Maria Alessandra Alisi

Effects of AF3442 [N-(9-ethyl-9H-carbazol-3-yl)-2-(trifluoromethyl)benzamide], a novel inhibitor of human microsomal prostaglandin E synthase-1, on prostanoid biosynthesis in human monocytes in vitro.

Inhibitors of microsomal prostaglandin (PG) E synthase-1 (mPGES-1) are being developed for the relief of pain. Redirection of the PGH(2) substrate to other PG synthases, found both in vitro and in vivo, in mPGES-1 knockout mice, may influence their efficacy and safety. We characterized the contribution of mPGES-1 to PGH(2) metabolism in lipopolysaccharide (LPS)-stimulated isolated human monocytes and whole blood by studying the synthesis of prostanoids [PGE(2), thromboxane (TX)B(2), PGF(2alpha) and 6-keto-PGF(1alpha)] and expression of cyclooxygenase (COX)-isozymes and down-stream synthases in the presence of pharmacological inhibition by the novel mPGES-1 inhibitor AF3442 [N-(9-ethyl-9H-carbazol-3-yl)-2-(trifluoromethyl)benzamide]. AF3442 caused a concentration-dependent inhibition of PGE(2) in human recombinant mPGES-1 with an IC(50) of 0.06microM. In LPS-stimulated monocytes, AF3442 caused a concentration-dependent reduction of PGE(2) biosynthesis with an IC(50) of 0.41microM. At 1microM, AF3442 caused maximal selective inhibitory effect of PGE(2) biosynthesis by 61+/-3.3% (mean+/-SEM, P<0.01 versus DMSO vehicle) without significantly affecting other prostanoids (i.e. TXB(2), PGF(2alpha) and 6-keto-PGF(1alpha)). In LPS-stimulated whole blood, AF3442 inhibited in a concentration-dependent fashion inducible PGE(2) biosynthesis with an IC(50) of 29microM. A statistically significant inhibition of mPGES-1 activity was detected at 10 and 100microM (38+/-14%, P<0.05, and 69+/-5%, P<0.01, respectively). Up to 100microM, the other prostanoids were not significantly affected. In conclusion, AF3442 is a selective mPGES-1 inhibitor which reduced monocyte PGE(2) generation also in the presence of plasma proteins. Pharmacological inhibition of mPGES-1 did not translate into redirection of PGH(2) metabolism towards other terminal PG synthases in monocytes. The functional relevance of this observation deserves to be investigated in vivo.

New anti-viral drugs for the treatment of the common cold

Human Rhinovirus (HRV) is the most important aetiologic agent of common cold in adults and children. HRV is a single-stranded, positive sense RNA virus and, despite the high level of conservation among different serotypes, sequence alignment of viral protease 3C with mammalian protease reveals no homology. Thus, protease 3C is an optimal target for the development of anti-HRV agents. In the present work we investigated the design, the synthesis and the development of new potential reversible inhibitors against HRV protease 3C. Docking studies on the crystallized structure of HRV2 protease 3C led us to the design and the synthesis of a series of 3,5 disubstituted benzamides able to act as analogues of the substrate. We also developed 1,3,5 trisubstituted benzamides where aromatic substitutions on the aryl ring led us to investigate the importance of pi-pi interaction on the stabilization of protease 3C-inhibitor complex. All structures were tested for enzymatic inhibition on HRV14 protease 3C. Results highlighted the inhibitory activity of compounds 13, 14, and 20 (91%, 81%, and 85% at 10 microM, respectively), with the latter exhibiting an ID(50) (dose that inhibits 50% of the viral cytopathic effect) on HRV-14=25 microg/ml.

Discovery and Pharmacological Profile of New 1H-Indazole-3-carboxamide and 2H-Pyrrolo[3,4-c]quinoline Derivatives as Selective Serotonin 4 Receptor Ligands

Since the discovery of the serotonin 4 receptor (5-HT(4)R), a large number of receptor ligands have been studied. The safety concerns and the lack of market success of these ligands have mainly been attributed to their lack of selectivity. In this study we describe the discovery of N-[(4-piperidinyl)methyl]-1H-indazole-3-carboxamide and 4-[(4-piperidinyl)methoxy]-2H-pyrrolo[3,4-c]quinoline derivatives as new 5-HT(4)R ligands endowed with high selectivity over the serotonin 2A receptor and human ether-a-go-go-related gene potassium ion channel. Within these series, two molecules (11 ab and 12 g) were identified as potent and selective 5-HT(4)R antagonists with good in vitro pharmacokinetic properties. These compounds were evaluated for their antinociceptive action in two analgesia animal models. 12 g showed a significant antinociceptive effect in both models and is proposed as an interesting lead compound as a 5-HT(4)R antagonist with analgesic action.

