Discovery of N-(2-aminophenyl)-4-(bis(2-chloroethyl)amino)benzamide as a potent histone deacetylase inhibitor

Article abstract of DOI:10.3389/fphar.2019.00957

Inhibition of histone deacetylases (HDACs) has been an important emerging therapy for the treatment of multiple cancers. However, the application of HDAC inhibitors is restricted by the limited potency against solid tumors. In order to discover novel HDAC inhibitors with potent antitumor activities, nitrogen mustard group was introduced to the structure of CI994. The derived molecule N-(2-aminophenyl)-4-(bis(2-chloroethyl)amino) benzamide (NA) exhibited enzyme inhibitory pattern of class I selectivity with IC₅₀ values of 95.2, 260.7, and 255.7 nM against HDAC1, HDAC2, and HDAC3, respectively. In the antiproliferative assay, NA exhibited 10.3-fold (2.66 μM) and 11.3-fold (1.73 μM) higher potency than did suberoylanilide hydroxamic acid (SAHA) (27.3 and 19.5 μM) in inhibition of A2780 and HepG2 cell growth, respectively. Further HepG2 cell-based cell cycle and apoptosis studies revealed that induction of the G2/M phase arrest and cell apoptosis contributes to the antitumor effects of NA. It is suggested that NA could be utilized as a lead compound in the development of bifunctional HDAC inhibitors for the treatment of solid tumors.

Full text of DOI:10.3389/fphar.2019.00957

ORIGINAL RESEARCH
published: 30 August 2019
doi: 10.3389/fphar.2019.00957
Discovery of N-(2-Aminophenyl)-4-
(bis(2-chloroethyl)amino)Benzamide
as a Potent Histone Deacetylase
Inhibitor
Lihui Zhang1†, Xiaoyang Li2†, Yiming Chen3, Minghui Wan3, Qixiao Jiang4, Li Zhang5,
C. James Chou6, Weiguo Song3* and Lei Zhang3*
1 School of Stomatology, Weifang Medical University, Weifang, China, 2 School of Medicine and Pharmacy, Ocean University
of China, Qingdao, China, 3 Department of Medicinal Chemistry, School of Pharmacy, Weifang Medical University, Weifang,
China, 4 School of Public Health, Qingdao University, Qingdao, China, 5 School of Pharmacy, Qingdao University, Qingdao,
China, 6 Department of Drug Discovery and Biomedical Sciences, College of Pharmacy, Medical University of South Carolina,
Charleston, SC, United States
Edited by:
Zhe-Sheng Chen,
St. John’s University, United States
Inhibition of histone deacetylases (HDACs) has been an important emerging therapy
for the treatment of multiple cancers. However, the application of HDAC inhibitors is
restricted by the limited potency against solid tumors. In order to discover novel HDAC
inhibitors with potent antitumor activities, nitrogen mustard group was introduced to the
structure of CI994. The derived molecule N-(2-aminophenyl)-4-(bis(2-chloroethyl)amino)
benzamide (NA) exhibited enzyme inhibitory pattern of class I selectivity with IC50 values
of 95.2, 260.7, and 255.7 nM against HDAC1, HDAC2, and HDAC3, respectively. In the
antiproliferative assay, NA exhibited 10.3-fold (2.66 μM) and 11.3-fold (1.73 μM) higher
potency than did suberoylanilide hydroxamic acid (SAHA) (27.3 and 19.5 μM) in inhibition
of A2780 and HepG2 cell growth, respectively. Further HepG2 cell-based cell cycle and
apoptosis studies revealed that induction of the G2/M phase arrest and cell apoptosis
contributes to the antitumor effects of NA. It is suggested that NA could be utilized as a
lead compound in the development of bifunctional HDAC inhibitors for the treatment of
solid tumors.
Reviewed by:
Fuming Xu,
University of Michigan,
United States
Shuaishuai Liu,
University of Maryland,
United States
Yi-Chao Zheng,
Zhengzhou University, China
*Correspondence:
Lei Zhang
leiqdu@foxmail.com
Weiguo Song
songwg@139.com
†These authors have contributed
equally to this work
Specialty section:
This article was submitted to
Experimental Pharmacology
and Drug Discovery,
Keywords: HDAC, inhibitor, nitrogen mustard, antitumor, selectivity
a section of the journal
INTRODUCTION
Frontiers in Pharmacology
Histone deacetylases (HDACs) are a family of enzymes that are responsible for the epigenetic
regulation of histone and more than 50 nonhistone proteins (Bernstein et al., 2000; De Ruijter
et al., 2003; Foglietti et al., 2006). Currently, four classes of HDACs including 18 different isoforms
have been identified in human (Zhang et al., 2015). Class I HDACs contain HDAC1, 2, 3 and 8,
which mostly exist in the nucleus. Class II HDACs shuttle between the cytoplasm and the nucleus
and are subdivided into IIa (HDAC4, 5, 7, and 9) and IIb (HDAC6 and 10). Class III HDACs known
as sirtuins (sirt1–7) are a group of NAD+-dependent enzymes. HDAC11 classified as class IV is
low homologous with either class I or class II HDACs. In contrast with class III, class I, II, and IV
HDACs are zinc-dependent HDACs with zinc ions in their catalytic sites.
