Aryl butenoic acid derivatives as a new class of histone deacetylase inhibitors: Synthesis, in vitro evaluation, and molecular docking studies

Article abstract of DOI:10.3906/kim-1305-56

New aryl butenoic acid derivatives have been synthesized by combining hydroxy- or methoxy-substituted phenyl rings as the capping group, with a double bond in the short linker as well as metal binding groups, enoic ester, and salts bearing either methyl o

Full text of DOI:10.3906/kim-1305-56

Turk J Chem
Turkish Journal of Chemistry
(2014) 38: 338 – 344
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http://journals.tubitak.gov.tr/chem/
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c
⃝ TUBITAK
doi:10.3906/kim-1305-56
Research Article
Aryl butenoic acid derivatives as a new class of histone deacetylase inhibitors:
synthesis, in vitro evaluation, and molecular docking studies
1
Peruze AYHAN ES¸IYOK , Ozlem SEVEN , Gu¨lu¨zar EYMUR , Gamze BORA TATAR ,
1
1
2
˙
¨
2
Didem DAYANGAC¸ ERDEN , Kemal YELEKC¸ I3,†, Hayat YURTER2,∗,†
,
˙
1,†,∗∗
˙
Ayhan Sıtkı DEMIR
1Department of Chemistry, Faculty of Arts and Sciences, Middle East Technical University, Ankara, Turkey
2Department of Medical Biology, Faculty of Medicine, Hacettepe University, Ankara, Turkey
3Department of Computational Biology and Bioinformatics, Faculty of Engineering and Natural Sciences,
˙
Kadir Has University, Istanbul, Turkey
Received: 24.05.2013
•
Accepted: 08.10.2013
•
Published Online: 14.03.2014
•
Printed: 11.04.2014
Abstract: New aryl butenoic acid derivatives have been synthesized by combining hydroxy- or methoxy-substituted
phenyl rings as the capping group, with a double bond in the short linker as well as metal binding groups, enoic
ester, and salts bearing either methyl or morpholine. These compounds have been shown to possess promising histone
deacetylase inhibition activities via in vitro fluorometric assay and molecular docking studies.
Key words: Histone deacetylase inhibition activity, aryl butenoic acid derivatives, molecular docking
1. Introduction
Histone acetylase (HAT) and histone deacetylase (HDAC) enzymes act in a competitive manner to determine
the histone acetylation levels that ultimately affect the high-order chromatin structure and gene activities
at a particular chromosomal region.1 HDACs reverse the activity of HATs by catalyzing the removal of
acetyl groups from lysine residues in the N-terminal tails of histones, leading to chromatin condensation and
posttranscriptional gene silencing. To date, 18 HDAC isoforms have been identified and grouped into 3 classes
based on homology to yeast HDACs (class I: 1, 2, 3, 8; class II: 4, 5, 6, 7, 9, 10; and class III: SIRT 1–
7). HDAC enzymes are gaining more attention, as they deacetylate not only histone but also nonhistone
proteins. HDAC inhibitors (HDIs) have been shown to be effective in the treatment of several diseases such as
cancer,2−4 neurodegenerative disorders like proximal spinal muscular atrophy,5−8 amyotrophic lateral sclerosis,
and multiple sclerosis.9−11
HDIs can be divided into several structural classes, including short chain fatty acids, hydroxamic acids,
epoxyketone-containing cyclic tetrapeptides, epoxyketone-containing cyclic tetrapeptides, and benzamides hy-
brid molecules. All of these molecules are active exclusively on HDAC class I and class II, by binding to
the zinc-containing catalytic domains of the enzymes with different modalities, affinities, and sites.12 Of these
subgroups, hydroxamic acids, including natural inhibitors like trichostatin A (TSA, 1)13 and synthesized ones
like suberoylanilide hydroxamic acid (SAHA, 2),14 are the most potent known HDAC inhibitors, as shown in
Figure 1.
∗Correspondence: herdem@hacettepe.edu.tr
† These authors contributed equally to this work.
∗∗ Deceased (1950–2012).
