Development of hydroxamate-based histone deacetylase inhibitors of bis-substituted aromatic amides with antitumor activities

Article abstract of DOI:10.1039/c9md00306a

Previously, we designed and synthesized a series of bis-substituted aromatic amide-based histone deacetylase (HDAC) inhibitors. In this study, we report the replacement of a bromine atom by different amides on the phenyl ring of the CAP region. Representa

Full text of DOI:10.1039/c9md00306a

View Article Online
View Journal
MedChemComm
The official journal of the European Federation for Medicinal Chemistry
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: F. Yang, D. Ge, L.
Han, Y. Yang, Y. Chen, H. Zhang and N. Zhao, Med. Chem. Commun., 2019, DOI: 10.1039/C9MD00306A.
Volume
Number
May 2017
8
5
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Pages 809-1132
MedChemComm
The official journal of the European Federation for Medicinal Chemistry
rsc.li/medchemcomm
Accepted Manuscripts are published online shortly after acceptance,
before technical editing, formatting and proof reading. Using this free
service, authors can make their results available to the community, in
citable form, before we publish the edited article. We will replace this
Accepted Manuscript with the edited and formatted Advance Article as
soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes to the
text and/or graphics, which may alter content. The journal’s standard
Terms & Conditions and the Ethical guidelines still apply. In no event
shall the Royal Society of Chemistry be held responsible for any errors
or omissions in this Accepted Manuscript or any consequences arising
from the use of any information it contains.
ISSN 2040-2511
COMMUNICATION
G. J. Sharples, S. L. Cobb et al.
Exploring the links between peptoid antibacterial activity and
toxicity
rsc.li/medchemcomm

Please do not adjust margins
Page 1 of 10
MedChemComm
View Article Online
DOI: 10.1039/C9MD00306A
ARTICLE
Development of Hydroxamate-Based Histone Deacetylase
Inhibitors of Bis-substituted Aromatic Amides with Antitumor
Activities
Received 00th January 20xx,
Accepted 00th January 20xx
Di Ge,1,# Lina Han,1,# Feifei Yang,1,2,#,* Na Zhao, Yang Yang,2 Hua Zhang,1,* and Yihua Chen2,*
DOI: 10.1039/x0xx00000x
Previously, we designed and synthesized a series of bis-substituted aromatic amide-based histone deacetylase (HDAC)
inhibitors. In this study, we report the replacement of bromine atom by different amides on the phenyl ring of the CAP
region. Representative compounds 9d and 10k exhibited low nanomolar IC50s against HDAC1, which were ten times lower
than the positive control SAHA. The IC50 of 9d against human A549 cancer cell line was 2.13 μM. Furthermore, 9d increased
the acetylation of histone H3 and H4 in a dose-dependent manner. Moreover, 9d significantly arrested A549 cells at G2/M
phase and induced A549 cells apoptosis. Finally, molecular docking investigation rationalized the high potency of
compound 9d.
Chidamide17) (Figure 1) have been approved by the US FDA for
the treatment of cutaneous T-cell lymphoma (CTCL),
1. Introduction
O
O
NH
Epigenetic modification plays an important role in the
treatment of cancers1,2. Protein lysine acetylation, regulated
by histone acetyltransferases (HATs) and histone deacetylases
(HDACs), can lead to changes in gene expression without
modifying the gene sequence3,4. HATs neutralize the positive
charge of lysine residues by adding an acetyl group to the N-
terminal tail of histone, which results in open and
transcriptionally active chromatin structure. Contrarily, HDACs
remove the acetyl group, leading to closed chromatin
structure and transcriptional repression5,6. The overexpression
of HDACs has been found in various cancers, which is
associated with the down-regulation expression of onco-
suppressor genes7. Meanwhile, some non-histone proteins
such as tubulin, HSP90, p53 and ER, also serve as substrates of
HDACs8-10. Over the past decades, HDACs have been validated
as practical targets for cancer treatments11.
HN
S
S
O
H
NH
O
O
N
NH
NHOH
O
O
O
1 (SAHA)
Approved (CTCL)
2 (FK-228)
Approved (CTCL, PTCL)
O
OH
N
H
N
O
H
N
H
S
OH
N
O
H
O
HN
3 (PXD-101)
Approved (PTCL)
4 (LBH-589)
Approved (multiple myeloma, MM)
O
O
OH
N
N
N
H
NH2
H
H
N
N
N
O
N
Until now, 18 HDAC isoforms have been found in humans
and can be divided into classes I, II, III and IV. Classes I (1, 2, 3,
8), II (4, 5, 6, 7, 9, 10) and IV (11) HDACs are Zn2+-dependent
enzymes, while class III HDACs (SIRT1−7) require NAD+ for
their activities12. Recent development of HDAC inhibitors
(HDACis) mainly focuses on Zn2+-dependent HDACs. So far, five
HDACis (SAHA13, FK-22814, PXD-10115, LBH-58916 and
F
5 (Chidamide)
Approved (PTCL)
6 (SB939)
Phase II
O
R1
R2
O
N
NH2
O
n
H
N
H
N
N
NHOH
O
O
7 (MS-275 )
Phase
8
I
Fig. 1 HDAC inhibitors approved or under clinical development.
a. 1School of biological science and technology, University of Jinan, Jinan, Shandong
Province 250022, China
peripheral T-cell lymphoma (PTCL) or multiple myeloma (MM).
A number of other inhibitors, such as 6 and 7, are also
processing at different stages of clinical investigations11,17,18
Structures of most common HDACis usually comprise three
parts: a zinc binding group (ZBG), a linker and a surface
b. 2Shanghai Key Laboratory of Regulatory Biology, The Institute of Biomedical
Sciences and School of Life Sciences, East China Normal University, Shanghai,
200241, China.
.
c. *Corresponding Authors: Dr. Yihua Chen or Dr. Hua Zhang or Dr. Feifei Yang, E-
mail: yhchen@bio.ecnu.edu.cn or bio_zhangh@ujn.edu.cn or
yee68511@126.com.
d. # These authors contribute equally to this research work.
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 2 of 10
ARTICLE
Journal Name
H
H
recognition group (CAP group)19,20
.
Recent structural
O
N
O
N
View Article Online
R
R
H
DOI: 10.1039/C9MD00306A
modifications of HDACis mainly focused on the CAP and linker
moieties to develop novel molecules with optimized activities
and selectivities21,22. In our previous study, we designed and
synthesized a series of bis-substituted aromatic amide based
hydroxamic acid HDACis23, among which, compound 8a
displayed mild inhibition against nuclear extract HDAC. In this
report, we aimed to seek more bis-substituted aromatic
amides HDACis with better affinity and physicochemical
properties based on the scaffold of 8a as shown in Fig. 2.
According to the preliminary results of docking studies of 8a
with HDAC1, we found that the meta-bromine in the A ring of
CAP region was in the cavity of the rim of HDAC enzyme, so we
replaced the bromine with different amides which could better
occupy the cavity and a panel of new HDACis were synthesized
and explored. Biological evaluations were also subsequently
performed to examine their mechanism of action and in vitro
antiproliferative activities in multiple cancer cell lines.
O
N
COOH
R
a
d
b, c
NH
N
OH
CHO
CHO
R'
R'
O
O
12
13a-k
14a-n
15a-n
H
N
H
O
O
N
R
N
R
N
e
f
OCH3
NHOH
R'
R'
O
O
O
O
16a-n
9a-b,d-e, 10b-k
Scheme 1 Reagents and conditions: (a) RNH2, EDC, HOBt, DMF; (b)
ArNH2, EtOH, 0 °C then to rt; (c) NaBH4, 0 °C then to rt; (d) Anhydride,
1,4-dioxane, reflux; (e) MeOH, Cat. SOCl2, reflux; (f) NH2OH•HCl, KOH,
MeOH.
The synthetic routes to compounds 9c, 10a and 10l were
illustrated in Scheme 2. The important intermediate 20a-b
were first synthesized via a similar procedure as 15a-n in
Scheme 1. Compound 21a-b were then obtained after
reduction of the nitro group of 20 and was further reacted
with different substituted carboxylic acids to furnish 22a-c
containing the extra amide functionality. Finally, treatment of
esters 22a-c with hydroxylamine gave the desired hydroxamic
acid derivatives 9c, 10a and 10l.
NO2
NO2
Fig. 2 Design strategy and general structures of the target compounds.
