Influence of side-chain changes on histone deacetylase inhibitory and cytotoxicity activities of curcuminoid derivatives

Article abstract of DOI:10.1016/j.bmcl.2020.127171

Using curcuminoids as lead compounds, fifty-nine curcuminoid derivatives with different side chains at the phenolic moiety were synthesized. All compounds were investigated for their histone deacetylase (HDAC) inhibitory activities. The potent pan-HDAC in

Full text of DOI:10.1016/j.bmcl.2020.127171

Bioorganic & Medicinal Chemistry Letters 30 (2020) 127171
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Influence of side-chain changes on histone deacetylase inhibitory and
cytotoxicity activities of curcuminoid derivatives
a
b
b
c
d
La-or Somsakeesit , Thanaset Senawong , Pakit Kumboonma , Somprasong Saenglee ,
b
a
a,⁎
Arunta Samankul , Gulsiri Senawong , Chavi Yenjai , Chanokbhorn Phaosiri
a
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
b
c
d
Natural Products Research Unit, Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand
Department of Applied Chemistry, Faculty of Science and Liberal Arts, Rajamangala University of Technology Isan, Nakhon Ratchasima 30000, Thailand
Ban Dong Subdistrict Administration Organization, Ubolratana District, Khon Kaen 40250, Thailand
A R T I C L E I N F O
A B S T R A C T
Keywords:
Curcumin
Turmeric
HDAC
Using curcuminoids as lead compounds, fifty-nine curcuminoid derivatives with different side chains at the
phenolic moiety were synthesized. All compounds were investigated for their histone deacetylase (HDAC) in-
hibitory activities. The potent pan-HDAC inhibitors were further tested against three human cancer cell lines
including Hela, HCT116 and MCF-7 with MTT-based assay. The bisethylamide 4z and the mono-sec-butyl de-
rivative 5j manifested good antiproliferative activities against HCT116 cancer cells with the IC50 values as
Anticancer
1
4.60 ± 1.19 μg/mL and 7.33 ± 0.98 μg/mL, respectively. Molecular docking study of both compounds with
Class I HDACs revealed that the compounds might bind tightly to the binding pocket of HDAC2. These findings
suggested that these compounds can be putative candidates for the development of anticancer drugs via in-
hibiting HDACs.
Histone acetyl transferases (HATs) and histone acetyl deacetylases
HDACs) are crucial enzymes in the regulation of gene expression. In
which can cause several side effects. Although, the hydroxamic acid is
the potent zinc-chelating functional group, its drawbacks arise from the
pharmacokinetics issues such as glucuronidation, sulfonation and en-
(
normal cells, there is a balance between histone acetylation and dea-
1
–3
18
cetylation.
The overexpression of HDACs have been found to cause
zymatic hydrolysis resulting in a short in vivo half-life. It also has
1
9
various diseases including cancer, diabetes, cardiac and neurodegera-
mutagenic properties. Therefore, several non-hydroxamic acid HDAC
4
–8
20–23
tive diseases.
To date, HDACs are divided into four groups. Class I
inhibitors have been designed and evaluated.
Following our in-
(
(
HDACs 1, 2, 3 and 8), class II (HDACs 4, 5, 6, 7, 9 and 10) and class IV
terest in this area, we have focused on investigating the non-toxic and
non-hydroxamic acid HDAC inhibitors with isoform selectivity. Hy-
droxycapsaicin (2) and curcumin (3) are examples of HDAC inhibitors
derived from chili and turmeric, respectively. Although the HDAC in-
hibitory activity of both compounds are not comparable to that of
SAHA (1), they have isoform selectivity and low toxicity to non-cancer
HDAC 11) HDACs all belong to the zinc-dependent enzymes, whereas
+
class III (SIRT1-7) HDACs are NAD -dependent enzymes. Various
HDAC isoforms are different in localizations, functions and tissue dis-
tributions. Class I HDACs play a crucial role in tumorigenesis through
9
,10
the formation of complexes with other proteins.
