Discovery of 1,2,4-oxadiazole-Containing hydroxamic acid derivatives as histone deacetylase inhibitors potential application in cancer therapy

Article abstract of DOI:10.1016/j.ejmech.2019.05.089

In this study, a series of novel HDAC inhibitors, using 1,2,4-oxadiazole-containing as the cap group, were synthesized and evaluated in vitro. Compound 14b, N-hydroxy-2-(methyl((3-(1-(4-methylbenzyl)piperidin-4-yl)-1,2,4-oxadiazol-5-yl)methyl)amino)pyrimidine-5-carboxamide, displayed the most potent histone deacetylase (HDAC) inhibition, especially against HDAC1, 2, and 3 with IC₅₀ values of 1.8, 3.6 and 3.0 nM, respectively. In vitro antiproliferative studies confirmed that 14b was more potent than SAHA, with IC₅₀ values against 12 types of cancer cell lines ranging from 9.8 to 44.9 nM. The results of Western blot assays showed that compound 14b can significantly up-regulate the acetylation of the biomarker his-H₃ and molecular docking analyses revealed the mode of action of compound 14b against HDAC1. The results of flow-cytometry analysis suggested that compound 14b induces cell cycle arrest at the G1 phase and has apoptotic effects. Further investigation of the activity of 14b on the primary cells of three patients, showed IC₅₀ values of 21.3, 61.1, and 77.4 nM. More importantly, an oral bioavailability of up to 53.52% was observed for 14b. An in vivo pharmacodynamic evaluation demonstrated that compound 14b can significantly inhibit tumor growth in a Daudi Burkitt's lymphoma xenograft model, with tumor inhibition rates of 53.8 and 46.1% observed at 20 and 10 mg/kg when administered p.o. and i.v., respectively. These results indicate that compound 14b may be a suitable lead for further evaluation and development as an HDAC inhibitor and a potent anticancer agent.

Full text of DOI:10.1016/j.ejmech.2019.05.089

Accepted Manuscript
Discovery of 1,2,4-oxadiazole-Containing hydroxamic acid derivatives as histone
deacetylase inhibitors potential application in cancer therapy
Zhuang Yang, Mingsheng Shen, Minghai Tang, Wanhua Zhang, Xue Cui, Zihao
Zhang, Heying Pei, Yong Li, Mengshi Hu, Peng Bai, Lijuan Chen
PII:
S0223-5234(19)30513-6
DOI:
https://doi.org/10.1016/j.ejmech.2019.05.089
EJMECH 11399
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 February 2019
Revised Date: 18 May 2019
Accepted Date: 31 May 2019
Please cite this article as: Z. Yang, M. Shen, M. Tang, W. Zhang, X. Cui, Z. Zhang, H. Pei, Y. Li, M.
Hu, P. Bai, L. Chen, Discovery of 1,2,4-oxadiazole-Containing hydroxamic acid derivatives as histone
deacetylase inhibitors potential application in cancer therapy, European Journal of Medicinal Chemistry
(2019), doi: https://doi.org/10.1016/j.ejmech.2019.05.089.
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Discovery of 1,2,4-Oxadiazole-Containing Hydroxamic Acid Derivatives as
Histone Deacetylase Inhibitors Potential Application in Cancer Therapy
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Zhuang Yang , Mingsheng Shen , Minghai Tang , Wanhua Zhang , Xue Cui , Zihao
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*, a
Zhang , Heying Pei , Yong Li , Mengshi Hu , Peng Bai , and Lijuan Chen
a
State Key Laboratory of Biotherapy and Cancer Center, West China Hospital,
Sichuan University and Collaborative Innovation Center of Biotherapy, Chengdu,
610041, China
b
Department of Hematology and Research Laboratory of Hematology, West China
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Hospital of Sichuan University, Chengdu 610041, China
* Corresponding author. State Key Laboratory of Biotherapy and Cancer Center,
West China Hospital, Sichuan University and Collaborative Innovation Center,
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Chengdu, Sichuan 610041, China.
Tel: +86-28-85164103; Fax: +86-28-85164060.
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E-mail address: chenlijuan125@163.com.
ORCID
Chen, Lijuan: 0000-0002-8076-163X
Yang, Zhuang: 0000-0002-8915-9944
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Abstract
In this study,
1,2,4-oxadiazole-containing as the cap group, were synthesized and evaluated in vitro.
Compound 14b,
a
series of novel HDAC inhibitors, using
N-hydroxy-2-(methyl((3-(1-(4-methylbenzyl)piperidin-4-yl)-1,2,4-oxadiazol-5-yl)meth
yl)amino)pyrimidine-5-carboxamide, displayed the most potent histone deacetylase
(HDAC) inhibition, especially against HDAC1, 2, and 3 with IC values of 1.8, 3.6
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and 3.0 nM, respectively. In vitro antiproliferative studies confirmed that 14b was
more potent than SAHA, with IC values against 12 types of cancer cell lines ranging
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from 9.8 to 44.9 nM. The results of Western blot assays showed that compound 14b
can significantly up-regulate the acetylation of the biomarker his-H and molecular
3
docking analyses revealed the mode of action of compound 14b against HDAC1. The
results of flow-cytometry analysis suggested that compound 14b induces cell cycle
arrest at the G1 phase and has apoptotic effects. Further investigation of the activity of
14b on the primary cells of three patients, showed IC values of 21.3, 61.1, and 77.4
5
0
nM. More importantly, an oral bioavailability of up to 53.52% was observed for 14b.
An in vivo pharmacodynamic evaluation demonstrated that compound 14b can
significantly inhibit tumor growth in a Daudi Burkitt's lymphoma xenograft model,
with tumor inhibition rates of 53.8 and 46.1% observed at 20 and 10 mg/kg when
administered p.o. and i.v., respectively. These results indicate that compound 14b may
be a suitable lead for further evaluation and development as an HDAC inhibitor and a
potent anticancer agent.
Keywords: HDAC; 1,2,4-oxadiazole; antiproliferative; anticancer.
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Introduction
Histone deacetylases (HDACs) are responsible for the deacetylation of lysine
residues in histone or non-histone substrates,[1, 2] the epigenetic targeting of which
has shown clinical benefits, especially for the treatment of cancer.[3] To date, 18
HDAC isoforms have been identified that are classified in four groups: classes I
(HDAC1, 2, 3, and 8); II (HDAC4, 5, 6, 7, 9, and 10); III (SIRT1-7); and IV
(HDAC11), depending on their sequence similarity, cellular localization, tissue
expression patterns, and enzymatic mechanism.[4, 5] HDAC inhibitors can mediate
cancer cell death by promoting reduced cell motility/migration, invasion,
angiogenesis, proliferation, induction of apoptosis, and inhibition of DNA repair.[6-9]
Currently, five HDAC inhibitors, vorinostat (SAHA),[10] romidepsin (FK-228),[11]
belinostat (PXD-101),[12] panobinostat (LBH589),[13] and chidamide, have been
approved for the treatment of cutaneous T-cell lymphoma (CTCL), peripheral T cell
lymphoma (PTCL), or multiple myeloma (MM).[14] In addition, many inhibitors are
currently in clinical trials for the treatment of hematological and/or solid tumors.[15]
However, these drugs still have a number of problems. For instance, LBH589 has a
“Highlights of prescribing information” boxed warning due to severe diarrhea
occurring in 25% of LBH589-treated patients, with severe and fatal cardiac ischemic
events, severe arrhythmias, and ECG changes also having occurred in patients
receiving LBH589 treatment.[16] Due to decades of synthetic efforts, more than
twenty candidates have entered clinical trials for a variety of disease treatments.[17]
Although the use of unselective HDAC inhibitors outside of oncology is limited due
to their side effects,[18-20] the benefit of isoform- or class-selective HDAC inhibitors
in cancer therapy is still under debate. To date, there is no clear clinical evidence that
isoform-selective HDAC inhibitors have sufficient efficacy while causing fewer
adverse effects.[18-20] However, it is particularly important to develop a novel
HDAC inhibitors with lower toxic side effects and better efficacy for cancer therapy.
