Novel histone deacetylase inhibitors induce growth arrest, apoptosis, and differentiation in sarcoma cancer stem cells

Article abstract of DOI:10.1021/acs.jmedchem.5b00126

Musculoskeletal sarcomas are aggressive malignancies of bone and soft tissues often affecting children and adolescents. Histone deacetylase inhibitors (HDACi) have been proposed to counteract cancer stem cells (CSCs) in solid neoplasms. When tested in hum

Full text of DOI:10.1021/acs.jmedchem.5b00126

Brief Article
pubs.acs.org/jmc
Novel Histone Deacetylase Inhibitors Induce Growth Arrest,
Apoptosis, and Differentiation in Sarcoma Cancer Stem Cells
Gemma Di Pompo,†,‡,# Manuela Salerno,†,‡,# Dante Rotili,§ Sergio Valente,§ Clemens Zwergel,§
Sofia Avnet,† Giovanna Lattanzi,∥ Nicola Baldini,*,†,‡ and Antonello Mai*,§,⊥
†Orthopaedic Pathophysiology and Regenerative Medicine Unit, Istituto Ortopedico Rizzoli (IOR), 40136 Bologna, Italy
‡Department of Biomedical and Neuromotor Sciences, University of Bologna, 40126 Bologna, Italy
§Department of Drug Chemistry and Technologies, Sapienza University of Roma, P.le A. Moro 5, 00185 Roma, Italy
∥Institute of Molecular Genetics, Unit of Bologna IOR, National Research Council of Italy, 40136 Bologna, Italy
⊥Pasteur InstituteCenci Bolognetti Foundation, Sapienza University of Roma, P.le A. Moro 5, 00185 Roma, Italy
S
* Supporting Information
ABSTRACT: Musculoskeletal sarcomas are aggressive malignan-
cies of bone and soft tissues often affecting children and
adolescents. Histone deacetylase inhibitors (HDACi) have been
proposed to counteract cancer stem cells (CSCs) in solid
neoplasms. When tested in human osteosarcoma, rhabdomyosar-
coma, and Ewing’s sarcoma stem cells, the new HDACi MC1742
(1) and MC2625 (2) increased acetyl-H3 and acetyl-tubulin levels
and inhibited CSC growth by apoptosis induction. At nontoxic
doses, 1 promoted osteogenic differentiation. Further investigation
with 1 will be done in preclinical sarcoma models.
acetylation in the differentiation process of mesenchymal
precursors,14 the hypothetical cell-of-origin of transformed
sarcoma cells.15 Therefore, we investigated the effects of
different HDACi in CSC cultures derived from human models
of osteosarcoma (OS), rhabdomyosarcoma (RMS), and
Ewing’s sarcoma (ES). We used MC1742 (1, Figure 1A),
belonging to the series of the uracil-based hydroxyamides
(UBHAs),16,17 MC2625 (2, Figure 1B), the aza-analogue of
INTRODUCTION
■
Musculoskeletal sarcomas are relatively rare, aggressive
malignancies of bone and soft tissues often affecting children
and young adults with devastating consequences both in terms
of morbidity and mortality.1 In recent years, a unique
population of cells, referred as cancer stem cells (CSCs),
have been retained responsible for cancer development and
progression, response to therapy, and metastatis in different
solid tumors,2 including sarcomas.3,4 Lysine acetylation and
deacetylation of histone and nonhistone proteins is a crucial
event in the epigenetic modulation of gene expression, cell
cycle progression, and signal transduction cascades.5,6 Novel
perspectives for the targeting of CSCs within sarcomas may
derive from the employment of HDACi. Some authors
provided evidence of the effectiveness of these compounds
against CSCs derived from different tumors. Specifically, it has
been reported that HDACi induce differentiation of breast
CSCs,7 promote apoptosis and cell cycle arrest in CSCs of head
and neck squamous cell carcinoma,8 and exert strong
antiproliferative effects on glioblastoma and colorectal
carcinoma CSCs.9,10 However, to date, no information about
their activity on CSCs derived from human sarcomas is
available. Nevertheless, some data indicate that these
compounds are effective in inhibiting the growth of native
sarcoma cells, as demonstrated by several preclinical stud-
ies.11−13 This is possibly due to the involvement of histone
Figure 1. Design of the novel HDACi 1 and 2 and structure of the
SIRTi 3 tested in this study.
Received: January 21, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jmedchem.5b00126
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Brief Article
aroylammino cinnamyl hydroxamates previously described by
us,19−22 and MC2141 (3, Figure 1C), a sirtuin (class III
HDAC) inhibitor active in the low micromolar range against
SIRT1 (IC50 = 9.8 μM) and SIRT2 (IC50 = 12.3 μM). The
classical UBHA scaffold16 in 1 was modified by converting the
C6-phenyl into a C6-(4-biphenyl) moiety, a structural change
which improved the anti-HDAC activity of UBHAs17 as well as
other hydroxamates.18,19 Compound 2 displays a 2,3-
diphenylpropanamide chain, related to some branched groups
highly efficient in improving potency and selectivity in HDAC
inhibition.22,23 The SIRTi 3 was described as able to induce
antiproliferative effects in different cancer cell lines including
human glioblastoma and colorectal carcinoma CSCs.10
RESULTS AND DISCUSSION
■
HDAC Inhibitory Enzyme Assays. The two novel HDACi
1 and 2 were tested against all the HDAC1−11 isoforms in 10-
dose IC50 mode with 3-fold serial dilution starting from 50 μM
solutions (Table 1). From these data, 1 emerged as a class I/
IIb-selective HDACi, being potent at submicromolar/nano-
molar level against class I HDACs (HDAC1−3, 8) and at
nanomolar level against class IIb HDACs (HDAC6, 10).
Against class IIa HDACs (HDAC4, 5, 7, 9), 1 did not show
appreciable inhibition at 50 μM, the maximum tested dose.
Differently, 2 behaved as a pan-HDACi, being efficient in
inhibiting all the 11 HDAC isoforms in the range 0.08−12 μM.
In particular, it showed the highest nanomolar inhibitory
potency against HDAC3 and HDAC6, inhibited HDAC8 at
submicromolar concentration, and it remained still active
against class IIa HDACs with IC50 values around 10 μM.
