Design, synthesis and biological evaluation of novel hydroxamates and 2-aminobenzamides as potent histone deacetylase inhibitors and antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2017.03.038

Many studies have indicated that histone deacetylase (HDAC) inhibitors are promising agents for the treatment of cancer. With the aim to search for novel potent HDAC inhibitors, we designed and synthesized two series of hydroxamates and 2-aminobenzamides

Full text of DOI:10.1016/j.ejmech.2017.03.038

Accepted Manuscript
Design, synthesis and biological evaluation of novel hydroxamates and 2-
aminobenzamides as potent histone deacetylase inhibitors and antitumor agents
Rui Xie, Yue Yao, Pingwah Tang, Guangyao Chen, Xia Liu, Fan Yun, Chunhui Chen,
Xinying Wu, Qipeng Yuan
PII:
S0223-5234(17)30195-2
10.1016/j.ejmech.2017.03.038
EJMECH 9298
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 September 2016
Revised Date: 19 March 2017
Accepted Date: 20 March 2017
Please cite this article as: R. Xie, Y. Yao, P. Tang, G. Chen, X. Liu, F. Yun, C. Chen, X. Wu, Q. Yuan,
Design, synthesis and biological evaluation of novel hydroxamates and 2-aminobenzamides as potent
histone deacetylase inhibitors and antitumor agents, European Journal of Medicinal Chemistry (2017),
doi: 10.1016/j.ejmech.2017.03.038.
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Design, synthesis and biological evaluation of novel hydroxamates and 2-aminobenzamides as
potent histone deacetylase inhibitors and antitumor agents
Rui Xie1, Yue Yao1, Pingwah Tang*, Guangyao Chen, Xia Liu, Fan Yun, Chunhui Chen, Xinying Wu
and Qipeng Yuan*
Medicinal Chemistry Research Division, Beijing Laboratory of Biomedical Materials, College of Life Science and
Technology, Beijing University of Chemical Technology, 15 Beisanhuan East Road, Beijing 100029, China
*Corresponding authors, E-mail addresses: tangpw@mail.buct.edu.cn; yuanqp@mail.buct.edu.cn
1 These two authors contributed equally
ABSTRACT:
Many studies have indicated that histone deacetylase (HDAC) inhibitors are promising agents
for the treatment of cancer. With the aim to search for novel potent HDAC inhibitors, we
designed and synthesized two series of hydroxamates and 2-aminobenzamides compounds as
HDAC inhibitors and antitumor agents. Those compounds were investigated for their HDAC
enzymatic inhibitory activities and in vitro anti-proliferation activities against diverse cancer cell
line (A549, HepG2, MGC80-3 and HCT116). Most of the synthesized compounds displayed potent
HDAC inhibitory activity and antiproliferative activity. In particular, Compound 12a,
N-(2-aminophenyl)-4-[(4-fluorophenoxy)methyl]benzamide , was shown to have the most HDAC
inhibitory activity (70.6% inhibition at 5μM) and antitumor activity with IC50 value of as low as
3.84μM against HepG2 human liver hepatocellular carcinoma cell line, more than 4.8–fold lower
than CS055 and 5.9–fold lower than CI994. HDAC isoform selectivity assay indicated 12a is a
potent HDAC2 inhibitor. Docking study of 12a suggested that it bound tightly to the binding
pocket of HDAC2. Further investigation showed that 12a could inhibit the migration and colony
formation of A549 cancer cells. Furthermore, 12a remarkably induced apoptosis and G2/M phase
cell cycle arrest in A549 cancer cells. Those results indicated that compound 12a could be a
promising candidate for treatment of cancer.
Keywords: Histone deacetylase inhibitor; hydroxamates; 2-aminobenzamides; Antiproliferation;
Molecular Docking;
1. Introduction
Cancer is a widespread disease with high mortality. Traditional antitumor drugs usually result in
seriously side effect because of lacking targeted therapies[1]. Histone deacetylase (HDACs) have
become promising targets in recent years for cancer therapy. Histone lysine acetylation and
deacetylation are important post-translational covalent modifications[2]. The acetylation of
histiones is balanced by histone acetyltransferases (HATs) and histone deacetylase (HDACs)[3-9].
Overexpressed HDACs catalyze the removal of acetyl groups from histones, resulting in chromatin
condensation and silencing tumor suppressor genes[10-15]. HDAC inhibitors have been shown to
cause differentiation and apoptosis of tumor cells[16]. Therefore, HDAC inhibitors are promising
agents for the treatment of cancer. 18 HDAC isozymes have been identified so far, which can be
divided into 4 classes: classＩ(HDACs 1,2,3 and 8), classⅡ (HDACs 4,5,6,7,9 and 10), classⅣ
(HDACs 11), and class Ⅲ(sirtuins 1–7). ClassＩ, classⅡ, and classⅣ are zinc-dependent HDACs,

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while class are NAD-dependent sirtuins. It has been shown that the dysregulation of classＩand
ⅡHDACs, especially classＩisozymes, has been associated with the process of tumor cells
proliferation, angiogenesis, invasion and metastasis[17-22].
To date, many HDAC inhibitors reached clinical trials, and five of them were approved for
clinical uses: Vorinostat (SAHA, 1) and Romidepsin (FK228,2) for use in cutaneous T-cell
lymphoma, Belinostat (PXD101,3) and Chidamide (CS055,4) both for use in peripheral T-cell
lymphoma, and panobinostat (LBH589, 5) for multiple myeloma. Many HDAC inhibitors such as
Tacedinaline (CI994, 6) ，(MS-275，7)，(MGCD-0103，8) are under clinical trials for the treatment
of different kinds of cancers(Fig.1)[23-29]. The common pharmacophore of these HDAC inhibitors
consists of 3 portions(Fig.1 ): (1) ZBG, a zinc binding group chelating Zinc at the bottom of HDACs
active site; (2) a CAP group, interacting with the surface of HDACs; (3) a linker connecting the ZBG
and the CAP group[30].
Fig. 1. FDA approved and clinical HDAC inhibitors.
In our previous studies, we have reported novel phenyl substituted SAHA-based hydroxamic
acid derivatives[31]. From our examples, we found that the CAP group has a crucial impact on the
antitumor activities of SAHA-based hydroxamic acid derivatives. In this report, we modified the
CAP group and the hydrophobic linker of our compounds, in order to obtain novel potential HDAC
inhibitors and search for more valuable candidates for the tumor therapy. Two series of
hydroxamates, and 2-aminobenzamides were synthesized and evaluated for their inhibitory
activities against HDACs and cancer cells. Docking studies of selected compounds binding to
HDAC were also discussed.
2. Results and discussion

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2.1. Chemistry
The synthetic route of target molecules is described in Schemes 1 . The key intermediate 1a or
1b was synthesized by starting compound A (amine or ether). Then, 1a or 1b were directly
converted to hydroxamic acids 2a, 3a, 2b or 3b by treating with hydroxylamine. In route (c) 1a or
1b was hydrolyzed and coupled with benzene-1,2-diamine to obtain compounds 5a-7a, 11a-19a,
5b-8b.
Scheme 1. Conditions: (a) K2CO3, KI/Butanone, reflux ; (b) KOH, NH2OH.HCl/MeOH, rt; (c) 1.NaOH/MeOH, 2.HCl,
0℃; (d) benzene-1,2-diamine, BOP, Et3N/DMF, rt;
2.2. Biological tests
2.2.1 HeLa Nuclear HDACs inhibition and antiproliferative assay
HDAC enzymatic inhibitory activities of the target compounds were evaluated using the Fluor
de Lys®-Green HDAC Assay kit (BML-AK530, Enzo® Life Sciences) with CS055 and CI994 as positive
control. Hela cell nuclear extracts were used as the source of the HDAC activity. Additionally, the
antiproliferative effects of the targeted compounds against A549 human lung cancer cells,
MGC80-3 human gastric cancer cells, and HepG2 human liver cancer cells were tested. The
results are listed in Table 1-2. According to the results, we found that both the hydrophobic linker
and cap group played a significant role in the inhibitory activities against HDACs and
antiproliferative activity. In general, compounds owning potent HDAC inhibitory activity, also have
better antiproliferative activity against diverse cancer cells. For hydroxamates, the compound 2b
with longer linker showed better enzyme inhibitory activity and intro antiproliferative activity
compared with shorter linker compound 2a. While 2-aminobenzamides, the compounds with
shorter linker (5a-7a) showed better enzyme inhibitory activity and intro antiproliferative activity
compared with longer linker compounds (5b-8b). In order to obtain more potent compounds, we
also modified R1 or R2 to other groups (3a,6a,7a,3b,6b,7b,8b), as a result, all of them is less