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AYHAN ES¸IYOK et al./Turk J Chem
O
O
O
H
N
OH
OH
N
H
N
H
O
Me2N
TSA, 1
SAHA, 2
Figure 1. Structures of the 2 popular hydroxamic acid HDAC inhibitors.13,14
With few exceptions, HDIs can be broadly characterized by a common pharmacophore that summa-
rizes key elements of inhibitor–enzyme interactions consisting of a capping group, linker, and metal-binding
moiety.15 The capping group is generally a phenyl ring, while the length of the carbon chain of the linker
changes and the metal-binding group is the group that interacts with the zinc. Phenolic carboxylic esters
constitute an interesting class of synthons with their diverse biological activities. With the earlier finding of
resveratrol exhibiting HDAC inhibition activity, phenolic compounds present interesting HDI alternatives.16
In this study, a new class of compounds, namely aryl butenoic acid derivatives, was designed in order
to explore the capacity of the phenolic carboxylic esters as HDAC inhibitors. Butenoic acid derivatives either
used as synthons for different pharmaceuticals or in their native forms possess several biological activities; they
are used for the treatment of dyslipidemia, atherosclerosis, and diabetes.17 A hydroxy- or methoxy-substituted
phenyl ring was employed as the capping group, a double bond was introduced in the short linker, and carboxylic
enoic ester was used as the metal-binding group. The ester moiety has also been functionalized with morpholine.
These compounds have been proven to be good HDIs with high inhibition capacity.
2. Results and discussion
Previously, resveratrol has been shown to possess HDAC inhibition activity (IC50 : 650 µM).16 This activity
has led to the suggestion that if one of the phenyl groups is replaced with the ester moiety as the metal-binding
group, then the resulting novel butenoic acid derivatives would have the same inhibition activity. To check this
hypothesis, several (E)-methyl 2-oxo-4-phenylbut-3-enoate derivatives (nonsubstituted or mono- and di-hydroxy
or methoxy-substituted derivatives) were synthesized. There are few strategies present in the literature for the
syntheses of (E)-methyl 2-oxo-4-phenylbut-3-enoate. This compound can be obtained as a side product of
the hydrolysis reaction of N-isopropyl-[2-(benzotriazol-1-yl)-2-phenoxy-4-phenyl-4-ethoxy]butyramide in EtOH-
H2 O18 or from potassium benzylidene pyruvate in hydrochloric acid.19−21 In this study, the second approach
was employed. Briefly, the potassium salts of benzylidene pyruvate were synthesized from benzaldehyde with
pyruvic acid, which were then converted to the corresponding esters with HCl. The potassium salts of (E)-methyl
2-oxo-4-arylbut-3-enoate and (E)-methyl 2-oxo-4-arylbut-3-enoate derivatives are shown in the Table.
Other derivatives of these compounds were also developed as potential HDAC inhibitors by functionalizing
the ester moiety with either nonsubstituted or substituted morpholine rings. Several amides were obtained,
starting from esters in condensation reactions of these 2 in the presence of trimethylaluminum. Methoxy
substituted derivatives of (E)-methyl 2-oxo-4-phenylbut-3-enoate were employed together with the morpholine
and trimethylaluminum in toluene.
To investigate the total HDAC inhibition activities biologically, synthesized compounds were screened
by in vitro fluorometric assay. Compounds were applied to a HeLa nuclear extract at a concentration of 500
µM and HDAC inhibition activities were calculated. Sodium butyrate (NaBA), which is a well-known HDI,
was used as a reference compound to compare inhibition activities of (E)-methyl 2-oxo-4-phenylbut-3-enoate
derivatives. Results are summarized in the Table.
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AYHAN ES¸IYOK et al./Turk J Chem
The best experimental result was obtained with (E)-methyl-4-(3,4-dihydroxyphenyl)-2-oxobut-3-enoate
(3h) and (E)-4-(4-methoxyphenyl)-1-(2,6-dimethylmorpholino)but-3-ene-1,2-dione (3j). P-substituted deriva-
tives have been shown to be better inhibitors compared to m- and o-substituted ones. Salts (3a, 3e, 3f, and
3g) were ineffective as inhibitors. Except for the salt derivatives, all the compounds that were tested showed
better inhibition compared to NaBA, a well-known HDI (20% experimental inhibition activity). Disubstituted
aryl butenoic acid derivatives were also shown to be effective.
When the methyl group was employed in the ester moiety, a higher inhibition occurred, emphasizing
this group’s zinc-binding ability. This effect has also been visualized from the enhanced activity of dimethyl
substituted morpholine compound 3j compared to the nonsubstituted morpholine compound 3i.