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Figure 1. Licensed HDAC inhibitors
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The pharmacophore models of most HDAC inhibitors always have three parts: a
2
+
cap group used as a selective vector, a ZBG group to bind with the Zn ion, and a
linker region that traditionally allows the ZBG group stretch into the catalytic binding
zone.[21-23] To date, modifications of the cap have been introduced that cause
changes in HDAC potency and selectivity profiles.[24-29] In our previous study, we
designed and synthesized a series of selective HDAC inhibitors by introducing
different heterocyclic groups as the cap group, such as purine and quinoline
groups.[30-32] Importantly, in 2014, Prof Antonello Mai et al reported a type of
1,3,4-oxadiazole-containing HDAC inhibitors with micromole antiproliferative
activities against a panel of cancer cell lines.[33] Guided by the results of previous
studies and available X-ray crystal structures[34, 35] or homology models generated
by our lab[31], in this study, we describe the synthesis, biological evaluation, and
modeling studies of
a
series of novel HDAC inhibitors that use
1,2,4-oxadiazole-containing derivatives as a cap group and exhibit nanomole
antiproliferative activities and remarkable bioavailability as potent anticancer agents.
2. Results and Discussion
2.1 Chemistry
9
5
The
synthetic
route
is
presented
in
Scheme
1-3.
t-Butyl
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4-cyanopiperidine-1-carboxylate (1) was first reacted with hydroxylamine
hydrochloride to generate compound 2. Then, compound 2 and chloroacetyl chloride
was reacted in the presence of triethylamine at room temperature, and without further
purification, the crude compound was heated in 1,4-dioxane under reflux to obtain the
intermediate compound 3. The linker group was introduced by coupling compound 3
with methyl 4-hydroxycinnamate to obtain compound 4, which was further
de-protected of the Boc group to yield compound 5. Subsequently, we used different
R-Cl or R-Br compounds to react with compound 5 to obtain compounds 6, which
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were directly converted to hydroxamic acid compounds 7 with NH OH.
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Scheme 1. Reagents and conditions: (a) H NOH·HCl, NaHCO , EtOH/H O (v/v, 5/1),
2
3
2
o
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14
reflux, overnight; (b) ClCH COCl, TEA, CH Cl , 0 C to rt, 2 h; Dioxane, reflux, 2 h;
2 2 2
(c) Methyl 4-hydroxycinnamate, CsCO , KI, MeCN, reflux, overnight; (d) CF COOH,
3
3
CH Cl , rt, 4 h; (e) R-Cl or R-Br, K CO , KI, MeCN, rt, 4 h; (f) NH OH, NaOH,
2
2
2
3
2
MeOH/CH Cl (v/v, 2/1), rt, 2 h.
2
2
To evaluate the influence of linker groups on HDAC inhibition, we also
synthesized a series of compounds using different commercially available compounds
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that were reacted with the intermediate compound 3. The resulting compounds 8 were
further treated as previous described in Scheme 1 to yield compounds 9 (Scheme 2).
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Scheme 2. Reagents and conditions: (a) CsCO , KI, MeCN, reflux, overnight; (b) i.
3
CF COOH, CH Cl , rt, 4 h; ii. R-Cl or R-Br, K CO , KI, MeCN, rt, 4 h; iii NH OH,
3
2
2
2
3
2
NaOH, MeOH/CH Cl (v/v, 2/1), rt, 2 h.
2
2
For the pyrimidine structure used as the linker region, compounds 13a-13b and
14a-14j were synthesized to modify the SAR (Scheme 2). Compound 10 was
synthesized by reacting compound 3 with methylamine, after which compound 10
was
coupled
with
ethyl-2-chloropyrimidine-5-carboxylate
and
ethyl
(E)-3-(2-chloropyrimidin-5-yl)acrylate to yield compounds 11 and 12, respectively.
Subsequently, compounds 11 and 12 were treated as described in Scheme 1 to yield
compounds 13a-13b and 14a-14j, respectively.
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Scheme 3. Reagents and conditions: (a) Methylamine, EtOH, rt, 2 h; (b) DIPEA,
MeOH, rt, 8 h; (c) i. CF COOH, CH Cl , rt, 4 h; ii. BnBr, K CO , KI, MeCN, rt, 4 h;
3
2
2
2
3
iii NH OH, NaOH, MeOH/CH Cl (v/v, 2/1), rt, 2 h.
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2.2 Evaluation of the vitro activities evaluation of synthesized compounds
The synthesized compounds were initially evaluated for their inhibitory activities
toward HDAC1 and HDAC6 at a concentration of 100 nM, the results of which are
presented in Tables 1, 2 and 3. As shown in Table 1, at 100 nM, compounds 7a-7p all
caused less than 50% inhibition of HDAC1, whereas more than 50% inhibition of
HDAC6 was observed. Compound 7a, which had an n-propyl chain introduced into
the cap group, showed 40 and 69% inhibition of HDAC1 and HDAC6 at 100 nM
respectively. However, the introduction of an unsaturated alkyl chain allyl group in
compoudn 7b caused a decrease in HDAC inhibition. Since the cap group promotes
the ability of compounds to achieve selective inhibition of HDACs, a series
substituted benzyl group were introduced. Compound 7c displayed 71% inhibition of
HDAC6 at 100 nM, whereas the inhibition of HDAC1 remained unchanged. When
introducing substituted groups to the benzyl group, the inhibitory activities showed
para- > meta- > ortho-position trend, such as compounds 7f>7e>7d, in which a
methyl groups were introduced, and 7i>7h>7g, in which fluorine atoms were
introduced. These results confirmed that para-position substitutions in these
compouds could increase their inhibition of HDAC1 and HDAC6. When
di-substituted benzyl groups were introduced, the ortho- and para- position-substituted
compound 7j was more effective than the meta- and para- position-substituted
compound 7k, with 35 and 17% inhibition of HDAC1 and 74 and 60% inhibition of
HDAC6 observed, respectively. In contrast with compounds 7g and 7i, introducing
the larger halogen chlorine (compound 7l) and bromine atom (compound 7m) caused
significant decreases in HDAC inhibition. Compounds 7o and 7p, which had
electron-withdrawing groups introduced, showed a reduction in HDAC1 and HDAC6
inhibition.
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Table 1. Inhibition of HDAC1 and HDAC6 by compounds 7a-7p (100 nM).
