N-(2-hydroxyphenyl)-2-propylpentanamide, a valproic acid aryl derivative designed in silico with improved anti-proliferative activity in HeLa, rhabdomyosarcoma and breast cancer cells

Article abstract of DOI:10.1080/14756366.2016.1210138

Epigenetic alterations are associated with cancer and their targeting is a promising approach for treatment of this disease. Among current epigenetic drugs, histone deacetylase (HDAC) inhibitors induce changes in gene expression that can lead to cell death in tumors. Valproic acid (VPA) is a HDAC inhibitor that has antitumor activity at mM range. However, it is known that VPA is a hepatotoxic drug. Therefore, the aim of this study was to design a set of VPA derivatives adding the arylamine core of the suberoylanilide hydroxamic acid (SAHA) with different substituents at its carboxyl group. These derivatives were submitted to docking simulations to select the most promising compound. The compound 2 (N-(2-hydroxyphenyl)-2-propylpentanamide) was the best candidate to be synthesized and evaluated in vitro as an anti-cancer agent against HeLa, rhabdomyosarcoma and breast cancer cell lines. Compound 2 showed a better IC₅₀ (μM range) than VPA (mM range) on these cancer cells. And also, 2 was particularly effective on triple negative breast cancer cells. In conclusion, 2 is an example of drugs designed in silico that show biological properties against human cancer difficult to treat as triple negative breast cancer.

Full text of DOI:10.1080/14756366.2016.1210138

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
N-(2-hydroxyphenyl)-2-propylpentanamide, a
valproic acid aryl derivative designed in silico
with improved anti-proliferative activity in HeLa,
rhabdomyosarcoma and breast cancer cells
Berenice Prestegui-Martel, Jorge Antonio Bermúdez-Lugo, Alma Chávez-
Blanco, Alfonso Dueñas-González, José Rubén García-Sánchez, Oscar Alberto
Pérez-González, Itzia Irene Padilla-Martínez, Manuel Jonathan Fragoso-
Vázquez, Jessica Elena Mendieta-Wejebe, Ana María Correa-Basurto, David
Méndez-Luna, José Trujillo-Ferrara & José Correa-Basurto
To cite this article: Berenice Prestegui-Martel, Jorge Antonio Bermúdez-Lugo, Alma Chávez-
Blanco, Alfonso Dueñas-González, José Rubén García-Sánchez, Oscar Alberto Pérez-González,
Itzia Irene Padilla-Martínez, Manuel Jonathan Fragoso-Vázquez, Jessica Elena Mendieta-Wejebe,
Ana María Correa-Basurto, David Méndez-Luna, José Trujillo-Ferrara & José Correa-Basurto
(2016): N-(2-hydroxyphenyl)-2-propylpentanamide, a valproic acid aryl derivative designed in
silico with improved anti-proliferative activity in HeLa, rhabdomyosarcoma and breast cancer
cells, Journal of Enzyme Inhibition and Medicinal Chemistry
To link to this article: http://dx.doi.org/10.1080/14756366.2016.1210138
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Date: 03 August 2016, At: 12:22

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
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2016 Informa UK Limited, trading as Taylor & Francis Group. DOI: 10.1080/14756366.2016.1210138
!
ORIGINAL ARTICLE
N-(2-hydroxyphenyl)-2-propylpentanamide, a valproic acid aryl
derivative designed in silico with improved anti-proliferative activity
in HeLa, rhabdomyosarcoma and breast cancer cells
1
1
2
3
Berenice Prestegui-Martel , Jorge Antonio Berm u´ dez-Lugo , Alma Ch a´ vez-Blanco , Alfonso Due n˜ as-Gonz a´ lez ,
4
5
6
Jos e´ Rub e´ n Garc ´ı a-S a´ nchez , Oscar Alberto P e´ rez-Gonz a´ lez , Itzia Irene Padilla-Mart ´ı nez , Manuel Jonathan
Fragoso-V a´ zquez , Jessica Elena Mendieta-Wejebe , Ana Mar ´ı a Correa-Basurto , David M e´ ndez-Luna ,
Jos e´ Trujillo-Ferrara , and Jos e´ Correa-Basurto
1
1
1
1
1
1
1
Laboratorio de Modelado Molecular y Bioinform a´ tica, Laboratorio de Bioqu ´ı mica, Laboratorio de Biof ´ı sica y Biocat a´ lisis, Secci o´ n de Estudios de
Posgrado e Investigaci o´ n, Escuela Superior de Medicina, Instituto Polit e´ cnico Nacional, Plan de San Luis y D ´ı az Mir o´ n, Ciudad de M e´ xico, M e´ xico,
2
3
Divisi o´ n de Investigaci o´ n B a´ sica, Instituto Nacional de Cancerolog ´ı a, Tlalpan, Secci o´ n XVI, Ciudad de M e´ xico, M e´ xico, Instituto de Investigaciones
4
Biom e´ dicas, Universidad Nacional Aut o´ noma de M e´ xico/Instituto Nacional de Cancerolog ´ı a, Ciudad de M e´ xico, M e´ xico, Laboratorio de Oncolog ´ı a
Molecular y Estr e´ s Oxidativo, Secci o´ n de Estudios de Posgrado e Investigaci o´ n, Escuela Superior de Medicina, Instituto Polit e´ cnico Nacional, Plan de
5
San Luis y D ´ı az Mir o´ n, Ciudad de M e´ xico, M e´ xico, Laboratorio de Oncolog ´ı a Experimental, Instituto Nacional de Pediatr ´ı a, Coyoac a´ n, Insurgentes
6
Cuicuilco, Ciudad de M e´ xico, M e´ xico, and Unidad Profesional Interdisciplinaria de Biotecnolog ´ı a, Instituto Polit e´ cnico Nacional, Barrio La Laguna
Ticom a´ n, Ciudad de M e´ xico, M e´ xico
Abstract
Epigenetic alterations are associated with cancer and their targeting is a promising approach Flexible docking, histone deacetylase
Keywords
for treatment of this disease. Among current epigenetic drugs, histone deacetylase (HDAC)
inhibitors induce changes in gene expression that can lead to cell death in tumors. Valproic acid
inhibitors, molecular modeling, valproic
acid, X-ray structure
(VPA) is a HDAC inhibitor that has antitumor activity at mM range. However, it is known that
VPA is a hepatotoxic drug. Therefore, the aim of this study was to design a set of VPA
derivatives adding the arylamine core of the suberoylanilide hydroxamic acid (SAHA) with
different substituents at its carboxyl group. These derivatives were submitted to docking
simulations to select the most promising compound. The compound 2 (N-(2-hydroxyphenyl)-2-
propylpentanamide) was the best candidate to be synthesized and evaluated in vitro as an anti-
cancer agent against HeLa, rhabdomyosarcoma and breast cancer cell lines. Compound 2
showed a better IC50 (mM range) than VPA (mM range) on these cancer cells. And also, 2 was
particularly effective on triple negative breast cancer cells. In conclusion, 2 is an example of
drugs designed in silico that show biological properties against human cancer difficult to treat
as triple negative breast cancer.
