2 Hz,
1H), 7.26 (d, J ¼ 8.0 Hz, 2H), 4.59 (s, 2H), 2.05 (ddd, J ¼ 13.8, 10.2,
6.0 Hz, 1H), 1.95 (ddd, J ¼ 13.7, 10.7, 5.8 Hz, 1H), 1.16 (q, J ¼ 5.9 Hz,
4.2.31. Methyl 4-((4-(4-(tert-butyl)benzyl)-2,5-dioxo-4-
phenylimidazolidin-1-yl)methyl) benzoate (31)
The title compound was synthesized from compound 24 and 1-
(bromomethyl)-4-(tert-butyl)benzene in a manner similar to that
described for the preparation of compound 4. White solid, yield:
2H), 0.85 (t, J ¼ 7.3 Hz, 3H). 13C NMR (101 MHz, DMSO)
d 175.01,
164.41, 156.19, 140.15, 139.18, 132.47, 129.03, 128.42, 127.73, 127.65,
125.84, 67.12, 41.44, 40.85, 17.23, 14.13. HRMS (AP-ESI) m/z, calcu-
lated for C20H21N3O4, ([M þ H]þ): 368.1605, found: 368.1602. HPLC
tR ¼ 9.52 min (96.96% purity).
94%, mp 199e201 ꢀC. 1H NMR (400 MHz, DMSO‑d6)
d 9.21 (s, 1H),
7.74 (d, J ¼ 8.3 Hz, 2H), 7.69 (d, J ¼ 8.4 Hz, 2H), 7.52e7.44 (m, 2H),
7.44e7.35 (m, 1H), 7.24 (d, J ¼ 8.4 Hz, 2H), 7.13 (d, J ¼ 8.3 Hz, 2H),
6.85 (d, J ¼ 8.2 Hz, 2H), 4.44 (d, J ¼ 16.0 Hz, 1H), 4.34 (d, J ¼ 16.0 Hz,
1H), 3.84 (s, 3H), 3.54 (d, J ¼ 13.7 Hz, 1H), 3.00 (d, J ¼ 13.7 Hz, 1H),
1.26 (s, 9H).
4.2.37. 4-((4-benzyl-2,5-dioxo-4-phenylimidazolidin-1-yl)methyl)-
N-hydroxybenzamide (37)
The title compound was synthesized from compound 27 in a
manner similar to that described for the preparation of compound
13. White solid, yield: 60%, mp 208e210 ꢀC. 1H NMR (400 MHz,
4.2.32. Methyl 4-((2,5-dioxo-4-phenethyl-4-phenylimidazolidin-1-
yl)methyl)benzoate (32)
DMSO‑d6) d 11.18 (s, 1H), 9.23 (s, 1H), 9.01 (s, 1H), 7.71e7.67 (m, 2H),
The title compound was synthesized from compound 24 and (2-
bromoethyl)benzene in a manner similar to that described for the
preparation of compound 4. White solid, yield: 72%, colorless oil. 1H
7.52 (d, J ¼ 8.1 Hz, 2H), 7.46 (t, J ¼ 7.5 Hz, 2H), 7.42e7.36 (m, 1H),
7.34e7.18 (m, 5H), 6.67 (d, J ¼ 8.1 Hz, 2H), 4.40e4.22 (m, 2H), 3.56
(d, J ¼ 13.5 Hz,1H), 3.07 (dd, J ¼ 13.7, 3.0 Hz,1H). 13C NMR (101 MHz,
12

T. Liang, J. Xue, Z. Yao et al.
European Journal of Medicinal Chemistry 221 (2021) 113526
DMSO)
d
173.98, 164.40, 155.53, 139.68, 139.54, 134.88, 131.93,
472.2230. HPLC tR ¼ 29.40 min (98.75% purity).
130.97, 129.79, 129.09, 128.60, 127.69, 127.41, 126.81, 126.12, 68.09,
44.15, 41.16. HRMS (AP-ESI) m/z, calculated for C24H21N3O4, ([M þ
H]þ): 416.1605, found: 416.1603. HPLC tR ¼ 15.07 min (99.67%
purity).
4.2.42. 4-((2,5-dioxo-4-phenethyl-4-phenylimidazolidin-1-yl)
methyl)-N-hydroxybenzamide (42)
The title compound was synthesized from compound 32 in a
manner similar to that described for the preparation of compound
13. White solid, yield: 56%, mp 132e134 ꢀC. 1H NMR (400 MHz,
4.2.38. 4-((4-(4-chlorobenzyl)-2,5-dioxo-4-phenylimidazolidin-1-
yl)methyl)-N- hydroxybenzamide (38)
The title compound was synthesized from compound 28 in a
manner similar to that described for the preparation of compound
13. White solid, yield: 52%, mp 220e222 ꢀC. 1H NMR (400 MHz,
DMSO‑d6)
d
11.15 (s, 1H), 9.27 (s, 1H), 9.01 (s, 1H), 7.75 (d, J ¼ 1.8 Hz,
1H), 7.68 (dd, J ¼ 8.3, 1.9 Hz, 2H), 7.57 (d, J ¼ 7.8 Hz, 2H), 7.45e7.40
(m, 2H), 7.36 (dd, J ¼ 8.0, 2.8 Hz, 1H), 7.25e7.29 (m, 4H), 7.19 (d,
J ¼ 7.0 Hz, 1H), 7.17e7.12 (m, 2H), 4.58 (s, 2H), 2.44 (q, J ¼ 4.8, 4.1 Hz,
2H), 2.36 (dd, J ¼ 10.2, 6.3 Hz, 1H), 2.33e2.21 (m, 1H). 13C NMR
DMSO‑d6)
d
11.13 (s, 1H), 9.19 (s, 1H), 9.00 (s, 1H), 7.66 (d, J ¼ 7.7 Hz,
2H), 7.57 (d, J ¼ 8.1 Hz, 2H), 7.46 (t, J ¼ 7.6 Hz, 2H), 7.39 (t, J ¼ 7.1 Hz,
1H), 7.27 (d, J ¼ 8.3 Hz, 2H), 7.19 (d, J ¼ 8.3 Hz, 2H), 6.78 (d,
J ¼ 8.1 Hz, 2H), 4.45e4.26 (m, 2H), 3.56 (d, J ¼ 13.5 Hz, 1H), 3.05 (d,
(101 MHz, DMSO) d 174.75, 164.40, 156.23, 140.87, 140.14, 138.90,
132.52, 129.15, 128.92, 128.60, 127.88, 127.75, 127.68, 126.56, 125.92,
66.94, 41.54, 35.37, 30.04. HRMS (AP-ESI) m/z, calculated for
J ¼ 13.6 Hz, 1H). 13C NMR (101 MHz, DMSO)
d
173.84, 164.59, 155.57,
C
25H23N3O4, ([M
þ
H]þ): 430.1761, found: 430.1759. HPLC
139.45, 139.29, 133.79, 132.65, 132.31, 129.14, 128.75, 128.58, 127.41,
tR ¼ 9.15 min (99.21% purity).
127.17, 126.10, 67.85, 43.29, 41.29. HRMS (AP-ESI) m/z, calculated for
C
24H20ClN3O4, ([M
þ
H]þ): 450.1215, found: 450.1211. HPLC
4.2.43. N-hydroxy-4-((4-(naphthalen-1-ylmethyl)-2,5-dioxo-4-
phenylimidazolidin-1-yl)methyl) benzamide (43)
The title compound was synthesized from compound 33 in a
manner similar to that described for the preparation of compound
13. White solid, yield: 67%, mp 162e164 ꢀC. 1H NMR (400 MHz,
tR ¼ 22.90 min (98.94% purity).
