Design, Synthesis, and Pharmacological Evaluation of Novel N-Acylhydrazone Derivatives as Potent Histone Deacetylase 6/8 Dual Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.5b01525

This manuscript describes a novel class of N-acylhydrazone (NAH) derivatives that act as histone deacetylase (HDAC) 6/8 dual inhibitors and were designed from the structure of trichostatin A (1). Para-substituted phenyl-hydroxamic acids presented a more potent inhibition of HDAC6/8 than their meta analogs. In addition, the effect of compounds (E)-4-((2-(4-(dimethylamino)benzoyl)hydrazono)methyl)-N-hydroxybenzamide (3c) and (E)-4-((2-(4-(dimethylamino)benzoyl)-2-methylhydrazono)methyl)-N-hydroxybenzamide (3f) on the acetylation of α-tubulin revealed an increased level of acetylation. These two compounds also affected cell migration, indicating their inhibition of HDAC6. An analysis of the antiproliferative activity of these compounds, which presented the most potent activity, showed that compound 3c induced cell cycle arrest and 3g induced apoptosis through caspase 3/7 activation. These results suggest HDAC6/8 as a potential target of future molecular therapies for cancer.

Full text of DOI:10.1021/acs.jmedchem.5b01525

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Article
Design, Synthesis and Pharmacological Evaluation of Novel N-
Acylhydrazone Derivatives as Potent Histone Deacetylase 6/8 Dual Inhibitors.
Daniel Alencar Rodrigues, Guilherme Ferreira-Silva, Ana Carolina Ferreira, Renan Fernandes,
J. K. Kwee, Carlos Maurício R Sant'Anna, Marisa Ionta, and Carlos Alberto Manssour Fraga
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 24 Dec 2015
Downloaded from http://pubs.acs.org on December 24, 2015
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Design, Synthesis and Pharmacological Evaluation of Novel NꢀAcylhydrazone Derivatives as
Potent Histone Deacetylase 6/8 Dual Inhibitors
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a, b
e
f
f
Daniel A. Rodrigues, Guilherme À. Ferreira-Silva, Ana C. S. Ferreira, Renan A. Fernandes,
f
a, d
e
*, a, b, c
Jolie K. Kwee, Carlos M. R. Sant’Anna, Marisa Ionta, Carlos A. M. Fraga
a
Laboratório de Avaliação e Síntese de Substâncias Bioativas (LASSBio), Instituto de Ciências
Biomédicas, Universidade Federal do Rio de Janeiro, PO Box 68023, 21941ꢀ902, Rio de Janeiro,
RJ, Brazil
b
Programa de PósꢀGraduação em Química, Instituto de Química, Universidade Federal do Rio de
Janeiro, 21941ꢀ909, Rio de Janeiro, RJ, Brazil
c
Programa de PósꢀGraduação em Farmacologia e Química Medicinal, Instituto de Ciências
Biomédicas, Universidade Federal do Rio de Janeiro, 21941ꢀ902, Rio de Janeiro, RJ, Brazil
d
Departamento de Química, Instituto de Ciências Exatas, Universidade Federal Rural do Rio de
Janeiro, 23970ꢀ000, Seropédica, RJ, Brazil
e
Laboratório de Biologia Animal Integrativa, Departamento de Biologia Celular e do
Desenvolvimento, Instituto de Ciências Biomédicas, Universidade Federal de Alfenas, 37130ꢀ
0
00, Alfenas, MG, Brazil
f
Coordenação de Pesquisa, Instituto Nacional de Câncer, 20231ꢀ050, Rio de Janeiro, RJ, Brazil
KEYWORDS: HDAC, Nꢀacylhydrazone, Cancer, Hepatocellular carcinoma
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ABSTRACT
This manuscript describes a novel class of Nꢀacylhydrazone (NAH) derivatives that act as
histone deacetylase (HDAC) 6/8 dual inhibitors and were designed from the structure of
trichostatin A (1). Para-substituted phenylꢀhydroxamic acids presented a more potent inhibition
of HDAC6/8 than their meta analogs. In addition, the effect of compounds (E)ꢀ4ꢀ((2ꢀ(4ꢀ
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(
dimethylamino)benzoyl)hydrazono)methyl)ꢀNꢀhydroxybenzamide (3c) and (E)ꢀ4ꢀ((2ꢀ(4ꢀ
dimethylamino)benzoyl)ꢀ2ꢀmethylhydrazono)methyl)ꢀNꢀhydroxybenzamide (3f) on the
(
acetylation of αꢀtubulin revealed an increased level of acetylation. These two compounds also
affected cell migration, indicating their inhibition of HDAC6. An analysis of the antiproliferative
activity of these compounds, which presented the most potent activity, showed that compound 3c
induced cell cycle arrest and 3g induced apoptosis through caspase 3/7 activation. These results
suggest HDAC6/8 as a potential target of future molecular therapies for cancer.
