Novel antiproliferative chimeric compounds with marked histone deacetylase inhibitory activity

Article abstract of DOI:10.1021/ml5000959

Given our interest in finding potential antitumor agents and in view of the multifactorial mechanistic nature of cancer, in the present work, taking advantage of the multifunctional ligands approach, new chimeric molecules were designed and synthesized by combining in single chemical entities structural features of SAHA, targeting histone deacetylases (HDACs), with substituted stilbene or terphenyl derivatives previously obtained by us and endowed with antiproliferative and pro-apoptotic activity. The new chimeric derivatives were characterized with respect to their cytotoxic activity and their effects on cell cycle progression on different tumor cell lines, as well as their HDACs inhibition. Among the other, trans-6 showed the most interesting biological profile, as it exhibited a strong pro-apoptotic activity in tumor cell lines in comparison with both of its parent compounds and a marked HDAC inhibition.

Full text of DOI:10.1021/ml5000959

Letter
pubs.acs.org/acsmedchemlett
Novel Antiproliferative Chimeric Compounds with Marked Histone
Deacetylase Inhibitory Activity
Elisa Giacomini,†,‡ Angela Nebbioso,§ Alfonso Ciotta,§ Cristina Ianni,† Federico Falchi,†,‡
Marinella Roberti,*,† Manlio Tolomeo,∥ Stefania Grimaudo,⊥ Antonietta Di Cristina,⊥
Rosaria Maria Pipitone,⊥ Lucia Altucci,§,# and Maurizio Recanatini†
†Department of Pharmacy and Biotechnology, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy
‡Department of Drug Discovery and Development, Italian Institute of Technology, Via Morego 30, 16163 Genova, Italy
§Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, Vico L. De Crecchio 7, 80138 Naples,
Italy
∥Interdepartmental Center of Research in Clinical Oncology and Department of Infectious Diseases, University of Palermo, Via del
Vespro 129, 90127 Palermo, Italy
⊥DiBiMIS, Laboratory of Molecular Pathology, Institute of Gastroenterology, University of Palermo, Piazza delle Cliniche 2, 90127
Palermo, Italy
#Institute of Genetics and Biophysics, IGB, Via Pietro Castellino 111, 80131 Naples, Italy
S
* Supporting Information
ABSTRACT: Given our interest in finding potential antitumor agents
and in view of the multifactorial mechanistic nature of cancer, in the
present work, taking advantage of the multifunctional ligands approach,
new chimeric molecules were designed and synthesized by combining
in single chemical entities structural features of SAHA, targeting histone
deacetylases (HDACs), with substituted stilbene or terphenyl
derivatives previously obtained by us and endowed with antiprolifer-
ative and pro-apoptotic activity. The new chimeric derivatives were
characterized with respect to their cytotoxic activity and their effects on
cell cycle progression on different tumor cell lines, as well as their
HDACs inhibition. Among the other, trans-6 showed the most interesting biological profile, as it exhibited a strong pro-apoptotic
activity in tumor cell lines in comparison with both of its parent compounds and a marked HDAC inhibition.
KEYWORDS: Multifunctional ligands, chimeric compound, stilbene, HDAC inhibition, antiproliferative activity
o address the biological complexity of cancer1 and to
unit, linking the CAP to a hydrophobic spacer and lying into
Tdevelop adequate therapeutic tools, an interesting and
the tunnel, and an enzyme inhibiting group, able to complex
the zinc ion, crucial for the catalysis at the bottom of the tunnel.
SAHA (suberoylanilide hydroxamic acid, Vorinostat, Zolinza)
(Figure 1A) was the first HDI approved by FDA (in 2006) for
the treatment of the rare cancer cutaneous T-cell lymphoma
(CTCL).8
On the other side, because of our ongoing interest in finding
new anticancer agents, we recently synthesized small libraries of
stilbenes and terphenyls (privileged structures)15−17 (Figure
1B) able to induce apoptosis and differentiation in leukemia
cells, after arresting the cell cycle in G0−G1 phase.18−20 Agents
that are able to block and/or kill cancer cells in G0−G1 phase
are currently considered of interest because most chemo-
therapeutic drugs available for the treatment of malignancies act
in the S or G2−M phase of the cell cycle, but not in G0−G1,
fruitful strategy is provided by the development of “multiple
ligands”, rationally designed single chemical entities able to
modulate multiple altered pathways, overcoming problems like
different pharmacokinetics and poor compliance.2−4 In the
present report, we applied this paradigm to develop novel
multifunctional ligands able to interfere with different
molecular pathways involved in neoplastic diseases, specifically
targeting some control mechanisms of epigenetics5,6 and cell
cycle progression.