Table. Calculated free energy of binding, inhibition constant and experimental HDAC inhibition activity values of the
synthesized compounds.
O
OR
O
R'
3
Calculated Free
Energy of Binding
( G, kcal/mol)
Calculated
Experimental
Compound
R’
R
Inhibition
HDAC Inhibition
Activities (%)
Constant (Ki, µM)
3a
3b
3c
3d
3e
3f
4-MeO
4-MeO
3-MeO
2-MeO
2-OH
K+
-10.98
-7.08
0.0089
6.50
3
CH3
CH3
CH3
K+
53
24
26
15
5
-7.33
4.23
-7.18
5.48
-10.68
-10.67
-10.29
-6.45
0.0142
0.0151
0.0286
18.67
3-OH
K+
3g
3h
3,4-(OH)2
3,4-(OH) 2
K+
4
CH3
60
N
N
O
3i
4-MeO
-7.34
4.14
51
O
3j
4-MeO
-7.82
1.86
59
N
N
O
3k
3l
3-MeO
2-MeO
-6.96
-6.96
7.89
7.86
30
33
O
It should be noted that there were some deviations in the results obtained from the experimental and
theoretical data. This might have resulted from the fact that in silico studies were carried out with HDAC8
only, while the HeLa nuclear extract contained a variety of HDAC enzymes.
When in vitro and in silico studies were both taken into consideration, the best inhibition was ob-
tained with (E)-4-(4-methoxyphenyl)-1-(2,6-dimethylmorpholino)but-3-ene-1,2-dione (3j), whose interaction
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AYHAN ES¸IYOK et al./Turk J Chem
with HDAC8 is shown in Figure 2. The supplementary Figures 1–12 contain 2-D and 3-D pictures of com-
pounds.
Figure 2. Compound 3j in the active site of the HDAC8 enzyme.
3. Experimental
3.1. General procedure for preparation of potassium salts
Pyruvic acid (0.0141 mol) and substituted benzaldehyde (0.0141 mol) were stirred in 1.5 mL of methanol in an
ice bath. To this mixture, 5 mL of the alkaline solution of KOH in methanol (1 g in 7.5 mL solvent) was added
at 0 ◦ C. Following the removal of the ice bath, the remaining portion of this solution was introduced to the
reaction mixture immediately. The resultant mixture was stirred for 1 h at 30 ◦ C and then overnight at 0 ◦ C.
A yellow potassium salt was gathered after being filtered and washed with cold methanol and then with ether.
The potassium salt was air-dried.19,20
3.2. General procedure for preparation of (E)-methyl-4-(aryl)-2-oxobut-3-enoate derivatives1
To generate hydrochloric acid, 7 mL acetyl chloride was added to 40 mL of methanol in an ice bath. The
potassium salt was added, the reaction mixture was stirred for 30 min, and then the ice bath was removed.
After 2 h, the mixture was stirred overnight at 65 ◦ C. The reaction mixture was removed and then 20 mL of
water was added and was extracted 2 times with 20 mL of dichloromethane. The organic phase was washed
with saturated sodium bicarbonate and then 20 mL of water. The organic phase was dried over anhydrous
magnesium sulfate and evaporated. The yellow crystals were obtained via recrystallization from ethanol or
methanol.20,21
3.3. General procedure of preparation morpholine amide derivatives
In an argon atmosphere, a solution of morpholino derivative (0.454 mmol) in 1.5 mL of toluene was added to
a solution of (E)-methyl-4-(aryl)-2-oxobut-3-enoate (0.454 mmol) in 3 mL of toluene, and then 0.2 mL of 2 M
triethylaluminum (in heptane) was added. The reaction mixture was stirred at 80 ◦ C for 30 h and cooled to
room temperature. Twenty-five milliliters of water was added and extracted 3 times with 25 mL of ethylacetate.
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Organic phases were combined and dried over magnesium sulfate, then evaporated. The products were purified
with column chromatography (hexane/ethyl acetate, 2:1 or 3:1).