Received: 06 May 2019
Accepted: 26 July 2019
Published: 30 August 2019
Citation:
Zhang L, Li X, Chen Y, Wan M,
Jiang Q, Zhang L, Chou CJ, Song W
and Zhang L (2019) Discovery
of N-(2-Aminophenyl)-4-(bis(2-
chloroethyl)amino)Benzamide as a
Potent Histone Deacetylase Inhibitor.
Front. Pharmacol. 10:957.
HDAC inhibitors (HDACIs) have been developed for treatment of epigenetic disorders, especially
cancer (Marks et al., 2001). Suberoylanilide hydroxamic acid (SAHA) (Richon et al., 1998) and FK228
doi: 10.3389/fphar.2019.00957
Frontiers in Pharmacology | www.frontiersin.org
1
August 2019 | Volume 10 | Article 957

Zhang et al.
Novel HDAC Inhibitors
(Ueda et al., 1994) exhibited therapeutic significance for the
treatment of refractory cutaneous T-cell lymphoma (CTCL) and
had been approved by US Food and Drug Administration (FDA).
Additionally, PDX101 (Yang et al., 2010) and LBH589 (Neri
et al., 2012) had been approved for the treatment of peripheral
T-cell lymphoma (PTCL) and multiple myeloma, respectively.
Chidamide (Dong et al., 2009) is a benzamide HDACI approved
by Chinese Food and Drug Administration (CFDA) for the
treatment of relapsed or refractory PTCL in 2015. Moreover,
there are also more than 15 HDACIs currently being evaluated
in clinical investigations for the treatment of tumor and other
diseases, administrated in single and combination doses (http://
www.clinicaltrials.gov).
e discovery of nitrogen mustard as an alkylating agent
initiated modern cancer chemotherapy in 1942. e alkylating
agents inhibit tumor by crosslinking two DNA strands, preventing
DNA replication and inducing cell death (Singh et al., 2018). e
relative lack of selectivity towards DNA of both tumor cell and
normal cell led to nitrogen mustard’s various adverse side effects
along with therapeutic effects. e aromatic nitrogen mustards
generated by replacing the methyl group in nitrogen mustard
with aromatic groups had been characterized with stabilized
nitrogen and reduced drug reactivities.
Development of bifunctional molecules by linking of aromatic
nitrogen mustards to the structure of targeted antitumor
agents is a promising strategy in anticancer drug development
(Chen et al., 2018). In the discovery of effective and safe antitumor
molecules, the aromatic nitrogen mustard fragment was employed
to the structure of HDAC inhibitor in the present study. First,
molecular design was performed by replacement of the acetyl
group of CI994, a potent HDACI being evaluated in phase II
clinical trials to a 4-(bis(2-chloroethyl)amino)benzoyl group
(Figure 1). e derived target molecule, N-(2-aminophenyl)-4-
(bis(2-chloroethyl)amino)benzamide (NA), was synthesized and
evaluated with enzymatic selectivity assay, in vitro cancer cell-
based screening, cell cycle, and apoptosis studies.
ethylene oxide. Subsequent substitution of chlorine for hydroxyl
group and carboxyl group deprotection afforded key intermediate
4-(bis(2-chloroethyl)amino)benzoic acid (e). Target compound
NA was derived by condensation of e with o-phenylenediamine.
RESULTS AND DISCUSSIONS
Enzyme Inhibitory Selectivity of NA
To determine the isoform selectivity of NA, an enzymatic activity
inhibition assay was performed against HDAC1, 2, 3, 4, 6, 7, 8,
and 9 (Figure 2). Compared with the nonselective inhibitor
SAHA and the class I selective MS275, NA also exhibited
inhibitory selectivity of class I HDACs with IC50 values of 95.2,
260.7, and 255.7 nM against HDAC1, HDAC2, and HDAC3,
respectively (Table 1). Nevertheless, NA exhibited much reduced
inhibitory activity against HDAC8, which also belongs to the
class I HDACs with IC50 value of over 5,000 nM. e IC50 values
of NA in inhibition of the class IIa HDAC4, HDAC7, and HDAC9
and the IIb HDAC6 were also over 5,000 nM, indicating high
selectivity of NA. CI994 was also reported to be a class I HDAC
inhibitor. However, in the subfamily composed of HDAC1, 2 and
3, CI994 was nonselective between HDAC1 (41 nM) and HDAC3
(46 nM), while it had relatively weak HDAC2 inhibitory activity
(147 nM). It is remarkable that NA is an HDAC1-selective
inhibitor with 2.74-fold selectivity versus HDAC2 and 2.69-
fold selectivity versus HDAC3, respectively. erefore, molecule
NA could be promising in the treatment of a specific disease by
targeting HDAC1 and in probing the functions of HDAC1 in the
disease development.