Thus, both of them deserved to be investigated in sarcoma
CSCs, 1 as more potent and selective HDACi, 2 as a broader
inhibitor including class IIa HDACs in its spectrum of action.
In addition, we included 310 in our studies as representative of a
specific class III HDAC (sirtuin) inhibitor.
CHEMISTRY
■
The synthetic routes followed for the preparation of 1 and 2 are
depicted in Scheme 1. The hydroxamate 1 was synthesized
a
Scheme 1
Screening of Cytotoxic Effects of 1−3 in Sarcoma
CSCs. The cytotoxicity of the investigated HDACi was
measured on CSCs generated from three different histotypes
of sarcoma: RMS (RD and A204 cell lines), OS (MG-63 and
HOS cell lines), and ES (SK-ES-1 and A673 cell lines). The
drug concentrations were arbitrarily chosen starting from a high
concentration (100 μM) and with dilutions until the lowest
concentration (0.1 μM). Compound cytoxicity was evaluated in
comparison to cells treated with DMSO alone in order to
exclude any nonspecific cytotoxicity caused by the vehicle. After
72 h of treatment, compounds exerted different effects on CSC
cultures (see the relative curves and half-maximal inhibitor
concentration (IC50) values in Supporting Information (SI),
Figure S1; p = 0.0209). 3 was effective on CSCs, although in
almost all cultures the effect was observed starting from 10 μM.
Adversely, 1 and 2 were able to significantly affect cell viability
even at 0.1 and 1 μM, depending on the cell line, showing a
clear dose-dependent activity and the lowest IC50 values for
each CSC culture, calculated by linear regression (SI, Figure
S1). To dissect the role of specific HDAC class inhibition in
sarcoma CSCs’ cytotoxicity, we tested also SAHA,24 the pan-
HDACi approved by FDA in 2006 for the treatment of CTCL,
MS-275, a class I-selective HDACi,25 and MC1568,26−30 a class
II-selective HDACi, on RD, MG-63, and SK-ES-1 cells. The
results and the IC50 values are shown in SI, Figure S2 (p =
0.0209; for MC1568, data not shown).
a
(a) (i) ClCOOEt, (Et)3N, THF, 0 °C, (ii) NH2OC(Me)2OMe, rt,
(iii) Amberlyst 15, MeOH, rt; (b) (i) SOCl2, 80 °C, (ii) Et3N,
anhydrous CH2Cl2, 0 °C; (c) P(Ph)3, Pd(OAc)2, n-Bu4NI, AcONa·
3H2O, butyl acrylate, anhydrous DMF, 140 °C; (d) 2N KOH, EtOH/
H2O, rt.
from the 5-((4-([1,1′-biphenyl]-4-yl)-6-oxo-1,6-dihydropyrimi-
din-2-yl)thio) pentanoic acid 417 by treatment with (i) ethyl
chloroformate and triethylamine, (ii) O-(2-methoxy-2-propyl)
hydroxylamine, and (iii) Amberlyst 15 ion-exchange resin in
methanol at room temperature (Scheme 1A). For the synthesis
of 2, 2-bromo-5-aminopyridine was treated with 2,3-diphenyl-
propanoyl chloride (previously prepared reacting 2,3-diphenyl-
propanoic acid with thionyl chloride at 80 °C) and triethyl-
amine in anhydrous dichlomethane to furnish N-(6-bromopyr-
idin-3-yl)-2,3-diphenylpropanamide 5. Afterward, the
intermediate 5 underwent Heck coupling reaction using butyl
acrylate, tetra-n-butyl ammonium iodide, sodium acetate
trihydrate, triphenylphosphine, and palladium acetate in
anhydrous N,N-dimethylformamide at 140 °C overnight to
afford the ester 6. By hydrolysis of 6, the acid 7 was obtained
and converted to the hydroxamate 2 through the method
already described for 1 (Scheme 1B).
Antiproliferative Effects of 1 and 2 on Sarcoma CSCs.
On the basis of the MTT results, 1 and 2 showed the best IC50
values and the highest cytotoxic activity. For this reason, their
antiproliferative effect on sarcospheres was confirmed by
determining the number of viable cells by a growth curve. A
more restricted range of concentrations (2, 1, and 0.5 μM) was
employed on the base of IC50 values calculated for each cell
histotype. As shown in Figure 2, all the concentrations of both
Table 1. IC50 Values (μM) of 1 and 2 against the HDAC1-11 Isoforms
IC50 vs HDAC (μM)
compd
1
2
3
4
5
6
7
8
9
10
11
1
2
0.10
1.42
0.11
1.77
0.02
0.08
>50
>50
0.007
0.01
>50
0.61
0.61
>50
0.04
1.8
0.1
11.7
9.37
8.77
10.6
10.2
B
DOI: 10.1021/acs.jmedchem.5b00126
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Journal of Medicinal Chemistry
Brief Article
Figure 2. Antiproliferative effect of 1 and 2 on different sarcoma CSC
cultures after 24, 48, and 72 h of treatment. The antitumor activity of 1
(A) and 2 (B) was determined by growth curves on sarcoma CSCs
after treatment with 0.5, 1, and 2 μM of compounds. *p < 0.05 vs
control.
drugs reduced cell proliferation in all CSC cultures in a dose-
dependent manner. In particular, 1 significantly inhibited cell
proliferation already after 48 h, with the exception of 0.5 μM
for A673 (Figure 2A; p < 0.05), showing the highest
effectiveness. The treatment with the lowest dose of 2 for 48
h significantly affected MG-63, RD, and SK-ES-1 viability, while
all CSCs were sensitive at 1 and 2 μM (Figure 2B; p < 0.05).
Nevertheless, all the 1 and 2 concentrations exerted significant
effects on all the CSC cultures after 72 h (Figure 2; p < 0.05).
Inhibition of HDAC Activity in Sarcoma CSCs.