History
Received 20 May 2016
Revised 27 June 2016
Accepted 28 June 2016
Published online 28 July 2016
Introduction
years are diagnosticated with cancer in developed countries, of
4
which 60% die . Pediatric leukemia represents 30% of the
It is thought that in the genesis of cancer there is a loss of ability
to control cell proliferation, which is secondary to altered genetic
1
material or a disruption of epigenetic processes . Cancer is a
pediatric cancer in which acute lymphoblastic leukemia (ALL)
has had improvements in the treatment yielding well results in
survival rate (90% in the latest studies) contrary to myeloid
major cause of mortality, in 2012 there was approximately
1
4 million new cases and 8 million cancer-related deaths,
affecting populations in all the countries and all the regions.
These estimates correspond to age-standardized incidence and
5
leukemia and other ALL . Due to the great number of pediatric
patients that die secondary to cancer disease and also, due to there
are several unknown factors associated to these diseases, the
European Pediatric Regulation has invited to develop drugs
focused to pediatric population alongside to research programs for
adults. However, there are several genetic and epigenetic factors
that could represent an important genomic project to find a
2
mortality rates of 182 and 102 per 100 000, respectively , being
3
% children . Each year, more than 160 000 children under 20
3
6
potential treatment or treatments in this kind of patients .
Address for correspondence: Jos e´ Correa-Basurto, Laboratorio de
Modelado Molecular y Bioinform a´ tica, Laboratorio de Bioqu ´ı mica,
Laboratorio de Biof ´ı sica y Biocat a´ lisis, Secci o´ n de Estudios de
Posgrado e Investigaci o´ n, Escuela Superior de Medicina, Instituto
Histones are octameric proteins formed by dimers of 2A, 2B, 3
and 4 subunits. In histones, the DNA material is bound and
therefore, the inter-histone interactions facilitate or limit the
´
cnico Nacional, Plan de San Luis y D ´ı az Mir o´ n, Ciudad de accessibility to genes promoters in DNA, which induce or inhibit
Polite
M e´ xico 11340, M e´ xico. E-mail: jcorreab@ipn.mx
7
gene transcription . The mechanisms include conformational

2
B. Prestegui-Martel et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
R3
R2
O
O
O
H
N
OH
N
H
HO
N
H
R1
O
Scheme 1. Design of VPA-aryl derivatives (center) by mixing valproic acid (VPA; left) with the arylamine core of the suberoylanilide hydroxamic acid
SAHA) with different substituents (right).
(
changes either DNA or histones due to their post-translational
On the other hand, suberoylanilide hydroxamic acid (SAHA)
modifications such as histone acetylation, methylation, phosphor- was the first FDA-approved HDAC inhibitor as anti-cancer agent
ylation, ubiquitination, etc., allowing that the DNA could be that is able to induce differentiation, and/or apoptotic cell death of
2
2
reached by transcription factors.
The acetylation and deacetylation of histone N-terminal Lys about the role of HDAC8 and its relationship with several cancer
transformed cells in vitro and in vivo . In addition, it is known
2
3,24
residues are the main regulators of interactions among histones, as disease, including the breast cancer .
well as between histones and DNA. These chemical changes are The aim of the present study was to design a set of VPA aryl
achieved by enzymes called histone acetyltransferases (HATs) and derivatives that maintain the SAHA core (aryl moiety) in an
8
histone deacetylases (HDACs) . HATs and HDACs are capable of attempt to improve the desired inhibitory effect with low toxicity
controlling genes by activating or silencing them under dynamic on non-tumorigenic cells. First, an in silico study was achieved
9
processes . HDACs constitute a family of zinc-dependent targeting HDAC8 in tridimensional (3D) model to select those
2
+
enzymes involved in the above processes that are divided into compounds that reached the catalytic site featured by Zn atom
four main groups. Group I is comprised of isoforms 1, 2, 3, and 8, and showed the best binding free energy values compared to VPA.
the second set includes isoforms 4, 5, 6, 7, 9, and 10 which are We obtained a promising compound named N-(2-hydroxyphenyl)-
distributed inside either cytoplasm or nucleus; the third group (or 2-propylpentanamide (2) (Scheme 1), which was chemically
sirtuins) are characterized by using NAD as cofactor instead of characterized by melting point, infrared spectroscopy (IR),
1
13
zinc; finally, group 4 contains isoform 11, which shares homology nuclear magnetic resonance spectroscopy ( H and C NMR),
with the group 2. The catalytic site of group 1 is highly conserved, mass spectrometry (MS) and X-ray studies. Subsequently, this
containing mainly aromatic amino acids (Phe, Tyr and Trp), an compound was tested as an anti-proliferative agent on cervical
+
2
acid amino acid (Asp), and a zinc ion (Zn ); the same cancer (HeLa cells), rhabdomyosarcoma and breast cancer cells,
composition is found in group 2 which gives great structural where it showed higher activity than VPA.
differences as high catalytic site volume. The mechanism of
removing the e-amino acetyl group from Lys residues of
nucleosomal histones is widely described.