Antiproliferative Activity of NA
A series of tumor cell lines including the lung cancer A549 and
Calu-3; breast cancer MDA-MB-231, MCF-7, and MDA-MB-468;
colon carcinoma LoVo and Colo205; ovarian cancer A2780 and
SKOV3; liver cancer HepG2; gastric cancer MKN45; pancreatic
cancer PNAC-1 cells; and leukemic U937 cells were cultured for
the antiproliferative assay of NA. Against most of the tested cell
lines, NA exhibited limited inhibitory activity than did SAHA
(Table 2). Remarkably, in the inhibition of the growth of A2780
and HepG2 cells, NA was revealed to be 10.3-fold (IC50 values
of 2.66 μM) and 11.3-fold (IC50 values of 1.73 μM) more potent
than was SAHA with IC50 values of 27.3 and 19.5, respectively.
e results suggested that NA may be further specifically used
Chemistry
Title molecule N-(2-aminophenyl)-4-(bis(2-chloroethyl)amino)
benzamide (NA) was synthesized according to the procedures
described in Scheme 1. e starting material 4-aminobenzoic acid
(a) was protected by methyl esterification, and the intermediate
methyl 4-(bis(2-hydroxyethyl)amino)benzoate (c) that followed
was derived by addition of two 2-hydroxyethyl groups using
FIGURE 1 | Design of NA from the structure of CI994.
Frontiers in Pharmacology | www.frontiersin.org
2
August 2019 | Volume 10 | Article 957

Zhang et al.
Novel HDAC Inhibitors
SCHEME 1 | Synthesis of compound NA. Reagents and conditions: (1) acetyl chloride, methanol; (2) ethylene oxide, acetic acid/water; (3) POCl3, toluene; (4)
concentrated hydrochloric acid; (5) N,N′-carbonyldiimidazole, trifluoroacetic acid, tetrahydrofuran.
FIGURE 2 | Inhibition curves of NA (A), MS275 (B), and SAHA (C) against various HDACs.
TABLE 1 | Enzyme inhibitory activity of NA compared with MS275 and SAHA (IC50, nM)a.
HDACIs
HDAC1
HDAC2
HDAC3
HDAC4
HDAC6
HDAC7
HDAC8
HDAC9
NA
MS275
SAHA
95.2
53.9
50.7
260.7
108.2
90.4
255.7
77.2
164.1
>5,000
>5,000
>5,000
>5,000
>5,000
169.5
>5,000
>5,000
>5,000
> 5,000
> 5,000
4,008
>5,000
>5,000
>5,000
aAssays were performed in replicate (n ≥ 2); the SD values are <10% of the mean.
for the treatment of liver cancer and ovarian cancer. Since the
application of classical HDACIs had been restricted by the lack
of therapeutic efficacy against solid tumors, the discovery of NA,
an HDAC1-selective inhibitor with potent solid tumor (ovarian
and liver cancer) inhibitory effects, is remarkable.
genetic mutations, dysregulated cell cycle resulted in uncontrolled
cell proliferation. To evaluate the effects of NA on cell cycle
distributions, HepG2 cells were treated with various doses of NA
and SAHA (1, 3, and 9 µM) for 6 h. As shown in Figure 3, both
NA (Figure 3A) and SAHA (Figure 3B) could regulate cell cycle
in a dose-dependent manner. It is revealed that NA treatment
leads to significantly higher accumulation of HepG2 cells at the
G2/M phase (25.98%, 35.05%, and 38.89% at concentrations of 1,
3, and 9 µM, respectively) compared with SAHA (23.98%, 25.89%,
and 27.02% at concentrations of 1, 3, and 9 µM, respectively).
Cell Cycle Analysis
e cell cycle consists of three distinct phases including the G0/
G1 phase, S phase, and G2/M phase. In tumor cells, as a result of
Frontiers in Pharmacology | www.frontiersin.org
3
August 2019 | Volume 10 | Article 957

Zhang et al.
Novel HDAC Inhibitors
TABLE 2 | Antiproliferative activity of NA compared with SAHA (IC50, μM)a.
further investigate the role of apoptosis in the antitumor effect
of NA, flow cytometry analysis was performed by staining
HepG2 cells with annexin V-fluorescein isothiocyanate (FITC)/
propidium iodide (PI). It is revealed that both NA (Figure 4A)
and SAHA (Figure 4B) treatment induced HepG2 cell apoptosis
in a dose-dependent manner. Afer treatment with difference
doses of NA (1, 3, and 9 µM), the percentage of apoptotic
cells was significantly increased from 3.52% of the control to
9.15%, 18.07%, and 37.39%, respectively, than that of SAHA
(4.52%, 6.24%, and 20.32% at concentrations of 1, 3, and 9 µM,
respectively). It is indicated that induction of cell apoptosis
contributes to the antitumor effect of NA.