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potent than compound 5a. Compound 5a is more potent than control compounds (CS055, CI994)
against HeLa nuclear HDACs and all three cancer cells. Therefore, further investigation of 5a as a
lead compound is needed.
Table 1
HDAC enzyme inhibition and antiproliferative activity data(IC50,μM) of compounds 2a-3a, 5a-7a
O
R2
N
H
R1
a
% Inhibitiona of HeLa
b
IC50 (μM)
nuclear HDACs
Compd.
R1
R2
0.625μM
5μM
A549
MGC80-3
HepG2
CS055
CI994
39.7%
32.5%
58.5%
49.4%
11.02±0.81 6.44±1.17
22.65±3.09
20.10±0.76 11.60±1.04 18.51±1.77
c
2a
-OH
-OH
3.0%
55%
27.99±3.06
／
29.55±8.04
7.77±0.86
3a
/
81.7%
8.93±0.70
／
5a
6a
7a
40.3%
26.0%
8.8%
61.8%
41.0%
27.0%
6.76±0.20
8.34±0.74
／
5.67±0.82
6.02±0.34
7.39±0.48
6.70±0.73
7.02±0.76
10.71±1.46
NH2
F
a The values represent the means of three independent experiments with SD less than 10%.
b IC50 values were obtained based on at least two independent experiments and expressed as means±SD.
c Not tested

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Table 2
HDAC enzyme inhibition and antiproliferative activity data(IC50,μM) of compounds 2b-3b, 5b-8b.
b
% Inhibitiona of HeLa nuclear
b
IC50 (μM)
HDACs
Compd.
R1
R2
0.625μM
39.7%
5μM
A549
MGC80-3
HepG2
CS055
CI994
58.5%
49.4%
11.02±0.81
6.44±1.17
22.65±3.09
32.5%
20.10±0.76 11.60±1.04 18.51±1.77
c
2b
3b
-OH
-OH
54.2%
56.4%
No
92.7%
89.1%
5.77±0.34
5.48±0.53
／
8.40±0.72
／
14.91±1.17
No
5b
6b
>32
>32
>32
>32
>32
>32
inhibition
inhibition
No
No
inhibition
inhibition
7b
8b
1.6%
33.6%
8.5%
／
14.21±0.7
13.50±1.67
No
13.43±0.64
>32
>32
inhibition
a The values represent the means of three independent experiments with SD less than 10%.
bIC50 values were obtained based on at least two independent experiments and expressed as means±SD.
c Not tested

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To optimize the lead compound 5a further, we performed a SAR study on the cap moiety by
Introducing different substituents to phenyl group. Target compounds 11a-19a were prepared
using the similar synthetic procedure of 5a, with replacement of the starting material by
corresponding substituted phenol. HDAC enzyme inhibition activity against HeLa nuclear HDACs
and antiproliferative activity against 4 kinds of cancer cells (A549 human lung cancer cells,
MGC80-3 human gastric cancer cells, HepG2 human liver cancer cells and HCT116 human
colorectal cancer cells) of these compounds were evaluated (Table 3). As shown in Table 3, Most
of the compounds exhibited stronger HDAC inhibition and anti-proliferative activities against
CS055 and CI994, which means that the substituents in phenyl ring of Cap group played a
significant role in the inhibitory activities against HeLa nuclear HDACs and cancer cells. Among
the compounds tested, 12a, 16a and 18a exhibited even better activities than 5a. However,
substitution with nitro, chloro, bromo or isopropyl in phenl ring of Cap group leads to a loss of
activity . The rim of the active pocket of HDAC is hydrophobic, however, nitro, chloro and bromo
are hydrophilic groups, which may be the reason why the substitutions with nitro, chloro, or
bromo in phenyl ring of Cap group lead to a loss of activity.
Table 3
HDAC enzyme inhibition and antiproliferative activity data(IC50,μM) of compounds 11a-19a
% Inhibitiona of HeLa nuclear
b
IC50 (μM
)
HDACs
Compd.
R
0.625μM
39.7%
32.5%
40.3%
5μM
58.5%
49.4%
61.8%
No inhibition
70.6%
26.3%
/
A549
MGC80-3
HepG2
HCT116
2.28±0.77
7.71±0.86
4.89±0.29
>32
CS055
CI994
5a
11.02±0.81 6.44±1.17
20.10±0.76 11.60±1.04
22.65±3.09
18.51±1.77
6.70±0.73
>32
-H
-NO2
6.76±0.20
>32
5.67±0.82
>32
11a
12a
13a
14a
15a
16a
17a
18a
19a
No inhibition
46.5%
-F
5.37±0.21
>32
4.97±0.65
>32
3.84±0.54
>32
3.86±0.39
17.33±2.74
>32
-Cl
18.7%
c
-Br
/
24.40±2.76
5.43±0.25
5.53±0.21
6.42±0.36
5.21±1.27
7.46±0.97
>32
>32
-CH3
45.4%
64.1%
69.4%
/
6.53±0.63
3.48±0.33
5.88±0.59
4.42±0.67
>32
6.59±0.60
4.20±0.51
6.96±1.31
4.75±0.89
>32
4.86±0.43
2.02±0.23
4.83±0.55
3.23±0.73
4.57±0.94
-CH3O
-CH2CH3
-CH2CH2CH3
-CH(CH3)2
46.9%
/
/
/
No inhibition
1.8%
a The values represent the means of three independent experiments with SD less than 10%.
bIC50 values were obtained based on at least two independent experiments and expressed as means±SD.
c Not tested

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2.2.2 HDAC isoform selectivity of 12a
To further investigate the HDAC isoforms selectivity, compound 12a with the most potent
HDAC inhibitory activity and antiproliferative activity was chosen to perform enzyme inhibitory
assays against a series of HDAC isoenzymes. We determined the IC50 values of compound 12a for
class
I representatives HDAC1 and HDAC2, and classⅡrepresentative HDAC6 with CI994 as
positive control (Table4). Results presented in Table 4 demonstrated that 12a is a potent HDAC2
inhibitor with IC50 of 0.57μM.
Table 4
In vitro inhibition of HDACs isoforms of 12a
IC50a(μM)
Compd.
Class I
ClassⅡ
HDAC1
HDAC2
HDAC6
CI994
12a
1.62±0.25
1.20±0.23
>40
1.27±0.20
0.57±0.09
>40
aIC50 values were obtained based on two independent experiments and expressed as means±SD.
2.2.3 Molecular docking
The proposed binding modes of compounds 12a and CI994 (Fig.2) in the active site of HDAC2
(PDB code 4LXZ) were investigated by docking analysis. Both 12a and CI994 could form 5
hydrogen bonds with amino acid residues GLY154, HIS145, HIS146, ASP181, and TYR308 in the
HDAC2 active site. The cap group of 12a could be well accommodated in the hydrophobic S
pocket of HDAC2, while CI994 could hardly achieve the same effect. Docking results of 12a to
HDAC2 could well explain its potent HDAC2 inhibitory activity.

