Development of hydroxamate-based histone deacetylase inhibitors containing 1,2,4-oxadiazole moiety core with antitumor activities

Article abstract of DOI:10.1016/j.bmcl.2018.11.027

Histone deacetylases (HDACs) has proved to be promising target for the development of antitumor drugs. In this study, we reported the design and synthesis of a class of novel hydroxamate-based bis-substituted aromatic amide HDAC inhibitors with 1,2,4-oxadiazole core. Most newly synthesized compounds displayed excellent HDAC1 inhibitory effects and significant anti-proliferative activities. Among them, compounds 11a and 11c increased acetylation of histone H3 and H4 in dose-dependent manner. Furthermore, 11a and 11c remarkably induced apoptosis in HepG2 cancer cells. Finally, the high potency of compound 11a was rationalized by molecular docking studies.

Full text of DOI:10.1016/j.bmcl.2018.11.027

Accepted Manuscript
Development of hydroxamate-based histone deacetylase inhibitors containing
1,2,4-oxadiazole moiety core with antitumor activities
Feifei Yang, Peipei Shan, Na Zhao, Di Ge, Kongkai Zhu, Cheng-shi Jiang,
Peifeng Li, Hua Zhang
PII:
S0960-894X(18)30894-1
https://doi.org/10.1016/j.bmcl.2018.11.027
BMCL 26138
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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17 October 2018
13 November 2018
Please cite this article as: Yang, F., Shan, P., Zhao, N., Ge, D., Zhu, K., Jiang, C-s., Li, P., Zhang, H., Development
of hydroxamate-based histone deacetylase inhibitors containing 1,2,4-oxadiazole moiety core with antitumor
activities, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.11.027
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Development of hydroxamate-based histone
deacetylase inhibitors containing 1,2,4-oxadiazole
moiety core with antitumor activities
,a
,b
Feifei Yang,† Peipei Shan,† Na Zhao,†, a Di Ge,a Kongkai Zhu,a Cheng-shi Jiang,a Peifeng Li*,b and
Hua Zhang*,a
a School of Biological Science and Technology, University of Jinan, Jinan, Shandong Province
250022, China
b Institute for Translation Medicine, Qingdao University, Qingdao, Shandong Province 266071,
China
† These authors contributed equally to this work.
*Corresponding Author: Dr. Hua Zhang or Dr. Peifeng Li, E-mail: bio_zhangh@ujn.edu.cn or
peifli@hotmail.com

ABSTRACT
Histone deacetylases (HDACs) has proved to be promising target for the development of
antitumor drugs. In this study, we reported the design and synthesis of a class of novel
hydroxamate-based bis-substituted aromatic amide HDAC inhibitors with 1,2,4-oxadiazole core.
Most newly synthesized compounds displayed excellent HDAC1 inhibitory effects and significant
anti-proliferative activities. Among them, compounds 11a and 11c increased acetylation of histone
H3 and H4 in dose-dependent manner. Furthermore, 11a and 11c remarkably induced apoptosis in
HepG2 cancer cells. Finally, the high potency of compound 11a was rationalized by molecular
docking studies.
Keywords:
1,2,4-oxadiazole, Hydroxamate, HDAC inhibitors, Structure-activity relationships, Antitumor
Epigenetics defines the heritable change of gene expression without modification of the DNA
nucleotide sequence1, 2. Histone lysine acetylation level regulated by HDACs and histone
acetyl-transferases (HATs) plays a key role in epigenetic modification of gene expression3. HDACs
catalyze the removal of acetyl groups from the  -amino lysine residues on histone tails, resulting in
chromatin conformation condensation associated with down-regulation of tumor suppressor genes4, 5.
In addition, HDACs also deacetylate many non-histone proteins, which influences the function,
protein-protein interaction and stability of these proteins6, 7. To date, 18 HDAC enzymes have been
identified and classified into four classes. Class I (HDACs 1, 2, 3, and 8), class II (HDACs 4, 5, 6, 7,
9 and 10) and class IV (HDAC11) HDACs are all belong to zinc-dependent enzymes, while class III
(SIRT1-7) belong to NAD+-dependent HDACs8, 9. The overexpression of HDACs have been found in

various cancers, making them attractive targets for anticancer therapy10, 11. Several studies have
demonstrated that Class I HDACs play a crucial role in regulating tumor growth, development and
apoptosis.
It has been confirmed that HDAC inhibitors (HDACis) significantly repress cancer cell
proliferation, angiogenesis, and metastasis while induce apoptosis, cell cycle arrest and DNA damage
through multiple antitumor pathways12, 13. Up to now, five HDACis, vorinostat (1 SAHA)14,
romidepsin (2 FK-228)15, belinostat (3 PXD-101)16, panobinostat (4 LBH-589)17 and chidamide (5
Epidaza)18, have been approved by FDA or CFDA for the treatment of cutaneous T-cell lymphoma
(CTCL), peripheral T-cell lymphoma (PTCL) or multiple myeloma (MM), and more than 20
HDACis, such as compounds 6 and 7, are at various stages of clinical trials as single agents or in
combination with other chemotherapeutic agents for the treatment of various types of tumors19, 20
.
However, most of the investigated HDACis display relatively weak efficacy against solid tumors.
The development of novel HDACis with improved potency against solid tumors is highly needed, to
expand their application in broad spectrum of cancer21.