Immunofluorescence and Western blotting were carried out
to demonstrate the inhibitory activity of the selected HDACi
on deacetylation of histone H3. MG-63 CSCs have been
chosen among all the CSC cultures as a representative model
because it resulted as being the most sensitive to the HDACi
antiproliferative effect. The 2 and 0.5 μM concentrations were
selected because they were the highest and the lowest used in
the growth assay, respectively. After 24 h of treatment with 1
and 2, a dose-dependent increase of acetylation, observable as
punctuate nuclear staining of acetyl-histone H3, was detected
by immunofluorescence analysis (Figure 3A). Accordingly,
protein level of acetyl-histone H3, detected by Western blot
(WB), was enhanced in HDACi-treated CSCs in respect to
untreated cells (Figure 3B, left). The densitometric analysis
showed that 1 was significantly effective both at 0.5 and 2 μM,
while 2 was effective only at 2 μM (Figure 3B, right; p =
0.0495), in agreement with their relative degrees of enzyme
inhibition. When tested with antiacetyl-tubulin antibodies by
WB as functional test for HDAC6 inhibition, again 1 was more
potent than 2 in increasing acetyl-tubulin levels (Figure 3B; p =
0.0495).
Figure 3. Inhibition of histone acetylation after HDACi treatment. (A)
Immunofluorescence staining of acetyl-histone H3 (acetyl K9) in
untreated cells (control, upper panel) or treated with 1 (middle panel)
and 2 (lower panel) at different concentrations (0.5 or 2 μM). Nuclei
were counterstained with DAPI. The merged images are shown in the
left column. Representative images, scale bar 10 μm. (B) Western
blotting of acetyl-H3 and acetyl-tubulin in MG-63 CSCs treated with
0.5 and 2 μM of 1 and 2 for 24 h (left, representative image) and
densitometric analysis (right) reported as index vs negative control
(bold line). *p < 0.05 vs control. Normalization to TBP.
cell proliferation. Treatment with 1 and 2 μM of 1 and 2
significantly induced apoptosis of all CSC cultures, with the
exception of A204 CSCs, that were less sensitive to treatment
with 2 (Figure 4B; *p < 0.05, **p < 0.01). In MG-63 CSCs, the
induction of apoptosis by 1 and 2 was demonstrated by
evaluating the presence of the molecular marker phosphatidyl
serine (PS) on the surface of the cytoplasmic membrane
(Figure S3 in SI).
Osteogenic Differentiation Induction in Sarcoma
CSCs. Differentiation therapy has been proposed as a
promising therapeutic strategy to revert chemoresistance of
CSCs in different tumor histotypes.31,32 To determine whether
differentiation occurred following the 1 and 2 cell treatment,
Alizarin Red S staining was carried out on MG-63 CSCs,
selected as representative OS model. Alizarin Red S selectively
binds to calcium salts, therefore the rate of mineralized nodule
formation is directly correlated to the amount of eluted dye,
detected by spectrophotometry. Because cytotoxicity of
compounds could negatively influence osteogenic differ-
Apoptosis Induction in Sarcoma CSCs. The Hoechst
33258 staining was used to verify if the selected compounds
were able to affect sarcoma CSC growth through apoptosis. As
shown by representative pictures of MG-63 CSCs, 1 and 2
generated an increase of the presence of apoptotic cells with
concentrated dense granular fluorescence compared to un-
treated cells, especially at 1 and 2 μM, whereas no relevant
apoptosis was detectable in control cells (Figure 4A).
The quantification of cells with apoptotic bodies revealed a
dose-dependent increase of apoptosis in all CSC cultures after
48 h of incubation with HDACi, consistent with the effect on
C
DOI: 10.1021/acs.jmedchem.5b00126
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Journal of Medicinal Chemistry
Brief Article
Figure 5. Mineralization assay after HDACi treatment. MG-63 CSCs
were treated with nontoxic doses of 1 and 2 under differentiating
conditions for 14 days. Quantification of Alizarin Red S staining
evaluated by dye elution with cetylpyridinium chloride, showing a
significant dose-dependent induction of mineral nodules formation
after 1 treatment. *p < 0.05 vs control.
MS-275 was less effective, and the class II-selective MC1568
inhibited the tested CSCs at 38.3 (MG-63), 1,733 (RD), and
50.7 (SK-ES-1) μM (data not shown), thus suggesting that
concurrent class I and IIb HDACs inhibition is crucial to obtain
anticancer effects in sarcoma CSCs. On these bases, the
antiproliferative activities of 1 and 2 against sarcospheres on the
six tested CSCs were determined. To demonstrate the HDAC
inhibition activity of 1 and 2 in cells, we detected an increase of
the acetyl-H3 and acetyl-tubulin levels after exposure of MG-63
CSCs, chosen as the most sensitive to the selected HDACi, to
both compounds. Notably, 1 resulted more effective than 2,
also at the lowest concentration, confirming its strong
antideacetylase activity. About apoptosis induction in sarco-
spheres, both 1 and 2 significantly enhanced the number of
apoptotic cells and induced PS as a molecular marker, the first
being again the most effective. We ultimately evaluated the
osteogenic differentiation induction of OS CSCs after exposure
to nontoxic concentrations of 1 and 2. As expected, 1 promoted
high cell differentiation evaluated in terms of mineral nodules
formation in vitro, whereas 2 was ineffective, probably due to its
lower anti-HDAC potency.
Figure 4. Apoptosis induction after HDACi treatment. Presence of
apoptotic CSCs after 48 h of exposure to 0.5, 1, and 2 μM of 1 and 2.
(A) Hoechst 33258 nuclear staining of MG-63 CSCs, showing the
basal level of cells with apoptotic bodies in untreated control (left) and
a dose-dependent increase in the number of apoptotic cells after
treatment with 1 (middle) and 2 (right). Representative pictures, scale
bar 50 μm. (B) Quantification of the percentage of apoptotic cells over
different fields in OS (top), RMS (middle), and ES (bottom) CSCs
evaluated after Hoechst 33258 staining. *p < 0.05 and **p < 0.01 vs
control.
In conclusion, this study provides an adequate background
about the in vitro efficacy of a novel uracil-based HDACi, 1, on
a panel of CSCs from different human sarcoma histotypes, thus
supporting the use of HDACi-based therapy also in these
neoplasms.
Further investigation is required to clarify its activity in in
vivo preclinical sarcoma models.
entiation and consequently cell ability to mineralize, nontoxic
concentrations of drugs were selected on the basis of IC50
values previously calculated. The Alamar Blue test, performed
at the mineralization end-point, confirmed that only the highest
concentration of 2 significantly inhibited cell growth (Figure S4
in SI; p < 0.05). The elution of the Alizarin Red S dye indicated
that 2 treatment did not improve mineralization with respect to
control cells. On the contrary, treatment with 1, starting from
the lowest dose (0.025 μM) up to the highest (0.5 μM),
successfully enhanced bone nodule formation in a significant
dose-dependent manner (Figure 5; p < 0.05).
