Thioether-based 2-aminobenzamide derivatives: Novel HDAC inhibitors with potent in vitro and in vivo antitumor activity

Article abstract of DOI:10.1016/j.ejmech.2019.05.007

Previously, we focused on a series of 2-aminobenzamide-based histone deacetylase (HDAC) inhibitors, compound 9 of which displayed potent HDAC inhibitory activity against HDAC1 and HDAC2, and moderate anti-proliferative activity against several cancer cell

Full text of DOI:10.1016/j.ejmech.2019.05.007

Accepted Manuscript
Thioether-based 2-aminobenzamide derivatives: Novel HDAC inhibitors with potent in
vitro and in vivo antitumor activity
Fan Yun, Chunhui Cheng, Sadeeq Ullah, Jie He, Mohamed Reda Zahi, Qipeng Yuan
PII:
S0223-5234(19)30416-7
DOI:
https://doi.org/10.1016/j.ejmech.2019.05.007
EJMECH 11317
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 March 2019
Revised Date: 4 May 2019
Accepted Date: 4 May 2019
Please cite this article as: F. Yun, C. Cheng, S. Ullah, J. He, M.R. Zahi, Q. Yuan, Thioether-based 2-
aminobenzamide derivatives: Novel HDAC inhibitors with potent in vitro and in vivo antitumor activity,
European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.05.007.
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Graphic abstracts

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Thioether-based 2-aminobenzamide Derivatives: Novel
HDAC Inhibitors with Potent In Vitro and In Vivo
Antitumor Activity
Fan Yun a †, Chunhui Cheng a †, Sadeeq Ullah a, Jie He a, Mohamed Reda Zahi b
Qipeng Yuana *
a:Key Laboratory of Biomedical Materials of Natural Macromolecules (Beijing University of Chemical
Technology), Ministry of Education. College of Life Science and Technology, Beijing University of Chemical
Technology, 15 Beisanhuan East Road, Beijing 100029, China.
b:Laboratory of Natural Products Chemistry and Biomolecules, University of Blida 1, BP 270 Blida, Algeria
† These two authors contributed equally to this work.
*E-mail addresses of the corresponding authors: yuanqp@mail.buct.edu.cn
ABSTRACT
Previously, we focused on a series of 2-aminobenzamide-based histone
deacetylase (HDAC) inhibitors, compound 9 of which displayed potent HDAC
inhibitory activity against HDAC1 and HDAC2, and moderate anti-proliferative
activity against several cancer cell lines. In the current study, we have designed and
synthesized a series of novel HDAC inhibitors based on thioether moiety with 9 as a
lead compound. Representative compounds12g and 12h showed apparently potent
anti-proliferative activities against five solid cancer cell lines: A549, HCT116, Hela,
A375 and SMMC7721, and low cytotoxicity against NIH 3T3 normal cells.
Especially, 12g and 12h also revealed potent HDAC inhibitory activity against
HDAC1, 2 and 3. In addition, the two compounds could arrest cell cycle in G2/M
phase and promote cell apoptosis. Moreover, they showed extended inhibition of
colony formation and effectively blocked cell migration towards A549 cancer cells.
Furthermore, 12g and 12h possessed better pharmacokinetic properties than the lead
compound 9. Benefiting from these results, we also explored 12g and 12h in the A549
xenografts model for in vivo antitumor activity. The in vivo experiment indicated that
12g and 12h could evidently augment antitumor activity (TGI=56.9% and 62.7%
respectively).
Key words: Thioether, HDAC inhibitors, 2-aminobenzamide, Pharmacokinetic
properties, Antitumor activity
1 Introduction
Cancer is one of the most serious epidemic health hazards, which is the second
major leading cause of death in the worldwide after cardiovascular diseases[1].

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Chemotherapy is an effective method to cure solid and hematological cancerous.
However, the application of these chemotherapeutic agents is limited due to their
systemic toxicity, dose-related adverse side effects, drug-resistance [2, 3], and the
lack of tumor targets [4]. Recently, histone deacetylases (HDACs) associated with
oncogenes and tumor suppressor genes have provided a promising target for the
treatment of various carcinomas [5-7]. HDACs and histone acetyltransferases (HATs)
are a pair of functional antagonism enzymes that mutually regulate the histone
acetylation in balance. HDACs regulate the deacetylation of lysine residues located
on core histones [8], while HATs had the opposite effect. They are involved in the
expression of related genes in cell growth, differentiation, senescence and
apoptosis[9-13]. Previous studies demostrated that, the overexpression of HDACs
silences tumor suppressor genes, resulting in cancer. Therefore, many researchers
have focused on developing prospective histone deacetylase inhibitors (HDACis) as
drug candidates.

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Figure. 1. Structures of representative HDAC inhibitors.
To date, several HDAC inhibitors were addressed for cancer treatment. SAHA
(Vorinostat) was the first HDAC inhibitor approved by U.S. Food and Drug
Administration (FDA) for cutaneous T cell lymphoma (CTCL) treatment (1, Figure.
1) [14]. In November 2009, FK228 (Romidespin) (2, Figure. 1) has also been
permitted by U.S.FDA for the treatment of CTCL. Additionally, LBH589
(Panobinostat, approved in February 2015) (3, Figure. 2) and PXD-101 (Belinostat,
approved in July 2014) (4, Figure. 1) were applied for the peripheral T cell
lymphoma (PTCL) and recurrent multiple myeloma (MM) treatment respectively
[14-17]. Moreover, CS055 (Chidamide) was approved by Chinese FDA for PTCL
treatment [18]. Besides that, phase trial of CI994 (5, Figure. 1)in treating patients
with advanced myeloma was completed on September 25, 2012, and MS275
(Entinostat) (6, Figure. 1) and MGCD-0103 (Methylgene) (7, Figure. 1) are at
different stages of clinical trials [19, 20]. The common pharmacophore models of
these HDAC inhibitors consist of three parts: (1) a zinc-binding group (ZBG), such as
hydroxamic acid and 2-aminobenzamide; (2) a cap group, generally a hydrophobic
and aromatic group; (3) a saturated or unsaturated linker portion, typically linear or
cyclic structures. The Cap-linker-ZBG combination was a classical HDAC inhibitor
model (Figure. 1) [21-23].
In our previous work, we discovered a series of HDAC inhibitors containing a
phenol cap group [24]. The most robust compound 9 revealed potent anti-proliferative
activity against A549, HCT116 and HepG2 cancer cells with IC50 of 5.37, 3.86 and
4.97 µM, respectively. Compound 9 also exhibited excellent inhibitory activity with
IC50 of 1.27 µM against HDAC1 and 0.57 µM against HDAC2. However, in vivo
evidence, compound 9 showed poor pharmacokinetic potential with short half-life and
fast metabolism, which limited its drug-forming property. We highlighted to optimize
the structure of 9 to enhance the pharmacokinetic property. Many studies have
illustrated that two carbon-sulfur bonds could be increased by an additional length of
∼0.8 Å, compared with the two carbon-oxygen bonds, and that the high polarization
of sulfur could improve water solubility. This may provide multidimensional
molecular diversity, and then modify chemical and biological properties[25-32].
Marson et al. reported that new HDAC inhibitors with aromatic thioether units, which
had the advantage of an adjustable oxidation level at sulfur for improving activity.
Moreover, aromatic thioether units could improve antiproliferative properties and
metabolic stability as shown for representative compounds 8a and 8b (Figure 2)[33].
Therefore, we designed and synthesized a series of novel HDAC inhibitors based on
thioether moiety according to the bioisosterism principle, and then evaluated in vitro
and in vivo tests for the antitumor activity.

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Figure. 2. Design strategy of the target compounds
2 Results and discussion
2.1 Chemistry
The synthetic route of target compounds was described in Scheme 1. The target
molecules were prepared by commercially available substituted thiol 10 as the starting
materials. Firstly, the initiating compound 10 was treated with 4-(bromomethyl)
benzoic acid or methyl (E)-3-(4-(bromomethyl)phenyl)acrylate to obtain intermediate
11 and 13, respectively (Scheme 1). Hydrolysis of ester 13 gave corresponding
compound 14, and then the acid 14 was treated with benzene-1, 2-diamine to achieve
the target compounds 15. Intermediate 11 was directly reacted with benzene-1,
2-diamine to synthesize the final products 12.