Class II HDACs in-
2
4–26
volve in a variety of biological processes and play key roles in activity-
cell line (Fig. 1).
1
1,12
dependent gene regulation in response to neural activity.
Currently,
The natural derived curcumin (3) was received a lot of attention
because of its variety of biological activities such as anti-inflammatory,
antioxidant, anticancer, anti-HIV, anti-angiogenic and antibacterial
four HDAC inhibitors including SAHA (vorinostat®, 1) from Merck,
romidepsin from Gloucester Pharmaceuticals, belinostat and panobi-
nostat have been approved by USFDA for the treatment of cutaneous T-
cell lymphoma, peripheral T-cell lymphoma and multiple mye-
2
7
activities. However, the simple structural with two phenolic func-
tional groups connected by a conjugated β–diketone system of cur-
1
3–16
loma.
Additionally, chidamide has been recently approved by
cumin (3) is unsuitable for clinical use due to its poor solubility, me-
1
7
28
Chinese FDA for curing relapsed or refractory PTCL. Most of the hy-
droxamic acid based HDAC inhibitors such as SAHA are pan-inhibitors
tabolic unstability and bioavailability.
Interestingly, minor
modifications of curcumin (3) lead to derivatives with improved
⁎
Corresponding author.
E-mail address: chapha@kku.ac.th (C. Phaosiri).
https://doi.org/10.1016/j.bmcl.2020.127171
Received 7 January 2020; Received in revised form 26 March 2020; Accepted 3 April 2020
Available online 04 April 2020
0960-894X/ © 2020 Elsevier Ltd. All rights reserved.

L.-o. Somsakeesit, et al.
Bioorganic & Medicinal Chemistry Letters 30 (2020) 127171
Fig. 1. Structures of known histone deacetylase inhibitors and curcuminoids.
Table 1
In vitro histone deacetylase inhibitory activity of the compounds at 100 μM.
Cpd
R
1
R
2
% *
Cpd
R
C
1
R
2
% *
3
H
H
33 ± 0.25
20 ± 0.48
13 ± 0.28
13 ± 0.19
34 ± 0.63
9 ± 0.04
8 ± 0.21
7 ± 0.17
8 ± 0.17
13 ± 0.26
13 ± 0.35
15 ± 0.24
14 ± 0.29
17 ± 0.29
10 ± 0.13
11 ± 0.26
11 ± 0.12
20 ± 0.56
12 ± 0.30
14 ± 0.13
9 ± 0.31
88 ± 1.47
9 ± 0.14
8 ± 0.17
5 ± 0.02
18 ± 0.02
7 ± 0.04
12 ± 0.01
15 ± 0.04
10 ± 0.18
5c
5d
5e
5f
2
H
H
5
H
7 ± 0.01
8 ± 0.10
9 ± 1.29
10 ± 0.06
8 ± 0.01
7 ± 0.01
7 ± 0.04
6 ± 0.06
15 ± 0.28
10 ± 0.31
30 ± 0.45
19 ± 0.44
15 ± 3.75
26 ± 0.17
9 ± 0.23
37 ± 0.10
14 ± 0.36
59 ± 1.43
25 ± 1.19
8 ± 0.01
30 ± 0.43
20 ± 0.35
17 ± 0.28
7 ± 0.12
17 ± 0.05
51 ± 0.83
31 ± 0.33
28 ± 0.30
18 ± 0.46
85 ± 0.62
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
5
5
a
b
c
CH
3
3
CH
3
C
2
H
5
9
CH
H
C