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Fig. 2. Predicted binding modes of compounds 12a and CI994 on HDAC2 (PDB id:4LXZ). Interactions between the
protein and the ligand are shown as yellow dotted lines. The ligands are shown as stick models in purple(CI994)
and red(12a). (A) Molecular surface of the HDAC2 binding pocket (B) Compound 12a interacted with the actives of
HDAC2. (C) Compound CI994 interacted with the actives of HDAC2.

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2.2.4 Migration, colony formation, apoptosis and cell cycle analysis of 12a
Fig. 3. Migration of A549 cancer cells. A549 cancer cells were treated with compounds 12a, 16a, CS055, CI994 or
vehicle for 48h, and their migration was determined using the scratch test. One representative of three
independent experiments is shown. Cells treated with vehicle (DMSO) were used as controls.
We selected compounds 12a and 16a to evaluate their effects on the migration and colony
formation of A549 cancer cells. As shown in Fig.3, compound 12a or 16a could inhibit the
migration of A549 cancer cells and even more potent than CS055 and CI994. As shown in Fig.4,
compound 12a or 16a could also inhibit the colony formation of A549 cancer cells in a dose
dependent manner, and remarkably more potent than CI994.
To investigate whether the antiproliferative activities of 12a are accompanied by enhanced
cancer cell apoptosis, flow cytometry assay were determined. As shown in Fig.5, 12a-treated
A549 cells exhibited a dose-dependent increase of apoptosis by 25.3% and 64.4% at 4 and 8μM,
respectively. The 64.4% induction of A549 cells apoptosis with incubation with 8μM of 12a was
significantly higher than that of CS055 (22.42% apoptotic cells at the same concentration) and
CI994 (12.58% apoptotic cells at the same concentration). These results showed that 12a is able
to remarkably induce apoptosis of the A549 cancer cells.
We further investigated the effect of compound 12a on cell cycle progression of A549 cancer
cells(Fig.6). In comparison with the control cells, 12a clearly arrested A549 cells in G2/M phase
with a concentration dependent manner. Compound 12a caused G2/M fraction increased from
9.1% in control samples to 37.74% in treated samples. In the meanwhile, the G0/G1 fraction
decreased from 75.77% to 54.03% and the S-phase fraction decreased from 15.13% to 8.23%,
While CS055 and CI994 showed no obvious effect on the cell cycle of A549 cancer cells at 2 μM
or 16 μM. These data suggested that compound 12a may induce G2/M phase arrest in A549
cancer cells.

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Fig. 4. Compound 12a and 16a inhibit A549 cancer cell colony formation.

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Fig. 5. Induction of apoptosis at 48h by compounds 12a, CS055 and CI994 in different concentrations in A549
cancer cells.

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Fig. 6. Effect of CS055, CI994, and 12a in A549 cell cycle progression.
3. Conclusions
In conclusion, two series of hydroxamates and 2-aminobenzamides derivatives have been
designed and synthesized as HDAC inhibitors. All targeted compounds were investigated for their
Hela cell nuclear extracts HDAC enzymatic inhibitory activities and in vitro antiproliferative
activities against diverse cancer cell lines. Most compounds exhibited potent HDAC inhibitory
activity and antiproliferative activities. Especially, compound 12a, which showed more potent
HDAC inhibition activity to CS055 and CI994, exhibited highly potent anti-proliferation effect,
particularly 5.9-fold enhancement compared with CS055 and 4.8-fold enhancement compared
with CI994 against HepG2 cancer cell. HDAC isoform inhibition assay indicated 12a is a potent
HDAC2 inhibitor. Docking results of 12a to HDAC2 could well explain its potent HDAC2 inhibitory
activity. Further investigation showed that 12a could inhibit the migration and colony formation
of A549 cancer cells and remarkably induced the G2/M phase cell cycle arrest and apoptosis in

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A549 cancer cells. By far, the existing data indicated that 12a is a potentially promising histone
deacetylase inhibitor for the therapy of cancer. The in vivo antitumor activity evaluation and
mechanism study of 12a are undertaken in our lab.
4. Experimental
4.1. Chemistry
All starting materials were obtained commercially and were used without further purification.
All reactions were carried out in oven-dried glassware. Dried nitrogen was used to purge the
reactor before the reaction and to protect the reaction during the entire operation. Melting
points were recorded on a XT5, manufactured by Beijing Keyiecopti Instrument Factory and
1
uncorrected. H NMR and 13C NMR spectra were recorded in DMSO-d6 on a Bruker AV-400
spectrometer (400MHz). The chemical shifts were reported in ppm relative to Me4Si as internal
standard. Mass spectra were obtained with a Waters Xevo G2 Qtof mass spectrometer. Thin layer
chromatography (TLC) on pre-coated plates with silica gel F254, purchased from Qingdao Haiyang
Chemical Co. Ltd., was employed to monitor the progress of the reaction. The purities of final
compounds were established to be above 95% pure using a Shimadzu LC-20AT system with
LC-20AT two-solvent delivery units: Solvent A: methanol. Solvent B: 0.1% H3PO4 aqueous solution.
A SPD-M20A DAD detector was set at λ = 254 nm. The temperature was 30℃. The system
included a SIL-20A auto sampler, a CTO-10ASVP column oven, a LC solution workstation Shimadzu,
Kyoto, Japan and an analytical reversed phase C18 column (4.6 × 250 mm, 5μm, Diamodsil). The
flow rate of solvent A with the gradient started at 20% of methanol and increased to 90% over 35
min.
4.1.1. General method for the synthesis of 2a,3a,2b,3b
Step-1: To an oven-dried, 100-mL, three-necked, round-bottomed flask equipped with a
thermometer, a vacuum-jacketed Dean-Stark trap topped with a reflux condenser fitted with a
T-joint inlet, glass stoppers, and a Teflon-coated magnetic stirring bar was sequentially charged
with compound A (8.7 mmol, 1 equiv.), compound B1 or B2 (8.7mmol), K2CO3 (17.4mmol), KI
(8.7mmol) and 30mL of 2-Butanone. The reaction was heated at reflux for 6-12h in an oil bath.
TLC analysis, using ethyl acetate: petroleum ether (1:12) as the eluent, indicated the complete
reaction. The reaction mixture was allowed to cool to room temperature and then poured into
200mL of ethyl acetate with vigorous stirring. Stirring was continued for an additional 10min, and
the resulting solid was then filtered off. The filtrate was concentrated to provide 1a or 1b as a
solid.
Step-2: To a 100-mL beaker equipped with a Teflon-coated magnetic stirring bar were
sequentially placed potassium hydroxide (74.3 mmol, 18equiv. ) and methanol (20mL). The
mixture was stirred until a complete dissolution was obtained. Hydroxylamine hydrochloride
(82.6 mmol, 20 equiv.) was added. After an additional 30 min of stirring at 35℃, the reaction
mixture was cooled to ambient temperature. 1a or 1b (4.1mmol, obtained from the step A) was
added. Stirring was continued for 30 min. TLC was employed to monitor the progress of the
reaction. After the complete reaction, the reaction mixture was poured into 600mL of ice-water
mixture with vigorous stirring leading to the formation of a solid. The resulting solid was collected
by vacuum filtration, and successively washed with water (2 X 10mL) and ether (2 X 10mL). After
having been dried in vacuo, the crude product was further purified by recrystallization from
acetonitrile to afford 2a,2b or 3b as a solid.