EXPERIMENTAL SECTION
Chemistry. Melting point were determined on a Buchi 530 melting
point apparatus and are uncorrected. H NMR spectra were recorded
■
1
at 400 MHz on a Bruker AC 400 spectrometer; reporting chemical
shifts in δ (ppm) units relative to the internal reference
tetramethylsilane (Me4Si). All compounds were routinely checked
by TLC and 1H NMR. TLC was performed on aluminum-backed silica
gel plates (Merck DC, Alufolien Kieselgel 60 F254) with spots
visualized by UV light. Yields of all reactions refer to the purified
products. All chemicals were purchased from Aldrich Chimica, Milan
(Italy), and were of the highest purity. Mass spectra were recorded on
a API-TOF Mariner by Perspective Biosystem (Stratford, TX, USA),
and samples were injected by an Harvard pump using a flow rate of 5−
10 μL/min, infused in the Electrospray system. Elemental analyses
were obtained by a PE 2400 (Perkin- Elmer) analyzer and have been
used to determine purity of the described compounds, that is, >95%.
Analytical results are within 0.40% of the theoretical values.
CONCLUSIONS
■
Here we show the effects of three HDACi, 1 (class I/IIb-
selective), 2 (nonselective), and 3 (class III-selective) on CSCs
generated from three different histotypes of human sarcomas
(RMS, OS, and ES). The first screening and the resultant IC50
values demonstrated a potent cytotoxic effect by the “classical”
HDACi 1 and 2 that reduced cell viability in some CSC
cultures even at the lowest tested concentration, 0.1 μM, while
the sirtuin inhibitor 3 was less effective. In the same test, SAHA
(a pan-HDACi) showed comparable (MG-63 cells) or lower
(RD and SK-ES-1 cells) potency than 1, the class-I selective
D
DOI: 10.1021/acs.jmedchem.5b00126
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Journal of Medicinal Chemistry
Brief Article
Synthesis of 5-((4-([1,1′-Biphenyl]-4-yl)-6-oxo-1,6-dihydropyrimi-
din-2-yl)thio)-N-hydroxypentanamide (1). To a 0 °C cooled solution
of 417 (0.53 mmol, 200 mg) in dry tetrahydrofuran (4 mL), ethyl
chloroformate (1.3 mmol, 138 mg, 0.12 mL) and triethylamine (1.38
mmol, 140 mg, 0.19 mL) were added and the mixture was stirred for
10 min. The solid was filtered off, and O-(2-methoxy-2-propyl)-
hydroxylamine (3.18 mmol, 0.24 mL) was added to the filtrate. The
resulting mixture was stirred at room temperature (rt) for 1 h then was
evaporated under reduced pressure, and the residue was diluted in
MeOH (2.5 mL). Amberlyst 15 ion-exchange resin (106 mg) was
added to the solution of the O-protected hydroxamate, and the
mixture was stirred at rt for 1 h. Afterward, the reaction was filtered
and the filtrate was concentrated in vacuum to give the crude 1, which
was purified by recrystallization; mp 202−204 °C; yield 48%.
Recrystallization system: methanol. 1H NMR (DMSO-d6) δ 1.69
(m, 4H, CH2CH2CH2CH2S), 2.02 (t, 2H, CH2CO), 3.25 (t, 2H,
CH2S), 6.72 (s, 1H, C5-H), 7.39−7.48 (m, 3H, benzene rings), 7.73−
7.83 (m, 4H, benzene rings), 8.14 (m, 2H, benzene rings), 8.69 (s, 1H,
NHOH), 10.37 (s, 1H, NHOH), 12.6 (s, 1H, uracil NH). 13C NMR
(DMSO-d6) δ 24.6, 31.7, 32.9, 36.1, 112.4, 127.1 (2C), 127.6, 127.9
(2C), 128.0 (2C), 129.2 (2C), 134.8, 140.8, 140.9, 160.6, 163.9, 167.7,
169.6 ppm. Anal. (C21H21N3O3S) % Calcd: C, 63.78; H, 5.35; N,
10.63; S, 8.11. Found (%) : C, 64.04; H, 5.40; N, 10.49; S, 8.01. MS
(ESI), m/z: 396 [M + H].
was added to a solution of butyl 3-(5-(2,3-diphenylpropanamido)-
pyridin-2-yl)acrylate 6 (1.26 mmol, 0.54 g) in ethanol (5 mL), and the
final solution was stirred at rt overnight. Then a 2 N HCl solution (5
mL) was added dropwise to the reaction, and the precipitated solid
was filtered, washed with water (3 × 10 mL) and dried to give the pure
7 as a colorless solid recrystallized by toluene; mp 123−125 °C; yield
83%. 1H NMR (DMSO-d6) δ 2.99−3.02 (dd, 1H, PhCHHCO−),
3.40−3.45 (dd, 1H, PhCHHCO−), 4.04−4.07 (t, 1H, PhCH2−),
6.66−6.70 (d, 1H, CH2CH2COOH), 7.15−7.45 (m, 11H, CH2
CH2COOH and benzene protons), 7.59−7.61 (d, 1H, pyridine
proton), 8.04−8.06 (d, 1H, pyridine proton), 8.70 (s, 1H, pyridine
proton), 10.51 (bs, 1H, NHCO), 12.00 (bs, 1H, COOH) ppm. 13C
NMR (DMSO-d6) δ 47.4, 48.5, 120.8, 122.9, 126.2, 127.7 (2C), 128.7
(2C), 129.5 (2C), 129.8 (2C), 130.6 (2C), 138.3, 139.5, 140.3, 142.5,
144.9, 148.9, 171.6, 172.7 ppm. MS (ESI), m/z: 371 [M − H]−.
