Novel pyrrole-containing histone deacetylase inhibitors endowed with cytodifferentiation activity

The International Journal of Biochemistry & Cell Biology 39 (2007) 1510–1522

Original research

Novel pyrrole-containing histone deacetylase inhibitors endowed with cytodifferentiation activity Antonello Mai a,∗ , Sergio Valente a , Dante Rotili a , Silvio Massa b , Giorgia Botta a , Gerald Brosch c , Marco Miceli d , Angela Nebbioso d , Lucia Altucci d,e,∗∗ a

c

Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Studi Farmaceutici, Universit`a degli Studi di Roma “La Sapienza”, P.le A. Moro 5, 00185 Roma, Italy b Dipartimento Farmaco Chimico Tecnologico, Universit` a degli Studi di Siena, via A. Moro, 53100 Siena, Italy Division of Molecular Biology, Biocenter, Innsbruck Medical University, Fritz-Preglstrasse 3, 6020 Innsbruck, Austria d Dipartimento di Patologia Generale, Seconda Universit` a degli Studi di Napoli, vico L. De Crecchio 7, 80138 Napoli, Italy e Centro di Oncogenomica AIRC, CEINGE Biotecnologia avanzata, Napoli Available online 4 April 2007

Abstract A novel series of aroyl-pyrrolyl-hydroxy-amides (APHAs) active as histone deacetylase (HDAC) inhibitors has been reported. The new derivatives were designed by replacing the benzene ring of the prototype 1 with both aromatic and aliphatic, monocyclic and polycyclic rings (compounds 3a–i), or by inserting a number of substituents on the methylene linker of 1 (compounds 4a–l). Compounds 3a–i and 4a–l were active at sub-micromolar level against the maize deacetylases HD1-B (class I), HD1-A (class II), and HD2. Tested at 5 ␮M against human HDAC1 and HDAC4, 3b, 4a, and 4j showed significant HDAC1 inhibition, whereas on HDAC4 only 4a was highly effective. On the human leukemia U937 cell line, the same compounds did not alter the cell cycle phases and failed in inducing apoptosis. However, they displayed granulocytic differentiation at 5 ␮M, with 3b being the most potent (76% CD11c positive cells). Tested to evaluate their effects on histone H3 and ␣-tubulin acetylation, 3b and 4a showed high H3 acetylation, whereas 4a and 4b were the most potent with ␣-tubulin as a substrate. © 2007 Elsevier Ltd. All rights reserved. Keywords: Pyrrole-containing compounds; Histone deacetylase inhibitors; Aroyl-pyrrolyl-hydroxyamides; Apoptosis; Granulocytic differentiation

1. Introduction Histone acetyltransferases (HATs) and histone deacetylases (HDACs) are key enzymes in chromatin remodelling, one of the epigenetic mechanisms for regulation of gene expression. HATs catalyze the acetylation at the N-terminal lysine residues of histone ∗

Corresponding author. Tel.: +39 06 4991 3392; fax: +39 06 491491. Corresponding author. Tel.: +39 081 566 7569; fax: +39 081 450 169. E-mail addresses: [email protected] (A. Mai), [email protected] (L. Altucci). ∗∗

1357-2725/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2007.03.020

tails, allowing the chromatin to fold in its relaxed status (euchromatin) that is transcriptionally active. Conversely, by removing the acetyl groups from the Lys residues at histone tails HDACs produce a condensed, transcriptionally silent form of chromatin (heterochromatin) (Cheung, Briggs, & Allis, 2000; Wu & Grunstein, 2000; Wolffe & Guschin, 2000; Kouzarides, 1999; Strahl & Allis, 2000). To date, the HDAC family is grouped into four classes, according to the homology of the mammalian with the corresponding yeast enzymes. Class I (HDAC1-3,8), class II (HDAC4-7,9,10), and class IV (HDAC11) HDACs, homologues of yeast RPD3 (class I/IV) and HDA1 (class II), are Zn-dependent

A. Mai et al. / The International Journal of Biochemistry & Cell Biology 39 (2007) 1510–1522

deacetylases, whereas class III HDACs (SIRT1-7, sirtuins) show homology with the yeast Sir2 and use NAD+ as co-factor for their deacetylase activity (Grozinger & Schreiber, 2002; Gregoretti, Lee, & Goodson, 2004; Verdin, Dequiedt, & Kasler, 2003; Blander & Guarente, 2004). Class I/II/IV HDACs take part to multiprotein, often multi-HDAC-containing complexes, in which some DNA binding proteins (such as Rb and Rb-like proteins, N-CoR, SMRT, MEF, MeCP2, sin3A, etc.) are involved, acting as transcriptional co-repressors by blocking the expression of some tumour-suppressor genes (Ng & Bird, 2000; Ferreira, Magnaghi-Jaulin, Robin, Harel-Bellan, & Trouche, 1988; Heinzel et al., 1997). Thus, a altered balance of the HAT/HDAC equilibrium may lead to the aberrant transcription of genes regulating cellular differentiation, cell cycle, and apoptosis. The treatment of such alterations with HDAC inhibitors (HDACis) can restore selective programs of cell differentiation and/or apoptosis, through the regulation of genes exerting a pleiotropic effect on key pathways involved in proliferation, apoptosis, DNA synthesis and repair, and protein turnover (Mai et al., 2005; Marks et al., 2001; Johnstone, 2002; Minucci & Pelicci, 2006). A number of HDACis are in clinical trials as anticancer agents (Kelly, O’Connor, & Marks, 2002), and the hybrid polar compound suberoylanilide hydroxamic