C
4
4
H
H
9
9
C
4
H
C
C
C
C
2
2
4
4
H
H
H
H
5
5
9
9
4
4
C
2
H
H
5
9
H
H
d
e
f
5g
5h
5i
C
4
H
9
4
C
4
H
H
sec-C
sec-C
H
4
4
H
9
9
sec-C
H
H
9
9
H
g
h
i
sec-C
sec-C
allyl
allyl
H
9
9
sec-C
H
4
H
9
5j
sec-C
allyl
4
H
H
5k
5l
allyl
allyl
H
propagyl
propagyl
j
5m
5n
5o
5p
5q
5r
5s
5t
benzyl
benzyl
k
l
propagyl
propagyl
benzyl
H
propagyl
H
H
benzyl
m
n
o
p
q
r
benzyl
benzyl
4-fluoro benzyl
H
4-fluoro benzyl
4-fluoro benzyl
4-tert-butyl benzyl
benzyl
H
4-fluoro benzyl
4-fluorobenzyl
4-tert-butyl benzyl
4-tert-butyl benzyl
4-fluoro benzyl
4-tert-butyl benzyl
H
CH
CH
H
2
2
CO
2
2
CH
3
3
CH
H
2
CO
2
CH
3
3
3
4-tert-butyl benzyl
CO
CH
H
5u
5v
6a
6b
6c
6d
6e
7a
7b
7c
8a
9
CH
CH
CH
H
2
2
2
CO
2
CH
s
CH
2
2
CO
2
2
3
CH
3
3
2
CH
2
CO
2
CH
3
CH
CH
CH
2
2
2
COOH
COOH
t
CH
CO
CH
H
CO
CO
2
2
H
CH
CH
3
3
2
CO CH
u
v
w
x
y
z
CH(CH
)CO
CH
3
CH(CH
3
)CO
2 3
CH
TBDMS
TBDMS
sec-C
4
9
H
TBDMS
H
CH
CH
CH
2
2
3
COOH
CONH
CH
CH
–
2
2
COOH
CH
2
2
COOH
CH
2
2
COOH
2
CONH
2
CH
CONH
2
CH
CONH
2
CH(CH
3
)CONH
2
CH(CH
3
)CONH
2
benzyl
–
zz
a
b
CH
CH
2
3
CONHNH
2
CH
CH
2
CONHNH
3
2
CH
CH
H
2
2
CO
2
2
CH
3
3
–
CO
CH
–
C
2
H
5
C
2
H
5
H
*
= Remaining HDAC activity.
2
9–34
stability and activity.
In this work, we continued with the mod-
were designed to improve the pharmacokinetic profiles from conjuga-
tion of glucuronide and sulfate of the phenolic group in the body by
substitution of the protons at phenolic groups with various aliphatic
and aromatic side chains. Besides improving stability, these substitu-
tions were also designed to increase inhibitor-enzyme binding via van
ifications of curcumin (3) to improve inhibitor-enzyme binding and
aimed to determine whether these compounds would be potent HDAC
inhibitors and manifest antiproliferative activities against cancer cells
as well as maintaining their low toxicity. The curcumin derivatives
2

L.-o. Somsakeesit, et al.
Bioorganic & Medicinal Chemistry Letters 30 (2020) 127171
Fig. 2. Structures of curcuminoid derivatives.
Table 2
In silico histone deacetylase inhibitory activity of the selected compounds.
Cpd
IC50 **
Class I HDACs
HDAC1
HDAC2
HDAC8
**
**
**
ΔG*
K
i
ΔG*
K
i
ΔG*
K
i
3
116 ± 0.22
0.77 ± 0.46
0.92 ± 0.19
0.90 ± 0.23
0.99 ± 0.34
−5.86
−6.39
−7.79
−8.22
−7.64
50.79
20.71
1.96
−5.47
−6.99
−7.37
−8.54
−8.15
97.67
7.47
3.99
0.55
1.06
−5.71
−6.81
−6.61
−8.26
−7.43
64.98
10.22
14.24
0.88
4
4
4
5
g
x
z
j
0.936
2.50
3.60
*
(kcal/mol), ** (μM).
der Waals, hydrogen bonds, hydrophobic, hydrophilic and ¶-¶ interac-
tions.
