Microwave-Mediated Hetero Diels-Alder reaction: Synthesis of biologically active compounds

Heterocyclic compounds represent almost two-thirds of all the known organic compounds: they are widely distributed in nature and play a key role in a huge number of biologically important molecules including some of the most significant for human beings. A powerful tool for the synthesis of such compounds is the hetero Diels-Alder reaction (HDA), that involve a [4+2] cycloaddition reaction between heterodienes and suitable dienophiles. Among heterodienes to be used in such six-membered heterocyclic construction strategy, 3-trialkylsilyloxy-2-aza-1,3-dienes (Fig 1) has been demonstrated particularly attractive. In this thesis work, HDA reactions between 2-azadienes and carbonylic and/or olefinic dienophiles, are described. Moreover, substitution of conventional heating by the corresponding dielectric heating as been explored in the frame of Microwave-Assisted-Organic-Synthesis (MAOS) which constitutes an up-to-grade research field of great interest both from an academic and industrial point of view. Reaction of the azadiene 1 (Fig 1) will be described using as dienophiles carbonyl compounds as aldehyde and ketones. The six-membered adducts thus obtained (Scheme 1) have been elaborated to biologically active compounds like 1,3-aminols which constitutes the scaffold for a wide range of drugs (Prozac®, Duloxetine, Venlafaxine) with large applications in the treatment of severe diseases of nervous central system (NCS). Scheme 1 The reaction provides the formation of three new stereogenic centres (C-2; C-5; C-6). The diastereoselective outcome of these reactions has been deeply investigated by the use of various combination of achiral and chiral azadienes and aliphatic, aromatic or heteroaromatic aldehydes. The same approach, basically, has been used in the synthesis of piperidin-2-one scaffold substituting the carbonyl dienophile with an electron poor olefin. Scheme 2 As a matter of fact, this scaffold is present in a very large number of natural substances and, more interesting, is a required scaffold for an huge variety of biologically active compounds. Activated olefins bearing one or two sulfone groups, were choose as dienophiles both for the intrinsic characteristic flexibility of the “sulfone group” which may be easily removed or elaborated to more complex decorations of the heterocyclic ring, and for the electron poor property of this dienophiles which makes the resulting HDA reaction of the type “normal electron demand”. Synthesis of natural compounds like racemic (±)-Anabasine (alkaloid of Tobacco’s leaves) and (R)- and (S)-Conhydrine (alkaloid of Conium Maculatum’s seeds and leaves) and its congeners, are described (Fig 2).

Ruolo dell'Enantiomero (R)-9-HSA nel controllo della proliferazione in una linea di Adenocarcinoma del Colon umano

9-hydroxystearic acid (9-HSA) is an endogenous lipoperoxidation product and its administration to HT29, a colon adenocarcinoma cell line, induced a proliferative arrest in G0/G1 phase mediated by a direct activation of the p21WAF1 gene, bypassing p53. We have previously shown that 9-HSA controls cell growth and differentiation by inhibiting histone deacetylase 1 (HDAC1) activity, showing interesting features as a new anticancer drug. The interaction of 9-HSA with the catalytic site of the 3D model has been tested with a docking procedure: noticeably, when interacting with the site, the (R)-9-enantiomer is more stable than the (S) one. Thus, in this study, (R)- and (S)-9-HSA were synthesized and their biological activity tested in HT29 cells. At the concentration of 50 M (R)-9-HSA showed a stronger antiproliferative effect than the (S) isomer, as indicated by the growth arrest in G0/G1. The inhibitory effect of (S)-9-HSA on HDAC1, HDAC2 and HDAC3 activity was less effective than that of the (R)-9-HSA in vitro, and the inhibitory activity of both the (R)- and the (S)-9-HSA isomer, was higher on HDAC1 compared to HDAC2 and HDAC3, thus demonstrating the stereospecific and selective interaction of 9-HSA with HDAC1. In addition, histone hyperacetylation caused by 9-HSA treatment was examined by an innovative HPLC/ESI/MS method. Analysis on histones isolated from control and treated HT29 confirmed the higher potency of (R)-9-HSA compared to (S)-9-HSA, severely affecting H2A-2 and H4 acetylation. On the other side, it seemed of interest to determine whether the G0/G1 arrest of HT29 cell proliferation could be bypassed by the stimulation with the growth factor EGF. Our results showed that 9-HSA-treated cells were not only prevented from proliferating, but also showed a decreased [3H]thymidine incorporation after EGF stimulation. In this condition, HT29 cells expressed very low levels of cyclin D1, that didn’t colocalize with HDAC1. These results suggested that the cyclin D1/HDAC1 complex is required for proliferation. Furthermore, in the effort of understanding the possible mechanisms of this effect, we have analyzed the degree of internalization of the EGF/EGFR complex and its interactions with HDAC1. EGF/EGFR/HDAC1 complex quantitatively increases in 9-HSA-treated cells but not in serum starved cells after EGF stimulation. Our data suggested that 9-HSA interaction with the catalytic site of the HDAC1 disrupts the HDAC1/cyclin D1 complex and favors EGF/EGFR recruitment by HDAC1, thus enhancing 9-HSA antiproliferative effects. In conclusion 9-HSA is a promising HDAC inhibitor with high selectivity and specificity, capable of inducing cell cycle arrest and histone hyperacetylation, but also able to modulate HDAC1 protein interaction. All these aspects may contribute to the potency of this new antitumor agent.