NO2
c
a, b
NH
N
OH
CHO
H3CO
H3CO
O
O
2. Results and discussion
17
18a-b
19a-b
2.1 Chemistry
NO2
NH2
The synthetic routes for compounds 9a-b, 9d-e and 10b-k
were depicted in Scheme 1. Compound 12 was coupled with
different substituted amines in the presence of EDC and HOBt
to generate 13a-k. The intermediates 14a-n were constructed
from substituted anilines with 13a-k through reductive
amination and were further condensed with pimelic acid
anhydride in 1,4-dioxane to yield acids 15a-n. Subsequent
esterification of 15a-n in methanol with catalytic amount of
thionyl chloride generated the corresponding esters 16a-n,
which were at last stirred with the freshly prepared
hydroxylamine in methanol to afford hydroxamic acids 9a-b,
9d-e and 10b-k.
e
d
N
OCH3
N
OCH3
H3CO
H3CO
O
O
O
O
20a-b
21a-b
O
O
HN
R
N
HN
R
N
g
f
OCH3
NHOH
H3CO
H3CO
O
O
O
O
22a-c
9c, 10a, 10l
Scheme 2 Reagents and conditions: (a) O-anisidine, EtOH, 0 °C then to
rt; (b) NaBH4, 0 °C then to rt; (c) Anhydride, 1,4-dioxane, reflux; (d)
MeOH, Cat. SOCl2, reflux; (e) Zn, CH3COOH, EtOH/H2O; (f) RCOOH, EDC,
HOBt, DMF; (g) NH2OH•HCl, KOH, MeOH.
2.2 HDAC Inhibition Assay
Initially, the effect of amide group at meta-position of A ring
was explored, compounds 9a-c and 10a-j with an arylalkyl
amino carbonyl group at the meta-position of A ring and
methoxy group at the ortho-position of B ring were assayed
against nuclear extract HDAC (mainly contain HDAC1 and 2)
with 1 (SAHA) as a reference drug. The assay results were
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Page 3 of 10
MedChemComm
Journal Name
shown as inhibition rates as listed in Table 1. Compounds with
ARTICLE
According to the results of our previous studyV,iecwoAmrtipcloe uOnnldines
mono-substitution and di-substitution on the nitrogen of with electron-donating groups at paDraO-Ip: o10s.i1t0io39n/C9oMf DB003r0in6Ag
amide (9a-9b) showed mild inhibitory activities. Excitingly, appeared to be optimal for increasing activity.23 Therefore, in
when a phenyl group was inserted to the terminal alkyl, most light of physicochemical properties and the aforementioned
10-series compounds (10a-c, 10g-h and 10j) exhibited better activity results derived from the substituent exploration of A
activities than the 9-series. In addition, the inversion of the ring, two compounds (9d and 10k) were further synthesized
amide bond seemed to show little influence to the HDAC and evaluated, and a dramatic increase of potency was
inhibition (9a vs 9c and 10a vs 10b). Thus, the amide bond was observed for both compounds, which could further confirm
retained in subsequent structural modifications. Analysis of the conclusion that compounds with para-substituents are
compounds (10b-f) indicated that carbon chain length superior to the corresponding ortho-position derivatives.
between nitrogen and benzene ring greatly affected the However, when the methoxyl of 9d was replaced with
inhibitory potency. Compounds 10b and 10c containing one or hydroxyl, the resulting compound 9e exhibited a decrease in
two methylene bridge showed better activities than their inhibitory activity, which testified the importance of methoxy
analogues with longer linker (10d-f). Furthermore, group for inhibitors. Meantime, compound 10l, the “inverse
introduction of electron-withdrawing groups to the terminal amide” version of amide 10k, exhibited slightly decreased
phenyl ring was beneficial for maintaining the inhibitory activity.
activities (10g & 10h vs 10i). Especially for the fluorine atom,
Table 2 Inhibitory activity of compounds 9d-e and 10k on HDAC
nuclear extract at 0.5 μM.
compound 10j also displayed much better activity than 10d.
Meanwhile, in consideration of the physicochemical properties
of target compounds, LogP values of these derivatives were
R1
predicted
through
the
ALOGPS
2.1
program
(http://146.107.217.178/lab/alogps/start.html). As shown in
Table 1, reports suggest that compounds with LogP between 1
and 3 could have good oral bioavailability24. The results
heralded that compound 9a may have the optimal absorption
properties in this test.
N
NHOH
O
O
R2
R1
R2
4´-OCH3
Inhibition%
LogP
2.85
Compd.
CH3CH2 NHCO-
51%
21%
53%
49%
46%
9d
CH3CH2 NHCO-
4´-OH
2.53
4.16
4.52
1.88
9e
Table 1 Inhibitory activity of compounds 9a-c and 10a-j on HDAC
nuclear extract at 0.5 μM.
4-F-Ph(CH2)2NHCO- 4´-OCH3
4-F-Ph(CH2)2CONH- 4´-OCH3
10k
10l
R1
N
NHOH
-
-
SAHA
O
O
OCH3
Compd.
R1
Inhibition%
LogP
Next, 9d and 10k with the best nuclear extract HDAC
inhibitory rates were further evaluated against HDAC1. Results
(Table 3) revealed that the two compounds showed significant
HDAC1 inhibitory activities, being >10 fold potent than SAHA.
Br-
8a
9a
21%
20%
20%
22%
37%
38%
38%
15%
16%
4%
4.05
2.75
3.44
3.18
3.98
3.74
4.21
5.06
5.60
6.07
4.12
4.71
4.47
5.12
1.88
CH3CH2NHCO-
(CH3CH2)2NCO-
CH3CH2CONH-
PhCH2CONH-
9b
9c
Table 3 Inhibition activity of tested compounds on HDAC1.
10a
10b
10c
10d
10e
10f
10g
10h
10i
IC50 (nM)
2.2±0.13
2.3±0.16
25.0±1.23
Compd.
9d
PhCH2NHCO-
10k
Ph(CH2)2NHCO-
Ph(CH2)4NHCO-
Ph(CH2)5NHCO-
Ph(CH2)6NHCO-
SAHA
The data were expressed as the mean ± SD of three independent
experiments.
4-F-Ph(CH2)2NHCO-
4-Cl-Ph(CH2)2NHCO-
4-OCH3Ph(CH2)2NHCO-
4-F-Ph(CH2)4NHCO-
--
The in vitro anti-proliferative activities of 9d and 10k against
three human tumor cell lines MDA-MB-231, MCF-7 and A549
were then evaluated using the SRB assay. As shown in Table 4,
it was found that A549 cells were more sensitive to
compounds 9d and 10k, as compared to the reference drug
SAHA. Compound 9d showed the strongest growth inhibition
toward A549 cells with an IC50 value of 2.13 M close to that
(2.93 M) of SAHA.
39%
36%
27%
34%
46%
10j
SAHA
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 4 of 10
ARTICLE
Journal Name
photographs from three independent experiments are shown. The
View Article Online
Table 4 Anti-proliferative activities of representative compounds
data were expressed as means ± SD of three separate experiments.
DOI: 10.1039/C9MD00306A
against tumor cellsa.
p values are for one-way analysis of variance (ANOVA). **p < 0.01,
***p < 0.001 vs control.
IC50 (M)
MCF-7
Compd.
MDA-MB-231
6.87±0.84
7.24±0.87
4.66±0.67
A549
2.4 Anti-migration activity
9d
6.96±0.84
7.17±0.86
6.17±0.56
2.13±0.33
4.05±0.61
2.93±0.47
10k
Investigating the effect of novel HDAC inhibitors against cancer
cell migration is valuable for the treatment of metastatic or
advanced cancers. Based on the previous results that the bis-
substituted aromatic amides showed potent activities against
tumor cell migration [23], compound 9d was then evaluated its
anti-metastatic effects via wound-healing migration assay. As
depicted in Figure 4, 9d could decrease the migration of A549
cells in a dose-dependent manner, with higher potency than
SAHA.
SAHA
a The inhibitory effects of individual compounds on the proliferation of
cancer cell lines were determined by the SRB assay. The data were
expressed as the mean ± SD of three independent experiments.
Moreover, to evaluate whether target compounds 9d and
10k show selectivity between cancer and non-cancer cells, the
in vitro cytotoxicity of them was further tested against three
normal cell lines: human breast epithelial cells (MCF-10A,
MCF-10F) and human lung epithelial cells (Beas-2B). As shown
in Table 5, the results indicated that both compounds
displayed no obvious toxicities against the three human
normal cells, and compound 9d behaved even better than
SAHA.
Table 5 Anti-proliferative activities of representative compounds
against normal cells.
IC50 (M)
MCF-10F
>100
Compd.
MCF-10A
>100
>50
Beas-2B
>100
>20
9d
>50
10k
>50
>50
>50
SAHA
2.3 Colony formation assay
Since compound 9d showed the best antiproliferative activity
against A549 cancer cell line among the investigated ones, we
further evaluated its inhibitory behavior via colony formation
assay, with SAHA as the positive control. As depicted in Figure
3, compound 9d resulted in a significant inhibition of the
colony formation more potently than SAHA. The results
suggested that 9d could at least partly inhibit the growth and
development of A549 cells.
