Synthesis and structural analysis of halogen substituted fibril formation inhibitors of Human Transthyretin (TTR)

Transthyretin (TTR), a β-sheet-rich tetrameric protein, in equilibrium with an unstable amyloidogenic monomeric form is responsible for extracellular deposition of amyloid fibrils, is associated with the onset of neurodegenerative diseases, such as senile systemic amyloidosis, familial amyloid polyneuropathy and familial amyloid cardiomyopathy. One of the therapeutic strategies is to use small molecules to stabilize the TTR tetramer and thus curb amyloid fibril formation. Here, we report the synthesis, the in vitro evaluation of several halogen substituted 9-fluorenyl- and di-benzophenon-based ligands and their three-dimensional crystallographic analysis in complex with TTR. The synthesized compounds bind TTR and stabilize the tetramer with different potency. Of these compounds, 2c is the best inhibitor. The dual binding mode prevalent in the absence of substitutions on the fluorenyl ring, is disfavored by (2,7-dichloro-fluoren-9-ylideneaminooxy)-acetic acid (1b), (2,7-dibromo-fluoren-9-ylideneaminooxy)-acetic acid (1c) and (E/Z)-((3,4-dichloro-phenyl)-methyleneaminooxy)-acetic acid (2c), all with halogen substitutions

Synthesis and cycloxygenase inhibitory properties of new naphthalene-methylsulfonamido, naphthalene-methylsulfonyl and tetrahydronaphthalen-methylsulfonamido compounds

We synthesized a series of new naphthalene derivatives: naproxen- and 6-methoxy naphthalene acetic acid-like 1–5. In these compounds the carboxylic function, typical of the classical NSAIDs, was replaced by a methylsulfonamido (1, 2 and 6a–c) or methylsulfonyl (3–5) group present in some selective COX-2 inhibitors. We also synthesized compounds 7 and 8 in which the naphthalene portion was substituted by tetrahydronaphthalene ring. Some of the new compounds were assayed for their enzymatic inhibitory activity towards cycloxygenase enzymes. Compounds 4 and 6b, at a concentration of 10 µM exhibit percentage inhibition values of 65%, 50% and 29%, 87% towards COX-2 and COX-1, respectively. The substitution of carboxylic group with a mehylsulfonamido or a methylsulfonyl groups does not allow to direct the selectivity versus to cycloxygenase enzymes

Ionic liquid-promoted green synthesis of biologically relevant diaryl thioethers

This study reports an ionic liquid (IL) promoted green method to obtain diaryl thioethers useful as key intermediates for the synthesis of matrix metalloproteinase (MMP) inhibitors. The synthetic pathway includes a sequential Ullmann reaction and Suzuki cross-coupling. The Ullmann conditions were optimized as regards the catalyst, the ionic liquid, reaction time, and temperature. Under optimal conditions (1-butyl-3-methylimidazolium bromide ([BMIM]Br) as solvent; catalyst, Cu; base, K2CO3; reaction time, 22 h; reaction temperature, 150°C) the aryl iodide conversion was 95%. The Suzuki cross-coupling was conducted in 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM]BF4) at 110°C, using Pd(PPh3)4 as catalyst and Na2CO3 as base. For both reactions, a combination of ILs with microwave (MW) irradiation determined a substantial improvement of reaction time and yields compared to conventional heating. This multi-step process reduces reaction times and removes organic solvents providing a more eco-friendly alternative for the synthesis of important pharmaceutical building blocks

N-(aroyl)-N-(arylmethyloxy)-α-alanines: selective inhibitors of aldose reductase

Aldose reductase (ALR2), a NADPH-dependent reductase, is the first and rate-limiting enzyme of the polyol pathway of glucose metabolism and is implicated in the pathogenesis of secondary diabetic complications. In the last decades, this enzyme has been targeted for inhibition but despite the numerous efforts made to identify potent and safe ALR2 inhibitors, many clinical candidates have been a failure. For this reason the research of new ALR2 inhibitors highly effective, selective and with suitable pharmacokinetic properties is still of great interest. In this paper some new N-(aroyl)-N-(arylmethyloxy)alanines have been synthesized and tested for their ability to inhibit ALR2. Some of the synthesized compounds exhibit IC50 in the low micromolar range and all have proved to be highly selective towards ALR2. The N-(aroyl)-N-(arylmethyloxy)-α-alanines are a promising starting point for the development of new ALR2 selective drugs with the aim of delaying the onset of diabetic complications