Figure 1. Structures of FDA approved HDACis and representative examples in clinical trials.
Most HDACis share the common three-motif pharmacophore model, comprising an aromatic
amide surface recognition group (usually called CAP group), a zinc binding group (ZBG), and a
linker part connecting ZBG and CAP22. Hydroxamic acid-based HDACis with branched CAP region
(8) have been demonstrated to have anticancer effects23-25. In previous report, we have identified a
series of novel hydroxamate-based HDACis of bis-substituted aromatic amides, with potent activities
against tumor growth and metastasis26. Detailed SAR analysis revealed that the branched CAP region
containing a p-substituted phenyl and two linked heteroaromatic groups is important for increasing
potency. Particularly, compound 9 containing pyrimidinylthiopheneyl exhibited nanomolar IC50

values toward HDACs as well as submicromolar activity against proliferation and migration of breast
cancer cells both in vitro and in vivo. To investigate whether the pyrimidinylthiopheneyl functionality
is essential for the anti-cancer activities of these compounds, we designed a series of
phenyloxadiazole containing bis-substituted aromatic amides hydroxamate based HDACis (Figure 2).
Figure 2. The design of novel 1,2,4-oxadiazole containing hydroxamate based HDACis
Oxadiazole derivatives generally display specific and high pharmacological properties, such as
anti-inflammatory27, antimicrobial28, anticonvulsant29 and anti-cancer30 activities. Researchers mainly
focus on 1,2,4-oxadiazole for its multi-applicability in medicinal chemistry 31. Recently, a number of
1,2,4-oxadiazole derivations have been reported to display anticancer activities, and the results
demonstrated that substituents at C-3 and C-5 of 1,2,4-oxadiazole ring were necessary for the good
activity32, 33. In this study, we used 1,2,4-oxadiazole to replace thiophene of compound 9, then a
series of 1,2,4-oxadiazole containing bis-substituted aromatic amides HDACis were synthesized and
evaluated for their antitumor activies.
The synthesis of compounds 10a-d was outlined in Scheme 1. Appropriately substituted
nitriles 12a-c were converted to 13a-c, which were then cyclized with bromoacetyl bromide in
refluxing tetrahydrofuran to obtain 1,2,4-oxadiazole compounds 14a-c. Intermediates 14a-c were

subsequently condensed with p-anisidine to produce compounds 15a-c, which were further
condensed with different anhydrides in 1,4-dioxane to yield acids 16a-d. Compounds 16a-d then
underwent esterification using catalytic amounts of SOCl2 in methanol to produce methyl esters
17a-d, Finally, treatment of methyl esters with hydroxylamine to afford the target compounds 10a-d.
Scheme 1. Synthesis of compounds 10a-d
Reagents and conditions: (a) NH2OH·HCl, Et3N, MeOH, 12h, 80-90%; (b) BrCH2COBr, THF,
reflux, 12h, 50-55%; (c) p-Anisidine, K2CO3, DMF, 3h, 35-40%; (d) anhydride, 1,4-dioxane, reflux,
5h, 70-80%; (e) MeOH, Cat.SOCl2, reflux, 1h, 90-95%; (f) NH2OH·HCl, KOH, MeOH, 2h, 20-40%.
The preparation route for compounds 11a-i was depicted in Scheme 2. Coupling of
p-anisidine and hydroxylamine hydrochloride gave 19, which was further condensed with pimelic
acid anhydride to yield acid 20. Subsequent transformations were carried out using similar protocols
as outlined in Scheme 1. At last, the ester compounds 23a-i were converted to corresponding 11a-i
by reacting with NH2OH in methanol.
Scheme 2. Synthesis of compounds 11a-i