4.2.39. N-hydroxy-4-((4-(4-methylbenzyl)-2,5-dioxo-4-
phenylimidazolidin-1-yl)methyl) benzamide (39)
The title compound was synthesized from compound 29 in a
manner similar to that described for the preparation of compound
13. White solid, yield: 55%, mp 211e213 ꢀC. 1H NMR (400 MHz,
DMSO‑d6)
d
11.13 (s, 1H), 9.20 (s, 1H), 9.00 (s, 1H), 8.25 (d, J ¼ 7.8 Hz,
1H), 7.91 (dd, J ¼ 7.0, 2.3 Hz, 1H), 7.88 (s, 1H), 7.83e7.77 (m, 2H),
7.56e7.46 (m, 4H), 7.46e7.33 (m, 5H), 6.47 (d, J ¼ 8.0 Hz, 2H), 4.29
DMSO‑d6)
d
11.18 (s, 1H), 9.21 (s, 1H), 9.01 (s, 1H), 7.70e7.66 (m, 2H),
(d, J ¼ 15.9 Hz, 1H), 4.24e4.12 (m, 2H), 3.56 (d, J ¼ 14.3 Hz, 1H). 13
C
7.52 (d, J ¼ 8.2 Hz, 2H), 7.49e7.43 (m, 2H), 7.42e7.35 (m, 1H), 7.09
(d, J ¼ 8.8 Hz, 2H), 7.01 (d, J ¼ 7.9 Hz, 2H), 6.72 (d, J ¼ 8.0 Hz, 2H),
4.32 (q, J ¼ 15.8 Hz, 2H), 3.50 (d, J ¼ 13.5 Hz,1H), 3.00 (d, J ¼ 13.6 Hz,
NMR (101 MHz, DMSO) d 174.13, 164.39, 155.55, 139.75, 139.39,
133.92, 132.73, 131.93, 131.35, 129.61, 129.15, 128.81, 128.73, 128.40,
127.28, 126.64, 126.35, 126.28, 126.01, 125.55, 125.27, 68.35, 55.38,
41.14. HRMS (AP-ESI) m/z, calculated for C28H23N3O4, ([M þ H]þ):
466.1761, found: 466.1761. HPLC tR ¼ 8.68 min (97.61% purity).
1H), 2.29 (s, 3H). 13C NMR (101 MHz, DMSO)
d 174.01,164.45,155.55,
139.74, 139.52, 136.65, 131.95, 131.74, 130.81, 129.15, 129.05, 128.59,
127.30, 127.02, 126.11, 68.11, 43.80, 41.18, 21.27. HRMS (AP-ESI) m/z,
calculated for C25H23N3O4, ([M þ H]þ): 430.1761, found: 430.1759.
HPLC tR ¼ 23.07 min (96.80% purity).
4.2.44. N-hydroxy-4-((4-(naphthalen-2-ylmethyl)-2,5-dioxo-4-
phenylimidazolidin-1-yl)methyl) benzamide (44)
The title compound was synthesized from compound 34 in a
manner similar to that described for the preparation of compound
13. White solid, yield: 69%, mp 248e250 ꢀC. 1H NMR (400 MHz,
4.2.40. N-hydroxy-4-((4-(4-methoxybenzyl)-2,5-dioxo-4-
phenylimidazolidin-1-yl)methyl) benzamide (40)
The title compound was synthesized from compound 30 in a
manner similar to that described for the preparation of compound
13. White solid, yield: 56%, mp 215e217 ꢀC. 1H NMR (400 MHz,
DMSO‑d6) d 11.00 (s, 1H), 9.30 (s, 1H), 8.97 (s, 1H), 7.88 (s, 1H),
7.82e7.70 (m, 5H), 7.56e7.44 (m, 4H), 7.43e7.31 (m, 2H), 7.15 (d,
J ¼ 8.1 Hz, 2H), 6.58 (d, J ¼ 8.0 Hz, 2H), 4.28 (dd, J ¼ 8.3, 1.7 Hz, 2H),
3.75 (d, J ¼ 13.5 Hz,1H), 3.25 (d, J ¼ 13.5 Hz,1H). 13C NMR (101 MHz,
DMSO‑d6)
d
11.12 (s, 1H), 9.16 (s, 1H), 9.00 (s, 1H), 7.67 (d, J ¼ 7.6 Hz,
2H), 7.51 (d, J ¼ 8.0 Hz, 2H), 7.45 (t, J ¼ 7.5 Hz, 2H), 7.38 (t, J ¼ 7.2 Hz,
1H), 7.12 (d, J ¼ 8.4 Hz, 2H), 6.79 (d, J ¼ 8.2 Hz, 2H), 6.71 (d,
J ¼ 8.0 Hz, 2H), 4.33 (q, J ¼ 15.9 Hz, 2H), 3.74 (s, 3H), 3.49 (d,
J ¼ 13.7 Hz, 1H), 2.99 (d, J ¼ 13.7 Hz, 1H). 13C NMR (101 MHz, DMSO)
DMSO) d 174.07, 164.47, 155.59, 139.61, 139.30, 133.20, 132.62,
132.58, 132.11, 129.71, 129.51, 129.15, 128.95, 128.72, 128.09, 127.99,
127.21, 126.75, 126.64, 126.42, 126.12, 68.14, 44.33, 41.23. HRMS
(AP-ESI) m/z, calculated for C28H23N3O4, ([M þ H]þ): 466.1761,
found:466.1760. HPLC tR ¼ 7.24 min (99.63% purity).
d
174.07, 164.59, 158.93, 155.67, 139.67, 139.55, 132.09, 132.01,
129.09, 128.63, 127.33, 126.96, 126.66, 126.08, 113.98, 68.22, 55.37,
43.45, 41.19. HRMS (AP-ESI) m/z, calculated for C25H23N3O5, ([M þ
H]þ): 446.1711, found: 446.1709. HPLC tR ¼ 30.37 min (99.53%
purity).
4.2.45. Methyl 4-((3-(1-methoxy-1-oxo-3-phenylpropan-2-yl)
ureido)methyl)benzoate (45)
The title compound was synthesized from methyl phenyl-
alaninate and methyl 4-(aminomethyl)benzoate in a manner similar
to that described for the preparation of compound 22. White solid,
4.2.41. 4-((4-(4-(tert-butyl)benzyl)-2,5-dioxo-4-
phenylimidazolidin-1-yl)methyl) -N-hydroxybenzamide (41)
The title compound was synthesized from compound 31 in a
manner similar to that described for the preparation of compound
13. White solid, yield: 65%, mp 200e202 ꢀC. 1H NMR (400 MHz,
yield: 71%, mp 132e134 ꢀC. 1H NMR (400 MHz, DMSO‑d6)
d 7.89 (d,
J ¼ 8.1 Hz, 2H), 7.36e7.22 (m, 5H), 7.21e7.14 (m, 2H), 6.65 (t,
J ¼ 6.1 Hz, 1H), 6.40 (d, J ¼ 8.3 Hz, 1H), 4.44 (td, J ¼ 8.2, 5.4 Hz, 1H),
4.25 (d, J ¼ 6.1 Hz, 2H), 3.84 (s, 3H), 3.61 (s, 3H), 3.00 (dd, J ¼ 13.7,
5.5 Hz, 1H), 2.90 (dd, J ¼ 13.7, 6.3 Hz, 1H).
DMSO‑d6) d 11.13 (s, 1H), 9.17 (s, 1H), 8.99 (s, 1H), 7.70e7.65 (m, 2H),
7.55 (d, J ¼ 8.1 Hz, 2H), 7.51e7.42 (m, 2H), 7.41e7.36 (m, 1H), 7.26 (d,
J ¼ 7.8 Hz, 2H), 7.12 (d, J ¼ 7.8 Hz, 2H), 6.83 (d, J ¼ 8.2 Hz, 2H), 4.34
(d, J ¼ 15.6 Hz, 2H), 3.53 (d, J ¼ 13.6 Hz,1H), 3.02 (d, J ¼ 13.7 Hz, 1H),
4.2.46. Methyl 4-((4-benzyl-2,5-dioxoimidazolidin-1-yl)methyl)
benzoate (46)
The solution of compound 45 (66.67 g, 180 mmol) in CH2Cl2 was
mixed with CH3ONa (19.45 g, 360 mmol) for 30 min. Filter and
wash the filtrate with 1 M HCl. Dry the organic solvent over with
anhydrous magnesium sulfate and then it was removed under
1.27 (s, 9H). 13C NMR (101 MHz, DMSO)
d
174.21, 164.38, 155.59,
149.84, 139.78, 139.57, 132.10, 131.88, 130.64, 129.10, 128.63, 127.41,
127.13, 126.03, 125.32, 67.98, 43.79, 41.29, 34.65, 31.65. HRMS (AP-
ESI) m/z, calculated for C28H29N3O4, ([M þ H]þ): 472.2231, found:
13

T. Liang, J. Xue, Z. Yao et al.
European Journal of Medicinal Chemistry 221 (2021) 113526
reduced pressure to give a white solid, yield: 88%, mp 136e138 ꢀC.
