**1. Introduction**

Marine biofouling is the undesired accumulation of micro and macroorganisms on submerged surfaces, including bacteria, algae, larvae, and adults of various phyla, and their by-products, in a dynamic process that begins immediately after water-submersion and takes hours to months to develop [1]. Biofouling formation is divided into four distinct phases: soon after the physical adherence of macromolecules, the process becomes biological, designated as the microfouling phase, in which a bacterial biofilm is responsible for the establishment of an appropriate surface for the subsequent macrofouling organisms to settle, first as spores and larvae which then develop into adults [2,3].

Marine biofouling creates risk to several industries such as aquaculture, power plants, and shipping, amongs<sup>t</sup> others [4,5]. Settlement on the vessel´s hull damages the rudder and propulsion systems [4,6], and leads to an increasing drag of up to 60%, requiring up to 40% higher fuel consumption, in addition to increased CO2 and SO2 emissions [7]. Moreover, hull biofouling and ballast water transfer are the main causes for the introduction and spread of nonindigenous marine species into ecosystems worldwide leading to environmental imbalances [8–12]. Antifouling (AF) methods are estimated to save the shipping industry around €60 billion/year in fuel [4]. The most e ffective AF coatings contain biocides, such as tributyltin (TBT) and tributyltin oxide (TBTO), which were proven to be harmful to non-target organisms and the environment [13]. In fact, the International Maritime Organization banned TBT from ship surfaces in 2008, sparking the demand for new generations of nontoxic or environmentally benign AF solutions [14–16].

In the last years, several reviews reported studies on natural products (NP) isolated from marine organisms with AF activity [17–25]. The quest for AF agents from marine sources started with 2-furanone bromine derivatives extracted from red algae that were reported to prevent fouling [26]. Oroidin, a bromopyrrole alkaloid with AF activity isolated from sponges, inspired the design of 50-synthetic analogs [27,28]. An interesting approach to create AF "living" paints was developed [29] using marine bacteria that were directly encapsulated into polyurethane coatings [30] and hydrogels [31]. Regarding actinomycetes as AF sources, lobocompactol, a diterpene from *Streptomyces cinnabarinus*, was active against the macroalgae *Ulva pertusa*, the diatom *Navicula annexa,* and the bacterium *Pseudomonas aeruginosa* [32,33]. The 6-benzyl and 6-isobutyl 2,5-diketopiperazine derivatives from *S. praecox* were also active against *U. pertusa* and *N. annexa* [34]. 2,5-Diketopiperazines from *S. fungicidicus* and a branched-chain fatty acid, 12-methyltetradecanoid acid, from *Streptomyces* sp. inhibited the barnacle *Balanus amphitrite* and the polychaeta *Hydroides elegans* larval attachment, respectively [35–37]. Quercetin, a flavonoid obtained from *S. fradiae* revealed activity against the cyanobacteria *Anabena sp.* and *Nostoc sp.*, and mussel *Perna indica* larvae [38]. To the best of our knowledge, ivermectin, a chemically modified form of avermectin, a macrolide isolated from *S. avermitilis*, commonly used to treat parasitic worms and as insecticide, is the only marketed AF agen<sup>t</sup> obtained from actinomycetes, although terrestrial, which is used in paints for macrofouling inhibition.

We focused on bioprospecting marine-derived actinomycetes as producers of biofouling inhibitors for the potential development of marine-derived sustainable antifouling products. Napyradiomycins are a class of hybrid isoprenoids and/or meroterpenoids known for their antimicrobial and anticancer activities [39,40]. The napyradiomycins reported herein were isolated from a marine-derived actinomycete collection obtained from ocean sediments collected o ff the Madeira Archipelago [41]. The napyradiomycin molecular network with antibiofilm statistical bioactivity prediction was reported in a recent study by our group [42]. Here, we describe the capacity of napyradiomycins isolated from *S. aculeolatus* to inhibit micro and macrofouling species, and evaluate their ecotoxicity using an in silico approach. Targeting the primary attachment phases of the fouling process would allow preventing accumulation of other marine species. We recently patented the use of napyradiomycin derivatives for marine antifouling paints and coatings [43].

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

### *2.1. Napyradiomycin Derivatives Description*

Ethyl acetate (EtOAc) extracts of *Streptomyces aculeolatus* PTM-029 and PTM-420 [41,42] were subjected to micro and macro antifouling bioassay-directed fractionation and isolation, first by silica flash chromatography and subsequently by C18 reversed-phase HPLC to yield nine samples comprising twelve napyradiomycin derivatives (**1**–**12**) (Figure 1). The structures of all compounds were established by HR-MS and interpretation of NMR spectroscopic data, especially 2D NMR (i.e., COSY, HSQC, HMBC, TOCSY experiments), and by comparing the data with those previously reported for napyradiomycins [39,44–49]. All isolated napyradiomycins (**1**–**12**) were previously reported and their structural characterization is described in the Supplementary Materials. Napyradiomycins SF2415B3 (**3**), 4-dehydro-4a-dechloro- napyradiomycin SF2415B3 (**7**), A80915A (**9**), A80915C (**10**), 4-dehydro-4a-dechloro- napyradiomycin A80915A (**12**) and A1 (**1**), 18-hydroxynapyradiomycin A1 (**2**), A2 (**4**), 16-oxonapyradiomycin A2 (**5**), 4-dehydro-4a-dechloro-16-oxonapyradiomycin A2 (**6**), B3 (**8**), 4-dehydro-4a-dechloro-napyradiomycin B3 (**11**) were isolated from strains PTM-029 and PTM-420, respectively. Interestingly, there was a marked difference in these two sets of napyradiomycins: all the napyradiomycins isolated from PTM-029 (**3**), (**7**), (**9**), (**10**), and (**12**) have a methyl group in the core structure at position 7, while the ones obtained from strain PTM-420 (**1**), (**2**), (**4**–**6**), (**8**), and (**11**) have a hydrogen atom in that position (Figure 1).

**Figure 1.** Chemical structures of napyradiomycins isolated from marine-derived *S. aculeolatus* strains PTM-029 (**3**), (**7**), (**9**), (**10**), and (**12**) and PTM-420 (**1**), (**2**), (**4**–**6**), (**8**), and (**11**).

Despite some of the napyradiomycins were isolated as a mixture (**3** and **7**), (**5** and **6**), (**8** and **11**), and (**9** and **12**) no further purification efforts were performed, since antifouling agents are commonly used as mixture of compounds. For example, ivermectin antifouling product is commercialized as a mixture of two homologous compounds, ~80% of ivermectin B1a, with an ethyl group at position C-26 and ~20% of ivermectin B1b, with a methyl group at C-26 [50].

### *2.2. Assessment of Napyradiomycin Derivatives Micro and Macrofouling Inhibitory Activity*

The antimicrofouling activity of napyradiomycins (**1**–**12**) was evaluated by testing their inhibitory activity on bacterial propagation and bacterial biofilm formation. Five species of marine bacteria, which are biofilm prolific and described as fouling effectors, including dominant primary colonizers of submerged surfaces [51], were chosen as models for our bioactivity assays, namely *Marinobacter* *hydrocarbonoclasticus* (DSM 8798), *Cobetia marina* (DSM 4741)*, Micrococcus luteus* (DSM 20030, ATCC 4698), *Pseudooceanicola batsensis* (DSM 15984) and *Phaeobacter inhibens* (DSM 17395) [52–58].