Histone deacetylase inhibitors (HDIs)7,8 have emerged as a
new class of promising cancer therapeutic agents and their
synergistic effects with many other drugs are well docu-
mented.9,10 Several HDIs belonging to different chemical
classes11 are currently in clinical trials as monotherapy or in
combination with chemo- or radiation therapy.12 According to
the well-known pharmacophore model for HDIs,13,14
a
Received: March 4, 2014
Accepted: July 7, 2014
Published: July 8, 2014
deacetylase inhibitor should bear a CAP group to interact
with the rim of the catalytic tunnel of the enzyme, a connection
© 2014 American Chemical Society
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Table 1. IC50 (μM SE) and AC50 (μM SE) of 3, 4, 5, cis-
6, and trans-6 and Their Parent Compounds SAHA, 1, cis-2,
and trans-2 Evaluated in K562 Cells after 48 h of Treatment
Figure 1. (A) SAHA. (B) Representative terphenyl and stilbene
derivatives endowed with pro-apoptotic or differentiating activity.
thus allowing a variable percentage of cells in G0−G1 phase to
escape from the cytotoxic effects of the therapy.
On the basis of these premises, following the multifunctional
ligands approach, we designed and synthesized novel chimeric
compounds as potential antitumor agents, by incorporating the
linker-Zn2+-binding motif fragment of SAHA into the selected
most biologically promising terphenyl 119 or stilbenes cis-2 and
trans-218 (Figure 1B). A new generation of more powerful
HDAC inhibitors could represent an opportunity for a clinical
use as single agent, unlike what happens now where the HDAC
inhibitors are mainly used in combination with other cytotoxic
agents.21
According to Morphy and Rankovic,2,3 the new chimeric
compounds 3, 4, cis-6, and trans-6 (Table 1) were “designed
in” by linking together the appropriate fragments via a suitable
connection unit, while a “merged” approach was applied for
chimeric compound 5 in which the selected frameworks were
integrated and overlapped in a common structure (Figure S1,
Supporting Information). The new derivatives were then
characterized with respect to their cytotoxic activity and their
effects on cell cycle progression and epigenetics on leukemia
Bcr-Abl-expressing K562, monoblastic U937, and breast cancer
MCF-7 cell line.
The desired chimeric compounds were synthesized as
described in the following. The synthetic strategy for
compounds 3 and 4 bearing a terphenyl fragment first
consisted of a PyBOP coupling between protected terphenyl
acid 7 and the appropriate amino methyl esters 8 and 9. The
resulting amides 10 and 11 were deprotected by tetrabuty-
lammonium fluoride (TBAF) giving 12 and 13, which reacted
with hydroxylamine hydrochloride in the presence of sodium
methoxide as a base to obtain the hydroxamic acid functions,
affording the final compounds 3 and 4, respectively (Scheme
1). Similarly, deprotection with TBAF of the terphenyl methyl
ester 14 gave the dihydroxy derivative 15, which was treated
with hydroxylamine hydrochloride in the same conditions
described above to give the desired compound 5 (Scheme 2).
Chimeric compounds cis-6 and trans-6 possessing the stilbene
fragment were prepared through amidation by use of DCC as
coupling reagent between suberic acid monomethyl ester
(commercially available) 16 and the aminostilbenes cis-2 and
trans-218 to obtain amides cis-17 and trans-17, respectively.
Conversion of the methyl ester to the hydroxamic acid moiety
afforded hybrid compounds cis-6 and trans-6 (Scheme 3).
Synthetic strategies for intermediates 7 and 14 (Scheme S1)
and 8 and 9 (Scheme S2) are given in the Supporting
Information.
a
b
Concentration (μM) able to inhibit 50% cell growth. Concentration
able to induce apoptosis in 50% of cells. All points were tested in
triplicate with error bars indicating the standard deviation.
The antiproliferative activity of each compound was
evaluated by counting cells with an automatic cell counter;
apoptosis was evaluated by annexin V test. The effects of
chimeric compounds on K562 cell cycle progression were
evaluated by flow cytometry after staining cells with propidium
iodide, and they were compared to the effects of the parent
compounds alone or in association (Figure S2, Supporting
Information). Among the chimeric hydroxamates 3−5 bearing
the terphenyl fragment, compound 3 containing the six
methylenes motif of SAHA exhibited an increased cytotoxic
Initially, we investigated the antiproliferative and the
proapoptotic activities of chimeric derivatives 3, 4, 5, cis-6
and trans-6 in comparison with their parent compounds SAHA,
1, cis-2, and trans-2 on K562 cells (Table 1).