2H), 5.11 (d, J ¼ 15.0 Hz, 1H), 4.62 (d, J ¼ 15.1 Hz, 1H), 4.51 (d,
J ¼ 15.1 Hz, 1H), 4.03 (s, 1H), 3.96 (d, J ¼ 15.0 Hz, 1H), 3.91 (s, 3H),
3.80 (s, 3H), 3.20e3.07 (m, 2H).
1H NMR (400 MHz, DMSO‑d6)
d
8.43 (s, 1H), 7.75 (d, J ¼ 8.3 Hz, 2H),
7.26 (dd, J ¼ 5.0, 1.9 Hz, 3H), 7.21e7.13 (m, 2H), 6.85 (d, J ¼ 8.2 Hz,
2H), 4.58e4.46 (m, 2H), 4.38 (d, J ¼ 16.1 Hz, 1H), 3.84 (s, 3H), 3.01
(dd, J ¼ 4.7, 2.4 Hz, 2H).
4.2.53. Methyl 4-((4-benzyl-3-(4-(tert-butyl)benzyl)-2,5-
dioxoimidazolidin-1-yl)methyl) benzoate (53)
The title compound was synthesized from compound 46 and 1-
(bromomethyl)-4-(tert-butyl)benzene in a manner similar to that
described for the preparation of compound 4. Colorless oil, yield:
4.2.47. Methyl 4-((4-benzyl-3-methyl-2,5-dioxoimidazolidin-1-yl)
methyl)benzoate (47)
The title compound was synthesized from compound 46 and
iodomethane in a manner similar to that described for the prepa-
ration of compound 4. White solid, yield: 93%, mp 62e64 ꢀC. 1H
93%. 1H NMR (400 MHz, CDCl3)
d
7.86 (d, J ¼ 8.3 Hz, 2H), 7.36 (d,
J ¼ 8.2 Hz, 2H), 7.24e7.20 (m, 1H), 7.19e7.13 (m, 2H), 7.11 (d,
J ¼ 8.2 Hz, 2H), 7.02 (dd, J ¼ 10.2, 7.7 Hz, 4H), 5.14 (d, J ¼ 15.0 Hz,1H),
4.62 (d, J ¼ 15.1 Hz, 1H), 4.51 (d, J ¼ 15.0 Hz, 1H), 4.06 (t, J ¼ 4.4 Hz,
1H), 4.01 (d, J ¼ 15.1 Hz, 1H), 3.92 (s, 3H), 3.20e3.07 (m, 2H), 1.32 (s,
9H).
NMR (400 MHz, CDCl3)
d
7.85 (dd, J ¼ 8.8, 2.5 Hz, 2H), 7.23e7.14 (m,
3H), 7.05 (J ¼ 8.1 Hz, 2H), 7.00 (J ¼ 8.2 Hz, 2H), 4.58 (d, J ¼ 15.1 Hz,
1H), 4.48 (d, J ¼ 15.1 Hz, 1H), 4.16 (t, J ¼ 4.4 Hz, 1H), 3.91 (s, 3H), 3.18
(d, J ¼ 4.4 Hz, 2H), 2.99 (s, 3H).
4.2.48. Methyl 4-((4-benzyl-2,5-dioxo-3-propylimidazolidin-1-yl)
methyl)benzoate (48)
The title compound was synthesized from compound 46 and 1-
iodopropane in a manner similar to that described for the prepa-
ration of compound 4. Colorless oil, yield: 56%. 1H NMR (400 MHz,
4.2.54. Methyl 4-((4-benzyl-2,5-dioxo-3-phenethylimidazolidin-1-
yl)methyl)benzoate (54)
The title compound was synthesized from compound 46 and (2-
bromoethyl)benzene in a manner similar to that described for the
preparation of compound 4. Colorless oil, yield: 61%. 1H NMR
CDCl3)
d
7.84 (d, J ¼ 7.8 Hz, 2H), 7.22e7.12 (m, 3H), 7.05 (d,
J ¼ 4.4 Hz, 2H), 6.99 (d, J ¼ 8.2 Hz, 2H), 4.58 (d, J ¼ 15.0 Hz, 1H), 4.47
(d, J ¼ 15.1 Hz, 1H), 4.27 (t, J ¼ 4.3 Hz, 1H), 3.91 (s, 3H), 3.77 (ddd,
J ¼ 14.1, 8.8, 7.3 Hz, 1H), 3.16 (d, J ¼ 4.4 Hz, 2H), 3.04 (ddd, J ¼ 13.9,
8.5, 5.1 Hz, 1H), 1.62e1.48 (m, 2H), 0.90 (t, J ¼ 7.4 Hz, 3H).
(400 MHz, CDCl3)
d
7.87 (d, J ¼ 8.1 Hz, 2H), 7.27 (d, J ¼ 2.3 Hz, 2H),
7.17 (q, J ¼ 6.9 Hz, 4H), 7.12e7.08 (m, 2H), 7.05e6.98 (m, 4H), 4.56
(d, J ¼ 15.0 Hz, 1H), 4.48 (d, J ¼ 15.0 Hz, 1H), 4.03e4.10 (m, 1H), 3.92
(s, 3H), 3.30e3.15 (m, 2H), 3.07 (qd, J ¼ 14.5, 4.5 Hz, 2H), 2.97e2.77
(m, 2H).
4.2.49. Methyl 4-((3,4-dibenzyl-2,5-dioxoimidazolidin-1-yl)
methyl)benzoate (49)
The title compound was synthesized from compound 46 and
(bromomethyl)benzene in a manner similar to that described for
the preparation of compound 4. Colorless oil, yield: 95%. 1H NMR
4.2.55. Methyl 4-((4-benzyl-3-(naphthalen-1-ylmethyl)-2,5-
dioxoimidazolidin-1-yl) methyl)benzoate (55)
The title compound was synthesized from compound 46 and 1-
(bromomethyl)naphthalene in a manner similar to that described for
the preparation of compound 4. White solid, yield: 88%, mp
(400 MHz, CDCl3)
d
7.87 (d, J ¼ 8.3 Hz, 2H), 7.37e7.31 (m, 3H),
7.24e7.21 (m, 1H), 7.20e7.13 (m, 4H), 7.07e7.00 (m, 4H), 5.17 (d,
J ¼ 15.1 Hz, 1H), 4.64 (d, J ¼ 15.0 Hz, 1H), 4.53 (d, J ¼ 15.0 Hz, 1H),
4.08e3.99 (m, 2H), 3.92 (s, 3H), 3.20e3.07 (m, 2H).
62e64 ꢀC. 1H NMR (400 MHz, CDCl3)
d 8.05e8.01 (m,1H), 7.95e7.84
(m, 4H), 7.52 (ddt, J ¼ 9.9, 7.4, 3.6 Hz, 3H), 7.43 (dd, J ¼ 8.2, 7.0 Hz,
1H), 7.32 (d, J ¼ 6.9 Hz, 1H), 7.22 (d, J ¼ 7.3 Hz, 1H), 7.16 (dd, J ¼ 8.4,
6.6 Hz, 2H), 7.03 (dd, J ¼ 7.6, 5.1 Hz, 3H), 5.72 (d, J ¼ 14.9 Hz, 1H),
4.67e4.49 (m, 3H), 3.93 (s, 3H), 3.78 (t, J ¼ 4.3 Hz, 1H), 3.22e3.04
(m, 2H).
