Design, synthesis and biological evaluation of novel coumarin-based hydroxamate derivatives as histone deacetylase (HDAC) inhibitors with antitumor activities

Article abstract of DOI:10.3390/molecules24142569

A series of novel coumarin-based hydroxamate derivatives were designed and synthesized as histone deacetylase inhibitors (HDACis). Selective compounds showed a potent HDAC inhibition with nM IC₅₀ values, with the best compound (10e) being nearly 90 times more active than vorinostat (SAHA) against HDAC1. Compounds 10e and 11d also increased the levels of acetylated histone H3 and H4, which is consistent with their strong HDAC inhibition. In addition, 10e and 11d displayed a higher potency toward human A549 and Hela cancer cell lines compared with SAHA. Moreover, 10e and 11d significantly arrested A549 cells at the G2/M phase and enhanced apoptosis. Molecular docking studies revealed the possible mode of interaction of compounds 10e and 12a with HDAC1. Our findings suggest that these novel coumarin-based HDAC inhibitors provide a promising scaffold for the development of new potential cancer chemotherapies.

Full text of DOI:10.3390/molecules24142569

molecules
Article
Design, Synthesis and Biological Evaluation of Novel
Coumarin-Based Hydroxamate Derivatives as Histone
Deacetylase (Hdac) Inhibitors with
Antitumor Activities
Feifei Yang 1 , Na Zhao 1, Jiali Song 1, Kongkai Zhu 1, Cheng-shi Jiang 1 , Peipei Shan 2,* and
Hua Zhang 1,
*
1
School of Biological Science and Technology, University of Jinan, Jinan 250022, China
Institute for Translation Medicine, Qingdao University, Qingdao 266071, China
Correspondence: bio_zhangh@ujn.edu.cn (H.Z.); shanpeipei@qdu.edu.cn (P.S.); Tel.: +053189736199 (H.Z.);
+053282991791 (P.S.)
2
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 27 May 2019; Accepted: 13 July 2019; Published: 15 July 2019
Abstract: A series of novel coumarin-based hydroxamate derivatives were designed and synthesized
as histone deacetylase inhibitors (HDACis). Selective compounds showed a potent HDAC inhibition
with nM IC50 values, with the best compound (10e) being nearly 90 times more active than vorinostat
(SAHA) against HDAC1. Compounds 10e and 11d also increased the levels of acetylated histone H3
and H4, which is consistent with their strong HDAC inhibition. In addition, 10e and 11d displayed
a higher potency toward human A549 and Hela cancer cell lines compared with SAHA. Moreover,
10e and 11d significantly arrested A549 cells at the G2/M phase and enhanced apoptosis. Molecular
docking studies revealed the possible mode of interaction of compounds 10e and 12a with HDAC1.
Our findings suggest that these novel coumarin-based HDAC inhibitors provide a promising scaffold
for the development of new potential cancer chemotherapies.
Keywords: coumarin; hydroxamate; HDAC inhibitors; antitumor growth; structure–activity relationship
1. Introduction
Coumarins, a class of plant secondary metabolites [
of benzene and 2–pyrone rings and exhibit various pharmacological properties, including
antidepressant [ ], antibacterial [ ], scavenging of reactive oxygen species (ROS) [ ],
anti-inflammatory [ ], anticholinesterase [ ], antithrombotic [ ] and anticancer activities [ ]. One of
1], are bicyclic heterocycles consisting
2
3
4
5
6
7
8,9
the most severe concerns in cancer treatment is related to the serious side effects of current
chemotherapies. However, coumarins and their analogues exhibit a very rare cardiotoxicity, dermal
toxicity, nephrotoxicity, hepatotoxicity and other adverse effects; therefore, they are generally considered
as a group of promising candidates in anticancer drug discovery [10,11]. Coumarins influence a
number of pathways in cancer, such as angiogenesis inhibition, kinase inhibition, carbonic anhydrase
inhibition, cell cycle arrest, aromatase inhibition, antimitotic activity, telomerase inhibition, sulfatase
inhibition and heat shock protein (HSP90) inhibition [1,12]. Numerous studies have confirmed the
possible use of coumarins in cancer therapies. For instance, coumarin (1), 7–hydroxycoumarin
(2), 6–nitro–7–hydroxycoumarin (3) and esculetin (4) (Figure 1) have been reported as potent
anti-proliferative agents both in vitro and in xenograft models [13,14]. Because of the potential
applications of coumarins in cancer treatment, extensive efforts have been made on the design and
synthesis of coumarin derivatives with an improved anticancer activity.
Molecules 2019, 24, 2569; doi:10.3390/molecules24142569
www.mdpi.com/journal/molecules

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Figure 1. Structures of representative coumarin derivatives, FDA-approved histone deacetylase
(HDAC) inhibitors and coumarin-based HDAC inhibitors.
Epigenetic changes in cancer are common and have been associated with pathogenesis and
molecular heterogeneity [15]. Histone deacetylases (HDACs) are overexpressed in a variety of
cancers and associated with tumorigenesis and development, thus making them attractive therapeutic
targets [16,17]. The inhibition of HDACs has been identified as a promising therapeutic approach [18].
HDACis have been shown to significantly suppress cell proliferation, angiogenesis, metastasis and
to lead to apoptosis through multiple mechanisms. These mechanisms include changes in the gene
expression and alterations of both histone and nonhistone proteins, which are a remarkable biological
phenomenon for the suppression of cancers [19–21].
To date, five HDACis, vorinostat (5, SAHA), romidepsin (6, FK228), belinostat (7, PXD101),
panobinostat (8, LBH589) and chidamide (9, CS055) (Figure 1), have been approved for the treatment of
cutaneous T cell lymphoma (CTCL), multiple myeloma (MM) or peripheral T cell lymphoma (PTCL),
and over 20 other inhibitors are in different phases of clinical trials, which certifies that the development
of novel HDACis is still one of the most important approaches for cancer treatment [22]. Despite their
rich structural diversity, HDACis generally have a common pharmacophore model comprising of
three motifs: a CAP region, a zinc-binding group (ZBG) and a linker part connecting CAP and ZBG.
The development of HDACis mainly focuses on the modifications of the three parts, especially at the
CAP and the linker regions, which define the activity and selectivity of inhibitors [23–25]. Nevertheless,
current HDACis exhibit a poor efficacy against solid tumors, and thus, the development of effective
HDACis that possess a high potency against solid tumors is still greatly needed [26].
HDACis have been employed in a number of efforts to generate hybrid agents with the purpose
of achieving synergistic effects [27]. Several coumarin-based HDAC inhibitors (9a, 9b and 9c) (Figure 1)