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a
3−5 was able to selectively block cells in G1 phase as 1 or the 1
and SAHA combination as we expected. Interesting results
were obtained with trans-6 and cis-6, designed by combining
the structural features of SAHA and two stilbenes, trans-2 and
cis-2, respectively, previously designed by us and endowed with
potent pro-apoptotic activity (Table 1).18 The cytotoxic activity
of hydroxamate cis-6 was markedly lower than that of parents
cis-2 and SAHA (Table 1). Noteworthy, trans-6 (IC50, 0.7 μM;
AC50, 5.0 μM) was more potent as cytotoxic agent than trans-2
(IC50, 2.5 μM; AC50, 20 μM) and SAHA (IC50, 1 μM; AC50, 10
μM). In particular, the ability to induce apoptosis in K562 cells
was two times higher than that observed with SAHA (Table 1).
The pattern of cell cycle distribution of K562 cells after
exposure to trans-6 and cis-6 showed that both compounds
behaved similarly to SAHA (block in G1 and G2−M phases)
and differently from the parent compounds alone (block in
G2−M) or in combination (prevalent block in G1), thus
suggesting they could share a mechanism of action similar to
that of SAHA (Figure S2, Supporting Information).
Scheme 1
a
Reagents and conditions: (a) PyBop, DIEA, CH2Cl2, 72h rt; (b) 1 M
TBAF, 2 h 0 °C to rt; (c) NH2OH·HCl, NaOMe 30%, MeOH, 24 h, 0
°C to rt.
Because of its interesting cytotoxic activity on K562 cells, we
considered trans-6 the best candidate for further investigations.
First, we studied this compound in two different cell lines: the
monoblastic U937 and the breast cancer MCF-7 cells. Once
again, trans-6 resulted to act similarly to SAHA as it was able to
induce a G1- and G2-block in U937 and MCF-7 cells,
respectively, with progressive cell death (Figure S3A,B,
Supporting Information). To better understand the progressive
cell death induction, a cytofluorimetric analysis was performed
to discriminate necrosis from apoptosis. As shown in Figure 2,
trans-6, similarly to SAHA, triggered an apoptotic death, but
with a 2-fold increase. These data corroborated and extended
the value of the AC50 shown in Table 1.
a
Scheme 2
a
Reagents and conditions: (a) 1 M TBAF, 2 h 0 °C to rt; (b)
NH2OH·HCl, NaOMe 30%, MeOH, 24 h, 0 °C to rt.
a
Scheme 3
Figure 2. Left: apoptosis/necrosis evaluation, by Annexin V/PI
doubling staining by flow cytometric assays on U937 cells treated with
5 μM trans-6 or SAHA (taken as positive control) for 48 h. All points
were tested in triplicate with error bars indicating the standard
deviation. Right: Western blot for caspase 8 after a stimulation of 24 h
for U937 cells and 48 h for MCF-7 cells; compounds were used at
concentration of 5 μM. ImageJ was used to quantify protein expression
levels. The ERKs signal accounts for equal loading.
a
Reagents and conditions: (a) HOBt, DCC, DMF, 48 h, rt; (b)
NH2OH·HCl, NaOMe 30%, MeOH, 3 h, 0 °C to rt.
and proapototic activity with respect to the parent compound 1
(IC50, 2.5 μM vs 20 μM; AC50, 16 μM vs >50 μM, respectively,
Table 1), while it was less potent than SAHA. Reducing the
alkyl chain to five methylenes as in 4 resulted in a decrease of
activity even though still higher if compared to 1, while
compound 5 lacking the methylene chain displayed the same
activity as parent terphenyl 1. These results suggested that the
alkyl hydroxamic chain conferred a better cytotoxic and pro-
apototic activity to the terphenyl scaffold.