4.2.50. Methyl 4-((4-benzyl-3-(4-chlorobenzyl)-2,5-
dioxoimidazolidin-1-yl)methyl)benzoate (50)
The title compound was synthesized from compound 46 and 1-
(bromomethyl)-4-chlorobenzene in
described for the preparation of compound 4. Colorless oil, yield:
95%. 1H NMR (400 MHz, CDCl3)
J ¼ 8.3 Hz, 2H), 7.25e7.22 (m, 1H), 7.18 (dd, J ¼ 8.2, 6.4 Hz, 2H), 7.04
(td, J ¼ 8.2, 1.8 Hz, 6H), 5.09 (d, J ¼ 15.2 Hz, 1H), 4.64 (d, J ¼ 15.1 Hz,
1H), 4.54 (d, J ¼ 14.9 Hz, 1H), 4.07e3.95 (m, 2H), 3.92 (s, 3H), 3.13
(qd, J ¼ 14.5, 4.6 Hz, 2H).
a manner similar to that
4.2.56. Methyl 4-((4-benzyl-3-(naphthalen-2-ylmethyl)-2,5-
dioxoimidazolidin-1-yl) methyl)benzoate (56)
The title compound was synthesized from compound 46 and 2-
(bromomethyl)naphthalene in a manner similar to that described for
the preparation of compound 4. White solid, yield: 65%, mp
d
7.88 (d, J ¼ 8.3 Hz, 2H), 7.30 (d,
76e77 ꢀC. 1H NMR (400 MHz, CDCl3)
d
7.88 (d, J ¼ 8.2 Hz, 2H), 7.82
(dd, J ¼ 9.0, 5.3 Hz, 3H), 7.78e7.74 (m, 1H), 7.50 (ddd, J ¼ 9.9, 5.2,
2.8 Hz, 3H), 7.28 (d, J ¼ 1.8 Hz, 1H), 7.19 (dd, J ¼ 8.3, 6.6 Hz, 2H),
7.11e7.03 (m, 4H), 5.33 (d, J ¼ 15.1 Hz, 1H), 4.66 (d, J ¼ 15.0 Hz, 1H),
4.56 (d, J ¼ 15.0 Hz, 1H), 4.17 (d, J ¼ 15.1 Hz, 1H), 4.05 (t, J ¼ 4.6 Hz,
1H), 3.92 (s, 3H), 3.21 (dd, J ¼ 14.5, 4.2 Hz, 1H), 3.10 (dd, J ¼ 14.5,
5.0 Hz, 1H).
4.2.51. Methyl 4-((4-benzyl-3-(4-methylbenzyl)-2,5-
dioxoimidazolidin-1-yl)methyl) benzoate (51)
The title compound was synthesized from compound 46 and 1-
(bromomethyl)-4-methylbenzene in a manner similar to that
described for the preparation of compound 4. Colorless oil, yield:
93%. 1H NMR (400 MHz, CDCl3)
(m, 1H), 7.20e7.11 (m, 5H), 7.03 (ddd, J ¼ 13.7, 8.1, 2.8 Hz, 5H), 5.14
(d, J ¼ 15.0 Hz, 1H), 4.62 (d, J ¼ 14.8 Hz, 1H), 4.51 (d, J ¼ 15.0 Hz, 1H),
4.10e3.95 (m, 2H), 3.91 (s, 3H), 3.22e3.05 (m, 2H), 2.34 (s, 3H).
d
7.86 (d, J ¼ 8.3 Hz, 2H), 7.30e7.20
4.2.57. Methyl 4-((4-benzyl-2,5-dioxo-3-(pyridin-2-ylmethyl)
imidazolidin-1-yl)methyl) benzoate (57)
The title compound was synthesized from compound 46 and 2-
(bromomethyl)pyridine in a manner similar to that described for the
preparation of compound 4. White solid, yield: 90%, mp
4.2.52. Methyl 4-((4-benzyl-3-(4-methoxybenzyl)-2,5-
dioxoimidazolidin-1-yl)methyl) benzoate (52)
The title compound was synthesized from compound 46 and 1-
(bromomethyl)-4-methoxybenzene in a manner similar to that
described for the preparation of compound 4. Colorless oil, yield:
171e173 ꢀC. 1H NMR (400 MHz, DMSO‑d6)
d
8.55 (d, J ¼ 4.8 Hz, 1H),
7.86e7.73 (m, 2H), 7.46e7.19 (m, 6H), 7.14e7.09 (m, 2H), 6.87 (d,
J ¼ 8.3 Hz, 2H), 4.93 (d, J ¼ 16.6 Hz, 1H), 4.66e4.55 (m, 3H), 4.46 (d,
J ¼ 16.3 Hz, 1H), 3.86 (s, 3H), 3.28 (dd, J ¼ 14.4, 4.7 Hz, 1H), 3.09 (dd,
J ¼ 14.4, 4.0 Hz, 1H).
84%. 1H NMR (400 MHz, CDCl3)
(m, 1H), 7.20e7.14 (m, 2H), 7.11e6.98 (m, 6H), 6.86 (d, J ¼ 8.6 Hz,
d
7.86 (d, J ¼ 8.3 Hz, 2H), 7.24e7.21
14

T. Liang, J. Xue, Z. Yao et al.
European Journal of Medicinal Chemistry 221 (2021) 113526
4.2.58. Methyl 4-((4-benzyl-2,5-dioxo-3-(pyridin-3-ylmethyl)
imidazolidin-1-yl)methyl) benzoate (58)
130.23, 129.17, 128.77, 128.56, 128.13, 127.50, 127.44, 126.93, 60.26,
44.66, 41.51, 33.66. HRMS (AP-ESI) m/z, calculated for C25H23N3O4,
([M þ H]þ): 430.1761, found: 430.1759. HPLC tR ¼ 27.15 min (99.72%
purity).
The title compound was synthesized from compound 46 and 3-
(bromomethyl)pyridine in a manner similar to that described for the
preparation of compound 4. White solid, yield: 75%, mp
172e174 ꢀC. 1H NMR (400 MHz, DMSO‑d6)
d
8.62e8.49 (m, 2H),
4.2.63. 4-((4-benzyl-3-(4-chlorobenzyl)-2,5-dioxoimidazolidin-1-
yl)methyl)-N- hydroxybenzamide (63)
The title compound was synthesized from compound 50 in a
manner similar to that described for the preparation of compound
13. White solid, yield: 76%, mp 171e173 ꢀC. 1H NMR (400 MHz,
7.79e7.72 (m, 2H), 7.45e7.37 (m, 1H), 7.31e7.15 (m, 4H), 7.07 (d,
J ¼ 7.8 Hz, 2H), 6.82 (d, J ¼ 8.3 Hz, 2H), 4.87 (d, J ¼ 15.8 Hz, 1H),
4.64e4.37 (m, 4H), 3.86 (s, 3H), 3.32 (dd, J ¼ 14.5, 4.0 Hz, 1H), 3.09
(dd, J ¼ 14.6, 3.9 Hz, 1H).
DMSO‑d6)
d 11.19 (s, 1H), 9.03 (s, 1H), 7.64e7.53 (m, 2H), 7.42 (d,
4.2.59. Methyl 4-((4-benzyl-2,5-dioxo-3-(pyridin-4-ylmethyl)
imidazolidin-1-yl)methyl) benzoate (59)
J ¼ 8.2 Hz, 2H), 7.35 (d, J ¼ 8.4 Hz, 2H), 7.28 (t, J ¼ 7.2 Hz, 1H), 7.21 (t,
J ¼ 7.3 Hz, 2H), 7.06 (d, J ¼ 8.2 Hz, 2H), 6.75 (d, J ¼ 8.3, 2H), 4.83 (dd,
J ¼ 15.7, 1.7 Hz, 1H), 4.65e4.40 (m, 3H), 4.38 (t, J ¼ 4.2 Hz, 1H), 3.28
(dd, J ¼ 14.5, 4.6 Hz, 1H), 3.07 (dd, J ¼ 14.5, 3.9 Hz, 1H). 13C NMR
The title compound was synthesized from compound 46 and 4-
(bromomethyl)pyridine in a manner similar to that described for the
preparation of compound 4. White solid, yield: 85%, mp
(101 MHz, DMSO)
d 172.10, 164.42, 156.41, 139.54, 135.90, 134.99,
184e185 ꢀC. 1H NMR (400 MHz, DMSO‑d6)
d
8.58e8.53 (m, 2H), 7.76
132.78, 132.08, 130.44, 130.18, 129.07, 128.77, 127.50, 127.44, 126.94,
(d, J ¼ 8.3 Hz, 2H), 7.34e7.31 (m, 2H), 7.29e7.17 (m, 3H), 7.08 (d,
J ¼ 7.7 Hz, 2H), 6.85 (d, J ¼ 8.2 Hz, 2H), 4.85 (d, J ¼ 16.5 Hz, 1H), 4.59
(t, J ¼ 15.9 Hz, 2H), 4.51 (t, J ¼ 4.2 Hz, 1H), 4.45 (d, J ¼ 16.1 Hz, 1H),
3.85 (s, 3H), 3.30 (dd, J ¼ 14.5, 3.9 Hz, 1H), 3.08 (dd, J ¼ 14.5, 4.0 Hz,
1H).