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were designed and demonstrated the validity of such a design strategy to develop novel HDACis [28–30].
The only hydroxamate HDACi 9a was just synthesized as a fluorescent probe for determining the
binding affinities (Kd) and the dissociation off-rates (Koff) of the HDAC-inhibitor complexes, while
the antitumor activities associated with this compound were not studied. Therefore, in the present
study, we incorporated coumarin into hydroxamate HDACis, and a series of new coumarin-based
hydroxamate HDACis were then synthesized. The rationale for the design of these compounds is
illustrated in Figure 2. We attempted the use of a substituted coumarin moiety as a CAP group for
enzyme surface interactions, different carbon chains as a linker region, and a hydroxamic acid group
as the ZBG. The synthesized compounds were evaluated for their inhibitory activities against HDAC1.
The cytotoxicity against three different cancer cell lines, including A549 (adenocarcinoma human
alveolar basal epithelial cells), Hela (human cervical cancer cell) and HepG2 (human liver cancer cells),
were also conducted. Docking studies were performed to explore the interaction between selective
compounds and HDAC1.
Figure 2. The design proposal of novel coumarins containing a hydroxamate moiety.
2. Chemistry
The routes used for the synthesis of the target compounds containing the coumarin moiety are
shown in Schemes 1–3. As depicted in Scheme 1, the intermediates 14a,b were synthesized from
2–hydroxyacetophenone derivatives 13a,b by reacting with diethyl carbonate and sodium hydride
(NaH) at 100 ◦C [31]. After the chlorination, 4–chloro coumarins 15a,b were further condensed with
different amino acid esters in ethanol (EtOH) to yield esters 16a–e according to procedure c, which
were then reacted with freshly prepared hydroxylamine (NH2OH) in methanol (MeOH) to give the
target compounds 10a–e.
Scheme 1. Synthesis of the compounds 10a
CH3Ph, 0 ◦C to reflux; (
) POCl3, TEBAC, CH3CN, reflux; (
and (d) NH2OH·HCl, KOH, MeOH.
–
e
. Reagents and conditions: (
a) Diethyl carbonate, NaH,
b
c) NH2(CH2)nCO2CH3, Et3N, EtOH, reflux;

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Scheme 2. Synthesis of compounds 11a
(b) Br(CH2)nCO2CH3, K2CO3, DMF; and (c) NH2OH·HCl, KOH, MeOH.
–e
. Reagents and conditions: (a) NaH, CH3Ph, 0 ◦C to reflux;
Scheme 3. Synthesis of compounds 12a,b. Reagents and conditions: (b) Br(CH2)7CO2CH3, K2CO3,
DMF; and (c) NH2OH·HCl, KOH, MeOH.
Compounds 11a
–
e
were prepared according to the methods described in Scheme 2. The intermediate
8) to
with NH2OH in methanol gave the target
14b was constructed from 13b, which was alkylated with brominated fatty acid esters (n = 4
yield ester compounds 17a
compounds 11a–e.
–
–e. Finally, treatment of 17a–e
Compounds 12a
7–hydroxycoumarins 18a
,
b
were obtained according to the methods described in Scheme 3. The substituted
were reacted with Br(CH2)7CO2Me in the presence of potassium carbonate
, which were converted to hydroxamic acids 12a with the treatment of NH2OH
,
b
to produce esters 19a
,
b
,b
in MeOH.
3. Results and Discussion
3.1. HDAC Inhibition Assay
The HDAC inhibitory activities of the target compounds were assayed using HDAC1. The results
were expressed as IC50 and summarized in Table 1, indicating that the HDAC1 inhibition activity was
linker-length-dependent. The potency of the compounds increased with an increasing linker length.
Compound 10e, containing a seven-methylene linker, showed the best activity (IC50 = 0.24 nM) among
its analogues and was 90 times more potent than SAHA (IC50 = 21.10 nM), which was consistent
with the classical feature of HDACis [32
,33]. It was also found that compounds with a 7–methoxy
substitution on the coumarin moiety were slightly more potent than those without the substitution
(10b vs. 10c), which manifested the suitability of this substituent group for the HDAC inhibitory effect.
Upon further modification, the nitrogen atom was replaced with an oxygen atom, and a series
of compounds 11a–e were synthesized with different linker lengths in order to further testify the
relationship between the linker length and HDAC1 inhibitory activities. As shown in Table 2,
the activities of the target compounds were improved with the elongation of the linker. For example,
compound 11d, with n = 7, showed the most potent inhibitory activity. However, the inhibitory activity
declined when n = 8. Then, for further modification, the seven-methylene linker was retained.
On the other hand, the linker and ZBG region were transferred to the C-7 position of the coumarin
moiety, and compounds 12a,b were synthesized. The inhibition activities of these two compounds were
approximately three-times better than SAHA (Table 3). In addition, when the 3–hydrogen atom was
replaced by methyl, the inhibitory activity was retained (12a vs. 12b). However, the two compounds
displayed decreased inhibitory activities compared with compound 11d. The results showed that the
substituted positions of the linker and ZBG region impacted the efficacy of the compounds.

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Table 1. HDAC1 inhibition activity of compounds 10a–e.
Compd.
R
X
n
HDAC1 IC50 (nM)
10a
10b
10c
10d
10e
2
5
5
6
7
–
> 100
H
H
OCH3
OCH3
OCH3
–
NH
NH
NH
NH
NH
–
9.11±0.20
6.43±0.16
2.51±0.12
0.24±0.01
21.10±1.05
SAHA
Assays were performed in replicate (n ≥ 3); the SD values are <20% of the mean.
Table 2. HDAC1 inhibition activity of compounds 11a–e.
Compd.
X
n
HDAC1 IC50 (nM)
11a
11b
11c
11d
11e
4
5
6
7
8
–
O
O
O
O
O
–
> 100
19.41 ± 2.21
5.61 ± 0.56
1.85 ± 0.16
7.33 ± 1.01
21.10 ± 1.05
SAHA
Assays were performed in replicate (n ≥ 3); the SD values are <20% of the mean.
Table 3. HDAC1 inhibition activity of compounds 12a,b.
Compd.
R0
Y
n
HDAC1 IC50 (nM)
12a
12b
SAHA
H
CH3
–
7
7
–
O
O
–
8.72 ± 1.03
6.88 ± 0.85
21.10 ± 1.05
Assays were performed in replicate (n ≥ 3); the SD values are <20% of the mean.
3.2. IC50 Values of HDAC Isoforms Inhibition of Potent Compounds
The selected compounds with a good HDAC1 inhibitory activity were also tested for their enzyme
inhibitory activity against HDAC1, HDAC2, HDAC4, HDAC5, HDAC6, HDAC8, and HDAC11,
in order to evaluate the selectivity of this series of compounds against HDAC isoforms (Table 4).
The results displayed that compounds 10e and 11d were pan-HDAC inhibitors, which were similar to
SAHA. The two compounds showed a more potent inhibition against class I and IIb HDAC isoforms
than against class IV and IIa ones. Particularly for HDAC1, HDAC2 and HDAC8, 10e exhibited about 88,
27 and 12 times, and 11d showed about 11, 60 and 107 times greater potency, respectively, than SAHA.
These results demonstrated that coumarin is an effective surface recognition cap for HDACs.