Reagents and conditions: (a) Bromoacetonitrile，K2CO3, DMF, 5h, 92.6%; (b) Pimelic acid
anhydride ， 1,4-dioxane, reflux, 4h, 92.1%; (c) MeOH, Cat.SOCl2, reflux, 1h, 90.5%; (d)
NH2OH·HCl, Na2CO3，EtOH/H2O, 12h, 65.8%; (e) RC6H4COCl, THF, reflux, 12h, 80-90%; (f)
NH2OH·HCl, KOH, MeOH, 2h, 20-40%.
On the basis of our previous results26, the series of bis-substituted aromatic amides
hydroxamate-based HDAC inhibitors were pan-HDAC inhibitors, and showed more potent inhibition
against class I HDAC isoforms especially HDAC1, so we chose HDAC1 for enzyme inhibition
assay. All synthesized compounds and the reference compound SAHA were detected for their ability
to inhibit HDAC1 at 20 nM. Results in Table 1 were shown as inhibition rates, indicating that the
four derivations (10a-d) showed weaker inhibitory activities than SAHA, compound 10d with five
methylene linker possessed stronger inhibitory rate than compound 10a, 10b and 10c with six

methylene linker, which was in line with our previous reported work26. Thus, the five methylene
linker length was retained in subsequent structural modifications.
Table 1 HDAC inhibition activity of compounds 10a-c
20 nM
Compd.
R
n
%inhibition on HDAC1
OCH3
CH3
H
6
6
6
5
35
30
40
50
60
10a
10b
10c
H
10d
SAHA
Assays were performed in replicate (n ≥ 3), the SD values are < 20% of the mean
Upon
further
modification,
3-aryl-5-alkyl-1,2,4-oxadiazole
was
converted
to
5-aryl-3-alkyl-1,2,4-oxadiazole to afford 11a. To further investigate how electron-withdrawing
groups (EWGs) or electron-donating groups (EDGs) of the benzene ring would affect potency,
compounds 11b-i were then synthesized and screened. The results in Table 2 displayed that all
derivations (11a-i) showed better inhibitory activities than SAHA. However, the potency of HDAC1
inhibition was only moderately affected by addition of EWGs or EDGs relative to that of 11a, with
no particular substituent-dependent trend being observed. To explore the optimal substitution position,
analysis of the activities of fluorine-substituted (11b-d) and methyl-substituted (11g-i) series revealed

that compounds with m-substitution (11c and 11h) showed better inhibition ratio than those with o- or
p-substitution.
Table 2 HDAC inhibition activity of compounds 11a-i
20 nM
Compd.
R
%inhibition on HDAC1
H
90
85
90
86
86
85
83
90
85
60
11a
11b
11c
2-F
3-F
11d
11e
4-F
4-Br
4-NO2
2-CH3
3-CH3
4-CH3
11f
11g
11h
11i
SAHA
Assays were performed in replicate (n ≥ 3), the SD values are < 20% of the mean
Three compounds (11a, 11c, 11h) with good HDAC1 inhibitory rates were further evaluated for
the IC50 values against HDAC1. All the three compounds showed promising activities with IC50

values in the nanomolar range (Table 3). Compound 11a was the most potent one, displaying IC50
value of 8.2 nM, which was nearly two fold lower than SAHA (IC50 = 15.0 nM). Compound 11c and
11h showed IC50 values of 10.5 and 12.1 nM, also lower than that of SAHA.
Table 3 IC50 values of HDAC1 inhibition of representative compounds
Compd.
IC50 (nM)
8.2
11a
11c
10.5
12.1
15.0
11h
SAHA
Assays were performed in replicate (n ≥ 3), the SD values are < 20% of the mean
Compounds 11a, 11c and 11h were further tested for their anti-proliferative activities against
human hepatocellular carcinoma cells (HCCLM3 and HepG2), SAHA was used as the reference
compound. The results are summarized in Table 4. All of these compounds showed excellent
anti-proliferative activities with the IC50 values in micromolar range (1.07 - 6.83 M) in both two cell
lines. Moreover, the results indicated that the inhibitory activities of these compounds on HepG2 are
more sensitive than HCCLM3 cells. Compounds 11a and 11c showed the better growth inhibition
with IC50 value of 1.07 M and 1.03 M in HepG2 cells, which was superior to SAHA obviously.
Table 4 Anti-proliferative activities of representative compounds against human hepatocellular
carcinoma cells
IC50 (M)
Compd.
HCCLM3
HepG2

5.19
6.56
6.83
6.52
1.07
1.03
1.68
4.50
11a
11c
11h
SAHA
Assays were performed in replicate (n ≥ 3), the SD values are < 20% of the mean
Given that the inhibition of HDACs by compounds 11a and 11c enhanced the tumor cell
anti-proliferative activities, the HDAC inhibitory effects of compounds on the level of acetylation of
histone H3 and H4 in HepG2 cells were determined by immunoblotting assays. HepG2 cells were
incubated with SAHA and compounds 11a and 11c (1.25, 2.5 and 5.0 M). As depicted in Figure 3,
both compounds can markedly increase the level of acetylated histone H3 and H4 in a
dose-dependent manner, which was consistent with the HDAC1 inhibition activities.