%
Inhibition
HDAC6
% Inhibition
Compounds
R
Compounds
R
HDAC1
40
HDAC1
HDAC6
75
7
a
Propyl
Allyl
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66
71
68
73
76
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73
7i
7j
4-F-Bn
2,4-difluoro-Bn
3,4-difluoro-Bn
2-Cl-Bn
38
35
17
8
7
b
34
74
7
c
Bn
35
7k
7l
60
7
d
2-Me-Bn
3-Me-Bn
4-Me-Bn
2-F-Bn
3-F-Bn
14
59
7
e
25
7m
7n
7o
7p
4-Br-Bn
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58
7
f
32
4-MeO-Bn
2-CN-Bn
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19
75
7
g
31
65
7
h
35
2-NO
-Bn
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Values are the averages of at least two independent experiments, SD < 10%.
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To evaluate the influence of the linker group, the synthesized compounds 9a-9f
(100 nM) were also assessed for their ability to inhibit HDAC1 and HDAC6. As
shown in Table 2, when the ethylene linkage was replaced by a saturated alkyl chain
(compounds 9a and 9b) their inhibitory activities were significantly decreased, in
contrast with the activities of compounds 7a and 7b, which have the same cap groups.
The inhibitory activities of compounds 9c and 9d, in which benzyl groups were
introduced to the cap groups, showed the same trends. The inhibitory activities of the
compounds were significantly decreased, introducing benzyl groups to the cap groups
was more favorable with respect to the inhibition of HDAC1 and HDAC6. The
inhibitory activities of 9e and 9f were effectively increased with the decrease in
carbon chain length. Taken together, these results suggest that the linker group is
important for the inhibition of HDACs.
a
Table 2. Inhibition of HDAC1 and HDAC6 by compounds 9a-9f (100 nM).
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78
%
inhibitory at 100 nM
compounds
R
n
HDAC1
HDAC6
13
9
a
Propyl
Allyl
Bn
2
2
2
2
1
5
9
b
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c
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Bn
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86
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92
93
94
95
96
97
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Values are the averages of at least two independent experiments, SD < 10%.
Based on the SAR analysis, the synthesized compounds 13a-13b and 14a-14j,
which had pyrimidine introduced into the linker region, were evaluated for their
inhibitory activities toward HDAC1 and HDAC6 at concentrations at 100 nM and for
their antiproliferative activities toward the Raji cell line, the results of which are
presented in Table 3. In contrast with compounds 7c and 7f, which have vinyl groups
in their linker regions, the inhibitory activities of compounds 13a and 13b toward
HDAC1 were significantly improved, by 65% and 61% respectively. When the vinyl
group was removed, compounds 14a and 14b showed increased inhibitory activities
toward HDAC1 at 100 nM, both 96%, and the antiproliferative IC values toward B
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cell lymphoma Raji cell line were 27.6 and 12.1 nM, respectively. Based on this result,
we further explored the effect of different substituted benzyl groups on the HDAC
inhibitory activity. The inhibition of HDAC1 and HDAC6 as well as the
antiproliferative activity of compound 14c was reduced after shifting the methyl
substitution to the meta position. Compounds 14d and 14e exhibit a similar SAR, with
the F atom being the better substitution. A comparison of the activities of compounds
14f and 14d shows that the Cl substitution is better than F, but when F and Cl are
simultaneously introduced into the molecule, the biological activity of 14j was
significantly reduced. When a 2,4-difluoro substitution was introduced, compound 14i
exhibited better antiproliferative activity, with an IC50 value 34.3 nM, although its
activity against HDAC1 and HDAC6 decreased. When the electron-withdrawing
groups CN (14g) or CF (14h) were introduced in the para position, the HDAC
3
inhibition and antiproliferative activities decreased. Based on the results of the SAR
analysis, compound 14b was selected as a seed compound for further research.
Table 3. Inhibition of HDAC1 and HDAC6 and antiproliferative activity toward the
a
Raji cell line of compounds 13a-13b and 14a-14j (100 nM).
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O
OH
OH
R
N
N
N
N
R
H
H
N
N
N
N
N
N
N
N
O
O
N
N
14a-14j
1
3a-13b
2
05
Compounds
R
% Inhibition
Raji
IC (nM)
HDAC1
65
HDAC6
55
5
0
1
3a
3b
4a
4b
4c
4d
4e
4f
4g
4h
H
547.3 ± 121.8
292.5 ± 79.4
27.6 ± 3.8
12.1 ± 1.9
47.3 ± 3.1
98.6 ± 8.6
22.0 ± 3.2
48.3 ± 5.7
116.6 ± 10.9
207 ± 32.3
34.3 ± 2.8
115.7 ± 15.7
1
4-Me
H
61
60
1
96
63
1
4-Me
3-Me
2-F
96
71
1
93
54
1
84
44
1
4-F
92
65
1
2-Cl
4-CN
93
55
1
68
38
1
4-CF
3
62
30
1
4i
2,4-F
88
66
1
4j
2-Cl-4-F
73
49
a
2
2
2
2
2
2
2
2
2
2
06
07
08
09
10
11
12
13
14
15
Values are the averages of at least two independent experiments, SD < 10%.
2.3 Compound 14b inhibitory activity toward HDAC1-11
Compound 14b was further evaluated for its activity toward the other HDAC
isoforms, the results of which are summarized in Table 4. Compound 14b was
effective toward class I HDAC1, 2, and 3, with IC values of 1.8, 3.6, and 3.0 nM
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respectively, which were more effective than SAHA. In contrast, the activity of
compound 14b toward class II and IV HDACs were relatively lower, especially
toward HDAC11, with an observed IC value of more than 10 µM.
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Table 4. HDAC inhibitory activity of compound 14b
a
Isoform
IC50 (nM)
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SAHA
20 ± 6
Class I
HDAC1
HDAC2
HDAC3
HDAC8
HDAC4
HDAC5
HDAC7
HDAC9
HDAC6
HDAC10
HDAC11
1.8 ± 0.2
3.6 ± 0.3
3.0 ± 0.8
260 ± 40
473 ± 30
540 ± 12
392 ± 18
441 ± 21
56 ± 1
29 ± 4
33 ± 2
682 ± 87
>10000
>10000
>10000
>10000
27 ± 2
Class IIa
Class IIb
102 ± 8
208 ± 19
>10000
Class IV
>10000
a
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
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: Compounds were tested in the 10-dose IC50 mode in duplicate with 3-fold serial
dilutions starting at 10 µM. The IC values are the means of at least two experiments.
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2.4 In vitro antiproliferative activity of 14b toward multiple tumor cell lines
To evaluate the antiproliferative activity of compound 14b, the IC50 values
toward 12 tumor cell lines were measured, including solid tumors (colon cancer
HCT116, ovarian cancer A2780s, SKOV3, breast cancer MCF-7, MDA-MB-231, and
liver cancer HepG2 cells) and hematological tumor (multiple myeloma ARD, MM1S,
RPMI-8226, B cell lymphoma Raji, Jeko-1, and Ramos cells), by MTT, with SAHA
used as the positive control. As presented in Table 5, compound 14b possessed more
potent inhibitory activity than SAHA. The IC values of compound 14b ranged from
5
0
9.8 to 44.9 nM, whereas SAHA was weakly active toward the tumor cell lines, with
IC s > 500 nM. Remarkably, compound 14b was equally effective toward the solid
5
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and hematological tumor cell lines, whereas the traditional HDAC inhibitors such as
SAHA are only effective toward hematological tumors. Those results confirmed that
compound 14b is a potent antitumor agent that could be used to treat solid tumors.