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Table 4. Inhibition activity (IC50) of the tested compounds on different HDAC isoforms.
IC50 (nM)
Class I
2
Class II
Class IV
Compound
1
8
4
5
6
11
10e
11d
SAHA
0.24 ± 0.01
1.85 ± 0.15
21.10 ± 1.05
0.68 ± 0.05
16.90 ± 0.01
1.85 ± 0.14
>1000
>1000
>1000
>1000
>1000
>1000
5.40 ± 1.00
5.67 ± 1.42
20.36 ± 3.92
>1000
>1000
>1000
<0.32
18.55 ± 0.13
195.90 ± 86.13
Assays were performed in replicate (n ≥ 3); the SD values are <20% of the mean.
3.3. Anti-Proliferative Activities against Three Cancer Cell Lines In Vitro
To investigate the anticancer activities, compounds 10e and 11d were then screened for their
anti-proliferative activity against three cancer cell lines, and the IC50 values were summarized in Table 5.
It was indicated that A549 and Hela cells were more sensitive to the selected compounds compared
to HepG2 cells. Notably, compounds 10e and 11d exhibited comparable or better anti-proliferative
activities when compared with SAHA against A549 and Hela cells.
Table 5. Anti-proliferative activities of representative compounds against different cancer cell lines.
IC50 (µM)
Compound
A549
Hela
HepG2
10e
11d
SAHA
1.31 ± 0.86
2.62 ± 1.53
2.86 ± 0.35
1.96 ± 0.74
0.56 ± 0.28
2.63 ± 0.87
>10
7.71 ± 2.07
4.51 ± 0.68
Assays were performed in replicate (n ≥ 3); the SD values are <20% of the mean.
3.4. Effects of Compounds 10e and 11d on Acetylated Histone Levels in A549 Cells
Based on the aforementioned results, we further investigated whether compounds 10e and 11d
induced the acetylation of histones in lung cancer cells at different concentrations. A549 cells were
incubated with the vehicle alone, and with SAHA, 10e and 11d (0.2, 0.5 and 1.0
µM) for 48 h, respectively.
The levels of acetylated histone H3 and H4 were analyzed by Western blotting assays with
β–actin as
the negative control. The results in Figure 3A showed that compounds 10e and 11d could increase the
expression of acetylated histone H3 and H4 in a dose-dependent manner. Meanwhile, Quantitative
analysis results in Figure 3B showed that the levels of acetyl–histone H3 and H4 in compounds 10e
and 11d treated groups were similar or even higher than those in the SAHA treated group (1.0 µM),
which was consistent with their HDAC inhibition activities.
3.5. Compounds 10e and 11d Enhanced Apoptosis in the A549 Cell Line
To investigate whether the potent anti-proliferative activities of compounds 10e and 11d were
related to enhancing the apoptosis of cancer cells, we carried out an annexin V fluorescein isothiocyanate
(FITC)/propidium iodide (PI) binding assay in A549 cancer cells. As shown in Figure 4A, compounds
10e and 11d caused a significant induction of apoptosis in a dose-dependent manner. 10e and 11d
induced 51.9% and 81.7% apoptosis in A549 cancer cells at 1.25
in A549 cancer cells at 2.5 M and induced 90.3% and 91.4% apoptosis in A549 cancer cells at 5
respectively. However, SAHA only induced 27.6%, 40.9% and 61.4% of apoptotic cells at 1.25, 2.5 and
M, respectively. The quantitative analysis results indicated that 10e and 11d were able to induce
more apoptosis in A549 cells than the positive control drug, SAHA (Figure 4B).
µM, induced 84.4% and 87.2% apoptosis
µ
µM,
5
µ

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Figure 3. Western blot analysis of the effects of compounds 10e and 11d on the acetylated histone
levels in A549 cells. ( ) A549 cells were treated with 10e 11d and SAHA for 48 h at the indicated
concentrations, and the levels of protein expression were detected using anti–acetyl–histone H3, H4
and anti– –actin antibodies, respectively. B–Actin was used as the loading control. ( ) Quantitative
analysis. The relative levels of Ac-H3 and Ac-H4 used to control –actin were determined by densimetric
SD of three separate experiments. * p < 0.05, ** p < 0.01,
A
,
β
B
β
scanning. The data are expressed as means
*** p < 0.001 vs. control.
±
Figure 4. Compounds 10e and 11d mediated A549 cell apoptosis in a concentration-dependent manner.
) Flow cytometry analysis. ( ) Quantitative analysis of the apoptotic cells. Data are expressed as
means SD of the percentages of apoptotic cells from three independent experiments. ** p < 0.01,
*** p < 0.001 vs. control.
(
A
B
±
3.6. Compounds 10e and 11d Induced Cell Cycle Arrest on A549 Cell Line
Next, we investigated whether the potent anti-proliferative activity of 10e and 11d resulted from
the induction of the cell cycle arrest. As shown in Figure 5A and 5B, 10e and 11d led to a significant
induction of the G2/M phase cell cycle arrest in a concentration-dependent manner. This was evidenced
by the increasing percentages of cells in the G2/M phase, accompanied by a proportionate reduction in
other phases of the cell cycle.

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Figure 5. Compounds 10e and 11d induced a A549 cancer cell cycle arrest. (A) Flow cytometry analysis.
(
B
) Representative histograms. Data are expressed as means SD of the percentages of A549 cells from
±
three independent experiments. * p < 0.05, *** p < 0.001 vs. control.
3.7. Theoretical Prediction of ADME Properties and Preliminary Toxicity Evaluation
For the purpose of achieving a better assessment of the druggability of these coumarin-based
hydroxamate HDAC inhibitors, several parameters, including the calculated LogP (cLogP), topological
polar surface area (tPSA), the number of hydrogen-bond acceptors and donors (n–ON and n–OHNH),
and the number of rotable bonds and molecular volumes were carried out for the prediction of
the ADME properties of the four compounds through the Molinspiration program (http://www.
molinspiration.com/cgi-bin/properties) [34]. The reports suggested that compounds which meet
the two criteria of (1) 10 or fewer rotatable bonds; (2) a polar surface area under 140 Å2 (or 12 or
fewer H-bond donors and acceptors) may have a good oral bioavailability [35]. As shown in Table 6,
compounds 10e and 11d both have 10 rotatable bonds, seven hydrogen bond acceptors (n–ON) and
≤
3 donors (n–OHNH). The polar surface area (tPSA) did not exceed 140 A2. The cLogP was in an
acceptable range ( 2 to 5). The above results indicated that the two compounds are in a reasonable
−
region for further development as potential drug candidates.
Table 6. Theoretical prediction of the ADME properties of representative compounds.
Compound
cLogP
MW
tPSA
n-ON
n-OHNH
nrotb
Volume (Å)
10e
11d
SAHA
3.18
3.38
2.47
348.40
349.38
264.32
100.80
98.00
78.42
7
7
5
3
2
3
10
10
8
323.55
320.13
255.64
3.8. Molecular Docking Studies
In order to determine the interaction mode between our compounds and HDACs, molecular
docking was performed using a validated molecular dock program (AutoDock 4.27) [36]. Compound
10e was docked into the active site of HDAC1 (PDB entry: 4BKX) (Figure 6). The results showed that
10e had a similar binding mode to SAHA in the active site of HDAC1: the cap group interacts with the
residues of the entrance region, the linker goes through the hydrophobic channel, and the hydroxamic
acid could chelate the catalytic zinc ion (Figure 6A). Compound 10e could form five hydrogen bonds
with ASP99, HIS28, HIS178, ASP176 and ASP264 residues in the active site of HDAC1 (Figure 6B).
It should be noted that the 7–methoxy group on the coumarin ring of compound 10e docked into the
hydrophobic pocket of the protein, which could account for the potency of this compound (Figure 6A).