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4.1.1.1 N-hydroxy-4-(phenoxymethyl)benzamide (2a)
1
White solid ; Yield 84%; HPLC Rt=23.19min; mp 130.0-131.4℃; H NMR (400MHz, DMSO-d6):
δ=7.77(d, J=8.0Hz, 2H), 7.45(d, J=8.0Hz, 2H), 7.30(t, J=7.6Hz, 2H), 7.02(d, J=8.1Hz, 2H), 6.95(t,
J=7.3Hz, 1H), 5.13(s, 2H); 13C NMR (400MHz, DMSO-d6): δ=68.68, 114.75, 120.73, 126.57, 127.10,
129.47, 133.97, 138.85, 158.20, 163.62 ppm; C14H13NO3, MS ( ES+) m/z: 244.09 (M+H)+.
4.1.1.2. N-hydroxy-4-({4-[(1E)-N-hydroxyethanimidoyl]phenoxy}methyl)benzamide (3a)
1
White solid; Yield 75%; HPLC Rt=21.72min; mp 176.7-178.2℃; H NMR (400MHz, DMSO-d6):
δ=11.01(s, 1H), 7.78(d, J=7.8Hz, 2H), 7.60(d, J=8.5Hz, 2H), 7.51(d, J=7.8Hz, 2H), 7.03(d, J=8.5Hz,
2H), 5.19(s, 2H), 2.12(s, 3H);13C NMR (400MHz, DMSO-d6): δ=11.42, 68.65, 114.61, 126.83, 126.93,
127.27, 129.75, 132.54, 139.83, 152.30, 158.51, 163.76 ppm; C16H16N2O4, MS ( ES+) m/z: 301.19
(M+H)+.
4.1.1.3. (2E)-N-hydroxy-3-[4-(phenoxymethyl)phenyl]acrylamide(2b)
1
White solid; Yield 89%; HPLC Rt=27.46min; mp 140.4-141.7℃; H NMR (400MHz, DMSO-d6):
δ=7.59(d, J=7.4Hz, 2H), 7.48(m, 3H), 7.30(t, J=7.4Hz, 2H), 7.01(d, J=7.8Hz, 2H), 6.95(t, J=7.0Hz,
1H), 6.5(d, J=15.8Hz, 1H), 5.12(s, 2H); 13C NMR (400MHz, DMSO-d6): δ=68.62, 114.75, 119.18,
120.76, 127.52, 128.03, 129.48, 134.33, 137.71, 138.43, 158.16, 162.60 ppm; C16H15NO3, MS ( ES+)
m/z: 270.13 (M+H)+.
4.1.1.4. (2E)-3-[4-(2,3-dihydro-1H-indol-1-ylmethyl)phenyl]-N-hydroxyacrylamide (3b)
1
Gray solid; Yield 57%; HPLC Rt=27.23min; mp 142.3-144.1℃; H NMR (400MHz, DMSO-d6):
δ=7.55(d, J=7.3Hz, 2H), 7.40(m, 3H), 7.05(d, J=6.8Hz, 1H), 6.99(t, J=7.4Hz, 1H), 6.58(m, 2H), 6.46(d,
J=15.8Hz, 1H), 4.29 (s, 2H), 3.27(t, J=8.0Hz, 2H), 2.91(t, J=8.0Hz, 2H); 13C NMR (400MHz,
DMSO-d6): δ=27.93, 52.27, 52.85, 106.94, 117.28, 118.77, 124.24, 127.03, 127.50, 128.43, 129.49,
133.67, 137.77, 139.84, 152.15, 162.63 ppm; C18H18N2O2, MS ( ES+) m/z: 295.1449 (M+H)+.
4.1.2. General method for the synthesis of 5a-7a, 5b-8b, 11a-19a
Step-1: Following the same procedure of 4.1.1. to afford 1a or 1b
Step-2: To a solution of 1a or 1b (1equiv.) in CH3OH, NaOH aqueous solution (2mol/L, 10 equiv.)
was added. The reaction was heated at reflux for 2h in an oil bath. The reaction mixture was
allowed to cool to room temperature. The reaction mixture was poured into 300mL of ice-water
mixture with vigorous stirring. Then, add hydrochloric acid aqueous solution (2mol/L, 15 equiv.)
to the mixture, leading to the formation of a solid. The resulting solid was collected by vacuum
filtration, successively washed with water (5 X 10mL) and ether (2 X 10mL) and dried in vacuo to
afford 4a or 4b as a solid.
Step-3: To a solution of acid 4a or 4b (1 equiv.) and (benzotriazol-1-yloxy)tris(dimethylamino)
phosphonium hexafluorophosphate (BOP) (1.2 equiv.) in anhydrous DMF was added
triethylamine (4 equiv.), and the mixture was stirred at 20℃ under nitrogen for 10 min.
Phenylene-1,2-diamine (1.2 equiv.) was then added and the solution stirred at room temperature
under nitrogen for 4h. The mixture was diluted with ethyl acetate , then washed with water,
saturated aqueous sodium hydrogen carbonate and last with brine. The organic layer was dried
over MgSO4, filtered, washed with MeOH, purified by recrystallization from acetonitrile to afford
5a-7a, 5b-8b as a solid.
4.1.2.1. N-(2-aminophenyl)-4-(phenoxymethyl)benzamide(5a)
1
Gray solid; Yield 51%; HPLC Rt=25.20min; mp 176.2-178.2℃; H NMR (400MHz, DMSO-d6):
δ=9.70 (s, 1H), 8.03(d, J=7.8Hz, 2H), 7.60(d, J=7.8Hz, 2H), 7.32(t, J=7.8Hz, 1H), 7.21(d, J=7.7Hz,
1H), 7.01(m, 4H), 6.82(m, J=7.8Hz,1H), 6.63(t, J=7.2Hz, 1H), 5.22(s,2H), 4.94(s, 2H); 13C NMR