To determine the antiproliferative efficacy of 14b toward primary cells, we
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obtained PB blasts from three AML patients (Supplementary Table S1) and evaluated
the antiproliferative activity of 14b toward these cells. Compound 14b effectively
inhibited cell proliferation, with IC50 values of 21.2, 77.4, and 61.13 nM observed.
These results prompted us to further study compound 14b.
Table 5. The activity of compound 14b against various human tumor cell lines and
three primary AML cell lines.
a
IC ± SD (nM)
5
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Tumor type
Cell line
1
4b
SAHA
Colon
HCT116
A2780s
16.0 ± 3.3
40.0 ± 19.5
20.5 ± 5.4
14.4 ± 3.5
25.6 ± 4.2
13.0 ± 28
44.9 ± 1.8
9.8 ± 1.1
720.0 ± 45.3
5541.0 ± 833.0
1130.0 ± 236.5
637.2 ± 56.4
1710.0 ± 230.8
514.5 ± 93.1
630.1 ± 177.5
590.3 ± 125.4
585.8 ± 116.5
867.4 ± 105.9
638.6 ± 137.4
514.6 ± 66.0
ND
Ovarian
SKOV3
Breast
MCF-7
MDA-MB-231
HepG2
Liver
Multiple myeloma ARD
MM1S
RPMI-8226
Raji
15.6 ± 4.4
12.1 ± 1.9
7.2 ± 1.7
B cell lymphoma
Jeko-1
Ramos
17.8 ± 0.5
21.2 ± 3.4
77.4 ± 8.2
61.13 ± 13.7
Patient #1
Patient #1
Patient #1
Primary
cells
AML
ND
ND
a
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: IC = concentration of the compound required to inhibit tumor cell proliferation by
50
50%. The data are expressed as the means ± SD from the dose-response curves of at
least three independent experiments. ND: Not determined.
2.5 Upregulation of the acetylation of the histone H and α-tubulin.
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Upregulation of the acetylation of histone H (substrate for HDAC 1, 2 and 3)
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and α-tubulin (substrate for HDAC6) are biomarkers of HDAC inhibition. To
investigate the ability of compound 14b in this context, western blot analysis of
α-tubulin and histone H acetylation in the MM1S cell line after 6 h of treatment with
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compound 14b or SAHA at 40, 200, 1000, and 5000 nM was performed, the results of
which are shown in Figure 2. Compound 14b could induce the formation of Ac-H3
and Ac-α-tubulin in a concentration-dependent manner, in agreement with the effects
of SAHA. Notably, compound 14b could significantly upregulate the acetylation of
histone H at 40 nM, whereas the acetylation level of α-tubulin could only be
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increased at high concentrations. This result confirmed the compound 14b is an
effective HDAC inhibitor.
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Figure 2. Western blot analysis of α-tubulin and histone H3 in the MM1S cell line
after 6 h of treatment with compound 14b or SAHA at 40, 200, 1000, and 5000 nM.
GAPDH was used as a loading control.
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2.6 Cell cycle and apoptotic induction assay
Previous studies have shown that the class I HDAC inhibitors promote the
induction of apoptosis, cell differentiation, and cell growth arrest. We further
evaluated compound 14b for its ability effect on cell cycle arrest and the induction of
apoptosis in B cell lymphoma Jeko-1 cells. The assays were performed at four
different doses (0, 10, 100 and 1000 nM) for 24 and 48 h, the results of which are
summarized in Figure 3. Consistent with previous reports, compound 14b induced
G1 cell cycle arrest in Jeko-1 cells after 24 h treatment in a dose-dependent manner
(Figure 3A and 3B). In addition, after 48 h of treatment, 14b displayed a clear
dose-dependent apoptotic effect in Jeko-1 cells, with the percentages of apoptosis
cells increasing from 6.41 up to 73.1% (Figure 3C and 3D). Thus, compound 14b can
block tumor cells in the G1-phase and induce tumor cell apoptosis in a
dose-dependent manner.
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Figure 3. (A) and (B) Jeko-1 cells were cultured with 14b (0, 10, 100, and 1000 nM)
for 24 h and the cell cycle distribution of these cells was analyzed. (C) and (D) Jeko-1
cells were cultured with 14b (0, 10, 100, and 1000 nM) for 48 h, and the cell
apoptosis induction of these cells was analyzed.
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2.7 Molecular docking
To identify potential interactions, compound 14b was docked into a crystal
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structure of HDAC1 (PDB code 4BKX) [36] using a previously reported method [30,
32, 37, 38]. In the predicted docking pose (shown in Figure 4), the hydroxyl group of
the hydroxamic tail transferred a proton to a neighboring basic residue and formed a
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penta-coordination bond to a catalytic Zn ion with the carbonyl group of the
hydroxamic tail, that involved residues, His178, Asp264, and Asp176. Simultaneously,
the NH of hydroxamic tail formed a hydrogen bond with the Gly149 residue. In the
linker region, the pyrimidine and methyl groups were formed three weak hydrogen
bonds with Gly149 and Asp99, with Asp99 also forming a pi-anion interaction with
1,2,4-oxadiazole. The piperidine ring allows the toluene ring to occupy a hydrophobic
pocket on the surface of the HDAC1 protein. In addition to this hydrophobic
interaction, the toluene formed a pi-donor hydrogen bonding interaction with His28,
while there is no such interaction in the docking simulation result of HDAC6 with
14b (Supplementary Figure S1). This result may explain the observed difference in
activity of compound 14b for HDAC1 over HDAC6. Therefore, these interactions
appeared to enhance the binding affinity of 14b for HDAC1.
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Figure 4. The binding models of 14b in HDAC1 crystal structure (PDB code 4BKX).
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2.8 Pharmacokinetic studies
HDAC inhibitors are well known to be limited by their low bioavailability,
which limits the administration of these compounds. To investigate the bioavailability
of compound 14b, it was administered to SD rats at 5 mg/kg both intravenously (i.v.)
and orally (p.o.), with blood samples subsequently analyzed for the concentration of
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14b using an LC-MS/MS system. As shown in Table 6, the oral bioavailability of 14b
was excellent in rats, up to 53.52%, suggesting that 14b is suitable both for i.v. and
p.o. dosing as a potent anticancer agent.
Table 6. Pharmacokinetic parameters tested in vivo
Route
14b
p.o.
i.v.
a
N
6
6
Dose (mg/kg)
5
5
-
1
-1 b
Cl (Lh kg )
11.87
6.33
433.26
954.91
0.39
22.31
29.13
231.89
240.96
1.01
c
V (L/kg)
ss
d
AUC (µg/L*h)
0
-t
e
Cmax (µg/L)
f
T1/2 (h)
g
F (%)
53.52
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: Numbers of rats. : Systemic clearance. : Volume of distribution following
d
intravenous dosing. : Area under the curve following intravenous dosing, integrated
drug concentration with respect to time and integrated drug concentration with respect
e
to time following oral dosing. : Maximum plasma concentration following
f
g
intravenous dosing. : Plasma half-life. : Percent of bioavailability.