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acid (SAHA) has been approved by FDA in 2006, October for the treatment of cutaneous T-cell lymphoma with the name of Vorinostat. Since 2001 we have been reporting a series of pyrrole-containing synthetic HDACis, the aroylpyrrolyl-hydroxy-amides (APHAs), that resembles in their structure the HDACi trichostatin A (TSA) and SAHA (Yoshida, Kijima, Akita, & Beppu, 1990; Richon et al., 1998; Massa et al., 2001; Mai et al., 2002). From structure–activity relationship studies, it emerged that the highest HDAC inhibiting activity is associated with: (i) the presence of an arylalkanoyl moiety at the C4 position of the pyrrole ring; (ii) the presence of the methyl substituent at the N1pyrrole; (iii) the insertion of a N-hydroxypropenamide chain at the pyrrole C2 position (Mai et al., 2003, 2004, 2006; Ragno et al., 2004). Among APHA derivatives, the 3-[1-methyl-4-phenylacetyl-1H-pyrrol2-yl]-N-hydroxy-2-propenamide 1 inhibited HDACs at submicromolar level, and in the maize HDAC system (HD1-B, homologue of class I, and HD1-A, homologue of class II HDACs) showed three-fold selectivity for class II. In murine erythroleukemia (MEL) cell assays, 1 showed antiproliferative and cytodifferentiating effects (Mai et al., 2003, 2004). Chemical manipulations performed on the 1 structure led to compound 2, strictly

Fig. 1. Chemical structures of the aroyl-pyrrolyl-hydroxy-amides (APHAs) 1, 2, and the novel compounds 3a–i and 4a–l.

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related to 1 and more potent both in enzymatic and in biological assays (Mai et al., 2006) (Fig. 1). In an alternative approach, we modified the lead compound 1 by replacing the phenyl ring of its C4-phenylacetyl portion with the bulkier 1-naphthyl, 2naphthyl, 3-benzothienyl, and 2-indanyl groups, as well as with differently sized cycloalkyl moieties (compounds 3a–i). Moreover, a wide series of alkyl substituents both saturated or unsaturated, straight or branched, with or without heteroatoms were inserted at the methylene connecting the phenyl and carbonyl groups of the C4phenacetyl group of 1, thus obtaining the compounds 4a–l (Fig. 1). The novel compounds 3a–i and 4a–l were tested against the maize HDACs HD1-B (class I) (K¨olle et al., 1999; Lechner et al., 2000), HD1-A (class II) (Brosch, Goralik-Schramel, & Loidl, 1996), and HD2 (Lusser, Brosch, Loidl, Haas, & Loidl, 1997), which is structurally quite different from mammalian HDACs and has been assigned to a class of its own. Selected derivatives 3b, 4a, 4b, and 4j were tested against human HDAC1 and HDAC4, and in the human leukaemia U937 cell line to determine their effects on cell cycle, apoptosis induction, and granulocytic differentiation. The differentiation activity of the novel APHAs was also evaluated in the human acute myeloid leukaemia NB4 cell line. Finally, the capability of 3b, 4a, 4b, and 4j to hyperacetylate histones H3 and ␣-tubulin (Haggarty, Koeller, Wong, Grozinger, & Schreiber) as well as to induce the expression of the cyclin dependent kinase inhibitor p21Waf1 (Sambucetti et al., 1999) in the U937 leukemia cells was assessed.

2. Materials and methods 2.1. Chemistry Acylation reactions between the ethyl 3-(1-methyl1H-pyrrol-2-yl)propenoate (Sinisterra, Mouloungui, Delmas, & Gaset, 1985) and the appropriate acyl chlorides under Friedel–Crafts conditions afforded the ethyl esters 5a–u, that were hydrolyzed in alkaline medium to furnish the corresponding propenoic acids 6a–u. Further reactions of 6a–u with ethyl chloroformate and triethylamine, followed by the addition of O(2-methoxy-2-propyl)hydroxylamine (Mori & Koseki, 1988) and then acidic hydrolysis with the Amberlyst 15 ion-exchange resin gave the desired hydroxamates 3a–i and 4a–l (Scheme 1). The 2-substituted phenylacetic acids useful for the synthesis of derivatives 4c–j,l were obtained by standard methods (phenylacetic acid, n-butyl lithium, diethylamine, alkyl halide at −70 ◦ C). Chemical and physical data for compounds 3a–i and 4a–l are listed in Table 1. Chemical and physical data for the intermediate compounds 5a–u and 6a–u are listed in Table 2. 2.2. Syntheses of compounds Melting points were determined on a Buchi 530 melting point apparatus and are uncorrected. Infrared (IR) spectra (KBr) were recorded on a Perkin-Elmer Spectrum One instrument. 1 H NMR spectra were recorded at 400 MHz on a Bruker AC 400 spectrometer; chemical shifts are reported in δ (ppm) units relative to the inter-

Scheme 1. Synthesis of compounds 3a–i and 4a–l. Reagents—a: (1) RCH(R1 )COOH, SOCl2 ; (2) AlCl3 , 1,2-dichloroethane; b: KOH, C2 H5 OH; c: ClCOOC2 H5 , (C2 H5 )3 N, THF; d: NH2 OC(CH3 )2 OCH3 ; e: Amberlyst 15, CH3 OH.

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Table 1 Chemical and physical data of compounds 3a–I and 4a–l Compound

R

R1

Melting point (◦ C)

Crystallization solvents

Yield (%)

3a 3b 3c 3d 3e 3f 3g 3h 3i 4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 4k 4l

1-Naphthyl 2-Naphthyl 3-Benzothienyl 2-Indanyl Cyclopentyl Cyclohexyl Cycloheptyl 2-Norbornyl 1-Adamantyl Phenyl Phenyl Phenyl Phenyl Phenyl Phenyl Phenyl Phenyl Phenyl Phenyl Phenyl Phenyl

H H H H H H H H H Methyl Ethyl iso-Propyl n-Propyl 2-Propenyl n-Butyl 2-Butenyl 3-Methyl-2-butenyl 2-Methylthioethyl 2-Methoxyethyl Phenyl Benzyl