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INTRODUCTION
Two families of enzymes regulate histone acetylation patterns: histone acetyltransferases
(HATs), which catalyze the transfer of an acetyl group to the lysine residues in the Nꢀterminal
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1
tails, and histone deacetylases (HDACs), which remove the acetyl groups of these proteins. The
balance between the acetylation and deacetylation of histones affects the structure of chromatin
because an imbalance between these processes disrupts the interaction of histones with DNA.
HDAC removes an acetyl group, resulting in a compact chromatin state and thereby silencing
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,2
gene expression. In addition, some HDACs regulate the function of nonꢀhistone proteins, e.g.,
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cytoplasmic proteins and transcription factors.
Eighteen HDACs have been identified in the human genome, and these enzymes can be
further divided into two large groups: zincꢀdependent HDACs and nicotinamide adenine
+
dinucleotide (NAD )ꢀdependent HDACs, which are known as sirtuins (class III). In addition, the
zincꢀdependent HDACs can be further divided into classes and subclasses: class I (HDAC1,
HDAC2, HDAC3 and HDAC8), class IIa (HDAC4, HDAC5, HDAC7 and HDAC9), class IIb
3,4
(
HDAC6 and HDAC10), and class IV (HDAC11).
HDACs have been identified as remarkable drug targets in a novel therapeutic approach
for the treatment of cancer and related diseases because HDAC inhibition results in the growth
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ꢀ8
arrest, differentiation and apoptosis of many transformed cells. At present, four HDAC
inhibitors (HDACI) have been approved through the Food and Drug Administration (FDA)
(
Figure 1). Vorinostat (SAHA) was approved in 2006 for the treatment of cutaneous T cell
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lymphoma (CTCL). Subsequently, romidepsin (FKꢀ228)
and belinostat (PXDꢀ101) were
approved for the treatment of peripheral Tꢀcell lymphoma (PTCL), and in 2015, panobinostat
13
(
LBHꢀ589) was approved for the treatment of multiple myeloma.
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Figure 1. Chemical structures of FDAꢀapproved HDAC inhibitors.
Although these compounds are structurally distinct, most HDACIs possess a wellꢀknown
pharmacophore comprising a zincꢀbinding group (ZBG), a linker and a cap group to allow
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,7,14,15
interactions at protein surface.
Notably, no selective HDACIs have been approved for
clinical use because all approved drugs have been characterized as panꢀinhibitors of many
different HDAC isoforms.
This manuscript describes the structural design, synthesis, in vitro pharmacological
profile, and molecular modeling of a novel class of Nꢀacylhydrazone (NAH) derivatives that act
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6ꢀ
as HDAC 6/8 dual inhibitors. Selective inhibitors of HDAC6 or HDAC8 are already described.
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8
The selective inhibition of HDAC6 has proved a useful strategy for the treatment of a wide
variety of solid and hematological tumors, wherein the antitumor effects observed through the
selective inhibition of HDAC6 may be due to multiple mechanisms related to modulation of
cytoplasmic proteins, that result in changes in the behavior of migration, motility and invasion of
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cells.
Furthermore, the selective inhibition of HDAC8, has been an attractive target for the
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treatment of neuroblastoma, Tꢀcell lymphoma and hepatocellular carcinoma (HCC).
Both
HDAC6 and HDAC8 have been related with increased breast cancer cells invasion, increasing
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the interest for compounds with such profile. It has been suggested that the dual inhibition of
HDAC6/8 approach has a great therapeutic potential, resulting in a large therapeutic window and
a profile of additive or synergistic effects which can benefit the treatment of various
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3,24
malignancies e.g., neuroblastoma, Tꢀcell lymphoma and HCC.
RESULTS AND DISCUSSION
Design. The compounds characterized in the present study were designed based on the structure
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of trichostatin A (TSA) (1), a natural panꢀHDAC inhibitor. The classical bioisosteric
replacement of two CH moieties with N was used as the primary molecular modification strategy
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6,27
for the synthesis of 1’s derivatives.
