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Natural compounds are characterized by a considerable chemical and structural diversity alongside to be considered as the most valuable and prolific source of new drugs up to now. Not surprisingly, nature remains a key inspirational factor in the search for new therapeutic agents. In this frame, marine environment holds a central role furnishing several secondary metabolites which possess peculiar functionality groups on their backbone and a wide ranging of biological activities. Despite this promising potential, the main disadvantage of marine-derived compounds is represented by their limited availability. Therefore, always more frequently marine metabolites are useful not only as effective drugs but also as inspiration for countless synthetic drugs. During the PhD activity, the whole of my work was managed in two different topics: a) Structural and pharmacological characterization of novel secondary metabolites isolated from marine organisms. This task implied the extensive application of most spectroscopic (mainly NMR) and spectrometric techniques, often assisted by computational studies, as well as the development of efficient method for stereochemical assignment, including chemical derivatization and synthesis. The whole of these studies led to the isolation of two novel alkaloids, polyaurine A and B, from the Caribbean ascidian Polycarpa aurata featuring unusual structural moieties. Polyaurine A with its peculiar N-methylguanidine group resulted in the impairment of the development of eggs-laid by Schistosoma mansoni. The chemical investigation of the sesquiterpenoid quinones/hydroquinones-rich sponge Dysidea avara afforded the isolation of avarone and avarol. According to the substantiated redox properties of different quinones against Plasmodium and the similarities in physio-pathological processes of that protozoon and the blood fluke Schistosoma, these derivatives were evaluated against malaria and some neglected tropical diseases (i.e. schistosomiasis and leishmaniasis) since there is an urgent need of new therapeutic alternatives to keep up with the widespread parasite resistance. Moreover, my PhD activity was also focused in completing the configurational assignment of phosphoeleganin, an acyclic phosphorylated polyketide with inhibitory activity against protein tyrosine phosphatase 1B. The univocal stereochemical elucidation required the synthesis of the 8,9-anti stereoisomers of the tetradecan-5,8,9-triol as diastereoisomeric model compounds directed to the application of UDB concept. Accordingly, the widening of NMR database as useful support for stereostructural elucidation has encouraged the synthesis of several simplified analogues of the marine metabolite. b) Design and synthesis of quinone molecules inspired by bioactive marine natural products for antiparasitic and cytotoxicity screenings. Taking into account the antiplasmodial effects of some marine compounds endowed with the thiazinoquinone bicyclic moiety and also the antischistosomal properties of several effective antimalarials, a rationalized chemical library of thiazinoquinones has been synthesised inspired by the marine cytotoxic aplidinone A. The adopted synthetic scheme reflected the compelling priority of an efficient and inexpensive procedure for neglected diseases. Some requirements appeared crucial for the antiparasitic activity: a specific regiochemistry of the thiazinoquinone system strongly affected the antiparasitic potency whereas the nature of the side-chain substituents on the quinone moiety could address the selective toxicity against mammalian cells or specific developmental stages of parasites. Computational studies provided useful insights on putative mechanism of action exerted by thiazinoquinones supporting the one-electron reduction bioactivation step with subsequent formation of a toxic semiquinone radical species. Overall these data clearly point out the thiazinoquinone scaffold as potential new lead structure for neglected disease drugs discovery. To further exploit the versatility of the synthetic protocol, a small series of prenylated quinones and related thiazinoquinones has been prepared in order to broaden the knowledge on the antiproliferative effects of these compounds. Among these, a geranylquinone derivative exerted a cytostatic activity through G0/G1 cell-cycle arrest in BxPC-3 cells.