Synthesis of 3-(5-(2,3-Diphenylpropanamido)pyridin-2-yl)-N-hy-
droxyacrylamide (2). To a cooled (0 °C) solution of 3-(5-(2,3-
diphenylpropanamido)pyridin-2-yl)acrylic acid 7 (0.32 mmol, 0.12 g)
in anhydrous THF (5 mL), ethyl chloroformate (0.35 mmol, 0.03 mL)
and triethylamine (0.39 mmol, 0.05 mL) were added and the resulting
mixture was stirred at 0 °C for 15 min. The solid was filtered and
washed with anhydrous THF (3 × 5 mL), and then O-(2-methoxy-2-
propyl)hydroxylamine (0.97 mmol, 0.07 mL) was added to the
solution at 0 °C and stirred at rt for 1 h. After this time, the solvent
was removed under vacuum, the residue was eluted with methanol (5
mL), and Amberlist 15 ion-exchange resin (0.032 g) was added to this
solution. The resulting mixture was stirred for 1 h, then the resin was
filtered and the solution concentrated to give 2 that was recrystallized
Synthesis of N-(2-Bromopyridin-5-yl)-2,3-diphenyl Propanamide
(5). Triethylamine (4.73 mmoli, 0.66 mL) and 2,3-diphenylpropanoyl
chloride (3.93 mmol, 0.96 g, previously prepared by reaction of the
corresponding acid with thionyl chloride (5 mL) at 80 °C for 1 h)
were added to a solution of 2-bromo-5-aminopyridine (3.93 mmol,
0.68 g) in anhydrous dichloromethane (10 mL) cooled at 0 °C. After
1.5 h, the reaction was quenched by water (50 mL) and extracted with
dichloromethane (3 × 50 mL). The collected organic phases were
washed with saturated sodium chloride solution (100 mL), dried, and
concentrated to obtain a solid residue that was purified by silica gel
chromatography eluting with ethyl acetate/chloroform 1/1 to afford
pure 5 as a colorless solid recrystallized by toluene; mp 131−133 °C;
1
by acetonitrile; mp 178−180 °C; yield 68%. H NMR (DMSO-d6) δ
2.98−3.03 (dd, 1H, PhCHHCO−), 3.40−3.45 (dd, 1H,
PhCHHCO−), 4.06−4.10 (t, 1H, PhCH2−), 6.78−6.82 (d, 1H,
CH2CH2CONHOH), 7.15−7.46 (m, 11H, benzene protons),
7.55−7.57 (d, 1H, pyridine proton), 8.06−8.08 (d, 1H, pyridine
proton), 8.74 (s, 1H, pyridine proton), 9.5 (bs, 1H, NHOH), 10.84
(bs, 1H, NHCO), 11.0 (bs, 1H, NHOH) ppm. 13C NMR (DMSO-d6)
δ 47.4, 48.5, 120.8, 122.9, 126.2, 127.7 (2C), 128.7 (2C), 129.5 (2C),
129.8 (2C), 130.6 (2C), 138.3, 139.5, 140.3, 142.5, 144.9, 148.9, 161.8,
172.7 ppm. Anal. (C23H21N3O3) % calcd: C, 71.30; H, 5.46; N, 10.85.
Found (%): C, 71.19; H, 5.41; N, 11.04. MS (ESI), m/z: 388 [M +
H]+.
1
yield 85%. H NMR (CDCl3) δ 3.02−3.07 (dd, 1H, PhCHHCO−),
3.55−3.61 (dd, 1H, PhCHHCO−), 3.80−3.84 (t, 1H, PhCH2−),
7.12−7.32 (m, 11H, pyridine benzene and −NHCO− protons), 7.91−
7.94 (m, 2H, pyridine protons), 8.00 (s, 1H, pyridine proton) ppm.
13C NMR (CDCl3) δ 47.5, 48.4, 126.2, 127.7, 128.5 (2C), 129.0, 129.5
HDAC1−11 Isoforms Inhibition Assay. See SI.
Biology: CSC Cultures. See SI.
Cell Viability Assay. See SI.
(2C), 129.8 (2C), 130.3, 130.7 (2C), 136.9, 138.4, 139.5, 141.9, 145.3,
173.0 ppm. MS (ESI), m/z 381 [M + H]+.
Cell Growth Assay. Single cells obtained after dissociation of
sarcospheres were plated in 12-well plates (50000/well) in complete
IMDM in order to induce cell adhesion. After 24 h, cells were
incubated with three concentrations (0.5, 1, and 2 μM) of 1 and 2 that
were selected on the basis of their IC50. After 24, 48, and 72 h, the
number of viable cells was evaluated by dye exclusion viability assay.
The percentage of growth inhibition was calculated in respect to
negative control cells (exposed to DMSO). The experiment was
repeated three times in duplicate.
Immunofluorescence. To confirm the activity of the HDACi in
CSCs, the acetylation level of histone H3 was detected by
immunofluorescence. Briefly, single MG-63 cells obtained after
dissociation of sarcospheres were seeded on glass coverslips in
complete IMDM in order to induce cell adhesion and then exposed to
0.5 and 2 μM of 1 and 2. After 10 h, the cells were fixed in 4%
paraformaldehyde, permeabilized in 0.15% Triton X-100, saturated
using 4% BSA in PBS at rt for 1 h, and incubated overnight with
antiacetyl-histone H3K9 (Abcam, Cambridge, UK) at 1:50 dilution in
PBS-4% BSA and then with a FITC-conjugated secondary antibody.
Slides were mounted in glycerol-DABCO and observed using a Nikon
Eclipse E600 microscope equipped with a digital camera. Images were
elaborated only for brightness and contrast using Adobe Photoshop 7.
Western Blotting. See SI.