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Figure 6. The proposed binding mode of compound 10e with HDAC1 (PDB 4BKX). (A) Molecular
surface of the HDAC1 binding pocket with 10e. (B) 10e interacted with the active site of HDAC1.
4. Materials and Methods
4.1. Chemistry: General Methods
The reagents were purchased from Energy Chemical Inc. Adamas-beta Ltd, J&K Inc. or
Aladdin-reagents Inc., and were used without further purification unless otherwise specified.
All reactions were carried out with the use of standard techniques under an inert atmosphere.
1H- and 13C-NMR spectra were generated on a Bruker 600 MHz instrument and obtained as CD3OD or
DMSO-d6 solutions, using residual solvent peaks as references (δH 3.31 and δC 49.0 ppm for CD3OD;
and δH 2.50 and δC 39.50 for DMSO-d6). Chemical shifts were shown in ppm and coupling constants (J)
in Hz. Standard abbreviations indicating spin multiplicities are given as follows: s (singlet), d (doublet),
t (triplet), q (quartet), br (broad) or m (multiplet). The high-resolution mass spectra were gathered on
an Agilent 6545 Q-TOF mass spectrometer operating in electrospray ionization (ESI) mode. HPLC
(Agilent Technologies 1260 Series) was employed for the purity determination, using the following
method: Eclipse XDB C18 column, 5
MeOH-H2O system, 40%−90% in 10 min, hold on 5 min, and then back to 40% in 5 min.
µ
m, 4.6 mm
×
150 mm, column temperature 40 ◦C, 1.5 mL/min
4.2. General Procedures for the Preparation of Target Compounds
N–hydroxy–3–((2–oxo–2H–chromen–4–yl)amino)propanamide (10a). To a solution of hydroxyl
amine hydrochloride (2.40 g, 34.8 mmol) in 10 mL◦MeOH, KOH (1.95 g, 34.8 mmol) was added.
◦
The reaction mixture was stirred for 10 min at 40 C, and was then cooled to 0 C and filtered.
3–((2–oxo–2H–chromen–4–yl)amino)propanoate methyl ester (320 mg, 1.1 mmol) was added to the
filtrate followed by KOH (195 mg, 3.5 mmol), after which the reaction was stirred at room temperature
for 30 min. The solvent was removed under reduced pressure, diluted with a saturated NH4Cl
aqueous solution, and extracted with EtOAc. The organic layer was washed with brine and dried
over Na2SO4. The resulting solution was evaporated under a reduced pressure and then purified by
column chromatography [eluting with EtOAc followed by 10:1 CH2Cl2/MeOH] to give compound
10a (105 mg, 34.5% yield). 1H-NMR (600 MHz, DMSO-d6)
J = 8.4 Hz, 1H), 7.76–7.75 (m, 1H), 7.59 (dd, J = 6.6, 6.0 Hz, 1H), 7.33–7.30 (m, 2H), 5.19 (s, 1H), 3.49–3.47
δ 10.50 (brs, 1H), 8.81 (brs, 1H), 8.00 (d,
(m, 2H), 2.36 (t, J = 6.6 Hz, 2H). 13C-NMR (150 MHz, DMSO-d6)
δ 166.90, 161.49, 153.06, 152.87, 131.92,
123.32, 122.38, 116.96, 114.40, 81.57, 40.04, 30.92. HRMS (ESI): calcd for [C12H12N2O4 + H] + 249.0870,
found 249.0882. HPLC purity: 99.6%, tR = 2.2 min.
Compounds 10b–e, 11a–e and 12a,b were prepared according to the procedure described for the
preparation of compound 10a.
N–hydroxy–6–((2–oxo–2H–chromen–4–yl)amino)hexanamide (10b) (31.4% yield). 1H-NMR (600 MHz,
DMSO-d6) 10.37 (brs, 1H), 8.68 (brs, 1H), 8.08 (d, J = 7.8 Hz, 1H), 7.66–7.64 (m, 1H), 7.60–7.57 (m,
δ