2.9 Antitumor activity in a Daudi xenograft model.
To determine the antitumor potency of compound 14b, we established a Burkitt's
lymphoma Daudi xenograft model. Compound 14b was administrated three times
over the cours of a week (10 mg/kg i.v. and 20 mg/kg p.o.). After the experiment was
completed, the organ tissues and tumors of the experimental animals were collected,
and the safety and effectiveness of compound 14b were evaluated by H&E staining
and immunohistochemistry analysis. As shown in Figure 5A and 5B, 14b promoted a
remarkable reduction in tumor growth, with 46.1% (10 mg/kg, i.v.) and 53.8% (20
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mg/kg, p.o.) TGI observed. It is worth noting that oral administration had a better
effect than intravenous administration, which may be related to a higher
bioavailability of compound 14b. Immunohistochemical analysis showed that
compared with the control group, the administration group exhibited significantly
increased expression of Ac-H in tumor tissues, inhibited the proliferation of tumor
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cells, and caused DNA damage of tumor cells, with the oral administration group
being more effective than the intravenous group. In addition, all treated groups did not
exhibit obvious abnormal behavior or significant toxic side effects, and H&E staining
of organ tissues showed that compound 14b did not cause organ damage in
experimental animals (Supplementary Figure S2). These findings suggest that
compound 14b can be used as a potential oral anticancer drug.
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Figure 5. Antitumor effect of 14b on the Daudi xenograft model. (A) Changes in
tumor volume. (B) Tumor weight results. (c) H&E and immunohistochemistry results
of tumor tissues.
3. Conclusion
In summary, in this study we developed a novel series of HDAC inhibitors with
containing 1,2,4-oxadiazole as cap group and hydroxamic acid as ZBG group. The
inhibitory activities of the synthesized compounds on the HDAC1 and 6 isoforms and
a SAR analysis were performed. The most potent compound (14b) displayed optimal
HDAC inhibitory activity, especially toward HDAC1, 2, and 3, with IC values 1.8,
5
0
3.6 and 3.0 nM, respectively, with the antiproliferative IC values ranging from 9.8 to
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0
44.9 nM against 12 diverse cancer cell lines from both hematological and solid tumors.
Importantly, 14b also showed excellent antiproliferative activity against primary AML
cells. The results of western blot analyses indicated that compound 14b can increase
the level of Ac-α-tubulin and Ac-H4 in a concentration-dependent manner, and
molecular docking analysis showed the mode of action of compound 14b with
HDAC1. Further assays confirmed that compound 14b could promote cell cycle arrest
and induce apoptosis, while pharmacokinetic studies showed an up to 53.52% oral
bioavailability of 14b. In vivo pharmacodynamics experiments showed that
compound 14b achieved 46.1 and 53.8% inhibition at 10 and 20 mg/kg when i.v. and
p.o. administered, respectively, with no significant side effects. These results show
that compound 14b represents a new scaffold to target HDAC for novel antitumor
drug discovery.
4. Experimental section
4.1 Chemistry
All the chemical solvents and reagents, which were analytically pure without
further purification, were commercially available. TLC was performed on 0.20 mm
Silica Gel 60 F254 plates (Qingdao Ocean Chemical Factory, Shandong, China).
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resonance ( C NMR) spectra were recorded on a Bruker Avance 400 spectrometer
(Bruke Company, Germany) or Varian spectrometer (Varian, Palo Alto, CA).
Chemical shifts are given in ppm relative to tetramethylsilane (TMS) as an internal
standard, where TMS = 0.00 ppm. Mass spectra (MS) were measured by Q-TOF
Priemier mass spectrometer (Micro mass, Manchester, UK). Room temperature (rt) is
o
within the range 20-25 C.
4.1.1. t-Butyl-4-(N'-hydroxycarbamimidoyl)piperidine-1-carboxylate (2)
The t-butyl-4-cyanopiperidine-1-carboxylate 1 (2.0 g, 9.51 mmol) was dissolved
in ethanol (25 mL) and hydroxylamine hydrochloride (0.91 g, 14.27 mmol), water (5
mL) and sodium bicarbonate (1.6 g, 19.02 mmol) were added. The reaction mixture
was stirred 1 h at ambient temperature and then heated overnight at reflux. After
cooling to room temperature, the EtOH was removed in vacuo, and the aqueous layer
was extracted with EtOAc (3 × 60 mL). The organic fractions were combined, washed
with water (3 × 80 mL), then brine, and dried (Na SO ). Removal of the sovent in
2
4
vacuo afforded t-butyl-4-(N'-hydroxycarbamimidoyl)piperidine-1-carboxylate 2.
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White solid, yield: 81.3%. H NMR (400 MHz, DMSO-d ) δ: 8.79 (s, 1H), 5.31 (s,
6
2H), 4.01–3.87 (m, 2H), 2.80–2.58 (m, 2H), 2.14 (tt, J = 11.7, 3.5 Hz, 1H), 1.72–1.62
+
(m, 2H), 1.49–1.40 (m, 2H), 1.39 (s, 9H). MS (ESI), m/z: 244.2 [M + H] .
4.1.2. t-Butyl-4-(5-(chloromethyl)-1,2,4-oxadiazol-3-yl)piperidine-1-carboxylate (3)
To a solution of the compound 2 (1.5 g, 6.17 mmol) in dichloromethane (25 mL)
was added triethylamine (1.7 mL, 12.33 mmol) and the mixture was cooled to 0 °C.
Chloroacetyl chloride (0.69 mL, 9.25 mmol) was slowly added dropwise over 5 min.
The reaction was stirred at 0 °C for 10 min and then at room temperature for 2 h. The
solvent was removed under vacuum and then the 1,4-dioxane solution was added and
heated to reflux for 2 h. After completion of the reaction, the solvent was extracted
with EtOAc, washed with water and brine, and dried with anhydrous Na SO . Then
2
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the solvent was removed under reduced pressure. The product was purified by Flash
Chromatography eluting with 1/5 EtOAc/PE (petroleum ether) to give the title
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compound 3. Light yellow solid, yield: 79.2%. H NMR (400 MHz, DMSO-d ) δ:
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5.08 (s, 2H), 3.99–3.88 (m, 2H), 3.06 (tt, J = 11.3, 3.8 Hz, 1H), 2.99–2.85 (m, 2H),
+
1.97–1.87 (m, 2H), 1.61–1.46 (m, 2H), 1.40 (s, 9H). MS (ESI), m/z: 302.2 [M + H] .
4.1.3.
t-Butyl-4-(5-((4-(3-methoxy-3-oxoprop-1-en-1-yl)phenoxy)methyl)-1,2,4-oxadiazol-3
-yl)piperidine-1-carboxylate (4)
Preparation of the compound 3 (1 g, 3.31 mmol), methyl 4-hydroxycinnamate
(0.65 g, 3.65 mmol), caesium carbonate (1.62 g, 4.97 mmol) and potassium iodide
(54.8 mg, 0.3 mmol) were dissolved in acetonitrile (20 mL) and heated overnight at
reflux. Reaction mixture was cooled and the solvent removed under vacuum.
Extracted with EtOAc, washed with solution of sodium carbonate (Na CO ) and brine,
2
3
and dried with anhydrous Na SO . Then the solvent was removed under reduced
2
4
1
pressure to provide the title compound 4. White solid, yield: 90.2%. H NMR (400
MHz, CDCl ) δ: 7.64 (d, J = 16.0 Hz, 1H), 7.50 (d, J = 8.7 Hz, 2H), 7.00 (d, J = 8.8
3
Hz, 2H), 6.34 (d, J = 16.0 Hz, 1H), 5.29 (s, 2H), 4.21–4.06 (m, 2H), 3.80 (s, 3H),
3.04–2.96 (m, 1H), 2.96–2.87 (m, 2H), 2.06–1.95 (m, 2H), 1.84–1.71 (m, 2H), 1.47 (s,
+
9H). MS (ESI), m/z: 444.4 [M + H] .