Fig. 4 Compound 9d inhibited the wound healing of A549 cell lines.
After a wound was made, cells were treated with different
concentrations of 9d and SAHA for 24 h. Representative photographs
from three independent experiments are shown. The data were
expressed as means ± SD of three separate experiments. p values
are for one-way analysis of variance (ANOVA). *p < 0.05, **p < 0.01 vs
control.
2.5 Western blot analysis
Considering the remarkable HDAC1 inhibitory potency,
compound 9d was progressed to western blot analysis by
immunoblotting assay. A549 cells were incubated with SAHA
and compound 9d (1.0, 2.5 and 5.0 μM). As shown in Figure 5,
9d could dramatically increase the levels of both acetyl-histone
H3 and H4 in a dose-dependent manner, which was consistent
with its HDAC1 inhibition activity. Compound 9d displayed
Fig. 3 Compound 9d inhibited the colony formation of A549 cells. After
treatment with different concentrations of 9d and SAHA in 6-well more potent activity to induce histone acetylation than SAHA.
plates for 7 days, cells were fixed with methanol and stained with 1%
crystal violet and the number of cell clones were counted. Experiments
were carried out in triplicate and repeated three times. Representative
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Page 5 of 10
MedChemComm
Journal Name
ARTICLE
stained with PI, followed by flow cytometry analysis. Representative
photographs from three independent experiments weVreiewsAhrtoicwlenO. nl(inBe)
DOI: 10.1039/C9MD00306A
Representative histograms. Data are expressed as means ± SD of the
percentages of A549 cells from three independent experiments. p values are
for one-way analysis of variance (ANOVA). *P < 0.05, **p < 0.01, ***p <
0.001 vs control. (C) The whole cell lysates were analyzed for the detection
of cyclin B and CDK1 using western blot. β-Actin was used as an equal
loading control. Representative photographs from three independent
experiments were shown. (D) Quantitative analysis. The relative levels of
CDK1 and Cyclin B used to control β-Actin were determined by densimetric
scanning. The data are expressed as means ± SD of three separate
experiments. p values are for one-way analysis of variance (ANOVA).*P <
0.05, **p < 0.01, ***p < 0.001 vs control. The blots are representative of
three independent experiments
2.7 Cell apoptosis analysis
To further determine whether the antiproliferative effects of
compound 9d was associated with enhanced apoptosis of cancer
cells, we carried out Annexin V-PE/7-AAD staining and flow
cytometry assay, and the percentages of apoptotic cells were
determined. A549 cancer cells were incubated with different
concentrations of 9d or SAHA for 72 h. As shown in Fig. 7, 9d
treated A549 cells exhibited
a dose-dependent increase of
Fig. 5 Western blot analysis of 9d on the effects of acetylated histone
levels in A549 cancer cells. A549 cells were treated with different
concentrations of 9d or SAHA for 24 h. Cell lystates were lysed,
collected and immunoblotted using western blotting. The relative
levels of each protein compared to control β-actin were determined
by densimetric scanning. Representative photographs from three
apoptosis by 30.03%, 56.44%, and 81.45% at 1.0 μM, 2.5 μM, and
5.0 μM, respectively. The 81.45% induction of A549 cells apoptosis
with 5.0 μM 9d was higher than 5.0 μM SAHA (75.64% apoptotic
cells). We also examined the cleavage states of caspase-3 and PARP
(Fig. 7C and D). It was revealed that 9d resulted in more cleavage of
independent experiments are shown. The data were expressed as both PARP and caspase-3 the SAHA treated group. Taken together,
means ± SD of three separate experiments. p values are for one-way
analysis of variance (ANOVA). **p < 0.01, ***p < 0.001 vs control.
these results proved that 9d treatment induced apoptosis
associated with cleavage of caspase 3 and PARP in A549 cells.
2.6. Cell cycle analysis
Cell cycle analysis of the most potent HDACi 9d was
investigated in A549 cancer cells. As shown in Fig. 6, in comparison
to the control group, compound 9d arrested A549 cells mainly in
G2/M phase (40.3% at 5 μM for 9d), which was obviously more
potent than SAHA (24.97% at 5 μM). We also evaluated the effect
of 9d treatment on cell-cycle–regulatory proteins, CDK1 and cyclin B,
which were involved in G2–M transition25. The results in Fig. 6
displayed that 9d treatment decreased CDK1 and cyclin B1 protein
levels.
Fig. 7 The effects of 9d on cell apoptosis in A549 cells. (A) Flow
cytometry analysis. Representative photographs from three independent
experiments were shown. (B) Quantitative analysis of apoptotic cells. Data
are expressed as means ± S.D. of the percentages of apoptotic cells from
three independent experiments. p values are for one-way analysis of
variance (ANOVA). *P < 0.05, **p < 0.01, ***p < 0.001 vs control. (C) A549
cells were incubated with, or without, 9d and SAHA at the indicated
concentrations for 48 h and the levels of protein expression were detected
using specific antibodies. Data shown are representative images of each
protein for three separate experiments. (D) Quantitative analysis of cleaved
caspase 3, and cleaved PARP. The relative levels of each protein compared
to control β-actin were determined by densimetric scanning. Data are
expressed as means ± SD from three separate experiments. p values are for
Fig. 6 The effects of 9d on cell cycle progression in A549 cells. (A) A549
cells were treated with vehicle (control), SAHA, or different doses of 9d and
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 6 of 10
ARTICLE
Journal Name
one-way analysis of variance (ANOVA). *P < 0.05, **p < 0.01, ***p < 0.001
vs control. The blots are representative of three independent experiments
B (10−15 min); flow rate of 1.5 mL/min. Purities of all
View Article Online
DOI: 10.1039/C9MD00306A
compounds for biological test were 95%.
4.2. General procedure for the preparation of target compounds
4.2.1 N1-(3-(ethylcarbamoyl)benzyl)-N7-hydroxy-N1-(2-methoxy-
2.8 Docking studies
phenyl) heptanediamide (9a)
Docking simulation was performed for the selected compound
9d which was docked into HDAC1. Besides chelating with Zn2+,
the hydroxamic acid group of 9d formed two hydrogen bonds
with His140 and His178, respectively (Figure 8). Moreover,
hydrogen bonding interaction was also observed between the
amide group of 9d and Leu271. In addition, the para-methoxy
group of 9d docked into the hydrophobic pocket of the surface
region of HDAC1, which could preliminarily explain the better
inhibitory activity of 9d than other analogues.
To a solution of hydroxyl amine hydrochloride (945 mg, 13.6
mmol) in 10 mL MeOH was added KOH (763 mg, 13.6 mmol).
Then the reaction mixture was stirred at 40 C for 10 min and
o
cooled to
0
°C and filtered. Compound methyl 7-((3-
amino)-7-
(ethylcarbamoyl)benzyl)(2-methoxyphenyl)
oxoheptanoate (300 mg, 0.68 mmol) was added to the filtrate
followed by KOH (76.3 mg, 1.36 mmol) at room temperature
for 30 min. The solvent was removed and extracted with EtOAc.
The organic layer was washed with saturated NH4Cl aqueous
solution and brine and dried over Na2SO4. The obtained
residue was finally purified by column chromatography [eluted
with EtOAc followed by 10:1 CH2Cl2-MeOH] to give compound
9a as a colorless oil (179 mg, 59.5% yield).
1H NMR (600 MHz, CD3OD) δ 7.67 – 7.65 (m, 2H), 7.35 - 7.31
(m, 3H), 7.06 (d, J = 8.4 Hz, 1H), 6.94 (d, J = 7.8 Hz, 1H), 6.88
(dd, J = 7.8, 7.8 Hz, 1H), 5.15 (d, J = 14.4 Hz, 1H), 4.54 (d, J =
14.4 Hz, 1H), 3.74 (s, 3H), 3.38 (q, J = 7.2 Hz, 2H), 2.11 – 2.04
(m, 2H), 2.03 (d, J = 7.2 Hz, 2H), 1.57 – 1.54 (m, 2H), 1.52 – 1.49
(m, 2H), 1.25 – 1.22 (m, 2H), 1.21 (t, J = 7.2 Hz, 3H). 13C NMR
(150 MHz, CD3OD) δ 176.15, 172.81, 169.84, 156.45, 139.27,
135.85, 133.01, 131.34, 131.14, 131.00, 129.38, 128.72, 127.15,
121.98, 113.38, 56.08, 52.75, 35.82, 34.46, 33.55, 29.60, 26.38,
25.96, 14.90. HRMS (ESI) m/z calcd for C24H31N3O5 [M + H]+
442.2336, found 442.2331. HPLC purity: 95.2%, tR = 4.7 min.