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Reagents and conditions: (a): K2CO3, KI / Butanone, reflux; (b): benzene-1,2-diamine,
BOP, Et3N/DMF, rt. (c): 1 NaOH / MeOH, reflux, 2 HCl, 0 ℃.
Scheme. 1. Synthesis route of target compounds.
2.2. Biological studies
2.2.1. In vitro anti-proliferative assay
The in vitro anti-proliferative efficacies of target compounds 12a-12n and
15a-15k along with the positive controls CS055 and CI994 were assayed with a
CCK-8 kit against five solid cancer cell lines including A549 (human lung cancer

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cells), A375 (human malignant melanoma cancer cells), HCT116 (human colorectal
cancer cells), HeLa (human cervical cancer cells) and SMMC7721 (human hepatoma
cancer cells). The inhibition rates at 2 µM and 8 µM were shown in Table S1.
According to the results, many synthesized samples showed potent inhibitory activity.
Compounds 15a-15k with the unsaturated longer linker displayed weaker
anti-proliferative activity than those with the shorter linker. Our current study also
exhibited that electron-donating substituents such as 3,5-dimethyoxyl (12e), 4-methyl
(12f), 4-methoxyl (12g), 4-ethyl (12h) and 3,5-dimethyl (12l), disclosed high
anti-proliferative activity while on the contrary, some electron-withdrawing groups
such as 3-fluorin(12j), 2- methoxycarbonyl(12k) did not.
Consequently, we selected these potent compounds to further evaluate their
antitumor inhibitory activities with CS055 and CI994 as the reference drugs. The
results were mentioned in Table 1. A number of tested derivatives exhibited excellent
anti-proliferative activities against five solid cancer cell lines, as evidenced by the low
IC50 values. Among the aforesaid compounds, 12g, 12h, 12l and 12m disclosed
admirable anti-proliferative activities with IC50 values that ranged from 0.97 µM to
3.80 µM against all the tested cancer cell lines compared with CI994 (6.39-16.45 µM).
Similarly, the IC50 values were recorded lower than CS055 for these four compounds
(1.57-8.63 µM) towards various carcinoma cell lines including: A549, A375 and
SMMC7721.
Table 1.
Anti-proliferative activities data (IC50, µM) of representative compounds against 5
different cancer cell lines.
Compd
IC50 (µM) a
SMMC7721
A549
A375
HCT116
Hela
b
b
b
/
/
/
/
/
3.75±0.86 12.65±3.15
12a
12b
12e
b
b
5.77±1.53
3.40±0.99
3.74±0.47
2.17±0.86
1.75±0.84
4.88±0.70
3.28±1.14
4.49±1.14
2.76±0.87
1.89±0.26
4.01±0.68
2.00±0.36
2.63±0.43
1.64±0.23
1.74±0.44
2.81±0.43
2.87±0.54
2.78±0.39
2.49±0.44
1.14±0.15
5.01±0.51
3.07±0.33
4.25±0.53
2.39±0.38
3.80±0.66
6.43±1.13
3.24±0.42
6.68±1.12
2.09±0.28
1.14±0.20
5.68±1.00
3.27±1.72
2.57±0.34
3.04±2.45
3.25±1.48
4.42±0.59
3.70±0.68
3.01±0.62
2.42±0.82
2.86±0.48
3.90±1.08
3.61±0.79
3.67±0.81
1.55±0.39
12f
12g
12h
12i
12j
12k
12l
12m
15b
15f
15g
15i
15j
CS055
CI994
b
/
/
b
2.89±0.16
0.97±0.26
b
b
1.15±0.20 15.74±1.94 10.92±2.69
/
/
/
/
/
/
/
/
b
b
b
1.19±0.34
0.64±0.19
0.83±0.46
1.30±0.76
6.81±4.50
3.67±0.81
1.55±0.39
/
/
/
/
b
b
b
b
b
b
b
b
/
/
b
b
b
/
/
4.77±1.32
8.63±1.77
14.06±2.98
1.57±0.15
3.42±0.31
16.45±4.34 9.64±1.63
6.39±1.14 13.43±1.70

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a Values are the mean of three separate experiments and expressed as means SD
b Not tested
2.2.2. HDAC inhibitory activity assay
To evaluate the inhibitory activity of HDAC inhibitors, four representative
compounds 12g, 12h, 12l and 12m were tested against four Class I isoforms (HDAC1,
2, 3 and 8) and one Class II isoform (HDAC6) with CS055 and CI994 as positive
control drugs. As shown in Table 2, the results revealed that 12g, 12h, 12l and 12m
possessed high inhibitory activities against HDAC1 ,2 and 3, but no inhibition of
HDAC6 and 8 (IC50 >32 µM) were observed. Especially, 12g and 12m demonstrated
dramatic inhibitory activity with IC50 values of 0.016 µM and 0.011 µM against
HDAC1 respectively. 12g, 12h, and 12l more effectively inhibited the HDAC2
activity with IC50 of 0.205, 0.144, 0.139 µM respectively in comparison with CS055
(0.310 µM). In addition, these four compounds also exhibited comparable or even
better HDAC3 inhibitory activity than that of CS055.
Table 2.
In vitro inhibition of HDACs of 12g, 12h, 12l and 12m.
IC50 of Hela
nuclear
IC50 of
HDAC1
(µM) a
IC50 of
HDAC2
(µM) a
IC50 of
HDAC3
(µM) a
IC50 of
HDAC6
(µM) a
IC50 of
HDAC8
(µM) a
Compd
extract(µM) a
0.136±0.11
0.215±0.14
>32
>32
0.016±0.004 0.205±0.38
0.071±0.097 0.144±0.26
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
12g
12h
12l
12m
CS055
CI994
10.72±4.07 0.020±0.002 0.139±0.04 0.381±0.25
1.995±0.78 0.011±0.005 0.441±0.33 0.274±0.21
0.900±0.15 0.190±0.070 0.310±0.08 0.234±0.06 7.230±1.21
1.810±0.14 1.670±0.210 1.920±0.68
>32
>32
a Values are the mean of at least three separate determinations.
2.2.3. Cytotoxicity
To measure inhibitory effects of 12g, 12h, 12l and 12m on non-cancerous cell line,
we chose the mouse embryonic fibroblast cells NIH 3T3 to further investigate the
cytotoxicity assay. The results were shown in Table 3. 12m exhibited higher
cytotoxicity with IC50 of 1.85 µM, as compared with CS055 and CI994 (IC50= 2.37
and 9.05 µM respectively). The IC50 values of 12g, 12h and 12l was tested with the
values of 4.04, 3.66 and 3.34 µM respectively, indicating their low cytotoxicity to the
positive drugs.
Table 3.
IC50 of compounds 12g, 12h, 12l and 12m against NIH 3T3 cell line.
Compd
CS055
CI994
12g
12h
12l
12m

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NIN 3T3
4.04
3.66
3.34
±0.67
1.85
±0.14
2.37
±0.46
9.05
±0.82
IC50 (µM) a ±0.86 ±0.93
a Values are the mean of three separate experiments and expressed as means
± SD
2.2.4. Molecular docking
To better understand the possible binding modes between targeting compounds
and these HDAC isoforms, we performed molecular docking analysis of 12g and 12h
in the active pocket of HDAC1(PDB code: 4BKX), HDAC2(PDB code: 4LXZ) and
HDAC6 (PDB code: 5G0G), respectively. The results were shown in Fig.3. The
2-aminobenzamide groups of 12g and 12h formed hydrogen bonding interactions
with HIS140, HIS141 and GLY149. Besides cheating with the zinc ion, 12g and 12h
formed extra hydrogen bond with PHE205 and ASP176 respectively with HDAC1
(Fig.3A and 3B). As expected, 12g and 12h were bound to HDAC2 via TYR308,
ASP181, HIS145, HIS146 and GLY154 (Fig.3C and 3D). To meet these binding
models of compounds and HDAC6, 12g and 12h have significantly weakened affinity
towards zinc ion in the active center and could not form hydrogen bonds (Fig.3 E and
3F).
Figure 3. Predicted binding models of 12g and 12h on HDAC1 (PDB code: 4BKX),
HDAC2 (PDB code:4LXZ) and HDAC6 (PDB code: 5G0G). Interactions between the