4.1.4.
Methyl-3-(4-((3-(piperidin-4-yl)-1,2,4-oxadiazol-5-yl)methoxy)phenyl)acrylate-2,2,2-
trifluoroacetate (5)
To a solution of intermediate 4 (1.25 g, 6.3 mmol) dissolved in dichloromethane
(10 mL) was added TFA (2.34 mL) and the solution stirred at room temperature for 4
h. After completion of the reaction, the solvent removed under vacuum afforded an
oily liquid which was added to a diethyl ether solution to precipitate a white solid.
The white solid formed was collected by filtration and dried under vacuum to give the
1
title compound 5. White solid, yield: 95.1%. H NMR (400 MHz, DMSO-d ) δ: 8.62 (s,
6
1H), 8.36 (s, 1H), 7.72 (d, J = 8.8 Hz, 2H), 7.63 (d, J = 16.0 Hz, 1H), 7.09 (d, J = 8.8
Hz, 2H), 6.54 (d, J = 16.0 Hz, 1H), 5.58 (s, 2H), 3.71 (s, 3H), 3.34–3.28 (m, 2H),
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3.27–3.18 (m, 1H), 3.10–2.98 (m, 2H), 2.17–2.05 (m, 2H), 1.91–1.77 (m, 2H). MS
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(ESI), m/z: 344.2 [M + H] .
4.1.5. General procedure for synthesis of compounds 6a-p
To a solution of the compound 5 (2.27 mmol) in acetonitrile (10 mL) was added
potassium carbonate (4.53 mmol) and potassium iodide (0.2 mmol). Halide (2.49
mmol) was then added to the reaction mixture. The reaction mixture was stirred at
room temperature for 4 h. The solvent removed under vacuum, extracted with EtOAc,
the organic layers were combined, washed with water and brine, dried with MgSO4,
and concentrated in vacuo. Flash chromatography (5% EtOAc/petroleum ether to 30%
EtOAc/petroleum ether) afforded the title compound 6a-p.
4.1.5.1.
Methyl-3-(4-((3-(1-propylpiperidin-4-yl)-1,2,4-oxadiazol-5-yl)methoxy)phenyl)acryla
1
te (6a). Pale yellow solid, yield: 83%. H NMR (400 MHz, CDCl ) δ: 7.64 (d, J =
3
16.0 Hz, 1H), 7.49 (d, J = 8.7 Hz, 2H), 7.00 (d, J = 8.8 Hz, 2H), 6.34 (d, J = 16.0 Hz,
1H), 5.28 (s, 2H), 3.80 (s, 3H), 3.05–2.96 (m, 2H), 2.89–2.78 (m, 1H), 2.43–2.31 (m,
2H), 2.19–1.87 (m, 6H), 1.62–1.50 (m, 2H), 0.91 (t, J = 7.3 Hz, 3H).
4.1.5.2.
Methyl-3-(4-((3-(1-allylpiperidin-4-yl)-1,2,4-oxadiazol-5-yl)methoxy)phenyl)acrylate
1
(6b). White solid, yield: 87%. H NMR (400 MHz, CDCl ) δ: 7.64 (d, J = 16.0 Hz,
3
1H), 7.49 (d, J = 8.8 Hz, 2H), 7.00 (d, J = 8.8 Hz, 2H), 6.34 (d, J = 16.0 Hz, 1H),
5.96–5.83 (m, 1H), 5.28 (s, 2H), 5.25–5.15 (m, 2H), 3.80 (s, 3H), 3.10–3.03 (m, 2H),
3.02–2.95 (m, 2H), 2.90–2.79 (m, 1H), 2.25–2.03 (m, 4H), 2.00–1.88 (m, 2H).
4.1.5.3.
Methyl-3-(4-((3-(1-benzylpiperidin-4-yl)-1,2,4-oxadiazol-5-yl)methoxy)phenyl)acryla
te (6c). White solid, yield: 88%. 1H NMR (400 MHz, DMSO-d ) δ: 7.71 (d, J = 8.8
6
Hz, 2H), 7.63 (d, J = 16.0 Hz, 1H), 7.35–7.28 (m, 4H), 7.27–7.21 (m, 1H), 7.09 (d, J
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= 8.8 Hz, 2H), 6.54 (d, J = 16.0 Hz, 1H), 5.55 (s, 2H), 3.71 (s, 3H), 3.48 (s, 2H),
2.87–2.75 (m, 3H), 2.14–2.03 (m, 2H), 1.97–1.87 (m, 2H), 1.78–1.61 (m, 2H). MS
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4.1.5.4.
Methyl-3-(4-((3-(1-(2-methylbenzyl)piperidin-4-yl)-1,2,4-oxadiazol-5-yl)methoxy)ph
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enyl)acrylate (6d). White solid, yield: 88%. H NMR (400 MHz, CDCl ) δ: 7.64 (d, J
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= 16.0 Hz, 1H), 7.49 (d, J = 8.8 Hz, 2H), 7.30–7.26 (m, 1H), 7.23–7.10 (m, 3H), 6.99
(d, J = 8.8 Hz, 2H), 6.33 (d, J = 16.0 Hz, 1H), 5.28 (s, 2H), 3.80 (s, 3H), 3.47 (s, 2H),
2.97–2.90 (m, 2H), 2.88–2.78 (m, 1H), 2.36 (s, 3H), 2.18–2.09 (m, 2H), 2.03–1.95 (m,
2H), 1.94–1.81 (m, 2H).
4.1.5.5.
Methyl-3-(4-((3-(1-(3-methylbenzyl)piperidin-4-yl)-1,2,4-oxadiazol-5-yl)methoxy)ph
1
enyl)acrylate (6e). White solid, yield: 81%. H NMR (400 MHz, CDCl ) δ: 7.64 (d, J
3
= 16.0 Hz, 1H), 7.49 (d, J = 8.6 Hz, 2H), 7.21 (t, J = 7.4 Hz, 1H), 7.16 (s, 1H), 7.12 (d,
J = 7.5 Hz, 1H), 7.07 (d, J = 7.3 Hz, 1H), 6.99 (d, J = 8.6 Hz, 2H), 6.33 (d, J = 16.0
Hz, 1H), 5.28 (s, 2H), 3.80 (s, 3H), 3.51 (s, 2H), 3.01–2.90 (m, 2H), 2.88–2.76 (m,
1H), 2.35 (s, 3H), 2.20–2.07 (m, 2H), 2.03–1.87 (m, 4H).
4.1.5.6.
Methyl-3-(4-((3-(1-(4-methylbenzyl)piperidin-4-yl)-1,2,4-oxadiazol-5-yl)methoxy)ph
1
enyl)acrylate (6f). White solid, yield: 85%. H NMR (400 MHz, CDCl ) δ: 7.64 (d, J
3
= 16.0 Hz, 1H), 7.49 (d, J = 8.7 Hz, 2H), 7.21 (d, J = 7.8 Hz, 2H), 7.13 (d, J = 7.7 Hz,
2H), 6.99 (d, J = 8.6 Hz, 2H), 6.33 (d, J = 16.0 Hz, 1H), 5.27 (s, 2H), 3.80 (s, 3H),
3.49 (s, 2H), 2.98–2.89 (m, J = 11.4 Hz, 2H), 2.87–2.75 (m, 1H), 2.34 (s, 3H), 2.16–
2.06 (m, 2H), 2.03–1.96 (m, 2H), 1.96–1.84 (m, 2H).
