European Journal of Medicinal Chemistry 70 (2013) 857e863
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Short communication
Azetidinoneeretinoid hybrids: Synthesis and differentiative effects Matteo Pori a,1, Paola Galletti a,1, Roberto Soldati a,1, Laura Calzà b, c, 2, Chiara Mangano c, 2, Daria Giacomini a, * a
Department of Chemistry “G. Ciamician”, University of Bologna, Via Selmi 2, 40126 Bologna, Italy Health Science and Technologies Interdepartmental Center for Industrial Research (HST-ICIR), University of Bologna, Via Tolara di Sopra 50, I-40064 Ozzano Emilia, Bologna, Italy c Department of Veterinary Medicine, University of Bologna, Via Tolara di Sopra 50/A, 40064 Ozzano Emilia, Bologna, Italy b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 May 2013 Received in revised form 12 September 2013 Accepted 29 September 2013 Available online 12 October 2013
As a part of a systematic investigation on the synthesis and biological activities of new b-lactam compounds, we examined b-lactam candidates 1, 2E and 2Z and their ability to induce cell proliferation or differentiation. Azetidinone 1 was chosen for its activity (previously evaluated) as selective HDAC8 inhibitor, whereas b-lactams 2E and 2Z were designed to have a hybrid retinoid-azetidinone structure, de novo synthesized and fully characterized. Biological activities were determined in cellular assays on neuroblastoma cells SH-SY5Y. Azetidinone 1, 2E and 2Z led to a moderate effect in decreasing SH-SY5Y cell proliferation and b-lactams 2E and 2Z induced an early stage differentiation. The present results uncovered a new role of specifically designed b-lactam compounds in epigenetic regulation. Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords: b-Lactams Retinoids Synthesis Cell differentiation HDAC inhibitors Epigenetic regulation
1. Introduction
b-Lactams have historically been viewed as an important class of antimicrobials since the discovery of naturally occurring penicillins and cephalosporins [1]. Recognition of the azetidin-2-one moiety as the key pharmacophoric component of b-lactam antibiotics initiated a flurry of synthetic activity to develop new structures, and nowadays new variants of monocyclic compounds (azetidinones) are showing new and specific biological activities [2]. In the last few decades their role as enzyme inhibitors has been expanded. b-Lactams are well-known as potent inhibitors of some enzymes that contain serine as the catalytic residue, including the bacterial penicillin binding proteins (PBPs), and Class A and Class C b-lactamases [3]. b-Lactams have also been appropriately modified to develop active site-directed and mechanism-based inhibitors of Human Leukocyte Elastase (HLE) [4], and in this field some new monocyclic b-lactams have shown important activities as HLE inhibitors [5], as new antibiotics towards resistant bacteria [6], and as inhibitors of platelet aggregation [7]. Recently, relevant studies
* Corresponding author. Tel.: þ39 051 209 9528; fax: þ39 051 209 9456. E-mail address: [email protected] (D. Giacomini). 1 Tel.: þ39 051 209 9450; fax: þ39 051 209 9456. 2 Tel.: þ39 051 2097 947; fax: þ39 051 2097 953. 0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejmech.2013.09.057
appeared on b-lactam compounds as anti-tumor drugs. Some arylb-lactam derivatives have been evaluated for cytotoxicity against a number of human tumor and normal cell lines [8]. In particular Nthiolated-b-lactams were found to induce DNA damage, cell growth arrest, and apoptosis in cultured human cancer cells, and antiproliferative effects on human breast cancer cells of some new azetidinones were evaluated [9]. Some bicyclic b-lactams were recently reported as inhibitors of Histone Deacetylases (HDACs), a family of proteins involved in the pattern of acetylation of chromatin proteins and thus in the regulation of gene expression [10]. We recently reported the synthesis of some azetidinone derivatives which showed good affinity and specificity towards histone deacetylases HDAC8 and HDAC6 [11]. Histone deacetylases are epigenetic regulators playing an important role in modulating gene transcription as well as protein function. Eighteen classical HDAC isoforms, divided into IV classes based on yeast deacetilases homology, are identified to date. HDAC inhibitors constitute a new class of promising anticancer drugs targeting one or more HDAC isoforms. HDAC6 overexpression was found in carcinoma of oral squamous tissue [12], although in breast cancer it correlates with better survival [13]. In lung, colon, and cervical cancer cell line the knockout of HDAC8 gene reduces cell proliferation [14]; in leukemia cell HDAC8 inhibitors leads to apoptosis [15]. The expression of HDAC8 (class I member) has been
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correlated with poor outcome in neuroblastoma [15], and in such cell line selective HDAC8 inhibitors induces differentiation and proliferation arrest [16]. Nowadays, there is great interest in the development of small molecules that regulate or modulate protein function and chemical genetic offers a valuable approach in this field [17]. Retinoids, as derivatives of Vitamin A, regulate key developmental pathways throughout life, by modulating the expression of many genes involved in cell proliferation, migration, differentiation and apoptosis, though the molecular mechanisms are not completely understood [18e21]. The effects of retinoic acid (RA) are mediated by the heterodimerization of nuclear RAR (Retinoic Acid Receptor), a family of ligand-dependent transcription factors, and RXR (Retinoid X Receptors), the 9-cis RA binding receptor [18]. To date, retinoids are known to activate or inhibit MAPK cascade, which is involved in both proliferation and growth arrest [22]. Some synthetic RA-derived molecules exert differentiative effect rather than apoptosis, blocking tumor progression in cancer cell lines [23,24]. In fact, retinoids are currently used as potential chemotherapeutic drugs in the treatment of several cancer types (neuroblastoma, breast cancer, lung cancer, rhabdomyosarcoma, and many others) [25e28]. For this reason, there is great interest in developing new synthetic molecules targeting retinoic acid receptors. As part of an interdisciplinary project on the development of new b-lactam derivatives with a modular structure designed for specific biological activities, the effect of the azetidinone 1 (Fig. 1), previously evaluated by us as specific HDAC8 inhibitor [11], was investigated on the proliferative activity of a neuron-like cell line. Moreover, we developed the synthesis of new b-lactams 2 with a hybrid retinoid-azetidinone structure and we investigated the retinoic acid-dependent differentiation of a neuron-like cell line. To our knowledge, only two example of b-lactams were reported in the literature with a differentiation-inducing activity on leukemia cells [29]. We report here the synthesis of the new hybrid retinoidb-lactams and preliminary results in biological evaluation of the three molecules on neuroblastoma cell line SH-SY5Y. 2. Results and discussion 2.1. Compound design and synthesis As mentioned above, we have previously reported an efficient synthesis of b-lactam 1 [11]. Its design was based on the pharmacophore model of HDAC inhibitors which consists in a modular structure with a cap-group that interacts with residues in the active site of the receptor and a linker group that sits in a hydrophobic channel and positions a metal-binding group which coordinates to the zinc ion in the active site. We demonstrated that the azetidinone ring can efficiently work as the zinc-binding group. Moreover, the substituent on the nitrogen atom allowed for the specificity against HDAC8 or HDAC6 inhibition. Azetidinone 1 was therefore chosen in the present study for its specific HDAC8 activity. Once the b-lactam ring was confirmed as an effective zincbinding group for HDAC, we developed a new hybrid structure retaining the b-lactam scaffold to target HDACs and functionalized with a retinoic side chain which could activate differentiative
Fig. 1. b-Lactam derivatives.