Experimental
1
0
However, there are differences among the patterns of epigen- Molecular modeling
etic changes which are HDAC isoform-specific. Thus, despite of
their highly conserved catalytic site, there are different residues
Docking and molecular dynamics simulations
1
1
that change their protein entrances , also, due to their cell In order to evaluate the plausible molecular recognition of 2 over
1
localization which modify their natural substrate differences . HDAC8 and the possible inhibitors here proposed, docking
2
2
5
HDACs inhibitors can suppress the activity of several HDACs by studies were carried out . Firstly, all the 2D chemical structures
increasing the expression of specific genes related to cell were drawn with IsisDraw program (http://mdl-isis-draw.updates-
morphology, metabolism, growth arrest, cell cycle arrest, differ- tar.com/es), submitted these structures to a previous pre-opti-
1
3,14
entiation, apoptosis and cell differentiation, among others
.
mization of the geometry and energy at AM1 semi-empirical
These HDAC inhibitors can be grouped into natural and synthetic level, employing HyperChem software (http://www.hyper.com/);
compounds. The natural compounds include trichostatin A (TSA), the complete geometrical and energetically optimization of the
apicidin, sodium butyrate, depudecin. Some synthetic compounds ligands was achieved at B3LYP6–31 level with Gaussian 98
2
6
are pyroxamide, analogs of TSA, oxamflatin, sulfonamide package , the outputs Z-matrix were converted into a .pdb input
hydroxamic acid, escriptaid, derivatives of phenylbutyrate and file to be docked with Molekel program (http://ugovaretto.githu-
1
5
valproic acid (VPA) . One of the most targeted HDACs is b.io/molekel/). Two HDAC8 crystal structures were retrieved
isoform 8, which is well known its role in cancer associated to from RCSB-PDB (ID: 1W22 and 2V5W), which were submitted
1
6
17
epigenetic changes . Eyal et al. determined the nonselective them to a refinement process prior to docking simulations.
inhibition of HDACs using structural isomers of VPA. They found Briefly, the crystal structures were stripped of additional
2
+
that 4-ene-valproic and 2-ene-valproic isomers showed IC50 molecules keeping the metallic atom Zn required to catalyst
values of 1.5 mM and 2.8 mM, respectively, compared to an IC
process in the orthosteric binding site. Afterwards, protein
of 1.5 mM for VPA . Similarly, a relationship between HDAC structures were submitted to Molecular Dynamics simulations
5
0
1
7
1
8,19
27
inhibition and cytotoxicity in tumor cells was found
. This with NAMD2.8 running a 10 ns of total simulation under
made researchers still searching the effectiveness of VPA standard conditions considering a 310 K temperature and 1 ATM
exploring the cell growth inhibition under apoptosis in endomet- pressure, which were kept constant with a time step of 2 fs using
rial cancer cells in vitro and in mice model without finding the Shake algorithm for all hydrogen atoms. Docking analysis
2
8
2
0
29
adverse effects . In addition, it has been exploited the pharma- were conducted with AutoDock4.0.1 and the used parameters
cological target of VPA under experimental procedures by were as follows: blind docking procedure with a grid box of
3
˚
comparing this drug with other more potent HDAC inhibitors, 126 ꢀ 126 ꢀ 126 A covering the entire orthosteric binding site
˚
these last show limitations such as toxicity, short half-life and low into the protein, spacing between each grid point of 0.375 A, as
2
bioavailability .
1
scoring sampling the Lamarckian Genetic algorithm was selected

DOI: 10.1080/14756366.2016.1210138
Valproic acid aryl derivative
3
5
'
Scheme 2. Synthesis of target compound 2.
Initially, VPA reacted with oxalyl chloride to
make an amide group by adding the ortho
hydroxy aniline. Reagents and conditions: (1)
oxalyl chloride and (2) o-aminophenol.
O
O
4'
3'
O
1
6'
5
3
(1)
(2)
2
OH
Cl
N
H
1'
2
'
4
OH
VPA
A
2
considering a randomized initial population of 100 individuals,
7
and a 1 ꢀ 10 cycles as maximum number of energy evaluations.
Table 1. X-ray data, collection and
refinement parameters for 2.
Synthesis
Empirical formula
Formula weight
Size (mm)
14 2
C H21NO
235.32
Materials and methods
0.14X0.19X0.51
Monoclinic
P2 /c (No. 14)
All chemicals (Sigma-Aldrich Qu ´ı mica, S.A. de C.V., Toluca de
Lerdo, M e´ xico) and solvents (Tecsiquim, S.A. de C.V., Ciudad de
M e´ xico, M e´ xico) were of reagent grade and were used as
received. Melting points (mp) was determined on an
Electrothermal IA 91000 apparatus (Electrothermal, Bibby
Scientific, Staffordshire, UK), these are uncorrected. IR spectra
were recorded on a Perkin Elmer Frontier FT-IR spectrometer
(PerkinElmer, Shelton, CT). Absorption values are expressed as
the number of waves (cm ), and in the present report only the
significant absorption bands are included. H and C NMR
spectra were obtained on a Varian Mercury 300 MHz spectrom-
eter (Varian, Inc., Palo Alto, CA) at 300 and 75.4 MHz,
Crystal system
Space group
1
˚
a (A)
8.0515(4)
˚
b (A)
9.0833(4)
19.0559(10)
90
98.740(2)
90
1377.45(12)
1.135
4
0.075 (Mo K
173(2)
4.978–50.57
11053
2512
6.83
1767
156
1.107
˚
c (A)
a ( )
ꢂ
ꢂ
b ( )
ꢂ
g ( )
3
V ( A˚ )
ꢁ1
3
ꢂ
Z-value
ꢃ (mm
T (K)
calcd (g/cm )
1
13
ꢁ1
)
a
)
ꢂ
2
Total reflections
y
range ( )
respectively, using deuterated dimethyl sulfoxide (DMSO-d ) as
6
the solvent. Electrospray Ionization (ESI) high resolution MS was
performed with a Bruker micrOTOF-Q II instrument (Bruker
Daltonik GmbH, Bremen, Germany).
Unique reflections
R
int (%)
I42s(I)
Parameters
Goodness of fit
(%)
min, ꢂmax (e A
N-(2-hydroxyphenyl)-2-propylpentanamide (2)
R (%), R
ꢂ
w
5.75, 19.51
ꢁ0.59, 0.67
1429149
-
ꢁ3
)
˚
Oxalyl chloride (3.71 mL, 34.6 mmol) was added dropwise to
ꢂ
CCDC deposition
VPA (5 g, 34.6 mmol) at 0 C. The reaction mixture was stirred for
8
h at the same temperature; then, it was allowed to warm at room
temperature under nitrogen to give 2-propylpentanoyl chloride
A) (Scheme 2). The flux of nitrogen was continued for 15 min
(
diffractometer (Bruker AXS GmbH, Karlsruhe, Germany)
˚
and was suspended for 15 min; this alternated process was carried
ꢂ
equipped with Mo-K
radiation (ꢁ ¼ 0.71073 A). The frames
a
out for 3 h, followed by cooling at ꢁ5 C. Hexane (3 mL) and
were integrated with the Bruker SAINT Software package using a
narrow-frame algorithm. Data were corrected for absorption
ortho hydroxy-aniline (3.74 g) were added and the mixture stirred
for 12 h before adding hexane (10 mL) and sodium bicarbonate
3
effects using the multi-scan method SADABS . The final
0
(3 g); subsequently, the mixture was stirred for 3 h. The compound
2
anisotropic full-matrix least-squares refine on F . The structure
was obtained by distillate hexane extraction and the solid formed
was filtered and washed with hexane. The synthesis of the
compound was monitored by thin layer chromatography using a
hex/AcOEt (8:2) mixture as the eluent, obtaining 2 as a white
3
1
was solved by direct methods using SHELXS-2013
3
of
the WINGX package . The final refinement was performed by
2
3
1
full-matrix least-squares methods using the SHELXL-2013
program. Crystal data, collection and refinement parameters
are given in Table 1. H atoms on C, N, and O were
positioned geometrically and treated as riding atoms with: CꢁH
solid in 85% yield. White solid, R ¼ 0.45 (Hex/AcOEt 8:2),
f
ꢂ
mp ¼ 56 ± 2 C. FT IR (ATR) ꢀ
3260, 3128, 3071, 2959, 2930,
1 1
866, 1631, 1545, 1499, 752 cm . H NMR (300 MHz, DMSO-
max
ꢁ
2
˚
˚
0
.95ꢁ0.99 A, Uiso(H) ¼ 1.2 eq(C) or 1.5 eq(C); OꢁH ¼ 0.84 A,
d ) d 9.73 (br, 1H, OH), 9.37 (s, 1H, NH), 7.60 (d, J ¼ 3 Hz, 1H,
6
˚
0
0
0
Uiso(H) ¼ 1.5 eq(O); NꢁH ¼ 0.88 A, Uiso(H) ¼ 1.2 eq(N).
H-6 ), 6.95 (t, J ¼ 3 Hz, 1H, H-5 ), 6.85 (d, J ¼ 6 Hz, 1H, H-3 ),
3
3
34
0
Platon and Mercury were used to prepare the material for
publication. Data collection and refinement parameters, atom
coordinates, bond lengths and bond angles have been deposited
with the Cambridge Crystallographic Data Center. The CCDC
deposition number is 1429149.
6
.76 (t, J ¼ 6 Hz,1H, H-4 ), 2.58 (q, J ¼ 3 Hz, 1H, H-2), 1.55–1.50
(
m, J ¼ 3 Hz, 2H, H-3), 1.36–1.24 (m, 6H, H-3, 2H-4), 0.87 (t,
1
3
J ¼ 3 Hz, 6H, 2H-5). C NMR (75.4 MHz, DMSO-d ) d 175.5
6
0 0 0 0
C-1), 148.7 (C-2 ), 126.8 (C-1 ), 125.4 (C-4 ), 123.1 (C-6 ), 119.5
(
(
0
0
C-5 ), 116.7 (C-3 ), 46.0 (C-2), 35.5 (2C-3), 20.7 (2C-4), 14.5
+
2C-5). ESI-MS calculated for C H NO : [M + Na] 258.1470.
(
Found: 258.1482.
1
4
21
2
Biological evaluation
Cell cultures
X-ray diffraction
Cancer cell lines HeLa (cervical cancer), MCF-7, MDA-MB-231,
Single crystal of 2 was ground by slow crystallization from SKBr3, MCF-10A (breast), A204 (rhabdomyosrcoma) were
hexanes. The X-ray data were collected on a Bruker D8 Venture obtained from ATCC, whereas Human Umbilical Vein

4
B. Prestegui-Martel et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
Endothelial Cells (HUVEC) were from primary culture. HUVEC Graphic Prism program V.6.0, through a logarithmic analysis of
were isolated from umbilical cord according to a previously four variables. The statistical analysis was realized with t de
3
5
described procedure . Cells were grown in M199 media with 1% student test, considering a p values of 0.05 difference significance
endothelial cell growth supplement, heparin, and 15% fetal bovine statistical, for comparative anti-proliferative effect between com-
serum. The donor of umbilical cord was recruited at the Hospital pound 2 and VPA.
Juarez of Mexico. The Ethics and Research Committee of the
Hospital Juarez of Mexico approved the participation of the donor
Results and discussion
and a written informed consent was obtained.
HeLa and A204 cells were cultured in DMEM medium (Gibco Molecular modeling
Lab, Grand Island, NY) in 5% bovine fetal serum. MCF-7 and
Docking simulations of VPA aryl derivatives on HDAC8
MDA-MB-231 cells were preserved in DMEM high glucose
(
4.5 g/L) medium with phenol red, 5% bovine fetal serum and The theoretical studies show several advantages during the
antibiotic 1X (streptomycin/penicillin 10000 U/mL, Gibco Lab, development of new chemical entities with potential biological
Grand Island, NY). MCF-10A cells were cultured in DMEM/F12 activity on a determinate target, such as those implicated in
medium without phenol red and SKBR3 were cultured in DMEM/ cancer. Based on the chemical knowledge of the ligands and/or
ꢂ
F12 with 10% bovine fetal serum. Cells were incubated at 37 C in their targets, it is possible to design molecules from combining
a humid atmosphere of 5% CO , keeping the environment at different pharmacophores able to reach different sites of the same
2
physiological pH.
protein or distinct proteins (bi-target or multi-target compounds),
In order to corroborate the biological effects of VPA (positive which could improve the anti-proliferative effect in situ, and
3
6
control) and explore those of 2, cell cultures were used to study consequently, in the improvement of the treatment efficiency .
cytotoxicity. For this purpose, 2 was evaluated in HeLa cells at the
same concentrations reported for VPA in this cell line.
In this work, we focused on the design and evaluation of a set
of VPA aryl derivatives with the aim of increasing the biological
potency of this drug, thus an aryl moiety was included at the
carboxyl group of VPA to get certain similarity with the aryl core
of SAHA molecule (Scheme 1), being the common target of these
Cell antiproliferative activity assays
Cervical cancer (HeLa) and rhabdomyosarcoma (A204) cell line. pharmacophores the HDAC8, since it has been shown that this
2
The effect of 2 and VPA on cell proliferation was measured with protein has several binding sites of recognition . Once the
1
4
the tetrazolium salt WST-1. First, 10 cells were plated in 96-well ligands were designed, they were submitted to docking simula-
ꢂ
plates and incubated for 24 h (37 C, 5% CO ) to allow them to tions on previously reported HDAC8 structures (PDB code: 1W22
2
adhere to the plate. Then different concentrations of the drug and 2V5W), and using the methodology previously reported for
solutions (2 and VPA) were prepared (from the stock solution) exploring the VPA recognition pattern on HDAC8 conform-
3
7
and added to the culture medium to treat the cells for 24 h. ations . Hence, the parameters used to select the best com-
Subsequently, the content of each plate was aspirated and 100 mL pounds, were the free energy values (Table 2) and the binding
of culture medium and 10 mL of WST-1 were added, followed by pose. The interaction of VPA aryl derivatives on HDAC8 in the
incubation for 4 h. Then, the plates were read with an ELISA plate docking simulations showed that the compounds 2, 18, 19, 20 and
reader at 520 nm. The IC50 was calculated by testing different 21 had the better affinity values (Figure 1), which were favored by
drug concentration-responses, plotting the values, and making a certain types of chemical substituents and their structural
curve on the University of Tokyo server (http://bsmdb.tmd.ac. JP conformation adopted through docking. Furthermore, these par-
3
000/) through a logarithmic analysis of four variables.
Furthermore, cell proliferation was measured by the 3-(4, 5- (Ki of HDAC1: 0.4 mM) .
dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) We decided to conduct a search for the binding properties of
ameters have shown certain relationship with experimental data
3
8
assay. Cells were treated as aforementioned and at the end of VPA with a region of the HDAC protein that Vannini et al.
the experimental period the medium was removed and replaced described in 2007 as the acetyl shuttle site (ASL), which was
3
9
with 100 mL of medium with MTT (0.5 mg/mL), but without removed during catalysis . This region is close to the stearic site,
phenol red, and incubated for 2.5 h. Then, the medium was in connection with it through a narrow passage that is probably
removed and replaced with 150 mL of DMSO. Optical density was responsible for removing the reaction product. In this search we
measured at 550 nm in a spectrophotometer (Sinergy Biotek found that there was a tendency of VPA to have higher affinity for
3
7
Instruments, Winooski, VT).
this ASL site than for the catalytic site in HDAC8 , and vice
Breast cancer cell lines. Initial concentrations used in this versa when VPA was tested with HDAC1, 2, and 3. For the
study were determined based on the previous results obtained members of Class II, the location or existence of this acetate
from the logarithmic curve. A 25 mM stock solution of 2 was release site is not clear. Based on this analysis, we proceeded to
prepared in dimethyl sulphoxide (DMSO) and a 25 mM stock search the drug library for other activity against HDAC8. We
solution of VPA in phosphate buffer solution (PBS). MCF-7 considered the aryl moiety of SAHA due to its structural stability
(ER+, PR + and HER2+), MDA-MB-231 (ER-, PR- and HER2), in the HDAC8 aromatic environment, in which Tyr100 serves to
SKBr3 (ER-, PR-, HER2+, GPER+) and MCF-10A (benign) cells engage the ligand in the protein-binding site
4
0,41
.
were treated with 800 mL of trypsin 0.05% (Invitrogen, Waltham,
MA) and counted with a Neubauer camera.
As can be seen, regardless of the substitution in each
compound, the orientation in space of the catalytic site is very
Each cell line was placed in 96-well plates at a density of similar (Figure 2, below). This is because the aliphatic chains
4
1
concentrations were prepared by dilution of the stock solution: site in all the cases, whereas the aromatic moiety reaches certain
ꢀ 10 cells per well and incubated overnight. Seven different stabilize the compound in the inlet of the throat of the catalytic
100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM and 400 mM. residues (as occurs for example with SAHA). The difference in
Cells were treated at each concentration for 24, 48, and 72 h. Each the affinity values depends on the type and amount of weak
concentration/time was tested in quadruplicate. The assay for the interactions caused by the substituted chemical group, which
interacts electrostatically with the zinc atom in HDAC8, thus
determination of IC50 was performed in triplicate.
The IC50 was calculated by testing different drug concentra- providing this protein with a partial negative charge (Figure 2,
tion-responses, plotting the values, and building a curve in left). In the current contribution, the VPA aryl derivatives show a

DOI: 10.1080/14756366.2016.1210138
Valproic acid aryl derivative
5
Table 2. Free energy values of the VPA aryl derivatives coupled on two crystal structures of HDAC8 retrieved from
the protein data bank (PDB) obtained by docking studies.
R3
R2
O
N
H
R1
1
2
R
3
R
Ligand
R
PDB ID: 1W22
PDB ID: 2V5W
VPA
—
H
OH
H
H
OCH
H
H
F
H
H
Cl
H
H
NO
H
H
CO
H
H
NH
H
H
—
—
H
H
OH
H
H
OCH
H
H
F
H
H
Cl
H
H
NO
H
H
CO
H
H
NH
H
—
H
H
H
OH
H
H
OCH
H
H
F
H
H
Cl
H
H
NO
H
H
CO
H
H
ꢁ7.27
ꢁ7.41
ꢁ8.83
ꢁ7.84
ꢁ8.21
ꢁ6.41
ꢁ7.22
ꢁ7.2
ꢁ7.0
1
2
3
4
5
6
7
8
9
ꢁ8.12
ꢁ8.25
ꢁ8.25
ꢁ7.86
ꢁ5.46
ꢁ7.45
ꢁ7.39
ꢁ7.45
ꢁ5.66
ꢁ7.2
3
3
3
ꢁ7.6
ꢁ6.27
ꢁ6.31
ꢁ6.44
ꢁ7.12
ꢁ6.6
10
11
12
13
14
15
16
17
18
19
20
21
22
ꢁ5.81
ꢁ6.32
ꢁ7.2
2
ꢁ7.57
ꢁ8.03
ꢁ9.07
ꢁ5.57
ꢁ8.12
ꢁ9.13
ꢁ9.22
ꢁ8.2
ꢁ5.79
ꢁ7.91
ꢁ6.97
ꢁ5.52
ꢁ6.58
ꢁ8.98
ꢁ5.15
ꢁ8.49
ꢁ8.6
2
2
2
2
2
2
2
NH
—
2
ꢁ8.72
ꢁ8.92
TSA
—
ꢁ8.39
−
−
−
−
−
−
4
5
6
7
8
9
Figure 1. Relationship of the binding energy
kcal/mol) of each compound tested in the
two crystal structures of HDAC8.
1W22
V5W
(
2
−
10
Ligands
binding pose behavior different than that observed for the agreement with the proposed structure, particularly, amide NH
3
lead compound . This means that the aryl addition induced a and phenolic OH are observed at 9.37 and 9.73 ppm in H NMR
7
1
ꢁ
1
SAHA binding site recognition, which explains the better spectrum and carbonyl appears at 175.5 ppm and at 1631 cm in
1
3
affinities for the derivatives compared to that observed for VPA.
Hence, based on docking results, it was shown that the
compound N-(2-hydroxyphenyl)-2-propylpentanamide (2) is one analysis, the corresponding structure is shown in Figure 3 and
C NMR and IR spectra, respectively.
Compound 2 gave single crystals suitable for X-ray diffraction
of the most promissory compounds according to its free energy selected geometric parameters are listed in the captions. Bond
values and non-bond interactions.
lengths and angles are characteristics of the amide bond. The
structure of 2 in the crystal is very similar to that adopted in the
high affinity conformation of HDAC obtained from docking. In
both cases, the amide group is out of the mean phenyl plane with
Chemistry
Compound 2 was successfully synthesized in 85% yield by O(17)C(8)N(7)C(1) and C(2)C(1)N(7)C(8) torsion angles of
chlorination of valproic acid (VPA) with oxalyl chloride to form 11.1(4) and ꢁ44.2(3) in the crystal and 0.06 and ꢁ82.01 in the
2-propylpentanoyl chloride (A) and subsequent amidation with docked conformation, respectively. In this last, the OH group is
ortho hydroxy aniline (Scheme 2). Analytical data are in twisted to the anti-conformer by hydrogen bonding with Tyr306

6
B. Prestegui-Martel et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
Figure 2. Interactions of compound 2, 18, 19, 20 and 21, (A) all ligands are capable of adopting the same binding pose. (B) 2 is making a coordination
+
with Zn . (C) and (D) show the cavity of HDAC8 reached by the target ligands.
2
of HDAC binding site, whereas in the crystal the OH is syn compound, resulting in 0.92 mM for 2 and 9.12 mM for VPA.
positioned to the benzene ring. The OHꢃꢃꢃO hydrogen bonding The IC50 value for VPA was calculated as previously reported
4
between phenolic donor and carbonyl amide acceptor, gives shape using a concentration 3 mM .
2
6
to a helix type structure in the crystal.
Once obtaining the IC50 for each drug, we seeded 1 ꢀ 10 cells
in 25 mL culture flasks, each with 5 mL of DMEM medium (alone
or in combination with the IC50 of one of the drugs). We made a
photographic follow-up of both time and effect. Whereas there
was an increase in the number of cells in the control, the cells in
the flasks with 2 and VPA maintained the same level from 0 to
Biological activity
In vitro antiproliferative activity of 2 on cervical cancer cells
(HeLa)
2
4 h (data not shown). This can be explained by a cytostatic effect
After standardizing the management of the cell line, we or a certain degree of apoptosis induced by the drugs.
proceeded to obtain information of the effect of 2 comparing Subsequently, an assay was conducted to detect apoptosis in
this with that of VPA. A concentration response curve was order to clarify the type of cell death that each drug caused. We
ꢁ
3
constructed under logarithmic units (100 mM to 1 ꢀ 10 mM for used an apoptosis detection kit (Takara Inc.) to evaluate cellular
ꢁ4
VPA, 10 mM to 1 ꢀ 10 mM for 2) by measuring the percentage DNA cut by endonucleases (typical of the apoptotic process but
of cell viability. The sigmoid curves reflect the concentration- not of necrosis). Fragmented DNA is identified by adding TdT
dependent effect of both compounds based on the decrease of cell (terminal deoxynucleotide transferase), which complements the
0
viability determined by the MTT assay (Figure 4A). Compound 2 chains on any 3 OH found. The nucleotides used are marked with
proved to be 10 times more potent than VPA. With the curve, it fluorescein and later stimulated with UV light. The control cells
was possible to calculate the IC50 on HeLa cells for each (untreated cells) were observed in red, showing no fluorescence.

DOI: 10.1080/14756366.2016.1210138
Valproic acid aryl derivative
7
Figure 3. Molecular structure of compound 2. Selected geometric
˚
parameters: bond lengths (A): O(2)–C(2) 1.366(3), O(17)–C(8)
ꢂ
1.245(2), N(7)–C(1) 1.426(3), N(7)–C(8) 1.337(3); bond angles ( ):
C(1)–N(7)–C(8) 127.36(17), O(2)–C(2)–C(1) 123.3(2), O(2)–C(2)–C(3)
1
N(7)–C(8)–C(9) 117.23(17); torsion angles ( ): C(2)–C(1)–N(7)–C(8)
–44.2(3), N(7)–C(1)–C(2)–O(2) –3.3(3), O(17)–C(8)–N(7)–C(1) 11.1(4),
C(9)–C(8)–N(7)–C(1) –167.9(2), O(17)–C(8)–C(9)–C(10)–59.7(3),
O(17)–C(8)–C(9)–C(13) 65.4(3), N(7)–C(8)–C(9)–C(13)–115.6(2).
17.7(2), O(17)–C(8)–N(7) 122.0(2), O(17)–C(8)–C(9) 120.81(19),
ꢂ
Figure 4. (A) Concentration-response curve of VPA and 2 in HeLa cells.
B) Concentration-response curve of VPA and 2 in the A204 cell line. All
data on cell proliferation after treatment are expressed as the percentage
of cell growth of treated versus untreated (control) cells. All assays were
determined by WST.
(
Whereas some slightly positive cells were found when using
Endothelial and MCF-10A cell lines (non-tumorigenic) were
the IC50 of 2 and VPA (in a time-dependent trend: 24 h548 h), used as a control because they are considered normal epithelial
the fluorescence was intense (more seen in the nucleus) when the cells. The effect of 2 was assayed at the same concentrations
IC99 concentration was employed for 4 h, indicating a strong previously used. With 250 mM of compound 2 (IC50 ¼213.7 mM),
apoptotic response (data not shown).
at 72 h of treatment cell proliferation decreased to 11.3% of that
found in the control cells (Figure 5C). This result indicates that
normal breast cells were more sensitive than breast cancer
cell lines.
In vitro antiproliferative activity of 2 on rhabdomyosarcoma
(A204) cell line
When we treated SKBr3 cells with 2, a higher inhibitor effect
on cell proliferation was found than with the other cell lines.
Hence, we used lower concentrations (0–250 mM) to find the IC .
VPA did not have any inhibitory effect. Rather, we observed a
slight increase in cell proliferation at the highest concentrations
used (Figure 5D).
The IC50 values of 2 were the following: 192.4 mM for MCF-7
cells, 283.0 mM for MDA-MB-231 cells, 213.7 mM for MCF-10A
cells, and 142.0 mM for SKBr3 cells. As reported in Table 3, 2
exhibits cytotoxic activity on all cell lines. This compound
showed activity at concentrations that could be considered
clinically acceptable. In contrast, at the same concentrations
Assays were conducted with the rhabdomyosarcoma (A204) cell
line to identify the activity of 2 and VPA. Since doxorubicin is
known to have activity against this type of tumor, it was added as
a positive control. Compound 2 and doxorubicin had a similar
profile on cell growth inhibition, while VPA showed approxi-
mately 100 times less potency (Figure 4B). The concentration-
response curves were created with the University of Tokyo server
to obtain the IC50 concentration for each compound: 36.42 mM for
50
2, 12.25 mM for VPA, and 12.22 mM for doxorubicin.
In vitro antiproliferative activity of 2 on breast cancer cell lines
MCF-7, MDA-MB-231, SKBr3, endothelial (Human Umbilical VPA was not able to exert an effect on breast cancer cell lines.
Vein Endothelial Cells (HUVEC) and MCF-10A cells were either These results suggest that 2 is capable of inhibiting cell growth of
untreated or treated with different concentrations of 2 and VPA estrogen responsive cells, with the greatest effect found in the
for 72 h (0–350 mM). After this time, it was found that the SKBr3 cell line.
concentrations of 2 that were employed suppressed MCF-7 cell
It is known that there are reports in which VPA exerts its
proliferation in a concentration-dependent manner, with the inhibitory activity of HDACs at concentrations higher than those
highest concentration decreasing cell to 20.8% of that found in found herein. In 2007, Fortunati et al. reported an IC50 of
the control cells (Figure 5A; p ¼ 0.001). However, with VPA cell 0.74 mM for VPA in MCF-7 cells and of 1.58 mM for this drug in
3
43
proliferation was maintained at about 100% regardless of the MDA-MB-231 cells (using 3 ꢀ 10 cells for eight days) .
concentration. A similar effect was observed in MDA-MB-231 Moreover, this group demonstrated that VPA strongly induces
4
3
cells treated for 72 h (Figure 5B), with 400 mM of 2 decreasing apoptosis in MCF-7 cells by activating caspase 8 . In the present
nc eo lle pf freo cl ti .f eration to 8.7% (p ¼ 0.003), and VPA showing virtually study the IC50 values are lower than those indicated by Fortunati,
suggesting that 2 has a greater effect on both cell lines than VPA

8
B. Prestegui-Martel et al.
A)125
00
J Enzyme Inhib Med Chem, Early Online: 1–10
(
(B) 150
1
1
00
75
50
25
0
5
0
2
VPA
2
VPA
0
0
100
200
300
400
0
100
200
300
ConcentrationmM
ConcentrationmM
(C)125
(D) 150
100
1
00
75
50
25
0
5
0
MCF-10A
Endothelial
2
VPA
0
0
100
200
300
400
0
50
100
150
200
250
ConcentrationmM
ConcentrationmM
Figure 5. Effect of 2 and VPA on cell proliferation in: (A) MCF-7 cells (0–350 mM concentration of the compounds), (B) MDA-MB-231 cells (0–
50 mM concentration of the compounds), (C) MCF-10A and endothelial cells (0–400 mM concentration of 2, and (D) SKBr3 cells (0–250 mM
concentration of the compounds). All data on cell proliferation after treatment are expressed as the percentage of cell growth of treated versus untreated
4
(
control) cells. All assays were determined by MTT. Results are expressed as the mean ± SD of three independent experiments (n ¼ 4).
Table 3. IC50 for 2 in breast cancer cell lines and non-tumorigenic cells.
MCF-7
ER+, HER2+
MDA-MB-231
ER-,HER2-
SKBr3
ER-, PR-,HER2+, GPER+
MCF-10A
GPER+
Cell estrogen receptor
IC50 (mM)
Epithelial
0.24
0.19
0.28
0.14
0.21
due to its ability to reach the SAHA site on HDAC8, or by acting VPA is clinically relevant because its anti-proliferative activity
on GPR30/GPER (a promissory target in drug design for current as an HDAC inhibitor has been recently described in breast cancer
46
chemotherapy) if compound were metabolized by the CYP450 patients, finding a mean plasma concentration of 86.3 mg/mL .
giving as result a dihydroxylated aryl moiety capable to interact In this study VPA was administrated with hydralazine, a
4
6
with the binding site onto GPER/GPR30. In 2012, Li et al. methylation inhibitor . In 2007, M u¨ nster et al. conducted a
demonstrated that concentrations of up to 3.2 mM of VPA had no clinical phase I study with 44 patients given 160 mg/kg/day of
effect on cell proliferation of MDA-MB-231 cells, although VPA, finding various adverse effects, such as drowsiness,
4
4
inhibition of cell migration was observed . This could explain hyponatremia, hypocalcemia, fatigue, hallucinations, dizziness,
7
4
why we did not find any changes in the proliferation of cells headache or dehydration . They found that the recommended
herein treated with VPA. phase II study dose is 140 mg/kg/day for 48 h, followed by
2
In 2011, Park et al. found that expression of HDAC4, 6 and 8 is 100 mg/m of epirubicin. There are no clinical reports in which
higher in MDA-MB-231 cells than MCF-7 cells, and that these VPA is administered as monotherapy for solid tumors. Hence, the
isoforms are correlated with an increased expression of current contribution demonstrates that the interaction of 2 with
metalloproteinase 9 (MMP-9), which is associated with cellular two therapeutic targets was able to inhibit cell growth with a
4
5
invasion . We observed that 2 had a significant effect on the lower concentration compared to the formerly reported combin-
decrease in cellular proliferation of MDA-MB-231, whose ation therapy. These data suggest that compound 2 is a promissory
characteristics is more aggressive than MCF-7 cells.
potential anticancer drug deserving further preclinical and clinical
By using in silico assays we demonstrated that 2 binds to development.
HDAC8 with greater affinity than VPA, evidenced by the Kd of
0
.33 mM versus 4.63 mM (2 versus VPA, respectively), calculated
from their free energy values (Table 2). These theoretical data
could explain the decrease in the proliferation of cancer cells used The chemical substitution of designed molecules that inhibit
presently.
HDAC8 isoform could result in a more efficient inhibition of this
Conclusions

DOI: 10.1080/14756366.2016.1210138
Valproic acid aryl derivative
9
enzyme than that of the starting compounds. In the present study
the compound 2 was designed and synthesized, which was the
result of adding a mono-substituted aryl core at the carboxyl
group of VPA to give an arylamide derivative (structural
similarity with SAHA). According to theoretical results, the
compound 2 apparently targets to HDAC8. Additionally, the
tested compound was able to inhibit the tumor cell proliferation in
vitro at a much lower concentration than that required for
achieving the same effect with VPA.
lymphoproliferation in the Fas-deficient MRL/lpr(ꢁ/ꢁ) murine
model of autoimmune lymphoproliferative syndrome (ALPS). Exp
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2
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5
06–15.
The authors have declared that no competing interests exist. The
present study was supported by grants from CONACYT (CB-
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