60.30, 43.95, 41.53, 33.67. HRMS (AP-ESI) m/z, calculated for
C
25H22ClN3O4, ([M
þ
H]þ): 464.1372, found: 464.1371. HPLC
tR ¼ 14.02min (97.32% purity).
4.2.64. 4-((4-benzyl-3-(4-methylbenzyl)-2,5-dioxoimidazolidin-1-
yl)methyl)-N- hydroxybenzamide (64)
4.2.60. 4-((4-benzyl-3-methyl-2,5-dioxoimidazolidin-1-yl)methyl)-
N-hydroxybenzamide (60)
The title compound was synthesized from compound 47 in a
manner similar to that described for the preparation of compound
13. White solid, yield: 73%, mp 171e173 ꢀC. 1H NMR (400 MHz,
The title compound was synthesized from compound 51 in a
manner similar to that described for the preparation of compound
13. White solid, yield: 72%, mp 170e172 ꢀC. 1H NMR (400 MHz,
DMSO‑d6)
d
11.18 (s, 1H), 9.02 (s, 1H), 7.55 (dd, J ¼ 8.3, 1.9 Hz, 2H),
7.34e7.14 (m, 7H), 7.06 (d, J ¼ 7.1 Hz, 2H), 6.74 (d, J ¼ 8.0 Hz, 2H),
4.83 (d, J ¼ 15.4 Hz, 1H), 4.54 (d, J ¼ 15.9 Hz, 1H), 4.38 (dd, J ¼ 15.7,
7.5 Hz, 2H), 4.28 (t, J ¼ 4.2 Hz, 1H), 3.25 (dd, J ¼ 14.4, 4.7 Hz, 1H),
3.06 (dd, J ¼ 14.4, 3.9 Hz, 1H), 2.30 (s, 3H). 13C NMR (101 MHz,
DMSO‑d6)
d
11.17 (s, 1H), 9.01 (s, 1H), 7.53 (d, J ¼ 8.2 Hz, 2H),
7.34e7.19 (m, 3H), 7.09 (dt, J ¼ 6.6, 1.6 Hz, 2H), 6.74 (d, J ¼ 8.2 Hz,
2H), 4.50 (d, J ¼ 15.9 Hz, 1H), 4.45 (t, J ¼ 4.3 Hz, 1H), 4.34 (d,
J ¼ 15.9 Hz, 1H), 3.22 (dd, J ¼ 14.4, 4.7 Hz, 1H), 3.09 (dd, J ¼ 14.4,
DMSO)
d 172.08, 164.43, 156.33, 139.57, 137.37, 135.01, 133.70,
3.9 Hz,1H), 2.93 (s, 3H). 13C NMR (101 MHz, DMSO)
d
172.39,164.40,
132.08, 130.23, 129.73, 128.76, 128.60, 127.49, 127.43, 126.91, 60.05,
156.30, 139.67, 135.25, 132.01, 130.12, 128.76, 127.43, 127.34, 126.89,
44.36, 41.48, 33.63, 21.19. HRMS (AP-ESI) m/z, calculated for
62.35, 41.28, 33.76, 28.45. HRMS (AP-ESI) m/z, calculated for
C
26H25N3O4, ([M
þ
H]þ): 444.1918, found: 444.1916. HPLC
C
19H19N3O4, ([M
þ
H]þ): 354.1448, found: 354.1446. HPLC
tR ¼ 15.41 min (98.93% purity).
tR ¼ 17.85min (98.65% purity).
4.2.65. 4-((4-benzyl-3-(4-methoxybenzyl)-2,5-dioxoimidazolidin-
1-yl)methyl)-N- hydroxybenzamide (65)
The title compound was synthesized from compound 52 in a
manner similar to that described for the preparation of compound
13. White solid, yield: 67%, mp 166e168 ꢀC. 1H NMR (400 MHz,
4.2.61. 4-((4-benzyl-2,5-dioxo-3-propylimidazolidin-1-yl)methyl)-
N-hydroxybenzamide (61)
The title compound was synthesized from compound 48 in a
manner similar to that described for the preparation of compound
13. White solid, yield: 71%, mp 86e88 ꢀC. 1H NMR (400 MHz,
DMSO‑d6)
d
11.18 (s, 1H), 9.02 (s, 1H), 7.54 (d, J ¼ 8.2 Hz, 2H),
DMSO‑d6)
d
11.18 (s, 1H), 9.01 (s, 1H), 7.58e7.53 (m, 2H), 7.24 (ddd,
7.35e7.19 (m, 5H), 7.06 (d, J ¼ 7.1 Hz, 2H), 6.93 (d, J ¼ 8.0 Hz, 2H),
6.73 (d, J ¼ 8.0 Hz, 2H), 4.81 (d, J ¼ 15.2 Hz, 1H), 4.53 (d, J ¼ 15.9 Hz,
1H), 4.36 (dd, J ¼ 15.6, 13.1 Hz, 2H), 4.27 (t, J ¼ 4.2 Hz, 1H), 3.75 (s,
J ¼ 14.2, 7.8, 6.0 Hz, 3H), 7.09 (d, J ¼ 7.0 Hz, 2H), 6.76 (d, J ¼ 8.0 Hz,
2H), 4.58 (t, J ¼ 4.3 Hz, 1H), 4.51 (d, J ¼ 15.8 Hz, 1H), 4.36 (d,
J ¼ 15.9 Hz, 1H), 3.54 (dt, J ¼ 13.8, 8.0 Hz, 1H), 3.24 (dd, J ¼ 14.5,
4.6 Hz, 1H), 3.16 (ddd, J ¼ 13.8, 8.5, 4.9 Hz, 1H), 3.06 (dd, J ¼ 14.5,
4.0 Hz, 1H), 1.73e1.42 (m, 2H), 0.83 (t, J ¼ 7.3 Hz, 3H). 13C NMR
3H), 3.26 (dd, J ¼ 14.4, 4.6 Hz, 1H), 3.06 (dd, J ¼ 14.4, 3.8 Hz, 1H). 13
C
NMR (101 MHz, DMSO)
d 172.10, 164.29, 159.31, 156.32, 139.57,
135.02, 132.06,130.24,130.08,128.75,128.63,127.49,127.42, 126.89,
114.56, 59.98, 55.57, 44.10, 41.45, 33.63. HRMS (AP-ESI) m/z,
calculated for C26H25N3O5, ([M þ H]þ): 460.1867, found: 460.1875.
HPLC tR ¼ 29.74 min (96.87% purity).
(101 MHz, DMSO)
d 172.45, 164.45, 156.02, 139.70, 135.20, 132.03,
130.10, 128.75, 127.43, 127.40, 126.94, 59.83, 42.42, 41.32, 33.78,
20.78, 11.53. HRMS (AP-ESI) m/z, calculated for C21H23N3O4, ([M þ
H]þ): 382.1761, found: 382.1762. HPLC tR ¼ 9.74 min (99.10% purity).