processes to mimic the action of retinoic acid (RA). The design of blactam 2 was based on a 4-alkylidene-b-lactam scaffold with demonstrated enhanced reactivity in protein inhibition [2a,5], and functionalized with a retinoid-amide side chain. The strategy is outlined in Scheme 1 and consists of a convergent synthesis with the coupling between the 4-alkylidene-azetidinone carboxy acid 3 and the retinoid amine 4 as the key step. 4-Alkyliden-azetidin-2-ones can be obtained using an original and well established protocol developed in our laboratory starting from 4-acetoxy-azetidinones and diazoesters in the presence of Lewis acids [30]. Given that 4-acetoxy azetidinone is commercially available, b-lactam 3 was obtained with benzyldiazoacetate and TiCl4 as outlined in Scheme 2. The 4-alkyliden-b-lactams 5 were obtained in 43% yields and in a E/Z diastereomeric ratio of 41/59. The diastereomeric mixture 5Z/5E was easily separated by flash chromatography. Hydrogenolysis of the isolated pure 5Z with Pd on carbon in THF-methanol 1:1 mixture gave 3 in quantitative yield. Retinoid amine 4 was obtained starting from the easily available b-ionone in a three step reaction (Scheme 3). Addition of lithium acetonitrile to b-ionone in THF at low temperature gave 6 [30], which underwent water elimination under ptoluensulfonic acid catalysis [31]. The nitrile 7 obtained as a E/Z mixture, was then reduced to give the amine 4EZ in 79% yield. Finally, with a carbodiimide coupling method, amides 2E and 2Z were obtained as a mixture and easily separated by flash chromatography. The 2E,4E and 2Z,4E configurations were assigned in the two isomers by NOE 1D analysis on the separated products 2E and 2Z (Scheme 4, and Supplementary material). In isomer 2E, the methyl group in the chain, chosen as the irradiated site, showed two NOE correlations with methylene and methine, respectively, indicating a close spatial relationship whereas for b-lactam 2Z NOE enhancements of the two vinylic CH were observed. The 2E,4E configuration for 2E, and the 2Z,4E configuration for 2Z were thus attributed. 2.2. Biology As said before, HDAC inhibitors are emerging as anti cancer drugs. In particular, HDAC8 inhibitors have shown to regulate proliferation in neuroblastoma cells [16]. As potential HDAC inhibitors, we evaluated the antiproliferative effect of azetidinones 1, 2E, and 2Z on undifferentiated SH-SY5Y. Moreover, as b-lactams 2E and 2Z have hybrid retinoid-azetidinone structures, we performed a set of experiments aiming to evaluate differentiative action of blactams 2E and 2Z compared to azetidinone 1 and retinoic acid treated cells. In fact, retinoids are currently used as potential chemotherapeutic drugs in the treatment of several cancer types including neuroblastoma due to their differentiative action [25e 28]. In order to assess azetidinone 1, 2E, and 2Z cytotoxicity, we calculated first the IC50 (50% inhibiting concentration) using MTT assay (Fig. 2). The MTT assay is a colorimetric assay for measuring the activity of cytosolic enzymes that reduce tetrazolium dye (MTT) in formazan salt in living cells. IC50 values were calculated and are reported above the curves in Fig. 2. The concentration used for subsequent experiments was chosen accordingly in order to obtain cytostatic effects rather than apoptosis or necrosis (Fig. 2). 2.2.1. Effect on proliferation In a preliminary analysis of biological effect on proliferation, undifferentiated SH-SY5Y cells were exposed to 7.5 microM of azetidinone 1, 10 microM of b-lactams 2E and 2Z for 24, 48, 72 and 96 h, the number of cells was counted daily and compared to control (vehicle treated). Fig. 3 shows the proliferation curve of control cells (ctrl, open circle), azetidinone 1 (black square), b-
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Scheme 1. Retro-synthetic route to retinoid b-lactam 2.
Scheme 2. Synthesis of 4-alkyliden-azetidinone 3.
Scheme 3. Synthesis of retinoid amine 4 and retinoid b-lactams 2E and 2Z.
Scheme 4. NOE 1D correlations in new retinoid-lactams for configuration assignments.
lactam 2E (black triangle) and 2Z treated cell (black rhombus). Significant differences were observed between the groups (twoway ANOVA, Dunnet’s multiple comparisons test). b-lactams 2E and 2Z determine a significative reduction in cell proliferation after 48 h of treatment (a: ctrl vs b-lactam 2E; b: ctrl vs b-lactam 2Z). All the compounds tested show an antiproliferative effect compared to control from 72 to 96 h (c: ctrl vs azetidinone 1 and b-lactam 2E, d: ctrl vs b-lactam 2Z, e: ctrl vs azetidinone 1 and b-lactam 2E p < 0.0001, f: ctrl vs b-lactam 2Z). Although the reduction is stable,
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Fig. 2. Cytotoxic evaluation of azetidinone 1, b-lactams 2E and 2Z on undifferentiated SH-SY5Y; IC50 ranges are reported above the curves. Data are shown as mean (normalized to control) SD.
doubling time calculated in 24e72 h range (Td) is affected exclusively in cells treated with b-lactam 2Z (TdCTRL ¼ 34,9 h; Td1 ¼ 39 h, Td2 ¼ 39.78, Td8 ¼ 51,25), whereas doubling time calculated in 24e 96 h range is not affected. These results indicated a slight effect of azetidinone 1, 2E and 2Z on SH-SY5Y proliferation. 2.2.2. Differentiation effect As they are derived from neuroblastoma, SH-SY5Y may be differentiated toward neural-like lineage. Retinoic acid is a powerful differentiating molecule inducing growth inhibition, neurites lengthening and branching. A six-days RA treatment induces neural-like differentiation as shown by neurites outgrowth (Fig. 4, panel B vs panel A; panel G vs panel F) visualized by bIIItubulin and NF200. Quantitative analysis was performed measuring the bIII-tubulin and NF200 network normalized according to the cells number. As hybrids retinoid-azetidinones, we investigated a possible differentiative effect of b-lactams 2E and 2Z compared to azetidinone 1 and RA treated SH-SY5Y. bIII-Tubulin is an early marker for differentiation toward neuronal lineage and the increase of bIII-tubulin area may be related to an early stage of differentiation. Neurofilament play important structural roles and influence axonal transport, neurite outgrowth and cell survival.
Sample micrographs of cell SH-SY5Y exposed to the vehicle (control), RA, azetidinone 1, and b-lactams 2E and 2Z are shown in Fig. 4, where panels AeE refers to b-tubulin-IR, and panels FeJ refers to neurofilament-IR, graph K and L to respective quantification of IR area/number of cells. While a differentiative effect of RA is evident in both bIII-tubulin and NF200 immunostaining (panels A vs panel B and panel F vs panel G, respectively) and azetidinone 1 does not display any differentiative effect (panels A vs panel C and panel F vs panel H), b-lactams 2E (panels A vs panel D and panel F vs panel I) and 2Z (panels A vs panel E and panel F vs panel J) induce a moderate differentiation, which is statistically significant in bIIItubulin staining (panels K and L, statistical analysis was performed with one-way ANOVA, Tukey post test). This effect could be possibly related to moderate structural analogies among RA and blactams 2E and 2Z. The data indicate a differentiative effect of blactams 2E and 2Z on SH-SY5Y, even though smaller when compared to RA. The absence in the b-lactams 2E and 2Z of an ionizable function, as like the carboxylic acid in RA, can explain the observed lower activity, as reported for some non-ionizable RA derivatives such as RA-esters or amides [32]. To bind effectively to the retinoic acid receptor (RAR), the polar terminus of the retinoid must be capable of efficient interactions, as observed in the crystal structure of RA-RAR complex [33]. The b-lactam scaffold, while it would not be considered a strong “polar terminus” in anchoring RAR, shows a positive effect on SH-SY5Y cell differentiation (blactams 2E and 2Z), and it deserves further investigation. 3. Conclusions As part of an interdisciplinary research project involving the synthesis of new monocyclic b-lactams specifically designed to target epigenetic regulators, we presented here an evaluation of three monocyclic azetidinones candidates 1, 2E and 2Z and their ability to interfere with cell proliferation or differentiation. Azetidinone 1 was chosen because of its specific inhibition of HDAC8, whereas 2E and 2Z were designed as innovative hybrid azetidinone-retinoid structures. Their de novo synthesis allowed for the anchoring of an alkylidenecarboxyl-side chain on the C4 position of the b-lactam core which was further condensed with a retinoid-amine. The new hybrids 2E and 2Z and the azetidinone 1 were tested in cellular assays on neuroblastoma cells SH-SY5Y. Azetidinone 1, b-lactams 2E and 2Z slightly decrease the proliferation rate in SH-SY5Y neuroblastoma cells, possibly due to the HDAC inhibition properties. The hybrid azetidinone-retinoids blactams 2E and 2Z have a differentiative effect, even if smaller when compared to RA. These preliminary results are worthy of a further evaluation and prompt us to currently work on the synthesis of a library of new hybrid azetidinone-retinoid molecules. 4. Experimental section 4.1. Chemistry
Fig. 3. Effect of azetidinones 1, 2E, and 2Z on SH-SY5Y proliferation. The rate of cells proliferation was determined as total cell number. Cells were incubated in the presence of 7.5 microM of azetidinone 1, 10 microM of 2E and 2Z and counted at 24, 48, 72, 96 h. Significant differences between groups were observed after 48 h of treatment. Statistical analysis was performed with two-way ANOVA, Dunnet’s multiple comparisons test (a: ctrl vs b-lactam 2E, p < 0.05; b: ctrl vs b-lactam 2Z, p < 0.01; c: ctrl vs azetidinone 1 and b-lactam 2E p < 0.001, d: ctrl vs b-lactam 2Z p < 0.0001, e: ctrl vs azetidinone 1 and b-lactam 2E p < 0.0001, f: ctrl vs b-lactam 2Z p < 0.001).