Synthesis of Butyl 3-(5-(2,3-Diphenylpropanamido)pyridin-2-
yl)acrylate (6). Triphenylphosphine (0.55 mmol, 0.143 g) and
palladium acetate (0.27 mmol, 0.06 g) were added under nitrogen
atmosphere to a solution of 5 (6.56 mmol, 2.50 g), tetra-n-
butylammonium iodide (6.56 mmol, 2.42 g), sodium acetate trihydrate
(17.05 mmol, 2.32 g), water (0.7 mL), and butyl acrylate (13.11 mmol,
2.0 mL) in anhydrous N,N-dimethylformamide (13.0 mL) and in
sealed tube. The resulting mixture was stirred at 140 °C overnight,
then the reaction was quenched by water (50 mL) and extracted with
ethyl acetate (3 × 50 mL). The collected organic phases were washed
with saturated sodium chloride solution (100 mL), dried, and
concentrated to obtain an oily residue that was purified by silica gel
chromatography eluting with ethyl acetate/n-hexane 1:2 to provide
pure 6 as a pale-yellow oil; yield 42%. 1H NMR (CDCl3) δ 0.95−0.99
( t , 3 H , O C H 2 C H 2 C H 2 C H 3 ) , 1 . 4 1 − 1 . 4 7 ( m , 2 H ,
OCH2CH2CH2CH3), 1.65−1.71 (m, 2H, OCH2CH2CH2CH3),
3.06−3.11 (dd, 1H, PhCHHCO−), 3.59−3.65 (dd, 1H,
PhCHHCO−), 3.78−3.81 (t, 1H, PhCH2−), 4.20−4.23 (t, 2H,
OCH2CH2CH2CH3), 6.77−6.81 (d, 1H, CH2CH2COOBu), 7.11−
7.38 (m, 12H, benzene protons, pyridine proton and NHCO), 7.59−
7.63 (d d, 1H, CH2CH2COOBu), 8.15−8.17 (d, 1H, pyridine
proton), 8.33−8.34 (d, 1H, pyridine proton) ppm. 13C NMR (CDCl3)
δ 13.9, 18.5, 31.5, 47.7, 48.4, 65.0, 116.5, 121.3, 125.9, 127.9 (2C),
128.7 (2C), 129.4 (2C), 129.8 (2C), 130.7 (2C), 138.5, 139.8 (2C),
140.6, 144.8, 149.0, 166.7, 172.7 ppm. MS (ESI), m/z: 429 [M + H]+.
Synthesis of 3-(5-(2,3-Diphenylpropanamido)pyridin-2-yl)acrylic
Acid (7). A solution of 2N potassium hydroxide (2.52 mmol, 0.14 g)
Apoptosis Analysis. Dissociated sarcospheres were seeded on
glass coverslips in complete IMDM in order to induce cell adhesion
and then exposed to 0.5, 1, and 2 μM of 1 and 2. Untreated cells were
considered as negative control. After 48 h, cells were fixed by
E
DOI: 10.1021/acs.jmedchem.5b00126
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Brief Article
methanol/acetic acid (3:1) for 5 min, hydrated with 7% NaHCO3
1:200 in Hank’s balanced salt solution (HBSS) at rt for 5 min, and
dark incubated in 2.25 μg/mL of Hoechst 33258 (Sigma-Aldrich) at rt
for 10 min. Cells were observed under fluorescence microscopy to
determine the number cells with apoptotic nuclear bodies over at least
five different fields at 20× objective. Results were expressed as
percentage of apoptotic cells over the fields.
Sapienza Ateneo Project 2013 (D.R.), PRIN 2012 (prot.2012C-
TAYSY) (D.R.), IIT-Sapienza Project (A.M.), FP7 Projects
BLUEPRINT/282510 and A-PARADDISE/602080 (A.M.).
ABBREVIATIONS USED
■
CSCs, cancer stem cells; CTCL, cutaneous T-cell lymphoma;
ES, Ewing’s sarcoma; FBS, fetal bovine serum; FICT-,
fluorescein isothiocyanate-; IMDM, Iscove’s Modified Dulbec-
co’s Medium; α-MEM, α-minimum essential medium; OS,
osteosarcoma; RMS, rhabdomyosarcoma; UBHAs, uracil-based
hydroxyamides
Osteogenic Differentiation. Osteogenic differentiation was
evaluated by in vitro assessing of cell mineralization ability of one
selected CSC culture. The 10000 cells derived from MG-63
sarcospheres were seeded in α-MEM (Sigma-Aldrich) supplemented
with 10% FBS (complete α-MEM) in order to induce cell adhesion.
After reaching a confluence of 70% cells, mineralization was induced
by mineralization medium (MM medium), consisting of complete α-
MEM supplemented with 10 mM β-glycerophosphate, 10−8
M
REFERENCES
(1) Montero Luis, A.; Per
Multidisciplinary management of soft tissue sarcomas. Clin. Transl.
■
dexamethasone, and 50 mg/mL L-ascorbic acid 2-phosphate (Sigma-
Aldrich). During the differentiation period, the cells were continuously
exposed to subcytotoxic concentrations of 1 and 2 (0.5, 0.1, 0.05, and
0.025 μM). After 14 days, cell viability was assessed by Alamar Blue
assay (see SI). Then, the cells were stained by Alizarin Red S, which
selectively binds to calcium salts, in order to evaluate the ability of cell
to deposit mineral nodules. Briefly, cells were fixed with 3.7%
paraformaldehyde at rt for 20 min, washed with PBS, and stained with
2% Alizarin Red S (Sigma-Aldrich), pH 4.2, in distilled H2O at rt for 1
h. The cells were washed thoroughly several times with distilled H2O
to remove unspecific staining. To quantify the content of calcium salts,
Alizarin Red dye was eluted by incubation with 10% cetylpyridinium
chloride in distilled H2O at rt for 15 min under gentle shaking. The
absorbance was measured at 570 nm with a microplate reader (Tecan
Infinite F200pro, Crailsheim, Germany). Data were expressed as OD
measured in treated sample in respect to negative control (MM
medium alone). The experiment was performed in triplicate.
Statistical Analysis. See SI.
́
́
ez Aguilar, D.; Lop
́
ez Martín, J. A.
Oncol. 2010, 12, 543−553.
(2) Jordan, C. T.; Guzman, M. L.; Noble, M. Cancer stem cells. N.
Engl. J. Med. 2006, 355, 1253−1261.
(3) Gibbs, C. P.; Kukekov, V. G.; Reith, J. D.; Tchigrinova, O.;
Suslov, O. N.; Scott, E. W.; Ghivizzani, S. C.; Ignatova, T. N.;
Steindler, D. A. Stem-like cells in bone sarcomas: implications for
tumorigenesis. Neoplasia 2005, 7, 967−976.
(4) Salerno, M.; Avnet, S.; Bonuccelli, G.; Eramo, A.; De Maria, R.;
Gambarotti, M.; Gamberi, G.; Baldini, N. Sphere-forming cell subsets
with cancer stem cell properties in human musculoskeletal sarcomas.
Int. J. Oncol. 2013, 43, 95−102.
