**1. Introduction**

Malaria and neglected tropical diseases (NTDs), a group of parasitic, bacterial, and viral infectious diseases (i.e., *Schistosoma* spp., *Leishmania* spp.), still have high morbidity and/or mortality rates worldwide. They a ffect more than one billion people and cause chronic illness, physical disability and/or deaths, especially in children and women of childbearing age, mostly in developing countries where they represent a serious hurdle to social and economic growth as well as a health problem [1,2]. Parasites belonging to di fferent species can a ffect humans and animals concurrently, a phenomenon referred to as multiparasitism, which poses additional diagnostic and therapeutic challenges [3]. The therapeutic strategies for malaria and NTDs are very limited; drug-resistance phenomena, toxicity profiles and drug administration procedures of the few available chemical entities are still challenging. In this view, a research aimed to discover new chemicals active against several parasites is crucial and the marine environment may be an important resource [4,5]. In order to cope with all the reported drawbacks and to limit the costs of the development of brand-new pharmaceutical strategies, several effective antimalarial drugs should be considered for the treatment of other underfunded parasitic diseases. For example, artemisinin and its derivatives, a potent class of antimalarial agents, have been proved to be beneficial for other infectious diseases such as schistosomiasis and leishmaniasis [6]. Furthermore, histone deacetylases (HDAC) inhibitors have been shown to have activity both against some *Plasmodium* species as well as *Leishmania* and *Schistosoma* parasites [7]. Importantly, the blood parasites, *Plasmodium* and *Schistosoma*, both feeding on human hemoglobin, can detoxify the free heme groups through the synthesis of insoluble hemozoin pigments [8]. The interference with hemozoin formation featured an important antischistosomal mechanism of action showed by the antimalarial quinine and quinidine [9].

In the frame of our research for new anti-parasitic chemical entities, we recently identified the thiazinoquinone sca ffold as a novel chemotype active against both *Plasmodium falciparum* and *Schistosoma mansoni* [10–14]. We developed this sca ffold by creating of a chemical library of thiazinoquinone derivatives designed on the model of aplidinones, natural products isolated from a marine invertebrate (Figure 1). Many compounds exhibited in vitro antiplasmodial activities against the D10 and W2 strains of *P. falciparum* [11,13] with IC50 in the low micromolar range. Through an integrated experimental (cyclic voltammetry) and theoretical approach, we demonstrated that the antiplasmodial and anticancer activity of a series of thiazinoquinone compounds was not related to their two electrons redox potential [11,12]. In particular, the antiplasmodial activity was found to depend on the ability of the compound to generate a semiquinone radical species able to form a stable adduct with heme [11]. This was later supported by the design and synthesis of other sets of new thiazinoquinone derivatives, indicating that the activity was related to the ability to form a specific semiquinone radical, and to the ability of this latter to transfer the radical by and hydrogen-radical shift to the R substituent [13]. In addition, several important SARs were obtained. First, the thiazinoquinone moiety was ascertained to be necessary for the antiplasmodial activity, since the corresponding quinone derivatives (e.g., derivatives lacking the 1,1-dioxothiazine moiety) were inactive. Second, the regiochemistry of the heterocyclic ring with respect to the substituents (a methoxyl group and an alkyl chain) on the quinone ring was revealed as crucial for the activity. Third, the nature and shape of the R- substituent were able to a ffect compound potency and selectivity.

**Figure 1.** Structures of aplidinones A, B and of thiazinoquinone derivatives.

Successively, we selected both some of the developed methoxy thiazinoquinones, and some ad hoc synthesized new derivatives with the aim of investigating the antischistosomal properties of this chemical scaffold. Compounds were thus tested against larval stage, adult worm couples and eggs of the platyhelminth *S. mansoni* [14]. Many of the tested molecules resulted active and, interestingly, as observed for the antiplasmodial activity, the effects against *S. mansoni* strongly depended on the regiochemistry of the heterocyclic ring, and from the nature and/or steric hindrance of the R- substituent. Computational studies indicated that semiquinone radical species could be involved also in the mode of action against *S. mansoni* impairing the redox equilibrium within the parasite. Importantly, the R- properties can affect both the pharmacodynamics and pharmacokinetics of the compounds [14].

In the course of a systematic chemical study of the macroflora and macrofauna of the coastal area of Turkey in the ˙ Izmir Bay (Aegean Sea), as a part of our ongoing search for bioactive marine-derived metabolites as leads for drug discovery [15–20], we isolated from the sponge *Dysidea avara* (Schmidt, 1862) the known sesquiterpene quinone avarone (**1**), along with its reduced form avarol (**3**, Figure 2) [21–23]. A wide range of pharmacological properties have been reported for the redox couple avarone (**1**) and avarol (**3**) including anti-tumor [24–26], anti-inflammatory [27–29], anti-mutagenic [30], anti-bacterial [31,32], anti-viral [33,34], anti-oxidant [23,35], anti-platelet [28], anti-psoriatic [36] and anti-biofouling [37,38] activities. Pharmacological studies on synthetic and semisynthetic derivatives of avarone have been previously reported, too [23,39–41]. Based on the above described extensive exploration of the thiazinoquinone scaffold as antiplasmodial and antischistosomal agen<sup>t</sup> [11,13,14], we used the quinone avarone (**1**) as chemical starting point to obtain the semisynthetic thiazinoquinone derivative, thiazoavarone (**2**, Figure 2). Compound **2**, as well as the natural metabolites **1** and **3**, were investigated in order to evaluate their in vitro activity against: (i) asexual stages of D10 and W2 strains and stage V gametocytes of *P. falciparum*, (ii) egg production, larval and adult stages of *S. mansoni*, (iii) promastigote and amastigote stages of *Leishmania infantum* and *Leishmania tropica*. Computational studies, including density functional theory (DFT) calculations, were performed in order to analyze the conformational and redox properties of the two natural metabolites (**1** and **3**), as well as those of the novel semisynthetic analogue **2**.

The obtained results shed light on the putative mechanism of action of the quinone/hydroquinone/thiazinoquinone compounds corroborating the hypothesis that their antiparasitic activity is related to the formation of a toxic semiquinone radical species. Noteworthy, thiazoavarone **2** resulted the most potent antimalarial thiazinoquinone developed by us, highlighting the important role for the activity played by the substituent of the 1,1-dioxo-1,4-thiazine ring.

**Figure 2.** Structure of avarone (**1**), the semisynthetic thiazoavarone (**2**) and avarol (**3**).

### **2. Results and Discussion**