4.1.1. General All reactions were performed under an inert atmosphere (N2). Commercial reagents (reagent grade, >99%) were used as received without additional purification. Anhydrous solvents (CH3CN, CH2Cl2, THF) were obtained commercially. 1 H and 13C NMR values were recorded on a INOVA 400, or a GEMINI 200 instrument with a 5 mm probe. All chemical shifts have been quoted relative to deuterated solvent signals, d in ppm and J in Hz. FT-IR: Nicolet 380 measured as films between NaCl, plates wavenumbers reported in cm1. TLC: Merck 60 F254. Column chromatography: Merck silica gel 200e300 mesh. GCeMS: Agilent
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Fig. 4. Effect of azetidinone 1, b-lactams 2E and 2Z on SH-SY5Y differentiation. bIII-tubulin (AeC) and neurofilament (DeF) were used to investigate a possible microtubule and intermediate filaments rearrangement. Panels A and F refer to vehicle treated cells; B and G to cells treated for 6 days with RA; C and F correspond to cells treated for 6 days with 7.5 microM of azetidinone 1, D and I correspond to cells treated for 6 days with 10 microM of b-lactam 2E; E and J correspond to cells treated for 6 days with 10 microM of b-lactam 2Z (Scale bar ¼ 10 micron). Graphs K and L refer to bIII-tubulin and neurofilament immunoreactive area, respectively, normalized for number of cells (mean SD). Statistical analysis was performed with one-way ANOVA, Tukey post test, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Technologies, column HP5 5% Ph-Me Silicon MS: Agilent Technologies MSD1100 single-quadrupole mass spectrometer, EI voltage 70 eV, gradient from 50 C to 280 C in 30 min. HPLC-MS, HPLC: Agilent Technologies HP1100, column ZOBRAX-Eclipse XDB-C8 Agilent Technologies, mobile phase: H2O/CH3CN, gradient from 30% to 80% of CH3CN in 8 min, 80% of CH3CN until 25 min, 0.4 mL/min MS: Agilent Technologies MSD1100 single-quadrupole mass spectrometer, full-scan mode from m/z 50 to m/z 2600, scan time 0.1 s in positive ion mode, ESI spray voltage 4500 V, nitrogen gas 35 psi, drying gas flow 11.5 mL/min, fragmentor voltage 20 V. Elemental analysis were performed on a Thermo Flash 2000 CHNS/O Analyzer. A Shimadzu UV-1601 PC spectrophotometer was used for spectrophotometric measurements. 4.1.2. Synthesis Azetidinone 5Z was prepared as previously reported [6b].
4.1.2.1. (Z)-2-(4-Oxoazetidin-2-ylidene)acetic acid (3). To a solution of 5Z (220 mg, 1.01 mmol) in a 10 mL mixture of THF/Methanol 1:1 was added Pd 10% wt on activated Carbon (44 mg). The solution was treated with H2 (1 atm) and after 2 h was filtered on celite and concentrated to give 3 (100 mg, 78%). Yellow solid: mp ¼ 162e163; 1H NMR (400 MHz, (DMSO d6): d ¼ 3.57 (s, 2H), 4.98 (s, 1H), 10.47 (brs, 1H), 11.94 (brs, 1H); 13C NMR (100 MHz, CD3OD): d ¼ 46.0, 92.2, 153.3, 169.7, 171.0; IR (neat): ṽ ¼ 3289, 2919, 1828, 1185 cm1; HPLC-MS (ESI): Rt ¼ min 1.66, m/z (%): 128 [M þ H]þ, 150 [M þ Na]þ, 679 (70) [2M þ Na]þ; Found C, 47.42; H, 3.92; N, 10.87%; C5H5NO3 requires C, 47.25; H, 3.97; N, 11.02%. 4.1.2.2. (E)-3-Hydroxy-3-methyl-5-(2,6,6-trimethylcyclohex-1-en-1yl)pent-4-enenitrile (6). To a stirred solution of acetonitrile (330 mL, 6.24 mmol) in dry THF (5 mL) at 78 C under inert atmosphere
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were added dropwise nButyllithium 2.5 M in THF (6.86 mmol, 2.74 mL) and thereupon a solution of b-ionone (1 g, 5.2 mmol) in dry THF (4 mL) were added over a period of 10 min. The solution was allowed to warm at room temperature and after 3 h the reaction was quenched with aqueous NH4Cl (10 mL) and extracted with EtOAc (3 10 mL). The organic extracts were dried over Na2SO4, filtered and concentrated in vacuo. Flash chromatography (cyclohexane/EtOAc: 80/20) of the extracts gave 6 (1.031 g, 85%). Pale yellow oil: 1H NMR (400 MHz, CDCl3): d ¼ 0.92 (s, 6H, C(CH3)2), 1.35e1.38 (m, 2H, C(CH3)2CH2CH2CH2), 1.41 (s, 3H, CH3C(OH)), 1.49e1.55 (m, 2H, C(CH3)2CH2CH2CH2), 1.58 (s, 3H, CH3C]C), 1.88e1.91 (m, 2H, C(CH3)2CH2CH2CH2), 2.56 (s, 2H, CH2CN), 3.34 (brs, 1H, OH), 5.45 (d, 1H, J ¼ 16.0 Hz, CH]CHC(OH), 6.11 (d, 1H, J ¼ 16.0 Hz, CH]CHC(OH)); 13C NMR (100 MHz, CDCl3): d ¼ 18.8, 20.9, 27.4, 28.3 (2 C), 31.6, 32.3, 33.6, 38.9, 70.8, 117.3, 127.1, 128.5, 136.0, 136.6; IR (neat); ṽ ¼ 3444, 3023, 2925, 2256, 1722, 1651, 1644, 1114 cm-1; GCeMS (EI): Rt ¼ 16.81 min, m/z: 233 [M]þ, 218 [M CH3]þ, 193 [M CH2CN]þ, 135, 109, 91, 69; Found C, 77.01; H, 9.98; N, 5.92%; C15H23NO requires C, 77.21; H, 9.93; N, 6.00%.
3H), 1.86 (s, 3H), 2.01 (m, 2H), 3.51 (s, 2H), 4.06 (m, 2H), 5.02 (s, 1H), 5.30 (brs, 1H, NH), 5.43 (m, 1H), 6.01 (d, J ¼ 16 Hz, 1H), 6.13 (d, J ¼ 16 Hz, 1H), 8.75 (brs, 1H, NH); 13C NMR (100 MHz, CDCl3): d ¼ 12.5, 19.2, 21.6, 28.8, 29.7, 32.9, 34.2, 37.3, 39.5, 44.9, 92.2, 124.9, 127.0, 129.0, 136.6, 137.5, 137.6, 146.6, 165.3, 166.3; IR (neat): ṽ ¼ 3367, 3250, 2958, 2922, 2853, 1815, 1692 cm1; HPLC-MS (ESI): Rt ¼ min 12.72, m/z (%): 329 [M þ H]þ; Found C, 72.98; H, 8.65; N, 8.41%; C20H22N2O2 requires C, 73.14; H, 8.59; N, 8.53%. 2Z: pale yellow syrup: Rf ¼ 0.5 (cyclohexane/EtOAc, 5/5); 1H NMR (400 MHz, CDCl3): d ¼ 1.26 (s, 6H), 1.47 (m, 2H), 1.63 (m, 2H), 1.70 (s, 3H), 1.91 (s, 3H), 2.02 (m, 2H), 3.51 (m, 2H), 4.04 (m, 2H), 4.99 (s, 1H), 5.25 (brs, 1H, NH), 5.36 (m, 1H), 6.22 (d, J ¼ 16 Hz, 1H), 6.38 (d, J ¼ 16 Hz, 1H), 8.72 (brs, 1H, NH); 13C NMR (100 MHz, CDCl3): d ¼ 19.2, 20.3, 21.7, 28.9, 29.7, 32.9, 34.1, 36.4, 39.5, 44.9, 92.3, 123.1, 129.0, 129.6, 129.7, 136.7, 137.7, 146.4, 165.3, 166.3; IR (neat): ṽ ¼ 3327, 2954, 2923, 2853, 1810, 1683 cm1; HPLC-MS (ESI): Rt ¼ min 11.21, m/z (%): 329 (65) [M þ H]þ, 351 (100) [M þ Na]þ, 679 (70) [2M þ Na]þ; Found C, 72.88; H, 8.65; N, 8.38%; C20H22N2O2 requires C, 73.14; H, 8.59; N, 8.53%.
4.1.2.3. (2ZE,4E)-3-Methyl-5-(2,6,6-trimethylcyclohex-1-enyl)penta2,4-dienenitrile (7EZ). A solution of 6 (400 mg, 1.72 mmol) and pToluensulfonic acid (33 mg, 0.172 mmol) in toluene (17 mL) was refluxed and the reaction was followed by TLC. After the completion of the reaction the solution was cooled at 0 C and washed with a solution of NaHCO3 5% and after with water. The organic extracts were dried over Na2SO4, filtered and concentrated in vacuo obtaining 352 mg (95%) of 7 as a E/Z:54/46mixture. Characterization resulted in agreement with the reported data [34].