(5) Mai, A.; Massa, S.; Rotili, D.; Cerbara, I.; Valente, S.; Pezzi, R.;
Simeoni, S.; Ragno, R. Histone deacetylation in epigenetics: an
attractive target for anticancer therapy. Med. Res. Rev. 2005, 25, 261−
309.
(6) Falkenberg, K. J.; Johnstone, R. W. Histone deacetylases and their
inhibitors in cancer, neurological diseases and immune disorders.
Nature Rev. Drug Discovery 2014, 13, 673−691.
(7) Salvador, M. A.; Wicinski, J.; Cabaud, O.; Toiron, Y.; Finetti, P.;
Josselin, E. The histone deacetylase inhibitor abexinostat induces
cancer stem cells differentiation in breast cancer with low Xist
expression. Clin. Cancer Res. 2013, 19, 6520−6531.
(8) Chikamatsu, K.; Ishii, H.; Murata, T.; Sakakura, K.; Shino, M.;
Toyoda, M.; Takahashi, K.; Masuyama, K. Alteration of cancer stem
cell-like phenotype by histone deacetylase inhibitors in squamous cell
carcinoma of the head and neck. Cancer Sci. 2013, 104, 1468−1475.
(9) Rotili, D.; Tarantino, D.; Nebbioso, A.; Paolini, C.; Huidobro, C.;
Lara, E.; Mellini, P.; Lenoci, A.; Pezzi, R.; Botta, G.; Lahtela-Kakkonen,
ASSOCIATED CONTENT
* Supporting Information
Experimental for HDAC assay, CSC cultures, cell viability
assay, Western blotting, annexin V-FICT assay, Alamar Blue
assay, and statistical analysis. The Supporting Information is
available free of charge on the ACS Publications website at
DOI: 10.1021/acs.jmedchem.5b00126.
■
S
AUTHOR INFORMATION
Corresponding Authors
*For N.B.: phone, +39 051 6366678; fax, +39 051 6366974; E-
mail, nicola.baldini@ior.it.
*For A.M.: phone, +39 06 49913392; fax, +39 06 49693268; E-
mail, antonello.mai@uniroma1.it.
■
M.; Poso, A.; Steinkuhler, C.; Gallinari, P.; De Maria, R.; Fraga, M.;
̈
Esteller, M.; Altucci, L.; Mai, A. Discovery of salermide-related sirtuin
inhibitors: binding mode studies and antiproliferative effects in cancer
cells including cancer stem cells. J. Med. Chem. 2012, 55, 10937−
10947.
Author Contributions
(10) Rotili, D.; Tarantino, D.; Carafa, V.; Paolini, C.; Schemies, J.;
#G. Di Pompo and M. Salerno share first authorship of this
Jung, M.; Botta, G.; Di Maro, S.; Novellino, E.; Steinkuhler, C.; De
̈
article.
Maria, R.; Gallinari, P.; Altucci, L.; Mai, A. Benzodeazaoxaflavins as
sirtuin inhibitors with antiproliferative properties in cancer stem cells.
J. Med. Chem. 2012, 55, 8193−8197.
Notes
The authors declare no competing financial interest.
(11) Wu, Z.; Ma, C.; Shan, Z.; Ju, Y.; Li, S.; Zhao, Q. Histone
deacetylase inhibitors suppress the growth of human osteosarcomas in
vitro and in vivo. J. BUON 2013, 18, 1032−1037.
(12) Kutko, M. C.; Glick, R. D.; Butler, L. M.; Coffey, D. C.; Rifkind,
R. A.; Marks, P. A.; Richon, V. M.; La Quaglia, M. P. Histone
deacetylase inhibitors induce growth suppression and cell death in
human rhabdomyosarcoma in vitro. Clin. Cancer Res. 2003, 9, 5749−
5755.
(13) Liu, S.; Cheng, H.; Kwan, W.; Lubieniecka, J. M.; Nielsen, T. O.
Histone deacetylase inhibitors induce growth arrest, apoptosis, and
differentiation in clear cell sarcoma models. Mol. Cancer Ther. 2008, 7,
1751−1761.
(14) Siddiqi, S.; Mills, J.; Matushansky, I. Epigenetic remodeling of
chromatin architecture: exploring tumor differentiation therapies in
ACKNOWLEDGMENTS
■
We thank Garland R. Marshall, Department of Biochemistry
and Molecular Biophysics, Washington University School of
Medicine, St. Louis, Missouri, USA, for critical reading of the
manuscript and scientific discussion. This work was supported
by FIRB grants (RBAP10447J to N.B. and RBFR10ZJQT to
A.M.) from the Italian Ministry of Education, University and
Research, by the “5 per mille” 2010 (N.B.) and RF-2010-
2318330 (A.M.) grant from the Italian Ministry of the Health,
Financial Support for Scientific Research, and by the Italian
Association for Cancer Research (AIRC 11426 to N.B.),
F
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Journal of Medicinal Chemistry
Brief Article
mesenchymal stem cells and sarcomas. Curr. Stem Cell Res. Ther. 2010,
5, 63−73.
(29) Colussi, C.; Rosati, J.; Straino, S.; Spallotta, F.; Berni, R.; Stilli,
D.; Rossi, S.; Musso, E.; Macchi, E.; Mai, A.; Sbardella, G.; Castellano,
S.; Chimenti, C.; Frustaci, A.; Nebbioso, A.; Altucci, L.; Capogrossi, M.
C.; Gaetano, C. Nε-Lysine acetylation determines dissociation from
GAP junctions and lateralization of connexin 43 in normal and
dystrophic heart. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 2795−2800.
(30) Lenoir, O.; Flosseau, K.; Ma, F. X.; Blondeau, B.; Mai, A.;
Bassel-Duby, R.; Ravassard, P.; Olson, E. N.; Haumaitre, C.;
Scharfmann, R. Specific control of pancreatic endocrine beta- and
delta-cell mass by class IIa histone deacetylases HDAC4, HDAC5, and
HDAC9. Diabetes 2011, 60, 2861−2871.
(31) Azzi, S.; Bruno, S.; Giron-Michel, J.; Clay, D.; Devocelle, A.;
Croce, M.; Ferrini, S.; Chouaib, S.; Vazquez, A.; Charpentier, B.;
Camussi, G.; Azzarone, B.; Eid, P. Differentiation therapy: targeting
human renal cancer stem cells with interleukin 15. J. Natl. Cancer Inst.