210–212 170–171 155–157 159–160 180–181 203–202 198–200 199–200 214–215 214–215 226–227 135–138 168–170 134–136 169–170 125–127 154–155 170–172 160–162 138–140 158–160

Benzene/acetonitrile Benzene/acetonitrile Benzene/acetonitrile Acetonitrile Benzene/acetonitrile Acetonitrile Benzene/acetonitrile Methanol Methanol Methanol Acetonitrile Benzene Benzene/acetonitrile Benzene Benzene/acetonitrile Benzene Benzene Benzene Benzene/acetonitrile Benzene/acetonitrile Benzene

65 47 53 65 67 56 54 49 58 64 60 57 63 59 61 66 54 57 69 63 68

Analytic results were within ±0.40% of the theoretical values.

nal reference tetramethylsilane (Me4 Si). All compounds were routinely checked by TLC and 1 H NMR. TLC was performed on aluminum-backed silica gel plates (Merck DC, Alufolien Kieselgel 60 F254 ) with spots visualized by UV light. All solvents were reagent grade and, when necessary, were purified and dried by standard methods. Concentration of solutions after reactions and extractions involved the use of a rotary evaporator operating at reduced pressure of ca. 20 Torr. Organic solutions were dried over anhydrous sodium sulphate. Analytical results are within ±0.40% of the theoretical values (by Carlo Erba 1106 elemental analyzer). All chemicals were purchased from Aldrich Chimica, Milan (Italy), or from Lancaster Synthesis GmbH, Milan (Italy), and were of the highest purity. General procedure for the synthesis of ethyl 3-(4acyl-1-methyl-1H-pyrrol-2-yl)-2-propenoates (5a–u). Example: ethyl 3-[4-(2,3-diphenylpropionyl)-1-methyl1H-pyrrol-2-yl]-2-propenoate (5u). Aluminum trichloride (15 mmol, 2 g) was slowly added to a cooled (0–5 ◦ C) solution of ethyl 3-(1-methyl-1H-pyrrol-2yl)-2-propenoate (Sinisterra et al., 1985) (7.5 mmol, 1.34 g) and 2,3-diphenylpropionyl chloride (previously prepared by heating the corresponding propionic acid (15.0 mmol, 3.39 g) with thionyl chloride (15 ml) for 1 h at 50 ◦ C) in 1,2-dicloroethane (100 ml). After stirring at room temperature for 30 min, the reaction mixture was poured onto crushed ice (100 g) and the pH of the solu-

tion was adjusted to 4 with 37% HCl. The organic layer was separated, and the aqueous one was extracted with chloroform (3 × 50 ml). The combined organic solution was washed with water (100 ml), dried and evaporated to dryness. The residual oil was purified by column chromatography on silica gel by eluting with a 1:20 mixture of ethyl acetate:chloroform. Compound 5u was obtained as pure solid and was recrystallized by diethyl ether. 1 H NMR (CDCl3 ) δ 1.30 (t, 3H, J = 7.12 Hz, COOCH2 CH3 ), 3.00 (dd, 1H, J = 11.7 Hz, J = 6.7 Hz, PhCH2 ), 3.55 (dd, 1H, J = 11.7 Hz, J = 7.8 Hz, PhCH2 ), 3.62 (s, 3H, N-CH3 ), 4.21 (q, 2H, J = 7.12 Hz, COOCH2 CH3 ), 4.40 (dd, 1H, J = 7.8 Hz, J = 6.7 Hz, PhCHCO), 6.14 (d, 1H, J = 15.7 Hz, CH CHCOOEt), 6.94 (s, 1H, pyrrole ␤-proton), 7.06–7.26 (m, 11H, benzene protons and pyrrole ␣-proton), 7.40 (d, 1H, J = 15.7 Hz, CH CHCOOEt); 13 C NMR (CDCl3 ) δ 14.2, 33.6, 38.1, 53.0, 61.4, 104.8, 122.3, 123.2, 124.3, 126.0, 127.7, 127.8, 128.7, 129.7, 130.6, 136.0, 139.5, 166.5, 199.3 ppm; IR (KBr) 1715 (CO), 1626 (CO) cm−1 . Analysis (C25 H25 NO3 , M.W. = 387.47): calcd. (%) C, 77.49; H, 6.50; N, 3.61; found (%) C, 77.77; H, 6.61; N, 3.37. General procedure for the synthesis of 3-(4-acyl1-methyl-1H-pyrrol-2-yl)-2-propenoic acids (6a–u). Example: 3-(4-cyclopentylacetyl-1-methyl-1H-pyrrol2-yl)-2-propenoic acid (6e). A mixture of 5e (4.94 mmol, 1.43 g), 2N potassium hydroxyde (19.76 mmol, 1.11 g,),

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Table 2 Chemical and physical data of compounds 5 and 6 Compound

R

R1

Melting point (◦ C)

Crystallization solvents

Yield (%)

5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 5m 5n 5o 5p 5q 5r 5s 5t 5u 6a 6b 6c 6d 6e 6f 6g 6h 6i 6j 6k 6l 6m 6n 6o 6p 6q 6r 6s 6t 6u

1-Naphthyl 2-Naphthyl 3-Benzothienyl 2-Indanyl Cyclopentyl Cyclohexyl Cycloheptyl 2-Norbornyl 1-Adamantyl Phenyl Phenyl Phenyl Phenyl Phenyl Phenyl Phenyl Phenyl Phenyl Phenyl Phenyl Phenyl 1-Naphthyl 2-Naphthyl 3-Benzothienyl 2-Indanyl Cyclopentyl Cyclohexyl Cycloheptyl 2-Norbornyl 1-Adamantyl Phenyl Phenyl Phenyl Phenyl Phenyl Phenyl Phenyl Phenyl Phenyl Phenyl Phenyl Phenyl