4.2. Cell culture and treatments
4.1.2.4. (2EZ,4E)-3-Methyl-5-(2,6,6-trimethylcyclohex-1-en-1-yl) penta-2,4-dien-1-amine (4). Amine 4 was prepared as reported in Ref. [35]; for a complete characterization: E/Z: 59/41. pale yellow oil, 1H NMR (400 MHz, CDCl3): d ¼ 0.97(s, 6H, C(CH3)2, trans), 0.98 (s, 6H, C(CH3)2, cis) 1.41e1.48 (m, 2H, CH2CH2C(CH3)2), 1.48e1.64 (m, 2H, CH2CH2C(CH3)2), 1.68 (s, 3H, CH2CH2C(CH3)]C, trans), 1.72 (s, 3H, CH2CH2C(CH3)]C, cis), 1.81 (s, 3H, CH3C]CH, trans), 1.88 (s, 3H, CH3C]CH, cis), 1.95e1.97 (m, 2H, CH2CH2C(CH3)]C), 2.47 (bs, 2H, NH2), 3.40 (d, 2H, J ¼ 6.8 Hz, CH2NH2), 5.37 (dd, 1H, J ¼ 6.4, 7.6 Hz, C]CHCH2NH2, cis), 5.45 (dd, 1H, J ¼ 6.4 Hz, 7.2 Hz, C]CHCH2NH2, trans), 5.94e6.01 (m, 2H, CH] CH, trans), 6.12 (d, 1H, J ¼ 15.6 Hz, CH]CH, cis), 6.30 (d, 1H, J ¼ 15.6 Hz, CH]CH, cis); 13C (100 MHz, CDCl3): d ¼ 12.2, 19.1, 21.6, 28.4 (2 C), 32.7, 34.1, 38.4, 39.3, 126.0, 128.5, 128.6, 129.0, 129.3, 130.1, 133.7, 135.0, 137.1, 137.5, 137.8; IR (neat); ṽ ¼ 3373, 2928, 2866, 1454, 1266 cm1; GCeMS (EI): Rt ¼ 16.09 (cis), 16.57 (trans) min, m/ z: 219 [M]þ, 204 [M CH3]þ, 187 [M CH2CN]þ, 162, 131, 119, 91, 83. 4.1.2.5. (2Z)-N-((2EZ,4E)-3-Methyl-5-(2,6,6-trimethylcyclohex-1enyl)penta-2,4-dienyl)-2-(4-oxoaze tidin-2-ylidene)acetamide (2E and 2Z). To a solution of b-lactam 3 (55 mg, 0.43 mmol) in dry DCM (4.5 mL) under inert atmosphere at 0 C were added amine 4EZ (94 mg, 0.43 mmol), DMAP (53 mg, 0.43 mmol) and after 10 min EDCl (82.5 mg, 0.43 mmol). The solution was stirred at 0 C for 45 min and after was allowed to warm at room temperature. The reaction was followed by TLC and after the disappearing of the starting materials was quenched with aqueous NH4Cl (10 mL) and extracted with DCM (3 5 mL). The organic extracts were dried over Na2SO4, filtered and concentrated in vacuo. The E/Z mixture was separated by flash chromatography (cyclohexane/EtOAc 70/30) affording 2E (62 mg) and 2Z (44 mg) in a overall yield of 75%. Configurations of the two diastereoisomers was assigned by NOE 1D studies. 2E: white syrup: Rf ¼ 0.4 (cyclohexane/EtOAc, 5/5); 1H NMR (400 MHz, CDCl3): d ¼ 1.00 (s, 6H), 1.46 (m, 2H), 1.62 (m, 2H), 1.68 (s,
Human neuroblastoma SH-SY5Y cell line were cultured in DMEM/F12 (Gibco-Invitrogen, Milan, Italy) supplemented with 10% fetal bovine serum (Euroclone, Milan, Italy), 1X MEM/NEAA (GibcoInvitrogen), 1X Penicillin-Streptomycin (Gibco-Invitrogen), NaOH 10 mM (Carlo Erba) and maintained at 37 C in a humidified 5% CO2 incubator. Azetidinone 1, b-Lactams 2, b-Lactams 2Z, and all-trans retinoic acid (RA, Sigma Aldrich) were dissolved in dimethylsulfoxide (DMSO, Sigma Aldrich) to an intermediate concentration of 100 mM (1 and 2E), and 16.6 mM (RA). Final dilutions (10 microM for b-lactam 2E, b-Lactams 2Z and RA, 7.5 microM for azetidinone 1) were done in the culture medium. A corresponding volume of DMSO was added to control cells (vehicle treated). To evaluate citotoxic effect exerted by Azetidinone 1, b-Lactams 2E and b-Lactams 2Z, SH-SY5Y cells were seeded at a density of 1 104 cells/well in 96 platic multiwells/dish; 24 h after seeding were treated for 24 h with Azetidinone 1, b-Lactams 2E and b-Lactams 2Z in the concentration range 1 microM to 1 mM and was performed MTT assay [36]. Cytotoxicity of compounds 1 and 2E and 2Z on SHSY5Y (IC50) was assessed in order to define the working concentration (IC501 ¼ 10e28 microM R2 ¼ 0.973; IC502E ¼ 38e65 microM R2 ¼ 9988; IC50 2Z ¼ 35e97 microM R2 ¼ 0.954) (Fig. 2). 4.2.1. Proliferation effect To evaluate the effect of Azetidinone 1, b-Lactams 2E and 2Z on proliferation, cells were seeded at a density of 1 104 cells/cm2 in 6 plastic multiwells dish and allowed to grow for 24 h before treatment. Twenty-four h after seeding, the medium was removed and fresh medium containing the drugs was added for additional 24, 48, 72 and 96 h. Rate of cells growth was determined counting viable cells using trypan blue exclusion assay. Doubling time (Td) was calculated during logarithmic growth phase. The experiment was repeated twice. Statistical analysis were performed with two-way ANOVA, Dunnet’s multiple comparisons test. 4.2.2. Differentiative effect Indirect immunofluorescence was used to investigate possible morphological changes of cytoskeleton due to Azetidinone 1, bLactams 2E and 2Z exposure. For this experiment, SH-SY5Y were seeded on glass coverslips coated with CultrexÒ Basement Membrane Extract (BME) (TrevigenÒ, Gaithersburg, MD, USA) at a density of 1 104 cells/cm2 and cultured in the previously described culture media. After 24 h of seeding, the medium was
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removed and was added fresh medium containing 7.5 microM azetidinone 1, 10 microM of b-lactam 2E, 10 microM of b-Lactams 2Z and 10 microM of RA. Cells were treated for additional 6 days and medium was replaced every 48 h. Untreated cells and cells treated with retinoic acid (RA) 10 microM were used respectively as nondifferentiated and differentiated controls [37]. After treatments, cells were washed in PBS and fixed with 4% paraformaldehyde in 0.1 M Sørensen phosphate buffer for 20 min at RT. Subsequently, fixed cells were blocked in PBS containing 0.3% Triton-X 100 (Merck, Darmstadt, Germany), 5% Donkey Normal Serum (Sigma Aldrich) for 1 h at RT, then incubated overnight at þ4 C in humid atmosphere with anti-neurofilament 200 (Sigma Aldrich) and anti- bIII-tubulin (R&D) antibodies diluted in blocking solution 1:400 and 1:1000 respectively. After rinsing twice in PBS for 10 min, cells were incubated for 30 min at 37 C with Dylight 488-labeled secondary antisera (Thermo Scientific) diluted in 0.3% Triton-X 100/PBS. For nuclear staining, cells were first washed in PBS then incubated 15 min with 1 mg/ml Hoechst 33258 in 0.2% Triton-X 100/PBS. After rinsing in PBS, coverslips containing cells were finally mounted in 1,4-phenylendiamine (Sigma Aldrich)/ glycerol 0.1%. Negative controls were performed by primary antibody omission. Images of SH-SY5Y cells were taken by Nikon Eclipse 600 microscope equipped with motorized z-stage control and F-view II CCD Cameras with 20 objective. Immunofluorescence staining was analyzed using the Nis-Elements Software 3.21.03 (Nikon). Analysis of bIII-tubulin and neurofilament 200 immunoreactive areas (IR) were executed applying “sharpen” and “detect edges” filters. For each image, nuclei were counted and IR/n of nuclei were calculated. The experiment was repeated twice and 3 samples for each experiment (at least 100 cells) were analyzed. Statistical analysis were performed with one-way ANOVA. Representative images were collected using a Nikon A1R confocal laser-scanning microscope, equipped with a Nikon PlanApo 40 lens.
[6]
[7] [8]
[9] [10] [11] [12] [13] [14]
[15] [16]
[17]
[18] [19] [20]
Acknowledgments
[21]
The authors DG, PG, and MP would like to thank Mrs Jessica Giacoboni for technical assistance. We are grateful to Dr. Francesca Paradisi (senior lecturer at the UCD School of Chemistry and Chemical Biology, University College Dublin) for revision of the English manuscript. This work was supported by MIUR (PRIN project), University of Bologna and POR-FESR Regione Emilia Romagna.
[22] [23] [24] [25] [26]
Appendix A. Supplementary data
[27] [28]
Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2013.09.057.