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1H), 7.33–7.30 (m, 2H), 5.14 (s, 1H), 3.22 (q, J = 6.0 Hz, 2H), 1.97 (t, J = 7.8 Hz, 2H), 1.66–1.61 (m, 2H),
1.58–1.53 (m, 2H), 1.37–1.31 (m, 2H). 13C-NMR (150 MHz, DMSO-d6)
169.02, 161.58, 153.10, 153.08,
δ
131.86, 123.26, 122.48, 116.95, 114.47, 81.16, 42.22, 32.23, 27.21, 26.19, 24.90. HR-MS (ESI): calcd for
[C15H18N2O4 + H] + 291.1339, found 291.1344. HPLC purity: 99.8%, tR = 4.1 min.
N–hydroxy–6–((7–methoxy–2–oxo–2H–chromen–4–yl)amino)hexanamide (10c) (28.9% yield). 1H-NMR
(600 MHz, DMSO-d6) δ 10.41 (brs, 1H), 8.68 (brs, 1H), 8.05 (d, J = 8.4 Hz, 1H), 7.68–7.66 (m, 1H), 6.89
(dd, J = 8.4, 2.4 Hz, 1H), 6.84 (d, J = 2.4 Hz, 1H), 4.98 (s, 1H), 3.83 (s, 3H), 3.20–3.15 (m, 2H), 1.96 (t,
J = 7.8 Hz, 2H), 1.63–1.58 (m, 2H), 1.55–1.50 (m, 2H), 1.34–1.30 (m, 2H). 13C-NMR (150 MHz, DMSO-d6)
δ
169.04, 162.09, 161.94, 154.86, 153.49, 123.93, 111.04, 107.66, 100.82, 79.13, 55.77, 42.11, 32.24, 27.33,
26.20, 24.93. HRMS (ESI): calcd for [C16H20N2O5 + H] + 321.1445, found 321.1450. HPLC purity: 99.5%,
tR = 5.0 min.
1
N–hydroxy–7–((7–methoxy–2–oxo–2H–chromen–4–yl)amino)heptanamide (10d) (30.1% yield). H-NMR
(600 MHz, DMSO-d6) δ 10.35 (brs, 1H), 8.67 (brs, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.54–7.52 (m, 1H), 6.91
(d, J = 8.4 Hz, 1H), 6.86–6.85 (m, 1H), 5.00 (s, 1H), 3.84 (s, 3H), 3.20–3.16 (m, 2H), 1.95 (t, J = 7.8 Hz,
2H), 1.63–1.58 (m, 2H), 1.52–1.48 (m, 2H), 1.37–1.32 (m, 2H), 1.31–1.28 (m, 2H). 13C-NMR (150 MHz,
DMSO-d6) δ 169.07, 162.08, 161.88, 154.85, 153.43, 123.68, 111.05, 107.59, 100.82, 79.19, 55.74, 39.92, 32.21,
28.34, 27.46, 26.27, 25.05. HRMS (ESI): calcd for [C17H22N2O5 + H] + 335.1601, found 335.1607. HPLC
purity: 99.1%, tR = 6.4 min.
N–hydroxy–8–((7–methoxy–2–oxo–2H–chromen–4–yl)amino)octanamide (10e) (25.4% yield). 1H-NMR
(600 MHz, DMSO-d6) δ 10.35 (brs, 1H), 8.67 (brs, 1H), 7.97 (d, J = 8.4 Hz, 1H), 7.53–7.51 (m, 1H), 6.91
(dd, J = 8.4, 2.4 Hz, 1H), 6.88–6.85 (m, 1H), 5.00 (s, 1H), 3.84 (s, 3H), 3.20–3.18 (m, 2H), 1.94 (t, J = 7.2 Hz,
2H), 1.62–1.59 (m, 2H), 1.50–1.48 (m, 2H), 1.34–1.30 (m, 4H), 1.26–1.23 (m, 2H). 13C-NMR (150 MHz,
DMSO-d6) δ 168.97, 162.08, 161.87, 154.85, 153.42, 123.66, 111.06, 107.59, 100.83, 79.19, 55.74, 39.92, 32.18,
28.55, 28.48, 27.56, 26.47, 25.06. HRMS (ESI): calcd for [C18H24N2O5 + H]+ 349.1758, found 349.1747.
HPLC purity: 99.4%, tR = 8.0 min.
N–hydroxy–5–((7–methoxy–2–oxo–2H–chromen–4–yl)oxy)pentanamide (11a) (31.2% yield). 1H-NMR
(600 MHz, CD3OD)
δ 7.78 (d, J = 8.4 Hz, 1H), 6.93 (dd, J = 8.4, 2.4 Hz, 1H), 6.88 (d, J = 2.4 Hz, 1H),
5.65 (s, 1H), 4.21 (t, J = 6.6 Hz, 2H), 3.88 (s, 3H), 2.20 (t, J = 7.2 Hz, 2H), 1.94–1.90 (m, 2H), 1.88–1.84
(m, 2H). 13C-NMR (150 MHz, CD3OD)
δ 172.48, 168.23, 165.97, 165.16, 156.42, 125.35, 113.63, 110.05,
101.40, 88.40, 70.28, 56.41, 33.21, 29.04, 23.23. HRMS (ESI): calcd for [C15H17NO6 + H] + 308.1129, found
308.1122. HPLC purity: 98.9%, tR = 5.8 min.
N–hydroxy–6–((7–methoxy–2–oxo–2H–chromen–4–yl)oxy)hexanamide (11b) (41.6% yield). 1H-NMR
(600 MHz, DMSO-d6)
6.95 (dd, J = 8.4, 2.4 Hz, 1H), 5.73 (s, 1H), 4.18 (t, J = 6.6 Hz, 2H), 3.86 (s, 3H), 1.99 (t, J = 7.2 Hz, 2H),
1.83–1.78 (m, 2H), 1.61–1.56 (m, 2H), 1.46–1.42 (m, 2H). 13C-NMR (150 MHz, DMSO-d6)
168.96, 165.40,
δ 10.36 (brs, 1H), 8.69 (brs, 1H), 7.69 (d, J = 8.4 Hz, 1H), 6.98 (d, J = 2.4 Hz, 1H),
δ
162.88, 162.10, 154.63, 123.93, 112.19, 108.35, 100.53, 87.88, 69.20, 55.92, 32.14, 27.17, 25.03, 24.77. HRMS
(ESI): calcd for [C16H19NO6 + H] + 322.1285, found 322.1285. HPLC purity: 99.2%, tR = 7.1 min.
N–hydroxy–7–((7–methoxy–2–oxo–2H–chromen–4–yl)oxy)heptanamide (11c) (39.7% yield). 1H-NMR
(600 MHz, CD3OD)
1H), 5.66 (s, 1H), 4.21 (t, J = 6.6 Hz, 2H), 3.89 (s, 3H), 2.12 (t, J = 7.2 Hz, 2H), 1.94–1.90 (m, 2H), 1.70–1.65
(m, 2H), 1.59–1.54 (m, 2H), 1.47–1.42 (m, 2H). 13C-NMR (150 MHz, CD3OD)
168.34, 166.04, 165.17,
δ 7.77 (d, J = 8.4 Hz, 1H), 6.95 (dd, J = 8.4, 2.4 Hz, 1H), 6.90 (d, J = 2.4 Hz,
δ
156.44, 125.31, 113.65, 110.11, 101.44, 88.33, 70.76, 56.42, 33.64, 29.76, 29.47, 26.72, 26.62. HRMS (ESI):
calcd for [C17H21NO6 + H] + 336.1442, found 336.1433. HPLC purity: 96.9%, tR = 18.8 min.
N–hydroxy–8–((7–methoxy–2–oxo–2H–chromen–4–yl)oxy)octanamide (11d) (45.8% yield). 1H-NMR
(600 MHz, CD3OD)
1H), 5.65 (s, 1H), 4.20 (t, J = 6.6 Hz, 2H), 3.89 (s, 3H), 2.10 (t, J = 7.2 Hz, 2H), 1.92–1.89 (m, 2H), 1.67–1.62
(m, 2H), 1.58–1.52 (m, 2H), 1.46–1.43 (m, 2H), 1.40–1.37 (m, 2H). 13C-NMR (150 MHz, CD3OD)
172.98,
δ 7.75 (d, J = 8.4 Hz, 1H), 6.94 (dd, J = 8.4, 2.4 Hz, 1H), 6.88 (d, J = 2.4 Hz,
δ

