**4. Conclusions**

We investigated the antiparasitic potential of the marine sesquiterpene avarone (**1**), its reduced form avarol (**3**), and thiazoavarone (**2**), a novel semisynthetic thiazinoquinone analogue obtained through a condensation reaction of **1** with hypotaurine, which resulted completely regioselective**.** Both the natural metabolites **1** and **3**, as well as the semisynthetic derivative **2** resulted active against D10 and W2 strains and late stage gametocytes of *P. falciparum*, against larval and adult developmental stages, and eggs of the platyhelminth *S. mansoni*, and also against promastigote and amastigote forms of *L. infantum* and *L. tropica*. The observed differences in the magnitude of the effects of the three molecules allowed us to draw some interesting conclusions, strongly supported by the results of the DFT analysis. In particular, the thiazinoquinone **2** resulted as significantly more potent than the quinone derivative **1** against D10 and W2 strains of *P. falciparum* as well as on the larval stage of *S. mansoni*. On the other hand, compound **1**, although lacking the 1,1-dioxo-1,4-thiazine ring, resulted still active on *P. falciparum* and on *Schistosoma* larval stage, contrarily to what previously observed for other 1,4-benzoquinone derivatives when compared to the corresponding thiazinoquinone analogues [13,14]. The calculated redox properties of **1** and **2** evidenced for **2** either a higher tendency to acquire one electron and a higher stability of the QH• radical. Noteworthy, thiazoavarone **2** resulted the most potent antimalarial thiazinoquinone developed by us.

The hydroquinone **3** resulted more active than the corresponding quinone **1** against all considered parasites, with the highest selectivity index with respect to mammalian cells. This led to hypothesize the presence in the studied parasites of a bioactivation reaction partner preferentially binding the reduced form (**3**) rather than the oxidized form (**1**). This putative bioactivation partner seems not to be present in human cells. Finally, comparison of the standard redox potential (E◦) and of the ionization potential (IP) of the QH- anion of **3** with those of the corresponding reduced form of **2**, indicated a higher propensity of **3** to be oxidized to the semiquinone radical and, thus, that the absence of the 1,1-dioxo-1,4-thiazine ring favours the one-electron oxidation reaction of **3**.

These results confirm the key role played by the 1,1-dioxo-1,4-thiazine ring on the electron affinity [11,13,14] and, mainly, corroborate the hypothesis that the compound activity is related to the formation of a toxic semiquinone radical species, which can be produced, upon a one-electron transfer reaction, starting both from quinone- and hydroquinone-based compounds. In the case of the antiplasmodial activity (on the erythrocytic stage of the parasite), our previous results indicated that the bioactivation partner can be represented by free heme (generated during hemoglobin digestion) [11,13]. On the contrary, in the case of *S. mansoni* as well as *Leishmania* parasites, at this stage, we do not know the bioactivation partner of the compounds. However, reactive oxygen species (ROS) generation was found to be involved in the pro-apoptotic mechanism of some natural marine derived thiazinoquinones against Jurkat cells [66]. Thus, it cannot be ruled out that the putative semiquinone radical species reacts with oxygen molecules in cells, generating ROS and future studies will be devoted to clarifying this issue. Moreover, the observed difference between the activity trend of **1**–**3** against the different parasites and their developmental stage may be related to morphological and/or metabolic differences in the targeted organism/stage. Thus, the selective toxicity against the different parasites and their developmental stages can be addressed, taking advantage of the possible bioactivation reaction partners to form the putative toxic radical (i.e., one-electron reduction or oxidation reaction).

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1660-3397/18/2/112/s1, Figure S1. HRESIMS spectrum of thiazoavarone (**2**), Figure S2. 1H NMR spectrum of thiazoavarone (**2**) in CDCl3, Figure S3. 1H-1H COSY spectrum of thiazoavarone (**2**) in CDCl3, Figure S4. HSQC spectrum of thiazoavarone (**2**) in CDCl3, Figure S5. HMBC spectrum of thiazoavarone (**2**) in CDCl3, Figure S6. HRESIMS spectrum of avarone (**1**), Figure S7. 1H-NMR spectrum of avarone (**1**) in CDCl3, Figure S8. HRESIMS spectrum of avarol (**3**), Figure S9. 1H-NMR spectrum of avarol (**3**) in CDCl3, Figure S10. Conformational enantiomers of thiazoavarone (**2**), Figure S11. Calculated pka values of avarol (**3**) (ACD/Percepta software), Figure S12. DFT conformer of **1**-**3**, and their radical species, Table S1. ΔEGM values and torsion angle values of the DFT conformers of avarol (**3**), Table S2. Hydrogen atoms suitable for an intramolecular radical shift, Table S3. cLogD values of **1**–**3**, Scheme S1. Reduction pathway of quinones in a protic solvent.

**Author Contributions:** Conceptualization, C.I., C.F. and M.M.; data curation, C.I., R.G., M.P., M.C., A.G., F.S., G.R., P.L., A.A., C.F. and M.M.; formal analysis, C.I., R.G., M.P., M.C., A.G., F.S., G.R., S.P. and S.A.; funding acquisition, G.R., C.F. and M.M.; investigation, C.I., R.G., M.P., M.C., A.G. and F.S.; methodology, C.I., R.G., M.P., M.C., G.R., S.P., N.B., C.F. and M.M.; resources, S.A.; writing—original draft, C.I., R.G., M.P., M.C., G.R., N.B., C.F. and M.M.; writing—review and editing, C.I., R.G., M.P., M.C., A.G., F.S., G.R., P.L., A.A., S.P., S.A., N.B., C.F. and M.M. All authors have read and agree to the published version of the manuscript.

**Funding:** This research was supported by Ministero dell'Istruzione, dell'Università e della Ricerca (MIUR), PRIN Projects 2010C2LKKJ\_006; 20154JRJPP\_004, by a gran<sup>t</sup> 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).

**Acknowledgments:** We wish to thank Asli Kacar and Burcu Omuzbuken for sample collection, and Arturo Facente for identifying the organism.

**Conflicts of Interest:** The authors declare no conflict of interest.