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protein and the ligand are shown as yellow dotted lines. (A, B) 12g and 12h interacted
with the active sites of HDAC1. (C, D) 12g and 12h interacted with the active sites of
HDAC2. (E, F) 12g and 12h interacted with the active sites of HDAC6. Molecular
surface of HDAC1, HDAC2 and HDAC6 binding pocket with compounds 12g and
12h were shown in the supporting information (SI).
2.2.5. Immunofluorescence assay
To explore whether the inhibitory action of these synthesized compounds was
related to the acetylation status of histone H3, 12g, 12h as well as reference drugs
such as CS055 and CI994 were assessed at 1 µM in A549 cancer cell line. The
analysis of Laser scanning confocal microscope was mentioned in Fig.4. 12g and 12h
with reference drugs positively traversed to the expression of H3K9ac. However,
owing to high HDAC expression of cancer cells [34-36], no fluorescent signal was
observed in the control group. The immunofluorescence results concluded that our
designed compounds could inhibit HDAC activity, which was linked with the
acetylation of histone H3K9.
DAPI
H3K9ac
Merge
12g a
12h a
CS055
a
CI994 a

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Control
Figure 4. Immunofluorescence images of H3K9ac, DAPI staining and merge in
untreated or treated A549 cells.
a Compounds was tested at 1 µM.
2.2.6. Cell apoptosis analysis
In order to confirm the efficacy of synthesized compounds towards cell death by
apoptosis analysis, in experimental exploring results, 12g and 12h were conducted to
induce the apoptosis in cancer cell line (A549). Flow cytometry analysis was shown
in Figure 5. It’s apparent from our evidence, that 12g and 12h promoted apoptosis
against A549 cells in a dose-dependent manner. The apoptosis rates of A549 cells
induced by 12g and 12h were 20.21 % and 18.08 % at 0.5 µM, respectively. The
apoptosis rates were 30.60 % and 21.12 % at 1 µM, and the apoptosis rates were
32.25 % and 23.25 % at 2 µM, respectively. However, the apoptosis rates of A549
cells induced by CS055 were 3.07 %, 6.26 % and 8.11 % at 0.5, 1, 2 µM, respectively.
The apoptosis rates induced by CI994 were 3.03 %, 9.79 % and 13.60 %, respectively.
It’s clearly concluded that 12g and 12h showed better apoptosis capability than the
positive drugs CS055 and CI994.

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Figure 5. Induction of apoptosis at 72 h by compounds 12g and 12h at different
concentrations in A549 cancer cells. The values represent the means of three separate
experiments with SD less than 10 %. * p <0.05 compared with the control group; ** p
<0.01 compared with the control group; *** p <0.001 compared with the control
group.
2.2.7. Cell cycle analysis
The designed compounds such as 12g and 12h were evaluated for the cell cycle
effect on cancer cell line (A549). The data was shown in Table 4. It’s clearly
disclosed that compounds 12g and 12h showed an obvious increase in the proportion
of cells in G2/M phase in a dose-dependent manner. As shown in Figure 6, the
percentage of cells in G2/M phase for 12g was increased from 18.07 % to 48.06 %;
that with 12h was from 53.55 % to 66.48 %. However, CS055 and CI994 as the
positive control drugs had no significant effect on the cell cycle of A549 cells.
Table 4.

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cell cycle analysis of cancer cell line (A549).
Proportion of A549 cells a
Compd concentration Compd concentration
Compd concentration
(2 µM)
Compd
(0.5 µM)
S
(1 µM)
S
G0/G1
G2/M G0/G1
G2/M G0/G1
S
G2/M
Control
12g
12h
73.35 12.83 13.82 73.35 12.83 13.82 73.35 12.83 13.82
64.38 17.55 18.07 41.07 20.63 38.3
33.89 12.56 53.55 30.39 9.74 59.87
39.3 12.64 48.06
24 9.52 66.48
CS055
CI994
75.24 6.97 17.79 74.07 7.26 18.67 75.53 9.43 15.04
76.99 7.64 15.37 75.86 6.73 17.41 77.38 7.37 15.25
a The values represent the means of three separate experiments with SD less than
10 %.
NC
12g 2 µM
12h 2 µM
CS055 2 µM
CI994 2 µM
Figure 6. Effect of 12g and 12h on A549 cancer cells progression
2.2.8. A549 cells migration assay
The inhibition action of cancer cell migration is an important characteristic
property of cancer treatment. To evaluate inhibitory migration activity, A549 cancer
cells were treated with 12g and 12h at 1 µM for 48 h. The results were mentioned in
Figure 7. Images of the wound were captured at 0, 24 and 48 h, respectively. It was
clearly observed that 12g and 12h suppressed cell migration more effectively than
CS055 and CI994, and finally concluded that 12g and 12h revealed significant
inhibitory capacity on A549 cells migration.
0h
24h
48h

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12g a
12h a
CS055 a
CI994 a
Control a
Figure 7. Migration of A549 cancer cells. A549 cancers cells were treated with 12g,
12h, CS055, CI994 and control (DMSO) for 48 h.
a Migration was investigated suing scratch test. Three independent experiments were
conducted.
2.2.9. Colony formation assay
Since the addressed compounds such as 12g and 12h demonstrated excellent
antitumor activities compared with CS055 and CI994 against A549 cancer cell line in
72 hours, we investigated to assess whether the compounds possess long-term
anti-proliferative activity for cancer cells. Accordingly, we further evaluated colony
formation of 12g and 12h at 0.5, 1 and 2 µM in A549 cells. The obtained results were
summarized in Figure 8. Compounds 12g and 12h exhibited the inhibition of colony
formation of A549 cancer cells in a dose dependent manner. 12g and 12h better
control the colony growth formation as compared to positive controls CS055 and
CI994.

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Figure 8. Compounds 12g and 12h inhibit A549 cancer cell colony formation.
2.2.10. Pharmacokinetic parameters
Though great progress towards in vitro antitumor activities of 12g and 12h has
been reported, the pharmacokinetic properties (PK) of 12g and 12h were tested in
ICR male mice following a dose of 4 mg/kg administration by intravenous injection
(iv) respectively. The PK parameters were shown in Table 5. 12g displayed half-life
(T1/2) of 2.24 h and an AUC0-t of 737 ng/mL×h. Similarly, the half-life (T1/2) of 12h
was 2.59h and AUC0-t was found to be 868 ng/mL×h. However, 9 displayed a short
half-life with the value of 0.56 h. The clearance rates of 12g and 12h were quite high
(CL= 90.1 and 76.3 mL/kg/min respectively), but were relatively lower than that of 9
(CL= 118 mL/kg/min). These data indicated that 12g and 12h exhibited better
pharmacokinetic properties than the lead compound 9, confirming the correct
optimization for 9.
Table 5.
Pharmacokinetic parameters of 12g, 12h and 9 in ICR mice. a
parameter
12g
12h
9

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T1/2 (h)
2.24
737
740
90.1
1.74
2.59
868
874
76.3
3.46
0.56
-
-
118
2.33
AUC0-t (ng/mL×h)
AUC0-inf (ng/mL×h)
CL (mL/kg/min)
Vdss (L/kg)
a
The pharmacokinetic parameters were determined after intravenous injection (4
mg/kg, n=3 at each time point) in ICR mice (males, weighted 29.7~37.4 g).
2.2.11. In vivo antitumor activity
A549 xenograft nude mice models were carried out to evaluate the in vivo
antitumor activity of compounds 12g and 12h against human lung cancer. When the
tumor volumes approximately reached to 95 mm3, the BALB/c female mice were
randomly assigned to four groups (n= 4). Mice were injected intraperitoneally with
12g, 12h and CI994 at a dose of 25 mg/kg twice a day for 21 days respectively. As
shown in Fig. 9A, all mice displayed no significant change in body weight. Tumor
growth inhibition (TGI) rates of 12g and 12h were 57.9 % and 62.7 % respectively,
whose antineoplastic activities were better than CI994 (TGI= 46.8 %). It was also
clearly observed from Figure 9B-C that 12g and 12h remarkably suppressed the
tumor growth as compared to CI994 and control groups. Moreover, the tumor size
visually demonstrated the potent in vivo anticancer activity of 12g and 12h (Fig.9D).
Figure.9. In vivo antitumor efficacy of compound 12g, 12h and CI994 in mice
(BALB/c nude mice) bearing A549 xenografts models. (A) Body weight and (B)
tumor volume measurements after 21 days of 12g and 12h administration. (C)