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168.33, 166.04, 165.15, 156.41, 125.27, 113.63, 110.10, 101.44, 88.32, 70.84, 56.42, 33.72, 29.99, 29.98, 29.54,
26.89, 26.65. HRMS (ESI): calcd for [C18H23NO6 + H] + 350.1598, found 350.1596. HPLC purity: 99.2%,
tR = 10.3 min.
N–hydroxy–9–((7–methoxy–2–oxo–2H–chromen–4–yl)oxy)nonanamide (11e) (42.3% yield). 1H-NMR
(600 MHz, DMSO-d6)
δ 10.33 (brs, 1H), 8.66 (brs, 1H), 7.69 (d, J = 8.4 Hz, 1H), 6.98 (d, J = 2.4 Hz,
1H), 6.96 (dd, J = 8.4, 2.4 Hz, 1H), 5.73 (s, 1H), 4.18 (t, J = 6.6 Hz, 2H), 3.85 (s, 3H), 1.94 (t, J = 7.2 Hz,
2H), 1.81–1.79 (m, 2H), 1.50–1.47 (m, 2H), 1.46–1.43 (m, 2H), 1.35–1.32 (m, 2H), 1.30–1.26 (m, 2H),
1.26–1.22 (m, 2H). 13C-NMR (150 MHz, DMSO-d6)
δ 169.07, 165.41, 162.87, 162.09, 154.62, 123.89, 112.20,
108.36, 100.54, 87.85, 69.29, 55.91, 32.23, 28.64, 28.56, 28.52, 27.95, 25.38, 25.08. HRMS (ESI): calcd for
[C19H25NO6 + H] + 364.1755, found 364.1761. HPLC purity: 99.0%, tR = 18.7 min.
N–hydroxy–8–((2–oxo–2H–chromen–7–yl)oxy)octanamide (12a) (37.6% yield). 1H-NMR (600 MHz,
DMSO-d6)
δ 10.34 (brs, 1H), 8.67 (brs, 1H), 7.99 (d, J = 9.0 Hz, 1H), 7.62 (d, J = 8.4 Hz, 1H),
6.98 (d, J = 2.4 Hz, 1H), 6.94 (dd, J = 8.4, 2.4 Hz, 1H), 6.28 (d, J = 9.0 Hz, 1H), 4.06 (t, J = 6.6 Hz, 2H), 1.94
(t, J = 7.2 Hz, 2H), 1.74–1.71 (m, 2H), 1.51–1.48 (m, 2H), 1.42–1.39 (m, 2H), 1.33–1.29 (m, 2H), 1.28–1.24
(m, 2H). 13C-NMR (150 MHz, DMSO-d6)
δ 169.08, 161.88, 160.32, 155.42, 144.35, 129.46, 112.71, 112.36,
112.22, 101.10, 68.25, 32.22, 28.49, 28.40, 28.38, 25.31, 25.04. HRMS (ESI): calcd for [C17H21NO5 + H] +
320.1492, found 320.1488. HPLC purity: 99.1%, tR = 8.9 min.
N–hydroxy–8–((4–methyl–2–oxo–2H–chromen–7–yl)oxy)octanamide (12b) (33.2% yield).
(600 MHz, DMSO-d6) 10.33 (brs, 1H), 8.66 (brs, 1H), 7.67 (d, J = 9.0 Hz, 1H), 6.96–6.94 (m, 2H), 6.20
(s, 1H), 4.07 (t, J = 6.6 Hz, 2H), 2.40 (s, 3H), 1.94 (t, J = 7.2 Hz, 2H), 1.75–1.70 (m, 2H), 1.52–1.47 (m,
2H), 1.43–1.38 (m, 2H), 1.35–1.30 (m, 2H), 1.28–1.23 (m, 2H). 13C-NMR (150 MHz, DMSO-d6)
169.09,
1H-NMR
δ
δ
161.79, 160.19, 154.77, 153.45, 126.44, 112.99, 112.43, 111.04, 101.12, 68.23, 32.22, 28.50, 28.41, 28.39, 25.32,
25.04, 18.13. HRMS (ESI): calcd for [C18H23NO5 + H] + 334.1649, found 334.1637. HPLC purity: 98.8%,
tR = 10.3 min.
Compounds 10a
–e and 11a–e have been published in patent no. CN 108658915A (Oct 16, 2018) [37].
4.3. HDAC1 Inhibitory Assay
The HDAC1 enzyme activity in vitro was determined by the protease-coupled assay. Different
concentrations of compounds were incubated with recombinant HDAC1 (BPS Biosciences, San Diego,
CA, USA) at room temperature for 15 min, followed by adding trypsin, as well as Ac-peptide-AMC
substrates, to initiate the reaction in a Tris-based assay buffer. Fluorescent AMC, released from the
substrate, was measured in SynergyMx (BioTek, VT, USA) using a filter set at excitation = 355 nm and
emission = 460 nm. The IC50 values were calculated by GraphPad Prism software (La Jolla, CA, USA).
(for details see Supplementary Materials)
4.4. In Vitro Anti-Proliferation Assay.
The in vitro anti-proliferation assay was operated as previously described [38]. In brief, the human
cancer cell lines, A549, HeLa and HepG2, were seeded into 96-well plates at the appropriate cell
densities. After incubation for 24 h, the cells were treated with various concentrations of tested
compounds for 48 h. Then, the 3–(4,5–dimethyl–2-thiazolyl)–2,5–diphenyl–2–H–tetrazolium bromide
(MTT) solution was added and co-incubated for another 4 h. The reactions were stopped by the addition
of DMSO solution, and the samples were measured at 490 nm by a microplate spectrophotometer
(Thermo Fisher Scientific, Waltham, MA USA). The IC50 was calculated using GraphPad Software.
Three independent experiments were carried out in triplicate. (for details see Supplementary Materials)
4.5. Western Blot Analysis
Western blotting was performed as previously described [39]. Briefly, human cancer cells were
lysed in a lysis buffer (50 mM Tris-HCl pH 8.0, 5 mM EDTA, 100 mM NaCl, 0.5% NP-40, 1 mM PMSF),

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centrifuged for 10 min at 10,000 g, and the insoluble debris were discarded. Cell lysates were further
analyzed with SDS-PAGE and western blotting with the indicated antibodies.
4.6. Cell Apoptosis Analysis
The cell apoptosis analysis was measured by annexin V FITC/PI assay using the Vybrant Apoptosis
Assay kit (Invitrogen, Carlsbad, CA, USA). Briefly, A549 cells (8
and compounds 10e or 11d for 72 h. The cells were then harvested and stained with annexin-binding
buffer, Alexa Fluor 488 annexin and propidium iodide for 15 min in the dark. After staining, 400 L of
×
104/well) were treated with DMSO
µ
1X annexin-binding buffer was added, mixed gently and kept on ice. The samples were measured
using a BD Biosciences FACS Aria flow cytometer.
4.7. Cell Cycle Analysis
The cell cycle analysis was carried out by estimating the DNA contents with flow cytometry. After
incubation with the indicated doses of 10e or 11d for 24 h, A549 cells were fixed in 70% ice-cold ethanol,
incubated overnight at –20 ◦C, stained with propidium iodide (PI)/Triton X-100 containing RNaseA
solution for 20 min at 37 ◦C, and then analyzed by FACS.
4.8. Molecular Docking Studies
Molecular docking studies were carried out with Autodock-4.27. For the docking calculations,
The HDAC1 crystal structure (PDB code: 4BKX) was retrieved from the Protein Data Bank
(www.pdb.org). Before docking, 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.
5. Conclusions
Coumarins have been identified as anticancer candidates. HDACis are one of the hot topics in
the field of antitumor research. In order to achieve an increased anticancer efficacy, a series of hybrid
compounds bearing coumarin and hydroxamic acid scaffolds have been designed and synthesized
as novel HDACis, and their biological activities have been evaluated in a series of in vitro assays.
Compound 10e showed the most potent inhibitory activity against HDAC1, with IC50 of 0.24 nM,
which was almost 90 times lower than SAHA (IC50 = 21.10 nM). In vitro cell growth inhibition assays
displayed that compound 10e and 11d exhibited better inhibitory activities against the human lung
cancer cell line A549 and cervical cancer cell line Hela than against the hepatocellular carcinoma cell
line HepG2. A western blotting analysis revealed that compounds 10e and 11d could upregulate the
levels of acetylated histone H3 and H4 in a dose-dependent manner. In addition, compound 10e and
11d were found to induce A549 cancer cell cycle arrest and enhance cancer cell apoptosis. Furthermore,
a molecular docking analysis exhibited a possible interaction mode of compounds 10e and 12a with
HDAC1. Taken together, all these data suggest that these novel coumarin-based HDAC inhibitors
could be promising candidates for the further development of novel antitumor agents.
Supplementary Materials: The following are available online. Raw spectroscopic data including HPLC analyses,
HRMS (ESI), and NMR (1H, 13C) spectra for new compounds 10a
–e, 11a–e and 12a, b. HDAC inhibition activity of
compounds 10b–e, 11a–e and 12a–b, Inhibition activity of compounds 10e and 11d on different HDAC isoforms,
Anti-proliferative activities of compounds 10e and 11d against different cancer cell lines.
Author Contributions: H.Z. and P.S. designed and guided the project and edited the paper. F.Y. conceived and
designed the study, coordinated the experimental work, and wrote the manuscript. N.Z and C.-S.J. participated in
the synthesis of target compounds and structural characterization. P.S. and J.S. performed biological experiments
and analyzed the data. K.Z. performed the molecular docking studies.
Funding: We acknowledge the financial support from National Natural Science Foundation of China (No. 81703360),
the Natural Science Foundation of Shandong Province (Nos. ZR2016HB43, JQ201721), the Young Taishan Scholars
Program (tsqn20161037), and the Shandong Talents Team Cultivation Plan of University Preponderant Discipline
(No. 10027).