ACCEPTED MANUSCRIPT
(400MHz, DMSO-d6): δ=68.50, 114.84, 116.11, 116.23, 120.83, 123.28, 126.47, 126.66, 127.15,
127.93, 129.50, 134.03, 140.58, 143.12, 158.12, 165.05 ppm; C20H18N2O2, MS (ES+) m/z: 319.1442
(M+H)+.
4.1.2.2. N-(2-amino-4-fluorophenyl)-4-(phenoxymethyl)benzamide (6a)
1
Gray solid; Yield 59%; HPLC Rt=29.07min; mp 158.8-160.0℃; H NMR (400MHz, DMSO-d6):
δ=9.61 (s, 1H), 8.01(d, J=6.7Hz, 2H), 7.59(d, J=6.7Hz, 2H), 7.32(m, 2H),7.05(m,4H), 6.56(d,
J=10.7Hz, 1H), 6.38(m, 1H), 5.24(m, 4H); 13C NMR (400MHz, DMSO-d6): δ=68.48, 101.28, 102.09,
114.83, 119.19, 120.83, 127,12, 127.94, 128.48, 128.58,129.50, 133.89, 140.60, 145.41, 145.53, 158.10,
159.83, 162.20,165.30 ppm; C20H17FN2O2, MS (ES+) m/z: 337.1347(M+H)+.
4.1.2.3. N-(2-aminophenyl)-4-[(5-bromo-2,3-dihydro-1H-indol-1-yl)methyl]benzamide (7a)
1
Gray solid; Yield 81%; HPLC Rt=32.20min; mp 171.0-171.9℃; H NMR(400MHz, DMSO-d6):
δ=9.66(s, 1H), 7.99(d, J=7.9Hz, 2H), 7.47(d, J=8.1Hz, 2H), 7.16(m, 3H), 6.99(t, J=7.5Hz, 1H), 6.80(d,
J=7.7Hz, 1H), 6.62(t, J=7.5Hz, 1H), 6.53(d, J=8.4Hz, 1H), 4.91(s,2H), 4.36(s, 2H), 3.35(m, 2H);
2.95(t, J=8.4Hz, 2H);13C NMR (400MHz, DMSO-d6): δ=27.69, 51.84, 52.78, 107.86, 108.38, 116.10,
116.22, 123.32, 126.43, 126.64, 127.01, 127.69, 127.95, 129.45, 132.52, 133.46, 141.46, 143.11,
151.38, 165.05;
C22H20BrN3O, MS (ES+) m/z:422.0866 (M+H)+.
4.1.2.4. (2E)-N-(2-aminophenyl)-3-[4-(phenoxymethyl)phenyl]acrylamide (5b)
1
Gray solid; Yield 64%; HPLC Rt=29.06min; mp 202.7-204.2℃; H NMR (400MHz, DMSO-d6):
δ=9.41(s, 1H), 7.60(m,5H), 7.32(m, 3H), 7.05(d, J=7.8Hz, 2H), 6.95(m, 3H), 6.77(d, J=7.8Hz,
1H),6.60(t, J=7.3Hz, 1H), 5.16(s, 2H), 4.97(s, 2H); 13C NMR (400MHz, DMSO-d6): δ=68.67, 114.77,
116.00, 116.27, 120.78, 122.41, 123.50, 125.75, 127.71, 128.10, 129.50, 134.37, 138.68, 139.15,
141.53, 158.22, 163.44 ppm; C22H20N2O2, MS ( ES+) m/z: 345.1604 (M+H)+.
4.1.2.5. (2E)-N-(3-aminopyridin-4-yl)-3-[4-(phenoxymethyl)phenyl]acrylamide (6b)
1
Yellow solid; Yield 73%; HPLC Rt=24.17min; mp 176.8-179.3℃; H NMR (400MHz, DMSO-d6):
δ=9.53(s,1H), 8.07(s,1H), 7.79(d, J=5.3Hz, 1H), 7.70(m, 4H), 7.54(d, J=7.9Hz, 2H), 7.32(t, J=7.9Hz,
2H), 7.03(d, J=7.9Hz, 2H), 6.97(m, 2H), 5.23(s, 2H), 5.16(s, 2H); C21H19N3O2, MS ( ES+) m/z:
346.1553 (M+H)+.
4.1.2.6.
(2E)-N-(2-aminophenyl)-3-[4-(3,4-dihydroisoquinolin-2(1H)-ylmethyl)phenyl]acrylamide(7b)
1
Gray solid; Yield 87%; HPLC Rt=12.55min; mp 148.4-150.7℃; H NMR(400MHz, DMSO-d6):
δ=9.56(s, 1H), 7.60(m, 3H), 7.43(m, 3H), 7.11(m, 3H), 7.01(m, 2H), 6.93(t, J=7.4Hz, 1H), 6.78(d,
J=7.9Hz, 1H), 6.59(t, J=7.4Hz, 1H), 5.03(s,2H), 3.68(s, 2H), 3.56(s, 2H); 2.83(m, 2H), 2.69(m,
2H);13C NMR (400MHz, DMSO-d6): δ=28.68, 50.26, 55.42, 61.48, 115.96, 116.18, 122.04, 123.59,
124.54, 125.44, 125.61, 125.95, 126.33, 127.60, 128.42, 129.22, 133.69, 134.05, 134.72, 139.28,
140.23, 141.46, 163.55;
C25H25N3O, MS (ES+) m/z: 384.2077 (M+H)+.
4.1.2.7. (2E)-N-(2-aminophenyl)-3-[4-(2,3-dihydro-1H-indol-1-ylmethyl)phenyl]acrylamide(8b)
1
Yellow solid; Yield 67%; HPLC Rt=30.30min; mp 151.4-152.9℃; H NMR (400MHz, DMSO-d6):
δ=9.59 (s, 1H), 7.60 (m, 3H), 7.42 (m, 3H), 7.02 (m, 3H), 6.92 (d, J=7.4Hz, 1H), 6.77 (d, J=7.7Hz, 1H),
6.59(m, 3H),5.04(s,2H), 4.30(s, 2H); 13C NMR (400MHz, DMSO-d6): δ=27.96, 52.33, 52.91, 106.95,
115.95, 116.16, 117.30, 122.13, 123.60, 124.25, 124.55, 125.60, 127.06, 127.72, 128.47, 129.49,
133.72, 139.18, 140.11, 141.48, 152.17, 163.54 ppm; C24H23N3O, MS ( ES+) m/z: 370.1920 (M+H)+.
4.1.2.8. N-(2-aminophenyl)-4-[(4-nitrophenoxy)methyl]benzamide (11a)
1
Yellow solid; Yield 59%; HPLC Rt=24.85min; mp 201.8-204.1℃; H NMR (400MHz, DMSO-d6):
δ=9.81(s, 1H), 8.24(d, J=9.2Hz, 2H), 8.06(d, J=8.1Hz, 2H), 7.60(d, J=8.1Hz, 2H), 7.27(m, 2H), 7.19(d,
J=7.7Hz, 1H), 6.97(m, 1H), 6.79(d, J=8.1Hz, 1H), 6.60(t, J=7.6Hz, 1H), 5.39(s,2H), 4.96(s, 2H); 13C
NMR (400MHz, DMSO-d6): δ=69.55, 115.46, 116.13, 123.28, 125.88, 126.40,126.70, 127.38, 128.09,
134.34, 139.28, 141.03, 143.13, 163.43, 164.92, ; C20H17N3O4, MS (ES+) m/z: 364.1299 (M+H)+.

