Phd in PHARMACEUTICAL SCIENCES

Thesis title: Design, synthesis, and biological evaluation of novel molecular scaffolds for single or multi-epigenetic targets to fight human diseases

In the present thesis, I describe the principal projects I developed during my PhD period in collaboration with the research group of Professor Antonello Mai. My research is based on design and synthesis of novel molecular scaffold for “single” or “multi” epigenetic targets to fight human diseases, in particular, solid or liquid cancers, Neglected Tropical Diseases (NTDs), and metabolic disorders. The epigenetic target I focused on are Histone Deacetylases (HDACs), Sirtuins, Lysine arginine methyl transferase 1 (LSD1) and Protein arginine methyl transferase 5 (PRMT5). Moreover, we projected and synthetized hybrid compounds able to inhibit HDAC/LSD1 and LSD1/PRMT5 simultaneously. Starting from the N-hydroxy-3-(4-(2-phenylbutanoyl)amino) phenyl)acrylamide (5b) previously described by us as a HDAC inhibitor, we prepared four aza-analogues, 6–8, 9b, as regioisomers containing the pyridine nucleus. A preliminary screening against mHDAC1 highlighted the N-hydroxy-5-(2-(2-phenylbutanoyl)amino)pyridyl)acrylamide (9b) as the most potent inhibitor. Thus, we further developed both pyridylacrylic- and nicotinic-based hydroxamates (9a, 9c–f, and 11a–f) and 2’- aminoanilides (10a–f and 12a–f), related to 9b, subsequently tested against all HDAC isoforms. Among them, the nicotinic hydroxamate 11d displayed sub-nanomolar potency (IC50: 0.5 nM) for HDAC4 and selectivity up to 34000 times over all the other tested HDAC isoforms. The 2’-aminoanilides were class I-selective HDAC inhibitors, generally more potent against HDAC3, with the nicotinic anilide 12d being the most effective (IC50HDAC3=0.113 μM). When tested in U937 leukemia cells, the hydroxamates 9e, 11c, and 11d blocked over 80% of cells in G2/M phase, whereas the anilides did not alter cell-cycle. In the same cell line, the hydroxamate 11c and the anilide 10b induced about 30% apoptosis, and the anilide 12c displayed about 40% cytodifferentiation. Finally, the most potent compounds in leukemia cells 9b, 11c, 10b, 10e, and 12c were also tested in K562, HCT116, and A549 cancer cells, displaying antiproliferative IC50 values at single-digit to sub-micromolar level. Based on our development of pyridine-based isomers as HDAC inhibitors, we synthetized a series of novel 5-acylamino-2-pyridylacrylic- and -picolinic hydroxamates and 2'-aminoanilides 27-30 as anticancer agents. The hydroxamate 27d provided HDAC3/6-selective inhibition with IC50 values of 80 and 11 nM, respectively, whereas the congener 27e behaved as inhibitor of HDAC1-3, -6, -8 and - 10 at dual-digit nanomolar level. This last class I/IIb-selective HDAC inhibitor 27e provided a very high antiproliferative activity (dual-digit nanomolar IC50 values) in both haematological and solid cancer cell lines. In leukaemia U937 cells, 27d and 30f induced remarkable cell death after 48 h, being more potent than the corresponding reference compounds SAHA and MS-275. The 2'-aminoanilide 30d was the best cytodifferentiating agent in U937 cells in a dose- and time-dependent manner (up to 35%), whereas the hydroxamates 5d and 27e and 2’-aminoanilide 30d were the most effective to induce p21 protein expression in U937 cells. Compounds 27d, 27e, 30d and 30f increased mRNA expression of p21, BAX and BAK while downregulating cyclin D1 and BCL-2, furthermore they also modulated several miRNAs involved in cell cycle regulation and apoptosis in U937 cells. Neglected tropical diseases (NTDs), including trypanosomiasis, leishmaniasis, and schistosomiasis, result in a significant burden in terms of morbidity and mortality worldwide every year. Current antiparasitic drugs suffer from several limitations such as toxicity, no efficacy toward all forms of the parasites’ life cycle, and/or induction of resistance. Histone-modifying enzymes play a crucial role in parasite growth and survival; thus, the use of epigenetic drugs has been suggested as a strategy for the treatment of NTDs. We tested structurally different HDACi 36-39, 27e, 40-43, which are chosen from our in-house library or newly synthesized, against Trypanosoma cruzi, Leishmania spp, and Schistosoma mansoni. Among them, 39 emerged as the most potent agent against all the tested parasites, but it was too toxic for the host cells, hampering further studies. The retinoic 2′-aminoanilide 42 was less potent than 39 in all parasitic assays, but as its toxicity