Cell line
Tumor type
NA
SAHA
A549
Calu-3
Lung cancer
Lung cancer
14.74
5.81
1.73
3.18
1.11
2.78
1.44
1.07
2.01
27.3
1.73
19.5
16.06
6.57
1.02
MDA-MB-231
MCF-7
MDA-MB-468
LoVo
Colo205
A2780
SKOV3
HepG2
Breast carcinoma
Breast carcinoma
Breast carcinoma
Colon carcinoma
Colon carcinoma
Ovarian cancer
Ovarian cancer
Liver cancer
7.64
18.71
6.33
10.37
33.81
2.66
3.91
1.73
MKN45
PNAC-1
U937
Gastric Cancer
Pancreatic cancer
Myeloid leukemia
14.35
8.52
1.21
Western Blotting studies
aAssays were performed in replicate (n ≥ 2); the SD values are <10% of the mean.
Western blotting analysis was performed to evaluate effects
of NA on the expression of apoptosis and cell cycle-related
proteins in tumor cells. Sequential activation of caspases such
as caspase-3 and caspase-9 plays a central role in apoptosis.
Cdc2 is a specific regulator in the cell cycle transition from
the G2 to M phase. erefore, effects of NA treatment on the
protein expression levels of caspase-3, cleaved caspase-3,
caspase-9, cleaved caspase-9, cdc2, and phosphorylated cdc2
were evaluated in HepG2 cells (Figure 5). e results revealed
that NA remarkedly increased the protein expression levels
of cleaved caspase-3 and caspase-9 and up-regulated the
phosphorylation of cdc2 than did SAHA. It is suggested that
NA promoted HepG2 cell apoptosis via activation of caspase-3
and caspase-9, and induced the G2/M phase arrest of HepG2
cells via activation of cdc2.
At the same time, reduced cell population at the G0/G1 phase
was detected from 55.60% (control) to 48.64% (1 µM), 41.61%
(3 µM), and 37.19% (9 µM) following treatment with NA, while
SAHA treatment had no significant effects in the population of
HepG2 cells at the G0/G1 phase. It is suggested that induction of
the G2/M phase arrest contributes to the antiproliferative effects
of molecule NA.
Cell Apoptosis Assay
Cancer is one of the scenarios where too little apoptosis occurs,
resulting in malignant cells that will not die. erefore, apoptosis
induction plays an important role in the treatment of cancer. To
FIGURE 3 | Cell cycle analysis of molecule NA on HepG2 cells. Cells were treated with NA (A) and SAHA (B) at concentrations of 1, 3, and 9 µM for 6 h. The results
were detected by flow cytometry analysis.
Frontiers in Pharmacology | www.frontiersin.org
4
August 2019 | Volume 10 | Article 957

Zhang et al.
Novel HDAC Inhibitors
the present study, nitrogen mustard group was introduced to
the structure of HDAC inhibitor (CI994), the derived molecule
that exhibited improved potency in the growth inhibition of
solid tumor cells (A2780 and HepG2) compared with SAHA.
It is suggested that the insufficient potency of HDACIs against
solid tumors could be overcame by development of bifunctional
molecules with pharmacophores of other anticancer drugs, such
as the nitrogen mustard group.
CONCLUSION AND DISCUSSION
In the discovery of HDAC inhibitors with potent antitumor
activity, nitrogen mustard group was introduced to the structure
of CI994. e derived molecule NA exhibited class I selectivity,
and especially HDAC1 inhibitory activity (with IC50 values of
95.2 nM) in the enzyme inhibitory assay. In the antiproliferative
assay, NA exhibited less potent activity in the inhibition of
the growth of most tested cells. However, in the inhibition of
A2780 and HepG2 cells, NA exhibited significantly improved
activities than did SAHA. Further, HepG2 cell-based cell cycle
and apoptosis analysis revealed the role of the G2/M phase arrest
and apoptosis in the antitumor effects of NA. Western blotting
revealed induction of cleaved caspase 3/9 and phosphorylation
of cdc2, further confirming the participation of apoptosis and
cell cycle arresting in NA-induced antitumor effects. Collectively,
a potent HDAC1 inhibitor (NA) was discovered, which could
be utilized as a potent lead compound in the development of
anticancer agents targeting solid tumors such as liver cancer.
Inhibition of HDACs is an effective strategy for the treatment
of cancer. A large number of HDAC inhibitors have been
designed, synthesized, and evaluated in the anticancer activity
tests. Until now, four HDAC inhibitors have obtained approval
from the US FDA for the treatment of cancer. However, most
HDAC inhibitors exhibited limited potency against solid tumors,
and none of the approved HDAC inhibitors showed significant
potency in clinical trials for the treatment of solid tumors. In
MATERIALS AND METHODS
All commercially available starting materials, reagents, and
solvents were used without further purification. All reactions
were monitored by thin-layer chromatography (TLC) with 0.25-
mm silica gel plates (60GF-254). UV light and ferric chloride
were used to visualize the spots. 1H NMR and 13C NMR spectra
were recorded on a Bruker DRX spectrometer at 500 MHz,
using tetramethylsilane (TMS) as an internal standard. High-
resolution mass spectra were performed in Shandong Analysis
and Test Center in Jinan, China. e derived target compound
(NA) is of 98.28% purity proved by high-performance liquid
chromatography (HPLC) analysis, which was performed on a
Waters Acquity H class HPLC instrument using an Inertsil ODS.3
column (150 mm × 4.6 mm). e mobile phase was acetonitrile–
water, and linear gradient elution (with H2O% from 5% to 90% in
3 min) was used with detection wavelength of 254 nm.
