Giuseppe Diamantini

Melatonin antagonizes apoptosis via receptor interaction in U937 monocytic cells

Abstract:  Among the non‐neurological functions of melatonin, much attention is being directed to the ability of melatonin to modulate the immune system, whose cells possess melatonin‐specific receptors and biosynthetic enzymes. Melatonin controls cell behaviour by eliciting specific signal transduction actions after its interaction with plasma membrane receptors (MT1, MT2); additionally, melatonin potently neutralizes free radicals. Melatonin regulates immune cell loss by antagonizing apoptosis. A major unsolved question is whether this is due to receptor involvement, or to radical scavenging considering that apoptosis is often dependent on oxidative alterations. Here, we provide evidence that on U937 monocytic cells, apoptosis is antagonized by melatonin by receptor interaction rather than by radical scavenging. First, melatonin and a set of synthetic analogues prevented apoptosis in a manner that is proportional to their affinity for plasma membrane receptors but not to their antioxidant ability. Secondly, melatonins antiapoptotic effect required key signal transduction events including G protein, phospholipase C and Ca2+ influx and, more important, it is sensitive to the specific melatonin receptor antagonist luzindole.

Synthesis, pharmacological characterization and QSAR studies on 2-substituted indole melatonin receptor ligands

A number of 6-methoxy-1-(2-propionylaminoethyl)indoles, carrying properly selected substituents at the C-2 indole position, were prepared and tested as melatonin receptor ligands. Affinities and intrinsic activities for the human cloned mt1 and MT2 receptors were examined and compared with those of some 2-substituted melatonin derivatives recently described by us. A quantitative structure activity relationship (QSAR) study of the sixteen 2-substituted indole compounds, 5a-k, 1, 8-11, using partial least squares (PLS) and multiple regression analysis (MRA) revealed the existence of an optimal range of lipophilicity for the C2 indole substituent. There are also indications that planar, electron-withdrawing substituents contribute to the affinity by establishing additional interactions with the binding pocket. No mt1/MT2 subtype selectivity was observed, with the relevant exception of the 2-phenethyl derivative 5e, which exhibited the highest selectivity for the h-MT2 receptor among all the compounds tested (MT2/mt1 ratio of ca. 50). Conformational analysis and superposition of 5e to other reported selective MT2 ligands revealed structural and conformational similarities that might account for the MT2/mt1 selectivity of 5e.

A Novel One-Pot Approach of Hexahydropyrrolo[2,3-b]indole Nucleus by a cascade addition/cyclization strategy: Synthesis of (±)-Esermethole

A practical and efficient synthesis of 3-substituted hexahydropyrrolo[2,3-b]indole is described. The addition/cyclization of 3-substituted indoles with alpha,beta-dehydroamino esters in the presence of a Lewis acid provides hexahydropyrrolo[2,3-b]indole adducts in good yields and stereoselectivities. This approach has been applied to the concise synthesis of esermethole employing an appropriately substituted indole and an N-acyl dehydroamino ester.

Synthesis of (E)-8-(3-Chlorostyryl)caffeine Analogues Leading to 9-Deazaxanthine Derivatives as Dual A2A Antagonists/MAO-B Inhibitors

A systematic modification of the caffeinyl core and substituents of the reference compound (E)-8-(3-chlorostyryl)caffeine led to the 9-deazaxanthine derivative (E)-6-(4-chlorostyryl)-1,3,5,-trimethyl-1H-pyrrolo[3,2-d]pyrimidine-2,4-(3H,5H)-dione (17f), which acts as a dual human A(2a) antagonist/MAO-B inhibitor (K(i)(A(2A)) = 260 nM; IC(50)(MAO-B) = 200 nM; IC(50)(MAO-A) = 10 μM) and dose dependently counteracts haloperidol-induced catalepsy in mice from 30 mg/kg by the oral route. The compound is the best balanced A(2A) antagonist/MAO-B inhibitor reported to date, and it could be considered as a new lead in the field of anti-Parkinsons agents. A number of analogues of 17f were synthesized and qualitative SARs are discussed. Two analogues of 17f, namely 18b and 19a, inhibit MAO-B with IC(50) of 68 and 48 nM, respectively, being 5-7-fold more potent than the prototypical MAO-B inhibitor deprenyl (IC(50) = 334 nM).

Indole-based analogs of melatonin: in vitro antioxidant and cytoprotective activities

Abstract:  The known neuroprotective actions of melatonin could be due to its antioxidant or radical scavenging activity, or they could be due to specific interactions of the indole with its receptors. A study of structure–activity relationships may provide useful information when a validated macromolecular target has not been (or is not) identified. A set of indole derivatives, with changes in the 5‐methoxy and acylamino groups, the side chain position and the lipophilic/hydrophilic balance, were selected and tested for their in vitro antioxidant potency in the ABTS (2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid disodium salt) and thiobarbituric acid reactive substances (TBARS) assays and for their cytoprotective activity against kainate excitotoxicity on cerebellar cell cultures. No quantitative model was able to relate the potencies obtained in the two antioxidant assays, probably because they are related to different physico‐chemical properties. However, the lipophilicity of the compounds and the antioxidant potency in the TBARS assay were linearly correlated. This may be due to improved access to the lipidic substrate, where the antioxidant action occurs. In the cytoprotection assay, most compounds showed potencies comparable with or lower than melatonin. An exception was N‐[2‐(5‐methoxy‐1H‐indol‐2‐yl)ethyl]acetamide (12), yielding, at 50 μm, percentages of cell vitality higher than 75%, while melatonin EC50 was 333 μm. No correlation was observed between cytoprotective and antioxidant potencies, nor with MT1 or MT2 receptor affinity. Compound 12 is a low‐affinity antagonist at melatonin membrane receptors, and one of the most potent compounds in the antioxidant assays; its cytoprotective potency and the absence of agonist activity at melatonin membrane receptors make it a valid candidate for further investigations.