Targeting Different Transthyretin Binding Sites with Unusual Natural Compounds

Misfolding and aggregation of the transthyretin (TTR) protein leads to certain forms of amyloidosis. Some nutraceuticals, such as flavonoids and natural polyphenols, have recently been investigated as modulators of the self-assembly process of TTR, but they generally suffer from limited bioavailability. To discover innovative and more bioavailable natural compounds able to inhibit TTR amyloid formation, a docking study was performed using the crystallographic structure of TTR. This computational strategy was projected as an adhoc inspection of the possible relationship between binding site location and modulation of the assembly process; interactions with the as-yet-unexplored epigallocatechin gallate (EGCG) sites and with the thyroxine (T4) pocket were simultaneously analyzed. All the compounds studied seem to prefer the traditional T4 binding site, but some interesting results emerged from the screening of an in-house database, used for validating the computational protocol, and of the Herbal Ingredients Targets (HIT) catalogue available on the ZINC database

Synthesis, experimental evaluation and molecular modelling of hydroxamate derivatives as zinc metalloproteinase inhibitors

Manuscript. Published version available in European Journal of Medicinal Chemistry, Vol. 108, 27 January 2016, pp 141–153Enzymes of the M4 family of zinc-metalloproteinases are virulence factors secreted from gram-positive or gram-negative bacteria, and putative drug targets in the treatment of bacterial infections. In order to have a therapeutic value such inhibitors should not interfere with endogenous zinc-metalloproteinases. In the present study we have synthesised a series of hydroxamate derivatives and validated the compounds as inhibitors of the M4 enzymes thermolysin and pseudolysin, and the endogenous metalloproteinases ADAM-17, MMP-2 and MMP-9 using experimental binding studies and molecular modelling. In general, the compounds are stronger inhibitors of the MMPs than of the M4 enzymes, however, an interesting exception is LM2. The compounds bound stronger to pseudolysin than to thermolysin, and the molecular modelling studies showed that occupation of the S2’ subpocket by an aromatic group is favourable for strong interactions with pseudolysin

Discovery of Dimeric Arylsulfonamides as Potent ADAM8 Inhibitors

The metalloproteinase ADAM8 is upregulated in several cancers but has a dispensable function under physiological conditions. In tumor cells, ADAM8 is involved in invasion, migration and angiogenesis. The use of bivalent inhibitors could impair migration and invasion, through the double binding to a homodimeric form of ADAM8 located on the cell surface of tumor cells. Herein we report the rational design and synthesis of the first dimeric ADAM8 inhibitors selective over ADAM10 and MMPs. Bivalent derivatives have been obtained by dimerizing the structure of a previously described ADAM17 inhibitor, JG26. In particular, derivative 2 showed to inhibit ADAM8 proteolytic activity in vitro and in cell-based assays, at nanomolar concentration. Moreover, it was more effective than the parent monomeric compound in blocking invasiveness in breast cancer MDA-MB-231 cell line, thus supporting our hypothesis about the importance of inhibiting the active homodimer of ADAM8

Discovery of a new selective inhibitor of A Disintegrin And Metalloprotease 10 (ADAM-10) able to reduce the shedding of NKG2D ligands in Hodgkin's lymphoma cell models