is considerably lower, it could be a promising starting structure for further development. In T. cruzi, compound 38 inhibited parasite growth at a single-digit micromolar level combined with moderate toxicity. In S. mansoni, 39’s close analogs 51-54 were tested in new transformed schistosomula (NTS) and adult worms displaying a high death induction against both parasite forms. Among them, 51 and 53 exhibited very low toxicity in human retinal pigment epithelial (RPE) cells, thus being promising compounds for further optimization. Schistosoma mansoni HDAC8 is a reliable target to fight schistosomiasis, and several inhibitors have been reported in the literature up to now. Nevertheless, only a few displayed selectivity over the human deacetylases and some exhibited very low or no activity against parasite larvae and/or adult worms. We report the in vitro enzyme and biological activity of a small library of HDAC inhibitors from our lab (1-12), in many cases (2, 4-7, 9-11) exhibiting submicromolar/nanomolar potency against smHDAC8 and diverse degrees of selectivity over hHDAC1 and/or hHDAC6. Compounds 55-58, 11e, 11d, 59-64 were tested against schistosomula and selected of them against the adult forms of S. mansoni to detect their effect on viability. Among them, 2, 8, and 9 showed the highest viability reduction for the larval stage with IC50 values around 1 µM, and 59, 60, and 61 displayed ~ 40-50% activity in adult worms at 10 µM, joined to moderate (59) to no (56, 60, 61) toxicity in human fibroblast MRC-5 cells. Also, in the class of histone deacetylases, Sirtuins are NAD+ -dependent protein involved in metabolic regulation and aging-related diseases. Nowadays, specific activators for seven human Sirtuin isoforms are considered as important chemical tools with a potential therapeutic application. Several activators have been described for Sirt1 and act via a unique N-terminal domain of this isoform. For most other Sirtuin isoforms, including mitochondrial Sirt3−5, no potent and specific activators have yet been identified. Our research team describes the identification and characterization of 1,4-dihydropyridine-based compounds that either act as pan Sirtuin activators or specifically stimulate Sirt3 or Sirt5. The activators bind to the Sirtuin catalytic cores independent of NAD+ and acylated peptides and stimulate turnover of peptide and protein substrates. The compounds also activate Sirt3 or Sirt5 in cellular systems regulating, e.g., apoptosis and electron transport chain. Starting from compound 79a, a moderate Sirt1/3 activator, we started our med-chem optimization campaign leading to the selective compounds 80a and 80s for Sirt3 and compound 80r for Sirt5. These results provide a scaffold for potent Sirtuin activation and derivatives specific for Sirt3 and Sirt5 as an excellent basis for further drug development. LSD1, another important epigenetic target, is highly involved in cancer. To date, several covalent and reversible LSD1 inhibitors are currently in clinical trials, including the well-characterized covalent inhibitor tranylcypromine (TCP). In a previous study my research group found that according to crystal structure analysis TCP-analogues decorated with long and hindered substituents were more potent and selective to inhibit LSD1 by filling the large catalytic cleft of LSD1. Therefore, we developed three different series of TCP derivatives, bearing aroyl- and arylacetyl-amino (93a-h), Z-amino acylamino (94a-o), or double-substituted benzamide (95a-n) residues at C4 or C3 position of TCP. Furthermore, we chemically manipulated the TCP scaffold to obtain different fragments (compounds 96a-i) suitable for the design of novel LSD1 inhibitors. When tested against LSD1, most of 93 and 95 exhibited IC50 values in the low nanomolar range, with 93e and 95a, d, f, g being the most selective respect to monoamine oxidases. In MV4-11 AML and NB4 APL cells, compounds 95 were the most potent, displaying up to sub-micromolar cell growth inhibition against both cell lines (95a) or against NB4 cells (95c). The most potent compounds in cellular assays were also able to induce the expression of LSD1 target genes, such as GFI-1b, ITGAM, and KCTD12, as functional read-out for LSD1 inhibition. Mouse and human intrinsic clearance data highlighted the high metabolic stability of compounds 95a, 95d and 95g. As regioisomers/bioisosteres of 125a, a 4-phenylbenzamide tranylcypromine (TCP) derivative previously disclosed by us, we report the synthesis and biological