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Comparison of the final tumor weights in each group after the 21 days treatment
period of 12g and 12h. ** p <0.01 compared with the control group. (D)
Representative photographs of tumors in each group.
3 Conclusion
We modified and optimized the lead compound 9, and generated novel HDAC
inhibitors based on thioether moiety. The addressed compounds were evaluated for
their in vitro biological activities, which demonstrated that 12g and 12h exhibited
significant in vitro antineoplastic activities. Importantly, 12g and 12h possessed better
pharmacokinetic properties than the lead compound 9, confirming the correct
optimization for 9. Moreover, compounds 12g and 12h significantly inhibited tumor
growth in A549 xenograft nude mice models. The highlighted compounds such as 12g
and 12h showed attractive and practical applications in the field of cancer diagnosis
and therapy.
4 Experimental
4.1 Chemistry
All starting materials were obtained commercially and were used without further
purification. Melting points were recorded on a XT5, manufactured by Beijing
Keyiecopti Instrument Factory and uncorrected. 1 H NMR and 13 C NMR spectra were
recorded in DMSO-d6 on a bruker AV-400 spectrometer (400 MHz). High-resolution
mass spectra (HRMS) were recorded using an Agilent 6540 UHD Q-TOF MS
equipped with a gas nebulizer probe, capable of analyzing ions up to m/z 6000. 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. All the final compounds were tested by HPLC, and the purity in every
case was ≥90 %. The reverse phase HPLC was conducted on Shimadzu LC-20AT
system with LC-20AT two-solvent delivery units, which was equipped with C18
column (5 mm, 4.6 mm × 250 mm). A SPD-M20A DAD detector was set at λ= 254
nm. The temperature was 35 . The mobile phase A was methanol, and mobile phase
B was water (0.1 % H3PO4). The 10 % of methanol was run at a flat rate of 1.0
mL/min over 20 min. Analyses indicated by the symbols of the elements or functions
were within 0.4 % of the theoretical values.
4.1.1 General method for the synthesis of 12a-12n;
Step-1: To a 100mL, one-necked, round-bottomed flask, sequentially charged
with compound 10 (4.5 mmol, 1equiv.), 4-(bromomethyl) benzoic acid (4.5 mmol),
K2CO3 (9 mmol), KI (4.5 mmol) and 30 mL of 2-Butanone. The reaction was heated
at reflux for 6-12 h in an oil bath. TLC analysis, using ethyl acetate: petroleum ether

ACCEPTED MANUSCRIPT
(1:3) as the eluent, indicated the complete reaction. The reaction mixture was allowed
to cool to room temperature and then poured into 200 mL of ethyl acetate with
vigorous stirring. Stirring was continued for an additional 10 min, and the resulting
solid was then filtered off. The filtrate was concentrated to provide 11 as a white
solid.
Step-2: To a 100mL one-necked, round-bottomed flask, sequentially charged with 11
(1.8 mmol, 1 equiv., obtained from step-1), DMF (15 ml), BOP (2.2 mmol, 1.2 equiv.)
and Triethylamine (7.2 mmol, 4 equiv.). The mixture was stirred for 10 min at r.t.
Ben- zene-1,2-diamine (2.2 mmol, 1.2 equiv.) was then added and the solution was
stirred at r.t. for another 6 h. The mixture was poured into 200 mL of saturated
aqueous sodium bicarbonate with vigorous stirring. Stirring was continued for an
additional 10 min, and the resulting solid was then filtered off. The filtrate was
washed by petroleum ether and purified by column chromatography to obtain
compound 12 as a solid.
4.1.1.1. N-(2-aminophenyl)-4-(((4-chlorophenyl)thio)methyl)benzamide (12a)
1
Gray solid; Yield 63.7 %. HPLC Rt = 2.931 min. mp: 168.5-169.2 ; H NMR
(400MHz, DMSO-d6): δ=9.62(1H, s), 7.92(2H, d, J=8.02Hz), 7.48(2H, d, J=7.22Hz),
7.36(4H, m), 7.17(1H, d, J=7.62Hz), 6.97(1H, t, J=7.62Hz), 6.79(1H, d, J=7.62Hz),
13
6.59(1H, t, J=8.02Hz), 4.89(2H, s), 4.33 (2H, s); C NMR (100MHz, DMSO-d6):
δ=165.43, 143.62, 141.41, 135.20, 133.88, 131.15, 130.69, 129.36, 129.10, 128.39,
127.15, 126.94, 123.73, 116.68, 116.55, 36.74ppm. HRMS (ESI): Calculated for
C20H17ClN2OS m/z (M+ H) +: 369.0829; found: 369.0833.
4.1.1.2. N-(2-aminophenyl)-4-(((4-fluorophenyl)thio)methyl)benzamide (12b)
1
Gray solid; Yield 78.5 %; HPLC Rt = 3.195 min. mp: 158.7-160.9 ; H NMR
(400MHz, DMSO-d6): δ=9.62(1H, s), 7.90-7.88(2H, d, J=8.42Hz), 7.41(4H, m),
7.15(3H, m), 6.96(1H, m), 6.76(1H, d, J=8.02Hz), 6.59(1H, t, J=8.42Hz), 4.88(2H, s),
4.28 (2H, s); 13C NMR (100MHz, DMSO-d6): δ=165.42, 162.70, 160.28, 143.62,
141.70, 133.77, 132.28, 132.20, 131.26, 129.09, 128.33, 127.15, 126.93, 123.73,
116.68, 116.61, 116.56, 116.39, 37.83ppm; HRMS (ESI): Calculated for
C20H17FN2OS m/z (M+H) +: 353.1125 ; found:353.1137.
4.1.1.3. N-(2-aminophenyl)-4-(((4-bromophenyl)thio)methyl)benzamide (12c)
Gray solid; Yield 83.2 %; HPLC Rt = 3.401 min. mp: 164.9-166.8 ;1H NMR
(400MHz, DMSO-d6): δ=9.61(1H, s), 7.89 (2H, d, J=8.82Hz), 7.46(4H, m), 7.31(2H,
d, J=8.82Hz), 7.13(1H, m), 6.96(1H, m), 6.78(1H, d, J=7.22Hz), 6.58(1H, t,
13
J=7.62Hz), 4.88 (2H, s), 4.33 (2H, s); C NMR (100MHz, DMSO-d6): δ=165.42,
143.62, 141.36, 135.79, 133.89, 132.23, 130.85, 130.05, 129.10, 128.71, 128.38,
127.14, 126.93, 123.72, 119.39, 116.66, 116.54, 36.55ppm;HRMS (ESI): Calculated
for C20H17BrN2OS m/z (M+ H)+: 413.0324 and 415.0303; found:413.0323 and
415.0305.
4.1.1.4. N-(2-aminophenyl)-4-(((4-(tert-butyl)phenyl)thio)methyl)benzamide (12d)