H H H H H H H H H Methyl Ethyl iso-Propyl n-Propyl 2-Propenyl n-Butyl 2-Butenyl 3-Methyl-2-butenyl 2-Methylthioethyl 2-Methoxyethyl Phenyl Benzyl H H H H H H H H H Methyl Ethyl iso-Propyl n-Propyl 2-Propenyl n-Butyl 2-Butenyl 3-Methyl-2-butenyl 2-Methylthioethyl 2-Methoxyethyl Phenyl Benzyl

115–117 113–115 101–103 50–52 Oil Oil Oil Oil 149–151 103–105 95–96 Oil Oil 60–61 Oil Oil Oil 170–172 160–162 120–122 59–60 258–260 245–246 208–210 220–221 180–182 195–196 200–202 >250 >250 164–165 152–153 84–86 138–139 88–89 129–130 134–135 >250 140–143 Oil 208–210 94–95

Diethyl ether Diethyl ether Cyclohexane/benzene Cyclohexane/benzene

59 50 63 65 61 58 54 61 62 56 66 64 60 71 67 59 62 57 58 63 61 77 85 82 85 89 73 68 73 69 73 76 72 86 84 80 75 82 74 78 94 85

Cyclohexane/benzene Diethyl ether Cyclohexane/benzene

Cyclohexane

Benzene Cyclohexane/benzene Diethyl ether Benzene/acetonitrile Acetonitrile Benzene/acetonitrile Acetonitrile Benzene Benzene Acetonitrile Benzene/acetonitrile Benzene/acetonitrile Benzene/acetonitrile Benzene Diethyl ether Benzene/acetonitrile Benzene Benzene Benzene Methanol Benzene Acetonitrile Diethyl ether

Analytic results were within ±0.40% of the theoretical values.

and ethanol (15 ml) was stirred at room temperature overnight. Afterwards, the solution was poured into water (50 ml) and extracted with ethyl acetate (3 × 20 ml). To the aqueous layer 2N HCl was added until the pH was 5, and the precipitate was filtered and recrystallized from benzene giving the pure compound 6e. 1 H NMR (DMSO-d6 ) δ 1.1–1.8 (m, 8H, cyclopentyl CH2 ), 2.2 (m, 1H, cyclopentyl CH), 2.69 (d, 2H, J = 10.4 Hz, CH2 CO), 3.76 (s, 3H, N-CH3 ), 6.25 (d, 1H, J = 15.7 Hz, CH CHCOOH), 7.15 (s, 1H, pyrrole

␤-proton), 7.55 (d, 1H, J = 15.7 Hz, CH CHCOOH), 7.80 (s, 1H, pyrrole ␣-proton), 12.3 (s, 1H, COOH); 13 C NMR (DMSO-d ) δ 27.9, 33.6, 35.9, 47.4, 104.8, 6 121.6, 123.2, 124.3, 130.6, 138.8, 170.6, 200.1 ppm; IR (KBr) 2960 (OH), 1683 (CO), 1609 (CO) cm−1 . Analysis (C21 H23 NO3 , M.W. = 337.41): calcd. (%) C, 74.75; H, 6.87; N, 4.15; found (%) C, 74.52; H, 6.78; N, 4.39. General procedure for the synthesis of 3-(4-acyl1-methyl-1H-pyrrol-2-yl)-N-hydroxypropenamides

A. Mai et al. / The International Journal of Biochemistry & Cell Biology 39 (2007) 1510–1522

(3a–h, 4a–l). Example: 3-[4-(2-phenylpent-4-enoyl)1-methyl-1H-pyrrol-2-yl)-N-hydroxy-2-propenamide (4e). Ethyl chloroformate (1.74 mmol, 0.17 ml) and triethylamine (1.88 mmol, 0.26 ml) were added to a cooled (0 ◦ C) solution of 6p (1.45 mmol, 0.45 g) in dry tetrahydrofuran (10 ml), and the mixture was stirred for 10 min. The solid was filtered off. To the filtrate was added O-(2-methoxypropyl)-hydroxylamine (Mori & Koseki, 1988) (4.35 mmol, 0.32 ml). The solution was stirred for 15 min at 0 ◦ C, then the mixture was evaporated under reduced pressure, the residue was eluted in methanol (10 ml) and to the solution of protected hydroxamate Amberlyst 15 (450 mg) was added, and the mixture was stirred at room temperature for 1 h. The resin was filtered and the filtrate was concentrated in vacuum to give compound 4e which was recrystallized from benzene. 1 H NMR (DMSO-d ) δ 2.38 (m, 1H, CH CHCH ), 2 2 6 2.77 (m, 1H, CH2 CHCH2 ), 3.68 (s, 3H, N-CH3 ), 4.44 (dd, 1H, J = 7.3 Hz, J = 6.8 Hz, PhCHCO), 4.89 (d, 1H, J = 10.2 Hz, CH2 CHCH2 ), 4.99 (d, 1H, J = 17.2 Hz, CH2 CHCH2 ), 5.65 (m, 1H, CH2 CHCH2 ), 6.21 (d, 1H, J = 15.7 Hz, CH CHCONHOH), 6.87 (s, 1H, pyrrole ␤-proton), 7.16–7.35 (m, 6H, benzene protons and CH CHCONHOH), 7.83 (s, 1H, pyrrole ␣-proton), 9.00 (s, 1H, OH), 10.80 (s, 1H, NH); 13 C NMR (DMSO-d6 ) δ 33.6, 50.6, 104.8, 116.4, 123.2, 124.3, 127.7, 129.3, 130.6, 134.4, 134.8, 136.4, 161.6, 199.3 ppm; IR (KBr) 3200 (NH), 1663 (CO), 1609 (CO) cm−1 . Analysis (C19 H20 N2 O3 , M.W. = 324.37): calcd. (%) C, 70.35; H, 6.21; N, 8.64; found (%) C, 70.59; H, 6.31; N, 8.42. 2.3. In vitro maize HD1-B, HD1-A, and HD2 enzyme inhibition Radioactively labeled chicken core histones were used as the enzyme substrate according to established procedures (Lechner et al., 1996; Brosch, Lusser, Goralik-Schramel, & Loidl, 1996; Kolle, Brosch, Lechner, Lusser, & Loidl, 1998). The enzyme liberated tritiated acetic acid from the substrate, which was quantified by scintillation counting. IC50 values are results of triple determinations. A 50 ␮l sample of maize enzyme (at 30 ◦ C) was incubated (30 min) with 10 ␮l of total [3 H]acetate-prelabeled chicken reticulocyte histones (2 mg/ml). Reaction was stopped by addition of 36 ␮l of 1 M HCl/0.4 M acetate and 800 ␮l of ethyl acetate. After centrifugation (10,000 × g, 5 min), an aliquot of 600 ␮l of the upper phase was counted for radioactivity in 3 ml of liquid scintillation cocktail. The compounds were tested at a starting concentration of 40 ␮M, and active substances were diluted further. TSA