[29]
References
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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Azetidinoneeretinoid hybrids: Synthesis and differentiative effects Matteo Pori a,1, Paola Galletti a,1, Roberto Soldati a,1, Laura Calzà b, c, 2, Chiara Mangano c, 2, Daria Giacomini a, * a
Department of Chemistry “G. Ciamician”, University of Bologna, Via Selmi 2, 40126 Bologna, Italy Health Science and Technologies Interdepartmental Center for Industrial Research (HST-ICIR), University of Bologna, Via Tolara di Sopra 50, I-40064 Ozzano Emilia, Bologna, Italy c Department of Veterinary Medicine, University of Bologna, Via Tolara di Sopra 50/A, 40064 Ozzano Emilia, Bologna, Italy b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 May 2013 Received in revised form 12 September 2013 Accepted 29 September 2013 Available online 12 October 2013
As a part of a systematic investigation on the synthesis and biological activities of new b-lactam compounds, we examined b-lactam candidates 1, 2E and 2Z and their ability to induce cell proliferation or differentiation. Azetidinone 1 was chosen for its activity (previously evaluated) as selective HDAC8 inhibitor, whereas b-lactams 2E and 2Z were designed to have a hybrid retinoid-azetidinone structure, de novo synthesized and fully characterized. Biological activities were determined in cellular assays on neuroblastoma cells SH-SY5Y. Azetidinone 1, 2E and 2Z led to a moderate effect in decreasing SH-SY5Y cell proliferation and b-lactams 2E and 2Z induced an early stage differentiation. The present results uncovered a new role of specifically designed b-lactam compounds in epigenetic regulation. Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords: b-Lactams Retinoids Synthesis Cell differentiation HDAC inhibitors Epigenetic regulation
1. Introduction
b-Lactams have historically been viewed as an important class of antimicrobials since the discovery of naturally occurring penicillins and cephalosporins [1]. Recognition of the azetidin-2-one moiety as the key pharmacophoric component of b-lactam antibiotics initiated a flurry of synthetic activity to develop new structures, and nowadays new variants of monocyclic compounds (azetidinones) are showing new and specific biological activities [2]. In the last few decades their role as enzyme inhibitors has been expanded. b-Lactams are well-known as potent inhibitors of some enzymes that contain serine as the catalytic residue, including the bacterial penicillin binding proteins (PBPs), and Class A and Class C b-lactamases [3]. b-Lactams have also been appropriately modified to develop active site-directed and mechanism-based inhibitors of Human Leukocyte Elastase (HLE) [4], and in this field some new monocyclic b-lactams have shown important activities as HLE inhibitors [5], as new antibiotics towards resistant bacteria [6], and as inhibitors of platelet aggregation [7]. Recently, relevant studies
* Corresponding author. Tel.: þ39 051 209 9528; fax: þ39 051 209 9456. E-mail address: [email protected] (D. Giacomini). 1 Tel.: þ39 051 209 9450; fax: þ39 051 209 9456. 2 Tel.: þ39 051 2097 947; fax: þ39 051 2097 953. 0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejmech.2013.09.057
appeared on b-lactam compounds as anti-tumor drugs. Some arylb-lactam derivatives have been evaluated for cytotoxicity against a number of human tumor and normal cell lines [8]. In particular Nthiolated-b-lactams were found to induce DNA damage, cell growth arrest, and apoptosis in cultured human cancer cells, and antiproliferative effects on human breast cancer cells of some new azetidinones were evaluated [9]. Some bicyclic b-lactams were recently reported as inhibitors of Histone Deacetylases (HDACs), a family of proteins involved in the pattern of acetylation of chromatin proteins and thus in the regulation of gene expression [10]. We recently reported the synthesis of some azetidinone derivatives which showed good affinity and specificity towards histone deacetylases HDAC8 and HDAC6 [11]. Histone deacetylases are epigenetic regulators playing an important role in modulating gene transcription as well as protein function. Eighteen classical HDAC isoforms, divided into IV classes based on yeast deacetilases homology, are identified to date. HDAC inhibitors constitute a new class of promising anticancer drugs targeting one or more HDAC isoforms. HDAC6 overexpression was found in carcinoma of oral squamous tissue [12], although in breast cancer it correlates with better survival [13]. In lung, colon, and cervical cancer cell line the knockout of HDAC8 gene reduces cell proliferation [14]; in leukemia cell HDAC8 inhibitors leads to apoptosis [15]. The expression of HDAC8 (class I member) has been
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correlated with poor outcome in neuroblastoma [15], and in such cell line selective HDAC8 inhibitors induces differentiation and proliferation arrest [16]. Nowadays, there is great interest in the development of small molecules that regulate or modulate protein function and chemical genetic offers a valuable approach in this field [17]. Retinoids, as derivatives of Vitamin A, regulate key developmental pathways throughout life, by modulating the expression of many genes involved in cell proliferation, migration, differentiation and apoptosis, though the molecular mechanisms are not completely understood [18e21]. The effects of retinoic acid (RA) are mediated by the heterodimerization of nuclear RAR (Retinoic Acid Receptor), a family of ligand-dependent transcription factors, and RXR (Retinoid X Receptors), the 9-cis RA binding receptor [18]. To date, retinoids are known to activate or inhibit MAPK cascade, which is involved in both proliferation and growth arrest [22]. Some synthetic RA-derived molecules exert differentiative effect rather than apoptosis, blocking tumor progression in cancer cell lines [23,24]. In fact, retinoids are currently used as potential chemotherapeutic drugs in the treatment of several cancer types (neuroblastoma, breast cancer, lung cancer, rhabdomyosarcoma, and many others) [25e28]. For this reason, there is great interest in developing new synthetic molecules targeting retinoic acid receptors. As part of an interdisciplinary project on the development of new b-lactam derivatives with a modular structure designed for specific biological activities, the effect of the azetidinone 1 (Fig. 1), previously evaluated by us as specific HDAC8 inhibitor [11], was investigated on the proliferative activity of a neuron-like cell line. Moreover, we developed the synthesis of new b-lactams 2 with a hybrid retinoid-azetidinone structure and we investigated the retinoic acid-dependent differentiation of a neuron-like cell line. To our knowledge, only two example of b-lactams were reported in the literature with a differentiation-inducing activity on leukemia cells [29]. We report here the synthesis of the new hybrid retinoidb-lactams and preliminary results in biological evaluation of the three molecules on neuroblastoma cell line SH-SY5Y. 2. Results and discussion 2.1. Compound design and synthesis As mentioned above, we have previously reported an efficient synthesis of b-lactam 1 [11]. Its design was based on the pharmacophore model of HDAC inhibitors which consists in a modular structure with a cap-group that interacts with residues in the active site of the receptor and a linker group that sits in a hydrophobic channel and positions a metal-binding group which coordinates to the zinc ion in the active site. We demonstrated that the azetidinone ring can efficiently work as the zinc-binding group. Moreover, the substituent on the nitrogen atom allowed for the specificity against HDAC8 or HDAC6 inhibition. Azetidinone 1 was therefore chosen in the present study for its specific HDAC8 activity. Once the b-lactam ring was confirmed as an effective zincbinding group for HDAC, we developed a new hybrid structure retaining the b-lactam scaffold to target HDACs and functionalized with a retinoic side chain which could activate differentiative
Fig. 1. b-Lactam derivatives.