We exploited the Nꢀacylhydrazone subunit (NAH), which
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8
has been identified as a privileged structure, for the design of new analogs (Figure 2).
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Figure 2. Design concept used to generate a novel class of NAH derivatives for the inhibition of
HDACs.
We replaced the unsaturation region conjugated to the hydroxamic acid with an
interphenylene linker. Although three regioisomers could be used for the new analogs (ortho-,
meta- and paraꢀsubstituted), this design primarily focused on the use of meta (2) and para (3)
analogs because orthoꢀsubstituted compounds find it difficult to enter the channel in the HDAC
structure.
We also explored the influence of alkylation at R and R (R and R = H or Me) because
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Nꢀmethylation of the NAH subunit could lead to conformational changes that may result in a
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different pharmacological profile.
Moreover, we evaluated carboxylic acids, esters and
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hydroxamic acids as zincꢀbinding groups to confirm the pharmacophoric behavior of these
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chelating subunits.
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Chemistry. The target NAH series 2aꢀ2f and 3aꢀ3f were synthesized according to the procedures
depicted in Schemes 1−4. The desired NAH derivatives were constructed using a convergent
route, divided into the synthesis of the two main building blocks, i.e., the acylhydrazines 6a and
6b and the respective aromatic aldehyde intermediates. Intermediate 6a was obtained at 54%
yield (2 steps) through the oxidative processing of aldehyde 4 as described by Yamada and coꢀ
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workers, who added iodine to a potassium hydroxide methanolic solution and then performed a
hydrazinolysis reaction using hydrazine hydrate in methanol (Scheme 1). The synthesis of Nꢀ
methylꢀacylhydrazine 6b was achieved through the protection of the terminal amino group of
intermediate 6a using phthalic anhydride followed by the baseꢀcatalyzed methylation of the
amide nitrogen with methyl iodide and the subsequent removal of the protecting group using
hydrazine hydrate in methanol (Scheme 1). After these three steps, acylhydrazine 6b was
obtained at 55% yield.
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The aromatic aldehydes 12a and 12b were prepared from the corresponding
benzaldehydes functionalized with ester groups. First, the aldehyde carbonyl group was
protected using 2,2ꢀdimethoxypropane under acid catalysis. A hydroxamic acid moiety was then
introduced after the treatment of the acetalꢀesters 10a and 10b with hydroxylamine under basic
conditions. Subsequently, the acetal group on hydroxamic acids 11a and 11b was hydrolyzed
using sulfuric acid diluted in acetone, generating the corresponding functionalized
benzaldehydes at yields ranging from 67 to 69% (three steps).
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Thus, the NAH derivatives 2a−2f and 3a−3f were prepared at high yields through the
acidꢀcatalyzed condensation of acylhydrazines 6a and 6b using the appropriate aromatic
aldehydes, as illustrated in Schemes 3 and 4.
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A careful analysis of the H NMR spectra of the NAH derivatives 2a−2f and 3a−3f
revealed the formation of a single diastereomer based on the presence of a single imine hydrogen
signal, reflecting the relative configuration of the isomer (E). Previous studies have described
this configuration for different bioactive Nꢀacylhydrazone and NꢀmethylꢀNꢀacylhydrazone
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30,33,34
derivatives, as determined through H NMR and Xꢀray analyses.
In addition, we performed
a NOEdiff (NOEꢀdifference spectroscopy) experiment using the NAH derivative 3c and the Nꢀ
methylꢀNAH 3f. The spectra were collected after irradiation at the imine hydrogen and the
substituent in the R position (3c = H and 3f = CH ). We observed a relationship indicating a
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structural proximity between these atoms, further confirming the relative configuration (E) at the
imine double bond of the NAH moiety (see Figures S15ꢀS18, Supporting information).
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HDAC inhibition. All of the Nꢀacylhydrazone derivatives from the first family, i.e., with a
hydrogen atom as R (see Figure 2), were assessed for their HDAC inhibitory activity. First, the
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5
inhibitory activity of NAH was screened through a nonꢀselective assay using rat liver HDACs.
Prior to this assay, we evaluated the solubility of these compounds in water (buffer pH 7.4) to
ensure that the inhibition percentage values were not incorrectly interpreted due to precipitation
of the compound in the reaction medium. Table 1 shows the inhibition percentage of rat liver
HDACs induced by the Nꢀacylhydrazones 2aꢀ2f and 3aꢀ3f at a concentration of 1 µM and the
solubility of these compounds in water.