FIGURE 4 | Pro-apoptotic effect of molecule NA. HepG2 cells were treated with NA (A) and SAHA (B) at concentrations of 1, 3, and 9 µM for 24 h. Then cells were
stained with annexin V-FITC/PI, and the results were detected by flow cytometry analysis.
Frontiers in Pharmacology | www.frontiersin.org
5
August 2019 | Volume 10 | Article 957

Zhang et al.
Novel HDAC Inhibitors
to the gummy residue and refluxed for 4 h. e pink solution was
filtered, and 4-(bis(2-chloroethyl)amino)benzoic acid (e) was
obtained by recrystallization of the filtered solid in ethyl acetate
(1.78 g, 68% yield). ESI-MS m/z: 263.1 [M + H]+.
N-(2-Aminophenyl)-4-(bis(2-chloroethyl)amino)benzamide
(NA). To a solution of compound e (2.0 g, 7.6 mmol) in
tetrahydrofuran (THF) (40 ml), CDI (1.8 g, 11 mmol) was added,
and the solution was refluxed for 3 h. N,N′-Carbonyldiimidazole
(6.5 g, 60 mmol) and trifluoroacetyl (TFA) were added with
stirring, and the mixture was kept for 20 h at room temperature.
en, the solvent was evaporated with the residue being taken up
in EtOAc (50 ml). e EtOAc solution was washed with 1 M of
citric acid (3 × 20 ml), NaHCO3 (3 × 20 ml), and brine (3 × 20 ml);
dried over MgSO4; and evaporated under vacuum. e desired
compound NA (1.35 g, 50% yield) was derived by crystallization
in EtOAc as white powder. High-resolution mass spectrometry
(HRMS) (AP-ESI) m/z calcd for C17H20Cl2N3O [M + H]+
352.0983, found 352.0979. 1H NMR (500 MHz, (CD3)2SO) δ 9.39
(s, 1H), 7.87 (d, J = 8.8 Hz, 2H), 7.14 (d, J = 7.6 Hz, 1H), 6.94 (t,
J = 7.6 Hz, 1H), 6.83 (d, J = 8.9 Hz, 2H), 6.77 (d, J = 7.8 Hz, 1H),
6.59 (t, J = 7.5 Hz, 1H), 4.82 (s, 2H), 3.95–3.66 (m, 8H). 13C NMR
(101 MHz, DMSO) δ 165.27, 149.38, 143.52, 130.04, 127.00,
126.57, 124.45, 122.63, 116.83, 116.68, 111.38, 52.27, 41.52 ppm.
HPLC retention time: 1.96 min, gradient eluted by CH3CN/H2O.
In Vitro HDAC Inhibitory Assay
All of the HDAC enzymes were bought from BPS Bioscience.
In vitro HDAC inhibition assays were conducted as previously
described. Briefly, 20 μl of recombinant HDAC enzyme solution
(HDAC1−9) was mixed with various concentrations of tested
compound (20 μl). e mixture was incubated at 30°C for 1 h
(for the time-dependent assay, the mixture was incubated for 15,
30, 60, and 90 min, respectively), and then 10 μl of fluorogenic
substrate (Boc-Lys (acetyl)-AMC (3 mM) for HDAC1, 2, 3, and 6,
Boc-Lys (trifluoroacetyl)-AMC (3 mM) for HDAC 4, 7, 8, and 9)
was added. Afer incubation at 30°C for 2 h, the catalytic reaction
was stopped by addition of 10 μl of developer containing trypsin
and trichostatin A (TSA). Afer 30 min, fluorescence intensity was
measured using a microplate reader at excitation and emission
wavelengths of 360 and 460 nm, respectively. e inhibition ratios
were calculated from the fluorescence intensity readings of tested
wells relative to those of control wells, and the IC50 curves and
values were determined by GraphPad Prism 6.0 sofware.
FIGURE 5 | NA induced apoptosis-related protein expression in HepG2
cell. HepG2 cells were treated with SAHA and NA at 1 µM for 24 h.
The apoptosis-related proteins cleaved caspase-3, caspase-9, and
cell cycle-related protein cdc2 were analyzed by western blotting.
β-Actin was used as an internal control. A representative immunoblot
from three independent experiments giving similar results was shown
for each western blot experiment. Densitometry was performed using
AlphaEaseFC-v4.0.0 program.
Methyl 4-aminobenzoate hydrochloric acid (b) has been
synthesized and described in our previous work.