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Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Dandriyal, J.; Singla, R.; Kumar, M.; Jaitak, V. Recent developments of C-4 substituted coumarin derivatives
as anticancer agents. Eur. J. Med. Chem. 2016, 119, 141–168. [CrossRef] [PubMed]
2.
Sashidhara, K.V.; Kumar, A.; Chatterjee, M.; Rao, K.B.; Singh, S.; Verma, A.K.; Palit, G. Discovery and
synthesis of novel 3-phenylcoumarin derivatives as antidepressant agents. Bioorganic Med. Chem. Lett.
2011, 21, 1937–1941. [CrossRef] [PubMed]
3.
4.
Rosselli, S.; Maggio, A.M.; Faraone, N.; Spadaro, V.; Morris-Natschke, S.L.; Bastow, K.F.; Lee, K.-H.; Bruno, M.
The Cytotoxic Properties of Natural Coumarins Isolated from Roots of Ferulago campestris (Apiaceae) and
of Synthetic Ester Derivatives of Aegelinol. Nat. Prod. Commun. 2009, 4, 1701–1706. [CrossRef] [PubMed]
Whang, W.K.; Park, H.S.; Ham, I.; Oh, M.; Namkoong, H.; Kim, H.K.; Hwang, D.W.; Hur, S.Y.; Kim, T.E.;
Park, Y.G.; et al. Natural compounds, fraxin and chemicals structurally related to fraxin protect cells from
oxidative stress. Exp. Mol. Med. 2005, 37, 436–446. [CrossRef] [PubMed]
5.
6.
7.
8.
Bansal, Y.; Sethi, P.; Bansal, G. Coumarin: A potential nucleus for anti-inflammatory molecules.
Med. Chem. Res. 2013, 22, 3049–3060. [CrossRef]
de Souza, L.G.; Renna, M.N.; Figueroa-Villar, J.D. Coumarins as cholinesterase inhibitors: A review.
Chem.-Biol. Interact. 2016, 254, 11–23. [CrossRef] [PubMed]
Amin, K.M.; Awadalla, F.M.; Eissa, A.A.M.; Abou-Seri, S.M.; Hassan, G.S. Design, synthesis and vasorelaxant
evaluation of novel coumarin-pyrimidine hybrids. Bioorganic Med. Chem. 2011, 19, 6087–6097. [CrossRef]
Basanagouda, M.; Jambagi, V.B.; Barigidad, N.N.; Laxmeshwar, S.S.; Devaru, V.; Narayana, C. Synthesis,
structure-activity relationship of iodinated-4-aryloxymethyl-coumarins as potential anti-cancer and
anti-mycobacterial agents. Eur. J. Med. Chem. 2014, 74, 225–233. [CrossRef] [PubMed]
Riveiro, M.E.; Moglioni, A.; Vazquez, R.; Gomez, N.; Facorro, G.; Piehl, L.; de Celis, E.R.; Shayo, C.; Davio, C.
Structural insights into hydroxycoumarin-induced apoptosis in U-937 cells. Bioorganic Med. Chem. 2008, 16,
2665–2675. [CrossRef] [PubMed]
9.
10. Riveiro, M.E.; Kimpe, N.D.; Moglioni, A.; Vazquez, R.; Monczor, F.; Shayo, C.; Davio, C. Coumarins: Old
Compounds with Novel Promising Therapeutic Perspectives. Curr. Med. Chem. 2010, 17, 1325–1338.
[CrossRef]
11. Bronikowska, J.; Szliszka, E.; Jaworska, D.; Czuba, Z.P.; Krol, W. The Coumarin Psoralidin Enhances
Anticancer Effect of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL). Molecules 2012, 17,
6449–6464. [CrossRef] [PubMed]
12. Thakur, A.; Singla, R.; Jaitak, V. Coumarins as anticancer agents: A review on synthetic strategies, mechanism
of action and SAR studies. Eur. J. Med. Chem. 2015, 101, 476–495. [CrossRef] [PubMed]
13. Finn, G.; Creaven, B.; Egan, D. Modulation of mitogen-activated protein kinases by 6-nitro-7-hydroxycoumarin
mediates apoptosis in renal carcinoma cells. Eur. J. Pharmacol. 2003, 481, 159–167. [CrossRef] [PubMed]
14. Kim, E.-K.; Kwon, K.-B.; Shin, B.-C.; Seo, E.-A.; Lee, Y.-R.; Kim, J.-S.; Park, J.-W.; Park, B.-H.; Ryu, D.-G.
Scopoletin induces apoptosis in human promyeloleukemic cells, accompanied by activations of nuclear
factor kappaB and caspase-3. Life Sci. 2005, 77, 824–836. [CrossRef] [PubMed]
15. Baretti, M.; Azad, N.S. The role of epigenetic therapies in colorectal cancer. Curr. Probl. Cancer 2018
.
[CrossRef] [PubMed]
16. Singh, A.; Patel, P.; Patel, V.K.; Jain, D.K.; Veerasamy, R.; Sharma, P.C.; Rajak, H. Histone Deacetylase
Inhibitors for the Treatment of Colorectal Cancer: Recent Progress and Future Prospects. Curr. Cancer
Drug Targets 2017, 17, 456–466. [CrossRef]
17. Bolden, J.E.; Peart, M.J.; Johnstone, R.W. Anticancer activities of histone deacetylase inhibitors. Nat. Rev.
Drug Discov. 2006, 5, 769. [CrossRef]
18. Sangwan, R.; Rajan, R.; Mandal, P.K. HDAC as onco target: Reviewing the synthetic approaches with SAR
study of their inhibitors. Eur. J. Med. Chem. 2018, 158, 620–706. [CrossRef]
19. Rajak, H.; Singh, A.; Dewangan, P.K.; Patel, V.; Jain, D.K.; Tiwari, S.K.; Veerasamy, R.; Sharma, P.C. Peptide
Based Macrocycles: Selective Histone Deacetylase Inhibitors with Antiproliferative Activity. Curr. Med. Chem.
2013, 20, 1887–1903. [CrossRef]