Hodgkin's lymphoma (HL) is the most common malignant lymphoma in young adults in the western world. This disease is characterized by an overexpression of ADAM-10 with increased release of NKG2D ligands, involved in an impaired immune response against tumor cells. We designed and synthesized two new ADAM-10 selective inhibitors, 2 and 3 based on previously published ADAM-17 selective inhibitor 1. The most promising compound was the thiazolidine derivative 3, with nanomolar activity for ADAM-10, high selectivity over ADAM-17 and MMPs and good efficacy in reducing the shedding of NKG2D ligands (MIC-B and ULBP3) in three different HL cell lines at non-toxic doses. Molecular modeling studies were used to drive the design and X-ray crystallography studies were carried out to explain the selectivity of 3 for ADAM-10 over MMPs

Specific ADAM10 inhibitors localize in exosome-like vesicles released by Hodgkin lymphoma and stromal cells and prevent sheddase activity carried to bystander cells

Shedding of ADAM10 substrates, like TNFa, MICA or CD30, is reported to affect both anti-tumor immune response and antibody-drug-conjugate (ADC)-based immunotherapy. Soluble forms of these molecules and ADAM10 can be carried and spread in the microenvironment by exosomes released by tumor cells. We reported new ADAM10 inhibitors able to prevent MICA shedding in Hodgkin lymphoma (HL), leading to recognition of HL cells by cytotoxic lymphocytes. In this paper, we show that the mature bioactive form of ADAM10 is released in exosome-like vesicles (ExoV) by HL cells and lymph node mesenchymal stromal cells (MSC). We demonstrate that ADAM10 inhibitors are released in ExoV by MSC or HL cells, endocytosed by bystander cells and localized in the endolysosomal compartment in HL MSC. ExoV released by HL cells can enhance MICA shedding by MSC, while ExoV from MSC induce TNFa or CD30 shedding by HL cells. Of note, ADAM10 sheddase activity carried by ExoV is prevented with the ADAM10 inhibitors LT4 and CAM29, pretreating either the ExoVproducing or the ExoV-receiving cells. In particular, both inhibitors reduce CD30 shedding maintaining the anti-tumor effects of the ADC Brentuximab-Vedotin or the anti-CD30 Iratumumab on HL cells. Thus, spreading of ADAM10 activity due to ExoV can result in the release of cytokines, like TNFa,a lymphoma growth factor, or soluble molecules, like sMICA or sCD30, that potentially interfere with host immune surveillance or immunotherapy. ADAM10 blockers can interfere with this process, allowing the development of anti-lymphoma immune response and/or efficient ADC-based or human antibody-based immunotherapy

ADAM10 new selective inhibitors reduce NKG2D ligand release sensitizing Hodgkin lymphoma cells to NKG2D-mediated killing

Hodgkin lymphoma (HL) resistant to conventional therapies is increasing, making of interest the search for new schemes of treatment. Members of the “A Disintegrin And Metalloproteases” (ADAMs) family, mainly ADAM10 or ADAM17, have been proposed as therapeutic targets in solid tumors and some ADAMs inhibitors have been shown to exert antitumor effects. We have previously described an overexpression of ADAM10 in HL, together with increased release of NKG2D ligands (NKG2D-L) and reduced activation of effector T lymphocytes with anti-lymphoma capacity. Aim of the present work was to verify whether inhibition of ADAM10 in HL cells could restore the triggering of NKG2D-dependent anti-lymphoma T cell response. As no selective ADAM10 blockers have been reported so far, we synthesized the two hydroxamate compounds LT4 and MN8 with selectivity for ADAM10 over metalloproteases (MMPs), LT4 showing higher specificity for ADAM10 over ADAM17. We show that (i) HL lymph nodes (LN) and cultured HL cells express high levels of the mature active membrane form of ADAM10; (ii) ADAM10 is the major sheddase for the NKG2D-L in HL cells; (iii) the new LT4 and MN8 compounds strongly reduce the shedding of NKG2D-L by HL cell lines and enhance the binding of NKG2D receptor; (iv) of note, these new ADAM10 inhibitors increase the sensitivity of HL cell lines to NKG2D-dependent cell killing exerted by natural killer and γδ T cells. Overall, the biologic activity of LT4 and MN8 appears to be more potent than that of the commercial inhibitor GI254023X