4.2.2 N1-(3-(diethylcarbamoyl)benzyl)-N7-hydroxy-N1-(2-methoxy-
Fig. 8 Proposed binding mode of compound 9d with HDAC1 (PDB
4BKX). (A) Molecular surface of the HDAC1 binding pocket with 9d. (B)
9d interacted with the active site of HDAC1.
3. Conclusion
In conclusion, a series of new bis-substituted aromatic amide
based hydroxamic acid HDACis were designed, synthesized and
evaluated for their antitumor activities. Two analogues 9d and
10k exhibited improved potency against HDAC1 compared
with SAHA. Moreover, compound 9d showed modest in vitro
antiproliferative effects towards three different types of
cancer cells, and increased the levels of acetylated histone H3
and H4 in dose dependent manner in A549 cells. Finally,
molecular modeling study was also performed to assess the
potential binding ability of 9d to HDAC1. The present work
afforded new HDACis that could be further investigated as
promising anticancer drug leads.
phenyl) heptanediamide (9b)
Compound 9b (35.9% yield) was prepared according to the
1
procedure described for the preparation of compound 9a. H
NMR (300 MHz, CDCl3) δ 10.33 (brs, 1H), 7.43 (s, 1H), 7.27 –
7.22 (m, 3H), 7.08 (d, J = 7.2 Hz, 1H), 6.90 (d, J = 8.4 Hz, 1H),
6.83 (dd, J = 8.4, 8.4 Hz, 1H), 6.74 (d, J = 8.4 Hz, 1H), 5.31 (d, J =
14.1 Hz, 1H), 4.28 (d, J = 14.1 Hz, 1H), 3.72 (s, 3H), 3.53 - 3.50
(m, 2H), 3.23 – 3.20 (m, 2H), 2.17 (t, J = 6.9 Hz, 2H), 2.02 (t, J =
6.9 Hz, 2H), 1.67 – 1.56 (s, 4H), 1.28 – 1.20 (s, 6H), 1.12 – 1.06
(m, 2H). 13C NMR (150 MHz, CD3OD) δ 176.05, 173.37, 172.79,
156.46, 139.44, 138.00, 131.26, 131.16, 131.13, 130.98, 129.65,
127.67, 126.33, 121.97, 113.42, 56.13, 52.60, 44.92, 40.85,
34.45, 33.54, 29.61, 26.37, 26.00, 14.41, 13.06. HRMS (ESI) m/z
calcd for C26H35N3O5 [M + H]+ 470.2649, found 470.2647. HPLC
purity: 95.4%, tR = 6.0 min.
4. Experimental section
4.1. Chemistry: general methods
All Reagents and solvents were purchased from Adamas-beta
Ltd., Aladdin-reagents Inc., or J&K Inc., and used without
further purification. All reactions were carried out using
4.2.3 N1-(3-(propionamido)benzyl)-N7-hydroxy-N1-(2-methoxy-
phenyl)heptanediamide (9c)
1
standard techniques and were monitored by TLC. H and 13C
Compound 9c (16.6% yield) was prepared according to the
NMR spectra were recorded on Bruker 300 or 600 MHz
instruments. The chemical shifts (δ) were reported in parts per
million (ppm) and coupling constants (J) were reported in Hz.
High resolution mass spectra were measured by Bruker
MicroTOF-Q II LCMS instrument operating in electrospray
ionization (ESI) mode. HPLC analyses were carried out on
Agilent Technologies 1260 Series, using the following condition:
Eclipse XDB C18 column, 5 m, 4.6×150 mm, column
temperature 40 oC; solvent A: water; solvent B: methanol;
gradient of 40−70% B (0−5 min), 70−90% B (5−10 min), 90−40%
1
procedure described for the preparation of compound 9a. H
NMR (300 MHz, DMSO-d6): δ 10.30 (br s, 1H), 9.81 (br s, 1H),
8.65 – 8.62 (m, 1H), 7.51 (d, J = 8.1 Hz, 1H), 7.40 (s, 1H), 7.33
(dd, J = 7. 5, 7. 2 Hz, 1H), 7.17 – 7.10 (m, 2H), 6.98 (d, J = 7. 5
Hz, 1H), 6. 87 (dd, J = 7. 5, 7. 2 Hz, 1H), 6.77 (d, J = 7. 2 Hz, 1H),
5.20 (d, J = 14.1 Hz, 1H), 4.12 (d, J = 14.1Hz, 1H), 3.77 (s, 3H),
2.30 (q, J = 7.5 Hz, 2H), 1.87 (t, J = 7.2 Hz, 2H), 1.47 – 1.42 (m,
2H), 1.39 –1.34 (m, 2H), 1.20 – 1.15 (m, 4H), 1.08 (t, J = 7.5 Hz,
6 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Page 7 of 10
MedChemComm
Journal Name
ARTICLE
3H). 13C NMR (150 MHz, CD3OD) δ 176.02, 175.41, 172.82, Compound 10b (37.2% yield) was prepared according to the
View Article Online
1
156.45, 139.96, 139.48, 131.43, 131.17, 131.04 ， 129.58, procedure described for the preparatioDnOoI:f10c.o10m3p9/oCu9nMdD090a3.06HA
125.42, 121.88, 121.37, 120.12, 113.34, 56.13, 52.72, 34.45, NMR (600 MHz, CD3OD) δ 7.73 – 7.69 (m, 1H), 7.68 (s, 1H),
33.54, 31.02, 29.57, 26.36, 25.98, 10.28. HRMS (ESI) m/z calcd 7.35 – 7.34 (m, 2H), 7.33 – 7.29 (m, 5H), 7.26 – 7.24 (m, 1H),
for C24H31N3O5 [M + H]+ 442.2336, found 442.2330. HPLC purity: 7.04 (d, J = 7.8 Hz, 1H), 6.95 (d, J = 7.8 Hz, 1H), 6.88 (dd, J = 7.8,
99.2%, tR = 5.2 min.
7.8 Hz, 1H), 5.12 (d, J = 14.4 Hz, 1H), 4.57 (d, J = 14.4 Hz, 1H),
4.55 (s, 2H), 3.71 (s, 3H), 2.11 – 2.05 (m, 2H), 2.02 (t, J = 7.8 Hz,
2H), 1.57 – 1.52 (m, 2H), 1.51 – 1.48 (m, 2H), 1.24 – 1.20 (m,
2H). 13C NMR (150 MHz, CD3OD) δ 176.14, 172.80, 169.94,
156.44, 140.21, 139.29, 135.63, 133.20, 131.33, 131.13, 130.98,
129.54, 129.45, 128.84, 128.52, 128.19, 127.30, 121.99, 113.36,
56.06, 52.77, 44.48, 34.45, 33.54, 29.59, 26.37, 25.95. HRMS
(ESI) m/z calcd for C29H33N3O5 [M + H]+ 504.2493, found
504.2511. HPLC purity: 96.9%, tR = 6.5 min.
4.2.4 N1-(3-(ethylcarbamoyl)benzyl)-N7-hydroxy-N1-(4-methoxy-
phenyl) heptanediamide (9d)
Compound 9d (49.1% yield) was prepared according to the
procedure described for the preparation of compound 9a. 1H
NMR (300 MHz, DMSO-d6) δ 10.31 (brs, 1H), 8.65 (brs, 1H),
8.43 (t, J = 5.1 Hz), 7.68 (d, J = 7.5 Hz, 1H), 7.63 (s, 1H), 7.36 (dd,
J = 7. 5, 7.5 Hz, 1H), 9.29 (d, J = 7.5 Hz, 1H), 7.04 (d, J = 8.7 Hz,
2H), 6.89 (d, J = 8.7 Hz, 2H), 4.85 (s, 2H), 3.72 (s, 3H), 3.26 (q, J
= 7.2 Hz, 2H), 2.05 (t, J = 7.5 Hz, 2H), 1.89 (t, J = 7.5 Hz, 2H),
1.50 – 1.45 (m, 2H), 1.41 – 1.36 (m, 2H), 1.19 – 1.15 (m, 2H),
4.2.8 N1-hydroxy-N7-(2-methoxyphenyl)-N7-(3-(phenethyl-
carbamoyl)benzyl) heptanediamide (10c)
1.10 (t, J = 7.2 Hz, 3H). 13C NMR (150 MHz, CD3OD) δ 175.76, Compound 10c (35.6% yield) was prepared according to the
172.81, 169.80, 160.86, 139.35, 136.10, 135.82, 132.78, 130.55, procedure described for the preparation of compound 9a. H
1
129.66, 128.51, 127.22, 115.84, 55.94, 53.84, 35.84, 34.88, NMR (300 MHz, DMSO-d6) δ 10.32 (s, 1H), 8.66 (s, 1H), 8.52 (t,
33.54, 29.62, 26.37, 26.20, 14.89. HRMS (ESI) m/z calcd for J = 5.7 Hz, 1H), 7.64 (d, J = 8.7 Hz, 2H), 7.35 (d, J = 7.8 Hz, 2H),
C24H31N3O5 [M + H]+ 442.2336, found 442.2342. HPLC purity: 7.31 – 7.28 (m, 2H), 7.24 (d, J = 9.0 Hz, 2H), 7.21 – 7.19 (m, 2H),
99.1%, tR = 4.6 min.