ACCEPTED MANUSCRIPT
1
Red solid; Yield 79.3 %; HPLC Rt = 3.592 min. mp: 113.0-114.2 ; H NMR
(400MHz, DMSO-d6): δ=9.61(1H, s), 7.89 (2H, d, J=8.82Hz), 7.49(2H, d, J=8.42Hz),
7.29(4H, m), 7.16(1H, d, J=7.62Hz), 6.96(1H, m), 6.78(1H, d, J=7.22Hz), 6.59(1H, t,
13
J=7.22Hz), 4.88 (2H, s), 4.29 (2H, s), 1.25 (9H, s); C NMR (100MHz, DMSO-d6):
δ=165.44, 149.16, 143.62, 141.93, 133.76, 132.75, 129.06, 128.88, 128.35, 127.14,
126.93, 126.34, 123.73, 116.67, 116.54, 37.02, 34.63, 31.50 ppm.HRMS (ESI):
Calculated for C24H26N2OS m/z (M+ H)+: 391.1845; found:391.1839.
4.1.1.5.N-(2-aminophenyl)-4-(((3,4-dimethoxyphenyl)thio)methyl)benzamide (12e)
Dark yellow solid; Yield 68.4 %; HPLC Rt = 3.442 min. mp: 125.0-127.7 ; H
1
NMR (400MHz, DMSO-d6): δ= 9.56(1H, s), 7.84(2H, s), 7.33(2H, s), 7.10(1H,
s),6.90-6.70(5H, m), 6.52(1H, m), 4.82(2H, s), 4.14(2H, s), 3.64(6H, s);13C NMR
(100 MHz, DMSO-d6): δ= 165.49, 149.31, 148.61, 143.60, 142.29, 133.62, 129.16,
128.28, 127.14, 126.94, 126.12, 123.77, 116.74, 116.61, 114.91, 112.64, 56.01, 38.73
ppm. HRMS (ESI): Calculated for C22H22N2O3S m/z (M+H) +:395.1430;
found:395.1430.
4.1.1.6. N-(2-aminophenyl)-4-((p-tolylthio)methyl)benzamide (12f)
Dark red solid; Yield 48.1 %; HPLC Rt = 3.493 min. mp: 140.2-141.0 ; H NMR
1
(400MHz, DMSO-d6): δ=9.62(1H, s), 7.88 (2H, d, J=8.42Hz), 7.44(2H, d, J=8.42Hz),
7.24(2H, d, J=8.42Hz), 7.12(3H, m), 6.96(1H, m), 6.78(1H, d, J=8.82Hz), 6.59(1H, t,
13
J=8.82Hz), 4.88 (2H, s), 4.29 (2H, s), 1.25 (9H, s); C NMR (100MHz, DMSO-d6):
δ=165.46, 143.61, 141.98, 136.17, 133.70, 132.25, 130.10, 129.75, 129.06, 128.30,
127.13, 126.91, 123.75, 116.67, 116.55, 37.37, 21.01ppm.HRMS (ESI): Calculated
for C21H20N2OS m/z (M+ H)+: 349.1375; found:349.1372.
4.1.1.7. N-(2-aminophenyl)-4-(((4-methoxyphenyl)thio)methyl)benzamide (12g)
White solid; Yield 54.8 %; HPLC Rt = 3.449 min. mp: 143.0-144.5 ;1H NMR
(400MHz, DMSO-d6): δ=9.61(1H, s), 7.87 (2H, d, J=8.42Hz), 7.37-7.28(4H, m),
7.14(1H, d, J=6.82Hz), 6.96-6.89(3H, m), 6.78(1H, d, J=8.42Hz), 6.59(1H, t,
13
J=7.62Hz), 4.88 (2H, s), 4.18 (2H, s), 3.73 (3H, s); C NMR (100MHz, DMSO-d6):
δ=165.45, 159.01, 143.61, 142.24, 133.59, 133.03, 129.06, 128.26, 127.14, 126.92,
125.72, 123.76, 116.69, 116.57, 115.13, 55.64ppmHRMS (ESI): Calculated for
C21H20N2O2S m/z (M+ H)+: 365.1324; found:365.1333.
4.1.1.8. N-(2-aminophenyl)-4-(((4-ethylphenyl)thio)methyl)benzamide (12h)
Dark red solid; Yield 60.3%; HPLC Rt = 3.550 min. mp: 150.8-151.7 ;1H NMR
(400MHz, DMSO-d6): δ=9.62(1H, s), 7.88 (2H, d, J=7.62Hz), 7.46(2H, d, J=8.02Hz),
7.26-7.15(5H, m), 7.12(3H, m), 6.96(1H, m), 6.78(1H, d, J=8.82Hz), 6.59(1H, t,
J=8.82Hz), 4.89 (2H, s), 4.27 (2H, s), 2.56 (2H, m), 1.14(3H, t, J=7.62Hz); 13C NMR
(100MHz, DMSO-d6): δ=165.45, 143.61, 142.42, 141.96, 133.72, 132.66, 129.59,
129.06, 128.92, 128.32, 127.13, 126.92, 123.75, 116.67, 116.55, 37.28, 28.10,
15.93ppmHRMS (ESI): Calculated for C22H22N2OS m/z (M+ H)+: 363.1532;
found:363.1523.

ACCEPTED MANUSCRIPT
4.1.1.9. N-(2-aminophenyl)-4-((phenethylthio)methyl)benzamide (12i)
Gray solid; Yield 40.3 %; HPLC Rt = 3.484 min. mp: 107.5-108.7 ;1H NMR
(400MHz, DMSO-d6): δ=9.63(1H, s), 7.93(2H, d, J=8.02Hz), 7.46(2H, d, J=8.02Hz),
7.3-7.15(6H, m), 6.96(1H, m), 6.79(1H, d, J=8.42Hz), 6.59(1H, t, J=7.62Hz), 3.83
(2H, s), 4.82(2H, t, J=7.22Hz), 2.62 (2H, t, J=8.02Hz);13C NMR (100MHz,
DMSO-d6): δ=165.51, 143.60, 142.89, 140.90, 133.55, 129.20, 128.97, 128.77,
128.38, 127.15, 126.92, 126.62, 123.79, 116.70, 116.57, 35.61, 35.10,
32.58ppm;HRMS (ESI): Calculated for C22H22N2OS m/z (M+ H)+: 363.1532;
found:363.1518.
4.1.1.10. N-(2-aminophenyl)-4-(((3-fluorophenyl)thio)methyl)benzamide (12j)
1
Dark red solid; Yield 77.3 %; HPLC Rt = 3.411 min. mp: 150.8-151.8 ; H NMR
(400MHz, DMSO-d6): δ=9.61(1H, s), 7.90(2H, d, J=7.62Hz), 7.51(2H, d, J=8.02Hz),
7.35-7.14(4H, m), 6.96(2H, m), 6.78(1H, d, J=8.42Hz), 6.58(1H, t, J=8.02Hz),
4.89(2H, s), 4.38(2H, s);13C NMR (100MHz, DMSO-d6): δ=165.44, 163.96, 161.52,
143.62, 141.24, 139.06, 133.94, 131.19, 129.11, 128.41, 127.14, 126.94, 124.36,
123.71, 116.66, 114.98, 114.75, 113.17, 112.96ppm;HRMS (ESI): Calculated for
C20H17FN2OS m/z (M+ H)+: 353.1125; found:353.1123.
4.1.1.11. methyl 2-((4-((2-aminophenyl)carbamoyl)benzyl)thio)benzoate (12k)
1
Dark red solid; Yield 78.6 %; HPLC Rt = 3.465 min. mp: 148.3-149.7 ; H NMR
(400MHz, DMSO-d6): δ=9.63(1H, s), 7.94-7.86(3H, m), 7.54(4H, m), 7.24-7.17(2H,
m), 6.96(1H, m), 6.78(1H, d, J=8.42Hz), 6.59(1H, t, J=7.62Hz), 4.89(2H, s), 4.38(2H,
s);13C NMR (100MHz, DMSO-d6): δ=166.55, 165.47, 143.61, 140.86, 133.98, 133.15,
131.22, 129.39, 128.44, 127.60, 127.14, 126.94, 126.79, 124.89, 123.72, 52.61,
35.77ppm; HRMS (ESI): Calculated for C22H20N2O3S m/z (M+ H)+: 393.1274;
found:393.1280.
4.1.1.12. N-(2-aminophenyl)-4-(((3,5-dimethylphenyl)thio)methyl)benzamide (12l)
Light yellow solid; Yield 73.2 %; HPLC Rt = 3.622 min. mp: 135.1-136.3 ; H
1
NMR (400MHz, DMSO-d6): δ=9.60(1H, s), 7.89(2H, d, J=8.42Hz), 7.48(2H, d,
J=8.02Hz), 7.16 (1H, d, J=8.02Hz), 6.97(3H, m), 6.80(2H, m), 6.59(1H, t, J=7.62Hz),
4.88(2H, s), 4.29(2H, s), 2.22(6H, s);13C NMR (100MHz, DMSO-d6): δ=165.47,
143.60, 141.83, 138.55, 135.75, 133.77, 129.12, 128.31, 128.06, 127.13, 126.92,
126.31, 123.73, 116.67, 116.54, 36.54, 21.25ppm;HRMS (ESI): Calculated for
C22H22N2OS m/z (M+ H)+: 363.1532; found:363.1530.
4.1.1.13. N-(2-aminophenyl)-4-(((4-(trifluoromethyl) pyrimidin-2-yl) thio) methyl)
benzamide (12m)
1
Dark red solid; Yield 60.5 %; HPLC Rt = 3.732 min. mp: 135.9-136.4 ; H NMR
(400MHz, DMSO-d6): δ=9.62(1H, s), 9.01(1H, s), 7.92(2H, d, J=5.61Hz), 7.74(1H, d,
J=4.81Hz), 7.60(2H, d, J=7.62Hz), 7.17(1H, d, J=6.82Hz), 6.97(1H, m), 6.79(1H, d,
J=8.82Hz), 6.59(1H, t, J=8.02Hz), 4.89(2H, s), 4.53(2H, s);13C NMR (100 MHz,
DMSO-d6): δ=172.38, 165.43, 161.76, 154.78, 154.42, 143.61, 141.36, 134.03,