4.2.66. 4-((4-benzyl-3-(4-(tert-butyl)benzyl)-2,5-
4.2.62. 4-((3,4-dibenzyl-2,5-dioxoimidazolidin-1-yl)methyl)-N-
hydroxybenzamide (62)
The title compound was synthesized from compound 49 in a
manner similar to that described for the preparation of compound
13. White solid, yield: 70%, mp 144e146 ꢀC. 1H NMR (400 MHz,
dioxoimidazolidin-1-yl)methyl)-N- hydroxybenzamide (66)
The title compound was synthesized from compound 53 in a
manner similar to that described for the preparation of compound
13. White solid, yield: 63%, mp 151e153 ꢀC. 1H NMR (400 MHz,
DMSO‑d6)
d
11.19 (s, 1H), 9.02 (s, 1H), 7.55 (dd, J ¼ 8.3, 2.6 Hz, 2H),
DMSO‑d6)
d
11.18 (s, 1H), 9.03 (s, 1H), 7.55 (dd, J ¼ 8.3, 2.4 Hz, 2H),
7.39 (d, J ¼ 8.0 Hz, 2H), 7.31e7.24 (m, 3H), 7.20 (t, J ¼ 7.5 Hz, 2H),
7.03 (d, J ¼ 6.9 Hz, 2H), 6.74 (d, J ¼ 8.3 Hz, 2H), 4.90e4.77 (m, 1H),
4.54 (d, J ¼ 15.9 Hz, 1H), 4.40 (dd, J ¼ 15.7, 10.3 Hz, 2H), 4.32 (t,
J ¼ 4.1 Hz, 1H), 3.27 (dd, J ¼ 14.4, 4.6 Hz, 1H), 3.07 (dd, J ¼ 14.5,
7.44e7.17 (m, 8H), 7.12e7.01 (d, J ¼ 7.8 Hz, 2H), 6.74 (d, J ¼ 8.0 Hz,
2H), 4.86 (d, J ¼ 15.5 Hz, 1H), 4.55 (d, J ¼ 15.9 Hz, 1H), 4.42 (dd,
J ¼ 20.5, 15.7 Hz, 2H), 4.33 (t, J ¼ 4.2 Hz, 1H), 3.27 (dd, J ¼ 14.5,
4.6 Hz, 1H), 3.07 (dd, J ¼ 14.4, 3.9 Hz, 1H). 13C NMR (101 MHz,
3.8 Hz, 1H), 1.28 (s, 9H). 13C NMR (101 MHz, DMSO)
d 172.09, 164.42,
DMSO)
d
172.10, 164.39, 156.41, 139.57, 136.82, 134.98, 132.08,
156.35, 150.55, 139.56, 134.95, 133.87, 132.07, 130.24, 128.73, 128.37,
15

T. Liang, J. Xue, Z. Yao et al.
European Journal of Medicinal Chemistry 221 (2021) 113526
127.48, 127.43, 126.91, 125.91, 60.26, 44.30, 41.48, 34.70, 33.63,
31.59. HRMS (AP-ESI) m/z, calculated for C29H31N3O4, ([M þ H]þ):
486.2387, found: 486.2384. HPLC tR ¼ 19.20 min (99.46% purity).
155.24, 149.05, 139.33, 137.52, 133.96, 132.09, 130.43, 128.76, 127.64,
127.39, 126.95, 122.99, 122.49, 88.48, 55.37, 45.04, 41.16. HRMS (AP-
ESI) m/z, calculated for C24H22N4O4, ([M þ H]þ): 431.1714, found:
431.1712. HPLC tR ¼ 7.43 min (95.26% purity).
4.2.67. 4-((4-benzyl-2,5-dioxo-3-phenethylimidazolidin-1-yl)
methyl)-N-hydroxybenzamide (67)
4.2.71. 4-((4-benzyl-2,5-dioxo-3-(pyridin-3-ylmethyl)
The title compound was synthesized from compound 54 in a
manner similar to that described for the preparation of compound
13. White solid, yield: 60%, mp 142e144 ꢀC. 1H NMR (400 MHz,
imidazolidin-1-yl)methyl)-N- hydroxybenzamide (71)
The title compound was synthesized from compound 58 in a
manner similar to that described for the preparation of compound
13. White solid, yield: 54%, mp 160e161 ꢀC. 1H NMR (400 MHz,
DMSO‑d6)
d
11.19 (s, 1H), 9.01 (s, 1H), 7.56 (dd, J ¼ 8.3, 1.7 Hz, 2H),
7.40e7.20 (m, 8H), 7.10 (d, J ¼ 8.0 Hz, 2H), 6.76 (d, J ¼ 8.0 Hz, 2H),
4.57e4.42 (m, 2H), 4.33 (d, J ¼ 15.9 Hz, 1H), 3.83 (ddd, J ¼ 14.8, 8.7,
6.8 Hz, 1H), 3.40 (s, 1H), 3.27 (dd, J ¼ 14.4, 4.6 Hz, 1H), 3.05 (d,
J ¼ 4.3 Hz, 1H), 2.90 (s, 1H), 2.83e2.71 (m, 1H). 13C NMR (101 MHz,
DMSO‑d6)
d
11.17 (s, 1H), 9.00 (s, 1H), 8.55 (d, J ¼ 2.3 Hz, 1H), 8.52
(dd, J ¼ 4.8, 1.6 Hz, 1H), 7.73 (dt, J ¼ 7.9, 2.0 Hz, 1H), 7.55 (d,
J ¼ 8.2 Hz, 2H), 7.38 (dd, J ¼ 7.9, 4.8 Hz, 1H), 7.31e7.17 (m, 3H), 7.06
(d, J ¼ 7.1 Hz, 2H), 6.76 (d, J ¼ 8.1 Hz, 2H), 4.84 (d, J ¼ 15.8 Hz, 1H),
4.61e4.49 (m, 2H), 4.44 (t, J ¼ 4.2 Hz, 1H), 4.39 (d, J ¼ 16.0 Hz, 1H),
3.30 (dd, J ¼ 14.4, 3.9 Hz,1H), 3.08 (dd, J ¼ 14.5, 3.9 Hz,1H). 13C NMR
DMSO)
d 172.31, 164.43, 155.85, 139.63, 138.98, 135.28, 132.08,
130.09, 129.19, 128.92, 128.79, 126.97, 126.87, 60.09, 42.30, 41.29,
34.04, 33.53. HRMS (AP-ESI) m/z, calculated for C26H25N3O4, ([M þ
H]þ): 444.1918, found: 444.1917. HPLC tR ¼ 11.26 min (98.77%
purity).
(101 MHz, DMSO)
d 172.13, 164.42, 156.47, 149.80, 149.27, 139.55,
136.32, 135.04, 132.61, 132.08, 130.14, 128.78, 127.49, 127.40, 126.95,
124.13, 60.49, 42.34, 41.53, 33.71. HRMS (AP-ESI) m/z, calculated for
C
24H22N4O4, ([M
þ
H]þ): 431.1714, found: 431.1708. HPLC
4.2.68. 4-((4-benzyl-3-(naphthalen-1-ylmethyl)-2,5-
tR ¼ 10.22 min (97.35% purity).
dioxoimidazolidin-1-yl)methyl)-N- hydroxybenzamide (68)
The title compound was synthesized from compound 55 in a
manner similar to that described for the preparation of compound
13. White solid, yield: 53%, mp 153e154 ꢀC. 1H NMR (400 MHz,
4.2.72. 4-((4-benzyl-2,5-dioxo-3-(pyridin-4-ylmethyl)
imidazolidin-1-yl)methyl)-N- hydroxybenzamide (72)
The title compound was synthesized from compound 59 in a
manner similar to that described for the preparation of compound
13. White solid, yield: 56%, mp > 300 ꢀC. 1H NMR (400 MHz,
DMSO‑d6)
d
11.20 (s, 1H), 9.04 (s, 1H), 8.18 (dd, J ¼ 6.6, 3.2 Hz, 1H),
8.00 (dd, J ¼ 6.4, 3.2 Hz, 1H), 7.94 (d, J ¼ 8.1 Hz, 1H), 7.71e7.48 (m,
6H), 7.26 (t, J ¼ 7.3 Hz, 1H), 7.18 (t, J ¼ 7.4 Hz, 2H), 7.04 (d, J ¼ 7.5 Hz,
2H), 6.79 (d, J ¼ 8.3 Hz, 2H), 5.34 (s, 1H), 4.95 (d, J ¼ 15.7 Hz, 1H),
4.55 (d, J ¼ 15.9 Hz, 1H), 4.42 (d, J ¼ 15.9 Hz, 1H), 4.20 (t, J ¼ 4.1 Hz,
DMSO‑d6)
d 11.17 (s, 1H), 9.00 (s, 1H), 8.55e8.52 (m, 2H), 7.56 (d,
J ¼ 8.1 Hz, 2H), 7.32e7.29 (m, 2H), 7.28e7.25 (m, 1H), 7.21 (t,
J ¼ 7.4 Hz, 2H), 7.09e7.04 (m, 2H), 6.78 (d, J ¼ 8.0 Hz, 2H), 4.82 (d,
J ¼ 16.6 Hz, 1H), 4.63e4.52 (m, 2H), 4.49 (t, J ¼ 4.3 Hz, 1H), 4.41 (d,
J ¼ 16.0 Hz, 1H), 3.29 (dd, J ¼ 14.5, 4.0 Hz, 1H), 3.07 (dd, J ¼ 14.5,
1H), 3.25 (dd, J ¼ 14.4, 4.7 Hz, 1H), 3.05 (dd, J ¼ 14.4, 3.7 Hz, 1H). 13
C
NMR (101 MHz, DMSO)
d 172.06, 164.46, 156.28, 139.57, 134.82,
134.01, 132.12, 131.96, 131.44, 130.26, 129.21, 129.08, 128.72, 127.50,
127.45, 127.09, 126.96, 126.56, 125.95, 123.83, 60.44, 42.95, 41.53,
33.86. HRMS (AP-ESI) m/z, calculated for C29H25N3O4, ([M þ H]þ):
480.1918, found: 480.1916. HPLC tR ¼ 6.93 min (99.67% purity).