consideration, the selected substitutions were introduced into de-
methoxycurcumin (5) to provide compounds 5a-5v. The mono-sec-
butyl derivative 5j showed the least remaining activity of HDAC and the
dicarboxylic 5v also exhibited very low remaining activity. The bisa-
cetamide derivatives of demethoxycurcumin (5) were synthesized,
however, they could not be obtained in good yields. For the minor
curcuminoid, bisdemethoxycurcumin (6), only selected derivatives
were synthesized based on the SAR of curcumin (3) and demethox-
ycurcumin (5). The dicarboxylic 6d still acted as the most potent in-
hibitor, nevertheless the mono-sec-butyl derivative 6c showed a weaker
inhibitory activity than 6d. The bisacetamide 6e could be obtained in a
satisfactory yield but it only showed the same order of inhibition with
6c. Interestingly, the Michael adduct 7a-7c were gained as by-products
from alkylation and benzylation of curcumin (3). It might come from a
trace amount of water in the reaction. The tetra-substituted methylester
8 was also obtained as the by-product. However, all by-products
showed moderate inhibition against HDACs. The octahydrocurcumin
Curcumin (3) as the major curcuminoid along with two minor
curcuminoids, demethoxycurcumin (5, R
1
= R
2
= H) and bisde-
methoxycurcumin (6, R
1
= R
2
= H) were obtained as previously de-
2
6
scribed. Derivatives 4a-4w were synthesized by reacting curcumin (3)
with K
2
CO
3
and the corresponding alkyl, benzyl as well as silyl halides
under reflux condition. Compound 4s was further hydrolyzed under a
basic condition to provide the dicarboxylic 4x. Trans-aminations of the
bismethylester 4s with ammonium hydroxide and hydrazine yielded
the bisacetamine 4y and 4zz, respectively. The bismethylester 4u was
also reacted with ammonium hydroxide to obtain the bisethylamide 4z.
The newly synthesized derivatives were fully characterized and the
spectroscopic data of the previously synthesized compounds were
3
0,35
compared to the literature.
The obtained compounds were initially
screened for HDAC inhibitory activity using the Fluor-de-Lys HDAC
2
6
activity assay kit. The remaining HDAC activity is shown in Table 1.
Except 4u, all derivatives showed good to excellent HDAC inhibitory
activity at 100 μM. The di-sec-butyl derivative 4g showed the best in-
hibition among the alkyl, benzyl and silyl derivatives of curcumin (3).
The dicarboxylic 4x was the most potent HDAC inhibitor, whereas the
bisethylamide 4z showed the same order of inhibition with 4g. Taking
the structure–activity relationship (SAR) of curcumin derivatives into a
3
6
(9) obtaining from the reduction of curcumin (3) exhibited only weak
inhibitory activity (Fig. 2). On the basic of these results, the most potent
derivatives 4g, 4x, 4z and 5j were further evaluated for their IC50 va-
lues as shown in Table 2. All the selected inhibitors exhibited the IC50
values in a submicromolar range which was lower than that of the lead
compound, curcumin (3).Table 3.
3

L.-o. Somsakeesit, et al.
Bioorganic & Medicinal Chemistry Letters 30 (2020) 127171
Molecular docking study was conducted with ClassI HDACs which
3
7
mainly involved in development of cancer. The available crystal
structures of HDAC1, HDAC2 and HDAC8 were obtained from the
Protein Data Bank. 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 50 runs using
AutoDockTools 1.5.4 (ADT) and AutoDock 4.2 programs and La-
3
8,39
marckian genetic algorithm search.