4.1.5.7.
Methyl-3-(4-((3-(1-(2-fluorobenzyl)piperidin-4-yl)-1,2,4-oxadiazol-5-yl)methoxy)phe
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nyl)acrylate (6g). White solid, yield: 76%. H NMR (400 MHz, CDCl ) δ: 7.64 (d, J =
3
16.0 Hz, 1H), 7.49 (d, J = 8.8 Hz, 2H), 7.43–7.37 (m, 1H), 7.26–7.20 (m, 1H), 7.13–
7.08 (m, 1H), 7.04 (d, J = 8.8 Hz, 1H), 6.99 (d, J = 8.8 Hz, 2H), 6.33 (d, J = 16.0 Hz,
1H), 5.28 (s, 2H), 3.80 (s, 3H), 3.61 (s, 2H), 3.01–2.92 (m, 2H), 2.86–2.77 (m, 1H),
2.23–2.14 (m, 2H), 2.04–1.98 (m, 2H), 1.97–1.88 (m, 2H).
4.1.5.8.
Methyl-3-(4-((3-(1-(3-fluorobenzyl)piperidin-4-yl)-1,2,4-oxadiazol-5-yl)methoxy)phe
1
nyl)acrylate (6h). White solid, yield: 80%. H NMR (400 MHz, CDCl ) δ: 7.64 (d, J =
3
16.0 Hz, 1H), 7.49 (d, J = 8.7 Hz, 2H), 7.32–7.27 (m, 1H), 7.14–7.07 (m, 2H), 7.00 (d,
J = 8.8 Hz, 2H), 6.98–6.90 (m, 1H), 6.33 (d, J = 16.0 Hz, 1H), 5.28 (s, 2H), 3.80 (s,
3H), 3.54 (s, 2H), 2.99–2.79 (m, 3H), 2.27–1.84 (m, 6H).
4.1.5.9.
Methyl-3-(4-((3-(1-(4-fluorobenzyl)piperidin-4-yl)-1,2,4-oxadiazol-5-yl)methoxy)phe
1
nyl)acrylate (6i). White solid, yield: 82%. H NMR (400 MHz, CDCl ) δ: 7.64 (d, J =
3
16.0 Hz, 1H), 7.49 (d, J = 8.7 Hz, 2H), 7.32–7.26 (m, 2H), 7.03–6.95 (m, 4H), 6.33 (d,
J = 16.0 Hz, 1H), 5.28 (s, 2H), 3.80 (s, 3H), 3.48 (s, 2H), 2.95–2.88 (m, 2H), 2.87–
2.77 (m, 1H), 2.15–2.07 (m, 2H), 2.04–1.97 (m, 2H), 1.95–1.85 (m, 2H).
4.1.5.10
Methyl-3-(4-((3-(1-(2,4-difluorobenzyl)piperidin-4-yl)-1,2,4-oxadiazol-5-yl)methoxy)
1
phenyl)acrylate (6j). White solid, yield: 86%. H NMR (400 MHz, CDCl ) δ: 7.64 (d,
3
J = 16.0 Hz, 1H), 7.49 (d, J = 8.8 Hz, 2H), 7.37 (dd, J = 15.1, 8.4 Hz, 1H), 6.99 (d, J
= 8.8 Hz, 2H), 6.89–6.75 (m, 2H), 6.33 (d, J = 16.0 Hz, 1H), 5.28 (s, 2H), 3.80 (s, 3H),
3.56 (s, 2H), 2.95–2.90 (m, 2H), 2.86–2.77 (m, 1H), 2.22–2.12 (m, 2H), 2.07–1.97 (m,
2H), 1.96–1.87 (m, 2H).
4.1.5.11.
Methyl-3-(4-((3-(1-(3,4-difluorobenzyl)piperidin-4-yl)-1,2,4-oxadiazol-5-yl)methoxy)
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phenyl)acrylate (6k). White solid, yield: 89%. H NMR (400 MHz, CDCl ) δ: 7.64 (d,
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J = 16.0 Hz, 1H), 7.50 (d, J = 8.8 Hz, 2H), 7.23–7.16 (m, 1H), 7.13–7.05 (m, 1H),
7.05–7.02 (m, 1H), 7.00 (d, J = 8.8 Hz, 2H), 6.34 (d, J = 16.0 Hz, 1H), 5.29 (s, 2H),
3.80 (s, 3H), 3.46 (s, 2H), 2.94–2.87 (m, 2H), 2.87–2.79 (m, 1H), 2.18–2.08 (m, 2H),
2.06–1.98 (m, 2H), 1.96–1.84 (m, 2H).
4.1.5.12.
Methyl-3-(4-((3-(1-(2-chlorobenzyl)piperidin-4-yl)-1,2,4-oxadiazol-5-yl)methoxy)phe
1
nyl)acrylate (6l). Pale yellow solid, yield: 79%. H NMR (400 MHz, CDCl ) δ: 7.64
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(d, J = 16.0 Hz, 1H), 7.50 (d, J = 8.7 Hz, 2H), 7.35 (d, J = 7.6 Hz, 1H), 7.25–7.14 (m,
3H), 7.00 (d, J = 8.7 Hz, 2H), 6.34 (d, J = 16.0 Hz, 1H), 5.29 (s, 2H), 3.80 (s, 3H),
3.66 (s, 2H), 3.14–2.94 (m, 2H), 2.91–2.76 (m, 1H), 2.38–2.18 (m, 2H), 2.13–1.84 (m,
4H).
4.1.5.13.
Methyl-3-(4-((3-(1-(4-bromobenzyl)piperidin-4-yl)-1,2,4-oxadiazol-5-yl)methoxy)ph
1
enyl)acrylate (6m). Yellow solid, yield: 81%. H NMR (400 MHz, CDCl ) δ: 7.64 (d,
3
J = 16.0 Hz, 1H), 7.49 (d, J = 8.6 Hz, 2H), 7.44 (d, J = 8.1 Hz, 2H), 7.21 (d, J = 8.1
Hz, 2H), 7.00 (d, J = 8.5 Hz, 2H), 6.33 (d, J = 16.0 Hz, 1H), 5.28 (s, 2H), 3.80 (s, 3H),
3.48 (s, 2H), 2.94–2.88 (m, 2H), 2.87–2.78 (m, 1H), 2.18–2.07 (m, 2H), 2.05–1.97 (m,
2H), 1.96–1.83 (m, 2H).
4.1.5.14.
Methyl-3-(4-((3-(1-(4-methoxybenzyl)piperidin-4-yl)-1,2,4-oxadiazol-5-yl)methoxy)p
1
henyl)acrylate (6n). White solid, yield: 86%. H NMR (400 MHz, CDCl ) δ: 7.64 (d,
3
J = 16.0 Hz, 1H), 7.49 (d, J = 8.7 Hz, 2H), 7.30–7.22 (m, 2H), 6.99 (d, J = 8.7 Hz,
2H), 6.86 (d, J = 8.5 Hz, 2H), 6.33 (d, J = 16.0 Hz, 1H), 5.28 (s, 2H), 3.80 (s, 3H),
3.80 (s, 3H), 3.52 (s, 2H), 3.02–2.92 (m, 2H), 2.89–2.78 (m, 1H), 2.24–1.88 (m, 6H).