7.10 (d, J = 8.4 Hz, 1H), 6.94 (d, J = 7.5 Hz, 1H), 6.87 (dd, J = 7.5,
7.5 Hz, 1H), 5.17 (d, J = 14.7 Hz, 1H), 4.34 (d, J = 14.7 Hz, 1H),
3.74 (s, 3H), 3.49 – 3.43 (m, 2H), 2.82 (t, J = 7.5 Hz, 2H), 2.05 –
1.94 (m, 2H), 1.87 (t, J = 7.5 Hz, 2H), 1.48 – 1.41 (m, 2H), 1.39 –
1.33 (m, 2H), 1.13 – 1.07 (m, 2H). 13C NMR (150 MHz, CD3OD) δ
178.31, 176.63, 175.97, 172.82, 169.54, 161.12, 135.98, 133.80,
133.40, 130.70, 130.54, 130.37, 130.06, 128.91, 127.01, 126.84,
124.26, 116.14, 115.97, 56.00, 49.85, 46.27, 34.66, 34.46,
33.51, 29.53, 26.36, 26.00. HRMS (ESI) m/z calcd for
C30H35N3O5 [M + H]+ 518.2649, found 518.2655. HPLC purity:
96.7%, tR = 6.9 min.
4.2.5 N1-(3-(ethylcarbamoyl)benzyl)-N7-hydroxy-N1-(4-hydroxy-
phenyl)heptanediamide (9e)
Compound 9e (58.5% yield) was prepared according to the
procedure described for the preparation of compound 9a. H
1
NMR (600 MHz, CD3OD) δ 7.69 (d, J = 7.2 Hz, 1H), 7.65 (s, 1H),
7.38 (dd, J = 7.8, 7.8 Hz, 1H), 7.34 (d, J = 7.8 Hz, 1H), 6.87 (d, J =
9.0 Hz, 2H), 6.74 (d, J = 9.0 Hz, 2H), 3.64 (s, 2H), 3.40 (q, J = 7.2
Hz, 2H), 2.14 (t, J = 7.8 Hz, 2H), 2.04 (d, J = 7.8 Hz, 2H), 1.60 –
1.57 (m, 2H), 1.54 – 1.51 (m, 2H), 1.27 – 1.24 (m, 2H), 1.21 (t, J
= 7.2 Hz, 3H). 13C NMR (150 MHz, CD3OD) δ 175.89, 172.82,
170.06, 158.66, 139.41, 136.06, 134.69, 132.81, 130.48, 129.64,
128.50, 127.20, 117.15, 53.87, 52.08, 34.84, 33.54, 29.63,
4.2.9 N1-hydroxy-N7-(2-methoxyphenyl)-N7-(3-((4-phenylbutyl)
carbamoyl)benzyl)heptanediamide (10d)
26.39, 26.23, 14.88. HRMS (ESI) m/z calcd for C23H29N3O5 [M + Compound 10g (34.6% yield) was prepared according to the
H]+ 428.2180, found 428.2177. HPLC purity: 95.6%, tR = 4.4 min. procedure described for the preparation of compound 9a. H
1
4.2.6 N1-(3-(2-phenylacetamido)benzyl)-N7-hydroxy-N1-(2-
NMR (600 MHz, CD3OD) δ 7.67 – 7.64 (m, 1H), 7.62 (s, 1H),
methoxy-phenyl)heptanediamide (10a)
7.34 – 7.29 (m, 3H), 7.24 (dd, J = 7.8, 7.8 Hz, 2H), 7.18 (d, J =
7.2 Hz, 2H), 7.14 (dd, J = 7.2, 7.2 Hz, 1H), 7.03 (d, J = 8.4 Hz, 1H),
6.94 (d, J = 7.8 Hz, 1H), 6.87 (dd, J = 7.8, 7.8 Hz, 1H), 5.13 (d, J =
14.4 Hz, 1H), 4.55 (d, J = 14.4 Hz, 1H), 3.72 (s, 3H), 3.38 (t, J =
7.2 Hz, 2H), 2.66 (t, J = 7.2 Hz, 2H), 2.10 – 2.06 (m, 2H), 2.03 (t,
J = 7.2 Hz, 2H), 1.70 – 1.67 (m, 2H), 1.65 – 1.61 (m, 2H), 1.56 –
1.53 (m, 2H), 1.52 – 1.49 (m, 2H), 1.24 – 1.19 (m, 2H). 13C NMR
(150 MHz, CD3OD) δ 176.13, 172.81, 170.01, 156.45, 143.61,
139.24, 135.85, 133.04, 131.32, 131.13, 130.99, 129.45, 129.39,
129.33, 128.73, 127.18. 126.77, 121.98, 113.36, 56.08, 52.74,
40.76, 36.52, 34.46, 33.56, 30.08, 30.06, 29.60, 26.38, 25.96.
HRMS (ESI) m/z calcd for C32H39N3O5 [M + H]+ 546.2962, found
546.2972. HPLC purity: 96.1%, tR = 7.8 min.
Compound 10a (41.2% yield) was prepared according to the
procedure described for the preparation of compound 9a. H
1
NMR (300 MHz, DMSO-d6): δ 10.31 (br s, 1H), 10.14 (br s, 1H),
8.66 (br s, 1H), 7.52 (d, J = 7. 2 Hz, 1H), 7.42 – 7.41 (m, 1H),
7.34 – 7.31 (m, 4H), 7.28 (d, J = 7. 2 Hz, 1H), 7.25 – 7.22 (m, 1H),
7.16 (dd, J = 7. 8, 7. 5 Hz, 1H), 7.10 (d, J = 7.5 Hz, 1H), 6.96 (d, J
= 7.8 Hz, 1H), 6.88 (dd, J = 7. 5, 7. 5 Hz, 1H), 6.81 (d, J = 7.5 Hz,
1H), 5.21 (d, J = 14.7 Hz, 1H), 4.15 (d, J = 14.7 Hz, 1H), 3.75 (s,
3H), 3.62 (s, 2H), 1.95 – 1.90 (m, 2H), 1.87 (t, J = 7.5 Hz,, 2H),
1.45 (t, J = 7.5 Hz,, 2H), 1.37 (t, J = 7.5 Hz,, 2H), 1.12 - 1.08 (m,
2H). 13C NMR (150 MHz, CD3OD) δ 176.03, 172.82, 172.34,
156.43, 139.82, 139.50, 136.86, 131.40, 131.12, 131.02, 130.14,
129.98, 129.60, 127.95, 125.67, 121.89, 121.50, 120.23, 113.31,
56.09, 52.73, 44.70, 34.42, 33.52, 29.55, 26.34, 25.96. HRMS
4.2.10 N1-hydroxy-N7-(2-methoxyphenyl)-N7-(3-((5-phenylpentyl)
carbamoyl)benzyl)heptanediamide (10e)
(ESI) m/z calcd for C29H33N3O5 [M + H]+ 504.2493, found Compound 10e (64.8% yield) was prepared according to the