The results in Table 2 indicate
that all compounds show better binding affinity with all HDAC isoforms
than curcumin (3). However, there were only 2–3 fold selectivity
among the isoforms. The bisethylamide 4z showed the lowest K values
i
for all Class I HDACs. Fig. 3A) shows that the strong inhibitor-enzyme
interactions consist of four hydrogen bonds between 4z and GLY32,
GLU103, TYR209 as well as LEU276 of HDAC2. The coordination of
2
+
amide functional group to the Zn ion can also be observed. The most
potent compounds, 4z and 5j, against HDAC2 in silico were further
specifically investigated in vitro. The IC50 values of 4z and 5j against
HDAC2 were 1.04 ± 0.29 μM and 1.13 ± 0.21 μM, respectively. To
complete the evaluation of these potent HDAC inhibitors, the anti-
proliferative activities were determined in human cervical cancer
(
HeLa), human colon cancer (HCT116) and human breast adenocarci-
noma cancer (MCF-7) cells. All compounds were less cytotoxic against
cancer cells and appeared to be less toxic to normal cells than curcumin
(
3). Even though compound 4z showed the high antiproliferative ac-
tivity among the derivatives, it appeared to be toxic towards the non-
cancer cells (Vero cells). Interestingly, compounds 4g and 5j performed
the better cytotoxicity against all cancer cells than compound 4z.
Moreover, both compounds also exhibited lower toxicity towards non-
cancer cells than the lead compound. After considering the binding
modes of 4g and 5j with HDAC2, it was clear that 5j showed more
interactions with HDAC2 than 4g. These major interactions of 5j and
HDAC2 consisted of the hydrogen bond to GLU103, ¶-¶ interaction with
2
+
HIS33, coordination to the Zn ion and hydrophobic interaction of the
sec-butyl group as shown in Fig. 3B. The western blot analysis of
compounds 4z and 5j was conducted with HCT116 cells. The results in
Fig. 4 show accumulation of acetylated H3. The significant accumula-
tion could be observed with compound 4z at 40 μg/mL.
In conclusion, curcuminoids were modified at the phenolic groups
to obtained fifty-nine derivatives which showed HDAC inhibitory ac-
tivity. At least, four derivatives were identified as potent HDAC in-
hibitors with antiproliferative activity. Both non-polar and polar func-
tional groups contributed to the increased HDAC-inhibitor binding. This
work brought to light problems with the metabolic stability of natural
compound with potent biological activities.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
Khon Kaen University is gratefully acknowledged for the financial
support of this work (Grant Number 6200014002). 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 La-or Somsakeesit is supported by
Rajamangala University of Technology Isan (RMUTI). Center of
Excellence for Innovation in Chemistry, Ministry of Higher Education,
Science, Research and Innovation is also acknowledged.
4

L.-o. Somsakeesit, et al.
Bioorganic & Medicinal Chemistry Letters 30 (2020) 127171
Fig. 3. The interaction modes of A) 4z and B) 5j in the active site of HDAC2.
Fig. 4. Western blot analysis. A. HCT116 cells were treated with absolute ethanol and DMSO (1:1 v/v) as control, 5j of 20, 40 and 60 μg/mL and 4z of 10, 20 and
4
2
0 μg/mL for 24 h. The cells were harvested and proteins were extracted and separated on SDS-PAGE. Expression of Ac-H3 were detected by immunoblotting. ERK1/
was used as a loading control in western blot analysis. The intensity of protein bans was quantitated by densitometric analysis. B. The bar graphs represent relative
fold of protein expression in HCT116 cells compared with internal control.
5

L.-o. Somsakeesit, et al.
Bioorganic & Medicinal Chemistry Letters 30 (2020) 127171
Appendix A. Supplementary data
18. Kelly WK, O’Connor OA, Krug LM, et al. J Clin Oncol 2005, 23, 3923.
1
2
2
9. Shen S, Kozikowski AP. ChemMedChem. 2016;11:15–21.
0. Jiao P, Jin P, Li C, et al. Bioorg Med Chem Lett. 2016;26:4679.
1. Lu A, Luo H, Shi M, et al. Bioorg Med Chem Lett. 2011;21:4924.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmcl.2020.127171.
22. Seidel C, Schnekenburger M, Zwergel C, et al. Bioorg Med Chem Lett. 2014;24:3797.
2
2
3. Botta CB, Cabri W, Cini E, et al. J Med Chem. 2011;54:2165.
4. Bora-Tatar G, Erden DD, Demir AS, Dalkara S, Yelekci K, Yurter HE. Bioorg Med
Chem. 2009;17:5219.
