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Apr 23, 2015 - Institute of Molecular Genetics, Unit of Bologna IOR, National ... Pasteur Institute Cenci Bolognetti Foundation, Sapienza University o...
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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 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 human osteosarcoma, rhabdomyosarcoma, 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.

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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 studies.11−13 This is possibly due to the involvement of histone © XXXX American Chemical Society

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

Figure 1. Design of the novel HDACi 1 and 2 and structure of the SIRTi 3 tested in this study. Received: January 21, 2015

A

DOI: 10.1021/acs.jmedchem.5b00126 J. Med. Chem. XXXX, XXX, XXX−XXX

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RESULTS AND DISCUSSION HDAC Inhibitory Enzyme Assays. The two novel HDACi 1 and 2 were tested against all the HDAC1−11 isoforms in 10dose 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/nanomolar 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. 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 panHDACi 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). 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

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 2,3diphenylpropanamide 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

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CHEMISTRY The synthetic routes followed for the preparation of 1 and 2 are depicted in Scheme 1. The hydroxamate 1 was synthesized Scheme 1a

(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. a

from the 5-((4-([1,1′-biphenyl]-4-yl)-6-oxo-1,6-dihydropyrimidin-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-diphenylpropanoyl chloride (previously prepared reacting 2,3-diphenylpropanoic acid with thionyl chloride at 80 °C) and triethylamine in anhydrous dichlomethane to furnish N-(6-bromopyridin-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).

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 11.7

>50 9.37

0.007 0.01

>50 8.77

0.61 0.61

>50 10.6

0.04 1.8

0.1 10.2

B

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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 dosedependent 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). 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 untreated 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

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 differC

DOI: 10.1021/acs.jmedchem.5b00126 J. Med. Chem. XXXX, XXX, XXX−XXX

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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 sarcospheres, 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. 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.

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.

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).

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EXPERIMENTAL SECTION

Chemistry. Melting point were determined on a Buchi 530 melting point apparatus and are uncorrected. 1H NMR spectra were recorded 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.

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CONCLUSIONS Here we show the effects of three HDACi, 1 (class I/IIbselective), 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 J. Med. Chem. XXXX, XXX, XXX−XXX

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Synthesis of 5-((4-([1,1′-Biphenyl]-4-yl)-6-oxo-1,6-dihydropyrimidin-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]. 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; yield 85%. 1H 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. 13 C NMR (CDCl3) δ 47.5, 48.4, 126.2, 127.7, 128.5 (2C), 129.0, 129.5 (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]+. Synthesis of Butyl 3-(5-(2,3-Diphenylpropanamido)pyridin-2yl)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-nbutylammonium 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, 3H, OCH2CH2CH2CH3), 1.41−1.47 (m, 2H, 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)

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-hydroxyacrylamide (2). To a cooled (0 °C) solution of 3-(5-(2,3diphenylpropanamido)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-2propyl)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 by acetonitrile; mp 178−180 °C; yield 68%. 1H 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]+. HDAC1−11 Isoforms Inhibition Assay. See SI. Biology: CSC Cultures. See SI. Cell Viability Assay. See SI. 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. 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

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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. 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 αMEM supplemented with 10 mM β-glycerophosphate, 10−8 M dexamethasone, and 50 mg/mL L-ascorbic acid 2-phosphate (SigmaAldrich). 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.

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Sapienza Ateneo Project 2013 (D.R.), PRIN 2012 (prot.2012CTAYSY) (D.R.), IIT-Sapienza Project (A.M.), FP7 Projects BLUEPRINT/282510 and A-PARADDISE/602080 (A.M.).

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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 Dulbecco’s Medium; α-MEM, α-minimum essential medium; OS, osteosarcoma; RMS, rhabdomyosarcoma; UBHAs, uracil-based hydroxyamides

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(1) Montero Luis, Á .; Pérez Aguilar, D.; López Martín, J. A. Multidisciplinary management of soft tissue sarcomas. Clin. Transl. 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, M.; Poso, A.; Steinkühler, 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. (10) Rotili, D.; Tarantino, D.; Carafa, V.; Paolini, C.; Schemies, J.; Jung, M.; Botta, G.; Di Maro, S.; Novellino, E.; Steinkühler, C.; De 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. (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

ASSOCIATED CONTENT

S 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.

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*For N.B.: phone, +39 051 6366678; fax, +39 051 6366974; Email, [email protected]. *For A.M.: phone, +39 06 49913392; fax, +39 06 49693268; Email, [email protected]. Author Contributions #

G. Di Pompo and M. Salerno share first authorship of this article. Notes

The authors declare no competing financial interest.

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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-20102318330 (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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