New histone deacetylase inhibitors and anticancer agents from Curcuma longa

Article abstract of DOI:10.1007/s00044-019-02414-5

The aims of this study were to explore histone deacetylase inhibitory and antioxidant activities of curcuminoids as well as derivatives of curcumin. Curcumin (6), demethoxycurcumin (7), dihydrocurcumin (8), bisdemethoxycurcumin (9), and hydroxycurcumin (1

Full text of DOI:10.1007/s00044-019-02414-5

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02414-5
ORIGINAL RESEARCH
New histone deacetylase inhibitors and anticancer agents from
Curcuma longa
1 ●
2 ●
2 ●
2 ●
1 ●
Pakit Kumboonma Thanaset Senawong Somprasong Saenglee Gulsiri Senawong La-or Somsakeesit
Chavi Yenjai1 Chanokbhorn Phaosiri
1
●
Received: 30 May 2019 / Accepted: 23 July 2019
©
Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
The aims of this study were to explore histone deacetylase inhibitory and antioxidant activities of curcuminoids as well as
derivatives of curcumin. Curcumin (6), demethoxycurcumin (7), dihydrocurcumin (8), bisdemethoxycurcumin (9), and
hydroxycurcumin (10) were isolated and tested against histone deacetylases in HeLa nuclear extract. Hydroxycurcumin (10)
showed the best inhibition among the isolated compounds. Some curcumin derivatives were also prepared and tested. The
potential derivatives were tested on five cancer cell lines. All compounds exhibited slightly weaker antiproliferative activities
against cancer cells and less toxic to non-cancer cells than curcumin (6). The least toxic derivative (17) exhibited the best
antiproliferative activity against human cervical cancer cell lines (HeLa) with the IC50 value of 4.69 ± 0.14 μM. The most
active histone deacetylase inhibitor (19) showed the highest potency against human colon cancer cell lines (HCT116) and the
selective binding to HDAC4 based on molecular docking experiments. Most derivatives possessed antioxidant activities
superior to curcumin. The results suggested potential candidates for anticancer agents.
●
●
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Keywords Turmeric Curcumin HeLa cell Anticancer Molecular docking
Introduction
differentiation, apoptosis, and tumorigenesis. Therefore,
HDACs have been validated as prominent therapeutic tar-
gets for a broad range of human disorders such as cancer,
diabetes, and brain disorders (Paris et al. 2008; Noh et al.
2009; Bertrand 2010; Manal et al. 2016). In humans,
HDACs are divided into four classes based on their
homology to yeast models, subcellular localization and
enzymatic activities. Eighteen mammalian isoforms of
HDACs have been reported (Micelli, Rastelli 2015). Class I
Acetylation and deacetylation of histone proteins play
important roles in the epigenetic regulation of transcription
in cells. Many disorders and metabolic diseases occur by
perturbation of the balance between histone acetyl-
transferases (HATs) and histone deacetylases (HDACs)
(
Konstantinopoulos et al. 2007; Weichert 2009). HDACs
have an important effect on gene transcription,
(HDAC1, 2, 3, and 8), class IIa (HDAC4, 5, 7, and 9), class
IIb (HDAC6 and 10) and class IV (HDAC11) HDACs are
zinc-ion-dependent metallo proteins. Class III (SIRT1-7)
HDACs are zinc-ion-independent and NAD -dependent
proteins. The different isoforms are mainly found in diverse
diseases such as cancer cells.
HDAC inhibitors (HDACi) have been reported and
categorized into five groups according to their chemical
structures including hydroxamic acid derivatives, cyclic
tetrapeptides, benzamides, electrophilic ketones, and car-
boxylic acids (Miller et al. 2003; Zhang et al. 2012; Ravi-
chandiran et al. 2015). In spite of the diverse structures, the
pharmacophore of most HDAC inhibitors consists of three
parts including a zinc-binding group, a hydrophobic linker,
and a surface recognition area. Up to now, the hydroxamic
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-019-02414-5) contains supplementary
material, which is available to authorized users.
+
*
Chanokbhorn Phaosiri
chapha@kku.ac.th
1
Natural Products Research Unit, Center of Excellence for
Innovation in Chemistry, Ministry of Higher Education, Science,
Research and Innovation (Implementation Unit-IU, Khon Kaen
University), Department of Chemistry, Faculty of Science, Khon
Kaen University, Khon Kaen 40002, Thailand
2
Natural Products Research Unit, Department of Biochemistry,
Faculty of Science, Khon Kaen University, Khon Kaen 40002,
Thailand

Medicinal Chemistry Research
acids have been reported to be the most potent group of
HDAC inhibitors of which suberoylanilide hydroxamic acid
Turmeric (Curcuma longa) is a rhizomatous perennial
plant of the ginger family. The rhizome of C. longa has
been used in indigenous medicine for the treatment of
inflammatory disorders. The major product from C. longa,
curcumin (6) is reported to have many interesting biological
activities including antioxidant (Ruby et al. 1995; Selvam
et al. 2005), antimycobacterial (Changtam et al. 2010), anti-
inflammatory (Chan et al. 1995), anticancer (Inano et al.