Table 1. HDAC inhibition, as assessed in rat liver HDACs, and aqueous solubility of each NAH
compound.
Compound % inhibition of
HDAC rat liver inhibition, Aqueous
a
b
c
HDAC (1 µM)
ꢀ11.0
IC (µM)
solubility (µM)
> 70
5
0
2
a
N.D.
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ꢀ4.6
N.D.
3.7
66.0
ꢀ5.8
1.2±0.047
N.D.
> 67
> 67
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d
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ꢀ10.8
33.4
ꢀ9.1
N.D.
2f
N.D.
> 70
> 77
1.7
3
3
a
N.D.
b
ꢀ10.6
90.4
ꢀ8.7
N.D.
3c
0.021±0.003
N.D.
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3
3
d
e
f
> 67
1.4
ꢀ11.6
90.4
N.D.
3
0.018±0.0001
> 64
a
The values presented are the average of two experiments. The data are shown as % inhibition
b
of HDAC. The IC values displayed are the mean of two experiments ± standard deviation in
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0
µM. The compounds were examined through a sixꢀpoint enzyme assay with a threeꢀfold serial
c
dilution starting from 3 µM for 2c and 1 µM for 3c and 3f. Determined using the
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spectrophotometric method developed by Schneider and coworkers.
determined.
N.D. = not
Compounds 2c, 2f, 3c and 3f presented 66.0, 33.4, 90.4 and 90.4% HDAC inhibition,
respectively. The compounds functionalized with carboxylic acid and esters groups were not
active at the concentrations used in the assay, consistent with previous studies that demonstrated
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4,38
that carboxylic acid derivatives are only active at high micromolar concentrations.
The rat
liver HDAC inhibition achieved by compounds 2c, 3c and 3f was determined (Table 1),
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revealing the potency for 3c and 3f, with IC values in the low nanomolar range of 21 and 18
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nM, respectively.
The selectivity profile of the most active NAH derivatives, namely 2c, 2f, 3c and 3f, was
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39
evaluated in the main human HDAC isoforms, namely class I and class IIb. Table 2 shows the
IC values obtained for these NAH derivatives against HDAC1, 2, 6 and 8.
5
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Table 2. Inhibitory profile of NAH derivatives 2c, 2f, 3c, 3f and trichostatin A (1) against human
HDAC 1, 2, 6 and 8.
a
Inhibition of human HDAC isoforms, IC (µM)
5
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Compound
HDAC 1
HDAC 2
HDAC6
HDAC8
0
.0085±0.001 0.052±0.008 0.009±0.002 0.36±0.015
>10.0
>10.0
0.39±0.04
2.2±0.23
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0.015±0.003 0.23±0.012
0.027±0.011 0.13±0.026
>3.0
>3.0
a
The IC values displayed are the mean of three experiments ± standard deviation in µM. The
5
0
compounds were examined through a sevenꢀpoint enzyme assay with a threeꢀfold serial dilution
starting from 10 µM for 2c and 2f and 3 µM for 3c and 3f.
Both compounds presented here were selective for HDAC6 and HDAC8, although 3c and
3
f were extremely potent, with IC values in the low nanomolar range. For this family of
50
compounds, we hypothesized that the reduced potency of the substituents in the meta position
was associated with hydroxamic acid, whereas the substituents in the para position generated
more potent compounds. The Nꢀmethylation of the meta analog (2c and 2f) series decreases their
ability to inhibit HDAC6. A similar finding was observed for the para analogs, although these
compounds can be considered nearly equipotent.
To evaluate the influence of the methyl group in the R position, we synthesized para
2
analogs harboring this methyl group. The synthesis of 3g and 3h is shown in Scheme 5. Using 4ꢀ
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acetylꢀbenzoic acid as the starting compound, we attempted to utilize the methodology applied
for the synthesis of the aldehyde intermediates; however, given the lower reactivity of ketones,
this strategy was not feasible. Therefore, we introduced the hydroxamic acid directly. The acid
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1
3 was subjected to acid chloride formation using oxalyl chloride. The intermediate was reacted
with hydroxylamine under basic conditions to generate acid 14 at 54% yield. The condensation
of the hydrazides (6a and 6b) was performed in ethanol under microwave irradiation using
AcOH as the catalyst, which led to the generation of 3g and 3h at yields of 45 and 38%,
respectively.