Synthesis, antioxidant activity and structure-activity relationships for a new series of 2-(N-acylaminoethyl)indoles with melatonin-like cytoprotective activity.

Abstract:  5‐Methoxy‐2‐(N‐acetylaminoethyl)indole (5d), a melatonin analogue derived from the transposition of the acetylaminoethyl side chain from C3 to C2 of the indole nucleus, had been previously characterized as a low affinity antagonist at MT1 and MT2 membrane receptors; this molecule is endowed with good in vitro antioxidant and cytoprotective potency in rat cerebellar cell cultures, comparable to or better than those of melatonin. In order to further investigate the role of structure–antioxidant activity relationships in cytoprotection, the structure of 5d was systematically modulated to design a new series of compounds. The 5‐methoxy group was replaced by substituents with different electronic and lipophilic properties and it was moved to a different position on the indole ring. Other modifications of the lead structure involved the methylation of the indole nitrogen or its replacement by a sulfur atom. The side chain was also modified either increasing its lipophilicity or introducing an ionisable acid group. The antioxidant activity of this set of compounds was evaluated by the ABTS and conjugated dienes (CD) assays, while their cytoprotection was evaluated against kainate‐induced cytotoxicity in cultured cerebellar neurons. In both antioxidant assays, the shift of the 5‐methoxy group to the 4‐position of the indole nucleus led to the most active radical scavenger (9), more potent than the parent compound and melatonin in the antioxidant tests, but much less effective as a cytoprotectant. Sharp structure–activity relationships were registered for cytoprotection, where the maintenance of the 5‐alkoxy‐2‐(N‐acylaminoethyl)indole scaffold appeared as the key feature to confer both antioxidant and cytoprotective activity to the structure. Some derivatives of the set, however, together with the most potent 5d, maintained a significant antioxidant and cytoprotective effect and could be employed as tools for in vivo pharmacological investigations on neuroprotective efficacy of melatonin‐related indoles.

Metastable ion studies in the characterization of melatonin isomers

The electron ionization mass spectrometric behaviour of melatonin (N-acetyl-5-methoxytryptamine) and of a series of its isomers has been studied with the aid of metastable ion experiments. The data show that the different positions of the substituents on the indole ring influence the fragmentation patterns and the relative abundances of the generated ions.

Antioxidant and cytoprotective activity of indole derivatives related to melatonin.

Melatonin (MLT) is known for its radical scavenger activity, which had been related to its ability to protect neuronal cells from different kinds of oxidative stress. In particular, MLT protects rat cerebellum granular cells from kainate-induced necrosis at concentrations higher than 100 microM, and is able to reduce lipoperoxidation induced by radical stress in rat brain homogenate at similar concentrations. On the other hand, MLT has nanomolar affinity for its membrane receptors (MT1 and MT2), and these are completely saturated at the high concentrations employed when the cytoprotective effect is observed. Other indole derivatives are also known to possess antioxidant and cytoprotective activity. In order to dissociate the cytoprotective effect of MLT from its receptor affinity, and to investigate the structure-activity relationships (SAR) between this effect and some potentially relevant chemical properties, we prepared a series of indole derivatives, where the structure of MLT was gradually modulated, varying the 5-methoxy group nature and position, the acylaminoethyl chain position, and by the introduction of lipophilic groups. These modifications resulted in a set of compounds having different receptor affinity and intrinsic activity, different lipophilicity, and different substitution at the indole nucleus. The compounds were tested for their antioxidant potency by the ABTS test and by inhibition of rat brain homogenate lipoperoxidation; their cytoprotective effect was also estimated from the inhibition of kainate-induced cellular death on rat cerebellum granular cells, and the results were evaluated by SAR comparison and QSAR analysis. An isomer of MLT resulted more potent and effective than MLT itself in the cytoprotection test, although it showed similar potency in the peroxidation test, and it was devoid of the ability to stimulate MT1 and MT2 receptors. This compound was selected as the lead compound for a further SAR study, devoted to the optimization of the cytoprotective effect and to the investigation on its mechanism.

Honey Flavonoids, Natural Antifungal Agents Against Candida Albicans

Candida albicans is the most prevalent fungal pathogen in humans. In this study, flavonoids from unprocessed multifloral honey were extracted and investigated their anticandidal activity in vitro. These results indicated that honey flavonoids inhibited Candida growth; however, they did not kill the yeasts and did not directly affect the cytoplasmic membrane. The viability tests of cells yeast with the fluorescent probe Sybr green I in combination with propidium iodide suggested that antifungal activity of honey flavonoids depended on their relative lipophilic properties and that they may reach a possible intracellular site of action without compromising membrane-associated functions.

Design and synthesis of melatonin receptors agonists and antagonists.

We review our work towards the design and synthesis of high-affinity melatonin (N-acetyl-5-methoxytryptamine) agonist and antagonist compounds. High affinity melatonergic agonists were obtained by shifting the melatonin side chain from C3 to N1 of the indole ring system. Conversely, by moving the side chain from C3 to C2 it was possible to obtain melatonin antagonist compounds, albeit of moderate affinity.