Type 2 diabetes mellitus (T2DM) is a complex disease characterized by impaired glucose homeostasis and serious long-term complications. First-line therapeutic options for T2DM treatment are monodrug therapies, often replaced by multidrug therapies to ensure that non-responding patients maintain target glycemia levels. The use of multitarget drugs instead of mono- or multidrug therapies has been emerging as a main strategy to treat multifactorial diseases, including T2DM. Therefore, modern drug discovery in its early stages aims to identify potential modulators for multiple targets; for this purpose, exploration of the chemical space of natural products represents a powerful tool. Our study demonstrates that avarone, a sesquiterpene quinone obtained from the sponge Dysidea avara, is capable of inhibiting in vitro PTP1B, the main negative regulator of the insulin receptor, while it improves insulin sensitivity, and mitochondria activity in C2C12 cells. We observe that when avarone is administered alone, it acts as an insulin-mimetic agent. In addition, we show that avarone acts as a tight binding inhibitor of aldose reductase (AKR1B1), the enzyme involved in the development of diabetic complications. Overall, avarone could be proposed as a novel natural hit to be developed as a multitarget drug for diabetes and its pathological complications.

Diabetes mellitus (DM) represents a complex and multifactorial disease that causes metabolic disorders with acute and long-term serious complications. The onset of DM, with over 90% of cases of diabetes classified as type 2, implies several metabolic dysfunctions leading to consider DM a worldwide health problem. In this frame, protein tyrosine phosphatase 1B (PTP1B) and aldose reductase (AR) are two emerging targets involved in the development of type 2 diabetes mellitus (T2DM) and its chronic complications. Herein, we employed a marine-derived dual type inhibitor of these enzymes, phosphoeleganin, as chemical starting point to perform a fragment-based process in search for new inhibitors. Phosphoeleganin was both disassembled by its oxidative cleavage and used as model structure for the synthesis of a small library of functionalized derivatives as rationally designed analogues. Pharmacological screening supported by in silico docking analysis outlined the mechanism of action against PTP1B exerted by a phosphorylated fragment and a synthetic simplified analogue, which represent the most potent inhibitors in the library.

Indole moiety is well-known as a superlative framework in many natural products and synthetic pharmaceuticals. Herein, we report an efficient procedure to synthesize a series of functionalized 2-methyl-1H-indole-3-carboxylate derivatives from commercially available anilines properly functionalized by different electron-withdrawing and -donating groups through a palladium-catalyzed intramolecular oxidative coupling. The conversion of a variety of enamines into the relevant indole was optimized by exposing the neat mixture of reactants to microwave irradiation, obtaining the desired products in excellent yields and high regioselectivity. The synthesized compounds were confirmed by 1H and 13C spectroscopic means as well as by high-resolution mass spectrometry.

The chemical investigation of the Mediterranean ascidian Clavelina lepadiformis has led to the isolation of a new lepadin, named lepadin L, and two known metabolites belonging to the same family, lepadins A and B. The planar structure and relative configuration of the decahydroquinoline ring of lepadin L were established both by means of HR-ESIMS and by a detailed as extensive analysis of 1D and 2D NMR spectra. Moreover, microscale derivatization of the new alkaloid lepadin L was performed to assess the relative configuration of the functionalized alkyl side chain. Lepadins A, B, and L were tested for their cytotoxic activity on a panel of cancer cell lines (human melanoma [A375], human breast [MDA-MB-468], human colon adenocarcinoma [HT29], human colorectal carcinoma [HCT116], and mouse myoblast [C2C12]). Interestingly, a deeper investigation into the mechanism of action of the most cytotoxic metabolite, lepadin A, on the A375 cells has highlighted its ability to induce a strongly inhibition of cell migration, G2/M phase cell cycle arrest and a dose-dependent decrease of cell clonogenity, suggesting that it is able to impair self-renewing capacity of A375 cells.

An in-depth study on the inhibitory mechanism on protein tyrosine phosphatase 1B (PTP1B) and aldose reductase (AR) enzymes, including analysis of the insulin signalling pathway, of phosphoeleganin, a marine-derived phosphorylated polyketide, was achieved. Phosphoeleganin was demonstrated to inhibit both enzymes, acting respectively as a pure non-competitive inhibitor of PTP1B and a mixed-type inhibitor of AR. In addition, in silico docking analyses to evaluate the interaction mode of phosphoeleganin with both enzymes were performed. Interestingly, this study showed that phosphoeleganin is the first example of a dual inhibitor polyketide extracted from a marine invertebrate, and it could be used as a versatile scaffold structure for the synthesis of new designed multiple ligands.