1
Analysis of the H NMR spectra of 3g and 3h revealed the formation of a single isomer
based on the presence of a single imino hydrogen signal, reflecting the (E)ꢀisomer. A NOEdiff
experiment (NOEꢀdifference spectroscopy) was performed for 3g. The spectra were collected
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after irradiation at the hydrogen of the methyl group at the imino position and the R position (3g
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=
H), allowing us to observe the structural proximity between these atoms (see Figures S19 and
S20, Supplementary information).
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Prior to the pharmacological assay, we evaluated the solubility of 3g and 3h and found
water solubility of 6.2 and 122 µM, respectively. The selectivity profile of 3g and 3h among
HDACs of classes I and IIb was then determined, and Table 3 shows the IC values of these
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compounds for HDAC1, 2, 6 and 8.
Table 3. Inhibitory profile of NAH derivatives 3g and 3h and trichostatin A (1) against human
HDAC 1, 2, 6 and 8.
Inhibition of human HDAC isoforms, IC (µM)
5
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Compound
HDAC 1
HDAC 2
HDAC6
HDAC8
0
.0085±0.001 0.052±0.008 0.009±0.002 0.36±0.015
>3.0
>3.0
>3.0
0.056±0.01 0.11±0.008
0.097±0.018 0.054±0.015
>3.0
a
The IC values displayed are the mean of three experiments ± standard deviation in µM. The
5
0
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compounds were examined through a sevenꢀpoint enzyme assay with a threeꢀfold serial dilution
starting from 3 µM.
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A methyl group in the R position decreased the potency for HDAC6 inhibition, whereas
2
the presence of this moiety increased the HDAC8 inhibitory ability. The compound with a
methyl group in the R and R positions (3h) showed greater potency for HDAC8 inhibition
1
2
compared with the other analogs.
The selectivity of these compounds is influenced by the catalytic site of the HDACs.
Although the catalytic site is highly conserved, the rims of the catalytic channel of the HDAC
classes I and II have different dimensions. Because the channel of HDAC6 is wider and
4
0
shallower than that of HDAC1, it is likely that aromatic linkers confer selectivity between these
isoenzymes, as previously described for phenylꢀlinked compounds, which have been
2
3,41
demonstrated to selectively inhibit HDAC6.
The HDAC8 isoenzyme exhibits high
conformational flexibility, thereby accommodating a large variety of ligands, including phenylꢀ
4
2
linked compounds.
To confirm the inhibitory effects of 3c and 3f on HDAC6, the A549 and Caluꢀ1 cell lines
43
were treated with these compounds, and the levels of acetylation in tubulin, a target of HDAC6,
were assessed through western blotting (Figure 3). Both compounds induced tubulin acetylation
in both tumor cell lines. Moreover, the effects of 3c and 3f on HDAC6 inhibition were found to
19
be correlated with cell migration. HDAC6 is considered the master regulator of cell migration,
and the inhibition of this enzyme abrogates cancer cell migration (Figure 4).
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Figure 3. HDACI enhances tubulin acetylation. Caluꢀ1 (A) and A549 (B) cells were treated with
compounds 3c and 3f for 24 h, and the levels of acetylated tubulin were assessed by western
blotting. GAPDH was used as a loading control. One representative of three independent
experiments is shown. Cells treated with vehicle (DMSO) were used as controls.
Figure 4. HDAC6 inhibition abrogates cell migration. Caluꢀ1 cells were treated with compounds
3c and 3f for 48 h, and their migration was determined using the scratch test. One representative
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of three independent experiments is shown. Cells treated with vehicle (DMSO) were used as
controls.
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Molecular modeling. To understand how compounds 3c and 3f bind to these two HDAC
isoforms, we performed molecular docking simulations of these compounds into HDAC6 and
HDAC8 structures. Compounds 3c and 3f, which differ by the presence of a methyl group at R1,
showed the best inhibition profiles, indicating that these two compounds represent the most
promising ligands investigated in the present study.