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and SAHA were used as the reference compounds, and blank solvents were used as negative controls. 2.4. Cellular assays. Cell lines and cultures U937 and NB4 cell lines were cultured in RPMI with 10% fetal calf serum, 100 U/ml penicillin, 100 ␮g/ml streptomycin and 250 ng/ml amphotericin-B, 10 mM HEPES and 2 mM glutamine. Cells were kept at the constant concentration of 200,000 cells per milliliter of culture medium. Human breast cancer ZR-75.1 cells were propagated in DMEM medium supplemented with 10% foetal calf serum and antibiotics (100 U/ml penicillin, 100 ␮g/ml streptomycin and 250 ng/ml amphotericin-B). In all biological assays, the cells of the control are representative of cells treated with vehicle. 2.5. Cell-based human HDAC1 and HDAC4 assays Cells (U937 cells for the HDAC1 assay; ZR75.1 cells for the HDAC4 assay) were lysed in IP buffer (50 mM Tris–HCl pH 7.0, 180 mM NaCl, 0.15% NP-40, 10% glycerol, 1.5 mM MgCl2, 1 mM NaMO4 , 0.5 mM NaF) with protease inhibitor cocktail (Sigma), 1 mM DTT and 0.2 mM PMSF for 10 min in ice and centrifugated at 14,700 × g for 30 min. Thousand micrograms of extracts were diluted in IP buffer up to 1 ml and pre-cleared by incubating with 20 ␮l A/G plus Agarose (Santa Cruz) for 30 min–1 h on a rocking table at 4 ◦ C. Supernatants were transferred into a new tube and the antibodies (3 ␮g) were added and IP was allowed to proceed overnight at 4 ◦ C on a rocking table. Antibodies used have been: HDAC1 (Abcam) and HDAC4 (Sigma). As negative control the same amount of protein extracts were immunoprecipitated with the corresponding purified IgG (Santa Cruz). The day after 20 ␮l A/G plus Agarose (Santa Cruz) were added to each IP and incubation was continued for 2 h. The beads were recovered by brief centrifugation and washed with cold IP buffer several times. The samples were then washed twice in PBS and resuspended in 20 ␮l of sterile PBS. The HDAC assay was carried out according to suppliers instructions (Upstate). Briefly, samples immunoprecipitated with the HDAC4 and HDAC1 or with purified IgG were pooled respectively to homogenize all samples. Ten microliters of the IP were incubated with a previously labelled 3 HHistone H4 peptide linked with streptavidine agarose beads (Upstate). In details, 120,000 CPM of the H43 H-acetyl-peptide was used for each tube and incubated in 1× HDAC buffer with 10 ␮l of the sample in presence or absence of HDAC inhibitors with a final volume of 200 ␮l. Those samples were incubated over night at

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37 ◦ C in slow rotation. The day after 50 ␮l of a quenching solution were added and 100 ␮l of the samples were counted in duplicate after a brief centrifugation in a scintillation counter. Experiments have been carried out in quadruplicate independently.

previously described (Nebbioso et al., 2005; Altucci et al., 2001). Western blots were shown for p21 (Transduction Laboratories, dilution 1:500) and total ERKs and Actine (Santa Cruz) were used to normalise for equal loading.

2.6. Cell cycle analysis on U937 cells

2.10. Histone H3 and α-tubulin acetylation in U937 cells

2.5 × 105 cells were collected and resuspended in 500 ␮l of a hypotonic buffer (0.1% Triton X-100, 0.1% sodium citrate, 50 ␮g/ml propidium iodide (PI), RNAse A). Cells were incubated in the dark for 30 min. Samples were acquired on a FACS-Calibur flow cytometer using the Cell Quest software (Becton Dickinson) and analysed with standard procedures using the Cell Quest software (Becton Dickinson) and the ModFit LT Version 3 Software (Verity) as previously reported (Nebbioso et al., 2005). All the experiments were performed three times in duplicate independently.

For determination of ␣-tubulin acetylation, 25 ␮g of total protein extracts were separated on a 10% polyacrylamide gel and blotted. Western blots were shown for acetylated ␣-tubulin (Sigma, dilution 1:500) and total ERKs and Actine (Santa Cruz, dilution 1:1000) were used to normalise for equal loading. For quantification of histone H3 acetylation, 100 ␮g of total protein extracts were separated on a 15% polyacrylamide gel and blotted. Western blots were shown for acetylated histone H3 (Upstate) and total tubulin (Sigma) was used to normalise for equal loading.