Methyl 4-(bis(2-hydroxyethyl)amino)benzoate (c). Methyl
4-aminobenzoate hydrochloric acid (b) (18.8 g, 100 mmol) was
dissolved in water (50 ml) and glacial acetic acid (50 ml). Ethylene
oxide (60 ml) was added with stirring, and the mixture was kept
for 24 h at room temperature. e clear yellow solution was
poured into water (100 ml), a slight excess sodium bicarbonate
was carefully added with stirring, a gummy precipitate was
obtained, which was extracted with ethyl acetate and dried over
MgSO4. e solvent was evaporated and recrystallized to give
desired compound c (18.2 g, 76% yield). Electrospray ionization–
mass spectrometry (ESI-MS) m/z: 240.3 [M + H]+.
MTT Assay
Antiproliferative activities of NA were evaluated by MTT
assay using SAHA as the positive control. e stock solutions
of tested compounds were diluted with culture medium. e
cells were seeded in 96-well plates at a density 5 × 103 cells per
well and incubated until confluency of 90–95%, and then each
well was treated with 100 µl of medium containing the desired
concentrations of tested compounds and incubated for 48 h.
MTT working solution of 20 µl (5 mg/ml) was then added to each
well and incubated for another 4 h. At the end of incubation, the
medium were carefully removed, and 200 µl of DMSO was added.
Methyl 4-(bis(2-chloroethyl)amino)benzoate (d). To
a
solution of compound c (2.40 g, 10 mmol) in toluene (30 ml),
phosphorus oxychloride (10 ml) was added and refluxed for 1 h.
e solution was evaporated until a clear gummy residue was
obtained (d). Concentrated hydrochloric acid (6 M) was added
Frontiers in Pharmacology | www.frontiersin.org
6
August 2019 | Volume 10 | Article 957

Zhang et al.
Novel HDAC Inhibitors
e optical density at 490 and 630 nm was then measured with a
microplate reader (Model 680, Bio-Rad). e percentage of cell
growth inhibition was calculated with the following equation: %
inhibition = [1 − (Sample group OD490 − Sample group OD630)/
(Control group OD490 − Control group OD630)] × 100%. e IC50
values were calculated with Origin 7.5 sofware, and standard
deviations of the IC50 values were obtained from at least three
independent experiments.
5% fat-free dry milk in 1 × Tris-buffered saline (TBST) for
2 h at room temperature. Membranes were then probed with
corresponding antibodies at 4°C overnight. Cleaved caspase-3
(#9661), caspase-3 (#9662), cleaved caspase-9 (#9505), caspase-9
(#9508), phospho-cdc2 (#4539), and cdc2 (#77055) antibodies
were obtained from Cell Signaling Technology (Danvers, MA).
Expression of human β-actin was detected with anti-β-actin
antibody (Proteintech, Rosemont, IL) as a loading control. e
membranes were then incubated with horseradish peroxidase
(HRP)-conjugated secondary antibody (Santa Cruz, CA) and
visualized with enhanced chemiluminescence (ECL)-detecting
reagents (ComWin Biotech Co., Beijing, China).
Cell Cycle Assay
HepG2 cells in logarithmic growth phase were seeded in 6-well plates
(6 × 105 cells/well) and incubated with different doses of NA and
SAHA (1, 3, and 9 µM) for 6 h. Cells were then washed twice with
cold phosphate-buffered saline (PBS) and fixed in 70% precooled
ethanol at 4°C for 12 h. Afer the fixation, cells were washed
again with PBS and stained with PI/RNase A for 30 min at room
temperature and eventually subjected to flow cytometry (CytoFLEX,
Beckman Coulter) for cell cycle distribution determination.
Statistical Analysis
Results were expressed as mean of ≥2 independent experiments
with SD values < 10% of the mean. SPSS 19.0 sofware was used
in the statistical analysis, and the means between two groups
were compared by one-way analysis of variance (ANOVA) with
Dunnett’s test. Values of P < 0.05 were considered to indicate
significant differences.
Annexin V/PI Detection
HepG2 cells in logarithmic growth phase were seeded in 6-well
plates (4×105 cells/well) and incubated with different doses of NA
and SAHA (1, 3, and 9 µM) for 24 h. Cells were then washed with
PBS, collected, resuspended with binding buffer from the annexin
V-FITC kit (ermo Fisher Co., USA) and then added with 5 µl of
annexin V-FITC and mixed gently. Afer 10 min of incubation, 1 µl
of PI was added to each sample and mixed gently. Afer incubation
at room temperature for another 20 min in the dark, cells were
subjected to flow cytometry (CytoFLEX, Beckman Coulter).
DATA AVAILABILITY
e raw data supporting the conclusions of this manuscript will
be made available by the authors, without undue reservation, to
any qualified researcher.
AUTHOR CONTRIBUTIONS
WS and LeZ designed the project. XL and CC performed the
enzymatic screening; YC synthesized the molecules; WH, LHZ
and QX performed the in vitro antitumor experiments. LHZ, QJ
and LiZ analyzed the data and wrote the manuscript.