To test HDAC inhibitory potential of all new chimeric
compounds, enzymatic assays were performed against human
recombinant (hr) enzymes (Table 2 and Figure S4A,
Supporting Information). Among the terphenyl derivatives,
the compound 4, containing the five methylene chain, although
exhibited an interesting inhibitory activity against hrHDAC1
(IC50, 0.9 μM), was completely inactive on hrHDAC4 as 3 and
5, demonstrating that the terphenyl scaffold is not a suitable
moiety for the anti-HDAC activity. At the same time the
Unfortunately the cell cycle analysis (Figure S2, Supporting
Information) revealed that none of the chimeric compounds
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Table 2. Human Recombinant HDAC1 Inhibitory Activity of Compounds 3−5, Compared with SAHA and Human
Recombinant HDAC4 and HDAC6 Inhibitory Activity of trans-6, cis-6, and SAHA
a
inhibition at 5 μM (% SE)
IC50 (μM) (95% confidence interval)
hrHDAC4
compounds
hrHDAC1
hrHDAC4
hrHDAC6
hrHDAC1
hrHDAC6
SAHA
68.9 1.73
20.4 2.12
62.1 1.87
33.1 2.86
91.7 1.19
88.3 2.0
21.5 0.25
40.7 0.84
0.6 (0.313−3,224)
15.7 (7.675−32.13)
0.9 (0.6941−1.088)
8.9 (6.496−12.34)
0.8 (0.493−1.163)
7.8 (5.88−12.06)
5.6 (5.31−7.45)
b
b
b
3
0
n.d.
n.d.
n.d.
b
b
b
4
0
n.d.
n.d.
n.d.
b
b
b
5
0
n.d.
n.d.
n.d.
trans-6
cis-6
39.1 0.78
68.1 1.3
50.2 1.40
74.7 2.0
9.8 (6.24−15.54)
7.6 (4.37−13.34)
1.3 (0.437−3.851)
1.6 (0.512−5.049)
2.1 (1.207- 3.614)
a
b
All points were tested in triplicate with error bars indicating the standard deviation. Not determined.
stilbene hydroxamate trans-6 and its cis isoform, cis-6, resulted
in being strong HDAC1 inhibitors (IC50, 0.8 and 2.1 μM,
respectively), and their effect was therefore tested on HDAC4
and 6 (Table 2 and Figure S4B, Supporting Information). Both
compounds at 5 μM were also able to inhibit these two
enzymes better than SAHA showing good IC50 values (IC50, 9.8
and 7.6 μM for HDAC4 and 1.3 and 1.6 μM for HDAC6,
respectively). In particular, trans-6 privileged HDAC1 inhib-
ition. However, cis-6 inhibited all tested enzymes at comparable
levels (Figure S4, Supporting Information).
HDAC6 inhibitor, induced a clear increment of the acetylation
level of the α-tubulin (Figure 3).
To better address the anticancer activity of trans-6,
proliferation and migration analysis were also performed in
MCF-7 cells in real-time mode. The inhibitory effects of trans-6
on the proliferation were showed within the 36 h of treatment
and were already evident in the early phases after 10 h (Figure
4). This trend was highlighted by the doubling time analysis,
Given the above results, again we considered trans-6 the best
candidate for further investigations. According to its HDAC
inhibitory action, trans-6 showed the ability to induce a high
histone hyper-acetylation in both U937 and MCF-7 cell lines
(Figure 3). Western blotting analyses showed a hyper-
Figure 4. (A) Proliferation analysis in real time of MCF-7 cells treated
with 5 μM SAHA or trans-6 as CI (cell index) vs time (left) within 36
h and as doubling time (right) within the interval 10−18 h. (B)
Migration analysis in real-time of MCF-7 cells treated with 5 μM
trans-6 as CI vs time (left) within 27 h and as slope within 24 h.
Figure 3. Western blot analyses carried out for the indicated targets in
U937 and MCF-7 cells after 24 h of treatment. ImageJ was used to
quantify protein expression. Histone H1, H4, and ERKs indicate equal
loading. SAHA and trans-6 were used at concentration of 5 μM.
which showed a strong decrease of this parameter both for
trans-6 and SAHA within the interval 10−18 h (Figure 3A).
Migration analyses showed a clear ability of trans-6 to inhibit
the tumor cell migration. The effects were more evident if
represented in slope values displaying a decrease of 25% within
24 h (Figure 4B).
