Carlo Mustazza

Development of nociceptin receptor (NOP) agonists and antagonists

The nociceptin opioid (NOP) receptor is the most recently discovered member of the family of the opioid receptors; its endogenous agonist is the peptide nociceptin. Due to the subsequent elucidation of its physiological role in both central and peripheral nervous system and in some non‐neural tissues, there is a rapidly growing interest in the pharmacological application of substances active on this receptor. Despite the current clinical use of a morphinane‐based NOP/MOP mixed ligand (buprenorphine) as an analgesic and in the treatment of drug addictions, so far just a few clinical trials have been made with selective NOP ligands. However, the perspective of their utilization is rapidly growing. Agonists can find applications in the treatment of neuropathic pain, anxiety, cough, drug addition, urinary incontinence, anorexia, congestive heart failure, hypertension; and antagonists for pain, depression, Parkinsons disease, obesity, and as memory enhancers. Besides peptide ligands, which are still subjected to many pharmacological investigations, many different chemical classes of NOP ligands have been discovered: piperidines, nortropanes, spiropiperidines, 4‐amino‐quinolines and quinazolines, and others. The new advances in establishing structure–activity relationships, also with the help of modeling studies, can permit the development of more active and selective molecules.

Synthesis and cholinesterase activity of phenylcarbamates related to Rivastigmine, a therapeutic agent for Alzheimer's disease.

In order to develop new cholinesterase agents effective against Alzheimers disease (AD) we synthesized some phenylcarbamates structurally related to Rivastigmine and evaluated their in vitro and in vivo biological activity. Among the compounds which displayed the most significant in vitro activity, 1-[1-(3-dimethylcarbamoyloxyphenyl)ethyl]piperidine (31b), in addition to a simple and cheaper synthesis, showed lower toxicity and very similar therapeutic index in comparison with Rivastigmine.

(E)-1-(Heterocyclyl or cyclohexyl)-2-[1,3,7-trisubstituted (xanthin-8-yl)] ethenes as A2a adenosine receptor antagonists

Some 1,3,7-trisubstituted-8-styrylxanthine analogues, with the aryl group replaced by a heterocycle or cyclohexane ring, were prepared and evaluated for their interaction with the A1 and A2a adenosine receptors. The highest degree of activity was displayed by the 1,3-dipropyl-7-methyl-8-[2-(3-thienyl)ethenyl]xanthine 4cdi, which was found to be a potent and selective A2a antagonist in binding assays (Ki = 19 nM, A1/A2a ratio = 30).

Characterization of Sildenafil analogs by MS/MS and NMR: A guidance for detection and structure elucidation of phosphodiesterase-5 inhibitors

Novel synthetic analogs of Sildenafil are constantly detected as adulterants in counterfeit drugs and dietary supplements. Their intake constitutes a serious health hazard as side effects are unknown. In this paper an investigation is carried out on NMR and MS/MS spectra of Sildenafil, Thiosildenafil, Acetildenafil and thirteen of their analogs: a list of key signals is reported and discussed with the intent to provide a tool that can help in detecting adulteration and in elucidating the structure of novel analogs. In this view extensive spectral data were reported, discussed and summarized in tables. A discussion on mass fragmentation and NMR chemical shifts is also provided to rationalize assignation. Moreover, a comprehensive information on the route of synthesis is provided for the benefit of those medicines control laboratories that need to synthesize analogs reference standards in-house.

Synthesis and pharmacological evaluation of bivalent antagonists of the nociceptin opioid receptor