2000; Thapliyal, Maru 2001), anti-acetylcholinesterase
(Premanand et al. 2009), and anti-HIV activities (Mazum-
der et al. 1995). However, several drawbacks of curcumin
come from low aqueous solubility, low absorption, and poor
bioavailability (Anand et al. 2008).Various structural
modification of curcumin have been carried out in order to
improve its bioavailability and activities (Khalil et al. 2014;
Ahsan et al. 2015; Sharma et al. 2015; Puneeth et al. 2016).
Curcumin can act as a good HDAC inhibitor with the IC50
value as 115 µM (Tatar et al. 2009). Nevertheless, there was
no report regarding HDAC inhibitory activities of curcumin
derivatives.
(
1, SAHA, Vorinostat), Belinostat, Chidamide and Pano-
binostat approved by the FDA for the treatment of lym-
phoma or myeloma (Mann et al. 2007; Banerjee et al.
2
019). However, the highly potent hydroxamic compounds
typically possess serious toxicities and poor metabolic sta-
bilities (Hahnen et al. 2008; Suzuki and Miyata 2005).
Moreover, these compounds also nonspecifically inhibit all
HDAC isoforms. All of these broad spectrum HDAC
inhibitors cause similar adverse effects such as fatigue,
nausea, and vomiting (Ontoria et al. 2009). Therefore,
development of novel HDAC inhibitors with safe, isoform-
selective and low toxicity still remain as a challenge
research. A variety of non-hydroxamic acids, for examples,
hydroxycapsaicin (2), [6]-gingerol (3), [6]-shogaol (4),
demethylated-[6]-shogaol (5), and curcumin (6) have been
investigated as HDAC inhibitors as shown in Figs 1 and 2
(
Senawong et al. 2015; Kumboonma et al. 2017; Tatar et al.
009). Even though their HDAC inhibitory activities are not
comparable to those of SAHA (1, IC = 110–370 nM)
2
In our previous report, the [6]-shogaol (4) and its deri-
vatives showed good HDAC inhibitory activities (Kum-
boonma et al. 2017). From the similar structure of [6]-
shogaol (4) to curcumin (6), the semi-synthetic derivatives
50
(
Gopalan et al. 2013), one of these compounds, hydro-
xycapsaicin (2) is accepted as a good inhibitor with low
toxicities towards normal cells.
Fig. 1 Structures and IC50 values of known histone deacetylase inhibitors
Fig. 2 Structures of minor
phenolic compounds from
Curcuma longa
Demethoxycurcumin (7)
Dihydrocurcumin (8)
Hydroxycurcumin (10)
Bisdemethoxycurcumin (9)

Medicinal Chemistry Research
Scheme 1 Synthesis of isoxazole, pyrazole, and N-(substituted) pyr-
azole derivatives of curcumin. Reagents and conditions: (I)
NH2OH·HCl, reflux, 6 h; (II) hydrazine hydrate, reflux, 8 h; (III) 4-
ydrazinobenzoic acid hydrochloride, reflux, 8 h; (IV) 2-hydrazino
benzothiazole, reflux, 8 h; and (V) 2,4-dinitrophenylhydrazine reflux,
8 h
Scheme 2 Synthesis of amino derivatives of curcumin. Reagents and conditions: (I) NH2OCH3·HCl, reflux, 6 h; (II) 2-aminothiophenol, reflux,
h; (III) o-phenylenediamine, reflux, 6 h; and (IV) aniline reflux, 6 h
6
Table 1 HDAC inhibitory activity of compounds at 100 µM
of curcumin (6) were further investigated in this work.
Molecular docking study, antiproliferative and antioxidant
activities of all compounds were also carried out to extend
the therapeutic potential.
Compounds %HDAC inhibition Compounds %HDAC inhibition
6
7
8
9
1
1
1
62
65
61
67
76
54
51
13
14
15
16
17
18
19
64
67
65
72
73
69
82
Results and discussion
0
1
2
Curcumin (6) as the major product along with four minor
phenolic compounds were extracted from the dried rhizome
powder of turmeric. The isoxazole and pyrazole derivatives
of curcumin were synthesized as depicted in Scheme 1. The
amino derivatives of curcumin were prepared according to
Scheme 2 by reacting curcumin (6) with methylamine
hydrochloride, 2-aminothiophenol, o-phenylenediamine,
and aniline to yield amino derivatives 16–19, respectively.
All compounds were initially screened at 100 μM, using the
™
Fluor-de-Lyse in vitro fluorescence activity assay kit and
measuring total HDAC activity in HeLa nuclear extract.