4.1.5.15.
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Methyl-3-(4-((3-(1-(2-cyanobenzyl)piperidin-4-yl)-1,2,4-oxadiazol-5-yl)methoxy)phe
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nyl)acrylate (6r). Pale yellow solid, yield: 75%. H NMR (400 MHz, CDCl ) δ: 7.66
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(d, J = 4.5 Hz, 1H), 7.63 (d, J = 3.5 Hz, 1H), 7.61–7.53 (m, 2H), 7.49 (d, J = 8.7 Hz,
2H), 7.35 (t, J = 7.4 Hz, 1H), 7.00 (d, J = 8.7 Hz, 2H), 6.33 (d, J = 16.0 Hz, 1H), 5.28
(s, 2H), 3.80 (s, 3H), 3.73 (s, 2H), 2.99–2.91 (m, 2H), 2.90–2.81 (m, 1H), 2.32–2.21
(m, 2H), 2.06–1.99 (m, 2H), 1.98–1.87 (m, 2H).
4.1.5.16.
Methyl-3-(4-((3-(1-(2-nitrobenzyl)piperidin-4-yl)-1,2,4-oxadiazol-5-yl)methoxy)phen
1
yl)acrylate (6p). Yellow solid, yield: 71%. H NMR (400 MHz, CDCl ) δ: 7.83 (d, J =
3
7.7 Hz, 1H), 7.68–7.61 (m, 2H), 7.58–7.52 (m, 1H), 7.50 (d, J = 8.7 Hz, 2H), 7.42–
7.36 (m, 1H), 7.00 (d, J = 8.7 Hz, 2H), 6.34 (d, J = 16.0 Hz, 1H), 5.28 (s, 2H), 3.86–
3.76 (m, 5H), 2.91–2.78 (m, 3H), 2.27–2.14 (m, 2H), 2.03–1.95 (m, 2H), 1.94–1.82
(m, 2H).
4.1.6. General procedure for synthesis of compounds 7a-p
The ester intermediate (1 mmol) was dissolved in dichloromethane and methanol
o
(1:2, 9 mL). The resulting solution was cooled to 0 C, and then hydroxylamine (50
wt% in water, 30 mmol) and sodium hydroxide (2 mmol) were added. At the
temperature, the reaction was stirred for 2 h. The solvent was then removed under
reduced pressure, and the obtained solid was dissolved in water, which was adjusted
to pH = 7–8 by acetic acid to precipitate a white solid. The white solid formed was
collected by filtration to afford the crude 7a-p and purified by flash chromatograph.
4.1.6.1.
N-Hydroxy-3-(4-((3-(1-propylpiperidin-4-yl)-1,2,4-oxadiazol-5-yl)methoxy)phenyl)a
o
1
crylamide (7a). White powder, yield: 62%, m.p.: 133-135 C. H NMR (400 MHz,
DMSO-d ) δ: 7.58–7.49 (m, 2H), 7.41 (d, J = 15.8 Hz, 1H), 7.08 (d, J = 8.7 Hz, 2H),
6
6.34 (d, J = 15.8 Hz, 1H), 5.29 (s, 2H), 3.10–3.00 (m, 1H), 2.88–2.79 (m, 2H), 2.23
(m, 2H), 2.08–1.99 (m, 4H), 1.79–1.64 (m, 2H), 1.49–1.36 (m, 2H), 0.84 (t, J = 7.4
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Hz, 3H). C NMR (101 MHz, DMSO-d ) δ: 183.08, 175.18, 166.80, 159.02, 129.50,
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128.83, 115.65, 61.26, 60.41, 52.95, 52.62, 34.16, 29.96, 29.54, 20.09, 20.05, 12.31.
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4.1.6.2.
3-(4-((3-(1-Allylpiperidin-4-yl)-1,2,4-oxadiazol-5-yl)methoxy)phenyl)-N-hydroxyacr
o
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ylamide (7b). Pale yellow powder, yield: 66%, m.p.: 127-129 C. H NMR (400 MHz,
DMSO-d ) δ: 7.58–7.49 (m, 2H), 7.41 (d, J = 15.8 Hz, 1H), 7.08 (d, J = 8.4 Hz, 2H),
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6.35 (dd, J = 15.8, 3.2 Hz, 1H), 5.89–5.75 (m, 1H), 5.30 (s, 2H), 5.22–2.06 (m, 2H),
3.11–3.00 (m,1H), 2.95 (d, J = 6.4 Hz, 2H), 2.88–2.78 (m, 2H), 2.13–1.89 (m,4H),
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1.80–1.61 (m, 2H). C NMR (101 MHz, DMSO-d ) δ: 183.03, 175.20, 173.46,
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166.81, 163.44, 136.08, 129.51, 117.90, 115.65, 61.49, 61.49, 61.25, 52.72, 52.38,
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33.98, 33.65, 29.87, 29.45. MS (ESI), m/z: 385.1 [M + H] .
4.1.6.3.
3-(4-((3-(1-Benzylpiperidin-4-yl)-1,2,4-oxadiazol-5-yl)methoxy)phenyl)-N-hydroxya
o
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crylamide (7c). White powder, yield: 71%, m.p.: 83-84 C. H NMR (400 MHz,
DMSO-d ) δ: 10.68 (s, 1H), 8.99 (s, 1H),7.53 (d, J = 8.3 Hz, 2H), 7.40 (d, J = 16.1 Hz,
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1H), 7.36–7.28 (m, 4H), 7.27–7.22 (m, 1H), 7.07 (d, J = 8.7 Hz, 2H), 6.34 (d, J = 15.8
Hz, 1H), 5.52 (s, 2H), 3.48 (s, 2H), 2.88–2.73 (m, 3H), 2.13–2.03 (m, 2H), 1.97–1.85
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(m, 2H), 1.78–1.61 (m, 2H). C NMR (101 MHz, DMSO-d ) δ: 175.20, 173.46,
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166.81, 158.63, 138.89, 129.54, 129.23, 128.63, 127.34, 115.63, 62.76, 61.24, 52.76,
+
33.64, 29.91. MS (ESI), m/z: 435.16 [M + H] .
4.1.6.4.
N-Hydroxy-3-(4-((3-(1-(2-methylbenzyl)piperidin-4-yl)-1,2,4-oxadiazol-5-yl)methox
o
1
y)phenyl)acrylamide (7d). White powder, yield: 72%, m.p.: 154-156 C. H NMR
(400 MHz, DMSO-d ) δ: 7.51 (d, J = 6.9 Hz, 2H), 7.36 (d, J = 14.9 Hz, 1H), 7.22 (d,
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J = 5.8 Hz, 1H), 7.18–7.14 (m, 2H), 7.07 (d, J = 8.1 Hz, 3H), 6.33 (d, J = 15.7 Hz,
1H), 5.29 (s, 2H), 3.43 (s, 2H), 3.15–3.04 (m, 1H), 2.87–2.75 (m, 2H), 2.32 (s, 3H),
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