The synthesis of two 5&prime, end (4-dimethylamino)azobenzene conjugated G-quadruplex forming aptamers, the thrombin binding aptamer (TBA) and the HIV-1 integrase aptamer (T30695), was performed. Their structural behavior was investigated by means of UV, CD, fluorescence spectroscopy, and gel electrophoresis techniques in K+-containing buffers and water-ethanol blends. Particularly, we observed that the presence of the 5&prime, (4-dimethylamino)azobenzene moiety leads TBA to form multimers instead of the typical monomolecular chair-like G-quadruplex and almost hampers T30695 G-quadruplex monomers to dimerize. Fluorescence studies evidenced that both the conjugated G-quadruplexes possess unique fluorescence features when excited at wavelengths corresponding to the UV absorption of the conjugated moiety. Furthermore, a preliminary investigation of the trans-cis conversion of the dye incorporated at the 5&prime, end of TBA and T30695 showed that, unlike the free dye, in K+-containing water-ethanol-triethylamine blend the trans-to-cis conversion was almost undetectable by means of a standard UV spectrophotometer.

The identification of molecules whose biological activity can be properly modulated by light is a promising therapeutic approach aimed to improve drug selectivity and efficacy on the molecular target and to limit the side effects compared to traditional drugs. Recently, two photo-switchable diastereomeric benzodiazopyrrole derivatives 1RR and 1RS have been reported as microtubules targeting agents (MTAs) on human colorectal carcinoma p53 null cell line (HCT 116 p53-/-). Their IC50 was enhanced upon Light Emitting Diode (LED) irradiation at 435&thinsp, nm and was related to their cis form. Here we have investigated the photo-responsive behavior of the acid derivatives of 1RR and 1RS, namely, d1RR and d1RS, in phosphate buffer solutions at different pH. The comparison of the UV spectra, acquired before and after LED irradiation, indicated that the trans&rarr, cis conversion of d1RR and d1RS is affected by the degree of ionization. The apparent rate constants were calculated from the kinetic data by means of fast UV spectroscopy and the conformers of the putative ionic species present in solution (pH range: 5.7&ndash, 8.0) were modelled. Taken together, our experimental and theoretical results suggest that the photo-conversions of trans d1RR/d1RS into the corresponding cis forms and the thermal decay of cis d1RR/d1RS are dependent on the presence of diazonium form of d1RR/d1RS. Finally, a photo-reaction was detected only for d1RR after prolonged LED irradiation in acidic medium, and the resulting product was characterized by means of Liquid Chromatography coupled to High resolution Mass Spectrometry (LC-HRMS) and Nuclear Magnetic Resonance (NMR) spectroscopy.

Neglected tropical diseases (NTDs), caused by several parasitic agents (Plasmodium spp., Schistosoma spp., Leishmania spp.), are still characterized by a high morbidity percentage worldwide. The therapeutic alternatives for neglected pathologies are very limited, first due to the drug-resistance phenomena but also due to unfavourable toxicity profiles and the difficult administration procedures of the few available chemical entities. A further drawback is represented by multiparasitism, namely the concurrent infestation of a single host by more than one parasite species, which poses additional diagnostic and therapeutic challenges [1]. In this view, a research aimed to discover a new chemical entity active against several parasites is crucial and the marine environment may be important resource [2]. As part of our own continuing search for new leads in the treatment of NTDs [3–5] we have explored the anti infective properties of the sponge derived metabolites avarone and avarol, as well as those of the semisynthetic thiazoavarone, obtained by converting the quinone avarone in the relevant thiazinoquinone. All compounds were shown to be active against the D10 and W2 strains of P. falciparum, the juvenile and adult forms of the worm S. mansoni, and both the promastigote and amastigote stages of L. infantum and L. tropica.