Figure 3. Western blot analysis of the effects of compounds 11a and 11c at different concentrations
on acetylated histone levels in HepG2 cells. A) Treatment resulted in upregulated expression of
acetylated H3 and H4 levels in dose dependent manner. B) Quantitative analysis. The levels of Ac-H3
and Ac-H4 relative to β-actin control were determined by densitometric scanning. (*P ≤ 0.05 vs.
SAHA treated group, n = 3)
To determine whether the inhibitory effects of target compounds on liver cancer cell
proliferation are accompanied by enhancing apoptosis, HepG2 cells were incubated with different
concentrations of target compounds and SAHA for 48 h. FITC-annexin V/propidium iodide (PI)
staining and flow cytometry assay were performed. As shown in Figure 4, compounds 11a and 11c

dose dependently induced apoptosis in HepG2 cells and the apoptosis percentage was even higher
than that of the positive control compound, SAHA.
Figure 4. Flow cytometry analysis. Compounds 11a and 11c mediated HepG2 cells apoptosis in
concentration dependent manner.
Reports suggest that compounds which meet the two criteria of (1) polar surface area (tPSA)
less than 140 Å2; (2) 12 or fewer H-bond donors and acceptors may have good oral bioavailability34,
35. Several parameters of target compounds were carried out for prediction of the ADME properties
through the molinspiration program (http://www.molinspiration.com/cgi-bin/properties). As shown in
Table 5, compounds 11a and 11c both have only nine hydrogen bond acceptors (n-ON) and two
donors (n-OHNH). tPSA did not exceed 140 A2. The calculated LogP (cLogP) in an acceptable range
(2 to 5). The above results indicated that compounds 11a and 11c are in a reasonable region for
further development as potential drug candidates.
Table 5. Theoretical Prediction of the ADME Properties of representative compounds

cLogP
2.77
MW
tPSA
117.79
117.79
78.42
n-ON
n-OHNH
Compd.
11a
438.48
456.47
264.32
9
9
5
2
2
3
2.91
11c
2.47
SAHA
To better explaining the effective HDAC1 inhibitory activity of compound 11a, the docking
assays (Autodock-4.27)36 were employed to analyze the binding model between 11a and HDAC1
(PDB code: 4BKX, chosen for its highest resolution). As shown in Figure 5, compound 11a and
SAHA shared the same binding mode (Figure 5A and 5B). The hydroxamate group of 11a could
chelate the zinc ion and could form two hydrogen bonds with GLY149 and HIS178 residues in the
active site of HDAC1. Meanwhile, the phenyl of compound 11a could also form π-π stacking
interaction with PHE 150 in the surface region of HDAC1. It is worth pointing out that the
4-methoxy group on the phenyl of compound 11a docked into the hydrophobic pocket of the surface
region of HDAC1, which could preliminarily rationalize the better inhibitory activity of 11a than
SAHA (Figure 5C).

Figure 5. Proposed binding mode of compound 11a and SAHA with HDAC1 (PDB 4BKX). A)
Molecular surface of the HDAC1 binding pocket with 11a. (B) Molecular surface of the HDAC1
binding pocket with SAHA. (C) 11a interacted with the active site of HDAC1.
Hydroxamic acid based HDACis with branched CAP region has been demonstrated to possess
anticancer effects. Based on previous research, a series of 1,2,4-oxadiazole containing bis-substituted
aromatic amides HDACis were synthesized and evaluated for their antitumor activities. Most newly
synthesized compounds displayed potent inhibitory activities against HDAC1. Several compounds
exhibited super anti-proliferative activities in HepG2 cell line, with an IC50 value range of 1.07-1.68
M, which were 2-4 fold lower than SAHA (IC50 = 4.50 M). Immunoblot analysis showed that 11a
and 11c increased the levels of acetylated histone H3 and H4 in dose dependent manner, confirming

its HDAC inhibitory action. Furthermore, compounds 11a and 11c could significantly induce cell
apoptosis. Finally, the high potency of compound 11a was interpreted by molecular docking study.
Taken together, these results suggest that these novel 1,2,4-oxadiazole containing HDAC inhibitors
could be identified as promising agents for hepatic carcinoma treatment.
Acknowledgments
We acknowledge the financial support from National Natural Science Foundation of China (No.
81703360), the Natural Science Foundation of Shandong Province (No. ZR2016HB43, JQ2017021),
the Young Taishan Scholars Program (tsqn20161037), and the Shandong Talents Team Cultivation
Plan of University Preponderant Discipline (No. 10027).

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Highlights:

Thirteen new hydroxamate-based bis-substituted aromatic amide HDAC inhibitors with
1,2,4-oxadiazole core were obtained.


The structures were elucidated by comprehensive spectroscopic analyses.
Three compounds displayed excellent HDAC1 inhibitory effects and significant anti-proliferative
activities.


Two compounds increased acetylation of histone H3 and H4 in dose-dependent manner.
Two compounds remarkably induced apoptosis in HepG2 cancer cells.