References
2
2
5. Senawong T, Wongphakham P, Saiwichai T, Phaosiri C, Kumboonma P. Turk J Biol.
2015;39:1.
1
2
3
4
5
6
7
. Bertrand P. Eur J Med Chem. 2010;45:2095.
6. Kumboonma P, Senawong T, Saenglee S, et al. Med Chem Res. 2019;28:1773.
. Miller TA, Witter DJ, Belvedere S. J Med Chem. 2003;46:5097.
. Micelli C, Rastelli G. Drug Discovery Today. 2015;20:718.
. Manal M, Chandrasekar MJN, Priya JG, Nanjan MJ. Bioorg Chem. 2016;67:18.
. Sharma S, Taliyan R. Pharmacol Res. 2016;113:320.
27. Anand P, Thomas SG, Kunnumakkara AB, et al. Biochem Pharmacol. 2008;76:1590.
28. Masters A, Multhaup CL, Simms G, Pottgiesser J, Martins RN, Beyreuther K, EMBO J
1985, 4, 2757.
. Abend A, Kehat I. Pharmacol Ther. 2015;147:55.
29. Changtam C, Hongmanee P, Suksamrarn A. Eur J Med Chem. 2010;45:4446.
3
3
0. Changtam C, Koning HP, Ibrahim H, Sajid MS, Gould MK, Suksamrarn A. Eur J Med
. Hahnen E, Hauke J, Trankle C, Eyupoglu IY, Wirth B, Blumcke I. Expert Opin Invest
Drugs. 2008;17:169.
Chem. 2010;45:941.
1. Banuppriya G, Sribalan R, Padmini V, Shanmugaiah V. Bioorg Med Chem Lett.
8
9
. Hu J, An B, Pan T, Li Z, Huang L, Li X. Bioorg Med Chem. 2018;26:5718.
. Banerjee S, Adhikari N, Amin SA, Jha T. Eur J Med Chem. 2019;164:214.
2016;26:1655.
1
1
1
1
1
0. Suzuki T. Chem Pharm Bull. 2009;57:897.
32. Okuda M, Hijikuro I, Fujita Y, et al. Bioorg Med Chem Lett. 2016;26:5024.
3
3. Konno H, Endo H, Ise S, et al. Bioorg Med Chem Lett. 2014;24:685.
1. Bürli RW, Luckhurst CA, Aziz O, et al. J Med Chem. 2013;56:9934.
2. Chang S, Young BD, Li S, Qi X, Richardson JA, Olsen EN. Cell. 2006;126:321.
3. Mann BS, Johnson JR, Cohen MH, Justice R, Pazdur R. Oncologist. 2007;12:1247.
4. Deal watch: Celgene acquires Gloucester Pharmaceuticals, gaining approved HDAC
inhibitor. Nat Rev Drug Disc 2010, 9, 94.
34. Das J, Pany S, Panchal S, Majhi A, Rahman GM. Bioorg Med Chem. 2011;19:6196.
3
5. Sribalan R, Kirubavathi M, Banuppriya G, Padmini V. Bioorg Med Chem Lett.
2015;25:4282.
36. Feng JY, Liu ZQ. J Agric Food Chem. 2009;57:11041.
37. Roche J, Bertrand P. Eur J Med Chem. 2016;121:451.
38. Sanner MF. J Mol Graph Model. 1999;17:57.
1
1
1
5. Campbell P, Thomas CM. J Oncol Pharm Pract. 2017;23:143.
6. Greig SL. Target Oncol. 2016;11:107.
3
9. Morris GM, Huey R, Lindstrom W, et al. J Comp Chem. 2009;30:2785–2791.
7. Lu X, Ning Z, Li Z, Cao H, Wang X. Intractable Rare Dis Res. 2016;5:185.
6