The results are illustrated in Table 1. Among the natural

Medicinal Chemistry Research
products, hydroxycurcumin (10) possessed the best %
HDAC inhibition value of 76%. Incorporation of functional
groups with strong interactions such as hydrogen bond, π–π,
and hydrophilic interactions can increase the binding affi-
nity between inhibitor and enzyme. Therefore, the iso-
xazole, pyrazole, and amino derivatives of curcumin were
designed and synthesized to improve the inhibitor-enzyme
binding efficiency via additional hydrogen bonds and
coordination to the zinc ion. No significant improvement
was observed from the isoxazole derivative 11 and the
pyrazole derivative 12. However, the pyrazole derivatives
with N-aromatic substitution 13–15 led to a moderately
increased activity as compared to curcumin, whereas the
amino derivatives of curcumin 16–19 showed a sig-
nificantly increased inhibition. The most active compound
among the amino derivatives of curcumin was 19 which
contained a phenylamino group at the Michael acceptor
position of curcumin with %HDAC inhibition of 82%.
In order to rationalize the in vitro results and to explore a
possibility of being HDAC isoform-selective inhibitors, the
selected compounds, curcumin (6), hydroxycurcumin (10),
and 19 were docked into the catalytic pockets of the
representative isoform of class I (HDAC2 and HDAC8) and
Class II (HDAC4 and HDAC7). The in silico results are
summarized in Table 2. The molecular docking method was
validated in all isoforms through the re-docking of SAHA
and TSA within the protein structures. In comparison to
curcumin (6), hydroxycurcumin (10) possessed the lowest
inhibition constant against HDAC2 (K of 10 = 0.98 µM). In
i
addition to coordination towards the zinc ion, the hydro-
philic interaction occurs between the methoxy group of 10
and the carbonyl group of Gly142 as shown in Fig. 3a.
Another binding is the close π–π stacking interaction
between the phenyl ring of 10 and Phe210. The previous
study indicated that HDAC2 are overexpressed in the cortex
and hippocampus of Alzheimer’s disease patients (Hu et al.
2018). Therefore, hydroxycurcumin (10) can be an attrac-
tive candidate for the treatment of neurodegenerative dis-
eases. Compound 19 showed the highest selectivity against
HDAC4 (K of 19 = 2.11 µM). The binding mode of 19 in
i
the HDAC4 binding cavity is depicted in Fig. 3b. The major
interaction between HDAC4 and compound 19 is the
coordination of the hydroxy group to the zinc ion with a
distance of 3.03 Å. Moreover, compound 19 binds to
HDAC4 via three hydrogen bonds. The hydrogen bonds
occur between the hydroxy group of 19 and the amino
group of Lys20 (2.16 Å) as well as the hydroxy group of 19
and the amino group of Gly331 (1.84 Å). In addition, the
carbonyl group of 19 can form another hydrogen bond with
the –NH belonging to the imidazole moiety of His198
Table 2 In silico histone deacetylase inhibitory activity of the selected compounds
Compounds
Class I HDACs
HDAC2
Class II HDACs
HDAC4
HDAC8
HDAC7
ΔG (kcal/mol)
Ki (μM)
ΔG (kcal/mol)
Ki
ΔG (kcal/mol)
Ki (μM)
ΔG (kcal/mol)
Ki (μM)
SAHA
TSA
6
−9.38
−0.75
−0.85
−0.19
−0.4
0.13
0.39
−0.74
−0.85
−0.46
−0.95
−0.37
1.78
−0.51
−0.39
−0.80
−0.62
−0.74
3.14
0.702
1.91
−0.70
−0.97
−0.79
−7.61
−0.93
12.36
1.44
0.326
18.38
8.01
9.54
10.63
2.64
1
1
0
9
0.98
13.99
2.11
20.23
3.97
8.26
Fig. 3 The interaction modes of
a 10 in the active site cavity of
HDAC2 and b 19 in the active
site cavity of HDAC4
A
B
PHE210
HIS198
Zn
2
.99
3
.03
Zn
1
.87
1.84
GLY331
(19)
(
10)
2
.16
3.16
LYS20
GLY142
HDAC2
HDAC4

Medicinal Chemistry Research
Table 3 In vitro antioxidant activities of the compounds
Compounds DPPHa RPb TAb Compounds DPPHa RPb TAb
6
7
8
9
1
1
1
a
6
11 43
16 45
12 46
25 60
12 52
22 51
17 30
13
14
15
16
17
18
19
5
10 28
30 28
22 35
15 54
15 38
10 39
14 42
11
7
8
7
11
24
7
14
6
0
1
2
6
5
7
b
IC50 (μM) EC50 (μM)
(
2.99 Å). The π–π interaction can also be observed between
the imidazole of His198 and the newly inserted amino-
phenyl group of 19. The histidine residues are important in
the mechanism of histone deacetylation as the zinc-ion
chelator. They also catalyze deprotonation of water mole-
cules to generate nucleophiles which can attack the acety-
lated lysine residues of histones as proposed (Bertrand
2
010; Manal et al. 2016). Therefore, the HDAC inhibitors
which can form strong interactions with the histone residues
will definitely reduce the binding affinity between co-factor,
natural substrate and enzymes. The class IIa HDAC inhi-
bitors are considered as a potential therapy for Huntington’s
disease (Bürli et al. 2013), so the HDAC4 selective inhi-
bitor, compound 19, should be further investigated. The
progression of multiple neurodegenerative diseases such as
Alzheimer’s and Huntington’s diseases relates to oxidative
stress which causes by the increased production of reactive
oxygen species (Tӧnnies and Trushina 2017). To explore
the antioxidative potency of the obtained HDAC inhibitors,
the antioxidant activities were tested and compound 19
appeared to be a good antioxidant as displayed in Table 3.