ACCEPTED MANUSCRIPT
129.38, 128.35, 127.14, 126.94, 123.70, 122.13, 119.39, 116.67, 116.54, 113.56,
34.52ppm.HRMS (ESI): Calculated for C19H15F3N4OS m/z (M+ H)+: 405.0998;
found:405.1006.
4.1.1.14. N-(2-aminophenyl)-4-(((5-(trifluoromethyl)pyridin-2-yl) thio) methyl)
benzamide (12n)
Dark red solid; Yield 54.3 %; HPLC Rt = 3.394 min. mp: 184.5-186.3 ;1H NMR
(400MHz, DMSO-d6): δ=9.66(1H, s), 8.85(1H, s), 8.02-7.92(3H, m), 7.58(3H, d,
J=7.22Hz), 7.17(1H, d, J=6.42Hz), 6.99(1H, t, J=6.82Hz), 6.82(1H, d, J=8.02Hz),
6.64(1H, t, J=7.22Hz), 4.57(2H, s);13C NMR (100 MHz, DMSO-d6): δ=165.32,
163.93, 146.55, 142.78, 141.57, 134.04, 133.92, 129.25, 128.44, 127.14, 126.96,
125.73, 124.13, 122.15, 121.94, 121.61, 117.29, 116.96, 34.87, 33.42ppm.HRMS
(ESI): Calculated for C20H16F3N3OS m/z (M+ H)+: 404.1045; found:404.1033.
4.1.2. General method for the synthesis of 15a-15k;
Step-1: To a 100 mL, one-necked, round-bottomed flask, sequentially charged with
compound 10 (7 mmol, 1 equiv), methyl (E)-3-(4-(bromomethyl) phenyl) acrylate (7
mmol), K2CO3 (14 mmol), KI (7 mmol) and 30 mL of 2-Butanone. The reaction was
heated at 40 for 6-12 h in an oil bath. TLC analysis, using ethyl acetate: petroleum
ether (1:10) as the eluent, indicated the complete reaction. The reaction mixture was
allowed to cool to room temperature and then poured into 200 mL of ethyl acetate
with vigorous stirring. Stirring was continued for an additional 10 min, and the
resulting solid was then filtered off. The filtrate was concentrated to provide 13 as a
white solid.
Step-2: To a 100 mL one-necked, round-bottomed flask, sequentially charged with 13
(1 mmol, 1 equiv, obtained from step-1), NaOH (10 mol), water and methanol
(v/v=1:1) 15 mL, the reaction was heated at 60 for 2 h in an oil bath. TLC analysis,
using ethyl acetate: petroleum ether (1:5) as the eluent, indicated the complete
reaction. The reaction mixture was cooled to room temperature, and the precipitate
was then filtered through a sintered glass funnel to afford 14 as a white solid.
Step-3: To a 100 mL one-necked, round-bottomed flask, sequentially charged with 14
(0.91 mmol, 1 equiv., obtained from step-2), DMF (15 mL), BOP (1 mmol, 1.2 equiv.)
and Triethylamine (3.6 mmol, 4 equiv.). The mixture was stirred for 10 min at r.t.
Ben- zene-1,2-diamine (1 mmol, 1.2 equiv.) was then added and the solution was
stirred at r.t. for another 6 h. The mixture was poured into 200 mL of saturated
aqueous sodium bicarbonate with vigorous stirring. Stirring was continued for an
additional 10 min, and the resulting solid was then filtered off. The filtrate was
washed by petroleum ether and purified by column chromatography to obtain
compound 15 as a solid.

ACCEPTED MANUSCRIPT
4.1.2.1. (E)-N-(2-aminophenyl)-3-(4-(((3,4-dimethoxyphenyl) thio) methyl) phenyl)
acrylamide (15a)
1
Light yellow solid; Yield 77.9 %; HPLC Rt = 3.523 min. mp: 129.0-131.1 . H
NMR (400MHz, DMSO-d6): δ= 9.39(1H, s), 7.53-7.49(3H, m), 7.35- 7.31(3H, m),
6.93-6.85(5H, m), 6.76(1H, d, J=8.02Hz), 6.58(1H, t, J=7.22Hz), 4.95(2H, s),
4.16(2H, s), 3.72(7H, d, J=10.83Hz);13C NMR (100 MHz, DMSO-d6): δ= 163.96,
149.24, 148.63, 142.05, 140.37, 139.68, 134.00, 129.95, 128.07, 126.23, 125.17,
123.91, 122.52, 116.74, 116.47, 115.07, 112.63, 55.95 ppm. HRMS (ESI): Calculated
for C24H24N2O3S m/z (M+H) +: 421.1587; found: 421.1580.
4.1.2.2.(E)-N-(2-aminophenyl)-3-(4-((phenethylthio)methyl)phenyl)acrylamide (15b)
Light yellow solid; Yield 68.8 %; HPLC Rt = 3.575min. mp: 103.4-105.4 ; 1H NMR
(400MHz, DMSO-d6): δ= 9.36(1H, s), 7.55-7.49(3H, m), 7.37-7.15(8H, m),
6.91-6.84(2H, m), 6.74(2H, d, J=6.82Hz), 6.55(2H, t, J=7.62Hz), 4.92(2H, s),
3.76(2H, s), 2.77(2H, t, J=7.22Hz), 2.61(2H, t, J=8.82Hz); 13C NMR (100 MHz,
DMSO-d6): δ= 163.99, 142.06, 140.98, 139.74, 133.91, 129.98, 128.96, 128.76,
128.18, 126.61, 126.24, 125.18, 123.98, 122.47, 116.76, 116.49, 35.66, 35.27, 32.67
+
ppm. HRMS (ESI): Calculated for C24H24N2OS m/z (M+H) :389.1688; found:
389.1679.
4.1.2.3. (E)-N-(2-aminophenyl)-3-(4-(((5-(trifluoromethyl)pyridin-2-yl) thio) methyl)
phenyl)-acrylamide (15c)
White solid; Yield 65.9 %; HPLC Rt = 3.454 min. mp: 205.4-207.5 ;1H NMR (400
MHz, DMSO-d6): δ=9.42(1H, s), 8.85(1H, s), 8.00(1H, s), 7.56(6H, m), 7.35(1H, m),
6.93(3H, m), 6.60(1H, m), 5.19(2H, m), 4.53(2H, s); 13C NMR (100 MHz, DMSO-d6):
δ=164.07, 163.94, 146.52, 141.77, 139.63, 139.55, 134.31, 134.03, 130.00, 128.26,
126.27, 125.74, 125.21, 124.11, 123.04, 122.66, 122.10, 121.90, 121.58, 116.98,
+
116.64, 33.51 ppm. HRMS (ESI): Calculated for C22H18F3N3OS m/z (M+H) :
430.1202; found: 430.1194.
4.1.2.4. (E)-N-(2-aminophenyl)-3-(4-(((3-fluorophenyl) thio) methyl) phenyl)
acrylamide (15d)
White solid; Yield 79.4 %; HPLC Rt = 3.473 min. mp: 182.7.0-184.2 ;1H NMR
(400 MHz, DMSO-d6): δ=9.37(1H, s), 7.51-7.47(3H, m), 7.42(2H, d, J=7.62Hz),
7.31(2H, m), 7.20(1H, d, J=9.63Hz), 7.14(1H, d, J=8.02Hz), 6.96(1H, m), 6.87(2H,
m), 6.74(1H, d, J=8.42Hz), 6.55(1H, t, J=8.02Hz), 4.92(2H, s), 4.30(2H, s); 13C NMR
(100 MHz, DMSO-d6): δ=163.94, 161.51, 142.07, 139.58, 139.24, 134.29, 131.17,
131.08, 129.91, 128.21, 126.25, 125.18, 124.37, 123.96, 122.73, 116.75, 116.48,
115.01, 114.78, 113.16, 112.95, 36.33ppm. HRMS (ESI): Calculated for
C22H19FN2OS m/z (M+H) +:379.1281; found: 379.1272.
4.1.2.5. (E)-N-(2-aminophenyl)-3-(4-(((3,5-dimethylphenyl) thio) methyl) phenyl)
acrylamide (15e)