ACCEPTED MANUSCRIPT
4.1.2.9. N-(2-aminophenyl)-4-[(4-fluorophenoxy)methyl]benzamide (12a)
1
White solid; Yield 62%; HPLC Rt=25.94min; mp 196.0-197.8℃; H NMR (400MHz, DMSO-d6):
δ=9.69 (s, 1H), 8.02(d, J=8.0Hz, 2H), 7.58d, J=8.0Hz, 2H), 7.20(m, 1H), 7.15(m, 2H), 7.06(m,2H),
6.99(d, J=7.8Hz, 1H), 6.81(m, 1H), 6.62(t, J=7.5Hz, 1H), 5.20(s,2H), 4.93(s, 2H); 13C NMR (400MHz,
DMSO-d6): δ=69.18, 115.72, 115.95, 116.11, 116.19, 116.22, 126.48, 126.67, 127.19, 127.93,134.07,
140.37, 143.12, 154.44, 157.79, 165.02 ; C20H17FN2O2, MS (ES+) m/z: 337.1347 (M+H)+.
4.1.2.10. N-(2-aminophenyl)-4-[(4-chlorophenoxy)methyl]benzamide(13a)
1
Gray solid; Yield 61%; HPLC Rt=28.96min; mp 191.6-193℃; H NMR (400MHz, DMSO-d6):
δ=9.68(s, 1H), 8.01(d, J=8.1Hz, 2H), 7.58(d, J=8.1Hz, 2H), 7.36(m, 2H), 7.19(d, J=7.7Hz, 2H), 7.07(m,
2H), 6.99(t, J=7.4Hz, 1H), 6.80(d, J=7.8Hz, 1H), 6.61(t, J=7.6Hz, 1H), 5.22(s,2H), 4.92(s, 2H); 13C
NMR (400MHz, DMSO-d6): δ=68.94, 116.08, 116.20, 116.65, 123.23, 124.55, 126.47, 126.66, 127.21,
127.94, 129.25, 134.12, 140.13, 143.12, 156.97, 164.99 ; C20H17ClN2O2, MS (ES+) m/z:353.1051
(M+H)+.
4.1.2.11. N-(2-aminophenyl)-4-[(4-bromophenoxy)methyl]benzamide(14a)
Gray solid; Yield 62%; HPLC Rt=29.80min; mp 201.4-202.6℃; 1H NMR(400MHz, DMSO-d6):
δ=9.71(s, 1H), 8.03(d, J=8.0Hz, 2H), 7.58(d, J=8.0Hz, 2H), 7.48(m, 2H), 7.21(d, J=7.6Hz, 1H), 7.01(m,
3H), 6.82(m, 1H), 6.63(d, J=7.3Hz, 1H), 5.22(s,2H), 4.93(s, 2H);13C NMR (400MHz, DMSO-d6):
δ=68.88, 112.28, 116.11, 116.23, 117.18, 123.26, 126.49, 126.67, 127.21, 127.96, 132.15, 134.13,
140.10, 143.13, 157.41, 165.01 ;
C20H17BrN2O2, MS (ES+) m/z:397.0549 (M+H)+.
4.1.2.12. N-(2-aminophenyl)-4-[(4-methylphenoxy)methyl]benzamide (15a)
1
White solid; Yield 53%; HPLC Rt=28.31min; mp 183.6-185.0℃; H NMR (400MHz, DMSO-d6):
δ=9.68 (s, 1H), 8.01(d, J=8.1Hz, 2H), 7.57(d, J=8.1Hz, 2H), 7.20(d, J=7.7Hz, 1H), 7.11(d, J=8.3Hz,
2H), 6.99(m,1H), 6.93(d, J=8.5Hz, 2H), 6.81(m, 1H), 6.62(t, J=7.5Hz, 1H), 5.18(s,2H), 4.92(s, 2H),
2.24(s,3H); 13C NMR (400MHz, DMSO-d6): δ=20.05, 68.57, 114.70 116.10, 116.22, 123.27, 126.46,
126.66, 127.09, 127.89, 129.49, 129.82, 133.95, 140.75, 143.12, 155.99, 165.04; C21H20N2O2, MS
(ES+) m/z: 333.1607 (M+H)+.
4.1.2.13. N-(2-aminophenyl)-4-[(4-methoxyphenoxy)methyl]benzamide (16a)
1
White solid; Yield 64%; HPLC Rt=24.84min; mp 173.2-174.7℃; H NMR (400MHz, DMSO-d6):
δ=9.68 (s, 1H), 8.01(d, J=8.0Hz, 2H), 7.57(d, J=8.0Hz, 2H), 7.20(d, J=7.6Hz, 1H), 6.98(m,3H), 6.88(m,
2H), 6.81(m, 1H), 6.62(t, J=7.5Hz, 1H), 5.15(s,2H), 4.92(s, 2H), 3.71(s,3H); 13C NMR (400MHz,
DMSO-d6): δ=55.32, 69.10, 114.59, 115.79, 116.10, 116.22, 123.27, 126.46, 126.66, 127.11, 127.88,
133.93, 140.83, 143.12, 152.09, 153.54, 165.04 ppm; C21H20N2O3, MS ( ES+) m/z: 349.1555
(M+H)+.
4.1.2.14. N-(2-aminophenyl)-4-[(4-ethylphenoxy)methyl]benzamide(17a)
1
Yellow solid; Yield 46%; HPLC Rt=30.78min; mp 171.9-173.8℃; H NMR (400MHz, DMSO-d6):
δ=9.68(s, 1H), 8.01(d, J=8.1Hz, 2H), 7.57(d, J=8.1Hz, 2H), 7.19(d, J=7.7Hz, 1H), 7.14(d, J=8.5Hz,
2H), 6.96(m, 3H), 6.81(m, 1H), 6.62(t, J=7.5Hz, 1H), 5.19(s,2H), 4.98(s, 2H), 2.54(m, 2H); 1.56(m,
2H), 1.16(t, J=7.5Hz, 3H);13C NMR (400MHz, DMSO-d6): δ=15.87, 27.26, 68.57, 114.71, 116.10,
116.23, 123.28, 126.46, 126.66, 127.08, 127.90, 128.64, 133.95, 136.04, 140.77, 143.08, 156.17,
165.03 ; C22H22N2O2, MS (ES+) m/z:347.1756 (M+H)+.
4.1.2.15. N-(2-aminophenyl)-4-[(4-propylphenoxy)methyl]benzamide(18a)
1
White solid; Yield 78%; HPLC Rt=33.20min; mp 171.5-173.0℃; H NMR (400MHz, DMSO-d6):
δ=9.68(s, 1H), 8.01(d, J=8.0Hz, 2H), 7.58(d, J=8.0Hz, 2H), 7.20(d, J=7.7Hz, 1H), 7.12(d, J=8.4Hz,
2H), 6.97(m, 3H), 6.81(d, J=7.8Hz, 1H), 6.62(t, J=7.5Hz, 1H), 5.18(s,2H), 4.92(s, 2H), 2.48(d,
J=7.7Hz, 2H); 1.56(m, 2H), 0.88(t, J=7.4Hz, 3H);13C NMR (400MHz, DMSO-d6): δ=13.56, 24.27,
36.35, 68.57, 114.60, 116.09, 116.21, 123.27, 126.45, 126.65, 127.09, 127.90, 129.22, 133.96, 134.39,
140.76, 143.11, 156.21, 165.03 ; C23H24N2O2, MS (ES+) m/z:361.1911 (M+H)+.
4.1.2.16. N-(2-aminophenyl)-4-[(4-isopropylphenoxy)methyl]benzamide(19a)

ACCEPTED MANUSCRIPT
1
Gray solid; Yield 53%; HPLC Rt=32.71min; mp 178.3-179.9℃; H NMR (400MHz, DMSO-d6):
δ=9.70(s, 1H), 8.03(d, J=7.8Hz, 2H), 7.58(d, J=8.1Hz, 2H), 7.18(m, 3H), 6.97(m, 3H), 6.82(d, J=8.1Hz,
1H), 6.62(t, J=7.6Hz, 1H), 5.19(s,2H), 4.93(s, 2H), 2.84(m, 1H); 1.18(d, J=6.9Hz, 6H);13C NMR
(400MHz, DMSO-d6): δ=24.08, 32.57, 68.58, 114.64, 116.11, 116.23, 123.29, 126.46, 126.65, 127.07,
127.14, 127.92, 133.97, 140.73, 140.80, 143.12, 156.22, 165.03 ; C23H24N2O2, MS (ES+)
m/z:361.1910 (M+H)+.
4.2. Biological materials and methods
4.2.1. HDAC Inhibition Assay
The HeLa Nuclear Extract HDAC inhibition assay was performed using Fluor de Lys®-Green
HDAC Assay kit (BML-AK530, Enzo® Life Sciences). HDAC1, 2 and 6 inhibition assay was performed
using Fluor de Lys® HDAC1 Assay kit (BML-AK511, Enzo® Life Sciences), Fluor de Lys® -Green
HDAC2 Assay kit (BML-AK512, Enzo® Life Sciences) and Fluor de Lys® HDAC6 Assay kit
(BML-AK516, Enzo® Life Sciences). 15μL of HDAC was mixed with 10μL of tested compounds.
Allow diluted Fluor de Lys®-Green Substrate and the microtiter plate to equilibrate to assay
temperature (37°C) for 5 minutes. add 25 μl diluted substrate to each well and mix thoroughly.
Incubate the plate at 37℃for enough time and then add 50μL Fluor de Lys® Developer to each
well to stop the HDAC reaction. Plate was Incubated at 25℃ for another 10 min and
fluorescence measurements were obtained using an EnSpire multimode plate reader
(PerkinElmer, USA).
4.2.2. Antiproliferative assay
Cell lines A549, MGC80-3, HepG2 and HCT116 were obtained from the Cell Resource Center,
Peking Union Medical College. A549 and HCT116 cells were cultured in RPMI1640 (Corning, USA)
contained 10% fetal bovine serum (FBS) (ThermoFisher, USA) and 1% penicillin/streptomycin.
MGC80-3 and HepG2 cells were cultured in DMEM (Corning, USA) contained 10% fetal bovine
serum (FBS) (ThermoFisher, USA) , and 1% penicillin/streptomycin. All cell lines were incubated
at 37℃ in a humidified atmosphere of 5% CO2. Cells in logarithmic phase were seeded in 96-well
culture plates at a density of 3000-4000 cells/well. After 12h, cells were treated with various
concentrations of compounds or solvent control. After 72h of incubation, 10μL of CCK-8 reagent
was added to each well, and the cells were incubated for an additional 1h. Absorbance was
measured at 450nm using an EnSpire multimode plate reader (PerkinElmer, USA). IC50 values
were calculated using percentage of growth versus untreated control.
4.2.3. Scratch test.
The scratch test method was used to estimate cell migration. The cells were seeded onto
12-well plates and treated with DMSO or compounds CS055, CI994, 12a and 16a. After tracing
the bottom of the wells, the cells were allowed to migrate. The image were captured after 48h
using an OPTEC BDS200 microscope.
4.2.4. Colony formation assay
A549 cells were seeded in 6-well plates at a density of 3000 cells per well. After 24h, cells were
treated with DMSO, CS055, CI994, 12a or 16a. After 7 days, Colonies were fixed with 3.7%
formaldehyde and stained with 0.1% crystal violet.