Finally, the MTT assay was carried out to gain more
details regarding the anticancer activities of the potent
HDAC inhibitors. The results in Tables 4 and 5 indicate that
the amino derivatives of curcumin 16–19 were less toxic to
non-cancer cells than the lead compound, even though
curcumin (6) was the most active against all cancer cells.
Compound 17, the least toxic compound, showed the best
activity against HeLa cells among the synthesized deriva-
tives. Moreover, compound 17 appeared to be about 16-fold
more selective towards the cancer cells than the non-cancer
cells, whereas curcumin (6) exhibited only three-fold
selectivity. Compounds 18 and 19 were the most potent
derivatives against MCF-7 and HCT116 cells, respectively.
Additionally, compound 18 displayed the strongest anti-
proliferative activity against the cholangiocarcinoma cells
as shown in Table 5. The least toxic compound against non-
cancer human bile-duct epithelial cells, compound 19,
exhibited antiproliferative activity against cholangiocarci-
noma cells without selectivity.

Medicinal Chemistry Research
Table 5 Antiproliferative activities of the potent HDAC inhibitors against cholangiocarcinoma cell lines
Cpd IC50 values (mean ± SD; n = 3 (μg/mL)
H-69 cells
KKU-100 cells
24 h
KKU-M214 cells
24 h 48 h
2
4 h
48 h
2.54 ± 0.09
72 h
48 h
3.28 ± 0.37
72 h
2.88 ± 0.14
72 h
3.64 ± 0.20
6
1
1
1
1
4.59 ± 0.29
2.37 ± 0.14 27.79 ± 0.93
6.95 ± 1.61
3.80 ± 0.06
6
7
8
9
22.98 ± 5.31 12.96 ± 2.02
9.94 ± 0.44 43.17 ± 6.56 14.29 ± 0.70 10.53 ± 1.02 28.04 ± 2.96 21.72 ± 1.82 14.55 ± 0.55
12.54 ± 3.21
15.43 ± 1.30
8.92 ± 0.81
8.13 ± 0.06
6.39 ± 1.13
> 100
17.15 ± 1.19 10.55 ± 0.27 33.96 ± 2.26
6.63 ± 0.81 5.50 ± 0.12 26.94 ± 0.70
8.63 ± 0.35
6.84 ± 1.08
7.00 ± 0.47
5.50 ± 0.37
6.62 ± 0.29 80.33 ± 9.66
23.58 ± 0.56 19.49 ± 0.61 17.37 ± 0.25 42.15 ± 0.59 19.93 ± 0.29 17.96 ± 0.42 37.71 ± 1.00 19.55 ± 1.76 17.39 ± 0.33
Numerous analogs of curcumin have been developed for
One FT-IR spectrophotometer. Mass spectra were deter-
mined using a Micromass Q-TOF 2 hybrid quadrupole
time-of-flight (Q-TOF) mass spectrometer with a Z-spray
ES source.
therapeutic purposes especially modification around
β-diketo scaffold. The replacement of β-diketo unit in cur-
cumin by isoxazole and pyrazole moieties can enhance
some biological activities and maintain their low toxicity.
However, there was unprecedented modification at the α,
β-unsaturated carbonyl group. Our newly synthesized
Michael-adduct derivatives of curcumin (16–19) performed
stronger HDAC inhibitory activity than curcumin (6), the
isoxazole derivative (11) and the pyrazole derivatives (12–
Plant material
The dried rhizome powder of turmeric was obtained from
the herbal drugstore at local market in Khon Kaen province,
Thailand. The powder was confirmed by a pharmacist as C.
longa.
1
5). Variety of nucleophiles could be introduced into the
Micael-acceptor curcumin in order to improve the inhibitor-
enzyme binding. This finding suggests another simple
approach to enhance biological activity for chemother-
apeutic application of a golden spice from mother nature.
Extraction and isolation
Six hundred grams of plant material were extracted three
times (3 × 1000 mL) with dichloromethane (CH Cl ). The
2
2
combinded extracts were evaporated to dryness under
vacuum to give the dichloromethane extract (90 g). The
dichloromethane extract was further isolated with normal
phase silica gel column chromatography and subsequently
eluted with three solvents (hexane, ethyl acetate (EtOAc),
and methanol (MeOH)) by gradually increasing polarity of
elution solvent system. The eluents were collected and
monitored by TLC to afford four fractions (DT –DT ). The
Conclusion
C. longa could serve as a great source of HDAC inhibitors
with partial isoform-selectivity. The core-structure mod-
ification of the natural compound, curcumin (6) provided
the series of derivatives with improved activities. The
cytotoxic activity of the active HDAC inhibitors might not
increase dramatically compared to the lead compound;
however, the non-toxicity of those compounds was pro-
mising. Therefore, the in vivo experiments of the potent
HDAC inhibitors will be further investigated.