3.4. In vitro HDAC inhibition activity screening
HDAC inhibition activity of synthesized compounds was screened by an in vitro fluorometric assay (BioVision)
according to the manufacturer’s protocol. Briefly, compounds at a 500 µM concentration were mixed with HeLa
nuclear extract, which contains all HDAC enzyme isoforms. HDAC fluorometric substrate [Boc-Lys(Ac)-AMC],
which contains an acetylated lysine side chain, was added to the mixture. Substrate sensitized by deacetylation
and the subsequent addition of lysine developer produced fluorophore. Fluorescence was measured with a mi-
croplate reader (Molecular Devices Spectramax M2) at excitation 350 nm and emission 440 nm. Screenings were
performed in triplicate. Data were analyzed according to decreasing fluorescence signal (arbitrary fluorescence
units). The values of treated samples were normalized to nontreated ones, which were set as 100%. NaBA was
used as a reference compound, which has 20% HDAC inhibition activity.
3.5. AutoDock
AutoDock uses a semiempirical force field based on the AMBER force field.22−25 It uses a molecular mechanics
model for enthalpic contributions, such as vdW and hydrogen bonding, and an empirical model for entropic
changes upon binding. Each component is multiplied by empirical weights obtained from the calibration against
a set of known binding constants. AutoDock uses a Lamarckian genetic algorithm for the conformational search.
For each molecule, 50 independent runs were performed. A total of 300 distinct ligand conformers were initially
generated and positioned randomly in the binding pocket. They had randomly assigned torsion angles to
rotatable bonds and a randomly assigned overall rotation. A maximum of 100 million energy evaluations was
allowed for each docking. A precalculated 3-dimensional energy grid of equally spaced discrete points was
generated prior to docking for a rapid energy evaluation, using the program AutoGrid.4 The grid box, with
˚
˚
˚
dimensions of 80 A × 80 A × 80 A, was centered near the Zn atom of the active site and covered the entire
˚
binding site and its neighboring residues. The distance between 2 grid points was set to 0.375 A.
3.5.1. Crystal structure of HDAC8
The crystal structures of human histone deacetylase, HDAC8 enzyme (PDB entry code: 1T64, complexed with
the inhibitor TSA), was extracted from the Protein Data Bank (http://www.rcsb.org). The enzyme structure
was cleaned of all water molecules and the irreversible inhibitor of TSA, as well as all noninteracting ions (except
zinc) before being used in the docking studies. To relieve the crystal structure tension and to make the protein
available to use in the AutoDock22 docking simulation program, the protein’s geometry was first optimized
and then submitted to the “Clean Geometry” toolkit of Discovery Studio (Accelerys, Inc.) for a more complete
check using a fast Dreiding-like force field. Missing hydrogen atoms were added based on the protonation state
of the titratable residues at a pH of 7.4. Ionic strength was set to 0.145 and the dielectric constant was set to
10.
3.5.2. Ligand setups
The 3D structures of ligand molecules were built, optimized at PM3 level, and saved in pdb format with the aid
of the molecular modeling program SPARTAN. The AutoDockTools (ADT) graphical user interface programs
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were also employed here to generate the docking input files of ligands. ADT helps the user easily set up
macromolecules (enzymes) and ligands for docking. It assigns the Gasteiger partial charges to each atom and
prepares 2 parameter files, namely a grid parameter file (gpf) and a docking parameter file (dpf). The detail of
the procedure is given elsewhere (http://autodock.scripps.edu).26
4. Conclusions
In this study, new aryl butenoic acid derivatives as HDAC inhibitors have been developed. A privileged structure
containing mono or multi methoxy- and hydroxyl-substituted phenyl rings with a carboxylic enoic ester moiety
were integrated as a zinc-binding group.
Calculated inhibition values of K salt of compounds 3a, 3e, 3f, and 3g are in the range of nanomolar
concentrations, which is not in agreement with the experimental values. One possible explanation is that, in
the calculations, bare carboxylic acid anions were strongly attracted by the zinc ion in the active center. On
the other hand, the carboxylate ion may behave differently in the aqueous environment in the active site of
the enzyme in in vivo experiments. All the other derivatives showed various degrees of experimental HDAC
inhibition activities parallel to computational values. Both morpholine and methyl-substituted enoic esters
showed excellent inhibition activity, revealing that both of these groups have zinc-binding capacities. As a
conclusion, inhibitor 3j both experimentally and computationally is the most promising candidate for further
HDAC inhibition studies.
Acknowledgment
This work was supported by the Scientific and Technological Research Council of Turkey, Research Project No.:
105G014. Patent Application No.: TR 2011 03844 A2.
Supplementary information is available. It includes the experimental details for the syntheses of com-
pounds, and 2-D and 3-D interaction images of the synthesized compounds (3a–3l).
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