Synthesis and Antifouling Activity Evaluation of Analogs of Bromosphaerol, a Brominated Diterpene Isolated from the Red Alga Sphaerococcus coronopifolius

Marine biofouling is an epibiotic biological process that affects almost any kind of submerged surface, causing globally significant economic problems mainly for the shipping industry and aquaculture companies, and its prevention so far has been associated with adverse environmental effects for non-target organisms. Previously, we have identified bromosphaerol (1), a brominated diterpene isolated from the red alga Sphaerococcus coronopifolius, as a promising agent with significant antifouling activity, exerting strong anti-settlement activity against larvae of Amphibalanus (Balanus) amphitrite and very low toxicity. The significant antifouling activity and low toxicity of bromosphaerol (1) motivated us to explore its chemistry, aiming to optimize its antifouling potential through the preparation of a number of analogs. Following different synthetic routes, we successfully synthesized 15 structural analogs (2–16) of bromosphaerol (1), decorated with different functional groups. The anti-settlement activity (EC50) and the degree of toxicity (LC50) of the bromosphaerol derivatives were evaluated using cyprids and nauplii of the cirriped crustacean A. amphitrite as a model organism. Derivatives 2, 4, and 6–16 showed diverse levels of antifouling activity. Among them, compounds 9 and 13 can be considered as well-performing antifoulants, exerting their activity through a non-toxic mechanism.


Introduction
Marine biofouling is an epibiotic biological process that is characterized by the attachment of various micro-and macro-organisms of the marine environment on submerged surfaces [1][2][3][4][5]. It is a build-up process in which the initial conditioning film formation occurring over swamped surfaces is followed by a biofilm adhesion caused by bacterial and algal cell colonization leading to the settlement of macrofouling organisms [6][7][8][9].
Marine biofouling represents a global phenomenon generating undoubtedly profound economic and ecological problems that needs to be addressed vigorously. It affects almost any kind of submerged surface, including aquaculture systems, coastal electric power stations, various underwater constructions, and marine vessels [10][11][12]. It has been and remains a major issue for the shipping industry with an outstanding financial cost, mandating huge capital investments for its control and efficient management, often associated until recently with detrimental environmental effects. The attachment of microbial slimes, algae, and marine sessile organisms, such as barnacles and mussels, on the rough surfaces of ship hulls results in increased weight and hydrodynamic frictional resistance, accounting for a tremendous increase in fuel consumption, greenhouse gas emissions, dry-docking time, vessel maintenance, and marine transport cost [13][14][15][16][17][18].
Several antifouling methods have been developed over the years, with the most effective being based mainly on biocidal chemicals incorporated in coatings and paints. The antifouling organotin-based paints that have been widely applied, such as tributyltin (TBT), have been proven very effective in preventing fouling [19][20][21][22]. However, they are characterized by severe toxicity for marine life and therefore are nowadays prohibited worldwide by the International Maritime Organization (IMO) [23]. Their replacement by copper-based coatings and booster biocides, such as diuron, Irgarol 1051, SeaNine 211, and zinc pyrithione, failed to adequately address the issue of environmental impact [24], and most of them have already been banned by many European countries [25]. Inert, silicon-based and polymer-based coatings that were subsequently applied as an alternative approach have been proven inefficient since they are expensive, difficult to apply, and not durable enough to provide long-lasting hull protection, while the environmental impact of their additives still remains undetermined [26].
Concerning marine ecosystem and human health, an ecological approach is unquestionably the only way forward for antifouling technology [27,28]. The ideal candidate for biofouling control should be environmentally safe, providing maximum fouling protection. The solution for alternative, effective, non-toxic antifouling agents could be lying in the chemistry of marine natural products [29][30][31][32][33]. In the very competitive marine ecosystem, organisms, such as seaweeds, sponges, corals, and other invertebrates, have developed various chemical and biological defense mechanisms to protect their surfaces from fouling. Secondary metabolites (e.g., sulfated polyphenols, steroids, terpenoids, and alkaloids) and biopolymers have been reported to be involved in different interactions of marine life for the repellence, inhibition, and suppression of settlement and growth of fouling organisms [33]. Many isolated compounds from marine prokaryotes and eukaryotes have shown antifouling activity, fulfilling the U.S. Navy Program standards of EC 50 values < 25.0 mg/L, with actual EC 50 values less than 5 mg/L. Among them, macroalgal secondary metabolites have been regarded as non-toxic antifouling agents with great potential [34,35].
In the framework of our research investigations, we have previously identified bromosphaerol (1), a brominated diterpene isolated from the red alga Sphaerococcus coronopifolius, as a promising agent with significant antifouling activity [36,37]. In these studies, bioassays conducted using larvae of the cirriped crustacean Amphibalanus (Balanus) amphitrite showed that bromosphaerol (1) exerted significant anti-settlement activity with an EC 50 value of 0.23 mg/L, combined with extremely low toxicity (LC 50 > 100 mg/L), resulting in an impressive therapeutic ratio (TR C = LC 50 /EC 50 ) of 434.78. The significant antifouling activity and low toxicity of bromosphaerol (1) motivated us to explore its chemistry, aiming to optimize its antifouling potential through the preparation of a number of analogs. Herein, we report the synthesis of a series of bromosphaerol derivatives, along with the evaluation of their antifouling activity.

Results and Discussion
In order to improve the antifouling potential of the brominated diterpene bromosphaerol (1), 15 structural analogs involving transformations at ∆ 1 double bond and positions C-11, C-16, and C-17 (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16) were designed and synthesized (Figures 1 and S1-S46). Our strategy for obtaining initial structure-antifouling activity relationships for bromosphaerol involved (a) introducing polar groups at C-1 and/or C-2 (2-6 and 8), (b) removing the C-11 hydroxyl group (9 and 10), and (c) substituting C-2 with functional groups (ester and oxime), while the ∆ 1 double bond was repositioned to C-1-C-10 to allow for the generation of an extended conjugated system (7 and 11-16). Initially, our synthetic efforts were focused on introducing an epoxide ring at the Δ 1 double bond of 1. This was achieved using p-chloroperbenzoic acid in dichloromethane, which resulted in a mixture of the two diastereomeric epoxides 2 and 3 ( Figure 2) in a 65:35 ratio and 75% overall yield, both of which were subsequently isolated in pure form. The configuration at the chiral centers C-1 and C-2 of 2 was deduced as 1S,2R on the basis of NOE enhancements of H-1 and H-2 with H3-19 and H3-20, respectively. Subsequently, we aimed at introducing a carbonyl functionality at C-1 or C-2. Therefore, bromosphaerol (1) was subjected to a hydroboration/oxidation sequence, using a borane tetrahydrofuran complex and sodium perborate, followed by pyridinium chlorochromate (PCC)-mediated oxidation of the generated alcohols. The above synthetic strategy resulted in a mixture of the ketone 4 and the precursor alcohol 5 due to incomplete oxidation, in a 1:0.7 ratio and a 41% yield. As a side product of the reaction, compound 6 was isolated in 45% yield ( Figure 2). Compound 6, bearing an oxygen bridge between C-1 and C-17, was formed during the hydroboration step through an SN2 nucleophilic attack of the C-1 epimer of 5 on the brominated carbon C-17 and could not be oxidized further upon PCC treatment. Initially, our synthetic efforts were focused on introducing an epoxide ring at the ∆ 1 double bond of 1. This was achieved using p-chloroperbenzoic acid in dichloromethane, which resulted in a mixture of the two diastereomeric epoxides 2 and 3 ( Figure 2) in a 65:35 ratio and 75% overall yield, both of which were subsequently isolated in pure form. The configuration at the chiral centers C-1 and C-2 of 2 was deduced as 1S,2R on the basis of NOE enhancements of H-1 and H-2 with H 3 -19 and H 3 -20, respectively. Subsequently, we aimed at introducing a carbonyl functionality at C-1 or C-2. Therefore, bromosphaerol (1) was subjected to a hydroboration/oxidation sequence, using a borane tetrahydrofuran complex and sodium perborate, followed by pyridinium chlorochromate (PCC)-mediated oxidation of the generated alcohols. The above synthetic strategy resulted in a mixture of the ketone 4 and the precursor alcohol 5 due to incomplete oxidation, in a 1:0.7 ratio and a 41% yield. As a side product of the reaction, compound 6 was isolated in 45% yield ( Figure 2). Compound 6, bearing an oxygen bridge between C-1 and C-17, was formed during the hydroboration step through an SN 2 nucleophilic attack of the C-1 epimer of 5 on the brominated carbon C-17 and could not be oxidized further upon PCC treatment. In a next step, we attempted to synthesize the bromohydrin derivatives 7a and 8a of 1 through treatment with N-bromoacetamide in the presence of perchloric acid in a mixture of water/dioxane. Instead, we isolated the α,β-unsaturated ketone 7, and the C-2 brominated compound 8 ( Figure 2). Compound 7 could be generated from the intermediate bromohydrin 7a through oxidation of the C-2 alcohol followed by E2 elimination of the C-1 bromine and subsequent formation of the C-1-C-10 double bond. Compound 7 was also isolated as the single product from the oxidation of the mixture of the diastereomeric epoxides 2 and 3 using Jones reagent. On the other hand, hypobromous acid generated in situ from NBA and perchloric acid reacted, with the Δ 1 double bond affording the 2α-bromo C-1 carbocation intermediate, which reacted further with the C-11 alcohol to produce the corresponding compound 8 bearing a bromine atom at C-2 and an additional fused furan ring composed of C-1, C-10, C-9, and C-11. The stereochemistry at the chiral centers C-1, C-2, and C-11 was deduced as 1R,2S,11R on the basis of the crosspeaks observed in the NOESY spectrum, as well as the measured coupling constants. In particular, the coupling constant measured between H-1 and H-10 (J = 11.8 Hz) allowed for the determination of the configuration of the oxygenated H-1 as axial, whereas the coupling constant measured between H-1 and H-2 (J = 3.2 Hz) allowed for the determination of the configuration of the bromomethine H-2 as equatorial. Additionally, the NOE correlations of H-1 with H-9, H3-11, and H-17b, as well as of H3-11 with H-9 and H-14, dictated that the stereochemistry at C-11 remained as R. This proposed mechanism is supported by the relative cis stereochemistry of H-1 and H-2 in compound 8. In the case of the formation of a bromonium ion intermediate, an anti-addition would be expected, resulting in a trans relative stereochemistry of H-1 and H-2.

OH
Furthermore, the regiomeric olefins 9 and 10 were formed in 70% and 10% yield, respectively, by an elimination reaction of the hydroxyl group at C-11 upon treatment of In a next step, we attempted to synthesize the bromohydrin derivatives 7a and 8a of 1 through treatment with N-bromoacetamide in the presence of perchloric acid in a mixture of water/dioxane. Instead, we isolated the α,β-unsaturated ketone 7, and the C-2 brominated compound 8 ( Figure 2). Compound 7 could be generated from the intermediate bromohydrin 7a through oxidation of the C-2 alcohol followed by E2 elimination of the C-1 bromine and subsequent formation of the C-1-C-10 double bond. Compound 7 was also isolated as the single product from the oxidation of the mixture of the diastereomeric epoxides 2 and 3 using Jones reagent. On the other hand, hypobromous acid generated in situ from NBA and perchloric acid reacted, with the ∆ 1 double bond affording the 2α-bromo C-1 carbocation intermediate, which reacted further with the C-11 alcohol to produce the corresponding compound 8 bearing a bromine atom at C-2 and an additional fused furan ring composed of C-1, C-10, C-9, and C-11. The stereochemistry at the chiral centers C-1, C-2, and C-11 was deduced as 1R,2S,11R on the basis of the cross-peaks observed in the NOESY spectrum, as well as the measured coupling constants. In particular, the coupling constant measured between H-1 and H-10 (J = 11.8 Hz) allowed for the determination of the configuration of the oxygenated H-1 as axial, whereas the coupling constant measured between H-1 and H-2 (J = 3.2 Hz) allowed for the determination of the configuration of the bromomethine H-2 as equatorial. Additionally, the NOE correlations of H-1 with H-9, H 3 -11, and H-17b, as well as of H 3 -11 with H-9 and H-14, dictated that the stereochemistry at C-11 remained as R. This proposed mechanism is supported by the relative cis stereochemistry of H-1 and H-2 in compound 8. In the case of the formation of a bromonium ion intermediate, an anti-addition would be expected, resulting in a trans relative stereochemistry of H-1 and H-2.
Furthermore, the regiomeric olefins 9 and 10 were formed in 70% and 10% yield, respectively, by an elimination reaction of the hydroxyl group at C-11 upon treatment of 1 with trimethylsilyl trifluoromethanesulfonate in the presence of acetic anhydride ( Figure 3). Compounds 9 and 10, named bromosphaerenes B and A, respectively, were previously isolated from S. coronopifolius by Fattorusso et al. (1983), and their structures were confirmed through dehydration of bromosphaerol (1) upon heating at 100 • C with phosphorus oxychloride-pyridine to afford bromosphaerene A (10) as the major product [38]. In contrast, our synthetic method provided preferentially bromosphaerene B (9).
1 with trimethylsilyl trifluoromethanesulfonate in the presence of acetic anhydride (Figure 3). Compounds 9 and 10, named bromosphaerenes B and A, respectively, were previously isolated from S. coronopifolius by Fattorusso et al. (1983), and their structures were confirmed through dehydration of bromosphaerol (1) upon heating at 100 °C with phosphorus oxychloride-pyridine to afford bromosphaerene A (10) as the major product [38]. In contrast, our synthetic method provided preferentially bromosphaerene B (9). The α,β-unsaturated ketone 7 was proven to be a valuable synthetic intermediate toward the synthesis of a number of analogs. Thus, the Horner-Wadsworth-Emmons reaction of 7 with triethyl phosphonoacetate afforded the unsaturated ester 11 in 90% yield ( Figure 4), as a mixture of E,Z geometric isomers (in a 6 to 4 ratio). Finally, a series of oxime derivatives (12)(13)(14)(15)(16) were obtained in good to excellent yields (56 to 98%), using ketone 7 and various substituted alkoxyamines in the presence of pyridine ( Figure 4).  The results of the settlement inhibition assay performed on cypris larvae of A. amphitrite and the mortality test carried out on the stage II nauplii of the same model organism for bromosphaerol derivatives 2, 4, and 6-16 are shown in Table 1, summarizing the EC50 values obtained for cyprids settlement test (after 72 h), the LC50 values obtained for cypris larvae mortality (after 72 h), and the LC50 values of naupliar mortality (after 48 h). According to the guidelines of the U.S. Navy Program that require an EC50 (settlement inhibition) value lower than 25 mg/L for a compound to be considered as a promising natural antifoulant, 9 of the 13 derivatives tested in this study meet this requirement. In particular, compounds 2, 4, 6, and 7, which bear an oxygen moiety at C-1 or C-2, exhibit similar EC50 values ranging from 10.44 to 8.75 mg/L. Conversely, the introduction of a bromine substituent at C-2 abolishes activity. This is also the case for the α,β-unsaturated ester analog 11. Interestingly, derivative 9, in which the hydroxyl at C-11 was eliminated to form the The α,β-unsaturated ketone 7 was proven to be a valuable synthetic intermediate toward the synthesis of a number of analogs. Thus, the Horner-Wadsworth-Emmons reaction of 7 with triethyl phosphonoacetate afforded the unsaturated ester 11 in 90% yield ( Figure 4), as a mixture of E,Z geometric isomers (in a 6 to 4 ratio). Finally, a series of oxime derivatives (12)(13)(14)(15)(16) were obtained in good to excellent yields (56 to 98%), using ketone 7 and various substituted alkoxyamines in the presence of pyridine ( Figure 4).

Br
1 with trimethylsilyl trifluoromethanesulfonate in the presence of acetic anhydride (Figure 3). Compounds 9 and 10, named bromosphaerenes B and A, respectively, were previously isolated from S. coronopifolius by Fattorusso et al. (1983), and their structures were confirmed through dehydration of bromosphaerol (1) upon heating at 100 °C with phosphorus oxychloride-pyridine to afford bromosphaerene A (10) as the major product [38]. In contrast, our synthetic method provided preferentially bromosphaerene B (9). The α,β-unsaturated ketone 7 was proven to be a valuable synthetic intermediate toward the synthesis of a number of analogs. Thus, the Horner-Wadsworth-Emmons reaction of 7 with triethyl phosphonoacetate afforded the unsaturated ester 11 in 90% yield (Figure 4), as a mixture of E,Z geometric isomers (in a 6 to 4 ratio). Finally, a series of oxime derivatives (12)(13)(14)(15)(16) were obtained in good to excellent yields (56 to 98%), using ketone 7 and various substituted alkoxyamines in the presence of pyridine ( Figure 4).  The results of the settlement inhibition assay performed on cypris larvae of A. amphitrite and the mortality test carried out on the stage II nauplii of the same model organism for bromosphaerol derivatives 2, 4, and 6-16 are shown in Table 1, summarizing the EC50 values obtained for cyprids settlement test (after 72 h), the LC50 values obtained for cypris larvae mortality (after 72 h), and the LC50 values of naupliar mortality (after 48 h). According to the guidelines of the U.S. Navy Program that require an EC50 (settlement inhibition) value lower than 25 mg/L for a compound to be considered as a promising natural antifoulant, 9 of the 13 derivatives tested in this study meet this requirement. In particular, compounds 2, 4, 6, and 7, which bear an oxygen moiety at C-1 or C-2, exhibit similar EC50 values ranging from 10.44 to 8.75 mg/L. Conversely, the introduction of a bromine substituent at C-2 abolishes activity. This is also the case for the α,β-unsaturated ester analog 11. Interestingly, derivative 9, in which the hydroxyl at C-11 was eliminated to form the The results of the settlement inhibition assay performed on cypris larvae of A. amphitrite and the mortality test carried out on the stage II nauplii of the same model organism for bromosphaerol derivatives 2, 4, and 6-16 are shown in Table 1, summarizing the EC 50 values obtained for cyprids settlement test (after 72 h), the LC 50 values obtained for cypris larvae mortality (after 72 h), and the LC 50 values of naupliar mortality (after 48 h). According to the guidelines of the U.S. Navy Program that require an EC 50 (settlement inhibition) value lower than 25 mg/L for a compound to be considered as a promising natural antifoulant, 9 of the 13 derivatives tested in this study meet this requirement. In particular, compounds 2, 4, 6, and 7, which bear an oxygen moiety at C-1 or C-2, exhibit similar EC 50 values ranging from 10.44 to 8.75 mg/L. Conversely, the introduction of a bromine substituent at C-2 abolishes activity. This is also the case for the α,β-unsaturated ester analog 11. Interestingly, derivative 9, in which the hydroxyl at C-11 was eliminated to form the exocyclic double bond, exhibited potent antifouling activity with an EC 50 < 0.5 mg/L. Surprisingly, the endocyclic elimination congener 10 was less potent (EC 50 = 3.87 mg/L). Furthermore, the activity of derivatives 12-16 was influenced significantly by the nature of the oxime functionality. Thus, the unsubstituted oxime compound 12 and the carboxy oxime derivative 14 were inactive, in contrast to the methoxy oxime analog 13 and the methyl ester congener of 14, compound 15, that possess EC 50 < 0.5 mg/L. This is also the case for dimethylaminoethyl oxime derivative 16 showing EC 50 < 0.5 mg/L. Thus, analogs 9, 13, 15, and 16 exhibit very promising antifouling efficacy.  As previously reported [36], although the therapeutic ratio (TR) is traditionally calculated by taking into account naupliar mortality, measuring mortality on the same larval stage on which settlement is evaluated (cypris larvae, competent larval stage) is also important since TR should actually indicate whether the mechanism of settlement inhibition is based on a toxic effect. In addition, the LC 50 on nauplii can be considered as a good index of toxicity against non-target organisms since nauplii are considered as a representative zooplankton organism. It is evident that naupliar response to the tested bromosphaerol derivatives is very different from that of cyprids; indeed, naupliar mortality occurs at lower concentrations than mortality of cyprids. Table 1 reports the TR values calculated taking into account both nauplii (TR N ) and cyprids (TR C ) mortality values. High TR values indicate a low toxicity anti-settlement mechanism for the tested compounds. For derivatives showing promising EC 50 values (9, 13, 15, and 16), the TR N is quite low (ranging from 2.38 to 3.62), while higher values are observed for TR C , especially for derivatives 9 and 13 (>100). Concerning cyprids mortality, 9 and 13 showed LC 50(cypris) values higher than the maximum tested concentration (50 mg/L). Looking at the two more promising in terms of antifouling efficacy derivatives (9 and 13), the TR N values are much lower than the TR C values. Taking into account only the TR N values, we can assert that analogs 9 and 13 are characterized by good antifouling properties (EC 50 < 0.5 mg/L) but may exhibit some toxicity against non-target organisms, as mentioned above. Conversely, considering the TR C values (>100) of 9 and 13, both derivatives can be considered as well-performing antifoulants, exerting their activity through a non-toxic mechanism.

Biological Material
Specimens of S. coronopifolius were collected by scuba diving in the bay of Palaiokastritsa, Corfu, Greece, at a depth of 15-40 m in August 2013. A voucher specimen of the alga has been deposited at the Herbarium of the Section of Pharmacognosy and Chemistry of Natural Products, Department of Pharmacy, National and Kapodistrian University of Athens (ATPH/MP0226).

Extraction and Isolation
Air-dried algal tissues (0.5 kg dry weight) were exhaustively extracted with mixtures of CH 2 Cl 2 /MeOH (3:1) at room temperature. Evaporation of the solvents in vacuo afforded a dark green oily residue (18 g) that was subjected to vacuum column chromatography over silica gel, using cHex with increasing amounts of EtOAc and subsequently EtOAc with increasing amounts of MeOH as the mobile phase to yield 15 fractions (1-15). Fraction 3 (cHex/EtOAc 80:20, 10.9 g) was subjected to preparative reversed-phase HPLC using MeOH 100% as the mobile phase to afford sphaerococcenol A (2.1 g) and bromosphaerol (1) (4.45 g).

Synthesis of Analogs 2 and 3
To a stirred solution of bromosphaerol (1) (19.3 mg, 0.043 mmol) in anhydrous CH 2 Cl 2 (0.8 mL), a solution of m-chloroperoxybenzoic acid 77% (14.45 mg, 0.065 mmol) in CH 2 Cl 2 (0.8 mL) was added dropwise at 0 • C, and the reaction mixture was stirred for 2 h at 25 • C. Upon completion of the reaction (monitored by TLC), saturated aqueous Na 2 SO 3 solution (2.0 mL) was added. The pH was adjusted to 7 by the addition of 5% aqueous NaHCO 3 solution, and the reaction mixture was extracted with CH 2 Cl 2 (3 × 15 mL). The combined organic layers were dried over anhydrous Na 2 SO 4 , concentrated under reduced pressure, dried in vacuo to afford a mixture of the diastereomeric epoxides 2 and 3 in a 65:35 ratio (15 mg, 75%), which was subjected to flash column chromatography (elution system: petroleum ether 40-65 • C/acetone, 40:60 to 95:10) and subsequently normal-phase HPLC using cHex/EtOAc (75:25) as eluent to afford 2 (9.5 mg) and 3 (3.8 mg) in pure form.

Synthesis of Analogs 7 and 8
To a stirred solution of bromosphaerol (1) (220 mg, 0.48 mmol) in anhydrous 1,4dioxane (1.4 mL), a solution of 70% HClO 4 (0.28 mL, 1.8 mmol) and water (1.4 mL) was added dropwise, followed by the addition of a suspension of N-bromoacetamide (0.13 g, 0.95 mmol) in water (0.5 mL). The reaction mixture was stirred for 2 h at 25 • C (the completion of the reaction was monitored by TLC), saturated aqueous Na 2 S 2 O 3 solution (1 mL) was added, and the resulting mixture was stirred for 30 min. Subsequently, the mixture was poured into ice water and extracted with CH 2 Cl 2 (3 × 15 mL). The combined organic layers were washed with NaHCO 3 , dried over anhydrous Na 2 SO 4 , and concentrated under reduced pressure. The residue was purified by flash column chromatography (elution system: petroleum ether 40-65 • C/acetone, 90:10) to afford compound 7 (141 mg, 40% yield) and 8 (152 mg, 38%).

Evaluation of Settlement Inhibitory Activity
Cypris larvae were obtained from laboratory cultures of the crustacean cirriped A. amphitrite brood stock. Twenty to thirty adult barnacles were reared in 800 mL aerated beakers containing filtered natural seawater (FNSW) at 20 ± 1 • C, with a 16 h:8 h light:dark (L:D) cycle. They were fed every two days with nauplii of Artemia salina (100 mL, 20-35 larvae/mL) and Tetraselmis suecica (100 mL, 2 × 10 5 cells/mL). Twenty beakers containing adults reared under the above-mentioned conditions produced nauplii throughout the year. Nauplii were collected with a 5 mL pipette by positioning the beaker near a light source and reared in 500 mL beakers containing 0.22 µm FNSW gently aerated at 28 ± 1 • C with a 16 h:8 h L:D cycle. Nauplii were fed every 48 h with T. suecica (5 × 10 5 cells/mL) until, after 5-6 days, they reached the cyprid stage.
Newly metamorphosed cyprids were filtered and maintained in 0.22 µm FNSW at 6 • C for 4 days before being used in settlement assays [41]. Settlement tests were performed by adding 15-20 cyprids (for each replicate) to 24-well polystyrene plates containing 2 mL of bromosphaerol derivatives at different concentrations (0, 0.5, 5, and 50 mg/L). Four replicates were prepared for each concentration of each derivative, and the reported results are the mean values of the four replicates. The 24-well plates were stored for 72 h at 28 • C with a 16:8 L:D cycle. After 24, 48, and 72 h, the number of settled, non-settled, and dead larvae was measured under a stereomicroscope. EC 50 values (concentration of bromosphaerol derivatives causing 50% settlement inhibition to exposed organisms) were calculated with the results obtained after 72 h. Additionally, at the same time, LC 50(cypris) values were calculated as the concentration of bromosphaerol derivatives causing 50% mortality to the exposed organisms.

Evaluation of Toxicity
Acute environmental toxicity of bromosphaerol derivatives was tested by using stage II nauplii of A. amphitrite. Nauplii were obtained from adult brood stock as described above, collected, and immediately filtered in 0.22 µm FNSW. The toxicity assay was set within 2-4 h from nauplii collection. The test was performed by adding 15 to 25 stage II nauplii to 24-well polystyrene plates containing 2 mL of bromosphaerol derivatives at different concentrations (0, 0.5, 5, and 50 mg/L). Four replicates were prepared for each concentration of each derivative, and the reported results are the mean values of the four replicates. The plates were stored for 48 h at 20 • C with a 16:8 L:D cycle. After 24 and 48 h, the number of dead larvae was observed under a stereomicroscope. LC 50(nauplii) values were calculated as the concentration of bromosphaerol derivatives causing 50% mortality to the exposed organisms after 48 h of contact.

Statistical Analysis
Settlement inhibition (EC 50 ) at 72 h and mortality (LC 50 ) values at 48 h (for nauplii) and 72 h (for cyprids) were calculated using trimmed Spearman-Karber analysis [42]. The therapeutic ratio (TN) was defined as LC 50 /EC 50 . This index was calculated using mortality values measured for larvae at naupliar stage (TR N ) and for larvae at cypris stage (TR C ).

Conclusions
Following different synthetic routes, we successfully synthesized 15 structural analogs (2-16) of bromosphaerol (1) decorated with different functional groups. The anti-settlement activity (EC 50 ) and the degree of toxicity (LC 50 ) of the bromosphaerol derivatives were evaluated using cyprids and nauplii of A. amphitrite as a model organism. Derivatives 2, 4, and 6-16 showed diverse levels of antifouling activity, with four of them (9, 13, 15, and 16) displaying a bioactivity comparable to the one of the naturally occurring molecule. Among them, compounds 9 and 13 can be considered as well-performing antifoulants, exerting their activity through a non-toxic mechanism. The chemical diversity of these derivatives provides new insights on structure-activity relationship studies regarding settlement inhibition, helping to understand the antifouling mechanism of bromosphaerol (1), while potentially serving as bioinspired alternatives in antifouling coatings and paints.
Funding: This research was partially supported by the Cooperation 2011 Action of the Operational Programme "Competitiveness and Entrepreneurship" with co-funding by the European Social Fund (ESF) and National Resources, Greece (project GSRT-EPANII-11ΣYN-5-1274 "MariPaints"). Part of the current work was financially supported by the project "OPENSCREEN-GR: An Open-Access Research Infrastructure of Chemical Biology and Target-Based Screening Technologies for Human and Animal Health Agriculture and the Environment" (2018-2020) (MIS 5002691), which is implemented under the Action "Reinforcement of the Research and Innovation Infrastructure", funded by the Operational Programme "Competitiveness, Entrepreneurship and Innovation" (NSRF 2014-2020) and co-financed by Greece and the European Union (European Regional Development Fund).

Data Availability Statement:
The data presented in this study are available in Supplementary Material.
Acknowledgments: Access to the infrastructure obtained by the research project "Center for the study and sustainable exploitation of Marine Biological Resources" (CMBR, MIS 5002670) in the framework of the National Roadmap for Research Infrastructures is gratefully acknowledged. The involvement of Stavroula Konstantopoulou and Thalia Sokali in the extraction and isolation of bromosphaerol is gratefully acknowledged.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.