processes to mimic the action of retinoic acid (RA). The design of blactam 2 was based on a 4-alkylidene-b-lactam scaffold with demonstrated enhanced reactivity in protein inhibition [2a,5], and functionalized with a retinoid-amide side chain. The strategy is outlined in Scheme 1 and consists of a convergent synthesis with the coupling between the 4-alkylidene-azetidinone carboxy acid 3 and the retinoid amine 4 as the key step. 4-Alkyliden-azetidin-2-ones can be obtained using an original and well established protocol developed in our laboratory starting from 4-acetoxy-azetidinones and diazoesters in the presence of Lewis acids [30]. Given that 4-acetoxy azetidinone is commercially available, b-lactam 3 was obtained with benzyldiazoacetate and TiCl4 as outlined in Scheme 2. The 4-alkyliden-b-lactams 5 were obtained in 43% yields and in a E/Z diastereomeric ratio of 41/59. The diastereomeric mixture 5Z/5E was easily separated by flash chromatography. Hydrogenolysis of the isolated pure 5Z with Pd on carbon in THF-methanol 1:1 mixture gave 3 in quantitative yield. Retinoid amine 4 was obtained starting from the easily available b-ionone in a three step reaction (Scheme 3). Addition of lithium acetonitrile to b-ionone in THF at low temperature gave 6 [30], which underwent water elimination under ptoluensulfonic acid catalysis [31]. The nitrile 7 obtained as a E/Z mixture, was then reduced to give the amine 4EZ in 79% yield. Finally, with a carbodiimide coupling method, amides 2E and 2Z were obtained as a mixture and easily separated by flash chromatography. The 2E,4E and 2Z,4E configurations were assigned in the two isomers by NOE 1D analysis on the separated products 2E and 2Z (Scheme 4, and Supplementary material). In isomer 2E, the methyl group in the chain, chosen as the irradiated site, showed two NOE correlations with methylene and methine, respectively, indicating a close spatial relationship whereas for b-lactam 2Z NOE enhancements of the two vinylic CH were observed. The 2E,4E configuration for 2E, and the 2Z,4E configuration for 2Z were thus attributed. 2.2. Biology As said before, HDAC inhibitors are emerging as anti cancer drugs. In particular, HDAC8 inhibitors have shown to regulate proliferation in neuroblastoma cells [16]. As potential HDAC inhibitors, we evaluated the antiproliferative effect of azetidinones 1, 2E, and 2Z on undifferentiated SH-SY5Y. Moreover, as b-lactams 2E and 2Z have hybrid retinoid-azetidinone structures, we performed a set of experiments aiming to evaluate differentiative action of blactams 2E and 2Z compared to azetidinone 1 and retinoic acid treated cells. In fact, retinoids are currently used as potential chemotherapeutic drugs in the treatment of several cancer types including neuroblastoma due to their differentiative action [25e 28]. In order to assess azetidinone 1, 2E, and 2Z cytotoxicity, we calculated first the IC50 (50% inhibiting concentration) using MTT assay (Fig. 2). The MTT assay is a colorimetric assay for measuring the activity of cytosolic enzymes that reduce tetrazolium dye (MTT) in formazan salt in living cells. IC50 values were calculated and are reported above the curves in Fig. 2. The concentration used for subsequent experiments was chosen accordingly in order to obtain cytostatic effects rather than apoptosis or necrosis (Fig. 2). 2.2.1. Effect on proliferation In a preliminary analysis of biological effect on proliferation, undifferentiated SH-SY5Y cells were exposed to 7.5 microM of azetidinone 1, 10 microM of b-lactams 2E and 2Z for 24, 48, 72 and 96 h, the number of cells was counted daily and compared to control (vehicle treated). Fig. 3 shows the proliferation curve of control cells (ctrl, open circle), azetidinone 1 (black square), b-
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859
Scheme 1. Retro-synthetic route to retinoid b-lactam 2.
Scheme 2. Synthesis of 4-alkyliden-azetidinone 3.
Scheme 3. Synthesis of retinoid amine 4 and retinoid b-lactams 2E and 2Z.
Scheme 4. NOE 1D correlations in new retinoid-lactams for configuration assignments.
lactam 2E (black triangle) and 2Z treated cell (black rhombus). Significant differences were observed between the groups (twoway ANOVA, Dunnet’s multiple comparisons test). b-lactams 2E and 2Z determine a significative reduction in cell proliferation after 48 h of treatment (a: ctrl vs b-lactam 2E; b: ctrl vs b-lactam 2Z). All the compounds tested show an antiproliferative effect compared to control from 72 to 96 h (c: ctrl vs azetidinone 1 and b-lactam 2E, d: ctrl vs b-lactam 2Z, e: ctrl vs azetidinone 1 and b-lactam 2E p < 0.0001, f: ctrl vs b-lactam 2Z). Although the reduction is stable,
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Fig. 2. Cytotoxic evaluation of azetidinone 1, b-lactams 2E and 2Z on undifferentiated SH-SY5Y; IC50 ranges are reported above the curves. Data are shown as mean (normalized to control) SD.
doubling time calculated in 24e72 h range (Td) is affected exclusively in cells treated with b-lactam 2Z (TdCTRL ¼ 34,9 h; Td1 ¼ 39 h, Td2 ¼ 39.78, Td8 ¼ 51,25), whereas doubling time calculated in 24e 96 h range is not affected. These results indicated a slight effect of azetidinone 1, 2E and 2Z on SH-SY5Y proliferation. 2.2.2. Differentiation effect As they are derived from neuroblastoma, SH-SY5Y may be differentiated toward neural-like lineage. Retinoic acid is a powerful differentiating molecule inducing growth inhibition, neurites lengthening and branching. A six-days RA treatment induces neural-like differentiation as shown by neurites outgrowth (Fig. 4, panel B vs panel A; panel G vs panel F) visualized by bIIItubulin and NF200. Quantitative analysis was performed measuring the bIII-tubulin and NF200 network normalized according to the cells number. As hybrids retinoid-azetidinones, we investigated a possible differentiative effect of b-lactams 2E and 2Z compared to azetidinone 1 and RA treated SH-SY5Y. bIII-Tubulin is an early marker for differentiation toward neuronal lineage and the increase of bIII-tubulin area may be related to an early stage of differentiation. Neurofilament play important structural roles and influence axonal transport, neurite outgrowth and cell survival.
Sample micrographs of cell SH-SY5Y exposed to the vehicle (control), RA, azetidinone 1, and b-lactams 2E and 2Z are shown in Fig. 4, where panels AeE refers to b-tubulin-IR, and panels FeJ refers to neurofilament-IR, graph K and L to respective quantification of IR area/number of cells. While a differentiative effect of RA is evident in both bIII-tubulin and NF200 immunostaining (panels A vs panel B and panel F vs panel G, respectively) and azetidinone 1 does not display any differentiative effect (panels A vs panel C and panel F vs panel H), b-lactams 2E (panels A vs panel D and panel F vs panel I) and 2Z (panels A vs panel E and panel F vs panel J) induce a moderate differentiation, which is statistically significant in bIIItubulin staining (panels K and L, statistical analysis was performed with one-way ANOVA, Tukey post test). This effect could be possibly related to moderate structural analogies among RA and blactams 2E and 2Z. The data indicate a differentiative effect of blactams 2E and 2Z on SH-SY5Y, even though smaller when compared to RA. The absence in the b-lactams 2E and 2Z of an ionizable function, as like the carboxylic acid in RA, can explain the observed lower activity, as reported for some non-ionizable RA derivatives such as RA-esters or amides [32]. To bind effectively to the retinoic acid receptor (RAR), the polar terminus of the retinoid must be capable of efficient interactions, as observed in the crystal structure of RA-RAR complex [33]. The b-lactam scaffold, while it would not be considered a strong “polar terminus” in anchoring RAR, shows a positive effect on SH-SY5Y cell differentiation (blactams 2E and 2Z), and it deserves further investigation. 3. Conclusions As part of an interdisciplinary research project involving the synthesis of new monocyclic b-lactams specifically designed to target epigenetic regulators, we presented here an evaluation of three monocyclic azetidinones candidates 1, 2E and 2Z and their ability to interfere with cell proliferation or differentiation. Azetidinone 1 was chosen because of its specific inhibition of HDAC8, whereas 2E and 2Z were designed as innovative hybrid azetidinone-retinoid structures. Their de novo synthesis allowed for the anchoring of an alkylidenecarboxyl-side chain on the C4 position of the b-lactam core which was further condensed with a retinoid-amine. The new hybrids 2E and 2Z and the azetidinone 1 were tested in cellular assays on neuroblastoma cells SH-SY5Y. Azetidinone 1, b-lactams 2E and 2Z slightly decrease the proliferation rate in SH-SY5Y neuroblastoma cells, possibly due to the HDAC inhibition properties. The hybrid azetidinone-retinoids blactams 2E and 2Z have a differentiative effect, even if smaller when compared to RA. These preliminary results are worthy of a further evaluation and prompt us to currently work on the synthesis of a library of new hybrid azetidinone-retinoid molecules. 4. Experimental section 4.1. Chemistry
Fig. 3. Effect of azetidinones 1, 2E, and 2Z on SH-SY5Y proliferation. The rate of cells proliferation was determined as total cell number. Cells were incubated in the presence of 7.5 microM of azetidinone 1, 10 microM of 2E and 2Z and counted at 24, 48, 72, 96 h. Significant differences between groups were observed after 48 h of treatment. Statistical analysis was performed with two-way ANOVA, Dunnet’s multiple comparisons test (a: ctrl vs b-lactam 2E, p < 0.05; b: ctrl vs b-lactam 2Z, p < 0.01; c: ctrl vs azetidinone 1 and b-lactam 2E p < 0.001, d: ctrl vs b-lactam 2Z p < 0.0001, e: ctrl vs azetidinone 1 and b-lactam 2E p < 0.0001, f: ctrl vs b-lactam 2Z p < 0.001).