On the basis of these results, it appears that the chimeric
hydroxamates bearing the terphenyl motif did not enhance the
biological activity of the parent compounds, losing the peculiar
block in G1 phase and showing a not significant HDAC
inhibitory profile. However, the stilbene structure proved to be
a valid scaffold for the design of strong HDAC inhibitors. Of
interest, the trans configuration of the stilbene architecture
acetylation of histone H3 in lysines 9 and 14 (H3K9 and
14ac) and of histone H4 in lysine 16 (H4K16ac). Remarkably,
the quantification of the signals showed a 6-fold against a 4-fold
increase of acetylation of histone H3 for trans-6 compared to
its parent compound SAHA, respectively. Taken together, these
results demonstrated that trans-6 was able to increase
acetylation of lysine residues on the N-terminal histones tails
of H3 and H4. Furthermore, trans-6 was also able to trigger a
repair pathway, causing the acetylation in lysine 382 of p53
(p53K382ac) and up-regulating the cell-cycle inhibitor
p21waf1/cip1 (Figure 3). As HDAC6 assay showed, trans-6, as a
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ACS Medicinal Chemistry Letters
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55.7, 56.4, 100.6, 105.3, 111.9, 121.1, 124.9, 128.2, 128.4, 129.7, 131.3,
seems to be an important feature in conferring a slight
selectivity toward the different isoforms of HDACs.
140.9, 141.0, 151.3, 162.5, 174.7 ppm; MS (ES) m/z 479 [M + Na]+.
In continuation of our research for innovative antitumor lead
candidates, we designed and synthesized some chimeric
compounds following the multifunctional ligands approach.
Even though the new molecules did not show a pharmacolog-
ically chimeric behavior, trans-6, obtained combining the
structural features of SAHA and a stilbene derivative previously
designed by us, was able to induce a stronger apoptosis in
K562, U937 and MCF-7 cells than both the parent compounds.
Moreover, it showed a marked HDAC inhibitory action and a
clear ability to inhibit the tumor cell migration. Our strategy
contributed to explore the chemical space around SAHA,
proving the stilbene structure as a valuable CAP group in
HDAC pharmacophore. Therefore, trans-6 could be considered
a suitable lead structure to develop new agents endowed with a
promising anticancer potential. Noteworthy, from a medicinal
chemistry point of view, the stilbene scaffold is a privileged
structure in which the biological relevance meets the synthetic
accessibility, allowing to rapidly obtain variously substituted
analogues, making the follow-up studies of the identified hits
more efficient.
ASSOCIATED CONTENT
* Supporting Information
■
S
Spectroscopic data for final compounds 3−5 and cis-6, and
synthesis and characterization of compounds 7−15 and cis- and
trans-17, elemental analyses, detailed biological protocols;
Schemes S1 and S2, Table S1, and Figures S1−S4. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*(M.Ro.) E-mail: marinella.roberti@unibo.it. Phone: +(39)
051 2099738.
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by a PRIN2009 and PRIN2012 Grant
from MiUR, Italy; EU: the Blueprint (contract no. 282510),
Epigenomics Flagship Project EPIGEN (MIUR-CNR); and the
Italian Association for Cancer Research (AIRC no. 11812).
EXPERIMENTAL METHODS
■
Chemistry. General Chemical Methods. Reaction progress was
monitored by TLC on precoated silica gel plates (Kieselgel 60 F254
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,
■
Merck) and visualized by UV254 light; hydroxamates were viewed by
staining with FeCl3 5% aqueous solution. Flash column chromatog-
raphy was performed on silica gel (particle size 40−63 μM, Merck).
When needed, silica was demetalled by suspending and standing
overnight in concentrated HCl, filtered and washed several times with
Et2O until free of chloride ions, and dried for 48 h at 120 °C. All
solvents were distilled prior to use. All reagents were obtained from
commercial sources and used without further purification. Unless
otherwise stated, all reactions were carried out under an inert
atmosphere. Compounds were named relying on the naming
algorithm developed by CambridgeSoft Corporation and used in
Chem-BioDraw Ultra 11.0. 1H NMR and 13C NMR spectra were
recorded on Varian Gemini at 400 and 100 MHz, respectively.
Chemical shifts (δH) are reported relative to TMS as internal standard.
Mass spectrum was recorded on a V.G. 7070E spectrometer or on a
Waters ZQ 4000 apparatus operating in electrospray (ES) mode.
Purity of compounds was determined by elemental analyses; purity for
all the tested compounds was ≥95% (see Supporting Information).
General Procedure for Chimeric Compounds 3−5, cis-6, and
trans-6. To cooled solutions of the appropriate methyl esters 12, 13,
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hydroxylamine hydrochloride NH2OH·HCl (10 equiv) and sodium
methylate solution 30% in MeOH (12.4 equiv) were added. The
reaction mixture was stirred for 3 to 24 h at room temperature then
cooled in an ice bath and acidified with 6 N HCl to pH 4. Water was
added to dissolve the salt, and the mixture was concentrated in vacuo
to remove MeOH/THF. The aqueous phase was extracted with
EtOAc (3 × 10 mL) and purified by flash chromatography on
demetalled silica gel.
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