Western Blot
Afer incubation with NA and SAHA for 24 h, cells were rinsed
with cold PBS, and then radioimmunoprecipitation assay
(RIPA) buffer was added, and protein samples were collected by
scrubbing and centrifuged at 14,000g for 10 min. Protein sample
concentrations were determined with Brandford assay. irty
micrograms of each sample was subjected to sodium dodecyl
sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Afer
electrophoresis, proteins were transferred to polyvinylidene
difluoride (PVDF) membrane (Millipore) and blocked with
FUNDING
is work was supported by National Natural Science Foundation
of China (Youth Found, Grant No. 81803343) and Natural
Science Foundation of Shandong Province (Youth Found, Grant
No. ZR2019QH005).
Chen, L. F., and Greene, W. C. (2004). Shaping the nuclear action of NF-kappa B.
Nat. Rev. Mol. Cell Biol. 5 (5), 392–401. doi: 10.1038/nrm1368
Chen, Y. M., Jia, Y. P., Song, W. G., and Zhang, L. (2018). erapeutic potential of
nitrogen mustard based hybrid molecules. Front. Pharmacol. 9, 1–2. doi: 10.3389/
fphar.2018.01453
REFERENCES
Bernstein, B. E., Tong, J. K., and Schreiber, S. L. (2000). Genomewide studies of
histone deacetylase function in yeast. Proc. Natl. Acad. Sci. U.S.A. 97 (25),
13708–13713. doi: 10.1073/pnas.250477697
Bian, J., Zhang, L. H., Han, Y. T., Wang, C. B., and Zhang, L. (2015). Histone
deacetylase inhibitors: potent anti-leukemic agents. Curr. Med. Chem. 22 (17),
2065–2074. doi: 10.2174/0929867322666150416094720
Bode, A. M., and Dong, Z. G. (2004). Post-translational modification of p53 in
tumorigenesis. Nat. Rev. Cancer 4 (10), 793–805. doi: 10.1038/nrc1455
Cantley, M. D., and Haynes, D. R. (2013). Epigenetic regulation of inflammation:
progressing from broad acting histone deacetylase (HDAC) inhibitors to
targeting specific HDACs. Inflammopharmacology 21 (4), 301–307. doi:
10.1007/s10787-012-0166-0
De Ruijter, A. J. M., Van Gennip, A. H., Caron, H. N., Kemp, S., and Van Kuilenburg, A. B.
P. (2003). Histone deacetylases (HDACs): characterization of the classical HDAC
family. J. Biochem. 370, 737–749. doi: 10.1042/bj20021321
Dong, M., Ning, Z., Newman, M. J., Xu, J., Dou, G., Cao, H., et al. (2009). Phase I
study of chidamide (CS055/HBI-8000), a novel histone deacetylase inhibitor,
in patients with advanced solid tumors and lymphomas. J. Clin. Oncol. 27 (15),
3529–3529. doi: 10.1200/jco.2009.27.15_suppl.3529
Foglietti, C., Filocamo, G., Cundari, E., De Rinaldis, E., Lahm, A., Cortese, R., et al.
(2006). Dissecting the biological functions of Drosophila histone deacetylases
Frontiers in Pharmacology | www.frontiersin.org
7
August 2019 | Volume 10 | Article 957

Zhang et al.
Novel HDAC Inhibitors
by RNA interference and transcriptional profiling. J. Biol. Chem. 281 (26),
17968–17976. doi: 10.1074/jbc.M511945200
inhibits histone deacetylases. Proc. Natl. Acad. Sci. U.S.A. 95 (6), 3003–3007.
doi: 10.1073/pnas.95.6.3003
Gallo, P., Latronico, M. V., Gallo, P., Grimaldi, S., Borgia, F., Todaro, M., et al.
(2008). Inhibition of class I histone deacetylase with an apicidin derivative
prevents cardiac hypertrophy and failure. Cardiovasc. Res. 80 (3), 416–424. doi:
10.1093/cvr/cvn215
Giandomenico, V., Simonsson, M., Gronroos, E., and Ericsson, J. (2003).
Coactivator-dependent acetylation stabilizes members of the SREBP family
of transcription factors. Mol. Cell. Biol. 23 (7), 2587–2599. doi: 10.1128/
MCB.23.7.2587-2599.2003
Gronroos, E., Hellman, U., Heldin, C. H., and Ericsson, J. (2002). Control of
Smad7 stability by competition between acetylation and ubiquitination. Mol.
Cell 10 (3), 483–493. doi: 10.1016/S1097-2765(02)00639-1
Jin, Y. H., Jeon, E. J., Li, Q. L., Lee, Y. H., Choi, J. K., Kim, W. J., et al. (2004).
Transforming growth factor-beta stimulates p300-dependent RUNX3
acetylation, which inhibits ubiquitination-mediated degradation. J. Biol. Chem.