4.0 Hz, 1H). 13C NMR (101 MHz, DMSO)
d 172.15, 164.40, 156.51,
150.25, 146.00, 139.53, 135.01, 132.09, 130.12, 128.79, 127.50, 127.41,
126.97, 123.10, 60.73, 43.64, 41.58, 33.71. HRMS (AP-ESI) m/z,
calculated for C24H22N4O4, ([M þ H]þ): 431.1714, found: 431.1713.
HPLC tR ¼ 7.25 min (96.10% purity).
4.2.69. 4-((4-benzyl-3-(naphthalen-2-ylmethyl)-2,5-
dioxoimidazolidin-1-yl)methyl)-N- hydroxybenzamide (69)
The title compound was synthesized from compound 56 in a
manner similar to that described for the preparation of compound
13. White solid, yield: 66%, mp 150e151 ꢀC. 1H NMR (400 MHz,
4.2.73. Methyl 4-((3-(1-methoxy-4-methyl-1-oxopentan-2-yl)
ureido)methyl)benzoate (73)
The title compound was synthesized from methyl leucinate and
methyl 4-(aminomethyl)benzoate in a manner similar to that
described for the preparation of compound 22. White solid, yield:
DMSO‑d6)
d
11.21 (s, 1H), 9.05 (s, 1H), 7.93 (dd, J ¼ 8.6, 2.9 Hz, 3H),
7.83 (s, 1H), 7.58 (d, J ¼ 8.2 Hz, 2H), 7.56e7.50 (m, 2H), 7.46 (dd,
J ¼ 8.5, 1.8 Hz, 1H), 7.29 (dd, J ¼ 7.8, 2.0 Hz, 1H), 7.22 (t, J ¼ 7.5 Hz,
2H), 7.10 (d, J ¼ 7.0 Hz, 2H), 6.79 (d, J ¼ 8.0 Hz, 2H), 5.05 (d,
J ¼ 15.6 Hz, 1H), 4.60 (dd, J ¼ 15.8, 10.6 Hz, 2H), 4.50e4.36 (m, 2H),
3.31 (dd, J ¼ 14.5, 3.9 Hz,1H), 3.09 (dd, J ¼ 14.4, 3.9 Hz, 1H). 13C NMR
82%, mp 89e91 ꢀC. 1H NMR (400 MHz, DMSO‑d6)
d 7.91 (d,
J ¼ 8.3 Hz, 2H), 7.37 (d, J ¼ 8.2 Hz, 2H), 6.54 (t, J ¼ 6.1 Hz, 1H), 6.44
(d, J ¼ 8.3 Hz, 1H), 4.29 (dd, J ¼ 6.2, 2.2 Hz, 2H), 4.19 (td, J ¼ 8.4,
6.3 Hz, 1H), 3.84 (s, 3H), 3.62 (s, 3H), 1.64 (dq, J ¼ 8.2, 6.4 Hz, 1H),
1.47 (ddd, J ¼ 8.0, 5.9, 2.0 Hz, 2H), 0.88 (dd, J ¼ 12.1, 6.6 Hz, 6H).
(101 MHz, DMSO)
d 172.17, 164.53, 156.49, 139.64, 135.09, 134.34,
133.41, 132.93, 132.15, 130.70, 130.25, 128.92, 128.81, 128.47, 128.23,
128.08, 127.51, 127.23, 127.00, 126.84, 126.58, 60.32, 44.85, 41.59,
33.80. HRMS (AP-ESI) m/z, calculated for C29H25N3O4, ([M þ H]þ):
480.1918, found: 480.1917. HPLC tR ¼ 9.26 min (95.71% purity).
4.2.74. Methyl 4-((4-isobutyl-2,5-dioxoimidazolidin-1-yl)methyl)
benzoate (74)
The title compound was synthesized from compound 73 in a
manner similar to that described for the preparation of compound
46. White solid, yield: 97%, mp 136e138 ꢀC. 1H NMR (400 MHz,
4.2.70. 4-((4-benzyl-2,5-dioxo-3-(pyridin-2-ylmethyl)
imidazolidin-1-yl)methyl)-N- hydroxybenzamide (70)
The title compound was synthesized from compound 57 in a
manner similar to that described for the preparation of compound
13. White solid, yield: 63%, mp 156e158 ꢀC. 1H NMR (400 MHz,
DMSO‑d6)
d
8.47 (s, 1H), 7.93 (d, J ¼ 8.2 Hz, 2H), 7.38 (d, J ¼ 8.2 Hz,
2H), 4.60 (s, 2H), 4.24e4.13 (m, 1H), 3.84 (s, 3H), 1.79 (ddd, J ¼ 9.1,
4.5,1.9 Hz,1H),1.54 (ddd, J ¼ 13.5, 9.1, 4.3 Hz,1H),1.43 (ddd, J ¼ 14.1,
9.5, 5.2 Hz, 1H), 0.89 (dd, J ¼ 6.6, 2.8 Hz, 6H).
DMSO‑d6)
d
11.18 (s, 1H), 9.01 (s, 1H), 8.53 (dd, J ¼ 5.1, 1.7 Hz, 1H),
4.2.75. Methyl 4-((4-isobutyl-3-methyl-2,5-dioxoimidazolidin-1-
yl)methyl)benzoate (75)
The title compound was synthesized from compound 74 and
iodomethane in a manner similar to that described for the prepa-
ration of compound 4. Colorless oil, yield: 93%. 1H NMR (400 MHz,
7.79 (td, J ¼ 7.7,1.8 Hz,1H), 7.54 (d, J ¼ 8.3 Hz, 2H), 7.45 (d, J ¼ 7.9 Hz,
1H), 7.32 (dd, J ¼ 7.5, 4.9 Hz, 1H), 7.28e7.23 (m, 1H), 7.17 (t,
J ¼ 7.5 Hz, 2H), 7.04e6.96 (m, 2H), 6.72 (d, J ¼ 8.0 Hz, 2H), 4.76 (q,
J ¼ 16.6 Hz, 2H), 4.54e4.34 (m, 3H), 3.34 (d, J ¼ 13.7 Hz, 1H), 3.16 (d,
J ¼ 13.8 Hz, 1H). 13C NMR (101 MHz, DMSO)
d
172.70, 164.39, 157.54,
CDCl3)
d
7.98 (d, J ¼ 8.3 Hz, 2H), 7.45 (d, J ¼ 8.3 Hz, 2H), 4.69 (d,
16

T. Liang, J. Xue, Z. Yao et al.
European Journal of Medicinal Chemistry 221 (2021) 113526
J ¼ 2.0 Hz, 2H), 3.90 (s, 3H), 3.87 (dd, J ¼ 6.4, 5.1 Hz,1H), 2.95 (s, 3H),
1.85 (dp, J ¼ 13.3, 6.7 Hz, 1H), 1.78e1.68 (m, 2H), 0.93 (d, J ¼ 6.6 Hz,
3H), 0.86 (d, J ¼ 6.5 Hz, 3H).
24.12, 23.33, 22.51. HRMS (AP-ESI) m/z, calculated for C22H19N3O4,
([M þ H]þ): 396.1918, found: 396.1919. HPLC tR ¼ 22.08 min (99.32%
purity).
4.2.76. Methyl 4-((4-isobutyl-2,5-dioxo-3-propylimidazolidin-1-yl)
methyl)benzoate (76)
Declaration of competing interest
The title compound was synthesized from compound 74 and 1-
iodopropane in a manner similar to that described for the prepa-
ration of compound 4. Colorless oil, yield: 81%. 1H NMR (400 MHz,
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.