1
4
fraction DT was an oil. The fraction DT was resolved by
1
2
flash silica gel column chromatography, using a gradient of
hexane-EtOAc (10:0 to 5:5) to provide three subfractions
(DT2–1 to DT2–3). The subfraction DT2–1 and DT2–2 were
purified by preparative thin layer chromatography (hexane-
EtOAC, 9:1) to obtain demethoxycurcumin (7, 0.05 g,
0.0083%) and dihydrocurcumin (8, 0.04 g, 0.00067%) as a
yellow solid. The fraction DT2–3 was subjected to normal
phase silica gel column chromatography, eluting with an
isocratic of CH Cl -MeOH (9:1) to afford curcumin (6,
Experimental
Reagents were purchased from commercial sources (Sigma-
Aldrich, Merck, and Carlo Erba). Reactions were monitored
using analytical TLC plates (Merck, silica gel 60 F254) and
compounds were visualized under ultraviolet light. Silica
gel grade 60 (230–400 mesh; Merck) was used for column
chromatography. The NMR spectra were recorded in the
individually indicated solvents on a Varian Mercury Plus
2
2
20 g, 3.33%) as an orange solid. The fraction DT was
3
further subjected to a normal phase silica gel column,
eluting with a step gradient of CH Cl -MeOH (10:0 to 7:3)
2
2
to yield two subfractions (DT3–1 and DT3–2). The subfrac-
tion DT3–2 was subjected to a normal phase column chro-
matography, eluting with an isocratic of CH Cl -MeOH
1
13
spectrometer operated at 400 MHz ( H) or 100 MHz ( C).
The IR spectra were obtained on Perkin Elmer Spectrum
2
2
(9:1) to produce bisdemethoxycurcumin (9, 1.0 g, 0.167%).

Medicinal Chemistry Research
The fraction DT was isolated with a normal phase silica gel
(4Z,6E)-5-Hydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)-
1-((2-mercaptophenyl)amino)hepta-4,6-dien-3-one (17): 82%.
R = 0.50 (5% MeOH in CH Cl ). IR (neat) υ 3288 (OH),
4
column, eluting with a step gradient of CH Cl -MeOH
2
2
(
10:0 to 7:3) to yield two subfractions (DT4–1 and DT4–2).
f
2
2
max
−1 1
The subfraction DT4–1 and DT4–2 were further purified by
1738 (C = O), 1560 (Ar), 1508 (Ar), 1265 (C–O) cm . H
preparative thin layer chromatography (CH Cl -MeOH,
NMR (CDCl , 400 MHz) δ 7.54 (dd, Ј = 2.0, 8.0 Hz, 1H,
2
2
3
9
:1) to gain hydroxycurcumin (10, 0.012 g, 0.002%). The
aromatic), 7.39 (m, 2H, aromatic), 7.14 (m, 3H, aromatic),
7.01 (dd, Ј = 2.0, 8.0 Hz, 1H, aromatic), 6.90 (s, 1H, aro-
matic), 6.76 (d, Ј = 8.0 Hz, 1H, aromatic), 6.69 (s, 2H, aro-
matic), 6.63 (d, Ј = 12.0 Hz, 1H ), 5.48 (s, 1H ), 4.71 (dd,
NMR data of all compounds were consistent with the pre-
vious report (Venkateswarlu et al. 2005).
6
4
Structural modifications
Ј = 4.0, 8.0 Hz, 1H ), 3.83 (s, OCH ), 3.72 (s, OCH ), 2.75
1 3 3
(
dd, Ј = 4.0, 12.0 Hz, 1H ), 2.61 (dd, Ј = 8.0, 12.0 Hz, 1H ).
2
2
1
3
To a solution of curcumin (6) (105 mg, 0.29 mmol) in
C NMR (CDCl , 100 MHz) δ 188.55 (C-3), 161.57 (C-5),
3
ethanol (5 mL) was added NH OH·HCl (40 mg, 0.57 mmol)
149.78 (C-3ʹʹ), 148.22 (C-4ʹʹ), 147.47 (C-3ʹ), 145.96 (C-4ʹ),
141.75 (C-1ʹʹʹ), 139.95 (C-7), 135.02 (C-1ʹʹ), 134.98 (C-1ʹ),
129.94 (C-3ʹʹʹ), 126.77 (C-6ʹʹʹ), 126.74 (C-5ʹʹʹ), 125.93 (C-6ʹʹ),
124.43 (C-6ʹ), 123.17 (C-4ʹʹʹ), 122.53 (C-5ʹʹ), 119.03 (C-5ʹ),
115.42 (C-2ʹʹʹ), 114.66 (C-2ʹʹ), 110.26 (C-2ʹ), 110.12 (C-6),
2
at room temperature. The mixture was refluxed for 6 h.