279 (28), 29409–29417. doi: 10.1074/jbc.M313120200
Kook, H., Lepore, J. J., Gitler, A. D., Lu, M. M., Wing-Man Yung, W., Mackay, J.,
et al. (2003). Cardiac hypertrophy and histone deacetylase-dependent
transcriptional repression mediated by the atypical homeodomain protein
Hop. J. Clin. Invest. 112 (6), 863–871. doi: 10.1172/JCI19137
Kovacs, J. J., Murphy, P. J. M., Gaillard, S., Zhao, X. A., Wu, J. T., Nicchitta, C. V.,
et al. (2005). HDAC6 regulates Hsp90 acetylation and chaperone-dependent
activation of glucocorticoid receptor. Mol. Cell 18 (5), 601–607. doi: 10.1016/j.
molcel.2005.04.021
Mai, A., Rotili, D., Valente, S., and Kazantsev, A. G. (2009). Histone deacetylase
inhibitors and neurodegenerative disorders: holding the promise.
Curr. Pharm. Des. 15 (34), 3940–3957. doi: 10.2174/138161209789649349
Margolis, D. M. (2011). Histone deacetylase inhibitors and HIV latency. Curr.
Opin. HIV and AIDS 6 (1), 25–29. doi: 10.1097/COH.0b013e328341242d
Marks, P. A., Rifind, R. A., Richon, V. M., Breslow, R., Miller, T., and Kelly, W. K.
(2001). Histone deacetylases and cancer: causes and therapies. Nat. Rev. Cancer
1 (3), 194–202. doi: 10.1038/35106079
Saiyed, Z. M., Gandhi, N., Agudelo, M., Napuri, J., Samikkannu, T., Reddy, P. V.,
et al. (2011). HIV-1 Tat upregulates expression of histone deacetylase-2
(HDAC2) in human neurons: implication for HIV-associated neurocognitive
disorder (HAND). Neurochem. Intern. 58 (6), 656–664. doi: 10.1016/j.
neuint.2011.02.004
Singh, R. K., Kumar, S., Prasad, D. N., and Bhardwaj, T. R. (2018). erapeutic
journery of nitrogen mustard as alkylating anticancer agents: historic to future
perspectives. Eur. J. Med. Chem. 151, 401–433. doi: 10.1016/j.ejmech.2018.04.001
Soragni, E., Xu, C., Cooper, A., Plasterer, H. L., Rusche, J. R., and Gottesfeld, J. M.
(2011). Evaluation of histone deacetylase inhibitors as therapeutics
for neurodegenerative diseases. Methods Mol. Biol. 793, 495–508. doi:
10.1007/978-1-61779-328-8_32
Ueda, H., Nakajima, H., Hori, Y., Fujita, T., Nishimura, M., Goto, T., et al.
(1994). Fr901228, a novel antitumor bicyclic depsipeptide produced by
Chromobacterium violaceum No-968. 1. Taxonomy, fermentation, isolation,
physicochemical and biological properties, and antitumor-activity. J. Antibiot.
47 (3), 301–310. doi: 10.7164/antibiotics.47.301
Yang, L., Xue, X. W., and Zhang, Y. H. (2010). Simple and efficient
synthesis of belinostat. Synth. Commun. 40 (17), 2520–2524. doi:
10.1080/00397910903277870
Ylisastigui, L., Archin, N. M., Lehrman, G., Bosch, R. J., and Margolis, D. M.
(2004). Coaxing HIV-1 from resting CD4 T cells: histone deacetylase
inhibition allows latent viral expression. AIDS 18 (8), 1101–1108. doi:
10.1097/00002030-200405210-00003
Zhang, L., Han, Y. T., Jiang, Q. X., Wang, C. B., Chen, X. H., Li, X. G., et al. (2015).
Trend of histone deacetylase inhibitors in cancer therapy: isoform selectivity or
multitargeted strategy. Res. Rev. 35 (1), 63–84. doi: 10.1002/med.21320
Conflict of Interest Statement: e authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Neri, P., Bahlis, N. J., and Lonial, S. (2012). Panobinostat for the treatment
of multiple myeloma. Exp. Opin. Invest. Drugs 21 (5), 733–747. doi:
10.1517/13543784.2012.668883
Patel, M. M., and Patel, B. M. (2018). Repurposing of sodium valproate in colon
cancer associated with diabetes mellitus: role of HDAC inhibition. Eur. J.
Pharm. Sci. 121, 188–199. doi: 10.1016/j.ejps.2018.05.026
Copyright © 2019 Zhang, Li, Chen, Wan, Jiang, Zhang, Chou, Song and Zhang.
is is an open-access article distributed under the terms of the Creative Commons
Attribution License (CC BY). e use, distribution or reproduction in other forums
is permitted, provided the original author(s) and the copyright owner(s) are credited
and that the original publication in this journal is cited, in accordance with accepted
academic practice. No use, distribution or reproduction is permitted which does not
comply with these terms.
Richon, V. M., Emiliani, S., Verdin, E., Webb, Y., Breslow, R., Rifind, R. A. et al.,
(1998). A class of hybrid polar inducers of transformed cell differentiation
Frontiers in Pharmacology | www.frontiersin.org
8
August 2019 | Volume 10 | Article 957