Molecules 2019, 24, 2569
14 of 15
20. Gao, S.; Li, X.; Zang, J.; Xu, W.; Zhang, Y. Preclinical and Clinical Studies of Chidamide (CS055/HBI-8000),
An Orally Available Subtype-selective HDAC Inhibitor for Cancer Therapy. Anti-Cancer Agents Med. Chem.
2017, 17, 802–812. [CrossRef]
21. Newbold, A.; Falkenberg, K.J.; Prince, H.M.; Johnstone, R.W. How do tumor cells respond to HDAC
inhibition? Febs J. 2016, 283, 4032–4046. [CrossRef] [PubMed]
22. Li, Y.; Seto, E. HDACs and HDAC Inhibitors in Cancer Development and Therapy. Cold Spring Harb.
Perspect. Med. 2016, 6. [CrossRef] [PubMed]
23. Jin, P.; Chen, X. Current Status of Epigenetics and Anticancer Drug Discovery. Anti-Cancer Agents Med. Chem.
2016, 16, 699–712. [CrossRef]
24. Manal, M.; Chandrasekar, M.J.N.; Priya, J.G.; Nanjan, M.J. Inhibitors of histone deacetylase as antitumor
agents: A critical review. Bioorganic Chem. 2016, 67, 18–42. [CrossRef] [PubMed]
25. Zhan, P.; Wang, X.; Liu, X.; Suzuki, T. Medicinal Chemistry Insights into Novel HDAC Inhibitors: An
Updated Patent Review (2012-2016). Recent Pat. Anti-Cancer Drug Discov. 2017, 12, 16–34.
26. Miao, J.-F.; Peng, Y.-F.; Chen, S.; Gao, W.-J.; Yang, Q.-X.; Zhu, P.; Guo, J.; Tao, J.; Luo, L.; Zhang, Y.;
et al. A novel harmine derivative, N-(4-(hydroxycarbamoyl) benzyl)-1-(4-methoxyphenyl)-9H-pyrido 3,4-b
indole-3-carboxamide (HBC), as histone deacetylase inhibitor: In vitro antiproliferation, apoptosis induction,
cell cycle arrest, and antimetastatic effects. Eur. J. Pharmacol. 2018, 824, 78–88. [CrossRef] [PubMed]
27. Musso, L.; Dallavalle, S.; Zunino, F. Perspectives in the development of hybrid bifunctional antitumour
agents. Biochem. Pharmacol. 2015, 96, 297–305. [CrossRef] [PubMed]
28. Singh, R.K.; Mandal, T.; Balasubramanian, N.; Cook, G.; Srivastava, D.K. Coumarin-suberoylanilide
hydroxamic acid as a fluorescent probe for determining binding affinities and off-rates of histone deacetylase
inhibitors. Anal. Biochem. 2011, 408, 309–315. [CrossRef] [PubMed]
29. Seidel, C.; Schnekenburger, M.; Zwergel, C.; Gaascht, F.; Mai, A.; Dicato, M.; Kirsch, G.; Valente, S.;
Diederich, M. Novel inhibitors of human histone deacetylases: Design, synthesis and bioactivity of
3-alkenoylcoumarines. Bioorganic Med. Chem. Lett. 2014, 24, 3797–3801. [CrossRef] [PubMed]
30. Abdizadeh, T.; Kalani, M.R.; Abnous, K.; Tayarani-Najaran, Z.; Khashyarmanesh, B.Z.; Abdizadeh, R.;
Ghodsi, R.; Hadizadeh, F. Design, synthesis and biological evaluation of novel coumarin-based benzamides
as potent histone deacetylase inhibitors and anticancer agents. Eur. J. Med. Chem. 2017, 132, 42–62. [CrossRef]
[PubMed]
31. Yang, L.; Hu, Z.; Luo, J.; Tang, C.; Zhang, S.; Ning, W.; Dong, C.; Huang, J.; Liu, X.; Zhou, H.-B. Dual functional
small molecule fluorescent probes for image-guided estrogen receptor-specific targeting coupled potent
antiproliferative potency for breast cancer therapy. Bioorganic Med. Chem. 2017, 25, 3531–3539. [CrossRef]
[PubMed]
32. Ahmad, M.; Aga, M.A.; Bhat, J.A.; Kumar, B.; Rouf, A.; Capalash, N.; Mintoo, M.J.; Kumar, A.; Mahajan, P.;
Mondhe, D.M.; et al. Exploring Derivatives of Quinazoline Alkaloid L-Vasicine as Cap Groups in the Design
and Biological Mechanistic Evaluation of Novel Antitumor Histone Deacetylase Inhibitors. J. Med. Chem.
2017, 60, 3484–3497. [CrossRef] [PubMed]
33. Peng, F.-W.; Xuan, J.; Wu, T.-T.; Xue, J.-Y.; Ren, Z.-W.; Liu, D.-K.; Wang, X.-Q.; Chen, X.-H.; Zhang, J.-W.;
Xu, Y.-G.; et al. Design, synthesis and biological evaluation of N-phenylquinazolin-4-amine hybrids as dual
inhibitors of VEGFR-2 and HDAC. Eur. J. Med. Chem. 2016, 109, 1–12. [CrossRef] [PubMed]
34. Perez-Cruz, F.; Vazquez-Rodriguez, S.; Joao Matos, M.; Herrera-Morales, A.; Villamena, F.A.; Das, A.;
Gopalakrishnan, B.; Olea-Azar, C.; Santana, L.; Uriarte, E. Synthesis and Electrochemical and Biological
Studies of Novel Coumarin- Chalcone Hybrid Compounds. J. Med. Chem. 2013, 56, 6136–6145. [CrossRef]
[PubMed]
35. Li, J.J.; Holsworth, D.D.; Hu, L.-Y. Molecular properties that influence the oral bioavailability of drug
candidates. Chemtracts 2003, 16, 439–442.
36. Morris, G.M.; Huey, R.; Lindstrom, W.; Sanner, M.F.; Belew, R.K.; Goodsell, D.S.; Olson, A.J. AutoDock4 and
AutoDockTools4: Automated docking with selective receptor flexibility. J. Comput Chem 2009, 30, 2785–2791.
[CrossRef] [PubMed]
37. Zhang, H.; Yang, F.; Shan, P.; Zhao, N.; Jiang, C.; Song, J.; Ge, D.; Han, L. Preparing method and application
of iso-hydroxamic acid compound containing coumarin structure. CN108658915A, 2018.
38. Li, R.; Wei, J.; Jiang, C.; Liu, D.; Deng, L.; Zhang, K.; Wang, P. Akt SUMOylation Regulates Cell Proliferation
and Tumorigenesis. Cancer Res. 2013, 73, 5742–5753. [CrossRef] [PubMed]

Molecules 2019, 24, 2569
15 of 15
39. Wang, C.; Ivanov, A.; Chen, L.; Fredericks, W.J.; Seto, E.; Rauscher, F.J.; Chen, J. MDM2 interaction with
nuclear corepressor KAP1 contributes to p53 inactivation. Embo J. 2005, 24, 3279–3290. [CrossRef] [PubMed]
Sample Availability: Samples of the compounds 10a–e, 11a–e and 12a,b are available from the authors.
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