ACCEPTED MANUSCRIPT
White solid; Yield 68.5 %; HPLC Rt = 3.727 min. mp: 186.3-187.5 ; 1H NMR (400
MHz, DMSO-d6): δ=9.39(1H, s), 7.52(3H, m), 7.40-7.32(3H, m), 6.94(4H, m),
13
6.73(2H, m), 6.56(1H, t, J=7.22Hz), 4.93(2H, s), 4.22(2H, s), 2.20(6H, s); C NMR
(100 MHz, DMSO-d6): δ=163.94, 142.06, 139.85, 139.64, 138.53, 135.89, 134.13,
129.92, 128.13, 128.07, 126.39, 126.23, 125.17, 123.97, 122.60, 116.73, 116.47,
36.81, 21.25 ppm. HRMS (ESI): Calculated for C24H24N2OS m/z (M+H) +:389.1688;
found: 389.1678.
4.1.2.6. (E)-N-(2-aminophenyl)-3-(4-(((4-fluorophenyl) thio) methyl) phenyl)
acrylamide (15f)
White solid; Yield 44.8 %; HPLC Rt = 3.432 min. mp: 178.9-179.8 ; 1H NMR (400
MHz, DMSO-d6): δ=9.42(1H, s), 7.54-7.49(3H, m), 7.40-7.34(5H, m), 7.15(2H, t,
J=9.02Hz), 6.93(2H, m), 6.76(1H, d, J=7.82Hz), 6.57(1H, t, J=7.42Hz), 4.95(2H, s),
4.22(2H, s);13C NMR (100 MHz, DMSO-d6): δ=163.94, 162.71, 160.29, 142.05,
139.71, 139.59, 134.16, 132.34, 132.26, 131.39, 131.36, 129.88, 128.13, 126.21,
125.15, 123.97, 122.67, 116.72, 116.58, 116.46, 116.37, 38.07 ppm. HRMS (ESI):
Calculated for C22H19FN2OS m/z (M+H) +:379.1281; found: 379.1286.
4.1.2.7. (E)-N-(2-aminophenyl)-3-(4-(((4-(tert-butyl) phenyl) thio) methyl) phenyl)
acrylamide (15g)
1
Yellow solid; Yield 52.9 %; HPLC Rt = 3.825 min. mp: 153.2-154.4 ; H NMR
(400MHz, DMSO-d6): δ=9.41(1H, s), 7.55-7.50(3H, m), 7.42-7.25(7H, m),
6.93-6.86(2H, m),6.77(1H, d, J=8.22Hz), 6.58(1H, t, J=7.42Hz), 4.95(2H, s), 4.23(2H,
13
s), 1.24(9H, s); C NMR (100 MHz, DMSO-d6): δ=163.94, 149.16, 142.05, 139.94,
139.64, 134.13, 132.90, 129.85, 128.92, 128.16, 126.32, 126.22, 125.15, 123.99,
122.61, 116.73, 116.48, 37.27, 34.62, 31.50 ppm. HRMS (ESI): Calculated for
C26H28N2OS m/z (M+H) +:417.2001; found: 417.1988.
4.1.2.8. (E)-N-(2-aminophenyl)-3-(4-((p-tolylthio)methyl)phenyl)acrylamide (15h)
1
White solid; Yield 58.6 %; HPLC Rt = 3.599 min. mp: 202.2-203.1 ; H NMR
(400MHz, DMSO-d6): δ=9.38(1H, s), 7.53-7.49(3H, m), 7.38-7.33(3H, m), 7.24(2H,
d, J=8.03Hz),7.12(2H, d, J=7.97Hz), 6.94-6.84(2H, m), 6.76(1H, d, J=8.15Hz),
13
6.58(1H, t, J=7.34Hz), 4.94(2H, s), 4.21(2H, s),2.25(3H, s); C NMR (100 MHz,
DMSO-d6): δ=163.94, 142.06, 140.02, 139.64, 136.15, 134.06, 132.41, 130.09,
129.86, 129.77, 128.11, 126.23, 125.17, 123.96, 122.57, 116.74, 116.47, 37.59, 21.02
ppm. HRMS (ESI): Calculated for C23H22N2OS m/z (M+H)+:375.1532;
found:375.1536.
4.1.2.9. (E)-N-(2-aminophenyl)-3-(4-((naphthalen-1-ylthio)methyl)phenyl)acrylamide
(15i)
1
White solid; Yield 61.5 %; HPLC Rt = 3.749 min. mp: 198.9-200.1 ; H NMR
(400MHz, DMSO-d6): δ=9.41(1H, s), 8.23(1H, d, J=7.88Hz), 7.93(1H, d, J=7.88Hz),
7.80(1H, d, J=8.35Hz), 7.61-7.34(10H, m), 6.93-6.85(2H, m), 6.76(1H, d, J=7.76Hz),
6.58(1H, t, J=7.53Hz), 4.95(2H, s), 4.34(2H, s);13C NMR (100 MHz, DMSO-d6):

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δ=163.93, 142.06, 139.59, 139.43, 134.22, 133.84, 133.25, 132.10, 129.98, 129.11,
128.14, 127.53, 127.28, 127.06, 126.84, 126.27, 125.16, 124.52, 123.97, 122.67,
116.72, 116.46, 37.32ppm. HRMS (ESI): Calculated for C26H22N2OS m/z (M+ H)
+:411.1532; found: 411.1531.
4.1.2.10. (E)-N-(2-aminophenyl)-3-(4-(((4-ethylphenyl) thio) methyl) phenyl)
acrylamide (15j)
1
White solid; Yield 55.5 %; HPLC Rt = 3.672 min. mp: 173.5-175.1 ; H NMR
(400MHz, DMSO-d6): δ=9.38(1H, s), 7.54-7.49(3H, m), 7.39-7.33(3H, m), 7.27(2H,
d, J=8.22Hz), 7.15(2H, d, J=8.22Hz), 6.93- 6.85(2H, m), 6.77(1H, d, J=7.82Hz),
6.58(1H, t, J=7.26Hz), 4.94(2H, s), 4.22(2H, s), 2.58-2.52(2H, m), 1.12(3H, t,
J=7.64Hz); 13C NMR (100 MHz, DMSO-d6): δ=163.94, 142.42, 142.06, 139.99,
139.65, 134.08, 132.81, 129.86, 129.62, 128.91, 128.13, 126.24, 125.17, 123.96,
122.57, 116.75, 116.48, 37.51, 28.11, 15.95 ppm. HRMS (ESI): Calculated for
C24H24N2OS m/z (M+H) +:389.1688; found: 389.1672.
4.1.2.11. (E)-N-(2-aminophenyl)-3-(4-(((4-chlorophenyl) thio) methyl) phenyl)
acrylamide (15k)
1
White solid; Yield 76.2 %; HPLC Rt = 3.599 min. mp: 192.7-194.5 ℃; H NMR
(400MHz, DMSO-d6): δ=9.39(1H, s), 7.55-7.49(3H, m), 7.41-7.33(7H, m),
6.91-6.84(2H, m), 6.76(2H, d, J=8.04Hz), 6.57(1H, t, J=7.16Hz), 4.94(2H, s),
4.28(2H, s); 13C NMR (100 MHz, DMSO-d6): δ=163.93, 142.06, 139.59, 139.42,
135.35, 134.23, 131.12, 130.70, 129.89, 129.35, 128.18, 126.25, 123.95, 122.69,
116.74, 116.47, 36.94 ppm. HRMS (ESI): Calculated for C22H19ClN2OS m/z (M+H)
+:395.0986, found:395.0989.
4.2. Biological materials and methods
4.2.1. Cell lines and cell culture
For cell culture experiments, A549, A375 and HCT116 cells were grown in
RPMI1640 containing 10 % fetal bovine serum (FBS) (ThermoFisher, USA) and 1 %
penicillin/streptomycin. Hela and SMMC7721 cells were grown in DMEM containing
10
%
fetal bovine serum (FBS) (ThermoFisher, USA) and
1
%
penicillin/streptomycin. The cells were grown at 37 ℃ under humidified air
supplemented with 5 % CO2 atmosphere.
4.2.2. CCK-8 cell assay
The cells were grown to 70 %-85 % confluence before next assays. the cell lines
were added into a 96-well plate with 4000 cells per well. After 12 h, 10 µL of various
concentrations of compounds were added into 96 wells. Following incubation for 72 h,
the mixture was incubated for another 1h by adding 10 µL of CCK-8 reagent. Then
absorbance was measured at 450 nm in an EnSpire multimode plate reader
(PerkinElmer, USA).