After completion based on TLC, the mixture was filtered
and the residue was washed with ethanol (10 mL) and dried
with anhydrous sodium sulfate. Evaporation of the com-
bined solvents gave a crude product. Purification of the
crude product by column chromatography (5% MeOH in
CH Cl ) gave a yellow solid of compound 11. The same
98.00 (C-4), 55.01 (C-1), 54.92 (OCH ), 40.15 (C-2). HRMS-
3
+
ESI (m/z) [M –H O + H] calcd for C H NO S 476.1531,
2
27 26
5
found 476.1574.
2
2
procedure was applied with the corresponding reagents to
convert curcumin (6) into 12–15. The NMR of all synthe-
sized compounds were in consistency with the earlier
reports (Mishra et al. 2008; Sahu et al. 2012; Jha et al. 2015;
Jordan et al. 2018).
(4Z,6E)-1-((2-Aminophenyl)amino)-5-hydroxy-1,7-bis(4-
hydroxy-3-methoxyphenyl)hepta-4,6-dien-3-one (18): 85%.
R = 0.55 (5% MeOH in CH Cl ). IR (neat) υ 3296 (OH),
f
2
2
max
−1 1
1738 (C = O), 1571 (Ar), 1508 (Ar), 1270 (C–O) cm . H
NMR (CDCl , 400 MHz) δ 7.36 (d, Ј = 16.0 Hz, 1H ), 7.15
3
7
To a solution of curcumin (6) (100 mg, 0.27 mmol) in
ethanol (5 mL) was added NH OCH ·HCl (34 mg,
(d, Ј = 2.0 Hz, 1H, aromatic), 7.02 (m, 4H, aromatic), 6.95 (d,
Ј = 8.0 Hz, 1H, aromatic), 6.89 (m, 1H, aromatic), 6.76 (m,
4H, aromatic), 6.61 (d, Ј = 16.0 Hz, 1H ), 3.88 (s, OCH ),
2
3
0.41 mmol) at room temperature. The mixture was refluxed
6
3
for 6 h. After completion based on TLC, the mixture was
filtered and the residue was washed with ethanol (10 mL) and
dried with anhydrous sodium sulfate. Evaporation of the
combined solvents gave a crude product. Purification of the
crude product by column chromatography (5% MeOH in
CH Cl ) gave a yellow solid of compound 16. The same
3.79 (s, OCH ), 2.78 (dd, Ј = 4.0, 8.0 Hz, 1H ), 2.67 (dd, Ј =
3
2
1
3
4.0, 16.0 Hz, 1H2). C NMR (CDCl , 100 MHz) δ 187.15
3
(C-3), 162.36 (C-5), 149.22 (C-3ʹʹ), 148.12 (C-4ʹʹ), 147.53 (C-
3ʹ), 145.75 (C-4ʹ), 139.83 (C-7), 138.85 (C-1ʹ), 136.19 (C-
1ʹʹ& C-2ʹʹʹ), 127.94 (C-1ʹʹʹ), 127.12 (C-6ʹʹ), 125.74 (C-6ʹ),
124.65 (C-5ʹʹʹ), 122.34 (C-4ʹʹʹ), 122.27 (C-6ʹʹʹ), 120.45 (C-5ʹʹ),
120.33 (C-5ʹ), 118.56 (C-2ʹ), 115.25 (C-2ʹʹ), 114.63 (C-3ʹʹʹ),
2
2
procedure was conducted with 2-aminothiophenol, o-pheny-
lenediamine, and aniline to convert curcumin (6) into 17–19.
110.08 (C-6), 109.73 (C-4), 64.97 (C-1), 54.98 (OCH ),
3
+
(
4Z,6E)-5-Hydroxy-1,7-bis(4-hydroxy-3-methox-
yphenyl)-1-(methoxyamino)hepta-4,6-dien-3-one (16): 80%.
R = 0.70 (10% MeOH in CH Cl ). IR (neat) υ 3272
40.44 (C-2). HRMS-ESI (m/z) [M –H O + H] calcd for
2
C H N O 459.1920, found 459.1970.
2
7 27 2 5
(4Z,6E)-5-Hydroxy-1,7-bis(4-hydroxy-3-methox-
yphenyl)-1-(phenylamino)hepta-4,6-dien-3-one (19): 80%.
R = 0.40 (5% MeOH in CH Cl ). IR (neat) υ 3355
f
2
2
max
(
OH), 1734 (C = O), 1583 (Ar), 1511 (Ar), 1273 (C–O) cm
−
1 1
.