2011, 103, 1884−1898.
(15) Xiao, W.; Mohseny, A. B.; Hogendoorn, P. C.; Cleton-Jansen, A.
M. Mesenchymal stem cell transformation and sarcoma genesis. Clin.
Sarcoma Res. 2013, 3, 10.
(16) Mai, A.; Massa, S.; Rotili, D.; Simeoni, S.; Ragno, R.; Botta, G.;
Nebbioso, A.; Miceli, M.; Altucci, L.; Brosch, G. Synthesis and
biological properties of novel, uracil-containing histone deacetylase
inhibitors. J. Med. Chem. 2006, 49, 6046−6056.
(17) Mai, A.; Perrone, A.; Nebbioso, A.; Rotili, D.; Valente, S.;
Tardugno, M.; Massa, S.; De Bellis, F.; Altucci, L. Novel uracil-based 2-
aminoanilide and 2-aminoanilide-like derivatives: histone deacetylase
inhibition and in-cell activities. Bioorg. Med. Chem. Lett. 2008, 18,
2530−2535.
(18) Mai, A.; Massa, S.; Valente, S.; Simeoni, S.; Ragno, R.; Bottoni,
P.; Scatena, R.; Brosch, G. Aroyl-pyrrolyl hydroxyamides: influence of
pyrrole c4-phenylacetyl substitution on histone deacetylase inhibition.
ChemMedChem 2006, 1, 225−237.
(19) Mai, A.; Massa, S.; Pezzi, R.; Valente, S.; Loidl, P.; Brosch, G.
Synthesis and biological evaluation of 2-, 3-, and 4-acylaminocinnamyl-
N-hydroxyamides as novel synthetic HDAC inhibitors. Med. Chem.
2005, 1, 245−254.
(32) Lombardo, Y.; Scopelliti, A.; Cammareri, P.; Todaro, M.; Iovino,
F.; Ricci-Vitiani, L.; Gulotta, G.; Dieli, F.; De Maria, R.; Stassi, G. Bone
morphogenetic protein 4 induces differentiation of colorectal cancer
stem cells and increases their response to chemotherapy in mice.
Gastroenterology 2011, 140, 297−309.
(20) Thaler, F.; Varasi, M.; Colombo, A.; Boggio, R.; Munari, D.;
Regalia, N.; Rozio, M. G.; Reali, V.; Resconi, A. E.; Mai, A.; Gagliardi,
S.; Dondio, G.; Minucci, S.; Mercurio, C. Synthesis and biological
characterization of amidopropenyl hydroxamates as HDAC inhibitors.
ChemMedChem 2010, 5, 1359−1372.
(21) Thaler, F.; Colombo, A.; Mai, A.; Amici, R.; Bigogno, C.;
Boggio, R.; Cappa, A.; Carrara, S.; Cataudella, T.; Fusar, F.; Gianti, E.;
di Ventimiglia, S. J.; Moroni, M.; Munari, D.; Pain, G.; Regalia, N.;
Sartori, L.; Vultaggio, S.; Dondio, G.; Gagliardi, S.; Minucci, S.;
Mercurio, C.; Varasi, M. Synthesis and biological evaluation of N-
hydroxyphenylacrylamides and N-hydroxypyridin-2-ylacrylamides as
novel histone deacetylase inhibitors. J. Med. Chem. 2010, 53, 822−839.
(22) Valente, S.; Tardugno, M.; Conte, M.; Cirilli, R.; Perrone, A.;
Ragno, R.; Simeoni, S.; Tramontano, A.; Massa, S.; Nebbioso, A.;
Miceli, M.; Franci, G.; Brosch, G.; Altucci, L.; Mai, A. Novel cinnamyl
hydroxyamides and 2-aminoanilides as histone deacetylase inhibitors:
apoptotic induction and cytodifferentiation activity. ChemMedChem
2011, 6, 698−712.
(23) Yang, F.; Zhang, T.; Wu, H.; Yang, Y.; Liu, N.; Chen, A.; Li, Q.;
Li, J.; Qin, L.; Jiang, B.; Wang, X.; Pang, X.; Yi, Z.; Liu, M.; Chen, Y.
Design and optimization of novel hydroxamate-based histone
deacetylase inhibitors of bis-substituted aromatic amides bearing
potent activities against tumor growth and metastasis. J. Med. Chem.
2014, 57, 9357−9369.
(24) Marks, P. A.; Breslow, R. Dimethyl sulfoxide to vorinostat:
development of this histone deacetylase inhibitor as an anticancer
drug. Nature Biotechnol. 2007, 25, 84−90.
(25) Knipstein, J.; Gore, L. Entinostat for treatment of solid tumors
and hematologic malignancies. Expert Opin. Invest. Drugs 2011, 20,
1455−1467.
(26) Mai, A.; Massa, S.; Pezzi, R.; Simeoni, S.; Rotili, D.; Nebbioso,
A.; Scognamiglio, A.; Altucci, L.; Loidl, P.; Brosch, G. Class II (IIa)-
selective histone deacetylase inhibitors. 1. Synthesis and biological
evaluation of novel (aryloxopropenyl)pyrrolyl hydroxamides. J. Med.
Chem. 2005, 48, 3344−3353.
(27) Nebbioso, A.; Manzo, F.; Miceli, M.; Conte, M.; Manente, L.;
Baldi, A.; De Luca, A.; Rotili, D.; Valente, S.; Mai, A.; Usiello, A.;
Gronemeyer, H.; Altucci, L. Selective class II HDAC inhibitors impair
myogenesis by modulating the stability and activity of HDAC-MEF2
complexes. EMBO Rep. 2009, 10, 776−782.
(28) Spallotta, F.; Rosati, J.; Straino, S.; Nanni, S.; Grasselli, A.;
Ambrosino, V.; Rotili, D.; Valente, S.; Farsetti, A.; Mai, A.; Capogrossi,
M. C.; Gaetano, C.; Illi, B. Nitric oxide determines mesodermic
differentiation of mouse embryonic stem cells by activating class IIa
histone deacetylases: potential therapeutic implications in a mouse
model of hindlimb ischemia. Stem Cells 2010, 28, 431−442.
G
DOI: 10.1021/acs.jmedchem.5b00126
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