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4.2.3. HDAC inhibition assay
The HDAC inhibitory activity assay was measured by Fluor de Lys®-Green
HDAC Assay kit (BML-AK530, Enzo® Life Sciences), HDAC1 Assay kit
(BML-AK511, Enzo® Life Sciences), HDAC2 Assay kit (BML-AK512, Enzo® Life
Sciences), HDAC3 Assay kit (BML-AK531, Enzo® Life Sciences), HDAC6 Assay
kit (BML-AK516, Enzo® Life Sciences) and HDAC8 Assay kit ((BML-AK518,
Enzo® Life Sciences). According to the manufacturer's guidelines, the various
concentrations of compounds and HDAC isoforms were mixed into a 96-well plate
and incubated at 37 for 10 min. Then the diluted HDAC substrate was added into
each well and incubated at 37 for 30 min, which initiated the deacetylation reaction.
The reaction was stopped by adding 50 µL of developer, and the mixture was
incubated at 37
for another 20 min. Fluorescence was measured by an EnSpire
multimode plate reader (PerkinElmer, USA).
4.2.4. Molecular docking
The molecular docking studies were implemented through Surflexdock in
Sybyl-X 2.1. (Tripos Inc.). The three-dimensional structure of HDAC1 (PDB code:
4BKX), HDAC2 (PDB code:4LXZ) and HDAC6 (PDB code: 5G0G) was performed
from the Protein Data Bank. The binding water and ligands were deleted and the polar
hydrogen was added. The molecular structures of compounds 12g and 12h were
optimized using Tripos force field. The molecular docking of 12g and 12h with
HDAC1, HDAC2 and HDAC6 was carried out with Surflex-Dock module.
4.2.5. Immunofluorescence test
A549 cells were seeded into glass bottom cell culture dish, incubated at 37 for
12 hours and treated with drugs 12g and 12h for 12 hours. Cells were fixed with 4 %
formaldehyde for 30 min at room temperature, and rinsed with 1 X PBS for 3 times.
Cells were permeabilized with blocking solution (0.3 % Terton X-100 and 3 % bovine
serum albumin (BSA) / PBS) for 60 min and rinsed 3 times in 1 X PBS for 5 min each.
Diluted primary antibody (abcam anti-Histone H3 (acetyl K9)) was applied into cells.
Cells were incubated overnight at 4 and rinsed three times in 1 X PBS for 5 min
each. The secondary antibody (abcam IgG H&L (Alexa Fluor® 488) was diluted and
added into wells. Following incubation for 1-2 h at room temperature in the dark,
samples were mixed with DAPI for 3 min, and rinsed three times in 1 X PBS for 2
min each. Confocal fluorescence microscope images were taken using Leica TCS SP8
confocal fluorescence microscope (Leica Microsystems, Germany).
4.2.6. Cell apoptosis analysis
A549 cells were added into 6-well plates with 4× 106 cells per well. After 12 h,
20 µL of various concentrations of compounds 12g and 12h were added into 6 wells.
After 48 h, cells were collected and resuspended in 1 X annexin-binding buffer. Cells
were stained with Annexin V-FITC (10ꢀ mg/ml) and propidium iodide, and incubated
for 15 min in dark. The cell apoptosis was analyzed by a MoFlo XDP flow cytometer
(Beckman Coulter, Inc.).

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4.2.7. Cell cycle analysis
A549 cells were added into 6-well plates with 106 cells per well. After 12 h, 20
µL of various concentrations of compounds were added into 6 wells. Following
incubation for 48 h, cells were collected, and then fixed with 75 % ethanol solution at
-20 for 24 h. Cells were incubated for 30 min by adding 200 µL of RNase, and then
stained with 200 µL of propidium iodide. The cell cycle was analyzed by a MoFlo
XDP flow cytometer (Beckman Coulter, Inc.).
4.2.8. Migration assay
A549 cell lines were added into a 12-well plate with 3 × 106 cells per well and
incubated for 12 h. After drawing a straight line at the bottom of wells, wells were
washed with phosphate-buffered saline. 10 µL of test compounds were added into 12
wells. Following incubation at 37 , 5 % CO2 atmosphere, the cells were allowed to
migrate. Images were generated with an OPTEC BDS200 microscope.
4.2.9. Colony formation assay
A549 cell lines were added into 6-well plates with 3000 cells per well. After 12 h,
20 µL of various concentrations of compounds were added into 6 wells. Following
incubation for 10-14 days, cells were fixed with 4 % Paraformaldehyde Fix Solution
for 15 min and stained with 0.1 % crystal violet.
4.2.10. Pharmacokinetic parameters
Compounds 12g and 12h were dissolved in a solution of DMSO: 40 %
SBE-β-CD = 20: 80 (v/v) for intravenous injection. Blood samples were collected
through retro-orbital plexus at 0.08, 0.25, 0.5, 1, 2, 4, 8, 12, 24 h after intravenous
injection. Plasma samples were placed in anticoagulant tubes containing EDTA-K2,
separated by centrifugation for 10 min and stored at -20 until analysis. An aliquot of
10 µL of sample was added with 200 µL ACN which contains Verapamil (5 ng/mL)
and Glibenclamide (50 ng/mL) for protein precipitation. The mixture was vortexed
for 10 min, and then centrifuged at 3700 rpm for 8 min. 80 µL of supernatant was
added with 80 µL water, then vortexed for 10 min. An aliquot of 10 µL of the mixture
was injected into the LC-MS/MS system. Pharmacokinetic parameters were estimated
by noncompartmental model using WinNonlin 8.0.
4.2.10 In vivo antitumor activity
All animal experiments were performed in compliance with the local ethics
committee. BALB/C nude female mice aged 5-6 weeks were provided by Vital River
Laboratory Animal Technology Co. Ltd (Beijing, China). 3× 106 human lung cancer
cells (A549) were injected subcutaneously into the right axilla region of female nude
mice. When the tumors reached approximately volume of 95 mm3, the mice were
randomized into treatment and control groups (4 mice per group). The treatment
groups received compounds 12g (25mg/kg), 12h (25mg/kg) and CI994 (25mg/kg) by
intraperitoneal administration twice a day for 21 days, and the control groups received

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equal volume of DMA & 5 % Solutol HS-15(5:95, v:v). The tumor size and body
weights were monitored every 3 days. Tumor volume (V) was calculated as
V=length× width2 /2. The tumor growth inhibition (TGI) was calculated using the
following formula:
TGI =100%× [1－(TVt(T)－TVinitial(T))/(TVt(C)－TVinitial(C))]
where TVt(T)and TVinitial(T) are the mean tumor volume measured at final time and at
initial time for the treatment groups, respectively, and TVt(C) and TVinitial(C) are the
mean tumor volume for the control group.
Acknowledgments
We are grateful to the National Science Foundation of China (Grant No. 21636001)
for their generous financial supports.
Appendix A. Supplementary data
Supplementary data related to this article will be found at xxxx.
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