2.7. FACS analysis of apoptosis on U937 cells 3. Results Apoptosis was measured with Annexin V/propidium iodide (PI) double staining detection (Roche and Sigma–Aldrich, respectively) as recommended by the suppliers; samples were analysed by FACS with Cell Quest technology (Becton Dickinson) as previously reported (Altucci et al., 2001). As second assay the caspase 3–7 detection (B-Bridge) was performed and quantified by FACS using the Cell Quest technology (Becton Dickinson). As third method, apoptosis was also quantified by the amount of the pre-G1 pick fragmentation. 2.8. Granulocytic differentiation on U937 cells Granulocytic differentiation was carried out as previously described (Altucci et al., 2001). Briefly, U937 cells were harvested and resuspended in 10 ␮l phycoerythrine-conjugated CD11c (CD11c-PE). Control samples were incubated with 10 ␮l PE conjugated mouse IgG1, incubated for 30 min at 4 ◦ C in the dark, washed in PBS and resuspended in 500 ␮l PBS containing PI (0.25 ␮g/mL). Samples were analysed by FACS with Cell Quest technology (Becton Dickinson). PI positive cells have been excluded from the analysis. 2.9. Determination of p21WAF1/CIP1 induction in U937 cells Hundred micrograms of total protein extracts were separated on a 15% polyacrylamide gel and blotted as

3.1. Enzyme inhibitory activities and structure–activity relationship The novel APHA compounds 3a–i and 4a–l were tested against the maize HDACs HD1-B (class I), HD1A (class II), and the specific form HD2. The results, expressed as IC50 (50% inhibitory concentration) values, are reported in Table 3. Among the new pyrrole derivatives 3a–i, all except 3h were more potent than 1 against HD1-B, with IC50 values ranging from 105 to 31 nM, while against HD1-A only 3a,b (IC50 = 33 (3a) and 19 (3b) nM) showed higher inhibiting activity than 1. Structure–activity relationship showed that to have an increase of anti-HD1-B activity, the phenyl ring of the C4-phenylacetyl moiety of 1 can be replaced by a wide range of both heterocyclic and aliphatic rings (with the exception of the 2-norbornyl substituent), whereas only the 1- and 2-naphthyl groups can efficiently replace the phenyl ring of 1 for the HD1A inhibiting activity. Against HD2, only the 2-naphthyl derivative 3b was more effective than the prototype 1. Differently from that observed with 3a–i, HD1-B inhibitory values for compounds 4a–l showed that only the insertion of a methyl (4a), ethyl (4b) or, to a lesser extent, 2-methoxyethyl (4j) group at the methylene of the C4-phenylacetyl moiety of 1 increased the inhibiting potency of the pyrrole derivatives of 10-fold (4a and 4b) or 1.6-fold (4j) respect to 1. On the contrary, the catalytic tunnel of HD1-A can accept a wider range of substituents

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Table 3 Maize HD1-B, HD1-A, and HD2 inhibitory activity of compounds 3a–i and 4a–l Compound

R

R1

IC50 ± S.D. (nM) HD1-B

3a 3b 3c 3d 3e 3f 3g 3h 3i 4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 4k 4l 1a

1-Naphthyl 2-Naphthyl 3-Benzothienyl 2-Indanyl Cyclopentyl Cyclohexyl Cycloheptyl 2-Norbornyl 1-Adamantyl Phenyl Phenyl Phenyl Phenyl Phenyl Phenyl Phenyl Phenyl Phenyl Phenyl Phenyl Phenyl

H H H H H H H H H Methyl Ethyl iso-Propyl n-Propyl 2-Propenyl n-Butyl 2-Butenyl 3-Methyl-2-butenyl 2-Methylthioethyl 2-Methoxyethyl Phenyl Benzyl

TSA SAHA

90 31 102 102 41 34 80 226 105 15 16 296 166 320 438 529 312 231 93 123 350 148

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

4.5 0.9 5.1 3 1.6 1.4 3.2 11.3 6.3 0.5 0.6 8.9 5 9.6 21.9 15.9 12.5 9.2 5.6 6.1 17.5 5.9

0.4 ± 0.01 28 ± 0.8

HD1-A 33 19 53 84 78 60 97 124 62 28 22 39 78 89 143 238 376 73 245 48 77 50

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.3 0.6 1.3 1.7 3.9 2.4 3.9 3.7 25 0.8 0.9 1.2 3.1 3.6 7.1 11.9 15 2.2 7.3 1.4 3.1 1.5

0.8 ± 0.03 178 ± 8.9

HD2 82 58 218 104 138 115 98 460 170 112 96 156 245 63 417 309 149 61 46 101 92 100

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3.3 2.9 10.9 6.2 5.5 6.9 5.9 23 8.5 5.6 3.8 6.2 4.9 1.9 16.7 12.4 4.5 3.7 1.8 5 3.7 4

7 ± 0.2 50 ± 2

Data represent mean values of at least three separate experiments. a Mai et al. (2004).

at the same position (methyl (4a), ethyl (4b), and isopropyl (4c) substituents increased up to 2.3-fold the anti-HD1-A activity of the derivatives, and the insertion of n-propyl, 2-methylthioethyl, phenyl, or benzyl groups produced only a little decrease of activity). The n-butyl and the unsaturated 2-butenyl and 3-methyl-2-butenyl as well as the 2-methoxyethyl group at the methylene position were detrimental for the HD1-A inhibitory action. The methyl-substituted (4a) and ethyl-substituted (4b) compounds were the most potent against HD1-A, with IC50 values of 28 and 22 nM, respectively. Against HD2, the insertion of allyl (4e), 2-methylthioehyl (4i), and 2methoxyethyl (4j) groups at the methylene position of 1 gave a increase of potency of the derivatives, with IC50 values ranging from 63 to 46 nM. Selected compounds 3b, 4a, 4b, and 4j were tested in vitro against human HDAC1 and HDAC4. HDAC1 and HDAC4 immunoprecipitates (IPs) were obtained from human leukemia U937 and breast cancer ZR-75.1 cell lysates, respectively, with the appropriate antibodies (anti-HDAC1 (Abcam) in U937, and anti-HDAC4 (Sigma) in ZR-75.1 cells), and the inhibition of the deacetylase activities with the new pyrroles at the con-