H NMR (CDCl , 400 MHz) δ 7.16 (d, Ј = 16.0 Hz, 1H ),
3
7
f
2
2
max
7
6
8
2
1
1
1
1
1
6
.05 (dd, Ј = 2.0, 8.0 Hz, 1H ), 6.93 (m, 5H, aromatic),
(OH), 1736 (C = O), 1509 (Ar), 1457 (Ar), 1271 (C–O) cm
2ʹʹ
−
1 1
.74 (d, Ј = 16.0 Hz, 1H ), 5.58 (s, 1H ), 4.68 (dd, Ј = 4.0,
. H NMR (CDCl , 400 MHz) δ 7.27 (m, 5H, aromatic),
6
4
3
.0 Hz, 1H ), 3.91 (s, OCH ), 3.90 (s, OCH ), 3.40 (s, 3H ),
7.10 (d, Ј = 8.0 Hz, 2H, aromatic), 6.79 (m, 3H, aromatic),
1
3
3
1ʹʹʹ
.90 (dd, Ј = 16.0, 16.0 Hz, 1H ), 2.73 (dd, Ј = 4.0,
6.74 (m, 2H, aromatic), 6.21 (d, Ј = 16.0 Hz, 1H ), 5.63
2
6
1
3
6.0 Hz, 1H2). C NMR (CDCl , 100 MHz) δ 192.02 (C-3),
(s, 1H ), 5.09 (dd, Ј = 4.0, 8.0 Hz, 1H ), 3.77 (s, OCH ),
3
4
1
3
64.70 (C-5), 147.34 (C-3ʹʹ), 146.83 (C-4ʹʹ), 146.47 (C-3ʹ),
45.84 (C-4ʹ), 137.52 (C-7), 129.52 (C-1ʹʹ), 128.23 (C-1ʹ),
21.68 (C-6ʹʹ), 121.27 (C-6ʹ), 117.35 (C-5ʹʹ), 114.89 (C-5ʹ),
14.38 (C-2ʹʹ), 110.68 (C-2ʹ), 109.56 (C-6), 100.47 (C-4),
3.25 (dd, Ј = 8.0, 16.0 Hz, 1H ), 2.74 (dd, Ј = 4.0, 16.0 Hz,
2
1
3
1H2). C NMR (CDCl , 100 MHz) δ 191.55 (C-3), 161.32
3
(C-5), 148.39 (C-1ʹʹʹ), 147.79 (C-3ʹʹ), 147.72 (C-4ʹʹ), 145.87
(C-3ʹ), 143.63 (C-4ʹ), 138.59 (C-7), 130.16 (C-1ʹʹ), 129.08
(C-1ʹ), 127.33 (C-6ʹʹ), 126.82 (C-6ʹ), 125.98 (C-3ʹʹʹ), 121.56
(C-4ʹʹʹ), 119.58 (C-5ʹʹ), 119.40 (C-5ʹ), 115.33 (C-2ʹʹʹ),
115.06 (C-2ʹʹ), 110.26 (C-2ʹ), 110.09 (C-6), 97.71 (C-4),
7.33 (C-1), 63.30 (C-1ʹʹʹ), 56.00 (OCH ), 44.95 (C-2).
3
+
HRMS-ESI (m/z) [M –H O + H] calcd for C H NO
2
22 24
6
3
98.1603, found 398.1617.

Medicinal Chemistry Research
6
5.03 (C-1), 55.41 (OCH ), 42.45 (C-2). HRMS-ESI (m/z)
chain atoms were added using the ADT program. Gasteiger
charges were calculated for the system. For ligand setup, the
molecular modeling program Hyperchem 8.0 was used to
build the ligands. These ligands were optimized with the
AM1 level. Molecular docking studies were performed for
3
+
[
M –H O + H] calcd for C H NO 444.1811, found
2
27 26
5
4
44.1870.
HDAC activity assay
50 runs using AutoDockTools 1.5.4 (ADT) and AutoDock
Natural compounds and the semi-synthetic derivatives were
evaluated for their ability to inhibit HDAC enzymes. Inhi-
bition of HDAC activity in vitro was assessed using the
Fluor-de-Lys HDAC activity assay kit (Biomol, Enzo Life
Sciences International, Inc., USA). The HeLa nuclear
extract provided with the kit was used as a source of HDAC
enzymes for in vitro study. TSA was used as the positive
control. The HeLa nuclear extract, substrate, buffer, and
inhibitors were incubated. Deacetylation of the substrate
followed with adding the developer generated a fluor-
ophore. The spectra Max Gemini XPS microplate spectro-
fluorometer (Molecular Devices, USA) was used to measure
fluorescence signal with excitation at 360 nm and emission
at 460 nm. A decrease in fluorescence signal indicated an
inhibition of HDAC activity. All experiments were carried
out in triplicate.
4.2 programs and Lamarckian genetic algorithm search
(Sanner 1999). A grid box size of 60 ×60 × 60 points with a
spacing of 0.375 Å between the grid points was imple-
mented and covered almost the entire HDAC protein sur-
face. For TSA and other inhibitors, the single bonds were
treated as active torsional bonds.
DPPH radical scavenging
The antioxidant activity of the obtained compounds were
measured in terms of hydrogen-donating or radical
scavenging ability, using the DPPH method (Kumboonma
et al. 2017). Gallic acid was used as a positive control.