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4.2.5. Cell apoptosis analysis
Cell apoptosis was determined using Vybrant Apoptosis Assay kit, (Invitrogen). Briefly, A549
cancer cells (1.6×105/well) were incubated in 6-well plates for 24h and then treated with DMSO,
CS055, CI994 or 12a. After 48h, cells were harvested and washed three times with prechilled PBS.
A 100μL volume of 1 X annexin-buiding buffer, 5μL of Alexa Fluor 488 annexin and 1μL of
propidium iodide (100μg/ml) were added. The cells were incubated for 15min in dark. After
staning, 400 µL of 1 X annexin-buiding buffer was added, mixed gently and kept on ice. The
samples were detected with a MoFlo XDP flow cytometer (Beckman Coulter, Inc.).
4.2.6. Cell cycle analysis.
A549 cancer cells (1.6×105/well) were incubated in 6-well plates for 24h and then treated
with DMSO, CS055, CI994 or 12a. After 48h, cells were harvested, and fixed with 70% ethanol in
phosphate buffer at -20℃ overnight. Cells were incubated with 200µL of DNAse-free RNase
(1mg/mL) for 30min，and stained by propidium iodide(PI) staining solution (100μg/ml). DNA
content was measured by a MoFlo XDP flow cytometer (Beckman Coulter, Inc.).
4.2.7. Molecular docking
Molecular docking calculations were carried out with Surflex-dock in Sybyl-X 2.0. (Tripos Inc.)
The three-dimensional structure of HDAC2 (PDB id:4LXZ) was taken from the Protein Data Bank.
For protein preparation, the water molecular and ligand were removed and hydrogen atoms were
added. The structures of CI994, 12a were optimized using the conjugated gradient method with
the Tripos force field with convergence criterion set at 0.001kcal/(A.mol). Other parameters
accepted the default values.
Acknowledgments
We are grateful to the National Science Foundation of China (Grant No. 21176018) for their
generous financial supports.
Appendix A. Supplementary data
References
[1] M. Dobbelstein, U. Moll, Targeting tumour-supportive cellular machineries in anticancer drug development,
Nat Rev Drug Discov, 13 (2014) 179-196.
[2] L.S. Kristensen, H.M. Nielsen, L.L. Hansen, Epigenetics and cancer treatment, Eur J Pharmacol, 625 (2009)
131-142.
[3] H.M. Prince, M.J. Bishton, S.J. Harrison, Clinical studies of histone deacetylase inhibitors, Clin Cancer Res, 15
(2009) 3958-3969.
[4] A.A. Lane, B.A. Chabner, Histone deacetylase inhibitors in cancer therapy, J Clin Oncol, 27 (2009) 5459-5468.
[5] T. Kouzarides, Chromatin modifications and their function, Cell, 128 (2007) 693-705.
[6] M.A. Glozak, E. Seto, Histone deacetylases and cancer, Oncogene, 26 (2007) 5420-5432.
[7] M.A. Glozak, N. Sengupta, X. Zhang, E. Seto, Acetylation and deacetylation of non-histone proteins, Gene, 363
(2005) 15-23.
[8] J.E. Bolden, M.J. Peart, R.W. Johnstone, Anticancer activities of histone deacetylase inhibitors, Nat Rev Drug
Discov, 5 (2006) 769-784.

ACCEPTED MANUSCRIPT
[9] P.A. Marks, R. Breslow, Dimethyl sulfoxide to vorinostat: development of this histone deacetylase inhibitor as
an anticancer drug, Nat Biotechnol, 25 (2007) 84-90.
[10] O. Witt, H.E. Deubzer, T. Milde, I. Oehme, HDAC family: What are the cancer relevant targets?, Cancer Lett,
277 (2009) 8-21.
[11] M.A. Dawson, T. Kouzarides, Cancer epigenetics: from mechanism to therapy, Cell, 150 (2012) 12-27.
[12] I.V. Gregoretti, Y.M. Lee, H.V. Goodson, Molecular evolution of the histone deacetylase family: functional
implications of phylogenetic analysis, J Mol Biol, 338 (2004) 17-31.
[13] K.J. Falkenberg, R.W. Johnstone, Histone deacetylases and their inhibitors in cancer, neurological diseases and
immune disorders, Nat Rev Drug Discov, 13 (2014) 673-691.
[14] M.A. Salvador, J. Wicinski, O. Cabaud, Y. Toiron, P. Finetti, E. Josselin, H. Lelievre, L. Kraus-Berthier, S. Depil, F.
Bertucci, Y. Collette, D. Birnbaum, E. Charafe-Jauffret, C. Ginestier, The histone deacetylase inhibitor abexinostat
induces cancer stem cells differentiation in breast cancer with low Xist expression, Clin Cancer Res, 19 (2013)
6520-6531.
[15] M. Haberland, R.L. Montgomery, E.N. Olson, The many roles of histone deacetylases in development and
physiology: implications for disease and therapy, Nat Rev Genet, 10 (2009) 32-42.
[16] R.W. Johnstone, Histone-deacetylase inhibitors: novel drugs for the treatment of cancer, Nat Rev Drug Discov,
1 (2002) 287-299.
[17] K.B. Glaser, J. Li, M.J. Staver, R.-Q. Wei, D.H. Albert, S.K. Davidsen, Role of Class I and Class II histone
deacetylases in carcinoma cells using siRNA, Biochem Biophys Res Commun, 310 (2003) 529-536.
[18] F.R. Fritzsche, W. Weichert, A. Roske, V. Gekeler, T. Beckers, C. Stephan, K. Jung, K. Scholman, C. Denkert, M.
Dietel, G. Kristiansen, Class I histone deacetylases 1, 2 and 3 are highly expressed in renal cell cancer, BMC Cancer,
8 (2008) 381.
[19] E. Verdin, F. Dequiedt, H.G. Kasler, Class II histone deacetylases: versatile regulators, Trends Genet, 19 (2003)
286-293.
[20] W. Weichert, A. Roske, V. Gekeler, T. Beckers, C. Stephan, K. Jung, F.R. Fritzsche, S. Niesporek, C. Denkert, M.
Dietel, G. Kristiansen, Histone deacetylases 1, 2 and 3 are highly expressed in prostate cancer and HDAC2
expression is associated with shorter PSA relapse time after radical prostatectomy, Br J Cancer, 98 (2008) 604-610.
[21] T. Yamaguchi, F. Cubizolles, Y. Zhang, N. Reichert, H. Kohler, C. Seiser, P. Matthias, Histone deacetylases 1 and
2 act in concert to promote the G1-to-S progression, Genes Dev, 24 (2010) 455-469.
[22] Y. Song, M. Shiota, S. Tamiya, K. Kuroiwa, S. Naito, M. Tsuneyoshi, The significance of strong histone
deacetylase 1 expression in the progression of prostate cancer, Histopathology, 58 (2011) 773-780.
[23] M. Dokmanovic, C. Clarke, P.A. Marks, Histone deacetylase inhibitors: overview and perspectives, Mol Cancer
Res, 5 (2007) 981-989.
[24] J. Roche, P. Bertrand, Inside HDACs with more selective HDAC inhibitors, Eur J Med Chem, 121 (2016)
451-483.
[25] P.A. Marks, Discovery and development of SAHA as an anticancer agent, Oncogene, 26 (2007) 1351-1356.
[26] P. Maiso, X. Carvajal-Vergara, E.M. Ocio, R. Lopez-Perez, G. Mateo, N. Gutierrez, P. Atadja, A. Pandiella, J.F. San
Miguel, The histone deacetylase inhibitor LBH589 is a potent antimyeloma agent that overcomes drug resistance,
Cancer Res, 66 (2006) 5781-5789.
[27] J.A. Plumb, P.W. Finn, R.J. Williams, M.J. Bandara, M.R. Romero, C.J. Watkins, N.B. La Thangue, R. Brown,
Pharmacodynamic response and inhibition of growth of human tumor xenografts by the novel histone
deacetylase inhibitor PXD101, Mol Cancer Ther, 2 (2003) 721-728.
[28] M. Fournel, C. Bonfils, Y. Hou, P.T. Yan, M.-C. Trachy-Bourget, A. Kalita, J. Liu, A.-H. Lu, N.Z. Zhou, M.-F. Robert,
MGCD0103, a novel isotype-selective histone deacetylase inhibitor, has broad spectrum antitumor activity in vitro