4.1.1. General All reactions were performed under an inert atmosphere (N2). Commercial reagents (reagent grade, >99%) were used as received without additional purification. Anhydrous solvents (CH3CN, CH2Cl2, THF) were obtained commercially. 1 H and 13C NMR values were recorded on a INOVA 400, or a GEMINI 200 instrument with a 5 mm probe. All chemical shifts have been quoted relative to deuterated solvent signals, d in ppm and J in Hz. FT-IR: Nicolet 380 measured as films between NaCl, plates wavenumbers reported in cm1. TLC: Merck 60 F254. Column chromatography: Merck silica gel 200e300 mesh. GCeMS: Agilent
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Fig. 4. Effect of azetidinone 1, b-lactams 2E and 2Z on SH-SY5Y differentiation. bIII-tubulin (AeC) and neurofilament (DeF) were used to investigate a possible microtubule and intermediate filaments rearrangement. Panels A and F refer to vehicle treated cells; B and G to cells treated for 6 days with RA; C and F correspond to cells treated for 6 days with 7.5 microM of azetidinone 1, D and I correspond to cells treated for 6 days with 10 microM of b-lactam 2E; E and J correspond to cells treated for 6 days with 10 microM of b-lactam 2Z (Scale bar ¼ 10 micron). Graphs K and L refer to bIII-tubulin and neurofilament immunoreactive area, respectively, normalized for number of cells (mean SD). Statistical analysis was performed with one-way ANOVA, Tukey post test, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Technologies, column HP5 5% Ph-Me Silicon MS: Agilent Technologies MSD1100 single-quadrupole mass spectrometer, EI voltage 70 eV, gradient from 50 C to 280 C in 30 min. HPLC-MS, HPLC: Agilent Technologies HP1100, column ZOBRAX-Eclipse XDB-C8 Agilent Technologies, mobile phase: H2O/CH3CN, gradient from 30% to 80% of CH3CN in 8 min, 80% of CH3CN until 25 min, 0.4 mL/min MS: Agilent Technologies MSD1100 single-quadrupole mass spectrometer, full-scan mode from m/z 50 to m/z 2600, scan time 0.1 s in positive ion mode, ESI spray voltage 4500 V, nitrogen gas 35 psi, drying gas flow 11.5 mL/min, fragmentor voltage 20 V. Elemental analysis were performed on a Thermo Flash 2000 CHNS/O Analyzer. A Shimadzu UV-1601 PC spectrophotometer was used for spectrophotometric measurements. 4.1.2. Synthesis Azetidinone 5Z was prepared as previously reported [6b].
4.1.2.1. (Z)-2-(4-Oxoazetidin-2-ylidene)acetic acid (3). To a solution of 5Z (220 mg, 1.01 mmol) in a 10 mL mixture of THF/Methanol 1:1 was added Pd 10% wt on activated Carbon (44 mg). The solution was treated with H2 (1 atm) and after 2 h was filtered on celite and concentrated to give 3 (100 mg, 78%). Yellow solid: mp ¼ 162e163; 1H NMR (400 MHz, (DMSO d6): d ¼ 3.57 (s, 2H), 4.98 (s, 1H), 10.47 (brs, 1H), 11.94 (brs, 1H); 13C NMR (100 MHz, CD3OD): d ¼ 46.0, 92.2, 153.3, 169.7, 171.0; IR (neat): ṽ ¼ 3289, 2919, 1828, 1185 cm1; HPLC-MS (ESI): Rt ¼ min 1.66, m/z (%): 128 [M þ H]þ, 150 [M þ Na]þ, 679 (70) [2M þ Na]þ; Found C, 47.42; H, 3.92; N, 10.87%; C5H5NO3 requires C, 47.25; H, 3.97; N, 11.02%. 4.1.2.2. (E)-3-Hydroxy-3-methyl-5-(2,6,6-trimethylcyclohex-1-en-1yl)pent-4-enenitrile (6). To a stirred solution of acetonitrile (330 mL, 6.24 mmol) in dry THF (5 mL) at 78 C under inert atmosphere
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were added dropwise nButyllithium 2.5 M in THF (6.86 mmol, 2.74 mL) and thereupon a solution of b-ionone (1 g, 5.2 mmol) in dry THF (4 mL) were added over a period of 10 min. The solution was allowed to warm at room temperature and after 3 h the reaction was quenched with aqueous NH4Cl (10 mL) and extracted with EtOAc (3 10 mL). The organic extracts were dried over Na2SO4, filtered and concentrated in vacuo. Flash chromatography (cyclohexane/EtOAc: 80/20) of the extracts gave 6 (1.031 g, 85%). Pale yellow oil: 1H NMR (400 MHz, CDCl3): d ¼ 0.92 (s, 6H, C(CH3)2), 1.35e1.38 (m, 2H, C(CH3)2CH2CH2CH2), 1.41 (s, 3H, CH3C(OH)), 1.49e1.55 (m, 2H, C(CH3)2CH2CH2CH2), 1.58 (s, 3H, CH3C]C), 1.88e1.91 (m, 2H, C(CH3)2CH2CH2CH2), 2.56 (s, 2H, CH2CN), 3.34 (brs, 1H, OH), 5.45 (d, 1H, J ¼ 16.0 Hz, CH]CHC(OH), 6.11 (d, 1H, J ¼ 16.0 Hz, CH]CHC(OH)); 13C NMR (100 MHz, CDCl3): d ¼ 18.8, 20.9, 27.4, 28.3 (2 C), 31.6, 32.3, 33.6, 38.9, 70.8, 117.3, 127.1, 128.5, 136.0, 136.6; IR (neat); ṽ ¼ 3444, 3023, 2925, 2256, 1722, 1651, 1644, 1114 cm-1; GCeMS (EI): Rt ¼ 16.81 min, m/z: 233 [M]þ, 218 [M CH3]þ, 193 [M CH2CN]þ, 135, 109, 91, 69; Found C, 77.01; H, 9.98; N, 5.92%; C15H23NO requires C, 77.21; H, 9.93; N, 6.00%.
3H), 1.86 (s, 3H), 2.01 (m, 2H), 3.51 (s, 2H), 4.06 (m, 2H), 5.02 (s, 1H), 5.30 (brs, 1H, NH), 5.43 (m, 1H), 6.01 (d, J ¼ 16 Hz, 1H), 6.13 (d, J ¼ 16 Hz, 1H), 8.75 (brs, 1H, NH); 13C NMR (100 MHz, CDCl3): d ¼ 12.5, 19.2, 21.6, 28.8, 29.7, 32.9, 34.2, 37.3, 39.5, 44.9, 92.2, 124.9, 127.0, 129.0, 136.6, 137.5, 137.6, 146.6, 165.3, 166.3; IR (neat): ṽ ¼ 3367, 3250, 2958, 2922, 2853, 1815, 1692 cm1; HPLC-MS (ESI): Rt ¼ min 12.72, m/z (%): 329 [M þ H]þ; Found C, 72.98; H, 8.65; N, 8.41%; C20H22N2O2 requires C, 73.14; H, 8.59; N, 8.53%. 2Z: pale yellow syrup: Rf ¼ 0.5 (cyclohexane/EtOAc, 5/5); 1H NMR (400 MHz, CDCl3): d ¼ 1.26 (s, 6H), 1.47 (m, 2H), 1.63 (m, 2H), 1.70 (s, 3H), 1.91 (s, 3H), 2.02 (m, 2H), 3.51 (m, 2H), 4.04 (m, 2H), 4.99 (s, 1H), 5.25 (brs, 1H, NH), 5.36 (m, 1H), 6.22 (d, J ¼ 16 Hz, 1H), 6.38 (d, J ¼ 16 Hz, 1H), 8.72 (brs, 1H, NH); 13C NMR (100 MHz, CDCl3): d ¼ 19.2, 20.3, 21.7, 28.9, 29.7, 32.9, 34.1, 36.4, 39.5, 44.9, 92.3, 123.1, 129.0, 129.6, 129.7, 136.7, 137.7, 146.4, 165.3, 166.3; IR (neat): ṽ ¼ 3327, 2954, 2923, 2853, 1810, 1683 cm1; HPLC-MS (ESI): Rt ¼ min 11.21, m/z (%): 329 (65) [M þ H]þ, 351 (100) [M þ Na]þ, 679 (70) [2M þ Na]þ; Found C, 72.88; H, 8.65; N, 8.38%; C20H22N2O2 requires C, 73.14; H, 8.59; N, 8.53%.
4.1.2.3. (2ZE,4E)-3-Methyl-5-(2,6,6-trimethylcyclohex-1-enyl)penta2,4-dienenitrile (7EZ). A solution of 6 (400 mg, 1.72 mmol) and pToluensulfonic acid (33 mg, 0.172 mmol) in toluene (17 mL) was refluxed and the reaction was followed by TLC. After the completion of the reaction the solution was cooled at 0 C and washed with a solution of NaHCO3 5% and after with water. The organic extracts were dried over Na2SO4, filtered and concentrated in vacuo obtaining 352 mg (95%) of 7 as a E/Z:54/46mixture. Characterization resulted in agreement with the reported data [34].