WOS: 000518664600043 PubMed ID: 32075136 The chemical analysis of the sponge Dysidea avara afforded the known sesquiterpene quinone avarone, along with its reduced form avarol. To further explore the role of the thiazinoquinone scaffold as an antiplasmodial, antileishmanial and antischistosomal agent, we converted the quinone avarone into the thiazinoquinone derivative thiazoavarone. The semisynthetic compound, as well as the natural metabolites avarone and avarol, were pharmacologically investigated in order to assess their antiparasitic properties against sexual and asexual stages of Plasmodium falciparum, larval and adult developmental stages of Schistosomamansoni (eggs included), and also against promastigotes and amastigotes of Leishmania infantum and Leishmania tropica. Furthermore, in depth computational studies including density functional theory (DFT) calculations were performed. A toxic semiquinone radical species which can be produced starting both from quinone- and hydroquinone-based compounds could mediate the anti-parasitic effects of the tested compounds. Ministero dell'Istruzione, dell'Universita e della Ricerca (MIUR), PRIN Projects [2010C2LKKJ_006, 20154JRJPP_004]; Regione Campania-POR Campania FESR 2014/2020 "Combattere la resistenza tumorale: piattaforma integrata multidisciplinare per un approccio tecnologico innovativo alle oncoterapie-Campania Oncoterapie" [B61G18000470007]; CNR (National Research Council)-CNCCS (Collezione Nazionale di Composti Chimici e Centro di screening) "Rare, Neglected and Poverty Related Diseases -Schistodiscovery Project" [DSB.AD011.001.003] This research was supported by Ministero dell'Istruzione, dell'Universita e della Ricerca (MIUR), PRIN Projects 2010C2LKKJ_006; 20154JRJPP_004, by a grant from Regione Campania-POR Campania FESR 2014/2020 "Combattere la resistenza tumorale: piattaforma integrata multidisciplinare per un approccio tecnologico innovativo alle oncoterapie-Campania Oncoterapie" (Project N. B61G18000470007) and by the CNR (National Research Council)-CNCCS (Collezione Nazionale di Composti Chimici e Centro di screening) "Rare, Neglected and Poverty Related Diseases -Schistodiscovery Project" (DSB.AD011.001.003).

Schistosomiasis is the most significant neglected tropical parasitic disease caused by helminths in terms of morbidity and mortality caused by helminths. In this work, we present the antischistosomal activity against Schistosoma mansoni of a rationally selected small set of thiazinoquinone derivatives, some of which were previously found to be active against Plasmodium falciparum and others synthesized ad hoc. The effects on larvae, juvenile, and adult parasite viability as well as on egg production and development were investigated, resulting in the identification of new multistage antischistosomal hit compounds. The most promising compounds 6, 8, 13, and 14 with a LC50 value on schistosomula from ∼5 to ∼15 μM also induced complete death of juvenile (28 days old) and adult worm pairs (7 weeks old) and a detrimental effect on egg production and development in vitro. Structure-activity relationships (SARs) were analyzed by means of computational studies leading to the hypothesis of a redox-based mechanism of action with a one-electron reduction bioactivation step and the subsequent formation of a toxic semiquinone species, similarly to what was previously observed for the antiplasmodial activity. Our results also evidenced that the selective toxicity against mammalian cells or parasites as well as specific developmental stages of a parasite can be addressed by varying the nature of the introduced substituents.

Several marine natural linear prenylquinones/hydroquinones have been identified as anticancer and antimutagenic agents. Structure-activity relationship studies on natural compounds and their synthetic analogs demonstrated that these effects depend on the length of the prenyl side chain and on the type and position of the substituent groups in the quinone moiety. Aiming to broaden the knowledge of the underlying mechanism of the antiproliferative effect of these prenylated compounds, herein we report the synthesis of two quinones 4 and 5 and of their corresponding dioxothiazine fused quinones 6 and 7 inspired to the marine natural product aplidinone A (1), a geranylquinone featuring the 1,1-dioxo-1,4-thiazine ring isolated from the ascidian Aplidium conicum. The potential effects on viability and proliferation in three different human cancer cell lines, breast adenocarcinoma (MCF-7), pancreas adenocarcinoma (Bx-PC3) and bone osteosarcoma (MG-63), were investigated. The methoxylated geranylquinone 5 exerted the highest antiproliferative effect exhibiting a comparable toxicity in all three cell lines analyzed. Interestingly, a deeper investigation has highlighted a cytostatic effect of quinone 5 referable to a G0/G1 cell-cycle arrest in BxPC-3 cells after 24 h treatment.