DMSO‑d6)
d
7.93 (d, J ¼ 8.3 Hz, 2H), 7.39 (d, J ¼ 8.2 Hz, 2H),
4.79e4.57 (m, 2H), 4.24 (t, J ¼ 4.9 Hz, 1H), 3.84 (s, 3H), 3.48 (dt,
J ¼ 14.1, 7.9 Hz, 1H), 3.05 (ddd, J ¼ 13.8, 8.2, 5.0 Hz, 1H), 1.84e1.72
(m, 1H), 1.65 (td, J ¼ 5.6, 4.9, 1.9 Hz, 2H), 1.61e1.42 (m, 2H),
0.95e0.84 (m, 6H), 0.84e0.77 (m, 3H).
Acknowledgement
We genuinely thank the Advanced Medical Research Institute,
Shandong University, for their help in the experiments. This work
was supported by the National Nature Science Foundation of China
(81874288, 92053105 and 82003590), the Shandong Provincial
Natural Science Foundation (ZR2019LZL004 and ZR2020QH342).
4.2.77. Methyl 4-((3-benzyl-4-isobutyl-2,5-dioxoimidazolidin-1-yl)
methyl)benzoate (77)
The title compound was synthesized from compound 74 and
(bromomethyl)benzene in a manner similar to that described for
the preparation of compound 4. Colorless oil, yield: 89%. 1H NMR
Abbreviations
(400 MHz, DMSO‑d6)
d
7.95 (d, J ¼ 8.3 Hz, 2H), 7.43 (d, J ¼ 8.3 Hz,
HL-60 cell human promyelocytic leukemia cell line
2H), 7.39e7.27 (m, 5H), 4.72e4.66 (m, 2H), 4.03 (d, J ¼ 4.7 Hz, 1H),
3.85 (s, 3H), 1.73e1.54 (m, 3H), 0.95e0.79 (m, 2H), 0.74 (d,
J ¼ 6.4 Hz, 3H), 0.67 (d, J ¼ 6.3 Hz, 3H).
HDACs
HATs
NOC-7
SAHA
Bax
histone deacetylases
histone acetyltransferases
a HDAC6 inhibitor
a pan-HDAC inhibitor
a pro-apoptotic protein of Bcl-2 family
triethylamine;
4.2.78. N-hydroxy-4-((4-isobutyl-3-methyl-2,5-dioxoimidazolidin-
1-yl)methyl)benzamide (78)
TEA
The title compound was synthesized from compound 75 in a
manner similar to that described for the preparation of compound
13. White solid, yield: 58%, mp 122e124 ꢀC. 1H NMR (400 MHz,
MF
THF
SAR
SEM
N,N-Dimethylformamide;
tetrahydrofuran
structure and activity relationship;
standard error of the mean
DMSO‑d6)
d
11.18 (s, 1H), 9.02 (s, 1H), 7.69 (d, J ¼ 8.3 Hz, 2H), 7.31 (d,
J ¼ 8.2 Hz, 2H), 4.64e4.52 (m, 2H), 4.14 (t, J ¼ 5.7 Hz, 1H), 2.86 (s,
3H), 1.82e1.72 (m, 1H), 1.64 (t, J ¼ 6.3 Hz, 2H), 0.88 (d, J ¼ 6.6 Hz,
KG1 cell human acute myeloid leukemia cell line;
NCIeH929 cell human multiple myeloma cell line;
Jurkat cell human acute T cell leukemia cell line;
3H), 0.81 (d, J ¼ 6.5 Hz, 3H). 13C NMR (101 MHz, DMSO)
d 173.55,
164.39, 156.33, 140.11, 132.43, 127.84, 127.56, 59.93, 41.65, 37.66,
HEL,
human erythroleukemia cell line;
28.31, 24.30, 23.29, 22.88. HRMS (AP-ESI) m/z, calculated for
HUVECs human umbilical vein endothelial cells
GEC-1 cell gastric epithelial cells
Tubastatin A a HDAC6 selective inhibitor
TUNEL assay terminal deoxynucleotidyl transferase (TdT)
deoxyuridine triphosphate (dUTP) Nick-End labeling
Assay
C
16H21N3O4, ([M
þ
H]þ): 320.1605, found: 320.1612. HPLC
tR ¼ 6.06 min (95.71% purity).
4.2.79. N-hydroxy-4-((4-isobutyl-2,5-dioxo-3-propylimidazolidin-
1-yl)methyl)benzamide (79)
The title compound was synthesized from compound 76 in a
manner similar to that described for the preparation of compound
13. White solid, yield: 66%, mp 89e91 ꢀC. 1H NMR (400 MHz,
PI
propidium iodide;
MOMP
TMRE
TSA
FBS
MTT
mitochondrial outer membrane permeabilization
tetramethylrhodamine ethyl ester
Trichostatin A, a potent pan-HDAC inhibitor
fetal bovine serum
3-(4,5-Dimethyl-2-Thiazolyl)-2,5-Diphenyl Tetrazolium
Bromide;
Dimethyl sulfoxide;
RIPA Lysis Buffer
Phenylmethylsulfonyl fluoride, a protease inhibitor
Sodium dodecyl sulfate
polyvinylidene fluoride;
glyceraldehyde-3-phosphate dehydrogenase
Tris Buffered saline Tween
electrochemiluminescence
phosphate buffer saline;
thin-layer chromatography
High-resolution mass spectra
melting point
DMSO‑d6)
d
11.18 (s, 1H), 9.02 (s, 1H), 7.70 (d, J ¼ 8.1 Hz, 2H), 7.31 (d,
J ¼ 8.0 Hz, 2H), 4.68e4.52 (m, 2H), 4.23 (t, J ¼ 5.6 Hz, 1H), 3.46 (dt,
J ¼ 14.1, 7.9 Hz, 1H), 3.04 (ddd, J ¼ 13.8, 8.3, 5.1 Hz, 1H), 1.76 (dt,
J ¼ 13.2, 6.6 Hz,1H),1.69e1.62 (m, 2H),1.61e1.41 (m, 2H), 0.99e0.77
(m, 9H). 13C NMR (101 MHz, DMSO)
d
173.53, 164.40, 156.10, 140.12,
DMSO
RIPA
PMSF
SDS
PVDF
GAPDH
TBST
ECL,
PBS
TLC
HRMS
mp
132.42, 127.80, 127.59, 57.74, 42.48, 41.60, 37.65, 24.17, 23.50, 22.68,
21.10, 11.53. HRMS (AP-ESI) m/z, calculated for C22H19N3O4, ([M þ
H]þ): 348.1918, found: 348.1921. HPLC tR ¼ 13.41 min (98.96%
purity).
4.2.80. 4-((3-benzyl-4-isobutyl-2,5-dioxoimidazolidin-1-yl)
methyl)-N-hydroxybenzamide (80)
The title compound was synthesized from compound 77 in a
manner similar to that described for the preparation of compound
13. White solid, yield: 67%, mp 101e102 ꢀC. 1H NMR (400 MHz,
DMSO‑d6)
d
11.20 (s, 1H), 9.04 (s, 1H), 7.72 (d, J ¼ 7.9 Hz, 2H),
7.43e7.25 (m, 7H), 4.70 (d, J ¼ 15.5 Hz, 1H), 4.66e4.58 (m, 2H), 4.38
(d, J ¼ 15.7 Hz, 1H), 4.02 (t, J ¼ 5.5 Hz, 1H), 1.62 (tdd, J ¼ 18.4, 13.9,
8.8 Hz, 3H), 0.74 (d, J ¼ 6.4 Hz, 3H), 0.68 (d, J ¼ 6.3 Hz, 3H). 13C NMR
Appendix A. Supplementary data
(101 MHz, DMSO)
d
173.32, 164.41, 156.61, 140.03, 137.05, 132.50,
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113526.
129.10, 128.23, 128.01, 127.89, 127.67, 58.29, 44.98, 41.86, 37.72,
17

T. Liang, J. Xue, Z. Yao et al.
European Journal of Medicinal Chemistry 221 (2021) 113526
infection is associated with oncogenic transformation in gastric cancer, Cell.
Physiol. Biochem. 42 (2017) 1326e1335.
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