1
504.2488. HPLC purity: 99.5%, tR = 6.7 min.
procedure described for the preparation of compound 9a. H
NMR (600 MHz, CD3OD) δ 7.65 – 7.63 (m, 1H), 7.62 (s, 1H),
7.36 – 7.30 (m, 3H), 7.22 (dd, J = 7.8, 7.2 Hz, 2H), 7.16 (d, J =
4.2.7 N1-(3-(benzylcarbamoyl)benzyl)-N7-hydroxy-N1-(2-methoxy-
phenyl)heptanediamide (10b)
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 8 of 10
ARTICLE
Journal Name
7.2 Hz, 2H), 7.12 (dd, J = 7.8, 7.2 Hz, 1H), 7.05 (dd, J = 8.4 Hz, 7.05 (d, J = 8.4 Hz, 1H), 6.93 (d, J = 7.8 Hz, 1H), 6.8V8ie(wdAdrt,icJle=O8nl.in4e,
DOI: 10.1039/C9MD00306A
1H), 6.94 (d, J = 7.8 Hz, 1H), 6.88 (dd, J = 7.8, 7.8 Hz, 1H), 5.15 8.4 Hz, 1H), 5.13 (d, J = 14.4 Hz, 1H), 4.54 (d, J = 14.4 Hz, 1H),
(d, J = 15.0 Hz, 1H), 4.54 (d, J = 15.0 Hz, 1H), 3.74 (s, 3H), 3.34 (t, 3.72 (s, 3H), 3.56 (t, J = 7.8 Hz, 2H), 2.89 (t, J = 7.2 Hz, 2H), 2.13
J = 7.2 Hz, 2H), 2.62 (t, J = 7.8 Hz, 2H), 2.11 – 2.04 (m, 2H), 2.02 – 2.04 (m, 2H), 2.03 (t, J = 7.2 Hz, 2H), 1.59 – 1.54 (m, 2H), 1.52
(t, J = 7.2 Hz, 2H), 1.69 – 1.64 (m, 2H), 1.63 – 1.60 (m, 2H), 1.58 – 1.48 (m, 2H), 1.25 – 1.20 (m, 2H). 13C NMR (150 MHz, CD3OD)
– 1.55 (m, 2H), 1.52 – 1.48 (m, 2H), 1.42 – 1.37 (m, 2H), 1.24 – δ 176.12, 172.81, 170.04, 156.46, 139.44, 139.31, 135.72,
1.20 (m, 2H). 13C NMR (150 MHz, CD3OD) δ 176.14, 172.78, 133.16, 133.12, 131.54, 131.32, 131.15, 130.98, 129.52, 129.41,
170.01, 156.46, 143.76, 139.26, 135.89, 133.00, 131.35, 131.14, 128.69, 127.16, 121.99, 113.39, 56.08, 52.72, 42.31, 35.80,
131.00, 129.43, 129.38, 129.27, 128.73, 127.17, 126.67, 121.98, 34.46, 33.56, 29.61, 26.38, 25.96. HRMS (ESI) m/z calcd for
113.38, 56.09, 52.75, 40.91, 36.76, 34.47, 33.54, 32.37, 30.27, C30H34ClN3O5 [M + H]+ 552.2260, found 552.2256. HPLC purity:
29.60, 27.55, 26.38, 25.96. HRMS (ESI) m/z calcd for 97.0%, tR = 7.5 min.
4.2.14 N1-hydroxy-N7-(3-((4methoxyphenethyl)carbamoyl)benzyl)-
N7-(2-methoxyphenyl)heptanediamide (10i)
C33H41N3O5 [M + H]+ 560.3119, found 560.3118. HPLC purity:
95.4%, tR = 8.2 min.
4.2.11 N1-hydroxy-N7-(2-methoxyphenyl)-N7-(3-((6-phenylhexyl)-
Compound 10i (37.3% yield) was prepared according to the
1
carbamoyl)benzyl)heptanediamide (10f)
procedure described for the preparation of compound 9a. H
Compound 10j (43.6% yield) was prepared according to the NMR (600 MHz, CD3OD) δ 7.63 – 7.62 (m, 1H), 7.60 (s, 1H),
1
procedure described for the preparation of compound 9a. H 7.34 – 7.32 (m, 3H), 7.15 (d, J = 9.0 Hz, 2H), 7.05 (d, J = 8.4 Hz,
NMR (300 MHz, DMSO-d6): δ 10.31 (br s, 1H), 8.65 (br s, 1H), 1H), 6.94 (d, J = 7.8 Hz, 1H), 6.88 (dd, J = 7.8, 7.8 Hz, 1H), 6.84
8.37 (t, J = 5.1 Hz, 1H), 7.66 (d, J = 7.2 Hz, 1H), 7.62 (s, 1H), 7.32 (d, J = 8.4 Hz, 2H), 5.14 (d, J = 14.4 Hz, 1H), 4.54 (d, J = 14.4 Hz,
(dd, J = 7.5, 7.5 Hz, 2H), 7.27 – 7.24 (m, 3H), 7.19 – 7.15 (m, 1H), 3.76 (s, 3H), 3.72 (s, 3H), 3.53 (t, J = 7.8 Hz, 2H), 2.83 (t, J =
3H), 7.09 (d, J = 7. 2 Hz, 1H), 6.95 (d, J = 7.5 Hz, 1H), 6.87 (dd, J 7.2 Hz, 2H), 2.12 – 2.07 (m, 2H), 2.03 (t, J = 7.2 Hz, 2H), 1.58 –
= 7.5, 7.5 Hz, 1H), 5.18 (d, J = 14.7 Hz, 1H), 4.35 (d, J = 14.7 Hz, 1.55 (m, 2H), 1.53 – 1.50 (m, 2H), 1.25 – 1.20 (m, 2H). 13C NMR
1H), 3.75 (s, 3H), 3.21 (q, J = 6.9 Hz, 2H), 2.59 (t, J = 7.8 Hz, 2H), (150 MHz, CD3OD) δ 176.13, 172.80, 170.02, 159.76, 156.46,
2.02 – 1.97 (m, 2H), 1.88 (t, J = 7.5 Hz, 2H), 1.55 (t, J = 7.8 Hz, 139.28, 135.84, 133.06, 132.54, 131.33, 131.14, 130.99, 130.82,
2H), 1.52 – 1.48 (m, 4H), 1.46 – 1.38 (m, 6H), 1.13 – 1.10 (m, 129.39, 128.69, 127.16, 121.99, 114.92, 113.39, 56.08, 55.65,
2H). 13C NMR (151 MHz, CD3OD) δ 176.14, 172.80, 170.00, 52.73, 42.82, 35.66, 34.47, 33.56, 29.61, 26.39, 25.96. HRMS
156.45, 143.90, 139.26, 135.89, 133.02, 131.33, 131.13, 130.99, (ESI) m/z calcd for C31H37N3O6 [M + H]+ 548.2755, found
129.40, 129.26, 128.73, 127.22, 127.18, 126.64, 121.97, 113.37, 548.2756. HPLC purity: 95.3%, tR = 6.7 min.
4.2.15 N1-(3-((4-(4-fluorophenyl)butyl)carbamoyl)benzyl)-N7-
56.08, 52.74, 40.99, 36.83, 34.46, 33.55, 32.69, 30.42, 30.00,
hydroxy-N1-(2-methoxyphenyl)heptanediamide (10j)
29.60, 27.91, 26.37, 25.96. HRMS (ESI) m/z calcd for
C34H43N3O5 [M + H]+ 574.3275, found 574.3287. HPLC purity:
97.4%, tR = 9.5 min.
Compound 10j (63.2% yield) was prepared according to the
procedure described for the preparation of compound 9a. H
NMR (300 MHz, DMSO-d6): δ 10.30 (br s, 1H), 8.64 (br s, 1H),
8.39 (t, J = 5.1 Hz, 1H), 7.65 (d, J =6.9 Hz, 1H), 7.62 (s, 1H), 7.33
1
4.2.12 N1-(3-((4-fluorophenethyl)carbamoyl)benzyl)-N7-hydroxy-
N1-(2-methoxyphenyl)heptanediamide (10g)
Compound 10g (74.4% yield) was prepared according to the – 7.30 (m, 3H), 7.21 (dd, J = 7.5, 7.5 Hz, 2H), 7.10 – 7.06 (m,
1
procedure described for the preparation of compound 9a. H 3H), 6.94 (d, J = 7.5 Hz, 1H), 6.84 (dd, J = 7.5, 7.5 Hz, 1H), 5.15
NMR (300 MHz , DMSO-d6): δ10.29 (br s, 1H), 8.63 (br s, 1H), (d, J = 14.7 Hz, 1H), 4.32 (d, J = 14.7 Hz, 1H), 3.73 (s, 3H), 3.25
8.47 (t, J = 5.7 Hz, 1H), 7.64 – 7.61 (m, 2H), 7.32 (d, J = 7.5 Hz, (q, J = 7.2 Hz, 2H), 2.58 (t, J = 7.2 Hz, 2H), 2.10 – 1.97 (m, 2H),
1H), 7.31 – 7.28 (m, 2H), 7.26 – 7.23 (m, 2H), 7.12 – 7.07 (m, 1.87 (t, J = 7.2 Hz, 2H), 1.55 – 1.50 (m, 4H), 1.40 – 1.32 (m, 4H),
3H), 6.94 (d, J = 7.5 Hz, 1H), 6.87 (dd, J = 7.5, 7.5 Hz, 1H), 5.14 1.16 – 1.11 (m, 2H). 13C NMR (150 MHz, CD3OD) δ 176.14,
(d, J = 15.0 Hz, 1H), 4.33 (d, J = 15.0 Hz, 1H), 3.73 (s, 3H), 3.47 – 172.82, 170.02, 162.69 (d, J = 240.5 Hz), 156.45, 139.51 (d, J =
3.42 (m, 2H), 2.81 (t, J = 7.8 Hz, 2H), 2.02 – 1.97 (m, 2H), 1.87 (t, 3.0 Hz), 139.27, 135.85, 133.04, 131.34, 131.14, 131.01 (d, J =
J = 7.2 Hz, 2H), 1.47 – 1.42 (m, 2H), 1.38 – 1.34 (m, 2H), 1.13 – 6.7 Hz), 129.41, 128.73, 127.71, 121.98, 115.84 (d, J = 21.0 Hz),
1.08 (m, 2H). 13C NMR (150 MHz, CD3OD) δ 176.14, 172.83, 113.37, 56.08, 52.75, 40.71, 35.62, 34.46, 33.56, 30.11, 30.00,
170.01, 163.05 (d, J = 240.5 Hz), 156.46, 139.31, 136.55 (d, J = 29.60, 26.38, 25.96. HRMS (ESI) m/z calcd for C32H38FN3O5 [M +
3.3 Hz), 135.76, 133.11, 131.55 (d, J = 7.8 Hz), 131.33, 131.15, H]+ 564.2868, found 564.2882. HPLC purity: 95.9%, tR = 7.7 min.