In the maritime industry, biofouling is a severe problem and represents a serious matter of economic losses worldwide. The sizable investments are made for the prevention of biofouling by marine related sectors, especially in the paint industries. It is known that antifouling applications contain heavy metal and biocides which are hazardous for the marine environment and, for this reason, new studies are focusing on the development of environmentally and friendly paints. The main target of our investigation was the identification of natural marine compounds from tunicate and sponge species, evaluating their capability to inhibit biofilm progress which is the first step of fouling process, and their plausible usage in industrial products, too. Therefore, in this study, the antibiofilm effects of four marine invertebrates, Styela plicata, Clavelina lepadiformis, Dysidea avara and Spirastrella sp., collected in different seasons along the coasts of Izmir Bay, were evaluated. The extracts from two tunicates (Styela plicata and Clavelina lepadiformis) and a sponge (Dysidea avara) were selected due to their interesting antibiofilm capacities. A bioactive-guided isolation method allowed us to identification of the active fractions which were tested for inhibition of bacterial adhesion at different concentration. The content of the active fractions was identified by NMR spectroscopy, LC-MS and HRMS analysis.

A small library of antiplasmodial methoxy-thiazinoquinones, rationally designed on the model of the previously identified hit 1, has been prepared by a simple and inexpensive procedure. The synthetic derivatives have been subjected to in vitro pharmacological screening, including antiplasmodial and toxicity assays. These studies afforded a new lead candidate, compound 9, endowed with higher antiplasmodial potency compared to 1, a good selectivity index when tested against a panel of mammalian cells, no toxicity against RBCs, a synergistic antiplasmodial action in combination with dihydroartemisinin, and a promising inhibitory activity on stage V gametocyte growth. Computational studies provided useful insights into the structural requirements needed for the antiplasmodial activity of thiazinoquinone compounds and on their putative mechanism of action.

Among the sub-phylum of Tunicate, ascidians represent the most abundant class of marine invertebrates, with 3000 species by heterogeneous habitat, that is, from shallow water to deep sea, already reported. The chemistry of these sessile filter-feeding organisms is an attractive reservoir of varied and peculiar bioactive compounds. Most secondary metabolites isolated from ascidians stand out for their potential as putative therapeutic agents in the treatment of several illnesses like microbial infections. In this review, we present and discuss the antibacterial activity shown by the main groups of ascidian-derived products, such as sulfur-containing compounds, meroterpenes, alkaloids, peptides, furanones, and their derivatives. Moreover, the direct evidence of a symbiotic association between marine ascidians and microorganisms shed light on the real producers of many extremely potent marine natural compounds. Hence, we also report the antibacterial potential, joined to antifungal and antiviral activity, of metabolites isolated from ascidian-associate microorganisms by culture-dependent methods.

The full absolute configuration assignment of phosphoeleganin (1), a recently discovered marine-derived phosphorylated polyketide with protein tyrosine phosphatase 1B inhibitory activity, was achieved. It was based on the synthesis of model diasteroisomeric compounds of the C-8–C-12 segment portion of phosphoeleganin, chiral derivatization methods, and application of the universal NMR database concept.

A deep study of the metabolic content of the tunicate Polycarpa aurata, collected from Indonesian coast, afforded the isolation of two novel alkaloids, polyaurines A (1) and B (2), along with two new p-substituted benzoyl derivatives (3 and 4) and four known compounds (5&ndash, 8). The structural elucidation of the new secondary metabolites was assigned by 1D, 2D NMR, and HRESIMS techniques. Computational studies resulted a useful tool to unambiguously determine in polyaurine B the presence of rarely found 1,2,4-thiadiazole ring. The effects of polyaurines A and B on mammalian cells growth and on the viability of different blood-dwelling Schistosoma mansoni (phylum: Platyhelminthes) stages, as well as egg production, were evaluated. Both compounds resulted not cytotoxic, interestingly some of the eggs produced by polyaurine A-treated adult pairs in vitro are smaller, deformed, and/or fragmented, therefore, polyaurine A could represent an interesting bioactive natural molecule to be further investigated.