4.2. Cell culture and treatments
4.1.2.4. (2EZ,4E)-3-Methyl-5-(2,6,6-trimethylcyclohex-1-en-1-yl) penta-2,4-dien-1-amine (4). Amine 4 was prepared as reported in Ref. [35]; for a complete characterization: E/Z: 59/41. pale yellow oil, 1H NMR (400 MHz, CDCl3): d ¼ 0.97(s, 6H, C(CH3)2, trans), 0.98 (s, 6H, C(CH3)2, cis) 1.41e1.48 (m, 2H, CH2CH2C(CH3)2), 1.48e1.64 (m, 2H, CH2CH2C(CH3)2), 1.68 (s, 3H, CH2CH2C(CH3)]C, trans), 1.72 (s, 3H, CH2CH2C(CH3)]C, cis), 1.81 (s, 3H, CH3C]CH, trans), 1.88 (s, 3H, CH3C]CH, cis), 1.95e1.97 (m, 2H, CH2CH2C(CH3)]C), 2.47 (bs, 2H, NH2), 3.40 (d, 2H, J ¼ 6.8 Hz, CH2NH2), 5.37 (dd, 1H, J ¼ 6.4, 7.6 Hz, C]CHCH2NH2, cis), 5.45 (dd, 1H, J ¼ 6.4 Hz, 7.2 Hz, C]CHCH2NH2, trans), 5.94e6.01 (m, 2H, CH] CH, trans), 6.12 (d, 1H, J ¼ 15.6 Hz, CH]CH, cis), 6.30 (d, 1H, J ¼ 15.6 Hz, CH]CH, cis); 13C (100 MHz, CDCl3): d ¼ 12.2, 19.1, 21.6, 28.4 (2 C), 32.7, 34.1, 38.4, 39.3, 126.0, 128.5, 128.6, 129.0, 129.3, 130.1, 133.7, 135.0, 137.1, 137.5, 137.8; IR (neat); ṽ ¼ 3373, 2928, 2866, 1454, 1266 cm1; GCeMS (EI): Rt ¼ 16.09 (cis), 16.57 (trans) min, m/ z: 219 [M]þ, 204 [M CH3]þ, 187 [M CH2CN]þ, 162, 131, 119, 91, 83. 4.1.2.5. (2Z)-N-((2EZ,4E)-3-Methyl-5-(2,6,6-trimethylcyclohex-1enyl)penta-2,4-dienyl)-2-(4-oxoaze tidin-2-ylidene)acetamide (2E and 2Z). To a solution of b-lactam 3 (55 mg, 0.43 mmol) in dry DCM (4.5 mL) under inert atmosphere at 0 C were added amine 4EZ (94 mg, 0.43 mmol), DMAP (53 mg, 0.43 mmol) and after 10 min EDCl (82.5 mg, 0.43 mmol). The solution was stirred at 0 C for 45 min and after was allowed to warm at room temperature. The reaction was followed by TLC and after the disappearing of the starting materials was quenched with aqueous NH4Cl (10 mL) and extracted with DCM (3 5 mL). The organic extracts were dried over Na2SO4, filtered and concentrated in vacuo. The E/Z mixture was separated by flash chromatography (cyclohexane/EtOAc 70/30) affording 2E (62 mg) and 2Z (44 mg) in a overall yield of 75%. Configurations of the two diastereoisomers was assigned by NOE 1D studies. 2E: white syrup: Rf ¼ 0.4 (cyclohexane/EtOAc, 5/5); 1H NMR (400 MHz, CDCl3): d ¼ 1.00 (s, 6H), 1.46 (m, 2H), 1.62 (m, 2H), 1.68 (s,
Human neuroblastoma SH-SY5Y cell line were cultured in DMEM/F12 (Gibco-Invitrogen, Milan, Italy) supplemented with 10% fetal bovine serum (Euroclone, Milan, Italy), 1X MEM/NEAA (GibcoInvitrogen), 1X Penicillin-Streptomycin (Gibco-Invitrogen), NaOH 10 mM (Carlo Erba) and maintained at 37 C in a humidified 5% CO2 incubator. Azetidinone 1, b-Lactams 2, b-Lactams 2Z, and all-trans retinoic acid (RA, Sigma Aldrich) were dissolved in dimethylsulfoxide (DMSO, Sigma Aldrich) to an intermediate concentration of 100 mM (1 and 2E), and 16.6 mM (RA). Final dilutions (10 microM for b-lactam 2E, b-Lactams 2Z and RA, 7.5 microM for azetidinone 1) were done in the culture medium. A corresponding volume of DMSO was added to control cells (vehicle treated). To evaluate citotoxic effect exerted by Azetidinone 1, b-Lactams 2E and b-Lactams 2Z, SH-SY5Y cells were seeded at a density of 1 104 cells/well in 96 platic multiwells/dish; 24 h after seeding were treated for 24 h with Azetidinone 1, b-Lactams 2E and b-Lactams 2Z in the concentration range 1 microM to 1 mM and was performed MTT assay [36]. Cytotoxicity of compounds 1 and 2E and 2Z on SHSY5Y (IC50) was assessed in order to define the working concentration (IC501 ¼ 10e28 microM R2 ¼ 0.973; IC502E ¼ 38e65 microM R2 ¼ 9988; IC50 2Z ¼ 35e97 microM R2 ¼ 0.954) (Fig. 2). 4.2.1. Proliferation effect To evaluate the effect of Azetidinone 1, b-Lactams 2E and 2Z on proliferation, cells were seeded at a density of 1 104 cells/cm2 in 6 plastic multiwells dish and allowed to grow for 24 h before treatment. Twenty-four h after seeding, the medium was removed and fresh medium containing the drugs was added for additional 24, 48, 72 and 96 h. Rate of cells growth was determined counting viable cells using trypan blue exclusion assay. Doubling time (Td) was calculated during logarithmic growth phase. The experiment was repeated twice. Statistical analysis were performed with two-way ANOVA, Dunnet’s multiple comparisons test. 4.2.2. Differentiative effect Indirect immunofluorescence was used to investigate possible morphological changes of cytoskeleton due to Azetidinone 1, bLactams 2E and 2Z exposure. For this experiment, SH-SY5Y were seeded on glass coverslips coated with CultrexÒ Basement Membrane Extract (BME) (TrevigenÒ, Gaithersburg, MD, USA) at a density of 1 104 cells/cm2 and cultured in the previously described culture media. After 24 h of seeding, the medium was
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removed and was added fresh medium containing 7.5 microM azetidinone 1, 10 microM of b-lactam 2E, 10 microM of b-Lactams 2Z and 10 microM of RA. Cells were treated for additional 6 days and medium was replaced every 48 h. Untreated cells and cells treated with retinoic acid (RA) 10 microM were used respectively as nondifferentiated and differentiated controls [37]. After treatments, cells were washed in PBS and fixed with 4% paraformaldehyde in 0.1 M Sørensen phosphate buffer for 20 min at RT. Subsequently, fixed cells were blocked in PBS containing 0.3% Triton-X 100 (Merck, Darmstadt, Germany), 5% Donkey Normal Serum (Sigma Aldrich) for 1 h at RT, then incubated overnight at þ4 C in humid atmosphere with anti-neurofilament 200 (Sigma Aldrich) and anti- bIII-tubulin (R&D) antibodies diluted in blocking solution 1:400 and 1:1000 respectively. After rinsing twice in PBS for 10 min, cells were incubated for 30 min at 37 C with Dylight 488-labeled secondary antisera (Thermo Scientific) diluted in 0.3% Triton-X 100/PBS. For nuclear staining, cells were first washed in PBS then incubated 15 min with 1 mg/ml Hoechst 33258 in 0.2% Triton-X 100/PBS. After rinsing in PBS, coverslips containing cells were finally mounted in 1,4-phenylendiamine (Sigma Aldrich)/ glycerol 0.1%. Negative controls were performed by primary antibody omission. Images of SH-SY5Y cells were taken by Nikon Eclipse 600 microscope equipped with motorized z-stage control and F-view II CCD Cameras with 20 objective. Immunofluorescence staining was analyzed using the Nis-Elements Software 3.21.03 (Nikon). Analysis of bIII-tubulin and neurofilament 200 immunoreactive areas (IR) were executed applying “sharpen” and “detect edges” filters. For each image, nuclei were counted and IR/n of nuclei were calculated. The experiment was repeated twice and 3 samples for each experiment (at least 100 cells) were analyzed. Statistical analysis were performed with one-way ANOVA. Representative images were collected using a Nikon A1R confocal laser-scanning microscope, equipped with a Nikon PlanApo 40 lens.
[6]
[7] [8]
[9] [10] [11] [12] [13] [14]
[15] [16]
[17]
[18] [19] [20]
Acknowledgments
[21]
The authors DG, PG, and MP would like to thank Mrs Jessica Giacoboni for technical assistance. We are grateful to Dr. Francesca Paradisi (senior lecturer at the UCD School of Chemistry and Chemical Biology, University College Dublin) for revision of the English manuscript. This work was supported by MIUR (PRIN project), University of Bologna and POR-FESR Regione Emilia Romagna.
[22] [23] [24] [25] [26]
Appendix A. Supplementary data
[27] [28]
Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2013.09.057.
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