centration of 5 ␮M was determined. SAHA and MS-275 (a class I-selective HDACi) were used as reference drugs. As depicted in Fig. 2, 3b, 4a, and 4j showed high HDAC1 inhibitory action (61, 54, and 59%, respectively), while 4b (23%) was less effective. In the HDAC4 assay, 4a was the most potent compound (75% inhibition), while 3b and 4j inhibited HDAC4 only for 35% and 38%, thus being to some extent weak class I-selective compounds. 3.2. Effects on human leukemia U937 cells. Cell cycle, apoptosis induction, and granulocytic differentiation Selected derivatives 3b, 4a, 4b, and 4j were tested on the human leukemia U937 cell line at 1 and 5 ␮M after 48 h of incubation to determine their effects on cell cycle, apoptosis induction, and granulocytic differentiation. SAHA was used as reference drug. In the U937 cells, while SAHA showed at 5 ␮M an arrest in the S phase, the tested pyrroles did not significantly alter the cell cycle phases, with only 3b showing when used at 5 ␮M a weak arrest in the G1 phase (Fig. 3).

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Fig. 2. Human HDAC1 and HDAC4 inhibitory activities exerted by selected APHAs used at 5 ␮M.

The apoptosis induction was measured using three different methods, such as Annexin V/PI double staining, caspase 3-7 activation and DNA fragmentation by FACS analyses, and was checked after 50 h of treatment of U937 cells with selected APHAs used at 1 and 5 ␮M (Figs. 4–6). The tested compounds showed low apoptosis induction both at 1 and 5 ␮M, whereas SAHA used as reference drug showed massive apoptosis in all the assays performed (90%) at 5 ␮M. Granulocytic differentiation was evaluated by CD11c expression level upon 48 h of stimulation with selected

APHAs at 1 and 5 ␮M. SAHA was used as reference drug. Among the tested derivatives, 3b at 5 ␮M showed the highest percent of CD11c positive PI negative cells (76%), superior to those of SAHA (48% at 1 ␮M and 32% at 5 ␮M). Also 4a and b at 5 ␮M displayed good cytodifferentiating activities (46 and 53%, respectively, of CD11c positive PI negative cells), better than SAHA at 5 ␮M (Fig. 7a). Moreover, differentiation was also evaluated after 72 h of treatment confirming the maturative potential of APHA compounds at 1 and 5 ␮M. Naturally, the PI positive cells have been excluded from

Fig. 3. Cell cycle analysis of selected APHAs in human U937 cells after 48 h. The concentrations used are indicated.

Fig. 4. Apoptosis induced on U937 cells by selected APHAs after 50 h, with the Annexin V/PI double staining method. The concentrations used are indicated.

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Fig. 5. Apoptosis induced on U937 cells by selected APHAs after 50 h, with the caspase 3-7 activities method. The concentrations used are indicated.

Fig. 6. Apoptosis induced on U937 cells by selected APHAs after 50 h. The concentrations used are indicated. The figure shows the DNA fragmentation measured as pre-G1 pick.

the analysis and therefore SAHA and MS275 have been represented only at 1 ␮M being too apoptogenic at 5 ␮M (Fig. 7b). Finally, we have been testing the cytodifferentiative capacity of these selected APHA compounds in the NB4 cell line, the prototype of acute myeloid leukaemia cell line. As shown in Fig. 8, we could confirm that APHAs induced overexpression of CD11c in NB4 cells after 72 h of treatment. Naturally, we also tested the activity of the well-known HDAC inhibitors SAHA and MS275 which were killing all NB4 cells before as reported previously (Nebbioso et al., 2005). Those

results not only confirmed the activity on differentiation of APHAs in U937 cells, but indicated that this action is also exerted in other leukaemia cells, although with different kinetics. 3.3. H3 histone and α-tubulin acetylation assays: induction of p21WAF1 To validate with functional assays the anti-HDAC effect of the novel APHAs, the capacity to induce acetylation of compounds 3b, 4a, 4b, and 4j was tested using

Fig. 7. Granulocytic differentiation showed by selected APHAs on U937 cells after 48 h (a) and 72 h (b) of stimulation. The concentrations used are indicated. Dead cells have been excluded from the analysis.

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(all at 5 ␮M) for 24 h. Then, the total protein extracts were separated and blotted, and Western blots have been performed for acetylated histone H3. To determine the ␣-tubulin acetylation extent and the p21 induction, the extracts were treated with the pyrroles (1 and 5 ␮M) and SAHA (5 ␮M), and then separated and blotted after 24 and 48 h. Western blots are shown for acetylated ␣-tubulin and p21. All western blots experiments are representative of duplicates. In acetylation assays, compounds 3b, 4a and to a lesser extent 4b showed acetylating effects on H3 histone. With ␣-tubulin as substrate, only 4a and 4b displayed high acetylating activity (greater than SAHA) whereas 3b was much less effective. In the same assays, 4j gave weak histone H3 acetylation, whereas influenced ␣-tubulin acetylation similar to SAHA (Fig. 9). As regards to p21 induction assay, none tested compound was able to induce p21 expression, the only compound showing weak activity in this assay being 3b (Fig. 9). 4. Discussion Fig. 8. Granulocytic differentiation showed by selected APHAs on NB4 cells after 72 h of stimulation. The concentrations used are indicated. Dead cells have been excluded from the analysis.

H3 histone and ␣-tubulin (a non-histone substrate) as substrates (Fig. 9). Moreover, the capability of induction of the cyclin dependent kinase inhibitor p21WAF1/CIP1 for the tested compounds was assessed too (Fig. 9). H3 Histone acetylation assay was performed by treating the U937 cells with the novel compounds or with SAHA

A new series of pyrrole-containing HDACis belonging to the APHA family (compounds 3a–i and 4a–l) have been reported. Such compounds were designed by replacing the benzene ring of the APHA prototype 1 with polycyclic, both aromatic and aliphatic rings (3a–i) or by inserting a number of alkyl/aryl substituents (4a–l) at the methylene position of 1. From the comparison of the maize deacetylase HD1-B (class I) and HD1-A (class II) inhibitory data, it emerges that for high HD1-B inhibition the 1 analogues can bear a lot (both small and large,

Fig. 9. Histone H3 acetylation, ␣-tubulin acetylation, and p21 induction assays by selected APHAs on U937 cells. Total ERKs and Actine expression levels stay for equal loading.