Different concentrations (1 mL) of the above compounds
were mixed with 3 mL DPPH radical solution in methanol
−
4
(1.0 × 10 M). The final concentration of DPPH reached
4
0
.6 × 10− M with methanol. The reaction mixtures were
MTT assay
shaken and incubated at room temperature for 30 min. At
the same time, the control was prepared as mentioned above
without any compounds. The absorbance of the solution
was measured at 517 nm. Radical scavenging activity was
expressed as the inhibition percentage and was calculated
via the following formula:
The MTT reduction assay was performed with non-cancer
(
(
Vero), human cervical cancer (HeLa), human colon cancer
HCT116), human breast adenocarcinoma cancer (MCF-7),
non-cancer human bile-duct epithelial (H-69), and cho-
langiocarcinoma (KKU-100 and KKU-M214) cell lines
according to the method previously described (Senawong
et al. 2015; Asgar et al. 2016; Kumnerdkhonkaen et al.
%
Radical scavenging activity
ÂÀ
Á
Ã
¼
Abscontrol À Abssample =Abscontrol  100:
2
018). Briefly, cells were seeded into 96-well plates. The
next day, cells were exposed to the selected compounds at
various concentrations and incubated for 24, 48, and 72 h.
After incubation, the culture medium was exchanged with
The decoloration was plotted against the samples, and a
logarithmic regression curve was established in order to
calculate the IC50 which is the amount of sample necessary
to decrease by 50% the absorbance of DPPH. All experi-
ments were carried out in triplicate. The IC50 value of gallic
acid was obtained as 21 µM which related to the previous
report (Assaashari et al. 2014).
1
10 µL of MTT (0.5 mg/mL in PBS medium) and further
incubated for 2 h. The amount of MTT formazan product
was determined after dissolved in DMSO by measuring its
absorbance with a microplate reader (Bio-Rad Laboratories,
USA) at a test wavelength of 550 nm and a reference
wavelength of 655 nm. The cell viability was expressed as a
percentage to the viable cells of control culture condition
and IC50 values of each group were calculated.
Reducing power
The reducing power of the obtained compounds was
determined by the Fe(III)-reduction (Oktay et al. 2003).
Different concentrations of the sample in DMSO 1 mL were
mixed with 0.2 M phosphate buffer (pH 6.6, 2.5 mL) and
1% (v/v) potassium ferricyanide (2.5 mL). The mixture was
incubated at 50 °C for 20 min. A portion (2.5 mL) of 10%
trichloroacetic acid was added to the mixture which was
then centrifuged at 3000 r.p.m. for 10 min. The upper layer
of solution 2.5 mL was mixed with DI water 2.5 mL and
Molecular docking studies
The crystal structures of HDAC2, HDAC4, HDAC7, and
HDAC8 [PDB entry code: 3MAX, 2VQW, 3C0Z, and
1
T64, respectively] were obtained from the Protein Data
Bank (available from http://www.rcsb.org, last accessed 30
October 2015). All water and non-interacting ions as well as
ligands were removed. Then, all missing hydrogen and side-
0.5 mL of 0.1% FeCl . The absorbance was measured at
3

Medicinal Chemistry Research
7
00 nm using a spectrophotometer. Ascorbic acid was used
Banerjee S, Adhikari N, Amin SA, Jha T (2019) Histone deacetylase 8
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(
as a positive control. The reducing power was displayed by
effective concentration of 0.5 absorbance. All experiments
were carried out in triplicate. Ascorbic acid showed the
EC50 value of 9 µM. The earlier study indicated the value to
be about 13 µM (Berber et al. 2014).
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Acknowledgements Khon Kaen University is gratefully acknowl-
edged for the financial support of this work (Grant Number
6
200014002). We also would like to thank Mr. Kittisak Poopasith for
the excellent NMR data. We are thankful to the Postdoctoral Training
Program, Graduate School, Khon Kaen University for providing a
fellowship to Dr. Somprasong Saenglee. A graduate fellowship given
to Pakit Kumboonma and La-or Somsakeesit is supported by Raja-
mangala University of Technology Isan (RMUTI).
Jha NS, Mishra S, Jha SK, Surolia A (2015) Antioxidant activity and
electrochemical elucidation of the enigmatic redox behavior of
curcumin and its structurally modified analogues. Electro Acta
Compliance with ethical standards
1
51:574–583
Jordan BC, Kumar B, Thilagavathi R, Yadhav A, Kumar P, Selvam C
2018) Synthesis, evaluation of cytotoxic properties of promising
Conflict of interest The authors declare that they have no conflict of
interest.
(
curcumin analogues and investigation of possible molecular
mechanisms. Chem Biol Drug Des 91:332–337
Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
Khalil MI, AL-Zahem AM, Qunaibit MM (2014) Synthesis, char-
acterization, and antitumor activity of binuclear curcumin-metal
(II) hydroxo complexes. Med Chem Res 23:1683–1689
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