Synthesis and structure-activity relationships of new acetylcholinesterase inhibitors: Morpholinoalkylcarbamoyloxyeseroline derivatives

Abstract Several new potent acetylcholinesterase inhibitors have been synthesised as potential drugs for the treatment of Alzheimers disease. Heptylphysostigmine (MF201) is a drug analogue of physostigmine under clinical evaluation. In order to obtain new physostigmine analogues, the methylcarbomoyloxy group was substituted with ω-morpholinoalkylcarbamoyloxy moieties of different chain lengths (C2–C12). Potent in vitro inhibition is seen when the chain length is composed of eight to twelve methylene groups. The inhibitory activity of the C10 and C11 is 7-fold greater with respect to heptylphysostigmine.

Synthesis of inositol phospholipids with thiophosphoester bonds

Abstract the synthesis of phosphatidylinositol (PI) analogues (±)1- O -(1- O -octadecanoyl-2- O -acetyl- rac -3-thioglycerylphosphoryl)- myo -inositol ( 7 ), (±)1- O -(1,2-di- O -octadecyl- rac -3-thioglycerylphosphoryl)- myo -inositol ( 14a ), (±)1- O -(1,2-di- O -octyl- rac -3-thioglycerylphosphoryl)- myo -inositol ( 14b ) and (±)1- O -(1- O -octadecyl-2- O -methyl- rac -3-thioglycerylphosphoryl)- myo -inositol ( 14c ) designed to show a novel mode of PI-phospholipase C (PI-PLC) inhibition, is described.

Hit Optimization of 5-Substituted-N-(piperidin-4-ylmethyl)-1H-indazole-3-carboxamides: Potent Glycogen Synthase Kinase-3 (GSK-3) Inhibitors with in Vivo Activity in Model of Mood Disorders

Novel treatments for bipolar disorder with improved efficacy and broader spectrum of activity are urgently needed. Glycogen synthase kinase 3β (GSK-3β) has been suggested to be a key player in the pathophysiology of bipolar disorder. A series of novel GSK-3β inhibitors having the common N-[(1-alkylpiperidin-4-yl)methyl]-1H-indazole-3-carboxamide scaffold were prepared taking advantage of an X-ray cocrystal structure of compound 5 with GSK-3β. We probed different substitutions at the indazole 5-position and at the piperidine-nitrogen to obtain potent ATP-competitive GSK-3β inhibitors with good cell activity. Among the compounds assessed in the in vivo PK experiments, 14i showed, after i.p. dosing, encouraging plasma PK profile and brain exposure, as well as efficacy in a mouse model of mania. Compound 14i was selected for further in vitro/in vivo pharmacological evaluation, in order to elucidate the use of ATP-competitive GSK-3β inhibitors as new tools in the development of new treatments for mood disorders.

Synthesis of some thiophosphate analogues (C-S-P) of phosphatidylinositol

Abstract the synthesis of analogues of phosphatidylinositol (PI), designed to show a novel mode of PI-phospholipase C (PI-PLC) inhibition, is described.

Identification of Novel Triazole-Based Nicotinamide Phosphoribosyltransferase (NAMPT) Inhibitors Endowed with Antiproliferative and Antiinflammatory Activity

Nicotinamide phosphoribosyltransferase (NAMPT) is a key enzyme involved in the recycling of nicotinamide to maintain adequate NAD levels inside the cells. It has been postulated to be a pharmacological target, as it is overexpressed in cancer cells as well as in inflammatory diseases. We describe the synthesis and characterization of a novel class of one-digit nanomolar NAMPT inhibitors based on in vitro characterization. The most active compound tested, 30c, displayed activity in xenograft and allograft models, strengthening the potential of NAMPT inhibitors as antitumoral drugs. Furthermore, in the present contribution we describe the ability of 30c to significantly improve the outcome of colitis in mice. Given that this is the first report of an effect of NAMPT inhibitors in colitis, this result paves the way for novel applications for this class of compounds.

A Novel Multi-step Virtual Screening for the Identification of Human and Mouse mPGES-1 Inhibitors.

We present here the development of a novel virtual screening protocol combining Structure‐based and Ligand‐based drug design approaches for the identification of mouse mPGES‐1 inhibitors. We used the existing 3D structural data of the murine enzyme to hypothesize the inhibitors binding mode, which was the starting point for docking simulations, shape screening, and pharmacophore hypothesis screening. The protocol allowed the identification of 16 mouse mPGES‐1 inhibitors with low micromolar activity, which, notably, also inhibit the human enzyme in the same concentration range. The inhibitors predicted binding mode is expected to be the base for the rational drug design of new potent dual species inhibitors of human and murine mPGES‐1.

Synthesis and cytotoxic activity of (±) octadecylphosphodithionyl-1-myo-inositol

Abstract the synthesis of the phosphatidylinositol (PI) analogue (±)-octadecylphosphodithionyl-1- myo -inositol is described. The cytotoxic activity on K562 erythroleukaemic cells and the effect on the activity of PI specific phospholipase C (PI-PLC) from human platelets are reported.