4.2.16 N1-(3-((4-fluorophenethyl)carbamoyl)benzyl)-N7-hydroxy-
130.99, 129.41, 128.70, 127.15, 121.99, 116.11 (d, J = 21.5 Hz),
N1-(4-methoxyphenyl)heptanediamide (10k)
113.39, 56.08, 52.73, 42.57, 35.67, 34.46, 33.56, 29.61, 26.38,
25.96. HRMS (ESI) m/z calcd for C30H34FN3O5 [M + H]+ 536.2555,
found 536.2563. HPLC purity: 96.6%, tR = 6.9 min.
Compound 10k (48.7% yield) was prepared according to the
procedure described for the preparation of compound 9a. H
NMR (300 MHz, DMSO-d6): δ 10.33 (br s, 1H), 8.67 (br s, 1H),
8.53 (t, J = 5.4 Hz, 1H), 7.65 (d, J = 7. 5 Hz, 1H), 7.61 (s, 1H),
1
4.2.13 N1-(3-((4-chlorophenethyl)carbamoyl)benzyl)-N7-hydroxy-
N1-(2-methoxyphenyl)heptanediamide (10h)
Compound 10h (62.5% yield) was prepared according to the 7.36 (dd, J = 7. 5, 7. 5 Hz, 1H), 7.31 – 7.23 (m, 3H), 7.10 (d, J =
1
procedure described for the preparation of compound 9a. H 9.0 Hz, 2H), 7.04 (d, J = 9.0 Hz, 2H), 6.89 (d, J = 8.7 Hz, 2H), 4.85
NMR (600 MHz, CD3OD) δ 7.63 - 7.61 (m, 1H), 7.59 (s, 1H), 7.34 (s, 2H), 3.72 (s, 3H), 3.47 – 3.40 (m, 2H), 2.80 (t, J = 7.5 Hz, 2H),
– 7.30 (m, 3H), 7.27 (d, J = 8.4 Hz, 2H), 7.24 (d, J = 8.4 Hz, 2H), 2.03 (t, J = 7.2 Hz, 2H), 1.88 (t, J = 7.2 Hz, 2H), 1.49 – 1.45 (m,
8 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Page 9 of 10
MedChemComm
Journal Name
ARTICLE
2H), 1.40 – 1.35 (m, 2H), 1.12 – 1.09 (m, 2H). 13C NMR (150
MHz, CD3OD) δ 175.73, 172.43, 169.98, 163.03 (d, J = 241 Hz),
160.84, 139.38, 136.54 (d, J = 3.2 Hz), 136.00, 135.80, 132.86,
131.55 (d, J = 8.0 Hz), 130.54, 129.70, 128.45, 127.23, 116.05
(d, J = 21.2 Hz), 115.84, 55.94, 53.83, 42.60, 35.67, 34.88,
33.42, 29.62, 26.38, 26.20. HRMS (ESI) m/z calcd for
C30H34FN3O5 [M + H]+ 536.2555, found 536.2556. HPLC purity:
97.2%, tR = 8.6 min.
experiment. After incubation for 24 h, the cells were treated
with various concentrations of tested coDmOpI:o1u0n.1d03s9f/oCr94M8D0h0.306A
View Article Online
Then the cells were fixed with 10% TCA for 1 h at 4 °C and
washed with distilled-water for five times. The plates were
allowed to air drying followed by being dyed with 0.4% SRB for
5 min at room temperature. After dying, the plates were
washed with 1% acetic acid and allowed to air drying. 150 μL
of 10 mM Tris-based solution was added to each well, and the
absorption at 540 nm was measured by microplate reader
(TECAN). The IC50 was calculated using GraphPad prism 7.0.
Three independent experiments were carried out in triplicate.
4.2.17
N1-(3-(3-(4-fluorophenyl)propanamido)benzyl)-N7-
hydroxy-N1-(4-methoxyphenyl)heptanediamide (10l)
Compound 10l (33.5% yield) was prepared according to the
1
procedure described for the preparation of compound 9a. H NMR
(600 MHz, DMSO-d6) δ 10.31 (brs, 1H), 9.88 (s, 1H), 8.65 (brs, 1H),
7.49 (d, J = 8.4 Hz, 1H), 7.37 (s, 1H), 7.28-7.25 (m, 2H), 7.17 (dd, J =
7.8, 7.8 Hz, 1H), 7.09 (dd, J = 8.4, 8.4 Hz, 2H), 7.06 (d, J = 8.4 Hz, 2H),
6.90 (d, J = 8.4 Hz, 2H), 6.79 (d, J = 7.2 Hz, 1H), 4.75 (s, 2H), 3.73 (s,
3H), 2.88 (t, J = 7.8 Hz, 2H), 2.59 (t, J = 7.2 Hz, 2H), 2.01 (t, J = 7.2 Hz,
2H), 1.89 (t, J = 7.2 Hz, 2H), 1.49-1.44 (m, 2H), 1.40 -1.35 (m, 2H),
1.15 -1.01 (m, 2H). 13C NMR (150 MHz, DMSO-d6) δ 171.97, 170.27,
169.04, 160.69 (d, J = 239.8 Hz), 158.28, 139.19, 138.33, 137.31 (d, J
= 3.0 Hz), 134.88, 130.02 (d, J = 7.8 Hz), 129.33, 128.59, 122.63,
118.38, 117.72, 114.96 (d, J = 20.8 Hz), 114.57, 55.24, 52.03, 38.00,
33.25, 32.13, 29.93, 28.21, 24.93, 24.68. HRMS (ESI) m/z calcd for
C30H34FN3O5 [M + H]+ 536.2555, found 536.2553. HPLC purity: 98.1%,
tR = 8.9 min.
4.4.2 Colony formation assay
A549 cells were plated into 35 mm dishes (800 cells per dish)
and treated with 9d or SAHA for one week and replenish every
24 h. After the removal of culture media, cells were washed
with PBS for three times prior to methanol fixation. The cells
were then immediately stained using 1% crystal violet
(Beyotime) for 15 min and washed with PBS for one time.
Images were collected using a scanning apparatus (Canon,
Japan). Experiments were carried out in triplicate and
repeated three times.
4.4.3 Wound healing assay
1.5x105 A549 cells were counted and plated in a 6-well dishes.
Cells were incubated overnight yielding confluent monolayers
for wounding. Wounds were made using a pipette tip and
photographs taken immediately (0 h), 12 h and 24 h after
wounding for A549 cells, respectively. The distance migrated
by the cell monolayer to close the wounded area during this
time period was measured via the Image J software (NIH, USA).
Results were expressed as the percent of wound clousure—
that is, the distance migrated 0 h minus the distance migrated
12 h or 24 h then relative to the distance migrated 0 h.
Experiments were carried out in triplicate and repeated three
times.
4.3 HDAC1 inhibitory assay
The in vitro HDAC1 inhibitory activity was determined by the
protease-coupled assay. Different concentrations of tested
compounds were incubated with recombinant HDAC1 (BPS
Biosciences, US) at room temperature for 15 min, followed by
addition of trypsin as well as Ac-peptide-AMC substrates to
initiate the reaction in Tris-based assay buffer. Fluorescent
AMC released from substrate was measured on SynergyMx
(BioTek, US) using filter sets as excitation = 355 nm and
emission = 460 nm. IC50 values were calculated by GraphPad
Prism software (California, USA).