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aromatic and aliphatic) of substituents by replacing the benzene ring, and must have a small group (methyl and ethyl, no larger!) as substituent at the methylene linkage. Instead, the prerequisites for efficient HD1-A inhibition are more stringent. HD1-A is able to accomodate only 1- and 2-naphthyl groups instead of benzene, and the other groups used in this study are only tolerated; moreover, HD1-A can accept methyl, ethyl, iso-propyl, and phenyl substituents at the methylene of the 1 scaffold, and can tolerate C3-carbon chain (both saturated and unsaturated), 2-methylthioethyl, and benzyl groups. These findings seem to show the possibility of a different size and shape for the catalytic tunnels of HD1-B (class I HDAC) and HD1-A (class II HDAC) enzymes, and can be useful for design and synthesis of new selective HDACis. Selected compounds (3b, 4a, 4b, and 4j) were tested against human HDAC1 and HDAC4 to confirm their deacetylase inhibiting activity in human systems in vitro. From IP assays data, 3b, 4a, and 4j were able to inhibit HDAC1 with % inhibition higher than 50%, whereas against HDAC4 IP only 4a was the most effective (75% of inhibition). Compounds 3b, 4a, 4b, and 4j were then tested on the human leukemia U937 cell line to evaluate their effects on cell cycle, apoptosis induction, and granulocytic differentiation. Whereas the tested compounds both at 1 and 5 ␮M did not show either alteration of the cell cycle phases or apoptosis induction, most of them at 5 ␮M were highly able to induce granulocytic differentiation, measured by the increase of the CD11c expression level. SAHA, tested as reference drug in the same assays, displayed a different behaviour, showing at 5 ␮M a dramatic block of the cell cycle in the S phase and a massive apoptosis measured with three different technical approaches (Nebbioso et al., 2005). These evidences indicate that cell cycle progression, differentiation and apoptosis could be considered as separable pathways which may require different epigenetic stimuli. In acetylation assays using histones H3 and ␣-tubulin as substrates, 3b showed high acetylating activity on histones and low activity on ␣-tubulin, thus sharing a slightly class I-selective HDACi behaviour. In the same assays, 4a and 4b were equally active in acetylating histones as well as ␣-tubulin, whereas 4j gave a signal of ␣-tubulin acetylation and only a faint blot with H3 histone, showing to be a slightly class IIb-selective inhibitor. Finally, the activity of selected APHA compounds have been measured as far as p21 induction is concerned. P21 is probably the best known target of HDAC inhibitors and its induction has been previously claimed as due to class I selective inhibition (Mai et al., 2005). Our results show

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a weak inducibility of p21 (Fig 9) whereas the APHA compounds are able to induce differentiation. These data would thereby support the hypothesis that p21 is dispensable for differentiation (although they do not exclude its participation in differentiation at least in some systems) as previously suggested by shRNA experiments (Nebbioso et al., 2005). Our findings are very exciting in consideration of the high therapeutic impact that is actually given to HDACis and more in general to epigenetic modulators. The possibility to design- and obtain-class selective HDAC inhibitors based on the data reported hereby has the fundamental aim to achieve more selective biological effects in neoplastic systems. Moreover, the fact that compounds able to induce a certain type of maturation in leukemia cells are minimally if at all affecting cell cycle and apoptosis at the indicated time points, suggests that at least in part differentiation may occur as an independent program. Agents able to selectively induce differentiation may show potential use alone or in combination with well-known cytodifferentiating agents such as retinoids, and thereby may find application for differentiation therapy of cancer. Acknowledgements This work was partially supported by grants from AIRC 2006 (A.M.), PRIN 2006 (A.M.), European Union (LSHC-CT2005-518417) (L.A.), and PRIN 2006 (L.A.). A.N. and M.M. have been financed within the LSHCCT2005-518417 EU contract. References Altucci, L., Rossin, A., Raffelsberger, W., Reitmair, A., Chomienne, C., & Gronemeyer, H. (2001). Retinoic acid-induced apoptosis in leukemia cells is mediated by paracrine action of tumor-selective death ligand TRAIL. Nat. Med., 7, 680–686. Blander, G., & Guarente, L. (2004). The Sir2 family of protein deacetylases. Annu. Rev. Biochem., 73, 417–435. Brosch, G., Goralik-Schramel, M., & Loidl, P. (1996). Purification of histone deacetylase HD1-A of germinating maize embryos. FEBS Lett., 393, 287–291. Brosch, G., Lusser, A., Goralik-Schramel, M., & Loidl, P. (1996). Purification and characterization of a high molecular weight histone deacetylase complex (HD2) of maize embryos. Biochemistry, 35, 15907–15914. Cheung, W. L., Briggs, S. D., & Allis, C. D. (2000). Acetylation and chromosomal functions. Curr. Opin. Cell Biol., 12, 326–333. Ferreira, R., Magnaghi-Jaulin, L., Robin, P., Harel-Bellan, A., & Trouche, D. (1988). The three members of the pocket proteins family share the ability to repress E2F activity through recruitment of a histone deacetylase. Proc. Natl. Acad. Sci. U. S. A., 95, 10493–10498. Gregoretti, I. V., Lee, Y.-M., & Goodson, H. V. (2004). Molecular evolution of the Histone deacetylase family: Functional implications of phylogenetic analysis. J. Mol. Biol., 338, 17–31.

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