First, we focused on HDAC8 because many crystallographic structures of this isoform
have been deposited in the Protein Data Bank (PDB). Due to the high flexibility of the HDAC8
active site, it was necessary to carefully select the most adequate structure for this docking
42
study. We searched for coꢀcrystals containing ligands with some structural similarity to the
tested compounds, particularly the phenylꢀlinked compounds. Two crystallographic structures
42
44
were selected: 1VKG (resolution: 2.20 Å) and 1W22 (resolution: 2.5 Å), which contain the
4'
3
3
ligands N ꢀhydroxyꢀN ,N ꢀdimethylꢀ5ꢀ(4ꢀmethylbenzamido)ꢀ[1,1'ꢀbiphenyl]ꢀ3,4'ꢀdicarboxamide
(CRAꢀ19156) and Nꢀhydroxyꢀ4ꢀ(Nꢀmethylꢀ5ꢀ(pyridinꢀ2ꢀyl)thiopheneꢀ2ꢀsulfonamido)benzamide
(NHB), respectively.
Similar positions were observed for the compounds after docking into both HDAC8
structures, although here we only show the docking models for the CRAꢀ19156 binding pocket
of HDAC8 (1VKG in PDB). As shown by the different conformational profiles of both ligands
presented in Figure 5 (see below), compounds 3c and 3f adopted different orientations in the
active site. Both compounds chelate with the zinc ion in the active site and form Hꢀbonds with
His142, His143 and Tyr306 (Figures 5A and 5B), which are important residues in the catalytic
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mechanism. Furthermore, these compounds undergo hydrophobic interactions through the
phenyl linker with residues in the pockets of the wall (Phe152 and Phe208). Compound 3c
undergoes hydrogen bonding between the NH moiety of its NAH group and the carboxylate of
residue Asp101, leaving the 4ꢀdimethylaminoꢀphenyl group in an essentially solventꢀexposed
orientation (Figure 5A), whereas 3f assumes a bent conformation, creating more contacts with
residues on the surface of HDAC8 (Figure 5B). Thus, the higher potency of 3f is likely driven by
entropy because interactions with the surface of the enzyme would dislocate more water
molecules to the bulk of the solvent.
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Figure 5. Predicted binding models of 3c and 3f in complex with HDAC8 (A, B) and HDAC6
(C, D), respectively. (A) and (B) Docking models of 3c and 3f in CRAꢀ19156 binding pocket of
HDAC8 (1VKG in PDB), respectively. (C) and (D) Docking models of 3c and 3f in the HDAC6
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homology model built from HDAC7 (1C0Z in PDB), respectively. Docking studies were
performed by using the program GOLD 5.1.
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Because there is no crystal structure for HDAC6, we constructed a homology model
4
5ꢀ47
using the SWISSꢀMODEL Workspace
with an HDAC7 isozyme structure (1C0Z, resolution
4
8
=
2.10 Å) as the template. For HDAC6, in addition to the pharmacophoric interactions, both
compounds have similar orientations in the active site, as shown in Figures 5C and 5D, reflecting
the equipotency between these compounds.
Antiproliferative activity. Considering the promising results previously obtained, we evaluated
the activity of some para analogs (3c, 3f, 3g, and 3h) and meta analogs (2c and 2f) on different
tumor cell lines derived from melanoma, breast cancer, lung cancer and hepatocellular carcinoma
(
HCC) tissues. The compounds were initially screened at 40 µM, and cell viability was assessed
after 48 h of treatment. The results showed that all of the tested compounds significantly reduced
the viability of MCFꢀ7, HTꢀ144, and HepG2 cells in culture; however, the HTꢀ144 and HepG2
cell lines were the most responsive to compounds 3c, 3f, 3g, and 3h (Figure 6). A doseꢀresponse
curve was obtained to determine the IC values, i.e., concentration necessary to inhibit 50% of
5
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cell growth. HTꢀ144 and HepG2 cells were treated with compounds 3c, 3f, 3g, and 3h at
different concentrations for 48 h. Lower IC values were observed for the HepG2 cultures
5
0
treated with compounds 3c and 3g (Table 4), suggesting the selectivity of these compounds for
certain hepatocellular carcinoma cells. These results are interesting because HCC is one of the
4
9
most malignant tumors, causing more than 600,000 deaths per year worldwide. In addition,
HDAC members, such as HDAC2, HDAC6, and HDAC8, are upregulated in HCC tissues and
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cell lines, indicating that their close association with the proliferative behavior and
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invasion/migration activity of hepatocellular carcinoma cells.
Although it is possible to observe discrepancy between the IC values in assays using
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human HDAC6 and HDAC8 for the compounds of 3c and 3f (low nanomolar) and the potency in
5
2
cellular assay (micromolar), similar behavior was observed by Bergman et al. The reduced
potency cannot be attributed to permeability problems since the treatment of A549 and Caluꢀ1
cells with nanomolar doses of 3c and 3f resulted in increased acetylation of αꢀtubulin. And
therefore these differences must be related to the selective inhibition of HDAC6/8.
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Figure 6. Cell viability determined using the MTS assay after 48 h of treatment with different
compounds at 40 µM. The significance of the differences compared with the control groups were
determined by ANOVA followed by Tukey’s postꢀtest. *** p < 0.001.
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Table 4. IC values (µM) determined from data obtained through the MTS assay.
50
Cell lines
HepG2
HT144
Compounds
3
c
f
9.48 ± 0.73
24.12 ± 0.87
11.52 ± 0.71
30.29 ± 1.38
21.18 ± 0.69
36.10 ± 0.52
19.99 ± 0.64
51.64 ± 4.19
3
3g
3h
Due to the drastic reduction in cell viability after treatment with compounds 3c and 3g
observed in HepG2 cell cultures, we investigated whether this event reflected the
antiproliferative or cytotoxic potential of these compounds. To this end, we performed flow
cytometry analyses to analyze the cell cycle progression of HepG2 cells treated with compounds
3c and 3g at concentrations of 5 and 10 µM for 24 and 48 h. Interestingly, the G2/M cell
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population was significantly increased (p < 0.001) in cultures treated for 24 and 48 h with
compound 3c at a concentration of 5 µM compared with the control group, whereas the
frequency of cells in the G0/G1 and S phases was decreased (Figure 7). We also observed a
gradual increase of the SubG1 population in cultures treated with compound 3c compared with
the control cultures. This last event may reflect cell cycle arrest during either the G2/M transition
or the M phase. Further studies are needed to elucidate the precise point of the cell cycle at
which arrest is induced by compound 3c. Studies have reported that the HDAC6 protein affects
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the function of cytoplasmic nonꢀhistone proteins, including tubulin.
In addition, disturbances
in the microtubule polarization dynamics lead to cell cycle arrest in the M phase, eventually
resulting in cell death. Zhang et al. (2003) reported that HDAC6 interacts with purified tubulin
and microtubules in vitro and that the inhibition of HDAC through TSA increases tubulin
55
acetylation. Blagosklonny et al. (2002) showed that HDAC inhibitors differentially induce
5
6
tubulin acetylation, mitotic arrest, and cytotoxicity. Because compound 3c presented selective
inhibitory activity for HDAC6/8, it is likely that the increased G2/M population reflects
alterations in microtubule dynamics. However, additional experiments are needed to confirm this
hypothesis.
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Figure 7. Cell cycle analysis of HepG2 cells treated with compounds 3c and 3g at concentrations
of 5 and 10 µM for 24 and 48 h. The significance of the differences compared with the control
results were determined by ANOVA followed by Tukey’s postꢀtest. *** p<0.001.
The cell cycle analysis of cultures treated with 3g revealed that this compound exhibits
prominent cytotoxic activity on HepG2 cells, as demonstrated by an increase in the frequency of
the SubG1 population accompanied by a gradual decrease in the G0/G1 and S populations
(Figure 7). Thus, we investigated the potential of compound 3g to induce apoptosis in HepG2
cells through flow cytometry. The frequency of TUNELꢀpositive cells was higher in cultures
treated for 24 h with compound 3g at concentrations of 5 and 10 µM compared with the
frequency observed in the control group (Figure 8A). We also evaluated the activation profile of
caspase 3/7 in cultures treated with compound 3g at a concentration of 5 µM, which resulted in
approximately 20% DNA fragmentation. The frequency of cells with activated caspase 3/7 was
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significantly higher in the treated cultures than in the control cultures (Figure 8B). These results
demonstrate the effective proꢀapoptotic activity of compound 3g on HepG2 cells after 24 h of
treatment. At the molecular level, HDAC inhibitors have been associated with both cell cycle
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regulation and apoptosis induction.
Previous studies have shown that HDAC inhibitors
regulate the expression of multiple apoptosis mediators, which subsequently induce caspase 3/7
5
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activation.
Figure 8. Frequency of apoptotic cells determined through a TUNEL assay (A), and the caspase
3/7 activation profile (B) in HepG2 cell cultures treated for 24 h with compound 3g. Doxorubicin
(DXR) was used as a positive control. The significance of the differences compared with the
control results were determined by ANOVA followed by Tukey’s postꢀtest. * p < 0.05 and *** p
< 0.001.
Chemical stability study. Based on the pharmacological data, compounds 3c, 3f, 3g and 3h are
promising for the study of HDAC6/8 inhibitors. However, the presence of a methyl group may
impart unique characteristics to these compounds, as will be discussed later. Furthermore, these
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compounds present an imine subunit, which may represent vulnerability to hydrolysis, leading to
instability and limiting the development of these compounds as candidate drugs. However, the
compounds in question are functionalized acylhydrazones, which are admittedly more stable than
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imines. Thus, to advance the development of the HDAC inhibitor compounds examined in the
present study, we examined the chemical stability of the most promising compounds from the
series, namely 3c, 3f, 3g and 3h, to evaluate the effect of Nꢀmethylation on the stability of these
compounds.
We conducted this study under two conditions, pH 2.0 and pH 7.4, to mimic the acidic
stomach and the neutral plasma environments, respectively. To this end, compounds 3c, 3f, 3g
and 3h were examined in the presence of acidic (pH 2) and neutral (pH 7.4) buffers under
stirring for 240 min at a controlled temperature (37 °C). The results are shown in Figure 9.
Figure 9. Chemical stability of compounds 3c, 3f, 3g and 3h. A) Chemical stability of
compounds 3c, 3f, 3g and 3h at pH 7.4. B) Chemical stability of compounds 3c, 3f, 3g and 3h at
pH 2. The experiments were conducted in triplicate, and the values shown represent the average
from the experiments.
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In the neutral medium (pH 7.4), all of the compounds were stable, with less than 15%
degradation (Figure 9A), as observed from the compound recovery percentage after 4 h.
However, in acidic buffer, all of the compounds underwent hydrolysis, although 3f showed more
stability, with a higher percentage recovery of 70% after 4h, whereas the compounds with a
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methyl group at the R position (3g and 3h) were highly unstable, and no significant quantities of
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these compounds were recovered after 30 min. Notably, we recovered 6% of compound 3g after
30 min, but after 4 h, we did not observe any amount of the compound (Figure 9B). The effect of
the methyl group on the stability of these compounds will be discussed later.
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The Nꢀmethylation conformational effect. As previously described, the Nꢀmethylation of Nꢀ
acylhydrazones changes the lowest energy conformation from sꢀcis to sꢀtrans, as shown in
Scheme 6. Additionally, to reduce unfavorable steric contacts between the methyl and carbonyl
groups, the modeling results showed a loss of coplanarity between the carbonyl and the NꢀN=C
moieties, with a dihedral angle for O=CꢀNꢀN of 166.43° in the Nꢀmethylated structures. It is
expected that a reduction of coplanarity would decrease conjugation between the nitrogen lone
pair and the carbonyl portion of the Nꢀacylhydrazone. The carbonyl carbon would become more
electron deficient, consistent with the chemical shift differences observed between the
compounds without the Nꢀmethyl group (2aꢀ2b, 3aꢀ3b and 3g; δ = 163 ppm) and the Nꢀ
methylated compounds (2dꢀ2f, 3dꢀ3f and 3h; δ = 169 ppm).
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Moreover, weakened conjugation with the carbonyl group increases the availability of the
electron pair on the nitrogen atom for conjugation with the imine portion of the Nꢀacylhydrazone
group. The observed effect of the increased electron density at the iminic carbon was a change in
the chemical shift value for the series with R = H from δ = 145 ppm (2aꢀ2c and 3aꢀ3c) to δ =
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38 ppm (2dꢀ2f and 3dꢀ3f). The presence of a methyl group at R yielded the opposite effect: to
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avoid unfavorable steric interactions, a conformational change occurs, leading to a reduction in
the conjugation of the Nꢀacylhydrazone subunit and thereby reducing the electronic density in
the imine carbon (δ = 152 ppm; 3g). Methylation at R and R further decreased conjugation,
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which was observed as a high chemical shift value for the imine carbon of δ = 167 ppm (3h).
The electron density on the imine carbon may reflect the chemical stability of the Nꢀ
acylhydrazones at acidic pH because a lower electron density at this carbon indicates that this
atom is more electrophilic and available for nucleophilic attack by water. This behavior was
demonstrated in the chemical stability studies of 3c, 3f, 3g and 3h, which revealed that
compound 3f was more chemically stable at pH 2.0.
Another important effect of the insertion of a methyl group is the increased water
solubility of Nꢀmethylated compounds (3f and 3h). Although the addition of a methyl leads to
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