4.4.4 Western blotting
4.4 Biological evaluation
After treatment with 9d for indicated times, cells were
harvested with RIPA buffer containing 150 mM sodium
chloride, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1%
SDS, 50 mM Tris and cocktails of protease and phosphatase
inhibitors for 10 min at room temperature and boiled for
another 10 min. Equal amounts of total proteins (35 μg)
underwent 15% SDS PAGE and were electroblotted onto the
polyvinylidene difluoride (PVDF) membrane. The membrane
was blocked with 5% (w/v) non-fat dry milk in PBS-Tween 20
(PBST; 0.05%) for 1 h and incubated with primary antibody
(1:1000 in PBST) at 4 ℃ overnight. After three washings in
PBST, the PVDF membrane was incubated with appropriate
horseradish peroxidase-conjugated secondary antibodies
(1:20000) for 1 h at room temperature. The immunoreactive
bands were developed with the ECL western blotting system.
β-actin were used as loading control. The relative quantity of
proteins was analyzed via the Image J software (NIH, USA).
4.4.5 Cell cycle analysis
4.4.1 In vitro anti-proliferation assay
Cell lines A549, MDA-MB-231, MCF-7 used in this study were
obtained from the American Type Culture Collection. The cells
were cultured in RPMI 1640 medium (HyClone, SH30809.01B)
supplemented with 10% fetal bovine serum (FBS) (HyClone,
SV30087.02) at 37 °C in a humidified atmosphere with 5% CO2.
Human breast epithelial cells (MCF-10A, MCF-10F) and human
lung epithelial cells (Beas-2B) were obtained from the Type
Culture Collection of the Chinese Academy of Sciences,
Shanghai, China. And the cells were cultured in MEM (HyClone,
SH30243.01B) supplemented with 10% fetal bovine serum (FBS)
(Gibco, 10270) at 37 °C in a humidified atmosphere with 5%
CO2.
SRB assay was performed according to the manufacturer’s
instruction (Sigma Aldrich). Tumor cells were seeded into 96-
well plates at the appropriate cell densities during the
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 9
Please do not adjust margins

Please do not adjust margins
MedChemComm
Page 10 of 10
ARTICLE
Journal Name
Cell cycle analysis was carried out by estimating DNA contents 2.
with flow cytometry. After incubation with the indicated doses
of 9d for 24 h, A549 cells were fixed in 70% ice-cold ethanol, 3.
incubated overnight at -20 C, stained with PI containing
RNaseA solution for 30 min at 37 C, and then analyzed by
C. B. Yoo and P. A. Jones, Nat. Rev. Drug Discov., 2006, 5,
37-50.
S. Kotian, L. Zhang, M. Boufraqech, K. Gaskins, S. K. Gara,
M. Quezado, N. Nilubol and E. Kebebew, Clin. Cancer Res.,
2017, 23, 5044-5054.
View Article Online
DOI: 10.1039/C9MD00306A
FACS.
4.
Y. C. Tseng, S. K. Kulp, I. L. Lai, E. C. Hsu, W. A. He, D. E.
Frankhouser, P. S. Yan, X. Mo, M. Bloomston, G. B.
Lesinski, G. Marcucci, D. C. Guttridge, T. Bekaii-Saab and
C. S. Chen, J. Natl. Cancer Inst., 2015, 107, djv274.
Y. Zhang, H. Fang, J. Jiao and W. Xu, Curr. Med. Chem.,
2008, 15, 2840-2849.
M. A. Dawson and T. Kouzarides, Cell, 2012, 150, 12-27.
A. M. A. Aboeldahab, E. A. M. Beshr, M. E. Shoman, S. M.
Rabea and O. M. Aly, Eur. J. Med. Chem., 2018, 146, 79-
92.
4.4.6 Cell apoptosis analysis
Cell apoptosis analysis was measured by annexin V FITC/PI
assay using Annexin v-PE/7-AAD Apoptosis Detection kit (BD).
Briefly, A549 cells (8 × 104/well) were treated with DMSO and 5.
compounds 9d for 72 h. The cells were then harvested and
stained with annexin binding buffer, Alexa Fluor 488 annexin 6.
and propidium iodide for 15 min in the dark. After staning, 400 7.
uL of 1X annexin-building buffer was added, mixed gently and
kept on ice. The samples were measured using an ACEA
Biosciences novocyte flow cytometer.
4.5 Molecular docking studies
8.
M. Haberland, R. L. Montgomery and E. N. Olson, Nat.
Rev. Genet., 2009, 10, 32-42.
9.
A. J. de Ruijter, A. H. van Gennip, H. N. Caron, S. Kemp and
A. B. van Kuilenburg, Biochem J., 2003, 370, 737-749.
M. Mrakovcic, L. Bohner, M. Hanisch and L. F. Frohlich,
Int. J. Mol. Sci., 2018, 19.
Y. Sun, F. Lu, Y. Sun, S. Yue and Y. Wang, Curr. Top. Med.
Chem., 2018, 18, 2420-2428.
O. Khan and N. B. La Thangue, Immunol. Cell Biol., 2012,
90, 85-94.
P. A. Marks and R. Breslow, Nat. Biotechnol., 2007, 25, 84-
90.
Molecular docking studies were carried out with Autodock-
4.27. For the docking calculations HDAC1 crystal structure
(PDB code: 4BKX) was retrieved from the Protein Data Bank
(www.pdb.org). For protein preparation, all the water
molecules were removed from HDAC1, and gasteiger partial
charges were assigned to the selected compounds and enzyme
atoms. The docking results were analyzed with the programs
AutoDockTools, 27 DOCKRES and VMD.
10.
11.
12.
13.
4.6 Statistical analyses
All experiments were performed in duplicate and repeated at
least three times. Each experimental value was expressed as
the mean ± standard deviation (SD). Data were analyzed by
14.
15.
16.
L. P. Yang, Drugs, 2011, 71, 1469-1480.
R. M. Poole, Drugs, 2014, 74, 1543-1554.
K. P. Garnock-Jones, Drugs, 2015, 75, 695-704.
K. Stenzel, A. Hamacher, F. K. Hansen, C. G. W. Gertzen, J.
Senger, V. Marquardt, L. Marek, M. Marek, C. Romier, M.
Remke, M. Jung, H. Gohlke, M. U. Kassack and T. Kurz, J.
Med. Chem., 2017, 60, 5334-5348.
Student's t-test between two groups and by one-way analysis 17.
of variance (ANOVA) followed by Bonferroni test for multiple
comparison. These analyses were performed using GraphPad
Prism Software Version 5.0 (GraphPad Software, Inc. CA). *, **
and *** indicated P < 0.05, P < 0.01 and P < 0.001, respectively. 18.
Y. Luan, J. Li, J. A. Bernatchez and R. Li, J. Med. Chem.,
2019, 62, 3171-3183.
19.
J. E. Bolden, M. J. Peart and R. W. Johnstone, Nat. Rev.
Drug Discov., 2006, 5, 769-784.
Conflicts of interest
20.
21.
22.
23.
F. Thaler and C. Mercurio, ChemMedChem, 2014, 9, 523-
526.
W. Chen, G. Dong, Y. Wu, W. Zhang, C. Miao and C. Sheng,
ACS Med. Chem. Lett., 2018, 9, 34-38.
M. Hassanzadeh, K. Bagherzadeh and M. Amanlou, J. Mol.
Graph. Model., 2016, 70, 170-180.
F. Yang, T. Zhang, H. Wu, Y. Yang, N. Liu, A. Chen, Q. Li, J.
Li, L. Qin, B. Jiang, X. Wang, X. Pang, Z. Yi, M. Liu and Y.
Chen, J. Med. Chem., 2014, 57, 9357-9369.
D. F. Veber, S. R. Johnson, H.-Y. Cheng, B. R. Smith, K. W.
Ward and K. D. Kopple, J. Med. Chem., 2002, 45, 2615-
2623.
The authors declare that there are no conflicts of interest.
Acknowledgements
This work was financially supported by the National Science
Foundation of China (No. 81703360, 31501122, 81673304),
Open Research Project Foundation of Shanghai Key Laboratory
of Regulatory Biology (2018LBR03), the Natural Science
Foundation of Shandong Province (No. ZR2016HB43,
JQ2017021), Shandong Excellent Young Scientist Award Fund
(BS2014SW031), the Young Taishan Scholars Program
(tsqn20161037), and the Shandong Talents Team Cultivation
Plan of University Preponderant Discipline (No. 10027).
24.
25.
M. B. Kastan and J. Bartek, Nature, 2004, 432, 316-323.
Notes and references
1.
M. Alzrigat, A. A. Parraga and H. Jernberg-Wiklund, Semin.
Cancer Biol., 2018, 51, 101-115.
10 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins