*3.1. General Methods*

Solvents: Carlo Erba (Pomezia, Rome, Italy). Commercial reagents: Sigma–Aldrich (Saint Louis, MO, USA). TLC: Silica Gel 60 F254, plates 5 × 20, 0.25 mm, Merck (Kenilworth, NJ, USA). Anhydrous solvents: Sigma-Aldrich-Merck. High-resolution ESI-MS analyses were performed on a Thermo LTQ Orbitrap XL mass spectrometer (Thermo-Fisher, San Josè, CA, USA). The spectra were recorded by infusion into the ESI (Thermo-Fisher, San Josè, CA, USA) source dissolving the sample in MeOH. 1H (500 MHz) and 13C (125 MHz) NMR spectra were recorded on an Agilent INOVA spectrometer (Agilent Technology, Cernusco sul Naviglio, Italy) equipped with a 13C enhanced HCN Cold Probe; chemical shifts were referenced to the residual solvent signal (CDCl3: δH = 7.26, δC = 77.0). For an accurate measurement of the coupling constants, the one-dimensional 1H NMR spectra were transformed at 64 K points (digital resolution: 0.09 Hz). Homonuclear (1H–1H) and heteronuclear (1H–13C) connectivities were determined by COSY and HSQC experiments, respectively. Two and three bond 1H-13C connectivities were determined by gradient 2D HMBC experiments optimized for a 2,3*J* of 8 Hz. <sup>3</sup>*J*H–H values were extracted from 1D 1H NMR. High performance liquid chromatography (HPLC) separations were achieved on a Shimadzu LC-10AT (Shimadzu, Milan, Italy) apparatus equipped with a Knauer K-2301 (LabService Analytica s.r.l., Anzola dell'Emilia, Italy) refractive index.

### *3.2. Collection, Extraction and Isolation*

Several fresh specimens of *D. avara* were collected along the coast of Narlidere, Bay of Izmir (Turkey, 38◦24-45 N 27◦8-18 E), in summer of 2017 and immediately frozen and stored at −25 ◦C until the use. The identification of fresh material was performed by Mr. Arturo Facente while a voucher specimen is deposited at Mugla University, Turkey.

The freshly thawed sponge (21.9 g dry weight after extraction) was homogenized and treated at room temperature with methanol (3 × 1 L) and, subsequently, with dichloromethane (3 × 1 L). The combined extracts were concentrated in vacuo to give an aqueous suspension that was subsequently extracted with BuOH. The butanol soluble material (5.4 g of a dark brown oil), obtained after evaporation of the solvent, was chromatographed on a RP-18 silica gel flash column using a gradient elution (water→methanol→chloroform). The fractions eluted with H2O/MeOH 2:8 (*v*/*v*) were chromatographed by HPLC on an RP-18 column (Luna, 3 μm C-18, 150 × 3.00 mm), using MeOH/H2O 95:5 as the eluent (flow 0.5 mL/min). This separation afforded 4.5 mg of pure avarol (**3**, *t*R = 5.4 min) and 26.8 mg of avarone (**1**, *t*R = 9.4 min), identified by comparison of its spectral properties with literature values [21–23].

Avarone (**1**): yellow powder; [α]D<sup>25</sup> = +2.6 (*c* 0.0014, CH3OH); 1H NMR (CDCl3) spectrum is reported in Supplementary Materials (Figure S6); HRMS (ESI): *m*/*z* 313.2156 [M + H]+ (calcd. for C21H29O2: 313.2162) (Figure S7).

*Synthesis of thiazoavarone (2)*. 20.3 mg of avarone (**1**, 0.065 mmol) were dissolved in 12 mL of a mixture of CH3CN/EtOH 1: 1 (v/v) and kept under stirring at room temperature; then, a solution of hypotaurine (7 mg, 0.065 mmol) in 2 mL of water was added dropwise together with a catalytic amount of salcomine added in portions. The mixture was stirred for 48 h at room temperature before removing the most of ethanol in vacuo and pouring the residue into water. The orange/yellow mixture was extracted with diethyl ether (60 mL × three times) and the organic phase was washed with brine, dried over sodium sulfate, filtered, and solvent was removed by rotary evaporator. The resulting mixture was chromatographed by HPLC on an RP-18 column (Luna, 3 μm C-18, 150 × 3.00 mm) eluting with MeOH/H2O 75:25 (*<sup>t</sup>*R = 31.6 min) and afforded the pure compound **2** (11 mg, 46%).

Thiazoavarone (**2**): orange powder; [α]D<sup>25</sup> = +19.2 (*c* 0.0035, CDCl3); 1H and 13C NMR data are reported in Table 1. 1D and 2D NMR data, Figures S2–S5; HRMS (ESI): m/z 440.1865 [M + Na]+ (calcd. for C23H31NO4SNa: 440.1866) (Figure S1).

Avarol (**3**): yellow powder; [α]D<sup>25</sup> = +12.4 (*c* 0.0018, CH3OH); 1H NMR (CDCl3) spectrum is reported in Supplementary Materials (Figure S8); HRMS (ESI): *m*/*z* 315.2337 [M + H]+ (calcd. for C21H31O2: 315.2319) (Figure S9).

### *3.3. P. falciparum Cultures and Drug Susceptibility Assay*

*P. falciparum* cultures were carried out according to Trager and Jensen with slight modifications [13]. The CQ-sensitive strain D10 and the CQ-resistant strain W2 were maintained at 5% hematocrit (human type A-positive red blood cells) in RPMI 1640 (EuroClone, Celbio) medium with the addition of 1% AlbuMax (Invitrogen, Milan, Italy), 0.01% hypoxanthine, 20 mM Hepes, and 2 mM glutamine at 37 ◦C in a standard gas mixture (1% O2, 5% CO2, and 94% N2). All compounds were dissolved in DMSO and then diluted with medium to achieve the required concentrations (final DMSO concentration <1%, non-toxic to the parasite). Drugs were placed in 96-well flat-bottomed microplates and serial dilutions made. Asynchronous cultures with parasitaemia of 1%–1.5% and 1% final hematocrit were aliquoted into the plates and incubated for 72 h at 37 ◦C. Parasite growth was determined spectrophotometrically (OD650) by measuring the activity of the parasite lactate dehydrogenase (pLDH), according to a modified version of the method of Makler in control and drug-treated cultures [13]. The antiplasmodial activity is expressed as 50% inhibitory concentrations (IC50); each IC50 value is the mean ± standard deviation of at least three separate experiments performed in duplicate.

### *3.4. Gametocytes Cultivation and Susceptibility Assay*

The transgenic *P. falciparum* 3D7 strain 3D7elo1-pfs16-CBG99 expressing the *Pyrophorus plagiophthalamus* CBG99 luciferase under a gametocyte specific promoter was used in all the experiments. Parasites were cultured and gametocytes obtained as previously described [54]. Late-stage gametocytes were exposed to compounds at day 11 after *N*-acetylglucosamine (NAG) addition. Gametocytes stages were counted in Giemsa stained smears and the percentage of stage V gametocytes was higher than 80%. Compounds were prepared by serial dilution, in 96-well plate, in complete medium. Plates were incubated for 72 h at 37 ◦C under 1% O2, 5% CO2, 94% N2 atmosphere. Luciferase activity was taken as measure of gametocytes viability, as previously described [55]. Briefly, drug-treated gametocyte samples at 2% haematocrit were transferred to 96-well black microplates and d-luciferin (1 mM in citrate buffer 0.1 M, pH 5.5) was added at a 1:1 volume ratio. Luminescence measurements were performed after 10 min with 500 ms integration time using a Sinergy 4 (Biotek) microplate reader. The IC50 was extrapolated from the non-linear regression analysis of the concentration–response curve.

### *3.5. In Vitro Promastigote Susceptibility Assays*

Promastigote stage of *L. infantum* strainMHOM/TN/80/IPT1 and *L. tropica* (MHOM/IT/2012/ISS3130) were cultured in Schneider's Drosophila medium (Lonza) supplemented with 10% heat-inactivated fetal calf serum (HyClone) at 24 ◦C.

The complete medium used for antileishmanial activity assay was RPMI (EuroClone) supplemented with 10% heat-inactivated fetal calf serum (EuroClone), 20 mM Hepes, and 2 mM l-glutamine. To estimate the 50% inhibitory concentration (IC50), the MTT (3-[4.5-dimethylthiazol-2-yl]-2.5-diphenyltetrazolium bromide) method was used. Compounds were dissolved in DMSO and then diluted with medium to achieve the required concentrations. Drugs were placed in 96-well round-bottom microplates and seven serial dilutions made. Amphotericin B was used as the reference anti-leishmanial drug. Parasites were diluted in complete medium to 5 × 10<sup>6</sup> parasites/mL and 100 μL of the suspension was seeded into the plates, incubated at 24 ◦C for 72 h and then 20 μL of MTT solution (5 mg/mL) was added into each well for 3 h. The plates were then centrifuged, the supernatants discarded and the resulting pellets dissolved in 100 μL of lysing buffer consisting of 20% (*w*/*v*) of a solution of SDS (Sigma), 40% of *<sup>N</sup>*,*<sup>N</sup>*-dimethylformamide (Merck) in H2O. The absorbance was measured spectrophotometrically at a test wavelength of 550 nm and a reference wavelength of 650 nm. The results are expressed as IC50 which is the dose of compound necessary to inhibit parasite growth by 50%; each IC50 value is the mean ± standard deviation of separate experiments performed in duplicate.

### *3.6. In Vitro Intracellular Amastigote Susceptibility Assays*

THP-1 cells (human acute monocytic leukemia cell line) were maintained in RPMI supplemented with 10% FBS (EuroClone), 50 μM 2-mercaptoethanol, 20 mM Hepes, 2 mM glutamine, at 37 ◦C in 5% CO2. For *Leishmania* infections, THP-1 cells were plated at 5 × 10<sup>5</sup> cells/mL in 16-chamber Lab-Tek culture slides (Nunc) and treated with 0.1 μM phorbol myristate acetate (PMA, Sigma) for 48 h to achieve differentiation into macrophages. Cells were washed and infected with metacyclic *L. infantum* promastigotes at a macrophage/promastigote ratio of 1/10 for 24 h. Cell monolayers were then washed and incubated in the presence of test compounds for 72 h. Slides were fixed with methanol and stained with Giemsa. The percentage of infected macrophages in treated and non-treated cells was determined by light microscopy.