evaluation of some (hetero)arylbenzoylamino TCP derivatives 125b-130, in which the 4-phenyl moiety of 125a was shifted at the benzamide C3 position or replaced by 2- or 3-furyl, 2- or 3-thienyl, or 4-pyridyl group, all at the benzamide C4 or C3 position. In anti-LSD1-CoREST assay, all the meta derivatives were more effective than the para-analogues, with the meta thienyl analogs 128b and 129b being the most potent (IC50 values ¼ 0.015 and 0.005 lM) and the most selective over MAO-B (selectivity indexes: 24.4 and 164). When tested in U937 AML and prostate cancer LNCaP cells, selected compounds 125a,b, 126b, 127b, 128b, and 129a,b displayed cell growth arrest mainly in LNCaP cells. Western blot analyses showed increased levels of H3K4me2 and/or H3K9me2 confirming the involvement of LSD1 inhibition in these assays. Cancer is a multifactorial disease caused by genetic and/or epigenetic alterations that lead to the malfunction of numerous pathways. By choosing a polypharmacological strategy where there is simultaneous inhibition of two or more targets involved in pathogenesis, an increase in therapeutic efficacy could be achieved compared with single target inhibition. Considering our long-lasting experience regarding HDAC and LSD1 inhibitors in concomitance with the convergence of both pathways, we developed dual hybrid LSD1/HDAC inhibitors by merging structure of the LSD1i GSK-2879552 with the HDAC-inhibiting ortho-aminoanilide function (146a-m), also including a substitution at the para position respect to amino function aimed to obtain metabolic stability or selective inhibition for HDAC1 and HDAC2 isoforms. In biochemical assays compounds 146a-m showed a submicromolar potency against LSD1 and a general preferred inhibition of class I HDACs. Among them, compound 146c exhibited selective inhibition against HDAC1 (IC50 = 0.048 µM; SI over HDAC3 = 364.6). Preliminary biological data showed that the dual LSD1/HDAC inhibitor 146c produced a significant increase in the pre-G1 phase when tested on lung (A549), colon (HCT-116), and breast (MCF7) cancer cell lines. Given the hybrid nature of 146c, we demonstrated MCF7 cells treated with 146c resulted in a strong increase of ac-H3 levels (mark for HDAC inhibition), and an increase of H3K4me2 (mark for LSD1 inhibition). Finally, we detected the effects of 146c on ER-α expression levels. As expected, 146c proved a significant dose-dependent decrease of also ERα levels. Other investigations on 146c to better characterize the molecular mechanism of its anticancer activity are in progress. Another dual hybrid inhibitors which we designed and synthetized are the LSD1/PRMT5 compounds 153a-f. LSD1 (lysine specific demethylase 1) and PRMT5 (protein arginine methyltransferase) are epigenetic targets involved in liquid and solid tumors. Recently, it has been shown that the catalytic activity of PRMT5 is implicated in the proliferation of MLL-AF9-rearranged cells of AML (acute myeloid leukemia); when inactivated, it inhibits colony formation by multiple oncogenic drivers (cyclin D1, c-MYC, NOTCH1), reduces the recruitment of co-activators (CBP, p300, MLL1) while increasing the recruitment of co-repressors (WNT/β-Catenin). PRMT5, therefore, governs the expression of pro-survival genes and its inhibition induces lymphoma cell death. Based on these results, we observed that irreversible LSD1 inhibitors preferentially kill MLL-AF9 PRMT5 +/- cells, thus suggesting a potential synergistic effect in leukemia models by dual inhibition of LSD1 and PRMT5. Indeed, by testing a combination of GSK-LSD1 (LSD1i) and GSK591 (PRMT5i) in a preliminary study, very strong synergistic antiproliferative effects were observed in MLL-AF9 SRSF2WT/WT leukemia cells. These results prompted us to design the first dual epigenetic hybrid LSD1-PRMT5 through the fusion of GSK591 and TCP (tranylcypromine), specifically by replacing cyclobutylamine directly with TCP or by using different spacers/connecting groups of amino acid nature. All newly synthesized compounds were tested in enzymatic assay against LSD1 and against PRMT isoforms 1, 5, 7. The most effective and specific inhibitors 153a will be tested in mouse models transfected with leukemic cell lines. To sum up, I prepared numerous epigenetic modulators which are currently at various stages regarding the biochemical and biological screening, however numerous compounds are promising candidates to fight human diseases, in particular, solid or liquid cancers, Neglected Tropical Diseases (NTDs), and metabolic disorders.