Bivalent ligands constituted by two identical pharmacophores structurally related to the Nociceptin Opioid Receptor (NOPr) antagonist JTC-801 were synthesized and their binding affinities for NOPr were evaluated. The novel ligands are formed by two modified JTC-801 units linked by di-iminic and di-aminic spacers with length ranging from three to ten methylene units. Moreover, the synthesis and the pharmacological characterization were extended to the corresponding univalent ligands. The latter compounds consisted in a single modified JTC-801 unit and an alkyl or alkylamino or alkylimino tail. The purpose of this study is to feature the location and surroundings of the allosteric binding site(s) of pharmacophores containing the 4-aminoquinoline structure. Most important, the bivalent ligands were exploited to reveal the eventual occurrence of a supramolecular receptorial architecture of the NOPr. All the bivalent derivatives 4 and 5 proved to be active in the nanomolar range with no outstanding dependence on the chain length. They showed potencies from three to ten times higher than the corresponding monomers. Consequently, results clearly indicated a positive role of the second pharmacophore in the ligand-protein interaction. The pharmacological profile of the monomers 7 and 8 clarified the contribution of the linker chain to NOP receptor affinity and suggested the presence of a lipophilic acidic site neighbouring the binding site of the JTC-like ligands. Selectivity of saturated compounds 5, 7, and 8 was tested by binding experiments on δ, κ and μ opioid receptors. Results indicated a general loss of selectivity as compared to JTC-801. In the [(35)S]GTPγS binding assay, all the compounds revealed antagonistic properties at the NOP Receptor. In conclusion the present study set the basis for a systematic investigation on the structural modifications that can be introduced into novel ligands for NOPr and helped to feature the surrounds of the allosteric site of NOPr.

Screening of preservatives by HPLC-PDA-ESI/MS: A focus on both allowed and recently forbidden compounds in the new EU cosmetics regulation

Commission regulation (EU) No 358/2014 amending the new regulation (EC) No 1223/2009 on cosmetics has prohibited the use of isopropyl-, isobutyl-, phenyl-, benzyl- and pentylparaben. Furthermore, Commission regulation (EU) No 1004/2014 has lowered the maximum permitted concentration of butyl- and propylparaben in cosmetics and it has also banned them in leave-on products designed for application on the nappy area of children under three years of age. A HPLC-PDA-ESI/MS method has been developed herein for the detection of seventeen preservatives, both the most utilised and the recently forbidden by the new EU regulations. The separation of these compounds, including benzoic acid and its derivatives in a 1.10 - 3.04 log Pow range, has been performed with a gradient elution on a Symmetry(®) C18 column (250×4.6mm i.d., particle size 5μm) with water and acetonitrile (0.1% formic acid) as mobile phase. Quantification has been carried out by HPLC-PDA. The method has been validated and successfully applied to the analysis of a large number of cosmetics with different functions like rinse-off and leave-on, or composition like skin, hair, face and oral products.

UV spectroscopic study of the interaction betweenα-,β-, andγ-cyclodextrins and pyridine derivatives

Complex formation betweenα-,β- andγ-cyclodextrins and some pyridine derivatives (hydroxy,O- andN-methyl) have been investigated by UV spectroscopy. Inclusion processes do not exert any noticeable influence on the UV absorption spectra of most of the guests which do not give rise to tautomeric equilibria or which show a strong prevalence for one of the tautomeric forms. In contrast, the equilibrium between the lactim and the lactim forms of 3-hydroxypyridine (which are present in comparable amounts in aqueous solution) was shown to be markedly affected by the inclusion process with a preferential complexation of the latter tautomer.

New fluorinated 1,4-bis-(arylaminomethyl)- and 1,4-bis-(arylaminomethylene)benzenes as fluorescent probes for amyloid plaques in Alzheimer's disease and transmissible spongiform encephalopathies

The research into selective ligands of misfolded protein plaques in Alzheimers disease and in transmissible spongiform encephalopathies led to compound 3, based on the Congo Red framework and bearing two trifluoromethoxy groups. Histochemical experiments on human brain tissues showed intense fluorescent staining of fibrillary deposits in both pathologies.

High resolution NMR conformational studies of new bivalent NOP receptor antagonists in model membrane systems.

The interaction of new bivalent NOP receptor antagonists with dodecyl phosphatidylcholine micelles and DMPC/cholesterol liposomes was investigated in solution by high resolution NMR. The ligands are structurally related to the NOP antagonist JTC-801 plus a propanediamine or heptanediamine spacer between the pharmacophoric units. Ligand internuclear distances were derived from 2D NOESY data and applied to molecular modelling calculations as conformational restraints. NMR experiments on micelles evidenced that the ligands closely approached the micelles but gave no hints on the preferential conformations of the interacting ligands. Results from NMR experiments in the presence of liposomes clearly indicated that both ligands strongly interacted with the bilayer assuming a preferential folded conformation with the quinoline arms superimposing on each other. The finding suggested that these strongly lipophilic pharmacophores could localize in the native receptorial membrane in the form of a depot, gaining access to the recognition site via the lipid bilayer.