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and in vivo, Mol Cancer Ther, 7 (2008) 759-768.
[29] C. Grant, F. Rahman, R. Piekarz, C. Peer, R. Frye, R.W. Robey, E.R. Gardner, W.D. Figg, S.E. Bates, Romidepsin: a
new therapy for cutaneous T-cell lymphoma and a potential therapy for solid tumors, Expert Rev Anticancer Ther,
10 (2010) 997-1008.
[30] T.A. Miller, D.J. Witter, S. Belvedere, Histone deacetylase inhibitors, J Med Chem, 46 (2003) 5097-5116.
[31] R. Xie, J.H. Shi, Y. Qu, P.W. Tang, X.Y. Wu, M. Yang, Q.P. Yuan, Synthesis and Anti-tumor Activities of Novel
Phenyl Substituted Suberoylanilide Hydroxamic Acid Derivatives Against Human Cancer Cells, Med Chem, 11
(2015) 636-648.

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Design, synthesis and biological evaluation of novel hydroxamates and 2-aminobenzamides as
potent histone deacetylase inhibitors and antitumor agents
1. Introduction
2. Results and discussion
2.1. Chemistry
2.2. Biological tests
2.2.1 HeLa Nuclear HDACs inhibition and antiproliferative assay
2.2.2 HDAC isoform selectivity of 12a
2.2.3 Molecular docking
2.2.4 Migration, colony formation, apoptosis and cell cycle analysis of 12a
3. Conclusions
4. Experimental
4.1. Chemistry
4.1.1. General method for the synthesis of 2a,3a,2b,3b
4.1.1.1 N-hydroxy-4-(phenoxymethyl)benzamide (2a)
4.1.1.2. N-hydroxy-4-({4-[(1E)-N-hydroxyethanimidoyl]phenoxy}methyl)benzamide (3a)
4.1.1.3. (2E)-N-hydroxy-3-[4-(phenoxymethyl)phenyl]acrylamide(2b)
4.1.1.4. (2E)-3-[4-(2,3-dihydro-1H-indol-1-ylmethyl)phenyl]-N-hydroxyacrylamide (3b)
4.1.2. General method for the synthesis of 5a-7a, 5b-8b, 11a-19a
4.1.2.1. N-(2-aminophenyl)-4-(phenoxymethyl)benzamide(5a)
4.1.2.2. N-(2-amino-4-fluorophenyl)-4-(phenoxymethyl)benzamide (6a)
4.1.2.3. N-(2-aminophenyl)-4-[(5-bromo-2,3-dihydro-1H-indol-1-yl)methyl]benzamide (7a)
4.1.2.4. (2E)-N-(2-aminophenyl)-3-[4-(phenoxymethyl)phenyl]acrylamide (5b)
4.1.2.5. (2E)-N-(3-aminopyridin-4-yl)-3-[4-(phenoxymethyl)phenyl]acrylamide (6b)
4.1.2.6. (2E)-N-(2-aminophenyl)-3-[4-(3,4-dihydroisoquinolin-2(1H)-ylmethyl)phenyl]acrylamide(7b)
4.1.2.7. (2E)-N-(2-aminophenyl)-3-[4-(2,3-dihydro-1H-indol-1-ylmethyl)phenyl]acrylamide(8b)
4.1.2.8. N-(2-aminophenyl)-4-[(4-nitrophenoxy)methyl]benzamide (11a)
4.1.2.9. N-(2-aminophenyl)-4-[(4-fluorophenoxy)methyl]benzamide (12a)
4.1.2.10. N-(2-aminophenyl)-4-[(4-chlorophenoxy)methyl]benzamide(13a)
4.1.2.11. N-(2-aminophenyl)-4-[(4-bromophenoxy)methyl]benzamide(14a)
4.1.2.12. N-(2-aminophenyl)-4-[(4-methylphenoxy)methyl]benzamide (15a)
4.1.2.13. N-(2-aminophenyl)-4-[(4-methoxyphenoxy)methyl]benzamide (16a)
4.1.2.14. N-(2-aminophenyl)-4-[(4-ethylphenoxy)methyl]benzamide(17a)
4.1.2.15. N-(2-aminophenyl)-4-[(4-propylphenoxy)methyl]benzamide(18a)
4.1.2.16. N-(2-aminophenyl)-4-[(4-isopropylphenoxy)methyl]benzamide(19a)
4.2. Biological materials and methods
4.2.1. HDAC Inhibition assay
4.2.2. Antiproliferative assay
4.2.3. Scratch test.
4.2.4. Colony formation assay
4.2.5. Cell apoptosis analysis
4.2.6. Cell cycle analysis.
4.2.7. Molecular docking
Acknowledgments

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Appendix A. Supplementary data
References

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1. Two series of hydroxamates and 2-aminobenzamides compounds were designed and
synthesized.
2. Synthesized compounds displayed potent HDAC inhibitory activity and antiproliferative activity.
3. The most potent compound 12a is a potent HDAC2 inhibitor with IC50 of 0.57μM
4. The most potent compound 12a displayed an IC50 values of 3.84-5.37μM against diverse
cancer cells
5. Compound 12a arrested A549 cancer cells in G2/M phase and induced 64.4% A549 cancer cells
apoptosis at 8μM.